JPS59205326A - Adjuvant containing abrine-a - Google Patents

Abstract

PURPOSE:To provide an adjuvant containing abrine-a and applicable as an injection. CONSTITUTION:The objective adjuvant effective for the remedy and preventive of infectious diseases caused by microorganisms such as bacteria, viruses, etc. or cancer, is obtained by using abrine-a (a component of abrines separated from the seed of a leguminous plant, Abrus cantoniensis) as a component. The necessary minimum amount of abrine-a in the preparation is 0.1ng per a man or animal, and the maximum dose is 10mug. Since abrine-a is soluble in water, it can be administered for immunization as a mixture with an antigen or vaccine by the subcutaneous, intramuscular, peritoneal or intrathoracic infusion. Furthermore, since the activity of abrine-a is maintained stably in the presence of galactose, it is preferable to add 1-100mM of galactose to the preparation for improving the stability of the injection and for providing a diluent for freeze- drying.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an immune enhancer containing abrin-a. Immune enhancers refer to substances that exhibit a strong enhancing effect on humoral or cellular immune response mechanisms as a function of their own, and in particular, immune enhancers that enhance the immune response to antigens when injected together with antigens. It is generally called an adjuvant.

Freund's complete adjuvant, aluminum hydroxide gel, etc. have traditionally been used as immune adjuvants, but these can cause a strong inflammatory reaction at the injection site, causing swelling, necrosis, and induration, so they should not be used in humans. It is not possible.

Freund's complete adjuvant consists of killed tuberculosis bacteria, a surfactant, and mineral oil, and in order to exhibit adjuvant activity, it is necessary to form a water-in-oil emulsion with an antigen aqueous solution or antigen suspension. Therefore, it takes time and effort to prepare the mixture.

Another disadvantage is that the liquid becomes viscous, making it inconvenient to inject.

In view of these circumstances, the present inventor conducted research in search of a substance that has an immune-enhancing effect on both humoral and cellular immune response mechanisms and is easy to mix and prepare with antigens. The present invention was completed based on the knowledge that the present invention can be used as an adjuvant substance that satisfies the requirements.

The present invention will be explained in detail below.

Abrin a is the legume plant Tuazuki (AbrusP).
It is a component contained in abrin isolated from the seeds of S. recatorius. That is, abrin is a mixture of abrin a, abrin b, abrin C, and abrin d, and these components have almost the same molecular weight but differ in toxicity and cell aggregation activity.

In addition, physicochemically, they exhibit different adsorption properties for galactose. Therefore, abrin-a is a substance that is fractionated from abrin and is toxic.

It is distinguished from other abrin components by its cell aggregation activity and adsorption properties for galactose. The following documents are referred to regarding the fractionation and various properties of abrin-a.

J., Y., Lin et al., Toxico
n Vol, 19. Pd2-51 (1981) The molecular weight fraction 9 and toxicity of each component in abrin are as follows.

The seed coat of the red bean is removed, the cotyledons are immersed in a 5% acetic acid aqueous solution, and the next day, after homogenization, the supernatant is collected by centrifugation at 10,000 g for 20 minutes. Add ammonium sulfate to the supernatant,
Collect the part that precipitates when the degree of saturation is 35% or more and 95%. This is 0. After dialyzing against 0.005 M phosphate buffer (pH 8,0) containing OOIM EDTA and removing ammonium sulfate, the dialyzed content is centrifuged at 10,000 a for 20 minutes and the supernatant is collected. This is referred to as a crude extract, which contains four types of abrin and Abrus Φ agglutinin.

Abrin a is isolated from the crude extract as follows.

The crude extract is first fractionated by 5epharose 4B affinity column chromatography.

All abrin and abulus Φagglutinins.

These lectins have the property of specifically binding to galactose residues, and 5epharose 4B is a polymer of galactose.

Other contaminant proteins that are adsorbed to the 5epharose 4 B column and have no binding properties can be removed. 0.2M
The crude extract solution was applied to a column of 5 epharose 4 B which had been sufficiently equilibrated with 0.005 M IJ acid buffer at pH 8.0 containing NaCl.

When elution is performed with this buffer, first, 5epha
Proteins that have no affinity for ros4B are eluted, and when measured by absorbance at 280 nm, the first peak ★\' appears, followed by 5epharose 4B.
A second peak appears, containing proteins with a weak affinity for . from the proteins that form this second peak.

Although abrin B and abrin C can be isolated by DEAE cellulose column chromatography, the details thereof will be omitted.

5epharose 4B column with 0.2M NaC
Wash thoroughly with 0.005M phosphate buffer containing 28
After the absorption at 0 nm is no longer detected, when a solution containing 0.1 M of F-galactose is run in this buffer, the protein adsorbed on this column is eluted, and a third sharp peak appears. Appear. This third peak pH
0 of 8,6. Dialyzed with OIM Tris buffer, adsorbed on a column of DEAE cellulose equilibrated with this buffer, and washed thoroughly to pH 8 containing 0.03M NaCl.
, 6 (7) 0, OIM When Tris buffer is passed, the protein eluted at this NaCl concentration can be detected by absorbance at 280 nm. This one is sodi to 0.196
Polyacrylamide gel electrophoresis (SDS-PAGE) containing um dodecyl 5ulfate showed a single band, which is abrin d. From this DEAE cellulose column.

After the absorbance of the protein eluted with 0.03M Mail was no longer observed, a 0.01M Tris buffer with a pH of 8.6 and a NaCl concentration of 0.2M was introduced.
A protein was found that eluted as a sharp peak.

Analysis by SDS-PAGE revealed that it was a mixture of two proteins. This 0.2M Na1l-containing T
The protein eluted with the RIS buffer was concentrated by ultrafiltration and simultaneously 0.005M at pH 7.2. 5ephadex which had been equilibrated with this buffer.
When applied to G-100 column chromatography, 280
Two peaks were obtained in the absorbance at nm, the first peak being Abrus agglutinin and the second peak being Abrus agglutinin.
It is.

Each of these four 1-sotoxin and abulus agglutinin in red bean was treated with 5% 2-mercaptoethanol at 45°C for 1 hour, followed by 0.1% SDS.
-When analyzed by PAGE, it is detected as two bands, so each molecule has two subunits ~
It is known to have an S-8 bonded structure.

The part that migrates quickly is called the A subunit, and the part that migrates slowly is called the B subunit. 0.1%SDS-
The molecular weight of abrin a by PAGE was compared to that of other abrins.
Table 1 shows a comparison of the molecular weights of sotoxin and Abulus agglutinin.

Table 1. Molecular weight of abrin and abulus red agglutinin and the molecular weight of each subunit ◆Protein Complete protein A subunit B subunit abrin a
63,000 28,000 35,
00077' IJ:yb 67.000
32,000 35,00077-! J n c
63,000 28,000 35,00
0ab! J 7 d 67.000 32,0
00 35,000, the molecular weight measured by 5DS-PAGE has an error range of ±5,000.

Although the four 1sotonns are almost the same in terms of molecular weight, as shown in Table 2, abrin-a exhibits strong toxicity, unlike the other three 1sotoxins and abulus agglutinin. In Table 2, CDF.

The value of LD, which was determined by intraperitoneally administering the drug once to mice and determining whether it was alive or dead two weeks later, is shown.

7- Table 2. Acute toxicity of abrin and abrin agglutinin in mice Toxicity LD 5°/average (intraperitoneal administration to mice) abrin a 1.4 μg abrin b 56 abrin C18 abrin d 35 abrin agglutinin 90 Now, abrin a will be described later. As shown by the experimental examples, the present inventors have revealed that the present invention has an immune-enhancing effect on both humoral and cellular immune response mechanisms. This finding is new.

It has the advantage of exhibiting sufficient activity with the other three abrins and a trace amount of about 1710 lysine. In the first place, it is known that the immune mechanism of 0 dorsal vertebral motion is established by two basic immune response mechanisms against infection and cancer. One is the cell-mediated immune response mechanism, which is mainly effective against infections caused by fungi, viruses, intracellular parasitic bacteria, etc., and against cancer.
The other one is the body fluid-8 immune response mechanism, which is mainly effective in preventing infection by viruses and extracellular parasitic bacteria.

Therefore, based on the above findings, the immunostimulant of the present invention, when administered to humans and animals, enhances immune responses against bacteria, viruses, other microorganisms, and cancer cells, and prevents infections caused by microorganisms such as bacteria and viruses, or cancer. Effective for treatment.

The minimum required dose of abrin-a in the immunostimulant of the present invention is 0.1 no per human or animal, regardless of body weight, and the maximum dose is 10 μg.

Furthermore, since abrin-a is water-soluble, the two immune enhancers of the present invention can be provided as an aqueous solution. Therefore, it can be mixed with an antigen and a vaccine and administered for immunization as a single unit. In this case, subcutaneous, intramuscular, intraperitoneal and intrathoracic administration is possible. The immunopotentiator of the present invention is an antigen,
Of course, it can be administered separately from the vaccine, and in addition to the above-mentioned routes of administration, intravenous administration and direct administration to the tumor site are also possible. It is sufficient to administer the drug twice a week or once every two weeks.

The specific dosage form of the immune enhancer of the present invention is mainly an injection. For example, physiological saline solution or pH 5.5 to pH
1μ of abrin-a in 0.1-1 mL of 8.0 buffer
The injection solution may be prepared so as to have a concentration of g~1*a/lIt. Moreover, if necessary, it can also be made into a freeze-dried injection. Abrin-a maintains its activity stably in the presence of galactose.

Adding 1mM to 100mM of galactose to the formulation is useful not only to maintain stability as an injection solution but also as an excipient for freeze-drying. Lactose can be used for similar purposes as galactose. Therefore, instead of galactose, lactose or lactose and galactose can be added to the formulation, and this is included as part of the additives in the immunopotentiator of the present invention. Note that the injection of the present invention can be manufactured according to a conventional method for manufacturing injections.

Since abrin-a maintains its adjuvant activity stably even after freeze-drying, if the antigen is also stable to freeze-drying, it is possible to freeze-dry a mixture of antigen and abrin-a. be.

The fact that abrin-a has a stronger immune adjuvant effect than other abrins, 1 sotoxin and ricin, will be explained by the following experimental examples.

Example Bovine serum albumin (BSA), a representative protein antigen
) to produce humoral antibodies against BSA.

And, as an indicator of cell-mediated immunity, delayed-type hypersensitivity reactions were measured and the efficacy of abrin-a was compared with that of other sotoxins and ricin. i.e. 2 weight 20-23g Q
10tta of BSA and 1 sotoxi of abrin were administered subcutaneously to each group of 6 mice.
0.11 of any of n or physiological saline containing ricin
After the injection of 1 L, each group of mice was subcutaneously immunized with a solution of the same composition 14 days later, and 7 days later, blood was injected from the fundus vascular plexus of the mice. b+1 was collected, added to 0.2 tnL of physiological saline, mixed, centrifuged, two supernatants were taken, and stored at -20°C. The dilution ratio of this serum was 5 times that of the original serum. Anti-BSA antibody titer is BSA
Sensitized Tannic Acid Treated Sheep Hemagglutination Reaction Method [Protein Nucleic Acid Enzyme, Reprint [Biochemistry of Immunology J Vol, 11゜Nα1
5.1506, 1966], the serum dilution factor is shown.

Delayed hypersensitivity reactions to BSA can be detected using the method of Robinson et al.
Robinson, J.H., 5cand., J.I.
mmunol, 5°299, 1976),
After blood collection, 10 μl of physiological saline containing 10 μg of BSA was injected into the ear shell of the mouse, and the increase in ear shell thickness was measured 24 hours later.

Humoral antibodies and cell-mediated immunity (delayed hypersensitivity reaction/DT
The measurement results for H) are shown in Table 3.

12- As shown in Experiment 1 of Table 3, the adjuvant activity of abrin a was equivalent to or greater than that of abrin b, abrin C, or abrin d at a dose of 1710. Furthermore, the adjuvant activity of abrin-C.

Table 2. As shown in Experiment 2, activity was observed even at doses of 0 and 1 ng, whereas with ricin, no adjuvant effect was observed at doses of 1r+g as shown in Table 2. Based on the second results, abrin-a was determined to be an adjuvant with a specific activity approximately 10 times higher than that of other abrins and ricin.

Example The adjuvant effect of abrin a on anti-tumor immunity was compared with that of another abrin, 1sotoxin.

Meth-A cancer cells transplanted into the peritoneal cavity of Bal b/c mice were collected, and after washing with Eagle's MBM medium, 40 μy of mitomin C was added to a suspension of 1 σ cells/NL of cancer cells.
/aL, incubated at 37°C for 1 hour, and washed. The cancer cells obtained in this way.

It is an antigen that does not show proliferative activity in the mouse body and can confer specific resistance to transplantation of Meth-A cancer cells, that is, it has activity as an inactivated cancer cell vaccine. In this experiment, 1×1σ mitomachine C processing Meth-
A 0.11 Eagle's MEM solution containing a constant amount of A cancer cells and each abrin was added to a female Bal weighing 20-23 g.
After immunizing b/c mice by subcutaneous injection, 14 days later, 1X10' live Meth-A cancer cells were implanted subcutaneously to attack them, and after another 3 weeks, the grown tumors were excised. The weight was measured. In addition.

Each group consisted of 6 animals. The results are shown in Table 4, showing that abrin-a had a tumor growth inhibition rate of just under 90% even at a log dose, indicating that it had a stronger adjuvant activity than other abrins at a minute amount.

Example 1 Preparation of Ampoule Containing Abrin A Crude extract of red bean was applied to Sθpharose 4B column.

DEAE c/l/mouth x column then 5ephad
Abrin a obtained by applying ex G100 column, 20
Pour 70ml of a solution containing 0u into a cellophane tube for dialysis and add 0.01M phosphate buffer (pH 6.0) containing 0.15M of 9 NaCl [hereinafter abbreviated as PBS (pH 6.0)] 1 to 51. I did dialysis at night. Remove the contents of the cellophane tube.

After centrifugation at 15.00 rpm for 10 minutes, the supernatant was
The absorbance at nm was measured and the E1% of abrin 280
nm to 15.9 (01snes et al.: J, Biol,
Chem, 249.803-810 (1974)
), dilute the two supernatants with PBS (pH 6,0) to obtain abrin 8500 μg/ml (7) solution.

This solution was sterilized through a 0.45μ filter, and IWI
A ampule for injection containing 100 μg of abrin a was prepared by dispensing 0.2 wI into L glass ampoules.

Example 2 Preparation of lyophilized vial containing abrin-a After measuring the absorbance at 280 nm in Example 1, PBS (pI-f
6.0) to make a solution of abrin a 1 yng/ml. To this, an equal volume of 0.2M D(+1 galactose solution) was added, followed by sterilization filtration with a 0.45μ filter.

0.2*j was dispensed into a freeze-drying vial with an internal volume of 2 ml and freeze-dried to create a freeze-dried vial containing 100 μg of abrin-a.

Example 3 The procedure was as described in Example 2, except that the same molar concentration of lactose was used in place of galactose in Example 2.

Example 4゜The concentration of abrin-a in Example 2 was changed to PBS (pH
6,0) and further diluted 10 times to 100 μa/m
A lyophilized vial containing 10 μq of abrin-a was prepared in the same manner as described in Example 2, except that a 10 μq of abrin-a solution was used.

patent applicant Koichizai Co., Ltd.

Claims (1)

[Scope of Claims] (1) An immune enhancer containing abrin-a (2) An immune enhancer according to claim 1, wherein the immune enhancer is an antitumor immunity enhancer (3) Antitumor immunity enhancer The immune enhancer according to claim 2, wherein the agent is an anti-Meth-A cancer cell immunity enhancer (4) The immune enhancer according to claims 1 to 3, wherein the immune enhancer is an injection. The concentration of enhancer (5) abrin-a is 1 μg to 1 my/
Immune enhancer (6) according to claims 1 to 4, which is at.Claims 1 to 5, which contain lactose or lactose and galactose as additives.
Immune enhancer described in section