JPS58203918A - Glycoprotein, its preparation and remedy for tumors - Google Patents

Abstract

NEW MATERIAL:CBX1 (carcino-breaker X1) [molecular weight: 70,000-90,000; color reactions: positive to Lowry protein reaction, ninhydrin reaction after hydrochloric acid hydrolysis; a white powder; soluble in water and aqueous sodium chloride, insoluble in benzene; saccharide content: 35-45%, isoelectric point: 4.3-6.2; adsorptive in 0.01M phosphate buffer solution at 7.2pH; stable for more than 24hr at 4 deg.C in an aqueous solution of 2.0, 7.0 and 11.0pH.] USE:Remedy for tumors. PREPARATION:Cells of human or warm-blood animals other than human such as reticuloendothelial cells, lymphoblasts, leukemic cells, or fibroblasts are suspended in an appropriate culture medium and an inducer such as phytohemagglutinin or concanavalin A2 is made to act directly on the cells to effect inductive formation of CBX1, which is collected from the suspension.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel glycoprotein obtained from an extract or a 1F culture medium drop of reticuloendothelial cells, lymphatacytes, leukemia cells, or SS blast cells of mixed-breed animals, a method for producing the same, and a method for producing the glycoprotein. The present invention relates to a tumor therapeutic agent containing as an active ingredient.

Currently, there is no complete drug treatment for tumors, and although many tumor treatments have been developed by many researchers around the world, there are still new therapeutic agents and multidrug combination therapy in clinical practice. is being attempted. Tumor therapeutic agents are broadly classified into cancer chemotherapeutic agents and cancer immunotherapeutic agents. Cancer chemotherapeutic agents are so-called cytotoxins that exert their effects by non-specifically suppressing cell proliferation, so they act not only on tumor cells but also on normal cells, causing leukopenia, infertility, and hair loss. Strict limits are placed on the amount of use of these drugs, as they exhibit extremely severe side effects such as teratogenicity and carcinogenesis.

In addition, cancer immunotherapeutics do not directly suppress the growth of tumor cells, but exert their therapeutic action by indirectly suppressing tumor growth by acting on the body's defense function, so they are compared to cancer chemotherapy drugs. Although serious side effects are extremely rare, tumor patients often do not have sufficient body defense functions remaining, and their therapeutic effects are not necessarily as sufficient as those of cancer chemotherapy drugs.

In the biological defense function! l! We have been conducting research for many years, believing that reticuloendothelial cells, which play an important role, may be producing substances that can treat tumors.

Already from reticuloendothelial cells, lymphotoxin, tumor necrosis factor,
Factors that can be expected to have a therapeutic effect on tumors, such as interferon, have been collected, and Grainger et al.
nger G, A, atal, , CeN
ular I amunolooy 13 8%
, pp. 388-402, 1978), Carswell et al.
,P roo,N atl,Acad,Sci,U,S
, A., vol. 72, pp. 3666-3670, 1975)
and Isaac et al. (l 5sacsJ 1, proc,
Ro, y, Soc, 3er, 3.

, Vol. 147, p. 268, 1957). In addition, recently, the present inventors have developed a mixed composition containing the aforementioned lymphotoxin and tumor necrosis factor from the culture medium of lymphoblasts grown in immunocompromised hamsters.
Breaking Factor, hereinafter abbreviated as CBF. ) was successfully isolated in large amounts and easily, and it was discovered that this CBF is effective against experimental tumors transplanted into animals! ! (Frail, morning edition, November 22, 1981) In the course of research on this CBF, the present inventors developed an anti-tumor glycoprotein (tumor-destroying substance x 1, ie, Carcino-Breaker X).

Hereafter, it will be abbreviated as GRX. ), but we have also found that the extracts or culture supernatants of reticuloendothelial cells, lymphoblasts, leukemia cells, or miscellaneous blast cells from exsanguinated animals contain the above-mentioned cytotoxic factors, etc. We have discovered a glycoprotein that is different from the substance of Completed the invention.

The glycoprotein of the present invention (hereinafter referred to as this substance)
i.e. Carcino-Breaker x.

and abbreviated as CBx+. ) details the physical, chemical, and biological properties of

■Molecular weight: The molecular weight was measured by gel filtration using Sephadex G-100 (Pharmacia) and 0.01M phosphate buffer (1) H7,2) as a solvent.
Its molecular weight is 70,000-90,000.

(2) Color reaction: Table 1 shows the results of testing the q color reaction of an aqueous solution of CBX+. The O-ly and ninhydrin reactions are described in Biochemical Experiments iJI, Volume 1, Protein Assay, 1971, the phenol sulfuric acid method, anthrone sulfuric acid, naphthol sulfuric acid, indole sulfuric acid, and tryptophan sulfuric acid reactions are described in Biochemical Experiments. The Holf reaction was carried out according to the method described in Lectures, Vol. 4, Carbohydrate Assay, 1971. The Holf reaction was carried out according to the method described in Biochemistry Experimental Lecture, Vol. 3, Lipid Assay, 1971.

As shown in Table 1, CBx indicates the yellow color of proteins and carbohydrates, but does not indicate the red color of lipids.

■Properties/Solubility: White powder, soluble in water, aqueous sodium chloride solution, and phosphate buffer, and almost insoluble in benzene, hexane, and chloroform.

■ Sugar content -: CBX+ sugar content" and its composition by Spiro (Spiro, H, A, , Methods in
Enzyiology, volume 13, pages 3-26,
As a result of measurement according to the method of 1966), the sugar content of this substance is 35-45%, and the sugar components are 23-28% hexose, 8-11% hexosamine, and 114-6% sial. .

■ Isoelectric point: When the isoelectric point of CBx 1 was measured in isoelectric focusing electrophoresis using Ampholine, it was 4.3 to 6.
．． It is 2.

(2) It is adsorbent in 0.01M phosphate buffer (pH 7.2) in a fractionation operation using Ulex Europeus agglutinin-bound Sephadex.

■pH2.0, I) H7.0 or pH11.

0 in an aqueous solution at 4°C for more than 24 hours, and at pH
CBx in an aqueous solution of 7.0 at 60°C for more than 3 hours.
The molecular weight and tumor cell damaging activity determined by the gel filtration method in No. 1 are stable.

■Selectively damages tumor cells without substantially damaging normal cells. The cytotoxicity of CBX 1 is 108
Cells were cultured with the addition of this substance for 48 hours at 37°C under aeration of oxygen gas containing 5% carbon dioxide, and the number of surviving cells that were not stained with trivan blue was counted and indicated by 50% growth inhibition M1 degree. Ta. One unit of this substance is a concentration that inhibits the proliferation of 105 KB cells by 50%.

CB They are clearly, clearly, and distinctly different substances. The molecular weight of lymphotoxin is α-lymphotoxin 70,
000-90,000, β-lymphotoxin 35°00
0-50,000, γ-lymphotoxin 1o, ooo~
It is known that there are 20,000 three species (
Edited by Cohen et al.
ymphokinens J, Academic
press.

(1979). Among these, β-lymphotoxin and γ-
Lymphotoxin clearly differs from CBX + in terms of its molecular weight. Furthermore, α-phospho2 todoxin has a molecule of 170.
000 to 90.000 glycoproteins reported by Granger et al.
et atl, Cel I amunol
ogy, vol. 38, pp. 388-402, 1978)
However, α-lymphotoxin prepared according to the method of Granger et al. exhibited adsorption properties different from that of CB .1. CBX is eluted in 7 courses of 0.08M, but CBX is not eluted in 7 courses of 0.08M, and CBX is eluted in 7 courses of 0.08M.
It is eluted in 7 courses of 1M. α-phosphorus 2F% toxin) LJ-Cass et al.
Al, J. Immunoloy, Vol. 109, p. 1233, 1972), it is not selective in its cytotoxicity and damages tumor cells and normal cells alike. However, CBX + is clearly tumor selective in its cytotoxicity, and α-lymphotoxin is different from CB X +.

Tumor necrosis factor selectively shows a damaging effect on tumor cells, has a molecular weight of 33,000-63,000, and a sugar content of 0% (Carswell, E.).

A,et al,,proc,NatlJcad,3
ci, U, S, A, , vol. 72, 3666-367
0, p. 0, 1975) or 40% molecule 139.0001 sugar-containing bun (Nihon Keizai Shimbun, morning edition, August 23, 1981), and both differ from CBX+ in molecular weight.

Furthermore, C [
It has a molecular weight of about 35,000 (Nihon Keizai Shimbun, morning edition, November 22, 1981), and is clearly different from csx I.

Furthermore, csxl is clearly different from interferon in that it has no antiviral effect and from CBX in terms of molecular weight. The molecular weight of CB x is 12.000-17.00
It is 0.

Next, the cells used in the present invention will be described.

Cells derived from human or non-human phlebotomy animals used in the present invention include reticuloendothelial cells, lymphoblasts, leukemia I1
m! Or I! Hanme I1m! Either primary culture or cultured cells can be used, and preferably, in order to provide csx 1 for human treatment,
Human-derived cells are safe in terms of treatment and side effects such as antigenicity caused by F. Examples of such cells include, for example, Miyoshi (Miyoshi, I.
, Nature, vol. 267, p. 843-8, p. 1
BA t-L-1 cells, King ALL-111 cells, and NALL-1 cells, reported by the Journal of Clinical Microbiology (LJ, 977).
CIin, M 1crobiol, , vol. 1, 11
6-117, 1975)
wa cells, Journal of Immunology (J.

111unolo (1, 113 volumes, 1334-1345
M-7002 cells, B-
7101 cells, 70x7000 cells (70tsusha), JBL cells, EBV-8a cells, EBV described in "Tissue Culture" (vol. 6, pp. 527-546, 1980)
-Wall cells, F f (V-I O cells, and other BA
LM2 cells, CCRF-8B cells (ATCCCCCl-1
20) etc. ml! , human-derived lymphocytes, macrophages, and cells that have been cultured by treating human-derived lymphocytes and macrophages with various viruses, drugs, radiation, etc. can be freely used.

In addition, examples of cells derived from exsanguinated animals other than humans include mouse 8ALB/C3T3 cells (70tsusha) and 11210 cells (J, Natl.
, Cancer Inst, vol. 13, p. 1328,
1953) and P388 cells (Scientific
Proceedings, pathologists
&B acteriolooists, vol. 33, 6
03, 1957), Mouse black tumor C1oneM-
3 (Flora), rat tumor LLC-WRC256 ()
In addition to hamster melanoma RPM11846 (Flora Inc.), lymphocytes and macrophages can be used; however, the cells that can be used in the present invention are not limited to those mentioned above.

Next, a method for generating CBxt according to the present invention will be described.

There are any methods for producing CBx from cells derived from human or non-human phlebotomy animals as described above, and CBx can be collected directly from the cells as they are, or by culturing and proliferating them. If specific COX 1 is desired, an inducer can be applied to those cells. For example, cells derived from human or non-human phlebotomy animals are suspended in an appropriate medium, an inducing agent is applied directly to the cells to induce the production of CBX+, and CBX+ is extracted from the suspension.
All you have to do is collect +.

CBX + inducers usually include, for example, phytohemagglutinin, concanavalin A1 bokweed mitogen, lectins such as ribopolysaccharide, polysaccharides such as phosphomannan and dextran phosphate, endotoxins, bacterial cell components, bacteria, and viruses. , nucleic acids, polynucleotides, etc., or two or more of them are used. Antigens are also CBx I inducers for sensitized cells.

The CBX 1 thus produced can be easily collected by known purification and separation methods, such as salting out, dialysis, filtration, centrifugation, concentration, and lyophilization. More advanced techniques include adsorption to ion exchangers, elution, gel filtration,
Electrophoresis, or using antibodies or Ulex Europeus agglutinin-conjugated Sephadex, etc.7
Finitei chromatography may also be used.

Next, we will describe a method for growing the cultured cells in the body of an animal.

The cultured cells derived from human or non-human phlebotomy animals used in the present invention may be reticuloendothelial cells, lymphoblasts, leukemia cells or fibrotic tJII cells;
Preferably, in order to provide CBx + for human treatment, human-derived cells are safe in terms of side effects such as antigenicity that may occur during treatment. As mentioned above, such cells include, for example, BAL, L-1 cells,
TAIL-1 cells, N A l-1-1 cells, Na1a
lWa cells, M-7002 cells, B-7101 cells, 7
07000 cells, BALB103T3 cells, L121
0 cells, P388 cells, lymphocytes, macrophages, etc. can be used freely.

In addition, warm-blooded animals to which such cells are directly transplanted or by inoculation into a diffusion chamber are those in which cultured cells derived from human or non-human phlebotomy animals can proliferate. - Animals of the same or different species, if any, may be used, for example, chickens, birds such as pigeons, dogs, cats, monkeys, rabbits. Mammals such as goats, pigs, horses, cows, guinea pigs, rats, hamsters, regular mice, and nude mice can be used.

In addition, when transplanting cultured cells derived from a different species of animal into these animals, there is a risk that an unfavorable immune reaction may occur, so in order to suppress that reaction as much as possible,
The animals used should be kept as young as possible, i.e. eggs,
Those for embryos, fetuses, or newborns, and those at a young age are more suitable.

In addition, the immune reaction can be suppressed by subjecting these animals to pretreatment such as, for example, injecting them with about 200 to 600 rem of X-rays or an immunosuppressant.

If the animal to be used is homologous to cultured cells derived from nude mice or non-human phlebotomy animals, the immune response is weak even if the cells are grown, so the pretreatment described above is required. This is particularly advantageous because cultured cells from human or non-human phlebotomized animals can be transplanted and rapidly proliferated without the need for human or non-human phlebotomy.

In addition, cells derived from humans or non-human hemorrhagic animals are first transplanted into hamsters and grown, and then these cells are further transplanted into nude mice. It is also possible to transplant the cells to stabilize cell proliferation and further increase the CBX 1M produced therefrom.

In this case, the transplant may be between the same species, the same genus, the same class, or the same phylum. The site within the animal body to which the human-derived cells are transplanted may be any site where the transplanted cells can proliferate, such as the urinary cavity, vein, abdominal cavity, or subcutaneous site.

In addition, a porous filtration layer that can block the passage of animal cells without directly transplanting cultured cells derived from humans or non-human animals into the animal body, for example,
Membrane filters having an angle of 0"7 to 10°3I - 1 Diffusion chambers of various known shapes and sizes equipped with ultrafiltration membranes or follower fibers are implanted in the animal's body, for example, in the abdominal cavity, and then removed from the animal's body. Any of the cultured human-derived cells described above can be grown within the chamber while being supplied with body fluids containing nutrients.

In addition, if necessary, a chamber in which the solution containing nutrients in this chamber is connected to body fluids in the animal body and perfused therein is attached to the surface of the animal body, for example, and the proliferation status of human-derived cells in the chamber is monitored. By making it possible to see through the chamber, and by making only a portion of the chamber removable and replaceable, it is possible to proliferate cells throughout the lifespan of the animal without having to sacrifice it, thereby further increasing the amount of cells produced per individual animal.

Methods using these diffusion chambers do not only allow for easy collection of established cell lines derived from human or non-human phlebotomized animals because they do not come into direct contact with animal cells, but also prevent undesirable immune reactions. Since there is little risk of causing cancer, there is no need for pretreatment to suppress immune reactions, and various types of animals can be used freely.

Transplanted animals should be maintained and managed as normal for the animal! This is advantageous because all you have to do is continue the process, and no special handling is required even after transplantation.

Human or non-human a! The purpose can usually be achieved within a period of 1 to 10 weeks for growing cells that have been established in automated culture. It has also been found that the number of cultured cells derived from human or non-human bloodletting animals obtained in this way reaches 101 to 1012 or more per animal.

In other words, the number of cultured cells derived from non-human fAI111 animals grown by the method for producing CBX1 used in the present invention is about 102 to 107 times the number of cells transplanted per individual animal, or about 10 times the number of cells transplanted per individual animal. This is about 10 to 10 times higher than when inoculated and grown in vitro in a nutrient medium, or more, which is extremely convenient for the production of GE3x.

Any method can be used to purify CBX1 from the cultured cells derived from human or non-human phlebotomy animals grown in this way. It can also be collected from the animal body in which it has grown. For example, from cultured cells derived from humans or non-human animals grown in suspension in ascites fluid in the peritoneal cavity, or from cells grown subcutaneously,
CBX t can be collected directly.

In addition, CBx 1 can be induced and produced by culturing cells derived from human or non-human bloodletting animals while growing them in the animal body or by taking them out of the body and treating them with an inducing agent in vitro. . For example, cultured cells derived from human or non-human bloodletting animals grown in ascites are sorted, and human or non-human I.
Excise a tumor containing cultured cells derived from an i-plane animal,
CBX is dispersed and the resulting cells are suspended in a nutrient medium kept at about 20 to 40°C to a cell concentration of about 10' to 10''/+el, and then treated with a CBX1 inducer to induce CBX. All you have to do is induce generation and collect it.

Additionally, if cultured cells from human or non-human animals are grown in a diffusion chamber, the grown cells can be left in the chamber or removed directly from the chamber, or CBX can also be collected by applying an inducing agent.

In addition, for example, CB can be first added to cultured cells derived from human or non-human animals while still in the animal body.
After Xt is induced to be produced, CBX is then applied outside the animal body to cultured cells derived from human or non-human bloodletting animals collected from specific parts or whole parts of the same animal.
CBX per individual animal to be used, such as by inductively producing CBX+, by using it again for inductively producing CBX+, or by increasing the number of cells that can be obtained by replacing the chamber that is implanted or connected to the animal body. +
It is also possible to further increase the production amount.

In addition, in order to generate CBx 1 by induction, the previously mentioned CB
An inducer of xt may be used, and the CBX1 thus produced may be obtained by the previously-mentioned known production separation method, rough production separation method, or the like.

As is clear from the experimental examples described later, CBxl is Hs°
Although it not only showed a selective growth-inhibitory effect on cells and markedly suppressed cancer metastasis, but was also effective against a wide variety of tumors, it was still lower than the doses that showed these pharmacological effects. CBxl is safe and extremely useful for tumor treatment even at sufficiently high doses. , can be used for oral administration preparations, intra-intestinal administration preparations, intravaginal administration preparations, etc.Also, CBx +
Considering its safety, the daily therapeutic amount for an adult is 0.5 to 500,000 units, although there is no particular limitation, and more preferably 0.5 to 500,000 units for topical application.
~5,000 units, 20 to 100,000 units for systemic injections such as intravenous injection and intramuscular injection, and 50 to 500,000 units for oral administration, and may be increased or decreased as appropriate depending on usage or symptoms. can.

CB X t can be formulated into pharmaceutical formulations in a conventional manner with any conventional pharmaceutical carriers, bases or excipients. Enteric-coated preparations such as capsules, tablets, and powders are available for light administration; intrarectal preparations are for rectal cattle; and injections are water-soluble injections or freeze-dried preparations that are dissolved in distilled water for injection. As for injections and external preparations, it is preferable to use them as ointments or lotions. In addition, it can also be used as eye drops, nasal drops, or inhalants.

The efficacy, toxicity, usage and dosage of CBxl thus produced are described below.

Experimental Example 1 Selective tumor cells with cytotoxic effects: Ni-8 (nasopharyngeal carcinoma) cells, Mx-1 (breast cancer) cells (distributed by Cancer Research Society, Dr. Shigeru Tsukagoshi, HEI)-
2 (pharyngeal cancer), HEL (liver cancer) cells (Dtsusha), normal small intestine (Intestine) 407 cells,
Girardi heart (Girardi f-1eart)
cells, Chang liver cells,
Yero (monkey kidney) cells, MOGK (canine kidney)
2 cells () Otsusha), 10S each, in advance.
After culturing for 4 hours, 10% of P2S5 and L1210 (white men's disease) cells (donated by Shigehiro Tsukagoe, Cancer Research Society) were immediately placed in Eagle's medium 1-1 containing 10% calf serum supplemented with the test substance. The cells were cultured for 48 hours at 37°C under aeration of oxygen gas containing 5% carbon dioxide. After completion of the culture, the number of surviving cells that were not stained with trivan blue was counted under an optical microscope, and the 50% cell-killing concentration of the test substance was calculated with the control as 100. The strength of the damaging effect of each test substance on tumor cells is
It was different between CB×1 and CBF. On the other hand,
Both α-lymphotoxin and mitomycin C showed non-selective damaging effects on normal cells and tumor cells.

Experimental Example 2 Effects on mice transplanted with Sarcoma 180 and Ehrlich cancer Cells weighing 25~
3×10 6 cells per 309 ddY male medium mouse were intraperitoneally transplanted, and the number of survival days was observed. Example 1 (described later)
CBx1 obtained in Production Example) was intravenously injected into 5 mice per group every day from the day after tumor implantation until immediately before death. The results are shown in Table 3, with the average survival days of the control group being 100.

From the results shown in Table 3 and above, CBx 1 showed a clear antitumor effect in both mice transplanted with Sarcoma 180 and Ehrlich's carcinoma.

Experimental Example 3 Effect on survival days of leukemia mice Body weight 2
1 per animal in the abdominal cavity of a BDF+ male of 0 to 25o.
0' mouse Shiromenkoi 1-1210 or 106 mouse Shiromenkoi P388 were transplanted, and the survival days were observed. CBx obtained in Example 2 (manufacturing example) described below with 5 animals per group
, was administered intraperitoneally every day from the day after transplantation until just before death.

The results are shown in Table 4, with the average survival days of the control group being 100.

From the results shown in Table 4 onwards, C3x is associated with murine Shiromen disease [121
0 and P388 showed clear antitumor effects in both tumor-bearing mice.

Experimental Example 4 Effect on survival days of lung cancer mice Body weight 20
2X into the right thigh muscle of a ~25g BDFi male
106 Lewis lung cancer ma were transplanted and the survival days were observed. Each group consisted of 6 animals, and C8x1 obtained in Example 3 (manufacturing example) described below was intravenously injected every day from the day after transplantation until just before death. The results are calculated based on the average survival days of the control group as 100.
It is shown in Table 5.

From the results shown in Table 5 and above, CBX 1 clearly showed antitumor effects in Lewis lung cancer-bearing mice.

Experimental Example 5 Effect on lung metastasis of cancer A piece of Lewis lung cancer 211 was subcutaneously transplanted into the back of BDF+ male male lineage of 1,120 to 30 o'clock in a group of 6 animals, and from 9 days later, for 12 days, the following examples will be described. CBX obtained in 1 (manufacturing example)
+ and CBF were intravenously injected once a day, and 21 days after transplantation, the amount of primary tumor III and the number of lung metastatic nodules were measured by Wexra (
Wexler.

H, ) method (J, Natl, Cancer I
n5 titute, Vol. 36, p. 641, 1966). The results are shown in Table 6.

Table 6 Results within the population are expressed as standard error above the mean.

*There is a statistically significant difference compared to the control group with a risk rate of 5% or less.

From the above results, csx I extremely well suppressed primary lung cancer and lung metastasis, whereas CBF had little effect on lung metastasis.

Experimental Example 8 Toxicity test (single administration) Groups of 10 BOF+ males weighing 20 to 25o were given intravenous injection of CBX+ obtained in Example 2 (manufacturing example) to be described later, and death occurred within 7 days. The number of strokes was observed. As a result, cs
Even after administering 110,000 mm/Kg, all 10 animals survived without any change in body weight or general symptoms.

Experimental Example 91 Sex test (Continuous administration for 30 days) Body weight 20-2
A group of 10 BDFI male males weighing 5 g were intravenously injected with csxl obtained in Example 1 (manufacturing example) described below for 30 days, and the number of death strokes, body Φ changes, and general symptoms were observed. . Body weight was measured between 9 a.m. and 10 a.m., and general symptoms were observed using Irwin's method (Science, 136).
Vol. 123, 1962), 10.20.30
I cycled that day. As a result, CBX,i,ooo
There were no deaths during 30 days after administration of units/KQ/day, and the weight gain curve showed no difference from the control group. Also,
Similar to the control group, no abnormalities were observed in general symptoms.

Examples are shown below, but the present invention is not limited thereto.

Example 1 (Production Example) Human peripheral lymphocytes (2 x 101") were suspended in 4.0001 Eagle's medium containing 10% calf IIh serum, and phytohemagglutinin (Difco) was added to a concentration of 5 ON/ml. ℃, under aeration of 11k gas containing 5% carbon dioxide, for 48 hours.

After culturing, the supernatant was added to 0.01M phosphate buffer (pH 7,
After dialysis at <2>, a 40-80% ammonium sulfate salting out fraction is obtained. After dialyzing this fraction again against the above-mentioned phosphorus@slow mm+, gel filtration was performed using Sephadex G~100 (Pharmacia) to obtain a fraction with molecules 1170.000 to 90,000 (7). It was made into a fraction.

The fractions were adsorbed on Sephadex bound to Ulex-Yo-Beus-Agglutinin (Maruzen Oil Co., Ltd.), eluted with 0.5M fucose-containing O, 01M phosphate buffer, and then dialysis was performed to remove the fucose. CBXl was eluted using the so-called gradient method, in which the concentration of phosphate buffer (pH 7, 2) was gradually lowered by absorbing Cef1dex bound to Urex Yo0Beus Agglutinin.In this way, CBx 10.1s.

get. The activity of CBXt thus obtained was 5.
000 vI, and the specific activity per 11 g of protein is 50.
It was about 000m/1 (It was.

Example 2 (manufacturing example) Human BALL-1 cells (formerly M1yos, Natllr
e.

267, pp. 843-844, 1977) 9X10”
1 was suspended in 1.800 ml of Eagle's medium containing 10% calf serum, 9 x 10' pfu of Sendai virus was added, and incubated at 37°C under aeration of oxygen gas containing 5% carbon dioxide for 48 hours.
After culturing for an hour, the supernatant was purified according to the method of Example 1.

In this way, CBX+1100P can be obtained.

The activity of CBxl thus obtained was 4°200
m position, and the specific activity was 42.000 m position/IN+.

Example 3 (Production example) 70 human cordoblasts 70,000 cells (Flora) 3x
600 ml of 10% calf I6]tII
Float in Eagle's medium, add phytohemagglutinin to a final concentration of 150 Pg/ggl, and incubate at 37°C for 50 minutes.
After culturing for 48 hours under aeration of oxygen gas containing % carbon dioxide, the supernatant was purified according to the method of Example 1. In this way C
You can get BX+10. The activity of C B X 1 thus obtained was about 250m, and the specific activity was about 25,000m.

Example 4 2 x 10" bovine peripheral blood lymphocytes were divided into 1.0001
% calf serum containing Eagle's medium, 37°C, 5%
Cultivate for 48 hours under aeration of carbon dioxide-containing oxygen gas. After the culture was completed, the culture supernatant was purified according to the method of Example 1.

In this way, UcBX 1 o, 00210 can be obtained. The activity of CBX+ thus obtained was 1
4 units, and the specific activity per ml/g of protein is 7,000
It was 1j/10.

Example 5 Human BAL L-1II cells grown in cell culture (
Miyos, Nature, vol. 267, 843-8
44, 1977) were suspended in 2.0001 Eagle's medium containing 10% calf serum, and incubated at 37°C.
Cultivate for 48 hours under aeration of oxygen gas containing 5% carbon dioxide.

After completion of the culture, the culture medium 111-natant was purified according to the method of Example 1. In this way, 10.2 ll1 g of CBX is obtained. The activity of C3x obtained in this way is 2
, 3°O units, and the specific activity per 111g of protein is 11
It was ゜5001st/n+o.

Example 6 (Water-soluble injection) CBx 1 100,000 sodium chloride 9.0 g Total amount in distilled water for injection 1.000 A+1 genuine, and. Get some salt! J '7 A e □ Measure and combine the ingredients, dissolve in distilled water for injection 5001, and make the total volume 1.000 ml with distilled water for injection.

This aqueous solution was passed aseptically through a membrane filter, and the filtrate was filled into sterilized glass containers at a rate of 2 ml and sealed tightly to obtain a water-soluble injection.

Example 7 (lyophilized injection) CBx + 100,000 units 20% human face-cleared albumin 101 Sodium chloride 9.0 g Total amount 1,000 w1 in distilled water for injection After weighing and mixing CB x + and sodium chloride, Dissolve in an aqueous solution of 5001 distilled water for injection and add a predetermined amount of human serum albumin, and then add 1.000 m of radiant heat distilled water.
Set to 1. This aqueous solution is aseptically filtered using a membrane filter, filled into 21 sterilized glass containers, and freeze-dried. This was sealed tightly to obtain a lyophilized powder preparation.

Example 8 (eye drops) CBX + 100,000 units Sodium chloride 5° Chlorobutanol 59 Total amount in distilled water for injection 1.000 slF Each component was weighed and dissolved in 9501 distilled water for injection. The total amount is 1.
00011 and aseptically passed through a membrane filter to obtain eye drops.

Example 9 (semi-annual) csx + 100.0001st position Polyethylene glycol 1500 250 a Poly]ethylene glycol 4000 approximately 750 g 1. ooo a Weigh the above ingredients, thoroughly grind the entire amount of CBX + and poly(1-ethylene glycol) 1500, and 500 g of polyethylene glycol 4000, and then add the remaining polyethylene glycol 4000 to bring the total amount to 1,000 g (
The mixture was thoroughly triturated as +, and a rectal half period of 5,000-0 was obtained by the melting method.

Example 10 (nasal drop) CBX + 100,000 units Sodium chloride 5g Chlorobutanol 5 Total amount in distilled water 1.0OOn+I Each of the above components was dissolved in distilled water of 9501 as a weighing component, and after cooling, the total amount was 1.
Dilute to 000ml and use as nasal spray.

Example 11 (enteric coated tablet) csx l 1.000,000 units lactose
64. OQ potato starch approx. 30. OQ polyvinyl alcohol 3.0g Macium stearate 3.0 1,ooo Q After weighing each of the above ingredients, mix the total amount of each of CBx j and lactose, and about half of the potato starch, and then add the remaining potato starch. Add the total amount to 94.0 (J) and mix uniformly. Add an aqueous solution of polyvinyl alcohol to this mixture and prepare granules by wet granulation method. After drying the granules and mixing magnesium stearate, Compression tableting was performed to produce tablets weighing 2001 fl g.The tablets were coated with methylcellulose phthalate to form enteric-coated tablets.

Example 12 (Ointment) CBx I 100,0001st liquid paraffin 10g Vaseline ・1000 1,000 CJ After weighing each of the above ingredients, CBX + is ground into liquid paraffin, and 500g of petrolatum is added and mixed well. . Add the remaining Vaseline to this and make the total amount 1.
000g and mixed well to prepare an ointment.

Patent applicant 11 J Pharmaceutical Co., Ltd. procedural amendment (method) % formula % 3゜Relationship with the case of the person making the amendment Patent applicant 5, supplementary + E order attached to the CJ: August 13, 1982 (Showa 57) (August 31, 2015) Please submit a clearly written detailed statement in typed print.

Claims (1)

[Claims] 1) Glycoprotein having the following properties: ■Molecular 1 near 0. ooo ~ 90.000 ■Fast color reaction: Lowry reaction shows the ν color of protein, peptide bond and amino acid coloring is shown in ninhydrin reaction after hydrochloric acid hydrolysis, phenol sulfuric acid reaction, anthon sulfuric acid reaction, indole Shows coloration of sugars by sulfuric acid reaction and tryptophan@acid reaction. ■Properties/Solubility: White powder, soluble in water, aqueous sodium chloride solution, and phosphate buffer, and almost insoluble in benzene, hexane, and chloroform. (2) Sugar content is 35-45%, and its composition is 23-28% hexose, 8-11% hexosamine, and 4-6% sialic acid. ■Isoelectric point: 4.3 to 6.2 ■Ulex eurobeus agglutinin ((〕le
x europeus agglutinin) in a fractionation operation using conjugated Cef1dex, 0.01M
Adsorbent in phosphate buffer (1)l-17,2). ■pH2.0, pi-17.0 or pHII. Stable for more than 24 hours at 4°C in an aqueous solution of
1) In an aqueous solution of l-17,0 at 60°C, 3
Stable over time. ■Selectively damages tumor cells without substantially damaging normal cells. 2) The following properties; ■Molecular weight near 0. OOO~90,000゜■ψcolor reaction;
Coloration of proteins is achieved by the Lowry reaction, coloration of peptide bonds and amino acids is shown by the ninhydrin reaction after hydrolysis with hydrochloric acid, and coloration of sugars is shown by the phenol-sulfuric acid reaction, anthrone sulfuric acid reaction, indole sulfuric acid reaction, and tryptophan sulfuric acid reaction. ■Properties/Solubility: White powder, soluble in water, thorium chloride aqueous solution and phosphorus 11111 solution, almost insoluble in benzene, hexane and chloroform, ■Sugar content is 35-45% , its composition is 23-28% hexose, 8-11% hexosamine, Sial I
I! 4-6%, ■ Isoelectric point: 4.3-6.2, ■ Urex eurobeus agglutinin (U Ie
x europeus agglutinin) in a fractionation operation using bound Sephadex, 0.01M
Adsorbent in phosphoric acid buffer H7,2>, pH2,o, I) H7,0 or pf-111°0
Stable for 24 hours or more at 4℃ in an aqueous solution of pH 7.0, and stable for 3 hours or more at 60℃ in an aqueous solution of pH 7.0. The glycoprotein is collected from cells derived from humans or non-human warm-blooded animals that have the ability to produce an anti-tumor glycoprotein that causes physical damage, either directly or by culturing and proliferating the glycoprotein. A method for producing an antitumor glycoprotein. 3>11alf[l The method for producing an antitumor glycoprotein according to claim 2, wherein the animal-derived cells are cultured. 4) I! Anti-Ill! according to claim 2, in which an inducing agent acts on cells derived from autophages. Method for producing glycoprotein. 5) Properties of F: ■Molecular (to) near 0,000 to -90,000° color reaction, reaction: O-ri reaction shows protein coloration, JJ
It shows the coloration of peptide bonds and amino acids in the ninhydrin reaction after iWI hydrolysis, and the coloration of sugars in the phenol-sulfuric acid reaction, anthrone sulfuric acid reaction, indole sulfuric acid reaction, and tryptophan sulfuric acid reaction. ■Properties/Solubility: White powder It is soluble in water, aqueous sodium chloride solution, and phosphate buffer, and almost insoluble in benzene, hexane, and triaroform. ■The sugar content is 35 to 45%, and its composition is 23 to 30% of hexose. 28%, hexosamine 8-11%, sialic acid 4-6%, ■ Isoelectric point: 4.3-6.2, ■ Urex iobeus acrutinin (U Ie
x europeus agglutinin) in a fractionation operation using bound Sephadex, 0.01M
Rinll! ! It is adsorbent in a liquid solution (pH 7.2>), is stable at 4°C for more than 24 hours in an aqueous solution of pH 2', 0, pH 7.0 or pH 11°0, and is stable in an aqueous solution of pH 7.0. , is E-stable for more than 3 hours at 60°C; 1) does not substantially damage normal cells, but selectively damages tumor cells; Cultured cells derived from a non-human animal may be transplanted directly into a phleblated non-human homologous or xenogeneic animal, or the cells may be transplanted into a cultured cell line derived from a warm-blooded animal, or may be transplanted in a cultured cell line derived from a warm-blooded animal. The animal is implanted with a diffusion chamber inoculated with
directly from cells obtained by proliferation, or
A method for producing an antitumor glycoprotein, which comprises further culturing and proliferating the glycoprotein to collect the glycoprotein. 6) The method for producing the anti-tumor I Chichiwa protein according to claim 5, which comprises acting on cultured cells derived from basal animals with an inducing agent. 7) A tumor therapeutic agent containing a glycoprotein having the following properties as an active ingredient: ■Molecular perspective; 70,000 to 90,000 ■Color reaction: Shows protein coloration by Lowry reaction;
It shows coloration of peptide bonds and amino acids in the hydrolytic ninhydrin reaction, and coloration of sugars in the phenol-sulfuric acid reaction, anthrone sulfuric acid reaction, indole-sulfuric acid reaction, and tryptophan-sulfuric acid reaction. ■Properties/Solubility: White powder, soluble in water, aqueous sodium chloride solution, and phosphate buffer, and almost insoluble in benzene, hexane, and chloroform. (2) The sugar-containing bottle is 35-45%, and its composition is 23-28% hexose, 8-11% hexosamine, and 4-6% sialic acid. ■Isoelectric point; 4.3 to 6.2 ■Ulex Yo0beus Agglutinin (LJ t
ex europeus agglutinin) in a fractionation operation using conjugated Cef7dex, 0.01
Adsorbent in M phosphate buffer (1)87.2). ■pH2.0, pH7.0 or gill 11. It is stable in an aqueous solution of O at 4°C for 24 hours or more, and stable in an aqueous solution of pH 7.0 at 60°C for 3 hours or more. ■Selectively damages tumor cells without substantially damaging normal cells.