JP4414163B2 - HSA-A24 binding cancer antigen peptide derived from PSA - Google Patents

Description

  The present invention relates to a PSA-derived HLA-A24-binding cancer antigen peptide.

  Cellular immunity, particularly cytotoxic T cells (hereinafter referred to as CTL), plays an important role in eliminating cancer cells, virus-infected cells, and the like by living bodies. CTL recognizes a complex formed by a peptide fragment of an antigenic protein derived from cancer or virus and a cell surface MHC class I molecule (referred to as HLA in the case of humans) by a T cell receptor, It specifically damages virus-infected cells and produces various cytokines to activate the immune system. Peptide fragments that form complexes with MHC class I molecules are called antigenic peptides and are usually about 8 to 11 amino acids long. Typical examples of cancer antigen proteins recognized by CTL include those described in Table 1 of Non-Patent Document 1. Specific examples include melanocyte tissue-specific proteins such as gp100, MART-1, and tyrosinase, and other cancer antigen proteins other than melanoma include HER-2 / neu, CEA, and PSA (non-patented). And tumor markers such as SART-1 and cyclophilin B derived from squamous cell carcinoma. These cancer antigen proteins, cancer antigen peptides and DNA encoding them, as well as virus-derived antigen proteins and antigen peptides are administered to enhance specific T cells in the body, so-called vaccine therapy is used for cancer and It is considered useful in the treatment and prevention of viral infections and the like. In fact, the clinical results using an antigen peptide derived from the cancer antigen protein MAGE-3 that is overexpressed in melanoma, lung cancer, head and neck cancer, etc. also suggest the importance of vaccine therapy in tumor elimination (Non-patent literature). 3).

  PSA is a glycoprotein that is specifically expressed even in normal prostate epithelial cells, but its expression level increases with canceration and becomes a cancer antigen protein, which is used as a diagnostic marker for cancer (PSA in patient serum) Measure value). Prostate cancer occupies the first to third ranks of male malignancies in both Western countries in terms of incidence and mortality, and has recently been increasing in Japan. Since prostate cancer is an androgen-sensitive cancer, treatment (endocrine therapy) for the purpose of androgen removal has been performed. However, even if prostate cancer is controlled in response to endocrine therapy at the start of treatment, more than half of the cases will transition to relapsed cancer, ie, androgen-refractory cancer, in five years. This androgen-dependent loss has become a major obstacle in endocrine treatment of prostate cancer. From such a background, development of vaccine therapy using a cancer antigen peptide derived from PSA is desired. Regarding PSA-derived cancer antigen peptides, antigen peptides having binding properties to HLA-A2 (see Non-Patent Document 4), antigen peptides having binding properties to HLA-A24 (see Patent Document 1 and Non-Patent Document 5) There are several known.

WO 02/47474 Immunity, vol.10: 281, 1999 J. Natl. Cancer. Inst., 89: 293, 1997 Int.J.Cancer., 80: 219,1999 J. Natl. Cancer. Inst., 89: 293, 1997 Int J. Cancer: 100,565-570 (2002)

  An object of the present invention is to provide a PSA-derived HLA-A24-binding cancer antigen peptide, use of the peptide as a cancer vaccine, and the like.

  The present inventors have intensively studied a PSA-derived HLA-A24-binding cancer antigen peptide that can be clinically applied as a cancer vaccine in cancer immunotherapy. It is known that the peptide sequence predicted to bind to the HLA-A24 antigen has regularity (motif), and recently, based on these regularity, the peptide sequence predicted to be able to bind to the HLA antigen is It is possible to search on the Internet by using, for example, NIH BIMAS software (http://bimas.dcrt.nih.gov/molbio/hla_bind/). However, the number of hit peptides is enormous, and the higher the score, the stronger the activity.

Based on empirical rules for various cancer antigen peptides that have been conventionally assayed in vivo, the present inventor has developed 10 types of PSA-derived peptides (5 types of natural peptides, modified to tyrosine at the second position). Five peptides, SEQ ID NOs: 1-10) were selected. HLA-A24-expressing transgenic mice (WO 02/47474, Int J. Cancer: 100,565-570 (2002)) were immunized with these selected peptides, and CTL inducibility in vivo was measured. As a result, it was revealed that all the peptides except for one peptide (SEQ ID NO: 9) showed good CTL inducing activity. This new finding has enabled a new cancer vaccine therapy that can induce PSA-specific CTLs in HLA-A24 positive cancer patients.
The present invention has been completed based on such findings.

That is, the present invention
(1) The following amino acid sequences:
Trp Val Pro Val Val Phe Leu Thr Leu (SEQ ID NO: 1),
Trp Tyr Pro Val Val Phe Leu Thr Leu (SEQ ID NO: 2),
Thr Trp Ile Gly Ala Ala Pro Leu Ile (SEQ ID NO: 3),
Thr Tyr Ile Gly Ala Ala Pro Leu Ile (SEQ ID NO: 4),
Asp Met Ser Leu Leu Lys Asn Arg Phe (SEQ ID NO: 5),
Asp Tyr Ser Leu Leu Lys Asn Arg Phe (SEQ ID NO: 6),
Lys Phe Met Leu Cys Ala Gly Arg Trp (SEQ ID NO: 7),
Lys Tyr Met Leu Cys Ala Gly Arg Trp (SEQ ID NO: 8), and
Val Tyr Leu Leu Gly Arg His Ser Leu Phe (SEQ ID NO: 10)
A peptide comprising any amino acid sequence selected from
(2) The peptide according to (1), comprising any amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, and 10.
(3) a modified amino acid sequence containing a modified amino acid residue in any amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8 and 10; and A peptide having HLA-A24-restricted CTL inducing activity,
(4) The peptide according to (3) above, comprising the amino acid sequence described in SEQ ID NO: 11 (except for the sequence corresponding to the amino acid sequences described in SEQ ID NOs: 1 and 2),
(5) The peptide according to (3) above, comprising the amino acid sequence described in SEQ ID NO: 12 (except for the sequence corresponding to the amino acid sequences described in SEQ ID NOs: 3 and 4),
(6) The peptide according to (3) above, comprising the amino acid sequence described in SEQ ID NO: 13 (except for the sequence corresponding to the amino acid sequences described in SEQ ID NOs: 5 and 6),
(7) The peptide according to (3) above, comprising the amino acid sequence described in SEQ ID NO: 14 (except for the sequence corresponding to the amino acid sequences described in SEQ ID NOs: 7 and 8),
(8) The peptide according to (3) above, comprising the amino acid sequence described in SEQ ID NO: 15 (excluding the sequence corresponding to the amino acid sequence described in SEQ ID NO: 10),
(9) The peptide according to (3) above, comprising any amino acid sequence selected from SEQ ID NOs: 16 to 51,
(10) consisting of a modified amino acid sequence containing a modified amino acid residue in any amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8 and 10; (3) to (9) the peptide according to any one of the above,
(11) The amino acid sequence described in SEQ ID NO: 11 (excluding sequences corresponding to the amino acid sequences described in SEQ ID NOS: 1 and 2), the amino acid sequence described in SEQ ID NO: 12 (however, SEQ ID NOs: 3 and 4) Except for the sequence corresponding to the amino acid sequence described in SEQ ID NO: 13) (except for the sequences corresponding to the amino acid sequences described in SEQ ID NO: 5 and 6), SEQ ID NO: 14. Amino acid sequence (excluding sequences corresponding to the amino acid sequences described in SEQ ID NOs: 7 and 8), amino acid sequence described in SEQ ID NO: 15 (excluding sequences corresponding to the amino acid sequence described in SEQ ID NO: 10) The peptide according to (10) above, consisting of any amino acid sequence selected from among the amino acid sequences set forth in SEQ ID NOs: 16 to 51,
(12) a polynucleotide encoding the peptide according to any one of (1) to (11),
(13) The polynucleotide according to (12), which encodes any amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, and 10.
(14) an expression vector containing the polynucleotide according to (12) or (13),
(15) A cell containing the expression vector according to (14),
(16) The method for producing a peptide according to any one of (1) to (11), wherein the cell according to (15) is cultured under conditions capable of expressing the peptide,
(17) an antibody that specifically binds to the peptide according to any one of (1) to (11),
(18) An antigen-presenting cell in which a complex of the cancer antigen peptide derived from the peptide according to any one of (1) to (11) and an HLA-A24 antigen is presented,
(19) A complex of a cancer antigen peptide consisting of any amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, and 10 and an HLA-A24 antigen is presented. The antigen-presenting cell according to (18),
(20) CTL that recognizes a complex of the cancer antigen peptide derived from the peptide according to any one of (1) to (11) and an HLA-A24 antigen,
(21) Recognizing a complex of a cancer antigen peptide consisting of any amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, and 10 and an HLA-A24 antigen CTL described in (20) above,
(22) The peptide according to any one of (1) to (11), the expression vector according to (14), the cell according to (15), the antigen-presenting cell according to (18) or (19), or the above (20) or a pharmaceutical composition comprising the CTL according to (21) and a pharmaceutically acceptable carrier,
(23) The pharmaceutical composition according to the above (22), which is used as an inducer of CTL,
(24) The pharmaceutical composition according to the above (22), which is used as a cancer vaccine,
(25) An HLA tetramer containing the peptide according to any one of (1) to (11) above and an HLA-A24 antigen, and (26) a diagnostic agent for the effect of a cancer vaccine containing the HLA tetramer according to (25) , Regarding.

  The present invention provides a PSA-derived HLA-A24-binding cancer antigen peptide, a polynucleotide encoding the peptide, a CTL inducer containing these peptides and polynucleotides, and the like. The CTL inducer of the present invention is useful as a cancer vaccine. The cancer vaccine of the present invention is applicable to many cancer patients who are HLA-A24 positive.

(I) Peptide of the Present Invention The peptide of the present invention is derived from human PSA (Biochem. Biophys. Res. Commun., 160: 903, 1989, NCBI database Accession No. M26663), and restricted in vivo by HLA-A24. It has sex CTL inducing activity (immunogenicity).
The peptide of the present invention has the property of being presented to antigen-presenting cells and inducing CTL in an HLA-A24 antigen-restricted manner in vivo. The characteristics can be examined by using an HLA-A24 model mouse (HLA-A24-expressing transgenic mouse; WO 02/47474, Int J. Cancer: 100,565-570 (2002)).

  As for the peptide containing any amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, and 10, the cancer antigen peptide derived from the peptide of the present invention is presented to the antigen-presenting cell. As long as it has the property of inducing CTL, it is not limited at all, but its length is usually 9-100, preferably 9-50, more preferably 9-30, still more preferably 9-20, and More preferably, it is 9-11 amino acid residues. Here, the cancer antigen peptide is defined as a peptide that induces CTL inducing activity that is presented to antigen-presenting cells.

  The peptide of the present invention can be synthesized according to a method used in ordinary peptide chemistry. As a synthesis method, literature (Peptide Synthesis, Interscience, New York, 1966; The Proteins, Vol 2, Academic Press Inc., New York, 1976; peptide synthesis, Maruzen (stock) ), 1975; Fundamentals and Experiments of Peptide Synthesis, Maruzen Co., Ltd., 1985; Drug Development Continued, Vol. 14, Peptide Synthesis, Hirokawa Shoten, 1991).

  The peptide of the present invention can also be produced using ordinary DNA synthesis and genetic engineering techniques based on the sequence information of the polynucleotide encoding the peptide of the present invention. Operations such as DNA synthesis, construction of various plasmids, transfection into hosts, culture of transformants and recovery of proteins from the culture are methods well known to those skilled in the art and methods described in the literature (Molecular Cloning, T. Maniatis et al., CSH Laboratory (1983), DNA Cloning, DM. Glover, IRL PRESS (1985)), or the method described in (II) below, etc.

  Hereinafter, the peptide of the present invention will be described in more detail.

(1) Peptide comprising any amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, and 10 As described above, the present invention includes SEQ ID NOs: 1, 2 , 3, 4, 5, 6, 7, 8, and 10 based on the new finding that peptides derived from PSA have CTL-inducing activity in vivo. Here, the peptides shown in SEQ ID NOs: 1, 3, 5, and 7 are PSA-derived natural peptides, and the peptides shown in SEQ ID NOs: 2, 4, 6, 8, and 10 are PSA-derived modified peptides. is there.
The knowledge that the novel peptides shown in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, and 10 surely show CTL-inducing activity in vivo has not been known. The peptide of the present invention containing any of these peptides is useful as an active ingredient of a CTL inducer in cancer immunotherapy and as an active ingredient of a cancer vaccine.

Specifically, the peptide of the present invention has the following amino acid sequence:
Trp Val Pro Val Val Phe Leu Thr Leu (SEQ ID NO: 1),
Trp Tyr Pro Val Val Phe Leu Thr Leu (SEQ ID NO: 2),
Thr Trp Ile Gly Ala Ala Pro Leu Ile (SEQ ID NO: 3),
Thr Tyr Ile Gly Ala Ala Pro Leu Ile (SEQ ID NO: 4),
Asp Met Ser Leu Leu Lys Asn Arg Phe (SEQ ID NO: 5),
Asp Tyr Ser Leu Leu Lys Asn Arg Phe (SEQ ID NO: 6),
Lys Phe Met Leu Cys Ala Gly Arg Trp (SEQ ID NO: 7),
Lys Tyr Met Leu Cys Ala Gly Arg Trp (SEQ ID NO: 8), and
One of Val Tyr Leu Leu Gly Arg His Ser Leu Phe (SEQ ID NO: 10) is included.

  More specific examples of the peptide of the present invention include the following peptides (1-1) to (1-3).

(1-1) Peptide consisting of any amino acid sequence selected from SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8 and 10 SEQ ID NO: 1, 2, 3, 4, 5 Specific examples of peptides consisting of any one of the amino acid sequences of 6, 7, 8, and 10 include the following cancer antigen peptides:
A cancer antigen peptide consisting of Trp Val Pro Val Val Phe Leu Thr Leu (SEQ ID NO: 1),
Cancer antigen peptide consisting of Trp Tyr Pro Val Val Phe Leu Thr Leu (SEQ ID NO: 2),
A cancer antigen peptide consisting of Thr Trp Ile Gly Ala Ala Pro Leu Ile (SEQ ID NO: 3),
A cancer antigen peptide consisting of Thr Tyr Ile Gly Ala Ala Pro Leu Ile (SEQ ID NO: 4),
Cancer antigen peptide consisting of Asp Met Ser Leu Leu Lys Asn Arg Phe (SEQ ID NO: 5),
A cancer antigen peptide consisting of Asp Tyr Ser Leu Leu Lys Asn Arg Phe (SEQ ID NO: 6),
A cancer antigen peptide consisting of Lys Phe Met Leu Cys Ala Gly Arg Trp (SEQ ID NO: 7),
A cancer antigen peptide consisting of Lys Tyr Met Leu Cys Ala Gly Arg Trp (SEQ ID NO: 8),
A cancer antigen peptide consisting of Val Tyr Leu Leu Gly Arg His Ser Leu Phe (SEQ ID NO: 10).

  These peptides can be produced by a general peptide synthesis method as described above. In addition, in vivo CTL-inducing activity can be measured by subjecting it to human model animals described in WO 02/47474 and Int J. Cancer: 100,565-570 (2002).

(1-2) Epitope peptide comprising any one of the amino acid sequences of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8 and 10 Recently, a peptide in which a plurality of CTL epitopes (antigen peptides) are linked ( Epitope peptide) has been shown to have CTL-inducing activity efficiently in vivo. For example, in Journal of Immunology 1998, 161: 3186-3194, a 30-mer peptide linked to a PSA-derived HLA-A2, -A3, -A11, B53-restricted CTL epitope is linked to each CTL epitope in vivo. It is described that a specific CTL was induced.

  It has also been shown that CTL is efficiently induced by a peptide (epitope peptide) in which a CTL epitope and a helper epitope are linked. Here, the helper epitope refers to a peptide having an action of activating CD4 positive T cells (Immunity., 1: 751, 1994). For example, HBVc128-140 derived from hepatitis B virus or TT947 derived from tetanus toxin. -967 and the like are known. CD4-positive T cells activated by the helper epitopes are thought to be important for anti-tumor immune responses because they exhibit effects such as induction and maintenance of CTL differentiation and activation of effectors such as macrophages. . As a specific example of a peptide in which such a helper epitope and a CTL epitope are arranged, for example, Journal of Immunology 1999, 162: 3915-3925 includes 6 types of HBV-derived HLA-A2 restricted antigen peptides, HLA-A11 restricted It is described that DNA (minigene) encoding a peptide composed of three types of antigen peptides and a helper epitope effectively induced CTL against each epitope in vivo. In fact, a peptide in which a CTL epitope (cancer antigen peptide consisting of positions 280 to 288 of melanoma antigen gp100) and a helper epitope (T helper epitope derived from tetanus toxin) is linked to clinical trials (Clinical Cancer Res., 2001, 7: 3012-3024).

Therefore, a peptide (epitope peptide) obtained by linking a plurality of epitopes including the cancer antigen peptide of the present invention as described in (1-1) above and having a CTL inducing activity in vivo is also included in the present invention. It can illustrate as a specific example of a peptide.
Here, when the epitope to be linked to the cancer antigen peptide of the present invention is a CTL epitope, the CTL epitope to be used is HLA-A0201, -A0204, -A0205, -A0206, -A0207, -A24, -A3, derived from PSA -A11, -B53, etc. include restricted CTL epitopes. A plurality of these CTL epitopes can be linked, and the length of one CTL epitope can be determined by analyzing antigen peptides bound to various HLA molecules (Immunogenetics, 41: 178, 1995), 8-14. The amino acid grade can be mentioned.

  When the epitope to be linked to the cancer antigen peptide of the present invention is a helper epitope, examples of the helper epitope to be used include HBVc128-140 derived from hepatitis B virus as described above and TT947-967 derived from tetanus toxin. The length of the helper epitope includes about 13 to 30 amino acids, preferably about 13 to 17 amino acids.

  More specifically, as an epitope peptide of the present invention, for example, one or more amino acid sequences of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, and 10 and a helper epitope Can be mentioned. More specifically, for example, one or more of the amino acid sequences of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, and 10 and a helper peptide derived from tetanus toxin (for example, Phe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg Val Pro Lys Val Ser Ala Ser His Leu Glu; SEQ ID NO: 52), SEQ ID NO: 1, 2, 3, 4, 5, 6, 7 One or more of the amino acid sequences of any of 8 and 10, and Ala Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu (SEQ ID NO: 53, Clinical Cancer Res., 2001, 7: 3012 -3024) and the like.

  Such a peptide in which a plurality of epitopes are linked (epitope peptide) can be produced by a general peptide synthesis method as described above. Moreover, based on the sequence information of the polynucleotide which codes the epitope peptide which connected these several epitopes, it can also manufacture using normal DNA synthesis and a genetic engineering technique. That is, by inserting the polynucleotide into a well-known expression vector, transforming the host cell with the obtained recombinant expression vector, culturing a transformant, and ligating a plurality of target epitopes from the culture. It can be produced by recovering the epitope peptide. These methods are described in the literature (Molecular Cloning, T. Maniatis et al., CSH Laboratory (1983), DNA Cloning, DM. Glover, IRL PRESS (1985)) as described above, and (II) below. Can be carried out according to the method described in the above.

  By subjecting the epitope peptide produced by linking the plurality of epitopes produced as described above to the human model animal described in WO 02/47474 and Int J. Cancer: 100,565-570 (2002), in vivo CTL-inducing activity can be measured.

(1-3) SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8 or 10 amino acid sequence, modified amino group of N-terminal amino acid or carboxyl group of C-terminal amino acid Peptide The amino group of the N-terminal amino acid or the carboxyl group of the C-terminal amino acid of the peptide of the present invention as exemplified in the above (1-1) to (1-2) can be modified.

  Here, examples of the amino group-modifying group of the N-terminal amino acid include 1 to 3 alkyl groups having 1 to 6 carbon atoms, phenyl group, cycloalkyl group, and acyl group. Specific examples of acyl groups include carbon. An alkanoyl group having 1 to 6 carbon atoms, an alkanoyl group having 1 to 6 carbon atoms substituted with a phenyl group, a carbonyl group substituted with a cycloalkyl group having 5 to 7 carbon atoms, an alkylsulfonyl group having 1 to 6 carbon atoms, phenyl Examples thereof include a sulfonyl group, an alkoxycarbonyl group having 2 to 6 carbon atoms, an alkoxycarbonyl group substituted with a phenyl group, a carbonyl group substituted with a cycloalkoxy having 5 to 7 carbon atoms, and a phenoxycarbonyl group.

  Examples of the peptide in which the carboxyl group of the C-terminal amino acid is modified include an ester form and an amide form. Specific examples of the ester form include an alkyl ester having 1 to 6 carbon atoms and a carbon number having 0 to 0 carbon atoms substituted with a phenyl group. 6 alkyl esters, cycloalkyl esters having 5 to 7 carbon atoms, and the like. Specific examples of amides include amides, amides substituted with one or two alkyl groups having 1 to 6 carbon atoms, phenyl An amide substituted with one or two alkyl groups having 0 to 6 carbon atoms substituted with a group, an amide containing a nitrogen atom of the amide group to form a 5- to 7-membered azacycloalkane, and the like .

(2) A peptide comprising a modified amino acid sequence containing a modified amino acid residue in any amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8 and 10 ( Modified peptide)
As described above, in the present invention, the PSA-derived natural peptide represented by SEQ ID NOs: 1, 3, 5, and 7 and the PSA-derived modified forms represented by SEQ ID NOs: 2, 4, 6, 8, and 10 are used. A new finding was obtained that the peptide has CTL-inducing activity in vivo. By further modifying the amino acid sequence of a peptide having such in vivo CTL inducing activity, a further modified peptide having equivalent or higher CTL inducing activity can be obtained. Therefore, in the present invention, a peptide comprising a modified amino acid sequence of the peptide shown in any of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, and 10 (hereinafter referred to as a modified peptide). Can also be provided.

That is, the present invention includes a modified amino acid sequence containing a modified amino acid residue in the amino acid sequence of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, and 10, and A peptide having HLA-A24-restricted CTL induction activity is provided.
More specifically, consisting of a modified amino acid sequence containing a modification of an amino acid residue in the amino acid sequence of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, and 10. The present invention also provides a peptide having HLA-A24-restricted CTL inducing activity.

  “Modification” of an amino acid residue in the present invention means substitution, deletion, and / or addition of one or several amino acid residues, preferably substitution. In the modification related to amino acid residue substitution, the number, position, and type of amino acid residues to be substituted are not particularly limited as long as they retain CTL inducing activity in vivo. Specific examples of the peptide having an amino acid sequence including such a modified amino acid sequence include the following peptides.

As the binding motif of HLA-A24, the second amino acid of the peptide consisting of 8-11 amino acids is tyrosine (Tyr), phenylalanine (Phe), methionine (Met) or tryptophan (Trp), and the C-terminal amino acid Is known to be phenylalanine (Phe), leucine (Leu), isoleucine (Ile), tryptophan (Trp) or methionine (Met) (J. Immunol., 152, p3913, 1994, Immunogenetics, 41, p178 1995, J. Immunol., 155, p4307, 1994). Therefore, in the modified peptide of the present invention, the amino acid at the second position and / or C-terminal of any amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, and 10 Residues can be substituted with amino acid residues that can be taken on the motif.
Specific examples include peptides containing the amino acid sequences listed below and having CTL-inducing activity in vivo (SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, and Except for sequences corresponding to the amino acid sequence described in 10).

Trp Xaa Pro Val Val Phe Leu Thr Xaa (where Xaa at position 2 is Val, Tyr, Phe, Met or Trp, and Xaa at position 9 is Leu, Phe, Ile, Trp or Met: SEQ ID NO: 11),
Thr Xaa Ile Gly Ala Ala Pro Leu Xaa (where Xaa at position 2 is Tyr, Phe, Met or Trp, and Xaa at position 9 is Leu, Phe, Ile, Trp or Met: SEQ ID NO: 12) ,
Asp Xaa Ser Leu Leu Lys Asn Arg Xaa (where Xaa at position 2 is Tyr, Phe, Met or Trp, and Xaa at position 9 is Leu, Phe, Ile, Trp or Met: SEQ ID NO: 13) ,
Lys Xaa Met Leu Cys Ala Gly Arg Xaa (where Xaa at position 2 is Tyr, Phe, Met or Trp, and Xaa at position 9 is Leu, Phe, Ile, Trp or Met: SEQ ID NO: 14) ,
Val Xaa Leu Leu Gly Arg His Ser Leu Xaa (where Xaa at position 2 is Tyr, Phe, Met or Trp, and Xaa at position 10 is Leu, Phe, Ile, Trp or Met: SEQ ID NO: 15 ).

  More specifically, the amino acid sequence described in SEQ ID NO: 11 (excluding sequences corresponding to the amino acid sequences described in SEQ ID NOS: 1 and 2), the amino acid sequence described in SEQ ID NO: 12 (however, SEQ ID NO: : Except for the sequence corresponding to the amino acid sequence described in 3 and 4), the amino acid sequence described in SEQ ID NO: 13 (except the sequence corresponding to the amino acid sequence described in SEQ ID NO: 5 and 6), SEQ ID NO: 14 (except for the sequences corresponding to the amino acid sequences described in SEQ ID NOs: 7 and 8), the amino acid sequence described in SEQ ID NO: 15 (however, corresponds to the amino acid sequence described in SEQ ID NO: 10) And a peptide having CTL inducing activity in vivo.

  As a preferred form of the modified peptide of the present invention related to the substitution, the C-terminal amino acid residue in the amino acid sequence of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, and 10 is used. And modified peptides substituted with amino acids that can be taken on the motif.

That is, the modified peptide of the present invention in this embodiment can include peptides having the amino acid sequences listed below and having CTL-inducing activity in vivo:
Trp Val Pro Val Val Phe Leu Thr Phe (SEQ ID NO: 16),
Trp Val Pro Val Val Phe Leu Thr Ile (SEQ ID NO: 17),
Trp Val Pro Val Val Phe Leu Thr Trp (SEQ ID NO: 18),
Trp Val Pro Val Val Phe Leu Thr Met (SEQ ID NO: 19),

Trp Tyr Pro Val Val Phe Leu Thr Phe (SEQ ID NO: 20),
Trp Tyr Pro Val Val Phe Leu Thr Ile (SEQ ID NO: 21),
Trp Tyr Pro Val Val Phe Leu Thr Trp (SEQ ID NO: 22),
Trp Tyr Pro Val Val Phe Leu Thr Met (SEQ ID NO: 23),

Thr Trp Ile Gly Ala Ala Pro Leu Phe (SEQ ID NO: 24),
Thr Trp Ile Gly Ala Ala Pro Leu Leu (SEQ ID NO: 25),
Thr Trp Ile Gly Ala Ala Pro Leu Trp (SEQ ID NO: 26),
Thr Trp Ile Gly Ala Ala Pro Leu Met (SEQ ID NO: 27),

Thr Tyr Ile Gly Ala Ala Pro Leu Phe (SEQ ID NO: 28),
Thr Tyr Ile Gly Ala Ala Pro Leu Leu (SEQ ID NO: 29),
Thr Tyr Ile Gly Ala Ala Pro Leu Trp (SEQ ID NO: 30),
Thr Tyr Ile Gly Ala Ala Pro Leu Met (SEQ ID NO: 31),

Asp Met Ser Leu Leu Lys Asn Arg Leu (SEQ ID NO: 32),
Asp Met Ser Leu Leu Lys Asn Arg Ile (SEQ ID NO: 33),
Asp Met Ser Leu Leu Lys Asn Arg Trp (SEQ ID NO: 34),
Asp Met Ser Leu Leu Lys Asn Arg Met (SEQ ID NO: 35),

Asp Tyr Ser Leu Leu Lys Asn Arg Leu (SEQ ID NO: 36),
Asp Tyr Ser Leu Leu Lys Asn Arg Ile (SEQ ID NO: 37),
Asp Tyr Ser Leu Leu Lys Asn Arg Trp (SEQ ID NO: 38),
Asp Tyr Ser Leu Leu Lys Asn Arg Met (SEQ ID NO: 39),

Lys Phe Met Leu Cys Ala Gly Arg Phe (SEQ ID NO: 40),
Lys Phe Met Leu Cys Ala Gly Arg Leu (SEQ ID NO: 41),
Lys Phe Met Leu Cys Ala Gly Arg Ile (SEQ ID NO: 42),
Lys Phe Met Leu Cys Ala Gly Arg Met (SEQ ID NO: 43),

Lys Tyr Met Leu Cys Ala Gly Arg Phe (SEQ ID NO: 44),
Lys Tyr Met Leu Cys Ala Gly Arg Leu (SEQ ID NO: 45),
Lys Tyr Met Leu Cys Ala Gly Arg Ile (SEQ ID NO: 46),
Lys Tyr Met Leu Cys Ala Gly Arg Met (SEQ ID NO: 47),

Val Tyr Leu Leu Gly Arg His Ser Leu Leu (SEQ ID NO: 48),
Val Tyr Leu Leu Gly Arg His Ser Leu Ile (SEQ ID NO: 49),
Val Tyr Leu Leu Gly Arg His Ser Leu Trp (SEQ ID NO: 50),
Val Tyr Leu Leu Gly Arg His Ser Leu Met (SEQ ID NO: 51).

  More specifically, a peptide comprising the amino acid sequence set forth in any of SEQ ID NOs: 16 to 51 and having CTL inducing activity in vivo can be mentioned.

In addition, cysteine residues are present in the amino acid sequences set forth in SEQ ID NOs: 7, 8, 14, and 40 to 47, which may be oxidized in a solution to form a disulfide bond. To avoid this, the cysteine residue is substituted with another amino acid residue, such as an alanine residue or serine residue, or is substituted with α-aminobutyric acid having a chemical structure similar to that of the cysteine residue. Are listed.
The modified peptide shown above can be produced by a general peptide synthesis method as described above. In addition, in vivo CTL-inducing activity can be measured by subjecting it to human model animals described in WO 02/47474 and Int J. Cancer: 100,565-570 (2002).

  Regarding the modified peptide of the present invention described above, a peptide in which a plurality of epitopes similar to (1-2) are linked, or a peptide modified with an amino group or a carboxyl group similar to (1-3) above. be able to.

  The peptide of the present invention as described above can be effectively used, for example, 1) as a CTL inducer described later and as an active ingredient of a cancer vaccine, and 2) in the production of antigen-presenting cells described later.

(II) Polynucleotide, Expression Vector, and Transformed Cell of the Present Invention The present invention also provides a polynucleotide encoding the peptide of the present invention. The polynucleotide encoding the peptide of the present invention may be in the form of DNA or RNA. These polynucleotides of the present invention can be easily produced based on the amino acid sequence information of the peptide of the present invention and the sequence information of the DNA encoded thereby. Specifically, it can be produced by ordinary DNA synthesis or PCR amplification.

Examples of such polynucleotides of the present invention include the following polynucleotides:
A polynucleotide encoding a peptide comprising Trp Val Pro Val Val Phe Leu Thr Leu (SEQ ID NO: 1);
A polynucleotide encoding a peptide comprising Trp Tyr Pro Val Val Phe Leu Thr Leu (SEQ ID NO: 2);
A polynucleotide encoding a peptide comprising Thr Trp Ile Gly Ala Ala Pro Leu Ile (SEQ ID NO: 3);
A polynucleotide encoding a peptide comprising Thr Tyr Ile Gly Ala Ala Pro Leu Ile (SEQ ID NO: 4),
A polynucleotide encoding a peptide comprising Asp Met Ser Leu Leu Lys Asn Arg Phe (SEQ ID NO: 5),
A polynucleotide encoding a peptide comprising Asp Tyr Ser Leu Leu Lys Asn Arg Phe (SEQ ID NO: 6),
A polynucleotide encoding a peptide comprising Lys Phe Met Leu Cys Ala Gly Arg Trp (SEQ ID NO: 7);
A polynucleotide encoding a peptide comprising Lys Tyr Met Leu Cys Ala Gly Arg Trp (SEQ ID NO: 8);
A polynucleotide encoding a peptide containing Val Tyr Leu Leu Gly Arg His Ser Leu Phe (SEQ ID NO: 10).

  Specifically, for example, an epitope peptide comprising any one of the amino acid sequences of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8 and 10 as described in the above (1-2) is encoded. A polynucleotide is mentioned. More specifically, for example, a peptide in which one or more of the amino acid sequences of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, and 10 is linked to a helper peptide. For example, one or more of the amino acid sequences of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, and 10 and a helper derived from tetanus toxin A polynucleotide encoding a peptide linked to a peptide (for example, Phe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg Val Pro Lys Val Ser Ala Ser His Leu Glu; SEQ ID NO: 52), SEQ ID NOS: 1, 2, One or more amino acid sequences of any of 3, 4, 5, 6, 7, 8, and 10 and Ala Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu (SEQ ID NO: 53, Clinical Cancer Res., 2001, 7: 3012-3024) and a polynucleotide encoding a peptide Kill.

By incorporating the polynucleotide of the present invention prepared above into an expression vector, a recombinant expression vector for expressing the peptide of the present invention can be prepared.
The expression vector used here can be appropriately selected according to the host to be used, the purpose, etc., and includes plasmids, phage vectors, virus vectors and the like.

  For example, when the host is Escherichia coli, examples of the vector include plasmid vectors such as pUC118, pUC119, pBR322, and pCR3, and phage vectors such as λZAPII and λgt11. When the host is yeast, examples of the vector include pYES2, pYEUra3 and the like. When the host is an insect cell, pAcSGHisNT-A and the like can be mentioned. When the host is an animal cell, plasmid vectors such as pKCR, pCDM8, pGL2, pcDNA3.1, pRc / RSV, and pRc / CMV, and viral vectors such as retrovirus vectors, adenovirus vectors, and adeno-associated virus vectors are listed. It is done.

The vector may appropriately have factors such as a promoter capable of inducing expression, a gene encoding a signal sequence, a selection marker gene, and a terminator.
Further, a sequence expressed as a fusion protein with thioredoxin, His tag, GST (glutathione S-transferase) or the like may be added so that isolation and purification can be facilitated. In this case, a GST fusion protein vector (such as pGEX4T) that has an appropriate promoter (such as lac, tac, trc, trp, CMV, or SV40 early promoter) that functions in the host cell, or a vector that has a tag sequence such as Myc or His. (PcDNA3.1 / Myc-His, etc.), and a vector (pET32a) that expresses a fusion protein with thioredoxin and His tag can be used.

By subjecting the polynucleotide of the present invention as described above or an expression vector containing the same to the human model animal described in WO 02/47474 and Int J. Cancer: 100,565-570 (2002), in vivo CTL-inducing activity can be measured.
The polynucleotide of the present invention or the expression vector containing it is effective, for example, in 1) production of the peptide of the present invention described later, 2) gene therapy described later, and 3) production of antigen-presenting cells described later. Can be used.

By transforming the host with the expression vector prepared above, a transformed cell containing the expression vector can be prepared.
Examples of the host used here include Escherichia coli, yeast, insect cells, animal cells and the like. Examples of E. coli include HB101 strain, C600 strain, JM109 strain, DH5α strain, AD494 (DE3) strain and the like of E. coli K-12 strain. Examples of yeast include Saccharomyces cerevisiae. Examples of animal cells include L929 cells, BALB / c3T3 cells, C127 cells, CHO cells, COS cells, Vero cells, and Hela cells. Insect cells include sf9.

  As a method for introducing the expression vector into the host cell, a normal method suitable for the host cell may be used. Specific examples include a calcium phosphate method, a DEAE-dextran method, an electroporation method, and a method using a lipid for gene transfer (Lipofectamine, Lipofectin; Gibco-BRL). After the introduction, a transformed cell in which the expression vector is introduced into the host cell can be selected by culturing in a normal medium containing a selection marker.

  The peptide of the present invention can be produced by continuing to culture the transformed cells obtained as described above under suitable conditions (where the peptide can be expressed). The obtained polypeptide can be further isolated and purified by general biochemical purification means. Examples of the purification means include salting out, ion exchange chromatography, adsorption chromatography, affinity chromatography, gel filtration chromatography and the like. In addition, when the polypeptide of the present invention is expressed as a fusion protein with the aforementioned thioredoxin, His tag, GST or the like, it can be isolated and purified by a purification method utilizing the properties of these fusion protein and tag. .

(III) Antibody of the present invention The present invention provides an antibody that specifically binds to the peptide of the present invention. The form of the antibody of the present invention is not particularly limited, and may be a polyclonal antibody using the peptide of the present invention as an immunizing antigen or a monoclonal antibody.
The antibody of the present invention is not particularly limited as long as it specifically binds to the peptide of the present invention as described above. Specifically, SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, An antibody that specifically binds to a cancer antigen peptide consisting of the amino acid sequence described in any of 8 and 10 can be mentioned.

  Methods for producing these antibodies are already well known, and the antibodies of the present invention can also be produced according to these conventional methods (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley and Sons. Section 11.12-11.13, Antibodies; A Laboratory Manual, Lane, H, D. et al., Cold Spring Harber Laboratory Press published New York 1989).

  Specifically, the peptide of the present invention (for example, a cancer antigen peptide consisting of the amino acid sequence described in any of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, and 10) is used as an immunogen. It is possible to immunize a non-human animal such as a rabbit and obtain it from the serum of the immunized animal according to a conventional method. On the other hand, in the case of a monoclonal antibody, the peptide of the present invention (for example, a cancer antigen peptide consisting of the amino acid sequence of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, and 10) is used. It can be obtained from hybridoma cells prepared by immunizing non-human animals such as mice and fusing the obtained spleen cells and myeloma cells (Current protocols in Molecular Biology edit. Ausubel et al. (1987 Publish. John Wiley and Sons. Section 11.4-11.11).

  Production of an antibody against the peptide of the present invention can also be performed by enhancing immunological reaction using various adjuvants depending on the host. Such adjuvants include Freund's adjuvant, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyol, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin and dinitrophenol, BCG ( There are human adjuvants such as Calumet-Guerin bacilli and Corynebacterium parvum.

  As described above, by appropriately immunizing an animal using the peptide of the present invention by a conventional method, an antibody that recognizes the peptide and an antibody that neutralizes the activity can be easily prepared. Examples of the use of the antibody include affinity chromatography and immunological diagnosis. The immunological diagnosis can be appropriately selected from immunoblotting, radioimmunoassay (RIA), enzyme immunoassay (ELISA), fluorescence or luminescence assay. Such immunological diagnosis is effective in diagnosing cancers in which the PSA gene is expressed, such as prostate cancer and breast cancer.

(IV) Antigen-presenting cell of the present invention The present invention provides an antigen-presenting cell in which a complex of a cancer antigen peptide derived from the peptide of the present invention and an HLA-A24 antigen is presented.
In the examples described below, CTL-inducing activity was observed by administration of the peptide of the present invention, which is a complex of the cancer antigen peptide derived from the peptide of the present invention and the HLA-A24 antigen in peripheral blood mononuclear cells. This shows that the presenting antigen-presenting cells were present, and CTLs specifically damaging the cancer cells presented by this complex were induced. Such antigen-presenting cells on which a complex of the HLA-A24 antigen and the cancer antigen peptide derived from the peptide of the present invention is presented are effectively used in cell therapy (DC therapy) described later.

  The antigen-presenting cell of the present invention may be an antigen-presenting cell in which a complex of the cancer antigen peptide derived from the peptide of the present invention and the HLA-A24 antigen is presented. Specifically, for example, SEQ ID NO: 1, Antigen presentation in which a complex of an HLA-A24 antigen and a cancer antigen peptide consisting of the amino acid sequence according to any of 2, 3, 4, 5, 6, 7, 8, and 10 is presented on the cell surface of dendritic cells Mention may be made of cells.

Antigen-presenting cells used in cell therapy isolate cells having antigen-presenting ability from cancer patients, and pulse these cells with the peptide of the present invention in vitro, or polynucleotides of the present invention and expression vectors containing the same Is introduced into the cell, and a complex of the HLA-A24 antigen and the cancer antigen peptide derived from the peptide of the present invention is presented on the cell surface. Here, the “cell having antigen-presenting ability” is not particularly limited as long as it is a cell that expresses the HLA-A24 antigen capable of presenting the peptide of the present invention on the cell surface. Dendritic cells are preferred.
In addition, the pulsed cell having the antigen-presenting ability may be the peptide of the present invention, or may be a polynucleotide encoding the peptide of the present invention or an expression vector containing the same. .

  The antigen-presenting cell of the present invention is isolated from, for example, a cell having antigen-presenting ability from a cancer patient, and the peptide of the present invention (for example, SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, and A cancer antigen peptide consisting of the amino acid sequence described in any one of 10) in vitro, and obtained by preparing a complex of the HLA-A24 antigen and the cancer antigen peptide derived from the peptide of the present invention (Cancer Immunol. Immunother., 46:82, 1998, J. Immunol., 158: p1796, 1997, Cancer Res., 59: p1184, 1999). When using dendritic cells, for example, lymphocytes are isolated from peripheral blood of cancer patients by Ficoll method, then non-adherent cells are removed, and adherent cells are cultured in the presence of GM-CSF and IL-4 to form dendritic cells. The antigen-presenting cell of the present invention can be prepared by inducing cells, culturing the dendritic cell with the peptide of the present invention, and pulsing.

Further, a polynucleotide encoding the peptide of the present invention (for example, the sequence described in any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, and 10) is contained in the cell having the antigen presenting ability When the antigen-presenting cell of the present invention is prepared by introducing a polynucleotide encoding a peptide) or an expression vector containing the same, if the polynucleotide is DNA, Cancer Res., 56: p5672,1996 and J .Immunol., 161: p5607,1998 etc. Further, antigen-presenting cells can be similarly prepared not only in the form of DNA but also in the form of RNA. In this case, it can be carried out with reference to J. Exp. Med., 184: p465, 1996 and the like.
The antigen-presenting cells of the present invention prepared as described above are effectively used in cell therapy (DC therapy) as an CTL inducer and an active ingredient of a cancer vaccine described later.

(V) CTL of the present invention
The present invention provides a CTL that recognizes a complex of a cancer antigen peptide derived from the peptide of the present invention and an HLA-A24 antigen.
In the examples described below, CTL-inducing activity was observed by administration of the peptide of the present invention, which is a complex of the cancer antigen peptide derived from the peptide of the present invention and the HLA-A24 antigen in peripheral blood mononuclear cells. This shows that the presenting antigen-presenting cells were present, and CTLs specifically damaging the cancer cells presented by this complex were induced. Such a CTL that specifically recognizes a complex of the HLA-A24 antigen and the cancer antigen peptide derived from the peptide of the present invention is effectively used in adoptive immunotherapy described later.

  The CTL of the present invention may be any one that specifically recognizes a complex of the cancer antigen peptide derived from the peptide of the present invention and the HLA-A24 antigen. Specifically, for example, SEQ ID NOs: 1, 2, Examples include CTLs that specifically recognize a complex of a cancer antigen peptide consisting of the amino acid sequence described in any of 3, 4, 5, 6, 7, 8, and 10 and an HLA-A24 antigen.

  The CTL used in adoptive immunotherapy isolates a patient's peripheral blood lymphocytes from the peptide of the present invention (eg, any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, and 10). Or a polynucleotide encoding the peptide of the present invention (for example, SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, and 10). (Polynucleotide encoding a peptide containing a sequence) or an expression vector containing the same, or in vitro stimulation (Journal of Experimental Medicine 1999, 190: 1669).

  The CTL of the present invention produced as described above is effectively used in adoptive immunotherapy as an active ingredient of a cancer vaccine.

(VI) Pharmaceutical composition as a cancer vaccine The peptide of the present invention described above, the expression vector containing the polynucleotide of the present invention, the cell containing the expression vector of the present invention, the antigen-presenting cell of the present invention, and the present invention These CTLs can be used as an inducer of CTLs, that is, an active ingredient of a cancer vaccine, by taking an appropriate form according to each substance. This will be specifically described below.

(6-1) Cancer vaccine comprising the peptide of the present invention as an active ingredient The peptide of the present invention has the ability to induce CTL, and the induced CTL is anticancerous through cytotoxicity or lymphokine production. The effect can be exerted. Therefore, the peptide of the present invention can be used as an active ingredient of a cancer vaccine for treating or preventing cancer. That is, the present invention provides a cancer vaccine (pharmaceutical composition as a cancer vaccine) containing the peptide of the present invention as an active ingredient. When the cancer vaccine of the present invention is administered to an HLA-A24 positive and PSA positive patient, a peptide (eg, SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, and Cancer antigen peptide consisting of the amino acid sequence of any one of 10), CTL specific for the complex of the presented peptide and HLA-A24 antigen can proliferate and destroy cancer cells, Accordingly, it is possible to treat or prevent cancer. The cancer vaccine of the present invention can be used for the prophylaxis or treatment of cancers associated with increased expression levels of the PSA gene, such as prostate cancer and breast cancer.
Therefore, the present invention provides, as another aspect, a method for treating or preventing cancer by administering an effective amount of the cancer vaccine of the present invention to HLA-A24 positive and PSA positive patients.

  Even if the cancer vaccine which uses the peptide of this invention as an active ingredient has a single CTL epitope as an active ingredient, the epitope peptide linked with other peptides (CTL epitope and helper epitope) becomes an active ingredient. It may be a thing. That is, in recent years, it has been shown that an epitope peptide in which a plurality of CTL epitopes (antigen peptides) are linked has efficient CTL inducing activity in vivo. For example, in Journal of Immunology 1998, 161: 3186-3194, an approximately 30-mer epitope peptide linked to a PSA-derived HLA-A2, -A3, -A11, B53-restricted CTL epitope (antigen peptide) was generated in vivo. It is described that CTL specific to each CTL epitope was induced. It has also been shown that CTL is efficiently induced by an epitope peptide in which a CTL epitope and a helper epitope are linked. When administered in the form of such an epitope peptide, it is taken up into the antigen-presenting cell, and then the individual antigen peptide generated by intracellular degradation binds to the HLA antigen to form a complex. Are present at high density on the surface of antigen-presenting cells, and CTL specific for this complex efficiently proliferate in the body and destroy cancer cells. In this way, treatment or prevention of cancer is achieved.

  In addition, the cancer vaccine comprising the peptide of the present invention as an active ingredient is administered together with a pharmaceutically acceptable carrier, for example, an appropriate adjuvant, or administered in a particulate dosage form so that cellular immunity is effectively established. can do. As adjuvants, those described in the literature (Clin. Microbiol. Rev., 7: 277-289, 1994) can be applied. Specifically, bacterial cell-derived components, cytokines, plant-derived components, hydroxylation Examples thereof include mineral gels such as aluminum, surfactants such as lysolecithin and pluronic polyol, polyanions, peptides, and oil emulsions (emulsion preparations). In addition, a liposome preparation, a particulate preparation bound to beads having a diameter of several μm, a preparation bound to lipid, and the like are also conceivable.

  Examples of the administration method include intradermal administration, subcutaneous administration, intramuscular administration, intravenous administration and the like. The dosage of the peptide of the present invention in the preparation can be adjusted as appropriate depending on the disease to be treated, the age, weight, etc. of the patient, but is usually 0.0001 mg to 1000 mg, preferably 0.001 mg to 1000 mg, more preferably 0.1 mg. ˜10 mg, which is preferably administered once every few days or months.

(6-2) DNA vaccine comprising an expression vector containing a polynucleotide encoding the peptide of the present invention as an active ingredient Not only the peptide of the present invention but also an expression vector containing a polynucleotide encoding the peptide, It can be an active ingredient of a DNA vaccine for the treatment or prevention of cancer. That is, the present invention provides a cancer vaccine (pharmaceutical composition as a cancer vaccine) containing, as an active ingredient, an expression vector containing a polynucleotide encoding the peptide of the present invention. In another embodiment, the present invention provides a method for treating or preventing cancer by administering an effective amount of the DNA vaccine of the present invention to HLA-A24 positive and PSA positive patients.

  In recent years, a polynucleotide encoding an epitope peptide in which a plurality of CTL epitopes (antigen peptides) are linked, or a polynucleotide encoding an epitope peptide in which a CTL epitope and a helper epitope are linked has been efficiently and efficiently induced in vivo. It has been shown to have For example, in Journal of Immunology 1999, 162: 3915-3925, DNA (minigene) encoding an epitope peptide linked with 6 types of HBV-derived HLA-A2 restricted antigen peptides, 3 types of HLA-A11 restricted antigen peptides, and a helper epitope. ) Effectively induced CTL against each epitope in vivo.

  Accordingly, a polynucleotide produced by ligating one or more polynucleotides encoding the peptide of the present invention and, optionally, also linking a polynucleotide encoding another peptide may be appropriately expressed. By incorporating into a vector, it can be used as an active ingredient of a cancer vaccine.

When applying the polynucleotide of the present invention as an active ingredient of a cancer vaccine (DNA vaccine), the following method can be used.
That is, as a method for introducing the polynucleotide of the present invention into a cell, a viral vector method and other methods (Nikkei Science, April 1994, pages 20-45, Monthly Pharmaceutical Affairs, 36 (1), 23- 48 (1994), Experimental Medicine Extra Number, 12 (15), (1994), and cited references thereof, etc.) can be applied.

As a method using a viral vector, for example, the DNA of the present invention is incorporated into a retrovirus, an adenovirus, an adeno-associated virus, a herpes virus, a vaccinia virus, a pox virus, a poliovirus, a symbis virus, or the like, and introduced. A method is mentioned. Of these, methods using retroviruses, adenoviruses, adeno-associated viruses, vaccinia viruses and the like are particularly preferred.
Examples of other methods include a method in which an expression plasmid is directly administered into muscle (DNA vaccine method), a liposome method, a lipofectin method, a microinjection method, a calcium phosphate method, an electroporation method, and the like. The method is preferred.

  In order for the polynucleotide of the present invention to actually act as a medicine, an in vivo method in which the polynucleotide-containing expression vector is directly introduced into the body, and certain cells from human beings are collected and the expression vector is applied to the cells outside the body. There is an ex vivo method to introduce and return the cells to the body (Nikkei Science, April 1994, 20-45, Monthly Pharmaceutical Affairs, 36 (1), 23-48 (1994), Experimental Medicine Extra Number, 12 (15 ), (1994), and references cited therein). In vivo methods are more preferred.

When administered by an in vivo method, it can be administered by an appropriate administration route according to the disease, symptom or the like for treatment. For example, it can be administered intravenously, artery, subcutaneously, intradermally, intramuscularly. When administered by the in vivo method, for example, it can take a pharmaceutical form such as a liquid, but is generally an injection containing the polynucleotide-containing expression vector of the present invention which is an active ingredient, and if necessary, Conventional carriers may be added. In addition, in the liposome or membrane fusion liposome (Sendai virus (HVJ) -liposome etc.) containing the polynucleotide-containing expression vector of the present invention, the form of liposome preparation such as suspension, freezing agent, centrifugal concentrated freezing agent, etc. can do.
The content of the polynucleotide-containing expression vector of the present invention in the preparation can be appropriately adjusted depending on the disease to be treated, the age, weight, etc. of the patient, but is usually 0.0001 mg to 100 mg, preferably 0.001 mg to 10 mg. The polynucleotide-containing expression vector of the invention is preferably administered once every several days to several months.

  By administering the polynucleotide-containing expression vector of the present invention as described above to a cancer patient, a polypeptide corresponding to the polynucleotide is highly expressed in antigen-presenting cells. Thereafter, individual cancer antigen peptides generated by intracellular degradation bind to HLA antigens to form complexes, which are presented at a high density on the surface of antigen-presenting cells, and are specific to this complex. CTL grows efficiently in the body and destroys cancer cells. As described above, treatment or prevention of cancer is achieved. A cancer vaccine comprising an expression vector containing the polynucleotide of the present invention as an active ingredient can be used for the prevention or treatment of cancers accompanied by an increase in the expression level of the PSA gene, such as prostate cancer and breast cancer.

(6-3) Cancer vaccine comprising the antigen-presenting cell of the present invention as an active ingredient The present invention provides a cancer vaccine comprising the antigen-presenting cell of the present invention as an active ingredient.
In recent years, cell therapy that isolates lymphocytes from peripheral blood of cancer patients, induces dendritic cells from them, and returns antigen-presenting cells prepared by pulsing peptides etc. in vitro to the patient by subcutaneous administration ( (DC therapy) has been reported (Cancer Immunol.Immunother., 46: 82,1998, J.Immunol., 158: p1796,1997, Cancer Res., 59: p1184,1999, Cancer Res., 56: p5672, 1996, J. Immunol., 161: p5607, 1998, J. Exp. Med., 184: p465, 1996). Therefore, the antigen-presenting cell of the present invention can be used as an active ingredient of a cancer vaccine in cell therapy.

The cancer vaccine containing the antigen-presenting cells of the present invention as an active ingredient preferably contains physiological saline, phosphate buffered saline (PBS), a medium, and the like in order to stably maintain the antigen-presenting cells. Examples of the administration method include intravenous administration, subcutaneous administration, and intradermal administration. The dosage is exemplified by the dosage described in the literature.
By returning the cancer vaccine to the body of the patient, specific CTLs can be efficiently induced in the body of HLA-A24-positive and PSA-positive patients, and cancer can be treated or prevented. The cancer vaccine comprising the antigen-presenting cell of the present invention as an active ingredient can be used for the prevention or treatment of cancers accompanied by an increase in the expression level of the PSA gene, such as prostate cancer and breast cancer.

(6-4) Cancer vaccine containing CTL of the present invention as an active ingredient The present invention provides a cancer vaccine (pharmaceutical composition as a cancer vaccine) containing the CTL of the present invention as an active ingredient. The CTL of the present invention is effectively used in the following adoptive immunotherapy.

  In melanoma, a therapeutic effect has been observed in adoptive immunotherapy in which the patient's own tumor-infiltrating T cells are cultured in large quantities outside the body and then returned to the patient (J. Natl. Cancer. Inst., 86: 1159, 1994). ). In mouse melanoma, spleen cells are stimulated in vitro with the cancer antigen peptide TRP-2, CTL specific for the cancer antigen peptide is proliferated, and the CTL is administered to the melanoma-transplanted mouse, thereby suppressing metastasis. (J. Exp. Med., 185: 453, 1997). This is based on the result of in vitro growth of CTL that specifically recognizes a complex of an antigen-presenting cell HLA antigen and a cancer antigen peptide. Therefore, a therapeutic method for increasing the cancer-specific CTL by in vitro stimulation of patient peripheral blood lymphocytes using the peptide of the present invention or the polynucleotide or expression vector of the present invention and then returning this CTL to the patient is It is considered useful. Therefore, the CTL of the present invention can be used as an active ingredient of a cancer vaccine in adoptive immunotherapy.

The cancer vaccine containing the CTL of the present invention as an active ingredient preferably contains physiological saline, phosphate buffered saline (PBS), a medium and the like in order to stably maintain CTL. Examples of the administration method include intravenous administration, subcutaneous administration, and intradermal administration. Moreover, as a dosage, the dosage of the said literature description is illustrated.
By returning the cancer vaccine to the body of the patient, the cytotoxic effect of CTLs on cancer cells is promoted in the body of HLA-A24-positive and PSA-positive patients, and cancer can be treated by destroying the cancer cells. . The cancer vaccine containing the CTL of the present invention as an active ingredient can be used for the prevention or treatment of cancer accompanied by an increase in the expression level of the PSA gene, such as prostate cancer and breast cancer.

The present invention also provides an HLA tetramer containing the peptide of the present invention and the HLA-A24 antigen.
Here, the HLA tetramer refers to a tetramer obtained by biotinylating a complex (HLA monomer) in which an α chain of an HLA antigen and β2 microglobulin are associated with a peptide (antigen peptide) and binding it to avidin. (Science 279: 2103-2106 (1998), Science 274: 94-96 (1996)). At present, HLA tetramers containing various antigen peptides are commercially available (for example, Medical and Biological Laboratories Co., Ltd.), and HLA tetramers containing the peptide of the present invention and the HLA-A24 antigen can be easily prepared. it can.
Specifically, for example, an HLA tetramer containing a peptide consisting of the amino acid sequence described in any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8 and 10 and an HLA-A24 antigen is mentioned. It is done.

  The HLA tetramer is preferably fluorescently labeled so that CTLs bound thereto can be easily selected or detected by known detection means such as flow cytometry and fluorescence microscope. Specific examples include HLA tetramers labeled with phycoerythrin (PE), fluorescein isothiocyanate (FITC), peridinin chlorophyll protein (PerCP), and the like.

  The HLA-A24 antigen (alpha chain of HLA-A24 antigen), which is a component of the HLA tetramer, is known from HLA-A2402 disclosed in Cancer Res., 55: 4248-4252 (1995) and Genbank Accession No. M64740. Based on the information of the base sequence, it can be easily cloned by conventional methods such as PCR.

  The β2 microglobulin that is a component of the HLA tetramer is preferably a human-derived β2 microglobulin. The human β2 microglobulin can be easily cloned by a conventional method such as PCR based on the known nucleotide sequence information of human β2 microglobulin disclosed in GenBank Acc. No. AB021288.

The method for preparing the HLA tetramer containing the above HLA tetramer components is well known in the literature (Science 279: 2103-2106 (1998), Science 274: 94-96 (1996), etc.). It becomes as follows.
First, an HLA-A24 α chain expression vector and a β2 microglobulin expression vector are introduced into E. coli or mammalian cells capable of expressing the protein and expressed. Here, it is preferable to use Escherichia coli (for example, BL21). The obtained monomer HLA-A24 complex and the peptide of the present invention are mixed to form a soluble HLA-peptide complex. Next, the sequence of the C-terminal part of the HLA-A24α chain in the HLA-peptide complex is biotinylated with a BirA enzyme. An HLA tetramer can be prepared by mixing the biotinylated HLA-peptide complex and fluorescently labeled avidin at a molar ratio of 4: 1. In each step, protein purification by gel filtration or the like is preferably performed.

Moreover, this invention provides the diagnostic agent of the effect of the cancer vaccine containing the HLA tetramer of this invention.
By using the diagnostic agent for the effect of the cancer vaccine containing the HLA tetramer of the present invention, the effect of the cancer vaccine can be diagnosed by detecting the peptide-specific CTL of the present invention. Specifically, a biological sample (test sample) is isolated from a test patient administered with a cancer vaccine, the HLA tetramer of the present invention is contacted with the biological sample, and the peptide-specific CTL of the present invention bound to the HLA tetramer is obtained. By determining the presence frequency or amount with a flow cytometer or the like, it can be diagnosed whether peptide-specific CTLs are induced or enhanced by administration of a cancer vaccine.

  EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, this invention is not limited at all by these Examples.

Selection of PSA-derived HLA-binding candidate peptide region The HLA-A24 binding motif (amino acid at the second position) in the amino acid sequence of PSA (Biochem. Biophys. Res. Commun., 160: 903, 1989, NCBI database Accession No. M26663) TLA, phenylalanine, methionine, or tryptophan, and the C-terminal amino acid is phenylalanine, tryptophan, leucine, isoleucine, or methionine) or a similar sequence thereof. Binding candidate peptide regions were selected. That is, peptide A (SEQ ID NO: 1) and its modified peptide B (SEQ ID NO: 2), peptide C (SEQ ID NO: 3) and its modified peptide D (SEQ ID NO: 4), peptide E: ( SEQ ID NO: 5) and its modified peptide F (SEQ ID NO: 6), peptide G: (SEQ ID NO: 7) and its modified peptide H (SEQ ID NO: 8), peptide I (SEQ ID NO: 9) And peptide J (SEQ ID NO: 10) of the variant thereof. Peptide A is PSA positions 2-10, Peptide C is positions 13-21, Peptide E is positions 102-110, Peptide G is positions 194-202, Peptide I is positions 72-81 It corresponds to. These peptides were synthesized by the Fmoc method.

Peptide A: Trp Val Pro Val Val Phe Leu Thr Leu (SEQ ID NO: 1)
Peptide B: Trp Tyr Pro Val Val Phe Leu Thr Leu (SEQ ID NO: 2)
Peptide C: Thr Trp Ile Gly Ala Ala Pro Leu Ile (SEQ ID NO: 3)
Peptide D: Thr Tyr Ile Gly Ala Ala Pro Leu Ile (SEQ ID NO: 4)
Peptide E: Asp Met Ser Leu Leu Lys Asn Arg Phe (SEQ ID NO: 5)
Peptide F: Asp Tyr Ser Leu Leu Lys Asn Arg Phe (SEQ ID NO: 6)
Peptide G: Lys Phe Met Leu Cys Ala Gly Arg Trp (SEQ ID NO: 7)
Peptide H: Lys Tyr Met Leu Cys Ala Gly Arg Trp (SEQ ID NO: 8)
Peptide I: Val Ile Leu Leu Gly Arg His Ser Leu Phe (SEQ ID NO: 9)
Peptide J: Val Tyr Leu Leu Gly Arg His Ser Leu Phe (SEQ ID NO: 10)

For each HLA-A24-binding candidate peptides selected by drug coordination and administration Example 1 peptide vaccine, ^{I-A b} binding helper peptide of murine MHC class II from tetanus toxin (FNNFTVSFWLRVPKVSASHLE, SEQ ID NO: 52) with, HLA -A24-expressing transgenic mice (WO 02/47474, Int J. Cancer: 100,565-570 (2002)) were immunized. That is, each synthetic peptide was adjusted to 40 mg / ml and the helper peptide was adjusted to 20 mg / ml with DMSO, and diluted to 2 mg / ml and 1 mg / ml with physiological saline. Next, a water-in-oil emulsion was prepared by mixing with an equal amount of incomplete Freund's adjuvant (Wako Pure Chemical Industries, Ltd.) using a glass syringe. 200 μl of the drug was immunized subcutaneously in the ridge of transgenic mice.

Preparation of spleen cells and in vitro re-stimulation experiment 7 days after the start of the in vitro re-stimulation experiment, the spleen was removed and scraped with a frosted portion of a slide glass to collect and prepare the spleen cells. A peptide immunized to a part of splenocytes treated with ACK buffer (0.15M NH ₄ Cl, 10 mM KHCO ₃ , 0.1 mM EDTA, pH 7.2-7.4) was pulsed at 100 μg / ml for 1 hour and 0.7 × 10 It seed | inoculated to 24-well plate by ⁶ piece / well. At this time, 7 × 10 ⁶ non-peptide pulses of splenocytes / well were simultaneously added and restimulation was performed at 37 ° C. (final peptide concentration 1 μg / ml). Culture medium containing 10% FCS, 10 mM HEPES, 20 mM L-glutamine, 1 mM sodium pyruvate, 1 mM MEM non-essential amino acid, 1% MEM vitamin, 55 μM 2-mercaptoethanol in RPMI1640 medium (CTM culture medium) Was re-stimulated in vitro using 10 ml.

Cytotoxicity test 5-6 days after the start of in vitro stimulation culture, a cytotoxicity test was performed according to a conventional method. EL4-A2402 / K ^b cells and peptide-pulsed EL4-A2402 / K ^b cells were used as target cells (T). The EL4-A2402 / K ^b cells used here were inserted into the expression vector pEF1 / myc-His A (Invitrogen) by inserting the gene encoding HLA-A2402 / K ^b (WO02 / 47474) into EL-4 It was prepared by transforming cells (ATCC T1B-39).
These cells were 1.875 MBq / 5 × 10 ⁵ cells and ⁵¹ Cr-labeled, and the peptide pulse was performed at 100 μg / ml for 1 hour (labeling time 2 hours, adding peptide 1 hour after the start of labeling). Spleen cells cultured in vitro were used as effector cells (E) and allowed to act at an E / T ratio of 80, 40, 20, 10 or 40, 20, 10, and the cytotoxic activity was determined by ⁵¹ Cr release assay (J. Immunol., 159: 4753, 1997). The results are shown in FIGS. In the figure, the vertical axis represents the cytotoxic activity (% specific lysis). In the figure, pep + indicates the result using a peptide pulsed cell as a target cell, and pep− indicates the result using a peptide non-pulsed cell as a target cell.

  As is clear from this figure, among the selected natural peptides, peptides A, C, E, and G are immunogenic, and among modified peptides, peptides B, D, F, H, and J are immunogenic (CTL inducing activity). It was shown to have The natural peptides A, E, and G were enhanced in immunogenicity by modification. On the other hand, natural peptide I has no immunogenicity, but has been modified to have immunogenicity.

  The present invention provides a PSA-derived HLA-A24-binding cancer antigen peptide, a polynucleotide encoding the peptide, a CTL inducer containing these peptides and polynucleotides, and the like. The CTL inducer of the present invention is useful as a cancer vaccine. The cancer vaccine of the present invention is applicable to many cancer patients who are HLA-A24 positive.

It is a graph which shows the result of having immunized the HLA-A24 expression transgenic mouse with the peptide A, and investigating specific CTL induction activity. In the figure, the vertical axis represents the cytotoxic activity (% Specific Lysis), and the horizontal axis represents the E / T ratio. The black circles show the results using target cells pulsed with peptide A, and the white circles show the results using peptide non-pulsed cells. It is a graph which shows the result of having immunized the HLA-A24 expression transgenic mouse with the peptide B, and investigating specific CTL induction activity. In the figure, the vertical axis represents the cytotoxic activity (% Specific Lysis), and the horizontal axis represents the E / T ratio. The black circles show the results using target cells pulsed with peptide B, and the white circles show the results using peptide non-pulsed cells. It is a graph which shows the result of having immunized the HLA-A24 expression transgenic mouse with the peptide C, and investigating specific CTL induction activity. In the figure, the vertical axis represents the cytotoxic activity (% Specific Lysis), and the horizontal axis represents the E / T ratio. The black circles show the results using target cells pulsed with peptide C, and the white circles show the results using peptide non-pulsed cells. It is a graph which shows the result of having immunized the HLA-A24 expression transgenic mouse with the peptide D, and investigating specific CTL induction activity. In the figure, the vertical axis represents the cytotoxic activity (% Specific Lysis), and the horizontal axis represents the E / T ratio. The black circles show the results using target cells pulsed with peptide D, and the white circles show the results using peptide non-pulsed cells. It is a graph which shows the result of having immunized the HLA-A24 expression transgenic mouse with the peptide E, and investigating specific CTL induction activity. In the figure, the vertical axis represents the cytotoxic activity (% Specific Lysis), and the horizontal axis represents the E / T ratio. The black circles show the results using target cells pulsed with peptide E, and the white circles show the results using peptide non-pulsed cells. It is a graph which shows the result of having immunized the HLA-A24 expression transgenic mouse with the peptide F, and investigating specific CTL induction activity. In the figure, the vertical axis represents the cytotoxic activity (% Specific Lysis), and the horizontal axis represents the E / T ratio. The black circles show the results using target cells pulsed with peptide F, and the white circles show the results using peptide non-pulsed cells. It is a graph which shows the result of having immunized the HLA-A24 expression transgenic mouse with the peptide G, and investigating specific CTL induction activity. In the figure, the vertical axis represents the cytotoxic activity (% Specific Lysis), and the horizontal axis represents the E / T ratio. The black circles show the results using target cells pulsed with peptide G, and the white circles show the results using peptide non-pulsed cells. It is a graph which shows the result of having immunized the HLA-A24 expression transgenic mouse with the peptide H, and investigating specific CTL induction activity. In the figure, the vertical axis represents the cytotoxic activity (% Specific Lysis), and the horizontal axis represents the E / T ratio. The black circles show the results using target cells pulsed with peptide H, and the white circles show the results using peptide non-pulsed cells. It is a graph which shows the result of having immunized the HLA-A24 expression transgenic mouse with the peptide I, and investigating specific CTL induction activity. In the figure, the vertical axis represents the cytotoxic activity (% Specific Lysis), and the horizontal axis represents the E / T ratio. The black circles show the results using the target cells pulsed with peptide I, and the white circles show the results using the non-peptide cells. It is a graph which shows the result of having immunized the HLA-A24 expression transgenic mouse with the peptide J, and investigating specific CTL induction activity. In the figure, the vertical axis represents the cytotoxic activity (% Specific Lysis), and the horizontal axis represents the E / T ratio. The black circles show the results using target cells pulsed with peptide J, and the white circles show the results using peptide non-pulsed cells.

Claims (13)

The following amino acid sequence:
Trp Val Pro Val Val Phe Leu Thr Leu (SEQ ID NO: 1),
Trp Tyr Pro Val Val Phe Leu Thr Leu (SEQ ID NO: 2),
Thr Trp Ile Gly Ala Ala Pro Leu Ile (SEQ ID NO: 3),
Thr Tyr Ile Gly Ala Ala Pro Leu Ile (SEQ ID NO: 4),
Asp Met Ser Leu Leu Lys Asn Arg Phe (SEQ ID NO: 5),
Asp Tyr Ser Leu Leu Lys Asn Arg Phe (SEQ ID NO: 6),
Lys Phe Met Leu Cys Ala Gly Arg Trp (SEQ ID NO: 7),
Lys Tyr Met Leu Cys Ala Gly Arg Trp (SEQ ID NO: 8), and
Val Tyr Leu Leu Gly Arg His Ser Leu Phe (SEQ ID NO: 10)
A peptide comprising any amino acid sequence selected from Polynucleotides encoding claim 1 Symbol placement peptide. An expression vector containing the polynucleotide according to claim 2 . A cell containing the expression vector according to claim 3 . The fourth aspect of the cells, characterized by culturing under conditions which allow expression of the peptide The method according to claim 1 Symbol placement peptide. Antibody that specifically binds to claim 1 Symbol placement peptide. Antigen-presenting cells complex between a cancer antigen peptide and HLA-A24 antigen of claim 1 Symbol placement of the peptide is presented. Recognizing a complex between a cancer antigen peptide and HLA-A24 antigen of claim 1 Symbol placement peptide CTL. Claim 1 Symbol placement peptide, the expression vector of claim 3, wherein, according to claim 4 cells, antigen presenting cells according to claim 7, or the CTL according to claim 8, and a pharmaceutically acceptable carrier A pharmaceutical composition containing. The pharmaceutical composition according to claim 9 , which is used as an inducer of CTL. The pharmaceutical composition according to claim 9 , which is used as a cancer vaccine. HLA tetramers containing the peptide and HLA-A24 antigen of claim 1 Symbol placement. The diagnostic agent of the effect of the cancer vaccine containing the HLA tetramer of Claim 12 .

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