JPH04293482A - Complex of superoxide dismutase and fc - Google Patents

Abstract

PURPOSE:To provide a superoxide dismutase having long half-life in blood and excellent safety and effective for the treatment of living tissue disorder caused by superoxide generated from oxygen molecule in the living body. CONSTITUTION:A superoxide dismutase having free amino group or thiol group is made to react with the Fc fragment of an immunoglobulin having free thiol group through a cross-linking agent (e.g. SMBU or BMH) to form a complex of these components.

Description

[Detailed description of the invention]

0001

[Field of Industrial Application] The present invention relates to superoxide dismutase, which is characterized by having a long half-life in blood, and more particularly relates to superoxide (O2 -
This invention relates to improving the persistence of superoxide dismutase in the blood by binding an Fc fragment, which is useful for the treatment of biological tissue damage caused by ).

0002

[Prior art] Superoxide dismutase (hereinafter referred to as S
(sometimes abbreviated as OD) is an enzyme that eliminates superoxide (O2 − ) generated in vivo. O2 − is extremely toxic to living tissues, and SO
D is effective against various tissue disorders caused by O2 − (e.g., osteoarthritis, chronic rheumatoid arthritis, damage caused by radiation, damage caused by ultraviolet rays, oxygen syndrome of prematurity, ischemic disease, myocardial infarction, etc.) It can be used as a therapeutic drug. As described above, SOD is very promising as a drug, but it has the disadvantage that when administered intravenously, it has a short half-life in the blood (about 5 minutes) and is rapidly metabolized. Therefore, the pharmacological effects of SOD are often not fully exerted when administered intravenously.

Various attempts have been made to improve the short blood half-life of SOD. For example, S.O.
A product in which a hydrophobic organic acid is bound to D and it is bound to albumin in the blood to delay excretion from the kidney [JP-A-63-211
Publication No. 238, Masayasu Inoue et al.: Clinical practice of free radicals, Japan Medical Museum, Vol. 2, 83 (1987), Masayasu Inoue et al.: BIO INDUSTRY Vol. 4, No.
4, 302 (1987)], polyethylene glycol in SOD [Pyatak, P. S. et al.
:Research Communications i
n Chemical Pathology and
Pharmacology, Vol. 29, No.
1,113 (1980)] and Ficoll [Petro
ne, W. F. et al. :Proceed
ings of National Academic
Science of America, Vol.
77, No. 2, 1159 (1980)], which is made into a macromolecule by attaching a polymer to it and delays its excretion from the kidneys.

However, in those modified using polyethylene glycol or Ficoll, the activity retention rate is (
(compared to SOD) by 50% or less. Furthermore, in any of the above cases, since the modified substance bound to SOD uses a substance that does not exist in the human body, safety remains a problem. From this point of view, human biological components are
Proposals have also been made to combine SOD with D to provide highly safe and stabilized SOD. As such, there is, for example, one in which human plasma protein is bound to SOD (Japanese Patent Application Laid-open No. 1-269490). However, its persistence in the blood in rats is still insufficient, and its molecular weight is too large, which may result in poor tissue permeability. In any case, it cannot necessarily be said that it is a practical method for pharmaceuticals.

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to provide an SOD characterized by a long half-life in blood. Another object of the present invention is to provide an SOD that is useful for the treatment of biological tissue damage caused by O2 − generated from oxygen molecules in the body, and that is fully satisfactory in terms of safety, antigenicity, and activity retention. It's about doing.

[0006]

SUMMARY OF THE INVENTION The present invention is a complex of superoxide dismutase and an immunoglobulin Fc fragment. Superoxide dismutase has a small molecular weight of about 32,000, so even if it is administered into the blood, it is quickly excreted from the kidneys. On the other hand, the blood half-life of immunoglobulin is very long, about 20 days. When IgG is separated into Fab fragments and Fc fragments and the blood half-life of each is examined, the half-life of Fab fragments is several hours, while that of Fc fragments is 43-1 hours.
There are 00 hours. Therefore, it is thought that the factor determining the length of the blood half-life of immunoglobulin is the Fc fragment. Therefore, the present inventors considered that the blood half-life could be significantly extended by binding an Fc fragment to SOD.

[0007] SO used for binding Fc fragments in the present invention
D includes SOD derived from various sources. For example, human SOD, SOD produced by genetic recombination technology, or SOD contained in animals such as cows, plants, and microorganisms can be used. Also,
Any of Cu.Zn-SOD present in mammalian cytoplasm, Mn-SOD present in mammalian mitochondria, and Fe-SOD present in bacteria can be used. Among these, human SOD and human SOD produced by genetic recombination technology are preferably used in order to eliminate antigenicity as a pharmaceutical preparation for humans. In addition, since Mn-SOD has low cell affinity, it is generally difficult to obtain good medicinal results, and Fe present in bacteria
- Antigenicity is a problem with SOD, so Cu・Zn-SO
D is preferably used.

[0008] Furthermore, the Fc fragment used in the present invention is also considered to be similar to the Fc fragment of human-derived immunoglobulin when administered to the human body.
use. For example, IgG1, 2, 3, 4, IgA1
, 2, Fc fragments of immunoglobulins such as IgM can be used.

[0009] SOD consists of two homologous subunits. The subunits are not bonded by disulfide bonds, but are very strongly bonded by hydrophobic bonds. SOD contains many amino acid residues in its molecule that can be chemically modified under mild conditions. For example, human red blood cell type SOD is
It has 4 thiol groups and 44 amino groups per molecule, which can be used to bind Fc fragments. Furthermore, the Fc fragment of IgG has a molecular weight of approximately 50,000 and consists of two homologous subunits. The subunits are connected by two disulfide bonds, and
There is one disulfide bond in each subunit. If these disulfide bonds are reduced to free thiol groups, these can be used to bind to SOD.

[0010] The complex of SOD and Fc fragment of the present invention is
It is produced by creating a crosslink between SOD and an Fc fragment. Conventionally, symmetrical divalent cross-linking agents such as glutaraldehyde have been used as cross-linking agents to cross-link proteins, but since these cross-linking agents perform complex reactions with various side chains of proteins, e.g. There is a drawback that crosslinks are formed within the SOD molecule, between SOD molecules, within the Fc molecule, and between Fc molecules, resulting in the formation of an extra complex in addition to the desired complex of SOD and Fc.

[0011] Therefore, in the complex of SOD and Fc fragment of the present invention, crosslinking agent to expose the SH group. Thereafter, SOD to which cross-linking agent X or Y is bound and Fc fragment with exposed SH groups are mixed, and SOD and Fc fragment are covalently bonded via cross-linking agent X or Y. In this case, it is necessary from the viewpoint of the stability of proteins such as SOD that all reactions be carried out under mild conditions (for example, room temperature, pH around neutrality).

[0012] Crosslinking agent X is a compound that crosslinks the NH2 group of SOD and the SH group of the Fc fragment.
It is expressed as Here, R1 is 1 that reacts with an amino group.
Examples of R1 include the following formula (I), (
III) or (IV)

[Chemical formula 3]

[C4]

Among them, the group represented by the above formula (I), which is characterized by specificity for amino groups and mild reaction conditions, is preferably used.

Further, R2 is a monovalent active group that reacts with a thiol group, and examples of such a group include the following formula (I
I) or (V) ~ (VIII)

[C6]

[C7]

[Chemical formula 8]

[Chemical formula 9]

Among these, the above formula (II) is characterized by specificity for SH groups and mild reaction conditions.
A group represented by is preferably used.

Further, A is a divalent organic group, such as -(CH2)n- (where n is 1
to 10), p-phenylene group, and m-phenylene group.

[0015] Furthermore, the crosslinking agent Y is a thiol group of SOD and F
It is a compound that crosslinks the thiol group of the c fragment, and R3-
It is represented by BR4. Here, R3 and R4 are monovalent active groups that react with a thiol group, and examples of these groups include the groups listed above as the group R2.
Similarly to 2, a group represented by the above formula (II) is preferably used. Like A, B is a divalent organic group, and examples thereof include the groups listed as the group A above.

[0016] When reacting SOD with Fc fragment, p
SOD in a buffer adjusted to pH 5-9, preferably pH 7.
After dissolving to 5-10 mg/ml, N-succinimidyl-4-(N-maleimido)butyrate (hereinafter SMBU
1 to 100 of a crosslinking agent such as
Add twice the mole and react, 0.5 per molecule of SOD
~5 active groups are introduced. On the other hand, the Fc fragment is 0.5 to 10 times the mole of SOD with the SH group exposed by a reducing agent and an active group introduced, preferably SOD:Fc
= 1:1 and react to create a complex of SOD and Fc.

The complex of SOD and Fc fragment of the present invention can be produced without using a crosslinking agent, for example, by using genetic recombination technology. For example, at the DNA level, SO
A fusion protein of SOD and Fc can be obtained by combining D and Fc and expressing it in E. coli or animal cells. It is easily inferred that this SOD and Fc fusion protein has the characteristics of the SOD and Fc fragment complex described in the present invention.

[0018]

[Examples] The present invention will be explained in more detail with reference to Examples below. Reference Example 1 (1) Purification of Fc fragment 90 ml of 0.02 M sodium phosphate buffer (pH 7.5) containing 900 mg of human IgG, 1.8 ml of 100 mM EDTA solution, 4.5 ml of 0.25 M
L-cysteine/HCl solution (pH 7.5), 9ml
A 1 mg/ml papain solution was sequentially added, and the reaction was carried out at 37° C. for 1 hour. After the reaction is complete, the reaction solution is mixed with protein A-
The mixture was passed through a Sepharose column (manufactured by Pharmacia) and the fractions eluted with distilled water were collected. The collected fractions were concentrated by ultrafiltration and then passed through a Sephadex G150 (fine) column (manufactured by Pharmacia) to collect a peak with a molecular weight of approximately 50,000 to obtain 20 ml of an Fc fragment of 1.5 mg/ml. When this Fc fragment was analyzed by SDS-polyacrylamide electrophoresis, it was recognized as a single band.

(2) Reducing disulfide bonds of Fc fragment 0.145M NaCl-0.
2-mercaptoethanol was added to 4.5 ml of 1M phosphate buffer (pH 7.4) (hereinafter abbreviated as PBS) to a final concentration of 50 mM, and reaction was performed at 37° C. for 1 hour. After the reaction, add 1mM EDTA and 0.1M Na.
Fc with reduced disulfide bonds was passed through Sephadex G25 (manufactured by Pharmacia) equilibrated with a sodium phosphate buffer (pH 7.2) containing Cl (hereinafter referred to as P-Buffer) to remove 2-mercaptoethanol. Got a piece. Add 0.1M Tr to this Fc fragment solution.
is・HCl, buffer containing 1mM EDTA (pH
8.2) (hereinafter abbreviated as T-Buffer) at 0.2mM
Add an equal amount of 5,5'-dithiobis(2-nitrobenzoic acid) (hereinafter abbreviated as DTNB) dissolved so that The molar concentration of was determined. As a result, Fc
There were 2.79 thiol groups per molecule. Theoretically, there should be four interchain disulfide bonds per Fc molecule, and under the reaction conditions described above,
This means that 2.79 of them were returned.

Reference Example 2 (1) Combination of SOD and SMBU P containing 35 mg of Cu・Zn-SOD derived from bovine red blood cells
- Add 30 μl of N-ethylmaleimide dissolved in ethanol at a concentration of 0.1 M to 5 ml of Buffer,
The reaction was carried out at room temperature for 30 minutes to block free thiol groups. Thereafter, it was passed through Sephadex G25 equilibrated with P-Buffer to remove unreacted N-ethylmaleimide, and 5.2 ml of SOD solution dissolved in P-Buffer at a concentration of 4.9 mg/ml was recovered. Next, SM dissolved in dimethylformamide at a concentration of 220 mg/ml
BU was added so that the molar ratio of SMBU to SOD was 10:1, and the reaction was carried out at room temperature for 1 hour. Then P-B
Unreacted SMBU was removed through Sephadex G25 equilibrated with buffer, and 6 ml of a conjugate of SOD and SMBU dissolved in P-Buffer at a concentration of 5.6 mg/ml was recovered.

[0021] SMBU is a crosslinking agent having active groups at both ends, one capable of reacting with an amino group and the other with a thiol group. In other words, SM bonded to the amino group of SOD
BU has an active group that can react with a thiol group. Therefore, by reacting the thiol group of appropriately diluted 2-mercaptoethanol with the active group of SMBU bound to SOD, and quantifying the decrease in the thiol group of 2-mercaptoethanol, the amount of SMBU bound per molecule of SOD can be measured.
The number of BU molecules can be determined. SOD and SMBU
A solution containing the conjugate and 0.1M NaCl, 2mM ED
Buffer solution (pH 5.5) containing TA (hereinafter referred to as N-Buffe)
2-mercaptoethanol suitably diluted with 2-mercaptoethanol (abbreviated as r) was mixed and reacted at room temperature for 20 minutes. After that, T-Bu
DTNB dissolved at 0.2 mM was added to the solution, and the mixture was allowed to stand at room temperature for 20 minutes to develop color. The absorbance at 412 nm was measured to determine the molar concentration of thiol groups. As a result of the above quantitative determination, the number of SMBU molecules per molecule of SOD was 3.

(2) P-Buffe containing 20 mg of Cu・Zn-SOD derived from bovine red blood cells combined with SOD and BMH
2-mercaptoethanol was added to 2 ml to give a final concentration of 0.1 M, and the reaction was carried out at 37°C for 1 hour. After that, it was passed through Sephadex G25 equilibrated with P-Buffer to remove 2-mercaptoethanol.
3 ml of SOD solution dissolved in P-Buffer at a concentration of 5.5 mg/ml was collected. The thiol groups in this SOD solution were quantified using DTNB in the same manner as in Reference Example 1 (2) and were found to be 1.27 per molecule of SOD.

Next, add 9.2 mg to dimethylformamide.
Bismaleimidohexane (hereinafter abbreviated as BMH) dissolved at a concentration of /ml was added so that the molar ratio of BMH to SOD was 10:1, and the reaction was carried out at room temperature for 1 hour. Sephadex G2 was then equilibrated with P-Buffer.
5 to remove unreacted BMH, and collect 4 ml of a conjugate of SOD and BMH dissolved in P-Buffer at a concentration of 3.4 mg/ml.

Example 1 (SOD using SMBU as a crosslinking agent)
and Fc fragment) 5.3 ml of P-Buffer containing 35 mg of the disulfide bond-reduced Fc fragment obtained in Reference Example 1 (2)
and 5.3 ml of P-Buffer containing 30 mg of the conjugate of SOD and SMBU obtained in Reference Example 2 (1) were mixed, and the mixture was reacted overnight at 4°C. After the reaction, the reaction solution was analyzed by SDS-polyacrylamide gel electrophoresis under reducing conditions, and it was found that one Fc chain was bound to each SOD subunit, and two Fc chains were bound to each SOD subunit.
A band of bound chains was observed (Figure 1).

Next, the reaction solution was treated with Sephadex G150 (superfine) 160m equilibrated with PBS.
The eluate was collected in 60 fractions of 2.75 ml each, and the absorbance at 280 nm (A280) of each fraction, Fc
The amount and SOD activity were measured (Figure 2). The Fc amount was measured by enzyme immunoassay, and the SOD activity was measured by Oyanagi's nitrite method [
Protein Nucleic Acid Enzyme Vol. 33 No. 16
(1988) 2906]. Fractions 24-27 and 28-31 were collected, and Cx. 1.Cx.
It was set as 2. As a result of analysis by SDS-polyacrylamide gel electrophoresis, Cx. 1 has two or more Fc fragments bound to the SOD1 molecule, and Cx. 2 was confirmed to be one Fc fragment bound to the SOD1 molecule (Fig. 1). When the SOD activity retention rate of the complex of SOD and Fc fragment was measured, it was 90% or more.

Example 2 (Preparation of a complex of SOD and Fc fragment using BMH as a crosslinking agent) 4 ml of P-Buffer containing 15 mg of the Fc fragment with reduced disulfide bonds obtained in Reference Example 1 (2),
Conjugate 12 of SOD and BMH obtained in Reference Example 2 (2)
P-Buffer containing mg was mixed and the reaction was carried out at 4°C overnight. After the reaction, the reaction solution was analyzed by SDS-polyacrylamide gel electrophoresis under reducing conditions, and a band with one Fc chain bound to each SOD subunit was observed (FIG. 3).

Next, the reaction solution was passed through a 160 ml column of Sephadex G150 (fine) equilibrated with PBS, the eluate was collected in 60 fractions of 2.75 ml each, and the absorbance at 280 nm of each fraction was measured in the same manner as in Example 1. , Fc amount, SO
D activity was measured (Figure 4). S with a peak of 27 fractions
A complex of OD and Fc fragment was observed.

Example 3 (Measurement of blood half-life of complex of SOD and Fc fragment) Cx. 1 and Cx. 2, the blood half-life in mice was measured. Three ICR mice (male) were intravenously injected with a complex of SOD and Fc fragment in an amount of 0.1 mg as SOD protein, and then injected with Cx. For 2, immediately after, 15 minutes, 30 minutes, 1 hour, 6 hours, 1 day, 2
day, 3 days later, Cx. For No. 1, blood was collected immediately, 30 minutes, 2 hours, 5 hours, 1 day, and 3 days later. As a control, the blood half-life of unmodified bovine SOD or a simple mixture of unmodified bovine SOD and Fc fragment was similarly measured. For these, the protein amount of SOD is 0.1
Blood was collected immediately, 5 minutes, 10 minutes, 20 minutes, and 40 minutes after the mg intravenous injection. After blood collection, the plasma is separated and the S in the plasma is
OD activity was measured in the same manner as in Example 1. FIG. 5 is a graph showing the attenuation of SOD activity in the blood when the SOD activity immediately after intravenous injection is taken as 100%.

In FIG. 5, the curve [SOD-(Fc)x] is Cx. The decay of SOD activity of 1 is expressed as a curve (SOD-Fc)
is Cx. The curve SOD represents the decay of the SOD activity of unmodified SOD, and the curve (SOD+Fc) represents the decay of the SOD activity of the unmodified SOD.
Figure 3 shows the attenuation of SOD activity of a simple mixture of D and Fc fragments. Modified Ma
Table 1 shows the results calculated using the rquardt method.

[0030]

[Table 1] From the above results, it can be seen that the blood half-life of the complex of SOD and Fc fragment is increased by 90 to 130 times compared to unmodified bovine SOD.

Example 4 (Pharmacological activity of complex of SOD and Fc fragment using mouse paraquat poisoning) When paraquat enters the body, it becomes paraquat cation radical, which reacts with O2 to produce superoxide (O2 - ). When paraquat is administered intraperitoneally to mice, this O
It is known that 2- destroys tissue cells and increases the activity of glutamate-oxaloacetate transaminase (hereinafter abbreviated as GOT) in the blood by 5 to 10 times the normal level. Therefore, an experiment was conducted to determine whether the increase in blood GOT activity caused by paraquat could be suppressed by administering a complex of SOD and Fc fragment into the blood. Cx. obtained in Example 1 was used as a sample to evaluate pharmacological activity. 1 and Cx. A mixture of 2 was used.

[0032] SOD and Fc were administered to 6 ICR mice (male).
The complex of fragments is 5 mg/kg as protein amount of SOD.
After intravenous injection, 15 minutes later, 100 mg/kg of paraquat (manufactured by Wako Pure Chemical Industries, Ltd.) was administered intraperitoneally, and immediately thereafter, for 2 hours, 4
After 6 hours, blood was collected from the tail. After blood collection, plasma was separated and GOT activity in the plasma was measured using an automatic analyzer. As a control, a group was also created in which 0.1 ml of PBS was intravenously injected instead of the complex of SOD and Fc fragment, and the same amount of paraquat was administered 15 minutes later. The results are shown in FIG. Figure 6
These results show that the group administered with the complex of SOD and Fc fragment clearly suppressed the increase in blood GOT activity compared to the control group. A similar experiment was conducted using SOD to which no Fc fragment was bound, but no effect of suppressing the increase in blood GOT activity was observed.

[0033]

Effects of the Invention According to the present invention, SOD has a long half-life in the blood, is effective in treating damage to biological tissue caused by O2 − generated from oxygen molecules in the body, and is highly safe.
can be provided.

[Brief explanation of the drawing]

FIG. 1: In Example 1, S
The reaction solution when OD and Fc fragment were reacted, Cx. 1 and Cx. 2 is an SDS-acrylamide gel electrophoresis image under reducing conditions of No. 2.

FIG. 2: SO in Example 1 with SMBU as a crosslinking agent.
280 nm of each fraction obtained by gel filtration of the reaction solution when D and Fc fragment were reacted with Sephadex G150.
FIG. 2 is a diagram showing absorbance, Fc concentration, and SOD activity.

[Figure 3] SOD using BMH as a crosslinking agent in Example 2
This is an SDS-polyacrylamide gel electrophoresis image of the reaction solution under reducing conditions when the Fc fragment was reacted with the Fc fragment.

FIG. 4: SOD using BMH as a crosslinking agent in Example 2
FIG. 3 is a diagram showing the absorbance at 280 nm, Fc concentration, and SOD activity of each fraction obtained by gel filtration of a reaction solution obtained by reacting Fc fragment with Sephadex G150.

FIG. 5 is a blood elimination curve in mice of the complex of SOD and Fc fragment obtained in Example 3.

FIG. 6 is a diagram showing the suppression of the increase in blood GOT activity in paraquat poisoning mice obtained in Example 4 by a complex of SOD and Fc fragment.

Claims (5)

[Claims] 1. A complex of superoxide dismutase and an immunoglobulin Fc fragment. [Claim 2] Superoxide dismutase is copper-based.
A complex according to claim 1, which is of the zinc type. 3. Generated by reacting the amino group of superoxide dismutase with an Fc fragment of immunoglobulin from which a thiol group has been released via a crosslinking agent X [here, the crosslinking agent X is R1 -A-R2 ( Here, R1 is a monovalent active group that reacts with an amino group, R2 is a monovalent active group that reacts with a thiol group, and A represents a divalent organic group. ], the composite according to claim 1. 4. Superoxide dismutase that has released a thiol group is reacted with an immunoglobulin Fc fragment that has released a thiol group via a crosslinking agent Y [here, the crosslinking agent Y is R3-B -R4 (where R
3, R4 is an active group that reacts with a thiol group, and B
represents a divalent organic group. ) represents. ], the composite according to claim 1. [Claim 5] Claim 3 or 4, wherein R1 is a group represented by the following formula (I) [Claim 1], and R2, R3 and R4 are groups represented by the following formula (II) [Claim 2] The complex described.