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WO2007013352A1 - Composition médicinale contenant un peptide se liant a une molécule hla-a24, issu d’une protéine apparentée a l’hormone parathyroïdienne - Google Patents

Composition médicinale contenant un peptide se liant a une molécule hla-a24, issu d’une protéine apparentée a l’hormone parathyroïdienne Download PDF

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Publication number
WO2007013352A1
WO2007013352A1 PCT/JP2006/314412 JP2006314412W WO2007013352A1 WO 2007013352 A1 WO2007013352 A1 WO 2007013352A1 JP 2006314412 W JP2006314412 W JP 2006314412W WO 2007013352 A1 WO2007013352 A1 WO 2007013352A1
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Prior art keywords
cancer
cells
peptide
hla
molecule
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PCT/JP2006/314412
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English (en)
Japanese (ja)
Inventor
Kyogo Itoh
Mamoru Harada
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Kurume University
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Publication of WO2007013352A1 publication Critical patent/WO2007013352A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/29—Parathyroid hormone, i.e. parathormone; Parathyroid hormone-related peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • composition containing peptide derived from HLA-A24 molecule-binding parathyroid hormone-related protein
  • the present invention relates to a pharmaceutical composition for treating or preventing cancer, particularly gastric cancer, renal cancer, colon cancer or cervical cancer, comprising a peptide derived from an HLA-A24 molecule-binding parathyroid hormone-related protein. .
  • Surgical excision and chemotherapy are effective treatments for many early stage cancer patients.
  • currently available treatments are not as effective for recurrent or advanced cancers.
  • the prognosis for cancer patients with distant metastases is very urgent, and there is an urgent need to provide new treatments for such patients.
  • One of the methods currently being investigated is specific immunotherapy, and peptide-based vaccines are considered to provide a convenient and attractive method for systemic immunity against cancer.
  • PTH-rP Parathyroid hormone-related protein
  • PTH-rP was so named because it is structurally similar to parathyroid hormone (PTH) (Non-patent Document 1).
  • PTH-rP is considered to be involved in malignant hypercalcemia (Non-patent Document 2).
  • PTH-rP is known to be a molecule that is expressed in 90% of early prostate cancers and is important for the development of bone metastases (Non-patent Documents 3 and 4). Therefore, this molecule is considered as a promising target molecule in immunotherapeutic treatment for cancer patients with bone metastasis (Non-patent Document 3).
  • PTH-rP-derived peptides that can be used in peptide-based immunotherapy for HLA-A24 or HLA-A2 molecule-positive prostate cancer patients (Patent Document 1, Non-Patent Documents 5 and 6). ).
  • Patent Document 1 International Publication No. 2005Z116056 Pamphlet
  • Non-Patent Document 1 Kemp, B.E., Moseley, J. M., Rodda, C. P., et al., Parathyroid hormon e-related protein of malignancy: active synthetic fragments. Science 1987; 238 (4833): 1568-70.
  • Non-Patent Document 2 Guise, TA, Parathyroid hormone-related protein and bone metastas es. Cancer 1997; 80 (8 Suppl): 1572—80.
  • Non-Patent Document 3 Francini, G., Scardino, A., Kosmatopoulos, K., et al., High-affinity H LA- A (*) 02.01 peptides from parathyroid hormone-related protein generate in vitro and in vivo antitumor CTL response without autoimmune side effects. J Immunol 200 2; 169 (9): 4840-9.
  • Non-Patent Document 4 Deftos, LJ, Barken, I., Burton, DW, RM Hoffman, and J. Geller, Direct evidence that PTHrP expression promotes prostate cancer progression in bone. Biochem Biophys Res Commun 2005; 327 (2): 468-72.
  • Non-Patent Document 5 Yao, A., Harada, M., Matsueda, S., et al., Identification of parathyroi d hormone-related protein-derived peptides immunogenic in human histocompatibilit y leukocyte antigen- A24 + prostate cancer patients.Br J Cancer 2004; 91 (2): 287-9 6.
  • Non-Patent Document 6 Yao, A., Harada, M., Matsueda, S., et al., New epitope peptides deriv ed from parathyroid hormone-related protein which have the capacity to induce pros tate cancer-reactive cytotoxic T lymphocytes in HLA— A2 + prostate cancer patients. Prostate 2005; 62 (3): 233—42.
  • Non-Patent Document 7 Hida, N., Maeda, Y., Katagiri, K., et al "A new culture protocol to d etect peptide— specific cytotoxic T lymphocyte precursors in the circulation. Cancar Immunol Immunother 2002; 51: 219- 28.
  • An object of the present invention is to provide a pharmaceutical composition containing a PTH-rP-derived peptide that can be used for peptide-based immunotherapy not only for prostate cancer but also for patients of various cancer types.
  • the present invention relates to a peptide derived from a parathyroid hormone-related protein that can bind to an HLA-A24 molecule and can be recognized by cytotoxic T cells, particularly the amino acid sequence shown in SEQ ID NO: 1 or 2.
  • a pharmaceutical composition for treating or preventing cancer is provided.
  • the present invention also provides a pharmaceutical composition for treating or preventing cancer (excluding prostate cancer), comprising a derivative of a peptide derived from a parathyroid hormone-related protein, wherein the derivative is SEQ ID NO: 1.
  • a drug that consists of an amino acid sequence in which substitution, deletion and Z or addition of 1 or 2 amino acids are introduced in the amino acid sequence shown in 2 and that can bind to the HLA-A24 molecule and is recognized by cytotoxic T cells
  • a composition is provided.
  • the present invention also relates to a cancer (excluding prostate cancer) comprising a vector capable of binding to an HLA-A24 molecule and expressing a peptide derived from a parathyroid hormone-related protein that can be recognized by cytotoxic T cells.
  • a pharmaceutical composition for treating or preventing is provided.
  • the present invention also relates to a peptide derived from a parathyroid hormone-related protein that can bind to an HLA-A24 molecule and can be recognized by cytotoxic T cells, particularly a peptide comprising the amino acid sequence shown in SEQ ID NO: 1 or 2.
  • cytotoxic T cells particularly a peptide comprising the amino acid sequence shown in SEQ ID NO: 1 or 2.
  • the present invention for the manufacture of a pharmaceutical composition for treating or preventing cancer (excluding prostate cancer).
  • the present invention administers to a patient a parathyroid hormone-related protein-derived peptide that can bind to an HLA-A24 molecule and can be recognized by cytotoxic T cells, particularly a peptide having the amino acid sequence shown in SEQ ID NO: 1 or 2.
  • a method of treating or preventing cancer except for prostate cancer).
  • the present invention also relates to a peptide derived from a parathyroid hormone-related protein that can bind to an HLA-A24 molecule and can be recognized by cytotoxic T cells, and an HLA-A24 molecule-positive cancer patient (provided that a prostate cancer patient is A method for inducing cancer-reactive cytotoxic T cells, and a kit for carrying out the method, comprising contacting with peripheral blood mononuclear cells collected from (except).
  • the present invention provides a parathyroid hormone-related protein-derived peptide that can bind to an HLA-A24 molecule and can be recognized by cytotoxic T cells, in an HLA-A24 molecule-positive cancer patient (provided that a prostate cancer patient is Preparing antigen-presenting cells capable of inducing cancer-reactive cytotoxic T cells, which are incorporated into cells having the ability to present antigens derived from (excluding) or introducing a vector expressing the peptide. Methods and kits for performing the methods are provided.
  • the invention's effect [0013] According to the present invention, specific immunotherapy of HLA-A24 molecule-positive cancer patients of various cancer types using PTH-rP-derived peptides has become possible. Since the HLA-A24 allele is found in 60% of Japanese, 20% of Kosasasus and 12% of Africans, the present invention is useful for the treatment or prevention of cancer in many cancer patients.
  • FIG. 1 shows the expression of PTH-rP mRNA in various cancer cell lines.
  • A Gastric cancer,
  • B Equine cervical cancer,
  • c Lung cancer,
  • d Breast cancer,
  • e Kidney cancer, and
  • £ Colon cancer cell lines are shown.
  • PC positive control
  • LNCaP prostate cancer cell line
  • NC negative control
  • FIG. 2A shows intracellular distribution of PTH-rP protein in various cancer cell lines.
  • SKG-I cervical cancer
  • QG56 lung cancer
  • R-27 breast cancer
  • MAMIYA renal cancer
  • the part surrounded by the white broken line represents the nucleus of the cell, and the surrounding white part represents the part stained with the anti-PTHrP antibody.
  • PTH-rP protein was found to be distributed in the cytoplasm in the form of dots.
  • FIG. 2B shows the results of examining intracellular PTH-rP protein expression of various cancer cell lines by flow cytometric analysis.
  • MKN-45 stomach cancer
  • SKG-1 cervical cancer
  • Q G56 lung cancer
  • R-27 breast cancer
  • KUR-ll renal cancer
  • DMAMIYA renal cancer
  • CO LO201 colon cancer
  • FIG. 3 shows the induction of cancer-reactive CTL from PBMC derived from cancer patients of various cancer types.
  • black circles indicate PTH-rP positive / HLA-A24 molecule positive target cells
  • white circles indicate PTH-rP positive / HLA-A24 negative target cells.
  • CD8 positive T cells derived from PBMC of cancer patients showed high levels of cytotoxic activity against PTH-rP positive / HLA-A24 molecule positive cancer cells.
  • FIG. 4A shows CD8-positive T cell-dependent cytotoxic activity against PTH-rP-positive / HLA-A24 molecule-positive cancer cells. Cytotoxic activity against PTH-rP positive / HLA-A24 molecule positive cancer cells was significantly suppressed by anti-HLA class I monoclonal antibody (mAb) but not by 1S anti-HLA class II mAb or anti-CD14 mAb .
  • mAb monoclonal antibody
  • FIG. 4B is a graph showing PTH-rP peptide-specific cytotoxic activity against PTH-rP positive / HLA-A24 molecule positive cancer cells.
  • PTH-rP positive / HLA-A24 molecule positive cancer cells Cytotoxic activity against CNS was significantly suppressed by C1R-A24 cells pulsed with the corresponding PTH-rP peptide. It was not suppressed by C1R-A24 cells pulsed with HIV peptide.
  • parathyroid hormone-related protein-derived peptide refers to one of the amino acid sequences of parathyroid hormone-related protein (PTH-rP).
  • PTH-rP parathyroid hormone-related protein
  • the PTH-rP-derived peptide used in the present invention is a peptide that can bind to the HLA-A24 molecule and be recognized by cytotoxic T cells.
  • HLA-A24 molecule means that the PTH-rP-derived peptide can form a complex with the HLA-A24 molecule and be presented on the cell surface.
  • amino acid sequence of a peptide that binds to an HLA molecule has regularity that depends on the type of HLA, and the amino acid sequence that conforms to the regularity is called a binding motif.
  • the binding motif for the HLA-A24 molecule refers to a sequence in which the second amino acid from the N-terminal is tyrosine or ferrolanine and the C-terminal amino acid is isoleucine, leucine, or ferrolanine.
  • Binding of a peptide having an HLA-A24 molecule-binding motif to an HLA-A24 molecule can be determined by computer analysis such as Bioinformatics and Molecular Analysis Section (NIH, Bethesda, MD) (Parker KC, et al., J Immunol, 152: 163-175, 1994).
  • cytotoxic T cells means that a PTH-rP-derived peptide has the ability to induce peptide-specific CTL.
  • Peptide-specific CTL inducing ability is, for example, by stimulating peripheral blood mononuclear cells (PBMCs) with PTH-rP-derived peptides, and the peptide-stimulated PBMCs react with antigen-presenting cells pulsed with the corresponding peptides. For example, whether or not IFN- ⁇ ) is produced can be measured by ELISA or the like.
  • the cytotoxic activity of the induced CTL can be confirmed by a 51 Cr release assay or the like.
  • the number of amino acid residues of PTH-rP-derived peptide is preferably within the range of 8 to 14, more preferably 8 to: L 1 and particularly preferably 9 or 10 It is.
  • the PTH-rP-derived peptide particularly preferably used in the present invention is SEQ ID NO: 1 (RYLTQETN KV, PTH-rP 102-111 peptide) and SEQ ID NO: 2 (KVETYKEQPL, PTH-rP 110-1 19 peptide).
  • a derivative of a PTH-rP-derived peptide refers to an amino acid sequence into which 1 or 2 amino acid substitutions, deletions, and Z or additions have been introduced in the amino acid sequence of the PTH-rP-derived peptide.
  • Substitution of amino acids does not change the properties of the peptide from the viewpoint of homologous amino acids (polar amino acids, nonpolar amino acids, hydrophobic amino acids, hydrophilic amino acids, positively charged amino acids, negatively charged amino acids and aromatics). Preferably between amino acids and the like). It is preferable that the amino acid deletion and addition be performed so that the number of amino acid residues of the derivative is 8: L1.
  • amino acid substitutions, deletions, and Zs or additions are preferably allowed on the HLA molecule-binding motif. That is, amino acid substitutions, deletions, and Zs or additions are made when the second amino acid from the N-terminus of the derivative's amino acid sequence is tyrosine or ferrolanine, and the C-terminal amino acid is isoleucine, leucine, or ferrolanine. It is preferable to do so.
  • Derivatives particularly suitable for the present invention include the substitution of the second amino acid from the N-terminal of the amino acid sequence of SEQ ID NO: 1 with ferrolanine and / or the C-terminal amino acid with isoleucine, oral lysine, or phenol.
  • a derivative consisting of an amino acid sequence substituted with luranin, and the second amino acid from the N-terminus of the amino acid sequence of SEQ ID NO: 2 is substituted with tyrosine or phenylalanine, and / or the C-terminal amino acid with isoleucine or phenolanine It is a substituted amino acid sequence ability derivative.
  • the amino acids constituting the PTH-rP-derived peptide and derivatives thereof may be natural amino acids or amino acid analogs, and amino acid analogs include N-acyl derivatives, 0-acylated products, esterified products of amino acids, Examples include acid amidated products and alkylated products.
  • PTH-rP-derived peptides and derivatives thereof may be modified in their constituent amino acids or carboxyl groups as long as the functions are not significantly impaired. Modifications include binding of formyl group, acetyl group, t-butoxycarbonyl group, etc.
  • Examples include those in which a methyl group, an ethyl group, a t-butyl group, a benzyl group or the like is bonded to the C-terminus or a free carboxyl group.
  • PTH-rP-derived peptides and derivatives thereof can be produced by ordinary peptide synthesis. Examples of such methods include Peptide Synthesis, Interscience, New York, 1966; The Proteins, Vol2, Academic Press Inc., New York, 1976; Peptide Synthesis, Maruzen Co., 1975; Maruzen Co., Ltd., 1985; Development of pharmaceuticals, 14th 'Peptide synthesis, Hirokawa Shoten, 1991).
  • the present invention uses a polypeptide comprising an amino acid sequence of a PTH-rP-derived peptide or derivative thereof, which can be fragmented in a cell to provide the peptide or derivative as a complex with an HLA molecule.
  • a polypeptide comprising an amino acid sequence of a PTH-rP-derived peptide or derivative thereof, which can be fragmented in a cell to provide the peptide or derivative as a complex with an HLA molecule.
  • the number of amino acid residues and the amino acid sequence of the polypeptide are arbitrary.
  • the pharmaceutical composition of the present invention may contain one type of PTH-rP-derived peptide or derivative thereof, or may contain two or more types of PTH-rP-derived peptide or derivative thereof. Also, the PTH-rP-derived peptides and Z or derivatives disclosed herein may be included in combination with other cancer antigen peptides. Since CTL of cancer patients is a collection of cells that recognize different cancer antigen peptides, it is more effective to use a combination of multiple types of cancer antigen peptides or derivatives thereof.
  • the pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier in addition to the PTH-rP-derived peptide or derivative thereof.
  • a pharmaceutically acceptable carrier cellulose, polymerized amino acid, albumin and the like can be used.
  • the pharmaceutical composition of the present invention may be a ribosome preparation, a particulate preparation bound to beads having a diameter of several meters, or a preparation bound to a lipid. It can also be administered together with adjuvants known to be used for conventional vaccine administration so that an immune response is effectively established.
  • the administration method is, for example, intradermal administration or subcutaneous administration.
  • the pharmaceutical composition of the present invention can be used as a cancer vaccine.
  • the dose can be adjusted as appropriate depending on the disease state, the age, weight, etc. of the individual patient.
  • the amount of peptide in the pharmaceutical composition is 0. OOOlmg to: L000mg, preferably 0. OOOlmg to: L00mg. More preferred Or OOOlmg ⁇ : LOmg, 0.01 ⁇ : LOmg, 0.1 ⁇ 5mg or 0.5 ⁇ 3mg. It is preferable to administer this once every few days, weeks or months for 1 to 3 years.
  • the pharmaceutical composition of the present invention can efficiently proliferate CTLs that specifically damage PTH-rP-expressing cancer cells, thereby treating or preventing cancer.
  • the cancer is selected from the group consisting of stomach cancer, renal cancer, colon cancer, cervical cancer, lung cancer and breast cancer, in particular, the group consisting of stomach cancer, kidney cancer, colon cancer and cervical cancer. It is suitable for the case.
  • PTH-rP is a factor that binds to an osteoblast receptor and stimulates bone formation and resorption.
  • PTH-rP produced by cancer cells is thought to be involved in hypercalcemia associated with cancer and metastatic bone lesions. Therefore, the pharmaceutical composition of the present invention that can proliferate CTLs that specifically damage PTH-rP-expressing cancer cells is particularly useful for cancers involving bone metastases.
  • the pharmaceutical composition of the present invention promotes injury of PTH-rP-expressing cancer cells and is therefore effective in preventing bone metastasis.
  • the pharmaceutical composition of the present invention can also be used for treating or preventing metastatic bone lesions of cancer.
  • metastatic bone lesion is meant a destructive or proliferative change that occurs in bone due to the metastasis of cancer cells to the bone.
  • the pharmaceutical composition of the present invention promotes the injury of PTH-rP-expressing cancer cells, thereby preventing metastasis of cancer cells to bone and suppressing the formation and progression of metastatic bone lesions.
  • the present invention also provides a pharmaceutical composition for treating or preventing cancer, comprising a vector that expresses a PTH-rP-derived peptide or derivative thereof that can bind to an HLA-A24 molecule and be recognized by cytotoxic T cells. Offer things. When the vector is expressed in antigen-presenting cells, a PTH-rP-derived peptide or derivative capable of inducing specific CTLs restricted to HLA-A24 forms a complex with the HLA-A24 molecule and is presented on the cell surface. This antigen-presenting cell can efficiently proliferate CTL that damages PTH-rP-expressing cancer cells.
  • the pharmaceutical composition can also be used to treat or prevent metastatic bone lesions of cancer.
  • the pharmaceutical composition is suitably used for a cancer selected from the group consisting of stomach cancer, renal cancer, colon cancer, cervical cancer, lung cancer and breast cancer, particularly from the group consisting of stomach cancer, kidney cancer, colon cancer and cervical cancer power.
  • the expressed PTH-rP-derived peptide preferably has the amino acid sequence shown in SEQ ID NO: 1 or 2.
  • Examples of vectors include various plasmids and viral vectors such as adenovirus, adeno-associated virus, retrovirus, vaccinia virus, etc. (Liu M, Acres B, Balloul JM, Bizouarne N, Paul b, bios P , Squiban P. ene—based vaccines and immotherapeutics. Proc Natl Acad Sci USA 101 Suppl, 14567-71, 2004). Methods for preparing vectors are well known in the art (Molecular Cloning: A laboraroy manual, 2nd dn. New York, Cold Spring Harbor Laboratory).
  • the vector-containing pharmaceutical composition of the present invention can be administered to a patient in order to express a PTH-rP-derived peptide or a derivative thereof in antigen-presenting cells in the patient.
  • the peptide or derivative may be expressed in, for example, a patient-derived rod-shaped cell outside the patient's body using the pharmaceutical composition, and the cell may be returned to the patient.
  • the dosage of the vector-containing pharmaceutical composition of the present invention is 0.1 ⁇ g to 100 mg, preferably 1 ⁇ g to 50 mg as the amount of force DNA that varies depending on the disease state, individual patient age, body weight, etc.
  • the Examples of the administration method include intravenous injection, subcutaneous administration, intradermal administration and the like.
  • the CTL inducing method of the present invention provides a CTL that specifically injures HLA-A24 molecule-positive cancer cells that express PTH-rP.
  • cancer reactivity means that it has the property of recognizing a complex of PTH-rP-derived peptide and HLA-A24 molecule on cancer cells and damaging the cells.
  • the cancer is selected from the group consisting of stomach cancer, renal cancer, colon cancer, cervical cancer, lung cancer and breast cancer, particularly the group consisting of stomach cancer, kidney cancer, colon cancer and cervical cancer.
  • Induction of CTLs can be achieved by, for example, inducing PBMCs collected from HLA-A24 molecule positive cancer patients in vitro in the presence of PTH-rP-derived peptides that can bind to HLA-A24 molecules and be recognized by cytotoxic T cells. Perform by culturing. This method is useful for adoptive immunotherapy in which cancer cells are damaged by returning CTL induced in the patient's body from which PBMCs were collected.
  • the CTL induction kit of the present invention is used for carrying out the CTL induction method.
  • the kit of the present invention contains one or more PTH-rP-derived peptides or derivatives thereof that can bind to the HLA-A24 molecule and can be recognized by cytotoxic T cells, and further contains an appropriate buffer or medium. But you can.
  • the antigen-presenting cell preparation method of the present invention provides an antigen-presenting cell for inducing CTL that damages HLA-A24 molecule-positive cancer cells expressing PTH-rP. This method is suitable when the cancer is selected from the group consisting of gastric cancer, renal cancer, colon cancer, cervical cancer, lung cancer and breast cancer, particularly from the group consisting of stomach cancer, renal cancer, colon cancer and cervical cancer.
  • Antigen-presenting cells can be prepared, for example, by combining PLA-rP-derived peptides capable of binding cells with antigen-presenting ability derived from HLA-A24 molecule-positive cancer patients with HLA-A24 molecules and being recognized by cytotoxic T cells. Incubate by binding and presenting the peptide to the cell surface HLA-A24 molecule.
  • a vector capable of expressing such a peptide may be introduced into a cell having antigen-presenting ability derived from an HLA-A24 molecule-positive cancer patient and expressed.
  • the PTH-rP derived peptide is preferably composed of the amino acid sequence shown in SEQ ID NO: 1 or 2.
  • Cells with antigen-presenting ability are, for example, rod-shaped cells, and culture plate adherent cells are separated from PBMC collected from patients, and the cells are cultured for about 1 week in the presence of IL-4 and GM-CSF. You can get it from Yuko.
  • the antigen-presenting cells prepared by the method of the present invention can induce CTL that specifically recognizes the complex of the peptide or derivative presented on the cell surface and the HLA molecule. Can promote the induction of CTL that damage HLA-A24 molecule-positive cancer cells that express PTH-rP.
  • the antigen-presenting cell preparation kit of the present invention is used to perform the antigen-presenting cell preparation method.
  • the kit of the present invention contains one or more PTH-rP-derived peptides or derivatives thereof that can bind to the HLA-A24 molecule and can be recognized by cytotoxic T cells, and further contain appropriate buffers and media. You can also
  • the present invention further relates to a PTH-rP-derived peptide or derivative thereof capable of binding to an HLA-A24 molecule and being recognized by cytotoxic T cells, for treating or preventing cancer (excluding prostate cancer).
  • a PTH-rP-derived peptide or derivative thereof capable of binding to an HLA-A24 molecule and being recognized by cytotoxic T cells, for treating or preventing cancer (excluding prostate cancer).
  • Use for the manufacture of a pharmaceutical composition is provided.
  • the present invention also includes administration of a PTH-rP-derived peptide or its derivative capable of binding to HLA-A24 molecule and being recognized by cytotoxic T cells to a patient (excluding prostate cancer).
  • MKN-7, MKN-28 and MKN-45 gastric adenocarcinoma
  • RERF-LC-AI and QG56 moonlight squamous cell carcinoma
  • 11-18, 1-87, LK87 and LC-1 lung adenocarcinoma
  • R-27 and CRL1500 breast cancer
  • KUR-11, RC30-14, Caki-1, MAMIYA and VMRC-RCW renal cell carcinoma
  • COLO201, COLO205, COLO320 and SW480 colon adenocarcinoma
  • MDA-MB-231 (breast cancer); HCT116 (colon adenocarcinoma); SKG-1, SKG-II, OMC-1 and SKG-IIIb (cervical squamous cell carcinoma); SKG-IIIa and OMC-4 (cervical cervix) Adenocarcinoma); and OMC-3 (ovarian cancer) cells were cultured in DMEM containing 10% FCS.
  • KWS gastric adenocarcinoma
  • MCF-7 and YMB-1-E (breast cancer)
  • SW620 colon adenocarcinoma) cells were cultured in 10% FCS-containing EMEM in a 37 ° C, 5% CO humidified environment .
  • RNAzol TM B Tel-Test Inc., Friendswood, TX, USA. Prepare cDNA using Superscript (Trademark) Preamplification System for First Strand cDNA Synthesis (Invitrogen) and use it for PTH-rP 5, -1 ⁇ Ding 1 ⁇ Ding 1 ⁇ Ji 1 ⁇ Ding 0 1 ⁇ ⁇ -3, (Sense, SEQ ID NO: 3) and 5, TGTCCTTGGAAGGTCTCTGCGC-3, (Antisense, SEQ ID NO: 4), ⁇ -actin, 5, -011 ⁇ ⁇ -3, (sense, SEQ ID NO: 5) and 5, -CGT ACATGGCTGGGGTGTTG-3 '(antisense, SEQ ID NO: 6) were used as primers.
  • Taq DNA polymerase is used to perform PCR at 95 ° C for 1 minute, 60 ° C for 1 minute, 72 ° C for 1 minute, 30 cycles using a DNA thermal cycler (iCycler, Bio-Rad Laboratories, Hercules, CA, USA). It was. PCR products were separated by electrophoresis on a 2% agarose gel. RT-PCR for ⁇ -actin mRNA was performed to assess the quality of the RNA used in the analysis.
  • TBS TBS (10 mM Tris, pH 7.4, 100 mM NaCl), fixed with 3.7% formaldehyde treated for 5 minutes at room temperature, washed with TBS, and then 3% BSA was treated for 15 minutes and blocked.
  • mAb mouse anti-PTH-rP monoclonal antibody
  • TBS TBS
  • mAb mouse anti-PTH-rP monoclonal antibody
  • FITC fluorescein isothiocyanate
  • IgG immunoglobulin G
  • PI odoprodigum Counterstained with. 1,4-Diazabicyclo- [2, 2, 2] -octane / glycerol was placed on the stained cells and observed with a confocal laser scanning microscope (Fluoview; Olympus).
  • PTH-rP derived peptides (PTH-rP 102-111: RYLTQ ETNKV (SEQ ID NO: 1) and PTH-rP 110-119: KVETYKEQPL (SEQ ID NO: 2)), influenza virus (Flu) virus with HLA-A24 binding motif Derived peptides (RFYIQMCYEL (SEQ ID NO: 7), EBV derived peptides (TYGPVFMCL (SEQ ID NO: 8)) and HIV derived peptides (both HIV peptides! /, U) (RYLR QQLLGI (SEQ ID NO: 9)) were used. All are dissolved in DMSO at a dose of 10 mg / ml. Solved
  • Non-patent Document 7 PBMC (1 X 10 5 cells / well) was incubated with 10 ⁇ g / ml of each peptide in a culture medium volume of 200 ⁇ 1 in a U-bottom 96-well microphone-mouth culture plate (Nunc, Roskilde, Denmark). .
  • the culture medium is 45% RPMI-1640, 45% AIM-V medium (Gibco B RL), 10% FCS, 100 U / ml interleukin (IL) -2 and 0.1 mM MEM non-essential amino acid solution (Gibco, BRL ).
  • PBMC stimulated in vitro with PTH-rP peptide was added for about 10 days with 100 U / ml IL-2 in a round bottom 96-well plate. Intercultured. Immediately before the cytotoxicity test, CD8 positive T cells were isolated using CD8 Positive Isolation Kit (Dynal, Oslo, Norway). The purified CD8 positive T cells were then tested for cytotoxic activity against tumor cells by a 6 hour 51 Cr release test. In a 96 round-bottomed well plate, 2000 51 Cr-labeled cells were cultured for each well at various effector cell Z target cell ratios.
  • Anti-HLA class I mAb W6 / 32: mouse IgG2a (BioLegend, C amino Santa Fe, San Diego, CA, USA), anti-HLA class II (HLA-DR) mAb (L243 : Mouse IgG2a) (provided by Prof. Taiji Nishimura of Kumamoto University) or anti-CD14 mAb (H14: Mouse IgG2a) (established at Kurume University Immunology) was added at a dose of 20 g / ml.
  • PTH-rP peptide-reactive CTL was confirmed by a cold target cell inhibition test.
  • 51 Cr-labeled target cells (2 X 10 3 cells / (Well) and CTL (4 ⁇ 10 4 cells / well) were cultured with 2 ⁇ 10 4 cold target cells.
  • a cancer cell line combined with a cancer type was used for 51 Cr-labeled target cells.
  • PTH-rP mRNA expression levels in gastric cancer, renal cancer, colon cancer, cervical cancer, lung cancer and breast cancer cell lines were examined by semiquantitative RT-PCR analysis (Fig. 1). Although expression levels differed depending on the cell line, it was found that PTH-rP mRNA was expressed in most cancer cell lines tested. In some cell lines, expression was stronger than LNCaP, a prostate cancer cell line used as a positive control (PC).
  • PC positive control
  • PTH-rP protein Fig. 2A
  • PTH-rP is expressed in SKG-I (cervical squamous cell carcinoma) (Fig. 2A-a), QG56 (pulmonary squamous cell carcinoma) (Fig. 2A-b) and MAMIYA (renal cell carcinoma) (Fig. 2A-d) cells.
  • SKG-I cervical squamous cell carcinoma
  • Fig. 2A-a QG56
  • MAMIYA renal cell carcinoma
  • PTH-rP protein was examined by flow cytometric analysis of intracellular staining (Fig. 2B).
  • the expression of PTH-rP protein is MKN-45 (gastric adenocarcinoma) (Fig. 2B—a), SKG-1 (cervical squamous cell carcinoma) (Fig. 2B—b), QG56 (pulmonary squamous cell carcinoma) (Fig. 2B) — C), and KU R-11 and MAMIYA (renal cell carcinoma) ( Figure 2B—e and f) higher in cells.
  • R-27 (breast cancer)
  • COLO201 colon adenocarcinoma)
  • PTH-rP 102-111 peptide is found in 2 of 5 patients with renal cancer, 2 of 5 patients with gastric cancer, 1 of 4 patients with colorectal cancer, and 4 patients with cervical cancer. Two of them were found to induce peptide-reactive CTL.
  • PTH-rP 110-119 peptide is also present in 2 of 5 patients with renal cancer, 2 of 5 patients with gastric cancer, 2 of 4 patients with colorectal cancer, and 4 of 4 Three of the patients with cervical cancer induced peptide-reactive CTL.
  • the following cell lines were positive for surface expression of the HLA-A24 molecule: KUR-11, MKN-45, COLO320 and SKG-1; —the following cell lines were negative for surface expression of the HLA-A24 molecule. RC30-14, MKN-28, COLO205 and OMC-1 (data not shown).
  • PBMC from 5 patients who produced significant levels of IFN- ⁇ in 2 above (Table 1, Pt # 1 (stomach cancer), Pt # 6 (kidney cancer), Pt # 12 (colon cancer), Pt # 13 (Colon cancer) and Pt # 15 (cervical cancer)) were repeatedly stimulated with the P TH-rP peptide indicated according to the culture protocol described above. And just before the test, these cell forces were also isolated CD8 positive T cells.
  • CD8 positive T cells stimulated with PTH-rP 102-111 peptide or PTH-rP 110-119 peptide are PTH-rP positive / HLA-A24 molecule positive cancer cells (KUR-11, MKN-45, COLO320 and And SKG-I), PTH-rP positive / HLA-A24 negative cancer cells (RC30-14, MKN-28, CO LO205 and OMC-1) and PTH-rP negative / HLA-A24 molecule positive PHA-induced T It showed a higher level of cytotoxic activity than did blast cells (Figure 3).
  • PTH-rP-derived peptides are useful in peptide-based immunotherapy not only for prostate cancer but also for cancer patients of various cancer types. It has been shown.

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Abstract

La présente invention concerne une composition médicinale visant à traiter ou à prévenir les cancers (à l’exception du cancer de la prostate), contenant un peptide capable de se lier à une molécule HLA-A24 et qui est issu d’une protéine apparentée à l’hormone parathyroïdienne reconnue par un lymphocyte T cytotoxique.
PCT/JP2006/314412 2005-07-29 2006-07-20 Composition médicinale contenant un peptide se liant a une molécule hla-a24, issu d’une protéine apparentée a l’hormone parathyroïdienne WO2007013352A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2000080100A (ja) * 1998-06-17 2000-03-21 Japan Tobacco Inc 副甲状腺ホルモン関連タンパクに対するヒトモノクローナル抗体
JP2003021631A (ja) * 2001-05-10 2003-01-24 Chugai Pharmaceut Co Ltd 骨転移抑制剤のスクリーニング方法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2000080100A (ja) * 1998-06-17 2000-03-21 Japan Tobacco Inc 副甲状腺ホルモン関連タンパクに対するヒトモノクローナル抗体
JP2003021631A (ja) * 2001-05-10 2003-01-24 Chugai Pharmaceut Co Ltd 骨転移抑制剤のスクリーニング方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
YAO A. ET AL.: "Identification of parathyroid hormone-related protein-derived peptides immunogenic in human histocompatibility leukocyte antigen-A24+ prostate cancer patients", BR. J. CANCER, vol. 91, no. 2, 2004, pages 287 - 296, XP002990847 *

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