TW201512223A - SMYD3 peptides and vaccines containing the same - Google Patents

Abstract

Peptide vaccines against cancer are described herein. In particular, isolated epitope peptides derived from the SMYD3 gene that elicit CTLs and thus are suitable for use in the context of cancer immunotherapy are provided. The inventive peptides encompass both SMYD3 -derived peptides and modified versions thereof, in which one, two, or several amino acids are substituted, deleted, inserted or added, provided such modified versions retain the requisite CTL inducibility of the original sequences. Further provided are polynucleotides encoding such peptides as well as pharmaceutical compositions that include any such peptides or polynucleotides as active ingredients. Antigen-presenting cells and isolated CTLs that target such peptides, as well as methods for inducing the antigen-presenting cell, or CTL are also provided. Furthermore, the present invention provides methods for the treatment and/or prophylaxis (i.e., prevention) of cancers (tumors), and/or the prevention of a metastasis or post-operative recurrence thereof, as well as methods for inducing CTLs, methods for inducing anti-tumor immunity, using the peptides derived from SMYD3, polynucleotides encoding the peptides, or antigen-presenting cells presenting the peptides, or the pharmaceutical compositions of the present invention.

Description

SMYD3 peptide and vaccine containing the same

The present invention relates to the field of biological sciences, and more particularly to the field of cancer treatment. In particular, the present invention relates to a novel peptide which is effective as a cancer vaccine and a medicament for treating and/or preventing tumors.

priority

The present invention claims the benefit of U.S. Provisional Application Serial No. 61/776,455, the entire disclosure of which is incorporated herein by reference.

CD8-positive cytotoxic T lymphocytes (CTLs) have been identified to recognize epitopes from tumor-associated antigens (TAAs) on major histocompatibility complex (MHC) class I molecules. The peptide is peptide, and then the tumor cells are killed. Since the melanoma antigen (MAGE) family has been found to be the first tumor-associated antigen, many other tumor-associated antigens have been found by immunological methods (Non-Patent Documents 1-2). Some of these tumor-associated antigens are currently undergoing clinical development as immunotherapeutic targets.

Suitable tumor-associated antigens are essential for cancer cell proliferation and survival. The use of such tumor-associated antigens as immunotherapeutic targets minimizes the risk of immune escape from cancer cells that are widely described, while cancer cells are free Evacuation escape is the result of therapeutic screening for immune screening, due to deletion, mutation or down regulation of tumor-associated antigens. Therefore, the identification of new tumor-associated antigens that induce an effective and specific anti-tumor immune response is the basis for further development. Therefore, clinical research on vaccination strategies for various forms of cancer is underway (Non-Patent Documents 3-10). Some clinical trials using these tumor-associated antigen-derived peptides have been reported to date. Unfortunately, these cancer vaccine trials have only shown a low objective response rate (Non-Patent Documents 11-13). Therefore, the need for new tumor-associated antigens as targets for immunotherapy is maintained.

SMYD3 (GenBank accession number NM_022743.2, NM_001167740 or AB057595), also known as ZNFN3A1, is a gene encoding a SET- and MYND-regional protein that was previously suggested to have histone methyltransferase activity. As a member of the RNA polymerase complex (Non-Patent Document 14, Patent Documents 1, 3), it plays a key role in transcriptional regulation. In addition to the testicles and skeletal muscles, SMYD3 was not expressed in normal organs, but was confirmed to be excessively expressed in liver cancer cells (HCC) and colorectal cancer cells (Non-Patent Document 14, Patent Document 1), and in breast cancer and bladder. Cancer (Patent Documents 4, 5, and 6) is regulated. Further, it has been shown that inhibition of the expression of the SMYD3 gene by siRNAs in a liver cancer (HCC) cell line results in significant growth inhibition (Patent Document 2). Taken together, this data suggests that SMYD3 can be a suitable target for cancer immunotherapy protocols, particularly cancer immunotherapy.

[Previous Technical Literature] [Patent Literature]

Patent Document 1: WO2003/027143

Patent Document 2: WO2004/076623

Patent Document 3: WO2005/071102

Patent Document 4: WO2006/092958

Patent Document 5: WO2007/004526

Patent Document 6: WO2008/152816

[Non-patent literature]

Non-Patent Document 1: Boon T, Int J Cancer 1993, 54(2): 177-80

Non-Patent Document 2: Boon T & van der Bruggen P, J Exp Med 1996, 183(3): 725-9

Non-Patent Document 3: Harris CC, J Natl Cancer Inst 1996, 88(20): 1442-55

Non-Patent Document 4: Butterfield LH et al., Cancer Res 1999, 59(13): 3134-42

Non-Patent Document 5: Vissers JL et al., Cancer Res 1999, 59(21): 5554-9

Non-Patent Document 6: van der Burg SH et al., J Immunol 1996, 156(9): 3308-14

Non-Patent Document 7: Tanaka F et al., Cancer Res 1997, 57(20): 4465-8

Non-Patent Document 8: Fujie T et al., Int J Cancer 1999, 80(2): 169-72

Non-Patent Document 9: Kikuchi M et al., Int J Cancer 1999, 81(3): 459-66

Non-Patent Document 10: Oiso M et al., Int J Cancer 1999, 81(3): 387-94

Non-Patent Document 11: Belli F et al., J Clin Oncol 2002, 20(20): 4169-80

Non-Patent Document 12: Coulie PG et al., Immunol Rev 2002, 188: 33-42

Non-Patent Document 13: Rosenberg SA et al., Nat Med 2004, 10(9): 909-15

Non-Patent Document 14: Hamamoto et al., Nat Cell Biol. 2004; 6(8): 761-40

The invention is based on, or at least in part based on, the discovery of novel peptides that are suitable targets for immunotherapy. Since TAAs are generally regarded as their own substances by the immune system, there is usually no innate immunogenicity, so it is still important to find the appropriate targets. In the course of the invention, SMYD3 (a typical amino acid sequence as set forth in SEQ ID NO: 60, 62 or 64; a typical nucleotide sequence as set forth in SEQ ID NO: 59, 61 or 63) (GenBank accession number NM_022743.2, NM_001167740 or AB057595)) has been shown to be specifically overexpressed in cancer, such as, but not limited to, colorectal cancer, liver cancer, breast cancer, and bladder cancer. Thus, the present invention focuses on SMYD3 as a candidate for cancer and/or tumor immunotherapy.

The present invention relates to, or at least partially relates to, specific antigenic decisions Identification of the peptide, with the ability to induce CTLs specific for SMYD3. The results of this description demonstrate that these peptides are HLA-A24 or HLA-A2-restricted epitope peptides that promote potent, specific immune responses against cells expressing SMYD3.

Accordingly, it is an object of the present invention to provide a SMYD3-derived peptide which can be used to stimulate CTLs in vitro , ex vivo or in vivo in a HLA-A24 or HLA-A2 restricted manner, or The individual can be administered directly to induce an anti-cancer immune response in vivo, such as, but not limited to, colorectal cancer, liver cancer, breast cancer, and bladder cancer.

The peptides of the invention are typically less than 15, 14, 13, 12, 11 or 10 amino acids in length. Preferred peptides are nine peptides and ten peptides. Particularly preferred nine peptides and ten peptides have amino acid sequences selected from the sequence identification numbers: 2, 4, 5, 6, 7, 8, 9, 13, 14, 16, 17, 19 and 22. Among these, a peptide having an amino acid sequence selected from the sequence identification numbers of 2, 4, 5, 6, 7, 8, 9, 13, 14, 16, 17, 19 and 22 is particularly preferred.

The present invention also encompasses a modified peptide having one of the amino acid sequences selected from the sequence identifiers: 2, 4, 5, 6, 7, 8, 9, 13, 14, 16, 17, 19 and 22. The amino acid sequence of two or more amino acids substituted, deleted, inserted and/or increased, but the resulting modified peptide still maintains the CTL inducing activity necessary for the original unmodified peptide.

In one embodiment, when the original peptide is a nine-peptide (eg, one of sequence identification numbers: 2, 4, 5, 6, 7, 8, 9, and 22), the length of the modified peptide is preferably 9 to 40 amino acids, such as 9 to 20 amino acids, such as 9 to 15 amino acids. Similarly, when the original peptide is a ten-peptide (eg, sequence identifier: The length of the modified peptide is preferably from 10 to 40 amino acids, such as from 10 to 20 amino acids, such as from 10 to 15 amino acids, of one of 13, 14, 16, 17 and 19.

The invention further encompasses an isolated polynucleotide encoding any of the peptides of the invention. These polynucleotides can be used to induce or prepare antigen-presenting cells (APCs) having cytotoxic T lymphocyte inducing ability. As with the peptide of the present invention, an individual such antigen-presenting cell can be administered to induce an immune response against cancer.

When administered to a body, the peptide of the present invention can be present on the surface of the antigen presenting cells to induce cytotoxic T lymphocytes that target the individual peptides. Accordingly, it is an object of the invention to provide a composition or medicament comprising one or more of the peptides of the invention or a polynucleotide encoding such a peptide. The compositions of the invention are useful for inducing cytotoxic T lymphocytes. This composition can be used for the treatment and/or prevention of cancer, and its metastasis or prevention of postoperative recurrence. Examples of cancers contemplated by the present invention include, but are not limited to, colorectal cancer, liver cancer, breast cancer, and bladder cancer.

The invention further contemplates pharmaceutical compositions comprising one or more peptides or polynucleotides of the invention. The pharmaceutical composition is preferably formulated for the treatment and/or prevention of cancer, particularly primary cancer, and/or prevention of metastasis or postoperative recurrence. In addition to the peptide or polynucleotide of the present invention, the pharmaceutical composition of the present invention may comprise an antigen presenting cell and/or an exosome which exhibits any of the peptides of the present invention as an active ingredient.

The peptide or polynucleotide of the present invention can be used to induce antigen-presenting cells which exhibit a complex of a human leukocyte antigen (HLA) and a peptide of the present invention on the surface, for example, by presenting an antigen from a body to a cell The invention is contacted with a peptide or by introducing a polynucleotide encoding a peptide of the present invention into an antigen-presenting cell. Such an antigen presenting cells have the ability to induce specific recognition on the cell surface The cytotoxic T lymphocytes of the cells of the currently labeled peptide, and thus can be used for the content of cancer immunotherapy. Accordingly, the present invention includes a method of inducing an antigen-presenting cell having a cytotoxic T lymphocyte-inducing ability and an antigen-presenting cell obtained by the method.

Furthermore, the invention also encompasses agents or compositions that induce antigen-presenting cells having cytotoxic T lymphocyte-inducing ability, including such peptides or polynucleotides of the invention.

A further object of the present invention is to provide a method for inducing cytotoxic T lymphocytes, the method comprising the steps of co-cultivating CD8-positive T cells with antigen-presenting cells which exhibit a complex of a HLA antigen and a peptide of the present invention. a step of co-culturing CD8-positive T cells with a surface-expressing HLA antigen and a complex of the peptide of the present invention, or introducing a T cell receptor (TCR) subunit or encoding each T cell A step of a polynucleotide of a subunit of a receptor, wherein the T cell receptor binds to a complex of the peptide of the present invention and an HLA antigen presented on the surface of the cell. The cytotoxic T lymphocytes obtained by such methods are effective for cancer, including, but not limited to, treatment and/or prevention of, for example, colorectal cancer, liver cancer, breast cancer, and bladder cancer. Accordingly, the present invention includes a method of inducing a cytotoxic T lymphocyte and a cytotoxic T lymphocyte obtained by the method.

Still another object of the present invention is to provide an isolated antigen-presenting cell which exhibits a complex of a HLA antigen and a peptide of the present invention on the surface. The invention further provides isolated cytotoxic T lymphocytes that target the peptide of the invention. This cytotoxic T lymphocyte can also be defined as a cytotoxic T lymphocyte capable of recognizing (or binding to) a complex of a peptide of the present invention and an HLA antigen on the cell surface. These antigens present cellular and cellular toxic T lymphocytes that can be used in cancer immunotherapy.

Yet another object of the present invention is to provide a method of inducing an immune response against cancer in an individual, the method comprising administering to the individual a composition comprising at least one of the following: (a) a peptide of the invention, encoding The polynucleotide of the peptide, (b) an antigen presenting cell or exocytosis of the peptide, and (c) a cytotoxic T lymphocyte recognizable to a cell exhibiting the peptide of the present invention.

One aspect of the present invention relates to a peptide of the present invention, a composition comprising the peptide, for use as a medicament.

The utility of the present invention extends to most diseases that are or are caused by excessive expression of SMYD3, such as, but not limited to, colorectal cancer, liver cancer, breast cancer, and bladder cancer.

More particularly, the present invention provides the following: [1] a single-isopeptide having cytotoxic T lymphocyte (CTL) inducibility, wherein the peptide comprises the following amino acid sequence (a) or (b): (a) an amino acid sequence of the SMYD3 immunologically active fragment; (b) an amino acid sequence having 1, 2 or more amino acid substitutions, deletions, insertions and/or additions in the amino acid sequence of the SMYD3 immunologically active fragment Wherein the CTL induced by the peptide has specific cytotoxicity for presenting cells derived from the SMYD3 fragment; [2] the peptide of [1], wherein the peptide comprises the following amino acid sequence (a) or (b) ): (a) an amino acid sequence selected from the group consisting of: 2, 4, 5, 6, 7, 8, 9, 13, 14, 16, 17, 19, and 22; (b) 1 in the amino acid sequence selected from the group consisting of: 2, 4, 5, 6, 7, 8, 9, 13, 14, 16, 17, 19 and 22 2 or a plurality of amino acid substituted, deleted, inserted and/or increased amino acid sequences; [3] the peptide of [2], wherein the peptide is the following oligopeptide (i) or (ii): (i) a peptide having one or two of the following characteristics: (a) a sequence identification number: 2, 4, 5, 6, 7, 8, 9, 13, 14, 17, or 19 amino acid sequence The N-terminal second amino acid is replaced by phenylalanine, tyrosine, methionine or tryptophan; and (b) the sequence number: 2, 4, 5, 6, 7, 8, 9, 13 The C-terminal amino acid of the 14, 17, or 19 amino acid sequence is substituted with phenylalanine, leucine, isoleucine, tryptophan or methionine; (ii) a peptide having the following One or two characteristics: (a) sequence identification number: the N-terminal second amino acid of the amino acid sequence of 8, 16, or 22 is substituted with leucine or methionine; and (b) sequence recognition No.: the C-terminal amino acid of the amino acid sequence of 8, 16 or 22 is substituted with lysine or leucine; [4] any of the peptides [1] to [3], wherein the winner The peptide is a nine-peptide or a ten-peptide; [5] the peptide of [4], wherein the peptide is selected from the sequence identification numbers: 2, 4, 5, 6, 7, 8, 9, 13, 14, 16 a group consisting of amino acid sequences of groups consisting of 17, 19 and 22; [6] an isolated polynucleotide encoding any of the peptides [1] to [5]; [7] a composition for inducing CTL And the composition comprising at least one active ingredient selected from the group consisting of: (a) any one of [1] to [5]; (b) a polynucleotide of [6]; (c) an antigen presenting cell (APC) having a surface of any of the peptides [1] to [5]; and (d) a surface [1] to [ 5] any of the peptides exosome; [8] a pharmaceutical composition for treating and/or preventing cancer, and/or preventing postoperative recurrence, wherein the composition comprises at least one selected from the group consisting of An active ingredient: (a) any one of [1] to [5]; (b) a polynucleotide of [6]; (c) any one of the peptides [1] to [5] on the surface. An antigen-presenting cell (APC); (d) a peptide exhibiting any of the peptides [1] to [5] on the surface; and (e) identifiable ones of [1] to [5] The CTL of the cell according to [8], wherein the pharmaceutical composition is formulated into an individual to which the HLA antigen is HLA-A24 or HLA-A2; [10] a substance which promotes CTL-inducing ability. A method of APC, wherein the method comprises the step of selecting from the group consisting of: (a) contacting the APC with any of the peptides [1] to [5], and (b) encoding [1] to [5] a polynucleotide of any peptide is introduced into APC; [11] a method for inducing CTL, wherein the method comprises The steps in the following group consisting of: (a) co-culturing CD8-positive T cells with APCs that exhibit a complex of HLA antigens with any of the peptides [1] to [5]; (b) CD8-positive T Cells and surfaces present HLA antigens with [1] to [5] The exosome of any peptide complex is co-cultured; (c) introducing a polynucleotide encoding a T cell receptor (TCR) subunit, or a polynucleotide encoding each TCR subunit into a CD8 positive T cell Wherein the TCR formed by the subunit can bind to a complex of any of the peptides and HLA antigens of [1] to [5] on the cell surface; [12] an isolated APC having a surface exhibiting an HLA antigen and [1] to [5] a complex of any peptide; [13] APC of [12], which is induced by the method of [10]; [14] an isolated CTL whose target [1] [15] to any of the peptides of [5]; [15] such as the CTL of [14], which is induced by the method of [11]; [16] a method for inducing an anti-cancer immune response in an individual, wherein the method includes The step of administering to the individual a composition comprising any of the peptides [1] to [5] or a polynucleotide encoding the peptide; [17] an antibody against any of the peptides of [1] to [5] or An immunologically active fragment; [18] a vector comprising a nucleotide sequence encoding any of the peptides [1] to [5]; [19] a host cell transformed or transfected with a vector of [18]; 20] A diagnostic kit comprising any one of [1] to [5] a peptide, a polynucleotide of [6], or an antibody or immunologically active fragment of [17]; [21] a method of screening for a peptide having cells which induce specific cytotoxicity against cells presenting a fragment derived from SMYD3 The ability of a toxic T lymphocyte (CTL), wherein the method comprises: (i) providing a candidate sequence that is replaced, deleted, inserted, and/or replaced by one, two or several amino acid residues from the original amino acid. Increased modified amino acid sequence Composition, wherein the original amino acid sequence is selected from the group consisting of: 2, 4, 5, 6, 7, 8, 9, 13, 14, 16, 17, 19, and 22; Choosing a candidate sequence that, in addition to SMYD3, does not have substantially significant homology to a peptide derived from any known human gene product; (iii) forming a candidate sequence selected in step (ii) The peptide is contacted with the antigen-presenting cell; (iv) contacting the antigen-presenting cell of step (iii) with CD8-positive T cells; and (v) identifying having the same or higher than the peptide consisting of the original amino acid sequence a cytotoxic T lymphocyte (CTL)-inducing ability peptide; [22] a pharmaceutical composition comprising any of the peptides [1] to [5]; [23] one of [1] to [5] The peptide is used as a drug; and [24] a polynucleotide of [6] or a carrier of [18] is used as a drug.

Alternatively, the present invention provides the following embodiments: [1] a single-isopeptide having cytotoxic T lymphocyte (CTL) inducibility, wherein the peptide-induced CTL has a cell exhibiting a fragment derived from SMYD3 Specific cytotoxicity, and the peptide includes the following amino acid sequence (a) or (b): (a) the amino acid sequence of the SMYD3 immunologically active fragment; (b) as in the amino acid sequence of (a) 1, 2 or a plurality of amino acid substitutions, deletions, insertions and/or additions, [2] the peptide of [1], wherein the peptide comprises the following amino acid sequence (a) or (b): (a ) from the serial identification number: 2, 4, 5, 6, 7, 8, 9, 13, 14 The amino acid sequence of the group consisting of 16, 17, 19 and 22; (b) having 1, 2 or several amino acid substitutions, deletions, insertions and/or additions in the amino acid sequence of (a) [3] The peptide of [2], wherein the peptide is the following oligopeptide (i) or (ii): (i) a peptide having one or two of the following characteristics: (a) N-terminal The two amino acids are phenylalanine, tyrosine, methionine or tryptophan; and (b) the C-terminal amino acid is phenylalanine, leucine, isoleucine, tryptophan or methyl sulfide Amino acid; (ii) a peptide having one or two of the following characteristics: (a) the N-terminal second amino acid is leucine or methionine; and (b) the C-terminal amino acid is hydrazine; Amino acid or leucine; [4] any of the peptides [1] to [3], wherein the peptide is a ninth peptide or a ten peptide; [5] one of the codes [1] to [4] An isolated polynucleotide of a peptide; [6] a composition for inducing CTL, wherein the composition comprises at least one active ingredient selected from the group consisting of: (a) any one of [1] to [4]; b) a polynucleotide of [5]; (c) an antigen presenting cell (APC) having a surface of any of the peptides [1] to [4]; and (d) a surface [1] to [ 4] either a peptide composition for treating and/or preventing primary cancer, and/or preventing metastasis or postoperative recurrence, or inducing an anti-cancer immune response, wherein the composition comprises At least one active ingredient in the following: (a) any one of [1] to [4]; (b) a polynucleotide of [5]; (c) an antigen-presenting cell having any of the peptides of [1] to [4] on the surface ( APC); (d) a CTL having any of the peptides [1] to [4] on the surface; and (e) a CTL capable of recognizing cells exhibiting any of the peptides [1] to [4] [8] The pharmaceutical composition according to [7], wherein the pharmaceutical composition is formulated into a body in which the HLA antigen is HLA-A24 or HLA-A2; [9] a method for promoting APC having CTL inducing ability; Wherein the method comprises the steps of: (a) contacting the APC with any of the peptides [1] to [4], and (b) the polynucleoside encoding the peptide of any of [1] to [4] Introduction of acid into APC; [10] A method of inducing CTL, wherein the method comprises the steps of: (a) presenting CD8-positive T cells with HLA antigen on the surface and any of the peptides [1] to [4] APC co-culture of the complex; (b) co-culturing CD8-positive T cells with exosomes whose surface exhibits a complex of HLA antigen and any of the peptides [1] to [4]; (c) encoding T cells A polynucleotide of a receptor (TCR) subunit or a polynucleotide encoding each TCR subunit is introduced into CD8 positivity In T cells, the TCR formed by the subunit can bind to the complex of any of the peptides and HLA antigens of [1] to [4] on the cell surface; [11] an isolated APC having a surface exhibiting HLA a complex of an antigen with any of the peptides [1] to [4]; [12] APC according to [11], which is induced by the method of [9]; [13] an isolated CTL which targets any of the peptides [1] to [4]; [14] such as [ 13] CTL, which is induced by the method of [10]; [15] A method of inducing an anti-cancer immune response in an individual, wherein the method comprises administering to the individual any of the peptides [1] to [4] Or a step of encoding a composition of the polynucleotide of the peptide; [16] an antibody against any of the peptides [1] to [4] or an immunologically active fragment thereof; [17] a vector comprising the coding [1] a nucleotide sequence of any of the peptides of [4]; [18] a host cell transformed or transfected with a vector of [17]; [19] a diagnostic kit comprising [1] to [4] Any one of the peptides, the polynucleotide of [5], or the antibody or immunologically active fragment of [16]; [20] a method for screening a peptide which induces specific cytotoxicity against the presentation of a fragment derived from SMYD3 The ability of a cell to be cytotoxic T lymphocyte (CTL), wherein the method comprises: (i) providing a candidate sequence that is replaced, deleted, by the original amino acid by one, two or several amino acid residues, Insert and / or add modifiers Amino acid sequence consisting of the original amino acid sequence selected from the group consisting of: 2, 4, 5, 6, 7, 8, 9, 13, 14, 16, 17, 19, and 22 a group; (ii) selecting a candidate sequence that, in addition to SMYD3, does not have substantially significant homology to a peptide derived from any known human gene product; (iii) selecting step (ii) The peptide formed by the candidate sequence is in contact with the antigen-presenting cell; (iv) contacting the antigen-presenting cell of step (iii) with the CD8-positive T cell; And (v) identifying a peptide having the same or higher cytotoxic T lymphocyte (CTL) inducing ability as the peptide consisting of the original amino acid sequence; [21] a pharmaceutical composition comprising [1] to [4] any of the peptides; [22] the use of any of the peptides [1] to [4] as a drug; and [23] the use of a polynucleotide of [5] or [17] as a drug .

Alternatively, the present invention also provides the following implementations:

[1] A single-span peptide having cytotoxic T lymphocyte (CTL) inducing ability, wherein the peptide comprises the following amino acid sequence (a) or (b): (a) selected from the sequence identifier: 2 The amino acid sequence of the group consisting of 4, 5, 6, 7, 8, 9, 13, 14, 16, 17, 19 and 22; (b) selected from the sequence identification numbers: 2, 4, 5 Substituting, deleting, inserting and/or increasing one, two or several amino acids in the amino acid sequence of groups consisting of 6, 7, 8, 9, 13, 14, 16, 17, 19 and 22 The amino acid sequence of [2], wherein the peptide is the following oligopeptide (i) or (ii): (i) a peptide having one or two of the following characteristics: a) sequence identification number: the N-terminal second amino acid of the amino acid sequence of 2, 4, 5, 6, 7, 8, 9, 13, 14, 17, or 19 is replaced with phenylalanine, cheese Amino acid, methionine or tryptophan; and (b) C-terminal of the amino acid sequence of the sequence number: 2, 4, 5, 6, 7, 8, 9, 13, 14, 17, or 19. The amino acid is replaced by phenylalanine, leucine, isoleucine, tryptophan or methionine; (ii) a peptide having one or two of the following characteristics: (a) Sequence identification number: N terminal amino acid sequences of 8, 16 or 22 second The amino acid is replaced with leucine or methionine; and (b) the C-terminal amino acid of the amino acid sequence of SEQ ID NO: 8, 16 or 22 is substituted with lysine or leucine.

[3] The peptide of [1] or [2], wherein the peptide is a nine-peptide or a ten-peptide; and [4] a peptide of [3], wherein the peptide is selected from the sequence identifier: The amino acid sequence consisting of 2, 4, 5, 6, 7, 8, 9, 13, 14, 16, 17, 19 and 22 is composed of amino acid sequences.

The objects and features of the present invention will become more fully apparent from the understanding of the appended claims. However, it is to be understood that the invention is not to be construed as limiting the invention.

In addition to the above, other objects and features of the present invention will become more apparent from the detailed description and appended claims. It is to be understood, however, that the invention is not intended to be

In particular, the invention as described herein with respect to the specific embodiments of the present invention is understood to be illustrative of the invention and is not to be construed as limiting. Various modifications and applications can be made by those skilled in the art without departing from the spirit and scope of the invention, as set forth in the appended claims. The other objects, features, advantages and advantages of the present invention are apparent from the description and appended claims. Such objectives, features, advantages and advantages are derived from the above-described combination of accompanying examples, materials, drawings and all references that are to be All reasonable inferences described are clear.

Various aspects and applications of the present invention will become apparent from the Detailed Description of the Drawings and the Detailed Description.

Figure 1 is a series of photographs including (a) to (1) plots depicting the results of IFN-γ Enzyme Linked Immunospot Analysis (ELISPOT) analysis of CTLs induced by SMYD3 peptide. CTLs in well number #1 were induced with SMYD3-A24-9-197 (SEQ ID NO: 2) (a), and #6 was induced with SMYD3-A24-9-326 (SEQ ID NO: 4) (b), No. #2 induced (c) with SMYD3-A24-9-138 (SEQ ID NO: 5), and #6 was induced with SMYD3-A24-9-130 (SEQ ID NO: 6) (d), number #7 SMYD3-A24-9-192 (SEQ ID NO: 7) induces (e), number #1 induces (f) with SMYD3-A24-9-118 (SEQ ID NO: 8), number #4 with SMYD3-A24- 9-301 (SEQ ID NO: 9) induces (g), number #2 induces (h) with SMYD3-A24-10-260 (SEQ ID NO: 13), number #3 with SMYD3-A24-10-266 ( SEQ ID NO: 14) induction (i), number #3 induced by SMYD3-A24-10-86 (SEQ ID NO: 17) (j) and number #8 with SMYD3-A24-10-138 (SEQ ID NO: 19) Induction (k) showed effective IFN-γ production, respectively, compared to the control group. The wells indicated by the boxes indicate the corresponding cell expansion to establish a CTL cell line with the wells. In contrast, no specific IFN-γ production was observed in CTL stimulated with SMYD3-A24-9-37 (SEQ ID NO: 1) (1), which is typical negative data. In the figure, "+" indicates the production of IFN-γ against the target cells pulsed with the appropriate peptide, and "-" indicates that the peptide is not pulsed with any peptide. The production of IFN-γ by the target cells.

Figure 2 is a series of photographs including (a) to (d) plots depicting the results of IFN-γ Enzyme Linked Immunospot Analysis (ELISPOT) analysis of CTLs induced by other peptides derived from SMYD3. CTLs in well number #6 were induced with SMYD3-A02-9-335 (SEQ ID NO: 22) (a), and #7 was induced with SMYD3-A02-9-118 (SEQ ID NO: 8) (b), And No. #4 induced (c) with SMYD3-A02-10-76 (SEQ ID NO: 16), showing effective IFN-γ production, respectively, as compared with the control group. The wells indicated by the boxes indicate the corresponding cell expansion to establish a CTL cell line with the wells. In contrast, no specific IFN-γ production was observed in CTL stimulated with SMYD3-A02-9-45 (SEQ ID NO: 20) (d), which is typical negative data. In the figure, "+" indicates the production of IFN-γ against a target cell pulsed with an appropriate peptide, and "-" indicates the production of IFN-γ against a target cell not pulsed with any peptide.

Figure 3 is a series of line graphs, including (a) to (f), depicting CTL cell lines via SMYD3-A24-9-197 (SEQ ID NO: 2) (a), SMYD3-A24-9-130 (SEQ ID NO: 6) (b), SMYD3-A24-9-192 (SEQ ID NO: 7) (c), SMYD3-A24-9-118 (SEQ ID NO: 8) (d), SMYD3-A24 -10-86 (SEQ ID NO: 17) (e) and SMYD3-A24-10-138 (SEQ ID NO: 19) (f) stimulation of IFN-γ production. The amount of IFN-γ produced by CTL was measured by IFN-γ enzyme-linked immunosorbent assay (ELISA). This result confirmed that the CTL cell strain established by each peptide stimulation showed an effective production of IFN-γ as compared with the control group. In the figure, "+" indicates the production of IFN-γ against a target cell pulsed with an appropriate peptide, and "-" indicates the production of IFN-γ against a target cell not pulsed with any peptide.

Figure 4 is a line graph depicting the production of IFN-γ stimulated by SMYD3-A02-9-335 (SEQ ID NO: 22) in CTL cell lines. The amount of IFN-γ produced by CTL was measured by IFN-γ enzyme-linked immunosorbent assay (ELISA). This result confirmed that the CTL cell strain established by the peptide stimulation showed an effective production of IFN-γ as compared with the control group. In the figure, "+" indicates the production of IFN-γ against a target cell pulsed with an appropriate peptide, and "-" indicates the production of IFN-γ against a target cell not pulsed with any peptide.

Figure 5 is a series of line graphs, including (a) to (c), depicting CTL cell lines via SMYD3-A24-9-197 (SEQ ID NO: 2) (a), SMYD3-A24-9-118 (SEQ ID NO: 8) (b) Production of IFN-γ by CTL colonies established by restriction dilution with SMYD3-A24-10-138 (SEQ ID NO: 19) (c). The amount of IFN-γ produced by CTL was measured by IFN-γ enzyme-linked immunosorbent assay (ELISA). This result confirmed that the CTL colonies established by each peptide stimulation showed efficient production of IFN-γ as compared with the control group. In the figure, "+" indicates the production of IFN-γ against a target cell pulsed with an appropriate peptide, and "-" indicates the production of IFN-γ against a target cell not pulsed with any peptide.

Figure 6 is a line graph depicting the production of IFN-γ from CTL colonies established by restriction dilution of SMYD3-A02-9-335 (SEQ ID NO: 22) in CTL cell lines. The amount of IFN-γ produced by CTL was measured by IFN-γ enzyme-linked immunosorbent assay (ELISA). This result confirmed that the CTL colonies established by the peptide stimulation showed efficient production of IFN-γ as compared with the control group. In the figure, "+" indicates the production of IFN-γ against a target cell pulsed with an appropriate peptide, and "-" indicates the production of IFN-γ against a target cell not pulsed with any peptide.

Figure 7 is a line diagram depicting the performance of SMYD3 and HLA-A*2402 Specific CTL activity of CTL colonies of target cells. COS7 cells transfected with HLA-A*2402 or full-length SMYD3 gene were used as control groups. The CTL colonies established with SMYD3-A24-9-197 (SEQ ID NO: 2) showed specific CTL activity against COS7 cells transfected with both SMYD3 and HLA-A*2402 (black diamonds). On the other hand, target cells resistant to HLA-A*2402 (triangles) or SMYD3 (circles) did not detect significant specific CTL activity.

Figure 8 is a line graph depicting specific CTL activity against CTL cell lines expressing SMYD3 and HLA-A*0201 target cells. COS7 cells transfected with HLA-A*0201 or full-length SMYD3 gene were used as control groups. The CTL cell line established with SMYD3-A02-9-335 (SEQ ID NO: 22) showed specific CTL activity against COS7 cells transfected with both SMYD3 and HLA-A*0201 (black diamond). On the other hand, target cells resistant to HLA-A*0201 (triangles) or SMYD3 (circles) did not detect significant specific CTL activity.

Although any methods or materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, the preferred materials, methods, and devices are described below. However, the description of the materials and methods is to be understood as illustrative only and not intended to be limiting. It is to be understood that the invention is not limited to the particular shapes, shapes, orientations, dimensions, materials, methodology, test procedures, and the like described herein, as such changes may be made by routine experimentation and/or optimization. In addition, the terminology used in the description is merely to be construed as a limitation of the specific embodiments or embodiments, and the scope of the invention is limited only by the scope of the accompanying claims.

The disclosures of the various publications, patents or patent applications mentioned in this specification are hereby incorporated by reference in their entirety. However, it is not understood as recognition The present invention is not earlier than the disclosure or earlier invention.

Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly used by those of ordinary skill in the art. In case of conflict, the specifications, including definitions, shall prevail. In addition, the materials, methods, and examples are merely illustrative and are intended to be limiting.

I. Definition

The terms "a" and "the" as used herein mean "at least one" unless otherwise specified.

The terms "isolated" and "purified" in connection with a substance (eg, a peptide, an antibody, a polynucleotide, etc.) mean that the substance does not substantially contain at least one substance that may be included in a natural source. Thus, an isolated or purified peptide refers to a peptide that is substantially free of cellular material such as carbohydrates, lipids, or other contaminating proteins derived from tissue derived from the peptide, or when chemically synthesized. It is virtually free of chemical precursors or other chemicals. The phrase "substantially free of cellular material" includes the preparation of a peptide which is isolated from the cellular components of its isolated cells or recombinantly produced. Thus, a peptide that is substantially free of cellular material, including a preparation of a multi-peptide, has less than about 30%, 20%, 10%, or 5% (by dry weight) of heterogeneous protein (also referred to herein as "Pollution protein"). When the peptide is produced recombinantly, it is also preferably substantially free of medium comprising a preparation of a peptide and having less than about 20%, 10%, or 5% by volume of the peptide preparation. Medium. When the peptide is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals, including a preparation of a peptide and involving a chemical precursor or other chemical that synthesizes the peptide. Less than about 30%, 20%, 10%, 5% of the peptide preparation (dry weight) meter). Specific peptides containing isolated or purified peptides, for example, by subjecting the protein preparation to sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and applying the gel to Komby's bright blue (Coomassie Brilliant Blue) is shown by the appearance of a single band such as staining. In a preferred embodiment, the peptides and polynucleotides of the invention are isolated or purified.

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of an amino acid residue. This term applies to amino acid polymers in which one or more amino acid residues may be modified residues, or non-naturally occurring residues, such as corresponding naturally occurring amino acids and corresponding naturally occurring An artificial chemical mimetic of an amino acid polymer.

The term "oligopeptide" as used herein refers to a peptide consisting of 20 or fewer amino acid residues, typically 15 amino acid residues or less. The term "nonapeptide" as used herein refers to a peptide consisting of 9 amino acid residues, and the term "nonapeptide" means a victory consisting of 10 amino acid residues. Peptide.

The term "amino acid" as used herein means naturally occurring and synthetic amino acids, and amino acid analogs and amino acid mimetics similar to those of naturally occurring amino acids. The amino acid can be an L-amino acid or a D-amino acid. Naturally occurring amino acids are encoded by the genetic code, as well as modified after translation in cells (eg, hydroxyproline, gamma-carboxy glutamic acid, and O-phosphoric acid). The term "amino acid analog" means having the same basic chemical structure as the naturally occurring amino acid (alpha carbon, carboxyl group, amine group and R group bonded to hydrogen) but having more than one modified R group or Modified backbone (e.g., homoserine, norleucine, methionine, hydrazine, methyl methionine methyl sulfonium salt). The word "amino acid mimetic" It means a chemical compound having a different structure but a similar function to a general amino acid.

The amino acid can be indicated herein by its general three-letter symbol or by the one-letter symbol suggested by the IUPAC-IUB Biochemical Nomenclature Association.

The terms "polynucleotide", "oligonucleotide" and "nucleic acid" are used interchangeably herein and, unless otherwise specified, are similar to those indicated by the generic one-letter code for the amino acid.

The terms "pharmaceutical" and "composition" are used interchangeably herein to mean a product containing a particular component in a particular amount, and any product that is directly or indirectly combined in that particular component. In addition, the modifier "medical" (such as "medicine" or "pharmaceutical composition") is intended to include products containing the active ingredient and any inert ingredients of the carrier, and is directly or indirectly combined and compounded. Or any product obtained by agglomerating any two or more of the components, or any product obtained by two or more other types of reactions or interactions of the components. Thus, the term "pharmaceutical agent" and "pharmaceutical composition" as used herein means any composition made by mixing a compound or substance of the present invention with a pharmaceutically or physiologically acceptable carrier.

The phrase "active ingredient" means a substance that has biological or physiological activity in an agent or composition. In particular, in the context of a medical composition, "active ingredient" refers to a substance that exhibits the pharmacological action of a target. For example, when a pharmaceutical agent or composition is used in the treatment or prevention of cancer, the active ingredient in the composition can directly or indirectly direct at least one biological or physiological action on cancer cells and/or tissues. Preferably, the effect comprises reducing or inhibiting the growth of cancer cells, damaging or killing cancer cells and/or tissues, and the like. In general, the indirect action of the active ingredient induces cellular toxic T lymphocytes that recognize or kill cancer cells. In match Before the party, the "active ingredient" is also called "bulk", "drug substance" or "technical product".

The term "pharmaceutically acceptable carrier" or "physiologically acceptable carrier" as used herein means a pharmaceutically or physiologically acceptable material, composition, substance, compound or carrier, including but not limited to Liquid or solid fillers, diluents, excipients, solvents or encapsulating materials.

In some embodiments, the pharmaceutical agents or compositions of the invention are particularly useful as a vaccine. In the context of the present invention, the term "vaccine" (also referred to as "immunological composition") refers to an agent or composition that has the function of ameliorating, enhancing and/or inducing anti-tumor immunity when inoculated into an animal.

If not otherwise defined, the term "cancer" refers to a cancer or tumor that overexpresses the SMYD3 gene, including but not limited to, colorectal cancer, liver cancer, breast cancer, and bladder cancer.

Unless otherwise defined, the terms "cytotoxic T lymphocytes", "cytotoxic T cells" and "CTL" are used interchangeably herein and, unless otherwise specified, mean a subgroup of T lymphocytes that can identify non-self. Cells (eg, tumors/cancer cells, cells infected with the virus) and induce death of such cells.

Unless otherwise defined, the term "HLA-A24" is used herein to mean a subtype of HLA-A24, including, but not limited to, HLA-A*2401, HLA-A*2402, HLA-A*2403, HLA-A. *2404, HLA-A*2407, HLA-A*2408, HLA-A*2420, HLA-A*2425 and HLA-A*2488.

Unless otherwise defined, the term "HLA-A2" is used herein to mean a subtype of HLA-A2, such as, but not limited to, HLA-A*0201, HLA-A*0202, HLA-A*0203, HLA- A*0204, HLA-A*0205, HLA-A*0206, HLA-A*0207, HLA-A*0210, HLA-A*0211, HLA-A*0213, HLA-A*0216, HLA-A*0218, HLA-A*0219, HLA- A*0228 and HLA-A*0250.

Unless otherwise defined, the term "set" as used herein is used with reference to a combination of reagents and other materials. It is recognized herein that the kit can include microarrays, wafers, indicia, and the like. The term "set" is not intended to be limited to a particular combination of reagents and/or materials.

When used in the context of a subject or patient, the phrase "individual (or patient's) HLA antigen is HLA-A24 or HLA-A2" means that the individual or patient carries a homozygous or heterozygous zygote The HLA-A24 or HLA-A2 antigen gene, and the HLA-A24 or HLA-A2 antigen appears as an HLA antigen in the cells of the individual or patient.

When the methods and compositions of the present invention indicate in the context that "treating" a cancer is useful, if it causes a clinical benefit, such as a reduction in the size of the cancer, a decrease in prevalence or metastatic potential, prolonged survival, metastasis or postoperative recurrence. Treatment, etc., is considered "effective". When the treatment is used prophylactically, "effectively" means delaying or preventing the formation of a cancer or preventing or reducing the clinical symptoms of the cancer. Efficacy is determined using any method known to be relevant for diagnosing or treating a particular tumor form.

If the methods and compositions of the present invention indicate in the context that "preventing" and "preventing" cancer are useful, these terms are used interchangeably herein to mean any reduction in the burden of mortality or morbidity due to disease. Prevention and prevention can occur at the "primary, secondary and tertiary levels of prevention". Primary prevention and prevention to avoid the development of disease, secondary and tertiary prevention and prevention levels include goals to prevent and prevent disease Progress and exhibits symptoms and activities that reduce the effects of established diseases by reverting function and reducing disease-related complications. Alternatively, prevention and prevention may include a wide range of therapies, the goal of which is to reduce the severity of a particular condition, such as reducing tumor proliferation and metastasis.

In the context of the present invention, treating and/or preventing cancer and/or preventing its metastasis or postoperative recurrence comprises any of the following steps: for example, surgical removal of cancer cells, inhibition of cancerous cell growth, tumor regression or recovery, induction Relieves and inhibits tumorigenesis, tumor recovery, and reduces or inhibits metastasis. Effective treatment and/or prevention of cancer reduces mortality and improves the prognosis of cancer-bearing individuals, reduces the level of tumor markers in the blood, and alleviates detectable symptoms associated with cancer. For example, reducing or ameliorating symptoms constitutes an effective treatment and/or prevention, including a 10%, 20%, 30% or more reduction, or a stable disease.

In the context of the present invention, the term "antibody" means an immunoglobulin and a fragment thereof which specifically react to a particular protein or its peptide. Antibodies can include human antibodies, primate antibodies, chimeric antibodies, bispecific antibodies, humanized antibodies, antibodies fused to other proteins or radiolabels, and antibody fragments. Furthermore, antibodies are used in the broadest sense herein, and in particular, a single antibody, a plurality of antibodies, and a multi-specific antibody (eg, a bispecific antibody) formed from at least two intact antibodies. And an antibody fragment which is capable of exhibiting a desired biological activity. "Antibody" stands for all types (eg, IgA, IgD, IgE, IgG, and IgM).

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art.

II. peptide

The peptide of the present invention described in detail below may be referred to as "SMYD3 peptide" or "SMYD3 peptide".

To demonstrate that the peptide derived from SMYD3 acts as an antigen recognized by cytotoxic T lymphocytes, analyze the peptide derived from SMYD3 (SEQ ID NO: 60, 62 or 64) to determine if it is a common HLA dual gene HLA. -A24 or antigen epitopes restricted by HLA-A2 (Date Y et al., Tissue Antigens 47: 93-101, 1996; Kondo A et al., J Immunol 155: 4307-12, 1995; Kubo RT et al., J Immunol 152: 3913-24, 1994).

Candidates for HLA-A24 binding peptides derived from SMYD3 were confirmed based on their binding affinity for HLA-A24. The following candidate peptides were identified: sequence identification number: 2 to 19.

As described above, after the T-cells were stimulated in vitro by the following peptide pulsed dendritic cells (DCs), the peptides of CTLs were successfully established: SMYD3-A24-9-197 (SEQ ID NO: 2), SMYD3 -A24-9-326 (SEQ ID NO: 4), SMYD3-A24-9-138 (SEQ ID NO: 5), SMYD3-A24-9-130 (SEQ ID NO: 6), SMYD3-A24-9- 192 (SEQ ID NO: 7), SMYD3-A24-9-118 (SEQ ID NO: 8), SMYD3-A24-9-301 (SEQ ID NO: 9), SMYD3-A24-10-260 (SEQ ID NO) : 13), SMYD3-A24-10-226 (SEQ ID NO: 14), SMYD3-A24-10-86 (SEQ ID NO: 17) and SMYD3-A24-10-138 (SEQ ID NO: 19).

Candidate for HLA-A2 binding peptide derived from SMYD3, based Confirmed by its binding affinity to HLA-A2. The following preferred peptides were identified: sequence identification number: 21 to 58.

In addition, the peptides of CTLs were successfully established after stimulation of T-cells in vitro with the following peptide pulsed dendritic cells (DCs): SMYD3-A02-9-335 (SEQ ID NO: 22), SMYD3- A02-9-118 (SEQ ID NO: 8) and SMYD3-A02-10-76 (SEQ ID NO: 16).

The CTLs established above showed potent specific CTL ability against target cells pulsed with each peptide. This result confirmed that SMYD3 is an antigen recognizable by CTLs, and the above peptide is a SMYD3 epitope peptide limited to HLA-A24 or HLA-A2, and therefore, through induction of CTL cytotoxicity, for HLA-A24 or HLA-A2 Positive patients are effective in cancer immunotherapy.

Since the SMYD3 gene is overexpressed in cancer cells and tissues, including, for example, colorectal cancer, liver cancer, breast cancer, and bladder cancer, and is almost not expressed in normal organs, it is a good immunotherapy target. Accordingly, the present invention provides a nine-peptide (a peptide composed of nine amino acids) and a ten-peptide (a peptide composed of 10 amino acids) corresponding to a CTL recognition epitope derived from SMYD3. Further, the present invention provides an isolated peptide which induces CTL, wherein the peptide consists of an immunologically active fragment of SMYD3. In certain embodiments, the invention provides peptides having amino acid sequences selected from the sequence identifiers: 2, 4, 5, 6, 7, 8, 9, 13, 14, 16, 19, and 22. . In a preferred embodiment, the peptide of the present invention is nine amino acid sequences comprising sequence identifiers: 2, 4, 5, 6, 7, 8, 9, 13, 14, 16, 17, 19 and 22. Peptide or ten peptides. Preferred embodiments of the peptide of the present invention include sequence identification numbers: 2, 4, 5, 6, 7, 8, 9, 13, 14, 16, The peptide consisting of the amino acid sequences of 17, 19 and 22.

The peptide of the present invention, particularly the nine peptide and the ten peptide of the present invention, may optionally have additional amino acid residues on the side, provided that the obtained peptide retains its cytotoxic T lymphocyte inducing ability. . This particular additional amino acid residue can be composed of any kind of amino acid, provided that the cytotoxic T lymphocyte inducing ability of the original peptide is not impaired. The invention therefore comprises a peptide having cytotoxic T lymphocyte inducing ability, in particular a peptide derived from SMYD3 (for example comprising a sequence identifier: 2, 4, 5, 6, 7, 8, 9, 13, 14, 16 a peptide of the amino acid sequence of 17, 19 or 22.). Such peptides are, for example, less than about 40 amino acids, often less than about 20 amino acids, typically less than about 15 amino acids. More specifically, the size of the peptide is preferably in the range of 10 to 40 amino acids, for example, in the range of 10 to 20 amino acids, for example, in the range of 10 to 15 amino acids.

Modifications of one, two or several amino acids in a peptide are generally known and do not affect the function of the peptide and, in some instances, even enhance the desired function of the original protein. In fact, modified peptides are known (ie, as one, two or several amino acid residues are modified (ie, substituted, added, deleted and/or inserted) compared to an original reference sequence The peptide consisting of the amino acid sequence) maintains the biological activity of the original peptide (Mark et al., Proc Natl Acad Sci USA 1984, 81: 5662-6; Zoller and Smith, Nucleic Acids Res 1982, 10:6487-500 ; Dalbadie-McFarland et al., Proc Natl Acad Sci USA 1982, 79: 6409-13). Thus, in one embodiment, the peptide of the present invention has a cytotoxic T lymphocyte eliciting ability and a sequence identification number from which one, two or even more amino acids are added, deleted, inserted and/or substituted. Both of the amino acid sequences in 2, 4, 5, 6, 7, 8, 9, 13, 14, 16, 17, 19 and 22. In other words, the peptide of the present invention has a cytotoxic T lymphocyte inducing ability and a sequence identification number in which one, two or even more amino acids are substituted, deleted, inserted and/or added: 2, 4, For the amino acid sequences of 5, 6, 7, 8, 9, 13, 14, 16, 17, 19 and 22, the modified peptide maintains the original reference peptide cytotoxic T lymphocyte evoking ability.

Those skilled in the art will appreciate that a small percentage of individual modifications (i.e., deletions, insertions, additions, and/or substitutions) to a single amino acid sequence that alters a single amino acid or an entire amino acid sequence tend to result in preservation of the original amine group. The characteristics of acid branching. Thus they are often referred to as "conservative substitution" or "conservative modification" in which a change in a protein results in a modified protein having the same function as the original protein. The conservative representation of providing functionally similar amino acids is well known in the art. Amino acid branching features which are conservatively required include, for example, hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C) , E, Q, G, H, K, S, T) and a branch having the following common functional groups or characteristics: an aliphatic branch (G, A, V, L, I, P); a hydroxyl group Chain (S, T, Y); a branch containing a sulfur atom (C, M); a branch containing a carboxylic acid and an amine (D, N, E, Q); an alkali-containing branch (R, K, H); And contains aromatic branches (H, F, Y, W). In addition, the following eight populations each comprise amino acids which are acceptable in the art to be conservatively substituted with each other: 1) alanine (A), glycine (G); 2) aspartic acid (D), bran Amino acid (E); 3) aspartic acid (N), glutamic acid (Q); 4) arginine (R), lysine (K); 5) isoleucine (I) , leucine (L), methionine (M), proline (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine (C), A Thiaminic acid (M) (see, for example, Creighton, Proteins 1984).

Such conservatively modified peptides are also considered to be peptides of the invention. However, the peptide of the present invention is not limited thereto, and may include a non-conservative modification as long as the modified peptide maintains the cytotoxic T lymphocyte-inducing ability of the original unmodified peptide. Furthermore, the modified peptide should not exclude the inducible cytotoxic T lymphocyte derived from the polymorphic variant, the interspecies homologues and the alleles of SMYD3. Peptide.

The amino acid residue can be inserted, substituted, deleted/and or added to the peptide of the present invention, or the amino acid residue can be deleted therefrom to achieve a higher binding affinity for the HLA antigen. In order to maintain the necessary cellular toxic T lymphocyte evoking ability, it is preferred to modify (ie, delete, insert, add, and/or replace) a small number (eg, 1, 2, or several) or a small percentage of amino acid. . The term "several" as used herein refers to 5 or fewer amino acids, such as 4 or 3 or less. The percentage of the modified amino acid is preferably 20% or less, more preferably 15% or less, even more preferably 10% or less, such as 1 to 5%.

When used in the context of cancer immunotherapy, the peptide of the present invention should be expressed on the surface of a cell or exosome in the form of a complex with an HLA antigen. Therefore, it is preferred to select a peptide which not only induces a cytotoxic T lymphocyte but also has a high binding affinity for an HLA antigen. To achieve this, the peptide can be modified by substitution, insertion, deletion and/or addition of amino acid residues. A modified peptide having improved binding affinity is produced. In addition to naturally occurring peptides, the rules for peptide sequences expressed by binding to HLA antigens (Kubo RT et al., J Immunol 1994, 152: 3913-24; Rammensee HG et al., Immunogenetics 1995, 41) :178-228; Kondo et al., J Immunol 1994, 155: 4307-12; Falk K, et al., Nature. 1991 May 23; 351 (6324): 290-6) are known and can be based on this Modification of the rules introduces the immunogenic peptide of the present invention.

For example, a peptide having a high HLA-A24 binding affinity tends to have a second amino acid from the N-terminus substituted with phenylalanine, tyrosine, methionine or tryptophan. Similarly, a peptide in which the C-terminal amino acid is substituted with phenylalanine, leucine, isoleucine, tryptophan or methionine tends to have high HLA-A24 binding affinity. Therefore, it is worthwhile to replace the second amino acid from the N-terminus with phenylalanine, tyrosine, methionine or tryptophan, and/or phenylalanine, leucine, isoleucine. The tryptophan or methionine is substituted with the amino acid at the C-terminus to increase the HLA-A24 binding affinity. Thus, a peptide having an amino acid sequence selected from the sequence identification numbers: 2, 4, 5, 6, 7, 8, 9, 13, 14, 17 and 19, wherein the amino acid sequence derived from the sequence identifier The second amino acid at the N-terminus is substituted with phenylalanine, tyrosine, methionine or tryptophan, and/or the C-terminal amino acid from the amino acid sequence of the sequence identifier is The substitution of phenylalanine, leucine, isoleucine, tryptophan or methionine is also included in the present invention. Further, the present invention includes a peptide comprising an amino acid sequence in which one, two or several amino acids are substituted, deleted, inserted and/or added to the sequence identification number: 2, 4, 5, The amino acid sequence of 6, 7, 8, 9, 13, 14, 17 and 19, which has one or two of the following characteristics: (a) N-terminal The second amino acid is phenylalanine, tyrosine, methionine or tryptophan; and the amino acid of terminal (b) is phenylalanine, leucine, isoleucine, tryptophan or Methionine. In a preferred embodiment, the peptide of the present invention comprises an amino acid sequence selected from the sequence identifiers: 2, 4, 5, 6, 7, 8, 9, 13, 14, 17 and 19, the N-terminal The two amino acids are substituted with phenylalanine, tyrosine, methionine or tryptophan, and/or the C-terminal amino acid is phenylalanine, leucine, isoleucine, tryptophan Or a methionine substituted amino acid sequence.

Similarly, a peptide having a high HLA-A2 binding affinity tends to have a second amino acid from the N-terminus substituted with leucine or methionine, and/or a C-substituted lysine or leucine Amino acid at the end. Therefore, it is worthwhile to replace the second amino acid from the N-terminus with leucine or methionine, and/or to replace the amino acid at the C-terminus with lysine or leucine to increase HLA-A2 binds affinity. Thus, a peptide having the amino acid sequence selected from the sequence identifiers: 8, 16, and 22, wherein the second amino acid from the N-terminus of the amino acid sequence of the sequence identifier is leucine or The methionine substitution, and/or the amino acid at the C-terminus of the amino acid sequence of the sequence identifier, is substituted with glutamic acid or leucine, and is also included in the present invention. Further, the present invention includes a peptide comprising an amino acid sequence in which one, two or several amino acids are substituted, deleted, inserted and/or added to the sequence identification numbers: 8, 16, and 22. The amino acid sequence of the peptide having one or two of the following characteristics: (a) the second amino acid at the N-terminus is leucine or methionine; and the amino acid at the C-terminus Proline or leucine. In a preferred embodiment, the peptide of the present invention comprises an amino acid sequence selected from the sequence identification numbers: 8, 16 and 22, wherein the second amino acid at the N-terminus is substituted with leucine or methionine. And/or the amino acid sequence of the C-terminal amino acid substituted with lysine or leucine.

Substitution is introduced not only at the terminal amino acid but also at the position recognized by the potential T cell receptor (TCR) of the peptide. Some studies have demonstrated that a peptide with an amino acid substitution can be equal to or better than the original, such as CAP1, p53 (264-272), Her-2/neu (369-377) or gp100 (209-217) (Zaremba Et al. Cancer Res. 57, 4570-4577, 1997, TK Hoffmann et al. J Immunol. (2002); 168(3): 1338-47., SODionne et al. Cancer Immunol immunother. (2003) 52: 199-206 and SO Dionne et al. Cancer Immunology, Immunotherapy (2004) 53, 307-314).

The invention also contemplates that the addition of one, two or several amino acids can also be added to the N and/or C terminus of the peptide of the present invention. Such modified peptides which retain the cytotoxic T lymphocyte inducing ability are also included in the present invention.

For example, the present invention provides an isolated peptide having a length of less than 15, 14, 13, 12, 11 or 10 amino acids having a cytotoxic T lymphocyte evoking ability and a group of the following: Amino acid sequence: (i) an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 4, 5, 6, 7, 8, and 9 wherein there are 1, 2 or several amine groups An acid-modified amino acid sequence, (ii) an amino acid sequence of (i), wherein the amino acid sequence has one or both of the following characteristics: (a) a second end of the N-terminus of the sequence identifier The amino acid is or modified to be an amino acid selected from the group consisting of phenylalanine, tyrosine, methionine and tryptophan; and (b) the C-terminal amine group of the sequence identifier Acid or modified to be a group of free amphetamine, leucine, isoleucine, tryptophan and methionine a monobasic acid, (iii) an amino acid sequence selected from the group consisting of SEQ ID NO: 8 and 22 wherein the amino acid sequence has 1, 2 or several amino acid modified; Ic) the amino acid sequence of (iii), wherein the amino acid sequence has one or two of the following characteristics: (a) the second amino acid of the N-terminus of the sequence identifier is or is modified to be free a monobasic acid of the group consisting of leucine and methionine; and (b) the C-terminal amino acid of the sequence identifier is or modified to be selected from the group consisting of lysine or leucine Group of monoamine acids.

Moreover, the present invention also provides an isolated peptide having a length of less than 15, 14, 13, 12 or 11 amino acids having cytotoxic T lymphocyte evoking ability and optionally free from the group consisting of Amino acid sequence: (i') an amino acid sequence of the group consisting of 13, 14, 17 and 19, wherein the amino acid sequence has 1, 2 or several amino acid modified amino acid sequences (ii') an amino acid sequence of (i'), wherein the amino acid sequence has one or both of the following characteristics: (a) the second amino acid of the N-terminus of the sequence identifier is or a monobasic acid modified to be a group consisting of free phenylalanine, tyrosine, methionine, and tryptophan; and (b) the C-terminal amino acid of the sequence identifier is or is modified to a group of monoamino acids consisting of free phenylalanine, leucine, isoleucine, tryptophan and methionine, (iii') selected from the group consisting of SEQ ID NO: 16. a group of amino acid sequences having 1, 2 or a plurality of amino acid modified amino acid sequences; (iv') an amino acid sequence of (iii'), wherein the amino acid sequence has one or two of the following characteristics: (a) the second amino acid of the N-terminus of the sequence identifier is or is modified a monobasic acid selected from the group consisting of leucine and methionine; and (b) the C-terminal amino acid of the sequence identifier is or modified to be free of lysine or leucine A group of amino acids of the group formed.

When these peptides are contacted or introduced into an antigen presenting cell, these peptides are processed in the antigen presenting cells to present a victory selected from the group consisting of (i) to (iv) and (i') to (iv'). The peptide is on it.

However, when the peptide sequence is identical to a portion of the amino acid sequence of an endogenous or exogenous protein having a different function, it may induce a negative effect, such as an autoimmune disease and/or an allergic syndrome against a specific substance. Therefore, it is desirable to first perform a homologous search using the available database to avoid the case where the sequence of the peptide conforms to the amino acid sequence of other proteins. When compared to the target peptide, the homologous search for the presence of a peptide having no peptide identical to or having 1 or 2 amino acids becomes clear, in order to increase its binding affinity to the HLA antigen, and/or The target amino acid can be modified by increasing its cytotoxic T lymphocyte inducing ability without any risk of side effects.

Although the peptide having high binding affinity for HLA antigen as described above is expected to be highly potent, the candidate peptide selected as the indicated high affinity performance is further tested for the performance of the cytotoxic T lymphocyte evoked ability. . The phrase "cytotoxic T lymphocyte-inducing ability" as used herein means the ability of a peptide to induce a cytotoxic T lymphocyte when expressed on an antigen-presenting cell. In addition, "cytotoxic T lymphocyte evoked ability" includes peptide-induced cytotoxicity T lymphocyte activation, cytotoxic T lymphocyte proliferation, to promote cytolysis by target cytotoxic T lymphocytes and to increase IFN-γ production by cytotoxic T lymphocytes.

Presenting cells (such as B-lymphocytes, macrophages, and dendritic cells) or more specifically dendritic cells derived from human peripheral blood mononuclear cells by inducing antigens carrying human MHC antigens, and stimulating peptide antigens After the cell stimulation is presented, the antigen presenting cells are mixed with CD8 positive T cells to induce cytotoxic T lymphocytes, and then IFN-γ produced and released by the cytotoxic T lymphocytes of the target cells can be measured to achieve cytotoxicity. Determination of T lymphocyte evoked ability. As such a reaction system, antigen-transgenic animals that have been produced to express human HLA can be used (for example, in Ben Mohamed L, Krishnan R, Longmate J, Auge C, Low L, Primus J, Diamond DJ, Hum Immunol 2000, 61 ( 8): 764-79, Induction of CTL response by a minimal epitope vaccine in HLA A*0201/DR1 transgenic mice:dependence on HLA class II restricted T(H)response). Alternatively, the target cells can be radiolabeled with 51Cr, and the cytotoxic activity of the cytotoxic T lymphocytes can be calculated from the radioactivity released from the target cells. Alternatively, the cytotoxic T lymphocyte-inducing ability can be measured by the antigen-presenting cells carrying the immobilized peptide, by measuring the IFN-γ produced and released by the cytotoxic T lymphocytes, and using the anti-IFN-γ single The antibody was evaluated to make the inhibition zone on the medium visible.

In addition to the above modifications, the peptide of the present invention may be linked to other peptides as long as the resulting linked peptide maintains the cytotoxic T lymphocyte inducing ability of the original peptide and, more preferably, maintains the essential HLA binding. ability. suitable Examples of "other" peptides include: the peptide of the present invention or a cytotoxic T lymphocyte-inducing peptide derived from other tumor-associated antigens. The peptide of the present invention may link one or more "other" peptides directly or indirectly via a linker. Suitable peptide internal linkers are well known in the art and include, for example, AAY (PMDaftarian et al., J Trans Med 2007, 5:26), AAA, NKRK (SEQ ID NO: 69) (RPMSutmuller) Et al., J Immunol. 2000, 165: 7308-7315) or K (S. Ota et al., Can Res. 62, 1471-1476, KSKawamura et al., J Immunol. 2002, 168: 5709-5715 ).

The above-mentioned linked peptide is referred to herein as "polytope", that is, two or more groups that have the potential to generate an immune response or immune response stimulating peptides can be joined together by various arrangements (eg, linkage, overlap). ). The polytype (or nucleic acid encoding the polytype) can be administered to, for example, an animal in a standard immunoassay procedure to test the ability of the polytype to stimulate, enhance, and/or elicit an immune response.

The peptides can be joined together directly, or via the use of flanking sequences to form a polytype, and the use of polytypes as vaccines is well known in the art (see, for example, Thomson et al., Proc. Natl. Acad. Sci USA 92(13): 5845-5849, 1995; Gilbert et al., Nature Biotechnol. 15(12): 1280-1284, 1997; Thomson et al., J Immunol. 157(2): 822-826 , 1996; Tarn et al., J Exp. Med. 171(1): 299-306, 1990). Polymorphisms containing different numbers of epitopes and combinations can be prepared and tested for cytotoxic T lymphocyte recognition and tested for increased immune response.

The peptide of the present invention can be further linked to other suitable substances, As long as it retains the cytotoxic T lymphocyte inducing ability of the original peptide. Such suitable materials may include: peptides, lipids, sugars and sugar chains, ethyl sulfonyl groups, natural and synthetic polymers, and the like. The peptide may comprise modifications such as saccharification, side chain oxidation or phosphorylation; as long as the modification does not impair the biological activity of the peptide described herein. Such types of modifications can be implemented to provide additional functionality (eg, targeting functions, and delivery functions), or to stabilize the multi-peptide.

For example, in order to increase the in vivo stability of a multi-peptide, it is known in the art to introduce a D-amino acid, an amino acid mimetic or an unnatural amino acid; this concept can also be applied to the multi-peptide. The stability of the peptide can be analyzed in a variety of ways. For example, peptidase and various biological mediators such as human plasma and serum can be used to test for stability (see, for example, Verhoef et al., Eur J Drug Metab Pharmacokin 1986, 11:291-302).

Furthermore, as described above, in the modified peptide obtained by substituting, deleting, inserting or adding one, two or several amino acid residues, the screening or selection may be the same as or different from the original peptide. More active. Accordingly, the invention also provides methods of screening or selecting modified peptides having the same or higher activity than the original peptide. For example, the method comprises the steps of: a: modifying (substituting, deleting, inserting and/or increasing) at least one amino acid residue of the peptide of the invention; b: determining the activity of the peptide modified in step a And c: screening or selecting a peptide having the same or higher activity than the original peptide.

Here, the activity to be analyzed may include MHC binding activity, APC or CTL inducing ability, and cytotoxic activity. The peptide activity is preferably cytotoxic Killing T lymphocyte inducing ability.

In a preferred embodiment, the invention provides a method of screening for a peptide having the ability to induce a cytotoxic T lymphocyte having a specific cytotoxicity against a cell derived from a fragment of SMYD3 Wherein the method comprises the steps of: (i) providing a candidate sequence which is substituted, deleted, inserted and/or increased by the original amino acid by one, two or several amino acid residues; a sequence consisting of the original amino acid sequence selected from the group consisting of: 2, 4, 5, 6, 7, 8, 9, 13, 14, 16, 17, 19, and 22; Ii) selecting a candidate sequence which, except for SMYD3, does not have substantially significant homology (or sequence identity) derived from any known human gene product; (iii) will be in step (ii) The peptide formed by the selected candidate sequence is contacted with the antigen-presenting cell; (iv) the antigen-presenting cell of step (iii) is contacted with CD8-positive T cells; and (v) the cytotoxic T lymphocyte-inducing ability is identified to be the same Or higher than the original amino acid sequence A peptide of a peptide.

When the peptide of the present invention comprises a cysteine residue (for example, sequence number: 2, 7, 8, or 14), the peptide tends to pass through a disulfide bond between the SH groups of the cysteine residue. Form a dimer. Thus, the invention extends to a peptide dimer having CTL inducibility. The two SYMD3 peptide monomers can be combined by a disulfide bond present in the monomer or added to the cysteine residue of the monomer to form the peptide dimer of the present invention. When one amino acid sequence of each peptide contains a cysteine residue Cys, a disulfide bond can be formed between the cysteine residues to form the oligopeptide of the present invention. Alternatively, if the amino acid sequence itself does not have a cysteine, a disulfide bond may be formed between the cysteine residues added to the amino acid sequence. For example, a cysteine residue (Cys) can be introduced into the C-terminus and/or N-terminus of each peptide to form a disulfide-dependent bond. In addition, one or more cysteine residues may also be inserted into the amino acid sequence of each peptide.

III. Preparation of SMYD3 peptide

The peptide of the present invention can be prepared using well-known techniques. For example, the peptide can be prepared by synthetic, recombinant DNA techniques or chemical synthesis. The peptide of the present invention can be synthesized individually or as a longer polypeptide comprising two or more peptides. The peptide can be isolated, that is, purified or isolated to be substantially free of other naturally occurring host proteins and fragments thereof, or any other chemical.

The peptide of the present invention may comprise modifications such as saccharification, side chain oxidation or phosphorylation; as long as such modifications do not impair the biological activity of the original peptide. Other illustrative modifications that may be used include the introduction of D-amino acids or other amino acid mimetics, for example to increase the serum half-life of the peptide.

The peptide of the present invention can be obtained by chemical synthesis depending on the selected amino acid sequence. For example, conventional peptide synthesis methods which can be used for synthesis include: (i) Peptide Synthesis, Interscience, New York, 1966; (ii) The Proteins, Vol. 2, Academic Press, New York, 1976; (iii) Peptide Synthesis (in) Japanese), Maruzen Co., 1975; (iv) Basics and Experiment of Peptide Synthesis (in Japanese), Maruzen Co., 1985; (v) Development of Pharmaceuticals (second volume) (in Japanese), Vol.14 (peptide synthesis), Hirokawa, 1991; (vi) WO99/67288; and (vii) Barany G. & Merrifield RB, Peptides Vol. 2, "Solid Phase Peptide Synthesis", Academic Press, New York , 1980, 100-118.

Alternatively, the peptide can be obtained by any genetic engineering method known to be used to produce a peptide (for example, Morrison J, J Bacteriology 1977, 132: 349-51; Clark-Curtiss & Curtiss, Methods in Enzymology (eds. Wu) Et al.) 1983, 101: 347-62). For example, a suitable vector harboring a polynucleotide encoding a target peptide in a expressible form (e.g., downstream of a regulatory sequence corresponding to a promoter sequence) is first prepared and transformed into a suitable host cell. Such vectors and host cells are also provided by the invention. The host cell is then cultured to produce the peptide of interest. The peptide can also be produced in vitro using an in vitro translation system.

When the peptide of the present invention is a peptide dimer, such a dimer can be formed by a known method. For example, if the peptide monomer comprises a pair of cysteine residues, for example, all protecting groups including the cysteine side chain are removed, and then the resulting monomer solution is subjected to air under alkaline conditions. The peptide dimer is prepared by air-oxidation or by adding an oxidizing agent under basic or acidic conditions to form a disulfide bond. The oxidizing agent includes, for example, iodine, dimethyl hydrazine (DMSO), and potassium ferricyanide.

When each peptide monomer includes two or more cysteine residues, the peptide dimer of the present invention can also be produced by the above method. In this case, isomers having different types of disulfide bonds can be obtained. Alternatively, the peptide of the present invention can be prepared by selecting a combination of protecting groups of the cysteine side chain. Dimer. The protecting group combination includes, for example, a combination of methylbenzyl (MeBzl) and acetaminomethyl (Acm), a combination of trityl (Trt) and acetaminomethyl (Acm), 3-nitro- Combination of 2-pyridylthio (Npys) with acetaminomethyl (Acm), in combination with S-tert-butyl (S-Bu-t) and acetaminomethyl (Acm). For example, in the case of a combination of methylbenzyl (MeBzl) and acetaminomethyl (Acm), the peptide dimer can be prepared by removing methylbenzyl (MeBzl) and on the side of cysteine. a protecting group other than the chain, and the resulting monomer solution is subjected to air-oxidation to form a disulfide bond between the de-protected cysteine residues, followed by deprotection with iodine and Oxidation forms a disulfide bond between the cysteine residues previously protected by acetaminomethyl (Acm).

IV. Polynucleotides

The present invention provides a polynucleotide encoding any of the aforementioned peptides of the present invention. Including polynucleotides derived from the naturally occurring SMYD3 gene (eg, GenBank Accession No. NM_022743.2 (SEQ ID NO: 59), NM_001167740 (SEQ ID NO: 61), or AB057595 (SEQ ID NO: 63), It has a nucleotide sequence that is conservatively modified. The phrase "conservatively modified nucleotide sequence" as used herein refers to a sequence encoding the same or substantially the same amino acid sequence. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids are encoded into any given protein. For example, the codons GCA, GCC, GCG, and GCU are all encoded as amino acid alanine. Thus, when a codon indicates alanine at each position, the codon can be altered to any corresponding codon without altering the encoded multi-peptide. This nucleic acid variation is a "silent variation", which is a conservatively modified variation. Each of the nucleic acids encoded herein as a peptide also recites all possible silent variations of the nucleic acid. In the technical field A person with ordinary knowledge will recognize each codon in a nucleic acid (except AUG and TGG, AUG is usually the only codon for methionine, TGG is usually the only codon for tryptophan) can be modified to produce Functionally identical molecules. Thus, each silent variation of a nucleic acid encoded as a peptide is implicitly recited in each disclosed sequence.

The polynucleotide of the present invention may be composed of DNA, RNA, and derivatives thereof. As is well known in the art, DNA molecules are suitably composed of a base such as naturally occurring bases A, T, C and G, and the T line is replaced by U in RNA. Those of ordinary skill in the art will recognize that non-naturally occurring bases are also included in the polynucleotide.

Polynucleotides of the invention can be encoded as a plurality of peptides of the invention with or without an intermediate amino acid sequence. For example, the intermediate amino acid sequence can provide a cleavage site (eg, an enzyme recognition sequence) of the polynucleotide or the translated peptide. Again, the polynucleotide may comprise any additional sequence for the coding sequence encoded as the peptide of the invention. For example, the polynucleotide may be a recombinant polynucleotide comprising a regulatory sequence required for expression of the peptide, or may be a expression vector (plastid) having a marker gene, and the like. In general, such recombinant polynucleotides can be prepared using conventional recombinant techniques, using, for example, polymerases and endonucleases to manipulate polynucleotides.

Both recombinant and chemical synthesis techniques can be used to produce the polynucleotides of the invention. For example, a polynucleotide can be produced by insertion into a suitable vector which can be expressed when transfected into a competent cell. Alternatively, the polynucleotide can be amplified using PCR techniques or expressed in a suitable host (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor). Laboratory, New York, 1989). Alternatively, polynucleotides can be synthesized using solid phase techniques as described by Beaucage SL & Iyer RP, Tetrahedron 1992, 48: 2223-311; Matthes et al., EMBO J 1984, 3: 801-5.

V.Exosome body (exosomes)

The present invention further provides intercellular vesicles, called exosome bodies, which exhibit a complex formed between the peptide of the present invention and the surface of human leukocyte antigen. For example, exosomes can be prepared by using the methods detailed in Japanese Patent Publication No. 11-510507 and WO99/03499 and using antigen-expressing cells obtained from a patient receiving treatment and/or avoidance. The exosome of the present invention can be vaccinated like a vaccine in a manner similar to the peptide of the present invention.

The HLA antigen form included in the complex must be compatible with the HLA antigen form of the individual in need of treatment and/or prevention. For example, in the Japanese ethnic group, HLA-A24 and HLA-A2, especially HLA-A*2402, HLA-A*0201 and HLA-A*0206 are common, and therefore are suitable for the treatment of Japanese patients. . The use of HLA-A24 or HLA-A2, which is often expressed in the Japanese and Caucasian populations, helps to obtain effective results, and subtypes such as HLA-A*2402, HLA-A*0201 and HLA-A* 0206 is also available. Generally, clinically, the HLA antigen form of the patient to be treated is pre-investigated, which can appropriately select a peptide having a high binding affinity for the specific antigen or exhibiting cytotoxic T lymphocyte-inducing activity via the antigen. Peptide. Furthermore, in order to obtain a peptide having both HLA antigen high binding affinity and cytotoxic T lymphocyte evokance, 1, 2 or several amine groups can be performed based on the amino acid sequence of the naturally occurring SMYD3 partial peptide. Acid substitution, insertion Enter, delete, and/or add.

When the exosome of the present invention possesses an HLA-A24 type HLA antigen, it has an amino acid selected from the sequence identification numbers: 2, 4, 5, 6, 7, 8, 9, 13, 14, and 19. Sequence peptides have particular utility.

Alternatively, when the exosome of the present invention possesses an HLA-A2 type HLA antigen, the peptide having the amino acid sequence selected from the sequence numbers: 8, 16, and 22 has a particular effect.

In some embodiments, the exosome of the present invention is a surface exhibiting a complex of a peptide of the present invention and an HLA-A24 or HLA-A2 antigen. In some exemplary embodiments, the exosome of the present invention exhibits a victory over the surface of an amino acid sequence having a sequence identifier of: 2, 4, 5, 6, 7, 8, 9, 13, 14, or 19. a peptide (or a modified peptide thereof) complexed with HLA-A24, or a peptide having the amino acid sequence of SEQ ID NO: 8, 16, or 22 (or a modified peptide thereof) and HLA-A2 Complex.

VI. Antigen presenting cells (APCs)

The present invention also provides an isolated antigen-presenting cell which exhibits a complex formed between the HLA antigen and the peptide of the present invention on its surface. The antigen presenting cells may be derived from a patient being treated and/or prevented, and may be administered as a vaccine by itself or in combination with other drugs including the peptide of the present invention, exosome or cytotoxic T lymphocytes. .

The antigen presenting cells are not limited to a particular type of cell. Examples of antigen-presenting cells include, but are not limited to, dendritic cells, Langerhans cells, macrophages, B cells, and activated T cells, which are known to represent proteinaceous antigens on their cell surface. Lymphocyte knowledge. Since dendritic cells are typical antigen-presenting cells, which have the strongest cytotoxic T lymphocyte-inducing activity in antigen-presenting cells, dendritic cells can be preferably used as the antigen-presenting cells of the present invention.

For example, antigen-presenting cells can be obtained by inducing dendritic cells derived from peripheral blood mononuclear cells and then in vitro ( ex vivo ) or in vivo ( in vivo ) contact (stimulation) with the peptide of the present invention. . When the peptide of the present invention is administered to a body, an antigen-presenting cell exhibiting the peptide of the present invention is induced in the body of the individual. Therefore, the antigen-presenting cells of the present invention can be obtained by collecting antigen-presenting cells from the individual after administering one or more peptides of the present invention to one body. Alternatively, the antigen-presenting cells of the present invention can be obtained by contacting antigen-presenting cells that have been collected from an individual with the peptide of the present invention.

The antigen presenting cells of the present invention can be administered to a body by itself or in combination with other drugs including the peptide, exocytosis or cytotoxic T lymphocytes of the present invention to induce an anti-cancer immune response in the individual. For example, administration in ex vivo may comprise the steps of: a: collecting antigen presenting cells from the first individual, b: contacting the antigen presenting cells of step a with the peptide of the invention, and c: step b The antigen present cells are administered to a second individual.

The first individual and the second individual may be the same individual or may be different individuals. The antigen presenting cells obtained from step b can be formulated and administered to vaccine for the treatment and/or prevention of cancer including, but not limited to, for example, colorectal cancer, liver cancer, breast cancer, and bladder cancer.

In the context of the present invention, one or more peptides of the invention may be used to make a pharmaceutical composition that induces APC. Provided herein for manufacturing inducible APC The method or process of the pharmaceutical composition preferably comprises the step of mixing or formulating the peptide of the present invention with a pharmaceutically acceptable carrier.

The invention also provides the use of the peptide of the invention to induce APC.

According to an aspect of the present invention, the antigen presenting cells of the present invention have a cytotoxic T lymphocyte evoking ability. In the context of antigen presenting cells, the expression "cytotoxic T lymphocyte evokengability" means that the antigen presenting cells induce cytotoxic T lymphocytes when contacted with CD8 positive T cells. Furthermore, "cytotoxic T lymphocyte evoking ability" includes antigen-presenting cells inducing cytotoxic T lymphocyte activation, cytotoxic T lymphocyte proliferation, and promoting the lysis of target cells by cytotoxic T lymphocytes. Increases the ability to produce IFN-[gamma] by cytotoxic T lymphocytes. In particular, the antigen presenting cells of the present invention have the ability to induce a cytotoxic T lymphocyte specific to SMYD3. The antigen presentation of the cytotoxic T lymphocyte-inducing ability can be prepared by a method comprising the step of transferring a polynucleotide encoding the peptide of the present invention to an antigen-presenting cell in vitro and the above method. cell. The introduced polynucleotide can be in the form of DNA or RNA. Examples of the introduction method include, without particular limitation, various methods generally performed in the field, and for example, lipofection, electroporation, and calcium phosphate methods can be used. More specifically, it can be performed as Reeves ME et al., Cancer Res 1996, 56: 5672-7; Butterfield LH et al., J Immunol 1998, 161: 5607-13; Boczkowski D et al., J Exp Med 1996 , 184: 465-72; described in Japanese Patent Publication No. JP2000-509281. By transferring a gene encoding the peptide of the present invention into an antigen-presenting cell, the gene undergoes transcription, translation, and the like in the cell, and the protein obtained thereafter is processed by MHC Class I or Class II, and is presented via a presentation pathway. Part of the peptide. Alternatively, the APC of the present invention can be prepared using a method comprising simply containing APC and the peptide of the present invention.

In some embodiments, the antigen presenting cells of the present invention are antigen presenting cells that exhibit an HLA-A24 or HLA-A2 antigen and a complex of the peptide of the present invention on its surface. In some exemplary embodiments, the antigen presenting cell of the present invention exhibits an amino acid sequence having a sequence identifier of: 2, 4, 5, 6, 7, 8, 9, 13, 14, 17 or 19 on its surface. a peptide (or a modified peptide thereof) complexed with HLA-A24, or a peptide having the amino acid sequence of SEQ ID NO: 8, 16, or 22 (or a modified peptide thereof) and HLA-A2 Complex.

VII. Cytotoxic T lymphocytes (CTLs)

The induction of anti-peptide of the present invention is any T-lymphocytes kill cells enhanced in vivo (in vivo) the immune response to the cancer cells of the subject, and thus for its part, can be similar to the embodiment of the peptides per se as a vaccine . The invention therefore provides an isolated cytotoxic T lymphocyte which is specifically induced or activated by any of the peptides of the invention.

Such cytotoxic T lymphocytes can be obtained by (1) administering the peptide of the present invention to one body; (2) presenting antigen-presenting cells from the individual to CD8-positive T cells or peripheral blood mononuclear cells. Lymphocytes are contacted with the peptide of the present invention in vitro (stimulation); (3) CD8-positive T cells or peripheral blood mononuclear lymphocytes are expressed in vitro and in vitro with the HLA antigen and the peptide of the present invention. The complex is present on the surface of the antigen presenting cells or exosome contacts; or (4) introducing a polynucleotide encoding a subunit of both T cell receptors or a polynucleoside encoding each T cell receptor subunit The acid, T cell receptor subunit can form a T cell receptor with the ability to bind to a complex of one of the HLA antigens on the cell surface and the peptide of the present invention. Such antigen-presenting cells or exosomes can be prepared by the above method. The details of the method of (4) are described in the paragraph below "VIII. T cell receptor (TCR)".

The cytotoxic T lymphocyte of the present invention may be derived from a patient who is treated and/or prevented, and by itself or in combination with other drugs including the peptide of the present invention for regulating effect, antigen presenting cells or exocytosis Can be cast. The obtained cytotoxic T lymphocytes are specifically resistant to the action of the target cells of the peptide of the present invention, for example, the same peptide for induction. The target cell may be a cell whose endogenous expression is SMYD3, such as a cancer cell, or a cell transfected with the SMYD3 gene; and the cell which exhibits the peptide of the present invention on the cell surface by stimulation with a peptide, may also be activated The goal of a cytotoxic T lymphocyte attack.

In some embodiments, the cytotoxic T lymphocytes of the invention are recognizable on cells whose surface exhibits a complex of HLA-A24 or HLA-A2 antigen and a peptide of the invention. In the context of cytotoxic T lymphocytes, the phrase "identifying a cell" means binding to a complex of the HLA-A24 or HLA-A2 antigen on the cell surface via the TCR and the peptide of the present invention via its TCR and showing resistance to this Specific cellular toxic T lymphocyte activity of cells. Herein, "specific cytotoxic T lymphocyte activity" means a cell which exhibits an anti-HLA-A24 or HLA-A2 antigen and a complex of the peptide of the present invention, but not other cells. Accordingly, the present invention encompasses cytotoxic cytotoxic T lymphocytes which exhibit cytotoxicity specific to cells exhibiting the peptide of the present invention. In some exemplary embodiments, the cytotoxic T lymphocytes of the present invention can be identified by HLA-A24 to have a sequence identifier of: 2, 4, 5, 6, 7, 8, 9, 13, 14, 17, or 19. A cell of a peptide of the amino acid sequence (or a modified peptide thereof), or identified by HLA-A2, having a sequence identifier: 8, 16 or A cell of a peptide of 22 amino acid sequence (or a modified peptide thereof). In a preferred embodiment, such cytotoxic T lymphocytes of the invention recognize cells expressing SMYD3 and HLA-A24 or HLA-A2 (eg, HLA-A2 positive cancer cells) (eg, HLA-A24 positive cancer) Cells) and showed cytotoxicity to this cell.

VIII. T cell receptor (TCR)

The invention also provides a composition comprising one or more polynucleotides encoding a T cell receptor subunit or a polynucleotide encoding each T cell receptor subunit, wherein the subunits are formed The T cell receptor is capable of forming a complex of the HLA antigen and the peptide of the present invention on the cell surface. The method of using it can also be expected. The subunit of the T cell receptor has the ability to form a T cell receptor that confers T cells specific to anti-tumor cells, and the tumor cells express SMYD3. The cytotoxic T lymphocytes can be identified by using the methods known in the art to encode the respective alpha- and beta-branched polynucleotides of the T cell receptor subunit of the cytotoxic T lymphocyte. Induced by the peptide of the present invention (WO2007/032255 and Morgan et al., J Immunol, 171, 3288 (2003)). For example, it is preferred to analyze T cell receptor subunits by a polymerase chain reaction method. The polymerase chain reaction primer for analysis may be, for example, (5'-gtctaccaggcattcgcttcat-3') as a 5' side primer (SEQ ID NO: 65), and a 3-TRa-C primer specific to the C region of the TCR alpha chain. (5'-tcagctggaccacagccgcagcgt-3') (SEQ ID NO: 66), 3-TRb-C1 primer (5'-tcagaaatcctttctcttgac-3') (sequence identification number: 67) specific to the C1 region of the TCR beta chain, or specific The 3-TRb-C2 primer (5'-ctagcctctggaatcctttctctt-3') (SEQ ID NO: 68) in the C2 region of the TCR beta chain is referred to as a 3' side primer, but is not limited thereto. The elicited T cell receptor can bind to the target cells exhibiting the peptide of the present invention with high affinity, and can efficiently kill target cells expressing the peptide of the present invention in vivo and in vitro as needed.

Polynucleotides encoding both T cell receptor subunits or polynucleotides encoding each of the T cell receptor subunits can be incorporated into a suitable vector, such as a retroviral vector. These vectors are well known in the art. Typically, the polynucleotide or vector comprising the same can be transferred to a T cell (e.g., a CD8 positive T cell), such as a T cell from a patient. Usefully, the present invention provides an off-the-shelf composition that allows rapid modification of T cells (or other mammalian T cells) possessed by a patient to rapidly and easily produce a modified cancer cell killing characteristic. T cells.

The specific T cell receptor against the peptide of the present invention has the ability to specifically recognize a complex of one of the peptides of the present invention and an HLA molecule, and to give T cell resistance when the T cell receptor is expressed on the surface of the T cell. The specific activity of the target cell of the complex of the peptide of the present invention and the HLA antigen. The essential activity can be confirmed by any known method, and the target cell can be specifically identified by introducing a cytotoxic T lymphocyte prepared by encoding a polynucleotide encoding such a T cell receptor subunit. . Preferred examples of such methods include, for example, HLA multimer staining analysis using HLA molecules and the peptide of the present invention, and ELISPOT analysis. By ELISPOT analysis, it was confirmed that the cytotoxic T lymphocytes prepared by the above method can specifically recognize the target cells, and the signals generated by the intracellular transmission are recognized. Further, it was confirmed by a known method that the cytotoxic T lymphocytes prepared as described above have specific cytotoxic activity against the target cells. Desirable examples of such methods, for example, determine cytotoxic activity against a target cell, such as a chromium release assay.

The invention also provides a cytotoxic T lymphocyte prepared by transduction of a polynucleotide encoding both a T cell receptor subunit or a polynucleotide encoding each of the T cell receptor subunits, wherein The T cell receptor formed by such a T cell receptor subunit can be combined with the SMYD3 peptide (eg, sequence identifiers: 2, 4, 5, 6, 7, 8, 9, 13, 14, 17 and 19) Binding to a complex of HLA-A24 antigen, it is also possible to bind to a complex of a peptide of sequence number: 8, 16 and 22 with an HLA-A2 antigen.

Transduced cytotoxic T lymphocytes can be self-directed to cancer cells in vivo and can be expanded in vitro by well-known culture methods (eg Kawakami et al., J Immunol., 142). , 3452-3461 (1989)). The cytotoxic T lymphocytes of the present invention can also be used to form a consensus immunological composition which is effective in treating or preventing cancer in one or both of the patients in need of treatment or protection (see WO2006/031221, the contents of which are incorporated herein by reference. ).

IX. Pharmaceutical composition

The invention also provides a pharmaceutical agent or composition comprising at least one active ingredient selected from the group consisting of: (a) a peptide of the invention; (b) a polynucleotide encoding the peptide of the invention in an expressible form; c) an antigen presenting cell of the invention; (d) an exosome of the invention; and (e) a cytotoxic T lymphocyte of the invention.

Since the expression of SMYD3 is particularly increased in cancer compared to normal tissues, examples include, but are not limited to, colorectal cancer, liver cancer, breast cancer, and bladder cancer, and the peptide or polynucleoside of the present invention is compared to normal tissues. Acid can be used Induction of an immune response against cancer or tumor cells and is therefore suitable for the treatment and/or prevention of cancer, and/or for the prevention of metastasis or its postoperative recurrence. Accordingly, the present invention provides a pharmaceutical composition or medicament formulated for the treatment and/or prevention of a primary cancer, and/or for preventing metastasis or post-operative recurrence thereof, the composition or medicament comprising one or more of the present invention The peptide or polynucleotide is the active ingredient. Alternatively, the peptide of the present invention may be expressed on the surface of any of the aforementioned exosome or cells, such as antigen presenting cells, for use as a pharmaceutical composition or medicament. Furthermore, the above cytotoxic T lymphocytes which are subject to any of the peptides of the present invention can also be used as an active ingredient of the pharmaceutical composition or medicament of the present invention.

Accordingly, the invention provides an agent or composition comprising at least one active ingredient selected from the group consisting of: (a) a peptide of the invention; (b) a polynucleotide encoding a peptide of the invention in an expressible form; The antigen of the present invention is a cell or an exocytosis; (d) an exosome of the present invention; and (e) a cytotoxic T lymphocyte of the present invention.

In this pharmaceutical or pharmaceutical composition, the peptide is present in a therapeutically or pharmaceutically effective amount.

A pharmaceutical composition or medicament of the present invention can also be used, for example, as a vaccine. In the context of the present invention, the phrase "vaccine" (also referred to as "immunogenic composition") means a medicament or composition that has an improvement, enhancement and/or induction of anti-tumor immunity by inoculation into an animal. ability. In other words, the invention provides a pharmaceutical agent or composition for inducing an anti-cancer immune response in an antibody.

The pharmaceutical composition or medicament of the present invention can treat and/or inhibit an individual or The patient's cancer, and / or prevention of metastasis or postoperative recurrence, the individual or patient includes humans and any other mammals, including, but not limited to, mice, rats, guinea pigs, rabbits, cats, dogs, sheep, goats, Pigs, cattle, horses, monkeys, baboons and chimpanzees, especially commercial animals or livestock. In certain embodiments, the pharmaceutical agents or compositions of the invention may be formulated to administer an HLA antigen to HLA-A24 or HLA-A2.

In other embodiments, the present invention further provides an active ingredient for use in the preparation of a pharmaceutical composition or medicament for treating and/or preventing cancer or neoplastic disease, and/or preventing metastasis or postoperative recurrence, wherein the active ingredient Selectively: (a) a peptide of the present invention; (b) a polynucleotide which is encoded in the expressible form of the peptide of the present invention; (c) an antigen presenting cell (APC) which exhibits the peptide of the present invention on the surface; (d) presenting the peptide of the present invention on the surface; and (e) the cytotoxic T lymphocyte of the present invention.

Alternatively, the present invention further provides for the use of an active ingredient for the treatment and/or prevention of cancer or tumor, and/or prevention of metastasis or postoperative recurrence, wherein the active ingredient is selected from: (a) the peptide of the present invention. (b) a polynucleotide which is expressed in a expressible form as a peptide of the present invention; (c) an antigen-presenting cell which exhibits the peptide of the present invention on the surface; (d) a surface-expressing peptide of the present invention; And (e) a cytotoxic T lymphocyte of the invention.

Alternatively, the invention further provides a preparation for treatment and/or prevention A method or process for treating a cancer or a tumor, and/or a pharmaceutical composition for preventing metastasis or recurrence thereof, wherein the method or process comprises formulating a pharmaceutically or physiologically acceptable carrier and an active ingredient, the active ingredient being selected From: (a) a peptide of the present invention; (b) a polynucleotide which is encoded in the expressible form of the peptide of the present invention; (c) an antigen-presenting cell which exhibits the peptide of the present invention on the surface; (d) on the surface Presenting the peptide of the present invention; and (e) the cytotoxic T lymphocytes of the present invention.

In other embodiments, the present invention further provides a method or process for preparing a pharmaceutical composition for treating and/or preventing cancer or a tumor, and/or preventing its metastasis or postoperative recurrence, wherein the method or process includes mixing An active ingredient and a pharmaceutically or physiologically acceptable carrier, wherein the active ingredient is selected from the group consisting of: (a) a peptide of the invention; (b) a polynucleotide which is encoded in the form of a peptide of the invention; An antigen presenting cell exhibiting the peptide of the present invention on the surface; (d) a sputum excipient that exhibits the peptide of the present invention on the surface; and (e) a cytotoxic T lymphocyte of the present invention.

In other embodiments, the invention also provides a method of treating and/or preventing cancer or a tumor, and/or preventing its metastasis or postoperative recurrence, wherein the method comprises administering to the subject at least one active ingredient: (a) a peptide of the present invention; (b) a polynucleotide which is expressed in a expressible form as a peptide of the present invention; (c) an antigen-presenting cell which exhibits a peptide of the present invention on the surface; (d) presents the present invention on the surface Peptide exosome; and (e) Cytotoxic T lymphocytes of the invention.

According to the present invention, peptides having sequence identifiers: 2, 4, 5, 6, 7, 8, 9, 13, 14, 17 and 19 are shown as HLA-A24 restricted epitope peptides, An effective and specific immune response against cancers expressing HLA-A24 and SMYD3 can be induced in one body, and the sequence identification numbers: 2, 4, 5, 6, 7, 8, 9, 13, 14, 17 and 19 are Shown as an HLA-A2 restricted epitope peptide, it induces an effective and specific immune response against cancers expressing HLA-A2 and SMYD3 in one body. Therefore, a pharmaceutical composition or medicament comprising any peptide selected from the amino acid sequence of sequence identification numbers: 2, 4, 5, 6, 7, 8, 9, 13, 14, 17 and 19 is particularly suitable for administration. The individual whose HLA antigen is HLA-A24. On the other hand, a pharmaceutical composition or agent comprising any of the peptides selected from the amino acid sequences of sequence identification numbers: 8, 16 and 22 is particularly suitable for administration to an individual having an HLA antigen of HLA-A2. The peptide content in such an agent or composition can be an amount effective to significantly induce an effective and specific immune response in a patient with cancer exhibiting SMYD3. Pharmaceutical compositions or agents containing a polynucleotide encoding any of the peptides (i.e., the polynucleotide of the present invention) are also suitable.

Cancers treated and/or prevented by the pharmaceutical compositions or agents of the invention include, but are not limited to, any of the cancers associated with SMYD3, including, for example, but not limited to, colorectal cancer, liver cancer, breast cancer, and bladder cancer. A cancer exhibiting HLA-A24 (i.e., an HLA-A24-positive cancer) or a cancer exhibiting HLA-A2 (i.e., an HLA-A2-positive cancer) is preferred.

In addition to the above active ingredients, the pharmaceutical composition or medicament of the present invention may contain other peptides having the ability to induce cytotoxic T lymphocytes against cancer cells, other polynucleotides encoding other peptides, and others exhibiting other peptides. Cells, and their analogs. Such "other" peptides that have the ability to induce cytotoxic T lymphocytes against cancer cells include, but are not limited to, peptides derived from cancer specific antigens (eg, established TAAs).

If desired, the pharmaceutical composition or medicament of the present invention may optionally include other therapeutic substances as additional active ingredients as long as the substance does not inhibit the active ingredient of the present invention, such as any of the peptides, polynucleotides, exosome, antigen of the present invention. Presents the anti-tumor efficacy of cellular or cytotoxic T lymphocytes. For example, the formulation may include anti-inflammatory substances, analgesics, chemotherapeutic agents, and the like. In addition to including other therapeutic substances in the agent itself, the agent of the invention may be administered sequentially or simultaneously with one or more other pharmaceutical compositions. The amount of the pharmaceutical and pharmaceutical composition will depend, for example, on the form of the pharmaceutical composition employed, the disease to be treated, and the dosage plan and route.

It will be appreciated by those skilled in the art that, in addition to the ingredients specifically recited herein, the pharmaceutical compositions or agents of the present invention may include other materials conventionally employed in the form of formulations discussed in the art.

In one embodiment of the invention, the pharmaceutical compositions or medicaments of the invention may be packaged in manufactured articles and kits that contain materials for the pathological condition of the disease being treated, such as cancer. The article of manufacture may comprise a container of any of the pharmaceutical compositions or medicaments of the invention having a label. Suitable containers include bottles, vials and test tubes. The container can be formed from a variety of materials such as glass or plastic. The label on the container indicates that the composition or agent is one or more of the conditions used to treat or prevent the disease. The label can also indicate medication instructions and the like.

In addition to the above-described containers, the kit comprising the pharmaceutical composition or medicament of the present invention may optionally include a second container for storing a pharmaceutically acceptable diluent. May include other materials that are needed from a business or user perspective, including Other buffer solutions, diluents, filters, needles, syringes and copies with instructions for use.

The pharmaceutical compositions or medicaments of the present invention may optionally be packaged in a package or dispenser device which may contain one or more unit dosage forms of the active ingredient. The package may for example comprise a metal or plastic foil, such as a blister pack. The package or dispenser may contain a dosing indication.

(1) A pharmaceutical composition comprising the peptide as an active ingredient:

The peptide of the present invention can be administered directly as a pharmaceutical composition or medicament, or can be formulated in a conventional formulation as needed. In the following examples, in addition to the peptide of the present invention, a carrier, an excipient or the like which is generally used for pharmaceutical use may be appropriately contained without particular limitation. Examples of the carrier include, but are not limited to, sterilized water, physiological saline, a phosphate buffer solution, a culture fluid, and the like. Furthermore, the pharmaceutical composition or medicament of the present invention may contain a stabilizer, a suspension, a preservative, a surfactant, and the like. The pharmaceutical composition or medicament of the present invention can be used for anti-cancer purposes.

The peptide of the present invention can be prepared in combination, which comprises two or more peptides of the present invention to induce cytotoxic T lymphocytes in vivo . The peptide combination can be in the form of a cocktail or can be conjugated to each other using standard techniques. For example, the peptide can be chemically linked or expressed as a single fused polypeptide. The peptides to be combined may be the same or different. By administering the peptide of the present invention, the peptide is present on the antigen-presenting cell at a high density via the HLA antigen, and then the cytotoxic T lymphocyte which specifically reacts with the complex formed between the present peptide and the HLA antigen is induced. . Alternatively, the antigen presenting cells (e.g., dendritic cells) are removed from the individual and then stimulated with the peptide of the present invention to obtain antigen presenting cells that exhibit any peptide of the present invention on the cell surface. These antigen-presenting cells can be re-administered into an individual to induce cytotoxic T lymphocytes in the individual, thereby increasing the invasion of tumor-associated endothelial cells.

A pharmaceutical composition or medicament comprising a therapeutic peptide comprising the peptide of the present invention as an active ingredient for the treatment and/or prevention of cancer may also comprise an adjuvant to effectively establish cellular immunity. Alternatively, the pharmaceutical composition or medicament may be administered with other active ingredients or may be formulated as granules for administration. An adjuvant refers to any compound, substance or composition that, when administered (or sequentially) with an immunologically active protein, enhances the immune response against the protein. Adjuvants contemplated herein include those described in the literature (Clin Microbiol Rev 1994, 7:277-89). Examples of suitable adjuvants include, but are not limited to, aluminum phosphate, aluminum hydroxide, alum, cholera toxin, salmonella toxin, IFA (Incomplete Freund's adjuvant), CFA (Fresh Complete Adjuvant ( Complete Freund's adjuvant)), ISCOMATRIX, GM-CSF, CpG, O/W emulsions, etc.

Further, a liposome formulation in which the peptide is linked to a micron-diameter bead, a granule formulation, and a lipid-linked formulation to the peptide can be generally used.

In another embodiment of the invention, a peptide of the invention may also be administered in the form of a pharmaceutically acceptable salt. Preferable examples of the salt include alkali metal salts, metal salts, organic base salts, and organic acid salts (for example, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, Oxalic acid, benzoic acid, methanesulfonic acid, etc.) and inorganic acid salts (for example, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.). As used herein, "pharmaceutically acceptable salts" means maintaining the biological effectiveness and properties of the compound and obtaining it from inorganic acids or bases such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, Ethane a salt that reacts with sulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like.

In some embodiments, the pharmaceutical compositions or agents of the invention may further comprise cells that induce cytotoxic T lymphocytes. Lipids have been defined as substances which induce cytotoxic T lymphocytes of antiviral antigens in vivo. For example, a palmitic acid residue can be attached to the epsilon- and alpha-amine groups of an amine acid residue, which is then linked to one of the peptides of the present invention. The lipidated peptide can be administered directly into the micelles or granules, incorporated into the liposomes or emulsified in an adjuvant. Another example of a lipid, E. coli lipoprotein, such as tripalmitoyl-S-glycerylcysteinyl-seryl-serine (P3CSS), when covalent It can be used to induce cytotoxic T lymphocytes when attached to a suitable peptide (see, for example, Deres et al., Nature 1989, 342: 561-4).

Examples of suitable methods of administration include, but are not limited to, oral, intradermal, subcutaneous, intramuscular, intraosseous, intraperitoneal, intravenous injection or the like. Administration may be by systemic administration or topical administration to adjacent areas of the target site (ie, direct injection). A single administration can be performed or by multiple administrations. The pharmaceutically or therapeutically effective amount of the peptide of the present invention can be administered to an individual who is required to treat a cancer or tumor that exhibits SMYD3. Alternatively, an amount of a peptide of the invention sufficient to enhance or induce an immune response mediated by a cytotoxic T lymphocyte and/or to induce a cytotoxic T lymphocyte against a cancer or tumor of SMYD3 can be administered to a cancer having SMYD3 expression. Individual. The peptide dose of the present invention can be appropriately administered depending on the disease to be treated, the age of the patient, the body weight, the administration method, and the like, and is usually 0.001 mg to 1,000 mg, for example, 0.01 mg to 100 mg, for example, 0.1 mg to 30 mg, for example, 0.1 mg to 10 mg, for example 0.5 mg to 5 mg, can be administered once every few days to several months. Those skilled in the art will be able to suitably select the appropriate dosage.

(2) A pharmaceutical composition comprising a polynucleotide as an active ingredient:

The pharmaceutical composition or medicament of the present invention may also comprise a nucleic acid which is encoded in an expressible form as a peptide of the present invention. Here, "in a form that can be expressed" means that a polynucleotide, when introduced into a cell, is expressed in vivo as a multi-peptide that induces anti-tumor immunity. In one illustrative embodiment, the nucleic acid sequence of the polynucleotide of interest includes the regulatory elements necessary to represent the polynucleotide. Polynucleotides can be assembled to achieve stable insertion into the genome of the target cell (see, for example, Thomas KR & Capecchi MR, Cell 1987, 51: 503-12, for the description of homologous recombination cassette vectors. See, for example, Wolff et al , Science 1990, 247: 1465-8; U.S. Patent Nos. 5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647; and WO 98/04720. Examples of DNA delivery techniques include "naked DNA", acceleration (bupivacaine, polymers, Peptide media delivery, delivery of cationic lipid complexes to particulate mediators ("gene guns") or pressure media (see, e.g., U.S. Patent No. 5,922,687).

The peptide of the present invention can also be expressed by a viral or bacterial vector. Examples of performance vectors include attenuating viral hosts such as vaccinia or fowl pox. This method involves the use of a vaccinia virus, for example, a vector to express the nucleotide sequence encoding the peptide. By introducing the host, the recombinant vaccinia virus exhibits an immunogenic peptide and thus causes an immune response. Vaccinia vectors and methods that are effective in the immunization step are described, for example, in U.S. Patent No. 4,722,848. Another vector is BCG (Bacille Calmette Guerin). The BCG vector is described in Stover et al., Nature 1991, 351: 456-60. For a variety of other vectors effective for therapeutic administration or immunization, such as a gland-and adenovirus-related vector, a retroviral vector, a Salmonella typhi vector, a detoxified anthrax toxin vector, and the like Obvious. See, for example, Shata et al., Mol Med Today 2000, 6: 66-71; Shedlock et al., J Leukoc Biol 2000, 68: 793-806; Hipp et al., In Vivo 2000, 14: 571-85.

Delivery of the polynucleotide to the patient's body can be direct, in this case, the patient can be directly exposed to the vector carrying the polynucleotide, or indirectly, in the example of which the multinucleus of interest is prior to in vitro The glucosinolate is transformed into cells, which are then transplanted to the patient. These two methods are known to be in vivo ( in vivo ) and in vitro ( ex vivo ) gene therapy.

For a general review of methods of gene therapy, see Goldspiel et al., Clinical Pharmacy 1993, 12: 488-505; Wu and Wu, Biotherapy 1991, 3: 87-95; Tolstoshev, Ann Rev Pharmacol Toxicol 1993, 33: 573-96 Mulligan, Science 1993, 260: 926-32; Morgan & Anderson, Ann Rev Biochem 1993, 62: 191-217; Trends in Biotechnology 1993, 11(5): 155-215). A method generally known in the art of recombinant DNA used in the present invention is described by Ausubel et al., in Current Protocols in Molecular Biology (John Wiley & Sons, NY, 1993; and Krieger, in Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY, 1990).

As with the administration of the peptide, the administration of the polynucleotide may be oral, intradermal, subcutaneous, intravenous, intramuscular, intraosseous, or intraperitoneal injection, or the like. Administration may be by systemic administration or topical administration (e.g., direct injection) to adjacent areas of the target location. A single administration can be performed or by multiple administrations. The pharmaceutically or therapeutically effective amount of a polynucleotide of the invention can be administered to an individual in need of treatment for a cancer that exhibits SMYD3. Alternatively, it can be sufficient to enhance or stimulate the cytotoxic T lymphocyte The amount of polynucleotide of the immune response of the globular media, and/or the amount of the polynucleotide of the invention that induces cytotoxic T lymphocytes against the cancer or tumor of SMYD3, administered to an individual having a cancer or tumor exhibiting SMYD3 . The dosage of the polynucleotide in a suitable vector or in a cell transformed with the polynucleotide encoding the peptide of the present invention can be suitably adjusted depending on the disease to be treated, the age of the patient, the body weight, the administration method, and the like. The peptide dosage of the invention is generally from 0.001 mg to 1000 mg, for example from 0.01 mg to 100 mg, for example from 0.1 mg to 30 mg, for example from 0.1 mg to 10 mg, for example from 0.5 mg to 5 mg, which can be administered once every few days to every few months. Those skilled in the art can readily identify suitable and desirable dosages.

X. Method for using peptide, exosome, antigen presenting cells and cytotoxic T lymphocytes

The peptides and polynucleotides of the invention can be used to prepare or induce antigen-presenting cells and cytotoxic T lymphocytes. The exosome and antigen presenting cells of the present invention can also be used to prepare or induce cytotoxic T lymphocytes. The peptide, polynucleotide, exosome and antigen presenting cells can be used in combination with any other compound as long as the added compound does not inhibit the cytotoxic T lymphocyte evoking ability. Thus, any of the above-described pharmaceutical compositions or medicaments of the invention can be used to prepare or induce cytotoxic T lymphocytes. In addition, pharmaceutical compositions or agents including peptides or polynucleotides can also be used to prepare antigen-presenting cells, as explained below.

(1) Method for inducing antigen presenting cells (APCs)

The present invention provides a method of inducing antigen-presenting cells having cytotoxic T lymphocyte-inducing ability using the peptide or polynucleotide of the present invention.

The method of the present invention comprises in vitro (in vitro), in vitro (ex vivo) or in vivo (in vivo) a step of contacting the cells with antigen presenting peptide of the present invention. For example, a method of ex vivo- inducing an antigen-presenting cell can include the steps of: a: collecting antigen-presenting cells from one body; and b: contacting the antigen-presenting cells of step a with the peptide of the present invention.

The antigen presenting cells are not limited to a particular kind of cells, and examples of antigen presenting cells include, but are not limited to, dendritic cells, Langerhans cells, macrophages, B cells, and activated T cells, which are known to be The cell surface expresses a proteinaceous antigen to be recognized by the lymphocytes. Dendritic cells are preferably used because they have the strongest cytotoxic T lymphocyte inducing ability in antigen presenting cells. Any of the peptides of the invention may be used in combination with one or more of the other peptides of the invention and/or one or more cytotoxic T lymphocyte-inducing peptides derived from tumor-associated antigens (TAAs) other than SMYD3.

On the other hand, when the peptide of the present invention is administered to a single body, the antigen presents cells to be contacted with the peptide in the body, thereby inducing antigen-presenting cells having cytotoxic T lymphocyte-inducing ability in the individual. Accordingly, the method of the present invention may comprise the step of administering a peptide of the present invention to a body to induce an antigen-presenting cell having a cytotoxic T lymphocyte-inducing ability in an individual. Similarly, when the polynucleotide of the present invention is administered in an expressible form to a single body, the peptide is expressed and is in contact with an antigen in vivo, thereby inducing cytotoxic T lymphocyte induction in the individual. The antigen of the ability presents the cells. Thus, the methods of the invention may comprise the step of administering a polynucleotide of the invention to a body to induce an antigen-presenting cell having cytotoxic T lymphocyte-inducing ability in an individual. "Expressible form" is described in the above paragraph "IX. Pharmaceutical composition (2) A pharmaceutical composition comprising a polynucleotide as an active ingredient.

The method of the present invention may further comprise the step of introducing a polynucleotide of the present invention into an antigen-presenting cell to induce an antigen-presenting cell having a cytotoxic T lymphocyte-inducing ability. For example, the method can include the steps of: a: collecting antigen presenting cells from one body; and b: introducing the polynucleotide encoding the peptide of the present invention into the antigen presenting cells of step a.

Step b can be performed as described in the aforementioned paragraph "VI. Antigen presenting cells".

Alternatively, the method of the invention may comprise the step of preparing an APC that specifically induces a cytotoxic T lymphocyte that exhibits anti-SMYD3 cytotoxicity, wherein the method may comprise one of the following steps: (a) presenting the antigen to the cell and the peptide of the invention In vitro , ex vivo or in vivo contact; and (b) introducing a polynucleotide encoding the peptide of the present invention into an antigen-presenting cell.

Alternatively, the method of the present invention may comprise the step of inducing an antigen-presenting cell having cytotoxic T lymphocyte-inducing ability, the method comprising the step of: (a) contacting the antigen-presenting cell with the peptide of the present invention. And (b) introducing a polynucleotide encoding the peptide of the present invention into the antigen-presenting cell.

In a preferred embodiment, the present invention provides a method of inducing or preparing an antigen-presenting cell having cytotoxic T lymphocyte-inducing ability, the method comprising one of the following steps: (a) presenting an antigen expressing HLA-A24 to a cell And a peptide having the amino acid sequence selected from the sequence identification numbers: 2, 4, 5, 6, 7, 8, 9, 13, 14, and 19 or a modified peptide thereof in vitro , ex vivo or in vivo contact; and (b) will encode alternative sequence identification numbers: 2, 4, 5, 6, 7, 8, 9, 13, 14, 17 and The peptide of one of the amino acid sequences of 19 or the polynucleotide of the modified peptide thereof is introduced into an antigen-presenting cell which expresses HLA-A24.

In another embodiment, the invention provides a method of inducing or preparing an antigen-presenting cell having cytotoxic T lymphocyte-inducing ability, the method comprising one of the following steps: (a) presenting an antigen expressing HLA-A2 to cells having selected from the SEQ ID NO: 8, 16 and one of 22 peptides in amino acid sequence or a modified peptide in vitro (in vitro), in vitro (ex vivo) or in vivo (in vivo) into contact; and (b) introducing a polynucleotide encoding a peptide having one of amino acid sequence sequences selected from the sequence identification numbers: 8, 16 and 22 or a modified peptide thereof into an antigen-presenting cell expressing HLA-A2.

The antigen-presenting cells induced by the above method exhibit the peptide on the surface thereof via HLA-A24 or HLA-A2, and can induce a specific cell having cells resistant to HLA-A24 and SMYD3 or cells expressing HLA-A2 and SMYD3. Toxic active cytotoxic T lymphocytes.

The method of the invention may in an in vitro (in vitro), in vitro (ex vivo) or in vivo (in vivo) embodiment. Preferably, the method of the invention can be carried out in vitro or ex vivo . An antigen-presenting cell for inducing an antigen-presenting cell having cytotoxic T lymphocyte-inducing ability, preferably expressing an HLA-A24 or HLA-A2 antigen (ie, an HLA-A24-positive antigen-presenting cell or HLA-A2-positive) The antigen presenting cells) is an antigen presenting cell. This antigen presenting cell can be prepared from peripheral blood mononuclear cells obtained from an individual having an HLA antigen of HLA-A24 or HLA-A2 by methods well known in the art. The antigen-presenting cell induced by the method of the present invention may be an antigen-presenting cell having a complex of a peptide of the present invention and an HLA antigen (HLA-A24 antigen or HLA-A2 antigen) on its surface. When the antigen-presenting cells induced by the method of the present invention are administered to a body to induce an anti-cancer immune response in the individual, the individual is preferably the same individual from which the antigen-presenting cells are obtained. However, the individual may also be different from the antigen presenting cell provider as long as the individual has the same HLA type as the antigen presenting cell provider.

In another embodiment, the invention provides an agent or composition for inducing an antigen presenting cell having the ability to induce cytotoxic T lymphocytes, the agent or composition comprising one or more peptides or polynucleosides of the invention acid.

In another embodiment, the invention provides the use of a peptide of the invention or a polynucleotide encoding the peptide in the manufacture of a reagent or a composition formulated to induce antigen presenting cells.

Alternatively, the present invention further provides the use of the peptide of the present invention or the polynucleotide encoding the peptide for inducing antigen-presenting cells having cytotoxic T lymphocyte-inducing ability.

(2) Method for inducing cytotoxic T lymphocytes

The present invention also provides a method of inducing cytotoxic T lymphocytes using the peptide, polynucleotide, exosome or antigen presenting cells of the present invention.

The invention also provides for the induction of cytotoxicity using a polynucleotide encoding a subunit of a T cell receptor or a polynucleotide encoding a subunit of each T cell receptor. In the method of killing a T lymphocyte, the T cell receptor formed by this unit can recognize (i.e., bind to) the cell-surface complex of the peptide of the present invention and the HLA antigen. Preferably, the method for inducing cytotoxic T lymphocytes comprises at least one step selected from the group consisting of: a: contacting CD8-positive T cells with antigen-presenting cells, the antigen exhibiting a combination of a HLA antigen on the surface and a peptide of the present invention. b: contacting CD8-positive T cells with exosome, which exhibits a complex of the HLA antigen and the peptide of the present invention on the surface; and c: a polynucleoside encoding the subunit of the T cell receptor The acid or a polynucleotide encoding a subunit of each T cell receptor is introduced into a CD8-positive T cell, wherein the T cell receptor formed by the subunit is capable of recognizing (binding to) the peptide of the present invention and the HLA antigen on the cell surface. Complex.

When the peptide, the polynucleotide, the antigen-presenting cell or the exosome of the present invention is administered to a body, the cytotoxic T lymphocyte is induced in the individual, and the immune response targeting the SMYD3 cancer cell is promoted. strength. Thus, the methods of the invention may comprise the step of administering to the individual a peptide, polynucleotide, antigen presenting cell or exosome of the invention.

Alternatively, by in vitro (ex vivo) or in vitro (in vitro) use them, may induce cytotoxic T lymphocytes, and after inducing cytotoxic T lymphocytes can be cytotoxic T lymphocytes activated by the return of The individual. For example, the method can include the steps of: a: collecting antigen presenting cells from one body; b: contacting the antigen presenting cells of step a with the peptide of the present invention; and c: combining the antigen presenting cells of step b with CD8 positive T cells to cultivate.

Anti-co-cultured with CD8-positive T cells in step c above The original presenting cells can also be prepared by transferring the polynucleotide of the present invention into an antigen presenting cell as described in the aforementioned paragraph "VI. Antigen presenting cells", however, the present invention is not limited thereto, and thus includes any An antigen-presenting cell whose surface effectively exhibits a complex of an HLA antigen and a peptide of the present invention.

A person skilled in the art can use an exosome having a surface exhibiting a complex of an HLA antigen and a peptide of the present invention in place of the antigen presenting cells described above. That is, the present invention may include the step of cocultivating the exosome having a complex of the HLA antigen and the peptide of the present invention on the surface and the peptide of the present invention. Such exosome bodies can be prepared by the method described in the above paragraph "V. Exosome". Appropriate antigen presenting cells of the method of the invention exhibit a complex of the peptide of the present invention with HLA-A24 or HLA-A2 on the surface.

For example, a peptide exhibiting HLA-A24 and an amino acid sequence selected from the sequence identification numbers: 2, 4, 5, 6, 7, 8, 9, 13, 14, 17 and 19 (or The antigen of the complex of the modified peptide) is presented as a cell or exosome, preferably used to induce cytotoxic T cells having specific cytotoxicity against cells expressing HLA-A24 and SMYD3, and exhibits HLA on the surface. -A2 and antigen-presenting cells having a complex of peptides (or modified peptides thereof) selected from the sequence identification numbers: 8, 16 and 22, presenting cells or exosomes, preferably utilized To induce cytotoxic T cells with specific cytotoxicity against cells expressing HLA-A2 and SMYD3.

Furthermore, the cytotoxic T lymphocytes of the present invention can also be induced by introducing a polynucleotide encoding a T cell receptor subunit or a polynucleotide encoding each T cell receptor subunit into a CD8 positive T cell, wherein The T cell receptor formed by this subunit can bind to the peptide of the present invention and the HLA antibody on the cell surface. The original compound. This transduction can be carried out as described in the aforementioned paragraph "VIII. T Cell Receptor (TCR)".

The methods of the invention can be practiced in vitro , ex vivo or in vivo . Preferably, the methods of the invention can be performed in vitro or ex vivo . CD8-positive T cells for the induction of cytotoxic T lymphocytes can be prepared from peripheral blood mononuclear cells obtained from one body by methods well known in the art. In a preferred embodiment, the provider of CD8 positive T cells can be an individual whose HLA antigen is HLA-A24 or HLA-A2. The cytotoxic T lymphocytes induced by the present invention can be identified as cells having a complex of a peptide of the present invention and an HLA antigen (for example, HLA-A24 or HLA-A2). This cytotoxic T lymphocyte can exhibit specific cytotoxicity against cells which exhibit a peptide of the present invention on the surface, and thus exhibits specific cytotoxicity against cells expressing SMYD3 (for example, cancer cells). When a cytotoxic T lymphocyte induced by the method of the present invention is administered to a body to induce an anti-cancer immune response in the individual, the individual is preferably the same as the individual who has obtained the CD8-positive T cell. However, the individual may also be different from the CD8 positive T cell provider as long as the individual has the same HLA type as the CD8 positive T cell provider.

Moreover, the invention also provides a method or process for making a pharmaceutical composition or agent for inducing a cytotoxic T lymphocyte, wherein the method or process comprises the step of mixing or formulating the peptide of the invention with a pharmaceutically acceptable carrier .

In another embodiment, the invention provides a composition or agent for inducing a cytotoxic T lymphocyte, wherein the composition or agent comprises one or more peptides, one or more polynucleotides of the invention One or more antigen presenting cells, and/or one or more exosome bodies.

In another embodiment, the invention provides the use of a peptide, polynucleotide, antigen presenting cell or exosome of the invention in the manufacture of a composition or agent for formulating a cytotoxic T lymphocyte.

Alternatively, the invention further provides the use of a peptide, polynucleotide, antigen presenting cell or exosome of the invention for inducing cytotoxic T lymphocytes.

XI. Method for inducing immune response

Moreover, the present invention provides a method of inducing an immune response against a disease associated with SMYD3. The diseases considered include cancer, and examples thereof include, but are not limited to, colorectal cancer, liver cancer, breast cancer, and bladder cancer. A cancer exhibiting HLA-A2 (i.e., an HLA-A2 positive cancer) or a cancer exhibiting HLA-A24 (i.e., an HLA-A24-positive cancer) is preferred.

The method of the invention may comprise the step of administering a composition comprising any of the peptides of the invention or a polynucleotide encoding the same. The methods of the invention also contemplate administration of a surface exhibiting any of the peptides of the invention, exosome or antigen presenting cells. For details, see the item "IX. Medicament or Composition", particularly the portion of the pharmaceutical composition of the present invention for use as a vaccine. Furthermore, the exosome and antigen presenting cells of the present invention which can be used in the method for inducing an immune response of the present invention are described in detail in "V. Exosome", "VI. Antigen presenting cells" and " X. Items of (1) and (2) using a peptide, an exosome, an antigen presenting cell and a cytotoxic T lymphocyte.

The invention also provides a method or process for making a pharmaceutical composition or medicament for inducing an immune response, wherein the method or process can include the step of mixing or formulating the peptide of the invention with a pharmaceutically acceptable carrier.

Alternatively, the method of the invention may comprise administering the invention comprising the following a step of a vaccine or a pharmaceutical composition or medicament: (a) a peptide of the present invention; (b) a polynucleotide which is encoded in the expressible form of the peptide of the present invention; (c) an antigen-presenting cell which exhibits the peptide of the present invention on the surface. (d) presenting the peptide of the present invention on the surface; or (e) the cytotoxic T lymphocyte of the present invention.

In the context of the present invention, these active ingredients can treat cancers that overexpress SMYD3. Examples of such cancers include, but are not limited to, colorectal cancer, liver cancer, breast cancer, and bladder cancer.

Therefore, prior to administration of the vaccine, pharmaceutical composition or medicament comprising the above-mentioned active ingredient, it is preferred to confirm the degree of expression of SMYD3 in the cancer cells or tissues collected in the treated individual compared to the normal collection from the same individual. Whether cells or tissues are increased. Thus, in one embodiment, the invention provides a method of treating a SMYD3 (excessive) manifestation of a cancer in a desired patient, the method comprising the steps of: i) determining SMYD3 in a biological sample obtained from an individual having the cancer to be treated Degree of performance; ii) compared to the degree of SMYD3 performance in the normal control group; and iii) administration of at least one of the above (a) to (e) individuals who have cancers that are overexpressed in the SMYD3 over normal control group .

Alternatively, the present invention may also provide a vaccine or pharmaceutical composition comprising at least one component selected from the above (a) to (e), which is administered to an individual having a cancer which overexpresses SMYD3. In other words, the invention further provides a method of identifying an individual treated with a multi-peptide of the invention, the method comprising determining the individual from A step of the degree of SMYD3 expression in a biological sample, wherein an increase in the degree of expression compared to the normal degree of control of SMYD3 indicates that the individual may have a cancer that can be treated with the peptide of the present invention.

Furthermore, in a preferred embodiment, the HLA type of the individual can be confirmed prior to administration of the peptide of the present invention. Preferably, the vaccine or pharmaceutical composition of the invention is administered to an HLA-A24 positive or HLA-A2 positive individual.

Any cell or tissue derived from an individual can be used to determine the extent of SMYD3 expression as long as it includes SMYD3 transcription or translation products. Examples of suitable samples include, but are not limited to, body tissues and fluids such as blood, saliva and urine. Preferably, the cell or tissue sample from the individual comprises a population of cells comprising epithelial cells, more preferably cancerated epithelial cells or derived from cancerous tissue epithelial cells. Moreover, if necessary, the cells can be purified from the obtained body tissue or liquid, and then used as a sample derived from the individual.

According to the present invention, the degree of SMYD3 expression in a biological sample obtained from a body can be determined. The degree of expression of SMYD3 at the level of transcription (nucleic acid) product can be detected using methods known in the art. For example, mRNA for SMYD3 can be quantified using a probe by hybridization methods (eg, Northern hybridization). The above detection can be performed on a wafer or array. An array is preferably used for detecting the degree of SMYD3 performance. This probe can be prepared by those skilled in the art using sequence information of SMYD3. For example, the cDNA of SMYD3 can be used as a probe. If desired, suitable markers can be used to label probes such as dyes, fluorescent materials and isotopes, and the degree of performance of SMYD3 can be detected as the intensity of the hybridization marker.

Moreover, SMYD3 can be used by using an amplification-based detection method (for example, RT-PCR) using primers. Transcription products were quantified. This primer can be prepared based on the sequence information available to SMYD3.

Specifically, the probe or primer used in the method is hybridized to the mRNA of SMYD3 under severe conditions, moderately severe conditions, and low stringency conditions. By "severe (heterozygous) conditions" it is meant herein that the probe or primer will hybridize to its target sequence, rather than other sequences. Strict conditions are sequence-dependent and will vary in different environments. Specific heterozygous sequences of longer sequences are observed at higher temperatures than shorter sequences. In general, the temperature of a stringent condition at a defined ionic strength and pH is about 5 ° C below the melting point (Tm) of a particular sequence. A Tm is a temperature at which 50% of the probe complementary to the target sequence is hybridized to the target sequence to form an equilibrium temperature (at a defined ionic strength and pH versus nucleic acid concentration). Since the target sequence is usually present in excess, at Tm, 50% of the probes are occupied to form a balance. In general, stringent conditions are pH values of 7.0 to 8.3 below 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salt), and for short probes or primers (eg, 10 to 50) The nucleotides have a temperature of at least about 30 ° C and, for longer probes or primers, a temperature of at least about 60 ° C. Desabilizing substances, such as formamide, may also be added to achieve stringent conditions.

Probes or primers of the invention are generally substantially purified oligonucleotides. Oligonucleotides generally comprise a region of a nucleotide sequence that is hybridized under stringent conditions to a continuous sense strand of a nucleic acid comprising a SMYD3 sequence, or an inverse meaning of a nucleic acid comprising a SMYD3 sequence. An anti-sense strand nucleotide sequence, or at least about 2000, 1000, 500, 400, 350, 300, 250, 200 of naturally occurring mutants of these sequences 150, 100, 50 or 25 bases. In particular, for example, in a preferred embodiment, an oligonucleotide having a length of 5 to 50 bases is used as an primer for amplifying a gene to be detected. More preferably, an oligonucleotide probe or primer having a specific length, typically 15 to 30 bases in length, is used to detect the mRNA or cDNA of the SMYD3 gene. The length can be at least 10 nucleotides, at least 12 nucleotides, at least 15 nucleotides, at least 20 nucleotides, at least 25 nucleotides, at least 30 nucleotides, and the probe The primers can be 5-10 nucleotides, 10-15 nucleotides, 15-20 nucleotides, 20-25 nucleotides, and 25-30 nucleotides in length. In a preferred embodiment, the length of the oligonucleotide probe or primer can be selected from 15-25 nucleotides. Analytical procedures, devices or reagents for gene detection using such oligonucleotide probes or primers are well known in the art (e.g., oligonucleotide microarrays or PCR). In these assays, the probe or primer may also include a tag or a linker sequence. Moreover, the probe or primer can be modified by a detectable label or an affinity ligand that can be captured. Alternatively, polynucleotides having hundreds of bases (e.g., about 100-200) to thousands (e.g., about 1000-2000) bases in length can also be used as probes in hybrid detection procedures. (eg, Northern blot analysis or cDNA microarray analysis).

Alternatively, the translation product of SMYD3 can be detected to confirm the individual treated by the method of the invention. For example, the amount of SMYD3 protein (eg, sequence identifier: 60, 62 or 64) can be detected. Methods for determining the amount of SMYD3 protein of a transcript include, but are not limited to, immunoassay methods using antibodies that specifically recognize the SMYD3 protein. The antibody may be single or multiple plants. Moreover, any fragment or modification of the antibody (e.g., chimeric antibody, scFv, Fab, F(ab')2, Fv, etc.) can be used to detect as long as the fragment or The modified antibody maintains the ability to bind to the SMYD3 protein. Methods of preparing such antibodies are well known in the art, and any method can be used to prepare such antibodies and their equivalents.

Another method for detecting the degree of expression of the SMYD3 gene based on the SMYD3 gene translation product can be measured by immunohistochemical analysis using an antibody against the SMYD3 protein. That is, in this measurement, strong staining indicates an increase in the presence/degree of the SMYD3 protein, and indicates a high degree of expression of the SMYD3 gene.

The degree of expression of the SMYD3 gene from an individual sample, if the degree of expression is higher than the degree of control of the SMYD3 gene (for example, the degree of expression in normal cells), may be confirmed as an increase, for example, 10%, 25%, or 50%, Or increase by more than 1.1 times, more than 1.5 times, more than 2.0 times, more than 5.0 times, more than 10.0 times or more.

The degree of control can be determined using samples previously collected or stored from one or more healthy individuals while confirming the cancer cells. Moreover, normal cells obtained from non-cancerous regions of organs having cancer to be treated can be used as normal controls. Alternatively, the degree of control can be determined by statistical methods based on the results of the degree of expression of the SMYD3 gene detected in a sample of an individual whose disease degree is known. Moreover, the degree of control can be derived from a database of performance profiles of previously tested cells. Moreover, according to one aspect of the invention, the degree of expression of the SMYD3 gene in a biological sample can be compared to a plurality of degrees of control identified from a plurality of reference samples. Preferably, the degree of control as determined by a reference sample of a tissue type similar to the biological sample from the individual is used. Moreover, it is preferred to use a standard value of the degree of expression of the SMYD3 gene in a group having a known disease stage (standard Value). This standard value can be obtained by any method known in the art. For example, the mean +/- 2 S.D. or the mean +/- 3 S.D. can be used as a standard value.

In the present specification, the degree of control determined to be a non-cancerous biological sample may be referred to as "normal control group degree". On the other hand, if the degree of control is measured from a cancerous biological sample, it is called "degree of canceration control".

The difference between the degree of expression of the sample and the degree of control can be normalized to the degree of expression of the controlled nucleic acid, such as the housekeeping gene, and the degree of expression of the controlled nucleic acid is known between the cancerous and non-cancerous states of the cell. No different. Control genes such as, but not limited to, beta-actin, glyceraldehyde-3-phosphate dehydrogenase, and riboprotein P1.

When the degree of performance of SMYD3 is increased as compared with the degree of normal control, the individual can be confirmed to have cancer treated by administration of the pharmaceutical composition or agent of the present invention.

The invention also provides a method of selecting an individual for use in the treatment of a cancer composition using the pharmaceutical composition or medicament of the invention described above, the method comprising the steps of: a) confirming the extent of SMYD3 expression in a biological sample obtained from an individual having cancer; b) comparing the degree of SMYD3 expression confirmed in step a) with the normal control degree; and c) when the degree of expression of SMYD3 is increased by ten compared to the normal control, the individual is selected to be treated with the pharmaceutical composition or medicament of the present invention. Individuals who treat cancer.

In some implementations, the above method may further be in the above steps Individuals having an HLA selected from HLA-A24 or HLA-A2 are identified before or after steps a)-c). The cancer therapy of the present invention is preferably directed to an individual having a cancer that overexpresses SMYD3 and has HLA-A24 or HLA-A2. Methods of HLA typing are well known in the art. For example, PCR-based methods for HLA dual genotyping are well known in the art. Antibodies specific for each HLA molecule are also suitable tools for confirming an individual's HLA type.

In one embodiment, the invention further provides a diagnostic kit comprising one or more peptides of the invention.

Cancer can be diagnosed by using the peptide of the present invention to detect antibodies against one or more peptides of the invention in a sample (e.g., blood) from an individual.

An individual is suspected of having cancer if the sample from the individual (eg, blood, tissue sample) contains an antibody against the peptide of the present invention and the antibody is confirmed to exceed the cutoff value of the control.

In another embodiment, the diagnostic kit of the invention can comprise an HLA molecule to which the peptide of the invention binds. A method of detecting antigen-specific cytotoxic T lymphocytes using an antigen peptide and an HLA molecule has been established (for example, Altman JD et al., Science. 1996, 274 (5284): 94-6). Therefore, the complex of the peptide of the present invention and the HLA molecule can be applied to a method for detecting a tumor cell-specific cytotoxic T lymphocyte, thereby enabling early detection of cancer recurrence and/or metastasis. Moreover, the diagnostic kit can be used for screening of individuals suitable for pharmaceutical compositions comprising the peptide of the present invention as an active ingredient, or for evaluation of the therapeutic efficacy of a pharmaceutical composition.

In particular, according to known methods (see, for example, Altman JD et al., Science. 1996, 274 (5284): 94-6), radiolabeled HLA can be prepared. An oligomeric complex of a molecule with a peptide of the invention, such as a tetramer. Diagnosis can be performed by using a complex, for example, by quantifying antigen-peptide specific cytotoxic T lymphocytes in peripheral blood lymphocytes from individuals suspected of having cancer.

The present invention further provides a diagnostic reagent and method for assessing an individual's immune response by using the peptide of the present invention. In one embodiment of the invention, the peptide of the invention is used as an agent for assessing or predicting an individual's immune response. This immune response will be assessed by immunizing antigen (ImmunoGen) (i.e., the peptide of the present invention) and immune competence (immunocompetent) cells in vitro (in vitro) or in the body (in vivo) into contact induction. In a preferred embodiment, the immunocompetent cells used to assess the immune response are selected from peripheral blood, peripheral blood lymphocytes (PBL), and peripheral blood mononuclear cells (PBMC). Methods for collecting or isolating such immunocompetent cells are well known in the art. In certain embodiments, any agent that results in antigen-specific CTL production that is identifiable and binds to the peptide epitope can be used. The peptide reagent does not need to be used as an immunogen. Analytical systems for such analysis include fairly new technological developments such as tetramer staining analysis, staining of intracellular lymphokines and interferon release assays or ELISPOT assays. In a preferred embodiment, the immunocompetent cells contacted with the peptide reagent can be antigen presenting cells, including dendritic cells.

For example, the peptides of the invention can be used in tetramer staining assays to assess peripheral blood mononuclear cells in the presence of antigen-specific cytotoxic T lymphocytes following exposure to tumor antigens or immunizing antigens. HLA tetrameric complexes can be used to directly visualize antigen-specific cytotoxic T lymphocytes (see, for example, Ogg et al., Science 279: 2103-2106, 1998; and Altman et al, Science 174:94-96, 1996), and measures the frequency of antigen-specific cytotoxic T lymphocyte populations in samples of peripheral blood mononuclear cells. The tetramer reagent using the peptide of the present invention can be produced as described below.

The peptide that binds to HLA is refolded in the presence of the corresponding HLA heavy chain and β2-microglobulin to produce a three molecule complex. In this complex, the carboxy terminus of the heavy chain is drawn in biotin at a location pre-designed into the protein. The avidin is then added to the complex to form a tetramer composed of a tri-molecular complex and streptavidin. The tetramer can be used to stain antigen-presenting cells by means of fluorescently marking avidin. Cells can then be identified using, for example, flow cytometry. Such analysis can be used for diagnostic and prognostic purposes. Cells identified by this procedure can also be used for therapeutic purposes.

The peptides of the present invention can also be used to make antibodies using techniques well known in the art (see, for example, CURRENT PROTOCOLS IMMUNOLOGY, Wiley/Greene, NY; and Antibodies A Laboratory Manual, Harlow and Lane, Cold Spring Harbor Laboratory Press, 1989). It can be used as an agent for diagnosing or detecting cancer. Such antibodies may include antibodies that recognize the peptide in the HLA molecular content, ie, antibodies that bind to the peptide-MHC complex.

The peptides and compositions of the present invention have a variety of additional uses, some of which are described herein. For example, the invention provides a diagnosis and detection of a disease characterized by the expression of SMYD3 multipeptide. For example, diagnosis can be performed by staining with a fluorescein-labeled HLA polyplex to allow direct quantification of antigen-specific T cells (eg, Altman, JD et al., 1996, Science 274:94; Altman, JD et al., 1993, Proc. Natl. Acad. Sci. USA 90: 10330). Intracellular lymphokines staining and interferon release assays or ELISPOT assays have also been proposed. Tetramer staining, intracellular lymphokine staining and ELISPOT analysis are clearly at least 10 times more sensitive than general analysis (Murali-Krishna, K. et al., 1998, Immunity 8: 177; Lalvani, A. et al., 1997, J. Exp. Med. 186: 859; Dunbar, PR et al., 1998, Curr. Biol. 8: 413). Pentamers (e.g., U.S. Patent Publication No. 2004-209295A), dextramers (e.g., WO 02/072631), and streptamers (e.g., Nature medicine 6.631-637 (2002) can also be used. ).

For example, in some embodiments, the invention provides a method of diagnosing or assessing an immune response in an individual administered a subject of at least one SMYD3 peptide of the invention, the method comprising the steps of: (a) suitable for inducing cells specific for the immunogen (i) detecting or determining the degree of induction of cytotoxic T lymphocytes induced in step (a); and (c) immunizing the individual under conditions of toxic T lymphocytes; The response is related to the degree of induction of cytotoxic T lymphocytes.

In the context of the present invention, the immunogen preferably comprises a SMYD3 peptide having the amino acid sequence of the sequence identifier: 2, 4, 5, 6, 7, 8, 9, 13, 14, 16, 17, 19 or 22. And at least one of the peptides having 1, 2 or more amino acid substitutions of the modified amino acid sequence. At this time, conditions suitable for inducing immunogen-specific cytotoxic T lymphocytes are well known in the art. For example, immunocompetent cells can be cultured in vitro in the presence of immunization to induce immunogen-specific Cytotoxic T lymphocytes. To induce immunogenic cytotoxic T lymphocytes, any stimulating factor can be added to the cell culture. For example, IL-2 is a preferred stimulator of cytotoxic T lymphocyte induction.

In some embodiments, the step of monitoring or assessing the immune response of an individual to be treated with a peptide cancer therapy can be performed before, during, and/or after treatment. In general, in a method of cancer therapy, an individual immunogenic peptide is repeatedly administered. For example, an immunogenic peptide can be administered weekly for 3-10 weeks. Thus, in a method of cancer therapy, an individual's immune response can be assessed or monitored. Alternatively, the step of assessing or monitoring the cancer treatment can be at the completion of the treatment.

According to the present invention, the promotion of induction of immunogenic-specific cytotoxic T lymphocytes is compared to the control group indicating that the individual being evaluated or diagnosed immunologically responds to the administered immunogen. Suitable control sets for assessing an immune response include, for example, when the immunocompetent cells are not contacted with any peptide or with a control peptide having an amino acid sequence other than the SMYD3 peptide (eg, any amino acid sequence) The degree of induction of cytotoxic T lymphocytes.

XII. antibody

The invention further provides antibodies that bind to the peptides of the invention. Preferred antibodies bind specifically to the peptide of the invention and do not bind (or weakly bind) to other peptides. Antibodies against the peptides of the invention can be provided for cancer diagnosis and prognosis analysis. Similarly, this antibody can be used in the treatment, diagnosis, and/or prognosis of cancer. Moreover, antibodies (eg, single chain antibodies) that are expressed intracellularly are therapeutically useful for treating cancers that are involved in the expression of SMYD3, such as, but not limited to, colorectal cancer, liver cancer, breast cancer, and bladder cancer.

The invention also provides for detecting and/or quantifying SMYD3 protein (preface) Various immunoassays of column identification numbers: 60, 62 or 64) or fragments thereof, including selection of free sequence identification numbers: 2, 4, 5, 6, 7, 8, 9, 13, 14, 16, 17, 19 and A peptide consisting of the amino acid sequence of the group consisting of 22. In the context of the present invention, the anti-SMYD3 antibody that binds to the SMYD3 peptide is preferably identified by a sequence number selected from the group consisting of: 2, 4, 5, 6, 7, 8, 9, 13, 14, 16, 17 a peptide consisting of one of the amino acid sequences of 19, and 22. The binding specificity of the antibody can be confirmed by inhibition test. In the presence of any of the multi-peptide fragments consisting of the amino acid sequences of the sequence identifiers: 2, 4, 5, 6, 7, 8, 9, 13, 14, 16, 19, and 22, When the binding between the antibody to be analyzed and the SMYD3 full-length multi-peptide is inhibited, such an antibody is considered to specifically bind the fragment. In the context of the present invention, such immunoassays are performed in a variety of immunoassay procedures well known in the art, including, but not limited to, various radio-immunoassays, immuno-chromatgraph techniques, Enzyme-linked immunosorbent assays (ELISA), enzyme-linked immunofluorescent assays (ELIFA), and the like.

Immunological but non-antibody analysis can also include T cell immunogenicity assays (inhibition or stimulation) as well as major histocompatibility complex (MHC) binding assays. Moreover, the present invention also contemplates immunoenvironment assays that detect cancers that exhibit SMYD3, examples of which include, but are not limited to, radio scintigraphic imaging using labeled antibodies of the invention. Such analysis provides clinical use in the detection, monitoring and prognosis of cancer manifested by SMYD3, for example but Not limited to, colorectal cancer, liver cancer, breast cancer, and bladder cancer.

The antibody of the present invention can be used in any form, for example, a single strain or a plurality of antibodies, and may further include an antiserum obtained by immunizing an animal, such as a rabbit, with the peptide of the present invention, all types of multiple strains or monoclonal antibodies. Human antibodies and humanized antibodies produced by genetic recombination.

The antibody of the present invention can recognize an amino acid sequence having a group consisting of the following sequence identification numbers: 2, 4, 5, 6, 7, 8, 9, 13, 14, 16, 17, 19 and 22. The peptide. Methods for synthesizing oligopeptides are well known in the art. After synthesis, the peptide can be selectively purified prior to use as an immunogen. In the context of the present invention, the oligopeptide (e.g., the octapeptide or the acetapeptide) can be conjugated or linked to enhance immunogenicity. Keyhole-limpet hemocyanin (KLH) is a well-known vector. Methods of joining keyhole limpet hemocyanin and peptides are well known in the art.

Alternatively, a gene encoding a peptide of the present invention or a fragment thereof can be inserted into a known expression vector and then transformed into a host cell as described herein. The desired peptide can be recovered from the outside or inside of the host cell by any standard method and can then be used as an antigen. Alternatively, the entire cell expressing the peptide or its cell extract or chemically synthesized peptide can be used as an antigen.

Any mammal can be immunized with the antigen, but compatibility of the parental cells for cell fusion is preferred. In general, animals of Rodentia, Lagomorpha or Primate can be used. Rodent animals include, for example, mice, rats, and hamsters. Animals of the rabbit-shaped family include, for example, rabbits. Primate animals include, for example, narrow-nose (old world monkey) monkeys, such as Macaca fascicularis, rhesus monkeys, Sacred baboon and chimpanzees.

Methods for immunizing animals with antigens are well known in the art. Intraperitoneal injection or subcutaneous injection of antigen is one of the standard methods for immunizing mammals. More specifically, the antigen may be diluted or suspended in a suitable amount of a phosphate buffer solution, physiological saline solution or the like. If desired, the antigen suspension can be combined with a suitable amount of a standard adjuvant, such as Freund's complete adjuvant, to make an emulsion and then administered to the mammal. Preferably, it is administered followed by an antigen mixed with a suitable amount of Freund's incomplete adjuvant every 4 to 21 days. A suitable vector can also be used for immunization. Following the above immunization, serum can be tested by standard methods for an increase in the amount of antibody required.

The polyclonal antibody against the peptide of the present invention can be prepared by collecting blood from an immunized animal that has been tested to increase the desired antibody in serum and isolating serum from blood by any conventional method. A plurality of antibodies may include a serum containing a plurality of antibodies, and a fraction of a plurality of antibodies isolated from the serum. For example, using an affinity column coupled to the peptide of the present invention, the portion is further purified using a Protein A or Protein G column, and the immunoglobulin G or M can be purified from only a portion of the peptide of the present invention.

To prepare a monoclonal antibody for use in the present invention, immune cells are collected from a mammal which has been immunized with an antigen and confirmed to have an increased degree of the desired antibody in the serum, as described above, and the immune cells are subjected to cell fusion. The immune cells used for cell fusion may preferably be obtained from the spleen. Other parental cells fused to the above-described immune cells include, for example, myeloma of a mammal, and preferably myeloma cells having characteristics obtained by drug-selected fused cells.

The above immune cells can be fused with myeloma cells according to known methods, for example, by Milstein et al. (Galfre and Milstein, Methods Enzymol 73: 3-46 (1981)).

Fusion tumors obtained from cell fusion are screened by culturing them in standard screening media, such as HAT medium (medium containing hypoxanthine, aminopterin, and thymidine). The cells are typically maintained in HAT medium for days to weeks for the time to allow death of other cells (non-fused cells) other than the desired fusion tumor. Thereafter, standard limiting dilutions can be performed to screen and replicate fusion tumors that produce the desired antibodies.

In addition to the above methods for preparing a fusion tumor and immunizing a non-human animal with an antigen, the peptide, the peptide-expressing cell or the cell extract thereof can be used to immunize human lymphocytes in vitro, such as human lymphocytes infected with Epstein-Barr virus. Thereafter, the immunized lymphocytes can be fused with a human-derived myeloma, such as U266, with ambiguous ability to produce a fusion of the desired human antibody, and the desired human antibody can be combined with the obtained peptide. (The published Japanese patent JPS 63-17688 has not been examined).

The obtained fusion tumor was then transplanted into the abdominal cavity of the mouse and ascites was extracted. The obtained monoclonal antibodies can be purified by, for example, ammonium sulfate precipitation, protein A or protein G column, DEAE ion exchange chromatography or an affinity column coupled to the peptide of the present invention. The antibody of the present invention can be used not only for purifying and detecting the peptide of the present invention, but also as a candidate for a promoter and/or an antagonist of the peptide of the present invention.

Genetic engineering techniques can also be used to recombinantly prepare and thus obtain monoclonal antibodies (see, for example, Borrebaeck and Larrick, Therapeutic Monoclonal Antibodies, published in the United Kingdom by MacMillan Publishers LTD (1990)). For example, the DNA encoding the antibody can be replicated from an immune cell, such as an antibody producing fusion tumor or an immunized lymphocyte, inserted into a suitable vector, and introduced into a host cell to produce a recombinant antibody. The invention also provides recombinant antibodies prepared as described above.

The antibody of the present invention may be a fragment of an antibody or a modified antibody as long as it binds to the peptide of the present invention. For example, the antibody fragment can be Fab, F(ab')2, Fv or single-chain Fv (scFv), wherein the Fv fragments from the heavy and light chains are joined by a suitable ligation sequence (Huston et al., Proc Natl) Acad Sci USA 85: 5879-83 (1988)). More particularly, antibody fragments can be produced by treating the antibody with an enzyme such as papain or pepsin. Alternatively, a gene encoding the antibody can be constructed, inserted into an expression vector and expressed in a suitable host cell (see, for example, Co et al., J Immunol 152: 2968-76 (1994); Better and Horwitz, Methods Enzymol 178:476 -96 (1989); Pluckthun and Skerra, Methods Enzymol 178: 497-515 (1989); Lamoyi, Methods Enzymol 121: 652-63 (1986); Rousseaux et al., Methods Enzymol 121: 663-9 (1986); Bird and Walker, Trends Biotechnol 9: 132-7 (1991)).

The antibody can be modified by binding to various molecules such as polyethylene glycol (PEG). The invention provides a modified antibody as described herein. Modified antibodies can be obtained by chemically modifying the antibody. These modifications are known in the art.

Alternatively, the antibody of the present invention can be obtained as a chimeric antibody, between a variable region derived from a non-human antibody and a fixed region derived from a human antibody, or a humanized antibody. The body includes a complementarity determining region from a non-human antibody, a framework work region (FR), and a fixed region derived from a human antibody. Such antibodies can be prepared according to known methods. Humanization can be performed by substituting the sequence corresponding to the human antibody by the sequence of the rodent complementarity determining region (see, for example, Verhoeyen et al., Science 239: 1534-1536 (1988)). Thus, such humanized antibodies are chimeric antibodies in which substantially less than the entire human variable region has been replaced by a corresponding sequence from a non-human species.

Whole human antibodies including human variable regions in addition to the framework and fixed regions can also be used. Such antibodies can be produced using a variety of techniques known in the art. For example, in vitro methods include the use of recombinant databases of human antibody fragments presented on phage (eg, Hoogenboom & Winter, J. Mol. Biol. 227:381 (1991)). Similarly, human antibodies can be made by introducing a human immunoglobulin locus (loci) into a transgenic animal, such as a mouse in which the endogenous immunoglobulin gene has been partially or completely deactivated. No. 6,150,584, 5,545,807; 5,545,806; 5,569,825;

Antibodies obtained from the above can be purified to homogeneity. For example, separation and purification of antibodies can be performed according to methods for separation and purification of general proteins. For example, by suitable selection and binding column chromatography, such as affinity column, filtration, ultrafiltration, salting out, dialysis, sodium lauryl sulfate, polyacrylamide gel electrophoresis (SDS polyacrylamide gel) The use of electrophoresis and isoelectric focusing can separate and isolate antibodies (Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory (1988)), but not Limited to this. Protein A column and protein G column can be used as affinity column. The protein A column used includes, for example, Hyper D, POROS, and Sepharose F.F. (Pharmacia).

In addition to affinity chromatography, examples of chromatographic techniques include, for example, ion exchange chromatography, hydrophobic chromatography, colloidal filtration, inverse chromatography, adsorption chromatography, etc. (Strategies for Protein Purification and Characterization) : A Laboratory Course Manual. Ed Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press (1996)). The chromatography step can be performed by liquid chromatography, such as HPLC and FPLC.

For example, the antigen binding activity of the antibody of the present invention can be measured using an enzyme-linked immunosorbent assay (ELISA), an enzyme immunoassay (EIA), a radioimmunoassay (RIA), and/or immunofluorescence to measure the absorbance. In an enzyme-linked immunosorbent assay, the antibody of the present invention is immobilized on a culture plate to provide the peptide of the present invention to the culture plate, followed by providing a sample containing the desired antibody, for example, a culture suspension of the antibody-producing cells or purified. Antibodies. Thereafter, an enzyme that recognizes the first antibody and is labeled, such as a second antibody to alkaline phosphatase, is provided, and then the culture plate is cultured. Next, after washing, an enzyme substrate such as p-nitrophenyl phosphate is added to the culture plate, and absorption is measured to evaluate the antigen binding activity of the sample. BIAcore (Pharmacia) can be used to assess the activity of the antibodies of the invention.

XII. Vector and host cells

The invention also provides vectors and host cells for introducing a nucleotide encoding a peptide of the invention. The vector of the present invention provides a vector, particularly DNA, as a polynucleotide in a host cell to express the peptide of the present invention, or to be a gene therapy The nucleotide of the present invention is administered.

When E. coli is selected as the host cell and the vector is amplified and produced in large quantities in E. coli (eg, JM109, DH5 alpha, HB101 or XL1Blue), the vector should have an "ori" suitable for amplification in E. coli . And a marker gene for screening for transgenic E. coli (for example, by, for example, ampicillin, tetracycline, kanamycin, chloramphenicol or the like) Drug screening for one of the drug resistance genes). For example, an M13-series vector, a pUC-series vector, pBR322, pBluescript, pCR-Script, or the like can be used. Moreover, pGEM-T, pDIRECT and pT7 can also be used for secondary selection and extraction of cDNA with the above vectors. A performance vector can be useful when the vector is used to produce a peptide of the invention. For example, a performance vector to be expressed in E. coli should have the above characteristics to be amplified in E. coli. When E. coli, such as JM109, DH5 alpha, HB101 or XL1 Blue, is used as the host cell, the vector should have a promoter, such as the lacZ promoter (Ward et al., Nature 341:544-6 (1989); FASEB J 6:2422-7 (1992)), araB promoter (Better et al., Science 240: 1041-3 (1988)), T7 promoter or analog, which is effective for expressing the desired gene in E. coli. in. In that regard, the above vector can be replaced with, for example, pGEX-5X-1 (Pharmacia), "QIAexpress system" (Qiagen), pEGFP and pET (in this case, the host preferably BL21, which expresses T7 RNA polymerase). Moreover, the vector may also contain a signal sequence for peptide secretion. A signal sequence that directs the secreted peptide to the periplasm of E. coli, such as the pelB signal sequence (Lei et al., J Bacteriol 169: 4379 (1987)). The manner in which the vector is introduced into the host cell of interest includes, for example, a calcium chloride method, and an electroporation method.

In addition to E. coli, for example, mammalian expression vectors (eg, pcDNA3 (Invitrogen) and pEGF-BOS (Nucleic Acids Res 18 (17): 5322 (1990)), pEF, pCDM8), expression vectors from insect cells (eg, , "Bac-to-BAC baculovirus expression system" (GIBCO BRL), pBacPAK8), plant-derived expression vectors (eg, pMH1, pMH2), expression vectors from animal viruses (eg, pHSV, pMV, pAdexLcw), a expression vector derived from retrovirus (for example, pZIpneo), a expression vector derived from yeast (for example, "Pichia Expression Kit" (Invitrogen), pNV11, SP-Q01) and from Bacillus subtilis. A performance vector (e.g., pPL608, pKTH50) can be used to produce the peptide of the present invention.

In order to express a vector in an animal cell, such as a CHO, COS or NIH3T3 cell, the vector should have a promoter necessary for expression in such a cell, such as the SV40 promoter (Mulligan et al., Nature 277: 108 (1979) , MMLV-LTR promoter, EF1 alpha promoter (Mizushima et al., Nucleic Acids Res 18: 5322 (1990)), CMV promoter, etc., and preferably a marker gene for screening for transformants (eg by drug) Screening (e.g., neomycin, anti-drug gene of G418) Examples of known vectors having these vector characteristics include, for example, pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following materials, methods, and examples are intended to be illustrative of the embodiments of the invention, and are not intended to limit the scope of the invention. As can be easily understood by those skilled in the art, The invention may be carried out or tested using methods and materials similar or equivalent to those shown in the present specification.

[Examples]

Example 1

Materials and Methods

Cell line

TISI, HLA-A*2402-positive B-lymocyte strains were purchased from IHWG Cell and Gene Bank (Seattle, WA). T2, HLA-A*0201 positive B-lymphocyte cell line, COS7, African green monkey kidney cell line were purchased from ATCC.

Candidate screening for peptides derived from SMYD3

Use "NetMHC3.2" in conjunction with the predictive server "BIMAS" (www-bimas.cit.nih.gov/molbio/hla_bind) (Parker et al., J Immunol 1994, 152(1): 163-75; Kuzushima et al , Blood 2001, 98(6): 1872-81) to predict the Nine Peptides and Tokapeptides derived from SMYD3 that bind to HLA-A*2402 or HLA-A*0201 molecules. These peptides were synthesized by standard solid phase synthesis using Biosynthesis (Lewisville, Texas) and purified by reverse high performance liquid chromatography (HPLC). The purity (>90%) and identity of these peptides were confirmed by analytical HPLC and mass spectrometry, respectively. The peptide was dissolved in dimethylsulfoxide (DMSO) at 20 mg/ml and stored at -80 °C.

Induction of cytotoxic T lymphocytes in vitro

Dendritic cells (DC) from mononuclear leukocytes are used as antigen-presenting cells to induce cytotoxic T lymphocyte responses against peptides present on human leukocyte antigen (HLA). Dendritic cells are produced in vitro as described in the previous literature (Nakahara S et al., Cancer Res 2003 Jul 15, 63(14): 4112-8). Specifically, peripheral blood mononuclear cells were isolated from a normal volunteer (HLA-A*2402 or HLA-A*0201 positive) in a Ficoll-Plaque (Pharmacia) solution via attaching to a plastic tissue culture plate (Becton Dickinson) The separation is therefore enriched as a mononuclear leukocyte fraction. The mononuclear white blood cell population is cultured at 1000 U/ml of granulocyte-macrophage colony-stimulating factor (GM-CSF) (R&D System) and 1000 U/ml of interleukin (interleukin, IL). -4 (R&D System) in the presence of 2% heat-deactivated autologous serum (AS) in AIM-V medium (Invitrogen). After 7 days of culture, the pulsarized cytokines were synthesized at 20 μg/ml in the presence of 3 μg/ml of β-2-microglobulin in AIM-V medium at 37 °C. Dendritic cells were induced for 3 hours. The resulting cells showed dendritic cell-associated molecules on the cell surface, such as CD80, CD83, CD86 and HLA Class II (data not shown). These dendritic cells pulsed with peptides were then deactivated by X-rays (20 Gy) and mixed with autologous CD8-positive T cells at a ratio of 1:20. CD8-positive T cells were isolated by CD8-positive T-cells. A positive screening of the group (Dynal) was obtained. These cultures were set in 48-well plates (Corning); each well containing 1.5 x 10 ⁴ warp dendritic cell peptide pulses, 3 x 10 ⁵ th CD8-positive T cells with 10ng / ml of IL-7 (R & D System ) in 0.5 ml of AIM-V/2% autologous serum medium. Three days later, IL-2 (CHIRON) was added to the culture to a final concentration of 20 IU/ml. On day 7 and day 14, T cells were further stimulated by dendritic cells pulsed with peptides. Dendritic cells were prepared each time in the same manner as above. On day 21, after the third round of peptide stimulation, the cytotoxic T lymphocytes were subjected to TISI cells pulsed with peptides (Tanaka H et al., Br J Cancer 2001 Jan 5, 84(1): 94- 9; Umano Y et al., Br J Cancer 2001 Apr 20, 84(8): 1052-7; Uchida N et al., Clin Cancer Res 2004 Dec 15, 10(24): 8577-86; Suda T et al ., Cancer Sci 2006 May, 97(5): 411-9; Watanabe T et al., Cancer Sci 2005 Aug, 96(8): 498-506).

Cytotoxic T lymphocyte expansion step

Use as described by Riddell et al. (Walter EA et al., N Engl J Med 1995 Oct 19, 333(16): 1038-44; Riddell SR et al., Nat Med 1996 Feb, 2(2): 216-23) A similar approach dilates cytotoxic T lymphocytes in culture. All 5 x 10 ⁴ cytotoxic T lymphocytes were suspended in 25 ml of AIM-V/5% autologous serum medium containing two human B lymphoblastoid cells at 40 ng/ml anti-CD3 monoclonal antibody ( In the presence of Pharmingen), it is deactivated by MMC. One day after the start of the culture, 120 IU/ml of IL-2 was added to the culture. On days 5, 8, and 11, fresh medium was supplied to AIM-V/5% autologous serum medium containing 30 IU/ml of IL-2 (Tanaka H et al., Br J Cancer 2001 Jan 5, 84 (1) ): 94-9; Umano Y et al., Br J Cancer 2001 Apr 20, 84(8): 1052-7; Uchida N et al., Clin Cancer Res 2004 Dec 15, 10(24): 8577-86; Suda T et al., Cancer Sci 2006 May, 97(5): 411-9; Watanabe T et al., Cancer Sci 2005 Aug, 96(8): 498-506).

Establishment of cytotoxic T lymphocyte selection (clone)

A 96-round-bottomed micro-valence plate (Nalge Nunc International) was diluted to have 0.3, 1 and 3 cytotoxic T lymphocytes per well. Cytotoxic T lymphocytes and two human B lymphoblastoid cells with 1 x 10 ⁴ cell wells per well, 30 ng/ml anti-CD3 antibody and 125 IU/ml IL-2 in total 150 μl per well The cells were cultured together in AIM-V medium containing 5% autologous serum. After 10 days, 50 μl/well of IL-2 was added to the medium to reach a final concentration of 125 IU/ml IL-2. The cytotoxic T lymphocyte activity was tested on day 14 and the cytotoxic T lymphocyte selection was expanded using the same method described above (Uchida N et al., Clin Cancer Res 2004 Dec 15, 10(24): 8577-86; Suda T et al., Cancer Sci 2006 May, 97(5): 411-9; Watanabe T et al., Cancer Sci 2005 Aug, 96(8): 498-506).

Specific cytotoxic T lymphocyte activity

To test specific cytotoxic T lymphocyte activity, an IFN-γ enzyme-binding immunospot (ELISPOT) assay was performed with IFN-γ enzyme-binding immunosorbent assay (ELISA). TISI (1 x 10 ⁴ /well) or T2 (1 x 10 ⁴ /well) of the peptide pulse was prepared as a stimulus cell. The cells cultured in 48 wells were used as responder cells. IFN-γ enzyme binding immunospot analysis and IFN-γ enzyme binding immunosorbent assay were performed according to the manufacturer's procedure.

Establishment of cells that strongly express the target gene and/or HLA-A24 or HLA-A*0201

The cDNA encoding the open reading frame of the target gene, HLA-A*2402 or HLA-A*0201 was amplified by PCR. The PCR amplification product is cloned into a performance vector. The plastids were transfected into COS7 using the lipofectamine 2000 (Invitrogen) according to the manufacturer's recommended procedure, which is the target gene, HLA-A*2402-null or HLA-A*0201-ineffective cell line. Two days after transfection, transfected cells were harvested as versene (Invitrogen) and target cells (5 x 10 ⁴ cells/well) analyzed for cytotoxic T lymphocyte activity were used.

result

Prediction of HLA-A24 binding peptide derived from SMYD3

Tables 1a and 1b show the high binding affinity ranking of HLA-A24 binding to HIX- and B-peptides of SMYD3. A total of 19 peptides with HLA-A24 binding activity were screened and analyzed to determine the epitope-determining peptide.

The starting position is the number of amino acids starting from the N-terminus of SMYD3.

The combined score is from "BIMAS".

The starting position is the number of amino acids starting from the N-terminus of SMYD3.

The combined score is from "BIMAS".

Prediction of HLA-A*0201 binding peptide derived from SMYD3

Tables 2a and 2b show the high binding affinity ranking of HLA-A*0201 binding to septin and ten peptides of SMYD3. A total of 39 peptides with HLA-A*0201 binding activity were screened and analyzed to determine the epitope peptide.

The starting position is the number of amino acids starting from the N-terminus of SMYD3.

The combined score is from "BIMAS".

The starting position is the number of amino acids starting from the N-terminus of SMYD3.

The combined score is from "BIMAS".

Induction of CTL of putative peptides derived from SMYD3 restricted by HLA-A*2402

CTLs derived from SMYD3 peptides were generated according to the procedures described in "Materials and Methods". The peptide-specific CTL activity was detected by IFN-γ ELISPOT assay (Fig. 1a-k). Well number #1 includes induction of (a) with SMYD3-A24-9-197 (SEQ ID NO: 2), number #6 with SMYD3-A24-9-326 (SEQ ID NO: 4) Induction (b), ## was induced with SMYD3-A24-9-138 (SEQ ID NO: 5) (c), and #6 was induced with SMYD3-A24-9-130 (SEQ ID NO: 6) (d) , #7 is induced with SMYD3-A24-9-192 (SEQ ID NO: 7) (e), and #1 is induced with SMYD3-A24-9-118 (SEQ ID NO: 8) (f), number #4 Induction of (S) with SMYD3-A24-9-301 (SEQ ID NO: 9), induction of (#) with SMYD3-A24-10-260 (SEQ ID NO: 13), number #3 with SMYD3-A24 -10-266 (SEQ ID NO: 14) induces (i), number #3 induces (j) with SMYD3-A24-10-86 (SEQ ID NO: 17), and number #8 with SMYD3-A24-10- 138 (SEQ ID NO: 19) induced (k), demonstrating that effective IFN-γ production was shown compared to the control group. On the other hand, although this peptide had the potential to bind to HLA-A*2402, no specific CTL activity was detected by stimulation with other peptides shown in Table 1a and Table 1b. Regarding the negative data, no specific IFN-γ production was observed in the SMYD3-A24-9-37 (SEQ ID NO: 1)-induced CTL (1). Taken together, this result suggests that the 11 screening peptides derived from SMYD3 can induce potent CTLs.

Induction of CTL of putative peptides derived from SMYD3 restricted by HLA-A*0201

CTLs derived from SMYD3 peptides were generated according to the procedures described in "Materials and Methods". The peptide-specific CTL activity was detected by IFN-γ ELISPOT assay (Fig. 2a-c). Hole number #6 was induced with SMYD3-A02-9-335 (SEQ ID NO: 22) (a), and #7 was induced with SMYD3-A02-9-118 (SEQ ID NO: 8) (b), with number # 4 (c) was induced with SMYD3-A02-10-76 (SEQ ID NO: 16), demonstrating that effective IFN-γ production was shown compared to the control group. On the other hand, only this peptide has the potential to bind to HLA-A*0201, as shown in Table 2a and Table 2b. Other peptide stimulations did not detect specific CTL activity. Regarding the negative data, no specific IFN-γ production was observed in the SMYD3-A02-9-45 (SEQ ID NO: 20)-induced CTL (d). Taken together, this result speculates that these three peptides derived from SMYD3 can induce potent CTLs.

Establishment of CTL cell lines and colonies resistant to SMYD3-derived peptides

The well number #1(a) containing SMYD3-A24-9-197 (SEQ ID NO: 2) and the well number containing SMYD3-A24-9-130 (SEQ ID NO: 6) were detected by IFN-γ ELISPOT assay. #6(b), well number #7(c) containing SMYD3-A24-9-192 (SEQ ID NO: 7), well number #1 containing SMYD3-A24-9-118 (SEQ ID NO: 8) (d), well number #3(e) containing SMYD3-A24-10-86 (SEQ ID NO: 17), and well number #8 containing SMYD3-A24-10-138 (SEQ ID NO: 19) ( f) shows cell expansion of the peptide-specific CTL activity and establishes a cell line. The CTL activity of these CTL cell lines can be detected by IFN-γ ELISA (Fig. 3). These cell lines confirmed the addition of SMYD3-A24-9-197 (SEQ ID NO: 2), SMYD3-A24-9-130 (SEQ ID NO: 6), SMYD3-A24-9-192 (SEQ ID NO: 7), SMYD3-A24-9-118 (SEQ ID NO: 8), SMYD3-A24-10-86 (SEQ ID NO: 17), and SMYD3-A24-10-138 (SEQ ID NO: 19) target cells of the peptide Target cells with no peptide added have potent IFN-γ production. Furthermore, CTL colonies were established by restriction-diluting CTL cell lines as described in "Materials and Methods", and IFN-γ of CTL colonies against target cell pulse peptides was measured by IFN-γ ELISA. produce. Effective IFN-γ production is in SMYD3-A24-9-197 (SEQ ID NO: 2) (a), SMYD3-A24-9-118 (SEQ ID NO: 8) (b), and SMYD3-A24- 10-138 (SEQ ID NO: 19) (c) stimulated CTL selection Observed (Figure 5).

Establishment of CTL cell lines and colonies resistant to SMYD3-derived peptides

The cell expansion showing the peptide-specific CTL activity in well number #6 containing SMYD3-A02-9-335 (SEQ ID NO: 22) was detected by IFN-γ ELISPOT assay and the cell strain was established. The CTL activity of these CTL cell lines can be detected by IFN-γ ELISA (Fig. 4). These cell lines confirmed that the target cells to which the SMYD3-A02-9-335 (SEQ ID NO: 22) peptide was added had potent IFN-γ production compared to the target cells to which no peptide was added. Furthermore, CTL colonies were established by restriction-diluting CTL cell lines as described in "Materials and Methods", and IFN-γ of CTL colonies against target cell pulse peptides was measured by IFN-γ ELISA. produce. Efficient production of IFN-[gamma] was observed in CTL colonies stimulated with SMYD3-A02-9-335 (SEQ ID NO: 22) (Fig. 6).

Specific CTL activity against target cells expressing SMYD3 and HLA-A*2402

Detection of CTL colonies established by anti-SMYD3-A24-9-197 (SEQ ID NO: 2) peptides produced the ability to recognize target cells expressing SMYD3 and HLA-A*2402 molecules. COS7 cells transfected with full-length SMYD3 and HLA-A*2402 genes (specific patterns of target cells expressing SMYD3 and HLA-A*2402 genes) were used as stimulator cells and transfected with SMYD3 or HLA-A *2402 of one of the cells served as a control group. As shown in Figure 7, CTL colonies stimulated with SMYD3-A24-9-197 (SEQ ID NO: 2) showed potent CTL activity against COS7 cells expressing SMYD3 and HLA-A* 2402. On the other hand, the control group did not find significant specific CTL activity. Therefore, this data clearly confirms that SMYD3-A24-9-197 (SEQ ID NO: 2) The peptide is endogenously processed and expressed on target cells with HLA-A*2402 molecules and can be recognized by CTLs. This result indicates that this peptide derived from SMYD3 can be used as a cancer vaccine for patients suffering from SMYD3 tumors.

Specific CTL activity against target cells expressing SMYD3 and HLA-A*0201

The ability to recognize target cells expressing SMYD3 and HLA-A*0201 molecules was examined by detecting CTL cell lines established by anti-SMYD3-A02-9-335 (SEQ ID NO: 22) peptide. COS7 cells transfected with full-length SMYD3 and HLA-A*0201 genes (specific patterns of target cells expressing SMYD3 and HLA-A*0201 genes) were used as stimulator cells and transfected with SMYD3 or HLA-A *0201 One of the cells served as a control group. As shown in Fig. 8, the CTL cell line stimulated with SMYD3-A02-9-335 (SEQ ID NO: 22) showed potent CTL activity against COS7 cells expressing SMYD3 and HLA-A*0201. On the other hand, the control group did not find significant specific CTL activity. Therefore, this data clearly confirms that the SMYD3-A02-9-335 (SEQ ID NO: 22) peptide is endogenously treated and expressed on target cells with HLA-A*0201 molecule and can be recognized by CTLs. This result indicates that this peptide derived from SMYD3 can be used as a cancer vaccine for patients suffering from SMYD3 tumors.

Homology analysis of antigenic peptides

SMYD3-A24-9-197 (SEQ ID NO: 2), SMYD3-A24-9-326 (SEQ ID NO: 4), SMYD3-A24-9-138 (SEQ ID NO: 5), SMYD3-A24- 9-130 (SEQ ID NO: 6), SMYD3-A24-9-192 (SEQ ID NO: 7), SMYD3-A24-9-118 (SEQ ID NO: 8), SMYD3-A24-9-301 (SEQ ID NO: 9), SMYD3-A24-10-260 (SEQ ID NO: 13), SMYD3-A24-10-226 (SEQ ID NO: 14), SMYD3-A24-10-86 (SEQ ID NO: 17) and SMYD3-A24-10-138 (SEQ ID NO: 19) Stimulated CTLs have significant and specific CTL activity. This result may be due to SMYD3-A24-9-197 (SEQ ID NO: 2), SMYD3-A24-9-326 (SEQ ID NO: 4), SMYD3-A24-9-138 (SEQ ID NO: 5), SMYD3-A24-9-130 (SEQ ID NO: 6), SMYD3-A24-9-192 (SEQ ID NO: 7), SMYD3-A24-9-118 (SEQ ID NO: 8), SMYD3-A24-9 -301 (SEQ ID NO: 9), SMYD3-A24-10-260 (SEQ ID NO: 13), SMYD3-A24-10-226 (SEQ ID NO: 14), SMYD3-A24-10-86 (SEQ ID The sequence of NO: 17) and SMYD3-A24-10-138 (SEQ ID NO: 19) is a homologue to a molecular peptide derived from other known sensitizing to the immune system.

To rule out this possibility, homology analysis of these peptides can be analyzed using the BLAST algorithm (http://blast.ncbi.nlm.nih.gov/Blast.cgi), showing no significant homology between the sequences. The results of the homology analysis revealed that SMYD3-A24-9-197 (SEQ ID NO: 2), SMYD3-A24-9-326 (SEQ ID NO: 4), SMYD3-A24-9-138 (SEQ ID NO: 5) ), SMYD3-A24-9-130 (SEQ ID NO: 6), SMYD3-A24-9-192 (SEQ ID NO: 7), SMYD3-A24-9-118 (SEQ ID NO: 8), SMYD3-A24 -9-301 (SEQ ID NO: 9), SMYD3-A24-10-260 (SEQ ID NO: 13), SMYD3-A24-1-226 (SEQ ID NO: 14), SMYD3-A24-10-86 ( SEQ ID NO: 17) is unique to the sequence of SMYD3-A24-10-138 (SEQ ID NO: 19), and as far as we know, these molecules are almost impossible to produce unintended immunity to certain unrelated molecules. reaction.

In summary, the novel HLA-A*2402 epitope peptide derived from SMYD3 can be applied to the field of cancer immunotherapy.

Homology analysis of antigenic peptides

CTLs stimulated with SMYD3-A02-9-335 (SEQ ID NO: 22), SMYD3-A02-9-118 (SEQ ID NO: 8) and SMYD3-A02-10-76 (SEQ ID NO: 16) were significant And specific CTL activity. This result may be due to SMYD3-A02-9-335 (SEQ ID NO: 22), SMYD3-A02-9-118 (SEQ ID NO: 8) and SMYD3-A02-10-76 (SEQ ID NO: 16) The sequence is a homologue to a molecular peptide derived from other known sensitizing immune systems.

To rule out this possibility, homology analysis of these peptides can be analyzed using the BLAST algorithm (http://blast.ncbi.nlm.nih.gov/Blast.cgi), showing no significant homology between the sequences. The results of the homology analysis revealed that SMYD3-A02-9-335 (SEQ ID NO: 22), SMYD3-A02-9-118 (SEQ ID NO: 8) and SMYD3-A02-10-76 (SEQ ID NO: 16) The sequences are unique, so as far as we know, these molecules are almost impossible to produce unintended immune responses to certain unrelated molecules.

In summary, the novel HLA-A*0201 epitope peptide derived from SMYD3 can be applied to the field of cancer immunotherapy.

[Industry Utilization]

The present invention provides novel epitopes that induce potent and specific anti-tumor immune responses and are applicable to a wide variety of cancers. This peptide can be used as a peptide vaccine for diseases related to SMYD3, for example, colorectal cancer, liver cancer, breast cancer, and bladder cancer.

While the invention has been described herein with reference to the specific embodiments, In the course of the routine experiment, those skilled in the art can immediately understand that various changes and modifications can be made without departing from the spirit and scope of the invention.

<110> Tumor therapy. Science Co., Ltd. (ONCOTHERAPY SCIENCE, INC.)

<120> SMYD3 peptide and vaccine containing the peptide

<130> ONC-A1303-TW

<150> US 61/776,455

<151> 2013-03-11

<160> 69

<170> PatentIn version 3.5

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<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> peptide derived from SMYD3

<400> 36

<210> 37

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> peptide derived from SMYD3

<400> 37

<210> 38

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> peptide derived from SMYD3

<400> 38

<210> 39

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> peptide derived from SMYD3

<400> 39

<210> 40

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> peptide derived from SMYD3

<400> 40

<210> 41

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> peptide derived from SMYD3

<400> 41

<210> 42

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> peptide derived from SMYD3

<400> 42

<210> 43

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> peptide derived from SMYD3

<400> 43

<210> 44

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> peptide derived from SMYD3

<400> 44

<210> 45

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> peptide derived from SMYD3

<400> 45

<210> 46

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> peptide derived from SMYD3

<400> 46

<210> 47

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> peptide derived from SMYD3

<400> 47

<210> 48

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> peptide derived from SMYD3

<400> 48

<210> 49

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> peptide derived from SMYD3

<400> 49

<210> 50

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> peptide derived from SMYD3

<400> 50

<210> 51

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> peptide derived from SMYD3

<400> 51

<210> 52

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> peptide derived from SMYD3

<400> 52

<210> 53

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> peptide derived from SMYD3

<400> 53

<210> 54

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> peptide derived from SMYD3

<400> 54

<210> 55

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> peptide derived from SMYD3

<400> 55

<210> 56

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> peptide derived from SMYD3

<400> 56

<210> 57

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> peptide derived from SMYD3

<400> 57

<210> 58

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> peptide derived from SMYD3

<400> 58

<210> 59

<211> 1469

<212> DNA

<213> Human (Homo sapiens)

<220>

<221> CDS

<222> (131)..(1240)

<400> 59

<210> 60

<211> 369

<212> PRT

<213> Human (Homo sapiens)

<400> 60

<210> 61

<211> 1641

<212> DNA

<213> Human (Homo sapiens)

<220>

<221> CDS

<222> (126)..(1412)

<400> 61

<210> 62

<211> 428

<212> PRT

<213> Human (Homo sapiens)

<400> 62

<210> 63

<211> 1622

<212> DNA

<213> Human (Homo sapiens)

<220>

<221> CDS

<222> (96)..(1382)

<400> 63

<210> 64

<211> 428

<212> PRT

<213> Human (Homo sapiens)

<400> 64

<210> 65

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> PCR primer for TCR analysis

<400> 65

<210> 66

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> PCR primer for TCR analysis

<400> 66

<210> 67

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> PCR primer for TCR analysis

<400> 67

<210> 68

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> PCR primer for TCR analysis

<400> 68

<210> 69

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 69

Claims (20)

A single-isopeptide having cytotoxic T lymphocyte (CTL) inducing ability, wherein the peptide comprises the following amino acid sequence (a) or (b): (a) selected from sequence identifiers: 2, 4 , the amino acid sequence of the group consisting of 5, 6, 7, 8, 9, 13, 14, 16, 17, 19 and 22; (b) selected from the sequence identification numbers: 2, 4, 5, 6 1, 1, or a plurality of amino acids in the amino acid sequence of groups consisting of 7, 8, 9, 13, 14, 16, 17, 19, and 22 are substituted, deleted, inserted, and/or added Amino acid sequence. The peptide according to claim 1, wherein the peptide is the following oligopeptide (i) or (ii): (i) a peptide having one or two of the following characteristics: (a) sequence recognition No.: The N-terminal second amino acid of the amino acid sequence of 2, 4, 5, 6, 7, 8, 9, 13, 14, 17, or 19 is substituted with phenylalanine, tyrosine, methyl sulfide Amino acid or tryptophan; and (b) C-terminal amino acid of the amino acid sequence of the sequence number: 2, 4, 5, 6, 7, 8, 9, 13, 14, or 19 is replaced Is phenylalanine, leucine, isoleucine, tryptophan or methionine; (ii) a peptide having one or two of the following characteristics: (a) sequence identification number: 8, 16 or 22 The N-terminal second amino acid of the amino acid sequence is substituted with leucine or methionine; and (b) the C-terminal amino acid of the amino acid sequence of sequence number 8, 8, or 22 is Replacement with proline or leucine. The peptide according to claim 1 or 2, wherein the peptide is a nine peptide or a ten peptide. The peptide according to claim 3, wherein the peptide is selected from the sequence identification numbers: 2, 4, 5, 6, 7, 8, 9, 13, 14, 16, 17, 19 and 22. Composed of a group of amino acid sequences. An isolated polynucleotide which encodes the peptide described in any one of claims 1 to 4. A composition for inducing a cytotoxic T lymphocyte (CTL), wherein the composition comprises at least one active ingredient selected from the group consisting of: (a) claiming any one of claims 1 to 4 (b) a polynucleotide according to claim 5; (c) an antigen presenting cell (APC) which exhibits the peptide of any one of claims 1 to 4; d) The peptide is exogenously expressed in any one of claims 1 to 4 on the surface. A pharmaceutical composition for treating and/or preventing cancer, and/or preventing postoperative recurrence, wherein the pharmaceutical composition comprises at least one active ingredient selected from the group consisting of: (a) Patent Application Nos. 1 to 4 a peptide according to any one of the preceding claims; (b) a polynucleotide according to claim 5; (c) an antigen presenting the peptide of any one of claims 1 to 4 on the surface Cell (APC); (d) surface-expressing the peptide excipients according to any one of claims 1 to 4; and (e) identifiable one of claims 1 to 4 Cytotoxic T lymphocytes (CTL) of the cells of the peptide. The pharmaceutical composition according to claim 7, wherein the pharmaceutical composition is formulated to be administered to an individual having an HLA antigen of HLA-A24 or HLA-A2. A method of promoting antigen presenting cells (APC) having cytotoxic T lymphocyte (CTL) inducing ability, wherein the method comprises the step of selecting from the group consisting of: (a) contacting antigen presenting cells (APC) The peptide according to any one of claims 1 to 4, and (b) introducing a polynucleotide encoding the peptide of any one of claims 1 to 4 into an antigen-presenting cell (APC). A method of inducing a cytotoxic T lymphocyte (CTL), wherein the method comprises the step of selecting a population consisting of: (a) presenting a CD8-positive T cell to a surface with an HLA antigen and claiming patent ranges 1 to 4 An antigen-presenting cell (APC) co-cultured with a complex of any of the peptides; (b) a CD8-positive T cell and a surface exhibiting an HLA antigen and the peptide of any one of claims 1 to 4 The exosome of the complex is co-cultured; and (c) a polynucleotide encoding a T cell receptor (TCR) subunit or a polynucleotide encoding each T cell receptor (TCR) subunit is introduced into the CD8 In the positive T cells, the T cell receptor (TCR) formed by the subunit can be bound to the complex of the peptide and the HLA antigen described in any one of claims 1 to 4 on the cell surface. An isolated antigen presenting cell (APC) having an HLA antigen on its surface and A composite of the peptides according to any one of claims 1 to 4 of the patent application. The antigen presenting cell (APC) according to claim 11, wherein the antigen presenting cell (APC) is induced by the method of Patent Application No. 9. An isolated cytotoxic T lymphocyte (CTL), the target of which is claimed in any one of claims 1 to 4. The cytotoxic T lymphocyte (CTL) according to claim 13, wherein the cytotoxic T lymphocyte (CTL) is induced by the method of claim 10 of the patent application. A method of inducing an anti-cancer immune response in an individual, wherein the method comprises the step of administering to the individual a composition comprising the peptide of any one of claims 1 to 4 or the polynucleotide encoding the peptide. An antibody or an immunologically active fragment thereof which is resistant to any of the peptides of any one of claims 1 to 4. A vector comprising a nucleotide sequence encoding the peptide of any one of claims 1 to 4. A host cell which is transfected or transfected with a vector of claim 17 of the patent application. A diagnostic kit comprising the peptide according to any one of claims 1 to 4, the polynucleotide of claim 5, or the antibody or immunologically active fragment thereof according to claim 16 of the patent application. . A method of screening for a peptide having the ability to induce specific cytotoxicity against a cytotoxic T lymphocyte (CTL) presenting a cell derived from a fragment of SMYD3, wherein the method comprises: (i) providing a candidate sequence, the candidate The sequence is one, two or several from the original amino acid The amino acid residue is substituted, deleted, inserted, and/or increased by the modified amino acid sequence, wherein the original amino acid sequence is selected from the sequence identifiers: 2, 4, 5, 6, and 7. a group consisting of 8, 9, 13, 14, 16, 17, 19, and 22; (ii) selecting a candidate sequence that, in addition to SMYD3, is not a peptide derived from any known human gene product Having substantially significant homology; (iii) contacting the peptide formed by the candidate sequence selected in step (ii) with the antigen presenting cell; (iv) contacting the antigen presenting cell of step (iii) with the CD8 positive T cell And (v) identifying a peptide having the same or higher cytotoxic T lymphocyte (CTL) inducing ability as the peptide consisting of the original amino acid sequence.