Biological response modifier having cell differentiation-induction activity and process for production thereof

There is provided a proteinaceous biological response modifier having the following properties: (a) molecular weight: 35,000 to 65,000; (b) isoelectric point: 5.0 to 6.1; (c) physiological action on human leukemia cells: to induce human leukemia to differentiate into macrophage-like cells; (d) physiological action on myeloid leukemia cells from mice: to induce myeloid leukemia cells from mice to differentiate into macrophage-like cells; (e) affinity to Concanavalin A Sepharose: not adsorbed; (f) affinity to Blue Sepharose Resin: not adsorbed; (g) pH-stability and thermostability: substantially not inactivated at pH 2 to 10, at 2.degree. C. for 6 hours; not inactivated at 56.degree. C. for 60 minutes; but inactivated by 30% at 70.degree. C. for 60 minutes; (h) sensitivity to enzymes: not inactivated by deoxyribonuclease; not inactivated by glycosidase; and inactivated by protease; (i) flow cytometry analysis: to concentrate cell division cycle of human leukemia cells to G.sub.0 /G.sub.1 phase. There is also provided a process for the production of the proteinaceous biological response modifier comprising culturing human leukemia cells in a differentiation medium in the presence of a substance capable of inducing the human leukemia cells to differentiate into macrophage-like cells; separating the macrophage-like cells from the culture medium; activating the macrophage-like cells in a production medium by a mitogen to enhance the production of the proteinaceous biological response modifier; and isolating the proteinaceous biological response modifier from the production medium. The biological response modifier has anti-tumor activity.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates to a novel purified biological response 
modifier having cell differentiation-induction activity which is herein 
referred to as Differentiation and Activation Factor and abbreviated as 
DAF and a process for the production thereof. 
2. Description of the Prior Art 
A phenomenon of cell differentiation is a process where cells capable of 
division and growth change into functionally matured cells responsible for 
the functions of an organism. For example, the processes are well known 
where hematopoietic stem cells constantly differentiate into matured blood 
cells such as leukocytes, erythrocytes, monocytes and macrophages. This 
differentiation and maturation process is controlled by various kinds of 
biological response modifiers, resulting in maintenance of the homeostasis 
of an organism. 
Conversely it is believed that leukemia cells are derived from 
hematopoietic stem cells, wherein the hematopoietic stem cells are 
tumorized into growing cells during the normal differentiation and 
maturation process of the stem cells. 
If such leukemia cells could be induced to redifferentiate into cells 
having normal functions, a new therapy of leukemia could be established on 
the basis of the differentiation principle. It is from such a point of 
view that recently, in addition to the conventional chemotherapy, 
radiation therapy and immunotherapy, a new type of therapy which 
detumorizes cancer cells into normal cells by redifferentiation of the 
cancer cells, has been actively investigated. 
Proteinaceous substances from mice which induce myeloid leukemia cells to 
differentiate and detumorize into macrophage-like cells were reported as a 
D-factor [J. Cell. Physiol., 74, 223 (1969)]. The D-factors obtained from 
the culture of murine embryo cells are considered to be glycoprotein with 
a molecular weight of 40,000 to 50,000 [Gann, 68, 435 (1977)]. When a 
laboratory strain of myeloid leukemia cells from mice was implanted in a 
mouse and the D-factors derived from murine embryo cells were 
administered, the implanted myeloid leukemia cells were induced to 
redifferentiate into normal cells, and a life-prolongation effect on the 
mouse was observed [Gan to Kagaku Ryoho, Vol 8, No. 1, 8 (1981)]. Thus the 
literature as mentioned above suggests the possibility of cancer therapy 
with these differentiation-induction factors. 
However, as the D-factors are proteinaceous substances derived from mice, 
they could cause an allergic response in a human body when administered. 
Therefore it is desirable to obtain a differentiation-induction factor 
derived from human stock. Proteinaceous differentiation-induction factors 
derived from human stock were produced by stimulation of the human 
peripheral lymphocyte with lectins, according to the J. National Cancer 
Institute, 67, 1225 (1981), Cancer Research, 42, 3928 (1982). However, 
none of the substantial properties of the differentiation-induction 
factors were described except that the factors were proteinaceous 
substances with a molecular weight of 25,000 and 40,000, as determined by 
gel filtration using Sephadex G-75 (Pharmacia Fine Chemicals A.B., 
Sweden). 
Conversely differentiation-induction factors found in the supernatant of a 
culture of human T-cell leukemia cells were reported to be proteinaceous 
substances with a molecular weight of 50,000 to 60,000, as determined by 
acrylamide gel electrophoresis. The activity of the factors was lost by 
about 60% to 90% during the isolation procedures [Summary of Japan Tissue 
Culture Communication, 43, (1983)]. 
As described above, regarding the differentiation-induction factors derived 
from human peripheral lymphocytes and human T-cell leukemia cells, only 
the molecular weights have been reported and other properties of the 
factors have not been reported since these factors have not been obtained 
in an amount sufficient to allow the investigation of their properties. 
As human peripheral lymphocytes can be obtained only from human blood, it 
is difficult to collect a large amount of the differentiation-induction 
factors from human peripheral lymphocytes and accordingly their industrial 
production is by no means easy. Also, since the maintenance of safety is 
expensive during the industrial culture of human T-cell leukemia cells 
which are infected with human T-cell leukemia virus (HTLV) and are 
proliferative, an economical production of the factors from human T-cell 
leukemia cells is very difficult. 
On the other hand, macrophages are known to produce and secrete many kinds 
of biological response modifiers and are important for immune response. 
However, as human functional macrophages cannot be cultured, industrial 
production of these modifiers is also impossible. 
Accordingly, it is desirable to establish a process for the production of a 
proteinaceous biological response modifier having 
differentiation-induction activity, wherein an established cell line 
capable of proliferating in vitro and not including a leukemia virus is 
used. 
SUMMARY OF THE INVENTION 
In accordance with the present invention, there is provided a new purified 
proteinaceous biological response modifier with differentiation-induction 
activity, having the following properties: 
(a) molecular weight: 35,000 to 65,000; 
(b) isoelectric point: 5.0 to 6.1; 
(c) physiological action on human leukemia cells: to induce human leukemia 
to differentiate into macrophage-like cells; 
(d) physiological action on myeloid leukemia cells from mice: to induce 
myeloid leukemia cells from mice to differentiate into macrophage-like 
cells; 
(e) affinity to concanavalin A Sepharose: not adsorbed; 
(f) affinity to Blue Sepharose resin: not adsorbed; 
(g) pH-stability and thermostability: substantially not inactivated at pH 2 
to 10, at 2.degree. C. for 6 hours; not inactivated at 56.degree. C. for 
60 minutes; but inactivated by 30% at 70.degree. C. for 60 minutes; 
(h) sensitivity to enzymes: not inactivated by deoxyribonuclease; not 
inactivated by ribonuclease; not inactivated by glycosidase; but 
inactivated by protease; 
(i) flow cytometry analysis: to concentrate cell division cycle of human 
leukemia cells to G.sub.0 /G.sub.1 phase. 
In accordance with the present invention, there is also provided a process 
for the production of the proteinaceous biological response modifier 
comprising: culturing human leukemia cells in a differentiation medium in 
the presence of a substance capable of inducing the human leukemia cells 
to differentiate into macrophage-like cells; separating the 
macrophage-like cells from the differentiation medium; activating the 
macrophage-like cells in a production medium by a mitogen to enhance the 
production of the proteinaceous biological response modifier; and 
isolating the proteinaceous biological response modifier from the 
production medium. 
Preferably, prior to culturing human leukemia cells in the differentiation 
medium, the cells are proliferated by culturing them in a proliferation 
medium.

DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The human differentiation and activation factor (DAF) according to the 
present invention is produced by human macrophage-like cells, and is 
capable of at least inducing human leukemia cells to differentiate into 
terminally differentiated functionally matured cells, resulting in 
inhibition of cell growth of the cells, and finally for the leukemia cells 
to die in vitro. According to a process of the present invention, a large 
amount of DAF can be produced by culturing a large amount of cells in an 
industrial cell culturing apparatus. 
Assay Method 
The DAF of the present invention was quantitatively analyzed by an in vitro 
assay method established by the inventors, wherein the extent of the 
differentiation-induction of human monocytic leukemia cell line THP-1 into 
macrophage-like cells was measured. A sample is diluted until it becomes 
two-fold of the volume, step by step, with a culture medium. Each 0.1 ml 
of a series of the diluted samples is added into each well of a flat 
bottom type microplate (Falcon). THP-1 cells are cultured for 2 days in 
RPMI-1640 medium supplemented with 10% of fetal calf serum (FCS), at a 
temperature of 37.degree. C..+-.0.5.degree. C. in an incubator conditioned 
to a 5% carbon dioxide concentration (5% CO.sub.2 -incubator). 
The cultured THP-1 cells are then washed with a phosphate buffer saline 
which is Ca.sup.++ and Mg.sup.++ free [PBS(-)]and suspended in RPMI-1640 
medium supplemented with 5% of FCS to a concentration of 2.times.10.sup.5 
cells/ml. Then 0.1 ml of the suspension is added to the each well of the 
microplate and cultured at 37.degree. C. in the 5% CO.sub.2 -incubator for 
48 hours. After cultivation, suspended cells are removed from the well, 
leaving the cells adhered to the bottom of the well. The microplate is 
rinsed twice with PBS(-) gently, and 0.1 ml of 0.5% gentian violet 
staining solution is then added to each well to stain the adhered cells. 
The microplate is thoroughly washed with tap water to remove excess dye, 
and dried after decanting the water. 0.1 ml of 2-methoxyethanol is added 
to each well of the microplate to extract the dye from the cells. After 
about one hour the difference between absorbance at 550 nm and 414 nm of 
the extract is measured by a double beam method using Titertek Multiskan 
(Flow Laboratories, U.S.A.). The absorptions for the samples are 
compensated by the absorptions for the controls. 
The absorption corresponds to the number of macrophage-like cells adhered 
to the bottom of each well of the microplate. 
The activity of DAF in a diluted sample providing an absorption of 0.1 is 
defined as one unit/ml. For example, if a diluted sample prepared by 
dilution of an original sample to a 32-fold volume provides an absorption 
of 0.1, the activity of the original sample is 32 units/ml. 
Properties of DAF 
(a) Molecular weight: 35,000 to 65,000. 
The molecular weight was determined by fractionating a sample by gel 
filtration with Sephadex G-100 equilibrated with Dulbecco's phosphate 
buffer solution (0.2 g/l potassium chloride, 0.2 g/ml potassium 
dihydrophosphate, 8 g/l sodium chloride, 1.15 g/l disodium phosphate, pH 
7.4), and comparing the DAF active band with standard proteins having a 
known molecular weight. 
(b) Isoelectric point: 5.0 to 6.1. 
The isoelectric point was determined by fractionating a sample by 
isoelectric chromatography according to a chromatofocusing method using a 
poly buffer exchanger (Pharmacia) and identifying the DAF active band. 
(c) Physiological action on human leukemia cells: to induce human leukemia 
cells to differentiate into macrophage-like cells. 
Human monocyte leukemia cells (THP-1) were cultured in the RPMI-1640 medium 
containing 10% FCS at 37.degree. C. in the 5% CO.sub.2 -incubation, the 
cultured cells were washed throughout with PBS(-), and the washed cells 
were suspended in the RPMI-1640 medium containing 5% FCS to the 
concentration of 2.times.10.sup.5 cells/ml medium. 0.5 ml of the 
suspension was added to each well of a 24 well flat bottom type cell 
culture plate (Falcon), 0.1 ml of the DAF sample and the same amount of 
the control solution were added to different wells and the cells were 
cultured in the 5% CO.sub.2 -incubation at 37.degree. C. for 4 days. 
Human promyelocytic leukemia cells (HL-60) were also cultured in the same 
manner as described above. 
Table 1 shows the degree of cell growth of the human leukemia cells. The 
addition of DAF caused the human leukemia cells to be induced to 
differentiate into macrophage-like cells, resulting in inhibition of cell 
growth of the cells. Conversely the human leukemia cells not treated with 
DAF were myeloblast-like and showed a higher cell concentration. 
TABLE 1 
__________________________________________________________________________ 
Strain of human 
leukemia cell 
Observation 
Presence of DAF 
Absence of DAF 
__________________________________________________________________________ 
THP-1 Number of cells 
2.1 .times. 10.sup.5 cells/ml 
10.7 .times. 10.sup.5 cells/ml 
Morphology 
Macrophage-like cells 
Myeloblast-like cells 
HL-60 Number of cells 
3.4 .times. 10.sup.5 cells/ml 
17.7 .times. 10.sup.5 cells/ml 
Morphology 
Macrophage-like cells 
Myeloblast-like cells 
__________________________________________________________________________ 
After culturing for 4 days, the properties of the human leukemia cells were 
tested. In the presence of DAF the human leukemia cells differentiated to 
macrophage-like cells in biological property as shown in Table 2. 
TABLE 2 
__________________________________________________________________________ 
Strain of Phagocytosis 
Candi- 
NBT- 
human leu- of dacidal 
reducing 
Adhesion 
Acid .beta.-glu- 
kemia cells 
DAF latex beads 
activity 
activity 
to vessel 
phosphatase 
curonidase 
__________________________________________________________________________ 
THP-1 Presence 
++++ +++ +++ ++++ +++ ++ 
Absence 
.+-. - .+-. - + + 
HL-60 Presence 
++++ ++ +++ ++++ +++ ++ 
Absence 
.+-. - .+-. - + + 
__________________________________________________________________________ 
Symbols -, .+-.,+ to ++++ show the degree of activity or ability. 
The above-mentioned properties, i.e., phagocytosis of polystyrene latex 
beads, candidacidal activity, adhesion to vessel, nitroblue tetrazolium 
dye (NBT) reducing activity, acid phosphatase activity and 
.beta.-glucuronidase activity are used as characteristics for the 
identification of macrophages, as described in, for example, Manual of 
Macrophage Methodology, Marcel Dekker Inc. New York, 1981. 
Comparisons between cells (THP-1) treated with DAF and cells (THP-1) not 
treated with DAF for NBT reducing activity and acid phosphatase activity 
are shown in FIGS. 1 and 2, respectively. As shown in the figures, the NBT 
reducing activity and the acid phosphatase activity increase with the time 
elapsed for the treated cells, but do not increase for the not treated 
cells. 
(d) Physiological action on myeloid leukemia cells from mice: to induce 
myeloid leukemia cells from mice to differentiate into macrophage-like 
cells. 
Myeloid leukemia cells M-1 from mice [J. Cell Physiol. 74,223 (1969)] were 
prepared by being cultured in the RPMI-1640 medium supplemented with 10% 
of horse serum for 3 days, and the cells were then suspended in the 
RPMI-1640 medium supplemented with 5% of horse serum to the concentration 
of 2.times.10.sup.5 cells/ml. 
The same tests were carried out as in the preceding paragraph (c), and 
showed the results as set forth in Table 3. The M-1 cells were induced to 
differentiate into macrophage-like cells in the presence of DAF, but were 
not induced in the absence of DAF. 
TABLE 3 
__________________________________________________________________________ 
Cell number Phagocytosis 
NBT-reducing 
Adhesion 
Acid .beta.-glu-curonidase 
DAF (cells/ml) 
Morphology 
of latex beads 
activity 
to vessel 
phosphatase 
activity 
__________________________________________________________________________ 
Presence 
4.4 .times. 10.sup.5 
Macrophage-like 
++++ +++ +++ ++ +++ 
cells 
Absence 
23.5 .times. 10.sup.5 
Myeloblast-like 
- .+-. - + + 
cells 
__________________________________________________________________________ 
Culture for 4 days 
Symbols -, .+-., + to ++++ show the degree of activity or ability. 
FIG. 3 shows the amount of increase of .beta.-glucuronidase activity 
produced by macrophage-like cells derived from the M-1 cells when the M-1 
cells were cultured in the presence of DAF for 4 days. 
Other properties of DAF of the present invention are described as follows: 
(1) When a PBS(-) solution containing DAF (173 units/ml) was added to a 
column of concanavalin A-Sepharose (Pharmacia) and the column was washed 
with PBS(-), the DAF was not adsorbed on the concanavalin A-Sepharose and 
passed through with the PBS(-). 
(2) When a PBS(-) solution containing DAF (173 units/ml) was added to a 
column of BlueSepharose CL-6B (Pharmacia) and the column was washed with 
PBS(-), the DAF was not adsorbed on the BlueSepharose CL-6B and passed 
through with the PBS(-). 
(3) The pH-stability and thermostability were tested. DAF was dissolved in 
the buffer solutions as set forth in Table 4, the solutions were allowed 
to stand at 2.degree. C. for 6 hours, neutralized to pH 7.4, and the 
residual activity of the DAF was measured. 
In addition, DAF was diluted in a phosphate buffer solution of pH 7.4 and 
the solutions were heated under different conditions. The results are 
shown in Tables 4 and 5. 
TABLE 4 
______________________________________ 
Residual 
activity 
pH Buffer solution (%) 
______________________________________ 
2.10 Citrate buffer 104 
4.08 Citrate-phosphate buffer 
96 
6.09 Citrate-phosphate buffer 
102 
7.40 Phosphate buffer 
100 
7.98 Tris-HCl buffer 91 
10.22 Carbonate buffer 
84 
______________________________________ 
TABLE 5 
______________________________________ 
Temperature Time Residual activity 
(.degree.C.) (minutes) 
(%) 
______________________________________ 
56 60 83 
70 15 94 
70 60 70 
96 3 91 
______________________________________ 
(4) Sensitivity to enzymes was measured according to a conventional method 
in which DAF was diluted in the buffer solutions as set forth in Table 6, 
subjected to the action of protease, nucleases or glycosidase and the 
residual activity of the DAF was measured. The results are shown in Table 
6. 
TABLE 6 
______________________________________ 
Residual 
Enzyme Buffer solution activity (%) 
______________________________________ 
DNase Tris-HCl buffer pH 7.5 
99 
(Deoxyribonuclease I) 
RNase Tris-HCl buffer pH 7.5 
95 
(Ribonuclease A) 
Glycosidase Sodium acetate buffer 
96 
(Neuraminidase) 
pH 5.0 
Protease Phosphate buffer not more 
(Pronase E) pH 7.4 than 6 
______________________________________ 
(5) The effect of DAF on the cell division cycle was tested by flow 
cytometry. DAF was diluted into the RPMI-1640 medium supplemented with 5% 
FCS to the concentration of 85 units/ml. Human monocytic leukemia cells 
were cultured in the medium prepared as above at 37.degree. C. for 3 days 
in the 5% CO.sub.2 -incubator. Nuclei in the cells were stained with 
acrydineorange dye, and analysed with a cell sorter system (Coulter 
Electronics). The DAF concentrated cell division phase to G.sub.0 /G.sub.1 
phase as shown in FIG. 4. The detailed analysis method is described in, 
for example, Proc. Natl. Acad. Sci., 73, 2881 (1976). 
Process for Production of DAF 
The process for the production of DAF according to the present invention 
comprises four steps, i.e., (i) a differentiation step where human 
leukemia cells are cultured in a differentiation medium in the presence of 
an inducer, to differentiate into macrophage-like cells; (ii) a separation 
step where the macrophage-like cells are separated from the 
differentiation medium; (iii) a production step where the macrophage-like 
cells are activated in a production medium by a mitogen to produce the 
target product DAF; and (iv) an isolation step where the DAF is isolated 
from the production medium. 
Although not essential for the present invention, a proliferation step 
where the starting human leukemia cells are proliferated in a 
proliferation medium to obtain a large amount of human leukemia cells may 
be preferably carried out prior to the differentiation step. 
(1) Human leukemia cells 
In the process of the present invention any kind of human leukemia cells 
which can be induced to differentiate into macrophage-like cells by an 
inducer described hereinafter can be employed. Such cells include leukemia 
cell lines derived from human stock, primary culture isolated from a human 
having leukemia and its established cell lines, which can be induced to 
differentiate into macrophage-like cells. To obtain a large amount of 
industrial culture, established cell lines capable of vigorous growth may 
be preferably used. More specifically, human monocytic leukemia THP-1 
cells [International J. Cancer 26, 171 (1980)], human promyelocytic 
leukemia HL-60 cells [Nature, 270, 347 (1977)], and human monocytic 
leukemia Mono-1 cells [Virchows Arch. A. Path. Anal. and Histol., 371, 15 
(1976)]etc., are preferably used. 
(2) Proliferation step 
As the proliferation medium, any medium which is usually employed for the 
culture of human cells can be utilized. Media including, for example, 
RPMI-1640 medium, Dulbecco's modified MEM medium, Eagle BME medium, Ham's 
medium, McCoy 5A medium, and Iscove's medium, etc., all of which are 
supplemented with about 0.5% to 20% of a serum such as fetal calf serum 
(FCS), newborn calf serum, calf serum and horse serum may be used. The 
compositions of such medium are described in Manual for Cell Culture, 
Kodan Sha, Japan, 1982. To prevent infection of the microorganisms, 
antibiotics such as 10 to 100 units/ml penicillin-G, 10 to 100 .mu.g/ml 
streptomycin sulfate and 40 to 60 g/ml kanamycin sulfate may be added to 
the medium. Additionally, to promote proliferation of the cells, the 
medium may be supplemented with cell growth promoting substances such as 
amino acids, vitamins, proteins such as albumin and casein, insulin and 
transferrin. To control the pH value and adjust the concentration of 
carbonate anion in the medium, buffering agents such as 10 to 60 mM 
4-(2-hydroxyethyl) -1-piperazinethansulphonate (HEPES) may be used. 
For the culturing of the cells, any vessel which allows growth of the cells 
can be used. 
When culturing the cells, human leukemia cells prepared as inoculum are 
suspended in the above-mentioned proliferation medium such as RPMI-1640 
medium supplemented with 10% FCS to the cell concentration of about 
1.times.10.sup.5 to 5.times.10.sup.5 cells/ml, preferably about 
2.times.10.sup.5 to 3.times.10.sup.5 cells/ml, and the cells are cultured 
for 2 to 6 days, preferably 3 to 4 days, at a temperature of 35.degree. to 
38.degree. C., preferably 37.degree. C., in a 5% CO.sub.2 -incubator. 
(3) Differentiation step 
The cells proliferated in the proliferation step are collected by a 
conventional method such as centrifugation. 
The cells thus obtained are then suspended in a differentiation medium. The 
differentiation medium is fundamentally the same as the proliferation 
medium, and a medium supplemented with 5% FCS is preferably used. The 
starting cell concentration is adjusted to typically about 
3.times.10.sup.5 to 30.times.10.sup.5 cells/ml, preferably about 
6.times.10.sup.5 to 18.times.10.sup.5 cells/ml. 
To the suspension, a substance capable of inducing human leukemia cells to 
differentiate into macrophage-like cells, i.e., an inducer, is then added. 
As the inducer, phorbolesters such as 12-0-tetradecanoylphorbol-13-acetate 
(TPA), phorbol-12,13-dibenzoate (PDB), and phorbol-12,13-didecanoate 
(PDD); and diterpene compound mezerein (MEZ) are used. The structures and 
properties of these compounds are described in Protein, Nucleic Acid and 
Enyzme, Vol 24, 999 (1979). The concentration of the inducer added to the 
medium is typically about 0.005 to 5 .mu.g/ml, preferably about 0.1 to 1 
.mu.g/ml. 
Differentiation culturing is carried out for about 1 to 72 hours, 
preferably 8 to 36 hours, at a temperature of 35 to 38.degree. C., 
preferably 37.degree. C. Macrophage-like cells differentiated from 
leukemia cells will adhere to the surface of a culture vessel. 
(4) Separation step 
The macrophage-like cells thus formed are separated from the 
differentiation medium by a conventional method such as centrifugation or 
filtration. Then the collected cells are thoroughly washed with an 
appropriate buffer solution such as PBS(-) to remove the differentiation 
medium from the macrophage-like cells. 
(5) Production step 
In this step the macrophage-like cells are activated by a mitogen to 
enhance the production of DAF. Note, without activation of the mitogen, a 
lower level of DAF is produced. 
The macrophage-like cells obtained in the separation step are then 
suspended in a production medium. 
The production medium can be substantially the same as the proliferation 
medium but in order to ensure easy purification of the DAF in the next 
isolation step, it is preferred that the production medium does not 
contain serum. To enhance the production of the DAF, the production medium 
may contain, in addition, about 0.1 to 10 mg/ml of protein such as albumin 
obtained from a calf or human serum. 
The mitogens which can be used as the activator include, for example, plant 
lectins and bacterial lipopolysaccharides. The lectins include 
succinylated concanavalin A, muramyldipeptide and phytohemagglutinin 
(Phaseolus vulgaris lectin). The bacterial lipopolysaccharides include 
those obtained from gram negative bacteria such as Escherichia coli, 
Serratia marcescens and Salmonella typhimurium. These mitogens are added 
to the medium in a concentration of typically about 1 to 50 .mu.g/ml, 
preferably about 2 to 10 g/ml. 
The production culturing is carried out at a temperature of about 
35.degree. C. to 38.degree. C., preferably 37.degree. C., for about 2 to 6 
days to produce DAF in the cultured medium. 
(6) Isolation step 
The cultured medium thus obtained is treated by a conventional method such 
as centrifugation or filtration to remove the cells and obtain a 
supernatant containing DAF. From the supernatant the DAF is isolated and 
purified according to the conventional biochemical isolation procedures 
such as ultrafiltration, salting out for concentration, dialysis for 
desalting, gel filtration, electrophoresis, ion exchange chromatography or 
a combination thereof. For example, the supernatant is concentrated by 
ultrafiltration, and the concentrate is fractionated by gel filtration 
with Sephacryl S-200 or S-300, or Sephadex G-75, G-100 or G-150 
(Pharmacia) to obtain active fractions. The combined active fraction is 
again concentrated by ultrafiltration, and the concentrate is fractionated 
with an anion exchanger such as DEAE-Sephadex, DEAE-Sepharose CL-6B or 
DEAE-Sephacel (Pharmacia) using a sodium chloride linear gradient elution 
method to obtain the active fraction. The combined active fraction is 
concentrated, desalted and lyophilized to obtain the product of this 
invention. 
Anti-tumor activity of DAF 
Tests were carried out to determine the anti-tumor activity of the DAF 
produced according to the above described process of the present 
invention. 
Several kinds of human leukemia cells were inoculated into RPMI-1640 medium 
supplemented with 10% FCS to a cell concentration of 1.0.times.10.sup.5 
cells/ml. To the cell suspension a phosphate buffer solution containing 93 
units/ml of DAF was added to the final concentration of 5 units/ml, and 
the cells were cultured at 37.degree. C. for 4 days in a 5% CO.sub.2 
-incubator. 
After cultivation, the number of cells was counted with a Coulter counter 
(Coulter Electronics, U.S.A.) and the percent depression of leukemia cell 
growth was calculated. The results are shown in Table 7. 
TABLE 7 
______________________________________ 
Number of Percent 
Human leukemia cells after de- 
cells Strain 4 days pression 
______________________________________ 
Human myelogenous 
K-562 1.10 .times. 10.sup.5 cells/ml 
0.65 
leukemia cells 
Human promyelocytic 
HL-60 0.95 -0.24 
leukemia cells 
Human monocytic 
THP-1 0.89 -0.76 
leukemia cells 
Human monocytic 
Mono-1 1.05 0.42 
leukemia cells 
______________________________________ 
##STR1## 
From the above described results, the DAF of the present invention is 
expected to act as an anti-tumor pharmaceutical on the basis of its 
differentiation-induction ability. Such a type of anti-tumor agent is not 
known at present. 
The following examples illustrate detailed embodiments of the present 
invention but in no way limit the invention. 
EXAMPLE 1 
A culture medium was prepared from RPMI-1640 medium powder by supplementing 
it with 10% of FCS (Flow), 50 units/ml of penicillin-G, 50 .mu.g/ml of 
streptomycin sulfate and 50 .mu.g/ml of kanamycin sulfate. Human monocytic 
leukemia cell line THP-1 cells were suspended in the medium to a cell 
concentration of 2.times.10.sup.5 cells/ml, and 10 ml of the suspension 
was added to each of 3 plastic culture vessels having a diameter of 10 cm 
(Falcon). After culturing at 37.degree. C..+-.0.5.degree. C. in a 5% 
CO.sub.2 -incubation for 4 days, the cell concentration reached 
12.times.10.sup.5 cells/ml. 
The cells were collected by centrifugation at 1000 rpm for 5 minutes, and 
resuspended in fresh RPMI-1640 medium supplemented with 5% of FCS to a 
cell concentration of 6.times.10.sup.5 cells/ml, and 10 ml of the 
suspension was added to each of 6 plastic culture vessels having a 
diameter of 10 cm. To each culture vessel, 0, 0.01, 0.05, 0.1, 0.5, and 10 
g/ml of TPA was added, and the cells were cultured to differentiate into 
macrophage-like cells for 20 hours at 37.degree. C..+-.0.5.degree. C. in a 
5% CO.sub.2 -incubator. 
The cells in each vessel were separately collected by centrifugation, 
thoroughly washed with PBS(-), and resuspended in 10 ml of RPMI-1640 
medium supplemented with 5% of FSC. 
To each suspension was added 20 .mu.g/ml of succinylated concanavalin A 
obtained from jack beans (E.Y. Laboratories) and the suspension was 
cultured at 37.degree. C..+-.0.5.degree. C. in a 5% CO.sub.2 -incubator 
for 4 days. The cultured cell suspension was then centrifuged to remove 
the cells. The obtained supernatant was subjected to an in vitro assay for 
DAF. The results are shown in Table 8. 
TABLE 8 
______________________________________ 
Concentration of TPA 
DAF produced 
.mu.g/ml units/ml 
______________________________________ 
0 4 
0.01 52 
0.05 70 
0.1 85 
0.5 103 
1.0 120 
______________________________________ 
EXAMPLE 2 
The same procedure for the cell proliferation step as in Example 1 was 
repeated to obtain THP-1 cells. 
For the differentiation step, the cells thus obtained were suspended in 350 
ml of RPMI-1640 medium supplemented with 5% of FCS to a cell concentration 
of 6.times.10.sup.5 cells/ml. The cell suspension was fractioned to to 10 
plastic culture vessels having a diameter of 15 cm (Falcon), and 
differentiation culturing was carried out by adding 0.2 .mu.g/ml of TPA to 
each vessel. 
The cells were collected by centrifugation, washed with PBS(-), and 
production culturing was carried out in the same manner as in Example 1 
for 4 days. 
The cultured medium was centrifuged to remove the cells, and 345 ml of a 
supernatant containing DAF was obtained. The supernatant was desalted with 
an ultrafilter (Millipore) and subjected to ion exchange chromatography 
with DEAE-Sepharose CL-6B (Pharmacia) equilibrated with a 10 mM HEPES 
buffer solution and using a 0 to 0.6 M sodium chloride concentration 
gradient elution to obtain active fractions. The combined active fractions 
were again desalted with an ultrafilter, subjected to gel filtration with 
Sephacryl S-200 (Pharmacia) equilibrated with pH 7.4 PBS(-) using PBS(-) 
as an eluent to obtain active fractions. The obtained fractions were 
measured for DAF activity. The active fractions showing a molecular weight 
of 35,000 to 65,000 were combined and concentrated with an ultrafilter to 
obtain 13 ml of the product containing 3,350 units of DAF according to 
this invention. 
EXAMPLE 3 
The same procedure for the cell proliferation step as in Example 1 was 
repeated to obtain THP-1 cells. 
For the differentiation step, the cells thus obtained were suspended in 
RPMI-1640 medium supplemented with 5% of FCS to a cell concentration of 
6.times.10.sup.5 cells/ml, and 10 ml of the cell suspension was put into 
each of 8 plastic culture vessels having a diameter of 10 cm. The 
differentiation culturing was carried out by adding the phorbol esters or 
mezerein as set forth in Table 9 to each vessel and culturing the cells 
for 24 hours. 
The production culturing was carried out in the same manner as in Example 
1, using 20 .mu.g/ml of succinylated concanavalin A. The supernatant 
obtained by centrifugation was subjected to an in vitro assay for DAF and 
the results shown in Table 9 were obtained. 
TABLE 9 
______________________________________ 
Concentration 
Differentiation 
of inducer DAF produced 
Inducer (.mu.g/ml) (units/ml) 
______________________________________ 
TPA 0.05 77 
0.5 115 
PDB 0.05 25 
0.5 153 
PDD 0.05 58 
0.5 89 
MEZ 0.05 111 
0.5 356 
______________________________________ 
EXAMPLE 4 
The same procedure for the cell proliferation step as in Example 1 was 
repeated to obtain the cells. For the differentiation step, the cells thus 
obtained were suspended in an Eagle's BME medium supplemented with 5% of 
FCS to a cell concentration of 8.times.10.sup.5 cells/ml, and 30 ml of the 
suspension was put into a plastic culture vessel having a diameter of 15 
cm. The differentiation culturing was carried out by adding 0.1 .mu.g/ml 
of TPA to the vessel for 24 hours at 37.degree. C. in a 5% CO.sub.2 
-incubator. 
For the production culturing, the cells were collected by centrifugation, 
thoroughly washed with PBS(-), resuspended in the Eagle's BME medium 
supplemented with 1 mg/ml of calf serum albumin, and 10 ml of the 
suspension was put into each of 3 plastic culture vessels having a 
diameter of 10 cm. After the addition of 5 .mu.g/ml of lipopolysaccharide 
obtained from the gram negative bacteria as set forth in Table 10 to the 
suspensions, the production culturing was carried out at 37.degree. C. for 
5 days in a 5% CO.sub.2 -incubator. The cultured media were centrifuged to 
remove the cells. The supernatants were subjected to an in vitro assay for 
DAF. The results are shown in Table 10. 
TABLE 10 
______________________________________ 
Origin of DAF produced 
lipopolysaccharide 
(units/ml) 
______________________________________ 
Escherichia coli 173 
Serratia marcescens 
181 
Salmonella typhimurium 
154 
______________________________________ 
EXAMPLE 5 
The same procedure for the cell proliferation step as in Example 1 was 
repeated to obtain the THP-1 cells. 
For the differentiation culturing, the cells thus obtained were suspended 
in RPMI-1640 medium supplemented with 5% of horse serum to a cell 
concentration of 8.times.10.sup.5 cells/ml. Then 0.2 .mu.g/ml of TPA was 
added to the suspension, and 10 ml of the suspension was put into each of 
4 plastic culture vessels having a diameter of 10 cm. The differentiation 
culturing was carried out at 37.degree. C. for 24 hours in a 5% CO.sub.2 
-incubator. 
For the production culturing, cells in each vessel were separately 
collected, washed with PBS(-) and resuspended in 10 ml of RPMI-1640 
supplemented with 1% of horse serum. To these suspensions the plant 
lectins and muramyldipeptide as set forth in Table 11 were added, and the 
production culturing was carried out at 37.degree. C. for 4 days in a 5% 
CO.sub.2 -incubator. The cultured media were centrifuged to remove the 
cells, and the supernatants obtained were subjected to an in vitro assay 
for DAF. The results are shown in Table 11. 
TABLE 11 
______________________________________ 
Concentration 
of lectin DAF produced 
Lectin (.mu.g/ml) (units/ml) 
______________________________________ 
Succinylated concanavalin A 
20 98 
Phytohemagglutinin 
20 79 
Muramyldipeptide 
20 95 
Not added -- 28 
______________________________________ 
EXAMPLE 6 
For the proliferation culturing, 150 ml of a medium having the composition 
as the one used in Example 1 was prepared in a 500 ml spinner flask 
(Bellco). Human promyelocytic leukemia cell line HL-60 cells were 
inoculated into the above-mentioned medium to a cell concentration of 
2.times.10.sup.5 cells/ml, and the flask was sealed. The cells were 
cultured at 37.degree. C. for 4 days with gentle stirring. 
For the differentiation culturing, the cultured cells were collected by 
centrifugation, suspended in fresh RPMI-1690 medium to a cell 
concentration of 12.times.10.sup.5 cells/ml, and 150 ml of the suspension 
was returned to the 500 ml spinner flask. Then 0.2 .mu.g/ml of mezerein 
was added to the flask which was then sealed. The differentiation 
culturing was carried out at 37.degree. C. for 20 hours with gentle 
stirring. 
For the production culturing, the cells were collected by centrifugation, 
thoroughly washed with PBS(-) and resuspended in 150 ml of RPMI-1640 
supplemented with 1 mg/ml of calf serum albumin, and simultaneously 10 
.mu.g/ml of succinylated concanavalin A was added to the suspension. The 
spinner flask containing the above described suspension was sealed, and 
the production culturing was carried out at 37.degree. C. for 4 days with 
gentle stirring. The medium cultured in the spinner flask contained 12,250 
units of DAF activity. 
EXAMPLE 7 
The cell proliferation culturing was carried out for 4 days in the same 
manner as in Example 1 to obtain human monocytic leukemia cell line Mono-1 
cells. 
For the differentiation culturing, the cells were suspended in fresh 
RPMI-1640 supplemented with 5% FCS to a cell concentration of 
8.times.10.sup.5 cells/ml. Then 30 ml of the suspension was put into each 
of 2 plastic culture vessels having a diameter of 15 cm, and 0.5 .mu.g/ml 
of TPA was added to each vessel, and the differentiation culturing was 
carried out at 37.degree. C. for 36 hours. 
For the production culturing, the cells in the each vessel were separately 
collected and resuspended in fresh RPMI-1640 medium. 10 .mu.g/ml of 
phytohemagglutinin was added to the vessel, and 5 .mu.g/ml of 
lipopolysaccharide obtained from E. coli was added to another vessel. The 
production culturing was carried out at 37.degree. C. for 3 days in a 5% 
CO.sub.2 -incubator. The cultured media were centrifuged to remove the 
cells, and the obtained supernatants were assayed for DAF activity. As a 
result, the culture added with phytohemagglutinin provided 31 units/ml of 
DAF activity and the culture added with lipopolysaccharide provided 15 
units/ml of DA activity.