JPS60209530A - Tumoricidal substance - Google Patents

Abstract

PURPOSE:To obtain a tumoricidal substance from supernatant liquid obtained by culturing a macrophage originated from mammals in a medium containing a composite protein originated from Sarcophaga peregrina and having lectin-like activity. CONSTITUTION:A composite protein exhibiting lectin-like activity and having a molecular weight of about 188,000 by gel-filtration and consisting of 4 subunits having a molecular weight of about 32,000 by SDS-gel electrophoresis and 2 subunits having a molecular weight of about 30,000 is prepared by pricking the body surface of a maggot or chrysalis of the early pupal stage of Sarcophaga peregrina, homogenizing the body for several minutes with a blender, and extracting, purifying and separating the objective substance. The composite protein is added to a medium for animal cell at a concentration of 1-100mug/ml, and a mammal- originated macrophage (e.g. mouse-originated monocytic leukemia cell strain J-774) is cultured in the medium. The objective tumoricidal substance which is a proteinous substance having a molecular weight of about 30,000-70,000 is separated from the supernatant liquid of the culture medium. The substance loses its activity by heating at 90 deg.C for 30min and loses about 20% of the activity at 56 deg.C for 30min.

Description

[Detailed description of the invention] The present invention is a method of killing! Concerning ulcerative substances.

As the number of cancer patients is rapidly increasing, there is a strong desire to develop more effective anticancer drugs. Chemotherapy agents, immunotherapy agents, and the like are used as anticancer drugs, but they have problems such as side effects.

Conventionally, mammalian-derived macrophages exhibit damage to tumor cells in vitro in the presence of various plant lectins, a so-called lectin-dependent macrophage-mediated cytotoxic response.
macrophage mediated cytot
oxicity; LDMC) was known to exist.

Therefore, the present inventors discovered the centinic fly (S.
A complex protein with lectin-like activity derived from Sarcophaga peregrina (referred to as Sarcophaga lectin).

], we investigated whether the above-mentioned LDI'IC activity could be induced, and it was revealed that mammalian-derived macrophages exhibited significant tumor cytotoxic activity when present in the coexistence of Lucophaga lectin. .

Furthermore, the present inventor stimulated mammalian-derived macrophages using the present Lucophaga lectin, and found that
that a novel tumoricidal substance can be obtained from the culture supernatant;
They also discovered that this substance could not be obtained when stimulated with plant lectins such as concanavalin A, and completed the present invention. That is, the present invention provides a method for treating mammalian-derived macrophages with stings on the body surface of the Sarcop fly.
aga pe-regrina) is obtained from the homogenate or body fluid of the larva or its early stage pupa, and has a molecular weight of approximately 188,000 as determined by gel filtration, and is 5O5-
It relates to a tumoricidal substance obtained by stimulation with Sarcophaga lectin, which is divided by gel electrophoresis into 4 molecules of subunits with a molecular weight of about 32,000 and 2 molecules of subunits with a molecular weight of about 30.0 (10).

The inventor has developed this substance into Tumor K11lir+B.
It was named l'actor (TKF).

The TKF of the present invention is produced by a centigrade fly (S.
It is obtained from the homogenate or body fluid of arcopaga peregrina) larvae or their early stage pupae, and has a molecular weight of about 188,000 by gel filtration method.
Cultivating mammalian-derived macrophages in a medium containing Sarcophaga lectin, which is separated into 4 subunit molecules with a molecular weight of about 32,000 and 2 subunit molecules with a molecular weight of about 30,000 by 5DS-gel electrophoresis,
Obtained by harvesting from culture.

The macrophages used in the present invention include those derived from mammals such as humans, mice, runts, cows, pigs, sheep, rabbits, and dogs, and which have the ability to produce the TKF of the present invention. Either is fine, but
In particular, J-774, P-, 338fl-1.

Pu5-1. ．． A mouse-derived monocytic leukemia cell line such as No. 8 or a mouse-derived myeloid leukemia cell line M1 can be used. Monocytes obtained from peripheral blood or macrophages obtained from organs can also be used.

Culturing is carried out under conditions normally used for culturing animal cells. That is, the animal cell culture medium (such as RPMI 1640) with or without the addition of serum (such as fetal bovine serum), preferably 37
It is carried out by culturing at ℃ for 2 to 24 hours. Moreover, a differentiation-inducing substance can also be present, if necessary.

Sarcophaga lectin is 0.1 μg/m 1
to several hundred μg/ml, preferably 1 to 100 μg/ml
It is sufficient to add l.

By centrifuging or decanting the culture fluid thus obtained, a culture supernatant containing the TKF of the present invention can be obtained. Purification of TKF from the culture supernatant is carried out by concentrating the culture supernatant as necessary and using conventional methods such as gel filtration, affinity chromatography, electrophoresis, dialysis, ultrafiltration, and ion exchange. , or preferably in any combination.

The Sarcophaga lectin used in the method of the present invention is obtained by injuring the body surface of the larvae of the centipede fly by a method such as puncturing it with an injection needle, homogenizing the larva for several minutes using a blender, and then extracting and purifying the larvae. Obtained by isolation. For example, the resulting homogenate is subjected to affinity chromatography using a column such as Sepharose CL6B, and after thoroughly washing the column with buffered saline, a galactose/8 solution or a lactose solution is used as a /8 synergist. Sarcophaga lectin is eluted when the adsorbed protein is eluted using -l. From this, the eluent is removed by dialysis or the like, and the product is freeze-dried to obtain a pure powder.

This Sarcophaga lectin is n1o-Gel P
-300 gel filtration method, the molecular weight was approximately 188
It was calculated as ゜000. This protein was determined by S-gel electrophoresis to have a molecular weight of approximately 32.000 and approximately 30.00.
(Since it is divided into two + subunits and the density ratio of the stained image is always 2:1, it is 32.0 + 10
4 molecules of male unit and 2 units of 30,000
It can be seen that it is made up of molecules.

The Sarcophaga lectin used in the present invention is thus extracted from the homogenate or body fluid of the larva or pupa in the early stage of axis formation of the sarcophagus fly that has been bitten on the body surface.
Although it is purified and isolated, it may be chemically synthesized or microbiologically produced using genetic engineering techniques.

The TKF of the present invention thus obtained is sensitive to trypsin and is inactivated by heating at 90°C for 30 minutes.
℃, approximately 20% deactivated by heating for 30 minutes, molecular weight approximately 30
,000 to about 70,000 (Sephadex G-20
It is a proteinaceous substance (measured by gel filtration method according to 0.0).

FIG. 1 shows the elution pattern of TKF by gel filtration with Sephadex G-200.

As shown in the experimental examples described below, the TKF of the present invention exhibits a cell-killing activity against mouse tumor cells that is about 1,000 times that of normal fetal cells, and therefore has the selectivity of acting only on tumor cells. , also exhibits excellent tumoricidal effects in 1 in vivo experiments on mice. It also has the advantage of not being species specific.

When the TKF of the present invention is used as a medicine, it can be mixed with carriers, activators, diluents, etc., and administered in the form of tablets, capsules, powders, granules, injections, ointments, suppositories, and the like. Although the dosage may vary depending on the type of tumor and symptoms, 0.1 to 100 mg per day for adults is usually appropriate.

The present invention will be explained below with reference to production examples, working examples, and experimental examples.

Production Example Production of Sarcophaga lectin The larvae of the centinic fly were used, which were prevented from forming an axial shell by wetting the body surface with water, and which were synchronized to the three lines.

To injure the body surface, the three-line larvae were placed on ice to slow their movement, placed on a glass plate, and the lower part of the first body cylinder was punctured with a hypodermic needle.

Two days after injury, 130 larvae per 1000 larvae.
Buffered physiological saline solution (hereinafter referred to as BIS) with the following composition of m1
It is called -1. ) was added, and the mixture was homogenized using a blender while cooling. After filtering the homogenate through gauze, the filtrate was centrifuged at 8000 rpm for 30 minutes. The obtained supernatant was applied to a Sepharose CL-6B column, and BIS-1
, followed by a buffered physiological saline solution (hereinafter referred to as BIS-
It is called 2. ), then B containing 0.2M galactose.
It was eluted with IS-1 solution.

Fractions exhibiting absorption at 280 nm were collected, dialyzed against deionized water, and then lyophilized to obtain Sarcophaga lectin as a white powder. The yield was 2 mg from 10,000 larvae.

Composition of BIS,-1: 130InM sodium chloride, 5mM potassium chloride, 1mM calcium chloride, 10mM)
(pH7', 9) Composition of BIS-2: 230mM sodium chloride, 5mM potassium chloride, 1mM calcium chloride, 10mM') Lis-(pH7,9) Example I Collection and purification of TKF Mouse-derived monocytes Cellular leukemia cells J-774 were grown 4x1 in RPM11640 medium containing 10% tallow serum (F'C3).
06 cells were suspended in a volume of 15 ml and cultured in a 5% COL-95% air atmosphere at 37°C for 53 hours on a 6 cm diameter plate. After culturing, centrifuge, remove the supernatant, and transfer the cells to F.
The cells were suspended in e2-free RPM11640 medium, centrifuged, the supernatant was removed, and the cells were washed. Washed cells
ml of Fe2-free RPM11640 medium, and added Sarcophaga lectin at 5-10 μg/ml.
37 in 5% Co, -95% air.
C. for 20 to 24 hours. Then, centrifuge and T
A culture solution containing KF was obtained.

This TKF-containing culture solution was applied to a Sephadex G-200 column equilibrated with buffered saline and eluted with the buffered saline. When the eluate was fractionated using a fraction collector and the activity of each fraction was measured using the assay method described below, the elution pattern shown in FIG. 1 was obtained. TKF was obtained by freeze-drying the active fraction.

When the sensitivity of this substance to trypsin was investigated, it was 68.7% when heated with trypsin 0.1 mg/ml at 27°C for 30 minutes, 81.4% when heated at 37°C for 30 minutes,
Furthermore, when heated with 1.0 mg/ml of tryfusin at 37°C for 130 minutes, it was inactivated by 87°7%. (Fe2
) in a MEM medium containing 2 to 3 x 10 6 cells/15 ml and cultured in a petri dish for 3 hours. Thereafter, tritium-labeled thymidine was added at a concentration of 1 to 0.5 μci/ml, and the cells were cultured at 37° C. in 5% C0L-95% air for 18 hours. After 18 hours, the culture medium was removed and the cells were
Calcium-imbalance buffered saline containing % EDTA [
After washing once with PBS (-)), add 3 ml of PBS (-) containing 0.02% and 0.05% trypsin per petri dish, and incubate for 3 minutes in 5% Coney 95% air.
It was kept at 7°C. Next, MliM medium containing a small amount of Fe2 was added, and the cells were centrifuged at 1000 rpm for 5 minutes at 4°C, the upper 1N was removed, and the cells were collected. The obtained cells were reduced to 1O%
It was suspended in HEM medium containing FC3.

The L-929 cells thus obtained were diluted with TKF solution to 2 x 10" cells/well, and a final solution of 200 μl was added to 5% Co.
Cultured for hours. After culturing, the radioactivity concentration in the supernatant was measured using a liquid scintillation counter.

Other target cells can be measured in the same manner.

Experimental Example I Selective cell killing effect of TKF Using the assay method of Example 2 above, L-9 of TKF was
When we investigated the cell killing effect on L-929 cells and mouse normal fetal fibroblast cells, we found that TK
1/1000 dilution of freeze-dried product of F is 25°3%,
A cell killing effect of 18.7% was observed in the 1/2000 diluted product. On the other hand, in Masatomi fetal fibroblast cells, 1/
Even at a dilution of 10, only 6.5% cell killing effect was observed.

Experimental Example 2 Tumor killing activity of TKF ICR mice were injected with 1×10'1 sarcoma 180 cells.
lil was intraperitoneally transplanted, and one day later, a 100 μm thick TKF saline solution was injected into the peritoneal cavity. 3 injections once a day
The mean survival days and number of cured cases were determined by repeating the test every day. The results were as shown in Table 1.

Table 1

[Brief explanation of drawings]

Figure 1 shows Sephadex G-200 of TKF of the present invention.
The elution pattern of gel filtration is shown. The vertical axis shows radioactivity concentration (cpm), and the horizontal axis shows both fraction numbers, A→ is blue dextran, B-→ is bovine serum albumin (molecular weight 66,200), C→ is ovalbumin (molecular weight 45,000), D- → indicates the elution position of α-chymotrypsin (molecular weight 25,000), and E-+ indicates the elution position of cytochrome C (molecular weight 12,500). Agent: Patent attorney Takamiyagi Sleeve of the winning procedure (spontaneous) September 1711, Showa 596 Manabu Shiga, Commissioner of the Patent Office 2, Name of the invention: Tumoricidal substance 3, Relationship with the Amendment 1 case Patent applicant residence Address: 13-35 Hirano-cho, Higashi-ku, Osaka Name: Furutomi Pharmaceutical Co., Ltd. (672) Mitsuhito Okuda 4, Agent Address: Inside Yoshitomi Pharmaceutical Co., Ltd., 3-35 Hirano-cho, Higashi-ku, Osaka 541 5. ?ili positive object 6, content of amendment l1lIi!- and the drawings are amended as follows: (1) "Inducible or not" on page 2, line 8 of the specification is changed to "
``Is it possible to induce it?'' (2) "Charcoal gas" on page 4, lines 6-7 of the same book is replaced with "carbon dioxide gas"
Correct. (3) Correct "homogenize" in line 1 of page 5 of the same evening to "homogenize". (4) The following statement is inserted between lines 10 and 11 on page 6 of the same book. [Figure 2 shows the elution pattern of TKF gel filtration with Sephacryl S-200. Figure 3 shows the elution pattern when T'KF was eluted with a saline solution applied with a concentration gradient in a high performance liquid chromatograph using a MonoQ column. (5) The following statement is inserted between the first and second lines of page 12 of the same book. [Figure 2 shows Sephacryl S-200 of TKF of the present invention.
The figure shows the elution bag from gel filtration. The vertical axis shows cell killing activity (%), the horizontal axis shows both numbers, a→ is bovine serum albumin (molecular weight 66200), b-・ is ovalbumin (molecular weight 4'5000), c-→ is α- The elution position of chymotrypsin (molecular weight 25 [109'>) is shown in Figure 3.
) and eluted with a saline solution with a concentration gradient. The vertical axis shows cell killing activity (%) and saline concentration (mM), and the horizontal axis shows both fraction numbers, 00-
indicates a graph of cell killing activity, and -...- indicates a graph of a saline concentration gradient. (6) Regarding the drawings, Figures 2 and 3 will be added as shown in the attached sheet. Above is Part 2: Drawing fraction flea No. 1973 Fraction flea also

Claims (1)

[Claims] The body surface of a mammal-derived macrophage was stabbed by a Sarcophaga peregrin.
a) From the homogenate or body fluid of the larva or its early axis, the molecular weight is approximately 1 by Kell filtration method.
88,000, 5O5-gel electrophoresis revealed that the molecular weight was 4 molecules of subunit with a molecular weight of about 32.000 and about 30.00. (
i:? by stimulation with a complex protein with lectin-like activity that splits into two molecules of +00 subunits. A tumoricidal substance having a molecular weight of about 30,000 to about 70,000.