JP2006122046A - Hla class ii-binding new cancer antigen peptide - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a peptide or the like being a help of cancer therapy applicable to wide tumors such as squamous cell carcinoma, applicable to many people in tumor patients even if the applicable tumor is limited and applicable to various kinds of tumors while complementing the therapy or the diagnosis of the tumor by identifying a new HLA class II-binding T-cell epitope derived from a tumor-associated antigen. <P>SOLUTION: The new HLA class II-binding epitope derived from a cancer-associated antigen SART2 is identified and isolated. The new HLA class II-binding epitope derived from the SART2 stimulates the activity and proliferation of CD4-positive T-lymphocyte. The gene product is a hopeful candidate for immunotherapeutic strategy for preventing, treating and diagnosing cancer patients. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to tumor-associated gene product fragments that are bound to and presented to T cells by HLA molecules, specifically peptides thereof, nucleic acid molecules encoding such peptides, and related antibodies and T cells. . They are useful in medicine, especially in the diagnosis and treatment of tumors.

  The process by which the mammalian immune system recognizes and reacts to foreign or foreign substances is complex. An important aspect of the system is the T cell response, which includes some mature T cells that are positive for CD4 or CD8 cell surface proteins. T cells recognize other cells via cell surface complexes on other cells of peptides and molecules called human leukocyte antigen (“HLA”) or major histocompatibility complex (“MHC”), and Can interact. Peptides are derived from large molecules that are processed by cells that also present HLA / MHC molecules. Interactions between T cells and HLA / peptide complexes are limited and require specific T cells for specific complexes of HLA molecules and peptides. In the absence of specific T cells, no T cell response is observed even in the presence of the partner complex. Similarly, if no specific complex is present, but T cells are present, no response is observed.

  T cell responses to foreign antigens include both cytolytic T cells and helper T cells. CD8 + cytotoxic or cytolytic T cells (CTLs) are T cells that lyse cells that, when activated, present the appropriate antigen presented by HLA class I molecules (eg, HLA-A2, HLA-A24, etc.) . CD4-positive helper T cells are macrophages and antigen-presenting B cells that present appropriate antigens by their surface HLA class II molecules (eg, HLA-DRB1 * 04, HLA-DRB1 * 07, HLA-DRB1 * 09, etc.) T cells that secrete cytokines to stimulate

  The mechanism by which T cells recognize foreign substances has also been implicated in cancer. For example, a number of cytolytic T cell (CTL) clones have been described that are directed against autologous melanoma. In some cases, the antigen recognized by these clones has been characterized. The expression products of these genes are processed into peptides which are then presented on the cell surface. This can lead to lysis of tumor cells by specific CTL.

  If a known specificity of a particular peptide for a particular HLA molecule is shown, then a particular peptide that binds one HLA molecule but not others can be predicted. This is important because different individuals have different HLA phenotypes. As a result, the identification of specific peptides as partners to specific HLA molecules has diagnostic and therapeutic problems, but these are only true for individuals with specific HLA phenotypes.

  The importance of CD4 positive T cells (helper T cells) in anti-tumor immunity has been demonstrated in animal models where these cells not only serve cooperative and effector functions, but are also important to retain immune memory (Reconsidered by Non-Patent Document 1).

In recent years, cancer antigen protein (SART2) restricted by HLA-A24 exists in squamous cell carcinoma with high onset frequency, and the presence of cancer antigen peptide recognized by host CTL has been reported (for example, Non-Patent Document 2, Patent Document) 1). Squamous cell carcinoma is one of the most common cancers in humans, and squamous cell carcinoma, especially in esophageal and lung cancers, is known to be relatively resistant to current chemotherapy and radiation therapy . At present, development of specific immunotherapy using tumor antigen peptides that activate CTL that attacks cancer antigens is expected.
JP 11-318455 A Topalian, “Current opinion in immunology”, 1994, Vol. 6, p741-745 Nakao, 12 others, “Journal of immunology”, 2000, 164, p. 2565-2574

  In recent years, specific immunotherapy using a tumor antigen peptide or the like that activates CTLs that attack cancer antigens has attracted attention, but it has been reported that sufficient therapeutic effects cannot be obtained in clinical trials. Therefore, discovery of peptides that can proliferate helper T cells is extremely important.

  The problem of the present invention is that it can be applied to a wide range of tumors such as squamous cell carcinoma, or can be applied to many patients with tumors even if the applicable tumors are limited, or complement the treatment and diagnosis of the tumors. However, it is to provide a peptide that helps cancer therapy that can be applied to various tumors.

  The present invention can be used for the treatment or diagnosis of conditions characterized by the expression of SART2.

  In the present invention, it was shown that the SART2 gene encodes a tumor rejection antigen, which is a peptide that induces helper T cells. This peptide, when presented by antigen presenting cells, effectively induces T cell activation and proliferation.

  The present invention provides isolated SART2 peptides that bind HLA molecules and functional variants of such peptides, wherein the functional variant comprises one or several amino acid additions, substitutions or deletions of the SART2 peptide sequence including. The present invention relates to isolated nucleic acid molecules encoding such peptides, expression vectors containing these nucleic acid molecules, host cells transfected with these nucleic acid molecules, and complexes of these peptides and peptides with HLA molecules Antibodies against are also provided. Also provided are T cells that recognize complexes of peptides and HLA molecules. A pharmaceutical composition, diagnostic agent, prophylactic agent, and therapeutic agent containing the molecule and the cell are additionally provided. Furthermore, all peptides to which T cells that recognize this peptide react are also provided.

Specifically, the present invention has the following configuration.
[1] A peptide consisting of 10 or more consecutive amino acid sequences of the amino acid sequence set forth in SEQ ID NO: 2 and stimulating T cell proliferation and activation.
[2] The peptide of claim 1, which binds to an HLA class II molecule.
[3] A peptide that binds to the following HLA class II molecule (a), (b), or (c):
(A) A peptide comprising the amino acid sequence represented by SEQ ID NO: 4, 6 or 8.
(B) A peptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 4, 6 or 8.
(C) A peptide that is a variant or derivative of the peptide according to (a).
[4] (1) a polynucleotide molecule represented by SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7, (2) a polynucleotide molecule encoding the peptide according to any one of [1] to [3], 3) In (1) or (2), the polynucleotide molecule in which one or more bases are substituted, deleted or added, and (4) the polynucleotide molecule in any one of (1) to (3) above A polynucleotide molecule selected from the group consisting of a polynucleotide molecule that hybridizes under stringent conditions.
[5] A leukocyte that recognizes the peptide according to any one of [1] to [3].
[6] An antibody against the peptide according to any one of [1] to [3].
[7] A pharmaceutical composition comprising the peptide according to any one of [1] to [3] as an active ingredient.
[8] A pharmaceutical composition comprising the polynucleotide molecule according to [4] as an active ingredient.
[9] A pharmaceutical composition comprising the leukocyte according to [5] as an active ingredient.
[10] A pharmaceutical composition comprising the antibody according to [6] as an active ingredient.
[11] A diagnostic agent for tumors comprising the peptide according to any one of [1] to [3] as an active ingredient.
[12] A tumor preventive agent comprising the peptide according to any one of [1] to [3] as an active ingredient.
[13] A tumor therapeutic agent comprising the peptide according to any one of [1] to [3] as an active ingredient.
[14] A tumor diagnostic agent comprising the polynucleotide molecule according to [4] as an active ingredient.
[15] A tumor diagnostic agent comprising the antibody according to [6] as an active ingredient.
[16] An isolated CD4-positive T cell that selectively binds to a complex of an HLA class II molecule and the peptide according to [1] to [3].
[17] The isolated CD4-positive T cell according to [16], which has an ability to damage cancer cells.
[18] An isolated antigen-presenting cell comprising a complex of an HLA class II molecule and the peptide according to [1] to [3].

  The present invention provides isolated SART2 peptides presented by HLA molecules that stimulate T cell proliferation and activation. One aspect of the present invention is an isolated peptide comprising the amino acid sequence of SEQ ID NO: 4, 6 or 8. The use of peptides presented by class I as well as antigenic peptides presented by class II may improve the efficacy of therapeutic anti-tumor vaccination. Such an effect is similarly exerted even in the case of a variant or derivative of the peptide of SEQ ID NO: 4, 6 or 8, wherein one or several amino acids are deleted from the amino acid sequence of SEQ ID NO: 4, 6 or 8. Even a peptide containing a substituted or added amino acid sequence can have the same efficacy. Further, DNA encoding the amino acid sequence of the peptide of the present invention and / or leukocytes and / or antibodies that recognize the peptide of the present invention are useful for tumor diagnosis, prevention, and treatment. Furthermore, an isolated CD4-positive T cell that binds to a complex of an HLA class II molecule and a peptide of the present invention, or an isolated antigen-presenting cell containing a complex of an HLA class II molecule and a peptide of the present invention is also a tumor. Useful for diagnosis, prevention and treatment. Furthermore, the knowledge about antigens presented by HLA class II molecules is useful for the evaluation of immune responses in cancer patients immunized with tumor antigens or recombinant viruses incorporating tumor antigen genes.

  It will be appreciated by those skilled in the art that peptides that have been processed into SART2 into a form that can bind to HLA are particularly those that bind to HLA class II and can be of any length or sequence.

  As demonstrated in the examples below, SART2 protein is properly processed, presented by HLA class II molecules, and effective to stimulate CD4 positive T cells. A peptide derived from SART2, eg, the peptide of SEQ ID NO: 4, 6 or 8, has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids added at one or both ends. Can do. The antigenic portion of such peptides is excised under physiological conditions for presentation by HLA, particularly HLA class II. It is also well known in the art that HLA class II peptide length can vary between about 10 amino acids and about 30 amino acids (Engelhard, Annual review of immunology, 1994). About 12 amino acids containing the amino acid sequence of SEQ ID NO: 4, 6 or 8 (or a part of the amino acid sequence of SEQ ID NO: 4, 6 or 8) as a peptide of the present invention. Peptides consisting of ˜about 30 amino acid residues are preferred. Renormalized HLA class II binding peptides have been identified, in which the peptides share a core sequence but have different amino acids at the amino and / or carboxyl termini. SART2 peptides having fewer amino acids than the peptides disclosed herein are also included in the present invention. Accordingly, additional SART2 HLA class II binding peptides as well as SART2 HLA class II binding peptides that are homologous to SART2 HLA class II binding peptides can be identified by those of skill in the art according to the procedures described herein.

  The techniques described in the examples can be utilized to identify HART class II binding peptides of SART2. Thus, recombinant SART2 can be introduced into, for example, antigen presenting cells, eg, dendritic cells of normal blood donors, by contacting the cell with a SART2 polypeptide or by introducing into the cell a nucleic acid molecule that directs expression of the SART2 protein. The protein (or fragment thereof) can be charged. The antigen-presenting cells can then be used to induce in vitro the activation and proliferation of specific CD4-positive T cells that recognize SART2 HLA class II binding peptides. The sequence of the peptide can then be determined, as described in the examples, by stimulating the cell with a peptide fragment of the SART2 protein used, for example, to stimulate activation and proliferation of CD4 positive T cells. Alternatively, a peptide derived from the SART2 protein can be loaded into antigen-presenting cells. Computer software for selecting HLA class II binding peptides is also available. HLA class II molecules have a pocket structure to which peptides can bind, and the chemical properties or structure sizes differ depending on the subtype of HLA class II. By profiling these pocket structures, it is possible to infer peptides that can interact with the structure. For example, TEPITOPE algorithm; (Sturniolo, 10 others, "Nature biotechnology, 1999, Vol. 17, p. 555-561, Manici, 10 others," The Journal “The Journal of experimental medicine”, 1999, Vol. 189, p. 871-876 ”, SYFPEITHI algorithm (Ramensee, 4 others,“ Immunogenetics ” ) ", 1999, Vol. 50, p.213-219), ProPred algorithm (Singh, et al.," Bioinformatics ", 2001, Vol. 17, p.1236-1237) Or RANKPEP algorithm (Raiche ( eche), 2 others, “Human immunology”, 2002, 63, p.701-709), peptides selected in this way are specific CD4 positive T Additional methods for selecting and testing peptides for HLA class II binding can be used in the assays described herein to induce cells, as well as for peptide identification. Is well known.

  As described above, the SART2 HLA class II-binding peptide referred to in the present invention includes a functional variant of the SART2 HLA class II-binding peptide. As used herein, a “variant” or “derivative” of an HLA class II binding peptide contains one or more modifications to the primary amino acid sequence of the HLA class II binding peptide, A peptide that retains the HLA class II and T cell receptor binding properties disclosed herein. Modifications that produce functional variants of SART2 HLA class II binding peptides include, for example: 1) protein-proteins such as SART2 HLA class II binding peptide properties, such as peptide stability in expression systems or HLA-peptide binding To enhance binding stability, 2) To provide a novel activity or property of SART2 to HLA class II binding peptides, eg, addition of an antigenic epitope such as a CTL epitope that induces CTL or addition of a detectable moiety Or 3) to provide different amino acid sequences that produce the same or similar T cell stimulatory properties. Modifications to HART class II binding peptides of SART2 can be made to nucleic acids encoding the peptides, examples of which include deletion, point mutation, truncation, amino acid substitution and amino acid addition. . Alternatively, modifications can be made directly to the polypeptide, eg, by cleavage, addition of a linker molecule, addition of a detectable moiety (eg, biotin), addition of a fatty acid, substitution of one amino acid with another amino acid, and the like. Variants can also be selected from libraries of peptides, which can be random peptides or peptides based on the sequence of the SART1 peptide containing substitutions at one or more positions. For example, peptide libraries can be used in competition assays with complexes of SART2 helper epitope peptides bound to HLA class II molecules (eg, dendritic cells loaded with HART class II binding peptides of SART2). . Peptides that compete for binding of SART2 peptides to HLA class II molecules are sequenced and used in other assays (eg, proliferation of CD4 positive T cells) to confirm suitability as a SART2 peptide functional variant. obtain. Other functional variants include peptidomimetic compounds. Peptidomimetics can be prepared for HLA class II binding and tested for stimulation of CD4 positive T cells.

  The amino acid sequence of the HLA class II binding peptide of SART2 can be of natural or non-natural origin. That is, they can contain native SART2 HLA class II binding peptide molecules, or the amino acid sequence retains the ability to stimulate helper T cells when presented and has the property of binding to HLA class II molecules. As long as it is retained, it may contain a modified sequence. For example, SART2 HLA class II binding peptides, in this context, express SART2 HLA class II binding peptides and unrelated amino acid sequences, labeled peptides, peptides isolated from patients with SART2 expressing cancer, SART2. It can be a fusion protein comprising a peptide isolated from cultured cells, a peptide conjugated to a non-peptide molecule, and other molecules comprising SEQ ID NO: 4, 6 or 8.

  Next, the binding of the mutant peptide to the HLA class II binding molecule and the stimulation of the T cells is determined by standard techniques. For example, as exemplified below, a mutant peptide can be contacted with an antigen presenting cell containing an HLA class II molecule that binds a SART2 peptide to form a complex of the mutant peptide and the antigen presenting cell. . This complex can then be contacted with a T cell that recognizes the HLA class II binding peptide of SART2 presented by the HLA class II binding molecule. T cells are obtained from patients with symptoms characterized by SART2 expression. Recognition of mutant peptides by T cells can be determined by measuring indicators of T cell stimulation, such as TNF or IFNγ production. Similar approaches can be performed for the identification and characterization of other SART2 HLA class II binding peptides. Binding of the mutant peptide to an HLA class II binding molecule and stimulation of T cells by the mutant peptide presented by the HLA class II binding molecule indicates that the mutant peptide is a functional mutant. The method may also include comparing the stimulation of T cells by the HLA class II binding peptide of SART2 and the stimulation of T cells by the functional variant as an assessment of the effectiveness of stimulation of T cells by the functional variant. . By comparing functional variants with SART2 HLA class II binding peptides, peptides can be prepared that exhibit increased T cell stimulatory properties.

  SART2 HLA class II binding peptide variants prepared by any of the methods described above are sequenced to determine the amino acid sequence, if necessary, and thus a nucleotide sequence encoding such a variant Can be estimated.

  Further part of the invention is a nucleic acid sequence encoding a SART2 HLA class II binding peptide or variant thereof, as well as other nucleic acid sequences that hybridize to nucleic acid molecules consisting of the above nucleotide sequences. Nucleic acid hybridization parameters can be found in references editing such methods, eg, Sambrook, “Molecular Cloning: A Laboratory Manual”, 2nd edition, Cold Spring Harbor. It can be found in the Cold Spring Harbor Laboratory Press, 1989, Vols. 1-3.

  The polynucleotide molecule of the present invention also includes “a polynucleotide molecule that hybridizes to the polynucleotide molecule of the present invention under stringent conditions”. Taking a DNA molecule as a representative example of a polynucleotide molecule, a “DNA molecule that hybridizes to a DNA molecule under stringent conditions” can be obtained, for example, by the method described in Molecular Cloning. Here, “hybridizes under stringent conditions” means, for example, 0.1 × SSC after heating at 42 ° C. in a solution of 6 × SSC, 0.5% SDS and 50% formamide. This shows that a positive hybridization signal is still observed even under conditions of washing at 68 ° C. in a solution of 0.5% SDS. There are conditions, reagents, etc. that can be used, but those skilled in the art are familiar with such conditions and are therefore not limited to these conditions.

  However, it should be understood that one skilled in the art can manipulate the conditions in a manner that allows unambiguous identification of homologues and alleles of nucleic acids encoding the HLA class II binding peptides of SART2 of the present invention. Those skilled in the art are also familiar with methods for screening cells and libraries for expression of such molecules that are routinely isolated and subsequent isolation and sequencing of related nucleic acid molecules.

  In general, homologues and alleles are typically each at least 90% amino acid identity and / or at least 75% nucleotide identity with the amino acid sequence of an HLA class II binding peptide of SART2 or a nucleic acid encoding such a peptide. Share gender. In some cases, homologues and alleles share at least 95% nucleotide identity and / or at least 90% amino acid identity, and in still other cases at least 90% nucleotide identity. And / or share at least 95% amino acid identity. Complements of the above nucleic acids are also encompassed by the present invention.

For screening for nucleic acids encoding HART class II binding peptides of SART2, nucleic acid hybridization, eg, Southern or Northern blots, can be performed using the above conditions in conjunction with a ³² P probe. After washing the membrane to which the DNA encoding the HLA class II binding peptide of SART2 has been transferred last, the membrane is placed against an X-ray film in order to detect the radioactive signal.

  The invention also encompasses the use of nucleic acid sequences comprising alternative codons that encode the same amino acid residue of the HART class II binding peptide of SART2. For example, as disclosed herein, the peptide QLNLKNVQRNLLILLHPQLLLLVDQIHL (SEQ ID NO: 4) is an HLA class II binding peptide of SART2. The leucine residue (second amino acid of SEQ ID NO: 4) can be encoded by the codons UUA, UUG, CUU, CUC, CUA and CUG. Each of the six codons is equivalent for the purpose of encoding a leucine residue. Thus, it will be apparent to one skilled in the art that any of the leucine-encoding nucleotide triplets can be used to direct a protein synthesizer in vitro or in vivo to incorporate a leucine residue. Similarly, nucleotide sequence triplets encoding other amino acid residues including the HART class II binding peptide of SART2 of SEQ ID NO: 4 include the following: CGA, CGC, CGG, CGT, AGA and AGG (arginine AAA and AAG (lysine codon); GUA, GUC, GUG and GUU (valine codon); GAA and GAG (glutamine codon); UUC and UUU (phenylalanine codon) and UAC and UAU (tyrosine codon). Other amino acid residues can be similarly encoded by the polynucleotide sequence. Accordingly, the present invention encompasses degenerate nucleic acids that differ from the native SART2 HLA class II binding peptide encoding nucleic acids in the codon sequence due to the degeneracy of the genetic code.

  The present invention also provides modified nucleic acid molecules comprising one or more nucleotide additions, substitutions and deletions. In preferred embodiments, these modified nucleic acid molecules and / or the polypeptides they encode are at least one activity or function of an unmodified nucleic acid molecule and / or polypeptide, such as antigenicity, enzymatic activity, receptor binding, MHC. It retains complex formation by binding of peptides by class I and class II molecules. In certain embodiments, the modified nucleic acid molecule encodes a modified polypeptide, preferably a polypeptide having conservative amino acid substitutions as described elsewhere herein. The modified nucleic acid molecule is structurally related to the unmodified nucleic acid molecule, and in preferred embodiments, the modified and unmodified nucleic acid molecule is sufficiently structurally unhybridized to hybridize under stringent conditions known to those skilled in the art. Associated with a modified nucleic acid molecule.

  For example, modified nucleic acid molecules that encode polypeptides having single amino acid changes can be prepared. Each of these nucleic acid molecules may have 1, 2, or 3 nucleotide substitutions that are incompatible with nucleotide changes corresponding to the degeneracy of the genetic code as described herein. Similarly, modified nucleic acid molecules can be prepared that encode polypeptides having two amino acid changes, which have, for example, 2 to 6 nucleotide changes. Numerous modified nucleic acid molecules similar to these are readily contemplated by those skilled in the art including, for example, substitution of nucleotides in codons encoding amino acids 2 and 3, 2 and 4, 2 and 5, 2, and 6, and the like. In the above example, each combination of two amino acids is included in the set of modified nucleic acid molecules as well as all nucleotide substitutions that encode amino acid substitutions. Encodes a polypeptide having additional substitutions (ie, 3 or more), additions or deletions (eg, by introduction of a stop codon or splice site (s)), as readily contemplated by those skilled in the art Additional nucleic acid molecules can also be prepared and are encompassed by the present invention. Any of the above nucleic acids or polypeptides can be tested by routine experimentation for retention of structural association or activity against the nucleic acids and / or polypeptides disclosed herein.

  The polynucleotide molecules of the present invention can take the form of DNA or RNA, which includes cDNA, genomic DNA, and synthetic DNA. DNA and RNA may be single-stranded or double-stranded, and in the case of single-stranded, both sense strand and antisense strand can be included.

  An ideal form of a SART2 polypeptide, such as SART2 or an HLA class II-binding peptide derived therefrom, can bind to at least one, preferably two, more preferably two or more HLA types, It has the property of allowing CD4 positive T cells to proliferate. For example, as shown in the Examples, the peptide of the present invention can bind to two HLA types and has the property of allowing proliferation of CD4-positive T cells.

  The present invention relates to prokaryotic (eg, E. coli) or eukaryotic (eg, dendritic cells, CHO cells, COS cells, yeast expression systems, intracellular parasite expression systems, lactic acid bacteria expression systems and insect cells in expression vectors. It should also be understood to encompass the use of this sequence to transfect host cells and cell lines that are recombinant baculovirus expression in). Expression vectors require that the relevant sequences, ie those described above, be operably linked to a promoter. Since human HLA molecules have been found to present SART2 HLA class II binding peptides, the expression vectors can also include nucleic acid sequences encoding HLA molecules (for other SART2 HLA class II binding peptides). Different HLA molecules can be used). In situations where the vector contains both coding sequences, it can be used to transfect cells that normally do not express either one.

  As used herein, a “vector” is any of a number of nucleic acids into which a desired sequence can be inserted by restriction and ligation for transport between different genetic environments or for expression in a host cell. It can be. Vectors are typically composed of DNA, but RNA vectors can also be used. Vectors include but are not limited to plasmids, phagemids and viral genomes. Cloning vectors can be replicated autonomously in the host cell or can be integrated into the genome, as well as the vector can be determined so that the new recombinant vector retains its ability to replicate in the host cell A cloning vector that is cut in a manner and is further characterized by one or more endonuclease restriction sites into which the desired DNA sequence can be ligated. In the case of a plasmid, replication of the desired sequence can occur many times if the plasmid increases copy number in the host bacterium, or only once per host before the host propagates by mitosis. In the case of phage, replication can occur actively during the lysis phase and passively during the lysogen phase. An expression vector is a vector into which a desired DNA sequence can be inserted by restriction and ligation so that it can be operably linked to regulatory sequences and expressed as an RNA transcript.

  The vector may further contain one or more marker sequences suitable for use in identifying cells that have been or have not been transformed or transfected with the vector. Markers include, for example, genes that encode proteins that increase or decrease resistance or sensitivity to antibiotics or other compounds, enzymes whose activity is detectable by standard assays known in the art (eg, β-galactosidase, Genes encoding alkaline phosphatase or luciferase), as well as genes that visually affect the phenotype of transformed, transfected cells, hosts, colonies or plaques (eg, green fluorescent protein). Preferred vectors are those that are capable of autonomous replication and expression of structural gene products present in the DNA segments to which they are operably linked.

  Preferably, the expression vector contains a sequence that targets a SART2 polypeptide, such as SART2 or an HLA class II binding peptide derived therefrom, to the endosome of a cell in which the protein or peptide is expressed. HLA class II molecules contain an invariant chain (Ii) that interferes with the binding of other molecules to the HLA class II molecule. This invariant chain is cleaved in the endosome, thereby allowing binding of the peptide by the HLA class II molecule. Accordingly, it is preferred that SART2 HLA class II binding peptides and precursors thereof (eg, SART2 proteins) are targeted to endosomes, thereby enhancing SART2 HLA class II binding peptides that bind to HLA class II molecules. Targeting signals for directing the molecule to the endosome are known in the art, and these signals can conveniently be incorporated into an expression vector so that a fusion protein containing the endosomal targeting signal is produced. San derson ("Proceedings of the National Academy of Sciences of the United States of America") 1995, 92, p. 7217-7221), Wu ("Proceedings of the National Academy of Science of the United States of America (Proceedings of the The National Academy of Sciences of the United States of America ", 1996, Vol. 92, pp. 11671-11675) and Thomson (" Journal of virology, 1998, 72 "). Vol., P. 2246-2252) is the endosome targeting signal (invariant Describes their use in the case where Ii and lysosomal-associated membrane protein LAMP-1) and direct the antigens into endosomes and / or lysosomes cell compartments.

  As used herein, coding sequences and regulatory sequences are “operably” linked when they are covalently linked in such a way as to place the expression or transcription of the coding sequence under the influence or control of the regulatory sequences. It is said to be done. When it is desired that the coding sequence be translated into a functional protein, induction of the promoter in the 5 'regulatory sequence results in transcription of the coding sequence, and the nature of the bond between the two DNA sequences is (1) the frameshift mutation Two DNA sequences, if not introduced, (2) do not interfere with the ability of the promoter region to direct the transcription of the coding sequence, or (3) do not interfere with the ability of the corresponding RNA transcript to be translated into protein It is said to be operably linked. Thus, a promoter region is operably linked to a coding sequence if the promoter region is capable of transcription of the DNA sequence so that the resulting transcript can be translated into the desired protein or polypeptide. Is done.

  The exact nature of the regulatory sequences required for gene expression can vary between species or cell types, but in general, if necessary, transcription and respectively, such as TATA box, capping sequence, CAAT sequence, etc. Contains 5 'untranscribed and 5' untranslated sequences involved in translation initiation. In particular, such 5 'non-transcribed regulatory sequences include a promoter region that includes a promoter sequence for transcriptional control of operably linked genes. Regulatory sequences can also include enhancer sequences or upstream activator sequences, if desired. The vectors of the present invention may optionally include a 5 'leader or signal sequence. Selection and design of appropriate vectors is within the ability and discretion of those skilled in the art.

  Expression vectors containing all the necessary elements for expression are commercially available and known to those skilled in the art (eg, Sambrook, “Molecular Cloning: A Laboratory Manual”). 2nd edition, Cold Spring Harbor Laboratory Press, 1989, Vols. 1-3). Cells are genetically engineered by introduction of heterologous DNA (or RNA) encoding SART2 HLA class II binding polypeptides into the cell. The heterologous DNA (or RNA) is placed under operable control of the transcriptional element to allow expression of the heterologous DNA in the host cell. As described herein, such expression constructs also optionally contain an endosomal targeting signal, preferably a nucleotide sequence encoding a human invariant chain or a targeting fragment thereof.

  Preferred systems for mRNA expression in mammalian cells are selectable, such as genes that confer G418 resistance (to facilitate selection of stable transfected cell lines), and human cytomegalovirus (CMV) enhancer-promoter sequences. A system such as pRc / CMV (available from Invitrogen, Carlsbad, CA) containing the marker. Also suitable for expression in primate or canine cell lines is the pCEP4 vector (Invitrogen), which contains an Epstein-Barr virus (EBV) origin of replication and facilitates the retention of the plasmid as a multicopy extrachromosomal element. is there. Another expression vector is the pEF-BOS plasmid containing the promoter of polypeptide elongation factor 1α that efficiently stimulates transcription in vitro. This plasmid has been described by Mizizuma et al. ("Nucleic acids research", 1990, Vol. 18, p. 5322) and its use in transfection experiments is e.g. (Demoulin et al., “Molecular and cellular biology, 1996, vol. 16, pp. 4710-4716.” Yet another preferred expression vector is Stanford- An adenovirus described by Stratford-Perricaudet et al., Which lacks E1 and E3 proteins ("The Journal of clinical investigation", 1992). 90, p. 626-630) Ad. The use of adenovirus as an eno.P1A recombinant has been disclosed by Warnier et al. ("International Journal of Cancer (International Journal of Cancer)" in intradermal injection in mice for immunization against P1A. journal of cancer) ", 1996, Vol. 67, pp. 303-310).

  When the term “disorder” or “symptom” is used herein, it refers to any pathological condition in which an HLA class II binding peptide of SART2 is expressed. Such disorders include cancers such as brain tumors including glioma, squamous cell carcinoma of the head, neck, lung, uterus or esophagus, melanoma, adenocarcinoma of the lung or uterus, and renal cancer.

  Several therapeutic approaches based on the disclosure are premised on inducing a response by the subject's immune system to SART2 HLA class II binding peptide presenting cells. One such approach is to target leukocytes, preferably the peptides of the present invention, that recognize complexes of SLA2 HLA class II binding peptides and HLA class II molecules to subjects with abnormal cells with tissue phenotypes. Specific autologous CD4 positive T cell administration. Developing such CD4 positive T cells in vitro is within the skill of the artisan. In general, a sample of cells taken from a subject, such as blood cells, proliferates in contact with cells that present the complex and are capable of inducing CD4 positive T cells. Target cells can be antigen presenting cells carrying HLA class II molecules, such as dendritic cells or B cells. Target cells can also be transfectants such as COS cells when a sample of cells is first sorted to isolate a CD4 positive T cell population. The above tetramer technology can be used for classification. These transfectants present the desired complex on their surface and stimulate their proliferation when combined with the CD4 positive T cells in question. COS cells, as well as other suitable host cells, are widely available. Specific production of CD4 positive T cells is described below. Clonal expandable autologous CD4 positive T cells are then administered to the subject. The CD4 positive T cells then stimulate the subject's immune response, thereby achieving the desired therapeutic goal.

  The above therapy assumes that at least some of the subject's abnormal cells present the relevant HLA / peptide complex. This is very easy because the art is very familiar with how to identify cells that present a particular HLA molecule, as well as cells that express DNA of the relevant sequence, in this case the SART2 sequence. Can be confirmed. The above therapy is not the only form of therapy available according to the present invention. CD4 positive T cells can also be raised in vivo using a number of approaches. One approach is the use of non-proliferating cells that express the complex. The cells used in this approach can be cells that normally express the complex, such as dendritic cells, or cells that have been transfected with one or both of the genes required for display of the complex. Similarly, vectors carrying one or both of the genes can be used. Viral or bacterial vectors are particularly preferred. For example, a nucleic acid encoding a SART2 HLA class II binding peptide can be operably linked to promoter and enhancer sequences that direct the expression of the SART2 HLA class II binding peptide in certain tissues or cell types. The nucleic acid can be incorporated into an expression vector. The expression vector may be an unmodified extracellular chromosomal nucleic acid, plasmid or viral genome constructed to allow insertion of exogenous nucleic acids such as those encoding HART class II binding peptides of SART2 . Nucleic acids encoding SART2 HLA class II binding peptides may also be inserted into the retroviral genome, thereby facilitating integration of the nucleic acid into the genome of the target tissue or cell type. In these systems, the gene is carried by microorganisms such as vaccinia viruses, generally poxviruses, adenoviruses, herpes simplex viruses, retroviruses or bacterial BCG, and in fact substances that "infect" host cells. The resulting cells present the complex and are recognized by autologous CD4 + T cells, which then grow.

  Similar effects can be achieved by combining SART2 HLA class II binding peptides with adjuvants to facilitate incorporation into HLA class II presenting cells in vivo. If larger than the HLA class II binding moiety, the HLA class II binding peptide of SART2 can be processed as necessary to produce the peptide partner of the HLA molecule. In general, a subject can receive an intradermal injection of an effective amount of a SART2 HLA class II binding peptide. Initial doses and subsequent booster doses can be administered according to immunization protocol standards in the art.

  Any of the above compositions or protocols may also include SART2 HLA class I binding peptides for induction of cytolytic T cell responses. For example, as demonstrated below, SART2 protein can be processed in cells to produce both HLA class I and HLA class II responses. By administering a SART2 peptide (or nucleic acid encoding such a peptide) that binds HLA class I and class II molecules, an improved immune response can be provided by inducing both T helper cells and T killer cells. .

  In addition, non-SART2 tumor associated peptides can also be administered to increase immune responses by HLA class I and / or class II. It is well established that cancer cells can express more than one tumor associated gene. One skilled in the art will determine whether a particular subject expresses an additional tumor associated gene, and then HLA class I derived from the expression product of such gene in the SART2 compositions and therapeutics and prophylactics described above. Inclusion of and / or HLA class II binding peptides is within the scope of routine experimentation.

  The present invention has a number of uses, as described herein, some of which are described herein. The following uses are described with respect to SART2 HLA class II binding peptides, but are equally applicable to other structurally or functionally related SART2 HLA class II binding peptides. First, the present invention enables those skilled in the art to diagnose diseases characterized by expression of HART class II binding peptides of SART2. These methods include determining the expression of a SART2 HLA class II binding peptide or a complex of a SART2 HLA class II binding peptide and an HLA class II molecule in a biological sample. Expression of a peptide or a complex of a peptide and an HLA class II molecule can be determined by assaying with a binding partner for the peptide or complex, eg, an antibody.

  The antibody against the peptide of the present invention or a variant / derivative thereof is described, for example, by Lane et al., “Antibodies; A Laboratory Manual, First Edition, Cold Spring Harbor. An antibody recognizing a tumor antigen protein by immunizing an appropriate animal by an appropriate method using the peptide of the present invention by a method described in “Cold Spring Harber Laboratory Press, 1989” or the like, or Antibodies that neutralize the activity can be easily prepared. Examples of the use of the antibody include affinity chromatography, cDNA library screening, immunological diagnosis, medicine and the like.

  A pharmaceutical composition comprising as an active ingredient a peptide of the present invention and / or a polynucleotide molecule of the present invention and / or a leukocyte that reacts with the peptide of the present invention and / or an antibody against the peptide of the present invention is a cancer that expresses SART2. By responding to cells and suppressing their growth, the effect of treating symptoms characterized by the expression of SART2 would be expected. These pharmaceutical compositions can be used as preventive agents for symptoms such as tumors by being administered before the symptoms characterized by the expression of SART2 occur. In addition, administration of these pharmaceutical compositions after the onset of symptoms characterized by the expression of SART2 can be a therapeutic agent for such symptoms, such as tumors. Furthermore, SART2 expression is characterized by using a combination of two or more appropriately selected peptides of the present invention, polynucleotide molecules of the present invention, leukocytes that react with the peptides of the present invention, and antibodies against the peptides of the present invention. The effect of enhancing the effect of treating the symptoms can be expected.

  Therapeutic and prophylactic agents also include naked DNA or RNA encoding SART2 HLA class II binding peptides or precursors thereof, which are produced in vitro and by injection, particle bombardment, nasal inhalation and other methods. Can be administered. “Naked nucleic acid” type therapeutic and prophylactic agents have been demonstrated to elicit immunological responses including the generation of CTLs specific for peptides encoded by the naked nucleic acid. Therapeutic and prophylactic agents also include nucleic acids packed in viruses, liposomes or other particles, such as polymer particles useful for drug delivery.

  As part of the immunization protocol, substances that enhance the immune response can be administered with nucleic acid or peptide components of cancer therapeutics and prophylactics. Such immune response enhancing compounds can be classified as adjuvants or cytokines. Adjuvants can enhance the immunological response by providing a reservoir of antigen (extracellular or in macrophages), activating macrophages and stimulating specific sets of lymphocytes. Many types of adjuvants are well known in the art. Specific examples include MPL (SmithKline Beecham), a salmonella Salnesella minnesota Re 595 lipopolysaccharide and the like obtained after acid hydrolysis; QS21 (SmithKline Beecham), pure QA purified from Quillja saponaria extract -21 saponin; DQS21 described in PCT application WO 96/33739 (SmithKline Beecham); QS-7, QS-17, QS-18 and QS-L1 (So, outside, 10 people, “Molecules and Cell (Molecules and cells), 1997, volume 7, p. 178-186); Freund's incomplete adjuvant; Freund's complete adjuvant; vitamin E; Montanide; alum; CpG oligonucleotides (eg, Kreig, 7 others, see “Nature”, Vol. 374, pages 546-549)); There are various water-in-oil emulsions prepared from biodegradable oils such as squalene and / or tocopherol. Preferably, the peptide is administered mixed with the DQS21 / MPL combination. The ratio of DQS21 to MPL is typically about 1:10 to 10: 1, preferably about 1: 5 to 5: 1, more preferably about 1: 1. Typically, for human administration, DQS21 and MPL are present in the vaccine formulation in the range of about 1 μg to about 100 μg. Other adjuvants are known in the art and can be used in the present invention (eg, by Goding, “Monoclonal Antibodies: Principles and Practice”, 2nd edition). 1986). Methods for preparing peptide and adjuvant mixtures or emulsions are well known to those skilled in vaccination.

  Other factors that stimulate the subject's immune response may also be administered to the subject. For example, other cytokines are also useful in vaccination protocols as a result of lymphocyte stimulatory properties. Numerous cytokines useful for such purposes are known to those of skill in the art, examples include interleukin-12 (IL-12), GM-, which have been shown to enhance the protective effects of vaccines. CSF, IL-18 and Flt3-Ligand.

  When administered, the therapeutic composition of the present invention is administered in a pharmaceutically acceptable preparation. Such preparations routinely contain pharmaceutically acceptable concentrations of salts, buffers, preservatives, compatible carriers, co-immune enhancing agents such as adjuvants and cytokines, and optionally other therapeutic agents. obtain.

  The present invention further provides a medicament containing the peptide relating to the tumor antigen of the present invention or a variant / derivative thereof. The tumor antigen protein and tumor antigen peptide of the present invention can be administered together with an adjuvant so as to effectively establish cellular immunity, or can be administered in a particulate dosage form. As the dosage form, a liposome preparation, a particulate preparation bound to beads having a diameter of several μm, a preparation bound to lipid (lipid), and the like are used. Also conceivable is a method of administering an antigen-presenting cell such as a dendritic cell or microphage pulsed with a tumor antigen peptide, or a cell into which DNA or RNA encoding the tumor antigen peptide of the present invention has been introduced. As a desirable form of the DNA or RNA to be introduced in this case, various helper epitopes including the peptide of the present invention or CTL epitopes such as tumor antigen peptide or tumor antigen-encoding DNA or RNA, or one or more in cells. A method of introducing the DNA or RNA by linking one or more of these DNAs or RNAs may be considered. It is also conceivable to introduce a peptide, polypeptide or protein into the cell instead of the DNA or RNA described above.

  The present invention further relates to a medicament containing the polynucleotide molecule or oligonucleotide molecule of the present invention or a chemical modification thereof. The “medicament” containing the polynucleotide molecule or oligonucleotide molecule of the present invention can treat or prevent a tumor by administering the DNA of the present invention to a tumor patient or the like, for example. As a method for administering the DNA of the present invention and introducing it into a cell, a viral vector method and other methods (Nikkei Science, April 1994, p. 20-45, Monthly Pharmaceutical Affairs, 1994, Vol. 36, Vol. 1, p.23-48, Experimental Medicine Extra Number, 1994, Vol. 12, No. 15, and references cited therein, etc.) can be applied.

  As a method using a viral vector, for example, the DNA of the present invention is incorporated into a retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, poxvirus, poliovirus, Sindbis virus or the like by incorporating the DNA of the present invention. 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.

  Plasmids having these polynucleotide molecules or oligonucleotide molecules of the present invention are also included in the scope of the present invention.

  In order for the gene encoding the tumor antigen peptide of the present invention to actually act as a medicine, an in vivo method in which the gene is directly introduced into the body, and certain cells from human beings are collected and introduced into the cells outside the body. There is an ex vivo method for returning the cells to the body (Nikkei Science, April 1994, p20-45, Monthly Pharmaceutical Affairs, 1994, Vol. 36, No. 1, p.23-48, Experimental Medicine Extra Number, 1994) , Vol. 12, No. 15, and references cited therein). In vivo methods are more preferred.

  When administered by an in vivo method, it can be administered by an appropriate route according to the disease, symptom or the like to be treated. For example, it can be administered intravenously, artery, subcutaneously, intramuscularly. When administered by an in vivo method, for example, it can be in the form of a pharmaceutical preparation such as a liquid, but is generally an injection containing the DNA of the present invention which is an active ingredient, and if necessary, a conventional carrier May be added. In addition, the liposome or membrane fusion liposome (Sendai virus (HVJ) -liposome etc.) containing the DNA of the present invention can be in the form of a liposome preparation such as a suspension, a freezing agent, and a centrifugal concentrated freezing agent. .

  The DNA content of the present invention in the preparation can be appropriately adjusted according to the disease to be treated, the age, weight, etc. of the patient, but is usually 0.0001 mg to 100 mg, preferably 0.001 mg to 10 mg as the DNA of the present invention. This is preferably administered once every few days to several months.

  The expression of a gene encoding a tumor antigen protein can be controlled using the antisense oligonucleotide of the present invention or a chemical modification thereof. By reducing the amount of tumor antigen protein produced by this method, autoimmune diseases can be treated or prevented. A medicament containing such an antisense oligonucleotide is also encompassed by the present invention.

  In the case of incorporating an antisense oligonucleotide into an expression plasmid, examples of a method for introducing this antisense oligonucleotide into a cell include the methods described in Experimental Medicine, 1994, Vol. 12, including liposomes and recombinant viruses. The method using such as. The expression plasmid of the antisense oligonucleotide is simply connected to the gene of the present invention in the reverse direction behind the promoter using a normal expression vector, that is, the gene of the present invention is transcribed in the 3 ′ to 5 ′ direction. Can be easily manufactured.

  A plasmid having such an antisense oligonucleotide is also encompassed by the present invention. When administering antisense oligonucleotides or their chemically modified products as they are, mix them with stabilizers, buffers, solvents, etc., and then use them at the same time as antibiotics, anti-inflammatory agents, anesthetics, etc. You can also. The preparation thus produced can be administered in various ways. The administration is preferably carried out every day or every few days to several weeks. In order to avoid such frequent administration, a sustained-release mini-pellet preparation can be prepared and implanted near the affected area. Or it is also possible to administer to a patient gradually gradually using an osmotic pump etc. The usual dose is adjusted so that the concentration at the site of action is 0.1 nM-10 μM.

  A medicament containing such an antisense oligonucleotide or a chemical modification thereof is also encompassed in the present invention.

  An antibody against the peptide of the present invention or a variant / derivative thereof can also be used for the treatment of tumors. For example, by administering an effective amount of an antibody against the peptide of the present invention or a variant / derivative thereof to a patient who has developed symptoms characterized by the expression of SART2, a desired response for cancer treatment can be induced. The dose of the antibody against the peptide of the present invention or a variant / derivative thereof 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 1000 mg, preferably 0.001 mg to 1000 mg. Yes, this is preferably administered once every few days to several months.

  A peptide of the present invention or a variant / derivative thereof, an antibody against the peptide of the present invention or a variant / derivative thereof, or a T cell induced by the peptide of the present invention or a variant / derivative thereof, such as an isolated CD4-positive T cell The therapeutic effect on cancer in isolated antigen-presenting cells such as dendritic cells can also be verified using animal models. For example, before or after transplanting tumor cells into immunodeficient mice (nude mice, skid mice, etc.) or human HLA transgenic mice, by administering peptides, antibodies or cells in an effective amount and with an optimal administration method, The therapeutic effect on cancer can be determined, but is not limited thereto. In this case, the dose of the antibody against the peptide of the present invention or a variant / derivative thereof or the peptide of the present invention or the variant / derivative thereof is usually 0.0001 mg to 1000 mg, preferably 0.001 mg to 1000 mg. It is preferably administered once every few days or months, and the dose of cells is usually 1 to 1,000,000, preferably 1 million to 1,000,000, and this is administered once every few days to several months. Is preferred.

  Where it is desirable to stimulate an immune response using the therapeutic composition of the present invention, this may be an increase in antibody titers in serum, clonal expansion of cytotoxic lymphocytes or some other desirable immunology. Stimulation of a humoral antibody response that triggers a sexual response. Depending on the mode of administration, doses of immunogen in the range of 1 nanogram / kilogram to 100 milligram / kilogram are considered effective. A preferred range appears to be 500 nanograms to 500 micrograms / kilogram. The absolute amount depends on various factors such as the substance selected for administration, whether the administration is a single dose or multiple doses, and individual patient parameters such as age, health status, size, weight and disease It will depend on the stage. These factors are well known to those skilled in the art and can be handled in routine experiments.

  Moreover, the diagnostic agent of the tumor which contains the peptide of this invention as an active ingredient can also be provided. That is, it is possible to immunologically diagnose symptoms characterized by the expression of SART2. The immunological diagnostic method can be appropriately selected from an immunoblot method, a radioimmunoassay method (RIA), an enzyme immunoassay method (ELISA), a fluorescence or luminescence assay method, and the like. For example, the antibody against SART2 in patient serum or plasma can be obtained by immobilizing the tumor antigen peptide of the present invention or a variant / derivative thereof and reacting with serum or plasma derived from a patient having symptoms characterized by the expression of SART2. The value can be measured.

  These isolated SART2 HLA class II binding peptides, proteins containing such peptides, or complexes of peptides and HLA class II molecules can be combined with an adjuvant-like substance to induce expression of the SART2 HLA class II binding peptides. Therapeutic agents and prophylactic agents useful in treating the characteristic disorder can be generated. In addition, the therapeutic and prophylactic agents can be prepared from cells that present SART2 HLA class II binding peptide / HLA complexes on their surface, such as dendritic cells, B cells, non-proliferative transfectants, and the like. In all cases where cells are used as therapeutic and prophylactic agents, they require cells transfected with a coding sequence for one or both of the components necessary to stimulate CD4 positive T cells, or require transfection It can be a cell that already expresses both molecules. For example, autoantigen-presenting cells can be isolated from a patient and processed to obtain cells that present a SART2 epitope in the association of HLA class I and HLA class II molecules. These cells can stimulate both CD4 positive and CD8 positive cell responses. Dendritic cells can also be loaded with HLA class I and HLA class II epitopes.

  Inoculating the peptide of the present invention or a variant / derivative thereof as a vaccine can provide a method for preventing tumors and a composition for prevention. For example, humoral immunity in which a tumor antigen peptide or a variant / derivative thereof is inoculated and the antibody action against the tumor antigen is expected, for example, antibody-dependent cytotoxicity, or the cellularity in which the T cell action is expected against the tumor antigen Tumor development can be prevented by promoting the induction of immunity.

  The present invention allows the provision of a therapeutic agent or method for one of ordinary skill in the art to treat a subject having a disorder characterized by expression of an HLA class II binding peptide of SART2. The treatment involves administering as a therapeutic agent a factor that increases the concentration of a HLA class II binding peptide of SART2 and an HLA class II molecule in a subject, and CD4 positive T cells that are specific for such a complex. The process of administering as a therapeutic agent is included. Factors useful for the above treatment include HART class II binding peptides of SART2 and functional variants thereof, endosome-targeted fusion proteins containing such SART2 peptides, nucleic acids expressing such proteins and peptides (nucleic acids Antigen-presenting cells carrying complexes of such peptides and HLA class II binding molecules, SART2 HLA class II binding peptides and HLA class II binding molecules, such as dendritic cells, B Examples include cells and non-proliferative transfectants. The present invention also allows one skilled in the art to selectively enrich a population of T cells for CD4 positive T cells specific for SART2 HLA class II binding peptides. Isolation of SART2 HLA class II binding peptides also allows isolation of nucleic acids encoding SART2 HLA class II binding peptides. The nucleic acids can be used to generate SART2 HLA class II binding peptides in vivo or in prokaryotic or eukaryotic host cells. The HLA class II binding peptide of isolated SART2 can be obtained using various methods well known to the skilled worker. For example, an expression vector can be introduced into the cell resulting in production of the peptide. In another method, the mRNA transcript can be microinjected or otherwise introduced into the cell, resulting in production of the encoded polypeptide. Translation of mRNA in cell-free extracts, such as reticulocyte lysate systems, can also be used to produce peptides. Peptides comprising SART2 HLA class II binding peptides of the invention can also be synthesized in vitro. One skilled in the art can readily follow known peptide isolation methods to produce HLA class II binding peptides of isolated SART2. Examples of these include, but are not limited to, immunochromatography, HPLC, size exclusion chromatography, ion exchange chromatography, and immunoaffinity chromatography.

  The dose of the tumor antigen peptide 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 1000 mg, preferably 0.001 mg to 1000 mg. Is preferably administered once every few days to several months.

  The preparations of the invention are administered in an effective amount. An effective amount is that amount of a pharmaceutical preparation that alone, or together with further doses, stimulates the desired response. When treating cancer, a desirable response is suppression of cancer progression. This may involve only temporarily slowing the progression of the disease, but more preferably involves permanently halting the progression of the disease. In the case of inducing an immune response, the desired response is an increase in antibodies or T cells that are specific for the SART2 immunogen (s) used. These desired responses can be monitored by routine methods or can be monitored by the diagnostic methods of the present invention discussed herein.

  The polynucleotide of the present invention includes a primer that can amplify a DNA region containing the peptide sequence of the present invention by PCR. The primer can be used to prepare RNA and cDNA from tissues, blood, etc. from patients with symptoms characterized by SART2 expression and make a diagnosis by PCR. Furthermore, a tumor can be diagnosed by Northern blotting using RNA prepared from a tissue or blood from a patient having a symptom characterized by SART2 expression and using the polynucleotide of the present invention as a probe. In addition, the DNA chip using the polynucleotide molecule of the present invention enables diagnosis of tumors using cDNA prepared from tissues, blood, etc. from patients with symptoms characterized by SART2 expression. These are one embodiment of a diagnostic agent for tumors containing the polynucleotide molecule of the present invention as an active ingredient, but the diagnostic agent for tumors comprising the polynucleotide molecule of the present invention as an active ingredient is not limited to such an embodiment.

  Furthermore, the antibody against the peptide of the present invention can provide a diagnostic agent for symptoms characterized by the expression of SART2. For example, it is possible to detect the expression of SART2 by reacting a tumor diagnostic agent containing an antibody against the peptide of the present invention as an active ingredient with a tissue derived from a patient having symptoms characterized by SART2 expression. Since SART2 is remarkably expressed in the tumor tissue, it is possible to diagnose the tumor by the above method.

  The immune response generated or enhanced by any of the treatments described herein can be monitored by various methods known in the art. For example, the presence of T cells specific for a given antigen can be detected by direct labeling of the T cell receptor with a soluble fluorogenic MHC molecular tetramer presenting an antigenic peptide (Dunbar, outer 5 Name, “Current biology”, 1998, Vol. 8, pp. 413-416). In short, soluble MHC class I molecules are folded in vitro in the presence of peptide antigens that bind β2-microglobulin and class I molecules. After purification, the MHC / peptide complex is purified and labeled with biotin. A tetramer is formed by mixing the biotinylated peptide-MHC complex with labeled avidin (eg, phycoerythrin) in a 4: 1 molar ratio. The tetramer is then contacted with a source of CTL such as peripheral blood or lymph nodes. The tetramer binds a CTL that recognizes the peptide antigen / MHC class I complex. Cells bound by the tetramer can be sorted by fluorescence activated cell sorting to isolate reactive CTL. The isolated CTL can then be expanded in vitro for use as described herein. The use of MHC class II molecules as tetramers has recently been demonstrated by Crawford et al. ("Immunity", 1998, Vol. 8, p. 675-682). Multimeric soluble MHC class II molecules were complexed with covalently linked peptides. Class II tetramers have been shown to bind to specific T cells with appropriate specificity and affinity. Thus, tetramers can be used to monitor both CD4 positive and CD8 positive cell responses to vaccination protocols.

  When administering the peptide of the present invention or a variant / derivative thereof, the effect can be enhanced by pulsing dendritic cells cultured in vitro.

  That is, dendritic cells are induced from bone marrow, umbilical cord blood or patient peripheral blood by granulocyte macrophage colony stimulating factor (GM-CSF) and IL-3 (or IL-4), and tumor-related peptide is added to the culture system Thus, tumor-specific dendritic cells can be induced. By administering an effective amount of this dendritic cell, a desired response for cancer treatment can be induced. More preferably, after inducing tumor-specific dendritic cells, T cells interact with the dendritic cells to proliferate and administer.

The cells used can be bone marrow and umbilical cord blood provided by healthy people, the patient's own bone marrow and peripheral blood, etc., but when using the patient's own autologous cells, it is highly safe and has serious side effects. It can also be expected to avoid it. Peripheral blood or bone marrow may be a fresh sample, a cryopreserved sample, or a cryopreserved sample. As the peripheral blood, whole blood may be cultured, or only the leukocyte component may be separated and cultured, but the latter is more efficient and preferable. Furthermore, mononuclear cells may be separated among the leukocyte components. Moreover, when originating in bone marrow or umbilical cord blood, the whole cells constituting the bone marrow may be cultured, or mononuclear cells may be separated and cultured from this. Peripheral blood, its leukocyte components, and bone marrow cells include mononuclear cells, hematopoietic stem cells or immature dendritic cells, CD4 positive cells, and the like that are the origin of dendritic cells. As long as the cytokine used has a property that has been confirmed to be safe and physiologically active, it does not matter whether it is a natural type or a genetically engineered type, and its production method is preferably ensured. Are used in the minimum amount required. The concentration of the cytokine to be added is not particularly limited as long as it is a concentration at which dendritic cells are induced, and is usually preferably about 10 to 1000 ng / mL, more preferably about 20 to 500 ng / mL as the total concentration of cytokines. The culture can be performed using a well-known medium usually used for culturing leukocytes. The culture temperature is not particularly limited as long as leukocyte growth is possible, but is most preferably about 37 ° C. which is the human body temperature. In addition, the gas environment during the culture is not particularly limited as long as leukocytes can grow, but it is preferable to aerate 5% CO ₂ . Further, the culture period is not particularly limited as long as a necessary number of cells are induced, but it is usually performed for 3 days to 8 weeks. As a device used for cell separation and culture, an appropriate device can be used as appropriate, but it is preferable that safety is confirmed for medical use and that the operation is stable and simple. In particular, for cell culture devices, stacked containers, multistage containers, roller bottles, spinner bottles, bag-type incubators, hollow fiber columns, etc. can be used regardless of general containers such as petri dishes, flasks, and bottles. .

  The dose of dendritic cells and T cells cultured in vitro with the peptide of the present invention or a variant / derivative thereof can be appropriately adjusted depending on the disease to be treated, the age, weight, etc. of the patient. It is preferable to administer this once every several days to several months.

  The peptide of the present invention or a variant / derivative thereof can induce a desired response to cancer treatment even when administered in vivo.

  An effective amount of the dendritic cell growth factor Flt3-Ligand or an alternative thereof is administered, and mRNA and RNA encoding the tumor-related peptide or peptide sequence thereof are administered. At this time, it is preferable to administer an effective amount of Imiguimod or CpG as an adjuvant to activate dendritic cells, more preferably Ontak which is a fusion protein derived from IL-2 and diphtheria toxin to remove CD25 positive cells. Can induce a desired response to cancer treatment.

Example 1: Induction of SART2-derived peptide epitope reactive CD4-positive T cells (1) Information on the amino acid sequence of human SART2 protein was obtained from GenBank. In order to predict a CD4-positive T cell antigen epitope, the amino acid sequence of human SART2 protein was analyzed using three computer prediction programs of SYFPEITHI algorithm, ProPred algorithm, and RANKPEP algorithm, and three types of peptides were selected (SEQ ID NO: 4: SART2 548-574, SEQ ID NO: 6: SART2 915-940, SEQ ID NO: 8: SART2 532-556).

(2) Peripheral blood is separated from healthy individuals with HLA-DRB1 * 09/14 positive, HLA-DRB1 * 04/09 positive, HLA-DRB1 * 04/15 positive, HLA-DRB1 * 14/15 positive, and Lymphocyte separation Layered on medium (OrganonpTeknika, Durham, NC) and centrifuged at 1,500 rpm for 20 minutes at room temperature. The phase containing PBMC was harvested and washed three times (or more) in cold phosphate buffer to obtain peripheral blood mononuclear cells (PBMC). The obtained PBMC was suspended in 20 ml of AIM-V medium (Life Technololgies, Inc., Grand Island, NY), and allowed to adhere in a culture flask (Falcon) for 2 hours under conditions of 37 ° C. and 5% CO ₂ . Non-adherent cells were used for T cell preparation, and adherent cells were used to prepare dendritic cells.

  Meanwhile, adherent cells were cultured in AIM-V medium in the presence of IL-4 (1000 U / ml) and GM-CSF (1000 U / ml). 6 days later, IL-4 (1000 U / ml), GM-CSF (1000 U / ml), IL-6 (1000 U / ml, Genzyme, Cambridge, MA), IL-1β (10 ng / ml, Genzyme, Cambridge, MA) The non-adherent cell population obtained after the AIM-V medium supplemented with TNF-α (10 ng / ml, Genzyme, Cambridge, MA) and further cultured for 2 days was used as dendritic cells.

(3) The prepared dendritic cells are suspended in AIM-V medium at a cell density of 1 × 10 ⁶ cells / ml, the selected peptide is added at a concentration of 10 mg / ml, and the temperature is 37 ° C. using a 24-well plate. The cells were cultured for 4 hours under 5% CO ₂ conditions. After incubation, X-ray irradiation (3000 rad), washed with AIM-V medium, 10% human AB serum (Nabi, Miami, FL), IL-6 (1000 U / ml) and IL-12 (10 ng / ml, Genzyme) , Cambridge, MA) and suspended in AIM-V medium, and 1 × 10 ⁵ cells were added per well of a 24-well plate. Further, the prepared T cell population was added at 1 × 10 ⁶ cells per well, and cultured under conditions of 37 ° C. and 5% CO ₂ . Seven days later, the respective culture supernatants were discarded, and the dendritic cells treated with each peptide obtained in the same manner as described above and then irradiated with X-rays were treated with 10% human AB serum (Nabi, Miami, FL) and IL-2 (10 U / The suspension was suspended in AIM-V medium containing ml, Genzyme, Cambridge, MA), and 1 × 10 ⁵ cells were added to each well of a 24-well plate and further cultured. The same operation was repeated 4 to 6 times every 7 days, and then stimulated T cells were collected, and induction of CD4 positive T cells was confirmed by flow cytometry.

Example 2: Determination of SART2-derived helper epitope that stimulates HLA-DRB1-positive CD4-positive T cells (1) Using HLA-DRB1 * 09 / 14-positive cells, this It was confirmed that T cells stimulated with the peptide shown in SEQ ID NO: 4, 6 or 8 which is the peptide of the invention are proliferating. In order to examine the specificity of the present T cells for the peptides shown in SEQ ID NO: 4, 6 or 8, dendritic cells pulsed with each peptide (each peptide was added in AIM-V medium at a concentration of 10 mg / ml, 5 × 10 ³ T cells stimulated with each peptide 4 to 6 times were added to 5 × 10 ⁴ cells and cultured for 10 hours under 37 ° C. and 5% CO ₂ conditions. The cells were cultured in AIM-V medium containing AB serum in a 96-well plate at 37 ° C. and 5% CO ₂ for 72 hours. It should be noted that 1 mCi of tritium thymidine was added to each culture solution 48 hours after the start of the culture. After culturing, the cells were collected on a glass filter with a cell harvester, and the amount of tritium thymidine incorporated was measured with a liquid scintillation counter. Moreover, the supernatant after 48-hour culture | cultivation was taken, and the production amount of IFN-gamma was measured by ELISA method. As a result, compared with the culture supernatant of the hole using the dendritic cells not pulsed with the peptide, it was remarkable in the culture supernatant of the hole using the dendritic cells pulsed with the peptide shown in SEQ ID NO: 4, 6 or 8. IFN-γ production was confirmed. (FIG. 1). Furthermore, as shown in FIG. 2, it was confirmed that T cells proliferated by stimulation of the peptide shown in SEQ ID NO: 4, 6 or 8. Therefore, the peptide shown in SEQ ID NO: 4, 6 or 8 is a T cell epitope peptide having the ability to specifically stimulate proliferation of HLA-DRB1 * 09/14 positive CD4 positive T cells and induce IFN-γ production. There was found.

(2) Next, the present peptide having the ability to stimulate proliferation of HLA-DRB1 * 09 / 14-positive T cells is an epitope that is naturally processed in antigen-presenting cells and presented on HLA-DR. We examined whether there was. As shown in FIG. 3, the expression of SART2 was confirmed in the glioma cell line, T98G, by RT-PCR. After T98G lysate was added to immature dendritic cells and digested to mature the dendritic cells, T cells stimulated with the peptide shown in SEQ ID NO: 4, 6 or 8 were stimulated by the dendritic cells. I researched. A pellet of 1.5 × 10 ⁶ T98G cells was freeze-thawed 7 times using liquid nitrogen and a hot water bath to prepare a cell lysate. On the other hand, peripheral blood is separated from healthy individuals of HLA-DRB1 * 09/14 positive, HLA-DRB1 * 04/09 positive, HLA-DRB1 * 04/15 positive, HLA-DRB1 * 14/15 positive, and Lymphocyte separation medium And centrifuged at 1,500 rpm at room temperature for 20 minutes. The phase containing PBMC was harvested and washed three times (or more) in cold phosphate buffer to obtain PBMC. The obtained PBMC was suspended in 20 ml of AIM-V medium and allowed to adhere in a culture flask (Falcon) at 37 ° C. under 5% CO ₂ for 2 hours. 1000 U / ml) and GM-CSF (1000 U / ml) were cultured for 6 days to prepare immature dendritic cells. Each cell lysate prepared was added to 5 × 10 ⁵ immature dendritic cells, and IL-4 (1000 U / ml), GM-CSF (1000 U / ml), IL-6 (1000 U / ml), IL- The cells were cultured in AIM-V medium supplemented with 1β (10 ng / ml) and TNF-α (10 ng / ml) for 2 days. At the same time, the peptide shown in SEQ ID NO: 4, 6 or 8 was added to immature dendritic cells, and the cell lysate of PBMC (prepared from 1.5 × 10 ⁶ cell pellets) to immature dendritic cells. Each of these was prepared, and IL-4 (1000 U / ml), GM-CSF (1000 U / ml), IL-6 (1000 U / ml), IL-1β (10 ng / ml) and TNF-α (10 ng / ml) were prepared. ml) was added for 2 days at 37 ° C. under 5% CO ₂ in AIM-V medium. Each cultured dendritic cell was irradiated with X-rays (3000 rad), washed with AIM-V medium, suspended in AIM-V medium containing 10% human AB serum, and 3. 3. ⁴ × 3 × 10 ⁴ were added. To these cells, 5 × 10 ⁴ T cells stimulated with the peptide shown in SEQ ID NO: 4, 6 or 8 were added and cultured at 37 ° C. under 5% CO ₂ for 72 hours. It should be noted that 1 mCi of tritium thymidine was added to each culture solution 48 hours after the start of the culture. After culturing, the cells were collected on a glass filter with a cell harvester, and the amount of tritium thymidine incorporated was measured with a liquid scintillation counter. As a result, HLA-DRB1 * 09/14 positive and HLA-DRB1 * 04/15 positive T cells stimulated with peptide SART2 548-574 shown in SEQ ID NO: 4 were stimulated by dendritic cells added with lysate of T98G cells. HLA-DRB1 * 09 / 14-positive and HLA-DRB1 * 04 / 09-positive T cells stimulated with the peptide SART2 915-940 shown in SEQ ID NO: 6 are T98G cells (FIGS. 4, 5, 6). HLA-DRB1 * 09/14 positive and HLA-DRB1 * 14 / stimulated by stimulation of dendritic cells supplemented with lysate (FIGS. 7, 8) and stimulated with peptide SART2 532-556 as shown in SEQ ID NO: 8 It was confirmed that 15-positive T cells proliferated by stimulation of dendritic cells to which lysate of T98G cells was added (FIGS. 9 and 10). Furthermore, since these reactions are inhibited by the addition of an anti-HLA-DR neutralizing antibody, the peptide shown in SEQ ID NO: 4, 6 or 8 is processed naturally in the antigen-presenting cells by the SART2 protein. It was revealed to be an epitope presented on the DR.

Example 3: Killer activity of SART2-derived peptide epitope-reactive CD4-positive T cells Whether HLA-DRB1 * 09 / 14-positive T cells stimulated with peptide SART2 532-556 shown in SEQ ID NO: 8 damage T98G cells Examined. To T98G cells, 1000 U / ml of IFN-g and 10 mg / ml of the peptide SART2 532-556 shown in SEQ ID NO: 8 were added and cultured overnight at 37 ° C. Thereafter, 50 μCi of chromium was added to 2 × 10 ⁵ T98G cells and cultured at 37 ° C. for 4 hours. After washing twice (or more) in cold phosphate buffer, the resulting T98G cells were suspended in RPMI medium containing 10% fetal bovine serum and prepared as target cells. In addition, 8 × 10 ⁴ , 2 × 10 ⁴ , and 5 × 10 ³ HLA-DRB1 * 09/14 positive T cells stimulated with the peptide SART2 532-556 represented by SEQ ID NO: 8 repeated 4 to 6 times The cells were mixed with 1 × 10 ³ target cells and cultured at 37 ° C. for 4 hours. After the culture, the culture supernatant was collected on a glass filter paper, and the amount of chromium released to the culture supernatant was measured with a liquid scintillation counter. As a control experiment, 1 × 10 ⁴ T98G cells (cold target cells) without addition of chromium were repeated 4 to 6 times with 8 × 10 ⁴ cells stimulated with the peptide SART2 532-556 shown in SEQ ID NO: 8, 2 × 10 ⁴ , 5 × 10 ³ HLA-DRB1 * 09/14 positive T cells and 1 × 10 ³ target cells were mixed and cultured at 37 ° C. for 4 hours. After the culture, the culture supernatant was collected on a glass filter paper, and the amount of chromium released to the culture supernatant was measured with a liquid scintillation counter. As shown in FIG. 11, HLA-DRB1 * 09 / 14-positive T cells stimulated with peptide SART2 532-556 shown in SEQ ID NO: 8 have killer activity that damages T98G cells that are cancer cells. It became clear that this activity was reduced in the presence of cold target cells, indicating that the killer activity was specific to cancer cells.

  Other aspects of the invention will be apparent to those skilled in the art and need not be repeated here. Each of the references cited herein is hereby incorporated by reference in its entirety.

It is a figure which shows the production of IFN-γ from T cells specific for the peptide shown in SEQ ID NO: 4, 6 or 8 derived from SART2. It is a figure which shows the proliferation of T cell specific to the peptide shown to sequence number 4, 6, or 8 derived from SART2. It is a figure which shows that the tumor antigen SART2 is expressing in the glioma cell line T98G cell. bp indicates the number of DNA bases. It is a figure which shows that the HLA-DRB1 * 09/14 positive CD4 positive T cell line induced | guided | derived with peptide SART2 548-574 derived from SART2 recognizes this peptide processed naturally from the tumor cell by the dendritic cell. . DC represents HLA-DRB1 * 09/14 positive dendritic cells, T98G Lysate represents T98G cell lysate, and PBMC Lysate represents HLA-DRB1 * 09/14 positive PBMC lysate. HLA-DRB1 * 09 / 14-positive CD4-positive T cell line induced with SART2-derived peptide SART2 548-574 recognizes this peptide naturally processed from tumor cells by dendritic cells in an HLA-DR-restricted manner FIG. DC is HLA-DRB1 * 09/14 positive dendritic cell, T98G Lysate is T98G cell lysate, PBMC Lysate is HLA-DRB1 * 09/14 positive PBMC lysate, aHLA-DR is an inhibitory antibody against HLA-DR Show. HLA-DRB1 * 04/15 positive CD4 positive T cell line induced by SART2 derived peptide SART2 548-574 recognizes this peptide naturally processed from tumor cells by dendritic cells in an HLA-DR-restricted manner FIG. DC represents HLA-DRB1 * 04 / 15-positive dendritic cells, T98G Lysate represents a cell lysate of T98G cells, and aHLA-DR represents an inhibitory antibody against HLA-DR. HLA-DRB1 * 09/14 positive CD4 positive T cell line induced by SART2 derived peptide SART2 915-940 recognizes this peptide naturally processed from tumor cells by dendritic cells in an HLA-DR-restricted manner FIG. DC is HLA-DRB1 * 09/14 positive dendritic cell, T98G Lysate is T98G cell lysate, PBMC Lysate is HLA-DRB1 * 09/14 positive PBMC lysate, aHLA-DR is an inhibitory antibody against HLA-DR Show. HLA-DRB1 * 04 / 09-positive CD4-positive T cell line induced with the SART2-derived peptide SART2 915-940 recognizes this peptide naturally processed from tumor cells by dendritic cells in an HLA-DR-restricted manner FIG. DC is HLA-DRB1 * 04/09 positive dendritic cell, T98G Lysate is T98G cell lysate, PBMC Lysate is HLA-DRB1 * 04/09 positive PBMC lysate, aHLA-DR is an inhibitory antibody against HLA-DR Show. HLA-DRB1 * 09 / 14-positive CD4-positive T cell line induced with the SART2-derived peptide SART2 532-556 recognizes this peptide naturally processed from tumor cells by dendritic cells in an HLA-DR-restricted manner FIG. DC is HLA-DRB1 * 09/14 positive dendritic cell, T98G Lysate is T98G cell lysate, PBMC Lysate is HLA-DRB1 * 09/14 positive PBMC lysate, aHLA-DR is an inhibitory antibody against HLA-DR Show. HLA-DRB1 * 14 / 15-positive CD4-positive T cell line induced with SART2-derived peptide SART2 532-556 recognizes this peptide naturally processed from tumor cells by dendritic cells in an HLA-DR-restricted manner FIG. DC is HLA-DRB1 * 14/15 positive dendritic cell, T98G Lysate is T98G cell lysate, PBMC Lysate is HLA-DRB1 * 14/15 positive PBMC lysate, aHLA-DR is an inhibitory antibody against HLA-DR Show. It is a figure which shows that the HLA-DRB1 * 09/14 positive CD4 positive T cell strain induced | guided | derived with peptide SART2 532-556 derived from SART2 has a cytotoxic activity with respect to a T98G cell.

Claims (18)

A peptide comprising 10 or more consecutive amino acid sequences of the amino acid sequence set forth in SEQ ID NO: 2 and stimulating T cell proliferation and activation. 2. The peptide of claim 1 that binds to an HLA class II molecule. A peptide that binds to the following HLA class II molecule of (a), (b) or (c):
(A) A peptide comprising the amino acid sequence represented by SEQ ID NO: 4, 6 or 8.
(B) A peptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 4, 6 or 8.
(C) A peptide that is a variant or derivative of the peptide according to (a). (1) a polynucleotide molecule represented by SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7, (2) a polynucleotide molecule encoding the peptide according to any one of claims 1 to 3, (3) ( In 1) or (2), a polynucleotide molecule in which one or more bases are substituted, deleted or added, and (4) stringent to any of the polynucleotide molecules of (1) to (3) above A polynucleotide molecule selected from the group consisting of a polynucleotide molecule that hybridizes under conditions. Leukocytes that recognize the peptide according to any one of claims 1 to 3. The antibody with respect to the peptide of any one of Claims 1-3. The pharmaceutical composition which contains the peptide of any one of Claims 1-3 as an active ingredient. A pharmaceutical composition comprising the polynucleotide molecule according to claim 4 as an active ingredient. A pharmaceutical composition comprising the leukocyte according to claim 5 as an active ingredient. A pharmaceutical composition comprising the antibody according to claim 6 as an active ingredient. The diagnostic agent of the tumor which contains the peptide of any one of Claims 1-3 as an active ingredient. The preventive agent of the tumor which contains the peptide of any one of Claims 1-3 as an active ingredient. The therapeutic agent of the tumor which contains the peptide of any one of Claims 1-3 as an active ingredient. A diagnostic agent for a tumor comprising the polynucleotide molecule according to claim 4 as an active ingredient. A diagnostic agent for a tumor comprising the antibody according to claim 6 as an active ingredient. An isolated CD4-positive T cell that selectively binds to a complex of an HLA class II molecule and the peptide of claims 1-3. The isolated CD4-positive T cell according to claim 16, which has an ability to damage cancer cells. An isolated antigen-presenting cell comprising a complex of an HLA class II molecule and the peptide according to claims 1 to 3.