JPWO2002010369A1 - tumor antigens - Google Patents

Abstract

(57) [Abstract] The present invention provides a tumor antigen that induces and/or activates HLA-A2-restricted tumor-specific cytotoxic T cells that are activated upon recognition of HLA-A2 and a tumor antigen peptide, a peptide or polypeptide derived from the tumor antigen, a polynucleotide encoding the peptide or a polynucleotide that is its complementary strand, a transformant comprising a recombinant vector containing the polynucleotide, and the like.

Description

Detailed Description of the Invention

TECHNICAL FIELD The present invention relates to tumor antigens, and more particularly to tumor-specific cytotoxic T cells.
a peptide or polypeptide recognized by said polypeptide;
a polynucleotide that encodes the polynucleotide or its complementary strand,
A recombinant vector containing the nucleotide, a transformant containing the recombinant vector,
Antibodies against peptides or polypeptides, said peptides or polypeptides
or a compound having an interaction with said polynucleotide, said peptide and/or
and a cytotoxic T cell inducer comprising said polypeptide, and one or more of these.
and a pharmaceutical composition comprising the polypeptide, a method for producing the polypeptide,
Method for screening compounds having interaction with peptides or polynucleotides
a method for inducing cytotoxic T cells using said peptide or said polypeptide;
Peptide or said polypeptide or a polynucleotide encoding said polypeptide
The present invention relates to a method for measuring leukocytes and a reagent kit for use in said method.
CTLs (hereinafter sometimes abbreviated as CTLs) are important
In cancer patients, the tumor area shows cytotoxic activity against tumor cells.
Infiltration of cytotoxic T cells has been observed (Arch. Surg., 126:2
00-205, 1990). The target molecule of this tumor-specific cytotoxic T cell (tumor
Tumor antigens (TMS) were first discovered in melanoma.
Tumor antigens are degraded within cells to form peptides consisting of 8 to 11 amino acids (tumor antigens).
The peptides are then converted into human leukocyte antigen (HLA) molecules, which are major histocompatibility antigens.
Cytotoxic T cells bind to HLA and tumor antigens and are presented on the surface of tumor cells.
It recognizes the complex consisting of the peptide and damages tumor cells.
T cells recognize tumor antigen peptides in an HLA-restricted manner. HLA is a cell membrane antigen that is expressed on almost all nucleated cells.
A is broadly divided into class I antigens and class II antigens, but they are anti-
The HLA recognized together with the original peptide is a class I antigen.
Hara is further classified into HLA-A, HLA-B, HLA-C, etc., and their genes
It has been reported that HLA-A subregion polymorphism is abundant.
The A-A2 allele is present in approximately 23% of African blacks and approximately 10% of Chinese.
53%, about 40% of Japanese, about 49% of North American Caucasians, and about 53% of South Americans.
It is found in approximately 38% of African Caucasians. Here, tumor antigens are tumor antigens that can induce tumor-specific cytotoxic T lymphocytes.
It also refers to a protein, polypeptide, or peptide contained in tumor cells.
The original peptide is a peptide generated by degradation of the tumor antigen within tumor cells,
Tumor-specific cytotoxicity by binding to HLA molecules and being presented on the cell surface
It means a peptide that can induce or activate T cells.
The site of the amino acid sequence capable of inducing tumor-specific cytotoxic T lymphocytes is designated as a tumor antigen.
Recently, many genes encoding tumor antigens that can be recognized by cytotoxic T cells have been identified.
The gene has been identified from the cDNA of human cancer cells (Science, 254:
1643-1647, 1991) (J. Exp. Med., 183:1185-
1192, 1996) (J. Immunol., 163:4994-5004,
1999). These include HER/neu (Proc. Natl. Acad. Sc
USA, 92:432-436, 1995), mutant cdk (Science
, 269:1281-1284, 1995), and mutant CASP-8 (J.E.
xp. Med., 186:785-793, 1997), etc., and
It is also found in tumor-specific immune responses, such as tumor rejection antigen genes and T cell antigen receptors (TCR).
The molecules involved have been shown to be effective in the treatment of melanoma, esophageal cancer, and other cancers over the past decade.
have been identified and are being used for specific immunotherapy of advanced or metastatic cancers.
Currently, in Europe and the United States, tumor antigen administration is used to activate cytotoxic T cells in cancer patients.
Cancer vaccine therapy is being developed to target melanoma-specific tumor antigens.
For example, the melanoma antigen gp10
The peptide was administered subcutaneously to melanoma patients, and interleukin-2 (IL-2)
When administered intravenously, tumor shrinkage was observed in 42% of patients (Natural
re Medicine, 4:321, 1998). Thus, tumor antigens
However, most of the identified tumor antigens are derived from melanoma, and the pathogenesis of the disease is unclear.
Such specific immunotherapy for common epithelial cancers and adenocarcinomas, such as pancreatic cancer, is
Pancreatic cancer accounts for 10% of the global cancer burden.
It is one of the leading causes of death, with approximately 27,000 deaths annually in the United States and approximately 50,000 deaths in Europe.
0,000 people die from pancreatic cancer. This huge number of deaths is primarily due to
This is due to the lack of effective treatments, the difficulty of diagnosis, and the aggressive nature of this cancer.
Only 1-4% of patients with liver cancer survive the disease, and the incidence and mortality rates are actually
Therefore, the development of new treatment methods, such as specific immunotherapy, is expected.
Furthermore, when considering the diversity of cancer, it is believed that the same tumor antigens are present in all cancer cells.
Of course, it is unlikely that a single tumor antigen is expressed to the same extent as the cell surface antigen.
Cancer vaccine therapy that activates cytotoxic T cells can also be used to treat cancers that have the tumor antigen.
However, tumor antigen-specific cytotoxicity is not yet achieved in cancer treatment.
To induce and activate immune T cells and achieve high therapeutic effects in response to cancer diversity.
Therefore, it is important to discover and utilize numerous new tumor antigens that correspond to the diversity of cancer.
DISCLOSURE OF THE INVENTION One aspect of the present invention is a method for producing a nucleotide sequence of a nucleotide sequence according to any one of SEQ ID NOs: 1 to 44 in the sequence listing.
One aspect of the present invention is a peptide having an amino acid sequence represented by any one of SEQ ID NOs: 45 to 53 in the sequence listing.
One aspect of the present invention is a polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NO: 1 to SEQ ID NO: 44 in the sequence listing.
and peptides consisting of the amino acid sequences of SEQ ID NO: 45 to SEQ ID NO: 53 in the sequence listing.
One or more polypeptides selected from the group consisting of the amino acid sequence according to any one of
One aspect of the present invention is a pharmaceutical comprising a peptide or polypeptide as set forth in any one of SEQ ID NO: 1 to SEQ ID NO: 44 in the sequence listing.
and peptides consisting of the amino acid sequences of SEQ ID NO: 45 to SEQ ID NO: 53 in the sequence listing.
One or more polypeptides selected from the group consisting of the amino acid sequence according to any one of
One aspect of the present invention is a cancer vaccine comprising a peptide or polypeptide of any one of SEQ ID NOs: 1 to 44 in the sequence listing.
and peptides consisting of the amino acid sequences of SEQ ID NO: 45 to SEQ ID NO: 53 in the sequence listing.
One or more polypeptides selected from the group consisting of the amino acid sequence according to any one of
and a method for treating pancreatic cancer, colon cancer, or gastric cancer, comprising administering to a subject a peptide or polypeptide of the formula (I) or (II) above.
One aspect of the present invention is a cancer vaccine comprising a compound represented by any one of SEQ ID NOs: 1 to 44 in the sequence listing.
and peptides consisting of the amino acid sequences of SEQ ID NO: 45 to SEQ ID NO: 53 in the sequence listing.
One or more polypeptides selected from the group consisting of the amino acid sequence according to any one of
One aspect of the present invention is a cytotoxic T cell inducer comprising a peptide or polypeptide of any one of SEQ ID NOs: 1 to 44 in the sequence listing.
and peptides consisting of the amino acid sequences of SEQ ID NO: 45 to SEQ ID NO: 53 in the sequence listing.
One or more polypeptides selected from the group consisting of the amino acid sequence according to any one of
A method for the treatment of cytotoxic T cells, characterized by using a peptide or polypeptide of
One aspect of the present invention is a method for inducing a gene encoding a nucleotide sequence of a target gene, which is a nucleotide sequence of a target gene, comprising a nucleotide sequence of a target gene, ...
A polynucleotide encoding a peptide or polypeptide consisting of the amino acid sequence
One aspect of the present invention is a nucleic acid sequence represented by any one of SEQ ID NOs: 54 to 62 in the sequence listing.
One aspect of the present invention is a polynucleotide having a sequence as set forth in any one of SEQ ID NO: 54 to SEQ ID NO: 62 in the sequence listing, or a complementary strand thereof.
The polynucleotide described above, wherein the polypeptide encoded by the polynucleotide
induces and/or is recognized by cytotoxic T cells
One aspect of the present invention is a polynucleotide or a complementary strand thereof, which ...
One aspect of the present invention is a polynucleotide that hybridizes under the above conditions.
hybridizes under stringent conditions with a nucleotide or its complementary strand
One aspect of the present invention is a recombinant vector containing the polynucleotide or its complementary strand, or the polynucleotide.
hybridizes under stringent conditions with a nucleotide or its complementary strand
One aspect of the present invention is a recombinant expression vector containing the polynucleotide or its complementary strand, or the polynucleotide.
hybridizes under stringent conditions with a nucleotide or its complementary strand
A recombinant vector or an expression recombinant vector containing a polynucleotide that acts
One aspect of the present invention is a transformant transformed with the vector.
hybridizes under stringent conditions with a nucleotide or its complementary strand
The recombinant vector containing the polynucleotide that induces the
and culturing the transformant.
In one aspect of the present invention, the peptide or polypeptide and/or H
Interacting with HLA-A2, it is activated by at least HLA-A2-restricted cytotoxic T cells.
a compound that enhances recognition of said peptide or said polypeptide by said antibody; and/or
A compound that interacts with the polynucleotide or its complementary strand to enhance its expression.
a method for screening a compound, comprising:
a recombinant vector or an expression recombinant vector,
and using at least one of the vector, the transformant, or the antibody.
In one aspect of the present invention, a method for screening the peptide or polypeptide and/or H
Interacting with HLA-A2, it is activated by at least HLA-A2-restricted cytotoxic T cells.
a compound that enhances recognition of said peptide or said polypeptide by said antibody; and/or
A compound that interacts with the polynucleotide or its complementary strand to enhance its expression.
a method for screening a compound, comprising:
a oligonucleotide or its complementary strand, said recombinant vector or said expression recombinant
At least one of the vector, the transformant, or the antibody is used.
In one aspect of the present invention, a compound is obtained by the screening method, characterized in that at least one of the peptides or polypeptides is
Compounds that enhance the recognition of HLA-A2-restricted cytotoxic T cells against
or a gene that interacts with the polynucleotide or its complementary strand to enhance its expression.
One aspect of the present invention is a compound comprising the peptide, the polypeptide, or the polynucleotide.
or a complementary strand thereof, the recombinant vector or the recombinant expression vector,
The antibody and/or the compound are contained in the transformant.
The present invention also provides a pharmaceutical composition for use in cancer treatment, comprising the pharmaceutical, the cancer vaccine, and the induction of cytotoxic T cells.
One aspect of the present invention is to use the peptide, polypeptide, or polynucleotide as an agent or pharmaceutical composition for cancer disease.
One aspect of the present invention is a method for quantitatively or qualitatively measuring a nucleotide.
A reagent kit used in a method for quantitatively or qualitatively measuring nucleotides.
and the peptide, polypeptide, polynucleotide or its complement.
One aspect of the present invention is a reagent kit comprising at least one of the peptide, polypeptide, or polynucleotide.
A reagent kit used in a method for quantitatively or qualitatively measuring nucleotides.
and the peptide, polypeptide, polynucleotide or its complement.
A reagent kit containing at least one of the chains or the antibody for cancer disease testing
BEST MODE FOR CARRYING OUT THE INVENTION The present inventors have identified tumor rejection antigen genes and
To identify the tumor antigen encoded by the gene, HLA-A2 was isolated from colon cancer patients.
HLA-A2-restricted, tumor-specific antibody that recognizes and activates tumor antigen peptides
Cytotoxic T cells were established (hereinafter, these cells may be referred to as OK-CTLp).
, encoding a tumor antigen that can be recognized by tumor-specific cytotoxic T lymphocytes (CTLs).
The gene responsible for this was isolated from a human pancreatic adenocarcinoma cell line,
It was isolated and identified from a cDNA library of Panc-1 cells.
X (Serological analysis of recombinant
cDNA expression libraries method (Proc.N
atl. Acad. Sci. USA, 92:11910-11813, 1995
) were identified as genes encoding tumor antigens, and similarly to the above, C
Furthermore, the tumors encoded by the obtained genes were identified.
Based on the tumor antigens, peptides having tumor antigen epitopes were found.
any peptide containing two or more amino acids linked to
Peptides are called polypeptides. For example, proteins are also called polypeptides in this specification.
Also included are short-chain peptides, also known as oligopeptides and oligomers.
Here, "recognize" means that something that recognizes something is recognized.
To distinguish the object being perceived from other substances and to recognize it, for example, to bind to the perceived object
In particular, in the present specification, it is meant that CTLs are capable of inhibiting tumor cells or tumor antigen peptides.
The term "recognizing a peptide" refers to the process by which CTL recognizes a tumor antigen peptide presented by HLA and binds to T cells.
"Activate" means to bind to a certain activity or
It is intended to further enhance or activate something or a condition that has an effect.
In particular, in the present specification, activation of CTLs means activation of CTLs by HLA.
By recognizing the antigen presented by the
means that CTLs show cytotoxicity against the target cells they recognize.
"Having" an activity or effect means changing from something or a state that has almost no activity or effect to something that has the activity or effect.
In particular, in the present specification, the term "antigen" refers to the ability to produce the desired effect.
Induction of specific CTL means the induction of specific CTLs in vitro or in vivo.
This means differentiating and/or proliferating CTLs that specifically recognize the antigen.
In addition, as used herein, an inducer of cytotoxic T cells is an agent that specifically recognizes a certain antigen.
There are no or very low percentages of CD8-positive T cells that recognize the
The condition suggests that a very large proportion of cytotoxic T cells that recognize the antigen are present.
(Isolation and Identification of Tumor Antigen Genes, Tumor Antigens, and Tumor Antigen Peptides) In the present invention, first, the OKT cells, which are HLA-A2-restricted cytotoxic T cells, are identified.
- Using CTLp as effector cells, tumor antigens capable of activating these cells are
, was isolated and identified using gene expression cloning.
Panc-1 cDNA and HLA-A0207 cDNA were transfected into COS-7
The co-transfection of the gene into cells, and among the cells in which the transgene was expressed, OK-CTLp
By selecting those that promote IFN-γ production from the
The specific method for identifying the gene encoding the tumor antigen is described in the Examples below.
As a result, seven antibodies were recognized by OK-CTLp in an HLA-A2-restricted manner.
A cDNA clone of SER was obtained from a cDNA library of the human pancreatic adenocarcinoma cell line CFPAC-1.
Genes encoding tumor antigens that have already been reported and detected by the EX method (Bi
ochem. Biophys. Res. Commun. , 281:936-94
4, 2001), two anti-tumor antibodies capable of activating CTL in an HLA-A2 restricted manner were identified.
The genes encoding the genotype, KM-PA-2 and KM-PA-4, were synthesized in the same manner as above.
The SEREX method is a method that can be used to detect tumor antigens.
However, among the more than 1,500 tumor antigens detected by this method, only cellular and liquid
MAGE-1, tyrosine kinase, and tyrosine kinase have been identified as tumor antigens that can induce immune responses.
Thus, the SEREX method was used to detect tumor antibodies.
Even if a tumor is identified as a tumor antigen, it does not necessarily mean that it can activate CTLs.
The tumor antigen genes KM-PA-2 and KM-PA-4 are HLA-A2 restricted.
It was first discovered in the present invention that the seven cDNA clones obtained from the Panc-1 cells described above were capable of activating TL.
These genes contained open reading frames (ORFs).
The base sequence is determined by the Sanger method (chain terminator method), and the base sequence
The amino acid sequence was deduced from these base sequences.
When a homology search was performed against existing databases such as GenBank,
Although there is high homology with the genes shown below, these are novel sequences.
The cDNA had the following structure:
The function of the seven obtained cDNA clones (clone
Among 1 to 7), clone 3 is the original clone obtained by the gene expression cloning method described above.
Wolf-Hirschhorn syndrome candidat
The base sequence of a gene highly homologous to the e2 protein (WHSC2) gene
was 25 bp shorter in the 5'-terminal region, the full-length cDNA was
From the cDNA library of c-1 cells, ³² The P-labeled clone was used as a probe.
The results were obtained by standard colony hybridization using the following:
The genes of the Panc-1 cells from which the clones 1 to 7 were derived were designated as genes 1 to 7, respectively.
7. The polypeptides consisting of the amino acid sequences encoded by each gene are called
These genes are also called gene products 1 to 7. The deduced amino acid sequences encoded by these genes are
The columns correspond to SEQ ID NOs: 45 to 51 in the sequence listing, and the base sequences correspond to SEQ ID NOs: 45 to 51 in the sequence listing.
The above genes 1 to 6 are shown in SEQ ID NOs: 54 to 60.
Gene 7 was registered with the National Institute of Genetics and the Japan DNA Data Bank (
The highly homologous genes shown in Table 1 were also found in KM-PA-2 and KM-PA-4.
The gene has already been reported (Biochem. Biophys. Res. Com
mun., 281:936-944, 2001). KM-PA-2 and KM-
The base sequences of PA-4 are shown in SEQ ID NO: 52 and SEQ ID NO: 53 in the sequence listing, respectively.
The amino acid sequences are shown in SEQ ID NO:61 and SEQ ID NO:62 in the sequence listing, respectively. The nucleotide sequence of gene 1 was obtained from GenBank (accession number: AF1551
The ubiquitin-conjugating enzyme variant Kua (UBE2V) gene registered in
The estimated amino acid length (aa) encoded by gene 1 is
, which was slightly longer than that of the UBE2V gene (three residues at positions 109-111).
The function of UBE2V has not been reported to date. The base sequence of gene 2 is the same as that of the heterogeneous nuclear ribonucleoprotein L gene (heterogene
ous nuclear ribonucleoprotein (HNRPL)
High homology was observed with the gene [accession number: NM001533].
However, the predicted amino acid length was similar to that of HNRP at positions 1-31 of the N-terminus.
The HNRPL gene product is a heterologous nuclear ribonucleoprotein.
The white matter complex is the base for the processing of pre-mRNA in the cytoplasm.
The base sequence of gene 3 is available from GenBank (accession number: AK0013
Wolf-Hirschhorn syndrome ca registered in 04)
High homology with the gene encoding the endodate 2 protein (WHSC2) was observed.
The WHSC2 gene is a genetic disorder that results from a partial deletion of the short arm of chromosome 4, causing mental and developmental disorders.
The phenotype of WHS, a multiple malformation syndrome characterized by
The base sequence of gene 4 is the same as that of the eIF-4E binding protein 1 gene (EIF4EBP1
High homology was observed with that of the gene encoding the nucleotide sequence of the present invention (accession number: NM004095).
The EIF4EBP1 gene product mediates insulin-dependent phosphorylation of 4E-BP1.
These factors are known as translation initiation factors and enable the formation of an active cap-binding complex.
The nucleotide sequence of gene 5 is based on a partially deduced sequence registered in GenBank.
Protein-activated protein kinase kinase (ppMAPkkk, activator
Session number: AJ242724) gene was found to be homologous to the gene.
The deduced amino acid sequence encoded by gene 5 is identical to that of the registered ppMAPkkk gene (
The N-terminal region is 230 aa and
The C-terminal end of gene 6 was 258 aa longer. The base sequence of gene 6 consisted of 6,707 bp and contained 2',5'-oligoadenilin.
OAS3 gene (accession number: NM
006187), but the 18th, 159th, 249th,
positions 287, 288, 316, 393-398, and 984
A total of 13 aa were different at the positions. 2-5 OAS3 is an IFN-inducible protein.
The base sequence of gene 7 is known as human cleavage and polyadenylation.
Denylation specificity factor (CPSF,
Accession number: U37012) (human cleavage and polyadenylation specificity factor
Although homology was found with positions 3701 to 4463 of the amino acid sequence of
The base sequence of the gene KM-PA-2 was 26 aa shorter than that of KIAA0124 and mouse b
Human homolog of lock of proliferation 1 (Bop1)
The KIAA0124 gene has a high homology to the 1466th base.
The amino acid residue at position 465 is a histidine (H).
However, in the gene KM-PA-2, the amino acids are adenine (A) and arginine (A), respectively.
The base sequence of the gene KM-PA-4 and the amino acid sequence encoded by the gene are
, and coactosin-like protein (CLP). To obtain tumor antigen peptides from the above nine genes encoding tumor antigens,
A peptide was designed and synthesized based on the amino acid sequence encoded by the above gene.
Regarding gene 7, the gene is a part of a gene consisting of a longer base sequence.
Therefore, the gene (CPSF) highly homologous to the gene is likely to encode
A peptide derived from the amino acid sequence was also designed and synthesized.
It is known that the amino acid sequence of a raw peptide contains motifs (regular sequences).
Therefore, first, we have to identify a molecule that has a motif (regular sequence) that can bind to HLA-A2.
The peptides that
) (Immunogenetics, 41:178, 1994) and obtained
The above gene generates different 9- to 11-mer peptides that match the motif.
The peptides were designed and synthesized based on the encoded amino acid sequence.
The recognition was performed using OK-CTLp or several clones obtained by cloning it by limiting dilution.
The OK-CTL clones were used, and IFN-γ produced by these CTLs was used as an index.
The OK-CTL clones recognized any of the above genes 1 to 7.
However, OK-CTLp recognizes all of genes 1 to 7.
It was found that this was a cell population that recognized multiple types of tumor antigens.
When examining the CTL activation ability of a peptide using a CTL clone,
A clone that recognizes the gene product encoding the peptide is used to
Of the synthesized peptides, 44 peptides (SEQ ID NOs: 1 to 44 in the sequence listing)
Tables 2 and 3) are recognized by OK-CTLp or OK-CTL clones.
Among these peptides, P1 to P2 stimulated IFN-γ production from CTLs.
5, P6 to P9, P10 to P13, P14 and P15, P16 to P18, and
P19 is gene 1, gene 2, gene 3, gene 4, gene 5, and
and a peptide consisting of a partial sequence of the amino acid sequence encoded by gene 6,
Genes highly homologous to each of these genes, UBE2V, HNPRL, WHSC2,
EIF4-EBP1, ppMAPkkk, and 2-5 are also encoded by OAS3.
In addition, P25, P26, P27, P28, P30 and P31 are
Amino acid sequence encoded by gene 7 and the CPSF gene highly homologous to gene 7
P20, P21, P22, P23, P24
P29, and P32 consist of partial sequences of the amino acid sequence specific to CPSF.
do. These tumor antigen peptides are presented on the cell surface by HLA-A2.
, T cell receptors (TCs) expressed on peptide-specific OK-CTL clones
R). Gene 1 and UBE2V, gene 2 and HNPRL
, or peptides derived from genes 6 and 2-5 OAS3 bind to TCR Vβ8.
1. OK-CTL expressing TCR Vβ3.2 or TCR Vβ14
The gene was recognized by the clones.
From gene 4 and EIF4EBP1, or gene 5 and ppMAPkkk
The peptides were TCR Vβ13.1, TCR Vβ8.1, or TCR Vβ13.1, respectively.
It was recognized by OK-CTL clones expressing R.Vβ18.
In addition, gene 1 and UBE2V, gene 2 and HNPRL, and gene
6 and 2-5. Each of the OK-CTL clones that recognize peptides derived from OAS3
The two have different complementarity-determining regions 3 (CDR3) (HLA class I molecules)
The TCR expressed a TCR with a nucleotide sequence (element responsible for binding to antigen epitopes in the groove of the
Gene 3 and WHSC2, gene 4 and EIF4EBP1, and gene
The OK-CTL clones that recognize peptides derived from ppMAPkkk were also
Furthermore, the 44 peptides recognized by OK-CTLp were
The autologous peripheral blood mononuclear cells (hereinafter abbreviated as PBMC) from the colon cancer patient from which OK-CTLp was obtained were used.
(may also be present), and/or HLA-A0201 ⁺ Cancer patients (pancreatic cancer patients,
HLA-A2-restricted tumor-specific C antigens were isolated from PBMCs of patients with colon cancer and gastric cancer.
CTLs were induced in vitro. The above CTLs were induced from peripheral blood mononuclear cells of cancer patients.
is HLA-A2 ⁺ Tumor cells were lysed in a dose-dependent manner.
HLA-A2 ⁻ Tumor cell RERF-LC-MS and HLA-A ⁺ However,
Autologous EBV-B cells derived from normal cells and T cells stimulated with PHA were not lysed.
Furthermore, the above CTLs were pulsed with the same peptide as that used for stimulation.
The above study revealed that 44 tumor anti-cancer drugs capable of activating OK-CTLp were effective against T2 cells in a dose-dependent manner.
Raw peptides are obtained, and these peptides are used to treat pancreatic cancer, colon cancer, and/or
can induce HLA-A2-restricted tumor-specific CTLs from PBMCs derived from gastric cancer patients.
In addition, we confirmed that the pancreatic adenocarcinoma cell line Panc-1 and the colon adenocarcinoma cell line SW6
20 both in OK-CTLp and in CTLs induced from PBMCs with the peptide.
This result suggests that pancreatic cancer and colon cancer are host-related.
These results suggest that the two tumor antigens share the same tumor antigen epitopes recognized by primary CTLs.
(Polypeptides and Peptides) The polypeptides of the present invention are derived from the human pancreatic adenocarcinoma cell line Panc-1.
The above genes 1 to 7 or genes obtained from the human pancreatic adenocarcinoma cell line CFPAC-1
From the amino acid sequence encoded by KM-PA-2 or KM-PA-4,
and preferably any one of SEQ ID NOs: 45 to 53 in the sequence listing.
These polypeptides are polypeptides consisting of the amino acid sequence set forth in
It can be used as a tumor antigen to induce and/or activate CTL.
The polypeptide is used to identify tumor antigen epitopes and obtain tumor antigen peptides.
The peptide according to the present invention can be prepared based on the amino acid sequence of the above polypeptide, for example,
For example, a peptide that matches the HLA-A2 restriction motif is designed, and the designed peptide is
those recognized by CTLs, for example, those that activate and/or induce CTLs
In the present invention, the peptide can be obtained by selecting a peptide having a
It binds to LA-A2, is presented on the surface of antigen-presenting cells, and is recognized by CTL.
It is sufficient that the antigen has the properties of a tumor antigen epitope that can be detected by the method, and the antigen is at least about
Five or more, preferably about seven or more, more preferably nine to ten amino acids
Particularly preferred is a peptide consisting of any one of SEQ ID NOS: 1 to 44 in the sequence listing.
These peptides are peptides consisting of the amino acid sequence described in any one of the following:
As a tumor antigen peptide, HLA-A2-restricted tumor-specific cytotoxic T cells
The polypeptide or peptide can be used to induce and/or activate CTLs.
Therefore, they may be used alone or in combination of two or more.
As described above, CTLs are a group of cells that recognize various antigens, and therefore,
It is recommended to use two or more of these in combination.
Mutations such as deletion, substitution, addition, or insertion of amino acids are introduced, and at least
and polypeptides or peptides recognized by HLA-A2-restricted CTLs.
The introduction of mutations such as deletions, substitutions, additions, or insertions is within the scope of the present invention.
The means are known per se, for example, the technique of Ulmer (Science, 219:66
6, 1983) can be used.
Does not change the basic properties of the peptide (physical properties, activity, immunological activity, etc.)
From this perspective, for example, homologous amino acids (polar amino acids, nonpolar amino acids, hydrophobic amino acids)
Neutral amino acids, hydrophilic amino acids, positively charged amino acids, negatively charged amino acids, aromatic amino acids
Interconversions between these available peptides (amino acids, etc.) are easily envisaged.
The peptides are modified by modifying their constituent amino groups or carboxyl groups, etc., to give them a distinctive function.
The polynucleotide of the present invention can be modified to the extent that it does not result in any significant changes.
-1 or genes 1 to 7 obtained from the human pancreatic adenocarcinoma cell line CFPAC-1 or
KM-PA-2 or KM-PA-4, SEQ ID NOs: 54 to 62 in the sequence listing
A polynucleotide consisting of any one of the base sequences described in any one of the preceding claims or its complementary strand.
The polynucleotide is any one of SEQ ID NOs: 1 to 44 in the sequence listing.
or a peptide having any one of the amino acid sequences set forth in SEQ ID NOs: 45 to 53.
Each of the polypeptides encoding the amino acid sequences described above is
Furthermore, the polynucleotide may be a complementary strand.
A small portion of the amino acid sequence of the peptide corresponding to the region encoding the tumor antigen epitope
A polynucleotide consisting of at least about 15 base sequences, preferably about 21 to 30 base sequences or more.
The polynucleotide may be a nucleotide and its complementary strand.
The selection and determination of the base sequence can be carried out, for example, by using a known protein expression system to generate expressed peptides.
Furthermore, the CTL inducibility and/or activation ability of the above polynucleotide can be confirmed by hybridizing the above polynucleotide under stringent conditions.
Polynucleotide molecules that are also included within the scope of the present invention.
Taking DNA molecules as a representative example, "DNA molecules undergo hyphenation under stringent conditions"
The "hybridizing DNA molecule" is, for example, a DNA molecule as described in Molecular Cloning
: A Laboratory Manual (Cold Spring Harbor Laboratory)
It can be obtained by the method described in (Rattley, 1989) or the like.
"Hybridizing under stringent conditions" means, for example, hybridization under conditions of 6x SSC, 0
After warming at 42°C in a solution of 5% SDS and 50% formamide, 0.
Even after washing in a solution of 1×SSC and 0.5% SDS at 68° C.,
This indicates that a positive hybridization signal is observed. When the polynucleotide is expressed in a cell having HLA-A2, it is possible to detect the HLA-A2.
It is possible to induce and/or activate LA-A2-restricted CTLs.
The polynucleotide has a poly(A) structure at its 3' end.
However, the number of poly(A) fragments is smaller than the coding region for amino acids that act as tumor antigens.
The number of poly(A)s contained in the polynucleotide does not particularly affect the position.
The above polynucleotides are not limited to the polypeptides or peptides of the present invention.
It provides genetic information useful for the production of nucleic acids, or as a reagent or
It can also be used as a standard product. (Recombinant Vector) By incorporating the above polynucleotide into an appropriate vector DNA,
The vector DNA to be used depends on the type of host and the
The vector DNA is extracted from naturally occurring vectors.
In addition to the above, there are also those that lack parts of DNA other than those necessary for proliferation.
For example, chromosomal, episomal and virally derived vectors, e.g., bacterial
Plasmid-derived, bacteriophage-derived, transposon-derived, yeast epitope-derived
derived from yeast chromosomal elements, derived from insertion elements, derived from yeast chromosomal elements, e.g., baculovirus
Rus, papovavirus, SV40, vaccinia virus, adenovirus, fowlpox
virus, pseudorabies virus, and viral vectors such as retroviruses
- and vectors combining them, such as plasmids and bacteriophages
Vectors derived from phage genetic elements, e.g., cosmids and phages
Depending on the purpose, expression vectors and cloning vectors can be used.
A recombinant vector carries the target gene sequence and information on replication and control.
Gene sequences, such as promoters, ribosome binding sites, terminators,
The components are a signal sequence, an enhancer, etc., and are synthesized by a method known per se.
The vector DNA is prepared by combining the polynucleotide of the present invention with the vector DNA.
The method for incorporating the nucleotide can be a method known per se. For example,
The DNA is then selected and treated to cut it at a specific site, and then the resulting vector is treated in the same way.
The method used is to mix the DNA with the DNA to be used and re-ligate it with ligase.
Alternatively, an appropriate linker may be ligated to the target polynucleotide, and the resulting product may be used as the target polynucleotide.
Alternatively, the desired gene can be inserted into the multicloning site of a vector suitable for the purpose.
The above-mentioned recombinant vector can be obtained by transforming the vector DNA incorporating the above-mentioned polynucleotide into a host known per se.
For example, Escherichia coli, yeast, Bacillus subtilis, insect cells, or animal cells can be cultured by a method known per se.
Transformants can be obtained by transformation with
A preferable system is integration into the chromosome, considering the stability of the gene.
However, an autonomous replication system using an extranuclear gene can be used more simply.
The vector DNA can be introduced into the host cell by, for example, the method described in Molecular C
loning: A Laboratory Manual (Sambrook et al.
Edited by Cold Spring Harbor Laboratory Press, Cold Spring
(Ling Harbor, New York, 1989).
Specifically, calcium phosphate transfection can be used.
DEAE-dextran mediated transfection, microinjection
ion, cationic lipid-mediated transfection, electroporation, transformation
Introduction, scrape loading, ballistic introduction
(ballistic introduction) and infection, etc.
(Production of Polypeptide or Peptide) If an expression vector is used as the vector DNA to be introduced into the transformant,
The polypeptide or peptide according to the present invention can be provided.
Transformants into which expression vector DNA containing the DNA fragment was introduced were transformed into each host.
The transformant is cultured under optimal culture conditions known per se.
The effect of the polypeptide or peptide according to the present invention, particularly the induction of at least CTL
and/or activating activity or the peptide produced in or outside the host.
The amount of the peptide or the transformant in the medium may be used as an index.
Subculture or batch culture may be carried out using the amount as an index. The peptide of the present invention can also be produced by a method known in ordinary peptide chemistry.
For example, Peptide Synthesis (Maruzen) 1975, "Peptide
Synthesis, Interscience, New York, 1996
" is an example, but of course, known methods are widely available. The recovery of the polypeptide or peptide according to the present invention can be carried out by
Using the CTL activation activity of the peptide as an index, molecular sieve and ion column chromatography were performed.
or in combination with other methods such as chromatography, or affinity chromatography.
can be purified and recovered by fractionation based on differences in solubility using ammonium sulfate, alcohol, etc.
More preferably, based on the information of these amino acid sequences,
The resulting polyclonal or monoclonal antibodies are used to produce antibodies against the
(Antibody) The antibody according to the present invention is produced using the above polypeptide or peptide as an antigen.
The antigen may be the above polypeptide or peptide itself or a fragment thereof.
It may be composed of at least 5, more preferably at least 8 to 10 amino acids.
To prepare an antibody specific to the above polypeptide or peptide,
a region consisting of an amino acid sequence unique to said polypeptide or said peptide
It is desirable that this amino acid sequence does not necessarily refer to the polypeptide or the peptide.
The amino acid sequence of the polypeptide or peptide does not necessarily have to be homologous to that of the
The exposed site on the outside of the three-dimensional structure is preferred, and the amino acid sequence of the exposed site is the primary structure
Even if the amino acid sequence is discontinuous, it is sufficient that the amino acid sequence is continuous at the exposed site.
The antibody is an antibody capable of immunologically binding to or recognizing the polypeptide or peptide.
The presence or absence of this binding or recognition can be determined by known antigen-antibody binding reactions.
To produce the antibody, a publicly known antibody production method can be used. For example,
The polypeptide or peptide according to the present invention may be administered alone in the presence or absence of an adjuvant.
The compound is administered alone or in combination with a carrier to an animal to stimulate humoral immunity and/or cellular immunity.
The carrier itself does not have any harmful effect on the host.
Otherwise, the polymer may be, but is not limited to, cellulose, polymerized amino acids, or albumin.
Examples of animals to be immunized include mice, rats, rabbits, goats, and horses.
Polyclonal antibodies can be prepared by isolating known antibodies from the serum of animals immunized with the above-mentioned methods.
The preferred method is immunoaffinity chromatography.
To produce monoclonal antibodies, the animals that have been immunized with the above methods are used.
Antibody-producing cells (e.g., lymphocytes from the spleen or lymph nodes) are harvested and purified.
into known immortalized cells (e.g., myeloma lines such as P3/X63-Ag8 cells)
For example, antibody-producing cells and permanently transforming them.
The proliferating cells are fused with the hybridoma cells by a method known per se to prepare hybridomas, which are then cloned.
and producing antibodies that specifically recognize the polypeptide or peptide.
Hybridomas that recognize and bind to the above polypeptide or peptide are selected, and antibodies are recovered from the culture medium of the hybridomas.
Clonal or monoclonal antibodies are used as purification antibodies, reagents, or labeled antibodies.
(Screening and compounds obtained by said screening) The above polypeptides or peptides, polynucleotides encoding them, and
and its complementary strand, and the transformant based on the information of these amino acid sequences and base sequences.
The cells or antibodies that immunologically recognize them can be used alone or in combination.
Screening of substances that can induce and/or activate CTLs by
The screening method provides an effective means for screening pharmaceuticals.
For example, as shown in the examples,
The activation of CTLs by antigen-presenting cells pulsed with tumor antigen peptides was investigated.
An experimental system is used to measure the amount of IFN-γ produced, and a test substance is added to the experimental system.
and a substance that induces and/or activates CTLs by
This experimental system can be used for screening or for selecting substances that enhance activation.
The screening method according to the present invention is
However, the present invention is not limited to the above. The present invention also covers compounds obtained by the above screening method.
The compound may be a polypeptide or peptide according to the invention and/or an HLA-
A2 to enhance the recognition of the polypeptide or peptide by CTL.
or a compound that interacts with the polynucleotide of the present invention to enhance its expression.
The compounds selected in this way can be compounds that have biological usefulness and toxicity.
By selecting the compound taking into consideration the balance of
(Pharmaceutical Composition) The polypeptide or peptide according to the present invention is a tumor antigen or a tumor antigen peptide.
As a target, it induces and/or induces HLA-A2-restricted tumor-specific cytotoxic T cells.
That is, the polypeptide or
A method for inducing CTLs characterized by using a peptide, and the polypeptide
The scope of the present invention also includes a CTL inducer containing the polypeptide or peptide of the present invention, a nucleic acid encoding the polypeptide, and a nucleic acid encoding the polypeptide.
the polynucleotides and their complementary strands, and their amino acid sequences and base sequences
A recombinant vector prepared based on the information, a recombinant vector transformed with the recombinant vector
a cell, or an antibody that immunologically recognizes the polypeptide or peptide,
Interacting with peptides or peptides and/or HLA-A2 to induce CTL
a compound that enhances recognition of said polypeptide or peptide by a target molecule, or said polypeptide
Compounds that interact with nucleotides to enhance their expression, either singly or in combination, are used.
By using them in combination, a pharmaceutical composition containing at least one of these
Specifically, for example, a pharmaceutical comprising the polypeptide or peptide according to the present invention can be provided.
Furthermore, a pharmaceutical composition containing the polypeptide or peptide according to the present invention may be
It can be used as a cancer vaccine.
Therefore, the polypeptide or peptide of the present invention can be administered in the presence of a suitable adjuvant.
It may be used alone or in the absence of a carrier.
As long as the substance itself does not have any harmful effect on the human body, it is not particularly limited, and examples include cellulose.
Examples of the dosage form include polypeptides known per se.
The dosage can be appropriately selected by applying the means for formulating the drug or peptide.
Although it varies depending on the degree of recognition of the peptide by CTL, it is generally the active form.
as a dose of 0.01 mg to 100 mg/day/adult human, preferably 0.1 mg to 10
The dose is 1 mg/day/adult human. This dose is administered once every few days to several months. Alternatively, a mononuclear cell fraction is collected from the peripheral blood of a patient, and the polypeptide of the present invention is administered.
or the mononuclear cells in which CTLs have been induced and/or activated by culturing with a peptide.
An effective cancer vaccine effect can also be obtained by returning the cell fraction to the patient's blood.
The mononuclear cell concentration during culture, the polypeptide or peptide of the present invention,
Culture conditions such as peptide concentration can be determined by simple experiments.
During the culture, substances that have lymphocyte proliferation ability, such as interleukin 2 (IL-2), are added.
When the polypeptide or peptide according to the present invention is used as a cancer vaccine,
Although a single polypeptide or peptide alone is effective as a cancer vaccine, multiple polypeptides or peptides
Several types of the above polypeptides or peptides can also be used in combination.
Because CTLs from cancer patients are a group of cells that recognize multiple tumor antigens,
Rather than using polypeptides or peptides as cancer vaccines,
In some cases, a more effective effect can be obtained by using the above-mentioned medicines, inducers of cytotoxic T cells, cancer vaccines, and pharmaceutical compositions.
Any of these is useful in the treatment of cancer diseases, such as pancreatic cancer, colon cancer, or gastric cancer.
Furthermore, a polynucleotide encoding the polypeptide of the present invention and its corresponding
The complement is also useful for gene therapy of cancer diseases, such as pancreatic cancer, colon cancer, or gastric cancer.
These polynucleotides are carried in vectors and directly introduced into the body.
There are two methods: one is to collect cells from a human and then introduce them ex vivo.
Vectors that can be used include retrovirus, adenovirus, and vaccinia.
Although viruses are known to be effective, retroviruses are recommended.
The dose is determined by the amount of the polynucleotide that is cloned by CTLs.
Although the degree of recognition of the polypeptide to be loaded varies, generally,
0.1 μg to 100 mg/day/as the content of DNA encoding the tumor antigen peptide
For an adult human, the dose is preferably 1 μg to 50 mg/day/adult human.
It is administered once every few months. (Method and reagent for diagnosis) The polypeptide or peptide according to the present invention, the polypeptide encoding said polypeptide,
Nucleotides and their complementary strands, as well as the polypeptides or peptides, are used in immunological studies.
The antibody that specifically recognizes the target protein can be used as a diagnostic marker or reagent by itself.
The present invention also provides a single or multiple-packaged container filled with one or more of these.
A reagent kit comprising one or more containers is also provided.
For example, a peptide or polypeptide, a polynucleotide, or an antibody known per se
A formulation method appropriate for each of the above may be introduced.
The diagnostic means may be, for example, a polypeptide of interest, or a polypeptide encoding the polypeptide of interest.
Using interactions or reactivities to determine the abundance of corresponding nucleic acids, and/or
or determining the biodistribution of said polypeptide or peptide in an individual.
and/or the presence of said polypeptide or peptide in a sample derived from an individual.
That is, the amount of the polypeptide of the present invention is determined.
The method defines a method for determining whether a gene or peptide, or a nucleic acid encoding the gene or peptides, is a diagnostic marker.
The amount of the polypeptide or peptide in the sample is measured either specifically or quantitatively.
Methods for quantitatively or qualitatively determining the nucleic acids encoding them are well known to those skilled in the art.
Such assays include radioimmunoassays.
B. Competitive binding assays, Western blot analysis, ELISA assays, etc.
Nucleic acids can also be synthesized by various methods, such as amplification, polymerase chain reaction (PCR), RT-PCR, etc.
PCR, RNase protection, Northern blotting and other hybridization
The sample to be measured can be cells from an individual, such as blood, urine, saliva, cerebrospinal fluid, tissue, etc.
Examples of the nucleic acid to be measured include biopsy or autopsy materials.
The nucleic acid is obtained from the sample by a known nucleic acid preparation method.
It may be used directly or may be subjected to PCR or other amplification methods prior to analysis.
RNA or cDNA may also be used.
The size change of the amplified product compared with the normal genotype indicates deletion and
The insertion can be detected by labeling the amplified DNA encoding the polypeptide.
Identifying point mutations by hybridizing to DNA containing the
The polypeptide of the present invention and the gene encoding said polypeptide can be detected by the above-mentioned assay method.
By detecting the mutation, loss, or increase of the DNA, it is possible to identify diseases associated with the polypeptide.
The present invention will be described in more detail below with reference to examples.
The present invention is not limited to the following examples. Example 1 (Establishment of HLA-A2-restricted CTL) An HLA-A2-restricted tumor-specific cytotoxic T lymphocyte line was established in a colon cancer patient (H
HLA-A0207/3101, HLA-B46/51, HLA-Cw1) tumors
The cells were established from infiltrating lymphocytes (TIL) (Int. J. Cancer, 81:45
9-466, 1999) (J. Immunol., 163:4997-5004
First, TILs obtained from a colon cancer patient were treated with 100 U/ml of recombinant human TILs.
Interleukin 2 (IL-2) was added and the cells were cultured for 50 days or more. Culture 7
A portion of the IL-2-activated TILs was collected daily and analyzed with various tumor or normal cells.
They were cultured together to measure the IFN-γ produced by the cancer cells and ⁵¹ Cr release
The CTL activity was assessed by a test (J. Immunol., 163:49
97-5004, 1999). IFN-γ was measured by enzyme-linked immunosorbent assay (ELISA).
On day 58 of culture, HLA-A2-restricted and tumor-specific CT was performed.
OK-CTLp, one of the sublines showing L activity, was obtained.
- CTLp is CD3 ⁺ CD4 ⁻ CD8 ⁺ 80% of cells have the phenotype of C
D3 ⁺ CD4 ⁺ CD8 ⁻ The cell population consists of 20% of cells with the phenotype
Using this OK-CTLp as effector cells, various cells such as tumor cells were
The cells are cultured together with target cells, and the cells are then subjected to cytotoxicity against the target cells. ⁵¹ Cr release
The test also showed that the activation of OK-CTLp was measured by the production of IFN-γ.
The results are shown in Figures 1 and 2. As shown in Figures 1 and 2, the obtained OK-CTLp
⁺ Panc-1 pancreatic adenocarcinoma cells and SW620 colon adenocarcinoma cells, HLA-A020
6 ⁺ KE3 esophageal squamous cell carcinoma (SCC) cells and HLA-A0207 ⁺ CA9
-22 oral SCC cells, producing IFN-γ and exhibiting sufficient cytotoxicity.
However, HLA-A2 ⁻ Tumor cells, QG56 lung adenocarcinoma cells, RER
F-LC-MC lung adenocarcinoma cells, COLO320 colon adenocarcinoma cells, and normal cell-derived
Autologous Epstein-Barr virus-transformed B cells (EBV-B) and phytohema
Glutinin (PHA) blasted autologous T cells (Autologous PHA-bl
It did not show cytotoxicity against leukocytes (e.g., leukocytes containing leukocyte antigens ... and leukocytes containing leukocyte antigens).
OK-CTLp were found to be positive for all HLA-A2 ⁺ Tumor cells (HLA-A0201
⁺ R27 breast adenocarcinoma cells, HAK-2 primary hepatocellular carcinoma cells, SK-MEL-5 melatonin cells,
SF126 astrocytoma cells, and SF126 astrocytoma cells, HLA-A0206 ⁺ PC9
Lung adenocarcinoma cells and HLA-A0207 ⁺ 1-87 lung adenocarcinoma cells and OMC-
The CTL activity was determined by lysing the 4 cervical SCC cells.
The antibody was blocked by a single antibody (mAb), anti-CD8 mAb, or anti-HLA-A2 mAb.
The inhibition was not observed by other mAbs (Figure 2).
OK-CTLp recognizes the above tumor cells in an HLA-A2-restricted manner and exhibits cytotoxicity.
The genotype of the HLA class I alleles of the tumor cells was determined as previously reported (J.I.
Mmunol., 163:4997-5004, 1999).
The HLA class I antigen type of the patient was determined using peripheral blood mononuclear cells (PBMC).
Furthermore, HLA-A2 subtypes were determined by sequence-specific oligonucleotides.
Nucleotide probe method and direct DNA sequencing
The surface phenotype of OK-CTLp was determined by full sequencing.
Anti-CD3 mAb labeled with fluorescein isothiocyanate (FITC), anti-CD
4 mAb, or anti-CD8 mAb (Nichirei), or anti-TCRαβ mAb (W
by direct immunofluorescence analysis with T31 (Becton Dickinson).
In addition, to examine the HLA restriction and specificity of OK-CTLp,
The antibodies used were anti-HLA class I mAb (W6/32, IgG2a), anti-HLA
-A2 mAb (BB7.2, IgG2b), anti-CD8 mAb (Nu-Ts/c,
IgG2a), anti-HLA class II mAb (H-DR-1, IgG2a), and
and anti-CD4 mAb (Nu-Th/i, IgG1).
CS-2, IgG2a) and anti-CD14 mAb (JML-H14, IgG1)
was used as an isotype-matched control mAb. Example 2 (Isolation and identification of cDNA clones encoding tumor antigens) The cDNA clones encoding the tumor antigens of Panc-1 tumor cells recognized by OK-CTLp were
The gene was cloned using a known gene expression cloning method (J. Immunol., 16
3:4997-5004, 1999).
Poly(A) of c-1 tumor cells ⁺ RNA was converted to cDNA and SalI-adapted.
The vector was ligated into the expression vector pCMV-SPORT-2 (Invit
The cDNA of HLA-A0207, HLA-A2402, or HLA-A2601 was inserted into the ribosomal RNA (manufactured by Irogen).
NA was obtained by reverse transcription polymerase chain reaction (RT-PCR) and expressed in eukaryotic cells.
The plasmid DNA pool or the Panc-1 cell cDNA library was cloned into the expression vector pCR3 (Invitrogen).
200 ng of the clone and 200 ng of HLA-A0207 cDNA were mixed together for 100 min.
The mixture was mixed in 1 μl of Opti-MEM (Invitrogen) for 30 minutes.
50 μl of this mixture was added to COS-7 cells (5 × 10 ³ ) plus 96-well U-bottom
The mixture was incubated in a microculture plate (Nunc) for 6 hours.
Then, RPMI-1640 medium containing 10% FCS was added.
The cells were cultured for 2 days, and OK-CTLp (5 × 10 ⁴ Cells) were added to each well.
After further incubation for 18 hours, 100 μl of the supernatant was taken and the produced
IFN-γ was measured by ELISA.
We investigated IFN-γ production by OK-CTLp using COS-7 cells as targets.
The value of the produced IFN-γ was subtracted from each measurement as background.
As a result, it is recognized by OK-CTL and promotes the IFN-γ production of the OK-CTL.
Seven cDNA clones were obtained. The base sequences of the seven obtained cDNA clones were determined by DNA sequencing.
ABI PRISM kit (Perkin-Elmer) was used. ^TM 377
DNA sequence was analyzed using a DNA Sequencer (Perkin-Elmer).
Deoxynucleotide sequencing was performed.
The amino acid sequences encoded by these clones were deduced from the base sequences.
A homology search was performed on the base sequence using GenBank.
The sequences are shown in Table 1. The seven cDNA clones obtained (clones 1 to 7)
Clone 3 is the original clone obtained by the gene expression cloning method.
Compared with the WHSC2 gene, which has a high sequence homology,
Since the cDNA was short, the full-length cDNA was cloned into a Panc-1 cell cDNA library.
-So, ³² The P-labeled clones were used as probes in standard colony hives.
As shown in Figure 3(A) and Figure 3(B), clones 1 to 7 were obtained by the chromatinization method.
It was recognized by OK-CTL and promoted IFN-γ production by OK-CTL.
HLA-A0207 cDNA and the cDNA clone used as a negative control
COS-7 cells co-transfected with the α-glucanase gene or any of the cDNA clones 1 to 7
One of these genes is co-transfected with the cDNA of HLA-A2402 or HLA-A2601.
The transfected COS-7 cells were not recognized by OK-CTLp and did not produce IFN-γ.
Furthermore, the concentrations of cDNA clones 1 to 6 were varied, and 100 ng of
Co-transfected with HLA-A0207 or HLA-A2402 cDNA in COS-7 cells
After gene transfer, IFN-γ production by OK-CTL was observed in a dose-dependent manner.
The mRNA expression of these genes was also examined by Northern blot analysis [Fig. 4(A) to (F)].
Similar expression patterns were observed, except for gene 5. These genes were expressed in cancer cells.
It is ubiquitously expressed in both normal and normal cells, but is not expressed in Panc-1 cells, SW620 cells, or any other cells.
The expression level in tumor cells, such as CA9-22 cells, was stimulated with PHA.
T cells and Epstein-Barr virus-transformed B cells (EBV-B)
The expression of gene 5 mRNA was significantly higher than that in normal cells.
It was almost impossible to detect ³² Colony hives using P-labeled clone 5
Redox also reduces the ⁶ Three clones were isolated from the cDNA library.
This is thought to be because the expression of gene 5 is rare, so that only one clone is obtained. Example 3 (Establishment of OK-CTL clones) CTLs that are activated by recognizing tumor antigens are derived from cells that recognize multiple tumor antigens.
Since it is considered that the OK-CTLp is a population, the above-mentioned OK-CTLp was cultured at limiting dilution (0.3,
0.5, 1, 2 and 4 cells/well) to clone OK-CTL clones.
(J. Immunol., 163: 4997-5004, 1999)
These clones were prepared by adding 100 ng/ml of any of the seven cDNA clones mentioned above.
The wells were co-transfected with 100 ng/well of HLA-A0207 cDNA.
The cells were cultured at a cell ratio of 1:1 with COS-7 cells or tumor cells, and their IFN-γ production was measured.
Clones having CTL activity were selected by measuring viability.
First, 300 CTL clones were isolated from the parent strain OK-CTLp by limiting dilution culture.
Of these, 80 CTL clones were HLA-A2-restricted tumor-specific.
Shows CTL activity and CD3 ⁺ CD4 ⁻ CD8 ⁺ and TCRαβ ⁺ Phenotype
Among them, 2, 3, 1, 3,
Two and four CTL clones were identified as clone 1, clone 2, and clone 3, respectively.
COS-7 cells expressing clone 3, clone 4, clone 5, and clone 6
In other words, the CTL clones showed reactivity against the tumor antigens recognized by the
Table 4 shows the data for 15 typical CTL clones.
This indicates that OK-CTLp, i.e., CTLs from cancer patients, are composed of multiple
It was suggested that this was a population of cells that recognized tumor antigens. Example 4 (Preparation of tumor antigen peptides and their CTL-inducing activity) Seven tumor antigens capable of inducing CTL in an HLA-A2-restricted manner obtained in Example 2
To obtain gene-derived tumor antigen peptides, a motif capable of binding to HLA-A2 was selected.
Regarding peptides having regular sequences,
2:163, 1994) (Immunogenetics, 41:178, 19
94) Based on the motifs obtained by the search, the amino acid sequence encoded by the above genes 1 to 7 is identified.
amino acid sequences and UBE2V, HNRPL, and WHS, which have high homology to these
C2, EIF4EBP1, ppMAPkkk, 2-5 OAS3, and CPS
From the amino acid sequence of F, different 9- to 11-mer peptides were designed and synthesized.
The purity of the obtained peptides was 70% or more.
The binding activity of the peptide to the HLA-A0201 molecule was examined using a T2 cell mutant.
(Cancer Res., 54:1071-1076, 1994).
T2 cells lack TAP, so HLA-A2 molecules do not bind to peptides.
First, the synthesized peptide (10 μM) was incubated with T2 cells.
Incubate for 3 hours at 26°C, then 5% CO ₂ -95% Air Condition
The mixture was incubated at 37°C for 3 hours to confirm that the peptide was cytosed by HLA-A2.
T2 cells were obtained by incubating these cells with anti-HLA-A2 mAb (BB).
7.2) and R-phycoerythrin (phycoerythrin
F(ab') ₂ Rabbit anti-mouse immunoglobulin (Ig
) (manufactured by DAKO) and analyzed by FACScan (Beckton Dickinson)
By analyzing the expression pattern using a ELISA kit (manufactured by NIH), the peptide-bound HLA was identified.
It was confirmed that the A0201 molecule was expressed on the cell surface. To test the recognition of the peptides by CTL, each peptide (10 μg/mL) was
T2 cells pre-pulsed with IFN-γ (M) were used as target cells (T), and OK-CTLp or
Alternatively, OK-CTL clones are used as effector cells (E) to induce target cells
The effector cells were incubated with the IgG1 antibody for 18 hours, and the supernatant was collected.
IFN-γ was measured by ELISA.
When using the OK-CTL clone, the E/T ratio was 10:1, and when using the OK-CTL clone, the E/T ratio was 2:
The test was carried out as No. 1. In addition, the OK-CTL clone was used to detect CTL of the peptide.
When examining the activation ability of a peptide to be examined, a nucleic acid encoding the peptide is used.
A clone that recognizes the gene product was used. T2 cells not pulsed with peptide
IFN-γ production of OK-CTLp or OK-CTL clones against
The ground was subtracted from each measurement, and the results are shown in Figures 5 to 10.
7, 8, 9, and 10 show gene 1 and UBE2V, gene 2, and gene 3, respectively.
Gene 2 and HNRPL, gene 3 and WHSC2, gene 4 and EIF4
EBP1, gene 5 and ppMAPkkk, and genes 6 and 2-5 O
The results for peptides derived from AS3 are shown. Furthermore, the concentrations of each peptide were varied when incubated with T2 cells.
The CTL clone activation ability was examined, and each peptide activated the CTL clone in a dose-dependent manner.
Gene 1 and UBE2V, gene 2 and H
NRPL, gene 3 and WHSC2, gene 4 and EIF4EBP1, genes
gene 5 and ppMAPkkk, and genes 6 and 2-5 from OAS3
Representative examples of peptides are shown in (A), (B), (C), (D), and (E) of Figure 11.
) and (F). Furthermore, peptides derived from gene 7 and CPSF were
The peptides derived from EBV and influenza virus are shown in Table 5.
The peptides derived from the IgG1 gene are positive controls capable of activating CTLs.
- It was found that CTL clones could be activated to produce IFN-γ. Example 5 (Induction of CTL from peripheral blood mononuclear cells of cancer patients by peptides) In Example 4, IFN-
Among the peptides that could promote γ production, P1 to P19 were analyzed using peripheral blood mononuclear cells from cancer patients.
The method for inducing CTLs using peptides was previously described.
(J. Exp. Med., 187: 277-288, 1998)
Cancer Res., 59:4056-4063, 1999). First, OK
Autologous peripheral blood mononuclear cells (PBMCs) from colon cancer patients who had obtained CTLp were incubated with the peptide
The cells were incubated with 10 μM of 10 μM ...
On the fourth day, the mice were pulsed with 10 μM of the same peptide for 2 hours and irradiated (30 Gray).
The cells were then restimulated using the autologous PBMCs as antigen-presenting cells (APCs).
On day 1, cells were harvested and examined for surface phenotype.
The recognition of the compound by the target cells is determined by the IFN produced when the compound is cultured together with the target cells.
The target cells were H-γ and H-γ.
LA-A2 ⁺ Tumor cells SW620 cells, CA9-22 cells, and Pan
The results are shown in Table 6. Of the 19 peptides, P1, P3, P5, P6, P8, P9, P10, and P
11, P13, P14, P15, P17, or P18.
The stimulated PBMCs express HLA-A2 ⁺ Tumor cells SW620 cells, CA9-2
2 cells and Panc-1 cells and produced significant levels of IFN-γ.
However, the PBMCs were HLA-A2 ⁻ Almost no tumor cells were recognized.
P2, P4, P7, P12, and P16 also bind to HLA-A2 ⁺ Tumor cell
PBMC stimulated with P19 induced CTLs that recognized either HLA-A or HLA-B.
It recognized not only tumor cells expressing HLA-2 but also tumor cells expressing other HLAs.
IFN-γ production from these CTLs was suppressed by anti-HLA class I mAb and anti-CD8 mAb.
b or anti-HLA-A2 mAb, but not other mAbs.
Furthermore, two HLA-A0201 ⁺ Patients (pancreatic cancer patients and colon cancer patients)
Peripheral blood mononuclear cells (PBMCs) were prepared from the blood of cancer patients and subjected to peptide-induced C
When TL induction was examined, similar results were obtained.
It has been found that tide can induce HLA-A2 restricted CTL from peripheral blood mononuclear cells of patients.
The peptide binding affinity to HLA-A0201 molecules was significantly higher than that of HLA-A2 molecules.
Relative mean fluorescence intensity (MFI)
The MFI of the positive or negative control was expressed as the MFI (e intensity).
The binding affinity of the peptide to the HLA-A0201 molecule was 898 and 490, respectively.
The binding affinity does not appear to correlate with the induction of CTLs by the peptides. Furthermore, the CTL activation ability of these 19 peptides was evaluated. ⁵¹ C
The toxicity to target cells was directly confirmed using the r-release test as an index.
The PBMCs from which the CTLs were induced were further treated with autologous APCs, IL-2, and
The cells were re-cultured and allowed to grow in the presence of the corresponding peptide. Approximately 21-28 days after re-culture
PBMCs were collected at 100°C, and their cytotoxicity was assessed by measuring IFN-γ and 6-hour ⁵¹ Cr play
The results are shown in Figures 12 to 17.
The stimulated PBMCs of cancer patients were HLA-A2 ⁺ Tumor cells were lysed
However, HLA-A2 ⁻ Tumor cell RERF-LC-MS and HLA-A2
Autologous EBV-B cells and PHA-stimulated T cells expressing EBV but derived from normal cells
Cells were not lysed except when stimulated with peptides P14, P15, and P17.
The PBMCs also showed cytotoxicity against autologous EBV-B cells.
PBMCs stimulated with 9 were highly cytotoxic to autologous EBV-B cells.
PBMC stimulated with these peptides showed a similar activity to the peptides used for PBMC stimulation.
The peptide caused dose-dependent cytotoxicity in T2 cells pulsed with the same peptide.
Representative examples are shown in (A) to (E) of Figure 18.
The peptides are expressed in peripheral blood mononuclear cells of cancer patients in an HLA-A2 restricted manner.
It was found that CTLs showing the above-mentioned activity can be induced. Example 6 (CTL induction from peripheral blood mononuclear cells of cancer patients by peptides) Among the tumor antigen peptides obtained in Example 4, peptides derived from gene 7 and CPSF were used.
Among peptides P20 to P32, six peptides (P21, P22) with purity of 95% or higher were selected.
2, P24, P26, P30, and P32) were cultured in human peripheral blood mononuclear cells (P
The in vitro CTL induction ability from the IFN-γ gene was examined using IFN-γ production as an indicator.
The PBMCs used were from 16 HLA-A2 positive cancer patients (4 with pancreatic cancer,
Peripheral blood samples were collected from 7 patients with gastric cancer and 5 patients with colon cancer, as well as 6 healthy volunteers.
First, 1 × 10 PBMCs were prepared. ⁵ 96-well U-bottom microculture
10 μM of each of the above peptides was added to each well of a scavenger plate (manufactured by Nunc).
Both were incubated in 200 μl of culture medium. The medium was 45% RPMI.
-1640, 45% AIM-V (Invitrogen), 10% fetal bovine serum
(FCS), 100 U/ml human interleukin-2, and 0.1 μM
From MEM non-essential amino acid solution (Invitrogen)
On the 4th and 7th days of culture, half of the medium was removed and the corresponding peptides were added.
On the 10th day of culture, the cells were collected and washed, and the corresponding
The peptide was reacted with pulsed T2 cells, and the amount of IFN-γ produced was measured.
In addition, the above cells stimulated with peptide and cultured for 10 days were cultured for another 10 days.
Cytotoxicity of the resulting cells against Panc-1 pancreatic adenocarcinoma cells (HLA-A2)
Sex was measured using a standard 6-hour E/T ratio of 10:1. ⁵¹ Measured by Cr release test,
The results were expressed as % specific lysis (Table 7). ⁻ tumor cells
The cytotoxicity against SSTW-9 tumor cells was measured as background.
, were subtracted from the above results. As a result, P21, P22, P23, P26, P30, and P32
The induction of HLA-A2-restricted CTL specific to each peptide from BMC was performed as described in the above 16.
Of the cancer patients, 31% (5/16), 38% (6/16), and 25% (
4/16), 31% (5/16), 44% (7/16), and 7% (1/16)
) was observed in patients with HBV.
CTL induction was observed in 50% (3/6) and 33% (2/6), respectively.
However, the other peptides did not induce CTLs from PBMCs of healthy individuals (Table 7).
Furthermore, the above CTLs induced from PBMCs of cancer patients using the peptides were
In addition to c-1 pancreatic adenocarcinoma cells, HLA-A2 ⁺ SW620 colon gland tumor cells
against cancer cells (HLA-A2/A24) and KWS gastric adenocarcinoma cells (HLA-A2)
Although it showed cytotoxicity to HLA-A2 ⁻ The tumor cells were SSTW9 gastric adenocarcinoma cells.
PHA that expresses HLA-A24 or HLA-A2 but is not a tumor cell
The blast T cells and EBV-transformed B cells were not lysed.
Recognition of tumor cells was achieved using anti-HLA class I mAb, anti-CD8 mAb, or anti-HLA
-A2 mAb but not the other mAbs. Example 7 (Isolation and identification of cDNA clones encoding tumor antigens) SEREX (Se) was isolated from a cDNA library of the pancreatic adenocarcinoma cell line CFPAC-1.
logical analysis of recombinant cD
NA expression libraries method (Proc. Natl.
Acad. Sci. USA, 92:11910-11813, 1995)
Genes encoding tumor antigens that have been detected and reported (Biochem. B
iophys. Res. Commun. , 281:936-944, 2001)
From the above, two antibodies capable of activating CTL in an HLA-A2-restricted manner were prepared in the same manner as in Example 2.
We discovered two tumor antigen genes, KM-PA-2 and KM-PA-4.
, KM-PA-2, KM-PA-4, KM-PA-5, KM-PA-14, KM
cDNA clones encoding KM-PA-15 and KM-PA-18 were
and inserted into the pBluescript vector digested with EcoRI and XhoI.
These cDNAs were then inserted into pCMV-SPORT2.
and co-expressed with HLA-A0207 or HLA-A2402 in COS-7 cells.
These COS-7 cells were used as target cells, and the resulting antibodies were injected with OK-CTLp.
As a result, KM-PA-2 or KM-PA-4 and HLA
COS-7 cells co-transfected with α-A0207 showed I-mediated activation of OK-CTLp.
FN-γ production was induced in a gene dose-dependent manner (Figures 19(A) and (B)).
The SEREX method can be used to detect tumor antigens.
Among the more than 1,500 tumor antigens detected in
The tumor antigens identified as soluble in the IL-1 receptor are MAGE-1, tyrosinase, and NYL.
Therefore, the tumor antigens identified by the SEREX method were
Even if a gene encodes a tumor antigen, it does not necessarily activate CTLs.
The genes KM-PA-2 and KM-PA-4 activate CTL in an HLA-A2-restricted manner.
It was first discovered in the present invention that tumor antigen peptides can be activated. Example 8 (Preparation of tumor antigen peptides and CTL induction activity from PBMCs of cancer patients) The tumor antigen genes KM-PA-2 and KM-PA-4 obtained in Example 7 were derived from
To obtain the tumor antigen peptide, KM-PA-2 and KM-PA-4 were used.
Based on the amino acid sequence to be encoded, different 9-10 mer
The peptides were designed and synthesized by a method known per se. The synthesized peptides were analyzed by immunohistochemistry using peripheral blood mononuclear cells from colon cancer patients from which OK-CTLp was obtained.
The CTL inducibility from the cells was examined in the same manner as in Example 6. As a result,
As shown in (A) and (B), peptides P33 to P41 (sequences P33 to P41) derived from KM-PA-2 were synthesized.
SEQ ID NOS: 33 to 41 in the table) and peptides P42 to P44 derived from KM-PA-4
PB stimulated in vitro with any of the following (SEQ ID NOS: 42 to 44 in the Sequence Listing)
MCs were prepared by pulsing the PBMCs with a peptide corresponding to the peptide used to stimulate the PBMCs.
2 cells [left panels of Figures 20(A) and (B)], or HLA-A2 ⁺ In tumor cells
Panc-1 cells (Fig. 20(A) and (B) right panel) and express IFN-
However, HLA-A2 ⁻ There was little reaction with tumor cells.
From these findings, it is clear that all of the above 12 peptides are HLA-specific peptides from PBMCs of cancer patients.
-A2-restricted antigen-specific CTL can be induced, and the induced CTL
It was found that the above peptide was recognized in an HLA-A2-restricted manner and IFN-γ was produced.
Furthermore, the cytotoxicity of these peptide-induced CTLs was investigated. ⁵¹ Cr
This was directly confirmed by a release test in the same manner as in Example 6. A representative example of the results is shown in Figure 21
As shown in Figure 21, P35, P39, or P40 inhibited the PB
CTLs induced from MCs are HLA-A2 ⁺ Panc-1 cells, which are tumor cells
and YPK-3 cells (lysis). However, HLA-A2 ⁻ tumor
PaCa-2 cells, EBV-B cell line OKAB2, and T cells stimulated with PHA.
In other words, the peptides did not lyse the cells from the peripheral blood mononuclear cells of cancer patients.
It was confirmed that CTLs exhibiting cytotoxicity in an HLA-A2-restricted manner could be induced.
In addition, OK-CTLp obtained from cancer patients other than colon cancer patients, such as pancreatic cancer patients,
CTLs were also induced from the PBMCs by the above peptides. Example 9: Expression of TCRs expressed on the cell surface of CTL clones that recognize the above peptides
To determine the phenotype, each CTL clone obtained in Example 3 was
5 x 10 ⁶ Each was purified using RNAzolTMB (TEL-TEST).
Total RNA was obtained from the cDNA prepared by First Strand cDNA synthesis.
SuperScript for anthesis ^TM Preamplific
The solution was prepared using a filtration system (manufactured by Invitrogen).
The single-stranded cDNA was purified using 22 different Vβ primers (Vβ1-20) and 3'
The Cβ primer was used for polymerase chain reaction (PCR) amplification.
Denaturation at 94°C for 1 minute, annealing at 58°C for 2 minutes, extension at 72°C for 3 minutes
The PCR product was transferred to the plasmid pCR2.
The DNA was inserted into the TA cloning system (Invitrogen) and then cloned into a large fragment.
Transform the plasmid into E. coli, select colonies, and extract the plasmid from the colonies.
The cDNA was prepared and sequenced. As a result, peptides derived from UBE2V and gene 1, HNPRL and gene
Reacts with peptides from gene 2 and 2-5 OAS3 and gene 6
Two CTL clones from each group were TCR Vβ8.1 and TCR Vβ3, respectively.
.2, and TCR Vβ14. In addition, WHSC2 and genes
3-derived peptides, EIF4EBP1 and gene 4-derived peptides, and p
CTL clones that recognize peptides derived from pMAPkkk and gene 5 were
TCR Vβ13.1, TCR Vβ8.1, and TCR Vβ18, respectively.
The peptides derived from UBE2V and gene 1, HNPRL and gene 2 were also expressed (Tables 8-1 and 8-2).
and 2-5 OAS3 and gene 6-derived peptides.
Each of the two CTL clones has a different complementarity-determining region 3 (CDR3) (
T having an element responsible for binding to an antigen epitope on the groove of an HLA class I molecule
CR. WHSC2 and gene 3-derived peptide, EIF4EB
Peptides derived from P1 and gene 4, and ppMAPkkk and gene 5
The CTL clones that recognize the original peptides also have different CDR3s.
The amino acid sequences of each CDR3 are shown in Table 6 with the underlined amino acids.
It is an array. Industrial Applicability The present invention makes it possible to induce HLA-A2-restricted cytotoxic T cells.
This makes it possible to perform specific immunotherapy for pancreatic cancer, colon cancer, gastric cancer, etc.
The A2 allele is present in 23% of African blacks, 53% of Chinese, 40% of Japanese,
49% of North American Caucasians and 38% of South American Caucasians
Therefore, the present invention is expected to make a great contribution to cancer treatment.
The present invention relates to the recognition by T cells of pancreatic cancer cells, colon cancer cells, gastric cancer cells, etc.
It will also make a significant contribution to basic research on children.

[Sequence List]

[Brief explanation of the drawings]

Figure 1 shows that OK-CTLp (HLA-A0207/A3101) binds to HLA-A2
Figure 2 shows that the recognition of the human pancreatic adenocarcinoma cell line Panc-1 by OK-CTLp and the resulting interferon-γ production are HLA-A2 restricted. Figure 3 shows that the cDNA clones 1 to 6 [
Figure 4 shows that OK-CTLp recognizes COS7 cells expressing clone 1 [(A) in the figure] and clone 7 [(B) in the figure] together with HLA-A2 in an HLA-A2-restricted manner. Figure 4 shows that cDNA clones 1 to 6 obtained from the human pancreatic adenocarcinoma cell line Panc-1 are recognized by OK-CTLp in a dose-dependent manner.
, (C), (D), (E), and (F) are UBE2V, HNRPL, respectively.
, WHSC2, EIF4EBP1, ppMAPkkk, and 2-5 OAS3
The figure shows that OK-CTLp recognizes COS7 cells expressing cDNA clones 1 to 6, which are highly homologous to the above, together with HLA-A2, in an HLA-A2-restricted manner.
The figure shows the amount of interferon γ produced by OK-CTLp when the 07 gene was expressed in target cells, and -◇- indicates the amount of interferon γ produced by OK-CTLp when the 07 gene was expressed in target cells together with the above-mentioned tumor antigen genes.
5 shows the amount of interferon γ produced by OK-CTLp when the tumor antigen gene 1, which has high homology to UBE2V obtained from the human pancreatic adenocarcinoma cell line Panc-1, was expressed in target cells.
Figure 6 shows that four peptides derived from the gene product of tumor antigen gene 2, which has high homology with HNRPL obtained from the human pancreatic adenocarcinoma cell line Panc-1, were recognized by OK-CTL clones.
Figure 7 shows that four peptides derived from the gene product of tumor antigen gene 3, which has high homology with WHSC2 obtained from the human pancreatic adenocarcinoma cell line Panc-1, were recognized by OK-CTL.
Figure 8 shows that two peptides derived from the gene product of tumor antigen gene 4, which has high homology to EIF4EBP1 obtained from the human pancreatic adenocarcinoma cell line Panc-1, were recognized by OK-CTL clones.
9 shows that three peptides derived from the gene product of tumor antigen gene 5, which has high homology to ppMAPkkk obtained from the human pancreatic adenocarcinoma cell line Panc-1, were recognized by OK-CTLp or OK-CTL clone.
Figure 10 shows that a peptide derived from the gene product of tumor antigen gene 6, which has high homology to OAS3, obtained from the human pancreatic adenocarcinoma cell line Panc-1, was recognized by OAS3.
Fig. 11 shows that tumor antigen peptides derived from tumor antigen gene products obtained from the human pancreatic adenocarcinoma cell line Panc-1 are recognized by OK-CTL clones in a dose-dependent manner.
A diagram showing representative peptides. In the figure, (A), (B), (C), and (D) are
D), (E), and (F) are UBE2V, HNRPL, and WHSC2, respectively.
Figure 12 shows that peptides derived from the gene products of tumor antigen genes 1 to 6, which have high homology to EIF4EBP1, ppMAPkkk, and 2-5 OAS3, are recognized by OK-CTLp. Figure 12 shows that three peptides derived from the gene product of tumor antigen gene 1, which has high homology to UBE2V, can induce CTLs with cytotoxicity against HLA-A2 ⁺ tumor cells from peripheral blood mononuclear cells of cancer patients.
- represents the human pancreatic adenocarcinoma cell line Panc-1 (HLA-A0201/1101);
◆- indicates human colon adenocarcinoma cell line SW620 (HLA-A0201/2402);
○- means HLA-A2 ^- lung adenocarcinoma cell line RERF-LC-MS (HLA-A1101
/1101), -△- indicates EBV-transformed autologous B cells (HLA-A0207/3
101), and -□- indicates PHA blast-transformed autologous T cells (HLA-A0207/3101
) are used in the descriptions of Figures 13 to 17 below. Figure 13 shows that two peptides derived from the gene product of tumor antigen gene 2, which is highly homologous to HNRPL, can induce CTLs with cytotoxicity against HLA-A2 ⁺ tumor cells from peripheral blood mononuclear cells of cancer patients. Figure 14 shows that two peptides derived from the gene product of tumor antigen gene 3, which is highly homologous to WHSC2, can induce CTLs with cytotoxicity against HLA-A2 ⁺ tumor cells from peripheral blood mononuclear cells of cancer patients. Figure 15 shows that two peptides derived from the gene product of tumor antigen gene 4, which is highly homologous to EIF4EBP1, can induce CTLs with cytotoxicity against HLA-A2 ⁺ tumor cells from peripheral blood mononuclear cells of cancer patients. Figure 16 shows that a peptide derived from the gene product of tumor antigen gene 5, which has high homology to ppMAPkkk, can induce CTLs with cytotoxicity against HLA-A2 ⁺ tumor cells from peripheral blood mononuclear cells of cancer patients. Figure 17 shows that a peptide derived from the gene product of tumor antigen gene 6, which has high homology to 2-5 OAS3, can induce CTLs with cytotoxicity against HLA-A2 ⁺ tumor cells from peripheral blood mononuclear cells of cancer patients. Figure 18 shows that a tumor antigen peptide can induce CTLs that exhibit cytotoxic activity in an HLA-A2-restricted and peptide dose-dependent manner from peripheral blood mononuclear cells of cancer patients. In the figures, (A), (B), (C), (D), and (E) represent UB, respectively.
E2V, HNRPL, WHSC2, EIF4EBP1, and ppMAPkkk
This shows that peptides derived from the gene products of tumor antigen genes 1 to 6, which have high homology to the above, induce CTLs that recognize the peptides in an HLA-A2-restricted manner from peripheral blood mononuclear cells of cancer patients.
-◇- indicates PHA blast-transformed autologous T cells. Figure 19 shows the tumor antigen gene KM- obtained from the human pancreatic adenocarcinoma cell line CFPAC-1.
The gene products of PA-2 ((A) in the figure) and KM-PA-4 ((B) in the figure)
Figure 20 shows that the tumor antigen gene KM-1 obtained from the human pancreatic adenocarcinoma cell line CFPAC-1 is recognized by OK-CTLp in an HLA-A2 restricted manner.
Peptides derived from PA-2 and KM-PA-4 [(A) and (B) in the figure, respectively]
) was pulsed with a peptide corresponding to the peptide used to stimulate the PBMC.
Figure 21 shows that CTLs with cytotoxicity against HLA-A2 ⁺ tumor cells Panc-1 (left panels in both (A) and (B)) and HLA-A2 + tumor cells Panc-1 (right panels in both (A) and (B)) can be induced from peripheral blood mononuclear cells of cancer patients.
CTLs induced from peripheral blood mononuclear cells of cancer patients by PA-2-derived peptides were
It is shown that tumor cells are lysed in an HLA-A2 restricted manner.

[Procedure Amendment] Submission of a copy of amendments under Article 34 of the Patent Cooperation Treaty (Official)

[Submission date] June 6, 2002 (2002.6.6)

[Procedural Correction 1]

[Name of document to be corrected] Statement

[Item to be amended] Claims

[Correction method] Change

[Contents of the amendment]

[Claims]

─────────────────────────────────────────────────────────Continued from the front page (51) Int.Cl. ⁷ Identification Symbol FI A61K 35/66 A61K 35/66 35/76 35/76 38/00 39/00 H 39/00 39/395 E 39/395 T 45/00 45/00 48/00 48/00 A61P 1/00 A61P 1/00 1/18 1/18 35/00 35/00 C07K 14/82 C07K 14/82 16/32 16/32 C12N 1/15 C12N 1/15 1/19 1/19 1/21 1/21 C12P 21/02 C 5/10 G01N 33/15 Z C12P 21/02 33/50 Z G01N 33/15 33/53 D 33/50 M 33/53 33/566 33/574 A 33/566 C12N 5/00 A 33/574 A61K 37/02 (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, I T, LU, MC, NL, PT, SE, TR), OA (BF , BJ, CF, CG, CI, CM, GA, GN, GQ, GW, ML, MR, NE, SN, TD, TG), AP (G H, GM, KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), UA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, B Y, BZ, CA, CH, CN, CO, CR, CU, CZ, DE, DK, DM, DZ, EC, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, I L, IN, IS, JP, KE, KG, KP, KR, KZ , LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI , SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (Note) This publication is based on the publication of the internationally published Japanese patent application (Japanese utility model registration application) related to this publication. The effect of the international publication of the Japanese patent application (Japanese utility model registration application) related to this publication arises pursuant to Article 184-10, Paragraph 1 of the Patent Act (Article 48-13, Paragraph 2 of the Utility Model Act) and is unrelated to this publication.

Claims (24)

[Claims] [Claim 1] A peptide consisting of an amino acid sequence set forth in any one of SEQ ID NO: 1 to SEQ ID NO: 44 in the sequence listing. 2. A polypeptide consisting of an amino acid sequence set forth in any one of SEQ ID NO: 45 to SEQ ID NO: 53 in the sequence listing. [Claim 3] A pharmaceutical comprising one or more peptides or polypeptides selected from a peptide consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 44 in the sequence listing and a polypeptide consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 45 to 53 in the sequence listing. [Claim 4] A cancer vaccine containing one or more peptides or polypeptides selected from a peptide consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 44 in the sequence listing and a polypeptide consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 45 to 53 in the sequence listing. [Claim 5] A cancer vaccine used in the treatment of pancreatic cancer, colon cancer, or gastric cancer, comprising one or more peptides or polypeptides selected from a peptide consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 44 in the sequence listing and a polypeptide consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 45 to 53 in the sequence listing. [Claim 6] An inducer of cytotoxic T cells comprising one or more peptides or polypeptides selected from a peptide consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 44 in the sequence listing and a polypeptide consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 45 to 53 in the sequence listing. [Claim 7] A method for inducing cytotoxic T cells, characterized by using one or more peptides or polypeptides selected from a peptide consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 44 in the sequence listing and a polypeptide consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 45 to 53 in the sequence listing. 8. A polynucleotide encoding a peptide or polypeptide consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 53 in the sequence listing, or a complementary strand thereof. 9. A polynucleotide set forth in any one of SEQ ID NO: 54 to SEQ ID NO: 62 in the sequence listing, or a complementary strand thereof. 10. A polynucleotide set forth in any one of SEQ ID NOs: 54 to 62 in the Sequence Listing, wherein the polypeptide encoded by said polynucleotide induces cytotoxic T cells and/or is recognized by cytotoxic T cells.
A polynucleotide or its complementary strand. 11. A polynucleotide which hybridizes under stringent conditions with the polynucleotide according to any one of claims 8 to 10 or its complementary strand. 12. A recombinant vector comprising the polynucleotide according to any one of claims 8 to 10 or its complementary strand, or a polynucleotide that hybridizes with said polynucleotide or its complementary strand under stringent conditions. 13. A recombinant expression vector comprising the polynucleotide according to any one of claims 8 to 10 or its complementary strand, or a polynucleotide that hybridizes with said polynucleotide or its complementary strand under stringent conditions. 14. A transformant transformed with a recombinant vector or an expression recombinant vector containing the polynucleotide or its complementary strand described in any one of claims 8 to 10, or a polynucleotide that hybridizes with said polynucleotide or its complementary strand under stringent conditions. [Claim 15] A method for producing the polypeptide described in claim 2, comprising a step of culturing a transformant transformed with an expression recombinant vector containing the polynucleotide described in any one of claims 8 to 10 or its complementary strand, or a polynucleotide that hybridizes to said polynucleotide or its complementary strand under stringent conditions. 16. An antibody that immunologically recognizes the peptide according to claim 1 or the polypeptide according to claim 2. 17. A peptide according to claim 1 or a polypeptide according to claim 2, which interacts with HLA-A2 to produce at least H
A method for screening for a compound that enhances the recognition of the peptide or polypeptide by LA-A2-restricted cytotoxic T cells, and/or a compound that interacts with the polynucleotide of any one of claims 8 to 11 or its complementary chain to enhance its expression, the method comprising: screening for a compound that enhances the recognition of the peptide or polypeptide by LA-A2-restricted cytotoxic T cells; screening for ... polynucleotide of any one of claims 8 to 11 or its complementary chain to enhance its expression, the method comprising: screening for a compound that enhances the recognition of the peptide or polypeptide by LA-A2-restricted cytotoxic T cells; screening for a compound that enhances the recognition of the polynucleotide of any one of claims 8 to 11 or its complementary chain to enhance its expression, the method comprising screening for a compound that enhances the recognition of the polynucleotide of any one of claims 8 to 11 or its complementary chain to enhance its expression, the method comprising screening for a compound that enhances the recognition of the polynucleotide of any one of claims 8 to 11 or its complementary chain to enhance its expression, the method comprising screening for a compound that
A screening method characterized by using at least one of the recombinant vector or expression recombinant vector described in claim 2 or 13, the transformant described in claim 14, or the antibody described in claim 16. 18. A peptide according to claim 1 or a polypeptide according to claim 2, which interacts with HLA-A2 to produce at least H
A method for screening for a compound that enhances the recognition of the peptide or polypeptide by LA-A2-restricted cytotoxic T cells, and/or a compound that interacts with the polynucleotide of any one of claims 8 to 11 or its complementary chain to enhance its expression, the method comprising: screening for a compound that enhances the recognition of the peptide or polypeptide by LA-A2-restricted cytotoxic T cells; screening for ... enhances the recognition of the peptide or polypeptide by LA-A2-re
A compound obtained by a screening method characterized by using at least one of the recombinant vector or expression recombinant vector described in claim 2 or 13, the transformant described in claim 14, or the antibody described in claim 16. 19. A compound that enhances the recognition by HLA-A2-restricted cytotoxic T cells of at least one of the peptides described in claim 1 or the polypeptides described in claim 2, or a compound that interacts with the polynucleotide or its complementary strand described in any one of claims 8 to 11 to enhance its expression. 20. A peptide according to claim 1, a polypeptide according to claim 2, a polynucleotide according to any one of claims 8 to 11 or a complementary strand thereof, a recombinant vector or an expression recombinant vector according to claim 12 or 13, a transformant according to claim 14,
A pharmaceutical composition for use in cancer treatment, comprising the antibody of claim 16 and at least one of the compounds of claim 18 or 19. 21. Use of the pharmaceutical composition according to claim 3, the cancer vaccine according to claim 4 or claim 5, the inducer of cytotoxic T cells according to claim 6, or the pharmaceutical composition according to claim 20 for the treatment of cancer. 22. A method for quantitatively or qualitatively measuring the peptide described in claim 1, the polypeptide described in claim 2, or the polynucleotide described in any one of claims 8 to 10. [Claim 23] A reagent kit for use in a method for quantitatively or qualitatively measuring the peptide described in claim 1, the polypeptide described in claim 2, or the polynucleotide described in any one of claims 8 to 10, comprising at least one of the peptide described in claim 1, the polypeptide described in claim 2, the polynucleotide described in any one of claims 8 to 11 or its complementary strand, or the antibody described in claim 16. [Claim 24] A reagent kit for use in a method for quantitatively or qualitatively measuring the peptide described in claim 1, the polypeptide described in claim 2, or the polynucleotide described in any one of claims 8 to 10, the reagent kit comprising at least one of the peptide described in claim 1, the polypeptide described in claim 2, the polynucleotide described in any one of claims 8 to 11 or its complementary strand, or the antibody described in claim 16, and use of the reagent kit in cancer disease testing.