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
urine
pro
cells
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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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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7151Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for tumor necrosis factor [TNF], for lymphotoxin [LT]
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52Cytokines; Lymphokines; Interferons
    • C07K14/525Tumour necrosis factor [TNF]

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

Fine Form

( 'The title of the invention>

Tumor 疡壊 death factor activity inhibiting substance and a manufacturing method thereof

':Technical field:'

: Invention isolation of a new substance and the substance Ru bovine inhibiting the activity of tumor 疡壊 death factor, rather relates to the purification: BACKGROUND click

Tumor 瘙壊 death factor (TN F) is administered Baci l lus C a 1 me 11 e -Gti er ί II <BCG> bacteria CD- 1 Swiss mice, administration of bacterial endotoxin (endotoxin) after 2 weeks its It is found in a physiologically active substance which appears in the blood when it is, Carswe in 1975! 1 et al. Report [EA Carswe! ! Et al., Pro at! .. Acad, Sci, USA, 3ot) t U 975:..] In the physiologically active protein, the amino acid sequences of Aggarwai et al in 1985 [E, B, Aggarwal et al., J. Biol diem 260, 2345 11985) It Ri has been revealed to J.

The Pennies et al., By irai et al. And Wang et al., The amino acid sequence and gene sequence of human k τ \ ι F is revealed [Panni ca et al, atur 312, 24 S hirai atur 313, 803;?. 1985;, Wang et al., Science 28 ^ 149

Initially from the antineoplastic lifting activity, TN F which development has been promoted as a cancer therapeutic, recently revealed various physiological activities, various functions in vivo are being elucidated: for example, a bacterial infection activity [B. Beutler et al., Science, 229, 869 (1985)] of the mediators in the ecology of endotoxin shock due to, induction of inflammatory reaction to vascular endothelial cells [J Gamble 'et al, P roc . X a † i. A cad. S c ί., USA, _82 ^ _ 8667 (1985)], Hatsujuku action [A. D are! io et al., J. Exp. Med. 163. 1443 (1986)] , Lee Ntariukin is one of the inflammation caused by substance:! [ 'PP Nawroth et al, J. Exp. Med. 1 _ 1363] Toki engagement clearly be Sorcerer There the like

There also, during holiday solution of certain diseases patient includes a number than in normal Ding NF, becoming clear that there is a close relationship content of the TN F and the pathology of the patient . in other words, Ri by the overproduction of TN F, diseases considered to deterioration of the condition has occurred many present: the administering to such diseases, a substance having an anti-TN F activity Ri, the improvement of the condition can be performed, the fact that it is easily inferred, for endotoxin sucrose click, as a substance having anti-TN F activity, administration of anti-F antibody, performed using animal , it has been reported that showed significant effects [B. BeuUer et al, Science, 299, 869; 1985)] - However, at present, the anti-TN F antibody other than human animal Yukari釆 of - also ;: in such an antibody for administration to humans has not been created only for large Q The of et believed to be problem, acute considered TN F is 閔与, in the inflammatory response of chronic, that immune complex formation is one of the factors that exacerbate the pathology least widely known, for such diseases, as a material that have a anti Ding NF action being ¾ anti Ding ヽ 'F antibodies for therapy is considered difficult. Therefore, anti Ding may exist naturally in the body fluids of human - if it is possible to have 闬 the F agent, against a disease TN F is Tokiazuka sufficiently, is used as the effective and safe therapeutics it is presumed to be the sell those UninaTsuta

With respect to such a TNF activity inhibiting substance (also referred to as \ F i down inhibitor formation), Peetre et al [Eur. J. Haemal c !, ϋ, 414 (1988), £ 2, -270 (1989) ", P. Sec ger et al.

[. J. Exp Med 16 :, 1 51 1988;.!., J. Bio !. Chen .. 2D4 1196b · 1989)] and, H E nge I mann;.. Ii.

Cheiii., 11974 (1989)] Ri by, these substances manufactured ft from urine has been reported that we row is its N-terminal amino acid sequences, then, amino acid sequences are determined Ding NF Riseata one [; 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 it is believed etc. would be one was present in the urine Te Π

, H. EagelmatiQ et Yuoru specific ¾ sequences -.. The two materials of molecular weight of about 30,000 are isolated from the urine (H. Enge lmann et ai, J. Biol Chem 265, 1531 ( 1990)! in addition, CA Smith et al. also, take discloses the amino acid sequence for the substance (CA Smith et ai, Science 248, 1019, 1990).

The present inventors have searched a novel TNF activity inhibitor Subeku, a result of our study, we have reached the present invention

< 'The purpose of the invention>

The invention shall be the object of the invention to provide a novel process for producing TNF activity inhibitor, compositions containing it and the material: the disclosure of Ku invention>

The invention Tsutsumi舍Oru the following invention. Sand Chi invention, H) tumors L 929 cell killing effect on cell necrosis factor and inhibition • 2) SDS- 3 4 molecular weight under reducing conditions in PAGE

K: is a 1 KD a,

'; 3'ί Ν amino acids from the end to the 1 ~U th a material represented by the following sequence Val-Ai a-Phe-Thr-Pro-T r-Ai a-Pro-Al a-Pro-T r is there

Substance of the present invention is characterized in that the N-terminal 11 th amino acid is Va! -AI a-Phe-T r-Pro-Tyr-Al a-Pro-Al a-? Ro-Thr substances invention, C. Peetre et al [Eur. J. Haematol. 11, 414 (and the material 1988 门 described, which have their reports at a molecular weight of from about 5 0 KD a, material of the present invention There differs in that 34 K two 1 KD a Sunru.

Moreover, P. Seck ger et al [J. Exp. Med. 167. 1551 (1988), The J. B io!. C em. 264, the materials described in 1196t ^ 989, in SDS-PAG £ the molecular weight is approximately equal: But, eluting Asetonito Lil concentration from ^ Iteiru reverse phase column in the final purification, also taking into account the condition that the column is different, different in that very different.

Also, H. Enge 1 Maun et [J. Biol C em _ 264, 11974 (1989;..] That the substance that is described, it is not at all homology to the amino acid sequence from its molecular weight and X-terminal in different:

.. Has, H. Engeimann et al and 7 Chem 265, the material TBPI listed in 153L 1990] in Bio, - \ ^ amino acid sequence from the end are completely different.

In addition, the TBP ϋ, they can not be recognized diversity of UNA end of silk amino acids I have reported, and the molecular weight is also different youth E

The Ding XF in the invention, Aggarwai et. Ίι. : · Chem 2345 (1985)] and Shirai et al. [Ature 313. 805:. 1985)] by Ri The reported Ding to F- is «, including the natural TN F and recombinase Jun door TNF

In the present invention, the L 929 cells, mouse

An established cell line derived from ί ibrcsarcoina, ATC C strain number is a cell that has been registered as a CCL one 1, the name L 929 (NCTC clone 929)

The molecular weight of the substance of the tree invention is the substance of the 3 '4 K two〗 KD a in the reduced state your Ru molecular weight in SDS- PAGE. This is a reduction state in SDS- PAGE with this, electrical by SDS ho Riakuriruami Dogeru before performing electrophoresis, a suitable reducing agent to support N'aru, for example, 2 - Merukaputoeta Bruno - le, in the presence of such Jichiosurei Torr, performed 100 ° C, the process for a few minutes, Jisurufu I in proteins after the de-binding is cleaved, it refers to performing electrophoresis - the invention embraces a set Narubutsu containing the tumor necrosis factor activity inhibiting substance

Substance of the present invention as possible out to obtain Ri by the purification of the urine. In particular, it can be obtained by purifying the urine of patients membranous proliferative glomerulonephritis,

Membrane proliferative glomerulonephritis, Clinically, immune complex deposition in the renal glomerulus were observed, further reduction in complement titer blood, thickening of the glomerular basement membrane, proliferation, etc. Mesangiumu cells is a kidney disease that is found but. this disease onset originals is still unknown, recently, Sai Kai down class in Mesangiumu cell proliferation type nephritis, Tokunyi interleukin [experimental medicine 2 involved has been suggested of 6, 11 (1989)]. Production of interleukin 6 to be induced by TNF Yai interleukin 1 have been reported in many cells [Experimental Medicine .2, 2: (1989), Modern Immunology 93 (1988)] it is speculated enhanced production of de is Okitsu also in this disease:

Water inventors blood Ding \ F content of various renal diseases patients. Place was boss, it was revealed that Ko滤 ¾ of TNF is contained in membrane proliferative glomerulonephritis patients blood with this disease patient urine was examined activity inhibitory ability Ding NF., it is found to be highly TNF activity inhibitory ability, the One.

Purification ion exchange, reverse phase column, as possible out to perform Ri by the combining purification operation such as Ding XF immobilized la.

That is, the present invention is a tumor 壌死 factor activity inhibiting substance manufactured method,

ia) by suction Chakuoyobi elution into Ion exchange columns and or reverse phase column urine ^ and ¾,

(The fraction that binds to 疡壊 death factors tumor Te cowpea adsorption and elution of the bi tumor 瘙壊 death factor immobilized column aliquoted,

ics; selecting fractions Ru suppress dimensions of L 929 cell-killing effect on cells of the tumor necrosis factor

It encompasses a manufacturing method which consists

Hereinafter, the present invention is described in detail: Preparation of the sample urine

Urine Hatsunyo, urine, preferably no Mawa or using any of the spot urine, later, in order to perform Baioa ,, / Si, it is one taken in a state as close as possible to a sterile or aseptic. 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 the Protea one Zi inhibitor development, etc. in order to prevent such may be added immediately after urine collection Iga, after urine collection, these additives if transition to rapidly frozen is not required. In the case of adding these additives, suitable operation, for example, dialysis, Beta¾ outside 沪過, gel ^ Ri by the operation of the over-like, Baioa sufficiently before performing Sosi, necessary to remove the added Addendum there is a

Purified from the specimen urine

Purification may be carried me to the usual method for protein purification. The urine concentrate, usually, than only observed low activity, it is preferable to first carry out the concentration. Is a concentration method, ultrafiltration over, freeze drying, may be performed based on the normal biochemical experimentation of salting, if ultrafiltration 沪過 performs salting, the target substance and molecular weight, it is necessary to examine the Shio澹 degree such that initiate precipitation advance:

After 瀵縮 is suitable purification procedure such as ion exchange, gel '沪過, in combination § full I two tea chromatography gate rough I over, isoelectric point electrostatic focusing, hydrophobic chromatography, reverse phase chromatography I Chief , it is possible to proceed with the purification

Materials of the present invention, after the concentration operation, TN F no rows by Ri separation and purification to be adsorbed on a column force ,, / off 'ring, then the particular § Ri by the elution with a reverse 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: , using 25 cm, Asetonito Lil If performed Asetonito Lil linear gradient of 1δ~ 37% of 150 minutes 溏度 2? It is those of ~ 28%,

Measurements of I'nyu F activity suppressing ability

Is a measure of ability to inhibit Ding lambda tau F activity, the system for measuring the TN F activity already known, may be added pressure to the test sample:. Onawachi, Dingヽ- the F §, Taioru killing effect on various tumor 塲 cells vitro which are known Chirete as a Risi method, cytostatic effect, Ya growth promoting effect of fatty acid metabolism _ suppression normal fibroblasts adipocyte 1 L - adhesion promoting effect and of 6 production inducing effect neutrophils to endothelial cells, Supaokisa I de secretion promoting effect, clotting hyperactivity effect of vascular endothelial cells, osteoclast effect on bone cells, IL in various cells one] the system for measuring the induction effect of the Ya-off 'Kuchisu Tagerandei down such that it is preferable that measured Sunru in cell killing effect of the particular L 929 cells available for certain cancer cells in in vivo Taisun Ru bleeding 壞死 effect, endotoxin sucrose, Suk of the induction effect, the heat generation efficiency But it is possible to use a system to measure, and activity in in vivo, the activity of many vitro, because there are many reactions that can be induced in substance other than TN P, Wherever possible simple and TN F other than the substance affected such have systems, for example the ', it is desirable to use a system for measuring the cytocidal activity against cancer cells in vitro

Tumor necrosis factor active inhibitors of the present invention, various

As a pharmaceutical group composition as for that to be used as therapeutic agents for Tokiren diseases include by tumor necrosis factor activity inhibitor of the present invention and effective active ingredient may allow other pharmaceutical in in those including responsible bodies 'Alejo' good

For preparing pharmaceutical compositions, such as a purpose of enhancing the reduction or physiological activity of the antigen of the active active ingredient and to tumor using «i Shiinko active inhibitors, for example Boriechiren glycol <PEG), dextran or poly DL- Aranin: it may be modified by known Borima such.

Is in the form of pharmaceutical compositions, injectable compositions, suppositories that although others include, as a injectable composition preferably used as a particularly intravenous sets Narubutsu is.

For injectable compositions are pharmaceutically effective amount and a mixture of pharmaceutically acceptable Cormorants Ru carrier of tumor necrosis factor activity suppression agent of the present invention, amino acid therein, sugars, cellulose derivatives , polyvinylpyrrolidones, is added to the general injectable compositions, such as inorganic compounds activator can also be used: to give these specific examples, are the amino acids, glycine, arginine, Aranin and salts, and the like that can be tolerated them of medicine biological to the like: are the sugars, Ma down two Lumpur, Lee * waves * ,, Ichiru, xylylene k one Norre, Chichi輕, 々'- 7 Les courses, etc. can be mentioned: Cal is a cellulose derivative Bokishimechi / receptacle / Lero Ichisu Juto Li um, methyl 'cell' mouth one scan, and the like: polyvinylpyrrolidones the molecularly weight 1 0, 000 1, 000, GOO Bolivia of Yurupirori Don mentioned, et al. Are:

Is an organic acid, Asukorubin acid, Kuen acids like beauty salts thereof are exemplified: is the inorganic compound 枸類-phosphate hydrogen Juto Li © beam, bicarbonate Juto Li © beam, acetic Juto there is such as Li ©-time

And a liquid for dissolving these excipients, injectable ^ distillation, there is a physiological saline for injection or injectable Ringer's solution

During other injections, stabilizers, surfactants, tonicity agents, soothing agents, preservatives, buffering agents and the like may optionally ^ is Ru. When shows these specific examples, and stabilizers pyromellitic sulfite acid tens ^ potassium is Te, antioxidants such as ϋ Asukorubin acid: DTA, is a surfactant chelating agent, and the like, such as Chio glycol agent, e Li Loubet DOO, Boriokishechi Le 'emissions derivatives non Ion surfactants such as there, 'sill isotonizing agent etc. the sodium chloride and the like.

Base down benzyl alcohol as a soothing agent, xylocaine, procaine, and the like.

Parabens as a preservative, Kurorobutano Ichiru, chloride benzalkonium Roh record Niu-time, thimerosal (Thimerosal) Hitoshigakyo is up.

As the buffer, Kuen acid, acetic acid, sodium © beam salts such as phosphoric acid.

<Effect of the invention>

According to the present invention provides novel substances that inhibit the cytotoxic effect to the cells of the tumor necrosis factor (TN F>, it believed TN F is 閬与 diseases, for example, E emission Dotoki Paperback click Ya burns at the time of the cane, Suk, acute liver failure, kidney failure and multi-organ failure, arthritis, SLE, Bechi, a number of autoimmune diseases of the V mildew, etc., rejection at the time of organ transplantation, Kawasaki disease, coagulation of DIC, etc. . the treatment of abnormal, can be utilized to diagnose the like also can provide a method for efficiently producing the substance Do ivy - Ku example>

Hereinafter, the present invention will be described in detail by way of examples, the present invention is not limited to the following examples 1

Purification Ding XF active inhibitor in the urine

(1) concentration by ultrafiltration 泸過

Concentrated membrane proliferative glomerulonephritis Ken dermatitis patients urine (sample 1) to ml Ri fractions on molecular weight〗 Man以 in ultrafiltration 沪過 by Bae Rico emissions (Mi Ripoa Co.), 10 m Tr !? pH8. 0 to '.', 'was one exchange (sample 2)

? 2 DAE - purification by Sep arose column

The concentrated urine sample was subjected to DEAE- Sepiiarose column of 4 S was ^ 1衡化at lOraM Tris pH8.0:? At a flow rate of 40ΰ ir .: zjir, after washing with 10m Tris pH8.0, 10mM Tri - lOOm at NaC! pH G, it performs the elution, a collection of 400 m! the Tsutsura Kusho down

Each Furakusho emissions of 1 m 1 a centriscreen con I 0 (Ami Con Corporation: In ultrafiltration沪過by> bar in PBS - after earth exchanged, 0 D 2s of each Tsu Rakushiyo down (a, L 929 cells measuring the ability to inhibit cell killing effect of Ding ヽ F against, and Earl the active fractions were 瀵縮 molecular weight of 10,000 or more fractions Ri by the Perricone, Tampa click ^ degree Π.8mg "ml of crude seminal ! product 103 m San § / was to give a '3,' to measure the ability to inhibit cell-killing effect of Ding NF against L 929 cells of the sample 3 <shown in the Table: '

; 3 Purification by reverse phase column

Then I) EAE crude product (Samburu 3, '1 Om to,::!. ° - Ding FA (k ij Furuoro acetate) 衡化 at the opposite ^ column (Protein C 4, VYDAC Co., 2: Ku subjected to 25 cm) ', from Asetoni Bok Lil 16% 40 min, with a linear gradient of 40, subjected to elution at a flow rate 5 ml Bruno Ffiin, similar 5 m!. Furakusho down, was subjected to preparative. the after repeating the operation of nine times, each fraction was lyophilized, the Furakusho down the 1 Di 1 click ', after melting into PBS, the Rakusho down the same elution time off' to Lumpur, 0 D 28. and L was measured inhibitory ability of cell killing effect of Ding NF for 929 cells. TNF activity inhibition Sunru 话性 were mainly observed in two peaks (peak one click E peak E)

(Shown in Figure 1 ". The cytotoxic effect of TXF to these L 929 cells are shown in Table 1.

Peak fractions E, after this, further subjected TNF § full I two Tikara beam, purification by reverse phase column, it was determined X-terminal amino acid sequence, Asp-Ser-Va! -X-Pro-G ! n- Gl -Lys-T rI I e-Hi s-Pro-Gi QX-Asn-Se rI ί e (X is an array called 477 a type Puroti Nshikuensa one beak did not et al observed residue> is obtained known TNF Lee down Hibita one or was estimated to be TNF Risepu evening shard, [I. "01 sson et al, Eur. J. Haemat .. 42, 270 (1989), H. Loetscher et al, Ce ! j _ ^ _ 351 (1990), T. J, Schai l et al., Ce l lJJ ^ 361 (1990; "

Purification by T λ 'F Afi two tea column

Next, 18IIP resulting reverse phase column a crude product of 7 m of (peak Π),] mg 4 TNF has couplings down gate of η.ι:! A i-Ge j 10 of {BIO-RAD Co. passed through). '] used herein, bets, is Rikonbi Jun preparative Τ-ヽF of specific activity 3.3 10 7 U., its amino acid sequence, Shirai et al

[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; purification by reverse phase column

Immediately after elution, the eluate 1.2 ml 0. c '0 chome FA _ 18'. § Se Tonito Lil equilibrated with reverse phase column (Protein C 4, \ YDAC Co., 0.46 25 cm) subjected to, of 150 minutes 18. . From Ri by the Asetonito Lil linear gradient of 37, in § Se Bok two k Lil 漶度 27-28% was subjected to elution, Mei Npiku were observed one by absorption of 215a, Re this was collected the fractions min the 示O Ding NF activity suppressing material for suppressing ability in Table 1 of the cell killing effect of F for ¾ cell of the final purified product a, which is referred to as final purified products a §, V Si

A Si Ding NF activity suppressing ability, using L 929 cells, Ding ヽ - systems Ruii & Giiiord to measure F activity [J. ί mmiuic ι, 125, 1671 (1980;] to, simultaneously with Ding NF Samburu conducted Ri by the fact the addition Oru: dimensions ie, 4 Bruno 10 9t force El plate; the z ml of L 929 cells were mixed with 2 u S π :! Kuchinomaishin D, seeded at 100 Ueru, and 5% C),, 3 7 Te: 2 hours incubation Oru ultrafiltration沪過to the cells, dialysis, or lyophilized, Ri by the remelting or the like, the San'aru 50 was replaced with PBS to cross Raa added at immediately after the further addition of 2 ng / 'ml of TN F solution (specific activity 3.3 1 G 7 U mg) 0 ^, was subjected to 18 hours culture at 5% C 0 2, 37 ° C. After culturing, click Risutarubaiore, '/ performs staining of viable cells by preparative, the stained cells were dissolved in 0.5 ¾ SDS, and the absorbance measured at 595Iotaiotaitaiota <

Based on the absorbance of the resulting 595am., It was calculated TN F- Inhibition rate. Calculation Deshiki of TNF _ I nh ibiti on rate is as shown below.

TNF - Inhibition rate

[0D 595] TNF + sample one [0D 595] TNF

10CM¾

[OD] No TF one [OD: 1 TNF each San'aru was calculated respectively TNF- In Mtion rate performs by Ria '' / Si to 1 Z2 dilution series. 30% of TNF - Iahibitioa give rate the reciprocal of the highest dilution of San'aru 1 defined unit and (U), it shows the TNF inhibiting activity in each purification step of the TN F active suppression substance in urine in Table 1 is there. 1

Purified fraction; activity total volume I total activity

! (ϋ / m 1) ■ ( ml):. (U urine undiluted) 3. 1, '10 fc molecular weight of 10,000 or more 4120 250 ί 1.0> 10-enriched fraction (Samburu 2)

DEAE crude active fraction 90 10 [delta]

Eight

Bi - click I :: Tikaramu - Roh

C4 reverse phase column § Se Bok two tri Λ υ Λ 1 υ Λ

27-28% eluted fractions

(Final purified product A) molecular weight of TNF activity inhibiting substance in urine

The final purified product A 35G χχϋ was lyophilized and redissolved in S of 230 JL St. This San'aru 9-!! 10 u of 2 XSDS- PAGE sample bar file one (1 mM Tr IS to -. HC] H6.8, 10% Sucrose 10% SDS, 0.25mg 'ml Bed B Moff We Nord Bull one :), 1 of 2 mercaptoethanol was added, 100 ° C, 5 minutes, after heating, 10 to 20 of SDS Poriaku Riruami de gradient gel (first reduction shaped, SDS - placed a total volume of PAGPL AT E 10 '20>, -) ir : at a constant current, 120 minutes, is the molecular weight markers was subjected to electrophoresis BI 〇 one RAD Inc. molecular weight Standards - using Low. After completion of electrophoresis was subjected to silver staining (lane loaded with its shows a sketch>. Sample 2 is a single-band Nomiga認 Merare molecular weight 34 K ± 1 KD a, TN F activity in urine inhibitor is, S]) S under reducing conditions - molecular weight on P AG E is, 34 K two 1 KL) a der Rukoto was confirmed.

The determination of N-terminal Amino acid sequence

At a final purified product A 1 ml lyophilized, to about 100, after performing 漶縮, Applied Bio Systems, Inc., 4 "type A protein Sea Kuen mono-, were performed analysis of N-terminal amino acid sequence .

Twice analysis, up to 11 th amino acids from the N-terminus, the same sequence is identified, this is the following sequence Vai- Aia- Phe- Thr-Pro- Tyr-Ala-Prc-Ala- Pro- I had a Thr:

<Cylinder single description of the drawings>

Figure 1 is a dissolution profiles when subjected to a reversed phase column D EAE crude product

2, SDS under reducing conditions of the final purified product A - shows the results of P AG E: <sequence listing>

SEQ ID NO: Interview

The length of the array: 1 1

The type of the sequence: amino acid

Topology: linear

- an array of categories: protein

The sequence:

Val Ala P e Thr Pro T r Ala Pro Ala Pro Thr 1 5 10.

Claims

■ range of 02 PCT / JP91 / 00920 20 claim and
1. (1) tumor necrosis factor, depressive won the cell-killing effect on L 929 cells,
(2) molecular weight in SDS-PAGE is in reduced state
Is a 34 K ^ 1 KD a,
(3) a substance amino acids from the N-terminus to the 1-11 th is represented by the following sequence Val-AI a-Phe-Thr-Pro-T r-Al a-Pro-Al a-Pro-T r.
2. materials ranging first claim of claim obtained by purifying the urine.
3. substances billed ranging second Claims those urine from patients membranous glomerulonephritis.
4. A method of manufacturing a tumor necrosis factor activity inhibiting substance
(A) was purified by adsorption and elution of the ion exchange column and or reverse phase column urine,
(B) was collected fractions amount to bind to I connexion tumor necrosis factor adsorption and elution of the tumor 疡壊 death factor immobilized column,
(Selecting a fraction to inhibit L 929 cell killing effect on cells 0 tumor necrosis factor
Manufacturing method which consists in
PCT/JP1991/000920 1990-07-11 1991-07-10 Tumor necrosis factor activity inhibitor and production thereof WO1992001002A1 (en)

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US6271346B1 (en) 1989-04-21 2001-08-07 Amgen Inc. TNF Receptors, TNF binding proteins and DNAs coding for them
US6306820B1 (en) 1996-12-06 2001-10-23 Amgen Inc. Combination therapy using a TNF binding protein for treating TNF-mediated diseases
US7238776B2 (en) 1989-04-21 2007-07-03 Amgen Inc. TNF binding proteins
WO2008054603A2 (en) 2006-10-02 2008-05-08 Amgen Inc. Il-17 receptor a antigen binding proteins
EP1992636A2 (en) 1999-11-12 2008-11-19 Amgen Inc. Process for correction of a disulfide misfold in Fc molecules
EP2002846A2 (en) 1996-12-06 2008-12-17 Amgen Inc. Combination therapy using an IL-1 inhibitor for treating IL-1 mediated diseases
EP2087908A1 (en) 2001-06-26 2009-08-12 Amgen, Inc. Antibodies to opgl
US7732587B2 (en) 1996-07-09 2010-06-08 Amgen Inc. Nucleic acids encoding truncated soluble tumor necrosis factor
WO2011046958A1 (en) 2009-10-12 2011-04-21 Amgen Inc. Use of il-17 receptor a antigen binding proteins
WO2013016220A1 (en) 2011-07-22 2013-01-31 Amgen Inc. Il-17 receptor a is required for il-17c biology
WO2015153144A1 (en) 2014-03-31 2015-10-08 Kirin-Amgen, Inc. Methods of treating nail and scalp psoriasis
US10072085B2 (en) 2010-01-15 2018-09-11 Kirin-Amgen, Inc. Method of treating psoriasis using an IL-17 receptor antibody formulation

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