JP2002356498A - Antigen peptide for synovial sarcoma - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a peptide capable of inducing cytotoxic T lymphocytes(CTL) targeting synoviosarcoma. SOLUTION: Items described in (1)-(6) are claimed in this invention; (1) a peptide expressed by the amino acid sequence expressed below: Pro Tyr Gly Tyr Asp Gln Ile Met Pro Lys or Gly Tyr Asp Gln Ile Met Pro Lys Lys, (2) a cancer vaccine containing the aforesaid peptide for synoviosarcoma, (3) an anticancer agent for synoviosarcoma containing the aforesaid peptide, (4) an application of the aforesaid peptide for inducing CLT targeting synoviosarcoma cells, (5) the CTL induced by the aforesaid peptide and (6) an anticancer agent for synoviosarcoma containing the CLT described in (5).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a peptide capable of inducing cytotoxic T cells (hereinafter referred to as CTLs) targeting synovial sarcoma cells. The present invention also relates to a cancer vaccine for synovial sarcoma and an anticancer agent for synovial sarcoma containing the peptide. Furthermore, the present invention relates to the use of said peptide for inducing CTL targeting synovial sarcoma cells, the obtained CTL and anticancer agents comprising said CTL.

[0002]

2. Description of the Related Art Synovial sarcoma is the second most common soft tissue sarcoma after malignant fibrous histiocytoma and liposarcoma in adults. Conventional treatment of synovial sarcoma is mainly surgical treatment, and unresectable metastatic lesions have been treated with chemotherapy or radiation treatment with anticancer drugs. However, even with these treatment methods, the 5-year survival rate of synovial sarcoma is 30% to 60%, and the prognosis is poor. In recent years, immunotherapy using a cancer antigen peptide capable of inducing CTL has been attempted in other malignant sarcomas, particularly in malignant melanoma, and its efficacy has been reported (Nestle, FO. Et al., Nat. Med., 1998,
4: 328-332 and Rosenberg, SA. Et al., Nat.Med., 1998,
4: 321-327). Immunotherapy using cancer antigen peptides is being established as one of the treatment methods for patients with advanced malignant melanoma.

[0003]

However, no cancer antigen peptide has been identified in synovial sarcoma, and immunotherapy using these has not been established. Therefore, an object of the present invention is to provide a cancer vaccine for synovial sarcoma and an anticancer agent for synovial sarcoma which can be used for immunotherapy of synovial sarcoma.

[0004]

Means for Solving the Problems The present inventors have noticed that immunological rejection of human cancer is mainly carried out by CTL, particularly CD8 (+) CTL. CD8 (+) CTL
Is the major histocompatibility complex on cancer cells (HL in humans)
Recognizes and activates the complex consisting of A) and the cancer antigen peptide presented on the HLA. Then, the activated CTL recognizes and attacks the cancer cell via the T cell antigen receptor on the cell surface. Therefore, if a synovial sarcoma antigen peptide is identified, it is used as a cancer vaccine for synovial sarcoma and an anticancer agent for synovial sarcoma,
Can be efficiently induced to prevent and treat synovial sarcoma. Synovial sarcomas are known to have specific chromosomal translocations. In the chromosomal translocation of synovial sarcoma, a translocation gene SYT-SSX, in which the SYT gene on chromosome 18 and the SSX gene on X chromosome are fused, is formed. This translocation gene SYT-SSX is composed of SYT protein (3
(87 amino acids) encodes a chimeric protein (457 amino acids) in which the C-terminal 8 amino acids of the SSX protein (188 amino acids) are substituted with the C-terminal 78 amino acids (Clark, J., Nature Genet., 1994, 7: 502-
508 and Crew, AJ., EMBO J., 1995, 14: 2333-234
0). The SYT gene is expressed in normal tissues, while the SSX gene is expressed in some malignant tumors (Tureci, O., Int. J. Cancer,
1998, 77: 19-23, dos Santos, NR., Cancer Res., 200
0, 60: 1654-1662 and de Leeuw, D., Hum. Mol. Gene
t., 1995, 4: 1097-1099) and only in testis. On the other hand, the translocation gene SYT-SSX is not expressed in normal cells even in the same synovial sarcoma patient, but 90% of synovial sarcomas
It is known that these are specifically expressed (Sandber
g, AA. and Bridge, JA., The Cytogenetics of Bone a
nd Soft Tissue Tumors, RG Landes Co .: Austin Texa
s). Therefore, it is considered that the polypeptide chain encoded by the gene on the SSX side from the translocation site of the translocation gene SYT-SSX has an amino acid sequence specific to synovial sarcoma.
Therefore, the present inventors conducted intensive studies on the possibility of various SYT-SSX gene products as antigens for immunotherapy which are common to many synovial sarcoma patients and in which autoimmunity against normal tissues is unlikely to occur. Have found that certain peptides can induce synovial sarcoma-specific CTL. The present invention has been made based on this finding. That is, the present invention provides: (1) a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or 2, and capable of inducing cytotoxic T cells targeting synovial sarcoma cells; (2) the above-mentioned (1) (3) an anticancer agent for synovial sarcoma comprising the peptide of (1); (4) for inducing cytotoxic T cells targeting synovial sarcoma cells (5) a cytotoxic T cell induced by the peptide according to (1), and (6) a cytotoxic T cell according to (5). An anticancer agent for synovial sarcoma;

[0005]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail.
The peptide capable of inducing a cytotoxic T cell targeting a cancer cell according to the present invention has the following sequence: Pro Tyr Gly Tyr Asp Gln Ile Met Pro Lys (SEQ ID NO: 1) or Gly Tyr Asp Gln Ile Met Pro Lys Lys (SEQ ID NO: 2).

The identification of the peptide of the present invention comprises the following steps: (1) Human major histocompatibility complex (MHC class I)
Providing a peptide derived from the SYT-SSX gene product having a sequence corresponding to the binding motif of HLA-A24, (2) adding the peptide to an antigen-presenting cell expressing HLA-A24; Obtaining an antigen presenting cell presenting the peptide by
(3) a step of stimulating T cells with the antigen-presenting cells to induce CTLs; and (4) a step of measuring the synovial sarcoma cytotoxicity of the induced CTLs.

The peptide of the present invention has 9 or 1 amino acid.
Since it is as small as 0, it can be synthesized by a general amino acid chemical synthesis method, for example, Fmoc method. It can also be synthesized using a commercially available amino acid synthesizer. The DNA encoding the amino acid sequence can be synthesized in a cell by incorporating it into an appropriate expression vector, for example, pcDNA3.1 (Invitrogen. Co.), and transfecting it into an appropriate cell, for example, DH5α (Gibco-BRL). it can.

[0008] The peptides of the present invention can be used to induce CTL targeting synovial sarcoma cells. The induced CTL recognizes and attacks the synovial sarcoma cells.
Therefore, the peptide of the present invention can be used as a cancer vaccine for synovial sarcoma and an anticancer agent for synovial sarcoma. A cancer to which the cancer vaccine for synovial sarcoma and the anticancer agent for synovial sarcoma of the present invention can be applied is a synovial sarcoma composed of synovial sarcoma cells presenting the peptide of the present invention by HLA-A24.
When the peptide of the present invention is used as a cancer vaccine and an anticancer agent, the peptide of the present invention can be used by itself or together with adjuvants, and can optionally contain a pharmaceutically acceptable carrier. Adjuvants include adjuvants for enhancing the immune response, such as incomplete (complete) adjuvants of Floyd, aluminum adjuvants and the like. Pharmaceutically acceptable carriers include, for example, diluents such as PBS and distilled water, and physiological saline. The cancer vaccine and anticancer agent of the present invention can be prepared by a method well-known in the art, such as a solution, an oil, an emulsion, a soft capsule, a hard capsule, a tablet,
It can be in the form of granules, solids and the like. The cancer vaccine and the anticancer agent of the present invention can be administered orally,
It can be administered parenterally or transdermally. For example, intravenous administration and intramuscular administration are mentioned. Dosages will usually vary depending on the weight of the patient, the nature and condition of the disease, but for use in adults may range up to 5-10 mg per day.
For example, when used by subcutaneous injection in adult cancer patients,
The amount is 100 to 1000 μg, preferably 100 to 200 μg per week.

[0009] The peptides of the present invention can also be used to induce CTLs targeting synovial sarcoma cells. Induction is performed, for example, according to the literature (Nabeta, Y. et al., Jpn. J. Ca
ncerRes. 2000. 91: 616-621). Specifically, the following steps: HLA-A
Providing a cell expressing H.24, adding the peptide of the present invention to the cell and presenting it on HLA-A24, stimulating T cells with cells presenting the peptide by HLA-A24 And inducing the T cells into CTLs for synovial sarcoma cells. Cells expressing HLA-A24 may be collected from a patient with synovial sarcoma, or may be prepared by introducing a gene encoding HLA-A24 into non-HLA-A24 expressing cells.

[0010] Since the obtained CTLs target synovial sarcoma cells, they can be used as anticancer agents. In this case, similarly to the above-mentioned anticancer agent containing the peptide of the present invention, it may contain a pharmaceutically acceptable carrier as appropriate and take various forms. The anti-cancer agent containing the CTL of the present invention can be parenterally administered in the same manner as the cancer vaccine and the anti-cancer agent containing the peptide of the present invention. The dosage usually varies depending on the weight of the patient, the nature and condition of the disease, and when used by subcutaneous injection in adult synovial sarcoma patients, is 100 to 1000 μg per week, preferably 100 to 1000 μg.
200 μg.

EXAMPLES Next, the effects of the present invention will be described specifically with reference to examples, but the present invention is not limited to the examples.

Example 1 Synovial sarcoma antigen peptide of the present invention
Production following amino acid sequence of: Pro Tyr Gly Tyr Asp Gln Ile Met Pro Lys ( SEQ ID NO: 1) or were synthesized two peptides having Gly Tyr Asp Gln Ile Met Pro Lys Lys ( SEQ ID NO: 2). The peptide is 9-
Solid-phase simultaneous multiple peptide synthesizer PSSM-8 based on fluorenylmethyloxycarbonyl (Fmoc) strategy
(Shimadzu) and then C18 reversed-phase high-performance liquid chromatography (HPLC) (Millipore)
And purified. Peptide purity and identity were determined by analytical HPLC and mass spectrometry, respectively. The peptide was dissolved in dimethyl sulfoxide at a concentration of 1.5 mg / ml.
And stored at -80 ° C.

Example 2 Synovial sarcoma antigen peptide of the present invention
In vitro induction of CTL using E. coli Peripheral blood mononuclear cells (PBMCs) were collected by centrifugation in a Ficoll-Conlay density gradient and then separated into adherent and non-adherent cells. AIM-V for adherent cells
(Gibco Co.), 100 ng / ml GM-CSF (N
ovartis Pharmaceuticals) and 10 IU / ml IL
-2 (Gibco-BRL). This cell was used as an antigen presenting cell (APC). Non-adherent cells were treated with 30-100 IU / ml of recombinant I in AIM-V.
Incubated with L-2 (Takeda Pharmaceutical). 7
On the 10th day, the peptide of the present invention (final concentration 30 μg / m
l) was added to APC, and one day later, APC was matured by adding recombinant TNF-α and IFN-α (Sumitomo Pharmaceutical). The APC was then irradiated with radiation. One day later, APC
And autologous non-adherent cells, AIM-V containing no IL-2
Mixed in. Two days after incubation, IL-2 (Takeda Pharmaceutical) was added to the culture at a final concentration of 100 IU / ml. Every 7 days, according to the same protocol as above, A
Responder cells were stimulated with self-adherent cells as PC. For each stimulation, the culture was supplemented with fresh medium containing 100 IU / ml IL-2. Day 28 CTL was used in the following activity tests.

[0013] was evaluated by ⁵¹ Cr cytotoxicity test cytotoxic potential of cytotoxic (1) Test method CTL of CTL in Example 3 the present invention. CTL target cells were labeled with 100 μCi of sodium chromate ( ⁵¹ Cr) at 37 ° C. for 2 hours, washed and resuspended. The effector cells (CTL) were transferred to a V-bottom microtiter plate Costar 3894 (Co
rning Incorporated), and the labeled target cells were added thereto at a concentration of 5 × 10 ³ cells / well to a volume of 0.2 ml. After 4 hours of incubation, 0.
Collect 1 ml of supernatant and use an automated gamma counter (LKB Wa
release was measured ⁵¹ Cr-by Llac). The measurement was performed in triplicate and the standard deviation was calculated. The percentage of specific cytotoxicity is calculated by dividing the percentage of specific ⁵¹ Cr release by the following formula: [(experimental value) − (spontaneous release value) / (maximum release value) − (spontaneous release value)] × 100. It was determined by calculation. Spontaneous release values were obtained from release values when target cells were incubated alone in the absence of effector cells. The maximum release value is 1% Nonidet, a surfactant.
-P40 (Nacalai Chemical Co.) to obtain the maximum release when incubated.

(2) Literature on cytotoxicity of CTL of the present invention (de Leeuw, B. et al., Hum. Mol. Genet., 2000, 4:10)
97-1099), four peptides corresponding to the binding motif of HLA-A24 shown in Table 1 according to the amino acid primary sequence of the SYT-SSX gene product described in Example 1 were obtained according to the method described in Example 1 ( (See FIG. 1).

Table 1. Test peptide

For convenience of the test, the four peptides were divided into a group X (peptide A and peptide B) containing the peptide of the present invention and a group Y (peptide C and peptide D) not containing the peptide of the present invention. Was. CTL was induced using the peptides of each group in the same manner as in Example 2. As CTL target cells, synovial sarcoma cells (HLA-A24 positive and SYT-SSX positive) collected from one synovial sarcoma patient and leukemia cell line K562 (American type cultur)
from the e collection) (HLA-A24 negative and SY
T-SSX negative) was used. According to the test method (1), the cytotoxicity of CTL was tested. FIGS. 2 and 3 show the results of ⁵¹ Cr cytotoxicity tests on CTLs induced using peptides in groups X and Y, respectively. The vertical axis shows the percentage of lysis of the target cells, and the horizontal axis shows the E / T ratio (effector cells (CTL) / target cells). CTLs induced by group X peptides showed high cytolysis on synovial sarcoma cells (O), but SYT-SSX and HLA
Low cell lysis was shown for leukemia cells not expressing -A24 (●) (FIG. 2). On the other hand, the CTLs induced by the Y group peptide showed low cytolyticity in all cells (FIG. 3). From these results, it was found that the CTL induced by the group X peptide containing the peptide of the present invention was H
It is understood that LA-A24-positive and SYT-SSX-positive cells have high cytotoxicity against synovial sarcoma cells.

Example 4 Presence of CTL specific for synovial sarcoma
Measurement of Frequency In order to examine the possibility that the peptide of the present invention can induce CTL even in synovial sarcoma patients other than those of Example 3, the presence frequency of CTL recognizing the peptide of the present invention in peripheral blood was determined by using multiple Measured in synovial sarcoma patients. The measurement was performed using an HLA tetramer. The HLA tetramer refers to a tetramer of an HLA molecule to which a synovial sarcoma antigen peptide is bound, and can specifically bind to CTL targeting synovial sarcoma. In this example, a fluorescent dye was bound to this HLA tetramer, and CTL bound to the HLA tetramer was detected by a flow cytometer. Literature (Altman, J. et al., Sc
ience, 1996, 274: 94-96), A peptide (invention, SEQ ID NO: 1), B peptide (invention, SEQ ID NO: 2) and C + D used in Example 3 as peptides to be bound were used. Three kinds of HLA tetramers were prepared using the peptides. Phycoerythrin (PE) was conjugated to the HLA tetramer as a fluorescent label according to the method described in the literature (supra, Altman, J. et al.). HLA-A2
Synovial sarcoma patients expressing HLA-4 and HLA-A2
Peripheral blood was collected from 9 healthy subjects expressing 4 and the frequency of peptide-specific T cells in each peripheral blood lymphocyte was measured. Specifically, the procedure was as follows. HLA-2
Peripheral blood of 4-positive synovial sarcoma patients and healthy subjects is centrifuged in a Ficoll-Conlay density gradient to obtain peripheral blood mononuclear cells (PB).
MC) were collected and incubated in AIM-V (Gibco Co.). After 3 to 5 days, the PBMCs were collected, washed with PBS, and the PE-labeled tetramer prepared by the above-described method was added.
The reaction was performed at 7 ° C. for 1 hour. After washing with PBS, the plate was reacted with an anti-CD8 antibody fluorescently labeled with FITC at 4 ° C. for 30 minutes. After washing with PBS, the flow cytometer (FACSCa
libur, Becton Dickinson). In the flow cytometer, the lymphocyte fraction is gated and FITC
Positive and PE-positive cells were defined as peptide-specific T cells bound to the tetramer. The frequency (percentage) of peptide-specific T cells in all lymphocytes was calculated with a flow cytometer. Table 2 shows the results.

Table 2. HLA tetramer binding cytotoxic T
Frequency of cells (%) In three out of six synovial sarcoma patients (patients 1, 3 and 4), the frequency of CTLs recognizing the HLA tetramer to which the A peptide of the present invention was bound was found to be higher in healthy subjects.
Was significantly increased as compared to the frequency of presence of B of the present invention
The peptide showed similar results. This indicates that CTLs were already induced in the patient's immune system by the SYT-SSX translocation gene product specifically expressed by synovial sarcoma cells,
This indicates that the CTL recognizes the A peptide or the B peptide. On the other hand, the frequency of CTLs recognizing the HLA tetramer to which the C + D peptide is bound is as follows:
Even in synovial sarcoma patients, the frequency of CTLs recognizing the A or B peptide-bound tetramer was lower than that of CTLs. This indicates that CTLs induced by the SYT-SSX translocation gene product do not recognize the C + D peptide,
The + D peptide indicates that it cannot induce CTL. Based on the above results, the peptide (A or B peptide) of the present invention was expressed by S-specifically expressed in synovial sarcoma cells.
It is understood that it is possible to recognize a part of the YT-SSX translocation gene product, ie, to induce CTL targeting synovial sarcoma cells.

[0019]

The peptide of the present invention can induce CTL targeting synovial sarcoma cells having HLA-A24 presenting the peptide of the present invention. HLA-A24
Is high, being 20 to 30% in Europeans and Americans, and particularly 50% or more in Japanese. Therefore, the cancer antigen peptide of the present invention can be used as a useful cancer vaccine for synovial sarcoma and an anticancer agent for synovial sarcoma.

[0020]

[Sequence List] SEQUENCE LISTING <110> Hokkaido Technology Licensing Office Co. Ltd. <120> A cancer antigen peptide derived from synovial sarcoma <130> y1i-0201 <140> <141> <160> 4 <170> PatentIn Ver. 2.1 <210> 1 <211> 10 <212> PRT <213> Homo sapiens <400> 1 Pro Tyr Gly Tyr Asp Gln Ile Met Pro Lys 1 5 10 <210> 2 <211> 9 <212> PRT <213> Homo sapiens <400> 2 Gly Tyr Asp Gln Ile Met Pro Lys Lys 1 5 <210> 3 <211> 9 <212> PRT <213> Homo sapiens <400> 3 Ala Trp Thr His Arg Leu Arg Glu Arg 1 5 < 210> 4 <211> 10 <212> PRT <213> Homo sapiens <400> 4 Ala Trp Thr His Arg Leu Arg Glu Arg Lys 1 5 10

[Brief description of the drawings]

FIG. 1 is a diagram showing a partial sequence of amino acids encoded by a translocation gene SYT-SSX.

FIG. 2 shows ⁵¹ C for CTL induced using a peptide derived from the gene product of SYT-SSX of the present invention.
It is a graph which shows the result of an r cell damage test.

FIG. 3 shows ⁵¹ Cr for CTL induced with a peptide derived from the control SYT-SSX gene product.
It is a graph which shows the result of a cytotoxicity test.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 35/00 C07K 7/06 C07K 7/06 C12N 5/00 E C12N 5/06 A61K 37/02 (72 ) Inventor Tetsu Kawaguchi 2-11-11-13, Nishioka, Toyohira-ku, Sapporo-city, Hokkaido 3F 18 Hiragishi, Toyohira-ku Mont Teverde Sumikawa 721 F Term (Reference) 4B065 AC20 BA30 BB01 BB19 BB40 BC01 BC50 CA44 4C084 AA02 AA07 BA01 BA08 BA17 BA23 CA59 NA14 ZA942 ZA962 ZB262 4C085 AA03 BB11 CC87 AE01 ZB94C AA11 AA20 AA30 BA15 CA40 DA86 EA22 EA28 FA34 FA41 FA58 GA25

Claims (6)

[Claims] 1. A peptide comprising an amino acid sequence represented by SEQ ID NO: 1 or 2 and capable of inducing cytotoxic T cells targeting synovial sarcoma cells. 2. A cancer vaccine for synovial sarcoma comprising the peptide according to claim 1. [3] An anticancer agent for synovial sarcoma comprising the peptide of [1]. 4. Cytotoxic T targeting synovial sarcoma cells
Use of the peptide of claim 1 for inducing cells. A cytotoxic T cell induced by the peptide of claim 1. 6. An anticancer agent for synovial sarcoma, comprising the cytotoxic T cell according to claim 5.