TW201831503A - Mphosph1 epitope peptides for th1 cells and vaccines containing the same - Google Patents

Abstract

Isolated MPHOSPH1-derived epitope peptides having Th1 cell inducibility are disclosed herein. Such peptides can be recognized by MHC class II molecules and induce Th1 cells. In preferred embodiments, such a peptide of the present invention can promiscuously bind to MHC class II molecules and induce MPHOSPH1-specific CTLs in addition to Th1 cells. Such peptides are thus suitable for use in enhancing immune response in a subject, and accordingly find use in cancer immunotherapy, in particular, as cancer vaccines. Also disclosed herein are polynucleotides that encode any of the aforementioned peptides, APCs and Th1 cells induced by such peptides and methods of induction associated therewith. Pharmaceutical compositions that comprise any of the aforementioned components as active ingredients find use in the treatment and/or prevention of cancers or tumors including, for example, bladder cancer and breast cancer.

Description

   MPHOSPH1 epitope peptide for TH1 cells and vaccine containing same   

The present invention relates to the field of biological sciences, and more specifically to the field of cancer treatment. In particular, the present invention relates to novel peptides, which are extremely effective as cancer vaccines and drugs for treating or preventing tumors, or for treating and preventing tumors.

[Priority]

This application claims a benefit based on Japanese Patent Application No. JP2016-224625 filed on November 18, 2016, the entire contents of which are incorporated herein by reference.

It has been demonstrated that CD8-positive cytotoxic T-lymphocytes (CTLs) recognize epitope-derived peptides derived from tumor-associated antigens (TAA) found on major histocompatibility complex (MHC) class I molecules, and have Kill the tumor cells. Since the discovery of the melanoma antigen (MAGE) family as the first case of TAA, many other TAAs have been mainly discovered using immunological methods (Non-Patent Documents 1, 2). Some of these TAAs are being used as targets for immunotherapy in clinical development.

TAA, which is indispensable for the proliferation and survival of cancer cells, is advantageous as a target for immunotherapy, because the use of this TAA can reduce cancer caused by deletion, mutation or down-regulation of TAA in the immune selection driven by treatment. The risk of cellular immune escape is minimized. Therefore, the identification of new TAAs capable of inducing effective and specific anti-tumor immune responses ensures further development. Currently, the clinical application of peptide vaccine strategies for many types of cancer is ongoing (Non-Patent Documents 3 to 10). To date, several clinical trials using these TAA-derived peptides have been reported. Unfortunately, these cancer vaccine trials have so far produced only low target response rates (Non-Patent Documents 11 to 13). Therefore, there is still a need in the art for new TAAs suitable as targets for immunotherapy.

Recently, the inventors of the present case have used a genome-wide cDNA microarray analysis to identify a cancer-testicle antigen, M-phase phosphoprotein1 (MPHOSPH1), which is often used in bladder cancer, breast cancer and many other Excessive expression in malignant tumors (Non-Patent Documents 14 to 17). By using small interfering RNA to suppress MPHOSPH1 performance, it significantly inhibits the growth of bladder cancer cells. Therefore, MPHOSPH1 is necessary for tumor growth and survival, which shows the clinical importance of these antigens as targets for antigen-specific cancer immunotherapy. The inventors also identified a highly immunogenic MPHOSPH1-derived short peptide (SP) capable of inducing HLA-A24 (A * 24: 02) -restricted CTLs from peripheral blood mononuclear cells (PBMCs) of bladder cancer patients (Patent Documents 1, 2, and Non-Patent Documents 18). Phase I / II clinical trials of combined immunotherapy with BCG and MPHOSPH1-derived SP vaccine in the bladder have shown promising safety and good immunogenicity in preventing the recurrence of non-muscle invasive bladder cancer and inducing CTL. (Non-Patent Document 18). Therefore, MPHOSPH1 is an attractive target molecule for cancer immunotherapy.

Tumor-specific CD4 ⁺ helper T (Th) cells, especially T-helper type 1 (Th1) cells, play a key role in efficiently inducing the antitumor immunity of CTL vectors (Non-Patent Document 19). Interferon (IFN) -γ and tumor necrosis facto (TNF) -α, which are mainly produced by Th1 cells, are critical to the induction and maintenance of long-term CTL response, and their key role is to maintain immune memory. Multiple sexual interactions help (Non-Patent Documents 20, 21, 22). IFN-γ secreted by Th1 cells also has a direct antitumor or antiangiogenic effect (Non-Patent Document 23). Furthermore, it has been shown that Th cells must pave the way for CTLs to enter the tumor site (Non-patent Document 24). Melief et al. Recently reported that human papillomavirus-derived synthetic LP naturally contains a CTL epitope as an attractive vaccine compound for the treatment of vulvar cancer and cervical cancer (Non-Patent Document 25). After injection of LP, the patient's DC takes up and processes the LP, and presents possible CTL and Th cell epitopes in the context of the patient's HLA class I and II molecules, respectively (Non-Patent Document 28). In addition, when combining radiation and chemotherapy, recent clinical studies have used a hybrid telomerase-derived helper-epitope vaccine called GV1001 to increase the survival of cancer patients (non- (Patent Documents 26, 27). Therefore, the identification of TAA-derived Th cell epitopes capable of activating specific Th1 cells is important for inducing effective tumor immunity in subjects suffering from tumors; ideally, designing an effective vaccine should include multiple epitopes, In order to stimulate both CTL and Th1 cells (Non-Patent Document 25). However, no such epitope derived from MPHOSPH1 has yet been identified.

[Quote list]

[Patent Literature]

[Patent Document 1] WO2008 / 047473

[Patent Document 2] WO2013 / 024582

[Non-patent literature]

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

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

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

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

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

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

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

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

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

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

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

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

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

[Non-Patent Document 14] Nishiu M, et al., Jpn J Cancer Res. 2002 Augl 93 (8): 894-901

[Non-Patent Document 15] Kanehira M, et al., Cancer Res. 2007; 67: 3276-85

[Non-Patent Document 16] Liu X, et al., Cancer Res. 2014; 74: 6623-34

[Non-Patent Document 17] Miyata Y, et al., Cancer Biomark. 2015; 15: 789-97

[Non-Patent Document 18] Obara W, et al, Jpn J Clin Oncol. 2012; 42: 591-600

[Non-Patent Document 19] Chamoto K, et al., Cancer Res 2004; 64: 386-90

[Non-Patent Document 20] Bevan MJ, Nat Rev Immunol 2004; 4: 595-602

[Non-Patent Document 21] Shedlock DJ, et al., Science 2003; 300: 337-9

[Non-Patent Document 22] Anders K, et al., Cancer Cell 2011; 17: 6847-57

[Non-Patent Document 23] Street SE et al. Blood 2001; 97: 192-7

[Non-Patent Literature 24] Bos R, et al., Cancer Res 2010; 70: 8368-77

[Non-Patent Document 25] Melief CJ, et al., E Nat Rev Cancer 2008; 8: 351-60

[Non-Patent Document 26] Brunsvig PF, et al., Clin Cancer Res 2011; 17: 6847-57

[Non-Patent Document 27] Kyte JA, et al., Clin Cancer Res 2011; 17: 4568-80

[Non-Patent Document 28] Kenter GG, et al., N Engl J Med 2009; 361; 1838-47

Bladder cancer is the most common malignancy related to the urinary system. Platinum-based chemotherapy remains standard care for first-line treatment of end-stage / metastatic urothelial carcinoma (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). Among those with metastatic bladder cancer receiving standard treatment with platinum-based chemotherapy, overall survival is estimated to be 9-15 months; bladder cancer stage IV has a five-year relative survival rate of about 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 some treatment options for patients who were resistant to chemotherapy or could not tolerate chemotherapy due to severe adverse reactions, and overall results were still very poor (Gupta S, et al., Supra). At present, systemic immunotherapy using programmed cell death protein 1 (PD-1)-and PD-1 ligand (PD-L1) -specific monoclonal antibodies, namely Nivolumab, respectively / Pembrolizumab and Atezolizumab, have been licensed for the treatment of urinary bladder cancer to achieve a significant improvement in response rate (Gupta S, et al., Supra; Zichi C, et al., Biomed Res Int 2017; 2017 (561874. Doi: 10.1155 / 2017/5618174)). Another recent clinical trial shows that MPHOSPH1-derived SP vaccine administered to UC in progress is safe, well tolerated, and efficiently activates CTL (Obara W, et al., Ann Oncol) 2017; 28 4; 798-803).

In the context of the present invention, the present inventors consider an ideal peptide vaccine line for cancer immunotherapy comprising a single peptide against epitopes of both CTL and Th1 cells, the epitopes of both of which are naturally occurring Close to each other.

For this reason, the inventors of this case devised a strategy to identify a novel epitope of MPHOSPH1-derived Th1 cells, which can be identified in the context of promiscuous HLA class II molecules and contains CTL epitopes, assuming that The identified epitopes will induce more efficient T cell-mediated tumor immunity. Computer algorithm for predicting HLA class II binding peptides and CTL epitope sequences known to be recognized by CTLs restricted by HLA-A24 (A * 24: 02) or HLA-A2 (A * 02: 01) To select candidate hybrid HLA-type II restricted epitopes containing CTL epitopes.

The invention is based at least in part on finding suitable epitope peptides as targets for immunotherapy that induces Th1 cell responses. It is recognized that the MPHOSPH1 gene is up-regulated in some types of cancer, including bladder cancer and breast cancer. The objective of the present invention is to further analyze the gene product of the MPHOSPH1 gene, and especially to be accessed by GenBank such as number NM_001284259 (sequence identification number: 5) or The gene code shown in No. NM_016195 (sequence identification number: 7) from GenBank accesses the polypeptide in sequence identification number: 6 or 8. In further research, the MPHOSPH1 gene product containing an epitope peptide that induces Th1 cells specific to the corresponding molecule was specifically selected. For example, peripheral blood mononuclear cells (PBMCs) obtained from healthy donors are stimulated with a hybrid HLA-DR and / or DP-binding peptide derived from human MPHOSPH1. Establish Th1 cells that identify HLA-DR or DP-positive target cells pulsed with individual candidate peptides, and identify HLA-DR and / or DP-restricted antigenic decisions that induce an effective and specific immune response against MPHOSPH1 Peptides. These results demonstrate that MPHOSPH1 has strong immunogenicity and that its epitope is effective for tumor immunotherapy via Th1 cell response vector. Further studies have shown that hybrid HLA-DR and / or DP-binding peptides containing at least one CTL epitope can also stimulate CTL responses in the same donor in a MPHOSPH1-specific manner. These results confirm that MPHOSPH1 is strongly immunogenic, and that MPHOSPH1-derived peptide contains both Th1 cells and CTL epitopes, and is effective for tumor immunotherapy systems via Th1 cells and CTL response vectors.

Therefore, an object of the present invention is to provide a peptide having Th1 cell inducing ability and an amino acid sequence selected from the sequence identification numbers: 1 to 2. The present invention contemplates modified peptides, that is, peptides having Th1 inducing ability and up to 30 amino acids in length and having a continuous amino acid sequence selected from the amino acid sequence of sequence identification number: 6 or 8 (MPHOSPH1), And functionally equivalent. Alternatively, the present invention provides a peptide having Th1 cell inducing ability and CTL inducing ability. In some specific examples, the peptides of the present invention respond to the sequence identification numbers: amino acid sequences of 1 to 2 or modified versions thereof, in which 2 or more amino acids are substituted, deleted, inserted, and / Or addition while still maintaining the ability to induce Th1 cells.

When the peptide is administered to a subject, the peptide of the present invention is preferably presented on the surface of one or more antigen-presenting cells (APC) to induce Th1 cells. When the peptide of the present invention further contains at least one CTL epitope, the APC will also process the peptide to present the CTL epitope generated from the peptide, thereby inducing CTLs targeted to each peptide. Therefore, another object of the present invention is to provide an APC that presents any of the peptides or fragments thereof of the present invention, and to provide a method for inducing APC.

Administration of one or more of the peptides of the present invention, or polynucleotides encoding such peptides, or APCs presenting such peptides or fragments thereof, will thus induce a potent anti-tumor immune response. Therefore, another object of the present invention is to provide a medicinal medicament or composition comprising, as an active ingredient, one or more of the following ingredients: (a) one or more peptides of the present invention, (b) one or more A plurality of polynucleotides encoding such peptides, and (c) one or more APCs of the invention. Such a pharmaceutical agent or composition of the present invention is particularly useful as a vaccine.

Another object of the present invention is to provide a method for treating and / or preventing cancer (i.e., tumor) (i.e. (avoiding) and / or preventing recurrence after surgery. It is also considered for inducing Th1 cells or for inducing anti-tumor immunity The method includes the step of administering one or more peptides, polynucleotides, APCs, or pharmaceutical agents or compositions of the present invention. Furthermore, the Th1 cells of the present invention are also found to be useful as vaccines against cancer Examples include, but are not limited to, bladder cancer and breast cancer.

Examples of objects specifically contemplated by the present invention include the following:

[1] An isolated peptide having a length of 10-30 amino acids and comprising a part of the amino acid sequence of the sequence identification number: 6 or 8, wherein the peptide includes an amine selected from the group consisting of Amino acid sequence: (a) a continuous amino acid sequence having more than 9 amino acids selected from the amino acid sequence of sequence identification number: 1 or 2; and (b) a monoamino acid sequence In which, one, two, or several amino acids in the amino acid sequence of (a) are substituted, deleted, inserted, and / or added, the peptide has the ability to induce Th1 cells.

[2] The isolated peptide of [1], wherein the peptide or a fragment thereof has the ability to bind to at least two MHC class II molecules.

[3] The isolated peptide of [2], wherein the MHC class II molecule is selected from the group consisting of HLA-DP2, HLA-DP5, HLA-DR4, and HLA-DR9.

[4] The isolated peptide according to any one of [1] to [3], wherein the peptide comprises an amino acid sequence of a peptide having MPHOSPH1-specific CTL inducing ability.

[5] The isolated peptide of [4], wherein the peptide comprises an amino acid sequence selected from the group consisting of: (a) a monoamino acid sequence selected from a sequence identification number: A group consisting of 1 to 2; and (b) an amino acid sequence in which one, two, or several amino acids are substituted, deleted, inserted, and / or in the amino acid sequence of (a) Attached.

[6] An isolated polynucleotide encoding a peptide as described in any one of [1] to [5].

[7] A composition for inducing at least one cell selected from the group consisting of:

(i) Th1 cells

(ii) CTL

(iii) the ability to induce APC in Th1 cells, and

(iv) an APC capable of inducing CTL; wherein the composition comprises one or more peptides such as any one of [1] to [5], or one or more polynucleotides encoding such peptides, Or a composition for inducing at least one type of cell selected from the group consisting of:

(i) Th1 cells

(ii) CTL

(iii) the ability to induce APC in Th1 cells, and

(iv) an APC capable of inducing CTL, wherein the composition comprises one or more peptides such as any one of [1] to [5], or one or more polynucleotides encoding such peptides.

[8] A pharmaceutical composition comprising at least one active ingredient selected from the group consisting of: (a) one or more peptides according to any one of [1] to [5]; (b) One or more polynucleotides such as [6]; (c) one or more APCs that present a peptide or fragment thereof as in any one of [1] to [5] on the surface; (d) one or more A variety of Th1 cells that recognize APCs that exhibit a peptide or fragment thereof as in any of [1] to [5] on the surface; and (e) any two or more of (a) to (d) above And the composition is formulated for use in a group selected from the group consisting of:

(i) Cancer treatment

(ii) Cancer prevention

(iii) preventing recurrence of cancer after surgery, and

(iv) a combination of any two or more of (i) to (iii) above.

[9] The pharmaceutical composition according to [8], wherein the composition is formulated to be administered to at least one selected from the group consisting of HLA-DP2, HLA-DP5, HLA-DR4, and HLA-DR9 Object of MHC class II molecules, or a pharmaceutical composition as in [8], wherein the composition is formulated for administration to at least one MHC class II molecule selected from the group consisting of HLA-DP2, HLA-DP5, HLA -Objects of a group consisting of DR4 and HLA-DR9.

[10] The pharmaceutical composition according to [8] or [9], wherein the composition further comprises one or more peptides capable of CTL induction.

[11] A composition for enhancing the immune response by MHC class II molecular mediators, the composition comprising at least one active ingredient selected from the group consisting of (a) one or more of the following as [1 A peptide according to any one of] to [5]; (b) one or more polynucleotides such as [6]; (c) one or more APCs, which appear on the surface as [1] to [5] A peptide or a fragment thereof according to any one; (d) one or more Th1 cells that recognize an APC that presents a peptide or a fragment thereof according to any one of [1] to [5] on the surface; and (e) ) A combination of any two or more of the above (a) to (d).

[12] A method for inducing APC, the APC being capable of inducing Th1 cells, the method comprising inducing APC in vitro, ex vivo, or in vivo, as in [1] to [5] Step of contacting any of the peptides.

[13] A method for inducing APC, the APC being capable of inducing CTL, the method comprising a step selected from the group consisting of: (a) inducing APC in vitro, ex vivo Or in vivo with a peptide as in any of [1] to [5]; and (b) a polynucleoside encoding a peptide as in any of [1] to [5] Acid was introduced into APC.

[14] A method for inducing Th1 cells, comprising the steps selected from the group consisting of: (a) co-culturing CD4 + T cells, and presenting MHC class II molecules on the surface, as in [1] to [5 APC of any of the peptides or fragments thereof; and (b) a polynucleotide encoding two T cell receptor (TCR) subunits, or a polymer encoding each TCR subunit. A nucleotide is introduced into a CD4 + T cell, wherein the TCR is capable of binding to a complex of a MHC class II molecule present on the cell surface and a peptide or a fragment thereof as in any of [1] to [5], or A method for inducing Th1 cells, comprising the steps selected from the group consisting of: (a) co-culturing CD4 + T cells, and presenting MHC class II molecules on the surface and any of [1] to [5] APC of a peptide or fragment thereof; and (b) introduction of a polynucleotide encoding two TCR subunits, or a multiple polynucleotide encoding each TCR subunit into CD4 + In T cells, the TCR is capable of binding to a complex of a MHC class II molecule and a peptide such as [1] to [5] or a fragment thereof on a cell surface if it is one of APCs.

[15] A method for inducing CTL cells, the method comprising the steps selected from the group consisting of: (a) co-culturing both CD4 + T cells and CD8 + T cells, and having been contacted as in [4] or [ 5] APC of the peptide; and (b) co-culture of CD8 + T cells, and APC that has been contacted with the peptide of [4] or [5].

[16] A method for enhancing an immune response by a MHC class II molecular medium, the method comprising the step of administering to a subject at least one active ingredient selected from the group consisting of: (a) one or more of the following as [ The peptide of any one of [1] to [5]; (b) one or more polynucleotides such as [6]; (c) one or more APCs that appear as [1] to [5 on the surface A peptide or a fragment thereof according to any one of []; (d) one or more Th1 cells that recognize an APC that presents a peptide or a fragment thereof according to any of [1] to [5] on a surface; and ( e) A combination of any two or more of the above (a) to (d).

[17] A detached APC showing on its surface a complex of a MHC class II molecule and a peptide or a fragment thereof according to any one of [1] to [5].

[18] An APC induced by a method such as [12] or [13].

[19] An isolated Th1 cell that recognizes APC that presents a peptide or a fragment thereof according to any one of [1] to [5] on the surface.

[20] A Th1 cell induced by the method as described in [14].

[21] The Th1 cell according to [20], wherein the α and β subunits of the TCR contain CDR3 composed of the amino acid sequences of sequence identification numbers 9 and 19, respectively.

[22] A method of inducing an immune response against cancer in a subject in need thereof, the method comprising the step of administering to the subject a composition containing at least one active ingredient selected from the group consisting of: ( a) one or more peptides as in any one of [1] to [5]; (b) one or more polynucleotides as in [6]; (c) one or more APCs which appear on the surface A peptide or a fragment thereof according to any one of [1] to [5]; (d) One or more Th1 cells that recognize a peptide or any of the peptides according to any one of [1] to [5] on a surface or The APC of the fragment thereof; and (e) a combination of any two or more of (a) to (d) above.

[23] An antibody or an immunologically active fragment thereof against a peptide according to any one of [1] to [5].

[24] A vector comprising a nucleotide sequence encoded as a peptide according to any one of [1] to [5].

[25] A host cell that has been transformed or transfected with an expression vector such as [24].

[26] A diagnostic kit comprising a peptide according to any one of [1] to [5], a polynucleotide such as [6] or an antibody such as [23].

[27] A method of assessing and / or monitoring a Th1 response specific to a peptide in an individual, the peptide comprising an amino acid sequence of sequence identification number: 1 or 2, the method comprising: (a) providing a A sample of an individual administered to a peptide, wherein the sample contains T cells; (b) detection of the presence of T cells in the sample, the T cells exhibiting TCR that binds to a complex of a MHC class II molecule and a peptide or a fragment thereof ; And (c) when the presence of T cells is detected in (b), induction of a peptide-specific Th1 response is shown.

[28] The method of [27], wherein the TCR includes a specific pair of α and β subunits, and each of the α and β subunits contains a CDR3 consisting of amino acid sequences of sequence identification numbers: 9 and 19, respectively.

As mentioned above, other objects or features of the present invention will become more apparent when reading the following detailed descriptions and the accompanying drawings and embodiments. However, it should be understood that the foregoing summary of the present invention and the following detailed description are examples of embodiments, and do not limit the present invention or other alternative embodiments of the present invention. In particular, when the present invention is described here with some specific embodiments, it can be understood that the description is for the purpose of illustrating the present invention and is not intended to limit the present invention. Those skilled in the art can think of various modifications and applications without violating the spirit and scope of the present invention, as described in the attached application patent. Likewise, from this summary and the specific embodiments described below, other objects, features, advantages, and advantages of the present invention are obvious and will be understood without difficulty by those skilled in the art. The above objectives, features, advantages, and advantages are obvious from the combination of the examples, materials, diagrams, and all reasonable inferences described therefrom, alone or considering the references introduced here.

Various aspects and applications of the present invention will become apparent to those having ordinary knowledge in the technical field after considering the simple description of the drawings and the detailed description of the present invention and the above-mentioned preferred embodiments.

[Figure 1-1] Figure 1 shows MPHOSPH1-derived and promiscuous HLA class II binding peptides, which contain CTL epitopes predicted by a recently developed computer algorithm. (A) Analysis of the amino acid sequence of human MPHOSPH1 protein using an algorithm. The number on the horizontal axis refers to the amino acid position at the N-terminus of the MPHOSPH1-derived 15-membered peptide. A lower percent identity rating refers to a stronger binding affinity for HLA class II molecules. (B) Synthesis of multiple HLA type II dual (DRB1 * 09: 01, DRB1 * 04: 05, and DRB1 * 15: 02) products with a low consistency percentage of 26 and 27 LPs, MPHOSPH1 _326-351 -LP (LP-2) and _{MPHOSPH1 272-298 -LP (LP-1)} ( FIG. 1A, black bars) and the _{HLA-A24 (MPHOSPH1-A24 278-286} ) or -A2 (MPHOSPH1- A2 _282-291 ) _-restricted CTL recognized 9-membered peptide.

[Figures 1-2] (C) Analysis of the amino acid sequence of human MPHOSPH1 protein using an algorithm. The number on the horizontal axis refers to the amino acid position at the N-terminus of the MPHOSPH1-derived 15-membered peptide. A lower percent identity rating refers to a stronger binding affinity for HLA class II molecules. The black bar indicates the position of the LP shown in Figure 1B.

[Figure 2A] Figure 2 shows the induction of MPHOSPH1-specific Th cells from healthy donors. (A) MPHOSPH1-specific Th cells are generated from DR9 ⁺ healthy donors (HD1) by stimulation with MPHOSPH1-LP1. The Th cells produced were stimulated again with autologous PBMC or L cells pulsed with MPHOSPH1-LP1. The number of Th cells produced by IFN-γ was analyzed by ELISPOT analysis. Representative data showing at least 3 independent experiments with similar results from HD1. The HLA class II genotype of donor HD1 is shown at the top of the bin. The underlined HLA class II dual gene encodes the HLA class II molecule that this peptide presents to Th cells. By stimulation with MPHOSPH1-LP1, MPHOSPH1-LP1 specific, HLA-DR9 restricted Th cells were generated from healthy donors (HD1).

[Figure 2B] (B) MPHOSPH1-LP1 specific and HLA-DP5 restricted Th cells were generated from healthy donors (HD3) by stimulation with MPHOSPH1-LP1.

[Fig. 2C] (C) MPHOSPH1-LP1 specific and HLA-DR4-restricted Th cells were generated from healthy donors (HD4) by stimulation with MPHOSPH1-LP1.

[Fig. 2D] (D) MPHOSPH1-LP2 specific and HLA-DR4-restricted Th cells were generated from healthy donors (HD1) by stimulation with MPHOSPH1-LP2.

[Figure 2E] (E) MPHOSPH1-LP2 specific and HLA-DP2-restricted Th cells were generated from healthy donors (HD2) by stimulation with MPHOSPH1-LP2.

[Fig. 2F] (F) MPHOSPH1-LP2 specific and HLA-DR4-restricted Th cells were generated from healthy donors (HD4) by stimulation with MPHOSPH1-LP2.

[Figure 3] Figure 3 shows MPHOSPH1-LP, which is naturally treated and presented by DC. HLA-DR4 restricted and MPHOSPH1-LP2 specific Th cells established from donor-HD4 identified autologous DCs loaded with recombinant MPHOSPH1 protein. Representative data from 2 independent experiments with similar results are shown.

[Figure 4] Figure 4 shows that MPHOSPH1-LP1 induces effective CTL cross-priming in vivo. HLA-A2 or HLA-A24 Tgm was immunized with MPHOSPH1-LP1 emulsified in IFA. After the second immunization vaccine MPHOSPH1-LP1 to by MPHOSPH1-A2 _282-291 SP or HIV-A2 SP (Figure 4A) and by MPHOSPH1-A24 _278-286 SP or HIV-A24 SP (Figure 4B ) Pulsed BM-DC stimulated mouse CD8 ⁺ T cells in the mucosal lymph nodes. The number of CD8 ⁺ T cells in mice producing IFN-γ was analyzed by ex vivo ELISPOT. Representative data showing at least 3 independent experiments with similar results.

[Fig. 5-1] Fig. 5 shows the production of various Th1-type cytokines in the MP-HOSPH1-LP-specific Th clone. (A-D) MPHOSPH1-LP-specific bulk Th cells or Th colonies were stimulated with autologous PBMCs with or without homologous peptide pulses. The concentration of the indicated cytokines in the culture supernatant was measured as described in the Materials and Methods section. Data are presented as the mean +/- SD of triplicate analysis. (A) MPHOSPH1-LP1 specific Th cell colonies were stimulated with autologous PBMC with or without homologous peptide pulses. (B) MPHOSPH1-LP1 specific bulk Th cells are stimulated with autologous PBMCs with or without homologous peptide pulses.

[Fig. 5-2] (C) MPHOSPH1-LP2 specific Th cell colonies were stimulated with autologous PBMC with or without homologous peptide pulses. (D) MPHOSPH1-LP2 specific bulk Th cells were stimulated with autologous PBMC with or without homologous peptide pulses.

[Fig. 6-1] Fig. 6 shows the results of TCR gene analysis of MPHOSPH1-LP1 specific Th cells. (A-B) Analysis of MPHOSPH1-LP1 specific T cell bank (repertoire) by deep cDNA sequencing of TCR-α and -β genes using next-generation sequencer. TCR-α is displayed at the top of the division, and TCR-β is displayed at the lower end of the division. The Y-axis refers to the number of times Th cells were stimulated with DC pulsed with homologous peptides. After stimulating the subject Th cells with irradiated PBMCs pulsed with homologous peptides 4 times, the colonization of MPHOSPH1-LP1 specific bulk Th cells was completed. The total stimulation frequency was 9 times. The X-axis refers to the top 10 frequent V-J-CDR3 sequences (details are shown in the right box). The Z axis refers to the frequency of individual V-J-CDR3 sequences.

[Fig. 6-2] (C) Mouse TCR-negative T cell line TG40, which expresses the full-length TCR-α and -β genes isolated from MPHOSPH1-LP1 specific T cell clones, The expression of peptide pulses specifically responded to L cells of HLA-DR, as shown by the cell surface manifestations of CD69 and CD137.

[Figure 7] Figure 7 shows the presence of MPHOSPH1-LP-specific Th cells in PBMCs of patients. Peripheral blood mononuclear cells (PBMC) isolated from patients with urinary epithelial cancer (UC) were stimulated in vitro with a mixture of MPHOSPH1-LP1 and MPHOSPH1-LP2 plus IL-2 and IL-7, The frequency of individual MPHOSPH1-LP specific T cells was evaluated using ELISPOT. Observe the specific responses of Th cells to MPHOSPH1-LP1 and MPHOSPH1-LP2 in five UC patients (A to F). HLA class II-restricted MPHOSPH1-LP-specific Th cells were confirmed by a blocking assay using a monoclonal antibody specific for HLA-DR or -DP.

Although any methods or materials similar or equivalent to those described herein can be used in the practice or testing of specific examples of the invention, the preferred materials, methods and devices are described below. However, before describing this material and method, it should be understood that such description is for understanding only and is not intended to be limiting. It should be understood that the invention is not limited to the particular sizes, shapes, directions, dimensions, materials, methodologies, test procedures, etc. described herein, as these may vary due to routine experimentation and / or optimization. In addition, the terms used in the description are only used to describe specific versions or specific examples, and are not intended to limit the scope of the present invention, which is limited only by the scope of the accompanying patent application.

The disclosures of each publication, patent, or patent application mentioned in this specification are specifically incorporated by reference in their entirety. However, it is not to be construed as an admission that the present invention is not entitled to antedate such disclosure or invention.

I. Definition

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

Unless specifically stated otherwise, the words "a" ("a" or "an") and "the" means "at least one".

The terms "isolated" and "purified" used in connection with a substance (eg, peptide, antibody, polynucleotide, etc.) mean that the substance does not substantially contain at least one substance that may be included in natural sources. Thus, an isolated or purified peptide means substantially free of cellular material such as carbohydrates, lipids, or other contaminating proteins from cell or tissue sources from which the peptide is derived, or substantially chemically synthesized when chemically synthesized. Does not contain chemical precursors or other chemicals.

The term "substantially free of cellular material" includes the preparation of a peptide in which the peptide is isolated from the cellular components of the cell in which it is isolated, or is produced recombinantly. Therefore, peptides that are substantially free of cellular material, including preparations of polypeptides, have less than about 30%, 20%, 10%, or 5% (on a dry weight basis) of a heterogeneous protein (also referred to herein as "Contaminated protein"). When the peptide is produced recombinantly, it is also preferably substantially free of culture medium, which includes the preparation of the peptide and has less than about 20%, 10%, or 5% by volume of the peptide preparation. Culture medium. When the peptide is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals, including preparations of the peptide and less than about 30%, 20%, 10% of the peptide preparation. %, 5% (by dry weight) of chemical precursors or other chemicals involved in the synthesis of the peptide. Specific peptides containing isolated or purified peptides can be obtained, for example, by performing sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis on the protein preparation and Coomassie brilliant blue (Coomassie). The appearance of a single band after Brilliant Blue) staining or similar colloids is shown. In a preferred embodiment, the peptides and polynucleotides of the present invention are isolated or purified.

The terms "polypeptide", "peptide" and "protein" are used herein interchangeably to refer to polymers of amino acid residues. This term applies to amino acid polymers in which one or more amino acid residues are modified residues, or non-naturally occurring residues, such as the corresponding naturally occurring amino acid and the corresponding naturally occurring amino acid Artificial chemical analogues of amino polymers.

The term "amino acid" herein means naturally occurring and synthetic amino acids, and analogs of amino acids and analogs of amino acids analogous to the action of naturally occurring amino acids. Naturally occurring amino acids are encoded by genetic codes and those that have been modified after translation in a cell (eg, hydroxyproline, γ-carboxyglutamic acid, and O -phosphoserine). The term "amino acid analog" means that it has the same basic chemical structure as the naturally occurring amino acid (α-carbon, carboxyl, amine, and R groups bonded to hydrogen) but has a modified R group or a modification (Eg, homoserine, n-leucine, methionine, sulfonium, methionine methyl phosphonium salt). The term "amino acid mimetic" means a chemical compound that has a different structure from a general amino acid but has a similar function.

Amino acids can be indicated here using their common three-letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Association.

The terms "gene", "polynucleotide" and "nucleic acid" are used interchangeably herein and, unless otherwise specified, are specified by their universally acceptable one-letter code.

The terms "agent" and "composition" are used interchangeably herein, and mean a product containing a specific ingredient in a specific amount, and any product obtained by directly or indirectly combining the specific ingredient in a specific amount. Such a term for a pharmaceutical composition is intended to include a product containing the active ingredient and any inactive ingredient that becomes a carrier, and obtained by directly or indirectly combining, compounding, or aggregating any two or more of these ingredients Any product, or any product obtained by dissociating 2 or more of these ingredients, or any product obtained by 2 or more other types of reactions or interactions in these ingredients. Therefore, the pharmaceutical composition of the present invention includes any composition prepared by mixing the compound of the present invention and a pharmaceutically or physiologically acceptable carrier.

The term "active ingredient" means a substance that is biologically or physiologically active in a composition. In particular, in the context of a pharmaceutical composition, "active ingredient" refers to a substance that exhibits the pharmacological effects of a target. For example, when a pharmaceutical composition is used in the treatment or prevention of cancer, the active ingredients in the composition can directly or indirectly direct at least one biological or physiological effect on cancer cells and / or tissues. Preferably, the effect includes reducing or inhibiting the growth of cancer cells, damaging or killing cancer cells and / or tissues. In general, the indirect action of the active ingredient is to induce an immune response of MHC class II molecular mediators. Prior to being formulated, "active ingredients" are also referred to as "bulks", "drug substances" or "technical products". As used herein, the terms "pharmaceutically acceptable carrier" or "physiologically acceptable carrier" refer to a pharmaceutically or physiologically acceptable material, composition, substance, compound, or carrier, including but not limited to Liquid or solid fillers, diluents, excipients, solvents or encapsulated materials.

Unless otherwise defined, the term "cancer" means a cancer expressing the MPHOSPH1 gene, and includes, for example, bladder cancer and melanoma. Cancers expressing the MPHOSPH1 gene are also referred to as cancers expressing the MPHOSPH1 gene, or cancers expressing the MPHOSPH1 gene.

Unless otherwise defined, the terms "T-lymphocyte" and "T-cell" are used interchangeably herein.

Unless otherwise defined, the terms "cytotoxic T-lymphocytes", "cytotoxic T-cells" and "CTL" are used interchangeably herein and, unless otherwise specified, means a subgroup of T-lymphocytes that can identify non-self Cells (e.g., tumor cells, virus-infected cells) and induce death of such cells. CTLs are differentiated from CD8 ⁺ T lymphocytes and can recognize peptides presented by MHC class I molecules.

Unless otherwise defined, the term "HLA-A24" herein means a type comprising the subtype HLA-A24. Examples of the subtype include but are not limited to 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.

Unless otherwise defined, the term "HLA-A2" here means representatively a subtype, examples of which include but are not limited to 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.

Unless otherwise defined, the terms "T helper type 1 cells" and "Th1 cells" are used interchangeably herein, and unless otherwise specified, refer to those capable of recognizing peptides presented by MHC class II molecules and related to cellular immunity CD4 ⁺ T lymphocytic subgroup. Unless otherwise defined, the terms "Th cell", "CD4 ⁺ T cell" and "CD4 ⁺ helper T cell" are also used interchangeably herein. Th1 cells secrete a variety of cytokines (e.g., IFN-γ, IL-2, TNF-β, GM-CSF, TNF-α, etc.) to help activate and / or stimulate other immune cells (such as CTL, macrophages) related to cellular immunity ).

Unless otherwise defined, the term "HLA-DP2" refers to a subtype, examples of which include, but are not limited to, HLA-DPB1 * 02: 01 and HLA-DPB1 * 02: 02.

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 term "HLA-DR4" refers to a subtype, examples of which include, but are not limited to, 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.

Unless otherwise defined, the term "HLA-DR9" refers to a subtype, examples of which include, but are not limited to, HLA-DRB1 * 09: 01, HLA-DRB1 * 09: 02, HLA-DRB1 * 09: 03, HLA-DRB1 * 09:04, HLA-DRB1 * 09: 05, HLA-DRB1 * 09: 06, HLA-DRB1 * 09: 07, HLA-DRB1 * 09: 08, and HLA-DRB1 * 09: 09.

Unless otherwise defined, the term "immune response via MHC class II molecular mediator" refers to the immune response induced by MHC class II molecules exhibiting peptides. "Immune response by MHC class II antigens" includes immune response induced by CD4 ⁺ T cells, especially Th1 cells. Examples of such immune responses include, but are not limited to, cytokine production (e.g., IFN-γ, IL-2, TNF-β, GM-CSF, TNF-α, etc.), and activation and / or stimulation of other immune cells (e.g., CTL, Macrophages, etc.).

Unless otherwise defined, the term "Th1 cells specific to MPHOSPH1" refers to Th1 cells specifically activated by APCs presenting peptides derived from MPHOSPH1, but not activated by other APCs.

Unless otherwise defined, the term "MPHOSPH1-specific CTL" refers to a CTL that specifically exhibits cytotoxicity against a target cell expressing MPHOSPH1. Unless otherwise defined, when used in the context of peptides, the term "CTL-inducing ability" refers to the ability of a peptide to induce CTL when presented on an APC. Unless otherwise defined, the term "set" as used herein refers to a combination of reagents and other materials. It is contemplated herein that this set may include microarrays, wafers, tags, 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" means an immunoglobulin and fragments thereof that specifically respond to a given protein or its peptide. Examples of antibodies may include human antibodies, primate antibodies, chimeric antibodies, bispecific antibodies, humanized antibodies, antibodies fused to other proteins or radiolabeled antibodies, and antibody fragments. Furthermore, antibodies are used here in the broadest sense, and specifically include intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (such as bispecific antibodies), and antibody fragments, the antibody fragments As long as the desired biological activity can be exhibited. "Antibody" stands for all types (eg IgA, IgD, IgE, IgG and IgM).

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

II. Peptide

The peptide of the present invention described in detail below may be referred to as "MPHOSPH1 peptide" or "MPHOSPH1 polypeptide".

To demonstrate that the peptide derived from MPHOSPH1 functions as an antigen recognized by Th1 cells, the peptide derived from MPHOSPH1 (sequence identification number: 6 or 8) was analyzed to determine whether it was confined by an MHC class II molecule Epitope. Candidates of hybrid MHC class II binding peptides derived from MPHOSPH1 were confirmed based on their binding affinities to HLA-DP2, HLA-DP5, HLA-DR4, and HLA-DR9. After stimulating CD4 ⁺ T cells in vitro by dendritic cells (DC) loaded with these peptides, Th1 cells were successfully established using each of the following peptides: MPHOSPH1 _272-298 / WVSFFEIYNEYIYDLFVPVSSKFQKRK (sequence identification number: 1) And MPHOSPH1 _326-351 / AYRLLKLGIKHQSVAFTKLNNASSRS (serial identification number: 2).

These above-mentioned established Th1 cells have shown strong specific Th1 cell activity in response to stimulation of APC by individual peptide pulses. Furthermore, the above peptides can stimulate Th1 cells restricted by HLA-DR and HLA-DP molecules (eg, HLA-DP2, HLA-DP5, HLA-DR4, and HLA-DR9) common in several Japanese populations. These results demonstrate that MPHOSPH1 is an antigen recognized by Th1 cells, and that the peptide line is confined by several HLA-type II molecules (such as HLA-DP2, HLA-DP5, HLA-DR4, and HLA-DR9). Of the epitope.

Some of the peptides identified above include a CTL epitope amino acid sequence that has the ability to induce CTL specific to MPHOSPH1, and as demonstrated herein, this peptide can induce CTL and Th1 cells specific to MPHOSPH1. These peptides are therefore suitable peptides that can be used to induce an immune response against cancers expressing MPHOSPH1. Because the MPHOSPH1 gene is overexpressed in most cancer tissue lines, including, for example, HCC and melanoma, it represents a good target for its line of immunotherapy.

Therefore, the present invention provides a peptide having the ability to induce Th1 cells specific to MPHOSPH1. The peptide of the present invention can bind to at least one MHC class II molecule and be presented on APC. Or a fragment of the peptide of the present invention can be bound to at least one MHC class II molecule and presented on APC. The peptide fragment can be generated by APC treatment. In a preferred embodiment, the peptide or fragment thereof of the present invention is capable of binding to two or more MHC class II molecules (eg, HLA-DP2, HLA-DP5, HLA-DR4, and HLA-DR9). In other words, the peptide of the present invention is capable of inducing Th1 cells restricted by 2 or more MHC class II molecules. In another specific example, the peptide of the present invention includes an amino acid sequence of a peptide having MPHOSPH1-specific CTL inducing ability. Typical examples of such peptides with MPHOSPH1-specific CTL inducing ability include peptides with amino acid sequences of sequence identification number: 3 or 4.

Since the binding grooves in the MHC class II molecules are open at both ends, the MHC class II binding peptides can be flexible in length. The core motif of the MHC class II molecule is composed of 9 amino acid residues, and the MHC class II binding peptide generally has other amino acid residues on the side of this core binding motif. The number of flanking amino acid residues is not limited. Therefore, not all amino acid residues of sequence identification numbers 1 or 2 are necessary for binding to MHC class II molecules. Therefore, the peptide of the present invention may be a peptide capable of inducing Th1 cells. Such a peptide includes an amino acid sequence selected from the group consisting of: (a) a continuous amino acid sequence having Longer than 9 amino acids selected from the amino acid sequence of the sequence identification number: 1 or 2; and (b) the amino acid sequence of (a), wherein the amino acid sequence has 1, 2 or Several amino acids are substituted, deleted, inserted, and / or added.

MHC class II binding peptides typically have 10-30 amino acids. When the amino acid sequence of the sequence identification number: 1 to 2 is composed of a part of the amino acid sequence of MPHOSPH1 (sequence identification number: 6 or 8), the peptide of the present invention may be the following [1] to [5] Peptide:

[1] An isolated peptide having a length of 10-30 amino acids and comprising a part of the amino acid sequence of the sequence identification number: 6 or 8, wherein the peptide includes an amine selected from the group consisting of Amino acid sequence: (a) a continuous amino acid sequence having more than 9 amino acids selected from the amino acid sequence of sequence identification number: 1 or 2; and (b) a monoamino acid sequence In which, the amino acid sequence of (a) has 1, 2, or several amino acids are substituted, deleted, inserted, and / or added, and the peptide has the ability to induce T helper type 1 (Th1) cells.

[2] The isolated peptide of [1], wherein the peptide or a fragment thereof has the ability to bind to at least two MHC class II molecules.

[3] The isolated peptide of [2], wherein the MHC class II molecule is selected from the group consisting of HLA-DP2, HLA-DP5, HLA-DR4, and HLA-DR9.

[4] The isolated peptide according to any one of [1] to [3], wherein the peptide comprises an amino acid sequence of a peptide having MPHOSPH1-specific CTL inducing ability.

[5] The isolated peptide of [4], wherein the peptide comprises an amino acid sequence selected from the group consisting of: (a) a monoamino acid sequence selected from a sequence identification number: A group of 1 to 2; and (b) an amino acid sequence, wherein (a) the amino acid sequence has one, two, or several amino acids which are substituted, deleted, inserted, and / or appended .

The peptide-induced Th1 cells of the present invention are specific to MPHOSPH1 line. Therefore, in some specific examples, the present invention provides a peptide having an amino acid length of less than 30, which is composed of a part of the amino acid sequence of the amino acid sequence of sequence identification number: 6 or 8, wherein the peptide includes a sequence The amino acid sequence of identification number 1 or 2.

In general, software programs such as Wang P, et al. 2008. PLoS Comput Biol are now available on the Internet, for example. 4 (4): e1000048.11: 568; and those described by Wang, P et al. 2010. BMC Bioinformatics can be used to calculate the binding affinity of various peptides to HLA antigens by computer simulation. The binding affinity to the HLA antigen can be, for example, 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: 304-14; Sturniolo T, et al. 1999. Nat Biotechnol. 17 (6): 555-61 and Nielsen M, et al. 2008. PLoS Comput Biol. 4 (7) e1000107 narrator measurement. Therefore, the present invention includes a peptide fragment of MPHOSPH1, which is predicted to bind to the identified HLA antigen using this known formula.

As mentioned above, the MHC class II binding peptide has flexibility in its length, and the amino acid sequence of sequence identification number 1 or 2 may optionally have additional amino acid residues on the side, provided that the obtained peptide is Retain the necessary Th1 cell induction capacity. Such peptides capable of inducing Th1 cells are generally less than about 30 amino acids, often less than about 29 amino acids, and often less than about 28 or 27 amino acids. The amino acid sequence located on the side of the amino acid sequence selected from the sequence identification numbers: 1 to 2 is not particularly limited and may include any kind of amino acid, provided that the amino acid sequence on this side does not weaken the original victory The Th1 cell inducing ability of the peptide is sufficient. In typical specific examples, such a side amino acid sequence may be selected from an amino acid sequence adjacent to the sequence identification number: 1 or 2 of the amino acid sequence: 6 or 8; however, the present invention is not limited to this. this. As described above, the present invention also includes a peptide capable of inducing Th1 cells, and an amino acid sequence selected from sequence identification numbers: 1 to 2.

On the other hand, because the core binding motif of MHC class II molecules consists of 9 amino acid residues, the full length of the amino acid sequence of sequence identification number: 1 or 2 is for binding to MHC class II molecules and inducing Th1 Cells are not necessary. Therefore, the peptide of the present invention may be in the form of a peptide having more than 9 consecutive amino acids from the amino acid sequence of sequence identification number 1 or 2, provided that the peptide retains the necessary Th1 cell inducing ability. The peptides capable of inducing Th1 cells are generally longer than about 10 amino acids, often more than 11 or 12 amino acids, and often longer than 13 or 14 amino acids. Therefore, the peptide of the present invention can be a peptide capable of inducing Th1 cells, and has an amino acid sequence from the sequence identification number: 1 or 2 which is longer than 9, 10, 11, 12, 13, or 14 consecutive amino groups. Amino acid sequences of acids.

In general, it is known that modifying one or more amino acids in a protein does not affect the function of the protein, or sometimes even enhances the desired function of the original protein. In fact, modified peptides (i.e., amino acids in which one, two, or several amino acid residues have been modified (i.e., substituted, deleted, inserted, or added) compared to the original reference sequence The peptides formed by the sequence) are known to retain the biological activity of the original peptides (Mark, et al., Proc Natl Acad Sci USA 1984, 81: 5662-6; Zoller and Smith, Nucleic Acids Res 1982, 10: 6487- 500; Dalbadie-McFarland et al., Proc Natl Acad Sci USA 1982, 79: 6409-13). Therefore, in a specific example of the present invention, the peptide of the present invention may have the ability to induce Th1 cells and one, two or more amino acids are added, inserted, deleted, and / or substituted from the sequence identification number: Both the amino acid sequences in 1 to 2. Alternatively, the peptide of the present invention may have the ability to induce Th1 cells, and have a monoamino acid sequence in which one, two, or more amino acid additions, insertions, deletions, and / or substitutions are made in the sequence identification number: 1 or 2 in. That is, in some specific examples, the peptides of the present invention may have the ability to induce Th1 cells and a monoamino acid sequence, in which the amino acid sequence identification number: 1 or 2 is performed one or two or is selected from the following Formation of the group: several modifications of additions, insertions, deletions and substitutions.

Those skilled in the art will recognize that the tendency of individual amino acids to be added or substituted by changing a single amino acid or a small percentage of amino acids results in the preservation of the original amino acid branch chain. Therefore they are often referred to as "conservative substitutions" or "conservative modifications", in which a change in a protein results in a protein with a similar function to the original protein. Conservative substitution tables providing functionally similar amino acids are well known in the art. Examples of characteristics of amino acid branches 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 branches with the following common functional groups or characteristics: an aliphatic branch (G, A, V, L, I, P); a hydroxyl-containing branch (S, T, Y); sulfur atom-containing branch (C, M); carboxylic acid and amine branch (D, N, E, Q); alkali branch (R, K, H); and aromatic Branches (H, F, Y, W). In addition, the following eight groups each contain amino acids that are conservatively substituted with each other: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3 ) Aspartic acid (N), glutamic acid (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 to be peptides of the present invention. However, the peptide of the present invention is not limited thereto, and may include non-conservative modifications as long as the modified peptide maintains the Th1 cell-inducing ability of the original peptide. Furthermore, modified peptides do not exclude polymorphic variants, interspecies homologues and MPHOSPH1 alleles, which can induce peptides in Th1 cells.

In order to maintain the Th1 cell-inducing ability, a small number (eg, one, two, or several) or a small percentage of amino acids can be modified (inserted, added, deleted, or substituted). The term "several" here means 5 or less amino acids, such as 4 or 3 or less. The percentage of the modified amino acid may be, preferably 20% or less, more preferably 15% or less, even more preferably 10% or 8%, less or 1 to 5%.

The preferred peptides of the present invention, that is, sequence identification numbers: 1 to 2 (MPHOSPH1 272-298- _LP , MPHOSPH1 _326-351-LP ), the homology analysis results confirm that these peptides and any other known human gene products The derived peptides do not have significant homology. Therefore, when used in immunotherapy, the potential for these peptides to produce an unknown or unwanted immune response can be significantly reduced. Therefore, these peptides are expected to be highly useful for eliciting immunity against MPHOSPH1 in cancer patients.

When used in the context of immunotherapy, the peptides or fragments thereof of the present invention should be presented on the surface of APC, preferably in the form of a complex with HLA class II antigen. Therefore, it is advisable to choose peptides that not only induce Th1 cells, but also have high binding affinity for HLA class II antigens. To this end, the peptide can be modified with substitutions, insertions, deletions and / or additions of amino acid residues to produce modified peptides with improved binding affinity.

The invention also contemplates the addition of one, two, or several amino acids to one or both of the N and C termini of the peptide. Such modified peptides having high HLA antigen-binding affinity and retaining Th1 cell-inducing ability are also included in the present invention.

For example, the present invention provides isolated peptides shorter than 31, 30, 29, 28, 27, or 26 amino acids, which bind to HLA class II antigens and have the ability to induce Th1 cells, and comprise selected from the sequence Identification number: 1, 2 or several amino acid modified amino acid sequences are included in the amino acid sequence in the group composed of 1 to 2.

When these peptides come into contact with APC or are introduced into APC, they are processed in APC and presented as processed fragments on APC. For example, the peptides of the present invention can be processed into fragments usually composed of 11-26 (usually 15-25) amino acid residues for presentation on the surface of APC.

However, when the peptide sequence is partly the same as the amino acid sequence of an endogenous or exogenous protein with different functions, it may cause side effects such as autoimmune disorders or allergic symptoms to specific substances. Therefore, homology searches can be performed initially using available databases to avoid situations where the peptide sequence matches the amino acid sequences of other proteins. When compared with naturally occurring target peptides, homology searches show no peptides that are the same or differ from the target peptide by 1 or 2 amino acids, the target peptide can be modified to enhance the HLA antigen Binding affinity, and / or enhancing its Th1 cell inducing ability and / or CTL inducing ability without the risk of such side effects.

Although the above peptides with high binding affinity for the HLA class II antigen are expected to be highly effective, according to candidate peptides with high binding affinity as an indicator, the existence of Th1 cell inducing ability is still further tested. Here, the term "Th1 cell-inducing ability" means that when contacted with an antigen-presenting cell (APC), the peptide imparts the ability to induce Th1 cells via the antigen-presenting cell (APC). In addition, "Th1 cell-inducing ability" includes peptide activation of Th1 cells and / or Th1 cell proliferation, and promotion of Th1 cell cytokine production, which includes IFN-γ production to help and / or stimulate other cells (such as CTL, Macrophage).

Confirmation of Th1 cell inducing ability is achieved by inducing APCs carrying human MHC antigens (such as B-lymphocytes, macrophages, and dendritic cells (DC)), preferably derived from human peripheral blood mononuclear white blood cells DC was mixed with CD4-positive T cells (CD4 ⁺ T cells) after stimulation with the peptide, and then the IFN-γ produced and released by the CD4 ⁺ T cells was measured. Alternatively, the Th1 cell-inducing ability of the peptide can be evaluated by the activation of CTL by Th1 cells. For example, CD4 ⁺ T cells are cultured with DCs stimulated with test peptides, and then mixed with CTL and target cells targeting CTL. The target cells can be calibrated with radioactivity such as ⁵¹ Cr, and the cytotoxic activity of CTLs activated by cytokines released from the Th1 cells can be calculated by calculating the radioactivity released from the target cells. Alternatively, Th1 cell-inducing activity can be measured by measuring the IFN-γ produced and released by Th1 cells in the presence of APC stimulated by the test peptide, and using an anti-IFN-γ monoclonal antibody to make the inhibitory area on the medium visible To evaluate.

In addition to the above modifications, the peptides of the present invention may be further linked to other substances, as long as the resulting linked peptide retains the Th1 cell-inducing ability of the original peptide. Examples of suitable substances include, for example, peptides, lipids, sugars and sugar chains, acetamyl, natural and synthetic polymers, and the like. The peptides of the present invention may include modifications, such as saccharification, side chain oxidation or phosphorylation, etc. The modifications provided will not impair the biological activity of the original peptide. These kinds of modifications can be implemented to provide additional functions (such as target function, and delivery function), or to stabilize the peptide.

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

The peptide of the present invention can be presented on the surface of APC as a complex together with HLA type II antigen, and then induce Th1 cells. The present invention therefore also includes peptides on the surface of APCs that form complexes with HLA class II antigens. APCs presenting the peptides of the present invention can be vaccinated.

The form of the HLA antigen included in the complex must match the form of the HLA antigen in the subject to be treated and / or prevented. For example, in the Japanese population, HLA-DP2, HLA-DP5, HLA-DR4, and HLA-DR9 are prevalent, so they are suitable for treating Japanese patients. In general, the HLA antigen type of the patient to be treated is pre-checked clinically to be able to appropriately select a peptide having the ability to bind to a specific HLA class II antigen. In a preferred embodiment, the peptide of the present invention can induce Th1 cells in a promiscuous manner. Here, when the peptide can induce Th1 cells restricted by at least two different MHC class II molecules, the peptide's Th1 cell inducing ability is referred to as "promiscuous". In other words, when a peptide is recognized by at least two different MHC class II molecules, this antigen recognition is considered "hybrid." When used in the context of peptides, the term "identified by at least 2 different MHC class II molecules" means that the peptide or fragment thereof can bind to at least 2 different MHC class II molecules. For example, MPHOSPH1 272-298- _LP (sequence identification number: 1) is recognized by HLA-DR9, HLA-DP5, and HLA-DR4, and MPHOSPH1 _326-351 -LP (sequence identification number: 2) is recognized by HLA-DR4 and HLA. -DP2 identification. These peptides are therefore typical examples of "hybrid" epitopes.

When using HLA-DR9, HLA-DP5 or HLA-DR4-positive APC, it is preferable to use a peptide having an amino acid sequence of sequence identification number: 1. When HLA-DR4 or HLA-DP2 positive APC is used, it is preferable to use a peptide having an amino acid sequence having a sequence identification number: 2.

Therefore, in a preferred embodiment, a peptide having an amino acid sequence of sequence identification number: 1 can be used to induce Th1 cells in a subject that has been identified to have HLA-DR9, HLA-DP5, or HLA-DR4 before induction. . Similarly, a peptide having an amino acid sequence of SEQ ID NO: 2 can be used to induce Th1 cells in a subject that has been identified to have HLA-DR4 or HLA-DP2 before induction.

III. Preparation of MPHOSPH1 peptide

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 by using recombinant DNA technology or chemical synthesis. The peptides of the present invention can be synthesized individually or made into longer polypeptides including two or more peptides. The peptides of the present invention can then be isolated, that is, purified or isolated, substantially free of other naturally occurring host proteins and fragments thereof, or other arbitrary chemicals.

The peptides of the invention may include modifications such as saccharification, side chain oxidation or phosphorylation; the modifications provided do not impair the biological activity of the original reference peptide. Other illustrative modifications include the introduction of D-amino acids or other amino acid mimetics. These modifications can be used to increase the serum half-life of the peptide.

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

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

IV. Polynucleotides

The present invention also provides a polynucleotide encoding any of the aforementioned peptides of the present invention. This includes polynucleotides derived from the naturally occurring MPHOSPH1 gene (GenBank Accession No. NM_001284259 (sequence identification number: 5) or NM_016195 (sequence identification number: 7)), and nucleotide sequences with conservative modifications. Of those. The term "conservatively modified nucleotide sequence" as used herein refers to a sequence encoded as the same or substantially the same amino acid sequence. Because of the degenerative nature of the genetic code, there are a large number of functionally identical nucleic acids that can encode any given protein. For example, the codons GCA, GCC, GCG, and GCU are all encoded as amino acid alanine. Therefore, when a codon indicates alanine at each position, the codon can be changed to any corresponding codon without changing the encoded polypeptide. This nucleic acid mutation is a "silent mutation", which is a conservatively modified mutation. Each nucleic acid encoding a peptide described herein also describes all possible silent variations of that nucleic acid. Those with ordinary knowledge in the technical field will recognize each codon in a nucleic acid (except for AUG, AUG is usually the only codon for methionine, and TGG is usually the only codon for tryptophan). Modifications to produce functionally identical molecules. Therefore, each silent variation of a nucleic acid encoded as a peptide is implicitly described in each disclosed sequence.

The polynucleotide of the present invention may be composed of DNA, RNA, and derivatives thereof. As is known in the art, DNA molecules are suitably composed of bases such as A, T, C, and G, and T is replaced with U in RNA. Non-naturally occurring bases recognized by those of ordinary skill in the art will also be included in the polynucleotide.

The polynucleotides of the invention can be encoded as a variety of peptides of the invention with or without intervening amino acid sequences. For example, the intervening amino acid sequence can provide a cleavage site for the polynucleotide or the translated peptide (eg, an enzyme recognition sequence). Furthermore, the polynucleotide may include any additional sequence for a coding sequence encoding a peptide of the present invention. For example, the polynucleotide may be a recombinant polynucleotide, which includes regulatory sequences required for expression of the peptide, or may be a expression vector (plasmid) with a marker gene, and the like. In general, such recombinant polynucleotides can be prepared using conventional recombinant techniques using, for example, polymerases and endonucleases to manipulate the polynucleotides.

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

V. Antigen presenting cells (APC)

The present invention also provides an antigen-presenting cell (APC) that appears on the surface as a complex formed between an HLA class II antigen and a peptide or fragment thereof of the present invention. The APC obtained by contacting with the peptide of the present invention can be obtained from a treated and / or prevented patient, and can be administered as a vaccine itself, or in combination with other drugs including the peptide of the present invention, Th1 cells or CTL Vote.

The APC is not limited to a specific type of cell and includes, but is not limited to, dendritic cells (DC), Langerhans cells, macrophages, B cells, and activated T cells, which are known to present proteins on the surface Sexual antigens, but will be recognized by lymphocytes. Because DC is a representative APC, it has the strongest Th1 cell-inducing activity in APC, so DC is useful as the APC of the invention.

Furthermore, in preferred embodiments, the peptides of the present invention also induce CTL responses regulated by MHC class I antigens and Th1 cell responses mediated by MHC class II antigens. In general, it is known that epitopes recognized by MHC class I antigens are shorter in length (eg, 8-10 amino acid residues) than MHC class II (15 or longer). Therefore, the processed products of some of the peptides of the present invention can cause the induction of CTL. In fact, MPHOSPH1 272-298- _LP (sequence identification number: 1) can induce recognizable fragments (YIYDLFVPVS: sequence identification number: 3), and MPHOSPH1 _272-298-LP (sequence identification number: 1) can induce recognizable fragments (IYNEYIYDL: sequence identification number: 4). Therefore, these peptides of the present invention can not only induce Th1 cells, but can also induce CTL after treating them in APC. In other words, APCs that have been in contact with the above peptides of the present invention will present these peptides with MHC class II antigens and simultaneously process these to present these fragments with MHC class I antigens. As a result, using the above peptides of the present invention, Th1 cells and CTLs can be induced.

For example, APC can be obtained by inducing DC from peripheral blood monocytes and then contacting (stimulating) the peptide of the present invention in vitro, in vitro or in vivo. When the peptide of the present invention is administered to the subject, an APC presenting the peptide of the present invention or a fragment thereof is induced in the body of the subject. The term "inducing an APC" includes contacting (stimulating) a cell with a peptide of the present invention to present a complex formed between a HLA class II antigen and a peptide or a fragment thereof of the present invention on a cell surface. Alternatively, after the peptide of the present invention is introduced into APC so that the APC presents the peptide or a fragment thereof, these APCs can be administered as a vaccine to the subject. For example, biological in vitro administration may include the following steps: (a) collecting APC from a first subject: (b) contacting the APC of step (a) with a peptide of the present invention, (c) step (b) of administering a second subject This should be loaded into the peptide's APC.

The first object and the second object may be the same individual or different individuals. Alternatively, according to the present invention, there is provided the use of the peptide of the present invention to produce a pharmaceutical composition for inducing APC. In addition, the present invention provides a method or treatment for manufacturing a pharmaceutical composition for inducing APC, wherein the method includes a step of mixing or formulating a peptide of the present invention with a pharmaceutically acceptable carrier. The present invention also provides a peptide of the present invention for inducing APC. The APC obtained in step (b) can be administered to the subject as a vaccine.

In one aspect of the present invention, the APC of the present invention has a high level of Th1 cell inducing ability. Here, the term "high level of Th1 cell inducing ability" refers to the level when APC is not in contact with the peptide or when it is not in contact with the peptide that does not induce Th1 cells. Here, in the context of APC, the term "Th1 cell inducing ability" refers to the ability of APC to induce Th1 cells when it comes into contact with CD4 ⁺ T cells. Such APCs having a high level of Th1 cell-inducing ability can also be prepared by a method including a step of transferring a polynucleotide encoding a peptide of the present invention to APC in vitro. The polynucleotide to be introduced may be in the form of DNA or RNA. Examples of the method of introduction include, but are not particularly limited to, various conventional methods performed in the field, such as lipid staining, electroporation, or calcium phosphate method. More specifically, published Japanese translations can be made in accordance with Cancer Res 1996, 56: 5672-7; J Immunol 1998, 161: 5607-13; J Exp Med 1996, 184: 465-72; International Publication No. 2000-509281 The implementation. By transferring the gene to APC, the gene will be transcribed, translated, etc. in the cell, and then the protein obtained will be processed by MHC class I or class II and proceeded through a presentation path to present the present invention. Peptide. Alternatively, the APC of the present invention can be prepared by a method including a step of contacting the APC with a peptide of the present invention.

In a preferred embodiment, the APC of the present invention may be an MHC class II molecule selected from the group consisting of HLA-DR9, HLA-DP5, and HLA-DR4 and the peptide of the present invention (including the amino group of sequence identification number: 1). Acid sequence) complex on its surface. In another specific example, the APC of the present invention may be a MHC class II molecule selected from HLA-DR4 and HLA-DP2 and a peptide of the present invention (including the amino acid sequence of SEQ ID NO: 2) APC on its surface. Preferably, HLA-DP2, HLA-DP5, HLA-DR4, and HLA-DR9 can each be HLA-DPB1 * 02: 01, HLA-DRB1 * 05: 01, HLA-DRB1 * 04: 05, and HLA-DPB1 * 09:01.

VI. T helper type 1 cells (Th1 cells)

Against the Th1 cells induced by any of the peptides of the present invention, it will strengthen the immune response of any effector cells. This arbitrary effector cells include CTLs that target cancer cells in the body, and can be used in a similar manner to this peptide itself vaccine. The invention therefore also provides isolated Th1 cells which are specifically induced or activated by any of the peptides of the invention.

Such Th1 cells can be obtained by (1) administering one or more peptides of the present invention to a subject, then collecting Th1 cells from the subject, and (2) contacting (stimulating) APC and CD4 with the peptides of the present invention in vitro ⁺ T cells or peripheral blood mononuclear cells, and then isolated Th1 cells, (3) contact the CD4 ⁺ T cells or peripheral blood mononuclear cells with the APC of the present invention in vitro, or (4) introduce a coded TCR subunit The two polynucleotides or polynucleotides encoded as TCR subunits are incorporated into CD4 ⁺ T cells, wherein the TCR can be bound to a complex of a MHC class II molecule and a peptide of the present invention. The APC according to the method (3) can be prepared by the above method. The details of method (4) will be detailed below under item "VII. T cell receptor (TCR)".

Th1 cells induced by stimulation with the APC of the present invention can be derived from a patient to be treated and / or prevented, and can be administered by themselves or in combination with other drugs including the peptide of the present invention to regulate Uses of effects. The Th1 cells thus obtained can activate and / or stimulate immune cells (eg, CTL, macrophages) responsible for cellular immunity. Such immune cells that can be activated by Th1 cells of the present invention include CTLs that exhibit cytotoxicity to target cells, such as cancer cells. For example, the target cells for this CTL may be cells that endogenously express MPHOSPH1 (such as cancer cells), or cells transfected with the MPHOSPH1 gene. In a preferred embodiment, the peptide of the present invention may include at least one amino acid sequence of a CTL epitope peptide, and in addition to inducing Th1 cells, it may also induce CTL against cells expressing MPHOSPH1, such as cancer cells. In this case, the peptide of the present invention can induce Th1 cells and CTLs simultaneously or sequentially in vitro, and the induced Th1 cells can effectively activate the induced CTLs. Therefore, the peptide including the amino acid sequence of at least one CTL epitope peptide is a peptide suitable for cancer immunotherapy.

Furthermore, the Th1 cells of the present invention secrete various cytokines (such as IFN-γ), which can activate and / or stimulate CTL against other target cells in an antigen-independent manner. Therefore, the Th1 cells of the present invention also help to enhance the CTL activity targeted to cells expressing TAA instead of MPHOSPH1. Therefore, the Th1 cells of the present invention are effective not only for immunotherapy for tumors expressing MPHOSPH1, but also for tumors expressing other TAAs, and peptides and APCs of the present invention.

In some specific examples, the Th1 cells of the present invention recognize cells that present a complex of HLA-DR or HLA-DP antigen and the peptide of the present invention. In the context of Th1 cells, the term "identifying a cell" refers to a complex of MHC class II molecules bound to the cell surface via its TCR and a peptide of the invention, and is activated in an antigen-specific manner. Here, the term "activated in an antigen-specific manner" refers to activation in response to a specific MHC class II molecule and peptide, and induces cytokine production of Th1 cells activated therefrom. In a preferred embodiment, HLA-DR and HLA-DP can be selected from the group consisting of HLA-DP2, HLA-DP5, HLA-DR4, and HLA-DR9.

VII. T cell receptor (TCR)

The invention also provides a composition comprising one or more polynucleotides encoding one or more peptides capable of forming a subunit of a T cell receptor (TCR); and providing the use thereof Method. This TCR subunit has the ability to form TCR, which provides MPHOSPH1 specificity for CD4 ⁺ T cells to APCs that counteract the presentation of MPHOSPH1 peptides. By using a method known in the technical field, it is possible to identify the α- and β-chain nucleic acids of TCR subunits of Th1 cells induced with the peptide of the present invention (WO2007 / 032255 and Morgan, et al., J Immunol 171, 3288 (2003)). The derivative TCR may bind to the APC presenting the MPHOSPH1 peptide with high affinity and, depending on the situation, mediate efficient cytokine production.

One polynucleotide / polynucleotides encoded as TCR subunits (i.e., a single polynucleotide encoded as both TCR subunits, or multiple polynucleosides encoded as separate TCR subunits The acid) may be contained in a suitable vector, such as a retroviral vector. Such vectors are well known in the art. The polynucleotide, or a vector comprising the polynucleotide, can generally be delivered into a CD4 ⁺ T cell, such as a CD4 ⁺ T cell from a patient. Advantageously, the present invention provides an off-the-shelf composition capable of rapidly modifying T cells (or T cells of another subject) possessed by a patient, and quickly and simply producing cells with excellent cancer cell killing. Modified T cells that are dead.

For example, in Th cells stimulated by MPHOSPH1-LP1 (MPHOSPH1 272-298- _LP ), TCR-α and -β subunits are added in a HLA-DR9 restricted manner, each of which has α and β subunits, respectively. CDR3 consisting of the amino acid sequences of sequence identification numbers: 9 and 19 (Figure 6). Therefore, for the Th1 induction of the present invention, polynucleotides encoding these amino acid sequences are preferred. Similarly, Th1 cells exhibit a TCR formed between α and β subunits, where each α and β subunit has a CDR3 consisting of the amino acid sequences of sequence identification numbers: 9 and 19, respectively. A preferred embodiment of the present invention. Generally, the antibody specificity of TCR depends mainly on its CDR3. Therefore, by replacing the α and β subunits of CDR3 with sequence identification numbers: 9 and 19, respectively, MPHOSPH1-LP1 specific TCR can also be reconstructed from known TCRs. Such a TCR with CDR3 colonized from another subunit can be referred to as a chimeric TCR. Methods for transplanting specific CDRs are well known in the art to which the present invention pertains.

The present invention further provides a Th1 cell, which is prepared by transducing a polynucleotide encoding both TCR subunits or a polynucleotide encoding each TCR subunit, wherein such TCR The subunit can bind to the MPHOSPH1 peptide (for example, sequence identification number: 1, on the background of HLA-DR9, HLA-DP5, or HLA-DR4; sequence identification number: 2, on the background of HLA-DR4 or HLA-DP2). The trait-introduced Th1 cells can be self-directed to cancer cells in vivo and can be expanded in vitro using well-known culture methods (for example, Kawakami et al., J Immunol., 142, 3452-3461 (1989)). The Th1 cells prepared as described above can be used for the formation of immunogenic compositions, and are useful for the treatment or avoidance of cancer in patients in need of treatment or protection.

VIII. Pharmaceutical agents or compositions

When the methods and compositions of the present invention indicate in context that it is useful to "treat" cancer, if it results in clinical benefits, such as reduced MPHOSPH1 gene expression in the subject, reduced cancer size, reduced prevalence, or reduced metastatic potential, The treatment is considered "effective." When the treatment is used prophylactically, "effective" means that it delays or prevents the formation of cancer or prevents or reduces the clinical symptoms of cancer. Effectiveness is determined using any method known to be relevant for the diagnosis or treatment of a particular tumor form.

If the methods and compositions of the present invention indicate that "preventing" and "preventing" cancer are useful in the context, these terms are used interchangeably herein to mean any activity that reduces the burden of mortality or morbidity due to disease. Prevention and prevention can occur at "primary, secondary and tertiary levels of prevention." Primary prevention and prevention prevents the development of disease, and secondary and tertiary prevention and prevention levels include goals to prevent and prevent disease progression and show symptoms and reduce the impact of established diseases by restoring function and reducing disease-related complications. active. Alternatively, prevention and prevention can include a wide range of therapies whose goals are to reduce the severity of a particular condition, such as reducing tumor proliferation and metastasis, and reducing angiogenesis.

In the context of the present invention, treating and / or preventing cancer and / or preventing its recurrence after surgery includes any of the following steps, such as surgically removing cancer cells, inhibiting the growth of cancerous cells, tumor regression or recovery, inducing remission, and Inhibit tumorigenesis, tumor recovery, and reduce or inhibit metastasis. Effective treatment and / or prevention of cancer reduces mortality and improves the prognosis of individuals with cancer, reduces the levels of tumor markers in the blood, and reduces detectable symptoms that accompany cancer. For example, reducing or improving symptoms constitutes effective treatment and / or prevention, including a 10%, 20%, 30% or more reduction, or stable disease.

As described above, the peptide-induced Th1 cells of the present invention can help immune cells responsible for cellular immunity. Such immune cells include not only CTLs against cancer cells expressing MPHOSPH1, but also CTLs against cancer cells expressing other TAAs, since cytokines secreted by Th1 cells can affect CTLs in an antigen-independent manner. Therefore, the present invention provides a pharmaceutical agent or composition comprising at least one of the peptides of the present invention. In the pharmaceutical agent or composition, the amount of the peptide is a therapeutically or pharmaceutically effective amount.

The pharmaceutical agent or composition of the present invention is useful in helping, stimulating, and / or enhancing any immune cells (such as CTL, macrophages) responsible for cellular immunity, because the Th1 cells induced by the pharmaceutical agent or composition of the present invention can secrete and affect The cytokine of any immune cell responsible for cellular immunity. Therefore, the agent or composition of the present invention is useful for any use that will enhance or promote an immune response mediated by such an immune cell including CTL. For example, because the agent or composition of the invention can enhance or promote an immune response against cancers or tumors mediated by such immune cells, the invention provides an agent or composition comprising at least one of the peptides of the invention for use in therapy and / Or prevent cancer. The amount of the peptide in such an agent or composition may be an amount effective to significantly enhance or stimulate an immune response in a subject suffering from a cancer expressing MPHOSPH1.

The invention also provides a reagent or composition for enhancing or stimulating an immune response mediated by MHC class I antigens, such as HLA-A2 and HLA-A24. In another specific example, the present invention further provides the use of the peptide of the present invention for the manufacture of an agent or composition for enhancing or stimulating an immune response mediated by a MHC class I antigen.

In a preferred embodiment, the MPHOSPH1-derived peptides identified in the context of the present invention can induce Th1 cells and counteract CTL of MPHOSPH1-expressing cells. The invention therefore also provides an agent or composition comprising at least one peptide of the invention for use in inducing CTLs against cancers or tumors exhibiting MPHOSPH1.

In addition, an agent or composition comprising at least one of the peptides of the present invention can be used to enhance or promote the immune response by MHC class II molecular agents.

Because MPHOSPH1 is manifested in some cancer types, including bladder cancer, breast cancer, and many other malignancy, it is more specific than normal tissues (WO2006 / 085684, Nishiu M, et al., Jpn J Cancer Res 2002; 93 (8): 894-901, Kanehira M, et al., Cancer Res. 2007; 67 (7): 3276-85 and data of microarray analysis in this case) (not shown)), the victory of the present invention The peptide or polynucleotide encoded as the peptide can be used for the treatment and / or prevention of cancer or tumor, and / or for preventing its recurrence after surgery. Therefore, the present invention provides a pharmaceutical agent or composition for the treatment and / or prevention of cancer or tumor, and / or preventing its recurrence after surgery, which comprises one or more peptides of the present invention, or encodes the peptides for this purpose. Polynucleotides of peptides as an active ingredient. Alternatively, the peptides of the present invention can be expressed on the surface of any of the aforementioned cells, such as APC, for use as a pharmaceutical agent or composition. In addition, the above-mentioned Th1 cells can also be used as an active ingredient of a pharmaceutical cancer or tumor agent or composition of the present invention.

In another specific example, the present invention also provides the use of an active ingredient for the manufacture of a pharmaceutical composition or medicament for treating cancer or tumor, the active ingredient is selected from the following: (a) a peptide of the present invention; Polynucleic acids in the form of such peptides disclosed herein; (c) APCs presenting the peptides or fragments thereof of the invention on their surfaces; and (d) Th1 cells of the invention.

Alternatively, the present invention further provides an active ingredient for treating cancer or tumor, the active ingredient is selected from the following: (a) a peptide of the present invention; (b) an expressible form of the peptide encoded by the peptide disclosed herein Polynucleic acids; (c) APCs presenting the peptides or fragments thereof of the invention on their surfaces; and (d) Th1 cells of the invention.

Alternatively, the present invention further provides a method or treatment for manufacturing a pharmaceutical composition or agent for treating cancer or tumor, wherein the method or treatment comprises a step of formulating a pharmaceutically or physiologically acceptable carrier with an active ingredient selected from the following : (A) a peptide of the present invention; (b) a polynucleic acid encoding such a peptide disclosed herein in a formable form; (c) an APC presenting the peptide of the present invention or a fragment thereof on its surface ; And (d) a Th1 cell of the invention.

In another specific example, the present invention also provides a method or treatment for manufacturing a pharmaceutical composition or agent for treating cancer or tumor, wherein the method or treatment includes mixing an active ingredient with a pharmaceutically or physiologically acceptable carrier. Step, wherein the active ingredient is selected from the following: (a) a peptide of the present invention; (b) a polynucleotide encoding such a peptide disclosed herein in a formable form; (c) presenting the present on its surface APCs of the invention peptides or fragments thereof; and (d) Th1 cells of the invention.

Alternatively, the pharmaceutical composition or such agent of the present invention can be used for one or both of preventing cancer or tumor and preventing recurrence after surgery.

The pharmaceutical agent or composition of the present invention is useful as a vaccine. In the context of the present invention, the term "vaccine" (also referred to as an immunogenic composition) refers to a composition having the function of inducing antitumor immunity through vaccination of an animal.

The pharmaceutical agent or composition of the present invention can be used for treating and / or preventing cancer or tumor, and / or preventing its recurrence or metastatic recurrence in a subject or patient after surgery. Examples of such objects include humans and other mammals, including but not limited to mice, rats, guinea pigs, rabbits, cats, dogs, sheep, goats, pigs, cattle, horses, monkeys, baboons and chimpanzees, especially commercially For important animals or livestock.

In the present invention, a peptide having an amino acid sequence selected from sequence identification numbers: 1 to 2 has been found to be composed of several HLA-DR and / or HLA-DP molecules (for example, HLA-DP2, HLA-DP5, HLA-DR4 and HLA-DR9) restricted hybrid Th1 cell epitopes and can be used as candidates for inducing an immune response that is effective and specific against cancer due to the immune response of MHC class II molecular mediators. The pharmaceutical agent or composition of the present invention thus comprising any one of these peptides having an amino acid sequence of SEQ ID NO: 1 to 2 is particularly suitable for having at least one selected from the group consisting of HLA-DRDP2, HLA-DP5 , HLA-DR4 and HLA-DR9 are administered as targets of MHC class II molecules. The same applies to a pharmaceutical agent or composition containing a polynucleotide encoding any of these peptides.

Or in a preferred embodiment, the peptide identified by the present invention can induce a CTL specific to MPHOSPH1 when administered to a subject having HLA-A2 or HLA-A24. Therefore, by administering the peptide of the present invention, in addition to the induction of Th1 cells, it is expected that a CTL response against cancers expressing MPHOSPH1 can be induced. In addition, the peptide of the present invention not only induces a CTL response against MPHOSPH1-expressing cells by treating it, but also enhances it by induction of Th1 cells as a medium. Therefore, in order to achieve the induction of both Th1 cells and MPHOSPH1-specific CTLs in the same subject, for example, when administering a peptide having an amino acid sequence of sequence identification number: 1, for example, the subject to be treated should have HLA-DR9, HLA-DP5 or HLA-DR4 as MHC class II molecules and HLA-A2 or HLA-A24 as MHC class I molecules.

In the present invention, it has been demonstrated that the peptides of the present invention promote an immune response mediated by MHC class II antigens, particularly in a combination of methods restricted by the type of HLA shown below:

MPHOSPH1-LP1: HLA-DR9, HLA-DP5, and HLA-DR4

MPHOSPH1-LP2: HLA-DR4 and HLA-DP2

Therefore, MPHOSPH1-LP1 and -LP2 and a peptide comprising any of the amino acid sequences of sequence identification numbers: 1 to 2 are beneficial for the treatment of cancer that expresses MPHOSPH1 in a patient having at least one HLA dual gene selected from The corresponding HLA subtype for this peptide is shown in the above combination. Alternatively, the present invention provides a pharmaceutical composition for treating a cancer expressing MPHOSPH1 in a patient, wherein the composition includes any one peptide selected from the group consisting of the peptides of the present invention, and wherein the patient has At least one HLA dual gene is selected from the corresponding HLA subtype of the peptide, which is shown in the above combination.

In addition, the present invention also provides the use of a peptide selected from the group consisting of the peptide of the present invention, which is a composition for treating a cancer that expresses MPHOSPH1 in a patient, the patient having At least one HLA dual gene is selected from the corresponding HLA subtype of the peptide, which is shown in the above combination. In addition, in some specific examples, the present invention provides a peptide, which is selected from the group consisting of the peptide of the present invention, and is used to treat cancers that express MPHOSPH1 in patients with at least one HLA dual gene. It is selected from the corresponding HLA subtype of the peptide, which is shown in the above combination. In other specific examples, the present invention provides a method for treating cancer expressing MPHOSPH1 in a patient, comprising the step of administering to the patient a peptide selected from the group consisting of the peptides of the present invention, wherein the disease A patient with at least one HLA dual gene is selected from the corresponding HLA subtype of the peptide in the above combination.

In addition, in some specific examples, the present invention provides a method for manufacturing or formulating a pharmaceutical composition for treating a cancer expressing MPHOSPH1 in a patient, wherein the composition is selected from the group consisting of a peptide of the present invention Any one of the peptides, and wherein the patient has at least one HLA dual gene selected from the corresponding HLA subtype of the peptide, which is shown in the above combination. The method of the present invention, for example, may include admixing or formulating any one peptide selected from the group consisting of the peptides of the present invention, and a pharmaceutically acceptable carrier.

As discussed above, it is known that Th1 cells are important for inducing effective tumor immunity in a host with tumor-bearing. The peptides of the present invention have the ability to induce Th1 cells in a HLA-restricted manner, and the specific HLA restriction pattern of each peptide of the present invention is shown above. Therefore, the present invention provides a composition for promoting or enhancing a Th1 cell response in a patient expressing cancer of MPHOSPH1, wherein the composition includes any one peptide selected from the group consisting of the peptides of the present invention, and The patient has at least one HLA dual gene selected from the corresponding HLA subtype of the peptide, as shown above.

In addition, the present invention also provides the use of a peptide selected from the group consisting of the peptides of the present invention, which is used to make a set of compounds to promote or enhance Th1 in patients with cancer expressing MPHOSPH1. Cell response. The patient has at least one HLA dual gene selected from the corresponding HLA subtype of the peptide, which is shown in the above combination. In addition, in some specific examples, the present invention provides a peptide selected from the group consisting of the peptides of the present invention, which is used to promote or enhance the Th1 cell response in patients with cancer expressing MPHOSPH1. The patient has At least one HLA dual gene is selected from the corresponding HLA subtype of the peptide and is shown in the above combination. In other specific examples, the present invention provides a method for promoting or enhancing a Th1 cell response in a patient expressing a cancer of MPHOSPH1, the method comprising administering to the patient one selected from the group consisting of a peptide of the present invention A peptide, wherein the patient has at least one HLA dual gene selected from the corresponding HLA subtype of the peptide in the above combination.

In addition, in some specific examples, the present invention provides a method for manufacturing or formulating a pharmaceutical composition that promotes or enhances a Th1 cell response in a patient expressing a cancer of MPHOSPH1, wherein the composition includes a compound selected from the group consisting of a peptide of the present invention Any one of the peptides in the group, and wherein the patient has at least one HLA dual gene selected from the corresponding HLA subtype of the peptide, is shown in the above combination. The method of the present invention, for example, may include admixing or formulating any one peptide selected from the group consisting of the peptides of the present invention, and a pharmaceutically acceptable carrier.

In another embodiment, the present invention provides a cancer immunotherapy induced by Th1 cells. The treatment strategy provided by the present invention is applicable and applicable to any cancer that is independent of MPHOSPH1 expression, as long as the immune cell line activated by cytokines secreted by Th1 cells targets cancer cells of the subject.

The cancer or tumor to be treated by the pharmaceutical agent or composition of the present invention includes any kind of cancer or tumor exhibiting MPHOSPH1, including but not limited to, for example, bladder cancer and breast cancer.

The pharmaceutical agent or composition of the present invention, in addition to the above-mentioned active ingredients, may also contain other peptides capable of inducing Th1 cells or CTLs, other polynucleotides encoded as other peptides, and other peptides or fragments thereof. Other cells etc. Examples of such "other" peptides having the ability to induce Th1 cells or CTLs include, but are not limited to, peptides derived from cancer-specific antigens, such as identified TAAs.

If necessary, the pharmaceutical agent or composition of the present invention may include other therapeutic substances as additional effective ingredients, as long as the substance does not inhibit the antitumor effect of the effective ingredients such as any of the peptides of the present invention. For example, the formulation may include anti-inflammatory agents, analgesics, chemotherapeutics, and the like. In addition to other therapeutic substances in the medicament itself, the medicament of the present invention may also be administered sequentially or simultaneously with one or more other pharmacological agents. The amount of the medicament and pharmacological composition depends on, for example, the form of the pharmacological agent used, the disease to be treated, and the time course and route of administration.

Those having ordinary knowledge in the technical field should understand that in addition to the ingredients specifically mentioned herein, the pharmaceutical agent or composition of the present invention may include other agents (e.g., fillers, binders) commonly used in the form of formulations in this technical field , Diluents, excipients).

In one embodiment of the present invention, the pharmaceutical agent or composition may be packaged in an article of manufacture and a kit, the kit containing a substance useful for treating a disease-causing condition such as cancer . The article of manufacture may include a container of any such pharmaceutical agent or composition with a label. Suitable containers include bottles, vials and test tubes. The container can be formed from a variety of materials, such as glass or plastic. The marking on the container must indicate that the agent is used to treat or prevent more than one condition of the disease. This mark may also indicate an administration instruction or the like.

In addition to the above container, the kit containing the pharmaceutical agent or composition of the present invention may optionally include a second container containing a pharmaceutically acceptable diluent. From the perspective of business and user needs, it can also include other materials, including other buffers, diluents, filters, needles, syringes, and packaging inserts for use instructions.

The pharmaceutical composition may be placed in a packaging or a dispensing device as necessary, and it may contain a dose of one or more units containing the active ingredient. The package comprises, for example, metal or plastic foil, such as a blister pack. The packaging or dispensing device may be accompanied by dosing instructions.

(1) A pharmaceutical agent or composition containing the peptide as an active ingredient:

The peptides of the present invention can be administered directly as a pharmaceutical agent or composition, or can be formulated by conventional formulation methods if necessary. In the latter, in addition to the peptide of the present invention, carriers, excipients, etc. which are generally used in medicine can be appropriately used without particular limitation. Examples of such carriers include, but are not limited to, sterile water, physiological saline, phosphate buffered saline, culture fluid, and the like. Furthermore, the pharmaceutical agent or composition may include a stabilizer, a suspending agent, a preservative, a surfactant, and the like, as necessary. The pharmaceutical agent or composition of the present invention can be used for anti-cancer applications.

The peptides of the present invention can be prepared in combination to include two or more peptides of the present invention to induce Th1 cells in vivo. The peptide combination can be in mixed (cocktail) form or joined to each other using standard techniques. For example, the peptide can be chemically linked or represented as a single fused polypeptide sequence. The peptides in this combination can be the same or different.

By administering the peptide of the present invention, the peptide or a fragment thereof is presented at a high density at the APC by the HLA class II antigen, and then the formation between the present peptide and the HLA class II antigen is induced. The complex is a Th1 cell that specifically responds. Alternatively, the APC (eg, DC) is removed from the subject and then stimulated with a peptide of the invention to obtain an APC that presents any of the peptides or fragments of the invention on its cell surface. These APCs were once again administered to the subject to induce Th1 cells in the subject, with the result that the invasion of tumor-associated endothelium can be increased.

The pharmaceutical agent or composition for the treatment and / or prevention of cancer or tumor comprising the peptide of the present invention as an active ingredient may also include an adjuvant known to effectively establish cellular immunity. Or this pharmaceutical agent or composition can be administered together with other active ingredients, and can be administered by granulation. An adjuvant means a compound that, when administered together (or continuously) with a protein having immunological activity, enhances the immune response to the protein. Adjuvants considered here include those described in the literature (Clin Microbiol Rev 1994, 7: 277-89). Examples of suitable adjuvants include, but are not limited to, aluminum phosphate, aluminum hydroxide, alum, cholera toxin, salmonella toxin, Incomplete Freund's adjuvant (IFA), Complete Freund's adjuvant , CFA), ISCOMatrix, GM-CSF, CpG, O / W emulsion, or the like.

Furthermore, a microlipid formulation, a granule formulation in which the peptide is bound to beads having a diameter of several micrometers, and a formulation in which the peptide is bound to the peptide can be conveniently used.

In another embodiment of the present invention, the peptide of the present 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, and organic acids such as acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, lemon Acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, etc.) and salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.). The term "pharmaceutically acceptable salt" as used herein means that the biological potency and properties of the compound are retained and can be obtained by contact with an inorganic acid or base such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, Salt obtained by the reaction of ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, etc.

In some embodiments, the pharmaceutical agent or composition of the present invention may further include a component that primes Th1 cells and selectively activates CTL. Lipids have been identified as agents capable of activating Th1 cells against viral antigens in vivo and selectively activating CTL agents. For example, a palmitic acid residue can be attached to the ε- and α-amino groups of an lysine residue and then linked to the peptide of the present invention. The lipidated peptide can then be administered directly as microcells or microparticles, encapsulated in microliposomes, or emulsified in an adjuvant. Another example of lipids activates Th1 cells and selectively initiates CTL reactions. When covalently attached to an appropriate peptide, E. coli lipoproteins, such as tripalmitinyl-S-glycolamine cysteamine, can be used. Pyro-seramine-serine (P3CSS) primes Th1 cells and selectively activates CTLs (see, eg, Deres et al. , Nature 1989, 342: 561-4).

Examples of suitable methods of administration may include, but are not limited to, oral, intradermal, subdermal, intramuscular, intramedullary, peritoneal, intravenous, etc., and systemic administration or local administration in the vicinity of the target site ( Ie direct injection). This administration may be implemented by a single administration or by multiple administrations. A pharmaceutical or therapeutically effective amount of this peptide of the present invention can be administered to a subject in need of treatment for a cancer that manifests MPHOSPH1. Alternatively, the peptides of the present invention sufficient to enhance or stimulate the immune response mediated by Th1 cells and / or induce CTLs against cancers or tumors expressing MPHOSPH1 can be administered to subjects suffering from cancers expressing MPHOSPH1. The dose of the peptide of the present invention can be appropriately adjusted depending on the disease to be treated, the age and weight of the patient, the method of administration, and the like. The dosage of the peptide may generally be 0.001 mg to 1,000 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 the peptide may be administered once a few days to several months. Those skilled in the art can easily select an appropriate and optimum dose.

(2) Pharmaceutical reagent or composition containing polynucleotide as an active ingredient:

The pharmaceutical agent or composition of the present invention may also include, in the form of expression, a polynucleotide encoding a peptide disclosed herein. The term "in expressable form" herein means that when a polynucleotide is introduced into a cell, it will appear in vivo as a polypeptide that induces antitumor immunity. In the specific example described, the nucleic acid sequence of the polynucleotide of interest includes regulatory elements required to express the polynucleotide. The polynucleotide can be equipped with sequences that help achieve stable insertion in the genome of the target cell (see, eg, Thomas KR & Capecchi MR, Cell 1987, 51: 503-12 for a description of homologous recombination cassette vectors. See also For example, Wolff et al. , Science 1990, 247: 1465-8; U.S. Patent Nos. 5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647; and WO 98/04720), examples of DNA-based delivery technologies include "naked DNA ", Facilitated (bupivacaine, polymer, peptide-mediated) delivery, cationic lipid complexes and particulate-based delivery (" gene gun ") or pressure delivery (see, e.g., U.S. Patent No. 5,922,687).

The peptides of the invention can also be expressed by viral or bacterial vectors. Examples of expression vectors include attenuated virus hosts, such as cowpox or fowlpox. This method involves using vaccinia virus, for example, as a vector to express a nucleotide sequence encoded as the peptide. By introduction into a host, the recombinant vaccinia virus will exhibit the immune-producing peptide and thus cause an immune response. Vaccinia vectors and methods useful in immunoassay procedures are described, for example, in U.S. Patent No. 4,722,848. Another carrier is BCG (Bacille Calmette Guerin). BCG vectors are described in Stover et al. , Nature 1991, 351: 456-60. A wide variety of other vectors useful for therapeutic administration or immunization, such as adenovirus and adenovirus-associated viral vectors, retroviral vectors, typhoid vectors, detoxifying anthrax toxin vectors, and the like are apparent. See, for example, Shata et al. , Mol Med Today 2000, 6: 66-71; Shedlock et al. , J Leukoc Biol 2000, 68: 793-806; Hipp et al. , In Vivo 2000, 14: 571-85.

Delivery of the polynucleotide to the patient can be performed directly, in which case the patient is directly exposed to the carrier carrying the polynucleotide, or indirectly, in which case the cell is first exposed to the polynucleotide of interest in vitro Transform and then transplant the cells into a subject. These two methods are each known as in vivo and in vitro gene therapy.

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

As with peptide administration, polynucleotides can be administered orally, intradermally, subdermally, intramedullarily, intraperitoneally, and / or intravenously, etc., and administered systemically or in the vicinity of the target site Local administration is useful. This administration may be implemented by a single administration or by multiple administrations. A pharmaceutically or therapeutically effective amount of this polynucleotide of the present invention can be administered to a subject in need of treatment for a cancer exhibiting MPHOSPH1. Alternatively, a polynucleotide of the present invention sufficient to enhance or stimulate an immune response mediated by Th1 cells and / or induce CTL against a cancer or tumor expressing MPHOSPH1 may be administered to a subject suffering from a cancer expressing MPHOSPH1. The dosage of the polynucleotide in a cell suitable for a carrier or a cell transformed with a polynucleotide encoding a peptide of the present invention can be appropriately adjusted depending on the disease to be treated, the age and weight of the patient, the method of administration, and the like. The dosage of the peptide may generally be 0.001 mg to 1,000 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 the peptide may be administered once a few days to several months. Those skilled in the art can easily select an appropriate and optimum dosage.

IX. Method for using the peptide, APC or Th1 cells

The peptide of the present invention and a polynucleotide encoded by the peptide can be used to induce APC and Th1 cells of the present invention. The APC of the present invention can also be used to induce Th1 cells of the present invention. The peptide, polynucleotide, and APC can be used in combination with any other compound as long as the additional compound does not inhibit the Th1 cell inducing ability. Therefore, any of the above-mentioned pharmaceutical agents or compositions of the present invention can be used to induce Th1 cells. In addition, the peptides or polynucleotides of the present invention that can be used to induce APC are described below.

(1) Method for inducing antigen-presenting cells (APC):

The present invention provides a method for inducing APC using the peptide of the present invention or a polynucleotide encoding the peptide. Induction of APC can be performed as described in the above item "V. Antigen-presenting cells". The present invention also provides a method for inducing APCs having the ability to induce Th1 cells, and this induction has also been mentioned in the above-mentioned item "V. Antigen-presenting cells".

Alternatively, the present invention provides a method for preparing APC having the ability to induce Th1 cells, wherein the method may include one of the following steps: (a) contacting the peptide of the present invention with APC in vitro, in vitro or in vivo; and (b ) The polynucleotide encoding the peptide of the present invention is introduced into APC.

Alternatively, the present invention provides a method for inducing APCs having the ability to induce Th1 cells, wherein the method comprises a step selected from the group consisting of (a) contacting a peptide of the present invention with an APC, and (b) encoding the The polynucleotide of the invention peptide is introduced into an APC.

The method of the present invention can be performed in vitro, ex vivo, or in vivo. Preferably, the method of the present invention can be performed in vitro or ex vivo. In a preferred embodiment, the APC for inducing APCs capable of inducing Th1 cells preferably exhibits at least one selected from the group consisting of HLA-DP2, HLA-DP5, HLA-DR4, and HLA-DR9 as MHC II APC-like molecules. This type of APC can be obtained from an object having at least one selected from the group consisting of HLA-DP2, HLA-DP5, HLA-DR4, and HLA-DR9 as an object of the MHC class II molecule by a technique known in the technical field. Preparation of blood mononuclear cells (PBMC). The APC induced by the method of the present invention may be a complex that presents a peptide of the present invention or a fragment thereof and an HLA class II antigen (such as HLA-DP2, HLA-DP5, HLA-DR4, and HLA-DR9) on its surface. APC. When the APC induced by the method of the present invention is administered to a subject to induce an immune response against cancer in the subject, the subject is preferably the same source as the APC. However, the object may be different from the APC donor as long as the object and the APC donor have the same HLA type.

In another specific example, the present invention provides a reagent or composition for inducing APC having Th1 cell inducing ability, and such reagent or composition includes one or more of the peptides or polynucleotides of the present invention.

In another specific example, the present invention provides the use of the peptide of the present invention or a polynucleotide encoding the peptide, which is used for manufacturing a reagent or a composition to be formulated for inducing APC.

Alternatively, the present invention further provides the use of the peptide of the present invention or a polynucleotide encoding the peptide, which is used to induce APCs having the ability to induce Th1 cells.

In a preferred embodiment, the peptide of the present invention can not only induce a Th1 response, but can also induce a CTL response after being treated in APC. Therefore, in a preferred embodiment, the APC prepared by the method of the present invention is also useful for inducing CTLs against cells expressing MPHOSPH1, including inducing CTLs against cancer cells. For example, when induced by a peptide containing the amino acid sequence of sequence identification number 3, APC expressing HLA-A2 is suitable for inducing MPHOSPH1-specific CTL. Alternatively, when induced by a peptide containing an amino acid sequence having the sequence identification number: 4, an APC expressing HLA-A24 is suitable for inducing MPHOSPH1-specific CTL.

(2) Method for inducing Th1 cells:

In addition, the present invention also provides a method for inducing Th1 cells using the peptide of the present invention, a polynucleotide encoded as the peptide, or APC presenting 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 peptide (ie, a TCR subunit) that can form a complex that recognizes the peptide of the present invention and an HLA class II antigen TCR. Preferably, the method for inducing Th1 cells comprises at least one selected from the following steps: (a) contacting CD4 ⁺ T cells with APC, and the antigen-presenting cells present HLA class II antigens on the surface thereof with the peptide of the present invention or A complex of fragments thereof; (b) introducing a polynucleotide encoding both TCR subunits or a polynucleotide encoding each of the TCR subunits into a CD4 ⁺ T cell, wherein the TCR can recognize or combine the victory of the present invention Complex of peptide or fragment thereof with HLA class II antigen.

When the peptide of the present invention is administered to a subject, Th1 cells are induced in the subject, and the immune response intensity is enhanced by MHC class II molecular mediators (for example, the immune response targeting the cancer cell). Alternatively, the peptide and the polynucleotide encoding the peptide can be used in the treatment of an organism in vitro, wherein the subject's APC, CD4-positive cells or peripheral blood mononuclear white blood cells and the peptide of the present invention are contacted in vitro (Stimulation) After Th1 cells are induced, activated Th1 cells return to the subject. For example, the method may include the following steps: (a) collecting APC from the subject; (b) contacting the APC of step (a) with the peptide of the present invention; (c) contacting the APC of step (b) with CD4 ⁺ T cells Mix and co-culture to induce Th1 cells, and (d) collect CD4 ⁺ T cells from the co-culture of step (c).

Furthermore, Th1 cells can be induced by introducing into the CD4 ⁺ T cells one polynucleotide encoding one of the TCR subunits or a plurality of polynucleotides encoding each TCR subunit, wherein the TCR can Complex of the peptide or fragment thereof bound to the HLA class II antigen of the present invention. Such transduction can be performed as described in item "VII. T cell receptor (TCR)".

The method of the invention can be performed in vitro, in vitro or in vivo. Preferably, the method of the present invention can be performed in vitro or in vitro. CD4 ⁺ T cells for inducing Th1 cells can be prepared from PBMC obtained from a subject by a method known in the art. In a preferred embodiment, the donor of the CD4 ⁺ T cells may be a subject having at least one member selected from the group consisting of HLA-DP2, HLA-DP5, HLA-DR4, and HLA-DR9 as a MHC class II molecule. The Th1 cell induced by the method of the present invention can be an APC capable of recognizing a complex of the peptide of the present invention or a fragment thereof and an HLA class II antigen on its surface. When Th1 cells induced by the method of the present invention are administered to a subject to induce an immune response against cancer in the subject (or an immune response by MHC class I molecular mediator), the subject is preferably derived from CD4 + T cells as The same object. However, the subject may be different from the source of the CD4 + T cells, as long as the subject has the same HLA type as the donor of the CD4 + T cells.

In a preferred embodiment, the peptide of the present invention can induce CTL against cells expressing MPHOSPH1, and induce Th1 cells. Therefore, the present invention further provides a method for inducing CTL, which comprises at least one step selected from the group consisting of: (a) co-culturing both CD4 + T cells and CD8 + T cells with APC that has been exposed to the peptide of the present invention ; And (b) co-culturing the CD8 + T cells with an APC that has been contacted with a peptide of the present invention.

In such a method for inducing CTL, the peptide of the present invention is processed in APC to generate a CTL epitope peptide, and the generated CTL epitope peptide is presented on the surface of APC.

Or according to the present invention, the use of the peptide of the present invention is provided for the manufacture of a pharmaceutical agent or composition that induces Th1 cells. In addition, the present invention provides a method or treatment for manufacturing a pharmaceutical agent or composition that induces Th1 cells, wherein the method comprises the step of mixing or formulating the peptide of the present invention with a pharmaceutically acceptable carrier. The present invention also provides a peptide of the present invention for inducing Th1 cells.

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

In the context of the present invention, cancers that overexpress MPHOSPH1 can be treated with these active ingredients. Examples of such cancers include, but are not limited to, bladder cancer and breast cancer. Therefore, before administering a vaccine or pharmaceutical composition containing this active ingredient, it is advisable to confirm whether the expression level of MPHOSPH1 has increased in cancer cells or tissues to be treated compared to normal cells of the same organ. Therefore, in a specific example, the present invention provides a method for treating (excessive) cancer expressing MPHOSPH1. The method may include the following steps: (i) measuring MPHOSPH1 expression in cancer cells or tissues obtained from a subject to be treated with cancer. Level; (ii) compare the performance level of this MPHOSPH1 with a normal control group; and (iii) administer at least one component of the group consisting of (a) to (d) above to those suffering from disease compared with the normal control group Subjects with cancer that overexpresses MPHOSPH1.

Alternatively, the present invention can provide a vaccine or a pharmaceutical composition for administration to a subject suffering from a cancer that overexpresses MPHOSPH1, which comprises at least one component selected from the group consisting of (a) to (d) above. In other words, the present invention further provides a method for identifying a subject to be treated with the MPHOSPH1 polypeptide of the present invention. The method includes a step of determining the expression level of MPHOSPH1 in cancer cells or tissues derived from the subject, wherein the gene is compared to a normal control of the gene The increased levels represent that this subject has a cancer that can be treated with the MPHOSPH1 polypeptide of the invention. The cancer treatment method of the present invention will be described in more detail below.

Furthermore, in a preferred embodiment, the HLA type of a subject can be identified before administration of the peptide of the present invention. For example, a subject identified as having HLA-DR9, HLA-DP5 or HLA-DR4 is preferably administered with a peptide having an amino acid sequence having a sequence identification number: 1. Alternatively, it is preferable to administer a peptide having an amino acid sequence having a sequence identification number: 2 to a subject identified as having HLA-DR4 or HLA-DP2.

Any cell or tissue derived from the subject can be used to determine MPHOSPH1-expression as long as it includes the target transcription or translation product of MPHOSPH1. Examples of suitable samples include, but are not limited to, body tissues and fluids, such as blood, saliva, and urine. Preferably, the cell or tissue sample from the subject contains a cell population including epithelial cells, more preferably cancerous epithelial cells or epithelial cells derived from tissue suspected of being cancerous. If necessary, the cells can be purified from the obtained body tissues and fluids and then used as a subject-derived sample.

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

According to the present invention, the expression level of MPHOSPH1 in cancer cells or tissues obtained from a subject can be determined. This level of performance can be determined at the transcription (nucleic acid) product level, using methods known in the art. For example, the mRNA of MPHOSPH1 can be quantified by hybridization using a probe (eg, northern hybridization). This inspection can be performed on a wafer, on an array, or the like. Using an array is better for detecting the performance level of MPHOSPH1. Those of ordinary skill in the art can use the sequence information of MPHOSPH1 to prepare such probes. For example, cDNA of MPHOSPH1 can be used as a probe. If necessary, the probe can be labeled with a suitable label such as a dye, a fluorescent substance, and an isotope, and the expression level of the gene can be detected by the intensity of the hybridized label.

Furthermore, the transcription product of MPHOSPH1 (for example, sequence identification number: 5 or 7) can be quantified using primers by amplification-based detection methods (for example, RT-PCR). Such primers can be prepared based on the sequence information available for the gene.

Specifically, the probe or primer for use in the present invention is hybridized to MPHOSPH1 mRNA under severe, moderately severe, or lowly severe conditions. As used herein, the term "stringent (hybrid) conditions" means that under these conditions, a probe or primer will hybridize to its target sequence but will not hybridize to other sequences. Stringent conditions are sequence dependencies and will be different under different conditions. Specific hybrids of longer sequences are observed at higher temperatures than shorter sequences. Generally speaking, the temperature of a severe condition is selected from a temperature about 5 degrees Celsius lower than the thermal melting point (Tm) at a defined ionic strength and pH of a specific sequence. The Tm refers to the temperature (at a defined ionic strength, pH, and nucleic acid concentration) at which hybridization between a probe that is 50% complementary to its target sequence and the target sequence reaches equilibrium. Since the target sequence is generally overexpressed, at Tm, 50% of the probes will be occupied by equilibrium. Usually harsh conditions are less than about 1.0M sodium ions, usually about 0.01 to 1.0M sodium ions (or other salts), pH 7.0 to 8.3, and for short probes or primers (e.g. 10 to 50 nucleosides) Acid), at least about 30 degrees Celsius, and at least about 60 degrees Celsius for longer probes or primers. Harsh conditions can also be achieved by adding destabilizing substances such as formamidine.

Alternatively, translation products can be detected to diagnose the invention. For example, the amount of MPHOSPH1 protein (sequence identification number: 6 or 8) can be determined. A method for confirming the protein quality of a translation product includes an immunoassay method using an antibody that specifically recognizes the protein. The antibody may be a single antibody or multiple antibodies. Furthermore, any fragment or modification of the antibody (such as a chimeric antibody, scFv, Fab, F (ab ') ₂ , Fv, etc.) can be used for detection, as long as the fragment or modified antibody retains the ability to bind to the MPHOSPH1 protein. can. Methods for preparing such antibodies for detecting proteins are well known in the art, and any method can be used in the present invention to prepare such antibodies and their equivalents.

In another method for detecting the expression level of the MPHOSPH1 gene based on the translation product, the intensity of staining can be determined using an antibody against the MPHOSPH1 protein using immunohistochemical analysis. That is, in this measurement, strong staining represents an increase in the presence / level of the protein, and at the same time, the MPHOSPH1 gene expression level is high.

The expression level of a target gene such as MPHOSPH1 gene in cancer cells, if the level is increased by 10%, 25%, or 50% compared to the control level of the target gene (such as horizontal cells in normal cells); or increased by more than 1.1 times , Higher than 1.5 times, higher than 2.0 times, higher than 5.0 times, higher than 10.0 times or more, it is decided to increase.

This control level can be determined at the same time as the cancer cells, and is performed by using a sample collected and stored from a subject in advance, and the subject's disease state (with or without cancer) is known. In addition, cells obtained from a non-cancerous region of an organ with cancer to be treated can be used as normal controls. Alternatively, the control level may be determined according to a statistical method of result analysis previously determined for the expression level of the MPHOSPH1 gene in a sample obtained from a subject with a known disease state. Furthermore, the control level can be derived from a database derived from the performance patterns of previously tested cells. In addition, according to one aspect of the present invention, the expression level of MPHOSPH1 gene in a biological sample can be compared with multiple control levels determined from multiple reference samples. It is preferred to use a control level determined from a reference sample derived from a tissue type similar to the biological sample obtained from the subject. In addition, it is preferable to use a standard value of the expression level of the MPHOSPH1 gene in a population whose disease state is known. The standard value can be obtained by any method known in the art. For example, a range of average +/- 2 S.D. or average +/- 3 S.D. can be used as the standard value.

In the context of the present invention, a control level determined from a known non-cancerous biological sample 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 called a "control level for canceration". The difference between the performance level of a sample and the control level can be normalized to the performance level of a control nucleic acid, such as a housekeeping gene, whose performance level is known not to vary depending on whether the cell is cancerous or non-cancerous. Exemplary control genes include, but are not limited to, β-actin, glyceraldehyde 3 phosphate dehydrogenase, and ribosomal protein P1.

When the expression level of the MPHOSPH1 gene is increased compared to the normal control level, or when the expression level of the MPHOSPH1 gene is close to or equal to the level of the cancerous control, the subject can be diagnosed as having cancer to be treated.

More specifically, the present invention provides a method for (i) diagnosing whether a subject has a cancer to be treated and / or (ii) selecting a subject for cancer treatment, the method comprising the following steps: The expression level of MPHOSPH1 in cancer cells or tissues obtained by the subject with the cancer to be treated; (b) comparing its expression level of MPHOSPH1 with the normal control level; (c) if the expression level of MPHOSPH1 is increased compared to the normal control level, then The subject is diagnosed as having a cancer to be treated; (d) if the subject is diagnosed as having a cancer to be treated in step (c), a subject for cancer treatment is selected.

Alternatively, the method may include the steps of: (a) determining the MPHOSPH1 expression level in a cancer cell or tissue sample obtained from a subject suspected of having a cancer to be treated; (b) comparing its MPHOSPH1 expression level with a cancerous control level; ( c) if the expression level of MPHOSPH1 is similar to or equivalent to a cancerous control level, the subject is diagnosed as having the cancer to be treated; and (d) if the subject is diagnosed as having the cancer to be treated in step (c) Then choose a subject for cancer treatment.

In some specific examples, after the steps (a)-(d) defined above, the method may further include identifying a group selected from the group consisting of HLA-DP2, HLA-DP5, HLA-DR4, and HLA-DR9. HLA object steps. The cancer therapy according to the present invention is preferably directed to a subject suffering from a cancer overexpressing MPHOSPH1 and having any one of HLA-DP2, HLA-DP5, HLA-DR4, and HLA-DR9. Methods of HLA typing are well known in the art. For example, PCR-based methods for genotyping HLA dual genotypes are well known. Antibodies specific to each HLA molecule are also appropriate tools in identifying the HLA type of a subject.

The present invention also provides a kit for identifying a subject with a cancer that can be treated with the MPHOSPH1 polypeptide of the present invention, which is also useful for assessing and / or monitoring the efficacy of specific cancer therapies, and more particularly cancer immunotherapy. useful. Examples of suitable cancers include, but are not limited to, bladder cancer and breast cancer. In particular, the set preferably includes at least one agent that detects the expression of the MPHOSPH1 gene in cancer cells derived from the subject, and the agent may be selected from the following group: (a) a reagent that detects mRNA of the MPHOSPH1 gene; (b) an agent for detecting the MPHOSPH1 protein; and (c) an agent for detecting the biological activity of the MPHOSPH1 protein.

Examples of reagents suitable for detecting the mRNA of the MPHOSPH1 gene may include a nucleic acid that specifically binds to or identifies the MPHOSPH1 mRNA, such as an oligonucleotide having a sequence complementary to the MPHOSPH1 mRNA. These kinds of oligonucleotides are, for example, primers and probes specific to the MPHOSPH1 mRNA. These kinds of oligonucleotides can be prepared according to methods well known in the art. If necessary, a reagent for detecting the MPHOSPH1 mRNA can be immobilized on a solid substrate. In addition, the kit may include more than one reagent for detecting the MPHOSPH1 mRNA.

On the other hand, examples of reagents suitable for detecting the MPHOSPH1 protein include antibodies to the MPHOSPH1 protein. The antibody may be a single antibody or multiple antibodies. Furthermore, any fragment or modification of the antibody (such as a chimeric antibody, scFv, Fab, F (ab ') ₂ , Fv, etc.) can be used as the reagent, as long as the fragment or modified antibody retains the ability to bind to the MPHOSPH1 protein. can. Methods for preparing these kinds of antibodies that detect 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 can generate molecular markers with signals via direct link or indirect calibration techniques. Labeling antibodies and labels and methods for detecting the binding of the antibodies to their targets are well known in the art, and arbitrary labels and methods can be used in the present invention. In addition, the kit may include more than one reagent for detecting the MPHOSPH1 protein.

The kit may contain more than one of the aforementioned agents. For example, a tissue sample obtained from a subject without or with cancer can be used as a useful control reagent. The kit of the present invention is required from a commercial and user perspective, and may further include other materials, including other buffers, thinners, filters, needles, syringes, and packaging inserts with instructions for use (such as paper, audio tapes, CD- ROM, etc.). These reagents are kept in labeled containers. Suitable containers include bottles, vials and test tubes. These containers can be formed from a variety of materials, such as glass or plastic.

In a specific example of the present invention, when the reagent is a probe against the MPHOSPH1 mRNA, the reagent may be immobilized on a solid substrate, such as a porous strip, to form at least one detection site. The measurement or detection area of the porous strip may include multiple sites, each containing a nucleic acid (probe). Test strips can also contain sites for negative and / or positive controls. Alternatively, the control site may be located on a strip separate from the test strip. Depending on the situation, the different detection sites may contain different amounts of immobilized nucleic acid, that is, a higher amount at the first detection site and a lower amount at the succeeding site. By adding a test sample, the number of sites showing a detectable signal provides a quantitative indication of the amount of MPHOSPH1 mRNA present in the sample. The detection position may be any shape suitable for detection, and is usually a rod or dot shape that spans the width of the test strip.

The kit of the present invention may further include a positive control sample or a MPHOSPH1 standard sample. The positive control sample of the present invention can be prepared by collecting a MPHOSPH1 positive sample and then analyzing its MPHOSPH1 level. Alternatively, purified MPHOSPH1 protein or polynucleotide may be added to cells that do not express MPHOSPH1 to form the positive sample or the MPHOSPH1 standard sample. In the present invention, the purified MPHOSPH1 may be a recombinant protein. The MPHOSPH1 level of the positive control sample is, for example, higher than the cutoff value.

X. Antibodies

The invention further provides antibodies that bind to the peptides of the invention. Preferred antibodies have antibodies that specifically bind to the peptides of the invention and do not (or weakly bind) to non-peptides of the invention. Alternatively, antibodies that bind to the peptides of the present invention, and homologues thereof. Antibodies against the peptides of the present invention are useful in cancer diagnostic and prognostic analysis methods and angiographic methods. Similarly, such antibodies are useful in the treatment, diagnosis, and / or prognosis of other cancers, as long as MPHOSPH1 is also expressed or overexpressed in cancer patients. In addition, antibodies (for example, single-chain antibodies) expressed intracellularly are useful for the treatment of cancers involving the expression of MPHOSPH1. Examples of cancers include, but are not limited to, bladder cancer and breast cancer.

The present invention also provides various immunoassays that detect and / or quantify the MPHOSPH1 protein (sequence identification number: 6 or 8) or a fragment thereof comprising a polypeptide consisting of amino acid sequences selected from the sequence identification numbers: 1 to 2 law. Such assays may include one or more anti-MPHOSPH1 antibodies that are capable of properly identifying and binding to MPHOSPH1 proteins or fragments. In the present invention, the anti-MPHOSPH1 antibody bound to the MPHOSPH1 polypeptide is preferably one that recognizes a polypeptide composed of an amino acid sequence selected from sequence identification numbers: 1 to 2, and more preferably excludes other peptides. The binding specificity of an antibody can be confirmed by an inhibition test. That is, when the binding between the antibody to be analyzed and the full-length MPHOSPH1 polypeptide is inhibited in the presence of a polypeptide having an amino acid sequence selected from the sequence identification numbers: 1 to 2, the antibody is considered "specific "Sexually bind" to the fragment. In the present invention, such an immunoassay is implemented in various immunoassay formats known in the technical field, including but not limited to various radioimmunoassays, immunochromatographic techniques, enzyme-linked immunosorbent assay (ELISA), enzyme Linked immunofluorescence analysis (ELIFA), etc.

Related immunological but non-antibody assays of the invention may include T cell immunogenicity assays (inhibitory or irritant), and MHC binding assays. In addition, the present invention also provides an immunocontrast method capable of detecting a cancer expressing MPHOSPH1, including but not limited to a radioscintigraphy method using a labeled antibody of the present invention. This analysis method can be used clinically to detect, monitor and predict the performance of MPHOSPH1 cancer, including but not limited to bladder cancer and breast cancer.

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

The peptides of the present invention are used as antigens to obtain antibodies, which can be derived from any animal species, but preferably derived from mammals such as humans, mice, rats, and more preferably humans. Human-derived peptides can be obtained from the nucleotide or amino acid sequences disclosed herein.

According to the present invention, the peptide as an immune antigen may be a complete peptide or a partial peptide of the polypeptide of the present invention. Examples of suitable partial peptides may include, for example, the amine (N) terminal or carboxy (C) terminal fragment of a peptide of the present invention.

Here, an antibody is defined as a protein that reacts with the full length or a fragment of the MPHOSPH1 peptide. In a preferred embodiment, the antibody of the present invention can recognize an MPHOSPH1 fragment peptide having an amino acid sequence selected from sequence identification numbers: 1 to 2. Methods for synthesizing oligopeptides are well known in the art. After synthesis, the peptide may be purified optionally before use as an immunogen. In the present invention, the oligopeptide (for example, 24 members or 26 members) may be conjugated or linked to a support to enhance immunogenicity. Keyhole-limpet hemocyanin (KLH) is a well-known carrier. The method of binding the peptides to KLH, is well known in the art.

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

Any mammal can be immunized with the antigen, but it is preferred to consider compatibility with the mother cell used for cell fusion. In general, rodents, rabbits or primates can be used. Animals of the rodent family include, for example, mice, rats, and hamsters. Rabbits include, for example, rabbits. Primates such as Catarrhini monkeys (Old World monkeys) such as Macaca fascicularis, macaques, baboons, chimpanzees.

Methods of immunizing animals with antigens are known in the art. Intraperitoneal or subcutaneous injection of antigen is the standard method for immunizing animals. More specifically, the antigen can be diluted and suspended in an appropriate amount of phosphate buffered saline (PBS), physiological saline, or the like. If desired, the antigen suspension can be mixed with an appropriate amount of a standard adjuvant, such as Freund's complete adjuvant, and the emulsion can be administered to a mammal. Preferably, an antigen mixed with an appropriate amount of Freund's incomplete adjuvant is administered several times every 4 to 21 days. Appropriate carriers can also be used for immunization. After immunization as described above, the expected increase in the amount of antibody in the serum can be checked by standard methods.

Polyclonal antibodies against the peptides of the present invention can be prepared by collecting blood from an immunized mammal whose serum is expected to increase in antibodies, and isolating the serum from the blood in a conventional manner. The polyclonal antibody includes a serum containing the polyclonal antibody and a fraction containing the polyclonal antibody can be isolated from the serum. Immunoglobulin G or M was never prepared by using, for example, an affinity column coupled to a peptide of the invention, and the fraction was further purified using a protein A or protein G column.

In order to prepare a monoclonal antibody for use in the present invention, immune cells are collected from a mammal immunized with the antigen, and the level of the desired antibody in the serum is checked as described above, and then cell fusion is performed. The immune cells used for cell fusion are preferably obtained from the spleen. Other preferred mother cells fused with the above-mentioned immune cells include, for example, mammalian myeloma cells, and more preferably myeloma cells having desired properties for selecting fused cells with drugs.

The above-mentioned immune cells and myeloma cells can be fused according to a known method, such as the method of Milstein et al. (Galfre and Milstein, Methods Enzymol 73: 3-46 (1981)).

The fusion tumor obtained by cell fusion can be selected by culturing it in a standard selection medium such as a HAT medium (a medium containing hypoxanthine, aminopterin, and thymine). Generally, the cell culture line is further cultured in the HAT medium for several days to several weeks, which is sufficient for the death of other cells (non-fusion cells) other than the desired fusion tumor. Standard limiting dilutions can then be performed to screen and breed fusion tumor cells that produce the desired antibody.

In addition to the above method, in which non-human animals are immunized with antigens to prepare fusion tumors, human lymphocytes, such as those infected with EB virus, can be immunized in vitro with peptides, peptide-expressing cells, or lysates thereof. Then, the immunized lymphocytes are fused with human-derived myeloma cells such as U266 that can divide indefinitely to obtain a fusion tumor that produces the desired human antibody capable of binding to the peptide (Unexamined Japanese Published Patent Application No. Zhao 63-17688).

The obtained fusion tumors were then transplanted into the abdominal cavity of mice and ascites fluid was extracted. The obtained monoclonal antibodies can be purified using, for example, ammonium sulfate precipitation, protein A or protein G columns, DEAE ion exchange chromatography, or affinity columns coupled with peptides of the present invention. The antibodies of the present invention can be used not only for purification and detection of the peptides of the present invention, but also as candidates for potentiators and antagonists of the peptides of the present invention.

Alternatively, immune cells such as immunized lymphocytes, antibody production can be immortalized with oncogenes and used to make monoclonal antibodies.

The obtained monoclonal antibodies can also be produced recombinantly using genetic engineering techniques (see, for example, Borrebaeck and Larrick, Therapeutic Monoclonal Antibodies, published by MacMillan Publishers LTD (1990) in the UK). For example, an antibody-encoding DNA can be cloned from an immune cell such as a fusion tumor or an immune lymphocyte producing the antibody, inserted into an appropriate vector, and introduced into a host cell to prepare a recombinant antibody. The present invention also provides a recombinant antibody prepared as described above.

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

Antibodies can be modified by conjugation to various molecules such as polyethylene glycol (PEG). The present invention provides such a modified antibody. The modified antibody can be obtained by chemically modifying the antibody. Such modification methods are well known in the art.

Alternatively, the antibodies of the invention may be obtained as chimeric antibodies derived from non-human antibody variable regions and human antibody-derived constant regions, or as humanized antibodies, including complementarity determining regions (CDRs) derived from non-human antibodies. Framework regions (FR) and invariant regions derived from human antibodies. Such antibodies can be prepared according to known techniques. Humanization can be performed by substituting a rodent CDR or CDR sequence as the corresponding sequence of a human antibody (see, eg, Verhoeyen et al. , Science 239: 1534-1536 (1988)). Such humanized antibodies are therefore chimeric antibodies in which substantially less than the entire human variable region has been replaced with a corresponding sequence derived from a non-human species.

Fully human antibodies that include human variable regions in addition to the human framework and constant regions can also be used. Such antibodies can be made using various techniques known in the art. For example, in vitro methods involve the use of a recombinant library of human antibody fragments that are presented on phages ( e.g., Hoogenboom & Winter, J. Mol. Biol. 227: 381 (1991). Similarly, human immunoglobulin loci can be introduced into a gene transfection Animal antibodies such as mice in which endogenous immunoglobulins have been partially or completely inactivated are used to prepare human antibodies. This method is described, for example, in US Patent Nos. 6,150,584; 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016.

The antibodies obtained above can be purified to homogeneity. For example, antibodies can be isolated and purified according to the isolation and purification methods used for general proteins. For example, column chromatography can be used with appropriate selection and combined use of isolation and antibody, column chromatography such as affinity chromatography, filtration, ultrafiltration, salting-out, dialysis, SDS polyacrylamide gel electrophoresis, etc. Electric point concentration method (Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory (1988)), but is not limited thereto. As the affinity column, a protein A column and a protein G column can be used. Exemplary protein A columns that can be used include, for example, Hyper D, POROS, and Sepharose F.F. (Pharmacia).

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

For example, measurement of absorbance, ELISA, enzyme immunoassay (EIA), radioimmunoassay (RIA), and / or immunofluorescence can be used to measure the antigen-binding activity of the antibodies of the invention. In the ELISA, the antibody of the present invention is immobilized on a plate, the peptide of the present invention is coated on the plate, and then a sample containing a desired antibody, such as a culture supernatant of a antibody-producing cell or a purified antibody, is coated. Then, the secondary antibody recognizing the primary antibody is labeled with an enzyme such as alkaline phosphatase, and then the plate is incubated. After washing, an enzyme substrate such as p-nitrophenyl phosphate is added to the plate, and the absorbance is measured to evaluate the antigen-binding activity of the sample. A fragment of the peptide, such as a C-terminal or N-terminal fragment, can be used as an antigen to evaluate the binding activity of the antibody. BIAcore (Pharmacia) can be used to assess the activity of the antibodies of the invention.

The above method can detect or measure the peptide of the present invention by exposing the antibody of the present invention to a sample presumed to contain the peptide of the present invention and detecting or measuring an immune complex formed by the antibody and the peptide.

Since the method for detecting or measuring the peptide of the present invention can specifically detect or measure a peptide, the method is useful in many experiments using the peptide.

For example, when a peptide of the invention is detected from cancer cells or tissues obtained from a patient, Th1 cells (or CTL cells) that are expected to combat them can be used as an effective tool for cancer immunotherapy.

XI. Vectors and host cells

The present invention also provides a vector and a host cell into which a nucleotide encoding a peptide of the present invention is introduced. The vector of the present invention is useful for carrying nucleotides, particularly DNA of the present invention, to a host cell to express a peptide of the present invention, or administration of the nucleotides of the present invention for gene therapy.

When selecting the host cell is E.coli and the carrier in E.coli (e.g. JM109, DH5α, HB101 or XLlBlue) amplification and production of a large number, the vector should have to be amplified in E.coli "ori" for selecting Transformed marker genes of E. coli (for example, resistance genes selected by drugs such as ampicillin, tetracycline, kanamycin, and chloramphenicol). For example, M13 series vectors, pUC series vectors, pBR322, pBluescript, pCR-Script, etc. can be used. In addition, pGEM-T, pDIRECT, and pT7 can also be used for colonization and extraction of cDNA and the above-mentioned vectors. When a vector is used to produce a protein of the invention, a performance vector is useful. For example, a vector to be expressed in E.coli should have the above-mentioned characteristics required to be amplified in E.coli . When E. coli such as JM109, DH5α, HB101 or XL1 Blue is used as a host cell, the vector should have a promoter such as the lacZ promoter (Ward et al. , Nature 341: 544-6 (1989); FASEB J 6: 2422- 7 (1992)), the araB promoter (Better et al. , Science 240: 1041-3 (1988)), the T7 promoter, and the like. It can efficiently express the desired gene in E.coli . In this regard, pGEX-5X-1 (Pharmacia), "QIAexpress system" (Qiagen), pEGFP and pET (in this case, the host is preferably BL21 expressing T7 RNA polymerase) may be used in place of the above vector. In addition, the vector may also contain signal sequences for peptide secretion. An exemplary signal sequence for secreting the peptide into the E.coli intercellular substance is the pelB signal sequence (Lei et al. , J Bacteriol 169: 4379 (1987)). Methods for introducing these vectors into a target host cell include, for example, a calcium chloride method and an electroporation method.

In addition to E. coli , for example, mammalian-derived expression vectors (e.g., pcDNA3 (Invitrogen) and pEGF-BOS (Nucleic Acids Res 18 (17): 5322 (1990)), pEF, pCDM8), insect cell-derived Expression vectors (e.g. `` Bac-to-BAC baculovirus expression system '' (GIBCO BRL), pBacPAK8), plant-derived expression vectors (e.g. pMH1, pMH2), animal virus-derived expression vectors (e.g. pHSV, pMV, pAdexLcw) , Expression vectors derived from retroviruses (e.g. pZIpneo), expression vectors derived from yeast (e.g. "Pichia Expression Kit" (Invitrogen), pNV11, SP-Q01), and those derived from Bacillus subtilis Expression vectors (eg, pPL608, pKTH50) are used to produce the peptides of the present invention.

In order to express a vector in animal cells such as CHO, COS or NIH3T3 cells, the vector should have a promoter such as the SV40 promoter required for expression in such cells (Mulligan et al. , Nature 277: 108 (1979)), MMLV-LTR promoter, EF1α promoter (Mizushima et al. , Nucleic Acids Res 18: 5322 (1990)), CMV promoter, etc., preferably with a marker gene for selection of a transformant (e.g., by a drug (e.g., new) Mycin, G418) resistance gene of choice). Examples of known vectors having these characteristics include, for example, pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13.

XII. Monitoring of Th1 response of peptide-mediated

Deep cDNA sequence analysis of the TCR-α and -β genes revealed that the frequency of specific gene pairs increased in Th cells stimulated with the peptides of the present invention. Therefore, when such a gene pair is detected in an individual vaccinated with the peptide vaccine of the present invention, it shows that the peptide-specific Th1 response is induced in the individual. Therefore, in Th cells stimulated with the peptides of the present invention, the increase in specific gene pairs can serve as a surrogate marker for monitoring or detecting Th1 responses in individuals after stimulation. In the context of the present invention, "peptide-specific Th1 response" is understood to mean that the TCR formed between the α and β subunit pair specifically recognizes the complex formed between the peptide of the present invention and the HLA molecule body. As discussed above, even after amino acid modification, the Th1 cell-inducing ability of the peptides defined by the specific sequences of the present invention can be maintained. Therefore, in addition to being stimulated with specific peptides, even when Th1 cells are induced by mutant peptides, their antigenic specificity is considered to be "peptide specificity", as long as the aforementioned TCR specifically recognizes the original peptide. The formation of such a complex.

For example, in Th cells stimulated by MPHOSPH1-LP1 (MPHOSPH1 272-298- _LP ), TCR-α and -β subunits are increased in a HLA-DR9 restricted manner, each TCR-α and -β subunit Each has a CDR3 consisting of the amino acid sequences of sequence identification numbers: 9 and 19 (Figure 6). Thus, the accumulation of Th cell populations in individuals shows that Th1 responses mediated by MPHOSPH1-LP1 were successfully induced in individuals vaccinated with MPHOSPH1-LP1 vaccine. The presence of CDR3 consisting of the amino acid sequences of sequence identification numbers: 9 and 19 can be detected by antibody-based analysis. In another embodiment of the present invention, specific TCR-α and -β subunit pairs can also be estimated by detecting polynucleotides encoding the TCR-α and -β subunits. For example, the nucleotide sequences with sequence identification numbers: 29 and 30 respectively represent polynucleotides encoding each of the CDR3 composed of the amino acid sequences of sequence identification numbers: 9 and 19. Therefore, each cDNA synthesized from mRNA encoding the alpha and beta subunits of MPHOSPH1-specific TCR may contain the nucleotide sequences of sequence identification numbers: 29 and 30, respectively. Such polynucleotides can be detected by PCR-based analysis.

In a preferred embodiment, the present invention provides a method for monitoring, assessing, or evaluating a peptide-specific Th1 response in an individual immunized with a peptide, comprising the steps of: (a) providing a peptide obtained from administration of the peptide A sample of an individual, wherein the sample comprises T cells; (b) detecting the presence of T cells in the sample, and the T cells exhibiting TCR that binds to a complex of a MHC class II molecule and a peptide or a fragment thereof; and (c) When the presence of T cells was detected in (b), induction of a peptide-specific Th1 response was shown.

For example, by detecting α- and β-subunits consisting of the amino acid sequences of sequence identification numbers: 9 and 19, it is possible to detect MPHOSPH1-LP1 (MPHOSPH1) induced in a HLA-DR9 restricted manner. _272-298-LP ) specific T cells.

In the present invention, as long as Th1 cells are contained in a sample, any biological sample taken from an individual can be used to monitor the Th1 response. For example, for the purposes of the present invention, blood or blood-derived samples can be used as biological samples. In the present invention, a blood-derived sample includes a cell population including T cells. The method for obtaining a cell population including T cells is well known to those having ordinary knowledge in the technical field to which the present invention pertains. Alternatively, in some embodiments, a tissue or lymph node infiltrated by T cells is also useful as a biological sample.

In some embodiments of the invention, a population of cells comprising T cells can be immobilized for use in antibody-based analysis in the evaluation of TCR subunits. After contacting the fixed cells, the antibody recognizes the CDR3 of the α and β subunits in the fixed cells, and when the antibody binds to both TCR subunits on a single cell, it can detect the formation of a specific pair of subunits TCR-antibody complex. Alternatively, in a nucleotide sequence-based analysis, the accumulation of amplicons synthesized from polynucleotides encoding TCR subunits in a single cell shows that a specific pair of subunits exists. Alternatively, a pair of specific polynucleotides encoding TCR subunits can be monitored by deep cDNA sequence analysis as shown in Fang H, et al, Oncoimmunology 3: e968467, 2015, which is used to immunize each peptide of a vaccine.

In this regard, it is well known that the structural diversity of TCRs on Th1 cells depends primarily on their CDR3. Therefore, antigen specificity also depends on CDR3. Therefore, CDR3 detection can be used to monitor the presence of a specific pair of α and β subunits on a single cell. In some embodiments of the invention, in order to monitor or evaluate the Th1 response, a specific pair of TCR-α and -β subunits can be detected at least one or more times after immunizing the vaccine. When the pair increases in a time-dependent manner through monitoring, it means that the Th1 response of the peptide-mediated agent in the individual is sufficiently induced. Alternatively, when this particular pair is detected at least once, it shows that a Th1 response occurs in the individual.

The invention is described in more detail below with reference to specific examples. However, the following materials, methods, and examples can assist those skilled in the art to make and use the specific examples of the present invention. These are only intended to explain aspects of the present invention, and thus do not limit the scope of the present invention. Those skilled in the art can easily understand that methods and materials similar or equivalent to those described herein can be used to implement or test the present invention.

[Example]

Materials and Methods

Cell line

The research protocol for collecting and using PBMCs from healthy donors (HD) and cancer patients was approved by the Institutional Review Board of Kumamoto University. PBMC were obtained from HD and cancer patients after written informed consent was obtained. As previously reported (Tomita Y, et al., Cancer Sci. 2011; 102 (1): 71-78; Omita 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. 2014; 3: e28100; Sayem MA, et al., Oncoimmunolgy. 2016; 5 (1): e1062209; Hirayama M, et al., Oncoimmunology. 2016; 5 (6): e1123368), producing human monocyte-derived DC and mouse BM-derived DC. Mouse fibroblast cell line (L-cell), which has been genetically engineered to express 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 / DPB1 * 02: 01), L- DP2; DP4 (DPA1 * 01: 03 / DPB1 * 04: 01), RM3-DP4; DP5 (DPA1 * 02: 02 / DPB1 * 05: 01), L-DP5; and DP9 (DPB1 * 09: 01) and L-DP9 was provided by Dr. Alessandro Sette (La Jolla Institute for Allergy and Immunology, CA, USA) and is used as APC.

Prediction of HLA class II binding peptides

In order to predict the potential hybrid HLA class II binding to human MPHOSPH1-derived peptides, the amino acid sequence of the human MPHOSPH1 protein was developed using a recently developed computer algorithm (IEDB analysis analysis resource, consensus method, http: /// tools.immuneepitope.org/analyze/html/mhc_II_binding.html) (Wang P, et al., BMC Bioinformatics 2010; 11: 568. Wang P, et al. PLoS Comput Biol 2008; 4: e1000048). This program analyzes 15 amino acid long sequence offsets to include the complete MPHOSPH1 protein. Select and synthesize 27 and 26 amino acid long peptides, MPHOSPH1 _272-298 -LP (MPHOSPH1-LP1; WVSFFEIYNEYIYDLFVPVSSKFQKRK) and MPHOSPH1 _326-351 -LP (MPHOSPH1-LP2; AYRLLKLGIKHQSVAFTKLNNASSRS), which is: 05. DRB1 * 09: 01 or DRB1 * 15: 02, DPB1 * 02: 01 and DPB1 * 05: 01 multiple HLA class II molecules encoded by dual genes have relatively low percent identity, and the above both contain MPHOSPH1- derivative 9 or 10 bits of the CTL epitope (MPHOSPH-A2 _282-291 and / or MPHOSPH1-A24 _278-286) (FIG. 1 and table 1).

The peptide-binding algorithm scores for the indicated HLA-type II molecules are expressed as repeating peptides derived from MPHOSPH1-derived every 15 members.

The peptide-binding algorithm scores for the indicated HLA-type II molecules are expressed as repeating peptides derived from MPHOSPH1-derived every 15 members.

Synthesis of peptides and recombinant proteins

Synthesis of 2 kinds of _{HLA-A2 (MPHOSPH1 A2-282-291} ) (Nishiu M, et al, Jpn J Cancer Res.2002 Aug; 93 (8):. 894-901) or _HLA-A24 (MPHOSPH1-A24 278- ₂₈₆ ) (Kanehira M, et al., Cancer Res. 2007; 67: 3276-85) as shown in human MPHOSPH1-derived SP and two LPs (MPHOSPH1-LP1 and MPHOSPH1-LP2) (MBL, Nagoya, Japan; purity >95%; Figure 1B). Human immunodeficiency virus (HIV) SPs bound to HLA-A2 (HIV-A2) or HLA-A24 (HIV-A24) were used as negative control SPs (Nishiu M, et al. Jpn J Cancer Res. 2002 Aug; 93 (8): 894-901; Tomita Y, et al., Cancer Sci 2011; 102: 697-705). The peptide was dissolved in dimethyl sulfene at a concentration of 10 mg / mL and stored at -80 ° C. Recombinant human full-length MPHOSPH1 protein and DEPDC1 protein were produced in Escherichia coli BL21 transformed with pET28a vector (Novagen, 69864-3) encoding MPHOSPH1 and DEPDC1 according to the manufacturer's instructions. Both MPHOSPH1 and DEPDC1 proteins were evaluated by polyacrylamide colloid electrophoresis (SDS-PAGE), and purified by HisTrap FF columns (GE Healthcare, Little Chalfont, Buckinghamshire, UK). DEPDC1 protein served as a negative control.

Generation of antigen-specific CD4 ⁺ T cells from healthy donors

The research plan for the collection and use of PBMCs derived from health donors was approved by the Human Test Committee of Kumamoto University. The inventor of this case obtained PBMC samples from 7 healthy donors with informed consent. The genotyping of HLA-A, DRB1 and DPB1 dual genes was performed in the HLA laboratory (Kyoto, Japan) (Table 2). Some modifications were made to the aforementioned manner (Tomita Y, et al., Clin Cancer Res 2013; 19: 4508-20; Tomita Y, et al., Int J Cancer 2014; 134: 352-66; Tomita Y, et al., OncoImmunology 2014; 3: e28100; Sayem MA, et al., OncoImmunology 2016; 5: e1062209; Hirayama M, et al., OncoImmunology 2016; 5: 6, e1123368; Inoue M, et al., Int J Cancer 2010; 127 : 1393-403) to induce antigen-specific CD4 ⁺ T cells. CD4 ⁺ T cells (Miltenyi Biotec, Auburn, CA, USA) were purified from PBMCs by positive screening with magnetic microbeads (Inoue M, et al. Int J Cancer 2010; 127: 1393-403). Mononuclear sphere-derived dendritic cells (DC) were produced from CD14 ⁺ cells in vitro as described above (Harao M, et al. Int J Cancer 2008; 123: 2616-25.) And used as APC to induce Antigen-specific CD4 ⁺ T cells. 10 micro-g / mL LP pulsed DC (1 X 10 ⁴ / well) for three hours and radiated DC (45 Gy), then in each well of a 96-well, flat-bottomed dish, supplemented with 200 micro-L at 5% without AIM-V supplemented with human blood plasma was mixed with CD4 ⁺ T cells (3 X 10 ⁴ / well). Seven days later, half of the culture broth was removed from each culture, and pulses containing recombinant human interleukin 7 (rhIL-7) pulsed with peptide (10micro-g / mL) and 5ng / mL were added. irradiated (50Gy) fresh medium (100micro-L / well) autologous PBMC (1 X 10 ⁵⁾ is. Two days after the second stimulation with peptide, rhIL-2 was added to each well (10 IU / mL). One week later, the specificity of stimulated CD4 ⁺ T cells in each well was analyzed in an IFN-γ ELISPOT analysis. T cells showing a specific response to cognate peptides were moved to 24-well plates at weekly intervals in a culture medium supplemented with rhIL-2 (20IU / mL) and rhIL-7 (5ng / mL) In the experiment, those T cells were restimulated with irradiated autologous PBMCs (1 × 10 ⁶ / well) pulsed with peptides. In some cases, T cells will be colonized by limiting dilution for further research as described above (Tabata H, et al. Hum Immunol 1998; 59: 549-60).

^a Th cells specific for MPHOSPH1-LP are derived from PMBCs from healthy donors (HD1, HD2, HD3, HD4, HD6 and HD7).

Evaluation of T cell responses to peptides and proteins

The IFN-γ enzyme linked immunospot assay (ELISPOT) (BD Biosciences) was used as described previously (Tomita Y, et al. Cancer Sci 2011; 102: 697-705) to evaluate Th cells for irradiated APC pulsed with peptides Immune response. Briefly, every ⁴ × 10 ⁴ bulk CD4 ⁺ T cells were analyzed when stimulated with irradiated PBMC (3 × 10 ⁴ cells / well) with peptide pulses, or every 1 × 10 ⁴ bulk CD4 ⁺ The frequency of peptide-specific CD4 ⁺ T cells producing IFN-γ when stimulated with L cells (5 × 10 ⁴ / well) with peptides pulsed by HLA-DP or HLA-DR-. In order to determine the restriction of antigen participation by HLA molecules, anti-HLA-AR monoclonal antibody (mAb) (L243, BioLegend), anti-HLA-DP mAb (B7 / 21, Abcam), anti-human HLA-DQ mAb (SPV-L3, Abcam) or anti-HLA class I mAb (W6 / 32, Abcam) to investigate the barriers to antigen-induced IFN-γ production. All mAbs were used at a final concentration of 5 micro-g / mL. All assessments of the IFN-γ ELISPOT analysis were performed in triplicate or duplication, and the results are presented as mean +/- standard deviation.

Assess MPHOSPH1-LP specific CD4 ⁺ T cell response in UC patients. At 37 ° C. in a final volume of 2 ml of AIM-V culture medium (Invitrogen) supplemented with 5% decomplemented human plasma in the presence of a mixture of MPHOSPH1-LP (20 micro-g / mL) in PBMCs (2 × 10 ⁶ / well, 24-well plate) taken from five UC patients were cultured at 37 ° C. IL-2 (20 U / ml) and IL-7 (5 ng / ml) were added on days 0 and 2. After seven days the cell culture was collected, and the cells washed with MPHOSPH1-LP alone as a control or LP moved ELISPOT plate (5-10 X 10 ⁴ / well) for 18 hours. As before, the number of MPHOSPH1-LP-specific CD4 ⁺ T cells was counted.

In vivo cross-start analysis

Dr. FALemonnier (Firat H, et al. Eur J Immunol 1999; 29: 3112-21; Jung Ko, et al., J Virol 2012; 86: 7616-24) kindly provided HLA-A2 and HLA-A24 (HHD) Transgenic mouse (Tgm). Mice were injected intradermally at the root of the tail with a solution of MPHOSPH1-LP1 (100 micro-g / mouse) emulsified in Freund's adjuvant (IFA) at intervals of seven days. Seven days after the second immunization with MPHOSPH1-LP1, CD8 ⁺ T cells were isolated from the mucosa lymph nodes by positive selection using magnetic microbeads (Miltenyi Biotec, Autum, CA, USA). By ex vivo ELISPOT analysis (Harao M, et al., Int J Cancer 2008; 123: 2616-25; Inoue M, et al., Immunol Lett 2009; 126: 67-72), calculated to pass MPHOSPH1 -A2 _282-291 MPHOSPH1-A24 _278-286 or derived from bone marrow DC (BM-DC, 2 X 10 4 / well) of the SP- pulse stimulation, the IFN-γ produced CD8 ⁺ T cells (1 X 10 ⁵ / Well).

Cytokine analysis

MPHOSPH1-LP- bulk specific Th or Th cells cloned body (1 X 10 ⁴ / well) and cultured in a 96-well plate with autologous peptide through pulses PBMC (3 X 10 ⁴ / well). Using the Bio-Plex system (Bio-Rad), as previously reported, the cytokine levels in the culture supernatant after 24 hours were measured according to the manufacturer's instructions.

TCR sequencing

Total RNA was isolated from MPHOSPH1-LP1-specific T cells using the RNeasy mini kit (Qiagen, Valencia, CA, USA). Using the protocol described earlier (Fang H, et al., Oncoimmunology. 2014; 3: 12: e968467; Choudhury NJ, et al., European urology focus. 2016; 2: 4: 445-52), After some modifications, a sequencing library for TRAV and TRBV was prepared, and 600 cycles of the Miseq Reagent Kit V3 (Illumina, Inc., San Diego, CA, USA) were used for sequencing on the IlluminaMiseq platform. Then, using the previously described algorithm (Fang H, et al., Oncoimmunology. 2014; 3: 12: e968467), the sequence reads were analyzed. In order to identify the V- (D) -J segment (V- (D) -J segment) in individual TRAV and TRBV sequence reads, 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), each sequence read in the FASTQ file A mapped sequence derived from IMGT / GENE-DB (Giudicelli V, et al., Nucleic acids research. 2005; 33 (Database issue): D256-61). In order to define the amino acid sequence of complementarity determining region 3 (CDR3) in TRAV and TRBV, it was identified that the conserved cysteine and The border of CDR3 formed by the conserved phenylalanine at the 5 'portion of the J fragment (Fang H, et al., Oncoimmunology. 2014; 3 (12); e968467). The nucleotide sequence between the conservative cysteine and phenylalanine was extracted to confirm the amino acid sequence of the CDR3 region.

TCR breeding and performance in TG40

As described previously (Hamana H, et al., Biochemical and Biophysical research communications. 2016; 474 (4): 709-14), one-step multiplex RT-PCR was used from T cell lines Amplify the TCR cDNA. The DNA sequence of the amplified PCR product was confirmed by direct sequencing. In order to analyze the antigen specificity of the obtained TCR cDNA, as described previously (Mou Z, et al., Science translational medicine. 2015; 7 (310): 310ra167; Kobayashi E, et al., Nature medicine. 2013; 19 ( 11): 1542-6), establish a TCR expression vector, and transfer it to Plat-E Cell (generally provided by Professor Toshio Kitamura, University of Tokyo) to generate a recombinant retrovirus. The produced TCR was transferred to TG40 cells (Ohno H, et al., Journal of immunology (Baltimore, Md) as previously described (Kobayashi E, et al., Nature medicine. 2013; 19 (11): 1542-6). : 1950) 1991; 146 (11): 3742-6). In the presence of 10 micro-g / mL of the antigen peptide, 1 X 10 ⁵ TCR-transferred TG40 cells and 1 X 105 ⁵ HLA-DR-restricted L cells in an atmosphere of 37 ° C and 5% CO ₂ Co-culture, and the expression of CD69 and CD137 on the cell surface was analyzed by flow cytometry.

Statistical Analysis

This case uses a two-tailed Student t test (bar graph) or a parameterless Mann-Whitney U test (scatter plot) to compare data. In all tests, differences in P-values less than 0.05 were considered statistically significant.

The result

Prediction and selection of CTL-epitope containing potential hybrid HLA class II binding MPHOSPH1 long peptide

In order to identify the potential hybrid HLA-type II binding Th cell epitope of MPHOSPH1, the amino acid sequence of MPHOSPH1 was first tested using a recently developed computer algorithm (Figure 1A, C and Table 1) (Wang P , et al. BMC Bioinformatics 2010; 11: 568; Wang P, et al., PLoS Comput Biol 2008; 4: e1000048). One peptide, MPHOSPH1 272-298- _LP (MPHOSPH1-LP1), is close to the CTL- epitope sequence, which is recognized by CTLs restricted by HLA-A2 and A24, while the other peptide, MPHOSPH1 _326-351 -LP (MPHOSPH1- LP2) does not contain the known CTL-epitope sequence (Figure 1B). Therefore, the inventors of the present case synthesized two candidate LPs, MPHOSPH1-LP1 and MPHOSPH1-LP2, and predicted that HLA class II molecules (HLA-DR4, HLA-DR9, HLA-DR15, HLA-DP2, and HLA) that are frequent in Japanese population -DP5) has strong binding affinity, and one of them contains a 9- or 10-membered peptide for subsequent analysis, which is identified by HLA-A2 or A24-restricted CTLs.

Identification of hybrid MPHOSPH1-derived Th cell epitopes

CD4 ⁺ T cells isolated from PBMCs from healthy donors were stimulated at weekly intervals with autologous DC pulsed with MPHOSPH1-LP1 or -LP2 and radiated PBMC. After at least 3 rounds of stimulation, the MPHOSPH1-LP specific response of the cultured CD4 ⁺ T cells was examined by IFN-γ ELISPOT analysis. In HLA-DR4 and DR9 positive healthy donors (HD1), the Th cells produced produced a significant amount of IFN-γ in a HLA-DR-dependent manner in response to MPHOSPH1-LP1 pulsed PBMC. Bulk Th cells specifically identify L-DR9 cells pulsed by MPHOSPH1-LP1 in a HLA-DR-dependent manner, rather than L-DR9 cells unpulsed, L-DR4 cells pulsed by MPHOSPH1-LP1-, or L-DR9. DR53 cells (Figure 2A). These results indicate that MPHOSPH1-LP1 is presented by HLA-DR9. To investigate whether MPHOSPH1-LP1 induces the response of Th cells restricted by other HLA class II molecules, CD4 ⁺ T cells (HD3 and HD4) from other healthy donors were tested. The present inventors confirmed that MPHOSPH1-LP1 produces HLA-DP5 (DPB1 * 05: 01) -restricted Th cells and HLA-DR4 (DRB1 * 04: 05) -restricted bulk Th cells (Figures 2B and 2C).

In HLA-DR4, DR9, and DR53-positive healthy donors (HD1), Th cells produced by stimulating PBMCs with MPHOSPH1-JP2 produced a significant amount of IFN-γ in a HLA-DR-dependent manner in response to MPHOSPH1 -LP2 pulsed PBMC. The Th cells in bulk specifically identify the L-DR4 cells pulsed by MPHOSPH1-LP2 in a HLA-DR-dependent manner, not the unpulsed L-DR4 cells, MP-HOSPH1-LP2 pulsed L-DR9 cells, or L- DR53 cells (Figure 2D). These results indicate that MPHOSPH1-LP2 is presented by HLA-DR4. To investigate whether MPHOSPH1-LP2 induces the response of Th cells restricted by other HLA class II molecules, CD4 ⁺ T cells from other healthy donors (HD2 and HD4) were tested. The inventors confirmed that stimulation with MPHOSPH1-LP2 will produce HLA-DP2 (DPB1 * 02: 01) -restricted Th cells and HLA-DR4 (DRB1 * 04: 05) -restricted bulk Th cells (Figures 2E and 2F) . Therefore, MPHOSPH1-LP2 binds to HLA-DP2 and HLA-DR4, which implies that MPHOSPH1-LP2 is also a hybrid Th-cell epitope presented by HLA class II molecules common in the Japanese population (Saito S, et al ., Tissue Antigens 2000; 56: 522-9; Mack SJ, et al., Tissue Antigens 2000; 55: 383-400). These results show that these two MPHOSPH1-LPs are promiscuous Th cell epitopes, as summarized in Table 3. Since these LPs are promiscuous Th cell epitopes presented by HLA class II common in Japanese populations (Table 4) (Zarour HM, et al., Cancer research. 2002; 62 (1): 213-8 Grabowska AK, et al., International journal of cancer. 2015; 136 (1): 212-24), so in many PBMCs of Japanese donors, the combination of these peptides induces MPHOSPH1-specific Th cell response.

The aggregation of MPHOSPH1-LP and the immune response of Th cells to MPHOSPH1-LP in five HDs.

^{a The} underlined HLA class II dual genes encode HLA class II molecules that present MPHOSPH1-LP to Th cells in HD; details of donor HLA dual genes are shown in Table 2.

^{b The} underlined and italicized sequence is the CTL epitope; the algorithm is used to predict the high affinity binding to HLA class II molecules, and the MPHOSPH1-LP2 sequence is selected; the HLA class II is bound to and is close to the known CTL antigen Decide on both and pick MPHOSPH1-LP1. aa: Amino acid, Negative: No positive data could be obtained without further progress, LP: Long peptide, HD: Healthy donor

The antigenic frequency of each HLA dual gene in the Japanese population is quoted from a previous report (Kobayashi, et al., Can res 2001; 61 (12): 4773-8)

MPHOSPH1-LP2 is naturally processed and presented by DC

Assess whether DCs ingest and process MPHOSPH1 protein to stimulate MPHOSPH1-LP specific Th colonies. DCs carrying recombinant MPHOSPH1 protein were prepared and used as APC in the IFN-γELISPOT analysis (Nishiu M, et al., Jpn J Cancer Res. 2002 Aug; 93 (8): 894-901; Harao M, et al. Int J Cancer 2008; 123: 2616-25). HLA-DR4-restricted MPHOSPH1-LP2 reactive Th colonies effectively identified DCs carrying the MPHOSPH1 protein, but did not identify DCs carrying the control protein, showing that this epitope was derived from MPHOSPH1 protein by natural processing and by HLA-DR4 Molecular presentation (Figure 3). These results show that the MPHOSPH1-LP2 line is naturally processed from the MPHOSPH1 protein and is presented by DC.

Th1 cytokines produced by Th cells induced by MPHOSPH1-LP

In order to characterize MPHOSPH1-LP-specific Th cells, the inventors measured various cytokines secreted by Th cells, which responded to stimulation of autologous PBMCs pulsed by their respective homologous peptides. After restimulation with homologous peptides, a relatively large amount of Th1 cytokines, such as IFN-γ, TNF-α, IL-2, and GM-CSF, but not IL-4 or IL-17, are specific to MPHOSPH1-LP Th cell production (Figure 5) shows that MPHOSPH1-LP has the ability to induce Th1-polarized Th cells. Therefore, the induction of Th cells via MPHOSPH1-LP is useful for antitumor therapy.

MPHOSPH1-LP1 specific bulk Th cells show converged TCR-α and -β genes

The next generation sequencer was used to analyze the MPHOSPH1-LP1 specific T cell bank by deep cDNA sequencing of the TCR-α and -β genes. After multiple stimulation of bulk Th cells, the frequency of use of a particular pair of TCR-α and -β genes increased in both bulk Th cells. Two LP-specific T cell colonies established from parental bulk T cell strains have been stimulated four or five times with peptides, showing the same pair of TCR-α and -β genes as bulk Th cells (6A and Figure 6B). TCR-α and -β genes isolated from MPHOSPH1-LP1 specific T cells isolated were introduced into mouse TCR-negative T cell line TG40, respectively, and the original antigenic peptide specificity and HLA-restriction of parental Th cells were successfully obtained. As shown by the upregulation of CD69 and CD137 detected by flow cytometry analysis (Figure 6C). These observations confirm the colonization of established T cells and the specificity of these TCR-α and -β chain pairs.

Cross-presentation of MPHOSPH1-LP effectively activates MPHOSPH1-SP-specific CD8 ⁺ T cells in vivo

The in vitro IFN-γ ELISPOT analysis was used to examine the initiation ability of MPHOSPH1-A2 _282-291 specific CTL and MPHOSPH1-A24 _278-226 specific CTL for MPHOSPH1-LP1. HLA-A2 and HLA-A24 Tgm were immunized twice with MPHOSPH1-LP1 emulsified in IFA. Isolated from the inoculated MPHOSPH1-LP1 vaccines HLA-A2 and HLA-A24 Tgm of specific CD8 ⁺ T cells produced IFN-γ, as to by MPHOSPH1-A2 _282-291 SP or MPHOSPH1-A24 _278-286 SP The pulsed BM-DC responds to the stimulus (Figures 4A and 4B). These results suggest that, after ingestion MPHOSPH1-LP1, the HLA-A2 and HLA-A24 Tgm in, the APC in vivo cross-priming can MPHOSPH1-A2 _282-291 SP-specific CTL and MPHOSPH1-A24 _278-286 specific CTL.

PBMCs isolated from UC patients identify some MPHOSPH1-LP

In order to test the induction of MPHOSPH1-LP-specific Th cell response in UC patients, the inventors of the present case stimulated PBMC isolated from late stage UC patients with LP in vitro. Patient characteristics are shown in Table 5. After stimulation, the frequency of individual MPHOSPH1-LP specific T cells was measured. MPHOSPH1-LP1 and MPHOSPH1-LP2 induced peptide-specific T cells from five patients. T-cell peptide-specific IFN-γ production was significantly inhibited by anti-HLA-DR mAb, but not by anti-HLA-DP mAb or anti-HLA class I mAb (Figure 7). These results indicate that IFN-γ is produced by MPHOSPH1-LP-specific and HLA-DR-restricted CD4 ⁺ T cells, suggesting that MPHOSPH1-LP1 and MPHOSPH1-LP2 not only induce antigen-specific Th cells from HD, but also from UC The patient's antigen-specific Th cells.

a Pooling of MPHOSPH1-LP-specific immune responses derived from PBMCs from five UC patients. The response of MPHOSPH1-LP-specific Th cells was measured using an IFN-γ enzyme-linked immunospot assay (ELISPOT). Briefly, patient-derived PBMCs were stimulated by MPHOSPH1-LP as shown. When the average number of IFN-γ spots exceeds 25 and is more than twice the background signal, the response is recorded as positive.

Discuss

Phase I / II clinical trials of combined immunotherapy with BCG and MPHOSPH1-derived SP vaccine in the bladder have shown promising safety and good immunogenicity in preventing the recurrence of non-muscle invasive bladder cancer and inducing CTL. (Obara W, et al., Clin Oncol 2012; 42: 591-600). Therefore, the inventors of the present case have tried to identify LPs that induce antigen-specific Th1 cells and CTLs to further develop better peptide-ephemeral immunotherapy.

The inventors of the present case identified two hybrid MPHOSPH1-derived Th cell epitope peptides, as summarized in Table 3. One of them contains HLA-A2 and A24 restricted CTL epitopes. The inventors of the present case confirmed that MPHOSPH1-LP2 was naturally processed from MPHOSPH1 protein and presented on the cell surface of DCs in the context of HLA class II molecules (Figure 3). It is suggested that CD4 ⁺ T cells induced by these two MPHOSPH1-LPs have beneficial properties 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, it should be considered that peptides can bind to multiple HLA class Class II molecules and a wider range of adaptability (Kobayashi H, et al., Cancer research. 2001; 61 (12): 4473-8; Zarou HM, et al., Cancer research. 2002; 62 (1): 213-8; Grabowska AK, et al., International journal of cancer. 2015; 136 (1): 212-24). The inventors of the present case have shown here that MPHOSPH1-LP is presented via HLA-DR4, HLA-DR9, HLA-DR15, HLA-DR53, HLA-DP2, and HLA-DP5, which implies that a combined vaccine with MPHOSPH1-LP Can cover most Japanese ethnic groups (Table 4). In fact, the inventors observed that MPHOSPH1-LP-specific Th cells are present in five UC patients, which suggests that MPHOSPH1-LP is widely useful in many of these patients, and the use of more Studies of various HLA Class II cancer patients.

MPHOSPH1-LP induced HLA-DP2, DP5, DR4, and DR9-restricted Th cells (Figure 2 and Table 3). In the Japanese and Pacific / Asian populations, HLA-DP2, DP5, DR4, and DR9 dual genes are frequent HLA class II dual genes, and HLA-A2 and A24 dual genes are also observed in those groups (Table 4) . In addition, the HLA-A2 or A24 Tgm _in, MPHOSPH1 272-298 -LP start and extending MPHOSPH1-A2 _282-291 vivo induction of specific CTL and MPHOSPH1-A24 _278-286 specific CTL, suggesting containing MPHOSPH1-SP Epitope MPHOSPH1-LP can induce MPHOSPH1-specific Th cells and CTLs. Therefore, the present inventors suggest that these immunogenic MPHOSPH1-LPs may have the potential to induce a stronger anti-tumor response.

Recent LP studies have shown that because of the long-term cross-presentation of LP, in terms of inducing anti-tumor CTL immunity, immunization vaccines containing natural CTL epitopes are superior to those consisting of minimal CTL epitopes (Melief CJ, et al., E Nat Rev Cancer 2008; 8: 351-60; Bijker MS, et al., Eur J Immunol 2008; 38: 1033-42; Chauvin JM, et al., J Immunol 2012; 188: 2102-10) . In HLA-A2 or A24 Tgm, MPHOSPH1-LP1 containing a known CTL epitope induces CTL epitope-specific CTLs in vivo through a cross-presentation pathway (Figure 4). Therefore, these peptide-based immunization vaccines can simultaneously induce MPHOSPH1-LP1 specific Th cells and CTLs in cancer patients.

MPHOSPH1-LP2 effectively induces those LP-specific Th cell responses (Figures 2D to F). However, they do not contain known HLA class I restricted CTL epitopes (Figure 1B). Recently, it has been reported that the phenomenon called epitope spreading is critical 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). In fact, patients receiving a therapeutic cancer (minimum CTL epitope peptide) immunization vaccine show clinical response and long-term survival. In those patients, responses of Th cells and CTLs to the same protein or many other epitopes of unrelated antigens that are not included in the immune vaccine were observed. This is because the immune vaccine induces CTL specific to the target antigen, and due to damage to tumor cells, other TAAs are released, taken up and presented by APC, to induce other TAA-specific T cell responses. If this is the case, the immunized vaccine of MPHOSPH1-LP2 without CTL epitopes induces LP-specific Th1 responses, as well as other anti-tumor Th1 and CTL responses. Furthermore, if the epitopes are dispersed to include neoantigen, it is an aberrant peptide encoded by a somatic missense mutation gene that is freely produced in cancer cells, and is anti-tumor. T-cell responses are more efficient because those neoantigens are non-self. In fact, recent studies have shown that there are many T cells specific for neoantigens, suggesting that the genes encoding the antigens have mutations and that the epitopes that have spread can also include neoantigens. Cancer antigen immunovaccination therapy using neoantigens has attracted a lot of attention (Kreiter S, et al., Nature. 2015; 520 (7549): 692-6; Linnemann C, et al., Nature medicine. 2015; 21 ( 1): 81-5). In addition to the induction of epitope diffusion, inducing and activating as many TAA-specific T cells as possible is the key to the success of cancer immunovaccination therapy. The immune vaccine formulations of MPHOSPH1-LP and SP can be promising immune vaccine formulations to be explored .

When the inventors of this case intend to use LPs containing Th cell epitopes for cancer immunotherapy, whether anti-tumor Th1 cells can be induced in the patient's body is a major problem. It has been reported that the phenotype of Th cells is not only affected by the surrounding cytokine environment (milieu) at different stages, but also by the affinity / avidity of the TCR and MHC peptide complex (Constant S, et al., The Journal of experimental medicine. 1995; 182 (5): 1591-6; Yamane H, et al., The Journal of experimental medicine. 2005; 202 (6): 793-804; van Panhuys N, et al., Immunity. 2014; 41 (1): 63-74; Hosken NA, et al., The Journal of experimental medicine. 1995; 182 (5): 1579-84). In other words, in the peptide-HLA class II complex that recognizes high-density signals that induce strong TCR, CD4 ⁺ T cells have a tendency to differentiate into Th1 cells, and in the opposite case, Th cells have a tendency Differentiate into Th2 cells. The MPHOSPH1-LP-specific Th cells induced by healthy donors from this study were all Th1-type cells, which mainly produced IFN-γ, TNF-α, GM-CSF, and IL-2 (Figure 5). Therefore, MPHOSPH1-LP can better induce Th1 type Th cell differentiation, which may be due to higher affinity for binding to HLA class II molecules, which can produce high-density LP-HLA class II complexes to stimulate durable and potential Ground-strength TCR media activation signal.

Instead of inducing various TAA-specific Th cells and CTLs by immunizing vaccines, direct introduction of genetically engineered T cells specific to TAA into cancer patients seems to be another effective anti-tumor therapy. In a clinical trial, Rosenberg et al. Introduced autologous T cells transduced with the TCR gene of NY-ESO-1 reactive CTL via retrovirus into disease with metastatic synovial cell sarcoma and melanoma Disease, which shows an objective clinical response (Robbins PF, et al., Clinical cancer research: an official journal of the American Association for Cancer Research. 2015; 21 (5): 1019-27). In this study, TCR use of MPHOSPH1-LP-specific Th cells, after repeated stimulation, respectively, extremely converged to a combination of TCRA and TCRB genes (Figures 6A-B). Each pair of TCR genes introduced into the mouse TCR-negative T cell line TG40 exhibited the specificity of the original T cells to their homologous ligands (Figure 6C), suggesting that patients expressing those TCR genes had autologous T Cell metastasis can be an effective therapy. The convergence of the TCR library of MPHOSPH1-LP-specific Th cells to a specific CDR sequence seems similar to recent observations by Dash et al. And Glanville et al., Compared to a random TCR library that was not selected to bind to a specific antigen peptide Antigen-specific TCRs share short sequence regions (motif) that are specifically enriched in CDRs (Dash P, et al., Nature. 2017; 547 (7661): 89-93; Glanville J, et al., Nature. 2017; 547 (7661): 94-8).

In summary, the inventors of the present case identified two immunogenic MPHOSPH1-LPs that can induce Th cells. One of them contains HLA-A2 restricted and HLA-A24 restricted CTL epitopes. The results suggest that MPHOSPH1-LP provides a useful tool for delivering MPHOSPH1-specific Th1 cells and CTLs. These findings support possible clinical trials of immunotherapy based on MPHOSPH1 peptides for many types of cancer, such as bladder cancer. In addition, a convergent TCR expressed on LP-specific Th cells may be useful for monitoring LP-specific Th cells in vitro and possibly in vivo.

Industrial availability

The present invention describes a Th1 cell epitope peptide derived from MPHOSPH1 that can induce an effective anti-tumor immune response and is therefore useful for a wide range of cancer types. This peptide guarantees the further development of a peptide vaccine against cancer, especially against cancers expressing MPHOSPH1. The peptides of the present invention can induce a Th1 cell response, and thus the cytokines secreted by Th1 cells can help or activate any immune cells that act as cellular immunity in an antigen-independent manner. Therefore, the immunotherapy strategy provided by the present invention can be applied to any disease including cancer, as long as the disease can be improved by the immune response of MHC class II molecular mediator. Specifically, the Th1 cells of the present invention can improve the immune response caused by CTL. Therefore, the peptide of the present invention is useful for enhancing the CTL response of a disease including cancer in a subject.

Furthermore, in a preferred embodiment, the peptide of the present invention also induces CTL and Th1 cells against cells expressing MPHOSPH1. Such a peptide of the present invention is also effective for treating diseases related to MPHOSPH1, such as cancer expressing MPHOSPH1, and more specifically, bladder cancer and breast cancer.

Although the present invention has been described in detail with reference to specific specific examples, it should be understood that the essence of the above description is exemplary and illustrative, and is provided for understanding the present invention and its preferred specific examples. Those with ordinary knowledge in the technical field can easily understand that various changes and modifications can be made without departing from the spirit and scope of the present invention through routine experiments. The boundaries and boundaries of the present invention are defined by the scope of the attached patent application.

<110> Cancer Therapy Science Co., Ltd.

<120> MPHOSPH1 epitope peptide for TH1 cells and vaccine containing the same

<130> ONC-A1608-TW

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Claims (28)

    A isolated peptide having a length of 10-30 amino acids and containing a sequence identification number: 6 or a part of an amino acid sequence, wherein the peptide comprises an amino acid sequence selected from the group consisting of : (A) a continuous amino acid sequence having more than 9 amino acids selected from the amino acid sequence of the sequence identification number: 1 or 2; and (b) a amino acid sequence, wherein In the amino acid sequence of (a), one, two or several amino acids are substituted, deleted, inserted and / or added, and the peptide has the ability to induce Th1 cells.         For example, the isolated peptide of item 1 of the patent application scope, wherein the peptide or a fragment thereof has the ability to bind to at least two MHC class II molecules.         For example, the isolated peptide of item 2 of the patent application scope, wherein the MHC class II molecule is selected from the group consisting of HLA-DP2, HLA-DP5, HLA-DR4, and HLA-DR9.         For example, the isolated peptide according to any one of claims 1 to 3, wherein the peptide comprises an amino acid sequence of a peptide having MPHOSPH1 specific CTL inducing ability.         For example, the isolated peptide of item 4 of the patent application scope, wherein the peptide comprises an amino acid sequence selected from the group consisting of: (a) a monoamino acid sequence selected from a sequence identification number: A group consisting of 1 to 2; and (b) an amino acid sequence in which one, two, or several amino acids are substituted, deleted, inserted, and / or in the amino acid sequence of (a) Attached.         An isolated polynucleotide encoding a peptide as described in any one of claims 1 to 5 of the patent application scope.         A composition for inducing at least one cell selected from the group consisting of: (i) Th1 cells; (ii) CTL; (iii) APC capable of inducing Th1 cells; and (iv) capable of APC that induces CTL; wherein the composition comprises one or more peptides according to any one of claims 1 to 5, or one or more polynucleotides encoding such peptides.         A pharmaceutical composition comprising at least one active ingredient selected from the group consisting of: (a) one or more peptides according to any one of claims 1 to 5 in the patent application scope; (b) one or A plurality of polynucleotides such as those in the scope of patent application No. 6; (c) one or more APCs that present on the surface a peptide or a fragment thereof as in any of the scopes of patent applications No. 1 to 5; (d) One or more Th1 cells, which recognizes an APC that presents a peptide or fragment thereof according to any one of claims 1 to 5 on the surface; and (e) any two or more of (a) to (d) above or More combinations; and this composition is formulated for use in a group selected from: (i) cancer treatment; (ii) cancer prevention; (iii) prevention of recurrence of cancer after surgery; And (iv) a combination of any two or more of (i) to (iii) above.         For example, the pharmaceutical composition under the scope of patent application No. 8 wherein the composition is formulated for use as MHC for having at least one member selected from the group consisting of HLA-DP2, HLA-DP5, HLA-DR4, and HLA-DR9. Targeted administration of Class II molecules.         For example, the pharmaceutical composition of claim 8 or 9, wherein the composition further comprises one or more peptides capable of CTL induction.         A composition for enhancing the immune response of MHC class II molecular mediators, the composition comprising at least one active ingredient selected from the group consisting of: (a) one or more of A peptide according to any one of 5 to 5 items; (b) one or more polynucleotides as claimed in item 6 of the patent application scope; (c) one or more APCs that appear on the surface as shown in claims 1 to 1 of the patent application scope The peptide or fragment thereof according to any one of 5 items; (d) one or more Th1 cells that recognize the APC that presents the peptide or fragment thereof according to any one of claims 1 to 5 on the surface; And (e) a combination of any two or more of (a) to (d) above.         A method for inducing APC, the APC being capable of inducing Th1 cells, the method comprising the step of contacting APC with a peptide of any one of claims 1 to 5 in vitro, in vitro or in vivo.         A method for inducing APC, the APC being capable of inducing CTL, the method comprising the steps selected from the group consisting of: (a) inducing APC in vitro, in vitro or in vivo, and as described in patent application scopes 1 to 5 Contacting a peptide according to any one of the items; and (b) introducing into the APC a polynucleotide encoding the peptide as described in any one of the claims 1 to 5 of the patent application scope.     A method for inducing Th1 cells, comprising the steps selected from the group consisting of: (a) co-culturing CD4 ⁺ T cells, and presenting MHC class II molecules on the surface and as described in claims 1 to 5 of the patent application scope APC of a complex of any one of the peptides or fragments thereof; and (b) introduction of a polynucleotide encoding two TCR subunits, or a polynucleotide encoding each TCR subunit into a CD4 ⁺ T Intracellular, where the TCR is capable of binding to a complex of a MHC class II molecule presented on the cell surface and a peptide or a fragment thereof according to any one of the claims 1 to 5 of the patent application. A method for inducing CTL cells, the method comprising the steps selected from the group consisting of: (a) co-cultivating both CD4 ⁺ T cells and CD8 ⁺ T cells, and having been contacted as claimed in patent application No. 4 or APC with 5 peptides; and (b) co-cultivation of CD8 ⁺ T cells, and APCs that have been exposed to peptides such as those in claims 4 or 5 of the patent application.     A method for enhancing an immune response by a MHC class II molecular medium, the method comprising the step of administering to a subject at least one active ingredient selected from the group consisting of: (a) one or more of The peptide of any one of items 1 to 5; (b) one or more polynucleotides as in the scope of patent application item 6; (c) one or more APCs that appear on the surface as in the scope of patent application scope 1 The peptide or fragment thereof according to any one of 5 to 5; (d) one or more Th1 cells that recognize the APC that presents the peptide or fragment thereof according to any one of claims 1 to 5 on the surface ; And (e) a combination of any two or more of (a) to (d) above.         A detached APC showing on its surface a complex of a MHC class II molecule and a peptide or a fragment thereof according to any one of claims 1 to 5 of the patent application.         An APC is induced by a method such as the one in claim 12 or 13.         A isolated Th1 cell that recognizes an APC that presents a peptide or fragment thereof as described in any one of claims 1 to 5 on the surface.         A Th1 cell is induced by a method as described in item 14 of the patent application.         For example, Th1 cells in the scope of application for patent No. 20, wherein the α and β subunits of the TCR include CDR3 consisting of the amino acid sequences of sequence identification numbers: 9 and 19.         A method of inducing an immune response against cancer in a subject in need thereof, the method comprising the step of administering to the subject a composition comprising at least one active ingredient selected from the group consisting of: (a) a Or one or more of the peptides according to any of claims 1 to 5; (b) one or more of the polynucleotides according to claim 6; (c) one or more APCs on the surface Presenting a peptide or a fragment thereof as in any of claims 1 to 5 in the scope of patent application; (d) One or more Th1 cells, which are identified on the surface as present in any of claims 1 to 5 in the scope of patent application APC of a peptide or a fragment thereof; and (e) a combination of any two or more of (a) to (d) above.         An antibody or an immunologically active fragment thereof is directed against a peptide such as any one of claims 1 to 5 of the scope of patent application.         A vector comprising a nucleotide sequence encoded as a peptide according to any one of claims 1 to 5.         A host cell that has been transformed or transfected with a performance vector such as that described in claim 24.         A diagnostic kit comprises a peptide as claimed in any one of claims 1 to 5, a polynucleotide as claimed in claim 6 or an antibody as described in claim 23.         A method for assessing and / or monitoring a Th1 response specific to a peptide in a body, the peptide comprising an amino acid sequence having a sequence identification number: 1 or 2, the method comprising: (a) providing A sample of the individual of the peptide, wherein the sample contains T cells; (b) detecting the presence of the T cells in the sample, which T cells appear to bind to MHC class II molecules and the peptide or TCR of the fragment complex; and (c) when the presence of the T cell is detected in (b), it shows the induction of a peptide-specific Th1 response.         For example, the method of claim 27, wherein the TCR includes a specific pair of α and β subunits, wherein each α and β subunit includes a CDR3 consisting of amino acid sequences of sequence identification numbers: 9 and 19, respectively. .