JPS5888322A - Remedy for malignant tumor containing target cell lysis factor and its production - Google Patents

Abstract

PURPOSE:Human-originating B-lymphocyte germule-like cells, capable of producing a taget cell lysis factor, are cultured to produce the titled remedy in a large amount easily. CONSTITUTION:A target cell lsysis factor (TCLF) of 10,000-100,000 molecular weight, containing 5-45% glycosides, is contained to give a remedy for malignant tumors. The TCLF is prepared by inoculating human-originating B-lymphocyte germule-like cells directly in a body of a warm-blooded animals other than human such as chicken, pigeon, dog, cat, monkey or propagating them in a diffusion chamber in which the cells can be fed the humor of the animal and by making a TCLF inducer act on the cells. The use of a combination of alpha-interferon inducer and gamma-interferon inducer as a TCLF inducer causes remarkable increase in TCLF production.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a malignant tumor therapeutic agent containing a target cytotoxic factor and a method for producing the same.

Target cytotoxic factor (Target Ce1l I,y
sis Factor.

Hereinafter, it will simply be abbreviated as TCLF. ) is mouse L-9
29 cells are the target cells, and this cell is damaged (Cyto-
toxicity) and kill and destroy the cells (Cy
Lymphotoxin, Tumoa necrosis factor, etc. are known as factors that cause necrosis).

Lymphotoxin is a ``lymphokine'' co-authored by Takashi Aoki et al.
, New Immunology Series 6. 1979, Igaku Shoin, '13
1oom, B, R, & Glade, P,' R,
Co-edited r In vitr.

methods in cell-mediated
immunity J Academicpress
, 1971, etc., for example, antigens are allowed to act on sensitized TIJ lymphocytes, or lymphotoxin inducers such as phytohemagglutinin and concanavalin A are used as mitogens in TIJ lymphocytes.
It is a proteinaceous substance that is induced to be produced inside and outside the cell by acting on J lymphocyte cells, and is the name given to a substance that has a cytotoxic function. Lymphotoxin is known to damage not only mouse L-929 cells but also human tumor cells and normal human cells. Also, Tsumoa Necrosis Factor is Carswel
l.

E, et al, , Pr, Nat, Acad,
Sci, USA, Vol. 72, No. 9.8
pp. 666-8670 (1975) and E. Pick, ed. Tumor Necrosis Factor in
uLymphokines” Vol, It.

1) I), 285-272, Academic pr.
ess, 1981, for example, 13acillus Calmet in rabbits.
te Qujrin (BCG), Corynebac
By parenterally administering T. terium parvum, t-endotoxin, and other necrosis factor inducers, the proteinaceous substances induced and produced by macrophage cells in the serum, which have the ability to cause hemorrhagic necrosis in MethA sarcoma, are affected. This is the name given. It is known that the Tumor necrosis factor damages not only mouse L-929 cells but also human tumor cells, killing and destroying them, but does not substantially cause any damage to human normal cells.

In this way, T
CLF has been expected to be a therapeutic agent for malignant tumors since it has a cytotoxic function against tumor cells. T.C.
There is almost no species specificity in LF, so rabbits,
TCLF prepared from cells derived from various animals such as rats
However, for use in human treatment, it is best to use human living cell sources as they are extremely safe and free from concerns about side effects such as antigenicity that may occur during treatment.

The present inventor has discovered that human TC can be easily carried out on an industrial scale.
We investigated the method for producing LF and conducted intensive research to determine whether TCLF is useful as a therapeutic agent for malignant tumors.

As a result, by allowing a TCLF-inducing agent to act on B-lymphoblastoid cells obtained by proliferating human-derived B-lymphoblastoid cells established in a cultured line with TCLF-producing ability, TCLF was highly active. It was discovered that TCLF can be easily produced in large quantities by purifying and collecting the induced product, and the present invention was completed by confirming that TCLF is an excellent therapeutic agent for malignant tumors.

The TCLF produced in this way has a molecular weight of about 1 to 1.
It is clear that it exists in the range of 0,000,000 and contains at least eight types of glycoproteins, and its action is
It was found that -929 cells have the ability to damage and kill human tumor cells, but do not substantially damage normal human cells.

Hereinafter, the method for manufacturing TCLF of the present invention will be explained in detail.

The cultured human-derived B lymphoblastoid cells used in the present invention are cultured in vitro according to conventional methods.
Cells grown under o) can be used. however,
In the case of the present invention, upon proliferation of B lymphoblastoid cells,
It is preferable to use B'J lymphoblastoid cells that have been transplanted directly into the body of a warm-blooded animal other than humans or inoculated into a diffusion chamber and grown while receiving a supply of body fluids from the warm-blooded animal.

That is, unlike in vitro proliferation, not only is the need for a nutrient medium containing expensive serum etc. unnecessary or can be greatly reduced, but maintenance and management during cell proliferation is also extremely easy. Inducibly generated TCLF
It is characterized by high activity.

Methods using warm-blooded animals other than humans include transplanting cultured human-derived B IJ lymphoblastoid cells into the body of a warm-blooded animal other than humans, or receiving a supply of body fluids from that animal. If the cells are housed in a diffusion chamber that can be used in a warm-blooded animal, and this chamber is implanted within the animal's body and the animal is reared normally, its cells can easily proliferate using the nutrient-containing body fluids provided by the warm-blooded animal's body. It is. Furthermore, in vitro
Compared to the case of proliferation in vitro), the proliferation of these cells is stable, the proliferation rate is high, the amount of cells obtained is large, and the TC per cell is
Another major feature is that the yield of LF is significantly increased.

The cultured human-derived cells used in the present invention may be BIJ lymphoblastoid cells that can be transplanted into the body of a warm-blooded animal other than humans and easily proliferate, and that also produce TCLF. For example, r Journal of Clinic
al Microbiology J Vol, 1.
Na described on pages 116-117 (1975)
ma1wa cells, 1. Written by Miyoshir Natu
re J Vol, 267, pp. 848-844 (19
BALL-1 cells, described in Tissue Culture, Vol. 6, No. 13, pp. 527-546 (1980)
JBL cells, EBV-3a cells, described in
EBV-Wa cells, EBV-HO cells, and other BAL
M2 cells, CCRF-8B cells (ATCCCCCL 1
Established cell lines such as 20), and furthermore, cells prepared by treating normal 331J cell lines with various viruses, drugs, radiation, etc. and culturing them can be used freely.

In addition, the gene having the TCLF-producing ability of these B lymphoblastoid cells can be transferred by means of cell fusion using polyethylene glycol, Sendai virus, etc., or by DNAIJ.
Treatment with genetic recombination methods using enzymes such as gauze, restriction enzymes (nucleases), and DNA polymerases can increase the growth rate and increase the TC per cell.
Cells with enhanced LF production ability may be used, and are not limited to the established cell lines described herein.

These cells can be freely used alone or in combination of two or more types in the process of inducing and producing TCLF, which will be described later. If necessary, leukocytes prepared from fresh human sources, for example, can be used in combination.

The warm-blooded animals used in the present invention may be any animal in which human-derived cells can proliferate, such as birds such as chickens and pigeons, dogs, cats, monkeys, rabbits, goats, pigs, horses, cows, guinea pigs, Mammals such as rats, hamsters, normal mice, and nude mice can be used.

Transplanting human-derived B IJ lymphoblastoid cells into these animals may cause an unfavorable immune reaction, so in order to suppress this reaction as much as possible, the animals used should be kept as young as possible, i.e. eggs, embryos, etc. Fetuses, newborns, and infants are preferred.

Furthermore, these animals may be subjected to pretreatment such as irradiation with X-rays or gamma rays at a dose of about 200 to 600 rem, or injection of antiserum or immunosuppressants to weaken the immune reaction before transplantation.

If the animal used is a nude mouse, the immune response is weak even if the animal is grown, so these pretreatments are not necessary, and cultured human-derived B IJ
This is particularly advantageous because the emboblastoid cells can be transplanted and rapidly proliferated.

In addition, human-derived B lymphoblastoid cells that have been cultured are first transplanted into hamsters and grown, and then these cells are further transplanted into nude mice. It is also possible to stabilize the proliferation of human-derived B-lymphoblastoid cells by transplanting them, or to increase the amount of TCLF induced therefrom.

In this case, transplantation may be carried out not only between the same species and the same genus, but also between cages and between the same phylum. The site within the animal body to which the human-derived BIJ tumor-like cells are transplanted may be any site where the transplanted cells can proliferate, such as the allantoic cavity, vein, peritoneal cavity, or subcutaneous site.

In addition, a porous filtration membrane capable of passing and blocking animal cells without directly transplanting human-derived cells into an animal body, such as a membrane filter-1 ultrafiltration having a pore size of 10-7 to 10-'m, can be used. Diffusion chambers of various known shapes and sizes equipped with membranes or follower fibers are implanted in the animal body, for example in the abdominal cavity, to contain nutrients from the animal body.
While being supplied with body fluid, any of the human-derived BIJ blastoid cells cultivated as described above can be grown in the chamber.

In addition, if necessary, a chamber in which the solution containing nutrients in this chamber is connected to body fluids in the animal body for perfusion is attached to the surface of the animal body (C), for example, and human-derived B lymph buds in the chamber are attached. It is possible to see through the proliferation state of globular cells, and by making it possible to attach and detach only a portion of this chamber, it is possible to proliferate cells to the fullest lifespan without slaughtering the animal, thereby reducing the amount of cells produced per individual animal. It can also be increased further.

These methods using diffusion chambers not only allow for easy collection of only human-derived B IJ lymphoblastoid cells, but also are undesirable, since human-derived B IJ lymphoblastoid cells do not come into direct contact with animal cells. Since there is little fear of causing an immune reaction, there is no need for pretreatment to suppress the immune reaction, and various warm-blooded animals can be used freely.

The transplanted animal can be maintained and managed simply by continuing the normal care and management of the animal, and is convenient because no special handling is required even after transplantation.

The purpose can usually be achieved within a period of 1 to 10 weeks for proliferating human-derived B IJ lymphoblastoid cells. It has also been found that the number of human-derived B lymphoblastoid cells obtained in this manner reaches about 107 to 1012 or more per animal.

In other words, the number of human-derived B lymphoblastoid cells proliferated by the method for producing TCLF used in the present invention is about 102-107 times the number of cells transplanted into the animal's side, or more. It also reaches about 101 to 10641 when inoculated and grown in vitro in a nutrient medium! , or even more, which is extremely advantageous for the production of TCLF.

Any method can be used to induce and produce TCLF from the human-derived B lymphoblastoid cells grown in this manner. It is also possible to cause the TCLF inducer to act in the animal body in which it has grown. For example, a TCLF inducer is directly applied to human-derived B lymphoblastoid cells grown in suspension in ascites in the peritoneal cavity, or to tumor cells generated subcutaneously.
LF may be induced and produced, and then TCLF may be purified and collected from the serum, ascites, or tumor.

In addition, human-derived proliferating cells are removed from the body of a non-human animal and treated with a TCLF inducer in vitro to induce TCLF.
can also be generated by induction.

For example, human-derived BIJ tumor-like cells grown in ascites are collected, or human-derived BIJ cells grown subcutaneously are collected.
Tumors containing J. lymphoblastoid cells are excised and dispersed, and the resulting cells are suspended in a nutrient medium maintained at approximately 20 to 40°C at a cell concentration of approximately 105 to 10'/m. TCLF may be induced and produced by the action of a TCLF inducer, and then purified and collected.

Furthermore, when human-derived B IJ lymphoblastoid cells are grown in a diffusion chamber, the grown cells can be left in the chamber or removed from the chamber and treated with TC.
TCLF can also be induced and produced by applying an LF inducer.

In addition, for example, after inducing and producing TCLF in the animal's body in the proliferated human-derived B lymphoblastoid cells,
Next, TC is applied to human-derived B IJ tumor-like cells collected from a specific part or the whole animal from the same animal in vitro.
A method of inductively producing LF, a method of using cells once used for inducibly producing TCLF twice or more for inductively producing TCL-F, or cells obtained by implanting or replacing a chamber to be connected in an animal body It is also possible to further increase the amount of TCLF produced per individual animal used, such as by increasing the number of animals used.

Examples of the TCLF inducer of the present invention include viruses, nucleic acids, polynucleotides, etc. known as α-interferon inducers, phytohemagglutinin, concanavalin A1 pokeweed mitogen, and ribopolysaccharide known as r-interferon inducers. Lido, endotoxin, polysaccharide, bacteria, etc. are used as appropriate, but it is usually more convenient to use an α-interferon inducer because highly active TCLF can be induced and produced.

Furthermore, for sensitized psychic cells, antigens are also TCLF inducers.

Furthermore, TCLF was obtained from human-derived B IJ lymphoblastoid cells.
In the induced production of α-
It has been found that the combined use of an interferon inducer and an r-interferon inducer significantly increases the amount of TCLF produced.

Furthermore, it has been found that not only TCLF is produced by these induced productions, but also human interferon, which is highly species-specific, is produced at the same time.

This makes it possible to simultaneously produce two or more valuable human physiologically active substances, and also to make advanced use of human-derived B lymphoblastoid cells, and to produce TCLF and human interferon in large quantities at low cost. This is extremely convenient from the point of view of supply.

TCLF thus induced and produced can be purified by known purification and separation methods, such as salting out, dialysis, filtration, centrifugation, concentration,
It can be easily purified and separated by freeze-drying, etc.
Can be collected. If a higher level of purification is required, the highest purity TCLF can be obtained by combining known methods such as adsorption-elution to ion exchangers, gel filtration, isoelectric point fractionation, and electrophoresis. Although it is possible to collect the sample, affinity chromatography using antibodies,
Affinity chromatography using concanavalin A-Sepharose is very convenient because highly purified TCLF can be produced extremely simply and quickly.

The TCLF produced in this way has a molecular weight of about 1 to 1.
The existence of at least eight types of glycoproteins in the range of 0,000,000 to 20,000, approximately 10,000 to 20,000, approximately 50,000 to 50,000, and approximately 70,000 to 90,000 has been revealed, and their effects have been demonstrated not only in mouse L-929 cells but also in humans. It has been found that although it has the ability to damage and kill tumor cells, it does not substantially damage normal human cells.

Therefore, the TCLF of the present invention can be advantageously used as a therapeutic agent for various human malignant tumors, which have hitherto been considered extremely difficult to treat, among preventive and therapeutic agents for TCLF-sensitive diseases such as malignant tumors.

The activity of TCLF was determined by Bloom, B, R, & Gl.
ade P, R.

Co-edited In vitro methods in c
ell-mediateaimmufty JA
According to the method for lymphotoxin reported in Academic Press (1971), mouse L-929 cells were used as target cells, and the number of surviving cells after a certain period of time was measured.

Interferon activity with high species specificity in humans
White Matter Nucleic Acid Enzyme J Vol, 20. No, 6.616
It was measured by the known plaque half-life method using human amnion-derived F'L cells as reported in 1975, p.

Hemagglutination titer is determined by J, E, 5alk's Journal.
al of.

Jmmunology J Vol, 49.87 pages,
(1944).

Next, Experiment A regarding the production of TCLF will be described.

[Experiment A] TCLF production ability test using cells grown in vitro or in vivo Experiment A-1 Proliferation in vitro Human-derived BIJ-blast-like cells BALL -1 cells in RPMI 1640 medium (pH 72) supplemented with 20% fetal bovine serum.
) and cultured in suspension at 37°C.

The obtained cells were placed in serum-free RPMI 1640 medium (
pH 12) and suspended in the same medium to approximately 1 x 10 cells.

Experiment A-2 Proliferation in vivo After injecting antiserum prepared from rabbits by a known method into newborn hamsters to weaken the immune response of the hamsters,
BALL-1 cells were subcutaneously transplanted, and then raised for 8 weeks in the usual manner. The subcutaneous tumor was removed and cut into small pieces, and dispersed and loosened in physiological saline. The obtained cells were cultured in serum-free RPMI 1640 medium (1)I('i[
2) and suspended in the same medium to approximately lx 10 /mj.

Experiment A-8 Production of TCLF Induce TCLF by using Sendai virus or phytohemagglutinin alone or in combination with Sendai virus and phytohemagglutinin to the suspension of BALL-1 cells obtained in Experiments A-1 and A-2. generated. That is, approximately 1×
Sendai virus was added to a suspension having a cell concentration of 10 μm at a rate of about 300 hemagglutinin titers per
5. Mata, which was kept at ℃ for 2 days to induce the production of TCLF.
In the case of phytohemagglutinin, about 50 μg of cell suspension was added and kept at 87° C. for 2 days to induce TCLF production. In addition, when Sendai virus and phytohemagglutinin are used together, Sendai virus is added with a hemagglutinating value of approximately 800 per portion and phytohemagglutinin is added with approximately 50 μg per portion, and the mixture is kept at 87°C for 2 days to conduct TC.
LF was induced to be produced. At this time, large amounts of α-interferon and r-interferon were simultaneously produced in addition to TCLF.

The TCLF production results are shown in Table 1.

(Note) The numerical value indicates the TCLF activity of Gotoshi.

However, the numbers in parentheses indicate human interferon activity per -.

As is clear from the results in Table 1, TCLF is also produced from cells grown in vitro.

However, the amount of TCLF induced and produced from cells grown in vivo is greater, about 10 times or more than that induced and produced in vitro. Furthermore, TCLF induced and produced by Sendai virus is equivalent to or higher than when phytohemagglutinin is used. In addition, focusing on the TCLF activity induced and produced when the TCLF inducer is Sendai virus alone or phytohemagglutinin alone, and the TCLF activity induced and produced when Sendai virus and phytohemagglutinin are used together, it is found that A synergistic effect between Sendai virus and phytohemagglutinin is observed both when using cells grown in vivo and when cells grown in vivo are used.

The degree of the synergistic effect is particularly remarkable when using pneumatic cells grown in vivo.

As is clear from the experimental results described above, among interferon inducers with high species specificity, α-interferon inducers and r-
Concomitant use of interferon inducers is advantageous.

Next, a manufacturing example of TCLF will be described.

Example A-1 A newborn 71 muster was injected with antiserum prepared from a rabbit using a known method to weaken the immune response of the hamster, and then human-derived lymphoblastoid cells BALL-1 were subcutaneously injected into the hamster.
Cells were transplanted and then reared for 8 weeks in the usual manner. A subcutaneous tumor of about 15 f was removed, cut into small pieces, and dispersed and loosened in physiological saline. The obtained cells were subjected to serum-free R.
The cells were washed with PMI 1640 medium (pH 72) and suspended in the same medium at a concentration of approximately 5×10 6 /d. To this suspension, approximately 200 µt of Sendai virus per LOOO hemagglutination titer and phytohemagglutinin were added, and 87
The mixture was kept at ℃ for 2 days to induce the production of TCLF. This was centrifuged at about 4°C and about i,ooor to remove the precipitate.
The obtained supernatant was dialyzed for 21 hours against physiological saline containing pH 72 and +101M phosphate buffer, and the filtrate obtained by further microfiltration was concentrated and lyophilized to obtain a powder containing TCLF activity. Ta. The TCLF activity obtained was about aooo million units per Notamuster. This product contained approximately 2 million units of human interferon.

Example A-2 Human-derived lymphoblastoid BALL-1 cells were cultured in Eagle's minimal basal medium (pH
74) and in vitro (in v.
Itro) was cultured in suspension. The obtained cells were washed with serum-free Eagle's minimal basal medium (pH 74),
Approximately I x 107. It was suspended so that it became Od. Add Sendai virus to this suspension - approximately i,o o per
o, hemagglutination titer and concanavalin A - about 5 per
μ2 was added and kept at 88° C. for 1 day to induce TCLF production. This was centrifuged at 4°C and about t, ooor, and the resulting supernatant was dialyzed for 15 hours against physiological saline containing αOIMIJ phosphate buffer at pH'Z2, and the filtrate was obtained by further microfiltration. was concentrated to obtain a solution containing TCLF activity. The TCLF activity obtained was approximately 4.5 million units per lt of suspension upon induction production. The human interferon activity of this solution is approximately 450 per 1 suspension during induction production.
It was in the thousands.

Example A-8 After transplanting human-derived lymphoblastoid cells Nama1wa cells into the peritoneal cavity of adult nude mice,
They were kept for a week. Newcastle disease virus with a hemagglutination value of approximately a000 was inactivated by ultraviolet light and then injected intraperitoneally into the peritoneal cavity, and the animals were sacrificed 24 hours later and the ascites fluid was collected. Thereafter, it was purified and concentrated in the same manner as in Example A-1 to obtain a powder having TCLF activity. Obtained TCLF
Activity was approximately 9 million units per nude mouse. This product contained approximately 5.2 million units of human interferon.

Example A-4 A grown normal mouse was pre-irradiated with approximately 400 rem of X-rays to weaken the mouse's immune capacity, and then human-derived B IJ blast-like cells C were cultured subcutaneously in the mouse.
CRF-8B cells were transplanted and then raised in the usual manner for 8 weeks. After removing approximately 102 tumors that occurred under the skin,
Cells were dispersed in the same manner as in Example A-1. After suspending the cells in the same manner as in Example A-1, approximately 500 hemagglutination titer per Sendai virus and 08 μ2 per concanavalin A were added to this suspension, and the mixture was heated at 37°C for 1 hour.
The cells were kept for 1 day to induce the production of TCLF.

Thereafter, it was purified and concentrated in the same manner as in Example A-1 to obtain a powder having TCLF activity. The TCLF activity obtained was 24 million units per mouse.

This product contained human interferon for approximately 19 million years.

Example A-5 Neonatal hamsters were transplanted with human-derived lymphoblastoid JBL cells in the same manner as in Example A-1, and then reared for 4 weeks in the usual manner. The tumor mass was loosened in the same manner as in Example A-1 to obtain a cell suspension of approximately 8×10 cells.

Approximately 1,000 red hemisphere agglutination titers per Sendai virus were added to this suspension and kept at 86°C for 2 days to induce the production of TCLF.The suspension was then purified and concentrated in the same manner as in Example A-2.
A concentrated solution having LF activity was obtained. The TCLF activity obtained was approximately 16 million units per No. muctor. This solution contained approximately 7 million units of human interferon per hamster.

Example A-6 Human-derived B lymphoblastoid cells EBV were cultured in a 10-volume plastic cylindrical diffusion chamber equipped with a membrane filter with a pore size of α5 microns.
-HO cells were suspended in physiological saline and implanted into the peritoneal cavity of an adult rat. This rat was
After a week of rearing, the diffusion chamber was removed.

The concentration of human-derived B IJ lymphoblastoid cells thus obtained is approximately 6 × 1 O, compared to approximately 10 × 1 O when grown in an in vitro nutrient medium in a carbon dioxide gas incubator.
It was found to be more than double. The cells were suspended in the same manner as in Example A-2, and about 500 cells per cell were added to the suspension.
Newcastle disease virus with hemagglutinating titer was inactivated in advance with ultraviolet rays, and about 100 μl of phytohemagglutinin was added, kept at 37°C for 2 days, and TC was added.
LF was induced to be produced. Thereafter, the powder was purified and concentrated in the same manner as in Example A-1 to obtain a powder having TCLF activity. The amount of TCLF obtained was approximately 5.4 million units per rat.

This product contained approximately 6.8 million units of human interferon.

Example A-7 A cultured human-derived BIJ lymphoblastoid BALL-1 cell was transplanted into a fertilized chicken egg that was kept at 37°C for 5 days, and then kept at 87°C for 1 week. The eggs were broken and the proliferating cells were collected. Example A-1
It was suspended at 5 x 10'/m in the same manner as above. Sendai virus with an agglutination titer of about 1,000 per cent was added to this suspension, kept at 87°C for 1 day to induce TCLF production, and then purified and concentrated in the same manner as in Example A-2 to activate TCLF. A concentrated solution having . The obtained TCLF activity was about 900,000 units per 10 fertilized eggs. This liquid contained approximately 350,000 units of human interferon per 10 fertilized eggs.

Example A-8 The TCLF-containing powder prepared by the method of Example A-1 was
, Bodo's report (Symposium on pr.
epa-ration, 5tandairizati
on and clinical use ofint
erferon, 11 th 1nternatio
nal Immunobiological Sympo
sium, 8 & 9 June 1977, Za
greb, Yugoslavia), interferon is removed by means such as adsorption/desorption to ion exchange, molecular weight fractionation by gel filtration, concentration and microfiltration,
Further, concentration and purification is performed by ammonium sulfate salting out, and then PH
74, +101. Phytohemagglutinin-Sepharose affinity chromatography was performed in M IJ phosphate buffer, and the adsorbed fraction was αIMN-acetyl-D-
Elution was performed with the above buffer containing galactosamine, and the resulting fraction was dialyzed against the above buffer, concentrated, and lyophilized to obtain TCL.
A powder containing F activity was obtained.

The TCLF thus obtained has a specific activity of 8QOOO
The unit was /■.

Furthermore, when molecular weight fractionation was performed by gel filtration method, T with a molecular weight of 70,000 to 90,000, a molecular weight of A5 to 50,000, and a molecular weight of 10,000 to 20,000 was found.
CLF was fractionated at an activity ratio of 1:1:2. This TCL
All F are glycoproteins, and their sugar content varies depending on the molecular weight, but is about 5 to 45%.

TCL of the present invention obtained as in the manufacturing examples described above
F can be used as TCLF alone, as a mixture of TCLF and interferon, or as TCLF containing one or more other substances, such as injection drugs, oral drugs, eye drops, nasal drops, and external drugs. etc. as T
It can be advantageously used as a preventive or therapeutic agent for CLF-sensitive diseases. A TCLF-sensitive disease is a disease that is prevented or treated by TCLF, for example,
Breast cancer, lung cancer, bladder cancer, uterine cancer, colon cancer, stomach cancer, leukemia,
It is a malignant tumor such as lymphoma or skin cancer.

Next, Experiment B will highlight the efficacy, toxicity, dosage, and dosage of TCLF.

[Experiment B] TCLF efficacy and toxicity test experiment B-
A piece of human breast cancer tissue is subcutaneously transplanted into the back of a nude mouse derived from 1 BALB/C. The tumor volume is about 200? F+771
TCLIF obtained by the method of Example A-8 from the period '
A mixed product with a molecular weight of 70,000 to 90,000, a molecular weight of 50,000 to 50,000, and a molecular weight of 10,000 to 20,000 (hereinafter simply referred to as TCLF mixed product) was
and 40 units/Kg, intravenously injected twice daily, 15
Mice were sacrificed on day 1 and tumor weights were measured. The results are shown in Table 2. As a control, TCLF-free physiological saline was intravenously injected.

Table 2 Experiment B-2 Each group had 10 BDF1 male mice weighing around 251 cm,
A Lewis lung carcinoma cut into 2+++s+ angles was subcutaneously transplanted into the back. From day 8 after transplantation, the TCLF mixture obtained by the method of Example A-8 and TCLF with a molecular weight of 10,000 to 20,000 were administered at 4 and 4 c units/Kt, respectively.

The drug was administered intravenously twice a day, and on the second day, the mice were sacrificed and tumor weights were measured.

The results are shown in Table 8. As a control, TCLF-free physiological saline was intravenously injected.

*There is a statistically significant difference compared to the control value when the risk rate is 5 or less.

Experiment B-8 Leukemia LL210 cells were transplanted into 20 BDF1 male mice weighing around 252 kg. From the first day after transplantation, the TCLF mixture was administered intravenously at 8c units/Kg/day and 800 units/KLi/day, respectively, once every 15 days or twice a day, and the survival rate decreased to 50%. We compared the number of days until As a control, TCLF-free physiological saline or mitomycin 05■/Kf1 day was similarly intravenously injected. The results are shown in Table 4.

Table 4 From the results shown in Table 4, even when the dose of the TCLF mixture is relatively small, increasing the administration method from once a day to twice a day is extremely effective.

Experiment B-4 20 BDF1 male mice weighing around g51i,
Leukemia P888 cells were transplanted into this. The TCLF mixture was intraperitoneally injected once a day every day for 80 days from the first day after transplantation, and the relationship between the dose of the TcLF mixture, survival days, and survival rate (4) was determined. As a control, TCLF-free physiological saline or mitomycin was intravenously injected at 05/day.

The results are shown in Table 5.

As is clear from the results in Table 5, the TCLF mixture was
Large doses of 1,000 units or more per day are extremely effective as preventive and therapeutic agents.

Experiment B-5 Acute Toxicity An acute toxicity test of the TCLF mixture obtained by the method of Example A-8 was conducted using mice 20 days after birth.
The toxicity of this TCLF mixture is extremely low, with an LD5o of tc4oo when injected intraperitoneally.

It turned out to be more than a unit.

Furthermore, using human normal cells and human tumor cells, the concentration of the TCLF mixture that inhibits cell growth by 50% was determined in vitro using a conventional method.

As a result, 1ntestine (407) cells, L4
ver (Chang) cells or Qirardi)
(In normal cells such as earth cells, both 2c (oo)
It has a high concentration of over 0 units/-.

In contrast, KB (nasopharyngeal carcinoma) cells, H, Ep 2 (throat muscle) cells, or HLE (lung cancer) cells each have 18 units/
The concentration was extremely low at -124 units/d and 88 units/d.

As is clear from the above experiments, the TCLF of the present invention is extremely safe in terms of its effective dose and can be used for TCLF-sensitive diseases.

Furthermore, when applied to malignant tumors, for example, a part of a patient's tumor is taken, treated with the TCLF of the present invention in vitro to increase the immunogenicity of the tumor, and then returned to the tumor patient's body. This malignant tumor can also be treated.

The daily dose of TCLF of the present invention for adults is 5 to 5 QOO
QOOO units, preferably for local application doses such as local injections and eye drops, 5-t, o o qo o o units, 10-fi00 QOOO units for ointments, 50-IQO, for systemic injections such as intravenous injections and intramuscular injections. The dosage is 0QOOO units, and in the case of oral administration, the dosage is 500 to 5QOOQOOO units, but the dosage can be increased or decreased as appropriate depending on the usage or symptoms.

If necessary, it can be prepared into a pharmaceutical preparation by a conventional method together with any conventional pharmaceutical carrier, base, or excipient, but considering the toxicity, effective amount, stability, etc. of TCLF, It is desirable to contain 5 or more units of TCLF per duram.

The form of the TCLF-containing preventive or therapeutic agent for TCLF-sensitive diseases of the present invention can be freely selected depending on the purpose.

Oral preparations include enteric preparations such as capsules, tablets, and powders; intrarectal preparations include rectal suppositories; injections include, for example, freeze-dried injections that are dissolved in distilled water for injection and used monthly; It can also be used as nasal or eye drops, or as an ointment.

Examples of formulations are shown below, but the formulations are not limited thereto.

Example B-1 Injection TCLF mixture 2CLO prepared by the method of Example A-8
The OO unit is dissolved in 200-mL physiological saline and aseptically filtered using a membrane filter.

The filtrate is filled into two sterilized glass containers and freeze-dried, and the containers are sealed tightly to obtain a freeze-dried powder preparation.

This product is suitable for the treatment of breast cancer, lung cancer, liver cancer, leukemia, etc.

Example B-2 Ointment After the TCLF mixture prepared by the method of Example A-8 was ground into a small amount of liquid paraffin according to a conventional method, vaseline was added to prepare an ointment of 50 units/f.

This product is suitable for treating skin cancer, breast cancer, lymphoma, etc.

Example B-1 Eye Drops Distilled water 8 oo mtt (!: β-phenylethyl alcohol 5 m/TCLF prepared by the method of Example A-6)
Salt was added to the mixture to make it isotonic with 40,000 units, and 1,000 units were mixed with distilled water to prepare eye drops.

This product is suitable for the treatment of retinoblastoma, etc.

Example B-4 Enteric coated tablet TC with a molecular weight of 10,000 to 20,000 prepared by the method of Example A-8
When compressing LF into tablets using a mixture of starch and maltose according to a conventional method, one tablet (100 cm) of this TCLF is used.
Tablets were prepared by containing 2,000 units per tablet, and the tablets were coated with methyl cellulose phthalate to make enteric-coated tablets.

This product is suitable for the treatment of colorectal cancer, colon cancer, liver cancer, etc.

patent applicant Manual continuation supplementary book December 29, 1980 Commissioner of the Patent Office Shima 1) Itsuki Haru L Incident display 1987 Patent Application No. 187627 2 Name of the invention Contains target cytotoxic factors Malignant tumor therapeutic agent and its manufacturing method a. Person making the amendment Relationship to the case Patent applicant Tun correction target Application form and statement

Claims (9)

[Claims] (1) A therapeutic agent for malignant tumor characterized by containing a target cytotoxic factor. (2) The target cytotoxic factor has a molecular weight of approximately 10,000 to 100,000 and approximately 5
The therapeutic agent for malignant tumor according to claim (1), which is a glycoprotein containing ~45% carbohydrate. (3) The target cytotoxic factor has a molecular weight of approximately 10,000 to 20,000 and approximately a5
50,000 and about 70,000 to about 70,000 to 90,000. The malignant tumor therapeutic agent according to claim 11 or (2), characterized in that it contains at least one or more of about 70,000 to about 70,000 to about 90,000. (4) Claims characterized in that each duram contains 5 or more units of the target cytotoxic factor (paragraph 11, (2))
The malignant tumor therapeutic agent according to item or item (3). (5) Claim (1) characterized in that the target cytotoxic factor is contained together with human interferon.
), (2), (3), or (4). (6) Claims (1), (2), and (3) characterized in that the target cytotoxic factor is produced by human-derived B IJ lymphoblastoid cells. , the malignant tumor therapeutic agent according to item (4) or item (5). (7) The target cytotoxic factor is directly transplanted or diffused into the body of a warm-blooded animal other than a human by directly transplanting cultured human-derived B IJ lymphoblastoid cells capable of producing the target cytotoxic factor. Human-derived OB obtained by inoculating into a chamber and growing while receiving body fluids from the warm-blooded animal
Claim C11, (2) characterized in that the target cytotoxic factor is purified and collected by applying a target cytotoxic factor inducing agent to IJ membrane blastoid cells.
), (3), (4), (5), or (6). (8) A method for producing a therapeutic agent for malignant tumor, which comprises containing a target cytotoxic factor having a molecular weight of about 10,000 to 100,000 and a carbohydrate content of about 5 to 45. (9) The method for producing a therapeutic agent for malignant tumor according to claim (8), characterized in that the target cytotoxic factor is contained at least Durham units. 0 [Claim (8) or (2) wherein the target cytotoxic factor is produced and purified from cultured human-derived BIJ lymphoblastoid cells;
9) A method for producing a therapeutic agent for malignant tumor according to item 9).