KR890000383B1 - Process for the production of human t-cell growth factor - Google Patents

Abstract

Process for prepn. of human T-cell growth factor comprises (a) implanting human-derived cells capable of producing hTCGF into warm blood animals except human or into a device which can supply nutritive body fluids of warm blood animals except human and proliferating the cells, and (b) gathering the proliferated cells and releasing hTCGF from the proliferated cells using a hTCGF inducer. The human cell can be selected from peripheral blood cell, spleen cell, amygdalade cell, amygdala tumor cell, liver cancer cell, lung cancer cell, and hybridized lymphoblastiod cell. The inducer contains mitogen, phytohaemagglutinin, concanavalin A, virus, nucleic acid and polynucleotide.

Description

Human T Cell Proliferation Factor

The present invention relates to a method for producing human T cell proliferation factor. Human T cell proliferation factor (hereinafter referred to as hTCGF) is a protein substance such as a hormone that shows the proliferation promoting effect of human T cells that can be prepared by human serum and is a kind of human cell proliferation promoting factor. T cells are important lymphocytes that are responsible for cellular immunity in vivo, for example, delayed allergy, tumor immunity, and the like.

Since hTCGF promotes and activates the proliferation of human T cells that act as such, it is not only used as a T cell proliferation promoter in vitro, but also as a medicine for the study of the treatment of diseases in asthma, malignant tumors, etc. It is expected to be used as an immunotherapeutic agent for diseases, and the establishment of mass production methods thereof is desired. T-cell proliferation factor is described by Narimatsu Hisashi, T Cell Proliferation Factor (TCGF) and Its Applications, Vol. As apparent from the description of 17, pages 2063 to 2077 (1980), nicknamed Interleukin, species dependence was not found in its action.

In treating human diseases, it is conceivable to use TCGF derived from animal cells other than the human body, but an undesirable antigen-antibody reaction such as anaphylactic shock is concerned. Therefore, to use in the treatment of the human body it is safe to use hTCGF derived from the living cells of the human body is excellent. Human serum has been known as a preparation of hTCGF. However, since human serum can be separated and prepared from fresh blood of the human body, it is difficult to preserve it, so it is very difficult to supply a large amount at a low price. For this reason, the production of hTCGF, which can be used for the prevention or treatment of human diseases, has not yet reached the industrial scale. MEANS TO SOLVE THE PROBLEM This inventor earnestly examined the manufacturing method of hTCGF which can be performed easily on an industrial scale. As a result, cells obtained by proliferating cells derived from a human body having hTCGF-producing ability using warm-blooded animals other than the human body had significantly higher production of hTCGF than cells obtained by tissue culture in vitro. The present invention was completed by finding that it reached about 2 to 10 times, or even more, per cell. That is, the present invention produces hTCGF from the cells obtained by transplanting cells derived from a human body having hTCGF-producing ability into a warm-blooded animal body other than the human body, or by proliferating under the fluid supply of the warm-blooded animal. It relates to a manufacturing method.

The method of the present invention can produce a large amount of hTCGF as compared to the case of tissue culture in the conventional in vitro, and it is unnecessary or considerably saves nutrient medium containing expensive serum, etc. Maintenance is extremely easy while doing so. In other words, cells derived from a human body having hTCGF-producing ability are transplanted into a warm-blooded animal body other than the human body, or housed in a chamber that can receive the supply of the animal body fluid, and is supplied from a warm-blooded animal body for normal breeding. By using the nutrient-containing body fluid, the cells can be easily proliferated. In addition, compared to the case of tissue culture in vitro, the cell proliferation is more stable, the fact that its growth rate is higher, a large number of cells can be obtained, and in addition, the production of hTCGF per cell increases. Can be mentioned.

The cells derived from the human body used in the present invention may be hTCGF acid-producing, and may be easily transplanted by transplantation into a warm-blooded animal other than the human body. For example, human normal cells such as human peripheral blood cells, human spleen cells, human tonsil cells; Human cancer cells such as human tonsil tumor cells, human liver cancer cells, human lung cancer cells; And cultured cells of the aforementioned cells are all preferably used in the present invention.

As known human-derived cells cultured, for example, KS-5, MOLT-3, MT-1, Mono-1, OUMS described in Tissue Culture Vol. 6, pages 527 to 546 (1980). You can choose -19 and so on. In addition, genes having hTCGF-producing ability of these cells may be, for example, cell fusion means using polyethylene glycol or Sendai virus, or genes using enzymes such as DNA ligase, restriction enzyme (nuclease), DNA polymerase, and the like. Introduced into cultured human lymphocyte-like cells that can be cultured more easily according to recombinant means, etc. is not only because of their high proliferation rate, but also about 2 to 10 times higher hTCGF production per cell, Or even more than that, it is especially preferable.

In addition, when cultured human lymphocyte-like cells cultured are used, when transplanted into warm-blooded animals other than the human body, it is easy to form soft varieties that are difficult to mix with the cells of the host animal, and are easy to disperse after extraction. Therefore, it is very advantageous to collect only human lymphocyte-like living cells. Such human lymphocyte-like cells are suitable cell lines derived from human leukemia or human malignant lymphoma, such as Namalwa cells, BALL-1 cells, NALL-1 cells, TALL-1 cells, JBL. Known human derived cell lines such as cells can be used particularly advantageously.

The warm-blooded animal used in the production method of the hTCGF of the present invention may be any cell that is derived from a human body having an hTCGF-producing ability, for example, a bird, a dog, a cat, a monkey, a goat, a pig, a cow, such as a chicken or a pigeon, Mammals such as horses, rabbits, malmots, mice, hamsters, common mice, and nude mice can be used.

Since transplantation of cells derived from human bodies into these animals may cause undesirable immune reactions, animals used to suppress the reaction as much as possible are as young as possible, ie eggs, embryos. It is preferable to use the fetus, or the newborn or juvenile animal. These animals may be transplanted by weakening the immune response after pretreatment such as irradiation with X-rays or X-rays of about 200 to 600 REM, or by injection of antisera or immunosuppressive agents. When the animal to be used is a Nude mouse, even if it is grown, the immune response is weak, and thus, it is particularly preferable because it can transplant cells derived from cultured human body without requiring such pretreatment, and can rapidly proliferate. In addition, cells derived from the human body, for example, are first transplanted into a hamster and then propagated, and then transplanted between warm-blooded animals other than the human body, such as transplanting the cells again to Nude mice, to stabilize the growth of human-derived cells more stably. In addition, it is also possible to arbitrarily increase the amount of hTCGF produced by the proliferated cells. In this case, it is possible to transplant not only allogeneic species and allogeneic livers, but also allograft and alumni.

The site in the animal body to which the cells derived from a human body are transplanted may be a site where the transplanted cells can proliferate, and can be freely selected, for example, urine fluid cavity, vein, abdominal cavity, subcutaneous. In addition, porous filtration membranes that can block the passage of animal cells without directly implanting human-derived cells into the animal body, for example, thin film filters having a diameter of about 10 ^-7 to 10 ^-5 m and ultrafiltration membranes. Alternatively, a diffusion chamber of various shapes and sizes known in which hollow fibers are installed is buried in an animal body, i.e., intraperitoneally, to receive a bodily fluid containing nutrients from the animal body, and cultured in the chamber by a known method. Any cell derived from a human body can grow.

In addition, if necessary, a diffusion chamber in which the bodily fluid containing the nutrients in the diffusion chamber is brought into contact with the body fluid in the animal body and flows down through the tube is attached to the surface of the animal, for example, to see the proliferation state of human-derived cells in the diffusion chamber. It is also possible to increase the cell production per animal individual by proliferating the cells by the end of their life without killing the animals by allowing them to be detached or exchanged only in the diffusion chamber.

Since the method of using a diffusion chamber does not directly contact human-derived cells, the cells derived from the human body are not only able to easily collect cells derived from the human body, but also less likely to cause an undesirable immune response. It does not require pretreatment and has the feature of freely using various warm-blooded animals. The maintenance of the transplanted animal is very good because it is possible to continue the normal breeding of the animal, and even after transplantation, no special handling is required. The period for proliferating cells derived from the human body is usually 1 to 20 weeks. If the cultured cells to be transplanted are tumor cells or otherwise lymphocyte-like cells, their proliferation rate is particularly high and can be achieved in a period of 1 to 5 weeks. It has also been found that the number of human derived cell numbers thus obtained reaches about 10 ⁷ to 10 ¹² or more per animal individual.

In other words, the human-derived cell number propagated by the method for producing hTCGF used in the present invention is about 10 ² to 10 ⁷ times or more than the number of cells transplanted per animal, and inoculated into nutrient medium in vitro. It is a very good method for the production of hTCGF because it reaches about 10 ¹ to 10 ⁶ times or more in the case of growth. Thus, the method of producing hTCGF from the living cells derived from human body which proliferated can be performed arbitrarily. For example, cells derived from the human body proliferated in a floating phase to the ascites in the abdominal cavity or collected or dispersed after subcutaneous growth and harvested, and the cells are collected at about 20 to 40 ° C. In the nutrient incubator maintained in the above, the cell concentration is raised to about 10 ⁴ to 10 ⁸ / ml to produce hTCGF. At this time, if necessary, a hTCGF inducer may be applied. The hTCGF inducer may be any substance capable of inducing the production of hTCGF from cells derived from the human body, which can be obtained by propagating using warm-blooded animals, for example, plant hemagglutinin, concanavalin A, foucid mitogem mitotic substances such as pokeweed mitogen, lipopolysaccharide and bacteria; Viruses; Hexane; Polynucleotides; It can be selected from the preceding triggers.

In addition, when hTCGF is produced, the hTCGF stabilizer may be added to stabilize the generated hTCGF and increase the production of hTCGF. The hTCGF thus produced can be easily purified and collected by performing known purification separation methods, for example, salting out, dialysis, filtration, centrifugation, concentrated freeze drying, and the like. In addition, when a high degree of purification is required, new well-known methods such as adsorption / desorption, gel filtration, affinity chromatography, isoelectric point fractionation, electrophoresis, etc. may be newly combined with ion exchangers. You can also harvest.

Of course, hTCGF prepared according to the present invention is not immunologically different from hTCGF prepared in the conventionally known human serum, and does not contain a pyrogenic substance or hepatitis virus. Therefore, hTCGF can be used alone or in combination with one or two or more substances such as vitamins, hormones, anticancer agents, etc., and can be advantageously used for the prevention or treatment of human diseases as oral medicines and injections. In addition, throughout the specification, the activity of hTCGF was modified according to the method described in Gillis S. et. Al., J. Immuno, Vol. 120, pages 2027-3032 (1978) in the binding assay of 3H-thymidine. Measured by.

That is, thymus cells from BALB / C mice were collected in microplates at 100 μl (10 ⁵ cells) per well, and 100 μl of the stepwise diluted hTCGF-containing solution was added thereto at 37 ° C. After daily in vitro culturing, 3H-thymidine was added at 0.5 μ Ci / well and after 4 hours, the amount of 3H-thymidine bound intracellularly was measured with a liquid scintillation counter. The active unit of hTCGF was a dilution factor of 5,000 cpm. In the following, the present invention has been described in more detail by enumerating two or three examples, but these examples are merely illustrative of the preferred embodiments of the present invention, and the present invention is not limited thereto.

Example 1

MT-1 cells cultured with human peripheral blood cells were implanted under grown Nude mice subcutaneously, and then grown for 3 weeks in a conventional manner. About 10 g of subcutaneous species were removed, cut, and suspended in physiological saline containing trypsin to disperse the cells. The cells were washed in serum-free RPMI 1640 medium (pH 7.2), then suspended in the same medium to a cell concentration of about 1 × 10 ⁵ / ml, and stored at 37 ° C. for 3 days to produce hTCGF. After the incubation was completed, the amount of hTCGF contained in the supernatant obtained by centrifuging the cell suspension for 30 minutes at about 8000 rpm was about 1600 units per 100 μl of the suspension. As a control, MT-1 cells were obtained using cells obtained by tissue culture in vitro at 37 ° C using Eagle's medium (pH 7.2) containing calf serum 1v / v% and meat extract 20v / v%. HTCGF was produced in the same manner as described above, and the yield was only about 40 units per 100 µl of the suspension.

Example 2

One way tumor the tumor cells and rimpa UGG shape or malba cells (Namalwa cell) was excised, sejeol, distributed from the patient to 140mM Nacl, 54mN KCl, 1mM NaH 2 PO 4, 10 respectively, in salt solutions containing 2mM CaCl ₂ ³ / Float to ml, and the above-mentioned salt solution containing Sendaivirus, previously inactivated by ultraviolet rays, is mixed below the freezing point, and then transferred to a constant temperature water bath at 37 ° C. after about 5 minutes, and then cell-fused by stirring for about 30 minutes. HTCGF acid production was introduced into subhocyte-shaped malamba cells. The lymphocyte-shaped namalba cells were transplanted into the intraperitoneal cavity of the grown Nude mice, and then bred for 5 weeks in a conventional manner. About 15 g of the produced species was extracted and treated in the same manner as in Example 1 except for using a medium to which 1 μg of plant hemagglutinin was added per ml to produce hTCGF. The yield of hTCGF per 100 μl of suspension was about 5,800 units.

In the same manner as in Example 1, cells obtained by fusion of lymphocyte-like namalba cells, which had been cell-fused for control, were produced using hTCGF, resulting in only about 90 units of production per 100 μl of suspension.

Example 3

Hamster-neonatal infants were previously injected with antiserum prepared by a known method from rabbits to attenuate the hamster's immune response, and then subcutaneously implanted with lymphocyte-like JBL cells into which hTCGF production was introduced according to the method of Example 2. The animals were bred for 3 weeks in the usual manner. About 18 g of the resulting type was extracted and treated in the same manner as in Example 1 except that 50 μg of concanavalin A was added per ml, and then treated with Eagle medium containing 20v / v% of meat extract instead of RPMI 1640 medium (pH7). HTCGF was produced in the same manner as in Example 1 except that .2) was used. The yield of hTCGF per 100 μl of suspension was about 1,200 units. Lymphocyte-like JBL cells, which were cell-fused for control, were cultured and grown in vitro in the same manner as in Example 1, whereby hTCGF was produced, resulting in only about 150 units of production per 100 μl of suspension.

Example 4

Except for using lymphocyte-shaped BALL-1 cells in place of lymphocyte-shaped namalba cells in a newborn mouse vein, the same procedure as in Example 2 was performed to transplant lymphocyte-shaped BALL-1 cells into which hTCGF-producing ability was introduced. Was bred for 4 weeks. About 35 g of the produced species was extracted and treated in the same manner as in Example 2 to produce hTCGF. The yield of hTCGF per 100 μL of suspension was about 5,300 units. On the other hand, as a control, the production of hTCGF by incubation in vitro resulted in only about 110 units of production per 100 μl of suspension.

Example 5

The grown mouse was irradiated with a gamma ray of about 400rem to attenuate the immune response of the mouse. Subsequently, mono-1 cells cultured with human peripheral blood cells were implanted subcutaneously, and grown for 4 weeks in a conventional manner. About 20 g of subcutaneous type was removed and treated in the same manner as in Example 3, and then hTCGF was produced. The yield of hTCGF per 100 μl of suspension was about 3,500 units. On the other hand, the culture was grown in vitro for control and the production of hTCGF was only about 30 units per 100 µl of the suspension.

Example 6

MT-1 cells used in Example 1 were suspended in physiological saline and embedded in the peritoneal cavity of mice grown in a plastic cylindrical diffusion chamber of about 10 ml with a thin-film filter having a diameter of about 0.5 microns. The mice were bred for 4 weeks in the usual manner and then the diffusion chamber was lifted. As such a cell concentration of human origin were obtained by the month of about 10 ⁹ / ml, a fact that leads to about 10 ^twice or more it was found that when cultured in a carbon dioxide incubator in vitro. The cells thus obtained were treated in the same manner as in Example 3 to produce hTCGF. The yield of hTCGF per 100 μl of suspension was about 3,200 units.

Example 7

Lymphocyte-like BALL-1 cells in which hTCGF acid production was introduced by the method of Example 4 were implanted into the fertilized eggs of chickens which were previously warmed at 37 ° C. for 5 days, and then stored at 37 ° C. for 1 week. The eggs were fertilized and proliferated cells were collected and treated in the same manner as in Example 1 to produce hTCGF. The yield of hTCGF per 100 μl of suspension was about 2,400 units.

Claims (16)

Human T-cell proliferation factor The human-derived cells having the ability to produce acid are transplanted into the warm-blooded animal, and the nutrient solution supplied to the animal is used to proliferate the cells derived from the human body, and the tumors formed in the animal body are extracted and crushed and proliferated. A method for producing human T cell growth factor (hTCGF), characterized by releasing human T cell growth factor from human cells. The method of claim 1, wherein the step of releasing the human T cell proliferation factor comprises acting on the proliferated human T cell proliferation factor inducing agent. The method of claim 1, wherein the cells derived from the human body having a human T cell proliferation factor production ability are human peripheral blood cells (peripheral blood cells), human splenocytes, human tonsil cells, human tonsil tumor cells, human liver cancer cells, human lung cancer cells And a method for producing human T cell proliferation factor, which is selected from the group consisting of cell lines of these cells. The method of claim 1, wherein the human-derived cells are KS-5, MOLT-3, MT-1, Mono-1, or OUMS-19 cells. The method according to claim 1, wherein the cells derived from the human body are hybridized human lymphoblastiod cells. The human T cell proliferation factor according to claim 5, wherein the lymphocyte-like cells are one selected from the group consisting of Namalwa cells, BALL-1 cells, NALL-1 cells, TALL-1 cells and JBL cells. Way. 3. The human T cell proliferation factor according to claim 2, wherein the human T cell proliferation factor inducing agent is one selected from the group consisting of mitogen, phytohaemagglutinin, concanavalin A, virus, hexane, polynucleotides, and mixtures thereof. Method of Preparation The human T-cell growth factor of claim 1, wherein the warm-blooded animals other than the human body are chickens, pigeons, dogs, cats, monkeys, goats, pigs, cattle, horses, rabbits, guinea pigs, mice, hamsters, mice, or nude mice. Manufacturing method. Cells derived from the human body having human T cell proliferation factor production ability are injected into a device capable of supplying nutrient fluids from warm-blooded animals other than the human body, and the nutrient fluids from warm-blooded animals are inserted into the warm-blooded animals or left in vitro. After supplying human-derived cells and breeding warm-blooded animals so that human-derived cells proliferate using nutrient fluids, the proliferated human-derived cells are collected to release human T-cell proliferation factors from the proliferated human-derived cells. Method for producing human T cell proliferation factor (hTCGF). The method of claim 9, wherein the cells derived from the human body having a human T cell proliferation factor production ability is human peripheral blood cells, human spleen cells, human tonsil cells, human tonsil abscess cells, human liver cancer cells, human lung cancer cells and culture of these cells A method for producing human T cell growth factor, which is one selected from the group consisting of chemotactic cells. The method of claim 9, wherein the human-derived cells are KS-5, MOLT-3, MT-1, Mono-1, or OUMS-19 cells. The method of claim 9, wherein the human-derived cells are lymphoblast-like cells. The method of claim 12, wherein the lymphocyte-like cells are one selected from the group consisting of namalba cells, BALL-1 cells, NALL-1 cells, TALL-1 cells, and JBL cells. The method of claim 9, wherein the step of releasing the human T cell proliferation factor comprises the step of interacting the proliferated human derived cells with human T cell proliferation factor inducing agent. The method according to claim 9, wherein the human T cell growth factor inducing agent is selected from the group consisting of mitogen, plant hemagglutinin, concanavalin A, virus, hexane, polynucleotide, and mixtures thereof. . The human T-cell growth factor of claim 9, wherein the warm-blooded animal other than the human body is a chicken, pigeon, dog, cat, monkey, goat, pig, cow, horse, rabbit, guinea pig, rat, hamster, mouse or nude mouse. Manufacturing method.