JP2518634B2 - Method for producing Hitstumore Necrosis Factor - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides Namalwa cells, BALL-1 cells, TALL-1 cells, NALL-1 cells, M-, which have the ability to produce human Tumor necrosis factor (hTNF).
7002 cells, B-7101 cells, and human-derived cells selected from cultured mononuclear cells, granular leukocyte cells, and lymphoblast cells are grown to produce hTNF, which is then purified and collected. The present invention relates to a method for producing human Tumor Necrosis Factor having the following physicochemical properties.

Substance Proteinaceous substance Purification method It can be purified by a series of purification methods including adsorption / desorption on an ion exchanger, molecular weight fractionation by gel filtration, and affinity chromatography with Con A Sepharose under salting out with ammonium sulfate.

Those purified from the specific activity the purification method, in addition to the L ₉₂₉ cells, actinomycin D was measured the survival cell number after incubation for 18 hours in the coexistence, active cells to one unit when killed 50% Approximately 350,000 units / mg when measured by the measuring method
The specific activity of the protein is shown.

Biological effect Shows an antitumor effect against malignant tumors and does not adversely affect normal cells.

 Has hemorrhagic necrotic potential for Meth A sarcoma.

It has cytotoxic activity against _L929 cells.

Acute toxicity LD ₅₀ when injected intraperitoneally in mice on day 20 was
200,000 units / kg or more.

Tumor Necrosis F
Actor, hereinafter abbreviated as TNF. ) Is "Proceedings," such as EACarswell.
Proceedings of the Nationa of the National Academy of Sciences
Academy of Sciences, USA) ", Vol. 72, No. 9, No. 3
666-3670 (1975) and E. Pick
Ed., "Lymphokines", Volume 2, 235
~ 272 pages, published by Academic Press (1981), etc., for example, in rabbits, bacilli Calmette-Guerin (BCG), Corynebacterium parvu (Corynebacterium parvu)
m), is a name given to a substance having a Meth A sarcoma hemorrhagic necrosis ability, which is a proteinaceous substance that is induced and produced in its serum by parenterally administering a TNF inducer such as endotoxin, In particular, it is known to have a cytotoxic function for tumor cells.

Because of these functions of TNF, TNF has been expected as a therapeutic agent for malignant tumors from the beginning of its discovery.

Although TNF is prepared from the serum of animals such as rabbits and rats and is said to have no species-specificity, the therapeutically effective treatment of humans is that hTNF is essentially derived from living human cells. It is extremely safe and excellent in terms of side effects such as antigenicity that occur in the above.

However, a method for producing hTNF has not been known so far, and its action and effect are also unknown. Furthermore, hTNF
It is not known at all whether it is effective in treating human malignancies.

The present inventor has studied a method for producing hTNF that can be easily carried out on an industrial scale, and has earnestly studied whether or not the hTNF is useful as a therapeutic agent for malignant tumor.

As a result, they have found that human-derived cells can produce highly active hTNF and completed the present invention. Further, it was also found that the purified and collected hTNF exhibited cytotoxic activity against various human malignant tumors in a relatively small amount, and that hTNF is highly safe.

In the method for producing hTNF of the present invention, cells having an ability to produce hTNF are grown to produce hTNF, which is then purified and collected. For example, human-derived cells having hTNF-producing ability, or cells into which a gene having human hTNF-producing ability of human-derived cells is introduced by means of cell fusion or gene recombination, etc. are grown and produced from these cells.
hTNF is purified, harvested and manufactured.

To describe a preferred production example of hTNF, cells of human origin are transplanted into a warm-blooded animal body other than human, or inoculated into a diffusion chamber while receiving a body fluid containing nutrients from the animal body. HTNF is induced and produced with high activity by proliferating and allowing the resulting cells to act on a TNF inducer in vivo or in vitro.
By purifying and collecting this, hTNF can be easily produced in a large amount.

Thus, when human-derived cells are grown using a warm-blooded non-human animal and supplied with body fluid, unlike the case where live cells are grown in vitro, expensive serum is used. Not only is a nutrient medium containing, for example, unnecessary or largely saved, it is also extremely easy to maintain and manage during cell growth, and the produced hTNF activity is high. That is, human-derived cells are transplanted into a warm-blooded animal body other than human, or they are housed in a diffusion chamber capable of receiving the body fluid of the animal, and this chamber is embedded in the animal body for normal breeding. Then, the cells can easily grow using the body fluid containing the nutrients supplied from the warm-blooded animal body. Further, compared to the case of growing in vitro, the growth of the cells is stable, the growth rate is high, the amount of cells obtained is large,
Another significant feature is that the amount of hTNF per cell is significantly increased.

As human-derived cells, they can easily proliferate and
Any substance can be used as long as it has the ability to produce hTNF. For example, “Journal of Clinical Microbiology (Jo
urnal of Clinical Miorcbiology) ”, Volume 1, 116
~ Namalva (Namalv) described on page 117 (1975)
a) Cell, BALL-1 cell, TALL-1 cell, NALL- described in I. Miyoshi, "Nature", Vol. 267, pp. 843-844 (1977). 1 cell, "The Journal of Immunology (The Jour
nal of Immunology), 113, 1334-1345 (19
1974), cell lines such as M-7002 cells and B-7101 cells described above, and normal mononuclear cells, granular leukocyte cells, etc. were treated with various viruses, drugs, radiation, etc., and cultured into cell lines. Cells etc. are used freely, and also hTNF of these cells
Genes with productivity, for example, means of cell fusion using polyethylene glycol or Sendai virus,
TNA ligase, restriction enzyme (nuclease), by means of gene recombination utilizing an enzyme such as DNA polymerase, etc., can be introduced into a culture-strained lymphoblastoid cell, microorganism, etc. that can be more easily subcultured, It may be used by increasing the growth rate or by increasing the hTNF producing ability per cell, and is not limited to the cells described in the present specification. These cells can be used alone or in combination of two or more in the steps up to the production of hTNF described later. If desired, this can be combined with leukocytes prepared from, for example, fresh human blood.

Non-human warm-blooded animals used for cell proliferation include:
It is sufficient that human-derived cells can grow, for example, birds such as chicken and pigeon, dog, cat, monkey, rabbit, goat, pig, horse, cow, guinea pig, rat,
Mammals such as hamsters, normal mice and nude mice can be used.

Since transplanting human-derived cells into these animals may cause undesired immune reactions, the animals used should be as young as possible, i.e. eggs, embryos, fetuses, or neonates, in order to suppress these reactions as much as possible. , Those in childhood are preferred.

In addition, even if these animals are pre-treated, for example, by irradiating with about 200 to 600 rem X-rays or gamma rays, or by injecting an antiserum or an immunosuppressant, the immune reaction is weakened and transplanted. Good.

If the animal to be used is an immunodeficient animal such as a nude mouse or a nude rat, even if it is a grown animal, the immune response is weak, so without the need for pretreatment of these,
It is particularly convenient because human-derived cells that have been cultured into a cell line can be transplanted and can rapidly proliferate.

In addition, human-derived cells that have been cultured into a culture strain are transplanted between warm-blooded animals other than humans, for example, by first transplanting them into hamsters and growing them, and then transplanting these cells into nude mice. It is also free to further stabilize the growth of human-derived cells and further increase the amount of hTNF produced by them.

In this case, not only the same species and the same genus but also the same rope and the same gate may be transplanted. The site in the animal body where the human-derived cells are transplanted may be any site where the transplanted cells can proliferate, and for example, a urinary cavity, vein, abdominal cavity, subcutaneous site and the like can be freely selected.

Further, a porous filter membrane capable of blocking the passage of animal cells without directly transplanting human-derived cells into an animal body, for example, a membrane filter having a pore size of about 10 ^{-7 to} 10 ^-3 m, an ultrafiltration membrane. Alternatively, a known diffusion chamber having various shapes and sizes provided with hollow fibers or the like is embedded in the animal body, for example, in the abdominal cavity, and the body fluid containing nutrients is supplied from the animal body while the diffusion chamber described above is provided. Any human-derived cells that have been made into a culture strain can be grown.

If necessary, the solution containing the nutrients in the chamber is connected to the body fluid in the animal body, and a chamber for perfusion is attached to, for example, the surface of the animal body, and the growth state of human-derived cells in the chamber is increased. It is also possible to make it possible to see through the cells, or to make it possible to replace and replace only this chamber portion so as to grow cells with a long life span without sacrificing the animals, thereby further increasing the cell production amount per animal.

In the method using these diffusion chambers, human-derived cells do not come into direct contact with animal cells, so not only human-derived cells can be easily collected, but also there is little concern that an undesired immune reaction will occur. It has the feature that various warm-blooded animals can be used freely without worrying about the pretreatment to suppress the disease.

The maintenance of the transplanted animal is convenient because normal breeding management of the animal may be continued, and no special handling is required even after the transplantation.

The period for growing human-derived cells is usually 1 to 10 weeks, and the purpose can be achieved. The number of human-derived cells thus obtained is about 10 per animal.
It was found to reach ^{7 to} 10 ¹² or more.

In other words, the number of human-derived cells thus proliferated is about 10 ^{2 to} 10 ⁷ times the number of cells transplanted per animal individual,
Or even more, about 10 to 10 ⁶ times as much as inoculating an in vitro nutrient medium to grow, or more, which is very convenient for the production of hTNF.

From the human-derived living cells thus grown, hT
The method of producing NF is free. It is also possible to require a TNF inducer in the animal body in which it proliferated.
For example, TNF inducer is directly acted on human-derived cells grown in suspension in ascites in the abdominal cavity or subcutaneously generated tumor cells to induce and produce hTNF, and then the serum thereof,
HTNF may be purified and collected from ascites or tumor.

In addition, human-derived proliferating cells are taken out from animal individuals,
In vitro production of hTNF can also be achieved by acting a TNF inducer in vitro. For example, human-derived cells grown in ascites are harvested, or tumors containing human-derived cells that are generated subcutaneously are harvested and harvested, and the obtained cells have a cell concentration in a nutrient medium kept at about 20 to 40 ° C. The cells may be suspended at a concentration of about 10 ^{5 to} 10 ⁸ cells / ml, and a TNF inducer may act on the suspension to induce hTNF production, which may be purified and collected.

Furthermore, when human-derived cells are grown in a diffusion chamber, the grown cells remain in the chamber,
Alternatively, it can be removed from the chamber and acted with a TNF inducer to induce hTNF production.

Also, for example, in expanded human-derived cells, hTNF is first produced in the animal body, and then hTNF is induced and produced outside the animal body in human-derived cells collected from a specific site or whole of the same animal individual. How to let or hTNF once
By using the cells used for the production of hTNF more than once, or by increasing the number of cells obtained by exchanging the chamber embedded or connected in the animal body. It is also possible to further increase the amount of hTNF produced.

As the TNF inducer, one kind or two kinds of BCG, Corynebacterium parvum, lipopolysaccharide, endotoxin, polysaccharide and the like are usually used. In general, first, to a warm-blooded animal other than humans transplanted with human-derived cells, for example, BCG, Corynebacterium parval, etc., is parenterally administered, and after a certain period of time, Alternatively, human-derived cells may be collected and, for example, one or more of lipopolysaccharide, endotoxin, and polysaccharide may be allowed to act in vitro to induce and produce hTNF.

The hTNF thus produced is a known purification method for protein biomaterials, for example, salting out, dialysis, filtration, centrifugation,
It can be easily purified, separated and collected by performing concentration, lyophilization and the like. When a higher degree of purification is required, known methods such as adsorption / elution on an ion exchanger, gel filtration and isoelectric focusing, electrophoresis, high performance liquid chromatography, affinity chromatography can be combined. Therefore, it is also possible to collect the highest purity hTNF.

The activity of hTNF is described in "Lymphokines", edited by E. Pick, Volume 2, pp. 235-272,
Published by Academic Press (1981)
, And the presence or absence of Meth A sarcoma hemorrhagic necrosis in BALB / c mice, and using L-929 cells,
A sample of hTNF was added thereto, and the number of surviving cells after culturing in the coexistence of actinomycin D for 18 hours was measured. In this case, a known method was used in which 1 unit was defined when 50% of the cells were dead.

 Examples of the present invention will be described below.

Example A human-derived mononuclear cell treated with SV-40 virus and cultured and cultured was transplanted subcutaneously to a hamster that was just after birth, and after being bred for 1 week by an ordinary method, live cells of BCG were intraperitoneally injected. To 1
0 ⁷ cells were injected and further raised for 2 weeks. About 15g subcutaneously
The tumor was excised, cut into small pieces, suspended in a physiological saline containing trypsin, and the cells were dispersed and fractionated. The cells were washed with a minimal basal medium of Eagle (pH 7.2) containing 5 v / v% of human serum, and the cell concentration was adjusted to about 5 × 10 ⁶ cells / ml in a medium of the same composition kept at 37 ° C. It was diluted so that E. coli-derived endotoxin was added at a rate of about 10 μg / ml and kept for 16 hours to induce hTNF production. This is centrifuged at 4 ° C. and about 1,000 × g to remove the precipitate,
The resulting supernatant is dialyzed for 21 hours against physiological saline containing 0.01M phosphate buffer at pH 7.2, and the filtrate obtained by microfiltration is concentrated and freeze-dried to have hTNF activity. A powder was obtained.
The resulting powder was presented to the 11th International Symposium on Immunobiology (Sympos) on interferon production, standardization and clinical use held in Zakreb on June 8 and 9, 1977.
ium on Preparation, Standardization and Clinical Us
e of Interferon, 11th International Immunobiologica
l Symposium, 8 & 9 June 1977, Zagreb, Yugoslavia) In accordance with the method reported by G.Bodo, adsorption / desorption on ion exchanger, molecular weight fractionation by gel filtration, concentration and microfiltration Remove interferon,
Further, it was purified by affinity chromatography using ammonium sulfate salting-out Con A Sepharose to obtain about 1,000,000 units of high-purity hTNF which is characterized by having hemorrhagic necrosis ability for MethA sarcoma and having no adverse effect on normal cells. The hTNF thus obtained had a specific activity of about 350,000 units / mg protein without the inclusion of the inducer used.

The method for purifying hTNF using the affinity chromatography with Con A Sepharose is as follows. For example, when hTNF is purified and collected from a crude hTNF preparation containing human lymphotoxin (hLT) as an impurity, the crude hTNF preparation is salted out with 90% saturated ammonium sulfate and the precipitate is separated with Con A Sepharose. Elute hTNF by loading it onto a packed column and decreasing the ammonium sulfate concentration linearly from 90% to 0%. On the other hand, hL
Although T does not elute under the above-mentioned conditions in which hTNF elutes, it can be eluted by using α-methylmannoside (α-MM) as an eluent. The elution pattern at this time is shown in the figure.

As is clear from this figure, Con
Since the elution patterns of hTNF and hLT for A sepharose are completely different, hLTF was added to the crude hTNF preparation to be purified.
Even in the case where both are mixed, it is possible to individually separate, purify and collect both.

The hTNF of the present invention can be easily distinguished from hLT because of the following physicochemical properties of hTNF.

Substance Proteinaceous substance Purification method It can be purified by a series of purification methods including adsorption / desorption on an ion exchanger, molecular weight fractionation by gel filtration, and affinity chromatography with Con A Sepharose under salting out with ammonium sulfate.

Those purified from the specific activity the purification method, in addition to the L ₉₂₉ cells, actinomycin D was measured the survival cell number after incubation for 18 hours in the coexistence, active cells to one unit when killed 50% Approximately 350,000 units / mg when measured by the measuring method
The specific activity of the protein is shown.

Biological effect Shows an antitumor effect against malignant tumors and does not adversely affect normal cells.

 Has hemorrhagic necrotic potential for Meth A sarcoma.

It has cytotoxic activity against _L929 cells.

Acute toxicity LD ₅₀ when injected intraperitoneally in mice on day 20 was
200,000 units / kg or more.

Hereinafter, the efficacy, toxicity, usage and dose of hTNF produced by the production method of the present invention will be described.

Experimental Example 1 A human breast cancer tissue piece is transplanted subcutaneously on the back of a BALB / c-derived nude mouse. From the time when the tumor volume was about 200 ml, 100 or 1,000 units / kg of hTNF obtained in the above-mentioned example was intravenously injected once each time, and the mouse was killed on the 15th day to measure the tumor weight. The results are shown in Table 1. The control is hTNF.
Non-containing physiological saline was intravenously injected.

Experimental Example 2 Male BDF ₁ mice weighing about 25 g were grouped into 10 mice, and Lewis lung cancer cut into 2 mm square was subcutaneously transplanted on the back. From the 8th day after transplantation, 100 or 1,000 hTNF obtained in the above-mentioned Example
Each unit / kg was intravenously injected once a day, and on the 21st day, the mouse was killed and the tumor weight was measured.

The results are shown in Table 2. As a control, physiological saline containing no hTNF was intravenously injected.

Experimental Example 3 Acute Toxicity An acute toxicity test of the hTNF obtained in the above-mentioned Example was carried out using 20-day-old mice, and the toxicity of hTNF was extremely low, and LD ₅₀ when injected intraperitoneally was 200,000 units / kg
It turned out to be the above.

As is clear from the above results, the hTNF of the present invention is
It is extremely safe even from its effective amount and can be advantageously used for the treatment of various malignant tumors.

The malignant tumor in the present invention is a disease that is prevented or treated by HTNF, for example, breast cancer, lung cancer, liver cancer, bladder cancer, uterine cancer, gastric cancer, colon cancer, leukemia, lymphoma, skin cancer, neuroblastoma, etc. It is a malignant tumor.

Furthermore, when applied to malignant tumors, for example, a part of the tumor of a patient is taken, treated with the hTNF of the present invention in vitro to enhance the immunogenicity of the tumor, and then returned to the body of the tumor patient. Thus, this malignant tumor can be treated.

The adult daily dose of the hTNF of the present invention is 1 to 50,00.
It is 0,000 units, preferably 1 to 1,000,000 units for topical injection and topical application such as eye drops, 10 to 5,000,000 units for ointments, 100 to 10,000,000 units for systemic injections such as intravenous injection and intramuscular injection, and oral administration 100 to
Although it is 50,000,000 units, it can be appropriately increased or decreased 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.

Considering the effective amount, toxicity, safety and the like, the amount used per formulation is preferably 1 unit or more, and more preferably 10 to 1,000,000,000 units per gram.

The shape of the therapeutic agent for malignant tumor containing hTNF of the present invention as an active ingredient can be freely selected according to the purpose.

Orally administrable agents, capsules, tablets, enteric-coated preparations such as powders, rectal intraoral agents are rectal suppositories, and injection agents are, for example, freeze-dried injection agents that are dissolved in distilled water for injection at the time of use, It can also be used as a nasal drop or eye drop, or as an ointment.

Reference examples of the preparation are shown below, but the preparation is not limited thereto.

Reference Example 1 Injectable solution 5 ml of hTNF prepared in the above-mentioned example was added to 200 ml of physiological saline.
Dissolve 00,000 units and filter aseptically using a membrane filter. Fill 2 ml each of the filtrate into a sterilized glass container, freeze-dry, and stopper tightly to give a freeze-dried powder formulation.

The product is suitable for treating breast cancer, lung cancer, liver cancer, leukemia and the like.

Reference Example 2 Ointment The HTNF prepared in the above Examples was ground to a small amount of liquid paraffin according to a conventional method, and then vaseline was added to give an ointment at 20,000 units / g.

The product is suitable for treating skin cancer, breast cancer, lymphoma and the like.

Reference Example 3 Eye Drops 800 ml of distilled water, 5 ml of β-phenylethyl alcohol, and 20,000,000 units of HTNF prepared in the above-mentioned example were added with sodium chloride to make it isotonic, and distilled water was adjusted to 1,000 ml to prepare eye drops.

 The product is suitable for treating retinoblastoma and the like.

Reference Example 4 Enteric coated tablet When the hTNF prepared in the above-mentioned Example was tableted using a mixture of starch and maltose according to a conventional method, hTNF was added in an amount of 200,000 units per product (100 mg). To produce a tablet, which was coated with methyl cellulose phthalate to give an enteric coated tablet.

The product is suitable for treating colorectal cancer, colon cancer, liver cancer and the like.

[Brief description of drawings]

The figure shows the elution pattern when hTNF and hLT were eluted using affinity chromatography with Con A Sepharose.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (56) References Takeo Kuroyanagi, 1st volume, “New Immunology Monographs (6) Lymphokine,” Sho 54-4-1, P.I. 88-102 Concer, 45, P.I. 1248-1253 (1980) Concer Letters, 6, P.P. 235-240 (1979) Molecular Immunology, 17, P.M. 613 ~ 623 (1980)

Claims (1)

(57) [Claims] 1. Namalwa cells, BALL-1 cells, and T capable of producing human Tumor necrosis factor (hTNF).
ALL-1 cells, NALL-1 cells, M-7002 cells, B-7101
Characterized in that cells and human-derived cells selected from cultured mononuclear cells, granular leukocyte cells and lymphoblast cells are grown to produce human Tumor necrosis factor, which is purified and collected A method for producing human Tumor Necrosis Factor having the following physicochemical properties: Substance Proteinaceous substance Purification method It can be purified by a series of purification methods including adsorption / desorption on an ion exchanger, molecular weight fractionation by gel filtration, and affinity chromatography with Con A Sepharose under salting out with ammonium sulfate. Those purified by the purification method of specific activity above, in addition to the L ₉₂₉ cells, actinomycin D was measured the survival cell number after incubation for 18 hours in the coexistence, active cells to one unit when killed 50% Approximately 350,000 units / mg when measured by the measuring method
The specific activity of the protein is shown. Biological effect Shows an antitumor effect against malignant tumors and does not adversely affect normal cells. Has hemorrhagic necrotic potential for Meth A sarcoma. It has cytotoxic activity against _L929 cells. Acute toxicity LD ₅₀ when injected intraperitoneally in mice on day 20 was 20
It is 000 units / kg or more.