WO2015152429A1 - Transplantation immune response suppression method - Google Patents

Transplantation immune response suppression method Download PDF

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WO2015152429A1
WO2015152429A1 PCT/JP2015/061045 JP2015061045W WO2015152429A1 WO 2015152429 A1 WO2015152429 A1 WO 2015152429A1 JP 2015061045 W JP2015061045 W JP 2015061045W WO 2015152429 A1 WO2015152429 A1 WO 2015152429A1
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transplantation
organ
antibody
cells
host
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PCT/JP2015/061045
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French (fr)
Japanese (ja)
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健二郎 松野
祐司 上田
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学校法人獨協学園獨協医科大学
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Priority to JP2016511659A priority Critical patent/JP6613412B2/en
Publication of WO2015152429A1 publication Critical patent/WO2015152429A1/en

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/22—Urine; Urinary tract, e.g. kidney or bladder; Intraglomerular mesangial cells; Renal mesenchymal cells; Adrenal gland
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/34—Muscles; Smooth muscle cells; Heart; Cardiac stem cells; Myoblasts; Myocytes; Cardiomyocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/37—Digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/37—Digestive system
    • A61K35/39—Pancreas; Islets of Langerhans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/37—Digestive system
    • A61K35/407—Liver; Hepatocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/42—Respiratory system, e.g. lungs, bronchi or lung cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/18—Drugs for disorders of the alimentary tract or the digestive system for pancreatic disorders, e.g. pancreatic enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • 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
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system

Definitions

  • the present invention relates to a method for specifically suppressing transplanted immune response using an organ preservation solution to which an antibody is added.
  • the transplanted immune response (FIG. 1) mainly includes [1] a graft-derived antigen-presenting tree that has migrated hematogenously from a graft (graft) or remained in the graft in a lymph organ of a host undergoing transplantation.
  • Dendritic cells form cell clumps with host T cells, directly present the transplanted antigen (direct sensitization, FIG. 2), and the rejection that the induced killer T cells attack the graft (host) vs graft reaction (HvG reaction)) [2]
  • the graft-derived migrated T cells are directly sensitized by the host DC cells (FIG. 3) and become killer T cells.
  • There is an attacking GvH disease graft vs host reaction, FIG. 4
  • DC cells or T cells graft leukocytes
  • FIG. 2 is a photograph of a host lymphoid tissue (lymph node) that has caused an HvG reaction, in which graft-derived DC cells (blue) form cell clumps with host T cells. It can be seen that a proliferative response (red) occurs.
  • FIG. 3 is a photograph of a host lymphoid tissue (lymph node) that has caused a GvH reaction. T cells derived from grafts (blue) form cell clusters with host DC cells (brown) and are activated T cells. It grows and grows (the nucleus is red).
  • FIG. 4 is a photograph of the host small intestine tissue that caused GvH disease. It can be seen that a large amount of graft-derived T cells (blue) infiltrated into the mucous membrane and caused destruction of the small intestine tissue.
  • An object of the present invention is to provide a method for specifically suppressing transplantation immune response using an organ preservation solution to which an antibody is added.
  • the present inventor binds to target cells in the transplanted organ by adding a specific removal antibody against grafted leukocytes to the organ preservation solution, causing it to react and washing away. It was found that only the target cells were reduced by the action of the antibody after transplantation, and that rejection and GvH disease did not occur, and the present invention was completed.
  • the present invention is as follows.
  • a method for treating an organ for transplantation which comprises storing the organ for transplantation in a preservation solution containing an anti-leukocyte antibody and then washing off the preservation solution before transplantation.
  • a method for suppressing a transplanted immune response wherein an organ for transplantation is stored in a storage solution containing an anti-leukocyte antibody, and then the storage solution is washed out before transplantation.
  • the transplantation immune response is GvH disease after organ transplantation.
  • the method according to (4), wherein the organ is liver, lung, or digestive tract.
  • the anti-leukocyte antibody is an anti-DC cell antibody or an anti-type II MHC antibody.
  • the transplantation immune response is an HvG reaction after organ transplantation.
  • the organ is liver, heart, lung, gastrointestinal tract, pancreas or kidney.
  • the present invention provides a method for specifically suppressing transplanted immune response.
  • the organ while organs for transplantation are refrigerated and preserved, the organ is pretreated by adding antibodies to leukocytes in the preservation solution, thereby suppressing transplantation immune responses such as GvH disease and rejection. it can. Therefore, the method of the present invention is extremely useful for organ transplantation medicine.
  • FIG. 1 is a schematic diagram showing the mechanism of transplantation antigen sensitization and transplantation immune response after organ transplantation.
  • FIG. 2 is a photograph of a host lymphoid tissue (lymph node) that has caused an HvG reaction by a graft-derived DC cell, in which the graft-derived DC cell (blue) forms a cell clump with the host T cell. Has a proliferative response (red).
  • FIG. 3 is a photograph of a host lymphoid tissue (lymph node) that has caused a GvH reaction. T cells derived from grafts (blue) form cell clusters with host DC cells (brown) and are activated T cells. Is growing and growing (nuclei are red).
  • FIG. 2 is a photograph of a host lymphoid tissue (lymph node) that has caused an HvG reaction by a graft-derived DC cell, in which the graft-derived DC cell (blue) forms a cell clump with
  • FIG. 4 is a photograph of the host small intestine tissue that caused GvH disease, in which a large amount of graft-derived T cells (blue) infiltrated into the mucosa and caused destruction of the small intestine tissue.
  • FIG. 5 is a photograph of host lymphoid tissue (spleen) in which an HvG reaction was caused by graft-derived DC cells in a rat liver transplantation model.
  • FIG. 6 is a graph showing that in a rat liver transplantation model, killer T cells (CD8 positive, blue) induced by HvG reaction infiltrate into the graft, and are actively proliferating (the nucleus is red) and activated. It is a photograph of liver tissue.
  • FIG. 1 is a photograph of liver tissue.
  • FIG. 7 is a schematic diagram (1) of the present invention showing suppression of GvH disease after liver transplantation by anti-T cell antibody.
  • FIG. 8 is a schematic diagram (2) of the present invention showing suppression of rejection of transplanted liver by an anti-DC cell antibody.
  • FIG. 9 is a graph showing significant suppression of GvH disease after liver transplantation by anti-T cell antibody in the individual body weight curve and survival period after liver transplantation. Experiments 1 and 2 compare the degree of immunosuppression of the host.
  • FIG. 10 is a schematic diagram showing suppression of rejection of transplanted liver by pre-administration of anti-graft MHC antibody in the host.
  • FIG. 11 is a photograph of a host lymphoid tissue (spleen) showing that graft DC cells have disappeared by the treatment of FIG. 10 or FIG.
  • FIG. 12 is a diagram showing an individual body weight curve and a survival period after liver transplantation. Pre-administration of anti-grafted MHC antibody in the host has been shown to suppress rejection of transplanted liver.
  • the liver of the graft, kidney, heart, lung, intestine, and blood vessels of most other organs are transported to the hospital where the host waits with the organ preservation solution filled and transplanted. It is customary. This organ preservation solution is to be perfused and washed out immediately before transplantation.
  • the present inventor added an antibody to the organ preservation solution, reacted, and then washed away the antibody-containing preservation solution, so that the antibody remained only bound to the target cells in the transplanted organ. It was found that only the target cells were reduced by the action of GvH, and GvH disease did not occur. Rejection has also been shown to be suppressed in a preliminary experiment with pre-administration of antibody host.
  • the organ to be treated is not particularly limited.
  • the liver respiratory organs (bronchi / lung), digestive tract (stomach, small intestine, large intestine), pancreas, heart, Or a kidney etc. are mentioned.
  • the organ may be all or part of the organ as long as it contains blood vessels (can perform vascular anastomosis).
  • each zone (S1 to S8) based on the sub-zone classification of Couinaud may be used.
  • the size can be set to a size that allows vascular anastomosis.
  • the heart and kidney it is the entire organ including the base of the large blood vessel.
  • the respiratory tract it ranges from partial transplantation including bronchi and blood vessels in the lung lobe to full lung transplantation performed with one lung and heart.
  • partial transplantation of a portion from the stomach to the large intestine that is more than a unit capable of vascular anastomosis.
  • the preservation solution used in the present invention includes beerspan solution (manufactured by Astellas Pharma), Custodiol solution (manufactured by Essential Pharmaceuticals), eurocollins solution (manufactured by Irom Pharmaceutical), etc. It is done. The temperature during storage is from 0 ° C to 4 ° C. If necessary, an additive such as a reduced glutathione solution can be added to the preservation solution.
  • the anti-leukocyte antibody means an antibody capable of removing T cells or DC cells, anti-T cell antibodies for T cells, anti-DC cell antibodies for DC cells (for example, anti-CD11c antibody, anti-CD103 antibody, Anti-CD205 antibody), anti-type II MHC antibody, and the like are subject to treatment in the present invention.
  • a method of adding the antibody to the preservation solution containing the organ or a method of using the preservation solution containing the antibody in advance for organ preservation can be employed.
  • the concentration of the antibody in the preservation solution can be appropriately changed according to the size and type of the organ and the titer of the antibody, and is, for example, 2-20 ⁇ g / g organ wet weight, and the lower the better.
  • the above-mentioned antibody is a commercial product, and can be obtained from eBioscience, BioLegend.
  • the method for preserving the organ varies depending on the size and type of the organ or the time until the patient requiring transplantation appears, but is, for example, 1 hour to 36 hours, preferably within 1 day.
  • the storage temperature is 0 ° C. to 4 ° C., preferably ice cooling (0 ° C. to 2 ° C.). If the patient undergoing the transplant is located remotely from the facility where the organ is stored, keep it in a storage container while moving. The storage condition at this time is that ice is placed outside the container in which the organ is placed, so that the internal temperature of the storage container is kept at 0 ° C. to 2 ° C. so as not to touch the organ directly.
  • Perfusion means washing the preservation solution by putting a washing solution into the organ.
  • the washing solution is not limited, for example, lactate Ringer solution is used, and the washing device is perfused at a rate of about 30 mL / min using a perfusion tube. In the case of the entire liver, 1 L of the washing solution is perfused from the portal vein. The kidney perfuses 10-40 mL per 250 g from the artery. Since the antibody is used at an appropriate concentration, if it becomes 1/100 or less, there is no side effect on the host, and the perfusion rate necessary for the normal organ preservation solution removal is sufficient.
  • “immune response” includes both GvH disease and rejection (HvG disease).
  • GvH disease is divided into four grades of acute illness depending on the severity, grade I to IV, and chronic illness is divided into mild, moderate, and severe three grades. It is theoretically possible to alleviate the above.
  • the organ to be a target for preventing GvH disease may be any of the above-mentioned organs.
  • an organ containing a large amount of white blood cells such as liver, lung, gastrointestinal tract (for example, intestine) is preferable.
  • Rejection means that the host refuses to graft.
  • the organ to be used for preventing rejection may be any of the above-mentioned organs, and for example, liver, lung, intestine, heart, kidney, pancreas and the like are preferable.
  • graft DC cells are also responsible for the liver, heart, kidney, pancreas, respiratory organ (lung) and gastrointestinal tract. If appropriate specific removal antibodies are found by this technique, they can be applied to these organs. Is possible.
  • the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.
  • Example 1 Specific prevention of GvH disease after liver transplantation by prior removal of graft T cells in Example 1, specific prevention of GvH disease after liver transplantation by prior removal of graft T cells was examined (FIG. 7).
  • the organ preservation solution (Biaspan solution, manufactured by Astellas) is usually cooled to an ice-cold temperature, it is assumed that it is an antibody that binds even at an ice-cold temperature and has an effect of removing target cells after transplantation.
  • a candidate antibody anti-rat T cell receptor ⁇ mouse monoclonal antibody
  • the average survival days and the time course of GvH pathology in the host were searched.
  • FIG. 9 shows the results of two experiments and shows the individual body weight curve and survival period after liver transplantation.
  • Experiment 1 is the result of the immunosuppression method (3 + 4 animals), the solid line (-) in the graph is the anti-T cell antibody added to the organ preservation solution, and the broken line (---) is the solvent only.
  • Added (4 + 5 animals).
  • the average survival time can be extended from about 20 days to 60 days or more by adding antibodies.
  • the candidate antibody can be added to the organ preservation solution of the graft, allowed to react, and after transplantation, the removal action in the host can be confirmed to determine the optimum amount.
  • liver transplantation under these conditions and comparing with an acute rejection control without antibody treatment, it is possible to retrieve the average number of days of survival and the time course of rejection pathology in the host.

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Abstract

Provided is a transplantation organ treatment method, or a transplantation immune response suppression method, characterized by: preserving an organ for transplantation in a preservation solution in the presence an anti-T-cell antibody, and then, washing the preservation solution away prior to the transplantation.

Description

移植免疫応答の抑制方法Method for suppressing transplanted immune response
　本発明は、抗体を添加した臓器保存液を用いた移植免疫応答の特異的抑制方法に関する。 The present invention relates to a method for specifically suppressing transplanted immune response using an organ preservation solution to which an antibody is added.
　臓器移植の臨床では、術後の移植免疫応答を抑制することが重要であるが、患者（ｈｏｓｔ、ホスト）はそのために免疫抑制剤を一生服用しなければならず、免疫力低下のためホストに易感染性や造血系腫瘍の増加が起こるだけでなく、医療費も高額となり、医療行政の深刻な課題の一つになっている。臨床で臓器移植が始まって以来、この問題を根本から解決する抑制法の開発が待望されてきた。
　移植免疫応答（図１）には主に、［１］移植を受けるホストのリンパ臓器において、移植片（ｇｒａｆｔ、グラフト）から血行性に遊走した、もしくはグラフト内に残存したグラフト由来の抗原提示樹状細胞（ＤＣ細胞）がホストＴ細胞と細胞集塊を形成し、直接、移植抗原を提示して（直接感作、図２）、誘導されたキラーＴ細胞がグラフトを攻撃する拒絶反応（ｈｏｓｔ　ｖｓ　ｇｒａｆｔ反応（ＨｖＧ反応））と、［２］逆に、グラフト由来の遊走したＴ細胞が宿主（ホスト）のＤＣ細胞により直接感作を受け（図３）、キラーＴ細胞となってホストを攻撃するＧｖＨ病（ｇｒａｆｔ　ｖｓ　ｈｏｓｔ反応、図４）があり、いずれもグラフト内に残っているグラフト白血球（ＤＣ細胞またはＴ細胞）の遊走や残存が原因となっている。 In the organ transplantation clinic, it is important to suppress the post-operative transplantation immune response. However, the patient (host, host) must take an immunosuppressant for the rest of the life, In addition to the increase in susceptibility and hematopoietic tumors, medical costs are high, which is one of the serious problems of medical administration. Since the beginning of organ transplantation in clinical practice, the development of a suppression method that fundamentally solves this problem has been awaited.
The transplanted immune response (FIG. 1) mainly includes [1] a graft-derived antigen-presenting tree that has migrated hematogenously from a graft (graft) or remained in the graft in a lymph organ of a host undergoing transplantation. Dendritic cells (DC cells) form cell clumps with host T cells, directly present the transplanted antigen (direct sensitization, FIG. 2), and the rejection that the induced killer T cells attack the graft (host) vs graft reaction (HvG reaction)) [2] Conversely, the graft-derived migrated T cells are directly sensitized by the host DC cells (FIG. 3) and become killer T cells. There is an attacking GvH disease (graft vs host reaction, FIG. 4), which is caused by migration and survival of graft leukocytes (DC cells or T cells) remaining in the graft.
　ここで詳述すると、図２は、ＨｖＧ反応を起こしたホストリンパ組織（リンパ節）の写真であり、グラフト由来のＤＣ細胞（青）がホストＴ細胞と細胞集塊を形成し，その中で増殖性応答（赤）を起こしていることが分かる。図３は、ＧｖＨ反応を起こしたホストリンパ組織（リンパ節）の写真であり、グラフト由来のＴ細胞（青）がホストのＤＣ細胞（茶）と細胞集塊を形成し，活性化したＴ細胞が大きくなり増殖している（核が赤）ことが分かる。図４は、ＧｖＨ病を起こしたホスト小腸組織の写真であり、大量のグラフト由来Ｔ細胞（青）が粘膜内に浸潤し、小腸組織の破壊を起こしていることが分かる。 More specifically, FIG. 2 is a photograph of a host lymphoid tissue (lymph node) that has caused an HvG reaction, in which graft-derived DC cells (blue) form cell clumps with host T cells. It can be seen that a proliferative response (red) occurs. FIG. 3 is a photograph of a host lymphoid tissue (lymph node) that has caused a GvH reaction. T cells derived from grafts (blue) form cell clusters with host DC cells (brown) and are activated T cells. It grows and grows (the nucleus is red). FIG. 4 is a photograph of the host small intestine tissue that caused GvH disease. It can be seen that a large amount of graft-derived T cells (blue) infiltrated into the mucous membrane and caused destruction of the small intestine tissue.
　上記［１］に関して、本発明者は、ラット肝移植モデルにおいて、グラフト由来のＤＣ細胞がホストの全身のリンパ臓器へ遊走し、遊走部位でホストのキラーＴ細胞を誘導して拒絶反応を促進する（図５，６）ことを証明した（Ｈｅｐａｔｏｌｏｇｙ　４７：１３５２，２００８（非特許文献１），Ｈｅｐａｔｏｌｏｇｙ　５６：１５３２，２０１２（非特許文献２））。また上記［２］に関しては、ヒトの肝臓移植症例では、特に親子間などのＨＬＡ半合致移植の時に、ホストの約１％が重度のＧｖＨ病を発症するといわれる。これはホストが免疫抑制されているので、グラフト由来のキラーＴ細胞が、抵抗されること無しに標的臓器であるホストの皮膚、消化管などを障害するためで、致命率も高い。 Regarding the above [1], in the rat liver transplantation model, the present inventor promotes rejection by inducing graft-derived DC cells to lymphatic organs throughout the host and inducing host killer T cells at the migration site. (FIGS. 5 and 6) (Hepatology 47: 1352, 2008 (Non-Patent Document 1), Hepatology 56: 1532, 2012 (Non-Patent Document 2)). Regarding the above [2], in human liver transplant cases, it is said that about 1% of the host develops severe GvH disease, particularly when HLA semi-match transplantation is performed between parents and children. Since the host is immunosuppressed, graft-derived killer T cells damage the target organ's skin, gastrointestinal tract and the like without resistance, and the fatality rate is also high.
　本発明は、抗体を添加した臓器保存液を用いた移植免疫応答の特異的抑制方法を提供することを目的とする。 An object of the present invention is to provide a method for specifically suppressing transplantation immune response using an organ preservation solution to which an antibody is added.
　本発明者は、上記課題を解決するため鋭意研究を行った結果、臓器保存液にグラフト白血球に対する特異的除去抗体を添加して反応させて洗い流すことにより、抗体は移植臓器内の標的細胞に結合したもののみ残り、移植後にはその抗体の作用で標的細胞のみ減少し、拒絶反応やＧｖＨ病が起こらなくなることを見出し、本発明を完成するに至った。 As a result of intensive studies to solve the above-mentioned problems, the present inventor binds to target cells in the transplanted organ by adding a specific removal antibody against grafted leukocytes to the organ preservation solution, causing it to react and washing away. It was found that only the target cells were reduced by the action of the antibody after transplantation, and that rejection and GvH disease did not occur, and the present invention was completed.
　すなわち、本発明は、以下の通りである。
（１）移植用臓器を、抗白血球抗体を含む保存液中で保存した後、移植前に当該保存液を洗い流すことを特徴とする、移植用臓器の処理方法。
（２）移植用臓器を、抗白血球抗体を含む保存液中で保存した後、移植前に当該保存液を洗い流すことを特徴とする、移植免疫応答の抑制方法。
（３）抗白血球抗体が、抗Ｔ細胞抗体である（１）又は（２）に記載の方法。
（４）移植免疫応答が臓器移植後のＧｖＨ病である（２）に記載の方法。
（５）臓器が肝臓、肺又は消化管である（４）に記載の方法。
（６）抗白血球抗体が、抗ＤＣ細胞抗体、又は抗ＩＩ型ＭＨＣ抗体である（１）又は（２）に記載の方法。
（７）移植免疫応答が臓器移植後のＨｖＧ反応である（２）に記載の方法。
（８）臓器が肝臓、心臓、肺、消化管、膵臓又は腎臓である（７）に記載の方法。 That is, the present invention is as follows.
(1) A method for treating an organ for transplantation, which comprises storing the organ for transplantation in a preservation solution containing an anti-leukocyte antibody and then washing off the preservation solution before transplantation.
(2) A method for suppressing a transplanted immune response, wherein an organ for transplantation is stored in a storage solution containing an anti-leukocyte antibody, and then the storage solution is washed out before transplantation.
(3) The method according to (1) or (2), wherein the anti-leukocyte antibody is an anti-T cell antibody.
(4) The method according to (2), wherein the transplantation immune response is GvH disease after organ transplantation.
(5) The method according to (4), wherein the organ is liver, lung, or digestive tract.
(6) The method according to (1) or (2), wherein the anti-leukocyte antibody is an anti-DC cell antibody or an anti-type II MHC antibody.
(7) The method according to (2), wherein the transplantation immune response is an HvG reaction after organ transplantation.
(8) The method according to (7), wherein the organ is liver, heart, lung, gastrointestinal tract, pancreas or kidney.
　本発明により、移植免疫応答の特異的抑制方法が提供される。本発明によれば、移植用臓器を冷蔵保存中に、保存液内に白血球に対する抗体を加えて臓器を前処理しておくことにより、ＧｖＨ病や拒絶反応などの移植免疫応答を抑制することができる。従って、本発明の方法は、臓器移植医療に極めて有用である。 The present invention provides a method for specifically suppressing transplanted immune response. According to the present invention, while organs for transplantation are refrigerated and preserved, the organ is pretreated by adding antibodies to leukocytes in the preservation solution, thereby suppressing transplantation immune responses such as GvH disease and rejection. it can. Therefore, the method of the present invention is extremely useful for organ transplantation medicine.
　図１は、臓器移植後の移植抗原感作と移植免疫応答の機序を示す模式図である。
　図２は、グラフト由来のＤＣ細胞によりＨｖＧ反応を起こしたホストリンパ組織（リンパ節）の写真であり、グラフト由来のＤＣ細胞（青）がホストＴ細胞と細胞集塊を形成し，その中で増殖性応答（赤）を起こしている。
　図３は、ＧｖＨ反応を起こしたホストリンパ組織（リンパ節）の写真であり、グラフト由来のＴ細胞（青）がホストのＤＣ細胞（茶）と細胞集塊を形成し，活性化したＴ細胞が大きくなり増殖（核が赤）している。
　図４は、ＧｖＨ病を起こしたホスト小腸組織の写真であり、大量のグラフト由来Ｔ細胞（青）が粘膜内に浸潤し、小腸組織の破壊を起こしている。
　図５は、ラット肝移植モデルにおいて、グラフト由来のＤＣ細胞によりＨｖＧ反応を起こしたホストリンパ組織（脾臓）の写真である。
　図６は、ラット肝移植モデルにおいて、ＨｖＧ反応により誘導されたキラーＴ細胞（ＣＤ８陽性、青）がグラフト内に浸潤して、盛んに増殖（核が赤）・活性化していることを示すグラフト肝組織の写真である。
　図７は、抗Ｔ細胞抗体による肝移植後ＧｖＨ病の抑制を示す本発明の模式図（１）である。
　図８は、抗ＤＣ細胞抗体による移植肝の拒絶反応の抑制を示す本発明の模式図（２）である。
　図９は、肝移植後の個体別体重曲線と生存期間で、抗Ｔ細胞抗体による肝移植後ＧｖＨ病の有意の抑制を示すグラフである。実験１と２はホストの免疫抑制の程度を変えて比較したものである。
　図１０は、抗グラフトＭＨＣ抗体のホスト内前投与による移植肝の拒絶反応の抑制を示す模式図である。
　図１１は、図１０または図８の処置によりグラフトＤＣ細胞が消失したことを示すホストリンパ組織（脾臓）の写真である。
　図１２は、肝移植後の個体別体重曲線と生存期間を示す図である。抗グラフトＭＨＣ抗体のホスト内前投与により、移植肝の拒絶反応の抑制が示されている。 FIG. 1 is a schematic diagram showing the mechanism of transplantation antigen sensitization and transplantation immune response after organ transplantation.
FIG. 2 is a photograph of a host lymphoid tissue (lymph node) that has caused an HvG reaction by a graft-derived DC cell, in which the graft-derived DC cell (blue) forms a cell clump with the host T cell. Has a proliferative response (red).
FIG. 3 is a photograph of a host lymphoid tissue (lymph node) that has caused a GvH reaction. T cells derived from grafts (blue) form cell clusters with host DC cells (brown) and are activated T cells. Is growing and growing (nuclei are red).
FIG. 4 is a photograph of the host small intestine tissue that caused GvH disease, in which a large amount of graft-derived T cells (blue) infiltrated into the mucosa and caused destruction of the small intestine tissue.
FIG. 5 is a photograph of host lymphoid tissue (spleen) in which an HvG reaction was caused by graft-derived DC cells in a rat liver transplantation model.
FIG. 6 is a graph showing that in a rat liver transplantation model, killer T cells (CD8 positive, blue) induced by HvG reaction infiltrate into the graft, and are actively proliferating (the nucleus is red) and activated. It is a photograph of liver tissue.
FIG. 7 is a schematic diagram (1) of the present invention showing suppression of GvH disease after liver transplantation by anti-T cell antibody.
FIG. 8 is a schematic diagram (2) of the present invention showing suppression of rejection of transplanted liver by an anti-DC cell antibody.
FIG. 9 is a graph showing significant suppression of GvH disease after liver transplantation by anti-T cell antibody in the individual body weight curve and survival period after liver transplantation. Experiments 1 and 2 compare the degree of immunosuppression of the host.
FIG. 10 is a schematic diagram showing suppression of rejection of transplanted liver by pre-administration of anti-graft MHC antibody in the host.
FIG. 11 is a photograph of a host lymphoid tissue (spleen) showing that graft DC cells have disappeared by the treatment of FIG. 10 or FIG.
FIG. 12 is a diagram showing an individual body weight curve and a survival period after liver transplantation. Pre-administration of anti-grafted MHC antibody in the host has been shown to suppress rejection of transplanted liver.
　以下、本発明を詳細に説明する。
　本発明者は、グラフト肝臓を保存液中で保存する際に、予めグラフト白血球に対する特異的除去抗体で処置しておけば、抗体が結合したグラフト白血球は、移植後すぐにホスト内で除去されるので、グラフト白血球が主因である拒絶反応もＧｖＨ病も選択的に抑制され予防できるのではないかという仮説を立てた。
　ヒト臓器移植において、このような抗体を移植後に使用する場合には、グラフト特異的であることが必要であり、テイラーメイドの抗体作製が前提となるため、あまり実用的ではない。ここで、ヒト臓器移植の方法を考察してみると、グラフトの肝臓、腎臓、心臓、肺、腸、他ほとんどの臓器の血管に臓器保存液を満したままホストの待つ病院に運び、移植することが通例である。この臓器保存液は、移植直前に臓器を灌流して洗い流す事になっている。 Hereinafter, the present invention will be described in detail.
When the present inventor preserves the grafted liver in a preservation solution, if it is previously treated with a specific antibody for removing the grafted leukocyte, the grafted leukocyte to which the antibody is bound is removed within the host immediately after transplantation. Therefore, it was hypothesized that both rejection and GvH disease, which are mainly caused by grafted leukocytes, could be selectively suppressed and prevented.
When such an antibody is used after transplantation in human organ transplantation, it is necessary to be graft-specific, and since it is premised on the production of a tailor-made antibody, it is not very practical. Here, when considering the method of human organ transplantation, the liver of the graft, kidney, heart, lung, intestine, and blood vessels of most other organs are transported to the hospital where the host waits with the organ preservation solution filled and transplanted. It is customary. This organ preservation solution is to be perfused and washed out immediately before transplantation.
　そこで本発明者は、この臓器保存液に抗体を添加して反応させた後、抗体含有保存液を洗い流すことにより、抗体は移植臓器内の標的細胞に結合したもののみ残り、移植後にはその抗体の作用で標的細胞のみ減少し、ＧｖＨ病が起こらなくなることを見出した。拒絶反応についても抗体ホスト内前投与の予備実験で抑制を見ている。 Therefore, the present inventor added an antibody to the organ preservation solution, reacted, and then washed away the antibody-containing preservation solution, so that the antibody remained only bound to the target cells in the transplanted organ. It was found that only the target cells were reduced by the action of GvH, and GvH disease did not occur. Rejection has also been shown to be suppressed in a preliminary experiment with pre-administration of antibody host.
１．移植用臓器
　本発明において、処理の対象となる臓器としては、特に限定されるものではないが、例えば肝臓、呼吸器（気管支・肺）、消化管（胃、小腸、大腸）、膵臓、心臓、又は腎臓などが挙げられる。
　臓器は、血管を含んでいる（血管吻合ができる）限り、その全部であってもよく、一部でもよい。
　例えば肝臓の場合、Ｃｏｕｉｎａｕｄ（クイノー）の亜区域分類に基づいたそれぞれの区域ごと（Ｓ１~Ｓ８）であってもよい。
Ｓ１：尾状葉、Ｓ２：左葉外側後区域、Ｓ３：左葉外側前区域、Ｓ４：左葉内側区域、Ｓ５：右葉前下区域、Ｓ６：右葉後下区域、Ｓ７：右葉後上区域、Ｓ８：右葉前上区域
　また、大きさは血管吻合ができる大きさとすることができる。
　心臓と腎臓の場合は、大血管基部を含む臓器全体となる。
　呼吸器の場合は、肺葉に気管支と血管を含む部分移植から片肺、心臓と一緒に行う心肺全体移植までとなる。
　消化管の場合は、胃から大腸までの一部で、血管吻合できる単位以上の部分移植となる。 1. Organ for transplantation In the present invention, the organ to be treated is not particularly limited. For example, the liver, respiratory organs (bronchi / lung), digestive tract (stomach, small intestine, large intestine), pancreas, heart, Or a kidney etc. are mentioned.
The organ may be all or part of the organ as long as it contains blood vessels (can perform vascular anastomosis).
For example, in the case of the liver, each zone (S1 to S8) based on the sub-zone classification of Couinaud may be used.
S1: caudate leaf, S2: left rear lateral rear region, S3: left lateral lateral front region, S4: left lateral lateral region, S5: right front lateral bottom region, S6: right lateral rear region, S7: right lateral leaf Upper area, S8: Upper area in front of right lobe Further, the size can be set to a size that allows vascular anastomosis.
In the case of the heart and kidney, it is the entire organ including the base of the large blood vessel.
In the case of the respiratory tract, it ranges from partial transplantation including bronchi and blood vessels in the lung lobe to full lung transplantation performed with one lung and heart.
In the case of the gastrointestinal tract, partial transplantation of a portion from the stomach to the large intestine that is more than a unit capable of vascular anastomosis.
２．保存液
　本発明において使用される保存液は、臓器移植に一般に使用されるビアスパン液（アステラス製薬社製）、クストディオール液（Ｅｓｓｅｎｔｉａｌ　Ｐｈａｒｍａｃｅｕｔｉｃａｌｓ社製）、ユーロコリンズ液（アイロム製薬社製）などが挙げられる。保存時の温度は０℃から４℃である。必要に応じて、保存液に還元グルタチオン溶液などの添加物を添加することもできる。 2. Preservation Solution The preservation solution used in the present invention includes beerspan solution (manufactured by Astellas Pharma), Custodiol solution (manufactured by Essential Pharmaceuticals), eurocollins solution (manufactured by Irom Pharmaceutical), etc. It is done. The temperature during storage is from 0 ° C to 4 ° C. If necessary, an additive such as a reduced glutathione solution can be added to the preservation solution.
３．抗白血球抗体
　本発明において、抗白血球抗体を用いて移植用臓器から除去する対象となる細胞は、Ｔ細胞、樹状細胞（ｄｅｎｄｒｉｔｉｃ　ｃｅｌｌ；ＤＣ細胞）などである。
　本発明において、抗白血球抗体とは、Ｔ細胞またはＤＣ細胞を除去できるものを意味し、Ｔ細胞については抗Ｔ細胞抗体、ＤＣ細胞については抗ＤＣ細胞抗体（例えば抗ＣＤ１１ｃ抗体、抗ＣＤ１０３抗体、抗ＣＤ２０５抗体）、抗ＩＩ型ＭＨＣ抗体などが本発明において処理の対象となる。
　抗体を保存液に含有させるには、例えば臓器を入れた保存液に抗体を添加する方法、あるいは予め抗体を含めた保存液をそのまま臓器保存に使用する方法のいずれを採用することもできる。保存液中の抗体の濃度は、臓器の大きさや種類と抗体の力価に応じて適宜変更することができ、例えば２−２０μｇ／ｇ臓器湿重量であり、低いほど良い。
　上記抗体は市販品であり、ｅＢｉｏｓｃｉｅｎｃｅ，ＢｉｏＬｅｇｅｎｄ社などから入手することができる。 3. Anti-leukocyte antibody In the present invention, cells to be removed from the organ for transplantation using the anti-leukocyte antibody are T cells, dendritic cells (DC cells), and the like.
In the present invention, the anti-leukocyte antibody means an antibody capable of removing T cells or DC cells, anti-T cell antibodies for T cells, anti-DC cell antibodies for DC cells (for example, anti-CD11c antibody, anti-CD103 antibody, Anti-CD205 antibody), anti-type II MHC antibody, and the like are subject to treatment in the present invention.
To contain the antibody in the preservation solution, for example, either a method of adding the antibody to the preservation solution containing the organ or a method of using the preservation solution containing the antibody in advance for organ preservation can be employed. The concentration of the antibody in the preservation solution can be appropriately changed according to the size and type of the organ and the titer of the antibody, and is, for example, 2-20 μg / g organ wet weight, and the lower the better.
The above-mentioned antibody is a commercial product, and can be obtained from eBioscience, BioLegend.
４．保存方法
　臓器を保存する方法は、臓器の大きさや種類、あるいは移植が必要な患者が登場するまでの時間に応じて異なるが、例えば、１時間~３６時間、好ましくは１日以内である。
　保存温度は、０℃~４℃、好ましくは氷冷（０℃~２℃）である。
　移植を受ける患者の所在地が、臓器が保存されている施設から遠隔地にある場合は、移動中は保存用容器に入れておく。このときの保存条件は、氷は臓器を入れている容器の外側に入れて、直接臓器に触れることのないようにして、保管容器の液中温度を０℃~２℃に保てるようにする。 4). Preservation method The method for preserving the organ varies depending on the size and type of the organ or the time until the patient requiring transplantation appears, but is, for example, 1 hour to 36 hours, preferably within 1 day.
The storage temperature is 0 ° C. to 4 ° C., preferably ice cooling (0 ° C. to 2 ° C.).
If the patient undergoing the transplant is located remotely from the facility where the organ is stored, keep it in a storage container while moving. The storage condition at this time is that ice is placed outside the container in which the organ is placed, so that the internal temperature of the storage container is kept at 0 ° C. to 2 ° C. so as not to touch the organ directly.
５．保存液の洗浄
　抗体を含む保存液で移植用臓器を保存した後は、移植前に保存液を灌流する。灌流とは、臓器内に洗浄液を入れて保存液を洗い流すことを意味する。
　洗浄液は限定されるものではないが、例えば乳酸リンゲル液を用い、洗浄用器具は灌流チューブを用いて、３０ｍＬ／分くらいの速度で灌流する。洗浄液量は肝臓全部の場合、１Ｌを門脈から灌流する。腎臓は２５０ｇあたり１０−４０ｍＬを動脈から灌流する。
　抗体は適正濃度を使用するため、１／１００以下になればホストへの副作用はなく、通常の臓器保存液除去に必要な灌流量で十分である。 5. Washing the preservation solution After storing the organ for transplantation with a preservation solution containing the antibody, the preservation solution is perfused before transplantation. Perfusion means washing the preservation solution by putting a washing solution into the organ.
Although the washing solution is not limited, for example, lactate Ringer solution is used, and the washing device is perfused at a rate of about 30 mL / min using a perfusion tube. In the case of the entire liver, 1 L of the washing solution is perfused from the portal vein. The kidney perfuses 10-40 mL per 250 g from the artery.
Since the antibody is used at an appropriate concentration, if it becomes 1/100 or less, there is no side effect on the host, and the perfusion rate necessary for the normal organ preservation solution removal is sufficient.
６．移植免疫応答の抑制
　上記のようにして処理された臓器を患者に移植することで、免疫応答を抑制することができる。従って、上記処理後の臓器をホストに移植しても、ホストにおける移植後の免疫応答を予防することができる。
　本発明において「免疫応答」とは、ＧｖＨ病及び拒絶反応（ＨｖＧ病）の両者を含む。
　ＧｖＨ病は、重症度に応じて急性病がグレードＩからＩＶの４段階に、慢性病が軽症、中等症、重症の３段階に分けられるが、本発明の処置を行うことで、それぞれ１，２段階以上緩和することが理論的に可能である。ＧｖＨ病を予防する対象となる臓器は、前記の臓器のいずれでもよいが、例えば肝、肺、消化管（例えば腸）などの白血球を多く含む臓器が好ましい。 6). Suppression of transplanted immune response By transplanting the organ treated as described above into a patient, the immune response can be suppressed. Therefore, even if the organ after the treatment is transplanted to the host, the immune response after the transplantation in the host can be prevented.
In the present invention, “immune response” includes both GvH disease and rejection (HvG disease).
GvH disease is divided into four grades of acute illness depending on the severity, grade I to IV, and chronic illness is divided into mild, moderate, and severe three grades. It is theoretically possible to alleviate the above. The organ to be a target for preventing GvH disease may be any of the above-mentioned organs. For example, an organ containing a large amount of white blood cells such as liver, lung, gastrointestinal tract (for example, intestine) is preferable.
　拒絶反応は、ホストがグラフトの生着を拒否することを意味する。拒絶反応を予防する対象となる臓器は、前記の臓器のいずれでもよいが、例えば肝、肺、腸、心、腎、膵臓などが好ましい。拒絶反応に関しては、肝臓、心臓、腎臓、膵臓、呼吸器（肺）や消化管に関してもグラフトＤＣ細胞が原因となるため、本手法で適切な特異的除去抗体が見つかればこれらの臓器にも応用可能である。
　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、本発明はこれら実施例に限定されるものではない。 Rejection means that the host refuses to graft. The organ to be used for preventing rejection may be any of the above-mentioned organs, and for example, liver, lung, intestine, heart, kidney, pancreas and the like are preferable. Regarding rejection, graft DC cells are also responsible for the liver, heart, kidney, pancreas, respiratory organ (lung) and gastrointestinal tract. If appropriate specific removal antibodies are found by this technique, they can be applied to these organs. Is possible.
Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.
グラフトＴ細胞事前除去による肝臓移植後ＧｖＨ病の特異的予防
　実施例１では、グラフトＴ細胞事前除去による肝臓移植後ＧｖＨ病の特異的予防を検討した（図７）。 Specific prevention of GvH disease after liver transplantation by prior removal of graft T cells In Example 1, specific prevention of GvH disease after liver transplantation by prior removal of graft T cells was examined (FIG. 7).
（１）ラット肝移植モデルでホストを術前に免疫抑制処置することにより、急性ＧｖＨ病が起こる条件を決定した。
（２）分離精製したドナーラット（種類：Ｌｅｗｉｓラット、年齢：８−１２週齢）のＴ細胞懸濁液に様々な特異的抗体を入れて４℃で反応させた後にホストに静脈投与し、ホスト内でドナーＴ細胞除去作用を持つ抗体のスクリーニングを行った。
　特異的抗体名と入手先：抗ラットＴ細胞受容体αβマウスモノクローナル抗体（ｅＢｉｏｓｃｉｅｎｃｅ社）、抗ラットＣＤ３マウスモノクローナル抗体（ＢｉｏＬｅｇｅｎｄ社）。
　ここで、臓器保存液（ビアスパン液、アステラス社製）は通常、氷冷温に冷却したものを用いるので、氷冷温でも結合し、移植後には標的細胞の除去効果がある抗体であることが前提となる。
（３）候補の抗体（抗ラットＴ細胞受容体αβマウスモノクローナル抗体）を使って、グラフトの臓器保存液中に加えて反応させて肝移植を行い、ＧｖＨ病を発症する抗体処置無しのコントロールと比較することにより、平均生存日数とホスト内のＧｖＨ病態の時間的経緯を検索した。 (1) The conditions under which acute GvH disease occurs were determined by immunosuppressing the host preoperatively in a rat liver transplantation model.
(2) Various specific antibodies were put into a T cell suspension of a separated and purified donor rat (type: Lewis rat, age: 8-12 weeks), reacted at 4 ° C., and then intravenously administered to the host. Screening for antibodies with donor T cell removal activity in the host was performed.
Specific antibody names and sources: anti-rat T cell receptor αβ mouse monoclonal antibody (eBioscience), anti-rat CD3 mouse monoclonal antibody (BioLegend).
Here, since the organ preservation solution (Biaspan solution, manufactured by Astellas) is usually cooled to an ice-cold temperature, it is assumed that it is an antibody that binds even at an ice-cold temperature and has an effect of removing target cells after transplantation. Become.
(3) A candidate antibody (anti-rat T cell receptor αβ mouse monoclonal antibody) is added to a graft organ preservation solution and reacted to perform liver transplantation, and control without antibody treatment that causes GvH disease. By comparing, the average survival days and the time course of GvH pathology in the host were searched.
（４）結果
（４−１）ホストの免疫抑制処置として、移植前に致死量以下の放射線照射（Ｘ線３Ｇｙ　一回照射）とＮＫ細胞およびＣＤ８細胞を除去する抗体（抗ラットＣＤ８αモノクローナル抗体，ｅＢｉｏｓｃｉｅｎｃｅ社）を投与することにより、ラット同種肝移植後のＧｖＨ病高率発症系を作製した。ここで使用する近交系ラットは、ヒトのＨＬＡ半合致移植モデルとして、親のグラフトを一代雑種の子供（主要組織適合遺伝子複合体ＭＨＣが半合致になる）に行う組み合わせにしている。
（４−２）ドナーＴ細胞を抗ラットＴ細胞受容体αβ抗体で４℃、１時間処置するとホスト内で除去作用があるが、抗ラットＣＤ３抗体では作用が弱いことを確認した。
（４−３）次に、臓器保存液（ビアスパン液、アステラス社製）に一般的な抗Ｔ細胞抗体（抗Ｔ細胞受容体αβ抗体、ｅＢｉｏｓｃｉｅｎｃｅ社製）を用いて本手法をおこなったところ、有意にＧｖＨ病を抑制できることを確認した。
　図９は、２つの実験の結果であり、肝移植後の個体別体重曲線と生存期間を示している。図９において、実験１は免疫抑制法の実験結果であり（３＋４匹）、グラフの実線（−）は臓器保存液中に抗Ｔ細胞抗体を添加したもの、破線（−−−）は溶媒のみ添加したもの（４＋５匹）である。平均生存期間は抗体添加により、約２０日が６０日以上へと延長できている。 (4) Results (4-1) As an immunosuppressive treatment of the host, an antibody (anti-rat CD8α monoclonal antibody, anti-rat CD8α monoclonal antibody, eBioscience) was administered to produce a GvH disease high-rate onset system after rat allogeneic liver transplantation. The inbred rat used here is a human HLA semi-match transplantation model in which the parent graft is made to a hybrid child (the major histocompatibility complex MHC is semi-match).
(4-2) It was confirmed that treatment of donor T cells with anti-rat T cell receptor αβ antibody at 4 ° C. for 1 hour has a removal effect in the host, but anti-rat CD3 antibody has a weak effect.
(4-3) Next, when this technique was performed using a general anti-T cell antibody (anti-T cell receptor αβ antibody, manufactured by eBioscience) in an organ preservation solution (Biaspan solution, manufactured by Astellas), It was confirmed that GvH disease can be significantly suppressed.
FIG. 9 shows the results of two experiments and shows the individual body weight curve and survival period after liver transplantation. In FIG. 9, Experiment 1 is the result of the immunosuppression method (3 + 4 animals), the solid line (-) in the graph is the anti-T cell antibody added to the organ preservation solution, and the broken line (---) is the solvent only. Added (4 + 5 animals). The average survival time can be extended from about 20 days to 60 days or more by adding antibodies.
グラフトＤＣ細胞事前除去による移植肝拒絶反応の特異的抑制
　本実施例では、グラフトＤＣ細胞事前除去による移植肝拒絶反応の特異的抑制の検討を行った（図８）。 Specific suppression of transplanted liver rejection by prior removal of grafted DC cells In this Example, specific suppression of transplanted liver rejection by prior removal of grafted DC cells was examined (FIG. 8).
（１）ヒトＤＣ細胞のほとんどはＩＩ型ＭＨＣ（ＭＨＣ　ＩＩ）が陽性であるが、肝細胞および非実質細胞は、Ｂ細胞やＫｕｐｆｆｅｒ細胞の一部を除き、ＭＨＣ　ＩＩ陰性である（Ａｍ　Ｊ　Ｐａｔｈｏｌ　１３３：８２，１９８８）。そこで予備実験として、ラット肝移植拒絶モデルで、グラフト樹状細胞を主に減少させる様々なグラフトＭＨＣ　ＩＩ特異的抗体をホストに前投与した後肝移植し、グラフトＤＣ細胞の遊走や拒絶反応が抑制されるかどうかを検索した。
（２）次に、候補の抗体（グラフトＭＨＣ　ＩＩに対する特異抗体）を使って、グラフトの臓器保存液中に加えて反応させて肝移植を行い、グラフトＤＣ細胞の遊走が抑制されるかどうかを検索した。
　さらに、本発明においては、候補の抗体を使って、グラフトの臓器保存液中に加えて反応させ、移植した後にホスト内での除去作用を確認し、至適量を決定することができる。
　この条件で肝移植を行い、抗体処置無しの急性拒絶コントロールと比較することにより、平均生存日数とホスト内の拒絶反応病態の時間的経緯を検索することができる。
（３）結果
（３−１）ホストにグラフトＭＨＣ　ＩＩに対する特異抗体（ＯＸ７６マウスモノクローナル抗体，ＡｂＤ　Ｓｅｒｏｔｅｃ社製）を腹腔内前投与した（図１０）。
　その結果、ほとんどのグラフトＤＣ細胞が消失し（図１１中央）、キラーＴ細胞の誘導抑制と拒絶反応の有意な遅延が起こるが、副作用としての肝障害は起こらないことを認めた（図１２、表１）。
（３−２）次に、臓器保存液（ビアスパン液、アステラス社製）に同じ抗体を用いて本手法をおこなったところ、投与量０．２ｍｇで十分な血行性遊走性グラフトＤＣ細胞の選択的除去効果を達成することができた（図１１右）。 (1) Most human DC cells are positive for type II MHC (MHC II), but hepatocytes and non-parenchymal cells are MHC II negative except for some of B cells and Kupffer cells (Am J Pathol). 133: 82, 1988). Therefore, as a preliminary experiment, in the rat liver transplant rejection model, various graft MHC II-specific antibodies that mainly reduce graft dendritic cells were pre-administered to the host and then transplanted to the liver to suppress the migration and rejection of graft DC cells. Searched for being.
(2) Next, using a candidate antibody (specific antibody against graft MHC II), add to the organ preservation solution of the graft and react to perform liver transplantation to determine whether migration of grafted DC cells is suppressed. searched.
Furthermore, in the present invention, the candidate antibody can be added to the organ preservation solution of the graft, allowed to react, and after transplantation, the removal action in the host can be confirmed to determine the optimum amount.
By performing liver transplantation under these conditions and comparing with an acute rejection control without antibody treatment, it is possible to retrieve the average number of days of survival and the time course of rejection pathology in the host.
(3) Results (3-1) A specific antibody against graf MHC II (OX76 mouse monoclonal antibody, manufactured by AbD Serotec) was administered intraperitoneally to the host (FIG. 10).
As a result, most of the grafted DC cells disappeared (center of FIG. 11), and killer T cell induction suppression and a significant delay of rejection occurred, but it was confirmed that liver damage as a side effect did not occur (FIG. 12, Table 1).
(3-2) Next, this technique was performed using the same antibody as an organ preservation solution (Biaspan solution, manufactured by Astellas). As a result, a sufficient dose of 0.2 mg was selected for hemodynamically migratory graft DC cells. The removal effect could be achieved (FIG. 11 right).
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　成熟期の日本で臓器移植手術は今後ますます増加し、厚生労働省の移植医療行政が格段に拡張することが予想される。ＧｖＨ病や拒絶反応を発症した時は、大量の薬剤投与が必要で深刻な副作用を伴う上に予後不良である。本方法は、ＧｖＨ病や拒絶反応をグラフト特異的に抑制できる簡便かつ安全な方法であり、免疫抑制剤の減量につながり移植医療に貢献できる。 】 Organ transplant surgery will continue to increase in mature Japan, and the transplant medical administration of the Ministry of Health, Labor and Welfare is expected to expand dramatically. When GvH disease or rejection occurs, a large amount of drug administration is required, with serious side effects and poor prognosis. This method is a simple and safe method capable of graft-specific suppression of GvH disease and rejection, leading to a reduction in the amount of immunosuppressant and contributing to transplantation medicine.

Claims (8)

  1. 移植用臓器を、抗白血球抗体を含む保存液中で保存した後、移植前に当該保存液を洗い流すことを特徴とする、移植用臓器の処理方法。 A method for treating an organ for transplantation, which comprises storing the organ for transplantation in a preservation solution containing an anti-leukocyte antibody and then washing off the preservation solution before transplantation.
  2. 移植用臓器を、抗白血球抗体を含む保存液中で保存した後、移植前に当該保存液を洗い流すことを特徴とする、移植免疫応答の抑制方法。 A method for suppressing a transplanted immune response, which comprises storing an organ for transplantation in a preservation solution containing an anti-leukocyte antibody and then washing off the preservation solution before transplantation.
  3. 抗白血球抗体が、抗Ｔ細胞抗体である請求項１又は２に記載の方法。 The method according to claim 1 or 2, wherein the anti-leukocyte antibody is an anti-T cell antibody.
  4. 移植免疫応答が臓器移植後のＧｖＨ病である請求項２に記載の方法。 The method according to claim 2, wherein the transplantation immune response is GvH disease after organ transplantation.
  5. 臓器が肝臓、肺又は消化管である請求項４に記載の方法。 The method according to claim 4, wherein the organ is a liver, a lung, or a digestive tract.
  6. 抗白血球抗体が、抗ＤＣ細胞抗体、又は抗ＩＩ型ＭＨＣ抗体である請求項１又は２に記載の方法。 The method according to claim 1 or 2, wherein the anti-leukocyte antibody is an anti-DC cell antibody or an anti-type II MHC antibody.
  7. 移植免疫応答が臓器移植後の拒絶反応である請求項２に記載の方法。 The method according to claim 2, wherein the transplanted immune response is rejection after organ transplantation.
  8. 臓器が肝臓、心臓、肺、消化管、膵臓又は腎臓である請求項７に記載の方法。 The method according to claim 7, wherein the organ is liver, heart, lung, gastrointestinal tract, pancreas or kidney.
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