DK158230B - Process for purifying a protein-like, physiologically active substance having an antineoplastic effect, and the protein-like, physiologically active substance which is purified in this process - Google Patents

Description

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Den foreliggende opfindelse angår en fremgangsmåde til rensning af et proteinlignende fysiologisk aktivt stof med antitumorvirkning.The present invention relates to a method of purifying a protein-like physiologically active substance with antitumor effect.

Der findes et antal rapporter om eksistensen af stoffer med fysiologiske virkninger såsom tumorcelledræbende evne, hvoraf typiske ek-5 sempler kort resumeres i det følgende.There are a number of reports on the existence of substances with physiological effects such as tumor cell killing ability, of which typical examples are briefly summarized below.

Currie et al. opdagede, at en faktor, der inhiberer formeringen af forskellige tumorceller, blev fremstillet ved administration af endotoxin til peritoneale exudatceller fra en normal rotte [J. Exp.Currie et al. discovered that a factor inhibiting the proliferation of various tumor cells was produced by administration of endotoxin to peritoneal exudate cells of a normal rat [J. Exp.

Med., bind 142, side 1600-1605 (1975)], hvorefter der blev foretaget 10 yderligere undersøgelser af denne faktor, hvorved det viste sig, at den væsentligste bestanddel i faktoren er arginase som beskrevet i Nature (London), bind 273, side 758-759 (1978).Med., Vol. 142, pages 1600-1605 (1975)], after which 10 further studies of this factor were performed, showing that the major component of the factor is arginase as described in Nature (London), Vol. 273, pp. 758-759 (1978).

Reed et al. opdagede også et proteinlignende stof med en molekylvægt på 45.000, der kunne dræbe dyrkede tumorceller såsom L-celler fra 15 dyrkede celler eller en dyrket mononuclear supernatant fra normale rotter og mennesker ved at anvende endotoxinbehandling, men den væsentligste bestanddel af dette stof er endnu ikke blevet isoleret og identificeret [J. Immunol., bind 115, side 395-404 (1975)].Reed et al. also discovered a protein-like substance with a molecular weight of 45,000 that could kill cultured tumor cells such as L cells from 15 cultured cells or a cultured mononuclear supernatant from normal rats and humans using endotoxin therapy, but the major component of this drug has not yet been isolated and identified [J. Immunol., Vol. 115, pp. 395-404 (1975)].

Carswell et al. opdagede, at serum fra CD-I schweiziske mus inficeret 20 med bacillus Calmette-Guérin (BCG) efter 2 uger efterfulgt af intravenøs injektion af endotoxin, har cytotoxisk virkning mod dyrkede L-celler, samt at det bevirker hæmorrhagisk necrose af transplanteret BALB/c-sarkom Meth A i (BALB/c x C57BL/6)F^ mus, og de kaldte det aktive stof i serummet TNF (Tumor Necrose Faktor) [Proc. Nat. Acad.Carswell et al. found that serum from CD-I Swiss mice infected 20 with bacillus Calmette-Guérin (BCG) after 2 weeks followed by intravenous injection of endotoxin has cytotoxic effect against cultured L cells, as well as causing hemorrhagic necrosis of transplanted BALB / c -sarcoma Meth A in (BALB / cx C57BL / 6) F ^ mice, and they called the active substance in the serum TNF (Tumor Necrosis Factor) [Proc. Night. Acad.

25 Sci. USA, bind 72 (nr. 9), side 3666-3670 (1975)]. De udførte endvidere delvis rensning af TNF fra dette serum og fik således TNF-frak-tioner, der var renset 20-30 gange i forhold til serummet, idet de rapporterede, at det aktive stof er et glycoprotein med en molekylvægt på ca. 150.000, der vandrer med a-globuliner i celluloseacetate-30 lektroforese [Proc. Nat. Acad. Sci. USA, bind 73 (nr. 2), side 381-385 (1976)].Sci. United States, Vol. 72 (No. 9), pages 3666-3670 (1975)]. They further performed partial purification of TNF from this serum, thus obtaining TNF fractions which were purified 20-30 times relative to the serum, reporting that the active substance is a glycoprotein having a molecular weight of approx. 150,000 migrating with α-globulins in cellulose acetate electrophoresis [Proc. Night. Acad. Sci. United States, Vol. 73 (No. 2), pages 381-385 (1976)].

Månnel et al. undersøgte den cytotoxiske faktors egenskaber ved anvendelse af det museserum, der fås ved den af Carswell et al. be- 2Månnel et al. investigated the cytotoxic factor properties using the mouse serum obtained from that of Carswell et al. be- 2

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skrevne metode som ovenfor beskrevet, hvorved det viste sig, at de eluerede fraktioner med phytotoxisk virkning varierede ved gelfiltrering afhængig af saltkoncentrationen i pufferen, og at den cy-totoxiske faktors molekylvægt var 55.000-60.000 i en puffer med en 5 høj saltkoncentration, hvorimod den var 125.000-150.000 ved aggregation i en puffer med lav saltkoncentration eller i serum [Infect.written method as described above, whereby it was found that the eluted fractions with phytotoxic effect varied by gel filtration depending on the salt concentration in the buffer and that the molecular weight of the cytotoxic factor was 55,000-60,000 in a buffer with a high salt concentration, whereas the was 125,000-150,000 by aggregation in a low salt buffer or serum buffer [Infect.

Immun., bind 28 (nr. 1), side 204-211 (1980)]. De rapporterede, at denne faktors isoelektriske punkt (pi-værdi) var 4,8. Men denne faktors virkning blev ikke undersøgt i et dyr (fx undersøgelser under 10 anvendelse af transplanteret Meth A sarkom i mus), hvorfor eksistensen af dens virkning in vivo ikke kan konstateres. Det er derfor umuligt at afgøre, om faktoren er identisk med det af Carswell et al. opdagede TNF. Endvidere nævnte de næsten intet om dens rensning og isolering, omend nogle af dens egenskaber blev undersøgt på baggrund 15 af cytotoxisk virkning mod L-celler.Immun., Vol. 28 (No. 1), pages 204-211 (1980)]. They reported that this factor's isoelectric point (pi value) was 4.8. However, the efficacy of this factor was not investigated in an animal (e.g., studies using 10 transplanted Meth A sarcoma in mice), so the existence of its effect in vivo cannot be ascertained. Therefore, it is impossible to determine whether the factor is identical to that of Carswell et al. discovered TNF. Furthermore, they mentioned almost nothing about its purification and isolation, although some of its properties were investigated on the basis of cytotoxic action against L cells.

Matthews et al. fremstillede TNF i en kanin, undersøgte egenskaberne hos kanin-TNF fra serummet og rapporterede, at kanin-TNF havde en molekylvægt i området på 40.000-50.000 målt ved gelfiltrering under anvendelse af Sephadex® G-200 [Br. J. Cancer, bind 38, side 302-309 20 (1978)]. De rapporterede imidlertid også, at molekylvægten var 67.000 ved hældende gradientpolyacrylamidgelelektroforese eller 39.000 ved gelfiltrering med Ultrogel AcA 44 [Br. J. Cancer, bind 42, side 416-422 (1980)]. Men eftersom de ikke anvendte nogen isoleret og renset prøve, er det ikke sikkert, om· TNF er et enkelt stof eller ikke.Matthews et al. prepared TNF in a rabbit, examined the characteristics of rabbit TNF from the serum and reported that rabbit TNF had a molecular weight in the range of 40,000-50,000 measured by gel filtration using Sephadex® G-200 [Br. J. Cancer, Vol. 38, pages 302-309 (1978)]. However, they also reported that the molecular weight was 67,000 by sloping gradient polyacrylamide gel electrophoresis or 39,000 by gel filtration with Ultrogel AcA 44 [Br. J. Cancer, Vol. 42, pages 416-422 (1980)]. However, since they did not use any isolated and purified sample, it is not certain whether TNF is a single substance or not.

25 Ruff et al. rapporterede, at kanin-TNF blev renset 2000 gange i forhold til serummet ved en serie af saltfraktionering, gelfiltrering, anionbytning og lectinaffinitetskromatografi. Molekylvægten for det fundne kanin-TNF blev anslået til at være 68.000 ved SDS-polyacrylamidgelelektroforese, 55.000 ved gelfiltrering ved Sepha-30 cryl-200 og 52.000 ved glycerolgradient-centrifugering [J. Immunol., bind 125 (nr. 4), side 1671-1677 (1980)].Ruff et al. reported that rabbit TNF was purified 2000 times relative to the serum by a series of salt fractionation, gel filtration, anion exchange and lectin affinity chromatography. The molecular weight of the found rabbit TNF was estimated to be 68,000 by SDS-polyacrylamide gel electrophoresis, 55,000 by gel filtration at Sepha-30 cryl-200 and 52,000 by glycerol gradient centrifugation [J. Immunol., Vol. 125 (No. 4), pp. 1671-1677 (1980)].

Kuli et al. fik tre slags tumorcelle-cytotoxin-fraktioner fra det museserum, der fås ved den af Carswell et al. beskrevne metode som ovenfor beskrevet, og udførte tests under anvendelse af transplante-35 ret Meth A-sarkom i mus, idet de rapporterede, at fraktionerne med en 3Kuli et al. obtained three types of tumor cell cytotoxin fractions from the mouse serum obtained by that of Carswell et al. described method as described above, and performed tests using transplanted Meth A sarcoma in mice, reporting that the

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molekylvægt på 160.000 inducerede tumornecrose, hvorimod fraktionerne med en molekylvægt på 225.000 og 50.000 ikke inducerede nogen tumornecrose [J. lammol., bind 126 (nr. 4), side 1279-1283 (1981)].molecular weight of 160,000 induced tumor necrosis, whereas the fractions of molecular weight of 225,000 and 50,000 did not induce any tumor necrosis [J. lammol., Vol. 126 (No. 4), pages 1279-1283 (1981)].

På trods af flere rapporter om eksistensen af forskellige fysiologisk 5 virksomme faktorer fås disse som ovenfor beskrevet i de fleste tilfælde ikke i tilstrækkelige mængder til udførlig undersøgelse af deres egenskaber. Eftersom der ikke udføres isolering, er det tillige ikke i i den nuværende situation sikkert, om de er kendte stoffer eller hidtil ukendte stoffer.In spite of several reports on the existence of various physiologically active factors, as described above, in most cases, these are not available in sufficient quantities for detailed examination of their properties. Also, since insulation is not carried out, it is not certain in the present situation whether they are known substances or novel substances.

10 Ved at anvende forskellige arter pattedyr har nærværende opfinder undersøgt produktiviteten og rensningen af et fysiologisk aktivt stof med antitumorvirkning, der induceres ved til et pattedyr at administrere et stof, som kan stimulere det reticuloendotheliale system, og injicere endotoxin i pattedyret, hvorved det har vist sig, at en ka-15 nin er bedst egnet til den praktiske anvendelse af det fysiologisk aktive stof som lægemiddel.Using various species of mammals, the present inventor has investigated the productivity and purification of a physiologically active substance with antitumor effect induced by administering to a mammal a substance capable of stimulating the reticuloendothelial system and injecting endotoxin into the mammal, thereby demonstrating say that a case is best suited for the practical use of the physiologically active substance as a drug.

Et af den foreliggende opfindelses formål er at tilvejebringe en praktisk anvendelig, særdeles værdifuld fremgangsmåde til rensning af et fysiologisk aktivt stof med antitumorvirkning, der induceres ved 20 til en kanin at administrere et stof, der kan stimulere det reticuloendotheliale system, og injicere endotoxinet i kaninen.It is an object of the present invention to provide a practically useful, extremely valuable method for purifying a physiologically active substance with antitumor effect induced by administering to a rabbit a substance capable of stimulating the reticuloendothelial system and injecting the endotoxin into the rabbit. .

Et andet af den foreliggende opfindelses formål er at tilvejebringe et hidtil ukendt fysiologisk aktivt stof med antitumorvirkning, der induceres ved til en kanin at administrere et stof, der kan stimulere 25 det reticuloendotheliale system, og injicere endotoxinet i kaninen.Another object of the present invention is to provide a novel physiologically active substance with antitumor effect induced by administering to a rabbit a substance which can stimulate the reticuloendothelial system and inject the endotoxin into the rabbit.

Disse formål opnås ved den foreliggende opfindelse, som angår en fremgangsmåde til rensning af et proteinlignende fysiologisk aktivt stof med antitumorvirkning og det proteinlignende fysiologisk aktive stof renset ved denne fremgangsmåde, idet stoffet fremstilles ved til 30 en kanin at administrere mindst ét stof, der kan stimulere det reticuloendotheliale system, og derefter injicere endotoxin fra en gramnegativ bakterie i kaninen, hvilken fremgangsmåde omfatter, at en legemsvæske, et serum, plasma eller ekstrakt af indre organer fra 4These objects are achieved by the present invention, which relates to a method of purifying a protein-like physiologically active substance with antitumor action and the protein-like physiologically active substance purified by this method, the substance being prepared by administering to at least one rabbit a substance capable of stimulating the reticuloendothelial system, and then injecting endotoxin from a gram-negative bacterium into the rabbit, comprising a body fluid, serum, plasma or extract of internal organs from 4

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kaninen indeholdende det proteinlignende fysiologisk aktive stof, som ikke har været underkastet ammoniumsulfatfraktionering, bringes i kontakt med en basisk anionbytter under anvendelse af en pufferopløsning med en pH-værdi på 6,0-9,0 og en saltkoncentration på ikke mere 5 end 0,2M, hvorved det fysiologisk aktive stof adsorberes til anion-bytteren, det adsorberede fysiologisk aktive stof elueres fra anion -bytteren med en pufferopløsning, der har en højere saltkoncentration, og eluatet, der indeholder det fysiologisk aktive stof efter koncentrering og eventuelt dialysering udsættes for gelfiltrering med en 10 gel, der egner sig til adskillelse af et stof med en molekylvægt i området 30.000-70.000, idet elueringsmidlet ved gelfiltreringen er en pufferopløsning med pH 6,0-9,0, for at opnå ovennævnte oprensede proteinlignende fysiologisk aktive stof.the rabbit containing the protein-like physiologically active substance which has not been subjected to ammonium sulfate fractionation is contacted with a basic anion exchanger using a buffer solution having a pH value of 6.0-9.0 and a salt concentration of not more than 5, 2M whereby the physiologically active substance is adsorbed to the anion exchanger, the adsorbed physiologically active substance is eluted from the anion exchanger with a buffer solution having a higher salt concentration, and the eluate containing the physiologically active substance after concentration and possibly dialyzing is subjected to gel filtration with a 10 gel suitable for separating a substance with a molecular weight in the range of 30,000-70,000, the eluent of the gel filtration being a buffer solution of pH 6.0-9.0, to obtain the above purified protein-like physiologically active substance.

For at fremstille det fysiologisk aktive stof ifølge opfindelsen (i 15 det følgende kaldet det fysiologisk aktive stof) injiceres først mindst ét stof, der kan stimulere det reticuloendotheliale system intravenøst eller intraperitonealt i en kanin i henhold til den af Carswell et al. [Proc. Nat. Acad. Sci. USA, bind 72 (nr. 9), side 3666-3670 (1975)] beskrevne metode. Som stoffer, der kan stimulere 20 det reticuloendotheliale system, anvendes sædvanligvis grampositive bakterier, protozoer eller gærarter, der administreres til kaninen i form af enten levende mikroorganismer, døde mikroorganismer (fx efter varmebehandling eller formalinbehandling) eller celleekstrakter fra mikroorganismer. Eksempler på grampositive bakterier omfatter Pro-25 pionibakterLer såsom Propionibacterium aenes (Corynebacterium parvum) eller Propionibacterium granulosum (Corynebacterium granulosum),To prepare the physiologically active substance of the invention (hereinafter referred to as the physiologically active substance), at least one substance that can stimulate the reticuloendothelial system intravenously or intraperitoneally in a rabbit according to that of Carswell et al. [Proc. Night. Acad. Sci. United States, Vol. 72 (No. 9), pages 3666-3670 (1975)]. As substances that can stimulate the reticuloendothelial system, gram-positive bacteria, protozoa or yeasts administered to the rabbit are usually administered in the form of either living microorganisms, dead microorganisms (e.g., after heat or formalin treatment) or cell extracts from microorganisms. Examples of gram-positive bacteria include Propionibacterium clays such as Propionibacterium aenes (Corynebacterium parvum) or Propionibacterium granulosum (Corynebacterium granulosum),

Mycobakterier såsom Bacillus Calmette-Guérin (BCG) eller Mycobacterium smegmatis og Nocardier såsom Nocardia erythropolis eller Nocardia gardneri. Som protozoer anvendes fx Plasmodium eller Toxo-30 plasma. Som gær anvendes sædvanligvis Zymosan, der er ekstraheret fra Saccharomyces cerevisiae eller andre. Der kan også anvendes syntetiske højmolekylære forbindelser såsom pyran-copolymer. 7-14 dage efter administration injiceres endotoxin fra en gramnegativ bakterie, fx et lipopolysaccharid, der fås fra Escherichia coli, 35 Pseudomonas aeruginosa eller Salmonella typhosa intravenøst i kaninen. 1,5-2 timer efter injektionen tages der legemsvæsker (fx asci-tesvæske eller lymfevæske) og/eller serum eller plasma fra kaninen, 5Mycobacteria such as Bacillus Calmette-Guérin (BCG) or Mycobacterium smegmatis and Nocardia such as Nocardia erythropolis or Nocardia gardneri. Plasmodium or Toxo-30 plasma are used as protozoa. As yeast, Zymosan extracted from Saccharomyces cerevisiae or others is usually used. Synthetic high molecular weight compounds such as pyran copolymer can also be used. 7-14 days after administration, endotoxin from a gram-negative bacterium, e.g., a lipopolysaccharide obtained from Escherichia coli, Pseudomonas aeruginosa or Salmonella typhosa, is injected intravenously into the rabbit. 1.5-2 hours after injection, body fluids (eg ascites or lymph fluid) and / or rabbit serum or plasma are taken, 5

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eller indre organer såsom lever, milt, etc. homogeniseres og eks-traheres med fysiologisk saltopløsning. Disse legemsvæsker, serum, plasma og/eller ekstrakter af indre organer kan anvendes som rå opløsninger af det nærværende fysiologisk aktive stof, men sædvanlig-5 vis anvendes serum eller plasma.or internal organs such as liver, spleen, etc. are homogenized and extracted with physiological saline. These bodily fluids, serum, plasma and / or extracts of internal organs can be used as raw solutions of the present physiologically active substance, but usually serum or plasma is used.

Undersøgelse af den fysiologiske virkning af det fysiologisk aktive stof udføres i overensstemmelse med nedenstående metoder.Examination of the physiological effect of the physiologically active substance is carried out according to the following methods.

a) Undersøgelse under anvendelse af L-cellera) Study using L cells

Denne undersøgelse udføres i overensstemmelse med den af Carswell et 10 al. [Proc. Nat. Acad. Sci. USA, bind 72 (nr. 9), side 3666-3670 (1975)] beskrevne metode. Som dyrkningsbeholder anvendes en plade, der fremstilles af Lymbro Chemical Co., Inc., (USA), og L-celler (S) dyrkes i "Eagle's minimal essential medium" (MEM medium) indeholdende ikke-essentielle aminosyrer og 10% varmeinaktiveret føtalt kalve-15 serum, 100 enheder/ml penicillin og 100 jug/ml streptomycin. Lige dele af en L-cellesuspension (1 x 10^ celler) og en seriefortyndet prøve blandes og inkuberes ved 37°C i 48 timer i luft, der indeholder 5% carbondioxid. Virkningen bestemmes ved at afsætte fortyndingen og antallet af levedygtige L-celler på en graf og beregne evnen til at 20 dræbe 50% af L-cellerne ud fra den fortynding, der svarer til 50% cytotoxicitet. Den fysiologiske virkning, der er nødvendig for at dræbe 50% af L-cellerne, defineres som 1 enhed.This study is conducted in accordance with that of Carswell et al. [Proc. Night. Acad. Sci. United States, Vol. 72 (No. 9), pages 3666-3670 (1975)]. As a culture vessel is used a plate manufactured by Lymbro Chemical Co., Inc., (USA), and L cells (S) are grown in "Eagle's minimal essential medium" (MEM medium) containing non-essential amino acids and 10% heat-inactivated fetal calf serum, 100 units / ml penicillin and 100 µg / ml streptomycin. Equal portions of an L-cell suspension (1 x 10 6 cells) and a series-diluted sample are mixed and incubated at 37 ° C for 48 hours in air containing 5% carbon dioxide. The effect is determined by plotting the dilution and the number of viable L cells on a graph and calculating the ability to kill 50% of the L cells from the dilution equivalent to 50% cytotoxicity. The physiological effect needed to kill 50% of the L cells is defined as 1 unit.

b) Undersøgelse under anvendelse af transplanteret Meth A-sarkom i mus 25 I henhold til den af Carswell et al. beskrevne metode (op. cit.) transplanteres 2 x 10-* BALB/c sarkom Meth A-celler intradermalt i armhulen på en (BALB/c x C57BL/6)F^ mus, og 7 dage senere udvælges mus med tumorer med en diameter på 7-8 mm, god vascularisering og ingen spontan central necrose til undersøgelse. En 0,5 ml's prøve 30 fortyndes med fysiologisk saltopløsning og injiceres gennem halevenen. Prøvens virkning bedømmes efter 24 timer i henhold til de nedenstående kriterier.b) Study using transplanted Meth A sarcoma in mice 25 According to that of Carswell et al. described method (op. cit.) 2 x 10- * BALB / c sarcoma Meth A cells are transplanted intradermally into the armpit of a (BALB / cx C57BL / 6) F ^ mouse, and 7 days later, mice with tumors with a diameter are selected at 7-8 mm, good vascularization and no spontaneous central necrosis for examination. A 0.5 ml sample 30 is diluted with physiological saline and injected through the tail vein. The effect of the test is evaluated after 24 hours according to the criteria below.

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(-) : ingen ændring (+) : lille hæmorrhagisk necrose (++) : moderat hæmorrhagisk necrose (central necrose, der breder sig over ca. 50% af tumorens over-5 flade) (+++) : udtalt hæmorrhagisk necrose (omfattende necrose, der efterlader en lille levedygtig rand langs tumorens periferi).(-): no change (+): minor haemorrhagic necrosis (++): moderate haemorrhagic necrosis (central necrosis extending over about 50% of the surface of the tumor) (+++): pronounced haemorrhagic necrosis ( extensive necrosis, leaving a small viable edge along the periphery of the tumor).

I det følgende beskrives fremgangsmåden til rensning ifølge opfin-10 delsen i detaljer.In the following, the process of purification according to the invention is described in detail.

Før det første trin, der består i at bringe stoffet i kontakt med en basisk aniohbytter, kan en rå opløsning af det fysiologisk aktive stof dialyseres mod en pufferopløsning, der skal anvendes på det tidspunkt, hvor stoffet bringes i kontakt med en anionbytter, eller 15 det kan fortyndes med en pufferopløsning med lav saltkoncentration.Prior to the first step of contacting the substance with a basic anion exchanger, a crude solution of the physiologically active substance may be dialyzed against a buffer solution to be used at the time of contact with an anion exchanger, or it can be diluted with a low salt buffer solution.

En rå opløsning af nærværende fysiologisk aktive stof kan bringes i kontakt med en basisk anionbytter ved enten søjlemetoden eller batch -metoden. Dette trin udføres ved at bringe den rå opløsning i kontakt med en basisk anionbytter under anvendelse af en pufferopløsning med 20 en pH-værdi på 6,0-9,0 og en saltkoncentration på 0,2M eller mindre for at få det fysiologisk aktive stof til at adsorbere til anion-bytteren, hvorefter anionbytteren vaskes med den samme pufferopløsning for at fjerne uadsorberede proteiner, hvorefter det fysiologisk aktive stof elueres med en pufferopløsning med en højere saltkon-25 centration (rensningstrin 1).A crude solution of the present physiologically active substance may be contacted with a basic anion exchanger by either the column method or the batch method. This step is accomplished by contacting the crude solution with a basic anion exchanger using a buffer solution having a pH of 6.0-9.0 and a salt concentration of 0.2M or less to obtain the physiologically active substance to adsorb to the anion exchanger, then the anion exchanger is washed with the same buffer solution to remove unadsorbed proteins, and then the physiologically active substance is eluted with a buffer solution of a higher salt concentration (purification step 1).

Typiske eksempler på anvendte basiske anionbyttere omfatter anion-byttere, der indeholder diethylaminogrupper såsom DEAE-Sephadex® A-50, DEAE-Sepharose CL-6B, DEAE-Sephacel (der alle fremstilles af Pharmacia Fine Chemicals AB, Sverige) og AIEC DE 52 (der fremstilles 30 af Whatman Ltd., England), anionbyttere, der indeholder aminoethyl-grupper såsom Servacel AE (der fremstilles af Serva Entwicklungslabor, BRD) og anionbyttere, der indeholder kvatemære aminoe thyl grupper såsom QAE-Sephadex®A-50, (der fremstilles af Pharmacia) og Cel-lex™ QAE (der fremstilles af Bio-Rad Laboratories, USA). De anvendte 7Typical examples of basic anion exchangers used include anion exchangers containing diethylamino groups such as DEAE-Sephadex® A-50, DEAE-Sepharose CL-6B, DEAE-Sephacel (all manufactured by Pharmacia Fine Chemicals AB, Sweden) and AIEC DE 52 ( produced by Whatman Ltd., England), anion exchangers containing aminoethyl groups such as Servacel AE (manufactured by Serva Entwicklungslabor, BRD) and anion exchangers containing quaternary aminoethyl groups such as QAE-Sephadex®A-50 (which manufactured by Pharmacia) and Cel-lex ™ QAE (manufactured by Bio-Rad Laboratories, USA). They used 7

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pufferopløsninger omfatter en fortyndet tris-saltsyrepuffer, en fortyndet phosphatpuffer og lignende. Der tilsættes fortrinsvis natriumchlorid eller kaliumchlorid for at indstille pufferopløsningens saltkoncentration. Indholdet af protein i et eluat bestemmes 5 ved den optiske densitet ved 280 nm. Koncentrationen af det fysiologisk aktive stof måles ved den cytotoxiske virkning mod L-celler som ovenfor beskrevet.buffer solutions include a dilute tris-hydrochloric acid buffer, a dilute phosphate buffer, and the like. Preferably sodium chloride or potassium chloride is added to adjust the salt solution of the buffer solution. The content of protein in an eluate is determined at the optical density at 280 nm. The concentration of the physiologically active substance is measured by the cytotoxic action against L cells as described above.

Selv om det ovennævnte trin kan udføres ved en enkelt kontakt med anionbytterne, foretrækkes det i tilfælde af søjlemetoden somme tider 10 at anvende rechromatografi.Although the above step can be performed by a single contact with the anion exchangers, in the case of the column method, it is sometimes preferable to use rechromatography.

Det i det ovennævnte trin vundne eluat, der indeholder det fysiologisk aktive stof, koncentreres på kendt måde såsom ved ultrafiltrering eller lyofilisering. Det således vundne koncentrat udsættes for gelfiltrering under anvendelse af en gel, der egner sig til adskil-15 lelse af et stof med en molekylvægt på 30.000-70.000 (rensningstrin 2). Som elueringsmiddel anvendes en pufferopløsning med en pH-værdi på 6,0-9,0. Saltkoncentrationen er ikke kritisk, men er fortrinsvis 0,15-2,OM. Gelerne til gelfiltrering omfatter Sephadex G-75, 100, 150 eller 200 (der fremstilles af Pharmacia), Sephacryl S-200 eller 300 20 (der fremstilles af Pharmacia), Bio-Gel P-30, 60, 100, 150 eller 200 (der fremstilles af Bio-Rad), CPG-10 (350 Å, 240 Å, 170 Å eller 120 Å) (der fremstilles af Electro-Nucleonics, Inc., USA) og lignende.The eluate obtained in the above step containing the physiologically active substance is concentrated in known manner such as by ultrafiltration or lyophilization. The concentrate thus obtained is subjected to gel filtration using a gel suitable for separating a substance having a molecular weight of 30,000-70,000 (purification step 2). As the eluent, a buffer solution having a pH of 6.0-9.0 is used. The salt concentration is not critical, but is preferably 0.15-2, OM. The gel filtration gels include Sephadex G-75, 100, 150 or 200 (manufactured by Pharmacia), Sephacryl S-200 or 300 20 (manufactured by Pharmacia), Bio-Gel P-30, 60, 100, 150 or 200 ( manufactured by Bio-Rad), CPG-10 (350 Å, 240 Å, 170 Å or 120 Å) (manufactured by Electro-Nucleonics, Inc., USA) and the like.

Eksempler på pufferopløsninger og salt er de samme som for de ovenfor beskrevne trin, der omfatter kontakt med en anioribytter.Examples of buffer solutions and salt are the same as for the steps described above which include contact with an anion exchanger.

25 De fraktioner, der indeholder det fysiologisk aktive stof, samles og koncentreres på kendt måde såsom ved ultrafiltrering eller lyofilisering. Dialysen af koncentratet af det fysiologisk aktive stof mod en fysiologisk saltopløsning giver en opløsning af stoffet, der er renset ca. 5000-10.000 gange i forhold til serummet eller plasmaet.The fractions containing the physiologically active substance are collected and concentrated in known manner, such as by ultrafiltration or lyophilization. The dialysis of the concentrate of the physiologically active substance against a physiological saline solution gives a solution of the substance which has been purified approx. 5000-10,000 times relative to the serum or plasma.

30 Den samlede virkningsvinding i de to trin i henhold til den undersøgelse, der anvender L-celler, er ca. 65-98¾.30 The overall efficacy gain in the two steps according to the study using L cells is approx. 65-98¾.

Den således fremstillede rensede opløsning af det fysiologisk aktive stof indstilles til en hensigtsmæssig pH-værdi og saltkoncentration ved dialyse eller gelfiltrering, steriliseres ved filtrering, op- 8The purified solution of the physiologically active substance thus prepared is adjusted to a suitable pH and salt concentration by dialysis or gel filtration, sterilized by filtration,

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varmes om nødvendigt, og lyofiliseres, hvorved fås et renset præparat af det fysiologisk aktive stof.if necessary, heated and lyophilized to give a purified preparation of the physiologically active substance.

Det rensede præparat i en mængde på ca. 3000 enheder har vist sig at have (++)-virkning i den undersøgelse, der anvender Meth A-sarkom som 5 ovenfor beskrevet. Det rensede præparat af det fysiologisk aktive stof har også vist sig at have cytotoxisk virkning mod forskellige dyrkede humane cancercellelinjer. Den procentvise cytotoxicitet 48 timer efter administration af 800 enheder af det rensede præparat er vist i tabel 1.The purified preparation in an amount of approx. 3000 units have been found to have (++) effect in the study using Meth A sarcoma as described above. The purified preparation of the physiologically active substance has also been shown to have cytotoxic effect against various cultured human cancer cell lines. The percent cytotoxicity 48 hours after administration of 800 units of the purified preparation is shown in Table 1.

10 Tabel 1Table 1

Cancercellelinjer % cytotoxicitet Medium * PC 10 69,1 a 15 KATO-III 67,9 b MK-7 65,8 aCancer cell lines% cytotoxicity Medium * PC 10 69.1 a 15 KATO-III 67.9 b MK-7 65.8 a

Rca 66,8 c W-2 75,9 a GOTO 61,3 b 20 SEKI 70,0 bRca 66.8 c W-2 75.9 a GOTO 61.3 b 20 SEKI 70.0 b

Kym-1 51,9 d MRK-l-nu 75,1 dKym-1 51.9 d MRK-1-nu 75.1 d

* a : “ 80% RPMI 1640 + 20% FCS* a: “80% RPMI 1640 + 20% FCS

25 b : 40% RPMI 1640 + 40% MEM + 20% FCS25 b: 40% RPMI 1640 + 40% MEM + 20% FCS

c : 80% MEM + 20% FCSc: 80% MEM + 20% FCS

d : 80% DM160 + 20% FCS.d: 80% DM160 + 20% FCS.

På den anden side har det rensede præparat af det fysiologisk aktive stof vist sig ikke at have nogen cytotoxisk virkning mod normale 30 celler såsom dyrkede fibroblastre fra mennesker og mus, selv ved en dosis på 2 x 10^ enheder.On the other hand, the purified preparation of the physiologically active substance has been found to have no cytotoxic effect against normal cells such as cultured human and mouse fibroblasts, even at a dose of 2 x 10 5 units.

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I de tests, hvor det rensede præparat af det fysiologisk aktive stof er blevet administreret til en BALB/c-mus med transplanteret Colon 26-adenocarcinom og en A/Jax-mus med transplanteret Neuro-2a-neuro-blastom, blev der endvidere iagttaget en signifikant vækstinhibering 5 og regression af tumorerne sammenlignet med en kontrolgruppe (en gruppe, til hvilken der administreredes fysiologisk saltopløsning).Furthermore, in the tests in which the purified preparation of the physiologically active substance has been administered to a BALB / c mouse with transplanted Colon 26 adenocarcinoma and an A / Jax mouse with transplanted Neuro-2a neuro-blastoma, a significant growth inhibition 5 and regression of the tumors compared to a control group (a group to which physiological saline was administered).

De udvoksede tumorer er i tilbagegang eller forsvandt uden at forårsage hæmorrhagisk necrose i nogle dyr.The adult tumors are in decline or disappear without causing hemorrhagic necrosis in some animals.

Det rensede præparat af det fysiologisk aktive stof har en fremragen-10 de antitumorvirkning, der ikke er særlig artsspecifik, og det kan anvendes som et antitumormiddel til behandling af maligne tumorer i et pattedyr, herunder et menneske.The purified preparation of the physiologically active substance has an excellent antitumor effect which is not very species specific and can be used as an antitumor agent for the treatment of malignant tumors in a mammal, including a human.

Det rensede præparat af det fysiologisk aktive stof administreres sædvanligvis parenteralt eller topisk i form af en vandig opløsning, 15 hvortil der eventuelt kan sættes et isotonisk middel såsom natriumch-lorid og/eller et pufringsmiddel såsom en phosphat. Den kliniske dosis af det rensede præparat af det fysiologisk aktive stof, der kan variere afhængig af administrationsvejen, lidelserne samt patientens vægt, er sædvanligvis på ca. 10^-10^ enheder pr. administration til 20 en voksen. Det rensede præparat af det fysiologisk aktive stof kan også anvendes sammen med andre antitumormidler såsom cyclophosphamid, mitomycin C, adriamycin og bleomycin.The purified preparation of the physiologically active substance is usually administered parenterally or topically in the form of an aqueous solution to which is optionally added an isotonic agent such as sodium chloride and / or a buffering agent such as a phosphate. The clinical dose of the purified preparation of the physiologically active substance, which may vary depending on the route of administration, the disorders as well as the patient's weight, is usually about 5%. 10 ^ -10 ^ units per. administration to 20 an adult. The purified preparation of the physiologically active substance may also be used with other antitumor agents such as cyclophosphamide, mitomycin C, adriamycin and bleomycin.

Det rensede præparat af det fysiologisk aktive stof, der fås som ovenfor beskrevet, kan yderligere udsættes for de nedenfor beskrevne 25 trin, hvorved det fysiologisk aktive stof kan isoleres: (3) Affinitetschromatografi på immobiliseret Cibacron Blue F3G-A; (4) Gelfiltrering; (5) Affinitetschromatografi på immobiliseret concanavalin A; 30 (6) Præparativ elektroforese på polyacrylamidgel og (7) Gelfiltrering.The purified preparation of the physiologically active substance obtained as described above can be further subjected to the steps described below, whereby the physiologically active substance can be isolated: (3) Affinity chromatography on immobilized Cibacron Blue F3G-A; (4) Gel filtration; (5) Affinity chromatography on immobilized concanavalin A; (6) Preparative electrophoresis on polyacrylamide gel and (7) Gel filtration.

Hvert af disse trin er detaljeret beskrevet nedenfor.Each of these steps is described in detail below.

Rensningstrin 3 10Purification step 3 10

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Det 1 trin 2 vundne koncentrat af det fysiologisk aktive stof udsættes for affinitetschromatografi på immobiliseret Cibacron Blue F3G-A (et farvestof, der fremstilles af Ciba-Geigy Corp.)· Immobili-5 sering af Cibacron Blue F3G-A på et underlag kan udføres på kendt måde som beskrevet af Bohme et al. i J. Chromatogr., bind 69, side 209-214 (1972), eller alternativt kan der anvendes en kommercielt tilgængelig adsorbent [fx Blue Sepharose CL-6B (der fremstilles af Pharmacia), eller Affi-Gel Blue (der fremstilles af Bio-Rad)]. Efter 10 at koncentratet af det fysiologisk aktive stof er blevet dialyseret mod en fortyndet pufferopløsning med en pH-værdi på 7,0-8,0 (fx phosphatpuffer eller tris-saltsyrepuffer), påføres det til det ovennævnte immobiliserede Cibacron Blue F3G-A. Herved adsorberes forurenende proteiner såsom albumin til immobiliseret Cibacron Blue F3G-15 A, og det fysiologisk aktive stof elueres i ikke-adsorberede fraktioner. Aktivitetsudbyttet ved dette trin er ca. 70-95% med en ca. 3 ganges forøgelse i renhed. Det samlede aktivitetsudbytte ved rensningstrinene 1-3 er ca. 56-93% med en forøgelse af renheden på ca.The 1 step 2 concentrate obtained from the physiologically active substance is subjected to affinity chromatography on immobilized Cibacron Blue F3G-A (a dye manufactured by Ciba-Geigy Corp.) · Immobilization of Cibacron Blue F3G-A on a substrate can be performed in known manner as described by Bohme et al. in J. Chromatogr., Vol. 69, pages 209-214 (1972), or alternatively, a commercially available adsorbent may be used [e.g., Blue Sepharose CL-6B (manufactured by Pharmacia), or Affi-Gel Blue (manufactured by Bio -Advice)]. After the concentrate of the physiologically active substance has been dialyzed against a diluted buffer solution having a pH of 7.0-8.0 (e.g., phosphate buffer or tris-hydrochloric acid buffer), it is applied to the aforementioned immobilized Cibacron Blue F3G-A. Hereby, contaminating proteins such as albumin are adsorbed to immobilized Cibacron Blue F3G-15A, and the physiologically active substance is eluted in non-adsorbed fractions. The activity yield at this step is approx. 70-95% with an approx. 3 times increase in purity. The total activity yield at the purification steps 1-3 is approx. 56-93% with an increase in purity of approx.

1,5 x 10^ - 3 x 104 gange.1.5 x 10 ^ - 3 x 104 times.

20 Rensningstrin 420 Purification step 4

De ikke-adsorberede fraktioner i trin 3 koncentreres, og koncentratet · udsættes for gelfiltrering under de samme betingelser som i trin 2.The non-adsorbed fractions in step 3 are concentrated and the concentrate · is subjected to gel filtration under the same conditions as in step 2.

Som underlag for gelfiltrering kan der anvendes Sephadex® G-75, 100, 150 eller 200 (fremstillet af Pharmacia), Bio-gel P-30, 60, 100, 150 25 eller 200 (fremstillet af Bio-Rad). De virksomme fraktioner samles, koncentreres og dialyseres mod en fortyndet phosphatpuffer eller tris-saltsyrepuffer med en pH-værdi på 7,0-8,0. Virkningsvindingen ved dette trin er cirka 70-95% med en cirka 3-4 ganges forøgelse i renhed.As a support for gel filtration, Sephadex® G-75, 100, 150 or 200 (manufactured by Pharmacia), Bio-gel P-30, 60, 100, 150 25 or 200 (manufactured by Bio-Rad) can be used. The active fractions are pooled, concentrated and dialyzed against a diluted phosphate buffer or tris-hydrochloric acid buffer having a pH of 7.0-8.0. The efficiency gain at this stage is about 70-95% with an approximately 3-4 fold increase in purity.

30 Det samlede aktivitetsudbytte ved rensningtrin 1-4 er ca. 47-88% med en forøgelse af renheden påca. 4,5 x 10^ - 9 x 10^ gange.The total activity yield at purification steps 1-4 is approx. 47-88% with an increase in purity onca. 4.5 x 10 ^ - 9 x 10 ^ times.

Rensningstrin 5 11Purification step 5 11

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Den i trin 4 vundne rensede opløsning udsættes derefter for affini-tetschromatografi ved anvendelse af inunobiliseret concanavalin A.The purified solution obtained in step 4 is then subjected to affinity chromatography using ununobilized concanavalin A.

Concanavalin A kan immobiliseres på kendt måde, eller der kan alter-5 nativt anvendes et kommercielt tilgængeligt immobiliseret concanavalin A (fremstillet af Sigma Chemical Co., U.S.A.) eller Con A-Sepha-rose CL-6B (fremstillet af Pharmacia). Den i trin 4 vundne rensede opløsning koncentreres og påføres på immobiliseret concanavalin A under anvendelse af en fortyndet pufferopløsning med en pH-værdi på 10 7,0-8,0 som anvendt i det samme trin, hvorefter, efter at søjlen er blevet vasket grundigt med den samme pufferopløsning, udføres elu-ering med den samme pufferopløsning, der indeholder 0,1M eller mere a-methyl-d-mannosid. Det fysiologisk aktive stof koncentreres i ikke-adsorberede fraktioner. Aktivitetsudbyttet i dette trin er ca. 60-80% 15 med en ca. 2-4 ganges forøgelse i renhed. Det samlede aktivitetsudbytte ved rensningstrin 1-5 er ca. 33-70% med en forøgelse af renheden på ca. 9,0 x 10^ - 3,6 x 10gange.Concanavalin A may be immobilized in known manner or alternatively a commercially available immobilized concanavalin A (manufactured by Sigma Chemical Co., U.S.A.) or Con A-Sepha-rose CL-6B (manufactured by Pharmacia) may be used. The purified solution obtained in step 4 is concentrated and applied to immobilized concanavalin A using a diluted buffer solution having a pH of 7.0 7.0-8.0 as used in the same step, after which the column has been thoroughly washed. with the same buffer solution, elution is performed with the same buffer solution containing 0.1M or more α-methyl-d-mannoside. The physiologically active substance is concentrated in non-adsorbed fractions. The activity yield in this step is approx. 60-80% 15 with an approx. 2-4 times increase in purity. The total activity yield at purification steps 1-5 is approx. 33-70% with an increase in purity of approx. 9.0 x 10 ^ - 3.6 x 10 times.

Rensningstrin 6Purification step 6

Den i trin 5 vundne opløsning, der indeholder det fysiologisk aktive 20 stof, koncentreres og udsættes for polyacrylamid-pladeelektroforese. Koncentratet af det fysiologisk aktive stof påføres på en 8% polya-crylamidgel, der fremstilles under anvendelse af et pladeelektrofore-seapparat model 221 (280 x 140 x 1,5 mm), der fremstilles af Bio-Rad Laboratories. Elektroforesen udføres ved en strømstyrke på ca. 70-100 25 mA. Efter migration skæres gelen i strimler, der hver har en bredde på 3 mm, og hver gelstrimmel ekstraheres med en fortyndet pufferopløsning med en pH-værdi på 7,0-8,0, der indeholder 1,0M natriumch-lorid, og de virksomme fraktioner samles og koncentreres. Aktivitets-udbyttet ved dette trin er ca. 5-10% med en ca. 15-20 ganges for-30 øgelse i renhed. Det samlede aktivitetsudbytte ved rensningstrin 1-6 er ca. 2,5-7,0% med en forøgelse af renheden på ca. 1,4 x 10® - 5,4 x 10® gange.The solution obtained in step 5 containing the physiologically active substance is concentrated and subjected to polyacrylamide plate electrophoresis. The concentrate of the physiologically active substance is applied to an 8% polyacrylamide gel prepared using a plate electrophoresis model 221 (280 x 140 x 1.5 mm) manufactured by Bio-Rad Laboratories. The electrophoresis is carried out at a current of approx. 70-100 25 mA. After migration, the gel is cut into strips each 3 mm wide and each gel strip extracted with a diluted buffer solution of pH 7.0-8.0 containing 1.0M sodium chloride and the active fractions are collected and concentrated. The activity yield at this step is approx. 5-10% with an approx. 15-20 times increase in purity. The total activity yield at purification steps 1-6 is approx. 2.5-7.0% with an increase in purity of approx. 1.4 x 10® - 5.4 x 10® times.

Rensningstrin 7 12Cleaning step 7 12

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Ved gelfiltrering i dette trin kan der anvendes den samme puffer, salt og gel, som der anvendes i trin 2. Men længden og diameteren af den søjle, der skal anvendes i dette trin, er henholdsvis længere og 5 mindre, end det er tilfældet med den søjle, der anvendes i trin 2. De virksomme fraktioner samles, koncentreres, dialyseres, steriliseres ved filtrering og lyofiliseres om nødvendigt, hvorved fås det hidtil ukendte fysiologisk aktive stof ifølge opfindelsen. Aktivitetsudbyttet ved dette trin er ca. 50-80% med en ca. 1,5-2,0 ganges for-10 øgelse i renhed. Det samlede aktivitetsudbytte ved rensningstrin 1-7 er ca. 1,6-5,6% med en forøgelse af renheden på ca. 2,8 x 10® - 8,1 x 10® gange.In gel filtration in this step, the same buffer, salt and gel as used in step 2. can be used. However, the length and diameter of the column to be used in this step are longer and 5, respectively, than is the case with The column used in step 2. The active fractions are collected, concentrated, dialyzed, sterilized by filtration and lyophilized if necessary to give the novel physiologically active substance of the invention. The activity yield at this step is approx. 50-80% with an approx. 1.5-2.0 times increase in purity. The total activity yield at purification steps 1-7 is approx. 1.6-5.6% with an increase in purity of approx. 2.8 x 10® - 8.1 x 10® times.

Det således fremstillede fysiologisk aktive stofs karakteristika blev målt, hvilket gav de nedenfor anførte resultater: 15 a) Molekylvægt 39.000 ±5.000 (ved SDS-polyacrylamidgelelektroforese og gelfiltrering) b) Isoelektrisk punkt pi 3,9 ± 0,3 20 c) Celluloseacetatelektroforese-mobilitet ΙΟ-4 - 10"6 cm2/V*sek.The characteristics of the physiologically active substance thus prepared were measured to give the results given below: 15 a) Molecular weight 39,000 ± 5,000 (by SDS-polyacrylamide gel electrophoresis and gel filtration) b) Isoelectric point pi 3.9 ± 0.3 20 c) Cellulose acetate electrophoresis mobility ΙΟ-4 - 10 "6 cm2 / V * sec.

d) Specifik aktivitet ved undersøgelse, hvor der anvendes L-celler mindst 0,5 x 10^ ehheder/mg protein.d) Specific activity of study using L cells at least 0.5 x 10 6 units / mg protein.

2 x 105 Meth A-sarkomceller blev tranplanteret intradermalt i arm-25 hulen på (BALB/c x C57BL/6)F^-mus og lades formere sig tilstrækkeligt til at danne faste tumorer, hvorefter det fysiologisk aktive stof blev administreret intravenøst (i en dosis, der svarede til 0,1-1 ng protein pr. mus), hvorved der viste sig (+) virkninger eller mere.2 x 10 5 Meth A sarcoma cells were transplanted intradermally into the armhole of (BALB / cx C57BL / 6) F 2 mice and allowed to proliferate sufficiently to form solid tumors after which the physiologically active substance was administered intravenously (in a dose equal to 0.1-1 ng of protein per mouse), showing (+) effects or more.

Af de ovennævnte karakteristika blev a) - c) målt i henhold til de 30 nedenstående metoder.Of the above characteristics, a) - c) were measured according to the 30 methods below.

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a) Bestemmelse af molekylvægt (i) I henhold til den af Segrest et al. [Methods in Enzymology, bind 28-B, s. 54-63 (1972)] påføres 5 pg af en prøve på SDS (natriumdode-cylsulfat)-polyacrylamidgel, og elektroforese foretages i SDS/ Tris- 5 glycinpuffer (pH-værdi 8,3). Kalibrering af molekylvægten udføres ved hjælp af et standardmolekylvægtudstyr (der fremstilles af Pharmacia).a) Determination of Molecular Weight (i) According to that of Segrest et al. [Methods in Enzymology, Vol. 28-B, pp. 54-63 (1972)] is applied to 5 µg of a sample on SDS (sodium dodecyl sulfate) polyacrylamide gel and electrophoresis in SDS / Trisglycine buffer (pH 8 , 3). Calibration of the molecular weight is carried out using a standard molecular weight equipment (manufactured by Pharmacia).

(ii) Ved at anvende en søjle (0,9 x 120 cm) af Sephadex® G-200 (der fremstilles af Pharmacia) foretages gelfiltrering med en pufferopløsning af 0,7M natriumchlorid/O,02M Tris-saltsyrepuffer (pH-værdi 10 7,8), hvor kalibrering af molekylvægten udføres ved anvendelse af standard-proteiner (ribonuclease A, chymotrypsinogen A, ovalbumin, aldolase, fremstillet af Pharmacia).(ii) Using a column (0.9 x 120 cm) of Sephadex® G-200 (manufactured by Pharmacia), gel filtration is performed with a buffer solution of 0.7M sodium chloride / 0.02M Tris-hydrochloric acid buffer (pH 10 7.8), wherein molecular weight calibration is performed using standard proteins (ribonuclease A, chymotrypsinogen A, ovalbumin, aldolase, manufactured by Pharmacia).

b) Bestemmelse af det isolektriske punktb) Determination of the isolectric point

Det isoelektriske punkt bestemmes ved at anvende apparatet for iso-15 elektrisk elektroforese, Ampholine (pi område 2,5-4,5), og 36% Ultro-dex (der alle er fremstillet af LKB Productor AB, Sverige). Dannelsen af pi-gradient udføres ved 340 V og 23 mA i 5-7 timer, hvorefter prøven påføres på pladen. Migration udføres ved op til 660 V og 120 mA i 5-7 timer. Strimler med en bredde på 1 mm fremstilles og eks-20 traheres med fysiologisk saltopløsning, og cytotoxisk virkning mod L-celler måles.The isoelectric point is determined by using the apparatus for iso-electric electrophoresis, Ampholine (in the range 2.5-4.5), and 36% Ultrodex (all manufactured by LKB Productor AB, Sweden). The formation of pi gradient is carried out at 340 V and 23 mA for 5-7 hours, after which the sample is applied to the plate. Migration is performed at up to 660 V and 120 mA for 5-7 hours. Strips 1 mm wide are prepared and extracted with physiological saline, and cytotoxic action against L cells is measured.

c) Mobilitet i elektroforesec) Mobility in electrophoresis

Under anvendelse af Separax-S (fremstillet af Fuji Photo Film Co.,Using Separax-S (manufactured by Fuji Photo Film Co.,

Ltd., Japan) som celluloseacetatmembran udføres elektroforese ved en 25 pH-værdi på 8,6 og en ionisk styrke på 0,06-0,07. Efter fuldførelse af migrationen fremstilles strimler med en bredde på 1 mm, der eks-traheres med fysiologisk saltopløsning og undersøges for cytotoxisk virkning mod L-celler for at bestemme mobiliteten.Ltd., Japan) as cellulose acetate membrane is electrophoresed at a pH of 8.6 and an ionic strength of 0.06-0.07. After completion of the migration, strips of 1 mm width are extracted, extracted with physiological saline solution, and examined for cytotoxic action against L cells to determine mobility.

Det fysiologisk aktive stof testes for cytotoxisk virkning mod for-30 skellige dyrkede humane cancercellelinjer. I tabel 2 vises resulta- 14The physiologically active substance is tested for cytotoxic action against various cultured human cancer cell lines. Table 2 shows results 14

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terne udtrykt i den tilsvarende proteinmængde, der er nødvendig for 50% cytotoxitet efter 48 timer.tes expressed in the corresponding amount of protein required for 50% cytotoxicity after 48 hours.

Tabel 2Table 2

Den nødvendige mængde 5 Cancercellelinjer for 50% cytotoxicitet (pg) Medium* PC 10 5 x 102 a KATO-III 9 x 102 b MK-7 9 x 102 a 10 Rca 6 x 102 c W-2 8 x 102 a GOTO 1 x 103 b SEKI 3 x 102 bRequired amount of 5 Cancer cell lines for 50% cytotoxicity (pg) Medium * PC 10 5 x 102 a CATO-III 9 x 102 b MK-7 9 x 102 a 10 Rca 6 x 102 c W-2 8 x 102 a GOTO 1 x 103 b SEKI 3 x 102 b

Kym-1 1 x 103 d 15 MRK-l-nu 7 x 102 dKym-1 1 x 103 d 15 MRK-l-nu 7 x 102 d

* a: 80% RPMI 1640 + 20% FCS* a: 80% RPMI 1640 + 20% FCS

b: 40% RPMI 1640 + 40% MEM + 20% FCSb: 40% RPMI 1640 + 40% MEM + 20% FCS

c: 80% MEM + 20% FCSc: 80% MEM + 20% FCS

20 d: 80% DM160 + 20% FCS20 d: 80% DM160 + 20% FCS

Den foreliggende opfindelse beskrives nærmere ved nedenstående eksempler.The present invention is further described by the following examples.

Eksempel 1Example 1

Hunkaniner med en vægt på 2,5-3 kg injiceredes med 50 mg formalin-25 dræbt Propionibacterium aenes (Corynebacterium parvum; Wellcome Research Laboratories, England) gennem ørevenen. 8 dage senere, injiceredes 100 pg endotoxin (lipopolysaccharid fra Escherichia coli 026:B6, fremstillet af Difco Laboratories, U.S.A.) igen gennem ørevenen, og 2 timer senere indsamledes blod fra hjertet. Det indsamlede 30 blod blev centrifugeret ved 5.000 rpm i 30 minutter for at fjerne blodceller og uopløselige faste stoffer. Fra 20 kaniner fik man 1200 ml serum med en aktivitet på 12.800 eriheder/ml.Female rabbits weighing 2.5-3 kg were injected with 50 mg of formalin-killed Propionibacterium aenes (Corynebacterium parvum; Wellcome Research Laboratories, England) through the ear vein. 8 days later, 100 µg of endotoxin (lipopolysaccharide from Escherichia coli 026: B6, manufactured by Difco Laboratories, U.S.A.) was re-injected through the ear vein and 2 hours later blood was collected from the heart. The collected blood was centrifuged at 5,000 rpm for 30 minutes to remove blood cells and insoluble solids. From 20 rabbits 1200 ml of serum were obtained with an activity of 12,800 units / ml.

1515

DK 158230 BDK 158230 B

Serummet blev fortyndet med 600 ml 0,02M Tris-saltsyrepuffer (pH-værdi 7,8) og påførtes langsomt på en søjle (6 x 36 cm) af DEAE-Sepharose CL-6B (Pharmacia) ækvilibreret med 0,02M Tris-saltsyrepuf-fer (pH-værdi 7,8), der indeholdt 0,1M natriumchlorid. Efter vaskning 5 af søjlen med 1000 ml ækvilibrerende puffer (0,02M Tris-saltsyrepuffer , pH-værdi 7,8, der indeholder 0,1M natriumchlorid), udførtes eluering med den NaCl-lineære gradient, der dannes ved en gradientblander, under anvendelse af 1,5 liter af 0,02M Tris-saltsyrepuffer (pH-værdi 7,8), der indeholder 0,1M natriumchlorid og 1,5 liter 0,02M 10 Tris-saltsyrepuffer (pH-værdi 7,8), der indeholder 0,3M natriumchlorid. Strømningshastigheden var 60 ml i timen, og der indsamledes fraktioner på hver 18 ml. De virksomme fraktioner blev samlet og koncentreret. Aktivitetsudbyttet ved dette trin var 92% med en 150 ganges forøgelse i renhed.The serum was diluted with 600 ml of 0.02M Tris hydrochloric acid buffer (pH 7.8) and slowly applied to a column (6 x 36 cm) of DEAE-Sepharose CL-6B (Pharmacia) equilibrated with 0.02M Tris hydrochloric acid buffer -fer (pH 7.8) containing 0.1M sodium chloride. After washing the column with 1000 ml equilibrating buffer (0.02M Tris-hydrochloric acid buffer, pH 7.8 containing 0.1M sodium chloride), elution was carried out with the NaCl linear gradient formed by a gradient mixer, using of 1.5 liters of 0.02M Tris hydrochloric acid buffer (pH 7.8) containing 0.1M sodium chloride and 1.5 liters of 0.02M 10 Tris hydrochloric acid buffer (pH 7.8) containing 0.3M sodium chloride. The flow rate was 60 ml per hour and fractions were collected every 18 ml. The active fractions were pooled and concentrated. The activity yield at this stage was 92% with a 150-fold increase in purity.

15 Derefter blev koncentratet dialyseret natten over med 0.005M phosp-hatpuffer (pH-værdi 7,4), der indeholdt 0,15M natriumchlorid, og gel-filtreret. En søjle (5 x 80 cm) af Sephacryl S-200 (Pharmacia) blev tilstrækkeligt ækvilibreret med den samme puffer, og koncentratet blev påført på søjlen, og elueringen blev udført med den samme 20 puffer. Strømningshastigheden var 60 ml i timen, og der indsamledes fraktioner på hver 10 ml. De vundne virksomme fraktioner blev koncentreret umiddelbart efter albuminfraktionen ved ultrafiltrering, hvilket gav et renset præparat af det fysiologisk aktive stof. I dette trin var aktivitetsudbyttet 92% med en 52 ganges forøgelse i 25 renhed. Den samlede aktivitetsvinding ved alle trinene var 85%, idet renheden var 7.800 gange så stor. Det rensede præparat af det fysiologisk aktive stof viste sig at have en specifik aktivitet på ca.Then, the concentrate was dialyzed overnight with 0.005M phosp hat buffer (pH 7.4) containing 0.15M sodium chloride and gel-filtered. A column (5 x 80 cm) of Sephacryl S-200 (Pharmacia) was sufficiently equilibrated with the same buffer and the concentrate was applied to the column and elution was performed with the same 20 buffer. The flow rate was 60 ml per hour and fractions were collected every 10 ml. The recovered active fractions were concentrated immediately after the albumin fraction by ultrafiltration to give a purified preparation of the physiologically active substance. In this step, the activity yield was 92% with a 52 fold increase in purity. The overall activity gain at all the stages was 85%, with the purity being 7,800 times that. The purified preparation of the physiologically active substance was found to have a specific activity of approx.

1,4 x 10^ eriheder/mg protein.1.4 x 10 5 erih / mg protein.

Fig. 1 og fig. 2 viser mønstrene af henholdsvis chromatografi på en 30 DEAE-Sepharose CL-6B-søjle i første trin og gelfiltrering på en Sephacryl S-200-søjle i det andet trin.FIG. 1 and FIG. 2 shows the patterns of chromatography respectively on a 30 step DEAE-Sepharose CL-6B column and gel filtration on a Sephacryl S-200 column in the second step.

DK 158230BDK 158230B

1616

Eksempel 2Example 2

Efter at 2.200 ml af et kaninserum, der indeholdt det fysiologisk aktive stof, var blevet fortyndet med 1.200 ml 0,02M Tris-saltsyre-puffer (pH-værdi 7,2), blev den resulterende opløsning langsomt 5 påført på en søjle (8 x 26 cm) af DEAE-Sepharose CL-6B ækvilibreret med 0,02M Tris-saltsyrepuffer (pH-værdi 7,2), der indeholdt 0,1M natriumchlorid. Derefter blev søjlen vasket med 1000 ml 0,02M Tris-saltsyrepuffer (pH-værdi 7,2), der indeholdt 0,13M natriumchlorid, og elueringen blev foretaget med NaCl-lineær gradient, der dannes med en 10 gradientblander, tinder anvendelse af 2,0 liter 0,02M Tris-saltsyre-puffer (pH-værdi 7,2), der indeholdt 0,15M natriumchlorid, og 2,0 liter 0,02M Tris-saltsyrepuffer (pH-værdi 7,2), der indeholdt 0,3M natriumchlorid. Strømningshastigheden var 90 ml i timen, og der indsamledes fraktioner på hver 18 ml.After 2,200 ml of a rabbit serum containing the physiologically active substance was diluted with 1,200 ml of 0.02M Tris-hydrochloric acid buffer (pH 7.2), the resulting solution was slowly applied to a column (8 x 26 cm) of DEAE-Sepharose CL-6B equilibrated with 0.02M Tris-hydrochloric acid buffer (pH 7.2) containing 0.1M sodium chloride. Then, the column was washed with 1000 ml of 0.02M Tris-hydrochloric acid buffer (pH 7.2) containing 0.13M sodium chloride, and the elution was done with NaCl linear gradient formed with a 10-gradient mixer, using 0 liter 0.02M Tris hydrochloric acid buffer (pH 7.2) containing 0.15M sodium chloride and 2.0 liter 0.02M Tris hydrochloric acid buffer (pH 7.2) containing 0 , 3M sodium chloride. The flow rate was 90 ml per hour and fractions were collected every 18 ml.

15 De samlede virksomme fraktioner blev dialyseret mod 0,02M Tris-saltsyrepuffer (pH-værdi 7,2), der indeholdt 0,1M natriumchlorid, og udsat for rechromatografi på en søjle (2,5 x 30 cm) af DEAE-Sepharose CL-6B ækvilibreret med den samme puffer. Det adsorberede fysiologisk aktive stof blev elueret med den NaCl-lineære gradient, der dannedes 20 med en gradientblander, under anvendelse af 350 ml ækvilibrerende puffer og 350 ml 0,02M Tris-saltsyrepuffer (pH-værdi), der indeholdt 0,3M natriumchlorid. Strømningshastigheden var 25 ml i timen, og der indsamledes fraktioner på hver 7 ml. De virksomme fraktioner blev samlet og koncentreret.The total effective fractions were dialyzed against 0.02M Tris hydrochloric acid buffer (pH 7.2) containing 0.1M sodium chloride and subjected to rechromatography on a column (2.5 x 30 cm) of DEAE-Sepharose CL -6B equilibrated with the same buffer. The adsorbed physiologically active substance was eluted with the NaCl linear gradient formed 20 with a gradient mixer using 350 ml equilibrating buffer and 350 ml 0.02M Tris hydrochloric acid buffer (pH) containing 0.3M sodium chloride. The flow rate was 25 ml per hour and fractions were collected every 7 ml. The active fractions were pooled and concentrated.

25 I næste trin påførtes koncentratet på en søjle af Sephacryl S-200 ækvilibreret med 0,01M Tris-saltsyrepuffer (pH-værdi 7,8), der indeholdt 1,0M natriumchlorid, og elueringen foretoges med den samme puffer. De virksomme fraktioner blev indsamlede og lyofiliseret, hvilket gav et renset præparat af det fysiologisk aktive stof med en 30 renhed, der var 10.000 gange højere en serummets. Aktivitetsudbyttet ved alle trinene var 80%.In the next step, the concentrate was applied to a column of Sephacryl S-200 equilibrated with 0.01M Tris-hydrochloric acid buffer (pH 7.8) containing 1.0M sodium chloride and eluted with the same buffer. The active fractions were collected and lyophilized to give a purified preparation of the physiologically active substance with a purity 10,000 times higher than that of the serum. The activity yield at all steps was 80%.

DK 158230 BDK 158230 B

Eksempel 3 17Example 3 17

Det i eksempel 1 vundne rensede koncentrat blev dialyseret mod 0,02M phosphatpuffer (pH-værdi 7,1) natten over og påført på en søjle (0,7 x 10 cm) af Blue Sepharose CL-6B ækvilibreret med den samme 5 puffer. Søjlen blev grundigt vasket med den samme puffer, og de adsorberede stoffer blev elueret med 50 ml 0,0211 phosphatpuffer (pH-værdi 7,1), der indeholdt 1,5M natriumchlorid. Strømningshastigheden var 2,5 ml i timen, og der indsamledes fraktioner på hver 3 ml. Der iagttoges ingen virkning i de adsorberede fraktioner, men alle virk-10 ninger blev genvundet i de ikke-adsorberede fraktioner. De virksomme fraktioner blev samlet og lyofiliseret. I dette trin var aktivitets-udbyttet 95% med en 3 ganges forøgelse i renhed. De samlede værdier ved alle trinene var 81% for aktivitetsudbyttet og 2,3 x 10^ gange for renhed.The purified concentrate obtained in Example 1 was dialyzed against 0.02 M phosphate buffer (pH 7.1) overnight and applied to a column (0.7 x 10 cm) of Blue Sepharose CL-6B equilibrated with the same 5 buffer. The column was thoroughly washed with the same buffer and the adsorbed was eluted with 50 ml of 0.0211 phosphate buffer (pH 7.1) containing 1.5M sodium chloride. The flow rate was 2.5 ml per hour and fractions were collected every 3 ml. No effect was observed in the adsorbed fractions, but all effects were recovered in the non-adsorbed fractions. The active fractions were pooled and lyophilized. In this step, the activity yield was 95% with a 3-fold increase in purity. The total values at all the steps were 81% for the activity yield and 2.3 x 10 ^ times for purity.

15 X næste trin opløstes det lyofiliserede produkt i 1 ml 0,02M Tris- saltsyrepuffer (pH-værdi 7,8), der indeholdt 0,7M natriumchlorid, og påførtes en søjle (2,6 x 95 cm) af Sephadex® G-75 (Pharmacia) ækvilibreret med den samme puffer. Strømningshastigheden var 20 ml i timen, og der indsamledes fraktioner på hver 10 ml. De virksomme fraktioner 20 blev samlet og koncentreret. I dette trin var aktivitetsudbyttet 95% med 3 ganges forøgelse i renhed. De samlede værdier ved alle trinene var 77% for aktivitetsudbyttet og 6,9 x 10^ gange for renhed.In the next X step, the lyophilized product was dissolved in 1 ml of 0.02 M Tris-hydrochloric acid buffer (pH 7.8) containing 0.7 M sodium chloride and applied to a column (2.6 x 95 cm) of Sephadex® G 75 (Pharmacia) equilibrated with the same buffer. The flow rate was 20 ml per hour and fractions were collected every 10 ml. The active fractions 20 were combined and concentrated. In this step, the activity yield was 95% with a 3-fold increase in purity. The total values at all the steps were 77% for the activity yield and 6.9 x 10 ^ times for purity.

Koncentratet blev derefter dialyseret mod 0,02M phosphatpuffer (pH-værdi 7,2), der indeholdt 4M magnesiumchlorid, og påførtes en søjle 25 (0,3 x 10 cm) af concanavalin A-Sepharose CL-6B (Pharmacia), ækvi libreret med den samme puffer, ved en strømningshastighed på 2,5 ml i timen. Efter at søjlen var blevet vasket grundigt med den samme puffer, udførtes elueringen med phosphatpufferen, der indholdt 0,1M a-methyl-d-mannosid. Virkningen blev genvundet i de ikke-adsorberede 30 fraktioner, og der iagttoges ingen virkning i de adsorberede fraktioner. De ikke-adsorberede fraktioner blev koncentreret og dialyseret mod phosphatpufferen, der indeholdt 0,15M natriumchlorid. I dette trin var aktivitetsudbyttet 70% med 2 ganges forøgelse i renhed. Det samlede aktivitetsudbytte var 54% med en forøgelse af renheden på 35 1,4 x 105 gange.The concentrate was then dialyzed against 0.02M phosphate buffer (pH 7.2) containing 4M magnesium chloride and applied to a column 25 (0.3 x 10 cm) of concanavalin A-Sepharose CL-6B (Pharmacia), equilibrated with the same buffer, at a flow rate of 2.5 ml per hour. After the column was thoroughly washed with the same buffer, elution was performed with the phosphate buffer containing 0.1M α-methyl-d-mannoside. The effect was recovered in the non-adsorbed fractions and no effect was observed in the adsorbed fractions. The non-adsorbed fractions were concentrated and dialyzed against the phosphate buffer containing 0.15M sodium chloride. In this step, the activity yield was 70% with a 2-fold increase in purity. The overall activity yield was 54% with an increase in purity of 35 1.4 x 105 times.

Claims (3)

1. Fremgangsmåde til rensning af et proteinlignende fysiologisk 25 aktivt stof med antitumorvirkning, der fremstilles ved til en kanin at administrere mindst ét stof, der kan stimulere det reticuloen-dotheliale system, og derefter injicere endotoxin fra en gram-negativ bakterie i kaninen, kendetegnet ved, at 30 (1) en legemsvæske, et serum, plasma eller ekstrakt af indre organer fra kaninen indeholdende det proteinlignende fysiologisk aktive stof, som ikke har været underkastet ammoniumsulfatfraktionering, bringes i kontakt med en basisk anioribytter under anvendelse af en pufferopløsning med en pH-værdi på 6,0-9,0 og en saltkoncentration på ikke mere DK 158230B end 0,2M, hvorved det fysiologisk aktive stof adsorberes til anion-bytteren, (2) det adsorberede fysiologisk aktive stof elueres fra anionbytteren med en pufferopløsning, der har en højere saltkoncentration, og 5 (3) eluatet, der indeholder det fysiologisk aktive stof, efter kon centrering og eventuelt dialysering udsættes for gelfiltrering med en gel, der egner sig til adskillelse af et stof med en molekylvægt i området på 30.000-70.000, idet elueringsmidlet ved gelfiltreringen er en pufferopløsning med en pH-værdi på 6,0-9,0, for at opnå ovennævnte 10 oprensede proteinlignende fysiologisk aktive stof.A method of purifying a protein-like physiologically active substance with antitumor action produced by administering to a rabbit at least one substance capable of stimulating the reticuloene-dothelial system and then injecting endotoxin from a gram-negative bacterium into the rabbit, characterized by (30) contacting a body fluid, serum, plasma, or extract of rabbit internal organs containing the protein-like physiologically active substance which has not been subjected to ammonium sulfate fractionation using a basic anionic exchanger using a buffer solution having a pH value of 6.0-9.0 and a salt concentration of no more DK 158230B than 0.2M, whereby the physiologically active substance is adsorbed to the anion exchanger, (2) the adsorbed physiologically active substance is eluted from the anion exchanger with a buffer solution which have a higher salt concentration, and 5 (3) the eluate containing the physiologically active substance, after concentration and possibly dialysis ng is subjected to gel filtration with a gel suitable for separation of a substance with a molecular weight in the range of 30,000-70,000, the eluent of the gel filtration being a buffer solution having a pH of 6.0-9.0 to obtain the above 10 purified protein-like physiologically active substance. 2. Fremgangsmåde ifølge krav 1, kendetegnet ved, at saltkoncentrationen af elueringsmidlet ved gelfiltreringen er 0.15-2M.Process according to claim 1, characterized in that the salt concentration of the eluent in the gel filtration is 0.15-2M. 3. Proteinlignende fysiologisk aktivt stof med antitumorvirkning, der 15 fremstilles ved til en kanin at administrere mindst ét stof, der kan stimulere det reticuloendotheliale system, og derefter injicere endotoxin fra en gram-negativ bakterie i kaninen og er renset ved fremgangsmåden ifølge krav 1 eller 2, kendetegnet ved, at det har følgende egenskaber: 20 a) Molekylvægt: 39.000 ± 5.000; b) Isoelektrisk punkt: pi 3,9 ± 0,3; c) Mobilitet i celluloseacetatelektroforese (pH-værdi 8,6): 10'^ - 10”® cm^/V»sek; d) Specifik aktivitet ifølge den i beskrivelsen definerede bi- 25 ologiske undersøgelse, der anvender L-celler: mindst 0,5 x 10^ enheder/mg protein, og e) Virkning ifølge den i beskrivelsen definerede biologiske undersøgelse, der anvender transplanteret Meth A-sarkom i (BALB/c x C57BL/6)F^-mus administreret intravenøst i en dosis, 30 der svarer til 0,1-1 ng protein pr. mus: (+) eller højere.An anti-tumor protein-like physiologically active substance prepared by administering to a rabbit at least one substance capable of stimulating the reticuloendothelial system, and then injecting endotoxin from a gram-negative bacterium into the rabbit and purified by the method of claim 1 or 2, characterized in that it has the following properties: a) Molecular weight: 39,000 ± 5,000; b) Isoelectric point: pi 3.9 ± 0.3; c) Mobility in cellulose acetate electrophoresis (pH 8.6): 10 '- 10 ”” cm cm / V »sec d) Specific activity according to the biological study defined in the specification using L cells: at least 0.5 x 10 5 units / mg protein, and e) Effect according to the biological study defined in the specification using transplanted Meth A sarcoma in (BALB / cx C57BL / 6) F 1 mice administered intravenously at a dose equal to 0.1-1 ng protein mice: (+) or higher.