JPS58225024A - Glycoprotein, its preparation and remedy for tumor - Google Patents

Abstract

NEW MATERIAL:Glycoprotein CBX3. Molecular weight: 7,000-9,000; color reactions: positive to Lowry's reaction showing the presence of protein, ninhydrin reaction showing the presence of peptide linkages and aminoacids after hydrochloric acid hydrolysis and phenol-sulfuric acid reaction showing the presence of saccharides; description: white powder; solubility: soluble in water, sodium chloride aqueous solution or the like, insoluble in benzene and others; saccharide content: 8-15%; stable in aqueous solution of 2, 7, and 11pH for more than 24hr, stable in aqueous solution of 7pH at 60 deg.C for more than 3hr; the aminoacid sequence on the N-terminal side is alanine-alanine-. USE:Remedy for tumors. PREPARATION:CBX3 is obtained directly from the extract of reticuloendothelia, lymphoblasts, leukemia cells or fibroblasts or after they are propagated by cultivation. Further, a large amount of CBX3 is desired, an inducer such as phytohemagglutinin is made to act on these cells directly and CBX3 is collected from its suspension.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides methods for extracting extracts or culture supernatants from reticuloendothelial cells, lymphocytes, leukemia cells, or cordoblasts of warm-blooded animals.
A novel glycoprotein with η and its properties! The present invention relates to the 1llll method and a therapeutic agent for ulcers containing this glycoprotein as an active ingredient.

Currently, there is no complete drug treatment for tumors, and although many tumor therapeutic agents have been developed by many researchers from all over the world, there are still very few new therapeutic agents in clinical practice. ) Multidrug therapy has been attempted. Tumor therapeutic agents are broadly classified into thermal chemotherapeutic agents and cancer immunotherapeutic agents. Thermochemotherapy agents are so-called cytotoxins and exert their effects by non-specifically suppressing cell proliferation. II It acts not only on tumor cells but also on normal cells, causing extremely serious side effects such as leukopenia, infertility, hair loss, malformations, and carcinogenesis, so strict limits are placed on its usage. .

In addition, cancer immunotherapy agents do not directly suppress the proliferation of liver cancer cells, but exert their therapeutic effects by indirectly suppressing tumor growth by acting on the body's defense function, so thermochemotherapy Although the occurrence of serious side effects is extremely low compared to chemotherapy drugs, in many cases tumor patients do not have sufficient biological defense functions, and their therapeutic effects (compared to hot chemotherapy drugs) Not enough.

The present inventors have continued their research for many years, believing that reticuloendothelial cells, which play an important role in the body's defense function, may produce 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 Grancito et al.
ranger, Q, A, etal. ,Cel
Carswell, F'.△. et al.
Proc, N al.

A catl. 3 ci. L), S. A.
, Volume 72, 3 6 6 6・～36
70, 1975) and Issac et al.
s, A. , Proc. Roy. Soc, S
Er.

B. , vol. 147, p. 268, 1957). Recently, the present inventors have also detected the above-mentioned lymphotoxin and tumor necrosis factor from the culture solution of lymphoid bulbs grown in immunocompromised hamsters.
The mixed composition containing tumor-destructive factor (C arc
ino- BreakinO factor. , hereinafter abbreviated as CBF. ), and it was announced that this CBF was effective against experimental tumors transplanted into animals (Yomiuri Shimbun, November 2, 1981).
(Morning edition) on the 2nd. In the course of research on CBF, the present inventors discovered that antitumor glycoproteins (till rupture substances x, xl, x2, i.e., (', arcino-Br
eeker x, x, , x2, hereinafter CBx
It is abbreviated as %CBX+ and CBx2. We have discovered a glycoprotein that has an antitumor effect (a), but we also found that phlebotomy fIJ
In extracts or culture supernatants of reticuloendothelial cells, lymphoblasts, and leukemia-like fibroblasts, we found a glycoprotein that was different from the above-mentioned substances. The present invention has been completed by discovering that the IIl tumor cell-killing action has a therapeutic action that can be characterized by one characteristic.

The glycoprotein of the present invention (hereinafter referred to as F, the present invention is a tumor-destroying substance x 3,
That is, it is called Carcino-Breaker x 3 and abbreviated as CBX 3. ) describes the physical, chemical, and biological properties of v1.

c℃ minute molecule; When the molecular weight was measured using 5OS-gel electrophoresis, the molecular weight was 7°000 to 9.0
It is 00.

(2) Komura reaction; Table 1 shows the results of testing the color reaction of an aqueous solution of C[3×3. The d'3, Lowry and ninhydrin reactions are described in Biochemistry Experiment Course, Volume 1, Protein Assay, 1971, phenol sulfuric acid method, anthrone sulfuric acid, naphthol sulfuric acid, indole sulfuric acid and 1
- Lip 1 - Fan sulfuric acid reaction is ([Method described in Kagaku Jikken Sabaza, Volume 4, Carbohydrate Standard Method, 1971.

The small reaction is 1, Higaku Experimental Course, Volume 3, Lipid Determination Method,
It was carried out according to the method described in 1971.

As shown above, csx 3 exhibits protein and carbohydrate coloration, but not lipid coloration.

■Properties/Solubility 1/I: White powder, water, chloride ゛)I
・It is soluble in aqueous solution and phosphoric acid solution, and Ben 1! It melts in water, hegisan, and chloroform.

■Sugar content: Sugar content m of cBX3
The sugar content of this substance was 8 to 15%, and the sugar content was roughly 6 to 10% hexose. %, hexo9mine 1~
・2%, sialic acid 1・3%.

φ) Carboxymethyl cellulose under 5M phosphate M buffer (pt-16.4) ()
In rΔn exchange chromatography, J5 was adsorbed to carboxymethyl cellulose ↑1.

■pLI2. O, pH 7.0 or rlLl 1 1
.

0 in an aqueous solution of J5 for 24 hours at 4°C.
Molecule m and tumor cell cytotoxic activity v1 of C B

(S11- It does not substantially damage normal cells, but selectively damages lung cancer cells. The cytotoxic layer of C B , 37℃, 5%
Under aeration of oxygen gas containing carbon dioxide gas (cultured for 48 hours, 1-
The number of surviving cells that were not stained by lipan blue was counted as 61 and expressed as a 50% growth-inhibiting concentration. 1 unit of this substance is 1
50% inhibition of KB cell proliferation! It is i# degree.

Φ) The amino acid sequence on the N-terminal side of the protein is alanine-alanine-.

The glycoprotein of the present invention, 1 or CB Lymphotoxin, tumor necrosis factor II, or CBF, which is removed from cells (1q), interferon (), C11×.CB
There is a clear distinction between X + and CBX 2 in the following points, and they are clearly different 71. Lymphoto4 herring is derived from its molecule ftt by α-lymphotogycin 70.
On(') 90,000, β-lympholinoxin 35,000-50.

000, γ-lymph 1-xin 10,000-2r),
It is known that 314i1 of 000 exists (
Edited by Cohen et al. [Biology of tide]
1. y+nphokinans J, Δcada
mic Press, 1979) is clearly different from CBX 3 based on its molecular weight.

In addition, the lymphoto-4 herring was identified by Rootsj et al. (1-u
cas, ;l, J, et al, J. r
mmunology, vol. 109, p. 1233, 1
972) reported that CB Lymphotoxin and different 4T
It's on.

Although the physicochemical properties of tumor necrosis factor are different from those reported, it selectively exerts a damaging effect on Il# tumor cells, and its molecular properties range from 33,000 to 63,000. Sugar content is 0% (CarswOll, E, △, cl
ill,, Proc, N atl, Δcad, 3
ci, IJ.

S, Δ1.72 volume, pages 3666-3670, 11.
975) and is clearly different from CBX in sugar content and molecular weight. Also, Mika I/shi! Micbael, R,R,a
nclGeOr(le, E, G,
, J, rnltnunolor+y
, vol. 125, pp. 1671-1677, 198
According to SDS gel electrophoresis method, the molecular weight is 68,000, the isoelectric heating is 5.1, and the pl-1
It has been reported that if it is less than 3, it will not adsorb to unstable soil and Con△-Sepharose. This result is also CBX 3,
In addition to differing from C in molecular weight, stability, and isoelectric point, CBx 3 is Con, A.

-They are different in this respect as they adsorb to Sepharose. Other reports (Matthews, N. & Wat
kins, J., F., Dr., J., ca.
According to Ncer Vol. 138, pp. 302-315, 1978), the molecular weight of tumor necrosis factor is 30,000-5.
0.000, and in further study (Mat+:he
ws, N, , Ota
t, Brit, ,), c
ancer, vol. 42, pp. 416-422, 1980
), its molecular weight was determined to be 67,000 by gel electrophoresis.
0, the isoelectric point is 5.1 to 5.2, and it is a protein that contains almost no sugar. This result is also C13x3,
The molecular weight, isoelectric point and sugar content m are also clearly different. There is also a report (E1 Honkeizai Shimbun, August 23, 1981, Fl, morning edition) that the molecular weight is 39°000 and 6 skewers 40%, but this molecular weight if; J: C' in sugar content. It's 11 x 3.

Furthermore, CBF has a molecular weight of about 35,000 (in 9-) TITIIN (Shizuoka Shimbun, November 221-1.9, 1981), and about 35,000 (F1 Hon Kousai Shimbun, 1981-1).
January 22F1, IJI I'll ) °c, which is clearly different from the glycoprotein of the present invention.

Furthermore, the glycoprotein of the present invention differs from interferon in that it does not have an antiviral effect, and is different from C13x, G Rx1. C.B.
It is clearly different from X2 in terms of molecular weight. CBx
, C13x I and Cl5X 2 have molecular weights of 12,000 and 17. (100,70,000-1・
90,000.40,000-5ri0.00(1-('
be.

The axle stock I1 of the CRX 3 of the present invention has a composition of 8...15%, as described in the first claim, and has a composition of 6...10% hexane, lX4-sol. 1 J min 1 to 2%, Sial M 1 to 3%, and the sugar content is 9-
114%, and the composition of A is 6 to 9% for l\1 sauce.
, hexosamint ~・2%, sialic acid 2・・3%, abutment 4''jl is 9・・・・1/I%−(・Yes, the composition of A is hexose 7・10%, hexosamine 1 ~2%
, those containing 1-2% of dinofluoric acid, etc. Also, C
The amino acid sequence from the N-terminal side of B
From the N-terminal side, the j′ amino acid sequence 1j, the one from the N'lanine to the alanine to the one from the N-terminal side to the one from the N-terminal side to the one from the N-terminal side to the 6th t3 of alanine-alanine-mebA-nin-threonine-alanine- and is included. To the isoelectric white of CRX 3-
) are 6.3 to 7, 8 and E3. 〇-9,2
There are b, a, and so on.

Next, regarding the cells used in the present invention).

The cells derived from warm-blooded animals other than human ζ used in the present invention (52) may be S-cells, lymphoid cells, leukemia cells or fibroblasts. Either primary Ji'4 cells or cultured cells can be used, preferably CI'1 for the treatment of humans.
When X3 is supplied, it is safe to use I31, human to human cells (') in terms of side effects such as antigenicity and other side effects that occur during treatment. 1
Miyoshi (M 1yoshi, I,, N8ture
267, pp. 8'13-4344, 1977).
L L -I II cell, NA3-1---1 cell, dii
--Naru of Clinical Microbiology AOG (J
, CIin, M 1crobiol 1.1 volume,
116-117, 1975)
alWa cells, Journal of Immunology (J,
I n+munolooy, 1 13 'l! -1
1334-1345, 1974)
7002 cells, B-7101 cells, Flora 7000 cells, rllJi culture'' (Volume 6, 527-
JBL cells, EF3V-8a Ill cells, EBV-Wa cells, E8V-1+011[1 cells, and other B A 1. ,
M2111 cells, CCRF-8B cells (ΔTCCGC
I-120> and other established cell lines, human-derived lymphocytes, macrophages, and human I-derived lymphocytes and macrophages treated with various viruses, drugs, radiation, etc. and cultured to form @ cells. Free to use.

In addition, examples of cells derived from wet animals outside of H1-1 include mouse BALB/C3T3 cells (]n size), mouse leukemia cells l-1210m1121 (J,
Natl, Cancer lnst, vol. 13,
1328, 1953) and P388 cells (3cien
tNic proceedings, Patho
logists & l”3acteriology
ts vol. 133, p. 603, 1957), mouse black tumor C1one M-3 (floran 1), rat incision L
IC-WRC256 (゛) Japan]), hamster melanoma RPM11846 (Flora), lymphocytes, macrophages, etc. can be used, but the cells that can be used in the present invention are limited to the above-6. It's not a thing.

Next, a method for producing CBX 3 of the present invention will be described.

There is no limit to the method of producing CBX:・'3 from cells derived from animals other than the above-mentioned Hi1~ or Hi1~, and CBX:・'3 can be produced directly from the cells or grown in culture. -'CG 1B ×s can be collected, but if a larger amount of CBX 3 is desired, an inducing agent can be applied to those cells. For example,
~ Or cells derived from non-human wet animals are suspended in an appropriate medium and an inducing agent is directly applied to the cells to induce CB
CBx3 was induced to be produced and CBx3 was collected from the suspension.
That's fine.

Inducers for CBX s typically include, for example,
Hemagglutinin, Konno J Navarin △, Pork Weed Mi1--gen, lectins such as riboboriritukalide, polysaccharides such as bosphomanpun, dextran phosphate, ■N1:1~Xin, bacterial cell components, ■1 bacteria, One or more of viruses, nucleic acids, and vorinucleotides are used. Furthermore, antigens are also CBx 3 H conductors for sensitized cells.

C f3
ii separation methods, such as salting out, dialysis, filtration, centrifugal force ml
It can be easily collected by performing , concentration, freeze-drying, etc. At a more advanced level, adsorption to a -7 ion exchanger, elution, gel filtration, electrophoresis, or affinity chroma 1 using an antibody or Sephadex conjugated to Urexus/Eurobeus/Agglutinin may be used. .

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

The cultured cells derived from animals other than Hi-I or I-- used in A (A) may be reticuloendothelial cells, lymphoblasts, leukemia cells, or fibroblasts; Preferably, to provide CI3X3 for the treatment of humans, cells derived from humans are safe in terms of side effects such as antigenicity that occur during treatment. As mentioned in, for example, B△L L
-1 cells, TAI+-1 cells, N A l-1-1 cells, Namalwa cells, M-7002 cells, 13-71
011Il cells, ) 1] 7000 cells, BΔl-R/
C3'F3 section 11. L, 1210 cells, l] 388 m
Cells, lymphocytes, Mac1-1 phages, etc. can be used freely.

In addition, such cells can be directly transplanted, or 1. l: 4,
The warm-blooded animal to be implanted by inoculating the cells into a diffusion chiton bar is the same type or one that is derived from a warm-blooded animal other than these 1- or HiI-, as long as the cultured cells can proliferate. Animals of different species may be used, such as birds such as ebay, chickens, birds, dogs, lil, eels, goats, pigs, horses, cows, guinea pigs, rats, etc.
Mammals such as hamsters, snail mice, and nude mice can be used.

<r J';, The transplantation of cultured cells derived from a different species of animal into these animals has the potential to cause an unfavorable immune reaction (・, I will not be able to explain the reaction of I). (J
To suppress the use of caps, animals can be kept in their juvenile state,
That is, eggs, liver, fetuses, or those in the newborn stage or infant stage are suitable.

In addition, these animals may be injected with, for example, 200-600 rem of 1 tux, 1 ml, and an immunosuppressant.
Pretreatment 4-1a, such as pretreatment, can also suppress immune reactions.

If the animal to be used is homologous to nude mice or cultured cells derived from warm-blooded animals other than humans, the immune response will be weak even if the cells are grown, so the pretreatment described above may not be necessary. Hi1～ or Hi1～ without J
Cultured cells from other warm-blooded animals can be transplanted,
It is particularly advantageous because it can rapidly grow and rust.

In addition, cells derived from warm-blooded animals other than humans or humans,
For example, first, after transplanting it to a hamster and increasing β,
These cells can be transplanted into warm-blooded animals other than humans (such as by transplanting them into nude mice).
CRX stabilized or generated from it
You are also free to increase 3114.

In this case, it is possible to transfer not only between the same booth or the same family, but also between the same class or class. Transplanting cells derived from human 1
h The site within the animal body may be any site where the transplanted cells can proliferate, such as the allantoic cavity, vein, II! You can choose freely, such as 2 cavities or subcutaneous.

In addition, it is possible to directly transplant cultivated cells derived from humans or warm-blooded animals other than humans into the animal body, and a porous pavement membrane that prevents the passage of animal cells, For example, a membrane filter with a pore diameter of 10-7-. For example, the human-derived cells cultured as described above are grown in a chamber that is implanted in the peritoneal cavity and receives a supply of body fluids containing nutrients from the animal body. You can use it.

Also, if necessary, the solution containing nutrients in this chamber can be connected to the body fluids inside the animal and perfused ()
For example, place a chamber on the surface of an animal's body. It is possible to see through the proliferation state of human-derived cells within the chamber.
It is also possible to make only part of the hair and hair part removable and replaceable, allowing goblet cells to proliferate without slaughtering the animal, thereby further increasing cell production per individual animal. can.

Methods using these diffusion chiton bars do not allow the established cell lines derived from humans or non-human equine animals to come into direct contact with animal cells, so not only can these cells be easily harvested, but they can also cause undesirable immune reactions. Since there are few concerns, there is no need for pretreatment to suppress immune reactions, and various warm-blooded animals can be used freely.

The maintenance and management of the transplanted animals is as follows: 1.
! This is convenient because all you have to do is follow +, and no special steps are required even after transplantation.

The period for growing cultured cells of humans or warm-blooded animals other than humans can usually achieve the objective within a period of 1 to 10 weeks. Hi1 obtained in this way
It was also found that the number of cells cultivated in one cultured cell line reached 10 to 1012 cells per animal, or more than I-, derived from warm-blooded animals other than .

(If the method for producing CRX3 used by Akira Kokumitsu is not limited to the production method of CRX3, it should be noted that
8 The number of cultivated cells is about 102 to 107 times the number of cells transplanted per individual animal, or more, and about 10 to 10 times more than the number of cells transplanted per animal, and about 10 to 10 times more than the number of cells transplanted per animal.
106 times or even more, which is extremely favorable for the production of CB X 3.

CB
You are free to decide how to avoid refining it.

It can also be collected from the animal's body where the disease has grown. For example, CB can be used directly from cultured cells derived from humans or warm-blooded animals other than humans, grown in floating form in ascitic fluid in the peritoneal cavity, or from cells grown subcutaneously.
All you have to do is collect X3.

In addition, J8 cultured cells derived from warm-blooded animals other than human 1~ or 1~ can be grown in the animal's body, or taken out of the body, and incubated with CB while avoiding the action of an inducing agent in vitro.
You can also avoid induced generation of X3. For example, cultured cells derived from a warm-blooded animal other than humans or humans grown in ascites are separated, and cells grown subcutaneously are cultured from a warm-blooded animal other than humans or humans. The IIIt tumor containing established cell lines is excised and dispersed, and the obtained cells are divided into approximately 20-
In a nutrient medium maintained at 40°C, the cell concentration was approximately 105-1
0'/ml (2), and add CB
CB x 3 can be induced and produced by applying the inducing agent No. 3, and then collected.

In general, when cultured cells from humans or other warm-blooded animals are grown in a diffusion chamber,
The proliferated cells can be left in the chamber, or taken out from the chamber and directly left, or treated with an inducing agent! 'You can collect CB x 3.

In addition, for example, cultured cells derived from humans or warm-blooded animals other than humans may be first cultured while in the animal body.
Once BX3 was induced to be produced, the same animal side...
A method for inducing and producing CRx 3 outside an animal body in cultured cells derived from a warm-blooded animal other than a human or a human, collected from a specific part or the whole body,
In addition, the amount of CBX3 produced per individual animal used can be further increased by covering the cells used for induced production of CBX3, or by increasing the number of cells obtained by replacing the chamber buried or connected to the animal body. You are also free to increase it.

In order to produce CB Advanced production separation methods may also be used.

As is clear from the experimental examples described below, CF3
Even though I had a five-year-old fissure, I received these pharmacological effects and took a dose that was sufficiently high compared to the dose.
It is safe and effective for example, human leukemia, gastrointestinal cancer, esophageal cancer, pharyngeal cancer, liver cancer, skin cancer, brain tumor, sarcoma,
It is extremely useful in the treatment of tumors such as lung cancer, adenocarcinoma, breast cancer, and uterine cancer. CBX 3 can be administered using the commonly used administration methods: gFJ preparations, eye drops, nasal drops, inhalants,
It can be used for external preparations, oral administration preparations, rectal administration preparations, intravaginal administration preparations, and the like. In addition, the daily therapeutic dose of CBX3 for adults is 0.5 to 500,000 units, although it is not particularly limited, considering its safety, and more preferably 0.5 to 500,000 units for topical application. 5-5.000
unit, intravenous t:I: 20-1r for systemic injection such as intramuscular injection, 0.000 unit for oral administration (50-500,000,500,000 unit, or increase or decrease as appropriate depending on symptoms) be able to.

CB X 3 can be formulated into pharmaceutical formulations in a conventional manner with any conventional pharmaceutical carriers, bases or excipients. Bonds [1 Administration: soluble formulations such as capsules, tablets, powders, etc. Direct administration: rectal suppositories; injections: water-soluble injections or distilled water for injection. Freeze-dried injections can be used by dissolving them in water, and for external use, they are preferably used as ointments or lotions.In addition, they can also be used as topical preparations, nasal drops, or inhalants. .

Hereinafter, experimental examples and examples of the glycoprotein of the present invention, namely CBX 3, will be described.

Experimental example 11111! ! KB (nasopharyngeal disease) cells, Mx-1 (breast cancer) cells, which are selective tumor cells with damaging effects (from Cancer Research Society, Dr. Shigeru Tsukagoshi! "4.), l-l L:
+]-2 (pharyngeal cancer), l-IEL- (liver cancer) cells (flora), normal cells in the small intestine (intestin
e) /I ()7 cells, Girardi heart (Girar
di l-1eart) cells, Chitong liver (Chan
GL! Ver) cell, Verl (V ero,
Lil kidney) cells, Mr) CK (canine kidney) cells (70tsusha)
After incubating for 10 s for 24 hours, P
Immediately, 105 cells of 388 and 11210 (donated by Shigeshina Tsukakoshi, Leukemia Cells (Cancer 1i1) Study Group) were added with the test substance, and then mixed with Eagle IP8-1 containing IC 10% calf serum.
... 1 for 48 hours at 37° C. under aeration of oxygen gas containing 5% carbon dioxide gas. After completion of the culture, the number of surviving cells that were not stained with trypan flu was counted under an optical microscope, and the 50% cell killing S degree of the test substance was subtracted from the control value as 100. The test substance is Example 2 (manufacturing example)
C B

Table 2 (1st experimental group) 1) Example 2 (WA example)
Table (Second Experiment) N11) C13X obtained in Example 5 (6 examples)
As is clear from the results of -, CB X 3 selectively damaged tumor cells without substantially damaging normal cells. On the other hand, My1~NishinC,
2T showed a detrimental effect on normal cells and tumor cells.

Experimental Example 2 Monkey]-7180 and T-Rich cancer Zhao transplanted mice had no effect on monkey loaf 180 cells, Ehrlich cancer cells weighed 25-
3 ('') 0 dd', 3X per mouse
10' cells were implanted intraperitoneally, and the number of survival days was observed.
The CBX3 obtained in 1 was administered to 5 mice per group (1 vein) until just before death. The results are shown in Tables 1 and 5, with the average survival days of the control group divided by 100.

Table 14 (First experiment [Y) Note 1) CBX obtained in Example 2 (manufacturing example) Table 5 (Second experimental group) Result of I-I J, S, CRXxl Marill = + -7180
Mice transplanted with both #3 and T-ritsu and cancer showed a clear anti-tumor effect.

Experimental Example 3 Influencer Φ2 on survival days of leukemia mice
0.~.21) g of RD F + string mouse intraperitoneally 105 murine leukemias 11210 or 101 per mouse
Mouse leukemia P388 cells were transferred to a tube, and the number of days of survival was observed. (51 Example 5 (Manufacturing example)
3 was intraperitoneally administered on the next day. The results are shown in Tables 6 and 7, with the average of the control group/4 (days being 100).

? 11) Based on the results of Cl5X3 obtained in Example 2 (Production Example), CB Indicated.

Experimental Example 4 Effect on survival days of lung cancer mice 2×10 Lewis lung cancer cells were transplanted into the right thigh muscle of a BDFt-based navy blue thread with a body weight of 20-25 (l), and 11 survived. was observed.One group was made up of 6 animals, and Example 21M3, which will be described later.
i! C I'3 obtained in Example i) or Example 5 (Production Example)
From the day after the transplant, 1-1 intravenous injections of x3 were administered until 11 days before the north face. The results are shown in Tables 8 and 9, with the average survival time of control U being 100.

11) Example 2 (manufactured filter example) obtained from IkoC13x3 Note 1
) In Example 5 (manufacturing example), CBX3 clearly showed an antitumor effect against lung cancer, based on the results of 1q C13X3 or more.

Experimental Example 5 J- Effect on Breast Cancer Mice Body weight 20-25 (l) BAL, B/c nude mice were subcutaneously treated with breast cancer (MX-1>2n+).
After 14 days of transplantation of the m-square piece, 1η of Example 2 ('!! fabrication example) or CRX 3 obtained in Example 5 (manufacturing example) was intravenously administered once a day for 1411 days. After sweating, the volume of the primary ulcer was measured 15 days after the start of administration. Results first
Not shown in Table 0 and Table 11.

41) Average Shinshi standard error Note 2) CBX3* obtained in Example 2 (manufacturing example) There is a statistically significant difference compared to the control group with a risk rate of 5% or less.

E1; At a risk rate of 1% or higher, compared to the control group, h
There is a difference in breath.

2.1:1) Average Shinshi standard error Note 2) Example 5 (Production example) C Obtained CBX 3;1;
There is a statistically significant difference compared to the control group with a risk rate of 5% or less.

Nido: F, lff17 compared to the control group with a risk rate of 1% or less
+ There is a scientifically significant difference.

Experimental Example 6 No effect on methyl 71 run i/ren induced tumors. 0.5 ml of 3-methylcholan 1-ren (olive oil solution) was subcutaneously applied to the lateral part of dd'/strain mice weighing 20-25 g.
About 60 days after subcutaneous injection of CB Then, the tumor volume was measured on the 21st day after the start of Ij injection. Results first
Shown in Table 2 d'3 and Table 13 Table 12 (1st experimental group) Note 1) Average 1 direct mark 4 shell difference Note 2> CBX obtained in Example 2 (IIJ model) 3 *There is a statistically significant difference compared to the control group with a risk rate of 5% or higher. '1;1; Risk rate 1%
There are statistically significant differences compared to the control group in the following cases.

Table 13 (Second Experimental Group) Note 1) Average 1110 Standard Error 12) Actual 1 Color Example 5 (Production Example> -cqy Cr5
x 3; 1z There is a theoretically significant difference compared to the control BY under a 5% risk condition.

:l::l: Compared to the control group at a risk rate of 1%,
There is a statistically significant difference.

From the results in 1- above, CBx 3 clearly showed an anti-lllli tumor effect against autologous tumors.

Experimental Example 7 Survival of Melanoma Mice] Effect on Number 10' mouse black Bφ1316 were subcutaneously implanted on the back of BnF+ MI mice weighing 20-25 g, and the number of survival days was observed. As 1 liver 74, Cox 3 obtained in Example 2 (manufacturing example) J:take described later was administered 1:1 intravenously (IQl) from the day after transplantation until immediately before death.The results were compared. !!', 4 In' average raw #It @100
As shown in Tables 14 and 15.

Table 14 (First experiment BY) Table 15 (Second experiment l!y>Note 1) Example 5
(Manufacturing example) CBX 3 or more result J stiff CB
3 clearly showed antitumor activity against melanoma.

Experimental Example 8 Toxicity test (single administration) A group of 10 f3 D F + strain mice weighing 20 to 25Q were used to test the mice obtained in Example 2 (%J missed case) or Example 5 (manufacturing example), which will be described later. 1. : CBx3 was intravenously injected and the number of deaths was observed for 7 days.

As a result, which CBX 3100,0001st/K
(Even if 1 was administered, there was no change in body weight or general symptoms.
All animals survived.

Experimental Example 9 Toxicity test (Continuous administration for 308 days) Body weight 20.
~2513 F31) F + strain Zhao mice were set at 10 mice per group, and Example 2 (manufacturing example) and (Yu Example 5 (
The OB Body weight was measured between 9:00 a.m. and 10:00 a.m., and general symptoms were observed using the Irwin method (S c
136, p. 123, 1962), on the 30th day 1 at 10.20°. As a result, any CBXi is 10,000171st/Kg,/11
There was no death within 30 days after administration, and the weight gain curve showed no difference from the control group. Also, as with the case study, no abnormalities were observed in general symptoms.

Example 1 (Production Example) 1 human to 2 peripheral blood lymphocytes x 1Q +o 4.0e
of Eagle's medium containing 10% calf serum, and
・Hemagglutinin (Difco) 50 pg2'mΩ
Incubate at 37°C for 48 hours in an oxygen gas venter containing 5% carbon dioxide. After 18 days of incubation, the supernatant was dialyzed against 0.01M phosphoric acid buffer (7.2).
A 40-80% ammonium sulfate salting out fraction is obtained. After dialyzing both of these fractions again against the phosphate buffer mentioned above, Rephadex G-100 (
Gel filtration was performed using Pharmacia, and the molecule
f17゜000-～. 9,000 fractions with IF7,
Three volumes of 71JCf3X, which were made up of three volumes of crude CB, were adsorbed onto a fi-1-hemagglutinin (round-necked 6 oil)-bound Sephas, and 0°5M N-
After elution with a 0.01M phosphate buffer containing acedel-θ-I tritrimine (pti 7.2), N-j was eluted by dialysis.
't? After removing blue [)-trichloride-IJ-mine, it was adsorbed to carboxymethyl cellulose under 0.05M phosphate buffer (pH 6,11), and the 1*0.5M chloride 11-11 containing .05M phosphorus M solution (pL1
The adsorbed fraction was eluted at >6.4 mL and 1 q of 1 mu of CBx3 was eluted.

This J: I got jqed (CI"3 X 3's life ↑
4 was 5°000 units.

Example 2 (Manufacturing Example) Antiserum prepared from rabbits by a known method was injected into a newborn baby's heel star to weaken the immune response of the heel star, and then human inoculation was administered subcutaneously. B△1. ．． l-1 cells (M
*VoslN, Nature t' 267 i, 8
43, p. 844, 197 (1), and reared for 3 weeks in the usual manner after A. A subcutaneous tumor measuring approximately 15° was removed, cut into small pieces, and dispersed in physiological saline to loosen it. After washing the obtained cells with serum-free Eagle's medium, 1 x 10" L?tl of the cells was washed with 150
．． 9x106 pfu of Sendai virus was added, and incubated at 37°C.
, after culturing for 48 hours under oxygen gas aeration containing 5% carbon dioxide,
The culture supernatant was diluted with N4 phosphoric acid solution (pl-1)
7. After dialysis in 2), remove the 7IO~.80% ammonium sulfate salting out fraction. This fraction was dialyzed against the phosphate buffer described in Sections 11 and 6i+, and then subjected to gel filtration using Hifatex G100 (Pharmacia).
000 fraction was made into 1q, and phase CBx was made into 3 fractions.
Three volumes of crude C1IX were adsorbed onto ConA (sigmane 1)-conjugated seno-lose, and 0.5M α・meroD
? Because of the 0.01M phosphorus M rice grain containing linnoside (rlN7.
2) After elution of F, dialysis was performed to remove α-methyl-1] mannoside, and to obtain carboxymethy/lz t=le EE −
0.05M in > R11M liquid (+1116
, 0 ) 'l suckt'i 8 u, 0 , 05 M
! Elute with J acid solution (pl-17,8) to 0.
2 mg of CB×3 was added to 1 q. The activity of 0Bx3 obtained by this method was 12,000 units, and its isoelectric point was 6.3.-.7.8.

Example 3 (manufacturing example) and 1 to 11 fibers [cells] 7000 cells (70?1
) 3x10'° pieces as 1. Phytohemagglutinin was suspended in 10% Oe of Eagle's medium at 5°C.
(Add to 1 Pg,,-' mg-(
After culturing for 48 hours at 37° C. under 5% carbon dioxide-containing oxygen gas, the tozuke was purified according to the method of Example 1. This J: CF3X30゜1 m (I can be obtained in this way.
21 was 3,100 units.

Example 4 (Production Example) 5 x 10 I0 bovine peripheral blood lymphocytes were suspended in 10 volumes of Eagle's medium containing 10% calf serum, and cultured for 48 hours at 37°C under aeration of 5% carbon dioxide. Ru. After the completion of the culture, the culture supernatant was purified according to the method of Example 1.
. In this way, CBX 30.1 In(l) can be removed.
The activity of 1.70 Example 5 (manufacturing example) Cell Jfr, human BAL L-1 enlarged with warts
1 cell (old Miyos, Nature, vol. 267, p. 843-844, 1977) 5 x 10'' cells
The cells were suspended in Eagle's medium containing 10% calf water and cultured at 37° C. for 48 hours under aeration of oxygen gas containing 55% carbon dioxide. After completion of the culture, the culture supernatant is dialyzed against 0.01M phosphate buffer (PI-17,2) to obtain a 710-80% ammonium sulfate salt fraction. Both fractions were dialyzed again against the aforementioned phosphate M solution, and then subjected to gel filtration using Hifadex G, 100 (Pharmacia) to obtain a molecular weight of 7,000.
-9.000 fractions were obtained. These two components were adsorbed onto senoalose bound to Lai 1 to hemagludinin (Saintets?) oil, and 0.5M N-acetyl-D-galactosamine was added.
,0. :l:, IM phosphate M buffer (11117,2)
After elution with
1 flame excluding limine, adsorbed under carboxymethyl cellulose, then 0.05M 1~lith buffer containing 0.5M chloride-J-Lium (11118.('))
2R gives μTE0, 1 mg of C/B
I got 3. The activity of C B
O = 9.2.

Example 6 (water-soluble injection) G [3X 3 20 (1.00 (l)
Moderate sodium chloride 18 (total amount 2.00 O in 1 fish water) n+j Example 2 (
After weighing and mixing CBX3 obtained in Production Example) and trichloride, add 1OOOn1ρ of hot distilled water for if kJ.
Dissolve in water and suspend with distilled water for injection for 2,000 m. l
Spelled out as l. This aqueous solution was filtered aseptically using a Menn 1 run filter, and the filtrate was placed in a sterilized glass container for 2 hours.
,Qm. L? Example 7 (lyophilized injection) 0 13 x 3,200,000 units 20% human serum albumin 20 mB1B]~Rium
Distilled water for i8g round trip m2,000
After weighing and mixing CBX 3 obtained in Example 5 (manufacturing example) and mono-lium chloride, an aqueous solution was prepared by adding a predetermined amount of human albumin to 1,000 mΩ of distilled water for injection. Dissolve in 2,O
Let it be OOmg. This aqueous solution is aseptically filtered using a membrane filter, filled into a sterilized glass container with 2 mU each, and lyophilized. This is tightly stoppered.
Freeze-dried powder formulation.

Example 8 (eye drops) Example 2 (manufacturing example) 1!1 Cl-3 X 3 200,0
00 medium puttrium chloride 10 g
9th [1 Buknor 10 (17, 1'
aJ for a Cji /l(ni'(total M2
, 000 mL Weigh and hang each component, 1.9
00...I dissolved in distilled water for injection. The total amount is 2,000
The volume was reduced to 0 ml and aseptically filtered using a Menn 7 run filter to obtain eye drops.

Example 9 (planing) CBX 3 10,00
0 medium poly]-Blengri]-ru 150 (125g polyethylene glycine]-ru = 1000 approx. 75j100
0 Hiki Weigh the following ingredients, and add CnXg obtained in Example 1 (manufacturing example) and Boliethylene Green 1-L 1500.
total amount of, and 50 of polyethylene glycol 4000
(Small I well and add remaining poly T ethylene green)
- Add 4,000 ml of water to bring the total amount back to 100 oz.<t
The rectal plane was prepared using the Melt 811 method to obtain a rectal plane of 5°00 On1g.

Example 10 (nasal drop) Example 5 (manufacturing example) Te'l! f CB X 3
200,0(to 11(
QIM Putrium 10 Riff 1:1
0 Athanol 109 Total amount with distilled water
2,000, ntll Weigh each of the above ingredients,
1,900 m, dissolved in G distilled water 1] After cooling, the total amount was 2,000 mj! Dilute to make a nasal spray.

Example 11 (enteric coated tablet) Example 5 (manufacturing example) r 11t, = c mouth x 3 210,000
Unit lactose 12.8 (1
Bofu 1-iyI powder approx. 6. OQ polyvinyl alcohol 600 mg S1) 7 Magnesium phosphate l\ 600 Ill (120, Og -1- After weighing each component, l* -/' CCB X in Example 5 (manufacturing example) 3 J3J: Mix the whole amount of lactose and about half of the starch from J5 Yohibote 1, and then add the remaining potato starch to make the total amount 18.80% and mix evenly 1. Add polyvinyl alcohol to this mixture. Add a permanent solution of and prepare granules using a wet granulation method.The granules are dried, mixed with stearin and magnesium, and then compressed into tablets with a suspended size of 200m0.
-ru. Enteric-coated tablets were prepared by 1-chewing methyl cellulose phthalate to this.

Example 12 (Ointment) CB
Approximately 9'J g00g After weighing each of the components listed in L, pour the CBX 3 obtained in Example 1 (manufacturing example))I Tritify with paraffin and add 50g
``Add Nohirin and mix well. Add the remaining Vaseline to this, add 1,000 ounces of Kinishi, and mix well to make an ointment.''

Patent applicant: Mochida Pharmaceutical Co., Ltd.

Claims (1)

[Claims] 1) Glycoprotein having the following properties: ■ Intermolecular: 7.000-=9.000 ■ Color reaction: The protein exhibits coloration due to the Lowry reaction, and hydrochloric acid hydrolysis Pi
In the ninhydrin reaction after I, the peptide bond d5 and 9 amino acids were shown, and the coloration of sugars was shown by the phenol-sulfuric acid reaction, the anthrone sulfuric acid reaction, the indole-sulfuric acid reaction, and the 1-lip 1-fan IlliIwi reaction. Solubility: White powder, soluble in water, aqueous sodium chloride solution, and phosphorus Mts buffer, and almost insoluble in benzene, hexadiene, and chloroform. ■Sugar content is 8-15%, and the composition is 6-10% of sauce, 1-2% of hexosamine, and cialis! 1~
It is 3%. (2) Ion exchange under 0.05M phosphate buffer (pt-1'6.4) using /J sulfoxymethyl cellulose [] Adsorbent to carboxymethyl cellulose in [] adsorption. ■l1ii 2,0,pl-17,0 or I)l
-111. Stable for 24 hours at 4°C in an aqueous solution of
Furthermore, it is stable in an aqueous solution of pH 7.0 at 60° C. for more than 3 hours. ■1 [Selectively damages tumor cells without substantially damaging normal cells. ■The amino acid sequence on the N-terminal side of the protein is alanine. 2) The following t (1 quality: ■Molecular weight; 7,000 to 9,000, ■♀Color reaction: Shows protein color due to Lowry reaction 1)
, J5 shows coloration of peptide bond J5 and amino acid in ninhydrin reaction after hydrochloric acid hydrolysis, fuconol sulfuric acid reaction, anthrone sulfuric acid reaction, indole sulfuric acid reaction, ]・
] Lip 1-fan sulfate anti-J shows coloration of saccharides,■
Properties/Solubility: White powder, soluble in water, aqueous solution of sodium to lithium chloride, and phosphate buffer, and not very soluble in benzene, benzene, and dichloroform. The content is 8 to 15%, and its composition is 6 to 10% hexose, 1 to 2% hexo1nomine, and 3% to sialic acid 1. ■Carboxymethyl 1? /0,05M using rerose
Phosphate buffer (pI-12,0, I]H7, which is adsorbent to carboxymethyl cellulose in ion-exchange chromatography under pf-16,4)
0 or 1) Hll. It is l-stable in an aqueous solution of pH 7.0 at 4°C for more than 24 hours, and stable in an aqueous solution of pH 7.0 at 60°C for more than 3 hours, ■Does not substantially damage normal cells; (2) The amino acid sequence on the N-terminal side of the protein that selectively damages tumor cells is alanine-alanine-1. An anti-tumor glycoprotein, characterized in that the glycoprotein is collected from cells derived from humans or non-human warm-blooded animals that have the ability to produce anti-tumor glycoproteins, either directly or by culturing and proliferating the glycoprotein. production lj method. 3> The method for producing an antitumor glycoprotein according to claim 2, wherein the cells derived from a wet animal are cultured. 4> Anti-tumor 11 glycoprotein glue according to claim 2, which acts on cells derived from warm-blooded animals with an inducing agent! I-building method. 5) The following properties; ■Molecular weight near, 000-9.0001 ■? Ou reaction: Shows the coloration of proteins by the Lowry reaction, shows the coloration of peptide bonds and amino acids in the ninhydrin reaction after n-acid hydrolysis, phenol sulfuric acid reaction, anthrone sulfuric acid reaction, indole sulfuric acid reaction, (→) 1/1 form/dissolution ↑11; White powder, soluble in water, 1- to 2-lium chloride aqueous solution, and phosphorus WjJM box solution; It is almost insoluble in hexane and chloroform, and has a sugar content of 8.15%, and a composition of hexose 6-10%, hexosamine 1-2%, and sialic acid 1-15%. 3%, (o, 05M phosphate buffer using Φ carboxymethylcellulose) 1-16.11) d'3 to l'
12.0.11 adsorbent to carboxymethyl cellulose in ion exchange chromatography
In an aqueous solution of 1-17.0 or 111°0, it is stable at 4°C for more than 24 hours, and 1) l-17
, is stable for more than 3 hours at 60°C in an aqueous solution of 0, (does not substantially damage normal cells, selectively damages tumor cells, +iit m on the N-terminal side of white matter. Amino acid sequence /)\, alanine-alanine-1, is a non-human cell line 18b derived from human 1 ~ or a wet animal other than hi 1 ~ that has the ability to produce an antitumor glycoprotein having the following: directly into the body of a warm-blooded animal of the same or different species, or an expanded cell inoculated with these cells so as to receive a supply of body fluids from that warm-blooded animal.
1. A method for producing an anti-tumor glycoprotein, which comprises the steps of implanting Inver into an animal and collecting the glycoprotein directly from the cells obtained with the aim of propagation, either directly or without culturing. 6) The method for producing anti-tumor 11 glycoprotein according to claim 5, which comprises causing an inducer to act on cultured J8 cells derived from warm-blooded animals. 7) A therapeutic agent for liver cancer containing a glycoprotein having the following properties as an active ingredient: ■Molecular 1: 7,000 to 9,000 ■Color reaction; shows coloration of the protein by a lip reaction;
In the ninhydrin reaction after hydrolysis with hydrochloric acid, the peptide bond 113 and the amino acid showed coloration, and phenol 1
The coloring of sugars is shown by the ijlt 1 reaction, anthrone sulfuric acid reaction, indole sulfuric acid reaction, and 1 to lipophon 7 sulfuric acid reaction. (1) Solubility: White powder, soluble in water, aqueous sodium chloride solution and phosphate buffer, and almost insoluble in benzene, hexane and chloroform. ■The sugar content is 8-15%, and its composition is 6-10% hexose, 1-2% hexo(nomine, 1-2% sialic acid)
・3%. ■Carboxymethylcellulose-less 5M phosphoric acid M solution (pl-1 6."I)-to-
It is adsorbent to carboxymethylyl[1-s] in ion-exchange media. ■11+-12.0, flH7.0bL, < is I) l-
111. stable for more than 24 hours at 4°C in an aqueous solution of
In addition, l) in an aqueous solution of l-17.0 at 60°C
Stable over time. ■Selectively damages tumor cells without substantially damaging normal cells. (2) The amino acid sequence on the N-terminal side of the protein is alanine-alanine-.