KR920007681B1 - Process for production of chemically modified lymphokine - Google Patents

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Description

Method for preparing chemically modified lymphokines

(항 비루스 활성분석)으로 표시되고, 대조군으로 사용된 rIFN-αA에 의해 수득된 결과는 ●(효소 면역분석) 및 ■(항비루스 활성분석)으로 표시된다.1 shows the delayed clearance effect in rat plasma described in Example 1 (iv). The measurement results obtained by the chemically modified IFN-α of the present invention prepared in Example 1 (i) were ○ (enzyme immunoassay) and

The results obtained by rIFN-αA used as a control (antiviral activity assay) are indicated by ● (enzyme immunoassay) and ■ (antivirus activity assay).

및 ●로 표시된 데이터는 각각 화합물 번호 8, 화합물 번호 2(표1) 및 대조 rIFN-αA의 효소 면역분석 데이터이다.2 shows the delayed clearance effect in rat plasma described in Example 3 (ii). △,

And the data marked with and ● are enzyme immunoassay data of Compound No. 8, Compound No. 2 (Table 1) and control rIFN-αA, respectively.

3 shows a formation scheme of the transgenic plasmid pHITtrp 1101-d2 described in Reference Example 3 (i).

4 shows a formation scheme of the expression plasmid pLC2 described in Reference Example 4 (i).

The present invention relates to a process for the preparation of chemically modified lymphokines, in particular the general formula R (-O-CH₂-CH₂-) n (I) (wherein R is a protecting group of a terminal oxygen atom, n is any positive integer), and relates to a process for producing a modified modified lymphokine with polyethyleneglycol.

Lymphokines such as interferon (hereinafter abbreviated IFNS) and interleukin-2 (hereinafter abbreviated IL-2) are clinically valuable for the treatment of viral infections and malignancies, and recent technological advances in genetic engineering Lymphokine can be produced in large quantities. However, the clearance of lymphokines administered in vivo is generally very short. In the case of lymphokines derived from atypical animals, antibodies are produced in some cases and may cause severe reactions such as anaphylaxis. Therefore, there is a need for technical development that delays the clearance of lymphokines used as medicaments and reduces their antigenicity while maintaining the activity of lymphokines. To achieve this goal, the analytical modification of lymphokines is a very effective means. Such chemical modifications delay the clearance of the living body, reduce antigenicity, and also increase physiological activity. From a practical standpoint, the chemical modification of lymphokines is of great importance.

In general, for chemically modified physiologically active proteins, there is a need for a method that can chemically modify such proteins while maintaining their physiological activity. Polyethylene glycol meryl ether is used for chemical modification of protein because it is not antigenic. Introduction of the material into the protein is generally carried out by mediation of cyanuricchloride. However, cyanuryl chloride itself is toxic, and its degradation products are likely to be toxic in vivo. Therefore, cyanuric chloride should be used with caution. Moreover, since this reaction requires an alkaline pH, the above-described modification method has a drawback that cannot be used for proteins that are inactivated under alkaline conditions.

US Pat. No. 4,002,531 describes a process for the preparation of monoalkyl polyethylene glycol derivatives of enzymes. However, the method described here uses sodium borohydride at pH 8.5 and, when used with lymphokines, can destroy the physiological activity of lymphokines and therefore cannot be considered an effective preparation method. In addition, there is no suggestion in the patent specification regarding the effect of delaying in vivo clearance of enzyme derivatives. Therefore, this effect is not known until now.

In addition, methods for introducing low molecular aldehydes, such as formaldehyde, acetaldehyde, benzaldehyde or pilipoxal, into physiologically active proteins in the presence of boron-containing reducing agents are known. Methods in Enzymology, 47, 467-478 (1977); Japanese Patent Laid-Open No. 154,596 / 1983] However, applying this method to lymphokines cannot achieve an effective delay of clearance. While no substantial reduction in antigenicity can be expected, the small molecule aldehydes introduced act as haptens to provide the above-mentioned Limpkin with immunogenicity.

The present inventors have extensively researched to complete the present invention in order to overcome the above drawbacks.

The present invention relates to the general formula R (-O-CH₂-CH₂-) n (I), wherein R is a protecting group of a terminal oxygen atom, directly bonded to one or more primary amino acids of the lymphokine molecular sieve, where n is any amount It provides a process for producing a chemically modified lymphokine having a polyethylene glycol (integer).

As used herein, "lymphokine" includes cytoimmunolytic soluble factors and physiological activity equivalent to those released from lymphocytes.

Therefore, lymphokines may be genetically engineered products, and products derived from various animals including the human body, and also include substances similar in structure and physiological activity.

For example, various interferons (interferon-α (IFN-α), interferon-β (IFN-), interferon-γ (IFN-γ)), IL-2, macrophage differentiation factor (MDF), macrophage activator (MAF), tissue plasminogen activator, and similar substances in structure and physiological activity may be mentioned.

Examples of similar substances in structure and physiological activity include substances having the structure of IFN-γ (PCT / JP84 / 00292, June 6, 1984 6), except that the N-terminus lacks 2-4 amino acids. Various IFN- [gamma] fragments lacking the C-terminal portion of [gamma] (e.g., 15K species: Korean Patent Application No. 5712/1984), IL- except that the N-terminal amino acid is lacking or the 4-amino acid from the N-terminus is insufficient A substance having the structure of IL-2, except that it lacks a substance having the structure of 2 (EPC (public) 91539) and one or more constituent amino acids, and with or without one or more substituted amino acids, eg For example, there is an IL-2 analogue (EPC (published) 104798) containing serine instead of the 125 th amino acid cysteine.

Among the lymphokines, IFN-α and IFN-γ [146 amino acids composed of 146 amino acids (EPC (public) 0089676)], two N-terminal amino acids lacking IFN-γ (IFN-γ d2) three N-terminal amino acids Insufficient IFN-γ (IFN-γ d3) and IL-2 are preferred.

The lymphokines used to practice 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 α-amino group and the ε-amino group of the lysine residue.

In the group represented by the above general formula (I), the terminal oxygen protecting group R is, for example, an alkyl or alkanoyl group. The alkyl group is preferably alkyl of 1 to 18 carbon atoms, more preferably lower (C1-4) alkyl such as methyl, ethyl, propyl, I-propyl, butyl, I-butyl, sec-butyl or t-butyl. Alkanoyl groups are preferably alkanoyl of 1 to 8 carbon atoms, more preferably lower (C1-6) alkanoyl such as formyl, acetyl, propionyl, butyryl, I-butyryl or caproyl. Positive integer n becomes like this. Preferably it is 500 or less, More preferably, it is 7-120.

The group of the general formula (1) preferably has a molecular weight of 25,000 or less, more preferably 350 to 6,000. In view of maintaining physiological activity and delaying clearance, the group of the general formula (I) has a molecular weight corresponding to 1 to 10%, more preferably 2 to 5% of the modified lymphokine molecular weight.

Chemically modified lymphokines according to the present invention have a group of general formula (I) attached directly to one or more of the primary amino groups of the corresponding lymphokines.

When the N-terminal α-amino acid is the only primary amino group in the modified lymphokines, the modified lymphokines have a group of formula (I) directly bonded to the amino group. When the modified lymphokines have one or more lysine residues in the molecule, the modified lymphokines have a group of formula (I) directly bonded to the ε-amino group by a few percent, preferably 15-80% (average). Have In this case, the N-terminal α-amino group may or may not have a group of formula (I) directly bonded thereto.

The chemically modified lymphokines according to the present invention may be formulated, for example, in the presence of a reducing agent, with the general formula R (-O-CH₂CH₂-) n-1 O-CH ₂ CHO (II) (wherein R and n are It can be prepared by reacting with the aldehyde (as defined).

Boron-containing reducing agents used in carrying out the reaction include sodium boro hydride and sodium cyanoboro hydride. Among them, sodium cyanoborohydride is more preferable in view of the selectivity of the reaction or the possibility of carrying out the reaction near neutral.

In carrying out the reaction, aldehyde (II) is used in an amount of about 1 to 10,000 moles per mole of lymphokine, and a boron-containing reducing agent is used in an amount of about 1 to 100 moles per mole of lymphokin. The degree of modification can be chosen as needed by varying the molar ratio between lymphokine and aldehyde (II). The solvent used to carry out the reaction may be any solvent as long as it does not interfere with the reaction, for example buffers such as phosphate or borate buffers are used. Organic solvents such as lower alkanols (e.g. methanol, ethanol, i-propanol) or acetonitrile may be added that do not inactivate the lymphokines or interfere with the reaction. This reaction can be carried out within a wide pH range of 3-14, but is preferably carried out near neutral (pH 6.5-7.5). The reaction temperature may be selected in a wide range of 0 ~ 80 ℃, in order not to cause denaturation of lymphokin is preferably carried out at 0 ~ 50 ℃. The reaction time is 0.5 to 100 hours, generally 10 to 80 hours is sufficient. Desired chemically modified lymphokines can be obtained by treating the reaction mixture by dialysis, salting out, ion exchange chromatography, gel filtration, high yield liquid chromatography, electrophoresis or other known protein purification methods. The degree of modification of the amino group can be calculated, for example, by acid degradation and amino acid analysis.

The above-mentioned aldehyde (II) can be prepared, for example, from ethylene glycol derivatives of the general formula R (-O-CH ₂ CH _2- ) n OH (III), wherein R and n are as defined above. have. The process for preparing aldehyde (II), which is advantageous in that little corresponding carboxylic acid is produced, is shown below.

Compound (III) is oxidized to pyridinium chlorochromate in a haloalkane solvent such as methylene chloride or chloroform. In this case, 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 DEG C, preferably at room temperature for 1 to 30 hours.

Compound (III) (n-1) was treated with potassium butoxide in t-butanol, reacted with bromo acetal and then acid such as organic acid (e.g. trifluoroacetic acid) or inorganic acid (e.g. hydrochloric or sulfuric acid) By treatment with a corresponding aldehyde (II) can be obtained in which the chain is longer by -O -CH ₂ CH ₂ -than compound (III). In this case, 10-30 mol of potassium t-butoxide per mole of compound (III) is added to the compound and dissolved, followed by addition of 3-15 mol of bromo acetal per mole of compound (III), followed by 10 to 80 ° C. React at 0.5 to 5 hours at. After the reaction mixture has been treated by a known method, the product is dissolved in a dilute aqueous solution of the acid described above and heated for 5 minutes to 2 hours.

In each case, the reaction mixture can be purified by methods known in the chemical art such as extraction, concentration, recrystallization, reprecipitation, chromatography and / or distillation.

Chemically modified lymphokines according to the present invention have useful physiological activities in comparison with the corresponding known unmodified lymphokines and are useful as medicaments and the like. The chemically modified lymphokines according to the present invention exhibit a delay in clearance in vivo as compared to known unmodified lymphokines, have low toxicity and antigenicity, and for the same purpose as for the known lymphokines. Can be used safely.

Chemically modified lymphokines according to the invention are usually orally or parenterally mammals (eg, monkeys, dogs, pigs, rabbits, mice, humans) in the form of suitable pharmaceutical compositions prepared using known carriers, diluents and the like. May be administered).

For example, when using the chemically modified IFN-α of the present invention as an antiviral agent, an adult may be administered by intravenous injection an amount of 1 × 10 ⁴ to 1 × 10 ⁹ international units once daily.

In the present specification, the abbreviation of an amino acid is abbreviated according to IUPAC-IUB (Biological Nomenclature Committee).

The transformant Escherichia coli 294 / pHITtrp 1101-d2 described in the following reference example has been deposited as IFO 14350 at the Japan Fermentation Research Institute (IFO), and Japan Institute of Commerce, Industry and Technology (FRI) has been deposited as FERM BP-703 under the Budapest Convention since June 6, 1984. In addition, the heteromorphic conversion agent was deposited on February 11, 1985, with the accession number of KAIST 850211-15311 to KAIST.

The strain Escherichia coli DH1 / pTF4 has been deposited with IFO 14299 at the Fermentation Research Institute in Japan and has been deposited with FRI as FERM BP-628 under the Budapest Convention since June 6, 1984. This strain was also deposited as KAIST 841101-13115 at KAIST on November 1, 1984.

The following examples and reference examples illustrate the invention in more detail, but do not limit the invention.

Example 1

Preparation of Polyethylene Glycol Methyl Ether-Modified IFN-α.

(i) A portion of 5 ml (4.8 mg protein) of IFN-α (rIFN-αA) solution was dialyzed in 0.2 M phosphate buffer (pH 7.0) and 0.15 M sodium chloride at 4 ° C. for 12 h. To the dialysate is added polyethylene glycol methyl ether aldehyde (average molecular weight 1,900) (260 mg) obtained in Reference Example 1. Then, sodium cyanoborohydride (140 mg) is added and the mixture is stirred at 37 ° C. for 40 hours. The reaction mixture is poured into Sephadex G-75 column (3.0 x 43.0 cm) and developed with 25 mM ammonium acetate buffer (pH 5.0) and 0.15 M sodium chloride. The eluate is collected 5 ml each. Combine the elution fractions (100-150 ml) containing the desired product. Analysis by Lowry's method using bovine serum albumin as standard shows protein content of 87 μg / ml in the combined fractions. The amino acid ratio in the acid hydrolyzate (6N hydrochloric acid, 110 ° C., 24 hours) is 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, decomposes. In view of the fact that rIFN-αA contains 11Lys residues, it can be concluded that about 41% of the Lys residues of the ε-amino group in the interferon were modified to polyethylene glycol methyl ether (average molecular weight 1,900). The titer of this product is 1.51 × 10 ⁷ international units / mg, as measured by enzyme immunoassay [Methods in Enzymology, 79, 589-595 (1981)], and the antiviral activity was determined by Journal of Virology 37,755-758 (1981). When measured by the method described in), it is 0.57 × 10 ⁷ international units / mg. This product (IFA-3) is used in the attention clearance test as described below.

(ii) rIFN-αA was treated in the same manner as (i) using 100 mg of polyethylene glycol methyl ether aldehyde and 100 mg of sodium cyanoborohydride obtained with the average molecular weight of 750 obtained in Reference Example 1 to obtain a protein content of 130 μg. 30 ml of a polyethylene glycol methyl ether-modified IFN-α solution at / ml are obtained. The amino acid ratio in the acid hydrolyzate (6N hydrochloric acid, 110 ° C., 24 hours) is 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, decomposes. The data indicate that about 57% of the Lys residues were modified at the ε-amino group. The enzyme immunoassay in the same manner as in (i) shows the titer of 5 × 10 ⁶ international units / mg, and the antiviral activity of the product is 0.14 × 10 ⁸ international units / mg.

(iii) repeating the method of (i) using 27 mg polyethylene glycol methyl ether aldehyde and 27 mg sodium cyanoborohydride to obtain 50 ml of polyethylene glycol methyl ether-modified IFN-α solution having a protein content of 45 μg / ml do. The amino acid ratio in the acid hydrolyzate (6N hydrochloric acid, 110 ° C., 24 hours) is 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); Ile, 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, decomposes. The data indicate that about 18% of the Lys residues were modified at the ε-amino group. In the enzyme immunoassay in the same manner as in (i), the titer is 1.09 × 10 ⁸ international units / mg, and the antiviral activity of this product is 1.53 × 10 ⁸ international units / mg.

(iv) The chemically modified IFN-α (IFA-3) of the present invention obtained in (i) above is administered to the femoral muscles of three 7-week-old female SD rats in an amount of 1.274 × 10 ⁶ units per capita. After the designated period of time, blood is collected from tail tailings and the IFN-α titer of plasma is measured by the enzyme immunoassay method and antiviral activity method described in Example 1 (i). A clear delay in clearance is observed when compared to the group administered unmodified interferon α (rIFN-αA) in an amount of 1.259 × 10 ⁶ units per capita. The above results are shown in FIG.

Example 2

250 mg of human serum albumin is added to 5 ml of the chemically modified IFN-α (IFA-3) solution of the present invention obtained in Example 1 (i). The resulting solution is filtered through a membrane filter (pore: 0.2λ μm), divided into 5 vials, lyophilized and stored. Before use, the contents of each vial are dissolved in 1 ml distilled water for injection.

Example 3

Preparation of polyethylene glycol methyl ether-modified IFN-α and alkanoyl-polyethylene glycol-modified IFN-α.

(i) The title compound is synthesized using the polyethylene glycol methyl ether aldehyde and the alkanoyl polyethylene glycol aldehyde obtained in Reference Example I and Reference Example 2 respectively, and the method of Example 1 is carried out. Various data of each synthesized derivative is shown in Table 1 and its amino acid analysis data is shown in Table 2.

(ii) The chemically modified IFN-α (Compound Nos. 2 and 8) obtained in (i) was femoral in the amount of 3.12 × 10 ⁶ units and 2.66 × 10 ⁶ units in 3 groups of 7-week-old female SD rats, respectively. Administered by intramuscular injection. Blood samples are taken from the tail vein at time intervals and the IFN-α titers of plasma are analyzed by enzyme immunoassay. Clearly, a delay in clearance is observed when compared to the group administered 3.82 × 10 ⁶ unmodified IFN-α. These results are shown in FIG.

TABLE 1

TABLE 2

Example 4

Preparation of Polyethylene Glycol Methyl Ether-Modified Interferon-γ.

(i) 5 ml portions (5.95 mg protein) of the interferon-γ prepared by recombinant DNA technology (hereinafter abbreviated as rIFN-γ: cf: EPC Publication No. 110044) were separated by Sephadex G-25 column (2.0 × 60.0 cm). ) And develop with 0.2 M phosphate buffer (pH 7.0). The eluate is fractionated 5 ml each. Combine fractions 11-13 and dilute to 100 ml with the same buffer. To this was added polyethylene glycol methyl ether aldehyde (average molecular weight 750) (225 mg) and further sodium cyano borohydride (300 mg). The mixture is stirred at 37 ° C. for 72 hours. The resulting precipitate is removed by centrifugation. Concentrate the supernatant to 10 ml using a diaflow membrane (Amicon). The concentrate is placed in Sephadex G-75 column (3.0 x 43.0 cm) and developed with 25 mM ammonium acetate buffer (pH 6.0) + 0.15 M sodium chloride + 10 mM glutathione. The eluate is fractionated 5 ml each. Combine fractions 17-24 containing the desired product.

The value measured by the Bradford method using bovine serum albumin as standard with the protein content of the combined fractions is 7.73 μg / ml. Acid hydrolyzate (6N hydrochloric acid, 110 ° C., 24 hours) shows the following amino acid analysis: 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, decomposes. Since rIFN- [gamma] contains 20Lys residues, the above results indicate that about 12% of Lys ε-amino groups in rIFN- [gamma] were modified by polyethylene glycol methyl ether (average molecular weight 750). The product has an antiviral activity of 1.3 × 10 ⁶ international units / mg. Administration of this product to rats clearly delays clearance in the blood. On the other hand, the precipitate was dissolved in 6M guanidine hydrochloride, dialyzed overnight at 4 ° C. with 25 mM ammonium acetate (pH 6.0) + 0.15M sodium chloride + 10 mM glutathione, followed by Sephadex G-75 gel filtration as above. . The purified fraction (25 ml) had a protein content of 126 μg / ml, and the amino acid analysis of the acid hydrolyzate (6N hydrochloric acid, 110 ° C., 24 hours) was as follows: 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); IIe, 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, decomposes.

It is assumed that the higher values of Glu and Gly than the theoretical ones are due to contamination by glutathione. Since rIFN- [gamma] contains 20 Lys- [epsilon] amino groups, the above results indicate that about 26.5% of Lys [epsilon] -amino groups in rIFN- [gamma] were modified by polyethylene glycol methyl ether.

(ii) rIFN- [gamma] was present in 2-mercaptoethanol (2%) by using polyethylene glycol methyl ether having an average molecular weight of 750 using 225 mg of aldehyde and 120 mg of sodium cyanoborohydride, and treating in the same manner as in (i). 30 ml of a polyethylene glycol methyl ether-modified rIFN- [gamma] solution with a protein content of 236 [mu] g / ml were obtained. Amino acid analysis of the acid hydrolyzate (6N hydrochloric acid, 110 ° C., 24 hours) is 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); Ile, 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, decomposes. The high values of Glu and Gly are presumably due to contamination with glutathione. Since rIFN-γ contains 20 Lys ε-amino groups, the above results indicate that about 50% of Lys ε-amino groups in rIFN-γ were modified by polyethylene glycol methyl ether.

Example 5

Preparation of Polyethylene Glycol Methyl Ether-Modified IFN-γ d2.

(i) 5 ml portion (4.95 mg protein) of IFN-γ d2 solution obtained in Reference Example 3 was placed in a Sephadex G-25 column (2.0 × 60.0 cm) and developed with 0.2 M phosphate buffer (pH 7.0). The eluate is fractionated 5 ml each. Combine fractions 11-13 and dilute to 100 ml with the same buffer. Polyethylene glycol methyl ether aldehyde (average molecular weight 750) (200 mg) is added to the diluent, and sodium cyanoborohydride (300 mg) is further added. The mixture is stirred at 37 ° C. for 72 hours. The resulting precipitate is removed by centrifugation. Concentrate the supernatant to 10 ml using a Diaflow membrane (Amicon). The concentrate is placed in a Sephadex G-75 column (3.0 x 43.0 cm) and developed with 25 mM ammonium acetate buffer (pH 6.0) + 0.15 M sodium chloride + 10 mM glutathione. The eluate is fractionated by 5 ml and the fractions containing the modified IFN- [gamma] d2 with polyethylene glycol methyl ether molecular sieves are collected and combined with the Lys ε-amino group of the molecule. When this product is administered to rats, the blood's tears are obviously delayed.

Meanwhile, the precipitate was dissolved in 6M guanidine hydrochloride and dialyzed overnight at 4 ° C. with 25 mM ammonium acetate buffer (pH 6.0) + 0.15M sodium chloride + 10 mM glutathione, followed by Sephadex G-75 gel filtration in the same manner as described above. Purify. Thus, a fraction containing a modified IFN- [gamma] d2 having a polyethylene glycol methyl ether molecular sieve is obtained with the Lys ε-amino group of the molecule.

Example 6

Preparation of Polyethyleneglycol Methylether-Modified IFN-γ d3.

(i) 5 ml portion (5.5 mg protein) of IFN-γ d3 solution obtained in Reference Example 4 was placed in a Sephadex G-25 column (2.0 × 60.0 cm) and developed with 0.2 M phosphate buffer (pH 7.0). The eluate is fractionated 5 ml each. Combine fractions 11-13, add polyethylene glycol methyl ether aldehyde (average molecular weight 750) (225 mg), and then add sodium cyanoborohydride (120 mg). The mixture is shaken at 37 ° C. for 24 hours. The reaction mixture is placed in Sephadex G-75 column (3.0 x 43.0 cm) and developed with 25 mM ammonium acetate buffer (pH 6.0). Thus, a fraction containing a modified IFN- [gamma] d3 having a polyethyleneglycol methyl ether molecular sieve is obtained with the Lys ε-amino group of the molecule. When this product is administered to rats, a clear delay in blood clearance is observed.

Example 7

Preparation of Polyethylene Glycol Methyl Ether Modified IL-2.

(i) 5 ml (5.0 mg protein) portion of interleukin-2 (hereinafter abbreviated γ IL-2) obtained in Reference Example 5 was dialyzed in 0.2 M phosphate buffer (pH 7.15) for 12 hours. Polyethylene glycol methyl ether aldehyde (average molecular weight 750) (97 mg) was added to the dialysate, and sodium cyanoborohydride (100 mg) was further added. The mixture is stirred at 37 ° C. for 24 hours. The resulting precipitate is removed by centrifugation. The supernatant is dialyzed in 5 mM ammonium acetate buffer (pH 5.0) for 5 hours. The dialysate is placed in a Sephadex G-75 column (3.0 x 43.0 cm) and developed in the same solvent system. The eluate is fractionated 5 ml each. Combine fractions 21-29 containing the desired product. The protein content measured by the Bradford method using bovine serum albumin as the standard for the combined fractions is 25 μg / ml.

Acid hydrolysates (6N hydrochloric acid, 110 ° C., 24 hours) show the following amino acid assays: 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, decomposes. Since rIL-2 contains 11Lys residues, the above results indicate that about 33.6% of Lys ε-amino groups were modified by polyethylene glycol methylether. Hinuma-dong's method for measuring growth of IL-2-dependent mouse natural killer cell lines (NKC 3) based on [3H] -thymidine uptake into DNA [Biochemical and Biphysical Research Communications, 109, 363-369 (1982) The IL-2 activity of the product measured by] is 22,998 units / mg. When rIL-2 has an activity of 40,000 units / mg, the product is estimated to maintain 57.7% activity. After administration of this product, the clearance of blood is obviously delayed.

Reference Example 1

Synthesis of Polyethylene Glycol Methyl Ether Aldehyde.

H(OH)₂)의 알데히드기로 인해 96.2ppm에서 흡광도를 나타낸다.(i) Polyethylene glycol methyl ether (5 g; average molecular weight 5,000) is dissolved in methylene chloride (100 ml) and pyridinium chlorochromate (330 mg) is added. The mixture is stirred at rt for 12 h. The reaction mixture is diluted twice with methylene chloride and placed in a Florisil column (6 × 10 cm), then the columns are washed sequentially with methylene chloride and chloroform and eluted with methanol-chloroform (1: 9). Fractions positive for the 2,4-dinitrophenylhydrazine test are combined and the solvent is distilled off under reduced pressure to give crystalline wax. Yield 1.5 g (30%). Thin layer chromatography: R _f = 0.08 (chloroform-methanol-acetic acid = 9: 1: 0.5, silica gel). ¹³ C-NMR spectrum is a hydration type (

The absorbance is obtained at 96.2 ppm due to the aldehyde group of H (OH) ₂ ).

(ii) Polyethylene glycol methyl ether (10 g, average molecular weight 5,000) is dissolved in t-butanol (100 ml), potassium t-butoxide (4.17 g) is added, followed by addition of bromoacetal (2.56 ml). The mixture is stirred at 40 ° C. for 2 hours. t-butanol is distilled off under reduced pressure, water is added to the residue, and then the aqueous mixture is extracted with chloroform (200 ml X 2). The extract is washed with water and dried over anhydrous sodium sulfate. Chloroform was distilled off under reduced pressure, petroleum benzine was added to the residue, and the resulting crystalline residue was collected by filtration and washed with ether to yield 9.5 g (95%) of the corresponding polyethylene glycol methyl ether diethyl acetal. do. 5 g portion of acetal is dissolved in 50 ml of 0.05 M trifluoroacetic acid, treated for 30 minutes in a circulating water bath, and lyophilized to have a chain length longer than that of the product obtained in (i) by -O-CH ₂ CH _2-. Polyethylene glycol methyl ether aldehyde is obtained.

H(OH)₂)의 알데히드기로 인해 96.2ppm에서 흡광도가 나타난다.(iii) Polyethylene glycol methyl ether (5.7 g; average molecular weight 1,900) is dissolved in methylene chloride (100 ml) and pyridinium chlorochromate (970 mg) is added. The mixture is stirred at room temperature for 12 hours, diluted with the same volume of methylene chloride and placed in a Florisil column (6.0 × 10.0 cm). The column is washed sequentially with methylene chloride and chloroform and eluted with 10% methanol / chloroform. Fractions positive for the 2,4-dinitrophenylhydrazine test are combined. The solvent is distilled off to obtain crystalline wax. Yield 1.8 g (30%). Thin layer chromatography: R _{f =} 0.10 (chloroform-methanol-acetic acid = 9: 1: 0.5, silica gel). ¹³ C-NMR spectrum is hydrated (

The absorbance appears at 96.2 ppm due to the aldehyde group of H (OH) ₂ ).

(iv) Polyethylene glycol methyl ether (19.5 g; average molecular weight 1,900) was dissolved in t-butanol (100 ml). Potassium t-butoxide (10.4 g) is added and bromoacetal (6.4 ml) is added. The mixture is stirred at 40 ° C. for 2 hours. t-butanol is distilled off under reduced pressure. Water is added to the residue and extracted with chloroform (200 ml x 2). The extract is washed with water and dried over anhydrous sodium sulfate. Chloroform is distilled off under reduced pressure, petroleum benzine is added to the residue, and the resulting crystalline residue is collected by filtration, washed with ether to give 8.5 g (89.5%) of acetal. 3 g of acetal was dissolved in 0.05 M trifluoroacetic acid, treated in a water bath for 30 minutes, and then lyophilized to have a chain length of -O-CH ₂ CH ₂ -longer than the product obtained in (i). Polyethylene glycol methyl ether aldehyde is obtained.

(v) Polyethylene glycol methyl ethers having average molecular weights of 750,550 and 350 are converted to the corresponding aldehydes by the same method as described above.

Reference Example 2

Synthesis of Alkanoyl Polyethylene Glycolaldehyde.

(i) 15 g of polyethylene glycol 1540 (Wako Pure Chemical Industries) (average molecular weight 1500) is dissolved in 50 ml of pyridine, and 1.85 ml of acetic anhydride is added to the solution. After the mixture is stirred at 40 ° C. for 2 hours and at room temperature for 16 hours, the solvent is distilled off under reduced pressure. The residue is dissolved in chloroform, the solution is washed with water, then the chloroform layer is dried over anhydrous sodium sulfate and the chloroform is distilled off under reduced pressure. The residue is dissolved in a small amount of chloroform, the petroleum benzine-ether (2: 1) mixture is added to the solution, and the mixture is left to yield 14 g (90%) of crystalline wax. 1.4 g portions of this wax are dissolved in 50 ml of methylene chloride and 300 mg of pyridinium chloromate is added. The resulting mixture is stirred at rt for 18 h. The reaction mixture is placed in a silica gel C-200 (Waco Pure Chemicals) column (3 x 50 cm), the column is washed with 5% methanol-chloroform (200 ml) and eluted with 10% methanol-chloroform. Fractions positive for 2,4-dinitrophenylhydrazine are combined and the solvent is distilled off under reduced pressure to obtain a macrocrystalline wax. Yield 580 mg (41%)

(ii) 20 g of polyethylene glycol 1000 (Wako Pure Chemical Industries) (average molecular weight 1000) is dissolved in 50 ml of methylene chloride, and 5.15 g of n-caproyl anhydride is added. The mixture is stirred at 70 ° C. for 2 hours. The solvent was distilled off, and the residue was eluted and purified with ethyl acetate-methanol (4: 1) using silica gel C-200 column (3 × 50 cm) to obtain 14.9 g (60%) of oil, which was then placed in a refrigerator. It is left to solidify. Then, in the same manner as in (i), continuous oxidation with pyridinium chlorochromate yields the corresponding aldehyde.

Reference Example 3

Preparation of IFN-γ d2.

(i) Transformant Preparation

IFN-γ expression plasmid pHITtrp 1101 [cf. EPC No. 110044, Example 2 (iii)] was digested with restriction enzymes AvaII and Pst I and the AvaII-PstI 1kb DND fragment containing the IFN-γ gene moiety was isolated. Protein Synthesis Starting Protein-Containing Oligonucleotide Adapters Chemically Synthesized by Phosphorester Method

CGATAATGTGCCAG

TATTACACGGTCCTG

Is linked to the AvaII sticky end of the DNA fragment using T4 DNA ligase.

Said adapter-binding gene was plasmid ptrp 771 [cf. Reference Example 2 () ii)] is inserted into the lower portion of the trp promoter of the DNA fragment obtained by cleavage using restriction enzymes CIa I and Pst I. This forms the expression plasmid pHITtrp 1101-d2, which encodes a Cys-Tyr-deficient IFN- [gamma] polypeptide (Figure 3).

Using this plasmid pHITtrp 1101-d2, Cohen et al. [Proc. Nat1. Acad. Sci. USA, 69, 2110 (1972), to transform Escherichia coli 294 to obtain the transformant Escherichia coli 294 / pHITtrp 1101-d2 carrying the plasmid.

(ii) transformant culture

Strain E. coli 294 / pHITtrp 1101-d2 carrying the plasmid formed in (1) above at 37 ° C. in an M9 medium containing 8 μg / ml tetracycline, 0.4% casaninoic acid and 1% glucose Incubate. When grown to KU 220, 3-β-indolylacrylic acid (IAA) is added at a concentration of 25 μg / ml, followed by further incubation for 4 hours. After incubation, cells are harvested by centrifugation and suspended in 1/10 volume of 0.5M Tris-HC1 (pH 7.6) containing 10% sucrose. To the suspension is added phenylmethylsulfonyl fluoride, NaCl, ethylenediaminetetraacetate (EDTA), spermidine and lysozyme at concentrations of 1 mM, 10 mM, 40 mM and 200 μg / ml, respectively. After standing at 0 ° C. for 1 hour, the suspension is treated at 37 ° C. for 3 minutes to obtain a degradation product.

The digest was centrifuged at 4 ° C. and 20,000 rpm (Serval Centrifuge, SS-34 Rotor) for 30 minutes to obtain a supernatant containing IFN-γ d2 polypeptide. This supernatant has an antiviral activity of 2.87 × 10 ⁸ U / L culture.

(iii) Purification of IFN-γ d2

18 ml of 0.1 M tris-hydrochloride buffer (pH 7.0) containing 7 M guanidine hydrochloride and 2 mM phenylmethylsulfonyl fluoride 5.9 g of cells obtained by the same method as (ii) above are suspended and stored frozen. The suspension is stirred at 14 ° C. for 4 hours and centrifuged at 10,000 × g for 30 minutes to yield 20 ml of supernatant. The supernatant was diluted with 260 ml of buffer (pH 7.4) containing 137 mM sodium chloride, 2.7 mM potassium chloride, 8.1 mM disodium phosphate and 1.5 mM monopotassium phosphate (hereinafter abbreviated to this buffer as PBS) and the dilution diluted to 1 ml /. Place in an antibody column (Mo γ 2-11.1, column volume 12 ml) at a flow rate of minutes. The column was washed with 60 ml of 20 mM sodium phosphate buffer (pH 7.0) containing 0.5 M guanidine hydrochloride and eluted with 36 ml of 20 mM sodium phosphate buffer (pH 7.0) containing 2 M guanidine hydrochloride to give 20 ml of antiviral active fraction. do.

Sephacryl S-200 (Pharmacia) column (2.6 × 94 cm, column volume 500 ml) pre-equilibrated with 25 mM ammonium acetate buffer (pH 6.0) containing 1 mM ethylenediaminetetraacetate, 0.15 M sodium chloride, 10 mM cysteine and 2 M guanidine hydrochloride To the above 20ml fraction, eluting with the same buffer to obtain 37ml of the antiviral active fraction.

결핍 IFN-γ 폴리펩티드(IFN-γ d2)는 5.9mg이며, 1.0×10⁷u/mg의 비활성을 갖는다.Obtained

The deficient IFN- [gamma] polypeptide (IFN- [gamma] 2) is 5.9 mg and has an inactivity of 1.0x10 ⁷ u / mg.

Reference Example 4

Preparation of IFN-γ d3

(i) Transformant preparation.

IFN- [gamma] expression plasmid pRC 23 / IFI-900 (cf. EPC Publication No. 0089676 Example 7) was digested with restriction enzymes NdeII and Nco I and a 710 bp NdeI-Nco containing IFN- [gamma] region. I DNA fragment (A) is isolated. On the other hand, plasmid pRC 23 is digested with restriction enzymes Bg1II and EcoR I, and 265 bp DNA fragment (B) containing the λ _p L promoter is isolated. Fragments (A) and (B) and chemically synthesized protein synthesis starting codon-containing oligonucleotides

결핍 IFN-γ 폴리펩티드를 코오딩하는 형질 발현 플라스미드 pLC 2를 형성한다(제2도). 이 플라스미드 pLC 2를 사용하여 코헨등의 방법에 의해 에스케리카아 콜리 RRI(pRK 248 cIts)를 형질 전환시켜 형질전환체(에스케리키아 콜리(이. 콜리) PRI(pLC 2, pRK 248 cIts)를 수득한다.Is linked to Nde I and EcoR I sticky ends using T4 DNA ligase. The DNA fragments thus obtained were treated with Nco I and Bg1 II, and then linked to plasmid pRc 23 / IFI-900.

Form the expression plasmid pLC 2 encoding the deficient IFN- [gamma] polypeptide (Figure 2). Using this plasmid pLC 2, Escherichia coli RRI (pRK 248 cIts) was transformed by Cohen et al. To transform the transformants (E. coli (E. coli) PRI (pLC 2, pRK 248 cIts). To obtain.

(ii) transformant culture

The strain carrying the plasmid formed in (i) was 50 ml of E. coli RRI (pLC 2, pRK 248 cIts) containing 1% bactotriptone, 0.5% yeast extract, 0.5% sodium chloride and 7 μg / ml tetracycline. Shake culture at 35 ° C. in liquid medium. Culture broth is transferred to 2.5 L M9 medium containing 0.5% casamino acid, 0.5% glucose and 7 μg / ml tetracycline and grown for 4 hours at 35 ° C and 3 hours at 42 ° C. Cells are harvested by centrifugation and stored at -80 ° C.

(iii) purification.

In 22 ml of 0.1 M tris-hydrochloride buffer (Ph 7.0) containing 7 M guanidine hydrochloride and 2 mM phenylmethylsulfonyl fluoroid, 7.1 g of the frozen cells obtained by the method (i) above are suspended. The suspension is stirred at 4 ° C. for 1 hour and centrifuged at 10,000 × g for 30 minutes to yield 24 ml of supernatant. 300 ml of PBS was added to the supernatant, and the diluted solution was added to an antibody column (Mo γ 2-11.1, column capacity 15 ml) at a flow rate of 1 ml / min. The column was diluted with 60 ml of 20 mM sodium phosphate buffer (pH 7.0) containing 0.5 M guanidine hydrochloride and eluted with 45 ml of 20 mM sodium phosphate buffer (pH 7.0) containing 2 M guanidine hydrochloride to give 25 ml antiviral active fraction. do. This fraction (25 ml) was pre-equilibrated Sephacryl S-200 (Pharmacia) column with 25 mM ammonium acetate buffer (pH 7.0) containing 1 mM ethylenediaminetetraacetic acid, 0.15 M sodium chloride, 10 mM cysteine and 2 M guanidine hydrochloride (2.6 ×). 94 cm; column volume 500 ml) and eluted with the same buffer to obtain 40 ml of an antivirus active fraction.

결핍 IFN-γ 폴리펩티드 IFN-γ d3는 7.0mg이며 2.72×10⁷IU/mg의 비활성을 갖는다.Thus obtained

Deficient IFN- [gamma] polypeptide IFN- [gamma] d3 is 7.0 mg and has an inactivity of 2.72x10 ⁷ IU / mg.

Reference Example 5

Preparation of aglycosylated human IL-2.

(i) transformant culture

In a 250 ml Erlenmeyer flask, 50 ml of 1% bactotrytol (Difco Lab, USA), 0.5% bactoest extract (Dofco Lab, USA), 0.5% sodium chloride and 7 μg / ml tetracycline (pH 7.0) was added. , And inoculated with E. coli DH1 / pTF4 (Korean Patent Application No. 7430/1984).

After overnight incubation at 37 ° C. in a swing rotor, the culture medium is transferred to a 5 bottle bottle fermentor containing 2.5 L M 9 medium containing 0.5% casamino acid, 0.5% glucose and 7 μg / ml tetracycline. Incubate at 37 ° C. for 4 hours with aeration and stirring, add 3-β-indolylacrylic acid (25 μg / ml), and further incubate for 4 hours. Cells are harvested from the obtained 2.5 L culture broth by centrifugation, frozen and stored at -80 ° C.

(ii) extraction

The cryostored cells (12.1 g) obtained above were treated with 7M guanidine hydrochloride and 0.1M Tris. It is uniformly suspended in 100 ml of extract (pH 7.0) containing HC1, the suspension is stirred at 4 ° C. for 1 hour, and the digest is centrifuged at 28,000 × g for 20 minutes to give 93 ml of supernatant.

(iii) Purification of IL-2 Protein.

The supernatant obtained above was 0.01M Tris. Dialysis in HC1 buffer (pH 8.5) and centrifugation at 19,000 × g for 10 minutes yield 94 ml of dialysis supernatant. 0.01M Tris this dialysis supernatant. Proteins are adsorbed in a column (50 ml volume) of DE 52 (DEAE-cellulose, Whatman, UK) equilibrated with HC1 buffer (pH 8.5). IL-2 is eluted by linear NaCl concentration gradient (0-0.15 M NaCl, 1 L). Concentrate the active fraction (53 ml) to 4.8 ml using a YM-5 membrane (Amicon, USA) and 0.1 M Tris. Gel filtration using a Sephacryl S-200 (Pharmacia, Sweden) column (500 ml volume) equilibrated with HC1 (pH 8.0) -1 M NaCl buffer. The obtained active fraction (28 ml) is concentrated to 2.5 ml using a YM-5 membrane. The concentrate is adsorbed on an Ultrapore RPSC (Altex, USA) column and high yield liquid chromatography is carried out using trifluoroacetic acid-acetonitrile as eluent.

Conditions: column, Ultrapore RPSC (4.6 × 75 mm); Column temperature, 30 ° C., eluent A, 0.1% trifluoroacetic acid-99.9% water; Eluent B, 0.1% trifluoroacetic acid-99.9% acetonitrile; Elution program, 0 minutes (68% A + 32% B) -25 minutes (55% A + 45% B) -35 minutes (45% A + 55% B) -45 minutes (30% A + 70% B) -48 min (100% B); Elution rate, 0.8 ml / min; Detection wavelength, 230 nm. The active fraction is collected at a residence time of about 39 minutes. Thus 0.53 mg of aglycosylated human IL-2 protein [inactive 40,000 U / mg; 30.6% active recovery from the starting material; 10 ml of a solution containing 99% purity of protein (measured by densimetry)] is obtained.

Claims (16)

Chemically modified with polyethyleneglycol of formula (I), directly bonded to at least one primary amino group of the lymphokine molecular sieve, characterized in that the lymphokine is reacted with an aldehyde of formula (II) in the presence of a reducing agent Method of preparing lymphokines.

(Wherein, R is a protecting group of the terminal oxygen atom, n is any positive integer.) The method of claim 1, wherein the lymphokine molecular sieve has a molecular weight of 5,000 to 50,000. The method of claim 2, wherein the lymphokine molecular sieve has a molecular weight of 10,000 to 30,000. The method of claim 1, wherein the lymphokine molecular sieve is an interferon, interleukin-2, macrophage differentiation factor, macrophage activator or substance similar in structure and physical activity. The method of claim 1, wherein the lymphokine molecular sieve is interferon-α, interferon-β, interferon-γ, interferon-γ d2, interferon-γ d3 or interleukin-2. The method of claim 1, wherein the lymphokine molecular sieve is interferon-α. The method of claim 1, wherein the lymphokine molecular sieve is interferon-γ. The method of claim 1, wherein the lymphokine molecular sieve is interleukin-2. The method of claim 1 wherein the polyethylene glycol has a molecular weight corresponding to 1-10% of the molecular weight of the lymphokine molecular sieve. The method of claim 1 wherein the polyethylene glycol has a molecular weight of 350-6,000. The method of claim 1, wherein R is alkyl or alkanoyl. The method of claim 1 wherein n is a positive integer from 7 to 120. The method according to claim 1, wherein the primary amino group is an N-terminal α-amino group or an ε-amino group of the lysine residue of the lymphokine molecular sieve. The method of claim 1 wherein the modified lymphokines have polyethylene glycol bound to an ε-amino group of 15-80% of the lysine residues of the lymphokine molecular sieve. The process of claim 1 wherein the reaction is performed near neutral. The method of claim 1 wherein the reducing agent is sodium cyanoborohydride.

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