JP2008260689A - Pharmaceutical composition containing peptide derived from HLA-A24 molecule binding parathyroid hormone related protein - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a medicinal composition that enables peptide-based immunotherapy of a cancer patient having a bone metastasis. <P>SOLUTION: The medicinal composition for treating or preventing cancers (excluding prostatic cancer) contains a peptide binding to an HLA-A24 molecule and originating in a parathyroid hormone-related protein or its derivative having functionally the same properties. The medicinal composition induces a PTH-rP peptide-specific and cancer-reactive CTL in various cancer type HLA-A24 molecule positive cancer patients and treats or prevents cancers. The medicinal composition enables peptide-based immunotherapy of many cancer patients, above all of a cancer patient having a bone metastasis. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  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 HLA-A24 molecule-binding parathyroid hormone-related protein.

  Surgical excision and chemotherapy are effective treatments for many early stage cancer patients. However, currently available treatment methods are not very effective for recurrent or advanced stage cancer. The prognosis for cancer patients with distant metastases is very poor, and it is urgent to provide new treatment methods for such patients. One of the methods currently under investigation is specific immunotherapy, and peptide-based vaccines are considered to provide a convenient and attractive method for systemic immunity against cancer.

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). Furthermore, it is known that PTH-rP is a molecule that is expressed in 90% of early prostate cancers and is important for the development of bone metastasis (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). The present inventors have identified PTH-rP-derived peptides that can be used for peptide-based immunotherapy for HLA-A24 or HLA-A2 molecule positive prostate cancer patients (Non-patent Documents 5 and 6).
Kemp, BE, Moseley, JM, Rodda, CP, et al., Parathyroid hormone-related protein of malignancy: active synthetic fragments.Science 1987; 238 (4833): 1568-70. Guise, TA, Parathyroid hormone-related protein and bone metastases.Cancer 1997; 80 (8 Suppl): 1572-80. Francini, G., Scardino, A., Kosmatopoulos, K., et al., High-affinity HLA-A (*) 02.01 peptides from parathyroid hormone-related protein generate in vitro and in vivo antitumor CTL response without autoimmune side effects. J Immunol 2002; 169 (9): 4840-9. 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. Yao, A., Harada, M., Matsueda, S., et al., Identification of parathyroid hormone-related protein-derived peptides immunogenic in human histocompatibility leukocyte antigen-A24 + prostate cancer patients. Br J Cancer 2004; 91 (2) : 287-96. Yao, A., Harada, M., Matsueda, S., et al., New epitope peptides derived from parathyroid hormone-related protein which have the capacity to induce prostate cancer-reactive cytotoxic T lymphocytes in HLA-A2 + prostate cancer patients. Prostate 2005; 62 (3): 233-42.

  An object of the present invention is to provide a pharmaceutical composition that enables peptide-based immunotherapy not only for prostate cancer but also for cancer patients of various cancer types having bone metastasis.

  The present inventors examined whether PTH-rP could be a common target molecule in specific immunotherapy for patients with various cancer types. First, the expression of PTH-rP at various mRNA and protein levels in various tumor cell lines was examined by RT-PCR, flow cytometry and immunocytochemistry. PTH-rP mRNA was expressed in most of gastric cancer, renal cancer, colon cancer, cervical cancer, lung cancer and breast cancer cell lines, and expression at the protein level could be confirmed by flow cytometry and immunocytochemistry. Next, peripheral blood mononuclear cells (PBMCs) of HLA-A24 molecule positive gastric cancer, renal cancer, colon cancer and cervical cancer patients were treated with two types of PTH-rP peptides (PTH-rP102-111 and PTH-rP110-119). Peptides) in vitro to examine whether cancer-reactive cytotoxic T lymphocytes (CTL) can be induced. These peptides were able to induce PTH-rP peptide-specific cancer-reactive CTLs from PBMCs of HLA-A24 molecule positive patients of various cancer types. The present invention has been completed based on the above results.

The present invention provides the following:
(1) A pharmaceutical composition for treating or preventing cancer (excluding prostate cancer), comprising a peptide derived from HLA-A24 molecule-binding parathyroid hormone-related protein or a derivative thereof having functionally equivalent properties;
(2) The pharmaceutical composition according to (1), wherein the cancer is selected from the group consisting of stomach cancer, renal cancer, colon cancer and cervical cancer;
(3) The pharmaceutical composition according to (1) or (2), wherein the cancer is bone metastatic.
(4) The pharmaceutical composition according to any one of (1) to (3), wherein the peptide has the amino acid sequence shown in SEQ ID NO: 1 or 2;
(5) The pharmaceutical composition according to any one of (1) to (4), which is for treating or preventing metastatic bone lesions of cancer;
(6) A medicament for treating or preventing cancer (excluding prostate cancer), comprising a vector expressing a peptide derived from an HLA-A24 molecule-binding parathyroid hormone-related protein or a derivative having functionally equivalent properties. Composition;
(7) HLA-A24 molecule-binding parathyroid hormone-related protein-derived peptide or its derivatives having functionally equivalent properties and peripheral blood samples collected from HLA-A24 molecule-positive cancer patients (excluding prostate cancer patients) A method of inducing cancer reactive cytotoxic T cells comprising contacting with a nuclear cell;
(8) For inducing cancer (but excluding prostate cancer) reactive cytotoxic T cells comprising a peptide derived from HLA-A24 molecule-binding parathyroid hormone-related protein or a derivative thereof having functionally equivalent properties kit;
(9) HLA-A24 molecule-binding parathyroid hormone-related protein-derived peptide or its derivative having functionally equivalent properties is capable of presenting antigens derived from HLA-A24 molecule-positive cancer patients (excluding prostate cancer patients). A method for preparing an antigen-presenting cell capable of inducing a cancer-reactive cytotoxic T cell, the method comprising incorporating the cell into a cell having cancer;
(10) HLA-A24 molecule-binding parathyroid hormone-related protein-derived peptide or a derivative thereof having functionally equivalent properties or a vector expressing the peptide or derivative, but a cancer-reactive cell (excluding prostate cancer) A kit for preparing an antigen-presenting cell capable of inducing a cytotoxic T cell.

  The present invention provides a pharmaceutical composition capable of treating or preventing cancer by inducing PTH-rP peptide-specific cancer-reactive CTL in patients with HLA-A24 molecule-positive cancer of various cancer types. The HLA-A24 allele is found in 60% of Japanese, 20% of Caucasians and 12% of Africans (Imanishi, T., Akazawa, T., Kimura, A., K. Tokunaga, and T. Gojobori, Allele and haplotype frewuencies for HLA and completment loci in various ethnic groups.In HLA1991, Tsuji, K., Aizawa, M., Sasazuki, T. (eds). Oxford: Oxford Scientific Publications 1992; Vol. 1: 1065-220) . The present invention thus enables peptide-based immunotherapy for many cancer patients.

In the present invention, “parathyroid hormone-related protein-derived peptide”, “PTH-rP-derived peptide” or “PTH-rP peptide” comprises a part of parathyroid hormone-related protein (PTH-rP) and is restricted by HLA-A24. It is a peptide that can induce peptide-specific CTLs. Such a peptide can be identified by, for example, the method described in Non-Patent Document 5 described above. To explain, first, a peptide having an HLA-A24 molecule binding motif is synthesized based on the amino acid sequence of PTH-rP, and PBMC collected from an HLA-A24 molecule positive cancer patient is stimulated with the peptide. Then, whether or not the peptide-stimulated PBMC produces a cytokine such as IFN-γ in response to the antigen-presenting cells pulsed with the corresponding peptide is determined by ELISA or the like to determine the CTL inducibility. The cytotoxic activity of the induced CTL can be confirmed by ⁵¹ Cr release assay or the like. The peptide derived from a parathyroid hormone-related protein preferably has 8 to 14 amino acid residues, more preferably 8 to 11, particularly preferably 9 or 10. In the present invention, a peptide having the amino acid sequence shown in SEQ ID NO: 1 or 2 is preferably used.

  In the present invention, a “peptide derivative” refers to a peptide having 1 or 2 substitutions, deletions and / or additions introduced into the amino acid sequence of the peptide. “Having a functionally equivalent property” means having a property capable of inducing a specific CTL in an HLA-A24-restricted manner. Whether or not “having functionally equivalent properties” can be determined according to the above method.

  Amino acid substitutions, deletions and / or additions are preferably allowed on the HLA molecule binding motif. When including substitution or addition by other amino acids, the other amino acids may be natural amino acids or amino acid analogs, and examples of amino acid analogs include N-acylated products, O-acylated products, esterified products, acid amidated products of amino acids, Examples include alkylated products. From the viewpoint of not changing the properties of the peptide, substitution between homologous amino acids (polar amino acids, nonpolar amino acids, hydrophobic amino acids, hydrophilic amino acids, positively charged amino acids, negatively charged amino acids, aromatic amino acids and the like) is preferable.

  The peptides and derivatives in the present invention may be modified in their constituent amino acids or carboxyl groups as long as the functions are not significantly impaired. Modifications include N-terminal and free amino groups bound to formyl, acetyl, t-butoxycarbonyl, etc., and C-terminal and free carboxyl groups to methyl, ethyl, t-butyl, and benzyl groups. And the like.

  The peptides and derivatives in the present invention 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; continued development of pharmaceuticals, Volume 14, Peptide Synthesis, Hirokawa Shoten, 1991).

  The present invention also includes a pharmaceutical composition comprising an amino acid sequence of a peptide derived from PTH-rP or a derivative thereof, and a peptide that can be fragmented in a cell to provide the peptide or derivative as a complex with an HLA molecule. Includes. As long as a PTH-rP-derived peptide or a derivative thereof can be provided, the number of amino acid residues and the amino acid sequence of the peptide are arbitrary.

  The pharmaceutical composition of the present invention may contain one kind of peptide or derivative, or may contain two or more kinds of peptides and / or derivatives in combination. Since CTLs of cancer patients are a collection of cells that recognize different cancer antigen peptides, it is more effective to use a combination of a plurality of types of peptides and / or derivatives. You may combine with cancer antigen peptides other than the peptide used by this invention.

  The pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier in addition to the peptide or derivative. As the carrier, cellulose, polymerized amino acid, albumin and the like can be used. A liposome preparation, a particulate preparation bound to beads having a diameter of several μm, a preparation bound to lipid, and the like may be used. Moreover, it can also administer with the adjuvant conventionally known to be used for vaccine administration so that an immune response may be effective. 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 appropriately adjusted depending on the disease state, the age, weight, etc. of the individual patient, but is usually 0.0001 mg to 1000 mg, preferably 0.0001 mg to 100 mg as the amount of the peptide or derivative in the pharmaceutical composition. More preferably 0.001 mg to 10 mg, 0.01 to 10 mg, 0.1 to 5 mg or 0.5 to 3 mg. It is preferable to administer this once every several 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 to treat or prevent cancer. In the pharmaceutical composition of the present invention, the cancer is selected from the group consisting of stomach cancer, kidney 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. PTH-rP is a factor that binds to osteoblast receptors 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 is particularly useful for cancer with bone metastasis. The pharmaceutical composition of the present invention is also effective in preventing bone metastasis.

  The pharmaceutical composition of the present invention can also be used to treat or prevent metastatic bone lesions of cancer. By “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 an HLA-A24 molecule-binding PTH-rP derived peptide or a derivative thereof having functionally equivalent properties. 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 CTLs that react with 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. . The peptide having the amino acid sequence shown in SEQ ID NO: 1 or 2 is preferable.

  Examples of the vector include various plasmids and viral vectors such as adenovirus, adeno-associated virus, retrovirus, vaccinia virus (Liu M, Acres B, Balloul JM, Bizouarne N, Paul S, Slos P, Squiban P. Gene- based vaccines and immunotherapeutics. Proc Natl Acad Sci USA 101 Suppl, 14567-71, 2004). Vector preparation methods are well known in the art (Molecular Cloning: A laboraroy manual, 2nd edn. New York, Cold Spring Harbor Laboratory).

  The pharmaceutical composition of the present invention comprising the vector may be administered to a patient to express the peptide or derivative in antigen-presenting cells in the patient, or suitable cells outside the body, such as dendritic cells derived from the patient, May be used to express said peptide or derivative. These methods are well known in the art (Hrouda D, Dalgleish AG. Gene therapy for prostate cancer. Gene Ther 3: 845-52, 1996).

  The dose varies depending on the disease state, the age and weight of each individual patient, and the DNA content is 0.1 μg to 100 mg, preferably 1 μg to 50 mg. Examples of the administration method include intravenous injection, subcutaneous administration, intradermal administration and the like.

  The CTL induction method of the present invention provides a CTL that specifically injures HLA-A24 molecule-positive cancer cells that express PTH-rP. In the present invention, “cancer reactivity” means that it has a property of recognizing a complex of a PTH-rP-derived peptide and an HLA-A24 molecule on a cancer cell and damaging the cell. In a preferred embodiment, the cancer is selected from the group consisting of stomach cancer, kidney 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. The CTL induction method of the present invention includes, for example, culturing PBMCs collected from HLA-A24 molecule-positive cancer patients in vitro in the presence of a PTH-rP-derived peptide capable of inducing peptide-specific CTLs restricted to HLA-A24. To do. This method is useful for adoptive immunotherapy in which cancer cells are damaged by returning CTLs induced in the body of a patient from whom PBMCs have been 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 capable of inducing peptide-specific CTLs restricted to HLA-A24, and may further contain an appropriate buffer or medium.

  The antigen-presenting cell preparation method of the present invention provides an antigen-presenting cell for inducing CTL that damages an HLA-A24 molecule-positive cancer cell that expresses PTH-rP. This method is suitable when the cancer is 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. The method for preparing an antigen-presenting cell of the present invention comprises, for example, pulsing a PTH-rP-derived peptide capable of inducing peptide-specific CTL in a HLA-A24-restricted manner into a cell having an antigen-presenting ability derived from an HLA-A24 molecule-positive cancer patient. Do more than you do. Alternatively, a vector capable of expressing such a peptide may be introduced into the cell and expressed. The peptide having the amino acid sequence shown in SEQ ID NO: 1 or 2 is preferable. Cells having antigen-presenting ability are, for example, dendritic cells, and can be prepared by separating cultured plate-adhered cells from PBMC collected from a patient and culturing them in the presence of IL-4 and GM-CSF for about 1 week. it can. Antigen-presenting cells prepared by the method of the present invention can induce CTL that specifically recognizes a complex of a peptide or derivative presented on the cell surface and an HLA molecule, and when administered to a patient Induction of CTLs that damage HLA-A24 molecule-positive cancer cells that express PTH-rP in the body can be promoted.

  The antigen-presenting cell preparation kit of the present invention is used for performing the antigen-presenting cell preparation method. The kit of the present invention contains one or more PTH-rP-derived peptides or derivatives thereof capable of inducing peptide-specific CTLs restricted to HLA-A24, and may further contain an appropriate buffer or medium.

  The present invention further includes cancer (except for prostate cancer), comprising administering to a patient a therapeutically effective amount of an HLA-A24 molecule-binding PTH-rP derived peptide or a derivative thereof having functionally equivalent properties. Provide a method of treating or preventing. The present invention also provides a pharmaceutical composition for treating or preventing cancer (excluding prostate cancer) of an HLA-A24 molecule-binding PTH-rP-derived peptide or a derivative thereof having functionally equivalent properties. Provide use to do.

The present invention will be described in more detail by the following examples, but the present invention is not limited by these examples in any way.
I. Materials and methods Cell lines
MKN-7, MKN-28 and MKN-45 (gastric adenocarcinoma); RERF-LC-AI and QG56 (lung 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); and COLO201, COLO205, COLO320 and SW480 (colon adenocarcinoma) cells, 10% FCS Incubated with RPMI-1640. MDA-MB-231 (breast cancer); HCT116 (colon adenocarcinoma); SKG-I, 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) and SW620 (colon adenocarcinoma) cells were cultured in EMEM containing 10% FCS in a 37 ° C., 5% CO ₂ humidified environment.

RT-PCR
Total RNA was isolated from cancer cell lines using RNAzol ^™ B (Tel-Test Inc., Friendswood, TX, USA). CDNA was prepared using ^{SuperScript TM Preamplification System for First Strand cDNA} Synthesis (Invitrogen), it 5'-TCTTCCTTCACCATCTGATCG-3 for PTH-rP '(sense) and 5'-TGTCCTTGGAAGGTCTCTGC-3' (the antisense) Β-actin was amplified using 5′-CTTCGCGGGCGACGATGC-3 ′ (sense) and 5′-CGTACATGGCTGGGGTGTTG-3 ′ (antisense) as primers. Taq DNA polymerase was used to perform PCR at 95 ° C. for 1 minute, 60 ° C. for 1 minute, 72 ° C. for 1 minute, 30 cycles with a DNA thermal cycler (iCycler, Bio-Rad Laboratories, Hercules, Calif., USA). PCR products were separated by electrophoresis on a 2% agarose gel. RT-PCR for β-actin mRNA was performed to assess the quality of the RNA used in the analysis.

Immunofluorescence microscopy analysis
Cells on 35-mm culture dishes are rinsed with TBS (10 mM Tris, pH 7.4, 100 mM NaCl), fixed with 3.7% formaldehyde for 5 minutes at room temperature, washed with TBS, then 15% BSA with 15% Treated for minutes and blocked. Mouse anti-PTH-rP monoclonal antibody (mAb) (1: 100 dilution, Ab-1; Oncogene Research Products) was treated for 1 hour at room temperature to stain the cells. After washing with TBS, cells were stained with fluorescein isothiocyanate (FITC) -conjugated anti-mouse immunoglobulin G (IgG) (1: 200 dilution; Molecular Probes) and counterstained with iodopropidium (PI). 1,4-Diazabicyclo- [2, 2, 2] -octane / glycerol was placed on the stained cells and observed with a confocal laser scanning microscope (Fluoview; Olympus).

Flow cytometry analysis
To examine PTH-rP protein expression, cells were harvested by trypsinization, resuspended in 10% FCS-RPMI and then fixed in 3% formaldehyde. After fixation, the cells were washed 3 times with PBS and incubated with 2.5 μg / ml rabbit polyclonal anti-PTH-rP antibody (1:75 dilution, H-137; Santa Cruz, CA, USA) for 1 hour at room temperature. Rabbit preimmune serum was used as a control. After washing with PBS, cells were stained with FITC-conjugated anti-rabbit IgG (1: 150 dilution; Molecular Probes) for 1 hour at room temperature. Flow cytometry was performed with an EPICS flow cytometer, and data was analyzed with EXPO32 analysis software (Beckman Coulter).

Preparation of patient-derived PBMC
Written consent was obtained from 18 cancer patients who participated in the study before the start of the study. None of these patients were infected with human immunodeficiency virus (HIV). 20 ml of peripheral blood was collected from each subject, and PBMCs were prepared by Ficoll-Conray density gradient centrifugation. The expression of HLA-A24 molecule on PBMC of cancer patients and healthy people was measured by flow cytometry.

Peptides All peptides have a purity exceeding 90% and were purchased from Biologica Co. (Nagoya). PTH-rP derived peptides (102-111: RYLTQETNKV and 110-119: KVETYKEQPL), influenza (Flu) virus derived peptides (RFYIQMCYEL), EBV derived peptides (TYGPVFMCL) and HIV derived peptides (RYLRQQLLGI) with HLA-A24 binding motif )It was used. All peptides were dissolved in DMSO at a dose of 10 mg / ml

Detection of peptide-reactive CTL in PBMC
Detection of peptide-reactive CTL in PBMC was performed according to a previously reported method (Non-patent Document 7). To explain, PBMC (1 × 10 ⁵ cells / well) were incubated with 10 μg / ml of each peptide in a volume of 200 μl of culture medium in a U-bottom 96-well microculture plate (Nunc, Roskilde, Denmark). The culture medium is 45% RPMI-1640, 45% AIM-V medium (Gibco BRL), 10% FCS, 100 U / ml interleukin (IL) -2 and 0.1 mM MEM non-essential amino acid solution (Gibco, BRL) Configured. Every 3 days, half of the culture medium was removed and replaced with fresh medium containing the corresponding peptide (20 μg / ml). On day 15 of culture, the cultured cells were divided into 4 wells, 2 of which were C1R-A24 cells pulsed with PTH-rP peptide (Dr. M. Takiguchi, Kumamoto University, Japan) and the other 2 wells were HIV The peptide was used against C1R-A24 cells pulsed. After 18 hours of incubation, the supernatant was collected and the level of IFN-γ was measured by ELISA.

Cytotoxicity test To obtain a sufficient number of cells to perform the cytotoxicity test, PBMC stimulated in vitro with PTH-rP peptide were combined with 100 U / ml IL-2 in a 96-well round bottom well plate. The culture was further continued for about 10 days. Immediately before the cytotoxicity test, CD8 positive T cells were isolated using a 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 bottom well plate, 2000 ⁵¹ Cr-labeled cells per well were cultured at various effector / target cell ratios. For antibody inhibition experiments, anti-HLA class I (W6 / 32: mouse IgG2a), anti-HLA class II (HLA-DR) (L243: mouse IgG2a), or anti-CD14 (H14: mouse IgG2a) mAb at the start of the study. Added at a dose of μg / ml.

Cold target cell inhibition test
The specificity of PTH-rP peptide-reactive CTL was confirmed by a cold target cell inhibition test. For example, ⁵¹ Cr-labeled target cells (2 x 10 ³ cells / well) and CTL (4 x 10 ⁴ cells / well) were cultured in 96 round bottom well plates with cold target cells of 2 x 10 ⁴ cells did. C1R-A24 cells pre-pulsed with either HIV peptide or the corresponding PTH-rP peptide were used as cold target cells. ^{As the 51} Cr-labeled target cells, cancer cell lines combined with cancer types were used.

Statistical significance of statistical data was examined by two-sided Student's t test. P values (*) less than 0.05 were considered statistically significant.

II. Result 1. Expression of PTH-rP in various cancer cell lines First, 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 higher than LNCaP, a prostate cancer cell line used as a positive control (PC).
Next, immunocytochemical analysis was performed to confirm the expression of PTH-rP protein (FIG. 2A). PTH-rP is expressed by SKG-I (cervical squamous cell carcinoma) (FIG. 2A-a), QG56 (squamous cell lung carcinoma) (FIG. 2A-b) and MAMIYA (renal cell carcinoma) (FIG. 2A-d) cells. In, it was expressed in punctate throughout the cytoplasm. On the other hand, the expression level was very low in R-27 (breast cancer) cells (FIG. 1-d) in which the expression of PTH-rP mRNA was weak in RT-PCR analysis (FIGS. 2A-c).
Furthermore, the expression of 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-I (cervical squamous cell carcinoma) (Fig. 2B-b), QG56 (pulmonary squamous cell carcinoma) (Fig. 2B). -C), and KUR-11 and MAMIYA (renal cell carcinoma) (FIGS. 2B-e and f) higher in cells. However, R-27 (breast cancer) (FIG. 2B-d) and COLO201 (colon adenocarcinoma) (FIG. 2B-g) cells show PTH-rP expression only at very low levels or PTH-rP expression. Negative.
The results obtained by these protein expression analyzes are consistent with the results obtained by PT-PCR analysis.

PTH-rP102-111 ペプチドは、5人の腎癌患者のうち2人、5人の胃癌患者のうち2人、4人の大腸癌患者のうち1人、および4人の子宮頸癌患者のうち2人において、ペプチド反応性CTLを誘導することがわかった。PTH-rP110-119 ペプチドもまた、5人の腎癌患者のうち2人、5人の胃癌患者のうち2人、4人の大腸癌患者のうち2人、4人の子宮頸癌患者のうち3人において、ペプチド反応性CTLを誘導した。
以上の結果は、これらPTH-rPペプチドが、様々な癌タイプの患者のPBMCからペプチド反応性CTLを誘導できることを示している。 2. Induction of PTH-rP peptide-reactive CTL derived from HLA-A24 molecule-positive patients of various cancer types We have previously obtained peptide-specific prostate cancer-reactive CTL from PBMC of HLA-A24 molecule-positive prostate cancer patients Two types of PTH-rP-derived peptides, PTH-rP102-111 and PTH-rP110-119, that can be induced were identified (Non-patent Document 5). To examine whether these peptides can induce PTH-rP peptide-specific CTLs in patients with various cancer types, the patient's PBMCs were stimulated in vitro with any of the PTH-rP peptides and then the peptide-stimulated PBMCs Were examined to produce IFN-γ in response to C1R-A24 cells pre-pulsed with the corresponding PTH-rP peptide or HIV peptide (Table 1). In this experiment, Flu and EBV derived peptides were used as controls. The results obtained by subtracting the IFN-γ produced in response to the HIV peptide as a background and showing the best response are shown (Table 1). Successful induction of peptide-specific CTL was judged positive when significant values (P <0.05, by two-sided Student's t test) were observed.

PTH-rP102-111 peptide is found in 2 of 5 renal cancer patients, 2 of 5 gastric cancer patients, 1 of 4 colorectal cancer patients, and 4 of cervical cancer patients. Two people were found to induce peptide-reactive CTL. PTH-rP110-119 peptide is also present in 2 out of 5 renal cancer patients, 2 out of 5 gastric cancer patients, 2 out of 4 colorectal cancer patients, out of 4 cervical cancer patients. In three, peptide-reactive CTLs were induced.
The above results indicate that these PTH-rP peptides can induce peptide-reactive CTLs from PBMCs of patients with various cancer types.

3. Cytotoxic activity of cancer patient-derived PTH-rP peptide-reactive CTLs against cancer cells Next, we examined whether PTH-rP peptide-reactive CTLs derived from cancer patients could show cytotoxic activity against cancer cells. . Based on RT-PCR results (Figure 1), the following cancer cell lines were used as target cells for cytotoxic activity studies: KUR-11 and RC30-14 (renal cell carcinoma); MKN-45 and MKN-28 ( Gastric adenocarcinoma); COLO320 and COLO205 (colon adenocarcinoma); SKG-I and OMC-1 (cervical squamous cell carcinoma). The following cell lines were positive for HLA-A24 molecule surface expression: KUR-11, MKN-45, COLO320 and SKG-I; whereas the following cell lines were negative for HLA-A24 molecule surface expression: : RC30-14, MKN-28, COLO205 and OMC-1 (data not shown). PBMCs from 5 patients who produced significant levels of IFN-γ in 2 above (Table 1, Pt # 1 (stomach cancer), Pt # 6 (kidney cancer), Pt # 12 (colon cancer), Pt # 13 (Colorectal cancer) and Pt # 15 (cervical cancer)) were repeatedly stimulated with the PTH-rP peptide indicated according to the culture protocol described above. Then, CD8 positive T cells were isolated from these cells immediately before the test.
PTH-rP102-111 peptide or PTH-rP110-119 peptide-stimulated CD8 positive T cells against PTH-rP positive / HLA-A24 molecule positive cancer cells (KUR-11, MKN-45, COLO320 and SKG-I) Than for PTH-rP positive / HLA-A24 negative cancer cells (RC30-14, MKN-28, COLO205 and OMC-1) and PTH-rP negative / HLA-A24 molecule positive PHA-induced T blasts Showed a high level of cytotoxic activity (FIG. 3).
Cytotoxic activity against PTH-rP positive / HLA-A24 molecule positive cancer cells was significantly suppressed by the addition of anti-HLA class I mAb, but was suppressed by addition of anti-HLA class II mAb or anti-CD14 mAb used as a control Not (FIG. 4A).
Furthermore, the cytotoxic activity against PTH-rP positive / HLA-A24 molecule positive cancer cells was significantly suppressed when C1R-A24 cells pulsed with the corresponding PTH-rP peptide were added as cold target cells. It was not observed by the addition of peptide pulsed C1R-A24 cells (FIG. 4B).
The above results indicate that PBMCs derived from various cancer patients stimulated with these PTH-rP peptides can damage the corresponding cancer cells. Moreover, it is shown that the cytotoxic activity observed in this experiment is mainly due to HLA class I-restricted PTH-rP peptide-specific CD8-positive T cells.
4). Summary From the above, it was shown that PTH-rP102-111 peptide and PTH-rP110-119 peptide are useful in peptide-based immunotherapy for cancer patients of various cancer types.

FIG. 1 is a drawing-substituting photograph showing the expression of PTH-rP mRNA in various cancer cell lines. (a) Gastric cancer, (b) Cervical cancer, (c) Lung cancer, (d) Breast cancer, (e) Renal cancer and (f) Colon cancer cell lines are shown. PC (positive control) is LNCaP (prostate cancer cell line) and NC (negative control) is PBMC derived from a healthy person. It was found that PTH-rP mRNA was expressed in most cancer cell lines. FIG. 2A is a drawing-substituting photograph showing the intracellular distribution of PTH-rP protein in various cancer cell lines. (a) SKG-I (cervical cancer), (b) QG56 (lung cancer), (c) R-27 (breast cancer) and (d) MAMIYA (renal cancer) cells. A portion surrounded by a white broken line represents a cell nucleus, and a surrounding white portion represents a portion stained with an anti-PTHrP antibody. PTH-rP protein was found to be distributed in the cytoplasm in a punctate manner. FIG. 2B is a diagram showing the results of examining intracellular PTH-rP protein expression of various cancer cell lines by flow cytometry analysis. (a) MKN-45 (gastric cancer), (b) SKG-I (cervical cancer), (c) QG56 (lung cancer), (d) R-27 (breast cancer), (e) KUR-11 (renal cancer) , (F) MAMIYA (renal cancer), and (g) COLO201 (colon cancer) cells. FIG. 3 is a graph showing the induction of cancer-reactive CTL from PBMC derived from cancer patients of various cancer types. In the graph, black circles indicate PTH-rP positive / HLA-A24 molecule positive target cells, and white circles indicate PTH-rP positive / HLA-A24 negative target cells. CD8-positive T cells derived from cancer patient PBMC exhibited a high level of cytotoxic activity against PTH-rP-positive / HLA-A24 molecule-positive cancer cells. FIG. 4A is a graph showing 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 inhibited by anti-HLA class I mAb, but not by anti-HLA class II mAb or anti-CD14 mAb. FIG. 4B is a graph showing PTH-rP peptide-specific 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 C1R-A24 cells pulsed with the corresponding PTH-rP peptide, but not by C1R-A24 cells pulsed with the HIV peptide There wasn't.

Claims (10)

  A pharmaceutical composition for treating or preventing cancer (excluding prostate cancer), comprising a peptide derived from an HLA-A24 molecule-binding parathyroid hormone-related protein or a derivative thereof having functionally equivalent properties.   The pharmaceutical composition according to claim 1, wherein the cancer is selected from the group consisting of stomach cancer, renal cancer, colon cancer and cervical cancer.   The pharmaceutical composition according to claim 1 or 2, wherein the cancer is bone metastatic.   The pharmaceutical composition according to any one of claims 1 to 3, wherein the peptide has the amino acid sequence shown in SEQ ID NO: 1 or 2.   The pharmaceutical composition according to any one of claims 1 to 4, which is for treating or preventing metastatic bone lesions of cancer.   A pharmaceutical composition for treating or preventing cancer (excluding prostate cancer), comprising a vector expressing a peptide derived from an HLA-A24 molecule-binding parathyroid hormone-related protein or a derivative thereof having functionally equivalent properties.   Peripheral blood mononuclear cells collected from HLA-A24 molecule-binding parathyroid hormone-related protein-derived peptides or their derivatives having functionally equivalent properties and HLA-A24 molecule-positive cancer patients (excluding prostate cancer patients) A method for inducing cancer-reactive cytotoxic T cells, which comprises contacting the cells.   A kit for inducing cancer (excluding prostate cancer) reactive cytotoxic T cells comprising a peptide derived from an HLA-A24 molecule-binding parathyroid hormone-related protein or a derivative thereof having functionally equivalent properties.   HLA-A24 molecule-binding parathyroid hormone-related protein-derived peptide or its functionally equivalent derivative is applied to cells with antigen-presenting ability derived from HLA-A24 molecule-positive cancer patients (excluding prostate cancer patients) A method for preparing an antigen-presenting cell capable of inducing a cancer-reactive cytotoxic T cell, comprising incorporating. HLA-A24 molecule-binding parathyroid hormone-related protein-derived peptide or a derivative thereof having functionally equivalent properties or a vector expressing the peptide or derivative reactive cancer (except for prostate cancer) reactive cytotoxicity T A kit for preparing an antigen-presenting cell capable of inducing a cell.

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