WO1985003868A1 - Chemically modified lymphokine and process for its preparation - Google Patents

Abstract

A chemically modified lymphokine having a group of R(-O-CH2CH2)n- (wherein R is a protective group for the terminal oxygen and n is an arbitrary positive integer) directly bound to at least one primary amino group of the molecule. It can be prepared by reacting lymphokine with an aldehyde of R(O-CH2CH2)n-1-O-CH2CHO (wherein R and n are as defined above) in the presence of a reducing agent, and is useful as medicine, etc.

Description

 One i one

 Specification

 Chemically modified lymphokine and method for producing the same

 Technical field

 The present invention relates to a chemically modified lymphokine and a method for producing the same.

 Background technology

Conventionally, useful lymphokines such as interferon (hereinafter sometimes abbreviated as IFN) or interleukin-2 (hereinafter sometimes abbreviated as IL-2) have been known. With the development of genetic recombination technology, it has become possible to synthesize these lymphokines in large quantities'. However, it is known that the clearance of lymphokines administered to a living body in vivo is generally very fast. If the lymphokine is obtained from a heterologous animal, there is a possibility that antibodies may be produced and serious symptoms may be caused. Therefore, when these are used as pharmaceuticals, it is desired to develop a technique for delaying clearance and further reducing antigenicity while maintaining their activity. To achieve this goal, chemically modifying lymphokines is a very effective method. That is, the chemical modification is expected to delay the clearance in vivo, weaken the antigenicity, and further enhance the physiological activity, and the chemical modification of lymphokines is of great practical significance. In general, when performing chemical modification of bioactive proteins, a method is required that can perform chemical modification while maintaining their bioactivity. Polyethylene glycol methyl ether itself is considered to have no antigenicity and is therefore used for chemical modification of proteins.However, the introduction of this substance into proteins is generally carried out using cyanuric chloride. It is a target. However, cyanuric chloride, which is simultaneously introduced as a conjugating group, has its own safety problems, and also has a problem in terms of the safety of its decomposition products in vivo. Not elucidated and its use needs to be used with caution. In addition, the reaction requires an alkaline PH, and alkaline deactivated proteins are disadvantageous in that this method cannot be used.

 U.S. Pat.No. 4,002,531 discloses a method for producing a monoalkyl polyethylene glycol derivative of an enzyme, but discloses a method using sodium borohydride with PH8.5 disclosed therein. When applied to liposomes, their physiological activities may be inactivated and cannot be an effective production method.In addition, the patent document does not even suggest the effect of delaying the clearance of enzyme derivatives in vivo, Is unknown.

 Furthermore, a method for introducing low-molecular-weight aldehydes such as formaldehyde, acetoaldehyde, benzylaldehyde, and pyridoxal into a bioactive protein in the presence of a boron-based reducing agent [Methods Enzymology, Vol. , 469-478 (1977)]; Japanese Patent Application Laid-Open No. 58-154596] is known. However, even if this method is applied to lymphokines, effective reduction of clearance is not achieved, and not only is the antigenicity not expected to decrease, but also the introduced low-molecular aldehyde acts as a hapten, and May render it immunogenic.

 The present inventors have conducted intensive research to solve these drawbacks and completed the present invention.

 Disclosure of the invention

 The present invention relates to at least one primary amino group in a molecule,

_{R- O - CH 2 - CH 2} ¾ group (I: R is a protecting group of the terminal oxygen, n Riuru positive integer changes to any) that provides a chemically modified lymphokines and their preparation becomes directly bonded to It is.

 In the present specification, lymphokines are soluble factors involved in cellular immunity released from lymphocytes and substances having a physiological activity equivalent thereto.

O PI Are collectively referred to.

 That is, lymphokines may be any of genetically engineered products, those derived from various animals including humans, synthetic products, and the like, and further include substances having a similar structure to these and having the same physiological activity.

 For example, various IFNs [IFN- 1 (IFN-), IFN-1 (1 FN-3), IFN7 (1 FN-r)], IL-2, macrophage differentiation factor (MD F), macula phage activator (MAF), tissue plasminogen activator (TPA), and substances having similar structures and similar physiological activities to these substances.

 As a substance having a structure similar to the above-mentioned lymphokine and having the same physiological activity, for example, IFN-7 in which two to four N-terminal amino acids are deleted (PCT / JP 84/00292, (Filed on June 6, 1984) and various IFN-fragments lacking the C-terminal portion (such as 15K species) US Patent Application No. 534,038, filed on March 20, 1983], and IL-12 Is a protein lacking one amino acid (EPC Publication No. 91539) or four amino acids (Japanese Patent Application No. 58-235638, filed on December 13, 1983) from its N-terminus, and its constituent amino acids Examples thereof include those in which a part of the acid is deleted or substituted with another amino acid, for example, those in which the cysteine at position 125 is substituted with serine (JP-A-59-93093).

 In particular, those with two or more N'-terminal amino acids (IFN-a d2) or three (F N-r d3), or IL-FN-, IFN-a or its N-terminal amino acid of phosphorus' — It is preferably 2,.

 The lymphokine of the present invention preferably has a molecular weight of 5,000 to 50,000, especially 10,000 to 30,000.

 Primary amino groups of lymphokines include the N-terminal monoamino group and the s-amino group of lysine residues.

CMPI To the group represented by the above (I), is a protective group of the terminal oxygen represented by R, an alkyl, Arukanoiru and the like, specifically as' alkyl, d - from _{1 8,} especially methyl, Echiru Lower alkyl such as, propyl, i-propyl, butyl, i-butyl, sec-butyl, and t-butyl (preferably C alkyl. Specific examples of the alkanoyl include, especially, holmil, acetyl, propionyl, butyryl, i-butyryl. Preferred are lower (Cis) alkanols, such as nitropropyl, etc. The positive integer represented by n is preferably 500 or less, particularly preferably 7 to 120.

 The molecular weight of the group represented by the formula (I) is preferably 250,000 or less, particularly preferably 350 to 6000. From the viewpoint of maintaining the physiological activity and delaying the clearance, a group represented by the formula (I) having a molecular weight of 1 to 10%, particularly 2 to 5% of the molecular weight of lymphokine is preferred.

 The chemically modified lymphokine of the present invention has a group represented by the formula (II) directly bonded to at least a part of a primary amino group of lymphokine. .

 When it has only an N-terminal α-amino group as a primary amino group, it has a group represented by the formula (I) directly bonded to the amino group. When the lymphokine molecule has one or more lysines, it is directly bonded to a part of the ε-amino group, preferably 15 to 80% (average) of the ε-amino group. It has a group represented by the formula (I). In this case, the terminal amino group may or may not have a group represented by the formula (I) directly bonded.

 The chemically modified lymphokine of the present invention is, for example,

R-o-CH ₂ CH ₂ ½ = TO-CH ₂ CH 0 (K: R and ιι are the same as defined above), and can be produced by reacting in the presence of a reducing agent. . Examples of the boron-based reducing agent used in this reaction include sodium borohydride, sodium cyanoborohydride, and the like. Among them, sodium cyanoborohydride can react at selectivity and near neutrality of the reaction. Is more preferable. .

 At the time of the reaction, the aldehyde (用 い) may be used in an amount of about ί to ΙΟ, per mole of the lymphokine, and the boron-based reducing agent may be used in an amount of about 1 to 100 times the mol of the aldehyde (Π). The degree of modification can be arbitrarily selected by reducing the molar ratio of aldehyde to aldehyde (II). The solvent used in the reaction may be any solvent as long as it does not hinder the reaction. Examples thereof include buffer solutions such as a phosphate buffer and a borate buffer. Further, an organic solvent such as lower alcohol (eg, methanol, ethanol, i-propanol) or acetonitrile which does not deactivate lymphokine and does not hinder the reaction may be added. The pH of the reaction can be in a wide range from 3 to 14, but is preferably around neutral (6.5 to 7.5). The reaction temperature may be any temperature at 0 ° to 80 ° C. so long as the lymphoid is not denatured, but is more preferably in the range of 0 ° to 50 ° C. The reaction time is Q. 5 to 72 hours, usually 3 to 30 hours. The reaction solution can be purified by a conventional protein purification method such as porcine, salt folding, ion exchange chromatography, gel filtration, high performance liquid chromatography, and electrophoresis to obtain a desired chemically modified lymphokine. The degree of modification of the amino group can be calculated by, for example, performing acid decomposition and then performing amino acid separation.

Wherein the aldehyde ([pi), for example _{R ~ 0- CH 2 C Η 2} ½0 Η: While (ΠΙ R and n are the same as defined) can be prepared from E Ji render recall derivative represented by the method below However, this is an advantageous production method with less by-product of the corresponding carboxylic acid.

That is, the compound (GO is halogenated such as methylene chloride, chloroform, etc.) Oxidizes with pyridinium chlorochromate in an alkyl solvent. In this case, pyridinium chlorochromate is used in an amount of 1 to 3 mol based on the compound (m),

— React at 10 ° -50 ° C, preferably at room temperature, for 1-30 hours.

Also, after treating the compound (m, where n-1) with t-butoxide in tert-butanol, it is reacted with promocetal, and then an organic acid (such as trifluoric acid) or an inorganic acid (such as hydrochloric acid). more compounds that be treated with an acid such as, sulfuric acid) (ΠΙ) from one 0 CH ₂ CH ₂ - can be prepared a long corresponding aldehyde of鎮長([pi). In this case, potassium t-butoxide is added and dissolved in an amount of 10 to 30 mol based on the compound (1), and bromoacetal is added in an amount of 3 to 15 mol based on the compound (dish). The reaction is carried out at 0.5 ° C. for 0.5 to 5 hours. After post-treatment by a conventional method, the mixture is dissolved in a dilute aqueous solution of the above acid and ripened for 5 minutes to 2 hours.

 Each reaction solution can be purified by ordinary chemical treatments such as extraction; concentration, recrystallization, reprecipitation, chromatography, and distillation.

 The chemically modified lymphokine of the present invention has the same useful physiological activity as the corresponding known unmodified lymphokine, and is useful as a pharmaceutical or the like.

 The chemically modified lymphokine of the present invention has a delayed in vivo clearance, and exhibits its activity effectively for a long time, has low toxicity and antigenicity, and has low toxicity and antigenicity as compared with the corresponding known unmodified lymphokine. It can be used safely for similar purposes and in similar usage.

 The chemically modified lymphokine of the present invention can be orally or parenterally administered to mammals (monkeys, dogs, pigs, rabbits, mice, and humans) orally or parenterally as appropriate pharmaceutical compositions using known carriers, diluents, and the like. Can be administered.

For example, when the chemically modified IFN- of the present invention is used as an antiviral agent, it is preferable to administer 1 × 10 ⁺ to 1 ^× 10 ^s international unit by intravenous injection once daily for an adult. . In the present specification, when an amino acid is represented by an abbreviation, it is based on IUPAC—Commission of Biological Nomenclature (IUB).

 The transformant Escherichia coli disclosed in the following Reference Examples

 (Escherichia coli) 294 / pH I Ttrp 1101-d2 was deposited with the Institute for Fermentation, Osaka, Japan under the deposit number IF 0-14350, from June 6, 1984 Deposited with the Research Institute of Microbial Technology (FRI :) under the accession number FE RM P-7658. Escherichia coli DH 1 ZPT F 4 was deposited at the Fermentation Research Institute under the accession number IF 0-14299 and became FR I on April 6, 1984.

 Deposited as F ERM · ΒP—628.

 Brief explanation of drawings

 FIG. 1 shows the effect of prolonging clearance in rat plasma disclosed in Example 1 (iv). 〇 (enzyme immunoassay) and □ (antiviral activity) are the control of the chemically modified IF Ν-α of the present invention obtained in Example 1 (ί) (enzyme immunoassay) and 騸 (antiviral activity) The measurement results of rIFN-A are shown below.

 FIG. 2 shows the effect of delaying clearance in rat plasma disclosed in Example 3 (Π). Δ and □ show the results of enzyme immunoassay of compounds NO. 8 and NO, 2 in Table 1, respectively, and Hata shows the results of enzyme immunoassay for rIFN-A as a control.

 Fig. 3 shows the construction diagram of the expression plasmid pH ITtr 1101-d2 disclosed in Reference Example 3 (i), and Fig. 4 shows the construction diagram of the expression plasmid PLC2 disclosed in Reference Example 4 (i). Shown respectively.

 BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described more specifically with reference to Examples and Reference Examples, but the present invention is not limited thereto. Example 1 Production of polyethylene glycol methyl ether-modified I FN-α:

 (i) Take 5 ml of the solution of IFN-a (rIFN-a A) (4.8 mg in terms of protein mass), and add 4 ° C to Q.2M phosphate buffer (PH7.0) + 0.15M salt. For 12 hours. The dialysate was removed, and the polyethylene glycol methyl ether aldehyde (average molecular weight 1900) (260 mg) obtained in Reference Example 1 was added thereto, followed by sodium cyanoborohydride (UOmg). C for 40 hours. The reaction solution was poured into a column of Sephadex G-75 (3. OX 43.0 cm), and developed with 25 mM ammonium acetate buffer (PH5.0) +0.15 M salt. Fractions (5 ml each) were collected, and fractions (100 to 150πι1) containing the desired product were collected. The protein content of this fraction was determined using bovine serum albumin as standard.

It was 84, "gZmi when measured by the (Lowry) method. The amino acid analysis values in the acid hydrolyzate (6 N hydrochloric acid, U0 ° C for 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, 4.5 (5); lie, 7.6 (8); L-eu, 21.0 (21); Tyr, 5.2 (5); Pe, 9.9 (10); Lys, 6.5; His, 3.8 (3) Arg, 9.1 (9); Cys, Trp, Decomposition, rl FN—a A contains 11 Lys, so from the above results, the e-amino group of Lys in interferon It was found that about% of the product had been modified with voletylene glycol methyl ether (average molecular weight: 1,900) This product was used in an enzyme immunoassay [Methods in Enzymology, Vol. 79, 589-595 ( 1981)]. The antiviral activity was determined to be 1.51 × 10 ⁷ international units / cm2, and the antiviral activity was 0 according to the method described in Journal of Virology, Vol. 37, pp. 755-758 (1981). .57 X 10 ⁷ IU was ZMG. This article a (IFA- 3) were subjected to clearance experiments in Raţ bets later.

(Π) Polyethylene dalicol meth having an average molecular weight of 750 obtained in Reference Example 1 Using lOOmg of Lutheraldehyde and lOOmg of sodium cyanoborohydride and treating rlFN-II in the same manner as (i), a solution of 130 g / ml of polyethylene glycol methyl ether-modified IFN-α with a protein mass of 130 g / ml was obtained. was gotten. The amino acid content in the acid hydrolyzate (6 N hydrochloric acid, 110, 24 hours) was as follows. : Asp, 12.1 (12); Thr.lO.l (lO); 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); lie, 7.4 (8); Leu, 21.0 (21); Thr, 5.1 (5); Phe, 9.6 (10); Lys, 4.7; His, 3.5 (3); Arg, 9.1 (9);

 Trp, 1.8 (2); Cys, degradation. Based on the results, this product is modified with about 57% of the Lys s'-amino group, and was measured by enzyme immunoassay as in (i).

In 5 X 1Q ⁸ international units / mg, anti-virus activity was riding in 0.14X10 ⁸ international units / mg _0, · '

 (iii) When the same treatment as in (i) was performed using 27 mg of polyethylene glycol methyl ether aldehyde having an average molecular weight of 750 and 27 mg of sodium cyanoborohydride obtained in Reference Example 1, a polyethylene glycol having a protein mass of 45 / g / ml was obtained. 50 ml of methyl ether modified IFN-a was obtained. The amino acid analysis values in the acid hydrolyzate (6 N hydrochloric acid, liO ° C, 24 hours) were as follows. : Asp, 13.6 (12); Thr, 10.4 (10); Ser, 14.9 (14); Glu, 26.6 (26); Pro, 5.5 (5); Gly, 6.1 (5); Ala, 8.3 (8 ); Val, 6.6 (7); Met, 5.2 (5);

lie, 7.4 (8); Leu, 21.0 (21); Tyr, 5.3 (5); Phe, 10.2 (10); Lys, 9.0; His, 3.6 (3); Arg, 9.1 (9); Trp, 2.3 (2); Cys, degradation From this result, it was found that about 8% of Lys was modified with the £ -amino group of Lys, and the result of enzyme immunoassay as in (i) 1.09 X 10 ⁸ international units Zmg The antiviral activity was 1.53 × 10 ⁸ international units / mg.

(iv) The chemically modified IFN— (IFA-3) of the present invention obtained in the above (i) was added to the thigh muscles of female 7-week-old SD rats in groups of 1.274 × 10 ⁶ units.

O PI Each animal was injected. After a certain period of time, blood was collected from the tail vein, and the IFN-α titer in the plasma was measured by the enzyme immunoassay described in Example 2 (i) and the antiviral activity. L.'259 x unmodified interpolated (rl FN—A)

Delaying the apparent clearance was observed with 10 ⁶ units Dzu' to Hi较the group administered.

 Figure 1 shows the results.

Example 2

 Example 1 Solution of Chemically Modified I FN—a (I FN—3) of the Present Invention Obtained in (〖)

 Dissolve 250 ml of human serum albumin in 5 ml. Filter this solution through a membrane filter (pore size: 0.201) and subdivide into 5 vials. Lyophilize aseptically, store and dissolve in 1 ml of distilled water for injection immediately before use.

Example 3 Production of polyethylene glycol methyl ether-modified IFN— and alkynylpolyethylene glycol-modified IFN—

 (i) The title compound was synthesized by the method described in Example 1 using the polyethylene glycol methyl ether aldehyde obtained in Reference Examples 1 and 2 or alkenylpolyethylene glycol alcohol aldehyde. Table 1 shows various data of the synthesized derivatives, and Table 2 shows the amino acid content.

(Ii) The chemically modified IFN-α (compound). 2 and 8) of the present invention obtained in (i) above were combined with 3.12 × 10 ^S units and 2.66 × 1 units in female female SD rats J, J, G Were injected into the thigh muscles of each group. After a certain time, blood was collected from the tail vein ', and the IFN- titer in the plasma was measured by enzyme immunoassay. Clear clearance delay was observed as compared to the group administered with 3.82 × 10 ⁸ units of unmodified IFN-a. Fig. 2 shows the results.

OMPI

Table 2

Phe 9.8 9.8 9.8 9.8 9.8 9.8 9.4 9.7 9.8 9.8

Take 5 ml (equivalent to 5.95 mg of protein) of a solution of the following rIFN-a: Abbreviation: EPC Publication No. 110044), pour it into a column of Sephadex G-25 (2.0 X 60.0 cm), It was developed with .2M phosphate buffer (PH7.0). Fractions were collected in 5 ml increments, and fractions 11 to 13 were collected. Dilute to 100 ml with the same buffer, add polyethylene glycol methyl ether aldehyde (average molecular weight 750) (225 mg), then sodium cyanoborohydride (300 mg) and shake at 37 ° C for 72 hours. Yes. The resulting precipitate was removed from the heart. The supernatant was concentrated to ^) n using Diaflo 1 (Namicon). This was poured into a column of Sephadex G-75 (3.0 × 43. Ocm), and developed with 25 mM ammonium acetate 'buffer (p.H 6.0) 4-0. I 5M salt + 10 mM glutathione. Fragments Nos. 17-24 containing the desired product were collected in 5 ml aliquots. The protein content of this fraction was determined to be 7.73 g / ml by the Bradford method using bovine serum albumin as a standard. The amino acid analysis values in the hydrolyzate (6 N hydrochloric acid, 110 ° C, 24 hours) were as follows.

Asp, 19.6 (20); Thr, 4.7 (5); Ser, 8.3 (11); Glu, 18.5 (18);

 Pro, 2.1 (2) G ly, 5.4 (5); Ala, 7.5 (8); Val, 8.4 (8);

Met, 3.7 (4) lie, 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, decomposition, rl FN-a contains 20 Lys, so the above results From the results, it was found that about 12% of the s-amino group of Lys in rIFN-r was modified with polyethylene glycol methyl ether (average molecular weight 750). The antiviral activity of this product was 1.3 × 10 ⁶ international units / mg. When this product was administered to rats, a clear delay in blood clearance was observed. On the other hand, the precipitate was dissolved in 6 M guanidine hydrochloride, and then subjected to transfection at 4 ° C against ammonium hydroxide (pH 6.0) + 0.15 M salt + 10 mM daltathione at 25 mM, and Sephadex as described above. G-75 gel filtration

O? R

,,;-, WIPO Made. The protein content of this fraction (25 ml) was S / ^ gZml, and the amino acid fraction of the acid hydrolyzate (6 N hydrochloric acid, U0 ° C, 24 hours) was as follows. Asp, 20.0 (20); Thr, 5.2 (5); Ser, 9.5 (11); G lu, 27.8 (18); Pro, 2.7 (2); Gly, 14.6 (5); Ala, 8.1 (8); Val, 8: 5 (8); Met, 4.3 (4); lie, 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, degradation, Glu and G values higher than theoretical values are likely due to glutathione contamination. Since rI FN-α contains 20 Lys s-amino groups, the above results show that about 26.5% of Lys s-amino groups in rI FN7 were modified with polyethylene glycol methyl ether. I knew it was being done.

 (ϋ) Using 225 mg of polyethylene glycol methyl ether aldehyde having an average molecular weight of 750 and 120 mg of sodium borohydride in the presence of 2-mercaptoethanol (2%), rlFN-a was prepared in the same manner as (ί :). After the treatment, 30 ml of a solution of polyethylene glycol methyl ether-modified rIFN-a having a protein content of 236 g / ml was obtained. The amino acid content of the acid hydrolyzate (6N hydrochloric acid, LltTC, 24 hours) was as follows. Asp, 20.0 (20); Thr, 5.2 (5); Ser, 9.6 (11): Glu, 33.6 (18); Pro, 1.8 (2); Gly, 19.9 (5); Ala, 8.2 (8); Val , 8.9 (8); Met, 4.6 (4); I le, 7.4 (7); Leu, 10.2 (10); Tyr, 5.9 (5); Phe, 10.7 (10): Lys, 10.2;

His, 2.3 (2); Arg, 7.9 (8); Trp, 0.6 (1); Cys, degradation, Glu and Gly values higher than theoretical values are likely due to glutathione contamination. Since rIFN-a contains 20 Lys ε-amino groups, the above results indicate that about 50% of Lys ε-amino groups in rFN-7 are polyethylene glycol. It was found to be modified with methyl ether.

Example 5 Polyethylene glycol methyl ether-modified IF 7—7 d 2 Manufacturing of

 (i) Take 5 ml (4.95 mg protein) of the IFN-7 d2 solution obtained in Reference Example 3 and pour it into a Sephadex G-25 column (2.0 × 60.0 cm), Develop with phosphate buffer (PH7.0). Take 5ml aliquots and collect fraction N0.11-13. Dilute to 100 ml with the same buffer, add poly (ethylene glycol) methyl ether aldehyde (average molecular weight 750) (200 mg), and then add sodium cyanoborohydride (300 mg) and shake at 37 ° C for 72 hours. . The precipitate that forms is removed by centrifugation. The supernatant is concentrated to 10 ml using Diaflo (manufactured by AMICON). This is poured into a power ram (3.0 × 43.0 cm) of Sefade, Sox G-75, and developed with 25 mM ammonium acetate buffer (pH 6.0) +0.15 M salt + 10 mM glutathione. Collect fractions of 5 ml each, and collect the fraction containing IFN-ad2 in which the s-amino group of Lys in the molecule has been modified with polyethylene glycol methyl ether. When this product is administered to rats, a marked delay in blood clearance is observed.

 On the other hand, the precipitate was dissolved in 6M guanidine hydrochloride, and then was allowed to pass through 25 mM ammonium acetate (PH 6.0) + 0.15M salt + ΜιΜΜdartathione at 4 ° C, followed by Sephadex G-75. Purification by gel filtration yields a fraction containing IFN-ad2 in which the ε-amino group of L in the molecule has been modified with polyethylene glycol methylate.

Example 6 Preparation of Polyethylene Glycol Methyl Ether Modified IFN—yd3

(i) Take 5 ml of the IFN-d3 solution obtained in Reference Example 4 (5.5 mg in terms of protein), pour into a column of Sephadex G-25 (2.0 × 60.0 cm), and add 0.2 M Develop with phosphate buffer (PH7.0). Collect fractions of 5 ml each and collect fractions N0.11-13. This was followed by polyethylene glycol methyl ether aldehyde (average molecular weight 750) (225 mg :), followed by sodium cyanoborohydride. Add shake (i20mg) and shake at 37 ° C for 24 hours. The reaction mixture was poured into a Sephadex G-75 column (3.0X43. Ocm), developed with 25 mM ammonium phosphate (PH 6.0), and the s-amino group of Lys in the molecule was modified with polyethylene glycol methyl ether. A fraction containing IFN-7d3 is obtained. When this product is administered to rats, a clear delay in blood clearance is observed.

Example 7 Production of Polyethylene Glycol Methyl Ether Modified IL-12 (i) 5 l of a solution of interleukin-12 (abbreviated as rIL-2) obtained in Reference Example 5 (S.Onig ) Was taken and subjected to a 12-hour pass through 0.2 M phosphate buffer (pH 7.15). To the dialysate were added polyethylene glycol methyl ether aldehyde (average molecular weight 750) (97 mg), and then sodium cyanoborohydride (lOOnig), and the mixture was stirred at 37 ° C for 24 hours. The formed precipitate was removed by centrifugation. The supernatant was passed through 5 mM ammonium phosphate buffer (PH5.0) for 5 hours. The translucent solution was applied to a Sephadex G-75 column (3.0 X 43.0 cni) and developed with the same solvent system. Fractions Nos. 21 to 29 containing the target substance were collected in fractions of .5 ml. The protein content of this fraction was 25 / zg / ml as determined by the Bradford method using bovine serum albumin as a standard. The amino acid analysis values of the acid hydrolyzate (6 N hydrochloric acid, U0 ° C, 24 hours) were as follows. 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); lie, 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, degradation, rI L-2 contains 11 values of Lys, so from the above results, Approximately 33.6% of the groups were found to be modified with polyethylene dalicol methyl ether. In this product, IL-12-dependent mouse nut Rarukira - growth ^[3 1? Cell line (NK C 3) - thymidine day swamp et al., Which is measured as an indicator of the interrupt taken to the DNA [Biochemical Anne Dobaiofi Jikari Lisa - Chikonmi Yunikeishon 109 Volume 363-369 According to the method described on page (1982)], the IL-2 activity was 22998 units Zmg. Assuming that rIL-12 is 40,000 units Zmg, it means that it retains 57.7% activity. When this product was administered to rats, a clear delay in blood clearance was observed.

Reference Example 1 Synthesis of polyethylene glycol methyl ether aldehyde

(i) Polyethylene glycol methyl ether (5 g, average molecular weight 5,000) was dissolved in methylene chloride (100 ml), chloro'pyridinium chromate (330 mg) was added, and the mixture was stirred at room temperature for 12 hours. Dilute the reaction solution with twice the volume of methylene chloride, pour it into a florisil column (6 x 10 cm), wash the column with methylene chloride and then with chloroform, and then add methanol in chloroform (1: 9). ). Positive fractions were collected by 2,4-dinitrophenylhydrazine test, and the solvent was distilled off under reduced pressure to obtain a crystalline wax. Yield: 1.5 g (30%), thin layer chromatography: Rf = 0.08 (cloth form: methanol: g but acid-9: 1: 0.5, silica gel), ¹³ C-1'11 Absorption of the aldehyde group was observed in the hydrated form (1 £ H (.0 H) ₂ ) in 96.2 PPM.

(ii) Polyethylene glycol methyl ether (10 g, average molecular weight 5,000) was dissolved in tertiary butanol (100 ml), and tertiary butoxide (4.17 g) was added, followed by bromoacetal (2.56 ml). In addition, I stir for 2 hours at 40 mm. Tertiary butanol was distilled off under reduced pressure, water was added to the residue, and the mixture was extracted with chloroform (200 ml × 2). It was washed with water and dried over anhydrous sodium sulfate. The chloroform was distilled off under reduced pressure, petroleum benzene was added to the residue, and the resulting crystalline residue was collected by filtration, washed with ether and washed with 9.5 g (95%) of the corresponding polyethylene glycol methyl ether getyl acetal. ) Was done. Take 5 g of this, dissolve in 50mU of 0.05M trifluorosulfonic acid, treat in boiling water for 30 minutes, freeze-dry, and reduce the chain length by-0—CH ₂ CH ₂ compared to that obtained in (〖). A long polyethylene glycol methyl ether aldehyde was obtained.

 (iii) Polyethylene glycol methyl ether (5.7 g, average molecular weight

900) was dissolved in methylene chloride (100 ml), pyridinium chlorochromate (970 mg) was added, and the mixture was stirred at room temperature for 12 hours. The reaction solution was diluted with methylene chloride, poured into a florisil column (6.0 × 10 Ocm), and the column was washed with methylene chloride and then with chloroform, and eluted with 10% methanol Z-chloroform. Fractions positive in the 2,4-dinitrylphenylhydrazine test were collected, and the solvent was distilled off to obtain a crystalline wax. Yield 1.½ (30%), thin layer chromatography: Rf = 0.10 (chloroform: methanol: acetic acid = 9: 1: (J.5, silylation gel) 96.2P PM by ¹³ C-NMR Hydrated in the form (1 £ H (0H) ₂ ), absorption of an aldehyde group was observed.

 (iv) polyethylene glycol methyl ether (l_9.5g, average molecular weight

1900) in tertiary butanol and GOml)

(10.4 g) was added, followed by bromacetal (6.4 ml), and the mixture was stirred at 40 ° C for 2 hours. Tertiary butanol was distilled off under reduced pressure, water was added to the residue, and the mixture was extracted with sigma-form (200nilx2). The reaction solution was washed with water, and then dried over anhydrous sodium sulfate. Chloroform was distilled off under reduced pressure, petroleum benzene was added to the residue, and the resulting crystalline residue was collected by filtration and washed with ether to obtain 8.5 g (89.5%) of acetal. Dissolve 3 g of this in 0.05 M trifluoroacetic acid, treat in boiling water for 30 minutes, freeze-dry, and-0-CH ₂ CH _2- longer polyethylene than that obtained in (m). Glycol methyl teraldehyde was obtained.

 (V) Polyethylene glycol methyl ester with an average molecular weight of 750, 550, 350.

ΟΜΡΙ One Tell also led to the corresponding aldehyde in the same way as above.

 Reference Example 2 Synthesis of alkenylpolyethylene glycol alcohol aldehyde

 (i) Dissolve 15 g of polyethylene glycol 1540 (manufactured by Wako Pure Chemical Industries) having an average molecular weight of 1500 in 50 ml of pyridine, add 1.85 ml of acetic anhydride, and stir.

 The reaction was carried out at 40 ° C for 2 hours and further at room temperature for 16 hours. After the reaction, the solvent was distilled off under reduced pressure. After dissolving in water, the water was dried over anhydrous sodium sulfate, and the water was distilled off under reduced pressure. The residue was dissolved in a small amount of chloroform, a petroleum benzene monoether (2: 1) mixture was added, and the mixture was allowed to stand, to obtain 14 g (90%) of crystalline wax. 1.4 g of this was dissolved in 50 ml of methylene chloride, 300 mg of pyridinium chlorochromate was added, and the mixture was stirred and reacted at room temperature for 18 hours. The reaction solution was passed through a column of silica gel C-200 (manufactured by Wako Pure Chemical Industries, Ltd.) (3 x 50 cm), washed with 5% methanol in single-mouthed form (200 ml), and eluted with 10% methanol-chloroform. A 2,4-dinitrophenylhydrazine test positive fraction was collected and the solvent was distilled off under reduced pressure to obtain a crystalline wax. Yield 580mg (41%)

 (ii) 20 g of polyethylene glycol 1000 (manufactured by Wako Pure Chemical Industries, Ltd.) having an average molecular weight of 1000 was dissolved in 50 tnU of methylene chloride, and 5.15 g of anhydrous n-caproic acid was added, followed by reaction at 70 ° C. for 2 hours. The solvent was distilled off, and the residue was purified using a silica gel C-200 (3 x 50 cm) column, eluting with ethyl acetate-methanol (4: 1), and 14.9 g (60% ). The aldehyde was obtained by oxidation with pyridinium chlorochromate in the same manner as in (i).

Reference Example 3 Production of I FN-7d2

 (i) Production of transformants

 Avail—Pst1 containing the IFN—y gene portion, which is obtained by digesting IFN—7 expression plasmid PH I Ttrp 1101 [EPC Publication No. 110044, Example 2 (ίίί)] with restriction enzymes Ava and PstI. A kb DNA fragment was collected. This DN

ΟΜΡΙ

レ 7> ,. — Oligonucleotide adapter containing the protein synthesis initiation codon chemically synthesized by the triester method in fragment A

 C GATAAT GT G C CAG

TATTACAC GGT C CTG

Was bound to the margin of Avail using T 4 DNA ligase.

 Plasmid Ptrp 7Π [see Example 2 (Π) in the above publication]]

Downstream of the trp promoter of the DNA fragment obtained by digestion with Clal and Pst I

 "1 2

The above-mentioned gene to which the above-mentioned adapter is bound is introduced, and an expression plasmid PHI Ttrp encoding the polypeptide of Cys-Tyr: missing IFN-α

U01—d2 was constructed (Fig. 3).

 Escherichia coli 294 was transformed with this plasmid PHI Ttrp1101-d2 according to the method of Cohen et al. [Proceding of National Academy of Sciences US A, Vol. 69, 2il0 (1972)]. A transformant containing this plasmid (Escherichia coli = E.

coli) 294 / pHI Ttrp1101-d2.

 (Π) Transformant culture

 The above (E. coli 294 / pH I Ttrp1101-d2 containing the plasmid constructed in (1) was converted to 37 ° C using 8 jiig ml of M9 medium containing tetracycline, 0.4% casamino acid, and 1% dulose. C. When the growth reached KU220, 3; 5 indolylacrylic acid (IAA) was added thereto at a concentration of 25 zg / mU, and the cells were further cultured for 4 hours. The body was collected and suspended in 0.05 M Tris-HCl pH 7.6 containing 1/10 volume of 10% sucrose, and the suspension was mixed with phenylmethylsulfonyl fluoride, NaC 1. ethylene diamine tetraacetate. (EDTA), spermidine, and lysozyme to 1 mM, 10 mM, 40 niM, and 200 gZml, respectively.

ΟΜΠ WWIIPPOO After standing for a period of time, the cells were treated at 37 ° C for 3 minutes to obtain a lysate.

 This lysate is centrifuged at 4 ° C, 20000rpm (with a S-34 rotor by Serval centrifuge).

After centrifugation for 30 minutes, a supernatant containing IFN-ad2 polypeptide was obtained.

When measuring the antiviral activity of the supernatant was 2.87 X 10 ⁸ U / £ broth ₀

 (iii) Purification of IFN-d2

5.9 g of the frozen cells obtained in the same manner as in the above (上 記) are suspended in 18 ml of a 0.1 M Tris-HCl buffer (pH 7.0) containing 7 M guanidine hydrochloride and 2 mM phenylmethylsulfonyl fluoride. After stirring for 1 hour at 4 minutes, the mixture was centrifuged at 10,000 X g for 30 minutes to obtain 20 mi of the supernatant. To this supernatant was added 260 ml of a buffer solution (PH7.4) (hereinafter abbreviated as PBS) consisting of 137 mM sodium chloride, 2.7 mM chloride, 8.1 mM sodium phosphate and 1.5 mM phosphate monolith. After dilution, the solution was applied to an antibody column (Mo ₇ 2-11.1, column volume 12 ml) at a flow rate of 1 l / min. After that, the column was washed with 60 ml of 20 mM sodium phosphate buffer (pH 7.0) containing guanidine hydrochloride, and then eluted with 36 nd of 20 mM sodium phosphate buffer (pH 7.0) containing 2 M guanidine hydrochloride. As a result, 20 nU of a fraction having antiviral activity was obtained.

 20 ml of this fraction was pre-equilibrated with 25 mM ammonium phosphate buffer (PH6.0) containing 1 mM ethylenediamintetraacetic acid, 0.15 M sodium chloride, 10 mM cysteine and 2 M guanidine hydrochloride (PH6.0), and Cefacryl S-200 (Pharmacia) (2.6 x 94 cm, column volume 500 ml) and eluted with the same buffer to obtain 37 ml of a fraction having antiviral activity.

 1 2

The obtained Cys- Tyr missing IFN-y Poribe Petit de of (I FN- § d2) is at 5.9mg specific activity was found to be (1.0xl0 ⁷ U / mg).

Reference Example 4 Production of IFN-rd3

OMPI one (i) Production of transformants

 I FN-Expression Brasmid PRC 23 '/ I F I-900 [EPG Publication No.

Limiting 0089676 Patent reference JP Example 7 Enzyme i ^T de I, digested with NcoI, I FN

The Ndel-Ncol 710 bp DNA fragment (A) containing the -a gene portion was collected. On the other hand, plasmid PR C23 was digested with restriction enzymes BglE and EcoRI,

A 265 bp DNA fragment (B) containing the APL promoter was collected. (A),

Oligonucleotide adapter containing a protein synthesis initiation codon obtained by chemical synthesis with (B) ''

 AATT CATG CAGGAT CCA GTACGT C CTAGGTAT

Was ligated to the margin of NdeI and EcoRI using T4DNA ligase. The DNA fragment obtained was treated with NcoI and BglII.

 . one two Three

 * Mid pR C 23 No I F I-900, Cys—Tyr—Cys missing I FN—

An expression plasmid PLC2 encoding 7 polypeptides was constructed. (No.

Fig. 4) Using this plasmid PLC2, E. coli RR I (PRK248 cits) was transformed according to the method of Cohen et al. [Supra], and a transformant Escherichia coli (E. coli) RR was used. I (pLC2, pR248cIts) was obtained.

 (ii) Culture of transformants

A liquid medium containing 1% pacttribubutone, 0.5% yeast extract, 0.5% salt, and 7 ^ gZml tetracycline was prepared from the plasmid-containing strain E. coli RRI (PLC2, PRK248cIts) containing the plasmid constructed in (i) above. Shaking culture was performed at 35 ° C for 12 hours in 50 ml. The culture was transferred to 2.5 JT of M9 medium containing 0.5% casamino acid, 0.5% glucose and 7 ml of tetracycline, and cultured at 35 ° C for 4 hours and then at 42 ° C for 3 hours. Collect cells by eccentric separation and store at -80 ° C did.

 (iii) Purification of IFN-d3

 Frozen cells obtained in the same manner as in (I) above. 7. Ig is 22 ml of 0.1 M Tris-HCl buffer (pH 7.0) containing 7 M guanidine hydrochloride and 2 mM phenylmethylsulfonyl fluoride. After stirring at 4 ° C for 1 hour, the mixture was subjected to eccentric separation at 10,000 X g for 30 minutes to obtain 24 ml of a supernatant. The supernatant was diluted with 300 ml of a buffer solution (pH 7.4) consisting of 137 mM sodium chloride, 2.7 mM chloride, 8.1 mM sodium phosphate and 1.5 mM phosphate, and diluted with the antibody. Mo 7 2 -11.1, column volume 15 ml) at a flow rate of 1 ml / min. Thereafter, the column was washed with 60 ml of 20 mM sodium phosphate buffer (pH 7.0) containing 0.5 M guanidine hydrochloride, and then eluted with 45 ml of 20 mM sodium phosphate buffer (pH 7.0) containing 2 M guanidine hydrochloride. Thus, 25 ml of a fraction having antiviral activity was obtained. Sephacryl S-200 (manufactured by Pharmacia) was preliminarily equilibrated with 25 ml of this fraction with a mM ammonium acetate buffer (PH 6.0) containing 1 mM ethylenediamine tetraacetic acid, 0.15 M sodium chloride, 10 mM cysteine and 2 M guanidine hydrochloride. ), And eluted with the same buffer to obtain 40 ml of a fraction having antiviral activity.

 1 <) 3

The obtained Cys- Tyr- Cys lack IFN- § of Poribe peptide (IF N- yd3) is located specific activity at 7.0mg was 2.72xl0 ⁷ U / mg.

Reference Example 5 Production of specific glycosylated human IL-2

 (i) Culture of transformants

 The transformant E. coli DHl / pTF4 [see the specification of Japanese Patent Application No. 58-225079 (filed on Nov. 28, 1983)] was prepared by transferring the tryptone (difuco labola) in a 250 ml Erlenmeyer flask. (Tries, America) 1%, Park toys

OM «I (Difco Laboratories, America) was inoculated into 50 ml of a liquid medium (pH 7.0) containing 0.5% of sodium chloride, 0.5% of salt and 7 zgZnd of tetracycline, and cultured at 37 ° C. with shaking for 1 °. The culture was transferred to a 5-pound jar fermenter containing 2.5J2 of M9 medium containing 0.5% casamino acid, 0.5% glucose and 7 g / ml tetracycline and transferred at 37 ° C for 4 hours, followed by 3-^-indolylacrylic acid (25 zg / nil), and the mixture was further cultured under aeration and agitation for 4 hours to obtain a culture solution 2.5J2. This culture wave was centrifuged, and the cells were collected, frozen at 180 ° C and stored. '

 (Π) Extraction

 The frozen cryopreserved bacterial cell obtained above was suspended uniformly in 100 mU of an extract (pH 7.0) containing 7 lg of guanidine hydrochloride J.1M Tris-HC1, and stirred at 4 ° C. for 1 hour. This lysate was centrifuged at 28, OOOxg for 20 minutes to obtain 93 ml of supernatant.

 (iii)-IL-1 protein; purification of white matter

 The supernatant obtained above was permeabilized with an O.iUM: Tris-HCl buffer (pH 8.5) and then eccentrically separated at 19,00.0 Xg for 10 minutes to obtain 94 ml of a permeate supernatant. The supernatant was passed through a DE 52 (DEAE-cellulose, Pittman, England) column (50 ml) equilibrated with OiM Tris-HCL buffer (PH8.5) to adsorb the protein. A linear gradient of NaCl concentration (0 to 15 MaCi, 12) was prepared to elute IL-2, and 53 ml of an active fraction was obtained.

The above active fraction (53 ml) was concentrated to 4.8 ml using a YM-5 membrane (Amicon, Ameri force) and equilibrated with 0.1 MT ris · HC 1 (ρΗ8.0)-1 M NaCl buffer. Gel filtration was performed using an S-200 (Pharmacia, Sweden) column (500 ml volume). 28 ml of the active fraction was concentrated to 25 ml with YM-5me / Bran. The resulting concentrate is adsorbed on an Ultrapore RP SC (Altex, USA) column, and high-performance liquid chromatography using trifluoroacetic acid-acetonitrile as an elution solvent. I did one. Column, Ultrapore RPSC (4.6X75IM); Column temperature, 30 ° C; Elution solvent Α, Ο. 1% trifluoroacetic acid-99.9% water; Elution solvent Β, 0.1% trifluoroacetic acid-99.9% acetonitrile; Elution program, 0 min (68% Α + 32% Β)-25 minutes (55% Α + 45% Β)-35 minutes (45% A + 55% 45)-45 minutes (30% A + 70% B)-48 minutes (100 % B); elution rate, 0.8 ml / min; detection wavelength, 230 nm _o The active fraction with a retention time of about 39 minutes was collected under these conditions, and non-glycosylated human IL-12 protein G.53 mg [specific activity, .OOOUZtng Thus, 10 ml of a solution containing 30.6% of the activity recovery from the starting material; purity of the protein, 99% (by densitometry) was obtained.

 Industrial applicability

-The chemically modified lymphokines produced by the present invention are useful as pharmaceuticals and the like. .

Claims

The scope of the claims At least one primary amino group in a molecule RO—A chemically modified lymphokine directly linked to a CH ₂ CH2¾ group (R is a terminal oxygen protecting group, n is a positive integer that can be arbitrarily changed). 2. Claims characterized in that lymphokine is reacted with an aldehyde represented by RO—CH ₂ CH ₂ ¾ = rO—GH ₂ CH0 (R and II are as defined above) in the presence of a reducing agent. 2. The method for producing the chemically modified lymphokine according to claim 1. -