USH1662H - Chemically modified lymphokine and production thereof - Google Patents

Chemically modified lymphokine and production thereof Download PDF

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USH1662H
USH1662H US07/519,280 US51928090A USH1662H US H1662 H USH1662 H US H1662H US 51928090 A US51928090 A US 51928090A US H1662 H USH1662 H US H1662H
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polyethylene glycol
ifn
glycol methyl
methyl ether
lymphokine
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Osamu Nishimura
Masahiko Fujino
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
    • C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
    • 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]
    • C07K14/55—IL-2
    • 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
    • C07K14/555—Interferons [IFN]
    • C07K14/56—IFN-alpha
    • 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
    • C07K14/555—Interferons [IFN]
    • C07K14/57—IFN-gamma
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • Lymphokines such as interferons (hereinafter sometimes abbreviated as IFNs) and interleukin-2 (hereinafter sometimes abbreviated as IL-2) are considered to be of clinical value for the treatment of viral infections and malignancies and recent technological advances in genetic engineering have made it in principle possible to produce such lymphokines on large scales. It is known that the clearance of lymphokines administered to the living body is in general very short. In the case of lymphokines derived from heterologous animals, it is anticipated that antibodies may be produced in some instances and cause severe reaction such as anaphylaxis. Therefore, technology development is desired which leads to delayed clearance of lymphokines used as drugs, with their activity retained, and further to decrease in their antigenicity.
  • IFNs interferons
  • IL-2 interleukin-2
  • U.S. Pat. No. 4,002,531 discloses a method of producing monoalkylpolyethylene glycol derivatives of enzymes.
  • the method disclosed therein which uses sodium borohydride at pH 8.5, when applied to lymphokines, may possibly destroy the physiological activity of lymphokines and therefore may not serve as an effective method of production.
  • said patent specification does not provide any suggestion as to a means for delaying the in vivo clearance of the enzyme derivatives and such a means in otherwise unknown in the prior art.
  • the present invention overcomes the above difficulties.
  • This is invention provides chemically modified lymphokines comprising a lymphokine moiety and at least one polyethylene glycol moiety of the formula R.paren open-st.O--CH 2 --CH 2 .paren close-st. n (I) wherein R is a protective group for the terminal oxygen atom and n is at least one, bonded directly to at least one primary amino group of the lymphokine moiety and a method of producing the same.
  • lymphokine includes soluble factors released from lymphocytes and involved in cellular immunity and substances equivalent thereto in physiological activity.
  • the lymphokines may be genetically engineered products, products derived from various animals including humans and further include substances similar in structure and in physiological activity thereto.
  • interferon- ⁇ interferon- ⁇
  • IFN- ⁇ interferon- ⁇
  • IFN- ⁇ interferon- ⁇
  • IFN- ⁇ interferon- ⁇
  • IFN- ⁇ interferon- ⁇
  • IFN- ⁇ interferon- ⁇
  • IL 2 interferon- ⁇
  • MDF macrophage differentiating factor
  • MAF macrophage activating factor
  • tissue plasminogen activator substances similar in structure and in physiological activity thereto.
  • Examples of said substances similar in structure and in physiological activity are substances having the structure of IFN- ⁇ except for the lack of 2 to 4 amino acids at the N-terminal thereof (U.S. pat. appl. Ser. No. 685,819 claiming the priority PCT/JP84/00292), various IFN- ⁇ fragments lacking in the C terminal portion of IFN- ⁇ (e.g. 15K species; U.S. pat. appln. Ser. No. 534,038), substances having the structure of IL-2 except for the lack of the N-terminal amino acid thereof (EPC (laid open) 91539) or the lack of 4 amino acids from the N-terminal portion (Japanese Patent Application 58-235638 filed Dec.
  • lymphokines IFN- ⁇ , IFN- ⁇ [consisting of 146 amino acids (EPC (laid open) 0089676)], IFN- ⁇ lacking in two N-terminal amino acids (IFN- ⁇ d2), IFN- ⁇ lacing in three N-terminal amino acids (IFN- ⁇ d3), and IL-2.
  • the lymphokines to be used in the practice of the invention preferably have a molecular weight of 5,000 to 50,000, more preferably 10,000 to 30,000.
  • the primary amino group of lymphokines includes the N-terminal ⁇ -amino group and the ⁇ -amino group of the lysine residue.
  • the terminal oxygen-protecting group R is, for example, an alkyl or alkanoyl group.
  • the alkyl group is preferably an alkyl of 1 to 18 carbon atoms, more preferably a lower (C 1-4 ) alkyl, such as methyl, ethyl, propyl, i-propyl, butyl, i-butyl, sec-butyl or t-butyl.
  • the alkanoyl group is preferably an alkanoyl of 1 to 8 carbon atoms, more preferably a lower (C 1-6 ) alkanoyl, such as formyl, acetyl, propionyl, butyryl, i-butyryl or caproyl.
  • the positive integer n is preferably not more than 500, more preferably 7 to 120.
  • the group of formula (I) preferably has a molecular weight of not more than 25,000, more preferably 350 to 6,000. From the viewpoints of physiological activity retention and clearance delaying effect, the group of formula (I) preferably has a molecular weight corresponding to 1 to 10%, more preferably 2 to 5% of the molecular weight of the lymphokine to be modified.
  • the chemically modified lymphokine according to the invention consists of a lymphokine moiety and the group of formula (I) directly bonded to at least one of the primary amino group of the lymphokine moiety.
  • the modified lymphokine When the N-terminal ⁇ -amino group is the only primary amino group in the lymphokine to be modified, the modified lymphokine has the group of formula (I) directly bonded to said amino group.
  • the modified lymphokine When the lymphokine to be modified has one or more lysine residues in its molecule, the modified lymphokine has the group of formula (I) directly bonded to some percentage, preferably 15 to 80% (on the average), of said ⁇ -amino groups. In this case, the N-terminal ⁇ -amino group may have or may not have the group of formula (I) directly bonded thereto.
  • the chemically modified lymphokines according to the invention can be produced, for example, by reacting a lymphokine with the aldehyde of the formula R.paren open-st.O--CH 2 CH 2 .paren close-st. n-1 O--CH 2 CHO (II) wherein R and n are as defined above, in the presence of a reducing agent.
  • sodium borohydride As the boron-containing reducing agent to be used as conducting the reaction, there may be mentioned sodium borohydride and sodium cyanoborohydride. Among them, more preferred is sodium cyanoborohydride from the viewpoint of selectivity of reaction or possibility of carrying out the reaction in the neighborhood of neutrality.
  • the aldehyde (II) is used in an amount of about 1 to 10,000 moles per mole of the lymphokine, and the boron-containing reducing agent is used in an amount of about 1 to 100 moles per mole of the lymphokine.
  • the degree of modification can be selected as desired by varying the mole ratio between lymphokine and aldehyde (II).
  • the solvent to be used in carrying out the invention may be any solvent which does not disturb the reaction and is, for example, a buffer such as a phosphate or borate buffer.
  • An organic solvent which does not inactivate lymphokines or disturb the reaction such as a lower alkanol (e.g.
  • the reaction may be conducted within a broad pH range of 3 to 14 but is preferably perfomred at about pH 7 (pH 6.5-7.5).
  • the reaction temperature may be selected within a broad range of 0° to 80° C., preferably 0° to 50° C., so as not to cause denaturation of lymphokines. A period of 0.5 to 100 hours, generally 10 to 80 hours, will be sufficient for the reaction.
  • the desired, chemically modified lymphokines can be obtained by purifying the reaction mixture by dialysis, salting out, ion exchange chromatography, gel filtration, high performance liquid chromatography, electrophoresis, or the like ordinary method of purifying proteins.
  • the degree of modification of the amino group or groups can be calculated by acid degradation followed by amino acid analysis, for instance.
  • the above-mentioned aldehyde (II) can be produced from an ethylene glycol derivative of the formula R.paren open-st.O--CH 2 CH 2 .paren close-st. n OH (III) wherein R and n are as defined above, for instance.
  • the following method of producing the same is advantageous in that the production of the corresponding byproduct carboxylic acid is little.
  • the compound (III) is oxidized with pyridinium chlorochromate in a haloalkane solvent such as methylene chloride or chloroform.
  • a haloalkane solvent such as methylene chloride or chloroform.
  • pyridinium chlorochromate is used in an amount of 1 to 3 moles per mole of compound (III) and the reaction is carried out at -10° to 50° C., preferably at room temperature, for 1 to 30 hours.
  • reaction mixture can be subjected to purification processes conventional in the field of chemistry, such as extraction, concentration, recrystallization, reprecipitation, chromatography and/or distillation.
  • the chemically modified lymphokines according to the present invention have useful physiological activities similar to those of the corresponding known, unmodified lymphokines and are useful as drugs, among other uses.
  • the chemically modified lymphokines according to the present invention exhibit delay in clearance in vivo as compared with the corresponding known, unmodified lymphokines and are low in toxicity and antigenicity and can be used safely for the same purposes and in the same manner as in the case of known lymphokines.
  • the chemically modified lymphokines according to the invention can usually by administered to mammals (monkey, dog, pig, rabbit, mouse, human) either orally or parenterally in the form of appropriate pharmaceutical compositions prepared by using carriers, diluents, etc., which are known in themselves.
  • chemically modified IFN- ⁇ according to the invention when used as an antiviral agent, is recommendably administered to human adults once a day by intravenous injection in a dose of 1 ⁇ 10 4 to 1 ⁇ 10 9 international units.
  • amino acids when referred to by abbreviations, are abbreviated according to IUPAC-IUB (Commision of Biological Nomenclature).
  • the transformant Escherichia coli 294/pHITtrp1101-d2 as disclosed hereinafter in a reference example has been deposited with Institute for Fermentation, Osaka (IFO) under the deposit number IFO-14350 and, since Jun. 6, 1984, with the Fermentation Research Institute (FRI), Agency of Industrial Science and Technology, Ministry of International Trade and Industry under the deposit number FERM BP-703 under Budapest Treaty.
  • strain Escherichia coli DH1/pTF4 has been deposited with the Institute for Fermentation, Osaka under the deposit number IFO-14299 and, since Apr. 6, 1984, with the FRI under the deposit number FERM BP-628 under Budapest Treaty.
  • FIG. 1 shows the clearance-delaying effect in rat plasma as disclosed in Example 1 (iv).
  • the measurement results obtained with the chemically modified IFN- ⁇ according to the invention as produced in Example 1 (i) are indicated by O (enzyme immunoassay) and ⁇ (antiviral activity assay), and the results obtained with rIFN- ⁇ A used as a control by ⁇ (enzyme immunoassay) and ⁇ (antiviral activity assay.
  • FIG. 2 shows the clearance-delaying effect in rat plasma as disclosed in Example 3 (ii).
  • the data indicated by ⁇ , ⁇ and ⁇ are the enzyme immunoassay data for compound No. 8, compound No. 2 (Table 1) and control rIFN- ⁇ A, respectively.
  • FIG. 3 shows the construction scheme for the expression plasmid pHITtrp1101-d2 disclosed in Reference Example 3 (i) and FIG. 4 the construction scheme for the expression plasmid pLC2 disclosed in Reference Example 4 (i).
  • the reaction mixture was poured into a Sephadex G-75 column (3.0 ⁇ 43.0 cm) and developed with 25 mM ammonium acetate buffer (pH 5.0) and 0.15M sodium chloride.
  • the eluate was collected in 5-ml portions. Eluate fractions (100-150 ml) containing the contemplated product were combined. Assaying by the Lowry method using bovine serum albumin as a standard revealed that the protein content in the combined fractions was 84 ⁇ g/ml.
  • Amino acid ratios in acid hydrolysate (6N hydrochloric acid, 110° C., 24 hours) were as follows: Asp, 12.2 (12); Thr, 10.4 (10); Ser, 16.0 (14); Glu, 24.8 (26); Pro, 6.0 (5); Gly, 6.3 (5); Ala, 8.6 (8); Val, 6.5 (7); Met, 4.0 (5); Ile, 7.6 (8); Leu, 21.0 (21); Tyr, 5.2 (5); Phe, 9.9 (10); Lys, 6.5; His, 3.8 (3); Arg, 9.1 (9); Cys, Trp, decomposed.
  • Amino acid ratios in acid hydrolysate (6N hydrochloric acid, 110° C., 24 hours) were as follows: Asp, 12.1 (12); Thr, 10.1 (10); Ser, 13.6 (14); Glu, 26.7 (26); Pro, 5.5 (5); Gly, 5.6 (5); Ala 8.4 (8); Val, 6.7 (7); Met, 5.5 (5); Ile, 7.4 (8); Leu, 21.0 (21); Tyr, 5.1 (5); Phe, 9.6 (10); Lys, 4.7; His, 3.5 (3); Arg, 9.1 (9); Trp, 1.8 (2); Cys, decomposed.
  • the above data indicate that about 57% of Lys residues had been modified at the ⁇ -amino group.
  • Enzyme immunoassay performed in the same manner as (i) gave the result 5 ⁇ 10 6 international units/mg, and the antiviral activity of the product was 0.14 ⁇ 10 8 international units/mg.
  • Example 1 To 5 ml of the solution of chemically modified IFN- ⁇ (IFA-3) of the invention as obtained in Example 1 (i), there is added 250 mg of human serum albumin. The resulting solution is filtered through a membrane filter (pore size: 0.2 ⁇ m) and distributed into 5 vials, followed by lyophilization and storage. The contents of each vial are dissolved in 1 ml of distilled water for injection just prior to use.
  • a membrane filter pore size: 0.2 ⁇ m
  • the acid hydrolysate (6N hydrochloric acid, 110° C., 24 hours) gave the following amino acid analysis values: Asp, 19.6 (20); Thr, 4.7 (5); Ser, 8.3 (11), Glu, 18.5 (18); Pro, 2.1 (2); Gly, 5.4 (5); Ala, 7.5 (8); Val, 8.4 (8); Met, 3.7 (4); Ile, 7.1 (7); Leu, 9.7 (10), Tyr, 5.3 (5); Phe, 9.7 (10); Lys, 17.6; His, 2.0 (2); Arg, 5.0 (8); Cys, Trp, decomposed.
  • the thus-purified fraction (25 ml) had a protein content of 126 ⁇ g/ml and amino acid analysis of the acid hydrolysate (6N hydrochloric acid, 110° C., 24 hours) gave the following values: Asp, 20.0 (20); Thr, 5.2 (5); Ser, 9.5 (11); Glu, 27.8 (18); Pro, 2.7 (2); Gly, 14.6 (5); Ala, 8.1 (8); Val, 8.5 (8); Met, 4.3 (4); Ile, 7.2 (7); Leu, 10.2 (10); Tyr, 5.8 (5); Phe, 10.1 (10); Lys, 14.7; His, 2.0 (2); Arg, 7.3 (8); Thr, 0.7 (1); Cys, decomposed.
  • the supernatant is concentrated to 10 ml using a Diaflow membrane (Amicon).
  • the concentrate is applied to a Sephadex G-75 column (3.0 ⁇ 43.0 cm) and developed with 25 mM ammonium acetate buffer (pH 6.0)+0.15M sodium chloride+10 mM glutathione.
  • the eluate is fractionated by 5 ml, and the fractions containing modified IFN- ⁇ d2 having the polyethylene glycol methyl ether moiety on the Lys ⁇ -amino group in the molecule are collected and combined. When this product is administered to rats, evident delay in clearance in blood is noted.
  • the precipitate is dissolved in 6M guanidine hydrochloride, dialyzed against 25 mM ammonium acetate buffer (pH 6.0)+0.15M sodium chloride+10 mM glutathione at 4° C. overnight, and purified by Sephadex G-75 gel filtration in the same manner as above.
  • a fraction containing modified IFN- ⁇ d2 having the polyethylene glycol methyl ether moiety on the Lys ⁇ -amino group in the molecule is obtained a fraction containing modified IFN- ⁇ d2 having the polyethylene glycol methyl ether moiety on the Lys ⁇ -amino group in the molecule.
  • the dialyzate was applied to a Sephadex G-75 column (3.0 ⁇ 43.0 cm) and developed with the same solvent system.
  • the eluate was fractionated in 5-ml portions.
  • the desired product-containing fractions Nos. 21-29 were combined.
  • the combined fraction had a protein content of 25 ⁇ g/ml as determined by the Bradford method using bovine serum albumin as a standard.
  • the acid hydrolysate (6N hydrochloric acid, 110° C., 24 hours) gave the following amino acid analysis values: Asp, 12.0 (12); Thr, 12.5 (13); Ser, 7.1 (8); Gly, 18.6 (18); Pro, 5.5 (5); Gly, 2.2 (2); Ala, 5.0 (5); Val, 3.7 (4); Met, 3.9 (4); Ile, 8.1 (8); Leu, 22.2 (22); Tyr, 3.0 (3); Phe, 6.0 (6); Lys, 7.3; His, 3.0 (3); Arg, 3.9 (4); Cys, Trp, decomposed.
  • the IFN- ⁇ expression plasmid pHITtrp1101 [cf. EPC (laid open) No. 110044, Example 2 (iii)] was digested with the restriction enzymes AvaII and PstI, and an AvaII-PstI 1 kb DNA fragment containing the IFN- ⁇ gene portion was isolated.
  • the protein synthesis start codon-containing oligonucleotide adapter ## chemically synthesized by the phosphotriester method was joined to the above DNA fragment at the AvaII cohesive end thereof using T4 DNA ligase.
  • the above adapted-joined gene was inserted into the DNA fragment obtained by cleavage of the plasmid ptrp771 [cf. above-cited publication, Example 2 (ii)] with the restriction enzymes ClaI and PstI, downstream from the trp promoter in said fragment.
  • the strain E. coli 294/pHITtrp 1101-d2 carrying the plasmid constructed in (i) above was cultivated in M9 medium containing 8 ⁇ g/ml of tetracycline, 0.4% of casamino acids and 1% of glucose at 37° C.
  • M9 medium containing 8 ⁇ g/ml of tetracycline, 0.4% of casamino acids and 1% of glucose at 37° C.
  • 3- ⁇ -indolylacrylic acid (IAA) was added to a concentration of 25 ⁇ g/ml. Thereafter, the cultivation was continued for further 4 hours. After cultivation, cells were harvested by centrifugation and suspended in 1/10 volume of 0.05M Tris-HCl (pH 7.6) containing 10% sucrose.
  • phenylmethylsulfonyl fluoride NaCl
  • EDTA ethylenediaminetetraacetate
  • the lysate was subjected to centrifugation at 4° C. and 20,000 rpm (Servall centrifuge, SS-34 rotor) for 30 minutes to given an IFN- ⁇ d2 polypeptide-containing supernatant.
  • This supernatant had an antiviral activity of 2.87 ⁇ 10 8 U/liter culture fluid.
  • This supernatant was diluted with 260 ml of a buffer (pH 7.4) comprising 137 mM sodium chloride, 2.7 mM potassium chloride, 8.1 mM disodium phosphate and 1.5 mM monopotassium phosphate (hereinafter such buffer being referred to by the abbreviation PBS) and the dilution was applied to an antibody column (Mo ⁇ 2-11.1, column volume 12 ml) at a flow rate of 1 ml/minute.
  • a buffer pH 7.4
  • PBS monopotassium phosphate
  • the column was then washed with 60 ml of 20 mM sodium phosphate buffer (pH 7.0) containing 0.5M guanidine hydrochloride and eluted with 36 ml of 20 mM sodium phosphate buffer (pH 7.0) containing 2M guanidine hydrochloride to give 20 ml of an antivirally active fraction.
  • This 20-ml fraction was applied to a Sephacryl S-200 (Pharmacia) column (2.6 ⁇ 94 cm, column volume 500 ml) equilibrated in advance with 25 mM ammonium acetate buffer (pH 6.0) containing 1 mM ethylenediaminetetraacetate, 0.15M sodium chloride, 10 mM cysteine and 2M guanidine hydrochloride, followed by eltuion with the same buffer. Thus was obtained 37 ml of the antivirally active fraction.
  • IFN- ⁇ polypeptide obtained weighed 5.9 mg and had a specific activity of 1.0 ⁇ 10 7 U/mg.
  • the IFN- ⁇ expression plasmid pRC23/IFI-900 [cf. Example 7 of the specification for a patent application under EPC as laid open under No. 0089676] was digested with the restriction enzymes NdeI and NcoI, and a 710 bp NdeI-NcoI DNA fragment (A) containing the IFN- ⁇ gene region was isolated. Separately, the plasmid pRC23 was digested with the restriction enzymes BglII and EcoRI, and a 265 bp DNA fragment (B) containing the ⁇ P L promoter was isolated.
  • the fragments (A) and (B) and the chemically synthesized, protein synthesis start codon-containing oligonucleotide ##STR4## were joined together using T4 DNA ligase, with the NdeI and EcoRI cohesive ends as the sites of joining.
  • the DNA fragment thus obtained was joined to the plasmid pRC23/IFI-900 after treatment with NcoI and BglII, to thereby construct an expression plasmid, pLC2, coding for the ##STR5## IFN- ⁇ polypeptide (FIG. 2).
  • the strain E. coli RRI (pLC2,pRK248 cIts) carrying the plasmid constructed in (i) above was shake-cultured at 35° C. in 50 ml of a liquid medium containing 1% Bactotryptone, 0.5% yeast extract, 0.5% sodium chloride and 7 ⁇ g/ml tetracycline.
  • the culture broth was transferred to 2.5 liters of M9 medium containing 0.5% casamino acid, 0.5% glucose and 7 ⁇ g/ml tetracycline, and grown at 35° C. for 4 hours and then at 42° C. for 3 hours. Cells were harvested by centrifugation and stored at -80° C.
  • This fraction (25 ml) was applied to a Sephacryl S-200 (Pharmacia) column (2.6 ⁇ 94 cm; column capacity 500 ml) equilibrated in advance with 25 mM ammonium acetate buffer (pH 6.0) containing 1 mM ethylenediaminetetraacetic acid, 0.15M sodium chloride, 10 mM cysteine and 2M guanidine hydrochloride, and eluted with the same buffer to give 40 ml of an antivirally active fraction.
  • IFN- ⁇ polypeptide IFN- ⁇ d3 weighed 7.0 mg and had a specific activity of 2.72 ⁇ 10 7 IU/mg.
  • E. coli DH1/pTF4 [U.S. pat. appln. Ser. No. 674,556] was inoculated into 50 ml of a liquid medium (pH 7.0) containing 1% Bacto tryptone (Difco Laboratories, USA), 0.5% Bacto yeast extract (Difco Laboratories, USA), 0.5% sodium chloride and 7 ⁇ g/ml tetracycline as placed in a 250-ml Erlenmeyer flask. After incubation at 37° C. overnight on a swing rotor, the culture medium was transferred to a 5-liter jar fermenter containing 2.5 liters of M9 medium containing 0.5% casamino acid, 0.5% glucose and 7 ⁇ g/ml tetracycline.
  • freeze-stored cells (12.1 g) obtained above were suspended uniformly in 100 ml of an extractant (pH 7.0) containing 7M guanidine hydrochloride and 0.1M Tris.HCl, the suspension was stirred at 4° C. for 1 hour and the lysate was centrifuged at 28,000 x g for 20 minutes. There was obtained 93 ml of a supernatant.
  • an extractant pH 7.0
  • the supernatant obtained above was dialyzed against 0.01M Tris.HCl buffer (pH 8.5) and then centrifuged at 19,000 x g for 10 minutes, giving 94 ml of a dialyzate supernatnat.
  • This dialyzate supernatant was applied to a DE 52 (DEAE-cellulose, Whatman, Great Britain) column (50 ml in volume) equilibrated with 0.01M Tris-HCl buffer (pH 8.5) for protein adsorption.
  • IL-2 was eluted making a linear NaCl concentration gradient (0-0.15M NaCl, 1 liter).
  • the active fractions (53 ml) were concentrated to 4.8 ml using a YM-5 membrane (Amicon, USA) and subjected to gel filtration using a Sephacryl S-200 (Pharmacia, Sweden) column (500 ml in volume) equibrated with 0.1M Tris.HCl (pH 8.0)-1M NaCl buffer.
  • the active fractions (28 ml) obtained were concentrated to 2.5 ml using a YM-5 membrane.
  • the concentrate was applied to a Ultrapore RPSC (Altex, USA) column for adsorption, and high performance liquid chromatography was performed using a trifluoroacetic acid-acetonitrile system as the eluent.

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US07/519,280 1984-03-06 1990-04-05 Chemically modified lymphokine and production thereof Abandoned USH1662H (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US07/519,280 USH1662H (en) 1984-03-06 1990-04-05 Chemically modified lymphokine and production thereof

Applications Claiming Priority (8)

Application Number Priority Date Filing Date Title
WOPCT/JP84/00085 1984-03-06
PCT/JP1984/000085 WO1985003934A1 (fr) 1984-03-06 1984-03-06 Proteine modifiee chimiquement et son procede de preparation
WOPCT/JP84/00575 1984-12-05
PCT/JP1984/000575 WO1985003868A1 (fr) 1984-03-06 1984-12-05 Lymphokine modifiee chimiquement et son procede de preparation
US70706385A 1985-03-15 1985-03-15
US91954486A 1986-10-14 1986-10-14
US40741989A 1989-09-14 1989-09-14
US07/519,280 USH1662H (en) 1984-03-06 1990-04-05 Chemically modified lymphokine and production thereof

Related Parent Applications (1)

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US40741989A Continuation 1984-03-06 1989-09-14

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USH1662H true USH1662H (en) 1997-07-01

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US07/519,280 Abandoned USH1662H (en) 1984-03-06 1990-04-05 Chemically modified lymphokine and production thereof

Country Status (5)

Country Link
US (1) USH1662H (ja)
JP (2) JPH0676439B2 (ja)
KR (1) KR920007681B1 (ja)
AU (1) AU2867784A (ja)
WO (2) WO1985003934A1 (ja)

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US9125880B2 (en) 2002-12-26 2015-09-08 Mountain View Pharmaceuticals, Inc. Polymer conjugates of interferon-beta with enhanced biological potency

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Publication number Priority date Publication date Assignee Title
US20040136952A1 (en) * 2002-12-26 2004-07-15 Mountain View Pharmaceuticals, Inc. Polymer conjugates of cytokines, chemokines, growth factors, polypeptide hormones and antagonists thereof with preserved receptor-binding activity
US20080058246A1 (en) * 2002-12-26 2008-03-06 Mountain View Pharmaceuticals, Inc. Polymer conjugates of cytokines, chemokines, growth factors, polypeptide hormones and antagonists thereof with preserved receptor-binding activity
US9125880B2 (en) 2002-12-26 2015-09-08 Mountain View Pharmaceuticals, Inc. Polymer conjugates of interferon-beta with enhanced biological potency

Also Published As

Publication number Publication date
JPS60226821A (ja) 1985-11-12
WO1985003934A1 (fr) 1985-09-12
JPS61178926A (ja) 1986-08-11
JPH0676439B2 (ja) 1994-09-28
WO1985003868A1 (fr) 1985-09-12
KR850006875A (ko) 1985-10-21
KR920007681B1 (ko) 1992-09-14
JPH0696599B2 (ja) 1994-11-30
AU2867784A (en) 1984-12-04

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