JP2019535270A - DEPDC1 epitope peptide for Th1 cells and vaccine containing the same - Google Patents

Abstract

Disclosed herein is an isolated DEPDC1-derived epitope peptide capable of inducing Th1 cells. Such peptides are recognized by MHC class II molecules and are capable of inducing Th1 cells. In a preferred embodiment, such a peptide of the invention is capable of binding promiscuous to MHC class II molecules and inducing DEPDC1-specific CTL in addition to Th1 cells. Such peptides are therefore suitable for use in enhancing an immune response in a subject and can therefore be used in cancer immunotherapy, especially as a cancer vaccine. Also disclosed herein are polynucleotides encoding any of the aforementioned peptides, APC and Th1 cells induced by such peptides, and methods of induction associated therewith. Pharmaceutical compositions containing any of the above components as an active ingredient are used, for example, in the treatment and / or prevention of cancer or tumors including bladder cancer and breast cancer. [Selection diagram] None

Description

The present invention relates to the field of biological science, and more particularly to the field of cancer therapy. In particular, the present invention relates to novel peptides that are extremely effective as cancer vaccines, and drugs for either or both of tumor treatment and prevention.
Priority This application claims the benefit of Japanese Patent Application No. 2006-224624 filed on November 18, 2016, the entire contents of which are incorporated herein by reference.

  CD8 positive cytotoxic T lymphocytes (CTL) recognize epitope peptides derived from tumor associated antigens (TAA) found on major histocompatibility complex (MHC) class I molecules and then kill tumor cells It has been shown. Since the discovery of the melanoma antigen (MAGE) family as the first example of TAA, many other TAAs have been discovered, primarily through immunological approaches (1). Some of these TAAs are currently under clinical development as targets for immunotherapy.

  TAA, essential for cancer cell growth and survival, is useful as a target for immunotherapy. Because the use of such TAA minimizes the widely described risk of immune evasion of cancer cells due to TAA deletion, mutation or down-regulation as a result of therapeutically performed immune selection. This is because it can be limited to the limit. Thus, the identification of novel TAAs that can induce strong and specific anti-tumor immune responses warrants further development. Therefore, clinical application of peptide vaccine strategies for various types of cancer is ongoing (Non-Patent Documents 3 to 10). To date, there are several reports of clinical trials using these TAA-derived peptides. Unfortunately, to date, clinical trials of these cancer vaccines have yielded only the low objective response rates that have been accepted in these cancer vaccine trials (Non-Patent Documents 11 to 13). Accordingly, there remains a need in the art for new TAAs that are suitable for use as immunotherapy targets.

In recent years, we have used cancer-testis antigens, DEP domain-containing 1 that are frequently overexpressed in bladder cancer, breast cancer, and some other malignancies using genome-wide cDNA microarray analysis. (DEPDC1) was identified (Non-Patent Documents 14 to 17). Bladder cancer cell growth is significantly inhibited by knockdown of DEPDC1 expression using small interfering RNA; therefore, DEPDC1 is essential for tumor growth and survival, which is antigen-specific. The clinical significance of these antigens as targets for cancer immunotherapy is shown. The present inventors have also been able to induce HLA-A24 (A ^* 24: 02) -restricted CTL from peripheral blood mononuclear cells (PBMC) of bladder cancer patients. A chain peptide (SP) was identified (Patent Document 1, Non-Patent Document 17). In a phase I / II clinical trial of combined immunotherapy with intravesical BCG vaccination and DEPDC1-derived SP, the vaccine induces promising clinical effects on prevention of recurrence of non-muscle invasive bladder cancer, as well as safety and CTL Demonstrated good immunogenicity (Non-patent Document 17). Therefore, DEPDC1 is an attractive target molecule for cancer immunotherapy.

Tumor-specific CD4 ⁺ helper T (Th) cells, particularly T helper type 1 (Th1) cells, play an important role in the effective induction of CTL-mediated anti-tumor immunity (Non-patent Document 18). Interferon (IFN) -γ and tumor necrosis factor (TNF) -α produced primarily by Th1 cells are essential for the induction and maintenance of long-lasting CTL responses and are important for the maintenance of immune memory. Support is provided through action (Non-Patent Documents 19, 20, and 21). IFN-γ secreted by Th1 cells also mediates direct anti-tumor or anti-angiogenic effects (Non-patent Document 22). Furthermore, it has been shown that Th cells may provide a path for CTL invasion at the tumor site (Non-patent Document 23). Recently reported that synthetic LPs derived from human papillomavirus naturally contain CTL epitopes as attractive vaccine compounds for the treatment of vulvar cancer and cervical cancer (24). . Following the injection of LP, the patient's DCs take up LPs, process them, and present possible CTL and Th cell epitopes associated with the patient's HLA class I and class II molecules, respectively (27). . In addition, recent clinical studies using a promiscuous telomerase-derived helper epitope vaccine called GV1001 have increased the survival of cancer patients when combined with radiation therapy and chemotherapy (25). , 26). Therefore, identification of TAA-derived Th cell epitopes that can activate specific Th1 cells is important for the induction of effective tumor immunity in tumor-bearing hosts; ideally, the design of effective vaccines Should contain multiple epitopes that stimulate both CTL and Th1 cells (24). However, such an epitope derived from DEPDC1 has not yet been identified.

WO2008 / 047473

Boon T. Int J Cancer 1993; 54 (2): 177-80 Boon T and van der Bruggen P. J Exp Med 1996; 183 (3): 725-9 Harris CC. J Natl Cancer Inst 1996; 88 (20): 1442-55 Butterfield LH, et al., Cancer Res 1999; 59 (13): 3134-42 Vissers JL, et al., Cancer Res 1999; 59 (21): 5554-9 van der Burg SH, et al., J Immunol 1996; 156 (9): 3308-14 Tanaka F, et al., Cancer Res 1997; 57 (20): 4465-8 Fujie T et al., Int J Cancer 1999; 80 (2): 169-72 Kikuchi M, et al., Int J Cancer 1999; 81 (3): 459-66 Oiso M, et al., Int J Cancer 1999; 81 (3): 387-94 Belli F, et al., J Clin Oncol 2002; 20 (20): 4169-80 Coulie PG, et al., Immunol Rev 2002; 188: 33-42 Rosenberg SA, et al., Nat Med 2004; 10 (9): 909-15 Kanehira M, et al., Oncogene 2007; 26: 6448-55 Harada Y, et al., Cancer Res 2010; 70: 5829-39. Kretschmer C, et al., Mol Cancer 2011; 10: 15 Obara W, et al., Clin Oncol 2012; 42: 591-600. Chamoto K, et al., Cancer Res 2004; 64: 386-90 Bevan MJ. Nat Rev Immunol 2004; 4: 595-602 Shedlock DJ and Shen H. Science 2003; 300: 337-9 Anders K, et al., Cancer Cell 2011; 20: 755-67 Street SE, et al., Blood 2001; 97: 192-7 Bos R, and Sherman LA. Cancer Res 2010; 70: 8368-77 Melief CJ, et al., Nat Rev Cancer 2008; 8: 351-60 Brunsvig PF, et al., Clin Cancer Res 2011; 17: 6847-57 Kyte JA, et al., Clin Cancer Res 2011; 17: 4568-80 Kenter GG, et al., N Engl J Med 2009; 361: 1838-47

  Bladder cancer is the most common malignancy involving the genitourinary system. Platinum-based chemotherapy remains the standard treatment for first line treatment of advanced / metastatic urothelial cancer (UC) (van der Maase H, et al. J Clin Oncol 1996; 156 (9 ): 3308-14; Gupta S, et al., Cancers 2017; 9 (2): pii: E15). In those who receive standard treatment with platinum-based chemotherapy, metastatic bladder cancer has an estimated overall survival of 9-15 months; the 5-year relative survival rate for stage IV bladder cancer is approximately 15% (Van der Maase H, et al., Supra; De Sanis M, et al., J Clin Oncol 2012; 30 (2): 191-9). Until recently, there were few treatment options for patients who were refractory to chemotherapy or who could not tolerate chemotherapy due to serious adverse reactions, and the overall outcome was still very poor (Gupta S , Et al., Supra). Currently, systemic immunotherapy using programmed cell death protein 1 (PD-1) specific monoclonal antibodies and PD-1 ligand (PD-L1) specific monoclonal antibodies (ie, nivolumab / pembrolizumab and atezolizumab, respectively) Approved for the treatment of bladder cancer and has achieved a significant improvement in response rate (Gupta S, et al., Supra; Zichi C, et al., Biomed Res Int 2017; 2017 (561874. Doi: 10.1155 / 2017/56618174)). Another recent clinical trial showed that the DEPDC1-derived SP vaccine administered to patients with advanced UC was safe, well tolerated and effectively activated CTL (Obara W, et al., Ann Oncol 2017; 28 (4): 798-803).

  In the context of the present invention, the inventors have found that an ideal peptide vaccine for cancer immunotherapy is a single poly-peptide containing both CTL and Th1 cell epitopes, both of which are naturally proximal to each other. It was thought to contain peptides.

To that end, we have designed a strategy to identify novel DEPDC1-derived Th1 cell epitopes that are recognized in the context of promiscuous HLA class II molecules, including CTL epitopes, and are thus characterized. The study proceeded under the assumption that the attached epitope more effectively induces T cell-mediated tumor immunity. Using computer algorithms to predict HLA class II binding peptides and known CTL epitope sequences recognized by HLA-A24 (A ^* 24 ^: 02) or HLA-A2 (A ^* 02 ^: 01) restricted CTLs, candidates Promiscuous HLA class II restricted Th1 cell epitopes containing CTL epitopes were selected.

  The present invention is based, at least in part, on the discovery of suitable epitope peptides that serve as targets for immunotherapy to induce a Th1 cell response. Since the DEPDC1 gene has been observed to be upregulated in many cancer types, including bladder cancer and breast cancer, the present invention relates to the gene product of the DEPDC1 gene, more specifically GenBank accession number NM_001114120.2. The polypeptide exemplarily shown in SEQ ID NO: 8 or 10 encoded by the gene of (SEQ ID NO: 7) or accession number NM — 01779.5 (SEQ ID NO: 9) is targeted for further analysis. The DEPDC1 gene product containing an epitope peptide that induces Th1 cells specific for the corresponding molecule was specifically selected for further testing. For example, peripheral blood mononuclear cells (PBMC) obtained from healthy donors were stimulated with promiscuous HLA-DR and / or DP binding peptides derived from human DEPDC1. HLA-DR and / or DP-restricted epitope peptide capable of establishing Th1 cells that recognize HLA-DR or DP positive target cells pulsed with each candidate peptide and inducing a strong and specific immune response against DEPDC1 Was identified. These results demonstrate that DEPDC1 has strong immunogenicity and its epitope is effective for tumor immunotherapy mediated through a Th1 cell response. Additional studies also revealed that promiscuous HLA-DR and / or DP binding peptides containing at least one CTL epitope can stimulate CTL responses specifically in the same donor. These results indicate that DEPDC1 is strongly immunogenic and that tumor DEPDC1-derived peptides containing both Th1 cell and CTL epitopes are mediated by both tumor cells and CTL responses. Confirm that it is effective.

  Therefore, it is an object of the present invention to provide a peptide having an ability to induce Th1 cells and an amino acid sequence selected from SEQ ID NOs: 1 to 4. The present invention is a modified peptide, that is, a length of up to 30 amino acids, and has Th1 cell inducibility having a continuous amino acid sequence selected from the amino acid sequences of SEQ ID NO: 8 or 10 (DEPDCl). Peptides, as well as functional equivalents thereof, are contemplated. Alternatively, the present invention also provides a peptide having both Th1 cell inducibility and CTL inducibility. In some embodiments, the peptides of the invention have the amino acid sequence of SEQ ID NO: 1 to 4 or the ability to induce Th1 cells, with 1, 2 or several amino acid substitutions, deletions, insertions and Corresponds to the modified version that is added.

  When administered to a subject, the peptides of the invention are preferably presented on the surface of one or more antigen presenting cells (APCs), which in turn induce Th1 cells. If the peptides of the present invention further comprise at least one CTL epitope, such APCs also present CTL epitopes generated from the peptides of the present invention and thus induce CTL targeting each peptide Process the peptide. It is therefore a further object of the invention to provide APCs that present any of the peptides of the invention or fragments thereof, as well as methods for inducing APCs.

Administration of one or more peptides of the invention or a polynucleotide encoding such a peptide, or APC presenting such a peptide or fragment thereof results in the induction of a strong anti-tumor immune response. Accordingly, it is yet another object of the present invention to provide a medicament or pharmaceutical composition containing one or more of the following as active ingredients:
(A) one or more peptides of the invention,
(B) one or more polynucleotides encoding such peptides, and (c) one or more APCs of the invention.
Such a medicament or pharmaceutical composition of the present invention is particularly useful as a vaccine.

  It is a still further object of the present invention to provide a method for the treatment and / or prophylaxis (ie prevention) of cancer (ie tumors) and / or prevention of its post-operative recurrence. Also contemplated are methods for inducing Th1 cells or inducing anti-tumor immunity comprising administering one or more peptides, polynucleotides, APCs or agents or pharmaceutical compositions of the invention. Furthermore, the Th1 cell of the present invention is also used as a vaccine against cancer, and examples of cancer include, but are not limited to, bladder cancer and breast cancer.

Examples of particularly contemplated purposes of the invention include the following:
[1] An isolated peptide having a length of 10 to 30 amino acids and comprising a part of the amino acid sequence of SEQ ID NO: 8 or 10,
(A) a continuous amino acid sequence having a length of more than 9 amino acids selected from the amino acid sequences of SEQ ID NO: 1, 2, 3 or 4; and (b) 1, 2 or several in the amino acid sequence of (a) A peptide comprising an amino acid sequence selected from the group consisting of amino acid sequences in which amino acids are substituted, deleted, inserted and / or added and having the ability to induce Th1 cells.
[2] The isolated peptide according to [1], wherein the peptide or a fragment thereof has an ability to bind to at least two types of MHC class II molecules.
[3] The isolated peptide according to [2], wherein the MHC class II molecule is selected from the group consisting of HLA-DR53, HLA-DR4, HLA-DR15, and HLA-DP5.
[4] The isolated peptide according to any one of [1] to [3], comprising an amino acid sequence of a peptide having DEPDC1-specific CTL inducing ability.
[5]
(A) an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 4; and (b) 1, 2 or several amino acids are substituted, deleted, inserted and / or added in the amino acid sequence of (a) The isolated peptide according to [4], comprising an amino acid sequence selected from the group consisting of:
[6] An isolated polynucleotide encoding the peptide according to any one of [1] to [5].
[7]
(I) Th1 cells,
(Ii) CTL,
(Iii) APC having the ability to induce Th1 cells, and (iv) APC having the ability to induce CTL.
A composition for inducing at least one cell selected from the group consisting of one or more peptides according to any one of [1] to [5], or encoding them A composition comprising one or more polynucleotides, or (i) Th1 cells,
(Ii) CTL,
(Iii) APC having the ability to induce Th1 cells, and (iv) APC having the ability to induce CTL.
A composition for inducing at least one cell selected from the group consisting of: one or more peptides according to any one of [1] to [5], or encoding them A composition comprising one or more polynucleotides.
[8]
(A) one or more peptides according to any one of [1] to [5];
(B) one or more polynucleotides according to [6];
(C) one or more APCs that present on their surface the peptide or fragment thereof according to any one of [1] to [5];
(D) one or more Th1 cells recognizing APC presenting on its surface the peptide or fragment thereof according to any one of [1] to [5]; and (e) from (a) above (D) containing at least one active ingredient selected from the group consisting of any two or more combinations of (d), and (i) treatment of cancer,
(Ii) cancer prevention,
(Iii) prevention of postoperative recurrence in cancer, and (iv) formulated for purposes selected from the group consisting of any two or more combinations of (i) to (iii) above , Pharmaceutical composition.
[9] Formulated for administration to a subject having at least one selected from the group consisting of HLA-DR53, HLA-DR4, HLA-DR15 and HLA-DP5 as MHC class II molecules [8] Or a pharmaceutical composition as described in or formulated for administration to a subject having at least one MHC class II molecule selected from the group consisting of HLA-DR53, HLA-DR4, HLA-DR15 and HLA-DP5 [8] The pharmaceutical composition according to [8].
[10] The pharmaceutical composition according to [8] or [9], further comprising one or more peptides having CTL inducibility.
[11] A composition for enhancing an immune response mediated by MHC class II molecules,
(A) one or more peptides according to any one of [1] to [5];
(B) one or more polynucleotides according to [6];
(C) one or more APCs that present on their surface the peptide or fragment thereof according to any one of [1] to [5];
(D) one or more Th1 cells recognizing APC presenting on its surface the peptide or fragment thereof according to any one of [1] to [5]; and (e) from (a) above A composition comprising at least one active ingredient selected from the group consisting of any two or more combinations of (d).
[12] A method for inducing APC having the ability to induce Th1 cells, comprising contacting APC with a peptide according to any one of [1] to [5] in vitro, ex vivo or in vivo Including a method.
[13] A method for inducing APC having the ability to induce CTL comprising:
(A) contacting the APC with the peptide according to any one of [1] to [5] in vitro, ex vivo or in vivo; and (b) according to any one of [1] to [5] A method comprising a step selected from the group consisting of introducing a polynucleotide encoding the peptide of APC into APC.
[14] A method for inducing Th1 cells,
(A) co-culturing CD4-positive T cells with APC that presents a complex of an MHC class II molecule and the peptide or fragment thereof according to any one of [1] to [5] on its surface And (b) introducing a polynucleotide encoding both T cell receptor (TCR) subunits, or a polynucleotide encoding each of the TCR subunits, into a CD4 positive T cell, wherein the TCR is Selected from the group consisting of steps capable of binding to a complex of an MHC class II molecule presented on the cell surface and a peptide or fragment thereof according to any one of [1] to [5] A method or a method for inducing Th1 cells, comprising the steps of:
(A) co-culturing CD4-positive T cells with APC that presents a complex of an MHC class II molecule and the peptide or fragment thereof according to any one of [1] to [5] on its surface And (b) introducing a single polynucleotide encoding both TCR subunits, or a plurality of polynucleotides each encoding a separate TCR subunit, into a CD4 positive T cell, wherein the TCR is Selected from the group consisting of steps capable of binding to a complex of an MHC class II molecule displayed on the cell surface of APC and the peptide or fragment thereof according to any one of [1] to [5] A method comprising the steps of:
[15] A method for inducing CTL,
(A) co-culturing both CD4 positive T cells and CD8 positive T cells with APC contacted with the peptide of [4] or [5]; and (b) CD8 positive T cells [4 Or a method selected from the group consisting of co-culturing with APC contacted with the peptide of [5].
[16] A method for enhancing an immune response mediated by MHC class II molecules comprising:
(A) one or more peptides according to any one of [1] to [5];
(B) one or more polynucleotides according to [6];
(C) one or more APCs that present on their surface the peptide or fragment thereof according to any one of [1] to [5];
(D) one or more Th1 cells recognizing APC presenting on its surface the peptide or fragment thereof according to any one of [1] to [5]; and (e) from (a) above (D)
Administering to the subject at least one active ingredient selected from the group consisting of any two or more combinations of:
[17] An isolated APC that presents on its surface a complex of an MHC class II molecule and the peptide or fragment thereof according to any one of [1] to [5].
[18] APC induced by the method according to [12] or [13].
[19] An isolated Th1 cell that recognizes the peptide or fragment thereof according to any one of [1] to [5] displayed on the surface of APC.
[20] Th1 cells induced by the method according to [14].
[21] The Th1 cell according to [20], wherein the α subunit and β subunit of the TCR contain CDR3 consisting of the amino acid sequences of SEQ ID NOs: 11 and 21, respectively.
[22] A method of inducing an immune response against cancer in a subject in need thereof,
(A) one or more peptides according to any one of [1] to [5];
(B) one or more polynucleotides according to [6];
(C) one or more APCs presenting the peptide or fragment thereof according to any one of [1] to [5] on its surface;
(D) one or more Th1 cells recognizing APC presenting on its surface the peptide or fragment thereof according to any one of [1] to [5]; and (e) from (a) above Administering to the subject a composition comprising at least one active ingredient selected from the group consisting of any two or more combinations of (d).
[23] An antibody against the peptide according to any one of [1] to [5] or an immunologically active fragment thereof.
[24] A vector comprising a nucleotide sequence encoding the peptide according to any one of [1] to [5].
[25] A host cell transformed or transfected with the expression vector according to [24].
[26] A diagnostic kit comprising the peptide according to any one of [1] to [5], the polynucleotide according to [6], or the antibody according to [23].
[27] A method for evaluating and / or monitoring a Th1 response specific for a peptide comprising an amino acid sequence of SEQ ID NO: 1, 2, 3 or 4 in a subject comprising:
(A) providing a sample obtained from a subject administered the peptide, wherein the sample comprises T cells;
(B) detecting the presence of a T cell expressing a TCR that binds to a complex of an MHC class II molecule and the peptide or fragment thereof in the sample; and (c) the presence of the T cell is (b) Wherein the method comprises induction of a Th1 response specific to the peptide when detected in.
[28] The method according to [27], wherein the TCR comprises a specific pair of α subunit and β subunit each comprising CDR3 consisting of the amino acid sequences of SEQ ID NOs: 11 and 21, respectively.

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

  Various aspects and applications of the present invention will become apparent to those skilled in the art after considering the following brief description of the drawings and detailed description of the invention and preferred embodiments thereof.

FIG. 1 shows a promiscuous HLA class II-binding peptide derived from DEPDC1 containing a CTL epitope as predicted by a recently developed computer algorithm. (A) The amino acid sequence of human DEPDC1 protein was analyzed using an algorithm. The numbers on the horizontal axis indicate the amino acid position at the N-terminal of the 15-mer peptide derived from DEPDC1. Lower consensus percentile ranking indicates stronger binding affinity to HLA class II molecules. The black bar indicates the position of the LP described in FIG. 1B. (B) DEPDC1 _191-213 − which is a 22-29 _mer LP with lower consensus percentile ranking for multiple HLA class II alleles (DRB1 ^* 09: 01, DRB1 ^* 04: 05, and DRB1 ^* ₁₅ : 02) products LP (LP-1), DEPDC1 _292-313 -LP (LP-2), DEPDC1 _301-329 -LP (LP-3), and DEPDC1 _613-634 -LP (LP-4), and HLA-A24, respectively DEPDC1-A24 _294-302 and DEPDC1-A2 _302-311 , which are 9-mer peptides recognized by restricted CTL or HLA-A2 restricted CTL, were synthesized. (C) The amino acid sequence of human DEPDC1 protein was analyzed using an algorithm. The numbers on the horizontal axis indicate the amino acid position at the N-terminal of the 15-mer peptide derived from DEPDC1. A lower consensus percentile rank indicates a stronger binding affinity for HLA class II molecules. The black bar indicates the position of the LP described in FIG. 1B.

FIG. 2 shows the induction of DEPDC1-specific Th cells from healthy donors. (A) DEPDC1-specific Th cells were generated from DR53 ⁺ healthy donor (HD1) by stimulation with DEPDC1-LP1. The generated Th cells were restimulated with autologous PBMC or L cells pulsed with DEPDC1-LP1. The number of IFN-γ producing Th cells was analyzed by ELISPOT assay. Representative data from at least three independent experiments with similar results obtained from HD1 are shown. The HLA class II genotype of donor HD1 is shown at the top of the panel. The underlined HLA-class II allele encodes an HLA-class II molecule that presents the peptide to Th cells. (B) DEPDC1-LP1-specific and HLA-DR4-restricted Th cells were generated from healthy donors (HD4) by stimulation with DEPDC1-LP1. (C) DEPDC1-LP2-specific and HLA-DR4-restricted Th cells were generated from healthy donors (HD1) by stimulation with DEPDC1-LP2. (D) DEPDC1-LP3-specific and HLA-DP5-restricted Th cells were generated from healthy donors (HD3) by stimulation with DEPDC1-LP3. (E) DEPDC1-LP3-specific and HLA-DR4-restricted Th cells were generated from healthy donors (HD4) by stimulation with DEPDC1-LP3. (F) DEPDC1-LP4-specific and HLA-DP5-restricted Th cells were generated from healthy donors (HD1) by stimulation with DEPDC1-LP4. (G) DEPDC1-LP4-specific and HLA-DR15 restricted Th cells were generated from healthy donors (HD5) by stimulation with DEPDC1-LP4. (H) DEPDC1-LP4-specific and HLA-DP5-restricted Th cells were generated from healthy donors (HD7) by stimulation with DEPDC1-LP4.

FIG. 3 shows the natural processing and presentation of DEPDC1-LP by DC. (A) HLA-DR4-restricted and DEPDC1-LP2-specific Th cells established from donor HD1 recognized autologous DC loaded with recombinant DEPDC1 protein. Representative data from two independent experiments that showed similar results are shown. (B) HLA-DR4-restricted and DEPDC1-LP3-specific Th cells established from donor HD4 recognized autologous DCs loaded with recombinant DEPDC1 protein. (C) Similarly, the HD7-derived HLA-DP5-restricted and DEPDC1-LP4-specific Th cell clones recognized autologous DC loaded with recombinant human DEPDC1 protein and those that pulsed DEPDC1-LP4. The control protein loaded was not recognized. The Th cell response was substantially reduced in the presence of anti-HLA-DP mAb, but not in the presence of anti-HLA-class I blocking Ab.

FIG. 4 shows that DEPDC1-LP2 induces effective cross-priming of CTL in vivo. HLA-A2 Tgm or HLA-A24 Tgm was immunized with DEPDC1-LP2 emulsified in IFA. After a second vaccination with DEPDC1-LP2, mouse CD8 ⁺ T cells in the inguinal lymph nodes were transferred to DEPDC1-A2 _302-311 SP or HIV-A2 SP (FIG. 4A) and DEPDC1-A24 _294-302 SP or HIV. -Stimulated with BM-DC pulsed with A24 SP (Figure 4B). The number of IFN-γ producing mouse CD8 ⁺ T cells was analyzed by ex vivo ELISPOT assay. Representative data from at least three independent experiments that showed similar results are shown.

FIG. 5 shows various Th1-type cytokine production in DEPDC1-LP specific Th clones. (AD) DEPDC1-LP specific bulk Th cells or Th clones were stimulated with autologous PBMC pulsed with or without cognate peptide. The concentrations of the indicated cytokines in the culture supernatant were measured as described in the “Materials and Methods” section. Data are shown as mean ± SD of triplicate assays. (A) DEPDC1-LP1-specific bulk Th cells were stimulated with autologous PBMC pulsed with or without cognate peptide. (B) DEPDC1-LP2-specific Th clones were stimulated with autologous PBMC pulsed with or without cognate peptide. (C) DEPDC1-LP3-specific bulk Th cells were stimulated with autologous PBMC pulsed with or without cognate peptide. (D) DEPDC1-LP3-specific bulk Th cells were stimulated with autologous PBMC pulsed with or without cognate peptide.

FIG. 6 shows the results of TCR gene analysis of DEPDC1-LP2-specific Th cells. (AB) The DEPDC1-LP2-specific T cell repertoire was analyzed by deep cDNA sequencing of TCR-α and TCR-β genes using a next generation sequencer. TCR-α is shown in the upper panel and TCR-β is shown in the lower panel. The Y axis shows the number of Th cell stimulations with DC pulsed with cognate peptides. Cloning of DEPDC1-LP2-specific bulk Th cells was performed after stimulation of bulk Th cells 5 times with irradiated PBMC pulsed with cognate peptides. The total stimulation frequency was 9 times. The X-axis shows VJ-CDR3 sequences (details are shown in the right square) with the top 10 frequencies, and the Z-axis shows the frequency of individual VJ-CDR3 sequences. (C) TCR-negative mouse T cell line TG40 expressing full length TCR-α and TCR-β genes isolated from DEPDC1-LP2-specific T cell clones was revealed by cell surface expression of CD69 and CD137. As shown, it specifically responded to HLA-DR expressing L cells pulsed with cognate peptides.

FIG. 7 shows the presence of DEPDC1-LP specific Th cells in the patient's PBMC. PBMCs isolated from urothelial cancer (UC) patients were stimulated in vitro with a mixture of DEPDC1-LP1, -2, -3, -4, and IL-2 and IL-7, and individual DEPDC1- The frequency of LP-specific T cells was assessed using ELISPOT. A Th cell response specific for DEPDC1-LP4 was observed in two UC patients (A, B). HLA class II restriction of DEPDC1-LP specific Th cells was determined by blocking assay using monoclonal antibodies specific for HLA-DR or HLA-DP.

DESCRIPTION OF EMBODIMENTS 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, the preferred methods, devices, and materials are now described. Describe. However, before describing the materials and methods of the present invention, the specific sizes, shapes, dimensions, materials, methodologies, protocols, etc. that the present invention describes herein will vary according to routine experimentation and optimization. It should be understood that the present invention is not limited to these as possible. Also, the terminology used herein is for the purpose of describing particular versions or aspects only and is not intended to limit the scope of the invention, which is limited only by the scope of the appended claims. It should also be understood that

  The disclosure of each publication, patent or patent application mentioned in this specification is expressly incorporated herein by reference in its entirety. However, nothing in this specification should be construed as an admission that the invention is not entitled to antedate such disclosure by the prior invention.

I. Definitions Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. However, in case of conflict, the present specification, including definitions, will control.

  As used herein, the terms “a”, “an”, and “the” mean “at least one” unless specifically indicated otherwise.

  The terms “isolated” and “purified” as used with respect to a substance (eg, peptide, antibody, polynucleotide, etc.) refer to at least one substance that may otherwise be contained in a natural source. Indicates that it does not contain substantially. Thus, an isolated or purified peptide is substantially free of cellular material such as carbohydrates, lipids or other contaminating proteins from the cell or tissue source from which the peptide is derived, or is chemically synthesized. Refers to a peptide that is substantially free of chemical precursors or other chemicals.

  The term “substantially free of cellular material” includes preparations of a peptide in which the peptide is cleaved from the cellular components of the cell from which it was isolated or recombinantly produced. Thus, a peptide that is substantially free of cellular material is a polypeptide that has less than about 30%, 20%, 10%, or 5% (dry weight) of a heterologous protein (also referred to herein as “contaminating protein”). Includes preparations. If the peptide is produced recombinantly, the peptide preferably includes substantially no medium, and includes a preparation of peptides comprising less than about 20%, 10% or 5% of the medium of the peptide preparation volume. . If the peptide is produced by chemical synthesis, the peptide is preferably substantially free of chemical precursors or other chemicals, and the chemical precursors or other chemicals involved in the synthesis of the peptide are peptide Includes peptide preparations comprising less than about 30%, 20%, 10% or 5% (dry weight) of the volume of the preparation. The fact that a specific peptide preparation contains an isolated or purified peptide means, for example, the appearance of a single band after sodium dodecyl sulfate (SDS) -polyacrylamide gel electrophoresis of the protein preparation and Coomassie brilliant blue staining of the gel. Can be indicated by In preferred embodiments, the peptides and polynucleotides of the invention are isolated or purified.

  The terms “polypeptide”, “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. In addition to natural amino acid polymers, the term refers to amino acid polymers in which one or more amino acid residues are modified residues, or non-natural residues such as artificial chemical mimetics of the corresponding natural amino acids. Also applies to the base.

  As used herein, the term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Natural amino acids are those encoded by the genetic code, as well as those post-translationally modified in cells (eg, hydroxyproline, γ-carboxyglutamate and O-phosphoserine). The phrase “amino acid analog” has the same basic chemical structure as natural amino acids (hydrogen, carboxy group, amino group, and α-carbon attached to the R group), but a modified R group or a modified backbone (eg, homoserine). , Norleucine, methionine, sulfoxide, methylmethioninesulfonium). The phrase “amino acid mimetic” refers to a compound that has a different structure than a common amino acid but functions similarly.

  Amino acids may be referred to by the commonly known three letter code or the one letter code recommended herein by the IUPAC-IUB Biochemical Nomenclature Committee.

The terms “gene”, “polynucleotide” and “nucleic acid” are used interchangeably herein, and are referred to by their generally accepted one-letter codes unless specifically indicated otherwise.
The terms “agent” and “composition” are used herein to refer to products that contain a specific amount of a specific component, as well as any product that results directly or indirectly from a combination of a specific amount of a specific component. Used interchangeably. Such term for a pharmaceutical composition refers to a product comprising an active ingredient and an inert ingredient that constitutes a carrier, and any combination of two or more ingredients, complexation or aggregation, or one of the ingredients. Alternatively, it is intended to encompass any product that results directly or indirectly from multiple dissociations or from one or more other types of reactions or interactions of components. Accordingly, the pharmaceutical compositions of the present invention encompass any composition made by admixing a compound of the present invention and a pharmaceutically or physiologically acceptable carrier.

  The term “active ingredient” as used herein refers to a substance in a biologically or physiologically active composition. In particular, in the context of pharmaceutical compositions, the term “active ingredient” refers to a component substance that exhibits the desired pharmacological effect. For example, in the case of a pharmaceutical composition for use in the treatment or prevention of cancer, the active ingredient in the composition is at least one biological or physiological effect on cancer cells and / or tissues, directly or indirectly. Can bring Preferably, such effects may include reducing or inhibiting cancer cell proliferation, damaging or killing cancer cells and / or tissues, and the like. Typically, the indirect action of the active ingredient is the induction of an immune response mediated by MHC class II molecules. Prior to formulation, the “active ingredient” may also be referred to as “bulk”, “API” or “API”. As used herein, the phrase “pharmaceutically acceptable carrier” or “physiologically acceptable carrier” includes liquid or solid extenders, diluents, excipients, solvents or encapsulating materials, but these Means a pharmaceutically or physiologically acceptable material, composition, substance or vehicle, which is not limited to

  Unless otherwise defined, the term “cancer” refers to cancers that express the DEPDC1 gene, including, for example, bladder cancer and breast cancer. A cancer that expresses the DEPDC1 gene is also referred to as a cancer that expresses DEPDC1 or a cancer that expresses a gene encoding DEPDC1.

  Unless otherwise defined, the terms “T lymphocyte” and “T cell” are used interchangeably herein.

Unless otherwise defined, the terms “cytotoxic T lymphocyte”, “cytotoxic T cell” and “CTL” are used interchangeably herein and, unless specifically indicated otherwise, non-self cells ( For example, it refers to a subgroup of T lymphocytes that can recognize and induce the death of such cells. CTLs can be distinguished from CD8 ⁺ T lymphocytes and recognize peptides presented by MHC class I molecules.

Unless otherwise defined, the term “HLA-A24” refers to the HLA-A24 type, including subtypes, examples of which include HLA-A ^* 24: 01, HLA-A ^* 24: 02, HLA-A ^*. 24:03, HLA-A ^* 24: 04, HLA-A ^* 24: 07, HLA-A ^* 24: 08, HLA-A ^* 24: 20, HLA-A ^* 24: 25 and HLA-A ^* 24: 88, but is not limited to.

Unless otherwise defined, the term “HLA-A2” as used herein typically refers to a subtype, examples of which include HLA-A ^* 02: 01, HLA-A ^* 02: 02, HLA-A ^* 02: 03, HLA-A ^* 02: 04, HLA-A ^* 02: 05, HLA-A ^* 02: 06, HLA-A ^* 02: 07, HLA-A ^* 02: 10, HLA- A ^* 02: 11, HLA-A ^* 02: 13, HLA-A ^* 02: 16, HLA-A ^* 02: 18, HLA-A ^* 02: 19, HLA-A ^* 02: 28 and HLA-A ^* 02:50 is included, but not limited to.

Unless otherwise defined, the terms “T helper type 1 cell” and “Th1 cell” are used interchangeably herein and, unless specifically indicated otherwise, recognize peptides presented by MHC class II molecules. Refers to a subgroup of CD4 ⁺ T lymphocytes that can be associated with cellular immunity. Unless otherwise defined, the terms “Th cell”, “CD4 ⁺ T cell” and “CD4 ⁺ helper T cell” are also used interchangeably herein. Th1 cells can be activated by various cytokines (eg, IFN-γ, IL-2, TNF-β, GM-CSF, Secretes TNF-α and the like).

Unless otherwise defined, the term “HLA-DR53” refers to a subtype, examples of which include HLA-DRB4 ^* 01: 01, DRB4 ^* 01: 02, DRB4 ^* 01: 03, DRB4 ^* 01: 04, DRB4 ^* 01: ^05, DRB4 ^* 01:06 and DRB4 ^* 01:07 include, but are not limited to these.

Unless otherwise defined, the term “HLA-DR4” refers to a subtype, examples of which include HLA-DRB1 ^* 04: 01, HLA-DRB1 ^* 04: 02, HLA-DRB1 ^* 04: 03, HLA-DRB1 ^* 04: 04, HLA-DRB1 ^* 04: 05, HLA-DRB1 ^* 04: 06, HLA-DRB1 ^* 04: 07, HLA-DRB1 ^* 04: 08, HLA-DRB1 ^* 04: 09, HLA-DRB1 ^* 04 : 10 and HLA-DRB1 ^* 04: 11, but are not limited to these.

Unless otherwise defined, the term “HLA-DR15” refers to a subtype, examples of which include HLA-DRB1 ^* 15: 01, HLA-DRB1 ^* 15: 02, HLA-DRB1 ^* 15: 03, HLA-DRB1 ^* 15: 04, HLA-DRB1 ^* 15: 05, HLA-DRB1 ^* 15: 06, HLA-DRB1 ^* 15: 07, HLA-DRB1 ^* 15: 08, HLA-DRB1 ^* 15: 09, HLA-DRB1 ^* 15 : 10 and HLA-DRB1 ^* 15: 11, but are not limited to these.

Unless otherwise defined, the term “HLA-DP5” refers to a subtype, examples of which include, but are not limited to, HLA-DPB1 ^* 05 ^: 01.

Unless otherwise defined, the phrase “immune response mediated by MHC class II molecules” refers to an immune response induced by presentation of peptides by MHC class II molecules. As used herein, “an immune response mediated by an MHC class II antigen” includes an immune response induced by CD4 ⁺ T cells, particularly Th1 cells. Examples of such immune responses include the production of cytokines (eg IFN-γ, IL-2, TNF-β, GM-CSF, TNF-α, etc.) and the activity of other immune cells (eg CTL, macrophages etc.) Include but are not limited to crystallization and / or stimulation.

  Unless otherwise defined, the phrase “Th1 cells specific for DEPDC1” refers to Th1 cells that are specifically activated by APCs that present peptides derived from DEPDC1, but are not activated by other APCs.

Unless otherwise defined, the phrase “DEPDCl-specific CTL” refers to a CTL that is specifically cytotoxic to a target cell that expresses DEPDC1.
Unless otherwise defined, the phrase “CTL inducibility” when used in connection with peptides refers to the ability of a peptide to induce CTL when presented on APC.
Unless otherwise defined, the term “kit” as used herein is used in reference to combinations of reagents and other materials. It is contemplated herein that a kit can include microarrays, chips, markers, and the like. The term “kit” is not intended to be limited to a particular combination of reagents and / or materials.

  In the context of the present invention, the term “antibody” refers to immunoglobulins and fragments thereof that are specifically reactive with the designated protein or peptide thereof. Antibodies can include human antibodies, primatized antibodies, chimeric antibodies, bispecific antibodies, humanized antibodies, antibodies fused to other proteins or radioactive labels, and antibody fragments. In addition, antibodies herein are used in their broadest sense, particularly intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), and antibodies so long as they exhibit the desired biological activity. Includes fragments. “Antibody” refers to all classes (eg, IgA, IgD, IgE, IgG and IgM).

  Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

II. Peptides The peptides of the present invention described in detail below may be referred to as “DEPDCl peptides” or “DEPDCl polypeptides”.
In order to demonstrate that peptides derived from DEPDC1 function as antigens recognized by Th1 cells, peptides derived from DEPDC1 (SEQ ID NO: 8 or 10) are promiscuously bound by MHC class II molecules. Analysis to determine whether it is an antigenic epitope. Candidate promiscuous MHC class II binding peptides derived from DEPDC1 were identified based on their binding affinity to HLA-DR53, HLA-DR4, HLA-DR15 and HLA-DP5. After in vitro stimulation of CD4 ⁺ T cells by dendritic cells (DC) loaded with these peptides, Th1 cells were successfully established using each of the following peptides:
DEPDC1 _191-213 / RYVILIYLQTILGVPSLEEVINP (SEQ ID NO: 1),
DEPDC1 _292-313 / TFEYYELFVNILVVCGIITVSD (SEQ ID NO: 2),
DEPDC1 _301-329 / NILVVCGIITVSSDRSGIHKIQDDPQSSK (SEQ ID NO: 3) and DEPDC1 _613-634 / NRRRKLQLLMRMISRSMSQNVDMP (SEQ ID NO: 4).

  These established Th1 cells described above showed potent specific Th1 cell activity in response to stimulation of APC pulsed with the respective peptides. In addition, the peptides inhibit Th1 cells that are restricted by several HLA-DR and HLA-DP molecules (eg HLA-DR53, HLA-DR4, HLA-DR15 and HLA-DP5) that are frequently found in Japanese. I was able to stimulate. These results herein show that DEPDC1 is the antigen recognized by Th1 cells and that the peptide is a number of HLA class II molecules (eg HLA-DR53, HLA-DR4, HLA-DR15 and HLA-DP5). ) To reveal the epitope peptide of DEPDC1 restricted to promiscuous.

  Some of the peptides identified above additionally include the amino acid sequence of a CTL epitope that has the ability to induce CTLs specific for DEPDC1, and as will be apparent herein, such peptides are Th1 In addition to cells, CTLs specific for DEPDC1 can also be induced. Thus, these peptides may be suitable peptides for the induction of an immune response against cancers that express DEPDC1. The DEPDC1 gene is a good target for immunotherapy because it is overexpressed in most cancer tissues including, for example, bladder cancer and breast cancer.

  Accordingly, the present invention provides peptides having the ability to induce Th1 cells specific for DEPDC1. The peptides of the invention can bind to at least one MHC class II molecule and can be presented on APC. Alternatively, a fragment of a peptide of the invention can bind to at least one MHC class II molecule and can be presented on APC. These fragments of the peptide can be generated by processing within APC. In preferred embodiments, the peptides of the invention or fragments thereof have the ability to bind to two or more types of MHC class II molecules (eg, HLA-DR53, HLA-DR4, HLA-DR15 and HLA-DP5). In other words, the peptides of the present invention may have the ability to induce Th1 cells that are restricted by two or more types of MHC class II molecules. In another aspect, the peptide of the present invention comprises the amino acid sequence of a peptide having the ability to induce DEPDC1-specific CTL. Typical examples of such peptides having the ability to induce DEPDC1-specific CTL include peptides having the amino acid sequence of SEQ ID NO: 5 or 6.

Since the binding groove in MHC class II molecules is open at both ends, the MHC class II binding peptide may be flexible in its length. The core binding motif for MHC class II molecules consists of 9 amino acid residues, and MHC class II binding peptides generally have other amino acid residues adjacent to the core binding motif. The number of adjacent amino acid residues is not limited. Thus, not all amino acid residues of SEQ ID NO: 1, 2, 3 or 4 are necessarily required for binding to MHC class II molecules. Thus, the peptides of the present invention can be peptides having the ability to induce Th1 cells, such peptides comprising an amino acid sequence selected from the group consisting of:
(A) an amino acid sequence having more than 9 consecutive amino acids from the amino acid sequence of SEQ ID NO: 1, 2, 3 or 4; and (b) 1, 2 or several amino acids are substituted, deleted, inserted and The amino acid sequence of (a) added / or.
MHC class II binding peptides are generally 10-30 amino acids in length. Since the amino acid sequence of SEQ ID NOs: 1 to 4 consists of a part of the amino acid sequence of DEPDC1 (SEQ ID NO: 8 or 10), the peptide of the present invention can be a peptide described in [1] to [5] below :
[1] An isolated peptide having a length of 10 to 30 amino acids and comprising a part of the amino acid sequence of SEQ ID NO: 8 or 10,
(A) a continuous amino acid sequence having a length exceeding 9 amino acids selected from the amino acid sequences of SEQ ID NOs: 1, 2, 3 or 4; and (b) 1, 2 or several amino acids are substituted or deleted. A peptide comprising an amino acid sequence selected from the group consisting of the amino acid sequence of (a) inserted and / or added, and having the ability to induce Th1 cells;
[2] The isolated peptide according to [1], wherein the peptide or a fragment thereof has an ability to bind to at least two types of MHC class II molecules;
[3] The isolated peptide according to [2], wherein the MHC class II molecule is selected from the group consisting of HLA-DR53, HLA-DR4, HLA-DR15 and HLA-DP5;
[4] The isolated peptide according to any one of [1] to [3], wherein the peptide comprises an amino acid sequence of a peptide having DEPDC1-specific CTL inducing ability; and [5]
(A) an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 4; and (b) the amino acid of (a) wherein 1, 2 or several amino acids are substituted, deleted, inserted and / or added. Array,
The isolated peptide of [4], comprising an amino acid sequence selected from the group consisting of:

Th1 cells induced by the peptides of the present invention are specific for DEPDC1.
Therefore, in some embodiments, the present invention provides a peptide of less than 30 amino acid residues consisting of a partial amino acid sequence of the amino acid sequence of SEQ ID NO: 8 or 10, comprising SEQ ID NO: 1, 2, 3 or 4 A peptide comprising an amino acid sequence is provided.

  In general, software programs currently available on the Internet, such as Wang P et al. , 2008. PLoS Compute Biol. 4 (4): e1000048.11: 568; and Wang P et al. , 2010. BMC Bioinformatics. The binding affinity between various peptides and HLA antigens can be calculated in silico using the software program described in. The binding affinity with the HLA antigen is described, for example, in Nielsen M and Lund O. 2009. BMC Bioinformatics. 10: 296; Nielsen M et al. , 2007. BMC Bioinformatics. 8: 238; Bui HH, et al. 2005. Immunogenetics. 57: 304-314; Sternio T et al. 1999. Nat Biotechnol. 17 (6): 555-561 and Nielsen M et al. , 2008. PLoS Compute Biol. 4 (7) e1000107. Accordingly, the present invention encompasses peptides of DEPDC1 that have been determined to bind to HLA antigens identified using such known programs.

  As described above, since the length of the MHC class II-binding peptide is flexible, the amino acid sequence of SEQ ID NO: 1, 2, 3 or 4 can be used as long as the resulting peptide retains the necessary Th1 cell inducibility. In some cases, it may be adjacent to an additional amino acid residue. Such peptides having the ability to induce Th1 cells are typically less than about 30 amino acids, often less than about 29 amino acids, usually less than about 28 or 27 amino acids. The specific amino acid sequence adjacent to the amino acid sequence selected from among SEQ ID NOs: 1 to 4 is not limited as long as such adjacent amino acid sequence does not impair the ability of the original peptide to induce Th1 cells. It can be composed of different types of amino acids. In an exemplary embodiment, such flanking amino acid sequences may be selected from among the amino acid sequences of SEQ ID NO: 8 or 10 that flank the amino acid sequence of SEQ ID NO: 1, 2, 3 or 4; Is not limited to this. Therefore, the present invention also provides a peptide having an ability to induce Th1 cells and an amino acid sequence selected from SEQ ID NOs: 1 to 4.

  On the other hand, the core binding motif for MHC class II molecules consists of 9 amino acid residues, so that the full length of the amino acid sequence of SEQ ID NO: 1, 2, 3 or 4 binds to MHC class II molecules and It is not necessary for the induction of Th1 cells. Accordingly, the peptide of the present invention takes the form of a peptide having more than 9 consecutive amino acids from the amino acid sequence of SEQ ID NO: 1, 2, 3 or 4 on condition that the necessary Th1 cell inducing ability is retained. be able to. Peptides having Th1 cell inducibility are typically more than about 10 amino acids, often more than 11 or 12 amino acids, usually more than 13 or 14 amino acids. Therefore, the peptide of the present invention has Th1 cell inducibility and has more than 9, 10, 11, 12, 13 or 14 consecutive amino acids from the amino acid sequence of SEQ ID NO: 1, 2, 3 or 4. It can be a peptide having an amino acid sequence comprising.

  It is generally known that modification of one, two or more amino acids in a protein does not affect the function of the protein and in some cases even enhances the desired function of the original protein. Indeed, from an altered peptide (ie an amino acid sequence in which one, two or several amino acid residues have been altered (ie substituted, added, deleted or inserted) compared to the original reference sequence) Is known to retain the biological activity of the original peptide (Mark et al., Proc Natl Acad Sci USA 1984, 81: 5562-6; Zoller and Smith, Nucleic Acids Res 1982). , 10: 6487-500; Dalbadie-McFarland et al., Proc Natl Acad Sci USA 1982, 79: 6409-13). Thus, in one aspect, a peptide of the invention may have both a Th1 cell inducibility and an amino acid sequence selected from SEQ ID NOs: 1 to 4, wherein 1, 2 or even more in this sequence Of amino acids are added, inserted, deleted and / or substituted. Alternatively, the peptide of the present invention has an ability to induce Th1 cells and amino acids in which 1, 2 or several amino acids are added, inserted, deleted and / or substituted in the amino acid sequence of SEQ ID NO: 1, 2, 3 or 4. It can have both sequences. That is, in some embodiments, the peptide of the present invention is selected from the group consisting of addition, insertion, deletion and substitution to the Th1 cell inducibility and the amino acid sequence of SEQ ID NO: 1, 2, 3 or 4. May have an amino acid sequence with one, two or several modifications.

Those skilled in the art will recognize that individual additions or substitutions to an amino acid sequence that modify a single amino acid or a small percentage of amino acids tend to conserve the properties of the original amino acid side chain. Thus, they are often referred to as “conservative substitutions” or “conservative modifications”, and modification of a protein results in a modified protein having a function similar to the original protein. Conservative substitution tables providing functionally similar amino acids are well known in the art. Examples of amino acid side chain properties 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 with the following common functional groups or 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 acid and amide-containing side chains (D, N, E, Q); base-containing side chains (R, K, H); and aromatic-containing side chains (H, F, Y, W). In addition, the following eight groups each contain amino acids that are conservative substitutions for each other:
1) Alanine (A), Glycine (G);
2) Aspartic acid (D), glutamic acid (E);
3) Asparagine (N), glutamine (Q);
4) Arginine (R), lysine (K);
5) Isoleucine (I), leucine (L), methionine (M), valine (V);
6) phenylalanine (F), tyrosine (Y), tryptophan (W);
7) serine (S), threonine (T); and 8) cysteine (C), methionine (M) (see, for example, Creighton, Proteins 1984).

  Such conservatively modified peptides are also considered peptides of the present invention. However, the peptides of the present invention are not limited thereto, and may include non-conservative modifications as long as the modified peptide retains the Th1 cell inducibility of the original peptide. Furthermore, modified peptides should not exclude polymorphic variants of DEPDC1, interspecies homologues, and allylic Th1 cell-inducing peptides.

  A small number (eg, 1, 2 or several) or a low percentage of amino acids can be modified (inserted, added, deleted and / or substituted) to retain the necessary Th1 cell inducibility. As used herein, the term “several” means 5 or fewer amino acids, such as 4 or 3 or fewer amino acids. The proportion of amino acids to be modified is preferably 20% or less, more preferably 15% or less, even more preferably 10% or 8% or less, or 1 to 5%.

Homology analysis of the preferred peptides of the present invention, ie SEQ ID NOs: 1 to 4 (DEPDC1 _191-213- LP, DEPDC1 _292-313- LP, DEPDC1 _301-329- LP and DEPDC1 _613-634- LP) Are not significantly homologous to peptides derived from other known human gene products. Thus, when used for immunotherapy, these peptides are significantly less likely to produce an unknown or undesirable immune response. Therefore, these peptides are expected to be extremely useful for inducing immunity against DEPDC1 in cancer patients.

  When used in connection with immunotherapy, the peptides of the invention or fragments thereof must be presented on the surface of the APC, preferably as a complex with the HLA class II antigen. It is therefore preferable to select peptides that not only induce Th1 cells but also have high binding affinity for HLA class II antigens. To that end, peptides can be modified by substitution, insertion, deletion and / or addition of amino acid residues to produce modified peptides with improved binding affinity.

  The present invention also contemplates the addition of 1-2 amino acids to either or both of the N-terminus and C-terminus of the peptides described above. Such modified peptides having high HLA antigen binding affinity and retaining the ability to induce Th1 cells are also included in the present invention.

  For example, in the present invention, 1, 2 or several amino acids are modified in an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 4, which binds to HLA class II antigen, has Th1 cell inducibility, Provided is an isolated peptide of less than 31, 30, 29, 28, 27, or 26 amino acids in length comprising the amino acid sequence of

  These peptides can also be processed in APC when brought into contact with or introduced into APC to present the processed fragment thereon. For example, the peptides of the present invention can be processed into fragments consisting of usually 11 to 26 (typically 15 to 25) amino acid residues presented on the surface of APC.

  However, if the peptide sequence is identical to part of the amino acid sequence of an endogenous or exogenous protein with different functions, it can induce negative side effects such as autoimmune diseases and / or allergic symptoms to certain substances. Therefore, it may be desirable to first perform a homology search using available databases to avoid situations where the sequence of the peptide matches the amino acid sequence of another protein. If the homology search reveals that the same peptide or a peptide having one or two amino acid differences does not exist in nature compared to the target peptide, the HLA without the risk of such side effects The peptide of interest can be modified to increase its binding affinity with the antigen and / or to increase its Th1 cell inducing ability and / or CTL inducing ability.

  Peptides with high binding affinity for the HLA class II antigens described above are expected to be very effective, but candidate peptides selected according to the presence of high binding affinity as an indicator will be further examined with respect to the presence of Th1 cell inducibility . As used herein, the phrase “Th1 cell inducing ability” refers to the ability of a peptide to confer to APC the ability to induce Th1 cells when contacted with APC. Furthermore, “Th1 cell inducibility” promotes Th1 cell-mediated cytokine production, including IFN-γ production, which induces Th1 cell activation and / or Th1 cell proliferation, and promotes other cells (eg, CTL, macrophages). The ability of the peptide to help and / or stimulate.

Confirmation of Th1 cell induction ability induced APC (eg, B lymphocyte, macrophage and dendritic cell (DC)) carrying human MHC antigen, preferably DC derived from human peripheral blood mononuclear leukocytes, and stimulated with peptide Later, it can be achieved by mixing with CD4 positive T cells (CD4 ⁺ T cells) and then measuring the IFN-γ produced and released by CD4 ⁺ T cells. Alternatively, the ability of the peptide to induce Th1 cells can be evaluated based on CTL activation by Th1 cells. For example, CD4 ⁺ T cells are co-cultured with DC stimulated with a test peptide and then mixed with CTL and CTL target cells. The target cells can be radiolabeled with ⁵¹ Cr or the like, and the cytotoxic activity of CTL activated by cytokine secreted from Th1 cells can be calculated from the radioactivity released from the target cells. Alternatively, Th1 cell inducibility is measured by measuring the released IFN-γ produced by Th1 cells in the presence of APC stimulated with the test peptide and visualizing the inhibition zone on the medium using anti-IFN-γ monoclonal antibody It can be evaluated by doing.

  In addition to the modifications described above, the peptides of the present invention can also bind to other substances as long as the resulting binding peptide retains the Th1 cell inducibility of the original peptide. Examples of suitable substances include, for example, peptides, lipids, sugars and sugar chains, acetyl groups, natural and synthetic polymers and the like. The peptides of the present invention may contain modifications such as glycosylation, side chain oxidation or phosphorylation as long as the modification does not impair the biological activity of the original peptide. These types of modifications can be performed to confer additional functions (eg, targeting and delivery functions) or to stabilize the peptide.

  For example, it is known in the art to introduce D-amino acids, amino acid mimetics or unnatural amino acids to increase the in vivo stability of the peptides; this concept can also be adapted to the peptides of the invention. . Peptide stability can be assessed in a number of ways. For example, stability can be tested using various biological media such as peptidases and human plasma and serum (see, eg, Verhoef et al., Eur J Drug Metapharma 1986, 11: 291-302).

  The peptides of the present invention can be presented on the surface of APC as a complex in combination with HLA class II antigen and then induce Th1 cells. Therefore, peptides that form a complex with the HLA class II antigen on the surface of APC are also included in the present invention. APC presenting the peptides of the invention can be inoculated as a vaccine.

The type of HLA antigen contained in the complex must match the type of subject in need of treatment and / or prevention. For example, in the Japanese, HLA-DR53, HLA-DR4, HLA-DR15 and HLA-DP5 are common and are therefore suitable for the treatment of Japanese patients. Typically, in clinical practice, the type of HLA antigen in patients in need of treatment is examined in advance, which allows for the appropriate selection of peptides that have the ability to bind to a particular HLA class II antigen. In a preferred embodiment, the peptides of the present invention are capable of inducing Th1 cells promiscuously. As used herein, the ability of a peptide to induce Th1 cells is “promiscuous” if the peptide can induce Th1 cells that are restricted by at least two different types of MHC class II molecules. In other words, such antigen recognition is considered “promiscuous” if the peptide is recognized by at least two different types of MHC class II molecules. When used in connection with peptides, the phrase “recognized by at least two different types of MHC class II molecules” indicates that the peptide or fragment thereof can bind to at least two different types of MHC class II molecules. For example, DEPDC1 _191-213- LP (SEQ ID NO: 1) is recognized by HLA-DR53 and HLA-DR4, DEPDC1 _292-313- LP (SEQ ID NO: 2) is recognized by HLA-DR4, and DEPDC1 _{301 -329-} LP (SEQ ID NO: 3) is recognized by HLA-DP5 and HLA-DR4, and DEPDC1 _613-634- LP (SEQ ID NO: 4) is recognized by HLA-DP5 and HLA-DR15. These peptides are therefore typical examples of “promiscuous” epitopes.

  When HLA-DR53 or HLA-DR4 positive APC is used, a peptide having the amino acid sequence of SEQ ID NO: 1 is preferably used. When HLA-DR4 positive APC is used, a peptide having the amino acid sequence of SEQ ID NO: 2 is preferably used. When HLA-DP5 or DR4-positive APC is used, a peptide having the amino acid sequence of SEQ ID NO: 3 is preferably used. When HLA-DP5 or DR15 positive APC is used, a peptide having the amino acid sequence of SEQ ID NO: 4 is preferably used.

  Thus, in a preferred embodiment, a peptide having the amino acid sequence of SEQ ID NO: 1 may be used for induction of Th1 cells in a subject identified as having HLA-DR53 or HLA-DR4 prior to induction. Similarly, a peptide having the amino acid sequence of SEQ ID NO: 2 can be used for the induction of Th1 cells in a subject identified as having HLA-DR4 prior to induction. Similarly, a peptide having the amino acid sequence of SEQ ID NO: 3 can be used for induction of Th1 cells in a subject identified as having HLA-DP5 or DR4 prior to induction. Similarly, a peptide having the amino acid sequence of SEQ ID NO: 4 can be used for induction of Th1 cells in a subject identified as having HLA-DP5 or DR15 prior to induction.

III. Preparation of DEPDC1 Peptides The peptides of the present invention can be prepared using well-known techniques. For example, the peptides of the present invention can be prepared synthetically using recombinant DNA techniques or chemical synthesis. The peptides of the invention can be synthesized individually or as longer polypeptides consisting of two or more peptides. The peptides of the present invention can then be isolated or purified so as to be substantially free of other natural host cell proteins and fragments thereof, or some other chemical.

  The peptides of the present invention may contain modifications such as glycosylation, side chain oxidation or phosphorylation as long as the modification does not impair the biological activity of the original reference peptide. Other exemplary modifications include incorporation of D-amino acids or other amino acid mimetics. These modifications can be used, for example, to increase the serum half-life of the peptide

The peptides of the present invention can be obtained through chemical synthesis based on selected amino acid sequences. Examples of conventional peptide synthesis methods that can be adapted to this synthesis include:
(I) Peptide Synthesis, Interscience, New York, 1966;
(Ii) The Proteins, Vol. 2, Academic Press, New York, 1976;
(Iii) "Peptide synthesis" (Japanese), Maruzen, 1975;
(Iv) “Basics and Experiments of Peptide Synthesis” (Japanese), Maruzen, 1985;
(V) “Development of drugs” (Japanese), Volume 14 (peptide synthesis), Hirokawa Shoten;
(Vi) WO 99/67288; 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 invention can be obtained by adapting any known genetic engineering method for making peptides (eg Morrison J, J Bacteriology 1977, 132: 349-51; Clark-Curtiss & Curtiss). , Methods in Enzymology (eds. Wu et al) 1983, 101: 347-62). For example, first, an appropriate vector carrying a polynucleotide encoding a peptide of interest in an expressible form (eg, downstream of a regulatory sequence corresponding to a promoter sequence) is prepared and transformed into an appropriate host cell. The host cell is then cultured to produce the peptide of interest. The peptides of the present invention can also be generated in vitro employing an in vitro translation system.

IV. Polynucleotides The present invention also provides polynucleotides that encode any of the aforementioned peptides of the present invention. These include polynucleotides derived from the natural DEPDC1 gene (GenBank accession number NM — 001114120 (SEQ ID NO: 7) or NM — 017779 (SEQ ID NO: 9)), as well as polynucleotides having conservatively modified nucleotide sequences. included. As used herein, the phrase “conservatively modified nucleotide sequence” refers to sequences that encode the same or essentially the same amino acid sequence. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode a given protein. For example, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be changed to any of the corresponding codons described above without changing the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one type of conservatively modified variations. Every nucleic acid sequence herein that encodes a peptide also represents every possible silent variation of the nucleic acid. Those skilled in the art recognize that each codon in a nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan) can be modified to yield a functionally identical molecule. To do. Accordingly, each silent variation of a nucleic acid that encodes a peptide is implicitly expressed in each disclosed sequence.

  The polynucleotide of the present invention can be composed of DNA, RNA, and derivatives thereof. As is well known in the art, DNA is suitably composed of bases such as A, T, C and G, where T is replaced with U in RNA. One skilled in the art will recognize that unnatural bases can also be included in the polynucleotide.

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

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

V. Antigen presenting cell (APC)
The present invention also provides an antigen presenting cell (APC) that presents on its surface a complex formed between an HLA class II antigen and a peptide of the present invention or a fragment thereof. The APC obtained by contacting with a peptide of the invention can be derived from a patient that is the subject of treatment and / or prevention, alone or in combination with other agents including the peptide, Th1 cells or CTLs of the invention. Can be administered as a vaccine.

  APCs are not limited to specific cell types, but are known to present proteinaceous antigens on their cell surfaces to be recognized by lymphocytes, dendritic cells (DC), Langerhans cells, macrophages, B Cells and activated T cells. Since DC is a representative APC having the strongest Th1 cell-inducing activity among APCs, DC is useful as the APC of the present invention.

Furthermore, in a preferred embodiment, the peptides of the invention can also induce CTL responses mediated by MHC class I antigens, as well as Th1 cell responses mediated by MHC class II antigens. In general, it is well known that the length of the epitope recognized by MHC class I antigens is shorter (eg, 8-10 amino acid residues) than that of MHC class II antigens (15 or more amino acid residues). Therefore, the processed product of the peptides of the present invention will result in the induction of CTL. Indeed, DEPDC1 _292-313- LP (SEQ ID NO: 2) induces a CTL that recognizes the fragment (EYYELFVNI: SEQ ID NO: 5), and DEPDC1 _292-313- LP (SEQ ID NO: 2) or DEPDC1 _301-329 -LP (SEQ ID NO: 3) induces a CTL that recognizes the fragment (ILVVCGIITV: SEQ ID NO: 6). Thus, the peptides of the present invention not only induce Th1 cells, but can also induce CTLs after processing in APC. In other words, APC in contact with the above peptides of the present invention present such peptides with MHC class II antigens and simultaneously present fragments thereof with MHC class I antigens. As a result, both Th1 cells and CTL can be induced by using the above-described peptides of the present invention.

For example, APCs are obtained by inducing DCs from peripheral blood mononuclear cells and then contacting (stimulating) them with the peptides of the invention in vitro, ex vivo or in vivo. When the peptide of the present invention is administered to a subject, APC presenting the peptide of the present invention or a fragment thereof is induced in the body of the subject. As used herein, the phrase “inducing APC” is formed between an HLA class II antigen and a peptide of the present invention or a fragment thereof by contacting (stimulating) APC with the peptide of the present invention. Presenting the complex on the surface of the APC. Alternatively, after introducing the peptide of the present invention into APC to present the peptide of the present invention or a fragment thereof to APC, APC can be administered to a subject as a vaccine. For example, ex vivo administration:
(A) recovering APC from the first subject;
(B) contacting the APC of step (a) with a peptide of the invention, and (c) administering the peptide loaded step (b) APC to a second subject.

  The first subject and the second subject may be the same individual or different individuals. Alternatively, according to the present invention, there is provided use of the peptide of the present invention for producing a pharmaceutical composition for inducing APC. In addition, the present invention provides a method or process for producing a pharmaceutical composition that induces APC, said method for mixing or formulating a peptide of the present invention with a pharmaceutically acceptable carrier. Including stages. Furthermore, the present invention also provides a peptide of the present invention for inducing APC. The APC obtained by step (b) can be administered to the subject as a vaccine.

As one aspect of the present invention, the APC of the present invention has a high level of Th1 cell inducing ability. In the present specification, in the phrase “high level Th1 cell inducing ability”, high level means Th1 cell inducing ability by APC not in contact with a peptide or APC in contact with a peptide that cannot induce Th1 cells. It is compared with the level. As used herein, the phrase “Th1 cell inducibility” when used in connection with APC refers to the ability of APC to induce Th1 cells when contacted with CD4 ⁺ T cells. Such APCs having a high level of Th1 cell inducibility can be prepared by a method comprising the step of transferring a polynucleotide encoding a peptide of the present invention to APCs in vitro. The gene to be introduced can be in the form of DNA or RNA. Examples of the method for introduction are not particularly limited, and include various methods conventionally performed in this field. For example, lipofection, electroporation, and calcium phosphate method can be used. More particularly, as described in Cancer Res 1996, 56: 5672-7; J Immunol 1998, 161: 5607-13; J Exp Med 1996, 184: 465-72; published patent publication 2000-509281. Can be implemented. By transferring the gene to APC, the gene undergoes transcription, translation, etc. in the cell, and then the obtained protein is processed by MHC class I or class II and proceeds to presentation of the peptide through the presentation pathway. Alternatively, the APC of the present invention can be prepared by a method that induces the step of contacting APC with the peptide of the present invention.

In a preferred embodiment, the APC of the present invention presents on its surface a complex of an MHC class II molecule selected from HLA-DR53 and HLA-DR4 and a peptide of the present invention (including the amino acid sequence of SEQ ID NO: 1). APC to In another embodiment, the APC of the present invention presents on its surface a complex of an MHC class II molecule selected from HLA-DR4 and the peptide of the present invention (including the amino acid sequence of SEQ ID NO: 2). It can be APC. In another embodiment, the APC of the present invention is an APC that presents a complex of HLA-DP5 and DR4 MHC class II molecules and the peptide of the present invention (including the amino acid sequence of SEQ ID NO: 3) on its surface. obtain. In another embodiment, the APC of the present invention is an APC that presents a complex of HLA-DP5 and DR15 MHC class II molecules and the peptide of the present invention (including the amino acid sequence of SEQ ID NO: 4) on its surface. obtain. Preferably, HLA-DR53, HLA-DR4, HLA-DR15 and HLA-DP5 are HLA-DRB4 ^* 01: 03, HLA-DRB1 ^* 04: 05, HLA-DRB1 ^* 15: 02 and HLA-DPB1 ^* 05, respectively. : 01.

VI. T helper type 1 cells (Th1 cells)
Th1 cells induced against any of the peptides of the present invention enhance the immune response of any of the effector cells, including CTL targeting cancer cells in vivo, and thus in a manner similar to the peptide itself, Useful as a vaccine. Thus, the present invention also provides isolated Th1 cells that are specifically induced or activated by any of the peptides of the present invention.

Such Th1 cells can be obtained by (1) administering one or more peptides of the present invention to a subject and recovering Th1 cells from the subject, (2) APC and CD4 ⁺ T cells or peripheral blood mononuclear leukocytes. Contacting (stimulating with) a peptide of the invention in vitro and then isolating Th1 cells, (3) contacting CD4 ⁺ T cells or peripheral blood mononuclear leukocytes with the APC of the invention in vitro, or (4) Introducing a polynucleotide encoding both TCR subunits or a polynucleotide encoding each of the TCR subunits into CD4 ⁺ T cells (TCR binds to a complex of MHC class II molecules and peptides of the invention) Can be obtained). Such APC for the method of (3) can be prepared by the method described above. Details of the method (4) will be described in the following section “VII. T cell receptor (TCR)”.

  Th1 cells induced by stimulation with APCs of the present invention may be derived from patients who are the subject of treatment and / or prevention, alone or other containing the peptides of the present invention to modulate action It can be administered in combination with a drug. The resulting Th1 cells can activate and / or stimulate immune cells (eg, CTL, macrophages) involved in cellular immunity. Such immune cells that can be activated by the Th1 cells of the present invention include CTLs that are cytotoxic to target cells such as cancer cells. For example, such CTL target cells can be cells that endogenously express DEPDC1 (eg, cancer cells) or cells that have been transfected with the DEPDC1 gene. In a preferred embodiment, the peptide of the present invention can include at least one amino acid sequence of a CTL epitope peptide, and can induce CTLs against DEPDC1-expressing cells such as cancer cells in addition to Th1 cells. In this case, the peptide of the present invention can induce Th1 cells and CTL simultaneously or sequentially in vivo, and the induced Th1 cells can effectively activate the induced CTL. Accordingly, such peptides comprising at least one amino acid sequence of a CTL epitope peptide are suitable peptides for cancer immunotherapy.

  Furthermore, the Th1 cells of the present invention secrete various cytokines (eg, IFN-γ) that activate and / or stimulate any CTL directed against other target cells in an antigen-independent manner. Therefore, the Th1 cell of the present invention can also contribute to enhancing CTL activity targeting cells expressing TAA other than DEPDC1. Accordingly, the Th1 cells of the present invention are useful not only for tumors that express DEPDC1, but also for immunotherapy for tumors that express other TAAs, as well as the peptides and APCs of the present invention.

  In some embodiments, a Th1 cell of the invention is a Th1 cell that recognizes a cell presenting a complex of an HLA-DR or HLA-DP antigen and a peptide of the invention. In the context of Th1 cells, the phrase “recognizes cells” means that a complex of a cell surface MHC class II molecule and a peptide of the present invention is bound and antigen-specifically activated via its TCR. Point to. As used herein, the phrase “antigen-specifically activated” refers to being activated in response to certain MHC class II molecules and peptides, and cytokine production from activated Th1 cells is Be guided. In a preferred embodiment, HLA-DR and HLA-DP may be selected from the group consisting of HLA-DR53, HLA-DR4, HLA-DR15 and HLA-DP5.

VII. T cell receptor (TCR)
The present invention also provides compositions containing one or more polynucleotides encoding one or more polypeptides capable of forming a T cell receptor (TCR) subunit, and methods of using the same I will provide a. Such TCR subunits have the ability to form TCRs that confer specificity for DEPDC1 to CD4 ⁺ T cells for APC presenting DEPDC1 peptides. By using methods known in the art, α- and β-chain nucleic acids can be identified as TCR subunits of Th1 cells induced by the peptides of the present invention (WO 2007/032255 and Morgan). et al., J Immunol, 171, 3288 (2003)). The derivative TCR can bind with high avidity to APC presenting the DEPDC1 peptide and in some cases can effectively mediate cytokines.

One or more polynucleotides encoding TCR subunits (ie, a single polynucleotide encoding both TCR subunits or a plurality of polynucleotides each encoding separate TCR subunits) are transferred to an appropriate vector, eg, retro It can be incorporated into a viral vector. These vectors are well known in the art. The polynucleotides or vectors containing them can be usefully transferred to CD4 ⁺ T cells, eg, patient-derived CD4 ⁺ T cells. Advantageously, the present invention allows for rapid modification of a patient's own T cells (or another subject's T cells) and produces modified T cells with superior cancer cell killing properties quickly and easily. Provide a readily available composition.

For example, a TCR-α subunit and a TCR-β subunit each having CDR3 consisting of the amino acid sequences of SEQ ID NOs: 11 and 21, respectively, in a HLA-DR4 restricted manner, DEPDC1-LP2 (DEPDC1 _292-313 -LP) Increased in Th cells stimulated by (FIG. 6). Therefore, polynucleotides encoding them may be preferred for Th1 cell induction of the present invention. Similarly, Th1 cells that express a TCR formed between an α subunit and a β subunit each having CDR3 consisting of the amino acid sequences of SEQ ID NOs: 11 and 21, are also preferred embodiments of the present invention. . In general, the antigen specificity of TCRs is primarily dependent on their CDR3. Accordingly, a DEPDC1-LP2-specific TCR can be reconstructed from a known TCR by replacing the CDR3 of the α subunit and β subunit with SEQ ID NOs: 11 and 21, respectively. Such a TCR with CDR3 grafted from another subunit can be referred to as a chimeric TCR. Methods for transplanting specific CDRs are well known in the art.

  The invention further provides Th1 cells prepared by transduction with a polynucleotide encoding both TCR subunits or a polynucleotide encoding each of the TCR subunits, wherein the TCR subunit is a DEPDC1 peptide ( For example, SEQ ID NO: 1 for HLA-DR53 or HLA-DR4, SEQ ID NO: 2 for HLA-DR4, SEQ ID NO: 3 for HLA-DP5 or DR4, SEQ ID NO: 4 for HLA-DP5 or DR15) Can be combined. Transduced Th1 cells can home to cancer cells in vivo and can be propagated in vitro by well-known culture methods (eg, Kawakami et al., J Immunol., 142, 3352-3461 (1989). )). Th1 cells prepared as described above can be used to form an immunogenic composition useful in treating or preventing cancer in a patient in need of treatment or prevention.

VIII. Drug or Pharmaceutical Composition As long as the methods and compositions of the invention have utility in connection with cancer “treatment”, the treatment is clinically beneficial, eg, reduced expression of the DEPDC1 gene, or cancer in the subject. A treatment is considered “effective” if it leads to a reduction in size, spread or metastatic potential. “Effective” when treatment is applied prophylactically means delaying or preventing the formation of cancer or preventing or reducing the clinical symptoms of cancer. Efficacy is determined in connection with any known method for diagnosing or treating a particular tumor type.
As long as the methods and compositions of the present invention have utility in connection with cancer “prevention” and prophylaxis, such terms are used herein to reduce mortality or morbidity burden due to disease. Used interchangeably to refer to any action that reduces. Prevention and prevention can be performed “at the primary, secondary and tertiary prevention levels”. Primary prevention (prevention and prophylaxis) avoids the development of the disease, while secondary and tertiary level prevention (prevention and prophylaxis) prevents the progression of the disease and the appearance of symptoms (prevention and prophylaxis) and restores function. Includes activities aimed at reducing the negative effects of existing diseases by reducing disease-related complications. Alternatively, prevention and prophylaxis includes a wide range of prophylactic treatments aimed at reducing the severity of certain disorders, for example, reducing tumor growth and metastasis, reducing angiogenesis.

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

  As described above, Th1 cells induced by the peptides of the present invention can help immune cells involved in cellular immunity. Since cytokines secreted by Th1 cells can affect CTLs in an antigen-independent manner, such immune cells include not only CTLs against cancer cells that express DEPDC1, but also other TAA-expressing cancers. CTLs for cells are also included. Accordingly, the present invention provides a medicament or pharmaceutical composition containing at least one peptide of the present invention. In a medicament or pharmaceutical composition, such peptides are present in a therapeutically or pharmaceutically effective amount.

  Since Th1 cells induced by the agents or compositions of the present invention can secrete cytokines that affect any immune cell involved in cellular immunity, the agents or pharmaceutical compositions of the present invention are cellular. It is useful to help, stimulate and / or enhance any immune cell involved in immunity (eg CTL, macrophages). Thus, the agents or compositions of the invention are useful for any purpose that enhances or promotes an immune response mediated by such immune cells, including CTLs. For example, because the agents or compositions of the present invention can enhance or promote an immune response against a cancer or tumor mediated by such immune cells, the present invention provides for the treatment and / or prevention of cancer. An agent or composition containing at least one of the peptides of the invention for use in is provided. The amount of peptide in such an agent or composition can be an amount effective to significantly enhance or stimulate an immune response in a subject bearing a cancer that expresses DEPDC1.

  The present invention also provides agents or compositions for enhancing or stimulating immune responses mediated by MHC class I antigens such as HLA-A2 and HLA-A24. In another aspect, the present invention further provides the use of a peptide of the present invention for the manufacture of a medicament or composition for enhancing or stimulating an immune response mediated by MHC class I antigens.

  In a preferred embodiment, the DEPDC1-derived peptide identified in the course of the present invention is capable of inducing CTL against Th1 cells as well as DEPDC1-expressing cells. Accordingly, the present invention also provides an agent or composition containing at least one of the peptides of the present invention for use in inducing CTL against a cancer or tumor that expresses DEPDC1.

  Furthermore, an agent or composition containing at least one of the peptides of the present invention can be used to enhance or promote an immune response mediated by MHC class II molecules.

  Since the expression of DEPDC1 is specifically elevated in several cancer types including bladder cancer, breast cancer and several other malignancies compared to normal tissues (WO 2006/085684, International Publication No. 2007/013665, Kanehira M, et al., Oncogene 2007; 26: 6448-55 and our microarray data (data not shown), encoding peptides of the invention or peptides of the invention The polynucleotide can be used for the treatment and / or prevention of cancer or tumor and / or for prevention of its post-operative recurrence. Accordingly, the present invention provides for the treatment and / or prevention of cancer or tumor and / or prevention of postoperative recurrence thereof, which contains one or more of the peptide of the present invention or the polynucleotide of the present invention as an active ingredient. A pharmaceutical or pharmaceutical composition for the use is provided. Alternatively, the peptides of the invention can be expressed on the surface of any of the cells, such as APC, for use as a drug or pharmaceutical composition. In addition, the Th1 cells can also be used as an active ingredient of the drug or pharmaceutical composition of the present invention.

In another aspect, the invention also in the manufacture of a pharmaceutical composition or medicament for treating cancer or tumor:
(A) the peptide of the present invention,
(B) a polynucleotide encoding the peptide as disclosed herein in an expressible form;
(C) APC which presents the peptide of the present invention or a fragment thereof on its surface, and (d) Use of an active ingredient selected from Th1 cells of the present invention.

Alternatively, the invention further provides for use in treating cancer or tumors,
(A) the peptide of the present invention,
(B) a polynucleotide encoding the peptide as disclosed herein in an expressible form;
(C) An APC that presents the peptide of the present invention or a fragment thereof on its surface, and (d) an active ingredient selected from the Th1 cells of the present invention.

Alternatively, the present invention further relates to a method or process for producing a pharmaceutical composition or medicament for treating cancer or tumor, comprising as an active ingredient:
(A) the peptide of the present invention,
(B) a polynucleotide encoding the peptide as disclosed herein in an expressible form;
(C) APC which presents the peptide of the present invention or a fragment thereof on its surface, and (d) an active ingredient selected from Th1 cells of the present invention and a pharmaceutically or physiologically acceptable carrier. A method or process comprising the steps of:

In another aspect, the present invention also provides a method or process for producing a pharmaceutical composition or medicament for treating cancer or tumor comprising:
(A) the peptide of the present invention,
(B) a polynucleotide encoding the peptide as disclosed herein in an expressible form;
(C) APC that presents the peptide of the present invention or a fragment thereof on its surface, and (d) an active ingredient selected from the Th1 cells of the present invention is mixed with a pharmaceutically or physiologically acceptable carrier. Also provided is a method or process comprising steps.

  Alternatively, the pharmaceutical composition or agent of the present invention may be used for either or both of preventing cancer or tumor and preventing its postoperative recurrence.

  The medicament or pharmaceutical composition of the present invention is used as a vaccine. In the context of the present invention, the phrase “vaccine” (also referred to as an immunogenic composition) refers to a composition that has the function of inducing anti-tumor immunity when inoculated into an animal.

  The medicament or pharmaceutical composition of the present invention can be used to treat and / or prevent cancer or tumor in a subject or patient and / or prevent its post-operative or metastatic recurrence. Examples of such subjects include humans and mice, rats, guinea pigs, rabbits, cats, dogs, sheep, goats, pigs, cows, horses, monkeys, baboons and chimpanzees, particularly commercially important animals or livestock. Other mammals are included, including but not limited to.

  In the course of the present invention, a peptide having an amino acid sequence selected from SEQ ID NOs: 1 to 4 may be converted into several HLA-DR and / or HLA-DP molecules (eg HLA-DR53, HLA-DR4, HLA- DR15 and HLA-DP5) are found to be promiscuous Th1 cell epitopes that are potent and specific immunity against cancer due to immune responses mediated by MHC class II molecules Can be a candidate that can induce a response. Therefore, an agent or pharmaceutical composition of the invention comprising any of these peptides having the amino acid sequence of SEQ ID NOs: 1 to 4 is HLA-DR53, HLA-DR4, HLA-DR15 and HLA as MHC class II molecules. -Particularly suitable for administration to a subject having at least one selected from among DP5. The same applies to drugs or pharmaceutical compositions comprising a polynucleotide encoding any of these peptides.

  Alternatively, in a preferred embodiment, the peptides identified in the course of the present invention can induce CTL specific for DEPDC1 when applied to a subject with HLA-A2 or HLA-A24. Therefore, it is further expected that the administration of the peptides of the present invention can induce a CTL response against cancers that express DEPDC1 in addition to the induction of Th1 cells. Furthermore, the peptides of the present invention can not only induce a CTL response against DEPDC1-expressing cells via its processing, but also enhance the CTL response by Th1 cell induction mediated thereby. Thus, to achieve induction of both Th1 cells and DEPDC1-specific CTL in the same subject, for example, if the subject to be treated is administered a peptide having the amino acid sequence of SEQ ID NO: 2 or 3, preferably It has HLA-DR4 or HLA-DP5 as an MHC class II molecule and HLA-A2 as an MHC class I molecule. Thus, in order to achieve induction of both Th1 cells and DEPDC1-specific CTL in the same subject, for example, when the subject to be treated is administered a peptide having the amino acid sequence of SEQ ID NO: 2, preferably the MHC class It has HLA-DR4 as the II molecule and HLA-A24 as the MHC class I molecule.

In the present invention, it has been confirmed that the peptides of the present invention promote an immune response mediated by MHC class II antigens, specifically in an HLA-type restricted mode in the combinations as shown below:
DEPDC1-LP1: HLA-DR53 and HLA-DR4
DEPDC1-LP2: HLA-DR4
DEPDC1-LP3: HLA-DP5 and HLA-DR4
DEPDC1-LP4: HLA-DP5 and HLA-DR15

  Thus, a peptide comprising DEPDC1-LP1, -LP2, -LP3 and -LP4 and any one of the amino acid sequences of SEQ ID NOs: 1 to 4 is selected from the HLA subtypes matching them shown in the combination above Useful for the treatment of cancers that express DEPDC1 in patients with at least one HLA allele. Alternatively, the present invention provides a pharmaceutical composition for the treatment of cancer that expresses DEPDC1 in a patient, the composition comprising any one of the peptides selected from the group consisting of the peptides of the present invention, The patient has at least one HLA allele selected from the HLA subtypes corresponding to the peptide shown in the above combination.

  Furthermore, the present invention also provides for the manufacture of a composition for the treatment of cancer that expresses DEPDC1 in a patient having at least one HLA allele selected from HLA subtypes corresponding to the peptides shown in the above combinations The use of a peptide selected from the group consisting of the peptides of the present invention is provided. Furthermore, in some embodiments, the present invention is used in the treatment of cancers that express DEPDC1 in patients having at least one HLA allele selected from HLA subtypes corresponding to the peptides shown in the above combinations. To this end, a peptide selected from the group consisting of the peptides of the present invention is provided. In a further aspect, the present invention provides a method for treating a cancer that expresses DEPDC1 in a patient, wherein the method is a peptide selected from the group consisting of the peptides of the present invention in the above combination, Administering to a patient having at least one HLA allele selected from the HLA subtype of the peptide.

  In addition, in some embodiments, the invention provides a method for the manufacture or formulation of a pharmaceutical composition for treating a cancer that expresses DEPDC1 in a patient, the composition comprising a peptide of the invention And the patient has at least one HLA allele selected from the HLA subtype corresponding to the peptide, as shown in the above combination. The methods of the invention can include, for example, a step for mixing or formulating any one of the peptides selected from the group consisting of the peptides of the invention and a pharmaceutically acceptable carrier.

  As described above, Th1 cells are well known to be important for the induction of effective tumor immunity in tumor-bearing hosts. The peptides of the present invention have the ability to induce Th1 cells in an HLA-restricted manner, and the specific HLA-restricted pattern of each peptide of the present invention is shown above. Accordingly, the present invention provides a composition that promotes or enhances a Th1 cell response to a cancer that expresses DEPDC1 in a patient, wherein the composition is any peptide selected from the group consisting of the peptides of the present invention. Including one, the patient has at least one HLA allele selected from the HLA subtypes corresponding to the peptides shown above.

  Furthermore, the present invention also comprises the use of a peptide selected from the group consisting of the peptides of the present invention for the manufacture of a composition for promoting or enhancing a Th1 cell response against a cancer expressing DEPDC1 in a patient. And the patient has at least one HLA allele selected from the HLA subtype corresponding to the peptide shown in the combination. Further, in some embodiments, the present invention provides a peptide selected from the group consisting of the peptides of the present invention for use in promoting or enhancing a Th1 cell response against a cancer that expresses DEPDC1 in a patient. And the patient has at least one HLA allele selected from the HLA subtype corresponding to the peptide shown in the combination. In a further aspect, the invention provides a method for promoting or enhancing a Th1 cell response against a cancer that expresses DEPDC1 in a patient, the method comprising a peptide selected from the group consisting of the peptides of the invention. Administering to a patient having at least one HLA allele selected from the HLA subtypes of the peptide in a combination as described above.

  In addition, in some embodiments, the present invention provides methods for producing or formulating a pharmaceutical composition for promoting or enhancing a Th1 cell response to a cancer that expresses DEPDC1 in a patient, The composition comprises any one of the peptides selected from the group consisting of the peptides of the present invention, and the patient has at least one HLA selected from the HLA subtypes corresponding to the peptides shown in the combination. Has allyl. The methods of the invention can include, for example, a step for mixing or formulating any one of the peptides selected from the group consisting of the peptides of the invention and a pharmaceutically acceptable carrier.

  In another aspect, the present invention provides cancer immunotherapy that relies on Th1 cell induction. The therapeutic strategy provided by the present invention can be applied to any cancer regardless of DEPDC1 expression, as long as immune cells activated by cytokines secreted from Th1 cells target the target cancer cells, It is valid.

  Cancers or tumors to be treated with the agents or pharmaceutical compositions of the present invention include any type of cancer or tumor that expresses DEPDC1, including but not limited to bladder cancer, breast cancer.

  The drug or pharmaceutical composition of the present invention comprises, in addition to the active ingredient, other peptides having the ability to induce Th1 cells or CTLs, other polynucleotides encoding the other peptides, the other peptides or fragments thereof Other cells presenting Examples of such “other” peptides that have the ability to induce Th1 cells or CTLs include, but are not limited to, peptides derived from cancer specific antigens (eg, identified TAAs).

  If necessary, the medicament or pharmaceutical composition of the present invention may optionally contain other therapeutic substances as long as the other therapeutic substances do not inhibit the antitumor action of any of the active ingredients such as the peptides of the present invention. It may be included as an active ingredient. For example, the formulation can include anti-inflammatory agents, analgesics, chemotherapeutic agents, and the like. In addition to including other therapeutic substances in the medicament itself, the medicament of the present invention can also be administered sequentially or simultaneously with one or more other pharmacological agents. The amount of pharmaceutical and pharmacological agent depends, for example, on the type of pharmacological agent used, the disease being treated, and the schedule and route of administration.

  Those skilled in the art will recognize that in addition to the ingredients specifically mentioned herein, the agent or pharmaceutical composition of the present invention may contain other agents (eg, bulking agents) routine in the art in view of the type of formulation of interest. , Binders, diluents, excipients, etc.).

  In one aspect of the invention, the agents or pharmaceutical compositions of the invention can be included in products and kits containing materials useful for treating the disease being treated, such as the pathological state of cancer. Said product may comprise a container of any of the labeled medicaments or pharmaceutical compositions of the invention. Suitable containers include bottles, vials and test tubes. The container can be formed from a variety of materials such as glass or plastic. The label on the container should indicate that the agent is used for the treatment or prevention of one or more conditions of the disease. The label may also indicate directions for administration and the like.

  In addition to the containers described above, the kit comprising the medicament or pharmaceutical composition of the present invention may further comprise a second container that optionally contains a pharmaceutically acceptable diluent. The second container may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes and package inserts along with instructions for use. .

  The drug or pharmaceutical composition can be packaged in a pack or dispenser device, which can contain one or more unit dosage forms containing the active ingredients, if desired. The pack may include a metal or plastic foil, such as a blister pack, for example. The pack or dispenser device can be accompanied by instructions for administration.

(1) Drug or pharmaceutical composition containing a peptide as an active ingredient:
The peptides of the invention can be administered directly as a drug or pharmaceutical composition or, if necessary, formulated by conventional formulation methods. In the latter case, in addition to the peptide of the present invention, carriers, excipients and the like that are usually used for drugs can be appropriately included without particular limitation. Examples of such carriers include, but are not limited to, sterilized water, physiological saline, phosphate buffer, culture fluid, and the like. In addition, the drug or pharmaceutical composition may contain stabilizers, suspensions, preservatives, surfactants and the like as required. The drug or pharmaceutical composition of the present invention can be used for anticancer purposes.

  The peptides of the invention can be prepared as a combination composed of two or more of the peptides of the invention that induce Th1 cells in vivo. The peptide combination may take the form of a cocktail or may be conjugated to each other using standard techniques. For example, the peptides can be chemically linked or expressed as a single fusion polypeptide sequence. The peptides in the combination may be the same or different.

  By administering the peptides of the present invention, the peptide or fragment thereof is presented at high density by the HLA class II antigen on the APC, and then into the complex formed between the presented peptide and the HLA class II antigen. In contrast, Th1 cells reacting specifically are induced. Alternatively, APC (eg, DC) is removed from a subject and then stimulated with a peptide of the invention to obtain APC that presents either the peptide of the invention or a fragment thereof on its surface. These APCs can be re-administered to the subject to induce Th1 cells in the subject, resulting in increased aggressiveness against the tumor associated endothelium.

  The drug or pharmaceutical composition for treating and / or preventing cancer or tumor containing the peptide of the present invention as an active ingredient may also contain an adjuvant known to effectively establish cellular immunity. Alternatively, the drug or pharmaceutical composition can be administered with other active ingredients or can be administered by formulating into granules. An adjuvant refers to a compound that enhances the immune response to the protein when administered together (or sequentially) with the protein having immunological activity. Adjuvants contemplated herein include those described in the literature (Clin Microbiol Rev 1994, 7: 277-89). Examples of suitable adjuvants include aluminum phosphate, aluminum hydroxide, alum, cholera toxin, salmonella toxin, incomplete Freund's adjuvant (IFA), complete Freund's adjuvant (CFA), ISCOMAtrix, GM-CSF, CpG, oil-in-water type Although emulsion etc. are included, it is not limited to these.

  In addition, liposomal preparations, granular preparations in which peptides are bound to beads having a diameter of several micrometers, and preparations in which lipids are bound to peptides may be advantageously used.

  In another aspect of the invention, the peptides of the invention can also be administered in the form of a pharmaceutically acceptable salt. Examples of preferred salts include salts with alkali metals, salts with metals, salts with organic bases, organic acids (eg acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, apple Salts with acid, oxalic acid, benzoic acid, methanesulfonic acid, etc.) and salts with inorganic acids (eg hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.). As used herein, the phrase “pharmaceutically acceptable salt” retains the biological effectiveness and properties of a compound and is an inorganic acid or base such as hydrochloric acid, hydrobromic acid, sulfuric acid, It refers to a salt obtained by reaction with nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like.

  In some embodiments, an agent or pharmaceutical composition of the invention may further comprise components that prime Th1 cells and optionally CTLs. Lipids have been identified as agents capable of priming Th1 cells and optionally CTLs in vivo against viral antigens. For example, palmitic acid residues can be linked to the ε-amino and α-amino groups of lysine residues and then linked to the peptides of the invention. The lipidated peptide can then be administered directly in micelles or particles, incorporated into liposomes, or emulsified in an adjuvant. As another example of lipid priming of Th1 cells and optionally CTL responses, E. coli lipoproteins such as tripalmitoyl-S-glyceryl cysteinyl seryl-serine (P3CSS) were covalently linked to the appropriate peptide. In some cases, it can be used to prime Th1 cells and optionally CTLs (see eg Deres et al., Nature 1989, 342: 561-4).

  Examples of suitable administration methods include oral, intradermal, subcutaneous, intramuscular, intraosseous, intraperitoneal and intravenous injection, as well as systemic administration or local administration in the vicinity of the target site (ie direct injection) However, it is not limited to these. Administration can be accomplished by a single dose or can be enhanced by repeated doses. A pharmaceutically or therapeutically effective amount of the peptide can be administered to a subject in need of treatment for a cancer that expresses DEPDC1. Alternatively, an amount of a peptide of the invention sufficient to enhance or stimulate an immune response mediated by Th1 cells and / or induce CTL against a cancer or tumor expressing DEPDC1 and a cancer expressing DEPDC1 Can be administered to a subject carrying. The dose of the peptide of the present invention can be appropriately adjusted according to the disease to be treated, the patient's age, body weight, administration method, etc., and is usually 0.001 mg to 1000 mg, such as 0.01 mg to 100 mg, such as 0.1 mg to It is 10 mg, for example, 0.5 mg to 5 mg, and can be administered once every several days to several months. One skilled in the art can readily determine an appropriate and optimal dose.

(2) Drug or pharmaceutical composition containing a polynucleotide as an active ingredient:
The medicament or pharmaceutical composition of the present invention may also contain a polynucleotide that encodes a peptide disclosed herein in a form capable of expression. As used herein, the phrase “in expressible form” means that the polynucleotide is expressed in vivo as a polypeptide that induces anti-tumor immunity when introduced into a cell. In an exemplary embodiment, the nucleic acid sequence of the polynucleotide of interest includes regulatory elements necessary for the expression of the polynucleotide. The polynucleotide may comprise what is necessary to achieve stable integration into the genome of the target cell (for a description of homologous recombination cassette vectors, see, for example, Thomas KR & Capecchi MR, Cell 1987, 51: 503-12. reference). For example, Wolff et al. , Science 1990, 247: 1465-8; U.S. Pat. Nos. 5,580,859; 5,589,466; 5,804,566; 5,739,118; , 736,524; 5,679,647; and WO 98/04720. Examples of DNA-based delivery technologies include “naked DNA”, facilitated (bupivacaine, polymer, peptide-mediated) delivery, cationic lipid complexes, and particle-mediated (“gene gun”) or pressure-mediated delivery. Included (see, eg, US Pat. No. 5,922,687).

  The peptides of the invention can also be expressed by viral or bacterial vectors. Examples of expression vectors include attenuated viral hosts such as vaccinia virus or fowlpox virus. This approach involves the use of vaccinia virus, for example as a vector expressing a nucleotide sequence encoding a peptide of the invention. After introduction into the host, the recombinant vaccinia virus expresses an immunogenic peptide, thereby eliciting an immune response. Vaccinia vectors and methods useful for immunization protocols are described, for example, in US Pat. No. 4,722,848. Another vector is BCG (Bacilli Calmette Guerin). The BCG vector is described by Stover et al. , Nature 1991, 351: 456-60. A wide variety of other vectors useful for therapeutic administration or immunization are evident, such as adenovirus vectors and adeno-associated virus vectors, retrovirus vectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, etc. . See, for example, Shata et al. MoI Med Today 2000, 6: 66-71; Shedlock et al. , J Leukoc Biol 2000, 68: 793-806; Hipp et al. , In Vivo 2000, 14: 571-85.

  Delivery of the polynucleotide to the subject may be direct, in which case the subject may be directly exposed to the vector carrying the polynucleotide, or indirectly, in which case the cells are first shed in vitro. Transform with the polynucleotide of interest and then transplant the cells into the subject. These two approaches are known as in vivo and ex vivo gene therapy, respectively.

  For a general review of gene therapy methods, see Goldspiel et al. , Clinical Pharmacy 1993, 12: 488-505; Wu and Wu, Biotherapy 1991, 3: 87-95; Tolstoshev, Ann Rev Pharmacol Toxicol 1993, 33: 573-96; Mulligan, Science 92-93; & 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 technology that can also be used in the present invention are described in eds. Ausubel et al. , Current Protocols in Molecular Biology, John Wiley & Sons, NY, 1993; and Krieger, Gene Transfer and Expression, A Laboratory Pressure, StockN, 19N.

  Similar to peptide administration, polynucleotide administration may be performed by oral, intradermal, subcutaneous, intravenous, intramuscular, intraosseous and / or intraperitoneal injection, etc., administered systemically or near the target site. Topical administration is also used. Administration can be performed by a single dose or can be enhanced by multiple doses. A pharmaceutically or therapeutically effective amount of a polynucleotide of the invention can be administered to a subject in need of treatment for a cancer that expresses DEPDC1. Alternatively, a sufficient amount of a polynucleotide of the invention expressing DEPDC1 is sufficient to enhance or stimulate an immune response mediated by Th1 cells and / or induce CTL against a cancer or tumor that expresses DEPDC1. Can be administered to subjects carrying cancer. The dosage of the polynucleotide in a cell transformed with a suitable carrier or polynucleotide encoding the peptide of the present invention can be appropriately adjusted according to the disease to be treated, the patient's age, weight, method of administration, etc. Is 0.001 mg to 1000 mg, such as 0.01 mg to 100 mg, such as 0.1 mg to 10 mg, such as 0.5 mg to 5 mg, and can be administered once every few days to once every several months. One skilled in the art can readily determine an appropriate and optimal dosage.

IX. Methods of using peptides, APC or Th1 cells The peptides of the invention and polynucleotides encoding such peptides can be used to induce APC and Th1 cells of the invention. The APC of the present invention can also be used to induce Th1 cells of the present invention. Peptides, polynucleotides and APCs can be used in combination with any other compound as long as any other compound does not inhibit their ability to induce Th1 cells. Thus, any of the aforementioned agents or pharmaceutical compositions of the present invention can be used to induce Th1 cells, in addition to those containing peptides and polynucleotides to induce APC as discussed below. Can be used.

(1) Method for inducing antigen-presenting cells (APC):
The present invention provides a method of inducing APC using a peptide of the present invention or a polynucleotide encoding the peptide of the present invention. Induction of APC can be performed as described above in the section “V. Antigen-presenting cells”. The present invention also provides a method for inducing APC having the ability to induce Th1 cells, and the induction of APC is also referred to in “V. Antigen-presenting cells”, supra.

Alternatively, the present invention is a method for preparing an APC having the ability to induce Th1 cells, comprising the following steps:
(A) contacting the APC with the peptide of the invention in vitro, ex vivo or in vivo; and (b) introducing one of the steps of introducing a polynucleotide encoding the peptide of the invention into the APC.
Alternatively, the present invention is a method for inducing APC having Th1 cell induction ability,
There is provided a method comprising: (a) contacting APC with a peptide of the present invention; and (b) introducing a polynucleotide encoding the peptide of the present invention into APC.

  The methods of the invention can be performed in vitro, ex vivo or in vivo. Preferably, the methods of the invention can be performed in vitro or ex vivo. In a preferred embodiment, the APC used for the induction of APC having Th1 cell inducibility is preferably at least selected from HLA-DR53, HLA-DR4, HLA-DR15 and HLA-DP5 as MHC class II molecules. It can be an APC that expresses one. Such APCs are from peripheral blood mononuclear cells (PBMC) obtained from subjects having at least one selected from HLA-DR53, HLA-DR4, HLA-DR15 and HLA-DP5 as MHC class II molecules. Can be prepared by methods well known in the art. APC induced by the method of the present invention presents on its surface a complex of the peptide of the present invention or a fragment thereof and an HLA class II antigen (eg, HLA-DR53, HLA-DR4, HLA-DR15 and HLA-DP5). APC to When administering to a subject an APC induced by the methods of the invention in order to induce an immune response against cancer in the subject, the subject is preferably the same subject from which the APC is derived. However, as long as the subject has the same HLA type as the APC donor, the subject may be a different subject than the APC donor.

  In another aspect, the present invention provides an agent or composition for use in inducing APC having Th1 cell inducibility, such agent or composition comprising one or more of the present invention. Includes peptides or polynucleotides.

  In another aspect, the invention provides the use of a peptide of the invention or a polynucleotide encoding a peptide of the invention in the manufacture of an agent or composition formulated to induce APC.

  Alternatively, the present invention further provides a peptide of the present invention or a polypeptide encoding the peptide of the present invention for use in inducing APC having Th1 cell inducibility.

  In a preferred embodiment, the peptides of the invention not only induce a Th1 response, but also induce a CTL response after being processed in APC. Thus, in a preferred embodiment, APC prepared by the methods of the present invention may also be useful for inducing CTL against DEPDC1-expressing cells, including cancer cells. For example, when induced by a peptide comprising the amino acid sequence of SEQ ID NO: 5, APC expressing HLA-A2 is suitable for inducing DEPDC1-specific CTL. Alternatively, when induced by a peptide comprising the amino acid sequence of SEQ ID NO: 6, APC expressing HLA-A24 is suitable for inducing DEPDC1-specific CTL.

(2) Method for inducing Th1 cells:
Furthermore, the present invention provides a method for inducing Th1 cells using APC presenting the peptide of the present invention, the polynucleotide encoding the peptide of the present invention, or the peptide of the present invention or a fragment thereof. The present invention also provides a method for inducing Th1 cells using a polynucleotide encoding a polypeptide capable of forming a TCR subunit that recognizes a complex of a peptide of the present invention and an HLA class II antigen. To do. Preferably, the method for inducing Th1 cells is:
(A) contacting a CD4 positive T cell with an APC that presents on its surface a complex of an HLA class II antigen and a peptide of the invention or a fragment thereof, and (b) the TCR is a peptide of the invention or a peptide thereof Introducing into the CD4 positive T cells a polynucleotide encoding both TCR subunits or a polynucleotide encoding each of the TCR subunits capable of recognizing or binding to the complex of the fragment and HLA class II antigen At least one stage selected from the group consisting of:

When the peptide of the present invention is administered to a subject, Th1 cells are induced in the subject's body, and an immune response mediated by MHC class II molecules (for example, an immune response targeting cancer cells) is enhanced. Alternatively, the peptides of the invention and polynucleotides encoding the peptides of the invention can be used in an ex vivo therapy, in which APC and CD4 positive cells from a subject, or peripheral blood mononuclear leukocytes are in vitro After contacting with the peptide of the present invention (stimulating with the peptide of the present invention) and inducing Th1 cells, the activated Th1 cells are returned to the subject. For example, the method is:
(A) recovering APC from the subject;
(B) contacting the APC of step (a) with a peptide of the invention;
(C) mixing the APC of step (b) with CD4 ⁺ T cells and co-culturing to induce Th1 cells; and (d) recovering CD4 ⁺ T cells from the co-culture of step (c) Stage.
Can be included.
In addition, Th1 cells may contain polynucleotides encoding both TCR subunits or polynucleotides encoding each of the TCR subunits, wherein the TCR can bind to a complex of a peptide of the invention or a fragment thereof and an HLA class II antigen. It can be induced by introducing nucleotides into CD4 positive T cells. Such transduction can be carried out as described above in the section “VII. T cell receptor (TCR)”.

  The methods of the invention can be performed in vitro, ex vivo or in vivo. Preferably, the methods of the invention can be performed in vitro or ex vivo. CD4-positive T cells used for the induction of Th1 cells can be prepared from PBMCs obtained from a subject by methods well known in the art. In a preferred embodiment, the CD4 positive T cell donor can be a subject having at least one selected from HLA-DR53, HLA-DR4, HLA-DR15 and HLA-DP5 as MHC class II molecules. The Th1 cell induced by the method of the present invention may be a Th1 cell capable of recognizing APC that presents a complex of the peptide of the present invention or a fragment thereof and an HLA class II antigen on its surface. When administering Th1 cells induced by the methods of the present invention to a subject to induce an immune response against cancer (or an immune response mediated by MHC class I molecules) in the subject, the subject is preferably CD4 positive The same subject from which the T cells are derived. However, as long as the subject has the same HLA type as the CD4 positive T cell donor, the subject may be a different subject than the CD4 positive T cell donor.

In a preferred embodiment, the peptides of the present invention can induce CTLs against DEPDC1-expressing cells as well as Th1 cells. Therefore, the present invention further provides a method for inducing CTLs:
(A) co-culturing both CD4 positive T cells and CD8 positive T cells with APC contacted with the peptide of the present invention; and (b) APC contacted with CD8 positive T cells with the peptide of the present invention. Co-culturing.
A method comprising at least one step selected from the group consisting of:

  In such methods of inducing CTL, the peptides of the invention are processed in APC to produce CTL epitope peptides, and the generated CTL epitope peptides are presented on the surface of APC.

  Alternatively, according to the present invention, there is provided use of the peptide of the present invention for producing a drug or pharmaceutical composition that induces Th1 cells. In addition, the present invention provides a method or process for producing an agent or pharmaceutical composition that induces Th1 cells, wherein the method mixes or formulates a peptide of the present invention with a pharmaceutically acceptable carrier. Including steps to do. Furthermore, the present invention also provides a peptide of the present invention for inducing Th1 cells.

CD4 ⁺ T cells induced by the methods of the present invention can be administered to a subject as a vaccine.

In the context of the present invention, cancers that overexpress DEPDC1 can be treated with these active ingredients. Examples of such cancers include but are not limited to bladder cancer and breast cancer. Therefore, before administration of a vaccine or pharmaceutical composition containing the active ingredient, to determine whether the expression level of DEPDC1 in the cancer cells or tissues to be treated is increased compared to normal cells of the same organ Is preferred. Accordingly, in one aspect, the invention is a method for treating a cancer that (over) expresses DEPDC1:
(I) measuring the expression level of DEPDC1 in a cancer cell or tissue obtained from a subject having a cancer to be treated;
(Ii) comparing the expression level of DEPDC1 with a normal control; and (iii) overexpression of DEPDC1 by comparing at least one component selected from the group consisting of (a) to (d) above with a normal control Administering to a subject with cancer.
Provides a method that may include:

  Alternatively, the present invention relates to a vaccine containing at least one component selected from the group consisting of (a) to (d) above for use in administration to a subject having a cancer that overexpresses DEPDC1. A pharmaceutical composition may be provided. In other words, the present invention further comprises a method for identifying a subject to be treated with a DEPDC1 polypeptide of the present invention comprising measuring the level of expression of DEPDC1 in a cancer cell or tissue from the subject, A method is provided wherein an increase in level compared to a normal control level of a gene indicates that the subject has a cancer that can be treated with a DEPDC1 polypeptide of the invention. The method for treating cancer of the present invention is described in more detail below.

  Furthermore, in a preferred embodiment, the subject's HLA type can be identified prior to administration of the peptides of the invention. For example, a peptide having the amino acid sequence of SEQ ID NO: 1 is preferably administered to a subject identified as having HLA-DR53 or HLA-DR4. Alternatively, the peptide having the amino acid sequence of SEQ ID NO: 2 is preferably administered to a subject identified as having HLA-DR4. Alternatively, the peptide having the amino acid sequence of SEQ ID NO: 3 is preferably administered to a subject identified as having HLA-DP5 or DR4. Alternatively, the peptide having the amino acid sequence of SEQ ID NO: 4 is preferably administered to a subject identified as having HLA-DP5 or DR15.

  Cells or tissues from any subject can be used to measure DEPDC1 expression so long as they contain the desired DEPDC1 transcription or translation product. Examples of suitable samples include, but are not limited to, body tissues and fluids such as blood, saliva and urine. Preferably, the subject-derived cell or tissue sample contains a cell population comprising epithelial cells, more preferably cancerous epithelial cells or epithelial cells derived from tissue suspected of being cancerous. Furthermore, if necessary, cells may be purified from the obtained body tissue and fluid, and then used as a subject-derived sample.

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

  According to the present invention, the expression level of DEPDC1 in a cancer cell or tissue obtained from a subject is measured. Expression levels can be measured at the transcription (nucleic acid) product level using methods known in the art. For example, DEPDC1 mRNA can be quantified using a probe by a hybridization method (eg, Northern hybridization). Detection can be performed on a chip or array. The use of an array is preferred for detecting the expression level of DEPDC1. One skilled in the art can prepare such a probe using the sequence information of DEPDC1. For example, DEPDC1 cDNA can be used as a probe. If necessary, the probe may be labeled with an appropriate label, such as a dye, fluorescent material, and isotope, and the expression level of the gene can be detected as the intensity of the hybridized label.

  In addition, transcripts of DEPDC1 (eg, SEQ ID NO: 7 or 9) can be quantified using primers by amplification-based detection methods (eg, RT-PCR). Such primers can be prepared based on available gene sequence information.

  Specifically, the probe or primer used in the method of the present invention hybridizes to DEPDC1 mRNA under stringent conditions, moderately stringent conditions or low stringent conditions. As used herein, the phrase “stringent (hybridization) conditions” refers to conditions under which a probe or primer hybridizes to its target sequence but not to other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Specific hybridization of longer sequences is observed at higher temperatures than shorter sequences. Generally, the temperature of stringent conditions is selected to be about 5 ° C. lower than the thermal melting temperature (Tm) for the specific sequence at a defined ionic strength and pH. Tm is the temperature at which 50% of a probe complementary to a target sequence (under a defined ionic strength, pH and nucleic acid concentration) hybridizes to the target sequence in equilibrium. Since the target sequence is generally present in excess, at Tm, 50% of the probe is occupied in equilibrium. Typically, stringent conditions are those with a salt concentration of pH 7.0-8.3 and less than about 1.0M sodium ion, typically about 0.01-1.0M sodium ion (or other And a temperature of at least about 30 ° C. for short probes or primers (eg, 10-50 nucleotides) and at least about 60 ° C. for longer probes or primers. Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.

Alternatively, the translation product may be detected for the diagnosis of the present invention. For example, the amount of DEPDC1 protein (SEQ ID NO: 9 or 11) can be measured. Methods for measuring the amount of protein as a translation product include immunoassays using antibodies that specifically recognize the protein. The antibody can be monoclonal or polyclonal. Furthermore, as long as the antibody fragment or the modified antibody retains the ability to bind to the DEPDC1 protein, any fragment or modified form of the antibody (eg, chimeric antibody, scFv, Fab, F (ab ′) ₂ , Fv, etc.) can be detected. Can be used. Methods for preparing these types of antibodies for the detection of proteins are well known in the art, and any method can be used in the present invention to prepare such antibodies and their equivalents.

  As another method for detecting the expression level of the DEPDC1 gene based on the translation product, the intensity of staining can be measured through immunohistochemical analysis using an antibody against the DEPDC1 protein. That is, in this measurement, intense staining refers to an increased presence / level of protein and at the same time a high expression level of the DEPDC1 gene.

  The expression level of a target gene, eg, DEPDC1 gene, in a cancer cell is increased by, for example, 10%, 25% or 50% from the control level of the target gene (eg, the level in normal cells) or more than 1.1 times , Greater than 1.5 times, greater than 2.0 times, greater than 5.0 times, greater than 10.0 times, or more may be determined to be increased.

  Control levels can be measured simultaneously with cancer cells by using a sample previously collected and stored from a subject with known disease state (cancerous or non-cancerous). In addition, normal cells obtained from non-cancerous areas of the organ having the cancer to be treated may be used as normal controls. Alternatively, the control level may be determined by statistical methods based on results obtained by analyzing the expression level of the DEPDC1 gene previously measured in a sample from a subject with a known disease state. Furthermore, control levels can be derived from a database of expression patterns from previously tested cells. Furthermore, according to one aspect of the invention, the expression level of the DEPDC1 gene in a biological sample can be compared to a plurality of control levels determined from a plurality of reference samples. It is preferred to use a control level determined from a reference sample derived from a tissue type similar to that of the biological sample from the subject. Furthermore, it is preferable to use a standard value for the expression level of the DEPDC1 gene in a population whose disease state is known. Standard values may be obtained by any method known in the art. For example, the mean ± 2S. D. Or mean ± 3S. D. Can be used as standard values.

  In the context of the present invention, a control level determined from a biological sample known to be non-cancerous is referred to as a “normal control level”. On the other hand, if the control level is determined from a cancerous biological sample, it is referred to as the “cancerous control level”. The difference between the expression level of the sample and the control level is normalized to the expression level of a control nucleic acid, such as a housekeeping gene, whose expression level is known not to vary depending on the cancerous or non-cancerous state of the cell. can do. Exemplary control genes include, but are not limited to, β-actin, glyceraldehyde 3-phosphate dehydrogenase, and ribosomal protein P1.

  A subject can be diagnosed as having a cancer to be treated if the expression level of the DEPDC1 gene is increased relative to a normal control level or similar / equivalent to a cancerous control level.

More particularly, the present invention is a method of (i) diagnosing whether a subject has a cancer to be treated and / or (ii) selecting a subject for cancer treatment:
(A) measuring the expression level of DEPDC1 in cancer cells or tissue obtained from a subject suspected of having a cancer to be treated;
(B) comparing the expression level of DEPDC1 with a normal control level;
(C) diagnosing the subject as having a cancer to be treated if the expression level of DEPDC1 is increased compared to a normal control level; and (d) in step (c), the subject is treated Selecting a subject for cancer treatment if it is diagnosed as having cancer to be done.
A method is provided.

Or such a method:
(A) measuring the expression level of DEPDC1 in cancer cells or tissue obtained from a subject suspected of having a cancer to be treated;
(B) comparing the expression level of DEPDC1 to a cancerous control level;
(C) diagnosing the subject as having a cancer to be treated if the expression level of DEPDC1 is similar or equivalent to the cancerous control level; and (d) in step (c), the subject is treated Selecting a subject for cancer treatment if it is diagnosed as having cancer to be done.
including.

  In some embodiments, such methods are selected from the group consisting of HLA-DR53, HLA-DR4, HLA-DR15 and HLA-DP5 after or before steps (a)-(d) as defined above. The method may further include identifying a subject having the HLA to be performed. The cancer therapy according to the present invention is preferred for a subject suffering from a cancer that overexpresses DEPDC1 and having any one of HLA-DR53, HLA-DR4, HLA-DR15 and HLA-DP5. HLA typing methods are well known in the art. For example, PCR-based methods for typing HLA alleles are well known. Antibodies specific for each HLA molecule are also suitable tools for identifying the HLA type of interest.

The present invention also provides kits for determining a subject suffering from cancer that can be treated with a DEPDC1 polypeptide of the present invention, such kits comprising a specific cancer therapy, more particularly May also be useful for assessing and / or monitoring the effects of cancer immunotherapy. Examples of suitable cancer descriptions include, but are not limited to, bladder cancer and breast cancer. More particularly, the kit preferably comprises at least one reagent for detecting the expression of the DEPDC1 gene in cancer cells from the subject, such reagent comprising:
(A) a reagent for detecting mRNA of the DEPDC1 gene;
(B) a reagent for detecting DEPDC1 protein; and (c) a reagent for detecting biological activity of DEPDC1 protein.

  Examples of reagents suitable for detecting DEPDC1 gene mRNA include nucleic acids that specifically bind to or identify DEPDC1 mRNA, eg, oligonucleotides having sequences complementary to a portion of DEPDC1 mRNA. It is. These types of oligonucleotides are exemplified by primers and probes specific for DEPDC1 mRNA. These types of oligonucleotides can be prepared based on methods well known in the art. If necessary, reagents for detecting DEPDC1 mRNA can be immobilized on a solid matrix. Furthermore, a plurality of reagents for detecting DEPDC1 mRNA may be included in the kit.

On the other hand, examples of reagents suitable for detecting DEPDC1 protein include antibodies to DEPDC1 protein. The antibody can be monoclonal or polyclonal. In addition, any fragment or modified form of the antibody (eg, chimeric antibody, scFv, Fab, F (ab ′) ₂ , Fv, etc.) may be used as long as the antibody fragment or modified antibody retains the ability to bind to DEPDC1 protein. Can be used as a reagent. Methods for preparing these types of antibodies for the detection of proteins are well known in the art, and any method can be used in the present invention to prepare such antibodies and their equivalents. Furthermore, the antibody may be labeled with a signal generating molecule by direct binding or indirect labeling techniques. Labels and methods for labeling antibodies and detecting binding of antibodies to their targets are well known in the art and any label and method may be utilized for the present invention. Furthermore, a plurality of reagents for detecting DEPDC1 protein may be included in the kit.

  The kit may include a plurality of the reagents. For example, a tissue sample obtained from a subject who does not have cancer or suffers from cancer can serve as a useful control reagent. The kit of the present invention includes a buffer, diluent, filter, needle, syringe and package insert (eg written, tape, CD-ROM, etc.) along with instructions for use, commercial and user perspective. To other materials desired. These reagents and the like can be held in a labeled container. Suitable containers include bottles, vials and test tubes. The container can be formed from a variety of materials such as glass or plastic.

  In one embodiment of the invention, when the reagent is a probe for DEPDC1 mRNA, the reagent can be immobilized on a solid matrix such as a porous strip to form at least one detection site. The measurement region or detection region of the porous strip may include a plurality of sites each containing a nucleic acid (probe). The test strip may also include sites for negative and / or positive controls. Alternatively, the control site can be located on a strip separated from the test strip. In some cases, different detection sites may contain different amounts of immobilized nucleic acid, ie, a higher amount at the first detection site and a lower amount at subsequent sites. After the addition of the test sample, the number of sites presenting a detectable signal provides a quantitative indicator of the amount of DEPDC1 mRNA present in the sample. The detection site can be configured in any suitable detectable shape and is typically in the form of a bar or dot across the width of the test strip.

  The kit of the present invention may further comprise a positive control sample or a DEPDC1 standard sample. A positive control sample of the invention can be prepared by taking a DEPDC1 positive sample and then assaying its DEPDC1 level. Alternatively, purified DEPDC1 protein or polynucleotide may be added to cells that do not express DEPDC1 to form a positive sample or DEPDC1 standard sample. In the present invention, purified DEPDC1 can be a recombinant protein. The DEPDC1 level of the positive control sample is above the cutoff value, for example.

X. antibody:
The present invention further provides antibodies that bind to the peptides of the present invention. Preferred antibodies specifically bind to the peptides of the invention and do not bind (or bind weakly) to other peptides. Alternatively, the antibody binds to a peptide of the invention as well as a homologue thereof. Antibodies against the peptides of the present invention can be used in cancer diagnostic and prognostic assays and imaging methods. Similarly, such antibodies can be used in the treatment, diagnosis and / or prognosis of other cancers as long as DEPDC1 is expressed or overexpressed in cancer patients. In addition, antibodies expressed within cells (eg, single chain antibodies) can be used therapeutically in treating cancers involving DEPDC1 expression, examples of which include bladder cancer and breast cancer However, it is not limited to these.

  The present invention also provides various methods for detection and / or quantification of DEPDC1 protein (SEQ ID NO: 8 or 10) or fragments thereof comprising a polypeptide consisting of an amino acid sequence selected from SEQ ID NOs: 1 to 4. Immunological assays are also provided. Such assays may optionally include one or more anti-DEPDCl antibodies that are capable of recognizing and binding to DEPDC1 protein or fragments thereof. In the present invention, an anti-DEPDC1 antibody that binds to a DEPDC1 polypeptide preferably recognizes a polypeptide consisting of an amino acid sequence selected from SEQ ID NOs: 1 to 4, preferably excluding other peptides. The binding specificity of the antibody can be confirmed by an inhibition test. That is, if binding between the antibody to be analyzed and the full-length DEPDC1 polypeptide is inhibited in the presence of any fragment polypeptide having an amino acid sequence selected from SEQ ID NOs: 1 to 4, the antibody binds to the fragment. Is specifically bound ". In the context of the present invention, such immunological assays include various types of radioimmunoassay, immunochromatographic techniques, enzyme linked immunoassay (ELISA), enzyme linked immunofluorescent assay (ELIFA), and the like. Are performed within various immunological assay formats well known in the art, including but not limited to.

  Related immunological but antibody-free assays of the invention can also include T cell immunogenicity assays (inhibitory or stimulatory) as well as MHC binding assays. In addition, immunological imaging methods capable of detecting cancers that express DEPDC1, including but not limited to radioscintigraphic imaging methods using labeled antibodies of the present invention, are also provided by the present invention. . Such assays can be used clinically in the detection, monitoring and prognosis of cancers that express DEPDC1, examples of such cancers include bladder cancer and breast cancer, It is not limited to.

  The present invention also provides antibodies that bind to the peptides of the present invention. The antibodies of the present invention can be used in any form, such as monoclonal antibodies or polyclonal antibodies, antisera obtained by immunizing animals such as rabbits with the peptides of the present invention, all classes of polyclonal and monoclonal antibodies , Human antibodies as well as humanized antibodies produced by genetic recombination.

The peptide of the present invention used as an antigen for obtaining an antibody can be derived from any animal species, but is preferably derived from a mammal such as a human, mouse or rat, more preferably a human. Human-derived peptides can be obtained from the nucleotide or amino acid sequences disclosed herein.
According to the present invention, the complete and partial peptides of the polypeptides of the present invention can serve as immunizing antigens. Examples of suitable partial peptides include, for example, an amino (N) terminal fragment or a carboxy (C) terminal fragment of a peptide of the invention.

  As used herein, an antibody is defined as a protein that reacts with either full-length DEPDC1 peptide or a fragment of DEPDC1 peptide. In a preferred embodiment, the antibody of the present invention can recognize a fragment peptide of DEPDC1 having an amino acid sequence selected from SEQ ID NOs: 1 to 4. Methods for synthesizing oligopeptides are well known in the art. After synthesis, the peptide may optionally be purified prior to use as an immunogen. In the present invention, an oligopeptide (eg, 24mer or 26mer) may be conjugated or linked to a carrier to enhance immunogenicity. Keyhole limpet hemocyanin (KLH) is well known as a carrier. Methods for conjugating KLH and peptides are also well known in the art.

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

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

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

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

To prepare a monoclonal antibody for use in connection with the present invention, immune cells are collected from a mammal immunized with an antigen and examined for elevated levels of the desired antibody in serum as described above and subjected to cell fusion. The immune cells used for cell fusion are preferably obtained from the spleen. Other preferred parent cells to be fused with the immune cells include, for example, mammalian myeloma cells, more preferably myeloma cells with acquired properties for selection of fusion cells with drugs.
The above immune cells and myeloma cells are obtained according to a known method, for example, Milstein et al. (Galfre and Milstein, Methods Enzymol 73: 3-46 (1981)).

  The hybridomas obtained by the resulting cell fusion can be selected by culturing them in a standard selection medium such as HAT medium (medium containing hypoxanthine, aminopterin and thymidine). Cell culture is typically continued in HAT medium for days to weeks, i.e. for a time sufficient to kill all other cells (non-fused cells) except the desired hybridoma. Subsequently, standard limiting dilutions can be performed to screen and clone hybridoma cells producing the desired antibody.

  In addition to the above methods of immunizing non-human animals with antigens to prepare hybridomas, human lymphocytes such as those infected with EB virus can be immunized in vitro with peptides, peptide-expressing cells or lysates thereof. The immunized lymphocytes can then be fused with human-derived myeloma cells that can divide indefinitely, such as U266, to produce hybridomas that produce the desired human antibody that can bind to the peptide (specifically Kaisho 63-17688).

The resulting hybridoma can then be transplanted into the abdominal cavity of a mouse to extract ascites. The obtained monoclonal antibody can be purified by, for example, ammonium sulfate precipitation, protein A or protein G column, DEAE ion exchange chromatography, or an affinity column to which the peptide of the present invention is bound. The antibodies of the present invention can be used not only for the purification and detection of the peptides of the present invention, but also as candidates for agonists and antagonists of the peptides of the present invention.
Alternatively, immune cells such as immune lymphocytes that produce antibodies may be immortalized by oncogenes and used to prepare monoclonal antibodies.

  Monoclonal antibodies obtained in this way can also be prepared by recombination using genetic engineering techniques (see, for example, Borrebaeck and Larrick, Therapeutic Monoclonal Antibodies, published in the United KingdomsMilDlM 90MlD). For example, DNA encoding an antibody can be cloned from immune cells, such as hybridomas or immune lymphocytes that produce antibodies, inserted into an appropriate vector, introduced into host cells, and recombinant antibodies can be prepared. The invention also provides a recombinant antibody prepared as described above.

The antibodies of the invention may be antibody fragments or modified antibodies, so long as the antibody fragments or modified antibodies bind to one or more of the peptides of the invention. For example, antibody fragments can be Fab, F (ab ′) ₂ , Fv, or single chain Fv (scFv) in which Fv fragments from H and L chains are linked by a suitable linker (Huston et al Proc Natl Acad Sci USA 85: 5879-83 (1988)). More particularly, antibody fragments can be generated by treating antibodies with enzymes such as papain or pepsin. Alternatively, a gene encoding the antibody fragment may be constructed, inserted into an expression vector and expressed in a suitable host cell (eg, Co et al., J Immunol 152: 2968-76 (1994); Better and Horwitz). , Methods Enzymol 178: 476-96 (1989); Pluckthun and Skerra, Methods Enzymol 178: 497-515 (1989); -9 (1986); Bird and Walker, Trends Biotechnol 9: 132-7 (1991)).

  An antibody can be modified by conjugation with a variety of molecules, such as polyethylene glycol (PEG). The present invention provides such modified antibodies. Modified antibodies can be obtained by chemically modifying antibodies. These modification methods are routine in the art.

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

  Fully human antibodies that contain human variable regions in addition to human framework and constant regions can also be used. Such antibodies can be generated using various techniques known in the art. For example, in vitro methods include the use of recombinant libraries of human antibody fragments displayed on bacteriophages (see, 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 genes have been partially or completely inactivated. This approach is described, for example, in US Pat. Nos. 6,150,584; 5,545,807; 5,545,806; 5,569,825; 5,625,126. No. 5,633,425; No. 5,661,016.

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

  Examples of suitable chromatography techniques, excluding affinity chromatography, include, for example, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, adsorption chromatography, etc. (Stratesies for Protein Purification and Characterization: A Laboratory Course Manual. Ed Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press (1996)). Chromatographic procedures can be performed by liquid phase chromatography such as HPLC and FPLC.

  For example, the antigen-binding activity of the antibody of the present invention can be measured using absorbance measurement, ELISA, enzyme immunoassay (EIA), radioimmunoassay (RIA) and / or immunofluorescence (IF). In ELISA, the antibody of the present invention is immobilized on a plate, the peptide of the present invention is applied to the plate, and then a sample containing the desired antibody, for example, a culture supernatant of antibody-producing cells or a purified antibody is applied. A secondary antibody labeled with an enzyme such as alkaline phosphatase that recognizes the primary antibody is then applied and the plate is incubated. Next, after washing, an enzyme substrate such as p-nitrophenyl phosphate is added to the plate, the absorbance is measured, and the antigen binding activity of the sample is evaluated. Fragments of peptides such as C-terminal or N-terminal fragments can be used as antigens for assessing antibody binding activity. BIAcore (Pharmacia) may be used to assess the activity of the antibody according to the invention.

In the above method, the antibody of the present invention is exposed to a sample presumed to contain the peptide of the present invention, and the immune complex formed by the antibody and the peptide is detected or measured to detect or measure the peptide of the present invention. Enable measurement.
Since the method for detecting or measuring a peptide according to the present invention can specifically detect or measure the peptide, this method can be used in various experiments using the peptide. For example, when the peptides of the present invention are detected in cancer cells or tissues obtained from patients, Th1 cells (or CTL cells) against them are expected to be an effective tool in cancer immunotherapy. The

XI. Vectors and Host Cells The present invention also provides vectors and host cells that introduce nucleotides encoding the peptides of the present invention. The vectors of the present invention are useful as carriers for the nucleotides of the present invention, particularly DNA, in host cells for expressing the peptides of the present invention or for administering the nucleotides of the present invention for gene therapy.

  If E. coli is selected as the host cell and the vector is amplified and produced in large quantities in E. coli (eg, JM109, DH5α, HB101 or XL1Blue), the vector is suitable for “origin” and transformation suitable for amplification in E. coli. Should have a suitable marker gene (for example, a drug resistance gene selected by a drug such as ampicillin, tetracycline, kanamycin, chloramphenicol, etc.). 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 be used for subcloning and extracting cDNA, similar to the vectors described above. When a vector is used to produce the protein of the present invention, an expression vector can be used. For example, an expression vector expressed in E. coli should have the above characteristics amplified in E. coli. When E. coli such as JM109, DH5α, HB101, or XL1 Blue is used as a host cell, the vector can be a promoter that can effectively express the desired gene in E. coli, such as the lacZ promoter (Ward et al., Nature 341: 544-6 (1989); FASEB J 6: 2422-7 (1992)), araB promoter (Better et al., Science 240: 1041-3 (1988)), T7 promoter and the like. In that regard, pGEX-5X-1 (Pharmacia), “QIAexpress system” (Qiagen), pEGFP and pET (in this case the host is preferably BL21 expressing T7 RNA polymerase), for example Can be used instead. In addition, the vector may also include a signal sequence for peptide secretion. An exemplary signal sequence that directs the peptide to be secreted into the periplasm of E. coli is the pelB signal sequence (Lei et al., J Bacteriol 169: 4379 (1987)). Means for introducing the vector into the target host cell include, for example, the calcium chloride method and the electroporation method.

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

  In order to express the vector in animal cells such as CHO, COS or NIH3T3 cells, the vector may be a promoter required for expression in such cells, such as the SV40 promoter (Mulligan et al., Nature 277: 108 (1979)), MMLV-LTR promoter, EF1α promoter (Mizushima et al., Nucleic Acids Res 18: 5322 (1990)), CMV promoter and the like, and preferably a marker gene for selecting a transformant (eg, drug (eg, neomycin, G418) ) Should carry the drug resistance gene selected by). Examples of known vectors having these characteristics include, for example, pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV and pOP13.

XII. Monitoring of peptide-mediated Th1 response Through deep cDNA sequence analysis of TCR-α and TCR-β genes, the frequency of a particular pair of genes was increased in Th cells stimulated by the peptides of the present invention. It was revealed. Thus, if such a gene pair is detected in a subject after vaccination with a peptide of the invention, it means that a peptide-specific Th1 response in the subject has been induced. Thus, an increase in a particular pair of genes in a Th cell stimulated by a peptide of the invention may be useful as a surrogate marker for monitoring or detecting a Th1 response in a subject after stimulation. In the context of the present invention, a “peptide-specific Th1 response” is a complex in which a TCR formed between a pair of α and β subunits is formed between a peptide of the present invention and an HLA molecule. It is understood to mean specifically recognizing the body. As discussed above, the Th1 cell inducibility of peptides defined by the specific sequences of the present invention can be maintained even after amino acid modification. Therefore, in addition to stimulation with a specific peptide, as long as the TCR specifically recognizes such a complex formed by the original peptide, even when Th1 cells are derived from the mutant peptide, Its antigen specificity is considered “peptide specific”.

For example, a TCR-α subunit and a TCR-β subunit each having CDR3 consisting of the amino acid sequences of SEQ ID NOs: 11 and 21, respectively, in a HLA-DR4 restricted manner, DEPDC1-LP2 (DEPDC1 _292-313 -LP) Increased in Th cells stimulated by (FIG. 6). Therefore, their accumulation in the Th cell population of the subject means that the Th1 response mediated by DEPDC1-LP2 was successfully induced in subjects vaccinated with DEPDC1-LP2. The presence of CDR3 consisting of the amino acid sequences of SEQ ID NOs: 11 and 21 can be detected by antibody-based analysis. In alternative aspects of the present application, specific pairs of TCR-α and TCR-β subunits can also be assessed by detecting the polynucleotides that encode them. For example, a polynucleotide encoding each of CDR3 consisting of the amino acid sequences of SEQ ID NOs: 11 and 21 is represented by the nucleotide sequences of SEQ ID NOs: 31 and 32, respectively. Accordingly, each of the cDNA synthesized from mRNA encoding the α and β subunits of the DEPDC1-specific TCR can comprise the nucleotide sequences of SEQ ID NOs: 31 and 32, respectively. Such polynucleotides can be detected by PCR-based analysis.

In a preferred embodiment, the present invention is a method for monitoring, assessing or assessing a peptide-specific Th1 response in a subject immunized with a peptide comprising:
(A) providing a sample obtained from a subject administered the peptide, wherein the sample comprises T cells;
(B) detecting the presence of a T cell expressing a TCR that binds to a complex of an MHC class II molecule and the peptide or fragment thereof in the sample; and (c) the presence of the T cell is (b) A method comprising the step of showing an induction of a Th1 response specific for said peptide when detected in. For example, T cells specific for DEPDC1-LP2 (DEPDC1 _292-313 -LP) in an HLA-DR4 restricted mode by detecting α and β subunits consisting of the amino acid sequences of SEQ ID NOs: 11 and 21 Can be detected.

  In the present invention, any biological sample obtained from a subject can be used to monitor a Th1 response as long as the sample contains Th1 cells. For example, blood or blood derived samples can be used as biological samples for the present invention. In the present invention, the blood-derived sample includes a cell population containing T cells. Methods for obtaining cell populations containing T cells are well known to those skilled in the art. Alternatively, in some embodiments, tissues or lymph nodes infiltrated with T cells are also useful as biological samples.

  In some embodiments of the invention, cell populations containing T cells can be fixed for antibody-based analysis in assessing TCR subunits. An antibody recognizes the α3 and β subunit CDR3 of the fixed cell after contact with the fixed cell, and when the antibody binds to both subunits of the TCR on a single cell, TCR-antibody complexes formed with unit pairs can be detected. Alternatively, in a nucleotide sequence-based analysis, the accumulation of amplicons synthesized from a polynucleotide encoding a TCR subunit in a single cell means that a particular pair of subunits is present. Alternatively, specific pairs of polynucleotides encoding TCR subunits for each of the peptides to be vaccinated can be obtained from Fang H, et al. , Oncoimmunology 3: e968467, 2015, can be monitored by deep cDNA sequence analysis.

In this aspect, it is well known that the structural diversity of TCRs on Th1 cells depends mainly on their CDR3. Consequently, the antigen specificity thereby is also dependent on CDR3. Therefore, the presence of a specific pair of α and β subunits on a single cell can be monitored through detection of CDR3.
In some embodiments of the invention, a specific pair of TCR-α subunit and TCR-β subunit is detected at least once or multiple times after vaccination to monitor or assess Th1 response. be able to. If the pair increases time-dependently through monitoring, it means that the peptide-mediated Th1 response in the subject is well induced. Alternatively, if a particular pair is detected at least once, it indicates that a Th1 response has occurred in the subject.

  In the following, the present invention will be described in more detail with reference to specific examples. However, the following experimental materials, methods and examples may help one skilled in the art to make and use certain aspects of the invention, but are intended only to illustrate aspects of the invention, Therefore, it does not limit the scope of the present invention in any way. Those skilled in the art will readily recognize that methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention.

Materials and Methods Cell lines A study protocol for collecting and using PBMC from healthy donors (HD) and patients with cancer was approved by the Kumamoto University Institutional Review Board. After obtaining written informed consent, PBMCs were obtained from patients with HD and cancer. Human monocyte-derived DCs and mouse BM-derived DCs were generated as previously reported (Tomita Y, et al., Cancer Sci. 2011; 102 (1): 71-78; Tomita Y, et al. , Clin Cancer Res. 2013; 19 (16): 4508-4520; Tomita Y, et al., Int J Cancer. 2014; 134 (2): 352-366; Tomita Y, et al., Oncoimmunology. : Sayem MA, et al., Oncoimmunology.2016; 5 (1): e1062209; Hirayama M, et al., Oncoimmunology.2016; 5 (6): e1233368).
^{DR4 (DRB1 * 04:05), L} -DR4; DR8 (DRB1 * 08:03), L-DR8; DR9 (DRB1 * 09:01), L-DR9; DR15 (DRB1 * 15:02), L- DR15; DR53 (DRB4 ^* 01: 03), L-DR53; DP2 (DPA1 ^* 01: 03 / DRB1 ^* 02: 01), L-DP2; DP4 (DPA1 ^* 01: 03 / DPB1 ^* 04: 01), RM3 Mouse fibroblasts genetically engineered to express DP4; DP5 (DPA1 ^* 02: 02 / DPB1 ^* 05: 01), L-DP5; and DP9 (DPB1 ^* 09: 01), L-DP9 The strain (L cells) was prepared by Dr. Alessandro Sette (La Jolla Institute for Allergy and Immu). (Nology, CA, USA) and used as APC.

Prediction of HLA class II binding peptides Computer algorithms (IEDB analysis resources, consensus method, http://tools.immunepepitope.org/) recently developed to predict potential promiscuous HLA class II binding human DEPDC1-derived peptides. (analyze / html / mhc_II_binding.html) (Wang P, et al., BMC Bioinformatics 2010; 11: 568. Wang P, et al. PLoS Compute Biol 2008; 4: e1000048). The program analyzed a 15 amino acid long sequence offset encompassing the entire DEPDC1 protein. Lower relative consensus percentile ranking for multiple HLA class II molecules encoded by DPB1 ^* 04: 05, DRB1 ^* 09: 01, DRB1 ^* 15: 02, DRB4 ^* 01: 03, and DPB1 ^* 05 ^: 01 alleles it is a 22-29 amino acid long peptide _{with, DEPDC1 191-213 LP (DEPDC1-LP1} ; RYVILIYLQTILGVPSLEEVINP ( SEQ ID _{NO: 1)), DEPDC1 292-313 -LP} (DEPDC1-LP2; TFEYYELFVNILVVCGYITVSD ( SEQ ID NO: 2)), DEPDC1 _301-329 -LP (DEPDC1-LP3; NILVVCGIITVSSDRSGIHKIQDDPQSSK (SEQ ID NO: 3)), and DEPDC 1 _613-634 -LP (DEPDC1-LP4; NRRKLQLLMRMISRMSQNVDMP (SEQ ID NO: 4)), two of which are 9-mer or 10-mer CTL epitopes from DEPDC1 (DEPDC1-A2 _302-311 and / or Or DEPDC1-A24 _294-302 ) was selected and synthesized (FIG. 1 and Table 1).

各々１５−ｍｅｒのＤＥＰＤＣ１由来のオーバーラップするペプチドについて、表示されているＨＬＡクラスＩＩ分子に対するペプチド結合アルゴリズムスコアを示す。

The peptide binding algorithm scores for the indicated HLA class II molecules are shown for overlapping peptides from each 15-mer DEPDC1.

各々１５−ｍｅｒのＤＥＰＤＣ１由来のオーバーラップするペプチドについて、表示されているＨＬＡクラスＩＩ分子に対するペプチド結合アルゴリズムスコアを示す。

The peptide binding algorithm scores for the indicated HLA class II molecules are shown for overlapping peptides from each 15-mer DEPDC1.

Synthetic peptides and recombinant proteins by HLA-A2 (DEPDC1-A2 _302-311 ) (Kanehira M, et al., Oncogene 2007; 26: 6448-55) or by HLA-A24 (DEPDC1-A24 _294-302 ) (Harada Y, et al., Cancer Res 2010; 70: 5829-39) Two human DEPDC1-derived SPs presented and four LPs (DEPDC1-LP1-4) were synthesized (MBL, Nagoya, Japan; purity) >95%; FIG. 1B). Human immunodeficiency virus (HIV) -SP (HIV-A2) binding to HLA-A2 or human immunodeficiency virus (HIV) -SP (HIV-A24) binding to HLA-A24 (Kanehira M, et al., Oncogene 2007; 26: 6448-55; Tomita Y, et al., Cancer Sci 2011; 102: 697-705) was used as a negative control SP. The peptide was dissolved in dimethyl sulfoxide at 10 mg / mL and stored at −80 ° C. Recombinant human full-length DEPDC1 protein and MPHOSPH1 protein were produced according to the manufacturer's instructions in E. coli BL21 transformed with the pET28a vector (Novagen, 69864-3) encoding DEPDC1 and MPHOSPH1. Both DEPDC1 protein and MPHOSPH1 protein were evaluated by SDS-PAGE and purified by HisTrap FF column (GE Healthcare, Little Chalfont, Buckinghamshire, UK). MPHOSPH1 protein was used as a negative control.

Generation of antigen-specific CD4 ⁺ T cells from healthy donors A study protocol for collecting and using PBMC from healthy donors was approved by the Kumamoto University Institutional Review Board. We obtained PBMC from 7 healthy donors with written informed consent. HLA-A, DRB1, and DPB1 alleles were genotyped at the HLA Institute (Kyoto, Japan) (Table 2). Induction of antigen-specific CD4 ⁺ T cells was performed as described previously (Tomita Y, et al., Clin Cancer Res 2013; 19: 4508-20; Tomita Y, with some modifications). Int J Cancer 2014; 134: 352-66; Tomita Y, et al., OncoImmunology 2014; 3: e28100; 5: 6, e1123368; Inoue M, et al., Int J Cancer 2010; 127: 1393-403; Harao M, et al., I t J Cancer 2008; 123: 2616-25). CD4 ⁺ T cells were purified from PBMC by positive selection with magnetic microbeads (Miltenyi Biotec, Auburn, CA, USA) (Inoue M, et al., Int J Cancer 2010; 127: 1393-403). Monocyte-derived dendritic cells (DCs) were generated from CD14 ⁺ cells by in vitro culture as previously described (Harao M, et al., Int J Cancer 2008; 123: 2616-25) Used as antigen presenting cells (APC) to induce antigen specific CD4 ⁺ T cells. DC (1 × 10 ⁴ cells / well) were pulsed with 10 μg / mL LP for 3 hours and irradiated (45 Gy), then 5% human complement-depleted plasma was added to each well of a 96-well flat bottom culture plate. Mixed with CD4 ⁺ T cells (3 × 10 ⁴ cells / well) in added 200 μL AIM-V. After 7 days, half of the medium was removed from each culture and irradiated (50 Gy) autologous PBMC (1 × 10 ⁵ ) pulsed with peptide (10 μg / mL) and 5 ng / mL recombinant human interleukin 7 (rhIL). Fresh medium (100 μL / well) containing -7) was added. Two days after the second stimulation with the peptide, rhIL-2 was added to each well (10 IU / mL). One week later, stimulated CD4 ⁺ T cells in each well were analyzed for specificity in an IFN-γ ELISPOT assay. T cells that showed a specific response to cognate peptides were transferred to 24-well plates and irradiated with peptide-pulsed radiation in medium supplemented with rhIL-2 (20 IU / mL) and rhIL-7 (5 ng / mL) Restimulated with autologous PBMC (1 × 10 ⁶ cells / well) at weekly intervals. In some cases, T cells were cloned by limiting dilution as described previously (Tabata H, et al., Hum Immunol 1998; 59: 549-60) for further studies.

^ａＤＥＰＤＣ１−ＬＰに特異的なＴｈ細胞は、健常ドナー（ＨＤ１〜ＨＤ７）のＰＢＭＣに由来した。

^a Th cells specific for DEPDC1-LP were derived from PBMCs of healthy donors (HD1-HD7).

Evaluation of T cell responses to peptides and proteins Th cell immune responses to peptide-pulsed irradiated APCs were previously described (Tomita Y, et al., Cancer Sci 2011; 102: 697-705). , Evaluated by IFN-γ enzyme-linked immunospot (ELISPOT) assay (BD Biosciences). Briefly, express 3 × 10 ⁴ bulk CD4 ⁺ T cells upon stimulation with peptide-pulsed irradiated PBMC (3 × 10 ⁴ ) or HLA-DP or HLA-DR pulsed with peptide The frequency of peptide-specific CD4 ⁺ T cells producing IFN-γ per 1 × 10 ⁴ bulk CD4 ⁺ T cells upon stimulation with L cells (5 × 10 ⁴ cells / well) was analyzed. Anti-HLA-DR monoclonal antibody (mAb) (L243, BioLegend), anti-HLA-DP mAb (B7 / 21, Abcam), anti-human HLA-DQ mAb ( Antigen-induced blocking of IFN-γ production was examined by adding SPV-L3, Abcam), or anti-HLA class I mAb (W6 / 32, Abcam). All mAbs were used at a final concentration of 5 μg / mL. All evaluations of the IFN-γ ELISPOT assay are performed in triplicate or duplicate and results are shown as mean ± SD.

Evaluation of DEPDC1-LP-specific CD4 ⁺ T cell responses in UC patients PBMCs obtained from 5 urothelial cancer (UC) patients were finalized in 2 ml AIM− supplemented with 5% human complement-depleted plasma. The cells were cultured in V medium (Invitrogen) at 37 ° C. in the presence of a mixture of DEPDC1-LP (20 μg / mL) (2 × 10 ⁶ cells / well, 24-well plate). IL-2 (20 U / ml) and IL-7 (5 ng / ml) were added on days 0 and 2. After 7 days of cell culture, the cells were harvested, washed and transferred to ELISPOT plates with individual DEPDC1-LP or control LP for 18 hours (5-10 × 10 ⁴ cells / well). The number of DEPDC1-LP specific CD4 ⁺ T cells was counted as described above.

In Vivo Cross-Priming Assay HLA-A2 and HLA-A24 (HHD) transgenic mice (Tgm) A. Courtesy of Dr. Lemonnier (Firat H, et al. Eur J Immunol 1999; 29: 3112-21; Jung KO, et al., J Virol 2012; 86: 7616-24). Mice were injected intradermally with a DEPDC1-LP2 solution (100 μg / mouse) emulsified in incomplete Freund's adjuvant (IFA) into the base of the tail once every 7 days. Seven days after the second vaccination with DEPDC1-LP2, CD8 ⁺ T cells were isolated from inguinal lymph nodes by positive selection with magnetic microbeads (Miltenyi Biotec, Auburn, CA, USA). Number of IFN-γ producing CD8 ⁺ T cells upon stimulation with bone marrow derived DCs (BM-DC, 2 × 10 ⁴ cells / well) pulsed with DEPDC1-A2 _302-311 SP or DEPDC1-A24 _294-302 SP ( 1 × 10 ⁵ cells / well) were counted by ex vivo ELISPOT assay (Harao M, et al., Int J Cancer 2008; 123: 2616-25; Inoue M, et al., Immunol Let 2009; 126: 67- 72).

Cytokine analysis DEPDC1-LP specific bulk Th cells or Th clones (1 × 10 ⁴ cells / well) were cultured in 96 well plates with autologous PBMC pulsed with cognate peptide (3 × 10 ⁴ cells / well). Cytokine levels in the culture supernatant after 24 hours were measured according to the manufacturer's instructions as previously reported using the Bio-Plex system (Bio-Rad).

Total RNA was isolated from DEPDC1-LP2 specific T cells using TCR sequencing RNeasy mini kit (Qiagen, Valencia, CA, USA). The TRAV and TRBV sequencing libraries were modified with several modifications to the previously described protocol (Fang H, et al., Oncoimmunology. 2014; 3 (12): e968467; 2016; 2 (4): 445-52) and sequencing on the Illumina Miseq platform using 600 cycles Miseq Reagent Kit V3 (Illumina, Inc., San Diego, CA, USA). It was used for. The sequence reads were then analyzed with a previously described algorithm (Fang H, et al., Oncoimmunology. 2014; 3 (12): e968467). To identify V- (D) -J segments in individual TRAV and TRBV sequencing reads, each of the sequence reads in the FASTQ file was converted to Bowtie2 aligner (Version 2.1.0) (Langmead B, et al , Nature methods.2012; 9 (4): 357-9; Lefranc MP, et al.Nucleic acids research.2005; 33 (Database issue): D593-7. , Et al., Nucleic acids research.2005; 33 (Database issue): D256-61). To define the amino acid sequence of complementarity determining region 3 (CDR3) in TRAV and TRBV, the conserved cysteine encoded in the 3 ′ portion of the V segment and the 5 ′ portion of the J segment form the boundary of CDR3 Conserved phenylalanine was identified (Fang H, et al., Oncoimmunology. 2014; 3 (12): e968467). The nucleotide sequence between both conserved cysteine and conserved phenylalanine was extracted to determine the amino acid sequence of the CDR3 region.

Cloning of TCR and expression in TG40 As previously described (Hamana H, et al., Biochemical and biophysical research communications. 2016; 474 (4): 709-14), using one-step multiplex RT-PCR TCR cDNA was amplified from the T cell line. The DNA sequence of the amplified PCR product was determined by direct sequencing. In order to analyze the antigen specificity of the obtained TCR cDNA, an expression vector for TCR was constructed and then transferred to Plat-E cells (provided courtesy of Professor Toshio Kitamura of the University of Tokyo) As previously described (Mou Z, et al. Science translational medicine. 2015; 7 (310): 310ra167; Kobayashi E, et al. Nature medicine. 2013; 19 (11): 1542-154. A replacement retrovirus was generated. The resulting TCR was analyzed as previously described (Kobayashi E, et al. Nature medicine. 2013; 19 (11): 1542-6), TG40 cells (Ohno H, et al. Journal of immunology (Baltiology, Balti. Md: 1950) .1991; 146 (11): 3742-6). 1 × 10 ⁵ TCR-transferred TG40 cells and 1 × 10 ⁵ restricted HLA-DR expressing L cells in the presence of 10 μg / mL antigen peptide overnight at 37 ° C., 5% CO ₂ atmosphere. After co-culture, the expression of CD69 and CD137 on the cell surface was analyzed with a flow cytometer.

Statistical analysis We compared the data by a two-tailed Student's t test (bar graph) or by a nonparametric Mann-Whitney U test (scatter plot). For all tests, differences with P values <0.05 were considered statistically significant.

Results Prediction and selection of potential promiscuous HLA class II binding DEPDC1 long chain peptides containing CTL epitopes To identify potential promiscuous HLA class II binding Th cell epitopes of DEPDC1, the amino acid sequence of DEPDC1 First investigated using developed computer algorithms (Wang P, et al. BMC Bioinformatics 2010; 11: 568; Wang P, et al., PLoS Compute Biol 2008; 4: e1000048) (FIGS. 1A, C, and Table 1). Are two areas _DEPDC1 191-213 -LP _(DEPDC1-LP1) and _DEPDC1 613-634 -LP _(DEPDC1-LP4) it does not include a known CTL epitope sequence. Is another of the two peptides _DEPDC1 292-313 -LP _(DEPDC1-LP2) and _DEPDC1 301-329 -LP _(DEPDC1-LP3) is, CTL recognized by HLA-A2 restricted CTL or HLA-A24-restricted CTL Proximal to the epitope (FIG. 1B). Therefore, we anticipate having strong binding affinity for high frequency HLA class II molecules (HLA-DR4, HLA-DR9, HLA-DR15, HLA-DP2, and HLA-DP5) in the Japanese population. Of these, two of which are DEPDC1-LP1-4 containing 9-mer or 10-mer peptides recognized by HLA-A2 restricted CTL or HLA-A24 restricted CTL, Synthesized for analysis.

Identification of promiscuous DEPDC1-derived Th cell epitopes CD4 ⁺ T cells isolated from healthy donor PBMCs were stimulated with autologous DC and irradiated PBMC pulsed with DEPDC1-LP1 at weekly intervals. After at least three stimulations, the DEPDC1-LP1-specific response of cultured CD4 ⁺ T cells was examined by IFN-γ ELISPOT assay.
In HLA-DR53 (DRB4 ^* 01 ^: 03) positive healthy donors (HD1), Th cells generated were significantly HIF-DR-dependent in response to PBMC pulsed with DEPDC1-LP1 in a significant amount of IFN-γ. Produced. Bulk Th cells specifically recognized L-DR53 cells pulsed with DEPDC1-LP1 in an HLA-DR-dependent manner, but did not pulse L-DR53 cells, L-DR4 cells pulsed with DEPDC1-LP1, and L-DR9 cells were not recognized (FIG. 2A). These results indicate that DEPDC1-LP1 was presented by HLA-DR53. To examine whether DEPDC1-LP1 induces a response in Th cells restricted by other HLA class II molecules, CD4 ⁺ T cells from other healthy donors (HD4) were tested. The present inventors confirmed that DEPDC1-LP1 generates HLA-DR4 (DRB1 ^* 04: 05 / −) restricted Th cells (FIG. 2B). Thus, DEPDC1-LP1 binds to HLA-DR53 and HLA-DR4, indicating that DEPDC1-LP1 is a high frequency HLA class II in the Japanese population (Saito S, et al., Tissue Antigens 2000; 56: 522-9; Mack SJ, et al., Tissue Antigens 2000; 55: 383-400), suggesting a promiscuous Th cell epitope.

DEPDC1-LP2 carrying known CTL epitopes can generate Th cells.
Th cells induced by DEPDC1-LP2 were derived from HD1. DEPDC1-LP2-specific Th cells derived from healthy donor HD1 (DRB1 ^* 04: 05/09: 01) specifically recognized L-DR4 cells pulsed with DEPDC1-LP2 (FIG. 2C), whereas DEPDC1-LP2 Did not recognize L-DR9 and L-DR53 pulsed with. Next, we confirmed that DEPDC1-LP2 was able to generate HLA-DR4-restricted bulk Th cells in this donor.

Similarly, DEPDC1-LP3 carrying known CTL epitopes can generate Th cells.
Th cells induced by DEPDC1-LP3 were derived from HD3 and HD4. In HLA-DP2 and DP5-positive healthy donors (HD3), the generated Th cells produced significant amounts of IFN-γ in response to PBMC pulsed with DEPDC1-LP3 in an HLA-DP-dependent manner. Next, we confirmed that DEPDC1-LP3 was able to generate HLA-DP5-restricted bulk Th cells in this donor (FIG. 2D). To examine whether DEPDC1-LP3 can induce a response in Th cells restricted by other HLA class II molecules, CD4 ⁺ T cells from other healthy donors (HD4) were tested. We confirmed that DEPDC1-LP3 was able to generate HLA-DR4 restricted bulk Th cells in this donor (FIG. 2E).

By conducting similar experiments, the present inventors can also generate IFN-γ producing cells specific for DEPDC1-LP4. Th cells induced by DEPDC1-LP4 were derived from HD1, HD5, and HD7. In DP5-positive healthy donors (HD1), the generated Th cells produced significant amounts of IFN-γ in response to PBMC pulsed with DEPDC1-LP4 in an HLA-DP-dependent manner. Next, we confirmed that DEPDC1-LP4 was able to generate HLA-DP5-restricted bulk Th cells in this donor (FIG. 2F). We also confirmed that DEPDC1-LP4 was able to generate HLA-DP5-restricted bulk Th cells in other healthy donors (HD7) (FIG. 2H). To examine whether DEPDC1-LP4 can induce a response in Th cells restricted by other HLA class II molecules, CD4 ⁺ T cells from other healthy donors (HD5) were tested. We confirmed that DEPDC1-LP4 was able to generate HLA-DR15 restricted bulk Th cells in this donor (FIG. 2G). These results indicate that DEPDC1-LP4 was presented by at least HLA-DP5 and HLA-DR15. These results demonstrate that these four DEPDC1-LPs are promiscuous Th cell epitopes, as summarized in Table 3. Because these LPs were promiscuous Th cell epitopes presented by HLA class II that are frequently observed in the Japanese population (Table 4) (Zarour HM, et al. Cancer research. 2002; 62 (1) Grabska AK, et al.International journal of cancer.2015; 136 (1): 212-24), a combination of these peptides has a DEPDC1-specific Th cell response in PBMC of many Japanese donors It is thought to induce.

ＤＥＰＤＣ１−ＬＰおよび５人のＨＤにおけるＤＥＰＤＣ１−ＬＰに反応するＴｈ細胞の免疫応答の概要
^ａ下線が引かれたＨＬＡクラスＩＩアリルは、ＨＤにおいてＤＥＰＤＣ１−ＬＰをＴｈ細胞に提示するＨＬＡクラスＩＩ分子をコードする；ドナーのＨＬＡアリルの詳細は、表２に示されている。
^ｂ下線が引かれた斜体太字の配列は、ＣＴＬエピトープである；ＤＥＰＤＣ１−ＬＰ１およびＤＥＰＤＣ１−ＬＰ４の配列は、アルゴリズムによって予測されたＨＬＡクラスＩＩ分子への高親和性結合に基づいて選択された；
ＤＥＰＤＣ１−ＬＰ２およびＤＥＰＤＣ１−ＬＰ３は、ＨＬＡクラスＩＩ結合の予測および公知のＣＴＬエピトープに近接していることの両方に基づいて選択された。ａ．ａ．：アミノ酸、陰性：陽性データを得られず、それ以上進めなかった、ＬＰ：長鎖ペプチド、ＨＤ：健常ドナー。

Overview of immune response of Th cells in response to DEPDC1-LP and DEPDC1-LP in 5 HD
^a Underlined HLA class II allele encodes an HLA class II molecule that presents DEPDC1-LP to Th cells in HD; details of the donor HLA allele are shown in Table 2.
^{b The} underlined italic bold sequences are CTL epitopes; the sequences of DEPDC1-LP1 and DEPDC1-LP4 were selected based on the high affinity binding to HLA class II molecules predicted by the algorithm;
DEPDC1-LP2 and DEPDC1-LP3 were selected based on both prediction of HLA class II binding and proximity to known CTL epitopes. a. a. : Amino acid, Negative: No positive data could be obtained and further progressed, LP: long chain peptide, HD: healthy donor.

日本人集団における各ＨＬＡアリルの抗原頻度は、以前の報告を参照した（Ｋｏｂａｙａｓｈｉ， ｅｔ ａｌ．， Ｃａｎ ｒｅｓ ２００１；６１（１２）：４７７３−８）。

The antigen frequency of each HLA allele in the Japanese population was referred to a previous report (Kobayashi, et al., Can res 2001; 61 (12): 4773-8).

DEPDC1-LP2, DEPDC1-LP3, and DEPDC1-LP4 are naturally processed and presented by DC to assess whether DCs take up and process DEPDC1 protein to stimulate DEPDC1-LP-specific Th clones did. DC loaded with recombinant DEPDC1 protein was prepared and used as APC in the IFN-γ ELISPOT assay (Kanehira M, et al. Oncogene 2007; 26: 6448-55; Harao M, et al., Int J Cancer 2008; 123: 2616-25). The HLA-DR4-restricted DEPDC1-LP2-reactive Th clone effectively recognized DC loaded with the DEPDC1 protein, but did not recognize DC loaded with the control protein, indicating that this epitope is from the DEPDC1 protein. It shows that it was naturally processed and presented by the HLA-DR4 molecule (FIG. 3A). HLA-DR4-restricted DEPDC1-LP3-reactive Th clones effectively recognized DC loaded with DEPDC1 protein in an HLA-DR-dependent manner, but did not recognize DC loaded with control protein, This epitope is also shown to have been naturally processed from the DEPDC1 protein and presented by the HLA-DR4 molecule (FIG. 3B). Similarly, the HLA-DP5-restricted DEPDC1-LP4 reactive Th clone effectively recognized DC loaded with the DEPDC1 protein in an HLA-DP-dependent manner, but did not recognize DC loaded with the control protein. This indicates that this epitope was also naturally processed from the DEPDC1 protein and presented by the HLA-DP5 molecule (FIG. 3C). These results suggest that DEPDC1-LP2, DEPDC1-LP3, and DEPDC1-LP4 are presented naturally processed from DEPDC1 protein by DC.

Th cells induced by DEPDC1-LP produced Th1 cytokines In order to characterize DEPDC1-LP-specific Th cells, we responded to stimulation with autologous PBMC pulsed with the respective cognate peptide. Various cytokines secreted by Th cells were measured. Relatively large amounts of Th1 cytokines, such as IFN-γ, TNF-α, IL-2, and GM-CSF, were produced by DEPDC1-LP-specific Th cells after restimulation with cognate peptides, but IL- No 4 or IL-17 was produced (FIG. 5), suggesting that DEPDC1-LP has the ability to induce Th cells polarized to Th1. Therefore, induction of Th cells with DEPDC1-LP may be useful for anti-tumor therapy.

The DEPDC1-LP2-specific bulk Th cells were expressed as follows: A repertoire of the DEPDC1-LP2-specific T cells that expressed the converged TCR-α and TCR-β genes, and the TCR-α and TCR-β using next-generation sequencers. The gene was analyzed by deep cDNA sequencing. After several stimulations of bulk Th cells, the frequency of a particular pair of TCR-α and TCR-β genes was increased in both bulk Th cells. Two LP-specific T cell clones established from the parent bulk T cell line, stimulated 4 or 5 times with the peptide, express the same pair of TCR-α and TCR-β genes as the bulk Th cells. (FIGS. 6A and 6B). Introduction of a pair of TCR-α and TCR-β genes, isolated from both DEPDC1-LP2-specific T cell clones, respectively, into the mouse TCR negative T cell line TG40 was detected by flow cytometric analysis. As revealed by the upregulation of CD69 and CD137, the original antigenic peptide specificity and HLA restriction of parental Th cells were successfully acquired (FIG. 6C). These observations confirmed the clonal nature of the established T cells and the specificity of these pairs of TCR-α and TCR-β chains.

Cross presentation of DEPDC1-LP effectively primes DEPDC1-SP specific CD8 ⁺ T cells in vivo DEPDC1-A2 _302-311 specific CTL and DEPDC1-A24 _294-302 specific CTL Was tested by ex vivo IFN-γ ELISPOT assay. HLA-A2 Tgm and HLA-A24 Tgm were immunized twice with DEPDC1-LP2 emulsified in IFA. CD8 ⁺ T cells isolated from HLA-A2 Tgm or HLA-A24 Tgm vaccinated with DEPDC1-LP2 were injected with BM-DC pulsed with DEPDC1-A2 _302-311 SP or DEPDC1-A24 _294-302 SP. IFN-γ was specifically produced in response to stimulation (FIGS. 4A and 4B). These results show that after uptake of DEPDC1-LP2, APC crossed DEPDC1-A2 _302-311 SP-specific CTL and DEPDC1-A24 _294-302- specific CTL in vivo in HLA-A2 Tgm and HLA-A24 Tgm. It suggests that it can be primed.

PBMCs isolated from UC patients recognized some of DEPDC1-LP In order to investigate the induction of DEPDC1-LP-specific Th cell responses in UC patients, we Isolated PBMC were stimulated with LP in vitro. Patient characteristics are shown in Table 5. After stimulation, the frequency of individual DEPDC1-LP specific T cells was measured. DEPDC1-LP4 induced peptide-specific T cells from two patients. Production of peptide-specific IFN-γ by T cells was significantly inhibited by anti-HLA-DR mAb, but not by anti-HLA-DP mAb or anti-HLA class I mAb (FIG. 7). These results demonstrate that IFN-γ was produced by DEPDC1-LP-specific and HLA-DR-restricted CD4 ⁺ T cells, indicating that DEPDC1-LP4 is not only from HD but UC This suggests that antigen-specific Th cells were also induced from patients who had the disease.

^ａＵＣを有する２人の患者に由来するＰＢＭＣのＤＥＰＤＣ１−ＬＰ特異的免疫応答の概要。ＤＥＰＤＣ１−ＬＰ特異的Ｔヘルパー（Ｔｈ）細胞の応答を、インターフェロン（ＩＦＮ）−γ酵素結合免疫スポットアッセイ（ＥＬＩＳＰＯＴ）を用いて測定した。
簡単に述べると、患者由来のＰＢＭＣを、表示されているＤＥＰＤＣ１−ＬＰで刺激した。ＩＦＮ−γスポットの平均数が、２５を超え、かつバックグラウンドシグナルの２倍よりも大きかった場合に、応答を陽性として記録した。

^a Summary of DEPDC1-LP-specific immune responses of PBMC from two patients with UC. The response of DEPDC1-LP specific T helper (Th) cells was measured using an interferon (IFN) -γ enzyme-linked immunospot assay (ELISPOT).
Briefly, patient-derived PBMC were stimulated with the indicated DEPDC1-LP. A response was recorded as positive if the average number of IFN-γ spots was greater than 25 and greater than twice the background signal.

Discussion In phase I / II clinical trial, combined immunotherapy with intravesical BCG vaccination and DEPDC1-derived SP vaccine induces promising clinical effects on prevention of recurrence of non-muscle invasive bladder cancer, as well as safety and CTL Have demonstrated good immunogenicity (Obara W, et al., Clin Oncol 2012; 42: 591-600). Therefore, we attempted to identify LPs that induce both antigen-specific Th1 cells and CTLs to further develop better peptide vaccine immunotherapy.

We identified four promiscuous DEPDC1-derived Th cell epitope peptides as summarized in Table 3. Two of them contain HLA-A2 and / or HLA-A24 restricted CTL epitopes. We confirmed that DEPDC1-LP2, DEPDC1-LP3, and DEPDC1-LP4 were naturally processed from DEPDC1 protein and presented on the cell surface of DC in the context of HLA class II molecules ( FIG. 3). This suggests that CD4 ⁺ T cells induced by these DEPDC1-LP have advantageous characteristics for cancer immunotherapy.

  Since MHC class II molecules are highly polymorphic, in order to clinically apply LPs that activate HLA class II-restricted Th cells to cancer vaccine therapy, the peptide must contain a plurality of HLA class II molecules. It should be considered that it can bind to the molecule and has a broader range of fit (Kobayashi H, et al., Cancer research. 2001; 61 (12): 4773-8; Zarour HM, et al. , Cancer research. 2002; 62 (1): 213-8; Grabowska AK, et al., International journal of cancer. 2015; 136 (1): 212-24). We present DEPDC1-LP by HLA-DR4, HLA-DR9, HLA-DR15 (FIG. 1-1), HLA-DR53, HLA-DP2, and HLA-DP5 (FIG. 1-2). This is demonstrated herein, suggesting that vaccination with the DEPDC1-LP combination could cover the majority of the Japanese population (Table 4). In fact, we have observed the presence of DEPDC1-LP-specific Th cells in two UC patients, suggesting that DEPDC1-LP may be widely useful in many of the patients, Further studies with a larger number of cancer patients with HLA class II should be conducted.

DEPDC1-LP induced HLA-DP5, DR4, DR53, and DR15-restricted Th cells (Figure 2 and Table 3). HLA-DP5, DR4, DR53, and DR15 alleles are very frequent HLA class II alleles in the Japanese and Pacific / Asian populations, and HLA-A2 and A24 alleles are also frequent in those populations. Observed (Table 4). In addition, DEPDC1-LP2 induces priming and proliferation of DEPDC1-A2 _302-311- specific CTL and DEPDC1-A24 _294-302- specific CTL in vivo in HLA-A2 or A24 Tgm (FIG. 4) This suggests that DEPDC1-LP carrying the DEPDC1-SP epitope can induce both DEPDC1-specific Th cells and CTLs. We therefore suggest that these immunogenic DEPDC1-LPs may have the potential to induce a stronger anti-tumor response.

  Recent studies of LP have vaccines containing natural CTL epitopes superior to vaccines composed of minimal CTL epitopes to induce anti-tumor CTL immunity due to the long-lasting cross-presentation of LP (Brunsvig PF, et al., Clin Cancer Res 2011; 17: 6847-57; Bijker MS, et al., Eur J Immunol 2008; 38: 1033-42; Chauvin JM, et al., J Immunol 2012; 188: 2102-10). DEPDC1-LP2, including known CTL epitopes, induced CTL epitope-specific CTL in vivo in HLA-A2 Tgm or A24 Tgm via a cross-presentation pathway (FIG. 4). Therefore, peptide vaccination can simultaneously induce both DEPDC1-LP2-specific Th cells and CTL in cancer patients.

  DEPDC1-LP1 and DEPDC1-LP4 effectively induced their LP-specific Th cell responses (FIGS. 2A-B and FH), but they do not contain known HLA class I-restricted CTL epitopes (FIG. 1B). Recently, a phenomenon called epitope expansion has been reported to be important in the induction of effective anti-tumor immune responses (Inderberg-Suso EM, et al., Oncoimmunology. 2012; 1 (5): 670-86. Corbiere V, et al., Cancer research. 2011; 71 (4): 1253-62). Indeed, patients who received a therapeutic cancer vaccine (minimal CTL epitope peptide vaccine) clearly showed clinical response and long-term survival. In those patients, both Th cell and CTL responses were observed against other epitopes of the same protein, or some unrelated antigens not included in the vaccine. This is because vaccination induces CTL specific for the target antigen and damages the tumor cells, so that other TAAs are released, taken up and presented by APC, and other TAA-specific T This is because a cellular response was induced. In this case, vaccination with DEPDC1-LP1, DEPDC1-LP4 without CTL epitopes would induce LP-specific Th1 responses along with other anti-tumor Th1 and CTL responses. Furthermore, if the epitope expands to include nascent antigens that are abnormal peptides derived from proteins encoded by somatic missense mutant genes generated in cancer cells, these nascent antigens are non-self The anti-tumor T cell response is believed to be more effective. In fact, recent studies have shown that there are many T cells specific for nascent antigens, which indicates that the gene encoding the antigen has a mutation and the expanded epitope may also include the nascent antigen. Suggests that. Cancer antigen vaccine therapy using nascent antigens has received much attention (Kreitter S, et al., Nature. 2015; 520 (7549): 692-6; Linnemann C, et al., Nature medicine. 2015; 21 (1): 81-5). In addition to induction of epitope expansion, vaccine formulations of DEPDC1-LP and SP have been investigated to induce and activate as many TAA-specific T cells as possible that are key to the success of cancer vaccine therapy. It may be promising to be done.

If we intend to use LPs with Th cell epitopes for cancer immunotherapy, one major concern is whether anti-tumor Th1 cells can be induced in the patient's body. is there. The Th cell phenotype has been reported to be influenced not only by the surrounding cytokine environment at the differentiation stage, but also by the affinity / binding power of TCR and MHC-peptide complexes (Constant S, et al. , The Journal of experimental medicine. 1995; 182 (5): 1591-6; Yamane H, et al., The Journal of experimental medicine. Ann. 803-N: 803; 2014; 41 (1): 63-74; Hosken NA, et al., The Journal of experimental medicine. 1995; 182 (5) : 1579-84). In other words, CD4 ⁺ T cells tend to differentiate into Th1 cells upon recognizing high density peptide-HLA class II complexes that induce strong TCR-mediated signals, and vice versa, Th cells are Th2 cells. There is a tendency to differentiate into cells. The DEPDC1-LP-specific Th cells derived from healthy donors in this study are all Th1-type cells and mainly produced IFN-γ, TNF-α, GM-CSF, and IL-2 (FIG. 5). . Therefore, DEPDC1-LP probably binds to HLA class II molecules with higher affinity, thereby producing a high density LP-HLA class II complex, which is a durable and potentially strong TCR. Due to the ability to stimulate mediated activation signals, DEPDC1-LP can preferentially cause Th1-type Th cell differentiation.

  In contrast to inducing various TAA-specific Th cells and CTLs by vaccination, the direct introduction of TAA-specific genetically engineered T cells into cancer patients is another effective anti-tumor Looks like a therapy. In a clinical trial, Rosenberg et al. Introduced an autologous T cell retrotransduced with the TCR gene of NY-ESO-1-reactive CTL into a patient with metastatic synovial cell sarcoma and melanoma. Clinical response has been demonstrated (Robbins PF, et al., Clinical cancer research: an official journal of the American Association for Cancer Research. 2015; 21 (5): 21 (5): In this study, TCR usage of DEPDC1-LP-specific Th cells was highly converged to one combination of TCRA and TCRB genes, respectively, after repeated stimulation (FIGS. 6A-B). Introduction of each pair of TCR genes into the mouse TCR-negative T cell line TG40 shows the original T cell specificity for their cognate ligand (FIG. 6C), which indicates that patients expressing their TCR gene This suggests that the transfer of autologous T cells can be an effective treatment. The convergence of the TEP repertoire of DEPDC1-LP-specific Th cells to those with a specific CDR sequence indicates that the antigen-specific TCR is compared to a random TCR repertoire that is not selected for binding to a specific antigen peptide. , Recent observations by Dash et al. And Granville et al. (Dash P, et al., Nature. 2017; 547 (7661): 89-93; that share a specifically enriched short sequence motif in the CDRs; Granville J, et al. Nature. 2017; 547 (7661): 94-8).

  In conclusion, we have identified four immunogenic DEPDC1-LPs that can induce Th cells. Two of them include the HLA-A2 restricted CTL epitope and the HLA-A24 restricted CTL epitope. Our results suggest that DEPDC1-LP provides a useful tool for the proliferation of both DEPDC1-specific Th1 cells and CTLs. These findings support potential clinical trials of DEPDC1 peptide-based immunotherapy for several types of cancer, such as bladder cancer. In addition, converged TCR expressed on LP-specific Th cells can be useful for monitoring LP-specific Th cells in vitro and possibly in vivo.

  The present invention describes a ThEP cell epitope peptide derived from DEPDC1 that can induce a strong anti-tumor immune response and thus has applicability to a wide variety of cancer types. Such peptides deserve further development as peptide vaccines against cancers, particularly those that express DEPDC1. The peptides of the present invention can induce a Th1 cell response, so that cytokines secreted by Th1 cells help or activate any immune cell involved in cellular immunity in an antigen-independent manner Can do. Therefore, the immunotherapy strategy provided by the present invention can be applied to any disease, including cancer, as long as the disease can be ameliorated by an immune response mediated by MHC class II molecules. In particular, the Th1 cells of the present invention can improve the immune response elicited by CTL. Therefore, the peptides of the present invention are beneficial in enhancing CTL responses to diseases including cancer in a subject.

  Furthermore, in a preferred embodiment, the peptides of the present invention can also induce CTLs against DEPDC1-expressing cells as well as Th1 cells. Such peptides of the invention may also be useful for the treatment of diseases associated with DEPDC1, such as cancers that express DEPDC1, more particularly bladder and breast cancer.

  Although the invention has been described herein in detail and with reference to specific embodiments thereof, the foregoing description is illustrative and explanatory in nature and is intended to illustrate the invention and preferred embodiments thereof. . Through routine experimentation, one skilled in the art will readily recognize that various changes and modifications can be made without departing from the spirit and scope of the invention as defined by the appended claims. Will do.

Claims (28)

An isolated peptide having a length of 10 to 30 amino acids and comprising a portion of the amino acid sequence of SEQ ID NO: 8 or 10,
(A) a continuous amino acid sequence having a length of more than 9 amino acids selected from the amino acid sequences of SEQ ID NO: 1, 2, 3 or 4; and (b) 1, 2 or several in the amino acid sequence of (a) A peptide comprising an amino acid sequence selected from the group consisting of amino acid sequences in which the amino acid is substituted, deleted, inserted and / or added, and having the ability to induce T helper type 1 (Th1) cells.   2. The isolated peptide of claim 1, wherein the peptide or fragment thereof has the ability to bind to at least two types of MHC class II molecules.   The isolated peptide of claim 2, wherein the MHC class II molecule is selected from the group consisting of HLA-DR53, HLA-DR4, HLA-DR15 and HLA-DP5.   The isolated peptide according to any one of claims 1 to 3, comprising an amino acid sequence of a peptide having DEPDC1-specific cytotoxic T lymphocyte (CTL) inducing ability. (A) an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 4; and (b) 1, 2 or several amino acids are substituted, deleted, inserted and / or added in the amino acid sequence of (a). 5. The isolated peptide of claim 4, comprising an amino acid sequence selected from the group consisting of:   An isolated polynucleotide encoding the peptide of any one of claims 1-5. (I) Th1 cells,
(Ii) CTL,
(Iii) an antigen presenting cell (APC) having the ability to induce Th1 cells; and (iv) an APC having the ability to induce CTLs.
A composition for inducing at least one cell selected from the group consisting of one or more peptides according to any one of claims 1 to 5, or one encoding them Or a composition comprising a plurality of polynucleotides. (A) one or more peptides according to any one of claims 1 to 5;
(B) one or more polynucleotides of claim 6;
(C) one or more APCs presenting on their surface the peptide or fragment thereof according to any one of claims 1 to 5;
(D) one or more Th1 cells recognizing APC presenting on their surface the peptide or fragment thereof according to any one of claims 1 to 5; and (e) from (a) to (d ) Containing at least one active ingredient selected from the group consisting of any two or more combinations of (i), and (i) treatment of cancer,
(Ii) cancer prevention,
(Iii) prevention of postoperative recurrence in cancer, and (iv) formulated for purposes selected from the group consisting of any two or more combinations of (i) to (iii) above , Pharmaceutical composition.   9. Formulated for administration to a subject having at least one selected from the group consisting of HLA-DR53, HLA-DR4, HLA-DR15 and HLA-DP5 as MHC class II molecules. Pharmaceutical composition.   The pharmaceutical composition according to claim 8 or 9, further comprising one or more peptides having CTL inducing ability. A composition for enhancing an immune response mediated by MHC class II molecules comprising:
(A) one or more peptides according to any one of claims 1 to 5;
(B) one or more polynucleotides of claim 6;
(C) one or more APCs presenting on their surface the peptide or fragment thereof according to any one of claims 1 to 5;
(D) one or more Th1 cells recognizing APC presenting on their surface the peptide or fragment thereof according to any one of claims 1 to 5; and (e) from (a) to (d ), Containing at least one active ingredient selected from the group consisting of any two or more combinations.   A method for inducing APC having the ability to induce Th1 cells, comprising contacting APC with a peptide according to any one of claims 1 to 5 in vitro, ex vivo or in vivo. A method for inducing APC having the ability to induce CTLs, comprising:
(A) contacting the APC with the peptide of any one of claims 1 to 5 in vitro, ex vivo or in vivo; and (b) encoding the peptide of any one of claims 1 to 5. A method comprising: selecting from the group consisting of introducing a polynucleotide to APC. A method for inducing Th1 cells comprising:
(A) co-culturing CD4 positive T cells with APC presenting on its surface a complex of an MHC class II molecule and a peptide or fragment thereof according to any one of claims 1 to 5; and (B) introducing a polynucleotide encoding both T cell receptor (TCR) subunits, or a polynucleotide encoding each of the TCR subunits, into a CD4 positive T cell, wherein the TCR is a cell Comprising a step selected from the group consisting of steps capable of binding to a complex of a MHC class II molecule presented on a surface and a peptide or fragment thereof according to any one of claims 1 to 5. Method. A method for inducing CTL, comprising:
(A) co-culturing both CD4 positive T cells and CD8 positive T cells with APC contacted with the peptide of claim 4 or 5; and (b) CD8 positive T cells in claim 4 or A method comprising a step selected from the group consisting of co-culturing with APC contacted with the peptide of claim 5. A method for enhancing an immune response mediated by MHC class II molecules comprising:
(A) one or more peptides according to any one of claims 1 to 5;
(B) one or more polynucleotides of claim 6;
(C) one or more APCs presenting on their surface the peptide or fragment thereof according to any one of claims 1 to 5;
(D) one or more Th1 cells recognizing APC presenting on their surface the peptide or fragment thereof according to any one of claims 1 to 5; and (e) from (a) to (d And) administering to the subject at least one active ingredient selected from the group consisting of any combination of two or more.   An isolated APC that presents on its surface a complex of an MHC class II molecule and a peptide or fragment thereof according to any one of claims 1-5.   APC induced by the method according to claim 12 or 13.   An isolated Th1 cell that recognizes the peptide or fragment thereof according to any one of claims 1 to 5 displayed on the surface of APC.   A Th1 cell induced by the method according to claim 14.   The Th1 cell according to claim 20, wherein the α subunit and β subunit of the TCR comprise CDR3 consisting of the amino acid sequences of SEQ ID NOs: 11 and 21, respectively. A method of inducing an immune response against cancer in a subject in need thereof,
(A) one or more peptides according to any one of claims 1 to 5;
(B) one or more polynucleotides of claim 6;
(C) one or more APCs presenting on their surface the peptide or fragment thereof according to any one of claims 1 to 5;
(D) one or more Th1 cells recognizing APC presenting on their surface the peptide or fragment thereof according to any one of claims 1 to 5; and (e) from (a) to (d ) Comprising administering to said subject a composition comprising at least one active ingredient selected from the group consisting of any two or more combinations of   An antibody against the peptide according to any one of claims 1 to 5, or an immunologically active fragment thereof.   A vector comprising a nucleotide sequence encoding the peptide according to any one of claims 1 to 5.   A host cell transformed or transfected with the expression vector of claim 24.   A diagnostic kit comprising the peptide according to any one of claims 1 to 5, the polynucleotide according to claim 6, or the antibody according to claim 23. A method for evaluating and / or monitoring a Th1 response specific for a peptide comprising the amino acid sequence of SEQ ID NO: 1, 2, 3 or 4 in a subject comprising:
(A) providing a sample obtained from a subject administered the peptide, wherein the sample comprises T cells;
(B) detecting the presence of a T cell expressing a TCR that binds to a complex of an MHC class II molecule and the peptide or fragment thereof in the sample; and (c) the presence of the T cell is (b) Wherein the method comprises induction of a Th1 response specific to the peptide when detected in.   28. The method of claim 27, wherein the TCR comprises a specific pair of alpha and beta subunits, each comprising CDR3 consisting of the amino acid sequences of SEQ ID NOs: 11 and 21, respectively.

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