JPH0643343B2 - Immunomodulator - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial field of application] The present invention relates to a blocked Fc fragment of human IgG (hereinafter, also referred to as a blocked Fc fragment), a blocked Fab fragment of human IgG (hereinafter, a blocked Fab fragment). Also called),
The present invention relates to an immunomodulator comprising a blocked L chain of human IgG (hereinafter also referred to as a blocked L chain) and a blocked H chain of human IgG (hereinafter also referred to as a blocked H chain) as main components.

[Conventional technology]

These series of blocked products are obtained by blocking the —SH group of the Fc fragment, Fab fragment, L chain and H chain with an appropriate group.

The currently known pharmacological actions on the above-mentioned series of blocked compounds, especially alkylated Fc fragment, alkylated Fab fragment, alkylated L chain and alkylated H chain, include anti-digestive ulcer action [Mimura, T. , Journal of Pharmacobio Dynamics (Mimura,
T. J. Pharm.Dyn.,) 6,397 (1983)], but their effects on the host immune system are completely unknown.

[Problems to be solved by the invention]

The present invention relates to a blocked Fc fragment, a blocked Fab fragment,
It is intended to provide a novel use of a human IgG-derived substance having a blocked L chain and a blocked H chain.

[Means for solving problems]

This time, the present inventors have found that a blocked Fc fragment, a blocked Fab fragment, a blocked L chain and a blocked H chain prepared from human IgG act in an immunopotentiating or immunosuppressive manner depending on the immune response state of the host. The present invention was completed by discovering that it has a so-called immunoregulatory effect.
That is, the present invention provides a blocked Fc fragment and a blocked Fa.
The present invention relates to an immunomodulator containing at least one selected from a b fragment, a blocked L chain and a blocked H chain as an active ingredient.

Collagen disease and rheumatic diseases have abnormal host immune responses, and it is considered that recognizing self-components and producing autoantibodies play an important role in the pathogenesis. Therefore, the treatment requires correction of the abnormal immune response. The subject of immunomodulators can be said to be a disease caused by immune dysfunction of the host.

The Fc and Fab fragments of human IgG have already been reported as known IgG fragments, for example, the report of Porter et al. [Biochem. J. , 73, 119 (1959)]. This human 1gG Fc fragment or Fab fragment is
It is a polypeptide chain having a molecular weight of 45,000 to 50,000 obtained by decomposing human-derived IgG with papain or plasmin, and its recovery method has been established by Porter et al.

Blocked F specified as an active ingredient in the present invention
The c fragment and the blocked Fab fragment are human IgG F
c. cleaves the disulfide bond of the fragment and Fab fragment,
It can be obtained by blocking the -SH group of each generated fragment.

Blocked Fc fragment and blocked F according to the present invention
The outline of a typical method for recovering an ab fragment is as follows.

PH containing a solution containing IgG (protein concentration 2-10%)
Adjust to 6-9, add plasmin or papain to this and treat at 20-40 ° C for 10-30 hours. Then, insoluble matter is removed from this treatment liquid, and undigested IgG and digested products are separated by gel filtration treatment. The digestion products are ion exchangers (CM-cellulose and DEAE
-Cellulose) to selectively adsorb the Fc fragment and Fab fragment of human IgG, elute and collect.

After recovering the Fc fragment and Fab fragment of human IgG, treatment with a reducing agent is performed to cleave the disulfide bond, and blocking (eg, alkylation) is performed to obtain a blocked (alkylated) Fc fragment and a blocked (alkylated) Fc fragment. Alkylated) Fab fragment is obtained.

As a reducing agent, 2-methylcaptoethanol (final concentration
0.75-5.25M), dithiothreitol, dithioerythritol (final concentration 0.01-0.068M) and the like are used.

The blocked Fc fragment or blocked Fab fragment is
It is prepared by blocking the SH group according to the usual method [Biochemistry, 7, 1950 (1968)]. The blocking is carried out by introducing a pharmacologically acceptable group such as the following, particularly an alkyl group or a substituted alkyl group, by a known means. In addition, in the present specification, the term “lower” usually means one having 1 to 4 carbon atoms.

Lower alkyl group: methyl, ethyl, n-propyl and the like N, N-di-lower alkylcarbamido-lower alkyl group: N, N-diethyl-carbamidomethyl lower alkoxycarbonyl group-lower alkyl: ethoxycarbonylmethyl, ethoxycarbonylethyl and the like carboxy -Lower alkyl group: carboxymethyl,
Carboxyethyl etc. Cyano-lower alkyl group: Cyanomethyl etc. β-amino-lower alkyl group: -CH ₂ CH ₂ NH ₂ etc. Benzoyl-lower alkyl group: -CH ₂ COC ₆ H ₅ etc. Carbamoyl-lower alkyl group: -CH ₂ CONH _{2 and} other sulfo groups On the other hand, human IgG-derived L chains and H chains are known Ig
It has already been reported as a constituent fragment of G, for example, there is a report by Freishmann et al. [Arch.Biochem.Biophys., Supp.
le. (1) .174 (1962)]. The L chain and H chain specified as the active ingredient in the present invention are human-derived IgG to IgG.
Molecular weight obtained by cleaving the disulfide bond of 23,000 ±
The polypeptide chains are 1,000 and 50,000 ± 1,500, and the recovery method thereof has been established by Freysmann et al. The outline of the typical L chain and H chain recovery method according to the present invention is as follows.

IgG to 0.55M Tris-HCl buffer, pH 8.2 about 2
Dissolve to a concentration of%. After passing the nitrogen gas gently, 2-
Add mercaptoethanol to a final concentration of 0.75M,
The reduction is carried out by leaving it at room temperature for 1 hour. Next, it was cooled in an ice-water bath, 0.75M monoiodoacetamide in the same amount as 2-mercaptoethanol was added, and the pH of the solution was kept at 8.0 by addition of trimethylamine, etc., and allowed to react for about 1 hour, then cooled to cold saline. Dialyse to remove excess reagents. In this reaction, the free SH group in the L chain or H chain is blocked. Then dialyzed against chilled 1M propionic acid
When the H chain and the H chain are dissociated and passed through a column of Sephadex G-75 equilibrated with 1M propionic acid, the L chain and the H chain are separated.
The chains elute as two separate peaks. After each of the L chain and H chain fractions is collected, a desired known treatment that can be provided as a medicine, such as dialysis, sterilization filtration, heat treatment, and freeze-drying, is performed.

The L chain and H chain used in the present invention are not limited to those obtained by the above-mentioned recovery method, and may be L chain or H chain produced according to other applicable methods. The SH group of these L chain and H chain is protected by a pharmacologically acceptable group such as blocked, particularly alkylated or sulfonated. Examples of the group that is substituted upon blocking are the same groups as described above.

These blocks can be manufactured by a method known per se or a method equivalent thereto.

Next, the blocked Fc fragment and blocked Fab of the present invention
Examples of experiments conducted to confirm the pharmacological action of the fragment, the blocked L chain and the blocked H chain and clinical tests, acute toxicity tests, doses, administration methods and the like are shown below.

Experimental Example 1 (Effect on antibody production) CDF ₁ mice (7 to 8 weeks old, male) were immunized with 2 × 10 ⁸ sheep red blood cells (SRBC) by intravenous administration, and 4 days later, the spleen was extracted, Antibody-producing cells (Hemolytic plaque
The number of forming cells) was measured to examine the influence of the test drug on antibody production. The number of antibody-producing cells can be measured by Cunningham
The method was followed [Immunolgy, 14, 599 (1968)]. The carbamoylmethylated Fc fragment, the carbamoylmethylated Fab fragment, the carbamoylmethylated L chain, and the carbamoylmethylated H chain as test drugs were intravenously injected immediately after the administration of SRB as an antigen. In the experiment, one group consisted of four animals. Table 1 shows the results.
Is.

Compared with the number of anti-SRBC antibody-producing cells in the spleen of the control group, carbamoylmethylated Fc fragment, carbamoylmethylated F
When the ab fragment, the carbamoylmethylated L chain and the carbamoylmethylated H chain were administered, remarkable enhancement of antibody production was observed.

Experimental Example 2 (Effect on host with reduced immune function) In preparing a state with reduced immune function in CDF ₁ mice (7-8 weeks old, male), an immunosuppressive agent was administered under the following experimental conditions. Azathioprine (40 mg / kg), cyclophosphamide (50 mg / kg), or betamethasone (1 mg / kg)
(kg) was administered 4 days, 3 days, and 2 days before the antigen stimulation.
The routes of administration of these drugs were all intraperitoneal injections. Then, simultaneously with antigen stimulation with sheep red blood cells (SRBC) (2 × 10 ⁸ ), carbamoylmethylated Fc as a test drug
Fragment, carbamoylmethylated Fab fragment, carbamoylmethylated L chain or carbamoylmethylated H chain was administered, the number of antibody-producing cells in the spleen on the 4th day was measured, and the effect on a host with impaired immune function was examined. In this experiment, levamisole, which is one of the immunomodulatory therapeutic agents, was selected as a control drug. Levamisole was given intraperitoneally and the other drugs were given intravenously. It should be noted that there were 4 animals in each group. Table 2 shows the above results.

Although the number of antibody-producing cells is reduced in the immunosuppressant-administered group compared to the untreated group, even in such a host with reduced immune function,
It was confirmed that the number of antibody-producing cells was increased in the carbamoylmethylated Fc fragment, carbamoylmethylated Fab fragment, carbamoylmethylated L chain and carbamoylmethylated H chain administration groups.

Experimental Example 3 (Action on host with enhanced immune function) It has been reported that administration of colchicine together with an antigen enhances antibody productivity due to inhibition of suppressor T cells [J. Exp.Med., 147, 1213 (1977)]. In this experiment, TNP as a hapten group was introduced into a keyhole limpet hemocyanin (KLH) in accordance with a method of a variety of methods [immunity experiment operation method p.1129 (1971), Japan Immunological Society], and so-called TNP- KLH was used as the antigen. TN
P-KLH (200 μg / mouse) was intraperitoneally administered to CDF ₁ mice together with bentonite as an adjuvant, and simultaneously colchicine (1 mg / kg) was intraperitoneally administered. Further, carbamoylmethylated Fc fragment and carbamoyl as test drugs were administered. Methylated Fab fragment, carbamoylmethylated L chain or carbamoylmethylated H chain was intravenously administered, and on the 6th day thereafter, the number of anti-TNP antibody-producing cells in the spleen was measured to examine the effect on the host with enhanced immune function. . The experimental group consisted of 4 animals. Table 3 shows the result.

The colchicine-treated control group had a clear increase in the number of antibody-producing cells as compared with the untreated state, which is a state in which it is considered that the so-called immune function is enhanced, but carbamoylmethylated Fc fragment, carbamoylmethyl Fab fragment,
The immune response of the carbamoylmethylated L chain and carbamoylmethylated H chain administration groups was a value close to that of the non-treated group (normal mouse), and it can be seen that there is a tendency to suppress the enhanced immune function of the host.

Experimental Example 4 (Effect on Adjuvant Arthritis as Rheumatoid Model) Using male Wistar rats weighing about 200 g, Mycobacterium butyricum (Difk), 0.5 mg / 50 μm, was suspended in liquid paraffin under the foot pad of the right hind leg under ether anesthesia.
l was injected. Carbamoylmethylated F as a test drug
The c fragment, carbamoylmethylated Fab fragment, carbamoylmethylated L chain or carbamoylmethylated H chain was intravenously administered for 20 days at a rate of 20 mg / kg / day from the day of adjuvant treatment. In addition, D-penicillamine, which is one of the therapeutic agents for rheumatism, was used as a control drug, and this was intraperitoneally injected. Then, the volume of both hind limbs was measured with a footpad edema volume device. The experiment was conducted with 7 animals per group. Table 4 shows the result.

Of the changes in the edema rate of the hind limbs treated with adjuvant, the cases of the 10th day and the 20th day are shown. For the delayed edema after the 7th day, the carbamoylmethylated Fc fragment was compared to the control group. , The carbamoylmethylated Fab fragment, the carbamoylmethylated L chain or the carbamoylmethylated H chain showed a clear inhibitory effect on adjuvant arthritis.

[Dose and administration method]

Blocked Fc fragment and blocked Fab that are active ingredients
From the results of the above test, it is preferable to administer 1 to 100 mg / kg of the fragment, the blocked L chain and the blocked H chain per day for an adult.

This drug can be administered in the form of either an injection or an oral preparation. When it is used as an injection, it is dissolved in distilled water for injection at the time of use. The method of administration is usually intravenous and intramuscular administration. When used as an oral preparation, it is administered as a capsule, tablet, powder, liposome preparation, oral liquid preparation, or the like. In the case of an oral preparation, it may be enteric coated. These are produced according to a method well known to those skilled in the art, for example, the method described in the Japanese Pharmacopoeia.

[Action / effect]

Blocked Fc fragment, blocked Fab according to the invention
The fragment, the blocked L chain and the blocked H chain have extremely low toxicity and their immunomodulatory action is remarkable.
It is considered to be extremely useful as a therapeutic / preventive agent (ie, immunomodulator) for diseases caused by immunodeficiency such as rheumatic diseases and collagen diseases. By the way, the immunomodulator according to the present invention is a concept including an anti-inflammatory agent because of its action on adjuvant arthritis.

[Reference examples / Examples]

Next, reference examples for producing a blocked Fc fragment and a blocked Fab fragment, and Examples of the present invention will be shown.

Reference Example 1 60% sodium azide was added to a 3% IgG solution (60 ml).
Add mg and adjust pH to 7.5 with 1N NaOH solution. Add plasmin to a final concentration of 4 cu / ml,
Digestion is performed at 35 ° C. for about 15 hours. After treatment
After adjusting the pH to 6.5 and leaving it at 4 ° C. for 1 hour, the insoluble matter is removed by centrifugation. Plasmin digestive fluid (about 60m
l) is injected into a column of Sephadex G-200 and subjected to gel filtration treatment to separate into undigested globulin (7S) and digested product (Fab + Fc). This digestion product is then contacted with a CM-cellulose column (pH 7.0), and F
Adsorb the ab and Fc fragments. After washing with a column, add 0.3M NaCl to 0.01M phosphate buffer (pH 7.0).
It is developed with a solvent added with and the Fab fragment and the Fc fragment are separately recovered.

Both fragments thus obtained were mixed with 0.05 M Tris-HCl buffer (pH 8.
It was dissolved in 2) at a concentration of about 2%, and 2-mercaptoethanol was added to a final concentration of 0.75 to 5.25 M to cleave the disulfide bond. Then add 0.75-5.25M iodoacetic acid,
After keeping the pH at 8.0 and reacting for 1 hour, Sephadex G
Excess sample was removed on a -25 column. Next, after dialyzing against physiological saline and performing sterilization filtration, each was made into a freeze-dried product.

Reference Example 2 2.5% IgG solution (20 ml; 0.02 M EDTA-0.05)
Add 5 mg of papain to M phosphate buffer (pH 7.5), and add 37
After digesting at C for 10 to 20 minutes, the mixture was cooled with ice water. After cooling, the insoluble matter was centrifuged, and the supernatant was fractionated by a Sephadex G-150 column to obtain a 7S fraction. This was treated with dithiothreitol at a pH of 7.5 to 8.0 and a final concentration of 0.01 M at room temperature for 2 hours, and then iodoacetamide was added to a final concentration of 0.2 M, and the mixture was reacted under ice cooling for 1 hour. This is 0.005 of pH8
It was dialyzed against M Tris-HCl and the resulting crystals were centrifuged. The supernatant was a crude blocked Fab fragment and the precipitated fraction was recovered as a blocked Fc fragment.

Reference Example 3 IgG was dissolved in 0.05 M Tris-HCl buffer (pH 8.2) at a concentration of about 2%, and 2-mercaptoethanol was added to a final concentration of 0.75 M to cleave the disulfide bond.
Next, 0.7 M iodoacetic acid was added, the pH was kept at 8.0, the reaction was carried out for 1 hour, and then the excess reagent was removed by a Sephadex G-25 column. Next, SDS (scdium dodecyl sulfat)
e) Sephadex G-200 column in the presence (4.0 × 12
0 cm) [solvent: 0.04 M SDS-0.05 M phosphate buffer (pH 8.0)] and measured at OD 280 mm to collect L chain and H chain fractions. Next, SDS was removed from the L chain fraction or the H chain fraction, dialyzed against physiological saline, and sterilized and filtered to obtain a freeze-dried product.

Next, examples of the present invention will be described.

Example 1 (oral preparation) (1) Human IgG-derived carbamoylmethylated Fc fragment, carbamoylmethylated Fab fragment, carbamoylmethylated L chain, or carbamoylmethylated H chain 5.0 mg (2) Fine particles for direct compression No.209 (manufactured by Fuji Chemical Co., Ltd.) 46.6 mg Magnesium metasilicate 20% Corn starch 30% Lactose 50% (3) Crystalline cellulose 24.0 mg (4) Carboxymethyl cellulose / calcium 4.0 mg (5) Magnesium stearate 0.4 mg ( All of 1), (3) and (4) are passed through a 100 mesh screen in advance. These (1), (3), (4) and (2) are each dried to reduce the water content to a constant value, and then mixed in the above-mentioned weight ratio using a mixer. Add (5) to the mixed powder that is homogenized and mix for a short time (30 seconds), and press the mixed powder into a tablet (punch: 6.3 mm).
φ, 6.0 mmR) to give tablets of 80 mg each.

If necessary, the tablets may be coated with a gastric-soluble film coating agent (eg, polyvinyl acetal diethylaminoacetate) or an edible coloring agent that is commonly used.

Example 2 (intravenous injection) (1) Human IgG-derived carbamoylmethylated Fc fragment, carbamoylmethylated Fab fragment, carbamoylmethylated L chain, or carbamoylmethylated H chain 50 mg (2) Glucose 100 mg (3 ) Saline solution 10 ml (3) are added with (1) and (2) in the above weight ratio and stirred to completely dissolve. The lysate is filtered using a membrane filter with a pore diameter of 0.45μ, and then sterilized and filtered again using a membrane filter with a pore diameter of 0.20μ. 10 filtrate
Aseptically dispense each ml into a vial, fill with nitrogen gas, and then seal to give an intravenous injection.

Example 3 (capsule) (1) Human IgG-derived carbamoylmethylated Fc fragment, carbamoylmethylated Fab fragment, carbamoylmethylated L chain, or carbamoylmethylated H chain 50 g (2) Lactose 935 g (3) Stearin Magnesium acid 15 g The above components are weighed and 1000 g in total are uniformly mixed, and 200 mg each of hardlatin capsules is filled with the mixed powder.

Example 4 (liposome preparation) Human IgG-derived carbamoylmethylated Fc fragment, carbamoylmethylated Fab fragment, carbamoylmethylated L chain or carbamoylmethylated H chain was added to 0.01M phosphate buffer (pH 7.2) containing 0.125M NaCl. Dissolve to a concentration of about 0.5%.

On the other hand, 100 mg of egg yolk phospholipid containing 0, 5, 10, 20% (w / w) phosphatidic acid was dissolved in 10 ml of chloroform, respectively, using a rotary evaporator.
A film of phospholipid was formed. In addition to the carbamoylmethylated Fc fragment, carbamoylmethylated Fab
1 ml of a solution of the fragment, carbamoylmethylated L chain or carbamoylmethylated H chain was added and shaken to form a closed fat body, and these drugs were incorporated to obtain a liposome preparation.

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Claims (3)

[Claims] 1. A blocked Fc fragment of human IgG, human I.
An immunomodulator comprising at least one selected from a blocked Fab fragment of gG, a blocked L chain of human IgG, and a blocked H chain of human IgG as an active ingredient. 2. The immunomodulator according to claim 1, which is in the form of a liquid preparation for intravenous, intramuscular or oral administration. 3. The immunomodulator according to claim 1, which is in the form of a powder, tablet, capsule or liposome preparation for oral administration.