KR20130043632A - Cluap1 peptides and vaccines including the same - Google Patents

Abstract

Described herein are peptide vaccines for cancer. Specifically, an epitope peptide derived from the CLUAP1 gene that induces CTL is provided. Antigen-presenting cells targeting the peptides and isolated CTLs, methods of inducing antigen presenting cells, or CTLs are also provided. The present invention also provides a pharmaceutical composition comprising a peptide derived from CLUAP1 as an active ingredient or a polynucleotide encoding a peptide as an active ingredient.
The present invention also provides a CTL induction method, anti-tumor immunity induction, a peptide derived from CLUAP1, a polynucleotide encoding the peptide, and an antigen presenting cell presenting the peptide, and a pharmaceutical. Provided are methods for the treatment and / or prophylaxis (eg, prevention) of cancer (tumor) with the composition, and / or the prevention of recurrence after surgery thereof.

Description

CLUAPAP1 peptide and vaccine containing same {CLUAP1 PEPTIDES AND VACCINES INCLUDING THE SAME}

The present invention relates to the field of biosciences and more specifically to the field of cancer treatment. In particular, the present invention relates to cancer vaccines and novel peptides that are very effective as drugs for the treatment and prevention of tumors.

preference

This application claims priority to US Provisional Application No. 61 / 322.099, filed April 8, 2010, the entire contents of which are incorporated herein by reference.

CD8 positive CTLs are epitope peptides derived from tumor-associated antigens (TAAs) found in major histocompatibility complex (MHC) class I molecules. It is known to kill tumor cells after being recognized. Since the discovery of the melanoma antigen (MAGF) family as the first example of TAA, many other TAAs have been discovered through immunological approaches [Boon T, Int J Cancer 1993 May 8, 54 (2): 177-80 ( Non Patent Literature 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 clinical development as targets for immunotherapy.

The identification of new TAAs that induce potent and specific anti-tumor immune responses has led to the development of peptide vaccine strategies for various types of cancers, leading to clinical trials [Harris CC, J Natl Cancer Inst 1996 Oct 16, 88 ( 20): 1442-55 (non-patent document 3); Butlerfield 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, some clinical trials using TAA derived peptides have been reported. Unfortunately, many of the recently attempted cancer vaccines showed only really low 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 remains a need for new TAAs as targets for immunotherapy.

To that end, CLUAP1 was analyzed through analysis of the expression profiles of colorectal cancer with a genome-wide cDNA microarray containing 23.040 genes. (Gen Bank Accession No: NM_015041 or NM_024793), clusterster-associated protein 1 has been identified as a gene that is frequently activated in colon cancer [Takahashi M et al., Oncogene, 2004 Dec. 9; 23 (57): 9289-94 (Non-Patent Document 14)]. Expression of CLUAP1 was observed to gradually increase in late S to G2 / M phase of the cell cycle and to return to basal level on G0 / G1. In addition, suppression of CLUAP1 expression by siRNA resulted in growth retardation of 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 of immunotherapy in many 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  Butlerfield 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 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

SUMMARY OF THE INVENTION [

The present invention is based, at least in part, on the discovery of novel peptides that can be used as targets for immunotherapy. Since a tumor-associated antigen (TAA) is generally recognized as "self" by the immune system and often does not have innate immunogenicity, the discovery of suitable targets is very important. As noted above, CLUAP1 [SEQ ID NO: 45 encoded by the gene of Gen Bank Accession No. NM_015041 or NM_024793 (eg, SEQ ID NO: 44) are breast cancer, cervical cancer, colon-colorectal cancer, esophageal cancer, gastric cancer, broad- It has been shown to be up-regulated in cancers including, but not limited to, gastric diffuse-type cancer, lymphoma, neuroblastoma and pancreatic cancer. Thus, the present invention has focused on CLUAP1 as a candidate target for cancer / tumor immunotherapy.

The invention also relates to the identification of specific epitope peptides of the CLUAP1 gene product with the ability to induce CTLs specific for CLUAP1. As described in detail below, peripheral blood mononuclear cells (PBMCs) obtained from healthy donors may be prepared using candidate peptides that bind to HLA-A * 2402 or HLA-A * 0201 derived from CLUAP1. Stimulated. Each candidate peptide was then pulsed with to establish CTL cell lines with specific cytotoxicity against HLA-A24 or HLA-A2 positive target cells. The results demonstrate that the peptide is an epitope peptide limited to HLA-A24 or HLA-A2 capable of inducing a strong and specific immune response against cells expressing CLUAP1. In addition, the results show that CLUAP1 induces a strong immune response and its epitope is an effective target for cancer / tumor immunotherapy.

It is therefore an object of the present invention to provide an isolated peptide or immunologically active fragment thereof which binds to the HLA antigen, in particular comprising CLUAP1 (SEQ ID NO: 45). These peptides have CTL inducibility, so they can be used to induce CTL ex vivo or can be used for breast cancer, cervical cancer, colon-rectal cancer, esophageal cancer, gastric cancer, broad-gastric cancer, lymph cancer, nerves. It may be administered to a subject to induce an immune response against cancers such as cell and pancreatic cancer. Preferred peptides are nonapeptides or decapeptides and more preferred are those having an amino acid sequence selected from SEQ ID NOs: 2 to 23 and 25 to 43. Among them, peptides having an amino acid sequence selected from SEQ ID NOs: 3, 4, 5, 8, 9, 10, 14, 15, 16, 18, 23, 30, 33, 34, 35, 36 and 38 induce particularly strong CTL Sex and so are particularly preferred.

Peptides of the invention are modified peptides in which one, two or more amino acids are substituted, deleted or added as long as the modified peptide maintains its original CTL inducibility. It includes.

The present invention also provides an isolated polynucleotide encoding any peptide of the present invention. These polynucleotides can be used for the induction or preparation of CTL inducible antigen-presenting cells (APCs) or can be administered to a subject to induce an immune response against cancer, such as a peptide of the invention. have.

When administered to a subject, the peptides of the invention are preferably presented on the surface of the APC to induce CTLs targeting each peptide. Therefore, one object of the present invention is to provide a CTL derived composition or formulation, such as one or more peptides of the present invention or a composition comprising a polynucleotide encoding such a peptide. The present invention provides a pharmaceutical composition comprising one or more peptides of the present invention or a polynucleotide encoding such a peptide, such as a composition designed to treat and / or prophylaxis cancer and prevent cancer recurrence after surgery ( Pharmaceutical compositions are also contemplated, and examples of such cancers include, but are not limited to, breast cancer, cervical cancer, colon-rectal cancer, esophageal cancer, gastric cancer, broad-gastric cancer, lymph cancer, neuronal cancer and pancreatic cancer.

Pharmaceutical compositions or formulations of the present invention may include APCs or exosomes presenting any peptide of the present invention instead of / in addition to the peptide or polynucleotide of the present invention as an active ingredient.

Peptides and polynucleotides of the invention can be used to express APCs presenting HLA antigens and peptide complexes of the invention on the surface, for example, by contacting an APC derived from an individual or introducing a polynucleotide encoding a peptide of the invention into the APC. Can be induced. Such APCs have high CTL inducibility for target peptides and are therefore useful for cancer immunotherapy. Accordingly, the present invention contemplates a method for inducing APC with CTL inducibility and an APC obtained by the method.

The present invention provides a method of co-culturing A8 or exosomes presenting one or more peptides of the present invention on CD8 positive cells and surfaces thereof, or T binding to the peptides of the present invention. It also provides a CTL induction method comprising introducing a gene comprising a polynucleotide encoding a T cell receptor (TCR) subunit polypeptide. CTLs obtained in this way are useful for the treatment and prevention of cancer, including but not limited to breast cancer, cervical cancer, colon-rectal cancer, esophageal cancer, gastric cancer, broad-gastric cancer, lymph cancer, neuronal cancer and pancreatic cancer. Do not. Therefore, the present invention includes the CTL obtained by the method of the present invention.

Another object of the present invention is to provide a method for inducing an immune response against cancer in a subject in need thereof, wherein the method comprises a CLUAP1 polypeptide or an immunologically active fragment thereof, a polynucleotide encoding a CLUAP1 polypeptide and a CLUAP1 polypeptide. Administering to the subject a composition or formulation comprising the exosomes or APCs present.

The applicability of the present invention may extend to any of a number of diseases related to or resulting from CLUAP1 overexpression, such as cancer, including cancer of the breast, cervical cancer, colon-rectal cancer, esophageal cancer, gastric cancer, broad spectrum- Gastric cancer, lymph cancer, neuronal cancer and pancreatic cancer are included, but are not limited to these.

More specifically, the present invention provides the following:

[1] an isolated peptide having CTL inducibility, characterized by a peptide consisting of the amino acid sequence of CLUAP1 or an immunologically active fragment thereof,

[2] The isolated peptide according to [1], characterized in that the peptide contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 23 and 25 to 43,

[3] The isolated peptide according to [1] or [2], wherein one, two or several amino acids are substituted, deleted or added.

[4] The isolated peptide according to [3], which is characterized by a peptide having one or both of the following characteristics with respect to HLA-A24:

(a) the second amino acid from the N-terminus is modified with an amino acid selected from the group consisting of phenylalanine, tyrosine, methionine or tryptophan;

(b) the C-terminus amino acid is an amino acid selected from the group consisting of phenylalanine, leucine, isoleucine, tryptophan or methionine, or modified with it.

[5] The isolated peptide according to [3], which has one or both of the following characteristics with respect to HLA-A2,

(a) the second amino acid from the N-terminus is selected from the group consisting of leucine and methionine;

(b) the C-terminal amino acid is selected from the group consisting of valine and leucine,

[6] The peptide according to any one of [1] to [5], wherein the peptide is a nonapeptide or a decapeptide,

[7] The isolated polynucleotide according to any one of [1] to [6], which encodes a peptide,

[8] The CTL induction composition according to any one of [1] to [6], wherein the composition is one or more peptides or a composition containing one or more polynucleotides according to [7].

[9] The treatment and / or treatment of cancer according to any one of [1] to [6], wherein the composition is one or more peptides or a composition containing one or more polynucleotides according to [7]. Pharmaceutical compositions for preventing and / or preventing recurrence after surgery of cancer,

[10] The pharmaceutical composition of [9], wherein the HLA antigen is formulated to be administered to an individual having HLA-A24 or HLA-A2.

[11] The pharmaceutical composition according to [9] or [10], wherein the composition is designed for treating cancer,

[12] A method of inducing antigen-presenting cell having CTL inducibility, comprising the step selected from the group consisting of:

(a) any one of [1] to [6], wherein the peptide is in contact with APC in vitro, ex vivo or in vivo;

(b) the induction of a polynucleotide according to any one of [1] to [6], which encodes a peptide for APC,

[13] CTL induction method by a method containing a step selected from the group consisting of:

(a) Co-culturing of CD8 positive T cells and APC according to any one of [1] to [6], which presents a complex of HLA antigen and peptide on its surface;

(b) the coculture of CD8 positive T cells with exosomes according to any one of [1] to [6], which presents a complex of HLA antigen and peptide on its surface;

(c) the induction of a gene according to any one of [1] to [6], wherein the gene contains a polynucleotide encoding a T cell receptor (TCR) subunit polypeptide that binds a peptide to a T cell,

[14] The method according to any one of [1] to [6], wherein the isolated APC presents a complex of the HLA antigen and the peptide on its surface;

[15] The method of [14], wherein the APC derived by the method of [12],

[16] The method according to any one of [1] to [6], wherein the isolated CTL is targeted to a peptide;

[17] The CTL derived from the method of [13], according to [16].

[18] A cancer according to any one of [1] to [6], comprising administering to the subject a composition comprising a peptide, an immunologically active fragment thereof, or a polynucleotide encoding the peptide or fragment Induction of immune response to

[19] The antibody or fragment thereof according to any one of [1] to [6],

[20] The vector of any one of [1] to [6], wherein the vector contains a nucleotide sequence encoding a peptide,

[21] The host cell of [20], which is transformed or transfected with an expression vector,

[22] A diagnostic kit containing the peptide of any of [1] to [6], the nucleotide of [7] or the antibody of [19],

[23] any one of [1] to [6], wherein SEQ ID NOs: 3, 4, 5, 8, 9, 10, 14, 15, 16, 18, 23, 30, 33, 34, 35, 36 and An isolated peptide selected from the group consisting of 38.

It is to be understood that the above summary and the following detailed description of the invention are examples and do not limit the invention or other alternative embodiments of the invention.

In addition, other objects and features of the present invention will become more fully apparent upon reading the following detailed description in conjunction with the accompanying drawings and examples. However, it is to be understood that the above summary and the following detailed description of the invention are examples, and do not limit the invention or other alternative embodiments of the invention. In particular, while the invention has been described herein with reference to many specific embodiments, the description is intended to illustrate the invention and not to limit the invention. Various modifications and adaptations may occur to those skilled in the art as described by the appended claims within the spirit and scope of the invention. Equally, other objects, features, benefits, and advantages of the present invention will become apparent from the specific examples described above and below, and will be readily apparent to those skilled in the art. Such objects, features, usefulness, and advantages will become apparent from the above, either alone or in conjunction with the accompanying examples, data, figures, and all reasonable inferences possible therefrom, or by consideration of the references incorporated herein.

Various aspects and applications of the present invention will become apparent to those skilled in the art in view of the brief description of the drawings, and the detailed description of the invention and its preferred embodiments are as follows.
1a-f.
1A-F consist of photographs (a)-(f) showing the results of IFN-gamma ELISPOT analysis of CTLs derived from CLUAP1 derived peptides. The following CTLs in the wells showed potent IFN-gamma production compared to the control: well number # 6 (a), CLUAP1-A24- stimulated with CLUAP1-A24-9-255 (SEQ ID NO: 3) Well number # 2 (b) stimulated with 9-283 (SEQ ID NO: 4), well number # 2 (c) stimulated with CLUAP1-A24-9-31 (SEQ ID NO: 5), CLUAP1-A24-9-168 ( Well number # 4 (d) stimulated with SEQ ID NO: 8), well number # 4 (e) stimulated with CLUAP1-A24-9-64 (SEQ ID NO: 9) and CLUAP1-A24-9-216 (SEQ ID NO: 10) Stimulated with well number # 1 (f). Square cells in the wells were expanded to establish CTL cell lines. In contrast, as a representative example of negative data, CTLs stimulated with CLUAP1-A24-9-258 (SEQ ID NO: 1) (k) did not show specific IFN-gamma production. "+" In the figure indicates IFN-gamma production for target cells added with the appropriate peptide and "-" indicates IFM-gamma production for target cells not added with any peptide.
1g-k]
1G-K consists of photographs (g)-(j) showing the results of IFN-gamma ELISPOT analysis of CTLs derived from CLUAP1 derived peptides. The following CTLs in the wells showed potent IFN-gamma production compared to the control: well number # 6 (g), CLUAP1-A24-10-91 stimulated with CLUAP1-A24-9-154 (SEQ ID NO: 14) Well number # 2 (h) stimulated with (SEQ ID NO: 15), well number # 4 (i) stimulated with CLUAP1-A24-10-104 (SEQ ID NO: 16), and CLUAP1-A24-10-125 (SEQ ID NO: Well number # 5 (j) stimulated with 18). Square cells in the wells were expanded to establish CTL cell lines. In contrast, as a representative example of negative data, CTLs stimulated with CLUAP1-A24-9-258 (SEQ ID NO: 1) (k) did not show specific IFN-gamma production. "+" In the figure indicates IFN-gamma production for target cells added with the appropriate peptide and "-" indicates IFM-gamma production for target cells not added with any peptide.
2
2 is 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: 9) (c) IFN-gamma ELISA assay showing IFN-gamma production of CTL cell lines stimulated with CLUAP1-A24-9-154 (SEQ ID NO: 14) (d) and CLUAP1-A24-10-91 (SEQ ID NO: 15) (e), respectively It consists of the line graph (a)-(e) which shows a result. The results demonstrate that CTL cell lines established with each peptide stimulation showed potent IFN-gamma production compared to the control. "+" In the figure indicates IFN-gamma production for target cells added with the appropriate peptide and "-" indicates IFM-gamma production for target cells not added with any peptide.
3
Figure 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 ID NO: 15) (c (A) to (c) showing the results of IFN-gamma ELISA analysis showing IFN-gamma production of CTL clones established by limiting dilution from CTL cell lines stimulated with). The results demonstrate that CTL clones, established with each peptide stimulus, showed potent IFN-gamma production compared to the control. "+" In the figure indicates IFN-gamma production for target cells added with each peptide and "-" indicates IFM-gamma production for target cells not added with any peptide.
[Figure 4]
4 is a line graph showing specific CTL activity against target cells exogenously expressing CLUAP1 and HLA-A * 2402. COS7 cells transfected with HLA-A * 2402 or full length CLUAP1 gene were prepared as controls. The CTL cell line established as CLUAP1-A24-9-255 (SEQ ID NO: 3) showed specific CTL activity against COS7 cells transfected with both CLUAP1 and HLA-A * 2402 (black lozenge). In contrast, no significant specific CTL activity was detected for target cells expressing HLA-A * 2402 (triangle) or CLUAP1 (circle), respectively.
5a-f.
5A-F are photographs (a)-(f) showing the results of IFN-gamma ELISPOT analysis of CTLs derived from CLUAP1 derived peptides. The following CTLs in the wells showed potent IFN-gamma production compared to the control: well number # 3 (a), CLUAP1-A02-9-99 (CLUAP1-A02-9-34 (SEQ ID NO: 23)) Well number # 6 (b) with SEQ ID NO: 30), well number # 2 (c) with CLUAP1-A02-10-153 (SEQ ID NO: 33), with CLUAP1-A02-10-85 (SEQ ID NO: 34) Well number # 2 (e) with well number # 3 (d), well number # 2 (e) with CLUAP1-A02-10-34 (SEQ ID NO: 35) and well number # 2 (f with CLUAP1-A02-10-33 (SEQ ID NO: 36)) )to be. Square cells in the wells were expanded to establish CTL cell lines. In contrast, as a representative example of negative data, CTLs stimulated with CLUAP1-A02-9-58 (SEQ ID NO: 24) (h) did not show specific IFN-gamma production. "+" In the figure indicates IFN-gamma production for target cells added with the appropriate peptide and "-" indicates IFM-gamma production for target cells not added with any peptide.
Figure 5g-h
5G-H are photographs (g) showing the results of IFN-gamma ELISPOT analysis of CTL induced with CLUAP1 derived peptides. The following CTLs in the wells showed potent IFN-gamma production compared to the control: well number # 5 (g) with CLUAP1-A02-10-80 (SEQ ID NO: 38). Square cells in the wells were expanded to establish CTL cell lines. In contrast, as a representative example of negative data, no specific IFN-gamma production was seen in CTL cell lines stimulated with CLUAP1-A02-9-58 (SEQ ID NO: 24) (h). "+" In the figure indicates IFN-gamma production for target cells added with the appropriate peptide and "-" indicates IFM-gamma production for target cells not added with any peptide.
6
Figure 6 is 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) (c) Stimulated with 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) (f) It consists of line graphs (a) to (f) showing the results of IFN-gamma ELISA analysis showing IFN-gamma production of the CTL cell line. The results demonstrate that CTL cell lines established with each peptide stimulation showed potent IFN-gamma production compared to the control. "+" In the figure indicates IFN-gamma production for target cells added with the appropriate peptide and "-" indicates IFM-gamma production for target cells not added with any peptide.
7
Figure 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: 33) (c) And a series of line graphs (a) to (d) showing IFN-gamma production of CTL clones established by limiting dilution from CTL cell lines stimulated with CLUAP1-A02-10-85 (SEQ ID NO: 34) (d). . The results demonstrate that CTL clones established with each peptide stimulus showed potent IFN-gamma production compared to the control. "+" In the figure indicates IFN-gamma production for target cells added with the appropriate peptide and "-" indicates IFN-gamma production for target cells not added with any peptide.
8
8 consists of line graphs (a) to (d) showing specific CTL activity for 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-A02-10-15 (SEQ ID NO: 33) (b), CLUAP1-A02-10-85 (SEQ ID NO: 34) (c), and CLUAP1- The CTL cell line established as A02-10-33 (SEQ ID NO: 36) (d) showed specific CTL activity against COS7 cells transfected with both CLUAP1 and HLA-A * 0201 (squares). In contrast, no significant specific CTL activity was detected for target cells expressing HLA-A * 0201 (triangle) or CLUAP1 (circle), respectively.

Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, preferred methods, devices, and materials are described herein. However, prior to describing the materials and methods of the present invention, the present invention is not limited to the specific sizes, shapes, dimensions, materials, methodologies, protocols described herein, which are common experiments and / or This can be due to optimization. Also, the terminology used in this description is intended to describe particular versions or embodiments only, and is not intended to limit the invention as defined by the following claims.

All publications, patents, or patent applications mentioned in this specification are specifically incorporated by reference in their entirety. However, nothing herein is deemed to approve of granting no precedence to the foregoing disclosure because the invention is prior art.

I. Definition

Unless defined otherwise, 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 case of conflict, the present specification, including definitions, will control.

Unless otherwise specified, the words "a", "an", and "the" used in the present invention mean "at least one".

As used herein, "polypeptide", "peptide" and "protein" refer to a polymer of amino acid residues. The term refers to non- such as artificial chemical mimetic (s) corresponding to naturally occurring amino acids, as well as residues in which one or more amino acid residues are modified, or naturally occurring amino acid polymers. Applied to amino acid polymers having naturally occurring residues.

The term "amino acid" as used herein refers to naturally occurring amino acids and synthetic amino acids as well as amino acid analogs and amino acid mimetics that function similarly to naturally occurring amino acids. The amino acid may be L-amino acid or D-amino acid. Naturally occurring amino acids are encoded by the genetic code as well as modified after translation in the cell (eg hydroxyproline, gamma-carboxyglutamate carboxyglutamate), O-phosphoserine]. The term "amino acid analog" refers to the same basic chemical structure as the naturally occurring amino acid (hydrogen, carboxyl group, amino group and R group). Have alpha carbon, but have one or more modified R groups or modified backbones (eg, homoserine, norleucine, methionine, sulfoxide) (sulfoxide), methionine methyl sulfonium] (compion). The term "amino acid mimetic" refers to chemical compounds that differ in structure from general amino acids but have similar functions.

Amino acids are represented herein by three letter symbols or one letter symbols recommended by the IUPAC-IUB Biochemical Nomenclatere Commission.

The terms "gene", "polynucleotide", "nucleotide", and "nucleic acid" as used interchangeably herein are generally similar to the above amino acids unless otherwise specified. It is indicated by a one-letter symbol that is recognized.

The term "composition" as used herein relates to a product comprising a specific amount of specific components as well as any product formed directly or indirectly from a combination of specific amounts of specific components. The term in relation to “pharmaceutical composition” refers to the combination, complexation or aggregation of any two or more components, or from the dissociation of one or more components, Or any product formed directly or indirectly from a reaction or interaction of one or more components with another kind, as well as products comprising an inert component constituting a carrier with the active component. will be. Thus, the term "pharmaceutical composition" in the context of the present invention relates to any composition made by admixing a compound of the present invention with a pharmaceutically or physiologically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" or "physiologically acceptable carrier" refers to a pharmaceutically or physiologically acceptable material, composition, or substance. (substance) or vehicle (carh), which carries an individual scaffolded polypharmacophore from one organ or part of the body to another organ or body part or Including but not limited to liquid or solid fillers, diluents, excipients, solvents or encapsulating materials involved in the transport.

Unless defined otherwise, the term "cancer" refers to a cancer or tumor that overexpresses the CLUAP1 gene, including breast cancer, cervical cancer, and colorectal cancer. ), Esophageal cancer, gastric cancer, gastric diffuse-type cancer, lymphoma, neuroblastoma and pancreatic cancer. Does not.

Unless defined otherwise, the terms "cytotoxic T lymphocyte", "cytotoxic T cell", and "CTL" are used interchangeably herein, unless otherwise specified. Means a sub-group of T lymphocytes capable of recognizing non-self cells (eg tumor / cancer cells, virus-infected cells) and inducing the death of such cells .

Unless defined otherwise, "HLA-A24" means HLA-A24 type, including subtypes such as HLA-A * 2402.

Unless defined otherwise, as used herein, "HLA-A2" typically refers to subtypes such as HLA-A * 0201 and HLA-A * 0206.

Unless defined otherwise, as used herein, "kit" means mixing a reagent with another material. Here, the kit may include a microarray, a chip, a marker, and the like. The term "kit" is not limited to any particular mix of reagents and / or materials.

To the extent that the methods and compositions of the present invention have utility in the context of "treatment" of cancer, such as reduced expression of the CLUAP1 gene or reduced size, prevalence or metastatic potential of the cancer in an individual. Treatment is considered to be "efficacious" if it results in clinical benefit. When treatment is used prophylactically, "effective" means to delay or prevent the formation of cancer or to prevent or alleviate the clinical sympton of the cancer. Effectiveness is determined with respect to known methods for diagnosing or treating a particular tumor type.

To reduce the burden of mortality or morbidity from disease within the scope of the methods and compositions of the present invention have utility in the context of "prevention" and "prophylaxis" of cancer. The terms are interchanged herein to refer to any activity that causes the activity. Prevention and prevention can occur at "primary, secondary and tertiary prevention levels". While primary prevention and prevention is the avoidance of disease development, secondary and tertiary prevention and prevention not only prevent and prevent disease progression and emergence, but also improve function and disease-related complications. By reducing activity and reducing the negative effects of established diseases. Alternatively, prevention and prevention may include a wide range of prophylactic therapies that alleviate the severity of certain disorders, such as reducing proliferation and metastasis of tumors.

In the context of the present invention, treating and / or preventing cancer and / or preventing recurrence after cancer surgery comprises one of the following steps; Surgical removal of cancer cells, inhibition of growth of cancer cells, involution or regression of tumors, induction of remission and suppression of cancer development, tumor regression, reduction or inhibition of metastasis. Effective treatment and / or prevention of cancer reduces mortality, improves the prognosis of individuals with cancer, reduces the level of tumor markers in the blood, and alleviates the perceived symptoms of cancer. By way of example, reduction or amelioration of symptoms means effective treatment and / or prophylaxis includes a 10%, 20%, 30% or more reduction, or stable disease.

In the context of the present invention, the term "antibody" refers to immunoglobulins and fragments thereof that specifically react to designated proteins and their peptides. Antibodies include human antibodies, primatized antibodies, chimeric antibodies, bispecific antibodies, humanized antibodies, other proteins or radiolabels, and antibodies. Fragments are included. The antibody is also used here in the broadest sense, in particular, a monoclonal antibody, a polyclonal antibody, a multispecific antibody formed from at least two complete antibodies (e.g. Specific antibodies), and one antibody fragment that exhibits the desired biological activity. "Antibody" refers to all classes (eg, Ig A, Ig D, Ig E, Ig G, Ig M).

Unless defined otherwise, 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 . Peptide

To demonstrate that a CLUAP1-derived peptide functions as an antigen recognized by CTL, CLUAP1 (SEQ ID NO) is used to determine if it is an antigen epitope restricted by HLA-A24 or A2, which often encounters the HLA allele. 45) Derived peptides were analyzed (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 was identified that binds to CLUAP1-derived peptides based on binding affinity with HLA-A24. The following peptides were examined as candidate peptides.

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),

CLUAP1-A24-10-63 (SEQ ID NO: 22).

In addition, after stimulating T-cells in vitro by pulsed (loaded) dendritic cells (DCs), CTLs were successfully established using each of the following peptides: It became.

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),

CLUAP1-A24-10-169-125 (SEQ ID NO: 18).

Candidate HLA-A2 was identified that binds to CLUAP1 derived peptides based on binding affinity with HLA-A2. The following peptides were examined as 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),

CLUAP1-A2-10-222 (SEQ ID NO: 43),

In addition, after stimulating T-cells by dendritic cells (DCs) to which the peptides were added (loaded) in vitro, CTLs were successfully established using each of 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),

CLUAP1-A2-10-80 (SEQ ID NO: 38),

The established CTLs exhibit potent specific CTL activity against target cells added with each peptide. The results of the present invention demonstrate that CLUAP1 is an antigen recognized by CTL and the peptide tested is a CLUAP1 epitope peptide restricted by HLA-A24 or HLA-A2.

CLUAP1 gene is overexpressed in cancer cells and tissues including but not limited to breast cancer, cervical cancer, colon-rectal cancer, esophageal cancer, gastric cancer, broad-gastric cancer, lymph cancer, neuronal cancer and pancreatic cancer, but is expressed in most normal organs This is a good target for cancer immunotherapy. Accordingly, the present invention provides nonapeptides (peptides consisting of nine amino acid residues) and decapeptides (peptides consisting of ten amino acid residues) corresponding to CTL-recognized epitopes of CLUAP1. Alternatively, the present invention provides an isolated peptide that binds to the HLA antigen and induces cytotoxic T lymphocytes (CTL), wherein the peptide has an amino acid sequence of SEQ ID NO: 45 and is an immunologically active fragment thereof. It is characteristic. Particularly preferred examples of nonapeptides and decapeptides of the present invention are selected from SEQ ID NOs: 3, 4, 5, 8, 9, 10, 14, 15, 16, 18, 23, 30, 33, 34, 35, 36, 38 Peptides having an amino acid sequence are included.

Generally, Parker KC et al., J Immunol 1994 Jan 1, 152 (1): 163-75 and Nielsen M et al., Protein Sci 2003; 12: 1007-17, currently available software programs, such as on the Internet, for example, can be used to calculate binding affinity between various peptides and HLA antigens in silico. 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: binding affinity with HLA antigens can be measured as described in e796; This is summarized by way of example in Lafuente EM et al., Current Phamarceutical Design, 2009, 15: 3209-3220. Methods for measuring binding affinity are described by way of example in the Journal of Immunological Methods, 1995, 185: 181-190 and Protein Science, 2009, 9: 1938-1846. Therefore, such a software program can be used to select fragments derived from CLUAP1 having high binding affinity for HLA antigens. Thus, the present invention relates to peptides consisting of any fragment derived from CLUAP1 that binds HLA antigens with this known program. Moreover, the peptide may comprise a full length peptide of CLUAP1.

Nonapeptides and decapeptides of the invention can adjoin additional amino acid residues so long as the resulting peptide maintains CTL inducibility. Additional amino acid residues may consist of any kind of amino acid, so long as it does not impair the CTL inducibility of the original peptide. Thus, the present invention includes peptides having binding affinity to HLA antigens, including CLUAP1 derived peptides. By way of example, these peptides are those that are less than about 40 amino acids, sometimes less than about 20 amino acids, usually less than about 15 amino acids.

In general, modification of one, two or more amino acids of any peptide does not affect the function of the peptide, and in some cases may enhance the desired function of the original protein. Indeed, a modified peptide (i.e., a peptide consisting of an amino acid sequence in which one, two or several amino acid residues have been modified (ie substituted, added, deleted or introduced) relative to the original reference sequence) retains the biological activity of the original peptide. Mark et al., Proc Natl Acad Sci USA 1984, 81: 5662-6; Zoller and Smith, Nucleic Acids Res 1982, 10: 6487-500; Dalbadie-McFarland et al., Proc Natl Acad Sci USA 1982, 79: 6409-13). Thus, in one embodiment of the invention, the peptides of the present invention are SEQ ID NOS: 3, 4, 5, 8, 9, 10, 14, 15, 16, 18, 23, 30, 33, 34, 35, Having an amino acid sequence selected from 36 and 38, wherein one, two or more amino acids are added, deleted and / or substituted.

Those skilled in the art recognize that individual additions, deletions or substitutions to amino acid sequences that change a single amino acid or a small percentage of amino acids tend to result in the preservation of the properties of the original amino acid side-chain. Thus, it is called "conservative substitution" or "conservative modification", where a change in the protein makes a modified protein with the same function as the original protein. Conservative substitution tables that provide functionally similar amino acids are well known in the art. Examples of preferred amino acid side chain features to conserve are hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E). , Q, G, H, K, S, T), and side chains having the following functional groups or common characteristics: 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 acids and amide-containing side chains (D, N, E, Q); Base containing side chains (R, K, H); Aromatic group containing side chains (H, F, Y, W). In addition, the following eight groups each contain amino acids that are considered conservative substitutions for one another in this field.

1) alanine (A), glycine (G);

2) aspartic acid (D), glutamic acid (E);

3) aspargine (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, eg, 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 them and consequently may include non-conservative modifications as long as the modified peptide maintains the CTL inducibility of the original peptide. In addition, CTL inducible polymorphic variants, interspecies homologues and CLUAP1 alleles should not be excluded from modified peptides.

To maintain the necessary CTL inducibility, small numbers (eg, one, two, or several) or low proportions of amino acids can be modified (introduced, added, deleted and / or substituted). In the present invention, "several" means five or fewer amino acids, for example four or three or fewer amino acids. The proportion of amino acids to be modified may preferably be 20% or less, more preferably 15% or less, even more preferably 10% or less or 1 to 5%.

When used in the context of immunotherapy, the peptides of the invention should be presented as complexes with the surface of cells or exosomes, preferably HLA antigens. Therefore, when selecting peptides, it is desirable to have high binding affinity to HLA antigens as well as CTL induction. To this end, peptides can be modified by substituting, introducing, and / or adding amino acid residues such that the modified peptide has improved binding affinity. In addition to naturally occurring peptides, the regularity of peptide sequences that appear by binding to HLA antigens is already known (J Immunol 1994, 152: 3913; Immunogenetics 1995, 41: 178; J Immunol 1994, 155: 4307). May be introduced into the immunogenic peptide of the present invention.

As an example, to increase HLA-24 binding affinity, the second amino acid from the N-terminus is phenylalanine, tyrosine, methionine or tryptophan and / or C. It may be desirable to replace the terminal amino acid with phenylalanine, leucine, isoleucine, tryptophan or methionine. Thus, a peptide having an amino acid sequence selected from SEQ ID NOs: 1 to 22, wherein the second amino acid from the N-terminus of the SEQ ID NO: amino acid sequence is substituted with phenylalanine, tyrosine, methionine or tryptophan and / or C of the SEQ ID NO: Peptides substituted at the end with phenylalanine, leucine, isoleucine, tryptophan or methionine are 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 the C-terminal amino acid with valine or leucine to increase HLA-A2 binding affinity. Thus, a peptide having an amino acid sequence selected from SEQ ID NO: 23 and 25 to 43, wherein a second amino acid from the N-terminus of the SEQ ID NO amino acid sequence is substituted with leucine or methionine and / or C- Peptides substituted at the end with valine or leucine, or methionine are included in the present invention.

Substitutions can be introduced at the terminal amino acids as well as possibly at the recognition T cell receptor (TCR) site of the peptide. Several studies, for example, CAP1, p53 _(264-272) , Her-2 / neu _(369-377) or gp100 _(209-217) (Zaremba et al., Cancer Res. 57, 4570-4577, 1997, TK Hoffmann et al., J Immunol. (2002) 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) demonstrated that amino acid substituted peptides may have the same or better function than the original peptide.

The present invention also notes that one, two or several amino acids may be added to the N and / or C-terminus of the peptide. Such modified peptides that have high HLA antigen binding affinity and maintain CTL inducibility are also included in the present invention.

However, if the peptide sequence is identical to a part of the amino acid sequence of an endogenous or exogenous protein having different functions, it is possible to cause an autoimmnune disorder or an allergy symptom to a specific substance. There is this. Therefore, it is desirable to perform a homology search using available databases to avoid situations where the peptide sequence matches the amino acid sequences of other proteins. If homology screening reveals that no peptide with one or two amino acid differences compared to the objective peptide is present, the binding affinity and / or CTL inducibility with the HLA antigen without risk of any side effects is revealed. The target peptide can be modified to increase.

As described above, peptides having high binding affinity with HLA antigens are expected to be very effective, but candidate peptides selected as indicators due to the presence of high binding affinity are further examined for the presence of CTL inducibility. The term "CTL inducible" as used herein means the ability of a peptide to induce cytotoxic T lymphocytes (CTL) when presented to antigen-presenting cells (APCs). "CTL inducible" also includes the ability to induce CTL activation, CTL proliferation of peptides, promote CTL lysis of target cells, and increase CTL IFN-gamma production.

Confirmation of CTL inducibility may include APCs carrying human MHC antigens (eg, B-lymphocytes, macrophage and dendritic cells, DCs) or more specifically human peripheral blood mononuclear cells. leukocyte) -derived dendritic cells are induced, stimulated with peptide, mixed with CD8 positive cells and then measured for IFN-gamma produced and released by CTL on target cells. As a reaction system, transgenic animals (eg, Ben Mohamed L, Krishnan R, Longmate J, Auge C, Low L, Primus J, Diamond DJ, Hum Immunol 2000) designed to express human HLA antigens 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) classII restricted T (H) response Can be used). For example, the target cells may be radiolabeled with ⁵¹ Cr or the like, and cytotoxic activity may be calculated from radioactivity emitted from the target cells. Alternatively, CTL inducibility is determined by measuring IFN-gamma produced and released by CTL in the presence of antigen-presenting cells (APCs) with immobilized peptides, and in media with anti-IFN-gamma monoclonal antibodies. It can be examined by visualizing the inhibition zone.

The CTL inducibility of the peptides described above was tested and found to be SEQ ID NOs: 3, 4, 5, 8, 9, 10, 14, 15, 16, 18, 23, 30, 33, 34, 35, 36 and 38 Nonapeptides or decapeptides selected from amino acid sequences have been shown to exhibit particularly high CTL inducibility and high binding affinity for HLA antigens. Accordingly, such peptides are illustrated as preferred embodiments of the present invention.

Moreover, the results of homology analysis show that these peptides do not have significant homology with peptides derived from any other known human gene product. Thus, the use of these peptides for immunotherapy reduces the likelihood of an unknown or unwanted immune response. Therefore, also from this aspect, these peptides are useful for inducing immunity to CLUAP1 in cancer patients. Thus, the peptide of the present invention is a peptide having 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 desirable.

In addition to the above-described modifications, the peptides of the invention can be linked with other peptides as long as the resulting peptide maintains the essential CTL inducibility of the original peptide. Examples of suitable other peptides include CTL inducible peptides derived from the peptides of the present invention or other TAAs: Linkers between suitable peptides are well known in the art, for example 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, non-CLUAP1 tumor associated antigen peptides can be used at about the same time to enhance immune responses through HLA class I and / or class II. It is well known that cancer cells can express one or more tumor associated genes. Thus, whether a particular individual expresses additional tumor associated genes and then comprising the HLA type I and / or type II binding peptides derived from the expression products of said genes in a CLUAP1 composition or vaccine according to the present invention is to this field. It is within the scope of conventional test methods in the universal expert.

Examples of HLA type I and HLA type II binding peptides are known to universal experts in the art (see, eg, Coulie, Stem Cells 13: 393-403, 1995) and can be used in the present invention in the same manner as disclosed herein. . Thus, universal experts in this field prepare nucleic acids that encode such peptides using standard procedures of polypeptide or molecular biology, including one or more CLUAP1 peptides and one or more non-CLUAP1 peptides. can do.

The linked peptide is herein a "polytope", ie two or more potential immunogenic or immunostimulating peptides that can be linked together in various arrangements (eg, concatenated, overlapping). Say a group of reactions. Polytops (or nucleic acids encoding polytops) may be administered, for example, to animals according to standard immunization protocols to test the effectiveness of the polytops to stimulate, enhance and / or elicit an immune response.

Peptides can be linked together to form polytops either directly or through the use of neighboring sequences, and the use of polytops 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 Tarn et al., J Exp. Med, 171 (1) 299-306, 1990). Polytops containing various numbers and combinations of epitopes can be prepared and used for the effect of enhancing cognitive and immune responses by CTLs.

Peptides of the invention can also be linked to other substances, so long as the resulting peptide maintains the essential CTL inducibility of the original peptide. Examples of suitable materials include: peptides, lipids, sugars and sugar chains, acetyl groups, natural and synthetic polymers, and the like. As long as the modification does not destroy the biological activity of the original peptide, the peptide may comprise modifications such as glycosylation, side chain oxidation, or phosphorylation. Modifications of this kind can be performed to provide additional functions (eg, targeting functions, delivery functions) or to stabilize the polypeptide.

For example, to increase the in vivo stability of a polypeptide, it is well known in the art to introduce D-amino acids, amino acid mimetics or non-natural amino acids; This concept can also be applied to the polypeptides of the present invention. The stability of the polypeptide can be examined in several ways. For example, various biological media such as peptidase and human plasma and serum can be used for stability testing (eg Verhoef et al., Eur J Drug Metab Pharmacokin 1986, 11 : 291-302).

In addition, as described above, among peptides modified by substitution, deletion or addition by one, two or several amino acid residues, those having the same or higher activity as compared to the original peptide can be screened or selected. The present invention therefore also provides a method for screening or selecting modified peptides having the same or higher activity compared to the original. Practical methods may include the following steps:

a: at least one amino acid residue of the peptide of the invention is substituted, deleted or added,

b: determining the activity of the peptide, and

c: selecting a peptide having the same or higher activity compared to the original.

Herein, the activity to be analyzed may include MHC binding activity, APC or CTL inducible and cytotoxic activity.

Herein, peptides of the present invention may be described as "CLUAP1 peptide (s)" or "CLUAP1 polypeptide (s)".

III . CLUAP1  Preparation of Peptides

The peptides of the present invention can be prepared using well known techniques. For example, the peptide can be prepared synthetically using recombinant DNA techniques or chemical synthesis. Peptides of the invention can be synthesized individually or as longer polypeptides comprising two or more peptides. The peptide may then be isolated, ie purified or isolated, with little other naturally occurring host cell protein and fragments thereof, or other chemicals.

Peptides of the invention can include modifications such as glycosylation, side chain oxidation, or phosphorylation, provided that the modifications do not destroy the biological activity of the original peptide. Other exemplary modifications may include, for example, incorporation of D-amino acids or other amino acid mimetics that can be used to increase the serum half life of the peptide.

Peptides of the invention can be obtained through chemical synthesis based on the selected amino acid sequence. Examples of common peptide synthesis methods that can be applied to the synthesis include:

(i) Peptide Synthesis, Interscience, New York, 1996;

(ii) The Proteins, Vol. 2, Academic Press, New York, 1976;

(iii) Peptide Synthesis (Japanese), Maruzen Co., 1975;

(iv) Basics and Experiment of Peptide Synthesis (Japanese), Maruzen Co., 1985;

(v) Development of Pharmaceuticals (second edition) (Japanese), Vol. 14 (peptide synthesis), Hirokawa, 1991;

(vi) WO99 / 67288; And

(vii) Barany G & Merrifield R.B., Peptides Vol. 2, "Solid Phase Peptide Synthesis", Academic Press, New York, 1980, 100-118.

Alternatively, the peptides of the present invention can be obtained by applying any known genetic engineering method to generate peptides (eg, Morrison J, J Bacteriology 1997, 132: 349-51; Clark-Curtiss & Curtiss , Methods in Enzymology (eds. Wu et al.) 1983, 101: 342-62). For example, first, a suitable vector comprising a polynucleotide encoding a target peptide in an expressible form (eg, downstream of a regulatory sequence corresponding to a promoter sequence) Is prepared and transformed into appropriate host cells. Such vectors and host cells are also provided in the present invention. The host cell is then cultured to produce the desired peptide. The peptides can be prepared in vitro by applying an in vitro translation system.

IV . Polynucleotide

The present invention also provides a polynucleotide encoding one of the aforementioned peptides of the invention. These include naturally occurring CLUAP1 [Gen Bank Accession No. 1 as well as polynucleotides having conservatively modified nucleotide sequences. NM_015041 or NM_024793 (eg, SEQ ID NO: 44)]. In the present invention, "conservatively modified nucleotide sequence" means a sequence encoding an identical or essentially identical amino acid sequence. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position of alanine determined by a codon, the codon can be altered to any of the corresponding codons, without altering the polypeptide to be encoded. Such nucleic acid variations are referred to as "silent variations" and are one type of conservatively modified variant. All nucleic acid sequences of the invention encoding peptides also exhibit all possible silencing of nucleic acids. One of the common techniques in this field will recognize that each codon of a nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is the only codon for tryptophan) can be modified to obtain functionally identical molecules. Thus, each silent variation of a nucleic acid encoding a peptide is implicitly described in each published 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 naturally occurring bases such as A, T, C, and G, where T is replaced by U in RNA. The expert will recognize that non-naturally occurring bases are also included in the polynucleotides.

The polynucleotide of the present invention can encode various peptides of the present invention with or without intervening between amino acid sequences. For example, the intervening amino acid sequence can provide a cleavage site (eg, an enzyme recognition sequence) of the polynucleotide or translated peptide. In addition, the polynucleotide may comprise any additional sequence to the coding sequence encoding the peptide of the invention. For example, the polynucleotide may be a recombinant polynucleotide including a regulatory sequence necessary for the 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 by engineering polynucleotides through conventional recombinant techniques using, for example, polymerases and endonucleases.

Recombinant and chemical synthetic techniques can be used to prepare the polynucleotides of the present invention. For example, polynucleotides can be prepared by inserting into a suitable vector that is transfected into and expressed in competent cells. Alternatively, polynucleotides can be amplified using PCR techniques or expression in an appropriate host (see, eg, Sambrook et al., Molecular Cloning; A Laboratory Mannual, Cold Spring Harbor Laboratory, New York, 1989). 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. Can be.

V. exosomes ( exsosomes )

The present invention also provides intracellular vesicles called exosomes which present on the surface a complex formed between the peptide of the invention and the HLA antigen. Exosomes are described, for example, in Japanese patent application Kohyo Publications No. It may be prepared using the methods described in Hei 11-510507 and WO99 / 03499 and may be prepared using APC obtained from patients subject to treatment and / or prophylaxis. Exosomes of the invention can be inoculated as vaccines in a manner similar to the peptides of the invention.

The type of HLA antigen contained in the complex should match the HLA antigen of the individual in need of treatment and / or prevention. For example, HLA-A24 and HLA-A2, in particular HLA-A * 2402, HLA-A * 0201 and HLA-A * 0206, are common in Japanese and will therefore be suitable for the treatment of Japanese patients. Use of type A24 or A2, which is highly expressed in Japanese and Caucasians, is desirable to achieve effective results, and subtypes such as HLA-A * 2402, HLA-A * 0201 and HLA-A * 0206 may also be used. Can be. In general, in clinical practice, HLA antigen types of patients in need of treatment have been tested in advance to properly select peptides having a high level of binding affinity for the specific antigen or inducing CTL induction by antigen presentation. It is possible. In addition, in order to obtain a peptide having both high binding affinity and CTL inducibility, substitution, introduction, deletion and / or addition of one, two or several amino acids based on the amino acid sequence of CLUAP1 partial peptide present in nature Can be performed.

When a type A24 HLA antigen is used for the exosomes of the invention, a peptide having one sequence selected from SEQ ID NOs: 1 to 22 is used.

Or when a type A2 HLA antigen is used for the exosomes of the invention, a peptide having one sequence selected from SEQ ID NOs: 23 and 25-43 is used.

VI . Antigen-presenting cells ( antigen - presenting cell , APC )

The invention also provides isolated antigen-presenting cells (APCs) that present on their surface a complex formed between the HLA antigen and the peptide of the invention. The APC may be derived from a patient who is the subject of treatment and / or prophylaxis and may be administered on its own or in combination with other drugs including the peptides, exosomes or CTLs of the invention.

The APC is not limited to a specific kind of cell and includes dendritic cells (DCs), Langerhans cells, macrophages, B cells, and activated T cells, which are cells that are recognized by lymphocytes. It is known to present proteinaceous antigens on the surface. Since DC is the representative APC that has the strongest CTL inducing action among APCs, dendritic cells can be used as APCs of the present invention.

For example, the APC of the present invention can be obtained by inducing DC from peripheral blood monocytes and then contacting (stimulating) the peptide of the present invention in vitro, ex vivo or in vivo. When the peptide of the invention is administered to a subject, APCs presenting the peptide of the invention are induced in the body of the subject. The term "APC induction" includes contacting (stimulating) a cell with a nucleotide encoding the peptide of the invention to present the peptide of the invention or a complex formed between the HLA antigen and the peptide of the invention on the cell surface. do. Therefore, the APC of the present invention can be obtained by collecting the APC from the subject after administering the peptide of the invention to the subject. Alternatively, the APC of the present invention can be obtained by contacting APC collected from an individual injected with the peptide of the present invention.

The APCs of the invention can be administered to a subject alone or in combination with peptides, exosomes or other drugs comprising the CTL of the invention to induce an immune response against cancer in the subject. For example, ex vivo administration may comprise the following steps:

a: collecting APC from the first entity,

b: contacting said peptide with the APC of step a, and

c: administering the APC of step b to a second subject.

The first object and the second object may be the same object or may be different objects. Or in accordance with the present invention, there is provided the use of a peptide of the invention for preparing a pharmaceutical composition for inducing antigen-presenting cells. The present invention also provides a method or process for preparing a pharmaceutical composition for inducing antigen-presenting cells. The present invention also provides a peptide of the invention for inducing antigen-presenting cells. The APC obtained in step b can be administered as a vaccine for the treatment and / or prevention of cancers such as breast cancer, cervical cancer, colon-rectal cancer, esophageal cancer, gastric cancer, broad-gastric cancer, lymph cancer, neuronal cancer and pancreatic cancer.

According to one aspect of the present invention, APC has a high level of CTL inducibility. In the term "high level of CTL inducibility", that high level is a CTL inducible level compared to the CTL inducible level of APC which is not in contact with the peptide or APC which is in contact with a peptide which cannot induce CTL. Such APCs with a high level of CTL inducibility can be prepared by the methods mentioned above as well as by introducing a polynucleotide encoding a peptide of the invention into the APC in vitro. The introduced gene may be in the form of DNA or RNA. Examples of the introduction method include, without particular limitation, various methods generally performed in the art, such as lipofection, electroporation, or calcium phosphate method. More specifically Cancer Res 1996, 56: 5672-7; J Immunol 1998, 161: 5607-13; J Exp Med 1996, 184: 465-72; It may be carried out by the method described in Published Japanese Translation of International Publication No.2000-509281. By transferring the gene to APC, the gene undergoes transcription, translation, etc. in the cell, and the obtained protein is then processed by MHC type I or type II, and the presentation pathway And in partial peptides of the invention.

VII . Cytotoxic T cells ( cytotoxic  T lymphocyte , CTL )

CTLs derived for any of the peptides of the present invention may be used as vaccines in a manner similar to the peptide itself, thereby enhancing the immune response targeting cancer cells in vivo. Accordingly, the present invention provides isolated CTLs specifically induced or activated by any peptide of the present invention.

(1) administering a peptide of the invention to a subject or (2) contacting (stimulating) peripheral blood mononuclear lymphocytes in vitro with a subject-derived APC, CD8 positive cells, or a peptide of the invention, or (3) HLA Contacting CD8 positive cells or peripheral blood mononuclear lymphocytes in vitro with APC or exosomes presenting the antigen and peptide complexes on their surface, or (4) a T cell receptor (TCR) subunit that binds to the peptides of the invention ( Such CTLs can be obtained by the introduction of genes containing polynucleotides encoding subunits). The APC or exosomes can be prepared by the above described method, the method of (4) is described in detail in "VIII. T cell receptor (TCR)" below.

The CTL of the present invention may be derived from a patient subject to treatment and / or prophylaxis and may be administered on its own or in combination with other drugs, including peptides or exosomes of the present invention for the purpose of modulating effects. The CTL obtained above acts specifically on target cells presenting the peptides of the invention, eg the same peptides used for induction. The target cell is a cell endogenously expressing CLUAP1, such as a cancer cell, or a cell transformed with the CLUAP1 gene; Cells presenting the peptide of the present invention on the cell surface by stimulation by the peptide may also be targets of activated CTL attack.

VIII . T cell receptor (T cell receptor , TCR )

The present invention also provides compositions comprising nucleic acids encoding polypeptides capable of forming subunits of TCR, and methods of using the same. TCR subunits have the ability to form TCRs that confer T cells specificity for tumor cells presenting CLUAP1. Methods known in the art can be used to identify alpha-chain and beta-chain nucleic acids that make up the TCR subunit of one or more peptide derived CTLs of the invention. (WO2007 / 032255 and Morgan et al., J Immunol, 171; 3288 (2003). For example, the PCR method is preferred for analyzing TCR. PCR primers for the assay are, for example, 5 'side primers. 5'-R primer (5'-gtctaccaggcattcgcttcat-3 ') (SEQ ID NO: 46) and 3-TRa-C primer specific for the TCR alpha chain C site (5'-tcagctggaccagccgcagcgt-3') (SEQ ID NO: 47) 3-TRb-C1 primer specific for the TCR beta chain C1 site (5'-tcagaaatcctttctcttgac-3 ') (SEQ ID NO: 48) or 3-TRbeta- specific for the TCR beta chain C2 site as the 3' side primer. C2 primer (5'-ctagcctctggaatcctttctctt-3 ') (SEQ ID NO: 49), but is not limited thereto. May be bonded with high affinity (avidity) to the target cell to present a federated, the effective killing of target cells presenting the peptides can optionally CLUAP1 mediated in vivo and in vitro.

Nucleic acids encoding TCR subunits can be incorporated into suitable vectors, such as retroviral vectors. These vectors are well known in the art. Nucleic acids or vectors containing them can be usefully introduced into T cells, such as T cells from a patient. Advantageously, the present invention is readily available (off-) allowing the rapid modification of the patient's (or other mammalian) T cells to produce quickly and easily modified T cells with the superior characteristics of killing cancer cells. the-shelf) composition.

Specific TCRs are receptors that can specifically recognize complexes of peptides and HLA molecules of the invention, which provide T cell specific activity against target cells when the TCRs are presented on the surface of T cells. Specific recognition of the complex can be performed by any known method, and preferred methods include, for example, HLA multimer staining assays using HLA molecules and peptides of the invention, and ELISPOT assays. By performing ELISPOT analysis, it can be confirmed that T cells expressing TCR on the cell surface recognize the cells by TCR, and that signals travel into the cells. When the complex is present on the T cell surface, the identification of the complex that can give T cell cytotoxic activity can be performed by known methods. Preferred methods include, for example, identification of cytotoxic activation against HLA positive target cells, such as chromium release assays.

The present invention provides, by way of example, SEQ ID NOs: 3, 4, 5, 8, 9, 10, 14, 15, 16 and 18, and also SEQ ID NOs: 23, 30, 33, in the context of HLA-A2, Also provided is a CTL prepared by transducing with a nucleic acid encoding a TCR subunit polypeptide that binds CLUAP1 peptides of 34, 35, 36, and 38.

Transduced CTLs can be hommed into cancer cells in vivo and proliferated by well-known in vitro culture methods (eg, Kawakami et al., J Immunol., 142, 3452-3461 (1989). )]. The CTL of the present invention can be used to construct immunological compositions useful for the treatment or prevention of cancer in patients in need of treatment or protection (WO2006 / 031221).

Prevention and prevention include any activity that reduces the pain of death or morbidity caused by the disease. Prevention and prevention can be referred to as "primary, secondary and tertiary prevention levels". Whereas primary prevention and prevention avoids the development of the disease, secondary and tertiary levels of prevention and prevention not only reduce the negative effects of the already established disease by improving function and reducing disease-related complications, but also the disease. It includes any activity aimed at preventing and preventing the development of symptoms and the appearance of symptoms. Alternatively, prevention and prevention include extensive prophylaxis aimed at alleviating the severity of certain diseases, such as reducing tumor proliferation and metastasis and reducing angiogenesis.

Treatments aimed at preventing cancer and / or preventing recurrence after its operation may include any of the following steps. Surgical removal of cancer cells, blocking the proliferation of cancerous cells, tumor regression or inhibition, inducing and reducing cancer development, tumor suppression, reduction or blocking metastasis, etc. Effectively treating and / or preventing cancer reduces mortality, improves the prognosis of cancer patients, reduces tumor marker levels in the blood, and alleviates the detective symptoms accompanying cancer. For example, the reduction or amelioration of symptoms consists of effective treatment and / or prevention, including a 10%, 20%, 30% or more reduction, or stable disease.

IX . Pharmaceutical Formulations or Compositions

CLUAP1 expression is specifically increased in cancers including, but not limited to, breast cancer, cervical cancer, colon-rectal cancer, esophageal cancer, gastric cancer, broad-gastric cancer, lymph cancer, neuronal cancer and pancreatic cancer as compared to normal tissue. As such, the peptides of the invention or polynucleotides encoding such peptides can be used to treat and / or prevent cancer and / or to prevent cancer from recurring after surgery. Accordingly, the present invention provides a pharmaceutical agent or composition for treating and / or preventing recurrence after surgery of cancer, the composition comprising one or more peptides or polynucleotides of the present invention as an active ingredient. Alternatively, the peptides of the present invention may be expressed on any surface of cells such as APCs or APCs used as pharmaceutical agents or compositions. In addition, the CTL which targets any of the peptides of the present invention can also be used as an active ingredient of the pharmaceutical formulation or composition of the present invention.

The pharmaceutical formulations and compositions of the present invention can also be used as a vaccine. In the context of the present invention, the term "vaccine" (also called an immunogenic composition) refers to a substance having the function of inducing anti-tumor immunity when administered to an animal. .

The pharmaceutical preparations or compositions of the present invention can be used in humans, mice, rats, guinea-pig, rabbits, cats, dogs, sheep, goats, pigs, cattle, horses, monkeys, baboons and Other mammals, including but not limited to chimpanzees, in particular individuals or patients including commercially important or domesticated animals, can be used for the treatment and / or prevention of cancer and / or the prevention of recurrence after the surgery.

In another embodiment, the invention also provides the use of an active ingredient selected from the following for the manufacture of a pharmaceutical composition or formulation for the treatment or prevention of cancer or a tumor:

(a) a peptide of the present invention;

(b) nucleic acids encoding peptides or the like disclosed herein in expressible form;

(c) an APC or exosome presenting the peptide of the invention on its surface; And

(d) Cytotoxic T cells of the present invention.

Alternatively, the present invention also provides active ingredients selected from the following for therapeutic or prophylactic use of cancer or tumors:

(a) a peptide of the present invention;

(b) nucleic acids encoding peptides or the like disclosed herein in expressible form;

(c) an APC or exosome presenting the peptide of the invention on its surface; And

(d) Cytotoxic T cells of the present invention.

Alternatively, the present invention also provides a method or process for preparing a pharmaceutical composition or formulation for treating or preventing cancer or a tumor, wherein the method or process is pharmaceutically or physiologically acceptable efficacy selected from Formulating a carrier with powder includes:

(a) a peptide of the present invention;

(b) nucleic acids encoding peptides or the like disclosed herein in expressible form;

(c) an APC or exosome presenting the peptide of the invention on its surface; And

(d) Cytotoxic T cells of the present invention.

In another embodiment, the present invention also provides a method or process for preparing a pharmaceutical composition or agent for treating or preventing cancer or a tumor, wherein the method or process is an pharmaceutically or Mixing a physiologically acceptable carrier with the active ingredient:

(a) a peptide of the present invention;

(b) nucleic acids encoding peptides or the like disclosed herein in expressible form;

(c) an APC or exosome presenting the peptide of the invention on its surface; And

(d) Cytotoxic T cells of the present invention.

According to the present invention, peptides having an amino acid sequence selected from SEQ ID NOs: 2 to 22 known as epitope peptides or candidates restricted to HLA-A24 and SEQ ID NOs: 23 and 25 to 43 known as epitope peptides or candidates limited to HLA-A2 are potent and Can induce specific immune responses. Therefore, the present invention includes any of the peptides having the amino acid sequences of SEQ ID NOs: 3, 4, 5, 8, 9, 10, 14, 15, 16, 18 and 23, 30, 33, 34, 35, 36 and 38 Pharmaceutical formulations or compositions of the invention are particularly suitable for administration to a subject whose HLA antigens are HLA-A24 and HLA-A2, respectively. The same applies to pharmaceutical formulations or compositions comprising polynucleotides encoding any of these peptides (ie, said polynucleotides of the invention).

Cancers to be treated with the pharmaceutical agents or compositions of the present invention include, without limitation, any cancer in which CLUAP1 is involved, for example overexpressed, such as breast cancer, cervical cancer, colon-rectal cancer, esophageal cancer, gastric cancer, Widespread-gastric, lymphatic, neuronal and pancreatic cancers.

In addition to the active ingredient, the pharmaceutical agent or composition of the present invention may include other peptides capable of inducing CTL against cancer cells, polynucleotides encoding other peptides, cells presenting the other peptides, and the like. Herein, other peptides having the ability to induce CTLs against cancerous cells may be exemplified by, but are not limited to, cancer specific antigens (eg, identified TAAs).

Unless the antitumor effect of any of the above active ingredients, eg, peptides of the present invention, is inhibited, the pharmaceutical formulation or composition of the present invention optionally includes other therapeutic substances as active ingredients. For example, the agent may include an anti-inflammatory agent or composition, analgesic agent, chemotherapeutic agent, and the like. In addition to including other therapeutic agents in the medicament, the medicament of the present invention may be administered sequentially or simultaneously with one or more other pharmaceutical agents or compositions. The amount of the medicament and pharmaceutical agent or composition depends, for example, on the type of pharmaceutical agent or composition used, the disease being treated, and the schedule and route of administration.

In addition to the components specifically mentioned herein, the pharmaceutical formulations or compositions of the present invention may include other formulations or compositions common in the art, which are in question as to the type of formulation.

In one embodiment of the invention, the pharmaceutical agent or composition may comprise products and kits containing substances useful for treating the pathology of the disease to be treated, such as cancer. The product may include a container of any one of the above pharmaceutical formulations or compositions with a label. Suitable containers include bottles, vials, and test tubes. The container may be made of various materials such as glass or plastic. The label on the container should indicate that it is an agent or composition used to treat or prevent one or more symptoms of the disease. Labels may also indicate instructions for administration and the like.

In addition to the container, the kit containing the pharmaceutical formulation or composition of the present invention may optionally further comprise a second container for storing a pharmaceutically-acceptable diluent. It may also include other buffers, diluents, filters, needles, syringes, and other materials that are desirable from a commercial and user standpoint.

If desired, the pharmaceutical composition may be presented as a pack or dispenser device containing one or more dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser instrument may be accompanied by instructions for administration.

(1) a pharmaceutical formulation or composition containing the peptide as an active ingredient

Peptides of the present invention can be administered directly as a pharmaceutical preparation or composition, or if necessary, prepared by conventional methods of preparation. In the latter case, the peptides of the present invention may be included as appropriate, without particular limitation, those commonly used in carriers, excipients and drugs, as well as peptides. Examples of such carriers are sterile water, saline, phosphate buffer, culture fluids and the like. In addition, the pharmaceutical formulation or composition may include a stabilizer, a suspension, a preservative, a surfactant, and the like, if necessary. The pharmaceutical agents or compositions of the present invention can be used for anticancer purposes.

Peptides of the invention can be prepared in combinations composed of two or more peptides of the invention for inducing CTL in vivo. The peptide combination may take the form of a cocktail or may be conjugated to each other using standard techniques. By way of example, the peptide may be expressed or chemically linked as a single fusion polypeptide sequence having one or several amino acids as a linker (eg, Lysine linker: KS Kawamura et al., J. Immunol. 2002, 168: 5709-5715). Peptides of the above combinations may be the same or different. By administering a peptide of the invention, the peptide is presented to the APC at high density by the HLA antigen, followed by the induction of a CTL that specifically responds to the complex consisting of the peptide and the HLA antigen presented. Alternatively, to obtain an APC presenting any of the peptides of the invention on its cell surface, the APC (eg, DC) can be removed from the subject and then stimulated with the peptides of the invention. These APCs can be administered back to the subject to induce CTL in the subject and consequently increased aggressiveness to the cancer-associated endothelial.

Pharmaceutical agents or compositions for treating and / or preventing cancer comprising the peptide of the present invention as an active ingredient may comprise an adjuvant known to effectively establish cellular immunity. Alternatively, the pharmaceutical composition may be administered with other active ingredients or may be formulated in granules. Adjuvants refer to any of the compounds, agents or compositions that, when administered with (or continuously) a protein with immunological activity, enhance an immune response to the protein. Adjuvants contemplated herein include those described in Clin Microbiol Rev 1994, 7: 277-89. Examples of suitable adjuvant include 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.

In addition, liposome formulations, granules with peptides with fine micrometer diameter beads bound, formulations with lipids bound with peptides can be conveniently used.

In another embodiment of the invention, the peptides of the invention may also be administered in the form of pharmaceutically acceptable salts. Preferred examples of such salts include alkali metal salts, salts with a metal, organic base salts, organic acid salts and inorganic acid salts. As used herein, "pharmaceutically acceptable salts" possess biological effectiveness and properties of the compound and include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid ( nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and its Salts obtained by reaction with the same inorganic acid or base.

In some embodiments, the pharmaceutical formulation or composition of the present invention comprises a component that first sensitizes CTL. Lipids have been identified as agents or compositions capable of priming CTLs in vivo against viral antigens. For example, palmitic acid residues can be attached to epsilon-amino and alpha-amino groups of lysine residues, followed by peptides of the invention. It can be connected with. Lipidated peptides can be administered directly into micelles or particles, incorporated into liposomes, or emulsified in adjuvant. As another example of a lipid that initially sensitizes a CTL reaction, tripalmitoyl-S-glycerylcysteinyl-seryl-serine (P3CSS) when covalently attached with an appropriate peptide There is the same E. coli lipoprotein (see, eg, Deres et al., Nature 1989, 342: 561-4).

The method of administration may be oral, intradermal, subcutaneous, intravenous and the like and systemic or topical administration near the target site. The administration can be carried out in a single or multiple doses. The dose of the peptide of the present invention can be appropriately adjusted according to the disease to be treated, the age of the patient, the weight of the patient, the method of administration, and the like, and usually 0.001 mg to 1,000 mg, such as 0.1 mg to 10 mg, once every few days to several months. May be administered. Experts in this field can choose the appropriate dosage appropriately.

(2) a pharmaceutical formulation or composition containing a polynucleotide as an active ingredient

The pharmaceutical agent or composition of the present invention may comprise a nucleic acid encoding a peptide disclosed herein in an expressible form. The term "expressable form" herein means that when introduced into a cell, the polynucleotide will be expressed in vivo as a polypeptide that induces anti-tumor immunity.

In the illustrated embodiment, the nucleic acid sequence of the polynucleotide of interest comprises regulatory elements necessary for the expression of the polynucleotide. The polynucleotide can be made to achieve stable insertion into the genome of the target cell [eg, Thomas KR & Capecchi MR. Cell 1987, 51: for a description fo homologous recombination cassette vectors. Wolff et al., Science 1990, 247: 1465-8; US Patent Nos.5.580.859; 5.589 .466; 5.804.566; 5.739.118; 5.736.524; 5.679.647; and WO98 / 04720). Examples of DNA-based delivery techniques include "naked DNA", easy delivery (bupivacaine, polymers, peptide-mediated), cationic lipid complexes and particles. -Mediated ("gene gun") or pressure-mediated delivery (see, eg, US Patent No.5.922.687).

The peptide of the present invention can also be expressed as a viral or bacterial vector. Examples of expression vectors include enhanced viral hosts such as vaccinia or fowlpox. Such methods include the use of vaccinia virus as a vector expressing nucleotide sequences encoding peptides. When introduced into a host, the recombinant vaccinia virus expresses an immune peptide and thereby induces an immune response. Vaccinia vectors and methods useful in the immunization protocol are described, for example, in U.S. It is described in Patent No.4.722.848. Another vector is BCG (Bacille Calmette Guerin). BCG vectors are described in Stover et al., Nature 1991, 351: 456-60. Other vectors useful for therapeutic administration or immunotherapy, such as adeno and adeno-associated virus vectors, retroviral vectors, salmonella typhi vectors, detoxified non-toxic anthraxtoxin) vectors, and the like, will vary. See, eg, Shata et al., Mol Med Today 2000, 6: 66-71; Shedlock et al., J Leukoc Biol 2000, 68: 793-806; See Hipp et al., In Vivo 2000, 14: 571-85.

Delivery of a polynucleotide to a patient can be either direct when the patient is directly exposed to the polynucleotide-carrying vector, or indirect when the cell is transplanted to the patient after the cell is first transformed into the polynucleotide of interest in vitro. Which can be These two methods are known as in vivo and ex vivo gene therapy, respectively.

General reviews of gene therapies are described in 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 generally known in the field of recombinant DNA that can be used in the present invention are described in 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.

As the method of administration, oral, intradermal, subcutaneous, intravenous, etc. and systemic administration or topical administration near the target site can be used. The administration can be carried out in a single or multiple doses. The dose of cells transformed with a suitable carrier or polynucleotide encoding the peptide of the present invention may be appropriately adjusted depending on such things as the disease to be treated, the age of the patient, the weight of the patient, the method of administration, and usually 0.001 mg to 1,000 mg. For example, 0.1 mg to 10 mg and may be administered once every few days to several months. Experts in this field can choose the appropriate dosage appropriately.

X. peptide, Exosome , APC  And CTL How to use

Peptides and polynucleotides of the invention can be used to prepare or induce APCs and CTLs. In addition, the exosomes and APCs of the invention can be used to induce CTLs. The peptides, polynucleotides, exosomes and APCs can be used in combination with any other compound so long as the compound does not inhibit its CTL inducibility. Thus, any of the aforementioned pharmaceutical formulations or compositions of the present invention can be used to induce CTLs. In addition, including these peptides and polynucleotides can also be used to induce APCs described below.

(1) Antigen-presenting cells (APC) induction method

The present invention provides a method of inducing APC with high CTL induction using the peptide or polypeptide of the present invention.

The method of the present invention contemplates the following steps of contacting the peptides of the present invention with APCs in vitro, ex vivo or in vivo. For example, the method of contacting the peptide and APC ex vivo may comprise the following steps:

a: collecting APC from the subject; And

b: contacting the peptide and the APC of step a.

APCs are not limited to specific types of cells and include dendritic cells, Langerhans cells, macrophages, B cells, and activated T cells that are known to express proteinaceous antigens on their cell surface for recognition by lymphocytes. Preferably DC can be used because of its strongest CTL induction between the APCs. Any peptide of the present invention may be used alone or in combination with other peptides of the present invention as the peptide of step b.

Alternatively, the peptide of the present invention can be administered to a subject for contacting the peptide and APC in vivo. As a result, APC with high CTL inducibility can be induced in the body of the individual. Accordingly, the present invention also contemplates a method of administering a peptide of the invention to a subject to induce APC in vivo. In addition, a polynucleotide encoding a peptide of the present invention in an expressible form such that the peptide of the present invention is expressed and contacts APC in vivo and consequently induces high CTL inducible APC in the body of the individual. It is possible to administer. Thus, the invention may also include administering a polynucleotide of the invention to a subject to induce APC in vivo. The phrase “expressible form” is defined in the section “IX. Pharmaceutical Substances, Compositions (2) Pharmaceutical substances, compositions containing polynucleotides as active ingredients ”.

In addition, the present invention also contemplates introducing a polynucleotide of the present invention into APC to induce CTL inducible APC. For example, the method may include the following steps:

a: collecting APC from an individual; and

b: introducing a polynucleotide encoding a peptide of the invention.

Step b can be performed as described in the section “VI. Antigen-presenting cell” above.

Alternatively, the present invention provides a method for preparing an antigen-presenting cell (APC) that induces specific CTL activity against CLUAP1, wherein the method 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 a peptide of the invention into APC.

(2) how to derive CTL

The invention also provides a method of inducing CTL using the peptides, polypeptides, or exosomes or APCs of the invention.

The present invention also provides a method of inducing CTL using a polynucleotide encoding a polypeptide capable of forming a T cell receptor (TCR) subunit that recognizes a complex of a peptide of the invention and an HLA antigen. Preferably, the method of inducing a CTL may comprise at least one step selected from:

a) contacting CD8-positive T cells with antigen-presenting cells and / or exosomes presenting complexes of HLA antigens and peptides of the invention on their surfaces; And

b) introducing a polynucleotide encoding a polypeptide capable of forming a TCR subunit that recognizes a complex of a peptide of the invention and an HLA antigen into a CD8 positive cell.

When peptides, polynucleotides, APCs, or exosomes of the invention are administered to a subject, CTLs are induced in the subject's body and the strength of the immune response against the targeted cancer cells is enhanced. Thus, the methods of the present invention contemplate the step of administering a peptide, polynucleotide, APC or exosome of the present invention to a subject.

Alternatively, it can be induced in their own use in vitro and after CTL induction, the activated CTL will be returned to the subject. For example, the method may include the following steps:

a: collecting APC from the subject;

b: contacting the peptide with the APC of step a; And

c: co-culture of CD8 positive cells with APC of step b.

APC co-cultured with CD8-positive cells in step c can be prepared by transforming APCs with a gene comprising a polynucleotide of the present invention as described in the section "VI. Antigen-presenting cells", but The invention includes, but is not limited to, all APCs that effectively present the complex of the peptide of the invention and HLA antigens on its surface.

Instead of the APC, exosomes presenting the complex of the peptide of the present invention and the HLA antigen on the surface can also be used. That is, the present invention may also include a method of co-cultured with an CD8-positive cell an exosome that presents on its surface a complex of an HLA antigen and a peptide of the invention. The exosomes may be prepared by the method described in the section "V. exosomes".

In addition, CTLs can be induced by introducing into a CD8-positive cell a gene comprising a polynucleotide encoding a TCR subunit that binds to a peptide of the invention. Such transduction may be performed as described in the section “VIII. T cell receptor (TCR)”.

The present invention also provides a method or process for preparing a CTL-inducing pharmaceutical substance or composition, the method comprising mixing or preparing a peptide of the present invention and a pharmaceutically acceptable carrier. .

(3) a method of inducing an immune response

The present invention also provides a method of inducing an immune response against a disease associated with CLUAP1. Suitable diseases include cancer, for example breast cancer, cervical cancer, colorectal cancer, esophageal cancer, gastric cancer, gastric diffuse-type cancer ), Lymphoma, neuroblastoma and pancreatic cancer.

The methods of the invention may comprise administering any peptide or substance or composition comprising a polynucleotide encoding them. In addition, the methods of the present invention contemplate the administration of exosomes or APCs presenting any peptide of the present invention. In particular, reference may be made to "IX. Pharmaceutical Substances or Compositions", in particular the part describing the use of the pharmaceutical substances or compositions of the invention as a vaccine. In addition, exosomes or APCs that can be applied to the methods of the present invention to induce an immune response are described in the above "V. exosomes", "VI. Antigen-presenting cells (APC)", and "X. peptides, exosomes (1) and (2) of "How to use APC and CTL".

The present invention also provides a method or process for preparing a pharmaceutical substance or composition for inducing an immune response, wherein the method comprises the steps of adding or formulating a peptide of the invention with a pharmaceutically acceptable carrier. It may include.

Alternatively, the methods of the present invention may comprise administering a vaccine or pharmaceutical composition comprising:

(a) a peptide of the present invention;

(b) a nucleic acid encoding a peptide disclosed herein in an expressible form;

(c) APCs or exosomes presenting the peptides of the invention on a surface; or

(d) Cytotoxic T cells of the present 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, colon-rectal cancer, esophageal cancer, gastric cancer, broad-gastric cancer, lymph cancer, neuronal cancer and pancreatic cancer. Thus, prior to administration of a vaccine or pharmaceutical composition containing the active ingredient, it is desirable to confirm that the expression level of CLUAP1 in the cells or tissues to be treated has increased compared to normal cells. Thus, in one embodiment, the present invention provides a method for treating cancer in a patient overexpressing CLUAP1 and in need thereof, wherein the method may comprise the following steps:

i) determining the expression level of CLUAP1 in cancer cells or tissues obtained from the individual having the cancer to be treated;

ii) comparing the expression level of CLUAP1 with normal control levels; And

iii) administering at least one component selected from (a) to (d) described above to a subject having a cancer in which CLUAP1 is overexpressed compared to a normal control.

Alternatively, the present invention provides a vaccine or pharmaceutical composition comprising at least one component selected from (a) to (d) described above for administration to a subject having a cancer in which CLUAP1 is overexpressed. In other words, the present invention provides a method for identifying a subject to be treated with a CLUAP1 polypeptide of the invention, the method comprising determining the expression level of CLUAP1 in a subject-derived cell or tissue, wherein Increased levels compared to normal control levels indicate that the subject may have cancer that may be treated with the CLUAP1 polypeptide of the invention. The method of treating the cancer of the present invention is described in more detail below.

Any subject-derived cancer cell or tissue can be used for the determination of the expression of CLUAP1 as long as it includes a transcription or translation product of CLUAP1. Suitable biological samples include, but are not limited to, body tissues and body fluids such as blood, sputum and urine. Preferably, the entity-derived cell or tissue comprises a population of cells comprising epithelial cells, more preferably epithelial cells derived from cancerous or suspected cancerous tissues. In addition, if desired, the cells can be purified from the obtained body tissues and body fluids and then used as individual-derived cells or tissues.

In the context of the present invention, control levels determined from biological samples known to be non-cancerous are referred to as "normal control levels." 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 sample expression level and the control expression level can be leveled to the expression level of the housekeeping gene whose expression level does not differ depending on the condition of the control nucleic acid, for example cancer or a non-cancer cell. Examples of control genes include, but are not limited to, beta-actin, glyceraldehyde 3 phosphate dehydrogenase, and ribosomal protein P1.

The individual to be treated by the method of the present invention is preferably a mammal. Examples of mammals include, but are not limited to, humans, non-human primates, mice, rats, dogs, cats, horses, and cattle.

According to the invention, the expression level of CLUAP1 in said cancer cells or tissues obtained from an individual can be determined. The expression level can be determined at the level of transcription (nucleic acid) product using methods known in the art. For example, mRNA of CLUAP1 can be quantified using probes as a hybridization method (eg Northern hybridization). Detection can be performed in a manner such as a chip or an array. Use of the array may be desirable for detecting the expression level of CLUAP1. Experts in this field can use the sequence information of CLUAP1 to prepare probes. For example, the cDNA of CLUAP1 can be used as a probe. If necessary, the probe can be labeled with appropriate labels, such as dyes, fluorescent materials and isotopes, and the expression level of the gene can be detected by the intensity of the hybridized label.

In addition, transcription products of the CLUAP1 (eg SEQ ID NO: 44) gene can be quantified using primers by amplification-based detection methods (eg RT-PCR). In addition, the primer can be prepared based on available sequence information of the gene.

In particular, the probes or primers used for the methods presented above hybridize with mRNA of CLUAP1 under stringent conditions, appropriately stringent or low stringency. As used herein, the term “stringent (hybridization) conditions” refers to the conditions under which a probe or primer hybridizes to its target sequence, but not to other sequences. Stringent conditions are sequence-dependent and will vary under different circumstances. Specific hybridization of longer sequences is observed at higher temperatures compared to shorter sequences. In general, the temperature of a stringent condition is selected to be about 5 [deg.] C lower than the thermal melting point (Tm) for the specific sequence at defined ionic strength and pH. The Tm is the temperature at which 50% of the probe complements equally to the target sequence (under defined ionic strength, pH and nucleic acid concentration). Since the target sequence is generally present in excess, 50% of the probes at Tm are equally occupied. In general, under stringent conditions the concentration of salt may be at a concentration that is less than about 1.0 M sodium ions, and in general, at pH 7.0 to 8.3, the temperature of about 0.01 to 1.0 M sodium ions (or other salts) may be at least for short probes or primers. About 30 ° C. (eg, 10-50 nucleotides) and at least about 60 ° C. for long probes or primers. Stringent conditions can be obtained by the addition of destabilizing compositions such as formamide.

The probes or primers may be of a particular 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 the probes and primers are 5-10 nucleotides. , 10-15 nucleotides, 15-20 nucleotides, 20-25 nucleotides and 25-30 nucleotides.

Alternatively, translational products 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 determined. Methods of determining the amount of protein as a translation product include immunoassays using specific antibodies that recognize the protein. The antibody may be monoclonal or polyclonal. In addition, any fragment or modification of an antibody (eg, chimeric antibody, scFv, Fab, F (ab ') ₂ , Fv, etc.) will be detected as long as the fragment or modified antibody retains its ability to bind CLUAP1 protein. Can be used for Antibodies to the peptides of the invention and fragments thereof are also provided herein. Methods of preparing these types of antibodies for the detection of proteins are well known in the art, and any method may be applied to preparing such antibodies and their equivalents in the present invention.

Since other methods for detecting CLUAP1 gene expression levels are based on their translation products, the intensity of staining can be measured through immunohistochemical analysis using antibodies to the CLUAP1 protein. That is, in this measurement, strong staining indicates increased presence / degree of protein / level and at the same time high expression level of CLUAP1 gene.

For example 10%, 25%, or 50%; Or a target gene, for example at least 1.1 times, at least 1.5 times, at least 2.0 times, at least 5.0 times, at least 10.0 times, or at a control level (eg, normal intracellular level) of the corresponding target gene. In cancer cells, the expression level of the target cell, for example, the CLUAP1 gene, may be determined to increase.

Control levels can be determined at the same time as cancer cells using samples collected and stored from an individual known as a disease state (cancerous or noncancerous). In addition, normal cells obtained from non-cancerous sites of organs having cancer to be treated can be used as normal controls. Or by simultaneously using a previously collected and stored sample. Alternatively, the control level can be identified by a statistical method based on the results obtained by analyzing the expression level of the CLUAP1 gene previously identified in a biological sample known to be in a disease state. In addition, control levels can be derived from expression pattern databases of previously tested cells. In addition, according to aspects of the present invention, the expression level of the CLUAP1 gene in a biological sample may be compared in plurality to the control level, which may be determined from a plurality of reference samples. It is desirable to determine a control from a reference sample derived from a tissue type similar to a biological sample. It is also preferable to use the reference value of the expression level of the CLUAP1 gene of the group known as the disease state. Baseline values can be obtained by any method known in the art. For example, mean +/- 2 S.D. Or average +/- 3 S.D. The range of can be used as a reference value.

When the expression level of the CLUAP1 gene is increased compared to the normal control level, or similar / equal to the cancer control level, the individual can be diagnosed as having cancer to be treated.

The invention also provides a method of (i) diagnosing a subject suspected of having a cancer to be treated, and / or (ii) selecting a subject for treating the cancer, the method comprising the following steps: :

a) determining the expression level of CLUAP1 in cells or tissues obtained from individuals suspected of having cancer to be treated;

b) comparing the expression level of CLUAP1 with normal control levels;

c) if the expression level of CLUAP1 has increased compared to the normal control level, diagnosing the subject as having cancer to be treated; And

d) if in step c) the individual is diagnosed as having the cancer to be treated, then selecting the individual for cancer treatment.

Alternatively, the method will include the following steps:

a) determining the expression level of CLUAP1 in cancer cells or tissues obtained from individuals suspected of having cancer to be treated;

b) comparing the expression level of CLUAP1 with a cancerous control level;

c) diagnosing the subject as having cancer to be treated if the level of CLUAP1 is comparable to or equivalent to the cancer control level; And

d) if in step c) the individual is diagnosed as having the cancer to be treated, then selecting the individual for cancer treatment.

The present invention provides a kit for diagnosing or determining a subject suffering from or suspected of having a cancer that can be treated with a CLUAP1 polypeptide of the invention, which tests for the diagnosis of cancer and / or cancer therapy, in particular cancer immunity. It may be useful to monitor the effectiveness of a therapy. Examples of suitable cancers that can be diagnosed or determined include, but are not limited to, breast cancer, cervical cancer, colon-rectal cancer, esophageal cancer, gastric cancer, broad-gastric cancer, lymph cancer, neuronal cancer and pancreatic cancer. More specifically, the kit will preferably comprise at least one reagent for detecting the expression of the CLUAP1 gene in subject-derived cancer cells, which reagent may be selected from:

(a) a reagent for detecting mRNA of the CLUAP1 gene;

(b) a reagent for detecting a CLUAP1 protein or immunological fragment thereof; And

(c) a reagent for detecting the biological activity of the CLUAP1 protein.

Examples of suitable reagents for detecting mRNA of CLUAP1 include nucleic acids such as oligonucleotides that specifically bind to CLUAP1 mRNA or identify CLUAP1 mRNA, eg, having a complementary sequence to a portion of CLUAP1 mRNA. Oligonucleotides of this type can be amplified with primers and probes specific for CLUAP1 mRNA. Oligonucleotides of this type can be prepared based on methods well known in the art. If necessary, reagents for detecting CLUAP1 mRNA can be immobilized on a solid matrix. In addition, one or more reagents for detecting CLUAP1 mRNA may be included in the kit.

On the other hand, examples of suitable reagents for detecting CLUAP1 protein or immunological fragments thereof include antibodies to the CLUAP1 protein or immunological fragments thereof. The antibody may be monoclonal or polyclonal. Also, any fragment or modification of such an antibody (eg, chimeric antibody, scFv, Fab, F (ab ′) ₂ , as long as the fragment or modified antibody retains the ability to bind CLUAP1 protein or immunological fragment thereof; Fv et al.] Can be used as a reagent. Methods of preparing this type of antibody for protein detection are well known in the art, and any method may be applied in the present invention to prepare such antibodies and their equivalents. In addition, antibodies can be labeled with signaling molecules through direct linkage or indirect labeling techniques. Methods for detecting and binding of antibodies to labels and antibody labels are well known in the art, and any label and method can be applied to the present invention. In addition, one or more reagents for detecting CLUAP1 protein may be included in the kit.

The kit may comprise one or more of the aforementioned reagents. In addition, the kits are secondary to detect reagents that bind to the solid matrix and probes for the CLUAP1 gene or antibodies to the CLUAP1 peptide, containers for the medium and cells to culture, positive and negative control reagents, and antibodies to the CLUAP1 peptide. It may include an antibody. For example, tissue samples obtained from individuals without or suffering from cancer may serve as useful control reagents. Kits of the present invention also include buffers, diluents, filters, needles, syringes, and other materials including instructions for use (eg, documents, tapes, CD-ROMs, etc.), from the preferred commercial and user standpoint. do. The reagents can be stored in labeled containers. Suitable containers will include bottles, vials and test tubes. The container may be made of various materials such as glass or plastic.

In some embodiments, when the probe for CLUAP1 mRNA is a reagent, the reagent may be immobilized on a solid matrix such as a porous strip consisting of at least one detection site. The measurement or detection of the porous strip may comprise a large number of each constituent nucleic acid (probe) sites. Test strips may also include sites for negative and / or positive controls. Alternatively, the control site may be located on a strip separated from the test strip. Alternatively, other detection sites may contain different amounts of immobilized nucleic acid, i.e., a higher amount of the first detection site and a lesser amount in the next site. When the test sample is added, the number of sites presenting a detectable signal provides a quantitative indicator of the amount of CLUAP1 mRNA present in the sample. The detection site may be of any suitably detectable shape and is generally within the width of the bar or dot spanning of the test strip.

Alternatively, the kits of the present invention may 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 examining their CLUAP1 levels. Alternatively, purified CLUAP1 proteins or polynucleotides can 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, above the cut-off value.

In an embodiment, the present invention also provides a diagnostic kit comprising a protein or an immunogenic fragment capable of specifically recognizing an antigen or fragment thereof.

Examples of partial peptides or immunogenic fragments of the protein of the invention contemplated herein are polypeptides consisting of at least 8, preferably 15, more preferably 20 contiguous amino acids in the amino acid sequence of the protein of the invention. It includes. Cancer can be diagnosed by detecting an antibody using a peptide (polypeptide) or protein of the present invention in a sample (e.g., blood, tissue). Methods for producing the proteins and peptides of the present invention are described above.

The method for diagnosing cancer can be performed by determining the difference between the amount of anti-CLUAP1 antibody and the amount in the corresponding control sample as described above. If the cell or tissue of the individual comprises an antibody against the expression product of gene (CLUAP1) and the amount of anti-CLUAP1 antibody is determined to be above the cut-off as compared to that in the normal control group, then the individual is a cancer It is estimated to suffer from.

In one embodiment, the diagnostic kit of the invention may comprise a peptide of the invention and an HLA molecule that binds to it. Appropriate 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). Thus, the complexes of the peptides and HLA molecules of the invention can be applied to detection methods for detecting tumor antigen specific CTLs, thus enabling early detection, recurrence and / or metastasis of cancer. In addition, it can be applied to the selection of an individual which can be applied together with pharmaceuticals containing the peptide of the present invention as an active ingredient, or to the examination of the therapeutic effect of a pharmaceutical product.

In particular, according to known methods (see, eg, Altman JD et al., Science. 1996. 274 (5284): 94-6), oligomeric complexes such as tetramers of radiolabeled HLA molecules and peptides of the invention are prepared. Can be. The complex will be used to quantify antigen-peptide specific CTLs in individual derived peripheral blood lymphocytes.

The present invention also provides methods or diagnostic reagents for assessing an individual's immune response using the peptide epitopes described herein. In one embodiment of the invention, the HLA restricted peptides described herein are used as reagents to assess or predict an immune response in a subject. The immune response to be evaluated is induced by contacting an immunogen with an immunocompetent cell in vitro or in vivo. In a preferred embodiment, cells with an immunocompetent to assess the immune response are in peripheral blood, peripheral blood lymphocytes (PBL) and peripheral blood mononuclear cells (PBMC). Can be selected. Methods of collecting or isolating such immune cells are well known in the art. In some embodiments, the agent or composition applied as a reagent will be a composition that results in the formation of antigen specific CTLs that bind to and recognize peptide epitopes. The peptide reagent may not be used as an immunogen. Test systems used for this assay include relatively recent technological developments such as tetramer, intracellular lymphokine and interferon secretion assays, or ELISPOT. In a preferred embodiment, the immune functional cells in contact with the peptide reagent may be antigen-presenting cells, including dendritic cells.

For example, the peptides of the present invention can be used in tetramer staining assays to test peripheral blood monocytes for the presence of antigen-specific CTLs when exposed to tumor cell antigens or immunogens. HLA tetramer complexes directly visualize antigen specific CTLs in samples of peripheral blood monocytes (Ogg et al., Science 279: 2103-2106, 1998; and Altman et al., Science 174: 94-96. 1996). ), Can be used to determine the frequency of antigen-specific CTL populations. Tetramer reagents using the peptides of the invention can be produced as described below.

Peptides that bind HLA molecules are refolded to produce trimolecular complexes in the presence of the corresponding HLA heavy chain and beta 2-microglobulin. In this complex, the carboxyl terminus of the heavy chain is biotinylated at the site that was previously engineered with the protein. Thereafter, streptoavidin is added to the complex to form a tetramer consisting of the trimolecular complex and streptoavidin. With fluorescence labeled streptoavidine, the tetramers can be used to stain antigen-specific cells. The cells are then identified by, for example, flow cytometry. The assay can be used for diagnostic or prognostic purposes. Cells identified by the above process can also be used for therapeutic purposes.

The present invention also provides reagents comprising the peptides of the invention to assess immune recall response (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 are analyzed for the presence of antigen-specific CTLs using specific peptides. Blood samples containing mononuclear cells are examined by incubating PBMC and stimulating the cells with the peptides of the invention. After an appropriate incubation period, for example, CTL activity in the proliferated cell population is assayed.

The peptide can be used as a reagent to assess the efficacy of the vaccine. PBMCs obtained from immunized patients can be analyzed using, for example, one of the above methods. Patients are classified into HLA type and peptide epitope reagents that recognize allele specific molecules presented in the patient are selected for analysis. Immunogenicity of the vaccine may be demonstrated by the presence of epitope-specific CTL in PBMC samples. Peptides of the invention can be used to make antibodies by 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) This can be useful as a reagent for diagnosing and monitoring cancer. The antibody may be an antibody that recognizes a peptide, in the context of an HLA molecule, that binds to a peptide-MHC complex.

The peptides and compositions of the present invention have many additional uses, some of which are described herein. For example, the present invention provides a method for diagnosing and detecting a disease characterized by expression of a CLUAP1 immunogenic polypeptide. The method involves determining the expression of a CLUAP1 HLA binding peptide, or a complex of CLUAP1 HLA binding peptide and HLA type I molecule in a biological sample. Expression of peptides or complexes of peptides and HLA class I molecules can be determined or detected by examination with the binding partner of the peptides or complexes. In a preferred embodiment, the binding partner for the peptide or complex is an antibody that specifically binds to and recognizes the peptide. Expression of CLUAP1 in a biological sample, eg, tumor biopsy, can be examined by a general PCR amplification protocol using CLUAP1 primers. Examples of tumor expression are shown here and further publications of exemplary conditions and primers for CLUAP1 can be found in WO2003 / 27322.

Preferred diagnostic methods involve contacting a biological sample isolated from an individual with a specific material for the CLUAP1 HLA binding peptide to detect the presence of the CLUAP1 binding peptide in the biological sample. As used herein, “contacting” refers to where the CLUAP1 HLA binding peptides present in the material and biological sample are sufficiently close to the reagents, such as concentration, temperature, time, This means placing the biological sample under appropriate conditions such as ionization intensity. In general, between a molecule and a cognate in the biological sample of the molecule (e.g., a protein and the protein recognizer receptor, an antibody and the antibody recognizer protein, nucleic acid and sequence complementary to the nucleic acid). Conditions for contacting a substance with a biological sample to facilitate specific cross-linking of the compounds are apparent to those skilled in the art. Exemplary conditions for facilitating specific interactions between molecules and their recognition bodies are described in U.S. Patent No. 5,108,921.

The diagnostic methods of the present invention can be performed in vivo and / or in vitro. Thus, the biological sample may be placed in vivo or in vitro in the present invention. For example, a biological sample can be a tissue in vivo, and a substance specific for the CLUAP1 immunogenic polypeptide can be used to detect the presence of the molecule in that tissue. Alternatively, biological samples can be collected or separated in vitro (eg, blood samples, tumor tissue tests, tissue extracts). In a particularly preferred example, the biological sample may be a cell containing sample, more preferably a sample comprising tumor cells collected from an individual being diagnosed or treated.

Alternatively, the diagnosis can be performed using a method that allows direct quantification of antigen-specific T cells via staining with fluorescently labeled HLA multiple binding complexes (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-gamma secretion assays or ELISPOTs are provided. Complex staining, intracellular lymphokine staining, and ELISPOT assays are all at least 10 times more sensitive than conventional tests (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: 314;). Pentamers (eg US 2004-209295A), dextramers (eg WO 02/072631), and streptamers (eg Nature Medicine 6. 631-637 ( 2002) may also be used.

For example, in some embodiments, the present invention provides a method comprising the following steps of diagnosing or evaluating an immunological response in an individual to which at least one CLUAP1 of a peptide of the invention is administered:

(a) contacting the immunogen with an immunocapable cell under suitable conditions of specific CTL induction for the immunogen;

(b) detecting or determining the level of CTL induction derived in step (a); and

(c) correlating the immune response of the individual with a CTL induction level.

In the present invention, the immunogen is a CLUAP1 peptide selected from amino acid sequences SEQ ID NOs: 2 to 23 and 25 to 43, peptides having such amino acid sequences, and peptides having such amino acid sequences modified by one, two or more amino acid substitutions. At least one of them. Meanwhile, suitable conditions for inducing immunogen specific CTLs are well known in the art. For example, immune cells can be cultured in the presence of an immunogen to induce immunogen specific CTLs. In order to induce immunogen specific CTLs, certain stimulating factors can be added to the cell culture. For example, IL-2 is the preferred stimulating factor for CTL induction.

In some embodiments, monitoring or evaluating the immunological response of the individual treated with the peptide cancer treatment may be performed before, during and / or after treatment. In general, during the protocol of cancer treatment, the immunological peptide is administered to a subject to be treated repeatedly. For example, the immunological peptide can be administered weekly for 3-10 weeks. Thus, an individual's immunological response can be assessed or monitored during a cancer treatment protocol. Or the step of evaluating or monitoring the immunological response to cancer treatment may be completion of the treatment protocol.

According to the present invention, increased immunogen-specific CTL induction compared to the control refers to an individual immunologically evaluated or diagnosed for the administered immunogen. Suitable controls for evaluating the immunological response include, for example, induction of CTL when immune-capable cells are contacted with a control peptide having an amino acid sequence without peptide or in addition to any CLUAP1 peptide (eg, random amino acid sequence). Include the level.

In a preferred embodiment, the immunological response of the individual is assessed in terms of sequence specificity by comparing the immunological response between each immunogen administered to the individual. In particular, even if a mixture of some types of CLUAP1 peptide is administered to an individual, the immune response may vary depending on the peptide. In such cases, peptides can be identified for individuals with higher responses by comparison of the immune response between each peptide.

XI . Antibody

The present invention provides antibodies that bind to the peptides of the invention. Preferred antibodies will specifically bind to the peptides of the invention and will not bind (or weakly bind) to non-peptides of the invention. Alternatively, the antibody binds to the peptide of the invention as well as its homologues. Antibodies to the peptides of the invention can be used in the diagnostic and prognostic methods of cancer, and in imaging methodology. Similarly, the antibodies can be used in the treatment, diagnosis and / or prognostic methods, imaging methodology of other cancers, as long as CLUAP1 is also expressed or overexpressed in cancer patients. In addition, antibodies expressed in cells (e.g., single chain antibodies) are cancers associated with the expression of CLUAP1, such as breast cancer, cervical cancer, colon-rectal cancer, esophageal cancer, gastric cancer, broad-gastric cancer, lymph cancer, neuronal cancer And pancreatic cancer, but may be used to treat cancer.

In addition, the present invention relates to CLUAP1 protein (SEQ ID NO: 45) or SEQ ID NOs: 3, 4, 5, 8, 9, 10, 14, 15, 16, 18, 23, 30, 33, 34, 35, 36, and 38. Various immunological assays are provided for detection and / or quantification of fragments thereof comprising polypeptides having selected amino acid sequences. The assay may suitably comprise one or more anti-CLUAP1 antibodies capable of binding to and recognizing a CLUAP1 protein or fragment thereof. In the present invention, the binding of the CLUAP1 polypeptide and the anti-CLUAP1 antibody is preferably SEQ ID NO: 3, 4, 5, 8, 9, 10, 14, 15, 16, 18, 23, 30, 33, 34, 35, 36 and It recognizes a polypeptide having an amino acid sequence selected from 38. The binding specificity of the antibody can be confirmed using an inhibition test. That is, a polypeptide of any fragment having an amino acid sequence selected from among the amino acid sequences of SEQ ID NOs: 3, 4, 5, 8, 9, 10, 14, 15, 16, 18, 23, 30, 33, 34, 35, 36, and 38 When binding between the antibody to be analyzed and the CLUAP1 polypeptide full length in the presence of is inhibited, the antibody shows specific binding to that fragment. In the present invention, the immunoassay is carried out within a variety of immunoassay types well known in the art, radioimmunoassay, immuno-chromatograph technique, ELISA (enzyme-linked immunosorbent assay). ), Various types of enzyme-linked immunofluorescent assays (ELIFAs), and the like.

In addition, the relevant immunological but non-antibody assays of the present invention may include MHC (major histocompatibility complex) binding assays as well as T cell immunogenicity assays (inhibitory or stimulatory). In addition, immunological imaging methods capable of detecting cancer expressing CLUAP1 are provided by the present invention and include, but are not limited to, radioscintigraphic imaging methods using labeled antibodies of the present invention. The assays detect, monitor cancers including, but not limited to, cancers expressing CLUAP1, such as breast cancer, cervical cancer, colon-rectal cancer, esophageal cancer, gastric cancer, broad-gastric cancer, lymph cancer, neuronal cancer, and pancreatic cancer. And clinically useful for prognosis.

The present invention provides an antibody that binds to a peptide of the present invention. Antibodies of the invention can be used in any shape, for example monoclonal or polyclonal antibodies, and also include antisera obtained by animals, eg rabbits, immunized with the peptides of the invention, and of all kinds Polyclonal and monoclonal antibodies, human antibodies, and humanized antibodies produced by genetic recombination.

The peptides of the invention used as antigens for obtaining antibodies may be derived from any kind of animal, but are preferably derived from mammals such as humans, mice, or rats, more preferably humans. Is derived from. Human-derived peptides can be obtained from the nucleotide or amino acid sequences disclosed herein.

According to the invention, the peptide used as an immunogenic antigen may be a complete protein or a partial peptide of the protein. For example, partial peptides may comprise amino (N) -terminal or carboxy (C) -terminal fragments of the peptides of the invention.

In the present invention, an antibody is defined as a protein that responds to the full length or fragment of a CLUAP1 peptide. In a preferred embodiment, the antibody of the invention comprises an amino acid sequence selected from SEQ ID NOs: 3, 4, 5, 8, 9, 10, 14, 15, 16, 18, 23, 30, 33, 34, 35, 36 and 38 Branches can recognize fragment peptides of CLUAP1. Methods of synthesizing oligopeptides are well known in the art. After synthesis, the peptides can be selectively purified before use as an immunogen. In the present invention, oligopeptides (eg 9 or 10 mers) can be linked or linked to a carrier to enhance immunogenicity. Keyhole-Limpet hemocyanin (KLH) is well known as a carrier. Methods of linking KLH and peptides are also well known in the art.

Alternatively, the gene encoding the peptide of the present invention or a fragment thereof can be inserted into a known expression vector and then used to transform the host cell as described herein. The desired peptide or fragment thereof can be recovered from the inside or outside of the host cell by any standard method and thereafter used as an antigen. Alternatively, whole cells expressing the peptides or lysates thereof or chemically synthesized peptides may be used as the antigen.

Some mammalian animals can be immunized with antigens, but are preferably compatible with the parental cells used for cell fusion. Generally, animals of rodentia, rabbit (Lagomorpha) or primate (primates) can be used. Rodent animals include, for example, mice, rats, and hamsters. Rabbits include, for example, rabbits. Primates include, for example, monkeys of Catarrhini (old world monkey) such as Macaca fascicularis, rhesus monkey, sacred baboon and chimpanzee. .

Methods of immunizing animals with antigens are well known in the art. Intraperitoneal injection or subcutaneous injection of antigen is the standard method for immunization of mammals. More specifically, the antigen may be diluted or suspended in an appropriate amount such as phosphate buffered saline (PBS), physiological saline, and the like. If desired, the antigen suspension can be mixed with an appropriate amount of standard adjuvant such as Freund's complete adjuvant, made into an emulsion and then administered to a mammalian animal. Preferably, the antibody is then administered multiple times with an appropriate amount of Freund's complete adjuvant every 4 to 21 days. Suitable carriers can also be used for immunization. After such immunization, the serum can be tested by standard methods for increasing the amount of the desired antibody.

Polyclonal antibodies to the peptides of the invention can be prepared by harvesting the blood of the immunized mammals tested for the increase in the desired antibody in the serum, and by separating the serum from the blood by any common method. Polyclonal antibodies not only comprise serum comprising polyclonal antibodies, but also fractions containing such polyclonal antibodies can be separated from serum. Immunoglobulin G or M can only be purified from fractions that recognize the peptide of the invention using, for example, an affinity column linked to the peptide of the invention, and using a Protein A or Protein G column. Can be prepared by.

To prepare monoclonal antibodies, immune cells are harvested from mammals immunized with antigen, in which the immune cells are examined for increased levels of the desired antibody in serum and cell fusion. Preferably, immune cells used for cell fusion are obtained from the spleen. Other preferred parent cells to be fused with the immune cells include bone marrow cells having the characteristics obtained for the selection of mammalian myeloma cells, more preferably cells fused by drugs.

The immune cells and bone marrow cells can be fused according to known methods, such as those of Milstein et al. (Galfre and Milstein, Method Enzymol 73: 3-46 (1981)).

The resulting hybridomas (hybridoma) obtained by the cell fusion are those in a standard selection medium such as HAT medium (medium containing hypoxanthine, aminopterin and thymidine). It can be selected by culturing. The cell culture generally lasts for days to weeks in HAT medium, which is sufficient time to kill all other non-fused cells except the desired hybridomas. Thereafter, standard limiting dilution is performed to clone and screen the hybridoma cells producing the desired antibody.

In addition to the above methods of immunizing non-human animals with antigens for the production of hybridomas, human lymphocytes such as humans infected with EB virus can be immunized in vitro using peptides, cells expressing peptides or their lysates. have. The immunized lymphocytes are then fused with infinitely dividing human derived bone marrow cells, such as U266, to produce hybridomas that produce the desired human antibodies that can bind to the peptide. (Unexamined Published Japanese Patent Application No. Sho 63-17688).

The obtained hybridomas are then implanted into the abdominal cavity of the mouse and the ascites is extracted. The obtained monoclonal antibody can be purified, for example, by methods such as ammonium sulfate precipitation, Protein A or Protein G columns, DEAE ion exchange chromatography or affinity columns to which peptides of the invention are linked. The antibodies of the present invention may be used not only for the purification and detection of the peptides of the present invention but also as candidates for the agonists and antagonists of the peptides of the present invention.

Alternatively, immune cells, such as immunized lymphocytes producing antibodies, can be immortalized by oncogene and used to produce monoclonal antibodies.

The monoclonal antibodies thus obtained can be produced recombinantly using genetic engineering techniques (see, eg, Borrebaeck and Larrick, Therapeutic Monoclonal Antibodies (1990), published in the UK by MacMillan Publishers LTD). For example, DNA encoding the antibody can be cloned from immune cells, such as hybridomas or immunized lymphocytes that produce the antibody, inserted into appropriate vectors, and introduced into host cells to make recombinant antibodies. have. In addition, the present invention provides the recombinant antibodies described above.

In addition, an antibody of the invention may be a fragment or modified antibody of an antibody as long as it binds one or more of the peptides of the invention. For example, the antibody fragment can be Fab, F (ab ') ₂ , Fv or single chain Fc (scFv), wherein the Fv fragments from the H and L chains are linked by appropriate linkers [Huston et al., Proc Natl Acad Sci USA 85: 5879-83 (1988). More specifically, antibody fragments can be produced by treating the antibody with an enzyme such as papain or pepsin. Alternatively, genes encoding the antibody fragments can be prepared, inserted into expression vectors and expressed in appropriate host cells (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); Bird and Walker, Trends Biotechnol 9: 132-7 (1991).

Antibodies can be modified by linking with various molecules, such as polyethylene glycol (PEG). The present invention provides such modified antibodies. Such modified antibodies can be obtained from chemically modified antibodies. Such modification methods are universal in this field.

Alternatively, the antibody of the present invention may be a variable region derived from a non-human antibody, a constant region derived from a human antibody, or a complementarity determining region (CDR) derived from a non-human antibody, or a FR (derived from a human antibody). It can be obtained as a chimeric antibody between humanized antibodies, including framework regions) and constant regions. The antibody may be prepared according to known techniques. Humanization can be performed by substituting a rodent CDR or CDR sequence for the corresponding sequence of a human antibody (see, eg, Verhoeyen et al., Science 239: 1534-1536 (1988)). Thus, the humanized antibody is a chimeric antibody, wherein significantly fewer domains than intact human variable domains have been replaced by corresponding sequences from the non-human species.

Human antibodies composed entirely of human variable regions as well as human frameworks and invariant regions may also be used. The antibodies can be produced using various techniques known in the art. For example, in vitro methods can be used in recombinant libraries of bacteriophages in which human antibody fragments are presented (eg, Hoogenboom & Winter, J. Mol. Biol. 227: 381 (1991). Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, eg, mice in which the endogenous immunoglobulin gene is partially or completely inactivated. The approach is described, for example, in U.S. Patent Nos. 6,150,584, 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016.

The antibody thus obtained can be purified to homogeneity. For example, the separation and purification of antibodies can be carried out according to the separation and purification methods used for common proteins. For example, the use of appropriately selected and mixed column chromatography, such as affinity chromatography, filtration, ultrafiltration, salting-out, dialysis, SDS polyacrylamide gel electrophoresis And by isoelectric focusing, the antibody can be separated and isolated (Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory (1988)]. Protein A and Protein G columns can be used as affinity columns. Exemplary Protein A columns used include, for example, Hyper D, POROS and Sepharose F. F. (Pharmacia).

Examples of chromatography except affinity include, for example, ion-exchange chromatography, hydrophobic chromatography, gel filtration, reverse-phase chromatography, adsorption. Chromatography and the like [Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press (1996). Chromatography procedures such as HPLC and FPLC can be performed by liquid-phase chromatography.

For example, measurement of absorbance, enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA), and / or immunofluorescence are used to measure the antigen binding activity of the antibody of the present invention Can be used. In an ELISA, an antibody of the present invention is immobilized on a plate, a peptide of the present invention is added to a plate, and a sample containing a desired antibody such as a culture supernatant or a purified antibody of the antibody producing cell is added. Then, the secondary antibody labeled with an enzyme such as alkaline phosphatase is recognized and the plate is cultured. Then, after washing, an enzyme substrate such as p-nitrophenyl phosphate is applied to the plate, and the degree of absorption is measured to evaluate the antigen binding activity of the sample. Fragments of peptides, such as C-terminal or N-terminal fragments, can be used as antigens to assess the binding activity of an antibody. Biacore [BIAcore (Pharmacia)] may be used to assess the activity of an antibody according to the present invention.

The method allows detection or measurement of the peptide of the invention and exposure or measurement of the immune complex formed by the antibody and the peptide by exposing the antibody of the invention to a sample assumed to contain the peptide of the invention.

Since the method of detecting or measuring a peptide according to the present invention can specifically detect or specify a peptide, this method will be useful in various experiments using the peptide.

XII . Vector and host cell

The invention also provides vectors and host cells into which the nucleotides encoding the peptides of the invention are introduced. The vectors of the invention will be used to retain nucleotides, particularly the DNA of the invention, in a host cell, and may be useful for expressing the peptides of the invention or for administering nucleotides of the invention for gene therapy.

When E. coli is the host cell and the vector is amplified and produced in bulk in E. coli (eg, JM109, DH5 alpha, HB101 or XL1Blue), the vector is "ori" to be amplified in E. coli. And drug resistance screened by drugs such as genetic markers for selecting transformed E. coli (eg, ampicillin, tetratracycline, kanamycin, chloramphenicol or the like). Gene]. For example, M13-series vectors, pUC-series vectors, pBR322, pBluescript, pCR-Script and the like can be used. In addition, pGEM-T, pDIRECT and pT7 can also be used for subcloning and extracting the vectors described above as well as cDNA. When the vector is used to produce the protein of the present invention, the expression vector is particularly useful.

For example, expression vectors expressed in E. coli must have the above characteristics to be amplified in E. coli. When using E. coli such as JM109, DH5 alpha, HB101 or XL1 Blue as host cells, the vector is a promoter that efficiently expresses the desired gene in E. coli, for example the lacZ promoter [Ward et al., Nature 341]. : 544-6 (1989); FASEB J 6: 2422-7 (1992)], araB promoter (Better et al., Science 240: 1041-3 (1988)), T7 promoter or the like. In this respect, for example, pGEX-5X-1 (Pharmacia), “QIAexpress system” (Qiagen), pEGFP and pET (in this case, the host cell is BL21 preferentially expressing T7 RNA polymerase) is the vector Can be used instead. In addition, the vector may include a signal sequence for peptide secretion. An example of a signal sequence that directs a 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.

E. coli, as well as, for example, expression vectors derived from mammals (eg, pcDNA3 (Invitrogen) and pEGF-BOS (Nucleic Acids Res 18 (17): 5322 (1990)), pEF, pCDM8), insects Expression vectors derived from cells (eg, "Bac-to-BAC baculovirus expression system" (GIBCO BRL), pBacPAK8), expression vectors derived from plants (eg pMH1, pMH2), animal viruses Expression vectors (eg pHSV, pMV, pAdexLcw), expression vectors derived from retroviruses (eg pZIpneo), expression vectors derived from yeast [eg, "Pichia Expression Kit" (Invitrogen), pNV11 , SP-Q01] and expression vectors derived from Bacillus subtilis (eg, pPL608, pKTH50) can be used to produce the polypeptides of the invention.

In order to express a vector in an animal cell, such as a CHO, COS or NIH3T3 cell, the vector is a promoter for expression in the cell, for example the SV40 promoter (Mulligan et al., Nature 277: 108 (1979)), MMLV- LTR promoter, EF1 alpha promoter (Mizushima et al., Nucleic Acids Res 18: 5322 (1990)), CMV promoters and the like, preferably genetic markers for screening transformants [eg, drugs drug resistance genes selected by (neomycin, G418). Examples of known vectors having this feature 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 those skilled in the art in making and using the same. The examples are not intended to limit the scope of the invention in any way.

Example

Materials and methods

Cell line

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

CLUAP1  Candidate Selection of Derived Peptides

CLUAP1-derived 9-mer and 10-mer peptides that bind to HLA-A * 2402 or HLA-A * 0201 molecules are “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 from Biosynthesis (Lewisville, Texas) according to a standard solid phase synthesis method and purified by reversed phase high performance liquid chromatography (HPLC). Purity (> 90%) and identity of the peptide were determined by analytical HPLC and mass spectrometry analysis, respectively. Peptides were dissolved at dimethyl sulfoxide (DMSO) at 20 mg / ml and stored at -80 ° C.

In vitro CTL  Judo

Monocyte-derived dendritic cells (DCs) were used as antigen-presenting cells (APCs) to induce CTL responses against peptides presented on human leukocyte antigens (HLA). DC was produced by known methods (Nakahara S et al., Cancer Res 2003 Jul 15, 63 (14): 4112-8). In particular, peripheral blood mononuclear cells (PBMC) isolated from normal volunteers (HLA-A * 2402 or HLA-A * 0201 positive) by Ficoll-Plaque (Pharmacia) solution were prepared from plastic tissue culture dishes (Becton Dickinson). Separation using attachment to yield monocyte fractions. Populations rich in monocytes were treated with AIM containing 1,000 U / ml of granulocyte-macrophage colony-stimulating factor (R & D System) and 2% heat-inactivated autologous serum (AS). Cultured in the presence of 1,000 U / ml of IL (interleukin) -4 (R & D System) in -V medium (Invitrogen). After 7 days of culture, 20 μg of each peptide synthesized to cytokine-induced DCs in the presence of 3 μg / ml of beta 2-microglobulin in AIM-V medium at 37 ° C. for 3 hours. / ml was imposed.

The resulting cells appeared to express DC-related molecules such as CD80, CD83, CD86 and HLA type II on their surface (data not shown). The peptide-loaded DC was inactivated by X-radiation (20 Gy) and mixed with its CD8 + T cells in a 1:20 ratio, by positive selection using a CD8 positive Isolation Kit (Dynal). Obtained. The culture was performed in 48-well plates (Corning); Each well contained 10 ng / ml of 1.5 × 10 ⁴ peptide-loaded DC, 3 × 10 ⁵ CD8 + T cells and IL-7 (R & D system) in 0.5 ml of AIM-V / 2% AS medium. After 3 days, IL-2 (CHIRON) was added to the culture so that the final concentration was 20 IU / ml. On days 7 and 14, the T cells were also stimulated with their peptide-loaded DCs.

The DC was prepared at each time in the same manner described above. CTLs were examined on peptide-pulsed TISI cells (A24) or T2 cells (A2) after the third peptide stimulation on day 21 (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  Multiplication process

CTLs were grown in culture using a method similar to that described by Riddell et al. (Walter EA et al., N Engl J Med 1995 Oct 19, 333 (16): 1038-44; Riddell SR et al., Nat Med 1996 Feb, 2 (2): 216-23). In the presence of 40 ng / ml of anti-CD3 monoclonal antibody (Pharmingen), a total of 5 x 10 ⁴ CTLs with two kinds of human B lymphoblasts inactivated by mitomycin C were added to AIM-V / Suspended in 25 ml of 5% AS medium. One day after the start of the culture, 120 IU / ml of IL-2 was added to the culture. On days 5, 8 and 11 fresh AIM-V / 5% AS medium containing 30 IU / ml of 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).

CTL  Establishment of clone

Dilutions were made to 96, round-bottomed micro titer plates (Nalge Nunc International) to 0.3, 1 and 3 CTL per well. CTL containing 5% AS, with two human B lymphoblastoid cell lines of 1 × 10 ⁴ cells / well, 30 ng / ml anti-CD3 antibody and IL-2 125 U / ml Incubated in a total of 150 μl / well of AIM-V medium. After 10 days, 50 μl / well of IL-2 was added to the medium so that the final concentration of IL-2 was 125 U / ml. CTL activity was tested at day 14 and CTL clones were propagated using the same method as 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  activation

In order to investigate specific CTL activity, interferon-gamma (IFN-γ) enzyme-linked immunospot (ELISPOT) assay and IFN-gamma enzyme-linked immunosorbent assay (ELISA) were performed. Specifically, peptide-added TISI (A24) or T2 (A2) (1 × 10 ⁴ / well) was prepared as stimulator cells. Cells cultured in 48 wells were used as response cells. IFN-gamma ELISPOT assays and IFN-gamma ELISA assays were performed according to the manufacturer's procedure.

The target gene and HLA -A24 or HLA Establishment of cells that allow expression of one or both of A2

CDNA encoding the target gene or the open reading frame of HLA-A24 or HLA-A2 was amplified by PCR. The PCR-amplified product was cloned into a vector. Using the lipofectamine 2000 (Invitrogen) according to the procedure provided by the manufacturer, the plasmid was transfected with the target gene and COS7, the HLA-A24 and HLA-A2 null cell lines. Two days after transfection, the transfected cells were harvested using versene (Invitrogen) and used as target cells for CTL activity testing (5 × 10 ⁴ cells / well).

Result 1

From cancer Increased CLUAP1  Expression

Total gene expression profile data obtained from various cancers using cDNA-microarray showed increased expression of CLUAP1 (GenBank Accession No. NM_015041 or NM_024793; for example SEQ ID NO: 44). CLUAP1 expression was compared to normal cells in 17 of 77 breast cancers, 15 of 19 cervical cancers, 12 of 19 colorectal cancers, and esophageal cancers. 12 out of 19, 7 out of 25 gastric cancers, 5 out of 5 gastric diffuse-type cancers, 5 out of 18 lymphomas, neuroblastoma Effectively increased in 2 out of 16 and 4 out of 9 pancreatic cancers (Table 1).

Compared to normal tissue In cancerous tissue  Observed CLUAP1 Percentage of Upward Adjustment Example Cancer / Tumor ratio Breast cancer 17/77 Cervical cancer 15/19 Colon-Rectal Cancer 12/19 Esophageal Cancer 12/19 Stomach cancer 7/25 Widespread-stomach cancer 5/5 Lymph cancer 5/18 Nerve cell cancer 2/16 Pancreatic cancer 4/9

Result 2

CLUAP1  origin HLA Prediction of Peptide Binding to -A24

Tables 2a and 2b show the 9-mers and 10-mers of CLUAP1 binding to HLA-A24 in order of high binding affinity. Peptides of SEQ ID NOs: 1 to 11 and SEQ ID NOs: 15 to 22 were predicted by BIMAS, and SEQ ID NOs: 12 to 14 were predicted by NetMHC 3.0. A total of 22 peptides with potential HLA-A24 binding capacity were selected and examined to determine the epitope peptides.

The starting position represents the number of amino acid residues from the N-terminus of CLUAP1.

The binding score and dissociation constant (Kd (nM)) are derived from "BIMAS" and "NetMHC3.0".

HLA Limited to -A * 2402 CLUAP1  As a derived predicted peptide CTL  Judo

CTLs for these peptides derived from CLUAP1 were prepared according to the protocol described in “Materials and Methods”. Peptide specific CTL activity was determined by IFN-gamma ELISPOT analysis (FIGS. 1A-J). The following well numbers showed potent IFN-gamma production compared to the control: well number # 6 (a), CLUAP1-A24-9-283 (SEQ ID NO: 4) stimulated with CLUAP1-A24-9-255 (SEQ ID NO: 3) # 2 (b) stimulated with # 2 (c) stimulated with CLUAP1-A24-9-31 (SEQ ID NO: 5), # 4 (d) stimulated with CLUAP1-A24-9-168 (SEQ ID NO: 8) , # 4 (e) stimulated with CLUAP1-A24-9-64 (SEQ ID NO: 9), # 1 (f) stimulated with CLUAP1-A24-9-216 (SEQ ID NO: 10), CLUAP1-A24-9-154 Stimulated with # 6 (g) stimulated with (SEQ ID NO: 14), # 2 (h) stimulated with CLUAP1-A24-10-91 (SEQ ID NO: 15), CLUAP1-A24-10-104 (SEQ ID NO: 16) # 4 (i), # 5 (j) stimulated with CLUAP1-A24-10-125 (SEQ ID NO: 18). On the other hand, although the peptide had possible binding activity to HLA-A * 2402, specific CTL activity could not be detected by the other peptide stimuli shown in Tables 2a and 2b. For example, as a representative example of negative data, no specific IFN-gamma production was observed from CTL stimulated with CLUAP1-A24-9-258 (SEQ ID NO: 1) (FIG. 1K). As a result, 10 peptides derived from CLUAP1 were selected as peptides capable of inducing strong CTLs.

CTL  Cell line establishment and CLUAP1  Clones for derived peptides ( clones )

Well number # 6 (a), CLUAP1-A24-9-168 (SEQ ID NO: 8) with CLUAP1-A24-9-255 (SEQ ID NO: 3) exhibited peptide specific CTL activity detected by IFN-gamma ELISPOT assay # 4 (b), # 4 (c) with CLUAP1-A24-9-64 (SEQ ID NO: 9), # 6 (d) with CLUAP1-A24-9-154 (SEQ ID NO: 14), CLUAP1-A24 In # 2 (e) with -10-91 (SEQ ID NO: 15), the cells were expanded and CTL cell lines were established with limiting dilution as described in the section “Materials and Methods” above. CTL activity of CTL cell lines was detected by IFN-gamma ELISA assay (FIGS. 2A-E). All CTLs show potent IFN-gamma production for target cells to which the corresponding peptide was added as compared to target cells to which no peptide was added. In addition, CTL clones were established by limiting dilution from CTL cell lines as described in the partial “Materials and Methods” above, and IFN-gamma production from CTL clones for peptide-pulsed target cells was analyzed by IFN-gamma ELISA analysis. Detected. Strong IFN-gamma production was stimulated with 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) in FIG. Detection was from CTL clones.

CLUAP1  And HLA Specific for target cells expressing -A * 2402 CTL  activation

The established CTL cell lines formed for these peptides were investigated for their ability to recognize target cells endogenously expressing CLUAP1 and HLA-A * 2402 molecules. Specific for full-length transfected COS7 cells (specific model for target cells exogenously expressing CLUAP1 and HLA-A * 2402 genes) of both CLUAP1 and HLA-A * 2402 molecules Red CTL activity was tested using CTL cell lines formed with the corresponding peptides. COS7 cells transformed with either the CLUAP1 gene or the full length of 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 for the control group. Thus, these data clearly demonstrate that the peptides of CLUAP1-A24-9-255 (SEQ ID NO: 3) are naturally expressed in target cells with the HLA-A * 2402 molecule and are recognized by CTL. These results indicate that such peptides derived from CLUAP1 may be suitable as cancer vaccines for patients with tumors expressing CLUAP1.

Of antigen peptide 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: No. 15), CTLs stimulated with CLUAP1-A24-10-104 (SEQ ID NO: 16) and CLUAP1-A24-10-125 (SEQ ID NO: 18) showed significant and specific CTL activity. The result is 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- The sequences of 91 (SEQ ID NO: 15), CLUAP1-A24-10-104 (SEQ ID NO: 16), and CLUAP1-A24-10-125 (SEQ ID NO: 18) differ from peptides derived from other molecules known to sensitize the human immune system. This may be due to the fact of having same-sex. To rule out this possibility, homology analyzes were performed. This peptide sequence was analyzed using the BLAST algorithm (http://www.ncbi.nlm.nih.gov/blast/blast.cgi), which did not show sequences with significant homology. The results of homology analysis were 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 The sequences of -10-91 (SEQ ID NO: 15), CLUAP1-A24-10-104 (SEQ ID NO: 16), and CLUAP1-A24-10-125 (SEQ ID NO: 18) are unique, so to our knowledge, this molecule Indicates that there is little chance of causing an unintended immune response to any unrelated molecule. In conclusion, new HLA-A24 epitope peptides derived from CLUAP1 have been identified. In addition, the results of the present invention demonstrate that the epitope peptide of CLUAP1 may be suitable for use in cancer immunotherapy.

Conclusion 3

CLUAP1  origin HLA Prediction of Peptides Binding to -A02

Tables 3a and 3b show the 9 and 10mer peptides of CLUAP1 binding to HLA-A02 in order of high binding affinity. A total of 21 peptides with potential HLA-A02 binding capacity were screened and examined to determine the epitope peptides.

The starting position represents the number of amino acid residues from the N-terminus of CLUAP1.

The binding score is derived from "BIMAS".

HLA -A * Limited to 0201 CLUAP1  By the resulting predicted peptide CTL  Judo

CTLs for CLUAP1 derived peptides were prepared according to the protocol of “Materials and Methods” above. Peptide specific CTL activity was determined by IFN-gamma ELISPOT assay (FIGS. 5A-G). The following well numbers demonstrated potent IFN-gamma production compared to the control: # 3 (a) with CLUAP1-A02-9-34 (SEQ ID NO: 23), with CLUAP1-A02-9-99 (SEQ ID NO: 30) # 6 (b), # 2 (c) with CLUAP1-A02-10-153 (SEQ ID NO: 33), # 3 (d) with CLUAP1-A02-10-85 (SEQ ID NO: 34), CLUAP1-A02- # 2 (e) with 10-34 (SEQ ID NO: 35), # 2 (f) with CLUAP1-A02-10-33 (SEQ ID NO: 36), and CLUAP1-A02-10-80 (SEQ ID NO: 38) # 5 (g). On the other hand, although the peptide had possible binding activity to HLA-A * 0201, no specific CTL activity was detected by the other peptide stimuli shown in Tables 3a and 3b. As a representative of negative data, no specific IFN-gamma production was observed from CTL stimulated with CLUAP1-A02-9-58 (SEQ ID NO: 24) (h). As a result, seven peptides derived from CLUAP1 were selected as peptides capable of inducing strong CTL.

CLUAP1  For the derived peptides CTL  Establishment of cell lines and clones

# 3 (a) with well number CLUAP1-A02-9-34 (SEQ ID NO: 23), CLUAP1-A02-9-99 (SEQ ID NO: 30), which showed peptide specific CTL activity detected by IFN-gamma ELISPOT assay # 6 (b) with CLUAP1-A02-10-153 (SEQ ID NO: 33), # 2 (c) with CLUAP1-A02-10-85 (SEQ ID NO: 34), CLUAP1- The cells of # 2 (e) with A02-10-34 (SEQ ID NO: 35) and # 2 (f) with CLUAP1-A02-10-33 (SEQ ID NO: 36) were proliferated, and the CTL cell line " Materials and methods. " CTL activity of these CTL cell lines was detected by IFN-gamma ELISA assay (FIGS. 6A-F). The CTL showed potent IFN-gamma production for target cells added with the peptide as compared to target cells without peptide addition. In addition, the CTL clones were established by limiting dilution from the CTL cell lines described in “Materials and Methods” described above, and IFN-gamma production was detected by IFN-gamma ELISA analysis from CTL clones against peptide added target cells. Strong IFN-gamma production was achieved by 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) (c) and CTL clones stimulated with CLUAP1-A02-10-85 (SEQ ID NO: 34) (d) were identified (FIGS. 7A-D).

CLUAP1  And HLA -A * 0201  Specific for exogenously expressed target cells CTL  activation

The established CTL cell lines and clones formed for each peptide were examined for their ability to recognize target cells endogenously expressing CLUAP1 and HLA-A * 0201. Full length of the CLUAP1 and HLA-A * 0201 genes for the peptides corresponding to the specific CTL activity for the transfected COS7 cells (specific model for target cells exogenously expressing the CLUAP1 and HLA-A * 0201 genes) The CTL cell lines and clones formed were tested. COS7 cells transfected with full length of CLUAP1 or HLA-A * 0201, respectively, were prepared as controls.

In Figure 8, the 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 clones stimulated with CLUAP1-A02-10-33 (SEQ ID NO: 36) (d) showed potent CTL activity against COS7 cells expressing both CLUAP1 and HLA-A * 0201. On the other hand, no significant specific CTL activity was detected for the control. Thus, these data are described in 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 clearly demonstrate that the peptides of CLUAP1-A02-10-33 (SEQ ID NO: 36) (d) are endogenously processed, presented with their HLA-A * 0201 molecules to their target cells, and recognized by CTL It was. These results indicate that the CLUAP1-derived peptide can be applied as a cancer vaccine for patients with tumors expressing CLUAP1.

Of antigen peptide 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-A02-10-80 (SEQ ID NO: 38) are significant and specific. CTL activity was shown. These results include 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-A02-10-80 (SEQ ID NO: 38) This may be due to homology with peptides derived from other molecules known to be sensitive. To rule out this possibility, these peptide sequences were applied to the BLAST algorithm (www.ncbi.nlm.nih.gov/blast/blast.cgi) queries that did not exhibit significant homologous sequences. Homology analysis was performed. The results of homology analysis were 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-A02-10-80 (SEQ ID NO: 38) are specific and. There is little chance that this molecule can cause an unintended immune response to any unrelated molecule.

In conclusion, a novel CLUAP1 derived HLA-A * 0201 epitope peptide was identified. It has also been demonstrated that the novel epitope peptides of CLUAP1 are applied for cancer immunotherapy.

The present invention provides new TAAs, in particular those derived from CLUAP1, which will induce potent and specific anti-tumor immune responses and have applicability to various types of cancer. Such TAA may be used for CLUAP1-related diseases, for example cancer, more specifically breast cancer, cervical cancer, colon-colorectal cancer, esophageal cancer, gastric cancer, It may be useful as a peptide vaccine against gastric diffuse-type cancer, lymphoma, neuroblastoma and pancreatic cancer.

While the invention has been described in detail herein in conjunction with specific embodiments thereof, it should be understood that the foregoing description is exemplary and illustrative, and is intended to illustrate the invention and the preferred embodiments. Through ordinary experimental methods, those skilled in the art will recognize that various changes and modifications can be readily made within the spirit and scope of the invention, and within the boundaries and limits defined by the appended claims. Accordingly, the metes and boundaries of the present invention are intended to be defined by the appended claims.

<110> ONCOTHERAPY SCIENCE, INC. <120> CLUAP1 PEPTIDES AND VACCINES INCLUDING THE SAME <130> 12FPI-09-07 <150> US 61 / 322,099 <151> 2010-04-08 <150> PCT / JP2010 / 006313 <151> 2010-10-26 <160> 49 <170> PatentIn version 3.5 <210> 1 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> An artificially synthesized peptide sequence <400> 1 Thr Tyr Leu Glu Lys Phe Gln Asn Leu   1 5 <210> 2 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> An artificially synthesized peptide sequence <400> 2 Glu Tyr Glu Lys Thr Glu Glu Glu Leu   1 5 <210> 3 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> An artificially synthesized peptide sequence <400> 3 Gln Tyr Asp Thr Tyr Leu Glu Lys Phe   1 5 <210> 4 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> An artificially synthesized peptide sequence <400> 4 Arg Phe Glu Glu Ala Lys Asn Thr Leu   1 5 <210> 5 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> An artificially synthesized peptide sequence <400> 5 Asn Phe Gly Leu Val Ser Glu Val Leu   1 5 <210> 6 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> An artificially synthesized peptide sequence <400> 6 Asn Phe Thr Glu Met Met Arg Ala Leu   1 5 <210> 7 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> An artificially synthesized peptide sequence <400> 7 Asn Phe Arg Thr Pro Asn Phe Gly Leu   1 5 <210> 8 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> An artificially synthesized peptide sequence <400> 8 Arg Thr Glu Ala Ile Ala Arg Pro Leu   1 5 <210> 9 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> An artificially synthesized peptide sequence <400> 9 Phe Phe Ile Lys Ala Ile Ala Gln Phe   1 5 <210> 10 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> An artificially synthesized peptide sequence <400> 10 Lys Ile Glu Lys Arg Lys Leu Glu Leu   1 5 <210> 11 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> An artificially synthesized peptide sequence <400> 11 Lys Pro Gln Thr Ala Met Glu Met Leu   1 5 <210> 12 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> An artificially synthesized peptide sequence <400> 12 Leu Tyr Gln Ala Asp Gly Tyr Ala Val   1 5 <210> 13 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> An artificially synthesized peptide sequence <400> 13 Gln Phe Met Ala Thr Lys Ala His Ile   1 5 <210> 14 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> An artificially synthesized peptide sequence <400> 14 Leu Tyr Asp Leu Leu Gly Met Glu Val   1 5 <210> 15 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> An artificially synthesized peptide sequence <400> 15 Gly Tyr Ala Val Lys Glu Leu Leu Lys Ile   1 5 10 <210> 16 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> An artificially synthesized peptide sequence <400> 16 Leu Tyr Asn Ala Met Lys Thr Lys Gly Met   1 5 10 <210> 17 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> An artificially synthesized peptide sequence <400> 17 Asn Phe Gly Leu Val Ser Glu Val Leu Leu   1 5 10 <210> 18 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> An artificially synthesized peptide sequence <400> 18 Lys Phe Lys Phe Asp Leu Gly Ser Lys Ile   1 5 10 <210> 19 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> An artificially synthesized peptide sequence <400> 19 Gly Tyr Pro Arg His Ile Ser Met Glu Asn   1 5 10 <210> 20 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> An artificially synthesized peptide sequence <400> 20 Asn Thr Leu Cys Leu Ile Gln Asn Lys Leu   1 5 10 <210> 21 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> An artificially synthesized peptide sequence <400> 21 Thr Tyr Leu Glu Lys Phe Gln Asn Leu Thr   1 5 10 <210> 22 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> An artificially synthesized peptide sequence <400> 22 Val Phe Phe Ile Lys Ala Ile Ala Gln Phe   1 5 10 <210> 23 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> An artificially synthesized peptide sequence <400> 23 Leu Val Ser Glu Val Leu Leu Trp Leu   1 5 <210> 24 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> An artificially synthesized peptide sequence <400> 24 Thr Glu Gln Asp Arg Val Phe Phe Ile   1 5 <210> 25 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> An artificially synthesized peptide sequence <400> 25 Lys Glu Leu Leu Lys Ile Thr Ser Val   1 5 <210> 26 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> An artificially synthesized peptide sequence <400> 26 Lys Leu Tyr Gln Ala Asp Gly Tyr Ala   1 5 <210> 27 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> An artificially synthesized peptide sequence <400> 27 Thr Leu Cys Leu Ile Gln Asn Lys Leu   1 5 <210> 28 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> An artificially synthesized peptide sequence <400> 28 Asn Leu Thr Tyr Leu Glu Gln Gln Leu   1 5 <210> 29 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> An artificially synthesized peptide sequence <400> 29 Lys Leu Lys Glu Glu Glu Lys Arg Leu   1 5 <210> 30 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> An artificially synthesized peptide sequence <400> 30 Lys Ile Thr Ser Val Leu Tyr Asn Ala   1 5 <210> 31 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> An artificially synthesized peptide sequence <400> 31 Ala Ile Lys Glu Ile Leu Thr Gln Val   1 5 <210> 32 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> An artificially synthesized peptide sequence <400> 32 Glu Leu Leu Lys Ile Thr Ser Val Leu   1 5 <210> 33 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> An artificially synthesized peptide sequence <400> 33 Ser Leu Tyr Asp Leu Leu Gly Met Glu Val   1 5 10 <210> 34 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> An artificially synthesized peptide sequence <400> 34 Lys Leu Tyr Gln Ala Asp Gly Tyr Ala Val   1 5 10 <210> 35 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> An artificially synthesized peptide sequence <400> 35 Leu Val Ser Glu Val Leu Leu Trp Leu Val   1 5 10 <210> 36 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> An artificially synthesized peptide sequence <400> 36 Gly Leu Val Ser Glu Val Leu Leu Trp Leu   1 5 10 <210> 37 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> An artificially synthesized peptide sequence <400> 37 Phe Met Ala Thr Lys Ala His Ile Lys Leu   1 5 10 <210> 38 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> An artificially synthesized peptide sequence <400> 38 Lys Leu Asn Thr Lys Lys Leu Tyr Gln Ala   1 5 10 <210> 39 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> An artificially synthesized peptide sequence <400> 39 Trp Leu Val Lys Arg Tyr Glu Pro Gln Thr   1 5 10 <210> 40 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> An artificially synthesized peptide sequence <400> 40 Thr Leu Gln Ser Val Arg Pro Cys Phe Met   1 5 10 <210> 41 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> An artificially synthesized peptide sequence <400> 41 Lys Val Met Arg Ile Ala Ile Lys Glu Ile   1 5 10 <210> 42 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> An artificially synthesized peptide sequence <400> 42 Leu Met Gln Gly Arg Pro Gly Lys Arg Ile   1 5 10 <210> 43 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> An artificially synthesized peptide sequence <400> 43 Leu Glu Leu Glu Arg Asn Arg Lys Arg Leu   1 5 10 <210> 44 <211> 1681 <212> DNA <213> Homo sapiens <400> 44 gccgtccaag ggtccattgg ttgccataga gatcgtcgag cgctgggcct gtgatcgctg 60 aggggcgagc agttgcgacc ctgggctcct ggggacctga gcgttatgtc tttccgcgac 120 ctccgcaatt tcacagagat gatgagagcc ctgggatacc ctcgacatat ttctatggaa 180 aatttccgta cacccaattt tggacttgta tctgaagtgc ttctctggct tgtgaaaaga 240 tatgagcccc agactgacat cccgcctgac gtggatactg aacaggaccg agttttcttc 300 attaaggcaa ttgcccagtt catggccacc aaggcacata taaaactcaa cactaagaag 360 ctttatcaag cagatgggta tgcggtaaaa gagctgctga agatcacatc tgtcctttat 420 aatgctatga agaccaaggg gatggagggc tctgaaatag tagaggaaga tgtcaacaag 480 ttcaagtttg atcttggctc aaagattgca gatttgaagg cagccaggca gcttgcgtct 540 gaaatcacct ccaaaggagc atctctgtat gacttgctcg gcatggaagt agagttgagg 600 gaaatgagaa cagaagccat tgccagacct ctggaaataa acgagactga aaaagtgatg 660 agaattgcaa taaaagagat tttgacacag gttcagaaga ctaaagacct gctcaataat 720 gtggcctctg atgaagctaa tttagaagcc aaaatcgaaa agagaaaatt agaactggaa 780 agaaatcgga agcgactaga gactctgcag agtgtcaggc catgttttat ggatgagtat 840 gagaagactg aggaagaatt acaaaagcag tatgacactt atctggagaa atttcaaaat 900 ctgacttatc tggaacaaca gcttgaagac catcatagga tggagcaaga aaggtttgag 960 gaagctaaaa acactctctg cctgatacag aacaagctca aggaggaaga gaagcgcctg 1020 ctcaagagtg gaagtaacga tgactcggac atagacatcc aggaggacga tgaatccgac 1080 agtgagttgg aagaaaggcg gctgcccaag ccacagacag ccatggagat gctcatgcaa 1140 ggaagacctg gcaaacgcat tgtgggcacg atgcaaggtg gagactccga tgacaatgag 1200 gactcggagg agagtgaaat tgacatggaa gatgatgatg acgaggatga cgatttggaa 1260 gacgagagca tttctctctc accaaccaag cccaatcgaa gggtccggaa atctgaaccc 1320 ctggatgaga gtgacaatga cttctgaccc ttttgccaag ggaccctggc agattaaaac 1380 cctcagactt gtaggtaaat gggaacttag aaggttagga aggtaacccc tgttttgttt 1440 actaagctgg ctggactcat gatcactgaa gcaatactta tttctgcttt agcctcctat 1500 gtttgcattc catgaagctt aaataagaat tgaagcaaat ccctaagatt tatttttttc 1560 caccttattt atcttctaaa acttgaggaa tgcatgtgtt cttagtgatt cacatccacg 1620 ggacaaaaac tcaagaagaa ataagagctg acgccacaca aaaaaaaaaa aaaaaaaaaa 1680 a 1681 <210> 45 <211> 413 <212> PRT <213> Homo sapiens <400> 45 Met Ser Phe Arg Asp Leu Arg Asn Phe Thr Glu Met Met Arg Ala Leu   1 5 10 15 Gly Tyr Pro Arg His Ile Ser Met Glu Asn Phe Arg Thr Pro Asn Phe              20 25 30 Gly Leu Val Ser Glu Val Leu Leu Trp Leu Val Lys Arg Tyr Glu Pro          35 40 45 Gln Thr Asp Ile Pro Pro Asp Val Asp Thr Glu Gln Asp Arg Val Phe      50 55 60 Phe Ile Lys Ala Ile Ala Gln Phe Met Ala Thr Lys Ala His Ile Lys  65 70 75 80 Leu Asn Thr Lys Lys Leu Tyr Gln Ala Asp Gly Tyr Ala Val Lys Glu                  85 90 95 Leu Leu Lys Ile Thr Ser Val Leu Tyr Asn Ala Met Lys Thr Lys Gly             100 105 110 Met Glu Gly Ser Glu Ile Val Glu Glu Asp Val Asn Lys Phe Lys Phe         115 120 125 Asp Leu Gly Ser Lys Ile Ala Asp Leu Lys Ala Ala Arg Gln Leu Ala     130 135 140 Ser Glu Ile Thr Ser Lys Gly Ala Ser Leu Tyr Asp Leu Leu Gly Met 145 150 155 160 Glu Val Glu Leu Arg Glu Met Arg Thr Glu Ala Ile Ala Arg Pro Leu                 165 170 175 Glu Ile Asn Glu Thr Glu Lys Val Met Arg Ile Ala Ile Lys Glu Ile             180 185 190 Leu Thr Gln Val Gln Lys Thr Lys Asp Leu Leu Asn Asn Val Ala Ser         195 200 205 Asp Glu Ala Asn Leu Glu Ala Lys Ile Glu Lys Arg Lys Leu Glu Leu     210 215 220 Glu Arg Asn Arg Lys Arg Leu Glu Thr Leu Gln Ser Val Arg Pro Cys 225 230 235 240 Phe Met Asp Glu Tyr Glu Lys Thr Glu Glu Glu Leu Gln Lys Gln Tyr                 245 250 255 Asp Thr Tyr Leu Glu Lys Phe Gln Asn Leu Thr Tyr Leu Glu Gln Gln             260 265 270 Leu Glu Asp His His Arg Met Glu Gln Glu Arg Phe Glu Glu Ala Lys         275 280 285 Asn Thr Leu Cys Leu Ile Gln Asn Lys Leu Lys Glu Glu Glu Lys Arg     290 295 300 Leu Leu Lys Ser Gly Ser Asn Asp Asp Ser Asp Ile Asp Ile Gln Glu 305 310 315 320 Asp Asp Glu Ser Asp Ser Glu Leu Glu Glu Arg Arg Leu Pro Lys Pro                 325 330 335 Gln Thr Ala Met Glu Met Leu Met Gln Gly Arg Pro Gly Lys Arg Ile             340 345 350 Val Gly Thr Met Gln Gly Gly Asp Ser Asp Asp Asn Glu Asp Ser Glu         355 360 365 Glu Ser Glu Ile Asp Met Glu Asp Asp Asp Asp Glu Asp Asp Asp Leu     370 375 380 Glu Asp Glu Ser Ile Ser Leu Ser Pro Thr Lys Pro Asn Arg Arg Val 385 390 395 400 Arg Lys Ser Glu Pro Leu Asp Glu Ser Asp Asn Asp Phe                 405 410 <210> 46 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence <400> 46 gtctaccagg cattcgcttc at 22 <210> 47 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence <400> 47 tcagctggac cacagccgca gcgt 24 <210> 48 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence <400> 48 tcagaaatcc tttctcttga c 21 <210> 49 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence <400> 49 ctagcctctg gaatcctttc tctt 24

Claims (23)

An isolated peptide having cytotoxic T lymphocyte (CTL) inducibility consisting of the amino acid sequence of CLUAP1 or an immunologically active fragment thereof.
The isolated peptide of claim 1, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-23 and 25-43.
The isolated peptide of claim 1, wherein one, two or several amino acids are substituted, deleted or added.
The isolated peptide of claim 3, wherein in relation to HLA-A24, the peptide has one or both of the following properties:
(a) the second amino acid from the N-terminus is an amino acid selected from the group consisting of phenylalanine, tyrosine, methionine or tryptophan or modified therewith; And
(b) The C-terminal amino acid is a modified or selected one consisting of phenylalanine, leucine, isoleucine, tryptophan or methionine.
The isolated peptide of claim 3, wherein in relation to HLA-A2, the peptide has one or both of the following properties:
(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 claim 1, wherein the peptide is a nonapeptide or a decapeptide. 7.
An isolated polynucleotide encoding the peptide of any one of claims 1 to 6.
A composition for inducing CTL comprising one or more peptide (s) of any one of claims 1 to 6, or one or more polynucleotide (s) of claim 7.
Treatment and / or prophylaxis of cancer comprising one or more peptide (s) of any one of claims 1 to 6 or one or more of the polynucleotide (s) of claim 7. A pharmaceutical composition for the prevention of relapse after surgery thereof.
The pharmaceutical composition of claim 9, wherein the pharmaceutical composition is formulated for administration to an individual whose HLA antigen is HLA-A24 or HLA-A2.
The pharmaceutical composition according to claim 9 or 10, wherein the pharmaceutical composition is formulated to treat cancer.
A method for inducing CTL inducible antigen-presenting cells (APCs) comprising a step selected from the group consisting of:
(a) contacting the peptide of any one of claims 1 to 6 with APC in vitro, ex vivo or in vivo; And
(b) introducing a polynucleotide encoding the peptide of any one of claims 1 to 6 into the APC.
A method of inducing a CTL by a method comprising a step selected from the group consisting of:
(a) Co-culture of CD8-positive T cells with APC presenting on its surface a complex of a HLA antigen and a peptide of any one of claims 1-6. Making;
(b) co-culture of CD8-positive T cells with an exosome presenting on its surface a complex of HLA antigen and the peptide of any one of claims 1 to 6; And
(c) introducing into a T cell a gene comprising a polynucleotide encoding a T cell receptor (TCR) subunit polypeptide that binds to the peptide of any one of claims 1 to 6 .
An isolated APC presenting on its surface a complex of an HLA antigen and a peptide of any one of claims 1 to 6.
15. The isolated APC of claim 14, which is derived by the method of claim 12.
An isolated CTL targeting the peptide of any one of claims 1 to 6.
The CTL according to claim 16, which is derived by the method of claim 13.
An immune response against cancer in a subject comprising administering to the subject a peptide comprising any one of claims 1 to 6, an immunologically active fragment thereof, or a composition comprising said peptide or polynucleotide encoding said fragment. How to induce.
An antibody or fragment thereof against the peptide according to any one of claims 1 to 6.
A vector consisting of a nucleotide sequence encoding a peptide according to any one of claims 1 to 6.
A host cell transformed or transfected with an expression vector according to claim 20.
A diagnostic kit comprising the peptide according to any one of claims 1 to 6, the polynucleotide of claim 7 or the antibody of claim 19.
The isolated peptide of claims 1 to 6 selected from the group consisting of SEQ ID NOs: 3, 4, 5, 8, 9, 10, 14, 15, 16, 18, 23, 30, 33, 34, 35, 36 and 38 .

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