JPH0214039B2 - - Google Patents

Description

[Detailed description of the invention]

In the production of lymphotoxin, the present invention involves direct transplantation of cultured human-derived cells that have the ability to produce lymphotoxin and that can be transplanted and proliferated into the body of a warm-blooded animal other than humans into the body of a warm-blooded animal other than humans. Or, the cells are inoculated into a diffusion chamber and grown while being supplied with the body fluids of the warm-blooded animal. The cells are then treated with a lymphotoxin inducer in vivo or in vitro to produce lymphotoxin. The present invention relates to a method for producing lymphotoxin, which comprises purifying and fractionating lymphotoxin. Lymphotoxins are co-authored by Ryuichi Aoki and others, “Lymphokines,” New Immunology Series 6, 1979, Igaku Shoin,
Bloom, BR & Glade, PR co-ed. “In vitro
"methods in cell-mediated immunity"
Academic Press, 1971. This is the name given to a protein-like substance that is induced to be produced inside and outside of cells by cells and has a cytotoxic function, and is known to have 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 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, we have developed cultured human-derived cells that have the ability to produce lymphotoxin and that can be transplanted and proliferated into the bodies of warm-blooded animals other than humans.
Instead of being inoculated and grown in a nutrient medium (in vitro), they can be transplanted into a warm-blooded non-human animal, or they can be inoculated into a diffusion chamber and supplied with nutrient-containing body fluids from that animal. By multiplying the cells and treating the resulting cells with a lymphotoxin inducer in vivo or in vitro, the lymphotoxin activity is higher than that obtained by culturing human-derived cells in vitro. The inventors discovered that lymphotoxin can be easily produced in large quantities by purifying and fractionating the induced product, and confirmed that the lymphotoxin is excellent as a therapeutic agent for malignant tumors, thereby completing the present invention. 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 that have the ability to produce lymphotoxin and can be transplanted and proliferated into the body of a warm-blooded animal other than humans are transplanted into the body of a warm-blooded animal other than humans, or If the animal is housed in a diffusion chamber that can receive a supply of body fluids, and this chamber is buried inside the animal's body and reared normally, the body fluids containing nutrients supplied by the warm-blooded animal's body will be utilized. Cells can easily proliferate. Furthermore, in vitro
Compared to when grown in vitro), the growth of these cells is stable, the growth rate is high, the amount of cells obtained is large, and the yield of lymphotoxin per cell is significantly increased. is also a major feature. The cultured human-derived cells used in the present invention may be those that can be transplanted into the body of a warm-blooded animal other than humans and easily proliferate, and that also have lymphotoxin production. Microbiology Vol.1” 116
- Namalva cells described in pages 117 (1975), BALL-1 cells described in "Nature Vol. 267" by I. Miyoshi, pages 843-844 (1977),
TALL-1 cells, NALL-1 cells, “Journal of
Immunology Vol.113” pages 1334-1345 (1974)
In addition to the M-7002 cells, B-7101 cells, etc. described,
Macrophages, fibroblasts, etc. are also freely used, and the genes with lymphotoxin-producing ability of these cells can be expressed using polyethylene glycol, for example.
Cell fusion methods using Sendai virus, DNA ligase, restriction enzymes (nucleases),
By genetic recombination using enzymes such as DNA polymerase, it can be introduced into cultured lymphoblast-like cells that can be more easily subcultivated to increase their proliferation rate and reduce the amount of lymphotoxin per cell. They may be used by increasing their productivity, 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 lymphotoxin, which will be described later.
If necessary, leukocytes prepared 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, horses, cows, guinea pigs, Mammals such as rats, 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. In addition, 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, etc., to weaken the immune response before transplantation. . 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, human-derived cells that have been cultured are first transplanted into hamsters and allowed to proliferate, and then these cells are further transplanted into nude mice.
It is also possible to stabilize the growth of human-derived cells by transplanting them between warm-blooded animals other than humans, and to increase the amount of lymphotoxin induced and produced from them. In this case, the transplant may be between the same species, the same genus, the same class, or the same 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 membranes that can block the passage of animal cells without directly transplanting human-derived cells into the animal body, such as membrane filters and ultrafiltration membranes with pore diameters of approximately 10 ^-7 to ^{10 -5} m, are also available. Alternatively, a diffusion chamber of various known shapes and sizes equipped with hollow eye bars or the like is buried in the animal's body, for example, in the abdominal cavity, and while receiving body fluids containing nutrients from the animal body, the above-mentioned Any cultured human-derived cells 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-based methods do not allow direct contact between human-derived cells and animal cells;
Not only can only human-derived cells be easily collected, but there is also little risk of causing undesirable immune reactions, so there is no need for pretreatment to suppress immune reactions, and various warm-blooded animals can be used freely. There is. 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 in a period of 1 to 10 weeks for growing 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
The number of cells transplanted per individual animal reaches approximately 10 ² to 10 ⁷ times, or more, and it reaches approximately 10 ¹ to 10 ⁶ times, or more, than the number of cells grown by inoculation in an in vitro nutrient medium. Therefore, it is extremely convenient for the production of lymphotoxin. Any method can be used to induce and produce lymphotoxin from human-derived living cells grown in this manner. The lymphotoxin-inducing agent can also be applied to the animal in which it has grown. For example, a lymphotoxin-inducing agent is directly applied to human-derived cells grown in suspension in ascites in the peritoneal cavity or tumor cells generated subcutaneously to induce the production of lymphotoxin, and then lymphotoxin is purified from the ascites or tumor. All you have to do is separate 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 grown in ascites are sorted, or a subcutaneous tumor containing human-derived cells is removed and sorted, and the resulting cells are approximately 20 to 40
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 used, such as by increasing the number of cells obtained by implanting or replacing the chamber connected to the animal body. As the lymphotoxin inducer, one or more of phytohemagglutinin, concanavalin A, pork weed mitogen, ribopolysaccharide, 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 way can be purified using known purification and separation methods such as salting out, dialysis,
By performing filtration, centrifugation, concentration, freeze-drying, etc., it can be easily purified and separated from interferon, which is induced and produced at the same time, and collected. If a higher level of purification is required, for example, adsorption-elution to 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 , very convenient. In this case, phytohemagglutinin is phytohemagglutinin-
Any phytohemagglutinin such as P, phytohemagglutinin-M, and phytohemagglutinin-E may be used. The lymphotoxin of the present invention can be used as a prophylactic or therapeutic agent for lymphotoxin-sensitive diseases. Lymphotoxin-sensitive diseases are diseases that are prevented or treated by lymphotoxin, such as malignant tumors such as breast cancer, lung cancer, liver cancer, bladder cancer, uterine cancer, colorectal cancer, gastric cancer, leukemia, lymphoma, and skin cancer. be. 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 It is also possible to treat this malignant tumor by returning it to the body of the patient. 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,
The existence of β- and γ-lymphotoxins has been described [Biology of the
Lymphokines” Academic Press (1979)]. The activity of lymphotoxin is determined by Bloom, BR &
Glade, PR co-editor “In vitro methods in cell−
mediated immunity” Academic Press (1971)
A known method of measuring the number of surviving cells after culturing for a certain period of time was used using mouse L cells as reported in . The efficacy, usage, and dosage will be explained below using experimental examples. Experimental Example 1 A piece of human breast cancer tissue is subcutaneously transplanted into the back of a BALB/C-derived nude mouse. From the time when the tumor volume was about 200 ^mm3 , α- and β obtained in Example 9 described later
-, γ-lymphotoxin mixture (hereinafter simply referred to as lymphotoxin) at 4 and 40 units/Kg, 1
Injected intravenously twice a day, and killed mice on the 15th day.
Tumor weight was measured. The results are shown in Table 1. As a control, lymphotoxin-free physiological saline was intravenously injected.

[Table] Experimental Example with Significant Differences 2 A group of 10 BDF ₁ male mice weighing around 25 g were subcutaneously implanted with Lewis lung carcinoma cut into 2 mm squares on the back. From the 8th day after transplantation, Example 9 described later
Lymphotoxin and γ-lymphotoxin obtained in 4 and 40 units/kg, respectively, were administered intravenously twice a day, and on the 21st day, the mice were sacrificed and tumor weights were measured. The results are shown in Table 2. As a control, lymphotoxin-free physiological saline was intravenously injected.

[Table] There is a significant difference.
Examples of the method for producing lymphotoxin of the present invention will be shown below. Example 1 After subcutaneously transplanting cultured BALL-1 cells into adult nude mice, they were cultured for 3 days in a normal manner.
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 washed with Eagle's minimal basal medium of PH 7.2 containing 5V/V% and kept at 37°C, and phytohemagglutinin was added to this. It was added at a rate of about 200 μg/ml and kept for 2 days to induce the production of lymphotoxin.
This was centrifuged at about 4°C and about 1000g to remove the precipitate, and the resulting supernatant was dialyzed for 21 hours against physiological saline containing PH7.2 and 0.01M phosphate buffer. The solution obtained through filtration was concentrated and lyophilized to obtain a powder containing lymphotoxin activity. The lymphotoxin activity obtained was approximately 360,000 units per nude mouse. Example 2 Cultured human-derived BALL-1 cells and TALL-1 cells were transplanted into the peritoneal cavity of grown nude mice, and then raised for 5 weeks in a conventional manner. 1 mg of phytohemagglutinin was injected into the abdominal cavity, and 24 hours later, the animals were sacrificed to obtain ascites. This is 4℃, about 1000
Centrifugation at
Dialysis was performed for 15 hours against physiological saline containing phosphate buffer, and the resulting solution was concentrated to obtain a solution containing lymphotoxin activity. The lymphotoxin activity obtained was approximately
It was 190,000 units. Example 3 Newborn hamsters were injected in advance with antiserum prepared from rabbits using a known method to weaken the hamster's immune capacity, and cultured human-derived JBL cells were subcutaneously transplanted into the hamsters, followed by regular injections. The animals were reared for 4 weeks using the following method. After about 30 g of tumor that had occurred under the skin was removed, the cells were dispersed in the same manner as in Example 1. The cells were incubated at PH7.4 containing 10V/V% calf serum.
After washing with RPMI 1640 medium, the cells were diluted to a cell concentration of approximately 2×10 ⁷ /ml in a medium of the same composition kept at 37°C. Concanavalin A500n for this
g/ml and Sendai virus hemagglutination titer 1000/ml and kept for 3 days to induce lymphotoxin production. Thereafter, it was purified and concentrated in the same manner as in Example 1 to obtain a solution having lymphotoxin activity. The lymphotoxin activity obtained was approximately
It was 1,960,000 units. Example 4 After pre-irradiating an adult normal mouse with X-rays at approximately 400 rem to weaken the immune system of the mouse, human-derived TALL was cultured subcutaneously in the mouse.
-1 cells were transplanted and then reared for 3 weeks in the usual manner. After about 10 g of tumor that had occurred under the skin was removed, the cells were dispersed in the same manner as in Example 1. These cells were treated in the same manner as in Example 2 to induce the production of lymphotoxin, and thereafter purified and concentrated in the same manner as in Example 2 to obtain a concentrate having lymphotoxin activity. The lymphotoxin activity obtained was approximately 310,000 units per normal mouse. Example 5 Neonatal rats were subcutaneously transplanted with cultured human-derived Namalva cells and then reared for 4 weeks in a conventional manner. After about 50 g of tumor that had occurred under the skin was removed, the cells were dispersed in the same manner as in Example 1. Next, lymphotoxin was induced and produced in the same manner as in Example 1 except that Maruyama vaccine was added at a rate of 1 μg/ml instead of phytohemagglutinin, and further purified and frozen in the same manner as in Example 1. After drying, a powder with lymphotoxin activity was obtained. The lymphotoxin activity obtained was approximately 410,000 units per rat. Example 6 Cultured MOLT-3 cells were first transplanted subcutaneously into hamsters by the method of Example 3, and the cells obtained by growing for 3 weeks were intraperitoneally injected into the peritoneal cavity of nude mice on the 10th day after birth. Re-implanted. After the nude mice were raised in a conventional manner for 5 weeks, ascites was collected and centrifuged to obtain proliferating cells. Example 1
After washing by the method described above, lymphotoxin was induced to be produced in the same manner as in Example 1, and then purified and concentrated in the same manner as in Example 2 to obtain a concentrated liquid having lymphotoxin activity. The lymphotoxin activity obtained was approximately 250,000 units per nude mouse. Example 7 Cultured human-derived JBL cells were suspended in physiological saline in a plastic cylindrical chamber with a capacity of about 10 ml and equipped with a membrane filter with a pore size of about 0.5 microns, and then grown into rat cells. It was implanted intraperitoneally. After raising these rats in the usual manner for 4 weeks,
I took out this chamber. The concentration of human-derived cells obtained in this way was about 5 x 10 ⁹ /ml, which is about the same as when grown in an in vitro nutrient medium in a carbon dioxide incubator.
It was found that the increase was more than 10 ³ times. These cells were treated in the same manner as in Example 1 to induce the production of lymphotoxin, purified and concentrated, and lyophilized to obtain a powder having lymphotoxin activity. The lymphotoxin activity obtained was approximately
It was 420,000 units. Example 8 Cultured human-derived NALL-1 cells were transplanted into fertilized chicken eggs kept at 37°C for 5 days, and then kept at 37°C for 1 week. After breaking the eggs, proliferating cells were collected, and 50% of the TALL-1 cells obtained in Example 4 were added to the cells, and then treated in the same manner as in Example 1 to induce lymphotoxin production.
Next, the product was purified and concentrated in the same manner as in Example 2 to obtain a concentrated solution having lymphotoxin activity. The lymphotoxin activity obtained was approximately 180,000 units per 10 fertilized eggs. Example 9 Lymphotoxin powder prepared by the method of Example 1 was prepared as reported by G. Bodo (Sympotium on preparation.
standardization and clinical use of
interferon, 11th International
Immunobiological Symposium.8＆9 June
1977, Zagreb.Yugoslavia), interferon was removed by means such as adsorption and desorption onto an ion exchanger, molecular weight fractionation by gel filtration, concentration and precision filtration, and further concentrated and purified by ammonium sulfate salting out. Phytohemagglutinin-Sepharose affinity chromatography was performed in 0.01M phosphate buffer at pH 7.4, and the adsorbed fraction was collected.
Elution was performed with the above buffer containing 0.1 MN-acetyl-D-galactosamine, and the resulting fraction was dialyzed against the above buffer, concentrated, and lyophilized to obtain a powder containing lymphotoxin activity. 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 35,000-50,000) and γ
-Three types of lymphotoxin (molecular weight 10,000 to 20,000) were separated at an activity ratio of approximately 1:1:2.

Claims (1)

[Scope of Claims] 1. Is it possible to transplant cultured human-derived cells that have the ability to produce lymphotoxin and proliferate by transplanting them into the body of a warm-blooded animal other than humans into the body of a warm-blooded animal other than humans? , or by inoculating the cells into a diffusion chamber and growing them while receiving the body fluids of the warm-blooded animal, and then treating the cells with a lymphotoxin inducer in vivo or in vitro to produce lymphotoxin. A method for producing lymphotoxin, which comprises purifying and preparatively separating the lymphotoxin. 2 The purification and preparative method is phytohemagglutinin-
The method for producing lymphotoxin according to claim 1, which is carried out by affinity column chromatography using Sepharose.