use of a peptide in the treatment of gastric, gastrointestinal, colorectal, pancreatic, lung or kidn

专利摘要:
immunotherapies against various tumors, including gastrointestinal and gastric cancer. the present invention relates to peptides, nucleic acids and cells for use in immunotherapeutic methods. in particular, the present invention relates to cancer immunotherapy. the present invention also relates to cytotoxic t-cell (ctl) peptide epitops associated with tumors, alone or in combination with other tumor-associated peptides, which serve as active pharmaceutical ingredients in vaccine compositions stimulating anti-tumor immune responses, to the present The invention relates to 95 new peptide sequences and their variations derived from class I molecules of human tumor cells usable in vaccine compositions to elicit antitumor immune responses.
公开号:BR112012023692B1
申请号:R112012023692
申请日:2011-03-15
公开日:2020-06-09
发明作者:Singh Harpreet；fritsche Jens；Lewandrowski Peter；Walter Steffen；Weinschenk Toni
申请人:Immatics Biotechnologies Gmbh；
IPC主号:

专利说明:

Descriptive Report of the Invention Patent for USE OF A PEPTIDE IN THE TREATMENT OF GASTRIC, GASTROINTESTINAL, COLORRETAL, PANCREATIC, PULMONARY OR RENAL CANCER.
[0001] The present invention relates to peptides, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to cancer immunotherapy. The present invention also relates to epitopes associated with CD8 + T-cell tumors, alone or in combination with other tumor-associated peptides, which serve as active pharmaceutical ingredients in vaccine compositions, which stimulate anti-tumor immune responses. The present invention relates to 33 new peptide sequences and their variations derived from HLA class I molecules of tumor cells can be used in vaccine compositions to elicit antitumor immune responses, in particular cytotoxic T cell (CTL) responses.
Background of the invention
[0002] Gastric cancer is a disease, in which malignant cells form in the lining of the stomach. Stomach or gastric cancer can develop anywhere in the stomach and can spread throughout the stomach and to other organs; particularly for the esophagus, lungs and liver. Stomach cancer is the fourth most common cancer in the world with 930,000 cases diagnosed in 2002. It is a disease with a high mortality rate (~ 800,000 per year) making it the second most common cause of cancer death worldwide after cancer pulmonary. It is more common in men and occurs more frequently in Asian and developing countries.
(http://www.who.int/mediacentre/factsheets/fs297/en/)
[0003] It represents approximately 2% (25,500 cases) of
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2/174 all new cases of cancer per year in the United States, but it is more common in other countries. It is the main type of cancer in Korea, with 20.8% of malignant neoplasms. In Japan, gastric cancer remains the most common cancer among men. Each year in the United States, approximately 13,000 men and 8,000 women are diagnosed with stomach cancer. Most of them are over 70 years old.
[0004] Stomach cancer is the fourth most common cancer in the world, after lung, breast, colon and rectal cancers. In addition, stomach cancer remains the second most common cause of cancer deaths. The American Cancer Society estimates that in 2007 there were approximately one million new cases, almost 70% of them in developing countries, and approximately 800,000 deaths (http://www.cancer.org/downloads/STT/Global Facts and Figures 20 07 rev2.pdf).
[0005] There are tremendous geographical variations in the incidence of this disease around the world. The rates of disease onset are highest in Asia and parts of South America and lowest in North America. The highest mortality rates were recorded in Chile, Japan, South America and the former Soviet Union.
[0006] Gastric cancer is often diagnosed at an advanced stage, because an exam is not performed in most parts of the world, except in Japan (and to a limited extent also in Korea) where early detection is often achieved. Thus, it remains an important challenge for healthcare professionals. Risk factors for gastric cancer are Helicobacter pilori (H. pilori) infection, smoking, high salt intake and other dietary factors. Some gastric cancers (1% to 3%) are associated with syndromes
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3/174 inherited from gastric cancer predisposition. Ecaderine mutations occur in approximately 25% of families with an autosomal dominant predisposition to spread gastric-type cancers. This subset of gastric cancer was called diffuse hereditary gastric cancer 12. It may be useful to provide genetic counseling and consider prophylactic gastrectomy in young, asymptomatic carriers of truncated germ-lines.
[0007] The stomach wall is composed of three layers of tissue: the mucosal layer (innermost), the muscular layer (in the middle), and the serosal layer (outermost). Gastric cancer begins in the cells lining the mucosal layer and, as it grows, extends to the outer layers. Four types of standard treatments are used. Treatment for gastric cancer may involve surgery, chemotherapy, radiation therapy or chemoradiation. Surgery is the primary treatment for gastric cancer. The goal of the surgery is to perform a complete resection with negative margins (R0 resection). However, approximately 50% of patients with locoregional gastric cancer cannot undergo an R0 resection. R1 indicates microscopic residual cancer (positive margins); and R2 indicates gross residual cancer (macroscopic), but not a distant disease. The outcome in the patient depends on the initial stage of cancer at diagnosis (NCCN Clinical Practice Guidelines in Oncology®).
[0008] The 5-year survival rate for curative surgical resection ranges from 30-50% for patients with stage II disease and from 10-25% for patients with stage III disease. These patients have a high probability of local and systemic relapse. Metastasis occurs in 80-90% of individuals with stomach cancer, with a six-month survival rate of 65% in those diagnosed in the early stages and less than 15% of those diagnosed in later stages.
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[0009] Thus, there is still a need for a new effective and safe treatment option for gastric cancer, prostate carcinoma, oral cavity carcinomas, oral squamous carcinoma (OSCC), acute myeloid leukemia (AML), HALT-induced MALT lymphoma pilori, colon carcinoma / colorectal cancer, glioblastoma, non-small cell lung cancer (NSCLC), cervical carcinoma, human breast cancer, prostate cancer, colon cancer, pancreatic cancers, ductal pancreatic adenocarcinoma, ovarian cancer, hepatocellular carcinoma , liver cancer, brain tumors of different phenotypes, leukemias such as acute lymphoblastic leukemia (ALL), lung cancer, Ewing's sarcoma, endometrial cancer, squamous cell carcinoma of the head and neck, laryngeal epithelial cancer, esophageal carcinoma, oral carcinoma, carcinoma of the urinary bladder, ovarian carcinomas, renal cell carcinoma, atypical meningioma, papillary thyroid carcinoma, brain tumors, carcinoma of ca salivary cancer, cervical cancer, extranodal T / NK cell lymphomas, Non-Hodgkins lymphoma and malignant solid tumors of the lung and chest as well as other tumors improving the well-being of patients without using chemotherapeutic agents or other agents that may lead to effects severe side effects.
[0010] The present invention incorporates peptides that stimulate the immune system and acts as anti-tumor agents in a non-invasive manner.
Summary of the invention
[0011] Stimulation of the immune response depends on the presence of antigens that are recognized as foreign by the host's immune system. The discovery of the existence of tumor-associated antigens has led to the possibility of using a host immune system to intervene in tumor growth. Various mechanisms for harnessing the humoral and cellular weapons of the
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5/174 immune systems are currently being explored in cancer immunotherapy.
[0012] Some specific elements of the cellular immune response are able to recognize and destroy tumor cells. The isolation of cytotoxic T cells (CTLs) from cell populations that infiltrate the tumor or, from peripheral blood, suggests that these cells play an important role in the natural immune defenses against cancer. CD8 positive T cells (TCD8 +) in particular, those that recognize Class I molecules of the Main Histocompatibility Complex (MHC) peptides, normally formed by 8 to 10 amino acid residues derived from proteins, defective ribosomal products (DRIPs) located in cytosols, they play an important role in this response. Human MHC molecules are also referred to as human leukocyte antigens (HLA).
[0013] There are two classes of MHC molecules: Class I MHC molecules that can be found in most cells that contain a nucleus. MHC molecules are composed of a heavy alpha chain and beta 2 microglobulin (MHC class I receptors) or an alpha and beta chain (MHC class II receptors), respectively. Its three-dimensional conformation results in a connecting groove, which is used for non-covalent interaction with peptides. Class I MHCs have peptides that result from proteolytic cleavage of predominantly endogenous proteins, DRIPs and larger peptides. Class II MHC molecules can be found predominantly in professional cells presenting antigens (APCs). They mainly feature peptides from exogenous or transmembrane proteins that are elevated by APCs during the course of endocytosis, and are subsequently processed. Class I MHC peptide and molecule complexes are recognized
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6/174 by CD8 positive cytotoxic T lymphocytes supporting the appropriate T cell (T cell receptor), where class II MHC peptide complexes and molecules are recognized by positive CD4 helper T cells supporting the appropriate TCR. It is known that TCR, peptide and MHC are thus present in a stoichiometric amount of 1: 1: 1.
[0014] For a peptide to elicit a cellular immune response, it needs to bind to an MHC molecule. This process depends on the alleles of the MHC molecule and the specific polymorphisms of the amino acid sequence of the peptide. The peptides that bind to MHCs class I normally have a length of 8-12 acid residues and contain two conserved residues (anchors) in their primary sequence of amino acids that interact with the corresponding MHC molecule binding gap. In this way, each MHC allele has a compulsory motif that determines which peptides can specifically bind to the compulsory fit.
[0015] In the MHC class I-dependent immune reaction, the peptides not only have to be able to bind to a certain class of MHC class I molecules being expressed by tumor cells, but they also have to be recognized by T cells supporting specific receptors T cell (TCR).
[0016] The antigens that are recognized by tumor-specific CTLs, that is, their epitopes, can be molecules derived from all classes of proteins, such as enzymes, receptors, transcription factors, etc. which are expressed and, in comparison with unchanged cells of the same origin, high-regulated in cells of the respective tumor.
[0017] The current classification of tumor-associated antigens (TAAs) contains the following important groups:
[0018] Testicular cancer antigens: the first TAAs ever
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7/174 identified that could be recognized by T cells belongs to this class, which was originally called cancer testis (CT) antigens because of the expression of its limbs in histologically different human tumors and, among normal tissues, only in spermatocytes / spermatogenia of testis and occasionally in placentas. Since testis cells do not express HLA class I and II molecules, these antigens cannot be recognized by T cells in normal tissues and, therefore, can be considered as immunologically tumor-specific. Known examples for CT antigens are members of the MAGE or NY-ESO-1 family. [0019] Differentiation antigens: these TAAs are shared between tumors and the normal tissue from which the tumor arose; most are found in normal melanomas and melanocytes. Many of these melanocyte related lineage proteins are involved in melanin biosynthesis and are therefore not tumor specific, but are nevertheless widely used for cancer immunotherapy. Examples include, but are not limited to, tyrosinase and Melan-A / MART-1 for melanoma or PSA for prostate cancer. [0020] Overexpressed TAAS: Genes encoding widely expressed TAAs have been detected in histologically different tumor types as well in many normal tissues, usually with lower level expression. It is possible that many of the epitopes processed and potentially presented by normal tissues are under the threshold level for T cell recognition, while their overexpression in tumor cells can trigger an anticancer response breaking the previously established tolerance. Prominent examples for this class of TAAs are Her-2 / neu, Survivin, Telomerase or WT1.
[0021] Tumor specific antigens: these rare TAAs arise from mutations of normal genes (such as β-catenin, CDK4, etc.).
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Some of these molecular changes are associated with neoplastic progression and / or transformation. Tumor-specific antigens can generally induce strong immune responses without inducing the risk for autoimmune reactions against normal tissues. On the other hand, these TAAs are in most cases only relevant to the exact tumor, in which they have been identified and are not normally shared between many individual tumors.
[0022] TAAs arising from abnormal post-translational changes: such TAAs may arise from proteins that are neither specific nor overexpressed in tumors, but nevertheless become associated with tumors by post-translational processes mainly active in tumors. Examples for this class arise from altered patterns of glycosylation leading to new epitopes in tumors such as for MUC1 or events such as protein junction during degradation that may or may not be tumor specific.
[0023] Oncoviral proteins: these TAAs are viral proteins that can play a critical role in the oncogenic process and, because they are foreign proteins (not of human origin), they can evoke a T cell response. Examples of such proteins are proteins of human papilloma type 16 viruses, E6 and E7, which are expressed in cervical carcinomas.
[0024] For proteins to be recognized by cytotoxic T lymphocytes as specific or associated tumor antigens and, for them to be used in a specific therapy, they need to meet certain prerequisites. The antigen must be expressed mainly by tumor cells and not by normal healthy tissues or preferably in small amounts. In addition, it is desirable that the respective antigen is present not only in a specific type of tumor, but also that it is present in high concentrations (ie, number of copies of the respective peptide per
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9/174 cell). Tumor-specific or associated antigens are often derived from proteins directly involved in transforming a normal cell into a tumor cell, due to a function, for example, in cell cycle control or suppression of apoptosis. In addition, targets downstream of proteins directly causative for a transformation can also be highly regulated and thus can be indirectly associated with a tumor. Such antigens indirectly associated with tumor may also be targets for improved vaccination (Singh-Jasuja H., Emmerich N. P., Rammensee H. G., Cancer Immunol. Immunother. 2004 Mar; 453 (3): 187-95). In both cases, the presence of epitopes in the antigen amino acid sequence is essential, since this peptide (immunogenic peptide), derived from a tumor-associated antigen, must induce a T cell response in vitro or in vivo.
[0025] Basically, any peptide capable of binding an MHC molecule can function as a T cell epitope. A prerequisite for inducing a T cell response in vitro or in vivo is the presence of a T cell with a corresponding TCR and the absence of immunological tolerance for this particular epitope.
[0026] Therefore, TAAs are a starting point for the development of an anti-tumor vaccine. The methods for identifying and characterizing TAAs are based on the use of CTL that can be isolated from healthy patients or people, or are based on the generation of differential transcription profiles or differential peptide expression patterns between tumors and normal tissues.
[0027] However, the identification of genes overexpressed in tumor tissues or human tumor cell lines, or selectively expressed in such tissues or cell lines, does not provide accurate information regarding the use of the antigens being
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10/174 transcripts of these genes in immune therapy. This is because only an individual subpopulation of epitopes of these antigens is suitable for such an application, since a T cell with a corresponding TCR must be present and the immunological tolerance for this particular epitope must be absent or minimal. It is therefore important to select only those peptides from overexpressed or selectively expressed proteins that are presented in relation to MHC molecules against which a functional T cell can be found. Such a functional T cell is defined as a T cell, since the stimulus with a specific antigen can be clonally expanded and can perform effector functions (effector T cell).
[0028] Helper T cells play an important role in coordinating the effective function of cytotoxic T cells in antitumor immunity. The helper T cell epitopes that elicit a Thi helper T cell response support the effector functions of CD8 + killer T cells, which include cytotoxic functions directed against tumor cells that have tumor-associated peptide / MHC complexes on cell surfaces. Thus, tumor-associated helper T-cell peptide epitopes, alone or in combination with other tumor-associated peptides, can serve as active pharmaceutical ingredients in vaccine compositions that stimulate anti-tumor immune responses.
Brief Description of the Drawings
[0029] Figure 1: Exemplary mass spectrum of CDC2-001 demonstrating its presentation in a primary tumor sample GC2464. NanoESI-LCMS was performed in a peptide basin eluted from sample GC 2464. The mass chromatogram for m / z 597.3501 ± 0.001 Da, z = 2 shows a peptide peak at 151.63 min retention time. B) The peak detected in the chromatogram of
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11/174 mass in 151.63 min revealed a m / z signal of 597.3501 in the MS spectrum. C) A collapsed mass spectrum induced by collision of the selected precursor m / z 597.3501 recorded in the nanoESI-LCMS experiment at the given retention time confirmed the presence of CDC2-001 in the GC2464 tumor sample. D) The fragmentation pattern of the synthetic reference peptide CDC2-001 was recorded and compared with the generated natural fragmentation pattern TUMAP and shown in C for sequential verification.
[0030] Figure 2: MRNA expression profiles of selected proteins in normal tissues and in 25 gastric cancer samples.
[0031] CDC2 (evidence kit ID: 203213_at)
[0032] ASPM (test kit ID: 219918_s_at)
[0033] Figure 3: Exemplary results of peptide specific in vitro immunogenicity class I TUMAPs. CD8 + T cells were prepared using artificial APCs loaded with relevant peptide (left panel) and irrelevant peptide (right panel), respectively. After three cycles of stimulation, the detection of peptide-reactive cells was performed by double staining with relevant and irrelevant A * 2402 multimers. The cells shown are pressed into live CD8 + lymphocytes and the numbers in the dashes represent percentages of multimer-positive cells.
Detailed description of the invention
[0034] As used in this document and unless otherwise stated, all terms are defined as given below. The term peptide is used here to denote a series of amino acid residues, connected to each other typically by peptide bonds between the alpha-amino and carbonyl groups of the adjacent amino acids. The peptides are preferably 9 amino acids in length, but can be as short as 8 amino acids in length.
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12/174 long and as long as 10, 11, 12, 13 or 14 amino acids in length.
[0035] The term oligopeptide is used here to denote a series of amino acid residues, connected to each other typically by peptide bonds between the alpha-amino and carbonyl groups of the adjacent amino acids. The length of the oligopeptide is not critical to the invention, as long as the correct epitope or epitopes are maintained on it. Oligopeptides are typically less than approximately 30 amino acid residues in length and greater than approximately 14 amino acids in length.
[0036] The term polypeptide is used to denote a series of amino acid residues, connected to each other typically by peptide bonds between the alpha-amino and carbonyl groups of the adjacent amino acids. The length of the polypeptide is not critical to the invention, as long as the correct epitope or epitopes are maintained on it. In contrast to the terms peptide or oligopeptide, the term polypeptide refers to molecules containing more than approximately 30 amino acid residues.
[0037] A peptide, oligopeptide, protein, or polynucleotide encoding for such a molecule is immunogenic (and thus is an immunogen within the present invention), if it is able to induce an immune response. In the case of the present invention, immunogenicity is more precisely defined as the ability to induce a T cell response. Thus, an immunogen would be a molecule that is capable of inducing an immune response, and in the case of the present invention, a molecule capable of induce a T cell response.
[0038] A T cell epitope requires a small peptide that is bound to a MHC class I receptor, forming a ternary complex (MHC class I alpha chain, beta-2 microglobulin and
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13/174 peptide) that can be recognized by a T cell bearing a T cell receptor that corresponds to the binding with the MHC / peptide complex with appropriate affinity. Binding peptides to MHC class I molecules are typically 8 to 14 amino acids in length, and more typically 9 amino acids in length. [0039] In humans, there are three different genetic loci that encode class I MHC molecules (human MHC molecules are also called human leukocyte antigens (HLA)): HLA-A, HLA-B, and HLA-C . HLA-A * 0l, HLA-A * 02, and HLA-A * 024 are examples of different MHC class I alleles that can be expressed from these loci.
[0040] Table 1: F frequencies of HLA * A024 expression and the most frequent HLA * A02402 serotypes. The frequencies are deduced from frequencies of Gf haplotypes in the American population, as adapted from Mori et al. (Mori et al 1017-27) using the formula Hardy-Weinberg F = 1- (1-Gf) ² . For more details, see Chanock and others (Chanock and others 1211-23).
[0041] The expression frequencies of HLA * 24 and A * 2402 serotypes in the world
Allele Population Allele frequency calculated phenotype A * 24 Philippines 65% A * 24 Russian Nenets 61% A * 2402 Japan 59% A * 24 Malaysia 58% A * 2402 Philippines 54% A * 24 India 47% A * 24 South Korea 40% A * 24 Sri Lanka 37% A * 24 China 32% A * 2402 India 29% A * 24 Western Australia 22% A * 24 USA 22%
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A * 24 Russian Samaras 20% A * 24 South America 20% A * 24 Europe 18% [0042] As used earlier, reference to a string of DNA includes both a DNA from single ribbon as a double ribbon.
Thus, the specific sequence, unless the context indicates otherwise, refers to the single-stranded DNA of such a sequence, the duplex of such a sequence with its complement (double-stranded DNA) and the complement of such a sequence. The term coding region refers to that portion of a gene that codes naturally or normally for the expression product of that gene in its natural genomic environment, that is, the region encoding in vivo for the native expression product of the gene .
[0043] The coding region may be from an unmuted (normal), mutated or altered gene, or even from a DNA sequence, or gene, fully synthesized in the methods used in the laboratory, well known in the art of synthesis of DNA.
[0044] The term nucleotide sequence refers to a deoxyribonucleotide heteropolymer.
[0045] The nucleotide sequence encoding for a particular peptide, oligopeptide, or polypeptide can occur naturally or they can be constructed synthetically. Generally, segments of DNA encoding the peptides, polypeptides, and proteins of this invention are assembled from cDNA fragments and short oligonucleotide linkers, or from a series of oligonucleotides, to provide a synthetic gene that is capable of being expressed in one unit recombinant transcriptional containing regulatory elements derived from a microbial or viral operon.
[0046] The term expression product means the polypeptide or protein that is the natural product of translation of the gene and any
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15/174 nucleic acid sequence encoding equivalent results of genetic code degeneration and, therefore, encoding for the same amino acid (s).
[0047] The term fragment, when referring to a coding sequence, means a portion of DNA comprising less than the complete coding region, whose expression product essentially retains the same biological function or activity as the expression product of the entire coding region.
[0048] The term DNA segment refers to a DNA polymer, in the form of a separate fragment or as a larger component of DNA, which has been derived from isolated DNA at least once in substantially pure form, that is, free of contaminated endogenous materials and in an amount or concentration that enables identification, manipulation and recovery of the segment and its nucleotide component sequences by standardized biochemical methods, for example, using a cloning vector. Such segments are provided in the form of an uninterrupted framed open reading by internal untranslated sequences, or introns, which are typically present in eukaryotic genes. Untranslated DNA sequences may be present downstream of the framed open reading, where it does not interfere with manipulation or expression of the coding regions.
[0049] The term primero means a short nucleic acid sequence that can be paired with a DNA strand and provides a free 3'OH terminal, in which a DNA polymerase begins a synthesis of a deoxyribonucleotide stream.
[0050] The term promoter means a region of DNA involved in binding RNA polymerase to initiate transcription.
[0051] The term isolated means that the material is removed from its original environment (for example, the natural environment, if it occurs
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16/174 of course). For example, a polynucleotide that occurs naturally or present in a living animal is not isolated, but the same polynucleotide or polypeptide - separated from some or all of the materials that coexist in the natural system - is isolated. Such polynucleotides could be part of a vector and / or such polynucleotides or polypeptides could be part of a composition, and still be isolated in such a vector or composition, which is not part of its natural environment.
[0052] Polynucleotides, and recombinant or immunogenic polypeptides, described in accordance with the present invention, can also be in purified form. The term purified does not require absolute purity; rather it is intended as a relative definition, and can include preparations that are highly purified or preparations that are only partially purified, as those terms are understood by professionals in the relevant field. For example, individual clones isolated from a cDNA library that have been purified in a conventional manner for electrophoretic homogeneity. Purification of the starting material or natural material to at least one order of magnitude, preferably two or three orders and, more preferably, four or five orders of magnitude is expressly contemplated. Furthermore, a claimed polypeptide having a purity of preferably 99,999%, or at least 99.99% or 99.9%; and yet desirably 99% by weight or greater is expressly contemplated.
[0053] The nucleic acids and polypeptide expression products described in accordance with the present invention, as well as expression vectors containing such nucleic acids and / or polypeptides, can be in enriched form. As used here, the term enriched means that the concentration of the material is at least approximately 2, 5, 10, 100, or 1000 times greater than its
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17/174 natural concentration (for example), advantageously 0.01% by weight, preferably at least approximately 0.1% by weight. Enriched preparations of approximately 0.5%, 1%, 5%, 10%, and 20% by weight are also contemplated. The sequences, constructs, vectors, clones, and other materials constituting the present invention can advantageously be in enriched or isolated form.
[0054] The term active fragment means a fragment that generates an immune response (that is, it has immunogenic activity) when administered, alone or optionally with a suitable adjuvant, to an animal, such as a mammal, for example, a rabbit or a mouse, or also a human being, such an immune response takes the form of stimulating a T cell response within the recipient animal, such as a human. On the other hand, the active fragment can also be used to induce a T cell response in vitro.
[0055] As used here, the terms portion, segment and fragment, when used in relation to polypeptides, refer to a continuous sequence of residues, such as amino acid residues, which form a larger sequence subset. For example, if a polypeptide is subjected to a treatment with any of the common endopeptidases, such as trypsin or chinotripsin, the oligopeptides resulting from such treatment would represent initial portions, segments or fragments of the polypeptide. This means that any such fragment will necessarily contain a segment as part of its amino acid sequence, fragment or portion, which is substantially identical, if not exactly identical, to a sequence of SEQ ID NO: 1 to 33, which correspond to the proteins naturally occurring, or proteins of SEQ ID NO: 1 to 33. When used in connection with polynucleotides, these terms refer to products produced by
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18/174 treatment of said polynucleotides with any of the common endonucleases.
[0056] According to the present invention, the term Percent Identity or Identical percent when referring to a sequence, means that a sequence is compared with a claimed or described sequence after the alignment of the sequence is compared (the Compared Sequence) with the claimed or described sequence (the Reference Sequence). The percent identity is then determined according to the following formula:
[0057] Percent identity = 100 [I - (C / R)]
[0058] Where C is the number of differences between the Reference Sequence and the Compared Sequence on the alignment length between the Reference Sequence and the Compared Sequence, in which
[0059] each base or amino acid in the Reference Sequence that does not have a corresponding base or amino acid aligned in the Compared Sequence and
[0060] each gap in the Reference Sequence and
[0061] each base or amino acid aligned in the sequence
Reference that is different from a base or amino acid aligned in the Compared Sequence, constitutes a difference; and R is the number of bases or amino acids in the Reference Sequence in addition to the length of the alignment with the Compared Sequence with any gap created in the Reference Sequence also being counted as a base or amino acid.
[0062] If an alignment exists between the Compared Sequence and the Reference Sequence for which the percent identity as calculated above is approx. equal to or greater than a minimum specified as Percent Identity, then the Compared String has the minimum specified to Identity percent
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19/174 that the alignments may exist, with the Identity percent calculated above being less than the Identity percent specified. [0063] The original peptides described here can be modified by replacing one or more residues in different, perhaps selective, areas within the peptide stream, if not otherwise determined. Such substitutions may be of a conservative nature, for example, where an amino acid is replaced by an amino acid of similar structure and characteristics, such as where a hydrophobic amino acid is replaced by another hydrophobic amino acid. Even more conservative would be the substitution of amino acids of the same or similar size and chemical nature, such as when a leucine is replaced by isoleucine. In studies of sequence variations in naturally occurring homologous protein families, some amino acid substitutions are more tolerated than others, and these often show similarities in size, charge, polarity and hydrophobicity between the original amino acid and its replacement, and this is the basis for the definition of conservative substitutions.
[0064] Conservative substitutions are defined here 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 residues, negatively charged and their starches (Asp, Asn, Glu, Gln); Group 3 - polar residues, positively charged (His, Arg, Lys); Group 4 - large, aliphatic, non-polar residues (Met, Leu, Ile, Val, Cys); and Group 5 - large, aromatic residues (Phe, Tyr, Trp).
[0065] Less conservative substitutions may involve replacing one amino acid with another that has similar characteristics, but is slightly different in size, such as
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20/174 replacement of an alanine with an isoleucine residue. Highly non-conservative substitutions may involve replacing an acidic amino acid with one that is polar, or even one that is basic in character. Such radical substitutions cannot, however, be dismissed as potentially ineffective, as chemical effects are not entirely predictable and radical substitutions may also result in splendid effects contrary to unpredictable simple chemical principles.
[0066] Naturally, such substitutions may involve different structures than ordinary L-amino acids. Thus, D-amino acids may be substituted for the L-amino acids generally found in the antigenic peptides of the invention and, but are still covered by the disclosure dealt with herein. In addition, amino acids having non-regular R groups (i.e., R groups different from those found in the 20 common natural protein amino acids) can also be used for substitution purposes to produce immunogenic immunogens and polypeptides according to the present invention.
[0067] If it is determined that substitutions in more than one position result in a peptide with substantially equivalent or greater antigenic activity as defined below, then combinations of these substitutions will be tested to determine whether the combined substitutions result in additive or synergistic effects on antigenicity of the peptide. In most cases, no more than four positions within the peptide would be simultaneously substituted.
[0068] The term T cell response means the proliferation and specific activation of effector functions induced by a peptide in vitro or in vivo. For MHC class I restricted CTLs, effector functions can be lysis of target cells that present peptide-pulsed, peptide-precursor-pulsed or natural,
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21/174 cytokine secretion, preferably Interferon-gamma, TNF-alpha, or peptide-induced IL-2, secretion of effector molecules, preferably peptide-induced granzymes or perforins, or degranulation.
[0069] Preferably, when CTLs specific for a peptide of SEQ ID NO: 1 to 33 are tested against the substituted peptides, the peptide concentration at which the substituted peptides achieve half of the maximum background lysis increase is not more than approximately 1 mM, preferably not more than approximately 1 μΜ, more preferably not more than approximately 1 nM, and even more preferably not more than approximately 100 pM, and most preferably preferably not more than approximately 10 pM . It is also preferred that the substituted peptide is recognized by CTLs from more than one individual, at least two, and more preferably three individuals.
Thus, the epitope of the present invention may be identical to tumor-associated or tumor-specific epitopes that occur naturally or may include epitopes that differ by no more than 4 residues of the reference peptide, as long as they have substantially antigenic activity. identical.
[0071] Immunotherapeutic improvements for treatment
[0072] Stimulation of the immune response depends on the presence of antigens that are recognized as foreign by the host's immune system. The discovery of the existence of tumor-associated antigens has led to the possibility of using a host immune system to intervene in tumor growth. Several mechanisms for harnessing humoral and cellular weapons of the immune system are currently being explored in cancer immunotherapy.
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[0073] Some specific elements of the cellular immune response are able to recognize and destroy tumor cells. The isolation of cytotoxic T cells (CTL) from cell populations that infiltrate the tumor or, from peripheral blood, suggests that these cells play an important role in the natural immune defenses against cancer. CD8 positive T cells in particular, those that recognize the Class I molecules of the Main Histocompatibility Complex (MHC) peptides, normally formed by 8 to 12 amino acid residues derived from proteins or ribosomal defective products (DRIPs) located in the cytosols, play an important role in that response. Human MHC molecules are also referred to as human leukocyte antigens (HLA).
[0074] Class I MHC molecules can be found in most cells containing a nucleus that has peptides that result from proteolytic cleavage of proteins, mostly endogenous, cytosolic or nuclear, DRIPs and larger peptides. However, peptides derived from endosomal compartments or exogenous sources are also often found in MHC class I molecules. This non-classical presentation of class I is referred to in the literature as cross-presentation.
[0075] For proteins to be recognized by cytotoxic T lymphocytes as specific or associated tumor antigens and, for them to be used in a specific therapy, they need to meet certain prerequisites. The antigen must be expressed mainly by tumor cells and not by normal healthy tissues or preferably in small amounts. In addition, it is desirable that the respective antigen is present not only in a specific type of tumor, but also that it is present in high concentrations (for example, number of copies of the respective
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23/174 peptide per cell). Tumor-specific or associated antigens are often derived from proteins directly involved in transforming a normal cell into a tumor cell, due to a function, for example, in cell cycle control or apoptosis. In addition, targets downstream of the proteins that are also directly causative for a transformation can also be highly regulated and thus indirectly associated with a tumor. Such antigens indirectly associated with a tumor may also be targets for an attempted vaccination. In both cases, the presence of epitopes in the amino acid sequence of the antigen is essential, since this peptide (immunogenic peptide), derived from a tumor-associated antigen, must induce a T cell response in vitro or in vivo.
[0076] Basically, any peptide capable of binding an MHC molecule can function as a T cell epitope. A prerequisite for inducing a T cell response in vitro or in vivo is the presence of a T cell with a corresponding TCR and the absence of immunological tolerance for this particular epitope.
[0077] Therefore, TAAs are a starting point for the development of an anti-tumor vaccine. The methods for identifying and characterizing TAAs are based on the use of CTL that can be isolated from healthy patients or people, or are based on the generation of differential transcription profiles or differential peptide expression patterns between tumors and normal tissues. (450-54; Weinschenk and others 5.818-27).
[0078] However, the identification of genes overexpressed in tumor tissues or human tumor cell lines, or selectively expressed in such tissues or cell lines, does not provide accurate information regarding the use of the antigens being transcribed from these genes in immune therapy. This is because only one
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24/174 individual subpopulation of epitopes of these antigens is convenient for such application, since a T cell with a corresponding TCR must be present and the immunological tolerance for this particular epitope must be absent or minimal. It is therefore important to select only those peptides from over-expressed or selectively expressed proteins that are presented in relation to MHC molecules against which a functional T cell can be found. Such a functional T cell is defined as a T cell, since the stimulus with a specific antigen can be clonally expanded and can perform effector functions (effector T cell).
[0079] Helper T cells play an important role in coordinating the effector function of cytotoxic T cells in antitumor immunity. The helper T cell epitopes that elicit a TH1 helper T cell response support the effector functions of CD8-positive killer T cells, which include cytotoxic functions directed against tumor cells that have tumor-associated peptide / MHC complexes on cell surfaces. Thus, tumor-associated helper T-cell peptide epitopes, alone or in combination with other tumor-associated peptides, can serve as active pharmaceutical ingredients in vaccine compositions that stimulate anti-tumor immune responses.
[0080] Since both types of responses, CD8 and CD4 dependent, jointly and synergistically contribute to the antitumor effect, the identification and characterization of tumor associated antigens recognized by any CD8 + CTLs (MHC class I molecule) or by positive CD4 CTLs (MHC class II molecule) are important in the development of tumor vaccines. It is, therefore, an object of the present invention, to provide peptide compositions containing peptide-binding peptides
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25/174 MHC complexes of any class.
[0081] Considering the serious side effects and expenses associated with cancer, better methods of diagnosis and prognosis are desperately needed. Therefore, there is a need to identify other factors that represent biomarkers for cancer in general and gastric cancer in particular. Therefore, there is a need to identify other factors that can be used in the treatment of cancer in general and gastric cancer in particular.
[0082] In addition, there is no established therapeutic project for gastric cancer patients with biochemical recurrence after radical prostatectomy, usually caused by residual tumor left in situ in the presence of locally advanced tumor growth. Currently, new therapeutic approaches are sought that provide lower morbidity with comparable therapeutic efficacy in relation to the available therapeutic approaches.
[0083] The present invention provides peptides that are useful in treating gastric cancer and other tumors that overexpress the peptides of the invention. Through mass spectrometry, these peptides have been proven to be naturally presented by HLA molecules in primary human gastric cancer samples (see example 1 and figure 1).
[0084] The source gene from which the peptides are derived has been shown to be highly overexpressed in gastric cancer, renal cell carcinoma, colon cancer, non-small cell lung carcinoma, adenocarcinoma, prostate cancer, benign neoplasm and melanoma malignant compared to normal tissues (see example 2, and figure 2) demonstrating a high degree of tumor association of the peptide, i.e. these peptides are intensely presented in tumor tissue, but not in normal tissues.
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[0085] HLA-bound peptides can be recognized by the immune system, specifically by T lymphocytes / T cells T cells can destroy cells presenting the recognized HLA / peptide complex, for example, gastric cancer cells presenting the derived peptides.
[0086] All peptides, which were compatible with the validation platform - see example 3 - of the present invention were shown to be able to stimulate T cell responses (see example 3 and figure 3). Thus, the peptides are useful for generating an immune response in a patient, in which tumor cells can be destroyed. An immune response in a patient can be induced by direct administration of the described peptides or suitable precursor substances (for example, elongated peptides, proteins, or nucleic acids encoding these peptides) to the patient, ideally in combination with an agent improving the immunogenicity (i.e. adjuvant). The immune response that stems from such therapeutic vaccination can be expected to be highly specific against tumor cells because the target peptides of the present invention are not presented in normal tissues in comparable copy numbers, preventing the risk of undesirable autoimmune reactions against normal cells on the patient.
[0087] The pharmaceutical compositions include the peptides, either in free form or in the form of a pharmaceutically acceptable salt. As used herein, a pharmaceutically acceptable salt refers to a derivative of the disclosed peptide where the peptide is altered by acid formation or by basic salts of the agent. For example, acid salts are prepared from the free base (usually where the neutral form of the drug has a neutral NH2 group), involving a reaction with suitable acid. Acids suitable for the preparation of acid salts include both organic acids, e.g.
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27/174 acetic, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methane sulfonic acid, ethane sulfonic acid, ptoluenesulfonic acid, salicylic acid and the like, as well as inorganic acids, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like. Conversely, the preparation of base salts from acidic molecules, which can be present in a peptide, is carried out using a pharmaceutically acceptable base such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, trimethylamino or similar.
[0088] In an especially preferred variant, the pharmaceutical compositions comprise peptides such as salts of acetic acid (acetates) or hydrochloric acid (chlorides).
[0089] In addition to being useful for treating cancer, the peptides of the present invention are also useful as diagnostics. Since the peptides were generated from gastric cancer cells and since it was determined that these peptides are not present in normal tissues, these peptides can be used to diagnose the presence of cancer.
[0090] The presence of the claimed peptides in tissue biopsies can assist a pathologist in the diagnosis of cancer. The detection of certain peptides by means of antibodies, mass spectrometry or other methods known in the art can tell the pathologist that the tissue is malignant or inflamed or generally sick. The presence of groups of peptides can enable the classification or subclassification of diseased tissues.
[0091] The detection of peptides in a diseased tissue specimen may enable the decision on the benefit of therapies involving the
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28/174 immune system, especially if T lymphocytes are known or expected to be involved in the mechanism of action. Loss of MHC expression is a well-described mechanism in which malignant or infected cells escape immunomonitoring. Thus, the presence of peptides shows that this mechanism is not exploited by the analyzed cells.
[0092] The peptides can be used to analyze lymphocyte responses against those peptides such as T cell responses or antibody responses against the complexed peptide or TUMAP for MHC molecules. These lymphocyte responses can be used as prognostic markers for decision in other therapy steps. These responses can also be used as substitute markers in immunotherapy attempts trying to induce responses by different means, for example, protein vaccination, nucleic acids, autologous materials, adoptive lymphocyte transfer. In gene therapy settings, lymphocyte responses against peptides can be considered when assessing side effects. Monitoring lymphocyte responses may also be a valuable tool for complementary examinations of transplantation therapies, for example, for the detection of transplantation against host and host against transplantation diseases.
[0093] Peptides can be used to generate and develop specific antibodies against MHC / peptide complexes. These can be used for therapy, attacking toxins or radioactive substances in diseased tissue. Another use of these antibodies may be targeting radionuclides in diseased tissue for imaging purposes such as PET. This use can help detect small metastases or determine the precise size and location of diseased tissues.
[0094] Furthermore, they can be used to verify a pathologist's diagnosis of cancer, based on a
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[0095] Table 2 shows the peptides according to the present invention, their respective SEQ ID No., the HLA alleles for which the respective peptides, and the source proteins from which these peptides may arise. All peptides bind the HLA A * 024 alleles.
Table 2: Peptides of the present invention
SEQ ID NO: Peptide code Sequence Source protein (s) 1 CDC2-001 LYQILQGIVF CDK1 2 ASPM-002 SYNPLWLRI ASPM 3 UCHL5-001 NYLPFIMEL UCHL5 4 MET-006 SYIDVLPEF MET 5 PROM1-001 SYIIDPLNL PROM1 6 MMP11-001 VWSDVTPLTF MMP11 7 MST1R-001 NYLLYVSNF MST1R 8 NFYB-001 VYTTSYQQI NFYB 9 SMC4-001 HYKPTPLYF SMC4 10 UQCRB-001 YYNAAGFNKL UQCRB 11 PPAP2C-001 AYLVYTDRL PPAP2C 12 AVL9-001 FYISPVNKL AVL9 13 NUF2-001 VYGIRLEHF NUF2 14 ABL1-001 TYGNLLDYL ABL1 15 MUC6-001 NYEETFPHI MUC6 16 ASPM-001 RYLWATVTI ASPM 17 EPHA2-005 VYFSKSEQL EPHA2 18 MMP3-001 VFIFKGNQF MMP3 19 NUF2-002 RFLSGIINF NUF2 20 PLK4-001 QYASRFVQL PLK4 21 ATAD2-002 KYLTVKDYL ATAD2 22 COL12A1-001 VYNPTPNSL COL12A1 23 COL6A3-001 SYLQAANAL COL6A3 24 FANCI-001 FYQPKIQQF FANCI 25 RPS11-001 YYKNIGLGF RPS11 26 ATAD2-001 AYAIIKEEL ATAD2 27 ATAD2-003 LYPEVFEKF ATAD2 28 HSP90B1-001 KYNDTFWKEF HSP90B1
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SEQ ID NO: Peptide code Sequence Source protein (s) 29 SIAH2-001 VFDTAIAHLF SIAH2 30 SLC6A6-001 VYPNWAIGL SLC6A6 31 IQGAP3-001 VYKVVGNLL IQGAP3 32 ERBB3-001 VYIEKNDKL ERBB3 33 KIF2C-001 IYNGKLFDLL KIF2C
0096] Other interesting HLA A * 024 peptides of the invention
SEQ IDN °: Peptide code Sequence Source protein (s) 34 CCDC88A-001 QYIDKLNEL CCDC88A 35 CCNB1-003 MYMTVSIIDRF CCNB1 36 CCND2-001 RYLPQCSYF CCND2 37 CCNE2-001 IYAPKLQEF CCNE2 38 CEA-010 IYPDASLLI CEACAM1, CEACAM5,CEACAM6 39 CLCN3-001 VYLLNSTTL CLCN3 40 DNAJC10-001 IYLEVIHNL DNAJC10 41 DNAJC10-002 AYPTVKFYF42 EIF2S3-001 IFSKIVSLF EIF2S3, LOC255308 43 EIF3L-001 YYYVGFAYL EIF3L, LOC340947 44 EPPK1-001 RYLEGTSCI EPPK1 45 ERBB2-001 TYLPTNASLSF ERBB2 46 GPR39-001 SYATLLHVL GPR39 47 ITGB4-001 DYTIGFGKF ITGB4 48 LCN2-001 SYNVTSVLF LCN2 49 SDHC-001 SYLELVKSL LOC642502, SDHC 50 PBK-001 SYQKVIELF PBK 51 POLD3-001 LYLENIDEF POLD3 52 PSMD14-001 VYISSLALL PSMD14 53 PTK2-001 RYLPKGFLNQF PTK2 54 RPS11-001 YYKNIGLGF RPS11 55 TSPAN1-002 VYTTMAEHF TSPAN1 56 ZNF598-001 DYAYLREHF ZNF598 57 ADAM10-001 LYIQTDHLFF ADAM10 58 MMP12-001 TYKYVDINTF MMP12 59 RRM2-001 YFISHVLAF RRM2 60 TMPRSS4-001 VYTKVSAYL TMPRSS4 61 TSPAN8-001 VYKETCISF TSPAN8
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[0097] In another embodiment of the invention, HLA-binding peptides against gastric cancer are also described. For people who have A * 02 and / or A * 24 positive, mixtures of the described peptides can be used for the treatment of gastric cancer. Preferred mixtures are 2 to 20 peptides and mixtures of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20 peptides .
SEQ ID NO: Peptide code Sequence Source protein (s) 62 DIO2-001 ALYDSVILL DIO2 63 IGF2BP3-001 KIQEILTQV IGF2BP3 64 LMNB1-001 LADETLLKV LMNB1 65 WNT5A-001 AMSSKFFLV WNT5A 66 FAP-003 YVYQNNIYL FAP 67 COPG-001 VLEDLEVTV COPG, COPG2, TSGA13 68 COL6A3-002 FLLDGSANV COL6A3 69 COL6A3-003 NLLDLDYEL COL6A3 70 COL6A3-004 FLIDSSEGV COL6A3 71 PSMC2-001 ALDEGDIAL PSMC2 72 UBE2S-001 ALNEEAGRLLL UBE2S 73 KIF11-001 ILSPTVVSI KIF11 74 ADAM8-001 KLLTEVHAA ADAM8 75 CCNB1-001 ALVQDLAKA CCNB1 76 CDC6-001 ILQDRLNQV CDC6 77 F2R-001 TLDPRSFLL F2R 78 OLFM4-001 TLDDLLLYI OLFM4 79 THY1-001 SLLAQNTSWLL THY1 80 CEP250-001 SLAEVNTQL CEP250 81 HIF1A-001 ALDGFVMVL HIF1A 82 KRAS-001 GVDDAFYTL KRAS 83 MET-001 YVDPVITSI MET 84 NCAPG-001 YLLSYIQSI NCAPG 85 NCAPG-002 QIDDVTIKI NCAPG 86 TOP-004 YLYGQTTTYL TOP2A 87 TOP-005 KLDETGNSL TOP2A 88 LAMC2-002 RLDDLKMTV LAMC2 89 AHR-001 LTDEILTYV AHR 90 CCNB1-002 ILIDWLVQV CCNB1
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SEQ ID NO: Peptide code Sequence Source protein (s) 91 CEACAM6-001 VLYGPDVPTI CEACAM6 92 COPB1-001 SIFGEDALANV COPB1 93 HMMR-001 KLLEYIEEI HMMR 94 TPX2-001 KILEDVVGV TPX2 95 TOP-001 KIFDEILVNA TOP2A, TOP2B
Cell division 2 protein (CDC2)
[0098] The serine / threonine kinase CDC2, also known as Cdk1 (cyclin 1 dependent kinase), has a key role in controlling the cell cycle. It is known as the main regulator of the G2-to-M transition. At the end of interphase, it binds type A cyclins. After breaking the nuclear envelope, type A cyclins are replaced by B cyclins, which form the mitosis-promoting factor (MPF) with Cdc2. MPF is essential for directing cells by mitosis.
[0099] The function of Cdc2 in mitosis is non-redundant and cannot be compensated for by the activity of other Cdks, such as Cdk2, 4 and 6. In contrast, Cdc2 has been reported to function also in other phases of the cell cycle such as in the transition G1-S, and is capable of substituting for the Cdks interface. Thus, Cdc2 has been proposed as the only essential cell cycle Cdk.
[00100] Cdc2 overexpression has been seen in several cancers, often corresponding to poor prognosis. Among them are prostate carcinoma, oral cavity carcinoma, oral squamous carcinoma (OSCC), acute myeloid leukemia (AML) (Qian and others), MALT lymphoma induced by H. pilori (Banerjee et al 217-25) and carcinoma of colon (Yasui and others 36-41). In gastric carcinoma, overexpression and / or improved activity was found and could have a causative role. Inhibitors of Cdc2 and other Cdks were considered to be drug candidates for cancer therapy (Shapiro 1770-83).
Abnormal triangular type protein associated with microcephaly (ASPM) [00101] Abnormal triangular type protein associated with microcephaly
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33/174 (ASPM) is the human orthology of the abnormal Drosophila axis (ASP). It is involved in the regulation of neurogenesis, and its mutation causes autosomal recessive primary microcephaly. ASPM is located at the axis poles during mitosis. Overexpression of ASPM has been suggested as a marker and potential therapeutic target in glioblastoma. SiRNA-mediated settlement prevents tumor cell proliferation and neural stem cell proliferation. Overexpression of ASPM can also predict improved invasive / metastatic potential, early tumor recurrence and poor prognosis in hepatocellular carcinoma. ASPM has been highly regulated in immortalized cells and cancer tissues of the human non-small cell lung (Jung, Choi, and Kim 703-13).
[00102] Matrix metalloproteases 3 (MMP3)
[00103] MMP3, also called progelatinase or stromelesin 1, is an endopeptidase that splits extracellular matrix (ECM) components such as fibronectin, laminin, elastin, the proteoglycan center protein and non-helical collagen regions. MMPs are important in several physiological processes requiring reorganization of ECM, such as cell migration during embryogenesis, tissue remodeling, vascularization, involution of the lactating breast and wound healing. MMP3 also has a role in platelet aggregation. Pathological conditions involving improved MMP3 expression and secretion include autoimmune inflammatory conditions and cancer.
[00104] MMP3 is overexpressed in some tumors, and has a role in the epithelial-mesenchymal transition (EMT). It may also contribute to the early stages of cancerogenesis, triggering epigenetic changes that result in the generation of a malignant phenotype (Lochter and others 180-93). Polymorphisms in the MMP3 promoter, which are associated with levels of expression, have been proven in
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34/174 influence risk and prognosis for some cancers such as esophageal adenocarcinoma (Bradbury and others 793-98) and oral squamous cell carcinoma (Vairaktaris and others 4095-100) (Liu and others 430-35). H. pilori positive gastric cancer patients with improved MMP3 and MMP7 serum levels showed greater lymph node invasion and shorter survival. In a group of 74 patients with gastric cancer, MMP3 was expressed in 27% of cases, (Murray et al. 791-97).
c-Met
[00105] C-Met mediates the potentially oncogenic activities of hepatocystic growth factor (HGF) / diffusion factor, including promoting cell growth, motility, survival, extracellular matrix dissolution, and angiogenesis. HGF binding activates the decreasing flow signaling events including Ras, phosphatidylinositol, 3'-kinase phospholipase and mitogen-activated protein kinase pathways (Dong et al. 5911-18; Furge et al. 10722-27; Furge, Zhang, and Vande Woude 5582-89; Montesano and others 355-65; Naldini and others 501-04; Ponzetto and others 4600-08). cMet is expressed predominantly in epithelial cells. Oncogenic activation of c-Met (also in non-epithelial malignant tissues) may result from amplification / overexpression, active mutations, acquisition of HGF / c-Met autocrine loops or constitutive phosphorylation (Di Renzo et al. 147-54; Ferracini and others 739-49; Fischer and others 733-39; Koochekpour and others 5391-98; Li and others 8125-35; Maulik and others 41-59; Qian and others 589-96; Ramirez and others 635-44; Tuck and others 225-32) (Nakaigawa and others 3,699-705). Constitutive activation of c-Met in transgenic mice overexpressing HGF promotes broad tumorigenesis (Takayama et al. 701-06; Wang et al. 1,023-34). Silent MET results in inhibiting tumor growth and metastasis (Corso et al. 684-93).
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MET amplification was associated with progression of human gastric cancer (Lin et al. 5680-89). (Yokozaki, Yasui, and Tahara 49-95). Ubiquitin carboxyl-terminal hydrolase L5 (UCHL5)
[00106] UCHL5, also known as ubiquitin carboxyl-terminal hydrolase (UCH37) or INO80R, is a proteasome-associated deubiquitinase. It dismantles chains of poly-ubiquitins attached to proteins at the distal end by splitting the isopeptide bond between the C-terminal Cys76 and Lys48 (Nishio and others 855-60). At its core, UCHL5 is associated with the Ino80 chromatin remodeling complex. When you turn on a proteasome, it becomes activated and can contribute to the regulation of transcription or DNA repair that was suggested to be mediated by Ino80 and proteasome.
[00107] Ubiquitin-specific proteases such as UCHL5 are involved in various processes such as cell cycle progression control, DNA differentiation, replication and repair, transcription, protein quality control, immune response and apoptosis. UCHL5 may contribute to the malignant transformation. Its activity has been shown to be highly regulated in human cervical carcinoma tissue compared to adjacent normal tissue. It can deubiquitinalize and thus stabilize the TGF-beta receptor and its decreasing mediators, the smears, thereby improving TGF-beta signaling. Enhanced TGF-beta signaling can act as a tumor promoter in advanced stages of cancer progression, although it has a dual function and can also be a tumor suppressor in early stages and before initiation (Bierie and Moses 29-40; Horton and others 138-43; Wicks and others 8080-84; Wicks and others 761-63).
Macrophage-stimulating protein receptor (MST1R)
[00108] The MST1R receptor (aka RON) is a member of the Met family of cell surface receptor tyrosine kinases and is expressed
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36/174 mainly in epithelial cells and macrophages. MST1R can induce cell migration, invasion, proliferation and survival in response to its ligands. Oncogenic properties have been proven in vitro as well as in live animal models, and it is often deregulated in human cancers (Dussault and Bellon, 2009). Clinical studies have shown that MST1R overexpression is associated with poor diagnosis and metastasis. The expression MST1R is significant in gastric carcinoma tissue and corresponding paraneoplastic tissue, but is not seen in normal gastric mucosa (Zhou et al. 236-40). Liquidation of MST1R in prostate cancer cells results in reduced endothelial cell chemotaxis in vitro, and reduced tumor growth and decreased microvessel density after orthotopic prostate transplantation in vivo. SiRNA-mediated MST1R settlement in a highly tumorigenic colon cancer cell line leads to reduced proliferation compared to control cells.
Kinesin-like protein (KIF2C)
[00109] KIF2C is a microtubule depolymerase regulating appropriate annex of kinetochoro-microtubule during axis formation. It is important for anaphase chromosome segregation and may be required to coordinate the sibling centromere separation principle. Disturbed annex of microtubule in chinetochore leads to desegregation of chromosome and aneuploid, which is observed in most solid tumors (Maney et al. 67-131; Moore and Wordeman 537-46). KIF2C is overexpressed in breast cancer cells (Shimo and others 62-70), colon cancer, colorectal cancer and gastric cancer (Nakamura and others 543-49). A gastric cancer cell line (AZ521) that stably expressed KIF2C has been shown to have increased proliferation and migration if
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37/174 compared to pseudo-transfected cells. Elevated expression of KIF2C in gastric cancer may be associated with lymphatic invasion, lymph node metastasis, and poor prognosis. Treatment of breast cancer cells with low interference RNA against KIF2C prevented their growth.
Structural maintenance of chromosome 4 proteins (SMC4) [00110] SMC proteins are chromosomal ATPases that have roles in the organization and dynamics of higher order chromosomes. SMC4 is a core component of the condensin complex that plays a role in chromatin condensation and has also been associated with nucleolar segregation, DNA repair, and maintenance of the chromatin framework. The SMC4 gene has been shown to be highly expressed in normal prostate and salivary gland, very weakly in the colon, pancreas, and intestine, and in no way in other tissues. RNA expression has been observed at high levels in many cancer cell lines and cancer specimens, including breast, prostate, colon and pancreatic cancer (Egland et al. 5,92934).
Type A Ephrin 2 (EPAH2) receiver
[00111] Efri receptors are a rare family of tyrosine receptor kinases (RTK) that have critical roles in embryonic, neuronal meta-pattern, and vascular development during normal embryogenesis. Stimulation of EphA2 by its ligand (ephrin-A1) results in self-photophosphorylation EphA2, the stimulus reverses the oncogenic transformation. Efri receptors and their ligands, ephrins, are often overexpressed in a wide variety of cancers. EphA2 is often overexpressed and functionally altered in aggressive tumor cells, and it is believed that it promotes tumor growth by improving cell adhesion to extracellular matrix, independent growth of anchorages and
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38/174 angiogenesis. Overexpression of EphA2 and EfrinaA-1 was shown in gastric carcinoma, corresponding to the depth of tumor invasion, tumor node metastasis (TNM) stages, lymph node metastasis and poor prognosis (Yuan et al. 2,410-17).
ATAD2
[00112] ATAD2 (also known as ANCCA) is a new member of the proteins of the AAA + ATPase family. It improves the transcriptional activity of the androgen receptor (AR) and estrogen receptor (ER), leading to the transcription of genes including IGF1R, IRS2, SGK1 and surviving (AR) and cyclin D1, c-myc and E2F1 (ER), respectively . It also improves the transcriptional activity of cMyc.
[00113] ATAD2 expression is high in several human tumors, such as breast cancer, prostate cancer and osteosarcoma. Expression was associated with poor prognosis.
AVL9
[00114] Surprisingly, this protein has been proven to be a source protein, but only very limited and poor data are available on the AVL9 protein and the function of the corresponding gene.
Collagen alpha-1 (XII) chain protein (Col12A1)
[00115] Alpha-1 (XII) collagen chain is a protein that is encoded in humans by the COL12A1 gene. This gene encodes the type XII alpha collagen chain, a member of the FACIT collagen family (collagen associated with fibril with interrupted triple helices). Type XII collagen is a homotrimer found in association with type I collagen, an association believed to modify the interactions between collagen fibrils I and the adjacent matrix. Alternately intertwined transcribed variants were identified encoding different isoforms.
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Collagen alpha-3 (VI) chain protein (COL6A3)
[00116] COL6A3 encodes the alpha-3 chain, one of the three type VI collagen alpha chains. The protein domains have been shown to bind extracellular matrix proteins, an interaction that explains the importance of this collagen in organizing matrix components. Remodeling of the extracellular matrix by collagen VI overexpression contributes to cisplatin resistance in ovarian cancer cells. The presence of collagen VI corresponded with the tumor level, a prognostic factor for ovarian cancer (Sherman-Baust et al. 377-86). COL6A3 is overexpressed in colorectal tumor (Smith and others 1452-64), salivary gland carcinoma (Leivo and others 104-13) and differentially expressed in gastric cancer (Yang and others 1033-40). COL6A3 has been identified as one of seven genes with tumor-specific entanglement variants. The validated changes in tumor-specific entanglement were highly consistent, enabling clear separation of normal and cancer samples and in some cases even different tumor stages (Thorsen et al. 1214-24).
Fanconi anemia, complementation group I (FANCI)
[00117] The FANCI protein locates chromatin in response to DNA damage and is involved in DNA repair (Smogorzewska and others 289-301). Mutations in the FANCI gene cause Fanconi anemia, a genetically heterogeneous recessive disorder characterized by cytogenetic instability, hypersensitivity to cross-linked DNA agents, increased chromosomal breakdown, and defective DNA repair. Alternative division of FANCI results in two variants of transcription encoding different isoforms.
Heat shock protein 90kDa beta 1 member (HSP90B1)
[00118] HSP90 (also known as glucose regulated protein 94, Grp94), member 1 is a human chaperone protein. She
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40/174 participates in processes associated with ER: translation, protein quality control and degradation associated with ER (ERAD), ER enhancing stress and linking calcium / calcium retention in ER (Christianson and others 272-82; Fu and Lee 741 -44). HSP90 contains the typical KDEL sequence for retained ER proteins, but it also appears on the surface of tumor cells (Altmeyer and others 340-49), as well as extracellularly. HSPs are known to be free of necrotic (but not apoptotic) cells and cells stressed by various stimuli such as heat shock and oxidative stress, and can occur in circulation (Basu et al. 1539-46; Tsan and Gao 27479). Extracellularly, HSP90 modulates (mainly stimulates) immune responses and is involved in antigen presentation. On the cell surface, it can serve as a receptor for pathogenic entry and / or signaling (Cabanes et al. 2827-38). In case of expression or release of a specific cell surface of the tumor, it may induce anti-tumor immunity (Zheng et al. 6,731-35). HSP90-based vaccines have been shown to be able to immunize against cancer and infectious diseases in both prophylactic and therapeutic protocols (reviewed in (Bolhassani and Rafati 1185-99; Castelli et al. 227-33; Murshid, Gong, and Calderwood 1019-30)).
[00119] However, HSP90 can also be considered as a target for tumor therapy as 1) it corresponds to tumor progression and leads to resistance towards apoptosis, also over irradiation or chemotherapy treatment, and 2) it is overexpressed in many tumors including GC, osteosarcoma (Guo et al 62-67), breast carcinoma (Hodorova et al 31-35). HSP90 overexpression is associated with aggressive behavior and poor prognosis in GC (Wang, Wang, and Ying 35-41; Zheng and others 1,042-49). Subregulation of HSP90 in GC leads to apoptosis of cancer cells (Sheu, Liu, and Lan e1096).
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Muc 6
[00120] MUC6 is expressed in mucous cells. Its primary function is known to be the protection of vulnerable epithelial surfaces from the harmful effects of constant exposure to a wide range of endogenous caustic or proteolytic agents (Toribara et al., 1997). MUC6 may also have a role in epithelial organogenesis (Reid and Harris, 1999). The expression MUC6 is found in normal gastric mucosa. It is overexpressed in some cancers as well as in adenoma and intestinal carcinoma, lung carcinoma (Hamamoto and others 891-96), colorectal polyps (Bartman and others 210-18), and breast carcinoma (Pereira and others 210-13), whereas it is not expressed in the respective normal tissues. The high rate of MUC6 expression in mucinous carcinoma suggests has been suggested to act as a barrier against cancerous extension resulting in its less aggressive biological behavior (Matsukita et al. 2636). MUC6 expression was lower in gastric carcinomas than in adenomas or normal mucosa and, conversely, corresponded to tumor size, depth of invasion, lymphatic and venous invasion, lymph node metastasis and UICC classification. MUC6 sub-regulation can contribute to the malignant transformation of gastric cells from epithelial cells and support the molecular bases of gastric carcinoma growth, invasion, metastasis and differentiation (Zheng et al. 817-23). There is also evidence that Helicobacter pilori infection, one of the major causes of gastric carcinoma, is associated with reduced expression of MUC6 (Kang et al. 29-35; Wang and Fang 425-31).
Quinetochlor protein Nuf2
[00121] The NUF2 gene (CDCA-1) encodes a protein that is highly similar to yeast Nuf2, a component of a conserved protein complex associated with the centromere. THE
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42/174 yeast Nuf2 disappears from the centromere during meiotic prophase when centromeres lose their connection to the body of the axis pole, and have a regulatory role in chromosome segregation. Survivin and hNuf2 csiRNAs have been proven to temporarily kill their mRNAs causing multinucleation and cell death by mitotic arrest, respectively (Nguyen and others 394-403). Nuf2 and Hec1 are required for the organization of sites connecting positive ends of stable microtubules to the outer plate that are necessary for the sustainable pole-directed forces required for bio-orientation in quinetocoros (DeLuca and others 519-31).
[00122] Nuf2 protein has been proven to be overexpressed in NSCLC, associated with poor prognosis (Hayama and others 1033948), and in cervical cancer (Martin and others 333-59). In surgically resected gastric cancer tissues (diffuse type, 6; intestinal type, 4), 2 variants of NUF2 were highly regulated. The alternative split variants detected in this study have been suggested to be potentially useful as diagnostic markers of novel and / or new targets for anticancer therapy (Ohem one and others 57-68).
[00123] NUF2-mediated SiRNA clearance has been shown to prevent cell proliferation and induction of apoptosis in NSCLC, ovarian cancer, cervical cancer, gastric cancer, colorectal cancer and glioma (Kaneko et al. 1,235-40).
Lipid phosphate phosphate hydrolase 2 (PPAP2C)
[00124] Phosphatidic acid phosphatases (PAPs) convert phosphatidic acid into diacylglycerol, and it works on de novo glycerolipid synthesis as well as phospholipase D-mediated receptor-activated signal transduction. Three alternately intertwined variants encoding different isoforms have been disclosed. PPAP2C is highly regulated in mesenchymal stem cells
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43/174 primary human adults (MSCs), and numerous human cancers. It may be required for increased cell proliferation. Overexpression of PPAP2C, but not a catalytically inactive mutant, caused premature S-phase entry, accompanied by premature accumulation of cyclin A. Liquidation reduces proliferation by delaying S-phase entries (Flanagan et al. 249-60).
40S ribosomal protein S11 is a protein (RPS11)
[00125] Ribosomes consist of a small 40S subunit and a large 60S subunit. Together, these subunits are composed of four RNA species and approximately 80 structurally distinct proteins. The RPS11 gene encodes a ribosomal protein that is a component of the 40S subunit. RPS11 was among six genes found in an examination for RNA-based fecal markers for the diagnosis of colorectal cancer. It was found specifically in fecal colonocytes derived from cancer patients (Yajima et al. 1,029-37).
Seven ubiquitin E3O protein ligases in homologous absentia 2 (SIAH2)
[00126] SIAH2 is an ubiquitin E3 ligase. Among its substrates are beta-catenin, TRAF2, and DCC (deleted in colorectal cancer) (Habelhah et al. 5756-65; Hu and Fearon 724-32; Nakayama, Qi, and Ronai 443-51). SIAH2 also leads to a degradation of the nuclear protein repp86, resulting in the abolition of mitotic arrest induced by overexpression of this protein (Szczepanowski et al. 485-90). SIAH2 has a tumor as well as properties that promote metastasis via at least two pathways, reviewed in (Nakayama, Qi, and Ronai 443-51): First, it leads to ubiquitination and protein degradation in the hypoxia response pathway, which leads to activity improved transcription of hypoxia-inducible factors (HIFs) (Nakayama, Qi,
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44/174 and Ronai 443-51) (Calzado et al 85-91). Second, it suppresses Sprouty2, a specific Ras / ERK signaling inhibitor. SIAH2 activity corresponds to the development of pancreatic tumor, probably due to its positive effect on signaling Ras (Nakayama, Qi, and Ronai 443-51).
[00127] Although the role of SIAH2 in cancer is partially controversial, some reports showing the association of low SIAH2 levels with poorer prognosis or response to therapy (Confalonieri et al. 2959-68) (Jansen et al. 263-71), others show a tumorigenic function (Frasor et al 13,153-57). Inhibition of SIAH2 was considered an anticancer treatment, as it managed to prevent the growth of xenotransplantation in mouse models with melanoma (Qi et al. 16713-18; Shah et al. 799-808), and in human lung cancer cell lines in mice naked (Ahmed et al. 1,606-29).
Sodium and chloride dependent taurine transporter (SLC6A6) [00128] SLC6A6 is a sodium and chloride dependent taurine transporter (TauT) (Han et al., 2006). Mice from the taurine transporter (taut - / -) liquidation suffer from chronic liver disease due to taurine deficiency, which can involve mitochondrial dysfunction (Warskulat et al., 2006). The expression of SLC6A6 is suppressed by the tumor suppressor gene p53 and is transactivated by proto-oncogenes such as WT1, c-Jun, and c-Myb. Overexpression of SLC6A6 protects renal cells from cisplatin-induced nephrotoxicity (Han et al., 2006; Han and Chesney, 2009). The expression of SLC6A6 mRNA was highly regulated by tumor necrosis factor alpha (TNF-alpha) in Caco-2 epithelial cells of the human intestine (Mochizuki et al., 2005).
Ubiquinol-cytochrome c reductase binding protein (UQCRB)
[00129] The protein encoded by the UQCRB gene is part of the
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45/174 ubiquinol-cytochrome oxidoreductase complex c. It binds ubiquinone and participates in electron transfer. Mutations in this gene are associated with complex mitochondrial deficiency III. A pseudogene has been described on the X chromosome.
[00130] The UQCRB gene can be a potential oncogen or a tumor suppressor gene in ductal pancreatic adenocarcinoma (Harada et al. 13-24). It was found to be overexpressed in hepatocellular carcinoma (Jia et al 1133-39)
Human epidermal growth factor 3 (ERBB3) receptor [00131] ERBB3 encodes a member of the epidermal growth factor (EGFR) receptor family of receptor tyrosine kinases. It is activated by neurogulins, by another ERBB and non-ERBB receptors as well as by other kinases, and by new mechanisms. Downstream, it interacts prominently with the 3 kinase / AKT phosphoinositol survival / mitogenic pathway, but also with GRB, SHC, SRC, ABL, rasGAP, SYK and the EBP1 transcription regulator (Sithanandam and Anderson 413-48). ERBB3 overexpression has been found in many cancers, including gastric cancer, where it can play a key causative role and negatively influence the prognosis (Kobayashi and others 1294-301) (Slesak and others 2,727-32). (Zhang and others 2112-18) found that ERBB3 overexpression was more frequent in the diffuse type (26.2%) of gastric cancer than in the intestinal type (5.0%). In both types, overexpression was associated with poor prognosis. Attempts to attack ERBB3 in cancer therapy include RNA aptamers to the extracellular domain (Chen et al. 9226-31), blocking their gene expression by synthetic transcription factors (Lund and others 9082-91), inhibitors of small- molecule such as the vitamin E isomer γ-tocotrienol (Samant and Sylvester 563-74), miRNA (Scott and others 1479-86) and siRNA (Sithanandam and others 1847-59).
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Prominine 1 (Proml)
[00132] Function: Prominin-1, also called CD133, has been identified as a specific molecule for CD34 + hematopoietic progenitor cells (Yin et al., 1997) and has proven to be a marker for normal stem cells and cancer stem cells (CSCs) from various fabrics. It is located mainly on plasma membrane protrusions, and may be involved in organizing membrane topology or maintaining the lipid composition of the plasma membrane. It has been suggested that a prominin-1 interlacing isoform called AC133-2 and the lack of a small exon of 27 amino acids may represent an even better stem cell marker (Mizrak et al., 2008; Bidlingmaier et al., 2008).
[00133] Only a small percentage of tumor cells are generally positive for prominin-1, as expected for a CSC marker. Depending on the type of tumor, the number of positive cells per tumor mass ranges from 1 to 15% and is mainly around 2%.
[00134] Prominina-1 has been associated with tumor formation, angiogenesis and chemoresistance (Zhu and others, 2009a) (Bruno and others, 2006; Hilbe and others, 2004) (Bertolini and others, 2009). However, prominin-1 positive cells may be accessible by the immune system, as they can be killed by NK cells (Castriconi et al., 2007; Pietra et al., 2009) and cytotoxic T cells (Tostam et al., 2009).
[00135] While for many cancer entities it has been shown that prominin-1 positive cells are functionally CSCs, and expression has often been associated with poor prognosis, there are still controversies. Some reports state that this is neither necessary nor sufficient to identify CSCs (Cheng and others,
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2009; Wu and Wu, 2009). Perhaps a combination of prominin-1 with other molecules such as CD44, or even multiple combinations such as prom1 (+), CD34 (+), CD44 (+), CD38 (-), CD24 (-) serve as the best markers for CSC (Zhu et al., 2009b; Fulda and Pervaiz, 2010)
[00136] In diffuse GC, PROM1 expression was suggested based on an in silico analysis (Katoh and Katoh, 2007) and overexpression in GC compared to normal stomach tissue at the protein level was detected by (Smith et al., 2008) . However, (Boegl and Prinz, 2009) reported that prominin-1 expression was reduced in GC, especially in more advanced stages, and claimed that prominin-1 expression previously corresponds with angiogenesis - which is also reduced in more advanced stages - more than with tumor growth. A study using GC cell lines (Takaishi et al., 2009) states that CD44, but not prominin-1, is a marker of CSC in GC.
Matrix metalloproteinase 11 (MMP11)
[00137] Like other MMPs, MMP11 is an endopeptidase with functions in processes requiring tissue remodeling, such as development, wound healing and scar formation. It may also negatively regulate fat homeostasis by reducing adipocyte differentiation. In contrast to other MMPs, it is not capable of splitting typical extracellular matrix molecules except collagen VI. However, other substrates have been identified such as alpha 2 macroglobulin, certain serine protease inhibitors (serpins) including alpha 1 antitrypsin, insulin-like growth factor-binding protein-1 and the laminin receptor. In cancer, MMP11 is mainly expressed in stromal cells surrounding the tumor tissue. This has been demonstrated for many tumor entities. It has been stated that MMP11 is over-expressed in
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48/174 stroma of most invasive human carcinomas, but rarely in sarcomas and other non-epithelial tumors. In most, but not all, MMP11 is expressed in stromal cells directly next to the tumor, whereas the tumor cells themselves, normal tissues and stromal cells distant from the tumor are negative. Higher levels of MMP11 correspond to a higher malignant phenotype / invasiveness and poor prognosis. However, in papillary thyroid carcinomas, MMP11 expression was inversely related to aggressive characteristics. MMP11 was found in tumor tissue as well as in the serum of gastric cancer patients, and the expression corresponded to metastasis (Yang et al.). Furthermore, (Deng and others 274-81) showed that MMP11 is highly expressed in tumor cell lines and primary gastric cancer tumor - in contrast to other types of cancer not exclusively in the stroma - and that it appears to improve the proliferation of tumor cell.
Nuclear transcription Y factor beta (NFYB) subunit
[00138] NFYB, also called CBF-B or CBF-UM is, in addition to NFYA and NFYC, a part of the basal heterotrimeric transcription factor NF-Y (also binding factor CCAAT or CBF) that binds to the CCAAT motifs - or the inverse motives, ATTGG, called Y box - in the promoters and enhancers of several genes. Among the NFY target genes are the MHC class II genes, the PDGF beta-receptor, various heat shock proteins, the hMLH1 repair gene without combination, and the alpha II topoisomerase.
[00139] NFYB is not a classic oncogene, however, its function may contribute to tumorigenesis. First, many cell-cycle genes such as cyclin A, cyclin B1, Aurora A and cdk1 are targets for NF-Y. The cells are trapped in G2 / phase M without functional NFYB. (Park et al) showed that over-regulation of cyclin B2 and other genes
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49/174 related to cell-cycle in colorectal adenocarcinoma are the result of NF-Y activity. Second, NF-Y activity neutralizes apoptosis. Cells lacking NF-Y undergo apoptosis due to p53 activation and reduced transcription of anti-apoptotic genes containing CCAAT boxes in their promoters, such as Bcl-2 (Benatti et al. 1415-28). Third, its tumorigenic properties are improved in combination with other transcription factors. For example, mutated p53 binds NF-Y and p300 proteins, increasing the expression of NF-Y-induced cell cycle genes.
ABL1
[00140] The protein tyrosine kinase c-Abl is transported between the nuclear and cytoplasmic compartments. Nuclear C-Abl is involved in cell growth inhibition and apoptosis, while cytoplasmic c-Abl may play a role in actin dynamics, morphogenesis and signaling induced by extracellular stimuli such as growth factors and integrin ligands. c-Abl cytoplasmic has been proven to promote mitogenesis.
[00141] The activity of c-Abl protein is downregulated by its SH3 domain, and deletion of the SH3 domain turns ABL1 into an oncogene. In chronic myeloid leukemia (CML), the gene is activated by translocation within the BCR (collective breakpoint region) gene on chromosome 22. This resulting fusion BCR-ABL protein locates the cytosol and allows cells to proliferate without being regulated by cytokines (Zhao and others). The activity of c-Abl is also highly regulated without solid tumors, as has been shown for breast carcinomas and NSCLC. Overexpression is not enough and constitutive kinase activity requires protein phosphorylation. In breast cancer cells, phosphorylation of c-Abl is induced by plasma membrane tyrosine kinase, including SFK, members of the EGFR family and the IGF-1 receptor. ABL fusion proteins were not
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50/174 detected in solid tumors (Lin and Arlinghaus, 2008). GLA has been shown to be expressed in gastric carcinoma and associated microvessels, suggesting a possible role in angiogenesis. Notably, the H. pilori cytotoxin-associated gene A (CagA) leads to activation of c-Abl, which consequently phosphorylates EGFR and thus blocks EGFR endocytosis (Bauer, Bartfeld, and Meyer 156-69). Several tyrosine kinase inhibitors are more or less specific for Abl. Imatinib (Gleevec) is used as a first line therapy for CML and has also been approved for patients with advanced gastrointestinal stromal tumors (GIST), as it also addresses KIT (Pytel and others 66-76) (Croom and Perry, 2003). Other inhibitors used for cancer therapy are Dasatinib and Nilotinib (Pytel et al 66-76) (Deremer, Ustun, and Natarajan 1956-75).
Kinase similar to polo 4 (Plk4)
[00142] The members of the pole kinase family (Plk1-4) are important during cell division, regulating several steps during mitosis. Plk4 is the organizer of the centriole and duplication training (Rodrigues-Martins et al. 1046-50). While Plk1 is a clear oncogene, Plk4's function in cancer is ambiguous. Subregulation as well as overexpression of Plk4 has been associated with cancer in humans, mice and flies (Cunha-Ferreira and others 43-49). For example, in colorectal cancer, Plk4 was said to be overexpressed, but a small group of patients showed strong Plk4 subregulation (Macmillan and others 729-40). This can be explained by the fact that both overexpression and Plk4 deficiency lead to aberrant formation of centriole, resulting in abnormal numbers of centrosome and structures that are often detected in tumor cells and contribute to mitotic aberrations that cause erroneous chromosome segregation and aneuploidy (Peel et al 834-43). (Kuriyama et al. 2014-23). (Korzeniewski and others 6668-75).
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The IQ motif contains protein 3 activating GTPase (IQGAP3)
[00143] IQGAPs participate in signaling cellular pathways as well as cytoskeletal architecture and cell adhesion. They have a sequence similar domain to RasGAPs and correspondingly binds to small GTPases. However (and despite their name), none of them have the ability to activate GTPase. For IQGAP1 and IQGAP2, they have been shown to stabilize the GTP junction state of Rac1 and Cdc42, and IQGAP3 has been suggested to stabilize activated Ras (Nojima et al 971-78; White, Brown, and Sacks 1817-24). Via their IQ domain they bind to calcium / calmodulin, and via the calponine homology domain to actin filaments (White, Brown, and Sacks 1817-24). (Wang and others 567-77) reports that IQGAP3 is expressed in the brain, where it is associated with actin filaments such as Rac1 and Cdc42. It accumulates in the distal region of axons and promotes the growth of Rac1 / Ccd42 dependent axons. IQGAPs have been implicated in cancer. IQGAP1 is considered an oncogene. It improves several cancer-related pathways, such as MAP kinase, beta-catenin and VEGF-mediated signaling and is overexpressed in many tumors. IQGAP2 appears to work best as a tumor suppressor and has been found to be reduced in gastric cancers with poor prognosis (White, Brown, and Sacks 1817-24). Little information is available on IQGAP3. (Skawran and others 505-16) classified it among the genes significantly high-regulated in hepatocellular carcinoma. Two studies report that IQGAP3 is expressed specifically in proliferating (Ki67 +) cells in the intestines, colon and liver of small mice (Nojima and others 971-78) (Kunimoto and others 621-31).
Coiled bulb domain containing 88a (CCDC88A)
[00144] CCDC88A is an Akt substrate that binds actin has a
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52/174 role in the actin organization and Akt-dependent cell motility in fibroblasts. The CCDC88A / Akt pathway is also essential in VEGF-mediated post-neonatal angiogenesis.
[00145] CCDC88A is also highly expressed in a variety of human malignant tissues, including the breast, colon, lung, and cervical carcinomas of the uterus. It plays an important role in tumor progression with aberrant activation of the Akt signaling pathway.
Cyclin B1 (CCNB1)
[00146] CCNB1 is induced during the G2 / M phase of mitosis and forms the mitosis promoting factor (MPF) together with cyclin-dependent kinase 1 (Cdk1) / Cdc2. Overexpression is found in a variety of cancers and is often associated with poor prognosis, for example, in breast cancer (Aaltonen et al., 2009; Agarwal et al., 2009; Suzuki et al., 2007), medulloblastoma (de et al.,
2008), NSCLC (Cooper et al., 2009), cervical cancer (Zhao et al., 2006), and others. It was one of the genes included in a signature of 11 genes that were considered to predict a short interval to disease recurrence in patients with 12 different types of cancers (Glinsky, 2006). No specific information on gastric cancer was found.
Cyclin D2 (CCND2)
[00147] CCND2 binds and activates, like other type D cyclins (D1 and D3), cyclin dependent kinase 4 (Cdk4) or Cdk6. This is required for G1 / S transition. CCND2 has been found to be overexpressed in many tumors, including testicular and ovarian tumors (Sicinski et al., 1996), hematological malignancies (Hoglund et al., 1996; Gesk et al., 2006), and gastric cancer, where it can be caused by H infection pilori, and associated with poor prognosis (Yu et al., 2003). (Yu and others, 2001) (Oshimo and others, 2003) (Takano and others,
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1999) (Takano et al., 2000).
Cyclin E2 (CCNE2)
[00148] CCNE2 binds and activates, like the other type E cyclin CCNE1, Cdk2. This activity reaches its maximum point in the G1 / S phase transition. Under healthy conditions, CCNE2 is not detectable in quiescent cells and can only be found in actively divided tissues (Payton and Coats, 2002). It is often expressed aberrantly in cancer, for example, in breast cancer, correlated with poor prognosis (Desmedt et al., 2006; Ghayad et al., 2009; Payton et al., 2002; Sieuwerts et al., 2006), and in metastatic prostate (Wu et al., 2009).
Carcinoembryonic cell adhesion molecules related to antigen 1.5 and 6 (CEACAM 1.5, and 6)
[00149] CEACAMs are membrane-anchored glycoproteins that mediate cell-cell interactions and activate integrin signaling pathways (Chan and Stanners, 2007). They can also serve as receptors for pathogens such as E. coli (Berger and others, 2004) (Hauck and others, 2006) and are involved in immune regulation (Shao and others, 2006).
[00150] CEACAM5 and CEACAM6 have procancerogenic functions. They prevent anonymity (Ordonez et al., 2000), promote metastasis (Marshall, 2003; Ordonez et al., 2000), and upset cell polarization and tissue architecture (Chan and Stanners, 2007). CEACAM1's role in cancer is ambiguous. It can be a tumor suppressor in the early stages, and contributes to the formation of metastasis, tumor immune escape and angiogenesis in more advanced stages (Hokari et al. 2007; Liu et al., 2007; Moh and Shen, 2009). Its functional role depends on the isoform, as CEACAM1 occurs in 11 interlacing variants, whose relationship determines the result it signals (Gray-Owen and Blumberg, 2006;
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Leung et al., 2006; Neumaier et al., 1993; Nittka et al., 2008). The relationship of the entanglement variants can be altered in cancer (Gaur et al., 2008).
[00151] CEACAM5 or CEACAM6 or both are overexpressed in up to 70% of all human tumors, often associated with poor prognosis (Chan and Stanners, 2007; Chevinsky, 1991). CEACAM5 serum is an accepted clinical marker for colon and rectal carcinoma, with high levels indicating poor prognosis or recurrence (Chevinsky, 1991; Goldstein and Mitchell, 2005). It has also been suggested as a marker for other entities including gastric cancer, however with limited prognostic power (Victorzon et al., 1995). CEACAM1 can be sub-regulated or over-regulated in cancer, depending on the entity (Kinugasa and others, 1998) (Dango and others, 2008) (Simeone and others, 2007). (Han et al., 2008) found abundant levels of CEACAM5 and CEACAM6 in nine gastric cancer cell lines, while CEACAM1 was undetectable. In contrast, an analysis of primary tumor samples from 222 patients showed stains or cytoplasmic or membranous ones for CEACAM1. The form of membrane link was related to improved angiogenesis (Zhou et al., 2009). The study by (Kinugasa et al., 1998) also showed an overregulation in gastric adenocarcinomas.
[00152] In some tumors, CEACAM1 is subregulated in tumor cells, which leads to VEGF overregulation, and VEGF or hypoxic conditions can induce CEACAM1 in the adjacent endothelium. Consequently, a monoclonal antibody against CEACAM1 blocked VEGF-induced endothelial tube formation (OliveiraFerrer et al., 2004; Tilki et al., 2006; Ergun et al., 2000).
[00153] Especially CEACAM5 was tested as a target for anticancer drugs, among others by vaccination approaches. These studies have shown that CEACAM5 can be a target of reactions
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55/174 cellular immune systems (Cloosen et al., 2007; Marshall, 2003). An overview of CEACAM5 T cell epitopes is provided in (Sarobe et al., 2004).
Chloride channel 3 (CLCN3)
[00154] CLCN3 is a Cl channel that can be barred in its volume and contributes to the regulatory volume decrease (RVD) that occurs as a reaction to an increase in cell volume in the case of conditions such as cell circulation or hypoosmosis (Lemonnier and others 2004; Sardini et al., 2003). However, this point is controversially discussed (Wang et al., 2004) and the channel that reduces the volume activated during apoptosis is different from CLCN3 (Okada et al., 2006).
[00155] The expression CLCN3 changes during the cell cycle, reaching its peak in the S phase (Wang et al., 2004). Currents of CLCN3 may be important in cancer-relevant processes in entities where CLCN3 is highly regulated, such as glioma: Tumor cells must be treated proliferatively in order to find hypoosomatic conditions, for example, in peritumoral edema (Ernest and others, 2005; Olsen and others, 2003; Sontheimer, 2008).
[00156] Furthermore, it has been reported that CLCN3 improves etoposide resistance by increasing acidification of the advanced stage endocytic compartment (Weylandt et al., 2007).
[00157] SiRNA-mediated CLCN3 clearance reduced the migration of nasopharyngeal carcinoma cells in vitro (Mao et al., 2008).
DNAJC10
[00158] DNAJC10 is a member of a supramolecular degradation complex associated with ER (ERAD) that recognizes and unveils incorrectly folded proteins for efficient retrotranslocation (Ushioda et al., 2008). The protein has been shown to have
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56/174 hepatocellular carcinoma (Cunnea et al., 2007). Liquidation of DNAJC10 by siRNA in neuroectodermal tumor cells increased the apoptotic response to the chemotherapeutic drug fenretinide (Corazzari et al., 2007). ERdj5 has been shown to decrease neuroblastoma cell survival by subregulation of the unveiled protein response (UPR) (Thomas and Spyrou, 2009).
The eukaryotic translation initiation factor 2, subunit 3 gamma (EIF2S3)
[00159] EIF2S3 is the largest subunit of a protein complex (EIF2) recruiting the initial methionyl-tRNA to the 40S ribosomal subunit (Clemens, 1997). The action of kinases that sub-regulates EIF activities, such as RNA-dependent protein kinase (PKR), can be proapoptotic and tumor suppressive (Mounir et al., 2009). In gastric cancer, higher levels of phosphorylated and dephosphorylated EIF2 were found, and a redistribution to the nucleus was observed. This deregulation points in the direction of an implication of eIF2alpha in gastrointestinal cancer (Lobo et al., 2000).
The eukaryotic translation initiation factor 3, subunit L (EIF3L) [00160] EIF3L is one of 10-13 subunits of EIF3, which is associated with the small ribosomal subunit. EIF3 has a role in preventing premature binding of the large ribosomal subunit. EIF3L is among the five subunits that were found to be non-essential for the formation of EIF3 (Masutani et al., 2007). A screen with an antisense library suggested that EIF3L subregulation improves the fluorouracil 5 antitumor activity in hepatocellular carcinoma cells (Doh, 2008). Epiplakin1 (EPPK1)
[00161] EPPK1 is a gene from the Plaquin family with functions largely unknown. Plaquin genes are known to function in the interconnection of cytoskeletal filaments and to
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57/174 anchor them in a junction of plasma associated with the membrane (Yoshida et al., 2008).
Protein G joined with receptor 39 (GPR39)
[00162] GPR39 is a united Gq protein receptor that has been found to be involved in metabolic and gastrointestinal function (Yamamoto et al., 2009). Its signaling activates cAMP and serum response elements (Holst et al., 2004). The endogenous ligand for GPR39 is probably zinc (Chen and Zhao, 2007). GPR39 is a new inhibitor of cell death, which may represent a therapeutic target with implications for processes involving apoptosis and endoplasmic reticulum stress such as cancer (Dittmer et al., 2008). GPR39 has been proven to be highly regulated in micromatrix both by human fetal HFK kidney and tumor similar to the blastema-enriched stem (Metsuyanim et al., 2009), and in a resistant hippocampal cell line against different cell death stimulators (Dittmer and others, 2008).
ERBB2 / HER2 / NEU
[00163] ERBB2 is a member of the EGFR receptor tyrosine kinase family. Its ligand is not known, but it is the preferred heterodimerization partner for other members of the HER family (Olayioye, 2001). In carcinomas, HER2 acts as an oncogene, mainly due to the high level amplification of the gene, which induces protein overexpression in the cell membrane and subsequent acquisition of beneficial properties for a malignant cell (Slamon et al., 1989). Overexpression is seen in a certain percentage of many cancers, including gastric cancer. In most cases, it is associated with poor prognosis (Song and others, 2010) (Yonemura and others, 1991) (Uchino and others, 1993) (Mizutani and others, 1993).
[00164] ERBB2 is the target of the monoclonal antibody trastuzumab
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58/174 (sold as Herceptin), which has been suggested as a treatment option for patients with advanced HER2-positive gastric cancer, in combination with chemotherapy (Meza-Junco et al., 2009; Van Cutsem et al., 2009). Another monoclonal antibody, Pertuzumab, which prevents dimerization of HER2 and HER3 receptors, is in the advanced stage of clinical trials (Kristjansdottir and Dizon, 2010). The selective overexpression of HER2 and HER3 in the two histological types of gastric cancers (intestinal type and diffuse type) is intensely associated with a poor prognosis (Zhang et al., 2009).
Beta-4 Integrin (ITGB4)
[00165] Integrins mediate cell adhesion as well as signal transduction from inside to outside and from outside to inside. The beta-4 subunit of integrin heterodimerizes with the alpha-subunit
6. The resulting integrin promotes the formation of hemidesmosomes between the intracellular keratin cytoskeleton and the base membrane (Giancotti, 2007). Beta-4 integrin has a dual function in cancer, as it can mediate stable adhesion on one side, and proinvasive signaling (including Ras / Erk and PI3K signaling) and angiogenesis on the other side (Giancotti, 2007; Raymond et al., 2007). It is overexpressed in many tumors as well as in angiogenic endothelial cells, often correlating with progression and metastasis. High levels were found in gastric cancer, particularly in stromal-invasive cells (Giancotti, 2007; Tani et al., 1996). However, it was low-regulated in gastric carcinoma of the non-differentiated type when the tumor invades deeper parts, perhaps due to the gradual transition from epithelial-mesenchymal, as beta-4 integrin is an epithelial integrin (Yanchenko et al., 2009).
Lipocalin (LCN2)
[00166] LCN2 or gelatinase-associated neutrophilic lipocalin (NGAL) is an iron regulatory protein that exists as a
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59/174 monomer, homodimer, or as a disulfite-linked heterodimer with MMP9 (Coles et al., 1999; Kjeldsen et al., 1993). Expression is increased in several cancers, in some cases associated with progression. Mechanically, it can stabilize MMP9 and alter cell-cell adhesion mediated by E-cadherin, thus increasing invasion. The MMP-9 and LCN2 complexes have been linked to poorer survival in gastric cancer (Kubben et al., 2007) (Hu et al., 2009). Although a clear pro-tumor effect has been observed in several tumors in humans, some studies have shown that LCN2 can prevent the pro-neoplastic factor HIF-1alpha, phosphorylation of FA kinase and also VEGF synthesis, thus suggesting, under alternative conditions, also LCN2, paradoxically, has an antitumor and antimetastatic effect in neoplasms of, for example, colon, ovary and pancreas. (Bolignano et al., 2009; Tong et al., 2008). LCN2 can be useful to prevent tumor angiogenesis, in addition to suppressing tumor metastasis, in cancers that show ras activation (Venkatesha et al., 2006).
The succinate dehydrogenase complex, subunit C (SDHC) [00167] SDHC is one of four nuclear-encoded subunits of succinate dehydrogenase (mitochondrial complex II), which transfers electrons from succinate to ubiquinone, yielding fumarate and ubiquinol. Succinate dehydrogenase deficiency can cause GISTs (McWhinney et al., 2007). Familial stromal gastrointestinal tumors can be caused by mutations in the SDHB, SDHC, and SDHD subunit genes, and abdominal paragangliomas associated with gastrointestinal tumors can be caused exceptionally by SDHC mutations (Pasini et al., 2008). Mutant SDHC protein in transgenic mice generates oxidative stress and can contribute to nuclear DNA damage, mutagenesis, and finally, tumorigenesis (Ishii et al., 2005). THE
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60/174 succinate dehydrogenase is considered a tumor suppressor (Baysal, 2003; Gottlieb and Tomlinson, 2005). Decreased levels of this enzyme complex can result in tumorigenesis (Eng et al., 2003).
PDZ binding kinase (PBK)
[00168] PBK is a MAPKK related to MEK3 / 6, which activates p38 MAP kinase, for example, below growth factor receptors (Abe et al., 2000; Ayllon and O'connor, 2007). JNK can be a secondary target (Oh et al., 2007). As PBK is expressed in the testes in adults (see below), a function in spermatogenesis has been proposed (Abe et al., 2000; Zhao et al., 2001). Apart from that, it contributes to the proliferation and resistance of apoptosis in tumor cells: It is phosphorized and activated during mitosis, which is necessary for axis formation and cytokinesis (Gaudet et al., 2000; Matsumoto et al., 2004; Park and others, 2009) (Abe and others, 2007). Other functions promoting anti-apoptotic growth and reaction include p53 subregulation and histone phosphorylation (Park et al., 2006; Zykova et al., 2006) (Nandi et al., 2007). PBK has been classified as a testicular cancer antigen (Abe et al., 2000; Park et al., 2006) and has been found to be overexpressed in many cancers.
Polymerase (directed by DNA) delta 3, accessory subunit (POLD3)
[00169] The DNA polymerase delta complex is involved in DNA replication and repair. It consists of the proliferating cellular nuclear antigen (PCNA), the multi-subunit replication factor C, and the subunit polymerase complex 4: POLD1, POLD2, POLD3, and POLD4 (Liu and Warbrick, 2006). POLD3 plays a crucial role in the efficient recycling of PCNA during pol dissociation-association cycles. delta during the stretching phase
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61/174 of DNA replication (Masuda et al., 2007).
Proteasome (prosoma, macropnea) 26S subunit, not ATPase, 14 (PSMD14)
[00170] PSMD14 is a component of the 26S proteasome. It belongs to the 19S complex (cover 19S; PA700), which is responsible for substrate ubiquitination during proteasomal degradation (Spataro et al., 1997). Overexpression of PSMD14 in mammalian cells affects cell proliferation and the response to cytotoxic drugs such as vinblastine, cisplatin and doxorubicin (Spataro et al., 2002). Suppression of PSMD14 siRNA in HeLa cells resulted in a reduction in cell viability and an increase in polyubiquitinated protein levels (Gallery et al., 2007). The low regulation of PSMD14 by siRNA had a considerable impact on cell viability, thus causing a cell arrest in the G0-G1 phase, ultimately leading to senescence (Byrne et al., 2010).
Proteasome (prosoma, macropnea) 26S subunit, ATPase, 2 (PSMC2)
[00171] PSMC2 is part of the 26S proteasome system. She is a member of the triple-A family of ATPases, which have an activity similar to chaperone. This subunit has shown to be able to interact with several of the basal transcription factors, that is, in addition to participating in proteasome functions, this subunit can participate in the regulation of transcription. It has been shown that the 26S proteasome system in the skeletal muscle can be activated by TNF-alpha (Tan et al., 2006). In transgenic HBx mice, which support the Hepatitis B regulator HBx gene in their germ line, and develop HCC, PSMC2 and other proteasome subunits are highly regulated in tumor tissues (Cui et al., 2006). The levels of mRNA for the ATPase PSMC2 subunit of the 19S complex increased in cancer cachexia (Combaret et al., 1999).
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Protein tyrosine kinase 2 (PTK2)
[00172] PTK2 is a non-receptor tyrosine kinase, which modulates integrin signaling and can promote tumor growth, progression and metastasis ((Giaginis et al., 2009); (Hauck et al., 2002); (Zhao and Guan, 2009)). PTK2 has been suggested to be a marker for carcinogenesis and cancer progression (Su et al., 2002; Theocharis et al., 2009; Jan et al., And / or 2009). Overexpression and / or increased activity occurs in a wide variety of human cancers including gastric cancer. PTK2 also transduces signals down the gastrin receptor, which contributes to the proliferation of gastric cancer cells (Li et al., 2008b). 8% of gastric carcinomas have been shown to contain the Epstein-Barr virus (EBV). Underlines of EBV-infected human gastric cancer cells showed increased PTK2 phosphorylation (Kassis et al., 2002). The level of PTK2 tyrosine phosphorylation in gastric epithelial cells is reduced by the Helicobacter pilori product from cagApositivo.
Tetraspanin 1 (TSPAN1) and tetraspanin 8 (TSPAN8)
[00173] TSPAN1 and TSPAN8 belong to the family of tetraspanins that are characterized by four transmembrane domains an intracellular terminus N and C and that have roles in a variety of processes including cell adhesion, motility, activation and tumor invasion. They often form large molecular complexes with other proteins such as integrins on the cell surface (Tarrant et al., 2003; Serru et al., 2000). The functions of TSPAN1 are still unknown and may include a role in secretion (Scholz et al., 2009). TSPAN1 is overexpressed in several cancers, often corresponding to stage, progression and worse clinical outcome. Notably, it has been confirmed to be overexpressed in 56.98% of 86 cases of gastric carcinoma, and the
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63/174 overexpression was definitely correlated with clinical stage, infiltration and lymph node state and negatively with survival rates and tumor differentiation level (Chen et al., 2008). TSPAN8 has been confirmed to be a gene associated with metastasis in many types of tumors (PMID: 16467180). In gastrointestinal cancer, TSPAN8 expression is associated with poor prognosis (PMID: 16849554).
Zinc Finger Protein 598 (ZNF598)
[00174] ZNF598 is a finger protein of zinc with function still unknown.
[00175] A disintegrin and a metalloproteinase 10 (ADAM10) [00176] ADAM10 has a role in angiogenesis, development and tumorigenesis. It is overexpressed in gastric carcinoma. Selective ADAM inhibitors against ADAM-10 are being tested clinically for the treatment of cancer. (PMID: 19408347) Matrix metalloproteinase 12 (MMP12)
[00177] MMP12 is a zinc endopeptidase, which degrades elastin and many other matrix- and non-matrix proteins and is involved in macrophage migration and inhibition of angiogenesis (Chakraborti et al., 2003; Chandler et al., 1996; Sang, 1998). It also plays a role in the pathological processes of tissue destruction such as asthma, emphysema and chronic obstructive pulmonary disease (COPD), rheumatoid arthritis and tumor growth (Cataldo et al., 2003; Wallace et al., 2008). MMP12 inhibitors are discussed as agents for treating these conditions (Churg et al., 2007; Norman, 2009). MMP12 is often overexpressed in cancer, where it can have ambiguous functions. While it may be involved in matrix dissolution and thus metastasis, it can also prevent tumor growth by producing angiostatin, which negatively influences angiogenesis. Improved expression of MMP12 was
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64/174 found for GC, and proved to be favorable: It corresponded negatively to the microvessel density, VEGF, level of tumor differentiation, vascular invasion, metastasis and lymph node recurrence. Patients with overexpression of MMP12 demonstrated a significantly better survival rate (Cheng et al., 2010; Zhang et al., 2007b; Zhang et al., 2007a) Ribonucleotide reductase M2 (RRM2)
[00178] RRM2 is one of two ribonucleotide reductase subunits, which generates ribonucleotide deoxyribonucleotides. RRM2 overexpression has been observed in tumors including gastric cancer and improves metastatic potential (PMID: 18941749) (PMID: 19250552) RRM2 settlement siRNA decreased tumor growth in several species (mouse, rat, monkey) (PMID: 17929316; PMID: 17404105).
Transmembrane protease, serine 4 (TMPRSS4)
[00179] TMPRSS4 is a type II transmembrane serine protease found on the cell surface that is highly expressed in various cancer tissues, including pancreatic, colon and gastric cancer. The biological functions of TMPRSS4 in cancer are not yet known. TMPRSS4 has four interlacing variants (Scott et al., 2001; Sawasaki et al., 2004). The expression in ovarian carcinoma corresponded to the stage (Sawasaki et al., 2004). TMPRSS4 is highly elevated in lung cancer tissues, and settlement of TMPRSS4 siRNA by treatment of low interference RNA in lung and colon cancer cell lines has been associated with reduced cell invasion and cell matrix adhesion as well as modulation cell proliferation (Jung et al., 2008).
Deiodinase, lodothyronine, type II (DIO2)
[00180] DIO2 converts prohormone thyroxine (T4) to tri
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65/174 3.3 ', 5 bioactive iodothyronine (T3). It is highly expressed in the thyroid, and activity of and / or expression has been found to be deregulated in thyroid cancers (de Souza Meyer et al., 2005) (Arnaldi et al., 2005). However, it has also been found in other tissues, such as normal lung and lung cancer (Wawrzynska et al., 2003), and in brain tumors (Murakami et al., 2000).
Growth factor 2 insulin-like protein 3 that binds mRNA 2 (IGF2BP3)
[00181] IGF2BP3 is mainly present in the nucleoli, where it binds IGF2 mRNA and represses its translation. It has a role in embryogenesis and is sub-regulated in adult tissues. In tumor cells it can be highly regulated and is thus considered an oncofetal protein (Liao et al. 2005). In many cancers including gastric cancer it has been found to be overexpressed, associated with poor prognosis (Jeng et al. 2009) (Jiang et al. 2006). Peptides derived from IGF2BP3 have been tested in cancer vaccination studies (Kono et al. 2009).
Blade B1 (LMNB1)
[00182] Lamina B1 is a nuclear lamina matrix protein and is involved with nuclear stability, chromatin structure and gene expression. In the early stages of apoptosis, the lamina is degraded (Neamati and others 1995) (Sato and others 2008b; Sato and others 2008a; Sato and others 2009). LMNB1 is expressed to some extent essentially in all normal somatic cells, and preliminary studies indicate that it can be reduced during the pathogenesis of some cancers, including gastric cancer (Moss et al. 1999). In other cancers, such as hepatocellular carcinoma, LMNB1 was found to be highly regulated and correlated definitively with stage, size and number of tumor nodules (Lim et al. 2002).
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Local family of integration of wingless type MMTV, member 5A [00183] WNT5A is a secreted signaling protein involved in developmental processes and oncogenesis. Canonical WNT5A signaling by curly receptors and LRP5 / LRP6 leading to maintenance of stem / progenitor cells, while non-canonical WNT5A signaling by curly receptors and ROR2 / PTK / RYK control tissue polarity leading to cell maintenance, cell adhesion or movement, for example, at the tumor stromal interface, leading to invasion (Katoh and Katoh, 2007). It can be a tumor suppressor in some cancers, but it is highly regulated in others including gastric cancer, where it contributes to progression and metastasis and leads to a poor prognosis (Li and others, 2010) (Yamamoto and others, 2009) (Kurayoshi and others, 2006).
Protein activating fibroblast, alpha (FAP)
[00184] FAP is an essential membrane gelatinase. Its putative serine protease activity may play a role in controlling fibroblast growth or epithelial-mesenchymal interactions during development, tissue repair and epithelial carcinogenesis (Scanlan et al. 1994). FAP has a potential role in the growth of cancer, metastasis and angiogenesis due to cell adhesion and migration processes, as well as rapid degradation of ECM components. It is present in tumor cells invading the ECM, in reactive fibroblasts associated with cancer, and in endothelial cells involved in angiogenesis, but not in inactive cells of the same type. (Dolznig and others 2005; Kennedy and others 2009; Rettig and others 1993; Rettig and others 1994; Scanlan and others 1994; Zhang and others 2010). The expression FAP was found in gastric cancer cells and associated stromal fibroblasts (Zhi and others 2010) (Chen and others 2006) (Mori and others 2004; Okada and others 2003). In a mouse model, cells expressing FAP,
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67/174 demonstrated to be a non-redundant, immunosuppressive component of the tumor microenvironment (Kraman et al. 2010). In tumor vaccination mouse models, FAP com has been used successfully as a target for CD8 + and CD4 + T cell responses (Loeffler et al. 2006; Wen et al. 2010) (Lee et al. 2005) (Fassnacht et al. 2005).
[00185] Coatomer protein complex, gamma subunit (COPG);
[00186] Coatomer protein complex, gamma 2 subunit (COPG2);
[00187] Coatomer protein complex, beta 1 subunit (COPB1)
[00188] COPG, COPG2 and COPB1 are subunits of the coatomero complex, also called coat protein complex 1 (COPI), which is associated with vesicles coated with non-clathrins. The vesicles lined by mediate Golgi retrograde transport back to the ER and intra-Golgi transport (Watson et al., 2004). They may also be involved in anterograde transport (Nickel et al., 1998). Retrograde trafficking regulates, among others, the EGFR-dependent nuclear transport of EGF, which binds to COPG (Wang et al., 2010). COPG has been proven to be overexpressed in lung cancer cells and microvascular lung endothelial cells associated with cancer (Park et al., 2008).
[00189] The sequence of COPG2 notably expressed is 80% identical to GOPG (Blagitko et al., 1999). COPG2 can form a complex similar to COP I in place of GOPG, which is probably functionally redundant (Futatsumori et al., 2000).
[00190] Liquidation of COPB1 in a cystic fibrous transmembrane conductance regulator (CFTR) expressing cell line suggested that the coatomero complex is involved in the trafficking of CRTR to the plasma membrane (Denning et al., 1992) (Bannykh and
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68/174 others, 2000).
Enzyme conjugating ubiquitin E2S (UBE2S)
[00191] UBE2S is an auxiliary factor of the complex promoting anaphase (APC), an E3 ubiquitin ligase that regulates mitotic output and G1 pointing cell cycle regulators. UBE2S stretches ubiquitin streams after the substrates are preubiquitinated by other components (Wu et al., 2010). UBE2S also points to VHL protein for proteasomal degradation, thus stabilizing HIF 1 alpha (Lim et al., 2008), and perhaps supporting proliferation, epithelial-mesenchymal transition, and metastasis (Chen et al., 2009) (Jung et al., 2006 ). UBE2S is overexpressed in several cancer entities.
Member of the kinesin family 11 (KIF11)
[00192] KIF11 is required for the assembly of a bipolar mitotic axis. It was considered highly regulated in several cancers, often corresponding to clinical and pathological parameters (Liu et al., 2010) (Peyre et al., 2010). Small molecule KIF11 inhibitors such as S-trityl-L-cysteine (STLC), developed as potentially anticancer drugs, arrest cells in mitosis and promote apoptosis of cancer cells (Tsui et al., 2009) (Wiltshire et al., 2010) (Ding and others, 2010). In the clinic, KIF11 inhibitors showed only modest activities (Kaan et al., 2010; Tunquist et al., 2010; Wiltshire et al., 2010; Zhang and Xu, 2008).
[00193] A disintegrin and metalloprotease domain 8 (ADAM8)
[00194] ADAM8 was initially considered to be an immunespecific ADAM, but it was also found in other cell types, often under conditions involving ECM inflammation and remodeling, including cancers and respiratory diseases such as asthma (Koller et al. 2009). Many ADAM species, including ADAM8, are
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69/174 expressed in human malignant tumors, where they are involved in the regulation of growth factor activities and integrin functions, leading to the promotion of cell growth and invasion, although their precise mechanisms are currently unclear (Mochizuki and Okada 2007 ). In mouse gastric tumors, ADAM8 and other ADAMs were increased, probably due to improved EGFR signaling (Oshima et al. 2011).
Homologous cell division cycle 6 (S. cerevisiae) (CDC6)
[00195] CDC6 is essential for the initiation of DNA replication. It is located in the nucleus during G1, but moves to the cytoplasm at the beginning of the S phase. CDC6 also regulates activation of replication of checkpoint by interaction with ATR (Yoshida and others 2010). Deregulation of CDC6 can cause inactivation of the INK4 / ARF area encoding three important tumor suppressor genes: p16INK4a and p15INK4b, both activators of the retinoblastoma pathway, and ARF, a p53 activator (Gonzalez et al. 2006). Liquidation of CDC6 siRNA could prevent proliferation and promote apoptosis (Lau et al. 2006). CDC6 is highly regulated in cancers including gastric cancer (Nakamura et al 2007) (Tsukamoto et al 2008).
Coagulation factor F2R II (thrombin) receptor (F2R)
[00196] F2R, also called a proteinase-activated receptor (PAR1) is a receptor attached to protein G. Signals by PAR1, PAR2, and PAR4 can regulate calcium release or activation of mitogen-activated protein kinase and lead to aggregation platelet, vascular rest, cell proliferation, cytokine release, and inflammation (Oikonomopoulou et al. 2010). F2R is believed to be involved in endothelial and tumor cell proliferation and angiogenesis, and is overexpressed in invasive and metastatic tumors
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70/174 of many types. The expression levels directly correspond to the degree of cancer invasiveness (Garcia-Lopez and others 2010) (Lurje and others 2010). In gastric carcinoma cells, F2R activation can trigger a series of responses that promote tumor cell growth and invasion, for example, overexpression of NFkappaB, EGFR, and Tenascin-C (TN-C) (Fujimoto et al. 2010) . Consequently, F2R expression in gastric cancer has been shown to be associated with the depth of wall invasion, peritoneal spread, and poor prognosis (Fujimoto et al. 2008). A mouse monoclonal anti-human antibody PAR1 (ATAP-2), which recognizes an epitope (SFLLRNPN) within the N-terminus of the thrombin receptor, has been described as well as the PARLL agonist peptide TFLLRNPNDK (Hollenberg and Compton 2002; Mari et al. 1996 ; Xu et al. 1995).
Olfactomedine 4 (OLFM4)
[00197] OLFM4, whose function is largely unknown, is overexpressed in the inflamed colonic epithelium and a number of human tumor types, especially those in the digestive system (Koshida et al., 2007). OLFM4 is a robust marker for stem cells in the human intestine and marks a subset of colorectal cancer cells (van der Flier et al., 2009). OLFM4 prevents the GRIM-19 protein that promotes apoptosis (Zhang et al., 2004) (Huang et al., 2010), regulates cell cycle and promotes S-phase transition in cancer cell proliferation. Furthermore, OLFM4 is associated with cancer adherence and metastasis (Yu et al., 2011b). Forced overexpression of OLFM4 in murine prostate tumor cells leads to faster tumor formation in a syngeneic host (Zhang et al., 2004). OLFM4 was found to be overexpressed in GC (Aung et al., 2006). The inhibition of OLFM4 expression could induce apoptosis in the presence of cytotoxic agent in
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71/174 gastric cancer cells (Kim et al., 2010). OLFM4 serum concentration in pre-surgical GC patients was also improved compared to healthy donors (Oue et al., 2009). OLFM4 has been identified as a single target gene for retinoic acids (RAs) and for the demethylating agent 5-aza-2'-deoxycytidine. These two agents have proven to be effective in treating certain myeloid leukemia patients (Liu et al., 2010).
Cell surface Thy-1 antigen (THY1)
[00198] Thy-1 (CD90) is a GPI-anchored glycoprotein found in many cell types including T cells, neurons, endothelial cells and fibroblasts. Thy-1 is involved in processes including adhesion, nerve regeneration, tumor growth, tumor suppression, migration, cell death, and T cell activation (Rege and Hagood 2006b; Rege and Hagood 2006a) (Jurisic and others 2010 ). Thy-1 appears to be an adult, but not embryonic, marker of angiogenesis (Lee et al. 1998). Furthermore, it was considered as a marker for several types of stem cells (mesenchymal stem cells, liver stem cells (oval cells) (Masson et al. 2006), keratinocyte stem cells (Nakamura et al. 2006), hematopoietic stem cells (Yamazaki and others 2009)). Thy1 is highly regulated in several cancers including gastric cancer and GISTs, for which it has been proposed as a marker (Yang and Chung 2008; Zhang et al. 2010) (Oikonomou et al. 2007). 250 kDa centrosomal protein (CEP250)
[00199] Cep250 has a role in the cohesion of centers organizing microtubules (Mayor et al, 2000). It is also called a protein associated with Nek2 centrosomal or C-Nap1, as it colocalizes with Nek2 serine / threonine kinase and is a substrate for it. Nek2 kinase and its substrates regulate the connection between centrosomes (Bahmanyar et al., 2008). At the beginning of mitosis,
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72/174 when centrosomes separate to form a bipolar axis, C-Nap1 is phosphorized and, therefore, dissociates from centrosomes. In vitro experiments showed that overexpression of Cep250 compromised microtubule organization in the centrosome (Mayor et al., 2002).
[00200] Hypoxia inducible factor 1, alpha subunit (basic helix-loop-helix transcription factor) (HIF1A)
[00201] HIF1A is the oxygen sensitive subunit of the inducible hypoxia factor (HIF), an active transcription factor under hypoxic conditions that are often found in tumors. It measured transcription of more than 60 genes involved in survival, glucose metabolism, invasion, metastasis and angiogenesis (for example, VEGF). HIF1 is overexpressed in many cancers, often associated with poor prognosis, and is considered an interesting target for pharmacological manipulation (Griffiths and others 2005; Quintero and others 2004; Stoeltzing and others 2004) (Zhong and others 1999).
[00202] In gastric cancer, HIF1A contributes to angiogenesis (Nam and others 2011), corresponds to tumor size, minor differentiation, shorter tumor stage survival (Qiu and others 2011) and metastasis (Wang and others 2010) (Han and others 2006; Kim and others 2009; Oh and others 2008; Ru and others 2007). It is also considered to generate resistance to chemotherapeutic drugs such as 5-FU via inhibition of drug-induced apoptosis and decreased accumulation of intracellular drug (Nakamura and others
2009) (Liu and others 2008). The HIF-1alpha 2-methoxy-estradiol inhibitor significantly reduced metastatic properties of gastric cancer cells (Rohwer et al. 2009).
[00203] Homologous viral oncogene of v-Ki-ras2 Kirsten rat sarcoma (KRAS)
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[00204] KRAS is a member of the small GTPase superfamily and a protooncogene involved in the first steps of many signal transduction pathways, such as MAPK and AKT-mediated pathways, which are potentially oncogenic. Unique amino acid substitutions lead to activating mutations, resulting in a transforming protein that plays a key role in several malignancies including gastric cancer (Capella et al., 1991). Oncogenic KRAS mutations are uncommon in gastric cancer. In a subset of gastric cancers, the KRAS site has been amplified, resulting in KRAS protein overexpression. Thus, gene amplification probably forms the molecular basis for KRAS overactivation in gastric cancer (Mita et al., 2009). KRAS mutant alleles contribute to the initiated boost of VEGF hypoxia (Kikuchi et al., 2009; Zeng et al., 2010). Mutated KRAS can also be detected in the serum or plasma of cancer patients and has thus been suggested as an easily accessible tumor marker (Sorenson, 2000). The KRAS-001 peptide is derived from only one of two entanglement variants - NP_004976 (188 amino acids) and not from the entanglement variant - NP_203524 (189 amino acids). The interlacing variants differ in their last exon, in which KRAS-001 is located.
[00205] Non-SMC I condensin complex, G subunit (NCAPG)
[00206] NCAPG is part of the condensin complex I, which is composed of structural maintenance of chromosomes (SMC) and non-SMC proteins, and regulates chromosome condensation and segregation during mitosis (Seipold et al., 2009). Overexpression of NCAPG has been found in numerous tumors including nasopharyngeal carcinoma (Li et al., 2010), hepatocellular carcinoma (Satow et al., 2010) and melanoma (Ryu et al., 2007). In between
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74/174 normal tissues, NCAPG showed higher expression in the testis. It has been suggested to be a possible proliferation marker and a potential prognostic indicator in cancer (Jager et al., 2000).
[00207] Topoisomerase (DNA) II alpha (TOP2A) and topoisomerase (DNA) II beta (TOP2B)
[00208] TOP2A and TOP2B encode highly homologous isoforms of a DNA topoisomerase, which controls and changes topological states of DNA during transcription and is involved in chromosome condensation, chromatid separation, replication and transcription. Topoisomerase is a target for several anticancer drugs, such as anthracyclines, and a variety of mutations have been associated with drug resistance (Kellner et al., 2002) (Jarvinen and Liu, 2006). TOP2A (not TOP2B) is essential for cell proliferation. It is located next to the HER2 oncogene and is amplified in a vast majority of breast tumors with amplified HER2, but also in those without HER2 amplification (Jarvinen and Liu, 2003), and in many other tumor entities. Also in a subset of gastric cancers, TOP2A was found to be amplified and overexpressed, often along with HER2 (Varis et al., 2002) (Liang et al., 2008).
Laminin, range 2 (LAMC2)
[00209] Laminins are the main non-collagenous constituents of base membranes. They are involved in cell adhesion, differentiation, migration, signaling, and metastasis. The gamma 2 chain together with the alpha 3 and beta 3 chains constitute laminin 5. LAMC2 promotes invasive growth of human cancer cells in vivo. It is highly expressed by human cancers in front of invasion, and the expression is related to poor prognosis (Tsubota et al., 2010). A laminin 5-generated MMP-2 cleavage product can activate EGFR signaling and promote
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75/174 cell (Schenk et al., 2003). In gastric carcinoma, LAMC2 can be induced by members of the EGFR family or by Wnt5a, and the invasive activity has been shown to depend on LAMC2 (Tsubota et al.,
2010) (Yamamoto et al., 2009).
Arila hydrocarbon receptor (AHR)
[00210] AHR binds planar aromatic hydrocarbons such as TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin), and mediates transcription of genes including xenobiotic-metabolizing enzymes such as cytochrome P450 enzymes. It also plays a role in cell cycle progression (Barhoover et al. 2010). AhR is believed to be partially associated with the tumor promoting dioxin activity, as it has professional-proliferative and anti-apoptotic functions, and can lead to deregulation of cell-cell contact, differentiation and improved motility (Watabe and others 2010) (Dietrich and Kaina 2010) (Marlowe and others 2008). AHR expression can be low-regulated by TGF-beta (Dohr and Abel 1997; Wolff and others 2001) and induced by Wnt or beta-catenin signaling (Chesire and others 2004). Overexpression of AHR has been found in many cancers including gastric cancer, where it corresponded with the frequent expression of CYP1A1 (Ma et al. 2006). AHR expression and nuclear translocation were higher in gastric cancer than in normal tissues, and the expression gradually increased during cancerogenesis (Peng et al. 2009a). Activation of the AhR pathway improves gastric cancer cell invasiveness probably by an induction of c-Jun-dependent MMP-9 (Peng et al. 2009b). In a mouse model, the expression of a constitutively active mutant of the aryl hydrocarbon receptor (CA-AhR) results in the development of stomach tumors, corresponding to increased mortality (Andersson et al. 2002; Kuznetsov et al.
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2005). AhR's role in cancer appears to be ambiguous, as some studies also point towards an activity that suppresses the tumor (Gluschnaider and others 2010) (Fan and others 2010).
Hyaluronan-mediated motility receptor (RHAMM) (HMMR) [00211] HMMR can occur on the cell surface, where it binds hyaluronic acid (HA) and interacts with the CD44 HA receptor. This interaction has a role in processes, such as cell motility, wound healing and invasion (Gares and Pilarski, 2000). Intracellularly, HMMR is associated with the cytoskeleton, microtubules, centrosomes and the mitotic axis and has a role in controlling the integrity of the mitotic axis. HMMR is overexpressed in various cancer tissues (Sohr and Engeland, 2008). HA has been suggested to protect cancer cells from immune attack. HA serum is often increased in metastatic patients (Delpech et al., 1997). HMMR has been identified as a promising tumor-associated antigen and a possible prognostic factor in AML and CLL. HMMR-derived peptides have been used in antileukemia vaccines. HMMR-001 has also been tested for in vitro immunogenicity, but not used for vaccination (Tzankov et al, 2011) (Greiner et al, 2010; Schmitt et al, 2008; Tabarkiewicz and Giannopoulos, 2010) (Greiner et al, 2005) . Overexpression of HMMR has also been found in several other cancers, often associated with poor prognosis. HMMR was also overexpressed in gastric cancer, often along with CD44, and has been suggested to facilitate invasion and metastasis (Li and others, 1999) (Li and others, 2000a) (Li and others, 2000b).
TPX2, associated with a microtubule, homologous (Xenopus laevis) (TPX2) [00212] TPRX2 is an expressed protein associated with proliferation in S, G (2) and M phases of the cell cycle and considered as a proliferation marker (Cordes and others , 2010).
[00213] It is required for normal microtubule nucleation, for
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77/174 example, for the assembly of mitotic axes. TPX2 recruits and activates Aurora A (Bird and Hyman, 2008; Moss et al., 2009). Phosphorylation of TPX2 with a kinase similar to Polo 1 increased its ability to activate Aurora A (Eckerdt et al., 2009). TPX2 is overexpressed in many types of tumors and often overexpressed with Aurora A (Asteriti et al., 2010). Examples where TPX2 overexpression (often associated with poor prognosis or advanced stage) were found are meningioma (Stuart and others, 2010), lung cancer (Kadara and others, 2009) (Lin and others, 2006; Ma and others, 2006) (Manda and others, 1999) and hepatocellular carcinoma (Shigeishi and others, 2009b) (Satow and others, 2010) (Wang and others, 2003).
[00214] Therefore, the present invention provides a peptide comprising a sequence that is selected from the group of SEQ ID NO: 1 to SEQ ID NO: 95 or a variant thereof, which is at least 80% homologous to SEQ ID N °: 1 to SEQ ID NO: 95 or a variant thereof, which induces T cells to cross-react with said peptide, and said peptide is not a full-body polypeptide.
Therefore, the present invention further provides a peptide comprising a sequence that is selected from the group of SEQ ID NO: 1 to SEQ ID NO: 95 or a variant thereof, which is at least 80% homologous to SEQ ID N °: 1 to SEQ ID NO: 95, where said peptide or variant thereof has a total length between 8 and 100 amino acids, preferably between 8 and 30 amino acids and most preferably between 8 and 14 amino acids.
[00216] The present invention is still related to the peptides previously described, having the ability to bind to a class I or II major human histocompatibility complex (MHC) molecule.
[00217] The present invention is still related to the peptides previously described, the peptide consisting of or
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78/174 consists essentially of an amino acid sequence according to SEQ ID NO: 1 to SEQ ID NO: 95.
[00218] The present invention is still related to the peptides previously described, where the peptide is modified and / or includes non-peptide bond.
[00219] The present invention is also related to the peptides previously described, where the peptide is a fusion protein, in particular, comprising N-terminal amino acids of the HLA-DR invariant chain associated with antigen (Ii).
[00220] The present invention is still related to a nucleic acid, encoding the previously described peptides, provided that the peptide is not the full human protein.
[00221] The present invention is still related to the previously described nucleic acid which is DNA, cDNA, PNA, CNA, RNA or combinations thereof.
[00222] The present invention is still related to a vector expression capable of expressing a previously described nucleic acid.
[00223] The present invention further relates to a peptide as described above, a nucleic acid as described above, or an expression vector as described previously for use in medicine.
[00224] The present invention still relates to a host cell consisting of a nucleic acid as described above or an expression vector as described above.
[00225] The present invention is still related to a host cell as described above, which is a cell presenting antigen.
[00226] The present invention also relates to the host cell described, in which the cell presenting antigen is a
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79/174 dendritic cell.
[00227] The present invention also relates to a method of producing a described peptide, which constitutes the culture of a host cell that isolates the peptide from the host cell or its culture agent.
[00228] The present invention also relates to an in vitro method for the production of activated cytotoxic T lymphocytes (CTL), the method comprising in vitro CTL contact with human MHC class I or II molecules loaded with antigen expressed on the surface of a suitable cell that has antigen for a period of time sufficient to shoot said CTL in a specific antigenic manner, in which said antigen is any peptide described.
[00229] The present invention also relates to a method according to the previous description, where the antigen is loaded onto MHC class I or II molecules expressed on the surface of a suitable cell-presenting antigen coming in contact with a sufficient amount of antigens with an antigen presenting cell.
[00230] The present invention also relates to a method according to the previous description, where the antigen is composed of cells that presents an expression vector capable of expressing a peptide containing SEQ ID N ° 1 to SEQ ID N ° 33 or said amino acid sequence variant.
[00231] The present invention also relates to activated cytotoxic T lymphocytes (CTL), produced by the described method, which selectively recognizes a cell that aberrantly expresses a polypeptide constituting a described amino acid sequence.
[00232] The present invention also relates to a method of killing target cells in a patient whose target cells aberrantly
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80/174 express a polypeptide composed of any described amino acid sequence, the method comprising administering to the patient an effective number of cytotoxic T lymphocytes (CTL), as defined.
[00233] The present invention also relates to the use of any described peptide, a nucleic acid as described, an expression vector as described, a cell as described, or an activated cytotoxic T lymphocyte as described as a drug or in the manufacture of a medicine.
[00234] The present invention also relates to a use as described, the medicine of which is a vaccine.
[00235] The present invention also relates to a use as described, the drug of which is active against cancer.
[00236] The present invention also relates to use as described, wherein said cancer cells are gastric, gastrointestinal, colorectal, pancreatic, pulmonary or renal cancer cells.
[00237] The present invention also relates to specific marker proteins, which can be used in the prognosis of gastric cancer.
[00238] In addition, the present invention also concerns the use of these new targets for the treatment of cancer.
[00239] As provided therein, proteins encoded by ABL1, ADAM10, AHR, CCND2, CDC6, CDK1, CEACAM1, CEACAM5, CEACAM6, CEACAM6, COL6A3, EIF2S3, LOC255308, EPHA2, ERBB2, ERBB3, F2R, FAPP90 , IGF2BP3, ITGB4, KIF2C, KRAS, LAMC2, LCN2, MET, MMP11, MMP12, MMP3, MST1R, NUF2, OLFM4, PROM1, RRM2, THY1, TMPRSS4, TOP2A, TSPAN1, WNT5A, HIF1A were described as PTK in gastric cancer, compared to normal gastric tissues and
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81/174 other vital tissues (eg kidney, liver, heart) in the literature. [00240] ABL1, ADAM10, ADAM8, AHR, ASPM, ATAD2, CCDC88A, CCNB1, CCND2, CCNE2, CDC6, CDK1, CEACAM1, CEACAM5, CEACAM6, CEACAM6, CLCN3, COL6A3, EPHA2, ERB2, ERB2, ERB2, ERB2 proteins , FAP, HIF1A, HMMR, HSP90B1, IGF2BP3, IQGAP3, ITGB4, KIF11, KIF2C, KRAS, LAMC2, LCN2, MET, MMP11, MMP3, MST1R, MUC6, NCAPG, NFYB, NUF2, PLFK4, PROM, , PTK2, RRM2, SIAH2, THY1, TOP2A, TPX2, TSPAN1, TSPAN8, UBE2S, UCHL5, and WNT5A have been confirmed to have an important role in tumorigenesis, as they are involved in malignant transformation, cell growth, proliferation, angiogenesis or invasion in normal fabric. Also for proteins encoded by DNAJC10, EIF2S3, EIF3L, POLD3, PSMC2, PSMD14, and TMPRSS4, there is some evidence for functions relevant to cancer.
[00241] Proteins encoded by PROM1, WNT5A, SMC4, PPAP2C, GPR38, OLFM4 and THY1 have been shown to be highly expressed and / or functionally important in cancer stem cells and / or stem cells. PROM1 has been discussed as a marker for gastric cancer stem cells, although the data is controversial. Cancer stem cells are a subpopulation of tumor cells with the potential self-renewal necessary for sustained tumor growth. These cells reside in specialized and highly organized structures, the so-called niches of cancer stem cells that are necessary to maintain the potential for self-renewal of cancer stem cells.
[00242] Overexpression of AHR, ASPM, ATAD2, CCNB1, CCND2, CCNE2, CDK1 (CDC2), CEACAM1, CEACAM5, CEACAM6, CEACAM6, COL6A3, EPHA2, ERBB2, ERBB3, F2R, FAP, proteins
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HIF1A, HMMR, HSP90B1, IGF2BP3, ITGB4, KIF11, KIF2C, KRAS, LAMC2, LCN2, LMNB1, MET, MMP11, MMP3, MST1R, MUC6, NCAPG, NUF2, OLFM4, PBK, PPAP2, PT2 TSPAN1, and WNT5A in tumors has been confirmed to be associated with advanced stages of the disease and poor prognosis for patients.
[00243] Therefore, the present invention provides methods of identifying animals, preferably humans, who are likely to have gastric cancer. In one mode, the probability determined is between 80% and 100%. Such a method comprises determining the level of at least one of the MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB and MUC6 proteins in a tumor sample from the tested animal. In one embodiment, the sample is obtained through radical surgery. In another embodiment, the sample is obtained by needle biopsy.
[00244] When the level of MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB or MUC6, as determined, is 20% or higher in cells in relation to the regulation determined in benign epithelial cells of the same sample, the animal subject is identified as probably having gastric cancer.
[00245] The more different proteins in the group consisting of MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB and MUC6 are highly regulated, the greater the possibility that the animal subject will be identified as having gastric cancer.
[00246] In one embodiment, the determination of the level of MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB or MUC6 is carried out in situ. In another modality, the determination of the level of MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB or MUC6 is carried out in situ. In yet another modality, determining the level of MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB or MUC6 is
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83/174 performed in vivo. In a preferred embodiment, the determination of the level of MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB or MUC6 is performed by Laser Capture Microscopy coupled to a Western blot.
[00247] In a particular mode, the determination of the level of MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB or MUC6 is performed with an antibody specific to MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB or MUC6 . In another such modality, the determination of the level of MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB or MUC6 is performed by PCR with a first specific for an MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9 mRNA coding, UQCRB or MUC6. In yet another modality, the determination of the level of MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB or MUC6 is performed with a specific nucleotide test for an mSTNA coding by MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9 , UQCRB or MUC6. In one of these personifications, the determination of the level of MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB or MUC6 is performed using a Northern blot. In another modality, the determination of the level of MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB or MUC6 is carried out through a ribonuclease protection assay. In other modalities, immunological tests, such as enzyme immunosorbent assays (ELISA), radioimmunoassays (RIA), and Western blots can be used to detect MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB and MUC6 polypeptides in a sample of body fluid (such as blood, serum, saliva, urine or peritoneal fluid). Biopsies, tissue samples and cell samples (such as ovaries, lymph nodes, ovarian surface scraps of epithelial cells, lung biopsies, liver biopsies, and any fluid sample containing cells, such as in
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84/174 peritoneal fluid, sputum, and pleural effusion) can be tested by disaggregating and / or solubilizing the tissue or cell sample and undergoing an immunoassay to detect polypeptides, such as ELISA, RIA, or Western blot. Such cells or tissue samples can also be analyzed by nucleic acid-based methods, for example, reversing the polymerase chain reaction (RT-PCR), Northern hybridization amplification, or slot or dot spotting. To visualize the distribution of tumor cells within a tissue sample, diagnostic tests that preserve the tissue structure of a sample, for example, immunostaining, in situ hybridization of RNA, or in situ RTPCR can be employed to detection of gastric cancer marker polypeptide or mRNA, respectively. By in vivo localization of tumor masses, imaging tests such as magnetic resonance imaging (MRI) can be used by introducing to the test subject an antibody that specifically binds a MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB or MUC6 polypeptide ( particularly a polypeptide cell located on the surface), where the antibody is conjugated or coupled to a paramagnetic marker (or other portion suitable for detection, depending on the imaging method used), alternatively, the location of a tumor marker specific unclassified antibody it can be detected by a secondary antibody coupled to a detectable portion.
[00248] Furthermore, the present invention also provides chimeric / fusion proteins / peptides comprising the MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB or MUC6 polypeptides, as well as fragments thereof, including functional, proteolytic and antigenic fragments.
[00249] The fusion partner or the sections of a hybrid molecule
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Appropriate 85/174 produce epitopes that stimulate CD4 ⁺ T cells. Epitopes that stimulate CD4 ⁺ are well known in the current state of the art, and include those identified in the tetanus toxoid. In a preferred variant, the peptide is a fusion protein, in particular comprising N-terminal amino acids of the HLA-DR invariant chain associated with the antigen (Ii). In another variant of the invention, the peptide is a truncated human protein or a protein fragment fusion protein and another polypeptide part, provided that the human part includes one or more amino acid sequences according to the invention.
[00250] Antibodies to MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB or MUC6 polypeptides for chimeric / fusion proteins comprising MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, ULCR, as well MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB or MUC6 polypeptide fragments, including proteolytic and antigenic fragments and for chimeric / fusion proteins / peptides containing these fragments are also an integral part of the present invention. In addition, methods of using these antibodies for the prognosis of cancer, and in particular, gastric cancer, are also part of the present invention.
[00251] The antibodies of the present invention can be polyclonal antibodies, monoclonal antibodies and / or chimeric antibodies. Immortal cell lines that produce a monoclonal antibody of the present invention are also part of the present invention.
[00252] A professional with normal skills in the field will understand that in some cases, greater expression of MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB or MUC6 as a tumor marker gene will indicate a worse prognosis for a test patient with gastric cancer. For example, relatively high levels
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86/174 of expression MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB or MUC6 can indicate a relatively large primary tumor, a greater tumor burden (for example, more metastases), or a relatively more malignant tumor phenotype.
[00253] The most different proteins in the group constituting MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB and MUC6 are overexpressed with the worst prognosis.
[00254] The diagnostic and prognostic methods of the invention involve the use of known methods, for example, antibody-based methods to detect MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB and MUC6 polypeptides as well as methods based on hybridization and / or nucleic acid amplification for mRNA detection of MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB and MUC6.
[00255] Furthermore, since rapid destruction of tumor cells generally results in the generation of autoantibodies, the gastric cancer tumor markers of the invention can be used in serological tests (for example, a patient's serum ELISA test) for detect autoantibodies against MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB or MUC6 in a patient. Levels of specific autoantibodies of MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB and MUC6 polypeptide are at least about three times greater (and preferably at least five or seven times greater, more preferably at least 10 times or 20 times greater) than in a control sample, are indicative of gastric cancer.
[00256] Intracellular MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB and MUC6 polypeptides, secreted and located on the cell surface can be used for the analysis of biopsies, for example, tissues or cell samples (including cells obtained from samples of liquids as cavity fluid
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87/174 peritoneal) to identify a tissue or cell biopsy as containing gastric cancer cells. A biopsy can be analyzed as an intact tissue or as a sample of whole cells, or the tissue or cell sample can be disaggregated and / or solubilized as necessary for the specific type of diagnostic test to be used. For example, biopsies or samples can be subjected to analysis of whole tissue or whole cell for MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB and MUC6 polypeptides or mRNA levels in situ, for example, using immunohistochemistry, hybridization in situ mRNA, or RT-PCR in situ. The skilled practitioner will know how to process tissues or cells for analysis of polypeptides or mRNA levels using immunological methods, such as ELISA, immunoblotting, or equivalent methods, or the analysis of nucleic acid-based mRNA levels using analytical methods, such as RT- PCR, Northern hybridization, or slot or dot spotting.
[00257] Kits to measure expression levels of MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB and MUC6.
[00258] The present invention provides kits for detecting a higher level of expression of MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB and MUC6 as a gastric cancer marker gene in a tested subject. A kit for the detection of gastric cancer marker polypeptide preferably containing an antibody that specifically binds to a chosen gastric cancer marker polypeptide. A gastric cancer marker mRNA detection kit preferably containing one or more nucleic acids (for example, one or more primers or oligonucleotide tests, DNA tests, RNA tests, or models for generating RNA tests), that hybridize specifically with MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB, and MUC6 mRNA.
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[00259] In particular, the antibody-based kit can be used to detect the presence of - and / or measure the level of MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB and MUC6 polypeptides that are specifically linked by the antibody or fragment their immunoreactive effect. The kit may include an antibody reactive with the antigen and a reagent for detecting a reaction of the antibody with the antigen. Such a kit may be an ELISA kit and may contain a primary and secondary control (for example, a certain amount of a specific gastric cancer marker polypeptide), and any other necessary reagents, such as groups detectable enzyme substrates and color reagents as described above. The diagnostic kit may alternatively be an immunoblot kit, usually containing the components and reagents described.
[00260] A nucleic acid based kit can be used to detect and / or measure the level of expression of MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB and MUC6 by detecting and / or measuring the amount of MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB and MUC6 mRNA in a sample, such as a tissue or cell biopsy. For example, an RT-PCR kit for detecting elevated expression of MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB and MUC6 preferably containing sufficient primers to carry out reverse transcription of gastric cancer marker mRNA to cDNA and amplification PCR of the gastric cancer marker cDNA, which preferably will also contain PCR control model molecules and primers to perform adequate positive and negative controls, and internal controls for quantization. A person of ordinary skill in the field will understand how to select the appropriate primers to perform reverse transcription and PCR reactions as well as the appropriate control reactions to be
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89/174 carried out. This guidance is found, for example, in F. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., 1997. Numerous RT-PCR variations are known in the art. Targeted delivery of immunotoxins to MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB and MUC6 can be used as therapeutic targets for the treatment or prevention of gastric cancer. For example, an antibody molecule that specifically binds to MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB and MUC6 polypeptides located on the cell surface, which can be conjugated to a radioisotope or other toxic compounds. Antibody conjugates are administered to the test subject so that the binding of the antibody to its cognate gastric cancer polypeptides results in the expected delivery of the therapeutic compound to gastric cancer cells, thereby treating ovarian cancer.
[00261] The therapeutic group can be a toxin, radioisotope, drug, chemical or a protein (see, for example, al Bera al. Pharmacokinetics and antitumor activity of a bivalent disulfidestabilized Fv immunotoxin with improved antigen binding to erbB2 Cancer Res. 59 : 4018-4022 (1999)). For example, the antibody can be linked to or conjugated to a bacterial toxin (eg, diphtheria toxin, pseudo-hormone exotoxin, cholera toxin) or plant toxin (eg, ricin toxin) for targeted delivery of the toxin to a cell expressing MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB and MUC6. This immunotoxin can be delivered to a cell and, by binding the gastric cancer marker polypeptide to the one located on the cell surface, the toxin conjugated to the specific antibody of the gastric cancer marker will be delivered to the cell.
[00262] In addition, for any MST1R, UCHL5 polypeptide,
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SMC4, NFYB, PPAP2C, AVL9, UQCRB and MUC6 for which there is a specific linker (for example, a linker that binds to a localized cell surface protein), the linker can be used in place of an antibody to target a toxic compound to the gastric cancer cell, as described above.
[00263] The term antibodies is used here in a broad sense and includes both polyclonal and monoclonal antibodies. In addition to intact immunoglobulin molecules, fragments or polymers of immunoglobulin molecules and the humanized versions of immunoglobulin molecules are also included in the concept of antibodies, as long as they have one of the desired properties (for example, specific binding being a marker polypeptide of gastric cancer, delivery of a cancer cell toxin that expresses a gastric cancer marker gene at a higher level and / or inhibits the activity of a gastric cancer marker polypeptide) described herein.
[00264] Whenever possible, the antibodies of the invention can be purchased from commercial sources. Antibodies of the invention can also be generated by well-known methods. The skilled practitioner will understand that the entire length of gastric cancer marker polypeptides or fragments thereof can be used to generate the antibodies of the invention. A polypeptide to be used to generate an antibody of the invention can be totally or partially purified from a natural source, or can be produced using recombinant DNA techniques. For example, a cDNA encoding an MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB or MUC6 polypeptide, or a fragment thereof, can be expressed in prokaryotic cells (for example, bacteria), or eukaryotic cells (for example, yeasts , insects, or mammalian cells), after which the recombinant protein can be
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91/174 purified and used to generate a preparation of monoclonal or polyclonal antibodies that specifically bind to the gastric cancer marker polypeptide used to generate the antibody.
[00265] Professionals, skilled in the art, know that the generation of two or more different sets of monoclonal or polyclonal antibodies maximizes the probability of obtaining an antibody with the specificity and affinity necessary for its use (for example, ELISA, immunohistochemistry, in vivo images, immunotoxin therapy). Antibodies are tested for the activity desired by known methods, in accordance with the purposes for which antibodies are to be used (eg, ELISA, immunohistochemistry, immunotherapy, etc., for further guidance on antibody generation and testing, see, for example, Harlow and Lane, antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988). For example, antibodies can be tested in ELISA, Western blot, immunohistochemistry of fixed formalin gastric cancer or frozen tissue sections. After their initial characterization in vitro, antibodies intended for therapeutic purposes or for use in in vivo diagnostics are tested according to known clinical testing methods.
[00266] The term monoclonal antibody, as used here, refers to an antibody obtained from a substantially homogeneous population of antibodies, that is, the individual antibodies, including the population, are identical, except for possible natural mutations that may be smaller quantities. The monoclonal antibodies here specifically include chimeric antibodies, in which part of the heavy and / or light chain, are identical or homologous to corresponding sequences
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92/174 in antibodies derived from a given species or belonging to a specific antibody class or subclass, while the rest of the chain (s) is / are identical or homologous to corresponding sequences in antibodies derived from another species or belonging to the other class or subclass of antibodies, as well as fragments of such antibodies, while exhibiting the desired antagonistic activity (US Pat. No.4, 816,567).
[00267] The monoclonal antibodies of the invention can be prepared using hybridoma methods. In a hybridoma method, a rat or other suitable host animal is usually immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that specifically bind to the immunizer. Alternatively, lymphocytes can be immunized in vitro.
[00268] Monoclonal antibodies can also be made using recombinant DNA methods, such as those described in U.
S. Pat. No. 4,816,567. DNA encoding the antibodies of the invention can be readily isolated and sequenced using standard procedures (for example, using oligonucleotide probes that are able to specifically bind to genes encoding murine antibody heavy and light chains).
[00269] In vitro methods are also suitable for the preparation of monovalent antibodies. Digestion of antibodies to produce fragments, especially Fab fragments, can be performed using routine techniques known in the profession. For example, digestion can be performed using papain. Examples of papain digestion are described in WO 94/29348, published on December 22, 1994, and U.S. Pat. No. 4,342,566. Papain digestion of antibodies normally produces two binding fragments of
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93/174 identical antigens, called Fab fragments, each with a single antigen binding site, and a residual Fe fragment. Pepsin treatment results in a fragment that has two sites - which combine antigen - and is still capable of cross-linking antigens.
[00270] Antibody fragments, whether attached to other sequences or not, may also include insertions, exclusions, substitutions or other selected modifications of specific regions or amino acid residues, provided that the fragment's activity is not significantly altered or decreased regarding unmodified antibodies or antibody fragments. These modifications may provide for some additional properties, such as to remove / add amino acids capable of disulfide binding, to increase its biological longevity, to alter its secretory characteristics, etc. In any case, the antibody fragment must have a bioactive property, such as mandatory activity, regulation of binding in the binding domain, etc. Functional or active regions of the antibodies can be identified by mutagenesis of a specific region of the protein, followed by expression and testing of the expressed polypeptide. Such methods are readily apparent to a physician skilled in the art and may include site-specific mutagenesis of the nucleic acid encoding the antibody fragment.
[00271] The antibodies of the invention can still comprise humanized antibodies or human antibodies. Humanized forms of non-humans (eg murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab 'or other antibody antigen binding substrates), which contain minimal immunoglobulin-derived sequences not human. Antibodies.
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Humanized 94/174 include human immunoglobulins (recipient antibodies) in which residues from a recipient's complementary determination region (CDR) are replaced by residues from a CDR of a non-human species (donor antibodies), such as mice, mice or the rabbit with the desired specificity, affinity and capacity. In some cases, table Fv (FR) of human immunoglobulin residues is replaced by corresponding non-human residues. Humanized antibodies can also include residues that are found in neither the recipient antibody nor the imported CDR or frame sequences. In general, the humanized antibody comprises substantially all of the variable domains, which are at least one in all, and typically two, where all or almost all CDR regions correspond to those of a non-human immunoglobulin and all or almost all FR regions are those of a consensus human immunoglobulin sequence. The optimally humanized antibody will also include at least part of the immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
[00272] Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are often referred to as import residues, which are usually taken from a variable domain import. Humanization can be done essentially by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Thus, as humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), where substantially less than an intact human variable domain
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95/174 has been replaced by the corresponding sequence of a non-human species. In practice, antibodies are typically humanized humanized antibodies in which some CDR residues and possibly some FR residues are replaced by residues from sites similar to rodent antibodies.
[00273] Transgenic animals (for example, mice), which are capable, after immunization, of producing a repertoire full of human antibodies, in the absence of endogenous production of immunoglobulins, can also be used. For example, it has been reported that homozygous deletion of the antibody heavy chain junction gene in chimeric and germline mutant mice has resulted in complete inhibition of endogenous antibody production. Transfer of the human germline immunoglobulin gene matrix in such a germline of mutant mice will result in the production of human antibodies in the face of the antigen challenge. Human antibodies can be produced in phage display libraries.
[00274] The antibodies of the invention are preferably administered to a patient in a pharmaceutically acceptable carrier. Typically, a suitable amount of a pharmaceutically acceptable salt is used in the formulation to make the formulation isotonic. Examples of the pharmaceutically acceptable carrier include saline, Ringer's solution and dextrose solution. The pH of the solution is preferably about 5 to about 8, and more preferably about 7 to about 7.5. Other carriers include sustained release preparations, such as semipermeable matrices of solid hydrophobic polymer containing the antibody, of which the matrices are in the form of in-mold articles, for example, films, liposomes or microparticles. It becomes evident that people skilled in the field that some carriers can be
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96/174 preferable, depending, for example, on the route of administration and the concentration of antibodies being administered.
[00275] Antibodies can be administered to the object, patient, or cell by injection (for example, intravenously, intraperitoneally, subcutaneously, intramuscularly), or by other methods, such as infusion, which ensures its delivery to the bloodstream of an effective way. Antibodies can also be administered intratumorally or peritumorally, to exert systemic and local therapeutic effects. Local or intravenous injection is preferred.
[00276] Effective and hourly doses to administer antibodies can be determined empirically, and making these determinations is within the skill of the art. Those skilled in the art will understand that the dosage of antibodies to be administered will vary depending, for example, on the subject receiving the antibodies, the route of administration, the type of antibody used and other drugs to be administered. A typical daily dose of the antibody used alone can vary from about 1 pg / kg to 100 mg / kg of body weight per day or more, depending on the factors mentioned above. After administration of an antibody to treat gastric cancer, the effectiveness of the therapeutic antibody can be assessed in a number of ways well known to the skilled practitioner. For example, the size, number and / or distribution of gastric cancer in a treatment subject receiving treatment can be controlled using standard tumor imaging techniques. A therapeutically administered antibody that stops tumor growth, results in tumor shrinkage, and / or prevents the development of new tumors, compared to the disease course - which occurred in the absence of antibody administration - is an effective antibody to treatment of gastric cancers.
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[00277] Because of the proteins ABL1, ADAM10, AHR, CCND2, CDC6, CDK1, CEACAM1, CEACAM5, CEACAM6, CEACAM6, COL6A3, EIF2S3, LOC255308, EPHA2, ERBB2, ERBB3, F2R, FAP, HMMR, HSP3, HSP3 , KIF2C, KRAS, LAMC2, LCN2, MET, MMP11, MMP12, MMP3, MST1R, NUF2, OLFM4, PROM1, RRM2, THY1, TMPRSS4, TOP2A, TSPAN1, WNT5A, HIF1A, and PTK2 have been shown to be less expressive at least a subset of gastric cancer tissues, compared to normal tissues, the inhibition of their expression or activity can be integrated into any therapeutic strategy to treat or prevent gastric cancer.
[00278] The principle of antisense therapy is based on the hypothesis that suppression of specific sequence of gene expression (through transcription and translation) can be achieved by intracellular hybridization between genomic DNA or mRNA and a complementary antisense species. The formation of such a hybrid nucleic acid duplex interferes with the transcription of the genomic DNA encoding the target tumor antigen, or processing / transport / translation and / or stability of the tumor antigen mRNA.
[00279] Anti-sense nucleic acids can be provided by a variety of approaches. For example, antisense oligonucleotides or antisense anti-RNA can be administered directly (for example, by intravenous injection) to a patient in a way that allows uptake into tumor cells. Alternatively, viral vectors or plasmids encoding antisense RNA (or fragments of RNA) can be introduced into cells in vivo. Antisense effects can also be induced by sense sequences, however, the extent of phenotypic changes is highly variable. Phenotypic changes induced by effective antisense therapy are evaluated according to the changes, for example, target mRNA levels,
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98/174 levels of target protein, and / or activity levels of the target protein. [00280] In a specific example, inhibition of gastric tumor marker function by antisense gene therapy can be accomplished by direct administration of the antisense gastric tumor marker RNA to a patient. The RNA antisense tumor marker can be produced and isolated by a standard technique, but it is more easily produced by in vitro transcription using an antisense cDNA tumor marker under the control of a high-efficiency promoter (for example, the T7 promoter). The administration of antisense tumor marker RNA to cells can be performed by any of the methods for direct administration of nucleic acids described below.
[00281] An alternative strategy to prevent MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB or MUC6 function using gene therapy involves intracellular expression of an antiMST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB or MUC6 antibody or UQCRB or MUC6 a portion of an anti-MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB or MUC6 antibody. For example, the gene (or gene fragment) that encodes a monoclonal antibody that specifically binds to an MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB or MUC6 polypeptide and inhibits its biological activity, is placed under transcriptional control of a (for example, tissue or tumor specific) regulatory sequence of the gene, within a nucleic acid expression vector. The vector is then administered to such a patient who is taken up by gastric cancer cells or other cells, which then secrete the anti-MST1R, UCHL5, SMC4 antibody, NFYB, PPAP2C, AVL9, UQCRB or MUC6 and thus block activity of the MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB and MUC6 polypeptide. Preferably, the MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB and MUC6 polypeptides
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99/174 are present on the extracellular surface of gastric cancer cells.
[00282] In the methods described above, which include the administration and absorption of exogenous DNA into cells of an individual (i.e., the transduction or transfection of the gene), the nucleic acids of the present invention can be in the form of naked DNA or the Nucleic acids can be a vector for delivering nucleic acids to cells for inhibiting gastric tumor marker protein expression. The vector may be a commercially available preparation, such as an adenovirus vector (Quantum Biotechnologies, Inc. Laval, Quebec, Canada). Delivery of the nucleic acid or vector to cells can be through a variety of mechanisms. As an example, delivery can be via liposomes, using commercially available preparations, such as LIPOFECTIN, LIPOFECTAMINE (GIBCO-25, Inc., Gaithersburg, Maryland), Superfect (Qiagen, Inc. Hilden, Germany) and TRANSFECTAM (Promega Biotec, Inc., Madison, Wisconsin), as well as other liposomes developed according to standard procedures in the art. In addition, the nucleic acid or vectors of the present invention can be delivered through in vivo electroporation, the technology for which it is available from Genetronics, Inc. (San Diego, California), as well as through a SONOPORATION machine (ImaRx Pharmaceutical Corp., Tucson, Arizona).
[00283] As an example, the delivery of the vector can be via a viral system, such as a retroviral vector system, which can package a retroviral recombinant genome. Recombinant retroviruses can be used to infect and thus deliver infected cells that inhibit the expression of MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB or MUC6 to the antisense nucleic acid. The exact method of introducing the altered nucleic acid
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100/174 in mammalian cells, of course, is not limited to the use of retroviral vectors. Other techniques are widely available for this procedure, including the use of adenoviral vectors, adeno-associated viruses (AAV) vectors, lentiviral vectors, pseudotype retroviral vectors. Physical transduction techniques can also be used, such as delivery of liposomes and mediated by receptors and other mechanisms of endocytosis. This invention can be used in conjunction with any of these or other used gene transfer methods.
[00284] Antibodies can also be used for in vivo diagnostic assays. Generally, the antibody is labeled with a radionucleotide (such as ¹¹¹ In, ⁹⁹ Tc, ¹⁴ C, ¹³¹ I, ³ H, ³² P or ³⁵ S), so that the tumor can be located using immunoscintiography. In one embodiment, the antibodies or fragments link the extracellular domains of two or more targets of MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB, and MUC6 and the affinity value (Kd) is less than 1x10pM.
[00285] Antibodies for diagnostic use can be labeled with detection probes by different imaging methods. Probe detection methods include, but are not limited to, fluorescence, light, confocal and electron microscopy; magnetic resonance imaging and spectroscopy, fluoroscopy, computed tomography and positron emission tomography. Suitable probes include, but are not limited to, fluorescein, rhodamine, eosin and other fluorophores, radioisotopes, gold, gadolinium and other lanthanides, paramagnetic iron, fluorine 18 and other positron-emitting radionuclides. In addition, the probes can be bi or multifunctional and can be detectable by more than one of the methods listed. These antibodies can be labeled directly or indirectly with the aforementioned tests. The test link for
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101/174 antibodies include covalent attachment of the probe, incorporation of the probe into the antibody, and covalent attachment of a chelating compound for attachment of the probe, among others well recognized in the art. By immunohistochemistry, the disease tissue sample can be fresh or frozen, or it can be soaked in paraffin and fixed with a preservative, such as formaldehyde. The fixed or embedded sections containing the sample are contacted with a labeled primary and secondary antibody antibody, where the antibody is used to detect the MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB and MUC6 proteins expressed in situ.
[00286] The present invention thus provides a peptide comprising a sequence which is selected from the group of SEQ ID NO: 1 to SEQ ID NO: 95 or a variant thereof that is 85%, preferably 90% and greater preferably 96%, homologous for SEQ ID NO: 1 to SEQ ID NO: 95 or a variant thereof that induces cross-reaction of T cells with said peptides.
[00287] The peptides of the invention have the ability to bind to a class I major human histocompatibility complex (MHC) molecule.
[00288] In the present invention, the term homologous refers to the degree of identity between sequences of two amino acid sequences, i.e. peptide or polypeptide sequences. The aforementioned homology is determined by comparing two sequences aligned under ideal conditions on the sequences being compared. The sequences to be compared may have an addition or deletion (for example, gap and the like) in the ideal alignment of the two sequences. This sequence homology can be calculated by creating alignment using, for example, the ClustalW algorithm. Sequence analysis software commonly available, more specifically, Vector NTI, GENETYX or
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102/174 analysis tools provided by public databases.
[00289] A person skilled in the art will be able to assess whether T cells induced by a variant of a specific peptide will be able to cross-react with the peptide itself (Fong and others 8809-14); (Appay and others 1805-14; Colombetti and others 2730-38; Zaremba and others 4570-77).
[00290] With a variant of the specific amino acid sequence, the inventors mean that in the side chains, for example, one or two of the amino acid residues are altered (for example, replacing them with the side chain of another amino acid residue that arose naturally occurring or a chain on the other side), such that the peptide is still capable of binding to an HLA molecule in substantially the same way as a peptide consisting of the amino acid sequence determined in SEQ ID NO: 1 to 33. For example , a peptide can be modified so that it at least maintains, if not improved, the ability to interact and bind to the compromising groove of a convenient MHC molecule, such as HLA-A * 02 or -DR, and in that way it less maintains, if not improves, the ability to connect to the activated CTL TCR.
These CTLs subsequently interbreed with cells and destroy cells expressing a polypeptide that contains the natural amino acid sequence of the cognate peptide as defined in aspects of the invention. As can be derived from scientific literature (Rammensee, Bachmann, and Stevanovic) and databases (Rammensee and others 213-19), certain positions of peptides that bind to HLA are typically anchor residues forming a center sequence serving the compromising motif of the HLA receptor, which is defined by polar, electrophysical, hydrophobic polypeptide chains and spatial properties constituting the compromising groove. So a skilled at
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103/174 art will be able to modify the amino acid sequences as stipulated in SEQ ID NO: 1 to 95, maintaining the known anchor residues, and could determine whether such variants retain the ability to bind to MHC class I molecules or II. The variants of the present invention retain the ability to bind to the activated CTL TCR, which may subsequently cross and kill cells expressing a polypeptide containing the natural amino acid sequence of the cognate peptide, as defined in aspects of the invention.
[00292] Those amino acid residues that do not substantially contribute to interactions with the T cell receptor can be modified by substitution with another amino acid whose integration does not substantially affect T cell reactivity and does not eliminate binding to the relevant MHC. Thus, apart from the given condition, the peptide of the invention can be any peptide (the inventors here including oligopeptide or polypeptide), which includes the amino acid sequences or a portion or variant thereof as given.
Table 3: Variants and motif of the peptides according to SEQ ID N °: 1 to 33
UCHL5-001 Peptide code N Y L P F I M AND LSEQ ID 3 Variants F F IF FF I Position 1 2 3 4 5 6 7 8 9MET-006 Peptide code s Y I D V L P AND FSEQ ID 4 Variants F L IF FF I Position 1 2 3 4 5 6 7 8 9
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UCHL5-001 Peptide code N Y L P F I M AND LPROM-001 Peptide code s Y I I D P L N LSEQ ID 5 Variants F F IF FF I Position 1 2 3 4 5 6 7 8 9 10 MMP11-001 Peptide code V W s D V T P L T F SEQ ID 6 Variants Y F L I Y L Y I F L F I Position 1 2 3 4 5 6 7 8 9MST1R-001 Peptide code N Y L L Y V s N FSEQ ID 7 Variants F L IF LF I Position 1 2 3 4 5 6 7 8 9NFYB-001 Peptide code V Y T T s Y Q Q ISEQ ID 8 Variants F L FF LF F Position 1 2 3 4 5 6 7 8 9SMC4-001 Peptide code H Y K P T P L Y FSEQ ID 9 Variants F L IF LF I
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UCHL5-001 Peptide code N Y L P F I M AND L Position 1 2 3 4 5 6 7 8 9 10 UQCRB-001 Peptide code Y Y N THE THE G F N K L SEQ ID 10 Variants F F I F F F I Position 1 2 3 4 5 6 7 8 9PPAP2C-001 Peptide code THE Y L V Y T D R LSEQ ID 11 Variants F F IF FF I Position 1 2 3 4 5 6 7 8 9AVL9-001 Peptide code F Y I s P V N K LSEQ ID 12 Variants F F IF FF I Position 1 2 3 4 5 6 7 8 9NUF2-001 Peptide code V Y G I R L AND H FSEQ ID 13 Variants F L IF LF I Position 1 2 3 4 5 6 7 8 9ABL1-001 Peptide code T Y G N L L D Y LSEQ ID 14 Variants F F IF FF I Position 1 2 3 4 5 6 7 8 9MUC-006 Peptide code N Y AND AND T F P H I
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UCHL5-001 Peptide code N Y L P F I M AND LSEQ ID 15 Variants F F LF FF L Position 1 2 3 4 5 6 7 8 9ASPM-001 Peptide code R Y L W THE T V T ISEQ ID 16 Variants F F LF FF L Position 1 2 3 4 5 6 7 8 9EPHA2-005 Peptide code V Y F s K s AND Q LSEQ ID 17 Variants F F IF FF I Position 1 2 3 4 5 6 7 8 9MMP3-001 Peptide code V F I F K G N Q FSEQ ID 18 Variants Y L IY LY I Position 1 2 3 4 5 6 7 8 9NUF2-002 Peptide code R F L s G I I N FSEQ ID 19 Variants Y L IY LY I Position 1 2 3 4 5 6 7 8 9PLK4-001 Peptide code Q Y THE s R F V Q LSEQ ID 20 Variants F F
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UCHL5-001 Peptide code N Y L P F I M AND L IF FF I Position 1 2 3 4 5 6 7 8 9ATAD2-002 Peptide code K Y L T V K D Y LSEQ ID 21 Variants F F IF FF I Position 1 2 3 4 5 6 7 8 9COL12A1-001 Peptide code V Y N P T P N s LSEQ ID 22 Variants F F IF FF I Position 1 2 3 4 5 6 7 8 9COL6A3-001 Peptide code s Y L Q THE THE N THE LSEQ ID 23 Variants F F IF FF I Position 1 2 3 4 5 6 7 8 9FANCI-001 Peptide code F Y Q P K I Q Q FSEQ ID 24 Variants F L IF LF I Position 1 2 3 4 5 6 7 8 9RSP11-001 Peptide code Y Y K N I G L G FSEQ ID 25 Variants F L IF L
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UCHL5-001 Peptide code N Y L P F I M AND LF I Position 1 2 3 4 5 6 7 8 9ATAD2-001 Peptide code THE Y THE I I K AND AND LSEQ ID 26 Variants F F IF FF I Position 1 2 3 4 5 6 7 8 9ATAD2-003 Peptide code L Y P AND V F AND K FSEQ ID 27 Variants F L IF LF I Position 1 2 3 4 5 6 7 8 9 10 HSP90B1-001 Peptide code K Y N D T F W K AND F SEQ ID 28 Variants F L I F L F I Position 1 2 3 4 5 6 7 8 9 10 SIAH2-001 Peptide code V F D T THE I THE H L F SEQ ID 29 Variants Y L I Y L Y I Position 1 2 3 4 5 6 7 8 9SLC6A6-001 Peptide code V Y P N W THE I G LSEQ ID 30 Variants F F IF FF I Position 1 2 3 4 5 6 7 8 9
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UCHL5-001 Peptide code N Y L P F I M AND LIQGAP3-001 Peptide code V Y K V V G N L LSEQ ID 31 Variants F F IF FF I Position 1 2 3 4 5 6 7 8 9ERBB3-001 Peptide code V Y I AND K N D K LSEQ ID 32 Variants F F IF FF I Position 1 2 3 4 5 6 7 8 9 10 KIF2C-001 Peptide code I Y N G K L F D L L SEQ ID 33 Variants F F I F F F I Position 1 2 3 4 5 6 7 8 9 10 CDC2-001 Peptide code L Y Q I L Q G I V F SEQ ID 1 Variants F L I F L F I Position 1 2 3 4 5 6 7 8 9ASPM-002 Peptide code s Y N P L W L R ISEQ ID 2 Variants F L FF LF F Position 1 2 3 4 5 6 7 8 9UCHL5-001 Peptide code N Y L P F I M AND LSEQ ID 3 Variants F
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UCHL5-001 Peptide code N Y L P F I M AND L F IF FF I Position 1 2 3 4 5 6 7 8 9MET-006 Peptide code s Y I D V L P AND FSEQ ID 4 Variants F L IF FF I Position 1 2 3 4 5 6 7 8 9PROM-001 Peptide code s Y I I D P L N LSEQ ID 5 Variants F F IF FF I Position 1 2 3 4 5 6 7 8 9 10 MMP11-001 Peptide code V W s D V T P L T F SEQ ID 6 Variants Y F L I Y L Y I F L F I Position 1 2 3 4 5 6 7 8 9MST1R-001 Peptide code N Y L L Y V s N FSEQ ID 7 Variants F L IF LF I Position 1 2 3 4 5 6 7 8 9NFYB-001 Peptide code V Y T T s Y Q Q I
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UCHL5-001 Peptide code N Y L P F I M AND LSEQ ID 8 Variants F L FF LF F Position 1 2 3 4 5 6 7 8 9SMC4-001 Peptide code H Y K P T P L Y FSEQ ID 9 Variants F L IF LF I Position 1 2 3 4 5 6 7 8 9 10 UQCRB-001 Peptide code Y Y N THE THE G F N K L SEQ ID 10 Variants F F I F F F I Position 1 2 3 4 5 6 7 8 9PPAP2C-001 Peptide code THE Y L V Y T D R LSEQ ID 11 Variants F F IF FF I Position 1 2 3 4 5 6 7 8 9AVL9-001 Peptide code F Y I s P V N K LSEQ ID 12 Variants F F IF FF I Position 1 2 3 4 5 6 7 8 9NUF2-001 Peptide code V Y G I R L AND H FSEQ ID 13 Variants F L
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UCHL5-001 Peptide code N Y L P F I M AND L IF LF I Position 1 2 3 4 5 6 7 8 9ABL1-001 Peptide code T Y G N L L D Y LSEQ ID 14 Variants F F IF FF I Position 1 2 3 4 5 6 7 8 9MUC-006 Peptide code N Y AND AND T F P H ISEQ ID 15 Variants F F LF FF L Position 1 2 3 4 5 6 7 8 9ASPM-001 Peptide code R Y L W THE T V T ISEQ ID 16 Variants F F LF FF L Position 1 2 3 4 5 6 7 8 9EPHA2-005 Peptide code V Y F s K s AND Q LSEQ ID 17 Variants F F IF FF I Position 1 2 3 4 5 6 7 8 9MMP3-001 Peptide code V F I F K G N Q FSEQ ID 18 Variants Y L IY L
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UCHL5-001 Peptide code N Y L P F I M AND LY I Position 1 2 3 4 5 6 7 8 9NUF2-002 Peptide code R F L s G I I N FSEQ ID 19 Variants Y L IY LY I Position 1 2 3 4 5 6 7 8 9PLK4-001 Peptide code Q Y THE s R F V Q LSEQ ID 20 Variants F F IF FF I Position 1 2 3 4 5 6 7 8 9ATAD2-002 Peptide code K Y L T V K D Y LSEQ ID 21 Variants F F IF FF I Position 1 2 3 4 5 6 7 8 9COL12A1-001 Peptide code V Y N P T P N s LSEQ ID 22 Variants F F IF FF I Position 1 2 3 4 5 6 7 8 9COL6A3-001 Peptide code s Y L Q THE THE N THE LSEQ ID 23 Variants F F IF FF I Position 1 2 3 4 5 6 7 8 9
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UCHL5-001 Peptide code N Y L P F I M AND LFANCI-001 Peptide code F Y Q P K I Q Q FSEQ ID 24 Variants F L IF LF I Position 1 2 3 4 5 6 7 8 9RSP11-001 Peptide code Y Y K N I G L G FSEQ ID 25 Variants F L IF LF I Position 1 2 3 4 5 6 7 8 9ATAD2-001 Peptide code THE Y THE I I K AND AND LSEQ ID 26 Variants F F IF FF I Position 1 2 3 4 5 6 7 8 9ATAD2-003 Peptide code L Y P AND V F AND K FSEQ ID 27 Variants F L IF LF I Position 1 2 3 4 5 6 7 8 9 10 HSP90B1-001 Peptide code K Y N D T F W K AND F SEQ ID 28 Variants F L I F L F I Position 1 2 3 4 5 6 7 8 9 10 SIAH2-001 Peptide code V F D T THE I THE H L F SEQ ID 29 Variants Y
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UCHL5-001 Peptide code N Y L P F I M AND LL I Y L Y I Position 1 2 3 4 5 6 7 8 9SLC6A6-001 Peptide code V Y P N W THE I G LSEQ ID 30 Variants F F IF FF I Position 1 2 3 4 5 6 7 8 9IQGAP3-001 Peptide code V Y K V V G N L LSEQ ID 31 Variants F F IF FF I Position 1 2 3 4 5 6 7 8 9ERBB3-001 Peptide code V Y I AND K N D K LSEQ ID 32 Variants F F IF FF I Position 1 2 3 4 5 6 7 8 9 10 KIF2C-001 Peptide code I Y N G K L F D L L SEQ ID 33 Variants F F I F F F I
[00293] Longer peptides may also be convenient. It is also possible that MHC class I epitopes, although normally between 8-11 amino acids in length, are generated by peptide processing of longer peptides or proteins that
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116/174 include the actual epitope. It is preferred that residues that border the actual epitope are residues that do not substantially affect the proteolytic cleavage necessary to expose the actual epitope during processing.
[00294] Thus, the present invention also provides peptides and variants, wherein the peptide or variants of MHC class I epitopes, have a total length between 8 and 100, preferably between 8 and 30, and most preferred between 8 and 14 , that is, 8, 9, 10, 11, 12, 13, 14 amino acids.
[00295] Naturally, the peptide or a variant according to the present invention will have the ability to bind to a molecule of the main human histocompatibility complex (MHC) class I. The binding of a peptide or a variant to an MHC complex can be tested by methods known in the art.
[00296] In a particularly preferred embodiment of the invention the peptide consists or essentially consists of an amino acid sequence according to SEQ ID NO: 1 to SEQ ID NO: 95.
[00297] Consisting essentially of will mean that a peptide according to the present invention, in addition to the sequence according to any SEQ ID NO: 1 to SEQ ID NO: 95 or a variant thereof contains terminally additional N and / or C extensions localized amino acids that are not necessarily part of the peptide that functions as an epitope for the epitope of MHC molecules.
[00298] However, these extensions can be important to provide an efficient introduction of the peptide - according to the present invention - into cells. In one embodiment of the present invention, the peptide is a fusion protein that includes, for example, the N 80-terminal amino acids of the antigen associated with the HLA-DR invariant chain (p33, hereinafter simply Ii) as derived from the NCBI, Accession number GenBank X00497.
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[00299] Furthermore, the peptide or variant can be further modified to improve stability and / or bind to MHC molecules to elicit a stronger immune response. Methods for such optimization of a peptide sequence are well known in the art and include, for example, the introduction of reverse peptide bonds or non-peptide bonds.
[00300] In inverse peptide-linked amino acid residues are not joined by peptide (-CO-NH-) connections, but the peptide bond is reversed. Such retro-inverse peptideomimetics can be created using methods known in the art, for example, such as those described in Meziere et al (1997) J. Immunol. 159, 32303237, incorporated herein by reference. This improvement involves the creation of pseudopeptides containing changes involving the vertebra, and not the orientation of lateral currents. Meziere et al (1997) shows that for MHC binding and helper T cell responses, these pseudopeptides are useful. Retro-reverse peptides, which contain NH-CO bonds rather than CO-NH peptide bonds, are much more resistant to proteolysis.
[00301] A non-peptide bond is, for example, -CH2-NH, CH2S-, -CH2CH2-, -CH = CH-, -COCH2-, -CH (OH) CH2-, and -CH2SO-. United States Patent 4,897,445 provides a method for the solid synthesis of non-peptide binding phase (-CH2-NH) in polypeptide streams that involve polypeptides synthesized by standard procedures and the non-peptide bond synthesized by reacting with an aldehyde amino and an amino acid in the presence of NaCNBH3.
[00302] The peptides constituting the sequences described above can be synthesized with additional chemical groups present at their amino and / or carboxylic terminals, to improve the stability, bioavailability and / or affinity of the peptides. For example, groups
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Hydrophobic 118/174 such as carbobenzoxyl, dansil, or tbutyloxycarbonyl groups can be added to the amino termini of the peptides. Likewise, an acetyl group or a group of 9 fluorenylmethoxy-carbonyl can be placed on the amino termini of the peptides. In addition, the hydrophobic group, t-butyloxycarbonyl or amino group can be added to the amino termini of the peptides.
[00303] Furthermore, the peptides of the invention can be synthesized in such a way that their steric configuration is altered. For example, the D-isomer of one or more of the peptide's amino acid residues can be used, instead of the normal L-isomer. Furthermore, at least one of the amino acid residues of the peptides of the invention can be replaced by one of the known amino acid residues that occur non-naturally. Changes such as these can serve to increase the stability, bioavailability and / or binding action of the peptides of the invention.
Similarly, a peptide or variant of the invention can be modified chemically by reacting with specific amino acids before or after synthesis of the peptide. Examples for such modifications are well known in the art and are summarized, for example, in R. Lundblad, Chemical Reagents for Protein Modification, 3rd ed. CRC Press, 2005, which is incorporated by reference. Chemical amino acid modification includes, but is not limited to, acylation modification, amidination, lysine pyridoxylation, reductive alkylation, trinitrobenzylation of amino groups with 2,4,6-trinitrobenzene sulfonic acid (TNBS), modification of starch groups carboxyl and sulfhydryl modification by acidic performatic oxidation of cysteine, cystic acid, formation of mercurial derivatives, formation of disulfide mixed with other thiol compounds, reaction with maleimide, carboxymethylation with iodoacetic acid or
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119/174 iodoacetamide and carbamoylation with cyanide at alkaline pH, although without limitation. In this regard, the knowledgeable person is referred to Chapter 15 of Current Protein Science Protocols, Eds. Coligan et al. (John Wiley and Sons NY 1995-2000) for more extensive methodology related to the chemical modification of proteins.
[00305] In summary, the modification of arginyl residues in proteins is often based on the reaction of vicinal dicarbonyl compounds such as phenylglyoxal, butanedione 2,3 and cyclohexanedion 1,2 to form an adult. Another example is the reaction of methylglioxal with arginine residues. Cysteine can be modified without concomitant modification of other nucleophilic sites such as histidine and lysine. As a result, a large number of reagents are available for modifying cysteine. The companies' websites, such as Sigma-Aldrich's (http://www.sigmaaldrich.com), provide information on specific reagents.
[00306] Selective reduction of disulfide bonds in proteins is also common. Disulfide bonds can be formed and can be oxidized during heat treatment of biopharmaceuticals.
[00307] Woodward's reagent K can be used to modify specific glutamic acid residues. N- (3- (dimethylamino) propyl) -N 'ethylcarbodiimide can be used to form intramolecular cross-links between a lysine residue and a glutamic acid residue.
[00308] For example, diethylpyrocarbonate is a reagent for the modification of histidyl residues in proteins. Histidine can also be modified to use 4-hydroxy-2-nonenal.
[00309] The reaction of lysine residues and other α-amino groups is, for example, useful in binding peptides to surfaces or in cross-linking proteins / peptides. Lysine is the poly (ethylene) glycol attachment site and the primary site of modification in protein glycosylation.
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[00310] Methionine residues in proteins can be modified with, for example, iodoacetamide, bromoethylamine, and chloramine T.
[00311] Tetranitromethane and N-acetylimidazole can be used for the modification of tyrosyl residues. Cross-linking through the formation of dithyrosine can be accomplished with hydrogen peroxide / copper ions.
[00312] Recent studies in the modification of tryptophan used Nbromosuccinimide, 2-hydroxy-5-nitrobenzyl or 3-bromo-3-methyl-2- (2-nitrophenylmercapto) -3H-indole (BPNS-eschatol) bromide.
[00313] Successful modification of therapeutic proteins and peptides with PEG is often associated with an extension of circulatory half-life whereas a cross-linking of proteins with glutaraldehyde, polyethylene glycol diacrylate and formaldehyde is used for the preparation of hydrogels. Chemical modification of allergens for immunotherapy is often carried out by carbamylation with potassium cyanide.
[00314] A peptide or variant, in which the peptide is modified or includes non-peptide bond is a preferred embodiment of the invention. Generally, peptides and variants (at least those containing peptide connections between amino acid residues) can be synthesized by the Fmoc-polyamido solid-phase peptide synthesis method as described by Lu et al. (1981) and references contained therein. Temporary N-amino group protection is provided by the 9-fluorenylmethyloxycarbonyl (Fmoc) group. Repetitive cleavage of this highly labile protecting group is ugly using 20% piperidine in N, N-dimethylformamide. The side current functionalities can be protected as its butyl ethers (in the case of serine threonine and tyrosine), butyl esters (in the case of glutamic acids and aspartic acids), butyloxycarbonyl derivative (in the case of histidine and
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121/174 lysine), derived from trityl (in the case of cysteine) and derived from 4-methoxy-2, 3,6-trimethylbenzenesulfonyl (in the case of arginine). Where glutamine or asparagine are C-terminal residues, use is made of the 4,4'-dimethoxybenzhydryl group to protect the side functions of current starch. The solid phase support is based on a polydimethyl acrylamide polymer consisting of the three dimethylacrylamide monomers (spine-monomer), bisacrylethylene diamine (crosslinker) and acryloylsarcosine methyl ester (functionalizing agent). The peptide-to-resin bound cleavage agent used is acid-labile hydroxymethyl-phenoxyacetic derivative. All amino acid derivatives are added as their preformed symmetric anhydride derivatives with the exception of asparagine and glutamine, which are added using an inverted procedure mediated by N, N-dicyclohexyl carbodiimide / 1hydroxybenzotriazole. All deprotection coupling reactions are controlled using ninhydrin, trinitrobenzene sulfonic acid or isotine testing procedures. Upon completion of synthesis, peptides are split from the resin backing with concomitant side-current removal protecting the groups by treatment with 95% trifluoroacetic acid containing a 50% radical interceptor mixture. Radical interceptors commonly used include etandythiol, phenol, anisole and water, the exact choice depending on the constituent amino acids of the peptide to be synthesized. A combination of solid phase and solution phase methodologies for peptide synthesis is also possible (see, for example, (Bruckdorfer, Marder, and Albericio 29-43) and the references cited there).
[00315] The trifluoroacetic acid is removed by evaporation in vacuo, with subsequent trituration with diethyl ether providing the crude peptide. Any radical interceptors present are removed by a simple extraction procedure in which the freeze-drying phase
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Aqueous 122/174 provides rough peptide from radical interceptors. Reagents for peptide synthesis are generally available from, for example, Calbiochem-Novabiochem (United Kingdom) Ltd., Nottingham NG7 2QJ, United Kingdom.
[00316] Purification can be performed by anyone, or a combination of techniques such as recrystallization, size exclusion chromatography, ion-exchange chromatography, hydrophobic interaction chromatography and (normally) high performance liquid chromatography in the phase reverse using, for example, acetonitrile / water slope separation.
[00317] Peptide analysis can be performed using thin layer chromatography, electrophoresis, in a private tube capillary electrophoresis, solid phase extraction (CSPE), high performance liquid chromatography in the reverse phase, amino acid analysis after hydrolysis acid and by rapid atom bombardment (FAB) mass spectrometric analysis, as well as MALDI and ESI-Q-TOF mass spectrometric analysis.
[00318] Another aspect of the invention provides a nucleic acid (for example, a polynucleotide) encoding a peptide or variant of the invention. The polynucleotide can be, for example, DNA, cDNA, PNA, CNA, RNA or combinations thereof, either single-stranded or double-stranded, or native or stabilized forms of polynucleotide, such as, for example, polynucleotides with a phosphorothioate spine , and may or may not contain introns as long as it encodes for the peptide. Naturally, only peptides containing naturally occurring amino acid residues are joined by naturally occurring peptide bonds, which can be encoded by a polynucleotide. An even more aspect of the invention provides an expression vector capable of expressing a polynucleotide according to the invention.
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[00319] A variety of methods have been developed to link polynucleotides, especially DNA, to vectors, for example, via complementary cohesive termini. For example, complementary homopolymer areas can be added to the DNA segment to be inserted into the DNA vector. The vector and DNA segment are then joined by hydrogen link between complementary homopolymer tails to form recombinant DNA molecules.
[00320] Synthetic linkers containing one or more restriction sites provide an alternative method of joining the DNA segment to vectors. Synthetic linkers containing a variety of restriction endonuclease sites are commercially available from a number of sources including International Biotechnologies Inc., New Haven, CN, USA.
[00321] A desirable method of modifying DNA - encoding the polypeptide of the invention - employs the polymerase chain reaction as described by (Saiki et al. 487-91). This method can be used to introduce DNA into a suitable vector, for example, by engineering at suitable restriction sites, or it can be used to modify DNA in other useful ways as is known in the art. If viral vectors are used, chickenpox or adenovirus vectors are preferred.
[00322] DNA (or in the case of retroviral vectors, RNA) can be expressed in a convenient host to produce a polypeptide constituting the peptide or variant of the invention. Thus, the DNA encoding the peptide or variant of the invention can be used according to known techniques, suitably modified in view of the teachings contained therein, to construct an expression vector, which is then used to transform an appropriate host cell for expression. and producing the polypeptide of the invention. Such techniques
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124/174 include those disclosed in US Patent No. 4440859, 4530901, 4582800, 4677063, 4678751,4704362, 4710463, 4757006, 4766075 and 4810648.
[00323] The DNA (or in the case of retroviral vectors, RNA) encoding the polypeptide constituting the compound of the invention can be joined to a wide variety of other DNA sequences for introduction into an appropriate host. The companion DNA will depend on its nature of the host, the manner in which the DNA is introduced into the host, and whether episomal maintenance or integration is desired.
[00324] Generally, DNA is inserted into an expression vector, such as a plasmid, in proper and corrected orientation by reading the frame for expression. If necessary, the DNA can be linked to the appropriate transcriptional and translational control nucleotide regulatory sequences recognized by the desired host, although such controls are generally available in the expression vector. The vector is then introduced into the host using standard techniques. Generally, not all hosts will be transformed by the vector. Therefore, it will be necessary to select for transformed host cells. A selection technique involves incorporating a DNA sequence into the expression vector, with any necessary control elements, which code for a selectable characteristic in the transformed cell, such as antibiotic resistance.
[00325] On the other hand, the gene for such a selectable trait may be in another vector, which is used to transform the desired host cell.
[00326] Host cells that have been transformed by the recombinant DNA of the invention are then cultured for a sufficient time and under appropriate conditions known to those
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125/174 professionals wearing the technique in view of the teachings described here to allow expression of the polypeptide, which can then be recovered.
[00327] Many expression systems are known, including bacteria (for example, E. coli and Bacillus subtilis), yeasts (for example, Saccharomyces cerevisiae), filamentous fungi (for example, Aspergillus spec.), Plant cells, animal cells and insect cells. Preferably, the system may be mammalian cells such as CHO cells available from the ATCC Cell Biology Collection.
[00328] A typical mammalian cell vector plasmid for constitutive expression constitutes the CMV or SV40 promoter with a convenient A-tail pole and a resistance marker, such as neomycin. An example is pSVL available from Pharmacia, Piscataway, NJ, USA. An example of an inducible mammalian expression vector is pMSG, also available from Pharmacia. Useful plasmid yeast vectors are pRS403-406 and pRS413-416 and are generally available from Stratagene Cloning Systems, La Jolla, CA 92037, USA. Plasmids pRS403, pRS404, pRS405 and pRS406 are yeast-integrating plasmids (YIps) and incorporate selectable yeast markers HIS3, TRP1, LEU2 and URA3. Plasmids pRS413416 are yeast centromere plasmids (Ycps). CMV promoter-based vectors (for example, those from Sigma-Aldrich) provide transient or stable expression, cytoplasmic expression or secretion, and N-terminus or C-terminus labeling in various combinations of FLAG, 3xFLAG, c-myc or MAT. These fusion proteins will allow the detection, treatment and analysis of the recombinant protein. Double-label fusions provide flexibility in detection.
[00329] The strong human cytomegalovirus regulatory region
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126/174 (CMV) generates high levels of constitutive protein expression as high as 1 mg / L in COS cells. For less potent cell lines, protein levels are typically ~ 0.1 mg / L. The presence of the SV40 origin of replication will result in high levels of DNA replication in the SV40 replication COS permissive cells. CMV vectors, for example, may contain the pMB1 (derived from pBR322) origin for replication in bacterial cells, the blactamase gene for selection of ampicillin resistance in bacteria, polyA hGH, and the f1 origin. Vectors containing the preprotripsin sequence leader (PPT) can direct the secretion of FLAG fusion proteins into the culture medium for purification using ANTIFLAG antibodies, resins, and dishes. Other vectors and expression systems are well known in the art for use with a variety of host cells.
[00330] The present invention also relates to a host cell transformed with a polynucleotide vector construct of the present invention. The host cell can be either prokaryotic or eukaryotic. Bacterial cells may preferably be prokaryotic host cells and, in some circumstances, typically an E. coli strain such as, for example, E. coli DH5 strains available from Bethesda Research Laboratories Inc., Bethesda, MD, USA, and RR1 available from the American Type Culture Collection (ATCC) of Rockville, MD, USA (ATCC 31343). Preferred host eukaryotic cells include yeast, insect and mammalian cells, preferably vertebrate cells such as those of a mouse, rat, monkey or human fibroblast cells and colon cell lines. Yeast host cells include YPH499, YPH500 and YPH501 which are generally available from Stratagene Cloning Systems, La Jolla, CA 92037, USA. Preferred mammalian host cells include
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127/174 Chinese hamster ovary (CHO) cells available from ATCC as CCL61, NIH / 3T3 from NIH Swiss mouse embryo cells available from CRCC 1658, monkey kidney-derived COS-1 cells available from ATCC as CRL 1650 and 293 cells that are human kidney embryonic cells. Preferred insect cells are Sf9 cells that can be transfected with baculovirus expression vectors. An overview regarding the choice of suitable host cells for expression can be found in, for example, the text by Paulina Balbás and Algeria Lorence Methods in Molecular Biology Recombinant Gene Expression, Reviews and Protocols, Part One, Second Edition, ISBN 978- 158829-262-9, and other literature known to the knowledgeable person.
[00331] Transformation of appropriate host cells with a DNA construct of the present invention is accomplished by well-known methods that typically depend on the type of vector used. With regard to the transformation of cells from the prokaryotic host, see, for example, Cohen et al. (1972) Proc. Natl. Acad. Sci. USA 69, 2110, and Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Transformation of yeast cells is described in Sherman et al. (1986) Methods In Yeast Genetics, A Laboratory Manual, Cold Spring Harbor, NY. The Beggs (1978) Nature 275,104-109 method is also useful. With reference to vertebrate cells, reagents useful in transferring such cells, for example, calcium phosphate and DEAE-dextran or liposome formulations, are available from Stratagene Cloning Systems, or Life Technologies Inc., Gaithersburg, MD 20877, USA. Electroporation is also useful for transfecting and / or transforming cells and is well known in the art for transforming yeast cells, bacterial cells, insect cells and vertebrate cells.
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[00332] Cells successfully transformed, i.e., cells containing a DNA construct of the present invention, can be identified by well-known techniques such as PCR. On the other hand, the presence of the protein in the supernatant can be detected using antibodies.
[00333] It will be appreciated that certain host cells of the invention are useful in the preparation of the peptides of the invention, for example, bacterial, yeast and insect cells. However, other host cells can be useful in certain therapeutic methods. For example, cells that have antigens, such as dendritic cells, can be sensibly used to express the peptides of the invention in such a way that they can be loaded into appropriate MHC molecules. Thus, the current invention provides a host cell constituting a nucleic acid or an expression vector according to the invention.
[00334] In a preferred embodiment, the host cell is a cell that has antigen, in particular a dendritic cell or cell that has antigen. APCs loaded with a recombinant fusion protein containing prostatic acid phosphatase (PAP) are currently under investigation for the treatment of prostate cancer (Sipuleucel-T) (Rini et al. 67-74; Small et al. 3,089-94).
[00335] Another aspect of the invention provides a method of producing a peptide or its variant, which constitutes the culture of a host cell that isolates the peptide from the host cell or its culture medium.
[00336] In another embodiment the peptide, nucleic acid or expression vector of the invention are used in medicine. For example, the peptide or its variant can be prepared for intravenous injection (i.v.), subcutaneous injection (s.c.), intradermal injection (i.d.), intraperitoneal injection (i.p.), intramuscular injection (i.m.). Methods
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Preferred peptide injection methods include s.c., i.d., i.p., i.m., and i.v. Preferred methods of DNA injection include i.d., i.m., s.c., i.p. and i.v. Doses, for example, between 50 pg and 1.5 mg, preferably 125 pg to 500 pg of peptide or DNA can be given and will depend on the respective peptide or DNA. The doses of this range have been used successfully in previous tests (Brunsvig et al. 1553-64; Staehler et al.).
[00337] Another aspect of the present invention includes an in vitro method for the production of activated T cells, the method comprising contacting T cells in vitro with human MHC molecules expressed on the surface of a suitable cell presenting antigen for a period of time sufficient to activate the T cell in a specific antigenic manner, in which said antigen is a peptide according to the invention. Preferably a sufficient amount of the antigen is used with an antigen presenting cell.
[00338] Preferably the mammalian cell lacks or has a reduced level of the function of the TAP peptide transporter. Convenient cells that lack a TAP peptide transporter include T2, RMA-S and drosophil cells. TAP is the carrier associated with antigen processing.
[00339] The human peptide carrying defective T2 cell line is available from the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, USA under Catalog No CRL 1992; the Schneider drosophila cell line is available from the ATCC under Catalog No CRL 19863; the mouse RMA-S cell line is described in Karre et al1985.
Preferably, prior to transfection, the host cell substantially does not express any MHC class I molecules. It is also preferable that the stimulator cell expresses an important molecule to provide a co-stimulating signal from
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130/174 T cells, like any B7.1, B7.2, ICAM-1 and LFA 3. The nucleic acid sequences of numerous MHC class I molecules and co-stimulating molecules are publicly available from the database GenBank and EMBL.
[00341] In the case of a class I MHC epitope being used as an antigen, T cells are CD8 positive CTL cells.
[00342] If an antigen presenting cell is transfected to express such an epitope, preferably the cell constitutes an expression vector capable of expressing a peptide containing SEQ ID NO: 1 to SEQ ID NO: 95 or the variant amino acid sequence.
[00343] A variety of other methods can be used to generate CTL in vitro. For example, the methods described in Peoples and others (1995) and Kawakami and others (1992) use autologous lymphocytes with tumor infiltration in the generation of CTL. Plebanski et al. (1995) uses peripheral autologous blood lymphocytes (PLBs) in the preparation of CTL. Jochmus et al. (1997) describes the production of autologous CTLs pulsating dendritic cells with peptide or polypeptide, or via infection with recombinant viruses. Hill et al (1995) and Jerome et al (1993) use B cells in the production of autologous CTL. In addition, macrophages pulsed with peptide or polypeptide, or infected with recombinant virus, can be used in the preparation of autologous CTL. S. Walter et al. 2003 describes the in vitro preparation of T cells using artificial antigen presenting cells (aAPCs), which is also a convenient way to generate T cells against the peptide of choice. In this study, aAPCs were generated by preforming MHC coupling: peptide complexes on the surface of polystyrene particles (microflanges) by biotin: streptavidin biochemistry. This system allows for accurate control of MHC density in aAPCs, which selectively elicits specific high or low avidity responses from T cell antigen with high efficiency.
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131/174 blood samples. Apart from MHC: peptide complexes, aAPCs must carry other proteins with co-stimulant activity such as anti-CD28 antibodies attached to their surface. Furthermore, such aAPC-based systems often require the addition of appropriate soluble factors, for example cytokines such as interleukin-12.
[00344] Halogenic cells can also be used in the preparation of T cells in a method described in detail in WO 97/26328, incorporated herein by reference. For example, in addition to drosophil cells and T2 cells, other cells can be used to present antigens such as CHO cells, baculovirus-infected insect cells, bacteria, yeast, infected vaccinia target cells. In addition, plant viruses can be used (see, for example, Porta et al. (1994)) which describes the development of cowpea mosaic virus as a high-throughput system for the presentation of foreign peptides.
[00345] Activated T cells that are directed against the peptides of the invention are useful in therapy. Thus, a further aspect of the invention provides activated T cells obtainable by the preceding methods of the invention.
[00346] Activated T cells, which are produced by the above method, selectively recognize a cell that aberrantly expresses a polypeptide comprising an amino acid sequence of SEQ ID NO: 1 to 95.
[00347] Preferably, the T cell recognizes the cell by interacting through its TCR with the HLA / peptide complex (e.g., ligand). T cells are useful in a method of eliminating target cells in a patient whose target cells aberrantly express a polypeptide constituting an amino acid sequence of the invention, in which the patient is administered a
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132/174 effective number of activated T cells. The T cells that were administered to the patient can be derived from the patient and activated as described above (ie, they are autologous T cells). Alternatively, T cells are not from the patient, but from another individual. Of course, it is preferred if the individual is a healthy individual. With a healthy individual the inventors mean that the individual is generally in good health, preferably with a competent immune system and, more preferably, does not suffer from any disease that can be readily tested and detected.
[00348] In vivo, the target cells for CD8 positive T cells according to the present invention can be tumor cells (which sometimes express MHC class I) and / or stromal cells around the tumor (tumor cells) (which sometimes also expresses MHC class I; (Dengjel et al. 4163-70)).
[00349] The T cells of the present invention can be used as active ingredients in a therapeutic composition. Thus, the invention also provides a method of killing target cells in a patient whose target cells aberrantly express a polypeptide composed of an amino acid sequence of the invention, the method comprising administering to the patient an effective number of T cells, as defined previously.
[00350] With aberrantly expressed the inventors also mean that the polypeptide is overexpressed compared to normal levels of expression or that the gene is silent in the tissue from which the tumor was derived, but in the tumor it is expressed. By overexpressed the inventors mean that the polypeptide is present at a level at least 1.2 times that present in normal tissue; preferably at least 2 times, and more preferably at least 5 times or 10 times the current level present in normal tissue.
[00351] T cells can be obtained by methods known in the art.
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133/174 art, for example, those described above.
[00352] The protocols for this adoptive transfer of so-called T cells are well known in the art and can be found, for example, in (Dudley and others 850-54; Dudley and others 2346-57; Rosenberg and others 889-97; Rosenberg et al 1676-80; Yee et al 16168-73); revised in (Gattinoni and others 383-93) and (Morgan and others).
[00353] Any molecule of the invention, that is, the peptide, nucleic acid, expression vector, cell, activated CTL, T cell receptor or nucleic acid coding is useful for the treatment of disorders, characterized by cells escaping from a immune response. Therefore, any molecule of the present invention can be used as a medicine or for the manufacture of a medicine. The molecule can be used by itself or in combination with other molecule (s) of the invention or (a) known molecule (s).
[00354] Preferably, the medicament of the present invention is a vaccine. It can be administered directly to the patient, in the affected organ or systematically i.d., i.m., s.c., i.p. and i.v., either applied ex vivo to cells derived from the patient or a human cell line that were subsequently administered to the patient, or used in vitro to select a subpopulation of immune cells derived from the patient, which are then readministered to the patient. If nucleic acid is administered to cells in vitro, it may be useful for cells if they are transfected in such a way as to coexpress immunostimulating cytokines, such as interleukin-2. The peptides can be substantially pure, or combined with an immunostimulatory adjuvant (see below), or used in combination with immunostimulatory cytokines, or be administered with a suitable delivery system, for example, liposomes. THE
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134/174 peptide can also be conjugated to an appropriate vehicle, such as the mollusk lock hemocyanin (KLH) or mannan (see WO 95/18145 and Longenecker 1993). The peptide can also be labeled, be a fusion protein or a hybrid molecule. The peptides whose sequences are given in the present invention are expected to stimulate CD4 or CD8 T cells. However, stimulation of CD8 CTLs is more efficient when there is help from CD4 helper T cells. Thus, for Class I MHC epitopes that stimulate CD8 CDL, the fusion partner or sections of a suitable hybrid molecule provides epitopes that stimulate CD4 positive T cells. Epitopes that stimulate CD4 and CD8 are well known in the art, and include those identified in the present invention.
[00355] In one aspect, the vaccine comprises at least one peptide having the amino acid sequence set out in SEQ ID NO: 1 to 33 and at least one additional peptide, preferably two to 50 years, more preferably two to 25 years , even more preferably two to 15 and even more preferably two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or thirteen peptides. The peptide (s) can be derived from one or more specific TAAs and can bind class I MHC molecules.
[00356] The polynucleotide can be substantially pure, or be contained in a convenient vector or delivery system. The nucleic acid can be DNA, cDNA, PNA, CNA, RNA or combinations thereof. Methods for designing and introducing a nucleic acid are well known in the art. An overview is provided, for example, by (Pascolo et al. 117-22). Polynucleotide vaccines are easy to prepare, but the mode of action of these vectors in inducing an immune response is not fully understood. Convenient vectors and delivery systems include viral DNA from and / or
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RNA, such as adenovirus-based systems, vaccinia virus, retrovirus, herpes virus, adeno-associated virus or hybrids containing elements from more than one virus. Non-viral delivery systems include cationic lipids and cationic polymers and are well known in the art of DNA delivery. Physical delivery, such as via a gene-gun, can also be used. The peptide or peptides encoded by the nucleic acid can also be a fusion protein, for example, with an epitope that stimulates T cells to the respective opposite CDR as noted above.
[00357] The medicament of the invention can also include one or more adjuvants. Adjuvants are substances that do not specifically enhance or enhance the immune response (for example, immune responses mediated by CTLs and helper T cells (Th)) to an antigen, and will therefore have to be considered useful for the drug of the present invention. Suitable adjuvants include, but are not limited to, 1018 ISS, aluminum salts, Amplivax®, AS15, BCG, CP-870893, CpG7909, CyaA, dSLIM, flagellin or TLR5 flagellin derivatives, FLT3 ligand, GM-CSF, IC30 , IC31, Imiquimod (ALDARA®), resiquimod, ImuFact IMP321, Interleukins such as IL-2, IL-13, IL-21, interferon-alpha-or beta, or pegylated, their derivatives, IS Patch, ISS, ISCOMATRIX, ISCOMs, JuvImmune, LipoVac, MALP2, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, water-in-oil and oil-in-water emulsions, OK-432, OM-174, OM-197 - MP-CE, ONTAK, OspA, PepTel® vector system, poly (lactide co-glycolide), microparticles based on [PLG] and dextram, talactoferrin SRL172, Virosomes and other viruses such as particles, YF-17D, VEGF trap, R848, beta-glucan, Pam3Cys, Aquila's QS21 stimulon, which is obtained from saponin, mycobacterial bacterial extracts and mimic cell wall synthesis, and other proprietary adjuvants rivers like
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Ribi's Detox, Quil, or Superfos. Adjuvants, such as Freund's or GM-CSF, are preferred. Various immunological adjuvants (eg, MF59) specific for dendritic cells and their preparations have been described previously (Allison and Krummel 93233). It is also possible to use cytokines. Several cytokines have been directly linked to the dendritic cell that influences migration to lymphoid tissues (eg, TNF), accelerating the maturation of dendritic cells in efficient antigen-presenting cells for T lymphocytes (eg, GM-CSF, IL- 1 and IL -4) (US Pat. No. 5849589, specifically incorporated herein by reference in its entirety) and acting as immunoadjuvants (for example, IL-12, IL-15, IL-23, IL-7, IFN-alpha IFN-beta) [Gabrilovich 1996].
[00358] CpG immunostimulatory oligonucleotides have also been reported to improve the effects of adjuvants in making a vaccine. Without being bound by theory, CpG oligonucleotides act by activating the innate (non-adaptive) immune system, through toll-like receptors (TLR), mainly TLR9. TLR9 activation of triggered CpG increases antigen-specific humoral and cellular responses to a wide variety of antigens, including peptide or protein antigens, live or dead viruses, dendritic cell vaccines, autologous cell vaccines and conjugated polysaccharides in both prophylactic vaccines and therapeutic. More importantly, it reinforces the maturation and differentiation of dendritic cells, resulting in a greater activation of Th1 cells and a strong generation of cytotoxic T lymphocytes (CTL), even in the absence of the help of CD4 T cells. The Th1 bias induced by TLR9 stimulation is maintained even in the presence of vaccine adjuvants, such as aluminum or Freund's incomplete adjuvant (IFA), which normally promotes a Th2 bias. CPG oligonucleotides show an even greater activity of
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137/174 adjuvant when formulated or co-administered with other adjuvants or in formulations such as microparticles, nanoparticles, lipid emulsions or similar formulations, which are especially necessary to induce a strong response when the antigen is relatively weak. They also speed up the immune response and allow antigen doses to be reduced by approximately two orders of magnitude, with comparable antibody responses to the full-dose vaccine without CpG in some experiments (Krieg 471-84). US Pat. No. 6,406,705 B1 describes the combined use of CPG oligonucleotides, non-nucleic acid adjuvants and an antigen to induce an antigen-specific immune response. A CPR TLR9 antagonist is the dSLIM (double Stem Loop Immunomodulator) by Mologen (Berlin, Germany), which is a preferred component of the pharmaceutical composition of the present invention. Other molecules that bind to TLR, such as TLR 7, TLR 8 and / or TLR 9 that bind to RNA can also be used.
[00359] Other examples of useful adjuvants include, but are not limited to chemically modified CpGs (eg, CPR, Idera), dsRNA analogs, such as Poly (I: C) and derivatives thereof (eg, AmpliGen®, Hiltonol® , poly- (ICLC), poly (IC-R), poly (I: C12U), DNAAmpliGen, DNA or non-CPG bacterial RNA, as well as small molecules and immunoactive antibodies such as cyclophosphamide, sunitiniba, Bevacizumab, Celebrex, NCX -4016, sildenafil, tadalafil, vardenafil, Sorafenib, temozolomide, temsirolimus, XL-999, CP547632, pazopanib, VEGF Trap, ZD2171, AZD2171, anti-CTLA4, other antibodies attacking key structures of the immune system (for example, anti-CD40, anti-TGFbeta, anti-TNFalpha receptor) and SC58175, which can act therapeutically and / or as an adjuvant The amounts and concentrations of adjuvants and additives useful in the context of the present invention can be easily determined
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138/174 by qualified craftsman without experimentation.
[00360] Preferred adjuvants are imiquimod, resiquimod, GM-CSF, cyclophosphamide, sunitinib, bevacizumab, interferon-alpha, CpG oligonucleotides and derivatives, poly- (I: C) and derivatives, RNA, sildenafil, and particulate formulations with PLG or virosomas.
[00361] In a preferred variant, the pharmaceutical composition according to the invention, the adjuvant is selected from the group of colony stimulating factors, such as granulocyte and macrophage colony stimulating factor (GM-CSF, Sargramostim), imiquimodod , resiquimod, and interferon-alpha.
[00362] In a preferred variant, the pharmaceutical composition according to the invention, the adjuvant is selected from the group of colony stimulating factors, such as granulocyte and macrophage colony stimulating factor (GM-CSF, Sargramostim), imiquimodo and resimiquimodo.
[00363] In a preferred variant, the pharmaceutical composition according to the invention, the adjuvant is imiquimod or resiquimod.
[00364] The composition is used for parenteral administration, for example, subcutaneously, intradermally or intramuscularly or for oral administration. For this, the peptides and other optional molecules are dissolved or suspended in a vehicle in acceptable pharmaceutical form, preferably in aqueous form. In addition, the composition may contain excipients, such as buffers, binding agents, disintegrating agents, diluents, flavorings, lubricants, etc. Peptides can also be administered together with immunostimulating substances such as cytokines. A complete list of excipients usable in this composition can be obtained, for example, from A. Kibbe, Handbook of Pharmaceutical Excipients, 3a. 2000, American Pharmaceutical Association and the pharmaceutical press. The composition can be used for prevention,
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139/174 prophylaxis and / or therapy of adenomatous or cancerous diseases. Exemplary formulations can be found in EP2113253.
[00365] The present invention provides a drug which is useful in treating cancer, in particular gastric cancer, renal cell, colon carcinoma, non-small cell lung carcinoma, adenocarcinoma, prostate cancer, benign neoplasm and malignant melanoma.
[00366] In addition, the present invention also includes a kit consisting of:
(a) a container containing a pharmaceutical composition, as described above, in solution or in lyophilized form;
(b) optionally, a second container containing a diluent or reconstituting solution for the lyophilized formulation; and (c) optionally, instructions for (i) use of the solution or (ii) reconstitution and / or use of the lyophilized formulation.
[00367] The kit can also include one or more buffers (iii), a diluent (iv), a filter (v), a needle (vi), or a syringe (v). The container should preferably be a bottle, vial, syringe or test tube, and it can be a multipurpose container. The pharmaceutical composition is preferably lyophilized.
[00368] Kits of the present invention preferably contain a lyophilized formulation of the present invention in a suitable container as well as instructions for reconstitution and / or use. Suitable containers include, for example, bottles, vials (for example, double-chamber vials), syringes (for example, double-chamber syringes) and test tubes. The container can be formed from a variety of materials such as glass or plastic. Preferably, the kit and / or the container contains instructions on the container or similar that indicate the directions
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140/174 for reconstitution and / or use. For example, the label may indicate that the lyophilized formulation should be reconstituted to concentrations of peptides, as described above. The label may also indicate that the formulation is intended or suitable for subcutaneous administration.
[00369] The container that holds the formulation can be a multipurpose bottle, which allows the repetition of administrations (for example, 2-6 administrations), of the reconstituted formulation. The kit can also include a second container, containing a suitable diluent (for example, sodium bicarbonate solution).
[00370] After mixing the diluent and the lyophilized formulation, the concentration at the end of peptide in the reconstituted formulation is preferably at least 0.15 mg / ml / peptide (= 75pg) and preferably not more than 3 mg / ml / peptide (= 1500pg). The kit can also include other materials, as desired from a commercial or user perspective, including other buffers, thinners, filters, needles, syringes, and packaging supplements with instructions for use.
[00371] Kits of the present invention may have a single container containing the formulation of pharmaceutical compositions according to the present invention, with or without other components (for example, other compounds or pharmaceutical compositions of these other compounds), or they may have separate containers for each component. [00372] Preferably, kits of the invention include a packaged formulation of the invention for use in combination with the co-administration of a second compound (as adjuvants (eg GM-CSF), a chemotherapeutic agent, a natural product, a hormone or antagonist, a antiangiogenesis agent or inhibitor, an apoptosis-inducing agent or a chelator) or a pharmaceutical composition thereof. Kit components can be pre
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141/174 complexed or each component can be deposited separately in separate containers before administration to a patient. The kit components can be supplied in one or more liquid solutions, preferably an aqueous solution, even more preferably, a sterile aqueous solution. The kit components can also be supplied as solids, which can be converted into liquids by adding suitable solvents, which should preferably be supplied in a separate container. [00373] The container of a therapeutic kit can be a vial, test tube, container, bottle, syringe, or any other storage medium for solids or liquids. Normally, when there is more than one component, the kit should contain a second bottle or other container, which allows to separate the dosage. The kit can also contain another container for a pharmaceutically acceptable liquid. Preferably, the therapeutic kit should contain an apparatus (for example, one or more needles, syringes, droppers, pipettes, etc.), which allows the correct administration of the agents of the invention that are components of the present kit.
[00374] The present formulation is suitable for the administration of peptides by any acceptable route, such as oral (enteric), nasal, ophthalmic, subcutaneous, intradermal, intramuscular, intravenous or transdermal. Preferably s.c. administration, and more preferably, i.d. Administration can be by infusion pump.
[00375] Since the peptides derived from the invention of MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB and MUC6 have been isolated from gastric cancer, the drug of the invention is preferably used to treat gastric cancer.
[00376] The present invention will now be described in the following examples, which describe its preferred embodiments without being limited, however, to these examples. For the purposes of this
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142/174 invention, all references cited herein become an integral part of this document by reference.
EXAMPLES
EXAMPLE 1:
[00377] Identification of tumor-associated peptides on the cell surface Tissue samples
[00378] Tumor tissues from patients were supplied by the Kyoto Prefectural University of Medicine (KPUM), Kyoto, Japan, and by the Graduate School of Medicine at Osaka City University (OCU), Osaka, Japan, and University Hospital from Tubingen, Germany. Expressed written consent from patients was given prior to surgery. The tissues were frozen by liquid nitrogen shock immediately after surgery and stored until the TUMAPs were isolated at -80 ° C.
Isolation of HLA peptides in tissue samples
[00379] Bowls of HLA peptides from tissue samples frozen by shock were obtained by immune precipitation of solid tissues according to a slightly modified protocol (Falk, K.1991; Seeger, FHT1999) using the specific W6 / 32 antibody HLA-A, HLA-B, HLA-C, the HLA-A * 02 antibody BB7.2, CNBr activated sepharose, acid treatment, and ultrafiltration.
Methods
[00380] The obtained HLA peptide basins were separated according to their hydrophobicity by reversed phase chromatography (nanoAcquity UPLC system, Waters) and the peptides to be diluted were analyzed in an LTQ-Orbitrap hybrid mass spectrometer (ThermoFisher Scientific) equipped with an ESI source. The peptide basins were loaded directly onto a micro-capillary fused silica analytical column (75 pm i.d. x 250 mm) packed with 1.7
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143/174 pm of reversed phase C18 material (Waters), applying a flow rate of 400 nL per minute. Subsequently, the peptides were separated using a 180-minute binary gradient in two steps of 10% to 33% B at a flow rate of 300 nL per minute. The gradient was composed of Solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile). A capillary tube coated with golden glass (PicoTip, New Objective) was used for introduction into the nanoESI source. The LTQOrbitrap mass spectrometer was operated in data dependent mode using a TOP5 strategy. In summary, a scanning cycle was started with a full high accuracy mass scan in the orbitrap (R = 30,000), which was followed by MS / MS scans also in the orbitrap (R = 7500) in the 5 most abundant ions of precursor with the dynamic exclusion of previously selected ions. The tandem mass spectra were interpreted by SEQUEST and by additional manual control. The peptide sequence identified was ensured by comparing the generated natural peptide fragmentation pattern with the fragmentation pattern of a synthetic identical sequence reference peptide. Figure 1 shows an exemplary spectrum obtained from tumor tissue for the MHC class I associated peptide CDC2-001 and its elution profile in the UPLC system.
EXAMPLE 2
[00381] Profile expression of genes encoding the peptides of the invention
[00382] Not all peptides identified as being presented on the surface of tumor cells by MHC molecules are suitable for immunotherapy, because most of these peptides are derived from normal cellular proteins expressed by many cell types. Only some of these peptides are associated with tumor and
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144/174 probably capable of inducing T cells with a high specificity of recognition for the tumor, from which they were derived. In order to identify such peptides and reduce the risk for vaccination-induced autoimmunity, the inventors focused on these peptides, which are derived from proteins that are overexpressed in tumor cells compared to most normal tissues.
[00383] The ideal peptide will be derived from a protein that is rare to the tumor and not present in any other tissue. To identify peptides that are derived from genes with an expression profile similar to the ideal gene, the identified peptides were assigned to the proteins and genes, respectively, from which they were derived, and expression profiles of these genes were generated. RNA sources and preparation
[00384] Surgically removed tissue specimens were provided from different clinical sites (see example 1), after express written consent was obtained from each patient. Tumor tissue specimens were frozen by snapping into liquid nitrogen immediately after surgery and later homogenized with mortar and pestle under liquid nitrogen. Total RNA was prepared from these samples using TRI Reagent (Ambion, Darmstadt, Germany) followed by RNeasy cleaning (QIAGEN, Hilden, Germany); both methods were performed according to the manufacturer's protocol.
[00385] Total healthy human tissue RNA was obtained commercially (Ambion, Huntingdon, United Kingdom; Clontech, Heidelberg, Germany; Stratagene, Amsterdam, Netherlands; BioChain, Hayward, CA, USA). The RNA of several individuals (between 2 and 123 individuals) was mixed, in such a way that the RNA of each individual was equally weighed. Leukocytes were isolated from blood samples from 4 healthy volunteers.
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[00386] The quality and quantity of all RNA samples were evaluated in an Agilent 2100 bioanalyzer (Agilent, Waldbronn, Germany) using the 6000 Pico LabChip kit (Agilent).
Micromatrix experiments
[00387] The gene expression analysis of all RNA samples from tumor and normal tissue was performed by Affymetrix Human Genome (HG) U133A or HGU133 Plus 2.0 micromatrix (Affymetrix, Santa Clara, CA, USA). All steps were performed according to the Affymetrix manual. In summary, double-stranded cDNA was synthesized from 5-8 pg of total RNA, using SuperScript RTII (Invitrogen) and the oligo-dT-T7 primer (MWG Biotech, Ebersberg, Germany) as described in the manual. An in vitro transcription was performed with the BioArray High Yield RNA transcript labeling kit (ENZO Diagnostics, Inc., Farmingdale, NY, USA) for the U133A series or with the GeneChip IVT (Affymetrix) labeling kit for the U133 Plus series 2.0, followed by cRNA fragmentation, hybridization, and stained with streptavidin-phycoerythrin and biotin anti-streptavidin antibody (Molecular Probes, Leiden, Netherlands). The images were scanned with the Agilent 2500A GeneArray scanner (U133A) or the Affymetrix Gene-Chip 3000 scanner (U133 Plus 2.0), and data were analyzed with the GCOS software (Affymetrix), using standard scenarios for all parameters. For normalization, 100 housekeeping genes provided by Affymetrix were used. Relative expression values were calculated from the signal diary relations provided by the software and the normal kidney sample was arbitrarily set to 1.0.
[00388] The expression profiles of the origin genes of the present invention, which are highly overexpressed in gastric cancer are shown in figure 2.
EXAMPLE 3
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[00389] In vitro immunogenicity of IMA941 MHC class I peptides presented
[00390] In order to obtain information on the immunogenicity of TUMAPs of the present invention, we carried out investigations with an in vitro stimulation platform in basin already described by (Walter, S, Herrgen, L, Schoor, O, Jung, G, Wernet, D, Buhring, HJ, Rammensee, HG, and Stevanovic, S; 2003, Cutting edge: predetermined avidity of human CD8 T cells expanded on calibrated MHC / anti-CD28-coated microspheres, J.Immunol., 171, 4974-4978) . With this system we were able to show positive immunogenicity results (ie specific T cell expansion) for 47 of 54 HLA-A * 2402 restricted tested TUMAPs and for 3 of 3 HLA-A * 0201 restricted tested TUMAPs of the invention, demonstrating that these peptides are T cell epitopes against which precursor CD8 + T cells exist in humans (Table 4).
In vitro preparation of CD8 + T cells
[00391] In order to perform in vitro stimuli by cells presenting artificial antigen (aAPC) loaded with peptide-MHC complex (pMHC) and anti-CD28 antibody, we first isolated CD8 T cells from fresh HLA-A * 24 leukapheresis products or layers HLA-A * 2 white blood cells from healthy donors obtained from the Tuebingen Blood Bank.
[00392] CD8 T cells were directly enriched or PBMCs (peripheral blood mononuclear cells) were isolated first using standard gradient separation medium (PAA, Colbe, Germany). Isolated CD8 lymphocytes or PBMCs were incubated until their use with T cell agent (TCM) formed by RPMI-Glutamax (Invitrogen, Karlsruhe, Germany) supplemented with 10% heat-inactivated AB human serum (PAN-Biotech, Aidenbach, Germany) , 100 U / ml penicillin / 100 pg / ml streptomycin
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147/174 (Cambrex, Colonia, Belgium), 1 mM sodium pyruvate (CC Pro, Oberdorla, Germany) and 20 pg / ml gentamicin (Cambrex). 2.5 ng / ml IL-7 (PromoCell, Heidelberg, Germany) and 10 U / ml IL-2 (Novartis Pharma, Nuremberg, Germany) of cytokines were also added to the TCM for this culture step. The isolation of CD8 + lymphocytes was performed by positive selection using MicroBeads CD8 (Miltenyi Biotec, Bergisch-Gladbach, Germany).
[00393] The generation of beads coated with pMHC / anti-CD28, the T cell stimuli and the revision were performed as previously described (Walter and others 4974-78) with minor modifications. In summary, HLA-A * 2402 and HLA-A * 0201 molecules loaded with peptide, biotinylated recombinants lacking a transmembrane domain and biotinylated in the heavy chain carboxy termini, were produced. The anti-human IgG2a CD28 Ab 9.3 (Jung, Ledbetter, and Muller-Eberhard 4611-15) purified from co-stimulant rat was chemically biotinylated using a Sulfo-N-hydroxysucinimidobiotin as recommended by the manufacturer (Perbio, Bonn, Germany). The beads used were formed by polystyrene particles coated with streptavidin with a size of 5.6 pm (Bangs Laboratories, Illinois, USA). The pMHC used as high and low immunogenic controls were A * 0201 / MLA-001 (modified Melan-A / MART-1 peptide ELAGIGILTV) and A * 0201 / DDX5-001 (DDLL5 YLLPAIVHI), respectively.
[00394] 800,000 beads / 200 pl were coated onto 96-wave corrugated dishes in the presence of 600 ng of anti-CD28 biotin plus 200 ng of relevant pMHC biotin (high density beads). The stimuli were initiated in 96 recipient holes by co-incubating 1x10 ⁶ CD8 + T cells with 2x10 ⁵ coated beads and washed in 200 pl TCM, supplemented with 5 ng / ml IL-12 (PromoCell) for 3-4 days at 37 ° C, 5% CO2 and 95% relative humidity. Half of the agent was
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148/174 then exchanged for fresh TCM supplemented with 80 U / ml IL-2 and incubation was continued for 3-4 days at 37 ° C. This stimulus cycle was performed a total of three times.
[00395] Finally, multimer analyzes were performed by staining cells with fluorescent multimers A * 0201 or A * 2402 HLA (produced as described by {Altman, 1996 ALTMAN1996 / id}) and CD8-FITC SK1 antibody clone (BD, Heidelberg, Germany ) or additionally with a viability marker (with blue / dead dye or violet dye (Invitrogen, Karlsruhe, Germany)), and were conducted on a FACSCalibur four-color cytometer or an LSRII SORP BD cytometer; eighteen colors, equipped with blue (488 nm), violet (405 nm), red (640 nm) and green (532 nm), respectively. Specific peptide cells were calculated as a percentage of CD8 + total T cells. The evaluation of the multimeric analysis was done using FCSExpress or FlowJo software (Tree Star, Oregon, USA). In vitro preparation of specific multimer + CD8 + lymphocytes was detected by appropriate gating and by comparison with negative control stimuli. Immunogenicity for a given antigen was detected if at least one in vitro evaluable recipient from a healthy donor was found to contain specific CD + 8 T cells after in vitro stimulation (ie the fraction of the multimer + cell population within this container made up of at least 1% of CD8 + cells, the frequency was at least 10 times more than the average of the respective negative controls (stimulus with spots with irrelevant multimers and spots with relevant multimers) and the cells were not located diagonally across the trace).
In vitro immunogenicity for IMA941 peptides
[00396] For 47 of 54 HLA-A * 2402 peptides tested and for 3 of 3 HLA-A * 0201 peptides tested, in vitro immunogenicity could be
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149/174 demonstrated by the generation of specific peptide T cell lines. Exemplary results of flow cytometry after TUMAP specific multimetric staining for two peptides of the invention are shown in Figure 3, along with a corresponding negative control. Results of 54 A * 2402 and 3 A * 0201 peptides of the invention are summarized in Table 4.
Table 4: In vitro immunogenicity of HLA class I peptides of the invention
[00397] The results of in vitro immunogenicity experiments conducted by Immatics show the percentage of donors tested positive and enclosures among the evaluable ones. At least four donors and 48 enclosures were available for each peptide.
SEQ IDN °: Antigen Positive / evaluable donors [%] positive / evaluable wells[%] 1 CDC2-001 83 28 2 ASPM-002 67 32 18 MMP3-001 11 1 4 MET-006 67 21 3 UCHL5-001 75 12 7 MST1R-001 50 13 33 KIF2C-001 17 2 9 SMC4-001 73 10 17 EPHA2-005 0 0 5 PROM1-001 83 26 6 MMP11-001 33 11 8 NFYB-001 50 7 16 ASPM-001 17 3 20 PLK4-001 60 5
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SEQ IDN °: Antigen Positive / evaluable donors [%] positive / evaluable wells [%] 14 ABL1-001 83 18 26 ATAD2-001 33 3 21 ATAD2-002 17 1 27 ATAD2-003 0 0 12 AVL9-001 100 31 22 COL12A1-001 0 0 23 COL6A3-001 0 0 24 FANCI-001 17 1 28 HSP90B1-001 50 7 15 MUC6-001 83 22 13 NUF2-001 100 50 19 NUF2-002 50 6 11 PPAP2C-001 83 29 25 RPS11-001 17 3 29 SIAH2-001 50 8 30 SLC6A6-001 17 1 10 UQCRB-001 83 24 31 IQGAP3-001 100 24 32 ERBB3-001 83 CCDC88A-001 0 0CCNB1-003 33 3CCND2-001 17 10CCNE2-001 0 0CEA-010 40 3
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SEQ IDN °: Antigen Positive / evaluable donors [%] positive / evaluable wells [%]CLCN3-001 33 6DNAJC10-001 50 15DNAJC10-002 33 3EIF2S3-001 17 1EIF3L-001 100 29EPPK1-001 17 1GPR39-001 50 6ITGB4-001 67 20LCN2-001 17 1SDHC-001 33 3PBK-001 0 0POLD3-001 67 7PSMD14-001 17 1PTK2-001 17 4TSPAN1-002 17 1ZNF598-001 83 17
[00398] The following peptides have already been described in other applications by Immatics and included in the IMA901 (MET-001 and TOP-001), IMA910 (MET-001 and TOP-001) and IMA950 (IGF2BP3-001) vaccines. As an example, MET-001 leads to extremely good in vivo reactions, the data can be seen as an indication for the clinical usefulness of the peptides of the invention,
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SEQ IDN °: Antigen Positive / evaluable donors [%] Positive / evaluable wells [%]IGF2BP3-001 50 21MET-001 67 42TOP-001 40 10
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权利要求:
Claims (2)
[1]
1. Use of a peptide selected from:
a) a peptide consisting of the sequence according to SEQ ID NO. 63;
b) the peptide according to a), wherein said peptide includes non-peptide bonds;
c) the peptide according to a), wherein said peptide is part of a fusion protein;
together with excipients or suitable vehicles, characterized by the fact that it is for the production of a drug for the treatment of cancer, in which said cancer is selected from gastric, gastrointestinal, colorectal, pancreatic, pulmonary or renal cancer.
[2]
Use according to claim 1, characterized by the fact that said fusion protein comprises Nterminal amino acids of the invariant chain (Ii) associated with HLA-DR antigen.

类似技术:

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公开号 | 公开日 | 专利标题

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法律状态:
2018-01-23| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2018-04-10| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-05-28| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |

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2019-07-09| B06T| Formal requirements before examination [chapter 6.20 patent gazette]|

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2019-12-10| B07A| Application suspended after technical examination (opinion) [chapter 7.1 patent gazette]|

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2020-04-07| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2020-06-09| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 15/03/2011, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US31570410P| true| 2010-03-19|2010-03-19|

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GBGB1004551.6A|GB201004551D0|2010-03-19|2010-03-19|NOvel immunotherapy against several tumors including gastrointestinal and gastric cancer|

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PCT/EP2011/053863|WO2011113819A2|2010-03-19|2011-03-15|Novel immunotherapy against several tumors including gastrointestinal and gastric cancer|

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