JPH0214038B2 - - Google Patents

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Publication number
JPH0214038B2
JPH0214038B2 JP57019560A JP1956082A JPH0214038B2 JP H0214038 B2 JPH0214038 B2 JP H0214038B2 JP 57019560 A JP57019560 A JP 57019560A JP 1956082 A JP1956082 A JP 1956082A JP H0214038 B2 JPH0214038 B2 JP H0214038B2
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JP
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Prior art keywords
cells
hirs
human
animal
approximately
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Lifetime
Application number
JP57019560A
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Japanese (ja)
Other versions
JPS58138395A (en
Inventor
Kaname Sugimoto
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Hayashibara Seibutsu Kagaku Kenkyujo KK
Original Assignee
Hayashibara Seibutsu Kagaku Kenkyujo KK
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Application filed by Hayashibara Seibutsu Kagaku Kenkyujo KK filed Critical Hayashibara Seibutsu Kagaku Kenkyujo KK
Priority to JP57019560A priority Critical patent/JPS58138395A/en
Priority to KR1019830000414A priority patent/KR900005860B1/en
Priority to IT47689/83A priority patent/IT1167074B/en
Priority to GB08303870A priority patent/GB2118560B/en
Priority to FR8302180A priority patent/FR2521588B1/en
Priority to CH805/83A priority patent/CH661745A5/en
Publication of JPS58138395A publication Critical patent/JPS58138395A/en
Publication of JPH0214038B2 publication Critical patent/JPH0214038B2/ja
Granted legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • 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

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Immunology (AREA)
  • Zoology (AREA)
  • General Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Veterinary Medicine (AREA)
  • Biotechnology (AREA)
  • Public Health (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Biophysics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Toxicology (AREA)
  • Microbiology (AREA)
  • Transplantation (AREA)
  • General Engineering & Computer Science (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Description

【発明の詳細な説明】 本発明は、ヒト免疫応答抑制因子の製造方法に
関する。DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing a human immune response suppressor.

ヒト免疫応答抑制因子（human immune
response suppressor、以後hIRSという）は、ヒ
ト由来の細胞から調製される免疫応答抑制因子で
ある。 human immune response suppressor
response suppressor (hereinafter referred to as hIRS) is an immune response suppressor prepared from human-derived cells.

免疫応答抑制因子は、Carl Waltenbaugh著
（1979年）、“Biology of the Lymphokines”（S.
Cohen、E.Pick、J.J.Oppenheim編）第422〜427
頁、Academic Press、New Yorkの記載から明
らかなように、抗原に非特異的に免疫応答の抑制
作用を示す体液性の蛋白性物質であることより、
免疫応答過敏症、例えばアレルギー、アナフイラ
キシーシヨツク、自己免疫疾患などの予防剤、治
療剤として、また組織移植、臓器移植などに際し
て起きる免疫拒絶反応の抑制剤などとしての利用
が期待されている。また、免疫応答抑制因子は、
その作用が種依存性を示さないことより、ヒト疾
患の治療に際し、ヒト以外の動物細胞由来の免疫
応答抑制因子を使用することも考えられるが、ヒ
トに供するには、ヒト生細胞由来のhIRSを使用
するのが安全であり、優れている。 Immune response suppressors are described in Carl Waltenbaugh (1979), “Biology of the Lymphokines” (S.
Cohen, E. Pick, JJ Oppenheim (eds.) No. 422-427
As is clear from the description in Page, Academic Press, New York, it is a humoral proteinaceous substance that suppresses immune responses non-specifically to antigens.
It is expected to be used as a preventive or therapeutic agent for immune response hypersensitivity, such as allergies, anaphylaxis, and autoimmune diseases, and as an agent for suppressing immune rejection reactions that occur during tissue and organ transplants. In addition, immune response suppressors are
Since its action is not species-dependent, it is possible to use immune response suppressors derived from non-human animal cells in the treatment of human diseases. is safe and superior to use.

しかしながら、ヒト疾患の予防や治療に使用し
得るhIRSの製造は、未だ工業的規模で実施され
るに至つていない。 However, the production of hIRS that can be used for the prevention and treatment of human diseases has not yet been carried out on an industrial scale.

本発明者は、工業的規模で容易に実施し得る
hIRSの製造方法を鋭意検討した。 The inventor has demonstrated that it can be easily implemented on an industrial scale.
We have intensively investigated the manufacturing method of hIRS.

その結果、意外にもhIRS産生能を有するヒト
由来の細胞をヒト以外の温血動物を利用して増殖
させて得た細胞が、インビトロでの組織培養で得
られる細胞よりもhIRSの産生が著しく高く、細
胞当り約２〜10倍、またはそれ以上にも達するこ
とを見いだし、本発明を完成した。 As a result, it was surprisingly found that cells obtained by growing human-derived cells capable of producing hIRS using non-human warm-blooded animals produced significantly more hIRS than cells obtained by in vitro tissue culture. The present invention was completed based on the discovery that the number of cells per cell is about 2 to 10 times higher, or even higher.

すなわち、本発明は、hIRS産生能を有し、か
つヒト以外の温血動物に移植して増殖するヒト由
来の細胞をヒト以外の温血動物体内に移植、また
はその温血動物の体液の供給を受けながら増殖さ
せ、得られる細胞からhIRSを産生させることを
特徴とするhIRSの製造方法に関するものである。 That is, the present invention relates to the transplantation of human-derived cells that have the ability to produce hIRS and that can be transplanted and proliferated into a warm-blooded animal other than humans into the body of a warm-blooded animal other than humans, or the supply of body fluids from that warm-blooded animal. The present invention relates to a method for producing hIRS, which is characterized in that hIRS is produced from the obtained cells by propagating the cells while receiving a

本発明の方法は、従来のインビトロで組織培養
する場合と比較して、大量のhIRSを生成できる
だけでなく、高価な血清などを含む栄養培地が不
要、または大幅に節約でき、更に細胞増殖中の維
持管理も極めて容易である。すなわち、hIRS産
生能を有し、かつヒト以外の温血動物に移植して
増殖するヒト由来の細胞をヒト以外の温血動物体
内に移植し、またはその動物の体液の供給を受け
ることのできるチヤンバーに収容し、通常の飼育
をすれば、温血動物体から供給される栄養物を含
有する体液を利用してその細胞が容易に増殖しう
るのである。更に、インビトロで組織培養する場
合と比較して、細胞の増殖が安定していること、
その増殖速度が大きいこと、大量の細胞が得られ
ること、更には細胞当りのhIRS産生量が大きい
ことが特徴である。 Compared to conventional in vitro tissue culture, the method of the present invention not only can produce a large amount of hIRS, but also eliminates or significantly saves nutrient media containing expensive serum, and furthermore, Maintenance and management is also extremely easy. In other words, human-derived cells that have the ability to produce hIRS and can be transplanted into a warm-blooded non-human animal and proliferate can be transplanted into the body of a warm-blooded non-human animal, or can be supplied with body fluids from that animal. When housed in a chamber and reared normally, the cells can easily proliferate using the nutrient-containing body fluids provided by the warm-blooded animal. Furthermore, cell proliferation is stable compared to in vitro tissue culture;
It is characterized by its high proliferation rate, ability to obtain a large amount of cells, and furthermore, the large amount of hIRS produced per cell.

本発明で使用するヒト由来の細胞は、hIRS産
生能を有し、かつヒト以外の温血動物の体内に移
植して容易に増殖するものであればよい。例え
ば、ヒト末梢血細胞、ヒト脾臓細胞、ヒト扁桃細
胞、或いはヒト扁桃腫瘍細胞、ヒト肝臓癌細胞、
ヒト脾臓腫瘍細胞、ヒト肺癌細胞など、更にはこ
れら細胞を培養株化させたものなどが好適であ
る。 The human-derived cells used in the present invention may be any cell that has hIRS-producing ability and that can be easily transplanted into the body of a warm-blooded animal other than humans and proliferate. For example, human peripheral blood cells, human spleen cells, human tonsil cells, human tonsil tumor cells, human liver cancer cells,
Suitable are human spleen tumor cells, human lung cancer cells, and cultured cell lines of these cells.

また、培養株化された公知のヒト由来の細胞と
しては、例えば、組織培養Vol.6、第527〜546頁
（1980年）に記載されているAd−Ｌ、HL−４、
L84−Ly、NPC−501、HB−７などが適宜選択
される。 In addition, known human-derived cells that have been cultivated include, for example, Ad-L, HL-4, which is described in Tissue Culture Vol. 6, pp. 527-546 (1980).
L84-Ly, NPC-501, HB-7, etc. are selected as appropriate.

また、これら細胞のhIRS産生能を持つ遺伝子
を例えば、ポリエチレングリコールやセンダイウ
イルスなどを利用する細胞融合の手段や、DNA
リガーゼ、制限酵素（ヌクレアーゼ）、DNAポリ
メラーゼなどの酵素を利用する遺伝子組み換えの
手段などによつて、より容易に継代培養しうる培
養株化されたヒトリンパ芽球様細胞に導入して使
用することは、その増殖速度が大きいだけでな
く、細胞当りのhIRS産生能が約２〜10倍、また
はそれ以上にも高まるので特に好都合である。 In addition, the genes capable of producing hIRS in these cells can be transformed by means of cell fusion using polyethylene glycol, Sendai virus, etc., or by DNA
To be used by introducing into cultured human lymphoblastoid cells that can be more easily subcultivated by means of genetic recombination using enzymes such as ligase, restriction enzymes (nucleases), and DNA polymerases. is particularly advantageous because it not only has a high proliferation rate but also increases the hIRS production capacity per cell by about 2 to 10 times or more.

また、培養株化されたヒトリンパ芽球様細胞を
利用すれば、ヒト以外の温血動物に移植する際、
その宿主動物の細胞と混りにくい軟腫瘤を形成し
易く、摘出後の分散も容易なので、ヒトリンパ芽
球様生細胞だけを採取するのにきわめて有利であ
る。 In addition, if cultured human lymphoblastoid cells are used, when transplanted into warm-blooded animals other than humans,
It is extremely advantageous for collecting only human lymphoblastoid living cells because it easily forms a soft tumor that is difficult to mix with the cells of the host animal and is easily dispersed after removal.

このようなヒトリンパ芽球様細胞には、ヒト白
血病もしくはヒト悪性リンパ腫由来の細胞株が適
しており、例えばナマルバ（Namalva）細胞、
BALL−１細胞、NALL−１細胞、TALL−１細
胞、JBL細胞などの公知ヒト由来細胞株が、特に
有利に使用しうる。 Cell lines derived from human leukemia or human malignant lymphoma are suitable for such human lymphoblastoid cells, such as Namalva cells,
Known human-derived cell lines such as BALL-1 cells, NALL-1 cells, TALL-1 cells, and JBL cells can be used particularly advantageously.

本発明のhIRSの製造方法に使用する温血動物
は、hIRS産生能を有するヒト由来の細胞が増殖
しうるものであればよく、例えば、ニワトリ、ハ
トなどの鳥類、イヌ、ネコ、サル、ヤギ、ブタ、
ウシ、ウマ、ウサギ、モルモツト、ラツト、ヌー
ドラツト、ハムスター、普通マウス、ヌードマウ
スなどの哺乳類などが使用できる。 The warm-blooded animal used in the method for producing hIRS of the present invention may be any animal that can proliferate human-derived cells capable of producing hIRS, such as birds such as chickens and pigeons, dogs, cats, monkeys, and goats. ,pig,
Mammals such as cows, horses, rabbits, guinea pigs, rats, nude rats, hamsters, normal mice, and nude mice can be used.

これら動物にヒト由来の細胞を移植すると、好
ましくない免疫反応を起すおそれがあるので、そ
の反応をできるだけおさえるために、使用する動
物は、できるだけ幼若な状態、すなわち卵、胚、
胎児、または新生期、幼少期のものの方が好まし
い。 When human-derived cells are transplanted into these animals, there is a risk of causing an unfavorable immune reaction, so in order to suppress that reaction as much as possible, the animals used should be kept as young as possible, i.e. eggs, embryos, etc.
Fetuses, newborns, and infants are preferred.

また、これら動物に例えば、約200〜600レムの
エツクス線若しくはガンマ線を照射するか、また
は抗血清若しくは免疫抑制剤などを注射するなど
の前処置をほどこして、免疫反応を弱めて移植し
てもよい。使用する動物がヌードマウスやヌード
ラツトなどの免疫不全動物の場合には、成長した
ものであつても免疫反応が弱いので、これらの前
処置を必要とすることなく、培養株化されたヒト
由来の細胞が移植でき、急速に増殖できるので特
に好都合である。 Alternatively, these animals may be subjected to pretreatment such as irradiation with approximately 200 to 600 rem of X-rays or gamma rays, or injection of antiserum or immunosuppressants to weaken the immune response before transplantation. good. If the animal used is an immunodeficient animal such as a nude mouse or nude rat, the immune response is weak even when the animal is grown, so cultured human-derived animals do not require these pretreatments. This is particularly advantageous because the cells can be transplanted and rapidly expanded.

また、ヒト由来の細胞を、例えば先ずハムスタ
ーに移植し増殖させた後、この細胞を更にヌード
マウスに移植するなどのように、ヒト以外の温血
動物間で移植して、ヒト由来の細胞の増殖をより
安定化したり、更にそれらから生成されるhIRS
量を増加させることも自由である。この場合、同
種間、同属間は勿論のこと同綱間、同門間移植で
あつてもよい。 In addition, human-derived cells can be transplanted into warm-blooded animals other than humans, such as by first transplanting human-derived cells into hamsters and growing them, and then transplanting these cells into nude mice. The hIRS produced from them can further stabilize proliferation.
You are also free to increase the amount. In this case, the transplant may be between the same species, the same genus, the same class, or the same phylum.

ヒト由来の細胞を移植する動物体内の部位は、
移植した細胞が増殖し得る部位であればよく、例
えば尿液腔、静脈、腹腔、皮下など自由に選ばれ
る。 The site in the animal body where human-derived cells are transplanted is
Any site can be selected as long as the transplanted cells can proliferate, such as the allantoic cavity, vein, peritoneal cavity, or subcutaneous region.

また、直接動物体内にヒト由来の細胞を移植す
ることなく、動物細胞の通過を阻止し得る多孔性
の濾過膜、例えば孔径約10-7〜10-5ｍを有するメ
ンブランフイルター、限外濾過膜またはホローフ
アイバーなどを設けた公知の各種形状、大きさの
拡散チヤンバーを動物体内、例えば腹腔内に埋設
して、動物体からの栄養物を含む体液の供給を受
けつつ、そのチヤンバー内で公知の培養株化され
たヒト由来の細胞を何れも増殖させることができ
る。 In addition, porous filtration membranes that can block the passage of animal cells without directly transplanting human-derived cells into the animal body, such as membrane filters and ultrafiltration membranes with a pore diameter of approximately 10 -7 to 10 -5 m, are also available. Alternatively, a diffusion chamber of various known shapes and sizes equipped with hollow eye bars or the like is buried in an animal's body, for example, in the abdominal cavity, and while receiving body fluids containing nutrients from the animal body, a known diffusion chamber is placed inside the chamber. Any cultured human-derived cells can be grown.

また、必要に応じて、この拡散チヤンバー内の
栄養物を含む体液を動物体内のそれと接続し潅流
させるようにした拡散チヤンバーを、例えば動物
体表に取付け、拡散チヤンバー内のヒト由来の細
胞の増殖状態を透視できるようにすることも、ま
た、この拡散チヤンバー部分のみを着脱交換でき
るようにして動物を屠殺せずに寿命一杯細胞を増
殖させて、動物個体当りの細胞生産量を更に高め
ることもできる。 In addition, if necessary, a diffusion chamber that connects and perfuses the body fluid containing nutrients in the diffusion chamber with that in the animal body may be attached to the surface of the animal body, and human-derived cells within the diffusion chamber may be grown. It is also possible to make it possible to see through the animal's condition, and to make it possible to attach and detach only the diffusion chamber part to allow cells to proliferate to the fullest lifespan without slaughtering the animal, thereby further increasing the amount of cells produced per individual animal. can.

これらの拡散チヤンバーを利用する方法は、ヒ
ト由来の細胞が動物細胞と直接接触しないので、
ヒト由来の細胞のみが容易に採取できるだけでな
く、好ましくない免疫反応を起す心配も少ないの
で、免疫反応を抑制する前処置の必要もなく、各
種温血動物を自由に利用できる特徴を有してい
る。 These diffusion chamber-based methods do not allow direct contact between human-derived cells and animal cells;
Not only can only human-derived cells be easily collected, but there is also little risk of causing undesirable immune reactions, so there is no need for pretreatment to suppress immune reactions, and various warm-blooded animals can be used freely. There is.

移植した動物の維持管理は、その動物の通常の
飼育を続ければよく、移植後と言えども特別の取
扱いは何ら必要としないので好都合である。ヒト
由来の細胞を増殖させるための期間は通常１〜20
週である。移植する培養株化された細胞が腫瘍細
胞であるかリンパ芽球様細胞である場合には、そ
の増殖速度が特に大であり、通常１〜５週の期間
で目的を達成することができる。このようにして
得られるヒト由来の細胞数は、動物個体当り約
107〜1012、またはそれ以上に達することも見い
出した。 The maintenance and management of transplanted animals is convenient because it is sufficient to continue the normal breeding of the animal, and no special handling is required even after transplantation. The period for growing human-derived cells is usually 1-20
It's a week. When the cultured cells to be transplanted are tumor cells or lymphoblastoid cells, their proliferation rate is particularly high, and the purpose can usually be achieved within a period of 1 to 5 weeks. The number of human-derived cells obtained in this way is approximately
10 7 to 10 12 or even higher.

換言すれば、本発明で使用するhIRSの製造方
法により増殖させたヒト由来の細胞数は、動物個
体当り移植した細胞数の約102〜107倍、またはそ
れ以上にも達し、インビトロで栄養培地に接種し
て増殖させる場合の約101〜106倍、またはそれ以
上にも達して、hIRSの製造にはきわめて好都合
である。 In other words, the number of human-derived cells grown by the method for producing hIRS used in the present invention reaches approximately 10 2 to 10 7 times or more than the number of cells transplanted per individual animal, and can be nourished in vitro. This is approximately 10 1 to 10 6 times or more than when propagating by inoculating into a culture medium, which is extremely convenient for producing hIRS.

増殖ヒト由来細胞は、hIRS産生に供する前に、
インビトロの栄養培地で適当期間、通常１〜４日
培養し、細胞代謝を整えてもよい。 Proliferated human-derived cells are grown before being subjected to hIRS production.
Cell metabolism may be adjusted by culturing in an in vitro nutrient medium for an appropriate period of time, usually 1 to 4 days.

このようにして増殖させたヒト由来の生細胞か
らhIRSを産生させる方法は自由である。 Any method can be used to produce hIRS from human-derived living cells grown in this manner.

例えば、腹腔内の腹水に浮遊状で増殖したヒト
由来の細胞を採取し、または皮下で増殖した腫瘤
を摘出し、分散させた後採取し、この細胞を約20
〜40℃に保つた栄養培地に細胞濃度が約104〜
108／mlになるように浮遊させてhIRSを産生させ
ればよい。この際、必要ならばhIRS誘導剤を作
用させてもよい。hIRS誘導剤は、温血動物を利
用して増殖させて得られるヒト由来細胞から
hIRSを誘導生成できる物質であれば何でもよく、
例えば、フイトヘマグルチニン、コンカナバリン
Ａ、ポークウイードミトーゲン、リポポリサツカ
リド、エンドトキシン、多糖類、細菌などのミト
ーゲンやウイルス、核酸、ポリヌクレオチドなど
が適宜選択される。 For example, human-derived cells that have grown in suspension in ascites in the peritoneal cavity are collected, or a tumor that has grown under the skin is removed, dispersed, and then collected.
The cell concentration in the nutrient medium kept at ~40°C is approximately 10 4 ~
hIRS may be produced by suspending it at a concentration of 10 8 /ml. At this time, an hIRS inducer may be used if necessary. hIRS inducers are derived from human-derived cells that are grown using warm-blooded animals.
Any substance that can induce and generate hIRS may be used.
For example, phytohemagglutinin, concanavalin A, porkweed mitogen, lipopolysaccharide, endotoxin, polysaccharides, mitogens such as bacteria, viruses, nucleic acids, polynucleotides, etc. are appropriately selected.

また、hIRS産生に際し、hIRS安定剤を添加
し、生成したhIRSの安定化を計つてhIRSの収量
を高めることも自由である。 Furthermore, during hIRS production, it is also possible to add an hIRS stabilizer to stabilize the generated hIRS and increase the yield of hIRS.

このようにして誘導生成されたhIRSは、公知
の精製分離法、例えば、塩析、透析、濾過、遠心
分離、濃縮、凍結乾燥などを行なうことによつて
容易に精製分離し、採取することができる。更
に、高度の精製を必要とする場合には、例えば、
イオン交換体への吸着・脱着、ゲル濾過、アフイ
ニテイクロマトグラフイー、等電点分画、電気泳
動などの公知の方法を更に組み合わせればよく、
最高純度のhIRSを採取することも可能である。 The hIRS induced and produced in this way can be easily purified and separated and collected by performing known purification and separation methods, such as salting out, dialysis, filtration, centrifugation, concentration, and freeze-drying. can. Furthermore, if a high degree of purification is required, for example,
It is sufficient to further combine known methods such as adsorption/desorption to ion exchangers, gel filtration, affinity chromatography, isoelectric focusing fractionation, and electrophoresis.
It is also possible to collect hIRS of highest purity.

本発明により製造したhIRSは、単独で、また
はこれに例えば、ビタミン、ホルモン、抗癌剤な
どその他の一種もしくは二種以上の物質を含有せ
しめ、内服薬、注射薬などとしてヒト疾病の予防
や治療に有利に利用できる。 The hIRS produced according to the present invention can be used alone or in combination with one or more other substances such as vitamins, hormones, and anticancer drugs, and can be used as an oral medicine, injection medicine, etc. to advantageously prevent or treat human diseases. Available.

なお、明細書を通じてhIRSの活性は、Robelt
R.Rich ＆ Carl W.Pierce、J.Immunol.、
Vol.112、第1360〜1368頁（1974年）に記載され
ている方法に準じてプラーク形成細胞の応答を抑
制する活性量で示した。 Throughout the specification, the activity of hIRS is defined by Robert
R.Rich & Carl W.Pierce, J.Immunol.
Vol. 112, pages 1360-1368 (1974), the activity was expressed as the amount of activity that suppresses the response of plaque-forming cells.

すなわち、C57BL／６マウスからの脾臓細胞
を１ml（107個）ずつになるようにペトリ皿にと
り、これに段階的に希釈したhIRS含有液0.1mlを
加え、インキユベートした後のプラーク数を測定
した。 Specifically, 1 ml (10 7 cells) of spleen cells from a C57BL/6 mouse were placed in a Petri dish, 0.1 ml of a serially diluted hIRS-containing solution was added, and the number of plaques after incubation was measured. .

hIRSの活性単位は、プラーク数が50％に減少
する希釈倍数とした。 The hIRS activity unit was the dilution factor that reduced the number of plaques by 50%.

以下、２〜３の実施例を挙げて更に詳細に説明
する。 Hereinafter, a more detailed explanation will be given with reference to a few examples.

実施例 １ 成長したヌードマウスの皮下に、ヒトアデノイ
ド細胞を培養株化させたAd−Ｌ細胞を移植した
後、通常の方法で３週間飼育した。皮下に生じた
腫瘤約12ｇを摘出して細切した後、トリプシン含
有生理食塩水に浮遊させ細胞を分散させた。Example 1 Ad-L cells, which are cultured human adenoid cells, were subcutaneously transplanted into adult nude mice, and then raised in a conventional manner for 3 weeks. Approximately 12 g of the tumor formed under the skin was removed and cut into small pieces, and then suspended in trypsin-containing physiological saline to disperse the cells.

この細胞を血清無含有RPMI 1640培地（PH
7.2）で洗浄した後、細胞濃度約１×106／mlにな
るように同培地に浮遊させ、これにコンカナバリ
ンＡをml当り約1μｇ加え、37℃で20時間保つて
hIRSを産生させた。この細胞浮遊液を約
8000rpmで30分間遠心分離し得られる上清に含ま
れるhIRSの量を測定したところ、浮遊液0.1ml当
り約2400単位であつた。 The cells were grown in serum-free RPMI 1640 medium (PH
After washing with 7.2), suspend the cells in the same medium to a concentration of approximately 1 x 10 6 /ml, add approximately 1 μg of concanavalin A per ml, and keep at 37°C for 20 hours.
hIRS was produced. This cell suspension is approx.
When the amount of hIRS contained in the supernatant obtained by centrifugation at 8000 rpm for 30 minutes was measured, it was approximately 2400 units per 0.1 ml of suspension.

対照として、Ad−Ｌ細胞を仔牛血清1v／ｖ％
及び肉エキス20v／ｖ％を含有するEagle培地
（PH7.2）を用い37℃、インビトロで組織培養して
得た対照の細胞を用いて、前記同様にhIRSを産
生せしめたところ、浮遊液0.1ml当り約90単位の
産生量にすぎなかつた。 As a control, Ad-L cells were treated with 1v/v% calf serum.
When hIRS was produced in the same manner as described above using control cells obtained by in vitro tissue culture at 37°C using Eagle medium (PH7.2) containing 20 v/v% meat extract, 0.1 The production amount was only about 90 units per ml.

実施例 ２ 脾臓腫瘍患者から摘出、細切、分散させた腫瘍
細胞とリンパ芽球様ナマルバ細胞（Namalva
Cell）とを140ｍＭ NaCl、54ｍＭ KCl、１ｍ
Ｍ NaH2PO4、２ｍＭ CaCl2を含有する塩類
溶液にそれぞれ約103／mlになるように浮遊させ、
これに予め紫外線で不活化したセンダイウイルス
を含有する前記塩類溶液を、氷冷下で混合し、約
５分後に37℃恒温水槽に移して、約30分間撹拌し
つつ細胞融合させ、リンパ芽球様ナマルバ細胞に
hIRS産生能を導入した。このリンパ芽球様ナマ
ルバ細胞を成長したヌードマウスの腹腔内に移植
した後、通常の方法で５週間飼育した。Example 2 Tumor cells and lymphoblastoid Namalva cells removed, cut into small pieces, and dispersed from a splenic tumor patient
Cell) and 140mM NaCl, 54mM KCl, 1m
Suspended in a saline solution containing M NaH 2 PO 4 and 2 mM CaCl 2 to a concentration of about 10 3 /ml, respectively.
The saline solution containing Sendai virus that had been previously inactivated with ultraviolet rays was mixed with this under ice-cooling, and after about 5 minutes, the mixture was transferred to a 37°C thermostatic water bath, and the cells were fused while stirring for about 30 minutes, resulting in lymphoblasts. similar to namalva cells
The ability to produce hIRS was introduced. These lymphoblastoid Namalva cells were intraperitoneally transplanted into adult nude mice, and then raised in the usual manner for 5 weeks.

生じた腫瘤約15ｇを摘出し、ml当り10μｇのフ
イトヘマグルチニンを加えた培地を使用した以外
は実施例１と同様に処理してhIRSを産生せしめ
た。浮遊液0.1ml当りのhIRS産生量は約12800単
位であつた。 Approximately 15 g of the resulting tumor mass was excised and treated in the same manner as in Example 1, except that a medium containing 10 μg of phytohemagglutinin per ml was used to produce hIRS. The amount of hIRS produced per 0.1 ml of suspension was approximately 12,800 units.

対照として細胞融合させたリンパ芽球様ナマル
バ細胞を実施例１と同様にインビトロで組織培養
して得た細胞を用いて、hIRSを産生せしめたと
ころ、浮遊液0.1ml当り約400単位の産生量にすぎ
なかつた。 As a control, hIRS was produced using cells obtained by in vitro tissue culture of fused lymphoblastoid Namalva cells in the same manner as in Example 1, and the production amount was approximately 400 units per 0.1 ml of suspension. It was nothing more than a simple thing.

実施例 ３ ハムスター新生児にウサギから公知の方法で調
製した抗血清を予め注射し、ハムスターの免疫反
応を弱めた後、その皮下に、実施例２の方法に準
じてhIRS産生能を導入したリンパ芽球様JBL細
胞を移植し、その後通常の方法で３週間飼育し
た。Example 3 Newborn hamsters were previously injected with antiserum prepared from rabbits using a known method to weaken the immune response of the hamsters, and then lymph buds into which hIRS-producing ability had been introduced according to the method of Example 2 were subcutaneously injected into newborn hamsters. Globoid JBL cells were transplanted and then reared for 3 weeks in the usual manner.

生じた腫瘤約18ｇを摘出し、RPMI 1640培地
に代えて、肉エキス20v／ｖ％を含有するEagle
培地（PH7.2）を用いたこと以外は実施例１と同
様に処理してhIRSを産生させた。浮遊液0.1ml当
りのhIRS産生量は約8500単位であつた。 Approximately 18 g of the resulting tumor was removed and replaced with RPMI 1640 medium using Eagle containing 20 v/v% meat extract.
hIRS was produced in the same manner as in Example 1 except that a medium (PH7.2) was used. The amount of hIRS produced per 0.1 ml of suspension was approximately 8500 units.

対照として細胞融合させたリンパ芽球様JBL細
胞を実施例１と同様にインビトロで培養増殖さ
せ、次いでhIRSを産生せしめたところ、浮遊液
0.1ml当り約240単位の産生量にすぎなかつた。 As a control, fused lymphoblastoid JBL cells were cultured and grown in vitro in the same manner as in Example 1, and then hIRS was produced.
The production amount was only about 240 units per 0.1 ml.

実施例 ４ ラツト新生児の静脈内へ、リンパ芽球様ナマル
バ細胞の代りにリンパ芽球様BALL−１細胞を用
いた以外実施例２と同様にしてhIRS産生能を導
入したリンパ芽球様BALL−１細胞を移植し、通
常の方法で４週間飼育した。Example 4 Lymphoblastoid BALL-1 cells with hIRS-producing ability were introduced into the veins of newborn rats in the same manner as in Example 2 except that lymphoblastoid BALL-1 cells were used instead of lymphoblastoid Namalva cells. One cell was transplanted and raised in the usual manner for 4 weeks.

生じた腫瘤約35ｇを摘出し、実施例２と同様に
処理してhIRSを産生せしめた。浮遊液0.1ml当り
のhIRS産生量は約9400単位であつた。 Approximately 35 g of the resulting tumor mass was excised and treated in the same manner as in Example 2 to produce hIRS. The amount of hIRS produced per 0.1 ml of suspension was approximately 9400 units.

これに対して、対照としてインビトロで培養増
殖させ、hIRSを産生せしめたものは、浮遊液0.1
ml当り約350単位の産生量にすぎなかつた。 On the other hand, as a control, the cells grown in vitro and allowed to produce hIRS had a suspension of 0.1
The production amount was only about 350 units per ml.

実施例 ５ 成長した普通マウスに、約400レムのガンマ線
を照射してマウスの免疫反応を弱めた後、その皮
下にヒトリンパ節細胞を培養株化させたHL−４
細胞を移植し、その後通常の方法で４週間飼育し
た。Example 5 A grown normal mouse was irradiated with approximately 400 rem of gamma rays to weaken the immune response of the mouse, and then human lymph node cells were cultured subcutaneously into the HL-4 cell line.
Cells were transplanted and then reared for 4 weeks in the usual manner.

皮下に生じた腫瘤約15ｇを摘出し、実施例３と
同様に処理してhIRSを産生せしめた。浮遊液0.1
ml当りのhIRS産生量は約3600単位であつた。 Approximately 15 g of the tumor formed under the skin was removed and treated in the same manner as in Example 3 to produce hIRS. Suspension liquid 0.1
The amount of hIRS produced per ml was approximately 3600 units.

これに対して、対照としてインビトロで培養増
殖せしめ次いでhIRSを産生させたものでは、浮
遊液0.1ml当り約80単位の産生量にすぎなかつた。 In contrast, when hIRS was produced after culturing in vitro as a control, the production amount was only about 80 units per 0.1 ml of suspension.

実施例 ６ 孔径約0.5ミクロンのメンブランフイルターを
設けた内容量約10mlのプラスチツク製円筒型拡散
チヤンバー内に、実施例１で用いたAd−Ｌ細胞
を生理食塩水に浮遊させ、これを成長したラツト
の腹腔内に埋設した。Example 6 The Ad-L cells used in Example 1 were suspended in physiological saline in a plastic cylindrical diffusion chamber with an internal capacity of approximately 10 ml and equipped with a membrane filter with a pore size of approximately 0.5 microns, and the cells were then incubated with grown rats. It was implanted intraperitoneally.

このラツトを通常の方法で４週間飼育した後、
この拡散チヤンバーを取り出した。これにより得
られたヒト由来の細胞濃度は、約109／mlにも達
し、インビトロの炭酸ガスインキユベーター中で
培養する場合の約102倍以上にも達することが判
明した。 After raising these rats in the usual manner for 4 weeks,
The diffusion chamber was removed. The concentration of human-derived cells thus obtained reached approximately 10 9 /ml, which was found to be approximately 10 2 times higher than when cultured in an in vitro carbon dioxide incubator.

こうして得た細胞を実施例３と同様に処理して
hIRSを産生せしめた。浮遊液0.1ml当りのhIRS産
生量は約4200単位であつた。 The cells thus obtained were treated in the same manner as in Example 3.
hIRS was produced. The amount of hIRS produced per 0.1 ml of suspension was approximately 4200 units.

実施例 ７ 予め37℃で５日間保温しておいたニワトリの受
精卵に実施例４の方法でhIRS産生能を導入した
リンパ芽球様BALL−１細胞を移植し、次いで37
℃に１週間保つた。Example 7 Lymphoblastoid BALL-1 cells into which hIRS-producing ability was introduced by the method of Example 4 were transplanted into fertilized chicken eggs that had been kept at 37°C for 5 days, and then 37°C
It was kept at ℃ for 1 week.

この卵を割卵して増殖細胞を採取し、実施例１
と同様に処理してhIRSを産生せしめた。浮遊液
0.1ml当りのhIRS産生量は約3200単位であつた。 The eggs were broken to collect proliferating cells, and Example 1
hIRS was produced in the same manner as above. floating liquid
The amount of hIRS produced per 0.1 ml was approximately 3200 units.

Claims (1)

【特許請求の範囲】 １ ヒト免疫応答抑制因子産生能を有し、かつヒ
ト以外の温血動物の体内に移植して増殖するヒト
由来の細胞をヒト以外の温血動物体内に移植し、
またはその温血動物の体液の供給を受けながら増
殖させ、得られる細胞からヒト免疫応答抑制因子
を産生せしめることを特徴とするヒト免疫応答抑
制因子の製造方法。 ２ ヒト免疫応答抑制因子を産生せしめるに際
し、得られる細胞にヒト免疫応答抑制因子誘導剤
を作用せしめることを特徴とする特許請求の範囲
第１項記載のヒト免疫応答抑制因子の製造方法。[Scope of Claims] 1. Transplanting into the body of a warm-blooded animal other than humans human-derived cells that have the ability to produce a human immune response suppressor and that can be transplanted and proliferated in the body of a warm-blooded animal other than human,
Or a method for producing a human immune response suppressor, which comprises growing the warm-blooded animal while receiving a supply of body fluid, and producing the human immune response suppressor from the obtained cells. 2. The method for producing a human immune response suppressor according to claim 1, which comprises allowing a human immune response suppressor inducer to act on the obtained cells when producing the human immune response suppressor.
JP57019560A 1982-02-12 1982-02-12 Production of human immune response suppression (hirs) factor Granted JPS58138395A (en)

Priority Applications (6)

Application Number Priority Date Filing Date Title
JP57019560A JPS58138395A (en) 1982-02-12 1982-02-12 Production of human immune response suppression (hirs) factor
KR1019830000414A KR900005860B1 (en) 1982-02-12 1983-02-03 Process for producing human immune response suppresor
IT47689/83A IT1167074B (en) 1982-02-12 1983-02-10 PROCEDURE TO PRODUCE THE IMMUNITY RESPONSE AGENT IN MAN AND PRODUCT OBTAINED
GB08303870A GB2118560B (en) 1982-02-12 1983-02-11 Process for producing human immune response suppressor
FR8302180A FR2521588B1 (en) 1982-02-12 1983-02-11 PROCESS FOR PRODUCING HUMAN IMMUNE RESPONSE SUPPRESSOR
CH805/83A CH661745A5 (en) 1982-02-12 1983-02-14 APPLICATION OF A PROCESS FOR THE PRODUCTION OF HUMAN IMMUNE RESPONSE SUPPRESSOR.

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
JP57019560A JPS58138395A (en) 1982-02-12 1982-02-12 Production of human immune response suppression (hirs) factor

Publications (2)

Publication Number Publication Date
JPS58138395A JPS58138395A (en) 1983-08-17
JPH0214038B2 true JPH0214038B2 (en) 1990-04-05

Family

ID=12002686

Family Applications (1)

Application Number Title Priority Date Filing Date
JP57019560A Granted JPS58138395A (en) 1982-02-12 1982-02-12 Production of human immune response suppression (hirs) factor

Country Status (6)

Country Link
JP (1) JPS58138395A (en)
KR (1) KR900005860B1 (en)
CH (1) CH661745A5 (en)
FR (1) FR2521588B1 (en)
GB (1) GB2118560B (en)
IT (1) IT1167074B (en)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS609795B2 (en) * 1980-12-11 1985-03-13 株式会社林原生物化学研究所 Method for producing human epidermal growth factor
JPS63126897A (en) * 1986-05-02 1988-05-30 Yoshitomi Pharmaceut Ind Ltd Immunosuppressive factor

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2016015B (en) * 1978-01-22 1982-05-06 Hayashibara Co Method of preparing interferon and preparations containing interferon
JPS5729294A (en) * 1980-07-30 1982-02-17 Hayashibara Biochem Lab Inc Preparation of human insulin
JPS6045848B2 (en) * 1980-07-31 1985-10-12 株式会社林原生物化学研究所 Method for producing human growth hormone

Also Published As

Publication number Publication date
FR2521588B1 (en) 1985-10-25
GB2118560A (en) 1983-11-02
IT1167074B (en) 1987-05-06
CH661745A5 (en) 1987-08-14
JPS58138395A (en) 1983-08-17
GB2118560B (en) 1985-06-05
KR840003419A (en) 1984-09-08
KR900005860B1 (en) 1990-08-13
IT8347689A0 (en) 1983-02-10
GB8303870D0 (en) 1983-03-16
FR2521588A1 (en) 1983-08-19

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