JP2008044848A - HLA-A24 restricted tumor antigen peptide - Google Patents

Abstract

PROBLEM TO BE SOLVED: To identify a tumor antigen peptide effective for HLA-A24 positive cervical cancer.
Any one of the following tumor antigen peptides: a peptide consisting of a specific amino acid sequence, or an amino acid sequence in which one or more amino acids are deleted, substituted and / or added in the amino acid sequence, and HLA- A peptide that binds to the A24 molecule and can be recognized by cytotoxic T cells. In particular, a tumor antigen peptide derived from the human papillomavirus type 16 E6 protein recognized by cytotoxic T lymphocytes and IgG antibodies of HLA-A24 positive cervical cancer patients.
[Selection figure] None

Description

  The present invention relates to an HLA-A24-restricted tumor antigen peptide, and more particularly, a tumor derived from human papillomavirus type 16 E6 protein recognized by cytotoxic T lymphocytes and IgG antibodies of HLA-A24-positive cervical cancer patients It relates to an antigenic peptide.

  In recent years, many genes and antigen-derived peptides encoding human cancer-related antigens recognized by T cells have been identified, and clinical trials of cancer vaccine therapy as immunotherapy have been carried out (Non-patent Documents 1-2). In addition, peptide vaccine therapy for various epithelial cancers has been performed by the group of the present inventors (Non-patent Documents 3-7). Cancer vaccine therapy has also been attempted for cervical cancer, and there are reports of effective cases, but many of the cancer antigen peptides used in these vaccine therapy are derived from self-antigen proteins that are also expressed to some extent in normal cells It was something to do. Therefore, to establish effective vaccine therapy against cervical cancer, it is necessary to identify useful novel antigenic peptides.

  Most cervical cancers are known to be infected with human papillomavirus (HPV) type 16 (hereinafter abbreviated as “HPV16”) or type 18 (hereinafter abbreviated as “HPV18”) (non-patented) Reference 8, 9). Furthermore, E6 and E7 proteins of these types of HPV are known to be involved in transformation to malignant transformation and maintenance by inactivating p53 and RB tumor suppressor genes, respectively (non-patented) Reference 10, 11). Therefore, immunotherapy targeting non-self antigen HPV16 or HPV18 E6 protein or E7 protein is considered to be a very effective treatment for cervical cancer. In fact, HPV16-specific CTLs have been identified in peripheral blood lymphocytes and tumor infiltrating lymphocytes of cervical cancer patients (Non-patent Documents 12-14). Further, several HPV16 E7 protein-derived antigenic peptides presented in the HLA-A2 molecule have been identified (Non-patent Documents 15-17). For HPV16 E6 and E7 protein-derived peptide fragments, various types of HLA molecules (A1, A2, A3, A11, A24, A29, B7, B8, B18, B27, B35, B44, B51 and B62) The possibility of the coupling | bonding of is suggested (patent document 1). However, in the same document, neither peptide-specific CTL induction by a peptide suggested to be capable of binding to the HLA-A24 molecule nor cytotoxic activity of the induced CTL against cervical cancer cells has been studied.

  Thus, the HPV16-derived E6 or E7-derived antigenic peptide that is presented on the HLA-A24 molecule expressed by approximately 60% of the Japanese and can induce cervical cancer-reactive CTL has not yet been identified. For development, there is a strong need to identify such peptides.

U.S. Patent No. 6,783,763 Boon, T. et al., Immunol Today 81: p267-268,1997 Rosenberg SA, Immunity 10: p281-287,1999 Tanaka S et al., J Immunother 26: p357-366,2003 Tsuda N et al., J. Immunother 27: p60-72,2004 Noguchi M, Prostate 57: p80-92, 2003 Mine T, Cancer Science 94: p548-556, 2003 Sato Y et al., Cancer Science 94: p802-808, 2003 Walboomers JM et al., J Pathol 181: p253-254, 1997 Munoz N, N Engl J Med 348: p518-527, 2003 Munger K et al., J Virol 63: p4417-4421, 1989 Von Knebel DM, etc., Int J Cancer 51: p831-834, 1992 Borsiewicz LK, etc., Lancet 347: p1523-1527, 1996 Lessing ME, Cancer Res 56: p582-588, 1996 Evans EM etc., Cancer Res. 57: p2943-2950, 1997 Nijman HW et al., J Immunother 14: p121-126, 1993 Kast WM, etc., J Immunol 152: p3904-3912, 1994 Lessing ME, J Immunol 154: p5934-5943, 1995

An object of the present invention is to identify an HPV16 E6 protein-derived antigenic peptide capable of inducing cervical cancer-reactive cytotoxic T cells (CTL) in HLA-A24-positive cervical cancer patients.
Another object of the present invention is to provide a means for preventing and treating cervical cancer using the above peptide.
Other objects of the present invention will become apparent throughout the specification.

In order to achieve the above-mentioned object, the present inventors prepared a plurality of candidate peptides according to certain criteria based on the amino acid sequence of the known HPV16 E6 protein, and the peptides were HLA-A24 positive children. By stimulating peripheral blood lymphocytes of patients with cervical cancer and determining the presence or absence of peptide-specific CTL-inducing activity, we succeeded in identifying an effective antigenic peptide against HLA-A24-positive cervical cancer.
That is, the present invention is as follows.
1. Any of the following tumor antigen peptides:
(1) a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or 2, or (2) an amino acid sequence in which one or more amino acids are deleted, substituted and / or added in the amino acid sequence shown in SEQ ID NO: 1 or 2 And a peptide that can be recognized by cytotoxic T cells in association with HLA-A24 molecules.
2. 2. The amino acid sequence of the peptide in (2) above includes substitution of the amino acid residue at the second position and / or C-terminal of the amino acid sequence shown in SEQ ID NO: 1 or 2 with another amino acid residue Tumor antigen peptide.
3. 3. The tumor antigen peptide according to 1, wherein the amino acid sequence of the peptide in (2) comprises substitution of tyrosine at the second position of the amino acid sequence shown in SEQ ID NO: 1 or 2 with phenylalanine, methionine, or tryptophan.
4). 3. The tumor antigen peptide according to 1, wherein the amino acid sequence of the peptide in (2) comprises substitution of the C-terminal leucine of the amino acid sequence shown in SEQ ID NO: 1 or 2 with phenylalanine, isoleucine, tryptophan or methionine.
5. A therapeutic or prophylactic agent for tumors, comprising as an active ingredient at least one selected from the tumor antigen peptides according to any one of 1 to 4 above.
6). 5. An antigen-presenting cell obtained by presenting a complex of an HLA-A24 molecule and the tumor antigen peptide described in any one of 1 to 4 above on the surface of an isolated cell having an antigen-presenting ability derived from a tumor patient.
7). 7. A therapeutic agent for tumors comprising the antigen-presenting cell according to 6 as an active ingredient.
8). A cytotoxic T cell that specifically recognizes a complex of an HLA-A24 molecule and the tumor antigen peptide described in any one of 1 to 4 above.
9. 9. A tumor therapeutic agent comprising the cytotoxic T cell according to the above 8 as an active ingredient.

  The tumor antigen peptide of the present invention forms a complex with the HLA-A24 molecule, and can effectively induce CTLs that specifically recognize the complex between the peptide and the HLA-A24 molecule. Moreover, since the tumor antigen peptide of the present invention is HLA-A24-restricted, which is positive in many Asians, particularly Japanese, diagnosis, prevention of HLA-A24-positive cervical cancer in these populations, It also has the effect of promoting the development of effective means for treatment and further prevention of metastasis.

  In the present specification, the “tumor antigen peptide” of the present invention refers to (1) the amino acid sequence set forth in SEQ ID NO: 1 or 2, or (2) one or more amino acids in the amino acid sequence deleted, substituted and / or Or HLA-, which consists of an added amino acid sequence and can be recognized by cytotoxic T cells in combination with HLA-A24 molecules, that is, can induce CTL effective against HLA-A24-positive cervical cancer A24-restricted tumor antigen peptide. In the present specification, a peptide having an altered amino acid sequence defined in (2) may be referred to as a “(tumor) peptide derivative”.

  In the context of the present invention, “can bind to HLA-A24 molecule and be recognized by CTL” means that the tumor antigen peptide of the present invention binds to HLA-A24 molecule to form a complex, It means that CTL can be recognized. In other words, the tumor antigen peptide of the present invention has a binding activity with the HLA-A24 molecule and an activity to induce peptide-specific CTL in the form of a complex with the HLA-A24 molecule. Means.

  In the context of the present invention, an “antigen-presenting cell” refers to a cell that presents a complex of HLA-A24-restricted antigen peptide and HLA-A24 molecule on the cell surface. Therefore, the antigen-presenting cell has a function of causing activation of CTL by presenting a complex of the HLA-A24-restricted tumor antigen peptide of the present invention and the HLA-A24 molecule on the cell surface. Such cells also include tumor cells that are targets for CTL cytotoxic effects.

  As described above, the tumor antigen peptide of the present invention is a peptide having the amino acid sequence shown in SEQ ID NO: 1 or 2, and an altered derived by deletion, substitution, and / or addition of at least one amino acid in the sequence. It is a peptide derivative having an amino acid sequence, and includes mutant peptides that are recognized by specific CTLs by binding to HLA-A24 molecules. The amino acid modification is specifically a deletion, substitution or addition in one to several, preferably one or two amino acids. When the mutation includes substitution or addition with 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, and esterified products of various amino acids. , Acid amidated products, alkylated products and the like. In order to obtain a derivative of the tumor antigen peptide of the present invention, one or more, preferably 1 to several, more preferably 1 or 2 amino acid residues of the amino acid sequence shown in SEQ ID NO: 1 or 2 are deleted. Or a candidate peptide substituted with or added with other amino acid residues or amino acid analogs, and assaying whether the complex of the candidate peptide and the HLA-A24 molecule is recognized by CTL By doing so, it can be identified.

The sequence of the antigen peptide presented by binding to the HLA antigen has regularity (motif). In the case of the HLA-A24 antigen (molecule), the second amino acid from the N-terminal of the peptide consisting of 8 to 11 amino acids Is known to be phenylalanine, tyrosine, methionine or tryptophan, and the C-terminal amino acid is known to be phenylalanine, leucine, isoleucine, tryptophan or methionine (Parker KC et al., J Immunol 152: 163-175, 1994, and Rammensee HG et al., Immunogenetics 41: 178-228, 1995). In addition, derivatives obtained by substituting amino acids that can be taken on such motifs with amino acids having similar properties may be acceptable as HLA-A24 antigen-binding peptides.
Therefore, when the peptide derivative derived from the amino acid sequence represented by SEQ ID NO: 1 or 2 contains a substitution with another amino acid, examples of such substitution include amino acid residues at the second position and / or the C-terminal of these sequences. Substitution with other amino acid residues as defined on the motif. Specifically, substitution of tyrosine at the second position in the amino acid sequence shown in SEQ ID NO: 1 or 2 with phenylalanine, methionine or tryptophan, and / or substitution of C-terminal leucine with phenylalanine, isoleucine, tryptophan or methionine. Can be mentioned. These peptide derivatives have an activity of binding to HLA-A24 molecule and being recognized by CTL.

  The tumor antigen peptide of the present invention not only binds itself to the HLA-A24 antigen as a peptide fragment and is presented on the surface of the antigen-presenting cell, but is appropriately fragmented in the target cell, and is represented by SEQ ID NO: 1 or 2. Also encompassed are peptides having the amino acid sequence shown or longer peptides having a modified amino acid sequence but capable of binding to the HLA-A24 antigen to give a peptide derivative that can be recognized by CTL. When the peptide itself binds to the HLA-A24 molecule, the length of the tumor antigen peptide of the present invention forms a complex with the HLA-A24 molecule and is presented on the surface of antigen-presenting cells to induce specific CTLs. The required length is usually 8 to 11 residues, preferably 9 to 10 residues.

  In addition, on the condition that it forms a complex with the HLA-A24 molecule and is presented on the surface of antigen-presenting cells and recognized by specific CTLs, the N-terminus and free amino group of the tumor antigen peptide have formyl Group, acetyl group, t-butoxycarbonyl (t-Boc) group and the like may be bonded, and the C-terminus and free carboxyl group of the tumor antigen peptide include methyl group, ethyl group, t-butyl group, benzyl group. A group or the like may be bonded. Furthermore, the tumor antigen peptide of the present invention may be modified so as to facilitate introduction into the living body.

Peptide Synthesis The tumor antigen peptide of the present invention can be prepared by ordinary peptide synthesis. As such a method, for example, literature (peptide synthesis, Interscience, New York, 1966; The Proteins, Vol2, Academic Press Inc., New York, 1976; peptide synthesis, Maruzen (Inc.), 1975; Basics and experiments of peptide synthesis, Maruzen Co., Ltd., 1985; Continued development of pharmaceuticals, Volume 14: Peptide synthesis, Hirokawa Shoten, 1991).

Recognition of HLA antigen-restricted CTL-recognized candidate peptide binding to HLA-A24 molecule and recognized by CTL can be examined, for example, according to the following general method.
(1) According to the method described in J. Immunol., 154, p2257, 1995, peripheral blood lymphocytes are isolated from humans positive for HLA-A24 antigen and stimulated by adding a candidate peptide in vitro. It is examined whether or not CTLs that specifically recognize HLA-A24 positive cells pulsed with peptides are induced. Here, the presence or absence of CTL induction is examined, for example, by measuring the amount of various cytokines (for example, IFN-γ) produced by CTL in response to antigen peptide-presenting cells by enzyme immunoassay (ELISA) or the like. Can do. Or a method of measuring the cytotoxicity of CTL against labeled antigen peptide presenting cells in ⁵¹ Cr ⁽⁵¹ Cr release assay, Int.J.Cancer, 58: p317,1994) can be examined by. HLA-A24 positive cells used in the above assay are generally available such as cervical cancer cell line SKG-IIIa (JCRB 0232), C1R-A24 cell, esophageal cancer cell line KE-4 (FERM BP-5955), etc. Cells.

(2) Furthermore, the HLA-A24 cDNA expression plasmid was introduced into COS-7 cells (ATCC No. CRL1651) and VA-13 cells (RIKEN Cell Bank), and the candidate peptide was pulsed against the obtained cells. It can also be examined by reacting an HLA-A24-restricted CTL line and measuring the amount of various cytokines produced by the cells (for example, IFN-γ) (J. Exp. Med., 187: 277, 1998). Various assay methods as described above are known to those skilled in the art, and are described in detail in WO97 / 46676, for example.

The binding affinity of the tumor antigen peptide derivative to the HLA-A24 molecule was determined by a competitive inhibition assay for binding of the derivative and a standard peptide (SEQ ID NO: 1 or 2) labeled with a radioisotope to the HLA antigen. It can be easily measured in the system (RTKubo et al., J. Immunol, 152: 3913, 1994).

  The tumor antigen peptide of the present invention is an antigen peptide derived from the HPV16 E6 protein, which is presented by binding to an HLA-A24 molecule possessed by a wide range of human subjects (for example, possessed by about 60% of Japanese people), Since it is recognized by CTL, it is useful for treatment, prevention, diagnosis and the like of tumors, particularly cervical cancer, in vivo and in vitro.

The present invention provides a therapeutic or prophylactic agent for tumors containing as an active ingredient at least one of the tumor antigen peptides of the present invention.
For use for the treatment or prevention of tumors, at least one or more of the tumor antigen peptides of the present invention are combined, and if necessary, administered to a patient in combination with other tumor antigen peptides. When the therapeutic or prophylactic agent for tumor of the present invention is HLA-A24 positive and is administered to a tumor antigen protein derived from the tumor antigen peptide of the present invention (positive for HPV16 E6 protein, cervical cancer patients, antigen-presenting cells Tumor antigen peptide derivatives are densely presented on the HLA-A24 molecule, and the presented HLA-A24 antigen complex-specific CTLs proliferate and destroy tumor cells, thereby treating the patient's tumor, or Tumor growth or metastasis can be prevented.

  The therapeutic or preventive agent for tumors containing the tumor antigen peptide of the present invention can be administered together with an adjuvant so as to effectively establish cellular immunity, or can be administered in a particulate dosage form. As the adjuvant, those described in the literature (Clin. Microbiol. Rev., 7: 277-289, 1994) can be applied. In addition, a preparation capable of efficiently presenting an exogenous antigen peptide derivative to the HLA antigen, such as a liposomal preparation, a particulate preparation bound to beads having a diameter of several μm, a preparation bound to lipid, and the like are also used. The administration method can be appropriately selected from intradermal administration, subcutaneous administration, intravenous injection and the like. The dose of the tumor antigen peptide derivative of the present invention can be appropriately adjusted according to the disease to be treated, the age, weight, etc. of the patient, but is usually 0.0001 mg to 1000 mg, preferably 0.001 mg to 1000 mg, more preferably. Is 0.1 mg to 10 mg, which is preferably administered once every few days to several weeks.

Moreover, antigen-presenting cells can be induced in vitro using the tumor antigen peptide of the present invention, and such cells are useful for tumor therapy and the like.
Accordingly, the present invention provides an antigen-presenting cell in which a complex of the HLA-A24 molecule and the tumor antigen peptide of the present invention is presented on the surface of an isolated cell having an antigen-presenting ability derived from a tumor patient.

The present invention also provides a therapeutic agent for tumors containing, as an active ingredient, antigen-presenting cells presenting a complex of the HLA-A24 molecule and the tumor antigen peptide of the present invention.
Here, the “cell having antigen-presenting ability” is not particularly limited as long as it is a cell expressing the HLA-A24 antigen capable of presenting the tumor antigen peptide of the present invention on the cell surface, but has particularly high antigen-presenting ability. Dendritic cells are preferred.

In order to prepare the antigen-presenting cell of the present invention, a cell having an antigen-presenting ability is isolated from a tumor patient, and the cell is pulsed with the tumor antigen peptide of the present invention in vitro to obtain an HLA-A24 antigen and the peptide derivative. A complex is made (Cancer Immunol. Immunother., 46:82, 1998).
The tumor therapeutic agent containing the antigen-presenting cell as an active ingredient preferably contains physiological saline, phosphate buffered saline (PBS), a medium, or the like in order to stably maintain the antigen-presenting cell. Examples of the administration method include intravenous administration, subcutaneous administration, and intradermal administration. When the above-mentioned tumor therapeutic agent is returned to the body of a patient, specific CTLs are efficiently induced in the body of a patient positive for HLA-A24-positive tumor antigen protein derived from the tumor antigen peptide of the present invention, thereby treating the tumor. Furthermore, metastasis can be prevented.

Furthermore, the utilization in the following adoptive immunotherapy is mentioned as an in vitro utilization method of the tumor antigen peptide derivative of this invention.
That is, in melanoma, a therapeutic effect has been observed in adoptive immunotherapy in which a patient's own tumor-infiltrating T cells are cultured in large quantities outside the body and returned to the patient (J. Natl. Cancer. Inst., 86 : 1159, 1994). In mouse melanoma, spleen cells were stimulated with tumor antigen peptide TRP-2 in vitro, CTL specific for the tumor antigen peptide was proliferated, and administration of the CTL to melanoma-transplanted mice was found to suppress metastasis. (J. Exp. Med., 185: 453, 1997). This is based on the result of in vitro proliferation of CTL that specifically recognizes a complex of an antigen-presenting cell HLA antigen and a tumor antigen peptide. The tumor antigen peptide of the present invention can be used to stimulate a patient's peripheral blood lymphocytes in vitro to increase tumor-specific CTL, and then return the CTL to the patient to treat the tumor.

The present invention also provides cytotoxic T cells that specifically recognize the complex of the HLA-A24 antigen and the tumor antigen peptide of the present invention.
Furthermore, this invention provides the therapeutic agent of the tumor which contains the said cytotoxic T cell as an active ingredient.
The therapeutic agent preferably contains physiological saline, phosphate buffered saline (PBS), a medium, or the like in order to maintain CTL stably. Examples of the administration method include intravenous administration, subcutaneous administration, and intradermal administration. By returning the above therapeutic agent to the patient's body, the CTLs promote tumor damage by CTL in the patient's body positive for HLA-A24-positive tumor antigen protein derived from the tumor antigen peptide of the present invention. By destroying, tumors can be treated and even metastasis can be prevented.

  Further, in order to use the tumor antigen peptide derivative of the present invention for tumor diagnosis, for example, an antibody against the tumor antigen peptide is prepared according to a conventional method, labeled as necessary, and used from a patient suspected of having a tumor. The presence or absence of a tumor can be diagnosed by detecting the presence of an antigen in the obtained sample (eg, blood, tumor tissue, etc.). Furthermore, it is possible to diagnose the presence or absence of a tumor by using the tumor antigen peptide itself of the present invention as a diagnostic agent and detecting the presence of an antibody in a sample such as blood or tumor tissue.

The present invention also uses the above-described tumor antigen peptide, an antigen-presenting cell presenting the tumor antigen peptide, and a cytotoxic T cell that specifically recognizes a complex of the tumor antigen peptide and HLA-A24 antigen. To provide a method for treating or preventing or diagnosing a tumor. Furthermore, the tumor antigen peptide derivative of the present invention is also useful as a research reagent in this technical field.
EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, this invention is not limited at all by these Examples.

Example 1 Preparation of HLA-A24 restricted tumor antigen peptide derived from HPV-16 E6 protein
I. Identification of tumor antigen peptides
I-1. Materials and experimental methods
Preparation of peripheral blood mononuclear cells (PBMC) 20 ml of blood is collected from cervical cancer patients and healthy individuals who are negative for human immunodeficiency virus (HIV), and peripheral blood lymphocytes and plasma are separated by Ficoll-Conrey density method. PBMCs were prepared.

Cell line
SKG-IIIa was used as an HPV16 positive and HLA-A24 positive cervical cancer cell line, and OMC-1 was used as an HPV16 positive and HLA-A24 negative cervical cancer cell line. Moreover, esophageal cancer cell line KE-4 was used as a HPV16 negative and HLA-A24 positive cancer cell line.

Peptides Based on the already reported HLA-A24 binding motif (Parker et al., Supra, and Ramensi et al., Supra), four types of antigen peptides derived from HPV16 and E6 protein were prepared. That is, positions 42-50 (VYDFAFQDL, SEQ ID NO: 3), positions 75-83 (EYRHYCYSL, SEQ ID NO: 1), positions 91-99 (QYNKPLCDL, SEQ ID NO: 4), and positions 91-100 of the amino acid sequence of HPV16-E6 It is a peptide having the amino acid sequence of (QYNKPLCDLL, SEQ ID NO: 2). The amino acid sequence of HPV16-E6 is disclosed in NCBI data bank: AJ388058.

Examination of peptide-specific CTL progenitor cells Peripheral blood mononuclear cells (PBMC) of cervical cancer patients were repeatedly stimulated with HPV16 E6 protein-derived antigenic peptide prepared according to the method described in the literature (Hida N, etc.) Cancer Immunol Immunother 51: 219-228, 2002). Specifically, the peptide (10 μg / ml) was added on the first day, and 45% RPMI-1640, 45% AIM-V medium (Gibcp BRL), 10% FCS was added in the presence of IL-2 (100 U / ml). After starting the culture at 37 ° C in the culture medium, the culture medium containing the doubled concentration of peptide (20 µg / ml) and IL-2 (100 U / ml) is half on days 3, 6, and 9 The culture broth was replaced. After 14 days, cultured lymphocytes were collected and C1R-A24 cells pulsed with the peptide used for stimulation (HLA-A * 2402-expressing subcell line of C1R lymphoma (Oiso M, et al., Int. J Cancer 81: 387-394, 1999)), and IFN-γ in the culture supernatant after 18 hours was measured by ELISA. As control peptides, two HIV peptides, an EBV-derived peptide (TYGPVFMCL, SEQ ID NO: 5) and a Flu-derived peptide (RFYIQMCYEL, SEQ ID NO: 6) having binding properties to the HLA-A24 molecule were used.

In order to examine the expression of HLA-A24 molecules on the cell surface of a flow cytometry cancer cell line, anti-HLA-A24 antibody (Cat # 0041HA, One Lambde Inc., Canoga Park, CA) as a primary antibody, secondary antibody Were stained with FITC-binding anti-mouse IgG and examined by flow cytometry.

PCR
After extracting total RNA from cancer cells, cDNA was synthesized using SuperScript (registered trademark) Preaplication system (Invitrogen). Subsequently, DNA was amplified using the following primers.
Sense: 5'-GTGTGTACTGCAAGCAACAG-3 '(SEQ ID NO: 7)
Antisense: 5'-GCAATGTAGGTGTATCTCCA-3 '(SEQ ID NO: 8)
As a control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified with the following primers.
Sense: 5'-ACAACAGCCTCAAGATCATCAG-3 '(SEQ ID NO: 9)
Antisense: 5'-GGTCCACCACTGACACGTTG-3 '(SEQ ID NO: 10)
PCR was performed routinely using TaqDNA polymerase for 30 cycles (54 ° C.) (Matsueda S. et al., Cancer Immunol Immunother., 53: 479-489, 2004).

Cytotoxicity Test The cytotoxic activity of CTL induced by peptide stimulation against HLA-A24 positive cervical cancer cell line infected with HPV16 was examined by ⁵¹ Cr release test. At the time of ⁵¹ Cr release test, anti-HLA-class I (W6 / 32, mouse IgG2a), anti-HLA-class II (H-DR-1, mouse IgG2a), anti-CD4 (Nu-Th / I, mouse IgG2a) Then, 10 μg / ml each of anti-CD8 (Nu-Ts / c, mouse IgG2a) antibody was added to examine cells responsible for cytotoxic activity. Furthermore, to confirm that the cytotoxic activity against cervical cancer cells depends on peptide-specific CTL, C1R-A24 not labeled with ⁵¹ Cr pulsed with peptide used for stimulation or control HIV peptide A non-radioactive inhibition assay (cold inhibition assay) was also performed.

Measurement of peptide-specific IgG Peptide-specific IgG antibodies followed the method already reported (Komatsu N et al., Scand J Clin Lab Invest 64: 1-11, 2004). Specifically, individual peptides were bound to colored carboxy beads (Luminex 100 (registered trademark)), the sample was reacted with peptide-bound beads, and reacted with biotin-labeled anti-human IgG (γ) antibody and streptavidin. . After the reaction, it was measured by a fluorescence flowmetry method (xMAP technique).

Statistically significant differences in statistical processing results were examined using a two-tailed student t-test, with P <0.05 being considered significant.

I-2. Result 1) CTL induction by HPV16-E6 protein-derived peptide
Four candidate peptides derived from the HPV16 E6 protein prepared based on the binding motif to the HLA-A24 molecule, and EBV and Flu-derived HLA-A24-binding peptides were used as controls (Table 1).
PBMCs from 10 HLA-A24 positive cervical cancer patients (Pt # 1 to Pt # 10) were stimulated with each peptide, and the induction of stimulating peptide-specific CTL was examined using IFN-γ production as an index did. Stimulation of PBMCs in HLA-A24-positive cervical cancer patients with peptides was performed according to the method described by Hida et al. (Supra). After 14 days, the amount of IFN-γ specific to the stimulated peptide released into the medium was examined by ELISA. Each peptide was examined in 4 wells. The results are shown in Table 1. The scores in Table 1 indicate the strength of binding of individual peptides to the HLA-A24 molecule.

表１に記載の通り、HPV16-E6 75-83ペプチドを用いた場合には10人中5人で、HPV16-E6 91-99ペプチドを用いた場合には、10人中2人、HPV16-E6 91-100ペプチドを用いた場合には、10人中4人の患者から得たPBMC試料でペプチド特異的CTLの誘導が認められた。HPV16-E6 42-50ペプチドの場合には、ペプチド特異的CTLは誘導されなかった。

As shown in Table 1, when HPV16-E6 75-83 peptide was used, 5 out of 10 people, and when HPV16-E6 91-99 peptide was used, 2 out of 10 people, HPV16-E6 When 91-100 peptide was used, induction of peptide-specific CTL was observed in PBMC samples obtained from 4 out of 10 patients. In the case of HPV16-E6 42-50 peptide, peptide-specific CTLs were not induced.

2) Cytotoxic activity of CTL stimulated with HPV16-E6 protein-derived peptide against cervical cancer 2-1) Expression of HPV16 and HLA-A24 molecules in cervical cancer cell lines Cervical cancer cells SKG-IIIa and OMC-1 FIG. 1A shows the results of examining the expression of HPV16 in the esophageal cancer cell line KE-4 by the PCR method. Moreover, the result of having analyzed the expression of the HLA-A24 molecule | numerator in cervical cancer cells SKG-IIIa and OMC-1 by the flow cytometry method is shown in FIG. 1B.
As shown in FIG. 1A, HPV16 infection was confirmed in cervical cancer cell lines SKA-IIIa and OMC-1, but HPV16 infection was not observed in esophageal cancer cell line KE-4 which is a negative control. It was. SKG-IIIa was positive for HLA-A24, but negative for OMC-1 (FIG. 1B).

2-2) Cytotoxic activity of CTL stimulated by HPV16-E6 peptide against cervical cancer cells
The cytotoxic activity of CTLs derived from cervical cancer patients induced by stimulation with HPV16-E6 75-83 peptide or HPV16-E6 91-100 peptide against cervical cancer cells was examined. PBMCs from 3 HLA-A24 positive cervical cancer patients (PT # 2, PT # 6, PT # 7) were stimulated with individual peptides and 3 types of target cells (HLA-A24 positive HPV16 positive SKG-IIIa, The cytotoxic activity against HLA-A24-negative HPV16-positive OMC-1 and HLA-A24-positive HPV16-negative PHA-stimulated T cells was examined in a ⁵¹ Cr release test. The results are shown in FIG.
As shown in FIG. 2, CTL induced by HPV16-E6 75-83 peptide from PBMCs of 3 HLA-A24 positive cervical cancer patients were HPV16 positive and HLA-A24 negative OMC-1 and PHA (phyto It showed higher cytotoxic activity against SKG-IIIa positive for both HPV16 and HLA-A24 than to HLA-A24 positive T cells induced by stimulation with hemagglutinin. Similar results were observed for CTL induced with HPV16-E6 91-100 peptide from PBMC of two HLA-A24 positive cervical cancer patients (Pt # 6, Pt # 7).

3) Dependence of CTL stimulated by HPV16-E6 protein-derived peptide on cervical cancer depends on class I-restricted peptide-specific CD8-positive T cells Stimulated and induced by two HPV16-E6 protein-derived peptides We examined the cell-dependent cytotoxic activity of CTLs against cervical cancer using antibody inhibition tests and a non-radioactive inhibition assay (cold inhibition assay) using competitive target cells. The results are shown in FIG. 3 and FIG.
In a ⁵¹ Cr release test in which PBMCs from two HLA-A24 positive cervical cancer patients (PT # 6, PT # 7) were stimulated with individual peptides and examined for cytotoxic activity against three target cells, anti-HLA -Class I (W6 / 32, mouse IgG2a), anti-HLA-class II (H-DR-1, mouse IgG2a), anti-CD4 (Nu-Th / I, mouse IgG2a) and anti-CD8 (Nu-Ts / c) , Mouse IgG2a) antibody was added at 10 μg / ml, and the effect on cervical cancer cells was examined (FIG. 3).

In addition, in order to confirm that the cytotoxic activity against cervical cancer cells depends on peptide-specific CTL, the peptide used for stimulation, or C1R-labeled with ⁵¹ Cr not pulsed with control HIV peptide. Non-radioactive inhibition assay was performed by adding A24 cells (FIG. 4).
As shown in FIG. 3, CTL induced by HPV16-E6 75-83 peptide or HPV16-E6 91-100 peptide against SKG-IIIa from two cervical cancer patients (Pt # 6 and Pt # 7) Cytotoxic activity was inhibited by the addition of anti-class I antibody and anti-CD8 antibody, but not by the addition of anti-class II antibody, anti-CD4 antibody or anti-CD14 antibody. Moreover, the cytotoxic activity against these SKG-IIIa was significantly suppressed by the addition of (cold) C1R-A24 cells not labeled with ⁵¹ Cr pulsed with the HPV16-E6 peptide used for stimulation. It was not suppressed when C1R-A24 cells not labeled with ⁵¹ Cr pulsed with HIV peptide were added (FIG. 4). These results indicate that the cytotoxic activity of CTL stimulated and induced by two types of HPV16-E6 protein-derived peptides against cervical cancer depends on class I-restricted peptide-specific CD8-positive T cells. .

4) Peptide-specific IgG antibody
IgG antibodies specific for the four HPV16-E6 protein-derived peptides (HPV16-E6 42-50, HPV16-E6 75-83, HPV16-E6 91-99 and HPV16-E6 91-100) are used in patients with cervical cancer Whether or not it was identified in the plasma of healthy persons was measured by fluorescence flowmetry. The results are shown in Table 2. In the table, each numerical value indicates the fluorescence intensity.

表２に記載の通り、子宮頸癌患者においては、HPV16-E6 42-50ペプチドとHPV16-E6 91-99ペプチドに対しては12人中3人で、HPV16-E6 91-100ペプチドに対しては12人中4人でペプチド反応性IgG抗体が同定できた。また、女性健常人においては、HPV16-E6 91-99ペプチドに対しては10人中1人で、HPV16-E6 91-100ペプチドに対しては10人中3人でペプチド反応性IgG抗体が同定できた。HPV16-E6 75-83ペプチドに対しては、子宮頸癌患者と女性健常人の両者においてペプチド特異的IgG抗体は同定できなかった。

As shown in Table 2, in cervical cancer patients, 3 out of 12 for HPV16-E6 42-50 peptide and HPV16-E6 91-99 peptide, and against HPV16-E6 91-100 peptide Were able to identify peptide-reactive IgG antibodies in 4 out of 12 people. In healthy females, 1 out of 10 HPV16-E6 91-99 peptides and 3 out of 10 HPV16-E6 91-100 peptides identified peptide-reactive IgG antibodies did it. For HPV16-E6 75-83 peptide, no peptide-specific IgG antibody could be identified in both cervical cancer patients and healthy females.

I-3. Conclusion As described in I-1 and I-2 above, HPV16 is capable of inducing cervical cancer-reactive CTLs by binding to HLA-A24 molecules expressed by approximately 60% of Japanese and presented by antigen-presenting cells. HPV16-E6 75-83 peptide and HPV16-E6 91-100 peptide were identified as antigen peptides derived from E6 protein. These peptides and derivatives thereof are useful for the purposes of the present invention. However, in a clinical study of cancer vaccine therapy conducted by the present inventors group, there was a tendency for survival to be prolonged in cases in which IgG antibodies were induced against the vaccinated peptide (Mine (Mine T) et al., Clin Cancer Res 10: 929-937, 2004).
Therefore, all of the tumor antigen peptides of the present invention induce HLA-A24-restricted CTL and are useful for the treatment and prevention of cervical cancer. Among them, HPV16-E6 91-100 peptide has an efficient ability to induce CTL. It is highly possible that the peptide is an antigenic peptide that is easily recognized by IgG antibodies and is expected to be extremely useful as an active ingredient in cancer vaccine therapy.

According to the present invention, a method for diagnosing, preventing or treating HLA-A24-positive cervical cancer that is positive in almost 60% of Asian races, particularly Japanese, and development of means for preventing metastasis are developed. Can greatly promote.

Photocopy of the results of PCR analysis of HPV16 expression of cervical cancer cells SKG-IIIa and OMC-1 and esophageal cancer cell line KE-4 and cervical cancer cells SKG-IIIa and OMC- FIG. 2 is a photocopy (B) showing the result of analyzing the expression of 1 HLA-A24 molecule by flow cytometry. Three types of target cells (HLA-A24-positive HPV16-positive SKG-IIIa, HLA-A24-negative HPV16-positive OMC-) induced by stimulating PBMCs from three HLA-A24-positive cervical cancer patients with each peptide it is a graph showing the examination results on the 1 and HLA-A24-positive HPV16-negative PHA-stimulated T cells) against the cytotoxic activity in a ⁵¹ Cr release test. Anti-HLA-class I (W6 / 32, mouse IgG2a) in a ⁵¹ Cr release test in which PBMCs from two HLA-A24-positive cervical cancer patients were stimulated with each peptide and examined for cytotoxic activity against three target cells ), Anti-HLA-class II (H-DR-1, mouse IgG2a), anti-CD4 (Nu-Th / I, mouse IgG2a) and anti-CD8 (Nu-Ts / c, mouse IgG2a) antibodies each 10 μg / ml In addition, it is a graph which shows the result of having examined the influence. In the same test as FIG. 3, in order to confirm the dependence of cytotoxic activity against cervical cancer cells on peptide-specific CTL, the peptide used for stimulation or labeled with ⁵¹ Cr pulsed with control HIV peptide was used. It is a graph which shows the result of the non-radioactive inhibition test performed by adding the C1R-A24 cell which is not.

Claims (9)

Any of the following tumor antigen peptides:
(1) a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or 2, or (2) an amino acid sequence in which one or more amino acids are deleted, substituted and / or added in the amino acid sequence shown in SEQ ID NO: 1 or 2 And a peptide that can be recognized by cytotoxic T cells in association with HLA-A24 molecules.   The amino acid sequence of the peptide in (2) above includes substitution of the amino acid residue at the second position and / or C-terminal of the amino acid sequence shown in SEQ ID NO: 1 or 2 with another amino acid residue. The tumor antigen peptide described.   The tumor antigen peptide according to claim 1, wherein the amino acid sequence of the peptide in (2) includes substitution of tyrosine at the second position of the amino acid sequence shown in SEQ ID NO: 1 or 2 with phenylalanine, methionine, or tryptophan.   The tumor antigen peptide according to claim 1, wherein the amino acid sequence of the peptide in (2) comprises substitution of C-terminal leucine of the amino acid sequence shown in SEQ ID NO: 1 or 2 with phenylalanine, isoleucine, tryptophan or methionine.   A therapeutic or prophylactic agent for tumors, comprising as an active ingredient at least one selected from the tumor antigen peptides according to any one of claims 1 to 4.   An antigen-presenting cell obtained by presenting a complex of an HLA-A24 molecule and the tumor antigen peptide according to any one of claims 1 to 4 on the surface of an isolated cell having an antigen-presenting ability derived from a tumor patient.   A therapeutic agent for a tumor comprising the antigen-presenting cell according to claim 6 as an active ingredient.   A cytotoxic T cell that specifically recognizes a complex of an HLA-A24 molecule and the tumor antigen peptide according to any one of claims 1 to 4.   A therapeutic agent for a tumor comprising the cytotoxic T cell according to claim 8 as an active ingredient.

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