HK1222121B - Novel immunotherapy against several tumors, such as lung cancer, including nsclc - Google Patents

Description

New immunotherapies for treating various tumors, such as lung cancer, including NSCLC

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 also relates to peptide epitopes of tumor-associated cytotoxic T lymphocytes (CTLs), used alone or in combination with other tumor-associated peptides, for use as active pharmaceutical ingredients in vaccine compositions to stimulate anti-tumor immune responses. The present invention relates to 67 novel peptide sequences, and their derivative sequences from human tumor cell HLA class I and HLA class II molecules, for use in eliciting anti-tumor immune responses in vaccine compositions.

Background of the Invention

Lung cancer is the leading cause of cancer-related death in both men and women. It is the most common cancer worldwide in terms of both incidence and mortality. In 2008, there were 1.61 million new cases of lung cancer and 1.38 million deaths from the disease, with the highest rates occurring in Europe and North America.

Since 1987, the number of women dying from lung cancer each year has exceeded the number of women dying from breast cancer. Between 1991 and 2003, the mortality rate for men continued to decline significantly, by approximately 1.9% per year. The lung cancer mortality rate for women is now stabilizing after decades of continuous increases. These trends in lung cancer mortality reflect the decline in smoking rates over the past 30 years.

According to the National Cancer Institute (NCI), there are expected to be approximately 230,000 new cases of lung cancer and 160,000 lung cancer deaths in the United States in 2013.

For ease of treatment, lung cancer is clinically categorized as either small cell carcinoma (13%, SCLC) or non-small cell carcinoma (87%, NSCLC). The prognosis is generally poor. Of all lung cancer patients, 15% survive five years after diagnosis. Diagnosis is usually at an advanced stage. At presentation, 30-40% of NSCLC cases are at stage IV, and 60% of SCLC cases are at stage IV.

Treatment options vary depending on the type of tumor (small cell or non-small cell) and stage, and may include surgery, radiation therapy, chemotherapy, and targeted biological therapies such as bevacizumab and erlotinib. For localized cancer, surgery is usually the treatment of choice. Recent studies have shown that chemotherapy after surgery improves survival in early-stage non-small cell lung cancer. Because the tumor is often found after it has spread, radiation therapy is often used along with chemotherapy, sometimes in combination with surgery. Chemotherapy, alone or in combination with radiation therapy, is the treatment of choice for small cell lung cancer; a high percentage of patients respond to this treatment, and some achieve long-term responses.

The 1-year survival rate for lung cancer has increased slightly, from 37% in 1975-1979 to 42% in 2002, primarily due to advances in surgical techniques and combination therapy. However, the 5-year survival rate for all stages of lung cancer combined is only 16%. For patients with localized tumors at diagnosis, the survival rate is 49%, but only 16% of lung cancers are diagnosed at this early stage.

Despite this, there is an urgent need for safe and effective new therapies to treat lung cancer, especially different phenotypes of non-small cell lung cancer (NSCLC), gastric cancer, and brain cancer, which can improve patients' health without overusing chemotherapy drugs or other drugs that can cause serious side effects.

The present invention utilizes peptides that can stimulate a patient's immune system and act as anti-tumor drugs in a non-invasive manner.

SUMMARY OF THE INVENTION

First, the present invention relates to a peptide consisting of an amino acid sequence selected from SEQ ID No. 1 to SEQ ID No. 65, SEQ ID No. 76 to SEQ ID No. 84 and SEQ ID No. 92 or a variant sequence thereof (at least 80% homologous, preferably 90% homologous (preferably at least 80% or at least 90% identical) to SEQ ID No. 1 to SEQ ID No. 65, SEQ ID No. 76 to SEQ ID No. 84 and SEQ ID No. 92), and the variant sequence can induce T cells to cross-react with the peptide (or a pharmaceutically acceptable salt thereof), wherein the peptide is a non-full-length polypeptide.

In addition, the present invention also relates to a peptide of the present invention consisting of 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, which is at least 80% homologous, preferably 90% homologous (preferably at least 80% or at least 90% identical) to SEQ ID No. 1 to SEQ ID No. 65, SEQ ID No. 76 to SEQ ID No. 84 and SEQ ID No. 92, wherein the total length of the peptide or variant thereof of SEQ ID No. 1 to SEQ ID No. 65, SEQ ID No. 78 to SEQ ID No. 84 and SEQ ID No. 92 is 8 to 100 amino acids (preferably 8 to 30, most preferably 8 to 14), and the total length of SEQ ID Nos. 76 and 77 is 12 to 100 amino acids (preferably 12 to 30, most preferably 12 to 18).

The following table lists the peptides of the present invention and their corresponding SEQ ID NOs, as well as the expected source proteins of these peptides. All peptides in Tables 1a, 1b, and 1c can bind to the HLA A*02 allele, and the peptides in Table 1d can bind to the HLA-DR allele.

The peptides in Table 1c can also be used for the diagnosis and/or treatment of gastric cancer and/or glioblastoma.

The class II peptides in Table 1d can also be used for the diagnosis and/or treatment of gastric cancer and other cancers that overexpress or overpresent MMP12 or POSTN.

Therefore, the present invention relates to a peptide of the present invention consisting of the sequence of SEQ ID No. 76 or a variant thereof (at least 80% homologous, preferably 90% homologous (preferably at least 80% homologous or at least 90% identical) to SEQ ID No. 76). The total length of the peptide or variant thereof is 12 to 100 amino acids (preferably 12 to 30, most preferably 12 to 18). The present invention relates to a peptide of the present invention consisting of the sequence of SEQ ID No. 76.

Furthermore, the present invention relates to a peptide of the present invention consisting of the sequence of SEQ ID No. 77 or a variant thereof (at least 80% homologous, preferably 90% homologous (preferably at least 80% homologous or at least 90% identical) to SEQ ID No. 77). The total length of the peptide or variant thereof is 12 to 100 amino acids (preferably 12 to 30, most preferably 12 to 18). The present invention relates to a peptide of the present invention consisting of the sequence of SEQ ID No. 77.

Table 1a: Peptides of the invention

Table 1b: Other peptides of the present invention

Table 1c: Other peptides also overexpressed in glioblastoma and/or gastric cancer

Table 1d: MHC class II peptides of the present invention

Table 1e: Other preferred peptides of the present invention that are overexpressed in other tumors

Table 1f: Other peptides of the present invention overexpressed in other tumors

The present invention also relates to peptides according to the present invention that can bind to human major histocompatibility complex (MHC) class I or class II molecules.

The present invention also relates to peptides according to the present invention, which consist of or essentially consist of the amino acid sequences of SEQ ID No. 1 to SEQ ID No. 65, SEQ ID No. 76 to SEQ ID No. 84 and SEQ ID No. 92.

The present invention also relates to peptides according to the invention which are modified and/or comprise non-peptide bonds.

The present invention also relates to peptides according to the invention, which are part of a fusion protein, in particular a protein fused to the N-terminal amino acids of the invariant chain (Ii) associated with the HLA-DR antigen, or to an antibody (or its sequence), such as a dendritic cell-specific antibody.

The present invention also relates to a nucleic acid encoding the peptide according to the present invention.

The present invention also relates to the nucleic acid according to the present invention, which is DNA, cDNA, PNA, RNA or a complex of DNA, cDNA, PNA, RNA.

The present invention also relates to an expression vector, which can express the nucleic acid according to the present invention.

The present invention also relates to a peptide according to the present invention, a nucleic acid according to the present invention or a pharmaceutical expression vector according to the present invention.

The present invention also relates to the antibody according to the present invention and a method for preparing the same.

The present invention also relates to a T cell receptor (TCR) according to the present invention, in particular a soluble TCR (sTCR), and a method for preparing the same.

The present invention also relates to a host cell comprising the nucleic acid according to the present invention, or an expression vector as described above.

The present invention also relates to a host cell according to the present invention, which must be an antigen presenting cell.

The present invention also relates to a host cell according to the present invention, wherein the antigen presenting cell is a dendritic cell.

The present invention also relates to a method for preparing the peptide according to the present invention, which comprises culturing the host cell according to the present invention, and separating the peptide from the host cell or its culture medium.

The present invention also relates to an in vitro method for preparing activated cytotoxic T lymphocytes (CTLs), which comprises contacting the in vitro CTLs with antigen-loaded human class I or class II MHC molecules (which are expressed on the surface of appropriate antigen-presenting cells for a sufficient time to activate the above-mentioned CTLs in an antigen-specific manner), wherein the antigen is any peptide according to the present invention.

The present invention also relates to a method according to the invention for loading an antigen onto a class I or class II MHC molecule expressed on the surface of a suitable antigen presenting cell (by contacting a sufficient amount of the antigen with the antigen presenting cell).

The present invention also relates to a method according to the present invention, wherein the antigen-presenting cell contains an expression vector capable of expressing the above-mentioned peptide comprising 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 above-mentioned variant amino acid sequence thereof.

The present invention also relates to activated cytotoxic T lymphocytes (CTLs) prepared by the method of the present invention, which can selectively recognize cells that abnormally express a polypeptide containing the amino acid sequence of the present invention.

The present invention also relates to a method for killing target cells that aberrantly express a polypeptide comprising any of the amino acid sequences described herein in a patient, comprising administering an effective number of cytotoxic T lymphocytes (CTL) to the patient according to the method described herein.

The present invention also relates to a medicament or any of the above peptides used in the production of a medicament, a nucleic acid according to the present invention, an expression vector according to the present invention, a cell according to the present invention or an activated cytotoxic T lymphocyte according to the present invention.

The present invention also relates to the use according to the present invention, wherein the medicament is a vaccine.

The present invention also relates to the use according to the invention, wherein the medicament has anti-tumor activity.

The present invention also relates to the use according to the present invention, wherein the cancer cells are lung cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, pancreatic cancer or renal cancer cells, and glioblastoma cells.

The present invention also relates to specific marker proteins and biomarkers based on the peptides according to the present invention, which can be used for the diagnosis and/or prognosis of lung cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, pancreatic cancer or renal cancer, and glioblastoma.

The present invention also relates to the use of the novel cancer treatment target.

The initiation of an immune response depends on antigens that are recognized as foreign by the host immune system. The discovery of tumor-associated antigens has raised the possibility of harnessing the host immune system to hinder tumor growth. Various mechanisms are currently being explored for cancer immunotherapy, leveraging both humoral and cellular responses.

Specific elements of the cellular immune response can specifically recognize and destroy tumor cells. Cytotoxic T lymphocytes (CTLs), isolated from tumor-infiltrating cell populations or peripheral blood, have been shown to play a crucial role in the innate immune defense against cancer. CD8+ T cells are particularly important in this response because they recognize peptide molecules bearing the major histocompatibility complex (MHC), typically composed of 8 to 10 amino acid residues derived from proteins or defective ribosomal products (DRIPS) in the cytosol. MHC molecules in humans are also known as human leukocyte antigens (HLA).

MHC molecules are divided into two classes: MHC class I molecules are present in most nucleated cells. MHC molecules consist of either one α heavy chain and one β-2 microglobulin (MHC class I receptor) or one α and one β heavy chain (MHC class II receptor). Their three-dimensional conformation forms a binding groove for non-covalent interactions with peptides. Peptides presented by class I MHC are primarily derived from endogenous proteins, DRIPs, and proteolytic cleavage of larger peptides. MHC class II molecules are found primarily on professional antigen-presenting cells (APCs), and the peptides they present are primarily derived from exogenous or transmembrane proteins taken up by APCs during endocytosis. Complexes of peptides with MHC class I molecules are recognized by CD8-positive cytotoxic T cells bearing the appropriate TCR (T cell receptor), while complexes of peptides with MHC class II molecules are recognized by CD4-positive helper T cells bearing the appropriate TCR. As is well known in the art, the TCR, peptide, and MHC are thus present in a stoichiometric ratio of 1:1:1.

CD4-positive helper T cells play a crucial role in initiating and maintaining effective CD8-positive cytotoxic T cell responses. Identifying the phenotype of CD4-positive T cells that respond to tumor-associated antigens (TAAs) is crucial for the development of therapeutics that stimulate antitumor immune responses (Kobayashi et al., 2002; Qin et al., 2003; Gnjatic et al., 2003). At the tumor site, helper T cells provide a pro-CTL cytokine milieu (Mortara et al., 2006) and attract effector cells such as CTLs, natural killer cells, macrophages, and granulocytes (Hwang et al., 2007).

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

Experiments in mammalian models (such as mice) have shown that even in the absence of CTL effector cells (such as CD8-positive T lymphocytes), CD4-positive T cells are sufficient to suppress tumor manifestation by inhibiting angiogenesis caused by interferon gamma (IFNγ) secretion.

Furthermore, studies have shown that CD4-positive T cells that recognize peptides derived from tumor-associated antigens presented by HLA class II molecules can prevent tumor progression by eliciting antibody (Ab) responses.

Unlike tumor-associated peptides that bind to HLA class I molecules, only a few class II ligands for tumor-associated antigens (TAAs) have been reported to date.

Because the constitutive expression of HLA class II molecules is generally limited to cells of the immune system, it was considered impossible to isolate class II peptides directly from primary tumors. However, Dengjel et al. successfully identified several MHC class II phenotypes directly from tumors (WO 2007/028574, EP 1 760 088 B1; (Dengjel et al., 2006)).

The antigens recognized by tumor-specific cytotoxic T lymphocytes (i.e., their phenotype) can be molecules derived from various proteins (e.g., enzymes, receptors, transcription factors, etc.) that are expressed and upregulated in the corresponding tumor cells (compared to the homologous unaltered cells).

Since both types of responses (CD8- and CD4-dependent, respectively) can work together to produce synergistic antitumor effects, the identification and characterization of tumor-associated antigens (recognized by CD8+ CTLs (ligand: MHC class I molecule + peptide phenotype) or CD4-positive helper T cells (ligand: MHC class II molecule + peptide phenotype)) are important for the development of antitumor vaccines.

The present invention also relates to two very useful novel MHC class II peptides (corresponding to SEQ ID NOs 76 and 77). These two peptides are particularly useful for the diagnosis and/or treatment of gastric cancer, NSCLC, and other cancers that overexpress and/or overpresent MMP12 and POSTN, respectively.

The present invention also relates to so-called length variants of novel MHC class II peptides (corresponding to SEQ ID NOs 76 or 77). As described above, the peptide corresponding to SEQ ID NO 76 comprises the amino acid sequence INNYTPDMNREDVDYAIR (MMP12 peptide), and the peptide corresponding to SEQ ID NO 77 comprises the amino acid sequence TNGVIHVVDKLLYPADT (POSTN-002 peptide). The length variants typically have N- and/or C-terminal extensions (1 to 5 amino acids, preferably 1 to 10 amino acids) or N- and/or C-terminal shortenings (1 to 5 amino acids). These variants are still capable of binding to MHC and eliciting the cellular immune responses described herein. Peptide binding to class II proteins is known to be independent of size, with lengths ranging from 11 to 30 amino acids. The peptide-binding groove in MHC class II molecules is open at both ends, allowing for binding of relatively long peptides. While the 9-residue "core" stretch is most important for peptide recognition, the flanking regions play an important role in the specificity of the peptide for the class II allele (see 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). Using a variety of existing software tools (such as those described above), one with the current level of skill can determine the binding motif and, from this, determine whether the MHC class II peptide can be extended and/or deleted relative to SEQ ID NO 76 or 77 to generate length variants.

To trigger (elicit) a cell-based immune response, a peptide must bind to an MHC molecule. This process depends on the MHC molecule allele and the specific polymorphism in the peptide's amino acid sequence. MHC class I binding peptides are typically 8-12 amino acid residues in length and typically contain two conserved residues ("anchor sites") that react with the binding groove of the corresponding MHC molecule. Each MHC allele thus contains a "binding motif" that determines which peptides will specifically bind to the binding groove.

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

The antigens recognized by tumor-specific cytotoxic T lymphocytes (i.e., their phenotype) can be molecules derived from various proteins (e.g., enzymes, receptors, transcription factors, etc.), which are expressed in the corresponding tumor cells and their expression is upregulated compared with the same source unchanged cells.

Current tumor-associated antigens are mainly divided into the following groups:

a) Tumor-testicular antigens: The first TAAs discovered to be recognized by T cells belong to this category. They were initially termed tumor-testicular (CT) antigens because members of this group are expressed in histologically distinct human tumors, while expression in normal tissues is restricted to testicular spermatocytes/spermatogonia and, rarely, the placenta. Because testicular cells do not express class I and class II HLA molecules, these antigens are not recognized by T cells in normal tissues and are therefore considered immunologically tumor-specific. Well-known CT antibodies include members of the MAGE family and NY-ESO-1.

b) Differentiation antigens: These antigens are present in both tumors and the normal tissues from which they arise, most commonly in melanomas and normal melanocytes. Many of these melanocyte lineage-associated proteins are involved in melanin biosynthesis and, therefore, are not tumor-specific, but are widely used in tumor immunotherapy. Examples include, but are not limited to, tyrosinase, Melan-A/MART-1 (for melanoma), and PSA (for prostate cancer).

c) Overexpressed TAAs: Genes encoding ubiquitously expressed TAAs have been found in a variety of histologically diverse tumors, as well as in many normal tissues, often at low expression levels. It is possible that many phenotypes processed and potentially presented by normal tissues are below the threshold for T cell recognition, but their overexpression in tumor cells can trigger anti-tumor responses by breaking previously established tolerance. Well-known antigens in this TAA class include Her-2/neu, survivin, telomerase, and WT1.

d) Tumor-specific antigens: These unique TAAs arise from mutations in normal genes (e.g., β-catenin, CDK4, etc.). Some of these molecular changes are associated with neoplastic transformation and/or progression. Tumor-specific antigens typically elicit a robust immune response and do not carry the risk of autoimmune reactions against normal tissues. Furthermore, these TAAs are often associated only with the specific tumor in which they are found and are not present across multiple tumors.

e) TAAs derived from aberrant post-translational modifications: These TAAs may arise from proteins that are neither specifically nor overexpressed in tumors but may become tumor-associated antigens through post-translational processing that is primarily active in tumors. Examples include antigens arising from altered glycosylation patterns that trigger new phenotypes in tumors (e.g., MUCI) or protein splicing during degradation, which may or may not be tumor-specific.

f) Oncovirus proteins: These TAAs are viral proteins that play a key role in tumorigenesis and, because they are foreign (non-human), can elicit T cell responses. These proteins include human papillomavirus type 16 proteins, E6, and E7 (expressed in cervical cancer).

For a protein to be recognized by cytotoxic T lymphocytes as a tumor-specific or tumor-associated antigen and used therapeutically, specific prerequisites must be met. The antigen must be expressed primarily by tumor cells and absent or expressed only at very low levels in normal healthy tissues. Alternatively, in another preferred embodiment, the peptide must be over-presented by tumor cells (compared to normal healthy tissues). It is particularly desirable if the relevant antigen is not only present in a particular tumor type but also present at high levels. Tumor-specific and tumor-associated antigens are often derived from proteins that directly participate in the transformation of normal cells into tumor cells (through functions such as cell cycle control and apoptosis inhibition). Furthermore, downstream targets of proteins that directly trigger transformation may be indirectly tumor-associated due to upregulation. Such indirect tumor-associated antigens can also serve as targets for vaccination (Singh-Jasuja et al., 2004). In both cases, the phenotype must be present in the antigen's amino acid sequence, as such peptides derived from tumor-associated antigens ("immunogenic peptides") should be able to elicit T cell responses in vitro or in vivo.

Essentially, any peptide that can bind to an MHC molecule can serve as a T cell phenotype. Prerequisites for eliciting a T cell response in vitro or in vivo are the presence of T cells with the corresponding TCR and the absence of immune tolerance to this specific phenotype.

In summary, TAA is the starting point for the development of tumor vaccines. The identification and characterization methods of TAA are based on the use of CTLs (isolated from patients or healthy subjects), or on the differentiation transcriptional profile or differentiation peptide expression profile between tumors and normal tissues. However, simply identifying genes that are overexpressed in tumor tissues or human tumor cell lines (or selectively expressed in such tissues or cell lines) cannot provide accurate information for the use of antigens transcribed from such genes as immunotherapy. The reason is that T cells with corresponding TCRs must exist, and immune tolerance for this specific phenotype must not exist or be extremely small, resulting in only individual phenotypic subpopulations of such genes being suitable for this purpose. Therefore, in an embodiment of the present invention, the selected over-presented or selectively presented peptides must have corresponding functional and/or proliferative T cells. The functional T cells are defined as T cells ("effector T cells") that can be clonally expanded and produce effector functions after stimulation with specific antigens.

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

Helper T cells play an important role in the effector function of CTLs in anti-tumor immunity. Helper T cell phenotypes that trigger T _H1 -type helper T cell responses can support the effector function of CD8-positive killer T cells. The cytotoxic function of CD8-positive killer T cells directly targets tumor cells presenting tumor-associated peptide/MHC complexes. Therefore, tumor-associated helper T cell peptide phenotypes (alone or in combination with other tumor-associated peptides) can be used as active pharmaceutical ingredients in vaccine compositions to stimulate anti-tumor immune responses.

The following discloses the use of the peptide proteins according to the present invention in other cancers.

ATP-binding cassette subfamily A (ABC1) member 13 (ABCA13)

In humans, the ATP-binding cassette (ABC) family of transmembrane transporters comprises at least 48 genes and seven subfamilies. The predicted ABCA13 protein consists of 5,058 amino acid residues, making it the largest ABC protein to date (Prades et al., 2002). Knight et al. determined that ABCA13 is expressed in the hippocampus and cortex of mice and humans, regions associated with schizophrenia and bipolar disorder (Knight et al., 2009). The ABCA13 gene is located on chromosome 7p12.3, a region implicated in a pancreatic genetic disorder (Shwachman-Diamond syndrome) and containing a locus involved in T-cell tumor infiltration and metastasis, making it a candidate target for these disorders (Prades et al., 2002).

Matrix metalloproteinase 12 (macrophage elastase) (MMP12)

MMP12, also known as human metalloproteinase (HME) or macrophage elastase (MME), is a zinc endopeptidase that degrades elastin. Its substrate range is broad, encompassing other matrix proteins (e.g., collagen, fibronectin, laminin, proteoglycans) and non-matrix proteins (e.g., α-1-antitrypsin). In asthma, emphysema, and chronic obstructive pulmonary disease (COPD), MMP12 is implicated in alveolar destruction and airway remodeling (Cataldo et al., 2003; Wallace et al., 2008). MMP12 is involved in macrophage migration and, through its ability to produce angiostatin from plasminogen, inhibits angiogenesis (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 menstruation (Chakraborti et al., 2003; Labied et al., 2009), but is also involved in pathological processes of tissue destruction.

Although data are based on a limited number of cases, there is ample literature demonstrating that MMP12 overexpression is common in cancer (Denys et al., 2004; Hagemann et al., 2001; Ma et al., 2009; Vazquez-Ortize et al., 2005; Ye et al., 2008). However, the data regarding the impact of MMP12 overexpression on clinical parameters and prognosis remain controversial. MMP12 may be involved in metastasis by participating in matrix lysis, while also inhibiting tumor growth through the production of angiostatin, thereby suppressing angiogenesis (Gorrin-Rivas et al., 2000; Gorrin-Rivas et al., 1998; Kim et al., 2004).

The role of MMP12 expression in lung cancer remains controversial. Overexpression of MMP12 in epithelial cells has been reported in inflammation-induced lung remodeling. MMP12 upregulation may play a role in the transition from emphysema to lung cancer (Qu et al., 2009). Animal studies have shown that expression of MMP12 in interstitial or macrophage cells can inhibit lung tumor growth (Acuff et al., 2006; Houghton et al., 2006). However, there are also reports that overexpression of MMP12 in lung tumors is associated with tumor recurrence, metastasis, and shorter recurrence-free survival (Cho et al., 2004; Hofmann et al., 2005).

Actin-binding protein (DST)

DST (BPAG1-e) encodes a focal adhesion protein belonging to the desmoplakin family. BPAG1-e is expressed in epithelial tissues and anchors keratin-containing intermediate filaments to hemidesmosomes (HDs). HDs are multiprotein adhesion complexes that promote epithelial-mesenchymal centromere attachment in stratified and complex epithelia (Litjens et al., 2006). Regulation of HD function is crucial for a range of biological processes, including wound healing and keratinocyte differentiation and migration, where cells detach from their substrates and acquire a motile phenotype, in tumor invasion (Litjens et al., 2006).

Malignant melanoma is one of the most aggressive tumor types. BPAG1 is expressed in human melanoma cell lines (A375 and G361) as well as in 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 have shown promise as diagnostic markers for melanoma (Shimbo et al., 2010). DST has also been 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).

Matrix remodeling-associated protein 5 (MXRA5)

MXRA5 encodes a proteoglycan that belongs to a gene family involved in ECM remodeling and cell-cell adhesion (Rodningen et al., 2008). Although the function of MXRA5 in cancer remains unclear, somatic mutations in MXRA5 have been identified in tumors from various tissues, such as skin, brain, lung, and ovary. RT-PCR microarray analysis of MXRA5 revealed its overexpression in colon cancer compared with normal colon tissue (13 colorectal cancers and 13 normal tissues) (Zou et al., 2002). In a recent study, MXRA5 was the second most frequently mutated gene in NSCLC (after TP53) (Xiong et al., 2012).

Cyclin-dependent kinase 4 (CDK4)/cyclin-dependent kinase 6 (CDK6)

CDK4 is a member of the Ser/Thr protein kinase family. As the catalytic subunit of the protein kinase complex, CDK4 is crucial for progression through the G1 phase of the cell cycle. Its activity is restricted to the G1-S transition of the cell cycle, and its expression is primarily controlled at the transcriptional level (Xiao et al., 2007). CDK4 and CDK6 enzymes, along with their regulatory factors (such as cyclins), play key roles in embryogenesis, homeostasis, and carcinogenesis (Graf et al., 2010).

CDK4 protein expression is significantly elevated in lung cancer tissue compared with normal tissue (P < 0.001). Patients with higher CDK4 expression have significantly shorter overall survival than those with lower CDK4 levels. Multivariate analysis demonstrated that CDK4 expression was an independent prognostic indicator of survival in lung cancer patients (P < 0.001). Furthermore, inhibition of CDK4 expression significantly increased expression of the cell cycle regulator p21 (Wu et al., 2011a). In lung cells expressing an endogenous K-Ras oncogene, ablation of CDK4 (but not CDK2 or CDK6) immediately triggered a senescence response. This response was not observed in lung tissue expressing a single CDK4 allele or other K-Ras-expressing tissues. Targeting the CDK4 allele in advanced tumors detectable by computed tomography also triggered senescence and delayed tumor progression (Puyol et al., 2010).

The heterogeneous nuclear ribonucleoprotein H1 (H) (HNRNPH1)/heterogeneous nuclear ribonucleoprotein H2 (H') (HNRNPH2) genes belong to a subfamily of ubiquitously expressed heterogeneous nuclear ribonucleoproteins (hnRNPs). hnRNPs are RNA-binding proteins that form complexes with heterogeneous nuclear RNA (hnRNA). These proteins associate with pre-mRNA in the nucleus and influence pre-mRNA processing as well as other aspects of mRNA metabolism and transport.

As a central splicing oncogenic switch, hnRNP H activity is implicated in the development and progression of gliomas, reflecting the reactivation of the stem cell landscape and mediating multiple key components of aggressive tumor behavior, including apoptosis escape and invasiveness (Lefave et al., 2011). Small interfering RNA-mediated knockdown of hnRNP H or A-Raf induces MST2-dependent apoptosis. In contrast, upregulation of hnRNP H or A-Raf expression blocks etoposide-induced apoptosis. Upregulation of hnRNP H/H' has been observed in a limited number of tissues that normally express low cytoplasmic levels of hnRNP H/H', such as pancreatic, hepatocellular, and gastric cancers (Honore et al., 2004).

Tetratricopeptide repeat, ankyrin repeat and coiled-coil containing 2 (TANC2)

The TANC family, which includes TANC1 and TANC2, was discovered in 2005 (Han et al., 2010). TANC family proteins are involved in the regulation of dendritic spines, spatial learning, and embryonic development. This is evidenced by the fact that TANC1 deficiency in mice reduces dendritic spine density in the hippocampus and impairs spatial learning, while TANC2 deficiency causes embryonic lethality. 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, with a significant portion localized to vesicle membranes (Han et al., 2010).

RING finger protein 213 (RNF213)

RNF213 encodes a protein containing a C3HC4-type RING finger domain, a specialized type of zinc finger that binds two zinc atoms and is thought to mediate protein-protein interactions.

A research group provided the first evidence that RNF213 is associated with genetic susceptibility to moyamoya disease (Liu et al., 2011b).

Another study showed that the RNF213 gene was associated with the genetic susceptibility of Han Chinese to moyamoya disease (Wu et al., 2012).

Solute carrier family 34 (sodium phosphate), member 2 (SLC34A2)

SLC34A2 is a pH-sensitive, sodium-dependent phosphate transporter. Upregulation of SLC34A2 in well-differentiated tumors reflects the cell differentiation process during ovarian cancer development and serves as a potential biomarker for ovarian cancer diagnosis and prognosis (Shyian et al., 2011). RT-PCR has demonstrated increased SLC34A2 expression in papillary thyroid carcinoma (Kim et al., 2010b). SLC34A2 expression is also significantly increased in breast cancer tissue compared to normal tissue (Chen et al., 2010a).

SET and MYND domain containing protein 3 (SMYD3)

Previous reports have shown that SMYD3, a histone H3 lysine 4-specific methyltransferase, plays a key role in the proliferation of colorectal cancer (CRC) and hepatocellular carcinoma (HCC). Another study also found elevated SMYD3 expression in a large proportion of breast cancer tissues. Similar to CRC and HCC, silencing SMYD3 with small interfering RNA targeting this gene inhibited breast cancer cell growth, suggesting that increased SMYD3 expression is also crucial 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 a key role in HeLa cell proliferation and migration/invasion and may serve as an effective therapeutic target for human cervical cancer (Wang et al., 2008b).

Aldosterone reductase family 1, member C1 (AKR1C1)/aldosterone reductase family 1, member C2 (AKR1C2) differ only by 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 AKR1C4, which is liver-specific) are expressed in various normal and diseased tissues and are therefore implicated in a variety of diseases, including lung, breast, prostate, and endometrial cancers, and myeloid leukemias (Brozic et al., 2011; Byrns et al., 2011). Sensitivity to cisplatin has been shown to correlate with AKR1C levels in lung cancer epithelial cell lines (Chen et al., 2010b) and NSCLC patients (Kuang et al., 2012; Stewart, 2010). Consequently, AKR1C overexpression has been shown to be a marker of poor prognosis and chemoresistance in human NSCLC (Wang et al., 2007). Overexpression of AKR1C2 has also been associated with prostate cancer progression (Huang et al., 2010). Depletion of AKR1C2 by RNAi inhibits tumorigenesis both in vitro and in vivo, suggesting that AKR1C2 siRNA may play a key role in inhibiting hepatocarcinogenesis (Dong-Dong, 2007).

Reticulin 1 (RCN1) EF-hand calcium-binding domain/Reticulin 3 (RCN3) EF-hand calcium-binding domain

RCN1 is a calcium-binding protein located in the lumen of the endoplasmic reticulum. Immunohistochemistry has shown that RCN is distributed in various organs in both fetuses and adults, primarily in endocrine and exocrine organs. Overexpression of RCN may play a role in tumorigenesis, tumor invasion, and drug resistance (Fukuda et al., 2007). RCN1 is a cell surface-bound protein expressed in both endothelial (EC) and prostate cancer (PCa) cell lines. RCN1 cell surface expression is upregulated by treatment of bone marrow endothelial cells with tumor necrosis factor α (Cooper et al., 2008). RCN1 is upregulated in colorectal cancer (CRC), localized in cancer cells or adjacent stromal cells (Watanabe et al., 2008). RCN3 is a member of the CREC (Cab45/reticulin/ERC45/calcein) family of multi-EF-handed Ca2+-binding proteins, which are localized in the secretory pathway (Tsuji et al., 2006). RCN3 has been suggested to be one of the important candidate genes in oligodendroglioma, although little is known about the function of RCN3 (Drucker et al., 2009).

Interleukin-8 (IL8)

IL8, a chemokine belonging to the CXC family, is a major mediator of inflammatory responses. Several cell types secrete this chemokine. It functions as a chemoattractant and is also a potent angiogenic factor. CXC (ELR+) chemokines similar to IL8 can induce angiogenesis, which may be important in cancers with an angiogenic phenotype, such as NSCLC (Arenberg et al., 1997). Recent studies have shown that tumor-derived IL8 can act as an attractant, prompting circulating tumor cells to home to the original tumor (breast cancer, colon cancer, and melanoma), resulting in a more aggressive tumor phenotype (Kim et al., 2009). IL-8 levels have been associated with lung cancer risk, even years before diagnosis (Pine et al., 2011). Activating KRAS or EGFR mutations can downregulate IL-8 expression in NSCLC; IL-8 expression levels are higher in males, smokers, elderly NSCLC patients, NSCLC with pleural involvement, and KRAS mutant adenocarcinomas; and in oncogenic KRAS-driven NSCLC, IL-8 plays a role in cell growth and migration (Sunaga et al., 2012).

G protein-coupled pyrimidine receptor P2Y 6 (P2RY6)

P2Y6 belongs to the G protein-coupled receptor family. This family comprises several receptor subtypes with varying (and sometimes overlapping) pharmacological selectivities for various adenosine and uracil nucleotides. P2Y6 subtypes are expressed at particularly high levels in the placenta, suggesting a crucial role for placental function. However, the cellular localization of P2Y6 in the placenta remains unclear. P2Y6 may play a crucial role in trophoblast development, differentiation, and neoplasia (Somers et al., 1999). Studies suggest that pyrimidine-activated P2Y receptors play a crucial role in inflammatory responses in the lung epithelium (Schafer et al., 2003).

HECT, UBA, and WWE domain-containing protein 1, an E3 ubiquitin-protein ligase (HUWE1)

HUWE1 encodes a member of the HECT E3 ubiquitin ligase family, in which the HECT domain is located at the C-terminus and contains an active site cysteine, thereby forming an intermediate ubiquitin-thioester bond.

ARF-BP1 (HUWE1) is a key mediator of both p53-independent and p53-dependent ARF tumor suppressor functions, making it a promising target for therapeutic intervention regardless of p53 status (Chen et al., 2005a). Inactivation of ARF-BP1 stabilizes p53 and induces apoptosis (Chen et al., 2006). HUWE1 (HectH9) is overexpressed in various 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 is significantly associated with prognostic factors (Confalonieri et al., 2009).

Versican (VCAN)

VCAN is a member of the aggrecan/versican mucin family. VCAN is known to associate with several molecules in the extracellular matrix, including hyaluronan, wrist-binding protein, fibulin-1, fibronectin, CD44, L-selectin, fibrillin, integrins, and connexins (Zheng et al., 2004). VCAN is expressed in various tissues, with high expression during early stages of tissue development and decreased expression during tissue maturation. Its expression is also elevated during wound healing and tumor growth (Ghosh et al., 2010). Knockdown of VCAN in 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 VCAN expression has been found in the preneoplastic stroma of early prostate and breast cancers, associated with aggressive tumor behavior (Yoon et al., 2002).

Drosha ribonuclease III (DROSHA)

Drosha is a class II RNase III enzyme responsible for initiating the processing of microRNAs, or short RNA molecules naturally expressed in cells. Drosha interacts with the RNA-induced silencing complex (RISC) to induce the cleavage of complementary messenger RNA (mRNA) as part of the RNAi pathway, thereby regulating a variety of other genes. MicroRNA molecules are primary transcripts of long RNAs, known as pri-miRNAs. Cleavage of pri-miRNAs by Drosha generates a stem-loop structure (approximately 70 base pairs long) known as pre-miRNA (Lee et al., 2003). Drosha is part of a protein complex known as the microprocessor complex, which also includes the double-stranded RNA-binding protein Pasha (also known as DGCR8) (Denli et al., 2004). Pasha is essential for Drosha activity and binds to single-stranded fragments of pri-miRNAs required for proper processing (Han et al., 2006). Human Drosha was cloned in 2000 and discovered to be a nuclear double-stranded RNA (dsRNA) ribonuclease involved in the processing of ribosomal RNA precursors (Wu et al., 2000). Drosha was the first human RNase III enzyme to be discovered and cloned. Two other human enzymes involved in miRNA processing and activation are Dicer and Argonaute proteins. Both Drosha and Pasha are located in the nucleus, where processing of both pri-miRNA and pre-miRNA occurs. The latter are further processed into mature miRNAs in the cytoplasm by the ribonuclease Dicer (Lee et al., 2003). Drosha and other miRNA-processing enzymes may have important implications for cancer prognosis (Slack and Weidhaas, 2008).

Platelet leukocyte C-kinase substrate homology domain-containing, family A (phosphatidylinositol-binding specificity) member 8 (PLEKHA8)

The gene for phosphatidylinositol-4-phosphate adaptor 2 (FAPP2 = PLEKHA8) encodes a cytoplasmic lipid transferase containing a platelet-leukocyte C-kinase substrate homology domain, which is involved in membrane vesicle maturation and transport from the trans-Golgi apparatus to the cytoplasmic membrane (Cao et al., 2009). Introduction of a ribozyme targeting the FAPP2 gene in colon cancer cells induced apoptosis in the tumor cells after administration of anti-Fas antibodies. Furthermore, apoptosis was significantly reduced in glioma and breast tumor cells transfected with FAPP2 siRNA (Tritz et al., 2009). Subsequent studies have highlighted the involvement of FAPP2 as a lipid transfer protein in glycosphingolipid metabolism within the Golgi complex (D'Angelo et al., 2012). FAPP2 plays a key role in the production of glycosphingolipids (GSLs): using its C-terminal domain, it transports newly synthesized GSLs away from the cis-Golgi membrane to the cytosol-facing glucosylceramide synthase for further anabolic processing (Kamlekar et al., 2013).

Acetyl-CoA carboxylase alpha (ACACA)

ACACA is a biotin-containing enzyme that catalyzes the carboxylation of acetyl-CoA to malonyl-CoA, a rate-limiting step in fatty acid synthesis (Tong and Harwood, Jr., 2006). ACACA has been found to be upregulated in various human cancers, making it a promising target for tumor intervention. Inhibitors developed for the treatment of metabolic diseases may serve as potential therapeutic agents for cancer treatment (Wang et al., 2010a). Two studies have demonstrated that RNA interference-induced silencing of ACACA results in growth inhibition and induces cell death to a degree comparable to that observed after silencing of FASN gene expression (Brusselmans et al., 2005; Chajes et al., 2006). TOFA (5-tetradecyloxy-2-furoic acid), an allosteric inhibitor of ACACA, is cytotoxic and induces apoptosis in lung cancer NCI-H460 cells and colon cancer cells HCT-8 and HCT-15 (Wang et al., 2009a). Soraphen A, another highly potent inhibitor of ACACA, blocks lipogenesis and promotes fatty acid oxidation in prostate cancer cells (Beckers et al., 2007). These findings suggest that, in addition to malonyl-CoA accumulation, inhibition of lipogenesis itself can trigger cancer cell death, and that ACACA may be a promising target for anti-tumor therapy (Brusselmans et al., 2005).

Integrin alpha 11 (ITGA11)

Integrins play a key role in a variety of cellular and developmental processes, including cell growth, differentiation, and survival, as well as tumorigenesis, cancer cell invasion, and metastasis. Integrin α11 (ITGA11/α11) is localized in stromal fibroblasts and is often overexpressed in NSCLC. α11 mRNA is overexpressed in both lung adenocarcinoma and squamous cell carcinoma (Wang et al., 2002). α11 has been reported to play a crucial role in fibroblast-promoting NSCLC cell growth in vitro, an activity mediated in part by regulating IGF2 expression (Zhu et al., 2007). Among the clinicopathological features 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 is associated with lymph node metastasis and poor prognosis (Chong et al., 2006).

Type XII collagen, alpha 1 (COL12A1)

The COL12A1 gene encodes the α chain of type XII collagen, which belongs to the FACIT (fibril-associating collagen with interrupted triple helices) collagen family. Type XII collagen is a homotrimer and a relative of type I collagen, an association that is thought to modify the interaction between collagen I fibrils and the surrounding machinery (Oh et al., 1992). COL12A1 may be involved in basement membrane regulation, forming specific molecular bridges between fibrils and other matrix components (Thierry et al., 2004). COL12A1 is expressed in the heart, placenta, lung, skeletal muscle, and pancreas (Dharmavaram et al., 1998), as well as in various connective tissues, including articular and epiphyseal cartilage (Gregory et al., 2001; Walchli et al., 1994; Watt et al., 1992). COL12A1 is downregulated in tumors with high microsatellite instability compared to stable groups with low or no microsatellite instability (Ortega et al., 2010).

Neutrophil-expressed elastase (ELANE)

Neutrophil elastase (or leukocyte elastase), also known as ELA2 (neutrophil elastase 2), is a serine protease belonging to the same family as chymotrypsin and exhibiting broad substrate specificity. Secreted by neutrophils in inflammatory settings, it destroys bacteria and host tissues (Belaaouaj et al., 2000). Human neutrophil elastase (ELANE) is a major pathogenic factor in chronic obstructive pulmonary disease and has recently been implicated in the progression of non-small cell lung cancer. The enzyme acts at multiple sites: (i) intracellular clearance of molecules such as the adaptor molecule insulin receptor substrate 1 (IRS-1); (ii) hydrolysis of receptors such as CD40 on the cell surface; and (iii) generation of elastin fragments (i.e., morphoelastokines) in the extracellular space. These fragments can potently stimulate tumor cell invasiveness and angiogenesis (Moroy et al., 2012). Neutrophil elastase directly induces tumor cell proliferation by entering an endosomal compartment in tumor cells (IRS-1), where neutrophil elastase degrades IRS-1 (Houghton et al., 2010).

Serine protease inhibitor, clade B (ovalbumin), member 3 (SERPINB3)

Squamous cell carcinoma antigen (SCCA), also known as SERPINB3, is a member of the high-molecular-weight serine protease inhibitor (serpin) family (Suminami et al., 1991). Elevated levels have been reported in head and neck tissues and other epithelial cancers (Torre, 1998). SCCA has also been reported to be overexpressed in tumors compared with pre-neoplastic tissues (Pontisso et al., 2004). Serpins B3/B4, particularly B4, may play a key role in epithelial dysplasia, particularly in patients with a high susceptibility to lung cancer (Calabrese et al., 2012). SCCA1 (SERPINB3) inhibits cell death induced by lysosomal damage and activates caspase 8 independently of the death receptor apoptosis pathway, sensitizing cells to endoplasmic reticulum stress (Ullman et al., 2011). Several findings suggest that SERPINB3 plays a key role in inducing epidermal barrier breakdown. SERPINB3 may be a key determinant of epithelial barrier function (Katagiri et al., 2010).

Kinesin family member 26B (KIF26B)

Kinesins belong to the motor protein class, which is present in eukaryotic cells. Kinesins move along microtubule filaments, powered by ATP hydrolysis (thus, kinesins are also ATPases). Kif26b, a kinesin family gene, is a downstream target of Sall1 (Nishinakamura et al., 2011). Kif26b is essential for kidney development by regulating the adhesion of interstitial cells contacting the ureteric bud. Overexpression of KiF26b in vitro leads to increased cell adhesion through interaction with non-muscle myosin (Terabayashi et al., 2012; Uchiyama et al., 2010).

Ankylosing kyphosis homolog (mouse) (ANKH)

ANKH (human homolog of ankylosing spondylosis) regulates the transport of inorganic pyrophosphate across the cell membrane (Wang et al., 2008a). Data indicate that ANKH expression and function are inhibited by hypoxia in vitro and in vivo, and this effect is regulated by HIF-1 (Zaka et al., 2009). The human ANKH gene is expressed in specific tissues, with the highest mRNA expression levels in the brain, heart, and skeletal muscle (Guo et al., 2001). Mutations in the ANKH gene cause autosomal dominant craniosynostosis (Kornak et al., 2010). ANKH is significantly upregulated in cervical cancer cell lines with amplification compared to cell lines without amplification (Kloth et al., 2007). Genomic amplification of the chromosome arm 5p region is common in SCLC, suggesting that this arm contains multiple oncogenes. Coe et al. reported microdeletions that were not detected by traditional screening methods, as well as TRIO and ANKH as novel putative tumor genes (Coe et al., 2005).

Nuclear RNA export factor 1 (NXF1)

In human cells, the mRNA export factor NXF1 is localized in the nucleoplasm and the nuclear pore complex (Zhang et al., 2011b). Transport of mRNA from transcription sites in the nucleus to translation sites in the cytoplasm is essential for eukaryotic gene expression (Kelly and Corbett, 2009). 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 within the nuclear pore complex (Kelly and Corbett, 2009). NXF1 is unique among nuclear transport factors in that it is a multidomain protein with no structural or mechanistic similarities to karyopherins, which transport protein cargo, tRNAs, and microRNAs through the nuclear pore complex. NXF1 supports mRNA export independently of the GTPase Ran (Gruter et al., 1998). Nuclear export of mRNPs is mediated by transport factors such as NXF1, which bind to the mRNP and mediate its translocation through the central channel of the nuclear pore (NPC) via a transient interaction with FG-nucleoporins (Wickramasinghe et al., 2010). mRNA transport can occur through both a bulk export pathway involving NXF1/TAP and a specialized pathway involving chromosome region maintenance protein 1 (CRM1) (Siddiqui and Borden, 2012).

Regulator of G protein signaling 4 (RGS4)

RGS4, as a GTPase-accelerating protein, regulates signaling at μ and δ opioid receptors (MOR and DOR, respectively). Opioid agonist-induced RGS4 reduction occurs through the ubiquitin-proteasome pathway and may play a role in maintaining cellular homeostasis during morphine dependence (Wang and Traynor, 2011). RGS4 plays an important role in regulating β-cell function (Ruiz et al., 2010). Xie et al. demonstrated that RGS4 could serve as a novel inhibitor of breast cancer migration and invasion, key steps in the metastatic cascade (Xie et al., 2009). RGS4 is overexpressed in thyroid cancer, and effective downregulation of its expression significantly reduces thyroid cancer cell viability, suggesting that RGS4 plays a key role in thyroid carcinogenesis (Nikolova et al., 2008). RGS4 is differentially expressed in human pancreatic tumor cell lines and has been found to serve as a marker gene for local tumor invasion and liver metastasis in pancreatic cancer (Niedergethmann et al., 2007). By selectively inhibiting G protein-mediated p38 MAPK activation, RGS4 overexpression can delay and alter the process of non-epithelial cell tube formation, thereby reducing epithelial proliferation, migration, and vascular endothelial growth factor (VEGF) expression (Albig and Schiemann, 2005).

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 2 diabetes and diabetic nephropathy (Zhang et al., 2004). Furthermore, the association of single nucleotide polymorphisms with GFPT2 suggests that genes involved in oxidative pathway regulation may be a major contributor to chronic renal failure in diabetes (Prasad et al., 2010). DNA methylation of the GFPT2 gene has been confirmed in primary acute lymphoblastic leukemia (ALL) samples (Kuang et al., 2008). GFPT2 plays a role in glutamine metabolism and is expressed more highly in mesenchymal cell lines. Glutamine metabolism may play an important role in tumor progression. Inhibitors of cellular metabolic pathways have the potential to be used as epigenetic therapies (Simpson et al., 2012).

Brain endothelial cell adhesion molecule (CERCAM)

CERCAM is located on the surface of epithelial cells (Starzyk et al., 2000). Its sequence is located on chromosome 9q34.11 (a candidate region on 9q) and is associated with familial idiopathic scoliosis (Miller et al., 2012). The CERCAM1 gene is widely transcribed in the nervous system and several secretory tissues, such as the salivary glands, pancreas, liver, and placenta (Schegg et al., 2009). CERCAM is structurally similar to the ColGalT enzymes GLT25D1 and GLT25D2.

Although its function is unclear, it appears to function differently from the related GLT25D1 protein and, unlike GLT25D1 and GLT25D2, does not function as a glycosyltransferase (Perrin-Tricaud et al., 2011).

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. This enzyme transfers N-acetylgalactosaminyltransferase (GalNAc) from UDP-GalNAc to a serine or threonine hydroxyl group on the target protein (Peng et al., 2010). GLNT2 is consistently expressed at low levels in nearly all human adenocarcinoma cell lines examined, including pancreatic, colon, gastric, and breast (Sutherlin et al., 1997). Research indicates that O-glucans and GALNT2 play key roles in a variety of physiological functions and human diseases. Single nucleotide polymorphisms in GALNT2 have been associated with the risk of epithelial ovarian cancer (Terry et al., 2010) and coronary artery disease (Willer et al., 2008). Aberrant glycosylation of cell surface glycoproteins, caused by altered glycosyltransferase activity, is often associated with cancer invasion and metastasis. GALNT2 is involved in the metastasis and invasion of gastric cancer (Hua et al., 2012), hepatocellular carcinoma (HCC) (Wu et al., 2011b), and human malignant gliomas (Liu et al., 2011a).

Heterogeneous nuclear ribonucleoprotein M (HNRNPM)

The HNRNPM gene belongs to a subfamily of ubiquitously expressed heterogeneous nuclear ribonucleoproteins (hnRNPs). HNRNPM is a major component of the human hnRNP complex, which influences pre-mRNA splicing by cleaving its own pre-mRNA (Hase et al., 2006) or by regulating alternative splicing of fibroblast growth factor receptor 2 (Hovhannisyan and Carstens, 2007). Proteomic analysis of purified spliceosomes in vitro has revealed the presence of HNRNPM in the pre-spliceosomal H complex and throughout spliceosomal assembly (Rappsilber et al., 2002; Wahl et al., 2009). HNRNPM participates in the spliceosomal machinery by interacting with the CDC5L/PLRG1 spliceosomal subcomplex (Lleres et al., 2010). In human cancer cells, some results indicate that cytoplasmic retention of IMP-3 and HNRNPM leads to a significant reduction in proliferation. The nuclear IMP-3-HNRNPM complex plays an important role in the efficient synthesis of CCND1, D3, and G1 and the proliferation of human cancer cells (Rivera et al., 2013).

Basic nuclear protein 1 (BNC1)

Basic nuclear protein (BNC) is a zinc-finger protein with a very restricted tissue distribution (Tseng, 1998). To date, BNC has been primarily found 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). Extensive evidence indicates that BNC acts as a cell-type-specific transcription factor for rRNA genes. The zinc fingers of BNC interact with three conserved sites in the rDNA promoter (Iuchi and Green, 1999; Tseng et al., 1999). Epigenetic regulation through CpG methylation plays a crucial role in tumorigenesis and the efficacy of cancer therapy. BNC1 is hypomethylated in radioresistant H1299 human NSCLC cells. Knockdown of BNC1 mRNA expression in H1299 cells also reduces the resistance of these cells to ionizing radiation (Kim et al., 2010a). Aberrant DNA methylation of BNC1 has also been found in chronic lymphocytic leukemia (CLL) samples (Tong et al., 2010). In renal cell carcinoma (RCC), BNC1 methylation is associated with a poorer prognosis regardless of tumor size, stage, or grade (Morris et al., 2010).

FK506 binding protein 10, 65 kDa (FKBP10)

FK506 binding protein 10 (FKBP10) belongs to the FKBP-like peptidyl-prolyl cis/trans isomerase family and is located in the endoplasmic reticulum. It acts as a molecular chaperone (Ishikawa et al., 2008; Patterson et al., 2000). FKBP10 is highly expressed during lung development and is reactivated in coordination with extracellular matrix proteins after lung injury (Patterson et al., 2005).

Frizzled zoster 1 (FZD1), Frizzled zoster 2 (FZD2), Frizzled zoster 7 (FZD7)

FZD2, FZD1, and FZD7 all belong to the "frizzled" gene family, members of which encode seven-transmembrane domain proteins that act as receptors for Wnt signaling proteins.

FZD2 gene expression is developmentally regulated, with high expression levels in embryonic kidney and lung, and in adult colon and ovary (Sagara et al., 1998; Zhao et al., 1995).

The FZD1 protein consists of a signaling protein, 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 lung, heart, kidney, pancreas, prostate, and ovary (Sagara et al., 1998). Expression of Frizzled 1 and 2 receptors has been found in breast cancer (Milovanovic et al., 2004).

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 downregulates APC function and enhances β-catenin-mediated signaling in poorly differentiated human esophageal cancer (Sagara et al., 1998; Tanaka et al., 1998).

Cardiac fast-twitch muscle Ca++-transporting ATPase 1 (ATP2A1), cardiac fast-twitch muscle Ca++-transporting ATPase 2 (ATP2A2)

Both genes (ATP2A1 and ATP2A2) encode SERCA Ca(2+)-ATPases. Sarcoplasmic reticulum (SR)1/ER calcium ATPases (SERCAs) are calcium pumps that couple ATP hydrolysis with calcium transport across the SR/ER membrane (MacLennan et al., 1997). SERCAs are encoded by three homologous genes: SERCA1 (ATP2A1), SERCA2 (ATP2A2), and SERCA3 (Wu et al., 1995). Some evidence suggests that SERCA may have direct effects on apoptosis, differentiation, and cell proliferation (Chami et al., 2000; Ma et al., 1999; Sakuntabhai et al., 1999).

Mutations in ATP2A1 (encoding SERCA1) cause certain forms of autosomal recessive Brody disease, characterized by impaired muscle relaxation during exercise (Odermatt et al., 1996).

ATP2A2 is an ATPase associated with Darier's disease, a rare autosomal dominant skin disorder characterized by abnormal keratinization and acantholysis (Huo et al., 2010). Germline variants in ATP2A2 predispose to lung and colon cancer, and ATP2A2 gene damage may contribute to tumorigenesis (Korosec et al., 2006). In small cell lung cancer (H1339) and lung adenocarcinoma (HCC) cell lines, ER Ca2+ levels are decreased compared to normal human bronchial epithelium. This decrease in Ca2+ levels correlates with decreased SERCA2 calcium pumping into the ER (Bergner et al., 2009). ATP2A2 has the potential to be a potential prognostic marker for patients with colorectal cancer (CRC). ATP2A2 has been found in circulating tumor cells (CTCs), and its overexpression is significantly associated with postoperative recurrence (Huang et al., 2012).

Laminin gamma 2 (LAMC2)

Laminins are a family of extracellular matrix glycoproteins that are the major non-collagenous components of the basement membrane and are widely involved in various physiological processes, including cell adhesion, differentiation, migration, signaling, neurite outgrowth, and metastasis. The LAMC2 gene encodes the laminin-5γ2 chain, which is part of laminin-5, a major component of the basement membrane. LAMC2 upregulation, mediated by promoter demethylation, is frequently observed in gastric cancer (Kwon et al., 2011). LAMC2 has been found to be overexpressed in vascular melanoma areas compared to avascular melanoma areas (Lugassy et al., 2009). LAMC2 is a marker for bladder cancer metastasis, and its expression level correlates with tumor grade (Smith et al., 2009b). Twenty-one of 32 non-SCLC cell lines (66%) showed co-expression of the LAMB3 and LAMC2 genes, compared to only one of 13 SCLC cell lines (8%). Co-expression of LAMB3 and LAMC2 genes was found in all four non-SCLC cell lines but was absent in the corresponding non-cancerous lung cell lines (Manda et al., 2000).

Heat shock protein 70 kDa 2 (HSPA2), heat shock protein 70 kDa 8 (HSPA8)

HSPA2 has been identified as a potentially tumor-promoting protein with aberrant expression 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). HSPA2 gene activity has also been observed in several human cancer cell lines (Scieglinska et al., 2008), and silencing of the HSPA2 gene in cancer cells leads to growth arrest and reduced oncogenic potential (Rohde et al., 2005; Xia et al., 2008). Furthermore, polymorphisms in the HSPA2 gene are associated with an increased risk of lung cancer (Wang et al., 2010b). In human breast, cervical, and urothelial bladder cancers, overexpression of HSPA2 is associated with increased cell proliferation, poor differentiation, and lymph node metastasis (Garg et al., 2010a; Garg et al., 2010b; Mestiri et al., 2001). The HSPA8 gene encodes the heat shock protein 70 (Hsc70) family, which includes both heat-induced and constitutively expressed members (Beckmann et al., 1990). Hsc70 functions as a molecular chaperone to assist in protein synthesis, folding, assembly, trafficking within cellular compartments, and degradation (Bukau and Horwich, 1998; Hartl and Hayer-Hartl, 2002). Hsc70 is expressed in both non-malignant and 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 led to the investigation of these proteins as potential clinical markers (Ciocca and Calderwood, 2005). This secreted hsc70 chaperone may play a role in cell proliferation, contributing to increased tumor growth in cancer cells overexpressing cathepsin D (Nirde et al., 2010). Furthermore, Ruisin et al. reported that polymorphisms in this gene are associated with lung cancer risk (Rusin et al., 2004).

Vacuole sorting protein 13 homolog B (yeast) (VPS13B)

VPS13B is a peripheral membrane protein localized to the Golgi complex, where it overlaps with the Golgi cis-matrix protein GM130. Consistent with its subcellular localization, VPS13B depletion by RNAi results in 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 critical region on chromosome 8q22 (Kolehmainen et al., 2003). Loss-of-function mutations in VPS13B cause recessive Cohen syndrome (Seifert et al., 2011). Mutations in VPS13B and other genes have been reported in gastric and colorectal cancers with minisatellite instability (An et al., 2012).

CSE1 chromosome segregation-like 1 (yeast) (CSE1L)

Research has shown that the cell death susceptibility 1L (CSE1L) gene regulates multiple cellular mechanisms, including the mitotic spindle checkpoint, proliferation, and apoptosis. CSE1L is present in both the cytoplasm and the nucleus. Nuclear CSE1L regulates the transcriptional activity of p53, a major tumor suppressor protein (Rao et al., 2011; Tanaka et al., 2007). Cytoplasmic CSE1L associates with microtubules, an association that has been shown to stimulate invadopodia extension and promote tumor cell migration (Tai et al., 2010). CSE1L is highly expressed in a variety of cancers, including benign and malignant skin melanocytic dysplasia (Boni et al., 1999), endometrial cancer (Peiro et al., 2001), ovarian cancer (Brustmann, 2004), breast cancer (Behrens et al., 2001), and urothelial carcinoma of the urinary tract and bladder (Chang et al., 2012), and studies have shown that its expression correlates with cancer progression. CSE1L silencing is expected to become a treatment for colon cancer (Zhu et al., 2013).

Dihydropyrimidinase-like 4 (DPYSL4)

Dihydropyrimidinase-related protein 4 (DPYSL4) is a known regulator of hippocampal neuronal development. DPYSL4 participates in the growth regulation, polarization, and differentiation of dental epithelial cells during tooth germ morphogenesis (Yasukawa et al., 2013). Studies have shown that DPYSL4 delays neurite outgrowth by inhibiting microtubule polymerization and have revealed a novel association with vimentin in nuclear condensation prior to neuronal death (Aylsworth et al., 2009). The p53 tumor suppressor gene, frequently mutated in various tumors, plays a crucial role in maintaining genomic integrity. DPYSL4 mRNA and protein expression are specifically induced by anticancer antigens in p53-enriched cells. DPYSL4 is an apoptosis-inducing factor regulated by p53 in response to DNA damage (Kimura et al., 2011).

Sec61γ subunit (SEC61G)

SEC61γ is a heterotrimeric protein channel composed of SEC61α, β, and γ subunits and is a member of the SEC61 translocon (Greenfield and High, 1999). The SEC61 complex forms a transmembrane pore, facilitating the translocation of nascent polypeptides into the ER lumen and the entry of transmembrane proteins into the ER bilayer (Osborne et al., 2005). SEC61γ is essential for tumor cell survival and cellular responses to ER stress (Lu et al., 2009). Knockdown of SEC61γ expression leads to apoptosis and blockade of EGFR/AKT survival signaling (Lu et al., 2009), as well as inhibition of tumor cell growth (Neidert et al., 2012).

ORM1-like protein 1 (Saccharomyces cerevisiae) (ORMDL1)

These human genes (ORMDL1, ORMDL2, and ORMDL3) are ubiquitously expressed in adult and embryonic tissues and encode transmembrane proteins anchored in the endoplasmic reticulum (ER). These genes are likely involved in protein folding in the ER. Genomic sequence analysis led Hjelmqvist et al. (2002) to locate the ORMDL1 gene to chromosome 2q32.2 (Hjelmqvist et al., 2002). ORMDL proteins are the primary regulators of ceramide biosynthesis in mammalian cells (Siow and Wattenberg, 2012). ORMDL1 is specifically downregulated in the presence of presenilin 1 (PS1) mutations (Araki et al., 2008).

Pecan-like protein 3 (Drosophila) (PCNXL3)

Pecan nutraceutical-like protein 3 (PCNXL3) is a multipass membrane protein belonging to the pecan nutraceutical family.

PCNXL3 is located in the chromosomal region 11q12.1-q13. Three novel human tumor-associated translocation breakpoints exist between markers D11S4933 and D11S546 in the 11q13 region. Therefore, PCNXL3 may be a 11q13-associated disease gene (van et al., 2000).

Small nuclear ribonucleoprotein 200kDa (U5) (SNRNP200)

Pre-mRNA splicing is catalyzed by the spliceosome (snRNP). The spliceosome is a specialized complex of RNA and protein subunits that removes introns from the transcribed pre-mRNA fragment. The spliceosome comprises the small nuclear RNA proteins (snRNPs) U1, U2, U4, U5, and U6, as well as approximately 80 conserved proteins (Maeder et al., 2009). SNRNP200 is expressed in the heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas (Zhao et al., 2009). Recently, mutations in SNRNP200 have been associated with autosomal recessive retinitis pigmentosa (adRP) (Benaglio et al., 2011; Liu et al., 2012).

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 (guard alpha motif) domains. SAMSN1 is expressed in hematopoietic tissues, muscle, heart, brain, lung, pancreas, endothelial cells, and myeloma. Endogenous SAMSN1 is upregulated in primary B cells stimulated by proliferation and proliferation induction, and in transduction experiments, SAMSN1 stimulates B cell differentiation into protoplasts (Brandt et al., 2010). Cell lines and primary cells isolated from patients with acute myeloid leukemia and multiple myeloma express SAMSN1 (Claudio et al., 2001). SAMSN1 is downregulated in the large cell lung cancer cell line Calu-6 (Yamada et al., 2008). SAMSN1 is differentially expressed in ulcerative colitis-associated carcinomas (Watanabe et al., 2011).

Signal transducer and activator of transcription 2, 113 kDa (STAT2)

STAT2 is a novel driver of colorectal and skin cancers, acting by increasing 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 of type I IFN-induced apoptosis. Importantly, defects in STAT2 expression or nuclear localization can reduce the efficacy of type I IFN immunotherapy (Romero-Weaver et al., 2010). Studies have found that STAT2 expression is lower in low-grade astrocytomas than in high-grade astrocytomas. These results demonstrate a clear relationship between STAT and PPARγ signaling and gliomas, further confirming the anticipated role of STATs in the growth and differentiation of these tumors (Ehrmann et al., 2008).

CCR4-NOT transcription complex subunit 1 (CNOT1)

The human CCR4-NOT deadenylase complex comprises at least nine enzymatic and nonenzymatic subunits. CNOT1 plays a crucial role in inhibiting the enzymatic activity of the CCR4-NOT complex and is therefore crucial for mRNA deadenylation and the control of mRNA decoys. CNOT1 depletion structurally and functionally disrupts the CCR4-NOT complex and leads to mRNA destabilization, increased translation, and ultimately ER stress-mediated apoptosis. Ito et al. hypothesized that CNOT1 promotes cell viability by ensuring CCR4-NOT deadenylase activity (Ito et al., 2011). SiRNA-mediated depletion of endogenous CNOT1 or other Ccr4-Not subunits in breast cancer cells resulted in deregulation of ERα target genes (increased induction of the ERα target genes TTF1 and c-Myc). These findings confirm the role of the human Ccr4-Not complex as a transcriptional repressor of nuclear receptor signaling, which has implications for the understanding of molecular pathways in cancer (Winkler et al., 2006).

Serine hydroxymethyltransferase 2 (mitochondrial) (SHMT2)

The SHMT2 gene encodes the mitochondrial form of a pyridoxal phosphate-dependent enzyme that catalyzes the reversible reaction from serine and tetrahydrofolate to glycine and 5,01-methylenetetrahydrofolate. Its encoded product primarily contributes to glycine synthesis. In polygenic diseases such as lung cancer, gene-gene interactions play a crucial role in determining phenotypic variability. Interactions between MTHFR677, MTHFR1298, and SHMT polymorphisms may significantly contribute to the genetic instability of lung cancer patients. Studies have shown that, in terms of cytogenetic alterations, the presence of MTHFR677, MTHFR1298, and SHMT allelic variants in lung cancer patients exposed to the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) can significantly increase cytogenetic damage in lymphocytes (Piskac-Collier et al., 2011). Pharmacogenomic studies investigated the impact of SHMT gene polymorphisms on the efficacy of 5-Fu and FOLFIRI treatment regimens in patients with colorectal cancer and showed a significant effect, resulting in changes in overall survival (Timar et al., 2006).

Jun B proto-oncogene (JUNB)

JunB is a member of the AP-1 (activator protein-1) family of dimeric transcription factors. The transcription factor AP-1 is involved in cell proliferation, transformation, and cell death (Shaulian and Karin, 2002). JunB may be regulated through the NF-κB pathway, and HGF-induced JunB upregulation may play a significant role in cell proliferation and invasion through MMP-9 expression (Lee and Kim, 2012). JunB has been shown to have oncogenic effects in lymphomas, particularly Hodgkin's lymphoma (Shaulian, 2010). JunB is an essential upstream regulator of p16 and participates in maintaining cellular senescence to prevent malignant transformation of T-cell carcinomas (TCA). Therefore, JunB plays a significant role in controlling prostate carcinogenesis (Konishi et al., 2008). JunB enhances tumor invasiveness and angiogenesis in VHL-deficient ccRCC (Kanno et al., 2012).

Transforming acidic frizzled coiled coil 3 (TACC3)

TACC3 exists in a complex containing ch-TOG (colon and liver tumor overexpressed gene) and clathrin, which can crosslink with microtubules in kinetochore fibers. TACC3 is expressed in certain proliferative tissues, such as the testis, lung, spleen, bone marrow, thyroid, and peripheral blood leukocytes. TACC3 expression is altered in certain human tumor types. In cells, TACC3 is also localized to centrosomes and spindle microtubules, but not to astral microtubules (Hood and Royle, 2011). TACC3 expression correlates with p53 expression; patients with tumors that highly express both TACC3 and p53 have a significantly worse prognosis than those with low immunostaining levels of both (P = 0.006). Studies have shown that increased TACC3 expression may indicate a proliferative advantage in NSCLC and promote tumor progression. TACC3 expression is a strong prognostic indicator for clinical outcome in NSCLC (Jung et al., 2006). TACC3 may be a negative regulator of the Notch signaling pathway (Bargo et al., 2010).

RAD54 homolog B (Saccharomyces 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 RAD54, which plays a key role in homologous chromosome recombination. Homologous chromosome recombination (HR) is essential for the accurate repair of DNA double-strand breaks (DSBs) (Sarai et al., 2008). Somatic mutations in the RAD54B gene are known to occur in cancer, and its knockout can cause chromosomal instability (CIN) in mammalian cells (McManus et al., 2009). In patients with GBM, increased RAD54B expression is associated with a shorter time to progression and poorer overall survival (Grunda et al., 2010).

Erythroid-enhancing factor 2 (EEF2)

EEF2 encodes a member of the GTP-binding translation elongation factor family. This protein is essential for protein synthesis. EEF2 promotes the GTP-dependent translocation of nascent protein chains from the A site to the P site of the ribosome. EEF2 is highly expressed in lung adenocarcinoma (LADC) but absent in adjacent non-tumor lung tissue. Studies have shown that eEF2 is an anti-apoptotic marker in LADC, as patients with high eEF2 expression have a significantly higher incidence of early-stage tumors and a significantly poorer prognosis. Silencing eEF2 expression increases mitochondrial elongation, autophagy, and sensitivity to cisplatin. Furthermore, eEF2 can be SUMOylated in LADC cells, and SUMOylation of eEF2 is associated with drug resistance (Chen et al., 2011a). EEF2 is an attractive target for cancer therapy because inhibition of EEF2 triggers a rapid cessation of protein synthesis, thereby inducing apoptosis and ultimately cell death. siRNA-induced EEF2 silencing produces specific cytotoxicity in tumor cells (Chen et al., 2011b; Wullner et al., 2008).

Cell cycle regulatory protein A2 (CCNA2)

CCNA2 belongs to a highly conserved family of cell cycle regulatory proteins. Cyclins are regulators of CDK kinases. Different cell cycle regulatory proteins display distinct expression and degradation patterns, thereby regulating the timing of various mitochondrial 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, accelerating the G1/S and G2/M transitions of the cell cycle (Honda et al., 2012). Mutations, amplifications, and overexpression of this gene can alter cell cycle progression and are commonly found in various tumors, potentially promoting tumorigenesis (Cooper et al., 2009; Kars et al., 2011; Kim et al., 2011; Tompkins et al., 2011). Furthermore, CCNA2 expression has been reported to confer poor prognosis in seven cancer types (Yasmeen et al., 2003), whereas cyclin A is associated with shorter survival (Dobashi et al., 1998).

Neuroepithelial transforming protein 1 (NET1)41

NET1 is part of the Rho family of guanine nucleotide exchange factors. Members of this family activate Rho proteins by catalyzing the exchange of GDP for GTP. The protein encoded by NET1 interacts with RhoA in the nucleus and plays a role in the repair of DNA damage after ionizing radiation.

The NET1 gene is expressed in breast adenocarcinoma cells (rather than opioid receptors) and may accelerate cell migration (Ecimovic et al., 2011). NET1 is upregulated in gastric cancer (GC) tissue and drives the invasive phenotype of this disease (Srougi and Burridge, 2011). NET1 plays a crucial role in GC cell migration and invasion, key processes in GC progression (Bennett et al., 2011). RhoC and NET1 are highly expressed in human prostate cancer after short-term endocrine therapy, suggesting that RhoC and NET1 may be promising therapeutic targets for endocrine therapy (Kawata et al., 2012).

Chromosome 11 open reading frame 24 (C11orf24)

C11orf24 was first discovered by Tweels et al. (2001). The C11orf24 gene has no known similarities to other genes, and its function remains unclear. Northern blot analysis revealed high expression of a 1.9-kb transcript in the heart, placenta, liver, pancreas, and colon, with low expression in the brain, lung, skeletal muscle, kidney, spleen, prostate, testis, ovary, and small intestine, and very low expression in the thyroid and leukocytes (Twells et al., 2001). The 449-amino acid C11orf24 protein is located in chromosomal region 11q13, which has been described as a multiple cancer susceptibility region (Gudmundsson et al., 2009; Purdue et al., 2011).

Regulator of chromosome condensation 1 (RCC1)

Regulatory factor for chromosome condensation 1 (RCC1) is a guanine nucleotide exchange factor for the Ran GTPase. Ran-GTP is generated by RCC1 on chromatin, a process crucial for nucleocytoplasmic transport, mitotic spindle assembly, and nuclear envelope formation (Hitakomate et al., 2010). Chromosomal binding of mitotic regulators such as RCC1, Mad2, and survivin has been shown to be essential for mitotic progression (Ho et al., 2008). Wong et al. found that nuclear RanGTP levels decrease during the early stages of apoptosis, correlating with RCC1 fixation on chromosomes. They therefore proposed that RCC reads the histone code generated by caspase-activated Mst1, thereby initiating apoptosis by reducing nuclear RanGTP levels (Wong et al., 2009).

Melanoma antigen F family protein, 1 (MAGEF1)

Most known members of the MAGE (melanoma-associated antigen) superfamily are expressed in tumors, testes, and embryonic tissues, a pattern described as cancer/testis expression (MAGE subgroup I). Peptides from MAGE subgroup I have been successfully used for 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, certain MAGE genes (MAGE subgroup II, such as MAGEF1) are ubiquitously expressed in all adult and embryonic tissues tested and in many 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, MAGEF1 overexpression was found in NSCLC (Tsai et al., 2007) and in 79% of patients in a Taiwanese colorectal cancer cohort (Chung et al., 2010).

Non-SMC condensin I complex, subunit D2 (NCAPD2)

Condensin is a heteropentameric complex originally discovered as a structural component of mitochondrial chromosomes. NCAPD2 is an essential component of the human condensin complex, which is required for mitochondrial chromosome condensation. NCAPD2 deficiency can affect chromosome alignment during metaphase and delay anaphase entry (Watrin and Legagneux, 2005). Recent linkage and association studies suggest that the chromosome 12p13 locus may harbor susceptibility variants for Alzheimer's disease (AD). Single-marker association studies revealed that two SNPs in NCAPD2 (rs7311174 and rs2072374) yielded nominally significant p-values (p = 0.0491 and 0.0116, respectively). These genetic analyses confirmed an association between the chromosome 12p13 locus and AD in Chinese individuals (Li et al., 2009).

Chromosome 12 open reading frame 44 (C12orf44)

By searching databases for orthologs of Drosophila Atg13-interacting proteins, Mercer et al. (2009) discovered human ATG101 (also known as C12orf44) (Mercer et al., 2009). The ATG101 gene is located on chromosome 12q13.13. This putative 218-amino acid protein is predicted to be a cytosolic hydrophilic protein (Hosokawa et al., 2009). Macroautophagy is a catabolic process involving the lysosomal degradation of cytoplasmic proteins, organelles, and macromolecules. ATG proteins such as ATG101 are required for the formation of autophagosomes, double-membrane vesicles that enclose and sequester cytoplasmic cargo before fusion with lysosomes. ATG101 (C12orf44) is essential for autophagy (Mercer et al., 2009).

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 (MIM 179710)-like domain (RLD). The 350-amino acid HECT domain is predicted to catalyze the formation of ubiquitin thioesters, which are subsequently transferred to a substrate. The RLD is predicted to function as an ornithine nucleotide exchange factor for small G proteins (Hochrainer et al., 2005). While ubiquitin E3 ligases are ubiquitinated in all tissues, expression is highest in the testis, specifically during spermatogenesis. HERC4 ligase is required for proper maturation and removal of cytoplasmic droplets, enabling sperm function (Rodriguez and Stewart, 2007).

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 and is involved in mRNA localization, turnover, and translational control. This protein contains several KH (K homology) domains. These domains are important for RNA binding and are known to be involved in RNA synthesis and metabolism. Its expression primarily occurs during embryonic development and has been found in certain tumors. Therefore, IGF2BP3 is considered an oncofetal protein (Liao et al., 2005). IGF2BP3 can promote tumor cell proliferation by stabilizing CD44 mRNA, thereby promoting IGF-II protein synthesis and inducing cell adhesion and invasion (Findeis-Hosey and Xu, 2012). Furthermore, IGF2BP3 expression has been studied in many human tumors, and there is increasing evidence that IGF2BP3 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 may be involved in angiogenesis (Suvasini et al., 2011; Chen et al., 2012). In lung adenocarcinoma, increased IGF2BP3 expression has been found in moderately or poorly differentiated adenocarcinomas, potentially contributing to their aggressive biological behavior (Findeis-Hosey et al., 2010; Beljan et al., 2012; Findeis-Hosey and Xu, 2012).

Cell division cycle 6 homolog (Saccharomyces cerevisiae) (CDC6)

The CDC6 protein acts as a regulator of the early steps of DNA replication. It is localized in the nucleus during G1 of the cell cycle but translocates to the cytoplasm at the onset of S phase. Furthermore, studies have suggested that CDC6 regulates replication checkpoint activation by interacting with ATR in higher eukaryotic cells (Yoshida et al., 2010). CDC6 is essential for DNA replication, and its deregulation is implicated in tumorigenesis. Studies have shown that downregulation of CDC6 by RNA interference (RNAi) inhibits cell proliferation and promotes apoptosis (Lau et al., 2006). CDC6 overexpression has been found in several cancer types, including 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 endothelioma (Romagnoli et al., 2009).

Fibroblast activation protein alpha (FAP)

Fibroblast activation protein (FAP) is a type II integral membrane glycoprotein that belongs to the serine protease family. The putative serine protease activity of FAPα and its induction in vivo suggest that this molecule may play a role in fibroblast growth or epithelial-mesenchymal interactions during development, tissue repair, and epithelial carcinogenesis (Scanlan et al., 1994). FAP expression is minimal or absent in most normal adult tissues and benign epithelial tumors. However, it is expressed in the stroma of over 90% of malignant breast, colorectal, lung, skin, and pancreatic tumors, fibroblasts in healing wounds, soft tissue sarcomas, and some embryonic mesenchymal cells. FAP may contribute to cancer growth and metastasis by participating in cell adhesion and migration and by rapidly degrading ECM components. Thus, FAP is present in tumor cells infiltrating the ECM and in endothelial cells involved in angiogenesis, but is absent in the same inactive cell types (Dolznig et al., 2005; Kennedy et al., 2009; Rettig et al., 1993; Rettig et al., 1994; Scanlan et al., 1994; Zhang et al., 2010a).

Wingless MMTV integration site family member 5A (WNT5A)

Wnt5a normally regulates various cellular functions, such as proliferation, differentiation, migration, adhesion, and polarization (Kikuchi et al., 2012) and is expressed in undifferentiated human embryonic stem cells (Katoh, 2008). WNT5A is classified as a non-transforming WNT family member, and its role in tumorigenesis remains controversial. WNT5A exhibits tumor suppressor activity in certain cancers (thyroid, brain, and colorectal cancer), but is aberrantly upregulated in lung, gastric, and prostate cancers (Li et al., 2010). Oncogenic WNT5A activates canonical WNT signaling in cancer stem cells to promote self-renewal and non-canonical WNT signaling at the tumor-stroma interface to promote invasion and metastasis (Katoh and Katoh, 2007). WNT5A expression has been reported in various tumors. For example, aberrant Wnt5a protein expression has been observed in 28% of prostate cancers, contributing to enhanced invasiveness (Yamamoto et al., 2010). In addition, overexpression of WNT5A has been reported to be associated with poor prognosis and/or increased tumor grade 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 is involved in tumorigenesis, while the non-canonical Wnt signaling pathway is involved in tumor progression (Yuzugullu et al., 2009).

TPX2 microtubule-associated protein homolog (Xenopus laevis) (TPX2)

TPX2 is a spindle assembly factor required for the normal assembly of the mitotic spindle and apoptotic microtubules, and for chromatin- and/or kinetochore-dependent microtubule nucleation (Bird and Hyman, 2008; Moss et al., 2009). Newly synthesized TPX2 is required for virtually all Aurora A activation and for complete p53 synthesis and phosphorylation in vivo during oocyte maturation (Pascreau et al., 2009). TPX2 is a cell cycle-associated protein that is overexpressed in many tumor types, including meningioma (Stuart et al., 2010), laryngeal squamous cell carcinoma (SCCL) (Cordes et al., 2010), oral squamous cell carcinoma (SCC) (Shigeishi et al., 2009), hepatocellular carcinoma (HCC) (Satow et al., 2010), pancreatic cancer (Warner et al., 2009), ovarian cancer (Ramakrishna et al., 2010), and lung squamous cell carcinoma (Lin et al., 2006; Ma et al., 2006). TPX2 is often co-overexpressed with Aurora-A, resulting in a novel functional unit with oncogenic properties (Asteriti et al., 2010). TPX2 expression is a prognostic marker in lung cancer (Kadara et al., 2009).

Hyaluronan-induced cell motility receptor (RHAMM) (HMMR)

The hyaluronan-induced cell motility receptor, RHAMM (HMMR), has diverse functions on cells and cell membranes. RHAMM is exported to the cell surface, where it binds to hyaluronan and interacts with the HA receptor CD44. Cell motility, wound healing, and invasion are all regulated by RHAMM (Sohr and Engeland, 2008). RHAMM is one of the hyaluronan (HYA) receptors (Gares and Pilarski, 2000). Furthermore, cancer cells contain binding sites for HYA (CD44, RHAMM, etc.), which may protect cancer cells from immune cell attack. Elevated serum HYA levels are often found in patients with metastatic disease (Delpeche et al., 1997). Furthermore, some researchers have proposed that the interaction of HYA with RHAMM (HMMR) and CD44 in cancer cells is a key factor promoting tumor progression and dissemination (Li et al., 2000b). Furthermore, RHAMM is overexpressed in several tumor 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)).

ADAM metallopeptidase domain 8 (ADAM8)

ADAM8 is a member of the ADAM (a disintegrin and metalloproteinase domain) family. Many ADAM types, including ADAM8, are expressed in human malignant tumors, where they participate in the regulation of growth factor activity and integrin function, thereby promoting cell growth and invasion (Mochizuki and Okada, 2007). ADAM8 expression is positively correlated with EGFR. Both are primarily expressed in the cytoplasm and cell membrane (Wu et al., 2008). ADAM8 is abundantly expressed in most studied lung cancers. Exogenous expression of ADAM8 enhances the migratory activity of mammalian cells, suggesting that ADAM8 may play an important role in lung cancer progression (Ishikawa et al., 2004). ADAM8 is associated with poor prognosis in lung cancer (Hernandez et al., 2010). Overexpression of ADAM8 is associated with shorter survival and is a good predictor of distant metastasis in RCC (Roemer et al., 2004b; Roemer et al., 2004a). The expression level and protease activity of ADAM8 are correlated with the invasive activity of glioma cells, suggesting that ADAM8 may play a significant role in brain cancer invasion (Wildeboer et al., 2006).

Collagen alpha-3(VI) chain protein (COL6A3)

COL6A3 encodes the α-3(VI) chain, one of the three α chains of type VI collagen. Studies have shown that its protein domain binds to extracellular matrix proteins, an interaction that may explain the important role this collagen plays in the organization of matrix components.

Overexpression of collagen VI leads to extracellular matrix remodeling, which increases cisplatin resistance in ovarian cancer cells. The presence of collagen VI correlates with tumor grade, a prognostic factor in ovarian cancer (Sherman-Baust et al., 2003). COL6A3 is overexpressed in colorectal cancer (Smith et al., 2009a) and salivary gland cancer (Leivo et al., 2005), and differentially expressed in gastric cancer (Yang et al., 2007). COL6A3 is one of seven tumor-specific splice variants identified. The confirmed tumor-specific splicing changes are highly consistent, allowing clear distinction between normal and cancerous samples and, in some cases, even between different tumor stages (Thorsen et al., 2008).

Thy-1 cell surface antigen (THY1)

Thy-1 (CD90) is a 25-37 kDa glycosylphosphatidylinositol (GPI)-anchored glycoprotein expressed by a variety of cell types, including T cells, thymocytes, neurons, endothelial cells, and fibroblasts. Thy-1 activation promotes 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, playing a crucial role in neural regeneration, metastasis, inflammation, and fibrosis (Rege and Hagood, 2006b; Rege and Hagood, 2006a). Furthermore, Thy-1 is expressed as an angiogenic marker in adults but not in embryos. The upregulation of Thy-1 by cytokines, but not growth factors, suggests a crucial role for inflammation in the etiology of adult angiogenesis (Lee et al., 1998). Compared to normal tissue or benign tumor tissue, Thy-1 is overexpressed in the nuclei of lung cancer cells, and this overexpression is a prognostic factor in NSCLC patients. Therefore, Thy-1 may be a novel latent malignancy marker in the pathogenesis of lung cancer (Chen et al., 2005b). Thy-1 can be considered as a surrogate marker for various stem cells, such as meningeal 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).

Type II iodide-containing adenine deiodinase (DIO2)

The protein encoded by the DIO2 gene belongs to the potassium iodide adenine deiodinase family and is highly expressed in the thyroid gland, potentially contributing significantly to the relative increase in thyroid T3 production seen in patients with Graves' disease and thyroid adenomas (Meyer et al., 2008; de Souza-Meyer et al., 2005). Its gene expression pattern differs significantly between upward- and downward-progressing nasopharyngeal carcinoma (NPC). DIO2 gene expression is higher in downward-progressing NPC (downward = distant metastasis) than in upward-progressing NPC (localized 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 present in brain tumors (Murakami et al., 2000). DIO2 activity is present in lung tissue and is similar to that in peripheral lung tissue and lung cancer (Wawrzynska et al., 2003).

Periostin (osteoblast-specific factor) (POSTN)

The POSTN gene encodes a protein with similarities to the fasciclin family, which is involved in cell survival and angiogenesis and is expected to become a tumor progression marker for various human cancer types (Ruan et al., 2009).

Overexpression of periostin or its mRNA has been found in many solid tumors, including breast cancer (Zhang et al., 2010b), colon cancer (Kikuchi et al., 2008), head and neck cancer (Kudo et al., 2006), pancreatic cancer (Kanno et al., 2008), papillary thyroid cancer (Puppin et al., 2008), prostate cancer (Tischler et al., 2010), ovarian cancer (Choi et al., 2010), lung cancer (Takanami et al., 2008), liver cancer (Utispan et al., 2010), and esophageal squamous cell carcinoma (Kwon et al., 2009). Periostin is abnormally overexpressed in lung cancer and is associated with angiogenesis, invasion, and metastasis (Takanami et al., 2008). Silencing periostin in A549 NSCLC cells inhibited tumor cell growth and reduced cell infiltration (Wu et al., 2013).

SLIT1 (slit homolog 1 (Drosophila)), SLIT2 (slit homolog 1 (Drosophila))

SLITs (SLIT1, SLIT2, and SLIT3) are a family of secreted proteins that mediate the positional interactions of cells with their environment during development through ROBO receptor signaling (Hinck, 2004). However, SLIT/ROBO signaling is not limited to development, and loss of these signals is likely to play a significant role in tumor progression (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 tumor samples (Sharma et al., 2007). Hypermethylation of SLIT2 is frequently found in NSCLC and is associated with various clinical features (Suzuki et al., 2013).

TLX3 (T-cell leukemia homeobox protein 3)

TLX3 (also known as RNX or HOX11L2) belongs to a family of orphan homeobox genes that encode DNA-binding nuclear transcription factors. Members of the HOX11 gene family are characterized by a highly conserved homology structure and a threonine-47 substitution for cytosine (Dear et al., 1993). TLX3 is uniquely expressed in the developing medulla oblongata. TLX3 is required for the normal formation of first-order visceral sensory neurons and most adrenergic neurons in the brainstem, particularly those involved in cardiovascular and respiratory physiology (Qian et al., 2001). TLX3 expression has been detected in 20% of pediatric and 13% of adult T-cell acute lymphoblastic leukemia samples (Cave et al., 2004), despite the gene not being involved in normal T-cell differentiation (Ferrando et al., 2004).

CEP192 (centrosomal protein 192 kDa)

Centrosomes play an important role in numerous cellular processes, including spindle formation and chromosome segregation. CEP192, a centrosomal protein, plays a key role in centrosome biogenesis and function in mammals, Drosophila, and Caenorhabditis elegans (Gomez-Ferreria et al., 2012). CEP192 stimulates the formation of the mitotic scaffolding upon which the gamma-tubulin ring complex and other proteins involved in microtubule nucleation and spindle assembly depend (Gomez-Ferreria et al., 2007).

ANKS1A (ankyrin repeat and guard alpha motif domain 1A)

Ankyrin repeat- and SAM domain-containing protein is a human protein encoded by the ANKS1A gene (Nagase et al., 1996). ANKS1A was first reported as a target and signaling mediator of receptor tyrosine kinases such as EGFR and PDGFR (Pandey et al., 2002) and more recently as an interacting partner of the receptor tyrosine kinase EphA8 (Shin et al., 2007). A recent study genotyping single nucleotide polymorphisms (SNPs) in 348 patients with advanced NSCLC identified 17 candidate SNPs that were most strongly associated with prognosis. These SNPs were located in the genomic region of the ANKS1A gene (Lee et al., 2013).

CEP250 (centrosomal protein 250 kDa)

The CEP250 gene encodes a core centrosomal protein required for centriole-centriole attachment during interphase of the cell cycle (Mayor et al., 2002). Using radiation hybridization analysis, Fry et al. (1998) localized CEP250 to the centromeric region of chromosome 20, near 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. Overexpression of CEP250 did not affect centrosome separation or cell division, but suggested that cell cycle-regulated activities could dissociate CEP250 from the centrosome (Mayor et al., 2002).

MDN1 (MDN1, midasin homolog (yeast))

MDN1, midasin homolog (yeast), is a protein encoded by the MDN1 gene in humans. Midastin is a single-copy gene that encodes a highly conserved protein of approximately 600 kDa in all eukaryotic organisms for which data are available. In humans, the gene is located at 6q15 and encodes a predicted protein of 5596 residues (632 kDa) (Garbarino and Gibbons, 2002). Recent studies have found that MDN1 is mutated in the luminal A subtype of breast cancer. MDN1 may play a role in the development of this aggressive subtype and its hormone resistance (Cornen et al., 2014).

OLFM1 (Olfactin 1)

OLFM1, also known as olfactory globulin, is a secreted glycoprotein belonging to the olfactory domain-containing protein family that plays a crucial role in regulating the generation of neural crest cells from the neural tube (Barembaum et al., 2000). Olfactory globulin 1 was originally discovered as a major component of the mucus layer that surrounds the chemosensory dendrites of olfactory neurons (Kulkarni et al., 2000). Olfactory globulin 1 protein expression is significantly elevated in lung adenocarcinoma compared with other histochemical types of lung cancer and normal lung tissue (Wu et al., 2010). Furthermore, OLFM1 is deregulated in endometrial carcinoma, Ewing sarcoma, and neuroblastoma (Wong et al., 2007; Allander et al., 2002; Khan et al., 2001).

BUB1B (benzimidazole budding inhibition relief protein 1 homolog beta (yeast))

BUB1B, also known as BubR1, is a core mitotic checkpoint component that binds to and inhibits the Cdc20-activated anaphase-promoting factor (APC/CCdc20). This factor is a ubiquitin E3 ligase that initiates anaphase by regulating the separase-mediated adhesion ring that binds sister chromatids (Baker et al., 2004). BubR1 promotes normal chromosome segregation not only by activating the mitotic checkpoint but also by regulating the chromosome-spindle attachment zone (Malureanu et al., 2009; Lampson and Kapoor, 2005). Impaired spindle checkpoint function has been found in various tumors. Mutations in BubR1 have been associated with aneuploidy syndrome, a rare human syndrome characterized by aneuploidy, tumor susceptibility, and multiple progeroid features, including shortened lifespan, growth and mental retardation, cataracts, and facial dysmorphisms (Matsuura et al., 2006).

PI4KA (phosphatidylinositol 4-kinase alpha)

Four different phosphatidylinositol 4-kinase (PI4K) isoforms are expressed in human cells. These isoenzymes (PI4KA, PI4KB, PI4K2A, and PI4K2B) catalyze the phosphorylation of phosphatidylinositol (PtdIns) on the cytoplasmic side of the cell membrane, generating phosphatidylinositol 4-phosphate (PtdIns4P) (Minogue and Waugh, 2012). PI4KA is primarily located in the endoplasmic reticulum (ER), and its activity appears to regulate both the formation of ER exit sites (Blumental-Perry et al., 2006) and the accumulation of PtdIns4P at the plasma membrane (Balla et al., 2008). One group has found that PI4KA mRNA is more abundant in HCC than in normal healthy tissue. This upregulation is significantly correlated with both the poor differentiation and active proliferation rate of HCC (Ilboudo et al., 2014).

AURKB (aurora kinase B)

Aurora B kinase is a protein that acts on the centromere of the mitotic spindle (Kim et al., 2011). Aurora B kinase is localized to microtubules near the centromere (Kunitoku et al., 2003). Aurora B kinase is overexpressed in various tumor cell lines, suggesting a role for this enzyme in tumorigenesis and has become a potential target for tumor diagnosis and treatment (Fu et al., 2007). Recently, a gene signature of five genes (TOP2A, AURKB, BRRN1, CDK1, and FUS) has been identified that is strongly associated with the prognosis of patients with NSCLC. Genes involved in chromosome condensation, such as AURKB, are likely associated with stem cell-like properties and may be a predictor of survival in lung adenocarcinoma (Perumal et al., 2012).

SLC3A2 (solute carrier family 3 (dibasic acidic and neutral amino acid transport activator), member 2)

SLC3A2 is the light subunit of the large neutral amino acid transporter (LAT1), also known as CD98 (clusters of differentiation 98) (Lemaitre et al., 2005). The CD98 heterodimer consists of a type II single-pass transmembrane heavy chain (CD98hc, also known as 4F2 antigen heavy chain or FRP-1, encoded by the SLC3A2 and Slc3a2 genes in humans and mice, respectively) of approximately 80–85 kDa, disulfide-bonded to a multipass light chain of approximately 40 kDa (Deves and Boyd, 2000). CD98hc functions to amplify integrin signaling and transport amino acids; both functions promote cell survival and proliferation (Cantor and Ginsberg, 2012). Many tumors express CD98hc (SLC3A2), and its expression is associated with poor prognosis in B-cell lymphomas. In addition, almost all studies on the expression of CD98hc or CD98 light chain in solid tumors have shown that their expression is associated with progressive or metastatic tumors (Kaira et al., 2009).

IFT81 (intraciliary transporter 81 homolog (Chlamydomonas))

Intraciliary transport (IFT), the transport of ciliary precursors (e.g., tubulin) from the cytoplasm to the ciliary tip, is involved in the construction of cilia, hair-like organelles found in most eukaryotic cells. IFT81 knockout and point mutation rescue experiments have shown that IFT81-mediated tubulin binding is required for cilia formation in human cells (Bhogaraju et al., 2013). IFT81 forms a core complex with IFT74/72 to assemble the IFT particle necessary for cilia formation (Lucker et al., 2005).

COG4 (oligomeric Golgi complex component 4)

The COG complex consists of eight subunits, COG1–8 (Ungar et al., 2002; Whyte and Munro, 2001), which can be divided into two subcomplexes: COG1–4 (Lobe A) and COG5–8 (Lobe B) (Ungar et al., 2005). The COG complex is involved in tethering Golgi-resident proteins, such as glycosylases, during vesicle recycling (Pokrovskaya et al., 2011). The COG4 gene is located on chromosome 16q22.1 (Reynders et al., 2009). Ungar et al. (2002) hypothesized that COG4 is essential for the structure and function of Golgi bodies and influences intracellular membrane trafficking (Ungar et al., 2002).

NCBP1 (nuclear cap binding protein subunit 1, 80 kDa)

The nuclear corona binding protein complex is an RNA-binding protein that binds to the 5' capsid of RNA polymerase II. Kataoka et al. (1994) reported the cloning of a gene encoding an 80 kD nuclear corona binding protein (NCBP1) from HeLa cell nuclear extracts, which appears to be involved in mRNA splicing and RNA export (Kataoka et al., 1994). By hybridization with genomic DNA from a somatic cell hybridization panel, Chadwick et al. (1996) located the NCBP1 gene at 9q34.1 (Chadwick et al., 1996).

NEFH (neurofilament heavy polypeptide)

NEFH, which encodes neurofilament heavy chain, is a major component of the neuronal cytoskeleton. The 200 kD gene for neurofilament heavy chain polypeptide (NEFH) is located on chromosome band 22q12.2 and is considered a presymptomatic DNA marker in families with neurofibromatosis type 2 (NF2). Loss or downregulation of NEFH has been reported in human autonomic neuromas or central neurocytomas (Mena et al., 2001; Segal et al., 1994). Furthermore, loss or reduction of 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). Notably, overexpression of NEFH can disrupt normal cellular structure and function and induce cell death (Szebenyi et al., 2002).

Detailed description of the invention

Unless otherwise specified, all terms used herein are defined as follows:

As used herein, the term "peptide" refers to a series of amino acid residues, typically connected to each other by peptide bonds between the alpha and carbonyl groups of adjacent amino acids. Peptides are preferably 9 amino acids in length, but can be as short as 8 amino acids or as long as 10, 11, 12, 13, or 14 amino acids. MHC class II peptides can be as long as 15, 16, 17, 18, 19, or 20 amino acids.

Furthermore, the term "peptide" shall include salts of a series of amino acid residues, which are typically linked to each other via peptide bonds between the alpha amino acid and carbonyl groups of adjacent amino acids. Such salts are preferably pharmaceutically acceptable salts.

The term "peptide" shall include "oligopeptide." As used herein, the term "oligopeptide" refers to a series of amino acid residues, typically linked to each other by peptide bonds between the alpha amino acid and carbonyl groups of adjacent amino acids. The length of the oligopeptide is not critical to the present invention, as long as the oligopeptide maintains the correct phenotype. Oligopeptides are typically less than about 30 amino acid residues and more than about 15 amino acids.

The term "peptide of the present invention" shall consist of the peptides corresponding to SEQ ID No. 1 to SEQ ID No. 92 as described above.

The term "polypeptide" refers to a series of amino acid residues, typically linked together by peptide bonds between the alpha and carbonyl groups of adjacent amino acids. The length of the polypeptide is not critical to the present invention, as long as the polypeptide maintains the correct phenotype. In contrast to "peptide" or "oligopeptide," the term polypeptide refers to molecules containing more than about 30 amino acid residues.

A peptide, oligopeptide, protein or polynucleotide is "immunogenic" (and therefore an "immunogen" within the context of the present invention) if the molecule it encodes can elicit an immune response. In the context of the present invention, immunogenicity is more specifically defined as the ability to induce a T cell response. Thus, an "immunogen" is a molecule that can induce an immune response (specifically, a molecule that can induce a T cell response in the present invention). On the other hand, an immunogen can be a peptide, a complex of a peptide and MHC, an oligopeptide and/or a protein that is used to generate antibodies or TCRs specific thereto.

The class I T cell "phenotype" refers to a short peptide bound to the class I MHC receptor, thereby forming a ternary complex (MHC class I alpha chain, beta-2-microglobulin, and peptide) that is recognized by T cells bearing the appropriate T cell receptor that binds the MHC/peptide complex with appropriate affinity. Peptides bound to MHC class I molecules are typically 8 to 14 amino acids in length, with 9 amino acids being the most common.

There are three different gene loci in humans that encode MHC class I molecules (human MHC molecules are also called human eukaryotic cell antigens (HLA): HLA-A, HLA-B, and HLA-C. HLA-A*01, HLA-A*02, and HLA-B*07 represent different MHC class I alleles that can be expressed from this locus.

Table 2: Expression frequency (F) of HLA*A02 and the most common HLA-DR serotypes.

Frequencies were estimated based on the haplotype frequency _Gf in the US population according to the method of Mori et al. (Mori et al., 1997), using the Hardy-Weinberg formula F = 1-(1- _Gf ) ² . Due to linkage disequilibrium, the combination of A*02 with certain HLA-DR alleles may result in an increase or decrease in their individual frequencies. For details, see Chanock et al. (Chanock et al., 2004).

Therefore, for therapeutic and diagnostic purposes, peptides that can bind to multiple different HLA class II receptors with appropriate affinity are highly desirable. Peptides that bind to several different HLA class II molecules are called promiscuous binders.

References to DNA sequences herein include both single-stranded and multi-stranded DNA. Therefore, unless otherwise specified, references to a specific sequence refer to single-stranded DNA of that sequence, its duplex (double-stranded DNA), and its complement. The term "coding region" refers to that portion of a gene that naturally or normally encodes the expression product of a gene in its natural genomic environment, such as a region that encodes the naturally expressed product of a gene in vitro.

The coding region can be from a non-mutated ("normal"), mutated or altered gene, or even from a DNA sequence or gene that has been fully synthesized in the laboratory using current DNA synthesis methods.

The term "nucleotide sequence" refers to a heteropolymer of deoxyribonucleic acids.

The nucleotide sequence encoding a specific peptide, oligopeptide or polypeptide may be naturally occurring or artificially synthesized. Generally speaking, DNA fragments encoding the peptides, polypeptides and proteins of the present invention are assembled from cDNA fragments and short oligonucleotide connectors, or from a series of oligopeptides, thereby generating a synthetic gene that can be expressed in a recombinant transcription unit (composed of regulatory elements derived from microbial or viral operons).

As used herein, the term "nucleotide encoding a peptide" refers to a nucleotide sequence encoding a peptide containing artificial start and stop codons suitable for the biological system in which the sequence is expressed.

The term "expression product" refers to a polypeptide or protein that is the natural translation product of a gene, or any equivalently encoded product of an amino acid sequence that results from the degeneracy of the genetic code and therefore encodes the same amino acids.

The term "fragment" when applied to a coding sequence refers to a portion of DNA that is smaller than the entire coding region, the expression product of which carries essentially the same biological function or activity as the expression product of the entire coding region.

The term "DNA fragment" refers to a polymer of DNA, either as an individual fragment or as a component of a larger DNA construct. The fragment is derived from DNA that has been fragmented at least once and is in essentially condensed form (i.e., free of contaminating endogenous material and in an amount or concentration that allows identification, manipulation, and recovery of the fragment or its amino acid sequence components by standard biochemical methods, such as cloning vectors). Such fragments appear as continuous open reading frames that are not interrupted by internal non-translated sequences or introns (typically present in eukaryotic genes). Non-translated DNA sequences may be present downstream of the open reading frame and do not interfere with manipulation or expression of the coding region.

The term "primer" refers to a short nucleotide sequence that pairs with a DNA strand to form a free 3'-OH end from which DNA polymerase initiates deoxyribonucleic acid chain synthesis.

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

The term "isolated" refers to a material that is removed from its original environment, such as its natural environment (if it is a naturally occurring material). For example, a naturally occurring polynucleotide or a polypeptide present in a living animal is not isolated material, while the same polynucleotide or polypeptide separated from some or all of the coexisting materials in the natural system is isolated material. Such a polynucleotide can be part of a vector and/or such a polynucleotide or polypeptide can be part of a component, but if the vector or component is not part of its natural environment, the polynucleotide or polypeptide is still isolated material.

The polynucleotides and recombinant or immunogenic polypeptides disclosed herein may also be "purified". The term "purified" does not require absolute purity, but is a relative definition that may encompass highly purified preparations or preparations that are only partially purified. Such terms are understood by those with the corresponding current technical level. For example, individual clones isolated from a cDNA library are typically purified to electrophoretic homogeneity. It is expressly provided that starting materials and natural materials should be purified to at least one order of magnitude (preferably 2 or 3 orders of magnitude, more preferably 4 or 5 orders of magnitude). In addition, it is expressly provided that the purity of the polypeptide is preferably 99.999%, or at least 99.99% or 99.9%; and even suitably, it is 99% or more by weight.

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

The term "active fragment" refers to a fragment that can be used alone or in combination with an appropriate adjuvant to produce an immune response (i.e., have immunogenic activity) in an animal (e.g., a rabbit or mouse, including mammals such as humans). This immune response is in the form of stimulating a T cell response in a recipient animal (e.g., a human). "Active fragments" can also be used to elicit T cell responses in vitro.

As used herein, the terms "portion," "segment," and "fragment," when applied to polypeptides, refer to a contiguous sequence of residues (e.g., amino acid residues) that constitutes a subset of a larger sequence. For example, upon treatment of a polypeptide with a common endopeptidase (e.g., trypsin or chymotrypsin), the oligopeptides produced by such treatment are portions, fragments, or fragments of the starting polypeptide. When applied to polynucleotides, these terms refer to the products of treatment of the polynucleotide with any endonuclease.

The term "percent identity" as used in the present invention, when applied to sequences, means that a sequence to be compared ("comparison sequence") is aligned and compared with a defined or reported sequence ("reference sequence"), and then the percentage identity is calculated using the following formula:

Percent identity = 100[1-(C/R)]

wherein C is the number of differences between the reference sequence and the comparison sequence in the length of the alignment between the reference sequence and the comparison sequence, wherein (i) each base or amino acid in the reference sequence for which the corresponding aligned base or amino acid does not exist in the comparison sequence, (ii) each gap in the reference sequence, and (iii) each base or amino acid in the reference sequence that differs from an aligned base or amino acid in the comparison sequence constitutes a difference, and (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 that are within the length of the comparison sequence (gaps in the reference sequence are also counted as bases or amino acids).

If there is an alignment between the comparison sequence and the reference sequence and the percentage identity between the two calculated as above is equal to or greater than the specified minimum percentage identity limit, then the comparison sequence can be considered to have the specified minimum percentage identity with the reference sequence, even though there may be alignments where the percentage identity calculated as above is lower than the specified percentage identity.

Unless otherwise stated, the original (unmodified) peptides referred to herein can be modified by substituting one or more residues at different (possibly selective) positions in the peptide chain.

These substitutions preferably occur at the termini of the amino acid chain. Such substitutions can be conservative, such as replacing one amino acid with another with similar structure or properties (e.g., replacing one hydrophobic amino acid with another). Substitutions between amino acids of the same or similar size and chemical properties are more conservative, such as replacing leucine with isoleucine. In studies of sequence variants within naturally occurring homologous protein families, some amino acid substitutions are more tolerated than others. Such substitutions are generally related to similar size, charge, polarity, and hydrophobicity between the original amino acid and the substituted amino acid. These properties form the basis for the definition of "conservative substitutions."

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

Less conservative substitutions might involve replacing an amino acid with one that has similar properties but differs in size, such as replacing an alanine residue with an isoleucine residue. Highly non-conservative substitutions might involve replacing an acidic amino acid with one that has polar or even basic properties. However, these "radical" substitutions should not be dismissed as potentially ineffective, as chemistry is not entirely predictable, and radical substitutions may produce unintended effects that cannot be predicted by single chemical principles.

Of course, such substitutions may involve structures that differ from common L-amino acids. Thus, even though D-amino acids may be substituted with L-amino acids common to the antigenic peptides of the present invention, the present invention still encompasses D-amino acids. Furthermore, amino acids with processable non-standard R groups (i.e., R groups other than the 20 common amino acids of natural proteins) may also be substituted to produce immunogens and immunogenic polypeptides according to the present invention.

If substitutions at multiple positions are found to produce peptides with substantially equivalent or enhanced antigenic activity (defined below), such substitution combinations should be tested to determine whether the substitutions have additive or synergistic effects on the antigenicity of the peptide. No more than four positions may be substituted simultaneously in the same peptide.

The peptides of the present invention may be extended by up to 4 amino acids, ie, 1, 2, 3 or 4 amino acids may be added to either end in any combination from 4:0 to 0:4.

Table 3 lists the types of peptide extension combinations allowed by the present invention:

The amino acids used for extension can be peptides or any other amino acids in the original sequence of the protein. The purpose of extension is to improve the stability or solubility of the peptide.

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 T cells, the effector function can be lysis of target cells pulsed with the peptide, pulsed with a peptide precursor, or presented with the native peptide, secretion of cytokines (preferably peptide-induced interferon gamma, TNF alpha, or IL-2), secretion of effector molecules (preferably peptide-induced granzymes or perforins), or degranulation.

Ideally, when detecting CTLs specific for peptides of SEQ ID No. 1 to SEQ ID No. 92 (as compared to the substituted peptide), the peptide concentration at which the substituted peptide achieves a maximum increase in lysis relative to background should not exceed about 1 mM, preferably not exceed about 1 μM, more preferably not exceed about 1 nM, even more preferably not exceed about 100 pM, and most preferably not exceed about 10 pM. The substituted peptide should preferably be recognized by CTL in at least one individual (a minimum of two, more preferably three).

Therefore, the phenotype of the present invention may be consistent with a naturally occurring tumor-associated or tumor-specific phenotype, and may also include a phenotype that differs from a reference peptide by no more than 4 residues, as long as the antigenic activity of the phenotype is substantially the same.

The initiation of an immune response depends on antigens that are recognized as foreign by the host immune system. The discovery of tumor-associated antigens has raised the possibility of harnessing the host immune system to hinder tumor growth. Various mechanisms are currently being explored for cancer immunotherapy, leveraging both humoral and cellular responses.

Specific elements of the cellular immune response can specifically recognize and destroy tumor cells. Cytotoxic T lymphocytes (CTLs), isolated from tumor-infiltrating cell populations or peripheral blood, play a crucial role in the innate immune defense against cancer. CD8+ T cells are particularly important in this response because they recognize cells carrying major histocompatibility complex (MHC) class I molecules, which typically consist of 8 to 10 amino acid residues derived from proteins or defective ribosomal products (DRIPS) in the cytosol. MHC molecules in humans are also known as human leukocyte antigens (HLA).

MHC class I molecules are present in most nucleated cells. The peptides they present are primarily derived from endogenous proteins, DRIPs, and proteolytic cleavage of larger peptides. However, peptides originating from endosomal compartments or exogenous sources are also frequently found on MHC class I molecules. This non-classical class I presentation is referred to in the literature as cross-presentation.

Because both types of responses (CD8- and CD4-dependent) can produce synergistic antitumor effects, the identification and characterization of tumor-associated antigens recognized by either CD8+ CTLs (ligand: MHC class I molecule + peptide phenotype) or CD4-positive helper T cells (ligand: MHC class II molecule + peptide phenotype) is crucial for the development of antitumor vaccines. Therefore, one of the objectives of the present invention is to propose peptide components that can bind to various types of MHC complexes.

Given the severe side effects and high costs of cancer treatment, there is an urgent need for better prognostic and diagnostic methods. Therefore, there is a need to identify additional factors that can serve as biomarkers for cancer, particularly lung cancer. Furthermore, there is a need to identify factors that can be used in tumor treatment, particularly for lung cancer.

The present invention provides peptides that can be used to treat cancer/tumors, preferably lung cancer, and most preferably non-small cell lung cancer (NSCLC), which can overexpress or uniquely express the peptides of the present invention. Mass spectrometry analysis has shown that such peptides can be naturally presented by HLA molecules in primary human lung cancer samples (see Example 1 and Figure 1).

The peptide's source gene/protein (also referred to as the "full-length protein" or "basic protein") is highly overexpressed in non-small cell lung cancer, gastric cancer (SEQ IDs Nos. 66 to 75), and glioblastoma compared to normal tissue (see Example 2; for NSCLC, see Figure 2), indicating a strong association between the source gene and tumors. Furthermore, the peptide itself is also significantly overexpressed in tumor tissue, but not in normal tissue (see Example 3 and Figure 3).

HLA-bound peptides are recognized by the immune system, particularly T lymphocytes, which can then destroy cells presenting the recognized HLA/peptide complex, such as lung cancer cells presenting the derived peptide.

Studies have shown that the peptides of the present invention can stimulate T cell responses and/or there is over-presentation, and therefore can be used to produce antibodies and/or TCRs involved in the present invention, in particular TCRs (see Example 4 and Figure 4). In addition, peptides complexed with corresponding MHC can be used to produce antibodies and/or TCRs involved in the present invention, in particular TCRs. The corresponding methods are well known to those skilled in the art and can also be found in the corresponding literature. Therefore, the peptides of the present invention help to produce an immune response that can destroy tumor cells in patients. An immune response can be elicited in patients by directly administering the peptides described herein or suitable precursor substances (such as extended peptides, proteins, or nucleotides encoding such peptides), preferably in combination with immunogenic drugs (such as adjuvants). The immune response generated by this therapeutic vaccination is expected to be highly specific to tumor cells because the target peptides of the present invention do not have a certain copy number in normal tissues, thereby preventing the risk of adverse autoimmune reactions to normal cells.

Pharmaceutical compositions include peptides in free form or as pharmaceutically acceptable salts. As used herein, "pharmaceutically acceptable salt" refers to a derivative of a peptide disclosed herein, wherein the peptide has been modified by forming an acid or base salt of the corresponding drug. For example, an acid salt can be prepared by reacting a free base (particularly a neutral form of a drug containing a neutral _-NH2 group) with a suitable acid. Suitable acids for preparing acid salts include organic acids (e.g., 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, acetylsalicylic acid, etc.) and inorganic acids (e.g., hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, etc.). Conversely, the preparation of base salts of acid groups that may be present in the peptide requires the use of a pharmaceutically acceptable base, such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine, etc.

In preferred embodiments, the pharmaceutical composition may include the peptide in the form of an acetate salt, a trifluoroacetate salt, or a hydrochloride salt.

In addition to being useful for cancer treatment, the peptides of the present invention may also be used for diagnosis. Since these peptides are produced in lung cancer cells and are absent or at low levels in normal cells, these peptides can be used for cancer diagnosis.

The presence of related peptides in biopsied tissue can aid pathologists in diagnosing cancer. Detection of specific peptides using antibodies, mass spectrometry, or other currently available methods can indicate to the pathologist whether the tissue is malignant, inflammatory, or a more general disease. Specific groups of peptides can be used to classify or subclassify diseased tissue.

Detection of peptides in diseased tissue specimens can predict benefit from immune system therapies, particularly when T lymphocytes are known or expected to be involved in their mechanism of action. Loss of MHC expression is a well-documented mechanism by which malignant cells can escape immune surveillance. Therefore, the presence of peptides indicates that the analyzed cells do not utilize this mechanism.

The peptides of the present invention are expected to be used to analyze the response of lymphocytes to such peptides, such as T cell responses or antibody responses to peptides or peptide-MHC molecule complexes. The above-mentioned lymphocyte responses can be used as prognostic markers to determine subsequent treatment steps. Such responses can also be used as surrogate markers in immunotherapy that aims to induce lymphocyte responses using various methods (e.g., immunization with proteins, nucleotides, autologous substances, and adoptive transfer of lymphocytes). In gene therapy, the response of lymphocytes to peptides can be considered in the assessment of side effects. Monitoring of lymphocyte responses is likely to be an effective tool for follow-up examinations (e.g., for the detection of graft-versus-host disease or host-versus-graft disease) in transplantation therapy.

The peptides of the present invention can be used to generate antibodies specific for MHC/peptide complexes. Such antibodies can be used therapeutically to target toxins or radioactive substances to diseased tissue. Another use of these antibodies is to target radionuclides to diseased tissue for imaging therapies such as PET. This application can help detect small metastases or determine the size and exact location of diseased tissue.

Therefore, another aspect of the present invention is a method for producing a recombinant antibody that can specifically bind to human major histocompatibility complex (MHC) I or II (complexed with an HLA-restricted antigen), the method comprising: immunizing genetically engineered non-human mammalian cells (capable of expressing the above-mentioned human MHC class I or class II) with a soluble form of an MHC class I or class II molecule complexed with the above-mentioned HLA-restricted antigen; isolating mRNA molecules from the above-mentioned antibody-producing non-human mammalian cells; generating a phage display library to display protein molecules encoded by the above-mentioned mRNA molecules; and isolating at least one phage from the above-mentioned phage display library, which phage can display the above-mentioned antibody that can specifically bind to MHC class I or class II (complexed with the above-mentioned HLA-restricted antigen).

Another aspect of the present invention is to provide an antibody that can specifically bind to human major histocompatibility complex (MHC) I or II (complexed with HLA-restricted antigens), which antibody is preferably a polyclonal antibody, a monoclonal antibody, a bispecific antibody and/or a chimeric antibody.

In addition, another aspect of the present invention relates to a method for producing the above-mentioned antibody that can specifically bind to human major histocompatibility complex (MHC) I or II (complexed with HLA-restricted antigens), the method comprising: immunizing genetically engineered non-human mammalian cells (capable of expressing the above-mentioned human MHC class I or class II) with a soluble form of MHC class I or class II molecules complexed with the above-mentioned HLA-restricted antigens; isolating mRNA molecules from the above-mentioned antibody-producing non-human mammalian cells; generating a phage display library to display protein molecules encoded by the above-mentioned mRNA molecules; and isolating at least one phage from the above-mentioned phage display library, which phage can display the above-mentioned antibody that can specifically bind to MHC class I or class II (complexed with the above-mentioned HLA-restricted antigens). Corresponding methods for producing the above antibodies, single-chain class I MHC, and other tools for producing the above 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, quantification, and in situdetection of human viral epitopes using peptide-specific, MHC-restricted human recombinant antibodies. J Immunol. 2003 Apr 15; 170(8): 4349-61, all of which are used in the present invention and are explicitly incorporated herein by reference in their entirety.

The antibody preferably binds to the complex with an affinity less than 20 nanomolar, and most preferably less than 10 nanomolar, and is therefore considered "specific" for purposes of the present invention.

Another aspect of the present invention is a method for producing a soluble T cell receptor that recognizes a specific peptide-MHC complex. Such a T cell receptor can be produced by specific T cell clones, and its affinity can be enhanced by mutations acting on the complementarity determining region. T cell receptors can be selected using phage display (US 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). For the purpose of stabilizing T cell receptors during phage display and in practical pharmaceutical applications, the α and β chains can be linked by non-native disulfide bonds, other covalent bonds (single-chain T cell receptors), or dimerization domains (see 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 GF, Wyer J, 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. 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 US 2013/0115191), effector cell recruitment domains such as anti-CD3 domains, etc. to produce specific functions on target cells. In addition, T cell receptors can be expressed in T cells used for adoptive transfer.

For more detailed information, see WO 2004/033685 A1 and WO 2004/074322 A1. The use of sTCR compositions is reported in WO 2012/056407 A1. More detailed information on receptor production is contained in WO 2013/057586 A1.

Additionally, such receptors could be used by pathologists to diagnose cancer in biopsy specimens.

Presentation profiles were calculated to select over-presented peptides, indicating moderate sample presentation and replicate variability. The profiles juxtapose relevant tumor samples with normal tissue samples (baseline). These profiles can then be used to score over-presentation by calculating p-values from a linear mixed-effects model (J. Pinheiro, D. Bates, S. DebRoy, Sarkar D., RCore team. Nlme: Linear and Nonlinear Mixed Effects Models. 2008), thereby adjusting for multiple testing using 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).

Mass spectrometry was used to identify and relatively quantify HLA ligands. HLA molecules from snap-frozen tissue samples were purified and HLA-associated peptides were isolated. The isolated peptides were separated and their sequences were identified using online nanoelectrospray ionization (nanoESI) liquid chromatography-mass spectrometry (LC-MS). The resulting peptide sequences were validated by comparing the fragmentation spectra of native TUMAPs recorded from NSCLC samples with those of corresponding synthetic reference peptides of the same sequence. Because the peptides were directly identified as ligands for HLA molecules in the primary tumor, these results provide direct evidence for the natural processing and presentation of the identified peptides in primary tumor tissue from NSCLC patients.

The patented discovery platform v2.1 (see US 2013-0096016, etc., incorporated herein by reference in its entirety) enables the identification and selection of relevant over-presented peptide vaccine candidates by directly quantifying HLA-restricted peptide levels in cancer tissues, compared to several different lung cancer tissues and organs. This functionality relies on the development of a label-free identification and quantification assay (using LC-MS data processed by the patented data analysis platform), combining sequence identification algorithms, spectral clustering, ion counting, retention time correction, charge state deconvolution, and normalization.

The presentation levels of each peptide and sample were established (including error estimates). Peptides that were exclusively presented in tumor tissue and those that were over-presented in tumor tissue (compared to non-cancerous tissues and organs) were discovered.

HLA-peptide complexes obtained from 50 snap-frozen NSCLC tumor tissue samples were purified, and HLA-associated peptides were separated and analyzed by LC-MS.

All TUMAPs included in this application were discovered in primary NSCLC tumor samples using the above-described method, confirming their presentation in primary NSCLC.

TUMAPs were quantified in various NSCLC tumor and normal tissues by counting ions in label-free LC-MS data. This method assumes that the LC-MS signal area of a peptide correlates with its abundance in the sample. All peptide quantification signals from various LC-MS experiments were normalized to their central tendency and the mean value for each sample and plotted as a bar graph, representing the presentation profile. The presentation profile incorporates various analytical methods, such as protein database searching, spectral clustering, charge state deconvolution (de-charging), retention time correction, and normalization.

Therefore, the present invention relates to a peptide consisting of 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 variant sequences thereof (which are at least 90% homologous (preferably identical) to SEQ ID No. 1 to SEQ ID No. 65, SEQ ID No. 76 to SEQ ID No. 84 and SEQ ID No. 92), wherein the variant sequence can induce T cells to cross-react with the peptide, wherein the peptide is a non-full-length polypeptide.

The present invention also relates to a peptide consisting of a sequence selected from SEQ ID No. 1 to SEQ ID No. 65, SEQ ID No. 76 to SEQ ID No. 84, and SEQ ID No. 92, or a variant thereof, which 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. The total length of the peptide or variant thereof is 8 to 100 amino acids (preferably 8 to 30, most preferably 8 to 14).

The present invention also relates to peptides according to the invention that bind to human major histocompatibility complex (MHC) class I or class II.

The present invention also relates to the peptides according to the present invention, which consist of (or essentially consist of) the amino acid sequences of SEQ ID No. 1 to SEQ ID No. 65, SEQ ID No. 76 to SEQ ID No. 84 and SEQ ID No. 92.

The present invention also relates to peptides according to the invention which are modified and/or comprise non-peptide bonds.

The present invention also relates to peptides according to the invention, which are part of a fusion protein, in particular a protein fused to the N-terminal amino acids of the invariant chain (Ii) associated with the HLA-DR antigen, or to an antibody (or its sequence), such as a dendritic cell-specific antibody.

The present invention also relates to a nucleic acid encoding a peptide according to the present invention (provided that the peptide is not a complete human protein).

The present invention also relates to the nucleic acid according to the present invention, which is in the form of DNA, cDNA, PNA, RNA or a combination of DNA, cDNA, PNA and RNA.

The present invention also relates to an expression vector, which can express the nucleic acid according to the present invention.

The present invention also relates to a peptide according to the present invention, a nucleic acid according to the present invention or a pharmaceutical expression vector according to the present invention.

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

The present invention also relates to a host cell according to the present invention, which must be an antigen presenting cell.

The present invention also relates to a host cell according to the present invention, wherein the antigen presenting cell is a dendritic cell.

The present invention also relates to a method for preparing the peptide according to the present invention, which comprises culturing the host cell according to the present invention, and separating the peptide from the host cell or its culture medium.

The present invention also relates to an in vitro method for preparing activated cytotoxic T lymphocytes (CTLs), which comprises contacting the in vitro CTLs with antigen-loaded human class I or class II MHC molecules (which are expressed on the surface of appropriate antigen-presenting cells for a sufficient time to activate the above-mentioned CTLs in an antigen-specific manner), wherein the antigen can be any peptide according to the present invention.

The present invention also relates to a method according to the invention, wherein an antigen is loaded onto a class I or class II MHC molecule expressed on the surface of a suitable antigen presenting cell (by contacting a sufficient amount of the antigen with the antigen presenting cell).

The present invention also relates to a method according to the present invention, wherein the antigen presenting cells contain an expression vector capable of expressing the peptide comprising the amino acid sequences 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 the variant amino acids thereof.

The present invention also relates to activated cytotoxic T lymphocytes (CTLs) prepared by the method of the present invention, which can selectively recognize cells that abnormally express a polypeptide containing the amino acid sequence of the present invention.

The present invention also relates to a method for killing target cells that aberrantly express a polypeptide comprising any of the amino acid sequences described herein in a patient, comprising administering an effective number of cytotoxic T lymphocytes (CTL) to the patient according to the method described herein.

The present invention also relates to a medicament or any of the above peptides, the nucleic acid according to the present invention, the expression vector according to the present invention, the cell according to the present invention or the activated cytotoxic T lymphocyte according to the present invention for use in the production of the medicament.

The present invention also relates to the use according to the present invention, wherein the medicament is a vaccine.

The present invention also relates to the use according to the invention, wherein the medicament has anti-tumor activity.

The present invention also relates to the use according to the present invention, wherein the cancer cells are lung cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, pancreatic cancer or renal cancer cells, and glioblastoma cells.

The present invention also relates to specific marker proteins and biomarkers based on the peptides according to the present invention, which can be used for the prognosis of lung cancer.

In addition, the present invention also relates to the use of the novel target for cancer treatment.

As used herein, "antibody" is a broad term that includes both polyclonal and monoclonal antibodies. In addition to intact or "whole" immunoglobulin molecules, the term "antibody" also includes fragments or multimers of such immunoglobulins, or humanized forms of immunoglobulin molecules, as long as they can produce the desired properties of the present invention (e.g., specific binding of a lung cancer marker polypeptide, delivery of a toxin to lung cancer cells expressing an elevated level of a lung cancer marker gene, and/or inhibition of the activity of a lung cancer marker polypeptide).

The antibodies of the present invention should be purchased from commercial sources whenever possible. The antibodies of the present invention can also be prepared using conventional methods. Those skilled in the art will appreciate that full-length lung cancer marker polypeptides or fragments thereof can be used to generate the peptides of the present invention. The polypeptides used to generate the antibodies of the present invention can be partially or completely purified from natural sources or produced using recombinant DNA technology.

For example, cDNAs encoding ABCA13, MMP12, DST, MXRA5, CDK4, HNRNPH, TANC2, 1RNF213, SMYD3, and SLC34A2, or any related polypeptides of SEQ ID No. 1 to SEQ ID No. 65, SEQ ID No. 76 to SEQ ID No. 84, or SEQ ID No. 92 (or fragments thereof) can be expressed in prokaryotic cells (e.g., bacteria) or eukaryotic cells (e.g., yeast, insect, or mammalian cells), after which the recombinant protein can be purified and used to produce monoclonal or polyclonal antibody preparations that can specifically bind to the lung cancer marker polypeptides used to generate antibodies in the present invention.

Those with the current state of the art know that generating at least two groups of monoclonal or polyclonal antibodies can ensure to the greatest extent that the resulting antibodies have the specificity and affinity required for their intended use (e.g., ELISA immunohistochemistry, in vivo imaging analysis, immunotoxin therapy). The desired activity of the antibody can be detected using known methods (e.g., ELISA, immunohistochemistry, immunotherapy, etc.) according to the intended use of the antibody; for more specific guidance on antibody generation and detection, see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988, new ^2nd edition 2013, etc.). For example, ELISA analysis, Western blotting, or immunohistochemical staining can be used to detect antibodies on formalin-fixed lung cancer or frozen tissue sections. After the initial in vitro characterization is completed, the antibodies planned for treatment or in vivo diagnosis should be tested using known clinical detection methods.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies in the population are identical except for possible naturally occurring mutations. Monoclonal antibodies herein specifically also include "chimeric" antibodies, in which a portion of the heavy and/or light chains are identical or homologous to corresponding sequences of antibodies obtained from a particular species or belonging to a particular antibody class or subclass, while the remaining chains are identical or homologous to corresponding sequences of antibodies obtained from another species or belonging to another antibody class or subclass (as well as fragments of such antibodies), so long as the antibody exhibits the desired anti-tumor activity (U.S. Patent No. 4,816,567, incorporated herein by reference in its entirety).

The monoclonal antibodies of the present invention can be prepared using the hybridoma method. When using the hybridoma method, mice or other appropriate host cells are generally immunized with an immunizing agent to allow lymphocytes to produce or generate antibodies that specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro.

Monoclonal antibodies can also be made by recombinant DNA methods (e.g., the method described in U.S. Patent No. 4816567). DNA encoding the monoclonal antibodies of the invention can be isolated and sequenced using conventional procedures (e.g., using oligonucleotide probes that bind to genes encoding mouse antibody heavy and light chains).

In vitro methods are also applicable to the preparation of monovalent antibodies. Antibodies can be broken down into their fragments (particularly Fab fragments) using conventional methods known in the art. For example, papain can be used for decomposition. WO94/29348, published on December 22, 1994, and U.S. Patent No. 4,342,566 both describe examples of papain decomposition. Papain decomposition of antibodies typically produces two identical antigen-binding fragments (called Fab fragments, each containing a single antigen-binding site) and a residual Fc fragment. Pepsin treatment can produce a fragment that contains two antigen-binding sites and can be cross-linked with an antigen.

Antibody fragments (whether or not other sequences are added) may also comprise insertions, deletions, substitutions or other selected modifications of specific regions or specific amino acid residues, as long as the activity of the fragment is not significantly changed or damaged compared to the unmodified antibody or antibody fragment. These modifications can produce certain additional properties, such as removing/adding amino acids that can form disulfide bonds to extend their biological lifespan or change their secretion characteristics. In any case, the antibody fragment should have biologically active properties, such as binding activity and regulation of binding to the binding domain. The functional or active region of the antibody can be identified by mutations in specific regions of the protein and then by quantitative expression and detection of the expressed polypeptide. Such methods are well known to those skilled in the art and may comprise mutations at specific sites of the nucleotides encoding the antibody fragment.

The antibodies of the present invention may also comprise humanized antibodies or human antibodies. The humanized form of a non-human (e.g., murine) antibody may be a chimeric immunoglobulin, an immunoglobulin chain, or its fragment (e.g., Fv, Fab, Fab', or other antigen-binding sequences of an antibody), comprising a minimal sequence derived from a non-human immunoglobulin. Humanized antibodies include human immunoglobulins (receptor antibodies), wherein the complementary determining region (CDR) residues of the receptor are replaced by residues with desired specificity, affinity, and ability in the CDRs of non-human species (donor antibodies) (e.g., mouse, rat, rabbit). In some cases, the Fv framework (FR) residues of the human immunoglobulin may be replaced by corresponding non-human residues. Humanized antibodies may also comprise residues that are not present in the receptor antibody, the imported CDR, or the framework sequence. Generally speaking, a humanized antibody comprises at least one (usually two) variable domain, wherein all or nearly all of the CDR regions correspond to the CDR regions of a non-human immunoglobulin, and all or nearly all of the FR regions are FR regions of a human immunoglobulin consensus sequence. The humanized antibody optimally also comprises at least a fragment of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

Methods for humanizing non-human antibodies are well known in the art. Generally speaking, a humanized antibody contains one or more amino acid residues that are derived from non-human pathways. Such non-human amino acid residues are often referred to as "input" residues and are typically derived from "input" variable domains. In essence, humanization can typically be achieved by replacing rodent CDRs or CDR sequences with corresponding human antibody sequences, and the resulting "humanized" antibodies are chimeric antibodies (U.S. Patent No. 4,816,567), in which a sequence smaller than a complete human variable domain is replaced by a corresponding sequence from a non-human species. In actual operation, humanized antibodies are typically human antibodies in which certain CDR residues (and possibly certain FR residues) are replaced by residues from similar sites in rodent antibodies.

Transgenic animals (e.g., mice) that can produce a full complement of human antibodies upon immunization even in the absence of endogenous immunoglobulins can be used. For example, homozygous deletion of the antibody heavy chain joining region gene in chimeric and germline mutant mice can lead to complete inhibition of endogenous antibody production. Transferring the human germline immunoglobulin gene array into the above-mentioned germline mutant mice can produce human antibodies upon antigen challenge. Human antibodies can also be produced in phage display libraries.

The antibodies of the present invention are preferably administered to a subject via a pharmaceutical carrier. An appropriate dose of a pharmaceutical salt is typically used in the formulation to render the formulation isotonic. Pharmaceutical carriers include physiological saline, Ringer's solution, and dextrose solution. The pH of the solution is preferably from about 5 to about 8, more preferably from about 7 to about 7.5. Other carriers include sustained-release formulations (e.g., a semipermeable matrix solution of a solid hydrophobic polymer containing the antibody), wherein the matrix is a tangible object, such as a membrane, liposome, or microparticle. It is well known to those skilled in the art that certain carriers may be more preferred (depending on, for example, the route of administration and the concentration of the antibody to be administered).

The antibody can be administered to the subject, patient, or cell by injection (e.g., intravenous, intraperitoneal, subcutaneous, or intramuscular) or by other routes (e.g., infusion) as long as the antibody can enter the bloodstream in an effective form. The antibody can also be administered intratumorally or peritumorally to produce local and systemic therapeutic responses. Local or intravenous administration is preferred.

The dosage and schedule of antibody administration can be determined empirically within the current state of the art. Those skilled in the art will appreciate that the dosage of antibodies will vary depending on the type of subject receiving the antibody, the route of administration, the specific type of antibody and other drugs used, and the like. Based on the above factors, common single-use daily doses of antibodies range from about 1 μg/kg per day to a maximum of 100 mg/kg body weight, and may be higher. After administration of antibodies for the treatment of lung cancer, the efficacy of the therapeutic antibodies can be evaluated in a variety of ways familiar to skilled personnel. For example, the size, number, and/or distribution of tumors in treated subjects can be monitored using standard tumor imaging techniques. If a therapeutic antibody can stop tumor growth, induce tumor shrinkage, and/or prevent the formation of new tumors (compared to the normal course of the disease without antibody treatment), then the antibody can be considered to be effective in treating lung cancer.

Since the non-tumor markers ABCA13 and MMP12 of the present invention are highly expressed in lung cancer cells and expressed at very low levels in normal cells, inhibiting the expression or polypeptide activity of ABCA13 and MMP12 can be used as part of a strategy for treating or preventing NSCLC.

The principle of antisense therapy is based on the following assumption: sequence-specific inhibition of gene expression (through transcription or translation) can be achieved by intracellular hybridization of genomic DNA or mRNA with a complementary antisense sequence. The formation of this hybrid nucleic acid duplex can interfere with the transcription of the DNA encoding the target tumor antigen or interfere with the processing/transport/translation and/or stability of the target tumor antigen mRNA.

Antisense nucleic acids can be delivered via a variety of routes. For example, antisense oligonucleotides or antisense RNA can be directly administered to a subject (e.g., by intravenous injection) in a form that can be taken up by tumor cells. Alternatively, a virus or plasmid vector encoding the antisense RNA (or RNA fragment) can be introduced into cells in vitro. Antisense effects can also be elicited by sense sequences; however, the magnitude of the phenotypic changes is highly variable. The phenotypic changes induced by effective antisense therapy can be assessed based on changes, for example, by target mRNA levels, target protein levels, and/or target protein activity levels.

For example, antisense gene therapy to inhibit lung tumor marker function can be achieved by directly administering antisense lung tumor marker RNA to a subject. Antisense tumor marker RNA can be produced and isolated using standard techniques, but can also be prepared ex vivo from antisense tumor marker cDNA under the control of a highly efficient promoter (e.g., the T7 promoter). Intracellular administration of antisense tumor marker RNA can be performed using any of the following direct nucleic acid administration methods.

Another gene therapy strategy for inhibiting ABCA13 and MMP12 function involves the intracellular expression of anti-ABCA13, MMP12 antibodies or anti-ABCA13, MMP12 antibody fragments. For example, a gene encoding a monoclonal antibody that specifically binds to and inhibits the biological activity of ABCA13, MMP12 polypeptides can be placed under the transcriptional control of specific (e.g., tissue- or tumor-specific) gene regulatory sequences within a nucleic acid expression vector. This vector is then administered to a subject for uptake by lung cancer cells or other cells, which then secrete the anti-ABCA13, MMP12 antibodies, thereby inhibiting the activity of the ABCA13, MMP12 polypeptides. The ABCA13, MMP12 polypeptides are preferably located on the extracellular surface of gastric cancer cells.

In the above-mentioned method of administering or taking up exogenous DNA into cells of a subject (eg, by gene transduction or transfection), the nucleotide of the present invention may be in the form of naked DNA, or the nucleotide may be delivered to the cells via a vector to inhibit the expression of gastric tumor marker protein. The vector can be a commercially available preparation, such as an adenoviral vector (Quantum Biotechnologies, Inc. (Laval, Quebec, Canada). Nucleic acid or vector delivery into cells can be achieved by a variety of mechanisms. For example, commercially available liposome preparations such as LIPOFECTIN, LIPOFECTAMINE (GIBCO-25BRL, Inc., Gaithersburg, Md.), SUPERFECT (Qiagen, Inc. Hilden, Germany), and TRANSFECTAM (Promega Biotec, Inc., Madison, Wis.) can be used for liposome delivery, as well as other liposomes prepared according to current standard procedures. In addition, the nucleotide or vector of the present invention can be delivered in vivo by electroporation, a technology available from Genetronics, Inc. (San Diego, Calif.), or by using a SONOPORATION instrument (ImaRx Pharmaceutical Corp., Tucson, Arizona).

For example, vector delivery can be performed via a viral system (e.g., a retroviral vector system that can package a recombinant retroviral genome). The recombinant retrovirus can then infect cells, thereby delivering antisense nucleotides that can inhibit the expression of ABCA13 and MMP12 to the infected cells. Of course, the specific method for introducing the altered nucleotides into mammalian cells is not limited to the use of retroviral vectors. Other commonly used techniques for this step include the use of adenoviral vectors, adenovirus-associated virus (AAV) vectors, lentiviral vectors, and pseudotyped viral vectors. Physical delivery techniques, such as liposome delivery and receptor-mediated and other cellular endocytosis mechanisms, can also be used. The present invention can be used in combination with the above or any other commonly used gene transfer methods.

Antibodies can also be used in in vivo diagnostic assays. Generally, antibodies can be labeled with radionucleotides (e.g., ¹¹¹ In, ⁹⁹ Tc, ¹⁴ C, ¹³¹ I, ³ H, ³² P, or ³⁵ S) to allow tumor localization using immunoscintigraphy. In one embodiment, the antibody or fragment thereof can bind to the extracellular domains of at least two ABCA13 and MMP12 targets with an affinity (Kd) of less than 1 x 10 μM.

Diagnostic antibodies can be labeled with probes suitable for detection by various imaging methods. Probe detection methods include, but are not limited to, fluorescence, natural light, confocal, and electron microscopy; magnetic resonance imaging and magnetic resonance spectroscopy; fluoroscopy, computed tomography, and positron emission tomography. Suitable probes include, but are not limited to, fluorescein, rhodamine, eosin, and other fluorescent groups, radioisotopes, gold, gadolinium or other lanthanides, paramagnetic ions, fluorine-18, and other positron-emitting radionuclides. Furthermore, the probes described above can be bifunctional or multifunctional and can be detected using the various methods described above. Methods for binding the probe to the antibody include covalent probe binding, probe incorporation into the antibody, probe binding induced by covalent chelate binding, and other commonly used techniques. Lesion tissue samples for immunohistochemical testing can be fresh or frozen, paraffin-embedded, or fixed with a preservative (e.g., formalin). Fixed or embedded sections are contacted with labeled primary or secondary antibodies, where the antibodies are used to detect in situ expression of ABCA13 and MMP12 proteins.

Therefore, the present invention provides a peptide composed of 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 their variant sequences (which 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), and the above sequence or its variant sequence can induce T cells to cross-react with the above peptide.

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

In the present invention, the term "homologous" refers to the degree of identity between two amino acid sequences (i.e., peptide or polypeptide sequences) (see percentage identity above). The "homology" described above is assessed by aligning the two sequences under optimal conditions above the sequence to be compared. Sequence homology can be calculated by establishing a sequence alignment using algorithms such as ClustalW. Commonly used sequence analysis software (e.g., Vector NTI, GENETYX) or other analysis tools are available in public databases.

The skilled artisan is able to assess whether T cells elicited by a specific peptide variant sequence can cross-react with the peptide itself (Fong et al., 2001); (Zaremba et al., 1997; Colombetti et al., 2006; Appay et al., 2006).

As used by the inventors, a "variant sequence" of a specific amino acid sequence refers to a peptide in which the side chains of one or two amino acid residues are altered (e.g., by replacing the side chains with those of another naturally occurring amino acid residue or with another side chain), but the peptide can still bind to HLA molecules in substantially the same manner as the peptides consisting of the specific amino acid sequences of SEQ ID No. 1 to SEQ ID No. 65, SEQ ID No. 76 to SEQ ID No. 84, and SEQ ID No. 92. For example, a modified peptide can at least maintain (even if not improve) the ability to react with and bind to the binding groove of an appropriate MHC molecule (e.g., HLA-A*02 or -DR) and at least maintain (even if not improve) the ability to bind to the TCR of activated CTLs.

These CTLs can further cross-react with cells and kill polypeptides that express the native amino acid sequence of the cognate peptides defined herein. It can be inferred from scientific literature (Rammensee et al., 1997) and databases (Rammensee et al., 1999) that certain positions within HLA-binding peptides are typical anchor residues, forming a core sequence that assembles into the binding motif of the HLA receptor. This process is determined by the polarity, electrophysical properties, hydrophobicity, and steric properties of the polypeptide chains that comprise the binding groove. Thus, one skilled in the art can modify the amino acid sequences corresponding to SEQ ID No. 1 to SEQ ID No. 65, SEQ ID No. 76 to SEQ ID No. 84, and SEQ ID No. 92 by maintaining known anchor residues and determine whether such variant sequences retain the ability to bind to MHC class I or II molecules. The variant sequences of the present invention can retain the ability to bind to the TCR of activated CTLs, which can further cross-react with and kill cells expressing polypeptides that contain the native amino acid sequence of the cognate peptides defined herein.

Amino acid residues that are not substantially involved in the interaction with the T cell receptor can be modified by substitution with another amino acid whose incorporation does not substantially affect T cell reactivity and does not eliminate binding to the relevant MHC. Thus, except for the above conditions, the peptides of the present invention may be any peptide (the inventors use this term to include both oligopeptides and polypeptides) comprising a given amino acid sequence or a fragment or variant sequence thereof.

Table 4: Variant sequences and motifs of peptides corresponding to SEQ ID NOs: 1, 2, 4, 5 and 7

Longer peptides may also be suitable. Although MHC class I phenotypes are typically 8-11 amino acids in length, it is possible that longer peptides or proteins may be generated from the processing of proteins that contain the actual phenotype. The residues flanking the actual phenotype are preferably residues that do not substantially affect the proteolytic cleavage required for exposure of the actual phenotype.

Accordingly, the present invention also provides peptides and variant sequences of the MHC class I phenotype, wherein the total length of the peptide or variant sequence is 8 to 100 amino acids (preferably 8 to 30 amino acids, most preferably 8 to 14, i.e., 8, 9, 10, 11, 12, 13 or 14 amino acids, and the length of the class II binding peptide can also be 15, 16, 17, 18, 19, 20, 21 or 33 amino acids).

Of course, the peptides or variant sequences of the present invention are capable of binding to human major histocompatibility complex (MHC) class I molecules. The binding of the peptides or variant sequences to MHC can be detected by known methods.

In a preferred embodiment of the present invention, such peptides consist of (or consist essentially of) the amino acid sequences of SEQ ID No. 1 to SEQ ID No. 65, SEQ ID No. 76 to SEQ ID No. 84 and SEQ ID No. 92.

"Essentially comprising" means that the peptides of the present invention and the sequences corresponding 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 their variant sequences contain additional N- or C-terminal amino acid extensions, and that the extensions are not necessary for the formation of the peptide phenotype required for the formation of the MHC molecule phenotype.

Nevertheless, such extensions may play an important role in the efficient introduction of the peptides according to the present invention into cells. In a preferred embodiment of the present invention, the peptide may be a fusion protein, such as the HLA-DR antigen-associated invariant chain (p33, hereinafter referred to as "Ii") containing 80 N-terminal amino acids (derived from NCBI, GenBank Accession No. X00497). In other fusions, the peptides of the present invention may be fused to the antibodies (or functional groups thereof) described herein (specifically, antibody sequences), thereby specifically targeting the aforementioned antibodies, such as dendritic cell-specific antibodies.

In addition, the peptide or variant sequence can be further modified to improve stability and/or binding strength to MHC molecules, thereby stimulating a stronger immune response. Such optimization methods of peptide sequences are commonly used in the art, such as introducing reversed peptide bonds or non-peptide bonds.

In a reverse peptide bond, the amino acid residues are not linked by the (-CO-NH-) bonds of the peptide, but by reverse peptide bonds. Such reverse peptidomimetics can be prepared by methods known in the art, such as those described in Meziere et al (1997) J. Immunol. 159, 3230-3237 (incorporated herein by reference). This method involves preparing pseudopeptides containing changes in the backbone (rather than the orientation of the side chains). Meziere et al (1997) showed that such pseudopeptides facilitate MHC binding and helper T cell responses. Reverse peptides containing NH-CO bonds instead of CO-NH peptide bonds are more resistant to proteolysis.

Non-peptide bonds include -CH2 _- NH, _-CH2S- , _-CH2CH2- , -CH=CH-, _-COCH2- , -CH(OH) _CH2- , _{-CH2SO- ,} etc. U.S. Patent No. 4,897,445 proposes a method for solid-phase synthesis of non-peptide bonds (-CH2 _- NH) in polypeptide chains, which involves synthesizing polypeptides through standard operations and synthesizing non-peptide bonds by reacting aminoaldehydes with amino acids in the presence of _NaCNBH3 .

Peptides comprising the above sequences can be synthesized by adding chemical groups to their amino and/or carboxyl termini to improve the stability, bioavailability, and/or affinity of the peptide. For example, a hydrophobic group such as a benzyloxycarbonyl group, a dansyl group, or a t-tert-butyloxyhydroxyl group can be added to the amino terminus of the peptide. Similarly, an acetyl group or a 9-fluorenylmethyloxycarbonyl group can be added to the amino terminus of the peptide. Furthermore, a hydrophobic group, a t-tert-butyloxyhydroxyl group, or an amino group can be added to the carboxyl terminus of the peptide.

The peptides of the present invention can also be synthesized by altering their spatial configuration. For example, the D-isomer of one or more amino acid residues of the peptide can be used (instead of the common L-isomer). In addition, at least one amino acid residue of the peptide of the present invention can be substituted with a common non-natural amino acid residue. Such alterations can improve the stability, bioavailability, and/or binding activity of the peptide of the present invention.

Similarly, the peptides or variant sequences of the present invention can be modified by chemical reactions with specific amino acids (either before or after the synthesis of the peptide). Examples of such modifications are well known in the art and are summarized, for example, in R. Lundblad, Chemical Reagents for Protein Modification (3rd ed. CRC Press, 2005), which is incorporated herein by reference. Chemical modifications of amino acids include, but are not limited to, acylation, guanidation, lysine pyridoxalation, reductive alkylation, trinitrophenylation of amino groups with 2,4,6-trinitrobenzenesulfonic acid (TNBS), amide modification of carboxyl groups, sulfhydryl modification by oxidation of cysteine to cysteic acid, formation of mercury derivatives, formation of mixed disulfides with other sulfhydryl compounds, reaction with maleimide, carboxymethylation by iodoacetic acid or iodoacetamide, and carbamoylation by cyanate in an alkaline environment. For more information on protein chemical modification, technicians can refer to Chapter 15 of Current Protocols in Protein Science, Eds. Coligan et al. (John Wiley and Sons NY1995-2000).

In short, modification of arginyl residues in proteins, such as those in the arginyl group, is generally based on the formation of adducts with adjacent dicarbon compounds (e.g., phenylglyoxal, 2,3-butanedione, and 1,2-cyclohexanedione). Another example is the reaction of methylglyoxal with arginine residues. Cysteine can be modified without concomitant modification of other nucleophilic sites (e.g., lysine and histidine), resulting in a wide variety of reagents available for cysteine modification. The websites of companies such as Sigma-Aldrich (http://www.sigma-aldrich.com) provide information on specific reagents.

Selective reduction of protein disulfide bonds, which can form and oxidize during thermal processing of biopharmaceuticals, is also common.

Woodward's reagent K can be used to modify specific glutamic acid residues. N-(3-(Dimethylamino)propyl)-N'-carbodiimide can be used to form intramolecular crosslinks between lysine and glutamic acid residues.

For example, diethyl pyrocarbonate can be used to modify histidine residues in proteins. Histidine can also be modified with 4-hydroxy-2-nonenal.

The reaction of lysine residues and other α-amino groups facilitates peptide binding to the surface of proteins/peptides or cross-linking with proteins/peptides, etc. Lysine is the attachment site for polyethylene glycol and the main modification site for protein glycosylation.

Methionine residues in proteins can be modified with iodoacetamide, bromoethylamine, and chloramine-T.

Tetranitromethane and N-acetylimidazole can be used to modify tyrosine residues. Cross-linking via dityrosine formation can be achieved using hydrogen peroxide/copper ions.

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

Successful PEGylation of therapeutic proteins and peptides often involves extending their circulation half-life. Protein crosslinking with glutaraldehyde, poly(ethylene glycol) acrylate, and formaldehyde is used to prepare hydrogels. Chemical modification of allergens for immunotherapy is often achieved through formylation with potassium cyanate ammonium.

In embodiments of the present invention, peptides or variants that have undergone peptide modification or contain non-peptide bonds are preferably used. Generally, peptides or variants (at least those containing peptide linkages between amino acid residues) can be synthesized using Fmoc-polyamide solid-phase peptide synthesis (as described by Lu et al. (1981) and references therein). 9-Fluorenylmethyloxycarbonyl (Fmoc) provides protection for sequential N-amino groups. Repeated cleavage of this highly base-labile protecting group is performed using a 20% piperidine solution of N,N-dimethylformamide. Side chain functions can be protected as butyl ethers (serine, threonine, and tyrosine), butyl esters (glutamic acid and aspartic acid), tert-butyloxyhydroxy derivatives (lysine and histidine), trityl derivatives (cysteine), and 4-methoxy-2,3,6-trimethylbenzenesulfonyl derivatives (arginine). If glutamic acid or aspartic acid is the C-terminal residue, the side chain amino function is protected using 4,4'-dimethylbibenzyl. The solid support is based on a polydimethylaminopropylamide polymer composed of three monomers: dimethylaminopropylamide (backbone monomer), bisacryloylethylenediamine (crosslinker), and acryloylsarcosine methyl ester (functionalizing agent). The peptide-resin cleavable linker used is an acid-sensitive 4-hydroxymethyl-phenoxyacetic acid derivative. All amino acid derivatives added are preformed symmetrical anhydride derivatives, with the exception of aspartic acid and glutamic acid, which are added using a reverse N,N-dicyclohexylcarbodiimide/hydroxybenzotriazole-mediated coupling procedure. All coupling and deprotection reactions are monitored using ninhydrin, trinitrobenzenesulfonic acid, or isocyanate detection procedures. After synthesis, the peptide is cleaved from the resin support and treated with 95% trifluoroacetic acid containing a 50% scavenger mixture to remove side-chain protecting groups. Common scavengers include ethanedithiol, phenol, anisole, and water, with the specific choice depending on the amino acids constituting the peptide being synthesized. It is also possible to combine solid-phase and solution-phase methods for peptide synthesis (see (Bruckdorfer et al., 2004) and references cited therein).

Trifluoroacetic acid was removed by vacuum evaporation, followed by trituration of the crude peptide with diethyl ether. Residual scavengers were removed using a single extraction procedure, and the liquid phase was lyophilized to produce the scavenger-free crude peptide. The reagents required for peptide synthesis were generally available from Calbiochem-Novabiochem (UK) Ltd, Nottingham NG7 2QJ, UK.

Purification can be achieved by one or a combination of the following techniques: recrystallization, molecular exclusion chromatography, ion exchange chromatography, hydrophobic interaction chromatography, and (usually) reversed-phase high performance liquid chromatography (using an acetonitrile/water gradient separation).

Thin layer chromatography, electrophoresis (particularly capillary electrophoresis), solid phase extraction (CSPE), reversed-phase high performance liquid chromatography, amino acid analysis after acid hydrolysis, fast atom bombardment (FAB) mass spectrometry, and MALDI and ESI-Q-TOF mass spectrometry can be used.

Another aspect of the present invention provides a nucleotide (e.g., a polynucleotide) that encodes a peptide or peptide variant of the present invention. This polynucleotide can be DNA, cDNA, PNA, RNA, or a combination thereof, and can be single-stranded or double-stranded, or a native or stabilized form of a polynucleotide, such as a polypeptide containing a phosphorothioate backbone. As long as it encodes the corresponding peptide, the polynucleotide may or may not contain introns. Of course, the polynucleotide can only encode peptides containing natural amino acid residues bound by natural peptide bonds. Another aspect of the present invention provides an expression vector that can express the polypeptide according to the present invention.

A variety of methods have been developed to connect polynucleotides (particularly DNA) to vectors, for example, via complementary cohesive ends. For example, complementary homopolymeric regions can be added to a DNA fragment that is inserted into the vector DNA. The vector and DNA fragment are then linked via hydrogen bonds between the complementary homopolymeric tails to form a recombinant DNA molecule.

Synthetic linkers containing one or more restriction sites are another method for connecting DNA fragments to vectors. Synthetic linkers containing multiple restriction sites can be purchased from various sources, such as International Biotechnologies Inc. New Haven, CN, USA.

One ideal method for modifying DNA encoding the polypeptides of the present invention utilizes the polymerase chain reaction, as described in (Saiki et al., 1988). This method can be used to introduce the DNA into a suitable vector (e.g., by manipulation of appropriate restriction sites), and can also be used to modify the DNA using conventional techniques. If a viral vector is used, poxvirus or adenovirus vectors are preferably used.

DNA (or RNA in the case of retroviral vectors) can be expressed in a suitable host to produce polypeptides containing the peptides or variant sequences of the present invention. Thus, DNA encoding the peptides or variant sequences of the present invention can be used to construct expression vectors according to known techniques (modified as appropriate based on the present disclosure). These vectors are then used to transform suitable host cells to express and produce the polypeptides of the present invention. These techniques include those described in U.S. Patents 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.

The DNA encoding the polypeptides of the compounds of the invention (RNA for retroviral vectors) can be combined with many other DNA sequences to introduce into a suitable host. The choice of partner DNA depends on the nature of the host, the mode of introducing the DNA into the host, and whether additional maintenance or integration is required.

In general, DNA is inserted into an expression vector (e.g., plasmid) in the appropriate direction and in the correct expression reading frame. If necessary, DNA can be connected to an appropriate transcription or translation regulatory nucleotide sequence (identified by the desired host), although such regulation already exists in the expression vector usually. Standard techniques are subsequently used to import the vector into the host. In general, not all hosts can be transformed by the vector. Therefore, it is necessary to select a successfully transformed host cell. A selection technique is provided to involve the use of any necessary control elements to incorporate a DNA sequence into the expression vector, which can encode the selectable characteristics (e.g., antibiotic resistance) of the transformed cell.

The gene for such selectable traits may also be located on another vector used to co-transform the desired host cell.

Host cells transformed with the recombinant DNA of the present invention are then cultured as described herein under appropriate conditions (which are well known to those skilled in the art) for a sufficient period of time to achieve expression of the polypeptide, which can then be recovered.

A variety of expression systems are known, including bacteria (e.g., E. coli, Bacillus subtilis), yeast (e.g., Saccharomyces cerevisiae), filamentous fungi (e.g., Aspergillus), plant cells, animal cells, and insect cells. The system is preferably mammalian cells, such as CHO cells in the ATCC Cell Biology Collection.

Typical mammalian cell vector plasmids for constitutive expression include CMV or SV40 promoters with an appropriate poly A tail and resistance markers, such as neomycin. For example, pSVL is available from Pharmacia, Piscataway, NJ, USA. Inducible mammalian expression vectors include pMSG (also available from Pharmacia). Useful yeast plasmid vectors include pRS403-406 and pRS413-416, which are generally available from Stratagene Cloning Systems, La Jolla, CA 92037, USA. pRS403, pRS404, pRS405, and pRS406 plasmids are yeast integrative plasmids (YIPs) that incorporate yeast selectable markers HIS3, TRP1, LEU2, and URA3. pRS413-416 plasmids are yeast centriole plasmids (Ycps). Vectors containing a CMV promoter, such as those available from Sigma-Aldrich, allow for cis- or stable expression, cytoplasmic expression or secretion, and N- or C-terminal tagging in various combinations of FLAG, 3xFLAG, c-myc, or MAT. These fusion proteins enable detection, purification, and analysis of recombinant proteins. Dual-tag fusions provide flexibility in detection.

The potent human cytomegalovirus (CMV) promoter regulatory region allows expression of constitutive proteins in COS cells at levels as high as 1 mg/L. Protein levels in less potent cell lines are typically around 0.1 mg/L. The presence of the SV40 origin of replication allows for high levels of DNA replication in SV40-permissive COS cells. CMV vectors can contain the pMB1 (pBR322 derivative) origin of replication for bacterial cells, the b-lactamase gene for ampicillin resistance selection in bacteria, the hGH polyA, and the f1 origin. Vectors containing a pretrypsinogen leader (PPT) sequence allow secretion of FLAG fusion proteins into the culture medium for purification using anti-FLAG antibodies, resins, and plates. Other vectors and expression systems utilize a variety of host cells commonly used in this art.

In another embodiment, two or more peptides or peptide variants of the invention are encoded and expressed sequentially (similar to a "beads on a string" structure). The peptides or peptide variants can then be linked or fused to stretching groups (e.g., LLLLLL) that link amino acids, even without any other peptides in between.

The present invention also relates to a host cell transformed by a polynucleotide vector structure of the present invention. The host cell can be a prokaryotic or eukaryotic cell. In some cases, bacterial cells are preferred prokaryotic host cells, typically strains of E. coli, such as E. coli DH5, available from Bethesda Research Laboratories Inc., Bethesda, MD, USA, and RR1, available from the American Type Culture Collection (ATCC) (Rockville, MD, USA (No ATCC 31343). Preferred eukaryotic host cells include yeast, insect, and mammalian cells, preferably vertebrate cells, such as mouse, rat, monkey, or human fibroblast or colon cell lines. Yeast host cells include YPH499, YPH500, and YPH501, which are commonly available from Stratagene Cloning Systems, La Jolla, CA 92037, USA. Preferred mammalian host cells include Chinese hamster ovary (CHO) cells (ATCC CCL61 cell line), NIH Swiss mouse embryonic cells NIH/3T3 (ATCC CRL 1658), monkey kidney-derived COS-1 cells (ATCC CRL 1650 cell line) and 293 cells (human eukaryotic kidney cells). Preferred insect cells include Sf9 cells (which can be transfected with baculovirus expression vectors). For a review of the selection of suitable host cells for expression, see the textbook "Molecular Biotechnology: Methods of Recombinant Gene Expression: Overview and Protocols" by Paulina Balbás and Argelia Lorence, 2nd edition, Part 1, ISBN 978-1-58829-262-9, as well as other references known to those skilled in the art.

Appropriate transformation of host cells using the DNA constructs of the present invention can be accomplished by conventional methods, which generally depend on the type of vector used. Information on prokaryotic host cell transformation can be found in 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. Yeast cell transformation methods are described in Sherman et al (1986) Methods in Yeast Genetics, A Laboratory Manual, Cold Spring Harbor, NY. The methods provided by Beggs (1978) Nature 275, 104-109 are also useful. For vertebrate cells, reagents useful for transfecting such cells (e.g., calcium phosphate and DEAE-ethyl dextran or liposome preparations) are available from Stratagene Cloning Systems or Life Technologies Inc., Gaithersburg, MD 20877, USA. Electroporation can also be used for cell transformation and/or transfection, and this method is commonly used in yeast cell, bacterial cell, insect cell and vertebrate cell transformation technology.

Successfully transformed cells (ie, cells containing the DNA construct of the present invention) can be identified by common techniques such as PCR, or by using antibodies to detect proteins in the supernatant.

Of note, certain host cells of the present invention (e.g., bacteria, yeast, and insect cells) can be used to produce the peptides of the present invention. However, other host cells may also be used in certain therapeutic methods. For example, antigen-presenting cells (e.g., dendritic cells) may be used to express the peptides of the present invention, which may then be loaded onto appropriate MHC molecules. Therefore, the present invention provides host cells or expression vectors comprising amino acids according to the present invention.

In a preferred embodiment, the host cell is an antigen presenting cell, in particular a dendritic cell or an antigen presenting cell. APCs loaded with a recombinant fusion protein containing prostatic acid phosphatase (PAP) (Sipuleucel-T) are currently being studied for the treatment of prostate cancer (Small et al., 2006; Rini et al., 2006).

Another aspect of the present invention also includes a method for preparing a peptide or a variant thereof, which comprises culturing host cells and isolating the peptide from the host cells or their culture medium.

In another embodiment, the peptide, nucleic acid or expression vector of the present invention is used in medicine. For example, the peptide or its variant can be prepared as an intravenous (i.v.) injection, subcutaneous (s.c.) injection, intradermal (i.d.) injection, intraperitoneal (i.p.) injection and intramuscular (i.m.) injection. Preferred methods of peptide injection include subcutaneous, intradermal, intraperitoneal and intravenous injection. Preferred methods of DNA injection include intradermal, intramuscular, subcutaneous, intraperitoneal and intravenous injection. The dose of peptide or DNA administered can be between 50 μg and 1.5 mg (preferably between 125 μg and 500 μg), depending on the type of peptide or DNA. This dose range has been successfully used in previous experiments (Walter et al Nature Medicine 18, 1254–1261 (2012)).

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

Ideally, mammals should lack or have reduced levels or function of the TAP peptide transporter. Suitable cells lacking the TAP peptide transporter include T2, RMA-S, and Drosophila cells. TAP is a transporter involved in antigen processing.

Human peptide-containing deletion T2 cell lines are available from the American Type Culture Collection (ATCC) (12301 Parklawn Drive, Rockville, Maryland 20852, USA) catalog number CRL 1992; Drosophila cell line Schneider cell line 2 is available from ATCC catalog number CRL 19863; and details of the mouse RMA-S cell line are available from Karre et al. 1985.

Ideally, the host cells prior to transfection should express substantially no MHC class I molecules. It is preferred to use a stimulatory cell that expresses molecules important for T cell co-stimulatory signals (e.g., B7.1, B7.2, ICAM-1, and LFA 3). Nucleic acid sequences for many MHC class I molecules and co-stimulatory molecules can be found in the public GenBank and EMBL databases.

If the MHC class I phenotype is used as the antigen, the T cells are CD8-positive CTLs.

If antigen-presenting cells are transfected to express the above phenotype, the cells preferably contain an expression vector capable of expressing peptides containing sequences 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 variant amino acid sequences thereof).

Several other methods can 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 to generate CTLs. Plebanski et al. (1995) used autologous peripheral blood lymphocytes (PLB) to generate CTLs. Jochmus et al. (1997) described the generation of autologous CTLs by pulsing dendritic cells with peptides or polypeptides (or by infection with recombinant viruses). Hill et al. (1995) and Jerome et al. (1993) used B cells to generate autologous CTLs. In addition, macrophages pulsed with peptides or polypeptides or infected with recombinant viruses can also be used to generate autologous CTLs. S. Walter et al. (2003) described the use of artificial antigen-presenting cells (aAPCs) to stimulate T cells in vitro, a method also suitable for generating T cells against a preferred peptide. In this study, aAPCs were generated by coupling preformed MHC/peptide complexes to the surface of polystyrene particles (microbeads) via a biotin/streptavidin biochemical mechanism. This system allows for precise control of MHC density within aAPCs, enabling the selective and efficient stimulation of high- or low-avidity antigen-specific T cell responses from blood samples. In addition to the MHC/peptide complex, aAPCs should also carry other co-stimulatory proteins (coupled to the aAPC surface), such as anti-CD28 antibodies. Furthermore, such aAPC systems often require the addition of appropriate soluble factors, such as cytokines like interleukin-12.

Allogeneic cells can also be used to prepare T cells, one method of which is described in detail in WO 97/26328 (incorporated herein by reference). For example, in addition to Drosophila cells and T2 cells, other cells can be used for antigen presentation, such as CHO cells, baculovirus-infected insect cells, bacteria, yeast, vaccinia-infected target cells, etc. Plant viruses can also be used (see Porta et al. (1994) for example), which describes the development of cowpea mosaic virus as a high-yield system for presenting exogenous peptides.

Activated T cells targeting the peptides of the present invention can be used for therapy. Therefore, another aspect of the present invention provides activated T cells obtainable by the above-described method of the present invention.

The activated T cells prepared by the above method can selectively recognize cells that abnormally express a polypeptide containing the amino acid sequence of SEQ ID No. 1 to SEQ ID No. 92 (preferably SEQ ID No. 1 to SEQ ID No. 65, SEQ ID No. 76 to SEQ ID No. 84 and SEQ ID No. 92).

Preferably, the T cells recognize cells by interacting (e.g., binding) with the HLA/peptide complex through their TCR. T cells can be used in a method for killing target cells, wherein the patient's target cells abnormally express a polypeptide comprising the amino acid sequence of the present invention, and the patient receives an effective number of activated T cells. The T cells received by the patient can be derived from the patient himself and activated by the above method (i.e., autologous T cells). The T cells may also be derived from another individual rather than the patient himself. Of course, the individual is preferably a healthy individual. The "healthy individual" used by the inventors refers to an individual who is in good overall health, preferably has a complete immune system, and more preferably does not suffer from a disease that is easily detected or discovered.

In vivo, the target cells of the CD8-positive T cells according to the present invention may be tumor cells (which in some cases express MHC class II) and/or stromal cells surrounding the tumor (tumor cells) (which in some cases may also express MHC class II; (Dengjel et al., 2006)).

The T cells of the present invention can be used as an active ingredient in a therapeutic composition. Therefore, the present invention also provides a method for killing target cells, wherein the target cells of the patient abnormally express a polypeptide comprising the amino acid sequence of the present invention, and the patient receives an effective number of activated T cells (as defined above).

The inventors use the term "abnormal expression" to refer to overexpression of a polypeptide compared to normal expression levels, or to silence of a gene expressed in a tumor in a tumor tissue specimen. The inventors use the term "overexpression" to refer to expression levels of a polypeptide that are at least 1.2 times, preferably at least 2 times, and more preferably at least 5 or 10 times, higher than those in normal tissue.

T cells can be prepared by methods known in the art, such as the methods described above.

Adoptive transfer of T cells is a common technique, reviewed in (Gattinoni et al., 2006) and (Morgan et al., 2006).

Any molecule of the present invention (i.e., peptide, nucleotide, antibody, expression vector, cell, activated CTL, T cell receptor or its encoding nucleotide) can be used to treat diseases characterized by cellular escape from an immune response. Therefore, any molecule of the present invention may be used as a medicament or for the production of a medicament. The above molecules can be used alone or in combination with other molecules of the present invention or known molecules.

Preferably, the pharmaceutical agent of the present invention is a vaccine. The vaccine can be administered directly to the patient's diseased organ or systemically via intradermal, intramuscular, subcutaneous, intraperitoneal, or intravenous administration; or it can act ex vivo on cells or human cell lines derived from the patient, which are then administered to the patient; or it can act ex vivo on a subset of immune cells derived from the patient, which are then re-administered to the patient. If the nucleic acid is administered to cells in vitro, it may be helpful to transfect the cells to co-express an immunostimulatory cytokine (e.g., interleukin-2). The peptide can be used substantially pure or in combination with an immunostimulatory adjuvant (see below), or administered via a suitable delivery system (e.g., liposomes). The peptide can also be conjugated to a suitable carrier (e.g., keyhole limpet hemocyanin or mannan) (see WO 95/18145 and Longenecker, 1993). The peptide can also be labeled, either as a fusion protein or a hybrid molecule. Peptides encoded by sequences described herein are expected to stimulate either CD4 or CD8 T cells, although stimulation of CD8 CTLs is more efficient with the assistance of CD4 helper T cells. Therefore, for the MHC class I phenotype that stimulates CD8 CTL, the use of fusion partners or hybrid molecular profiles can appropriately generate a phenotype that stimulates CD4 positive T cells. CD4 and CD8 stimulatory phenotypes are common in the relevant technical field, including the phenotypes proposed in the present invention.

In one aspect, the vaccine comprises a peptide encoded by at least one amino acid sequence of SEQ ID No. 1 to SEQ ID No. 92, and at least one additional peptide, preferably 2 to 50 additional peptides, 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. The peptides may be derived from one or more specific TAAs and may bind to MHC class I molecules.

In another aspect, the vaccine comprises a peptide encoded by at least one of the amino acid sequences of SEQ ID No. 1 to SEQ ID No. 65, SEQ ID No. 76 to SEQ ID No. 84, and SEQ ID No. 92, and at least one additional peptide, preferably 2 to 50 additional peptides, 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. The peptides may be derived from one or more specific TAAs and may bind to MHC class I molecules.

The polypeptide can be substantially pure or contained within a suitable vector or delivery system. The nucleotide can be DNA, cDNA, PNA, RNA, or a combination of DNA, cDNA, PNA, and RNA (Pascolo et al., 2005). Polynucleotide vaccines are easy to prepare, but the mode of action of such vectors in inducing an immune response is not fully understood. Suitable vectors and delivery systems include viral DNA and/or RNA, such as adenovirus, vaccinia virus, retrovirus, herpes virus, adeno-associated virus systems, or hybrids containing elements of multiple viruses. Non-viral delivery systems include cationic lipids and cationic polymers, which are common in DNA delivery technologies. Physical delivery methods, such as "gene guns," can also be used. The peptide encoded by the nucleic acid can be a fusion protein, such as a fusion protein containing a T cell phenotype that stimulates the corresponding CDR of the T cell.

The pharmaceutical compositions of the present invention may also contain one or more adjuvants. Adjuvants are substances that can non-specifically enhance or strengthen immune responses to antigens (e.g., immune responses mediated by CTLs and helper T ( _TH ) cells) and are therefore considered useful for the pharmaceutical compositions of the present invention. Suitable adjuvants include, but are not limited to, 1018ISS, aluminum salts, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, flagellin or TLR5 ligands of flagellin, FLT3 ligands, GM-CSF, IC30, IC31, imiquimod, ImuFact IMP321, interleukins such as IL-2, IL-13, IL-21, interferon-α or interferon-β or their PEGylated derivatives, IS patches, ISS, ISCOMATRIX, ISCOMs, LipoVac, MALP2, MF59, monophosphoryl lipid A, alliin IMS 1312, alliin ISA 206, alliin ISA 50V, ISA-51, water-in-oil and oil-in-water emulsions, OK-432, OM-174, OM-197-MP-EC, ONTAK, OspA, carrier systems, polyglycolic acid copolymers [PLG] and dextran microspheres, lactoferrin SRL172, virosomes or other virus-like particles, YF-17D, VEGF traps, R848, β-glucan, Pam3Cys, Aquila QS21 provirin (derived from saponins), mycobacterial extracts and synthetic bacterial cell wall mimics, and other proprietary adjuvants such as Ribi's Detox, Quil, or Superfos. Preferred adjuvants include Freund's or GM-CSF. Several immunoadjuvants (e.g., MF59) and their formulations for dendritic cells have been reported (Allison and Krummel, 1995; Allison and Krummel, 1995). Cytokines can also be used. Several cytokines have been found to directly affect the migration of dendritic cells to lymphoid tissues (e.g., TNF-), thereby 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, specifically incorporated herein by reference in its entirety) and functioning as immune adjuvants (e.g., IL-12, IL-15, IL-23, IL-7, IFN-α, IFN-β) (Gabrilovich, 1996).

There are also reports that CpG immunostimulatory oligonucleotides can enhance the effects of adjuvants in vaccines. Without being bound by theory, CpG oligonucleotides can activate the innate (non-adaptive) immune system through Toll-like receptors (TLRs), primarily TLR9. CpG-triggered TLR9 activation can enhance antigen-specific humoral or cellular responses to a range of antigens, including peptide or protein antigens, live or killed viruses, dendritic cell vaccines, autologous cell vaccines, and polysaccharide conjugates in prophylactic and therapeutic vaccines. More importantly, this activation can promote dendritic cell maturation and differentiation, triggering an increase in T _H1 cell activation and the production of potent cytotoxic T lymphocytes (CTLs) (even in the absence of CD4 T cell assistance). Even in the presence of incomplete Freund's adjuvant (IFA), which typically promotes T _H2 bias, the T _H1 bias triggered by TLR9 stimulation can still be maintained. CpG oligonucleotides can produce higher adjuvant activity when administered in combination with other adjuvants or in microparticles, nanoparticles, lipid emulsions, or similar formulations, which is particularly necessary for eliciting a stronger response to relatively weak antigens. CpG oligonucleotides can also accelerate the immune response and ensure that the antigen dose is reduced by about 2 orders of magnitude, and the antibody response generated is comparable to the full-dose vaccine in certain trials that do not use CpG (Krieg, 2006). U.S. Patent No. 6406705B1 describes the use of CpG oligonucleotides, non-nucleotide adjuvants, and antigens in combination to elicit an antigen-specific immune response. A CpG TLR9 antagonist is dSLIM (double stem loop immunomodulator) from Mologen (Berlin, Germany), which is a preferred component of the pharmaceutical composition of the present invention. Other TLR binding molecules, such as RNA-binding TLR 7, TLR8, and/or TLR9, can also be used.

Other useful adjuvants also include, but are not limited to, chemically modified CpGs (e.g., CpR, Idera), dsRNA analogs (e.g., poly(I:C)) and its derivatives (e.g., poly(ICLC), poly(IC-R), poly(I:C12U)), non-CpG bacterial DNA or RNA, and immunologically active small molecules and antibodies, such as cyclophosphamide, sunitinib, Celebrex, NCX-4016, sildenafil, tadalafil, vardenafil, sorafenib, temozolomide, tadalafil, XL-999, CP-547632, pazopanib, VEGF trap, ZD2171, AZD2171, anti-CTLA4, other antibodies against key immune system structures (e.g., anti-CD40, anti-TGFβ, anti-TNFα receptor), and SC58175 (which can act therapeutically and/or as adjuvants). The amounts and concentrations of adjuvants and additives useful in the present invention can be readily determined by one skilled in the art without undue experimentation.

Preferred adjuvants include imiquimod, resiquimod, GM-CSF, cyclophosphamide, sunitinib, bevacizumab, interferon-α, CpG oligonucleotides and derivatives, poly (I: C) and derivatives, RNA, sildenafil, and particulate formulations containing PLG or virosomes.

In a preferred embodiment, the excipients in the pharmaceutical composition of the present invention are selected from a group of colony stimulating factors, such as granulocyte-macrophage colony stimulating factor (GM-CSF, desertgrastim), cyclophosphamide, imiquimod, resiquimod and interferon-α.

In a preferred embodiment, the excipients in the pharmaceutical composition of the present invention are selected from the group consisting of colony stimulating factors, such as granulocyte-macrophage colony stimulating factor (GM-CSF, desertgrastim), cyclophosphamide, imiquimod and resiquimod.

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

More preferred adjuvants are allicin MS 1312, allicin ISA 206, allicin ISA 50V, allicin ISA-51, poly-ICLC and anti-CD40 mAb or a combination of the above adjuvants.

This composition is used for enteral administration, such as subcutaneous, intradermal, intramuscular or oral administration. For convenient administration, the peptide or other molecules are dissolved or suspended in a pharmaceutical, preferably liquid carrier. In addition, the pharmaceutical composition may contain excipients (such as buffers, binding agents, explosives, diluents, flavor enhancers, lubricants, etc.). The peptide can also be co-administered with an immunostimulating substance (such as a cytokine). A more extensive list of excipients available for this pharmaceutical composition can be found in A.Kibbe, Handbook of Pharmaceutical Excipients, ^3rd Ed., 2000, American Pharmaceutical Association and pharmaceutical press, etc. This composition can be used for the prevention and/or treatment of adenomatous or cancerous diseases. Typical preparations can be found in EP2113253, etc.

Nevertheless, based on the number and physicochemical properties of the peptides of the present invention, further studies are needed to generate specific combination formulations of peptides, in particular combination formulations containing more than 20 peptides that are stable for more than 12 to 18 months.

The present invention provides a medicament that can be used to treat cancer, in particular non-small cell lung cancer, gastric cancer, renal cell carcinoma, colon cancer, adenocarcinoma, prostate cancer, benign tumors and malignant melanoma.

The present invention also relates to the following kit:

(a) a package containing the above-mentioned pharmaceutical composition, which may be in solution or lyophilized dosage form;

(b) (Optional) Secondary packaging containing a diluent or reconstitution solution for the lyophilized preparation;

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

The kit may further comprise one or more of (iii) a buffer, (iv) a diluent, (v) a filter, (vi) a needle, or (v) a syringe. The packaging is preferably a vial, a phial, a syringe, or a test tube, and may be multi-purpose packaging. The pharmaceutical composition is preferably a lyophilized formulation.

The pharmaceutical packaging of the present invention preferably comprises the lyophilized formulation of the present invention (with suitable packaging and instructions for reconstitution and/or use). Suitable packaging includes vials, vials (e.g., double-chamber vials), syringes (e.g., double-chamber syringes), and test tubes. Ideally, the kit and/or packaging should include instructions for reconstitution and/or use on or within the packaging. For example, the label may indicate that the lyophilized formulation should be reconstituted to the above-mentioned peptide concentration. The label may further indicate that the formulation is for or intended for subcutaneous administration.

The packaging for storing the preparation can be a multi-use vial to facilitate repeated administration (e.g., 2-6 administrations) of the reconstituted preparation. The kit may contain another package containing an appropriate diluent (e.g., sodium bicarbonate solution).

The peptide concentration in the final reconstituted formulation obtained by mixing the diluent and the reconstituted formulation is preferably at least 0.15 mg/mL/peptide (=75 μg) and not more than 3 mg/mL/peptide (=1500 μg). The kit may also contain other sales and user materials, including other buffers, diluents, filters, needles, syringes, and instructions for use.

The kit of the present invention may be a single package of the pharmaceutical composition preparation according to the present invention, with or without other components (such as other compounds or pharmaceutical compositions of these compounds), or may provide separate packages for each component.

Ideally, the kit of the present invention comprises a formulation of the present invention for administration in combination with another compound (e.g., an adjuvant (GM-CSF, etc.), a chemotherapeutic drug, a natural product, a hormone or antagonist, an anti-angiogenic agent or angiogenesis inhibitor, an apoptosis inducer or a chelating agent) or a pharmaceutical composition thereof. Prior to administration to the patient, the components of the kit may be pre-mixed, or separate packaging may be provided for each component. The components of the kit may be one or more liquid solutions, preferably aqueous solutions, more preferably sterile aqueous solutions. The components of the kit may also be solids, which may be converted to liquids by adding an appropriate solvent, which should be stored in another separate package.

The packaging of the therapeutic kit can be a vial, test tube, flask, medicine bottle, syringe or any other device for encapsulating a solid or liquid. Typically, if there is more than one component, the kit will contain another vial or other packaging to facilitate separate administration. The kit may also contain another package for storing a medicinal liquid. Ideally, the therapeutic kit should contain a device (e.g., one or more needles, syringes, eye droppers, pipettes, etc.) for administering the medicament of the present invention in the kit components.

The present preparation allows the peptide to be administered by any acceptable route (e.g., oral (enteral), intranasal, intraocular, subcutaneous, intradermal, intramuscular, intravenous or transdermal). Subcutaneous administration is preferred, and intradermal administration is most preferably performed using an infusion pump.

Since the peptides of the present invention are isolated from NSCLC, the pharmaceutical agent of the present invention is preferably used to treat NSCLC.In a preferred embodiment, since the peptides of the present invention derived from ABCA13 and MMP12 are isolated from NSCLC, the pharmaceutical agent of the present invention is preferably used to treat NSCLC.

The peptides with sequences of SEQ ID Nos. 78 to 92 are isolated from Merkel cell carcinoma and can therefore be used to treat Merkel cell carcinoma.

The present invention will now be described in the following examples (describing preferred embodiments thereof), but is not limited to these examples. For the purposes of the present invention, all references are incorporated herein by reference in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Presentation of ABCA13-001 in the primary tumor sample NSCLC898: Representative mass spectrum of ABC13-001. NanoESI-LCMS was performed on a peptide pool eluted from NSCLC sample 898. The mass spectrum of m/z 543.8318 ± 0.001 Da, z = 2 shows a peptide peak with a retention time of 86.36 min. B) The peak detected at 86.36 min in the mass spectrum represents the m/z 543.8318 signal in the MS spectrum. C) The collisional decay mass spectrum of the selected precursor at m/z 543.8318 recorded at the specific retention time in the nanoESI-LCMS experiment confirms the presence of ABCA13-001 in the NSCLC898 tumor sample. D) The fragmentation pattern of the synthetic ABCA13-001 reference peptide was recorded and compared with the fragmentation pattern of the native TUMP generated in C for sequence verification.

Figure 2: 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: Presentation profiles of selected HLA class I molecules. The expression profiles for each peptide were calculated, showing the mean expression level and replicate variance across samples. The profiles are presented for the relevant tumor samples alongside the normal tissue sample (baseline).

a)ABCA13-001

b)DST-001

c)MXRA5-001

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. The dot plots represent the population of MHC multimers double-positive for the stimulating peptide (left panel) and the corresponding negative control stimulation (right panel).

Figure 5: Binding properties of POSTN-002 and MMP12-002 to the investigated HLA haplotypes. Shown are 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 at 37°C for 24 h: The graph shows the percentage of binding scores of intact HLA-POSTN-002 and HLA-MMP12-002 complexes with the corresponding HLA molecules after 24 h at 37°C.

Figure 7: Representative vaccine-induced CD4 T cell responses to CEA-006 in a Class II ICS assay. Following in vitro priming, PBMCs from patient 36-031 were analyzed to examine CD4 T cell responses to CEA-006 (top panel) and mock (bottom panel) at time point pool V8/EOS. Cells were stimulated with the corresponding peptides and stained for cell viability, anti-CD3, anti-CD-8, anti-CD4, and effector markers (from right to left: CD154, TNF-α, IFN-γ, IL-2, IL-10).

Figure 8: Immunogenicity of various class II peptides. Shown are the immune response rates to five class II peptides (IMA950 peptide in 16 patients and IMA910 peptide in 71 patients) as measured by ICS.

Example

Example 1:

Identification and Quantification of Tumor-Associated Peptides Presented on Cell Surfaces

tissue samples

Patient tumor samples were provided by the University of Heidelberg, Heidelberg, Germany. All patients provided written informed consent before surgery. Immediately after surgery, tissues were snap-frozen in liquid nitrogen and stored at −80°C until isolation of TUMAPs.

Isolation of HLA peptides from tissue samples

HLA peptide pools were obtained from snap-frozen samples by immunoprecipitation from solid tissues according to a slightly modified protocol (Falk, K., 1991; Seeger, F.H.T., 1999) using the HLA-A*02-specific antibody BB7.2, the HLA-A, -B, C-specific antibody W6/32, CNBr-activated agarose, acid treatment, and ultrafiltration.

method

The obtained HLA peptide pool was separated based on hydrophobicity using reversed-phase chromatography (Acquity UPLC system, Waters), and the eluted peptides were analyzed using an LTQ-Orbitrap hybrid mass spectrometer (ThermoElectron) equipped with an ESI source. The peptide pool was directly loaded onto a sintered silica microcapillary analytical column (75 μm i.d. x 250 mm) packed with 1.7 μm C18 reversed-phase material (Waters) at a flow rate of 400 nL/min. The peptides were then separated using a two-step binary gradient elution from 10% to 33% B at a flow rate of 300 nL/min for 180 min. The gradient consisted of solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile). A gold-coated glass column (PicoTip, New Objective) was used for introduction into the nanoESI source. The LTQ-Orbitrap mass spectrometer was operated in data-dependent mode using a TOP5 strategy. Briefly, a full scan with high mass accuracy was performed in the Orbitrap to initiate the scan cycle (R = 30,000), followed by MS/MS scans of the five most abundant precursor ions (R = 7,500), also in the Orbitrap. Tandem mass spectrometric interpretation was performed using SEQUEST and other artificial controls. The identified peptide sequence was confirmed by comparing the generated fragmentation spectrum of the native peptide with that of a synthetic reference peptide of the same sequence. Figure 1 shows a typical chromatogram and elution profile of the MHC class I-associated peptide ABCA13-001 in tumor tissue on the UPLC system.

Label-free relative LC-MS quantification (i.e., extraction and analysis of LC-MS features) was performed using ion counting (Mueller et al. 2007a). This method assumes that the LC-MS signal area of a peptide is correlated with its abundance in the sample. The extracted features were further processed using charge state deconvolution and retention time correction (Mueller et al. 2007b; Sturm et al. 2008). Finally, all LC-MS features were cross-referenced with sequence identification results to integrate quantitative data from different samples and tissues with peptide presentation profiles. To account for technical and biological variability among replicates, quantitative data were double-normalized according to central tendency. Thus, each identified peptide could be associated with quantitative data, enabling relative quantification across samples and tissues. Furthermore, all quantitative data obtained from candidate peptides were manually reviewed to ensure data consistency and verify the accuracy of the automated analysis. The expression profile of each peptide was calculated, and the sample mean expression and replicate variance were presented. This profile juxtaposes the relevant tumor samples with normal tissue samples (baseline).

The presentation spectrum of a typical overexpressed peptide is shown in Figure 3.

Example 2

Determination of expression profile of the peptide encoding gene of the present invention

Not all peptides that can be recognized by MHC molecules as presented on the surface of tumor cells are suitable for immunotherapy. This is because the majority of such peptides are derived from normal cellular proteins expressed by a wide range of cell types. Only a very small number of such peptides are tumor-associated and have the potential to induce T cells with high specificity for their tumor of origin. To identify such peptides and minimize the risk of vaccine-induced autoimmunity, the inventors focused on peptides derived from proteins that are overexpressed in tumor cells compared to most normal tissues.

Ideally, peptides should be derived from proteins that are unique to the tumor of interest and not present in any other tissue. To identify peptides derived from genes with expression profiles similar to the ideal profile, the identified peptides were juxtaposed with their source proteins and genes, and expression profiles of these genes were generated.

RNA source and preparation

Surgical resection tissue specimens were provided by the University of Heidelberg, Heidelberg, Germany (see Example 1). All patients provided written informed consent before surgery. Immediately after surgery, tumor tissue specimens were snap-frozen in liquid nitrogen and then homogenized using a mortar and pestle in liquid nitrogen. Total RNA was prepared from these samples using TRI reagent (Ambion, Darmstadt, Germany) and subsequently purified using RNeasy (QIAGEN, Hilden, Germany); all methods were performed according to the manufacturer's instructions.

Total RNA from healthy human tissues was purchased from Ambion, Huntingdon, UK; Clontech, Heidelberg, Germany; Stratagene, Amsterdam, Netherlands; and BioChain, Hayward, CA, USA. RNA from multiple individuals (2 to 123 individuals) was pooled to equalize the RNA content of each individual.

All RNA samples were qualitatively and quantitatively analyzed using the RNA 6000 Pico LabChip kit (Agilent) and an Agilent 2100 Bioanalyzer (Agilent, Waldbronn, Germany).

Microarray experiments

Gene expression analysis of all tumor and normal tissue RNA samples was performed using Affymetrix Human Genome (HG) U133A or HG-U133 Plus 2.0 oligonucleotide microarrays (Affymetrix, Santa Clara, CA, USA). All procedures were performed according to Affymetrix specifications. 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 manufacturer's instructions. In vitro transcription was performed using the Bioarray High Yield RNA Transcript Labeling Kit (ENZO Diagnostics, Inc., Farmingdale, NY, USA) (for U133A arrays) or the GeneChip IVT Labeling Kit (Affymetrix) (for U133Plus 2.0 arrays), followed by cRNA strand fragmentation, hybridization, and staining using streptavidin-phycoerythrin and biotinylated anti-streptavidin antibodies (Molecular Probes, Leiden, Netherlands). Images were then scanned using an Agilent 2500A Gene Array Scanner (U133A) or an Affymetrix GeneChip Scanner 3000 (U133Plus 2.0), and data were analyzed using GCOS software (Affymetrix) (all parameters were set to default). A panel of 100 housekeeping genes provided by Affymetrix was used. Relative expression values were calculated using the signal log ratio provided by the software, and the normal kidney sample value was arbitrarily set to 1.0.

Typical expression profiles of the source genes of the present invention that are highly overexpressed or uniquely expressed in non-small cell lung cancer are shown in FIG2 .

Example 4

In vitro immunogenicity of MHC class I-presented peptides against NSCLC

To investigate the immunogenicity of the TUMAPs described herein, we used an in vitro T cell priming assay, repeatedly stimulating CD8+ T cells with artificial antigen-presenting cells (aAPCs) loaded with peptide/MHC complexes. Using this method, we determined the immunogenicity of nine HLA-A*0201-restricted TUMAPs described herein to date, demonstrating that these peptides are T cell-specific and that their CD8+ precursor T cells exist in humans (Table 4).

In vitro priming of CD8+ T cells

For in vitro stimulation using 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 (from healthy donors who gave informed consent at Transfusion Medicine Tuebingen, Germany) by positive selection using CD8 microbeads (Miltenyi Biotec, Bergisch-Gladbach, Germany).

Isolated CD8+ lymphocytes or PBMCs were cultured until use in T cell culture medium (TCM) containing RPMI-GutaMax (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). 2.5 ng/ml IL-7 (PromoCell, Heidelberg, Germany) and 10 U/ml IL-2 (Novartis Pharma, Nürnberg, Germany) were also added to the TCM at this step.

A well-defined in vitro system (four different pMHC molecules per stimulation condition and eight pMHC molecules per readout condition) was used for pMHC/anti-CD28-coated bead generation, T cell stimulation, and readout.

All pMHC complexes used for aAPC loading and cell readouts were generated by UV-induced MHC ligand exchange (Rodenko et al., 2006, with minor modifications). To determine the amount of pMHC monomers obtained by exchange, we performed a streptavidin sandwich ELISA according to the method of (Rodenko et al., 2006).

Purified co-stimulatory mouse IgG2a anti-human CD28 antibody 9.3 (Jung et al., 1987) was chemically biotinylated using thio-N-hydroxysuccinimidyl-biotin as recommended by the manufacturer (Perbio, Bonn, Germany). The microbeads used were 5.6 μm diameter streptavidin-coated polystyrene particles (Bangs Laboratories, Illinois, USA).

The pMHC used for positive and negative control stimulations were A*0201/MLA-001 (peptide ELAGIGILTV obtained from modified Melan-A/MART) and A*0201/DDX5-001 (DDX5YLLPAIVHI obtained from DDX5), respectively.

800,000 microbeads/200 μl were coated in a 96-well plate (supplemented with 4 x 12.5 ng of different biotin-pMHCs). After washing, 200 μl of 600 ng of biotin anti-CD28 was added. Stimulation was initiated by incubating 1 x 10 ⁶ CD8+ T cells with 2 x 10 ⁵ washed, coated microbeads in 200 μl of TCM supplemented with 5 ng/ml IL-12 (PromoCell) at 37°C for 3-4 days in the 96-well plate. Half of the medium was then replaced with fresh TCM supplemented with 80 U/ml IL-2, and incubation was continued at 37°C for 3-4 days. This stimulation cycle was repeated three times. For pMHC multimer readout (eight different pMHCs per condition), a two-dimensional combinatorial encoding method coupled to five different fluorochromes was used (as previously described with minor modifications) (Andersen et al., 2012). Finally, multimer analysis was performed using a live/dead near-infrared dye (Invitrogen, Karlsruhe, Germany), CD8-FITC monoclonal antibody clone SK1 (BD, Heidelberg, Germany), and fluorescent pMHC multimers. The analysis was performed using a BD LSRII SORP cytometer equipped with appropriate lasers and filters. The number of peptide-specific cells was calculated as a percentage of total CD8+ cells. Multimer analysis was evaluated using FlowJo software (Tree Star, Oregon, USA). In vitro priming of specific multimer+ CD8+ lymphocytes was determined by comparison with negative control stimulation. The presence of a specific CD8+ T cell line in the evaluable in vitro stimulation wells was confirmed after in vitro stimulation (i.e., at least 1% of the CD8+ T cells in that well were specific multimer+ and the number of specific multimer+ cells was at least 10-fold higher than the median number of negative control stimulation cells).

In vitro immunogenicity of NSCLC peptides

The in vitro immunogenicity of the tested HLA class I peptides can be demonstrated by the generation of peptide-specific T cell lines. Figure 4 shows representative flow cytometry results for two peptides of the present invention after staining with TUMAP-specific multimers (with corresponding negative control results attached). The results for the 25 peptides of the present invention are summarized in Table 5.

Table 5: In vitro immunogenicity of HLA class I peptides of the present invention

Typical results of in vitro immunogenicity tests performed by the applicant for the peptides of the present invention. <20% = +; 20%-49% = ++; 50%-70% = +++; and >70% = ++++

Example 5

Peptide synthesis

All peptides were synthesized using standard, well-established solid-phase peptide synthesis methods (using the Fmoc-strategy). After purification by preparative RP-HPLC, physiologically compatible counterions (e.g., trifluoroacetate, acetate, ammonium, or chloride) were incorporated by ion exchange procedures.

The peptides were identified and their purity was determined by mass spectrometry and RP-HPLC analysis. The peptides obtained after the ion exchange procedure were white or off-white lyophilized products with a purity ranging from 90% to 99.7%.

All TUMAPs are preferably administered in the form of trifluoroacetate or acetate, although other salts are also possible. The assay in Example 4 uses the trifluoroacetate form of the peptide.

Example 6

UV-ligand exchange

The immunogenicity of candidate peptides for the vaccine according to the present invention was further tested using an in vitro priming assay. The various peptide-MHC complexes required for this assay were generated by UV-ligand exchange, in which UV-sensitive peptides were dissociated by UV irradiation and exchanged with the candidate peptides being assayed. Only candidate peptides that effectively bind to and stabilize peptide-sensing MHC molecules prevented dissociation of the MHC complexes. The yield of the exchange reaction was determined by ELISA assay for the light chain (β2m) of the stabilized MHC complexes. This assay was essentially based on the method 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.).

96-well MAXISorp plates (NUNC) were coated with 2 μg/ml streptavidin in PBS at room temperature overnight, washed four times for 30 minutes at 37°C, and blocked with 2% BSA in blocking buffer for 30 minutes. Renatured HLA-A*0201/MLA-001 monomers were used as standards (ranging from 8 to 500 ng/ml). The peptide-MHC monomers in the UV exchange reaction were diluted 100-fold in blocking buffer. Samples were incubated at 37°C for 1 hour, washed four times, and incubated with 2 μg/ml HRP-conjugated anti-β2m for 1 hour at 37°C, washed again, and finally detected with TMB solution (flow _stopped with _NH₂SO₄ ). Absorbance was measured at 450 nm.

Table 6: UV-ligand exchange

Candidate peptides that exhibit a high exchange yield (i.e., greater than 40%, preferably greater than 50%, more preferably greater than 70%, and most preferably greater than 80%) are generally preferred for the generation and production of antibodies or fragments thereof and/or T cell receptors or fragments thereof because they have sufficient affinity for MHC molecules and can prevent dissociation of the MHC complex.

Example 7

Binding and immune activity of selected MHC class II peptides

HLA class II proteins are divided into three major isotypes: HLA-DR, -DP, and DQ, which are encoded by a variety of haplotypes. The diverse combinations of α- and β-chains increase the diversity of HLA class II proteins in any given population. Therefore, selected HLA class II TUMAPs must bind to a variety of different HLA-DR molecules (i.e., exhibit broad binding capacity) in order to elicit effective T cell responses in a significant percentage of patients.

The broad binding of POSTN-002 and MMP12-002 to multiple HLA-DR haplotypes and the stability of the formed complexes were assessed by in vitro binding assays performed by an external service provider, as described below.

Materials and methods

Peptide List

List of tested HLA-DR haplotypes

The seven HLA-DR haplotypes tested were selected based on their frequency in HLA-A*02 and HLA-A*24 positive North American populations (Tables 7.1 and 7.2).

Data were derived from an analysis of 1.35 million HLA-typed volunteers enrolled in the National Bone Marrow Donor Program (Mori et al., 1997). The analytic population was further divided into the following racial groups: White Americans (N = 997,193), African Americans (N = 110,057), Asian Americans (N = 81,139), Hispanics (N = 100,128), and American Indians (N = 19,203).

Table 7.1 Frequencies of haplotypes in the HLA-A*02 positive North American population: haplotypes analyzed are highlighted in grey.

Table 7.2 Frequency of haplotypes in the HLA-A*24-positive North American population: haplotypes analyzed are highlighted in gray.

Detection principle

The ProImmune MHC-Peptide Binding Assay measures the ability of each candidate peptide to bind to a selected HLA class II haplotype and stabilize the HLA-peptide complex. This assay involves in vitro assembly of candidate peptides with specific HLA class II proteins. The extent of peptide incorporation into HLA molecules is assessed by the presence or absence of the native conformation in the assembled HLA-peptide complex at time 0 (after the renaturation procedure is complete) (the so-called "assembly rate").

The binding ability of candidate peptides to specific HLA molecules is compared with that of a known very strong binding peptide (positive control) to calculate the corresponding MHC-peptide binding score. The positive control peptide is selected and provided by ProImmune (based on its experience with each HLA haplotype).

In addition to the affinity of the peptide for a specific HLA molecule, the long-term stability of the formed HLA-peptide complex is also crucial for generating an immune response. To this end, the formed HLA-peptide complex was incubated at 37°C for 24 hours to detect its presence. The binding score of the formed MHC-peptide complex at 24 hours was then calculated as a percentage of the binding score immediately after renaturation (i.e., time 0) to assess the stability of the formed MHC-peptide complex.

result

MHC-peptide binding analysis of POSTN-002 and MMP12-002 demonstrated that both peptides bound to a wide range of HLA haplotypes. Of the seven HLA haplotypes studied, POSTN-002 formed complexes with five, and MMP12-002 with four (Figure 5). Neither peptide bound to HLA-DR3 or HLA-DR6. The measured binding scores ranged from 0.02% to 2.5% of the positive control and were significantly higher than those of the non-binding peptides.

Stability analysis of the formed HLA-POSTN-002 and HLA-MMP12-002 complexes showed that, among the seven HLA-peptide complexes studied, three and two, respectively, were stable at 37°C for 24 h ( FIG. 6 ).

By comparing the binding scores of peptides with those of known immunogenic peptides, the immunogenicity of the peptide (based on its ability to bind to HLA molecules) can be inferred. Therefore, five well-studied peptides with confirmed immunogenicity were selected for this comparison. The immunogenicity of these peptides was determined ex vivo in blood samples from vaccinated patients (using intracellular cytokine staining (ICS) on CD4 somatic cells).

In principle, the ICS assay assesses the quality of specific T cells through their effector function. Therefore, peripheral blood mononuclear cells (PBMCs) are cultured in vitro and then restimulated with the test peptide, a reference peptide, and a negative control (in this case, MOCK). The restimulated cells are then stained for the production of FN-γ, TNF-α, IL-2, and IL-10, and for expression of the costimulatory molecule CD154. The cells are then counted using flow cytometry (Figure 7).

Immunogenicity analysis showed that 16 patients immunized with IMA950 peptides (BIR-002 and MET-005) produced a 100% immune response, while 71 patients immunized with IMA910 peptides (CEA-006, TGFBI-004, MMP-001) produced an immune response ranging from 44% to 86%.

To compare the binding scores of POSTN-002 and MMP12-002 with those of the IMA910 and IMA950 peptides, all peptides were tabulated according to their binding scores for each HLA-DR haplotype studied (Tables 8.1, 8.2, 8.3, 8.4, and 8.5).

Table 8.1 Binding scores of POSTN-002 and MMP12-002 to HLA-DR1 (compared to binding scores of class II peptides of known immunogenicity): Results for POSTN-002 and MMP12-002 are highlighted in grey.

Table 8.2 Binding scores of POSTN-002 and MMP12-002 to HLA-DR2 (compared to binding scores of class II peptides of known immunogenicity): Results for POSTN-002 and MMP12-002 are highlighted in grey.

Table 8.3 Binding scores of POSTN-002 and MMP12-002 to HLA-DR4 (compared to binding scores of class II peptides of known immunogenicity): Results for POSTN-002 and MMP12-002 are highlighted in grey.

Table 8.4 Binding scores of POSTN-002 and MMP12-002 to HLA-DR5 (compared to binding scores of class II peptides of known immunogenicity): Results for POSTN-002 and MMP12-002 are highlighted in grey.

Table 8.5 Binding scores of POSTN-002 and MMP12-002 to HLA-DR7 (compared to binding scores of class II peptides of known immunogenicity): Results for POSTN-002 and MMP12-002 are highlighted in grey.

Binding scores for POSTN-002 and MMP12-002 compared to other known immunogenic class II peptides indicate that the binding abilities of both peptides are mostly in the lower middle portion of the table (with the exception of HLA-DR2). Binding abilities for both peptides to HLA-DR2 are in the upper half of the table, with MMP12-002 being the strongest binding candidate. Based on this analysis, both peptides are expected to induce an immune response.

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Claims (30)

1. A peptide comprising the sequence of SEQ ID No.2 (KIQEMQHFL). 2. The peptide according to claim 1, having the ability to bind to human major histocompatibility complex (MHC) class I or II molecules. 3. The peptide according to claim 1, wherein the peptide is modified and/or contains non-peptide bonds. 4. The peptide of claim 1, wherein the peptide is part of a fusion protein and is fused to the N-terminal amino acid of the HLA-DR antigen-associated invariant chain (Ii). 5. A nucleic acid encoding the peptide of claim 1. 6. The nucleic acid according to claim 5, wherein it is DNA, PNA, RNA or a combination thereof. 7. The nucleic acid according to claim 5 is cDNA. 8. An expression vector capable of expressing the nucleic acid of any one of claims 5-7. 9. A host cell comprising the nucleic acid of any one of claims 5-7 or the expression vector of claim 8. 10. The host cell according to claim 9, wherein it is an antigen-presenting cell. 11. The host cell according to claim 10, wherein the antigen-presenting cell is a dendritic cell. 12. A pharmaceutical composition comprising the peptide of any one of claims 1-4, and at least one other component selected from pharmaceutically acceptable carriers and/or excipients, immunostimulants or immunomodulatory substances. 13. A method for preparing the peptide according to any one of claims 1-4, the method comprising culturing the host cell according to claim 9, and isolating the peptide from the host cell or its culture medium. 14. A method for preparing activated cytotoxic T lymphocytes (CTLs) or helper T cells (Th cells) in vitro, the method comprising contacting CTLs or Th cells with human class I or II MHC molecules loaded with antigens in vitro for a sufficient period of time, the MHC molecules being expressed on the surface of suitable antigen-presenting cells, thereby activating CTLs or Th cells in an antigen-specific manner, wherein the antigen is the peptide of claim 1. 15. The method of claim 14, wherein the antigen is loaded into a class I or class II MHC molecule by contacting a sufficient amount of the antigen with an antigen-presenting cell, the class I or class II MHC molecule being expressed on the surface of a suitable antigen-presenting cell. 16. The method of claim 15, wherein the antigen-presenting cell contains an expression vector capable of expressing the peptide of claim 1. 17. Activated cytotoxic T lymphocytes (CTLs) or helper T cells (Th cells) prepared by the method of any one of claims 14 to 16, wherein the CTLs or Th cells selectively recognize a cell that aberrantly expresses a polypeptide containing the amino acid sequence given in claim 1. 18. A method for in vitro preparation of a TCR or sTCR or a fragment thereof specific to the peptide of claim 1, the method comprising cloning the variable domain of an activated cytotoxic T lymphocyte (CTL) or helper T cell (Th cell) of claim 17, and expressing the TCR or sTCR or a fragment thereof in a suitable host and/or expression system. 19. A separate binding agent that specifically binds to the peptide of claim 1, or a complex of the peptide of claim 1 and an MHC molecule. 20. The binder according to claim 19, wherein it is a protein, peptide, or nucleic acid. 21. The binder of claim 19, wherein the protein or peptide is an antibody or a fragment thereof, a TCR or sTCR or a fragment thereof. 22. An isolated T-cell receptor capable of reacting with an HLA ligand, the amino acid of which is shown in SEQ ID No. 1. 23. Use of an effective amount of the activated cytotoxic T lymphocytes (CTLs) or helper T cells (Th cells) of claim 17 in the preparation of a medicament for killing non-small cell lung cancer (NSCLC), lung cancer, gastric cancer and/or glioblastoma cells in patients, wherein the cells aberrantly express or present a polypeptide containing the amino acid sequence given in any one of claims 1-4. 24. Use of the peptide of any one of claims 1-4, the nucleic acid of any one of claims 5-7, the expression vector of claim 8, the host cell of any one of claims 9-11, the activated cytotoxic T lymphocyte or helper T cell of claim 17, the binder of any one of claims 19 to 21, or the T cell receptor of claim 22 in the preparation of a medicament for treating cancer. 25. The use according to claim 24, wherein the drug is a vaccine. 26. The use according to claim 24, wherein the cancer is non-small cell lung cancer (NSCLC), lung cancer, gastric cancer, and/or glioblastoma. 27. The use according to claim 24, wherein the drug is used for human infusion cell therapy. 28. An autologous or allogeneic human cytotoxic T cell (CTL) or helper T cell (Th cell) transfected with the T cell receptor of claim 22. 29. A pharmaceutical composition comprising: (a) Selected from the following entities: (a1) The peptide according to any one of claims 1-4, (a2) The T cell receptor of claim 22 (a3) The nucleic acid according to any one of claims 5-7, (a4) The expression vector according to claim 8 (a5) The host cell according to any one of claims 9-11, (a6) The activated cytotoxic T lymphocytes (CTLs) or helper T cells (Th cells) as described in claim 17; (b) Pharmaceutically acceptable carriers, and optionally (c) Selected pharmaceutically acceptable excipients, buffers, binders, explosives, diluents, and flavor enhancers. Lubricants and at least one other component among immune stimulants or immune modulators. 30. The pharmaceutical composition according to claim 12 or 29, wherein the immunostimulatory or immunomodulatory substance is selected from cytokines, immunomodulators, adjuvants, and therapeutic substances having immunomodulatory properties.