JP2018070469A - HIF-1α-DERIVED PEPTIDE CAPABLE OF BINDING TO HLA-A24 MOLECULE AND RECOGNIZED BY CELL-MEDIATED IMMUNITY AND RENAL CELL CARCINOMA VACCINE CONTAINING SAME - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide peptides derived from HIF-1α or modified peptides thereof, which bind to HLA-A24 molecule, are recognized by cell-mediated immunity, can be conveniently prepared, and are useful for treatment and prevention of cancer diseases such as renal cell carcinoma.SOLUTION: A peptide recognized by cellular immunity comprises a skeleton represented by the amino acid sequence of Tyr Tyr His Ala Leu Asp Ser Asp His Leu or a modified amino acid sequence with one or two of the amino acids of the amino acid sequence deleted, substituted and/or added.SELECTED DRAWING: None

Description

  The present invention relates to a peptide which is suitable as an active ingredient for the prevention or treatment of cancer diseases, particularly renal cell carcinoma, and which is recognized by cellular immunity thereof, a nucleic acid molecule encoding the same and a vector thereof, cancer diseases, particularly renal cell carcinoma. Preparations for use in the prevention or treatment of cancer, and biological preparations containing antigen-presenting cells or renal cell carcinoma-reactive cytotoxic T cells presenting the complex of the peptide and human leukocyte antigen-A24 on the cell surface It is about.

  More specifically, the present invention relates to an HIF-1α-derived peptide that can bind to an HLA-A24 molecule and is recognized by cellular immunity, useful for the treatment or prevention of patients with HLA-A24-positive renal cell carcinoma, and The present invention relates to a pharmaceutical preparation such as a renal cell carcinoma vaccine.

  Renal cell carcinoma (hereinafter also referred to as RCC) is more likely to occur in older adults, and is a tumor of tubule cells that has become cancerous. Then it was found by chance during a close examination of other diseases.

  Metastatic RCC (hereinafter also referred to as mRCC) as a treatment method for cancerous angiogenesis in addition to general cancer treatment methods such as cancer removal and chemotherapy and radiation therapy The molecular therapy is becoming common. However, although these treatment methods have a certain degree of anticancer effect, a sufficient treatment effect cannot be obtained, so that the complete cure is not achieved. Therefore, it is indispensable to use in combination with other treatment methods such as immunotherapy as the cancer progresses. Immunotherapy using ILs such as interleukin (hereinafter also referred to as IL) -2 and cytokines such as IFNs such as interferon (hereinafter also referred to as IFN) -α has been introduced to these patients with metastatic renal cell carcinoma. Although it has been applied, it still has limited therapeutic effects.

  Under such circumstances, renal cell carcinoma-related antigen and human leukocyte antigen (hereinafter also referred to as HLA) class I-restricted and renal cell carcinoma-responsive cytotoxic T cells (cytotoxic T lymphocytes) : Cytotoxic T lymphocytes (hereinafter also referred to as CTLs), some of these peptides have been identified and have already been used in clinical trials. Among these antigens, carbonic anhydrase 9 (hereinafter also referred to as CA9) is overexpressed in approximately 90% of all renal cell carcinoma types, and has attracted attention as a cancer vaccine candidate. In Non-Patent Documents 1 and 2, the present inventors have disclosed, as peptide vaccines for renal cell carcinoma, CA9 antigen-derived peptides, vascular endothelial growth factor receptor (hereinafter also referred to as VEGFR1) -derived peptides. Has been proposed. Furthermore, for renal cancer, we started to develop a vaccine using the peptide, conducted clinical research using the peptide vaccine CA9 vaccine and VEGFR1 vaccine, and reported on its safety and usefulness.

  Based on the clinical experience of vaccine therapy with these peptide vaccines, there is a wide variety of immune responses such as cancer-responsive cytotoxic T-cell responses due to the diversity of individual patients' immune status and the variety of cancers that are therapeutic targets. It has become clear that there are cases in which once effective, it becomes ineffective due to immune escape or tolerance. Only some of the vaccinated patients with renal cell carcinoma have a clinical effect and have shown limited efficacy in clinical cases, but treatment methods such as this vaccine therapy require further improvement. It was suggested that there was.

  In view of the current situation, a host that targets multiple molecules based on the development, differentiation, and growth of renal cancer, rather than targeting one specific antigen, and has cancers with different properties. There is a need for a tailor-made vaccine therapy that suits your needs.

  In fact, the present inventors blocked the reaction between erythropoietin (hereinafter abbreviated as Epo) and erythropoietin receptor (hereinafter abbreviated as EpoR) by administration of a peptide, thereby maximizing the survival of cancer cells. We have found an EpoR-derived peptide that is useful for a treatment that kills oxygen by cutting off oxygen, which is an important factor (Patent Document 1), and has made a step forward to tailor-made vaccine therapy.

    Thus, further development of a peptide useful for cancer vaccine therapy for renal cell cancer patients is strongly demanded for the spread of tailor-made vaccine therapy.

H Uemura, K Fujimoto, M Tanaka, et al., “A phase I trial of vaccination of CA9-derived peptides for HLA-A24-positive patients with cytokine-refractory metastatic renal cell carcinoma.”, Clinical Cancer Research, 2006; 12 (6) March 15, 2006, p.1768-1775 K Yoshimura, T Minami, M Nozawa, H Uemura, “Phase I clinical trial of human vascular endothelial growth factor receptor 1 peptide vaccines for patients with metastatic renal cell carcinoma.”, British Journal of Cancer, (2013) 108, p.1260 -1266

JP2015-136293A

The present inventors have developed HIF-1α (Hypoxia Inducible Factor (Hypoxia Inducible Factor) applicable as an anti-cancer vaccine for HLA-A24 positive (positive may be expressed as “ ⁺ ”) renal cell carcinoma patients. ) -1α) -derived peptides (peptides involved in oxygen-dependent degradation of hypoxia-inducible factors involved in tumor malignancy and resistance to treatment), and found highly active peptides and examined their effectiveness. Thus, the present invention has been completed.

  The present invention has been made to solve the above-mentioned problems, and is a HIF-1α-derived peptide or a modified peptide thereof, which binds to the HLA-A24 molecule and is recognized by cellular immunity and can be easily prepared as renal cell carcinoma. Peptides useful for the treatment and prevention of cancer diseases such as, nucleic acid molecules encoding the same and vectors and transformants thereof, pharmaceutical preparations for the prevention or treatment of cancer diseases such as renal cell carcinoma, and the peptides To provide a biological preparation for the prevention or treatment of renal cell carcinoma, which contains antigen-presenting cells or renal cell carcinoma reactive cytotoxic T cells presenting a complex of HCV and human HLA-A24 on the cell surface Objective.

Wherein the peptide has been made in order to achieve the purpose of, HIF-l [alpha] peptide comprising the amino acid sequence shown in _278-287, and one or two amino acid substitutions in the amino acid sequence shown in HIF-1α _278-287, deletion, And / or a derivative thereof having a functionally equivalent property to a peptide consisting of an amino acid sequence into which an addition has been introduced and consisting of the amino acid sequence shown in HIF-1α _278-287 . That is, the peptide of the present invention comprises the amino acid sequence of SEQ ID NO: 5 in the sequence listing shown in three letter code (corresponding to the amino acid sequence of 278 to 287 of YYHALDSDHL: HIF-1α in one letter code), It consists of a skeleton represented by either a modified amino acid sequence in which one or two amino acids have been deleted, substituted and / or added, and is recognized by cellular immunity.

  This peptide binds to the HLA-A24 molecule and is recognized by the cellular immunity of, for example, renal cell carcinoma.

  In addition, this peptide functions as a programmed death ligand 1-inducing peptide.

  For the present invention, a nucleic acid molecule having a base sequence encoding the peptide may be used.

  Alternatively, a vector in which the nucleic acid molecule is incorporated may be used.

  A transformant obtained by introducing the vector into a host cell may be used.

  The pharmaceutical preparation of the present invention comprises a peptide represented by at least one of the amino acid sequence of SEQ ID NO: 5 and a modified amino acid sequence in which one or two amino acids are deleted, substituted and / or added. It is contained as an active ingredient.

  This pharmaceutical preparation may be used as an inducer for renal cell carcinoma reactive cytotoxic T cells.

  Moreover, this pharmaceutical preparation may be used as an inducer for antigen-presenting cells of CD8-positive T cells.

  Furthermore, this pharmaceutical preparation may be used as a therapeutic or prophylactic agent for HLA-A24 positive renal cell carcinoma.

  This pharmaceutical preparation may be used as a cancer vaccine when the active ingredient is the peptide.

  The antigen-presenting cell of the present invention is a peptide represented by at least one of the amino acid sequence of SEQ ID NO: 5 and a modified amino acid sequence in which one or two amino acids are deleted, substituted and / or added. Is introduced into a cell having antigen-presenting ability collected from a patient with HLA-A24-positive renal cell carcinoma, and a complex of the peptide and HLA-A24 is presented on the cell surface.

  The renal cell carcinoma reactive cytotoxic T cell of the present invention comprises an amino acid sequence of SEQ ID NO: 5 and a modified amino acid sequence in which one or two amino acids are deleted, substituted, and / or added. A peptide represented by at least one of the above is induced by contact with peripheral blood mononuclear cells collected from a patient with HLA-A24-positive renal cell carcinoma.

  The biologic of the present invention comprises a peptide represented by at least one of the amino acid sequence of SEQ ID NO: 5 and a modified amino acid sequence in which one or two amino acids are deleted, substituted and / or added. And antigen-presenting cells that are induced by introduction into peripheral blood mononuclear cells collected from patients with HLA-A24-positive renal cell carcinoma and present the complex of the peptide and HLA-A24 on the cell surface Contains at least one induction cell selected from induction cells and induction cells composed of renal cell carcinoma reactive cytotoxic T cells induced by bringing the peptide into contact with the collected cells It is.

  Since the peptide of the present invention can bind to the HLA-A24 allele and is recognized by cellular immunity of cancer patients, particularly HLA-A24 positive renal cell carcinoma patients, it is useful for the treatment and prevention of cancer diseases, particularly renal cell carcinoma. It is. According to this peptide, renal cell carcinoma reactive cytotoxic T cells can be induced in an HLA-restricted manner. This peptide is derived from HIF-1α existing in the living body or modified or derived based on its skeleton, and binds to a desired binding site in the living body to express an action effect. There are few, reliable and safe.

  Using the sequence of this peptide, a nucleic acid molecule encoding it and a vector thereof and a transformant thereof can be prepared and used to produce the peptide in vitro or produce it in vivo.

  Further, by using this peptide or a nucleic acid molecule encoding the peptide, a pharmaceutical preparation for the prevention or treatment of renal cell carcinoma, for example, an inducer for renal cell carcinoma reactive cytotoxic T cells, CD8 positive T cells As an inducing agent for antigen-presenting cells, a therapeutic or prophylactic agent for HLA-A24-positive renal cell carcinoma, and a cancer vaccine, it can be used for cancer therapy, particularly for treatment of HLA-A24-positive renal cell carcinoma.

  Furthermore, a biological preparation for the prevention or treatment of renal cell cancer containing antigen presenting cells or renal cell carcinoma reactive cytotoxic T cells presenting the complex of this peptide and human HLA-A24 on the cell surface is prepared. Thus, it can be used for treatment of HLA-A24 positive renal cell carcinoma patients.

  These inventions have expanded the options for peptide-based immunotherapy, particularly cancer vaccine therapy, for HLA-A24 positive renal cell carcinoma patients. According to the present invention, in the treatment of HLA-A24-positive renal cell carcinoma patients for which several cancer vaccine candidate peptides have been identified to date, further therapeutic effects can be obtained by adding HIF-1α involved in cancer growth to the target. It is expected to contribute to the improvement.

FIG. 1 shows the expression of HIF-1α in an RCC (renal cell carcinoma) cell line to which the present invention is applied. FIG. 1A shows RCC cancer cell line and LNCaP (human prostate cancer) cell line cultured in the presence or absence of DFO (deferoxamine) for 24 hours, and immunoblotting the expression of HIF-1α in whole cell lysate. Shows the result of the test. β-actin is a control example. FIG. 1B shows that KPK-13, KK-RCC6 and LNCaP cell lines were cultured under normoxic (20% O ₂ ) or hypoxic (1% O ₂ ) for 24 hours in whole cell lysates and nucleic acid extraction. The result of having tested the expression of HIF-1 (alpha) in a thing by the immunoblotting is shown. β-actin and E2F-1 are control examples. FIG. 1C shows cell surface expression of HLA-A24 molecule analyzed by flow cytometry. The white outline shows the result of staining without using the anti-HLA-A24 monoclonal antibody. FIG. 2 shows the cytotoxicity of CTL (cytotoxic T cells) from HLA-A24 ⁺ renal cell carcinoma patients stimulated with HIF-1α-derived peptides to which the present invention is applied. FIG. 2A shows the results of analysis of peripheral blood mononuclear cells stimulated with the peptide of patient # 5 in a 5-hour ⁵¹ Cr release assay for their cytotoxicity against the target. Peripheral blood mononuclear cells stimulated with these peptides were also tested in peptide-specific cytotoxicity in the presence or absence of cold target cells designated for cytotoxicity against KPK-13 cells. FIG. 2B similarly shows the results of testing CD8 ⁺ T cells from PBMC (peripheral blood mononuclear cells) stimulated with peptides of HLA-A24 ⁺ patients (# 15) in terms of their cytotoxicity. Peripheral blood mononuclear cells stimulated with these peptides were also tested in peptide-specific cytotoxicity in the presence or absence of cold target cells designated for cytotoxicity against KPK-13 cells.

  Hereinafter, although the form for implementing this invention is demonstrated in detail, the scope of the present invention is not limited to these forms.

[Peptide of the present invention]
Details of the peptide to which the present invention is applied are as follows.

  A preferred form of the peptide of the present invention is a HIF-1α-derived peptide, which is represented by the amino acid sequence of SEQ ID NO: 5 in the sequence listing and is recognized by cellular immunity. This peptide is capable of binding to the HLA-A24 allele.

  “Recognized by cellular immunity” means that the peptide is recognized by specific cytotoxic T cells, that is, peptide-specific cytotoxic T cells can be induced. “Peptide can bind to HLA-A24 molecule” means that the peptide can form a complex with the HLA-A24 molecule and be presented on the cell surface.

The peptide represented by the amino acid sequence of SEQ ID NO: 5 has been screened as a new HIF-1α peptide candidate for HLA-A24 positive renal cell carcinoma patients. As a result of the screening, HIF has the potential to induce peptide-specific and renal cell carcinoma reactive cytotoxic T cells from peripheral blood mononuclear cells (hereinafter also referred to as PBMCs) of HLA-A24-positive renal cell carcinoma patients. -1α peptide HIF-1α _278-287 (single letter YYHALDSDHL: SEQ ID NO: 5) was identified. This peptide has an amino acid prediction motif that binds to the HLA-A24 molecule, that is, the amino acid residue at the second sequence position from the N-terminal side is the Y residue, and the tenth sequence position or C-terminal from the N-terminal side. When the amino acid residue at the sequence position is an L residue, it is extremely compatible.

  Therefore, these HIF-1α-derived peptides can be applied as active ingredients of peptide-derived anticancer vaccines for HLA-A24-positive renal cell carcinoma patients.

Since HIF-1α is expressed in several types of cancer, including renal cell carcinoma, this molecule may be a valuable target for cancer immunotherapy. In fact, among the HIF-1α-derived peptides, HIF-1α _278-287 (SEQ ID NO: 5) is suggested to be effective for various types of cancer, and is particularly effective for renal cell carcinoma.

  The HIF-1α-derived peptide represented by the amino acid sequence of SEQ ID NO: 5 is useful for cancer vaccine therapy for HLA-A24-positive renal cell carcinoma patients so that it can be used for peptide vaccine therapy from among many peptides. Specifically, the HIF-1α-derived peptide was selected and identified as follows.

  In general, it is known that a peptide that binds to the HLA-A24 molecule has a regular and unique amino acid sequence that depends on the type of HLA. Such a regular and unique amino acid sequence is called a binding motif. Whether or not any peptide can bind to the HLA-A24 molecule is determined by, for example, the website BIMAS (Bioinformatics and Molecular Analysis Section, Computational Bioscience and Engineering Laboratory, Division of Computer Research & Technology, NIH, http: // www -bimas.cit.nih.gov/) can be predicted by computer analysis.

  There is no restriction | limiting in particular as a method of confirming whether a peptide specific cytotoxic T cell can be induced | guided | derived, It can select suitably according to the objective. For example, peripheral blood mononuclear cells are removed from blood collected from HLA-A24-positive renal cell carcinoma patients, and peripheral blood mononuclear cells stimulated with the peptide react with antigen-presenting cells pulsed with this peptide to give IFN-γ. And a method of confirming by measuring whether or not cytokines are produced. There is no restriction | limiting in particular as a method of measuring this cytokine, According to the objective, it can select suitably, For example, it can measure by enzyme-linked immunosorbent assay (Enzyme-Linked ImmunoSorbent Assay: ELISA) etc. .

The method for confirming the cytotoxic activity of the induced cytotoxic T cells is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include a method for confirming by ⁵¹ Cr release test. It is done.

  The peptide of the present invention is preferably a HIF-1α-derived peptide represented by the amino acid sequence of SEQ ID NO: 5, but one or two amino acids of these sequences are deleted, substituted and / or modified. There is no particular limitation as long as it is a derivative of a HIF-1α-derived peptide consisting of a skeleton represented by any of the amino acid sequences, recognized by cellular immunity, and capable of binding to the HLA-A24 allele, and is appropriately selected according to the purpose. be able to.

  The position at which one or two amino acids are deleted, substituted, and / or added is not particularly limited and may be appropriately selected depending on the purpose.

  As the substitution, substitution between homologous amino acids is preferable in that the properties of the peptide are not changed. The homologous amino acid can be appropriately selected in view of, for example, a polar amino acid, a nonpolar amino acid, a hydrophobic amino acid, a hydrophilic amino acid, a positively charged amino acid, a negatively charged amino acid, and an aromatic amino acid.

  The amino acid to be substituted or added for constituting the peptide may be a natural amino acid or an amino acid analog. That is, a functional group such as an amino group or a carboxyl group may be modified or protected by an amino acid analog as long as the effect of having the peptide skeleton and being recognized by cellular immunity is not impaired. There is no restriction | limiting in particular as an amino acid analog, According to the objective, it can select suitably, For example, the N-acylated product, O-acylated product, esterified product, acid amidated product, alkylated product etc. of an amino acid etc. are mentioned. For example, a formyl group, acetyl group, t-butoxycarbonyl group or the like bonded to the N-terminal or free amino group, or a methyl group, ethyl group, t-butyl group, benzyl group or the like to the C-terminal or free carboxyl group And the like.

  The peptide is preferably a peptide that is recognized by both cellular immunity and humoral immunity. This peptide has high immunogenicity and is recognized by humoral immunity in that it is expected to be excellent in cytotoxic T cell inducing ability, particularly in renal cell carcinoma reactive cytotoxic T cell inducing ability. Is preferred. “Recognized by humoral immunity” means that peptide-specific immunoglobulin (Ig) G is present in vivo, that is, peptide-specific IgG is detected from plasma. There is no restriction | limiting in particular as a method of measuring specific IgG in plasma, According to the objective, it can select suitably, For example, it can measure by ELISA etc.

  Furthermore, the peptide is also excellent in the ability to induce antigen-presenting cells in which a complex of the peptide and HLA-A24 is presented on the cell surface. Therefore, it is useful as an active ingredient of cancer vaccines, particularly HLA-A24 positive renal cell cancer vaccines.

  There is no restriction | limiting in particular as a manufacturing method of the said peptide, A well-known method can be selected suitably. For example, peptide solid phase synthesis method by Fmoc method using amino protecting group of amino acid (Fmoc group: 9-fluorenylmethyloxycarbonyl group), peptide liquid phase synthesis method in which protected amino acids are coupled with a condensing agent in a batch type, Examples include a synthesis method using a commercially available automatic peptide synthesizer.

  The peptide may be produced by fragmenting a polypeptide containing the amino acid sequence in a cell. There is no restriction | limiting in particular as said polypeptide, According to the objective, it can select suitably.

  The peptide is a HIF-1α-derived peptide that is considered to be involved in cancer growth and programmed death 1, and can bind to HLA-A24 molecule and is recognized by cellular immunity. For example, HLA-A24 molecule positive kidney cells It can be suitably used for cancer vaccine therapy for cancer patients. This peptide is a programmed death ligand 1 derived peptide.

[Nucleic acid molecule of the present invention]
Details of the nucleic acid molecule to which the present invention is applied are as follows.

  The nucleic acid molecule is not particularly limited as long as it is composed of a base sequence encoding the peptide of SEQ ID NO: 5, and can be appropriately selected according to the purpose. The nucleic acid molecule of the present invention encoding the peptide may be produced in vivo.

  This nucleic acid molecule can be suitably used as a vector for forming a transformant.

  The nucleic acid molecule is not particularly limited, and a known method can be appropriately selected. Examples thereof include a chemical synthesis method using a commercially available nucleic acid synthesizer utilizing the phosphoramidite method, and an enzymatic synthesis method utilizing replication or transcription reaction with a polymerase.

[Vector of the present invention]
Details of the vector to which the present invention is applied are as follows.

  The vector of the present invention contains at least a nucleic acid molecule encoding the peptide of SEQ ID NO: 5, and further contains other elements as necessary.

  There is no restriction | limiting in particular as said vector, According to the objective, it can select suitably, For example, a plasmid, a viral vector, etc. are mentioned. Specific examples of the viral vector include adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, vaccinia virus vectors, and the like.

  The method for preparing the vector is not particularly limited, and can be prepared by appropriately selecting a known method such as gene recombination.

[Transformant of the present invention]
The details of the transformant to which the present invention is applied are as follows.

  The vector expresses the peptide when introduced into a host cell such as an antigen presenting cell. The antigen-presenting cell presents a complex of the peptide and the HLA-A24 molecule on the cell surface. The antigen-presenting cells can efficiently proliferate cytotoxic T cells that specifically damage renal cell carcinoma cells.

  The peptide or the vector is suitably used for a pharmaceutical preparation for the prevention or treatment of renal cell carcinoma, or a biological preparation containing induced cells induced into antigen-presenting cells or renal cell carcinoma reactive cytotoxic T cells. Is available.

[Pharmaceutical preparation of the present invention]
Details of the pharmaceutical preparation to which the present invention is applied are as follows.

  This pharmaceutical preparation is a pharmaceutical composition for preventing or treating cancer, particularly renal cell carcinoma, and contains at least one of the peptide, the nucleic acid molecule, and the vector, and further contains other additional components as necessary. Including.

  There is no restriction | limiting in particular as content of the said peptide in the pharmaceutical formulation, the said nucleic acid molecule, and the said vector, According to the objective, it can select suitably. In this pharmaceutical preparation, it may consist only of the active ingredient of either the peptide or the vector. In this pharmaceutical preparation, the peptide, the nucleic acid molecule, and the vector may be used singly or in combination of two or more. In addition, each of the peptide and the vector may be used alone or in combination of two or more.

  The additive component in the pharmaceutical preparation is not particularly limited as long as the effects of the present invention are not impaired, and can be appropriately selected according to the purpose. Examples thereof include a pharmaceutically acceptable carrier. The said additive component may be used individually by 1 type, and may use 2 or more types together. Specific examples of the carrier include cellulose, polymerized amino acid, albumin and the like. There is no restriction | limiting in particular as content of the said additive component, According to the objective, it can select suitably.

  This pharmaceutical preparation may be used in a form in which other active ingredients coexist. Moreover, this pharmaceutical formulation may be used in the state mix | blended in the pharmaceutical which uses another component as an active ingredient.

  Since cytotoxic T cells of renal cell carcinoma patients are a collection of cells that recognize various cancer antigen peptides, the pharmaceutical preparation may be used in combination with, for example, a CA9 antigen-derived peptide or a VEGFR1-derived peptide. .

  There is no restriction | limiting in particular as a dosage form of this pharmaceutical formulation, A well-known dosage form can be selected suitably, For example, a solid agent, a semi-solid agent, a liquid agent etc. are mentioned. Pharmaceutical preparations of these dosage forms can be produced according to a conventional method.

  This pharmaceutical preparation prevents cancer as an inducer to cancer reactive cytotoxic T cells, particularly renal cell carcinoma reactive cytotoxic T cells, or as an inducer to antigen-presenting cells of CD8 positive T cells.・ It may be used for treatment.

  This pharmaceutical preparation is preferably used as a cancer vaccine.

  This pharmaceutical preparation is used for the prevention or treatment of cancer, particularly renal cell carcinoma, and there are no particular restrictions on the administration method, dose, administration timing, administration interval, and administration target, and it can be selected appropriately according to the purpose. .

  This pharmaceutical preparation is preferably administered together with an adjuvant that has been conventionally used for vaccine administration so that an immune response is effectively established. Examples of the administration method include intradermal administration and subcutaneous administration. Etc.

  The dosage is not particularly limited and may be appropriately selected in consideration of the disease state, age, weight, constitution, presence / absence of administration of a pharmaceutical preparation containing other ingredients as active ingredients, etc. it can.

  There is no restriction | limiting in particular as this dose when this pharmaceutical formulation contains the said peptide, According to the objective, it can select suitably, For example, 0.0001 mg-1,000 mg are illustrated as a peptide amount.

  There is no restriction | limiting in particular also as this dosage in case this pharmaceutical formulation contains the said nucleic acid molecule or the said vector, According to the objective, it can select suitably, For example, 0.1 microgram-100 mg etc. are mentioned as DNA amount. It is done. Examples of the administration method include intravenous injection, subcutaneous administration, and intradermal administration.

  The administration time and administration interval are not particularly limited and may be appropriately selected depending on the purpose. For example, the administration may be performed once, once every several days, or once every several weeks to several months. Administration for 1 to 3 years.

  There is no restriction | limiting in particular as said administration object, According to the objective, it can select suitably, For example, a human is mentioned.

[Antigen-presenting cell of the present invention]
Details of the antigen-presenting cell to which the present invention is applied are as follows.

  The antigen-presenting cell is derived by in vitro introduction of the peptide and / or the vector into a cell having antigen-presenting ability collected from a patient with HLA-A24-positive renal cell carcinoma, A complex of the peptide and HLA-A24 is presented on the cell surface.

  In addition, the said pharmaceutical formulation which has the said peptide is administered to a patient, especially a renal cell carcinoma patient, The treatment which induces | guides | derives an antigen presentation cell so that the complex of the said peptide and HLA-A24 may be shown on the cell surface. As a method, it may be used.

[Method for inducing antigen-presenting cells]
In the method for inducing the antigen-presenting cell, the peptide and / or the vector is brought into contact with an antigen-presenting cell collected from a patient with HLA-A24-positive renal cell carcinoma by a contacting step. The complex of the peptide and HLA-A24 is presented on the cell surface. The method for inducing the antigen-presenting cell includes, for example, at least a step of introducing a peptide and / or the vector by a contacting step, and further includes other steps as necessary. More specifically, in the contact step, the collected cells having antigen-presenting ability may be directly contacted in vitro, or may be contacted by gene transfer, in the presence of the peptide. The cells may be cultured. By the contact process, it is induced into antigen-presenting cells. This method includes at least a contact step, and may further include other steps as necessary. Other steps are not particularly limited as long as the effects of the present invention are not impaired, and can be appropriately selected depending on the purpose.

  The antigen-presenting cell that presents on the cell surface the complex of the peptide and HLA-A24 molecule prepared by the contacting step induces cytotoxic T cells that damage HLA-A24 molecule-positive renal cell carcinoma cells. obtain.

  In the method of inducing antigen-presenting cells, an antigen-presenting cell-inducing agent containing only the peptide as an active ingredient or further containing other ingredients as required is used as a pharmaceutical preparation. In the method for inducing antigen-presenting cells, only the vector may be used as an active ingredient, or an antigen-presenting cell-inducing agent containing other components as necessary may be used as a pharmaceutical preparation.

  Among the methods for inducing antigen-presenting cells, the method for introducing at least one of the peptide and the vector into cells having the antigen-presenting ability is not particularly limited and can be appropriately selected according to the purpose.

  The antigen-presenting cells can be obtained, for example, by culturing cells having antigen-presenting ability derived from HLA-A24 molecule positive renal cell carcinoma patients together with the peptide. Alternatively, for example, the vector can be obtained by introducing the vector into a cell having antigen-presenting ability derived from an HLA-A24 molecule-positive renal cell carcinoma patient and expressing the peptide.

  There is no restriction | limiting in particular as a cell which has the said antigen presentation ability, According to the objective, it can select suitably, For example, a dendritic cell etc. are mentioned. The method for preparing the dendritic cells is not particularly limited and can be appropriately selected according to the purpose. For example, cells that adhere to a culture plate are isolated from peripheral blood mononuclear cells collected from a patient, and the cells For example, in the presence of interferon (IL) -4 and granulocyte monocyte colony stimulating factor (GM-CSF).

  The antigen-presenting cells obtained by the method for preparing antigen-presenting cells are, for example, returned to the body of an individual from which peripheral blood mononuclear cells have been collected, and promote induction of renal cell carcinoma reactive cytotoxic T cells in the body. It can be preferably used for suppressing renal cell carcinoma.

[Antigen-presenting cell inducer]
This antigen-presenting cell inducer contains at least the peptide and / or the vector, and further contains other components as necessary. The said peptide and the said vector may be used individually by 1 type, and may use 2 or more types together. Moreover, about each of the said peptide and the said vector, you may use individually by 1 type and may use 2 or more types together. This antigen-presenting cell inducer is for preparing antigen-presenting cells that present a complex of the peptide and the HLA-A24 molecule on the cell surface.

  The content of at least one of the peptide and the vector in the antigen-presenting cell inducer is not particularly limited and can be appropriately selected depending on the purpose. The antigen-presenting cell inducer may be composed of at least one of the peptide and the vector.

  Other components in the antigen-presenting cell inducer are not particularly limited as long as the effects of the present invention are not impaired, and can be appropriately selected according to the purpose. Examples thereof include a medium and a buffer solution. The said other component may be used individually by 1 type, and may use 2 or more types together. There is no restriction | limiting in particular as content of the said other component in the said antigen presentation cell inducer, According to the objective, it can select suitably.

[Antigen-presenting cell induction kit]
The method for inducing antigen-presenting cells may be performed using a kit for inducing antigen-presenting cells. This antigen-presenting cell induction kit is an antigen-presenting cell induction kit for preparing an antigen-presenting cell that presents a complex of the peptide and HLA-A24 molecule on the cell surface, and the renal cell carcinoma reactivity The other elements including at least a cytotoxic T cell inducer and further including other elements as necessary are not particularly limited as long as the effects of the present invention are not impaired, and may be appropriately selected according to the purpose. Examples thereof include a medium and a buffer solution. The said other elements may be used individually by 1 type, and may use 2 or more types together. The kit for inducing antigen-presenting cells can be suitably used for the method for preparing antigen-presenting cells described above.

  The antigen-presenting cells obtained by the method for inducing antigen-presenting cells are, for example, returned to the body of the individual from which the cells having antigen-presenting ability were collected, and in the body by the antigen-presenting cells, renal cell carcinoma reactive cytotoxicity T It can be suitably used for promoting cell induction and suppressing renal cell carcinoma.

[Renal cell carcinoma reactive cytotoxic T cells of the present invention]
Details of the renal cell carcinoma reactive cytotoxic T cells to which the present invention is applied are as follows.

  This renal cell carcinoma reactive cytotoxic T cell is derived by bringing the peptide into contact with peripheral blood mononuclear cells collected from an HLA-A24 positive renal cell carcinoma patient.

  The pharmaceutical preparation having the peptide is administered to a patient, particularly a renal cell carcinoma patient, and contacted with peripheral blood mononuclear cells in vivo to induce renal cell carcinoma reactive cytotoxic T cells. It may be used as a treatment method.

[Method of Inducing Renal Cell Carcinoma Reactive Cytotoxic T Cells]
The method of inducing renal cell carcinoma reactive cytotoxic T cells comprises contacting the peptide with peripheral blood mononuclear cells collected from a patient with HLA-A24-positive renal cell carcinoma by a contact step, It induces renal cell carcinoma reactive cytotoxic T cells. The method for inducing renal cell carcinoma reactive cytotoxic T cells may be, for example, directly contacting in vitro with cells collected from peripheral blood mononuclear cells by the contact step, The cells may be cultured in the presence of the peptide. The contacting step can induce cytotoxic T cells that damage HLA-A24 molecule-positive renal cell carcinoma cells. This method includes at least a contact step, and may further include other steps as necessary. Other steps are not particularly limited as long as the effects of the present invention are not impaired, and can be appropriately selected depending on the purpose.

  The cytotoxic T cell is “renal cell carcinoma responsive” means that the cytotoxic T cell recognizes a complex of a cancer antigen peptide on the renal cell carcinoma cell and an HLA molecule, and the cell. It has the ability to injure.

  In the method of inducing renal cell carcinoma reactive cytotoxic T cells, renal cell carcinoma reactive cytotoxic T cells are induced by using only the peptide as an active ingredient or, if necessary, other ingredients. Pharmaceutical preparations that are agents are used.

  The obtained renal cell carcinoma reactive cytotoxic T cell is returned to the body of an HLA-A24-positive renal cell carcinoma individual from which peripheral blood mononuclear cells have been collected, for example, to suppress renal cell carcinoma. Can be suitably used.

[Renal cell carcinoma reactive cytotoxic T cell inducer]
This renal cell carcinoma reactive cytotoxic T cell inducer contains at least the peptide, and further contains other components as necessary. The said peptide may be used individually by 1 type, and may use 2 or more types together.

  There is no restriction | limiting in particular as content of the said peptide in the said renal cell carcinoma reactive cytotoxic T cell inducer, According to the objective, it can select suitably. The renal cell carcinoma reactive cytotoxic T cell inducer may consist of only the peptide.

  The other components in the renal cell carcinoma reactive cytotoxic T cell inducer are not particularly limited as long as the effects of the present invention are not impaired, and can be appropriately selected according to the purpose. Liquid and the like. The said other component may be used individually by 1 type, and may use 2 or more types together. There is no restriction | limiting in particular as content of the said other component in the said renal cell carcinoma reactive cytotoxic T cell inducer, According to the objective, it can select suitably.

[Kit for inducing renal cell carcinoma reactive cytotoxic T cells]
The method for inducing renal cell carcinoma reactive cytotoxic T cells may be performed using a kit for inducing renal cell carcinoma reactive cytotoxic T cells. This kit for inducing renal cell carcinoma reactive cytotoxic T cells includes at least the renal cell carcinoma reactive cytotoxic T cell inducer, and further contains other elements as necessary. The other elements are not particularly limited as long as the effects of the present invention are not impaired, and can be appropriately selected according to the purpose. Examples thereof include a medium and a buffer solution. The said other elements may be used individually by 1 type, and may use 2 or more types together. The kit for inducing renal cell carcinoma reactive cytotoxic T cells can be suitably used for the method for inducing renal cell carcinoma reactive cytotoxic T cells.

  The renal cell carcinoma reactive cytotoxic T cells obtained by the method for inducing renal cell carcinoma reactive cytotoxic T cells are, for example, returned to the body of the individual from whom the peripheral blood mononuclear cells were collected. It can be suitably used for adoptive immunotherapy that damages the skin.

[Biologic of the present invention]
A biologic to which the present invention is applied contains induced cells composed of the antigen-presenting cells and / or renal cell carcinoma reactive cytotoxic T cells, and is administered to patients, particularly renal cell carcinoma patients. Used in cancer prevention and treatment.

[Pharmaceutical preparation of the present invention / antigen-presenting cell / method of preventing or treating cancer using a biological preparation containing renal cell carcinoma reactive cytotoxic T cells]
The peptide, the nucleic acid molecule, the vector, and a pharmaceutical preparation containing any of them can be administered to an individual to prevent the onset of cancer in the individual, particularly renal cell cancer, or to suffer from renal cell cancer. Individuals can be treated. Therefore, a method for preventing or treating cancer, particularly renal cell carcinoma, using this pharmaceutical preparation is performed by administering to the individual at least one of the peptide and the vector.

  In addition, the method for preventing or treating renal cell carcinoma may be performed by administering a biological preparation containing the renal cell carcinoma reactive cytotoxic T cells and antigen-presenting cells prepared by induction to an individual.

  Hereinafter, embodiments to which the present invention is applied will be described in detail.

(Test Example 1: Search from binding motif of HIF-1α-derived peptide)
First, in order to increase the options for peptide vaccine therapy, HIF-1α-derived peptides useful for cancer vaccine therapy for HLA-A24 positive renal cell carcinoma patients were searched. Specifically, the inventors searched for a peptide derived from HIF-1α and capable of binding to the HLA-A24 molecule and recognized by cellular immunity.

  Parker KC, Bednarek MA, Coligan JE. “Scheme for ranking potential HLA-A2 binding peptides based on independent binding of individual peptide side-chains.”, Journal of Immunology 1994 (152), p.163-175, and Abiko K, Mandai M, Hamanishi J, Yoshioka Y, Matsumura N, Baba T, et al. “PD-L1 on tumor cells is induced in ascites and promotes peritoneal dissemination of ovarian cancer through CTL dysfunction.”, Clinical Cancer Research 2012 (19), Based on the description of p.1363-1374, computer analysis was performed based on the binding motif for the HLA-A24 allele. The outline is abbreviated. Using BIMAS, five types of peptides with the highest binding scores were selected based on the binding scores calculated from HLA class I molecules based on the predicted half-life of dissociation. It is predicted that the higher the binding score, the more strongly the binding motif is expressed and the activity is expressed by computer analysis. The calculation results are shown in Table 1.

(Test Example 2: Induction of cytotoxic T cells from peripheral blood mononuclear cells)
It was investigated whether the five types of HIF-1α-derived peptides in Table 1 could induce peptide-specific cytotoxic T cells from peripheral blood mononuclear cells of HLA-A24-positive renal cell carcinoma patients.

[Samples from donors of patients and healthy individuals]
Peripheral blood mononuclear cells for this study were obtained from donors of renal cell carcinoma patients and healthy individuals who had their consent submitted. Donors included HLA-A24 positive patients, but no human immunodeficiency virus (HIV) infection. 30 ml of peripheral blood was collected, and peripheral blood mononuclear cells were prepared by Ficoll-Conley density gradient centrifugation. All samples were stored frozen until use. This test was conducted with the approval of the Kinki University Research Ethics Review Board.

[Cell line]
C1R-A24 is a C1R lymphoma subline cell line that stably expresses the HLA-A ^* 24: 02 gene. SKRC-1 cells, SKRC-44 cells, and SKRC-52 cells are all RCC cell lines, and were readily provided by Dr. Kazuhiro Yoshikawa of Aichi Medical University. KPK-13 cells and KK-RCC6 cells were kindly provided by Dr. Hiromitsu Mimata of Oita University School of Medicine and Dr. Yoshiyuki Tsuji of Kagawa University Graduate School of Medicine, respectively. LNCaP is a human prostate cancer cell line, and LNCaP-A24 is a stable transfectant expressing HLA-A24 ⁽¹⁾ . All RCC cell lines were clear cell carcinomas and were cultured in 10% fetal calf serum (FCS) using RPMI 1640 medium (Invitrogen).
(1) Yao A, Harada M, Matsueda S, et al. Identification of parathyroid hormone-related protein-derived peptides immunogenic in human histocompatibility leukocyte antigen-A24 ⁺ prostate cancer patients. Brit J Cancer, 91: 287-296, 2004.

〔peptide〕
Five types of peptides in Table 1 were used. Epstein-Barr virus (EBV) -derived peptide (single letter notation TYGPVFMSL: SEQ ID NO: 6) and human immunodeficiency virus (HIV) -derived peptide (same notation RYLRRDQQLL: SEQ ID NO: 7) are combined into HLA-A24 molecule. Used as a bound control peptide. In addition, all the peptides of the five types of peptides in Table 1 and the control group peptides were purchased from Eurofin Genomics Co., Ltd., dissolved in dimethyl sulfoxide (DMSO) and used as a 10 mg / ml solution. .

[Method for inducing peptide-specific cytotoxic T cells from peripheral blood mononuclear cells]
Which of the five HIF-1α-derived peptides in Table 1 prepared based on the binding motif for the HLA-A24 molecule is a peptide-specific cell from a peripheral blood mononuclear cell collected from a patient with HLA-A24-positive renal cell carcinoma It was determined whether to induce cytotoxic T cells. The method for detecting peptide-specific cytotoxic T cells using these five types of HIF-1α-derived peptides as test peptides and EBV-derived peptides as control peptides is described in Brahmer JR, Tykodi SS, Chow LQM, Hwu WJ, Topalian SL, Hwu P, et al. “Safety and activity of anti-PD-L1 antibody in patients with advanced cancer.”, The New England Journal of Medicine, 2012 (366), p.2455-2464 Improved.

Peripheral blood mononuclear cells (5 × 10 ⁴ cells / well) were mixed with 10 μg / ml of each HIF-1α-derived peptide in a U-bottom 96-well (well) microculture plate (Nunc). Incubated in. The culture medium is 45% RPMI 1640 medium (Invitrogen), 45% AIM-V medium (Gibco), 10% fetal calf serum, MEM non-essential amino acid solution (GibcoBRL) 100-fold dilution, and Consists of 1% penicillin / streptomycin. Eight wells were used for each peptide. The day after the start of the culture, 100 μl of culture medium containing 50 U / ml IL-2 was added. On day 8 of culture, 100 μl of medium was removed and supplemented with 100 μl of fresh medium containing the corresponding HIF-1α-derived peptide (10 μg / ml). On day 9 of culture, medium containing 50 U / ml IL-2 100 μl was added. On the 17th day of culturing, the cells cultured in a new medium were separated from 1 well to 4 wells. Two wells are for C1R-A24 cells pulsed with the corresponding HIF-1α-derived peptide, and the other two wells are for C1R-A24 cells pulsed with the HIV peptide (control). After 18 hours of incubation, supernatants were collected and IFN-γ production levels were measured by ELISA. The induction of peptide-specific cytotoxic T cells was judged to be active only when the following two criteria were satisfied. Criterion (i) is that both wells stimulated with the corresponding HIF-1α peptide are 50 pg / ml or more, and criterion (ii) is that the two wells stimulated with each corresponding HIF-1α peptide are It is 1.2 times or more than the average value of two wells stimulated with the control HIV peptide.

  Thus, C1R-A24 in which peripheral blood mononuclear cells were stimulated in vitro with each of the HIF-1α-derived peptides or stimulated with EBV-derived peptides, and the peripheral blood mononuclear cells were pulsed with the corresponding peptides. Whether or not IFN-γ was produced in response to cells was examined. Table 2 below shows the induction results of peptide-specific cytotoxic T cells from peripheral blood mononuclear cells of HLA-A24-positive renal cell carcinoma patients.

As shown in Table 2, each HIF-1α-derived peptide of HIF-1α _37-46 , HIF-1α _65-73 , HIF-1α _91-99 , HIF-1α _118-127, and HIF-1α _278-287 is Among 12 samples from 12 HLA-A24 positive renal cell carcinoma patients, 2 samples, 1 sample, 2 samples, 2 samples and 4 samples respectively induced corresponding peptide-reactive cytotoxic T cells from peripheral blood mononuclear cells. . As a control, EBV peptide induced peptide-specific cytotoxic T cells from peripheral blood mononuclear cells in 3 samples out of 12 samples from the same patient. The HIF-1α _278-287 peptide (corresponding to SEQ ID NO: 5) had a high activity expression ratio of 4/12 producing IFN-γ. Based on this finding, it was _judged that this HIF-1α _278-287 peptide is most likely to produce peptide-specific cytotoxic T cells from peripheral blood mononuclear cells of HLA-A24-positive renal cell carcinoma patients. . In addition, it turned out that the thing with a high binding score calculated from the HLA class I molecule based on the estimated half-life of dissociation is not high in activity.

(Test 3: Flow cytometry (FACS) analysis for HIF-1α expression study)
Prior to performing the cytotoxic activity test, the expression of HIF-1α in the RCC cell line was examined. The cells were stained with polyethylene (PE) -carrying anti-HIF-1α monoclonal antibody (manufactured by eBioscience). Isotype-matched PE-loaded mouse IgG (BD Bioscience) was used as a control. In order to examine the expression of the HLA-A24 molecule, the cells were stained with an anti-HLA-A24 monoclonal antibody (manufactured by One Lambda) followed by a FITC-carrying goat anti-mouse IgG (H + L) antibody (manufactured by KPL). Analysis was carried out using FACS Calibur (manufactured by BD Biosciences). The result is shown in FIG.

FIG. 1 shows the expression of HIF-1α in an RCC (renal cell carcinoma) cell line to which the present invention is applied. FIG. 1A shows RCC cancer cell line and LNCaP (human prostate cancer) cell line cultured in the presence or absence of DFO (deferoxamine) for 24 hours, and immunoblotting the expression of HIF-1α in whole cell lysate. Shows the result of the test. β-actin is a control example. FIG. 1B shows that KPK-13, KK-RCC6 and LNCaP cell lines were cultured under normoxic conditions (20% O ₂ ) or hypoxic conditions (1% O ₂ ) for 24 hours in whole cell lysates and The result of having tested the expression of HIF-1 (alpha) in a nucleic acid extract by the immunoblotting is shown. β-actin and E2F-1 are control examples. FIG. 1C shows cell surface expression of HLA-A24 molecule analyzed by flow cytometry. The white background shows the result of staining without using the anti-HLA-A24 monoclonal antibody.

As a result, as shown in FIG. 1A, SKRC-44 cells were strongly HIF-1α positive even under 20% O ₂ normoxia conditions, and HIF− in KPK-13 and KK-RCC6 cells. 1α expression was low. When cancer cells are treated with DFO, DFO is an iron chelator and may stabilize HIF-1α against proteolysis ⁽²⁾ , and all RCC cell lines HIF-1α expression was increased in whole cell lysates. The same was true for LNCap (human prostate cancer). Furthermore, as shown in FIG. 1B, HIF-1α expression was increased in whole cell lysates of KPK-13 and KK-RCC6 under hypoxic conditions of 1% O ₂ . For expression in their cell nuclei, HIF-1α expression was detected in all three cell lines, whether under normoxic or hypoxic conditions. Considering that HIF is proteasome-dependently degraded ⁽³⁾ , and considering that HLA class I molecules and processed peptides are presented on the cell surface ⁽⁴⁾ , these results indicate that HIF- It suggests that 1α is reliably processed in the RCC cell line and that peptides derived from HIF-1α can be presented with HLA-A24 molecules on the cell surface. On the other hand, as shown in the flow cytometry of FIG. 1C, KPK-13 cells expressed HLA-A24 molecules, and KK-RCC6 cells were negative for these molecules. In addition, we tested LNCaP and LNCaP-A24 (LNCaP transfectants expressing HLA-24 (LNCaP transfectants prior to testing HLA-24-restricted RCC-reactive T cells derived from peptides ^{). 1)} The expression of HLA-A24 molecule on it was tested.
(1) Yao A, Harada M, Matsueda S, et al. Identification of parathyroid hormone-related protein-derived peptides immunogenic in human histocompatibility leukocyte antigen-A24 ⁺ prostate cancer patients. Brit J Cancer, 91: 287-296, 2004.
(2) Triantafyllou A, Liakos P, Tsakalof A, Georgatsou E, Simos G, Bonanou S. Cobalt induces hypoxia-inducible factor-1alpha (HIF-1alpha) in HeLa cells by an iron-independent, but ROS-, PI-3K -and MAPK-dependent mechanism.Free Radic Res, 40: 847-56, 2006.
(3) Cockman ME, Masson N, Mole DR, et al. Hypoxia inducing factor-alpha binding and ubiquitylation by the von Hipple-Lindau tumor suppressor protein.J Biol Cehm, 275: 25733-41, 2000.
(4) Rammensee HG, Flak K, Rotzschke O. Peptides naturally presented by MHC class I molecules.Annu Rev Immunol 1993; 11: 213-244.

(Test 4: Cytotoxic activity test of T cells stimulated with HIF-1α _278-287 peptide against HLA-A24 ⁺ RCC cells)
Next, cytotoxic T cells induced by stimulation with HIF-1α _278-287 peptide in vitro were HLA-A24 positive cells (KPK-13) and HLA-A24 negative cells (KK-RCC6), and prostate cancer. It was measured whether it showed cytotoxic activity with respect to a derived cell line cell (LNCaP) and HLA-A24 positive LNCaP cell (LNCaP-A24). Peripheral blood mononuclear cells from 7 HLA-A24 positive renal cell carcinoma patients were stimulated in vitro with HIF-1α _278-287 peptide, and after CD8 positive T cell purification, cytotoxic activity against each cell Was examined.

Specifically, all or purified CD8 positive T cells were used as effector cells in order to perform cytotoxic activity test of cytotoxic T cells by standard 5 hour ⁵¹ Cr release assay (release test). CD8 positive T cells were prepared as a negative group from peptide-stimulated peripheral blood mononuclear cells as follows. Peptide-stimulated peripheral blood mononuclear cells are first stained with an anti-CD56 monoclonal antibody (mIgG1: DIACLONE, manufactured by Besankov Cedex), followed by Dynabeads anti-mouse IgG (manufactured by Invitrogen), Dynabeads anti-CD19 (manufactured by Invitrogen), and Dynabeads. Treated with anti-CD4 (Invitrogen). Dynabeads binding cells were magnetized and negatively separated. After removing CD4 positive T cells, B cells, and NK cells, the percentage of CD8 positive T cells was approximately 80-85% during the test (data not shown). Target cells were cultured under 1% O ₂ for 24 hours immediately before the cytotoxicity test.

As target cells, KPK-13 cells, which are HLA-A24 molecule-positive renal cell carcinoma cell lines, and KK-RCC6 cells, which are HLA-A24 molecule-negative renal cell carcinoma cell lines, are used, or HLA-A24 is expressed. LNCaP-A24 cells, which are human prostate cancer cell lines, and LNCaP cells that are HLA-A24 molecule negative, and the cytotoxic activity of the CD8-positive T cells (also referred to as effector cells) against these is as follows: And tested. In a round bottom type 96-well plate, 2000 ⁵¹ Cr target cells per well were cultured together with effector cells at a predetermined effector cell / target cell ratio. ⁵¹ Cr release was calculated according to the following equation.
Specific lysis (%) = (release in subject-spontaneous release) x 100 / (maximum release-spontaneous release)
“Spontaneous release” is determined from the supernatant when ⁵¹ Cr-labeled target cells are incubated without effector cells, and “maximum release” is 1% Triton X (made by Wako Pure Chemical Industries, Ltd.) without effector cells. ) And ⁵¹ Cr-labeled target cells.

The statistical significance of the cytotoxic activity test was judged using a two-sided student t test. If the P value was less than 0.05, it was regarded as statistically significant ( ^* P <0.05). Only the results of positive cytotoxic activity tests are summarized in FIG. In FIG. 2, the vertical axis indicates lysis (%), and the horizontal axis indicates the ratio of effector cells to target cells (E / T: effector cells / target cells).

FIG. 2A1 shows cells against the four different targets by 5 hour ⁵¹ Cr release test for purified CD8 positive T cells from HIF-1α _278-287 peptide stimulated peripheral blood mononuclear cells of HLA-A24 positive renal cell carcinoma patients. It is a test result of injury activity. Thereafter, peripheral blood mononuclear cells from HLA-A24-positive renal cell carcinoma patients are limited in availability, so peripheral blood mononuclear cells were obtained from additional HLA-A24-positive renal cell carcinoma patients. Similarly, additional cells were obtained for LNCaP-A24 cells, which are human prostate cancer cell lines expressing HLA-A24, and LNCaP cells that are HLA-A24 molecule negative. In these studies, peripheral blood mononuclear cells stimulated in vitro were negatively purified as CD8 positive cells prior to cytotoxicity testing. FIG. 2B1 shows the cells against the four different targets according to the 5-hour ⁵¹ Cr release test for purified CD8 positive T cells from HIF-1α _278-287 peptide stimulated peripheral blood mononuclear cells of HLA-A24 positive renal cell carcinoma patients. It is a test result of injury activity. That is, FIG. 2B1 can be said to be the reproducibility test of FIG. 2A1.

As is apparent from FIG. 2A1, CD8 positive T cells derived from HIF-1α _278-287 peptide-stimulated peripheral blood mononuclear cells from HLA-A24 positive renal cell carcinoma patients are more responsive to KPK− than to KK-RCC6 cells. The level of cytotoxic activity was higher for 13 cells. Similarly, CD8 positive T cells derived from HIF-1α _278-287 peptide stimulated peripheral blood mononuclear cells from HLA-A24 positive renal cell carcinoma patients are LNCaP, a human prostate cancer cell line expressing HLA-A24. -The level of cytotoxic activity was higher for A24 cells than for LNCaP cells negative for HLA-A24 molecules.

Furthermore, CD8-positive T cells derived from HIF-1α _278-287 peptide-stimulated peripheral blood mononuclear cells from HLA-A24-positive renal cell carcinoma patients were also more _effective against KPK-13 cells than other target cells. However, the cytotoxic activity level was higher for LNCaP-A24 cells, and the reproducibility of this test was confirmed (see FIG. 2B1).

These results indicate that the HIF-1α _278-287 peptide has the ability to induce cytotoxic T cells specifically against HLA-A24 positive HIF-1α _278-287 expressing RCC cells. Therefore, it is shown that peripheral blood mononuclear cells stimulated with these HIF-1α _278-287- derived peptides or equivalent derivatives thereof exert cytotoxic activity against renal cell carcinoma cells in an HLA-A24-restricted manner. . In addition, these HIF-1α _278-287- derived peptides or their equivalent derivatives can induce renal cell carcinoma reactive cytotoxic T cells in HLA-A24 positive renal cell carcinoma patients, and can be used for specific immunotherapy, particularly cancer vaccine therapy. Indicates usefulness.

(Test 5: Cold inhibition test of T cells stimulated with HIF-1α _278-287 peptide against HLA-A24 ⁺ RCC cells)
The peptide-dependent cytotoxic activity of peptide-stimulated CTL (cytotoxic T cells) was examined by cold inhibition test. The cold inhibition test was performed as follows.

In a round-bottom 96-well plate, ⁵¹ Cr-labeled target cells (KPK-13 cells; 2,000 cells / well) and the above-described effector cells (2 × 10 ⁴ cells / well) were added to any one of the following ⁵¹ Cr. The cells were cultured with unlabeled cold target cells (4 × 10 ⁴ cells / well), and specific lysis (%) was determined in the same manner as in Test Example 4. As a control, specific lysis (%) was determined in the same manner even when the cold target cells not labeled with ⁵¹ Cr were not used. The results are shown in FIGS. A2 and B2.
- ⁵¹ Cr unlabeled Cold Target cells -
(1) C1R-A24 cells pulsed with the HIV-derived peptide (2) C1R-A24 cells pulsed with the HIF-1α _278-287 peptide In this study, prepared from patients with HLA-A24 molecule-positive renal cell carcinoma Effector cells obtained using peripheral blood mononuclear cells (PBMC) were examined.

The horizontal axis of FIGS. A2 and B2 indicates “specific lysis (%)” (% lysis), and the vertical axis indicates, in order from the top, “control” (none) as a cold target cell not labeled with ⁵¹ Cr. When “C1R-A24 cells pulsed with HIV-derived peptides” are used (C1R-A24 / HIV), “C1R-A24 cells pulsed with HIF-1α _278-287 peptide” as cold target cells not labeled with ⁵¹ Cr The result when (C1R-A24 / HIF-1 ₂₇₈ ) is used is shown.

As shown in FIG. A2, the cytotoxic activity of PBMCs derived from HLA-A24 molecule positive patients stimulated with HIF-1α _278-287 peptide against KPK13 cells was unlabeled with HIF-1α _278-287 peptide pulsed. Although significantly suppressed by addition of C1R-A24 cells, it was not suppressed by unlabeled C1R-A24 cells pulsed with HIV-derived peptides. Similarly, the same result was reproduced also about the cytotoxic activity with respect to KPK13 cell of PBMC derived from the added HLA-A24 molecule positive patient (FIG. B2). The results of this cold inhibition test indicate that the peptide-stimulated PBMC has a high cytotoxicity against the HLA-A24 molecule-positive renal cell carcinoma cell line due to peptide-specific CD8-positive T cells.

  The above results show that the HIF-1α-derived peptide of the present invention can induce renal cell carcinoma reactive cytotoxic T cells in HLA-A24 molecule-positive renal cell carcinoma, and is useful for specific immunotherapy, particularly cancer vaccine therapy. It shows that. It is believed that the HIF-1α-derived peptide of the present invention can increase the options for peptide vaccine therapy.

  The HIF-1α peptide of the present invention is useful as an active ingredient of a pharmaceutical preparation such as a peptide-derived anticancer vaccine for HLA-A24-positive renal cell carcinoma patients. These HIF-1α peptides can help treatment or prevention such as peptide-derived anticancer vaccines for renal cell carcinoma patients. Since the expression of HIF-1α is widely observed in various types of epithelial cancers, these HIF-1α peptides can treat or prevent not only renal cell carcinoma but also other types of malignant tumors. Applicable to.

  The HIF-1α-derived peptide of the present invention or an equivalent derivative thereof, a nucleic acid molecule encoding the peptide and a vector thereof, a pharmaceutical preparation used for the prevention or treatment of renal cell carcinoma, and a complex of the peptide and HLA-A24 Biological preparations containing antigen-presenting cells presented on the cell surface or renal cell carcinoma reactive cytotoxic T cells are suitable for the prevention or treatment of renal cell carcinoma, particularly in patients with HLA-A24-positive renal cell carcinoma. It shows that it can induce cell cancer reactive cytotoxic T cells and is useful for specific immunotherapy, particularly cancer vaccine therapy.

  The present invention has expanded the options for peptide-based immunotherapy, particularly cancer vaccine therapy, for HLA-A24 positive renal cell carcinoma patients. According to the present invention, the therapeutic effect is further improved by adding HIF-1α, which is considered to be involved in cancer growth, in the treatment of HLA-A24 positive renal cell carcinoma patients for which several cancer vaccine candidate peptides have been identified to date. It is expected to contribute to

Claims (11)

  It consists of a skeleton represented by either the amino acid sequence of SEQ ID NO: 5 and a modified amino acid sequence in which one or two amino acids are deleted, substituted and / or added, and is recognized by cellular immunity. A peptide characterized by that.   The peptide according to claim 1, wherein the peptide is recognized by the cellular immunity of renal cell carcinoma by binding to human leukocyte antigen-A24 molecule.   The peptide according to claim 2, which is a programmed death ligand 1-inducing peptide.   Containing as an active ingredient a peptide represented by at least one of the amino acid sequence of SEQ ID NO: 5 and a modified amino acid sequence in which one or two amino acids are deleted, substituted and / or added. A pharmaceutical preparation characterized.   The pharmaceutical preparation according to claim 4, which is an inducer for renal cell carcinoma reactive cytotoxic T cells.   The pharmaceutical preparation according to claim 4, which is an inducer of antigen-presenting cells of CD8-positive T cells.   The pharmaceutical preparation according to any one of claims 4 to 6, which is a therapeutic or prophylactic agent for human leukocyte antigen-A24 positive renal cell carcinoma.   The pharmaceutical preparation according to any one of claims 4 to 7, wherein the active ingredient is the peptide and a cancer vaccine.   A peptide represented by at least one of the amino acid sequence of SEQ ID NO: 5 and a modified amino acid sequence in which one or two amino acids of these sequences are deleted, substituted and / or added is human leukocyte antigen-A24 positive The present invention is characterized in that it is induced by introduction into a cell having antigen-presenting ability collected from a patient with renal cell carcinoma and presenting the complex of the peptide and human leukocyte antigen-A24 on the cell surface. Antigen-presenting cells.   A peptide represented by at least one of the amino acid sequence of SEQ ID NO: 5 and a modified amino acid sequence in which one or two amino acids of these sequences are deleted, substituted and / or added is human leukocyte antigen-A24 positive A renal cell carcinoma reactive cytotoxic T cell, which is derived from contact with peripheral blood mononuclear cells collected from a patient with renal cell carcinoma. A peptide represented by at least one of the amino acid sequence of SEQ ID NO: 5 and a modified amino acid sequence in which one or two amino acids of these sequences are deleted, substituted and / or added is human leukocyte antigen-A24 positive Induced cells consisting of antigen-presenting cells introduced on peripheral blood mononuclear cells collected from patients with renal cell carcinoma and presenting the complex of the peptide and human leukocyte antigen-A24 on the cell surface ,
It contains at least one of induced cells selected from induced cells composed of renal cell carcinoma reactive cytotoxic T cells induced by contacting the peptide with the collected cells. Biologics.