WO1992001002A1 - Tumor necrosis factor activity inhibitor and production thereof - Google Patents

Tumor necrosis factor activity inhibitor and production thereof Download PDF

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Publication number
WO1992001002A1
WO1992001002A1 PCT/JP1991/000920 JP9100920W WO9201002A1 WO 1992001002 A1 WO1992001002 A1 WO 1992001002A1 JP 9100920 W JP9100920 W JP 9100920W WO 9201002 A1 WO9201002 A1 WO 9201002A1
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Prior art keywords
necrosis factor
tumor necrosis
tnf
urine
pro
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PCT/JP1991/000920
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French (fr)
Japanese (ja)
Inventor
Jun Suzuki
Kenji Yone
Noriyuki Tsunekawa
Yataro Ichikawa
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Teijin Limited
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    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7151—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for tumor necrosis factor [TNF], for lymphotoxin [LT]
    • 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
    • C07K14/525—Tumour necrosis factor [TNF]

Definitions

  • Tumor necrosis factor activity inhibitor and method for producing the same
  • the present invention relates to a novel substance that suppresses the activity of tumor necrosis factor, and the isolation and purification of the substance.
  • Tumor necrosis factor was administered to a CD-1 Swiss mouse with Bacillus Ca1me11e-Gtier II (BCG) bacteria, and two weeks later with bacterial endotoxin (endotoxin) It was discovered as a biologically active substance that appeared in the blood when it was killed, and Carswe! 1 reports [EA Carswe! ! La Pro at! Acad, Sci., USA, 3ot) t U 975:] A bioactive protein whose amino acid sequence was determined in 1985 by Aggarwai et al. [E, B, Aggarwal et al., J. Biol. Diem. 260, 2345 11985). It has been clarified by J.
  • TNF TNF-1 fibroblast growth factor
  • B. Beutler et al., Science, 229, 869 (1985) Induction of inflammatory response to vascular endothelial cells [J Gamble 'et al., Proc. Xa ⁇ i. A cad. Sc ⁇ ., USA, _82 ⁇ _ 8667 (1985)], ripening [A. D are! Io et al., J. Exp. Med. 163. 1443 (1986)], Interleukin, one of the substances causing inflammation :! ['PP Nawroth et al., J. Exp. Med. 1 _ 1363]
  • Noshika has been prepared and is not suitable for administration to the human body Such antibodies are considered to be a major problem.
  • immune complex formation is at least a factor that worsens the condition.
  • anti-Din'F antibody as a substance having anti-NF activity for such diseases. Therefore, if a human body fluid could contain an anti-Fing agent that could be present naturally, it would be a sufficient, effective and safe treatment for TNF-fighting diseases.
  • TNF activity inhibitor also referred to as ⁇ F inhibitor
  • Peetre et al. [Eur. J. Haemalc !, ⁇ , 414 (1988), £ 2, -270 (1989)], P. Secger La
  • the present inventors have conducted research to search for a novel TNF activity inhibitor, and have reached the present invention.
  • An object of the present invention is to provide a novel TNF activity inhibitor, a composition containing the same, and a method for producing the same.
  • the present invention encompasses the following inventions. That is, the present invention relates to H) suppressing the cell killing effect of tumor necrosis factor on L929 cells. • 2) SDS-PAGE has a reduced molecular weight of 34
  • amino acids from the 1st to the Uth from the ⁇ terminus are substances represented by the following sequence Val-Aia-Phe-Thr-Pro-Tr-Aia-Pro-Ala-Pro-Tr is there
  • the substance of the present invention is characterized in that the 11th amino acid from the N-terminus is Va! -AIa-Phe-Tr-Pro-Tyr-Ala-Pro-Ala-? Ro-Thr.
  • the substances of the present invention are described in C. Peetre et al. [Eur. J. Haematol. 11, 414 (1988 ⁇ ). However, the difference is that it is 34 K2 1 KDa.
  • amino acid sequence from the- ⁇ ⁇ end is completely different from the substance TBPI described in H. Engeimann et al., 7. Bio, Chem. 265, 153L 1990].
  • Ding XF is described by Aggarwai et al. Chem. 2345 (1985)] and Shirai et al. [Ature 313. 805: 1985)]. Including natural TNF and recombinant TNF
  • L 929 cells are mice
  • ATC C strain number is CCL-11, registered under the name L 929 (NCTC clone 929)
  • the molecular weight of the substance of the present invention is a substance having a reduced molecular weight in SDS-PAGE of 3'4K / 200 KDa.
  • the reduced state in SDS-PAGE refers to the electrical properties of SDS polyacrylamide gel.
  • a suitable reducing agent for example, 2-mercaptoethanol, dithiothreitol, etc., at 100 ° C for several minutes to obtain the disulfide in the protein.
  • a suitable reducing agent for example, 2-mercaptoethanol, dithiothreitol, etc.
  • the substance of the present invention can be obtained by purifying urine.
  • it can be obtained by purifying the urine of patients with membranous proliferative glomerulonephritis,
  • Membranous proliferative glomerulonephritis clinically involves the deposition of immune complexes in the renal glomeruli, lowering blood complement value, thickening of the glomerular basement membrane, and proliferation of mesangial cells.
  • sycaines especially interleukin 6
  • the inventor of the present invention determined the F content in the blood of patients with various renal diseases and found that high blood levels of TNF were contained in the blood of patients with membranous proliferative glomerulonephritis. Finally, using the urine of the patient with this disease, we examined the ability to inhibit the activity of DNF and found that it had a high ability to inhibit TNF activity.
  • Purification can be performed by combining purification operations such as ion exchange, reversed phase column, and XF immobilization.
  • the present invention relates to a method for producing a tumor necrosis factor activity inhibitor
  • the urine may be any of primary urine, urine collection, and spot urine.
  • urine is collected under aseptic or aseptic conditions as much as possible in order to carry out a bioa / se / se later.
  • a protease inhibitor may be added immediately after urine collection, but these additives are not required if urine is immediately transferred to frozen storage. When these additives are added, it is necessary to remove the additives sufficiently by performing appropriate operations, for example, dialysis, ⁇ extracellular filtration, gel filtration, etc.
  • Purification may be performed by an ordinary protein purification method. Since a low activity is usually observed in the urine stock solution, it is preferable to first concentrate the urine solution.
  • the enrichment method may be based on ordinary biochemical experiment methods such as ultrafiltration, lyophilization, salting out, etc., but in the case of ultrafiltration, salting out, etc., the target substance It is necessary to check in advance the molecular weight and the degree of saltiness that initiates precipitation:
  • the substance of the present invention is subjected to separation and purification by adsorbing to a column to which TNF has been applied after the above-mentioned concentration operation, and then specific eluting by elution with a reversed-phase column.
  • cell is a substance contained in the fraction in two k Lil concentration cell), column nits Lil concentration used varies depending ⁇ cell Tonito Lil ⁇ gradient, Protein C 4, VYDA and 'Company, 0.46: , 25 cm, and a 150-minute linear gradient of 1 ⁇ to 37% acetonitrile, 2? ⁇ 28%,
  • a test sample may be added to a known system for measuring TNF activity.
  • Cell killing effect on various tumor cells in vitro known as the Lysie method, cell growth inhibitory effect, fatty acid metabolism of fat cells _ inhibitory effect
  • Growth promoting effect of normal fibroblasts and 1 L-6 production inducing effect Neutrophil adhesion promoting effect on endothelial cells, superoxide secretion promoting effect, vascular endothelial cell coagulation activity enhancing effect, osteoclasting effect on bone cells, IL in various cells
  • a system for measuring the inducing effect of ⁇ 'mouth mouth staglandins is available. It is particularly preferable to measure the cell killing effect on L929 cells.
  • the tumor necrosis factor activity inhibitor of the present invention includes various types of
  • the pharmaceutical composition used as a therapeutic agent for the disease of the sword is a pharmaceutical composition containing the substance for suppressing tumor necrosis factor activity of the present invention as an active ingredient, and is other pharmaceutically acceptable. It includes a carrier and is good
  • tumors to be used as active ingredients may be used to reduce the antigenicity of the inhibitor of death factor activity or to enhance the physiological activity.
  • polyethylene glycol ⁇ PEG polyethylene glycol ⁇ PEG
  • dextran it can be modified by a known polymer such as poly-DL-alanine.
  • Examples of the form of the pharmaceutical composition include an injectable composition, a suppository and the like, and it is particularly preferable to use the injectable composition as an intravenous composition.
  • an injectable composition it is a mixture of a pharmaceutically effective amount of the tumor necrosis factor activity-inhibiting substance of the present invention and a pharmaceutically acceptable carrier, including amino acids, saccharides, and cellulose.
  • a pharmaceutically acceptable carrier including amino acids, saccharides, and cellulose.
  • activators generally added to injectable compositions, such as derivatives, polyvinylpyrrolidones, and inorganic compounds. Specific examples thereof include glycine and arginine as amino acids. , Alanine and their pharmaceutically acceptable salts, etc.
  • the sugars include mannitol, wild boar, il, xyli, oleoresin, milk, and -7'-coose.
  • Cellulose derivatives include carboxymethyi / rese / relo-t-tris, methyl 'cell'-mouth, etc .: Polyvinylpyrrolidones have a molecular weight of 10 000 ⁇ 1,000, GOO Bolibulpyrrolidone:
  • Examples of the organic acids include ascorbic acid, citric acid, and the salts thereof.
  • Examples of the inorganic compound calcium include 10% of hydrogen phosphate, 10% of hydrogen carbonate, and 10% of acetate. There is a room
  • Solutions for dissolving these excipients include injection ⁇ distillation, saline for injection or Ringer's solution for injection.
  • injections may contain stabilizers, surfactants, tonicity agents, soothing agents, preservatives, buffers, etc. as required.
  • Antioxidants such as deuterium pyrosulfite and perscorbic acid: chelating agents such as DTA and thioglycol agents, etc.
  • Examples of surfactants include phosphorylates and boroxixylene derivatives
  • Non-ionic surfactants such as Examples of the tonicity agent include sodium chloride and the like.
  • Soothing agents include benzyl alcohol, xylocaine, procaine and the like.
  • Preservatives include parabens, chlorobutanol, benzanolone chloride, and thimerosal.
  • buffer examples include sodium salts such as citric acid, acetic acid, and phosphoric acid.
  • a novel substance that suppresses the cell killing effect of tumor necrosis factor (TNF) on cells is provided, and a disease considered to be exerted by TNF, for example, endotoxin shock ⁇ ⁇
  • TNF tumor necrosis factor
  • Many autoimmune diseases such as acute burn, acute liver failure, renal failure and multiple organ failure, rheumatism, SLE, Behcet, V Grade disease at the time of burn, rejection at organ transplantation, coagulation of Kawasaki disease, DIC, etc. It can be used for treating abnormalities, diagnosing abnormalities, etc.
  • Urine from a patient with membranous proliferative glomerulitis was concentrated by ultra-filtration with a silicone (Millipore) to a fraction with a molecular weight of 10,000 or more to a volume of 10 ml Tr !? Changed '.' and 'to pH 8.0 (Sample 2)
  • the concentrated urine sample was applied to a 4 S DEAE-Sepiiarose column equilibrated with ⁇ OraM Tris pH 8.0 ⁇ 1 : Flow rate 40 ⁇ ir .: After washing with 10m Tris pH 8.0 in zjir, 10mM Tri? -LOOm Elution was carried out with NaC! PH G, and the mixture was collected at 400 m!
  • Table 1 shows the cell killing effect of TXF on these L929 cells.
  • the peak fraction was further purified by a TNF affinity column and a reversed-phase column, and the X-terminal amino acid sequence was determined.Asp-Ser-Va! -X-Pro-G ! n-Gl-Lys-T rI I e-His-Pro-Gi QX-Asn-Se rI ⁇ e (X is the residue that was not recognized in the 477A type protein sequencer without beak) It was presumed to be a known TNF inhibitor or a TNF receptor fragment, [I. "01 sson et al., Eur. J. Haemat .. 42, 270 (1989); H. Loetscher et al., Ce. j _ ⁇ _ 351 (1990), T.J, Schail, et al., Cell JJ ⁇ 361 (1990;)
  • TNF_Inhibtition rate is as follows.
  • the final purified product A35G ⁇ was freeze-dried and redissolved in 230 JL St of S. This Sanal 9 ⁇ ! 10 u 2 XSDS—PAGE sample buffer (1 mM Tris — HC] H6.8, 10% Sucrose. 10% SDS, 0.25 mg 'ml bromphenol buffer :), 1 2— After adding mercaptoethanol and heating at 100 ° C for 5 minutes, apply the whole amount to a 10-20 SDS polyacrylamide gradient gel (first character, SDS-PAGPL AT E 10'20>, —) ir. : A fixed Electrophoresis was performed for 120 minutes at a current, and BI-RAD Molecular Weight Standards-Low was used as a molecular weight marker.
  • Figure 1 shows the elution profile of the DEAE crude product when applied to a reversed-phase column.
  • Figure 2 shows the results of SDS-PAGE under reduced conditions of the final purified product A: ⁇ Sequence list>

Abstract

A novel tumor necrosis factor activity inhibitor which inhibits the cytocidal effect of a tumor necrosis factor and has a molecular weight of about 34 kDa and a sequence of 11 amino acids at the N-terminus of Val-Ala-Phe-Thr-Pro-Tyr-Ala-Pro-Ala-Pro-Thr.

Description

細 書  Book
('発明の名称〉 ('Title of invention>
腫疡壊死因子活性抑制物質及びその製造方法  Tumor necrosis factor activity inhibitor and method for producing the same
'：技術分野:' ':Technical field:'
： 発明は腫疡壊死因子の活性を抑制し う る新しい物質 及びその物質の単離、 精製に関する く：背景技術ク  The present invention relates to a novel substance that suppresses the activity of tumor necrosis factor, and the isolation and purification of the substance.
腫瘙壊死因子 （ TN F ) は C D— 1 Swiss マウスに Baci l lus C a 1 m e 11 e -Gti e r ί II 〈 B C G 〉 菌を投与し、 そ 2週間後に細菌内毒素 （エンド トキシン ） を投与した際 に血中に現われる生理活性物質と して発見され、 1975年 に Carswe！ 1らが報告 [ E. A. Carswe！！ ら、 Pro at！. Acad, Sci. , USA, 3ot)t U 975: ] した生理活性蛋白 質で、 そのアミノ酸配列は 1985年に Aggarwaiら [ E, B, Aggarwalら、 J. Biol. diem. 260, 2345 11985) J によ り明らかにされている .  Tumor necrosis factor (TNF) was administered to a CD-1 Swiss mouse with Bacillus Ca1me11e-Gtier II (BCG) bacteria, and two weeks later with bacterial endotoxin (endotoxin) It was discovered as a biologically active substance that appeared in the blood when it was killed, and Carswe! 1 reports [EA Carswe! ! La Pro at! Acad, Sci., USA, 3ot) t U 975:] A bioactive protein whose amino acid sequence was determined in 1985 by Aggarwai et al. [E, B, Aggarwal et al., J. Biol. Diem. 260, 2345 11985). It has been clarified by J.
また Pennies ら、 i r a iらおよび Wangらによって、 ヒ k τ\ι Fのアミノ酸配列および遺伝子配列が明らかにさ れ [ Panni ca ら、 atur 312, ?24 . S h i r a i atur 313, 803 ;1985; , Wangら、 Science 28 ^ 149  Pennies et al., Irai et al., And Wang et al. Have revealed the amino acid sequence and gene sequence of hen k τ \ ιF [Pannica et al., Atur 312,? 24. Shirai atur 313, 803; 1985;, Wang Et al., Science 28 ^ 149
当初その抗腫揚活性から、 癌治療薬と して 開発が進められていた TN Fは、 最近種々の生理活性が 明らかにされ、 生体内での諸機能が解明されつつある： 例えば、 細菌感染によるエンドトキシンショ ックの生 態内のメディエイターと しての活性 [B. Beutlerら、 Science, 229, 869 ( 1985) ] 、 血管内皮細胞への炎症反 応の惹起 [ J Gamble 'ら、 P r o c . X a† i . A cad. S c ί . , USA, _82^_ 8667 ( 1985) ] 、 発熟作用 [ A. D are! ioら、 J. Exp. Med. 163. 1443 ( 1986) ] 、 炎症の起因物質の ひとつであるイ ンターリウキン：！ ['P. P. Nawrothら、 J. Exp. Med. 1 _ 1363 ] などとの鬨係が明らかにされつ つある Initially, due to its anti-swelling activity, it was The development of TNF has recently revealed various physiological activities, and its functions in vivo are being elucidated: For example, as a mediator in the state of endotoxin shock caused by bacterial infection [B. Beutler et al., Science, 229, 869 (1985)], Induction of inflammatory response to vascular endothelial cells [J Gamble 'et al., Proc. Xa † i. A cad. Scί., USA, _82 ^ _ 8667 (1985)], ripening [A. D are! Io et al., J. Exp. Med. 163. 1443 (1986)], Interleukin, one of the substances causing inflammation :! ['PP Nawroth et al., J. Exp. Med. 1 _ 1363]
また、 ある種の病気の患者の休液中には丁 N Fが通常 よ り多く含まれており、 その TN Fの含有量とその患者 の病態には深い関係があることが明らかになりつつある . すなわち、 TN Fの過剰産生によ り、 病態の悪化が生じ ていると考えられる疾患が多く存在する： このような疾 患に対して、 抗 TN F作用を有する物質を投与すること によ り、 病態の改善が行なえうることは、 容易に推察が できる 事実、 エンドトキシンショ クに対しては、 抗 TN F作用を有する物質と して、 抗 F抗体の投与が、 動物を用いて行なわれ、 顕著な効果を示したことが報告 されている [B. BeuUerら、 Science, 299, 869 ;1985)] - しかし、 現在のところ、 抗 TN F抗体はヒ ト以外の動物 由釆の-も ;:のしか作成されておらず人体に対する投与には このような抗体では大きな問題があると考えられる さ らに、 TN Fが閔与していると考えられる急性、 慢性の 炎症反応においては、 免疫複合体の形成が、 少なく とも 病状を悪化させる要因のひとつであることが広く知られ ており、 このような疾患に対して、 抗丁 N F作用を有す る物質と して、 抗丁ヽ' F抗体を治療のために ¾いること は難しいと考えられる、. そこで、 ヒ トの体液 に天然に 存在し うる抗丁- F作用物質を闬いることができれば、 TN Fが鬨与している疾患に対して、 充分、 有効かつ安 全な治療薬と して使用し うるものであると推察されるよ うになつた In addition, it is becoming clear that patients with certain illnesses have more than normal NF in their fluids during resting, and that the TNF content is closely related to the patient's condition. In other words, there are many diseases that are considered to be exacerbated by the overproduction of TNF: by administering substances having anti-TNF action to such diseases. In fact, it can be easily inferred that the disease condition can be improved.In fact, administration of anti-F antibody to endotoxin shock is performed using animals as a substance having an anti-TNF action. However, it has been reported that the antibody had a remarkable effect [B. BeuUer et al., Science, 299, 869; 1985)]-However, at present, anti-TNF antibodies are also derived from animals other than humans. ;: Noshika has been prepared and is not suitable for administration to the human body Such antibodies are considered to be a major problem.In addition, in acute and chronic inflammatory reactions, which are thought to be caused by TNF, immune complex formation is at least a factor that worsens the condition. It is widely known that it is difficult to use anti-Din'F antibody as a substance having anti-NF activity for such diseases. Therefore, if a human body fluid could contain an anti-Fing agent that could be present naturally, it would be a sufficient, effective and safe treatment for TNF-fighting diseases. Came to be inferred to be usable as medicine
かかる T N F活性抑制物質 （ \ Fイ ン ヒビターと も いう ） に関しては、 Peetre ら [ Eur. J. Haemal c!, ϋ, 414 (1988), £2, -270 (1989)」 、 P. Sec gerら Regarding such a TNF activity inhibitor (also referred to as \ F inhibitor), Peetre et al. [Eur. J. Haemalc !, ϋ, 414 (1988), £ 2, -270 (1989)], P. Secger La
[ J. Exp. Med. 16：, 1 51 !1988; , J. Bio!. Chen.. 2D4. 1196b · 1989) ] 及び、 H . E n g e I m a n n ；. ii . [J. Exp. Med. 16 :, 151! 1988 ;, J. Bio !. Chen .. 2D4. 1196b · 1989)] and H. EngeI mann; .ii.
Cheiii. , 11974 ( 1989) ] によ り、 尿から ft製が行な われたことが報告されている これらの物質はその N末 端アミ ノ酸配列と、 その後、 アミ ノ酸配列が決定され 丁 N F リセァタ一 [； T. J. Scha!！ ら、 CeH 61, 3tl (1 90S. H. L o e t s c li e rら、 C e！！ ？ i .351 U 900 ク アミ ノ 酸配列とがー致し、 丁 ド リセプタ一の一部が切断さ Π て尿中に存在したものであろう ど考えられている Cheiii., 11974 (1989)] reported that ft-products were produced from urine.These amino acids were sequenced at the N-terminal amino acid sequence of these substances. Ding NF Resata I [; TJ Scha! Al, CeH 61, 3tl (1 90S . H. L oetsc li er et al, C e !!? I .351 U 9 0 0 click match amino acid sequence is over, the cutting of a part of Ding de receptors oneて い る It is thought that it was present in urine
、 H. EagelmatiQらは特定の ¾配列を有ォる-分 子量約 3万の 2つの物質を尿より単離している （H. Enge lmann et ai, J. Biol. Chem. 265, 1531 ( 1990) ! また、 C. A. Smithらも、 かかる物質についてそのアミ ノ 酸配列を開示している （ C. A. Smith et ai, Science 248, 1019, 1990 ) 。 H. EagelmatiQ et al. Have a specific sequence Two substances with a molecular weight of about 30,000 have been isolated from urine (H. Engelmann et ai, J. Biol. Chem. 265, 1531 (1990)! Also, CA Smith et al. Discloses the acid sequence (CA Smith et ai, Science 248, 1019, 1990).
そこで本発明者は、 新規な T N F活性抑制物質を探索 すべく、 研究を進めた結果、 本発明に到達した  Thus, the present inventors have conducted research to search for a novel TNF activity inhibitor, and have reached the present invention.
〈発明の'目的〉 <Purpose of invention>
本発明は新規な T N F活性抑制物質、 それを含有する 組成物及び該物質の製造方法を提供することを目的とす る :， く発明の開示〉  SUMMARY OF THE INVENTION An object of the present invention is to provide a novel TNF activity inhibitor, a composition containing the same, and a method for producing the same.
本発明は下記発明を包舍ォる . すな ち本発明は、 H) 腫瘍壊死因子の L 929 細胞への殺細胞効果を抑制し •2) S D S— P A G Eにおける分子量が還元状態で 3 4 The present invention encompasses the following inventions. That is, the present invention relates to H) suppressing the cell killing effect of tumor necrosis factor on L929 cells. • 2) SDS-PAGE has a reduced molecular weight of 34
K： 1 K D aであり、 K: 1 KDa
';3'ί Ν末端から 1 〜U番目までのアミノ酸が次の配列 Val-Ai a-Phe-Thr-Pro-T r-Ai a-Pro-Al a-Pro-T r で表わされる物質である  '; 3'ί The amino acids from the 1st to the Uth from the Ν terminus are substances represented by the following sequence Val-Aia-Phe-Thr-Pro-Tr-Aia-Pro-Ala-Pro-Tr is there
本発明の物質は、 N末端から 11番目のアミ ノ酸が Va!-AI a-Phe-T r-Pro-Tyr-Al a-Pro-Al a-?ro-Thr である ことを特徴とする 発明の物質は、 C. Peetre ら [Eur. J. Haematol. 11, 414 (1988门 記載の物質とは、 その分子量において 彼等の報告しているものが約 5 0 K D a、 本発明の物質 が 34 K二 1 K D aという点で相違寸る . The substance of the present invention is characterized in that the 11th amino acid from the N-terminus is Va! -AIa-Phe-Tr-Pro-Tyr-Ala-Pro-Ala-? Ro-Thr. The substances of the present invention are described in C. Peetre et al. [Eur. J. Haematol. 11, 414 (1988 门). However, the difference is that it is 34 K2 1 KDa.
また、 P. Seck gerら [ J. Exp. Med. 167. 1551 (1988) , J. B i o ! . C em. 264, 1196t ^989 に記載さ れている物質とは、 S D S— P A G £における分子量は ほぼ等しいものである： しかし、 最終精製に^いている 逆相カラムからの溶出ァセトニト リル濃度が、 カラムが 異なるという条件を考慮しても、 大きく異なるという点 で相違する。  Also, the substance described in P. Seckger et al. [J. Exp. Med. 167. 1551 (1988), J. Bio !. Cem. 264, 1196t ^ 989] The molecular weights are about the same: however, the difference is that the concentration of acetonitrile eluted from the reversed-phase column in the final purification is very different, taking into account the different columns.
ま 、 H. Enge 1 maunら [ J. Biol. C em. _ 264, 11974 (1989； ] に記載されている物質とは、 その分子量及び X 末端からのアミノ酸配列に全く相同性がないという点で 異なる：  Also, the substance described in H. Enge 1 maun et al. [J. Biol. Cem. _264, 11974 (1989;)] has no homology in its molecular weight and amino acid sequence from the X-terminal. In different:
た、 H. Engeimannら し7. Bioに Chem. 265, 153L 1990 ] に記載されている物質 T B P Iとは、 -\ ^端から のアミノ酸配列が全く異なる。 In addition, the amino acid sequence from the-\ ^ end is completely different from the substance TBPI described in H. Engeimann et al., 7. Bio, Chem. 265, 153L 1990].
また、 T B P ϋとは、 彼らが報告しているよ うな 末 绢アミノ酸の多様性が認められず、 かつ、 分子量も若ェ 異なる  In addition, unlike TBP, they do not show any amino acid diversity as reported, and their molecular weight differs slightly.
発明において丁 X Fとは、 Aggarwaiら . ίι。：· Chem. 2345 (1985) ] や Shiraiら [ ature 313. 805 :1985) ] によ り報告されている丁 F— «であり、 天然 TN F及びリ コンビ十ン ト T N Fを含む In the invention, Ding XF is described by Aggarwai et al. Chem. 2345 (1985)] and Shirai et al. [Ature 313. 805: 1985)]. Including natural TNF and recombinant TNF
本発明において、 L 929 細胞とは、 マウス  In the present invention, L 929 cells are mice
ί ibrcsarcoina由来の樹立細胞株であり、 ATC C株番号 は C C L一 1 、 名称 L 929 ( N C T C clone 929 ) と して登録されている細胞である ib Established cell line derived from ibrcsarcoina, ATC C strain number is CCL-11, registered under the name L 929 (NCTC clone 929)
木発明の物質の分子量は S D S— P A G Eにお る 分子量が還元状態で 3' 4 K二 〗 K D aの物質である . こ こで S D S— P A G Eにおける還元状態とは、 S D Sホ リァクリルァミ ドゲルにて電気泳動を行なう以前に、 サ ンアルを適当な還元剤、 例えば、 2 -メルカプトエタ ノ —ル、 ジチオスレィ トール等の存在下にて、 100 °C、 数 分間の処理を行なって、 タンパク中のジスルフ ィ ド結 を開裂させた後、 電気泳動を行なう ことをいう - 本発明は、 該腫瘍壊死因子活性抑制物質を含有する組 成物を包含する  The molecular weight of the substance of the present invention is a substance having a reduced molecular weight in SDS-PAGE of 3'4K / 200 KDa. Here, the reduced state in SDS-PAGE refers to the electrical properties of SDS polyacrylamide gel. Prior to electrophoresis, the sanal is treated in the presence of a suitable reducing agent, for example, 2-mercaptoethanol, dithiothreitol, etc., at 100 ° C for several minutes to obtain the disulfide in the protein. Refers to performing electrophoresis after cleaving the bond-the present invention encompasses a composition containing the tumor necrosis factor activity inhibitor.
本発明の物質は尿を精製することによ り得ることがで きる。 特に、 膜性増殖性糸球体腎炎患者の尿を精製する ことにより得ることができる，  The substance of the present invention can be obtained by purifying urine. In particular, it can be obtained by purifying the urine of patients with membranous proliferative glomerulonephritis,
膜性増殖性糸球体腎炎は、 臨床的には、 腎糸球体への 免疫複合体の沈着が認められ、 さらに血中補体価の低下、 糸球体基底膜の肥厚、 メサンギゥム細胞の増殖等が認め られる腎疾患である . 本症発症原図はいまだ不明である が、 最近、 メサンギゥム細胞増殖型腎炎にはサイ カイ ン類、 特にィ ンターロイキン 6の関与が示唆されている [実験医学 2 , 11 (1989) ] 。 イ ンターロイキン 6の産 生は、 T N Fやイ ンターロイキン 1 によって誘導される ことが、 多くの細胞で報告されており [実験医学.2 , 2： ( 1989) , 現代免疫学 93 ( 1988) ] 本疾患においても ドの産生亢進がおこつていることが推測される： Membranous proliferative glomerulonephritis clinically involves the deposition of immune complexes in the renal glomeruli, lowering blood complement value, thickening of the glomerular basement membrane, and proliferation of mesangial cells. The origin of this disease is still unknown, but it has recently been suggested that sycaines, especially interleukin 6, are involved in mesangial cell proliferative nephritis. [Experimental medicine 2, 11 (1989)]. It has been reported in many cells that the production of interleukin 6 is induced by TNF and interleukin 1 [Experimental Medicine.2, 2: (1989), Modern Immunology 93 (1988)]. It is presumed that hyperactivity of dode also occurs in this disease:
水発明者は、 種々の腎疾患患者の血中丁 \ F含量を . 定したところ、 膜性増殖性糸球体腎炎患者血中に高滤¾ の T N Fが含有されていることを明らかと した さつに 本疾患患者尿を用いて、 丁 N Fの活性抑制能 .を検討した ところ、 高い T N F活性抑制能があること 見出し のである、.  The inventor of the present invention determined the F content in the blood of patients with various renal diseases and found that high blood levels of TNF were contained in the blood of patients with membranous proliferative glomerulonephritis. Finally, using the urine of the patient with this disease, we examined the ability to inhibit the activity of DNF and found that it had a high ability to inhibit TNF activity.
精製はイオン交換、 逆相カラム、 丁 X F固定化 ラ 等の精製操作を組み合わせることによ り行なう ことがで きる。  Purification can be performed by combining purification operations such as ion exchange, reversed phase column, and XF immobilization.
すなわち本発明は、 腫瘍壌死因子活性抑制物質 製 方法であって、  That is, the present invention relates to a method for producing a tumor necrosis factor activity inhibitor,
ia) 尿をィオン交換カラム及び 又は逆相カラムへの吸 着及び溶出によって^ ¾し、 ia) urine is absorbed and eluted into the ion exchange column and / or reverse phase column,
(bi 腫瘙壊死因子固定化カラムへの吸着及び溶出によつ て腫疡壊死因子に結合する画分を分取し、 (The fraction that binds to the tumor necrosis factor by adsorption and elution to the bitumor
ic; 腫瘍壊死因子の L 929 細胞への殺細胞効果を抑制寸 る画分を選択する ic; Select fractions that suppress tumor necrosis factor's cytocidal effect on L929 cells
ことからなる製造方法を包含する Comprising a manufacturing method comprising:
以下、 本発明を詳述する： 検体尿の調製 Hereinafter, the present invention will be described in detail: Preparation of sample urine
尿は、 初尿、 蓄尿、 スポッ ト尿のいずれを用いてもか まわない 好ましくは、 後に、 バイオア、、/セィを行なう ために、 できるだけ無菌もしくは無菌に近い状態で採取 されたものがよい。 採取した尿は、 すみやかに冷凍保存 し、 使用直前に融解して'用いることが望ま しい カビゃ バクテリアの繁殖を抑えるために、 N a. N 3 や抗生物質、 また、 ァロテア一ゼによる分解を防ぐためにプロテア一 ゼィ ンヒビター等を、 尿採取直後に添加してもかまわな いが、 尿採取後、 速やかに冷凍保存に移行すればこれら の添加物は不要である。 それらの添加物を添加した場合 には、 適当な操作、 例えば、 透析、 β¾外沪過、 ゲル ^過 等の操作によ り、 バイオア、ソセィ を行なう前に充分、 添 加物を除去する必要がある The urine may be any of primary urine, urine collection, and spot urine. Preferably, urine is collected under aseptic or aseptic conditions as much as possible in order to carry out a bioa / se / se later. Collected urine, quickly frozen and stored, correct desired to use melted immediately before use 'in order to suppress the growth of Kabiya bacteria, N a. N 3 and antibiotics, also the degradation by Arotea Ichize In order to prevent this, a protease inhibitor may be added immediately after urine collection, but these additives are not required if urine is immediately transferred to frozen storage. When these additives are added, it is necessary to remove the additives sufficiently by performing appropriate operations, for example, dialysis, β extracellular filtration, gel filtration, etc. There is
検体尿からの精製 Purification from sample urine
精製は、 通常のタンパク質の精製法に って行なえば よい。 尿原液では、 通常、 低い活性しか認められないの で、 まず濃縮を行なうことが好ましい。 濃縮法と しては、 限外 過、 凍結乾燥、 塩析等の通常の生化学実験法に基 づいて行なえばよいが、 限外沪過、 塩析等を行なう場合 には、 目的 する物質の分子量や、 沈殿を開始する塩澹 度等をあらかじめ調べておく必要がある：  Purification may be performed by an ordinary protein purification method. Since a low activity is usually observed in the urine stock solution, it is preferable to first concentrate the urine solution. The enrichment method may be based on ordinary biochemical experiment methods such as ultrafiltration, lyophilization, salting out, etc., but in the case of ultrafiltration, salting out, etc., the target substance It is necessary to check in advance the molecular weight and the degree of saltiness that initiates precipitation:
瀵縮後は、 適当な精製操作、 例えばイオン交換、 ゲル' 沪過、 ァフ ィ二ティークロマトゲラフ ィー、 等電点電 泳動、 疎水クロマトグラフィー、 逆相クロマトグラフ ィ 一等を組み合わせて、 精製を進めることができる After reduction, appropriate purification procedures such as ion exchange, gel filtration, affinity chromatography, isoelectric focusing Purification can be performed by a combination of electrophoresis, hydrophobic chromatography, reverse phase chromatography, etc.
本発明の物質は、 上記濃縮操作の後、 TN Fが力、、/フ' リングしたカラムに吸着させることによ り分離精製を行 ない、 その後、 逆相カラムによる溶出によ り特定のァセ ニ k リル濃度の画分中に含まれる物質である . セ ）、 ニト リル濃度は使用するカラム、 ァセ トニト リル澹度勾 配によって異なるが、 Protein C 4 , V Y D Aし'社、 0.46：、 25cmを使用し、 150 分間の 1δ〜 37%のァセトニト リル直線勾配を行なった場合はァセトニト リル溏度 2？〜 28%のものである， The substance of the present invention is subjected to separation and purification by adsorbing to a column to which TNF has been applied after the above-mentioned concentration operation, and then specific eluting by elution with a reversed-phase column. . cell is a substance contained in the fraction in two k Lil concentration cell), column nits Lil concentration used varies depending § cell Tonito Lil澹度gradient, Protein C 4, VYDA and 'Company, 0.46: , 25 cm, and a 150-minute linear gradient of 1δ to 37% acetonitrile, 2? ~ 28%,
Ί'Ν F活性抑制能の測定 Measurement of Ί'Ν F activity inhibitory ability
丁 λτ F活性の抑制能の測定法と しては、 既に知られて いる TN F活性の測定を行なう系に、 被験サンプルを添 加すればよい.： ォなわち、 丁 ヽ - Fのァ、リセィ法と して知 ちれている vitroの種々の腫塲細胞に対ォる殺細胞効 果、 細胞増殖抑制効果、 脂肪細胞の脂肪酸代謝 _抑制効果 正常線維芽細胞の増殖促進効果や 1 L - 6産生誘導効果 好中球の内皮細胞への付着促進効果や、 スーパーォキサ ィ ド分泌促進効果、 血管内皮細胞の凝固活性亢進効果、 骨細胞への破骨効果、 種々の細胞での I L一 ] ゃフ'口ス タゲランデイ ン類の誘導効果を測定する系が利用できる 特に L 929 細胞への殺細胞効果で測定寸ることが好適で ある また、 in vivo においてもある種の癌細胞に対寸 る出血壞死効果、 エンドトキシンショ 、ソクの誘導効果、 発熱効果を測定する系を利用することができる しかし、 in vivo での活性や、 多くの vitroでの活性は、 TN P以外の物質でも誘導しうる反応が多くあるため、 でき るだけ単純で、 かつ、 TN F以外の物質の影響を受けな い系、 例えは'、 in vitroの癌細胞に対する殺細胞活性を 測定する系を用いることが望ましい As a method for measuring the ability to inhibit λ τ F activity, a test sample may be added to a known system for measuring TNF activity. Cell killing effect on various tumor cells in vitro, known as the Lysie method, cell growth inhibitory effect, fatty acid metabolism of fat cells _ inhibitory effect Growth promoting effect of normal fibroblasts and 1 L-6 production inducing effect Neutrophil adhesion promoting effect on endothelial cells, superoxide secretion promoting effect, vascular endothelial cell coagulation activity enhancing effect, osteoclasting effect on bone cells, IL in various cells [1] A system for measuring the inducing effect of ゃ 'mouth mouth staglandins is available. It is particularly preferable to measure the cell killing effect on L929 cells. Size However, in vivo and many in vitro activities can be induced by substances other than TNP. Because there are many possible reactions, it is desirable to use a system that is as simple as possible and that is not affected by substances other than TNF, e.g., a system that measures cell killing activity against cancer cells in vitro
本発明の腫瘍壊死因子活性抑制物質は、 各種の  The tumor necrosis factor activity inhibitor of the present invention includes various types of
鬨連疾患の治療剤と して使用される そのための医薬組 成物と しては、 本発明の腫瘍壊死因子活性抑制物質を有 効活性成分と して含み、 その他製薬学的に許容しうる担 体を含むもので'あれほ'よい The pharmaceutical composition used as a therapeutic agent for the disease of the sword is a pharmaceutical composition containing the substance for suppressing tumor necrosis factor activity of the present invention as an active ingredient, and is other pharmaceutically acceptable. It includes a carrier and is good
医薬組成物を調製する場合、 有効活性成分と して使用 する腫 «i死因子活性抑制物質の抗原性の低減あるいは 生理活性の増強などを目的と して、 例えばボリエチレン グリ コール 〈 P E G ) 、 デキストラン又はポリー D L— ァラニン：などの公知のボリマーによって修飾することも できる。  When a pharmaceutical composition is prepared, tumors to be used as active ingredients may be used to reduce the antigenicity of the inhibitor of death factor activity or to enhance the physiological activity. For example, polyethylene glycol <PEG), dextran Alternatively, it can be modified by a known polymer such as poly-DL-alanine.
医薬組成物の形態と しては、 注射用組成物、 坐剤その 他が挙げられるが、 注射用組成物と しては特に静注用組 成物と して使用するのが好ましい .  Examples of the form of the pharmaceutical composition include an injectable composition, a suppository and the like, and it is particularly preferable to use the injectable composition as an intravenous composition.
注射用組成物の場合は、 本発明の腫瘍壊死因子活性抑 制物質の薬学的有効量及び製薬学的に許容しう る担体の 混合物であり 、 その中にはアミ ノ酸、 糖類、 セルロース 誘導体、 ポリビニルピロリ ドン類、 無機化合物類などの 一般的に注射用組成物に添加される賦活剤を用いること もできる： それらの具体例をあげると、 アミ ノ酸類と し ては、 グリシン、 アルギニン、 ァラニン及びそれらの薬 学的に許容できる塩等があげられる： 糖類と しては、 マ ン ニ ール、 イ ノシ 、、一ル 、 キシリ k一ノレ、 乳輕 ， 々'- 7レ コース等があげられる： セルロース誘導体と してはカル ボキシメチ/レセ /レロ一ス十ト リ ウム、 メチル'セル'口一ス 等があげられる： ポリビニルピロリ ドン類と しては分子 量 1 0 , 000〜1 , 000 , G O O のボリビュルピロリ ドンがあげら れる： In the case of an injectable composition, it is a mixture of a pharmaceutically effective amount of the tumor necrosis factor activity-inhibiting substance of the present invention and a pharmaceutically acceptable carrier, including amino acids, saccharides, and cellulose. It is also possible to use activators generally added to injectable compositions, such as derivatives, polyvinylpyrrolidones, and inorganic compounds. Specific examples thereof include glycine and arginine as amino acids. , Alanine and their pharmaceutically acceptable salts, etc. Examples of the sugars include mannitol, wild boar, il, xyli, oleoresin, milk, and -7'-coose. Cellulose derivatives include carboxymethyi / rese / relo-t-tris, methyl 'cell'-mouth, etc .: Polyvinylpyrrolidones have a molecular weight of 10 000 ~ 1,000, GOO Bolibulpyrrolidone:
有機酸類と しては、 ァスコルビン酸、 クェン酸類等 びそれらの塩があげられる： 無機化合枸類と してはリ ン 酸水素十ト リ ゥム、 炭酸水素十ト リ ゥム、 酢酸十ト リ ゥ ムなどがある  Examples of the organic acids include ascorbic acid, citric acid, and the salts thereof. Examples of the inorganic compound calcium include 10% of hydrogen phosphate, 10% of hydrogen carbonate, and 10% of acetate. There is a room
これら賦形剤を溶解させる液と して 、 注射^蒸留 、 注射用生理食塩水又は注射用リ ンゲル液がある  Solutions for dissolving these excipients include injection ^ distillation, saline for injection or Ringer's solution for injection.
その他注射液中には、 安定剤、 界面活性剤、 等張化剤、 無痛化剤、 防腐剤、 緩衝剤などが必要に応じて^ され る . これらの具体例を示すと、 安定剤と してはピロ亜硫 酸十 ^ リウム、 ϋ ーァスコルビン酸等の抗酸化剤 ： D T A、 チォグリコール剤等のキレート剤等があげられる 界面活性剤と しては、 ホ リ ルベー ト、 ボリォキシェチ レ'ン誘導体等の非ィオン性界面活性剤等があ · ' しる 等張化剤としては塩化ナトリウム等が挙げられる。 Other injections may contain stabilizers, surfactants, tonicity agents, soothing agents, preservatives, buffers, etc. as required. Antioxidants such as deuterium pyrosulfite and perscorbic acid: chelating agents such as DTA and thioglycol agents, etc. Examples of surfactants include phosphorylates and boroxixylene derivatives Non-ionic surfactants such as Examples of the tonicity agent include sodium chloride and the like.
無痛化剤としてはべンジルアルコール、 キシロカイン、 プロカイン等が挙げられる。  Soothing agents include benzyl alcohol, xylocaine, procaine and the like.
防腐剤と してはパラベン類、 クロロブタノ一ル、 塩化 ベンザノレコニゥム、 チメロサール （Thimerosal ) 等が挙 げられる。  Preservatives include parabens, chlorobutanol, benzanolone chloride, and thimerosal.
緩衝剤としては、 クェン酸、 酢酸、 リン酸等のナトリ ゥム塩等が挙げられる。  Examples of the buffer include sodium salts such as citric acid, acetic acid, and phosphoric acid.
〈発明の効果〉 <The invention's effect>
本発明によれば腫瘍壊死因子 （ TN F〉 の 細胞 への殺細胞効果を抑制する新規な物質を提供し、 TN F が閬与していると考えられる疾患、 例えば、 ェン ドトキ シンショ ックゃ火傷時のショ 、ソク、 急性肝不全、 腎不全 や多臓器不全、 リウマチ、 S L E、 ベーチ 、 Vト病等の 多くの自己免疫疾患、 臓器移植時の拒絶反応、 川崎病、 D I C等の凝固異常の治療、 診断等に利用できる。 また 上記物質の効率的な製造方法を提供することが可能とな つた - く実施例〉  According to the present invention, a novel substance that suppresses the cell killing effect of tumor necrosis factor (TNF) on cells is provided, and a disease considered to be exerted by TNF, for example, endotoxin shockシ ョ Many autoimmune diseases such as acute burn, acute liver failure, renal failure and multiple organ failure, rheumatism, SLE, Behcet, V Grade disease at the time of burn, rejection at organ transplantation, coagulation of Kawasaki disease, DIC, etc. It can be used for treating abnormalities, diagnosing abnormalities, etc. In addition, it has become possible to provide an efficient method for producing the above substances.
以下、 実施例を掲げて本発明について詳細に説明する が、 本発明は以下の実施例に限定されるものではない 実施例 1 Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited to the following Examples. Example 1
尿中丁 X F活性抑制物質の精製 Purification of urinary XF inhibitor
(1) 限外泸過による濃縮  (1) Concentration by ultrafiltration
膜性増殖性糸球体賢炎患者尿 （サンプル 1 ) を ぺリコ ン (ミ リポア社） による限外沪過で分子量〗 万以 上の画分を り mlまで濃縮、 10m Tr!？ pH8. 0 に ' .' ， ' 一交換した （サンプル 2 ) Urine from a patient with membranous proliferative glomerulitis (sample 1) was concentrated by ultra-filtration with a silicone (Millipore) to a fraction with a molecular weight of 10,000 or more to a volume of 10 ml Tr !? Changed '.' and 'to pH 8.0 (Sample 2)
2? D A E - Sep arose カラムによる精製  2? D A E-Separose column purification
濃縮尿サンプルを lOraM Tris pH8.0 にて^1衡化した 4 S の D E A E— Sepiiarose カラムにかけた： 流速 40ΰ ir.： zjirにて、 10m Tris pH8.0 で洗った後、 10mM Tri?- lOOm NaC! pH Gにて、 溶出を行ない、 400 m!ザつつラ クショ ンを集めた The concentrated urine sample was applied to a 4 S DEAE-Sepiiarose column equilibrated with ^ OraM Tris pH 8.0 ^ 1 : Flow rate 40ΰ ir .: After washing with 10m Tris pH 8.0 in zjir, 10mM Tri? -LOOm Elution was carried out with NaC! PH G, and the mixture was collected at 400 m!
各フラクショ ンの 1 m 1をセント リ コン ί 0 (アミ コン社： > による限外沪過で、 P B Sにバ - アー交換した後、 各 っラクシヨ ンの 0 D 2s(, と、 L 929 細胞に対する丁ヽ F の殺細胞効果の抑制能を測定し、 活性画分をアールし、 ペリコンによ り分子量 1万以上の画分を瀵縮し、 タンパ ク^度 Π.8mg " mlの粗精製品 103 m!を得た サン ァ /レ ' 3 ,' サンプル 3の L 929 細胞に対する丁 N Fの殺細胞効果 の抑制能を測定した 〈表】 に示す :' 1 ml of each fraction was centrifuged to PBS with an ultrafiltration by Centricon ί0 (Amicon:>), and then exchanged with PBS for 0D2s ( , and L929 cells). Was measured for its ability to suppress the cell killing effect of D-F on the active fraction, and the active fraction was rounded off, the fraction with a molecular weight of 10,000 or more was reduced with Pellicon, and the protein was purified at a concentration of 8.8 mg "ml. Sampling / product '103,' which obtained 103 m! Of the product, was measured for its ability to suppress the cell-killing effect of Ding NF on L929 cells of sample 3, as shown in Table.
;3 逆相カラムによる精製 ; 3 Purification by reversed-phase column
次に I) E A E粗精製品 （サンブル 3 、' 1 Om！を、 ：.： °- 丁 F A ( k ij フルォロ酢酸 ) にて 衡化した逆^カラム ( Protein C 4 、 V Y D A C社、 2 ：く 25 cm)' にかけ、 40 分間のァセトニ卜 リル 16%から、 40 の直線勾配で、 流 速 5 mlノ ffiin にて溶出を行ない、 5 m! . フラクショ ンに て、 分取を行なった。 この操作を 9回繰り返した後、 各 フラクションを凍結乾燥した， 各フラクショ ンを 1 Di 1ク'、 P B Sに融解した後、 同じ溶出時間の ラクショ ン をフ' ールし、 0 D 28。 と L 929 細胞に対する丁 N Fの殺細胞 効果の抑制能を測定した。 T N F活性を抑制寸る话性は 主に 2つのピーク （ピ一ク ェ ， ピーク E ) に認められたNext, I) Reverse ^ column equilibrated with EAE crude product (Samble 3, '1 Om!) With:.: ° -D FA (kij fluoroacetic acid) (Protein C4, VYDAC, 2: 25 cm) '' and eluted from 16% of acetonitrile for 40 minutes with a linear gradient of 40 at a flow rate of 5 ml of ffiin. Then, fractionation was performed. After this procedure was repeated 9 times, each fraction was freeze-dried. Each fraction was thawed in 1 Di 1 'and PBS, and fractions with the same elution time were filtered out, and 0 D 28 was obtained . And the ability to inhibit the cell killing effect of Ding NF on L929 cells were measured. The activity of suppressing TNF activity was mainly observed in two peaks (peak and peak E).
(図 1 に示す》 。 これらの L 929 細胞に対する T X Fの 殺細胞効果を表 1 に示す。 (Shown in FIG. 1) Table 1 shows the cell killing effect of TXF on these L929 cells.
ピーク ェの画分は、 この後、 T N Fァフ ィ二ティカラ ム、 逆相カラムによる精製をさらに行ない、 X末端アミ ノ酸配列を決定したところ、 Asp-Ser-Va!-X-Pro-G! n- Gl -Lys-T r-I I e-Hi s-Pro-Gi Q-X-Asn-Se r-I ί e ( Xは 477 A型プロティ ンシークェンサ一でビークが認めら なかった残基〉 という配列が得られ、 公知の T N Fイ ン ヒビタ一、 あるいは T N Fリセプ夕ー断片であると推測 された， [ I." 01 sson ら、 Eur. J. Haemat.。 42, 270 ( 1989) , H. Loetscherら、 Ce ! j_^_ 351 (1990 ) , T. J, Schai lら、 Ce l lJJ^ 361 (1990； 」  The peak fraction was further purified by a TNF affinity column and a reversed-phase column, and the X-terminal amino acid sequence was determined.Asp-Ser-Va! -X-Pro-G ! n-Gl-Lys-T rI I e-His-Pro-Gi QX-Asn-Se rI ί e (X is the residue that was not recognized in the 477A type protein sequencer without beak) It was presumed to be a known TNF inhibitor or a TNF receptor fragment, [I. "01 sson et al., Eur. J. Haemat .. 42, 270 (1989); H. Loetscher et al., Ce. j _ ^ _ 351 (1990), T.J, Schail, et al., Cell JJ ^ 361 (1990;)
T λ' Fァフィ 二ティーカラムによる精製  Purification using T λ 'F affinity column
次に、 18ΙΪΠ得られた逆相カラム粗精製品 （ピーク Π ) のうち 7 m!を、 ] mgの T N Fがカップリ ン ゲした 4 η.ι: の A i-Ge j 10 { BIO-RAD 社） に通した . ここで用いた '】、 ト、は比活性3.3 107 U . のリコンビ十ン ト Τ—ヽ Fであり、 そのアミノ酸配列は、 Shiraiらの文献 Next, 7 μm of the crude product (peak Π) obtained from 18ΙΪΠ of the reversed-phase column was coupled with 4 mg of TNF. Through the the A i-Ge j 10 {BIO -RAD Co.). '] Used herein, bets, is Rikonbi Jun preparative Τ-ヽF of specific activity 3.3 10 7 U., The amino acid sequence , Shirai et al.
[ ature 313, 803 ( 85) ] に記載されているものと同 一である P B S - 0.02?o K a N 3 を流して、 充分、 非 特異的吸着成分を除去した後、 25 ク ン酸 100 N a C ] 一 0.02% N a N 3 pH2. 5 によ り溶出を行なった . [Ature 313, 803 (85) ] is the same as those described in PBS -? 0.02 o K by flowing a N 3, sufficient, after removing the non-specific adsorption component 25 click phosphate 100 N a C] was carried out by Ri eluted with an 0.02% N a N 3 pH2. 5.
(5； 逆相カラムによる精製  (5; Purification by reverse phase column)
溶出直後に、 溶出液 1.2 mlを 0. c'0丁 F A _ 18 '。ァセ トニト リルで平衡化した逆相カラム （ Protein C 4 、 \ Y D A C社、 0.46 25cm) にかけ、 150 分間の 18。。から 37 のァセトニト リル直線勾配によ り、 溶出を行なった ァセ 卜二 k リル漶度 27〜28%において、 215a の吸収で メイ ンピークが 1つ認められ、 その画分を分取した こ れを最終精製品 Aとよぶ この最終精製品 Aの ¾ 胞に対する Fの殺細胞効果の抑制能を表 1 に示ォ 丁 N F活性抑制物質のァ、 Vセィ Immediately after elution, the eluate 1.2 ml 0. c '0 chome FA _ 18'. Apply to a reversed-phase column (Protein C4, \ YDAC, 0.46 25 cm) equilibrated with acetonitrile for 150 minutes. . From 37 to 28% of the eluted acetonitrile at a concentration of 27 to 28%, one main peak was observed at 215a, and the fraction was collected. Is referred to as final purified product A. The ability of this final purified product A to suppress the cell killing effect of F on cells is shown in Table 1.
丁 N F活性抑制能のア セィは、 L 929 細胞を用いて、 丁ヽ— F活性を測定する Ruii & Giiiordの系 [ J. ί mmiuic ι , 125, 1671 ( 1980； ] に、 丁 N Fと同時にサンブルを添加 ォることによ り行なった： 寸なわち、 9t力エルプレート に4 ノ 10; z mlの L 929 細胞を、 2 u S π:! クチノマイシン Dと混合し、 100 ゥエルで播種、 5 % Cし) , , 37て:にて 2時間培養ォる この細胞に限外 沪過、 透析、 もしくは凍結乾燥、 再融解等によ り、 バツ ラァーを P B Sに置換したサンアル 50 を加え、 直後 に 2 ng /'mlの TN F溶液 (比活性 3.3 1 G 7 U mg) 0 ^ をさらに添加し、 5%C 02 , 37°Cにて 18時間培 養を行なった。 培養後、 ク リスタルバイオレ、' / トによる 生細胞の染色を行ない、 染色された細胞を 0.5 ¾ S D S にて溶解し、 595ιιηι の吸光度を測定した < Assays for the ability to inhibit Ding NF activity were performed simultaneously with Ding NF in the Ruii & Giiiord system [J. ίmmiuic ι, 125, 1671 (1980;)], which measures D-F activity using L929 cells. This was done by adding a sample: 4 to 10; z ml of L929 cells were mixed with 2 uS π :! cutinomycin D in a 9 t force plate and seeded at 100 μl. Incubate for 2 hours at 5% C),, 3 7 By filtration, dialysis, or lyophilization, re-thawing, etc., add Sanal 50 in which the buffer has been replaced with PBS, and immediately thereafter, add a 2 ng / 'ml TNF solution (specific activity 3.3 1 G 7 U mg) 0 ^ Was further added, and the cells were cultured at 5% CO 2 and 37 ° C for 18 hours. After culturing, live cells were stained with Crystal Violet, and the stained cells were lysed with 0.5 ¾ SDS and the absorbance at 595ιιηι was measured <
得られた 595am の吸光度をもとに.、 TN F— Inhibition rate を算出した。 T N F _ I nh i b i t i on rate の算 出式は、 以下に示すとおりである。  Based on the obtained absorbance of 595am, the TNF-inhibition rate was calculated. The formula for calculating TNF_Inhibtition rate is as follows.
T N F - Inhibition rate T N F-Inhibition rate
[ 0D595 ] TNF+サンプル一 [ 0D595 ] TNF [0D 595 ] TNF + Sample [0D 595 ] TNF
10CM¾ 10CM¾
[OD ] T F なし一 [OD: 1 TNF 各サンアルは 1 Z2稀釈系列によ りァ '' /セィ を行ない それぞれ T N F— In Mtion rate を算出した。 30%の T N F - Iahibitioa rate を与えるサンアルの最大稀釈 の逆数を 1ユニッ ト （ U ) と規定し、 尿中 TN F活性抑 制物質の各精製ステップにおける T N F抑制活性を示し たものが表 1である。 1 [OD] TF None [OD: 1 TNF Each sample was subjected to a '' / see using a 1Z2 dilution series to calculate the TNF-In Mtion rate. The reciprocal of the maximum dilution of Sanal that gives a TNF-Iahibitioa rate of 30% is defined as 1 unit (U), and the TNF inhibitory activity in each purification step of urinary TNF inhibitor is shown in Table 1. is there. 1
精製画分 ； 活 性 総液量 I総活性  Purified fraction; Total activity I Total activity
！ (ϋ/m 1 ) ■ ( ml ) ： ( U 尿原液 ) 3. 1 、'. 10fc 分子量 1万以上 4120 250 ί 1.0 > 10 濃縮画分 （サンブル 2 ) ! (ϋ / m 1) ■ (ml): (U urine stock solution) 3.1, '. 10 fc molecular weight 10,000 or more 4120 250 ί 1.0> 10 Concentrated fraction (Samble 2)
DEAE粗精製活性画分 90 10 δ  DEAE crude purification active fraction 90 10 δ
8つ Eight
Figure imgf000019_0001
Figure imgf000019_0001
ビ-ク ィ::ティカラム- ノ  Beak :: Tee column
C4逆相カラム ァセ卜二トリ Λ υ Λ 1 υ Λ  C4 reversed-phase column acetate 二 υ Λ 1 υ Λ
27-28%溶出画分  27-28% eluted fraction
(最終精製品 A ) 尿中 T N F活性抑制物質の分子量  (Final purified product A) Molecular weight of urinary TNF inhibitor
最終精製品 A 35G χχϋ を凍結乾燥し、 230 J.L St の Sに再溶解した。 このサンアル 9 ·!！ に 10 u の 2 X S D S— P A G Eサンプルバ ファ一 （ 1 mM Tr i s — H C ] H6.8, 10 % Sucrose. 10% S D S , 0.25mg ' mlブ ロモフヱノールブル一：） 、 1 の 2—メルカプトエタ ノールを加え、 100 °C、 5分間、 加熱した後、 10〜20 の S D Sポリァク リルアミ ド勾配ゲル （第一化字、 S D S - P A G P L AT E 10 ' 20〉 に全量をのせ、 — )ir:A定 電流にて、 120 分間、 泳動を行なった 分子量マーカー と しては B I 〇一 R A D社 Molecular Weight Standards - Lowを用いた。 電気泳動終了後、 銀染色を行なった （図 2にそのスケッチを示す〉 。 サンプルをのせたレーンに は、 分子量 34 K ± 1 K D aのシングルバンドのみが認 められ、 尿中の TN F活性抑制物質は、 還元状態での S ]) S - P AG E上での分子量は、 34 K二 1 K L) aであ ることが確認された。 The final purified product A35Gχχϋ was freeze-dried and redissolved in 230 JL St of S. This Sanal 9 ·! 10 u 2 XSDS—PAGE sample buffer (1 mM Tris — HC] H6.8, 10% Sucrose. 10% SDS, 0.25 mg 'ml bromphenol buffer :), 1 2— After adding mercaptoethanol and heating at 100 ° C for 5 minutes, apply the whole amount to a 10-20 SDS polyacrylamide gradient gel (first character, SDS-PAGPL AT E 10'20>, —) ir. : A fixed Electrophoresis was performed for 120 minutes at a current, and BI-RAD Molecular Weight Standards-Low was used as a molecular weight marker. After electrophoresis, silver staining was performed (the sketch is shown in Fig. 2) In the lane on which the sample was placed, only a single band with a molecular weight of 34 K ± 1 KDa was observed, and TNF activity in urine was observed. The inhibitory substance was confirmed to have a molecular weight of 34 K-21 KL) a on S]) S-PAGE in the reduced state.
N末ァミノ酸配列の決定 Determination of N-terminal amino acid sequence
最終精製品 A 1 mlを凍結乾燥にて、 約 100 まで、 漶縮を行なった後、 Applied Bio Systems 社製、 4" A 型プロテインシークェンサ一で、 N末アミノ酸配列の分 析を行なった。  After freeze-drying 1 ml of the final purified product A to reduce to about 100, the N-terminal amino acid sequence was analyzed using a 4 "A-type protein sequencer manufactured by Applied Bio Systems. .
2回の分析で、 N末端より 11番目のアミ ノ酸まで、 同 一の配列が同定され、 これは以下の配列 Vai- Aia- Phe- Thr-Pro- Tyr-Ala-Prc-Ala- Pro-Thr を有していた：  In the two analyses, the same sequence was identified from the N-terminus to the amino acid at position 11, which is the following sequence: Vai-Aia-Phe-Thr-Pro-Tyr-Ala-Prc-Ala-Pro- Had Thr:
〈図面の筒単な説明〉 <Single description of drawings>
図 1は、 D EAE粗精製品を逆相カラムにかけた時の 溶出プロフアイルである  Figure 1 shows the elution profile of the DEAE crude product when applied to a reversed-phase column.
図 2は、 最終精製品 Aの還元状態下における S D S - P AG Eの結果を示したものである： 〈配列表〉 Figure 2 shows the results of SDS-PAGE under reduced conditions of the final purified product A: <Sequence list>
配列番号 ： ュ SEQ ID NO:
配列の長さ ： 1 1 Array length: 1 1
配列の型 ： アミノ酸 Sequence type: amino acid
トポロジー ： 直鎖状  Topology: linear
—配列の種類 ： タンパク質 —Sequence type: Protein
配列 ： Array:
Val Ala P e Thr Pro T r Ala Pro Ala Pro Thr 1 5 10 .  Val Ala Pe Thr Pro Tr Ala Pro Ala Pro Thr 1 5 10.

Claims

■ 02 PCT/JP91/00920 20 請求の範囲 · ■ 02 PCT / JP91 / 00920 20 Claims ·
1. (1) 腫瘍壊死因子の、 L 929 細胞への殺細胞効果を抑 制し、 1. (1) Suppresses the cell-killing effect of tumor necrosis factor on L929 cells,
(2) S D S— P A G Eにおける分子量が還元状態で  (2) When the molecular weight of SDS-PAGE is reduced
34 K ^ 1 K D aであり、  34 K ^ 1 K Da
(3) N末端から 1〜11番目までのアミノ酸が次の配列 Val-AI a-Phe-Thr-Pro-T r-Al a-Pro-Al a-Pro-T r で表わされる物質。  (3) A substance in which the 1st to 11th amino acids from the N-terminal are represented by the following sequence: Val-AIa-Phe-Thr-Pro-Tr-Ala-Pro-Ala-Pro-Tr.
2. 尿を精製することにより得られる請求の範囲第 1項 記載の物質。  2. The substance according to claim 1, which is obtained by purifying urine.
3. 尿が膜性増殖性糸球体腎炎患者由来のものである請 求の範囲第 2項記載の物質。 3. The substance according to claim 2, wherein the urine is derived from a patient with membranous proliferative glomerulonephritis.
4. 腫瘍壊死因子活性抑制物質の製造方法であって 4. A method for producing a tumor necrosis factor activity inhibitor,
(a) 尿をイオン交換カラム及び 又は逆相カラムへの 吸着及び溶出によって精製し、  (a) urine is purified by adsorption and elution on an ion exchange column and / or a reversed phase column,
(b) 腫疡壊死因子固定化カラムへの吸着及び溶出によ つて腫瘍壊死因子に結合する画分を分取し、  (b) Separating the fraction binding to tumor necrosis factor by adsorption and elution on the tumor-necrosis factor-immobilized column,
(0 腫瘍壊死因子の L 929 細胞への殺細胞効果を抑制 する画分を選択する  (0 Select fractions that suppress tumor necrosis factor's cell killing effect on L929 cells
ことからなる製造方法  Manufacturing method comprising:
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US7238776B2 (en) 1989-04-21 2007-07-03 Amgen Inc. TNF binding proteins
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US7732587B2 (en) 1996-07-09 2010-06-08 Amgen Inc. Nucleic acids encoding truncated soluble tumor necrosis factor
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US6271346B1 (en) 1989-04-21 2001-08-07 Amgen Inc. TNF Receptors, TNF binding proteins and DNAs coding for them
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