NO832811L - PROCEDURE FOR THE PREPARATION OF ANTI-TUMOR GYCLOPROTEIN - Google Patents

Abstract

Reduced molecular weight anti-tumor glycoproteins obtained by degrading at least one starting anti-tumor glycoprotein. This in turn originates from an extract or a culture supernatant of reticuloendothelial cells, lymphoblasts, leukemia cells or fibroblasts from warm-blooded animals. Reduced molecular weight anti-tumor glycoproteins are prepared by reacting the starting anti-tumor glycoprotein with a reducing agent and then recovering one or more reduced molecular weight anti-tumor glycoproteins.

Description

The present invention relates to a method for producing an anti-tumor glycoprotein with a smaller molecular weight than that of the starting material, which comprises reducing the molecular weight of an anti-tumor glycoprotein with a larger molecular weight by treatment with a reducing agent, and then isolating anti-tumor glycoproteins with a reduced molecular weight from the reaction mixt.

Despite the fact that many therapeutic agents against tumors

has so far been presented by a number of researchers in the world, the cure rate of tumors with such therapeutic agents against tumors is still very low, and the development of further improved therapeutic agents is strongly sought.

One purpose of the present invention is to provide a method for the production of anti-tumor glycoproteins.

Another object of the invention is to provide a method for producing anti-tumor glycoproteins with smaller molecular weights from an anti-tumor glycoprotein with a larger molecular weight.

A further object of the invention is to provide a method for producing CB in high yield.

Another aim of the invention is to provide a method for producing CB^ in a high yield.

A further aim of the invention is to provide a method for producing CB in high yield.

The present invention provides a method for the production of one or more anti-tumor glycoproteins with a lower molecular weight than that of the starting material, which consists in reacting an anti-tumor glycoprotein with a larger molecular weight

weight with a reducing agent and then obtained the anti-It tumor glycoproteins with a reduced molecular weight from rejac-

tion mixture. '

With the method according to the invention, an anti-tumour glycoprotein CB with a desired molecular weight can be produced in high yield.

It has been found that reticuloendothelial cells, which play an important role in the biophylaotic mechanism, could possibly provide a substance that can cure tumors after long continuous and intense studies on this subject. It was now found that four types of anti-tumor glycoproteins with different molecular weights from an extract or culture supernatant of reticuloendothelial cells, lymphoblasts, leukemia cells or fibroblasts from warm-blooded animals, which was named Carcino-Breaker X (molecular weight 12,000 - 17,000), X1 (molecular weight 70,000 - 90,000), X2 (molecular weight

-40,000 - 50,000) and X3 (molecular weight 7,000 - 9,000) (See Japanese Patent Applications Nos. 28992/1982, 28993/1982, 87674/1982, 87675/1982, 10846/1982 and the concurrent

US Patent Application No. 467,840 of February 18, 1983. These glycoproteins are hereinafter referred to as CBX, CB^, CBX209, respectively CBx3', and in cases where they are referred to collectively, CB).

Since CBX - CBX2 is present in mixture in an extract or a culture supernatant of reticuloendothelial cells, lymphoblasts, leukemic cells or fibroblasts from warm-blooded animals, it has been difficult to effectively obtain exclusively a "carcino-breaker" with a desired molecular weight, "especially those with less molecular weights, eg CBX or CBX2 in larger amounts.

The present invention is aimed at an efficient process for the production and purification of CBX, CB^ and/or CB^ and as a result arrived at that by degrading treatment of an anti-tumor glycoprotein (i.e. a "carcino-breaker") obtained from an extract or a culture supernatant of

reticuloendothelial cells, lymphoblasts, leukemia cells and fibroblasts from warm-blooded animals with a reducing

agent, one obtains from this small reaction mixture an anti- ^ tumor glycoprotein with a molecular weight of 40,000 - 50,000, i.e. CBX2/ an anti-tumor glycoprotein with a molecular weight of 12,000 - 17,000, i.e. CBX and/or an anti-tumor glycoprotein with a molecular weight of 7,000 - 9,000, i.e. CB^

easily and in a high yield, which constitutes the present invention.

The terms CBX, CBxl, CBx2 and CBx3 used in this application are defined as "carcino-breaker" anti-tumor glycoproteins which are referred to in the previous section and with the following properties:

CBX:

(1) Molecular weight: 12,000 - 17,000; (2) Color reactions: shows a color indicating proteins in the Lowry reaction, shows a color reaction typical of peptide bonds and amino acids in ninhydrin reaction after hydrolysis with hydrochloric acid, and shows a s color reaction typical of sugars in the phenol - sulfuric acid reaction, anthrone - the sulfuric acid reaction, the indole - sulfuric acid reaction and the tryptophan - sulfuric acid reaction; (3) Appearance and solubility: white powder soluble in water, aqueous sodium chloride and phosphate buffer and slightly soluble in benzene, hexane and chloroform; (4) Sugar content: 27 - 33%, sugar composition 17 - 10% hexoses, 5 - 7% hexosamines and 5 - 6% sialic acids; (5) Isoelectric point: 4.3 - 7.3; (6) Adsorbability: adsorbable on Ulex europeus agglutinin-corrected Sefadex in 0.01 M phosphate buffer (pH 7.2); (7) Stability: stable in an aqueous solution of pH 2.0, pH \ 7.0 or pH 11.0 at 4°C for 24 hours or longer^gg i an aqueous solution of pH 7.0 at 60°C for 3 hours or longer;

( 8) Cytotoxicity: selectively destroys tumor cells without i

significant degree to destroy normal cells; and

(9) Differentiation: induces differentiation of tumor cells, i.e. transfers tumor cells to normal cells.

<CB>X1<:>

(1) Molecular weight: 70,000 - 90,000; (2) Color reactions: shows a color typical of proteins in the Lowry reaction, shows a color typical of peptide bonds and amino acids in ninhydrin reaction after hydrolysis with hydrochloric acid, and shows a color typical of sugars in phenol - sulfuric acid reactions, anthrone - sulfuric acid reactions, indole - sulfuric acid reaction and tryptophan - the sulfuric acid reaction; (3) Appearance and solubility: white powder soluble in water, aqueous sodium chloride and phosphate buffer and slightly soluble in benzene, hexane and chloroform; (4) Sugar content: 35 - 45%, sugar composition 23 - 28% hexoses, 8 - 11% hexosamines and 4 - 6% sialic acids; (5) Isoelectric point: 4.3-6.2; (6) Adsorbability: Adsorbable on Ulex europeus agglutinin-conjugated Sefadex in 0.01 M phosphate buffer (pH 7.2); (7) Stability: stable in an aqueous solution of pH 2.0, pH 7.0 or pH 11.0 at 4°C for 24 hours or longer and in an aqueous solution of pH 7.0 at 60°C for 3 hours or

longer; and

1(8) Cytotoxicity: selectively destroys tumor cells

to significantly destroy normal cells. '

CBX2 :

(1) Molecular weight: 40,000 - 50,000; (2) Color reactions: shows a color reaction typical of proteins in the Lowry reaction, shows a color reaction typical of peptide bonds and amino acids in the ninhydrin reaction after hydrolysis with hydrochloric acid, and shows a color typical of sugar in the phenol - sulfuric acid reaction, anthrone - sulfuric acid reaction, indole - sulfuric acid reaction and tryptophan - the sulfuric acid reaction; (3) Appearance and solubility: white powder soluble in water, aqueous sodium chloride and phosphate buffer and slightly soluble in benzene, hexane and chloroform; (4) Sugar content: 30 - 37%, sugar composition 20 - 23% hexoses, 6 - 8% hexosamines and 4 - 6% sialic acids; (5) Isoelectric point: 4.2 - 7.3; (6) Adsorbability: adsorbable on Ulex europeus agglutinin-conjugated Sefadex in 0.01 M phosphate buffer (pH 7.2); (7) Stability: stable in an aqueous solution of pH 2.0, pH 7.0 or pH 11.0 at 4°C for 24 hours or longer and in an aqueous solution of pH 7.0 at 60°C for 3 hours or longer; and (8) Cytotoxicity: selectively destroys tumor cells without significantly destroying normal cells.

<CB>X3<:>

(1) Molecular weight: 7,000 - 9,000;

!

(2) Color reactions: shows a color typical of proteins in the Lowry reaction, shows a color typical of peptide bonds and amino acids in the ninhydrin reaction after hydrolysis with hydrochloric acid, and shows a color typical of sugars in the phenol - sulfuric acid reaction, anthrone - sulfuric acid reaction, indole - the sulfuric acid reaction and the tryptophan - sulfuric acid reaction; (3) Appearance and solubility: white powder soluble in water, aqueous sodium chloride and phosphate buffer and slightly soluble in benzene, hexane and chloroform;

(4) Sugar content: 8 - 15%, of which the composition is

6 - 10% hexoses, 1 - 2% hexosamines and 1 - 3% sialic acids; (5) Adsorbability: adsorbable on carboxymethyl cellulose in ion exchange chromatography with carboxymethyl cellulose in 0.05 M phosphate buffer (pH 6.4); (6) Stability: stable in an aqueous solution of pH 2.0, pH 7.0.or pH 11.0 at 4°C for 24 hours or longer and in an aqueous solution of pH 7.0 at 60°C in 3 hours or longer; (7) Cytotoxicity: selectively destroys tumor cells without significantly destroying normal cells; and (8) The amino acid sequence of the N terminal portion of the protein is alanine-alanine-.

The present invention is generally carried out as follows:

An anti-tumor glycoprotein CB with a larger molecular weight than the desired molecular weight, which is separated and purified from a cell extract or a culture supernatant, is dissolved at a suitable concentration of e.g. 10-10,000 ug/ml and is broken down by reaction with a suitable amount of reducing agent. The reaction mixture is dialysed, the dialysate is salted out and the formed

'precipitates are filtered out. These problems are then resolved

ing in a small amount of physiological saline or a buffered solution and after dialysis the solution is gel filtered

with Sefadex G-50, which gives an anti-tumor glycoprotein of the desired molecular weight.

The term "degradation" here means a treatment which converts a glycoprotein with a larger molecular weight into a glycoprotein with a smaller molecular weight by chemically breaking the intramolecular bonds such as disulfide bonds.

The starting material in the present invention is preferably CB^^ or CB^', although CBX can also be used when CB^ is a desired product. Although it is preferred as starting material to use a solution of purified CBX^ and/or CB^ in buffer, physiological saline, distilled water, etc., it is also possible to use a cell extract or a culture supernatant containing CB per se.

CB can be prepared from an extract or a culture supernatant of reticuloendothelial cells, lymphoblasts, leukemia cells or fibroblasts from warm-blooded animals. Details of this process for producing anti-tumor activity of CB are described in US patent application 467,840,

of 18 February 1983.

Examples of reducing agents that can be used in the present invention include thiol compounds such as 2-mercaptoethanol, dithiothreitol, dithioerythritol, thioglycolic acid, monothiophosphoric acid, etc., metal hydrogen complexes such as sodium borohydride, tertiary phosphines such as tri-n-butylphosphine, tris-diethylaminoethylphosphine, etc., sulfites such as sodium sulfite, cyanic acid salts such as cyanogen bromide , metal ions such as silver ion, etc. These compounds are such as are generally used for the cleavage and removal of proteinaceous high molecular weight contaminants,

for breaking down higher dimensional structures in a protein and analyzing the protein's basic structure, etc.

Proteins treated with these compounds often lose their biological activity, but it is surprising that ne<j>d-

.the refractive treatment is possible without loss of the anti-tumor activity of the present invention. The conditions for carrying out this invention, such as the concentration of the reducing agent, the treatment temperature, the treatment type, the treatment pH, etc., may be suitably is chosen depending on the purpose, e.g. about CBOr CBV_. shall X X j be obtained •■ alone or both shall be obtained at the same time, etc. The reducing agent can be used in a concentration of '10 -9 -5-9 to 10 M, preferably e.g. 10 to 10 M 2-mercaptoethanol, 10~2 - IO-5 M dithiothreitol, 10~<2>- 10~<6>M dithio-1—6 —2 —5 erythritol, 10~ - 10 M monothiophosphoric acid, 10 - 10 M — 1 —6 sodium borohydride or 10 - 10 M thioglycolic acid. The treatment temperature is not particularly critical as long as the glycoproteins are not denatured, and 0-37°C, preferably temperatures around 15-25°C can be used. The treatment time can be "chosen correctly so that you get a CB with the desired molecular weight, which is generally in the range of 10 minutes to 2 hours. The pH is preferably chosen in the slightly acidic to neutral range, i.e. between approx. pH 5.5 and 7.5. When carrying out the degradation treatment, a surface-active agent, e.g. an ionic surfactant such as sodium didecyl sulfate (SDS), a nonionic surfactant such as Triton X, etc. be to- present in the reaction mixture to increase the yield. The optimum concentration of the surfactant varies depending on the starting material in the degradation treatment, preferably in the range from 0.001 - 10%. The ionic surfactants include anionic, cationic and amphoteric ones. Examples of suitable anionic surfactants are SDS, salts of cholic acid, etc. Examples of suitable cationic surfactants are dodecyltrimethylammonium bromide, etc., and examples of suitable amphoteric surfactants are deoxylysolecithin, etc. Examples of suitable non-ionic surfactants are Triton, Tween type, Span type, etc. The surfactants in agents can be used individually or in mixtures. The present invention is described in more detail by the following non-limiting examples.

Example 1

a) Production of starting materials.

Higher molecular weight CB (i.e. CBX1, CBx2 and CBX)

used as starting materials in examples 1-7 were prepared.

Human lymphocytes (2 x 10 cells) were resuspended in 4,000 ml of Eagle's medium containing 10% calf serum and, after addition of phytohemagglutinin (Difco Co.) at a final concentration of 50 µg/ml, cultured at 37°C for 48 h~in.en 5 % C02, 9 5 % air atmosphere. The supernatant of the culture medium was then dialysed against 0.01 M phosphate buffer (pH 7.2), and a fraction that was salted out with 40 - 80% ammonium sulfate was obtained from the dialysate. This fraction was dialyzed again against the same phosphate buffer and then gel filtered on Sefadex G-100 (Pharmacia Co.).

Crude CBjq, CBX2°9CBxernoldtes 1 fractions with molecular weights of 70,000 - 90,000, 40,000 - 50,000 and 12,000 - 17,000 respectively.

Crude CBX was adsorbed onto Ulex europeus agglutinin (Maruzen Oil Co.)-conjugated Sefadex, and eluted with 0.01 M phosphate buffer containing 0.5 M fucose. After removal of fucose by dialysis, CBjq was adsorbed again on Ulex europeus agglutinin-conjugated Sefadex, followed by elution by the gradient method using phosphate buffer (pH 7.2), during which purified CBX^ was eluted. Purified CBjq with specific activity 50,000 units per ml was obtained in a total amount of 5,000 units in one operation.

Crude CBx2 was purified in the same way and purified CBx2 with . 'specific activity of 20.8 00 units/ml was obtained in a...

total quantity of 5,200 units in one operation.

Raw CBV was also purified in the same way and purified CB with a specific activity of 10,000 units/ml was obtained in a total amount of 10,000 units in one operation. Purified CBV with a specific activity of 7,500 units/mg was also obtained in a total amount of 150 units from the same number of human lymphocytes and by following the same procedure except that cytohemagglutinin was not present in the culture medium.

The activity is expressed through the specificity based on the concentration that inhibits 50% of the proliferation of 10 5 cells of KB cells taken as a unit.

b) Decomposition treatment.

200,000 units of CBxl (molecular weight 70,000 - 90,000) were

dissolved in physiological saline and reacted for one hour by

-7

room temperature in the presence of 5 x 10 M 2-mercaptoethanol. After the reaction, the reaction mixture was dialyzed and salted out with 80% ammonium sulfate, and the precipitated fraction was filtered off. This fraction was dissolved in physiological saline and gel filtered with Sefadex G-50. Fractions corresponding to the molecular weight 40,000 - 50,000;

12,000 - 17,000 and 7,000 - 9,000 were collected and called fraction A, fraction B and fraction C respectively. The above-mentioned procedures were also repeated in the presence of 0.05% SDS whereby the same fractions were obtained.

The physiological properties of these fractions A, B and C were investigated and they turned out to be CB, CBX and CBx3 respectively - The results were summarized in table 1.

1) According to the method of Seikagaku Jikken Koza, Vol. 1 (Tan-pakushitsu Teiryohou), 1971-2) According to the method of Seikagaku Jikken Koza, Vol. 4 (To-shitsu Teiryohou), 1971 3) According to the method of Seikagaku Jikken Koza, Vol. 3 (Shi-shitsu Teiryohou), 1971 4) Spiro, H.A., Methods in Enzymology, Vol. 8, pp. 3 - 26, 1966 5) Isoelectrofocusing using "Amfolin" (available from LKB Produkter AB, Sweden) 6) 10 5 cells were cultured in 1 ml of Eagle's medium containing 10% calf serum and each fraction at 37°C for 48 hours in a 5%, 95% air atmosphere, after which the number of live

cells that did not stain with Tryptophan blue were counted and the concentration of the fraction at which 50% of the cells are killed was taken as a measure of cytotoxicity.

The activity yield for each fraction in Example 1 is shown in Table 2.

Example 2 200,000 units of CBv0 were dissolved in physiological saline and -7 reacted for 1 hour at room temperature in the presence of 5 x 10 M 2-mercaptoethanol. After the reaction, this was dialysed and salted out with ammonium sulphate, which gave a fraction which was gel filtered with Sefadex G-50. Fractions with molecular weight 12,000 - 17,000 (fraction B) and 7,000 - 9,000 (fraction C) were collected. The above procedures were repeated also in the presence of 0.05% SDS. The fractions were identified as CB^ and CB^ in the manner described in Example 1. The yields of CB^ and CB^-j obtained as a result are shown in the table. 3.

Example 3

-7

200,000 units of CBV were treated with 5 x 10 M 2-mercaptoethanol and a fraction with a molecular weight of 7,000 - 9,000 (fraction C) was obtained in a similar manner as in Example 1. The fraction was identified as CBX^as in Example 1. Obtained yields of CBx3 are shown in table 4.

Example 4

A mixture of 100,000 units of CBX1 and 100,000 units of CBx2 was treated with 5 x 10 7M 2-mercaptoethanol in a similar manner as in example 1. The yield of CBx2/CBX and CBX3 obtained is shown in table 5.

Example 5

A mixture of 100,000 units of CBV, and 100,000 units of CB„

_ 7 Al X was treated with 5 x 10 M 2-mercaptoethanol in a similar manner as in example 1. Yields of CBvo, CBV and CBvr> are shown X^ X Xj

in table 6.

Example 6

A mixture of 100,000 units of CBX and 100,000 units of CBx2 was treated with 5 x 10 7 M 2-mercaptoethanol in a similar manner as in example 1. The yield of CBX and CBX3 obtained is shown in table 7.

Example 7

A mixture of 100,000 units CBX1, 100,000 units CBx2 and 100,000 units CB was treated with 5 x 10~<7>M 2-

mercaptoethanol in a similar way as in example 1. Obtained yields of CBx2, CBX and CBx3 are shown in table 8.

Example 8

A mixture containing 100,000 units of CBV, 100,000 units of CBx2, 9100,000 units of CBX was treated with each reducing agent listed in Table 9 by a similar method to Example 1. The yields of CBX2, CBX and CBX3 obtained are shown in Table 9.

Example 9

1 x 10"<L1>cells human BALL-1 cells (Miyoshi,' Nature,

Vol. 267, pp. 843-844, 1977) which had proliferated subcutaneously

in nude mice were resuspended in 20 L Eagle's medium containing 10% calf serum and, after addition of 1 x 10 8pfu Sendai virus, cultured at 37°C in a 5% CC>2, 95% air atmosphere in

48 hours. The resulting culture supernatant was used as starting material. This culture supernatant contained— ~ a mixture of CBX1, CBx2,' CBX and CBX3. The degradation treatment was performed on this culture supernatant by the addition of 10 - 6 M 2-mercaptoethanol and 0.1% SDS at room temperature for one hour. The reaction mixture was dialyzed and salted out with ammonium sulfate which gave a fraction which was gel filtered with Sefadex C-50 and gave CBx2, CBX and CBx3 - The results are shown in table 10.

Example 10

The procedure from Example 9 was repeated, except that

-7

5 x 10 M 2 -mercaptoethanol was used as reducing agent and each compound listed in Table 11 at a given concentration was used as surfactant. The results are shown in table 11.

Example 11

5 x 10 9 cells of bovine peripheral lymphocytes were resuspended in 1000 ml of Eagle's medium containing 10% calf serum and cultured at 37°C in a 5% CO 2 , 95% air atmosphere for 48 hours. The resulting culture supernatant was used as starting material. This culture supernatant contained a mixture of CB^, CBX2, CB^ and CB^* Degradation treatment was performed on this culture supernatant by addition of

-3

10 M dithiothreitol at room temperature for 2 hours, followed by

by similar procedures as in Example 9, giving CBx2'CB^ and CBX3- The results are shown in Table 12.

Example 12

; 3 x 10"^ cells of human fibroblast Flow 7,000 cells

T (Flow Co.) was resuspended in 6,000 ml of Eagle's medium containing 10% calf serum and, after addition of phytohema-I gglutinin giving a final concentration of 50 µg/ml, cultured at 37°C in a 5% CO 2 , 95% air atmosphere for 48 hours. The culture supernatant obtained therefrom was used as a starting material. This culture supernatant contained a mixture of CBxl, CBx2 in CBX and CBX3. Digestion treatment was performed on this culture supernatant in the presence of 0.1 M sodium borohydride at room temperature for 2 hours, followed by similar methods as in Example 9, yielding CBX2 CB^- and CBx3 . The results are shown in table 13.

Example 13

2 x 10^ cells of human peripheral lymphocytes were resuspended in 4,000 ml of Eagle's medium containing 10% calf serum and, after addition of phytohemagglutinin to a final concentration of 50 µg/ml, cultured at 37°C in a 5% CO 2 , 95% air atmosphere in 48 hours. The culture supernatant thus obtained was used as a starting material. This culture supernatant contained a mixture of CBX^, CBX2, CBX and CB. Degradation treatment was performed on this culture supernatant by addition of 5 x 10 II dithioerythritol at room temperature for 1 hour, followed by similar methods as in Example 9, yielding CBx2, CBX, and CBx3" The results are shown in Table 14. Example 141x 10" ^ human Ball-1 cells propagated by cell culture were cultured in 2,000 ml of Eagle's medium containing 10% calf serum and cultured at 37°C in a 5% CO 2 , 95% air atmosphere for 48 hours. The culture supernatant thus obtained was used as a starting material. This culture supernatant contained a mixture of CB^, CBx2, CB^ and CB^. Digestion treatment was performed on this culture supernatant in the presence of 5 x 10 2 M 2 -mercaptoethanol at room temperature for either 2 hours or 5 minutes, and followed by similar methods as in Example 9, yielding CBx 2 , CBX and CB 2 . The results are shown in table 15.

When the degradation treatment was carried out for 5 minutes, CBx2 and CBX could be obtained in high yields, while 2 hours of degradation

ning treatment gave CBV and CB _ in high yields.

X X ,3

Example 15 Purification of CBx2, CBX and CBX^

Crude CBx2, CBX and CBx3 obtained in the preceding examples can be further purified if desired in the usual manner used for the purification of biological substances.

CBx2 and CBX were e.g. purified by the method described in Example 1 a) above for the preparation of starting materials using Urex europeus agglutinin-conjugated

Isefadex. Purified CBV~ and CBV preparations with s^esi-- A —I were obtained

fig activities of 2,400 units/mg and 2,500 units/mg.

CBx3 was purified e.g. by adsorption onto concanavalin A-conjugated Cephalose, followed by elution with 0.01 M

phosphate buffer (pH 7-2) containing 0.5 M α-methyl-D-mannoside. After removal of α-methyl-D-mannoside by dialysis, the solution was applied to carboxymethylcellulose equilibrated with 0.05 M phosphate buffer (pH 6.0), followed by elution with 0.05 M phosphate buffer (pH 7.8). This resulted in a purified CBX3 preparation with a specific activity of 2,100 units/mg.

Efficacy, toxicity, method of use and dosage of CB from the invention

Experiment 1 Selectivity of the cytotoxic effect

Samples of 10^ cells from each of several tumor cell lines - included KB cells (nasopharyngeal cancer), MX-1 cells (breast cancer, provided by Dr. Shigery Tsukagoshi, Cancer Institute), HEp-2 cells (laryngeal cancer) and HEL cells (hepatoma, Flow Co.) and from normal cell lines including visceral 407 cells, Girardi heart cells, Chang liver cells, Vero cells (monkey kidney) and MDCK cells (dog kidney)

(Flow Co.), all of which were precultured for 24 h, and 10<5> cells of each of P388 and L1210 cells (leukemia, provided by Dr. Shigeru Tsukagoshi, Cancer Institute), which were used immediately - were all cultured for 1 ml of Eagle's medium containing 10% calf serum and each test substance at 37°C for 48 hours in a 5% CO 2 , 95% air atmosphere. Then the number of living cells that were not stained by Trypro-fan blue was counted under a light microscope and the concentration of the test substance at which 50% of the cells were killed was calculated against a control set as 100. As test substances, CB^ r CB^ and CBx3 prepared in Example 15, a-, 3- and y-lymphotoxins prepared by a known method (Granger G.A. et al., Cellular Immunology, Vol. 38, pp. 388-402 (1978)) and Mitomycin C. One unit of the lymphotoxin is expressed by a common index which is based on cytotoxicity--

teten on mouse L cells (Eds., Bloom, B.R. & Grade, P.R. "In Vitro Methods in Cell-mediated Immunity", Academic Press, 1979). The results are shown in table 16.

As can be seen from the above results, CB destroys

selectively tumor cells without significantly causing any damage to normal cells. In contrast, both a-, 0- and y-lympho-toxin and Mitomycin C did not show selective cytotoxicity towards normal cells and tumor cells.

Experiment 2 Influence on mice transplanted with Sarcoma

180 or Ehrlich Tumor

Male mice (ddy strain) weighing 25-30 g were intraperitoneally transplanted with 3 x 10 cells per animal Sarcoma 180 or Ehrlich acites tumor and the survival time in days was observed. CBX, CB^°g (-Bx3 prepared in Example 13 was given intravenously to groups of 5 mice daily from 1 day after transplantation until death. The results expressed in percentages of average survival days of the experimental groups in relation to the control group are shown in Table 17.

Experiment 3 Influence on the survival day of mice with lung carcinoma

Male mice of the BDF strain weighing 20-25 g were transplanted with 2 x 10 6 cells of Lewis lung carcinoma intramuscularly in the right thigh, and survival days were observed. CB , CBv0

X2\Z

and CBx3 prepared in Example 15 were given intravenously to groups of 6 mice daily from one day after transplantation

until death. The results expressed in percentages of survival days for the experimental groups in relation to a control group are listed in table 18.

Experiment 4 Influence on lung metastasis in cancer

Groups of male mice of the BDF^ strain weighing 20-30 g, 6 animals in each group, were transplanted subcutaneously in the back with a 2 mm square segment of Lewis lung cancer. CB^, CB^ and CB^^ prepared in Example 15 were given intravenously a

once a day for 12 days from the 9th day after the transplant.

[ On the 21st day after transplantation, the mass of the primary tumor was isolated and weighed, and the number of metastases in

IN

the lung of the animal was calculated according to H. Wexler's method (J. Nati. Cancer Institute, Vol. 36, 641 1966). The results are shown in table 19.

Notes) The results in the table are expressed as (mean) + (standard deviation)

Statistically different from the control group with a significance level of p ^ 5%.

X ^-Statistically different from the control group with a significance level of p ^ 1%.

As the results above clearly show, CBX, CB^ and CBx3 successfully suppressed primary lung cancer and its lung metastasis.

Experiment 5 Toxicity test (single administration)

Three groups of male mice of the BDF^ strain weighing 20-25 g, 10 animals in each group, received intravenous administration of 10,000 units/kg CBX, 10,000 units/kg CBx2, respectively

or 100,000 units/kg CB.., and the animals were observed in the 7th

today is. All 10 animals in the respective groups survived without showing any change in body weight and general condition

despite the fact that so many units of glycoproteins were given.

Experiment 6 Toxicity test (30 days continuous

administration)

Male mice of the BDF-^ strain weighing 20-25 g, 10 animals in each group, were intravenously administered CBX, CBx2 or CB^ for 30 days, and the number of dead animals, change in body weight and general conditions were observed. The body weight was measured between "9 and 10 o'clock in the morning, and observation of the general condition was carried out on the 10th, 20th and 30th days according to Arvien's method (Science, Vol. 36, p. 123 (1962)). As a result, there were no dead animals in these 30 days when 1,000 units/kg/day of CBX or CBx2, or 10,000 units/kg/day of CBx3 were given, and the weight gain curve was about the same as that of the control group. Furthermore, the general condition was found to be normal as in the control group.

As can be seen in the experiments described above, CBX, CBx2 and CBX3 selectively suppress the growth of tumor cells, and furthermore not only noticeably suppress the cancer metastases, but are also extremely effective against various tumors and are nevertheless very safe to use even at higher doses than the dose that causes pharmaceutical effect. Therefore, CBX, CBx2 and CBx3 are very useful for the therapy of various tumors such as stomach cancer, lung cancer, hepatoma, colon cancer, breast cancer, uterine cancer, leukaemia, etc. Furthermore, relatively low molecular weight forms of CB (CBX, CBx2, CBX^) can be derived from relatively higher molecular weight forms of CB (CBX, CB^ CBx2) by treatment with a reducing agent without impairing their ability to act on tumors.

Claims (13)

1. Method for the production of an anti-tumor glycoprotein with a reduced molecular weight, characterized by carrying out degradation treatment on at least one starting anti-tumor glycoprotein obtained from an extract or a culture supernatant of reticuloendothelial cells, lymphoblasts, leukemia cells or fibroblasts of warm-blooded animals by reacting the starting anti-tumor glycoprotein with a reducing agent, and then recovering at least one anti-tumor glycoprotein of reduced molecular weight. 2. Method according to claim 1, characterized in that glycoprotein CB^ is used as starting anti-tumor glycoprotein, and as anti-tumor glycoprotein with reduced molecular weight at least one of anti-tumor glycoproteins is obtained the proteins CBx2, CBX and CB^. 3. Method according to claim 2, characterized in that the glycoprotein CB^ is obtained from a culture supernatant of reticuloendothelial cells, lymphoblasts, leukemia cells or fibroblasts from warm-blooded animals by salting out the supernatant and fractionating a precipitate from this by gel filtration, which gives a. fraction with a molecular weight of 70,000 - 90,000 and then adsorb and elute this fraction on Ulex europeus agglutinin-conjugated Sefadex. 4. Method according to claim 1, characterized in that one uses as starting anti-tumor glycoprotein CBx2 'ocJ s° m anti-tumor glycoprotein with reduced molecular weight isolates at least one of CBX and CBX3" 5. Method according to claim 4, characterized in that the starting anti-tumor glycoprotein CBX2 is obtained from a culture supernatant of reticuloendothelial cells, lymphoblasts, leukemia cells or fibroblasts of warm •bloody animals by salting out the supernatant and fractionating <1> a precipitate from this by gel filtration, which gives a fraction with a molecular weight of 40,000 - 50,000 and then adsorbing this fraction and eluting it on Ulex europeus agglutinin-conjugated Sefadex. 6. Method according to claim 1, characterized in that CBX is used as the starting anti-tumor glycoprotein, and the glycoprotein with "reduced molecular weight is obtained <CB>X3 . 7. Method according to claim 6, characterized in that starting anti-tumor glycoprotein CBX is obtained from a culture supernatant of reticuloendothelial cells, lymphoblasts, leukemia cells or fibroblasts from warm-blooded animals by salting out the supernatant and fractionating a precipitate from this by gel filtration, which gives a "fraction with a molecular weight of 12,000 - 17,000, and then ad- sorb and elute this fraction on Ulex europeus agglutinin-conjugated Sefadex. 0. Method according to claim 1, 2, 4 or 6, characterized in that the reducing agent is at least one selected from the group of a thiol compound, a metal hydride complex, a tertiary phosphine, a sulfite, a cyanate and a metal ion. 9. Method according to claim 8, characterized in that the thiol compound is selected from the group 3-mercaptoethanol, dithiothreitol, dithioerythritol, thioglycolic acid and monothiophosphoric acid. 10. Method according to claim 1 or 8, characterized in that the degradation treatment is carried out in the presence of a surfactant. 11. Method according to claim 10, characterized in that the surfactant is selected from the group ! sodium dodecyl sulfate, sodium cholate, dodecyltrimethylammonium- : bromide, deoxylysolecithin, "Triton X", "Tween 20" and "Span 60". 12. Method according to claim 1 or 8, characterized in that the decomposition treatment is carried out at a temperature between 0 and 37°C in a slightly acidic to neutral pH range. 13. Method according to claim 12, characterized in that the temperature is chosen between 15 and 25°C.