JPS63258896A - Modified interleukin-2 - Google Patents

Abstract

NEW MATERIAL:A modified interleukin-2 (IL-2) modified with a polyethylene glycol having a molecular weight of 6X10<3>-2X10<4>, keeping the IL-2 activity and composed of a linked product produced by bonding the amino group of IL-2 to the carboxyl group of a terminal carboxylated polyethylene glycol through an amide bond or a linked product obtained by bonding the amino group of IL-2 to the terminal hydroxymethyl group of a polyethylene glycol through a crosslinking agent. USE:Remedy for cancer, bacterial or viral infection diseases, autoimmune diseases, etc. PREPARATION:The objective modified IL-2 can be produced e.g. by oxidizing terminal hydroxymethyl group of a polyethylene glycol into carboxyl group using a platinum-palladium-carbon catalyst and reacting the group with the amino group of IL-2 in the presence of a condensation agent.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to modified interleukin-2 (IL-2) which is highly stable in vivo.

Prior Art IL-2 is a soluble protein produced by lectin- or antigen-activated T cells that can modulate lymphocyte activity and promote long-term culture of antigen-specific effector T lymphocytes in vitro. It is. ! L-2 is also known to promote thymocyte division and activate cytotoxic 7972 spheres. Therefore, this phosphorus/arboviral substance is useful for strengthening humoral and cellular immunity and restoring a state of lack of immunity to normal. ! These immunoactivities of L-2 are useful in the treatment of cancer, bacterial or viral infections, autoimmune diseases, immunodeficiencies, and the like.

When such protein factors, including IL-2, are used for therapeutic purposes, their stability in the body is often a problem. The main factors are thought to be 1) decomposition by proteases in the body, 2) elimination via the glomerulus of the kidney, 3) elimination of foreign substances by antigen-antibody reactions, etc. When using host-derived protein factors,
Since they do not exhibit antigenicity, 1) and 2) are the main factors, but 2) in particular is particularly fatal because it depends on the molecular weight of the protein factor, and those with small molecular weights are particularly deadly. In fact, recombinant human IL produced by genetic recombination method
It is thought that after administering -2 to humans, it rapidly disappears from the blood. In such cases, even if the drug is expected to be effective if it is present in a sufficient amount at the target site,
In order to not show sufficient effect or to show effect,
It is expected that large doses will be required.

Therefore, if the stability of the protein factor in the blood could be increased by some method, it would be possible to more effectively exhibit its effects and reduce the side effects caused by large doses.

One known method for increasing the stability of protein factors is to bind non-immunogenic water-soluble polymers to protein factors. This method covers the molecular surface of the protein factor with a water-soluble polymer to reduce its degradability by tetratease, and by increasing the molecular weight of the protein factor, In the case of a protein factor that is antigenic, the antigenicity is reduced by covering the antigenic site.

For IL-2, Nandini V, Katr.
s et al. (Proc, Natl, Aead, Set
, 84.1487 (1987)), but with an average molecular weight of 5
Modified interleukin 2t was prepared by bonding 000 heptanomethoxypolyethylene glycol to interleukin 2 via glutaric anhydride, and it was reported that its anticancer effect was enhanced and clearance was decreased.

Problems to be Solved by the Invention Conjugates of protein factors and non-immunogenic water-soluble polymers have been proven to be useful in some enzymes, insulin, etc. (for example, F, FeDa!). $ et al.
42087), but in this case, it is important to bind the protein factor so that it has its original physiological activity. In order for a protein factor to express its physiological activity, it is necessary that the protein maintains its original higher-order structure and that its binding site for its substrate or receptor is open. . Even if the former holds, the latter
This contradicts the purpose of modification, which is to cover the molecular interface of protein factors, and is expected to be difficult. If the substrate is a small enzyme, it is possible to overcome this difficulty relatively easily, but Reseg! In the case of a protein factor that expresses its physiological activity by binding to -, it is quite difficult to modify it while maintaining its physiological activity. IL-2 is T
It is known that IL-2 expresses its activity by binding to a receptor on cells, but the receptor molecule is also a polymer and has been modified to not interfere with binding to the receptor and to provide stability to IL-2. It was expected that it would be difficult to implement.

As an example of binding polyethylene glycol to IL-2, there is a report by Nandlni et al. mentioned above, but the third
As shown in the table, when the total molecular weight of the bound polyethylene glycol was less than 10,000, the half-life was not sufficiently extended.

Means for Solving the Problems As a result of extensive research into modified IL-2''4c-2'' that maintains IL-2 activity and increases stability in the body, the present inventors discovered that IL-2 The inventors have discovered that a combination of polyethylene glycol and polyethylene glycol having an average molecular weight of eooo or more is suitable for this purpose, and based on this knowledge, they have completed the present invention.

That is, the modified IL-2 of the present invention is a conjugate of the following a) or b) that retains IL-2 activity.

a) A conjugate in which an amide bond is formed between the amino group of IL-2 and the carboxyl group of polyethylene glycol whose terminal hydroxymethyl group has been converted to a carboxyl group.

b) A conjugate in which the amino group of IL-2 and the hydroxymethyl group at the end of polyethylene glycol are bonded via a crosslinking agent.

Polyethylene glycol that binds to IL-2 is
In order to extend the half-life of 2 and improve its stability in the living body, it is preferable that the molecular weight is in the range of 6 x 10 to 2 x 10', particularly in the range of 104 to 2 x 104. In addition, in order to maintain IL-2 activity, the number of polyethylene glycol bonds in one molecule of IL-2 is 1 to 2.
molecule is appropriate. The polyethylene glycol used here includes not only copolymers of ethylene oxide, but also derivatives thereof such as monoalkyl ethers and monoacylated products, and as described later,! It also includes those modified at one or both ends for the purpose of binding to L-2.

As for the method of binding IL-2 and polyethylene glycol, one must choose a method that does not impair IL-2 activity! The method of bonding the amino group of L-2 with polyethylene glycol was proven to be effective.

One of the seven methods is to convert the terminal hydroxymethyl group of polyethylene glycol into a carboxyl group, and react this as an active ester with the amino group of IL-2 to form an amide bond. /Methods for converting the terminal end of lyethylene glycol into a carboxyl group include: 1) oxidation method (Japanese Unexamined Patent Publication No. 141219/1983); 2)
Method for condensing divalent fatty acids with anhydrides, (Eurs P
olym-J% Dan 1177 (1983)) 3) Method of alkylation with halodanated carbic acid visiting conductor (M
akromOL Ch@m,s 182 t1379,
(1981) ), etc. Furthermore, as active esters, those normally used for pegtide synthesis can be suitably used, such as N
-Hydroxysuccinimide ester, N-hydroxyphthalimide ester (J, A, C, S,.

86.1839 (1964)), p-nitrophenyl ester, and the like.

Another method is to use a cross-linking agent, and the cross-linking agent in this case may include no, ranidrophenyl, cyanine fluoride (J, Biol, Chem, 252.3
578), the purpose of which is achieved in this case by reacting activated polyethylene glycol, obtained by previously reacting these reagents with polyethylene glycol, with IL-2.

During the reaction, the pH is 6 to 10, preferably 6.5 to 8.
．． Add 0.01 of IL-2 to the buffer solution prepared to give a concentration of 5.
The activated polyethylene glycol was added to 1% of IL-2 after dissolution at a concentration of ~1%, preferably 0.05-0.5%.
The amount of the reaction is 0.5 to 20 times the mole, preferably 1 to 10 times the mole. At this time, it is possible to control the molecular weight of IL-2.

The modified IL-2 produced in this way is not a single molecule, but one in which polyethylene glycol has a different number of bonds per IL-21 molecule, and two or more IL-2 molecules are cross-linked with polyethylene glycol. However, if necessary, it can be purified and used by a method commonly used for protein purification.

In the present invention, human IL-2 used as a manufacturing raw material
IL-2 is derived from human cells, human IL-2 obtained from microorganisms and animal cells by genetic engineering techniques, and there are no restrictions on the production method. It also includes those whose basic molecular skeleton is original human IL-2, such as those with substitutions, and those with addition or deletion of amino acid residues at the N-terminus or C-terminus.

Example Hereinafter, the present invention will be explained in detail by way of examples.

Example 1 A polyethylene glycol derivative (with an average molecular weight of 3 .400 ) 3.4 II
(0,001 mol), N-hydroxysuccinimide 0
．． 46 N (0,004 mol) to 300 d of N, N-
After dissolving in dimethylformamide, 0.84 J (0,004 mol) of dicyclohexyl ciimide was added and stirred overnight at room temperature.

The produced dicyclohexyl urea was separated by filtration, 600 ml of diethyl ether was added to the filtrate, and the produced crystals of the succiimidyl derivative were filtered, washed with ether and dried to obtain 3.6Ii of white crystals of the activated derivative.

Human IL-2 (R-IL-2) produced by genetic recombination using Escherichia coli in sodium phosphate buffer (0.1M, pi-17.2) solution (2rn9/d) 1
d(0,13μmob) KThe activated derivative aqueous solution (4o/d) obtained above was mixed with 62 pi (0,65a
rnoL) and allowed to react at room temperature for 1 hour.

I'L-2 activity was determined by 93H-thymidine incorporation analysis using CTLL cells (-Eri Uo Shizui J @ Immuno
1.1202027 (197B)).

As a result, while the reactive agent IL-2 was 5 x 10' units/■ protein, after the reaction, it was 4 x 10' units/■ protein.
'Unit/η protein was largely preserved.

The molecular weight distribution of the reaction solution was determined by HPLC using a TSKG3000SW (manufactured by Toyo Soda) column, and the IL-2
The activity is shown in Figure 1.

The reaction solution was further purified by column chromatography using Sephadex G-75 (manufactured by Pharmacia) or high-speed GPC (manufactured by Toyo Soda). The chromatogram and IL-2 activity of each fluxin are shown in FIG.

Three peaks were obtained, of which peak C was found to be unreacted IL-2. Regarding the other two peaks, the molecular weight according to SDS-/lyacrylamide gel electrophoresis, the number of isoelectric points of polyethylene glycol (
Table 1 shows the results of examining the number of bound PEGs) and specific activity.

Table 1 Comparative Example I R-IL-2ON-ethylmorpholine acetate buffer (0,
1M, pH 8,0) solution (21jlg/1117) 0.
5m (0,065 μmoA) was added with 2.4 da (0.65 μmoA) of the activated derivative described in Example IK and reacted for 2 hours at room temperature.
The mother turn analyzed by SW K is shown in Figure 3. The IL-2 activity at this time was 5% of that before the reaction.

Example 2 Polyethylene glycol with an average molecular weight of 31-14,000 14.9t-15IIL in dimethylformamide
Dissolve at 0°C, add succinic anhydride 300rn9, and add 1
The reaction was carried out at 00°C for 3 hours. The reaction solution was added to 50 d of diethyl ether, and the resulting precipitate was filtered and washed to obtain disuccinylated polyethylene glycol 13.9
I got 3jl.

This was dissolved in dimethylformamide (30j17) at 50°C and cooled to 30°C to give N-hydroxyco)2.2 acid io, 286□, 2. Hello, hello.

5131°C g of diimide was added and stirred at 30°C for 3 hours.

The precipitate was separated by filtration, and the filtrate was poured into 1501 diethyl ether. The resulting precipitate was filtered and washed to obtain activated ethylene glycol 13.43τ-9.

The obtained activated polyethylene glycol aqueous solution (40~
/ag) 20μJt-R-IL-2 in sodium phosphate buffer (0.1M, PH7.2) solution (2■/ml) 1
00 μl and allowed to react at room temperature for 1 hour.

The reaction solution was mixed with CM-Sepharose (Pharmamia fM),
A modification in which one or two polyethylene glycols are bound to one molecule of IL-2, purified by column chromatography using high-speed GPC (manufactured by Toyo Ichidasha)!
L-2 was prepared.

Example 3 Polyethylene glycol (average molecular i 14000
) was used as a starting material to obtain an activated polyethylene glycol derivative having para-nitrophenylcarnylated terminals.

R-IL
-2 sodium phosphate buffer (0.1M.

It was added to 330 μl of voice 7.2) solution (1,7 instant/d) and allowed to react at room temperature for 5 hours. - Purification was performed in the same manner as in Example 2 to obtain modified IL-2.

Example 4 Polyethylene glycol 10j with an average molecular weight of 14,000
' was dissolved in 50-t@rt-tutyl alcohol at 70°C, 2.4 g of ethyl bromoacetate was added in the presence of potassium butoxide 71, and the mixture was reacted at 80°C for 4 hours.

The solvent was distilled off under reduced pressure, and the residue was extracted with chloroform to obtain polyethylene glycol in which the terminal ethyl group was oxymethylated. This was saponified in an aqueous solution of caustic soda having a pH of 13, and extracted with chloroform to obtain 7501 ng of polyethylene glycol in which the cal-terminus was xymethylated.

An activated polyethylene glycol derivative was obtained using N-hydroxysuccinimide in the same manner as in Example 1, and this was further reacted with R-IL-2.

Purification was performed in the same manner as in Example 2 to obtain modified IL-2.

Example 5 Modification in which polyethylene glycol with a molecular weight of 6000 was bonded in the same manner as in Example 1! L-2 was obtained.

Example 6 Modified IL-2 bound to polyethylene glycol having a molecular weight of 6000 was obtained in the same manner as in Example 2.

Example 7 In the same manner as in Example 3, molecular weight 6000 or 200
Modified IL-2 with 00 polyethylene glycol attached
I got it.

Example 8 Modified IL-2 bound to polyethylene glycol having a molecular weight of 20,000 was obtained in the same manner as in Example 4.

Example 9 Modifications prepared in Examples 1-8! CTL specific activity of L-2
It was measured by L assay and the results are shown in Table 2.

2nd Table 1 3400 1~2 4×103
802-3 1XIO' 20 # 0 5X10 Zo.

214000 1 4×103 80' 2
3×103 60 314000 1 4.5×103 902 2.5
X107 50 414000 1 3.75x107 75' 2
1X107 20 56000 1 3.25x107 651 2 2
X107 40 6 6000 1 5x107 1002 3x1
07 60 7 6000 1 4.5x107 902 4x10
7s.

20000 1 4X107 80 22.5x107 50 820000 1 2.25X107 45' 2
1.25x107 2r Example 1 〇 Group of 4 animals, average weight of about 303jl, SD strain - 200μl'Ft of normal saline:

Polyethylene glycol-modified IL-2 obtained in 1 to 8
200 μl of 0.05 M phosphate buffer (pH 7.0) containing 100 μl of protein and combined with 0.1 M NaCl.
E (containing 100 μ9 equivalent in terms of IL-2 protein) 1
, respectively, were injected into the tail vein. 2.0 after administration of IL-2 and modified IL-2;
5.0, 10.20, 30, 40.60°120.24
After 0,480 minutes, approximately 0.5-
Blood samples were collected over time, serum fractions were collected according to a conventional method, and the serum fractions were extracted from each serum sample as shown in Table 3.
0.151”<-IL-Z?0
0.2.52 14000 1
1.623.9 3 14000 1 0.8
24.0 4 14000 1 1.4
' 2 3.15
6000 1 0.521.1 6 6000 1 0.42
” 1.0 7 6000 1 0.3'
2 1.420000
1 2.123.4 8 20000 1 2.2'
2 5.7 Example 11 The antitumor activity test was conducted, for example, as follows.

I x 10' of mouse myeloma cells x 5563
3H/41・M IJ acid buffer saline CPBS)
Prepared at (PH7,2), 0.4 Da/kP once,
The animals were administered intraperitoneally or subcutaneously every 3 days for a total of 4 times. The tumor size (major axis x minor axis) was measured 3 weeks after the start of administration, and the tumor inhibition rate was determined. The tumor inhibition wheel was calculated using the following formula, where the average size of the modified IL-2 administration group was defined as human and the average tumor size of the non-administration group was defined as B.

In addition, the mouse survival rate after 5 weeks was measured and the complete cure rate was calculated. The results are shown in Table 4.

No. 4 Table 0.4 to cram school x 4 times (subcutaneous administration) 92.9% 5
780.4 da/yu x 4 times (intraperitoneal administration) 99.4%
415 non-administration group 0% 015 Detailed explanation of the invention K, modified interleukin-2 of the present invention
is extremely useful industrially because it maintains the inherent physiological activity of IL-2 while increasing its stability in the body, so there is no need to worry about side effects due to large doses, etc., and it can be easily produced.

[Brief explanation of the drawing]

Figure 1 shows modified interleukin 2, TSK-G30.
This is an analytical chromatogram using 00SW. The interleukin-2 activity was also recorded.

Claims (3)

[Claims] (1) Modified interleukin-2 according to a) or b) below, which is modified with polyethylene glycol having a molecular weight in the range of 6 x 10^3 to 2 x 10^4 and retains interleukin-2 activity. a) A conjugate in which an amide bond is formed between the amino group of interleukin-2 and the carboxyl group of polyethylene glycol whose terminal hydroxymethyl group has been converted to a carboxyl group, b) The amino group of interleukin-2 A conjugate in which the terminal hydroxymethyl group of polyethylene glycol and polyethylene glycol are bonded via a crosslinking agent. (2) The modified interleukin-2 according to claim 1, wherein one to two molecules of polyethylene glycol are bound to one molecule of interleukin-2. (3) Modified interleukin-2 according to claim 1, wherein the crosslinking agent has a carbonyl group.