JPH0696599B2 - Chemically modified lymphokine and method for producing the same - Google Patents

Description

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

BACKGROUND ART Conventionally, useful lymphokines such as interferon (hereinafter sometimes abbreviated as IFN) or interleukin-2 (hereinafter sometimes abbreviated as IL-2) have been known, but in recent years, gene recombination technology has been known. With the development of, it has become possible to synthesize a large amount of these lymphokines. However, it is known that the in vivo clearance of lymphokines administered to a living body is generally very fast. In addition, when the lymphokine is obtained from a different animal, it is expected that there is a risk that antibodies will be produced and serious symptoms will occur in some cases. Therefore, when these are used as pharmaceuticals, it is desired to develop a technique for delaying the clearance and reducing the antigenicity thereof while maintaining the activity. To achieve this purpose, the method of chemically modifying lymphokines is a very effective means. That is, chemical modification is expected to delay the above-mentioned clearance in vivo, reduce antigenicity, and enhance physiological activity, and the chemical modification of lymphokines is of great practical significance.

Problems to be Solved by the Invention Generally, when chemically modifying a bioactive protein,
There is a need for a method that can be chemically modified while retaining their physiological activity. Polyethylene glycol methyl ether is used for the chemical modification of proteins because it is believed that polyethylene glycol itself does not have antigenicity, but the method using cyanuric chloride is generally used to introduce the substance into proteins. is there. However, cyanuric chloride, which is introduced as a bonding group at the same time, has a problem in safety per se, and the safety of its degradation product in vivo has not been elucidated, and its use must be done with caution. Also, the reaction requires a pH on the alkaline side, and this method cannot be applied to alkaline proteins that are easily deactivated.

Further, U.S. Pat.No. 4,002,531 discloses a method for producing a monoalkyl polyethylene glycol derivative of an enzyme,
When the method using sodium borohydride at pH 8.5 disclosed therein is applied to lymphokines, there is a possibility that the physiological activity thereof may be inactivated, and the method cannot be an effective production method. There is no suggestion of a delaying effect of clearance in vivo on the substance, and its effect is unknown.

Furthermore, a method of introducing a low molecular weight aldehyde such as formaldehyde, acetaldehyde, benzaldehyde, and pyridoxal into a physiologically active protein in the presence of a boron-based reducing agent [Method in Enzymology, Vol. 47, 469-47].
8 (1977)]; JP-A-58-154596]. However, even if the method is applied to lymphokines, effective clearance delay is not achieved, and reduction in antigenicity is not expected, and the introduced low-molecular-weight aldehyde acts as a hapten to immunogen the lymphokines. There is a possibility to give a sex.

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

Means for Solving the Problem The present invention provides for at least one primary amino group in a molecule,
RO-CH ₂ -CH _{2 n} groups (I: R is the terminal oxygen protecting group, n represents 7
A chemically modified lymphokine and a method for producing the same.

In the present specification, lymphokines collectively refer to soluble factors involved in cell-mediated immunity released from lymphocytes and substances having physiological activities equivalent to those.

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

For example, various IFNs [interferon-α (IFN-α),
Interferon-β (IFN-β), interferon-γ (IFN-γ)], IL-2, macrophage differentiation factor (MD
F), macrophage activator (MAF), tesch plasminogen activator (TAP), and substances similar in structure to these substances and having similar physiological activity.

As a substance having a structure similar to that of the above lymphokine and having similar physiological activity, for example, IFN-γ lacking 2 to 4 of its N-terminal ammonoic acid (PCT / JP84 / 00292 specification, June 1984 6) Japanese application) and various I without C-terminal part
FN-γ fragment (15K species, etc.)
No. 9-196498, filed Sep. 19, 1984], and IL
-2, one amino acid (EPC Publication 91539 gazette) or four amino acids (Japanese Patent Application No. 58-235) from its N-terminus.
No. 638, filed Dec. 13, 1983), in which some of the constituent amino acids are further deleted or replaced with other amino acids, for example, cysteine at position 125 is replaced with serine. And the like (Japanese Patent Laid-Open No. 59-93093). Especially, lymphokine is IFN-α, IFN
-Γ [consisting of 146 amino acids, for example EPC Publication No. 00
89676] or a deletion of two N-terminal amino acids thereof (IFN-γd2) or 3
It is preferable that it is deficient (IFN-γd3) or IL-2.

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

Examples of the primary amino group of lymphokines include an α-amino group at the N-terminal and an ε-amino group of a lysine residue.

Regarding the group represented by (I) above, examples of the terminal oxygen protecting group represented by R include alkyl and alkanoyl, and specific examples of alkyl include C _1-18 , particularly methyl, ethyl and propyl. , i-propyl, butyl, i-butyl, sec-butyl, t-butyl etc. lower (C _1-4 ).
Alkyl is preferred. Specifically as alkanoyl,
Preferred are C _1-8 , especially lower (C _1-6 ) alkanoyl such as formyl, acetyl, propionyl, butyryl, i-butyryl, caproyl. n is a positive integer of 7 to 120.

The molecular weight of the group represented by the formula (I) is preferably 25,000 or less, and particularly preferably 350 to 6000. From the viewpoint of maintaining physiological activity and delaying clearance, a group represented by the formula (I) having a molecular weight of 1 to 10%, especially 2 to 5% of the molecular weight of lymphokine is preferable.

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

When it has only the N-terminal α-amino group as the primary amino group, it has a group represented by the formula (I) directly bonded to the amino group. 1 in the lymphokine molecule
When it has more than one lysine, those having a group represented by the formula (I) directly bonded to a part of the ε-amino group, preferably 15 to 80% (average) of the ε-amino groups. In this case, the N-terminal α-amino group may or may not have a group represented by the formula (I) directly bonded.

Chemically modified lymphokines according to the invention is, for example, lymphokine and _{RO-CH 2 CH 2 n-} 1 O-CH 2 CHO (II: R and n are the same as defined) in the presence of sodium cyanoborohydride and aldehydes represented by It can be produced by reacting below.

In the reaction, the aldehyde (II) may be used in an amount of about 1 to 10,000 times the molar amount of the lymphokine, and the boron-based reducing agent may be used in the amount of about 1 to 100 times the molar amount of the aldehyde (II).
The degree of modification can be arbitrarily selected by increasing or decreasing the molar ratio of lymphokine and aldehyde (II).
The solvent used in the reaction may be any solvent as long as it does not interfere with the reaction, and examples thereof include buffer solutions such as a phosphate buffer solution and a borate buffer solution. Further, an organic solvent such as lower alkanol (eg, methanol, ethanol, i-propanol), acetonitrile or the like which does not inactivate the lymphokine and does not hinder the reaction may be added. PH of reaction is 3 ~
A wide range of 14 is possible, but near neutrality (pH 6.5 to 7.5)
Is desirable. The reaction temperature may be 0 ° to 80 ° C. as long as the lymphokine does not denature, but 0 ° to 50 ° C.
Is more preferable. A reaction time of 0.5 to 100 hours, usually about 10 to 80 hours is sufficient. The reaction solution can be purified by an ordinary protein purification method such as dialysis, salting out, ion exchange chromatography, gel filtration, high performance liquid chromatography, and electrophoresis to obtain the desired chemically modified lymphokine. The degree of modification of the amino group can be calculated, for example, by performing amino acid analysis after acid decomposition.

The above-mentioned aldehyde (II) is, for example, RO-CH ₂ CH _{2
Although it} can be produced from the ethylene glycol derivative represented by _n OH (III: R and n have the same meanings as above), the following method is an advantageous production method in which the corresponding carboxylic acid is less by-produced.

That is, the compound (III) is oxidized with pyridinium chlorochromate in an alkyl halide solvent such as methylene chloride or chloroform. In this case, pyridinium chlorochromate was used in an amount of 1 to 3 mol based on the compound (III),
The reaction is carried out at a temperature of 50 ° C to 50 ° C, preferably room temperature for 1 to 30 hours.

Further, the compound (III, but n-1) is treated with potassium t-butoxide in t-butanol and then reacted with bromoacetal, followed by organic acid (trifluoroacetic acid etc.) or inorganic acid (hydrochloric acid, sulfuric acid etc.), etc. The corresponding aldehyde (II) having a longer —OCH ₂ CH ₂ — chain length can be produced from the compound (III) by treating with the acid (1). in this case,
First, 10 to 30 mol of potassium t-butoxide was added to and dissolved in the compound (III), and bromoacetal was added to the compound (III) in an amount of 3 to 15 mol, and the temperature was adjusted to 10 °.
After reacting at -80 ° C for 0.5 to 5 hours and after-treatment by a conventional method,
It is dissolved in a dilute aqueous solution of the above acid and heated for 5 minutes to 2 hours.

All of the above reaction solutions were extracted, concentrated, recrystallized, reprecipitated,
It can be purified by usual chemical treatments such as chromatography and distillation.

The chemically modified lymphokine of the present invention has useful physiological activity similar to that of the corresponding known unmodified lymphokine, and is useful as a drug or the like.

The chemically modified lymphokine of the present invention has a delayed in vivo clearance as compared with the corresponding known unmodified lymphokine, and not only exhibits its activity effectively for a long time, but also has low toxicity and antigenicity, and is not known as a known lymphokine. It can be safely used for similar purposes and in similar usages.

The chemically modified lymphokine of the present invention is usually orally or parenterally administered to mammals (monkey, dog, pig, rabbit, mouse, human) as an appropriate pharmaceutical composition using a carrier, a diluent and the like known per se. be able to.

For example, when the chemically modified IFN-α of the present invention is used as an antiviral agent, it is preferable to administer 1 × 10 ⁴ to 1 × 10 ⁹ international units of an adult once a day by intravenous injection.

In the present specification, when an abbreviation for an amino acid is used, IU
PAC-IUB (Commission of Biological Nomenclature)
It is based on the abbreviation.

In addition, the transformant Escherichia coli 294 / pH I Ttrp 1101-d2 disclosed in the following Reference Example is the Institute for Fementation, Osak
Deposit number IFO-14350 in a) from June 6, 1984, Ministry of International Trade and Industry, Industrial Technology Institute, Institute of Microbial Technology (FRI)
Deposited under accession number FERM BP-703.

In addition, Escherichia coli DHI / pTF4 has been assigned to the Fermentation Research Institute as a deposit number IFO-14299 from FR 6 April 1984.
Deposited to I as FERM BP-628.

Actions and Examples The present invention will be described in more detail with reference to the following examples and reference examples, but the present invention is not limited thereto.

Example 1 Polyethylene glycol methyl ether modified IF
Production of N-α (i) Take 5 ml of IFN-α (rIFN-αA) solution (4.8 mg in terms of protein amount) and use 0.2 M phosphate buffer (pH 7.0) + 0.15 M
It was dialyzed against salt for 12 hours at 4 ° C. The dialysate was taken out, and polyethylene glycol methyl ether aldehyde (average molecular weight 1900) (260 mg) obtained in Reference Example 1 was added to it.
Then, sodium cyanoborohydride (140 mg) was added, and the mixture was stirred at 37 ° C for 40 hr. The reaction solution was poured into a Sephadex G-75 column (3.0 × 43.0 cm) and developed with 25 mM ammonium acetate buffer (pH 5.0) +0.15 M sodium chloride.
Fractions containing 5 ml each are collected (100-150 m
l) collected. The protein content of this fraction was 84 μg / ml when measured by the Lowry method using bovine serum albumin as a standard. In addition, acid decomposition products (6N hydrochloric acid, 110 ° C 24
The amino acid analysis values during time) were as follows. : As
p, 12.2 (12); Thr, 10.4 (10); Ser, 16.0 (14); Glu, 24.
8 (26); Pro, 6.0 (5); Gly, 6.3 (5); Ala8.6 (8); V
al, 6.5 (7); Met, 4.5 (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, degradation, 1 for rIFN-αA
Since one Lys is included, from the above results, about 41% of the ε-amino group of Lys in interferon α is polyethylene glycol methyl ether (average molecular weight 1,900).
It was found to be modified with. This product is an enzyme-linked immunosorbent assay [Method in Enzymology, Volume 79, 589-595].
Page (1981)], 1.51 x 10 ⁷ international units / mg,
Journal of Birology, Vol. 37, pp. 755-758 (1981)
The antiviral activity measured according to the method described in 1.
^{It was 7} international units / mg. This product (IFN-3) was subjected to a clearance experiment in rats described below.

(Ii) rIFN was prepared in the same manner as in (i), except that 100 mg of polyethylene glycol methyl ether aldehyde having an average molecular weight of 750 obtained in Reference Example 1 and 100 mg of sodium cyanoborohydride were used.
When -αA was treated, 30 ml of a solution of polyethylene glycol methyl ether modified IFN-α having a protein amount of 130 μg / ml was obtained. The amino acid analysis values in the acid decomposition product (6N hydrochloric acid, 110 ° C, 24 hours) were as follows. : Asp, 12.1 (12); Th
r, 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); M
et, 5.5 (5); Ile, 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, decomposition. From these results, this product shows that about 57% of Lys's ε-amino group is modified,
As a result of measurement by the enzyme immunoassay as in (i), the result was 5 × 10 ⁶ international units / mg, and the antiviral activity was 0.14 × 10 ⁸ international units / mg.

(Iii) When 27 mg of polyethylene glycol methyl ether aldehyde having an average molecular weight of 750 obtained in Reference Example 1 and 27 mg of sodium cyanoborohydride were used in the same manner as in (i), polyethylene glycol methyl ether modification with a protein amount of 45 μg / ml was performed. 50 ml of IFN-α was obtained. Acid degradation products (6N hydrochloric acid, 110 ° C, amino acid analysis in 9 hours at 24 hours were as follows: Asp, 13.6 (12); Thr, 10.4 (10); Ser, 14.9 (1
4); Glu, 26.6 (26); Pro, 5.5 (5); Gly, 6.1 (5); Al
a, 8.3 (8); Val, 6.6 (7); Met, 5.2 (5); Ile, 7.4
(8); Leu, 21.0 (21); Thr, 5.3 (5); Phe, 10.2 (1
0); Lys, 9.0; His, 3.6 (3); Arg, 9.1 (9); Trp, 2.3
(2); Cys, decomposition. From these results, this product has about 18% modified ε-amino group of Lys. As a result of measurement by enzyme immunoassay as in (i), 1.09 × 10 ⁸ international units The antiviral activity in mg / mg was 1.53 × 10 ⁸ international units / mg.

(Iv) The chemically modified IFN-α (IFN of the present invention obtained in (i) above.
-3) was injected into the thigh muscles of female 7-week-old SD rats in groups of 1.274 × 10 ⁶ units, 3 groups per group. After a certain time,
Blood was collected from the tail vein and IFN-α titer in plasma was determined in Example 1.
It was measured by the enzyme immunoassay and the antiviral activity described in (i). Unmodified interferon α (rIFN-αA)
A clear delay was observed in comparison with the group administered with 1.259 × 10 ⁶ units each.

The results are shown in FIG.

Example 2 The chemically modified IFN-α (IFN-of the present invention obtained in Example 1 (i)
Add 250 mg of human serum albumin to 5 ml of the solution of 3) and dissolve. The solution is filtered through a membrane filter (pore size: 0.2 μm) and divided into 5 vials.
Aseptically freeze-dry and store, just before use, distilled water for injection
Dissolve in 1 ml and use.

Example 3 Polyethylene glycol methyl ether modified IF
Production of N-α and IFN-α Modified with Alkanoyl Polyethylene Glycol (i) Using the polyethylene glycol methyl ether aldehyde or alkanoyl polyethylene glycol aldehyde obtained in Reference Examples 1 and 2, the method described in Example 1 was used. The compound was synthesized. Various data of the synthesized derivatives are shown in Table 1 and amino acid analysis values are shown in Table 2.

(Ii) 3.12 × 10 ⁶ units and 2.66 × 10 ⁶ units of the chemically modified IFN-α (Compound Nos. 2 and 8) of the present invention obtained in (i) above, and the thighs of female 7-week-old SD rats Each group was injected with 3 animals per group. After a fixed time, blood was collected from the tail vein, and the IFN-α titer in plasma was measured by the enzyme immunoassay. Unmodified I
A clear delay was observed in comparison with the group administered with 3.82 × 10 ⁶ units of FN-α.

These results are shown in FIG.

Example 4 Production of polyethylene glycol methyl ether-modified interferon-γ (i) Interferon-γ protein produced by gene recombination technology (hereinafter abbreviated as rIFN-γ: EPC Publication No. 110044)
5 ml of the solution (refer to Japanese Patent Laid-Open Publication No. 5) (5.95 mg in terms of protein amount) was poured into a Sephadex G-25 column (2.0 × 60.0 cm) and developed with 0.2 M phosphate buffer (pH 7.0). 5 ml
Fractions No. 11 to 13 were collected. Dilute to 100 ml with the same buffer and add polyethylene glycol methyl ether aldehyde (average molecular weight 750) (225 m
g) and then sodium cyanoborohydride (300 mg) were added, and the mixture was shaken at 37 ° C for 72 hours. The resulting precipitate was removed by centrifugation. The supernatant was concentrated to 10 ml using Diaflow (manufactured by Amicon). This is Sephadex G-75
Column (3.0 × 43.0 cm), and developed with 25 mM ammonium acetate buffer (pH 6.0) +0.15 M sodium chloride + 10 mM glutathione. Fractions No. 17-24 containing the desired product were collected by collecting 5 ml each. The protein content of this fraction is based on Bradford (Br
It was 7.73 μg / ml when measured by the Adford method. The analytical values of amino acids in the acid decomposition product (6N hydrochloric acid, 110 ° C, 24 hours) were as follows. Asp, 19.6 (20); Thr, 4.7 (5); Se
r, 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); I
le, 7.1 (7); Leu, 9.7 (10); Thr, 5.3 (5); Phe, 9.7
(10); Lys, 17.6; His, 2.0 (2); Arg, 5.0 (8); Cys, Tr
p, decomposition, rIFN-γ contains 20 Lys, so
From the above results, about 1 of the ε-amino group of Lys in rIFN-γ was
It was found that 2% 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 6M guanidine hydrochloride, then dialyzed against 25mM ammonium acetate (pH 6.0) + 0.15M salt + 10mM glutathione overnight at 4 ° C, and then subjected to Sephadex G-75 gel filtration as above. Purified. The protein content of this fraction (25 ml) was 126 μg / ml, and the amino acid analysis value of the acid decomposition product (6N hydrochloric acid, 110 ° C., 24 hours) was as follows. Asp, 20.0 (20); Thr, 5.2 (5); Se
r, 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); I
le, 7.2 (7); Leu, 10.2 (10); Thr, 5.8 (5); Phe, 10.1
(10); Lys, 14.7; His, 2.0 (2); Arg, 7.3 (8); Thr, 0.
The values of 7 (1); Cys, degradation, Glu and Gly are higher than the theoretical values, probably because of the contamination of glutathione. 20 for rIFN-γ
Since the number of Lys ε-amino groups is included, it was found from the above results that about 26.5% of Lys ε-amino groups in rIFN-γ were modified with polyethylene glycol methyl ether. .

(Ii) Using 225 mg of polyethylene glycol methyl ether aldehyde having an average molecular weight of 750 and 120 mg of sodium cyanoborohydride in the presence of 2-mercaptoethanol (2%), rIFN-γ was treated in the same manner as in (i) to obtain 236 μg / m 2.
30 ml of a solution of polyethylene glycol methyl ether-modified rIFN-γ with a protein mass of 1 was obtained. The amino acid analysis values of the acid decomposition product (6N hydrochloric acid, 110 ° C, 24 hours) were as follows. Asp, 20.0 (20); Thr, 5.2 (5); Ser, 9.6 (11); G
lu, 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); L
eu, 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, decomposition, and Glu and Gly values higher than the theoretical values are considered to be due to contamination with glutathione. rIFN-γ contains 20 Lys ε-
Since the amino group is contained, rIFN-
It was found that about 50% of the ε-amino group of Lys in γ was modified with polyethylene glycol methyl ether.

Example 5 Polyethylene glycol methyl ether modified IF
Production of N-γd2 (i) 5 ml of the solution of IFN-γd2 obtained in Reference Example 3 (4.95 mg in terms of protein amount) was taken and poured into a Sephadex G-25 column (2.0 × 60.0 cm), 0.2 M Phosphate buffer (pH 7.
Expand with 0). Fraction 5 ml each, fraction No.11 ~ 1
Collect 3. Dilute to 100 ml with the same buffer, add polyethylene glycol methyl ether aldehyde (average molecular weight 750) (200 mg) and then sodium cyanoborohydride (300 mg), and shake at 37 ° C. for 72 hours. The precipitate formed is removed by centrifugation, and the supernatant is concentrated to 10 ml using Diaflow (Amicon). Pour this into a Sephadex G-75 column (3.0 x 43.0 cm) and
M ammonium acetate buffer (pH 6.0) + 0.15M salt + 10mM
Deploy with glutathione. Aliquot 5 ml and Lys in the molecule
The fractions containing IFN-γd2 whose ε-amino group was modified with polyethylene glycol methyl ether were collected. When this product is administered to rats, a clear delay in blood clearance is observed.

On the other hand, the precipitate was dissolved in 6M guanidine hydrochloride, dialyzed against 25mM ammonium acetate (pH 6.0) + 0.15M salt + 10mM glutathione at 4 ° C overnight, and purified by Sephadex G-75 gel filtration as above. Then, a fraction containing IFN-γd2 in which the ε-amino group of Lys in the molecule is modified with polyethylene glycol methyl ether is obtained.

Example 6 Polyethylene glycol methyl ether modified IF
Production of N-γd3 (i) 5 ml of the solution of IFN-γd3 obtained in Reference Example 4 (5.5 mg in terms of protein mass) was taken and poured into a column (2.0 × 60.0 cm) of Sephadex G-25, 0.2 M phosphate buffer (p
Deploy in H7.0). Fraction No. 11
Collect ~ 13. Polyethylene glycol methyl ether aldehyde (average molecular weight 750) (225 mg), then sodium cyanoborohydride (120 mg) were added to this, and the temperature was 37 ° C.
Then, shake it for 24 hours. The reaction solution is Sephadex G-75.
Column (3.0 × 43.0 cm) and developed with 25 mM ammonium acetate (pH 6.0), the ε-amino group of Lys in the molecule was modified with polyethylene glycol methyl ether.
A fraction containing IFN-γd3 is obtained. When this product is administered to rats, a clear delay in blood clearance is observed.

Example 7 Polyethylene glycol methyl ether modified IL
-2 (i) Interleukin-2 (rIL obtained in Reference Example 5)
5 ml of solution (abbreviated as -2) (5.0 mg in terms of protein amount) was taken and dialyzed for 12 hours against 0.2 M phosphate buffer (pH 7.15). Polyethylene glycol methyl ether aldehyde (average molecular weight 750) (97 mg) and then sodium cyanoborohydride (100 mg) were added to the dialysate, and the mixture was kept at 37 ° C for 24 hours.
Stir the time. The formed precipitate was removed by centrifugation. The supernatant was dialyzed against 5 mM ammonium acetate buffer (pH 5.0) for 5 hours. The dialyzed solution is Sephadex G-
It was applied to 75 columns (3.0 × 43.0 cm) and developed with the same solvent system. Fraction No. 21 containing the desired product in 5 ml fractions
Collected ~ 29. The protein content of this fraction was determined using Bradford (Bradfor
It was 25 μg / ml when measured by the method d). In addition, acid decomposition products (6
The amino acid analysis values for N hydrochloric acid at 110 ° C for 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); Ile, 8.1
(8); Leu, 22.2 (22); Thr, 3.0 (3); Phe, 6.0 (6);
Lys, 7.3; His, 3.0 (3); Arg, 3.9 (4); Cys, Trp, decomposition,
Since rIL-2 contains 11 Lys, the above results show that about 33.6% of the ε-amino group of Lys in rIL-2 is modified with polyethylene glycol methyl ether. It was Regarding this product, the growth of IL-2 dependent mouse natural killer cell line (NKC3) was confirmed by [ ³ H] -thymidine
According to the method of Hinuma et al. [Biochemical and Biophysical Research Communication Vol. 109, pp.363-369 (1982)], which measures uptake into DNA as an index, I
The L-2 activity was determined to be 22998 units / mg. rIL-2
This means that it retains 57.7% activity, which is 40,000 units / mg. 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), pyridinium chlorochromate (330 mg) was added, and the mixture was stirred at room temperature for 12 hours.
Stir the time. The reaction mixture was diluted with twice the volume of methylene chloride, poured into a Florisil column (6 × 10 cm), washed with methylene chloride and then with chloroform, and then eluted with methanol-chloroform (1: 9). 2,4
Fractions positive in the dinitrophenylhydrazine test were collected and the solvent was evaporated under reduced pressure to give a crystalline wax. Yield 1.5g (30%), thin layer chromatography: Rf = 0.08
(Chloroform: Methanol: Acetic acid = 9: 1: 0.5, silica gel), ¹³ C-NMR hydrated form to 96.2 PPM The absorption of aldehyde groups was confirmed by.

(Ii) Dissolve polyethylene glycol methyl ether (10 g, average molecular weight 5,000) in tertiary butanol (100 ml),
Potassium tertiary butoxide (4.17 g) was added, then bromacetal (2.56 ml) was added, and the mixture was stirred at 40 ° C for 2 hr. The tertiary butanol was distilled off under reduced pressure, water was added to the residue, and then the mixture was extracted with chloroform (200 ml × 2). It was 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 give the corresponding polyethylene glycol methyl ether diethyl acetal.
9.5 g (95%) was obtained. 5 g of this was taken, dissolved in 50 ml of 0.05 M trifluoroacetic acid, treated in boiling water for 30 minutes and then lyophilized to give -O-CH ₂ C more than that obtained in (i).
Polyethylene glycol methyl ether aldehyde having a long chain length of only H ₂ was obtained.

(Iii) polyethylene glycol methyl ether (5.7 g,
Dissolve the average molecular weight of 1,900 in methylene chloride (100 ml),
Pyridinium chlorochromate (970 mg) was added, and the mixture was mixed at room temperature for 1
Stir for 2 hours. The reaction mixture was diluted with methylene chloride, poured into a Florisil column (6.0 × 10.0 cm), washed with methylene chloride, and then with chloroform.
Elute with% methanol / chloroform. Fractions positive by the 2,4-dinitrophenylhydrazine test were collected and the solvent was evaporated to give a crystalline wax. Yield 1.8g (30
%), Thin layer chromatography: Rf = 0.10 (chloroform: methanol: acetic acid = 9: 1: 0.5, silica gel), ¹³ C-N
Hydrated to 96.2PPM with MR The absorption of aldehyde groups was confirmed by.

(Iv) Polyethylene glycol methyl ether (19.5g,
An average molecular weight of 1900) was dissolved in tertiary butanol (100 ml), potassium tertiary butoxide (10.4 g) and then bromacetal (6.4 ml) were added, and the mixture was stirred at 40 ° C. for 2 hours. The tertiary butanol was distilled off under reduced pressure, water was added to the residue, and then the mixture was extracted with chloroform (200 ml × 2). The reaction solution was washed with water and then dried over anhydrous sodium sulfate. Chloroform was evaporated under reduced pressure, petroleum benzine 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. 3 g of this was dissolved in 0.05 M trifluoroacetic acid, treated in boiling water for 30 minutes, and then lyophilized to give -O-CH ₂ CH ₂ than that obtained in (iii).
₂ - only chain length long polyethylene glycol methyl ether aldehyde was obtained.

(V) Polyethylene glycol methyl ether having an average molecular weight of 750,550,350 was introduced into the corresponding aldehyde in the same manner as above.

Reference Example 2 Synthesis of alkanoyl polyethylene glycol aldehyde (i) Polyethylene glycol 1540 having an average molecular weight of 1500
(Wako Pure Chemical Industries, Ltd.) Dissolve 15 g in pyridine 50 ml and add acetic anhydride 1.8
5 ml was added, and the mixture was reacted with stirring 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 chloroform and washing with water, the chloroform layer was dried over anhydrous sodium sulfate, and chloroform was distilled off under reduced pressure. Dissolve the residue in a small amount of chloroform, add a petroleum benzine-ether (2: 1) mixture and let it stand.
4 g (90%) were obtained. A 1.4 g portion of this was dissolved in 50 ml of methylene chloride, 300 mg of pyridinium chlorochromate was added, and the mixture was reacted at room temperature for 18 hours while stirring. The reaction solution was passed through a silica gel C-200 (Wako Pure Chemical Industries) column (3 × 50 cm), washed with 5% methanol-chloroform (200 ml), and then eluted with 10% methanol-chloroform. 2,
The 4-dinitrophenylhydrazine test positive fractions were collected and the solvent was distilled off under reduced pressure to obtain a crystalline wax. Yield 58
0mg (41%) (ii) Polyethylene glycol 1000 with an average molecular weight of 1000
(Wako Pure Chemical Industries, Ltd.) 20 g dissolved in methylene chloride 50 ml, anhydrous n
-Caproic acid (5.15 g) was added, and the mixture was reacted at 70 ° C for 2 hours.
The solvent was distilled off, and the residue was purified by silica gel C-200 (3 x 50 cm) column, eluting with ethyl acetate-methanol (4: 1), and solidified in a refrigerator to obtain 14.9 g (60%) of an oily substance. Obtained. It was oxidized with pyridinium chlorochromate in the same manner as in (i) to obtain an aldehyde.

Reference Example 3 Production of IFN-γd2 (i) Production of transformant IFN-γ expression plasmid pHITtrp1101 [EPC Publication No. 110044]
No. 2 (iii) of the gazette] was digested with restriction enzymes AvaII and PstI to separate an AvaII-Pst 1 kb DNA fragment containing the IFN-γ gene portion. To this DNA fragment, an oligonucleotide adapter CGATAATGTGCCAG TATTACACGGTCCTG containing a protein synthesis initiation codon chemically synthesized by the triester method was ligated to a margin of AvaII using T4 DNA ligase.

Plasmid ptrp771 See Example 2 (ii) of the above-mentioned publication]
By inserting the above gene to which the above adapter is bound downstream of the trp promoter of the DNA fragment obtained by cutting with the restriction enzymes ClaI and PstI, An expression plasmid pHITtrp 1101-d2 encoding a missing IFN-γ polypeptide was constructed (Fig. 3).

This plasmid pHI Ttrp 1101-d2 was used to transform E. coli 294 according to the method of Cohen et al. [Procedure of National Academy of Sciences USA, Vol. 69, page 2110 (1972)].
And a transformant containing this plasmid (Escherichia coli = E.coli) 294 / pHITtrp
1101-d2 was obtained.

(Ii) Cultivation of transformant E. coli 294 / containing the plasmid constructed in (i) above
pHITtrp1101-d2 was 8 μg / ml tetracycline, 0.4%
The cells were cultured at 37 ° C. using M9 medium containing casamino acid and 1% glucose, and when the growth reached KU220, 3β indolylacrylic acid (IAA) was added at 25 μg / ml to further culture for 4 hours. After culturing, centrifuge to collect cells and collect
It was suspended in 0.05 M Tris-HCl pH 7.6 containing 1/10 volume of 10% sucrose. Add phenylmethylsulfonyl fluoride, NaCl, ethylenediaminetetraacetate (EDT
A), spermidine, lysozyme 1 mM, 10 mM, 40 respectively
mM and 200 μg / ml were added, and the mixture was allowed to stand at 0 ° C. for 1 hour and then treated at 37 ° C. for 3 minutes to obtain a lysate.

This lysate was centrifuged at 4 ° C., 20000 rpm (serval centrifuge SS-34 rotor for 30 minutes to obtain a supernatant containing IFN-γd2 polypeptide. The antiviral activity of this supernatant was 2.87 ×. It was 10 ⁸ U / culture medium.

(Iii) Purification of IFN-γd2 5.9 g of frozen cells obtained in the same manner as in (ii) above was added to 18 ml of 0.1 M Tris-HCl buffer (pH 7.0) containing 7 M guanidine hydrochloride and 2 mM phenylmethylsulfonyl fluoride. After suspending and stirring at 4 ° C. for 1 hour, centrifugation was performed at 10,000 × g for 30 minutes to obtain 20 ml of supernatant. A buffer solution (pH 7.4) consisting of 137 mM sodium chloride, 2.7 mM potassium chloride, 8.1 mM disodium phosphate and 1.5 mM monopotassium phosphate is added to this supernatant.
260 ml (hereinafter abbreviated as PBS) was added to dilute and applied to an antibody column (Moγ2-11.1, column volume 12 ml) at a flow rate of 1 ml / min. Then, 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 36 ml of 20 mM sodium phosphate buffer (pH 7.0) containing 2 M guanidine hydrochloride, 20 ml fractions with antiviral activity were obtained.

20 ml of this fraction was previously added to 1 mM ethylenediaminetetraacetic acid,
Sephacryl S-200 (Pharmacia) equilibrated with a 25 mM ammonium acetate buffer (pH 6.0) containing 0.15 M sodium chloride, 10 mM cysteine and 2 M guanidine hydrochloride.
Column (2.6 × 94 cm, column volume 500 ml) and eluted with the same buffer to obtain 37 ml of a fraction having antiviral activity.

Got here Polypeptide (IFN-γd2) was 5.9 mg, and the specific activity was (1.0 × 10 ⁷ U / mg).

Reference Example 4 Production of IFN-γd3 (i) Production of transformant IFN-γ expression plasmid pRC23 / IFI-900 [EPC Publication No. 0089]
No. 676, refer to Example 7] was digested with restriction enzymes NdeI and NcoI, and an NdeI-NcoI 710 bp DNA fragment (A) containing the IFN-γ gene portion was collected. On the other hand, the plasmid pRC23 was digested with the restriction enzymes Bgl II and EcoRI to obtain 265b containing the λPL promoter.
The p DNA fragment (B) was collected. Oligonucleotide adapter AATTCATGCAGGATCCA GTACGTCCTAGGTAT containing a protein synthesis initiation codon obtained by chemical synthesis with (A) and (B) was ligated to the margins of NdeI and EcoRI using T4 DNA ligase. The obtained DNA fragment was ligated to the plasmid pRC23 / IFI-900 obtained by treating with NcoI and BglII, An expression plasmid pLC2 encoding the polypeptide of was constructed. (FIG. 4) Escherichia coli RRI (pRK248 cIts) was transformed with this plasmid pLC2 according to the method of Cohen et al. [Supra] and the transformant Escherichia coli was transformed.
coli = E. coli) RRI (pLC2, pRK248cIts) was obtained.

(Ii) Culture of transformant Strain E. coli RRI containing the plasmid constructed in (i) above
(PLC2, pRK248cIts) was shake-cultured at 35 ° C. for 12 hours in 50 ml of a liquid medium containing 1% bactotryptone, 0.5% yeast extract, 0.5% sodium chloride and 7 μg / ml tetracycline. The culture solution was transferred to M9 medium 2.5 containing 0.5% casamino acid, 0.5% glucose and 7 μg / ml tetracycline, and kept at 35 ° C. for 4 hours.
Then, it was incubated at 42 ° C. for 3 hours. The cells were collected by centrifugation and stored at -80 ° C.

(Iii) Purification of IFN-γd3 7.1 g of frozen cells obtained by the same method as in (ii) above was added to 22 ml of 0.1 M Tris-HCl buffer (pH 7.0) containing 7 M guanidine hydrochloride and 2 mM phenylmethylsulfonyl fluoride. After suspending and stirring at 4 ° C. for 1 hour, centrifugation was performed at 10,000 × g for 30 minutes to obtain 24 ml of supernatant. A buffer solution containing 137 mM sodium chloride, 2.7 mM potassium chloride, 8.1 mM disodium phosphate and 1.5 mM monopotassium phosphate (pH 7.
4) Add 300 ml to dilute, and add antibody column (Mo γ2-11.1,
A column volume of 15 ml) was applied at a flow rate of 1 ml / min. After that, 0.
The column was washed with 60 ml of 20 mM sodium phosphate buffer (pH 7.0) containing 5M guanidine hydrochloride, and then 45m of 20 mM sodium phosphate buffer (pH 7.0) containing 2M guanidine hydrochloride.
Elution with l gave 25 ml of fraction with antiviral activity.
25 ml of this fraction was previously added to 1 mM ethylenediaminetetraacetic acid,
Sephacryl S-200 (Pharmacia) equilibrated with a 25 mM ammonium acetate buffer (pH 6.0) containing 0.15 M sodium chloride, 10 mM cysteine and 2 M guanidine hydrochloride.
Column (2.6 × 94 cm) and column volume of 500 ml were eluted with the same buffer to obtain 40 ml of a fraction having antiviral activity.

Got here Polypeptide (INF-γd3) is 7.0 mg, and the specific activity is
It was 2.72 × 10 ⁷ U / mg.

Reference Example 5 Production of non-glycosylated human IL-2 (i) Culture of transformant Transformant E, coil DHI / pTF 4 [Japanese Patent Application No. 58-225079 (Showa Sho)
(Applied on November 28, 1983) [Refer to the description] in a 250 ml Erlenmeyer flask Bacto tryptone (Difco Laboratories, USA) 1%, Bacto yeast extract (Difco Laboratories, USA) 0.5%, salt 0.5% and tetracycline Liquid medium (pH 7.0) 50m containing 7μg / ml
The resulting mixture was inoculated into 1 liter and cultivated at 37 ° C. overnight under rotary shaking. This culture was transferred to a 5-volume jar fermenter containing M9 medium 2.5 containing 0.5% casamino acid, 0.5% glucose and 7 μg / ml tetracycline, and transferred to 37 ° C. for 4 hours, followed by 3-β-indolylacrylic acid (25 μg / ml). ml) was added, and the mixture was further cultivated with aeration and stirring for 4 hours to obtain a culture solution 2.5. The culture was centrifuged to collect the cells, which were frozen and stored at -80 ° C.

(Ii) Extraction 12.1 g of the cryopreserved cells obtained above was added to 7 M guanidine hydrochloride, 0.1 M
The suspension was uniformly suspended in 100 ml of an extract (pH 7.0) containing Tris · HCI, and stirred at 4 ° C for 1 hour. This lysate at 28,000 xg
Centrifugation for 20 minutes gave 93 ml of supernatant.

(Iii) Purification of IL-2 protein The supernatant obtained above was dialyzed against 0.01M Tris.HCl buffer (pH 8.5) and then centrifuged at 19,000 xg for 10 minutes to dialyzate the supernatant 94.
I got ml. This dialysis supernatant is mixed with 0.01 M Tris / HCl buffer (pH
After adsorbing the protein through a DE52 (DEAE-cellulose, Whatman, England) column (50 ml volume) equilibrated with 8.5), a linear gradient of NaCl concentration (0 to 0.15 M NaCl, 1) was prepared to produce IL- 2 was eluted and 53 ml of an active fraction was obtained.

53 ml of the above active fraction was concentrated to 4.8 ml using YM-5 membrane (Amicon, USA), and 0.1M Tris · HC
Sephacryl S-200 (Pharmacia, Sweden) column (500 equilibrated with 1 (pH8.0) -1M NaCl buffer)
gel filtration was carried out using the (ml volume). 28 ml of active fraction was added to YM-
Concentrated to 25 ml with 5 membranes. The obtained concentrated liquid was adsorbed on an Ultrapore RPSC (Altex Co., USA) column and subjected to high performance liquid chromatography using a trifluoroacetic acid-acetonitrile system as an elution solvent. Column, Ultrapore RPSC (4.6 x 75 mm); Column temperature, 30
Elution solvent A, 0.1% trifluoroacetic acid-99.9% water; Elution solvent 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 minutes (100% B); elution rate, 0.8 ml / min;
Detection wavelength, 230 nm. Under these conditions, the active fraction with a retention time of about 39 minutes was collected and 0.53 mg of non-glycosylated human IL-2 protein [specific activity, 40,000 U / mg, activity recovery from starting material, 30.6%; protein purity, 99 % (According to densitometry)] was obtained.

Effect of the Invention The chemically modified lymphokines of the present invention maintain the physiological activity of lymphokines and delay the clearance in vivo,
Antigenicity is also reduced.

[Brief description of drawings]

FIG. 1 shows the effect of delaying clearance in rat plasma disclosed in Example 1 (iv). ○ (enzyme immunoassay) and □ (antiviral activity) are ● (enzyme immunoassay) and ■ of the chemically modified IFN-α of the present invention obtained in Example 1 (i).
(Antiviral activity) shows the measurement results of rINF-αA as a control. FIG. 2 shows the effect of delaying clearance in rat plasma disclosed in Example 3 (ii). △ and □ are the first
In the compounds NO.8 and NO.2 in the table, ● indicates rINF− as a control
The result of enzyme immunoassay for αA is shown. FIG. 3 shows the expression plasmid pHIT t disclosed in Reference Example 3 (i).
rp1101-d2 shows the construction diagram, and FIG. 4 shows the construction diagram of the expression plasmid pLC2 disclosed in Reference Example (i) i.

Claims (3)

[Claims] To claim 1 wherein at least one primary amino group in the molecule, RO ^- CH ₂ CH _{2 n} group (R is the terminal oxygen protecting group, n represents a positive integer of 7-120) becomes directly bonded to Chemically modified lymphokine. 2. Lymphokine and RO ^-- CH ₂ CH _{2 n-1} O ^-- CH ₂ CH
At least one in the molecule, characterized by reacting with an aldehyde represented by O (R is a terminal oxygen protecting group, n is a positive integer of 7 to 120) in the presence of sodium cyanoborohydride; a primary amino group, RO ^- CH ₂ CH _{2 n} group (R and n are the same defined above) preparation of chemically modified lymphokines formed by bonding directly. 3. The method according to claim 2, wherein the reaction is carried out near neutrality.