EP0153357A1 - Process for producing human interleukine 1 and related drugs - Google Patents

Process for producing human interleukine 1 and related drugs

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Publication number
EP0153357A1
EP0153357A1 EP19840903069 EP84903069A EP0153357A1 EP 0153357 A1 EP0153357 A1 EP 0153357A1 EP 19840903069 EP19840903069 EP 19840903069 EP 84903069 A EP84903069 A EP 84903069A EP 0153357 A1 EP0153357 A1 EP 0153357A1
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EP
European Patent Office
Prior art keywords
interieukin
liquid phase
cells
line
concentration
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP19840903069
Other languages
German (de)
French (fr)
Inventor
Hiro Wakasugi
Françoise HAREL-BELLAN
Marie-Christine Dokhelar
Thomas Tursz
Didier Fradelizi
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Institut National de la Sante et de la Recherche Medicale INSERM
Original Assignee
Institut National de la Sante et de la Recherche Medicale INSERM
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Application filed by Institut National de la Sante et de la Recherche Medicale INSERM filed Critical Institut National de la Sante et de la Recherche Medicale INSERM
Publication of EP0153357A1 publication Critical patent/EP0153357A1/en
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/545—IL-1
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to a process for the production of human interieukin 1; it also relates to medicaments or medicinal compositions the active substance of which comprises human interieukin 1.
  • Human interieukin 1 is a protein hormone of fundamental importance in triggering the immune response; its molecular weight is around 20,000 to 25,000 and it is represented by three biochemical forms, the isoelectric points of which are respectively at pHs of 5.7, 6, 6 and 7.
  • Interieukin 1 constitutes one of the two signals - the other being provided by the antigen or the mitogen - which trigger the secretion, by T lymphocytes, * of 1 interieukin 2 which amplifies the effector phase of immunity by causing the multiplication of lymphocytes your T Helper and T cytotoxic.
  • monocytes normal peripheral blood by using as an inducer muramyl dipeptide.
  • the object of the invention is therefore, above all, to remedy Dier * these disadvantages and provide pro ⁇ means to allow the manufacture of human interieukin 1 as pure as possible and in quantities to consider its application as a drug.
  • the inventors had the merit of finding that it was possible to unexpectedly induce the secretion of very pure interieukin 1, that is to say unaccompanied by the factors listed above, from human leukemic lines, lymphomatous, of hematopoietic and lymphoid origin, which do not normally secrete interieukin 1, these lines being established in continuous culture, preferably in liquid medium; these lines include in particular those identified by the codes HL-60 and K562; the inducer is an agent of the group comprising, on the one hand, those which have the properties of induction of cell differentiation, in particular the phorbols and the phorbol-esters, in particular 12-0-tetra- decaroyl-phorbol-15-acetate (or TPA) and, on the other hand, those belonging to other families such as that of teleocydine.
  • TPA 12-0-tetra- decaroyl-phorbol-15-acetate
  • an effective concentration is established in the liquid phase, that is to say sufficient to obtain an industrially exploitable production of interieukin, in cells belonging to at least one of the human leukemia lines defined above,
  • the secretion of interieukin 1 is induced by the cells in question by introducing into the medium an effective amount, that is to say sufficient to trigger the secretion of interieukin, by at least one inductor chosen from the group also defined above,
  • the interieukin 1 is separated from the medium, that is to say from the liquid phase, and
  • the liquid phase consists of the culture medium in which the cells of the selected human leukemia line are normally propagated; this medium can comprise approximately 10% of fetal calf serum and approximately 90% of the composition designated by the code RPMI-1640 and the constituents of which are indicated in the publication "In vitro" 9, 6 (1974).
  • the line selected is that known under the code HL-60 and the concentration of the liquid phase in constituent cells of this line is from 10 5 to 10 8 cells / ml, preferably around 4.10 6 cells per ml of liquid phase.
  • the line selected is that known under the code K562 and the concentration of the liquid phase in constituent cells of this line is from 10 ⁇ to 10 ° cells / ml, preferably close 3.10 ⁇ cells per ml of liquid phase.
  • the concentration of the liquid phase in inducer constituted by TPA is from 1 ng to 100 ⁇ g / ml, preferably from 0.5 to 2 ⁇ g / ml of liquid phase.
  • the temperature of the liquid phase is 34 to 38 ° C, preferably 37 ° C and the medium thus defined is maintained under the conditions in question for the secretion of interieukin 1 for a sufficient time to
  • the culture medium containing the human interleukin 1 formed is treated by a concentration process, preferably precipitation with ammonium sulphate, centrifugation and re-solution in the minimum of volume of distilled water.
  • the human 1-interieukin 1 is separated by subjecting the concentrated liquid phase to solid phase chromatography, preferably on a column of the Ultragel AcA 54 type (IBF, France) , the eluent being chosen from the group comprising saline buffers, preferably 0.5 M NaCl, sodium phosphate pH 7.2 added with a stabilizing molecule, preferably polyethylene glycol 6000 (MERCK).
  • saline buffers preferably 0.5 M NaCl, sodium phosphate pH 7.2
  • a stabilizing molecule preferably polyethylene glycol 6000 (MERCK).
  • the interieukin 1 separated from the liquid phase containing the cells is purified, using a specific immunoabsorption by antibodies calling into question the general teachings of the techniques of immunoabsorption by antibody; these general teachings are described in relation to other biological products, including in erleukin 2 and in ⁇ terferons, in Robb et al. Immunobiology 161: 21 (1982).
  • the invention also relates to the extraction from the above lines of the messenger RNA coding for the molecule of interieukin 1, the manufacture of a cDNA from this messenger RNA and the introduction by implementation of the techniques of genetic engineering of this cDNA in an appropriate microorganism, then the manufacture of interieukin 1 by extraction from the supernatant collected at the end of any process comprising the
  • the liquid phase in which the cells of the HL-60 line are found consists of the above-defined composition RPMI 1640, supplemented with antibiotics, L-glutamine and 10% fetal calf serum (FFS).
  • RPMI 1640 supplemented with antibiotics, L-glutamine and 10% fetal calf serum (FFS).
  • FFS fetal calf serum
  • the experiments are carried out in preferably thermostated ovens and the quantities of liquid phase used in each experiment are 5-10 ml per point of the experiment.
  • the appearance of molecules carrying the activity of interieukin 1 in the liquid phase and the increase in the concentration of these molecules is followed by titration of aliquots of the liquid phase, these aliquots being removed at 24-hour intervals. hours, then subjected to the so-called "Costimulator assay” test described in detail by MELTZER MS and OPPENHEIM 3 .3. in 3. Immunol. 118, pages 77-82 (1977).
  • mouse thymocytes placed at a concentration of 1.5 ⁇ 10 4 cells / ml in a medium constituted by the composition RPMI 1640 and 1% of fetal calf serum, are brought into contact a suboptimal dose (that is to say a dose forty times lower than the optimal dose, itself equal to 100 ⁇ g / ml of medium) of phytohemagglutinin-A or PHA; under these conditions, the thymocytes in question are incapable of multiplying.
  • a suboptimal dose that is to say a dose forty times lower than the optimal dose, itself equal to 100 ⁇ g / ml of medium
  • the addition of interleukin 1 induces their proliferation.
  • the measurement of the proliferation of thymocytes therefore reflects the intensity of the activity of interieukin 1 in the liquid phase, for example of the culture medium containing the line studied.
  • each aliquot part taken from the liquid phase is therefore introduced into a known volume of the medium containing the thymocytes in the presence of PHA "
  • the actual measurement of proliferation is carried out 48 hours later by incorporating an amount of 0.1 to 50 ⁇ Ci, preferably 0.5 to 2 ⁇ Ci of tritiated thymidine which is normally incorporated by D.N.A. ; the value thus determined of the activity in interieukin 1 at a given time in the liquid phase containing the cells of the line studied is expressed in counts per minute (c.p.m.).
  • a second set of identical experiments is carried out by replacing the HL-60 cells with
  • the monocytes are extracted from the blood by the techniques described in CHOUAIB et al-i Journal of I munology (1982) 129: 2463 and suspended at the concentration of 10 ° cells / ml in a medium constituted by the composition RPMI 1640 and 1% SVF; tnuramyl dipeptide is introduced into this medium at a concentration of 10 ⁇ g / ml and the whole is kept at a temperature of 37 ° C. for 48 hours.
  • the monocytes or cells of the lines studied are removed, for example by centrifugation, respectively, the three supernatants thus collected are concentrated by precipitation with ammonium sulfate and the molecules of interieukin 1 are isolated by column chromatography.
  • AcA 54 (IBF, France); the eluent used is sodium phosphate pH 7.3, 0.5 M NaCl, added with a stabilizing molecule, namely PEG 6000 at 0.1 ° / oo.
  • the "costimulator assay” test determines the "interieukin 1 activities" of successive elution fractions.
  • interleukins 1 are in fact concentrated at two levels of the elution profile, that is to say in two groups of fractions, respectively 16 to 20 and 32 to 38.
  • a second identification was carried out by determining the isoelectric point of the interieukin 1 molecules secreted by the HL-60 line and the same was done for that produced by the monocytes.
  • this interieukin is, however, purer, that is to say accompanied by fewer factors of the interferon, BCGFD or CSF type, given the absence of other contaminating cell types, in particular T lymphocytes, always present in monocyte preparations and which are the cells producing these factors.
  • Such production can also be carried out by cultivating the microorganisms into which the cDNA made from the messenger RNA extracted from the selected lines has been introduced.
  • compositions based on the interleukin thus produced are in the form of injectable preparations, preparations for external applications and preparations for oral and rectal administration.
  • compositions contain an effective amount of interleukin 1, that is to say sufficient to trigger the immune response; they also contain an acceptable non-toxic and pharmaceutical vehicle.

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Toxicology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

On induit la secrétion d'interleukine 1 humaine à partir de lignées leucémiques humaines, lymphomateuses, d'origine hématopoiétique et lymphoïde, ne secrétant normalement pas l'interleukine 1, au moyen d'un inducteur choisi parmi ceux qui possèdent les propriétés d'induction de la différentiation cellulaire, notamment les phorbols et les phorbol-esters.The secretion of human interleukin 1 is induced from human leukemic lines, lymphomatous, of hematopoietic and lymphoid origin, normally not secreting interleukin 1, by means of an inducer chosen from those which have the properties of induction cell differentiation, especially phorbols and phorbol-esters.

Description

Procédé de production d' interieukine 1 humaine et médicaments correspondants. Process for the production of human interieukin 1 and corresponding medicaments.
L'invention a pour objet un procédé de produc¬ tion d' interieukine 1 humaine ; elle vise également les médicaments ou compositions médicamenteuses dont la substance active comporte de 1' interieukine 1 humaine.The invention relates to a process for the production of human interieukin 1; it also relates to medicaments or medicinal compositions the active substance of which comprises human interieukin 1.
L ' interieukine 1 humaine est une hormone protéi- que d'importance fondamentale dans le déclenchement de la réponse immunitaire ; son poids moléculaire est voi- sin de 20.000 à 25.000 et elle est représentée par trois formes biochimiques dont les points isoélectriques se situent respectivement à des pH d'environ 5,7, 6 , 6 et 7. L ' interieukine 1 constitue l'un des deux signaux --l'autre étant apporté par l'antigène ou le mitogène— qui déclenchent la sécrétion, par les lymphocytes T, *de 1 ' interieukine 2 qui amplifie la phase effectrice de l'immunité en provoquant la multiplication des lymphocy¬ tes T Helper et T cytotoxiques.Human interieukin 1 is a protein hormone of fundamental importance in triggering the immune response; its molecular weight is around 20,000 to 25,000 and it is represented by three biochemical forms, the isoelectric points of which are respectively at pHs of 5.7, 6, 6 and 7. Interieukin 1 constitutes one of the two signals - the other being provided by the antigen or the mitogen - which trigger the secretion, by T lymphocytes, * of 1 interieukin 2 which amplifies the effector phase of immunity by causing the multiplication of lymphocytes your T Helper and T cytotoxic.
On sait produire 1' interieukine 1 humaine en in- duisant sa sécrétion par les macrophages/monocytes (ci- après on retiendra le terme monocytes) normaux du sang périphérique en ayant recours en tant qu'inducteur au muramyl dipeptide.It is known to produce human interieukin 1 by inducing its secretion by normal macrophages / monocytes (hereinafter the term monocytes) normal peripheral blood by using as an inducer muramyl dipeptide.
Les principaux inconvénients de ce procédé connu sont notamment celui d'être d'un rendement faible et ce¬ lui de conduire à un produit relativement impur ; en ef¬ fet, ce procédé implique le recours à de multiples don¬ neurs de sang et sous-entend le recours à des procédés complexes de séparation des monocytes ; il conduit à des surnageants contenant non seulement de 1' interieukine 1 mais égaLement de 1 ' interieukine 2 et d'autres facteurs tels que 1 ' interf ron, le BCGFD (ou B cellgrowth factor) et le CSF (ou colony stimulating factor).The main disadvantages of this known method include that of being a low yield and it ¬ it to lead to a product relatively impure; in fact, this process involves the use of multiple blood donors and implies the use of complex processes for the separation of monocytes; it leads to supernatants containing not only interieukin 1 but also interieukin 2 and other factors such as interfon, BCGFD (or B cellgrowth factor) and CSF (or colony stimulating factor).
L'invention a donc pour but, surtout, de remé- dier à* ces inconvénients et de fournir des moyens pro¬ pres à permettre la fabrication d' interieukine 1 humaine la plus pure possible et en des quantités permettant d'envisager son application en tant que médicament.The object of the invention is therefore, above all, to remedy Dier * these disadvantages and provide pro¬ means to allow the manufacture of human interieukin 1 as pure as possible and in quantities to consider its application as a drug.
Or, les Inventeurs ont eu le mérite de trouver qu'il était possible d'induire, de façon inattendue, la sécrétion d' interieukine 1 très pure, c'est-à-dire non accompagnée des facteurs énumérés ci-dessus, à partir de lignées leucémiques humaines, lymphomateuses, d'origine hematopoiétique et lymphoîde, qui ne sécrètent normale¬ ment pas l' interieukine 1, ces lignées étant établies en culture continue, de préférence en milieu liquide ; ces lignées comprennent notamment celles identifiées par les codes HL-60 et K562 ; l'inducteur est un agent du groupe comprenant, d'une part, ceux qui possèdent les proprié¬ tés d'induction de la differentiation cellulaire, notam¬ ment les phorbols et les phorbol-esters, en particulier le 12-0-tétra-decaroyl-phorbol-15-acétate (ou TPA) et, d'autre part, ceux appartenant à d'autres familles tel¬ les que celle de la téléocydine.However, the inventors had the merit of finding that it was possible to unexpectedly induce the secretion of very pure interieukin 1, that is to say unaccompanied by the factors listed above, from human leukemic lines, lymphomatous, of hematopoietic and lymphoid origin, which do not normally secrete interieukin 1, these lines being established in continuous culture, preferably in liquid medium; these lines include in particular those identified by the codes HL-60 and K562; the inducer is an agent of the group comprising, on the one hand, those which have the properties of induction of cell differentiation, in particular the phorbols and the phorbol-esters, in particular 12-0-tetra- decaroyl-phorbol-15-acetate (or TPA) and, on the other hand, those belonging to other families such as that of teleocydine.
Il s'ensuit que le procédé conforme à l'inven¬ tion de production d ' interieukine 1 humaine est caracté¬ risé par le fait que, successivement,It follows that the process according to the invention for the production of human interieukin 1 is characterized by the fact that, successively,
- on établit en phase liquide une concentration efficace, c'est-à-dire suffisante pour obtenir une pro¬ duction d' interieukine industriellement exploitable, en cellules appartenant à au moins l'une des lignées leucé¬ miques humaines définies ci-dessus,an effective concentration is established in the liquid phase, that is to say sufficient to obtain an industrially exploitable production of interieukin, in cells belonging to at least one of the human leukemia lines defined above,
- on induit la sécrétion d' interieukine 1 par les cellules en question en introduisant dans le milieu une quantité efficace, c'est-à-dire suffisante pour dé¬ clencher la sécrétion d' interieukine, en au moins un in¬ ducteur choisi dans le groupe également défini ci-des¬ sus,- the secretion of interieukin 1 is induced by the cells in question by introducing into the medium an effective amount, that is to say sufficient to trigger the secretion of interieukin, by at least one inductor chosen from the group also defined above,
- on maintient pendant un laps de temps suffi¬ sant, c'est-à-dire tel que la quantité d' interieukine 1- It is maintained for a sufficient period of time, that is to say such that the amount of interieukin 1
produite permette d'envisager une exploitation indus¬ trielle, les conditions nécessaires à la sécrétion d'in- terleukine 1, produced allows to envisage an industrial exploitation, the conditions necessary for the secretion of interleukin 1,
- on sépare 1 ' interieukine 1 du milieu, c'est- à-dire de la phase liquide, etthe interieukin 1 is separated from the medium, that is to say from the liquid phase, and
- éventuellement, on la purifie et on la condi¬ tionne en vue de son utilisation future.- Optionally, it is purified and conditioned for its future use.
Dans un mode de réalisation avantageux du susdit procédé, la phase liquide est constituée par le milieu de culture dans lequel sont normalement propagées les cellules de la lignée leucémique humaine sélectionnée ; ce milieu peut comprendre environ 10 % de sérum de veau foetal et environ 90 % de la composition désignée par le code RPMI-1640 et dont les constituants sont indiqués dans la publication "In vitro" 9 , 6 (1974).In an advantageous embodiment of the above process, the liquid phase consists of the culture medium in which the cells of the selected human leukemia line are normally propagated; this medium can comprise approximately 10% of fetal calf serum and approximately 90% of the composition designated by the code RPMI-1640 and the constituents of which are indicated in the publication "In vitro" 9, 6 (1974).
Dans un autre mode de réalisation avantageux du susdit procédé, la lignée sélectionnée est celle connue sous le code HL-60 et la concentration de la phase li¬ quide en cellules constitutives de cette lignée est de 105 à 108 cellules/ml, de préférence voisine de 4.106 cellules par ml de phase liquide.In another advantageous embodiment of the above process, the line selected is that known under the code HL-60 and the concentration of the liquid phase in constituent cells of this line is from 10 5 to 10 8 cells / ml, preferably around 4.10 6 cells per ml of liquid phase.
Dans un autre mode de réalisation avantageux du susdit procédé, la lignée sélectionnée est celle connue sous le code K562 et la concentration de la phase liqui- de en cellules constitutives de cette lignée est de 10^ à 10° cellules/ml, de préférence voisine de 3.10^ cel¬ lules par ml de phase liquide.In another advantageous embodiment of the above process, the line selected is that known under the code K562 and the concentration of the liquid phase in constituent cells of this line is from 10 ^ to 10 ° cells / ml, preferably close 3.10 ^ cells per ml of liquid phase.
Dans un autre mode de réalisation avantageux du susdit procédé, la concentration de la phase liquide en inducteur constitué par le TPA est de 1 ng à 100 μg/ml, de préférence de 0,5 à 2 μg/ml de phase liquide.In another advantageous embodiment of the above process, the concentration of the liquid phase in inducer constituted by TPA is from 1 ng to 100 μg / ml, preferably from 0.5 to 2 μg / ml of liquid phase.
Dans un autre mode de réalisation avantageux du susdit procédé, la température de la phase liquide est de 34 à 38°C, de préférence de 37°C et le milieu ainsi défini est maintenu aux conditions en question de sécré¬ tion d' interieukine 1 pendant une durée suffisante pourIn another advantageous embodiment of the above process, the temperature of the liquid phase is 34 to 38 ° C, preferably 37 ° C and the medium thus defined is maintained under the conditions in question for the secretion of interieukin 1 for a sufficient time to
O PI que sa concentration en interieukine 1 atteigne une valeur palier à partir de laquelle elle n'augmente pratiquement plus, cette durée étant avantageusement de 2 à 10, de préférence de 3 à 5 jours. Dans un autre mode de réalisation avantageux du susdit procédé, le milieu de culture contenant l'inter- leukine 1 humaine formée est traité par un procédé de concentration, de préférence précipitation au sulfate d'ammonium, centrifugation et remise en solution dans le minimum de volume d'eau distillée.O PI that its concentration in interieukin 1 reaches a plateau value from which it practically does not increase any more, this duration advantageously being from 2 to 10, preferably from 3 to 5 days. In another advantageous embodiment of the above process, the culture medium containing the human interleukin 1 formed is treated by a concentration process, preferably precipitation with ammonium sulphate, centrifugation and re-solution in the minimum of volume of distilled water.
Dans un autre mode de réalisation avantageux du susdit procédé, on sépare 1' interieukine 1 humaine for¬ mée en soumettant la phase liquide concentrée à une chromatographie en phase solide, de préférence sur co- lonne du type Ultragel AcA 54 (IBF, France), l'éluant étant choisi dans le groupe comprenant les tampons salins, de préférence NaCl 0,5 M, phosphate de sodium pH 7,2 additionné d'une molécule stabilisante, de préfé¬ rence polyéthylène glycol 6000 (MERCK). Dans un autre mode de réalisation avantageux du susdit procédé, on purifie 1' interieukine 1 séparée de la phase liquide contenant les cellules, en ayant re¬ cours à une immunoabsorption spécifique par anticorps mettant en cause les enseignements généraux des techni- ques d' immunoabsorption par anticorps ; ces enseigne¬ ments généraux sont décrits en rapport avec d'autres produits biologiques, dont l' in erleukine 2 et les in¬ terférons, dans Robb et al. Immunobiology 161:21 (1982). L'invention vise également l'extraction à partir des susdites lignées de l'ARN messager codant pour la molécule d' interieukine 1, la fabrication d'un cADN à partir de cet ARN messager et l'introduction par mise en oeuvre des techniques du génie génétique de ce cADN dans un microorganisme approprié, puis la fabrication de l' interieukine 1 par extraction à partir du surnageant recueilli à l'issue de tout procédé comportant laIn another advantageous embodiment of the above process, the human 1-interieukin 1 is separated by subjecting the concentrated liquid phase to solid phase chromatography, preferably on a column of the Ultragel AcA 54 type (IBF, France) , the eluent being chosen from the group comprising saline buffers, preferably 0.5 M NaCl, sodium phosphate pH 7.2 added with a stabilizing molecule, preferably polyethylene glycol 6000 (MERCK). In another advantageous embodiment of the abovementioned method, the interieukin 1 separated from the liquid phase containing the cells is purified, using a specific immunoabsorption by antibodies calling into question the general teachings of the techniques of immunoabsorption by antibody; these general teachings are described in relation to other biological products, including in erleukin 2 and in¬ terferons, in Robb et al. Immunobiology 161: 21 (1982). The invention also relates to the extraction from the above lines of the messenger RNA coding for the molecule of interieukin 1, the manufacture of a cDNA from this messenger RNA and the introduction by implementation of the techniques of genetic engineering of this cDNA in an appropriate microorganism, then the manufacture of interieukin 1 by extraction from the supernatant collected at the end of any process comprising the
îJRË OMPI culture dudit microorganisme.îJRË WIPO culture of said microorganism.
D'autres caractéristiques du procédé conforme à l'invention de production d 'interieukine 1 humaine appa¬ raissent dans la suite de la description. Pour illustrer les indications qui précèdent, on décrit un ensemble d'essais qui ont permis la mise au point des susdites conditions préparatoires, les lignées étudiées dans le cadre de ces essais étant les lignées K562 et HL-60. Ces lignées K562 et HL-60 sont conservées sous ces noms au C.I.R.C. (O.M.S.) à Lyon.Other characteristics of the process according to the invention for the production of human interieukin 1 appear in the following description. To illustrate the above indications, a set of tests is described which allowed the development of the above preparatory conditions, the lines studied in the context of these tests being lines K562 and HL-60. These lines K562 and HL-60 are kept under these names at C.I.R.C. (O.M.S.) in Lyon.
Parallèlement et à titre de référence, on con¬ duit une expérience de préparation d' interieukine 1 hu¬ maine par le procédé connu à partir de monocytes de sang périphérique.At the same time and by way of reference, an experiment is carried out for the preparation of human interieukin 1 by the known process using peripheral blood monocytes.
Dans une première série d'expériences, on étudie pour la lignée HL-60,In a first series of experiments, we study for the HL-60 line,
- l'influence de la concentration de la phase liquide contenant les cellules en inducteur, en l'occur- rence le TPA,- the influence of the concentration of the liquid phase containing the cells as an inducer, in this case TPA,
- l'influence de la durée de l'expérience pour une température de la phase liquide dont l'optimum se situe entre 36 et 38°C etthe influence of the duration of the experiment for a temperature of the liquid phase, the optimum of which is between 36 and 38 ° C. and
- l'influence de la concentration de la phase liquide en cellules de la lignée HL-60.- the influence of the concentration of the liquid phase in cells of the HL-60 line.
On réalise trois expériences pour l'étude de l'influence de chacun de ces paramètres.Three experiments are carried out to study the influence of each of these parameters.
La phase liquide dans laquelle se trouvent les cellules de la lignée HL-60 est constituée par la com- position RPMI 1640 susdéfinie, additionnée d'antibio¬ tiques., de L-glutamine et de 10 % sérum de veau foetal (SVF).The liquid phase in which the cells of the HL-60 line are found consists of the above-defined composition RPMI 1640, supplemented with antibiotics, L-glutamine and 10% fetal calf serum (FFS).
Les expériences sont effectuées dans des étuves de préférence thermostatées et les quantités de phase liquide utilisées dans chaque expérience sont de 5-10 ml par point de l'expérience. L'apparition des molécules portant l'activité d ' interieukine 1 dans la phase liquide et l'augmentation de la concentration de ces molécules est suivie par ti- tration de parties aliquotes de la phase liquide, ces parties aliquotes étant prélevées à intervalles de 24 heures, puis soumises au test dit "Costimulator assay" décrit en détail par MELTZER M.S. et OPPENHEIM 3 .3 . dans 3 . Immunol. 118, pages 77-82 (1977).The experiments are carried out in preferably thermostated ovens and the quantities of liquid phase used in each experiment are 5-10 ml per point of the experiment. The appearance of molecules carrying the activity of interieukin 1 in the liquid phase and the increase in the concentration of these molecules is followed by titration of aliquots of the liquid phase, these aliquots being removed at 24-hour intervals. hours, then subjected to the so-called "Costimulator assay" test described in detail by MELTZER MS and OPPENHEIM 3 .3. in 3. Immunol. 118, pages 77-82 (1977).
Dans le cadre de ce test, des thymocytes de sou- ris, placés à une concentration de 1,5 x 10^ cellules/ml dans un milieu constitué par la composition RPMI 1640 et 1 % de sérum de veau foetal, sont mis en présence d'une dose suboptimale (c'est-à-dire à une dose quarante fois inférieure à la dose optimale, elle-même égale à 100 μg/ml de milieu) de phytohémagglutinine-A ou PHA ; dans ces conditions, les thymocytes en question sont in¬ capables de se multiplier. Par contre, l'addition d'in- terleukine 1 induit leur prolifération.As part of this test, mouse thymocytes, placed at a concentration of 1.5 × 10 4 cells / ml in a medium constituted by the composition RPMI 1640 and 1% of fetal calf serum, are brought into contact a suboptimal dose (that is to say a dose forty times lower than the optimal dose, itself equal to 100 μg / ml of medium) of phytohemagglutinin-A or PHA; under these conditions, the thymocytes in question are incapable of multiplying. On the other hand, the addition of interleukin 1 induces their proliferation.
La mesure de la prolifération des thymocytes traduit donc l'intensité de l'activité d' interieukine 1 de la phase liquide, par exemple du milieu de culture contenant la lignée étudiée.The measurement of the proliferation of thymocytes therefore reflects the intensity of the activity of interieukin 1 in the liquid phase, for example of the culture medium containing the line studied.
En vue de cette mesure, chaque partie aliquote prélevée de la phase liquide est donc introduite dans un volume connu du milieu contenant les thymocytes en pré¬ sence de PHA»In view of this measurement, each aliquot part taken from the liquid phase is therefore introduced into a known volume of the medium containing the thymocytes in the presence of PHA "
La mesure proprement dite de la prolifération est effectuée 48 heures plus tard par l'incorporation d'une quantité de 0,1 à 50 μCi, de préférence de 0,5 à 2 μCi de thymidine tritiée qui est normalement incorpo¬ rée par le D.N.A. ; la valeur ainsi déterminée de l'ac¬ tivité en interieukine 1 à un moment donné de la phase liquide contenant les cellules de la lignée étudiée est exprimée en coups par minute (c.p.m.). Un deuxième ensemble d'expériences identiques est réalisé en remplaçant les cellules de HL-60 par desThe actual measurement of proliferation is carried out 48 hours later by incorporating an amount of 0.1 to 50 μCi, preferably 0.5 to 2 μCi of tritiated thymidine which is normally incorporated by D.N.A. ; the value thus determined of the activity in interieukin 1 at a given time in the liquid phase containing the cells of the line studied is expressed in counts per minute (c.p.m.). A second set of identical experiments is carried out by replacing the HL-60 cells with
OMPI cellules de la lignée K562.WIPO cells of the K562 line.
Les résultats des mesures ainsi effectuées sont réunis dans les tableaux suivants I, II et III, illus¬ trés respectivement par les graphiques des figures 1, 2 et 3.The results of the measurements thus carried out are collated in the following tables I, II and III, illustrated respectively by the graphs of FIGS. 1, 2 and 3.
TABLEAU I Variation de l'activité d'interieukine 1 de la phase liquide en fonction de sa concentration en TPATABLE I Variation of the activity of interieukin 1 in the liquid phase as a function of its TPA concentration
Sur le graphique de la figure 1, la variation de l'activité d'interieukine 1 en fonction de la concentra¬ tion en TPA est représentée pour la lignée HL-60 par la courbe A, pour la lignée K562 par la courbe B.In the graph in FIG. 1, the variation in the activity of interieukin 1 as a function of the concentration in TPA is represented for the line HL-60 by the curve A, for the line K562 by the curve B.
Il résulte du tableau I et du graphique corres¬ pondant de la figure 1 que la concentration optimale en TPA est voisine de 1 μg/ml.It follows from Table I and the corresponding graph of Figure 1 that the optimal concentration of TPA is close to 1 μg / ml.
TABLEAU II Variation de l'activité d' interieukine 1 de la phase liquide en fonction de la durée de l'expérience exprimée en joursTABLE II Variation of the activity of interieukin 1 of the liquid phase as a function of the duration of the experiment expressed in days
Sur le graphique de la figure 2 , la variation de l'activité d' interieukine 1 en fonction de la durée de l'expérience est représentée par la courbe C pour la li¬ gnée HL-60 et par la courbe D pour la lignée K562.In the graph in Figure 2, the change in the activity of interieukin 1 as a function of the duration of the experiment is represented by curve C for the line HL-60 and by curve D for the line K562.
Il résulte de ce tableau II et du graphique cor¬ respondant de la figure 2 qu'il n'y a pas d'intérêt à poursuivre l'expérience après 4 jours ; l'activité dé¬ croît dès le cinquième jour.It follows from this table II and from the corresponding graph of FIG. 2 that there is no point in continuing the experiment after 4 days; activity decreases on the fifth day.
TABLEAU III Variation de l'activité d' interieukine 1 du milieu de culture en fonction de la concentration en cellules leucémiques exprimée en millions de cellules/ml de milieuTABLE III Variation of the activity of interieukin 1 of the culture medium as a function of the concentration of leukemic cells expressed in millions of cells / ml of medium
Sur le graphique de la figure 3, la variation de l'activité d ' interieukine 1 en fonction de la concentra¬ tion en cellules productrices dans le milieu est repré¬ sentée par la courbe E pour la lignée HL-60 et par la courbe F pour la lignée K562.In the graph in FIG. 3, the variation in the activity of interieukin 1 as a function of the concentration in producer cells in the medium is represented by the curve E for the line HL-60 and by the curve F for line K562.
Il résulte de ce tableau III et du graphique correspondant de la figure 3 que la concentration opti¬ male se situe vers 3.10° cellules/ml de milieu pour la lignée K562 et vers 4.106 cellules/ml pour la lignée HL-60.It follows from this Table III and from the corresponding graph in FIG. 3 that the optimum concentration is around 3.10 ° cells / ml of medium for the K562 line and around 4.10 6 cells / ml for the HL-60 line.
Pour identifier les molécules portant l'activité interieukine 1 produites par les lignées K562 et HL-60 , on les compare à 1 ' interieukine sécrétée par les monocy¬ tes activés par le uramyl dipeptide.To identify the molecules carrying the interieukin 1 activity produced by the K562 and HL-60 lines, they are compared with the interieukin secreted by the monocytes activated by uramyl dipeptide.
ÎRË Les monocytes sont extraits du sang par les techniques décrites dans CHOUAIB et al-i Journal of I munology (1982) 129:2463 et mis en suspension à la concentration de 10° cellules/ml dans un milieu consti- tué par la composition RPMI 1640 et 1 % SVF ; on intro¬ duit dans ce milieu du tnuramyl dipeptide à une concen¬ tration de 10 μg/ml et on maintient l'ensemble à une température de 37°C pendant 48 heures.ÎRE The monocytes are extracted from the blood by the techniques described in CHOUAIB et al-i Journal of I munology (1982) 129: 2463 and suspended at the concentration of 10 ° cells / ml in a medium constituted by the composition RPMI 1640 and 1% SVF; tnuramyl dipeptide is introduced into this medium at a concentration of 10 μg / ml and the whole is kept at a temperature of 37 ° C. for 48 hours.
Parallèlement, on conduit respectivement pour les lignées HL-60 et K562 :At the same time, we conduct respectively for lines HL-60 and K562:
- une expérience de production drinterieukine 1 à partir de cellules de la lignée HL-60 mises en suspen¬ sion à la concentration de 4.10° cellules par ml de la phase liquide définie plus haut et on maintient cette suspension à 37°C pendant 4 jours en présence de 1 μg de TPA par ml,- a production experience r interleukin 1 from cell line HL-60 put in suspen¬ sion at a concentration of 4.10 ° cells per ml of the liquid phase defined above and maintained this suspension at 37 ° C for 4 days in the presence of 1 μg of TPA per ml,
- une expérience de production d' interieukine 1 à partir de cellules de la lignée K562 mises en suspen¬ sion à la concentration de 3.10^ cellules par ml de la phase liquide définie plus haut et on maintient cette suspension à 37°C pendant 4 jours en présence de 1 μg de TPA par ml.an experiment for producing interieukin 1 from cells of the K562 line put in suspension at the concentration of 3.10 ^ cells per ml of the liquid phase defined above and this suspension is maintained at 37 ° C. for 4 days in the presence of 1 μg of TPA per ml.
On élimine, par exemple par centrifugation , res¬ pectivement les monocytes ou cellules des lignées étu- diées, on concentre les trois surnageants ainsi recueil¬ lis par précipitation au sulfate d'ammonium et on isole les molécules d' interieukine 1 par chromatographie sur colonne de AcA 54 (IBF, France) ; l'éluant utilisé est le phosphate de Na pH 7,3, NaCl 0,5 M, additionné d'une molécule stabilisante, à savoir PEG 6000 à 0,1 °/oo.The monocytes or cells of the lines studied are removed, for example by centrifugation, respectively, the three supernatants thus collected are concentrated by precipitation with ammonium sulfate and the molecules of interieukin 1 are isolated by column chromatography. AcA 54 (IBF, France); the eluent used is sodium phosphate pH 7.3, 0.5 M NaCl, added with a stabilizing molecule, namely PEG 6000 at 0.1 ° / oo.
On détermine par le test dit "Costimulator assay" les "activités interieukine 1" des fractions d'élutioή successives.The "costimulator assay" test determines the "interieukin 1 activities" of successive elution fractions.
Dans le tableau IV, on a réuni les valeurs mesu- rées de cette activité, toujours exprimée en c.p.m., pour chacune des fractions recueillies, les étapes deIn Table IV, we have gathered the measured values of this activity, always expressed in c.p.m., for each of the fractions collected, the stages of
OMPI chromatographie et d'élution étant réalisées pour les trois cultures dans les mêmes conditions.WIPO chromatography and elution being carried out for the three cultures under the same conditions.
TABLEAU IVTABLE IV
La variation de l'activité d' interieukine 1 en fonction du n° de la fraction chromatographique étudiée est représentée respectivement par les courbes G et H pour les lignées HL-60 et K562 et par la courbe K pour les monocytes.The variation in the activity of interieukin 1 as a function of the number of the chromatographic fraction studied is represented respectively by the curves G and H for the lines HL-60 and K562 and by the curve K for the monocytes.
L'examen des chiffres réunis dans ce tableau IV et plus encore celui des courbes du graphique de la fi¬ gure 4 font apparaître l'identité entre les interleuki- nes 1 produites par les cellules des lignées HL-60 et K562 d'une part, et celle produite par les monocytesExamination of the figures gathered in this table IV and even more that of the curves of the graph of FIG. 4 reveal the identity between the interleukins 1 produced by the cells of the lines HL-60 and K562 on the one hand , and that produced by monocytes
OMPI d ' autre part .WIPO on the other hand .
Ces interleukines 1 sont en fait concentrées à deux niveaux du profil d'élution, c'est-à-dire dans deux groupes de fractions, respectivement 16 à 20 et 32 à 38. On a mesuré par la méthode de L. FISHER,These interleukins 1 are in fact concentrated at two levels of the elution profile, that is to say in two groups of fractions, respectively 16 to 20 and 32 to 38. We measured by the method of L. FISHER,
Laboratory Techniques in Biochemistry and MolecularLaboratory Techniques in Biochemistry and Molecular
Biology,Vol 1,part. ,11, North Holland Publishing CompanyBiology, Vol 1, part. , 11, North Holland Publishing Company
- Amsterdam - (cité dans "Gel filtration Theory and- Amsterdam - (cited in "Gel filtration Theory and
Practice" édité par Pharmacia), les poids moléculaires des interleukiπes 1 correspondant respectivement aux fractions 16 à 20 et 32 à 38.Practice "edited by Pharmacia), the molecular weights of interleukiπes 1 corresponding respectively to fractions 16 to 20 and 32 to 38.
On a trouvé : une valeur voisine de 70.000 pour celle contenue dans les fractions 16 à 20 et on pense qu'il s'agit ici d' interieukine 1 complexée avec des protéines,We have found: a value close to 70,000 for that contained in fractions 16 to 20 and it is believed that this is interieukin 1 complexed with proteins,
- une valeur voisine de 17.000 pour les frac¬ tions 32 à 38.- a value close to 17,000 for fractions 32 to 38.
On a procédé à une deuxième identification par la détermination du point isoélectrique des molécules d' interieukine 1 sécrétées par la lignée HL-60 et on a procédé de même pour celle produite par les monocytes.A second identification was carried out by determining the isoelectric point of the interieukin 1 molecules secreted by the HL-60 line and the same was done for that produced by the monocytes.
Pour ce faire, on a utilisé la méthode de B.T. RAD0LA, décrite dans Biochemical Biophysical Acta, 386, p 181 (1975) .To do this, the method of B.T. RAD0LA, described in Biochemical Biophysical Acta, 386, p 181 (1975), was used.
On a pu déterminer ainsi que, tant pour l'inter- leukine produite par la lignée HL-60, que pour celle produite par les monocytes, il s'agit en fait de trois formes biochimiques de même poids moléculaire focalisant respectivement aux pH d'environ 5,7, d'environ 6 , 6 et d ' environ 7.It was thus possible to determine that, both for the inter-leukin produced by the HL-60 line, and for that produced by the monocytes, these are in fact three biochemical forms of the same molecular weight focusing respectively at the pH of about 5.7, about 6, 6 and about 7.
Les courbes L et M apparaissant respectivement sur les figures 5 et 6 et représentant la variation de l'activité d ' interleukine en fonction du numéro de fraction d'un gradient de pH , respectivement pour les monocytes et pour la lignée HL-60, montrent en effet desThe curves L and M appearing respectively in FIGS. 5 and 6 and representing the variation of the activity of interleukin as a function of the fraction number of a pH gradient, respectively for the monocytes and for the line HL-60, show indeed
OMPI valeurs de focalisation extrêmement proches.WIPO extremely close focus values.
Il résulte de ce qui précède qu'il est possible, à l'aide du procédé conforme à l'invention, de fabriquer de 1 ' interieukine 1 très semblable à celle classiquement obtenue à partir de monocytes.It follows from the above that it is possible, using the method according to the invention, to manufacture 1 interieukin 1 very similar to that conventionally obtained from monocytes.
On peut prévoir que cette interieukine est tou¬ tefois plus pure, c'est-à-dire accompagnée de moins de facteurs du type interféron, BCGFD ou CSF, étant donné l'absence d'autres types cellulaires contaminants, en particulier lymphocytes T, toujours présents dans les préparations de monocytes et qui sont les cellules productrices de ces facteurs.It can be predicted that this interieukin is, however, purer, that is to say accompanied by fewer factors of the interferon, BCGFD or CSF type, given the absence of other contaminating cell types, in particular T lymphocytes, always present in monocyte preparations and which are the cells producing these factors.
De plus, la mise" en oeuvre du procédé conforme à l'invention permet de produire 1 ' interieukine 1 humaine en des quantités permettant son application en tant que médicament.In addition, setting "out the method according to the invention produces 1 'human interleukin 1 in amounts permitting its application as a medicament.
En effet, cette production n'est plus tributaire d'un plus ou moins grand nombre de donneurs de sang.Indeed, this production is no longer dependent on a greater or lesser number of blood donors.
Bien au contraire, les possibilités de culture offertes par les lignées sélectionnées autorisent le re¬ cours à des quantités de cellules illimitées, donc per¬ mettent d'envisager une production industrielle.On the contrary, the cultivation possibilities offered by the selected lines allow the use of unlimited quantities of cells, therefore allow industrial production to be envisaged.
Celle-ci pourrait être réalisée comme suit :This could be done as follows:
- croissance des lignées cellulaires en fermen- teur,- growth of cell lines as a farmer,
- induction de la production d' interieukine 1 en grands volumes,- induction of the production of interieukin 1 in large volumes,
- concentration des surnageants (par exemple par ultrafiltration) , - purification de la molécule en plusieurs étapes comprenant une étape de filtration sur gel, une étape d' isoélectrofocalisation, etc.- concentration of the supernatants (for example by ultrafiltration), - purification of the molecule in several stages comprising a gel filtration stage, an isoelectric focusing stage, etc.
Une telle production peut également être mise en oeuvre en cultivant les microorganismes dans lesquels a été introduit le cADN fabriqué à partir du ARN messager extrait des lignées sélectionnées.Such production can also be carried out by cultivating the microorganisms into which the cDNA made from the messenger RNA extracted from the selected lines has been introduced.
OMPI Les compositions pharmaceutiques à base de l'in- terleukine ainsi produite se présentent sous la forme de préparations injectables, préparations pour applications externes et préparations pour administration orale et rectale.WIPO The pharmaceutical compositions based on the interleukin thus produced are in the form of injectable preparations, preparations for external applications and preparations for oral and rectal administration.
Ces compositions comportent une quantité d'in- terleukine 1 efficace, c'est-à-dire suffisante pour dé¬ clencher la réponse immunitaire ; elles comportent également un véhicule non toxique et pharmaceutique acceptable.These compositions contain an effective amount of interleukin 1, that is to say sufficient to trigger the immune response; they also contain an acceptable non-toxic and pharmaceutical vehicle.
L'application thérapeutique essentielle de ces compositions pharmaceutiques est celle de médicament coopérant au déclenchement de la réponse immunitaire, ce qui ouvre un immense champ d'utilisations cliniques po- tentielles comparable à celui de 1 ' interieukine 2.The essential therapeutic application of these pharmaceutical compositions is that of medicament which cooperates in triggering the immune response, which opens up an immense field of potential clinical uses comparable to that of interieukin 2.
Quoi qu'il en soit et quel que soit le mode de réalisation adopté, on dispose ainsi d'un procédé de production d ' interieukine 1 humaine dont les caractéris¬ tiques et avantages par rapport à l'art antérieur résul- tent suffisamment de ce qui précède pour, qu'il soit inu¬ tile d'insister à ce sujet et qui est donc propre à fournir en quantités industriellement exploitables et sous une forme très pure, ladite interieukine 1 dont les propriétés rappelées plus haut en font un principe thé- rapeutique très intéressant.Anyway and whatever the embodiment adopted, there is thus a process for the production of human interieukin 1 whose characteristics and advantages compared with the prior art result sufficiently from this. which precedes for, that it is useless to insist on this subject and which is therefore suitable for supplying in industrially exploitable quantities and in a very pure form, said interieukin 1 whose properties recalled above make it a principle the- very interesting rapeutique.
Comme il va de soi et comme il résulte d'ail¬ leurs déjà de ce qui précède, l'invention ne se limite nullement à ceux de ses modes d'application et de réa¬ lisation qui ont été plus particulièrement envisagés ; elle en embrasse, au contraire, toutes les variantes.As is obvious and as it results from their already from the foregoing, the invention is in no way limited to those of its modes of application and of realization which have been more particularly envisaged; on the contrary, it embraces all its variants.
O PI O PI

Claims

REVENDICATIONS 1. Procédé de production d' interieukine 1 hu¬ maine est caractérisé par le fait que, successivement, CLAIMS 1. Process for producing interieukin 1 hu¬ maine is characterized by the fact that, successively,
- on établit en phase liquide une concentration efficace en cellules appartenant à au moins une lignée leucémique humaine, lymphomateuse, d'origine hematopo¬ iétique et lymphoîde ne sécrétant pas normalement l'in- terleukine 1, cette lignée étant établie en culture continue, de préférence en milieu liquide, - on induit la sécrétion d' interieukine 1 par les cellules de la susdite lignée en introduisant dans le milieu une quantité efficace en au moins un induc¬ teur choisi dans le groupe comprenant, d'une part, ceux qui possèdent les propriétés d'induction de la diffé- rentiation cellulaire, notamment les phorbols et les phorbol-esters et, d'autre part, ceux appartenant à d'autres familles telles que celle de la téléocydine,an effective concentration of cells belonging to at least one human leukemic, lymphomatous line, of hematopoietic and lymphoid origin which does not normally secrete interleukin 1 is established in the liquid phase, this line being established in continuous culture, preferably in liquid medium, - the secretion of interieukin 1 is induced by the cells of the above-mentioned line by introducing into the medium an effective amount of at least one inductor chosen from the group comprising, on the one hand, those which have the cell differentiation induction properties, in particular phorbols and phorbol-esters and, on the other hand, those belonging to other families such as that of teleocydine,
- on maintient pendant un laps de temps suffi¬ sant les conditions nécessaires à la sécrétion d'inter- leukine 1,- the conditions necessary for the secretion of inter-leukin 1 are maintained for a period of time sufficient,
- on sépare 1' interieukine 1 du milieu, c'est- à-dire de la phase liquide, etthe interieukin 1 is separated from the medium, that is to say from the liquid phase, and
- éventuellement,* on la purifie et on la condi¬ tionne en vue de son utilisation future. - optionally, * it is purified and it is conditioned for its future use.
2. Procédé selon la revendication 1, caracté¬ risé par le fait que la phase liquide dans laquelle sont suspendues les cellules de la lignée leucémique sélec¬ tionnée est constituée par le milieu de culture dans le¬ quel ces cellules sont normalement propagées. 2. Method according to claim 1, characterized by the fact that the liquid phase in which the cells of the selected leukemic line are suspended is constituted by the culture medium in which these cells are normally propagated.
3, Procédé selon l'une des revendications 1 et3, Method according to one of claims 1 and
2 , caractérisé par le fait que la lignée sélectionnée est celle connue sous le code HL-60, la concentration de la phase liquide en cellules constitutives de cette li¬ gnée étant de 105 à 108 cellules/mi; de préférence voi- sine de 4.10^ cellules/ml de phase liquide.2, characterized in that the selected line is that known under the code HL-60, the concentration of the liquid phase in cells constituting this line being from 10 5 to 10 8 cells / mi; preferably around 4.10 ^ cells / ml of liquid phase.
4. Procédé selon l'une des revendications 1 et4. Method according to one of claims 1 and
OMPI 2, caractérisé par le fait que la lignée sélectionnée est celle connue sous le code K562, la concentration de la phase liquide en cellules constitutives de cette li¬ gnée étant de 10^ à 108 cellules/ml, de préférence voi¬ sine de 3.10° cellules/ml de phase liquide.WIPO 2, characterized in that the selected line is that known under the code K562, the concentration of the liquid phase in cells constituting this line being from 10 ^ to 10 8 cells / ml, preferably around 3.10 ° cells / ml of liquid phase.
5. Procédé selon l'une des revendications 1 à5. Method according to one of claims 1 to
4, caractérisé par le fait que l'inducteur sélectionné est le 12-0-tétra-decaroyl-phorbol-13-acétate (ou TPA), la concentration de la phase liquide en TPA étant de4, characterized in that the inductor selected is 12-0-tetra-decaroyl-phorbol-13-acetate (or TPA), the concentration of the liquid phase in TPA being
1 ng/ml à 100 μg/ml, de préférence de 0,5 à 2 μg/ml.1 ng / ml to 100 μg / ml, preferably 0.5 to 2 μg / ml.
6. Procédé selon l'une des revendications 1 à6. Method according to one of claims 1 to
5, caractérisé par le fait que les conditions de sécré¬ tion d' interieukine 1 sont maintenues jusqu'au moment à partir duquel la concentration de la phase liquide en interieukine 1 n'augmente plus.5, characterized in that the conditions for the secretion of interieukin 1 are maintained until the moment from which the concentration of the liquid phase in interieukin 1 no longer increases.
7. Procédé selon l'une des revendications 3 ou 4 et selon les revendications 5 et 6, caractérisé par le fait que les conditions de sécrétion de 1' interieukine 1 sont maintenues pendant 2 à 10, de préférence pendant 3 à 5 jours.7. Method according to one of claims 3 or 4 and according to claims 5 and 6, characterized in that the secretion conditions of one interieukin 1 are maintained for 2 to 10, preferably for 3 to 5 days.
8. Procédé selon l'une des revendications 1 à 7, caractérisé par le fait que 1' interieukine 1 est sé¬ parée de la phase liquide, après avoir retiré de celle- ci les cellules de la lignée sélectionnée, en soumettant ladite phase liquide à une chromatographie en phase so¬ lide, de préférence sur colonne de AcA 54, l'éluant étant choisi dans le groupe comprenant les tampons salins.8. Method according to one of claims 1 to 7, characterized in that 1 'interieukine 1 is separated from the liquid phase, after having removed therefrom the cells of the selected line, by subjecting said liquid phase solid phase chromatography, preferably on an AcA 54 column, the eluent being chosen from the group comprising saline buffers.
9. Procédé selon les revendications 1 et 8, ca¬ ractérisé par le fait que l'on purifie. 1' interieukine 1 séparée de la phase liquide en ayant recours à une immu¬ noabsorption spécifique par anticorps.9. Method according to claims 1 and 8, ca¬ acterized in that it is purified. 1 'interieukin 1 separated from the liquid phase by using a specific immunoabsorption by antibodies.
10. Procédé de production d' interieukine 1 ca¬ ractérisé par le fait qu'on extrait cette interieukine 1 du surnageant recueilli à l'issue de tout procédé com¬ portant la culture d'un microorganisme dans lequel a été10. Process for the production of interieukin 1, characterized by the fact that this interieukin 1 is extracted from the supernatant collected at the end of any process comprising the culture of a microorganism in which
5TTRËX O PI introduit préalablement, par mise en oeuvre des techni¬ ques du génie génétiquej le cADN fabriqué à partir de l'ARN messager codant pour la molécule d' interieukine 1 extrait des lignées HL-60 ou K562.5TTRËX O PI introduced beforehand, by using genetic engineering techniques, the cDNA made from messenger RNA coding for the molecule of interieukin 1 extracted from the HL-60 or K562 lines.
11. Composition pharmaceutique comportant, d'une part, à titre de médicament coopérant au déclenchement de la réponse immunitaire, une quantité efficace d'in- terleukine 1 humaine notamment préparée par mise en oeu¬ vre de l'un des procédés selon les revendications 1 à 10 et, d'autre part, tout véhicule non toxique et physiolo- giquement acceptable. 11. Pharmaceutical composition comprising, on the one hand, as a medicament which cooperates in triggering the immune response, an effective amount of human interleukin 1, in particular prepared by implementing one of the methods according to the claims 1 to 10 and, on the other hand, any non-toxic and physiologically acceptable vehicle.
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FR8313399 1983-08-17

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US5342614A (en) * 1984-12-21 1994-08-30 Otsuka Pharmaceutical Co., Ltd. Method of treating arthritus or inflammation with IL-1β or derivatives thereof
DK172052B1 (en) * 1984-12-21 1997-09-29 Otsuka Pharma Co Ltd Antitumor active substance, method for its preparation, drug containing the substance, gene encoding the substance, vector containing the gene as well as recombinant microorganism transformed with such a vector
US6107465A (en) * 1984-12-21 2000-08-22 Otsuka Pharmaceutical Co., Ltd. IL-1β and derivatives thereof and drugs
US5831022A (en) * 1986-02-18 1998-11-03 Hoffmann-La Roche Inc. Purification of recombinant human IL-1α
US5008374A (en) * 1986-03-14 1991-04-16 Otsuka Pharmaceutical Co., Ltd. IL-1α derivatives and drugs
AU5355790A (en) * 1989-04-19 1990-11-16 Cetus Corporation Multifunctional m-csf proteins and genes encoding therefor

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