JP2013523083A - CLUAP1 peptide and vaccine containing the same - Google Patents

Abstract

This specification describes peptide vaccines against cancer. In particular, an epitope peptide derived from the CLUAP1 gene that induces CTL is provided. Also provided are antigen presenting cells and isolated CTL targeting such peptides, and methods for inducing the antigen presenting cells or CTLs. The present invention further provides a pharmaceutical composition comprising a peptide derived from CLUAP1 or a polynucleotide encoding the peptide as an active ingredient. Furthermore, the present invention relates to a method for treating and / or preventing cancer (tumor) and / or a method for preventing postoperative recurrence thereof, a peptide derived from CLUAP1, a polynucleotide encoding the peptide, Alternatively, the present invention provides a method for inducing CTLs and a method for inducing anti-cancer immunity using the antigen-presenting cells presenting the peptide, or the pharmaceutical composition of the present invention.

Description

  The present invention relates to the field of biological science, more specifically to the field of cancer therapy. In particular, the present invention relates to novel peptides that are very effective as cancer vaccines, and drugs for treating and preventing tumors.

Priority This application claims the benefit of, filed on April 8, 2010, US Provisional Application No. 61 / 322,099, the entire contents of which are incorporated herein by reference.

  CD8 positive CTL has been demonstrated to recognize an epitope peptide derived from a tumor associated antigen (TAA) on a major histocompatibility complex (MHC) class I molecule and then kill tumor cells. Many other TAAs have been discovered by immunological approaches since the discovery of the melanoma antigen (MAGE) family as the first example of TAA (Boon T, Int J Cancer 1993 May 8, 54 (2): 177-80 (Non-patent Document 1); Boon T & van der Bruggen P, J Exp Med 1996 Mar 1, 183 (3): 725-9 (Non-patent Document 2)), some of these TAAs are Currently in the process of clinical development as a target for immunotherapy.

  The identification of novel TAAs capable of inducing strong and specific anti-tumor immune responses ensures further development of peptide vaccination strategies and advances in clinical research against various types of cancer (Harris CC, J Natl Cancer Inst 1996 Oct 16, 88 (20): 1442-55 (Non-Patent Document 3); Butterfield LH et al., Cancer Res 1999 Jul 1, 59 (13): 3134-42 (Non-Patent Document 4); Vissers JL et al ., Cancer Res 1999 Nov 1, 59 (21): 5554-9 (Non-Patent Document 5); van der Burg SH et al., J Immunol 1996 May 1, 156 (9): 3308-14 (Non-Patent Document 6) ); Tanaka F et al., Cancer Res 1997 Oct 15, 57 (20): 4465-8 (Non-patent Document 7); Fujie T et al., Int J Cancer 1999 Jan 18, 80 (2): 169-72 (Non-patent document 8); Kikuchi M et al., Int J Cancer 1999 May 5, 81 (3): 459-66 (Non-patent document 9); Oiso M et al., Int J Cancer 1999 May 5, 81 ( 3): 387-94 (Non-patent Document 10) To date, clinical trials using these TAA-derived peptides Unfortunately, many current cancer vaccine trials have shown low objective response rates (Belli F et al., J Clin Oncol 2002 Oct 15, 20 (20): 4169-80 (Non-Patent Document 11); Coulie PG et al., Immunol Rev 2002 Oct, 188: 33-42 (Non-Patent Document 12); Rosenberg SA et al., Nat Med 2004 Sep, 10 (9): 909-15 ( Non-patent document 13) Therefore, there is still a need for new TAAs as immunotherapy targets.

  To this end, expression profile analysis of colorectal cancer using a genome-wide cDNA microarray containing 23,040 genes was performed by CLUAP1 (GenBank accession number NM_0155041 or NM_024793), clusterin-associated protein 1 (clusterin-associated). protein 1) was identified as a gene that is frequently transactivated in colon cancer (Takahashi M et al., Oncogene. 2004 Dec 9; 23 (57): 9289-94). It has been observed by Northern blot analysis that CLUAP1 expression gradually increases from the late S phase to the G2 / M phase of the cell cycle and returns to the basal level in the G0 / G1 phase. Furthermore, suppression of CLUAP1 by siRNA has been shown to result in growth delay in transfected tumor cells. Overexpression of CLUAP1 has also been observed in osteosarcoma, ovarian cancer, and lung cancer (Ishikura H et al., Int J Oncol. 2007 Feb; 30 (2): 461-7 (Non-patent Document 15)). Taken together, these facts suggest that CLUAP1 may be a useful target for cancer immunotherapy in multiple cancers.

Boon T, Int J Cancer 1993 May 8, 54 (2): 177-80 Boon T & van der Bruggen P, J Exp Med 1996 Mar 1, 183 (3): 725-9 Harris CC, J Natl Cancer Inst 1996 Oct 16, 88 (20): 1442-55 Butterfield LH et al., Cancer Res 1999 Jul 1, 59 (13): 3134-42 Vissers JL et al., Cancer Res 1999 Nov 1, 59 (21): 5554-9 van der Burg SH et al., J Immunol 1996 May 1, 156 (9): 3308-14 Tanaka F et al., Cancer Res 1997 Oct 15, 57 (20): 4465-8 Fujie T et al., Int J Cancer 1999 Jan 18, 80 (2): 169-72 Kikuchi M et al., Int J Cancer 1999 May 5, 81 (3): 459-66 Oiso M et al., Int J Cancer 1999 May 5, 81 (3): 387-94 Belli F et al., J Clin Oncol 2002 Oct 15, 20 (20): 4169-80 Coulie PG et al., Immunol Rev 2002 Oct, 188: 33-42 Rosenberg SA et al., Nat Med 2004 Sep, 10 (9): 909-15 Takahashi M et al., Oncogene. 2004 Dec 9; 23 (57): 9289-94 Ishikura H et al., Int J Oncol. 2007 Feb; 30 (2): 461-7

  The present invention is based at least in part on the discovery of novel peptides that may serve as targets for immunotherapy. Since TAA is generally perceived as “self” by the immune system and therefore often has no innate immunogenicity, discovery of a suitable target is extremely important. As mentioned above, CLUAP1 (encoded by GenBank accession number NM — 05041 or NM — 024793 gene (eg, SEQ ID NO: 44), SEQ ID NO: 45) is breast cancer, cervical cancer, colorectal cancer, esophageal cancer, gastric cancer Have been identified as being upregulated in cancers including, but not limited to, invasive gastric cancer, lymphoma, neuroblastoma, pancreatic cancer. Therefore, the present invention focuses on CLUAP1 as a target candidate for cancer / tumor immunotherapy.

The invention further relates to the identification of specific epitope peptides of the CLUAP1 gene product that have the ability to induce CTLs specific for CLUAP1. As detailed below, peripheral blood mononuclear cells (PBMCs) obtained from healthy donors were stimulated with HLA-A ^* 2402 or HLA-A ^* 0201 binding candidate peptides derived from CLUAP1. Thereafter, CTL lines having specific cytotoxicity against HLA-A24 or HLA-A2 positive target cells pulsed with each candidate peptide were established. These results demonstrate that these peptides are HLA-A24 restricted or HLA-A2 restricted epitope peptides that can induce a strong and specific immune response against cells expressing CLUAP1. . Furthermore, these results indicate that CLUAP1 has strong immunogenicity and that its epitope is an effective target for cancer / tumor immunotherapy.

  Accordingly, it is an object of the present invention to provide isolated peptides that bind to HLA antigens, particularly those comprising CLUAP1 (SEQ ID NO: 45) or immunologically active fragments thereof. These peptides are expected to have CTL inducibility and can therefore be used to induce CTL ex vivo, or breast cancer, cervical cancer, colorectal cancer, esophageal cancer, gastric cancer, invasive gastric cancer, It can also be administered to a subject for the induction of an immune response against cancers such as lymphoma, neuroblastoma, pancreatic cancer and the like. Preferred peptides are nonapeptides or decapeptides, more preferably those having an amino acid sequence selected from among SEQ ID NOs: 2-23 and 25-43. Among them, it has an amino acid sequence selected from SEQ ID NO: 3, 4, 5, 8, 9, 10, 14, 15, 16, 18, 23, 30, 33, 34, 35, 36, and 38 Peptides exhibit a particularly strong ability to induce CTL and are therefore particularly preferred.

The peptides of the present invention include those in which one, two, or more amino acids are substituted, deleted, or added as long as the resulting modified peptide retains the original ability to induce CTLs .
The invention also provides an isolated polynucleotide encoding any peptide of the invention. These polynucleotides can be used to induce or prepare APCs with CTL inducing ability, or, similar to the peptides of the present invention, can be administered to a subject to induce an immune response against cancer. it can.

  When administered to a subject, the peptides of the present invention are preferably presented on the surface of the APC to induce CTL targeting each peptide. Accordingly, one object of the present invention is to provide compositions or substances that induce CTLs, such compositions encoding one or more peptides of the present invention or such peptides. Including polynucleotides. The present invention further contemplates a pharmaceutical composition comprising one or more peptides of the present invention or a polynucleotide encoding such a peptide, wherein such composition comprises cancer treatment and / or prevention. Such as cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, gastric cancer, invasive gastric cancer, lymphoma, neuroblastoma, pancreatic cancer Including but not limited to these. The pharmaceutical composition or substance of the present invention may comprise, as an active ingredient, an APC or exosome that presents any of the peptides of the present invention instead of or in addition to the peptide or polynucleotide of the present invention.

  The peptides and polynucleotides of the present invention can be obtained by, for example, bringing an APC derived from a subject into contact with the peptide, or introducing a polynucleotide encoding the peptide of the present invention into the APC, whereby the HLA antigen and the peptide of the present invention are mixed. APC can be induced which presents the complex on its surface. Such APC has a high CTL inducing ability for the target peptide and is therefore useful for cancer immunotherapy. Accordingly, the present invention contemplates both methods for inducing APC having CTL inducibility as well as APCs obtained by such methods.

  The invention also provides a method for inducing CTL, ie co-culturing CD8 positive cells with APC or exosomes that present one or more peptides of the invention on their surface, or peptides of the invention Also provided is a method comprising the step of introducing a gene comprising a polynucleotide encoding a T cell receptor (TCR) subunit polypeptide that binds to. CTLs obtained by such methods include, but are not limited to, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, gastric cancer, invasive gastric cancer, lymphoma, neuroblastoma, pancreatic cancer treatment And may be useful in prevention. The present invention therefore encompasses CTLs obtained by the method.

Furthermore, a method for inducing an immune response against cancer in a subject in need thereof, comprising a CLUAP1 polypeptide or an immunologically active fragment thereof, a composition or substance, a polynucleotide encoding a CLUAP1 polypeptide It is another object of the present invention to provide such a method comprising administering to said subject an exosome or APC presenting a CLUAP1 polypeptide.
The applicability of the present invention covers any of several diseases, such as cancer, associated with or resulting from CLUAP1 overexpression, and examples of cancer are not limited to these Breast cancer, cervical cancer, colorectal cancer, esophageal cancer, gastric cancer, invasive gastric cancer, lymphoma, neuroblastoma, pancreatic cancer.

More specifically, the present invention provides the following:
[1] An isolated peptide having the ability to induce CTL, comprising an amino acid sequence of CLUAP1 or an immunologically active fragment thereof,
[2] The isolated peptide of [1], comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 23 and 25 to 43,
[3] The isolated peptide of any one of [1] and [2], wherein one, two, or several amino acids are substituted, deleted, or added
[4] In the context of HLA-A24, the isolated peptide of [3] having one or both of the following characteristics:
(A) the second amino acid from the N-terminus is modified to be an amino acid selected from the group consisting of phenylalanine, tyrosine, methionine, or tryptophan; and (b) the C-terminal amino acid is phenylalanine, leucine, An amino acid selected from the group consisting of isoleucine, tryptophan, or methionine, or modified to be the amino acid;
[5] In the context of HLA-A2, the isolated peptide of [3] having one or both of the following characteristics:
(A) the second amino acid from the N-terminus is selected from the group consisting of leucine and methionine; and (b) the C-terminal amino acid is selected from the group consisting of valine and leucine.
[6] The isolated peptide of any one of [1] to [5], which is a nonapeptide or a decapeptide,
[7] An isolated polynucleotide encoding the peptide of any one of [1] to [6],
[8] A composition for inducing CTL, comprising one or more peptides of any one of [1] to [6], or one or more polynucleotides of [7] ,Composition,
[9] A pharmaceutical composition for treating and / or preventing cancer and / or preventing its recurrence after surgery, comprising one or more of any one of [1] to [6] Or a pharmaceutical composition comprising one or more polynucleotides of [7],
[10] The pharmaceutical composition of [9], formulated for administration to a subject whose HLA antigen is HLA-A24 or HLA-A2,
[11] The pharmaceutical composition of [9] or [10], formulated for the treatment of cancer,
[12] A method for inducing an antigen-presenting cell (APC) having CTL inducing ability, comprising a step selected from the group consisting of:
(A) contacting APC with any one peptide of [1] to [6] in vitro, ex vivo or in vivo; and (b) encoding any one peptide of [1] to [6] Introducing a polynucleotide to APC,
[13] A method for inducing CTL by a method comprising a step selected from the group consisting of:
(A) co-culturing CD8-positive T cells with APC that presents a complex of HLA antigen and any one of the peptides [1] to [6] on its surface;
(B) co-culturing CD8 positive T cells with exosomes presenting a complex of HLA antigen and any one of peptides [1]-[6] on its surface; and (c) [1 Introducing a gene comprising a polynucleotide encoding a T cell receptor (TCR) subunit polypeptide that binds to any one of the peptides of [6] to T cells;
[14] An isolated APC presenting a complex of the HLA antigen and any one of the peptides of [1] to [6] on its surface,
[15] APC of [14] introduced by the method of [12],
[16] An isolated CTL that targets the peptide of any one of [1] to [6],
[17] CTL of [16], induced by the method of [13],
[18] A method for inducing an immune response against cancer in a subject, the peptide according to any one of [1] to [6], an immunologically active fragment thereof, or a polynucleotide encoding the peptide or the fragment Administering to the subject a composition comprising:
[19] An antibody or a fragment thereof against any one of the peptides of [1] to [6],
[20] A vector comprising a nucleotide sequence encoding the peptide of any one of [1] to [6],
[21] A host cell transformed or transfected with the expression vector of [20],
[22] Diagnostic kit comprising the peptide of any one of [1] to [6], the nucleotide of [7], or the antibody of [19], and [23] SEQ ID NO: 3, 4, 5, 8 , 9, 10, 14, 15, 16, 18, 23, 30, 33, 34, 35, 36, and 38, any one of [1] to [6] Peptide.

  It should be understood that the foregoing summary of the invention and the following detailed description are exemplary, and are not restrictive of the invention and other alternative aspects of the invention.

  In addition to the foregoing, other objects and features of the invention will become more fully apparent when the following detailed description is read in conjunction with the accompanying drawings and examples. However, it is to be understood that both the foregoing summary of the invention and the following detailed description are exemplary embodiments, and are not restrictive of the invention or other alternative embodiments of the invention. In particular, while the invention is described herein with reference to certain specific embodiments, it is understood that the description is illustrative of the invention and is not to be construed as limiting the invention. Like. Various modifications and applications can occur to those skilled in the art without departing from the spirit and scope of the invention as described by the appended claims. Similarly, other objects, features, benefits and advantages of the present invention will become apparent from the present summary and specific embodiments described below and will be readily apparent to those skilled in the art. Such objects, features, benefits and advantages can be found in the accompanying examples, data, drawings, and references cited alone or incorporated herein, in conjunction with any reasonable inferences derived from them. It will be clear in consideration.

Various aspects and applications of the present invention will become apparent to those skilled in the art from consideration of the brief description of the drawings and detailed description of the invention and preferred embodiments thereof.
FIGS. 1a-f consist of a series of photographs (a)-(f) showing the results of an IFN-γ ELISPOT assay in CTL induced with a CLUAP1-derived peptide. CTL in the following wells showed strong IFN-γ production compared to controls: well # 6 (a) stimulated with CLUAP1-A24-9-255 (SEQ ID NO: 3), CLUAP1-A24- # 2 (b) stimulated with 9-283 (SEQ ID NO: 4), # 2 (c) stimulated with CLUAP1-A24-9-31 (SEQ ID NO: 5), CLUAP1-A24-9-168 ( # 4 (d) stimulated with SEQ ID NO: 8), # 4 (e) stimulated with CLUAP1-A24-9-64 (SEQ ID NO: 9), and CLUAP1-A24-9-216 (SEQ ID NO : # 1 (f) stimulated by 10). Cells in the wells indicated by the rectangular frame were grown to establish CTL lines. In contrast, as an example of typical negative data, specific IFN-γ production from CTL stimulated with CLUAP1-A24-9-258 (SEQ ID NO: 1) was not shown (k). In the figure, “+” indicates IFN-γ production for target cells pulsed with an appropriate peptide, and “−” indicates IFN-γ production for target cells not pulsed with any peptide. FIGS. 1g-k consist of a series of photographs (g)-(j) showing the results of an IFN-γ ELISPOT assay in CTL induced with a CLUAP1-derived peptide. CTL in the following wells showed strong IFN-γ production compared to controls: well # 6 (g) stimulated with CLUAP1-A24-9-154 (SEQ ID NO: 14), CLUAP1-A24- # 2 (h) stimulated with 10-91 (SEQ ID NO: 15), # 4 (i) stimulated with CLUAP1-A24-10-104 (SEQ ID NO: 16), and CLUAP1-A24-10-125 Stimulated with (SEQ ID NO: 18) # 5 (j). Cells in the wells indicated by the rectangular frame were grown to establish CTL lines. In contrast, as an example of typical negative data, specific IFN-γ production from CTL stimulated with CLUAP1-A24-9-258 (SEQ ID NO: 1) was not shown (k). In the figure, “+” indicates IFN-γ production for target cells pulsed with an appropriate peptide, and “−” indicates IFN-γ production for target cells not pulsed with any peptide. FIG. 2 shows CLUAP1-A24-9-255 (SEQ ID NO: 3) (a), CLUAP1-A24-9-168 (SEQ ID NO: 8) (b), CLUAP1-A24-9-64 (SEQ ID NO. CTL strains stimulated with NO: 9) (c), CLUAP1-A24-9-154 (SEQ ID NO: 14) (d), and CLUAP1-A24-10-91 (SEQ ID NO: 15) (e) Consists of a series of line graphs (a)-(e) showing the results of an IFN-γ ELISA assay demonstrating IFN-γ production. These results demonstrate that the CTL lines established by stimulation with each peptide show strong IFN-γ production compared to the control. In the figure, “+” indicates IFN-γ production for target cells pulsed with an appropriate peptide, and “−” indicates IFN-γ production for target cells not pulsed with any peptide. FIG. 3 shows CLUAP1-A24-9-255 (SEQ ID NO: 3) (a), CLUAP1-A24-9-64 (SEQ ID NO: 9) (b), and CLUAP1-A24-10-91 (SEQ A series of line graphs (a)-showing the results of an IFN-γ ELISA assay demonstrating IFN-γ production of CTL clones established by limiting dilution from CTL lines stimulated with ID NO: 15) (c) (C). These results demonstrate that CTL clones established by stimulation with each peptide show strong IFN-γ production compared to controls. In the figure, “+” indicates IFN-γ production for target cells pulsed with each peptide, and “−” indicates IFN-γ production for target cells not pulsed with any peptide. FIG. 4 is a line graph showing specific CTL activity against target cells that exogenously express CLUAP1 and HLA-A ^* 2402. COS7 cells transfected with HLA-A ^* 2402 or full-length CLUAP1 gene were prepared as controls. A CTL line established using CLUAP1-A24-9-255 (SEQ ID NO: 3) showed specific CTL activity against COS7 cells transfected with both CLUAP1 and HLA-A ^* 2402 ( Black diamond). On the other hand, no significant specific CTL activity was detected against target cells expressing either HLA-A ^* 2402 (triangle) or CLUAP1 (circle). FIGS. 5a-f consist of a series of photographs (a)-(f) showing the results of an IFN-γ ELISPOT assay in CTL induced with a CLUAP1-derived peptide. CTL in the following wells showed strong IFN-γ production compared to controls: well number # 3 (a), CLUAP1-A02 with CLUAP1-A02-9-34 (SEQ ID NO: 23) # 6 (b) using -9-99 (SEQ ID NO: 30), # 2 (c) using CLUAP1-A02-10-153 (SEQ ID NO: 33), CLUAP1-A02-10-85 # 3 (d) using (SEQ ID NO: 34), # 2 (e) using CLUAP1-A02-10-34 (SEQ ID NO: 35), and CLUAP1-A02-10-33 (SEQ ID # 2 (f) using NO: 36). Cells in the wells indicated by the rectangular frame were grown to establish CTL lines. In contrast, as an example of typical negative data, specific IFN-γ production from CTL stimulated with CLUAP1-A02-9-58 (SEQ ID NO: 24) was not shown (h). In the figure, “+” indicates IFN-γ production for target cells pulsed with an appropriate peptide, and “−” indicates IFN-γ production for target cells not pulsed with any peptide. FIGS. 5g-h consist of a series of photographs (g) showing the results of an IFN-γ ELISPOT assay in CTL induced with a peptide derived from CLUAP1. CTL in the following wells showed strong IFN-γ production compared to the control: well number # 5 (g) with CLUAP1-A02-10-80 (SEQ ID NO: 38). Cells in the wells indicated by the rectangular frame were grown to establish CTL lines. In contrast, as an example of typical negative data, specific IFN-γ production from CTL stimulated with CLUAP1-A02-9-58 (SEQ ID NO: 24) was not shown (h). In the figure, “+” indicates IFN-γ production for target cells pulsed with an appropriate peptide, and “−” indicates IFN-γ production for target cells not pulsed with any peptide. 6 shows CLUAP1-A02-9-34 (SEQ ID NO: 23) (a), CLUAP1-A02-9-99 (SEQ ID NO: 30) (b), CLUAP1-A02-10-153 (SEQ ID NO. NO: 33) (c), CLUAP1-A02-10-85 (SEQ ID NO: 34) (d), CLUAP1-A02-10-34 (SEQ ID NO: 35) (e), and CLUAP1-A02-10 -33 (SEQ ID NO: 36) consisting of a series of line graphs (a)-(f) showing the results of an IFN-γ ELISA assay demonstrating IFN-γ production of CTL lines stimulated with (f) . These results demonstrate that the CTL lines established by stimulation with each peptide show strong IFN-γ production compared to the control. In the figure, “+” indicates IFN-γ production for target cells pulsed with an appropriate peptide, and “−” indicates IFN-γ production for target cells not pulsed with any peptide. FIG. 7 shows CLUAP1-A02-9-34 (SEQ ID NO: 23) (a), CLUAP1-A02-9-99 (SEQ ID NO: 30) (b), CLUAP1-A02-10-153 (SEQ ID NO. NO: 33) (c), and IFN-γ production of CTL clones established by limiting dilution from CTL lines stimulated with CLUAP1-A02-10-85 (SEQ ID NO: 34) (d), It consists of a series of line graphs (a) to (d). These results demonstrate that CTL clones established by stimulation with each peptide show strong IFN-γ production compared to controls. In the figure, “+” indicates IFN-γ production for target cells pulsed with an appropriate peptide, and “−” indicates IFN-γ production for target cells not pulsed with any peptide. FIG. 8 consists of a series of line graphs (a)-(d) showing specific CTL activity against target cells exogenously expressing CLUAP1 and HLA-A ^* 0201. COS7 cells transfected with HLA-A ^* 0201 or full-length CLUAP1 gene were prepared as controls. CLUAP1-A02-9-99 (SEQ ID NO: 30) (a), CLUAP1-A02-10-153 (SEQ ID NO: 33) (b), CLUAP1-A02-10-85 (SEQ ID NO: 34) (C), and CTL lines established using CLUAP1-A02-10-33 (SEQ ID NO: 36) (d) are against COS7 cells transfected with both CLUAP1 and HLA-A ^* 0201. Specific CTL activity was shown (squares). On the other hand, no significant specific CTL activity was detected against target cells expressing either HLA-A ^* 0201 (triangle) or CLUAP1 (circle).

DESCRIPTION OF EMBODIMENTS Although any methods and materials similar or equivalent to those described herein can be used in practicing or testing aspects of the present invention, the preferred methods, devices, and materials are now described. Describe. However, before describing the materials and methods of the present invention, the specific sizes, shapes, dimensions, materials, methodologies, protocols, etc. described herein may be varied according to routine experimentation and optimization. As such, it should be understood that the invention is not limited thereto. The terminology used in the description is for the purpose of describing particular types or embodiments only and is not intended to limit the scope of the invention which is limited only by the appended claims. It should also be understood.
All publications, patents, or patent applications mentioned in this specification are expressly incorporated herein by reference in their entirety. However, nothing in this specification should be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

I. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. However, in the case of conflict, the present specification, including definitions, will control.
As used herein, the words “a”, “an”, and “the” mean “at least one” unless otherwise specified.
The terms “polypeptide”, “peptide”, and “protein” are used interchangeably herein and refer to a polymer of amino acid residues. The term refers to an amino acid polymer that is a non-natural residue such as one or more amino acid residues that may be modified, or an artificial chemical mimic of the corresponding natural amino acid, As well as natural amino acid polymers.

  As used herein, the term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. The amino acid may be either an L-amino acid or a D-amino acid. Natural amino acids are amino acids encoded by the genetic code and amino acids that are post-translationally modified in cells (eg, hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine). The phrase “amino acid analog” has the same basic chemical structure as a natural amino acid (hydrogen, carboxy group, amino group, and α-carbon attached to the R group), but one or more modified R groups or modifications And a compound having a structured skeleton (for example, homoserine, norleucine, methionine, sulfoxide, methionine methylsulfonium). The phrase “amino acid mimetic” refers to a compound that has a structure that is different from a common amino acid but that has a similar function.

Amino acids may be referred to herein by their commonly known three-letter or one-letter code as recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
The terms “gene”, “polynucleotide”, “nucleotide”, and “nucleic acid” are used interchangeably herein and are generally accepted as well as amino acids unless otherwise specified. Referenced by character code.

  As used herein, the term “composition” is intended to encompass products containing a specific amount of a specific component, as well as any product that results directly or indirectly from a combination of a specific amount of a specific component. . Such term for “pharmaceutical composition” refers to a product comprising the active ingredient and any inert ingredients that make up the carrier, as well as combinations, complexation, or aggregation of any two or more ingredients. From any dissociation of one or more components or from any other type of reaction or interaction of one or more components is intended to encompass any product. Thus, in the context of the present invention, the term “pharmaceutical composition” refers to any composition made by mixing a compound of the present invention and a pharmaceutically or physiologically acceptable carrier. As used herein, the phrase “pharmaceutically acceptable carrier” or “physiologically acceptable carrier” refers to a subject scaffolded in a polypharmacophore from another organ or body part. Pharmaceutically or physiologically acceptable including, but not limited to, liquid or solid fillers, diluents, excipients, solvents, or encapsulating materials involved in transporting or transporting to an organ or body part Means a material, composition, substance or medium.

Unless otherwise specified, the term “cancer” refers to a cancer or tumor that overexpresses the CLUAP1 gene, examples of which include breast cancer, cervical cancer, colorectal cancer, esophageal cancer, gastric cancer, invasive type This includes, but is not limited to, gastric cancer, lymphoma, neuroblastoma, pancreatic cancer.
Unless otherwise specified, the terms “cytotoxic T lymphocyte”, “cytotoxic T cell”, and “CTL” are used interchangeably herein, and unless otherwise specified, Refers to a subgroup of T lymphocytes that can recognize autologous cells (eg, tumor / cancer cells, virus-infected cells) and induce the death of such cells.
Unless otherwise specified, the term “HLA-A24” refers to the HLA-A24 type, including subtypes such as HLA-A ^* 2402.
Unless otherwise specified, the term “HLA-A2” as used herein typically refers to subtypes such as HLA-A ^* 0201 and HLA-A ^* 0206.

Unless otherwise specified, the term “kit” as used herein is used in reference to combinations of reagents and other materials. It is contemplated herein that a kit can include a microarray, a chip, a marker, and the like. The term “kit” is intended to be not limited to a particular combination of reagents and / or substances.
As long as the methods and compositions of the present invention are useful in the context of cancer “treatment”, such as reducing the expression of the CLUAP1 gene or reducing the size, spread, or metastatic potential of a cancer in a subject, etc. A treatment is considered “effective” if it provides the clinical benefit of. When the treatment is applied prophylactically, “effective” means that the treatment delays or prevents the formation of cancer or prevents or alleviates the clinical symptoms of the cancer. Efficacy is determined in connection with any known method for diagnosing or treating a particular tumor type.

  As long as the methods and compositions of the present invention are useful in the context of cancer “prevention and prophylaxis”, such terms are used interchangeably herein, and mortality or morbidity due to disease. Refers to any action that reduces the load of. Prevention and prevention may be performed at “primary, secondary, and tertiary prevention levels”. Primary prevention and prevention (prophylaxis) avoid the development of disease, whereas secondary and tertiary prevention (prevention and prophylaxis) is to prevent disease progression and appearance of symptoms. In addition, it includes actions aimed at reducing the adverse effects of existing diseases by restoring function and reducing disease-related complications. Alternatively, prevention and prophylaxis can include a wide range of prophylactic therapies aimed at reducing the severity of certain disorders, eg, reducing tumor growth and metastasis.

  In the context of the present invention, the treatment and / or prevention of cancer and / or prevention of its recurrence after surgery includes the following stages: surgical removal of cancer cells, inhibition of proliferation of cancerous cells, tumors Or any of the stages such as regression or regression, induction of remission and suppression of cancer development, tumor regression, and reduction or inhibition of metastasis. Effective treatment and / or prevention of cancer reduces mortality, improves the prognosis of individuals with cancer, decreases the level of tumor markers in the blood, and detectable symptoms associated with cancer To ease. For example, symptom reduction or amelioration constitutes effective treatment and / or prevention and includes 10%, 20%, 30%, or more of a reduced or stable disease.

In the context of the present invention, the term “antibody” refers to immunoglobulins and fragments thereof that specifically react with the designated protein or peptide thereof. Antibodies can include human antibodies, primatized antibodies, chimeric antibodies, bispecific antibodies, humanized antibodies, antibodies fused with other proteins or radiolabels, and antibody fragments. In addition, antibodies are used herein in a broad sense, specifically including complete monoclonal antibodies, polyclonal antibodies, and multispecific antibodies (eg, bispecific antibodies) formed from at least two complete antibodies. In addition, antibody fragments are included as long as they exhibit the desired biological activity. “Antibody” refers to all classes (eg, IgA, IgD, IgE, IgG, and IgM).
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

II. In order to demonstrate that peptides derived from the peptide CLUAP1 function as antigens recognized by CTL, peptides derived from CLUAP1 (SEQ ID NO: 45) were analyzed and they are HLA alleles, which are normally found HLA alleles. It was determined whether it is an antigenic epitope constrained by A24 or 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).

Candidate HLA-A24 binding peptides derived from CLUAP1 were identified based on their binding affinity for HLA-A24. The following peptides are considered as candidates:
CLUAP1-A24-9-244 (SEQ ID NO: 2),
CLUAP1-A24-9-255 (SEQ ID NO: 3),
CLUAP1-A24-9-283 (SEQ ID NO: 4),
CLUAP1-A24-9-31 (SEQ ID NO: 5),
CLUAP1-A24-9-8 (SEQ ID NO: 6),
CLUAP1-A24-9-26 (SEQ ID NO: 7),
CLUAP1-A24-9-168 (SEQ ID NO: 8),
CLUAP1-A24-9-64 (SEQ ID NO: 9),
CLUAP1-A24-9-216 (SEQ ID NO: 10),
CLUAP1-A24-9-335 (SEQ ID NO: 11),
CLUAP1-A24-9-86 (SEQ ID NO: 12),
CLUAP1-A24-9-71 (SEQ ID NO: 13),
CLUAP1-A24-9-154 (SEQ ID NO: 14),
CLUAP1-A24-10-91 (SEQ ID NO: 15),
CLUAP1-A24-10-104 (SEQ ID NO: 16),
CLUAP1-A24-10-31 (SEQ ID NO: 17),
CLUAP1-A24-10-125 (SEQ ID NO: 18),
CLUAP1-A24-10-17 (SEQ ID NO: 19),
CLUAP1-A24-10-289 (SEQ ID NO: 20),
CLUAP1-A24-10-258 (SEQ ID NO: 21) and CLUAP1-A24-10-63 (SEQ ID NO: 22).

In addition, after in vitro stimulation of T cells with dendritic cells (DC) pulsed (loaded) with these peptides, CTLs were successfully established using the following peptides:
CLUAP1-A24-9-151-255 (SEQ ID NO: 3),
CLUAP1-A24-9-152-283 (SEQ ID NO: 4),
CLUAP1-A24-9-153-31 (SEQ ID NO: 5),
CLUAP1-A24-9-156-168 (SEQ ID NO: 8),
CLUAP1-A24-9-157-64 (SEQ ID NO: 9),
CLUAP1-A24-9-158-216 (SEQ ID NO: 10),
CLUAP1-A24-9-163-154 (SEQ ID NO: 14),
CLUAP1-A24-10-166-91 (SEQ ID NO: 15),
CLUAP1-A24-10-167-104 (SEQ ID NO: 16) and CLUAP1-A24-10-169-125 (SEQ ID NO: 18).

Candidate HLA-A2 binding peptides derived from CLUAP1 were identified based on their binding affinity for HLA-A2. The following peptides are considered candidate peptides:
CLUAP1-A2-9-34 (SEQ ID NO: 23),
CLUAP1-A2-9-95 (SEQ ID NO: 25),
CLUAP1-A2-9-85 (SEQ ID NO: 26),
CLUAP1-A2-9-290 (SEQ ID NO: 27),
CLUAP1-A2-9-265 (SEQ ID NO: 28),
CLUAP1-A2-9-297 (SEQ ID NO: 29),
CLUAP1-A2-9-99 (SEQ ID NO: 30),
CLUAP1-A2-9-188 (SEQ ID NO: 31),
CLUAP1-A2-9-96 (SEQ ID NO: 32),
CLUAP1-A2-10-153 (SEQ ID NO: 33),
CLUAP1-A2-10-85 (SEQ ID NO: 34),
CLUAP1-A2-10-34 (SEQ ID NO: 35),
CLUAP1-A2-10-33 (SEQ ID NO: 36),
CLUAP1-A2-10-72 (SEQ ID NO: 37),
CLUAP1-A2-10-80 (SEQ ID NO: 38),
CLUAP1-A2-10-41 (SEQ ID NO: 39),
CLUAP1-A2-10-233 (SEQ ID NO: 40),
CLUAP1-A2-10-183 (SEQ ID NO: 41),
CLUAP1-A2-10-343 (SEQ ID NO: 42) and CLUAP1-A2-10-222 (SEQ ID NO: 43).

In addition, after in vitro stimulation of T cells with dendritic cells (DC) pulsed (loaded) with these peptides, CTLs were successfully established using the following peptides:
CLUAP1-A2-9-34 (SEQ ID NO: 23),
CLUAP1-A2-9-99 (SEQ ID NO: 30),
CLUAP1-A2-10-153 (SEQ ID NO: 33),
CLUAP1-A2-10-85 (SEQ ID NO: 34),
CLUAP1-A2-10-34 (SEQ ID NO: 35),
CLUAP1-A2-10-33 (SEQ ID NO: 36) and CLUAP1-A2-10-80 (SEQ ID NO: 38).

  These established CTLs show potent specific CTL activity against target cells pulsed with each peptide. These results herein demonstrate that CLUAP1 is an antigen recognized by CTL and that the peptides tested are HLA-A24 or HLA-A2-restricted CLUAP1 epitope peptides.

  The CLUAP1 gene is overexpressed in cancer cells and tissues including but not limited to breast cancer, cervical cancer, colorectal cancer, esophageal cancer, gastric cancer, invasive gastric cancer, lymphoma, neuroblastoma, pancreatic cancer. It is an excellent target for cancer immunotherapy because it is not expressed in most normal organs. Accordingly, the present invention provides a nonapeptide (a peptide composed of 9 amino acid residues) and a decapeptide (a peptide composed of 10 amino acid residues) corresponding to an epitope derived from CLUAP1 recognized by CTL. To do. Alternatively, the present invention relates to an isolated peptide that binds to an HLA antigen and induces cytotoxic T lymphocytes (CTL), which has the amino acid sequence of SEQ ID NO: 45 or an immunologically active fragment thereof A peptide is provided. Particularly preferred examples of nonapeptides and decapeptides of the invention include SEQ ID NOs: 3, 4, 5, 8, 9, 10, 14, 15, 16, 18, 23, 30, 33, 34, 35, 36, And peptides having an amino acid sequence selected from among 38.

  Generally, software programs such as those described in Parker KC et al., J Immunol 1994 Jan 1, 152 (1): 163-75 and Nielsen M et al., Protein Sci 2003; 12: 1007-17, such as on the Internet The binding affinity between various peptides and HLA antigens can be calculated in silico using currently available software programs. The binding affinity with the HLA antigen is, for example, Parker KC et al., J Immunol 1994 Jan 1, 152 (1): 163-75, Kuzushima K et al., Blood 2001, 98 (6): 1872-81, Larsen MV et al. BMC Bioinformatics. 2007 Oct 31; 8: 424, Buus S et al. Tissue Antigens., 62: 378-84, 2003, Nielsen M et al., Protein Sci 2003; 12: 1007-17, and Nielsen M et al. PLoS ONE 2007; 2: e796, which are summarized in, for example, Lafuente EM et al., Current Pharmaceutical Design, 2009, 15, 3209-3220 Yes. Methods for measuring binding affinity are described, for example, in Journal of Immunological Methods, 1995, 185: 181-190; and Protein Science, 2000, 9: 1838-1846. Therefore, using such a software program, it is possible to select a fragment derived from CLUAP1 having a high binding affinity with the HLA antigen. Therefore, the present invention encompasses a peptide composed of any fragment derived from CLUAP1 that binds to an HLA antigen according to such a known program. Further, such peptides can include the full-length peptide of CLUAP1.

  The nonapeptides and decapeptides of the present invention can be flanked by additional amino acid residues as long as the resulting peptide retains its ability to induce CTL. The additional amino acid residues can be composed of any type of amino acid as long as they do not impair the ability of the original peptide to induce CTL. Accordingly, the present invention encompasses peptides having binding affinity for HLA antigens, including CLUAP1-derived peptides. Such peptides are, for example, less than about 40 amino acids, often less than about 20 amino acids, and usually less than about 15 amino acids.

  In general, modification of one, two, or more amino acids in a peptide does not affect the function of the peptide and in some cases even enhances the desired function of the original protein. Indeed, modified peptides (ie amino acids in which one, two, or several amino acid residues have been modified (ie substituted, added, deleted or inserted) compared to the original reference sequence) Peptides composed of sequences) are known to retain 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 aspect, the peptides of the invention may have CTL inducibility and have SEQ ID NO, wherein one, two, or even more amino acids are added, deleted, and / or substituted. : Having an amino acid sequence selected from among 3, 4, 5, 8, 9, 10, 14, 15, 16, 18, 23, 30, 33, 34, 35, 36, and 38.

One skilled in the art recognizes that individual additions, deletions, or substitutions to an amino acid sequence that alter a single amino acid or a small percentage of amino acids tend to conserve the properties of the original amino acid side chain. Will. Thus, they are often referred to as “conservative substitutions” or “conservative modifications”, where a change in the protein results in a modified protein having a function similar to the original protein. Conservative substitution tables providing functionally similar amino acids are well known in the art. Examples of amino acid side chain properties that are desirably conservative 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), as well as side chains having the following functional groups or characteristics in common: aliphatic side chains (G, A, V, L, I, P); hydroxyl group-containing side chains (S, T, Y); sulfur atom-containing side chains (C, M); carboxylic acid and amide-containing side chains (D, N, E, Q); base-containing side chains (R, K, H); and aromatic group-containing side chains (H, F, Y, W). In addition, the following eight groups each contain amino acids recognized in the art as conservative substitutions for each other:
1) Alanine (A), Glycine (G);
2) Aspartic acid (D), glutamic acid (E);
3) Asparagine (N), glutamine (Q);
4) Arginine (R), Lysine (K);
5) Isoleucine (I), leucine (L), methionine (M), valine (V);
6) phenylalanine (F), tyrosine (Y), tryptophan (W);
7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, for example, Creighton, Proteins 1984).

Such conservatively modified peptides are also considered peptides of the present invention. However, the peptides of the present invention are not limited to these, and may contain non-conservative modifications as long as the resulting modified peptides retain the ability of the original peptide to induce CTL. Furthermore, the modified peptides do not exclude polymorphic variants of CLUAP1, interspecies homologues, and CTL inducible peptides of alleles.
Preferably a small number (eg 1, 2, or several) or a small percentage of amino acids are modified (insertions, additions, deletions and / or substitutions) in order to retain the required CTL inducibility can do. As used herein, the term “several” means 5 or fewer amino acids, such as 4 or 3 or fewer. The percentage of amino acids that are modified is preferably 20% or less, more preferably 15% or less, even more preferably 10% or less, or 1 to 5%.

  When used in the context of cancer immunotherapy, the peptides of the invention should be presented on the surface of cells or exosomes, preferably as a complex with HLA antigens. Therefore, it is preferred to select peptides that not only induce CTLs but also include high binding affinity for HLA antigens. For this purpose, the peptide can be modified by substitution, insertion and / or addition of amino acid residues to obtain a modified peptide with improved binding affinity. In addition to naturally presented peptides, the regularity of the sequence of peptides presented by binding to HLA antigens is known (J Immunol 1994, 152: 3913; Immunogenetics 1995, 41: 178; J Immunol 1994 155: 4307), modifications based on such regularity can be introduced into the immunogenic peptides of the present invention.

  For example, in order to increase the binding affinity with HLA-A24, the second amino acid from the N-terminus is substituted with phenylalanine, tyrosine, methionine, or tryptophan, and / or the C-terminal amino acid is phenylalanine, leucine, isoleucine, Substitution with tryptophan or methionine may be desirable. Accordingly, a peptide having an amino acid sequence selected from SEQ ID NOs: 1 to 22, wherein the second amino acid from the N-terminal of the amino acid sequence of SEQ ID NO is substituted with phenylalanine, tyrosine, methionine, or tryptophan And / or a peptide in which the C-terminus of the amino acid sequence of SEQ ID NO is substituted with phenylalanine, leucine, isoleucine, tryptophan, or methionine is included in the present invention.

  Alternatively, it may be desirable to replace the second amino acid from the N-terminus with leucine or methionine and / or to replace the C-terminal amino acid with valine or leucine to increase HLA-A2 binding affinity. . Therefore, a peptide having an amino acid sequence selected from SEQ ID NO: 23 and 25 to 43, wherein the second amino acid from the N-terminal of the amino acid sequence of SEQ ID NO is substituted with leucine or methionine And / or a peptide in which the C-terminus of the amino acid sequence of SEQ ID NO is substituted with valine, leucine or methionine.

Substitutions can be introduced not only at the terminal amino acid, but also at the potential T cell receptor (TCR) recognition site of the peptide. Some studies have shown that peptides with amino acid substitutions, such as CAP1, p53 _(264-272) , Her-2 / neu _(369-377) , or gp100 _(209-217) are equivalent to the original function Or has demonstrated that it may have better function (Zaremba et al. Cancer Res. 57, 4570-4577, 1997, TK Hoffmann et al. J Immunol. (2002) Feb 1; 168 (3): 1338-47., SO Dionne et al. Cancer Immunol immunother. (2003) 52: 199-206, and SO Dionne et al. Cancer Immunology, Immunotherapy (2004) 53, 307-314).
The present invention also contemplates that the addition of one, two, or several amino acids can also be added to the N-terminus and / or C-terminus of the peptides of the present invention. Such modified peptides having high HLA antigen binding affinity and retaining CTL inducibility are also included in the present invention.

  However, if the peptide sequence is identical to part of the amino acid sequence of an endogenous or exogenous protein with different functions, side effects such as autoimmune disorders or allergic symptoms to certain substances can be induced. Therefore, in order to avoid the situation where the peptide sequence matches the amino acid sequence of another protein, it is preferable to perform a homology search using an available database. If the homology search reveals that there is not even one peptide that differs in one or two amino acids compared to the peptide of interest, without any risk of such side effects, The peptide of interest may be modified to increase its binding affinity with the antigen and / or to increase its CTL inducibility.

  As described above, peptides having high binding affinity for HLA antigens are expected to be very effective. However, candidate peptides selected using the presence of high binding affinity as an index are determined for the presence or absence of CTL inducing ability. Check further. As used herein, the phrase “CTL inducibility” refers to the ability of a peptide to induce cytotoxic T lymphocytes (CTLs) when presented on antigen presenting cells (APCs). Further, “CTL inducibility” includes the ability of a peptide to induce CTL activation, induce CTL proliferation, promote lysis of target cells by CTL, and increase CTL IFN-γ production.

Confirmation of the ability to induce CTLs induces APCs (eg, B lymphocytes, macrophages, and dendritic cells (DC)) carrying human MHC antigens, or more specifically DCs derived from human peripheral blood mononuclear leukocytes. This is accomplished by stimulation with peptides, mixing with CD8 positive cells, and then measuring IFN-γ produced and released by CTL against target cells. As a reaction system, a transgenic animal prepared to express human HLA antigen (for example, BenMohamed L, Krishnan R, Longmate J, Auge C, Low L, Primus J, Diamond DJ, Hum Immunol 2000 Aug, 61 (8 ): 764-79, Related Articles, Books, Linkout Induction of CTL response by a minimal epitope vaccine in HLA A ^* 0201 / DR1 transgenic mice: dependent on MHC (HLA) class II restricted T (H) response Can be used. For example, target cells may be radiolabeled with ⁵¹ Cr or the like, and cytotoxic activity may be calculated from the radioactivity released from the target cells. Alternatively, by measuring IFN-γ produced and released by CTL in the presence of APC carrying the immobilized peptide and visualizing the zone of inhibition on the medium using anti-IFN-γ monoclonal antibody, Inducibility can be evaluated.

As a result of examining the CTL inducing ability of the peptide as described above, SEQ ID NOs: 3, 4, 5, 8, 9, 10, 14, 15, 16, 18, 23, 30, 33, 34, 35, 36, It was found that nonapeptides or decapeptides selected from among the amino acid sequences represented by and 38 show a particularly high CTL inducing ability in addition to a high binding affinity for HLA. These peptides are therefore preferred exemplary embodiments of the present invention.
Furthermore, the results of homology analysis showed that these peptides do not have significant homology with peptides derived from any other known human gene products. Thus, the potential for an unknown or undesirable immune response when used in immunotherapy is low. Therefore, also from this aspect, these peptides are useful for inducing immunity against CLUAP1 in cancer patients. Accordingly, the peptides of the present invention preferably have SEQ ID NOs: 3, 4, 5, 8, 9, 10, 14, 15, 16, 18, 23, 30, 33, 34, 35, 36, and 38. A peptide having an amino acid sequence selected from among them.

  In addition to the above modifications, the peptides of the present invention can be linked to other peptides as long as the resulting linked peptide retains the necessary CTL inducibility of the original peptide. Examples of other suitable peptides include CTL inducing peptides derived from the peptides of the invention or other TAAs. Suitable interpeptide linkers are well known in the art, such as AAY (PM Daftarian et al., J Trans Med 2007, 5:26), AAA, NKRK (RPM Sutmuller et al., J Immunol. 2000, 165: 7308-7315), or K (S. Ota et al., Can Res. 62, 1471-1476, KS Kawamura et al., J Immunol. 2002, 168: 5709-5715).

  For example, a tumor associated antigen peptide that is not CLUAP1 can be used substantially simultaneously to increase immune responses via HLA class I and / or class II. It is well established that cancer cells can express two or more tumor-related genes. Thus, determining whether a particular subject expresses an additional tumor associated gene, and subsequently in a CLUAP1 composition or vaccine according to the invention, HLA class I and / or derived from the expression product of such gene Inclusion of HLA class II binding peptides is within the routine experimentation of one of ordinary skill in the art.

  Examples of HLA class I and HLA class II binding peptides are known to those skilled in the art (see, eg, Coulie, Stem Cells 13: 393-403, 1995) and in a manner similar to that disclosed herein. It can be used in the present invention. Accordingly, those skilled in the art can use one or more CLUAP1 peptides and one or more non-CLUAP1 peptides, or nucleic acids encoding such polypeptides, using standard techniques in molecular biology. And can be easily prepared.

The above linking peptides are referred to herein as “polytopes”, ie, two or more potential immunogenic or immune responses that can be linked in various configurations (eg, linked, overlapping). A group of stimulatory peptides. A polytope (or nucleic acid encoding the polytope) can be administered to a standard immunization protocol, for example, to an animal to test the effectiveness of the polytope in promoting, enhancing, and / or inducing an immune response.
Peptides can be linked directly or by the use of flanking sequences to form polytopes, and the use of polytopes as vaccines is well known in the art (eg, 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; see Tarn et al., J Exp. Med. 171 (1): 299-306, 1990). Polytopes containing various numbers and combinations of epitopes can be prepared and tested for recognition by CTLs and for efficacy in increasing immune responses.

The peptide of the present invention can be linked to other substances as long as the resulting linked peptide retains the necessary CTL inducing ability of the original peptide. Examples of suitable materials include, for example, peptides, lipids, sugars and sugar chains, acetyl groups, natural and synthetic polymers, and the like. The peptide may include modifications such as glycosylation, side chain oxidation, or phosphorylation provided that the biological activity of the original peptide is not impaired by the modification. Such types of modifications can be made to confer additional functions (eg, targeting and delivery functions) or to stabilize the peptide.
For example, it is known in the art to introduce D-amino acids, amino acid mimetics, or unnatural amino acids to increase the in vivo stability of the polypeptide, and this concept is also compatible with the polypeptides of the invention. obtain. Polypeptide stability can be assayed in several ways. For example, peptidases and various biological media such as human plasma and serum can be used to test stability (eg, Verhoef et al., Eur J Drug Metab Pharmacokin 1986, 11: 291-302). See).

Further, as described above, among the modified peptides that are substituted, deleted, or added by one, two, or several amino acid residues, the same or more than the original peptide. Those having high activity can be screened or selected. Accordingly, the present invention also provides a method for screening or selecting for a modified peptide having the same or higher activity compared to the original. An exemplary method may include the following steps:
a: substituting, deleting or adding at least one amino acid residue of a peptide of the invention,
b: measuring the activity of the peptide; and c: selecting a peptide having the same or higher activity compared to the original.
As used herein, activities assayed can include MHC binding activity, APC or CTL inducibility, and cytotoxic activity.
Herein, the peptides of the present invention can also be described as “CLUAP1 peptide” or “CLUAP1 polypeptide”.

III. Preparation of CLUAP1 peptides The peptides of the invention can be prepared using well-known techniques. For example, peptides can be prepared synthetically using recombinant DNA techniques or chemical synthesis. The peptides of the present invention can be synthesized individually or as longer polypeptides comprising two or more peptides. The peptide can then be isolated, i.e. purified or isolated so that it is substantially free of other natural host cell proteins and fragments thereof, or any other chemicals.
The peptides of the present invention may contain modifications such as glycosylation, side chain oxidation, or phosphorylation, as long as the modification does not impair the biological activity of the original peptide. Other exemplary modifications include the incorporation of D-amino acids or other amino acid mimetics that can be used, for example, to increase the serum half-life of the peptide.

The peptide of the present invention can be obtained by chemical synthesis based on the selected amino acid sequence. Examples of conventional peptide synthesis methods that can be adapted to the synthesis include the following:
(I) Peptide Synthesis, Interscience, New York, 1966;
(Ii) The Proteins, Vol. 2, Academic Press, New York, 1976;
(Iii) “Peptide Synthesis” (Japanese), Maruzen, 1975;
(Iv) “Basics and Experiments of Peptide Synthesis” (Japanese), Maruzen, 1985;
(V) “Continued Drug Development” (Japanese), Volume 14 (Peptide Synthesis), Hirokawa Shoten, 1991;
(Vi) WO 99/67288; and (vii) Barany G. & Merrifield RB, Peptides Vol. 2, "Solid Phase Peptide Synthesis", Academic Press, New York, 1980, 100-118.

  Alternatively, any known genetic engineering method for producing peptides can be adapted to obtain the peptides of the invention (eg Morrison J, J Bacteriology 1977, 132: 349-51; Clark-Curtiss & Curtiss, Methods in Enzymology (eds. Wu et al.) 1983, 101: 347-62). For example, first, an appropriate vector having a polynucleotide encoding the peptide of interest in a form that can be expressed (eg, downstream of a regulatory sequence corresponding to a promoter sequence) is prepared and transformed into an appropriate host cell. Such vectors and host cells are also provided by the present invention. The host cell is then cultured to produce the peptide of interest. In vitro translation systems can be incorporated to produce peptides in vitro.

IV. Polynucleotides The present invention also provides polynucleotides that encode any of the aforementioned peptides of the present invention. These include polynucleotides derived from the native CLUAP1 gene (GenBank accession number NM_0155041 or NM_024793 (eg, SEQ ID NO: 44)) and polynucleotides having conservatively modified nucleotide sequences thereof. As used herein, the phrase “conservatively modified nucleotide sequence” refers to sequences that encode the same or essentially the same amino acid sequence. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any particular protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at any position where an alanine is specified by a codon, the codon can be changed to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid mutations are “silent mutations” and are a type of mutation conservatively modified. Every nucleic acid sequence herein that encodes a peptide also represents every possible silent variation of the nucleic acid. Those of ordinary skill in the art can modify each codon in a nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan) to obtain a functionally identical molecule. You will recognize. Accordingly, each silent variation of a nucleic acid that encodes a peptide is implicitly described in each disclosed sequence.
The polynucleotide of the present invention may be composed of DNA, RNA, or derivatives thereof. As is well known in the art, DNA molecules are composed of bases such as natural bases A, T, C, and G, and T is replaced with U in RNA. One skilled in the art will recognize that unnatural bases are also included in the polynucleotide.

  A polynucleotide of the present invention may encode multiple peptides of the present invention with or without intervening amino acid sequences. For example, the intervening amino acid sequence can provide a cleavage site (eg, an enzyme recognition sequence) for the polynucleotide or translated peptide. Furthermore, the polynucleotide may comprise any additional sequence to the coding sequence that encodes a peptide of the invention. For example, the polynucleotide may be a recombinant polynucleotide containing regulatory sequences necessary for expression of the peptide, or may be an expression vector (plasmid) having a marker gene or the like. In general, such recombinant polynucleotides can be prepared, for example, by manipulating the polynucleotide by conventional recombinant techniques using polymerases and endonucleases.

  The polynucleotides of the present invention can be produced using either recombinant techniques or chemical synthesis techniques. For example, a polynucleotide can be made by inserting into an appropriate vector, which can be expressed when transfected into competent cells. Alternatively, polynucleotides can be amplified using PCR techniques or expression in a suitable host (see, eg, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1989). I want to be) Alternatively, polynucleotides can be synthesized using solid phase techniques as described in Beaucage SL & Iyer RP, Tetrahedron 1992, 48: 2223-311; Matthes et al., EMBO J 1984, 3: 801-5. it can.

V. Exosomes The present invention further provides intracellular vesicles called exosomes that present complexes formed between the peptides of the present invention and HLA antigens on their surface. Exosomes can be prepared using, for example, the methods described in detail in Japanese Patent Publication No. 11-510507 and WO99 / 03499, and an APC obtained from a patient to be treated and / or prevented can be prepared. Can be prepared. The exosomes of the present invention can be vaccinated as a vaccine in the same manner as the peptides of the present invention.

The type of HLA antigen contained in the complex must match that of the subject in need of treatment and / or prevention. For example, in the Japanese population, HLA-A24 and HLA-A2, especially HLA-A ^* 2402, HLA-A ^* 0201, and HLA-A ^* 0206 are widely common and are therefore suitable for the treatment of Japanese patients It is thought that. The use of A24 or A2 types that are highly expressed among Japanese and Caucasians is preferred to obtain effective results, and subtypes such as A ^* 2402, HLA-A ^* 0201, and HLA-A ^* 0206 are also used. used. Typically, peptides that have a high level of binding affinity for a particular antigen or have the ability to induce CTLs by antigen presentation by pre-examining the type of HLA antigen of the patient in need of treatment at the clinic Appropriate selection is possible. Furthermore, in order to obtain a peptide having both high binding affinity and CTL inducibility, one, two, or several amino acid substitutions, insertions, deletions based on the amino acid sequence of the natural CLUAP1 partial peptide And / or additions can be made.

When A24 type HLA antigen is used for the exosome of the present invention, a peptide having a sequence selected from SEQ ID NOs: 1 to 22 is used.
Alternatively, when using the A2 type HLA antigen for the exosome of the present invention, a peptide having a sequence selected from SEQ ID NO: 23 and 25-43 is used.

VI. Antigen presenting cell (APC)
The present invention also provides isolated antigen presenting cells (APCs) that present complexes formed with HLA antigens and peptides of the present invention on their surface. APC may be derived from a patient to be treated and / or prevented and can be administered as a vaccine alone or in combination with other drugs, including peptides, exosomes, or CTLs of the present invention. .
APC is not limited to a particular type of cell, including dendritic cells (DCs), Langerhans, known to present proteinaceous antigens on their cell surface as recognized by lymphocytes. Cells, macrophages, B cells, and activated T cells are included. Since DC is a representative APC having the strongest CTL-inducing activity among APCs, DC is used as the APC of the present invention.

  For example, the APCs of the invention can be obtained by inducing DCs from peripheral blood monocytes and then contacting (stimulating) them with the peptides of the invention in vitro, ex vivo, or in vivo. When the peptide of the present invention is administered to a subject, APC presenting the peptide of the present invention is induced in the subject's body. The phrase “inducing APC” is formed between an HLA antigen and a peptide of the present invention when a cell is contacted with (stimulated with) the peptide of the present invention or a nucleotide encoding the peptide of the present invention. Presenting the complex on the cell surface. Therefore, the APC of the present invention can be obtained by administering the peptide of the present invention to a subject and then recovering the APC from the subject. Alternatively, the APC of the present invention can be obtained by contacting APC recovered from a subject with the peptide of the present invention.

To induce an immune response against cancer in a subject, the APCs of the invention can be administered to the subject alone or in combination with other drugs, including peptides, exosomes, or CTLs of the invention. For example, ex vivo administration may include the following steps:
a: recovering APC from the first subject;
b: contacting the APC of step a with the peptide; and c: administering the APC of step b to a second subject.

The first object and the second object may be the same individual or different individuals. Alternatively, according to the present invention, there is provided use of the peptide of the present invention for producing a pharmaceutical composition for inducing antigen-presenting cells. In addition, the present invention provides a method or process for producing a pharmaceutical composition that induces antigen-presenting cells. Furthermore, the present invention also provides a peptide of the present invention for inducing antigen-presenting cells. The APC obtained by step b is a vaccine for treating and / or preventing cancer such as breast cancer, cervical cancer, colorectal cancer, esophageal cancer, gastric cancer, invasive gastric cancer, lymphoma, neuroblastoma, pancreatic cancer. possible.
According to one aspect of the present invention, APC has a high level of CTL inducibility. The high level in the term “high level of CTL inducibility” is compared to the level of CTL inducibility by APC not contacted with a peptide, or APC contacted with a peptide that may not induce CTL. is there. Such an APC having a high level of CTL inducibility can be prepared by a method comprising the step of introducing a polynucleotide encoding the peptide of the present invention into APC in vitro in addition to the above method. The gene to be introduced may be in the form of DNA or RNA. Various methods conventionally introduced in the art, such as, but not limited to, examples of methods of introduction, including lipofection, electroporation, and the calcium phosphate method can be used. More specifically, it is described in Cancer Res 1996, 56: 5672-7; J Immunol 1998, 161: 5607-13; J Exp Med 1996, 184: 465-72; published patent publication 2000-509281. As such, it can be implemented. By introducing the gene into APC, the gene undergoes transcription, translation, etc. in the cell, and then the obtained protein is processed by MHC class I or class II, and the partial peptide of the present invention passes through the presentation pathway. Presented.

VII. Cytotoxic T lymphocyte (CTL)
CTLs induced against any of the peptides of the invention can be used as vaccines in a manner similar to the peptides themselves to enhance immune responses targeting cancer cells in vivo. Accordingly, the present invention provides isolated CTLs that are specifically induced or activated by any of the peptides of the present invention.
Such CTLs (1) administer a peptide of the invention to a subject, or (2) contact APC and CD8 positive cells or peripheral blood mononuclear leukocytes from the subject with the peptide of the invention in vitro. Or (3) contacting CD8 positive cells or peripheral blood mononuclear leukocytes in vitro with APC or exosomes that present complexes of HLA antigen and the peptide on their surface, or (4) It can be obtained by introducing a gene containing a polynucleotide encoding a T cell receptor (TCR) subunit that binds to the peptide of the present invention. Such APCs or exosomes can be prepared by the method described above, and details of the method (4) are described in the section “VIII. T cell receptor (TCR)” below.

  The CTLs of the present invention may be derived from the patient to be treated and / or prevented and administered alone or in combination with other drugs comprising the peptides or exosomes of the present invention for the purpose of modulating the effect. Can be administered. The obtained CTLs act specifically on target cells presenting the peptides of the present invention, for example the same peptides used for induction. The target cell may be a cell that endogenously expresses CLUAP1, such as a cancer cell, or a cell that has been transfected with the CLUAP1 gene, and that presents the peptide on the cell surface upon stimulation with the peptide of the present invention. Can also be a target for activated CTL attacks.

VIII. T cell receptor (TCR)
The invention also provides compositions comprising a nucleic acid encoding a polypeptide capable of forming a T cell receptor (TCR) subunit, and methods of using the same. The TCR subunit has the ability to form a TCR that confers to the T cells specificity for tumor cells presenting CLUAP1. Using methods known in the art, alpha and beta chain nucleic acids can be identified as TCR subunits of CTLs derived from one or more peptides of the invention (WO2007 / 032255, And Morgan et al., J Immunol, 171, 3288 (2003)). For example, the PCR method is preferable for analyzing TCR. PCR primers for analysis include, for example, a 5′-R primer (5′-gtctaccagcatcatgcttcat-3 ′) (SEQ ID NO: 46) as a 5 ′ primer and a TCR α chain C region as a 3 ′ primer. Specific 3-TRa-C primer (5'-tcagctggaccacagccgcagcgt-3 ') (SEQ ID NO: 47), 3-TRb-C1 primer specific for TCRβ chain C1 region (5'-tcaagaattccttctctctgac-3') (SEQ ID NO: 48), or a 3-TRβ-C2 primer specific for the TCRβ chain C2 region (5′-cttagcctctgaatccttttcttt-3 ′) (SEQ ID NO: 49), but is not limited thereto Do not mean. The TCR derivative can bind with high avidity to target cells presenting the CLUAP1 peptide, and optionally mediates efficient killing of target cells presenting the CLUAP1 peptide in vivo and in vitro.

  The nucleic acid encoding the TCR subunit may be incorporated into a suitable vector, such as a retroviral vector. These vectors are well known in the art. The nucleic acids or vectors containing them may be introduced into T cells, eg, patient-derived T cells. Advantageously, the present invention provides for the rapid and easy generation of modified T cells with superior cancer cell killing properties by rapid modification of the patient's own T cells (or another mammalian T cell). An easily available composition is provided that enables.

  Specific TCR refers to the specific recognition of the complex of the peptide of the present invention and the HLA molecule when TCR is displayed on the surface of T cells, and specific activity against target cells is given to T cells. It is a receptor that can be imparted. Specific recognition of the complex can be confirmed by any known method, and preferred examples include HLA multimer staining analysis using HLA molecules and peptides of the present invention, and ELISPOT assay. By performing an ELISPOT assay, it is possible to confirm that T cells expressing TCR on the cell surface recognize the cells by the TCR and that signals are transmitted intracellularly. Confirmation that the complex described above can impart cytotoxic activity to T cells when the complex is present on the surface of T cells can also be performed. Preferred methods include, for example, measurement of cytotoxic activity against HLA positive target cells, such as a chromium release assay.

The present invention also relates to CLUAP1 peptides with SEQ ID NOs: 3, 4, 5, 8, 9, 10, 14, 15, 16, and 18 in connection with HLA-A24, and also with HLA-A2. Also provided in the context are CTLs prepared by transduction of nucleic acids encoding TCR subunit polypeptides that bind to the CLUAP1 peptides of SEQ ID NOs: 23, 30, 33, 34, 35, 36, and 38.
Transduced CTL can home to cancer cells in vivo and can be propagated in vitro by well-known culture methods (eg, Kawakami et al., J Immunol., 142, 3452-3461 ( 1989)). The CTLs of the present invention can be used to form immunogenic compositions useful for cancer therapy or prevention in patients in need of treatment or prevention (WO 2006/031221).

  Prevention and prophylaxis include any act that reduces the mortality or morbidity burden due to the disease. Prevention and prevention can be performed “at the primary, secondary, and tertiary prevention levels”. Primary prevention and prophylaxis avoid the development of disease, whereas secondary and tertiary prevention (prevention and prophylaxis) prevents disease progression and appearance of symptoms (prevention and In addition to prophylaxis), it includes acts aimed at reducing the adverse effects of existing diseases by restoring function and reducing disease-related complications. Alternatively, prevention and prophylaxis include a wide range of prophylactic treatments aimed at reducing the severity of certain disorders, for example, reducing tumor growth and metastasis, reducing angiogenesis.

  Treatment for cancer prevention and / or prevention of post-operative recurrence includes surgical removal of cancer cells, inhibition of cancerous cell growth, tumor regression or regression, induction of remission and Includes any of the stages such as suppression of cancer development, tumor regression, and reduction or inhibition of metastasis. Effective treatment and / or prevention of cancer reduces mortality, improves the prognosis of individuals with cancer, decreases the level of tumor markers in the blood, and detectable symptoms associated with cancer To ease. For example, symptom reduction or amelioration constitutes an effective treatment, and / or prevention includes 10%, 20%, 30%, or more of a reduced or stable disease.

IX. Expression of the pharmaceutical substance or composition CLUAP1 includes breast cancer, cervical cancer, colorectal cancer, esophageal cancer, gastric cancer, invasive gastric cancer, lymphoma, neuroblastoma, pancreatic cancer as an example compared to normal tissue Is specifically elevated in cancers not limited thereto, the peptides of the invention or polynucleotides encoding such peptides can be used to treat and / or prevent cancer and / or prevent its recurrence after surgery. Can be used. Accordingly, the present invention provides a pharmaceutical substance or composition for the treatment and / or prevention of its post-operative recurrence, such composition comprising one or more of the peptides or polynucleotides of the present invention As an active ingredient. Alternatively, the peptides of the invention can be expressed on the surface of any of the aforementioned exosomes or cells such as APC for use as a pharmaceutical substance or composition. In addition, the above-mentioned CTL targeting any of the peptides of the present invention can also be used as an active ingredient of the pharmaceutical substance or composition of the present invention.
The pharmaceutical substances and compositions of the invention may also be useful as vaccines. In the context of the present invention, the phrase “vaccine” (also referred to as “immunogenic composition”) refers to a substance that has the function of inducing anti-tumor immunity when inoculated into an animal.

  The pharmaceutical substances or compositions of the invention are human and mice, rats, guinea pigs, rabbits, cats, dogs, sheep, goats, pigs, cows, horses, monkeys, baboons, and chimpanzees, especially commercially important Used to treat and / or prevent cancer and / or prevent its recurrence after surgery in a subject or patient including but not limited to animals or any other mammal including livestock it can.

In another aspect, the present invention also provides the use of an active ingredient selected from among the following in the manufacture of a pharmaceutical composition or substance for treating or preventing cancer or tumor:
(A) the peptide of the present invention;
(B) a nucleic acid encoding the peptide disclosed herein in an expressible form;
(C) APC or exosome presenting the peptide of the invention on its surface; and (d) the cytotoxic T cell of the invention.

Alternatively, the present invention further provides an active ingredient selected from the following for use in the treatment or prevention of cancer or tumor:
(A) the peptide of the present invention;
(B) a nucleic acid encoding the peptide disclosed herein in an expressible form;
(C) APC or exosome presenting the peptide of the invention on its surface; and (d) the cytotoxic T cell of the invention.

Alternatively, the present invention further relates to a method or process for producing a pharmaceutical composition or substance for treating or preventing cancer or tumor, wherein the active ingredient is selected from the following as an active ingredient: A method or process comprising the steps of formulating with a pharmaceutically or physiologically acceptable carrier is provided:
(A) the peptide of the present invention;
(B) a nucleic acid encoding the peptide disclosed herein in an expressible form;
(C) APC or exosome presenting the peptide of the invention on its surface; and (d) the cytotoxic T cell of the invention.

In another aspect, the present invention also provides a method or process for producing a pharmaceutical composition or substance for treating or preventing cancer or tumor, comprising an active ingredient selected from A method or process comprising the step of admixing with a pharmaceutically or physiologically acceptable carrier is provided:
(A) the peptide of the present invention;
(B) a nucleic acid encoding the peptide disclosed herein in an expressible form;
(C) APC or exosome presenting the peptide of the invention on its surface; and (d) the cytotoxic T cell of the invention.

  According to the present invention, a peptide having an amino acid sequence selected from SEQ ID NOs: 2 to 22 is an HLA-A24 restricted epitope peptide capable of inducing a strong and specific immune response or a candidate thereof. And a peptide having an amino acid sequence selected from SEQ ID NO: 23 and 25-43 is an HLA-A2 restricted epitope peptide or a candidate thereof capable of inducing a strong and specific immune response. Was found. Thus, any of these peptides having the amino acid sequence of SEQ ID NO: 3, 4, 5, 8, 9, 10, 14, 15, 16, 18 and 23, 30, 33, 34, 35, 36, 38 Are particularly suitable for administration to a subject whose HLA antigens are HLA-A24 and HLA-A2, respectively. The same applies to pharmaceutical substances or compositions comprising a polynucleotide encoding any of these peptides (ie, a polynucleotide of the invention).

  Cancers to be treated by the pharmaceutical agents or compositions of the present invention include, but are not limited to, any cancer in which CLUAP1 is involved (eg, overexpressed), including, for example, breast cancer, children Examples include, but are not limited to, cervical cancer, colorectal cancer, esophageal cancer, gastric cancer, invasive gastric cancer, lymphoma, neuroblastoma, pancreatic cancer.

The pharmaceutical substance or composition of the present invention includes, in addition to the above-mentioned active ingredient, other peptides having the ability to induce CTL against cancerous cells, other polynucleotides encoding the other peptides, the other Other cells presenting the peptide may be included. As used herein, other peptides that have the ability to induce CTLs against cancerous cells are exemplified by, but are not limited to, cancer specific antigens (eg, identified TAAs).
If necessary, the pharmaceutical substance or composition of the present invention can be used as an active ingredient unless the other therapeutic substance inhibits the anti-tumor effect of the active ingredient such as any of the peptides of the present invention. May optionally be included. For example, the formulation can include anti-inflammatory substances or anti-inflammatory compositions, analgesics, chemotherapeutics, and the like. In addition to other therapeutic substances in the medicament itself, the medicaments of the present invention can also be administered sequentially or simultaneously with one or more other pharmacological substances or compositions. The amount of pharmaceutical and pharmacological substance or composition depends, for example, on the type of pharmacological substance or composition used, the disease to be treated, and the schedule and route of administration.

In addition to the ingredients specifically mentioned herein, the pharmaceutical substance or composition of the present invention may contain other substances or compositions customary in the art in view of the type of formulation in question. It should be understood that objects can also be included.
In one aspect of the invention, the pharmaceutical substance or composition of the invention can be included in products and kits containing materials useful for treating the condition of the disease to be treated, eg, cancer. The product may comprise a container of any of the pharmaceutical substances or compositions of the invention having a label. Suitable containers include bottles, vials, and test tubes. The container may be formed from a variety of materials such as glass or plastic. The label on the container surface should indicate that the substance or composition is used for the treatment or prevention of one or more conditions of the disease. The label may also indicate directions for administration and the like.

The kit containing the pharmaceutical substance or composition of the present invention may optionally further comprise a second container containing a pharmaceutically acceptable diluent in addition to the container described above. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and packaging enclosures with instructions for use.
If desired, the pharmaceutical composition can be provided in a pack or dispenser device that may contain one or more unit dosage forms containing the active ingredients. The pack may include metal foil or plastic foil, such as a blister pack. The pack or dispenser device can be accompanied by instructions for administration.

(1) Pharmaceutical Substance or Composition Containing Peptide as Active Ingredient The peptide of the present invention may be directly administered as a pharmaceutical substance or composition or, if necessary, formulated by a conventional formulation method May be used. In the latter case, in addition to the peptide of the present invention, carriers, excipients and the like that are usually used for drugs can be included as needed without particular limitation. Examples of such carriers are sterilized water, physiological saline, phosphate buffer, culture solution and the like. Furthermore, the pharmaceutical substance or composition may contain stabilizers, suspensions, preservatives, surfactants and the like as required. The pharmaceutical substance or composition of the present invention can be used for anticancer purposes.

  In order to induce CTLs in vivo, the peptides of the invention can be prepared as a combination composed of two or more of the peptides of the invention. The peptide combination may take the form of a cocktail or may be conjugated to each other using standard techniques. For example, the peptide may be chemically coupled or may have one or more amino acids as a linker (eg, lysine linker: KS Kawamura et al. J. Immunol. 2002, 168: 5709-5715). It may be expressed as a single fusion polypeptide sequence. The peptides in the combination may be the same or different. By administering the peptides of the present invention, the peptides are presented at high density on the APC by the HLA antigen and then react specifically to the complex formed between the presented peptide and the HLA antigen. CTL is induced. Alternatively, APC (eg, DC) is removed from the subject and then stimulated with a peptide of the invention to obtain APC that presents any of the peptides of the invention on the cell surface. These APCs can be re-administered to a subject to induce CTL within the subject, resulting in increased aggressiveness against the tumor associated endothelium.

  A pharmaceutical substance or composition for the treatment and / or prevention of cancer comprising the peptide of the present invention as an active ingredient may also contain an adjuvant known to efficiently establish cellular immunity. . Alternatively, the pharmaceutical composition may be administered with other active ingredients or may be administered by formulation into granules. An adjuvant refers to any compound, substance, or composition that, when administered with (or sequentially) a protein having immunological activity, enhances the immune response to 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, incomplete Freund's adjuvant (IFA), complete Freund's adjuvant (CFA), ISCOMatrix , GM-CSF, CpG, O / W emulsion and the like.

Furthermore, liposome preparations, granule preparations in which peptides are bound to beads having a diameter of several micrometers, and preparations in which lipids are bound to peptides may be used advantageously.
In another aspect of the invention, the peptides of the invention may also be administered in the form of a pharmaceutically acceptable salt. Preferred examples of the salt include a salt with an alkali metal, a salt with a metal, a salt with an organic base, a salt with an organic acid, and a salt with an inorganic acid. As used herein, a “pharmaceutically acceptable salt” refers to a compound that retains the biological effectiveness and properties of the compound and is hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, methanesulfonic acid. , A salt obtained by a reaction with an inorganic acid or an inorganic base such as ethanesulfonic acid, p-toluenesulfonic acid or salicylic acid.

  In some embodiments, the pharmaceutical agent or composition of the invention may further comprise a component that primes CTL. Lipids have been identified as substances or compositions that can stimulate CTLs in vivo against viral antigens. For example, palmitic acid residues can be attached to the ε and α amino groups of lysine residues and then linked to the peptides of the invention. The lipidated peptide can then be administered directly in the form of micelles or particles, administered in liposomes, or emulsified in an adjuvant. As another example of lipid stimulation of the CTL response, E. coli lipoproteins such as tripalmitoyl-S-glycerylcysteinyl-seryl-serine (P3CSS) when covalently linked to the appropriate peptide Can be used to stimulate CTLs (see, eg, Deres et al., Nature 1989, 342: 561-4).

  The method of administration may be oral, intradermal, subcutaneous, intravenous injection, etc., and systemic administration or local administration in the vicinity of the target site. Administration can be by a single dose or can be boosted by multiple doses. The dose of the peptide of the present invention can be appropriately adjusted according to the disease to be treated, the patient's age, weight, administration method, etc., and is usually 0.001 mg to 1,000 mg, for example, 0.1 mg to 10 mg. Yes, it can be administered once every few days to several months. Those skilled in the art can appropriately select an appropriate dose.

(2) A pharmaceutical substance or composition comprising a polynucleotide as an active ingredient The pharmaceutical substance or composition of the present invention may also comprise a nucleic acid encoding the peptide disclosed herein in a form capable of being expressed. As used herein, the phrase “in an expressible form” means that a polynucleotide is expressed in vivo as a polypeptide that induces anti-tumor immunity when introduced into a cell. In an exemplary embodiment, the nucleic acid sequence of the polynucleotide of interest includes regulatory elements necessary for expression of the polynucleotide. The polynucleotide can be provided with the necessary ones so that stable insertion into the genome of the target cell is achieved (for descriptions of homologous recombination cassette vectors, see eg, Thomas KR & Capecchi MR, Cell 1987 51: 503-12, also see, for example, Wolff et al., Science 1990, 247: 1465-8, U.S. Patent Nos. 5,580,859, 5,589,466, 5,804. No. 5,739,118; 5,736,524; 5,679,647; and WO 98/04720). Examples of DNA-based delivery technologies include “naked DNA”, facilitated (bupivacaine, polymer, peptide-mediated) delivery, cationic lipid complexes, and particle-mediated (“gene gun”) or pressure-mediated (See, for example, US Pat. No. 5,922,687).

  The peptides of the present invention can also be expressed by viral vectors or bacterial vectors. Examples of expression vectors include attenuated viral hosts such as vaccinia virus or fowlpox virus. This approach involves the use of vaccinia virus, for example, as a vector to express nucleotide sequences encoding peptides. When introduced into a host, recombinant vaccinia virus expresses an immunogenic peptide, thereby eliciting an immune response. Vaccinia vectors and methods useful for immunization protocols are described, for example, in US Pat. No. 4,722,848. Another vector is BCG (Bacilli Calmette Guerin). BCG vectors are described in Stover et al., Nature 1991, 351: 456-60. A wide variety of other vectors useful for therapeutic administration or immunization, such as adenovirus vectors and adeno-associated virus vectors, retrovirus vectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, etc. are apparent . See, eg, 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. I want to be.

Delivery of the polynucleotide to the patient may be direct, in which case the patient may be directly exposed to a vector carrying the polynucleotide, or indirectly, in which case first in vitro. The cells are transformed with the polynucleotide of interest, and then the cells are transplanted into the patient. Each of these two approaches is known as in vivo and ex vivo gene therapy.
For a general review of gene therapy methods, 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. Methods commonly known in the field of recombinant DNA technology that can also be used in the present invention are eds. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, NY, 1993; and Krieger, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY, 1990.

  The administration method may be oral, intradermal, subcutaneous, intravenous injection or the like, and systemic administration or local administration in the vicinity of the target site is used. Administration can be by a single dose or can be boosted by multiple doses. The dosage of the polynucleotide in a suitable carrier, or the dosage of the polynucleotide in a cell transformed with a polynucleotide encoding a peptide of the invention will depend on the disease being treated, the age, weight of the patient, method of administration, etc. It is usually 0.001 mg to 1000 mg, such as 0.001 mg to 1000 mg, such as 0.1 mg to 10 mg, and can be administered once every few days to once every several months. Those skilled in the art can appropriately select an appropriate dose.

X. Methods Using Peptides, Exosomes, APCs, and CTLs The peptides and polynucleotides of the invention can be used to prepare or derive APCs and CTLs. CTLs can also be induced using the exosomes and APCs of the present invention. Peptides, polynucleotides, exosomes, and APCs can be used in combination with any other compound as long as they do not inhibit their ability to induce CTL. Therefore, CTLs can be induced using any of the aforementioned pharmaceutical substances or compositions of the present invention, and in addition, APCs can be used as described below using those containing the aforementioned peptides and polynucleotides. It can also be guided.

(1) Method for Inducing Antigen Presenting Cells (APC) The present invention provides a method for inducing APC having high CTL inducing ability using the peptide or polynucleotide of the present invention.
The methods of the present invention comprise contacting APC with the peptides of the present invention in vitro, ex vivo or in vivo. For example, a method of contacting APC with the peptide ex vivo may comprise the following steps:
a: recovering APC from the subject; and b: contacting the APC of step a with the peptide.

  APCs are not limited to specific types of cells, DCs, Langerhans cells, macrophages, B cells, and activities known to present proteinaceous antigens on their cell surfaces as recognized by lymphocytes Including T cells. Since it has the strongest CTL inducing ability among APCs, DC can be preferably used. Any peptide of the invention can be used alone or with other peptides of the invention.

  On the other hand, when the peptide of the present invention is administered to a subject, APC comes into contact with the peptide in vivo, and as a result, APC having high CTL inducing ability is induced in the body of the subject. Accordingly, the present invention includes the step of administering to the subject a peptide of the present invention. Similarly, when the polynucleotide of the present invention is administered to a subject in an expressible form, the peptide of the present invention is expressed in vivo and contacts APC, so that APC having a high ability to induce CTLs in the subject's body. Be guided. Accordingly, the present invention can also include the step of administering a polynucleotide of the present invention to a subject. The “expressible form” is described in the section “IX. Pharmaceutical substance or composition, (2) Pharmaceutical substance or composition comprising a polynucleotide as an active ingredient” above.

The present invention may also include the step of introducing the polynucleotide of the present invention into APC in order to induce APC having CTL inducibility. For example, the method may include the following steps:
a: recovering APC from the subject; and b: introducing a polynucleotide encoding a peptide of the invention.
Step b can be performed as described above in section "VI. Antigen-presenting cells".
Alternatively, the present invention provides a method for preparing an antigen presenting cell (APC) that specifically induces CTL activity against CLUAP1, which may comprise one of the following steps:
(A) contacting APC with a peptide of the invention in vitro, ex vivo, or in vivo; and (b) introducing a polynucleotide encoding the peptide of the invention into APC.

(2) Method for Inducing CTL The present invention also provides a method for inducing CTL using the peptide, polynucleotide, exosome, or APC of the present invention.

The present invention also provides a method for inducing CTLs using a polynucleotide encoding a polypeptide capable of forming a T cell receptor (TCR) subunit that recognizes a complex of the peptide of the present invention and an HLA antigen. To do. Preferably, the method for inducing CTL may comprise at least one step selected from the following:
a) contacting a CD8 positive T cell with an antigen presenting cell and / or an exosome that presents a complex of the HLA antigen and the peptide of the present invention on its surface; and b) the peptide of the present invention and the HLA antigen. Introducing into a CD8-positive cell a polynucleotide encoding a polypeptide capable of forming a TCR subunit that recognizes the complex.
When the peptide, polynucleotide, APC, or exosome of the present invention is administered to a subject, CTL is induced in the subject's body, and the intensity of an immune response targeting cancer cells is enhanced. Accordingly, the methods of the present invention comprise administering to the subject a peptide, polynucleotide, APC, or exosome of the present invention.

Alternatively, CTLs can be induced by using them ex vivo, and activated CTLs can be returned to the subject after induction of CTLs. For example, the method may include the following steps:
a: recovering APC from the subject;
b: contacting the APC of step a with the peptide; and c: co-culturing the APC of step (b) with CD8 positive cells.

APC co-cultured with CD8-positive cells in the above step c can also be prepared by introducing a gene containing the polynucleotide of the present invention into APC as described above in the section “VI. Antigen-presenting cells”. However, the present invention is not limited to this, and includes any APC that effectively presents a complex of the HLA antigen and the peptide of the present invention on its surface.
Instead of such APC, an exosome that presents a complex of the HLA antigen and the peptide of the present invention on its surface can also be used. That is, the present invention can also include a step of co-culturing exosomes that present a complex of the HLA antigen and the peptide of the present invention on its surface. Such exosomes can be prepared by the methods described above in the section “V. Exosomes”.

Furthermore, CTL can be induced by introducing a gene containing a polynucleotide encoding a TCR subunit that binds to the peptide of the present invention into CD8 positive cells. Such transduction can be performed as described above in the section “VIII. T Cell Receptor (TCR)”.
In addition, the present invention provides a method or process for producing a pharmaceutical substance or composition that induces CTL, comprising mixing or formulating a peptide of the present invention with a pharmaceutically acceptable carrier. A method of including is provided.

(3) Method for Inducing an Immune Response Furthermore, the present invention provides a method for inducing an immune response against a disease associated with CLUAP1. Suitable diseases include cancer, examples of which include breast cancer, cervical cancer, colorectal cancer, esophageal cancer, gastric cancer, invasive gastric cancer, lymphoma, neuroblastoma, pancreatic cancer. It is not limited.

The methods of the present invention can include administering a substance or composition comprising any of the peptides of the present invention or any of the polynucleotides encoding them. The methods of the present invention also contemplate administration of exosomes or APCs that present any of the peptides of the present invention. For details, see the section “IX. Pharmaceutical substances or compositions”, in particular the part describing the use of the pharmaceutical substances or compositions of the invention as vaccines. In addition, exosomes and APCs that can be used in the methods of the present invention to induce an immune response are the above-mentioned “V. exosomes”, “VI. Antigen presenting cells (APCs)”, and “X. peptides, exosomes”. , APC, and CTL method "(1) and (2).
The present invention also includes a method or process for producing a pharmaceutical substance or composition that induces an immune response, comprising mixing or formulating a peptide of the present invention with a pharmaceutically acceptable carrier. Provide a way to get.

Alternatively, the method of the invention may comprise administering a vaccine or pharmaceutical composition comprising:
(A) the peptide of the present invention;
(B) a nucleic acid encoding the peptide disclosed herein in an expressible form;
(C) APC or exosome presenting the peptide of the invention on its surface; or (d) the cytotoxic T cell of the invention.

In the context of the present invention, cancers that overexpress CLUAP1 can be treated with these active ingredients. Examples of such cancers include but are not limited to breast cancer, cervical cancer, colorectal cancer, esophageal cancer, gastric cancer, invasive gastric cancer, lymphoma, neuroblastoma, pancreatic cancer. . Thus, before administering a vaccine or pharmaceutical composition comprising an active ingredient, it may be ascertained whether the expression level of CLUAP1 in the cells or tissues to be treated is enhanced compared to normal cells of the same organ preferable. Thus, in one aspect, the present invention provides a method for treating a cancer that (over) expresses CLUAP1 in a patient in need thereof, such method may comprise the following steps:
i) determining the expression level of CLUAP1 in cells or tissues obtained from a subject having a cancer to be treated;
ii) comparing the expression level of CLUAP1 with a normal control; and iii) overexpressing CLUAP1 with respect to at least one component selected from (a) to (d) above compared to a normal control, Administering to a subject with cancer.

  Alternatively, the present invention provides a vaccine or pharmaceutical composition comprising at least one component selected from the above (a) to (d), which is administered to a subject having a cancer that overexpresses CLUAP1. . In other words, the invention further comprises a method for identifying a subject to be treated with a CLUAP1 polypeptide of the invention, comprising determining the expression level of CLUAP1 in a cell or tissue from the subject, Provided is a method wherein the level is elevated compared to a normal control level of the gene, indicating that the subject may have a cancer that can be treated with a CLUAP1 polypeptide of the invention. The method for treating cancer of the present invention will be described in more detail below.

  Any cell or tissue from the subject can be used to measure CLUAP1 expression so long as it includes the desired CLUAP1 transcript or translation product. Examples of suitable samples include, but are not limited to, body tissue and body fluids such as blood, sputum, and urine. Preferably, the subject-derived cell or tissue sample comprises an epithelial cell, more preferably a cancerous epithelial cell, or a cell population comprising an epithelial cell derived from a tissue suspected of cancer. Furthermore, if necessary, cells may be purified from the obtained body tissue and body fluid, and then used as a subject-derived sample.

In the context of the present invention, a control level determined from a biological sample that has been found not to be cancerous is referred to as a “normal control level”. On the other hand, if the control level is determined from a cancerous biological sample, it is referred to as the “cancerous control level”. The difference between the expression level of the sample and the control level is changed to the expression level of a control nucleic acid, for example a housekeeping gene, whose expression level is known not to vary depending on the cancerous or non-cancerous state of the cell. It can be normalized to. Exemplary control genes include, but are not limited to, β-actin, glyceraldehyde 3-phosphate dehydrogenase, and ribosomal protein P1.
The subject to be treated by the method of the present invention is preferably a mammal. Exemplary mammals include, but are not limited to, for example, humans, non-human primates, mice, rats, dogs, cats, horses, and cows.

According to the present invention, the expression level of CLUAP1 in cells or tissues obtained from a subject may be determined. Expression levels can be determined at the transcription (nucleic acid) product level using methods known in the art. For example, CLUAP1 mRNA can be quantified using a probe by a hybridization method (eg, Northern hybridization). The detection can be performed on a chip or an array. The use of an array may be preferred for detecting the expression level of CLUAP1. One skilled in the art can prepare such probes using the sequence information of CLUAP1. For example, CLUAP1 cDNA can be used as a probe. If necessary, the probe may be labeled with an appropriate label such as a dye, fluorescent substance, and isotope, and the expression level of the gene may be detected as the intensity of the hybridized label.
Furthermore, the transcription product of CLUAP1 (eg, SEQ ID NO: 44) may be quantified using a primer by a detection method based on amplification (eg, RT-PCR). Such primers may be prepared based on available sequence information for the gene.

  Specifically, the probe or primer used in the method of the present invention hybridizes to CLUAP1 mRNA under stringent conditions, moderately stringent conditions, or low stringent conditions. As used herein, the phrase “stringent (hybridization) conditions” refers to conditions under which a probe or primer hybridizes to its target sequence but not to other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Specific hybridization of longer sequences is observed at higher temperatures than shorter sequences. Generally, the temperature of stringent conditions is selected to be about 5 ° C. lower than the melting temperature (Tm) of the specific sequence at a given ionic strength and pH. Tm is the temperature (at a given ionic strength, pH, and nucleic acid concentration) at which 50% of the probe complementary to the target sequence hybridizes with the target sequence at equilibrium. Since target sequences are generally present in excess, at Tm, 50% of the probe is occupied at equilibrium. Typically, stringent conditions include a salt concentration of less than about 1.0 M sodium ions at pH 7.0-8.3, typically about 0.01-1. The conditions are 0 M and the temperature is at least about 30 ° C. for short probes or primers (eg, 10-50 nucleotides) and at least about 60 ° C. for longer probes or primers. Stringent conditions may also be achieved with the addition of destabilizing substances such as formamide.

  The probe or primer may be of a specific size. The size may range from 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 probe and primer sizes may range from 5-10 nucleotides, 10-15 nucleotides, 15-15 It may range from 20 nucleotides, 20-25 nucleotides, and 25-30 nucleotides.

  Alternatively, the translation product can be detected for the diagnosis of the present invention. For example, the amount of CLUAP1 protein (SEQ ID NO: 45) or an immunological fragment thereof can be measured. Methods for measuring the amount of protein as a translation product include immunoassays using antibodies that specifically recognize the protein. The antibody may be monoclonal or polyclonal. Furthermore, as long as the fragment or modified antibody retains the binding ability to the CLUAP1 protein, any fragment or modified antibody (eg, chimeric antibody, scFv, Fab, F (ab ′) 2, Fv, etc.) is used for detection. be able to. Such antibodies or fragments thereof against the peptides of the present invention are also provided by the present invention. Methods for preparing such types of antibodies for detecting proteins are well known in the art and any method in the present invention may be used to prepare such antibodies and their equivalents. Can do.

As another method for detecting the expression level of the CLUAP1 gene based on its translation product, the intensity of staining may be measured by immunohistochemical analysis using an antibody against the CLUAP1 protein. That is, in this measurement, strong staining indicates an increase in the presence / level of the protein and at the same time a high expression level of the CLUAP1 gene.
The level of expression of a target gene, eg, CLUAP1 gene, in a cancer cell is increased by, for example, 10%, 25%, or 50% from a control level of the target gene (eg, a level in normal cells); or It can be determined that it has risen when it rises to over 1.1 times, over 1.5 times, over 2.0 times, over 5.0 times, over 10.0 times, or more.

The control level can be determined at the same time as the cancer cells by using a sample that has been previously collected and stored from a subject with known disease state (cancerous or non-cancerous). In addition, normal cells obtained from non-cancerous areas of the organ having the cancer to be treated may be used as normal controls. Alternatively, the control level may be determined by statistical methods based on results obtained by analyzing a predetermined expression level of the CLUAP1 gene in a sample from a subject whose disease state is known. Good. Further, the control level can be derived from a database of expression patterns from previously tested cells. Furthermore, according to one aspect of the present invention, the expression level of the CLUAP1 gene in a biological sample can be compared to a plurality of control levels determined from a plurality of reference samples. It is preferred to use a control level determined from a reference sample derived from a tissue type similar to that of the biological sample from the subject. Furthermore, it is preferable to use a reference value for the expression level of the CLUAP1 gene in a population whose disease state is known. The reference value can be obtained by any method known in the art. For example, the average value +/− 2S. D. Or the average value +/- 3 S.I. D. Can be used as a reference value.
A subject can be diagnosed with a cancer to be treated if the expression level of the CLUAP1 gene is elevated compared to a normal control level or is similar / equivalent to a cancerous control level.

The invention also provides (i) a method for diagnosing whether a subject is suspected of having a cancer to be treated and / or (ii) a method for selecting a subject for cancer treatment, said method May include the following steps:
a) determining the expression level of CLUAP1 in cells or tissues obtained from a subject suspected of having a cancer to be treated;
b) comparing the expression level of CLUAP1 with a normal control level;
c) diagnosing the subject as having a cancer to be treated when the expression level of CLUAP1 is elevated compared to a normal control level; and d) the cancer to be treated by the subject in step c) Selecting the subject for cancer treatment if diagnosed as having.

Alternatively, such a method may include the following steps:
a) determining the expression level of CLUAP1 in cells or tissues obtained from a subject suspected of having a cancer to be treated;
b) comparing the expression level of CLUAP1 to a cancerous control level;
c) diagnosing the subject as having a cancer to be treated if the expression level of CLUAP1 is similar or equivalent to a cancerous control level; and d) treating the subject in step c) Selecting the subject for cancer treatment when diagnosed with having cancer.

The present invention also provides a diagnostic kit for diagnosing or determining a subject suffering from or suspected of having a cancer that can be treated with the CLUAP1 polypeptide of the present invention, the diagnostic kit also comprising It may also be useful for assessing the prognosis of cancer and / or monitoring the effectiveness or applicability of cancer therapy, particularly cancer immunotherapy. Examples of suitable cancers include but are not limited to breast cancer, cervical cancer, colorectal cancer, esophageal cancer, gastric cancer, invasive gastric cancer, lymphoma, neuroblastoma, pancreatic cancer . More particularly, the kit may preferably comprise at least one reagent for detecting the expression of the CLUAP1 gene in a subject-derived cell, such reagent selected from the following group:
(A) a reagent for detecting mRNA of the CLUAP1 gene;
(B) a reagent for detecting the CLUAP1 protein or immunological fragment thereof; and (c) a reagent for detecting the biological activity of the CLUAP1 protein.

  Examples of reagents suitable for detecting CLUAP1 gene mRNA include nucleic acids that specifically bind to or identify CLUAP1 mRNA, such as oligonucleotides having a complementary sequence to a portion of CLUAP1 mRNA. Can be included. This type of oligonucleotide is exemplified by primers and probes specific for CLUAP1 mRNA. Such types of oligonucleotides can be prepared based on methods well known in the art. If necessary, a reagent for detecting CLUAP1 mRNA can be immobilized on a solid substrate. In addition, more than one reagent for detecting CLUAP1 mRNA can be included in the kit.

On the other hand, examples of reagents suitable for detecting CLUAP1 protein or immunological fragments thereof may include antibodies to CLUAP1 protein or immunological fragments thereof. The antibody may be monoclonal or polyclonal. Furthermore, any fragment or modification of the antibody (eg, chimeric antibody, scFv, Fab, F (ab ′) ₂ , Fv) as long as the fragment or modified antibody retains the ability to bind to the CLUAP1 protein or immunological fragment thereof. Etc.) can also be used as reagents. Methods for preparing such types of antibodies for detecting proteins are well known in the art and any method in the present invention may be used to prepare such antibodies and their equivalents. Can do. Furthermore, the antibody can be labeled with a signal generating molecule by direct linking or indirect labeling techniques. Labels and methods for labeling antibodies and detecting antibody binding to a target are well known in the art, and any label and method can be used in the present invention. In addition, more than one reagent for detecting CLUAP1 protein can be included in the kit.

  The kit can include two or more of the aforementioned reagents. The kit further detects solid substrates and reagents for binding probes to the CLUAP1 gene or antibodies to the CLUAP1 peptide, media and containers for culturing cells, positive and negative control reagents, and antibodies to the CLUAP1 peptide. A secondary antibody may be included. For example, a tissue sample obtained from a subject without cancer or suffering from cancer can serve as a useful control reagent. The kit of the present invention includes commercial solutions and uses, including buffers, diluents, filters, needles, syringes, and packaged enclosures (eg, documents, tapes, CD-ROMs, etc.) with instructions for use. It may further include other materials desirable from a person's point of view. These reagents and the like can be held in a labeled container. Suitable containers can include bottles, vials, and test tubes. The container may be formed from a variety of materials such as glass or plastic.

  In one embodiment of the present invention, when the reagent is a probe for CLUAP1 mRNA, the reagent can be immobilized on a solid substrate such as a porous strip to form at least one detection site. The measurement or detection region of the porous strip can include multiple sites, each containing a nucleic acid (probe). The test strip can also include sites for negative and / or positive controls. Alternatively, the control site can be located on a separate strip from the test strip. Optionally, the different detection sites may contain different amounts of immobilized nucleic acid, ie, a higher amount of immobilized nucleic acid at the first detection site and a lower amount of immobilized nucleic acid at subsequent sites. When a test sample is added, the number of sites that exhibit a detectable signal provides a quantitative indicator of the amount of CLUAP1 mRNA present in the sample. The detection site can be configured in any shape that can be suitably detected and is typically in the shape of a bar or dot across the width of the test strip.

  The kit of the present invention may further comprise a positive control sample or a CLUAP1 standard sample. Positive control samples of the present invention can be prepared by collecting CLUAP1 positive samples and then assaying their CLUAP1 levels. Alternatively, purified CLUAP1 protein or polynucleotide may be added to cells that do not express CLUAP1 to form a positive sample or CLUAP1 standard sample. In the present invention, purified CLUAP1 may be a recombinant protein. The CLUAP1 level of the positive control sample is for example higher than the cutoff value.

In one aspect, the present invention further provides a diagnostic kit comprising a protein or a partial protein thereof that can specifically recognize the antibody of the present invention or an immunogenic fragment thereof.
Examples of partial peptides and immunogenic fragments of the protein of the invention contemplated herein include at least 8, preferably 15, and more preferably 20 contiguous sequences in the amino acid sequence of the protein of the invention. Polypeptides composed of the amino acids are included. Cancer can be diagnosed by detecting an antibody in a sample (for example, blood, tissue) using the protein or peptide (polypeptide) of the present invention. The method for preparing the peptide or protein of the present invention is as described above.

  The method of diagnosing cancer of the present invention can be carried out by measuring the difference between the amount of anti-CLUAP1 antibody and the amount of anti-CLUAP1 antibody in the corresponding control sample as described above. The subject cell or tissue contains an antibody against the expression product (CLUAP1) of the gene, and the amount of this anti-CLUAP1 antibody is determined to be higher than the level of the cutoff value compared to the amount of anti-CLUAP1 antibody in the normal control. The subject is suspected of having cancer.

In another embodiment, the diagnostic kit of the present invention may comprise a peptide of the present invention and an HLA molecule bound thereto. Suitable methods for detecting antigen-specific CTLs using antigenic peptides and HLA molecules have already been established (eg, 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 detection method for detecting tumor antigen-specific CTL, thereby enabling early detection of cancer recurrence and / or metastasis. It becomes possible. Furthermore, it can be used for selecting a subject to which a medicine containing the peptide of the present invention as an active ingredient can be applied, or for evaluating the therapeutic effect of the medicine.
Specifically, according to known methods (see, for example, Altman JD et al., Science. 1996, 274 (5284): 94-6), a tetramer of a radiolabeled HLA molecule and a peptide of the present invention, etc. The oligomer complex can be prepared. This complex can be used to quantify antigen peptide-specific CTL in peripheral blood lymphocytes derived from a subject suspected of having cancer.

  The present invention further provides methods or diagnostic reagents for assessing a subject's immunological response using the peptide epitopes described herein. In one aspect of the invention, an HLA-restricted peptide as described herein can be used as a reagent for evaluating or predicting a subject's immune response. The immune response to be assessed can be induced by contacting the immunogen with immunocompetent cells in vitro or in vivo. In preferred embodiments, immunocompetent cells for assessing an immunological response may be selected among peripheral blood, peripheral blood lymphocytes (PBL), and peripheral blood mononuclear cells (PBMC). Methods for recovering or isolating such immunocompetent cells are well known in the art. In certain embodiments, the substance or composition used as a reagent may be a composition that can result in the production of antigen-specific CTLs that recognize and bind to peptide epitopes. It is not necessary to use peptide reagents as immunogens. Assay systems used for such analysis include relatively recent technological developments such as tetramers, intracellular lymphokine staining, and interferon release assays, or ELISPOT assays. In a preferred embodiment, the immunocompetent cells that are contacted with the peptide reagent may 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 for the presence of antigen-specific CTL after exposure to tumor cell antigens or immunogens. The HLA tetramer complex was used to directly visualize antigen-specific CTL (eg, Ogg et al., Science 279: 2103-2106, 1998; and Altman et al, Science 174: 94-96, 1996). See), the frequency of antigen-specific CTL populations in samples of peripheral blood mononuclear cells can be measured. Tetrameric reagents using the peptides of the present invention can be made as described below.

  Peptides that bind to the HLA molecule are refolded in the presence of the corresponding HLA heavy chain and β2-microglobulin to produce a trimolecular complex. In this complex, the site created in the protein in advance of the carboxy terminus of the heavy chain is biotinylated. Streptavidin is then added to the complex to form a tetramer composed of a trimolecular complex and streptavidin. This tetramer can be used to stain antigen-specific cells by the fluorescently labeled streptavidin approach. The cells can then be identified, for example, by flow cytometry. Such an analysis can be used for diagnostic or prognostic purposes. Cells identified by this technique can also be used for therapeutic purposes.

  The present invention also provides reagents for assessing immune recall responses comprising the peptides of the present invention (eg, Bertoni et al, J. Clin. Invest. 100: 503-513, 1997, and Penna et al. , J Exp. Med. 174: 1565-1570, 1991). For example, patient PBMC samples from individuals with cancer to be treated can be analyzed for the presence of antigen-specific CTL using specific peptides. A blood sample containing mononuclear cells can be evaluated by culturing PBMC and stimulating the cells with the peptide of the present invention. After an appropriate culture period, the expanded cell population can be analyzed, for example, for CTL activity.

  You may use this peptide as a reagent for evaluating the effectiveness of a vaccine. PBMC obtained from a patient vaccinated with an immunogen can be analyzed, for example, using any of the methods described above. The patient's HLA type is determined and a peptide epitope reagent that recognizes allele-specific molecules present in the patient is selected for analysis. The immunogenicity of the vaccine can be demonstrated by the presence of epitope-specific CTL in the PBMC sample. The peptides of the present invention can also be used to generate antibodies using techniques well known in the art (eg, CURRENT PROTOCOLS IN IMMUNOLOGY, Wiley / Greene, NY; and Antibodies A Laboratory Manual, Harlow and Lane). , Cold Spring Harbor Laboratory Press, 1989), this antibody can be used as a reagent for diagnosing, detecting, or monitoring cancer. Such antibodies can include antibodies that recognize peptides in the context of HLA molecules, ie, antibodies that bind to peptide-MHC complexes.

  The peptides and compositions of the present invention have several additional uses, some of which are described herein. For example, the present invention provides a method for diagnosing or detecting a disorder characterized by expression of a CLUAP1 immunogenic polypeptide. Such a method comprises measuring the expression of a CLUAP1 HLA binding peptide or a complex of a CLUAP1 HLA binding peptide and an HLA class I molecule in a biological sample. Expression of a peptide or complex of a peptide and an HLA class I molecule can be measured or detected by assaying with the peptide or the binding partner of the complex. In a preferred embodiment, the binding partner of a peptide or complex may be an antibody that recognizes and specifically binds to the peptide. Expression of CLUAP1 in biological samples such as tumor biopsies can also be tested by standard PCR amplification protocols using CLUAP1 primers. Examples of tumor expression are presented herein and further disclosure of exemplary conditions and primers for CLUAP1 amplification can be found in WO2003 / 27322.

  A preferred diagnostic method comprises contacting a biological sample isolated from a subject with a substance specific for a CLUAP1 HLA binding peptide to detect the presence of the CLUAP1 HLA binding peptide in the biological sample. As used herein, “contacting” refers to a biological sample, for example at a concentration, so that a specific interaction between the agent and the CLUAP1 HLA-binding peptide present in the biological sample is possible. Means to be placed in close proximity to the agent under appropriate conditions of temperature, time and ionic strength. In general, the conditions for contacting an agent with a biological sample include the molecule and its homologue in the biological sample (eg, protein and its receptor homologue, antibody and its protein antigen homologue, nucleic acid and its complement). Conditions known to those skilled in the art to promote specific interactions with the homologous sequence homologues). Exemplary conditions for promoting specific interactions between a molecule and its congeners are described in US Pat. No. 5,108,921 issued to Low et al.

  The diagnostic methods of the invention can be performed in one or both in vivo and in vitro. Thus, the biological sample can be located in vivo or in vitro in the present invention. For example, the biological sample can be an in vivo tissue and an agent specific for the CLUAP1 immunogenic polypeptide can be used to detect the presence of such molecules in the tissue. Alternatively, the biological sample can be collected or isolated in vitro (eg, blood sample, tumor biopsy, tissue extract). In particularly preferred embodiments, the biological sample may be a sample containing cells, more preferably a sample containing tumor cells taken from a subject to be diagnosed or treated.

  Alternatively, diagnosis can be made using methods that allow direct quantification of antigen-specific T cells by staining with a fluorescein-labeled HLA multimeric complex (eg, Altman, JD et al., 1996). Science 274: 94; Altman, JD et al., 1993, Proc. Natl. Acad. Sci. USA 90: 10330). Intracellular lymphokine staining and interferon-γ release assays, or ELISPOT assays are also provided. Multimeric staining, intracellular lymphokine staining, and ELISPOT assays all appear to be at least 10 times more sensitive than the more conventional assays (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 (eg, US 2004-209295A), dextramers (eg, WO 02/072631), and streptamers (eg, Nature medicine 6.631-637 (2002)) can also be used.

For example, in some embodiments, the present invention provides a method for diagnosing or assessing an immunological response in a subject that has been administered at least one of the CLUAP1 peptides of the present invention, the method comprising the steps of: Including:
(A) contacting the immunogen with immunocompetent cells under conditions suitable for induction of CTL specific for the immunogen;
(B) detecting or determining the induction level of CTL induced in step (a); and (c) correlating the subject's immunological response with the CTL induction level.

  In the present invention, the immunogen comprises (a) a CLUAP1 peptide selected from among the amino acid sequences of SEQ ID NOs: 2 to 23 and 25 to 43, a peptide having such an amino acid sequence, and such an amino acid sequence At least one of the peptides modified by one, two or more amino acid substitutions. On the other hand, conditions suitable for induction of immunogen-specific CTL are well known in the art. For example, immunocompetent cells may be cultured in vitro in the presence of immunogen to induce immunogen specific CTL. Any stimulating factor may be added to the cell culture for the purpose of inducing immunogen specific CTL. For example, IL-2 is a preferred stimulator for CTL induction.

In some embodiments, monitoring or assessing the immunological response of a subject treated with peptide cancer therapy can be performed before, during, and / or after treatment. Generally, during a cancer therapy protocol, the immunogenic peptide is administered repeatedly to the subject being treated. For example, the immunogenic peptide may be administered weekly for 3-10 weeks. Thus, the subject's immunological response can be evaluated or monitored during a cancer therapy protocol. Alternatively, the step of assessing or monitoring the immunological response to cancer therapy may be at the completion of the therapy protocol.
In accordance with the present invention, the induction of immunogen-specific CTL is enhanced compared to controls, such that the subject being assessed or diagnosed has responded immunologically to the administered immunogen. Indicated. Suitable controls for assessing an immunological response include, for example, a control peptide when the immunocompetent cells are not contacted with the peptide or have an amino acid sequence (eg, a random amino acid sequence) that is different from any CLUAP1 peptide The CTL induction level when in contact with is included.

  In one preferred embodiment, a subject's immunological response is assessed in a sequence specific manner by comparing the immunological response between each immunogen administered to the subject. In particular, even when a mixture of several types of CLUAP1 peptides is administered to a subject, the immunological response may vary depending on the peptide. In that case, by comparing the immunological response between each peptide, the peptide to which the subject exhibits a stronger response can be identified.

XI. Antibodies The present invention further provides antibodies that bind to the peptides of the present invention. Preferred antibodies specifically bind to peptides of the invention and do not bind (or bind weakly) to non-peptides of the invention. Alternatively, the antibody binds to a peptide of the invention and homologues thereof. Antibodies against the peptides of the invention can be used in cancer diagnostic and prognostic assays, and imaging methodologies. Similarly, such antibodies can be used in the treatment, diagnosis, and / or prognosis of other cancers as long as CLUAP1 is also expressed or overexpressed in cancer patients. Furthermore, antibodies expressed in cells (eg, single chain antibodies) can be used therapeutically in the treatment of cancers involving CLUAP1 expression, examples of such cancers include breast cancer, Examples include, but are not limited to, cervical cancer, colorectal cancer, esophageal cancer, gastric cancer, invasive gastric cancer, lymphoma, neuroblastoma, pancreatic cancer.

  The present invention also includes CLUAP1 protein (SEQ ID NO: 45), or SEQ ID NO: 3, 4, 5, 8, 9, 10, 14, 15, 16, 18, 23, 30, 33, 34, 35, Various immunological assays are provided for detecting and / or quantifying fragments thereof comprising a polypeptide having an amino acid sequence selected from among 36 and 38. Such assays can optionally include one or more anti-CLUAP1 antibodies that can recognize and bind to the CLUAP1 protein or fragment thereof. In the context of the present invention, an anti-CLUAP1 antibody that binds to a CLUAP1 polypeptide is preferably SEQ ID NOs: 3, 4, 5, 8, 9, 10, 14, 15, 16, 18, 23, 30, 33. , 34, 35, 36, and 38 are recognized. The binding specificity of the antibody can be confirmed by an inhibition test. That is, the binding between the analyzed antibody and the full-length CLUAP1 polypeptide is SEQ ID NO: 3, 4, 5, 8, 9, 10, 14, 15, 16, 18, 23, 30, 33, 34, When inhibited in the presence of any fragment polypeptide having an amino acid sequence selected from among 35, 36, and 38, this antibody is shown to specifically bind to the fragment. In the context of the present invention, such immunological assays include various types of radioimmunoassay, immunochromatographic techniques, enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunofluorescence assay (ELIFA), etc. Is performed within a variety of immunological assay formats well known in the art, including but not limited to.

  Immunological but non-antibody related assays of the invention can also include T cell immunogenicity assays (inhibitory or stimulatory) and MHC binding assays. In addition, the present invention contemplates immunological imaging methods capable of detecting cancers that express CLUAP1, examples of which include radioscintigraphic imaging methods that use the labeled antibodies of the present invention, including: It is not limited to. Such assays can be used clinically in the detection, monitoring, and prognosis of cancers that express CLUAP1, examples of such cancers include breast cancer, cervical cancer, colorectal cancer, Examples include, but are not limited to esophageal cancer, gastric cancer, invasive gastric cancer, lymphoma, neuroblastoma, pancreatic cancer.

The present invention also provides antibodies that bind to the peptides of the present invention. The antibody of the present invention can be used in any form, for example, as a monoclonal antibody or a polyclonal antibody, and further, an antiserum obtained by immunizing an animal such as a rabbit with the peptide of the present invention, all classes of polyclonal antibodies. It may include antibodies and monoclonal antibodies, human antibodies, and humanized antibodies produced by genetic recombination.
The peptide of the present invention used as an antigen for obtaining an antibody can be derived from any animal species, but is preferably derived from a mammal such as a human, mouse, or rat, more preferably a human. Human-derived peptides can be obtained from the nucleotide or amino acid sequences disclosed herein.

According to the present invention, a peptide used as an immunizing antigen may be a complete protein or a partial peptide of the protein. The partial peptide can comprise, for example, an amino (N) terminal fragment or a carboxy (C) terminal fragment of the peptide of the invention.
As used herein, an antibody is defined as a protein that reacts with either the full length or a fragment of the CLUAP1 peptide. In a preferred embodiment, the antibody of the invention is in SEQ ID NO: 3, 4, 5, 8, 9, 10, 14, 15, 16, 18, 23, 30, 33, 34, 35, 36, and 38. A fragment peptide of CLUAP1 having an amino acid sequence more selected can be recognized. Methods for synthesizing oligopeptides are well known in the art. After synthesis, the peptide may optionally be purified before use as an immunogen. In the context of the present invention, oligopeptides (eg 9 mer or 10 mer) may be bound or linked to a carrier in order to increase immunogenicity. As a carrier, keyhole limpet hemocyanin (KLH) is well known. Methods for conjugating KLH and peptides are also well known in the art.

  Alternatively, a gene encoding a peptide of the invention or a fragment thereof can be inserted into a known expression vector, which is then used to transform host cells as described herein. The desired peptide or fragment thereof can be recovered from the exterior or interior of the host cell by any standard method and can later be used as an antigen. Alternatively, whole cells expressing a peptide or a lysate thereof, or a chemically synthesized peptide may be used as an antigen.

  Any mammal can be immunized with the antigen, but preferably the compatibility with the parental cell used for cell fusion is taken into account. In general, Rodentia, Lagomorpha, or Primate animals can be used. Rodent animals include, for example, mice, rats, and hamsters. Rabbits include, for example, rabbits. Primate animals include, for example, monkeys of the Catarrini (Old World monkey) such as cynomolgus monkeys (Macaca fascicularis), rhesus monkeys, baboons, and chimpanzees.

  Methods for immunizing animals with antigens are known in the art. Intraperitoneal or subcutaneous injection of antigen is a standard method for immunizing mammals. More specifically, the antigen is diluted with an appropriate amount of phosphate buffered saline (PBS), physiological saline or the like and suspended. If desired, the antigen suspension can be mixed with an appropriate amount of a standard adjuvant, such as Freund's complete adjuvant, emulsified and administered to a mammal. Thereafter, the antigen mixed with an appropriate amount of Freund's incomplete adjuvant is preferably administered several times every 4 to 21 days. A suitable carrier may be used for immunization. After immunization as described above, serum can be examined by standard methods for increasing amounts of the desired antibody.

  Polyclonal antibodies against the peptides of the present invention can be prepared by recovering blood from the immunized mammal examined for an increase in the desired antibody in the serum and separating the serum from the blood by any conventional method. . Polyclonal antibodies may include serum containing polyclonal antibodies, and fractions containing polyclonal antibodies may be isolated from the serum. Immunoglobulin G or M can be obtained from a fraction that recognizes only the peptide of the present invention by using, for example, an affinity column to which the peptide of the present invention is bound, and further using this protein A or protein G column. It can be purified and prepared.

In order to prepare a monoclonal antibody, immune cells are collected from a mammal immunized with an antigen, confirmed for an increase in the level of a desired antibody in serum as described above, and subjected to cell fusion. The immune cells used for cell fusion can be preferably obtained from the spleen. Other preferred parent cells to be fused with the above immune cells include, for example, mammalian myeloma cells, and more preferably myeloma cells that have acquired properties for selection of fusion cells with drugs.
According to a known method, for example, the method of Milstein et al. (Galfre and Milstein, Methods Enzymol 73: 3-46 (1981)), the above immune cells and myeloma cells can be fused.

  The resulting hybridomas by cell fusion can be selected by culturing them in a standard selective medium such as HAT medium (medium containing hypoxanthine, aminopterin, and thymidine). Cell culture typically continues in HAT medium for days to weeks, a period sufficient for all other cells (non-fused cells) except the desired hybridoma to die. Thereafter, standard limiting dilution can be performed to screen and clone hybridoma cells producing the desired antibody.

  In addition to the above method of immunizing non-human animals with antigens to prepare hybridomas, human lymphocytes such as lymphocytes infected with EB virus can be transformed with peptides, cells expressing peptides, or lysates thereof. It is also possible to immunize in vitro. Subsequently, the lymphocyte after immunization is fused with an infinitely-dividable human-derived myeloma cell such as U266 to obtain a hybridoma that produces a desired human antibody capable of binding to the peptide (Japanese Patent Laid-Open No. Sho). 63-17688).

  Subsequently, the obtained hybridoma is transplanted into the abdominal cavity of the mouse, and ascites is extracted. The obtained monoclonal antibody can be purified by, for example, ammonium sulfate precipitation, protein A or protein G column, DEAE ion exchange chromatography, or an affinity column to which the peptide of the present invention is bound. The antibodies of the present invention can be used not only for purification and detection of the peptides of the present invention but also as candidates for agonists and antagonists of the peptides of the present invention.

Alternatively, immune cells that produce antibodies, such as immunized lymphocytes, can be immortalized by oncogenes and used to prepare monoclonal antibodies.
The monoclonal antibodies thus obtained can also be prepared recombinantly using genetic engineering techniques (see, eg, Borrebaeck and Larrick, Therapeutic Monoclonal Antibodies published in the UK by MacMillan Publishers LTD (1990)). Wanna) For example, DNA encoding an antibody is cloned from an immune cell such as an antibody-producing hybridoma or immunized lymphocyte, inserted into an appropriate vector, and then introduced into a host cell to prepare a recombinant antibody. Can do. The present invention also provides a recombinant antibody prepared as described above.

Furthermore, the antibody of the present invention may be an antibody fragment or a modified antibody as long as it binds to one or more of the peptides of the present invention. For example, an antibody fragment may be Fab, F (ab ′) ₂ , Fv, or a single chain Fv (scFv) in which Fv fragments from H and L chains are linked by a suitable linker (Huston et al. al., Proc Natl Acad Sci USA 85: 5879-83 (1988)). More specifically, an antibody fragment can be prepared by treating an antibody with an enzyme such as papain or pepsin. Alternatively, a gene encoding the antibody fragment can be constructed, inserted into an expression vector, and expressed in a suitable host cell (eg, 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); see Bird and Walker, Trends Biotechnol 9: 132-7 (1991)).
Antibodies can be modified by conjugation with various molecules such as polyethylene glycol (PEG). The present invention provides such modified antibodies. Modified antibodies can be obtained by chemically modifying antibodies. These modification methods are routine in the art.

  Alternatively, the antibody of the present invention is a chimeric antibody between a variable region derived from a non-human antibody and a constant region derived from a human antibody, or a complementarity determining region (CDR) derived from a non-human antibody and a human antibody. It can also be obtained as a humanized antibody comprising a framework region (FR) derived from and a constant region. Such antibodies can be prepared according to known techniques. Humanization can be performed by replacing the corresponding sequence of a human antibody with a rodent CDR or CDR sequence (see, eg, Verhoeyen et al., Science 239: 1534-1536 (1988)). . Accordingly, such humanized antibodies are chimeric antibodies in which substantially less than a human variable domain has been replaced by the corresponding sequence from a non-human species.

  In addition to human framework and constant regions, fully human antibodies that also contain human variable regions can be used. Such antibodies can be generated using various techniques known in the art. For example, in vitro methods include the use of recombinant libraries of human antibody fragments displayed on bacteriophages (eg, Hoogenboom & Winter, J. Mol. Biol. 227: 381 (1991)). Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, such as mice, in which the endogenous immunoglobulin genes are partially or completely inactivated. This approach is described, for example, in US Pat. Nos. 6,150,584, 5,545,807; 5,545,806; 5,569,825; 5,625,126; 633,425; 5,661,016.

  The antibody obtained as described above may be purified to homogeneity. For example, separation and purification of antibodies can be performed according to separation and purification methods used for general proteins. For example, but not limited to, the appropriate use of column chromatography such as affinity chromatography, filters, ultrafiltration, salting out, dialysis, SDS polyacrylamide gel electrophoresis, and isoelectric focusing By selection and combination, antibodies can be separated and isolated (Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory (1988)). Protein A columns and protein G columns can be used as affinity columns. Exemplary protein A columns to be used include, for example, Hyper D, POROS, and Sepharose F. F. (Pharmacia).

  Exemplary chromatography includes, in addition to affinity chromatography, for example, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase 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)). Chromatographic techniques can be performed by liquid phase chromatography such as HPLC and FPLC.

  For example, the antigen-binding activity of the antibody of the present invention is measured using absorbance measurement, enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunosorbent assay (EIA), radioimmunoassay (RIA), and / or immunofluorescence. Can be measured. In the case of ELISA, the antibody of the present invention is immobilized on a plate, the peptide of the present invention is added to the plate, and then a sample containing a desired antibody such as a culture supernatant of antibody-producing cells or a purified antibody is added. A secondary antibody that recognizes the primary antibody and is labeled with an enzyme such as alkaline phosphatase is then added and the plate is incubated. Subsequently, after washing, an enzyme substrate such as p-nitrophenyl phosphate is added to the plate, the absorbance is measured, and the antigen binding activity of the sample is evaluated. In order to evaluate the binding activity of an antibody, a peptide fragment such as a C-terminal or N-terminal fragment may be used as an antigen. BIAcore (Pharmacia) may be used to evaluate the activity of the antibody of the present invention.

By exposing the antibody of the present invention to a sample considered to contain the peptide of the present invention, and detecting or measuring an immune complex formed by the antibody and the peptide, the peptide of the present invention can be obtained by the above method. Can be detected or measured.
Since the method for detecting or measuring a peptide according to the present invention can specifically detect or measure a peptide, this method can be used in various experiments using the peptide.

XII. Vectors and host cells The present invention also provides vectors and host cells into which nucleotides encoding the peptides of the present invention have been introduced. The vectors of the invention can be used to retain the nucleotides of the invention, in particular DNA, in host cells, to express the peptides of the invention, or to administer the nucleotides of the invention for gene therapy.

  If E. coli is the host cell and the vector is amplified in large quantities in E. coli (eg, JM109, DH5α, HB101, or XL1Blue), the vector is “origin of replication” for amplification in E. coli. And a marker gene (for example, a drug resistance gene selected by a drug such as ampicillin, tetracycline, kanamycin, chloramphenicol) for selecting transformed E. coli. For example, M13 vectors, pUC vectors, pBR322, pBluescript, pCR-Script, and the like can be used. In addition, pGEM-T, pDIRECT, and pT7 can also be used for cDNA subcloning and extraction, similar to the vectors described above. When the vector is used for production of the protein of the present invention, an expression vector can be used.

  For example, an expression vector to be expressed in E. coli must have the above characteristics in order to amplify in E. coli. When E. coli such as JM109, DH5α, HB101, or XL1 Blue is used as the host cell, the vector can be a promoter that can efficiently express the desired gene in E. coli, such as the lacZ promoter (Ward et al., Nature 341: 544-6 (1989); FASEB J 6: 2422-7 (1989)), araB promoter (Better et al., Science 240: 1041-3 (1988)), T7 promoter and the like. In this regard, for example, pGEX-5X-1 (Pharmacia), “QIAexpress system” (Qiagen), pEGFP, and pET (where the host is preferably BL21 expressing T7 RNA polymerase) of the above vector. Can be used instead. Furthermore, the vector may contain a signal sequence for peptide secretion. An exemplary signal sequence that causes the peptide to be secreted into the periplasm of E. coli is the pelB signal sequence (Lei et al., J Bacteriol 169: 4379 (1987)). Means for introducing the vector into the target host cell include, for example, the calcium chloride method and the electroporation method.

  In addition to E. coli, for example, expression vectors derived from mammals (for example, pcDNA3 (Invitrogen) and pEGF-BOS (Nucleic Acids Res 18 (17): 5322 (1990)), pEF, pCDM8), expression from insect cells Vectors (eg, “Bac-to-BAC baculovirus expression system” (GIBCO BRL), pBacPAK8), plant-derived expression vectors (eg, pMH1, pMH2), animal virus-derived expression vectors (eg, pHSV, pMV, pAdexLcw) ), Retrovirus-derived expression vectors (eg, pZIpneo), yeast-derived expression vectors (eg, “Pichia expression kit” (Invitrogen), pNV11, SP-Q01), and Bacillus subtilis Expression vectors (e.g., pPL608, pKTH50) can be used in producing a polypeptide of the present invention.

In order for a vector to be expressed in animal cells such as CHO, COS, or NIH3T3 cells, the vector must be a promoter required for expression in such cells, such as the SV40 promoter (Mulligan et al., Nature 277: 108 (1979 )), MMLV-LTR promoter, EF1α promoter (Mizushima et al., Nucleic Acids Res 18: 5322 (1990)), CMV promoter and the like, and preferably a marker gene (eg drug (eg , Neomycin, G418). Examples of known vectors having these characteristics include, for example, pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13.
The following examples are presented to illustrate the present invention and to assist one of ordinary skill in making and using the present invention. This example is not intended in any way to limit the scope of the invention.

Materials and methods
TISI, a cell line HLA-A ^* 2402-positive B lymphoblastoid cell line, was purchased from IHWG Cell and Gene Bank (Seattle, WA). HLA-A ^* 0201 positive B lymphoblastoid cell line T2 and African green monkey kidney cell line COS7 were purchased from ATCC.

Candidate selection of CLUAP1-derived peptides 9-mer and 10-mer peptides derived from CLUAP1 that bind to HLA-A ^* 2402 or HLA-A ^* 0201 molecules are linked to binding prediction software “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)) and “NetMHC 3.0” (www. cbs.dtu.dk/services/NetMHC/) (Buus et al. (Tissue Antigens., 62: 378-84, 2003), Nielsen et al. (Protein Sci., 12: 1007-17, 2003, Bioinformatics, 20 (9): 1388-97, 2004)). These peptides were synthesized by Biosynthesis (Lewisville, Texas) by standard solid phase synthesis and purified by reverse phase high performance liquid chromatography (HPLC). The purity (> 90%) and identity of the peptides were determined by analytical HPLC and mass spectrometry, respectively. The peptide was dissolved in dimethyl sulfoxide (DMSO) at 20 mg / ml and stored at −80 ° C.

In vitro, CTL-induced monocyte-derived dendritic cells (DC) were used as antigen presenting cells to induce cytotoxic T lymphocyte (CTL) responses to peptides presented on human leukocyte antigen (HLA). DCs were generated in vitro as described elsewhere (Nakahara S et al., Cancer Res 2003 Jul 15, 63 (14): 4112-8). Specifically, peripheral blood mononuclear cells (PBMC) isolated from healthy volunteers (HLA-A ^* 2402 or HLA-A0201 positive) with Ficoll-Plaque (Pharmacia) solution were transformed into plastic tissue culture dishes (Becton Dickinson) and separated as monocyte fractions. 1,000 U / ml granulocyte-macrophage colony-stimulating factor (R & D System) and 1,000 U / ml interleukin in AIM-V medium (Invitrogen) containing 2% heat-inactivated autoserum (AS) The population enriched for monocytes was cultured in the presence of (IL) -4 (R & D System). After 7 days of culture, cytokine-induced DCs were pulsed with 20 μg / ml of each synthetic peptide in AIM-V medium at 37 ° C. for 3 hours in the presence of 3 μg / ml β2-microglobulin.

The generated cells appeared to express DC related molecules such as CD80, CD83, CD86, and HLA class II on their cell surface (data not shown). These peptide-pulsed DCs were then inactivated by X-ray irradiation (20 Gy) and mixed in a 1:20 ratio with autologous CD8 + T cells obtained by positive selection using CD8 Positive Isolation Kit (Dynal). These cultures were prepared in 48-well plates (Corning), each well containing 1.5 × 10 ⁴ peptide-pulsed DC, 3 × in 0.5 ml AIM-V / 2% AS medium. 10 ⁵ CD8 + T cells and 10 ng / ml IL-7 (R & D System) were included. After 3 days, IL-2 (CHIRON) was added to these cultures to a final concentration of 20 IU / ml. On days 7 and 14, T cells were further stimulated with peptide-pulsed autologous DCs.

  DCs were prepared each time by the same method as above. On day 21, CTL was tested on peptide-pulsed TISI cells (A24) or T2 cells (A2) after the third peptide stimulation (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).

CTL proliferation procedure 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) CTLs were grown in culture using methods similar to those described. In the presence of 40 ng / ml anti-CD3 monoclonal antibody (Pharmingen), a total of 5 × 10 ⁴ CTLs with 25 ml AIM-V / Suspended in 5% AS medium. One day after the start of culture, 120 IU / ml IL-2 was added to the culture. On days 5, 8, and 11, fresh AIM-V / 5% AS medium containing 30 IU / ml IL-2 was fed to the culture (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).

Establishing CTL clones Dilutions were performed in 96 round-bottom microtiter plates (Nalge Nunc International) at CTL 0.3, 1, and 3 / well. CTL was combined with 2 human B lymphoblastoid cell lines at 1 × 10 ⁴ cells / well, 30 ng / ml anti-CD3 antibody, and 125 U / ml IL-2 for a total of 150 μl / well of 5% AS. The cells were cultured in a containing AIM-V medium. Ten days later, 50 μl / well of IL-2 was added to the medium so that the final concentration of IL-2 reached 125 U / ml. CTL activity was tested on day 14 and CTL clones were propagated 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 CTL activity To investigate specific CTL activity, an interferon (IFN) -γ enzyme-linked immunospot (ELISPOT) assay and an IFN-γ enzyme-linked immunosorbent assay (ELISA) were performed. Specifically, peptide-pulsed TISI (A24) or T2 (A2) (1 × 10 ⁴ cells / well) was prepared as stimulating cells. Cultured cells in 48 wells were used as responder cells. IFN-γ ELISPOT assay and IFN-γ ELISA assay were performed according to the manufacturer's procedure.

A cDNA encoding the target gene and an HLA-A24 or HLA-A2 open reading frame was established by PCR. The cells were found to forcibly express either or both of HLA-A24 and HLA-A2. The PCR amplification product was cloned into a vector. The plasmid was transfected into the target gene and COS7, an HLA-A24 and HLA-A2-null cell line, using Lipofectamine 2000 (Invitrogen) according to the manufacturer's recommended procedure. Two days after transfection, the transfected cells were harvested using Versen (Invitrogen) and used as target cells (5 × 10 ⁴ cells / well) for CTL activity assay.

Result 1
Enhanced expression of CLUAP1 in cancer Extensive gene expression profile data obtained from various cancers using cDNA microarrays increases expression of CLUAP1 (GenBank accession number NM_0155041 or NM_024793; eg, SEQ ID NO: 44) It became clear that CLUAP1 expression was found in 17 of 77 breast cancers, 15 of 19 cervical cancers, 12 of 19 colorectal cancers, 12 of 19 esophageal cancers, and 25 of gastric cancers. 7 cases, 5 cases of 5 invasive gastric cancers, 5 cases of 18 lymphomas, 2 cases of 16 neuroblastomas, 4 cases of 9 cases of pancreatic cancer, corresponding normal tissues Certainly increased compared to (Table 1).

Table 1. Ratio of cases in which up-regulation of CLUAP1 was observed in cancerous tissues compared to corresponding normal tissues

Result 2
CLUAP1-derived HLA-A24 binding peptide prediction tables 2a and 2b show the 9 and 10 mer peptides of CLUAP1 binding to HLA-A24 in descending order of binding affinity. SEQ ID NO: 1-11 and SEQ ID NO: 15-22 peptides were predicted by BIMAS, and SEQ ID NO: 12-14 peptides were predicted by NetMHC 3.0. To determine the epitope peptides, a total of 22 peptides potentially having HLA-A24 binding ability were selected and examined.

Table 2a HLA-A24 binding 9mer peptide derived from CLUAP1

開始位置は、ＣＬＵＡＰ１のＮ末端からのアミノ酸残基の番号を示す。
結合スコアおよび解離定数［Ｋｄ（ｎＭ）］は「ＢＩＭＡＳ」および「ＮｅｔＭＨＣ３．０」に由来する。 Table 2b: HLA-A24 binding 10mer peptide derived from CLUAP1

The start position indicates the amino acid residue number from the N-terminus of CLUAP1.
The binding score and dissociation constant [Kd (nM)] are derived from “BIMAS” and “NetMHC3.0”.

Induction of CTLs by HLA-A ^* 2402-restricted CLUAP1-derived predicted peptides CTLs for CLUAP1-derived peptides were generated according to the protocol described in "Materials and Methods". Peptide-specific CTL activity was measured by IFN-γ ELISPOT assay (FIGS. 1a-j). The following well numbers showed strong IFN-γ production compared to control wells: well number # 6 (a), CLUAP1-A24 stimulated with CLUAP1-A24-9-255 (SEQ ID NO: 3). # 2 (b) stimulated with -9-283 (SEQ ID NO: 4), # 2 (c) stimulated with CLUAP1-A24-9-31 (SEQ ID NO: 5), CLUAP1-A24-9-168 # 4 (d) stimulated with (SEQ ID NO: 8), # 4 (e) stimulated with CLUAP1-A24-9-64 (SEQ ID NO: 9), CLUAP1-A24-9-216 (SEQ ID NO # 1 (f) stimulated with 10), # 6 (g) stimulated with CLUAP1-A24-9-154 (SEQ ID NO: 14), CLUAP1-A24-10-91 (SEQ ID O: Stimulated with 15) # 2 (h), CLUAP1-A24-10-104 (SEQ ID NO: 16) stimulated # 4 (i), and CLUAP1-A24-10-125 (SEQ ID NO: 18) ) Stimulated with # 5 (j). On the other hand, although other peptides shown in Tables 2a and 2b may have binding activity with HLA-A ^* 2402, specific CTL activity is not measured by stimulation with these peptides. It was. As a typical example of negative data, no specific IFN-γ production was observed from CTL stimulated with CLUAP1-A24-9-258 (SEQ ID NO: 1) (FIG. 1k). As a result, it was shown that 10 kinds of peptides derived from CLUAP1 were screened as peptides capable of inducing strong CTL.

Establishment of CTL lines and clones for CLUAP1-derived peptides As described in the “Materials and Methods” section above, well number # 6 (a) using CLUAP1-A24-9-255 (SEQ ID NO: 3), # 4 (b) using CLUAP1-A24-9-168 (SEQ ID NO: 8), # 4 (c) using CLUAP1-A24-9-64 (SEQ ID NO: 9), CLUAP1-A24- # 6 (d) using 9-154 (SEQ ID NO: 14) and # 2 (e) using CLUAP1-A24-10-91 (SEQ ID NO: 15) by IFN-γ ELISPOT assay Cells that showed detected peptide-specific CTL activity were grown and CTL lines were established by limiting dilution. The CTL activity of these CTL lines was measured by IFN-γ ELISA assay (FIGS. 2a-e). All CTL lines showed strong IFN-γ production against target cells pulsed with the corresponding peptide compared to target cells that were not pulsed with the peptide. In addition, CTL clones were established by limiting dilution from CTL lines as described in “Materials and Methods”, and IFN-γ production from CTL clones against peptide-pulsed target cells was measured by IFN-γ ELISA assay. In FIG. 3, CLUAP1-A24-9-255 (SEQ ID NO: 3), CLUAP1-A24-9-64 (SEQ ID NO: 9), and CLUAP1-A24-10-91 (SEQ ID NO: 15) Strong IFN-γ production was measured from stimulated CTL clones.

Specific CTL activity against target cells that exogenously express CLUAP1 and HLA-A ^* 2402 Established CTL lines produced against these peptides are targeted cells that endogenously express CLUAP1 and HLA-A ^* 2402 molecules We investigated the ability to recognize. Specific CTL activity against COS7 cells (specific model of target cells exogenously expressing CLUAP1 and HLA-A ^* 2402 genes) transfected with both full-length CLUAP1 and HLA-A ^* 2402 molecule genes, corresponding peptides CTL strains produced by COS7 cells transfected with either the full-length CLUAP1 gene or HLA-A ^* 2402 were prepared as controls. In FIG. 4, CTL stimulated with CLUAP1-A24-9-255 (SEQ ID NO: 3) showed potent CTL activity against COS7 cells expressing both CLUAP1 and HLA-A ^* 2402. On the other hand, no significant specific CTL activity was detected relative to the control. Thus, these data clearly demonstrated that CLUAP1-A24-9-255 (SEQ ID NO: 3) is naturally expressed on target cells with HLA-A ^* 2402 molecules and is recognized by CTL. . These results indicate that this peptide derived from CLUAP1 may be suitable as a cancer vaccine for patients with tumors that express CLUAP1.

Homology analysis of antigen peptides CLUAP1-A24-9-255 (SEQ ID NO: 3), CLUAP1-A24-9-283 (SEQ ID NO: 4), CLUAP1-A24-9-31 (SEQ ID NO: 5) CLUAP1-A24-9-168 (SEQ ID NO: 8), CLUAP1-A24-9-64 (SEQ ID NO: 9), CLUAP1-A24-9-216 (SEQ ID NO: 10), CLUAP1-A24- 9-154 (SEQ ID NO: 14), CLUAP1-A24-10-91 (SEQ ID NO: 15), CLUAP1-A24-10-104 (SEQ ID NO: 16), and CLUAP1-A24-10-125 ( CTL stimulated with SEQ ID NO: 18) showed significant and specific CTL activity As a result, CLUAP1-A24-9-255 (SEQ ID NO: 3), CLUAP1-A24-9-283 (SEQ ID NO: 4), CLUAP1-A24-9-31 (SEQ ID NO: 5), CLUAP1 -A24-9-168 (SEQ ID NO: 8), CLUAP1-A24-9-64 (SEQ ID NO: 9), CLUAP1-A24-9-216 (SEQ ID NO: 10), CLUAP1-A24-9- 154 (SEQ ID NO: 14), CLUAP1-A24-10-91 (SEQ ID NO: 15), CLUAP1-A24-10-104 (SEQ ID NO: 16), and CLUAP1-A24-10-125 (SEQ ID NO: 14) The sequence of NO: 18) is derived from other molecules known to sensitize the human immune system There is a possibility that due to the fact that the peptide and is homologous. To eliminate this possibility, homology analysis was performed on these peptide sequences as queries using the BLAST algorithm (www.ncbi.nlm.nih.gov/blast/blast.cgi) No sequences with significant homology were found. As a result of this homology analysis, CLUAP1-A24-9-255 (SEQ ID NO: 3), CLUAP1-A24-9-283 (SEQ ID NO: 4), CLUAP1-A24-9-31 (SEQ ID NO: 5), CLUAP1-A24-9-168 (SEQ ID NO: 8), CLUAP1-A24-9-64 (SEQ ID NO: 9), CLUAP1-A24-9-216 (SEQ ID NO: 10), CLUAP1- A24-9-154 (SEQ ID NO: 14), CLUAP1-A24-10-91 (SEQ ID NO: 15), CLUAP1-A24-10-104 (SEQ ID NO: 16), and CLUAP1-A24-10- The sequence of 125 (SEQ ID NO: 18) is unique, and therefore the present invention In the knowledge of al, these molecules have shown that there is little possibility of causing unintended immunologic response to unrelated molecules.
In conclusion, a novel HLA-A24 epitope peptide derived from CLUAP1 has been identified. Furthermore, the results herein demonstrate that CLUAP1 epitope peptides may be suitable for use in cancer immunotherapy.

Result 3
CLUAP1-derived HLA-A02 binding peptide prediction tables 3a and 3b show the 9 and 10 mer peptides of CLUAP1 binding to HLA-A02 in descending order of binding affinity. In order to determine the epitope peptides, a total of 21 peptides that could have HLA-A02 binding ability were selected and examined.

Table 3a: HLA-A02 binding 9mer peptide derived from CLUAP1

開始位置は、ＣＬＵＡＰ１のＮ末端からのアミノ酸残基の番号を示す。
結合スコアは「ＢＩＭＡＳ」に由来する。 Table 3b: HLA-A02 binding 10mer peptide derived from CLUAP1

The start position indicates the amino acid residue number from the N-terminus of CLUAP1.
The binding score is derived from “BIMAS”.

Induction of CTLs by predicted peptides derived from HLA-A ^* 0201 restricted CLUAP1 CTLs against CLUAP1-derived peptides were generated according to the protocol described in "Materials and Methods". Peptide-specific CTL activity was measured by IFN-γ ELISPOT assay (FIGS. 5a-g). The following well numbers showed strong IFN-γ production compared to control wells: well number # 3 (a), CLUAP1-A02 with CLUAP1-A02-9-34 (SEQ ID NO: 23) # 6 (b) using -9-99 (SEQ ID NO: 30), # 2 (c) using CLUAP1-A02-10-153 (SEQ ID NO: 33), CLUAP1-A02-10-85 # 3 (d) using (SEQ ID NO: 34), # 2 (e) using CLUAP1-A02-10-34 (SEQ ID NO: 35), CLUAP1-A02-10-33 (SEQ ID NO # 2 (f) using 36), and # 5 (g) using CLUAP1-A02-10-80 (SEQ ID NO: 38). On the other hand, other peptides shown in Tables 3a and 3b measure specific CTL activity when stimulated with these peptides, even though they may have binding activity with HLA-A ^* 0201. I couldn't. As a typical example of negative data, specific IFN-γ production was not observed from CTL stimulated with CLUAP1-A02-9-58 (SEQ ID NO: 24) (h). As a result, it was shown that 7 types of peptides derived from CLUAP1 were screened as peptides capable of inducing strong CTLs.

Establishment of CTL lines and clones for CLUAP1-derived peptides Well number # 3 (a), CLUAP1 using CLUAP1-A02-9-34 (SEQ ID NO: 23) as described in the “Materials and Methods” section above. # 6 (b) using -A02-9-99 (SEQ ID NO: 30), # 2 (c) using CLUAP1-A02-10-153 (SEQ ID NO: 33), CLUAP1-A02-10 # 3 (d) using -85 (SEQ ID NO: 34), # 2 (e) using CLUAP1-A02-10-34 (SEQ ID NO: 35), and CLUAP1-A02-10-33 ( SEQ ID NO: 36) shows peptide specific CTL activity detected by IFN-γ ELISPOT assay in # 2 (f) Cells were grown and established CTL lines by limiting dilution. CTL activity of these CTL lines was measured by IFN-γ ELISA assay (FIGS. 6a-f). These CTL lines showed strong IFN-γ production against target cells pulsed with the corresponding peptide compared to target cells that were not pulsed with the peptide. In addition, CTL clones were established by limiting dilution from CTL lines as described in “Materials and Methods” and IFN-γ production from CTL clones against peptide-pulsed target cells was measured by IFN-γ ELISA assay. CLUAP1-A02-9-34 (SEQ ID NO: 23) (a), CLUAP1-A02-9-99 (SEQ ID NO: 30) (b), CLUAP1-A02-10-153 (SEQ ID NO: 33) Strong IFN-γ production was measured from CTL clones stimulated with (c), and CLUAP1-A02-10-85 (SEQ ID NO: 34) (d) (FIGS. 7a-d).

Specific CTL activity against target cells that exogenously express CLUAP1 and HLA-A ^* 0201 Established CTL lines and clones produced for each peptide were used to target CLUAP1 and HLA-A ^* 0201 molecules endogenously. The ability to recognize cells was examined. Specific CTL activity against COS7 cells transfected with both full-length CLUAP1 and HLA-A ^* 0201 genes (a specific model of target cells exogenously expressing CLUAP1 and HLA-A ^* 0201 genes) is determined by the corresponding peptides. CTL strains and clones produced were tested. COS7 cells transfected with either full length CLUAP1 or HLA-A ^* 0201 were prepared as controls.

In FIG. 8, CLUAP1-A02-9-99 (SEQ ID NO: 30) (a), CLUAP1-A02-10-153 (SEQ ID NO: 33) (b), CLUAP1-A02-10-85 (SEQ ID CTL lines stimulated with NO: 34) (c), and CLUAP1-A02-10-33 (SEQ ID NO: 36) (d) were directed against COS7 cells expressing both CLUAP1 and HLA-A ^* 0201. It showed strong CTL activity. On the other hand, no significant specific CTL activity was detected relative to the control. Therefore, according to these data, CLUAP1-A02-9-99 (SEQ ID NO: 30) (a), CLUAP1-A02-10-153 (SEQ ID NO: 33) (b), CLUAP1-A02-10-85 (SEQ ID NO: 34) (c), and CLUAP1-A02-10-33 (SEQ ID NO: 36) (d) peptides are endogenously processed and expressed on target cells with HLA-A ^* 0201 molecules And was clearly demonstrated to be recognized by CTL. These results indicated that these peptides from CLUAP1 may be available for application to cancer vaccines for patients with tumors that express CLUAP1.

Homology analysis of antigen peptides CLUAP1-A02-9-34 (SEQ ID NO: 23), CLUAP1-A02-9-99 (SEQ ID NO: 30), CLUAP1-A02-10-153 (SEQ ID NO: 33) CLUAP1-A02-10-85 (SEQ ID NO: 34), CLUAP1-A02-10-34 (SEQ ID NO: 35), CLUAP1-A02-10-33 (SEQ ID NO: 36), and CLUAP1-A02 CTL stimulated with -10-80 (SEQ ID NO: 38) showed significant and specific CTL activity. As a result, CLUAP1-A02-9-34 (SEQ ID NO: 23), CLUAP1-A02-9-99 (SEQ ID NO: 30), CLUAP1-A02-10-153 (SEQ ID NO: 33), CLUAP1 -A02-10-85 (SEQ ID NO: 34), CLUAP1-A02-10-34 (SEQ ID NO: 35), CLUAP1-A02-10-33 (SEQ ID NO: 36), and CLUAP1-A02-10 It may be due to the fact that the sequence of -80 (SEQ ID NO: 38) is homologous to peptides derived from other molecules known to sensitize the human immune system.

  To eliminate this possibility, homology analysis was performed on this peptide sequence as a query using the BLAST algorithm (http://www.ncbi.nlm.nih.gov/blast/blast.cgi). However, no sequences with significant homology were found. As a result of this homology analysis, CLUAP1-A02-9-34 (SEQ ID NO: 23), CLUAP1-A02-9-99 (SEQ ID NO: 30), CLUAP1-A02-10-153 (SEQ ID NO: 33), CLUAP1-A02-10-85 (SEQ ID NO: 34), CLUAP1-A02-10-34 (SEQ ID NO: 35), CLUAP1-A02-10-33 (SEQ ID NO: 36), and CLUAP1 -The sequence of A02-10-80 (SEQ ID NO: 38) is unique and, therefore, to the best of our knowledge, these molecules are unintended immunological to certain unrelated molecules. It indicates that there is almost no possibility of causing a response.

In conclusion, we have identified a novel HLA-A ^* 0201 epitope peptide derived from CLUAP1. Furthermore, it was demonstrated that the epitope peptide of CLUAP1 may be applicable to cancer immunotherapy.

INDUSTRIAL APPLICABILITY The present invention provides novel TAAs, particularly novel TAAs derived from CLUAP1 that can induce potent and specific anti-tumor immune responses and have applicability to a wide range of cancer types. . Such TAAs are peptide vaccines for diseases related to CLUAP1, such as cancer, more specifically breast cancer, cervical cancer, colorectal cancer, esophageal cancer, gastric cancer, invasive gastric cancer, lymphoma, neuroblastoma, pancreatic cancer As useful.

  Although the invention has been described in detail herein with reference to specific embodiments thereof, the foregoing description is exemplary and explanatory in nature and is intended to describe the invention and preferred embodiments thereof. It should be understood that it is intended. Through routine experimentation, one skilled in the art will readily recognize that various changes and modifications can be made therein without departing from the spirit and scope of the invention, the boundaries of which are Specified by the scope of

Claims (23)

  An isolated peptide having the ability to induce CTL, comprising the amino acid sequence of CLUAP1 or an immunologically active fragment thereof.   The isolated peptide of claim 1, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2-23 and 25-43.   3. An isolated peptide according to any one of claims 1 and 2, wherein one, two or several amino acids are substituted, deleted or added. 4. The isolated peptide of claim 3 having one or both of the following characteristics in the context of HLA-A24:
(A) the second amino acid from the N-terminus is modified to be an amino acid selected from the group consisting of phenylalanine, tyrosine, methionine, or tryptophan; and (b) the C-terminal amino acid is phenylalanine, leucine, An amino acid selected from the group consisting of isoleucine, tryptophan, or methionine, or modified to be the amino acid. 4. The isolated peptide of claim 3 having one or both of the following characteristics in the context of HLA-A2:
(A) the second amino acid from the N-terminus is selected from the group consisting of leucine and methionine; and (b) the C-terminal amino acid is selected from the group consisting of valine and leucine.   The isolated peptide according to any one of claims 1 to 5, which is a nonapeptide or a decapeptide.   An isolated polynucleotide encoding the peptide of any one of claims 1-6.   A composition for inducing CTL, comprising one or more peptides according to any one of claims 1 to 6, or one or more polynucleotides according to claim 7. object.   A pharmaceutical composition for the treatment and / or prevention of cancer and / or prevention of its recurrence after surgery, comprising one or more peptides according to any one of claims 1 to 6, Or a pharmaceutical composition comprising one or more polynucleotides according to claim 7.   10. The pharmaceutical composition of claim 9, formulated for administration to a subject whose HLA antigen is HLA-A24 or HLA-A2.   The pharmaceutical composition according to claim 9 or 10, which is formulated for the treatment of cancer. A method for inducing antigen-presenting cells (APCs) capable of inducing CTL, comprising a step selected from the group consisting of:
(A) contacting APC with the peptide of any one of claims 1-6 in vitro, ex vivo, or in vivo; and (b) encoding the peptide of any one of claims 1-6. Introducing a polynucleotide to APC. A method for inducing CTLs by a method comprising a step selected from the group consisting of:
(A) co-culturing CD8 positive T cells with APC presenting a complex of HLA antigen and the peptide of any one of claims 1 to 6 on its surface;
(B) co-culturing CD8 positive T cells with exosomes presenting on their surface a complex of HLA antigen and the peptide of any one of claims 1-6; and (c) A step of introducing a gene comprising a polynucleotide encoding a T cell receptor (TCR) subunit polypeptide that binds to the peptide according to any one of 1 to 6 into a T cell.   An isolated APC presenting on its surface a complex of an HLA antigen and the peptide of any one of claims 1-6.   15. APC according to claim 14, introduced by the method according to claim 12.   An isolated CTL that targets the peptide of any one of claims 1-6.   The CTL of claim 16, wherein the CTL is derived by the method of claim 13.   A method for inducing an immune response against cancer in a subject comprising the peptide according to any one of claims 1 to 6, an immunologically active fragment thereof, or a polynucleotide encoding the peptide or the fragment. Administering a product to the subject.   An antibody or a fragment thereof against any of the peptides according to any one of claims 1 to 6.   A vector comprising a nucleotide sequence encoding the peptide of any one of claims 1-6.   A host cell transformed or transfected with the expression vector of claim 20.   A diagnostic kit comprising the peptide according to any one of claims 1 to 6, the nucleotide according to claim 7, or the antibody according to claim 19.   SEQ ID NO: 3, 4, 5, 8, 9, 10, 14, 15, 16, 18, 23, 30, 33, 34, 35, 36, and 38, selected from the group 7. The isolated peptide according to any one of 6.

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