Glycoproteins and processes for their production

A glycoprotein obtained from the cells of human or non-human warm-blooded animals having an anti-tumor effect and characterized by the following properties: PA1 (a) molecular weight: in the range from 7,000 to 90,000 by Sephadex gel filtration or SDS gel electrophoresis; PA1 (b) color reactions: it exhibits a color indicating proteins in the Lowry reaction, exhibits a color indicating peptide bonds and amino acids in the ninhydrin reaction after hydrolysis with hydrochloric acid, and exhibits a color indicating sugars in the phenol-sulfuric acid reaction, the anthrone-sulfuric acid reaction, the indole-sulfuric acid reaction and the tryptophane-sulfuric acid reaction; PA1 (c) appearance and solubility: white powder soluble in water, aqueous sodium chloride and phosphate buffer, and sparingly soluble in benzene, hexane and chloroform; PA1 (d) sugar content: sugar content is 8-45%, 6-28% of the total sugar being hexoses, 1-11% being hexosamines and 1-6% being sialic acids; PA1 (e) stability: stable in an aqueous solution of pH 2.0, pH 7.0 or pH 11.0 at 4.degree. C. for 24 hours or longer and in an aqueous solution of pH 7.0 at 60.degree. C. for 3 hours or longer; and PA1 (f) cytotoxicity: it selectively damages tumor cells without substantially damaging normal cells.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention relates to novel glycoproteins obtained from an extract or a 
supernatant of culture medium of reticulo-endothelial cells, lymphoblasts, 
leukemia cells or fibroblasts of warm-blooded animals, processes for their 
production and therapeutic agents for malignant tumors which contain such 
glycoproteins singly or in combination as an active ingredient. 
2. Description of the Prior Art 
There has been known no perfect therapy for tumors, and in spite of the 
fact that many therapeutic agents for tumors have hitherto been developed 
by a number of researchers in the world, there have been many attempts to 
use new therapeutic agents and multi-agent combination treatments in the 
clinical field. 
Therapeutic agents for tumors are roughly classified into two categories, 
chemotherapeutic agents and immunotherapeutic agents. Chemotherapeutic 
agents, also known as cytotoxic substances, manifest their effect by 
nonspecifically suppressing cell growth and hence are toxic not only to 
tumor cells but also to normal cells, and show serious adverse reactions 
such as leukocytopenia, acyesis, alopecia, teratism, malignant neoplasms, 
etc.; consequently, there is a strict restriction on the dosage. On the 
other hand, since the immunotherapeutic agents manifest their therapeutic 
effect by indirectly inhibiting tumor growth through acting upon the 
biophylactic functions and not by directly inhibiting the growth of the 
tumor cells, there is far less danger for serious adverse reactions as 
compared with chemotherapeutic agents. However, tumor patients do not 
often retain enough biophylactic functions and therefore the therapeutic 
effect of immunotherapeutic agents is not always satisfactory as compared 
with that of chemotherapeutic agents. 
The present inventors conceived that the reticulo-endothelial cells which 
play an important role in biophylactic functions produce a substance which 
is effective for treating tumors, and have been searching for this 
substance. 
Several factors considered as promising therapeutic agents for tumors, e.g. 
Lymphotoxin, Tumor Necrosis Factor, Interferon, etc., have been obtained 
from reticulo-endothelial cells, as reported by Granger, G. A. et al., 
Cellular Immunology, Vol. 38, 338-402 (1978); Carswell, E. A. et al., 
Proc. Natl. Acad. Sci. U.S.A., Vol. 72, 3666-33670 (1975); and Issacs, A. 
et al., Proc. Roy. Soc. Ser. B., Vol. 147, 268 (1975), respectively. 
Further, the present inventors have recently discovered a simple method 
for isolating a large amount of Carcino-Breaking Factor (hereinafter 
referred to as CBF) as a mixture which contains the aforesaid Lymphotoxin, 
Tumor Necrosis Factor, etc. from a culture of lymphoblasts grown in 
hamsters whose immune response had been suppressed, and have reported that 
this CBF is effective against experimental tumors transplanted to an 
animal (The Yomiuri, morning issue, Nov. 22, 1981). 
During the course of the research on CBF, the present inventors have 
discovered that glycoproteins which differ from the aforesaid cytotoxic 
factors such as Lymphotoxin, Tumor Necrosis Factor, CBF, etc. are present 
in an extract or a supernatant of culture medium of reticulo-endothelial 
cells, lymphoblasts, leukemia cells or fibroblasts of warm-blooded 
animals, and are characterized by a very strong and selective cytotoxic 
effect against tumor cells. The present inventors have also established 
several processes for producing such glycoproteins without difficulties. 
SUMMARY OF THE INVENTION 
An object of this invention is to provide novel glycoproteins having an 
anti-tumor activity. 
Another object of this invention is to provide glycoproteins having an 
anti-tumor activity which are harvested from an extract or a supernatant 
of culture medium of reticulo-endothelial cells, lymphoblasts, leukemia 
cells or fibroblasts of warm-blooded animals. 
A further object of this invention is to provide processes for producing 
anti-tumor glycoproteins from an extract or a supernatant of culture 
medium of reticulo-endothelial cells, lymphoblasts, leukemia cells or 
fibroblasts of warm-blooded animals. 
A still further object of this invention is to provide therapeutic agents 
for tumors which contain such anti-tumor glycoproteins singly or in 
combination as an active ingredient. 
Accordingly, this invention relates to a glycoprotein having the following 
properties: 
(a) molecular weight: in the range from 7,000 to 90,000 by Sephadex gel 
filtration or SDS gel electrophoresis; 
(b) color reactions: it exhibits a color indicating proteins in the Lowry 
reaction, exhibits a color indicating peptide bonds and amino acids in the 
ninhydrin reaction after hydrolysis with hydrochloric acid, and exhibits a 
color indicating sugars in the phenol-sulfuric acid reaction, the 
anthrone-sulfuric acid reaction, the indole-sulfuric acid reaction and the 
tryptophane-sulfuric acid reaction; 
(c) appearance and solubility: while powder soluble in water, aqueous 
sodium chloride and phosphate buffer and sparingly soluble in benzene, 
hexane and chloroform; 
(d) sugar content is 8-45%, 6-28% of the total sugar being hexoses, 1-11% 
being hexosamines and 1-6% being sialic acids; 
(e) stable in an aqueous solution of pH 2.0, pH 7.0 or pH 11.0 at 4.degree. 
C. for 24 hours or longer and in an aqueous solution of pH 7.0 at 
60.degree. C. for 3 hours or longer; and 
(f) it selectively damages tumor cells without substantially damaging 
normal cells.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
Since the glycoproteins of this invention may be divided into four 
fractions with different molecular weights and sugar contents, these 
glycoproteins are classified according to the difference in molecular 
weight throughout the specification; one fraction with a molecular weight 
of 12,000-17,000 is referred to as Carcino-Breaker X (hereinafter referred 
to as CB.sub.X ; this also applies to the rest), one with a molecular 
weight of 70,000-90,000 as CB.sub.X1, one with a molecular weight of 
40,000-50,000 as CB.sub.X2 and one with a molecular weight of 7,000-9,000 
as CB.sub.X3. Where all of CB.sub.X, CB.sub.X1, CB.sub.X2 and CB.sub.X3 
are to be generally considered, "CB" is used as a general term therefor. 
Physical, chemical and biological properties of glycoproteins of the 
invention 
CB.sub.X 
(a) Molecular weight: When measured by gel filtration using Sephadex G-100 
(Pharmacia Co.) and 0.01M phosphate buffer (pH 7.2) as a solvent, the 
molecular weight is 12,000-17,000. 
(b) Color reactions: The results of the tests on the CB.sub.X aqueous 
solution for color reactions are shown in Table 1-1. The Lowry reaction 
and the ninhydrin reaction were conducted according to the procedures 
described in Seikagaku Jikken Koza, Vol. 1, Quantitative Method of 
Proteins, 1971. The phenol-sulfuric acid reaction, the anthrone-sulfuric 
acid reaction, the naphthol-sulfuric acid reaction, the indole-sulfuric 
acid reaction and the tryptophane-sulfuric acid reaction were conducted 
according to the procedures described in Seikagaku Jikken Koza, Vol. 4, 
Quantitative Method of Sugars, 1971. The Holff reactions were conducted 
according to the procedures described in Seikagaku Jikken Koza, Vol. 3, 
Quantitative Method of Lipids, 1971. 
TABLE 1-1 
______________________________________ 
Color Reaction Color Indication 
______________________________________ 
Lowry Blue Peptide bonds 
Ninhydrin Purple blue Amino acids 
Phenol-sulfuric acid 
Brown Sugars 
Anthrone-Sulfuric acid 
Greenish blue 
Sugars 
.alpha.-Naphthol-Sulfuric acid 
Purple Sugars 
Indole-Sulfuric acid 
Brown Sugars 
Tryptophane-Sulfuric acid 
Purple brown 
Sugars 
Holff Colorless No lipids 
______________________________________ 
As shown above, CB.sub.X exhibits colors indicating proteins and sugars, 
but does not exhibit a color indicating lipids. 
(c) Appearance and solubility: White powder soluble in water, aqueous 
sodium chloride and phosphate buffer, and sparingly soluble in benzene, 
hexane and chloroform. 
(d) Sugar content: According to the method of Spiro (Spiro, H. A., Methods 
in Enzymology, Vol. 8, 3-26 (1966)), the sugar content of CB.sub.X is 
27-33%, and its sugar composition is 17-20% of hexoses, 5-7% of 
hexosamines and 5-6% of sialic acids. 
(e) Isoelectric point: When measured by isoelectrofocusing on Ampholine, 
its isoelectric point is 4.2-7.3. 
(f) Adsorbability: Adsorbable on Ulex europeus agglutinin-conjugated 
Sephadex in 0.01M phosphate buffer (pH 7.2). 
(g) Stability: Stable with respect to molecular weight by gel filtration 
and to cytotoxic activity against tumor cells in an aqueous solution of pH 
2.0, pH 7.0 or pH 11.0 at 4.degree. C. for 24 hours or longer and in an 
aqueous solution of pH 7.0 at 60.degree. C. for 3 hours or longer. 
(h) Cytotoxicity: It selectively damages tumor cells without substantially 
damaging normal cells. 
The cytotoxicity of CB.sub.X was measured by culturing 10.sup.5 cells of 
tumor cells or normal cells in 0.2 ml of a medium in the presence of this 
substance at 37.degree. C. for 48 hours in a 5% CO.sub.2, 95% air 
atmosphere, and counting the number of viable cells not stained with 
Trypan Blue, and expressed by the concentration at which increase of the 
cells in number was inhibited by 50%. One unit of CB is defined to be the 
amount of the substance at which the growth of 10.sup.5 nasopharynx cancer 
of KB cell is inhibited by 50%. 
(i) Differentiation: Induces differentiation of tumor cells, that is, 
recovers the tumor cells to normal cells in a test according to the method 
of Hozumi et al (Hozumi, et al., Cancer Research, Vol. 40, 2919-2924 
(1980)), employing myelogenous leukemia cells M-1. 
CB.sub.X1 
(a) Molecular weight: When measured by gel filtration using Sephadex G-100 
and 0.01M phosphate buffer (pH 7.2) as a solvent, the molecular weight is 
70,000-90,000. 
(b) Color reactions: The results of the tests on the CB.sub.X1 aqueous 
solution for color reactions are shown in Table 1-2. 
TABLE 1-2 
______________________________________ 
Color Reaction Color Indication 
______________________________________ 
Lowry Blue Peptide bonds 
Ninhydrin Purple blue Amino acids 
Phenol-sulfuric acid 
Brown Sugars 
Anthrone-Sulfuric acid 
Greenish blue 
Sugars 
.alpha.-Naphthol-Sulfuric acid 
Purple Sugars 
Indole-Sulfuric acid 
Brown Sugars 
Tryptophane-Sulfuric acid 
Purple brown 
Sugars 
Holff Colorless No lipids 
______________________________________ 
As shown above, CB.sub.X1 exhibits colors indicating proteins and sugars, 
but does not exhibit a color indicating lipids. 
(c) Appearance and solubility: White powder soluble in water, aqueous 
sodium chloride and phosphate buffer, and sparingly soluble in benzene, 
hexane and chloroform. 
(d) Sugar content: According to the method of Spiro, supra, the sugar 
content of CB.sub.X1 is 35-45%, and its sugar composition is 23-28% of 
hexoses, 8-11% of hexosamines and 4-6% of sialic acids. 
(e) Isoelectric point: When measured by isoelectrofocusing on Ampholine, 
its isoelectric point is 4.3-6.2. 
(f) Adsorbability: Adsorbable on Ulex europeus agglutinin-conjugated 
Sephadex in 0.01M phosphate buffer (pH 7.2). 
(g) Stability: Stable with respect to molecular weight by gel filtration 
and to cytotoxic activity against tumor cells in an aqueous solution of pH 
2.0, pH 7.0 or pH 11.0 at 4.degree. C. for 24 hours or longer and in an 
aqueous solution of pH 7.0 at 60.degree. C. for 3 hours or longer. 
(h) Cytotoxicity: It selectively damages tumor cells without substantially 
damaging normal cells. The cytotoxicity of CB.sub.X1 was measured by 
procedures described with respect to CB.sub.X. 
CB.sub.X2 
(a) Molecular weight: When measured by gel filtration using Sephadex G-100 
and 0.01M phosphate buffer (pH 7.2) as a solvent, the molecular weight is 
40,000-50,000. 
(b) Color reactions: The results of the tests on the CB.sub.X2 aqueous 
solution for color reactions are shown in Table 1-3. 
TABLE 1-3 
______________________________________ 
Color Reaction Color Indication 
______________________________________ 
Lowry Blue Peptide bonds 
Ninhydrin Purple blue Amino acids 
Phenol-sulfuric acid 
Brown Sugars 
Anthrone-Sulfuric acid 
Greenish blue 
Sugars 
.alpha.-Naphthol-Sulfuric acid 
Purple Sugars 
Indole-Sulfuric acid 
Brown Sugars 
Tryptophane-Sulfuric acid 
Purple brown 
Sugars 
Holff Colorless No lipids 
______________________________________ 
As shown above, CB.sub.X2 exhibits colors indicating proteins and sugars, 
but does not exhibit a color indicating lipids. 
(c) Appearance and solubility: White powder soluble in water, aqueous 
sodium chloride and phosphate buffer, and sparingly soluble in benzene, 
hexane and chloroform. 
(d) Sugar content: According to the method of Spiro, supra, the sugar 
content of CB.sub.X2 is 30-37%, and its sugar composition is 20-23% of 
hexoses, 6-8% of hexosamines and 4-6% of sialic acids. 
(e) Isoelectric point: When measured by isoelectrofocusing an Ampholine, 
its isoelectric point is 4.2-7.3. 
(f) Adsorbability: Adsorbable on Ulex europeus agglutinin-conjugated 
Sephadex in 0.01M phosphate buffer (pH 7.2). 
(g) Stability: Stable with respect to molecular weight by gel filtration 
and to cytotoxic activity against tumor cells in an aqueous solution of pH 
2.0, pH 7.0 or pH 11.0 at 4.degree. C. for 24 hours or longer and in an 
aqueous solution of pH 7.0 at 60.degree. C. for 3 hours or longer. 
(h) Cytotoxicity: It selectively damages tumor cells without substantially 
damaging normal cells. The cytotoxicity of CB.sub.X2 was measured by 
procedures described with respect to CB.sub.X. 
CB.sub.X3 
(a) Molecular weight: When measured by SDS gel electrophoresis, the 
molecular weight is 7,000-9,000. 
(b) Color reactions: The results of the tests on the CB.sub.X3 aqueous 
solution for color reactions are shown in Table 1-4. 
TABLE 1-4 
______________________________________ 
Color Reaction Color Indication 
______________________________________ 
Lowry Blue Peptide bonds 
Ninhydrin Purple blue Amino acids 
Phenol-sulfuric acid 
Brown Sugars 
Anthrone-Sulfuric acid 
Greenish blue 
Sugars 
.alpha.-Naphthol-Sulfuric acid 
Purple Sugars 
Indole-Sulfuric acid 
Brown Sugars 
Tryptophane-Sulfuric acid 
Purple brown 
Sugars 
Holff Colorless No lipids 
______________________________________ 
As shown above, CB.sub.X3 exhibits colors indicating proteins and sugars, 
but does not exhibit a color indicating lipids. 
(c) Appearance and solubility: White powder soluble in water, aqueous 
sodium chloride and phosphate buffer, and sparingly soluble in benzene, 
hexane and chloroform. 
(d) Sugar content: According to the method of Spiro, supra, the sugar 
content of CB.sub.X3 is 8-15%, and its sugar composition is 6-10% of 
hexoses, 1-2% of hexosamines and 1-3% of sialic acids. 
(e) Adsorbability: Adsorbable on carboxymethylcellulose in an ion exchange 
chromatography in 0.05M phosphate buffer (PH 6.4) using 
carboxymethylcellulose. 
(f) Stability: Stable with respect to molecular weight by gel filtration 
and cytotoxic activity agaist tumor cells in an aqueous solution of pH 
2.0, pH 7.0 or pH 11.0 at 4.degree. C. for 24 hours or longer and in an 
aqueous solution of pH 7.0 at 60.degree. C. for 3 hours or longer. 
(g) Cytotoxicity: It selectively damages tumor cells without substantially 
damaging normal cells. The cytotoxicity of CB.sub.X3 was measured by the 
procedures described with respect to CB.sub.X. 
(h) The amino acid sequence of the N terminal of the protein portion is 
Alanine-Alanine-. 
The glycoproteins of this invention have common characteristics in color 
reactions, appearance, solubility, stability, effect on tumor cells, etc., 
but they differ from each other with respect to molecular weight and sugar 
content, and therefore the respective substances may be distinguished from 
one another. 
The glycoproteins of this invention are clearly distinguished from 
Lymphotoxin, Tumor Necrosis Factor, a mixture thereof (i.e. CBF) or 
Interferon, all of which are obtained from reticulo-endothelial cells, 
lymphoblasts, leukemia cells or fibroblasts, with regard to the following 
features. Thus they are evidently different substances. 
More specifically, Lymphotoxin is known to be present in three different 
types depending on the molecular weight, i.e. .alpha.-Lymphotoxin having a 
molecular weight of 70,000-90,000, .beta.-Lymphotoxin having a molecular 
weight of 35,000-50,000 and .gamma.-Lymphotoxin having a molecular weight 
of 10,000-20,000 (Eds., Cohen et al., Biology of the Lymphokinase, 
Academic Press, 1979). With respect to the molecular weight, CB.sub.X 
resembles .gamma.-Lymphotoxin, CB.sub.X1 resembles .alpha.-Lymphotoxin and 
CB.sub.X2 resembles .beta.-Lymphotoxin. However, Lymphotoxin, as reported 
by Lucas et al (Lucas, Z. J. et al., J. Immunology, Vol. 109, 1233 
(1972)), has little selectivity in cytotoxic effect and causes damage to 
normal cells as well as to tumor cells. In contrast, the cytotoxic effect 
of the glycoproteins of this invention is selective to tumor cells, and 
thus they are clearly different from Lymphotoxin. Moreover, the 
glycoproteins of this invention are different from Lymphotoxin in 
adsorbability and stability. More specifically, while Lymphotoxin prepared 
according to the method of Granger et al (Granger, G. A. et al, Cellular 
Immunology, Vol. 38, 388-402 (1978)) is not or only weakly adsorbed on 
Ulex europeus agglutinin-conjugated Sephadex in 0.01M phosphate buffer, 
the glycoproteins of this invention are adsorbed thereon. Furthermore, the 
glycoproteins of this invention are stable in aqueous solutions of pH 2.0, 
pH 7.0 and pH 11.0 at 4.degree. C. for 24 hours or longer, and also are 
stable at pH 7.0 at 60.degree. C. for 3 hours or longer. In constrast, 
Lymphotoxin loses its activity by 60% or more after it is maintained at 
56.degree. C. for 4 hours. 
Tumor Necrosis Factor exhibits a selective cytotoxic effect on tumor cells, 
and it has a molecular weight of 33,000-63,000 and a sugar content of 0% 
(Carswell, E. A. et al, Proc. Natl. Acad. Sci. U.S.A., Vol. 72, 3666-3670 
(1975)) or it has a molecular weight of 39,000 and a sugar content of 40% 
(The Nippon Keizai Shimbun, Morning issue, Aug. 23, 1981). Both are 
different from CB.sub.X, CB.sub.X1 and CB.sub.X3 with respect to molecular 
weight and from CB.sub.X2 with respect to sugar content. 
Further, CBF, which contains these cytotoxic factors in combination, has a 
molecular weight of about 35,000 (The Nippon Keizai Shimbun, Morning 
Issue, Nov. 22, 1981) and differs from the glycoproteins of this invention 
with respect to molecular weight. 
Finally, the glycoproteins of this invention are different from Interferon 
in that the former do not possess antiviral activity. 
Cells employed for producing glycoproteins of the invention 
The source cells originated from human or non-human warm-blooded animals 
for use in this invention may be any of reticuloendothelial cells, 
lymphoblasts, leukemia cells and fibroblasts, and they may be employed 
either in a primary culture or in an established cell line. Preferably, 
cells of human origin are desirable and safe because they elicit fewer 
antigenicity induced reactions and other adverse reactions with respect to 
use of CB in the treatment of human diseases. As such cells, any cells may 
be chosen from, for example, BALL-1 cells, TALL-1 cells and NALL-1 cells 
reported by Miyoshi (Miyoshi, I., Nature, Vol. 267, 843-844 (1977)), 
Namalwa cells described in Journal of Clinical Microbiology (J. Clin. 
Microbiol., Vol. 1, 116-117 (1975)), M-7002 cells and B-7101 cells 
described in Journal of Immunology (Vol. 113, 1334-1345 (1974)), Flow 7000 
cells (Flow Co.), JBL cells, EBV-Sa cells, EBV-Wa cells and EBV-HO cells 
described in "The Tissue Culture" (Vol. 6, 527-546 (1980)), established 
cell lines such as BALM 2 cells, CCRF-SB cells (ATCC CCL 120) etc., human 
lymphocytes and macrophages, as well as the cells of an established cell 
line from human lymphocytes and macrophages treated with various viruses, 
drugs, radiation, etc. 
As the source cells originated from non-human warm-blooded animals, any 
cells may be chosen from, for example, mouse BALB/C 3T3 cells (Flow Co.), 
mouse leukemia cells such as L1210 cells (J. Natl. Cancer Inst., Vol. 13, 
1328 (1953)) and P388 cells (Scientific Proceedings, Pathologists & 
Bacteriologists, Vol. 33, 603 (1957)), mouse melanoma clone M-3 (Flow 
Co.), rat tumor LLC-WRC 256 (Flow Co.), hamster melanoma RPMI 1846 cells 
(Flow Co.), and lymphocytes, macrophages, etc. It should be understood 
that the cells which may be employed in this invention are not restricted 
to those described above. 
Process for producing glycoproteins (CB) of the invention 
The process for producing CB by cells originated from human or non-human 
warm-blooded animals may be chosen from known methods for producing active 
substances from cells, and it may be harvested either directly from the 
cells or after the cells have been cultured, or, if a larger amount of CB 
is desired, the cells may be exposed to one or more inducers. For example, 
the cells originated from human or non-human warm-blooded animals may be 
suspended in an appropriate medium and directly exposed to inducer to 
produce CB which may then be harvested from the medium. 
As the inducer for CB, generally one or more substances chosen from the 
following may be used: lectins such as phytohemagglutinin, concanavalin A, 
pokeweed mitogen, lypopolysaccharides, polysaccharides such as 
phosphommanan, dextran phosphate, endotoxins, microbial cell components, 
bacteria, viruses, nucleic acids, polynucleotides, etc. Further, for the 
antigen-sensitized cells, corresponding antigens also serve as inducers 
for CB. 
CB thus produced may be easily isolated by known purification methods, such 
as salting out, dialysis, filtration, centrifugation, concentration, 
lyophilization, etc. If higher purification is desired, it may be achieved 
by adsorption and elution on an ion exchange resin, gel filtration, 
electrophoresis or affinity chromatography using, for example, antibody- 
or Ulex europeus agglutinin-conjugated Sephadex. 
If CB is to be obtained in a large quantity, the cells of the established 
cell line may be grown in the body of warm-blooded animals as is now 
explained. 
The established cell lines originated from human or non-human warm-blooded 
animals may be any of reticuloendothelial cells, lymphoblasts, leukemia 
cells and fibroblasts. Cell lines of human origin are especially desirable 
and safe because they elicit fewer antigenicity induced reactions and 
other adverse reactions with respect to use of CB in the treatment of 
human diseases. As such cell lines, any cell lines may be employed, as 
described above, for example, BALL-1 cells, TALL-1 cells, NALL-1 cells, 
Namalwa cells, M-7002 cells, B-7101 cells, Flow 7000 cells, BALB/C 3T3 
cells, L1210 cells, P388 cells, lymphocytes, macrophages, etc. 
When these cells are to be grown in warm-blooded animals' bodies, 
transplantation of such cells may be carried out directly or, as described 
hereinbelow, indirectly by inoculating a chamber with said cells and 
placing the chamber into the body. The warm-blooded animals into which 
such cells are transplanted may be of the same or different species as 
long as the established cell line originated from human or non-human 
warm-blooded animals can grow therein. For example, fowls such as chickens 
and pigeons and mammals such as dogs, cats, monkeys, goats, pigs, horses, 
bovines, rabbits, guinea pigs, rats, hamsters, ordinary mice, or nude mice 
may be employed. 
When cultured cells originated from an animal of different species are 
transplanted into one of these animals, there is a possibility of 
undesirable immunological reactions. Therefore, animals in the most 
immature state, e.g. eggs, foetuses, embryos, or onatals or infant 
animals, are suitably employed so that the possibility of immunological 
reactions is minimized. In addition, the immunological reactions may also 
be suppressed by pre-treatments, for example, by exposing these animals to 
X-ray of 200-600 REM, or injecting them with immunosuppressive agents. 
When the animal to be used as the host is a nude mouse or of the same 
species as the source of cells to be transplanted, immunological reactions 
are weak and therefore such cells may be transplanted thereinto and grown 
rapidly without any pretreatment, and therefore the use of such cells is 
especially convenient. 
Alternatively, constant growth of the cells may also be assured and the 
amount of CB produced therefrom may be increased by transplanting cells 
from one warm-blooded animal to another warm-blooded animal, for example, 
by transplanting cells originated from human or non-human warm-blooded 
animals into hamsters for growth and then retransplanting said cells into 
nude mice. In such cases, transplantation may be conducted between the 
same class or division as well as between the same species or genus. 
The site to which the cells originated from human or non-human warm-blooded 
animals are to be transplanted may be any site where the transplanted 
cells can grow, for example the allantoic cavity, veins, the abdominal 
cavity, or subcutaneous sites may be freely chosen. 
Instead of directly transplanting and growing established cell lines 
originated from human or non-human warm-blooded animals in the body of 
warm-blooded animals, any of the above mentioned established cell lines 
may be inoculated and grown in a conventional diffusion chamber of various 
shapes and sizes which is placed, for example, in the peritoneal cavity of 
the body of a warm-blooded animal. The diffusion chamber is designed to 
enable said cells to grow by facilitating the uptake of body fluid of the 
animal as nutrients. The chamber is also provided with porous filter 
membranes, for example, membrane filters with pore sizes of about 
10.sup.-7 -10.sup.-3 m, ultrafilters or hollow fibers, which prevent the 
migration of cells out of the chamber and allow the body fluid as 
nutrients to enter the chamber. 
If necessary, the diffusion chamber may be designed and placed, for 
example, on the surface of the animal body, so as to connect the nutrient 
fluid in the chamber with the body fluid of the animal and circulate them, 
so that the growth of the cells inoculated in said chamber can be observed 
through a view window. The diffusion chamber can also be designed so that 
it can be disconnected from the animal body, enabling cells to be grown 
over the whole life span of the animal, thus increasing the yield of cells 
per animal. 
The method involving the use of these diffusion chambers has further 
advantages; that is, since the cells of the established cell lines 
originated from human or non-human warm-blooded animals are not brought 
into direct contact with the animal cells, such cells may be easily 
harvested. Because of the lower possibility of causing undesirable 
immunological reactions, various warm-blooded animals can be used without 
the need for pre-treatment of the animals for immunosuppression. 
The animals to which the cells have been transplanted may be fed and 
maintained in the usual way for the animal, and no special care is 
required even after transplantation, a very convenient feature. 
The period required for growth of the cells of the established cell lines 
originated from human or non-human warm-blooded animals is generally 1-10 
weeks. The number of cells thus obtained has been found to be about 
10.sup.7 -10.sup.12 cells or more per animal. 
In other words, the process according to this invention for producing CB is 
extremely advantageous for producing CB, because the established cell 
lines originated from warm-blooded animals are multiplied by about 
10.sup.2 -10.sup.7 fold or more over the number of the cells directly 
inoculated to animal, or about 10-10.sup.8 fold or more compared to the 
case wherein the cells were cultured in a nutrient medium. 
The production of CB from the grown cells of an established cell line 
originated from human or non-human warm-blooded animals may be conducted 
in various manners. They may also be harvested directly from the body in 
which such cells have been grown. For example, CB may be harvested 
directly from the cells obtained by growing the transplanted cells of the 
established cell lines originated from human or non-human warm-blooded 
animals in ascites as a suspension or by growing them subcutaneously. 
Alternatively, the production of CB may be conducted by using an inducer 
after growing the established cell lines originated from human or 
non-human warm-blooded animals in the body of an animal by applying the 
inducer either directly in vivo or in vitro after taking the cells out of 
the body. For example, the cells of an established cell line originated 
from human or non-human warm-blooded animals, which have been grown in 
ascites and harvested therefrom, or those isolated and dissociated from a 
subcutaneous tumor comprising the cells of an established cell line 
originated from human or non-human warm-blooded animals, may be suspended 
in a nutrient medium kept at about 20.degree.-40.degree. C. to give a cell 
concentration of about 10.sup.5 -10.sup.8 cells per ml, and then exposed 
to a CB inducer, thereby inducing the production of CB which may then be 
harvested. 
Further, where the cells of an established cell line originated from human 
or non-human warm-blooded animals are grown in a diffusion chamber, the 
cells may be directly harvested from the chamber, of they may be harvested 
after once removing from the chamber either directly or even after 
exposure to one or more inducers. 
Furthermore, the yield of CB per animal may be even further increased by 
employing, for example, the following methods: a method wherein the cells 
of an established cell line originated from human or non-human 
warm-blooded animals which have been grown in the body of another animal 
are exposed to an inducer to induce the production of CB in situ, and then 
the grown cells, which have been harvested from a specific site or the 
whole of the same animal body, are exposed to an inducer to induce the 
production of CB; a method wherein the used cells are again exposed to an 
inducer to induce the production of CB; a method wherein a diffusion 
chamber placed in or connected to the animal body is replaced by a new one 
to increase the number of the obtained cells; and the like. 
For inducing the production of CB, any inducer for CB described above may 
be employed, and the thus produced CB may be fractionated into the 
respective CB.sub.X, CB.sub.X1, CB.sub.X2 and CB.sub.X3 having the 
specified molecular weights by using the above-described known separating 
and purifying procedures. 
Effectiveness, toxicity, method of use and dosage of CB of the invention 
Experiment 1: Selectivity of the Cytotoxic Effect 
Samples of 10.sup.5 cells of each of tumor cell lines including KB cells 
(nasopharynx cancer), MX-1 cells (breast cancer, supplied from Dr. Shigeru 
Tsukagoshi, Cancer Institute), HEp-2 cells (throat cancer) and HEL cells 
(hepatoma, Flow Co.) and of normal cell lines including intestine 407 
cells, Girardi heart cells, Chang Liver cells, Vero cells (monkey kidney) 
and MDCK cells (dog kidney) (Flow Co.), all of which had been precultured 
for 24 hours respectively, and 10.sup.5 cells of each of P388 and L1210 
cells (leukemia, supplied from Dr. Shigeru Tsukagoshi, Cancer Institute), 
which were used immediately, were each cultured in 1 ml of Eagle's medium 
containing 10% calf serum and each test substance at 37.degree. C. for 48 
hours in a 5% CO.sub.2, 95% air atmosphere. Thereafter the number of the 
viable cells not stained by Trypan Blue was counted under a light 
microscope, and the concentration of the test substance at which 50% of 
the cells are killed was calculated against a control taken as 100. 
Employed as the test substances were CB.sub.X obtained in Example 10, 
CB.sub.X1 obtained in Example 16, CB.sub.X2 obtained in Example 20, 
CB.sub.X3 obtained in Example 26 or 29, mixture of CB.sub.X and CB.sub.X1, 
mixture of CB.sub.X, CB.sub.X1, CB.sub.X2 and CB.sub.X3, mixture of 
CB.sub.X2 and CB.sub.X3, .alpha.-, .beta.- and .gamma.-Lymphotoxins 
obtained by a known method (Granger, G. A. et al, Cellular Immunology, 
Vol. 38, 388-402 (1978)), CBF separated from CB.sub.X in Example 1 and 
Mitomycin C. One unit of the Lymphotoxins and CBF is expressed by a 
conventional index which is based on the cytotoxicity 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 Tables 2-1 to 
2-4. 
TABLE 2-1 
__________________________________________________________________________ 
Concentration for 50% Inhibition 
of Growth 
.gamma.-Lympho- 
Cell CB.sub.X 
toxin CBF Mitomycin C 
Name Species 
(unit/ml) 
(unit/ml) 
(unit/ml) 
(.mu.g/ml) 
__________________________________________________________________________ 
Tumor 
KB Human 
1.0 16 18 34 
Cells 
HEp-2 
Human 
1.6 5.6 24 17 
HEL Human 
1.1 -- 33 26 
MX-1 Human 
1.9 -- 3.5 32 
L1210 
Mouse 
3.0 -- -- 43 
P388 Mouse 
3.3 -- -- 36 
Normal 
Intes- 
Human 
&gt;1,000 
80.0 &gt;20,000 
32 
Cells 
tine 407 
Girardi 
Human 
&gt;1,000 
-- &gt;20,000 
45 
Heart 
Chang 
Human 
&gt;1,000 
8.0 &gt;20,000 
19 
Liver 
Vero Monkey 
650 
-- -- 52 
MDCK Dog 520 
-- -- 41 
__________________________________________________________________________ 
(Note) 
"--" means the experiment has not been conducted. 
TABLE 2-2 
__________________________________________________________________________ 
Concentration for 50% Inhibition of Growth 
.alpha.-Lympho- 
.beta.-Lympho- 
Cell CB.sub.X1 
CB.sub.X2 
toxin toxin CBF Mitomycin C 
Name Species 
(unit/ml) 
(unit/ml) 
(unit/ml) 
(unit/ml) 
(unit/ml) 
(.mu.g/ml) 
__________________________________________________________________________ 
Tumor 
KB Human 
1.0 1.0 19 21 18 34 
Cells 
Hep-2 
Human 
1.5 1.4 4.8 7.2 24 17 
HEL Human 
1.3 1.5 -- -- 33 26 
MX-1 Human 
1.8 1.6 -- -- 3.5 32 
L1210 
Mouse 
3.2 3.4 -- -- -- 43 
P388 Mouse 
3.2 3.2 -- -- -- 36 
Normal 
Intes 
Human 
&gt;1,000 
&gt;1,000 
76.0 92.0 &gt;20,000 
32 
Cells 
tine 407 
Girardi 
Human 
&gt;1,000 
&gt;1,000 
-- -- &gt;20,000 
19 
Heart 
Chang 
Human 
&gt;1,000 
&gt;1,000 
9.6 8.8 &gt;20,000 
19 
Liver 
Vero Monkey 
630 
640 
-- -- -- 52 
MDCK Dog 550 
530 
-- -- -- 41 
__________________________________________________________________________ 
(Note) 
"--" means the experiment has not been conducted. 
TABLE 2-3 
______________________________________ 
Concentration for 50% Inhibition 
of Growth 
Mitomycin 
Cell CB.sub.X3 *.sup.(1) 
CB.sub.X3 *.sup.(2) 
C 
Name Species (unit/ml) 
(unit/ml) 
(.mu.g/ml) 
______________________________________ 
Tumor KB Human 1.0 1.0 35 
Cells HEp-2 Human 1.5 1.7 18 
HEL Human 1.3 1.4 25 
MX-1 Human 1.8 1.7 30 
L1210 Mouse 3.1 2.9 44 
P388 Mouse 3.4 3.5 37 
Normal Intes- Human &gt;1,000 &gt;1,000 35 
Cells tine 407 
Girardi Human &gt;1,000 &gt;1,000 44 
Heart 
Chang Human &gt;1,000 &gt;1,000 21 
Liver 
Vero Monkey 620 580 50 
MDCK Dog 510 540 44 
Primary Rat 870 910 61 
Culture 
Rat 
Liver 
______________________________________ 
(Notes) 
*.sup.(1) CB.sub.X3 obtained in Example 26 
*.sup.(2) CB.sub.X3 obtained in Example 29 
TABLE 2-4 
______________________________________ 
Concentration for 50% Inhibition 
of Growth 
Cell A*.sup.(1) 
B*.sup.(2) 
C*.sup.(3) 
Name Species (unit/ml) 
(unit/ml) 
(unit/ml) 
______________________________________ 
Tumor KB Human 1.0 1.0 1.0 
Cells HEp-2 Human 1.5 1.4 1.6 
HEL Human 1.6 1.7 1.9 
MX-1 Human 1.8 1.7 1.6 
L1210 Mouse 3.0 3.2 3.0 
P388 Mouse 3.1 3.4 3.2 
Normal Intes- Human &gt;1,000 &gt;1,000 &gt;1,000 
Cells tine 407 
Girardi Human &gt;1,000 &gt;1,000 &gt;1,000 
Heart 
Chang Human &gt;1,000 &gt;1,000 &gt;1,000 
Liver 
Vero Monkey 610 590 580 
MDCK Dog 580 610 600 
______________________________________ 
(Notes) 
*.sup.(1) Mixture of CB.sub.X and CB.sub.X1 
*.sup.(2) Mixture of CB.sub.X, CB.sub.X1, CB.sub.X2 and CB.sub.X3 
*.sup.(3) Mixture of CB.sub.X2 and CB.sub.X3 
As evident from the above results, CB similar to CBF selectively damages 
tumor cells without substantially causing any damage to the normal cells. 
However, the intensities of the cytotoxic effect on the respective tumors 
were different between CB and CBF. In contrast, both .alpha.- and 
.beta.-Lymphotoxin and Mitomycin C showed a nonselective cytotoxicity to 
the normal cells and the 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.times.10.sup.6 cells per animal of Sarcoma 180 or Ehrlich ascites 
tumor and the period of survival in days was observed. CB.sub.X obtained 
in Example 6, CB.sub.X1 obtained in Example 15, CB.sub.X2 obtained in 
Example 20 and CB.sub.X3 obtained in Example 26 or 29 were intravenously 
administered to groups of 5 mice daily from one day after transplantation 
until death. The results are expressed in percentages of the average 
survival days to that of the control group and shown in Tables 3-1 to 3-3. 
TABLE 3-1 
______________________________________ 
Average 
Survival 
Tumor Test Substance Daily Dose 
Days (%) 
______________________________________ 
Mouse CB.sub.X 1.2 unit/kg 
111 
Sarcoma 4 unit/kg 
131 
180 12 unit/kg 
164 
Mitomycin C 0.5 mg/kg 140 
Cyclophosphamide 
20 mg/kg 172 
Ehrlich CB.sub.X 1.2 unit/kg 
141 
Ascites 4 unit/kg 
159 
Tumor 12 unit/kg 
187 
Mitomycin C 0.5 mg/kg 168 
Cyclophosphamide 
20 mg/kg 212 
______________________________________ 
TABLE 3-2 
______________________________________ 
Average 
Survival 
Tumor Test Substance Daily Dose 
Days (%) 
______________________________________ 
Mouse CB.sub.X1 1 unit/kg 
113 
Sarcoma 3 unit/kg 
133 
180 10 unit/kg 
160 
CB.sub.X2 1 unit/kg 
110 
3 unit/kg 
135 
10 unit/kg 
159 
Mitomycin C 0.5 mg/kg 138 
Cyclophosphamide 
20 mg/kg 170 
Ehrlich CB.sub.X1 1 unit/kg 
139 
Ascites 3 unit/kg 
164 
Tumor 10 unit/kg 
187 
CB.sub.X2 1 unit/kg 
135 
3 unit/kg 
161 
10 unit/kg 
189 
Mitomycin C 0.5 mg/kg 164 
Cyclophosphamide 
20 mg/kg 206 
______________________________________ 
TABLE 3-3 
______________________________________ 
Average 
Survival 
Tumor Test Substance 
Daily Dose 
Days (%) 
______________________________________ 
Mouse CB.sub.X3 *.sup.(1) 
1 unit/kg 
110 
Sarcoma 3 unit/kg 
129 
180 10 unit/kg 
157 
CB.sub.X3 *.sup.(2) 
1 unit/kg 
108 
3 unit/kg 
131 
10 unit/kg 
150 
Mitomycin C 0.5 mg/kg 142 
Ehrlich CB.sub.X3 *.sup.(1) 
1 unit/kg 
139 
Ascites 3 unit/kg 
155 
Tumor 10 unit/kg 
181 
CB.sub.X3 *.sup.(2) 
1 unit/kg 
132 
3 unit/kg 
157 
10 unit/kg 
179 
Mitomycin C 0.5 mg/kg 166 
______________________________________ 
(Notes) 
*.sup.(1) CB.sub.X3 obtained in Example 26 
*.sup.(2) CB.sub.X3 obtained in Example 29 
As clearly seen in the results above, CB showed a significant anti-tumor 
effect on mice to which both Sarcoma 180 and Enrlich tumor had been 
transplanted, respectively. 
Experiment 3: Influence on the Survival Days of Leukemic Mice 
BDF.sub.1 -strain male mice weighing 20-25 g were intraperitoneally 
transplanted with 10.sup.5 cells per animal of mouse leukemia L1210 or 
10.sup.6 cells per animal of mouse leukemia P338, and the period of 
survival in days was observed. CB.sub.X obtained in example 10, CB.sub.X1 
obtained in Example 16, CB.sub.X2 obtained in Example 20 and CB.sub.X3 
obtained in Example 26 or 29 were intraperitoneally administered to groups 
of 5 mice, either daily from one day after the transplantation until death 
(for CB.sub.X, CB.sub.X1 and CB.sub.X2) or once on the day following the 
transplantation (for CB.sub.X3). The results are expressed in percentages 
of the average survival days to that of the control group and set forth in 
Tables 4-1 to 4-3. 
TABLE 4-1 
______________________________________ 
Average 
Survival 
Tumor Test Substance 
Daily Dose 
Days (%) 
______________________________________ 
Mouse CB.sub.X 0.4 unit/kg 
105 
Leukemia 1.2 unit/kg 
123 
L1210 4 unit/kg 
151 
Mitomycin C 0.5 mg/kg 128 
Cyclophosphamide 
20 mg/kg 172 
Mouse CB.sub.X 0.4 unit/kg 
113 
Leukemia 1.2 unit/kg 
128 
P388 4 unit/kg 
144 
Mitomycin C 0.5 mg/kg 133 
Cyclophosphamide 
20 mg/kg 147 
______________________________________ 
TABLE 4-2 
______________________________________ 
Average 
Survival 
Tumor Test Substance 
Daily Dose 
Days (%) 
______________________________________ 
Mouse CB.sub.X1 3 unit/kg 
108 
Leukemia 10 unit/kg 
122 
L1210 30 unit/kg 
149 
CB.sub.X2 1 unit/kg 
110 
3 unit/kg 
121 
10 unit/kg 
151 
Mitomycin C 0.5 mg/kg 136 
Cyclophosphamide 
20 mg/kg 149 
Mouse CB.sub.X1 1 unit/kg 
110 
Leukemia 3 unit/kg 
126 
P338 10 unit/kg 
147 
CB.sub.X2 0.3 unit/kg 
109 
1 unit/kg 
125 
3 unit/kg 
151 
Mitomycin C 0.5 mg/kg 130 
Cyclophosphamide 
20 mg/kg 145 
______________________________________ 
TABLE 4-3 
______________________________________ 
Average 
Survival 
Tumor Test Substance 
Daily Dose Days (%) 
______________________________________ 
Mouse CB.sub.X3 *.sup.(1) 
10 unit/kg 
109 
Leukemia 30 unit/kg 
120 
L1210 100 unit/kg 
149 
CB.sub.X3 *.sup.(2) 
10 unit/kg 
111 
30 unit/kg 
121 
100 unit/kg 
146 
Mitomycin C 5.0 mg/kg 132 
Mouse CB.sub.X3 *.sup.(1) 
10 unit/kg 
122 
Leukemia 30 unit/kg 
145 
P388 100 unit/kg 
176 
CB.sub.X3 *.sup.(2) 
10 unit/kg 
120 
30 unit/kg 
148 
100 unit/kg 
171 
Mitomycin C 5.0 mg/kg 141 
______________________________________ 
(Notes) 
*.sup.(1) CB.sub.X3 obtained in Example 26 
*.sup.(2) CB.sub.X3 obtained in Example 29 
As clearly seen in results above, CB showed a significant anti-tumor effect 
on both tumor-bearing mice with mouse leukemia L1210 and P388, 
respectively. 
Experiment 4: Influence on the Survival Days of Lung Carcinoma bearing Mice 
BDF.sub.1 -strain male mice weighing 20-25 g were transplanted with 
2.times.10.sup.6 cells of Lewis's lung carcinoma intramuscularly to the 
right thigh, and the survival days were observed. CB.sub.X obtained in 
example 7, CB.sub.X1 obtained in Example 17, CB.sub.X2 obtained in Example 
22 and CB.sub.X3 obtained in Example 26 or 29 were intravenously 
administered to groups of 6 mice daily from one day after the 
transplantation until death. The results are expressed in percentages of 
the average survival days to that of the control group and set forth in 
Tables 5-1 to 5-2. 
TABLE 5-1 
______________________________________ 
Average 
Survival 
Test Substance Daily Dose 
Days (%) 
______________________________________ 
CB.sub.X 1.2 unit/kg 104 
4 unit/kg 112 
12 unit/kg 146 
Mitomycin C 0.5 mg/kg 121 
Cyclophosphamide 20 mg/kg 163 
______________________________________ 
TABLE 5-2 
______________________________________ 
Average 
Survival 
Test Substance Daily Dose 
Days (%) 
______________________________________ 
CB.sub.X1 1 unit/kg 107 
3 unit/kg 113 
10 unit/kg 145 
CB.sub.X2 1 unit/kg 109 
3 unit/kg 121 
10 unit/kg 143 
CB.sub.X3 *.sup.(1) 
1 unit/kg 115 
3 unit/kg 143 
10 unit/kg 157 
CB.sub.X3 *.sup.(2) 
1 unit/kg 111 
3 unit/kg 136 
10 unit/kg 159 
Mitomycin C 0.5 mg/kg 120 
Cyclophosphamide 20 mg/kg 164 
______________________________________ 
(Notes) 
*.sup.(1) CB.sub.X3 obtained in Example 26 
*.sup.(2) CB.sub.X3 obtained in Example 29 
Experiment 5: Influence on the Survival Period in Days of Melanoma bearing 
Mice 
BDF.sub.1 -strain male mice weighing 20-25 g had transplanted 
subcutaneously in their back 10.sup.6 cells per animal of mouse melanoma B 
16, and the survival days were observed. CB.sub.X obtained in example 10 
and CB.sub.X3 obtained in Example 26 or 29 were intravenously administered 
to groups of 7 mice daily from one day after the transplantation until 
death. The results are expressed in percentages of the average survival 
days of the control group and set forth in Tables 6-1 to 6-2. 
TABLE 6-1 
______________________________________ 
Average 
Survival 
Test Substance Daily Dose 
Days (%) 
______________________________________ 
CB.sub.X 1.2 unit/kg 112 
4 unit/kg 135 
12 unit/kg 180 
Mitomycin C 0.5 mg/kg 138 
Cyclophosphamide 20 mg/kg 158 
______________________________________ 
TABLE 6-2 
______________________________________ 
Average 
Survival 
Test Substance Daily Dose 
Days (%) 
______________________________________ 
CB.sub.X3 *.sup.(1) 
1 unit/kg 110 
3 unit/kg 131 
10 unit/kg 178 
CB.sub.X3 *.sup.(2) 
1 unit/kg 116 
3 unit/kg 129 
10 unit/kg 176 
Mitomycin C 0.5 mg/kg 140 
______________________________________ 
(Notes) 
*.sup.(1) CB.sub.X3 obtained in Example 26 
*.sup.(2) CB.sub.X3 obtained in Example 29 
As clearly seen in the results above, CB evidently showed an anti-tumor 
effect on the mice bearing mouse melanoma B 16. 
Experiment 6: Influence on the Lung Metastasis of Cancer 
Groups of BDF.sub.1 -strain male mice weighing 20-30 g, 6 animals in each 
group, had transplanted subcutaneously into their backs 2 mm square 
segments of Lewis's lung cancer. CB.sub.X obtained in Example 6, CB.sub.X1 
obtained in Example 15, CB.sub.X2 obtained in Example 20 and CBF obtained 
were administered intravenously once a day for 12 days from the 9th day 
after transplantation. On the 21st day after transplantation, the mass of 
primary tumor was isolated and weighed, and the number of the metastasized 
nodes in the animal's lungs was calculated according to the method of 
Wexler, H. (J. Natl. Cancer Institute, Vol. 36 641 (1966)). The results 
are set forth in Tables 7-1 to 7-2. 
TABLE 7-1 
______________________________________ 
No. of 
Tumor Metasta- 
Experi- Weight sized Nodes 
ment Test substance 
Daily Dose 
(g) in Lung 
______________________________________ 
1 Control 9.6 .+-. 1.9 
29.2 .+-. 7.3 
CB.sub.X 4 unit/kg 
5.1 .+-. 1.6* 
6.8 .+-. 3.2* 
40 unit/kg 
3.0 .+-. 0.3** 
0.4 .+-. 0.4** 
Cyclophos- 20 unit/kg 
3.4 .+-. 0.7** 
0.4 .+-. 0.2** 
phamide 
2 Control 7.3 .+-. 0.3 
29.2 .+-. 1.4 
CB.sub.X 4 unit/kg 
4.1 .+-. 1.2* 
6.7 .+-. 2.1* 
40 unit/kg 
2.3 .+-. 0.2** 
0.5 .+-. 0.2** 
CBF 4 unit/kg 
6.6 .+-. 0.6 
22.0 .+-. 5.0 
40 unit/kg 
4.2 .+-. 0.4* 
21.4 .+-. 4.5 
______________________________________ 
TABLE 7-2 
______________________________________ 
Tumor No. of Metasta- 
Weight sized Nodes in 
Test substance 
Daily Dose 
(g) Lung 
______________________________________ 
Control 7.8 .+-. 0.5 
29.6 .+-. 1. 
CB.sub.X1 3 unit/kg 
4.3 .+-. 1.3* 
7.3 .+-. 2.0* 
30 unit/kg 
2.4 .+-. 0.3** 
0.5 .+-. 0.3** 
CB.sub.X2 3 unit/kg 
4.9 .+-. 1.3* 
7.1 .+-. 1.9* 
30 unit/kg 
2.1 .+-. 0.5** 
0.7 .+-. 0.3** 
CBF 3 unit/kg 
6.8 .+-. 0.6 
23.0 .+-. 5.2 
30 unit/kg 
4.3 .+-. 0.5* 
22.4 .+-. 4.4 
Cyclophosphamide 
20 unit/kg 
3.5 .+-. 0.6** 
0.6 .+-. 0.3** 
______________________________________ 
(Notes) 
The results in the tables are expressed as (average) .+-. (standard error 
*Statistically different from the control group at a significance level o 
p .ltoreq. 5%. 
**Statistically different from the control group at a significance level 
of p .ltoreq. 1%. 
As clearly seen in the results above, CB.sub.X very successfully suppressed 
the primary lung cancer and its lung metastases, whereas CBF had almost no 
effect on the lung metastases. 
Experiment 7: Effect on Inducing Differentiation of Tumor Cells 
According to the method of Hozumi, M. et al (Cancer Research, Vol, 40, 
2919-2924 (1980)), 5.times.10.sup.5 cells of acute myelogenous leukemia 
cells M-1 (supplied from Dr. Motoo Hozumi, Saitama Cancer Center) were 
suspended in 1 ml of Eagle's medium containing 10% calf serum and also 
containing amino acids and vitamins in amounts twice the ordinary levels, 
to which each test substance had been added, and cultured at 37.degree. C. 
for 48 hours in a 5% CO.sub.2, 95% air atmosphere. Thereafter, the cells 
were resuspended in a medium containing 0.2% polystyrene latex particles 
(Dow Chemical Co.) and incubated at 37.degree. C. for 4 hours. Then the 
total number of cells which phagocytotized the particles and the total 
number of cells were counted under a light microscope, and the 
differentiation rate was calculated from the ratio of these cells. The 
results are set forth in Table 8. 
TABLE 8 
______________________________________ 
Differentiation 
Test Substance 
Concentration 
Rate (%) 
______________________________________ 
Control 1 
CB.sub.X 0.004 unit/ml 8 
0.04 unit/ml 11 
0.4 unit/ml 18 
Dexamethasone 
20.0 ng/ml 25 
______________________________________ 
CB.sub.X exhibited the effect on inducing differentiation. 
Experiment 8: Pyrogen Test 
According to the method described in the Japanese Pharmacopeia, CB.sub.X 
obtained in Example 3 was intravenously administered to white rabbits at a 
dose of 100 units per animal. The results of measurement of the rectal 
temperature up to 3 hours later using a thermocouple type thermometer are 
set forth in Table 9. 
TABLE 9 
______________________________________ 
Rectal Temperature pre- and post-CB.sub.X 
Injection (.degree.C.) 
Weight Before 1 hour 2 hours 
3 hours 
Rabbit 
(kg) Injection 
Later Later Later 
______________________________________ 
A 2.0 38.90 38.70 38.80 38.80 
B 2.0 38.90 38.90 38.97 38.97 
C 2.0 39.25 39.25 39.22 39.30 
______________________________________ 
Experiment 9: Influence on Breast Cancer bearing Mice 
BALB/C strain nude mice weighing 20-25 g, 6 animals in each group, had 
transplanted subcutaneously into their backs 2 mm square segments of human 
breast cancer MX-1. CB.sub.X3 obtained in Example 26 or 29 was 
intravenously administered into the mice for 14 days from the 14th day 
after the transplantation. On the 15th day after the first administration, 
the volume of the primary tumor was measured. The results are set forth in 
Table 10. 
TABLE 10 
______________________________________ 
Tumor Volume*.sup.(1) 
Test substance 
Daily Dose 
(cm.sup.3) 
______________________________________ 
Control 9.7 .+-. 2.2 
CB.sub.X3 *.sup.(2) 
1 unit/kg 7.6 .+-. 1.3 
3 unit/kg 4.3 .+-. 1.3* 
10 unit/kg 2.2 .+-. 0.9** 
CB.sub.X3 *.sup.(3) 
1 unit/kg 7.3 .+-. 1.2 
3 unit/kg 4.6 .+-. 1.4* 
10 unit/kg 2.0 .+-. 1.0** 
Mitomycin C 0.5 mg/kg 4.4 .+-. 1.1* 
______________________________________ 
(Notes) 
*.sup.(1) (Average Value) .+-. (Standard Error) 
*.sup.(2) CB.sub.X3 obtained in Example 26 
*.sup.(3) CB.sub.X3 obtained in Example 29 
*Statistically different from the control group at a significance level o 
p .ltoreq. 5%. 
**Statistically different from the control group at a significance level 
of p .ltoreq. 1%. 
Experiment 10: Influence on Methylcholanthrene-Induced Tumor 
3-Methylcholanthrene dissolved in olive oil was subcutaneously injected to 
the lateral abdomen of ddY-strain mice, weighing 20-25 g, 8 animals in 
each group, at 0.5 mg per mouse. CB.sub.X3 obtained in Example 26 or 29 
was intravenously administered to the mice once a day for 21 days from 
about 60 days after the 3-Methylcholanthren injection. On the 21st day 
after the first CB.sub.X3 administration, the volume of the tumor was 
measured. The results are set forth in Table 11. 
TABLE 11 
______________________________________ 
Tumor Volume*.sup.(1) 
Test Substance 
Daily Dose 
(cm.sup.3) 
______________________________________ 
Control 10.5 .+-. 2.3 
CB.sub.X3 *.sup.(2) 
1 unit/kg 8.5 .+-. 1.4 
3 unit/kg 5.4 .+-. 1.4* 
10 unit/kg 2.5 .+-. 0.7** 
CB.sub.X3 *.sup.(3) 
1 unit/kg 8.9 .+-. 1.5 
3 unit/kg 5.1 .+-. 1.3* 
10 unit/kg 2.2 .+-. 0.8** 
Mitomycin C 0.5 mg/kg 6.6 .+-. 1.3* 
______________________________________ 
(Notes) 
*.sup.(1) (Average Value) .+-. (Standard Error) 
*.sup.(2) CB.sub.X3 obtained in Example 26 
*.sup.(3) CB.sub.X3 obtained in Example 29 
*Statistically different from the control group at a signifcance level of 
p .ltoreq. 5%. 
**Statistically different from the control group at a significance level 
of p .ltoreq. 1%. 
As clear from the above results, CB.sub.X3 evidently shows an anti-tumor 
effect on spontaneous tumors. 
Experiment 11: Toxicity Test (Single Administration) 
BDF.sub.1 -strain male mice weighing 20-25 g, 10 animals in each group, 
were intravenously administered the CB and the number of dead animals was 
observed for 7 days. As a result, all 10 animals survived without showing 
any change in body weight and general conditions, even when administered 
10,000 unit/kg of CB.sub.X, CB.sub.X1, or CB.sub.X2 or 100,000 unit/kg of 
CB.sub.X3. 
Experiment 12: Toxicity Test (30-Day Continuous Administration) 
BDF.sub.1 -strain male mice weighing 20-25 g, 10 animals in each group, 
were intravenously administered the CB for 30 days, and the number of dead 
animals, the change in body weight and the general conditions were 
observed. The body weight was weighed between 9 a.m. and 10 a.m., and the 
observation of the general conditions was conducted on the 10th, 20th and 
30th days according to the method of Arvien (Science, Vol. 36, 123 
(1962)). As a result, there was no dead animal in these 30 days when 1,000 
unit/kg/day of CB.sub.X, CB.sub.X1 or CB.sub.X2, or 10,000 unit/kg/day of 
CB.sub.X3 was administered, and the weight gain curve was more or less the 
same as that of the control group. Further, the general conditions were 
found to be normal as in the control group. 
As can be seen in the experiments described above, CB selectively 
suppresses the growth of tumor cells, and moreover, it not only remarkably 
suppresses the cancer metastasis but also is extremely effective against 
various tumors and is still very safe even at doses higher than the dose 
at which the pharmaceutical effect would be manifest. Therefore, CB is 
extremely useful for therapy of various tumors such as stomach cancer, 
lung cancer, hepatoma, colon cancer, breast cancer, uterus cancer, 
leukemia, etc. 
CB may be administered in the form of conventional preparations, such as 
injections, eye drops, nasal drops, inhalants, topical preparations, oral 
preparations, rectal preparations, vaginal preparations etc. The daily 
therapeutic dose of CB for an adult is not particularly restricted because 
of the high safety thereof, but generally it is 0.5-500,000 units, 
preferably 0.5-5,000 units for topical application, 20-100,000 units for 
systemic administration, such as intravenous injection, intramuscular 
injection etc., and 50-500,000 units for oral administration. The dose may 
be suitably adjusted depending on the method of use or the severity of the 
diseases. The preparation may contain each of CB.sub.X, CB.sub.X1, 
CB.sub.X2 and CB.sub.X3 alone or in combination with each other in any 
desired ratio. 
CB may be formulated into pharmaceutical preparations by any conventional 
method using pharmaceutically acceptable carriers, bases, excipients, etc. 
Preferably, it is employed as an oral preparation such as an enteric 
preparation, e.g. capsules, tablets, powder, etc.; a rectal preparation 
such as a rectal suppository, an injection such as an aqueous injection, 
or reconstitutable preparation of lyophilized powder for dissolution in 
distilled water for injection before use; and a topical preparation such 
as an ointment, lotion etc. In addition, it may be used as eye drops, 
nasal drops, or inhalants. 
Examples of solid carriers and excipients usable advantageously herein 
include common excipients such as lactose, mannitol, corn starch and 
potato starch; binders such as crystalline cellulose, cellulose 
derivatives, arabic gum, corn starch and gelatin; disintegrators such as 
corn starch, potato starch and calcium carbohydroxymethylcellulose; and 
lubricants such as talc and magnesium stearate. Examples of liquid 
carriers usable advantageously herein include distilled water for 
injection, physiological saline solution, vegetable oils for injection and 
glycols such as propylene glycol and polyethylene glycol. 
Examples are given below, but this invention is not intended to be 
restricted thereto. 
EXAMPLE 1 
Human lymphocytes (2.times.10.sup.10 cells) were suspended in 4,000 ml of 
Eagle's medium containing 10% calf serum and cultured at 37.degree. C. for 
48 hours in a 5% CO.sub.2, 95% air atmosphere. Thereafter, the supernatant 
of the culture medium was dialyzed against 0.01M phosphate buffer (pH 
7.2), and a fraction salted out with 40-80% ammonium sulfate was obtained 
from the dialyzate. This fraction was redialyzed against said phosphate 
buffer and then subjected to gel filtration using Sephadex G-100 
(Pharmacia Co.). A fraction of a molecular weight of 12,000-17,000 was 
collected, which was designated as the crude CB.sub.X fraction, while the 
earlier eluted fraction was designated as the crude CBF fraction. The 
crude CB.sub.X fraction was adsorbed on Ulex europeus agglutinin (Maruzen 
Oil Co.)-conjugated Sephadex, eluted with 0.01M phosphate buffer 
containing 0.5M fucose. After removing fucose by dialysis, CB.sub.X was 
adsorbed again on the Ulex europeus agglutinin-conjugated Sephadex, 
followed by elution by gradient method using phosphate buffer (pH 7.2), 
whereby purified CB.sub.X was eluted. A total of 0.02 mg of CB.sub.X was 
obtained. The total activity of the obtained CB.sub.X was 150 units as 
determined by the above described method. Thus the specific activity of 
the purified CB.sub.X was 7,500 unit/mg. 
EXAMPLE 2 
Bovine lymphocytes (2.times.10.sup.9 cells) were suspended in 1,000 ml of 
Eagle's medium containing 10% calf serum and cultured at 37.degree. C. for 
48 hours in a 5% CO.sub.2, 95% air atmosphere. Thereafter, the supernatant 
of the culture medium was subjected to the procedures in Example 1 for 
purification of CB.sub.X and 0.01 mg of CB.sub.X was obtained. The total 
activity of the obtained CB.sub.X was 20 units thus the specific activity 
of the purified CB.sub.X was 2,000 unit/mg. 
EXAMPLE 3 
Mouse lymphocytes (5.times.10.sup.10 cells) were suspended in 5,000 ml of 
Eagle's medium containing 10% calf serum, and cultured at 37.degree. C. 
for 48 hours in a 5% CO.sub.2, 95% air atmosphere. Thereafter, the 
supernatant of culture medium was subjected to the procedures in Example 1 
for purification of CB.sub.X, and 0.12 mg of CB.sub.X was obtained. The 
total activity of the obtained CB.sub.X was 400 units, thus the specific 
activity of the purified CB.sub.X was 3,333 unit/mg. 
EXAMPLE 4 
BALL-1 cells (human cell line, 1.times.10.sup.10 cells), which had been 
precultured, were suspended in 2,000 ml of Eagle's medium containing 10% 
calf serum, and cultured at 37.degree. C. for 48 hours in a 5% CO.sub.2, 
95% air atmosphere. Thereafter, the supernatant of the culture medium was 
subjected to the procedures in Example 1 for purification of CB.sub.X, and 
0.7 mg of CB.sub.X was obtained. The total activity of the obtained 
CB.sub.X was 4,000 units, thus the specific activity of the purified 
CB.sub.X was 5,714 unit/mg. 
EXAMPLE 5 
Flow 7000 cells (human fibroblasts line, 3.times.10.sup.9 cells), which had 
been grown by cell culture, were suspended in 600 ml of Eagle's medium 
containing 10% calf serum, and cultured at 37.degree. C. for 48 hours in a 
5% CO.sub.2, 95% air atmosphere. Thereafter, the supernatant of the 
culture medium was subjected to the procedures in Example 1 for 
purification of CB.sub.X, and 0.005 mg of CB.sub.X was obtained. The total 
activity of the obtained CB.sub.X was 10 units, thus the specific activity 
of the purified CB.sub.X was 2,000 unit/mg. 
EXAMPLE 6 
Human lymphocytes (2.times.10.sup.10 cells) were suspended in 4,000 ml of 
Eagle's medium containing 10% calf serum, and after adding 
phytohemagglutinin (Difco Co.) at a final concentration of 50 .mu.g/ml, 
cultured at 37.degree. C. for 48 hours in a 5% CO.sub.2, 95% air 
atmosphere. Thereafter, the supernatant of the culture medium was 
subjected to the procedures in Example 1 for purification of CB.sub.X, and 
1.0 mg of CB.sub.X was obtained. The total activity of the obtained 
CB.sub.X was 10,000 units, thus the specific activity of the purified 
CB.sub.X was 10,000 unit/mg. 
EXAMPLE 7 
Flow 7000 cells human fibroblasts line, 3.times.10.sup.9 cells) were 
suspended in 600 ml of Eagle's medium containing 10% calf serum, and after 
adding phytohemagglutinin at a final concentration of 50 .mu.g/ml, 
cultured at 37.degree. C. for 48 hours in a 5% CO.sub.2, 95% air 
atmosphere. Thereafter, the supernatant of the culture medium was 
subjected to the procedures in Example 1 for purification of CB.sub.X, and 
60 .mu.g of CB.sub.X was obtained. The total activity of the obtained 
CB.sub.X was 480 units, thus the specific activity of the purified 
CB.sub.X was 8,000 unit/mg. 
EXAMPLE 8 
TALL-1 cells (human cell line, 9.times.10.sup.9), which had been grown by 
cell culture, were suspended in 800 ml of Eagle's medium containing 10% 
calf serum, and after adding phytohemagglutinin at a final concentration 
of 50 .mu.g/ml, cultured at 37.degree. C. for 48 hours in a 5% CO.sub.2, 
95% air atmosphere. Thereafter, the supernatant of the culture medium was 
subjected to the procedures in Example 1 for purification of CB.sub.X, and 
0.8 mg of CB.sub.X was obtained. The total activity of the obtained 
CB.sub.X was 7,500 units, thus the specific activity of the purified 
CB.sub.X was 9,375 unit/mg. 
EXAMPLE 9 
Adult mice were pre-treated by irradiating with X-ray of about 400 REM to 
suppress their immune responses, and then TALL-1 cells (human origin) were 
transplanted into them subcutaneously. Thereafter, the mice were fed for 3 
weeks. The mass of tumor that subcutaneously formed weighing about 10 g 
was isolated, minced and dissociated in a physiological saline solution 
containing trypsin, then the dispersed cells were collected. These cells 
were treated according to the method in Example 1 to obtain CB.sub.X. The 
yield of CB.sub.X was about 190 units per mouse. 
EXAMPLE 10 
BALL-1 cells (human cell line, 9.times.10.sup.9 cells) were suspended in 
1,800 ml of Eagle's medium containing 10% calf serum, and, after adding 
9.times.10.sup.6 pfu (plaque forming units) of Sendai virus (HVJ), 
cultured at 37.degree. C. for 48 hours in a 5% CO.sub.2, 95% air 
atmosphere. Thereafter, the supernatant of the culture medium was 
subjected to the procedures in Example 1 for purification of CB.sub.X, and 
720 .mu.g of CB.sub.X was obtained. The total activity of the obtained 
CB.sub.X was 7,920 units, thus the specific activity of the purified 
CB.sub.X was 11,000 unit/mg. 
CB.sub.X obtained above was dissolved in physiological saline at a 
concentration of 1 mg/ml, and optical rotation of the solution at 598 nm 
(Na. D line) was measured at 26.5.degree.-28.5.degree. C. by a polarimeter 
(Nihon Bunko DIP-181) using a microcell of 10 mm in light path. The 
optical rotation of physiological saline as a control was assured to be 
zero. CB.sub.X showed levo-rotation. 
CB.sub.X (10 .mu.g) obtained above was prepared into a microtablet with 
potassium bromide powder to measure IR spectrum of CB.sub.X. The 
integrated measurement (60 times) was carried out by Fourier transform 
infrared spectrophotometer fX-6201 (Analect Instruments Co.). The result 
is shown in FIG. 1. 
EXAMPLE 11 
BALL-1 cells (human origin) were transplanted subcutaneously into adult 
nude mice which were then fed for 3 weeks. The resultant mass of tumor 
that formed subcutaneously weighing about 10 g each was isolated, minced, 
and then dissociated in a physiological saline solution containing 
trypsin, after which the dispersed cells were collected. These cells were 
washed with Eagle's medium containing 5% human serum, then 
2.times.10.sup.9 cells thereof were suspended in 2,000 ml of the same 
medium and cultured at 37.degree. C. for 48 hours in a 5% CO.sub.2, 95% 
air atmosphere. Thereafter, the supernatant of the culture medium was 
subjected to the procedures in Example 1 to obtain CB.sub.X. The yield of 
CB.sub.X was about 200 units per nude mouse. 
EXAMPLE 12 
JBL cells (human cell line) were suspended in physiological saline, and the 
suspension was then placed in a plastic cylindrical diffusion chamber 
having a capacity of about 10 ml and fitted with a membrane filter having 
a pore size of about 0.5 microns, and this chamber was placed in the 
peritoneal cavity of an adult rat. The rat was fed for 4 weeks, and the 
chamber was removed therefrom. 
The cell concentration of the human cells thus obtained was found to be 
about 5.times.10.sup.9 cells per ml, which represents about 10.sup.3 fold 
or more times the concentrations obtained by culturation in vitro in a 
nutrient medium in a 5% CO.sub.2, 95% air atmosphere. 
A total of 1.times.10.sup.10 JBL cells obtained by the method described 
above were suspended in 4,000 ml of Eagle's medium containing 10% calf 
serum, and cultured at 37.degree. C. for 48 hours in a 5% CO.sub.2, 95% 
air atmosphere. Thereafter, the supernatant of the culture medium was 
subjected to the procedures in Example 1 to obtain CB.sub.X. The yield of 
CB.sub.X was about 350 units per rat. 
EXAMPLE 13 
BALL-1 cells (human origin) were transplanted subcutaneously into adult 
nude mice, which were then fed for 5 weeks. Thereafter, each mouse was 
intraperitoneally injected with 1 mg of phytohemagglutinin, and sacrificed 
24 hours after the injection, and ascites was collected. The ascites were 
centrifuged at 4.degree. C. and 1,000 g, and the obtained supernatant was 
dialyzed against a physiological saline solution containing 0.01M 
phosphate buffer (pH 7.2) for 15 hours. The solution was further 
ultrafiltered with a membrane filter and the filtrate was concentrated to 
obtain a solution containing CB.sub.X. The amount of CB.sub.X was about 
8,000 units per nude mouse. 
EXAMPLE 14 
NALL-1 cells (human cell line) were suspended in physiological saline and 
poured into a plastic cylindrical diffusion chamber having a capacity of 
about 10 ml and fitted with a membrane filter with a pore size of about 
0.5 microns, and this chamber was placed in the peritoneal cavity of an 
adult rat. This rat was fed for 4 weeks, and then the chamber was removed. 
The cells thus grown were washed with Eagle's medium containing 5% human 
serum, and resuspended in the same medium at a cell concentration of about 
5.times.10.sup.6 cells per ml. The suspension was supplemented with about 
200 .mu.g/ml of phytohemagglutinin, and the mixture was incubated at 
37.degree. C. for 2 days to induce the production of CB.sub.X. CB.sub.X 
thus produced was purified and concentrated as described in Example 1, and 
it was further lyophilized to obtain a powder of CB.sub.X. The yield of 
CB.sub.X was about 15,000 units per rat. 
EXAMPLE 15 
According to the procedures described in Example 6, human lymphocytes were 
cultured, and the supernatant of the culture medium was subjected to 
purification to obtain a purified fraction with a molecular weight of 
70,000-90,000. This fraction was designated as the CB.sub.X1. The total 
activity of the 0.1 mg of purified CB.sub.X1 was 5,000 units, thus the 
specific activity of the purified CB.sub.X1 was 50,000 unit/mg. 
Optical rotation of CB.sub.X1 obtained above was measured as described in 
Example 10. CB.sub.X1 showed dextro-rotation. 
IR measurement of CB.sub.X1 obtained above was carried out as described in 
Example 10. The result is shown in FIG. 2. 
EXAMPLE 16 
According to the procedures described in Example 10, BALL-1 cells were 
cultured, and the supernatant of the culture medium was subjected to 
purification to obtain a purified CB.sub.X1. The total activity of the 100 
.mu.g of purified CB.sub.X1 obtained was 4,200 units, thus the specific 
activity of the purified CB.sub.X1 was 42,000 unit/mg. 
EXAMPLE 17 
According to the procedures described in Example 7, Flow 7000 cells were 
cultured, and the supernatant of the culture medium was subjected to 
purification to obtain purified 10 .mu.g of CB.sub.X1. The total activity 
of the obtained CB.sub.X1 was 250 units, thus the specific activity of the 
purified CB.sub.X1 was 25,000 unit/mg. 
EXAMPLE 18 
According to the procedures described in Example 2, bovine lymphocytes were 
cultured, and the supernatant of the culture medium was subjected to 
purification to obtain 0.002 mg of purified CB.sub.X1. The total activity 
of the obtained CB.sub.X1 was 14 units, thus the specific activity of the 
purified CB.sub.X1 was 7,000 unit/mg. 
EXAMPLE 19 
According to the procedures described in Example 4, BALL-1 cells were 
cultured, and the supernatant of the culture medium was subjected to 
purification to obtain 0.2 mg of purified CB.sub.X1. The total activity of 
the obtained CB.sub.X1 was 2,300 units, thus the specific activity of the 
purified CB.sub.X1 was 11,500 unit/mg. 
EXAMPLE 20 
According to the procedures described in Example 6, human lymphocytes were 
cultured, and the supernatant of the culture medium was subjected to 
purification to obtain a purified fraction with a molecular weight of 
40,000-50,000. This fraction was designated as the CB.sub.X2. The total 
activity of the 0.25 mg of purified CB.sub.X2 obtained was 5,200 units, 
thus the specific activity of the purified CB.sub.X2 was 20,800 unit/mg. 
EXAMPLE 21 
According to the procedures described in Example 10, BALL-1 cells were 
cultured, and the supernatant of the culture medium was subjected to 
purification to obtain 75 .mu.g of purified CB.sub.X2. The total activity 
of the obtained CB.sub.X2 was 10,000 units, thus the specific activity of 
the purified CB.sub.X2 was 133,333 unit/mg. 
Optical rotation of CB.sub.X2 obtained above was measured as described in 
Example 10. CB.sub.X2 showed dextro-rotation. 
IR measurement of CB.sub.X2 obtained above was carried out as described in 
Example 10. The result is shown in FIG. 3. 
EXAMPLE 22 
According to the procedures described in Example 7, Flow 7000 cells were 
cultured, and the supernatant of the culture medium was subjected to 
purification to obtain 20 .mu.g of purified CB.sub.X2. The total activity 
of the obtained CB.sub.X2 was 500 units, thus the specific activity of the 
purified CB.sub.X2 was 25,000 unit/mg. 
EXAMPLE 23 
According to the procedures described in Example 2, bovine lymphocytes were 
cultured, and supernatant of the culture medium was subjected to 
purification to obtain 0.001 mg of purified CB.sub.X2. The total activity 
of the obtained CB.sub.X2 was 40 units, thus the specific activity of the 
purified CB.sub.X2 was 40,000 unit/mg. 
EXAMPLE 24 
According to the procedures described in Example 4, BALL-1 cells were 
cultured, and supernatant of the culture medium was subjected to 
purification to obtain 0.25 mg of purified CB.sub.X2. The total activity 
of the obtained CB.sub.X2 was 2,900 units, thus the specific activity of 
the purified CB.sub.X2 was 11,600 unit/mg. 
EXAMPLE 25 
Human lymphocytes (2.times.10.sup.10 cells) were suspended in 4000 ml of 
Eagle's medium containing 10% calf serum, and after adding 
phytohemagglutinin at a concentration of 50 .mu.g/ml the suspension was 
cultured at 37.degree. C. for 48 hours in a 5% CO.sub.2, 95% air 
atmosphere. Thereafter, the supernatant of the culture medium was dialyzed 
against 0.01M phosphate buffer (pH 7.2), and a fraction which was salted 
out with 40-80% ammonium sulfate was obtained from the dialyzate. This 
fraction was dialyzed again against said phosphate buffer and then 
subjected to gel filtration using Sephadex G-100 to obtain a fraction 
having a molecular weight of 7,000-9,000, which was designated as the 
crude CB.sub.X3 fraction. 
The crude CB.sub.X3 fraction was adsorbed on phytohemagglutinin-conjugated 
Sephalose, eluted with 0.01M phosphate buffer (pH 7.2) containing 0.5M 
N-acetyl-D-galactosamine. After removing N-acetyl-D-galactosamine by 
dialysis, the resultant solution was applied to carboxymethylcellulose 
equilibrated with 0.05M phosphate buffer (pH 6.4), followed by elution 
with 0.5M phosphate buffer (pH 6.4) containing 0.5M sodium chloride. Thus, 
0.1 mg of CB.sub.X3 was obtained. The total activity of the obtained 
CB.sub.X3 was 5,000 units. 
EXAMPLE 26 
Newborn hamsters were pre-treated by injection with antiserum prepared from 
rabbit in a conventional method so as to reduce their immune responses as 
much as possible, and then had transplanted subcuntaneously into them 
BALL-1 cells. They were then fed for 3 weeks. The mass of tumors that 
formed subcutaneously and weighing about 15 g was isolated, minced and 
dissociated in physiological saline. After washing the obtained cells with 
serum-free Eagle's medium, 1.times.10.sup.11 cells thereof were suspended 
in 150 l of Eagle's medium containing 10% calf serum, and, after adding 
9.times.10.sup.6 pfu of Sendai virus (HVJ), cultured at 37.degree. C. for 
48 hours in a 5% CO.sub.2, 95% air atmosphere. The supernatant of the 
culture medium was dialyzed against 0.01M phosphate buffer (pH 7.2) and a 
fraction which was salted out with 40-80% ammonium sulfate was obtained 
from the dialyzate. This fraction was dialyzed again against said 
phosphate buffer and then subjected to gel filtration using Sephadex G-100 
obtain a fraction having a molecular weight of 7,000-9,000, which was 
designated as the crude CB.sub.X3 fraction. This crude CB.sub.X3 fraction 
was adsorbed on concanavalin A-conjugated Sephalose, and eluted with 0.01M 
phosphate buffer (pH 7.2) containing 0.5M .alpha.-methyl-D-mannoside. 
After removing the .alpha.-methyl-D-mannoside by dialysis, the solution 
was applied to carboxymethylcellulose equilibrated with 0.5M phosphate 
buffer (pH 6.0), followed by elution with 0.5M phosphate buffer (pH 7.8). 
The total activity of the 0.2 mg of CB.sub.X3 obtained was 12,000 units, 
and its isoelectric point was 6.3-7.8. 
EXAMPLE 27 
Flow 7000 cells (3.times.10.sup.10 cells) were suspended in 1.0 l of 
Eagle's medium containing 10% calf serum, and, after adding 
phytohemagglutinin at a final concentration of 50 .mu.g/ml, cultured at 
37.degree. C. for 48 hours in a 5% CO.sub.2, 95% air atmosphere. The 
supernatant of the culture medium was subjected to the procedures in 
Example 26 for purification of CB.sub.X3, and 0.1 mg of CB.sub.X3 was 
obtained. The total activity of the obtained CB.sub.X3 was 3,100 units. 
EXAMPLE 28 
Bovine lymphocytes (5.times.10.sup.10 cells) were suspended in 10 l of 
Eagle's medium containing 10% calf serum, and cultured at 37.degree. C. 
for 48 hours in a 5% CO.sub.2, 95% air atmosphere. Thereafter, the 
supernatant of culture medium was subjected to the procedures in Example 
26 for purification of CB.sub.X3, and 0.1 mg of purified CB.sub.X3 was 
obtained. The total activity of the obtained CB.sub.X3 was 1,700 units. 
EXAMPLE 29 
BALL-1 (5.times.10.sup.11 cells), which had been grown by cell culture, 
were suspended in 100 l of Eagle's medium containing 10% calf serum, and 
cultured at 37.degree. C. for 48 hours in a 5% CO.sub.2, 95% air 
atmosphere. Thereafter, the supernatant of culture medium was dialyzed 
against 0.01M phosphate buffer (pH 7.2), and a fraction which was salted 
out with 40-80% ammonium sulfate was obtained. This fraction was dialyzed 
again against said phosphate buffer and then subjected to gel filtration 
using Sephadex G-100 to obtain a fraction with a molecular weight of 
7,000-9,000. This fraction was adsorbed on phytohemagglutinin-conjugated 
Sephalose, and eluted with 0.01M phosphate buffer (pH 7.2) containing 0.5M 
N-acetyl-D-galactosamine. After removing the N-acetyl-D-galactosamine by 
dialysis, the dialyzed solution was applied to carboxymethylcellulose 
equilibrated with 0.05M Tris buffer (pH 8.0), followed by elution with 
0.05M Tris buffer (pH 8.0) containing 0.5M sodium chloride, whereby 0.1 mg 
of purified CB.sub.X3 was obtained. The total activity of CB.sub.X3 
obtained was 8,200 units and its isoelectric point was 8.0-9.2. 
Optical rotation of CB.sub.X3 obtained above was measured as described in 
Example 10. CB.sub.X3 did not show optical rotation. 
IR measurement of CB.sub.X3 obtained above was carried out as described in 
Example 10. The result is shown in FIG. 4. 
EXAMPLE 30: (AQUEOUS INJECTIONS) 
______________________________________ 
CB.sub.X 100,000 units 
Sodium chloride 9 g 
Distilled water for injection to make 
1,000 ml 
______________________________________ 
The CB.sub.X and sodium chloride were weighed and mixed, then dissolved in 
500 ml of distilled water for injection, and the total volume was adjusted 
to 1,000 ml with distilled water for injection. This aqueous solution was 
filtered under sterile conditions using a membrane filter, and 2 ml each 
of the filtrate was placed into sterilized glass containers and sealed to 
prepare aqueous injections. 
EXAMPLE 31-33 
Procedures similar to those in Example 30 were carried out for CB.sub.X1, 
CB.sub.X2 and CB.sub.X3 to prepare aqueous injections respectively. 
EXAMPLE 34: (LYOPHILIZED INJECTIONS) 
______________________________________ 
CB.sub.X 100,000 units 
20% Human serum albumin 10 ml 
Sodium chloride 9 g 
Distilled water for injecton to make 
1,000 ml 
______________________________________ 
The CB.sub.X and sodium chloride were weighed and mixed, then dissolved in 
a solution obtained by adding the predetermined amount of the human 
albumin to 500 ml of distilled water for injection, and the total volume 
was adjusted to 1,000 ml with distilled water for injection. This solution 
was filtered under sterile conditions with a membrane filter, and 2 ml 
each of the filtrate was placed into sterilized glass containers, 
lyophilized, and sealed to prepare lyophilized powders for injection. 
EXAMPLE 35-37 
Procedures similar to those in Example 34 were carried out for CB.sub.X1, 
CB.sub.X2 and CB.sub.X3 to prepare lyophylized powders for injection 
respectively. 
EXAMPLE 38: (EYE DROP) 
______________________________________ 
CB.sub.X 100,000 units 
Sodium chloride 5 g 
Chlorobutanol 5 g 
Distilled water for injection to make 
1,000 ml 
______________________________________ 
The above ingredients were weighed and dissolved in 950 ml of distilled 
water for injection. The total volume was adjusted to 1,000 ml, and the 
solution was filtered under sterile conditions using a membrane filter to 
make an eye drop preparation. 
EXAMPLE 39-41 
Procedures similar to those in Example 38 were carried out for CB.sub.X1, 
CB.sub.X2 and CB.sub.X3 to make eye drop preparations respectively. 
EXAMPLE 42: (SUPPOSITORIES) 
______________________________________ 
CB.sub.X 100,000 units 
Polyethylene glycol 1500 
250 g 
Polyethylene glycol 4000 
Ca. 750 g 
1,000 g 
______________________________________ 
The above ingredients were weighed and the whole amounts of the CB.sub.X 
and polyethlene glycol 1500 and 500 g of the polyethylene glycol 4000 were 
mixed thoroughly, after which the remaining polyethylene glycol 4000 was 
added to give the total weight of 1,000 g, further mixed thoroughly and 
made into 5,000 mg rectal suppositories by the melting method. 
EXAMPLE 43-45 
Procedures similar to those in Example 42 were carried out for CB.sub.X1, 
CB.sub.X2 and CB.sub.X3 to prepare rectal suppositories respectively. 
EXAMPLE 46: (NASAL DROP) 
______________________________________ 
CB.sub.X 100,000 units 
Sodium chloride 5 g 
Chlorobutanol 5 g 
Distilled water to make 
1,000 ml 
______________________________________ 
The above ingredients were weighed and dissolved in 950 ml of distilled 
water. The resultant solution was adjusted to the total volume of 1,000 ml 
with distilled water to prepare a solution for nasal drop. 
EXAMPLE 47-49 
Procedures similar to those in Example 46 were carried out for CB.sub.X1, 
CB.sub.X2 and CB.sub.X3 to prepare a solutions for nasal drop 
respectively. 
EXAMPLE 50: (ENTERIC COATED TABLETS) 
______________________________________ 
CB.sub.X 1,000,000 
units 
Lactose 64 g 
Potato starch ca. 30 g 
Polyvinyl alcohol 3 g 
Magnesium stearate 3 g 
100 g 
______________________________________ 
The above ingredients were weighed respectively, the whole of the CB.sub.X 
and lactose and about half amount of the potato starch were mixed; then 
the remaining potato starch was added to a mixture so as to give the total 
weight of 94 g; and the mixture was mixed to achieve homogenity. To the 
resultant mixture was added an aqueous polyvinyl alcohol solution, and 
granules were prepared by the wet pelletizing method. The granules were 
dried, mixed with the magnesium stearate, and compressed into 200 mg 
tablets. The tablets were coated with methyl cellulose phthalate to 
prepare enteric coated tablets. 
EXAMPLE 51-53 
Procedures similar to those in Example 50 were carried out for CB.sub.X1, 
CB.sub.X2 and CB.sub.X3 to prepare enteric coated tablets respectively. 
EXAMPLE 54: (OINTMENT) 
______________________________________ 
CB.sub.x 100,000 units 
Liquid paraffin 10 g 
Vaseline (Trademark) ca. 1,000 
g 
1,000 g 
______________________________________ 
The above ingredients were weighed respectively, then the CB.sub.X was 
thoroughly kneaded with the liquid paraffin, 500 g of the Vaseline 
(Trademark) was added thereto, and mixed thoroughly. To the mixture was 
gradually added the remaining Vaseline (Trademark) to give the total 
weight of 1,000 g, and the mixture was thoroughly mixed to prepare an 
ointment. 
EXAMPLE 55-57 
Procedures similar to those in Example 54 were carried out for CB.sub.X1, 
CB.sub.X2 and CB.sub.X3 to prepare ointments respectively.