JPH0446928B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to an anti-lymphotoxin-sensitive disease agent containing lymphotoxin. Lymphotoxin is co-authored by Ryuichi Aoki et al., “Lymphokine”, New Immunology Series 6, Igaku Shoin, 1979,
Bloom, BR & Glade, PR co-editor “In vitro
"methods in cell-mediated immunity"
Academic Press, 1971. This is a name given to a proteinaceous substance that is induced to be produced inside and outside the cell by causing cell damage, and has a cytotoxic function.It is known that it has a cytotoxic function, especially against tumor cells. It is. Because of these functions of lymphotoxin, lymphotoxin has been expected to be a therapeutic agent for malignant tumors since its discovery. Lymphotoxin has been prepared from lymph cells of various animals including humans, but in order to be used for human treatment, it is extremely important to derive it from living human cells in order to avoid side effects such as antigenicity that may occur during treatment. It's safe and it's good. Live human cells that have traditionally been used to prepare lymphotoxin include white blood cells. However, leukocytes are prepared by separating them from fresh human blood, and it is difficult to preserve them, and it is extremely difficult to supply them in large quantities at low cost. For these reasons, the production of lymphotoxin that can be used for human treatment has not yet been carried out on an industrial scale. The present inventors have investigated a method for producing lymphotoxin that can be easily implemented on an industrial scale, and have conducted intensive research to determine whether the lymphotoxin is useful as a therapeutic agent for malignant tumors. As a result, instead of inoculating cultured human-derived cells into nutrient media and growing them in vitro, they can be transplanted into non-human warm-blooded animals or inoculated into a diffusion chamber. Lymphotoxin is induced and produced with high activity by allowing the cells to proliferate while being supplied with body fluids containing nutrients from the animal body, and by acting on the obtained cells with a lymphotoxin inducing agent in vivo or in vitro. The inventors discovered that lymphotoxin can be easily produced in large quantities by purifying and fractionating it, and completed the present invention by confirming that the lymphotoxin is excellent as a therapeutic agent for malignant tumors. Unlike when living cells are grown in vitro, the method for producing lymphotoxin used in the present invention not only eliminates or significantly saves nutrient media containing expensive serum, but also Maintenance and management during proliferation is extremely easy,
Moreover, it is characterized by high induced lymphotoxin activity. That is, cultured human-derived cells are transplanted into the body of a warm-blooded animal other than humans, or they are housed in a diffusion chamber that can receive the body fluids of that animal, and this chamber is buried within the animal body. However, with normal husbandry, the cells can easily proliferate using the nutrient-containing body fluids provided by the warm-blooded animal. Furthermore, compared to in vitro proliferation, the proliferation of these cells is stable, the proliferation rate is high, the amount of cells obtained is large, and the yield of lymphotoxin per cell is high. Another major feature is that there is a marked increase in The cultured human-derived cells used in the present invention may be any cell that can be transplanted into a warm-blooded animal other than humans and easily proliferate and has the ability to produce lymphotoxin. Microbiology
Volume 1” pages 116-117 (1975)
Namalva cells, I, Miyoshi, “Nature
BALL-1 cells, TALL-1 cells, NALL-1 cells described in "Vol. 267" pages 843-844 (1977)
Cell, “Journal of Immunology Vol.113” 1334
- M-7002 cells described on page 1345 (1974), B-
In addition to 7101 cells, macrophages, fibroblasts, etc. are also used freely, and the lymphotoxin-producing genes of these cells can be transferred using cell fusion methods using polyethylene glycol, Sendai virus, etc., DNA ligase, etc. By genetic recombination using enzymes such as restriction enzymes (nucleases) and DNA polymerases, the cells are introduced into cultured lymphoblastoid cells that can be more easily subcultured, and their growth rate is controlled. The cells may be used by increasing the lymphotoxin production ability per cell, and are not limited to the established cell lines described herein. During the process, they can be freely used alone or in combination of two or more. If necessary, leukocytes purified from fresh human blood, for example, can also 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, cows, guinea pigs, and rats. Mammals such as , hamsters, normal mice, and nude mice can be used. Transplanting human-derived cells into these animals may cause an unfavorable immune reaction, so in order to suppress such reactions as much as possible, the animals used should be kept as young as possible, i.e. eggs, embryos, fetuses, or newborns. Preferably from childhood. Alternatively, 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 response before transplantation. good. When the animals used are nude mice, the immune response is weak even when they are grown, so cultured human-derived cells can be transplanted without the need for these pretreatments, and the cells can be rapidly transplanted. This is particularly advantageous since it can be propagated. In addition, cultured human-derived cells may be transplanted between warm-blooded animals other than humans, for example, first transplanted into hamsters and allowed to proliferate, and then transplanted into nude mice. It is also possible to further stabilize the proliferation of human-derived cells or to increase the amount of lymphotoxin induced therefrom. In this case, transplantation may be performed not only between the same species and the same genus, but also between the same order and phylum. The site within the animal body to which human-derived cells are transplanted may be any site where the transplanted cells can proliferate, such as the allantoic cavity, vein, abdominal cavity, or subcutaneous site. In addition, porous filtration membranes that can block the passage of animal cells without directly transplanting human-derived cells into the animal body, such as membrane filters with pore diameters of about 10 ^-7 to ^{10 -3} m, ultraviolet filters, etc. Diffusion chambers of various known shapes and sizes equipped with filtration membranes or hollow eye bars are buried in the animal's body, for example in the abdominal cavity, and the chamber is supplied with body fluids containing nutrients from the animal body. Any of the human-derived cells cultivated as described above can be grown. If necessary, a chamber that connects and perfuses the solution containing nutrients within the chamber with body fluids within the animal's body is attached to the surface of the animal's body, for example, to check the proliferation status of human-derived cells within the chamber. It is also possible to make it transparent, or to make only this chamber part removable and replaceable, allowing cells to proliferate throughout the lifespan of the animal without having to sacrifice it, thereby further increasing the amount of cells produced per individual animal.
These diffusion chamber methods do not allow human-derived cells to come into direct contact with animal cells, making it easy to collect only human-derived cells. There is no need for pre-treatment to inhibit the use of this method, and it has the advantage of being able to freely utilize various warm-blooded animals. 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 cells. It has also been found that the number of human-derived cells obtained in this manner reaches about 10 ⁷ to ^{10 12} or more per animal. In other words, the number of human-derived cells grown by the method for producing lymphotoxin used in the present invention is approximately 10 ² to 10 ⁷ of the number of cells transplanted per individual animal.
It is very convenient for the production of lymphotoxin, reaching about 10 ¹ to 10 ⁶ cultures or more when inoculated and grown in vitro in a nutrient medium. Any method can be used to induce and produce lymphotoxin from human-derived living cells grown in this manner. Lymphotoxin derivatives can also be made to act within the animal's body in which it has grown. For example, lymphotoxin derivatives are induced to produce lymphotoxin by directly acting on human-derived cells grown in suspension in ascites fluid in the peritoneal cavity or tumor cells generated subcutaneously, and then lymphotoxin is purified from the ascites fluid or tumor. Just take it. Furthermore, human-derived proliferating cells can be removed from an animal body and lymphotoxin-inducing agents can be applied to the cells in vitro to induce lymphotoxin production. For example, human-derived cells proliferated in ascites are sorted, or tumors containing human-derived cells generated subcutaneously are removed and dispersed, and the resulting cells are divided into approximately 20 to 40 cells.
The cell concentration is approximately 10 ⁵ - 10 ⁸ /ml in the nutrient medium kept at ℃.
Lymphotoxin can be induced and produced by suspending the protein and treating it with a lymphotoxin inducing agent, which can then be purified and fractionated. Furthermore, when human-derived cells are grown in a diffusion chamber, the grown cells can be left in the chamber or removed from the chamber and treated with a lymphotoxin-inducing agent to induce lymphotoxin production. In addition, for example, lymphotoxin is first induced to be produced in grown human-derived cells in the animal body, and then lymphotoxin is induced outside the animal body in human-derived cells collected from a specific part or the whole of the same animal. a method of producing lymphotoxin, and a method of using cells once used for the induced production of lymphotoxin two or more times,
Alternatively, it is also possible to further increase the amount of lymphotoxin produced per individual animal by embedding it in the animal body or by replacing the connected chamber to increase the number of cells obtained. As the lymphotoxin inducer, one or more of phytohemagglutinin, concanavalin A, porkweed mitogen, lipopolysaccharide, endotoxin, polysaccharides, mitogens such as bacteria, viruses, and nucleic acids are usually used. Antigens are also lymphotoxin inducers for sensitized cells. The lymphotoxin induced and produced in this manner is purified by known purification and separation methods, such as salting out, dialysis, filtration, centrifugation, concentration, and freeze-drying, to combine with interferon, etc., which was induced and produced at the same time. It can be easily purified, separated, and collected. If a higher level of purification is required, for example, adsorption-elution on an ion exchanger, gel filtration, isoelectric point fractionation, electrophoresis, ion exchange chromatography, high performance liquid chromatography, column chromatography. Although it is possible to collect the highest purity lymphotoxin by combining known methods such as Therefore, it is very convenient. In this case, phytohemagglutinin is phytohemagglutinin-P,
Any phytohemagglutinin may be used, such as phytohemagglutinin-M and phytohemagglutinin-E. Lymphotoxin has a molecular weight of 7 to 9.
Three types: 10,000, 35,000 to 50,000, and 1 to 20,000, namely α,
It has been reported that β- and γ-lymphotoxins exist [Biology of the
Lmphokines” Academic Press (1979)]. The activity of lymphotoxin was determined by Bloom, BR & Glade, P.
Co-edited by R. “In vitro methods in cell-mediated”
A known method was used to measure the number of surviving cells after culturing for a certain period of time using mouse L cells as reported in "Immunity" Academic Press (1979). Lymphotoxin, which is the active ingredient of the anti-lymphotoxin-sensitive disease agent of the present invention, can be prepared, for example, by the following production example. After transplanting the cultured BALL-1 cells subcutaneously into adult nude mice, they were cultured for 3 days using the usual method.
They were kept for a week. Approximately 10 g of tumor that had occurred under the skin was excised and cut into small pieces, and then suspended in trypsin-containing physiological saline to disperse and separate the cells. These cells were added to human serum.
Cells were diluted to a cell concentration of approximately 5 x 10 ⁶ /ml in a medium of the same composition that had been washed with Eagle's minimum basal medium of PH 7.2 containing 5 v/v% and kept at 37°C, and phytohemagglutinin was added to this medium. In addition at a rate of approximately 20 μg/ml, 2
The cells were kept for several days to induce lymphotoxin production.
This was centrifuged at 4°C and approximately 1000 g to remove the precipitate, and the resulting supernatant was dialyzed for 21 hours against physiological saline containing PH7.2 and 0.01M phosphate buffer, followed by microfiltration. The resulting filtrate was concentrated and lyophilized to obtain a powder containing lymphotoxin activity. The powder obtained was reported by G. Bodo (Symposium on
preparation, standardization and clinical use
of interfer on, llth International
Immunobiological Symposium 889 June 1977,
Zagreb. It was purified by chromatography to obtain highly pure lymphotoxin. The lymphotoxin thus obtained had a specific activity of 30,000 units/mg. Furthermore, molecular weight fractionation by gel filtration revealed that α-lymphotoxin (molecular weight 70,000 to 90,000), β
- lymphotoxin (molecular weight 35,000 to 50,000), and γ
-Three types of lymphotoxin (molecular weight 10,000 to 20,000) were separated at an activity ratio of approximately 1:1:2. The effectiveness, toxicity, usage, and dosage of lymphotoxin, which is the active ingredient of the present invention, will be explained below. Experimental Example 1 A piece of human breast cancer tissue was subcutaneously transplanted into the back of a BALB/C-derived nude mouse. α-, β-, γ- obtained in the above production example from the time when the tumor volume was approximately 200 ^mm3 .
A lymphotoxin mixture (hereinafter simply referred to as lymphotoxin) was intravenously injected twice a day at 4 and 40 units/kg, and on the 15th day, the mice were sacrificed and tumor weights were measured. The results are shown in Table 1. As a control, lymphotoxin-free physiological saline was intravenously injected.

[Table] *There is a statistically significant difference compared to the control value at a risk rate of 5% or less.
Experimental Example 2 A group of 10 BDF ₁ male mice weighing approximately 25 g were subcutaneously implanted with Lewis lung carcinoma cut into 2 mm squares on the back. From the 8th day after transplantation, lymphotoxin and γ-lymphotoxin obtained in the above-mentioned production example were intravenously injected twice a day at 4 and 40 units/kg, respectively, and on the 21st day, the mice were killed and the tumor weight was determined. was measured. The results are shown in Table 2. As a control, lymphotoxin-free physiological saline was intravenously injected.

[Table] Scientifically significant difference.
Experimental Example 3 Acute Toxicity When the lymphotoxin obtained in the above production example was tested for acute toxicity using mice 20 days after birth, the toxicity of this lymphotoxin was extremely low.
The LD ₅₀ when injected intraperitoneally was found to be more than 10,000 units/Kg. Experimental Example 4 Tumor Cell Growth Suppression Effect After culturing 10 ⁵ human-derived tumor cells shown in Table 3 for 24 hours in advance, the test substance was added to Eagle medium containing 10% calf serum or RPMI1640 containing 10% calf serum. The cells were cultured in 1 ml of medium at 37°C for 48 hours under aeration of oxygen gas containing 5% carbon dioxide. After completion of the culture, the number of surviving cells that were not stained with trypan blue was counted under an optical microscope, and the growth inhibition rate (%) was determined using the following formula. Growth inhibition rate = (1 - number of surviving cells in treatment group/number of surviving cells in control group) x 100

[Table] As described above, lymphotoxin clearly showed a growth-inhibiting effect on various human-derived tumor cells. As is clear from the above experiments, the lymphotoxin of the present invention is extremely safe in terms of its effective dose and can be used for lymphotoxin-sensitive diseases. A lymphotoxin-sensitive disease is a disease that is treated with lymphotoxin, that is, a disease that is completely cured and whose symptoms are alleviated by lymphotoxin.
It is also a disease whose recurrence is prevented, and may be a malignant tumor such as breast cancer, lung cancer, liver cancer, bladder cancer, uterine cancer, stomach cancer, colon cancer, leukemia, lymphoma, or skin cancer. Furthermore, when applied to malignant tumors, for example, a part of a patient's tumor is taken and treated with the lymphotoxin of the present invention in vitro to increase the immunogenicity of the tumor, and then treated with the tumor patient. It is also possible to treat this malignant tumor by returning it to the body of the patient.
The daily dose for adults of the lymphotoxin of the present invention is 5 to 500,000 units, preferably 5 to 1000 units for local application such as local injection and eye drops, 10 to 1000 units for ointment, and 10 to 1000 units for systemic administration such as intravenous injection and intramuscular injection. 50-2000 units for injection, 500-2000 units for oral administration
The dosage is 500,000 units, but it 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. The form of the lymphotoxin-containing anti-lymphotoxin-sensitive disease agent of the present invention can be freely selected depending on the purpose. Enteric-coated preparations such as capsules, tablets, and powders are available for oral administration, and rectal suppositories are available for intrarectal administration.
Examples of injections include freeze-dried injections that are dissolved in distilled water for injection before use, nasal or eye drops, and ointments. Examples of formulations are shown below, but the formulations are not limited thereto. Example 1 Injection Lymphotoxin 20000 prepared in the above production example
Dissolve the unit in 200 ml of physiological saline and filter aseptically using a membrane filter. Fill 2 ml of the filtrate into sterilized glass containers, freeze-dry, and seal 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 2 Ointment After the lymphotoxin prepared in the above production example was dissolved into a small amount of varafine according to a conventional method, vaseline was added to prepare an ointment with a concentration of 50 units/g. This product is suitable for treating skin cancer, breast cancer, lymphoma, etc. Example 3 Eye drops: 800 ml of distilled water, 5 ml of β-phenylethyl alcohol, and lymphotoxin prepared in the above production example.
Salt was added to make the solution isotonic to 40,000 units, and the volume was made up to 1,000 ml with distilled water to prepare eye drops. This product is suitable for the treatment of retinoblastoma, etc. Example 4 Enteric-coated tablet When compressing the γ-lymphotoxin prepared in the above production example into tablets using a mixture of starch and maltose according to a conventional method, the amount of lymphotoxin was adjusted to 2000 units per tablet (100 mg) of the product. Enteric-coated tablets were prepared by coating the tablets with methyl cellulose phthalate. This product is suitable for the treatment of colorectal cancer, colon cancer, liver cancer, etc.

Claims (1)

[Claims] 1 Transplant cultured human-derived cells with lymphotoxin-producing ability into the body of a warm-blooded animal other than humans, and grow the cells while receiving body fluids from the warm-blooded animal. An anti-lymphotoxin-sensitive disease agent containing lymphotoxin as an active ingredient by purifying and fractionating lymphotoxin produced in vitro by the action of a lymphotoxin inducer.