JP6784964B2 - EZH2-derived peptide useful for cancer vaccine therapy for HLA-A3 supertype allele-positive prostate cancer patients - Google Patents

Description

The present invention relates to EZH2-derived peptides useful for cancer vaccine therapy useful for the treatment or prevention of HLA-A3 supertype allele-positive prostate cancer patients.

Prostate cancer is a common cancer in older men. Androgen depletion therapy is transiently successful for prostate cancer, but there is no effective treatment for recurrence as castration-resistant or bone metastatic prostate cancer. Specific immunotherapy may be a promising option for such patients. To date, many cancer antigenic peptides that can be used for specific immunotherapy for prostate cancer patients have been identified. However, since the frequency of HLA (human leukocyte antigen: also referred to as human leukocyte antigen) -A2 and -A24 allele (allele: hereinafter also referred to as allele) is high worldwide, the conventional cancer antigen peptide is HLA-A2. Or, most of them were for patients who were positive for the -A24 allele (positive is sometimes referred to as " ⁺ ").

For HLA class I alleles, HLA-A2, -A3, -B7 and -44 supertype alleles have been proposed based on their structural homology and peptide bond motif analysis. Of these, HLA-A3 supertype alleles are found in 38% of Caucasians, 53% of Chinese, 46% of Japanese, and 43% of North American African Americans and Hispanics. Nevertheless, the cancer antigen peptide vaccines that can be used to treat patients with HLA-A3 supertype allergen-positive prostate cancer are limited (for example, the peptide derived from a prostate-related protein disclosed in Patent Document 1, disclosed in Patent Document 2). SART3-derived peptides and Lck-derived peptides disclosed in Patent Document 3).

On the other hand, reported examples of EZH2-derived peptides that can be used in peptide-based anticancer vaccines for cancer patients having the HLA-A2 or -A24 molecule can be found in Patent Document 4 and Non-Patent Document 1, etc. ..

However, there have been no cases in which EZH2-derived peptides have been used as vaccines for HLA-A3 supertype allele ⁺ prostate cancer patients.

Based on the experience of vaccine therapy with the peptide vaccine, the immune response (for example, CTL response) is diverse depending on the variety of immune status of individual patients and the variety of cancers to be treated, and cases that were once effective. Was also found to be ineffective by immune escape or tolerance. In view of this evidence, hosts that target multiple molecules based on the development, differentiation, and proliferation of prostate cancer, rather than targeting a single fixed antigen, and carry cancers of different properties. It seems that tailor-made vaccine therapy suitable for the patient is needed.

Therefore, there is a strong demand for the development of peptides useful for cancer vaccine therapy for prostate cancer patients.

International Publication No. 2007/000935 International Publication No. 2008/007711 International Publication No. 2009/022652 Japanese Unexamined Patent Publication No. 2005-170799

Ogata R, et al. “Identification of Polycomb Group Protein Enhancer of Zeste Homolog2 (EZH2)-Derived Peptides Immunogenic in HLA-A24 + Prostate Cancer Patients.” Prostate 2004; 60: 273-281. Itoh Y, et al., “New peptides of the polycomb group protein enhancer of zeste homolog 2 with the potential to induce cancer-reactive cytotoxic T lymphocytes in human leukocyte antigen-A2 + prostate cancer patients.” Oncol Rep 2007; 18: 1231- 1237.

The present invention has been made to solve the above problems, and an object of the present invention is to provide a peptide useful for cancer vaccine therapy for prostate cancer patients. Another object of the present invention is to provide a pharmaceutical composition capable of inducing specific cell-mediated immunity and useful for the prevention or treatment of prostate cell cancer. Further, the present invention provides a prostate cancer-reactive cytotoxic T cell inducer capable of inducing prostate cancer-reactive cytotoxic T cells and a method for inducing prostate cancer-reactive cytotoxic T cells using the same. The purpose is to provide.

The present invention made to achieve the above object is a peptide derived from EZH2 (enhancer of zeste homolog 2: hereinafter also referred to as zesta homologue enhancer 2), which can bind to the human leukocyte antigen HLA-A3 supertype allele. And to provide a peptide that can be recognized by cell-mediated immunity. Specifically, the present invention is shown below:
<1> The present invention is a partial peptide derived from EZH2, which is a prostate cancer antigen peptide capable of binding to the human leukocyte antigen HLA-A3 supertype allele and recognizable by cell-mediated immunity, or a derivative thereof having equivalent properties. Is.

<2> In the present invention, one or two amino acid substitutions, deletions and / or additions are introduced in (a) a peptide consisting of the amino acid sequence shown in SEQ ID NO: 5 or (b) the amino acid sequence shown in SEQ ID NO: 5. The peptide according to <1>, which is a peptide having an amino acid sequence and having functionally equivalent properties to the peptide consisting of the amino acid sequence shown in SEQ ID NO: 5.

<3> In the present invention, HLA-A belonging to the HLA-A3 supertype is HLA-A selected from the group consisting of HLA-A11, HLA-A31 and HLA-A33, <1> or <2>. The peptide according to.

<4> For the present invention, a nucleic acid molecule consisting of a base sequence encoding the peptide according to any one of <1> to <3> may be used.

<5> For the present invention, a vector containing the nucleic acid molecule described in <4> may be used.

<6> The present invention comprises a pharmaceutical composition for preventing or treating prostate cancer, which comprises the peptide according to any one of <1> to <3> and at least one of the vectors described in <5>. It may be a thing.

<7> The present invention is the pharmaceutical composition according to <6>, which is a cancer vaccine.

<8> The present invention is a step of contacting peripheral blood mononuclear cells (PBMC) collected from a patient with HLA-A3 supertype allergen-positive prostate cancer with the peptide according to any one of <1> to <3>. Is a method for inducing prostate cancer-reactive cytotoxic T cells (CTL), which comprises.

<9> The present invention is a prostate cancer-reactive cytotoxic T cell inducer containing the peptide according to any one of <1> to <3>.

<10> The present invention is a step of causing cells having an antigen-presenting ability derived from an HLA-A3 supertype allergen-positive prostate cancer patient to incorporate the peptide according to any one of <1> to <3>, or < 5> is a method for preparing an antigen-presenting cell that presents a complex of an EZH2-derived peptide or a derivative thereof and an HLA-A3 supertype allergen on the cell surface, which comprises the step of introducing the vector according to 5>.

<11> The present invention is an antigen-presenting cell preparation agent for preparing an antigen-presenting cell that presents a complex of an EZH2-derived peptide or a derivative thereof and an HLA-A3 supertype allele on the cell surface.
It may be an antigen-presenting cell preparation agent containing the peptide according to any one of <1> to <3> and at least one of the vectors described in <5>.

The present invention has expanded the options for peptide-based immunotherapy, especially prostate cancer vaccine therapy, for patients with HLA-A3 supertype allele-positive prostate cancer. According to the present invention, further therapeutic effect can be obtained by adding EZH2, which is considered to be involved in cancer growth, to the target in the treatment of patients with HLA-A3 supertype allele-positive prostate cancer for which several cancer vaccine candidate peptides have been identified so far. It is expected to contribute to improvement.

Since the peptide of the present invention can bind to the HLA-A3 supertype allele and is recognized by the cell-mediated immunity of cancer patients, especially HLA-A3 supertype allele-positive prostate cancer patients, it is suitable for the treatment and prevention of cancer diseases, especially prostate cancer. It is useful. According to this peptide, prostate cancer-reactive cytotoxic T cells can be induced in an HLA-restricted manner. This peptide is derived from EZH2 existing in the living body or modified or induced based on the skeleton thereof, and binds to a desired bind site in the living body to exert an action effect, and thus has few side effects. It is reliable and safe.

The sequence of this peptide can be used to prepare a nucleic acid molecule encoding the same and a vector thereof, which can be used for producing the peptide in vitro or in vivo.

In addition, using this peptide and the nucleic acid molecule encoding it, a pharmaceutical preparation for the prevention or treatment of prostate cancer, for example, an inducer to prostate cancer-reactive cytotoxic T cells, an antigen of CD8-positive T cells As an inducer to presenting cells, a therapeutic or prophylactic agent for HLA-A3 supertype allergen-positive prostate cancer, and a cancer vaccine, it can be used for cancer treatment, particularly HLA-A3 supertype allergen-positive prostate cancer treatment.

Furthermore, a biologic for the prevention or treatment of prostate cancer containing an antigen-presenting cell or a prostate cancer-reactive cytotoxic T cell presenting a complex of this peptide and a human HLA-A3 supertype allele on the cell surface was prepared. It can be useful for the treatment of HLA-A3 supertype allergen-positive prostate cancer patients.

(A) is a gel migration chart showing the expression of EZH2 protein in LNCaP cells (androgen-sensitive human prostate adenocarcinoma cells: hereinafter also referred to as androgen-dependent prostate cancer cells). In (b), three LNCaP transfectants are examined by flow cytometry (hereinafter, also referred to as fluid cell measurement method) for their surface expression of HLA-A11, -A31, or -A33 molecules. It is a figure. The EZH2-derived peptide to which the present invention is applied stimulates peripheral blood mononuclear cells (PBMC) from HLA-A3 supertype allele ⁺ prostate cancer patients, and the peptide-reactive CTL induced thereby is applied to the prostate-derived cancer cell line. It is a figure which investigated whether the cytotoxic activity can be exhibited. The cytotoxic activity of HLA-A3 supertype _allergen ⁺ PBMC derived from prostate cancer cells stimulated with the EZH2 _733-741 peptide of SEQ ID NO: 5 to which the present invention is applied to LNCaP-A11, -A31 and -A33 is HIV. It is a figure showing that it was significantly suppressed by the addition of unlabeled C1R-A11, -A31 and -A33 cells pulsed with EZH2 _733-741 peptide as compared with the peptide pulse (C1R: HLA human B cell line). Refers to a cell line that expresses only HLA alleles).

Hereinafter, embodiments for carrying out the present invention will be described in detail, but the scope of the present invention is not limited to these embodiments.

(peptide)
The peptide of the present invention is a partial peptide derived from EZH2, which is a prostate cancer antigen peptide that can bind to the human leukocyte antigen HLA-A3 supertype allele and can be recognized by cell-mediated immunity, or a derivative peptide thereof having equivalent properties. Is. Specifically, it is one of the following (a) and (b).
(A) A peptide consisting of the amino acid sequence shown in EZH2 _733-741 (SEQ ID NO: 5).
(B) A peptide _{consisting of} an amino acid sequence in which one or two amino acids are deleted, substituted, or added in the amino acid sequence represented by EZH2 _733-741 (SEQ ID NO: 5).

In the present invention, "the peptide can bind to the HLA-A3 supertype allele" means that the peptide can form a complex with the HLA-A3 supertype allele and be presented on the cell surface. In general, peptides that bind to HLA molecules are known to have a regular amino acid sequence that depends on the type of HLA, and the regular amino acid sequence is called a binding motif. Whether or not a peptide can bind to the HLA-A3 supertype allele is determined by computer analysis such as on the website (Bioinformatics and Molecular Analysis Section, Computational Bioscience and Engineering Laboratory, Division of Computer Research & Technology, NIH). Can be predicted.

In the present invention, "recognized by cell-mediated immunity" means that the peptide is recognized by specific cytotoxic T cells (hereinafter, also referred to as CTL), that is, the peptide-specific CTL can be induced. To say. The method for confirming whether or not peptide-specific CTL can be induced is not particularly limited and may be appropriately selected depending on the intended purpose. For example, peripheral blood mononuclear cells stimulated with peptide (hereinafter referred to as “Peptide-stimulated”). A method for confirming by measuring whether or not PBMC (also referred to as PBMC) produces cytokines such as interferon (hereinafter, also abbreviated as IFN) -γ in response to antigen-presenting cells pulsed with the peptide. Can be mentioned. The method for measuring the cytokine is not particularly limited and may be appropriately selected depending on the intended purpose. For example, it is measured by ELISA (enzyme-linked immunosorbent assay: hereinafter also referred to as enzyme-linked immunosorbent assay). Can be done. The method for confirming the induced cytotoxic activity of CTL is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include a method for confirming by a ⁵¹ Cr release test.

The peptide consisting of the amino acid sequence represented by (a) EZH2 _733-741 (SEQ ID NO: 5) is an EZH2-derived peptide and corresponds to the 733th to 741st amino acid sequence in EZH2.

In the amino acid sequence represented by SEQ ID NO: 5 in (b), the peptide consisting of the amino acid sequence in which one or two amino acids are deleted, substituted, or added can be bound to the HLA-A3 supertype allele. The peptide is not particularly limited as long as it is a peptide recognized by cell-mediated immunity, and can be appropriately selected depending on the intended purpose.

The positions of the deletion, substitution, and addition are not particularly limited and may be appropriately selected depending on the intended purpose. As the substitution, it is preferable to substitute between homologous amino acids in that the properties of the peptide are not changed. The homologous amino acids can be selected from the viewpoints of, for example, polar amino acids, non-polar amino acids, hydrophobic amino acids, hydrophilic amino acids, positively charged amino acids, negatively charged amino acids, aromatic amino acids and the like.

The deletions, substitutions, and additions are the binding motifs of the HLA-A3 supertype allele, and the second amino acid from the N-terminus of the peptide amino acid sequence is alanine, serine, isoleucine, valine, threonine, methionine, Alternatively, it is leucine, and it is preferable that the 9th amino acid from the N-terminal is lysine or arginine.

The amino acids constituting the peptide may be natural amino acids or amino acid analogs. The amino acid analog is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include N-acylated product, O-acylated product, esterified product, acid amidated product and alkylated product of amino acid.

Further, the peptide may be modified with its constituent amino acids, carboxyl groups and the like as long as the effects of the present invention are not impaired. The modification is not particularly limited and may be appropriately selected depending on the intended purpose. For example, a formyl group, an acetyl group, a t-butoxycarbonyl group or the like bonded to an N-terminal or a free amino group, C. Examples thereof include those in which a methyl group, an ethyl group, a t-butyl group, a benzyl group and the like are bonded to a terminal or free carboxyl group.

The peptide is recognized by both cell-mediated immunity and humoral immunity. The peptide is expected to have high immunogenicity and excellent CTL-inducing ability, and is preferably recognized by humoral immunity. In the present invention, "recognized by humoral immunity" means that peptide-specific IgG is present in the living body, that is, peptide-specific IgG is detected in plasma. The method for measuring specific IgG in plasma is not particularly limited and may be appropriately selected depending on the intended purpose. For example, it can be measured by an ELISA method or the like.

The method for producing the peptide is not particularly limited, and a known method can be appropriately selected.

The peptide of the present invention may be produced by fragmenting a polypeptide containing the amino acid sequence in the cell. The polypeptide is not particularly limited and may be appropriately selected depending on the intended purpose.

The peptide is an EZH2-derived peptide that is considered to be involved in cancer growth, can bind to the HLA-A3 supertype allele, and is recognized by cell-mediated immunity. Therefore, for example, in patients with HLA-A3 supertype allele-positive prostate cancer. It can be suitably used for cancer vaccine therapy.

(Nucleic acid molecule)
The nucleic acid molecule of the present invention is not particularly limited as long as it has a base sequence encoding the peptide, and can be appropriately selected depending on the intended purpose.

The nucleic acid molecule can be suitably used for, for example, a vector described later.

(vector)
The vector of the present invention contains at least the nucleic acid molecule of the present invention, and further contains other elements as required. The vector is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include plasmids and viral vectors. Specific examples of the virus vector include an adenovirus vector, an adeno-related virus vector, a retrovirus vector, and a vaccinia virus vector.

The method for preparing the vector is not particularly limited, and a known method can be appropriately selected and prepared.

The vector expresses the peptides of the invention when introduced into antigen-presenting cells. Then, the antigen-presenting cell presents a complex of the peptide and the HLA-A3 supertype allele on the cell surface. The antigen-presenting cells can efficiently proliferate CTLs that specifically damage prostate cancer cells.

The vector can be suitably used for a pharmaceutical composition for preventing or treating prostate cancer, which will be described later, and an antigen-presenting cell preparation agent.

(Pharmaceutical composition for prevention or treatment of prostate cancer)
The pharmaceutical composition for the prevention or treatment of prostate cancer of the present invention contains at least one of the peptide of the present invention and the vector of the present invention, and further contains other components as necessary.

The content of the peptide and the vector in the pharmaceutical composition for preventing or treating prostate cancer is not particularly limited and may be appropriately selected depending on the intended purpose.

The pharmaceutical composition for the prevention or treatment of prostate cancer may consist of at least one of the peptide and the vector. The peptide and the vector may be used alone or in combination of two or more. Further, each of the peptide and the vector may be used alone or in combination of two or more.

The other components in the pharmaceutical composition for preventing or treating prostate cancer are not particularly limited as long as the effects of the present invention are not impaired, and can be appropriately selected depending on the intended purpose. For example, they are pharmaceutically acceptable. Examples include the carrier to be obtained. The other components may be used alone or in combination of two or more. Specific examples of the carrier include cellulose, polymerized amino acids, albumin and the like.

The content of the other components in the pharmaceutical composition for preventing or treating prostate cancer is not particularly limited and may be appropriately selected depending on the intended purpose.

The pharmaceutical composition for preventing or treating prostate cancer may be used in combination with a pharmaceutical composition containing another ingredient as an active ingredient. Further, the pharmaceutical composition for preventing or treating prostate cancer may be used in a state of being blended in a medicine containing another ingredient as an active ingredient.

Since the CTL of a prostate cancer patient is a collection of cells that recognize various cancer antigen peptides, the pharmaceutical composition for preventing or treating prostate cancer is, for example, derived from Lck-derived peptide (Patent Document 3) and / or SART3. It may be used in combination with a peptide (Patent Document 2).

The dosage form of the pharmaceutical composition for preventing or treating prostate cancer is not particularly limited, and a known dosage form can be appropriately selected, and examples thereof include solid preparations, semi-solid preparations, and liquid preparations. Pharmaceutical compositions for the prevention or treatment of prostate cancer in these dosage forms can be produced according to a conventional method.

The pharmaceutical composition for preventing or treating prostate cancer is preferably used as a cancer vaccine.

The administration method, dose, administration time, administration interval, and administration target of the pharmaceutical composition for preventing or treating prostate cancer are not particularly limited and may be appropriately selected depending on the intended purpose.

The pharmaceutical composition for the prevention or treatment of prostate cancer is preferably administered together with an adjuvant known to be conventionally used for vaccination so that an immune response can be effectively established. For example, intradermal administration, subcutaneous administration and the like can be mentioned.

The dose is not particularly limited, and should be appropriately selected in consideration of the disease state, age, body weight, constitution of the individual to be administered, and the presence or absence of administration of a pharmaceutical composition containing other ingredients as active ingredients. Can be done. When the pharmaceutical composition for preventing or treating prostate cancer contains the peptide, the dose is not particularly limited and may be appropriately selected depending on the intended purpose. For example, the amount of peptide is 0.0001 mg to 1 000 mg and the like can be mentioned.

The dose when the pharmaceutical composition for preventing or treating prostate cancer contains the vector is not particularly limited and may be appropriately selected depending on the intended purpose. For example, the amount of DNA is 0.1 μg to 100 mg or the like. Can be mentioned. 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 intended purpose. For example, once every few weeks to several months, administration may be continued for 1 to 3 years. Be done.

The administration target is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include humans.

(Prostate cancer prevention or treatment method)
The peptide and the vector can be administered to an individual to prevent the development of prostate cancer in the individual or to treat an individual suffering from prostate cancer. Therefore, the present invention is a method for preventing or treating prostate cancer, which comprises administering to an individual at least one of the peptide and the vector. Also related.

Further, as the method for preventing or treating prostate cancer, the prostate cancer-reactive cytotoxic T cells induced by the method for inducing prostate cancer-reactive cytotoxic T cells described later can be returned to an individual, or the antigen-presenting cells described later can be used. It may be carried out by returning the antigen-presenting cells prepared by the preparation method to an individual.

(Method for inducing prostate cancer-reactive cytotoxic T cells)
The method for inducing prostate cancer-reactive cytotoxic T cells of the present invention includes at least a contact step and, if necessary, other steps.

<Contact process>
The contact step is a step of contacting peripheral blood mononuclear cells (PBMC) collected from a patient with HLA-A3 supertype allele-positive prostate cancer with the peptide of the present invention. By the contact step, cytotoxic T cells (CTL) that damage HLA-A3 supertype allele-positive prostate cancer cells can be induced.

When the cytotoxic T cell is "prostate cancer reactive", the cytotoxic T cell recognizes a complex of a cancer antigen peptide and an HLA molecule on the prostate cancer cell and damages the cell. It means having the ability.

As the peptide, a peptide consisting only of the peptide may be used, or a prostate cancer-reactive cytotoxic T cell inducer described later may be used.

The contact method is not particularly limited and may be appropriately selected depending on the intended purpose. For example, peripheral blood mononuclear cells (PBMC) collected from the HLA-A3 supertype allele-positive prostate cancer patient can be selected. Examples thereof include a method of culturing in vitro in the presence of the peptide.

<Other processes>
The 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 intended purpose.

The prostate-reactive cytotoxic T cells obtained by the method for inducing prostate cancer-reactive cytotoxic T cells are, for example, returned to the body of an individual from which peripheral blood mononuclear cells have been collected and adopted to damage the prostate cancer cells. It can be suitably used for immunotherapy.

(Prostate cancer reactive cytotoxic T cell inducer)
The prostate cancer-reactive cytotoxic T cell inducer of the present invention contains at least the peptide of the present invention, and further contains other components as necessary. The peptide may be used alone or in combination of two or more. The content of the peptide in the prostate cancer-reactive cytotoxic T cell inducer is not particularly limited and may be appropriately selected depending on the intended purpose. The prostate cancer reactive damaging T cell inducer may consist only of the peptide.

The other components of the prostate cancer-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 depending on the intended purpose. For example, the above-mentioned prostate cancer. The same as those described in the item of other components of the pharmaceutical composition for prevention or treatment of. The other components may be used alone or in combination of two or more. The content of the other components in the prostate cancer-reactive cytotoxic T cell inducer is not particularly limited and may be appropriately selected depending on the intended purpose.

The prostate cancer-reactive cytotoxic T cell-inducing agent can be suitably used in the above-mentioned method for inducing prostate cancer-reactive cytotoxic T cells.

(Prostate cancer reactive cytotoxic T cell induction kit)
The kit for inducing prostate cancer-reactive cytotoxic T cells of the present invention contains at least the prostate cancer-reactive cytotoxic T cell-inducing agent of the present invention, 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 depending on the intended purpose. Examples thereof include a medium and a buffer solution. The other elements may be used alone or in combination of two or more. The prostate cancer-reactive cytotoxic T cell induction kit can be suitably used for the above-mentioned method for inducing prostate cancer-reactive cytotoxic T cells.

(Method of preparing antigen-presenting cells)
The antigen-presenting cells of the present invention include at least an introduction step and, if necessary, other steps.

The introduction step is a step of introducing at least one of the peptide of the present invention and the vector of the present invention into cells having an antigen-presenting ability collected from a patient with HLA-A3 supertype allele-positive prostate cancer. By the introduction step, an antigen-presenting cell that presents a complex of an erythropoietin receptor-derived peptide and an HLA-A3 supertype allele on the cell surface can be prepared. The antigen-presenting cells can induce cytotoxic T cells that damage HLA-A3 supertype allele-positive prostate cancer cells.

As the peptide, a peptide consisting only of the peptide may be used, or an antigen-presenting cell preparation agent described later may be used. As the vector, a vector consisting of only the vector may be used, or an antigen-presenting cell preparation agent described later may be used. 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 may be appropriately selected depending on the intended purpose.

The antigen-presenting cells are obtained, for example, by culturing cells having an antigen-presenting ability derived from a patient with HLA-A3 supertype allele-positive prostate cancer together with the peptide. Further, for example, it can be obtained by introducing the vector into cells having an antigen-presenting ability derived from a patient with HLA-A3 supertype allele-positive prostate cancer and expressing the peptide.

The cell having an antigen-presenting ability is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include dendritic cells. The method for preparing the dendritic cells is not particularly limited and may be appropriately selected depending on the intended purpose. For example, cells to be adhered to a culture plate are separated from peripheral blood mononuclear cells collected from a patient, and the cells are separated. Examples include a method of culturing the cells in the presence of IL-4 and GM-CSF for about 1 week.

<Other processes>
The 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 intended purpose.

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 to promote the induction of prostate cancer-reactive cytotoxic T cells in the body. Can be suitably used for.

(Antigen-presenting cell preparation agent)
The antigen-presenting cell preparation agent of the present invention is an antigen-presenting cell preparation agent for preparing an antigen-presenting cell that presents a complex of an erythropoietin receptor-derived peptide and an HLA-A3 supertype allele on the cell surface. , At least one of the peptides of the invention, and the vectors of the invention, and further comprises other components as needed.

The peptide and the vector may be used alone or in combination of two or more. Further, each of the peptide and the vector may be used alone or in combination of two or more. The content of at least one of the peptide and the vector in the antigen-presenting cell preparation agent is not particularly limited and may be appropriately selected depending on the intended purpose. The antigen-presenting cell preparation may consist of at least one of the peptide and the vector.

The other components of the antigen-presenting cell preparation are not particularly limited as long as the effects of the present invention are not impaired, and can be appropriately selected depending on the intended purpose. For example, the above-mentioned pharmaceutical for preventing or treating prostate cancer. Examples thereof include those described in the section of other components of the composition. The other components may be used alone or in combination of two or more. The content of the other components in the antigen-presenting cell preparation is not particularly limited and may be appropriately selected depending on the intended purpose.

The antigen-presenting cell preparation agent can be suitably used in the above-mentioned method for preparing antigen-presenting cells.

(Kit for preparing antigen-presenting cells)
The antigen-presenting cell preparation kit of the present invention is an antigen-presenting cell preparation kit for preparing an antigen-presenting cell that presents a complex of an erythropoietin receptor-derived peptide and an HLA-A3 supertype allele on the cell surface. It contains at least the prostatic cancer-reactive cytotoxic T cell inducer of the present invention and, if necessary, other elements.

The other elements are not particularly limited as long as the effects of the present invention are not impaired, and can be appropriately selected depending on the intended purpose. Examples thereof include a medium and a buffer solution. The other elements may be used alone or in combination of two or more.

The antigen-presenting cell preparation kit can be suitably used for the above-mentioned method for preparing antigen-presenting cells.

Hereinafter, test examples of the present invention will be described, but the present invention is not limited to these test examples.

<Materials and methods>
<Patients> Peripheral blood mononuclear cells (PBMC) were obtained from prostate cancer patients and healthy donors (HD) who provided written informed consent. Subjects included HLA-A11 ⁺ , -A31 ⁺ or -A33 ⁺ patients; PBMCs from HLA-A3 ⁺ or -A68 ⁺ patients had an extremely low frequency of them in the Japanese population ( Not available for 1.6 and 0.5% respectively) [Aizawa M. The Proceedings of the 3rd Asia-Oceania histocompatibility workshop conference. Oxford University Press 1986; 1090-1103]. None of the participants were infected with the human immunodeficiency virus (HIV). 30 milliliters of peripheral blood was obtained. PBMCs were then prepared by Ficoll-Conley density gradient centrifugation. All samples were cryopreserved until use. The Kinki University Ethics Review Committee has approved the research protocol (Approval No. 19-10).

<Cell line>
C1R-A11, C1R-A31 and C1R-A33 are C1R lymphoma sublines strongly transfected with HLA-A11: 01 (A31: 01) (A33: 03 gene), respectively. Expression of HLA-A11, -A31 and -A33 molecules on these subsystems has been reported [Takedatsu H, Shichijo S, Katagiri K, Sawamizu H, Sata M, Itoh K: Identification of peptide vaccine candidates sharing among HLA. -A3 +, -A11 +, -A31 +, -A33 + cancer patients. Clin Cancer Res 2004; 10: 1112-1120]. LNCaP-A11, LNCaP-A31 and LNCaP-A33 are LNCaP subseries expressing each of the HLA-A11, -A31 and -A33 molecules [Minami T, Matsueda S, Takedatsu H, Tanaka M, Noguchi M, Uemura. H, Itoh K, Harada M: “Identification of SART3-derived peptides having the potential to induce cancer-reactive cytotoxic T lymphocytes from prostate cancer patients with HLA-A3 supertype alleles.” Cancer Immunol Immuother 2007; 56: 689-698]. All of these cell lines were maintained in RPMI 1640 (Invitrogen) with 10% FCS. PrEC is a normal prostate epithelial cell line purchased from Lonza, Walkersville, MD, USA.

<Peptide>
All peptides derived from EZH2 were prepared based on the motif that binds to the HLA-A3 supertype allele [Parker KC, Bednarek MA, Coligan JE. “Scheme for ranking potential HLA-A2 binding peptides based on independent binding of individual peptide side-chains. ”J Immunol 1994; 152: 163-175]. Simply put, peptide binding scores are HLA class I as obtained from BIMAS (Bioinformatics and Molecular Analysis Section, Computational Bioscience and Engineering Laboratory, Division of Computer Research & Technology, NIH, bimas.cit.nih.gov). Calculated based on the predicted half-life of dissociation from the molecule, and five peptides from EZH2 (SEQ ID NOs: 1-5) were selected. They are shown below:

In addition, the amino acid sequence of the peptide used for the control that binds to the HLA-A3 supertype allele is shown below:

All peptides were purchased from (Eurofin Genomics, Inc., Tokyo, Japan) and dissolved in DMSO at a dose of 10 mg / ml.

<Induction of peptide-specific CTL from PBMC>
Assays for the detection of peptide-specific CTLs were performed by previously reported methods with some modifications [Hida N, Maeda Y, Katagiri K, Takasu H, Harada M, Itoh K: “A” simple culture protocol to detect peptide-specific cytotoxic T lymphocyte precursors in the circulation. ”Cancer Immunol Immunother 2002; 51: 219-228]. PBMCs (5 cells x 104 cells / well) were cultured in 100 μL of medium on U-bottom 96-well subculture plates (Nunc, Roskilde, Denmark) at 10 μg / ml of each peptide. The medium consisted of 45% RPMI 1640, 45% AIM-V medium (Gibco BRL, Gaithersburg, MD, USA), 10% FCS and MEM non-essential amino acid solution (x100 dilution, Gibco BRL). Eight wells were used for each peptide. The next day, 100 μL of medium containing 50 U / ml IL-2 was added. On day 8, 100 μL of medium was removed, fresh medium containing the corresponding peptide (10 μg / ml) was replenished, and on day 9 it contained 50 U / ml IL-2. 100 μL of medium was added. On day 17 of culturing, it was replaced with fresh medium. The cultured cells were then separated from one well into four wells: two wells for stimulation with corresponding peptide pulses C1R-A11, C1R-A31, or C1R-A33 cells and two other wells. Was used for stimulation with control HIV peptide pulses C1R-A11, C1R-A31 or C1R-A33 cells. After 18 hours of culture, the supernatant was collected and the level of IFN-γ was determined by ELISA. Peptide-specific induction of CTL was positive only when the following two criteria were met:
(I) Both IFN-γ levels in the two wells stimulated with the corresponding peptide were ≥50 pg / ml, and (ii) IFN in the two wells stimulated with the corresponding peptide. Both -γ levels were 1.2-fold or more higher than the intermediate values of INF-γ levels in the two wells stimulated with the control HIV peptide.

<Flow cytometric analysis>
To investigate the expression of the HLA-A3 supertype allele, LNCaP transfectants were used as anti-HLA-A11 mAbs (Cat. No. 0284HA; One Lambda Inc., Monoclonal CA, USA), anti-HLA- Stained with either A31 mAb (Cat. No. 0273HA; One Lambda) or anti-HLA-A33 mAb (Cat. No. 0612HA; One Lambda), followed by FITC-conjugated goat anti-mouse IgG (H + L) antibody ( It was stained with KPL, Gaithersburg, MD, USA).

<Immune blotting assay>
Cells were lysed in M-PER reagents (Price, Rockford, IL, USA) with a protease inhibitor mixture (Nacalai Tesque). The protein concentration was determined using the Coomasie Plus Protein Bradford kit (Pierce). NuPAGE gel (4-12% or 12%; Life Technologies, Carlsbad, CA, USA) was used for protein separation. The protein was then immobilized on a PVDF membrane (Life Technologies) using the iBlot transfer method (Life Technologies). Membranes were blocked for 30 minutes with TBST (tris basic salt at 0.1% tween 20) containing Blocker BSA solution (1x) (Pierce). Culturing with the primary antibody was performed overnight at 4 ° C. using TBST containing Blocker BSA (1x). The following primary antibodies were used: anti-EZH2 (Cell Signaling Technology, Danvers, MA, USA) and anti-β-actin (BioLegend, San Diego, CA, USA) antibodies. After washing, membranes were cultured with alkaline phosphatase-conjugated secondary antibody at room temperature for 30 minutes. The protein band was visualized using CDP stellate chemiluminescence and imaged with LAS-4000 (FujiFilm, Tokyo, Japan).

<Cytotoxicity test>
To test the cytotoxicity of CTLs by a standard 6-hour ⁵¹ Cr release test, CD8 ⁺ T cells were negatively purified from peptide-stimulated PBMCs as follows: Peptide-stimulated PBMCs First stained with anti-CD56 mAb (mIgG1: DIACLONE, Besankov Cedex, France), then stained with Dynabeads anti-mouse IgG (Invitrogen), Dynabeads anti-CD19 (Invitrogen) and Dynabeads anti-CD4 (Invitrogen). Cells bound to Dynabeds were magnetized and removed negatively. After depletion of CD4 ⁺ T cells, B cells and natural killer cells, the percentage of CD8 ⁺ T cells was approximately 80-85% throughout the experiment (data not shown). Phytohaemagglutinin (PHA) -activated T cells were used as negative controls for HLAA-11 ⁺ , -A31 ⁺ and -A33 ⁺ normal target cells. 2000 cells labeled with ⁵¹ Cr per well were cultured with effector cells in 96 round well plates at the indicated effector / target ratios. The specific ⁵¹ Cr release was calculated by the following equation:
Specific solubility% = (test sample free-natural free) x 100 / (maximum free-natural free)
Maximum liberation was determined by the supernatant of a sample cultured in 1% Triton X (Wako Pure Chemical Industries, Osaka, Japan).

A cold inhibition assay was performed to test whether peptide-stimulated CD8 ⁺ T cells were responsible for cytotoxicity against LNCaP transfectant cells. Simply put, ⁵¹ Cr-labeled target cells (2 x 10 ³ cells / well) in a 96-well-round-bottom well plate with purified CD8 ⁺ T cells (2 x 10 ⁴ cells / well). 4 × 10 cultured with ⁴ unlabeled cold target cells. C1R-A11, C1R-A31 and C1R-A33 cells pre-pulsed with either the control HIV peptide or the corresponding EZH2 peptide were used as cold target cells.

<Statistical processing>
Statistical significance of the data was determined using a two-sided Student's t-test. P-values (probabilities) less than 0.05 were considered statistically significant.

Example 1
<Induction of CTL specific to EZH2 peptide from PBMC of prostate cancer patients with HLA-A3 supertype allele>
Based on binding motifs for HLA-A11, -A31 and -A33 molecules [Parker KC, Bednarek MA, Coligan JE. “Scheme for ranking potential HLA-A2 binding peptides based on independent binding of individual peptide side-chains.” J Immunol 1994; 152: 163-175], five peptides derived from EZH2 (listed in Table 3 below) were prepared and which peptides were HLA-A11 ⁺ , -A31 ⁺ , or -A33 ⁺ prostate cancer. It was determined whether peptide-specific CTLs from the patient's PBMC could be induced (Table 4).

<1> That is, first, the binding scores of the five partial peptides of EZH2 for the HLA-A3 supertype allele were calculated (Table 3). As a result, the partial peptide EZH2 _733-741 (SEQ ID NO: 5) showed the highest binding score of 12, suggesting that it is most likely to bind to the HLA-A3 supertype allele.

In Table 3, a) Peptide bond score is the expected half-life of dissociation from HLA class I molecules obtained from the website (Bioinformatics and Molecular Analysis Section, Computational Bioscience and Engineering Laboratory, Division of Computer Research & Technology, NIH). Calculated based on (half-time). The binding score for HIV peptides was not calculated. This is because the peptide consisted of the 11-mer of amino acids.

<2> Next, the inducibility of peptide-reactive CTLs from PBMCs of HLA-A11 ⁺ , -A31 ⁺ and -A33 ⁺ patients and healthy donors was tested. PBMCs are C1R-A11, C1R-A31, or C1R-A33 stimulated in vitro with each partial peptide (5 types) derived from EZH2, or either EBV or influenza peptide, and pulsed with the corresponding peptide. Cells were tested with their IFN-γ production items depending on the cells. Eight wells were used for each peptide. The results are shown in Table 4.

In Table 4, PBMCs were stimulated in vitro with each of the indicated peptides and peptide-specific reactivity was tested. Only positive results are shown in Table 4.

The results in Table 4 determined that peptide-specific induction of CTL was positive only when the following two criteria were met: (i) IFN-γ in two wells stimulated with the corresponding peptide. The levels are both ≥50 pg / ml, and (ii) the IFN-γ levels in the two wells stimulated with the corresponding peptide are both INF- in the control HIV in the two wells stimulated with the peptide. It is ≥1.2 times higher than the average of γ levels. With regard to HD (healthy _donor), both _{EZH2 23-31} and _{EZH2 70-78} peptides to induce specific CTL peptides from ^HLA-A11 + HD. The EZH2 _73-81 and EZH2 _699-707 peptides induced peptide-specific CTLs from HLAA31 ⁺ HD. The EZH2 _733-741 peptide then induced a peptide-specific CTL from HLA-A33 ⁺ HD. For prostate cancer patients, PBMCs from 4 patients were tested for each HLA-A3 supertype allele. As a result, the EZH2 _733-741 peptide most efficiently induced peptide-reactive CTLs from PBMCs in patients with HLA-A3 supertype allele ⁺ prostate cancer. The inducing effect of the EZH2 _733-741 peptide was higher than that of the control EBV or influenza peptide. Peptide-specific CTL positive induction was observed in 2 patients with HLA-A11, 2 patients with HLA-A31, and patients with HLA-33 out of 4 patients with HLA-A3 supertype allele. It was induced by two patients. Compared to the EZH2 _733-741 peptide, the other four EZH2 peptides were less successful in inducing peptide-specific CTLs. Overall, these results show that, of the five candidates, the EZH2 _733-741 peptide is the best peptide capable of producing a peptide-specific CTL from HLA-A3 supertype allele ⁺ PBMC in prostate cancer patients. Indicates that there is. Therefore, this EZH2 _733-741 peptide was selected as a candidate peptide, and then a cytotoxicity test was conducted.

Example 2
<Cytotoxicity of CTLs reactive with EZH2 _733-741 peptide against HLA-A3 supertype allele ⁺ and EZH2-expressing prostate cancer cells>

CD8 ⁺ CTL pulsed with EZH2 _733-741 peptide, which showed induction of peptide-specific CTL in Example 1, was LNCaP, LNCaP-A11, LNCaP-A31 and LNCaP-A33 in a 6-hour ⁵¹ Cr release test. Confirm cytotoxicity against.

First, before testing the cytotoxicity to HLA-A3 supertype allele ⁺ and prostate cancer cells expressing EZH2, the protein expression of EZH2 and HLA-A3 supertype allele was confirmed. The result is shown in FIG. 1 (a). FIG. 1 (a) is a gel electrophoresis diagram showing the expression of EZH2 protein in LNCaP cells to which the present invention is applied. EZH2 protein expression in prostate cancer cell lines LNCaP, PC3, normal prostate epithelial cells PrEC, and PHA-activated T cell blasts was examined by immunoblotting. Expression of β-actin was used as a control. As a result, the prostate cancer cell lines LNCaP and PC3, as well as PHA-activated T cell blasts, expressed EZH2 at the protein level. In contrast, its expression was very low in the prostate normal epithelial cell line PrEC.

Next, three LNCaP transfectants to which the present invention is applied are tested for their surface expression of HLA-A11, -A31, or -A33 molecules by flow cytometry. It was. The result is shown in FIG. 1 (b). These cells were first stained with anti-HLA-A11, anti-HLA-A31, or anti-HLA-A33 mAbs, followed by FITC-conjugated anti-mouse IgG mAbs. White outline represents the first stain without mAb. LNCaP substrains strongly transfected with the HLA-A11, -A31 and -A33 plasmids were positive for each A3 supertype allele.

Next, it was determined _{whether the} MP (multinucleated leukocyte) in vitro stimulation-induced CTL with the EZH2 _733-741 peptide exhibits cytotoxicity against prostate cancer cells expressing HLA-A3 supertype allele ⁺ and EZH2.
PBMCs from HLA-A3 supertype allele ⁺ prostate cancer patients are in vitro stimulated by the EZH2 _733-741 peptide. After purification for CD8 ⁺ T cells, their cytotoxicity to the LNCaP subline expressing any molecule of HLA-A11, -A31, or -A33 was examined. The obtained results are shown in FIG.

In FIG. 2, PBA-stimulated PHA-stimulated T cell blasts from HLA-A3 supertype allele ⁺ HDs were used as normal control cells. Purified CD8 ⁺ T cells of PBMC from HLA-A11 ⁺ patients (patients Pt # 1 and Pt # 2) (they were in vitro stimulated by the EZH2 _733-741 peptide) were parental LNCaP cells and HLA. It showed higher levels of cytotoxicity to LNCaP-A11 cells than to -A11 ⁺ T cell blasts. Patients Pt # 7 and Pt # 13 in FIG. 2 correspond to patients Pt # 2 and Pt # 4 for HLA-A11 in Table 4. Similarly, compared to parental LNCaP cells, HLAA31 ⁺ or HLA-A33 ⁺ T cell blasts, stimulated and purified with the EZH2 _733-741 peptide of PBMC from HLA-A31 ⁺ or HLA-A33 ⁺ patients. CD8 ⁺ T cells each exhibited higher cytotoxicity against LNCaP-A31 and LNCaP-A33 cells. Patients Pt # 34 and Pt # 35 for HLA-A31 in FIG. 2 correspond to patients Pt # 1 and Pt # 4 for HLA-A31 in Table 4, and patients Pt # 14 for HLA-A33 in FIG. And Pt # 30 correspond to patients Pt # 1 and Pt # 2 for HLA-A33 in Table 4.
These results indicate that the EZH2 _733-741 peptide has the potential to induce prostate cancer-reactive CTLs from PBMCs in HLA-A3 supertype allele ⁺ prostate cancer patients.

To confirm the specificity of cytotoxicity, a cold inhibitory cytotoxicity assay was then performed using purified CD8 ⁺ T cells. The obtained results are shown in FIG.
As a result, as shown in FIG. 3, HLA-A11 ⁺ , HLA-A31 ⁺ or HLA-A33 ⁺ stimulated and purified with the EZH2 _733-741 peptide, CD8 ⁺ T cell LNCaP-A11, LNCaP from the patient. Cytotoxicity to -A31, or LNCaP-A33 cells corresponds to the corresponding EZH2 _733- when added with C1R-A11, C1R-A31, or C1R-A33 cells pulsed and unlabeled with the HIV peptide. Significantly suppressed by the addition of C1R-A11, C1R-A31 or C1R-A33 cells pulsed with ₇₄₁ peptide and unlabeled.

These results show that the cytotoxicity of PBMCs stimulated with EZH2 _733-741 peptide from HLA-A3 supertype allele ⁺ prostate cancer patients to HLA-A3 supertype allele ⁺ prostate cancer cells is at least partially. It suggests that it was reduced to EZH2 _733-741 peptide-specific CD8 ⁺ T cells.

<Discussion>
To date, EZH2-derived peptides that can be used in peptide-based anti-cancer vaccines for cancer patients with HLA-A2 or -A24 molecules have been reported (eg, Patent Document 4, Non-Patent Documents). 2 and Non-Patent Document 4), there is no example in which an EZH2-derived peptide is used as a vaccine for HLA-A3 supertype allergic ⁺ prostate cancer patients. The present invention shows that the EZH2 _733-741 peptide has the potential to induce a CTL that is unique to the peptide from PBMC in HLA-A3 supertype allele ⁺ prostate cancer patients and is responsive to prostate cancer. It was. The EZH2 _733-741 peptide more efficiently induced peptide-specific CTLs from PBMCs in HLA-A3 supertype allele ⁺ prostate cancer patients as compared to control EBV or Flu (influenza) peptides. In addition, the cold inhibition assay showed that PBMCs from HLA-A3 supertype alleles ⁺ prostate cancer patients stimulated with EZH2 _733-741 peptide were cytotoxic to HLA-A3 supertype ⁺ EZH2 expressing prostate cancer cells. , Depends on CD8 ⁺ T cells specific to peptides. The results of these series of inventions show that the EZH2 _733-741 peptide can be applied as an anticancer vaccine for HLA-A3 supertype allele ⁺ prostate cancer patients.

The present invention has identified new EZH2 peptide candidates applicable to HLA-A3 supertype allele ⁺ prostate cancer patients. Combined with the known EZH2 peptide for HLA-A2 ⁺ or -A24 ⁺ prostate cancer patients, the EZH2 peptide panel is a peptide for hormone refractory and metastatic prostate cancer patients in various minority populations. It makes it possible to promote anti-cancer vaccines based on.

Claims (6)

EZH2 a derived peptide, capable of binding to human leukocyte antigen HLA-A3 supertype allele, and Ri Oh a peptide that is recognized by cellular immunity, the peptide, SEQ ID NO: comprising an amino acid sequence shown in 5 HLA-A, which is a pharmaceutical composition for preventing or treating prostate cancer and belongs to the HLA-A3 supertype, is selected from the group consisting of HLA-A11, HLA-A31 and HLA-A33. HLA-A, a pharmaceutical composition . The pharmaceutical composition according to claim 1 , which is a cancer vaccine. Prostate Cancer Reactive Cytotoxic T Cells (CTL) comprising the step of contacting peripheral blood mononuclear cells (PBMC) collected from a patient with HLA-A3 supertype allergen-positive prostate cancer with the peptide according to claim 1 . Guidance method. A prostate cancer-reactive cytotoxic T cell inducer comprising the peptide according to claim 1 . A composite of an EZH2-derived peptide or a derivative thereof and an HLA-A3 supertype allele, which comprises a step of incorporating the peptide according to claim 1 into cells having an antigen-presenting ability derived from an HLA-A3 supertype allele-positive prostate cancer patient. A method of preparing antigen-presenting cells that present the body to the cell surface. An antigen-presenting cell preparation agent for preparing an antigen-presenting cell that presents a complex of an EZH2-derived peptide or a derivative thereof and an HLA-A3 supertype allele on the cell surface, and comprises the peptide according to claim 1. An antigen-presenting cell preparation agent characterized by.

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