WO2012017678A1 - 免疫機能の強化剤 - Google Patents
免疫機能の強化剤 Download PDFInfo
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- WO2012017678A1 WO2012017678A1 PCT/JP2011/004455 JP2011004455W WO2012017678A1 WO 2012017678 A1 WO2012017678 A1 WO 2012017678A1 JP 2011004455 W JP2011004455 W JP 2011004455W WO 2012017678 A1 WO2012017678 A1 WO 2012017678A1
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Definitions
- the present invention relates to an immune function enhancer. More specifically, the present invention relates to an immune function enhancer for mammals other than humans, including activated lymphocytes derived from other families, a therapeutic agent for neonatal weak child syndrome in mammals other than humans, and these agents. It relates to the manufacturing method.
- Herbivores represented by cattle and horses cannot receive immune substances such as antibodies and immune cells from the mother animal through the placenta during the fetal period. For this reason, neonates in these animals have weak immune function and may have neonatal frail syndrome. The neonates of these animals usually ingest immune substances such as antibodies and immune cells via colostrum, thereby promoting the function of the newborn's own immune cells. However, newborns with neonatal frail syndrome are less susceptible to colostrum absorption and are more susceptible to infections, and in severe cases can be fatal. In addition, because there are infectious diseases in livestock that are transmitted through colostrum and cause serious medical conditions, in order to prevent infection, live colostrum is not given directly from mother cows to newborns for the purpose of infection prevention. It is not uncommon for people to deal with processed colostrum that contains no cells and is less immune.
- Activating lymphocyte therapy is known as one of immunotherapy for cancer of small animals including humans, dogs and cats.
- Activated lymphocyte therapy is the activation and amplification of own peripheral blood lymphocytes outside the body and back into the body.
- JP-A-2004-2312 discloses a preparation containing activated lymphocytes derived from others who have the same HLA (human leukocyte type antigen). Since this preparation contains activated lymphocytes, it is said to be effective for the prevention and treatment of autoimmune diseases.
- HLA human leukocyte type antigen
- rare beef cattle such as Japanese black beef and thoroughbreds with good pedigree have a high probability of suffering from neonatal frail syndrome. For this reason, brand cattle and thoroughbreds have a high mortality rate during the neonatal period.
- serious side effects may occur when components derived from other animals are administered.
- an object of the present invention is to provide a strengthening agent for immune function of mammals excluding humans other than self, with few side effects. This enhancer of immune function is particularly effective as an agent for treating frail syndrome in newborn animals. Moreover, an object of this invention is to provide the method of manufacturing such an agent. A further object of the present invention is to provide a method for enhancing the immune function of animals.
- the present invention is surprisingly surprising in mammals other than humans, particularly those in which antibodies and immune cells do not migrate from the mother to the fetus via the placenta, even when activated lymphocytes other than self are used. This is based on the findings of the example that the immune function of other animals can be enhanced without serious side effects, and is particularly effective in the treatment of frail syndrome in newborn animals.
- the first aspect of the present invention relates to a strengthening agent for immune function of other animals, which is the same species as that animal and contains activated lymphocytes derived from tissues of mammals other than humans as an active ingredient.
- a preferred embodiment of the agent of the first aspect of the present invention is an activation wherein activated lymphocytes derived from mammalian tissues other than humans are activated by one or both of interleukin-2 and anti-CD3 antibody. It is a lymphocyte.
- the mammal is an animal in which antibodies and immune cells do not migrate from the mother to the fetus via the placenta, specifically a cow or a horse.
- a preferred embodiment of the agent of the first aspect of the present invention is used as an agent for treating frail syndrome of a neonate of an animal in which antibodies and immune cells do not migrate from the mother to the fetus via the placenta.
- An example of frail syndrome is infection.
- bovine or horses no serious side effects were observed even when activated lymphocytes from other families were administered.
- newborn cattle or horses born in a weak constitution are likely to suffer from infections such as diarrhea and pneumonia, and are highly likely to die without treatment.
- the second aspect of the present invention relates to a method for producing a strengthening agent for immune function of other animals.
- This method comprises the steps of isolating lymphocytes from mammals other than humans, proliferating and activating the isolated lymphocytes to obtain activated lymphocytes, and formulating with the obtained activated lymphocytes. Including.
- the step of obtaining activated lymphocytes comprises the step of culturing lymphocytes in the presence of either or both of interleukin-2 and anti-CD3 antibody.
- a preferred embodiment of the method of the second aspect of the present invention is that the enhancer of the immune function of the xenogeneic animal is the same species as that of mammals other than humans, particularly those in which antibodies and immune cells are not transferred from the mother to the fetus via the placenta. It is a therapeutic agent for animal neonatal frail syndrome, which is used to treat frail syndrome in other-born neonates.
- the third aspect of the present invention relates to a method for enhancing the immune function of mammals other than humans, especially animals in which antibodies and immune cells do not migrate from the mother to the fetus via the placenta.
- the method includes the step of administering lymphocytes isolated, expanded and activated from an animal to an animal of the same species as the animal and from another home.
- a preferred embodiment of the third aspect of the present invention administers lymphocytes grown and activated to neonates of mammals other than humans, particularly those in which antibodies and immune cells do not migrate from the mother to the fetus via the placenta. Is. That is, this embodiment strengthens the neonatal immune function and treats frail syndrome.
- a preferred embodiment of the third aspect of the present invention is an animal in which neonatal antibodies and immune cells are not transferred from the mother to the fetus via the placenta, up to 1 year or less immediately after birth, more preferably 3 months or less immediately after birth.
- Administering proliferated and activated lymphocytes Animals within the first few months of life are highly immune-tolerant, and so do not cause much rejection when given activated lymphocytes from other homes. For this reason, the agent of this invention can be used effectively in order to strengthen the immune function of the animal of several months of age.
- an enhancer of immune function of other animals with few side effects in animals in which antibodies and immune cells do not migrate from the mother to the fetus via the placenta.
- This enhancer of immune function is particularly effective as an agent for treating frail syndrome in newborn animals.
- the method of manufacturing such an agent can be provided.
- FIG. 1 is a graph showing a proliferation curve of active lymphocytes.
- FIG. 2 is a graph showing changes in peripheral blood mononuclear cells (PBMC).
- FIG. 3 is a graph showing changes in CD3-positive T lymphocytes.
- FIG. 4 is a graph showing changes in B lymphocytes.
- FIG. 5 is a graph showing changes in IgM type B lymphocytes.
- FIG. 6 is a graph showing changes in natural killer cells (NK cells).
- FIG. 7A is a graph showing changes in CD4 + cells.
- FIG. 7B is a graph showing changes in CD4 + CD45R ⁇ cells.
- FIG. 8A is a graph showing changes in CD8 + cells.
- FIG. 8B is a graph showing changes in CD8 + CD45R ⁇ cells.
- 9A, 9B, 9C and 9D are graphs showing the expression levels of the IL-2 gene, IL-4 gene, IL-6 gene and IFN- ⁇ gene, respectively.
- the first aspect of the present invention relates to a strengthening agent for immune function of other animals, which is the same species as that animal and contains activated lymphocytes derived from tissues of mammals other than humans as an active ingredient.
- Allogeneic animals mean the same species and so-called allogenic.
- the same species as an animal means a horse if the subject from whom the lymphocytes were collected is a horse.
- Cross-animal means non-self.
- An example of an allogeneic animal is a mother and a child (the subject from whom lymphocytes are collected is a mother and the subject to whom an agent containing activated lymphocytes is administered is a pup).
- Another example of an allogeneic animal is a father and son.
- the animals have no serious side effects even when they are transferred from other families. For this reason, the allogeneic animal may be the same kind of animal that is not related.
- Mammals include not only humans that transfer sufficient antibodies through the placenta, but also animals that do not transfer antibodies or immune cells from the mother to the fetus through the placenta.
- Examples of animals in which antibodies and immune cells do not transfer from the mother to the fetus through the placenta are cattle, horses, sheep, goats, pigs, and pandas.
- the agent of the present invention is effectively used for animals in which such antibodies and immune cells do not migrate from the mother to the fetus through the placenta (hereinafter also referred to as “target animals”).
- the target animal cannot transfer any immune substances including antibodies and immune cells to the fetus through the mother placenta during the fetal period.
- the agent of the present invention can be preferably used for cattle or horses.
- Cattle have a high probability of suffering from neonatal frailty syndrome (WCS) occurring in newborn calves. WCS develops because the immune function (particularly the function of T lymphocytes among immune cells) has decreased since the birth of a cow. Therefore, the agent of the present invention is particularly effective for the treatment of neonatal frail syndrome of cattle. Brand cattle newborns are more likely to die of infection. As shown in the examples described later, since the immune function of bovine neonates can be enhanced by the agent of the present invention, the agent of the present invention should be used effectively to prevent infection of bovine neonates. Can do. Horses, like cows, cannot transfer immune substances through the placenta of the mother. Therefore, the agent of the present invention is effective for the treatment of equine neonatal frail syndrome.
- WCS neonatal frailty syndrome
- activated lymphocytes derived from animal tissues in which antibodies and immune cells do not migrate from the mother to the fetus via the placenta may be obtained by separating and culturing lymphocytes from the peripheral blood of the animal.
- animal tissues include bone marrow cells such as bone marrow stem cells.
- the activated lymphocytes are preferably activated lymphocytes activated by, for example, interleukin-2 or anti-CD3 antibody.
- the present invention is effective in that when activated lymphocytes are administered to cattle or horses, it is possible to administer activated lymphocytes derived from another family whose leukocyte antigen does not match that of the self.
- activated lymphocytes derived from another family whose leukocyte antigen does not match that of the self.
- the probability of matching leukocyte antigens between individuals is low. Serious side effects occur when activated lymphocytes from other homes with different leukocyte antigens are administered.
- autologous activated lymphocytes are often used to treat infectious diseases using activated lymphocytes. Only when the leukocyte antigen was found to match that of its own, activated lymphocytes from other families with the same leukocyte antigen as its own were used.
- activated lymphocyte preparations can prevent neonatal infections, and are effective in treating neonatal frail syndrome, which has been particularly problematic.
- activated lymphocytes when activated lymphocytes are administered to cattle or horses, it is possible to administer activated lymphocytes from other homes that do not match the leukocyte antigens, so that the step of examining leukocyte antigens can be omitted. Further, since it is not necessary to match the leukocyte antigens, mass production of an activated lymphocyte of a certain individual enables mass production of the activated lymphocyte preparation, thereby reducing the manufacturing cost.
- This method comprises the steps of isolating lymphocytes from an animal in which antibodies and immune cells do not migrate from the mother to the fetus via the placenta, and proliferating and activating the isolated lymphocytes to obtain activated lymphocytes. Using the activated lymphocytes.
- a method for producing activated lymphocytes for example, a method obtained by appropriately modifying the method disclosed in Japanese Patent Application Laid-Open No. 2004-2312 may be adopted.
- a preferred embodiment of this method involves culturing the isolated lymphocytes in the presence of interleukin-2 or a factor that promotes T cell proliferation (eg, anti-CD3 antibody). As demonstrated by the examples described later, when IgG1 type is used as the anti-CD3 antibody, activated lymphocytes can be activated effectively.
- gelatin can be added to the culture medium of lymphocytes. Gelatin promotes lymphocyte activation. In addition, when gelatin is added to the medium, gelatin serves to protect activated lymphocytes. Furthermore, as will be described later, since preparations containing activated lymphocytes to be administered to cattle or horses can be stored frozen, gelatin can function as a buffer during frozen storage.
- the gelatin pork skin gelatin, beef bone gelatin, fish bone or fish leather gelatin can be used.
- Gelatin can be added in an amount of 0.1 to 10 parts by weight, preferably 1 to 5 parts by weight, based on 100 parts by weight of the culture medium. Gelatin is mixed into the medium in the usual manner.
- the culture temperature may be 37 ° C. to 42 ° C., preferably 39 ° C. to 42 ° C.
- the temperature of 39 ° C. to 42 ° C. or lower is higher than the normal culture temperature. However, the temperature of 39 ° C. or more and 42 ° C.
- lymphocytes that are more active against cattle or horses can be obtained by culturing lymphocytes to be administered to cattle or horses at a temperature of 39 ° C. or higher and 42 ° C. or lower.
- the reinforcing agent of the present invention may be stored frozen after production. For example, when a newborn is born, it may be thawed and administered.
- the agent of this invention may be provided as a kit which prepared as a liquid agent and frozen with the male sperm of a brand cow or a racehorse (Thoroughbred).
- male sperm and activated lymphocyte preparations as a kit, it is possible to sell activated lymphocyte preparations that are relatively free of side effects. That is, since there is an agent containing active lymphocytes derived from the father of a newborn born by fertilization using the sperm contained in such a kit, the agent of the present invention is administered with relatively few side effects. be able to. Since the breeding cattle and the stallion are limited among the brand cattle and the thoroughbred, the kit of the present invention can be used effectively.
- the dosage of the preparation mainly composed of activated lymphocytes may be appropriately increased or decreased depending on the type of animal in which the target antibody or immune cells do not migrate from the mother to the fetus via the placenta.
- the lymphocyte dosage per kg body weight is 1 ⁇ 10 2 or more and 1 ⁇ 10 9 or less.
- the dosage of the preparation per administration may be such that the lymphocyte dosage is 1 ⁇ 10 3 or more and 1 ⁇ 10 11 or less, or 1 ⁇ 10 4 or more and 1 ⁇ 10 10 or less.
- a well-known dosage form can be adopted for the agent of the present invention.
- a preferred drug system of the agent of the present invention is an injection.
- An example of this agent is an injection in which an appropriate amount of active lymphocytes is dispersed in a physiological saline solution containing 0.01 to 5% by volume of serum or serum albumin.
- An example of a method for administering an injection is intravenous injection.
- An example of administration frequency is from once / day to once / month.
- the third aspect of the present invention relates to a method for enhancing the immune function of an animal in which antibodies and immune cells do not migrate from the mother to the fetus via the placenta.
- the method includes the step of administering lymphocytes isolated, expanded and activated from an animal to an animal of the same species as the animal and from another home.
- lymphocytes that are proliferated and activated are administered to a newborn of an animal in which antibodies and immune cells do not migrate from the mother to the fetus via the placenta. That is, this embodiment strengthens the neonatal immune function and treats frail syndrome.
- a preferred embodiment of the third aspect of the present invention is an animal in which antibodies and immune cells are not transferred from the mother to the fetus via the placenta, and is preferably 1 year or less immediately after birth, more preferably 3 months or less immediately after birth.
- the neonate is administered proliferated and activated lymphocytes. Animals within the first few months of life are highly immune-tolerant, and so do not cause much rejection when given activated lymphocytes from other homes. For this reason, the agent of this invention can be used effectively in order to strengthen the immune function of the animal of several months of age.
- activated lymphocytes were obtained by collecting lymphocytes from bovine peripheral blood and activating the collected lymphocytes using IL-2 and anti-bovine CD3 antibody. .
- IL-2 and anti-bovine CD3 antibody were obtained by collecting lymphocytes from bovine peripheral blood and activating the collected lymphocytes using IL-2 and anti-bovine CD3 antibody.
- Anti-bovine CD3 antibody (MM1A (IgG1), manufactured by VRRD) was diluted to a concentration of 2.5 ⁇ l / ml with sterile phosphate buffered saline (PBS), and the surface area was 75 cm 2 . 10 ml was injected into a flask (manufactured by Sumitomo Bakelite). This antibody diluted solution was spread evenly on the bottom of the flask, and allowed to stand in a refrigerator (4 ° C.) until use to solidify. At the time of use, it was taken out from the refrigerator, and the antibody diluted solution was removed by suction, followed by washing three times with sterilized PBS. Finally, the remaining remaining solution was sufficiently removed by suction to prepare an activated lymphocyte culture flask.
- PBS sterile phosphate buffered saline
- peripheral blood 20 ml of peripheral blood was collected from the jugular vein of cattle using a heparinized blood collection tube. Peripheral blood was centrifuged at 1,600 rpm for 10 minutes, and the separated plasma was collected into a new sterile tube.
- RPMI-1640 medium manufactured by Wako Pure Chemical Industries, Ltd.
- GE Ficoll-Paque
- 10 ml of diluted blood is layered on 3 ml, centrifuged at 1,600 rpm for 30 minutes at 22 ° C., and a peripheral blood mononuclear cell (PBMC) layer containing lymphocytes is collected and RPMI-1640 Washed once with medium.
- PBMC peripheral blood mononuclear cells
- the culture was started. After 3 days of culture, 20 ml of LAM-2 medium was added, and 40 ml of LAM-2 medium was added 2 days later (after 5 days of culture). Two days later (7 days after culturing), it was confirmed that the cells sufficiently proliferated, and the entire amount of the cell suspension was transferred to the LAM-3 medium. Thereafter, the cells were allowed to stand in a carbon dioxide incubator for 5 to 10 days until the proliferated cells were collected.
- the culture temperature may be 37 ° C. to 42 ° C., and the culture can be preferably performed in a relatively high temperature range of 39 ° C. to 42 ° C.
- the bag culture container (LAM-3) containing the culture solution in which the cultured lymphocytes are suspended is thoroughly stirred, and the cell suspension is transferred from the sample port to the centrifuge tube.
- the cells were collected and centrifuged at 1,600 rpm for 7 minutes, and cultured lymphocytes were collected. Thereafter, 0.1% autologous serum (centrifuged plasma from whole blood was stored refrigerated for 1 day or more and then inactivated by incubation at 56 ° C. for 30 minutes.
- the monoclonal antibodies used for the measurement were FITC-labeled anti-bovine CD8 antibody (9ACT80C: manufactured by VRRD), PE-labeled anti-bovine CD4 antibody (CACT183B: manufactured by VRRD), PE-labeled anti-canine CD21 antibody (MCA1781PE, cellotech).
- FITC-labeled anti-bovine CD8 antibody 9ACT80C: manufactured by VRRD
- PE-labeled anti-bovine CD4 antibody CACT183B: manufactured by VRRD
- PE-labeled anti-canine CD21 antibody MCA1781PE, cellotech
- anti-bovine CD3 antibody M1A, manufactured by VRRD
- anti-bovine ⁇ T cell antibody WC-1, manufactured by Serotech
- anti-B-B7 (CD21-like) antibody G25A, VMRD
- FIG. 1 is a graph showing a proliferation curve of active lymphocytes.
- Cultured lymphocytes were collected within 2 days from the birth date (0 day of age) of the newborn calf and transferred from the jugular vein of the newborn calf.
- the dose of activated lymphocytes was about 1 ⁇ 10 9 .
- These newborn calves were used as activated lymphocyte administration groups.
- a group of newborn calves that did not receive activated lymphocytes was used as a control group.
- all newborn calves were fed a commercially available powdered colostrum formulation after birth.
- Peripheral blood was collected for a total of 8 times, analysis of leukocyte fraction in peripheral blood, measurement of lymphocyte surface type, and analysis of cytokine gene expression level by stimulation with PHA (phytohemagglutinin).
- peripheral blood lymphocyte surface type In the same manner as in Example 1, the peripheral blood lymphocyte surface type was measured.
- FIG. 2 is a graph showing changes in peripheral blood mononuclear cells (PBMC).
- FIG. 3 is a graph showing changes in CD3-positive T lymphocytes.
- FIG. 4 is a graph showing changes in B lymphocytes.
- FIG. 5 is a graph showing changes in IgM type B lymphocytes.
- FIG. 6 is a graph showing changes in natural killer cells (NK cells).
- PBMC peripheral blood mononuclear cells
- the number of peripheral blood mononuclear cells PBMC
- the number of CD3-positive T lymphocytes the number of CD8 killer T cells
- the number of NK cells are the same for both the activated lymphocyte administration group and the control group. It can be seen that it gradually increases later. However, these cell proliferations are higher in the activated lymphocyte administration group than in the control group after the second vaccination.
- the activated lymphocyte-administered group showed that B lymphocytes (MHC class-II + CD14 ⁇ B cells) and IgM type B cells 3 days and 6 days after the second vaccination compared to the control group. It can be seen that lymphocytes (CD21 + IgM + B cells) were significantly increased.
- FIG. 7A is a graph showing changes in CD4 + cells.
- FIG. 7B is a graph showing changes in CD4 + CD45R ⁇ cells.
- FIG. 8A is a graph showing changes in CD8 + cells.
- FIG. 8B is a graph showing changes in CD8 + CD45R ⁇ cells.
- FIG. 7A, FIG. 7B, FIG. 8A, and FIG. 8B show that these cell proliferations are higher in the activated lymphocyte administration group than in the control group. This indicates that the activated lymphocytes in this example enhanced the immune function of the newborn calf.
- these cells increase gradually after the first vaccination and increase after the second vaccination. This indicates that the antigen responsiveness of the cells is increased by the first vaccination, and the responsiveness to the antigen is higher than that in the second vaccination.
- PBMC peripheral blood mononuclear cells
- cDNA is synthesized using the extracted mRNA, and real-time PCR was performed. This was carried out using ⁇ -actin (actin) as an internal standard gene. 7700 Sequence according to the method reported using SYBR Green PCR Master Mix (Applied Biosystems, CA, USA). Real-time PCR was performed using a Detector.
- PCR conditions are as follows: Step One Plus TM Real-Time PCR The instruction manual of System (Applied Biosystem Japan) was followed. A sample was set in this apparatus, 50 ° C. for 30 minutes and 95 ° C. for 15 minutes were each performed once, and the reaction with 95 ° C. for 15 minutes and 60 ° C. for 1 minute was repeated 45 times.
- the target gene and the forward and reverse primers for PCR were as shown in Table 2 below.
- FIG. 9A, B, C and D are graphs showing the expression levels of IL-2 gene, IL-4 gene, IL-6 gene and IFN- ⁇ gene, respectively.
- all cytokine genes had a higher cytokine expression level in the group to which activated lymphocytes were administered than in the target group before and after the second vaccination, but on the 6th day. It can be seen that the amount of cytokine expression is lower in the group administered activated lymphocytes than in the subject group. This indicates that the activated lymphocytes in this example enhanced the immune function of newborn calves early.
- activated lymphocytes derived from a mother horse were obtained, and an agent containing active lymphocytes was administered to a newborn foal.
- Activated lymphocytes are prepared in the same manner as in Example 2 except that lymphocytes are collected using maternal horse-derived peripheral blood and the collected lymphocytes are activated using IL-2 and anti-horse antibodies. did. These agents containing activated lymphocytes were administered to newborn foals in the same manner as in Example 2. These newborn foals were used as the activated lymphocyte administration group. On the other hand, a group of neonates that did not receive activated lymphocytes was used as a control group. All newborn calves were given a commercially available powdered colostrum formulation after birth to eliminate the effects of colostrum intake from the mother horse.
- Peripheral blood was collected for a total of 8 times, analysis of leukocyte fraction in peripheral blood, measurement of lymphocyte surface type, and analysis of cytokine gene expression level by stimulation with PHA (phytohemagglutinin).
- the agent of the present invention and the method for producing the same can be used in the pharmaceutical industry as a strengthening agent for an immune function of an animal in which antibodies and immune cells do not migrate from the mother to the fetus through the placenta, or a therapeutic agent for the neonatal weak child syndrome.
- the animal immune function enhancing method of the present invention can be used in veterinary medicine.
- Sequence number 1 Primer sequence number 2: Primer sequence number 3: Primer sequence number 4: Primer sequence number 5: Primer sequence number 6: Primer sequence number 7: Primer sequence number 8: Primer sequence number 9: Primer sequence number 10: Primer sequence number
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Abstract
Description
この実施例では,ウシ末梢血からリンパ球を採取し,採取したリンパ球をIL-2及び抗ウシCD3抗体を用いて活性化することで,活性化リンパ球を得た。以下,各工程について説明する。
抗ウシCD3抗体(MM1A(IgG1),VNRD社製)を滅菌したリン酸緩衝食塩液(PBS)で2.5μl/mlの濃度に希釈し,表面積75cm2のフラスコ(住友ベークライト社製)に10ml注入した。この抗体希釈液をフラスコの底面にまんべんなく広げ,冷蔵庫(4℃)で使用時まで静置し,固相化した。使用時に冷蔵庫から取り出し,抗体希釈液を吸引除去後,滅菌PBSで3回洗浄し,最後に洗浄した残りの液を十分に吸引除去し,活性化リンパ球培養用フラスコを調製した。
インターロイキン(IL)-2を700U/ml含有するLAM-1培地(株式会社ケーナインラボ製)に体積比2.5%仔牛血清(JRHバイオサイエンス社製,放射線滅菌済み)を含むよう添加して,調製した。培養工程の途中で使用するIL-2を175U/ml含むLAM-2培地(株式会社ケーナインラボ製)及びIL-2を175U/mlを含むLAM-3培地(ガス透過性バッグ入り,全量750ml,株式会社ケーナインラボ製)は,既成ものを使用した。
ヘパリン処理した採血管を用いて,ウシの頸静脈から末梢血全血20mlを採血した。末梢血を1,600回転で10分間遠心分離し,分離された血漿を新しい滅菌チューブに分取した。血球成分が含まれる遠心分離の沈さに,総量が全血量の2倍である40mlになるようにRPMI-1640培地(和光純薬社製)を加えて希釈し,Ficoll-Paque(GE社製)3mlに対し希釈血液10mlを重層して,1,600回転,30分,22℃の条件で遠心を行い,リンパ球を含む末梢血単核球(PBMC)層を回収し,RPMI-1640培地で1回洗浄した。この操作で,約1×107個の末梢血単核球(PBMC)が得られた。
回収したPBMCを,IL-2を700U/ml及び体積比2.5%の仔牛血清を含むLAM-1培地20ml中に懸濁させ,抗ウシCD3抗体を固相化したフラスコ中に入れて,培養を開始した。培養3日後にLAM-2培地を20ml添加し,さらにその2日後(培養5日後)にLAM-2培地を40ml添加した。その2日後(培養7日後)に細胞が十分に増殖したことを確認し,LAM-3培地に細胞浮遊液の全量を移した。以降,増殖した細胞を回収するまでの5~10日間,炭酸ガスインキュベーター内に静置した。培養温度は,37℃から42℃であれば良く,好ましくは,39℃から42℃の比較的高温域で培養することができる。
投与予定が確定した培養リンパ球は,培養リンパ球が浮遊した培養液が入ったバッグ培養容器(LAM-3)をよく攪拌し,サンプルポートから細胞浮遊液を遠心管に回収して,1,600回転,7分間の条件で遠心し,培養リンパ球を回収した。その後,0.1%自己血清(全血から遠心分離した血漿を1日以上冷蔵保存した後,56℃で30分間インキュベーションし非働化した。さらに1日以上冷蔵保存した後,3,000回転,20分間の条件で遠心し,その上清を自己血清とした。)を含む生理食塩液で2回洗浄し,最後に1%自己血清を含む生理食塩液50mlに浮遊させ,注射筒に充填し,投与製剤とした。なお,培養リンパ球浮遊培養液中のエンドトキシン測定(トキシカラーテスト,生化学工業社製)とトリプトソーヤ寒天培地(ニッスイ社製)による細菌培養を実施し,投与前に安全性を確認した。
上記により得られた培養リンパ球5×105個を試験管に分取し,PBSを加えて1回洗浄後上清を除去して,直接染色の場合FITC(フルオレセインイソチオシアネート)またはPE(フィコエリスリン)標識モノクローナル抗体10μlを,間接染色の場合非標識モノクローナル抗体1μgをそれぞれ加えよく撹拌した。冷蔵庫内で30分間静置の後,PBSで1回洗浄した。間接染色の場合,次に適当に希釈したFITC標識ヒツジ抗マウス抗体(セロテック社製)50μlを加えよく撹拌し,冷蔵庫内に30分間静置した。その後,PBSで1回洗浄し,最後にシース液0.5mlを加え,よく撹拌し,CyAn(Dako社製)で各抗体に反応した表面抗原を測定した。測定に用いたモノクローナル抗体は,直接染色にはFITC標識抗ウシCD8抗体(9ACT80C:VNRD社製),PE標識抗ウシCD4抗体(CACT183B:VNRD社製),PE標識抗イヌCD21抗体(MCA1781PE,セロテック社製),間接染色には抗ウシCD3抗体(MM1A,VNRD社製),抗ウシγδT細胞抗体(WC-1,セロテック社製),抗B-B7(CD21-like)抗体(GB25A,VMRD),を用いた。対照として非特異的反応を測定するため,FITC標識抗マウスIgG1抗体(Dako社製)及びPE標識抗マウスIgG1抗体(Dako社製)を用いた。その結果を,表1に示す。
培養リンパ球を,新生子牛の出生日(0日齢)から2日以内に回収し,新生子牛の頸静脈から移入した。活性化リンパ球の投与量は,約1×109個であった。これらの新生仔牛を活性化リンパ球投与群とした。一方,新生仔牛のうち活性化リンパ球を投与しなかった群を対照群とした。そして,全ての新生子牛に対し,母牛からの初乳摂取による影響を排除するため,出生後に市販の粉末初乳製剤を給与した。その後,2週齢後に1回目ワクチン接種,6週齢後に2回目ワクチン接種をそれぞれ実施し,0,3,7日齢,2週齢とその3日後,6週齢とその3日後及び6日後の計8回について末梢血の採血を行い,末梢血中白血球の分画の解析及びリンパ球表面型の測定,PHA(フィトヘムアグルチニン)刺激によるサイトカイン遺伝子発現量の解析を行った。
実施例1と同様にして,末梢血中リンパ球の表面型を測定した。
ヘパリン添加血液から比重液を用いて末梢血単核球(PBMC)を分離し,mRNAを抽出した。従来の方法を参考に,抽出したmRNAを用いてcDNAを合成し,real-time
PCRを実施した。内部標準遺伝子としてβ-アクチン(actin)を用いて実施した。SYBR Green PCR Master Mix(Applied Biosystems, CA, USA)を利用して報告されている方法に準じて7700 Sequence
Detectorを用いてreal-time PCRを実施した。
System (Applied Biosystem Japan)の取扱説明書に従った。この装置にサンプルをセットし,50℃30分間,95℃15分間をそれぞれ1回行い,95℃15分間,60℃1分間を1サイクルとした反応を45回繰り返した。
配列番号2:プライマー
配列番号3:プライマー
配列番号4:プライマー
配列番号5:プライマー
配列番号6:プライマー
配列番号7:プライマー
配列番号8:プライマー
配列番号9:プライマー
配列番号10:プライマー
Claims (13)
- ヒトを除く哺乳動物の組織由来の活性化リンパ球を有効成分として含む,前記動物と同種であり他家動物の免疫機能の強化剤。
- 前記動物の組織由来の活性化リンパ球が,インターロイキン-2及び抗CD3抗体のいずれか又は両方により活性化された活性化リンパ球である,請求項1に記載の剤。
- 前記ヒトを除く哺乳動物が,抗体や免疫細胞が母体から胎子へ胎盤を介して移行しないヒトを除く哺乳動物である,請求項1に記載の剤。
- 前記ヒトを除く哺乳動物が,ウシ又はウマである,請求項1に記載の剤。
- 請求項1に記載の剤であって,
前記動物の新生子の虚弱症候群を治療するための剤として用いられる剤。 - 前記虚弱症候群が感染症を含む,請求項5に記載の剤。
- ヒトを除く哺乳動物からリンパ球を分離する工程と,
分離されたリンパ球を増殖及び活性化し活性化リンパ球を得る工程と,
得られた活性化リンパ球を用いて製剤する工程と
を含む,
他家動物の免疫機能の強化剤の製造方法。 - 前記活性化リンパ球を得る工程が,
インターロイキン-2及び抗CD3抗体のいずれか又は両方の存在下に分離されたリンパ球を培養する工程を含む,
請求項7に記載の製造方法。 - 前記動物がウシであり,活性化リンパ球を得る工程が,抗ウシCD3抗体の存在下に分離されたリンパ球を培養する工程を含み,
前記抗ウシCD3抗体が,IgG1型の抗体である,
請求項7に記載の製造方法。 - 前記他家動物の免疫機能の強化剤が,
前記動物と同種であり他家新生子の虚弱症候群を治療するための,前記動物の新生虚弱子症候群の治療剤である,
請求項7に記載の製造方法。 - ヒトを除く哺乳動物から分離され,増殖及び活性化されたリンパ球を前記動物と同種であり他家の動物に投与する工程を含む,
前記動物の免疫機能強化方法。 - 前記リンパ球を投与する工程は,前記動物の新生子に増殖及び活性化されたリンパ球を投与する工程である請求項11に記載の方法。
- 前記リンパ球を投与する工程は,生後直後から1年以下の前記動物に増殖及び活性化されたリンパ球を投与する工程である請求項11に記載の方法。
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JP2011546484A JP5006992B2 (ja) | 2010-08-06 | 2011-08-05 | 免疫機能の強化剤 |
NZ603641A NZ603641A (en) | 2010-08-06 | 2011-08-05 | Immunological function enhancing agent |
EP11814313.0A EP2601957B1 (en) | 2010-08-06 | 2011-08-05 | Immunological function enhancing agent |
US13/697,851 US20130071437A1 (en) | 2010-08-06 | 2011-08-05 | Immunological function enhancing agent |
AU2011287108A AU2011287108B2 (en) | 2010-08-06 | 2011-08-05 | Immunological function enhancing agent |
CA2799516A CA2799516C (en) | 2010-08-06 | 2011-08-05 | Immunological function enhancing agent |
US14/826,022 US20150342995A1 (en) | 2010-08-06 | 2015-08-13 | Immunological function enhancing agent |
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US14/826,022 Division US20150342995A1 (en) | 2010-08-06 | 2015-08-13 | Immunological function enhancing agent |
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WO2016013041A1 (ja) * | 2014-07-22 | 2016-01-28 | 株式会社がん免疫研究所 | 末梢循環癌細胞の検出方法および検出装置 |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20030044387A1 (en) * | 2000-06-09 | 2003-03-06 | Teruaki Sekine | Method and activated lymphocyte preparations for preventing recurrence of carcinoma |
EP1352955A2 (en) * | 2002-04-08 | 2003-10-15 | Lymphotec Inc. | HLA matching donor-originating activated lymphocytes to be used in prevention/treatment of tumors, infectious diseases and autoimmune diseases, treatment method achieved by using the lymphocytes, formula having the lymphocytes as a main constitutuent thereof, method for manufacturing the formula and preparation kit to be used to prepare the formula |
Family Cites Families (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6534055B1 (en) * | 1988-11-23 | 2003-03-18 | Genetics Institute, Inc. | Methods for selectively stimulating proliferation of T cells |
US5858358A (en) * | 1992-04-07 | 1999-01-12 | The United States Of America As Represented By The Secretary Of The Navy | Methods for selectively stimulating proliferation of T cells |
EP0831860B1 (en) * | 1995-05-25 | 2004-10-20 | Baxter International Inc. | Allogeneic cell therapy for cancer following allogeneic stem cell transplantation |
DK0969865T3 (da) * | 1996-05-23 | 2007-03-19 | Scripps Research Inst | Systemer til præsentation af MHC-antigener af klasse II og fremgangsmåder til aktivering af CD4+-T-celller |
US6867041B2 (en) * | 2000-02-24 | 2005-03-15 | Xcyte Therapies, Inc. | Simultaneous stimulation and concentration of cells |
DK1257632T3 (da) * | 2000-02-24 | 2008-01-28 | Xcyte Therapies Inc | Samtidig stimulering og opkoncentrering af celler |
US20030235908A1 (en) * | 2000-02-24 | 2003-12-25 | Xcyte Therapies, Inc. | Activation and expansion of cells |
US7048922B2 (en) * | 2002-05-29 | 2006-05-23 | Demao Yang | Stimulation of hematopoiesis by ex vivo activated immune cells |
US7435592B2 (en) * | 2003-05-13 | 2008-10-14 | Immunovative Therapies, Ltd. | Compositions for allogeneic cell therapy |
ES2482141T3 (es) * | 2006-04-13 | 2014-08-01 | Immunovative Therapies, Ltd. | Terapia con células alogénicas para el tratamiento de una infección oportunista |
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Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20030044387A1 (en) * | 2000-06-09 | 2003-03-06 | Teruaki Sekine | Method and activated lymphocyte preparations for preventing recurrence of carcinoma |
EP1352955A2 (en) * | 2002-04-08 | 2003-10-15 | Lymphotec Inc. | HLA matching donor-originating activated lymphocytes to be used in prevention/treatment of tumors, infectious diseases and autoimmune diseases, treatment method achieved by using the lymphocytes, formula having the lymphocytes as a main constitutuent thereof, method for manufacturing the formula and preparation kit to be used to prepare the formula |
JP2004002312A (ja) | 2002-04-08 | 2004-01-08 | Lymphotec:Kk | 腫瘍・感染症および自己免疫疾患の予防・治療用hla一致他人由来活性化リンパ球および該リンパ球を主成分とする製剤ならびに該製剤の製造方法、該製剤調製用キット |
Non-Patent Citations (4)
Title |
---|
OHTSUKA, H. ET AL.: "Changes in peripheral leukocyte populations of weak calf syndrome of Japanese Black calves", JOURNAL OF VETERINARY MEDICAL SCIENCE, vol. 65, no. 7, 2003, pages 793 - 796, XP009174312 * |
See also references of EP2601957A4 |
SLAVIN, S. ET AL.: "Immunotherapy for resistant hematologic malignancies using matched or mismatched rIL-2 activated donor lymphocytes positively selected for CD56+ after allogeneic stem cell transplantation for allogeneic cell therapy without GVHD", BLOOD, vol. 102, no. 11, 2003, pages 400B, XP009175363 * |
TAKASU, M. ET AL.: "Thymic hypoplasia in Japanese black calves with stillbirth/perinatal weak calf syndrome", JOURNAL OF VETERINARY MEDICAL SCIENCE, vol. 70, no. 11, 2008, pages 1173 - 7, XP055098233 * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2016013041A1 (ja) * | 2014-07-22 | 2016-01-28 | 株式会社がん免疫研究所 | 末梢循環癌細胞の検出方法および検出装置 |
JP6052756B2 (ja) * | 2014-07-22 | 2016-12-27 | 株式会社がん免疫研究所 | 末梢循環癌細胞の検出方法および検出装置 |
KR101922322B1 (ko) | 2014-07-22 | 2018-11-26 | 가부시키가이샤 간멘에키켄큐쇼 | 말초 순환 암세포의 검출 방법 및 검출 장치 |
Also Published As
Publication number | Publication date |
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NZ603641A (en) | 2014-11-28 |
EP2601957B1 (en) | 2019-04-10 |
CA2799516A1 (en) | 2012-02-09 |
EP2601957A4 (en) | 2014-03-12 |
JP5006992B2 (ja) | 2012-08-22 |
EP2601957A1 (en) | 2013-06-12 |
AU2011287108B2 (en) | 2014-08-28 |
US20150342995A1 (en) | 2015-12-03 |
US20130071437A1 (en) | 2013-03-21 |
JPWO2012017678A1 (ja) | 2013-10-03 |
AU2011287108A1 (en) | 2012-12-06 |
CA2799516C (en) | 2016-01-05 |
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