JP4925033B2 - Stress-induced anti-apoptotic molecule (IEX-1) derived peptide - Google Patents

Description

  The present invention relates to a stress-induced anti-apoptotic molecule (IEX-1) -derived peptide that can be used in immunotherapy for cancer patients. Specifically, the present invention relates to an IEX-1-derived peptide that is recognized by cytotoxic T cells (CTL) and induces a specific CTL, a polypeptide containing the peptide, a cancer vaccine containing the peptide, and the like.

  Gastric cancer is one of the most universally occurring malignant tumors in the world (References 1-6). The disease has a generally good prognosis at an early stage, but in advanced cancer, despite recent advances in existing therapies such as surgical resection, chemotherapy, and radiation therapy, Prognosis is very bad. Therefore, there is a need to develop new therapies such as new specific immunotherapy to treat patients with advanced stage gastric cancer.

In recent years, it has become clear that human tumors express antigenic peptides recognized by CTLs, and such peptides are used as peptide vaccines for cancer patients with HLA-A2 or A24 alleles. (References 1-7). Many reports have been made on epitope peptides recognized by HLA-A2 or A24-restricted CTL (References 8-11), and the present inventors have recently also been treated with peptide vaccine treatment for patients with severe gastric cancer of HLA-A2 or A24. Reported an increase in the overall survival rate (Reference 4).
On the other hand, despite the relatively wide expression of HLA-A33 alleles in various races around the world (References 14 and 15), there are reports on antigens and peptides recognized by HLA-A33-restricted CTLs. Very limited (12, 13). This has hindered the development of peptide-based specific immunotherapy for HLA-A33 ⁺ cancer patients.

IEX-1 (immediate early response gene X-1) (also known as p22 / PRG1 (Reference 20), Dif-2 (Reference 21), or mouse homolog gyl96 (Reference 22)) is a stress-inducing gene. Involved in the regulation of cell cycle progression and apoptosis. IEX-1 plays an important role in cell resistance to apoptosis induced by various pro-apoptotic factors such as TNF and Fas (Ref. 23) and accelerates cell cycle progression in several cell lines (References 24-26) have been reported. In addition, the anti-apoptotic effect of IEX-1 on T-lymphocyte activation-induced cell death in vivo has been reported (Reference 27).
IEX-1 expression is rapidly activated by several cellular stresses including irradiation, growth factors, viral infections, inflammatory cytokines such as TNF-α and IL-1β, lipopolysaccharides, and steroid hormones. Obtain (Reference 27). Although IEX-1 was originally identified as an NF-κB / rel target gene (Reference 23), the IEX-1 promoter contains consensus sequences for other transcription factors such as p53, SP-1 and c-Myc. Are included (Reference 7). For example, it has been shown that a mutation in the tumor suppressor gene p53 common to tumor cells increases IEX-1 expression (Reference 29).
As described above, it has been suggested that IEX-1 is involved in malignant transformation of tumor cells, but no IEX-1 epitope peptide that functions as an antigenic peptide in tumor cells has been known.

An object of the present invention is to provide an antigenic peptide useful for developing a cancer vaccine for HLA-A33 ⁺ cancer patients.

  The present inventors established an HLA-A33-restricted CTL line from tumor tissue-infiltrating lymphocytes (TIL) of patients with severe gastric cancer, and three HLA-A33 binding epitopes derived from IEX-1 were recognized by this cell line. Identified as a tumor antigen. Furthermore, the present invention was completed by confirming that these antigenic epitopes induce peptide-specific CTLs in PBMC (peripheral blood mononuclear cells) of patients with malignant tumor diseases.

That is, the present invention includes the following.
(1) A peptide derived from a stress-induced anti-apoptotic molecule (IEX-1) or a mutant peptide thereof that is recognized by cytotoxic T cells (CTL) and induces a specific CTL.
(2) The peptide according to (1), wherein CTL recognizes HLA-A33 restricted.
(3) The peptide according to (1) or (2), comprising 8 to 11 consecutive amino acid residues.
(4) A peptide consisting of the amino acid sequence shown in any one of SEQ ID NOs: 1 to 3, or one or more amino acids in the amino acid sequence shown in any of SEQ ID NOs: 1 to 3 are deleted, substituted and / or added The peptide according to any one of (1) to (3), which is a peptide that has a specific amino acid sequence and induces a specific CTL by binding to an HLA-A33 molecule.
(5) A polypeptide comprising the peptide according to any one of (1) to (4) above.
(6) An antigen-presenting cell obtained by presenting a complex of the HLA-A33 molecule and the peptide according to any one of (1) to (4) on the surface of an isolated cell having an antigen-presenting ability.
(7) A nucleic acid molecule encoding the peptide according to any one of (1) to (4) above or the polypeptide according to (5).
(8) A vector containing the nucleic acid molecule according to (7) above.
(9) The peptide according to any one of (1) to (4), the polypeptide according to (5), the antigen-presenting cell according to (6), the nucleic acid molecule according to (7), or the description according to (8) A pharmaceutical composition for inducing specific CTL, comprising a vector.
(10) The pharmaceutical composition according to (9), which is a cancer vaccine.
(11) An IEX-1 reactive CTL that recognizes a complex of the peptide according to any one of (1) to (4) above and HLA, or a complex presented on an antigen-presenting cell according to (6) .
(12) A method for inducing IEX-1-reactive CTL using the peptide according to any one of (1) to (4), the polypeptide according to (5), or the antigen-presenting cell according to (6).
(13) An antibody that specifically recognizes the peptide according to any one of (1) to (4) or the polypeptide according to (5).
(14) A pharmaceutical composition for inducing specific CTL, comprising the polypeptide according to the following 1) or 2):
1) a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 4, or 2) an amino acid sequence comprising one or more amino acids deleted, substituted and / or added in the amino acid sequence shown in SEQ ID NO: 4, and HLA A polypeptide that gives a peptide that binds to the A33 molecule and induces a specific CTL.
(15) The pharmaceutical composition according to (14), which is a cancer vaccine.

  The present invention enables IEX-1-based immunotherapy suitable for the treatment of malignant tumors, particularly HLA-A33 positive cancer patients.

The figure which shows the characteristic of the HLA-A33 restriction | limiting 850B-CTL cell line established from the tumor infiltration lymphocyte (TIL) of a gastric adenocarcinoma patient (HLA-A ^* 2402 / A ^* 3303, B7 / B44, Cw7 / Cw14). It is. A shows the result of examining the recognition ability with respect to various target cells based on the production of IFN-γ in various E: T ratios (effector cells: target cells). B shows the results of cytotoxic activity against various target cells tested by a 6 hour ⁵¹ Cr release test at different E: T ratios. C is a figure which shows the difference in the reactivity with respect to HLA-A33 ⁺ LC-1 cell of 850B-CTL in the inhibition experiment using various mAb. FIG. 5 shows HLA-A33 restricted recognition of identified tumor antigen genes by the 850B-CTL cell line. Based on IFN-γ production, reactivity of 850B-CTL to COS7 cells co-transfected with clones obtained by gene expression cloning method from LC-1 lung adenocarcinoma cell cDNA library and HLA-A3303 or HLA-A2601 gene And examined. FIG. 2 shows IEX-1 expression at the mRNA and protein levels. A is a reproduction of photographs showing the results of examining the expression of IEX-1 mRNA in various normal tissues by Northern blot analysis using a ³² P-labeled IEX-1 probe. B is a copy of a photograph showing autoradiography in Northern blot analysis of mRNA expression of IEX-1 gene in normal and cancer cells. C to E are photomicrographs showing the results of immunohistochemical examination of IEX-1 expression in various tumor tissues at the protein level. C shows stomach cancer tissue, D shows breast cancer, and E shows lung cancer staining. It is a figure which shows the identification of the antigenic epitope in IEX-1. Eight candidate peptides that could have strong binding activity for HLA-A33 were selected and tested by computer analysis based on the HLA-A33 binding motif of the deduced amino acid sequence of IEX-1. In A, these eight candidate peptides were cultured with C1R-A33 cells (C1R human multiple myeloma cells stably transfected with HLA-A ^* 3303 cDNA), and 850B-CTL was further added. The results of measuring the production of IFN-γ in the culture supernatant by ELISA after culturing was shown. B shows the concentration of three peptides (IEX 47-56, IEX 61-69, and IEX 65-73 peptides) and one control peptide that induced significant levels of IFN-γ production, and IEX-1 reactive CTL inducing activity. The result of examining the relationship is shown. It is a figure which shows the cellular response with respect to a peptide. C1R-A33 cells stimulated with IEX47-56, IEX61-69, and IEX65-73 peptides in HLA-A33 ⁺ epithelial cancer patients and HLA-A33 ⁺ healthy individuals and pulsed with the corresponding peptides or HIV peptides as negative controls Stimulated with C1R-A33 cells pulsed with and tested for cellular responses based on IFN-γ producing activity. FIG. 2 is a view showing cytotoxicity of peptide-induced CTL. LC-1 (HLA-A33 ⁺ IEX-1 ⁺ ), QG56 (HLA-A33 ^- IEX-1 ⁺ ) and HGC27 (HLA-A33 ^- IEX-1 ⁻ ) cells were cultured with IL-2 alone and different E The ⁵¹ Cr release test was conducted for 6 hours at a T ratio to examine cytotoxic activity. It is a figure which shows the result of the cytotoxicity inhibition test by a monoclonal antibody. Using anti-HLA class I, anti-HLA class II, anti-CD8 (Nu-Ts / c, IgG2a), anti-CD4 (Nu-Th / i, IgG1) and anti-CD14 (JML-H14, IgG2a) mAb as a control, The inhibitory effect on the cytotoxicity of peptide-stimulated PBMC was examined. It is a figure which shows the result of the competition test of specific cytotoxicity. Unlabeled C1R cells were pulsed with the corresponding peptide or HIV peptide (negative control) and subjected to ⁵¹ Cr release test (unlabeled vs. labeled cells = 10: 1). A ⁵¹ Cr release test was conducted at an E / T ratio of 10: 1.

The present invention will be described in further detail.
HLA-restricted CTL line HLA-restricted CTL line is IL-2 which is a cytokine that collects lymphocytes (TIL) infiltrating tumor tissues of cancer patients having the target HLA allele and promotes T cell proliferation. It is obtained by culturing and growing together. This method is well known in the art.
Gene Expression Cloning Method Tumor antigens recognized by HLA restricted CTL lines are identified by gene expression cloning methods. In this method, the protein encoded by the cDNA library is transiently expressed in mammalian cells, the cDNA encoding the target protein specifically recognized by the established CTL line is screened, and the gene encoding the protein Is to be isolated. In the present invention, screening is performed using IFN-γ produced when an established CTL line recognizes a protein-expressing cell as an index.

Peptides and polypeptides The IEX-1-derived peptides of the present invention are peptides that can bind to HLA and induce specific CTLs. The peptide of the present invention preferably comprises 8 to 11 consecutive amino acid residues of IEX-1. Specific examples of such a partial peptide of IEX-1 include IEX 47-56, (SEQ ID NO: 1), IEX 61-69 (SEQ ID NO: 2), or IEX 65-73 (SEQ ID NO: 3). The entire nucleotide sequence and deduced amino acid sequence of the IEX-1 gene are registered in GeneBank as accession number NM_003897 (SEQ ID NO: 4). Other IEX-1-derived peptides that induce specific CTLs can be readily determined and selected by methods according to the examples herein.

  The phrase “can bind to HLA to induce specific CTL” means that the peptide of the present invention binds to HLA to form a complex, and CTL can recognize the complex. In other words, it means that the peptide of the present invention has an activity of inducing peptide-specific CTL in the form of a complex with HLA, as well as binding activity with HLA. A preferred HLA in the present invention is HLA-A33.

  The present invention also includes a mutant peptide of an IEX-1-derived peptide capable of inducing specific CTL by binding to HLA, and having a similar CTL inducing ability. Mutations can be introduced into the IEX-1-derived peptide of the present invention by deletion, substitution, addition, insertion, or the like of one or several amino acids, and the means are well known in the art. . 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 variant of the peptide of the present invention, for example, 1 to several amino acid sequences represented by SEQ ID NOs: 1 to 3, preferably 1 to 4, more preferably 1 to 3, more preferably 1 Alternatively, a candidate peptide in which two amino acid residues are deleted, substituted with another amino acid residue or amino acid analog, or added thereto is synthesized, and the candidate peptide is combined with the HLA-A33 molecule. It can be identified by assaying whether the body is recognized by CTL.

The assay can be performed, for example, according to the CTL induction method described below. That is, when a candidate peptide is added and stimulated in vitro, it is examined whether CTL that specifically recognizes HLA-A33 positive cells pulsed with the candidate peptide is induced. Here, the presence or absence of CTL induction is examined, for example, by measuring the amount of various cytokines (IFN-γ, etc.) 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. Examples of HLA-A33 positive cells used in the assay include HLA-A33 positive cells described in Examples.
The number of amino acid residues of the mutant peptide may be any number as long as it is displayed on the antigen-presenting cell surface and has a property as a CTL recognition epitope, and is usually at least about 8 and preferably about 9 or more. 12 or less, preferably 11 or less, and more preferably 10 or less. Particularly preferred is 9 to 10 amino acid residues.

The present invention further includes a polypeptide containing the IEX-1-derived peptide of the present invention or a mutant peptide thereof having specific CTL inducing activity. The number of amino acids constituting the polypeptide is not particularly limited, and follows the common general technical knowledge in the field to which the present invention belongs. Usually, the length is about 100 or less amino acid residues, preferably about 50 or less, more preferably about 30 or less.
The polypeptide of the present invention can be fragmented in HLA-A33 positive cells to give a peptide fragment having specific CTL inducing activity. Such a polypeptide contains a partial sequence corresponding to the peptide of the present invention represented by SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, or a sequence corresponding to a variant of the peptide of the present invention. Is particularly preferred.

Furthermore, the peptides and polypeptides of the present invention can be altered by modifying the constituent amino acids or carboxyl groups to such an extent that the function is not significantly impaired. For example, a formyl group, an acetyl group, a t-butoxycarbonyl (t-Boc) group or the like may be bonded to the N-terminus or a free amino group, and the C-terminus or free carboxyl group of an antigen peptide may be A methyl group, an ethyl group, a t-butyl group, a benzyl group or the like may be bonded. Furthermore, the peptide of the present invention may be modified so as to facilitate introduction into the living body.
In general, it is known that there are different motifs (regular sequences) depending on the type of HLA in the amino acid sequences of tumor antigen peptides that bind to HLA molecules, and mutations or modifications are permitted on the motifs. It is preferable that From the viewpoint of not changing the basic properties (physical properties, functions, physiological activities or immunological activities, etc.) of peptides or polypeptides in the introduction of mutations, for example, homologous amino acids (polar amino acids, nonpolar amino acids, hydrophobicity) Mutual substitution between amino acids, hydrophilic amino acids, positively charged amino acids, negatively charged amino acids, aromatic amino acids, etc.) is readily envisioned.
The peptides and polypeptides of the present invention can be produced by general known methods in peptide chemistry.

“Antigen-presenting cell (APC)” means a cell that presents a complex of HLA and an antigen peptide on the cell surface. Therefore, the antigen-presenting cell has a function of bringing about activation of HLA-A33-restricted CTL by presenting the complex of the tumor antigen peptide of the present invention and the HLA-A33 molecule on the cell surface. Such cells also include tumor cells that are targets for CTL cytotoxic effects.
The antigen-presenting cell of the present invention presents a complex of the HLA-A33 molecule and the tumor antigen peptide of the present invention on the surface of an isolated cell having the ability to present antigen. Such cells can also be induced in vitro. Specifically, cells having the antigen-presenting ability of HLA-A33 positive are pulsed with the tumor antigen peptide of the present invention, and HLA-A33 and the peptide are used together. It is obtained by presenting the complex on the cell surface. The “cell having antigen-presenting ability” is not particularly limited as long as it is a cell that expresses the HLA-A24 antigen capable of presenting the tumor antigen peptide of the present invention on the cell surface. Dendritic cells are preferred. In particular, antigen-presenting cells in which a complex of an HLA-A33 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 an HLA-A33-positive tumor patient is preferable.

Nucleic Acid Molecules The nucleic acid molecules of the present invention comprise single-stranded (including complementary strands) and double-stranded polypec- tides encoding amino acid sequences of the IEX-1-derived peptide of the present invention or a mutant peptide thereof and a polypeptide comprising the peptide. Contains nucleotides. The nucleic acid molecule of the present invention may be DNA or RNA. Peptides having the amino acid sequence encoded by these nucleic acid molecules are recognized by CTLs themselves and can activate the CTLs or give peptide fragments having such activities, and can function as tumor antigens.
Further, the nucleic acid molecule of the present invention may be a polynucleotide comprising at least 24 bases corresponding to the region encoding the peptide of the present invention and its complementary strand. Such a polynucleotide can be selected, for example, by confirming the expressed peptide using a known protein expression system.

Antibody The antibody of the present invention may be either a monoclonal antibody or a polyclonal antibody. A peptide or polypeptide that specifically recognizes an epitope peptide or polypeptide comprising at least 5 consecutive amino acid residues in the amino acid sequence of one peptide or polypeptide selected from the IEX-1-derived peptides or polypeptides of the present invention It is.
Antibodies can be generated using these epitope peptides, which epitope peptides are composed of at least 5, preferably at least 8-10 amino acids. The present invention also includes a peptide consisting of at least 5 amino acid residues and a nucleic acid molecule encoding the peptide.
The antibody of the present invention can induce production by immunizing, for example, mice, rats, rabbits, goats and the like, in the presence or absence of an adjuvant, with the epitope peptide alone or in a form bound to a carrier. The obtained polyclonal antibody can be recovered from the serum by a known method.
On the other hand, monoclonal antibodies can be produced by fusing antibody-producing cells collected from animals that have induced immune responses as described above with permanently proliferating cells. This method is well known in the art.
These polyclonal and monoclonal antibodies can be used as purifying antibodies, reagents, labeling markers and the like. Moreover, it may be humanized and used for treatment.

Pharmaceutical Composition The pharmaceutical composition of the present invention comprises an IEX-1-derived peptide, mutant peptide, polypeptide, antigen-presenting cell, peptide or polyporoid presenting a complex of an HLA molecule and a peptide on the cell surface. It can be prepared by using a nucleic acid molecule encoding a peptide, a recombinant vector prepared based on the nucleotide sequence information of the nucleic acid molecule, or an antibody of the present invention alone or in combination.
Specifically, the IEX-1-derived peptide of the present invention or a variant thereof, a polypeptide containing the peptide, and antigen-presenting cells can be used as a cancer vaccine. Although one type of peptide is effective as a cancer vaccine, it is preferable to use a combination of multiple types of peptides. This is because the CTL of a cancer patient is a population of cells that recognize a plurality of different types of tumor antigens, and it is expected that it is more effective to use a combination of a plurality of types of tumor antigens as a cancer vaccine. Because. A plurality of types of peptides according to the present invention may be used in combination with other peptides.

  The cancer vaccine of the present invention can be used alone or in combination with a pharmaceutically acceptable carrier in the presence or absence of a suitable adjuvant. The carrier is not limited as long as it does not adversely affect the human body. For example, cellulose, polymerized amino acid, albumin and the like can be used. As the dosage form, a known dosage form for the peptide preparation can be selected. The dose varies depending on the recognizability by CTL, the disease to be treated, the age of the patient, the body weight, etc. In the case of peptides, the active substance is usually 0.0001 mg to 1000 mg, preferably 0.001 mg to 1000 mg, more preferably 0.1 mg to 100 mg, more preferably 0.1 to 10 mg / day / adult human. This is administered once every few days or several weeks or months.

  The pharmaceutical composition of the present invention can also be used to incorporate a nucleic acid sequence encoding the peptide of the present invention into an appropriate vector and introduce it in vivo or ex vivo. Examples of the vector include retrovirus, adenovirus, vaccinia virus and the like, but a retrovirus system is preferable. Although the dose varies depending on the recognition by CTL, the DNA content is 0.1 μg-100 mg / day / adult human, preferably 1 μg-50 mg / day / adult human. This is administered once every few days or months.

The IEX-1-reactive CTL and the induction method “IEX-1-reactive CTL” mean a CTL that recognizes and induces a complex of the IEX-1-derived peptide of the present invention or a variant thereof and HLA. . The CTL is HLA-A33 restricted CTL.
Such CTLs can be derived, for example, from peripheral blood mononuclear cells (PBMC) of HLA-A33 ⁺ gastric cancer patients using the peptides according to the present invention.
That is, HLA-A33 ⁺ gastric cancer patient's PBMC is incubated with antigen-presenting cells (APC) pulsed with the peptide of the present invention to induce CTL, and IFN-γ production is evaluated as an index. Furthermore, the induced CTL activity can be confirmed by a ⁵¹ Cr release test or the like using tumor cytotoxicity as an index.

  The above method can be used for adoptive immunotherapy in which antigen-specific CTLs induced in vitro are returned to the body of a patient to injure tumor cells. 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 amounts outside the body and returned to the patient (J. Natl. Cancer. Inst., 86 : 1159, 1994). In mouse melanoma, spleen cells are stimulated with tumor antigen peptide TRP-2 in vitro, CTL specific for the tumor antigen peptide is proliferated, and the CTL is administered to melanoma transplanted mice 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 is 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.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not restrict | limited in any meaning by the following Example.

The following cancer cell lines were used in the present invention.
Gastric adenocarcinoma MKN-28, MKN-45, SSTW-9, KATO-III, KWS, and HGC-27; lung adenocarcinoma LC-1; lung squamous cell carcinoma QG-56; head and neck cancer KUMA-1; SW620 and COLO201; pancreatic adenocarcinoma Panc-1; human chronic myeloid leukemia K562. The HLA class I genotype of these tumor cells has been shown previously (10, 11).
The expression of HLA class I or HLA-A33 antigen on these cells is anti-HLA class I (W6 / 32) monoclonal antibody (mAb) (recognizing a single region of the HLA class I molecule), or anti-HLA-A33 mAb. (Recognizing the polymorphic region of the HLA-A33 molecule) (IgM, One Lamda, Canoga Park, Calif.) And measured by flow cytometry on a FACScan (Becton Dickinson, San Jose, Calif.).
For statistical analysis, a two-sided Student t test was used in all of the present invention.

Example 1
(Establishment of 850B-CTL strain)
HLA-A33-restricted and tumor-specific CLT strain (850B-CTL) has a TIL of 10% FCS (Equitech Bio) for patients with severe gastric adenocarcinoma (HLA-A ^* 2402 / A ^* 3303, B7 / B44, Cw7 / Cw14). , Ingram, TX), 100 U / ml IL-2 (Shionogi Pharmaceutical, Osaka, Japan), and 10 μg / ml PHA (Difco, Detroit, MI) -containing culture medium (45% RPIM1640 medium, 45% AIM-V medium; Life Technologies, It was established by incubation for 14 days in Walkersville, MA, followed by further culturing for more than 30 days in the presence of irradiated (30 Gy) allogeneic peripheral blood mononuclear cells (PBMC) as support cells. The phenotype of this CTL line was examined by immunological fluorescence test using FITC-conjugated anti-CD3, CD4, or CD8 monoclonal antibody (mAb) and found to be CD3 ⁺ CD4 ⁻ CD8 ⁺ (> 95%) (data) Not shown).

(Characteristics of 850B-CTL strain)
The 850B-CTL strain was characterized according to literature methods (Reference 9).
The 850B-CTL cell line was tested at various E: T ratios (effector cells: target cells) for the ability to produce IFN-γ by recognizing target cells. The value represents the average of triplicate measurements by ELISA (detection limit 10 pg / ml). As shown in FIG. 1A, this CTL line produced significant levels of IFN-γ by recognizing HLA-A33 ⁺ epithelial cancer cells, LC-1 and KUMA-1, but it was found in HLA-A33 ^- target cells. There was no reaction.

Next, the cytotoxic activity of 850B-CTL against various target cells was tested by a 6 hr ⁵¹ Cr release test at different E: T ratios. This method is known (Reference 9). The value represents the average of triplicate measurements. 850B-CTL showed stronger cytotoxicity against HLA-A33 ⁺ LC-1 and KUMA-1 cells, but HLA-A33 ^- target cells, COS7 cells, NK target cell lines, K562, or healthy donors None of the HLA-A33 ⁺ PHA-activated normal T cells (PHA immature cells) obtained from human PBMCs.

Furthermore, the reactivity of 850B-CTL was examined by an inhibition experiment using mAb. Anti-HLA class I (W6 / 32, IgG2a), anti-CD8 (Nu-Ts / c, IgG2a), anti-HLA-A24 (0041HA, IgG2a), anti-CD4 (Nu-Th / i, IgG1), anti-HLA class IB , C (B1-23, IgG2a), and anti-HLA class II (H-DR, IgG2a) mAb (20 μg / ml) were used in the same manner as described in the literature (10, 11). Anti-CD14 (JML-H14, IgG1) or anti-CD13 (MCS2, IgG2a) was prepared as an isotype matched control mAb.
IFN-γ production from 850B-CTL by recognition of HLA-A33 ⁺ LC-1 cells was inhibited by anti-HLA class I and anti-CD8 mAb (20 μg / ml), but anti-HLA-B, C, anti-HLA class It was not inhibited by II, anti-HLA-A24, anti-CD4, or an irrelevant isotype-matched anti-CD13 or anti-CD14 mAb (FIG. 1C).
These results indicate that the 850B-CTL line shows HLA-A33 restricted cytotoxicity against tumor cells but not normal cells.

Example 2
(Identification of IEX-1 gene)
A gene encoding a tumor antigen recognized by the 850B-CTL strain was identified by a gene expression cloning method (Reference 9).
LC-1 lung adenocarcinoma cell poly (A) ⁺ RNA was converted to cDNA, SalI adapters were ligated and inserted into the expression vector pSV-SPORT-6 (Invitrogen, San Diego, Calif.). HLA-A ^* 3303 or HLA-A ^* 2601 cDNA was prepared by RT-PCR from RNA recovered from KUMA-1 or KE4 cells, respectively, and inserted into a eukaryotic cell expression vector pCR3 (Invitrogen) for cloning. Both the plasmid DNA pool or clone (200 ng) of the LC-1 cDNA library, and HLA-A ^* 3303 or HLA-A ^* 2601 (negative control) cDNA (200 ng), 1 μl Lipofectamine (Invitrogen) and 120 μl Opti Mix for 40 minutes in MEM (Invitrogen). COS7 cells (5 × 10 ³ ) were incubated with 50 μl of this mixture for 6 hours, followed by addition of 150 μl of RPMI 1640 medium containing 10% FCS. After culturing for 2 days, 850B-CTL (2 × 10 ⁵ cells / well) was added, followed by incubation for 18 hours, and 100 μl of the supernatant was recovered, and IFN-γ was measured by a duplicate test by ELISA (Reference 9).

In the primary screen, all 1 × 10 ⁵ clones from the LC-1 cDNA library were tested for their ability to stimulate IFN-γ production by 850B-CTL after transfection into COS7 cells with HLA-A ^* 3303 cDNA. That is, the cDNA pool (1 × 10 ⁵ clones) was divided in duplicate into about 2000 different wells in 96 flat bottom plates (expected number of clones in each well: 100 clones / well). The primary screen resulted in significant levels of IFN-γ production in 10 different wells. As a secondary screen, each cDNA pool obtained by cloning from positive wells was divided in duplicate into about 200 different wells of 96 flat bottom plates and tested for IFN-γ production stimulating activity.
After secondary screening, two positive clones for further assay were identified. DNA sequencing was performed by the dideoxynucleotide sequencing method using a DNA sequencing kit and an ABI PRISM 377 DNA sequencer (Perkin-Elmer, Foster, CA). The following examination was performed on one of the isolated genes.

As shown in FIG. 2, COS7 cells transfected with clone 1 and HLA-A ^* 3303 induced IFN-γ production in 850B-CTL in a dose-dependent manner, but clone 1 and HLA-A as a negative control. ^* Cells transfected with 2601 were not induced. In contrast, COS7 cells transfected with either clone 1 or HLA-A ^* 3303 alone were not recognized by 850B-CTL (data not shown). Furthermore, other clones used as negative controls from the LC-1 cDNA library were unable to induce IFN-γ production in 850B-CTL when transfected into COS7 cells with HLA-A ^* 3303 (data) Not shown). This suggests that clone 1 encodes a tumor antigen that is specifically recognized by 850B-CTL.
A search of GeneBank revealed that the nucleotide sequence of clone 1 was identical to the sequence of IEX-1 (Reference 17) reported as a stress-induced anti-apoptotic gene.

Example 3
(Expression of IEX-1 mRNA and protein in normal and cancerous tissues)
Expression of IEX-1 mRNA in various tumors or normal tissues (Multiple Tissue Northern Blots, Clontech, Tokyo, Japan) was examined by Northern blot analysis using a ³² P-labeled IEX-1 probe according to previously described methods (references) 9) (FIG. 3A, lane 1: brain, lane 2: heart, lane 3: skeletal muscle, lane 4: colon, lane 5: thymus, lane 6: spleen, lane 7: kidney, lane 8: liver, lane 9: Small intestine, lane 10: placenta, lane 11: lung, lane 12: PBL). A β-actin probe was used as a control. As shown in FIG. 3A, a band of approximately ˜1.3 kb was clearly detected in all normal tissues except the brain (lane 1), and the heart (lane 2), kidney (lane 7), lung (lane 11). ), Or peripheral blood lymphocytes (PBL) (lane 12), and particularly low expression in the thymus (lane 5), spleen (lane 6), liver (lane 8), or small intestine (lane 9) .

Next, mRNA expression of IEX-1 gene in normal and cancer cells was examined by Northern blot analysis (FIG. 3B, lane 1: PBL, lane 2: MKN45, lane 3: MKN28, lane 4: SSTW, lane 5: HGC27. Lane 6: LC-1, Lane 7: QG56, Lane 8: KUMA-1, Lane 9: Panc-1, Lane 10: SW620, Lane 11: COLO201, Lane 12: KATO-III). IEX-1 except for the HCG27 gastric cancer cell line (lane 5), stomach (lanes 2-4, 12), lungs (lanes 6, 7), head and neck (lane 8), pancreas (lane 9), and large intestine (lane 9) It was highly expressed in most of the adenocarcinoma and SCC cell lines derived from various organs including lanes 10 and 11).
These results imply that this gene is expressed in all cancers and normal tissues.

Furthermore, the expression of this gene in various tumor tissues was examined at the protein level. Expression of IEX-1 protein was performed on formalin-fixed paraffin-embedded tissue sections using a Ventana Medical Systems automation device (Tucson, AZ) with anti-IEX-1 antibody (Santa-Cruz biotechnology, Santa-Cruz, CA). Assessed by immunohistochemistry.
Representative staining in gastric cancer tissue is shown (FIG. 3C). In gastric cancer, IEX-1 protein expression was selectively enhanced in cancer cells but not in surrounding normal epithelium or connective tissue. IEX-1 protein was also highly and selectively expressed in various types of cancer tissues including breast cancer (FIG. 3D), lung cancer (FIG. 3E), and colon cancer (data not shown). 3C to 3E, IEX-1 protein is stained brown, and normal cells and connective tissue are not stained brown. For convenience, some of these spots stained brown are indicated by arrows.
From the above results, it can be said that IEX-1 is one of the ideal target molecules in the treatment of cancer.

Example 4
(Identification of IEX-1-derived antigenic peptide recognized by 850-B CTL)
The following experiment was conducted to identify a peptide that can be recognized by CTL as an antigenic epitope of IEX-1.
Among peptide sequences that may have a motif (References 18 and 19) for binding to the HLA-A33 molecule in the deduced amino acid sequence of IEX-1, computer analysis (Bioinformatics and Molecular Analysis Section (BIMAS), NIH, Bethesda, MD) used 8 different peptides with stronger binding activity to HLA-A33. Peptides with> 95% purity were obtained from BioSynthesis, Lewisville, TX. For peptide binding assays, RMA-S-A33 cells (RMA-S tap (transporter involved in peptide processing) stably transfected with HLA-A ^* 3303 cDNA) mouse lymphoma cells (Hiroko Takedatsu, et al., Identification of Peptide Vaccine Candidates Sharing Among HLA-A3, -A11, -A31, and -A33 Cancer Patients., Clin Can Res, 2004, in press) (1 × 10 ⁴ cells / well). Was incubated for 18 hours at 26 ° C. After washing with PBS, cells (1 × 10 ⁶ cells) were suspended in Opti-MEM (containing 3 μg / ml human β2-microglobulin and 10 μg / ml peptide) followed by 26 ° C. Incubated for 3 hours and 3 hours at 37 ° C. After washing with PBS, cells were incubated with anti-HLA-A33 mAb for 30 minutes at 4 ° C. Followed by incubation with FITC-conjugated rabbit anti-mouse IgM antibody (Cappel, Aurora, OH) for 30 minutes at 4 ° C. Cells were analyzed by FACScan and binding activity was assessed by mean fluorescence intensity (MFI) .26 Cells pulsed with TRP2-197 peptide (reference peptide) and non-pulsed cells were also used at 0 ° C. As shown in Table 1, all of the 8 peptides were RMA-S-A33 cells with slightly different affinity. Could be combined.

For detection of antigenic peptides recognized by the 850B-CTL line, C1R-A33 cells (C1R human multiple myeloma cells stably transfected with HLA-A ^* 3303 cDNA transfected (Hiroko Takedatsu, et al , Clin Can Res, 2004, in press) with the indicated concentration of peptide, 2 hours later, 850B-CTL (2 × 10 ⁵ cells / well) was added and incubated for another 18 hours. -Γ production was measured by ELISA.
IFN-γ production by 850B-CTL in response to peptide non-loaded C1R-A33 cells was subtracted from that value as background. Values represent the average of triplicate tests.

Three of these peptides, IEX 47-56, IEX 61-69, and IEX 65-73, induced significant levels of IFN-γ production in a dose-dependent manner (FIGS. 4A, 4B). The optimal concentration of the three peptides to load on HLA-A33 transfected C1R cells varied over the range of 0.1-1 μM for each peptide (FIG. 4B), but by RMA-S-A33 cells It was not dependent on their determined binding affinity for HLA-A33 molecules (Table 1).
Based on the above results, IEX47-56, IEX61-69, and IEX65-73 were identified as IEX-1-derived antigenic peptides recognized by the 850B-CTL strain.

Example 5
(Induction of CTL by IEX-1-derived peptide)
Regarding the HLA-A33-restricted and tumor-specific CTL inducing ability of IEX47-56, IEX61-69, and IEX65-73 peptides, HLA-A33 ⁺ epithelial cancer patients (n = 4, gastric cancer patients (2), lung cancer patients (1 ), Prostate cancer (1)) and HLA-A33 ⁺ healthy individuals (HD) PBMC.
HLA-A33 ⁺ cancer patients and HLA-A33 ⁺ healthy human PBMC (1 × 10 ⁵ / well) were combined with each peptide (10 μM) in 200 μl of IL-2 containing culture medium in a 96-well microculture plate (Nunc, Roskiide, Denmark). Incubated (Reference 13). On day 14, peptide-stimulated PBMC (80-120 × 10 ⁴ / well) were collected separately from each well, washed and divided into 4 equal parts. Two were stimulated with C1R-A33 cells loaded with the corresponding peptide and the other two were C1R-A33 cells loaded with the negative control HIV peptide. After 18 hours, supernatants were collected and tested for their IFN-γ producing activity.
A representative example of four patients is shown in FIG. IFN-γ production against HIV peptides (<50 pg / ml) was subtracted as background. Cancer patient-derived PBMC stimulated with these three peptides recognized HLA-A33 transfected C1R cells loaded with the corresponding peptide in most cases and produced significant amounts of IFN-γ (FIG. 5). In contrast, PBMCs obtained from 5 HD did not produce significant amounts of IFN-γ against them (data not shown).

Next, the tumor cytotoxic activity of peptide-derived CTLs was examined.
Cells capable of producing a significant amount of IFN-γ were collected, cultured for additional 10-14 days with IL-2 alone, and subjected to a ⁵¹ Cr release test at different E: T ratios for 6 hours (Reference 13). ^{LC-1 (HLA-A33 +} IEX-1 +), QG56 (HLA-A33 - IEX-1 +), and ^{HGC27 (HLA-A33 - IEX-} 1 -) for the measurement of the cytotoxic activity. As a control, PBMCs from cancer patients stimulated with a non-antigenic IEX-1-derived peptide, IEX 43-51, were used. Values indicate the average of triplicate measurements.
As shown in FIG. 6, PBMC stimulated with IEX-1 derived peptides showed a significant level of cytotoxicity against HLA-A33 ⁺ IEX-1 ⁺ LC-1 tumor cells, but HLA-A33. ^- it did not show against HGC27 or QG56 cells. IEX43-51 (negative control peptide) did not show specific CTL activity. This result suggests that IEX47-56, IEX61-69, and IEX65-73 are antigenic epitope peptides capable of inducing specific CTL in an HLA-A33-restricted manner in PBMC of epithelial cancer patients.

Furthermore, the cytotoxic restraint and peptide specificity were confirmed by inhibition tests and competition tests.
Inhibition tests include anti-HLA class I (W6 / 32, IgG2a), anti-HLA class II (H-DR, IgG2a), anti-CD8 (Nu-Ts / c, IgG2a), anti-CD4 (Nu-Th / i, IgG1) (20 μg / ml) was used. Anti-CD14 (JML-H14, IgG2a) mAb was used as a control.
The cytotoxicity of these peptide-stimulated PBMCs was significantly inhibited by anti-HLA class I or anti-CD8 antibodies in all cases tested, but not by other mAbs (FIG. 7).
In competition experiments, and the ratio of non-labeled C1R cells pulsed with the corresponding peptide or HIV peptide (negative control) to ^{a 51 Cr} release test unlabeled cells versus labeled cells were added at 10: 1. A ⁵¹ Cr release test was conducted at an E / T ratio of 10: 1. Values represent the mean ± SD of specific injury activity (%).
Cytotoxicity was inhibited by adding corresponding peptide pulsed C1R-A33 cells, but not in HIV peptide pulsed cells (FIG. 8).
These results suggest that peptide-specific CTL activity is exerted by CD8 + T cells mainly in HLA-A class I restriction.

The present invention discloses that IEX-1 encodes a tumor antigenic epitope recognized by HLA-restricted and tumor-specific CTL established from T cells infiltrating gastric adenocarcinoma. . The present invention also discloses that the IEX-1-derived antigenic peptide can induce tumor-specific CTL in an HLA-restricted manner in PBMC cultures of cancer patients.
Since IEX-1 is also expressed in normal tissues, particularly heart, kidney, lung and PBL, these organs may be adverse events of specific immunotherapy with IEX-1-derived antigenic epitopes. However, the present invention shows that both the 850-BCTL strain and the CTL induced by the IEX-1-derived peptide lyse HLA-A33 ⁺ tumor cells, while PHA is present in culture despite the presence of an excess of the corresponding peptide. It was revealed that activated normal HLA-A33 ⁺ T cells were not damaged. In clinical trials using peptide vaccines derived from tumor antigens conducted by the inventors, no serious adverse events have been observed even though some tumor antigens are widely expressed in normal tissues or organs. (References 4-7).
Therefore, the above results suggest that the IEX-1-derived peptide of the present invention can be used as a peptide vaccine suitable for cancer treatment.

In addition, radiation and some chemotherapeutic drugs have been reported to induce IEX-1 expression at relatively high levels (Refs. 26, 30), thus targeting the IEX-1 molecule of the present invention. Specific immunotherapy can be a novel and attractive method especially for the treatment of patients suffering from chemotherapy or radiation resistant cancer.
HLA-A33 is one of the most common HLA-A alleles in Asians and blacks and is found in 13% of Japanese, 14% of Koreans, 14% of whites, and 16% of blacks ( References 14, 15). IEX-1 is highly expressed in cancer tissues. Therefore, the antigenic peptides of the present invention will be widely available for specific immunotherapy against HLA-A33 ⁺ cancer patients.

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Claims (10)

  A peptide comprising the amino acid sequence shown in any one of SEQ ID NOs: 1 to 3.   A peptide comprising an amino acid sequence in which one amino acid is substituted in the amino acid sequence shown in any one of SEQ ID NOs: 1 to 3, and which binds to an HLA-A33 molecule and induces a CTL specific for the peptide. 3. An antigen-presenting cell obtained by presenting a complex of an HLA-A33 molecule and the peptide according to claim 1 or 2 on the surface of an isolated cell having an antigen-presenting ability. A nucleic acid molecule encoding the peptide according to claim 1 or 2 . A vector containing the nucleic acid molecule according to claim 4 . The peptide according to claim 1 or 2, antigen-presenting cells according to claim 3, claim 4 nucleic acid molecule according or claim 5 comprising the vector of the peptides or pharmaceutical specific CTL induction to the antigens, Composition. The pharmaceutical composition according to claim 6 , which is a cancer vaccine. An IEX-1 reactive CTL that recognizes a complex of the peptide according to claim 1 or 2 and HLA, or a complex presented to an antigen-presenting cell according to claim 3 . A method for inducing IEX-1 reactive CTL in vitro using the peptide according to claim 1 or 2 or the antigen-presenting cell according to claim 3 . An antibody having the peptide according to claim 1 or 2 as an epitope.

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