WO2020166729A1 - T cell vaccine - Google Patents

T cell vaccine Download PDF

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WO2020166729A1
WO2020166729A1 PCT/JP2020/006949 JP2020006949W WO2020166729A1 WO 2020166729 A1 WO2020166729 A1 WO 2020166729A1 JP 2020006949 W JP2020006949 W JP 2020006949W WO 2020166729 A1 WO2020166729 A1 WO 2020166729A1
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treatment
cells
antigen
pathogen
vaccine
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French (fr)
Japanese (ja)
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健二郎 松野
祐司 上田
祐介 北沢
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学校法人獨協学園獨協医科大学
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Publication of WO2020166729A1 publication Critical patent/WO2020166729A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/145—Orthomyxoviridae, e.g. influenza virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4648—Bacterial antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants

Definitions

  • the present invention relates to a T cell vaccine that induces neutralizing antibodies in lymphoid organs in multiple locations.
  • DST (donor specific blood transfusion/donor-specific blood transfusion) is a treatment method of inducing immune tolerance by administering a donor (allo) blood to a host before organ transplantation (Non-patent document 1: Clin Transplant 25:317). , 2011). Performing DST before kidney transplantation since the 1970s suppresses rejection reaction in a donor-specific manner. From clinical practice, antibodies against donor histocompatibility antigen (MHC antigen) can be easily produced by a single blood transfusion. Although it has been reported, side effects may occur, so it has almost disappeared due to the advent of immunosuppressive drugs that can easily treat rejection reactions. Therefore, by what component in the allo antibody response (AFC response) blood, where, how happen, made antibodies is not yet elucidated or with what effects, including immunosuppression ..
  • AFC response allo antibody response
  • Non-patent document 2 Cell Transplant 21:581). , 2012; Non-patent document 3: Cell Transplant 19:765, 2010; Non-patent document 4: Arch Histol Cyto 73:1, 2010; Non-patent document 5: Hepatology 56: 1532, 2012).
  • the allo-immune response is presented by direct sensitization in which donor antigen-presenting dendritic cells (DCs) directly associate with host T cells to present donor MHC antigens, and MHC antigens derived from donor cells are taken up by host DCs and presented. It is said to be caused by indirect sensitization (Non-Patent Document 8: Immunity 14:357, 2001).
  • AFC response (humoral immunity) is induced by Th2 helper T cells and follicular helper T cells (Follicular helper T cells/Tfh), and the GATA-3 gene, which is a cytokine or transcription factor such as IL-4 and IL-10. It has been reported that the expression of the above is dominant (Non-patent document 9: Immunity 30:324, 2009).
  • the antibody production response by DST mainly involves an immune response by indirect sensitization in the spleen.
  • the immune response in the spleen occurs in the periarterial lymphocyte sheath (PALS/T cell region) in the white pulp of the spleen, where DC is localized, and T and B cells are used for immune surveillance. It is known that most of them recirculate throughout the body, always migrate from blood into PALS and stay in PALS for a while, and T cells further associate with DC of PALS to confirm antigen information (Non-Patent Document 10). : Arch Histol Cyto 73:1, 2010).
  • a vaccine against a pathogen is usually administered intramuscularly or subcutaneously to induce the production of neutralizing antibodies mainly in the spleen. Therefore, when the spleen function is low or the spleen is removed, the usual vaccine effect cannot be expected so much.
  • the present inventor has conducted extensive studies to solve the above-mentioned problems, and as a result, by labeling allo-T cells with a pathogen antigen and inoculating this to an individual, not only the spleen but also lymph nodes throughout the body are multifocal.
  • the inventors have found that a neutralizing antibody is induced in Escherichia coli and have completed the present invention.
  • the present invention is as follows.
  • a vaccine against the pathogen antigen in an allogeneic recipient individual which comprises donor-derived T cells that have undergone histocompatibility antigen expression suppression treatment, activation suppression treatment, and pathogen antigen labeling treatment.
  • the vaccine according to (1), wherein the treatment for suppressing the expression of the histocompatibility antigen is RNA interference treatment for the histocompatibility antigen gene or knockout treatment of the gene.
  • the vaccine according to (1) or (2), wherein the activation suppression treatment is an antimetabolite or DNA synthesis inhibitor treatment or irradiation treatment.
  • the vaccine according to any one of (1) to (3), wherein the pathogen is a virus or a bacterium.
  • the vaccine according to (4), wherein the virus is influenza virus.
  • a neutralizing antibody inducer in an allogeneic recipient individual which comprises donor-derived T cells that have undergone histocompatibility antigen expression suppression treatment, activation suppression treatment, and pathogen antigen labeling treatment.
  • the neutralizing antibody inducer according to (7), wherein the treatment for suppressing the expression of the histocompatibility antigen is RNA interference treatment for the histocompatibility antigen gene or knockout treatment of the gene.
  • the present invention provides a T cell vaccine and a neutralizing antibody inducer.
  • the T cell vaccine of the present invention is capable of inducing neutralizing antibodies in multiple points.
  • FIG. 3 is a schematic diagram of a staining method for specific antibody-producing cells in Examples.
  • FIG. 5 is a diagram showing an experimental result of Example 1.
  • semi-allo T cells a system in which parental T cells are administered to a first-generation hybrid F1 recipient
  • a specific antibody against the labeled antigen phycoerythrin (PE) was detected in serum (left graph, red arrow), and cervical lymph node section Antibody-producing cells (blue, right panel) appeared on the top, but alloantibodies did not appear (black arrow).
  • the allo T cells a large amount of alloantibodies appeared, but the PE antibody titer was low.
  • the present inventor has found that donor T cells migrate to the spleen PALS after blood transfusion and, after associating with the host DC, transfer the donor MHCI antigen in some form and cause indirect sensitization most efficiently there, resulting in Th2 and Tfh
  • the inventor has come up with the hypothesis that antigen-specific antibodies are induced and efficiently produced.
  • the present invention relates to a vaccine against a pathogen in an allogeneic recipient individual and a neutralizing antibody inducer, which comprises donor-derived T cells that have undergone histocompatibility antigen expression suppression treatment, activation suppression treatment, and pathogen antigen labeling treatment.
  • Allogeneic (allo) T cells like autologous T cells, have the ability to recirculate to systemic lymphoid organs and undergo hematogenous migration, but efficiently mobilize tissue-resident dendritic cells via T cell receptors. It was found that when stimulated well, alloantibodies against the type I histocompatibility antigen (MHC) of the T cell itself were easily induced. This finding is different from autologous T cells.
  • the present inventor utilizes the above findings to label allo T cells (donor-derived T cells) with a pathogen antigen such as influenza, and administer this to recipients other than self (donor) to obtain only the spleen. It was found that multiple neutralizing antibodies are induced in the normal lymph node. Since there are hundreds of lymph nodes in humans, the vaccine of the present invention capable of inducing neutralizing antibody in systemic lymph nodes is highly efficient and can be used as a vaccine of a new concept.
  • the T cells used in the present invention are collected from one individual. After this T cell is subjected to the above treatment, it is administered to another individual who is the same species as the one individual but is allogeneic. Therefore, the T cells used are referred to herein as "allo T cells.”
  • T cells (allo T cells) collected from donor blood are subjected to histocompatibility antigen expression suppression treatment, activation suppression treatment, and pathogen antigen labeling treatment.
  • the thus treated allo-T cells are administered to an allogeneic individual (recipient) different from the donor.
  • the expression suppressing treatment of the histocompatibility antigen means an alloantigenicity suppressing treatment, and as the treatment for suppressing the expression of the histocompatibility antigen, for example, RNA interference treatment to the histocompatibility antigen gene or knockout treatment of the gene is performed.
  • RNA interference treatment to the histocompatibility antigen gene or knockout treatment of the gene is performed.
  • synthetic nucleic acid molecules capable of suppressing gene expression by RNA interference (RNAi) include siRNA (small interfering RNA), microRNA (miRNA) and shRNA (short hairpin RNA).
  • siRNA can be designed based on criteria well known in the art. For example, as the target segment of the mRNA of the target histocompatibility antigen gene, a continuous 15 to 30 bases, preferably 19 to 25 bases segment starting with AA, TA, GA or CA can be selected.
  • the siRNA has a GC ratio of 30 to 70%, preferably 35 to 55%.
  • siRNA is produced as a single-stranded hairpin RNA molecule that folds on its own nucleic acid to produce a double-stranded portion.
  • siRNA molecules can be obtained by conventional chemical synthesis, but can also be produced biologically using expression vectors containing sense and antisense siRNA sequences.
  • a method of ligating siRNA synthesized in vitro with plasmid DNA and introducing it into cells a method of annealing double-stranded RNA, or the like can be adopted.
  • ShRNA is an RNA molecule having a stem-loop structure in which a part of a single strand forms a complementary strand with another region. Therefore, shRNA is designed such that a part thereof forms a stem-loop structure. For example, if the sequence of a certain region is sequence A and the complementary strand to sequence A is sequence B, sequence A, spacer, sequence B are linked in this order so that these sequences exist in one RNA strand, and It is designed to have a length of 45 to 60 bases. Sequence A is a sequence of a partial region of the target histocompatibility antigen gene, and the target region is not particularly limited, and any region can be used as a candidate. The length of the sequence A is 19 to 25 bases, preferably 19 to 21 bases.
  • miRNA is a single-stranded RNA having a length of about 20 to 25 bases existing in cells, and is a kind of ncRNA (non coding RNA) which is considered to have a function of regulating the expression of other genes. .. miRNA exists as a nucleic acid forming a hairpin structure that is generated by being processed when it is transcribed into RNA and suppresses the expression of a target sequence. Since miRNA is also an inhibitory nucleic acid based on RNAi, it can be designed and synthesized according to shRNA or siRNA.
  • a histocompatibility antigen gene can also be knocked out.
  • the knockout method include, but are not limited to, knockout by CRISPR/Cas9.
  • the siRNA treatment, the gene knockout treatment with CRISPR/Cas9, or the like can be performed by a method described in a known document (Cancer Cell Int. 13:112, 2013; Clin Cancer Res 23:2255, 2017).
  • the T cell activation suppressing treatment means a treatment for removing the risk of T cell onset of GvH disease, and the activation suppressing treatment includes an antimetabolite or a DNA synthesis inhibitor treatment, or irradiation treatment.
  • an antimetabolite or a DNA synthesis inhibitor treatment or irradiation treatment.
  • ⁇ Antimetabolite or DNA synthesis inhibitor Folic acid analogs: methotrexate, pemetrexed, pralatrexate, etc. Purine analogs: mercaptopurine, thioguanine, cladribine, fludarabine, etc. Pyrimidine analogs: cytarabine, fluorouracil, tegafur, gemcitabine, etc. Antibiotics: mitomycin C, actinomycin, doxorubicin, etc. Alkylating agents: cyclophosphamide, melphalan, thiotepa, busulfan, etc. Platinum preparations: cisplatin, iproplatin, carboplatin, etc. Topoisomerase inhibitors: irinotecan, nogitecan, etoposide, anthracycline drugs, etc.
  • the amount of the antimetabolite or the DNA synthesis inhibitor to be used is 20 ⁇ g/5 ⁇ 10 7 /ml for mitomycin C, treated at 37° C. for 30 minutes, and other appropriate amount is used. This treatment can be performed by a method described in a known document (Hepatology 56: 1532, 2012).
  • the radiation dose of X-rays, gamma rays, etc. is 10 to 50 Gy, preferably 15 Gy per 10 8 cells. These treatments can be performed by a method described in a known document (Arch Pathol Lab Med 142:662, 2018).
  • the pathogen used as an antigen is not particularly limited and includes viruses, bacteria, protozoa and the like.
  • the antigen labeling is performed by preparing a gene vector of the target antigen and using a gene transfer technique into T cells.
  • influenza virus examples include influenza virus, hepatitis virus, herpes zoster, measles/rubella, papilloma (HPV), human immunodeficiency (HIV) virus and the like.
  • bacteria examples include pneumococcus, meningococcus, diphtheria, tetanus, pertussis, tuberculosis, and the like.
  • protozoa examples include malaria.
  • the allo T cell vaccine is prepared by treating donor-derived T cells with (a) histocompatibility antigen expression suppression treatment, (b) activation suppression treatment, and (c) pathogen antigen labeling treatment, in the order of It is not particularly limited.
  • the above processes may be performed in the order of (a), (b) and (c), or may be performed in another order.
  • a pathogen antigen is bound to an antibody that specifically recognizes T cells such as an anti-CD4 antibody to prepare a complex of the antibody and the pathogen antigen, and the complex is bound to a donor T cell.
  • alloimmunity that causes GvH disease or induction of alloantibody production in the recipient of donor T cells (allo T cells) is lost, but a new function of antigen transporting ability is acquired.
  • cells specialized for antigen transport capable of delivering the pathogen antigen to the recipient dendritic cells of lymphoid organs throughout the body without producing an alloimmune response have been produced.
  • the cells can be administered to any recipient of different MHC and in that sense can be regarded as "standardized" allo-T cells. This standardized allo-T cell can be used as a completely new type of vaccine that is relatively safe and versatile.
  • the allo-T cells that have been subjected to the above treatment are administered to the recipient allogeneic target individual.
  • the vaccine of the present invention can be used as a pharmaceutical composition for a disease associated with the antigen, depending on the antigen used.
  • the pharmaceutical composition of the present invention can be administered according to the form of parenteral administration such as injection.
  • parenteral administration such as injection.
  • local injection into the abdominal cavity and the like are exemplified.
  • the administration method examples include intravenous administration and intraperitoneal administration.
  • the dose is appropriately selected according to the administration route, administration subject, age, body weight, sex of the patient, symptoms and other conditions.
  • the daily dose of allo-T cells used as a vaccine is about 10 7 cells/ml to 10 9 cells/ml in the case of intravenous administration, preferably about 5 ⁇ 10 7 cells/ml to 5 ⁇ 10 8 cells/ml, It can be administered once a day or divided into several times.
  • the vaccine of the present invention can induce neutralizing antibodies systemically and multifocally not only in humans with normal spleen function but also in humans with decreased or splenectomy. Therefore, the allo T cell of the present invention can be used as a neutralizing antibody inducer.
  • the F1 rat system can be said to be a model similar to the system using siRNA.
  • autologous T cells cannot induce an antibody response, it is a necessary condition in the present invention to use allo T cells.

Abstract

A vaccine against a pathogen in an allogeneic recipient individual, said vaccine containing T cells derived from a donor that have been subjected to a treatment for suppressing the expression of a histocompatibility antigen, a treatment for suppressing the activation thereof and a treatment for labeling the target antigen.

Description

Ｔ細胞ワクチンT cell vaccine
　本発明は、リンパ器官で多所性に中和抗体を誘導するＴ細胞ワクチンに関する。 The present invention relates to a T cell vaccine that induces neutralizing antibodies in lymphoid organs in multiple locations.
　ＤＳＴ（ｄｏｎｏｒ　ｓｐｅｃｉｆｉｃ　ｂｌｏｏｄ　ｔｒａｎｓｆｕｓｉｏｎ／ドナー特異的輸血）とは、臓器移植前にドナー（アロ）血液を宿主に投与し、免疫寛容を誘導する治療法である（非特許文献１：Ｃｌｉｎ　Ｔｒａｎｓｐｌａｎｔ　２５：３１７，２０１１）。１９７０年代から腎移植の前にＤＳＴを行うと、拒絶反応がドナー特異的に抑制されること　ドナー組織適合抗原（ＭＨＣ抗原）に対する抗体が一回の輸血だけでも簡単に作られることが臨床現場から報告されたが、副作用が起こることもあるため、拒絶反応を容易に治療できる免疫抑制剤の登場によりほとんど行われなくなっている。そのため、アロ抗体産生応答（ＡＦＣ応答）が血液中の何の成分により、どこで、どのようにおこるのか、作られた抗体が免疫抑制を含めどのような作用を持つかについては未だに解明されていない。 DST (donor specific blood transfusion/donor-specific blood transfusion) is a treatment method of inducing immune tolerance by administering a donor (allo) blood to a host before organ transplantation (Non-patent document 1: Clin Transplant 25:317). , 2011). Performing DST before kidney transplantation since the 1970s suppresses rejection reaction in a donor-specific manner. From clinical practice, antibodies against donor histocompatibility antigen (MHC antigen) can be easily produced by a single blood transfusion. Although it has been reported, side effects may occur, so it has almost disappeared due to the advent of immunosuppressive drugs that can easily treat rejection reactions. Therefore, by what component in the allo antibody response (AFC response) blood, where, how happen, made antibodies is not yet elucidated or with what effects, including immunosuppression ..
　本発明者は、これまでｉｎ　ｖｉｖｏ免疫学の観点からラットの移植免疫応答における免疫担当細胞の動態と機能を臓器レベルで免疫組織学的に解析してきた（非特許文献２：Ｃｅｌｌ　Ｔｒａｎｓｐｌａｎｔ　２１：５８１，２０１２；非特許文献３：Ｃｅｌｌ　Ｔｒａｎｓｐｌａｎｔ　１９：７６５，２０１０；非特許文献４：Ａｒｃｈ　Ｈｉｓｔｏｌ　Ｃｙｔｏｌ　７３：１，２０１０；非特許文献５：Ｈｅｐａｔｏｌｏｇｙ　５６：１５３２，２０１２）。近年では、免疫応答のメカニズムの解明のために、近親交配系でのＧｖＨ病ラットモデルを用い、チミジンアナログであるＥｄＵ（Ｅｔｈｙｎｙｌ　ｄｅｏｘｙｕｒｉｄｉｎｅ）を用いた多重蛍光免疫染色法による免疫組織学とフローサイトメトリー（ＦＣＭ）を並行しておこなう定性定量解析法を確立した（非特許文献６：Ｈｉｓｔｏｃｈｅｍ　Ｃｅｌｌ　Ｂｉｏｌ　１４４：１９５，２０１５）。 The present inventor has previously analyzed the dynamics and functions of immunocompetent cells in the transplantation immune response of rats from the viewpoint of in vivo immunology at an organ level by immunohistological analysis (Non-patent document 2: Cell Transplant 21:581). , 2012; Non-patent document 3: Cell Transplant 19:765, 2010; Non-patent document 4: Arch Histol Cyto 73:1, 2010; Non-patent document 5: Hepatology 56: 1532, 2012). In recent years, in order to elucidate the mechanism of immune response, immunohistochemistry and flow cytometry by a multiple fluorescence immunostaining method using a thymidine analog EdU (Ethynyl deoxyuridine) using a GvH disease rat model in an inbred system. A qualitative quantitative analysis method for performing (FCM) in parallel was established (Non-patent document 6: Histochem Cell Biol 144:195, 2015).
この手法により抗原貪食の提示の細胞間相互作用や免疫性増殖応答が、どこで、どの程度起こるかの定性定量解析が可能となり、免疫応答のメカニズムが解析できるようになった。そこで、本研究の予備実験としてＤＳＴ後に宿主の免疫応答を解析したところ、主に脾臓でアロＡＦＣ応答が起こること、抗体はドナーＩ型ＭＨＣ抗原（ＭＨＣＩと省略）に対するものであることが明らかになり（非特許文献７：Ｉｎｔ　Ｉｍｍｕｎｏｌ　３０：５３，２０１８）、ドナー血液成分中、白血球、特にＴ細胞分画が有効で、赤血球などのそれ以外の成分は無効であることがわかった。 By this method, qualitative and quantitative analysis of where and to what extent the cell-cell interaction of presentation of antigen phagocytosis and immune proliferative response occur became possible, and the mechanism of immune response became possible. Therefore, when the host immune response was analyzed after DST as a preliminary experiment of this study, it was revealed that an allo-AFC response mainly occurs in the spleen and that the antibody is against the donor type I MHC antigen (abbreviated as MHCI). (Non-patent document 7: Int Immunol 30:53, 2018), it was found that leukocytes, particularly T cell fraction, were effective in the donor blood components, and other components such as red blood cells were ineffective.
　アロ免疫応答は、ドナー抗原提示樹状細胞（ＤＣ）が宿主Ｔ細胞と直接会合してドナーＭＨＣ抗原を提示する直接感作と、ドナー細胞由来のＭＨＣ抗原が宿主のＤＣに取り込まれて提示される間接感作により起こるとされる（非特許文献８：Ｉｍｍｕｎｉｔｙ　１４：３５７，２００１）。ＡＦＣ応答（液性免疫）については、Ｔｈ２ヘルパーＴ細胞や濾胞ヘルパーＴ細胞（Ｆｏｌｌｉｃｕｌａｒ　ｈｅｌｐｅｒ　Ｔ　ｃｅｌｌｓ／Ｔｆｈ）により誘導され、ＩＬ−４，ＩＬ−１０などのサイトカインや転写因子であるＧＡＴＡ−３遺伝子の発現が優位になることが報告されている（非特許文献９：Ｉｍｍｕｎｉｔｙ　３０：３２４，２００９）。 The allo-immune response is presented by direct sensitization in which donor antigen-presenting dendritic cells (DCs) directly associate with host T cells to present donor MHC antigens, and MHC antigens derived from donor cells are taken up by host DCs and presented. It is said to be caused by indirect sensitization (Non-Patent Document 8: Immunity 14:357, 2001). AFC response (humoral immunity) is induced by Th2 helper T cells and follicular helper T cells (Follicular helper T cells/Tfh), and the GATA-3 gene, which is a cytokine or transcription factor such as IL-4 and IL-10. It has been reported that the expression of the above is dominant (Non-patent document 9: Immunity 30:324, 2009).
血液中にＤＣはほとんど存在しないので、ＤＳＴによる抗体産生応答は、主に脾臓内での間接感作による免疫応答が関与していることが予測される。ここで、脾臓での免疫応答は白脾髄内の動脈周囲リンパ球鞘（ＰＡＬＳ／Ｔ細胞領域）で起こり、そこにＤＣが局在している事、Ｔ及びＢ細胞は免疫監視のために多くが全身を再循環しており、常に血液からＰＡＬＳ内に遊走してＰＡＬＳにしばらく留まる事、Ｔ細胞はさらにＰＡＬＳのＤＣと会合し抗原情報を確認する事がわかっている（非特許文献１０：Ａｒｃｈ　Ｈｉｓｔｏｌ　Ｃｙｔｏｌ　７３：１，２０１０）。 Since DC are rarely present in blood, it is expected that the antibody production response by DST mainly involves an immune response by indirect sensitization in the spleen. Here, the immune response in the spleen occurs in the periarterial lymphocyte sheath (PALS/T cell region) in the white pulp of the spleen, where DC is localized, and T and B cells are used for immune surveillance. It is known that most of them recirculate throughout the body, always migrate from blood into PALS and stay in PALS for a while, and T cells further associate with DC of PALS to confirm antigen information (Non-Patent Document 10). : Arch Histol Cyto 73:1, 2010).
　ところで、病原病原体に対するワクチンは、通常、筋肉内又は皮下投与して、主に脾臓で中和抗体の産生を誘導するものである。そのため、脾臓機能が低い場合や摘脾した場合は通常のワクチン効果があまり期待できない。 By the way, a vaccine against a pathogen is usually administered intramuscularly or subcutaneously to induce the production of neutralizing antibodies mainly in the spleen. Therefore, when the spleen function is low or the spleen is removed, the usual vaccine effect cannot be expected so much.
　上記の状況下、多所性に中和抗体を誘導するワクチンの開発が望まれていた。 Under the above circumstances, it was desired to develop a vaccine that induces neutralizing antibodies in multiple places.
　本発明者は、上記課題を解決するために鋭意検討を行った結果、アロＴ細胞に病原体抗原を標識し、これを個体に接種することにより、脾臓のみならず全身のリンパ節で多所性に中和抗体を誘導することを見出し、本発明を完成するに至った。 The present inventor has conducted extensive studies to solve the above-mentioned problems, and as a result, by labeling allo-T cells with a pathogen antigen and inoculating this to an individual, not only the spleen but also lymph nodes throughout the body are multifocal. The inventors have found that a neutralizing antibody is induced in Escherichia coli and have completed the present invention.
　すなわち、本発明は以下の通りである。
（１）組織適合抗原の発現抑制処理、活性化抑制処理、及び病原体抗原の標識処理がされたドナー由来Ｔ細胞を含む、同種異型レシピエント個体における前記病原体抗原に対するワクチン。
（２）組織適合抗原の発現抑制処理が、組織適合抗原遺伝子に対するＲＮＡ干渉処理又は当該遺伝子のノックアウト処理である（１）に記載のワクチン。
（３）活性化抑制処理が、代謝拮抗剤若しくはＤＮＡ合成阻害剤処理又は放射線照射処理である（１）又は（２）に記載のワクチン。
（４）病原体がウイルス又は細菌である（１）~（３）のいずれか１項に記載のワクチン。
（５）ウイルスがインフルエンザウイルスである（４）に記載のワクチン。
（６）リンパ器官で多所性に中和抗体を誘導する、（１）~（５）のいずれか１項に記載のワクチン。 That is, the present invention is as follows.
(1) A vaccine against the pathogen antigen in an allogeneic recipient individual, which comprises donor-derived T cells that have undergone histocompatibility antigen expression suppression treatment, activation suppression treatment, and pathogen antigen labeling treatment.
(2) The vaccine according to (1), wherein the treatment for suppressing the expression of the histocompatibility antigen is RNA interference treatment for the histocompatibility antigen gene or knockout treatment of the gene.
(3) The vaccine according to (1) or (2), wherein the activation suppression treatment is an antimetabolite or DNA synthesis inhibitor treatment or irradiation treatment.
(4) The vaccine according to any one of (1) to (3), wherein the pathogen is a virus or a bacterium.
(5) The vaccine according to (4), wherein the virus is influenza virus.
(6) The vaccine according to any one of (1) to (5), which induces neutralizing antibody in multiple sites in lymphoid organs.
（７）組織適合抗原の発現抑制処理、活性化抑制処理、及び病原体抗原の標識処理がされたドナー由来Ｔ細胞を含む、同種異型レシピエント個体における中和抗体誘導剤。
（８）組織適合抗原の発現抑制処理が、組織適合抗原遺伝子に対するＲＮＡ干渉処理又は当該遺伝子のノックアウト処理である（７）に記載の中和抗体誘導剤。
（９）活性化抑制処理が、代謝拮抗剤若しくはＤＮＡ合成阻害剤又は放射線照射処理である（７）又は（８）に記載の中和抗体誘導剤。
（１０）病原体がウイルス又は細菌である（７）~（９）のいずれか１項に記載の中和抗体誘導剤。
（１１）ウイルスがインフルエンザウイルスである（１０）に記載の中和抗体誘導剤。
（１２）リンパ器官で多所性に中和抗体を誘導する、（７）~（１１）のいずれか１項に記載の中和抗体誘導剤。 (7) A neutralizing antibody inducer in an allogeneic recipient individual, which comprises donor-derived T cells that have undergone histocompatibility antigen expression suppression treatment, activation suppression treatment, and pathogen antigen labeling treatment.
(8) The neutralizing antibody inducer according to (7), wherein the treatment for suppressing the expression of the histocompatibility antigen is RNA interference treatment for the histocompatibility antigen gene or knockout treatment of the gene.
(9) The neutralizing antibody inducer according to (7) or (8), wherein the activation suppression treatment is an antimetabolite, a DNA synthesis inhibitor, or irradiation treatment.
(10) The neutralizing antibody inducer according to any one of (7) to (9), wherein the pathogen is a virus or a bacterium.
(11) The neutralizing antibody inducer according to (10), wherein the virus is influenza virus.
(12) The neutralizing antibody inducer according to any one of (7) to (11), which induces neutralizing antibodies in multiple sites in lymphoid organs.
　本発明により、Ｔ細胞ワクチン及び中和抗体誘導剤が提供される。本発明のＴ細胞ワクチンは、多所性に中和抗体を誘導することが可能である。 The present invention provides a T cell vaccine and a neutralizing antibody inducer. The T cell vaccine of the present invention is capable of inducing neutralizing antibodies in multiple points.
実施例の実験方法を示す図である。It is a figure which shows the experimental method of an Example. 実施例の特異的抗体産生細胞の染色法の模式図である。FIG. 3 is a schematic diagram of a staining method for specific antibody-producing cells in Examples. 実施例１の実験結果を示す図である。セミアロＴ細胞投与（親のＴ細胞を一代雑種Ｆ１レシピエントに投与する系）により、標識抗原のｐｈｙｃｏｅｒｙｔｈｒｉｎ（ＰＥ）に対する特異抗体が血清中に検出され（左グラフ、赤矢印）、頚部リンパ節切片上に抗体産生細胞（青、右図）が出現したが、アロ抗体は出なかった（黒矢印）。一方、アロＴ細胞ではアロ抗体が大量に出るが、ＰＥ抗体価は低かった。FIG. 5 is a diagram showing an experimental result of Example 1. By the administration of semi-allo T cells (a system in which parental T cells are administered to a first-generation hybrid F1 recipient), a specific antibody against the labeled antigen phycoerythrin (PE) was detected in serum (left graph, red arrow), and cervical lymph node section Antibody-producing cells (blue, right panel) appeared on the top, but alloantibodies did not appear (black arrow). On the other hand, in the allo T cells, a large amount of alloantibodies appeared, but the PE antibody titer was low.
　本発明者は、ドナーＴ細胞が輸血後に脾臓ＰＡＬＳに遊走し、宿主のＤＣと会合した後にドナーＭＨＣＩ抗原を何らかの形で受け渡し、そこで最も効率よく間接感作を起こし、その結果、Ｔｈ２やＴｆｈが誘導され、効率的に抗原特異的な抗体が産生されるという仮説を着想した。 The present inventor has found that donor T cells migrate to the spleen PALS after blood transfusion and, after associating with the host DC, transfer the donor MHCI antigen in some form and cause indirect sensitization most efficiently there, resulting in Th2 and Tfh The inventor has come up with the hypothesis that antigen-specific antibodies are induced and efficiently produced.
　本発明は、組織適合抗原の発現抑制処理、活性化抑制処理及び病原体抗原の標識処理がされたドナー由来Ｔ細胞を含む、同種異型レシピエント個体における前記病原体に対するワクチン及び中和抗体誘導剤に関する。
　同種異系（アロ）Ｔ細胞は、自己Ｔ細胞と同様に全身のリンパ器官に再循環し血行性遊走する能力を持っているが、Ｔ細胞受容体を介して組織在住の樹状細胞を効率よく刺激すると、当該Ｔ細胞自身のＩ型組織適合抗原（ＭＨＣ）に対するアロ抗体が容易に誘導されることを見出した。この知見は、自己Ｔ細胞とは異なるものである。 The present invention relates to a vaccine against a pathogen in an allogeneic recipient individual and a neutralizing antibody inducer, which comprises donor-derived T cells that have undergone histocompatibility antigen expression suppression treatment, activation suppression treatment, and pathogen antigen labeling treatment.
Allogeneic (allo) T cells, like autologous T cells, have the ability to recirculate to systemic lymphoid organs and undergo hematogenous migration, but efficiently mobilize tissue-resident dendritic cells via T cell receptors. It was found that when stimulated well, alloantibodies against the type I histocompatibility antigen (MHC) of the T cell itself were easily induced. This finding is different from autologous T cells.
　本発明者は上記知見を利用して、アロＴ細胞（ドナー由来のＴ細胞）にインフルエンザなどの病原体抗原を標識し、これを自己（ドナー）以外のレシピエントに投与することにより、脾臓のみならずリンパ節で多所性に中和抗体を誘導することを見出した。リンパ節はヒトで数百個あるので、全身のリンパ節で中和抗体を誘導できる本発明のワクチンは効率が高く、新しい概念のワクチンとして利用できる。
　ここで、本発明において使用されるＴ細胞は、一の個体から採取される。このＴ細胞に上記処理を施した後、当該一の個体とは同種であるが他の個体（つまり同種異系の個体）に投与する。従って、使用するＴ細胞を本明細書では「アロＴ細胞」という。 The present inventor utilizes the above findings to label allo T cells (donor-derived T cells) with a pathogen antigen such as influenza, and administer this to recipients other than self (donor) to obtain only the spleen. It was found that multiple neutralizing antibodies are induced in the normal lymph node. Since there are hundreds of lymph nodes in humans, the vaccine of the present invention capable of inducing neutralizing antibody in systemic lymph nodes is highly efficient and can be used as a vaccine of a new concept.
Here, the T cells used in the present invention are collected from one individual. After this T cell is subjected to the above treatment, it is administered to another individual who is the same species as the one individual but is allogeneic. Therefore, the T cells used are referred to herein as "allo T cells."
　本発明においては、ドナー血液から採取されたＴ細胞（アロＴ細胞）に対し、組織適合抗原の発現抑制処理、活性化抑制処理、及び病原体抗原の標識処理を行う。このような処理がされたアロＴ細胞を、当該ドナーとは異なる同種異系個体（レシピエント）に投与する。 In the present invention, T cells (allo T cells) collected from donor blood are subjected to histocompatibility antigen expression suppression treatment, activation suppression treatment, and pathogen antigen labeling treatment. The thus treated allo-T cells are administered to an allogeneic individual (recipient) different from the donor.
　組織適合抗原の発現抑制処理とは、アロ抗原性の抑制処理を意味し、組織適合抗原の発現を抑制するための処理としては、例えば組織適合抗原遺伝子に対するＲＮＡ干渉処理又は当該遺伝子のノックアウト処理が挙げられる。
　ＲＮＡ干渉（ＲＮＡｉ）により遺伝子発現を抑制し得る合成核酸分子としては、例えばｓｉＲＮＡ（ｓｍａｌｌ　ｉｎｔｅｒｆｅｒｉｎｇ　ＲＮＡ）、マイクロＲＮＡ（ｍｉＲＮＡ）及びｓｈＲＮＡ（ｓｈｏｒｔ　ｈａｉｒｐｉｎ　ＲＮＡ）が挙げられる。 The expression suppressing treatment of the histocompatibility antigen means an alloantigenicity suppressing treatment, and as the treatment for suppressing the expression of the histocompatibility antigen, for example, RNA interference treatment to the histocompatibility antigen gene or knockout treatment of the gene is performed. Can be mentioned.
Examples of synthetic nucleic acid molecules capable of suppressing gene expression by RNA interference (RNAi) include siRNA (small interfering RNA), microRNA (miRNA) and shRNA (short hairpin RNA).
　ｓｉＲＮＡは、当分野において周知の基準に基づいて設計できる。例えば、標的となる組織適合抗原遺伝子のｍＲＮＡの標的セグメントは、好ましくはＡＡ、ＴＡ、ＧＡ又はＣＡで始まる連続する１５~３０塩基、好ましくは１９~２５塩基のセグメントを選択することができる。ｓｉＲＮＡのＧＣ比は、３０~７０％、好ましくは３５~５５％である。
　ｓｉＲＮＡは、二本鎖部分を生成するために自身の核酸上で折り畳む一本鎖ヘアピンＲＮＡ分子として生成される。 siRNA can be designed based on criteria well known in the art. For example, as the target segment of the mRNA of the target histocompatibility antigen gene, a continuous 15 to 30 bases, preferably 19 to 25 bases segment starting with AA, TA, GA or CA can be selected. The siRNA has a GC ratio of 30 to 70%, preferably 35 to 55%.
siRNA is produced as a single-stranded hairpin RNA molecule that folds on its own nucleic acid to produce a double-stranded portion.
　ｓｉＲＮＡ分子は、通常の化学合成により得ることができるが、センス及びアンチセンスｓｉＲＮＡ配列を含有する発現ベクターを用いて生物学的に生成することも可能である。
　ｓｉＲＮＡを細胞に導入するには、ｉｎ　ｖｉｔｒｏで合成したｓｉＲＮＡをプラスミドＤＮＡに連結してこれを細胞に導入する方法、２本鎖ＲＮＡをアニールする方法などを採用することができる。 siRNA molecules can be obtained by conventional chemical synthesis, but can also be produced biologically using expression vectors containing sense and antisense siRNA sequences.
In order to introduce siRNA into cells, a method of ligating siRNA synthesized in vitro with plasmid DNA and introducing it into cells, a method of annealing double-stranded RNA, or the like can be adopted.
　ｓｈＲＮＡは、一本鎖の一部の領域が他の領域と相補鎖を形成されたステムループ構造を有するＲＮＡ分子である。従って、ｓｈＲＮＡは、その一部がステムループ構造を形成するように設計する。例えば、ある領域の配列を配列Ａとし、配列Ａに対する相補鎖を配列Ｂとすると、配列Ａ、スペーサー、配列Ｂの順でこれらの配列が一本のＲＮＡ鎖に存在するように連結し、全体で４５~６０塩基の長さとなるように設計する。配列Ａは、標的となる組織適合抗原遺伝子の一部の領域の配列であり、標的領域は特に限定されるものではなく、任意の領域を候補にすることが可能である。そして配列Ａの長さは１９~２５塩基、好ましくは１９~２１塩基である。 ShRNA is an RNA molecule having a stem-loop structure in which a part of a single strand forms a complementary strand with another region. Therefore, shRNA is designed such that a part thereof forms a stem-loop structure. For example, if the sequence of a certain region is sequence A and the complementary strand to sequence A is sequence B, sequence A, spacer, sequence B are linked in this order so that these sequences exist in one RNA strand, and It is designed to have a length of 45 to 60 bases. Sequence A is a sequence of a partial region of the target histocompatibility antigen gene, and the target region is not particularly limited, and any region can be used as a candidate. The length of the sequence A is 19 to 25 bases, preferably 19 to 21 bases.
　さらに、本発明は、マイクロＲＮＡ（ｍｉＲＮＡ）を用いて上記遺伝子の発現を抑制することができる。ｍｉＲＮＡとは、細胞内に存在する長さ２０~２５塩基ほどの１本鎖ＲＮＡであり、他の遺伝子の発現を調節する機能を有すると考えられているｎｃＲＮＡ（ｎｏｎ　ｃｏｄｉｎｇ　ＲＮＡ）の一種である。ｍｉＲＮＡは、ＲＮＡに転写された際にプロセシングを受けて生じ、標的配列の発現を抑制するヘアピン構造を形成する核酸として存在する。
　ｍｉＲＮＡも、ＲＮＡｉに基づく阻害性核酸であるため、ｓｈＲＮＡ又はｓｉＲＮＡに準じて設計し合成することができる。 Furthermore, the present invention can suppress the expression of the above genes using microRNA (miRNA). miRNA is a single-stranded RNA having a length of about 20 to 25 bases existing in cells, and is a kind of ncRNA (non coding RNA) which is considered to have a function of regulating the expression of other genes. .. miRNA exists as a nucleic acid forming a hairpin structure that is generated by being processed when it is transcribed into RNA and suppresses the expression of a target sequence.
Since miRNA is also an inhibitory nucleic acid based on RNAi, it can be designed and synthesized according to shRNA or siRNA.
　また、本発明においては、組織適合抗原遺伝子をノックアウトすることもできる。ノックアウトする方法としては、例えばＣＲＩＳＰＲ／Ｃａｓ９によるノックアウトなどが挙げられるが、これらに限定されるものではない。
　ｓｉＲＮＡ処理又はＣＲＩＳＰＲ／Ｃａｓ９によるｇｅｎｅ　ｋｎｏｃｋｏｕｔ処理などは、公知文献に記載の方法（Ｃａｎｃｅｒ　Ｃｅｌｌ　Ｉｎｔ．１３：１１２，２０１３；Ｃｌｉｎ　Ｃａｎｃｅｒ　Ｒｅｓ２３：２２５５，２０１７）により行うことができる。 Moreover, in the present invention, a histocompatibility antigen gene can also be knocked out. Examples of the knockout method include, but are not limited to, knockout by CRISPR/Cas9.
The siRNA treatment, the gene knockout treatment with CRISPR/Cas9, or the like can be performed by a method described in a known document (Cancer Cell Int. 13:112, 2013; Clin Cancer Res 23:2255, 2017).
　Ｔ細胞の活性化抑制処理とは、Ｔ細胞のＧｖＨ病発症などのリスクを除去する処理を意味し、活性化抑制処理としては、代謝拮抗剤若しくはＤＮＡ合成阻害剤処理、又は放射線照射処理が挙げられる。本発明において使用可能な代謝拮抗剤及びＤＮＡ合成阻害剤、並びに放射線照射を以下に例示する。 The T cell activation suppressing treatment means a treatment for removing the risk of T cell onset of GvH disease, and the activation suppressing treatment includes an antimetabolite or a DNA synthesis inhibitor treatment, or irradiation treatment. To be The antimetabolites and DNA synthesis inhibitors that can be used in the present invention, and irradiation are exemplified below.
＜代謝拮抗剤又はＤＮＡ合成阻害剤＞
　葉酸類似体：メトトレキサート、ペメトレキセド、プララトレキサート等
　プリン類似体：メルカプトプリン、チオグアニン、クラドリビン、フルダラビン等
　ピリミジン類似体：シタラビン、フルオロウラシル、テガフール、ゲムシタビン等
　抗生物質：マイトマイシンＣ、アクチノマイシン、ドキソルビシン、エピルビシン等
　アルキル化剤：シクロフォスファミド、メルファラン、チオテパ、ブスルファン等
　白金製剤：シスプラチン、イプロプラチン、カルボプラチン等
　トポイソメラーゼ阻害薬：イリノテカン、ノギテカン、エトポシド、アントラサイクリン系薬剤等 <Antimetabolite or DNA synthesis inhibitor>
Folic acid analogs: methotrexate, pemetrexed, pralatrexate, etc. Purine analogs: mercaptopurine, thioguanine, cladribine, fludarabine, etc. Pyrimidine analogs: cytarabine, fluorouracil, tegafur, gemcitabine, etc. Antibiotics: mitomycin C, actinomycin, doxorubicin, etc. Alkylating agents: cyclophosphamide, melphalan, thiotepa, busulfan, etc. Platinum preparations: cisplatin, iproplatin, carboplatin, etc. Topoisomerase inhibitors: irinotecan, nogitecan, etoposide, anthracycline drugs, etc.
　代謝拮抗剤又はＤＮＡ合成阻害剤の使用量は、マイトマイシンＣならば２０μｇ／５ｘ１０７／ｍｌで３７℃３０分間処理、他は適正量を用いる。この処理は、公知文献に記載の方法（Ｈｅｐａｔｏｌｏｇｙ　５６：１５３２，２０１２）により行うことができる。 The amount of the antimetabolite or the DNA synthesis inhibitor to be used is 20 μg/5×10 7 /ml for mitomycin C, treated at 37° C. for 30 minutes, and other appropriate amount is used. This treatment can be performed by a method described in a known document (Hepatology 56: 1532, 2012).
＜放射線照射処理＞
　Ｘ線、ガンマ線等
　放射線照射量は、１０８個の細胞あたり１０~５０Ｇｙ、好ましくは１５Ｇｙである。こらの処理は、公知文献に記載の方法（Ａｒｃｈ　Ｐａｔｈｏｌ　Ｌａｂ　Ｍｅｄ　１４２：６６２，２０１８）により行うことができる。 <Radiation treatment>
The radiation dose of X-rays, gamma rays, etc. is 10 to 50 Gy, preferably 15 Gy per 10 8 cells. These treatments can be performed by a method described in a known document (Arch Pathol Lab Med 142:662, 2018).
　本発明において、抗原として使用する病原体は特に限定されるものではなく、ウイルス、細菌、原虫などが挙げられる。抗原標識には、標的抗原の遺伝子ベクターを作製し、Ｔ細胞への遺伝子導入技術を用いて行う。 In the present invention, the pathogen used as an antigen is not particularly limited and includes viruses, bacteria, protozoa and the like. The antigen labeling is performed by preparing a gene vector of the target antigen and using a gene transfer technique into T cells.
　ウイルスとしては、インフルエンザウイルス、肝炎ウイルス、帯状疱疹、麻疹・風疹、パピローマ（ＨＰＶ）、ヒト免疫不全（ＨＩＶ）ウイルスなどが挙げられる。
　細菌としては、肺炎球菌、髄膜炎菌、ジフテリア菌、破傷風菌、百日咳菌、結核などが挙げられる。
　原虫としては、マラリアなどが挙げられる。 Examples of the virus include influenza virus, hepatitis virus, herpes zoster, measles/rubella, papilloma (HPV), human immunodeficiency (HIV) virus and the like.
Examples of bacteria include pneumococcus, meningococcus, diphtheria, tetanus, pertussis, tuberculosis, and the like.
Examples of the protozoa include malaria.
　アロＴ細胞ワクチンは、ドナー由来Ｔ細胞を、（ａ）組織適合抗原の発現抑制処理、（ｂ）活性化抑制処理及び（ｃ）病原体抗原の標識処理することにより調製されるが、その順序は特に限定されるものではない。上記処理を（ａ）、（ｂ）、（ｃ）の順序で行ってもよく、別の順序でもよい。また（ｃ）については、抗ＣＤ４抗体などＴ細胞を特異的に認識する抗体に、病原体抗原を結合させて当該抗体と病原体抗原との複合体を作製し、これをドナーＴ細胞に結合させることもできる。 The allo T cell vaccine is prepared by treating donor-derived T cells with (a) histocompatibility antigen expression suppression treatment, (b) activation suppression treatment, and (c) pathogen antigen labeling treatment, in the order of It is not particularly limited. The above processes may be performed in the order of (a), (b) and (c), or may be performed in another order. Regarding (c), a pathogen antigen is bound to an antibody that specifically recognizes T cells such as an anti-CD4 antibody to prepare a complex of the antibody and the pathogen antigen, and the complex is bound to a donor T cell. Can also
　このような処理により、ドナーＴ細胞（アロＴ細胞）のレシピエントにＧｖＨ病やアロ抗体産生誘導などを起こすアロ免疫能という本来の機能が失われるが、抗原輸送能という新しい機能を獲得することとなる。これにより、アロ免疫応答を起こさずに病原体抗原を全身のリンパ器官のレシピエント樹状細胞に届けることができる抗原輸送専門の細胞を作製したことになる。本細胞はＭＨＣの異なるどのレシピエントにも投与可能であり、その意味で「標準化」したアロＴ細胞と位置づけることができる。この標準化アロＴ細胞は、比較的安全で汎用性が高い全く新しいタイプのワクチンとして使用することが可能である。 By such treatment, the original function of alloimmunity that causes GvH disease or induction of alloantibody production in the recipient of donor T cells (allo T cells) is lost, but a new function of antigen transporting ability is acquired. Becomes As a result, cells specialized for antigen transport capable of delivering the pathogen antigen to the recipient dendritic cells of lymphoid organs throughout the body without producing an alloimmune response have been produced. The cells can be administered to any recipient of different MHC and in that sense can be regarded as "standardized" allo-T cells. This standardized allo-T cell can be used as a completely new type of vaccine that is relatively safe and versatile.
　上記処理が行われたアロＴ細胞は、レシピエントである同種異系の対象個体に投与する。これにより、同種異系個体では、リンパ器官で多所性に標識抗原に対する中和抗体を誘導することが可能となる。
　本発明のワクチンは、使用する抗原に応じて、当該抗原に関連する疾患に対する医薬組成物として使用することもできる。本発明の医薬組成物は、注射剤等の非経口投与剤などの形態に応じて投与することができる。好ましくは、静脈注射のほか、腹腔等への局部注射等が例示される。 The allo-T cells that have been subjected to the above treatment are administered to the recipient allogeneic target individual. As a result, in allogeneic individuals, it becomes possible to induce neutralizing antibodies against the labeled antigen at multiple sites in lymphoid organs.
The vaccine of the present invention can be used as a pharmaceutical composition for a disease associated with the antigen, depending on the antigen used. The pharmaceutical composition of the present invention can be administered according to the form of parenteral administration such as injection. Preferably, in addition to intravenous injection, local injection into the abdominal cavity and the like are exemplified.
　投与方法としては、静脈投与、腹腔内投与などが挙げられる。
　投与量は、投与経路、投与対象、患者の年齢、体重、性別、症状その他の条件により適宜選択される。ワクチンとして使用されるアロＴ細胞の一日投与量としては、静脈投与の場合は１０７個／ｍｌ~１０９個／ｍｌ、好ましくは５ｘ１０７個／ｍｌ~５ｘ１０８個／ｍｌ程度であり、１日１回投与することもでき、数回に分けて投与することもできる。
　本発明のワクチンは、脾臓機能が正常なヒトだけでなく、低下または脾摘したヒトであっても全身性・多所性に中和抗体を誘導できる。
　従って、本発明のアロＴ細胞は、中和抗体誘導剤として使用することができる。 Examples of the administration method include intravenous administration and intraperitoneal administration.
The dose is appropriately selected according to the administration route, administration subject, age, body weight, sex of the patient, symptoms and other conditions. The daily dose of allo-T cells used as a vaccine is about 10 7 cells/ml to 10 9 cells/ml in the case of intravenous administration, preferably about 5×10 7 cells/ml to 5×10 8 cells/ml, It can be administered once a day or divided into several times.
The vaccine of the present invention can induce neutralizing antibodies systemically and multifocally not only in humans with normal spleen function but also in humans with decreased or splenectomy.
Therefore, the allo T cell of the present invention can be used as a neutralizing antibody inducer.
　以下、実施例により本発明をさらに具体的に説明する。但し、本発明の範囲はこれらの実施例により限定されるものではない。
［実施例１］ Hereinafter, the present invention will be described more specifically with reference to Examples. However, the scope of the present invention is not limited to these examples.
[Example 1]
　方法
　親ラットのＴ細胞に、抗原としてＦＩＴＣそのもの、または抗ＣＤ４抗体に結合させたｐｈｙｃｏｅｒｙｔｈｒｉｎを標識後、マイトマイシンＣ処理の後、脾摘した一代雑種Ｆ１ラットに静脈内投与し、７日後に種々のリンパ節と血清を採取した。
リンパ節は切片上に特異的な抗体産生細胞を可視化し、血清はフローサイトメーターで特異抗体の定量をおこなった（図１）。
　すなわち、凍結切片上で、まずｐｈｙｃｏｅｒｙｔｈｒｉｎやＦＩＴＣを標識したノーマルマウスＩｇＧを、抗原特異的ＡＦＣの抗体存在部位に結合させた。次に酵素（アルカリホスファターゼ）標識した抗マウスＩｇＧを反応させた後、酵素発色させて可視化した（図２）。 Method After labeling FITC itself as an antigen or phycoerythrin bound to an anti-CD4 antibody on T cells of parental rats, mitomycin C treatment was followed by intravenous administration to splenectomized first-generation hybrid F1 rats. Nodes and serum were collected.
In the lymph node, specific antibody-producing cells were visualized on the section, and in the serum, the specific antibody was quantified by a flow cytometer (Fig. 1).
That is, on a frozen section, first, normal mouse IgG labeled with phycoerythrin or FITC was bound to the antibody-existing site of the antigen-specific AFC. Next, an enzyme (alkaline phosphatase)-labeled anti-mouse IgG was reacted, and then enzyme color was developed for visualization (FIG. 2).
　その結果、特異的抗体産生細胞が複数のリンパ節で検出され、血清には特異抗体を認めたが、ドナー細胞の増殖性応答もアロＭＨＣ抗体も検出されなかった。一方、抗原標識自己Ｔ細胞は抗体応答を誘導しなかった（図３）。 As a result, specific antibody-producing cells were detected in multiple lymph nodes, and specific antibodies were found in serum, but neither proliferative response of donor cells nor allo-MHC antibody was detected. On the other hand, antigen-labeled autologous T cells did not induce an antibody response (Fig. 3).
　父親（Ａ系）のＴ細胞をＦ１ラットに投与したセミアロの組み合わせの場合、Ｆ１（Ｂ系ｘＡ系）は両親のＭＨＣＩを共発現するため、ドナーＴ細胞のＭＨＣ（Ａ系）を認識できない。そのため、ＭＨＣＩに対する反応が起こらないが、ドナーＴ細胞はＴ細胞受容体を介して組織在住の樹状細胞上に発現する母親のＭＨＣ（Ｂ系）を認識し、相互作用を起こして樹状細胞を活性化できるため、結果として標識抗原に対する抗体産生を誘導できたものと考えられる。
ｓｉＲＮＡなどでＭＨＣＩ発現を抑制したドナーＴ細胞はＭＨＣＩに対する反応を理論的に起こさないはずなので、Ｆ１ラットの系はｓｉＲＮＡを用いる系と類似なモデルといえる。一方、自己Ｔ細胞は抗体応答を誘導できないため、アロＴ細胞を用いることが本発明における必要条件である。 In the case of a semi-allo combination in which T cells of a father (A line) are administered to F1 rats, F1 (B line xA line) co-expresses MHC of the parents, and therefore MHC (A line) of the donor T cells cannot be recognized. Therefore, the reaction to MHCI does not occur, but the donor T cell recognizes the maternal MHC (B system) expressed on the tissue-resident dendritic cell through the T cell receptor, and interacts with the dendritic cell. It is considered that, as a result, it was possible to induce antibody production against the labeled antigen as a result.
Since donor T cells that suppress MHCI expression with siRNA or the like should not theoretically cause a reaction to MHCI, the F1 rat system can be said to be a model similar to the system using siRNA. On the other hand, since autologous T cells cannot induce an antibody response, it is a necessary condition in the present invention to use allo T cells.

Claims (12)

  1. 組織適合抗原の発現抑制処理、活性化抑制処理、及び病原体抗原の標識処理がされたドナー由来Ｔ細胞を含む、同種異型レシピエント個体における前記病原体抗原に対するワクチン。 A vaccine against the pathogen antigen in an allogeneic recipient individual, which comprises a donor-derived T cell that has been subjected to histocompatibility antigen expression suppression treatment, activation suppression treatment, and pathogen antigen labeling treatment.
  2. 組織適合抗原の発現抑制処理が、組織適合抗原遺伝子に対するＲＮＡ干渉処理又は当該遺伝子のノックアウト処理である請求項１に記載のワクチン。 The vaccine according to claim 1, wherein the treatment for suppressing the expression of the histocompatibility antigen is RNA interference treatment for the histocompatibility antigen gene or knockout treatment of the gene.
  3. 活性化抑制処理が、代謝拮抗剤若しくはＤＮＡ合成阻害剤処理又は放射線照射処理である請求項１又は２に記載のワクチン。 The vaccine according to claim 1 or 2, wherein the activation suppression treatment is an antimetabolite or DNA synthesis inhibitor treatment or irradiation treatment.
  4. 病原体がウイルス又は細菌である請求項１~３のいずれか１項に記載のワクチン。 The vaccine according to any one of claims 1 to 3, wherein the pathogen is a virus or a bacterium.
  5. ウイルスがインフルエンザウイルスである請求項４に記載のワクチン。 The vaccine according to claim 4, wherein the virus is influenza virus.
  6. リンパ器官で多所性に中和抗体を誘導する、請求項１~５のいずれか１項に記載のワクチン。 The vaccine according to any one of claims 1 to 5, which induces neutralizing antibodies in multiple places in lymphoid organs.
  7. 組織適合抗原の発現抑制処理、活性化抑制処理、及び病原体抗原の標識処理がされたドナー由来Ｔ細胞を含む、同種異型レシピエント個体における中和抗体誘導剤。 A neutralizing antibody inducer in an allogeneic recipient individual, comprising a donor-derived T cell that has been subjected to histocompatibility antigen expression suppression treatment, activation suppression treatment, and pathogen antigen labeling treatment.
  8. 組織適合抗原の発現抑制処理が、組織適合抗原遺伝子に対するＲＮＡ干渉処理又は当該遺伝子のノックアウト処理である請求項７に記載の中和抗体誘導剤。 The neutralizing antibody inducer according to claim 7, wherein the treatment for suppressing the expression of the histocompatibility antigen is RNA interference treatment for the histocompatibility antigen gene or knockout treatment of the gene.
  9. 活性化抑制処理が、代謝拮抗剤若しくはＤＮＡ合成阻害剤又は放射線照射処理である請求項７又は８に記載の中和抗体誘導剤。 The neutralizing antibody inducer according to claim 7 or 8, wherein the activation suppressing treatment is an antimetabolite, a DNA synthesis inhibitor, or irradiation treatment.
  10. 病原体がウイルス又は細菌である請求項７~９のいずれか１項に記載の中和抗体誘導剤。 The neutralizing antibody inducer according to any one of claims 7 to 9, wherein the pathogen is a virus or a bacterium.
  11. ウイルスがインフルエンザウイルスである請求項１０に記載の中和抗体誘導剤。 The neutralizing antibody inducer according to claim 10, wherein the virus is influenza virus.
  12. リンパ器官で多所性に中和抗体を誘導する、請求項７~１１のいずれか１項に記載の中和抗体誘導剤。 The neutralizing antibody inducer according to any one of claims 7 to 11, which induces neutralizing antibodies in multiple places in lymphoid organs.
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