Novel peptides and peptide compositions for immunotherapy of lung cancer, including non-small cell lung cancer and other cancers
阅读说明：本技术 用于肺癌(包括非小细胞肺癌和其他癌症)免疫治疗的新型肽和肽组合物 (Novel peptides and peptide compositions for immunotherapy of lung cancer, including non-small cell lung cancer and other cancers ) 是由 安德里亚·马尔 托尼·温斯切尼克 奥利弗·施尔 延斯·弗里切 哈普瑞特·辛格 克劳迪娅·瓦 于 2016-04-22 设计创作，主要内容包括：本发明涉及用于免疫治疗方法的肽、蛋白质、核酸和细胞。特别是,本发明涉及癌症的免疫疗法。本发明还涉及单独使用或与其他肿瘤相关肽(刺激抗肿瘤免疫反应或体外刺激T细胞和转入患者的疫苗复合物的活性药物成分)联合使用的肿瘤相关T细胞(CTL)肽表位。与主要组织兼容性复合体(MHC)分子结合的肽或与此同类的肽也可能是抗体、可溶性T细胞受体和其他结合分子的靶标。(The present invention relates to peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods. In particular, the invention relates to immunotherapy of cancer. The invention also relates to tumor-associated T Cell (CTL) peptide epitopes, used alone or in combination with other tumor-associated peptides that stimulate an anti-tumor immune response or stimulate T cells in vitro and the active pharmaceutical ingredient of the vaccine composition transferred into the patient. Peptides that bind to or are cognate to Major Histocompatibility Complex (MHC) molecules may also be targets for antibodies, soluble T cell receptors, and other binding molecules.)
1. A peptide comprising an amino acid sequence selected from the group consisting of SEQ ID No.1 to SEQ ID No.110, and variant sequences thereof which are at least 88% homologous to SEQ ID No.1 to SEQ ID No.110, wherein said variant binds to Major Histocompatibility Complex (MHC) and/or induces T cell cross-reactivity with the variant peptide, wherein said peptide is not a full-length polypeptide.
2. The peptide of claim 1, wherein said peptide has the ability to bind to an MHC class-I or-II molecule, wherein said peptide, when bound to said MHC, is capable of being recognized by CD4 and/or CD8T cells.
3. The peptide or variant thereof according to claim 1 or 2, wherein the amino acid sequence comprises a continuous stretch of amino acids according to any one of SEQ ID No.1 to SEQ ID No. 110.
4. The peptide or variant thereof according to any of claims 1 to 3, wherein said peptide or variant thereof has an overall length of from 8 to 100 amino acids, preferably from 8 to 30 amino acids, more preferably from 8 to 16 amino acids, most preferably the peptide consists or consists essentially of an amino acid sequence according to any of SEQ ID No.1 to SEQ ID No. 110.
5. The peptide or variant thereof according to any of claims 1 to 4, wherein said peptide is modified and/or comprises non-peptide bonds.
6. The peptide or variant thereof according to any of claims 1 to 5, wherein said peptide is part of a fusion protein, in particular comprising the N-terminal amino acid of the HLA-DR antigen associated invariant chain (Ii).
7. A nucleic acid encoding a peptide or variant thereof according to any one of claims 1 to 6, optionally linked to a heterologous promoter sequence.
8. An expression vector for expressing a nucleic acid according to claim 7.
9. A recombinant host cell comprising a peptide according to claims 1 to 6, a nucleic acid according to claim 7, an expression vector according to claim 8, wherein said host cell is preferably an antigen presenting cell, such as a dendritic cell.
10. The peptide or variant thereof according to any one of claims 1 to 6, the nucleic acid according to claim 7, the pharmaceutically acceptable expression vector according to claim 8 or according to claim 9.
Background
The majority of cancer-related deaths in men and women are lung cancers. Worldwide, lung cancer is the most common cancer in terms of both incidence and mortality. In 2012, there were over 180 million new cases (13% of total cancer morbidity) and 160 million people died of lung cancer (20% of total cancer mortality). Lung cancer is the leading cause of cancer death in men in 87 countries and women in 26 countries. Over one third of the newly diagnosed cases are in china. The areas of highest incidence are north america, europe and east asia (World Cancer Report, 2014).
Women who died of lung cancer annually since 1987 outperformed breast cancer. Mortality rates in men continue to decline, approximately 1.9% per year, from 1991 to 2003. Female lung cancer mortality is leveling off after decades of continuous growth. These trends in lung cancer mortality reflect a decline in smoking rates over the last 30 years.
According to the data of the National Cancer Institute (NCI), 23 million new cases and 16 million deaths from lung cancer are expected in the us in 2013.
According to previous experience, small cell lung cancer is distinguished from non-small cell lung cancer (NSCLC), which includes histological types of adenocarcinoma, squamous cell carcinoma and large cell carcinoma. However, in the last decade, the distinction between adenocarcinoma and squamous cell carcinoma has been increasingly recognized due to major differences in genetics and response to specific treatments. Lung cancer is therefore further classified according to molecular subtype, depending on the specific genetic alterations that drive and maintain lung tumorigenesis (Travis et al, 2013).
The prognosis is generally poor. Of all lung cancer patients, 10% -15% survive five years after diagnosis. Poor survival in lung cancer patients is due, at least in part, to metastasis in 80% of patients at the time of diagnosis, and distant metastasis in more than half of patients (SEER Stat artifacts, 2014). At the time of onset, 30-40% of NSCLC cases are in stage IV and 60% of SCLC cases are in stage IV.
The 1-year relative survival rate for lung cancer is 35% during 1975-1979, and rises slightly to 44% by 2010, mainly due to improvements in surgical techniques and the use of combination therapies. However, all stages of lung cancer, combined, have a 5-year survival rate of only 17%. Survival rates for cases where disease is still localized at the time of detection are 54%; but only 16% of lung cancers are diagnosed at this early stage (SEER Stat facts, 2014).
Treatment options are determined by the type (small cell and non-small cell lung cancer) and stage of the cancer, including surgery, radiation therapy, chemotherapy, targeted biological treatments, e.g.Andfor localized cancer, surgical treatment is often selected. Recent studies have shown that post-operative chemotherapy can improve survival of early stage non-small cell lung cancer. Since the disease usually has spread when it is discovered, radiation and chemotherapy are often used, sometimes in combination with surgery. Single chemotherapy or a combination of chemoradiotherapy is a common option for treating small cell lung cancer; with this protocol, a significant proportion of patients achieve remission, which in some cases is persistent in response to surgery (S3-leitline Lungenkarzinom, 2011).
Advanced lung cancer also develops resistance to traditional chemotherapy. However, recent research advances have relied on histological and genetic exciting advances in therapeutic approaches. The examination level is: adjuvant chemotherapy trials aimed at distinguishing between codon 12 and 13 mutations of KRAS and different amino acid substitutions determined by codon 12 specific mutations (Shepherd et al, 2013).
In order to expand the therapeutic regimens for NSCLC, different immunotherapeutic approaches have been or are being investigated. Although vaccination with L-BLP25 or MAGEA3 failed to demonstrate the vaccine-mediated survival advantage in NSCLC patients, allogeneic cell-derived vaccines have shown promising results in clinical studies. In addition, further vaccination trials against gangliosides, epidermal growth factor receptor and several other antigens are currently in progress. Another strategy to enhance a patient's anti-tumor T cell response involves the use of specific antibodies to block inhibitory T cell receptors or their ligands. Several of these antibodies, including ipilimumab, nivolumab, pembrolizumab, MPDL3280A, and MEDI-4736, are currently being evaluated in clinical trials for their therapeutic potential in NSCLC (Reinmuth et al, 2015).
Given the severe side effects and costs associated with treating cancer, it is often necessary to identify factors that can be used to treat cancer, particularly lung cancer, including NSCLC. It is also often necessary to determine factors that represent biomarkers of cancer, particularly lung cancer, in order to better diagnose cancer, assess prognosis and predict treatment success.
Cancer immunotherapy represents one option for cancer cell-specific targeting, while minimizing side effects. Cancer immunotherapy utilizes the presence of tumor-associated antigens.
The current classification of Tumor Associated Antigens (TAAs) mainly includes the following groups:
a) cancer-testis antigen: the first identified TAA that T cells can recognize belongs to this class of antigens, which were originally called cancer-testis (CT) antigens because their members are expressed in histologically distinct human tumors, in normal tissues, only in spermatocytes/spermatogonia of the testis, and occasionally in the placenta. Since testis cells do not express HLA class I and class II molecules, these antigens are not recognized by T cells in normal tissues and are therefore immunologically considered to be tumor-specific. Well known examples of CT antigens are members of the MAGE family and NY-ESO-1.
b) Differentiation antigen: both tumor and normal tissues (from which the tumor originates) contain TAAs. Most known differentiation antigens are found in melanoma and normal melanocytes. Many of these melanocyte lineage associated proteins are involved in melanin biosynthesis, and thus these proteins are not tumor specific, but are still widely used in cancer immunotherapy. Examples include, but are not limited to, tyrosinase for melanoma and PSA for Melan-A/MART-1 or prostate cancer.
c) Overexpressed TAA: genes encoding widely expressed TAAs have been detected in histologically distinct tumors as well as in many normal tissues, generally at low levels. It is likely that many epitopes processed and potentially presented by normal tissues are below the threshold level of T cell recognition, and their overexpression in tumor cells can trigger an anti-cancer response by breaking the previously established tolerance. Typical examples of such TAAs are Her-2/neu, survivin, telomerase or WT 1.
d) Tumor specific antigens: these unique TAAs arise from mutations in normal genes (e.g., β -catenin, CDK4, etc.). Some of these molecular changes are associated with neoplastic transformation and/or progression. Tumor specific antigens generally induce strong immune responses without risking autoimmune responses in normal tissues. On the other hand, these TAAs are in most cases only associated with the exact tumor on which they are confirmed, and are not usually shared between many individual tumors. Peptide tumor specificity (or relatedness) may also occur if the peptide is derived from a tumor (associated) exon, in the case of proteins containing tumor-specific (associated) isoforms.
e) TAA resulting from aberrant post-translational modifications: such TAAs may be produced by proteins that are neither specific nor overexpressed in tumors, but which still have tumor-related properties (the association results from post-translational processing that is primarily active against tumors). Such TAAs arise from alterations in the variant glycosylation pattern, resulting in the tumor developing a novel epitope for MUC1 or in degradation processes resulting in events such as protein splicing, which may or may not be tumor specific.
f) Oncoviral proteins: these TTAs are viral proteins that may play a key role in carcinogenesis and, since they are foreign proteins (non-human proteins), are capable of triggering T cell responses. Examples of such proteins are human papilloma virus type 16 proteins, E6 and E7, which are expressed in cervical cancer.
T cell-based immunotherapy targets peptide epitopes derived from tumor-associated or tumor-specific proteins presented by Major Histocompatibility Complex (MHC) molecules. The antigen recognized by tumor-specific T lymphocytes, i.e. the epitope thereof, can be molecules derived from all protein types, such as enzymes, receptors, transcription factors, etc., which are expressed in the cells of the respective tumor and whose expression is usually upregulated compared to homologous unaltered cells.
MHC molecules are of two classes: MHC class I and MHC class II. MHC class I molecules consist of an alpha heavy chain and beta-2-microglobulin, and MHC class II molecules consist of an alpha chain and a beta chain. Its three-dimensional configuration forms a binding pocket for non-covalent interaction with the peptide.
MHC class I molecules are found on most nucleated cells. They presented peptides generated by cleavage of mainly endogenous proteins, defective ribosomal products (DRIP) and larger peptides. However, peptides derived from endosomal structures or from exogenous sources are also often found on MHC class I molecules. This non-classical way of presenting class I molecules is known in the literature as cross-presentation (Brossartand Bevan, 1997; Rock et al, 1990). MHC class II molecules are found predominantly on professional Antigen Presenting Cells (APCs) and present predominantly, for example, peptides of exogenous or transmembrane proteins that are occupied by APCs during endocytosis and subsequently processed.
Complexes of peptides and MHC class I are recognized by CD8 positive T cells bearing the corresponding T Cell Receptor (TCR), while complexes of peptides and MHC class II molecules are recognized by CD4 positive helper T cells bearing the corresponding TCR. Thus, it is well recognized that TCR, peptide and MHC are presented in a 1: 1 stoichiometric ratio.
CD 4-positive helper T cells play an important role in inducing and maintaining an effective response of CD 8-positive cytotoxic T cells. The recognition of a Tumor Associated Antigen (TAA) derived CD4 positive T cell epitope may be important for the development of pharmaceutical products capable of eliciting anti-tumor immune responses (Gnjatic et al, 2003). At the tumor site, T helper cells maintain a cytokine environment friendly to cytotoxic T Cells (CTL) (Mortara et al, 2006) and attract effector cells such as CTL, Natural Killer (NK) cells, macrophages and granulocytes (Hwang et al, 2007).
In the absence of inflammation, MHC class II molecule expression is primarily restricted to immune system cells, especially professional Antigen Presenting Cells (APCs), e.g., monocytes, monocyte derived cells, macrophages, dendritic cells. Expression of MHC class II molecules is found in tumor cells of cancer patients (Dengjel et al, 2006).
The elongated (longer) peptides of the invention can serve as MHC class II active epitopes.
MHC-II epitope-activated helper T cells play an important role in orchestrating CTL effector functions of anti-tumor immunity. Helper T cell epitopes that trigger TH1 cell responses support effector functions of CD8 positive killer T cells, including cytotoxic functions that act directly on tumor cells (tumor-associated peptide/MHC complexes are displayed on the surface of such tumor cells). Thus, the tumor-associated T helper cell epitopes, used alone or in combination with other tumor-associated peptides, can serve as active pharmaceutical ingredients of vaccine compounds for stimulating anti-tumor immune responses.
Mammalian (e.g., mouse) models have shown that CD4 positive T cells can suppress angiogenesis sufficiently to suppress tumor expression by secreting interferon-gamma (IFN γ) even in the absence of CD8 positive T lymphocytes (Beatty and Paterson, 2001; Mumberg et al, 1999). There is no evidence for CD4T cells as direct anti-tumor effectors (Braumulleret al, 2013; Tran et al, 2014).
Since constitutive expression of HLA class II molecules is usually restricted to immune cells, it was previously considered impossible to isolate class II peptides directly from primary tumors. However, Dengjel et al succeeded in directly recognizing multiple MHC class II epitopes in tumors (WO 2007/028574, EP 1760088B 1).
Since both CD 8-dependent and CD 4-dependent responses together and synergistically contribute to anti-tumor effects, the identification and characterization of tumor-associated antigens recognized by CD8+ T cells (ligand: MHC class I molecule + peptide epitope) or CD4 positive T helper cells (ligand: MHC class II molecule) is important for the development of tumor vaccines.
For a peptide to trigger (elicit) a cellular immune response by an MHC class I peptide, it must also bind to an MHC molecule. This process relies on alleles of MHC molecules and specific polymorphisms of peptide amino acid sequences. MHC class I-binding peptides are typically 8-12 amino acid residues in length and typically comprise two conserved residues ("anchors") in their sequence that interact with the corresponding binding groove of the MHC molecule. Thus, each MHC allele has a "binding motif" to determine which peptides are capable of specifically binding to the binding groove.
In an MHC class I dependent immune response, peptides not only bind to certain MHC class I molecules expressed by tumor cells, but they must then be recognized by a T cell-loaded specific T Cell Receptor (TCR).
Special conditions must be met for the proteins recognized by T lymphocytes as tumor-specific or related antigens and for therapy. The antigen should be expressed predominantly by tumor cells and not by normal healthy tissue, or in relatively small amounts. In a preferred embodiment, the peptide should be over-represented in tumor cells compared to normal healthy tissue. Preferably, the corresponding antigen is not only present in a tumor, but also in high concentrations (i.e., the number of copies of the corresponding peptide per cell). Tumor-specific and tumor-associated antigens are often derived from proteins that are directly involved in the transformation of normal cells into tumor cells, which occurs due to their function in cell cycle control or apoptosis inhibition. In addition, downstream targets of these proteins that directly lead to a transformation event may be upregulated and thus may be indirectly associated with the tumor. These indirect tumor-associated antigens may also be targets for vaccination methods (Singh-Jasuja et al, 2004). It is essential that epitopes are present in the amino acid sequence of the antigen to ensure that such peptides from tumour associated antigens ("immunogenic peptides") can cause T cell responses in vitro or in vivo.
Basically, any peptide that binds to an MHC molecule may serve as a T cell epitope. The prerequisite for inducing an in vitro or in vivo T cell response is the presence of T cells with the corresponding TCR and the absence of immune tolerance to this particular epitope.
Thus, TAAs are the starting point for development based on T cell therapies, including but not limited to tumor vaccines. Methods for identifying and characterizing TAAs are generally based on the use of T cells in patients or healthy subjects, or on the generation of differential transcriptional profiles or differential expression patterns between tumor and normal tissue peptides. However, the identification of genes that are overexpressed or selectively expressed in tumor tissues or human tumor cell lines does not provide accurate information on the antigens transcribed using these genes in immunotherapy. This is because only a portion of the epitopes of these antigens are suitable for this application, since T cells with the corresponding TCR must be present and immune tolerance to this particular epitope must be absent or minimal. Therefore, in a very preferred embodiment of the invention, it is important to select only those peptides that are over-or selectively presented for situations where functional and/or proliferative T cells are found. Such functional T cells are defined as T cells that are clonally expanded upon stimulation with a specific antigen and are capable of performing effector functions ("effector T cells").
Where targeting is to a peptide-MHC by a particular TCR (e.g. a soluble TCR) and an antibody or other binding molecule (scaffold) according to the invention, immunogenicity of the potential peptide is of minor importance. In these cases, presentation is the determining factor.
Brief introduction to the invention
In a first aspect of the invention, the invention relates to a peptide comprising a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 110, or an amino acid sequence of the group of SEQ ID NO: 1 to SEQ ID NO: 110 (wherein the variant binds to MHC and/or induces T cells to cross-react with the peptide), or a pharmaceutically acceptable salt thereof (wherein the peptide is not a potentially full-length polypeptide), which has at least 77%, preferably at least 88% homology (preferably at least 77% or at least 88% identical).
The invention further relates to a peptide of the invention comprising a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 162 is preferably SEQ ID NO: 1 to SEQ ID NO: 110, or a sequence identical to one of the group of SEQ ID NO: 1 to SEQ ID NO: 110, wherein the total length of the peptide or variant thereof is from 8 to 100, preferably from 8 to 30, most preferably from 8 to 20 amino acids.
The following table shows the peptides according to the invention, their respective SEQ ID NOs, and possible source (potential) genes for these peptides. 3 all 3 peptides 3 in 3 tables 3 1 3 and 3 4 3 bound 3 to 3 HLA 3- 3 a 3 02 3. 3 3 all 3 peptides 3 in 3 table 3 2 3 bound 3 to 3 HLA 3- 3 a 3 x 3 24 3. 3 All peptides in tables 3 and 5 bind to HLA-DR. The peptides in tables 4 and 5 were previously disclosed in large lists as high error rates or calculated using algorithms as a result of high throughput screening, but had no previous association with cancer. The peptides in tables 6, 7 and 8 are other peptides that can be used in combination with other peptides of the present invention. The peptides in tables 9 and 10 may also be used in the diagnosis and/or treatment of various other malignant diseases involving the overexpression or over-presentation of each potential polypeptide.
Table 1: peptides of the invention
Table 2: other peptides of the invention
Table 3: HLA-DR peptide of the invention
Table 4: other peptides of the invention, previously not known to be associated with cancer
Sequence ID number
Sequence of
Gene ID
Formal gene symbol
94
ILVDWLVQV
9133
CCNB2
95
KIIGIMEEV
2956
MSH6
96
AMGIAPPKV
9129
PRPF3
97
TLFPVRLLV
79888
LPCAT1
98
VLYPHEPTAV
29980，5523
DONSON，PPP2R3A
99
ALFQRPPLI
1736
DKC1
100
KIVDFSYSV
701
BUB1B
101
LLLEILHEI
30001
ERO1L
102
SLLSELQHA
115362
GBP5
103
KLLSDPNYGV
79188
TMEM43
104
SLVAVELEKV
25839
COG4
105
IVAESLQQV
6772
STAT1
106
SILEHQIQV
4173
MCM4
107
ALSERAVAV
10213
PSMD14
108
TLLDFINAV
55236
UBA6
109
NLIEVNEEV
221960，51622
CCZ1B，CCZ1
Table 5: other HLA-DR peptides of the invention have not previously been known to be associated with cancer
Table 6: other peptides for personalized cancer therapy
Table 7: other peptides for use in, e.g., personalized cancer therapy
Table 8: HLA-DR peptides for use in, e.g., personalized cancer therapy
Sequence ID number
Sequence of
Gene ID
Formal gene symbol
162
TNGVIHVVDKLLYPADT
10631
POSTN
The invention also relates generally to the use of the peptides of the invention for the treatment of proliferative diseases, e.g., brain, breast, colorectal, esophageal, kidney, liver, ovary, pancreas, prostate, stomach, melanoma, merkel cell, leukemia (AML, CLL), non-hodgkin lymphoma (NHL), esophageal including gastroesophageal junction cancer (OSCAR), gall bladder and bile duct cancer (GBC _ CCC), bladder cancer (UBC), uterine cancer (UEC).
Particularly preferred are peptides of the invention (alone or in combination) selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 110. More preferably the peptides (alone or in combination) are selected from the group comprising SEQ ID NO: 1 to SEQ ID NO: 14 (see table 1) and SEQ ID NO: 23 to SEQ ID NO: 47 (see table 2) and for the immunotherapy of lung cancer (including NSCLC), brain cancer, breast cancer, colorectal cancer, esophageal cancer, kidney cancer, liver cancer, ovarian cancer, pancreatic cancer, prostate cancer, stomach cancer, melanoma, merkel cell carcinoma, leukemia (AML, CLL), non-hodgkin lymphoma (NHL), esophageal cancer including gastroesophageal junction cancer (OSCAR), gallbladder cancer and bile duct cancer (GBC _ CCC), bladder cancer (UBC), uterine cancer (UEC), preferably lung cancer including NSCLC.
As shown in tables 9, 9-2 and tables 10, 10-2 below, many of the peptides of the present invention are also found in other tumors and thus may be used for immunotherapy for other indications. See also figure 1 and example 1.
Table 9: 3 the 3 HLA 3- 3 a 3 02 3 peptides 3 of 3 the 3 invention 3 and 3 their 3 particular 3 use 3 in 3 other 3 proliferative 3 diseases 3, 3 in 3 particular 3 other 3 cancerous 3 diseases 3. 3 The table shows that for selected peptides of other tumor types, they were found to be over-presented (specifically presented) in tumor samples assayed at greater than 5%, or in tumor samples assayed at greater than 5% and the geometric mean tumor to normal tissue ratio was greater than 3.
Table 9-2: 3 the 3 HLA 3- 3 a 3 02 3 peptides 3 of 3 the 3 invention 3 and 3 their 3 particular 3 use 3 in 3 other 3 proliferative 3 diseases 3, 3 in 3 particular 3 other 3 cancerous 3 diseases 3 ( 3 table 3 9 3 revision 3) 3. 3 The table (e.g., table 9) shows that for selected peptides of other tumor types, they were found to be over-presented (specifically presented) in tumor samples assayed at greater than 5%, or in tumor samples assayed at greater than 5% and the geometric mean tumor to normal tissue ratio was greater than 3. Over-presentation is defined as higher presentation in tumor samples compared to the highest presented normal samples. The normal tissues presented by the degree of history are: adipose tissue, adrenal gland, blood cells, blood vessels, bone marrow, brain, cartilage, esophagus, eye, gall bladder, heart, kidney, large intestine, liver, lung, lymph node, nerve, pancreas, parathyroid gland, peritoneum, pituitary gland, pleura, salivary gland, skeletal muscle, skin, small intestine, spleen, stomach, thymus, thyroid, trachea, ureter, bladder.
SCLC ═ small cell lung cancer, RCC ═ kidney cancer, CRC ═ colon or rectal cancer, GC ═ stomach cancer, HCC ═ liver cancer, PC ═ pancreatic cancer, PrC ═ prostate cancer, BRCA ═ breast cancer, MCC ═ merkel cell carcinoma, OC ═ ovarian cancer, NHL ═ non-hodgkin lymphoma, AML ═ acute myeloid leukemia, CLL ═ chronic lymphocytic leukemia.
Table 10: 3 the 3 HLA 3- 3 a 3 x 3 24 3 peptides 3 of 3 the 3 invention 3 and 3 their 3 particular 3 use 3 in 3 other 3 proliferative 3 diseases 3, 3 in 3 particular 3 other 3 cancerous 3 diseases 3. 3 The table shows that for selected peptides of other tumour types, they were found to be over-represented (specifically represented) in tumour samples assayed at above 5%, or in tumour samples assayed at above 5% and the ratio of the geometric mean tumour to normal tissue was greater than 3.
Table 10-2: 3 the 3 HLA 3- 3 a 3 x 3 24 3 peptides 3 of 3 the 3 invention 3 and 3 their 3 particular 3 use 3 in 3 other 3 proliferative 3 diseases 3, 3 in 3 particular 3 other 3 cancerous 3 diseases 3 ( 3 table 3 10 3 revision 3) 3. 3 The table (e.g., table 10) shows that for selected peptides of other tumor types, they were found to be over-presented (specifically presented) in tumor samples assayed at greater than 5%, or in tumor samples assayed at greater than 5% and the geometric mean tumor to normal tissue ratio was greater than 3. Over-presentation is defined as higher presentation in tumor samples compared to the highest presented normal samples. The normal tissues presented by the degree of history are: adrenal gland, artery, brain, heart, kidney, large intestine, liver, lung, pancreas, pituitary, skin, spleen, stomach, thymus.
GC-gastric cancer, HCC-liver cancer.
Thus, another aspect of the invention relates to at least one peptide of the invention according to any one of SEQ id nos. 7, 14, 15, 18, 94, 95, 97, 98, 101, 102, 105, 106, 111, 112, 117, 118, 120, 121, 122, 123, 126, 127, 128, 130, 131, 132, 136, 138, 139, 143, 146, 147, 150, 28, 29, 42, 47, 50, 54, 56, 59, 66, 67 and 161 in combination with a preferred embodiment peptide for use in the treatment of renal cancer.
Thus, another aspect of the invention relates to the use of at least one peptide according to the invention according to any one of SEQ id nos. 8, 9, 15, 16, 20, 94, 98, 100, 103, 104, 111, 114, 117, 118, 120, 127, 129, 132, 135, 138, 139, 145, 149, 150, 151, 29, 36, 37, 41, 45, 54, 59, 70, 73, 79, 80 and 82 in combination with a peptide of a preferred embodiment for the treatment of brain cancer.
Thus, another aspect of the present invention relates to at least one peptide according to the present invention according to any one of SEQ id nos. 2, 4, 18, 94, 105, 113, 114, 115, 117, 120, 124, 126, 128, 130, 131, 132, 134, 137, 138, 144, 146, 149, 153, 26, 31, 33, 36, 41, 42, 44, 49, 50, 56, 58, 63, 67, 77, 78, 85, 159, 160 and 161, in combination with a peptide of a preferred embodiment for use in the treatment of gastric cancer.
Thus, another aspect of the invention relates to the use of at least one peptide according to the invention according to any one of SEQ id nos 2, 7, 11, 13, 94, 96, 98, 99, 100, 111, 113, 114, 115, 116, 117, 118, 120, 121, 122, 123, 124, 125, 126, 128, 129, 130, 131, 132, 137, 138, 139, 144, 145, 146, 149 and 152 in combination with a peptide of a preferred embodiment for the treatment of colorectal cancer.
Thus, another aspect of the invention relates to the use of at least one peptide according to the invention according to any one of SEQ id nos. 7, 8, 9, 11, 15, 16, 18, 19, 20, 21, 94, 96, 98, 99, 101, 104, 111, 113, 114, 115, 117, 118, 119, 120, 121, 126, 129, 131, 132, 135, 136, 138, 139, 143, 149, 150, 152, 26, 27, 28, 29, 37, 38, 39, 41, 44, 46, 50, 51, 52, 56, 58, 59, 60, 61, 62, 63, 66, 67, 69, 70, 71, 72, 73, 75, 76, 77, 79, 81, 82, 84 and 161 in combination with a preferred embodiment peptide for the treatment of liver cancer.
Thus, another aspect of the invention relates to the use of at least one peptide according to the invention according to any one of SEQ id nos 1, 2, 3, 4, 13, 18, 96, 101, 103, 104, 105, 112, 113, 114, 115, 117, 119, 120, 121, 123, 124, 125, 126, 128, 131, 132, 133, 135, 136, 137, 138, 139, 143, 146 and 156 in combination with a peptide of a preferred embodiment for the treatment of pancreatic cancer.
Thus, another aspect of the invention relates to at least one peptide of the invention according to any one of SEQ id nos. 8, 10, 16, 18, 114, 128, 139, 143, 153, 27, 37, 41, 43, 53, 59, 61, 67, 72, 76, 78, 80, 82 and 84 in combination with a preferred embodiment peptide for use in the treatment of prostate cancer.
Thus, another aspect of the invention relates to at least one peptide according to the invention according to any one of SEQ id nos 9, 15, 96, 97, 120 and 127 in combination with a peptide of a preferred embodiment for use in the treatment of leukemia (AML, CLL).
Thus, another aspect of the invention relates to the use of at least one peptide according to the invention according to any one of SEQ id nos 1, 3, 4, 5, 7, 13, 16, 18, 101, 102, 105, 112, 113, 115, 119, 124, 126, 128, 133, 145 and 156 in combination with a preferred embodiment of the peptide for the treatment of breast cancer.
Thus, another aspect of the invention relates to at least one peptide according to the invention according to any one of SEQ id nos 95, 98, 100, 104, 138, 149 and 151 in combination with a peptide of a preferred embodiment for use in the treatment of meikel cell carcinoma.
Thus, another aspect of the invention relates to at least one peptide according to the invention according to any one of SEQ id nos. 4, 5, 9, 16, 19, 20, 94, 98, 112, 115, 117, 118, 128, 130, 132, 134, 138, 139, 144, 146 and 148 in combination with a peptide of a preferred embodiment for use in the treatment of melanoma.
Thus, another aspect of the invention relates to at least one peptide according to the invention according to any one of SEQ id nos. 4, 8, 9, 10, 16, 18, 94, 98, 99, 100, 101, 102, 104, 105, 111, 113, 114, 115, 117, 118, 120, 121, 123, 124, 125, 126, 128, 129, 130, 131, 132, 134, 137, 138, 139, 142, 143, 144, 148, 149, 150 and 152 for use in combination with a preferred embodiment peptide in the treatment of ovarian cancer.
Thus, another aspect of the invention relates to at least one peptide according to the invention according to any one of SEQ id nos 5, 9, 13, 18, 19, 21, 95, 102, 104, 105, 113, 114, 115, 119, 120, 123, 124, 125, 127, 129, 130, 132, 133, 137, 138, 139, 141, 143, 144, 145, 146, 149, 150, 151, 153, 155, 156 and 157 in combination with a preferred embodiment peptide for use in the treatment of esophageal cancer.
Thus, another aspect of the invention relates to at least one peptide according to the invention according to any one of SEQ id nos 13, 25, 113, 114, 115, 120, 121, 128, 159 and 161, preferably in combination for the treatment of breast lung cancer (including NSCLC).
Thus, another aspect of the invention relates to the use of the peptides of the invention-preferably in combination for the treatment of a proliferative disease selected from the group of lung cancer (including NSCLC), brain cancer, breast cancer, colorectal cancer, esophageal cancer, kidney cancer, liver cancer, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, melanoma, merkel cell carcinoma, leukemia (AML, CLL).
The invention also relates to peptides of the invention having the ability to bind to Major Histocompatibility Complex (MHC) I or to molecules in elongated form, e.g.MHC-class II molecules of varying length.
The invention further relates to a peptide according to the invention, wherein said peptide(s) (each peptide) consists or essentially consists of a peptide according to seq id no: 1 to SEQ ID NO: 162, preferably SEQ ID NO: 1 to SEQ ID NO: 110.
The invention further relates to a peptide according to the invention, wherein said peptide is modified and/or comprises non-peptide bonds.
The invention further relates to a peptide of the invention, wherein the peptide is part of a fusion protein, in particular fused to the N-terminal amino acid of the invariant chain (Ii) associated with the HLA-DR antigen, or to an antibody (e.g. a dendritic cell specific antibody) or a sequence of an antibody.
The invention further relates to a nucleic acid encoding a peptide according to the invention. The invention further relates to a nucleic acid according to the invention, being DNA, cDNA, PNA, RNA, and possibly a combination thereof.
The invention further relates to an expression vector capable of expressing and/or expressing the nucleic acid of the invention.
The invention further relates to a peptide according to the invention, a nucleic acid according to the invention or a pharmaceutically acceptable expression vector according to the invention for the treatment of a disease, in particular for the treatment of cancer.
The invention further relates to specific antibodies to the peptides of the invention or to the peptide complexes (containing MHC) described in the invention and to methods for producing these antibodies.
The invention further relates to T Cell Receptors (TCRs) of the invention, in particular soluble TCRs (sTCRs) and cloned TCRs processed into autologous or allogeneic T cells, as well as methods of making these TCRs and methods of making NK cells bearing said TCRs or said TCR cross-reactivity.
Antibodies and TCRs are further embodiments of the present immunotherapeutic uses of the peptides according to the invention.
The invention further relates to a host cell comprising a nucleic acid according to the invention or an expression vector as described above. The invention further relates to a host cell of the invention which is an antigen presenting cell, preferably a dendritic cell.
The invention further relates to a method for formulating a peptide of the invention, said method comprising culturing a host cell of the invention and isolating the peptide from said host cell or its culture medium.
The invention further relates to the method of the invention wherein the antigen is carried on a human MHC class I or II molecule expressed on the surface of a suitable antigen presenting cell or artificial antigen presenting cell by binding to a sufficient amount of antigen from a suitable antigen presenting cell.
The invention further relates to a method of the invention, wherein the antigen-presenting cell is selected from the group consisting of cells expressing a polypeptide comprising SEQ ID No.1 to SEQ ID No.110, preferably SEQ ID No.1 to SEQ ID No.14, and SEQ ID No.23 to SEQ ID No.: 47, or a variant amino acid sequence.
The invention further relates to a T-cell promoter produced by the method of the invention, wherein the T-cell selectively recognizes a cell that expresses a polypeptide comprising an amino acid sequence of the invention.
The invention further relates to a method of killing target cells in a patient, wherein the target cells in the patient abnormally express a polypeptide comprising any of the amino acid sequences of the invention, the method comprising administering to the patient an effective amount of T cells produced by the method of the invention.
The invention further relates to the use of any of said peptides, nucleic acids of the invention, expression vectors of the invention, cells of the invention, priming T lymphocytes of the invention as a medicament or in the manufacture of a medicament, T cell receptors or antibodies or other peptide-and/or peptide-MHC binding molecules. The agent preferably has anti-cancer activity.
Preferably, the agent is a soluble TCR or antibody based cell therapy drug, vaccine or protein. Preferably, the agent is a cell therapy drug, vaccine or protein obtained from a soluble TCR or antibody (e.g., a sTCR comprising an anti-CD 3 antibody or portion thereof).
The invention also relates generally to the use of the invention, wherein the cancer cell is a lung cancer (including NSCLC) brain cancer, breast cancer, colorectal cancer, esophageal cancer, kidney cancer, liver cancer, ovarian cancer, pancreatic cancer, prostate cancer, stomach cancer, melanoma, merkel cell cancer, leukemia (AML, CLL), non-hodgkin lymphoma (NHL), esophageal cancer including gastroesophageal junction cancer (OSCAR), gallbladder and bile duct cancer (GBC, CCC), bladder cancer (UBC), uterine cancer (UEC) cell, preferably a lung cancer cell.
The invention further relates to a biomarker, herein referred to as "target", based on a peptide of the invention, which can be used for the diagnosis of cancer, preferably lung cancer (including NSCLC). The marker may be over-presented by the peptide itself or over-expressed by the corresponding gene. The marker may also be used to predict the likelihood of success of a treatment, preferably an immunotherapy, most preferably an immunotherapy targeting the same target recognized by the biomarker. For example, antibodies or soluble TCRs can be used to stain tumor sections to detect the presence or absence of the relevant peptide complexed to MHC.
Alternatively, the antibody has further effector functions, such as an immunostimulatory domain or a toxin.
The invention also relates to the use of these novel targets in the treatment of cancer.
Collagen alpha-3 (VI) chain protein (COL6A3) -COL6A3 encodes the alpha-3 chain, which is one of the 3 alpha chains of collagen VI. The protein domain has been shown to bind to extracellular matrix proteins, an interaction that may indicate the importance of this collagen in the tissue matrix components. The extracellular matrix is reconstituted by expressing collagen VI that promotes cisplatin resistance in ovarian cancer cells. The presence or absence of collagen VI is associated with tumor grade, a prognostic factor for ovarian cancer (Sherman-Baust et al, 2003). COL6a3 was overexpressed in colorectal tumors (Smith et al, 2009a), salivary adenocarcinoma (Leivo et al, 2005), and differentially expressed in gastric cancer (Yang et al, 2007). COL6a3 has been identified as one of seven genes containing tumor-specific splice variants. The validated tumor-specific splicing changes are highly consistent, enabling clear differentiation between normal and cancer samples, and even clear differentiation between different stages of the tumor (Thorsen et al, 2008).
Solute Carrier family 6 (amino acid transport) member 14(SLC6A14) -SLC6A14 encodes solute carrier family 6 member 14(SLC6A 14). SLC6a14 is an amino acid transporter, a member of solute carrier family 6. Members of this family are sodium and chloride dependent amino acid/neurotransmitter transporters. SLC6a14 transports neutral and cationic amino acids. Transporters are expressed at low levels in normal tissues (Sloan and Mager, 1999). SLC6a14 was demonstrated to be upregulated in cervical (Gupta et al, 2006), colorectal (Gupta et al, 2005) and Estrogen Receptor (ER) positive breast cancer (Karunakaran et al, 2011) tissues and cell lines, as well as hepatoma cells (Fuchs et al, 2004). Although SLC6A14 is expressed very poorly in the corresponding normal tissues/cells, cancer cells up-regulate SLC6A14 to meet their increased demand for these amino acids. alpha-methyl-DL-tryptophan (alpha-MT), a selective blocker of SLC6a14, induces amino acid deprivation and leads to apoptosis in ER positive breast cancer cell lines (Karunakaran et al, 2011).
Dual specificity phosphatase 4(DUSP4) -the protein encoded by the DUSP4 gene is a member of the dual specificity protein phosphatase subfamily. DUSP4 inactivates ERK1, ERK2 and JNK, is expressed in various tissues, and is located in the nucleus. According to reports, DUSP4 (alias MKP2) is overexpressed in malignant samples compared to non-malignant breast cancer samples (Wang et al, 2003). In colon cancer patient microarray datasets, DUSP4 expression was found to be differentially expressed, with highest expression in BRAF mutant tumors. In addition, high DUSP4 is associated with poor overall survival (De, V et al, 2013).
Glycoprotein (transmembrane) nmb (GPNMB) -gene GPNMB encodes a type I transmembrane glycoprotein. GPNMB has been shown to be expressed in most different cancer types and primarily increases tumor invasiveness by promoting tumor cell migration, invasion and metastasis formation. At the molecular level, GPNMB has been shown to increase the expression of MMP-2, 3 and 9, and is itself regulated by p53 (Metz et al, 2005; Metz et al, 2007; Rose et al, 2007; Fiorentii et al, 2014). High levels of GPNMB are further associated with a decrease in overall lifetime of SCLC, GBM and ccRCC (Qin et al, 2014; Li et al, 2014; Kuanet al, 2006).
Keratin, type II cytoskeleton 80(KRT80) -KRT80 encodes keratin 80(KRT 80). KRT80 is found in almost all types of epithelial cells, associated with late tissue or cell differentiation. KRT80 containing intermediate filaments is located near the cell edge of desmoplasm and KRT80 is cytoplasmic only in cells entering terminal differentiation (Langbein et al, 2010).
The chromosome structure maintenance protein 4(SMC4) -SMC4 protein is a core component of a cohesin protein, plays a role in chromatin condensation, and is also associated with nucleolar segregation, DNA repair and maintenance of chromatin scaffolds (Cervantes et al, 2006).
Solute carrier family 1 (glutamate/neutral amino acid transporter) member 4(SLC1a4) -SLC1a4 is an amino acid transporter that mediates small neutral sodium amino acid dependent exchange (reviewed in Kanai et al, 2013). SLC1a4 was described as being expressed by significantly more esophageal adenocarcinoma than squamous cell carcinoma (Younes et al, 2000). SLC1a4 expression in prostate cancer cells showed an increase in response to androgen treatment (Wang et al, 2013 a).
Keratin 5(KRT5), keratin 6A (KRT6A), keratin 6B (KRT6B), keratin 6C (KRT6C) -KRT5, KRT6A, KRT6B and KRT6C are proteins of homologous keratin, and they are intermediate silk proteins. Keratins are widely used as marker proteins for tumor diagnosis because their expression patterns are related to the tissue of origin of malignant tumors (review (Karantza, 2011)). Normally, KRT6A and KRT6B appear to inhibit cell migration by sequestering, thereby inhibiting the activity of the migrating-philic Src kinase. Whether this mechanism is also applicable to cancer cells has not been studied yet (Rotty and count, 2012). KRT5/6 staining was proposed as one of several markers to distinguish poorly differentiated adenocarcinomas from squamous cell carcinomas in NSCLC (Zhao et al, 2014 b; Xu et al, 2014). KRT5/6 was also negative for pulmonary neuroendocrine tumors (Zhang et al, 2014).
Chemokine (C-C motif) ligand 18 (lung and initiation regulation) (CCL18) -this antibacterial gene is one of several cysteine-cysteine (CC) cytokine genes that cluster on chromosome 17. The cytokines encoded by this gene showed chemotactic activity for naive T cells, CD4+ and CD8+ T cells and unactivated lymphocytes, but no chemotactic activity for monocytes or granulocytes. Upregulation of CCL18 levels in tumor tissues and blood has been described in cancer, and CCL18 serum levels have been proposed as biomarkers for several tumor types. In various cases, it was shown that advanced tumor types are associated with poor prognosis (e.g., gastric cancer (Wu et al, 2013a), breast cancer (Chen et al, 2011; Narita et al, 2011), prostate cancer (Chen et al, 2014), bladder cancer (Urquidi et al, 2012)). Serum levels of CCL18 were increased in NSCLC patients compared to healthy controls. In addition, increased serum levels predict a decrease in survival time in adenocarcinoma patients (Plones et al, 2012). CCL18 is part of a 12-protein serum biomarker, and NSCLC can be identified (Ostroffet al., 2010).
Matrix metallopeptidase 12 (macrophage elastase) (MMP12) -MMP12, also known as Human Metallopeptidase (HME) or Macrophage Metallopeptidase (MME), is a zinc endopeptidase whose capacity to degrade elastin is recognized. In addition, it has a wide range of substrates, extending to other matrix proteins (e.g., collagen, fibronectin, laminin, proteoglycans) and non-matrix proteins (e.g., alpha-1-antitrypsin). In asthma, emphysema and Chronic Obstructive Pulmonary Disease (COPD), MMP12 may contribute to alveolar destruction and airway remodeling (Cataldo et al, 2003; Wallace et al, 2008). MMP12 is implicated in macrophage migration and, because it produces angiostatin from plasminogen, it helps to inhibit angiogenesis (Chakraborti et al, 2003; Chandler et al, 1996; Sangg, 1998). Like other metalloproteases, MMP12 is involved in physiological processes such as: embryogenesis, wound healing and menstrual cycle (Chakraborti et al, 2003; Labied et al, 2009), but also participate in the pathological process of tissue destruction. Although the data is based on a small number of patients in some cases, there is ample evidence in the literature that MMP12 is often overexpressed in cancer (Denys et al, 2004; Hagemann et al, 2001; Ma et al, 2009; Vazquez-oriz et al, 2005; Ye et al, 2008). However, data on the impact of MMP12 on clinical parameters and prognosis is controversial. Although it may be involved in matrix dissolution and thus metastasis, tumor growth may also be inhibited by the production of angiostatin, which negatively affects angiogenesis (Gorrin-Rivas et al, 2000; Gorrin Rivas et al, 1998; Kim et al, 2004). For lung cancer, the consequences of MMP12 expression are controversial. According to reports, MMP12 was overexpressed in epithelial cells in lung remodeling induced by inflammation. MMP12 upregulation may play a role in emphysema to lung cancer transition (Qu et al, 2009). Animal studies have shown that stroma or macrophages express MMP12 to inhibit the growth of lung tumors (Acuff et al, 2006; Houghton et al, 2006). However, it has also been reported that lung tumor MMP12 overexpression is associated with post-resection recurrence, metastatic disease, and a short recurrence-free survival (Cho et al, 2004; Hofmann et al, 2005).
Lysosomal associated membrane protein 3(LAMP3) -LAMP3 is a type I transmembrane protein found in lysosomal compartments, with a small cytoplasmic domain and a highly glycosylated luminal domain (Wilke et al, 2012). LAMP3 up-regulation was reported in several cancers, however, it has not been demonstrated that tumor cells express LAMP3 themselves. LAMP3(+) DCs are particularly detected in aggressive tumor margin clusters containing proliferating T lymphocytes and are thus proposed to reflect local anti-tumor immune responses, for example, for renal cell carcinoma (middlel et al, 2010), esophageal squamous cell carcinoma (Liu et al, 2010), colorectal cancer (Yuan et al, 2008; Sandel et al, 2005) and melanoma (Ladanyi et al, 2007). Meta-analysis of the transcript data indicated that low lung cancer LAMP3 expression levels may be associated with a shorter overall survival (Lindskog et al, 2014).
The protein encoded by the centromeric protein N (cennp) -cennp gene forms part of a nucleosome-associated complex and is important for kinetochore assembly. Cennp recognizes one centromere-specific histone variant (CENP- ｃA), and thus requires the determination of the recruitment site for many other centromere proteins (Carroll et al, 2009). Depletion of the cennp and the Nucleosome Associated Complex (NAC) does not impair bipolar spindle formation but leads to a defect in chromosome plate assembly (McClelland et al, 2007). Cennp is recruited to DNA double strand breaks along with other NAC proteins, and therefore this complex is proposed to play a role in DNA repair (Zeitlin et al, 2009).
Procollagen lysine, 2-oxoglutarate 5-dioxygenase 2(PLOD2) -the protein encoded by this gene is a membrane-bound homodimer enzyme localized to the retention vacuole of the rough endoplasmic reticulum. The coding region mutation of the gene is associated with the bruck syndrome. PLOD2 was described as being up-regulated in colorectal cancer (Nicastri et al, 2014), multiple myeloma (Slany et al, 2014) and cervical cancer (Rajkumar et al, 2011) and correlated with the formation of bone metastases (Blanco et al, 2012). Studies have shown that elevated PLOD2 expression is associated with poor prognosis in glioblastoma (Dong et al, 2005) as well as breast cancer (Gilkes et al, 2013) and hepatocellular carcinoma, where it is also associated with increased tumor size and the formation of intrahepatic metastases (Noda et al, 2012).
Matrix metalloproteinase 1(MMP1) -MMP1 are a member of the Matrix Metalloproteinase (MMP) family. Generally, MMPs play an important role in the regulation of vascular function, remodeling and angiogenesis. Through degradation of the ECM and other extracellular molecules, they promote migration and invasion of endothelial cells and vascular smooth muscle cells and affect vascular cell proliferation and apoptosis (Chen et al, 2013). MMP1 overexpression has been described in several cancer types and is associated with angiogenesis, invasion, and poor prognosis. For example, elevated MMP1 levels have been described as an independent factor in colon cancer survival (Langenskiold et al, 2013), with MMP1 expression in tumors and stroma being correlated with tumor progression and poor prognosis in breast cancer (Bostrom et al, 2011). MMP1 levels have been shown to be elevated in plasma and tumor tissues of lung cancer patients and to be associated with later stages and decreased survival (Li et al, 2010 b). A meta-analysis demonstrated that the MMP1-16071G/2G polymorphism was associated with an increased risk of developing lung cancer (Xiao et al, 2012).
Keratin 10(KRT10), keratin 12(KRT12), keratin 13(KRT13), keratin 14(KRT14), keratin 15(KRT15), keratin 16(KRT16), keratin 17(KRT17), keratin 19(KRT19) -homologous keratin KRT10, KRT12, KRT13, KRT14, KRT15, KRT16, KRT17, and KRT19 are intermediate silk proteins. Some keratins are associated with stem cell characteristics, for example KRT14 is considered a cancer stem cell marker (Hatina and Schulz, 2012; Schalken and van, 2003). KRT15 was used as a marker to identify and localize epidermal stem cells (Adhikary et al, 2013; Troy et al, 2011), and KRT17 was expressed in stem cells of the hair's basal lamina protruberans (Bragulla and Homberger, 2009). The expression patterns of different keratins were analyzed for different cancer types and reported both up and down. For example, high levels of KRT17 correlate with poor prognosis (Wang et al, 2013 b; Escorbar-Hoyos et al, 2014) and later stage (Kim et al, 2012). For KRT13, most studies indicate down-regulation in cancerous tissues (Hourihan et al, 2003; Ida-Yonemochi et al, 2012), the expression of KRT13 seems to be replaced by the expression of KRT17 during squamous cell transformation (Mikami et al, 2011). Both up-and down-regulation in cancer was confirmed by different studies for KRT10 and KRT 15. KRT19 is often overexpressed in many cancer types and is associated with metastasis and poor survival (Zong et al, 2012; Leeet al, 2012). KRT12 is expressed in corneal epithelial cells. The cornea shows down-regulation of keratin 12 (considered as a differentiation marker) (Zhang et al, 2010 b).
Mucin 16, cell surface (MUC16) -MUC16, is the largest of several membrane-bound mucins. MUC16 is a single transmembrane protein with a highly glycosylated extracellular domain. MUC16 is a tumor-associated antigen that lyses from the surface of ovarian cancer cells, shed into the blood and serves as a mature biomarker for monitoring the growth of ovarian cancer (Bafna et al, 2010). Increased expression levels of MUC16 have been demonstrated in lung squamous cell carcinoma (Wang et al, 2014). Furthermore, high MUC16 serum levels are associated with shortened survival in NSCLC patients (Yu et al, 2013; Cedres et al, 2011). In combination with other biomarkers, MUC16 may be part of a lung cancer subtype gene expression marker (Li et al, 2012).
Integrin α -2(CD49B, α 2 subunit of the VLA-2 receptor) (ITGA2) -ITGA2 encodes the α subunit of the transmembrane receptor for collagen and related proteins. A small number of studies report that ITGA2 is deregulated in cancer, which contains evidence of elevated and decreased levels: in pancreatic ductal adenocarcinoma, ITGA2 is hypomethylated and overexpressed, with elevated expression associated with poor prognosis (nos et al, 2014). In contrast, ITGA2 down-regulation in prostate cancer has been demonstrated (Shaikhibrahim et al, 2011). In breast and prostate cancer, decreased ITGA2 expression is associated with metastasis formation and poor survival (Ramirez et al, 2011).
Olfactin-like 2B (OLFML2B) -OLFML2B belongs to the olfactin protein family, which is an extracellular glycoprotein mainly involved in chemosensory cilia differentiation, early neurogenesis, neural tube dorsalization, neuromuscular signaling, synaptic vesicle exocytosis and the pathogenesis of glaucoma. OLFM2B transcript was detectable in different tissues of mice, including lung, stomach and prostate, but not in liver (Furutani et al, 2005). The OLFML2B gene maps to chromosome 1q23.3, which has been shown in association studies to be a susceptibility gene site for schizophrenia (Puri et al, 2007).
The thirty-four peptide repeat domain 13(TTC13) -TTC13 belongs to the thirty-four peptide repeat (TPR) domain protein family. The TPR domain appears to be important for chaperone function, cell cycle, transcription and protein transport, and proteins containing the TPR motif are often associated with polyprotein complexes (Blatch and Lassle, 1999). The TCC13 gene maps to chromosome 1q 42.2. Chromosome 1q42.2-43 was described in a related analytical study as a putative susceptibility gene locus for prostate cancer (Berthon et al, 1998), but this could not be confirmed in further studies for a larger patient population (Singh, 2000; Gibbs et al, 1999).
Cytokinesis factor 2(DOCK2) -the protein encoded by the DOCK2 gene belongs to the CDM protein family. DOCK2 is known to be an important factor in lymphocyte migration and chemotaxis. Exome and whole genome sequencing studies confirmed the presence of DOCK2 intragenic mutations in colorectal, esophageal adenocarcinoma, and pancreatic intraductal papillary mucinous tumors (Yu et al, 2014; Dulaket al, 2013; Furukawa et al, 2011). Furthermore, DOCK2 was shown to be differentially expressed in the pediatric astrocytoma samples and therefore may represent a relevant therapeutic target for this disease (Zhao et al, 2014 a).
Poliovirus receptor-associated 1 (herpes virus entry mediator C) (PVRL1) -PVRL1 encodes an adhesion protein that plays a role in epithelial and endothelial cell adhesion junctions and tight junctions of tissues. The PVRL1 gene maps to chromosome 11q23, a region that is found to be amplified in adenoid cystic carcinoma (Zhang et al, 2013). Due to the important function of cell adhesion, PVRL1 is involved in the regulation of cell invasion and migration properties and epithelial-mesenchymal transition, which are important processes of tumorigenesis. PVRL1 was identified as part of the marker profile of cervical cancer squamous cell carcinoma subtypes (imodomes et al, 2010). PVRL1 expression was found to increase in thyroid tumors and further in papillary thyroid carcinomas relative to normal thyroid tissue (Jensen et al, 2010). Expression of PVRL1/2 correlates with a better prognosis of acute myeloid leukemia (Graf et al, 2005).
FK506 binding protein 10, 65kDa (FKBP10) -FK 506-binding protein 10(FKBP10) belongs to the FKBP-type peptidyl prolyl cis/trans isomerase family. It is located in the endoplasmic reticulum and acts as a chaperone (Ishikawa et al, 2008; Patterson et al, 2000). It is highly expressed in lung development and can be re-initiated in a coordinated manner by extracellular matrix proteins following lung injury (Patterson et al, 2005).
ATP-binding cassette, subfamily C (CFTR/MRP), member 1(ABCC1) -the protein encoded by the ABCC1 gene is a member of the ATP-binding cassette (ABC) transporter superfamily. ABC proteins transport a variety of molecules across intracellular and extracellular membranes. ABCC1 plays an important role as a drug efflux pump in normal and tumor cells (Chen and Tiwari, 2011). Several studies describe the overexpression of ABCC1 in different tumor types, and in many cases ABCC1 expression levels were found to correlate with tumor staging, metastasis and poor prognosis (e.g., in breast, prostate and lung cancers) (Deeley et al, 2006). One study on chinese patients determined that SNPs of the ABCC1 gene increased susceptibility to NSCLC (Yin et al, 2011). Another study reported a correlation between the ABCC1 SNP and progression-free survival of NSCLC patients (lamb et al, 2014).
Arachidonic acid 15-lipoxygenase type B (ALOX15B) -ALOX15B encodes a member of the lipoxygenase family of structurally related non-heme iron dioxygenases involved in fatty acid hydroperoxide production. The role of ALOX15B (more commonly referred to as 15LOX-2) and its enzymatic product 15-S-hydroxy acid (15S-HETE) in tumor development has been most extensively studied in prostate cancer. Several studies have demonstrated that the expression level of ALOX15B and the production level of 15S-HETE are both reduced in prostate cancer compared to normal tissues or cell lines (Hu et al, 2013; shapecell et al, 2001). In normal lung, ALOX15B expression is restricted to type II lung cells. Expression is described as an inverse correlation between increased NSCLC, ALOX15B levels and tumor grade and tumor cell proliferation index (Gonzalez et al, 2004).
Sphingomyelin phosphodiesterase, acid-like 3B (SMPDL3B) -SMPDL3B, is a sphingomyelin phosphodiesterase expressed in podocytes and whose expression is associated with diabetic nephropathy and focal segmental glomerulosclerosis. Decreased expression of SMPDL3B in kidney disease is associated with cytoskeletal remodeling and apoptosis (Merscher and Fornoni, 2014). The SMPDL3B gene maps to chromosome 1p 35.3.
Glutamine-fructose-6-phosphate transaminase 2(GFPT2) -GFPT2 is involved in neurite outgrowth, early neuronal cell development, neuropeptide signaling/synthesis and neuroreceptors (Tondreau et al, 2008). Genetic variation of GFPT2 was associated with type II diabetes and diabetic nephropathy (Zhang et al, 2004). Furthermore, the relevance of GFPT2 SNPs suggests that genes involved in the modulation of the oxidative pathway may be the major cause of diabetic chronic renal insufficiency (Prasad et al, 2010). DNA methylation of the GFPT2 gene was verified in major Acute Lymphoblastic Leukemia (ALL) samples. Patients with multiple CpG island methylation have poor overall survival (Kuang et al, 2008). GFPT2 plays a role in glutamine metabolism, with higher expression observed in mesenchymal cell lines. Glutamine metabolism may play an important role in tumor progression and an inhibitor of a cellular metabolic pathway may be an epigenetic therapy (Simpson et al, 2012).
DEAD (Asp-Glu-Ala-Asp) frame helicase 5(DDX5) -DDX5(P68) is an ATP-dependent RNA helicase that plays a role in splicing, rRNA processing and ribosome biogenesis, miRNA processing, and transcriptional regulation. DDX5 is a transcriptional co-promoter of a variety of factors that play a role in the development of cancer, such as androgen receptor, p53, and Runx 2. DDX5 has been shown to be overexpressed in many different cancer types, such as colorectal, breast, prostate, glioma, hepatocellular, and leukemia (Dai et al, 2014; Fuller-Pace, 2013).
Enolase 1, (α) (ENO1) -ENO1 gene encodes enolase α (ENOA), one of three enolase proteins, the others being enolase- β and enolase- γ, respectively. ENO1/ENOA overexpression has been demonstrated in many cancer types (Capelloet al, 2011). ENOA is a metalloenzyme which plays a role in glycolysis for the synthesis of phosphoenolpyruvate.
Increased levels of ENOA are associated with poor survival in NSCLC patients (Chang et al, 2006). Likewise, another study demonstrated that ENO1 is up-regulated in a group of lung adenocarcinoma patients with poor prognosis (Pernemalm et al, 2013). ENOA has been demonstrated to be a tumor-associated antigen, and anti-ENOA antibodies as well as specific ENOA T cells have been detected in pancreatic cancer patients (cappella et al, 2009). ENOA autoantibodies have also been detected in NSCLC patients, with ENOA expression having been demonstrated to increase in NSCLC tissues (He et al, 2007; Li et al, 2006).
The killer lectin-like receptor subfamily D, member 1(KLRD1) -KLRD1, more commonly referred to as CD94, is associated with the NKG2 molecule to form heterodimers that are expressed on Natural Killer (NK) cells and Cytotoxic T Lymphocytes (CTLs). Inhibitory receptor KLRD1(CD 94): NKG2A was shown to be overexpressed in tumor-infiltrating lymphocytes, such as renal cell carcinoma and cervical cancer, which may lead to an impaired anti-tumor immune response (Schlepten et al, 2003; Sheu et al, 2005). Likewise, HLA-E-KLRD 1(CD 94): overexpression of NKG2A ligand on tumor cells may also contribute to tumor immune escape (boscard et al, 2012; Gooden et al, 2011).
The collagen, type XII, α 1(COL12a1) -COL12a1 gene encodes the α chain of collagen type XII, a family member of FACIT (fiber-related collagen with discontinuous triple helical regions) collagen. Type XII collagen, a homotrimer found in type I collagen, is thought to modify the interaction between collagen I fibers and the surrounding matrix (Oh et al, 1992). COL12a1 may be involved in basement membrane regulation, providing specific molecular bridges between fibrils and other matrix components (Thierry et al, 2004). COL12A1 is expressed in the heart, placenta, lung, skeletal muscle and pancreas (Dharmavaram et al, 1998) and in a variety of connective tissues including joint and epiphyseal cartilage (Gregory et al, 2001; Walchli et al, 1994; Watt et al, 1992). COL12a1 was down-regulated in tumors of high microsatellite instability compared to the stable group of lower or no microsatellite instability (Ortega et al, 2010).
ATP-binding cassette, subfamily a (ABC1), member 13(ABCA13) -in humans, the ATP-binding cassette (ABC) family of transmembrane transporters has at least 48 genes and 7 gene subfamilies. The predicted ABCA13 protein consists of 5058 amino acid residues, and thus is the largest ABC protein described to date (Prades et al, 2002). Knight et al determined that ABCA13 protein is expressed in the hippocampus and cortex of mice and humans, both of which are associated with schizophrenia and bipolar disorder (Knight et al, 2009). The ABCA13 gene maps to chromosome 7p12.3, a region that contains both genetic diseases affecting the pancreas (Shwachman-Diamond syndrome) and sites involved in T cell tumor invasion and metastasis (INM7), and is therefore a targeted candidate for these diseases (Prades et al, 2002).
Cyclin B2(CCNB2) -CCNB2 is one of several cyclins associated with the major cell cycle regulating kinase CDK1(CDC 2). Cyclin protein levels are transcriptionally regulated during the cell cycle, providing varying levels of activity and specificity of CDK1, thereby controlling cell cycle progression. The expression of cyclin B2 is regulated by the tumor suppressor genes p53 and BRCA1, which act by inhibiting cyclin B2 transcription (Quaas et al, 2012; det al, 2011). CCNB2 upregulation is described in several tumor types, such as cervical cancer (Espinosa et al, 2013; Rajkumar et al, 2011), bladder cancer (Lu et al, 2010), colorectal cancer (Park et al, 2007), astrocytoma (Liu et al, 2013), and glioblastoma (Hodgson et al, 2009). The level of CCNB2 expression correlates with a poor prognosis of breast cancer and was determined as an independent prognostic marker for survival (Shubbar et al, 2013). CCNB2 is overexpressed in NSCLC (Hofmann et al, 2004), and is determined to be an independent predictor of poor prognosis in patients with lung adenocarcinoma, but not a predictor of squamous cell carcinoma (Takashima et al, 2014).
MutS homolog 6(MSH6) -MSH6 encodes a member of the DNA mismatch repair MutS family. MSH proteins, including MSH6, recognize errors in genomic sequence during replication to prevent replication of damaged strands and repair single strand breaks (Conde-Perezprina et al, 2012). Among several cancers, MSH6 mutations and erroneous DNA mismatch repair mechanisms (MMR) are described (e.g., colorectal cancer (Sameer et al, 2014; Vilar and Gruber, 2010; silvera et al, 2009; kastronos and Syngal, 2007; Davidson, 2007), pancreatic cancer (Solomon et al, 2012), ovarian cancer (Xiao et al, 2014), breast cancer (Mahdi et al, 2013)).
PRP3 pre-mRNA processing factor 3 homolog (saccharomyces cerevisiae) (PRPF3) -PRPF3 encodes pre-mRNA processing factor 3. PRPF3 mediates recruitment of nuclear RNA decay mechanisms to spliceosomes (Nag and Steitz, 2012). PRPF3 is upregulated in hepatocellular carcinoma by the fetal/cancer-specific splice variant of the transcription factor HNF4 α (Niehof and Borlak, 2008).
Lysophosphatidylcholine (LPC) is catalyzed by lysoegg phosphatidyltransferase 1(LPCAT1) -LPCAT1 to convert to phosphatidylcholine. In addition, LPCAT1 is capable of converting lysopaf (alkylated LPC) into Platelet Activating Factor (PAF). LPCAT1 overexpression has been described in colorectal cancer (Mansilella et al, 2009), hepatocellular carcinoma (Morita et al, 2013), breast cancer (Abdelzaher and Mostafa, 2015), prostate cancer (Xu et al, 2013; Grupp et al, 2013; Zhouet al, 2012), and lung cancer (Wu et al, 2013 b). LPCAT1 overexpression promotes cell proliferation, migration, and invasion in vitro (Morita et al, 2013).
A downstream neighbor of a SON (DONSON) DONSON encodes the downstream neighbor of the SON (DONSON). DONSON is a centrosomal protein whose levels are regulated during the cell cycle, peaking at S phase. DONSON is required for the formation of an appropriate mitotic spindle and appears to play a role in the DNA damage response (Fuchs et al, 2010). There is currently no cancer-related literature.
Inhibition of benzimidazole budding disambiguates homolog 1 β (yeast) (BUB1B) -BUBlB encodes serine/threonine kinase B, a serine/threonine-protein kinase, at the step of BUB1 mitosis. It functions as a mitotic regulator, ensuring accurate chromosome segregation by its action in the mitotic step and establishing proper microtubule centromere adhesion. BUB1B was reported to be both up-and down-regulated in expression in various tumors. Overall, more literature reports that BUB1B is overexpressed in cancers, associations of tumor progression and poor prognosis are also described, such as nasopharyngeal carcinoma (Huang et al, 2012a), tonsil carcinoma (Hannisdal et al, 2010), breast cancer (Maciejczyk et al, 2013), ovarian epithelial carcinoma (Lee et al, 2009), and pancreatic adenocarcinoma (Gladhaug et al, 2010). Also, a decrease in BUB1B protein was associated with longer survival of prostate cancer (Cirak et al, 2013).
Oligomeric Golgi complex 4(COG4) -COG4 are components of oligomeric protein complexes, and are involved in the structure and function of the Golgi apparatus. Interaction studies have shown that COG4, as a core component of this complex, plays an important role in the assembly/function of the complex (Loh and Hong, 2004). COG subunits COG4, 6 and 8 can interact with defined golgi SNARE and participate in the definition of vesicle sorting specificity within the golgi (Willett et al, 2013). In addition, the COG complex has been shown to regulate maintenance of the golgi glycosylation machinery (Pokrovskaya et al, 2011).
Proteasome (precursor, megalin) 26S subunit, a non-atpase, 14(PSMD14) -PSMD14 is the 26S proteasome, a component of a polyprotein complex that targets destruction of proteins through ubiquitin pathway degradation. The 19S complex in vivo PSMD14 protein complex (19S cap; PA700) is responsible for matrix deubiquitination during proteasome degradation (Spataro et al, 1997). Aberrant expression and dysfunction of proteasome subunits are involved in malignant transformation and cellular resistance to various cytotoxic drugs. Overexpression of PSMD14 in mammalian cells affects cell proliferation and response to cytotoxic drugs such as vinblastine, cisplatin and doxorubicin (Spataro et al, 2002). Down-regulation of siRNA-transfected PSMD14 had a considerable effect on cell viability, causing cell arrest at stages G0-G1, ultimately leading to senescence (Byrne et al, 2010).
RAD54 homolog B (saccharomyces cerevisiae) (RAD54B) -DNA repair and recombination protein RAD54B is a protein encoded by the RAD54B gene in humans. RAD54 binds to double-stranded DNA and exhibits atpase activity in the presence of DNA. The human RAD54B protein is a paralogue of the RAD54 protein and plays an important role in homologous recombination. Homologous Recombination (HR) is important for the accurate repair of DNA Double Strand Breaks (DSB) (Sarai et al, 2008). It is known that the knock-down of the somatic mutation gene RAD54B in cancer leads to Chromosomal Instability (CIN) in mammalian cells (McManus et al, 2009). Elevated gene expression of RAD54B is associated with a shorter time to disease progression and poor OS in GBM patients (Grunda et al, 2010).
Frizzled family receptor 1(FZD1), frizzled family receptor 2(FZD2), frizzled family receptor 7(FZD7) -genes FZD2, FZD1, and FZD7 are all from the "frizzled" gene family; members of this gene family encode 7-transmembrane domain proteins, which are receptors for Wnt signaling proteins. The expression of the FZD2 gene appears to be developmentally regulated, with high levels of expression in fetal kidney, lung, and adult colon and ovary (Sagara et al, 1998; Zhao et al, 1995). The FZD1 protein contains a signal peptide, an N-terminal extracellular region cysteine-rich domain, 7 transmembrane domains, and a C-terminal PDZ domain binding motif. FZD1 transcript is expressed in various tissues including lung, heart, kidney, pancreas, prostate and ovary (Sagara et al, 1998). As a result, expression of frizzled 1 and 2 receptors was found to be upregulated in breast cancer (Milovanovic et al, 2004). The FZD7 protein contains an N-terminal signal sequence, 10 cysteine residues (typical cysteine-rich extracellular domain of Fz family members), 7 transmembrane domains, and an intracellular C-terminal tail with a PDZ domain binding motif. In poorly differentiated human esophageal cancers, FZD7 gene expression may down-regulate APC function, enhancing β -catenin-mediated signaling (Sagara et al, 1998; Tanaka et al, 1998).
The wingless MMTV integration site family, member 5A (WNT5A) -generally, WNT5A modulates a variety of cellular functions such as proliferation, differentiation, migration, adhesion, and polarity (Kikuchi et al, 2012). It is expressed in undifferentiated human embryonic stem cells (Katoh, 2008). WNT5A was classified as a non-transforming WNT family member, and its role in tumorigenesis remains unclear. It shows tumor inhibitory activity in certain cancers (thyroid, brain, breast and colorectal), but is abnormally upregulated in lung, stomach and prostate cancers (Li et al, 2010 a). Oncogenic WNT5A initiates classical WNT signaling for self-renewal in cancer stem cells and non-classical WNT signaling for invasion and metastasis at the tumor stromal interface (Kaohand Kaoh, 2007). Expression of WNT5A was described in various tumor entities. For example, aberrant protein expression of Wnt5a, which promotes tumor invasiveness, was observed in 28% of prostate cancer cases (Yamamoto et al, 2010). Furthermore, WNT5A overexpression has been described as being associated with poor prognosis and/or increased tumor grade in ovarian cancer (Badiglian et al, 2009), melanoma (Da Forno et al, 2008; Weerarata et al, 2002), GBM (Yu et al, 2007), lung cancer (Huang et al, 2005) and pancreatic cancer (Ripka et al, 2007). In HCC, it appears that the canonical Wnt signaling pathway contributes to tumor initiation and atypical signaling to tumor progression (Yuzugullu et al, 2009).
Fibroblast activation protein alpha (FAP) -Fibroblast Activation Protein (FAP) is a type II integral membrane glycoprotein belonging to the serine protease family. The putative serine protease activity of FAP α and its in vivo induction pattern may suggest that this molecule plays a role in controlling fibroblast growth or epithelial-mesenchymal interaction during development, tissue repair and epithelial carcinogenesis (Scanlan et al, 1994). Most normal adult tissues and benign epithelial tumors show little or no detectable expression of FAP. However, FAP expression is detected in more than 90% of the stroma, wound-healed fibroblasts, soft tissue sarcomas and some fetal mesenchymal cells of malignant breast, colorectal, lung, skin and pancreatic tumors. FAP plays a potential role in cancer cell growth and metastasis through the process of cell adhesion and migration, as well as rapid degradation of ECM components. Thus, it is present in tumor cells that invade the ECM and endothelial cells involved in angiogenesis, but is not expressed in the same type of inactive cells (Dolznig et al, 2005; Kennedy et al, 2009; Rettia et al, 1993; Rettig et al, 1994; Scanlan et al, 1994; Zhang et al, 2010 a).
Cyclin B1(CCNB1) -CCNB1 encodes cyclin B1, one of several mitotic cyclins that are associated with CDK1/CDC2 to promote mitotic progression. CNB1 overexpression is described in many cancer types and is associated with poor tumor progression and prognosis, such as colorectal cancer (Li et al, 2003), breast cancer (Aaltonen et al, 2009; Agarwal et al, 2009), NSCLC (Cooper et al, 2009), and esophageal squamous cell carcinoma (Huang et al, 2014). Furthermore, in gastric cancer, CCNB1 expression is associated with regional lymph node metastasis and poor clinical prognosis (begsami et al, 2010; Fujita et al, 2012). Antibodies to CCNB1 were detected in lung or prostate cancer patients and were proposed as biomarkers for early detection of lung cancer (Egloff et al, 2005; Zhang et al, 2003).
ATP enzyme, Ca++Transport, myocardium, rapid contraction 1(ATP2A1), ATPase, Ca++Transport, myocardium, rapid contraction 2(ATP2a2) -both genes (ATP2a1 and ATP2a2) encode SERCA Ca (2+) -atpase. Sarcoplasmic Reticulum (SR)1/ER calcium ATPase (SERCA) is a calcium pump that binds ATP hydrolysis and calcium transporters throughout the SR/ER membrane (MacLennan et al, 1997). SERCA is encoded by three homologous genes: SERCA1(ATP2a1), SERCA2(ATP2a2) and SERCA3(Wu et al, 1995). Some evidence has emerged suggesting that SERCA may also contribute to apoptosis, differentiation and cell proliferation The reproductive process has a direct impact (Chami et al, 2000; Ma et al, 1999; Sakuntabhai et al, 1999). Mutations in ATP2a1 encoding SERCA1 result in some autosomal recessive forms of brodif's disease characterized by an aggravated impairment of muscle relaxation during exercise (odimatt et al, 1996). ATP2a2 is an atpase associated with follicular keratosis, a rare autosomal dominant skin disorder characterized by abnormal keratinization and acantholysis (Huo et al, 2010). Germline changes in ATP2a2 may lead to predisposition to lung and colon cancer, and impairment of the ATP2a2 gene may be involved in canceration (Korosecet al, 2006). In small cell lung cancer (H1339) and adenocarcinoma lung cancer (HCC) cell lines, ER Ca2+ -levels were reduced compared to normal human bronchial epithelium. A decrease in Ca2+ -content is associated with a decrease in SERCA 2 expression pumping calcium into the ER (Bergneret al, 2009). ATP2a2 may be a potential prognostic marker for colorectal cancer (CRC) patients. It was detected in Circulating Tumor Cells (CTCs) and postoperative recurrence was significantly associated with gene overexpression (Huang et al, 2012 b).
Fibronectin 1(FN1) -FN1 encodes fibronectin, a glycoprotein, which exists in plasma in the form of soluble dimers, on the cell surface in the form of dimers or multimers, and in the extracellular matrix. It was reported that in most tumors, FN1 was predominantly expressed by cancer-associated fibroblasts (CAF) and endothelial cells, but not by tumor cells (Berndt et al, 2010). Elevated levels of FN1 have been reported in some cancer types and have been associated with poor prognosis or cancer progression, such as gallbladder cancer (Cao et al, 2015), prostate cancer (von et al, 2013), and renal cell carcinoma (Steffens et al, 2012; waales et al, 2010). FN1 has also been implicated in the stimulation of lung cancer pathogenesis, including cell growth, chemoresistance, and inhibition of apoptosis (reviewed in Ritzenthaler et al, 2008).
Insulin-like growth factor 2mRNA binding protein 3(IGF2BP3) -IGF2BP3 is a member of the insulin-like growth factor-IImRNA binding protein family, involved in mRNA localization, translation, and translational control. The protein contains several KH (potassium homologous) domains that play an important role in RNA binding and are known to be involved in RNA synthesis and metabolism. Expression occurs primarily during embryonic development and is described in some tumors. Thus, IGF2BP3 is considered to be an oncofetal protein (Liao et al, 2005). IGF2BP3 may promote tumor cell proliferation by enhancing IGF-II protein synthesis and inducing cell adhesion and invasion by stabilizing CD44mRNA (Findeis-Hosey and Xu, 2012). Furthermore, IGF2BP3 expression has been studied in many human tumors, with increasing evidence that it mediates migration, infiltration, cell survival and tumor metastasis (Jenget 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 also be involved in angiogenesis (Suvasini et al, 2011; Chen et al, 2012). In lung adenocarcinomas, a higher frequency of IGF2BP3 expression can be detected in moderate or poorly differentiated adenocarcinomas, which may be associated with aggressive biological behavior (Findeis-Hosey et al, 2010; Beljan et al, 2012; Findeis-Hosey and Xu, 2012).
Laminin, γ 2(LAMC2) -laminin (a family of extracellular matrix glycoproteins) is the major non-collagenous component of basement membrane. They are involved in a variety of biological processes including cell adhesion, differentiation, migration, signaling, neurite outgrowth and metastasis. The LAMC2 gene encodes laminin-5 γ 2 chain (part of laminin-5), which is one of the major components of the basal membrane region. LAMC2 is often up-regulated by promoter demethylation in gastric cancer (Kwon et al, 2011). LAMC2 was found to be overexpressed in areas of anisotropic and ischemic melanoma (Lugassy et al, 2009). LAMC2 is a biomarker of bladder cancer metastasis, the expression level of which correlates with tumor grade (Smith et al, 2009 b). The genes LAMB3 and LAMC2 were co-expressed in 21 of 32 NSCLC cell lines (66%), but only in 1 of 13 SCLC cell lines (8%). Coexpression of the LAMB3 and LAMC2 genes was also observed in all 4 primary NSCLC cells examined, but not in the corresponding non-cancerous lung cells (Manda et al, 2000).
Brain endothelial cell adhesion molecule (CERCAM) -CERCAM is localized to the surface of endothelial cells (Starzyk et al, 2000), mapped to chromosome 9q34.11, a candidate region on 9q, identified as associated with familial idiopathic scoliosis (Miller et al, 2012). The CEECAM1 gene is widely transcribed in the nervous system and in several secretory tissues, such as salivary glands, pancreas, liver and placenta (Schegg et al, 2009). The CERCAM protein is structurally similar to the ColGalT enzymes GLT25D1 and GLT25D 2. However, although its function is still unknown, it appears to be functionally distinct from the related GLT25D1 protein, which does not have the function of glycosyltransferases such as the GLT25D1 and GLT25D2 proteins (Perrin-Tricaud et al, 2011).
Matrix remodeling-associated protein 5(MXRA5) -MXRA5, also known as adlican, encodes an adhesion proteoglycan, belonging to a group of genes involved in extracellular matrix remodeling and cell-cell adhesion (Rodningen et al, 2008). Although the role of MXRA5 in cancer is unknown, MXRA5 somatic mutations have been identified in tumors from various tissues (e.g., skin, brain, lung, and ovary). RT-PCR on adlican (MXRA5) confirmed microarray results of overexpression in colon cancer compared to normal colon tissue (13 colorectal tumors and 13 normal tissues) (Zou et al, 2002). In a recent study, matrix remodeling-associated protein 5 was the second most common mutant gene in NSCLC (first major TP53) (Xionget al, 2012).
ADAM Metallopeptidase Domain 8(ADAM8) -ADAM8 is a member of the ADAM (disintegrin and metalloprotease domain) family. Many ADAM species, including ADAM8, are expressed in human malignancies and they are involved in the regulation of growth factor activity and integration function, leading to promotion of cell growth and invasion (Mochizuki and Okada, 2007). The expression of ADAM8 was positively correlated with EGFR. Both are expressed predominantly in the cytoplasm and on the cell membrane (Wu et al, 2008). ADAM8 was abundantly expressed in the vast majority of lung cancers tested. Exogenous expression of ADAM8 increased the migratory activity of mammalian cells, suggesting that ADAM8 may play a significant role in the development of lung cancer (Ishikawa et al, 2004). ADAM8 is associated with poor prognosis in lung cancer (Hernandez et al, 2010). ADAM8 overexpression was associated with a short patient survival, a good predictor of remote transfer of RCC (Roemer et al, 2004 b; Roemer et al, 2004 a). Furthermore, the expression level and protease activity of ADAM8 correlated with glioma cell invasion activity, suggesting that ADAM8 may play a significant role in brain cancer tumor invasion (willeboer et al, 2006).
The melanoma antigen family F, 1(MAGEF1) -MAGE (melanoma associated antigen) superfamily of most known members are expressed in tumor, testis and fetal tissues, which is described as cancer/testis expression pattern (MAGE subgroup I). Peptides of MAGE subgroup I have been successfully used for peptide and DC vaccination (Nestle et al, 1998; Marchand et al, 1999; Marchand et al, 1995; Thurner et al, 1999). In contrast, some MAGE genes (MAGE subgroup II), such as MAGEF1, are ubiquitously expressed in all adult and fetal tissues tested, but also in many tumor types, including ovarian, breast, cervical, melanoma and leukemia (Nestle et al, 1998; Marchand et al, 1999; Marchand et al, 1995; Thurner et al, 1999). Nevertheless, overexpression of MAGEF1 could be detected in NSCLC (Tsai et al, 2007) and the 79% taiwan colorectal cancer patient cohort (Chung et al, 2010).
Small ribonucleoprotein 200kDa (U5) (SNRNP200) -pre-mRNA splicing is catalyzed by the spliceosome, which is a complex of specialized RNA and protein subunits that eliminates the intron from the pre-mRNA segment that is transcribed. Spliceosomes consist of the small nuclear RNA protein (snRNP) U1, U2, U4, U5 and U6 together with approximately 80 conserved proteins. SNRNP200 is a gene required for U4/U6 duplex helication, a gene essential for catalytic activation of spliceosomes (Maeder et al, 2009). SNRNP200 expression was detected in heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas (Zhao et al, 2009 a). SNRNP200 mutations have recently been found to be associated with autosomal dominant retinitis pigmentosa (adrP) (Benagllio et al, 2011; Liu et al, 2012).
TPX2, microtubule-associated, homolog (xenopus) (TPX2) -TPX2 is a spindle assembly factor. It is required for normal assembly of microtubules during mitotic spindle and apoptosis. TPX2 is required for chromatin and/or centromere-associated microtubule nucleation (Bird and Hyman, 2008; Moss et al, 2009). Newly synthesized TPX2 is required for nearly all Aurora a activation and for the full synthesis and phosphorylation of p53 in vivo during oocyte maturation (Pascreau et al, 2009). TPX2 is a cell cycle-associated protein that is overexpressed in a variety of tumor types, such as meningiomas (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 tumors (Warner et al, 2009), ovarian cancer (ramakrishne et al, 2010), lung squamous cell carcinoma (Lin et al, 2006; Ma et al, 2006). It is frequently co-overexpressed with Aurora-a, forming a new functional unit with oncogenic properties (Asteritiet al, 2010). TPX2 expression is a prognostic indicator for lung cancer (Kadara et al, 2009).
The beta-induced transforming growth factor, 68kDa (TGFBI) -TGFBI, was first identified as a TGF-beta-inducible gene for human lung adenocarcinoma cell lines. It encodes secreted extracellular matrix proteins that are thought to act on cell adhesion and extracellular matrix components. Normally, TGFBI expression is found predominantly in fibroblasts, keratinocytes and muscle cells (Bae et al, 2002). TGFBI is overexpressed in several solid tumors, such as colon (Kitahara et al, 2001), pancreatic (Schneider et al, 2002) and renal (Ivanov et al, 2008). TGFBI is down-regulated in lung cancer (Zhao et al, 2004; Shao et al, 2006), reducing the metastatic potential of lung tumor cells, promoting apoptotic cell death when overexpressed (Zhao et al, 2006). In NSCLC samples, a strong correlation between elevated TGFBI expression and response to chemotherapy was observed (Irigoyen et al, 2010).
Cyclin-dependent kinase 4(CDK 4)/cyclin-dependent kinase 6(CDK6) -CDK4 are members of the serine/threonine protein kinase family. It is the catalytic subunit of the protein kinase complex and is important for the progression of the G1 phase of the cell cycle. The activity of this kinase is limited to the G1 to S phase transition phase during the cell cycle, with its expression being mainly controlled at the transcriptional level (Xiao et al, 2007). CDK4 and CDK6 enzymes and their modulators (e.g., cyclins) play a key role in embryonic development, homeostasis, and carcinogenesis (Graf et al, 2010). In lung cancer tissues, the expression level of CDK4 protein was significantly elevated (P < 0.001) relative to normal tissues. The overall survival time of patients with higher expression of CDK4 was significantly shorter than that of patients with low expression of CDK 4. Multifactorial analysis has shown that CDK4 expression levels are an independent prognostic indicator of survival in lung cancer patients (P < 0.001). In addition, inhibition of CDK4 expression also significantly increased the expression of the cell cycle regulator p21 gene (Wu et al, 2011). Ablation of CDK4 instead of CDK2 or CDK6 induced an immediate senescence response in lung cells expressing endogenous K-ras oncogenes. Such a response would not occur in the lungs expressing the single allele Cdk4 nor in other K-ras gene expressing tissues. Computer tomography detectable late stage tumor targeting Cdk4 alleles also induced senescence and prevented tumor progression (Puyol et al, 2010).
Pluripotent glycan (VCAN) -the VCAN gene is a member of the aggrecan/pluripotent glycan family. VCAN is known to be associated with several molecules in the extracellular matrix, including hyaluronic acid, tenascin, fibronectin, CD44 and L-selectin, fibrillin, integrin and desmin (Zheng et al, 2004). VCAN is expressed in a variety of tissues. It is highly expressed at an early stage of tissue development, and its expression is reduced after tissue maturation. Its expression is also elevated during wound repair and tumor growth (Ghosh et al, 2010). Human lung adenocarcinoma (a549) cells knocked-down VCAN by RNA interference significantly inhibited tumor growth in vivo, but not in vitro (Creighton et al, 2005). VCAN is a direct target of the p53 gene. High expression of VCAN is also found in the peritumoral stromal tissue of early prostate and breast cancers and is associated with aggressive tumor behavior (Yoon et al, 2002).
Ubiquitin conjugating enzyme E2S (UBE2S) -UBE2S is an APC cofactor that promotes mitotic withdrawal. Its consumption prolongs drug-induced mitotic arrest and inhibits mitotic slippage (Garnett et al, 2009). UBE2S is overexpressed in common human cancers. In esophageal cancer, UBE2S was significantly associated with the degree of tumor burden. Its positivity is associated with poor response and poor survival of neoadjuvant therapy (Chen et al, 2009). In the UBE2S promoter, early growth response-1 (EGR-1) and Serum Response Factor (SRF) binding sites were identified. Overexpression of these factors increases the expression of UBE2S required for cancer cell proliferation (Lim et al, 2008).
SET and MYND domain containing protein 3(SMYD3) -it has been reported that upregulation of SMYD3 (histone H3 lysine 4 specific methyltransferase) plays a key role in the proliferation of colorectal cancer (CRC) and hepatocellular carcinoma (HCC). Another study showed that SMYD3 expression was also elevated in the vast majority of breast cancer tissues. Like CRC and HCC, SMYD3 suppression of this gene by small interfering RNAs leads to inhibition of breast cancer cell growth, suggesting that increased expression of SMYD3 is also essential for breast cancer cell proliferation (Hamamoto et al, 2006). Knockdown of SMYD3 by RNA interference down-regulates the expression of c-Met and inhibits HGF-induced cell migration and invasion (Zou et al, 2009). SMYD3 plays a key role in HeLa cell proliferation and migration/invasion, and it may be a useful therapeutic target for human cervical cancer (Wang et al, 2008).
Dystonia protein (DST) -DST (BPAG1-e) encodes a member of the thrombolysin family of adhesion junction plaque proteins. BPAG1-E is expressed in epithelial tissue anchoring keratin-containing intermediate filaments to Hemidesmosomes (HDS). HD is a multi-protein adhesion complex that promotes the adhesion of epithelial substrates in stratified and complex epithelial cells. The regulation of its function is crucial in various biological processes, such as differentiation and migration of keratinocytes and tumor invasion during wound healing, during which cells detach from the stroma and acquire a motile phenotype (Litjens et al, 2006). Malignant melanoma is one of the most aggressive tumor types. BPAG1 was expressed in human melanoma cell lines (a375 and G361) and normal human melanocytes. The level of anti-BPAG 1 autoantibodies in the serum of melanoma patients was significantly increased (p < 0.01) compared to the serum of healthy volunteers. anti-BPAG 1 autoantibodies may be a promising marker for melanoma diagnosis (Shimbo et al, 2010). DST is associated with breast cancer invasion (Schuetz et al, 2006). The BPAG1 gene may be involved in proliferation, apoptosis, invasion and metastasis of nasopharyngeal carcinoma (NPC) (Fang et al, 2005).
Solute carrier family 34 (sodium phosphate), member 2(SLC34a2) -SLC34a2 is a pH-sensitive sodium-dependent phosphate transporter. The up-regulation of SLC34A2 gene expression in highly differentiated tumors may reflect the cell differentiation process during ovarian carcinogenesis, and can be used as a potential marker for diagnosis and prognosis of ovarian cancer (Shyian et al, 2011). RT-PCR demonstrated increased expression of SLC34a2 in papillary thyroid carcinomas (Kim et al, 2010 b). Gene expression of SLC34a2 was also significantly increased in breast cancer tissues compared to normal tissues (Chen et al, 2010).
Cytoadhesin C (tenascin) (TNC) -cytoadhesin C (TNC) is an extracellular matrix protein that is highly upregulated in processes closely related to increased migration activity, such as: embryo development (Bartsch et al, 1992), wound healing (Mackie et al, 1988) and tumor progression (Chiquet-Ehrismann, 1993; Chiquet-Ehrismann and Chiquet, 2003). In addition, TNC is abundantly expressed in tumor vessels with a high proliferation index, suggesting that TNC is involved in tumor angiogenesis (Kim et al, 2000). TNC overexpression has been further reported in the following cancers: colon cancer (De et al, 2013); adenoid cystic carcinoma, which is associated with the worst prognosis (Siu et al, 2012); nasopharyngeal fibroangioma, which may promote angiogenesis (Renkonen et al, 2012); advanced melanoma (Fukunaga-Kalabis et al, 2010); pancreatic cancer, which exerts proliferative, migratory and metastatic effects (Paron et al, 2011).
Endoplasmic reticulum calcium binding protein 1, EF-hand calcium binding domain (RCN1), endoplasmic reticulum calcium binding protein 3, EF-hand calcium binding domain (RCN3) -endoplasmic reticulum calcium binding protein 1 is a calcium binding protein located in the lumen of the ER. Immunohistochemical examination confirmed that RCN is widely distributed in various organs of fetuses and adults, mainly in endocrine and exocrine organs. RCN overexpression may play a role in tumorigenesis, tumor invasion and drug resistance (Fukuda et al, 2007). Endoplasmic reticulum calcium binding protein 1(RCN1) is a cell surface associated protein in Endothelial (EC) and prostate cancer (PCa) cell lines. RCN1 expression on the cell surface was upregulated by tumor necrosis factor α treatment of bone marrow endothelial cells (Cooper et al, 2008). RCN1 is upregulated in colorectal cancer (CRC), localized to cancer cells or stromal cells in the vicinity of cancer cells. It may be a new candidate CRC marker (Watanabe et al, 2008). RCN3 is a member of the family of various EF-chiral calcium binding proteins CREC (Cab 45/endoplasmic reticulum calcium binding protein/ERC 45/calcoelenterazine) that are located in the secretory pathway (Tsuji et al, 2006). In oligodendroglioma, RCN3 was shown to be a potentially important candidate gene. Although little is known about the function of RCN3 (Drucker et al, 2009).
Basic nucleoprotein 1(BNC1) -basic nucleoprotein is a zinc finger protein with a highly restricted tissue distribution (Tseng, 1998). To date, basic nucleoproteins have been detected predominantly in stratified squamous epithelial basal keratinocytes (skin, oral epithelium, esophagus, vagina and cornea) and testicular and ovarian gametocytes (Tseng and Green, 1994; Weiner and Green, 1998). There is now considerable evidence that basic nucleoproteins are cell-type specific transcription factors for the rRNA gene (rDNA). The zinc finger of the basic nucleoprotein interacts with three evolutionarily conserved sites within the rDNA promoter (Iuchi and Green, 1999; Tseng et al, 1999). Epigenetic regulation via CpG methylation plays an important role in tumorigenesis as well as in cancer therapeutic response. BNC1 is hypomethylated in radiation resistant H1299 human non-small cell lung cancer (NSCLC) cell line. Inhibition of BNC1 mRNA expression in H1299 cells also reduces the resistance of these cells to ionizing radiation (Kimet al, 2010 a). Aberrant DNA methylation of BNC1 was also detected in Chronic Lymphocytic Leukemia (CLL) samples (tongt al., 2010). In Renal Cell Carcinoma (RCC), methylation of BNC1 is associated with poor prognosis, regardless of tumor size, stage or grade (Morris et al, 2010).
Transformation, acid-containing coiled coil protein 3(TACC3) -TACC3, which are microtubules cross-linked in the centromere fibers, is present in a complex of CH-TOG (genes overexpressed in colon and liver tumors) and entactin. TACC3 is expressed in certain proliferative tissues, including testis, lung, spleen, bone marrow, thymus and peripheral blood leukocytes. TACC3 expression is altered in certain human tumor types. In cells, TACC3 is located in centrosomes and spindle microtubules, rather than in star microtubules (Hood and Royle, 2011). Expression of TACC3 was associated with P53 expression, and patients with high expression of TACC3 and P53 in tumors had significantly poorer prognosis than patients with low expression levels in both tumors as measured by immunostaining (P ═ 0.006). This suggests that an increase in TACC3 may contribute to NSCLC proliferation and tumor progression, while TACC3 expression is a strong prognostic indicator of clinical outcome of NSCLC (Jung et al, 2006). Tacc3 may be a negative regulator of the Notch signaling pathway (Bargo et al, 2010).
Hickory nut like 3 (drosophila) (PCNXL3) -hickory nut like protein 3(PCNXL3) is a multiple-pass membrane protein; it belongs to the pecan family. The PCNXL3 gene was mapped to the chromosome 11q12.1-q13 region. Three new human tumor-associated translocation breakpoints were located in the region of chromosome 11q13 between markers D11S4933 and D11S 546. Thus, PCNXL3 may be an 11q 13-associated disease gene (van et al, 2000).
The Drosha enzyme, ribonuclease type III (Drosha) -Drosha enzyme is a class 2 ribonuclease type III enzyme responsible for initiating the processing of micrornas (mirnas) that regulate a variety of other genes by interacting with the RNA-induced silencing complex (RISC) to induce the division of complementary messenger RNAs (mrnas) that are part of the RNAi pathway, from short RNA molecules that are naturally expressed by the cell. Microrna molecules are synthesized as long RNA primary transcripts (called pri-mirnas) that are cleaved by Drosha to generate a characteristic stem-loop structure (called pre-miRNA) that is about 70 base pairs long (Lee et al, 2003). Drosha exists as part of a protein complex called a microprocessing complex, which also contains the double-stranded RNA binding protein Pasha (also known as DGCR8) (Denli et al, 2004), which is essential for Drosha activity and is capable of binding the single-stranded fragment of the pri-miRNA required for proper processing (Han et al, 2006). The human Drosha enzyme was cloned in 2000, when it was designated as a nuclear dsRNA ribonuclease involved in the processing of ribosomal RNA precursors (Wu et al, 2000). The Drosha enzyme is the first human ribonuclease type III enzyme discovered and cloned. Two other human enzymes involved in miRNA processing and activity are Dicer enzyme and Argonaute protein. Both Drosha and Pasha are located in the nucleus where pri-miRNA is processed to pre-miRNA. The latter is then further processed by the ribonuclease Dicer enzyme into the cytoplasmic mature miRNA (Lee et al, 2003). Drosha enzymes and other miRNA processing enzymes may be important enzymes for cancer prognosis (Slack and Weidhaas, 2008).
Cell division cycle 6 homolog (saccharomyces cerevisiae) (CDC6) -CDC6 protein served as a regulator in the early steps of DNA replication. It is located in the nucleus during the G1 phase of the cell cycle, but begins to be transported into the cytoplasm during the S phase. In addition, CDC6 regulates replication step activation in higher eukaryotes by interacting with ATR (Yoshida et al, 2010). CDC6 is critical for DNA replication and its dysregulation is involved in tumorigenesis. It was found that CDC6 down-regulation by RNA interference (RNAi) prevented cell proliferation and promoted apoptosis (Lau et al, 2006). CDC6 was found to be overexpressed in several cancers. The types of cancers that overexpress CDC6 are gastric cancer (Tsukamoto et al, 2008), brain tumors (Ohta et al, 2001), oral squamous cell carcinoma (Feng et al, 2008), cervical cancer (Wang et al, 2009), and malignant mesothelioma (Romagnoli et al, 2009).
Deiodinase, methyladenine iodide, type II (DIO2) — the protein encoded by the DIO2 gene belongs to the family of iodothyronine deiodinases. It is highly expressed in the thyroid gland and may significantly contribute to the relative increase in thyroid T3 production in patients with graves' disease and thyroid adenoma (Meyer et al, 2008); (de Souza Meyer et al, 2005)). Gene expression patterns differ significantly between the ascending and descending progressive forms of nasopharyngeal carcinoma (NPC). Expression of DIO2 gene is higher in downward-progressing cancers (downward ═ distant metastasis) than in upward-progressing cancers (cranial base local growth and invasion), which may be closely related to the metastatic potential of NPC (Liang et al, 2008 a). DIO2mRNA as well as DIO2 activity is expressed in brain tumors (Murakami et al, 2000). D2 activity is present in the lung and resembles peripheral lung and lung cancer tissues (Wawrzynska et al, 2003).
Kinesin family member 26B (KIF26B) -kinesin is a protein belonging to a class of motor proteins found in eukaryotic cells. Kinesin moves along microtubules and gains strength by ATP hydrolysis (thus kinesin is an atpase). Kif26b, a kinesin family gene, is a downstream target of Salll (Nishinakamura et al, 2011). Kif26b is critical for kidney development because it regulates the adhesion of mesenchymal cells in contact with ureteral buds. Kif26b overexpression in vitro resulted in increased cell adhesion through interaction with non-muscle myosin (Terabayashi et al, 2012; Uchiyama et al, 2010).
Serpin, the serpin inhibitor, clade B (ovalbumin), member 3(SERPINB3) -Squamous Cell Carcinoma Antigen (SCCA), also known as SERPINB3, is a member of the high molecular weight family of serpins (suminaet al, 1991). High levels of expression have been reported in head and neck tissue cancers and other epithelial cancers (Torre, 1998). According to reports, SCCA is overexpressed in tumor tissues compared to peritumoral tissues, suggesting its role as a potential marker for histological detection of HCC (Pontisso et al, 2004). The serpin B3/B4, and in particular the serpin B4, appear to play an important role in abnormal epithelial cell proliferation. The assessment of the serpin B3/B4 may be of prognostic value in predicting disease progression, particularly for patients with increased susceptibility to lung cancer (calabree et al, 2012). SCCA1(SERPINB3) on the one hand inhibits lysosomal injury-induced cell death and on the other hand sensitizes cells to ER stress by initiating caspase-8 independent of the death receptor apoptotic pathway (Ullman et al, 2011). Several findings indicate that SERPINB3 plays an important role in the guidance of epidermal barrier disruption. SERPINB3 may be a key determinant of epidermal barrier function (Katagiri et al, 2010).
Cyclin-dependent kinase 1(CDK1) -CDC2 (cell division cycle 2), also known as p34CDC2 or CDK1 (cyclin-dependent kinase 1), belongs to the serine/threonine protein kinase family CDK, playing a key role in cell cycle regulation (Vermeulen et al, 2003). Overexpression of CDC2 was found in a number of cancers, although expression of other cyclins (e.g., cyclins) was deregulated more frequently according to the data of (Vermeulen et al, 2003). Overexpression of CDC2 in NSCLC is described (Xu et al, 2011; Zhang et al, 2011). Perumal et al (2012) reported that CDC2 overexpression is associated with poor prognosis (Perumal et al, 2012). In addition, one study indicated that CDC2 may be used clinically as a predictor of early non-small cell lung cancer recurrence (Kubo et al, 2014).
Collagen, type XI, α 1(COL11a1) -COL11a1 encodes one of the two α chains of type XI collagen (smaller fibrillar collagen). According to reports, COL11a1 is up-regulated in several cancers, e.g. colorectal cancer (freere et al, 2014), breast cancer (Ellsworth et al, 2009), gastric cancer (Zhao et al, 2009b), bladder tumors (Ewald et al, 2013). Expression of COL11a1 in ovarian cancer is associated with cancer recurrence and poor survival. Knockdown of COL11A1 reduced cell migration, invasion and tumor progression in mice in vitro (Cheon et al, 2014; Wu et al, 2014 b). Based on microarray analysis results, COL11a1 was found to be differentially expressed in lung tissue in lung cancer patients in non-smoking women compared to healthy controls (Lv and Wang, 2015).
Collagen, type I, α 2(COL1a2) -COL11a2 encodes the α -philic 2 chain of type I collagen, with a triple helix structure comprising two α 1 chains and one α 2 chain. In gastric cancer samples, COL1A2 was found to be up-regulated compared to normal tissue (Yan et al, 2014; Yang et al, 2007) and associated with later staging (Yasui et al, 2004). COL1a2 was reported to be upregulated in osteosarcoma (Wu et al, 2014a), advanced bladder cancer (Fang et al, 2013), head and neck/oral squamous cell carcinoma (HNOSCC) (Yeet al, 2008) and medulloblastoma (the most common malignant brain tumor in children) (Liang et al, 2008 b).
Periostin, osteoblast specific factor (POSTN) -POSTN, which encodes proteins with similarities to fasciculated protein family, is involved in cell survival and angiogenesis, has become a promising marker for tumor progression in various types of human cancers (Ruan et al, 2009). Periostin protein or mRNA high expression is detected in most solid tumors, including breast cancer (Zhang et al, 2010c), 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 (tischeler 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 highly expressed in lung cancer, and is associated with angiogenesis, invasion, and metastasis (Takanami et al, 2008). The silencing of periostin in a549 non-small cell lung carcinoma (NSCLC) cells inhibits tumor cell growth and reduces cell invasion (Wu et al, 2013 c).
AT hook-containing, DNA binding motif 1(AHDC1) -the gene encodes a protein that contains two AT-hooks that may play a role in DNA binding. Mutations in this gene are associated with a visual deficit in the brain (Bosch et al, 2015). Using whole genome sequencing, new truncation mutations of AHDC1 were identified in patients with language hypoevolutism, low muscle tone and sleep apnea as indicative of cluster manifestations. Mutations are most likely responsible for this genetic syndrome (Xia et al, 2014).
Apoptosis-inducing factor, mitochondrially-related, 2(AIFM2) -this gene encodes a flavoprotein oxidoreductase that binds single-stranded DNA and is thought to promote apoptosis in the presence of bacterial and viral DNA. AIFM2 is not well characterized, but limited evidence suggests that it may act as a tumor suppressor. AIFM2 expression is activated by the tumor suppressor p53, and ectopic expression of p53 has been shown to induce apoptosis. In addition, AIM2 expression was shown to be down-regulated in a range of human tumors including kidney, stomach, colorectal and other cancer samples (Ohiro et al, 2002; Wu et al, 2004). However, in knockout mouse models, AIFM2 is not required for p 53-dependent tumor inhibition (Mei et al, 2006). In cell culture, AIFM2 is involved in mediating adenosine-induced apoptosis (Yang et al, 2011).
Chromosome 6 open reading frame 132(C6orf132) -C6orf132 encodes chromosome 6 open reading frame 132. Gene C6orf132 is located on chromosome 6p21.1 (Mungall et al, 2003). The function of this gene is not known.
CCZ1 (Saccharomyces cerevisiae) (CCZ1), CCZ1 (Saccharomyces cerevisiae) (CCZ1B) -CCZ1 encodes CCZ1 (Saccharomyces cerevisiae) homologous genes for transport and biosynthesis of vacuolar proteins. CCZ1B encodes the gene homolog B (Saccharomyces cerevisiae) involved in CCZ1 vacuolar protein trafficking and biosynthesis. CCZ1 and CCZ1B were identified (by comparison of proteomics) as human genes that are evolutionarily conserved among c. The genes CCZ1 and CCZ1B are located on chromosome 7p22.1 (Hillier et al, 2003). CCZ1 appears to play a role in lysosomal biogenesis and phagosome maturation by recruiting the gtpase RAB7a7 in the phagosome (Nieto et al, 2010). The CCZ1B gene is an uncharacterized gene.
Collagen, type V, α 2(COL5a2) — the gene encodes the α chain of one of the low abundance fibrillar collagens. According to reports, COL5a2 was upregulated in colorectal cancer tissue samples compared to adjacent non-cancerous tissue (Fischer et al, 2001). Corresponding samples of Ductal Carcinoma In Situ (DCIS), Invasive Ductal Carcinoma (IDC) and breast cancer patient stroma showed elevated expression of COL5a2 in IDC (Vargas et al, 2012). In osteosarcoma, COL5a2 was reported to be up-regulated, playing an important role in tumorigenesis (Wu et al, 2014).
Aggregation subfamily member 12(COLEC12) -this gene encodes a member of the C-lectin family, which has a collagen-like sequence and a carbohydrate recognition domain. The COLEC12 protein is a scavenger receptor, a cell surface glycoprotein that displays multiple functions related to host defense. The COLEC12 gene was shown to be a candidate biomarker for the potential of undifferentiated thyroid carcinoma (Espenal-Enriquez et al, 2015). COLEC12 was differentially expressed in HER2 positive breast cancer cell line BT474, likely contributing to the efficacy of trastuzumab (von der Heyde et al, 2015).
The subunits of the integrins complex, gamma 1(COPG1) -COPG1, encode the protein subunits of the integrins complex 1 (COPI). The COPI vacuole mediates retrograde transport (from the Golgi apparatus back to the ER) and intracolonic transport. The cytoplasmic precursor of the COPI-coat is a heptameric integrins complex, which can be considered to consist of two sub-complexes. The first consists of β -, γ -, δ -and ξ -COP subunits, which are distantly homologous to the AP clathrin linker subunit (Watson et al, 2004). EGF-dependent nuclear trafficking of EGFR is regulated by retrograde trafficking from the golgi to the ER, which involves the association of EGFR with gamma-COP (one of the subunits of the COPI set of voxels) (Wang et al, 20l0 b). By immunohistochemical methods, coprg 1 was demonstrated to be abundantly expressed in lung cancer-derived endothelial cells and cancerous lung cells (Park et al, 2008).
CSNK2A 2-Casein kinase II subunit alpha-promoter is an enzyme encoded by the CSNK2A2 gene in humans. A retrospective study showed that CSNK2a1 may be a useful prognostic marker independent of lymph node metastasis status after complete resection of NSCLC patients (Wang et al, 2010 c). CSNK2a2 is associated with tumor progression in advanced human colorectal cancer (Nibbe et al, 2009).
Expression of dendritic cell seven-way transmembrane protein (DCSTAMP) -this gene encodes a seven-way transmembrane protein that is expressed predominantly in dendritic cells. The encoded protein is involved in a series of immune functions of dendritic cells. DCSTAMP has been identified as a gene that is differentially expressed in papillary thyroid carcinomas (Lee et al, 2009), and subsequently demonstrated elevated expression levels in these samples (Kim et al, 2010).
Congenital dyskeratosis 1, dyskherin (DKC1) -DKC1 gene functions in two different complexes. Dyskerin mediates uridine modification on ribosomal and micronucleus RNA and stabilization of the telomerase RNA component (TERC). In human tumors, dyskherin expression was found to be associated with both rRNA modification and TERC levels (Penzo et al, 2015). Furthermore, dyskherin overexpression is associated with poor prognosis in various tumor types (e.g. HCC) (Liu et al, 2012).
Bispecific tyrosine (Y) -phosphorylation regulated kinase 2(DYRK 2)/bispecific tyrosine (Y) -phosphorylation regulated kinase 4) (DYRK4) -DYRK2 and DYRK4 belong to the DYRK protein kinase family (a 5-member mammalian family) which are involved in the regulation of cell differentiation, proliferation and survival (Papadopoulos et al, 2011). DYRK2 controls epithelial-mesenchymal transition of breast cancer by degrading Snail (Mimoto et al, 2013). DYRK2 modulates p53 to induce apoptosis and enhance response to DNA damage: upon exposure to genotoxic stress, DYRK2 translocates to the nucleus and initiates p53 by phosphorylation (Meulmeester and Jochemsen, 2008; Taira et al, 2007). The DYRK4 gene maps to chromosome 12p13.32, which is described as a susceptibility gene for CRC because the CCND2 gene is affected (Jia et al, 2013; Peters et al, 2013). Some studies have highlighted a role for DYRK4 in neuronal differentiation (Leypoldt et al, 2001; Slepak et al, 2012).
ERO 1-like (saccharomyces cerevisiae) ERO1L-ERO 1-like protein a is a protein encoded by the ERO1L gene in humans. ERO 1-alpha is an oxidase enzyme that is induced in the endoplasmic reticulum and under hypoxic conditions. ERO 1-a is overexpressed in a variety of tumor types. Furthermore, cancer-associated ERO1- α regulates expression of MHC class I molecules by oxidative folding (Kukita et al, 2015). Studies have shown that expression of hERO 1-a in cancer cells correlates with a poorer prognosis and is therefore likely a prognostic factor in breast cancer patients (Kutomi et al, 2013). In natural human tumors, ERO1L mRNA is specifically induced in hypoxic microenvironments consistent with upregulation of VEGF expression. Studies have shown that reduction of ERO1L production by siRNA results in significant inhibition of VEGF secretion, impaired proliferative capacity and enhanced apoptosis (May et al, 2005).
The sequence similarity family 83, member a (FAM83A) -FAM83A, was identified as elevated in several different cancer tissue types (cipiano et al, 2014). However, the function of FAM83A is not clear (Boyer et al, 2013). FAM83A was predicted to be a tumor-specific gene in lung cancer, and its expression in lung cancer samples was experimentally confirmed. Expression is particularly high in adenocarcinomas (Li et al, 2005). Others have reported a correlation with lung cancer disease progression (Liu et al, 2008).
Fragile X mental retardation, autosomal homolog 1(FXR1) -the protein encoded by the FXR1 gene is an RNA-binding protein that interacts with functionally similar proteins FMR1 and FXR 2. FXR1 is dysregulated in expression in a variety of human diseases, including cancer. FXR1 acts as an oncogene, which may increase proliferation, migration and invasion of cancer cells (Jin et al, 2015). FXR1 is a novel oncogene of NSCLC and FXR1 regulates its function in a manner that forms a novel complex with two other oncogenes, protein kinase C, iota (PRKCI) and epithelial transformation 2(ECT2), in two identical amplicons of lung cancer cells (Qian et al, 2015 b). Increased expression of FXR1 in NSCLC is reported to be a candidate biomarker predicted to be poor survival and may represent a new therapeutic target. Furthermore, FXR1 expression was associated with poor clinical outcomes in a variety of human cancers, suggesting that this RNA binding protein is more widely involved in cancer progression (Qian et al, 2015 a).
G2/M phase specific E3 ubiquitin protein ligase (G2E3) -G2/M phase specific E3 ubiquitin-protein ligase is an enzyme encoded by the G2E3 gene in humans. G2E3 shuttles between the cytoplasm and nucleus, focusing on the nucleoli and relocating to the nucleoplasm in response to DNA damage. G2E3 is a bifunctional ubiquitin ligase that is required to prevent apoptosis during early embryogenesis (Brooks et al, 2008). Several findings indicate that G2E3 is a molecular determinant of DNA damage response and cell survival, and its depletion sensitizes tumor cells to DNA damage therapy (Schmidt et al, 2015 b). In addition, loss of G2E3 triggers apoptosis and reduces cancer cell proliferation. Thus, G2E3 acts as a survival factor (Schmidt et al, 2015 a).
Guanylate binding protein 5(GBP5) -human guanylate binding protein 5(hGBP5) belongs to the interferon-gamma inducible large gtpase family, and is well known for its high sensitivity due to proinflammatory cytokines (Wehner and Herrmann, 2010). hGBP5 is present in three splice variants, forming two different proteins, wherein the C-terminus of the tumor-specific protein is truncated by 97 amino acids (Fellenberg et al, 2004).
Glutaminase (GLS) the GLS gene encodes K-type mitochondrial glutaminase. Glutaminase (GLS) converts glutamine to glutamate, and plays a key role in cancer cell metabolism, growth, and proliferation. Several studies have shown that GLS is required for tumorigenesis and to support small molecules, and that GLS gene suppression is a possible approach for GLS tumor cell independent targeting for cancer therapy (Xiang et al, 2015). Transient knockdown of GLS splice variants indicates that GAC loss has the most adverse effect on NSCLC tumor cell growth (van den Heuvel et al, 2012). In colon cancer (Huang et al, 2014a), hepatocellular carcinoma (Yu et al, 2015) and Pancreatic Ductal Adenocarcinoma (PDA) (Chakrabarti et al, 2015), expression of GLS1 is up-regulated and correlated with clinical pathology.
Heat shock 70kDa protein 2(HSPA2) -HSPA2 has been identified as a potential oncogenic protein expressed at normal levels in a subset of human cancers, such as breast cancer (Mestiri et al, 2001), cervical cancer (Garg et al, 2010a), urothelial cancer (Garg et al, 2010c), nasopharyngeal cancer (Jalbout et al, 2003) and malignancies (chouchanet et al, 1997). A certain degree of HSPA2 gene activity was also observed in cell lines from several human cancers (Sceglinska et al, 2008), whereas silencing of the HSPA2 gene in cancer cells resulted in growth arrest and reduced tumorigenicity (Rohde et al, 2005; Xia et al, 2008). Furthermore, HSPA2 gene polymorphisms are associated with an increased risk of developing lung cancer (Wang et al, 2010 a). Overexpression of HSPA2 is associated with increased cell proliferation, poor differentiation, and lymph node metastasis in breast, cervical and urinary bladder urothelial cancers (Garg et al, 2010 a; Garg et al, 2010 c; Mestii et al, 2001).
The heat shock 70kDa protein 8(HSPA8) -HSPA8 gene encodes a member of the heat shock protein 70 family Hsc70, which family includes heat-induced and constitutively expressed members. HSPA8 binds to nascent polypeptides to promote correct protein folding (Beckmann et al, 1990). HSC70 acts as a molecular chaperone, assisting in protein synthesis, folding, assembly, transport and degradation between compartments (Bukau and Horwich, 1998; Hartl and Hayer-Hartl, 2002). HSC70 is expressed in non-malignant breast and breast cancer cells (Kao et al, 2003; Vargas-Roig et al, 1998), and HSP/HSC70 is overexpressed in chemotherapy-resistant cancer cells (Ciocca et al, 1992; Lazaris et al, 1997) leading to a study of the possible clinical indices of these proteins (Ciocca and Calderwood, 2005). This secreted hsc70 chaperone plays a potential role in cell proliferation, possibly leading to a higher proportion of tumor growth in cancer cells overexpressing cathepsin D (Nirde et al, 2010). Furthermore, Russin et al reported a correlation between this gene polymorphism and lung cancer risk (Rusin et al, 2004).
Heat shock 70kDa protein 1A (HSPA1A) -HSPA1A, also known as HSP72, was shown to be strongly upregulated in cancer and to play a key role in tumor cell growth by inhibiting both the p 53-dependent and p 53-independent senescence pathways (Sherman, 2010). It is overexpressed in RCC (Atkins et al, 2005) and gastrointestinal cancer (Wang et al, 2013a), with the latter showing a significant correlation with the progression, infiltration and presence of lymph nodes and distant metastases.
Heat shock 70kDa protein 1B (HSPA1B) -HSPA1B, also known as HSP70-2, encodes a testis-specific heat shock protein 70-2, essential for the growth of spermatocytes and cancer cells (Hafield and Lovas, 2012). Various studies have shown that HSP70-2 plays an important role in disease progression in cervical cancer (Garg et al, 2010b), renal cell carcinoma (Singh and Suri, 2014) and bladder cancer, with intragenic polymorphisms associated with gastric cancer development (Ferrer-Ferrer et al, 2013). Some functional HSPA1B variants are associated with lung cancer risk and survival. These Hsp70 gene variants may provide useful biomarkers for predicting the risk and prognosis of lung cancer (Szondy et al, 2012; Guo et al, 2011).
Heat shock 70kDa protein 1-like (HSPA1L) -heat shock 70kDa protein 1L is a protein encoded by the HSPA1L gene on chromosome 6 in humans. Although it shares close homology with HSPA1A and HSPA1B, the regulation is different and not heat inducible (Ito et al, 1998). Intragenic polymorphisms are associated with susceptibility and prognosis of prostate cancer (Sfar et al, 2010) and susceptibility to hepatocellular carcinoma (Medhi et al, 2013).
Heat shock 70kDa protein 6(HSP70B ') (HSPA6) -Heat Shock Protein (HSP) 70B' is a strictly inducible human HSP70 chaperone protein with little or no basal expression levels in most cells (Noonan et al, 2007). HSPA6, also known as heat shock protein 70B', was shown to be upregulated in glioblastoma cells (Huang et al, 2014B) by Y15 treatment and heat shock of head and neck cancer cells (Narita et al, 2002). High levels of HSPA6 may be associated with early recurrence of HCC (Yang et al, 2015).
Heat shock protein 70kDa 7 (HSPA 70B) (HSPA7) -HSPA7 is a pseudogene.
The HSPA (heat shock 70kDa) binding protein, cytoplasmic chaperone 1(HSPBP1) -heat shock binding protein HSPBP1 is a member of the Hsp70 chaperone family. HspBP1 is a helper chaperone that binds to and regulates the chaperone Hsp 70. Both HspBP1 and Hsp70 levels were significantly higher in the serum of breast cancer patients than in the serum of healthy individuals (Souza et al, 2009). HSPBP1 is overexpressed in leukemia patients (sedackova et al, 2011). HspBP1 is up-regulated in human HCV-HCC, an increase that is associated with increased Hsp70 levels (Yokoyama et al, 2008).
The GTP-containing initiator protein IQ motif 1(IQGAP1) -IQGAP1, also known as p195, is a ubiquitously expressed protein encoded by the IQGAP1 gene in humans. IQGAP1 is a key mediator of several different cellular processes, particularly cytoskeletal rearrangement. Recent studies have demonstrated the potential role of IQGAP1 in cancer, as evidenced by the over-expression of IQGAP1 and the different membrane localization observed in a range of tumors (Johnson et al, 2009). IQGAP1 overexpression may play an important role in pancreatic cancer development and progression (Wang et al, 2013 c). Inhibition of IQGAP1 expression reduces tumor cell growth, migration and invasion in Esophageal Squamous Cell Carcinoma (ESCC). (Wang et al, 2014 c). Furthermore, increased expression of IQGAP1 during differentiation of ovarian cancer stem-like cells (CSC-LCS) is involved in aggressive cellular behavior, which may lead to metastasis of ovarian cancer (Huang et al, 2015 a).
The integrin, β 6(ITGB6) -ITGB6, is a subtype of integrin, is expressed only on the surface of epithelial cells, and is a receptor for extracellular matrix proteins (Weinacker et al, 1994). One study found that ITGB6 was expressed in increased relative to normal tissue in the 10 tumor types studied. The highest frequency of ITGB6 expression was reported for squamous cell carcinoma of uterus, skin, esophagus, head and neck. Notably, blockade mediated by the ITGB6 antibody inhibited tumor progression in vivo (VanAarsen et al, 2008). ITGB6 has been developed as a target for tumor-specific drug delivery and enhances the therapeutic efficacy of colon cancer (liaang et al, 2015; Zhao-Yang et al, 2008). In breast cancer, high expression of ITGB6, either mRNA or protein, is associated with poor survival and increased distant metastasis. The ITGB6 targeting antibody inhibited tumor growth in a mouse model of breast cancer (Allen et al, 2014).
Lysine (K) -specific demethylase 6B (KDM6B) -KDM6B, also known as JMJD3, is a histone demethylase encoded by the KDM6B gene in humans. KDM6B affects transcriptional regulation by demethylation of the histone 3 lysine 27 residue. KDM6B low expression is an independent predictor of poor prognosis for patients with surgical resection of colorectal cancer (P ═ 0.042) (Yokoyamaet al, 2008). Furthermore, KDM6B overexpression inhibited cell growth by initiating mitochondrion-dependent apoptosis and by attenuating the NSCLC cell invasion metastasis cascade (Ma et al, 2015). KDM6B, on the other hand, was expressed at high levels in renal clear cell carcinoma (ccRCC) and positively correlated with ccRCC prognosis difference. KDM6B knockdown inhibited ccRCC tumor formation in vitro (Li et al, 2015). Furthermore, deregulation of KDM6B may contribute to glioma formation by inhibiting the p53 pathway, resulting in terminal differentiation block (Ene et al, 2012).
Keratin 9, type I (KRT9) -keratin 9 is a type I keratin protein encoded in humans by the KRT9 gene. It is found only in the terminally differentiated epidermis of the palms and soles of the feet. Mutations encoding the protein lead to epidermal palmoplantar keratosis (Reis et al, 1994). KRT9 is upregulated in HCCs. This overexpression may play a crucial role in HCC metastasis and may serve as a potential serum marker for predicting HCC metastasis (Fu et al, 2009).
LINE1 retrotransposable element 1(L1RE1) -L1RE1 gene, also known as LRE1, encodes "LINE" (long interspersed nuclear element) retrotransposable element (LRE), a mobile DNA sequence containing autonomous transposon activity. According to reports, the LINE1 retrotransposon family is hypomethylated in many cancers, reflecting the ubiquitous methylation status of the genome (Ostertag and Kazazian, jr., 2001). A long interspersed nuclear element repeat region LRE1, located at 22q11-q12, is an indicator of universal methylation status invariance (Chalithagorn et al, 2004; Ostertag and Kazazian, Jr., 2001). Some data indicate that LRE1 relative methylation is an independent epigenetic biomarker of Head and Neck Squamous Cell Carcinoma (HNSCC) (Hsiung et al, 2007).
Laminin, β 3(LAMB3) -LAMB3 encodes the β 3 subunit of laminin, which together with the α and γ subunits forms laminin 5. LAMB3 is upregulated in Papillary Thyroid Carcinoma (PTC) (Barros-Fillho et al, 2015), cervical squamous cell carcinoma (cervical SCC) (Yamamoto et al, 2013), and Oral Squamous Cell Carcinoma (OSCC) (Tanis et al, 2014). Gene array and bioinformatics analysis suggest that LAMB3 is a key gene involved in lung cancer. The gene knockdown inhibits invasion and metastasis of human lung cancer cells in vitro and in vivo. LAMB3 is overexpressed in lung cancer patients, the expression of which is associated with lymph node metastasis (Wang et al, 2013 b).
Lysosomal protein transmembrane 5 (lamtm 5) -lamtm 5 gene encodes intracellular vesicle membrane protein associated with lysosomes. LAPTM5 is aberrantly methylated in lung cancer, methylation being associated with tumor differentiation status (cortex et al, 2008). Lamtm 5 positive vesicles are closely associated with programmed cell death that occurs during spontaneous regression of neuroblastoma (Inoue et al, 2009). The CD1e protein is involved in the presentation of lipid antigens in dendritic cells. LATPM5 controls CDle ubiquitination or production of soluble lysosomal CDle proteins (Angenieux et al, 2012).
Mini-chromosome maintenance complex component 4(MCM4) -the protein encoded by the MCM4 gene is one of the highly conserved mini-chromosome maintenance proteins (MCM) that are essential for initiating replication of eukaryotic genomes. MCM4 is down-regulated in bladder cancer (Zekri et al, 2015) and differentially expressed in lung adenocarcinoma compared to normal lung tissue (Zhang et al, 2014). MCM4 overexpression is associated with shorter survival in breast cancer patients (Kwok et al, 2015).
Minichromosome maintenance complex component 5(MCM5) -MCM5 is involved in DNA replication and cell cycle regulation. High expression levels of MCM5 were demonstrated to correlate with progression and poor prognosis of oral squamous cell carcinoma (Yu et al, 2014), cervical cancer (Das et al, 2013), gastric cancer (Giaginis et al, 2011), and colon cancer (Burger, 2009).
Melanotropin (MREG) MREG plays a role in intracellular melanosome distribution through modulation of retrograde microtubule-dependent melanosome transport (Wu et al, 2012) (Ohbayashi et al, 2012). In addition, MREG also plays a role in pigment incorporation into melanin regulation (Rachel et al, 2012). In estrogen receptor positive breast cancer cells, MREG shows miRNA-26 targeting through its 3' non-coding region. However, the direct involvement of MREG in miRNA-26 mediated cell proliferation cannot be demonstrated (Tan et al, 2014).
NODAL Modulator 1(NOMO1)/NODAL Modulator 2(NOMO2)/NODAL Modulator 3(NOMO3) -NOMO1, NOMO2 and NOMO3 genes are three highly similar genes located in the p-arm replication region of chromosome 16. These genes encode closely related proteins that may have the same function. NOMO1 was identified as an overexpressed gene in the cutaneous T-cell lymphoma (CTCL) cell line HuT78 (Lange et al, 2009). NOMO1 is an antagonist of Nodal signaling. Nodal is a signaling factor of the transforming growth factor-beta (TGF- β) superfamily, which plays a key role in spinal development (Haffner et al, 2004).
Nucleoporin 153kDa (NUP153) -nucleoporin 153(NUP153), a component of the Nuclear Pore Complex (NPC), is involved in the interaction of NPC with the nuclear lamina. Nup153 depletion induces dramatic cytoskeletal rearrangements that impair migration of human breast cancer cells (Zhou and Pante, 2010). NUP153 nucleoporins regulate specific protein distribution between the nucleus and cytoplasm, and interestingly, include the transduction factor SMAD2 for TGF β signaling (Xu et al, 2002). Recently, some analyses have shown a possible novel oncogenic function of the pancreatic cancer nucleopore protein NUP153 (apparently by modulating TGF β signalling) (Shain et al, 2013).
PERP, TP53 apoptosis effector (PERP) -PERP is a p53/p63 regulatory gene encoding desmosomal proteins that play key roles in cell-cell adhesion and tumor suppression. Loss of PERP expression is associated with transition to Squamous Cell Carcinoma (SCC) and increased local recurrence in oral SCC patients (Kong et al, 2013). PERP expression is reduced in many human breast cancer cell lines (durek et al, 2012). Some studies have shown that Perp deficiency contributes to carcinogenesis by enhancing cell survival, desmosome loss, and inflammation (Beaudry et al, 2010). PERP is an apoptosis-related target of the p53 gene, and activation alone is sufficient to induce apoptotic pathways, leading to cell death (Chen et al, 2011).
Putative homeodomain transcription factor 1(PHTF 1)/putative homeodomain transcription factor 2(PHTF2) -PHTF1 (putative homeodomain transcription factor) are putative homeogenes located in the human genome 1P 11-P13. This gene is evolutionarily conserved and is expressed primarily in the testis (Manuel et al, 2000). As a transcription factor, the PHTF1 gene is mainly involved in and regulates biological processes such as DNA-dependent transcription. The overexpression of PHTF1 is responsible for regulating cell proliferation and apoptosis in T-cell acute lymphoblastic leukemia (T-ALL) cell lines. PHTF1 may be a tumor suppressor-like gene used as a therapeutic target for triggering the PHTF1-FEM1b-Apaf-1 apoptotic pathway (Huang et al, 2015 b).
The putative homeodomain transcription factor 2 is a protein encoded by the PHTF2 gene in humans. PHTF2 is expressed primarily in muscle and is located in the human genome 7q11.23-q21(Manuel et al, 2000).
Contains pleckstrin homology domain, member 1(PLEKHM1) family M (having RUN domain) -the protein encoded by the PLEKM1 gene is essential for bone resorption and may play an important role in osteoclast vesicle trafficking. Mutations in this gene have been associated with autosomal recessive osteopetrosis type 6(OPTB6) (van et al, 2004). Studies have shown that pleckm 1 is a candidate susceptibility gene for epithelial ovarian cancer (Permuth-Wey et al, 2013).
Phospholipid transfer protein (PLTP) -phospholipid transfer protein (PLTP) plays an important role in the regulation of inflammation. Some data indicate that PLTP has anti-inflammatory capacity in macrophages (Vuletic et al, 2011). Furthermore, PLTP is essential in regulating the association of triacyl lipid a with lipoproteins, leading to a prolongation of its residual time and an amplification of its pro-inflammatory and anti-cancer properties (Gautier et al, 2010). PLTP is differentially expressed in breast cancer patients, possibly associated with a chemotherapeutic response (Chen et al, 2012).
Protein phosphatase 2, regulatory subunit B ", α (PPP2R3A) -one of the regulatory subunits of protein phosphatase 2 encoded by this gene. Protein phosphatase 2 (previously named type 2A) is one of the four major serine/threonine phosphatases involved in negative control of cell growth and division (ruddiger et al, 2001). PPP2R3A is frequently methylated in childhood Acute Lymphoblastic Leukemia (ALL) (Dunwell et al, 2009).
PTC7 protein phosphatase homolog (Saccharomyces cerevisiae) (PPTC7) -PPTC7 encodes a PTC7 protein phosphatase homolog, located on chromosome 12q 24.11. PPTC7 was recently identified as a new susceptibility gene in response to environmental toxins (Zhu et al, 2015).
Protein kinases, DNA activated catalytic Polypeptide (PRKDC) PRKDC encodes the catalytic subunit of DNA-dependent protein kinase (DNA-PK), a member of the PI3/PI4 kinase family. Studies have shown that PRKDC can stabilize c-Myc oncoprotein via the Akt/GSK3 pathway (An et al, 2008). The initiation of PRKDC is positively correlated with HCC proliferation, genomic instability and microvascular density, and negatively correlated with apoptosis and patient survival (Evert et al, 2013).
The proteasome (precursor, megalin) subunit, alpha-type, 4(PSMA4) -PSMA4 encodes the protease subunit alpha 4, which cleaves peptides in an ATP/ubiquitin dependent process in the non-lysosomal pathway. The single nucleotide polymorphism of the PSMA4 gene is related to the risk of lung cancer of Chinese Han population (Wang et al, 2015). On the other hand, it has been reported that the single nucleotide polymorphism of PSMA4 gene is not a major contributor to non-small cell lung cancer susceptibility (Yongjun Zhang et al, 2013). Furthermore, PSMA4 was observed to be overexpressed in lung tumors compared to normal lung tissue. Down-regulation of PSMA4 expression reduces protease activity and induces apoptosis (Liu et al, 2009).
Protein tyrosine phosphatase, non-receptor type 13(PTPN13) -this gene encodes a member of the Protein Tyrosine Phosphatase (PTP) family. PTPs are signaling molecules that regulate a variety of cellular processes, including cell growth, differentiation, mitotic cycle, and oncogenic transformation. PTPN13 was found to interact with the Fas receptor and therefore likely to play a role in Fas-mediated apoptosis. In addition, PTPN13 interacts with gtpase promoter proteins and thus can act as a regulator of the Rho signaling pathway. In hematological malignancies, PTPN13 has contradictory effects, inhibiting or promoting tumor growth in lymphomas and myeloid leukemias, respectively (Wang et al, 2014 b). This can be explained by the ability of PTPN13 to counteract oncogenic tyrosine kinase activity and its inhibition to interact with Fas death receptors (Freiss and Chalbos, 2011). In breast cancer, PTPN13 is considered to be a unique marker of breast tumors in response to antiestrogens and is a potential therapeutic target for the initiation of tumor apoptosis stimuli (Freiss et al, 2004). Inhibition of Fas/PTPN13 binding may provide a good target for the development of anticancer drugs (Takahashi and Kataoka, 1997).
RAS p21 protein activator 2(RASA2) -RAS p21 protein activator 2 encodes a member of the gtpase promoter GAP1 family. As an inhibitor of RAS function, RASA2 enhances the weak intrinsic gtpase activity of RAS proteins, resulting in inactive GDP-bound forms of RAS, thereby controlling cell proliferation and differentiation. RASA2 could theoretically be used as either an oncogene or as a tumor suppressor depending on the precise genetic alteration, its location within the gene and the effect it exerts on protein function (Friedman, 1995). Under mild stress conditions, RASA2 is cleaved by caspase-3, which forms fragments, called N-fragments, that stimulate anti-death signaling. When caspase-3 activity is further increased, this results in a fragment, designated N2, which no longer protects the cells. On the other hand, full-length RASA2 contributes to Akt activity by avoiding the influence of inactivated phosphatases (cailliu et al, 2015). In breast cancer, caspase-3, which should be activated, may contribute to the inhibition of metastasis by the production of fragment N2 (Barras et al, 2014). RASA2 was identified as a tumor suppressor gene that was mutated in 5% melanoma (araafeh et al, 2015).
Immunoglobulin k J region recombination signal binding protein (RBPJ) -immunoglobulin k J region recombination signal binding protein encodes a transcriptional regulator important for the Notch signaling pathway. RBPJ acts as an inhibitor when not bound to Notch protein and acts as an activator when bound to Notch protein. It is thought to act by recruiting chromatin remodeling complexes containing histone deacetylation or histone acetylase proteins to the Notch signaling pathway. Xenograft mouse models showed that RBPJ knockdown inhibits tumorigenicity and reduces tumor volume, suggesting that tissue hypoxia can promote Smoothened transcription by up-regulating RBPJ to induce proliferation, invasiveness and tumorigenesis of pancreatic cancer cells (Onishi et al, 2016). The effect of RBPJ knockdown leading to a significant decrease in cell growth was also found in prostate and lung cancer cells, suggesting that RBPJ expression may be a promising therapeutic modality for the treatment of human cancer (Xue et al, 2015; Lv et al, 2015). Furthermore, RBPJ overexpression promotes anchorage-dependent growth of rhabdomyosarcoma cells (Nagao et al, 2012). RBPJ-mediated Notch signaling is also critical for dendritic cell-dependent anti-tumor immune responses (Feng et al, 2010).
In 9-like sterile α motif domain (SAMD9L) -SAMD9L encodes a vector containing the 9-like sterile α motif domain and is located on chromosome 7q 21.2. SAMD9 and SAMD9L genes share a common gene structure and encode proteins with 60% amino acid identity, suggesting an inhibitory effect on inflammatory pathways. SAMD9L localized to early endosomes, acting as an endosomal fusion facilitator. Haploinsufficiency of SAMD9L gene contributes to bone marrow transformation, and SAMD9L was identified as a candidate myeloid tumor suppressor gene (Nagamachiet al., 2013). SAMD9L knockdown significantly promoted cell proliferation and colony formation in hepatocyte cancer cell lines because the silence in SAMD9L transitioned the G1-S phase of cell cycle progression and resulted in an increase in the activity of the Wnt/β -catenin pathway. Recent findings underscore the tumor-inhibiting effect of somatic mutations leading to inactivation of SAMD9L and reduced expression in human cancers (wangetal, 2014 a). SAMD9L showed a significant reduction in expression in T and B cell populations of metastatic melanoma patients compared to healthy control populations (Critchley-Thorne et al, 2007).
Splicing factor 3B, subunit 3, 130kDa (SF3B3) -SF3B3 encodes subunit 3 of the splicing factor 3B protein complex. SF3B3 overexpression is significantly associated with overall survival and endocrine in estrogen receptor positive breast cancer (Gokmen-polar, 2015).
Surfactant protein a1(SFTPA 1)/surfactant protein a2(SFTPA2) — these genes encode lung surfactant proteins, which are members of the C-type lectin subfamily known as collectins. SFTPA binds to lipids and specific carbohydrate moieties found on microbial surfaces and plays a crucial role in surfactant homeostasis and defense against respiratory pathogens. Mutations in these genes are associated with idiopathic pulmonary fibrosis. Lung cancer specific gene markers, containing the SFTPA1 and SFTPA2 genes, accurately discriminate lung cancer from other cancer samples (Peng et al, 2015). EGFR mutations are significantly more common in lung adenocarcinomas with SFTPA expression than those without (Jie et al, 2014). SFTPA inhibits lung cancer progression by modulating the polarization of tumor-associated macrophages (Mitsuhashi et al, 2013). Expression of the lung epithelial cell mutation SFTPA2 resulted in latent TGF- β 1 and TGF- β 1 mediated EMT secretion (Maitra et al, 2012). Furthermore, the development of prostate cancer may be associated with decreased SFTPA levels (Kankavi et al, 2014).
The proteins of solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocating molecule) member 31(SLC25A 31)/solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocating molecule) member 4(SLC25A 4)/solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocating molecule) member 5(SLC25A 5)/solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocating molecule) member 6(SLC25A6) -solute carrier family 25 were ADP/ATP vectors, exchanging cytosolic ADP for matrix ATP in mitochondria. They act as a gate hole to translocate ADP/ATP and form homodimers embedded in the inner mitochondrial membrane. Cells overexpressing this gene family have been shown to exhibit an anti-apoptotic phenotype. The inhibition expression of the gene family shows that the gene family can induce apoptosis and inhibit tumor growth. Although SLC25a4 is preferentially present in differentiated tissues and is present specifically for muscle and brain, SLC25a5 is expressed in hyperplastic tissues (e.g., tumors). SLC25a6 is ubiquitously expressed, with SLC25a31 present in hepatocytes and germ cells (Dolce et al, 2005). In particular SLC25a5 contributes to canceration. Since SLC25a5 expression is closely related to tumor mitochondrial bioenergy, this is a promising target for cancer personalized therapy and development of anti-cancer strategies (Chevrollier et al, 2011). Furthermore, stable overexpression of SLC25a31 protected cancer cells from lonidamine and staurosporine apoptosis, independent of Bcl-2 expression. Thus, dichotomy was found in the human SLC25 isoform subfamily, with SLC25a4 and SLC25a6 isoforms having pro-apoptotic function, whereas SLC25a5 and SLC25a31 isoforms desensitize cell death-inducing stimuli (gallerre et al, 2010).
SP140 nuclear protein (SP140) -SP140 encodes the SP140 nuclear protein and is located on chromosome 2q 37.1. SP140 was shown to be upregulated in squamous cell carcinoma of the larynx (Zhou et al, 2007). SP140 is associated with chronic lymphocytic leukemia (net al., 2010), multiple myeloma (Kortum et al., 2015) and acute promyelocytic leukemia (Blochet al., 1996).
The signal transducer and transcriptional activator 1, 91kDa (STAT1) -STAT1 is activated by tyrosine phosphorylation in response to all interferons (Decker et al, 2002) and contributes to Th1 cell differentiation (Schulz et al, 2009). On a molecular level, STAT1 inhibits the proliferation of mouse and human tumor cells receiving IFN- γ treatment by its ability to increase expression of the cyclin-dependent kinase inhibitor p21Cip1 or decrease expression of c-myc (Ramana et al, 2000). The anti-tumor activity of STAT1 is further supported by its ability to inhibit angiogenesis and tumor metastasis in mouse models (Huang et al, 2002). The increased levels of STAT1 mRNA are shown to be part of the molecular signature, and are well correlated with the prognosis of metastasis in hormone receptor-negative and triple-negative breast cancer patients (Yau et al, 2010).
Transmembrane protein 43(TMEM43) -this gene encodes transmembrane protein 43. The defect of this gene is responsible for familial arrhythmogenic right ventricular dysplasia type 5(ARVD5), a disease also known as arrhythmogenic right ventricular cardiomyopathy type 5(ARVC 5). ARVD is a genetic disease characterized by ventricular tachycardia, heart failure, sudden cardiac death and fibro-adipose replacement cardiomyocytes (silam et al, 2014). TMEM43 may have an important role in the maintenance of nuclear membrane structure in the nuclear membrane by tissue protein complexes (Bengtsson and Otto, 2008).
Topoisomerase (DNA) II α 170kDa (TOP 2A)/topoisomerase (DNA) II β 180kDa (TOP2B) -TOP2A and TOP2B encode a highly homologous subset of DNA topoisomerases that are capable of controlling and altering the topological state of DNA during transcription. Such ribozymes are involved in processes such as chromosome condensation, separation of chromatids, and reduction of torsional stress generated during DNA transcription and replication. TOP2A is essential for cell proliferation and is highly expressed in fast growing cells, whereas TOP2B is not essential for growth and has recently been shown to be involved in treatment-related secondary malignancies (Toyoda et al, 2008). TOP2A was found to be overexpressed in several cancer types (e.g., malignant pleural mesothelioma (Roe et al, 2010), malignant schwannoma (Kresse et al, 2008), lung adenocarcinoma cells (Kobayashi et al, 2004), bladder cancer (Simonet et al, 2003), glioblastoma (van den Boom et al, 2003)). TOP2B is involved in DNA transcription, replication, recombination and mitosis and, in addition to TOP1, represents a second NUP98 fusion partner gene belonging to the topoisomerase gene family (Nebral et al, 2005).
Tryptase α/β 1(TPSAB 1)/tryptase β 2(TPSB2) -tryptase α/β 1(TPSAB1) and tryptase β 2(TPSB2) are expressed by mast cells together with the other two tryptase isoforms. Tryptase has been shown to be a mediator involved in the pathogenesis of asthma and other allergic and inflammatory diseases. Tryptase secreted by mast cells has a pro-angiogenic function and contributes to tumor angiogenesis. Tryptase acts through activation of protease activated receptor-2 (PAR-2) and additionally contributes to extracellular matrix degradation, thereby also promoting vascular growth. In addition, the appearance of tryptase-positive mast cells in tumor tissue is associated with angiogenesis in several cancer types (amendola et al, 2014). Tryptase-positive mast cell levels are reported to be elevated in prostate cancer, associated with microvascular density, tumor stage and shorter survival (Nonomura et al, 2007; Stawerski et al, 2013). Similarly, tryptase-positive mast cells are also associated with tumor stage and angiogenesis in gastric cancer (Zhao et al, 2012; Ribatti et al, 2010) and lung adenocarcinoma (Imada et al, 2000; Takanami et al, 2000).
Triplex motif-containing 11(TRIM11) -Triplex motif-containing protein 11 is a protein encoded by TRIM11 gene in human. TRIM11 is known to be involved in the development of the central nervous system and to disrupt the stability of human peptides (inhibitors of Alzheimer's neurone injury) (Niikura et al, 2003). TRIM11 was overexpressed in high grade gliomas, promoting cell proliferation, invasion, metastasis and glial tumor growth (Di et al, 2013).
Transient receptor potential cation channel, subfamily M, member 2(TRPM2) — the protein encoded by this gene is a calcium-permeable cation channel regulated by intracellular free ADP-ribose. TRPM2 may be involved in mediating apoptosis under certain conditions (Ishii et al, 2007; Cao et al, 2015). However, its effect on cell growth proliferation is less clear and may depend on cell culture conditions and the expression of alternatively spliced isoforms (Chen et al, 2014). In melanoma and prostate cancer, tumor-rich TRPM2 antisense transcripts have been identified that correlate with apoptosis and clinical outcome (Orfanelli et al, 2015).
Tubulin gamma complex-associated protein 3(TUBGCP3) -tubulin gamma complex-associated protein 3 is part of a multi-subunit gamma tubulin complex and is essential for microtubule nucleation in eukaryotic cells (Lynch et al, 2014). The cytoplasmic gamma tubulin complex is directed to the centrosome or other microtubule tissue center by a group of so-called gamma tubulin complex binding proteins (Schiebel, 2000). It was found that expression of the TUBGCP3 transcript was significantly increased in glioblastoma cells compared to normal human astrocytes, and TUBGCP3 immunoreactivity was significantly increased compared to normal brain. TUBGCP3 is also associated with microvascular proliferation and signaling pathways interactions, leading to a malignant phenotype (drabehoo et al, 2015). Furthermore, TUBGCP3 was found to be significantly higher expressed in the near tetraploid than in the diploid mantle cell lymphoma samples (Neben et al, 2007).
Ubiquitin-like modulator promoter 6(UBA6) -ubiquitin-like modulator promoter 6 is a protein encoded by the UBA6 gene in humans. UBA6 is the ubiquitin promoter with the greatest expression in testis. Furthermore, it is essential for a cellular response to DNA damage (Moudry et al, 2012).
Heterophilic and polytropic retroviral receptor 1(XPR1) -XPR1 is a multipass membrane molecule comprising an amino-terminal SPX domain 180 residues in length (designated SYG1, PHO81, and XPR 1). XPR1 has been reported to mediate phosphate export (Giovannini et al, 2013). Increased transcription of XPR1 mRNA was found following osteoclast differentiation (Sharma et al, 2010). Initially, XPR1 was described as a retroviral receptor, used by two gamma retroviruses, heterophilic and polytropic MLV (X-MLV and P-MLV), and could infect human cells as well as various other species, such as mice and birds (Kozak, 2010; Martin et al, 2013).
Zinc finger-containing BED type 5(ZBED5) -zinc finger BED type 5 is characterized by encoding sequences derived primarily from a physical-like DNA transposon, but it does not appear to be an active DNA transposon because it is not flanked by inverted terminal repeats. ZBED5 was associated with the Buster DNA transposon and was phylogenetically separated from the other ZBEDs. The ZBED genes are widely expressed in spinal tissues, which together regulate a significant diversity of functions (Hayward et al, 2013).
The zinc finger protein 697(ZNF697) -ZNF697 gene encodes a zinc finger protein 697, which is located on chromosome 1p12 and may play a role in DNA binding (Yu et al, 2011).
Whether or not an immune response is stimulated depends on the presence of antigens that are recognized as foreign by the host immune system. The discovery that the presence of tumor associated antigens increases the likelihood of using the host immune system to interfere with tumor growth. Currently, various mechanisms of utilizing the humoral and cellular immune systems for immunization are being explored for cancer immunotherapy.
Specific elements of the cellular immune response specifically recognize and destroy tumor cells. T-cells isolated from tumor infiltrating cell populations or peripheral blood suggest that these cells play an important role in the natural immune defense of cancer. In particular, CD8 positive T cells play an important role in this response, TCD8+ recognizes class I molecules contained in peptides carried by the Major Histocompatibility Complex (MHC) of typically 8 to 10 amino acid residues derived from proteins or defective ribosomal products (DRIP) located in the cytoplasm. Human MHC molecules are also known as human leukocyte-antigens (HLA).
Unless otherwise indicated, all terms used herein are defined as follows.
The term "T cell response" refers to the specific spread and initiation of effector functions induced by a peptide in vitro or in vivo. For MHC class I-restricted cytotoxic T cells, the effector function may be lysis of peptide pulsed, peptide precursor pulsed or native peptide presented target cells, secretion of cytokines, preferably peptide induced interferon- γ, TNF- α or IL-2, secretion of effector molecules, preferably peptide induced granzyme or perforin, or degranulation.
The term "peptide" as used herein refers to a series of amino acid residues, typically joined by peptide bonds between the alpha-amino and carbonyl groups of adjacent amino acids. These peptides are preferably 9 amino acids in length, but may be 8 amino acids in length to as short as 10, 11, 12 or 13 amino acids in length or longer, and in the case of MHC class II peptides (elongate variants of the peptides of the invention) may be 14, 15, 16, 17, 18, 19 or 20 amino acids in length or longer.
Thus, the term "peptide" shall include salts of a series of amino acid residues, typically linked by peptide bonds between the alpha-amino and carbonyl groups of adjacent amino acids. Preferably, the salt is a pharmaceutically acceptable salt of the peptide, for example: chloride or acetic acid (trifluoroacetic acid) salt. It must be noted that the salts of the peptides of the invention are substantially different from the peptides in their in vivo state, since they are not salts in vivo.
The term "peptide" shall also include "oligopeptides". The term "oligopeptide" as used herein refers to a series of amino acid residues, typically linked by peptide bonds between the alpha-amino and carbonyl groups of adjacent amino acids. The length of the oligopeptide is not critical to the present invention, as long as the correct epitope is maintained in the oligopeptide. Generally, oligopeptides are less than about 30 amino acid residues in length and longer than about 15 amino acids in length.
The term "polypeptide" refers to a series of amino acid residues, typically joined by peptide bonds between the alpha-amino and carbonyl groups of adjacent amino acids. The length of the polypeptide is not critical to the present invention, so long as the correct epitope is maintained. The term "polypeptide" as opposed to the term peptide or oligopeptide refers to a molecule comprising more than about 30 amino acid residues.
A peptide, oligopeptide, protein or nucleotide encoding such a molecule is "immunogenic" (and thus an "immunogen" in the present invention) if it induces an immune response. In the context of the present invention, a more specific definition of immunogenicity is the ability to induce a T cell response. An "immunogen" is therefore a molecule capable of inducing an immune response, and in the context of the present invention, a molecule capable of inducing a T cell response. In another aspect, the immunogen may be a peptide, a complex of a peptide with MHC, and/or a protein for increasing specific antibody or TCR resistance.
A class I T cell "epitope" requires a short peptide that binds to the MHC class I receptor, thereby forming a ternary complex (MHC class I α chain, β -2-microglobulin and peptide) that can be recognized by T cells loaded with a matched T cell receptor in combination with an MHC/peptide complex with appropriate affinity. Peptides that bind to MHC class I molecules are typically 8-14 amino acids in length, most typically 9 amino acids in length.
In humans, there are three distinct genetic loci that encode MHC class I molecules (human MHC molecules are also designated Human Leukocyte Antigens (HLA)): HLA-A, HLA-B and HLA-C. HLA-A01, HLA-A02 and HLA-B07 are examples of different MHC class I alleles that can be expressed from these gene loci.
Table 11: HLA-A02 and HLA-A24 and the frequency of expression F of the most common HLA-DR serotypes. The frequency was adapted according to Hardy-Weinberg formula F ═ 1(1-Gf)2 used by Mori et al (Mori et al, 1997) and was derived from the haplotype frequency across the us population. Due to linkage disequilibrium, a 02 or a 24 combinations within certain HLA-DR alleles may be concentrated or less frequent than their expected single frequency. For details, see Chanock et al (Chanock et al, 2004).
The peptides of the invention, preferably when incorporated into the vaccines of the invention as described herein, bind to HLA-a 02 and HLA-a 24. The MHC class II peptides of the present invention bind to several different HLA class II molecules and are referred to as promiscuous binders (pan-binding peptides). Vaccines may also include pan-bound MHC class II peptides. Thus, the vaccines of the invention can be used to treat cancer in patients positive for a x 02 or a x 24, but not because of the extensive tuberculous nature of these peptides, MHC class II allotypes must be selected.
If the a 02 peptides of the invention are combined with the a 24 peptides of the invention, a higher proportion of the patient population can be treated than the MHC class I alleles alone. Although less than 50% of patients in most populations are resolved by individual alleles, a vaccine of the invention containing both HLA-A24 and HLA-A02 epitopes can treat at least 60% of patients in any relevant population. In particular, in each region, at least one of these alleles of the patient in the following ratios has a positive effect: U.S. 61%, western europe 62%, china 75%, korea 77%, japan 86% (by weight)www.allelefrequencies.netCalculation).
In a preferred embodiment, the term "nucleotide sequence" refers to a heteropolymer of deoxynucleotides.
The nucleotide sequence encoding a particular peptide, oligopeptide or polypeptide may be a natural nucleotide sequence or a synthetic nucleotide sequence. Generally, DNA fragments encoding peptides, polypeptides, and proteins of the invention are composed of cDNA fragments and short oligonucleotide linkers, or a series of oligonucleotides, to provide a synthetic gene that can be expressed in a recombinant transcription unit comprising regulatory elements derived from a microbial or viral operon.
The term "nucleotide encoding of a peptide" as used herein refers to encoding a peptide with a nucleotide sequence, wherein the peptide comprises artificial (man-made) start and stop codons compatible with the biological system that will express the sequence by the dendritic cell or another cellular system used to generate the TCR.
Reference herein to a nucleic acid sequence includes both single-stranded and double-stranded nucleic acids. Thus, unless otherwise indicated herein, for example with respect to DNA, a particular sequence is a single-stranded DNA of that sequence, a duplex (double-stranded DNA) of that sequence with its complement, and the complement of that sequence.
The term "coding region" refers to that portion of a gene that naturally or normally encodes the expression product of the gene in its natural genomic environment, i.e., the region that encodes the natural expression product of the gene in vivo.
The coding region may be derived from a non-mutated ("normal") gene, a mutated gene or an aberrant gene, and may even be derived from a DNA sequence, and may well be synthesized in the laboratory using DNA synthesis methods well known in the art.
The term "expression product" refers to a polypeptide or protein that is the translation product of any nucleic acid sequence encoded equivalent by the degeneracy of genes and genetic codes and thus encoding the same amino acids.
The term "fragment," when referring to a coding sequence, refers to a portion of DNA that contains a non-complete coding region, the expression product of which has substantially the same biological function or activity as the expression product of the complete coding region.
The term "DNA fragment" refers to a DNA polymer, either in the form of individual fragments or as a component of a larger DNA construct, which is obtained in substantially pure form, i.e., free of contaminating endogenous material, from DNA that has been isolated at least once, and in quantities or concentrations that enable the identification, manipulation and recovery of the fragment and its component nucleotide sequences using standard biochemical methods, e.g., using cloning vectors. Such fragments exist in the form of open reading frames (not interrupted by internal untranslated sequences) or introns (usually present in eukaryotic genes). The untranslated DNA sequence may be present downstream of the open reading frame where it does not interfere with the manipulation or expression of the coding region.
The term "primer" refers to a short nucleic acid sequence that can pair with a DNA strand and provide a free 3' -OH terminus where DNA polymerase begins to synthesize a strand of deoxyribonucleic acid.
The term "promoter" refers to a region of DNA that is involved in the binding of RNA polymerase to initiate transcription.
The term "isolated" means that a substance is removed from its original environment (e.g., the natural environment if it occurs naturally). For example, a native nucleotide or polypeptide in a living animal is not isolated, but a nucleotide or polypeptide isolated from some or all of the coexisting materials in the native system is isolated. Such polynucleotides may be part of a vector and/or such polynucleotides and polypeptides may be part of a composition, and as the vector or composition is not part of its natural environment, it remains isolated.
The polynucleotides and recombinant or immunogenic polypeptides disclosed in the present invention may also be present in "purified" form. The term "purified" does not require absolute purity; it is a relative definition and may include highly purified or partially purified preparations, as those skilled in the relevant art will understand. For example, each clone isolated from a cDNA library that has been purified by conventional methods to have an electrophoretic isotype. It is expressly contemplated that the starting material or natural substance may be purified by at least one order of magnitude, preferably two or three orders of magnitude, more preferably four or five orders of magnitude. Furthermore, it is expressly contemplated that the purity of the polypeptide is preferably 99.999%, or at least 99.99% or 99.9%; even more suitably 99% by weight or more.
Nucleic acid and polypeptide expression products disclosed according to the invention, as well as expression vectors comprising such nucleic acids and/or polypeptides, may exist in "concentrated form". The term "concentrated" as used herein means that the concentration of a material is at least about 2, 5, 10, 100 or 1000 times its natural concentration, advantageously 0.01%, preferably at least 0.1% by weight. Concentrated formulations of about 0.5%, 1%, 5%, 10% and 20% by weight are also specifically contemplated. The sequences, configurations, vectors, clones, and other materials comprising the present invention may advantageously be present in concentrated or isolated form. The term "active fragment" refers to a fragment that generates an immune response (i.e., has immunogenic activity), typically a fragment of a peptide, polypeptide or nucleic acid sequence, whether administered alone or optionally together with a suitable adjuvant or in a carrier to an animal, such as a mammal, e.g., a rabbit or mouse, also including a human; this immune response takes the form of stimulating a T cell response in a recipient animal (e.g., a human). Alternatively, an "active fragment" may also be used to induce an in vitro T cell response.
The terms "portion", "segment" and "fragment" as used herein when used in relation to a polypeptide refer to a contiguous sequence of residues, such as amino acid residues, the sequence of which forms a subset of a larger sequence. For example, if a polypeptide is treated with any endopeptidase (e.g., trypsin or chymotrypsin), the oligopeptide resulting from the treatment will represent a portion, segment, or fragment of the starting polypeptide. When used in relation to a polynucleotide, these terms refer to the product resulting from treatment of the polynucleotide with any endonuclease.
According to the present invention, the term "percent identity" or "percent identity", if referring to a sequence, means that the sequence to be compared ("the compared sequence") is compared to the sequence or sequences of claims after alignment of the sequence to be compared ("the reference sequence"). The percent equivalence is then calculated according to the following formula: percent equivalence of 100[1- (C/R) ]
Wherein C is the number of differences between the reference sequence and the compared sequence over the alignment length between the reference sequence and the compared sequence, wherein
(i) Each base or amino acid sequence in the reference sequence has no corresponding aligned base or amino acid in the compared sequence;
(ii) each gap in the reference sequence, an
(iii) Each aligned base or amino acid in the reference sequence is different from the aligned base or amino acid in the aligned sequence, i.e., a difference is formed and
(iiii) alignment must begin at position 1 of the alignment sequence;
and R is the number of bases or amino acids in the reference sequence that produce any gaps in the reference sequence over the length of alignment of the reference sequence to the compared sequence, also counted as one base or amino acid.
If there is an alignment between the "aligned sequence" and the "reference sequence" that is approximately equal to or greater than the specified minimum percent of identity as calculated above, then the aligned sequence has the specified minimum percent of identity with the reference sequence, although there may be alignments where the percent of identity as calculated above herein is less than the specified percent of identity.
Thus, as mentioned above, the present invention proposes a peptide comprising a sequence selected from SEQ ID NO: 1 to SEQ ID NO: 110, or a sequence identical to SEQ ID NO: 1 to SEQ ID NO: 110, or a variant thereof which induces a T cell cross-reaction with the peptide, having 88% homology. The peptides of the invention have the ability to bind to a class II molecule of the Major Histocompatibility Complex (MHC) I or an elongated version of the peptide.
In the present invention, the term "homology" refers to the degree of identity between two amino acid sequences (see percent identity above, e.g., peptide or polypeptide sequences. the "homology" as described above is determined by aligning two sequences adjusted under ideal conditions with the sequences to be compared.
One skilled in the art can assess whether T cells induced by a particular peptide variant cross-react with the peptide itself (Apay et al, 2006; Colombetti et al, 2006; Fong et al, 2001; Zaremba et al, 1997).
By "variant" of a given amino acid sequence, the inventors mean that the side chain of one or two amino acid residues, etc., is altered by substitution with the side chain of another natural amino acid residue or other side chain, so that such a peptide can still bind to an HLA molecule in substantially the same manner as a peptide comprising the given amino acid sequence (consisting of SEQ ID NO: 1 to SEQ ID NO: 110). For example, a peptide may be modified to at least maintain (e.g., not increase) its ability to interact with and bind to the binding groove of a suitable MHC molecule such as HLA-a 02 or-DR, and to at least maintain (e.g., not increase) its ability to bind to a TCR of a T-cell promoter.
These T cells can then cross-react with cells expressing the polypeptide (which comprises the natural amino acid sequence of the homologous peptide as defined in the present invention) and with killer cells. As described in the scientific literature and databases (Rammensee et al, 1999; Godkin et al, 1997), certain sites of HLA-A binding peptides are usually anchor residues, which form a core sequence that is commensurate with the binding motif of HLA-binding grooves, whose definition is determined by the polarity, electrophysical, hydrophobic and steric properties of the polypeptide chains that make up the binding groove. Thus, one skilled in the art can modify seq id No: 1 to SEQ ID NO: 110, and determining whether the variants retain the ability to bind to MHC class I or II molecules. The variants of the invention retain the ability to bind to the TCR of initiating T cells which can then cross-react with and kill cells expressing a polypeptide comprising the native amino acid sequence of the cognate peptide as defined herein.
The original (unmodified) peptides disclosed herein may be modified by substitution of one or more residues at different (possibly selective) positions within the peptide chain, if not otherwise indicated. Preferably, these substitutions are at the end of the amino acid chain. Such substitutions may be conservative, for example, where one amino acid is substituted for another amino acid of similar structure and characteristics, such as where one hydrophobic amino acid is substituted for another. More conservative substitutions are those between amino acids of the same or similar size and chemical nature, for example, leucine substituted with isoleucine. In the study of sequence variations in the native homologous protein family, certain amino acid substitutions tend to be more tolerant than others, and these amino acids tend to show similarity correlations between the size, charge, polarity and hydrophobicity of the original amino acid, which underlies the identification of "conservative substitutions".
Herein, conservative substitutions are defined as exchanges within one of the following five groups: group 1-small aliphatic, non-polar or slightly polar residues (Ala, Ser, Thr, Pro, Gly); group 2-polar, negatively charged residues and their amides (Asp, Asn, Glu, Gln); group 3-polar, positively charged residue (His, Arg, Lys); group 4-the bulky aliphatic nonpolar residue (Met, Leu, Ile, Val, Cys) and group 5-the bulky aromatic residue (Phe, Tyr, Trp).
Less conservative substitutions may involve the substitution of one amino acid with another having similar characteristics but differing in size, such as: alanine is substituted with an isoleucine residue. Highly non-conservative substitutions may involve the substitution of one acidic amino acid with another amino acid having polar or even basic properties. However, such "aggressive" substitutions cannot be considered ineffective and are not considered because the chemical action is not fully predictable, and aggressive substitutions may bring about unexpected contingent effects in their simple chemical principles.
Of course, such substitutions may involve structures other than the normal L-amino acid. Thus, D-amino acids may be substituted by L-amino acids commonly found in the antigenic peptides of the present invention and remain within the scope of this disclosure. In addition, non-standard amino acids (i.e., in addition to the common natural proteinogenic amino acids) may also be used for substitution purposes to produce immunogens and immunogenic polypeptides according to the present invention.
If substitutions at more than one position are found to result in a peptide having an antigenic activity substantially equal to or greater than the value defined below, the combination of substitutions is tested to determine whether the combined substitutions produce a additive or synergistic effect on the antigenicity of the peptide. The number of positions within the peptide that are simultaneously substituted cannot exceed 4 at the most.
A peptide consisting essentially of the amino acid sequence referred to herein may have one or two non-anchor amino acids (see below in relation to the anchor motif) exchanged, without the situation that the ability to interact with human Major Histocompatibility Complex (MHC) -class I or II molecules is substantially altered or adversely affected compared to the unmodified peptide. In another embodiment, one or two amino acids may be exchanged with their conserved exchange partners (see below) in a peptide consisting essentially of the amino acid sequences described herein, without the situation that the ability to human Major Histocompatibility Complex (MHC) -class I or II molecules is substantially altered or adversely affected compared to the unmodified peptide.
These amino acid residues that do not substantially interact with the T cell receptor can be modified by substituting other amino acids that hardly affect the T cell response and do not interfere with the binding to the relevant MHC. Thus, except for certain limiting conditions, a peptide of the invention may be any peptide comprising a given amino acid sequence or portion or variant thereof (this term as used by the inventors includes oligopeptides or polypeptides).
Table 12: according to SEQ ID NO: 1. preferred variants and motifs of HLA-A02 peptides of 2 and 4
Table 12B: according to SEQ ID NO: preferred variants and motifs of HLA-A02 peptides of 13
Position of
1
2
3
4
5
6
7
8
9
SEQ ID 13
F
L
F
D
G
S
A
N
L
Variants
V
I
A
M
V
M
I
M
M
A
A
V
A
I
A
A
A
V
V
V
I
V
V
A
T
V
T
I
T
T
A
Q
V
Q
I
Q
Q
A
Table 13: according to SEQ ID NO: 23. preferred variants and motifs of HLA-A24 peptides of 24 and 25
Longer (elongated) peptides may also be suitable. MHC class I epitopes (typically 8 to 11 amino acids in length) may result from processing of peptides from longer peptides or proteins containing the actual epitope. Residues flanked by actual epitopes are preferably residues that hardly affect the proteolytic cleavage required to expose the actual epitope during processing.
The peptides of the invention may be elongated by up to four amino acids, i.e. 1, 2, 3 or 4 amino acids, and may be added to either end in any combination between 4: 0 and 0: 4. The elongated combinations of the present invention are shown in Table 14.
Table 14: elongated combinations of the peptides of the invention
The stretched/extended amino acid may be the pro-sequence peptide of the protein or any other amino acid. Elongation may be used to enhance the stability or solubility of the peptide.
Thus, the epitope described herein may be identical to a native tumor-associated epitope or tumor-specific epitope, and may also include different peptides of no more than four residues from a reference peptide, so long as they have substantially the same antigenic activity.
In an alternative embodiment, one or both sides of the peptide are elongated by more than 4 amino acids, preferably up to a total length of 30 amino acids. This can result in MHC class II binding peptides. Binding to MHC class II peptides can be tested by methods known in the art.
Thus, the invention proposes peptides and variants of MHC class I epitopes, wherein the total length of the peptide or antibody is between 8 and 100, preferably between 8 and 30, most preferably between 8 and 14 amino acids in length (i.e. 10, 11, 12, 13, 14 amino acids, and if a class II binding peptide is elongated, 15, 16, 17, 18, 19, 20, 21 or 22 amino acids in length).
Of course, the peptides or variants of the invention are capable of binding to human Major Histocompatibility Complex (MHC) class I or II molecules. Binding of the peptide or variant to the MHC complex can be tested using methods known in the art.
Preferably, when peptide-specific T cells of the invention are tested in comparison to the substituted peptide, the peptide concentration is no more than about 1mM, preferably no more than about 1. mu.M, more preferably no more than about 1nM, even more preferably no more than about 100pM, and most preferably no more than about 10pM, if the solubility of the substituted peptide increases to half of the maximum relative to the background peptide. Also preferably, the substituted peptide is recognized by more than one T cell, a minimum of 2, more preferably 3.
In a particularly preferred embodiment of the invention, the peptide consists or consists essentially of a sequence according to SEQ ID NO: 1 to SEQ ID NO: 110, or a pharmaceutically acceptable salt thereof.
Substantially consisting of "means a peptide of the invention, except that according to SEQ ID NO: 1 to SEQ ID NO: 110 or a variant thereof, and further comprises amino acids located in other N-and/or C-terminal extensions, which are not necessarily capable of forming peptides as epitopes of MHC molecules.
However, these extended regions are important for efficiently introducing the peptide of the present invention into cells. In one embodiment of the present invention, the peptide is a part of a fusion protein, and contains 80N-terminal amino acids of HLA-DR antigen-associated invariant chain (p33, hereinafter referred to as "Ii") derived from NCBI and GenBank accession number X00497, and the like. In other fusions, the peptides of the invention may be fused to the sequences of the antibodies, or functional portions thereof, particularly human antibodies, as described herein, for specific targeting by the antibodies, or, for example, into dendritic cell-specific antibodies as described herein.
In addition, the peptide or variant may be further modified to improve stability and/or binding to MHC molecules, thereby eliciting a stronger immune response. Such optimization methods for peptide sequences are well known in the art and include, for example, the introduction of trans-peptide bonds and non-peptide bonds.
In the trans peptide bond amino acid, the peptide (-CO-NH-) is not linked to its residue, but its peptide bond is reversed. Such reverse-reverse mimetic peptides (retro-inverso peptidomimetics) can be prepared by methods known in the art, for example: the method described in Meziere et al (Meziere et al, 1997), incorporated herein by reference. This method involves the preparation of a mimetic peptide comprising a change in the backbone (rather than in the side chains). Studies by Meziere et al (Meziere et al, 1997) show that these mimetics are favorable for MHC binding and helper T cell responses. The reverse peptide with NH-CO bond to replace CO-NH peptide bond has greatly raised hydrolysis resistance.
Non-peptide bonds are-CH 2-NH, -CH2S-、-CH2CH2-、-CH＝CH-、-COCH2-、-CH(OH)CH2-and-CH2SO-, and the like. U.S. Pat. No. 4897445 proposes non-peptide bonds (-CH) in polypeptide chains2-NH) by standard procedures and by means of an aminoaldehyde and a NaCNBH-containing enzyme3The amino acids of (a) interact to synthesize non-peptide bonds.
Peptides containing the above sequences may be synthesized with other chemical groups at the amino and/or carboxy terminus of the peptide, thereby improving stability, bioavailability, and/or affinity of the peptide. For example, a hydrophobic group such as benzyloxycarbonyl or dansyl, or a tert-butoxycarbonyl group may be added to the amino terminus of the peptide. Similarly, acetyl or 9-fluorenylmethyloxycarbonyl may be located at the amino terminus of the peptide. Furthermore, a hydrophobic group, a t-butyloxycarbonyl group, or an amino group may be added to the carboxy terminus of the peptide.
In addition, all peptides of the present invention may be synthesized to change their spatial configuration. For example, it is possible to use the right-hand forms of one or more amino acid residues of these peptides, usually not the left-hand forms thereof. Further, at least one amino acid residue of the peptide of the present invention may be substituted with a known non-natural amino acid residue. Such changes may contribute to increased stability, bioavailability and/or binding of the peptides of the invention.
Also, the peptide or variant of the present invention may be chemically modified by reaction of specific amino acids before or after synthesis of the peptide. Examples of such modifications are well known in the art, and are summarized, for example, in "chemical reagents for Protein Modification" (3rd ed. crc Press, 2004) (Lundblad, 2004) by r.lundblad, which is incorporated herein by reference. Although there is no limitation to the chemical modification method of amino acids, it includes (but is not limited to) modification by the following methods: acylation, amidination, lysine pyridoxylation, reductive alkylation, trinitrophenylation of amino groups with 2, 4, 6-trinitrobenzenesulfonic acid (TNBS), amino modification of carboxyl groups and sulfhydryl groups by oxidation of cysteine performic acid to cysteic acid, formation of labile derivatives, formation of mixed disulfide compounds with other sulfhydryl compounds, reaction with maleimide, carboxymethylation with iodoacetic acid or iodoacetamide, carbamoylation with cyanate at basic pH. In this connection, the skilled worker refers to the extensive methods In connection with the chemical modification of proteins described In chapter 15 of Current Protocols In Protein Science (eds. Coligan et al (John Wiley and Sons NY 1995-2000)) (Coligan et al, 1995).
Briefly, the arginyl residues of modified proteins and the like are often based on the reaction of ortho-dicarbonyl compounds (e.g., benzaldehyde, 2, 3-butanedione, and 1, 2-hexenedione) to form adducts. Another example is the reaction of methylglyoxal with an arginine residue. Cysteine can be modified without concomitant modification at nucleophilic sites such as lysine and histidine. Thus, there are a number of reagents available for cysteine modification. The website of Sigma-Aldrich (http:// www.sigma-Aldrich. com) etc. contains information on the specific reagents.
Selective reduction of disulfide bonds in proteins is also common. Disulfide bonds may be formed and oxidized in biopharmaceutical heat treatments. Wood wade reagent K can be used to modify specific glutamic acid residues. N- (3-dimethylaminopropyl) -N' ethyl-carbodiimide can be used to form intramolecular cross-links of lysine and glutamic acid residues. For example: diethylpyrocarbonate is an agent that modifies histidine residues in proteins. Histidine can also be modified with 4-hydroxy-2-nonenal. The reaction of lysine residues with other alpha-amino groups, for example, facilitates peptide binding to the surface or cross-linking of the protein/peptide. Lysine poly is the attachment point for poly (ethylene) glycol and is also the primary modification site for protein glycosylation. Methionine residues of proteins can be modified by iodoacetamide, bromoethylamine, chloramine T, and the like.
Tetranitromethane and N-acetylimidazole can be used for the modification of tyrosine residues. The crosslinking via the di-tyrosine can be accomplished by hydrogen peroxide/copper ions.
Recent studies on tryptophan modification have used N-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide or 3-bromo-3-methyl-2- (2-nitrobenzenesulfo-l) -3H-indole (BPNS-skatole).
Successful modification of therapeutic proteins and peptides containing polyethylene glycol often results in prolonged circulation half-lives when crosslinking of the protein with glutaraldehyde, polyethylene glycol diacrylate and formaldehyde is used to formulate hydrogels. Chemical modification of allergens for immunotherapy is often achieved by carbamoylation of potassium cyanate.
A peptide or variant wherein the peptide is modified or contains non-peptide bonds, preferably an embodiment of the invention. In general, peptides and variants (containing at least peptide linkages between amino acid residues) can be synthesized using the solid phase peptide synthesis Fmoc-polyamide model disclosed by Lukas et al (Lukas et al, 1981) and the references cited therein. The fluorenylmethyloxycarbonyl (Fmoc) group provides temporary protection for the N-amino group. The cleavage was repeated using the highly base-sensitive protecting group in 20% dimethylpiperidine in N, N-dimethylformamide. The side chain function may be protected due to their butyl ethers (in the case of serine threonine and tyrosine), butyl esters (in the case of glutamic acid and aspartic acid), t-butyloxycarbonyl derivatives (in the case of lysine and histidine), trityl derivatives (in the case of cysteine) and 4-methoxy-2, 3, 6-trimethylbenzenesulfonyl derivatives (in the case of arginine). As long as glutamine and asparagine are C-terminal residues, the side chain amino function protection is provided by a 4, 4' -dimethoxydiphenyl group. The solid support is based on a polydimethylacrylamide polymer, which is composed of three monomers, dimethylacrylamide (backbone monomer), bisacryloylethylene diamine (crosslinker) and N-acryloylsarcosine methyl ester (functional agent). The peptide-resin coupling agent used is an acid-sensitive 4-hydroxymethylphenoxyacetic acid derivative. All amino acid derivatives were added as their preformed symmetrical anhydride derivatives, except asparagine and glutamine, which were added using a reversed N, N-dicyclohexylcarbodiimide/1-hydroxybenzotriazole mediated coupling procedure. All coupling and deprotection reactions were monitored using ninhydrin, nitrobenzenesulfonic acid, or isotin test procedures. After completion of the synthesis, the peptide was cleaved from the resin support with concomitant removal of the side chain protecting groups using trifluoroacetic acid at a concentration of 95% containing 50% scavenger mix. Commonly used scavenger mixtures include ethanedithiol, phenol, anisole and water, the exact choice being based on the amino acid composition of the synthetic peptide. Furthermore, solid-phase and liquid-phase methods in combination are possible for peptide synthesis (see, for example, Bruckdorfer et al, 2004 and references cited therein).
Trifluoroacetic acid was removed by evaporation in vacuo, followed by titration with diethyl ether bearing the crude peptide. Any scavenger mixture present was purged using a simple extraction procedure (after lyophilization of the aqueous phase, which produced peptides without scavenger mixture). Peptide synthesis reagents are generally available from Calbiochem-Novabiochem (Nonburg, England).
Purification can be carried out by any one or a combination of the following techniques, such as: recrystallization, size exclusion chromatography, ion exchange chromatography, hydrophobic interaction chromatography, and (typically) reversed phase high performance liquid chromatography (e.g., using an acetonitrile/water gradient separation).
Peptide analysis can be performed using thin layer chromatography, electrophoresis, particularly capillary electrophoresis, solid phase extraction (CSPE), reverse phase high performance liquid chromatography, amino acid analysis after acid hydrolysis, Fast Atom Bombardment (FAB) mass spectrometry, and MALDI and ESI-Q-TOF mass spectrometry.
To select for over-presented peptides, a presentation graph is calculated showing the amount of bit-present as well as the variation in replication in the sample. This feature allows the baseline values of the relevant tumor entity samples to be aligned with those of normal tissue samples. Each of the above features can be incorporated into the overpresentation score by calculating the p-value that adjusts the linear mixture effect model (Pinheiro et al, 2015), thus adjusting the multiple test by false discovery rate (Benjamini and Hochberg, 1995).
For identification and relative quantification of HLA ligands by mass spectrometry, HLA molecules from shock frozen tissue samples are purified and HLA-related peptides are isolated. The isolated peptides were separated and identified by an on-line nano-electrospray-ionization (nanoESI) liquid chromatography-spectroscopy (LC-MS) experiment. The peptide sequences thus generated were verified by comparing the pattern of fragments of native TUMAP recorded in lung cancer (NSCLC) samples (N91 a × 02 positive samples, N80 a × 24 positive samples) with the pattern of fragments of corresponding synthetic reference peptides of the same sequence. Since these peptides were directly identified as ligands for HLA molecules of primary tumors, these results provide direct evidence for the determination of the natural processing and presentation of peptides on primary cancer tissues obtained from 155 patients with lung cancer (NSCLC).
Discovering a pipelinev2.1 (see, e.g., US 2013-. This is achieved by the following method: the label-free differential quantification method is developed using LC-MS acquisition data processed by proprietary data analysis pipelines, in combination with sequence recognition algorithms, spectral clustering, calculating ions, retention time adjustment, state of charge convolution and normalization.
Levels of presentation were established for each peptide and sample, including error estimates. Peptides that are abundantly presented in tumor tissues and peptides that are excessively presented in tumor and non-tumor tissues and organs have been identified.
HLA peptide complexes from lung cancer (NSCLC) tissue samples were purified and HLA-related peptides were isolated and analyzed using LC-MS (see examples). All TUMAPs contained in the present application were identified using methods of primary lung cancer (NSCLC) sampling, confirming their presentation on primary lung cancer (NSCLC).
TUMAP determined on multiple lung cancers (NSCLC) and normal tissues was quantified using an ion-counting method with unlabeled LC-MS data. This method assumes that the LC-MS signal region of the peptide correlates with its abundance in the sample. All quantitative signals for peptides in various LC-MS experiments were normalized on a central trend basis, averaged per sample, and combined with a human histogram (referred to as a presentation graph). The presentation graph integrates different analytical methods, such as: protein database retrieval, spectral clustering, state of charge convolution (neutralization) and retention time calibration and normalization.
In addition, a pipeline is foundv2.x direct absolute quantification of MHC-peptides, preferably HLA-restricted peptides, on cancer or other infected tissues. Briefly, the total cell count is calculated from the total DNA content of the tissue sample being analyzed. The total peptide amount of TUMAP in tissue samples was determined by nanoLC-MS/MS as the ratio of native TUMAP and known amount of isotopically labeled version of TUMAP, referred to as internal standard. TUMAP separation efficiency determination method: the peptide: MHC for all selected TUMAPs were added to tissue lysates at the earliest time point of the TUMAP isolation procedure and detected by nanoLC-MS/MS after completion of peptide isolation. Total cell counts and total peptide amounts were calculated from three measurements per tissue sample. The peptide-specific separation efficiency was calculated as the average of 10 spiking experiments measured in triplicate (see example and table 22).
The present invention proposes a therapeutic benefit for the treatment of cancerous tumors, preferably lung cancer that presents either in excess or only the peptides of the invention. These peptides were directly revealed by mass spectrometry, but were naturally presented by HLA molecules in human primary human lung cancer (NSCLC) specimens.
Many of the source genes/proteins from which peptides are highly overexpressed in cancer (also designated as "full-length proteins" or "potential proteins") compared to normal tissues-the "normal tissues" to which the invention relates are healthy lung cells or other normal tissue cells, indicating a high association of the tumor with these source genes (see example 2). Furthermore, these peptides are also over-presented themselves in tumor tissue ("tumor tissue" in connection with the present invention refers to a sample from a lung cancer (NSCLC) patient), but not in normal tissue (see example 1).
HLA-binding peptides are recognized by the immune system, particularly T lymphocytes. T cells can destroy cells presenting the recognized HLA/peptide complex (e.g., lung cancer cells presenting peptide derived therefrom).
All peptides of the invention have been shown to have the ability to stimulate a T cell response and are presented in excess and thus can be used to prepare antibodies and/or TCRs of the invention, e.g., soluble TCRs (see example 3 and example 4). Furthermore, peptides, when combined with the corresponding MHC, may also be used to prepare the antibodies, specific binding fragments thereof, antibody-like binders and/or TCRs, in particular stcrs, of the invention. The respective methods are well known to the skilled worker and can be found in the respective literature. Thus, the peptides of the invention can be used to generate an immune response in a patient, thereby enabling the destruction of tumor cells. The immune response in a patient can be induced by direct administration of the peptide or precursor (e.g., an elongated peptide, protein, or nucleic acid encoding such peptide) to the patient, preferably in combination with an agent that enhances immunogenicity. The immune response derived from this therapeutic vaccine is expected to be highly specific against tumor cells, since the target peptides of the invention present a smaller number of replications on normal tissues, preventing the risk of the patient of an adverse autoimmune response against normal cells.
In another aspect of the invention, a method of making a soluble T cell receptor (sTCR) that recognizes a specific peptide-MHC complex is provided. Such soluble T cell receptors can be generated from specific T cell clones, and their affinity can be increased by complementarity determining region-targeted mutagenesis. For the purpose of T cell receptor selection, phage display may be used (us 2010/0113300, (likdy et al, 2012)). For the purpose of stabilizing T cell receptors during phage display and when actually used as drugs, the alpha and beta chains may be linked by non-native disulfide bonds, other covalent bonds (single chain T cell receptors), or by dimerization domains (Boulter et al, 2003; Card et al, 2004; Willcox et al, 1999). T cell receptors can be linked to toxins, drugs, cytokines (see US 2013/0115191), domains recruit effector cells, such as anti-CD 3 domains, etc., in order to perform specific functions on target cells. In addition, it may be expressed in T cells for adoptive transfer. Further information can be found in WO 2004/033685a1 and WO 2004/074322a 1. Combinations of stcrs are described in WO2012/056407a 1. Other methods of preparation are disclosed in WO 2013/057586a 1.
"pharmaceutical composition" refers to a composition suitable for use in a medical facility for the human body. Preferably, the pharmaceutical compositions are sterile and manufactured according to GMP guidelines.
The pharmaceutical composition comprisesPeptide in isolated form or in the form of a pharmaceutically acceptable salt (see also above). As used herein, "pharmaceutically acceptable salt" refers to a derivative of the disclosed peptide wherein the peptide is modified by making acid or base salts of pharmaceutical agents. For example, with a free base which reacts with a suitable acid (usually where the neutral drug has a neutral-NH group)2Group) to prepare the acid salt. Suitable acids for preparing the acid salts include organic acids such as: acetic, propionic, hydroxy, pyruvic, oxalic, malic, malonic, succinic, maleic, fumaric, tartaric, citric, benzoic, cinnamic, mandelic, methanesulfonic, benzenesulfonic, salicylic, and the like, as well as inorganic acids such as: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. In contrast, base salt formulations of acidic groups that may be presented on a peptide are prepared using pharmaceutically acceptable bases, such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine, and the like.
In a particularly preferred embodiment, the pharmaceutical composition comprises the peptide in the form of acetic acid (acetate), trifluoroacetate or hydrochloric acid (chloride).
The agent of the invention is preferably an immunotherapeutic agent, for example, a vaccine. The vaccine may be administered directly to the affected organ of the patient, either systemically by i.d., i.m., s.c, i.p., and i.v. injection, or applied in vitro to cells from the patient or a cell line thereof (which are then injected into the patient), or in vitro to a subpopulation of cells from immune cells from the patient (which are then re-administered to the patient). If the nucleic acid is injected into the cells in vitro, it may be beneficial to transfect the cells to co-express an immunostimulatory cytokine (e.g., interleukin-2). The peptides may be administered entirely alone, in combination with an immunostimulating adjuvant (see below), or in combination with an immunostimulating cytokine, or in a suitable delivery system (e.g. liposomes). The peptide may also be conjugated to form a suitable carrier, such as Keyhole Limpet Hemocyanin (KLH) or mannoprotein (see WO 95/18145 and (Longenecker et al, 1993)). The peptide may also be labeled, may be a fusion protein, or may be a hybrid molecule. Peptides of the sequences given in the present invention are expected to stimulate CD4 or CD8T cells. However, CD8T cells stimulated more effectively with the help of CD 4T-helper cells. Thus, a fusion partner or fragment of a hybrid molecule provides an appropriate epitope for stimulation of CD4 positive T cells for stimulation of MHC class I epitopes of CD8T cells. CD 4-and CD 8-stimulating epitopes are well known in the art and include the epitopes identified in the present invention.
In one aspect, the vaccine comprises a polypeptide comprising at least SEQ ID NO: 1 to SEQ ID NO: 110, preferably 2 to 50, more preferably 2 to 25, even more preferably 2 to 20, most preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 peptides. The peptide may be derived from one or more specific TAAs and may bind to an MHC class I molecule
In another aspect, the invention features a nucleic acid (e.g., a polynucleotide) encoding a peptide or peptide variant of the invention. A polynucleotide may be, for example, DNA, cDNA, PNA, RNA, or combinations thereof, which may be single-and/or double-stranded, or a native or stabilized form of a polynucleotide (e.g., a polynucleotide having a phosphorothioate backbone), and which may or may not contain an intron so long as it encodes a peptide. Of course, polynucleotides can only encode peptides that incorporate natural peptide bonds and contain natural amino acid residues. In another aspect, the invention features an expression vector for expressing a polypeptide according to the invention.
For the ligation of polynucleotides, various methods have been developed, particularly for DNA, ligation can be performed by a method of supplementing a vector with a ligatable end or the like. For example, a complementary homopolymer track can be added to the DNA fragment, after which the DNA fragment is inserted into the vector DNA. The vector and DNA fragment are then bound by hydrogen bonding of the complementary homopolymer tail, thereby forming a recombinant DNA molecule.
Synthetic linkers containing one or more cleavage sites provide an alternative method for ligating DNA fragments into vectors. Synthetic linkers containing various restriction endonucleases are commercially available through a variety of tubes, including from International Biotechnology Inc., New Haven, CN, USA.
A desirable modification method for DNA encoding a polypeptide of the present invention is the polymerase chain reaction method employed by Saiki et al (Saiki et al, 1988). This method can be used to introduce the DNA into a suitable vector (e.g., by designing appropriate cleavage sites), and can also be used to modify the DNA by other useful methods known in the art. If viral vectors are used, either poxvirus vectors or adenoviral vectors are preferred.
The DNA (or RNA in the case of retroviral vectors) may then be expressed in a suitable host to produce a polypeptide comprising a peptide or variant of the invention. Thus, a DNA encoding a peptide or variant of the invention may be used according to known techniques, suitably modified by the methods described herein, to construct an expression vector, which is then used to transform a suitable host cell, thereby expressing and producing a polypeptide of the invention. Such techniques include those disclosed in, for example, U.S. patents 4,440,859, 4,530,901, 4,582,800, 4,677,063, 4,678,751, 4,704,362, 4,710,463, 4,757,006, 4,766,075, and 4,810,648.
DNA (or RNA in the case of retroviral vectors) encoding a polypeptide comprising a compound of the invention may be added to a variety of other DNA sequences for introduction into a suitable host. The companion DNA will depend on the nature of the host, the manner in which the DNA is introduced into the host, and whether it needs to be maintained episomally or bound to each other.
In general, the DNA can be attached to an expression vector (e.g., a plasmid) in the proper orientation and correct expression reading frame. If necessary, the DNA may be linked to corresponding transcriptional and translational regulatory control nucleotide sequences recognized by the desired host, although such control functions are typically present in expression vectors. The vector is then introduced into the host by standard methods. In general, not all hosts will be transformed by the vector. Therefore, it is necessary to select transformed host cells. The selection method involves the insertion of a DNA sequence encoding a selectable attribute (e.g., antibiotic resistance) in the transformed cell into the expression vector using any necessary control elements.
Alternatively, the gene having such selective properties may be on another vector which is used to co-transform the desired host cell. Host cells transformed with the recombinant DNA of the invention are then cultured under suitable conditions, as described herein, familiar to those skilled in the art, for a time sufficient to express the peptide that can be recovered thereafter.
There are many known expression systems, including bacteria (e.g., E.coli and Bacillus subtilis), yeasts (e.g., yeast), filamentous fungi (e.g., Aspergillus), plant cells, animal cells, and insect cells. The system may preferably be mammalian cells, such as CHO cells from the ATCC Cell Biology Collection (Cell Biology Collection).
Typical mammalian cell constitutive expression vector plasmids include the CMV or SV40 promoter with a suitable poly-A tail, and resistance markers (e.g., neomycin). An example is pSVL obtained from Pharmacia (Piscataway, New Jersey, USA). An example of an inducible mammalian expression vector is pMSG, also available from Pharmacia. Useful yeast plasmid vectors are pRS403-406 and pRS413-416, generally available from Stratagene cloning Systems, La Jolla, CA 92037, USA. Plasmids pRS403, pRS404, pRS405 and pRS406 are yeast integrative plasmids (YIp) with the insertion of the yeast selectable markers HIS3, TRP1, LEU2 and URA 3. The pRS413-416 plasmid is a yeast centromere plasmid (Ycp). The CMV promoter-based vector (e.g., from Sigma-Aldrich) provides transient or stable expression, cytoplasmic expression or secretion, as well as N-terminal or C-terminal markers in various combinations of FLAG, 3xFLAG, C-myc, or MATN. These fusion proteins can be used for detection, purification and analysis of recombinant proteins. Dual label fusion provides flexibility for detection.
The strong human Cytomegalovirus (CMV) promoter regulatory region resulted in expression levels of constitutive proteins in COS cells as high as 1 mg/L. For weaker cell lines, protein levels are generally below 0.1 mg/L. The presence of the SV40 replication origin will result in high levels of DNA replication in SV40 replication competent COS cells. For example, CMV vectors may comprise the origin of pMB1 (a derivative of pBR 322) replication in bacterial cells, the calcium-lactamase gene for ampicillin resistance selection in bacteria, the origin of hGH polyA and f 1. Vectors containing preproinsulin leader (PPT) sequences can be secreted into the medium for purification using anti-FLAG antibodies, resins and plate-directed FLAG fusion proteins. Other vectors and expression systems for use with a variety of host cells are well known in the art.
In another embodiment, two or more peptides or peptide variants of the invention are encoded and, thus, expressed in a sequential order (similar to a "string of beads" construct). To achieve this, the peptides or peptide variants may be linked or fused together by extensions of linker amino acids (e.g., LLLLLL), or may be linked without any additional peptide between them. These constructs are also useful in cancer therapy, and induce immune responses involving MHC class I and MHC class II molecules.
The invention also relates to a host cell transformed with the polynucleotide vector construct of the invention. The host cell may be a prokaryotic cell or a eukaryotic cell. In some instances, bacterial cells are preferred prokaryotic host cells, typically E.coli strains, e.g., E.coli strain DH5 (obtained from Bethesda Research Laboratories, Inc. (Bethesda, Md., USA)) and RR1 (obtained from American type culture Collection (ATCC, Rockville, Md., USA), ATCC No. 31343). Preferred eukaryotic host cells include yeast, insect and mammalian cells, preferably vertebrate cells, such as: mouse, rat, monkey or human fibroblasts and colon cancer cell lines. Yeast host cells include YPH499, YPH500 and YPH501, and are generally available from Stratagene Cloning Systems, Inc. (La Jolla, CA92037, USA). The preferred mammalian host cells include CCL61 cells from ATCC as Chinese Hamster Ovary (CHO) cells, CRL 1658 cells from ATCC as NIH Swiss mouse embryo cells NIH/3T3, CRL 1650 cells from ATCC as monkey kidney-derived COS-1 cells, and 293 cells from human embryonic kidney cells. The preferred insect cell is Sf9 cell, which can be transfected with baculovirus expression vector. A summary of the selection of suitable host cells for Expression can be found in textbooks (Paulina Balb and old Argelia Lorence. Methods in Molecular Biology recombination Gene Expression, Reviews and Protocols, Part One, Second Edition, ISBN 978-1-58829-262-9) and other documents known to the person skilled in the art.
Transformation of a suitable host cell containing a DNA construct of the invention may be accomplished using well known methods, generally depending on the type of vector used. For the transformation of prokaryotic host cells, see, for example, Cohen et al (Cohen et al, 1972) and (Green and Sambrook, 2012). Transformation of yeast cells is described in Sherman et al (Sherman et al, 1986). The methods described in Beggs (Beggs, 1978) are also useful. For vertebrate cells, reagents for transfecting these cells, etc., e.g., calcium phosphate and DEAE-dextran or liposome formulations, are available from Stratagene Cloning Systems, Inc. or Life Technologies, Inc. (Gaithersburg, MD 20877, USA). Electroporation may also be used to transform and/or transfect cells, and is a well-known method used in the art for transforming yeast cells, bacterial cells, insect cells, and vertebrate cells.
Successfully transformed cells (i.e.cells containing the DNA construct of the invention) can be identified by well-known methods, such as PCR. Alternatively, proteins present in the supernatant may be detected using antibodies.
It will be appreciated that certain host cells of the invention are useful for the production of peptides of the invention, such as bacterial cells, yeast cells and insect cells. However, other host cells may be useful for certain therapeutic approaches. For example, antigen presenting cells (e.g., dendritic cells) can be used to express the peptides of the invention, allowing them to be loaded with the corresponding MHC molecules. Thus, the present invention provides a host cell comprising a nucleic acid or expression vector of the invention.
In a preferred embodiment, the host cell is an antigen-presenting cell, in particular a dendritic cell or an antigen-presenting cell. On 29/4/2010, recombinant fusion proteins containing Prostatic Acid Phosphatase (PAP) were approved by the U.S. Food and Drug Administration (FDA) for treatment of asymptomatic or mildly symptomatic metastatic HRPC (Rini et al, 2006; Small et al, 2006).
In another aspect, the invention features a method of formulating a peptide and variants thereof, the method including culturing a host cell and isolating the peptide from the host cell or culture medium thereof.
In another embodiment, the peptide, nucleic acid or expression vector of the invention is used in medicine. For example, the peptide or variant thereof may be prepared as an intravenous (i.v.) injection, a subcutaneous (s.c.) injection, an intradermal (i.d.) injection, an intraperitoneal (i.p.) injection, an intramuscular (i.m.) injection. Preferred methods of peptide injection include s.c., i.d., i.p., i.m., and i.v. injections. Preferred methods of DNA injection are i.d., i.m., s.c., i.p., and i.v. injection. For example, 50. mu.g to 1.5mg, preferably 125. mu.g to 500. mu.g, of peptide or DNA is administered, depending on the particular peptide or DNA. The above dosage ranges were successfully used in previous trials (Walter et al, 2012).
Polynucleotides used for active immunization may be in substantially purified form, or may be coated onto a carrier or delivery system. The nucleic acid may be DNA, cDNA, PNA, RNA, or a combination thereof. Methods for the design and introduction of such nucleic acids are well known in the art. For example, there is a summary thereof in the literature (Teufel et al, 2005). Polynucleotide vaccines are readily prepared, but the mode of action of these vectors to induce an immune response is not fully understood. Suitable vectors and delivery systems include viral DNA and/or RNA, such as systems based on adenovirus, vaccinia virus, retrovirus, herpes virus, adeno-associated virus, or mixed viruses containing more than one viral element. Non-viral delivery systems, including cationic liposomes and cationic polymers, are well known in the art for DNA delivery. Physical delivery systems, such as by "gene gun," can also be used. The peptide or nucleic acid encoding the peptide can be a fusion protein, e.g., containing an epitope that stimulates T cells to perform the above-described CDRs.
The agents of the invention may also include one or more adjuvants. Adjuvants are those substances that non-specifically enhance or potentiate an immune response (e.g., an immune response to an antigen mediated by CD 8-positive T cells and helper T (TH) cells and are therefore considered useful for the agents of the invention AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, flagellin or flagellin-derived TLR5 ligand, FLT3 ligand, GM-CSF, IC30, IC31, imiquimodresiquimod, ImuFactIMP321, interleukins IL-2, IL-13, IL-21, interferon alpha or beta, or polyethylene glycol derivatives thereof, IS Patch, ISS, ISCOMATRIX, ISCOMs,Lipovac, MALP2, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, oil-in-water and water-in-oil emulsions, OK-432, OM-174, OM-197-MP-EC, ONTAK, OspA, and mixtures thereof,Carrier system, polylactide-based composite glycolide [ PLG]And dextran microparticles, recombinant human lactoferrin SRL172, viral and other virus-like particles, YF-17D, VEGF trap, R848, β -glucan, Pam3Cys, QS21 stimulators from Aquila corporation derived from saponin, mycobacterial extracts, and bacterial cell wall synthesis mimics, as well as other proprietary adjuvants such as: ribi's Detox, Quil or Superfos. Preferred adjuvants are, for example: freund's adjuvant or GM-CSF. Several dendritic cell-specific immunoadjuvants (e.g. MF59) and methods for their preparation have been described (Allison and Krummel, 1995). Cytokines may also be used. Some cytokines directly affect dendritic cell migration to lymphoid tissues (e.g., TNF-), accelerate dendritic cell maturation to effective antigen presenting cells for T lymphocytes (e.g., GM-CSF, IL-1, and IL-4) (U.S. patent No. 5849589, particularly the fully incorporated form thereof and herein), and serve as immunological adjuvants (e.g., IL-12, IL-15, IL-23, IL-7, IFN- α, IFN- β) (Gabrilovich et al, 1996).
CpG immunostimulatory oligonucleotides are reported to enhance the effect of adjuvants in vaccines. Without being bound by theory, CpG oligonucleotides can function by initiating the innate (non-adaptive) immune system through Toll-like receptors (TLRs), mainly TLR 9. CpG-induced TLR9 activation enhances antigen-specific humoral and cellular responses to a variety of antigens, including peptide or protein antigens, live or killed viruses, dendritic cell vaccines, autologous cell vaccines, and polysaccharide conjugates in prophylactic and therapeutic vaccines. More importantly, it enhances dendritic cell maturation and differentiation, leading to enhanced activation of TH1 cells and enhanced Cytotoxic T Lymphocyte (CTL) production, even with the loss of CD4T cell repertoire. The activation-induced TH1 bias of TLR9 was maintained even in the presence of vaccine adjuvants such as: alum or Freund's incomplete adjuvant (IFA) which normally promotes TH2 migration. CpG oligonucleotides, when prepared or co-administered with other adjuvants or formulations such as microparticles, nanoparticles, fat emulsions or the like, exhibit enhanced adjuvant activity, which is particularly necessary to induce a strong response when the antigen is relatively weak. They also accelerated the immune response, reducing antigen dose by about two orders of magnitude, and in some experiments, produced similar antibody responses to full dose vaccines without CpG (Krieg, 2006). U.S. Pat. No. 4, 6406705, 1 describes the use of CpG oligonucleotides, non-nucleic acid adjuvants and antigens in combination to elicit an antigen-specific immune response. One CpG TLR9 antagonist is dlim (dual stem-loop immunomodulator) from Mologen, berlin, germany, which is a preferred ingredient of the pharmaceutical composition of the present invention. Other TLR-binding molecules, such as: the RNA binds TLR7, TLR8, and/or TLR 9.
Other examples of useful adjuvants include, but are not limited to, chemically modified CpG (e.g., CpR, Idera), dsRNA mimetics, e.g., Poly (I: C) and its derivatives (e.g., AmpliGen, Hiltonol, Poly- (ICLC), Poly (IC-R), Poly (I: C12U)), non-CpG bacterial DNA or RNA, and immunologically active small molecules and antibodies, e.g.: cyclophosphamide, sumicitizumab,Celecoxib, NCX-4016, sildenafil, tadalafil, vardenafil, sorafenib, temozolomide, temsirolimus, XL-999, CP-547632, pazopanib, VEGF Trap, ZD2171, AZD2171, anti-CTLA 4, other antibody targeting major structures of the immune system (e.g., anti-CD 40, anti-TGF β, anti-TNF α receptor) and SC58175, all of which may have therapeutic effects and/or act as adjuvants. The skilled artisan can readily determine the amounts and concentrations of adjuvants and additives useful in the present invention without undue experimentation.
Preferred adjuvants are particulate formulations of anti-CD 40, imiquimod, resiquimod, GM-CSF, cyclophosphamide, sunitinib, bevacizumab, interferon alpha, CpG oligonucleotides and derivatives, poly (I: C) and derivatives, RNA, sildenafil and PLG or viral particles.
In a preferred embodiment of the pharmaceutical composition of the invention, the adjuvant is selected from the group consisting of colony stimulating factor-containing preparations, such as granulocyte macrophage colony stimulating factor (GM-CSF, sargrastim), cyclophosphamide, imiquimod, resiquimod and interferon- α.
In a preferred embodiment of the pharmaceutical composition of the invention, the adjuvant is selected from the group consisting of colony stimulating factor-containing preparations, such as granulocyte macrophage colony stimulating factor (GM-CSF, sargrastim), cyclophosphamide, imiquimod and resimiquimod. In a preferred embodiment of the pharmaceutical composition of the invention, the adjuvant is cyclophosphamide, imiquimod or resiquimod. More preferred adjuvants are Montanide IMS 1312, Montanide ISA 206, Montanide ISA50V, Montanide ISA-51, poly-ICLCAnd anti-CD 40mAB or a combination thereof.
The composition can be administered by parenteral injection, such as subcutaneous injection, intradermal injection, intramuscular injection, or oral administration. For this purpose, the peptides and other selective molecules are dissolved or suspended in a pharmaceutically acceptable carrier, preferably an aqueous carrier. In addition, the composition may comprise adjuvants such as: buffers, binders, impactors, diluents, flavorants, lubricants, and the like. These peptides may also be used in combination with immunostimulatory substances, such as: a cytokine. Further adjuvants which can be used in such compositions are known from Handbook of pharmaceutical excipients (Kibbe, 2000) et al, by a. The combination is useful for the prevention, prophylaxis and/or treatment of adenoma or cancerous disease. For example, there are exemplified formulations in EP 2112253.
It is important to recognize that the immune response elicited by the vaccine of the present invention attacks cancer at different cellular stages and at different stages of development. And different cancer-related signaling pathways are attacked. This has the advantage over other vaccines that target only one or a few targets, which may lead to easy adaptation of the tumor to attack (tumor escape). Furthermore, not all individual tumors express the same pattern of antigens. Thus, the combination of several tumor-associated peptides ensures that each tumor bears at least some of the targets. The composition is designed in such a way that it is expected that each tumor can express several antigens and cover several independent pathways required for tumor growth and maintenance. Thus, the vaccine can be readily "off-the-shelf" for use in a larger patient population. This means that patients pre-selected for vaccine treatment can be restricted to HLA typing without any additional biomarker assessment of antigen expression, but still ensure that multiple targets are simultaneously challenged by an induced immune response, which is important for therapeutic efficacy (Banchereau et al, 2001; Walter et al, 2012).
The term "scaffold" as used herein refers to a molecule that specifically binds to a (e.g. antigenic) determinant. In one embodiment, the scaffold is capable of directing the entity (e.g., the (second) antigen-binding moiety) to which it is attached to a target of interest, e.g., to a specific type of tumor cell or tumor substrate bearing an antigenic determinant (e.g., a complex of a peptide and MHC according to the present application). In another embodiment, the scaffold is capable of initiating a signaling pathway through its target antigen (e.g., a T cell receptor complex antigen). Scaffolds include, but are not limited to, antibodies and fragments thereof, antigen-binding regions of antibodies comprising antibody heavy chain variable regions and antibody light chain variable regions, bound proteins including at least one ankyrin repeat motif and Single Domain Antigen Binding (SDAB) molecules, aptamers, (soluble) TCRs, and (modified) cells, such as allogeneic or autologous T cells. To assess whether a certain molecule is a scaffold bound to a target, a binding assay can be performed.
By "specific" binding is meant that the scaffold binds better to the peptide-MHC complex of interest than to other native peptide-MHC complexes to the extent that a scaffold possessing an active molecule capable of killing cells bearing a particular target is not capable of killing another cell not bearing the particular target but presenting one or more other peptide-MHC complexes. If the cross-reactive peptide-MHC peptide is not native, i.e., not from the human HLA-polypeptide group, binding to other peptide-MHC complexes is not critical. Assays to assess target cell killing are well known in the art. They should be carried out with target cells (primary cells or cell lines) or cells loaded with peptides, which contain unaltered peptide-MHC presentation, in order to reach the level of native peptide-MHC.
Each scaffold may include a label that detects binding to the scaffold by determining the presence or absence of a signal provided by the tag. For example, the scaffold may be labeled with a fluorescent dye or any other suitable cellular marker molecule. Such marker molecules are well known in the art. For example, fluorescent labeling by a fluorescent dye may provide visualization of the bound aptamer by fluorescence or laser scanning microscopy or flow cytometry.
Each scaffold can be conjugated to a second active molecule (e.g., IL-21, anti-CD 3, anti-CD 28).
For further information on polypeptide scaffolds see, for example, the background section of WO 2014/071978A1, incorporated herein by reference.
The invention also relates to aptamers. Aptamers (see, e.g., WO2014/191359 and references cited therein) are short single-stranded nucleic acid molecules that can fold into a defined three-dimensional structure and recognize a specific target structure. They appear to be suitable alternatives for the development of targeted therapies. Aptamers have been shown to selectively bind complex targets with high affinity and specificity.
Aptamers that recognize cell surface molecules have been identified over the past decade and provide a means for developing diagnostic and therapeutic methods. Aptamers are promising candidates for biomedical applications, since they have been shown to be almost non-toxic and immunogenic. In fact, aptamers, such as prostate specific membrane antigen recognition aptamers, have been successfully used for targeted therapy and have been shown to function in xenografts in vivo models. In addition, it is recognized that aptamers to specific tumor cell lines have also been identified.
DNA aptamers can be selected to reveal a broad spectrum of signature properties of various cancer cells, particularly those from solid tumors, while non-tumorigenic and predominantly healthy cells are not recognized. If the aptamers identified not only recognize tumor-specific subtypes, but also interact with a range of tumors, this makes the aptamers suitable as so-called broad-spectrum diagnostic and therapeutic tools.
Furthermore, studies of cell binding behavior with flow cytometry showed that aptamers displayed good affinity in the nanomolar range.
Aptamers are used for diagnostic and therapeutic purposes. In addition, it may also be shown that some aptamers are taken up by tumor cells and thus can enter tumor cells as molecular excipients for targeted delivery of anticancer agents, such as siRNA.
Aptamers can be selected against targets of complexes such as cells and tissues and peptide complexes and MHC molecules according to the current invention comprising, preferably including, a sequence according to any of SEQ ID NO 1 to SEQ ID NO 110 using the cellular SELEX (systematic evolution of ligands by exponential enrichment) technique.
The peptides of the invention are useful for the generation and development of specific antibodies against MHC/peptide complexes. These antibodies are useful in therapy to target toxins or radioactive substances to diseased tissue. Another use of these antibodies is to target radionuclides to diseased tissue for imaging purposes (e.g., PET). This may help to detect small metastases or to determine the size and accurate location of diseased tissue.
Thus, another aspect of the invention is directed to a method of generating a recombinant antibody that specifically binds to class I or II human Major Histocompatibility Complex (MHC) complexed with an HLA-restricted antigen, the method comprising: immunizing a genetically engineered non-human mammal comprising a molecule expressing said Major Histocompatibility Complex (MHC) class I or II with a soluble form of a (MHC) class I or II molecule complexed with an HLA-restricted antigen; separating the mRNA molecules from antibodies raised to said non-human mammalian cells; generating a phage display library displaying protein molecules encoded by said mRNA molecules; and separating at least one bacteriophage from said phage display library, said at least one bacteriophage displaying said antibody specifically binding to said human Major Histocompatibility Complex (MHC) class I or II complexed to an HLA-restricted antigen.
Another aspect of the invention provides an antibody that specifically binds to a class I or II human Major Histocompatibility Complex (MHC) complexed to an HLA-restricted antigen, wherein the antibody is preferably a polyclonal antibody, a monoclonal antibody, a bispecific antibody and/or a chimeric antibody.
Corresponding methods for producing such antibodies and single chain class I major histocompatibility complexes, as well as other tools for producing such antibodies, are disclosed in WO 03/068201, WO 2004/084798, WO 01/72768, WO 03/070752, and publications (Cohen et al, 2003 a; Cohen et al, 2003 b; Denkberg et al, 2003), all references being incorporated herein by reference in their entirety for the purposes of the present invention.
Preferably, the binding affinity of the antibody to the complex is less than 20 nanomolar, preferably less than 10 nanomolar, which is also considered to be "specific" in the context of the present invention.
The present invention relates to a peptide comprising a sequence selected from SEQ ID NO: 1 to SEQ ID NO: 110 or a sequence of the group of SEQ ID NO: 1 to SEQ ID NO: 110, or a variant which induces a T cell cross-reaction with said variant peptide, wherein said peptide is not substantially a full-length polypeptide.
The invention further relates to a peptide comprising a sequence selected from SEQ ID NO: 1 to SEQ ID NO: 110, or a sequence identical to one of the group of SEQ ID NO: 1 to SEQ ID NO: 110, wherein the total length of the peptide or variant is from 8 to 100, preferably from 8 to 30, most preferably from 8 to 14 amino acids.
The invention further relates to peptides of the invention having the ability to bind to a Major Histocompatibility Complex (MHC) class I or II molecule.
The invention further relates to a peptide according to the invention, wherein the peptide consists or consists essentially of a sequence according to SEQ ID NO: 1 to SEQ ID NO: 110.
The invention further relates to a peptide of the invention, wherein the peptide is (chemically) modified and/or comprises non-peptide bonds.
The invention further relates to a peptide of the invention, wherein the peptide is part of a fusion protein, in particular comprising the N-terminal amino acid of HLA-DR antigen associated invariant chain (Ii), or wherein the peptide is fused to an antibody, e.g., a dendritic cell specific antibody.
The invention further relates to a nucleic acid encoding a peptide according to the invention, with the proviso that the peptide is not a complete (fully) human protein.
The invention further relates to a nucleic acid according to the invention, being DNA, cDNA, PNA, RNA, and possibly a combination thereof.
The invention further relates to an expression vector capable of expressing the nucleic acid of the invention.
The invention further relates to a peptide according to the invention, a nucleic acid according to the invention or a pharmaceutical expression vector according to the invention, in particular for the treatment of lung cancer.
The invention further relates to a host cell comprising a nucleic acid according to the invention or an expression vector according to the invention.
The invention further relates to a host cell of the invention which is an antigen presenting cell, preferably a dendritic cell.
The invention further relates to a method for formulating a peptide of the invention, said method comprising culturing a host cell of the invention and isolating the peptide from said host cell or its culture medium.
The invention further relates to methods of the invention wherein an antigen is loaded with an MHC class I or II molecule expressed on the surface of a suitable antigen-presenting cell by binding to a sufficient amount of the antigen comprising the antigen-presenting cell.
The invention further relates to the method of the invention, wherein the antigen presenting cell comprises an expression vector capable of expressing a polypeptide comprising SEQ ID NO: 1 to SEQ ID NO: 110 or the variant amino acid sequence.
The invention further relates to a T-cell promoter produced by the method of the invention, wherein the T-cell selectively recognizes a cell that aberrantly expresses a polypeptide comprising an amino acid sequence of the invention.
The invention further relates to a method of killing target cells in a patient, wherein the target cells in the patient abnormally express a polypeptide comprising any of the amino acid sequences of the invention, the method comprising administering to the patient an effective amount of a T cell of the invention.
The invention further relates to any of said peptides, a nucleic acid according to the invention, an expression vector according to the invention, a cell according to the invention, a use of a T lymphocyte according to the invention for initiating cytotoxicity as a medicament or for the manufacture of a medicament. The invention further relates to a use according to the invention, wherein the medicament is effective against cancer.
The invention further relates to a use according to the invention, wherein the medicament is a vaccine. The invention further relates to a use according to the invention, wherein the medicament is effective against cancer.
The invention also relates generally to the use of the invention, wherein the cancer cell is a lung cancer cell or other solid or hematological tumor cell, such as: brain cancer, breast cancer, colorectal cancer, esophageal cancer, kidney cancer, liver cancer, ovarian cancer, pancreatic cancer, prostate cancer, stomach cancer, melanoma, merkel cell carcinoma, leukemia (AML, CLL), non-hodgkin lymphoma (NHL), esophageal cancer including gastroesophageal junction cancer (OSCAR), gallbladder and bile duct cancer (GBC, CCC), bladder cancer (UBC), and uterine cancer (UEC) cells.
The invention further relates to a specific marker protein and biomarker, herein referred to as "target", based on the peptides of the invention, which can be used for diagnosing and/or judging the prognosis of lung cancer. The invention also relates to these novel targets for use in cancer therapy.
The term "antibody" is defined herein in a broad sense to include both polyclonal and monoclonal antibodies. In addition to intact or "whole" immunoglobulin molecules, the term "antibody" also includes fragments (e.g., CDR, Fv, Fab, and Fc fragments) or polymers of these immunoglobulin molecules and humanized immunoglobulin molecules, so long as they exhibit any of the desired properties of the invention (e.g., specific binding of a lung cancer marker (poly) peptide, delivery of a toxin to lung cancer cells at increased levels of lung cancer cell marker gene expression, and/or inhibition of the activity of a lung cancer marker polypeptide).
Antibodies of the invention may be purchased from commercial sources whenever possible. The antibodies of the invention may also be prepared using known methods. The skilled artisan will appreciate that full length lung cancer marker polypeptides or fragments thereof may be used to prepare antibodies of the invention. The polypeptides used to produce the antibodies of the invention may be partially or wholly purified from natural sources or may be produced using recombinant DNA techniques.
For example, a cDNA of the invention encoding a peptide, e.g., a peptide according to SEQ ID NO: 1 to SEQ ID NO: 110, or a variant or fragment thereof, may be expressed in prokaryotic (e.g., bacterial) or eukaryotic cells (e.g., yeast, insect or mammalian cells), after which the recombinant protein may be purified and used to produce a monoclonal or polyclonal antibody preparation that specifically binds to the lung cancer marker polypeptide used to produce the antibodies of the invention.
One skilled in the art will recognize that two or more different sets of monoclonal or polyclonal antibodies maximize the likelihood of obtaining an antibody of the specificity and affinity (e.g., ELISA, immunohistochemistry, in vivo imaging, immunotoxin therapy) required for the intended use. Antibodies for therapeutic or in vivo diagnostic use are tested according to their use by known methods (e.g., ELISA, immunohistochemistry, immunotherapy, etc.; further guidance in producing and testing antibodies is available, see, e.g., Greenfield, 2014(Greenfield, 2014.) for example, the antibodies can be detected by ELISA or immunoblotting, immunohistochemistry staining formalin-fixed lung cancer tissue or frozen tissue sections.
The term "monoclonal antibody" as used herein refers to an antibody obtained from a large homogeneous population of antibodies, i.e., a population of antibodies consisting of identical antibodies, except for natural mutations that may be present in minor amounts. The monoclonal antibodies described herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chains are identical (homogeneous) to the corresponding sequences of antibodies obtained from a particular species or antibodies belonging to a particular antibody type and class, while the remaining chains are identical (homogeneous) to the corresponding sequences of antibodies obtained from other species or antibodies belonging to a particular antibody type and sub-class, and fragments of such antibodies, so long as they exhibit the desired antagonistic activity (U.S. patent No. 4816567, which is incorporated herein in its entirety by reference).
The monoclonal antibodies of the invention may be made using hybridoma methods. In the hybridoma method, a mouse or other appropriate host animal is typically primed with an immunizing agent to elicit or produce antibodies that will specifically bind to the immunizing agent. Alternatively, lymphocytes may be immunized in vitro.
Monoclonal antibodies can also be made by recombinant DNA methods, such as: as described in U.S. patent No. 4816567. DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that specifically bind to genes encoding the heavy and light chains of murine antibodies).
In vitro methods are also suitable for the production of monovalent antibodies. Digestion of antibodies to produce fragments of antibodies, particularly Fab fragments, can be accomplished using conventional techniques known in the art. Digestion may be accomplished, for example, by using papain. Examples of papain digestion are described in WO 94/29348 and U.S. Pat. No. 4342566. Papain digestion of antibodies typically produces two identical antigen-binding fragments, called Fab fragments (each having an antigen binding site) and a residual Fc fragment. Pepsin treatment produced an aF (ab ') 2 fragment and a pFc' fragment.
Antibody fragments, whether attached to other sequences or not, may include insertions, deletions, substitutions, or other selective modifications of particular regions or particular amino acid residues, provided that the activity of the fragment is not significantly altered or impaired compared to the unmodified antibody or antibody fragment. These modifications may provide some additional attributes, such as: amino acids that can bind to disulfide bonds are deleted/added to increase their biological life, alter their secretory properties, etc. In any case, the antibody fragment must possess bioactive properties such as: binding activity, modulating binding capacity of the binding domain, and the like. The functional or active region of an antibody can be determined by genetic mutation of a particular region of the protein, subsequent expression and testing of the expressed polypeptide. Such methods are well known to those skilled in the art and may include site-specific genetic mutations in the nucleic acids encoding the antibody fragments.
The antibody of the present invention may further include a humanized antibody or a human antibody. Humanized forms of non-human (e.g., murine) antibodies are chimeric antibody immunoglobulins, immunoglobulin chains or fragments thereof (e.g., Fv, Fab' or other antigen binding sequences of an antibody) which contain minimal sequence derived from the non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a Complementarity Determining Region (CDR) of the recipient are substituted with residues from a CDR of a non-human species (donor antibody), such as mouse, rat or rabbit having the specificity, affinity and capacity therefor. In some cases, Fv Framework (FR) residues of the human immunoglobulin are substituted for corresponding non-human residues. Humanized antibodies may also include residues found in neither the recipient antibody nor the imported CDR or framework sequences. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of the same sequence of a human immunoglobulin. Ideally, the humanized antibody will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
Methods for humanizing non-human antibodies are well known in the art. In general, humanized antibodies have one or more amino acid residues introduced from a source that is non-human. These non-human amino acid residues are often referred to as "import" residues, and are typically obtained from an "import" variable domain. Humanization can be essentially accomplished by substituting rodent CDRs or CDR sequences with corresponding human antibody sequences. Thus, such "humanized" antibodies are chimeric antibodies (U.S. patent No. 4816567) in which substantially less than an entire human variable domain is replaced by a corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
Transgenic animals (e.g., mice) that are immunized to produce fully human antibodies in the absence of endogenous immunoglobulin can be used. For example, it is described that homozygous deletion of antibody heavy chain junction region genes in chimeric and germline mutant mice results in complete inhibition of endogenous antibody production. Transfer of human germline immunoglobulin gene arrays in such germline variant mice will result in the production of human antibodies following antigen challenge. Human antibodies can also be produced in phage display libraries.
The antibodies of the invention are preferably administered to a subject in the form of a pharmaceutically acceptable carrier. Generally, an appropriate amount of a pharmaceutically acceptable salt is used in the formulation to render the formulation isotonic. Examples of pharmaceutically acceptable carriers include physiological saline, ringer's solution and dextrose solution. The pH of the solution is preferably about 5 to 8, more preferably about 7 to 7.5. In addition, the carrier may also include sustained release formulations such as: semipermeable matrices of solid hydrophobic polymers containing the antibody, wherein the matrices are in the form of shaped articles, such as: a film, liposome, or microparticle. It is well known to those skilled in the art that certain carriers may be more preferred depending, for example, on the route of administration and the concentration of the antibody.
The antibody can be administered to a subject, patient, or cell by injection (e.g., intravenously, intraperitoneally, subcutaneously, intramuscularly) or by other means such as infusion, ensuring that it is delivered to the blood in an effective form. These antibodies can also be administered by intratumoral or peritumoral routes, thereby exerting local and systemic therapeutic effects. Topical or intravenous injection is preferred.
Effective dosages and schedules for administration of the antibodies can be determined empirically, and making such determinations is within the skill of the art. Those skilled in the art will appreciate that the dosage of antibody that must be administered will vary depending on factors such as: the subject receiving the antibody, the route of administration, the antibody used, and the particular type of drug being used. Typical daily dosages of antibody used alone may range from about 1. mu.g/kg up to 100mg/kg body weight or more, depending on the factors mentioned above. After administration of the antibody, preferably for the treatment of lung cancer, the efficacy of the therapeutic antibody can be assessed by different methods well known to the skilled person. For example: the size, number and/or distribution of lung cancer in a subject receiving treatment can be monitored using standard tumor imaging techniques. An antibody administered as a result of treatment prevents tumor growth, causes tumor shrinkage, and/or prevents the development of new tumors, as compared to the course of disease in the absence of administration of the antibody, such an antibody being an effective antibody for treating lung cancer.
In another aspect of the invention, a method of making a soluble T cell receptor (sTCR) that recognizes a specific peptide-MHC complex is provided. Such soluble T cell receptors can be generated from specific T cell clones, and their affinity can be increased by complementarity determining region-targeted mutagenesis. For the purpose of T cell receptor selection, phage display may be used (us 2010/0113300, (likdy et al, 2012)). For the purpose of stabilizing T cell receptors during phage display and when actually used as drugs, the alpha and beta chains may be linked by non-native disulfide bonds, other covalent bonds (single chain T cell receptors), or by dimerization domains (Boulter et al, 2003; Card et al, 2004; Willcox et al, 1999). T cell receptors may be linked to toxins, drugs, cytokines (see US 2013/0115191), domain recruitment effector cells such as anti-CD 3 domains, etc., in order to perform specific functions on target cells. In addition, it may be expressed in T cells for adoptive transfer. Further information can be found in WO 2004/033685a1 and WO 2004/074322a 1. Combinations of stcrs are described in WO 2012/056407a 1. Further methods of preparation are disclosed in WO 2013/057586a 1.
In addition, the peptides and/or TCRs or antibodies or other binding molecules of the invention can be used to validate the pathologist's diagnosis of cancer on the basis of biopsy samples.
The antibodies or TCRs may also be used in vivo diagnostic assays. Generally, antibodies are labeled with radionuclides (e.g.:111In、99Tc、14C、131I、3H、32p or35S) so that immunoscintigraphy can localize tumors. In one embodiment, the antibody or fragment thereof binds to two or more target extracellular domains of proteins selected from the group consisting of the proteins described above, and has an affinity value (Kd) of less than 1X 10. mu.M.
Diagnostic antibodies can be labeled by various imaging methods using probes suitable for detection. Probe detection methods include, but are not limited to, fluorescence, light, confocal and electron microscopy methods; magnetic resonance imaging and spectroscopy techniques; fluoroscopy, computed tomography, and positron emission tomography. Suitable probes include, but are not limited to, fluorescein, rhodamine, eosin and other fluorophores, radioisotopes, gold, gadolinium and other rare earths, paramagnetic iron, fluorine-18, and other positron emitting radionuclides. In addition, the probe may be bifunctional or multifunctional, and detection may be performed using more than one of the methods described above. These antibodies can be labeled with the probes directly or indirectly. The linking of antibody probes, including covalent attachment of probes, fusion of probes into antibodies, and covalent attachment of chelating compounds to bind probes, among other methods well known in the art. For immunohistochemistry, diseased tissue samples may be fresh or frozen or may be embedded in paraffin and fixed with a preservative such as formalin. The fixed or embedded sections include samples contacted with labeled primary and secondary antibodies, wherein the antibodies are used to detect in situ protein expression.
Another aspect of the invention includes a method for preparing activated T cells in vitro, comprising contacting T cells in vitro with antigen loaded human MHC molecules expressed on the surface of a suitable antigen-presenting cell for a period of time sufficient to activate T cells in an antigen-specific manner, wherein the antigen is a peptide according to the invention. Preferably, a sufficient amount of antigen is used with the antigen presenting cells.
Preferably, the TAP peptide transporter is absent or reduced in level or function in mammalian cells. Suitable cells lacking the TAP peptide transporter include T2, RMA-S, and Drosophila cells. TAP is a transporter associated with antigen processing.
Human peptide-carrying human deficient cell line T2, catalog number CRL1992, from American type culture Collection (ATCC, 12301ParklawnDrive, Rockville, Maryland 20852, USA); the ATCC catalogue CRL 19863, which is subordinate to the Drosophila cell line Schneider No. 2 strain; mouse RMA-S cell line Ljunggren et al (Ljunggren and Karre, 1985).
Preferably, the host cell does not substantially express MHC class I molecules prior to transfection. The stimulator cells also preferably express molecules that play an important role in T cell costimulatory signaling, e.g., any of B7.1, B7.2, ICAM-1, and LFA 3. Nucleic acid sequences for a number of MHC class I molecules and co-stimulatory molecules are publicly available from GenBank and EMBL databases.
When MHC class I epitopes are used as one antigen, the T cells are CD8 positive T cells.
If the antigen presenting cell is transfected to express such an epitope, preferred cells include an expression vector capable of expressing a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 110 or a variant amino acid sequence.
Several other methods can be used to generate T cells in vitro. For example, autologous tumor-infiltrating lymphocytes can be used to generate CTLs. Plebanski et al (Plebanski et al, 1995) used autologous peripheral blood lymphocytes (PLB) to make T cells. Alternatively, it is also possible to pulse dendritic cells with peptides or polypeptides or to make autologous T cells by infection with recombinant viruses. In addition, B cells can be used to prepare autologous T cells. In addition, macrophages pulsed with peptides or polypeptides or infected with recombinant viruses may be used to formulate autologous T cells. Walter et al (Walter et al, 2003) describe the in vitro priming of T cells by using artificial antigen presenting cells (aapcs), which is also a suitable method for generating T cells that act on selected peptides. In the present invention, according to biotin: streptomycin biochemical method streptomycin is prepared by mixing preformed MHC: peptide complexes were coupled to polystyrene particles (microspheres) to generate aapcs. The system enables precise regulation of MHC density on aapcs, which allows for the selective priming of high-potency antigen-specific T cell responses of high or low avidity in blood samples. In addition to MHC: in addition to peptide complexes, aapcs should also carry other proteins containing co-stimulatory activity, such as anti-CD 28 antibodies coupled to the surface. In addition, such aAPC-based systems often require the addition of appropriate soluble factors, e.g., cytokines such as interleukin-12.
T cells can also be prepared from allogeneic cells, a process which is described in detail in WO 97/26328, incorporated herein by reference. For example, in addition to Drosophila cells and T2 cells, other cells may be used to present peptides, such as CHO cells, baculovirus-infected insect cells, bacteria, yeast, vaccinia-infected target cells. Furthermore, plant viruses can also be used (see, for example, Porta et al (Porta et al, 1994) which describes the development of cowpea mosaic virus as a highly productive system for presenting foreign peptides.
The activated T cells are directed against the peptides of the invention and contribute to the treatment. Thus, in another aspect of the invention, there are provided primed T-cells prepared by the methods of the invention described above.
The T-promoter cells prepared by the above method will selectively recognize the expression of a peptide comprising SEQ ID NO: 1 to seq id NO 110.
Preferably, the T cell recognizes the cell by interacting with (e.g., binding to) the TCR of its HLA/peptide-containing complex. T cells are cells useful in a method of killing a target cell in a patient, wherein the target cell abnormally expresses a polypeptide comprising an amino acid sequence of the invention. Such patients are administered an effective amount of priming T cells. The T cells administered to the patient may be derived from the patient and primed as described above (i.e., they are autologous T cells). Alternatively, the T cells are not derived from the patient, but from another person. Of course, it is preferred that the donor is a healthy person. By "healthy individual" we mean a person who is generally in good condition, preferably has a qualified immune system, and more preferably is free of any disease that can be easily tested or detected.
According to the present invention, the in vivo target cells of CD 8-positive T cells may be tumor cells (sometimes expressing MHC-class II antigens) and/or stromal cells (tumor cells) surrounding the tumor (sometimes also expressing MHC-class II antigens; (Dengjel et al, 2006)).
The T cells of the invention are useful as active ingredients in therapeutic compositions. Accordingly, the present invention also provides a method of killing target cells in a subject, wherein the target cells in the subject aberrantly express a polypeptide comprising an amino acid sequence of the invention, the method comprising administering to the subject an effective amount of a T cell as described above.
The meaning of "aberrantly expressed" as used by the inventors also includes that the polypeptide is overexpressed compared to the normal expression level, or that the gene is not expressed in the tissue derived from the tumor but is expressed in the tumor. "overexpression" refers to a level of polypeptide that is at least 1.2 fold higher than in normal tissue; preferably at least 2-fold, more preferably at least 5 or 10-fold, that of normal tissue.
T cells can be prepared by methods known in the art (e.g., as described above).
T cell secondary transfer protocols are well known in the art. A review can be found in: gattioni et al, and Morgan et al (Gattinone et al, 2006; Morgan et al, 2006).
Another aspect of the invention includes the use of peptides complexed with MHC to generate T cell receptors, the nucleic acids of which are cloned and introduced into host cells, preferably T cells. The genetically engineered T cells can then be delivered to a patient for cancer therapy.
Any of the molecules of the invention (i.e., peptides, nucleic acids, antibodies, expression vectors, cells, priming T cells, T cell receptors, or encoding nucleic acids) is useful in treating diseases characterized by cells that escape the immune response. Thus, any of the molecules of the present invention may be used as a medicament or in the manufacture of a medicament. Such molecules may be used alone or in combination with other or known molecules of the present invention.
The invention also relates to a kit comprising:
(a) a container containing the above pharmaceutical composition in the form of a solution or lyophilized powder;
(b) optionally a second container containing a diluent or reconstitution solution in the form of a lyophilized powder; and
(c) optionally (i) instructions for use of the solution or (ii) for use of the recombinant and/or lyophilized formulation.
The kit further comprises one or more of (iii) a buffer, (iv) a diluent, (v) a filtrate, (vi) a needle, or (v) a syringe. The container is preferably a bottle, vial, syringe or test tube, and may be a multi-purpose container. The pharmaceutical composition is preferably lyophilized.
The kit of the present invention preferably comprises a lyophilized formulation in a suitable container together with reconstitution and/or instructions for use. Suitable containers include, for example, bottles, vials (e.g., dual chamber bottles), syringes (e.g., dual chamber syringes), and test tubes. The container may be made of a variety of materials, such as glass or plastic. The kit and/or container preferably has a container or instructions for the container indicating the direction of reconstitution and/or use. For example, the label may indicate that the lyophilized dosage form will reconstitute to the peptide concentration described above. The label may further indicate that the formulation is for subcutaneous injection.
The container holding the formulation may use a multi-purpose vial of penicillin such that the reconstituted dosage form may be administered repeatedly (e.g., 2-6 times). The kit may further comprise a second container containing a suitable diluent, such as a sodium bicarbonate solution.
After mixing the dilution and the lyophilized formulation, the final concentration of peptide in the recombinant formulation is preferably at least 0.15 mg/mL/peptide (═ 75 μ g), and not more than 3 mg/mL/peptide (═ 1500 μ g). The kit may also include other materials that are commercially and user-friendly, including other buffers, diluents, filtrates, needles, syringes, and package inserts with instructions for use.
The kit of the present invention may have a separate container containing a formulation of a pharmaceutical composition of the present invention with or without other ingredients (e.g., other compounds or pharmaceutical compositions thereof), or with different containers for each ingredient.
Preferably, the kits of the present invention comprise a formulation of the present invention packaged for use in combination with a second compound (e.g., an adjuvant (e.g., GM-CSF), a chemotherapeutic agent, a natural product, a hormone or antagonist, an anti-angiogenic or inhibitory agent, an apoptosis inducing agent, or a chelator) or pharmaceutical composition thereof. The components of the kit may be pre-complexed or each component may be placed in a separate, distinct container prior to administration to a patient. The components of the kit may be one or more solutions, preferably aqueous solutions, more preferably sterile aqueous solutions. The components of the kit may also be in solid form, converted to a liquid upon addition of a suitable solvent, and preferably placed in a different container.
The container of the treatment kit may be a vial, test tube, flask, bottle, syringe, or any other means for holding a solid or liquid. Typically, where there is more than one component, the kit will contain a second vial or other container of penicillin, so that it can be dosed separately. The kit may also contain another container for holding a medicinal liquid. Preferably, the treatment kit will contain a device (e.g., one or more needles, syringes, eye droppers, pipettes, etc.) such that the medicament of the present invention (the composition of the present kit) can be injected.
The pharmaceutical formulations of the present invention are suitable for administration of the peptides by any acceptable route, such as oral (enteral), intranasal, intraocular, subcutaneous, intradermal, intramuscular, intravenous or transdermal administration. Preferably, the administration is subcutaneous, most preferably intradermal, and may also be by infusion pump.
Since the peptide of the present invention is isolated from lung cancer, the agent of the present invention is preferably used for treating lung cancer.
The invention further relates to a method for preparing a personalized medicine for an individual patient, comprising: manufacturing a pharmaceutical composition comprising at least one peptide selected from a pre-screened TUMAP reservoir, wherein the at least one peptide used in the pharmaceutical composition is selected to be suitable for an individual patient. In one embodiment, the pharmaceutical composition is a vaccine. The method can also be modified to produce T cell clones for downstream applications such as: TCR spacers or soluble antibodies and other therapeutic options.
"personalized medicine" refers to treatments that are specific to an individual patient and will only be used for that individual patient, including personalized active cancer vaccines and adoptive cell therapies using autologous tissue.
As used herein, "depot" shall refer to a group or series of peptides that have been subjected to immunogenic pre-screening and/or over-presented in a particular tumor type. The term "depot" does not imply that the particular peptide included in the vaccine has been pre-manufactured and stored in physical equipment, although such a possibility is contemplated. It is expressly contemplated that the peptides may be used in the new manufacture of each individual vaccine, and may also be pre-manufactured and stored. The repository (e.g., database format) consists of tumor-associated peptides that are highly overexpressed in tumor tissues of various HLA-A HLA-B and HLA-C allele lung cancer patients. It may contain peptides including MHC class I and MHC class II or elongated MHC class I peptides. In addition to tumor-associated peptides collected from several lung cancer tissues, the pools may also contain HLA-a 02 and HLA-a 24 labeled peptides. These peptides allow quantitative comparison of the TUMAP-induced T cell immunity, leading to important conclusions about the capacity of vaccines to respond to tumors. Second, they can serve as important positive control peptides from "non-self" antigens in the absence of any vaccine-induced T cell responses from the patient's "self" antigen TUMAP. Third, it may also allow conclusions to be drawn on the immune function status of the patient.
TUMAP from the repository was identified using a functional genomics approach combining gene expression analysis, mass spectrometry and T-cell immunologyThis approach ensures that only TUMAPs that are actually present in a high percentage of tumors but are not or only rarely expressed in normal tissues are selected for further analysis. For the initial peptide selection, lung cancer samples of patients and blood of healthy donors were analyzed in a progressive manner:
1. determination of HLA ligands for malignant materials by mass spectrometry
2. Genome-wide messenger ribonucleic acid (mRNA) expression analysis was used to determine genes that are overexpressed in malignant tumor tissue (lung cancer) compared to a range of normal organs and tissues.
3. The determined HLA ligands are compared to gene expression data. Peptides over-or selectively presented on tumor tissue, preferably the selectively expressed or over-expressed genes detected in step 2, encode TUMAP that is considered a suitable candidate for a polypeptide vaccine.
4. Literature search to determine more evidence to support the relevance of peptides identified as TUMP
5. The correlation of overexpression at the mRNA level was determined by the selected TUMAP retest at step 3 in tumor tissue and the absence (or infrequent) of detection in healthy tissue.
6. To assess whether it is feasible to induce a T cell response in vivo by the selected peptides, in vitro immunogenicity assays were performed using human T cells from healthy donors as well as lung cancer patients.
In one aspect, the peptides are screened for immunogenicity prior to being added to the repository. For example, but not limited to, methods for determining the immunogenicity of peptides incorporated into a depot include in vitro T cell priming, specifically: CD8+ T cells from healthy donors were repeatedly stimulated with artificial antigen presenting cells loaded with peptide/MHC complexes and anti-CD 28 antibodies.
This method is preferred for rare cancers and patients with rare expression profiles. In contrast to the cocktail containing polypeptides currently developed as fixed components, the depot allows a higher degree of matching of the actual expression of antigens in tumors to the vaccine. In a multi-objective approach, each patient will use a selected single peptide or combination of several "off-the-shelf" peptides. Theoretically, one approach based on selecting, for example, 5 different antigenic peptides from a 50 antigenic peptide library could provide about 170 million possible pharmaceutical product (DP) components.
In one aspect, the peptides are selected for use in a vaccine based on the suitability of the individual patient and using the methods of the invention described herein or below.
HLA phenotypic, transcriptional and peptidological data were collected from tumor material and blood samples of patients to determine the peptides most appropriate for each patient and containing a "repository" and patient-unique (i.e., mutated) TUMAP. Those peptides selected are selectively or over-expressed in patient tumors and, possibly, exhibit strong in vitro immunogenicity if tested with patient individual PBMCs.
Preferably, a method of determining the peptide comprised by the vaccine comprises: (a) identifying a tumor associated peptide (TUMAP) presented by a tumor sample from an individual patient; (b) aligning the peptides identified in (a) with a repository (database) of such peptides; and (c) selecting at least one peptide from a repository (database) associated with tumor associated peptides identified in the patient. For example, TUMAPs presented in tumor samples can be identified by: (a1) comparing expression data from a tumor sample to expression data from a normal tissue sample corresponding to the tissue type of the tumor sample to identify proteins that are over-or aberrantly expressed in the tumor tissue; and (a2) correlating the expression data with MHC ligand sequences bound to MHC class I and/or class II molecules in the tumor sample to determine MHC ligands derived from proteins overexpressed or abnormally expressed by the tumor. Preferably, the sequence of the MHC ligand is determined by: the MHC molecules isolated from the tumor sample are eluted with the peptide and the eluted ligand is sequenced. Preferably, the tumor sample and the normal tissue are obtained from the same patient.
In addition to, or as an alternative to, using a repository (database) model for peptide selection, TUMAPs may be identified in new patients and then included in vaccines. As an example, candidate TUMAPs in a patient may be identified by: (a1) comparing expression data from a tumor sample to expression data from a normal tissue sample corresponding to the tissue type of the tumor sample to identify proteins that are over-or aberrantly expressed in the tumor tissue; and (a2) correlating the expression data with MHC ligand sequences bound to MHC class I and/or class II molecules in the tumor sample to determine MHC ligands derived from proteins overexpressed or abnormally expressed by the tumor. As another example, the method of identifying a protein may comprise a mutation that is unique to the tumor sample relative to the corresponding normal tissue of the individual patient, and the TUMAP may be identified by specifically targeting the variation. For example, the genome of a tumor and corresponding normal tissue can be sequenced by whole genome sequencing methods: to find non-synonymous mutations in the protein coding region of the gene, genomic DNA and RNA were extracted from tumor tissue and normal non-mutated genomic germline DNA was extracted from Peripheral Blood Mononuclear Cells (PBMC). The NGS method used is limited to the re-sequencing of protein coding regions (exome re-sequencing). For this purpose, exon DNA from human samples was captured using a target sequence enrichment kit supplied by the supplier, followed by sequencing using HiSeq2000 (Illumina). In addition, mRNA from the tumor was sequenced to directly quantify gene expression and confirm that the mutant gene was expressed in the patient's tumor. Millions of sequence reads are obtained and processed through software algorithms. The output list contains mutations and gene expression. Tumor specific somatic mutations were determined by comparison to PBMC-derived germline changes and optimized. Then, for the purpose of storage it is possible to test newly identified peptides for immunogenicity as described above and select candidate TUMAPs with appropriate immunogenicity for use in vaccines.
In an exemplary embodiment, the peptides contained in the vaccine are determined by: (a) identifying a tumor associated peptide (TUMAP) presented from a tumor sample from an individual patient using the method described above; (b) aligning the peptides identified in (a) with a repository of tumor-bearing (as compared to corresponding normal tissue) immunogenic and over-presenting pre-screening peptides; (c) selecting at least one peptide from a repository associated with identified tumor-associated peptides in a patient; and (d) optionally selecting at least one newly identified peptide from (a) for confirmation of its immunogenicity.
In an exemplary embodiment, the peptides contained in the vaccine are determined by: (a) identifying a tumor associated peptide (TUMAP) presented by a tumor sample from an individual patient; and (b) selecting at least one newly identified peptide in (a) and confirming its immunogenicity.
Once the peptides for the individualized peptide vaccine are selected, the vaccine is produced. The vaccine is preferably a liquid formulation comprising the individual peptides dissolved in between 20-40% DMSO, preferably about 30-35% DMSO, e.g., about 33% DMSO.
Each peptide listed in the product was dissolved in DMSO. The choice of the concentration of the individual peptide solutions depends on the amount of peptide to be included in the product. The single peptide-DMSO solutions were mixed equally to achieve a solution containing all peptides at a concentration of 2.5mg/ml per peptide. The mixed solution was then diluted 1: 3 with water for injection to a concentration of 0.826mg/ml per peptide in 33% DMSO. The diluted solution was filtered through a 0.22 μm sterile screening procedure. Thereby obtaining the final bulk solution.
The final bulk solution was filled into vials and stored at-20 ℃ prior to use. One vial contained 700 μ L of solution with 0.578mg of each peptide. Of which 500 μ L (about 400 μ g of each peptide) will be used for intradermal injection.
The peptides of the invention are useful in diagnosis as well as in the treatment of cancer. Since peptides are produced by lung cancer cells and it has been determined that these peptides are absent or present at low levels in normal tissues, these peptides can be used to diagnose the presence or absence of cancer.
A biopsy of a tissue from a blood sample and a peptide according to claim, useful for the diagnosis of cancer by a pathologist. Detection of certain peptides by antibodies, mass spectrometry, or other methods known in the art allows a pathologist to determine whether the tissue sample is malignant or inflammatory or diseased in general, and may also be used as a biomarker for lung cancer. Presentation of the peptide groups allows for classification or further subclassification of the diseased tissue.
Detection of peptides in diseased specimens allows for the determination of the benefit of immune system treatment methods, particularly if T-lymphocytes are known or predicted to be involved in the mechanism of action. Loss of MHC expression is a mechanism that adequately accounts for which infected malignant cells evade immune surveillance. Thus, presentation of the peptide indicated that the analyzed cells did not utilize this mechanism.
The peptides of the invention can be used to assay lymphocyte responses to peptides (e.g., T cell responses), or antibody responses to peptides complexed with peptides or MHC molecules. These lymphocyte responses can be used as prognostic indicators to determine whether further treatment should be undertaken. These responses can also be used as surrogate indicators in immunotherapy aimed at inducing lymphocyte responses in different ways, such as vaccination, nucleic acids, autologous material, adoptive transfer of lymphocytes. In gene therapy, the reaction of lymphocytes to peptides can be taken into account in the assessment of side effects. Lymphocyte response monitoring may also be a valuable tool in follow-up examinations of transplantation therapies, e.g., for detecting graft-versus-host and host-versus-graft disease.
The invention will now be illustrated by the following examples describing preferred embodiments, and with reference to the accompanying drawings, but without being limited thereto. All references cited herein are incorporated by reference for the purposes of this invention.
In the context of the figures, it is,
FIGS. 1A-D show the over-presentation of various peptides in normal tissues and NSCLC samples. FIGS. 1E-F show all cell lines, normal tissues and cancer tissues, in which typical peptides were detected (FVFSFPVSV, SEQ ID NO: 4(A × 02) and YYTKGFALLNF, SEQ ID NO: 29(A × 24)). FIG. 1A-Gene: SLC6a14, peptide: FLIPYAIML (A02; SEQ ID NO: 2) -tissue from left to right: adipose tissue 1, adrenal gland 3, artery 5, bone marrow 3, brain 8, breast 3, colon 13, duodenum 1, esophagus 7, gall bladder 2, heart 5, kidney 16, leukocyte sample 4, liver 21, lymph node 1, ovary 1, pancreatic cancer 7, peripheral nerve 2, peritoneum 1, pituitary 1, placenta 1, pleura 3, rectus muscle 6, salivary gland 2, skeletal muscle 3, skin 3, small intestine 2, spleen 4, stomach 7, testis 3, thymus gland 2, thyroid gland 3, ureter 1, uterus 2, vein 2, lung 46, NSCLC 91. The peptide is also found in pancreatic, gastric, colorectal, esophageal cancers (not shown). FIG. 1B-Gene: COL6a3, peptide: FLFDGSANL (A02; SEQ ID NO: 13) -tissues from left to right: adipose tissue 1, adrenal gland 3, artery 5, bone marrow 3, brain 8, breast 3, colon 13, duodenum 1, esophagus 7, gall bladder 2, heart 5, kidney 16, leukocyte sample 4, liver 21, lymph node 1, ovary 1, pancreatic cancer 7, peripheral nerve 2, peritoneum 1, pituitary 1, placenta 1, pleura 3, rectus muscle 6, salivary gland 2, skeletal muscle 3, skin 3, small intestine 2, spleen 4, stomach 7, testis 3, thymus gland 2, thyroid gland 3, ureter 1, uterus 2, vein 2, lung 46, NSCLC 91. The peptide is also found in prostate cancer, breast cancer, colorectal cancer, liver cancer, melanoma, ovarian cancer, esophageal cancer, pancreatic cancer, and gastric cancer (not shown). FIG. 1C-Gene: CCL18, peptide: VYTSWQIPQKF (A24; SEQ ID NO: 23) -tissues from left to right: 2 adrenal gland, 1 artery, 4 brain, 1 breast, 5 colon, 1 heart, 13 kidney, 9 liver, 3 pancreas, 1 pituitary, 2 rectus muscle, 3 skin, 1 spleen, 12 stomach, 1 thymus, 2 uterus, 9 lung, 80 NSCLC. The peptide is also found in prostate cancer, gastric cancer (not shown). FIG. 1D-Gene: cennp, peptide: RYLDSLKAIVF (A24; SEQ ID NO: 28) -tissues from left to right: 2 adrenal gland, 1 artery, 4 brain, 1 breast, 5 colon, 1 heart, 13 kidney, 9 liver, 3 pancreas, 1 pituitary, 2 rectus muscle, 3 skin, 1 spleen, 12 stomach, 1 thymus, 2 uterus, 9 lung, 80 NSCLC. The peptide is also found in liver cancer, stomach cancer, RCC (not shown). FIG. 1E-Gene: DUSP4, peptide: FVFSFPVSV (A02; SEQ ID NO: 4) -tissues from left to right: 5 pancreatic cell lines, 3 skin, 15 normal tissue (2 esophagus, 7 lung, 3 spleen, 3 stomach), 126 cancer tissue (1 brain cancer, 2 breast cancer, 5 colon cancer, 5 esophagus cancer, 2 gall bladder cancer, 8 kidney cancer, 5 liver cancer, 58 lung cancer, 11 ovary cancer, 9 pancreas cancer, 2 prostate cancer, 1 rectum cancer, 4 skin cancer, 12 stomach cancer, 1 testicular cancer). This group of normal tissues was identical to A-B, but no tissues tested were shown. FIG. 1F-Gene: PLOD2, peptide: YYTKGFALLNF (A24; SEQ ID NO: 29) -tissues from left to right: 30 cancer tissues (1 brain cancer, 3 kidney cancer, 2 liver cancer, 22 lung cancer, 2 stomach cancer). This group of normal tissues was identical to C-D, but no tissues tested were shown. Fig. 1G shows the excess presentation of a x 24 peptide in normal tissues and NSCLC samples. Gene: LAMP3, peptide: RFMDGHITF (A24; SEQ ID NO: 25) -tissues from left to right: 2 adrenal gland, 1 artery, 4 brain, 1 breast, 5 colon, 1 heart, 13 kidney, 9 liver, 3 pancreas, 1 pituitary, 2 rectus muscle, 3 skin, 1 spleen, 12 stomach, 1 thymus, 2 uterus, 9 lung, 80 NSCLC. The peptide is also found in prostate cancer, gastric cancer (not shown).
FIG. 2 shows representative expression profiles (relative expression compared to normal kidney) of the source genes of the invention, which are highly or exclusively over-expressed in a range of normal tissue pancreatic cancers as well as in 38 lung cancer samples. Organization from left to right: adrenal gland, artery, bone marrow, brain (whole), breast, colon, esophagus, heart, kidney (triplicate), white blood cells, liver, lung, lymph node, ovary, pancreas, placenta, prostate, salivary gland, skeletal muscle, skin, small intestine, spleen, stomach, testis, thymus, thyroid, bladder, cervix, uterus, vein, 1 normal (healthy) lung sample, 38 NSCLC samples. A) SMC4, B) LAMB 3; C) MMP12, and D) LAMP 3.
Figure 3 shows exemplary immunogenicity data: flow cytometry results after staining of peptide specific multimers. A) SLC1A4-001(SEQ ID No.12), B) IGF2BP3-001(SEQ ID No.120), C) LAMC2-001(SEQ ID No.121), D) COL6A3-008(SEQ ID No.13), and E) LAMP3-001(SEQ ID No. 25).
Figure 4 shows the results of antigen stimulation of CD4+ T cell proliferation: the figure shows the number of positive donors per peptide.
FIG. 5 shows that an exemplary vaccine against CEA-006 induces a CD4T cell response in a class II ICS assay. After in vitro priming, PBMCs from patients 36-031 were analyzed for CD4T cell response to CEA-006 (upper panel) and simulated at the V8/EOS pool time point (lower panel). Cells were stimulated with the corresponding peptides and stained with viability, anti-CD 3, anti-CD 8, anti-CD 4 and effector markers (from right to left: CD154, TNF-. alpha., interferon-. gamma., IL-2, IL-10), respectively. Live CD4T cells were analyzed to understand the proportion of one or more effector positive cells.
Figure 6 shows the immunogenicity of control class II peptides: this figure shows the immune response rates of 5 class II peptides tested with ICS against the IMA950 peptide in 16 patients and against the IMA910 peptide in 71 patients.
Examples
Example 1
Identification and quantification of cell surface presented tumor associated peptides
Tissue sample
Tumor tissues of patients were obtained from the university of Heidelberg hospital and Munich university Hospital. Normal (healthy) tissue is available from Bio-optics Inc., CA, USA, BioServe, Beltsville, MD, USA, Capita Bioscience Inc., Rockville, MD, USA, Genetist Inc., Glendale, CA, USA, Rinware university Hospital, Hedelberg Hospital, Kyoto Fuli university Hospital (KPUM), Osaka City University (OCU), Munich university Hospital, ProteoGenex Inc., Culver City, CA, USA, and Tibingen Hospital.
All patients received written informed consent prior to surgery or autopsy. Immediately following resection, the tissue was cold shocked and stored at-70 ℃ or below prior to isolation of the TUMAP.
Isolation of HLA peptides from tissue samples
With minor modifications to the protocol (Falk et al, 1991; Seeger et al, 1999), HLA peptide libraries from cold shock tissue samples were obtained by immunoprecipitation of solid tissues using HLA-A02-specific antibody BB7.2, HLA-A, HLA-B, HLAC-specific antibody W6/32, CNBr-activated agarose gel, acid treatment and ultrafiltration.
Mass spectrometric analysis
The obtained HLA peptide library was separated by reverse phase chromatography (Waters) according to its hydrophobicity, and the eluted peptides were analyzed by LTQ-velos fusion hybridization Mass Spectrometry (ThermoElectron) equipped with an electrospray source. The peptide library was directly loaded into an analytical fused silica microcapillary column (75 μm id x250mm) filled with 1.7 μm C18 reverse phase material (Waters) using a flow rate of 400nL per minute. Subsequently, the peptides were separated using a two-step 180 min binary gradient from solvent B at a concentration of 10% to 33% at a flow rate of 300nL per minute. The gradient was made from solvent a (water with 0.1% formic acid) and solvent B (acetonitrile with 0.1% formic acid). Gold coated glass capillary (PicoTip, New Objective) was used for introduction into the nanoliter electrospray source. The LTQ-Orbitrap mass spectrometer was operated in a data dependent mode using the TOP 5(TOP5) strategy. In short, a scan cycle is first started at orbitrap with a high accurate mass complete scan (R30000), followed by MS/MS scanning of the 5 most abundant precursor ions in orbitrap with the dynamic exclusion technique of the previously selected ions (R7500). Tandem mass spectra were interpreted in sequence and another manual controller. The resulting fragmentation pattern of the natural peptide was compared to the fragmentation pattern of a reference peptide of the same synthetic sequence, ensuring the identified peptide sequence.
Label-free relative LC-MS quantification was performed by ion counting (i.e. by LC-MS functional extraction and analysis) (Mueller et al, 2007). This method assumes that the LC-MS signal region of the peptide correlates with its abundance in the sample. The extracted features are further processed by state-of-charge deconvolution and retention time calibration (Mueller et al, 2008; Sturm et al, 2008). Finally, all LC-MS features were cross-referenced with sequence identification results to combine the quantitative data of different samples and tissues with peptide presentation features. Quantitative data were normalized in a two-layer fashion from the pooled data to account for both technical and biological replicative variation. Thus, each identified peptide can be correlated with quantitative data, thereby allowing a relative quantification between the sample and the tissue. In addition, all quantitative data obtained for candidate peptides were manually checked to ensure data consistency and to verify the accuracy of automated analysis. For each peptide, a presentation graph is calculated showing the mean presentation of the sample as well as the variation in replication. These features allow for the baseline values of lung cancer samples versus normal tissue samples.
The presentation profile of an exemplary over-presenting peptide is shown in fig. 1. The scores for presentation of exemplary peptides are shown in tables 15 and 16.
Table 15: and (5) presenting scores. The table lists HLA-a x 02 peptides that are highly over-presented at the tumor (++) compared to a series of normal tissues, at the tumor (++) compared to a series of normal (healthy) tissues, or at the tumor (+) compared to a series of normal tissues.
Table 16: and (5) presenting scores. The table lists HLA-a x 24 peptides that are highly over-represented (++) on tumors compared to a series of normal tissues, (+ +) on tumors compared to a series of normal tissues, or (+) on tumors compared to a series of normal tissues.
Example 2
Expression profiling of genes encoding the peptides of the invention
An over-presentation or specific presentation of one peptide on tumor cells compared to normal cells is sufficient for its effective use in immunotherapy, some peptides are tumor-specific, although their source proteins are also present in normal tissues. However, mRNA expression profiling increases the safety of other levels in the selection of immunotherapeutic target peptides. Particularly for treatment options with high safety risks, such as affinity matured TCRs, the ideal target peptide will be derived from a protein that is unique to the tumor and does not appear in normal tissues.
RNA source and preparation
Surgically excised tissue specimens were provided as described above (see example 1) after obtaining written informed consent from each patient. Tumor tissue specimens were snap frozen immediately after surgery, and then homogenized with a pestle and mortar in liquid nitrogen. Total RNA was prepared from these samples using TRI reagent (Ambion, Darmstadt, germany) followed by clearance with RNeasy (QIAGEN, Hilden, germany); both methods were performed according to the manufacturer's protocol.
Total RNA in healthy human tissue is commercially available (Ambion, Huntingdon, UK; Clontech, Heidelberg, Germany; Stratagene, Amsterdam, the Netherlands; BioChain, Hayward, Calif., USA). The RNAs of several persons (2 to 123 persons) were mixed so that the RNAs of each person were equally weighted.
The quality and quantity of all RNA samples were evaluated on an Agilent 2100Bioanalyzer analyzer (Agilent, Waldbronn, Germany) using the RNA 6000Pico LabChip Kit (Agilent).
Microarray experiments
All tumor and normal tissue RNA samples were analyzed for gene expression using Affymetrix Human Genome (HG) U133A or HG-U133Plus 2.0Affymetrix oligonucleotide chips (Affymetrix, Inc., Santa Clara, Calif., USA). All steps were performed according to the Affymetrix manual. Briefly, double-stranded cDNA was synthesized from 5-8. mu.g of RNA using SuperScript RTII (Invitrogen) and oligo-dT-T7 primer (MWG Biotech, Ebersberg, Germany) as described in the manual. The in vitro transcription was accomplished by performing U133A assay with the BioArray High Yield RNA transcription labeling Kit (ENZO Diagnostics, Farmingdale, NY, USA) or U133Plus 2.0 assay with the GeneChip IVT labeling Kit (Affymetrix), followed by disruption, hybridization and staining with streptavidin-phycoerythrin and biotinylated anti-streptomycin protein antibody (Molecular Probes, Leiden, Netherlands). The images were scanned with an Agilent2500A general array Scanner (U133A) or Affymetrix Gene-Chip Scanner 3000(U133Plus 2.0), and the data were analyzed with GCOS software (Affymetrix, Inc.) with all parameters set by default. To achieve standardization, 100 housekeeping genes (housekeeping genes) provided by Affymetrix were used. Relative expression values were calculated using the software given signal log ratio, with the value for normal kidney tissue samples arbitrarily set at 1.0. The expression profile of the representative source gene of the present invention highly overexpressed in lung cancer is shown in fig. 2. Further representative gene expression scores are shown in tables 17 and 18.
Table 17: expressing the score. The table lists HLA-a x 02 peptides of genes that are highly overexpressed (+++) on tumors compared to a series of normal tissues, highly overexpressed (++) on tumors compared to a series of normal tissues, or (+) on tumors compared to a series of normal tissues.
Table 18: expressing the score. The table lists HLA-a x 24 peptides of genes that are highly overexpressed (+++) on tumors compared to a series of normal tissues, highly overexpressed (++) on tumors compared to a series of normal tissues, or (+) on tumors compared to a series of normal tissues.
Sequence ID number
Sequence of
Peptide codes
Gene expression
23
VYTSWQIPQKF
CCL18-001
+++
24
NYPKSIHSF
MMP12-005
+++
25
RFMDGHITF
LAMP3-001
+++
26
RYLEKFYGL
MMP12-006
+++
28
RYLDSLKAIVF
CENPN-001
++
29
YYTKGFALLNF
PLOD2-002
+
30
KYLEKYYNL
MMP1-001
+
31
SYLDKVRAL
KRT-008
+
34
VFMKI)GFFYF
MMP1-002
+
35
TYNPEIYVI
ITGA2-002
+
38
VFLNRAKAVFF
GPNM-003
+
39
KFLEHTNFEF
DOCK2-001
+
43
RYTLHINTL
ALOX15B-001
+
47
RYISPDQLADL
EN01-001
+
159
TYKYVDINTF
MMP12-004
+++
161
LYQILQGIVF
CDC2-001
+
Example 3
MHC-I presenting peptidesIn vitro immunogenicity of
To obtain information on the immunogenicity of the TUMAPs of the present invention, the inventors performed studies using an in vitro T cell expansion assay based on repeated stimulation with artificial antigen presenting cells (aapcs) loaded with peptide/MHC complexes and anti-CD 28 antibodies. In this way, the inventors could show that the 84 HLA-a x 02 restricted TUMAPs of the present invention up to now are immunogenic, indicating that these peptides are T cell epitopes against human CD8+ precursor T cells (table 19).
CD8+ T cell priming in vitro
For in vitro stimulation with artificial antigen-presenting cells loaded with peptide-MHC complexes (pMHC) and anti-CD 28 antibodies, the inventors first obtained healthy donor CD8 microbeads from University clinics Mannheim, Germany (Miltenyi Biotec, Bergisch-Gladbach, Germany) and isolated CD8+ T cells by positive selection of fresh HLA-A02 product after leukopheresis.
PBMC and isolated CD8+ lymphocytes were cultured in T cell culture medium (TCM) before use, including RPMI-Glutamax (Invitrogen, Karlsruhe, Germany) supplemented with 10% heat-inactivated human AB serum (PAN-Biotech, Aidenbach, Germany), 100U/ml penicillin/100. mu.g/ml streptomycin (Cambrex, Cologne, Germany), 1mM sodium pyruvate (CC Pro, Oberdorla, Germany) and 20. mu.g/ml gentamycin (Cambrex). At this step, 2.5ng/ml IL-7(Promocell, Heidelberg, Germany) and 10U/ml IL-2(Novartis Pharma, N ü rnberg, Germany) were also added to the TCM.
Generation of pMHC/anti-CD 28 coated beads, stimulation and readout of T cells were performed in a highly defined in vitro system using four different pMHC molecules per stimulation condition and 8 different pMHC molecules per readout condition.
Purified co-stimulatory mouse IgG2a anti-human CD28 antibody 9.3(Jung et al, 1987) was chemically biotinylated using N-hydroxysuccinimide biotin recommended by the manufacturer (Perbio, Bonn, Germany). The beads used were streptavidin-coated polystyrene particles (Bangs laboratories, Ill., USA) of 5.6 μm. The pMHC used for the positive and negative control stimulators were A.times.0201/MLA-001 (peptide ELAGIGILTV (SEQ ID NO.163) modified from Melan-A/MART-1) and A.times.0201/DDX 5-001 (YLLPAIVHI (SEQ ID NO.164) obtained from DDX 5), respectively.
800.000 beads/200. mu.l were packed in 96-well plates containing 4X 12.5ng of different biotin-pMHC, washed, and then 600ng of biotin anti-CD 28 was added in a volume of 200. mu.l. Co-culture of 1X10 in 200. mu.l TCM with 5ng/ml IL-12(Promocell) at 37 ℃6CD8+ T cells and 2x105The coated beads were washed for 3 days, thereby initiating stimulation. Thereafter, half of the medium was exchanged with fresh TCM supplemented with 80U/ml IL-2 and incubated at 37 ℃ for 4 days. This stimulation cycle was performed a total of 3 times. For pMHC multimer readout using 8 different pMHC molecules per condition, the two-dimensional combinatorial coding approach was used as described previously (Andersen et al, 2012), slightly modified, encompassing coupling to 5 different fluorescent dyes. Finally, multimer analysis was performed with Live/dead near IR dye (Invitrogen, Karlsruhe, Germany), CD8-FITC antibody clone SK1(BD, Heidelberg, Germany) and fluorescent pMHC multimers. For the analysis, a BD LSRII SORP cytometer equipped with a suitable laser and screening procedure was used. Peptide-specific cells were calculated as a percentage of total CD8+ cells. Multimer analysis results were evaluated using FlowJo software (Tree Star, Oregon, usa). The in vitro priming of specific multimers + CD8+ lymphocytes was tested in comparison to the negative control stimulation group. Immunogenicity of a given antigen is tested if at least one evaluable in vitro stimulated well in a healthy donor is found to contain a specific CD8+ T cell strain after in vitro stimulation (i.e. the well contains at least 1% of specific multimers + CD8+ T cells and the percentage of specific multimers + is at least 10 times the median of negative control stimulation).
In vitro immunogenicity of lung cancer peptides
For the tested HLA class I peptides, their immunogenicity in vitro can be demonstrated by the generation of peptide-specific T cell lines. Exemplary results of flow cytometry assays of TUMAP-specific multimers after staining 3 peptides of the invention are shown in FIG. 3, and also contain corresponding negative control information. The results for the 84 peptides of the invention are summarized in Table 19.
Table 19: in vitro immunogenicity of the HLA-a 02 peptides of the invention.
Exemplary results of in vitro immunogenicity experiments performed by the applicants on the peptides of the invention. + < 20% >; 20% -49% ++; 50% -69% ++; 70% + 70 +++
Table 20: in vitro immunogenicity of the HLA-a 24 peptides of the invention.
Exemplary results of in vitro immunogenicity experiments performed by the applicants on the peptides of the invention. + < 20% >; 20% -49% ++; 50% -69% ++; 70% + 70 +++
Example 4
Synthesis of peptides
All peptides were synthesized by standard, well-accepted solid-phase peptide synthesis using the Fmoc strategy. The identity and purity of each peptide has been determined using mass spectrometry and RP-HPLC analysis. White to off-white peptides were obtained with > 85% purity by lyophilization (trifluoroacetate). All TUMAPs are preferably administered as the trifluoroacetate or acetate salt, although other pharmaceutically acceptable salt forms are possible.
Example 5
MHC binding assay
The candidate peptides of the present invention based on T cell therapy were further tested for their MHC binding capacity (affinity). The individual peptide-MHC complexes are generated by UV-ligand exchange, wherein the UV-sensitive peptides are cleaved after UV irradiation and exchanged with the relevant peptide for analysis. Only candidate peptides that can effectively bind and stabilize a peptide-accepting MHC molecule can prevent dissociation of the MHC complex. To determine the yield of the exchange reaction, an ELISA assay based on the detection of the stable MHC complex light chain (. beta.2m) was performed. The assays were generally performed according to the method described by Rodenko et al (Rodenko et al, 2006).
96-well Maxisorp plates (NUNC) were coated overnight at 2ug/ml pronase in PBS at room temperature, washed 4-fold and blocked in 2% BSA with blocking buffer for 1 hour at 37 ℃. Folded HLA-a 02: 01/MLA-001 monomer as standard substance, covering the range of 15-500 ng/ml. The peptide-MHC monomer of the uv exchange reaction was diluted 100-fold in blocking buffer. The samples were incubated at 37 ℃ for 1 hour, washed four times, incubated at 37 ℃ for 1 hour with 2ug/ml HRP conjugated anti-. beta.2m, washed again, and detected with NH2SO4 blocked TMB solution. The absorption was measured at 450 nm. Candidate peptides that exhibit high exchange yields (preferably greater than 50%, most preferably greater than 75%) are generally preferred when generating and producing antibodies or fragments thereof and/or T cell receptors or fragments thereof, because they exhibit sufficient affinity for MHC molecules and prevent dissociation of MHC complexes.
Table 21: MHC class I binding fraction
＜20％＝+；20％-49％＝++；50％-75％＝+++；＞＝75％＝++++
Sequence ID
Peptide codes
Peptide exchange
31
KRT-008
+++
45
GFPT2-002
+++
51
PLE-001
+++
55
PERP-001
+++
60
FLJ44796-001
+++
80
FXR1-001
+++
Example 6
Absolute quantification of cell surface presented tumor associated peptides
The production of adhesives, such as antibodies and/or TCRs, is a laborious process that can be performed against only a few selected targets. In the case of tumor-associated and specific peptides, selection criteria include, but are not limited to, exclusion of the presentation and concentration of peptides presented on the cell surface. In addition to the isolation and relative quantification of peptides as described in example 1, the inventors also analyzed the absolute peptide copy number per cell. Quantitation of the copy number of TUMAPs per cell in solid tumor samples requires absolute quantitation of the isolated TUMAPs, efficiency of TUMAP isolation, and cell counts of the analyzed tissue samples. The experimental procedure is as follows.
Nano LC-MS/MS peptide quantitation
For accurate quantification of peptides by mass spectrometry, an internal standard method was used to generate a calibration curve for each peptide. Internal standards are dual isotopically labeled variants of each peptide, i.e., 2 isotopically labeled amino acids were incorporated in the TUMAP synthesis. It differs only qualitatively from tumor-associated peptides, but not in other physicochemical properties (Anderson et al, 2012). An internal standard was incorporated into each MS sample and all MS signals were normalized to the internal standard MS signal to balance potential technical differences between MS experiments. The calibration curves were plotted with at least three different matrices, i.e., HLA peptide eluate from a natural sample similar to a conventional MS sample, and each preparation was measured in duplicate MS runs. For evaluation, MS signals were normalized to internal standard signals and calibration curves were calculated by logistic regression. For quantification of tumor associated peptides from tissue samples, each sample was also spiked with an internal standard, the MS signal was normalized to the internal standard and quantified using the peptide calibration curve.
Efficiency of peptide/MHC separation
For any protein purification process, the isolation of proteins from a tissue sample is associated with some loss of the relevant proteins. To determine the efficiency of the TUMAP separation, peptide/MHC complexes were generated for all TUMAPs selected as absolute quantification. To be able to distinguish the native peptide/MHC complex from the sample, a monoisotopically labeled version of TUMAP was used, i.e. 1 isotopically labeled amino acid was incorporated during TUMAP synthesis. These complexes are incorporated into freshly prepared tissue lysates, e.g., at the earliest possible time point during the TUMAP isolation, and then captured as native peptide/MHC complexes in subsequent affinity purifications. Therefore, measuring the recovery of single-marker TUMAPs can lead to conclusions regarding the separation efficiency of individual TUMAPs. Separation efficiency was analyzed using a small number of samples, and these tissue samples were comparable. In contrast, the separation efficiency differs between individual peptides. This indicates that the separation efficiency, although measured in only a limited number of samples, can be extrapolated to any other tissue preparation. However, since separation efficiency cannot be extrapolated from one peptide to the other, it is necessary to analyze each TUMAP separately.
Determination of cell count in solid, frozen tissue
To determine the cell number of tissue samples quantified by absolute peptides, the inventors used DNA content analysis. This method is applicable to a wide range of samples from different sources, most importantly frozen samples (Forsey and Chaudhuri, 2009; Alcoser et al, 2011; Silva et al, 2013). During the peptide isolation protocol, tissue samples were processed into homogeneous lysates from which a small aliquot of the lysate was taken. The samples were aliquoted in triplicate and DNA was isolated therefrom (QiaAmp DNA Mini Kit, Qiagen, Hilden, Germany). The total DNA content of each DNA isolation was quantified using a fluorescence-based DNA quantification Assay (Qubit dsDNA HS Assay Kit, Life Technologies, Darmstadt, Germany) at least twice in duplicate. To calculate the cell number, a standard curve of DNA from a single aliquot of healthy blood cells was generated, using a series of specified cell numbers. The standard curve was used to calculate the total cell content of the total DNA content per DNA isolation. The average total cell count of the tissue samples used for peptide isolation was calculated taking into account the known volume of lysate aliquots and the total lysate volume.
Peptide copy number per cell
Using the data from the previous experiments, the inventors calculated the number of copies of TUMAP per cell as the total peptide amount divided by the total cell count of the sample, and then divided by the separation efficiency. The cell copy number of the selected peptides is shown in table 22.
Table 22: absolute copy number. The table lists the results of absolute peptide quantification in NSCLC tumor samples. Median copy number per cell for each peptide is expressed as: <100 ═ +; 100 ═ + +; 1,000+ + +; 10,000 ═ + + + + +. Sample numbers are prompted in which to provide the evaluated high quality MS data.
Example 6
HLA class II T cell proliferation assay
The following experiment summarizes the results of T cell proliferation assays for selected MHC class II TUMAPs. 9 of the 10 peptide antigens tested were positively immunogenic. 11 of the 21 evaluable T cell samples showed a positive response to at least one peptide. Individual peptide antigens stimulate CD4+ T cell proliferation in up to 6 donors. These numbers are comparable to the five reference peptide results tested in the same assay and demonstrate the immunogenicity of most patients in the context of clinical vaccine trials. Thus, it can be concluded that the new test peptides are also likely to induce T cell responses in vaccine trials.
To summarize the potential of selected peptides, in particular as alternative vaccines, T cell proliferation was analyzed using the commercial T cell proliferation assay of promimune to determine their immunogenicity in vitro.
Healthy donor CD 8-depleted blood cell samples were tested with the selected peptides. Peptides that induce CD4+ T cell proliferation may lead to the development of helper T cell immune responses, and therefore, these peptides are considered immunogenic. Proliferation of CD4+ T cells was determined using carboxysuccinimidyl ester (CFSE) labeling. In proliferating cells, CFSE is evenly distributed to dividing cells. Thus, proliferation in the analyzed cells can be measured as a decrease in CFSE fluorescence.
Table 23 selected HLA class II peptides.
Principle of testing
Peripheral Blood Mononuclear Cell (PBMC) samples from healthy human donors were selected from the proammune cell bank based on HLA-DRB1 allele expression. CD8+ T cells were depleted from donor blood samples prior to use to avoid false positive reactions. The remaining CD4+ T cells were labeled with CFSE, followed by incubation with 5 μ M of each selected peptide. Each peptide was tested in six replicate wells. Background measurements were performed in six unstimulated control wells per plate.
After 7 days incubation, cells were co-stained with anti-CD 4 antibody and analyzed by flow cytometry. The extent of proliferation was determined by measuring the extent of decrease in CFSE intensity.
Flow cytometer data was evaluated using FlowJo software (Tree Star, inc.). Flow cytometry analysis results as CD4+The ratio of the non-significant population to the total CD4+ population. The extent of proliferation was expressed as the percentage of stimulation above background, i.e. the proportion of antigen-stimulated CD4+ CFSE dim cells minus the proportion of unstimulated control wells CD4+ CFSE dim cells. For each sample, the mean of each replicate was calculated, along with the corresponding standard error of the mean (SEM).
Washing selection of donors
Donors were selected for HLA-DRB1 allele expression. Two other HLA class II sites (DQ and DP) were not included in the analysis. The relevant DRB1 allele was selected based on the SYFPEITHI algorithm based on predicted peptide binding frequency (rammenee et al, 1999). For HLA-DR, binding is defined by an SYFPEITHI binding prediction score equal to or greater than 18. This binding threshold score was defined based on binding score analysis of published known promiscuous HLA-DR ligands (table 24).
Table 24: the SYFPEITHI predicted scores for HLA-DR binding of peptides bound to several HLA-DR alleles were experimentally demonstrated. Only the prediction scores are shown if the experiments demonstrate binding to the indicated DR alleles. If high resolution information of the DR allele is missing, the label is x. If the experiment demonstrated that one allele was bound, the SYFPEITHI score of 23/26 (89%) > -18.
All DRB1 genes with binding frequency of more than 20% in all selected peptides were required to be included in the donor group of proammune. An additional requirement was for 4 other rare DRB1 alleles (DRB1 x 10: 01, DRB1 x 16: 01, DRB1 x 08: 01 and DRB1 x 13: 03). The combined donor groups are shown in table 26.
Table 25 binding capacity of selected peptides to various HLA-DRB1 alleles containing known binding motifs: SYFPEITHI score exceeded 17, a binding event was counted as 1. The last column shows the percentage of binding events for all selected peptides.
Table 26 donor group. HLA-DRB1 allele distribution for 21 selected donors
In vitro immunogenicity results
Antigen-stimulated proliferation of CD4+ T cells was considered an indicator of immunogenicity in vitro and was studied using a commercial T cell proliferation assay from prolmmone. The extent to which antigen stimulates CD4+ T cell proliferation is expressed as the percentage of stimulation above background. Stimulating responses above 0.02% above background, SEM ═ 2 (i.e., two standard error values greater than background) were considered positive.
Out of 10 selected peptide antigens, 9 tested positive (with the exception of FN 1-002). 11 of the 21 evaluable T cell samples showed positive responses to at least one peptide (figure 4). Individual peptide antigens stimulate CD4+ T cell proliferation in up to 6 donors.
Comparison of immunogenicity in vivo and in vitro
The T cell proliferation assay included 5 peptides with known in vivo immunogenicity as a positive control. For the in vivo immunogenicity of these peptides, blood samples from patients vaccinated with these peptides were used in clinical trials using the Intracellular Cytokine Staining (ICS) method of CD4T cells.
In principle, ICS assays analyze the quality of specific T cells associated with effector function. Thus, peripheral mononuclear cells (PBMCs) were re-stimulated in vitro with the relevant peptide, the reference peptide and the negative control (here mock). After re-stimulation of the cells for staining to check for IFN-. gamma.TNF-. alpha.IL-2 and IL-10 production and expression of the co-stimulatory molecule CD154, stained cells were counted on a flow cytometer (FIG. 5).
Immunogenicity analysis showed 100% immune response to vaccination with IMA950 peptides (BIR-002 and MET-005) in 16 patients (study IMA950-101) and 44% to 86% immune response to vaccination with IMA910 peptides (CEA-006, TGFBI-004 and MMP-001) in 71 patients (study IMA910-101) (FIG. 6).
In vitro immunogenicity results for peptides with known in vivo immunogenicity were compared to selected peptides (table 27). Analysis showed that the positive control peptide stimulated CD4+ T cell proliferation in 7 out of 21 donor samples that received the study. The intensity of the mean stimulatory response ranged from 0.09 to 0.31% above background in up to 4 donor samples per peptide. For example, the stimulation intensity for BIR-002 was 0.24%. BIR-002 was considered to be highly immunogenic in various clinical trials. In one clinical trial against 19 evaluable patients expressing different HLA-DR alleles, BIR-002 was tested as a component of a prostate cancer-specific peptide vaccine (Feyerabend et al, 2009). 16 (84%) patients had a CD4+ T cell response against BIR-002 (Widenmeyer et al, 2008), indicating their high immunogenic potential. In the IMA950 test, 100% (N ═ 16) of patients showed an immune response to BIR-002.
By comparison, the selected peptides in the current assay stimulated CD4+ T cell proliferation in a total of 11 donor specimens studied, except FN 1-002. Thus, in up to 6 donors per peptide, the intensity of the mean stimulatory response ranged from 0.19 to 0.48% above background. These values are similar to the intensity of the highly immunogenic peptide BIR-002 stimulating response. Interestingly, for all positive control peptides, the fraction of positive donor samples for in vitro immunogenicity assays (range: 4-19%) was much lower than the fraction of patients in clinical trials who were immune-responsive to these peptides (range: 44-100%). This observation indicates that current in vitro immunogenicity assay settings are fairly conservative and likely underestimates the immunogenicity of peptides in a clinical setting. Thus, it is expected that 9 out of 10 peptides studied are likely to elicit an immune response in vivo in most patients in clinical trials.
Table 27. T cell proliferation assay results for selected peptides and positive control peptides with known in vivo immunogenicity.
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More specifically, the present application provides the following:
1. a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID No.1 to SEQ ID No.110, and variant sequences thereof which are at least 88% homologous to SEQ ID No.1 to SEQ ID No.110, wherein said variant binds to Major Histocompatibility Complex (MHC) and/or induces T cell cross-reactivity with the variant peptide, wherein said peptide is not a full-length polypeptide.
2. The peptide according to claim 1, wherein the peptide has the ability to bind to an MHC class-I or-II molecule, wherein the peptide, when bound to the MHC, is capable of being recognized by CD4 and/or CD8T cells.
3. The peptide or variant thereof according to item 1 or 2, wherein the amino acid sequence comprises a continuous stretch of amino acids of any one of SEQ ID No.1 to SEQ ID No. 110.
4. The peptide or variant thereof according to any of claims 1 to 3, wherein said peptide or variant thereof has an overall length of from 8 to 100 amino acids, preferably from 8 to 30 amino acids, more preferably from 8 to 16 amino acids, most preferably the peptide consists or consists essentially of an amino acid sequence according to any of SEQ ID No.1 to SEQ ID No. 110.
5. The peptide or variant thereof according to any one of claims 1 to 4, wherein said peptide is modified and/or comprises non-peptide bonds.
6. The peptide or variant thereof according to any of claims 1 to 5, wherein the peptide is part of a fusion protein, in particular comprising the N-terminal amino acid of the HLA-DR antigen associated invariant chain (Ii).
7. A nucleic acid encoding a peptide or variant thereof according to any one of claims 1 to 6, optionally linked to a heterologous promoter sequence.
8. An expression vector for expressing a nucleic acid according to the one described in item 7.
9. A recombinant host cell comprising a peptide according to item 1 to 6, a nucleic acid according to item 7, an expression vector according to item 8, wherein said host cell is preferably an antigen presenting cell, such as a dendritic cell.
10. The peptide or variant thereof according to any one of claims 1 to 6, the nucleic acid according to claim 7, the expression vector according to claim 8 or the expression vector according to claim 9 for pharmaceutical use.
11. A method of producing a peptide or variant thereof according to any one of items 1 to 6, the method comprising culturing a host cell according to item 9, which presents a peptide according to items 1 to 6 or expresses a nucleic acid according to item 7 or carries an expression vector according to item 8, and isolating the peptide or variant thereof from the host cell or culture medium thereof.
12. An in vitro method for producing activated T lymphocytes, the method comprising contacting in vitro T cells with antigen loaded human class I or II MHC molecules expressed on the surface of a suitable antigen-presenting cell or an artificial construct mimicking an antigen-presenting cell for a period of time sufficient to activate the T cells in an antigen-specific manner, wherein the antigen is a peptide according to any one of claims 1 to 4.
13. An activated T lymphocyte, produced by the method described in item 12, that selectively recognizes a cell that presents a polypeptide comprising an amino acid sequence given in any one of items 1 to 4.
14. A method of killing a targeted cell in a patient, wherein the targeted cell presents a polypeptide comprising an amino acid sequence given in any one of items 1 to 4; a method of administration comprising administering to a patient an effective amount of T cells as defined in item 13.
15. An antibody, in particular a soluble or membrane-bound antibody, which specifically recognizes a peptide or variant thereof according to any of items 1 to 5, preferably a peptide or variant thereof according to any of items 1 to 5 when bound to an MHC molecule.
16. Use of a peptide according to any one of items 1 to 6, a nucleic acid according to item 7, an expression vector according to item 8, a cell according to item 9 or a T lymphocyte with toxicity promoting properties according to item 13 or an antibody according to item 15 in the treatment of cancer or in the manufacture of an anti-cancer medicament.
17. The use according to claim 16, wherein the cancer is selected from lung cancer, brain cancer, breast cancer, colorectal cancer, esophageal cancer, kidney cancer, liver cancer, ovarian cancer, pancreatic cancer, prostate cancer, stomach cancer, melanoma, merkel cell carcinoma, leukemia (AML, CLL) and other tumors that overexpress a protein derived from a peptide according to any of SEQ ID No.1 to SEQ ID No. 110.
18. A kit, comprising:
(a) a container comprising a pharmaceutical composition comprising a peptide or variant according to any one of items 1 to 6, a nucleic acid according to item 7, an expression vector according to item 8, a cell according to item 10, a T-priming lymphocyte according to item 13 or an antibody according to item 15 in solution or lyophilized form;
(b) optionally, a second container containing a diluent or reconstitution solution in the form of a lyophilized powder;
(c) optionally, at least one more peptide selected from the group consisting of SEQ ID No.1 to SEQ ID No.162, and
(d) optionally instructions for (i) using the solution or (ii) reconstituting and/or using the lyophilized powder form.
19. The kit of claim 18, further comprising one or more of (iii) a buffer, (iv) a diluent, (v) a filtrate, (vi) a needle, or (v) a syringe.
20. The kit according to claim 18 or 19, wherein the peptide is selected from the group consisting of SEQ ID No.1 to SEQ ID No. 110.
21. A method for producing a personalized anti-cancer vaccine, the method comprising:
a) identifying a tumor associated peptide (TUMAP) presented by a tumor sample from the individual patient;
b) comparing the peptides identified in a) with peptides from a depot that have been subjected to an immunogenic pre-screening and/or over-presented in a tumor compared to normal tissue.
c) Selecting at least one peptide in a repository that matches a TUMAP identified in a patient; and
d) formulating a personalized vaccine based on step c).
22. The method of clause 21, wherein the TUMAP is identified by:
a1) comparing the expression data of the tumor sample with expression data of a normal tissue sample corresponding to the tissue type of the tumor sample to identify proteins that are overexpressed or abnormally expressed in the tumor sample; and
a2) correlating the expression data with MHC class I and/or class II molecule-bound MHC ligand sequences in the tumor sample to identify protein-derived MHC ligands that are overexpressed or aberrant in the tumor.
23. The method of item 21 or 22, wherein the sequence of the MHC ligand is determined by: the MHC molecules isolated from the tumor sample are eluted with the peptide and the eluted ligand is sequenced.
24. The method according to any one of claims 21 to 23, wherein the normal tissue sample corresponding to the tumor sample of the type is obtained from the same patient.
25. The method of any one of items 21 to 24, wherein the peptides comprised by the repository are identified based on the following steps:
performing genome-wide messenger ribonucleic acid (mRNA) expression analysis by highly parallel methods, such as microarrays or sequencing-based expression profiling, comprising identifying genes that are overexpressed in malignant tissue compared to normal tissue;
ab. selecting the peptide encoded by the gene specifically expressed or overexpressed as detected in step aa, and
determining induction of an in vivo T cell response by the selected peptide, comprising an in vitro immunogenicity assay using human T cells of a healthy donor or the patient; or
ba. identifying HLA ligands from the tumor sample by mass spectrometry;
performing genome-wide messenger ribonucleic acid (mRNA) expression analysis by highly parallel methods, such as microarrays or sequencing-based expression profiling, comprising identifying genes that are overexpressed in malignant tissue compared to normal tissue;
comparing the identified HLA ligands to said gene expression data;
bd. selecting the peptide encoded by the gene specifically expressed or overexpressed as detected in step bc;
be. retesting selected TUMAP from step bd on tumor tissue, its absence or infrequent detection on healthy tissue, and determining a correlation of overexpression at the mRNA level; and
bf. the induction of in vivo T cell responses was determined by the selected peptides, including in vitro immunogenicity assays using human T cells from healthy donors or the patients.
26. The method according to any one of claims 21 to 25, wherein whether the repository comprises peptide immunogenicity is determined by a method comprising in vitro immunogenicity testing, individual HLA-binding patient immune monitoring, MHC multimer staining, ELISPOT analysis and/or intracellular cytokine staining.
27. The method of any one of claims 21 to 26, wherein said repertoire comprises a plurality of peptides selected from the group consisting of SEQ ID No.1 to SEQ ID No. 162.
28. The method of any one of items 21 to 27, further comprising the step of: identifying at least one mutation unique to said tumor sample as compared to corresponding normal tissue of the individual patient, and selecting a peptide associated with the mutation and for inclusion in a vaccine or for use in generating cell therapy.
29. The method of item 28, wherein the at least one mutation is identified by whole genome sequencing.
30. A T cell receptor, preferably a soluble or membrane bound T cell receptor reactive with an HLA ligand, wherein said ligand consists of an amino acid sequence at least 75% homologous to an amino acid sequence selected from the group consisting of SEQ ID No.1 to SEQ ID No. 110.
31. The T cell receptor of claim 30, wherein the amino acid sequence is at least 88% homologous to SEQ ID No.1 to SEQ ID No. 110.
32. The T cell receptor of claim 30 or 31, wherein the amino acid sequence comprises any one of SEQ ID No.1 to SEQ ID No. 110.
33. The T cell receptor according to any one of items 30 to 32, wherein the T cell receptor is provided as a soluble molecule and optionally has further effector function, such as an immunostimulatory domain or a toxin.
34. A nucleic acid encoding a TCR according to any one of claims 30 to 33, optionally linked to a heterologous promoter sequence.
35. An expression vector capable of expressing a nucleic acid according to item 34.
36. A host cell comprising a nucleic acid according to item 34 or encoding an antibody according to item 15 or an expression vector according to item 35, wherein the host cell is preferably a T cell or NK cell.
37. A method of producing a T cell receptor according to any one of items 30 to 33, said method comprising culturing a host cell according to item 36 and isolating said T cell receptor from the host cell and/or its culture medium.
38. A pharmaceutical composition comprising at least one active ingredient selected from the group consisting of
a) A peptide selected from the group consisting of SEQ ID No.1 to SEQ ID No. 110;
b) a T cell receptor which reacts with the peptide and/or peptide MHC complex according to a);
c) a fusion protein consisting of the peptide according to a) and the 1 st to 80 th N-terminal amino acids of the HLA-DR antigen-associated invariant chain (Ii);
d) a nucleic acid encoding any one of a) to c) or an expression vector comprising said nucleic acid.
e) Comprising d a host cell expressing the vector in a cell,
f) an activated T lymphocyte obtainable by a method comprising contacting a T cell in vitro with a peptide of a) expressed on the surface of a suitable antigen-presenting cell for a time sufficient to activate said T cell in an antigen-specific manner; and methods of transferring these activated T cells into autologous or other patients;
g) an antibody or soluble T cell receptor reactive with a peptide and/or peptide-MHC complex of a) and/or providing a peptide according to a) and possibly modified by fusion with an immune initiation domain or toxin,
h) an aptamer recognizing a peptide selected from the group comprising SEQ ID No.1 to SEQ ID No.110 and/or a complex of a peptide selected from the group comprising SEQ ID No.1 to SEQ ID No.162 and an MHC molecule,
i) a conjugated or labelled peptide or scaffold according to any of a) to h) and a pharmaceutically acceptable carrier, or a pharmaceutically acceptable excipient and/or stabiliser.
39. An aptamer that specifically recognizes a peptide or a variant thereof according to any of items 1 to 4, preferably a peptide or a variant thereof according to any of items 1 to 4 that binds to an MHC molecule.
Sequence listing
<110> Imamatix Biotechnology Ltd
<120> novel peptides and peptide compositions for immunotherapy of lung cancer, including non-small cell lung cancer and other cancers
<130> I32809WO
<150> US 62/152,258
<151> 2015-04-24
<150> GB 1507030.3
<151> 2015-04-24
<160> 177
<170> PatentIn version 3.5
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Lys Leu Leu Pro Tyr Ile Val Gly Val
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Phe Leu Ile Pro Tyr Ala Ile Met Leu
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Phe Leu Tyr Asp Val Val Lys Ser Leu
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Phe Val Phe Ser Phe Pro Val Ser Val
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Ala Leu Thr Ser Thr Leu Ile Ser Val
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Ser Leu Gln Gly Ser Ile Met Thr Val
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Asn Leu Leu Gln Val Leu Glu Lys Val
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Ala Leu Leu Asn Ile Leu Ser Glu Val
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Ala Leu Ser Gly Thr Leu Ser Gly Val
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Lys Met Ala Gly Ile Gly Ile Arg Glu Ala
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Tyr Leu Asn Val Gln Val Lys Glu Leu
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Ile Val Asp Arg Thr Thr Thr Val Val
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Phe Leu Phe Asp Gly Ser Ala Asn Leu
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Leu Ile Gln Asp Arg Val Ala Glu Val
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Glu Leu Asp Arg Thr Pro Pro Glu Val
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Leu Ile Phe Asp Leu Gly Gly Gly Thr Phe Asp Val
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Thr Leu Leu Gln Glu Gln Gly Thr Lys Thr Val
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Ile Leu Leu Thr Glu Gln Ile Asn Leu
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Val Leu Thr Ser Asp Ser Pro Ala Leu
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Leu Met Thr Lys Glu Ile Ser Ser Val
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Val Leu Ser Ser Gly Leu Thr Ala Ala
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Asn Leu Ile Asn Gln Glu Ile Met Leu
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Val Tyr Thr Ser Trp Gln Ile Pro Gln Lys Phe
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Asn Tyr Pro Lys Ser Ile His Ser Phe
1 5
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Arg Phe Met Asp Gly His Ile Thr Phe
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Arg Tyr Leu Glu Lys Phe Tyr Gly Leu
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Arg Tyr Pro Pro Pro Val Arg Glu Phe
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Arg Tyr Leu Asp Ser Leu Lys Ala Ile Val Phe
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Tyr Tyr Thr Lys Gly Phe Ala Leu Leu Asn Phe
1 5 10
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Lys Tyr Leu Glu Lys Tyr Tyr Asn Leu
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Ser Tyr Leu Asp Lys Val Arg Ala Leu
1 5
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Glu Tyr Gln Pro Glu Met Leu Glu Lys Phe
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Thr Tyr Ser Glu Lys Thr Thr Leu Phe
1 5
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Val Phe Met Lys Asp Gly Phe Phe Tyr Phe
1 5 10
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Thr Tyr Asn Pro Glu Ile Tyr Val Ile
1 5
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Tyr Tyr Gly Asn Thr Leu Val Glu Phe
1 5
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Arg Tyr Leu Glu Tyr Phe Glu Lys Ile
1 5
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Val Phe Leu Asn Arg Ala Lys Ala Val Phe Phe
1 5 10
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Lys Phe Leu Glu His Thr Asn Phe Glu Phe
1 5 10
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Ile Tyr Asn Pro Ser Met Gly Val Ser Val Leu
1 5 10
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Thr Tyr Ile Gly Gln Gly Tyr Ile Ile
1 5
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Val Tyr Val Thr Ile Asp Glu Asn Asn Ile Leu
1 5 10
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Arg Tyr Thr Leu His Ile Asn Thr Leu
1 5
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Ile Tyr Asn Gln Ile Ala Glu Leu Trp
1 5
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Lys Phe Leu Glu Ser Lys Gly Tyr Glu Phe
1 5 10
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Asn Tyr Thr Asn Gly Ser Phe Gly Ser Asn Phe
1 5 10
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Arg Tyr Ile Ser Pro Asp Gln Leu Ala Asp Leu
1 5 10
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Tyr Tyr Tyr Gly Asn Thr Leu Val Glu Phe
1 5 10
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Gln Tyr Leu Phe Pro Ser Phe Glu Thr Phe
1 5 10
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Leu Tyr Ile Gly Trp Asp Lys His Tyr Gly Phe
1 5 10
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Asn Tyr Leu Leu Glu Ser Pro His Arg Phe
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Ser Tyr Met Glu Val Pro Thr Tyr Leu Asn Phe
1 5 10
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Ile Tyr Ala Gly Gln Trp Asn Asp Phe
1 5
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Ala Tyr Lys Asp Lys Asp Ile Ser Phe Phe
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Ile Tyr Pro Val Lys Tyr Thr Gln Thr Phe
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Arg Tyr Phe Pro Thr Gln Ala Leu Asn Phe
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Ser Tyr Ser Ile Gly Ile Ala Asn Phe
1 5
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Val Tyr Phe Lys Pro Ser Leu Thr Pro Ser Gly Glu Phe
1 5 10
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His Tyr Phe Asn Thr Pro Phe Gln Leu
1 5
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Ser Tyr Pro Ala Lys Leu Ser Phe Ile
1 5
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Arg Tyr Gly Ser Pro Ile Asn Thr Phe
1 5
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Ala Tyr Lys Pro Gly Ala Leu Thr Phe
1 5
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Leu Tyr Ile Asn Lys Ala Asn Ile Trp
1 5
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Val Tyr Pro Leu Ala Leu Tyr Gly Phe
1 5
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Ile Tyr Gln Arg Trp Lys Asp Leu Leu
1 5
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Asp Tyr Ile Pro Gln Leu Ala Lys Phe
1 5
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Ile Phe Leu Asp Tyr Glu Ala Gly His Leu Ser Phe
1 5 10
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Arg Tyr Leu Phe Val Val Asp Arg Leu
1 5
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Thr Tyr Ala Ala Leu Asn Ser Lys Ala Thr Phe
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Val Tyr His Ser Tyr Leu Thr Ile Phe
1 5
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Thr Tyr Leu Thr Asn His Leu Arg Leu
1 5
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Tyr Tyr Val Asp Lys Leu Phe Asn Thr Ile
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Arg Tyr Leu His Val Glu Gly Gly Asn Phe
1 5 10
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Glu Tyr Leu Pro Glu Phe Leu His Thr Phe
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Ala Tyr Pro Asp Leu Asn Glu Ile Tyr Arg Ser Phe
1 5 10
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Val Tyr Thr Glx Ile Gln Ser Arg Phe
1 5
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Arg Tyr Leu Glu Ala Gly Ala Ala Gly Leu Arg Trp
1 5 10
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Ile Tyr Thr Arg Val Thr Tyr Tyr Leu
1 5
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Arg Tyr Gly Gly Ser Phe Ala Glu Leu
1 5
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Ala Tyr Leu Lys Glu Val Glu Gln Leu
1 5
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Lys Tyr Ile Glu Ala Ile Gln Trp Ile
1 5
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Phe Tyr Gln Gly Ile Val Gln Gln Phe
1 5
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Glu Tyr Ser Asp Val Leu Ala Lys Leu Ala Phe
1 5 10
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Thr Phe Asp Val Ala Pro Ser Arg Leu Asp Phe
1 5 10
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Pro Phe Leu Gln Ala Ser Pro His Phe
1 5
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Leu Ser Ala Asp Asp Ile Arg Gly Ile Gln Ser Leu Tyr Gly Asp Pro
1 5 10 15
Lys
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Glu Gly Asp Ile Gln Gln Phe Leu Ile Thr Gly Asp Pro Lys Ala Ala
1 5 10 15
Tyr Asp Tyr
<210> 88
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Asn Pro Val Ser Gln Val Glu Ile Leu Lys Asn Lys Pro Leu Ser Val
1 5 10 15
Gly
<210> 89
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Lys Leu Tyr Ile Gly Asn Leu Ser Glu Asn Ala Ala Pro Ser
1 5 10
<210> 90
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Asp Ala Val Gln Met Val Ile Thr Glu Ala Gln Lys Val Asp Thr Arg
1 5 10 15
<210> 91
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Val Ala Arg Leu Pro Ile Ile Asp Leu Ala Pro Val Asp Val Gly Gly
1 5 10 15
Thr Asp
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Asn Lys Pro Ser Arg Leu Pro Phe Leu Asp Ile Ala Pro Leu Asp Ile
1 5 10 15
Gly Gly Ala Asp
20
<210> 93
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Ser Arg Pro Gln Ala Pro Ile Thr Gly Tyr Arg Ile Val Tyr Ser Pro
1 5 10 15
Ser Val
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Ile Leu Val Asp Trp Leu Val Gln Val
1 5
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Lys Ile Ile Gly Ile Met Glu Glu Val
1 5
<210> 96
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Ala Met Gly Ile Ala Pro Pro Lys Val
1 5
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Thr Leu Phe Pro Val Arg Leu Leu Val
1 5
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Val Leu Tyr Pro His Glu Pro Thr Ala Val
1 5 10
<210> 99
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Ala Leu Phe Gln Arg Pro Pro Leu Ile
1 5
<210> 100
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Lys Ile Val Asp Phe Ser Tyr Ser Val
1 5
<210> 101
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Leu Leu Leu Glu Ile Leu His Glu Ile
1 5
<210> 102
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Ser Leu Leu Ser Glu Leu Gln His Ala
1 5
<210> 103
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Lys Leu Leu Ser Asp Pro Asn Tyr Gly Val
1 5 10
<210> 104
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Ser Leu Val Ala Val Glu Leu Glu Lys Val
1 5 10
<210> 105
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Ile Val Ala Glu Ser Leu Gln Gln Val
1 5
<210> 106
<211> 9
<212> PRT
<213> Intelligent people
<400> 106
Ser Ile Leu Glu His Gln Ile Gln Val
1 5
<210> 107
<211> 9
<212> PRT
<213> Intelligent people
<400> 107
Ala Leu Ser Glu Arg Ala Val Ala Val
1 5
<210> 108
<211> 9
<212> PRT
<213> Intelligent people
<400> 108
Thr Leu Leu Asp Phe Ile Asn Ala Val
1 5
<210> 109
<211> 9
<212> PRT
<213> Intelligent people
<400> 109
Asn Leu Ile Glu Val Asn Glu Glu Val
1 5
<210> 110
<211> 15
<212> PRT
<213> Intelligent people
<400> 110
Ile Gln Leu Ile Val Gln Asp Lys Glu Ser Val Phe Ser Pro Arg
1 5 10 15
<210> 111
<211> 9
<212> PRT
<213> Intelligent people
<400> 111
Ser Leu Tyr Lys Gly Leu Leu Ser Val
1 5
<210> 112
<211> 9
<212> PRT
<213> Intelligent people
<400> 112
Val Leu Ala Pro Leu Phe Val Tyr Leu
1 5
<210> 113
<211> 9
<212> PRT
<213> Intelligent people
<400> 113
Phe Leu Leu Asp Gly Ser Ala Asn Val
1 5
<210> 114
<211> 9
<212> PRT
<213> Intelligent people
<400> 114
Ala Met Ser Ser Lys Phe Phe Leu Val
1 5
<210> 115
<211> 9
<212> PRT
<213> Intelligent people
<400> 115
Tyr Val Tyr Gln Asn Asn Ile Tyr Leu
1 5
<210> 116
<211> 9
<212> PRT
<213> Intelligent people
<400> 116
Lys Ile Gln Glu Met Gln His Phe Leu
1 5
<210> 117
<211> 9
<212> PRT
<213> Intelligent people
<400> 117
Ile Leu Ile Asp Trp Leu Val Gln Val
1 5
<210> 118
<211> 9
<212> PRT
<213> Intelligent people
<400> 118
Ser Leu His Phe Leu Ile Leu Tyr Val
1 5
<210> 119
<211> 9
<212> PRT
<213> Intelligent people
<400> 119
Ile Val Asp Asp Ile Thr Tyr Asn Val
1 5
<210> 120
<211> 9
<212> PRT
<213> Intelligent people
<400> 120
Lys Ile Gln Glu Ile Leu Thr Gln Val
1 5
<210> 121
<211> 9
<212> PRT
<213> Intelligent people
<400> 121
Arg Leu Leu Asp Ser Val Ser Arg Leu
1 5
<210> 122
<211> 9
<212> PRT
<213> Intelligent people
<400> 122
Lys Leu Ser Trp Asp Leu Ile Tyr Leu
1 5
<210> 123
<211> 9
<212> PRT
<213> Intelligent people
<400> 123
Gly Leu Thr Asp Asn Ile His Leu Val
1 5
<210> 124
<211> 9
<212> PRT
<213> Intelligent people
<400> 124
Asn Leu Leu Asp Leu Asp Tyr Glu Leu
1 5
<210> 125
<211> 9
<212> PRT
<213> Intelligent people
<400> 125
Arg Leu Asp Asp Leu Lys Met Thr Val
1 5
<210> 126
<211> 9
<212> PRT
<213> Intelligent people
<400> 126
Lys Leu Leu Thr Glu Val His Ala Ala
1 5
<210> 127
<211> 10
<212> PRT
<213> Intelligent people
<400> 127
Ile Leu Phe Pro Asp Ile Ile Ala Arg Ala
1 5 10
<210> 128
<211> 9
<212> PRT
<213> Intelligent people
<400> 128
Thr Leu Ser Ser Ile Lys Val Glu Val
1 5
<210> 129
<211> 9
<212> PRT
<213> Intelligent people
<400> 129
Gly Leu Ile Glu Ile Ile Ser Asn Ala
1 5
<210> 130
<211> 9
<212> PRT
<213> Intelligent people
<400> 130
Lys Ile Leu Glu Asp Val Val Gly Val
1 5
<210> 131
<211> 9
<212> PRT
<213> Intelligent people
<400> 131
Ala Leu Val Gln Asp Leu Ala Lys Ala
1 5
<210> 132
<211> 10
<212> PRT
<213> Intelligent people
<400> 132
Ala Leu Phe Val Arg Leu Leu Ala Leu Ala
1 5 10
<210> 133
<211> 9
<212> PRT
<213> Intelligent people
<400> 133
Arg Leu Ala Ser Tyr Leu Asp Lys Val
1 5
<210> 134
<211> 9
<212> PRT
<213> Intelligent people
<400> 134
Thr Leu Trp Tyr Arg Ala Pro Glu Val
1 5
<210> 135
<211> 9
<212> PRT
<213> Intelligent people
<400> 135
Ala Ile Asp Gly Asn Asn His Glu Val
1 5
<210> 136
<211> 9
<212> PRT
<213> Intelligent people
<400> 136
Ala Leu Val Asp His Thr Pro Tyr Leu
1 5
<210> 137
<211> 9
<212> PRT
<213> Intelligent people
<400> 137
Phe Leu Val Asp Gly Ser Trp Ser Val
1 5
<210> 138
<211> 11
<212> PRT
<213> Intelligent people
<400> 138
Ala Leu Asn Glu Glu Ala Gly Arg Leu Leu Leu
1 5 10
<210> 139
<211> 9
<212> PRT
<213> Intelligent people
<400> 139
Ser Leu Ile Glu Asp Leu Ile Leu Leu
1 5
<210> 140
<211> 9
<212> PRT
<213> Intelligent people
<400> 140
Thr Leu Tyr Pro His Thr Ser Gln Val
1 5
<210> 141
<211> 9
<212> PRT
<213> Intelligent people
<400> 141
Asn Leu Ile Glu Lys Ser Ile Tyr Leu
1 5
<210> 142
<211> 12
<212> PRT
<213> Intelligent people
<400> 142
Val Leu Leu Pro Val Glu Val Ala Thr His Tyr Leu
1 5 10
<210> 143
<211> 9
<212> PRT
<213> Intelligent people
<400> 143
Ala Ile Val Asp Lys Val Pro Ser Val
1 5
<210> 144
<211> 10
<212> PRT
<213> Intelligent people
<400> 144
Lys Ile Phe Asp Glu Ile Leu Val Asn Ala
1 5 10
<210> 145
<211> 9
<212> PRT
<213> Intelligent people
<400> 145
Ala Met Thr Gln Leu Leu Ala Gly Val
1 5
<210> 146
<211> 9
<212> PRT
<213> Intelligent people
<400> 146
Phe Gln Tyr Asp His Glu Ala Phe Leu
1 5
<210> 147
<211> 9
<212> PRT
<213> Intelligent people
<400> 147
Val Leu Phe Pro Asn Leu Lys Thr Val
1 5
<210> 148
<211> 9
<212> PRT
<213> Intelligent people
<400> 148
Ala Leu Phe Gly Ala Leu Phe Leu Ala
1 5
<210> 149
<211> 10
<212> PRT
<213> Intelligent people
<400> 149
Lys Leu Val Glu Phe Asp Phe Leu Gly Ala
1 5 10
<210> 150
<211> 9
<212> PRT
<213> Intelligent people
<400> 150
Gly Val Leu Glu Asn Ile Phe Gly Val
1 5
<210> 151
<211> 9
<212> PRT
<213> Intelligent people
<400> 151
Ala Val Val Glu Phe Leu Thr Ser Val
1 5
<210> 152
<211> 9
<212> PRT
<213> Intelligent people
<400> 152
Ile Leu Gln Asp Arg Leu Asn Gln Val
1 5
<210> 153
<211> 9
<212> PRT
<213> Intelligent people
<400> 153
Ala Leu Tyr Asp Ser Val Ile Leu Leu
1 5
<210> 154
<211> 9
<212> PRT
<213> Intelligent people
<400> 154
Ile Leu Phe Glu Ile Asn Pro Lys Leu
1 5
<210> 155
<211> 9
<212> PRT
<213> Intelligent people
<400> 155
Ala Leu Asp Glu Asn Leu His Gln Leu
1 5
<210> 156
<211> 9
<212> PRT
<213> Intelligent people
<400> 156
Thr Val Ala Glu Val Ile Gln Ser Val
1 5
<210> 157
<211> 9
<212> PRT
<213> Intelligent people
<400> 157
Lys Leu Phe Gly Glu Lys Thr Tyr Leu
1 5
<210> 158
<211> 9
<212> PRT
<213> Intelligent people
<400> 158
Lys Leu Asp Glu Thr Asn Asn Thr Leu
1 5
<210> 159
<211> 10
<212> PRT
<213> Intelligent people
<400> 159
Thr Tyr Lys Tyr Val Asp Ile Asn Thr Phe
1 5 10
<210> 160
<211> 9
<212> PRT
<213> Intelligent people
<400> 160
Ser Tyr Leu Gln Ala Ala Asn Ala Leu
1 5
<210> 161
<211> 10
<212> PRT
<213> Intelligent people
<400> 161
Leu Tyr Gln Ile Leu Gln Gly Ile Val Phe
1 5 10
<210> 162
<211> 17
<212> PRT
<213> Intelligent people
<400> 162
Thr Asn Gly Val Ile His Val Val Asp Lys Leu Leu Tyr Pro Ala Asp
1 5 10 15
Thr
<210> 163
<211> 10
<212> PRT
<213> Intelligent people
<400> 163
Glu Leu Ala Gly Ile Gly Ile Leu Thr Val
1 5 10
<210> 164
<211> 9
<212> PRT
<213> Intelligent people
<400> 164
Tyr Leu Leu Pro Ala Ile Val His Ile
1 5
<210> 165
<211> 15
<212> PRT
<213> Intelligent people
<400> 165
Thr Leu Gly Glu Phe Leu Lys Leu Asp Arg Glu Arg Ala Lys Asn
1 5 10 15
<210> 166
<211> 17
<212> PRT
<213> Intelligent people
<400> 166
Thr Phe Ser Tyr Val Asp Pro Val Ile Thr Ser Ile Ser Pro Lys Tyr
1 5 10 15
Gly
<210> 167
<211> 16
<212> PRT
<213> Intelligent people
<400> 167
Ser Gln Asp Asp Ile Lys Gly Ile Gln Lys Leu Tyr Gly Lys Arg Ser
1 5 10 15
<210> 168
<211> 16
<212> PRT
<213> Intelligent people
<400> 168
Ser Pro Gln Tyr Ser Trp Arg Ile Asn Gly Ile Pro Gln Gln His Thr
1 5 10 15
<210> 169
<211> 15
<212> PRT
<213> Intelligent people
<400> 169
Thr Pro Pro Ile Asp Ala His Thr Arg Asn Leu Leu Arg Asn His
1 5 10 15
<210> 170
<211> 15
<212> PRT
<213> Intelligent people
<400> 170
Lys Ile Phe Tyr Val Tyr Met Lys Arg Lys Tyr Glu Ala Met Thr
1 5 10 15
<210> 171
<211> 15
<212> PRT
<213> Intelligent people
<400> 171
Arg Lys Val Ala Glu Leu Val His Phe Leu Leu Leu Lys Tyr Arg
1 5 10 15
<210> 172
<211> 15
<212> PRT
<213> Intelligent people
<400> 172
Phe Phe Pro Val Ile Phe Ser Lys Ala Ser Ser Ser Leu Gln Leu
1 5 10 15
<210> 173
<211> 15
<212> PRT
<213> Intelligent people
<400> 173
Gly Asp Asn Gln Ile Met Pro Lys Ala Gly Leu Leu Ile Ile Val
1 5 10 15
<210> 174
<211> 15
<212> PRT
<213> Intelligent people
<400> 174
Thr Ser Tyr Val Lys Val Leu His His Met Val Lys Ile Ser Gly
1 5 10 15
<210> 175
<211> 18
<212> PRT
<213> Intelligent people
<400> 175
Val Leu Leu Lys Glu Phe Thr Val Ser Gly Asn Ile Leu Thr Ile Arg
1 5 10 15
Leu Thr
<210> 176
<211> 17
<212> PRT
<213> Intelligent people
<400> 176
Lys Val Pro Ile Lys Trp Met Ala Leu Glu Ser Ile Leu Arg Arg Arg
1 5 10 15
Phe
<210> 177
<211> 13
<212> PRT
<213> Intelligent people
<400> 177
Ala Lys Ala Val Ala Ala Trp Thr Leu Lys Ala Ala Ala
1 5 10