CH652745A5 - PROCESS FOR THE PRODUCTION OF HUMAN ADRENOCORTICOTROPIC HORMONE. - Google Patents

Description

The present invention relates to a process for the production of human adrenocorticotropic hormone, hACTH.

This hormone, also known by the abbreviation hACTH, is a hormone secreted by the anterior lobe of the pituitary gland, which stimulates the synthesis of steroid hormones and the secretion of human insulin.

The incumbent studied processes for the mass production of hACTH. These efforts have led to the unexpected discovery that human lymphoblastoid cells capable of producing this hormone are suitable for the massive production of hACTH, due to the very high values of their multiplication speed and their production of hACTH per cell.

The process mentioned at the start is therefore characterized according to the invention by the characterizing part of claims 1.

The method according to the invention, in addition to an increased production of hACTH, does not require or requires much less nutritive medium containing expensive serum for cell multiplication; it makes maintenance of the balance of the culture medium, during cell multiplication, much easier than in the case of tissue cultures in vitro. In particular, it is possible to easily multiply any human lymphoblastoid cell capable of producing hACTH, by using a nutritive liquid from the organism of a warm-blooded animal, by transplantation of the cells into the organism of the animal. or by suspending them in a conventional diffusion chamber designed to receive the nutritive liquid from the organism, the animal being fed in the usual way. In addition, the method also provides more stable and abundant cell multiplication, with increased production of hACTH per cell.

The invention further relates to a method according to the preamble of claim 5, said method being characterized by the characterizing part of this claim.

As far as usable human lymphoblastoid cells are concerned, any lymphoblastoid cells can be taken, provided that they produce hACTH and multiply in the body of the warm-blooded animal. The most preferred human lymphoblastoid cells are those into which genetic sites have been introduced, controlling the production of hACTH, human cells capable of producing this hormone, for example cells which naturally produce hACTH, such as those anterior pituitary lobe, pituitary tumor cells or chromophobic adenoma, or other human cells that produce ectopic hACTH, such as pancreatic carcinoma cells reaching islets of Langerhans, or lung carcinoma cells. The introduction can be done by cell fusion using polyethylene glycol or the Sendai virus, or alternatively by a technique of genetic recombination with DNA-ligase, nuclease and DNA polymerase, or by human lymphoblastoid cells producing ectopic hACTH.

As the use of human lymphoblastoid cells causes the formation of a massive tumor that is easy to disintegrate when the cells are transplanted into the organism of a warm-blooded animal and that the massive tumor is hardly contaminated by the cells of the Host animal, multiplied living human lymphoblastoid cells can be easily harvested.

As warm-blooded animals, any animal can be used as long as human lymphoblastoid cells develop in its body. For example, it is advantageous to use volatiles, such as chicken or pigeon, and mammals, such as dog, cat, monkey, goat, pig, cow, horse, rabbit, guinea pig, rat, hamster, mouse or naked mouse. As the transplantation of human lymphoblastoid cells to the animal causes an undesirable immune reaction, the use of a newborn or very young animal, preferably at the earliest possible stage, for example of egg, embryo or of fetus, is desirable. To reduce the immune reaction as much as possible, the animal can be treated before cell transplantation by irradiation with x or y rays at a rate of about 200 to 600 rems or by injection of an antiserum or a prepared immunosuppressive agent according to a conventional process. As the naked mouse, when used as a warm-blooded animal, exhibits a weaker immune reaction even in the adult state, it is practically possible to transplant all human lymphoblastoid lines, provided that they multiply rapidly, without the need for pre-treatment.

Stabilized cell multiplication and increased hACTH production can be achieved by repeated transplantation with one or more combinations of different warm-blooded animals; the objectives can be achieved by first implanting human lymphoblastoid cells in hamsters so that they multiply, and then re-implanting them in naked mice. Repeat transplantation can be done with animals of the same class or group or the same species or genus.

Human lymphoblastoid cells can be implanted in any site of the animal on the only condition that they multiply there: for example, implantation can be carried out in the allantoic cavity or by intravenous, intraperitoneal or subcutaneous routes .

Apart from direct cell transplantation in the organism of an animal, it is possible to multiply lympho-

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any conventional human blastoids capable of producing hACTH, using the nutritive liquid supplied by the body of a warm-blooded animal, by inclusion, for example intraperitoneally, in the body of the animal d '' a conventional diffusion chamber of appropriate shape and size provided with a porous filter membrane, an ultrafilter or hollow fibers having pores of approximately 10 ~ to 10 ~ m in diameter preventing contamination of the chamber by host animal cells. As the diffusion chamber can, if necessary, be designed to be able to be placed for example on the body of the host animal and to allow the circulation of the animal's liquid in the chamber, the observation of the cell suspension contained in the room by one or more transparent side windows with one or more walls of the room and the replacement and exchange with a new room, cell production per host can be increased during the life of the animal, without that there is no need to sacrifice it. In addition, when using such a diffusion chamber, as there is no immune reaction due to the absence of direct contact of human cells with the cells of the host animal, various warm-blooded animals can be used as hosts, without pretreatment to reduce their immune responses, and multiplied human lymphoblastoid cells can be easily harvested.

We can easily feed the host animal, in which we have implanted human lymphoblastoid cells in the usual way,

even after cell transplantation, and no special care is needed.

Cell multiplication usually reaches a maximum in 1 to 20 weeks after cell transplantation. The number of human lymphoblastoid cells obtained per host can be between approximately 107 and 1012 or more. In other words, the number of human lymphoblastoid cells implanted in the animal's body increases by about 102 to 107 times or more or from about 10 to 106 times or more compared to what the obtained by the tissue culture method in vitro with a nutrient medium; therefore, human lymphoblastoid cells are suitable for the production of hACTH.

For the induction of hACTH, any method can be used in which the human lymphoblastoid cells obtained release this hormone. For example, the human lymphoblastoid cells obtained by suspension are multiplied in suspension in the ascites fluid and by harvesting in this ascites fluid, or by the extraction of the massive tumor formed under the skin, and by harvesting after disintegration of the massive tumor, to obtain a cell concentration of approximately 104 to 10 s cells / ml in a nutritive medium preheated to a temperature of approximately 20 to 40 ° C; then they are subjected to the action of an inducer of adrenocorticotropic hormone at this temperature, for approximately 1 to 50 h, to induce the production of this hormone.

As useful inducers of adrenocorticotropic hormone, any inducer capable of inducing the release of this hormone by human lymphoblastoid cells can be used. The preferred adrenocorticotropic hormone inducers are vasopressin, atropine methylnitrate, insulin, glucagon, a bacterial endotoxin, serotonin and propanolol.

The licensee discovered that after induction of hACTH a large amount of melanocyte stimulating human hormone, hereinafter abbreviated as hMSH, and human lipotropin hLPH, is produced simultaneously.

One can easily collect hACTH, hMSH and hLPH according to purification and separation techniques using conventional operations, such as salting out, dialysis, filtration, centrifugation, concentration and lyophilization. If a more purified preparation is desirable, it can be obtained by combining the aforementioned techniques with other conventional operations, such as adsorption and desorption with ion exchange, gel filtration, affinity chromatography, isoelectric point fractionation. and electrophoresis.

The hACTH preparation thus obtained can be used advantageously alone or in combination with one or more agents for administration by injection, externally, internally or for diagnostic purposes in the prevention and treatment of diseases. human, such as pituitary insufficiency.

In the present description, the production of hACTH is determined according to a radioimmunological method, as described by S. Matsukura et al. in "J. Lab. Clin. Med. ”, Vol. 77, pp. 490-500 (1971); it is expressed by weight per milliliter of cell suspension.

Also the productions of hMSH and hLPH are determined by a radioimmunological method, as described by K. Abe et al. in "J. Clin. Invest. ”, Vol. 46, pp. 1906-1916 (1967). The total amount of the hormones is first determined with a standard of human ß-MSH, then the weight ratio of the hormones is calculated from the amounts of human ß-MSH and ß-LPH isolated by gel filtration.

Embodiments of the invention are described below.

Example 1:

Human lung carcinoma cells obtained by extraction are suspended together from a patient suffering from pulmonary carcinoma, followed by chopping, and human leukemic lymphoblastoid cells of the Namalwa line in a container with a saline solution 140 mmol in NaCl, 54 mmol in KCl, 1 mmol in NaH2PO4 and 2 mmol in CaCl2, to obtain concentrations of each cell type of approximately 101 cells / ml. The frozen cell suspension is mixed with a fresh preparation of the same saline solution containing Sendai virus previously inactivated by ultraviolet irradiation; the whole is placed in an oven at 37 ° C for 5 min, after mixing, then stirred for 30 min to effect cell fusion; the hACTH production capacity of human lung carcinoma cells is thus introduced into the human leukemic lymphoblastoid line. After cloning, by a conventional method, of the hybridoma cell strain capable of producing hACTH, this strain is implanted in naked adult mice which are then fed in the usual way for 5 weeks. The massive tumors formed under the skin, each weighing about 15 g, are extracted and broken up by chopping and suspended in a physiological saline solution containing trypsin. After washing the cells with Earle's medium 199 (pH 7.2) added with 10% by volume of fetal calf serum, the cells are resuspended, at a concentration of approximately 10 ° cells / ml, in a preparation fresh from the same medium containing 0.1 (μg of glucagon / ml as an inducer, then incubated at 35 ° C. for 20 h to induce the production of hormones. Then, the cells are subjected to an ultrasound treatment and the hACTH, hMSH and hLPH in the supernatant.

The production of hACTH is 250 ng / ml of cell suspension, that of hMSH of 580 jig / ml and that of hLPH of 40 ng / ml.

Control cells are obtained by implantation of human cells of pulmonary carcinoma into adult naked mice, by feeding the animals in the usual manner for 5 weeks, by extraction of the massive tumors formed under the skin and weighing approximately 5 g each and by disintegration of these tumors. These control cells obtained are treated as above to induce the production of hormones. The production of hACTH is then only 600 ng / ml of cell suspension, that of hMSH of 1.2 μg / ml and that of hLPH of 350 ng / ml.

Example 2:

As in example 1, human cells of chromophobic adenoma are obtained, obtained by extraction from a patient suffering from chromophobic adenoma of the pituitary gland and by hashing,

with human leukemia lymphoblastoid cells of the JBL line to introduce the production capacity of hACTH

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human chromophobic adenoma cells in the human leukemic lymphoblastoid line. After cloning, by a conventional method, of the strain of hybridoma cells capable of producing hACTH, these hybridoma cells are implanted subcutaneously in newborn hamsters, to which an antiserum has previously been injected prepared on the rabbit to reduce their immune reactions; the animals are fed in the usual way for 3 weeks. The massive tumors formed under the skin and weighing about 10 g each are extracted, they are disintegrated by chopping and suspended in a physiological saline solution containing collagenase. After washing the cells with minimal Eagle essential medium (pH 7.2) added with 5% by volume of human serum, the cells are resuspended, at a concentration of approximately 10 cells / ml, in a fresh preparation of the same medium containing 3 | iU of vasopressin / ml as inducer; incubated at 37 ° C for 15 h to induce the production of hormones. The production determined as in Example 1 is for the hACTH of 380 ng / ml of cell suspension, for the hMSH of 740 ng / ml and for the hLPH of 510 fj.g / ml.

The control cells obtained by implanting human chromophobic adenoma cells in newborn hamsters, by feeding the animals in the usual way for 3 weeks, by extracting massive tumors are treated in a similar manner, to induce the production of hormones. formed under the skin and weighing about 3 g each and by disintegration of these tumors. The production of hACTH per milliliter is only 750 ng / ml, that of hMSH 560 ng and that of hLPH 430 ng.

Example 3:

Newborn rats are implanted intravenously with human leukemic lymphoblastoid cells of the Ball-1 line, to which the ability to produce hACTH of human carcinoma human cells has been conferred, as in Example 1; the rats are fed in the usual way for 4 weeks. The massive tumors formed weighing approximately 30 g each are extracted and disaggregated. After washing with RPMI 1640 medium (pH 7.4) supplemented with 10% by volume of fetal calf serum, the cells are resuspended, at a concentration of approximately 107 cells / ml, in a fresh preparation of the same medium 6 mmol in serotonin (inducer), then incubated at 30 ° C for about 40 d. The production of hACTH is 730 µg / ml cell suspension and that of hMSH 940 µg / ml.

Control cells obtained by implantation of human lung carcinoma cells in newborn rats are treated as above, by feeding the animals in the usual manner for 4 weeks, by extraction of the massive tumors formed weighing approximately 5 g each and by disintegration. of these tumors. The production of hACTH is then only 650 ng / ml of cell suspension and that of hMSH of 1.8 μg / ml.

Example 4:

After irradiating adult mice with an X-ray dose equivalent of approximately 400 rems to reduce their immune reactions, the animals are implanted subcutaneously with human leukemia lymphoblastoid cells of the Nall-1 line, to which the hACTH production capacity of human chromophobic adenoma cells, as in Example 2, and they are fed in the usual way for 3 weeks. Massive tumors formed under the skin and weighing about 15 g each are extracted and disaggregated. The cells are treated as in Example 2 to induce the production of hormone. The production of hACTH is 480 ng / ml of cell suspension and that of hLPH of 350 ng / ml.

Control cells obtained by implantation of human chromophobic adenoma cells in the mouse are treated as above. The feeding of the animals takes place in the usual way for 3 weeks, after which one proceeds to the extraction of the massive tumors formed weighing about 5 g each and to the disintegration of these tumors. The production of hACTH is only 520 ng / ml of cell suspension, that of hLPH is 790 ng / ml.

Example 5:

Human leukemia lymphoblastoid cells of the Tall-1 line are suspended in physiological saline, to which the hACTH production capacity of human lung carcinoma cells has been conferred, in the same manner as in example 1; the solution is introduced into a cylindrical plastic diffusion chamber with an internal volume of approximately 10 ml and provided with a filter membrane having pores of approximately 0.5 nm. After inclusion of the chamber in the peritoneum of an adult rat, the animal is fed in the usual way for 4 weeks, then the chamber is removed. The density of human cells in the chamber obtained in the above operation is approximately 7 x 108 cells / ml, which is approximately at least 102 times greater than that obtained by culture in vitro with an incubator. to C02. The human cells thus obtained are treated as in Example 3 to induce the formation of hormones. The production of hACTH is 280 ng / ml of cell suspension, that of hMSH of 770 ng / ml and that of hLPH of 160 ng / ml.

Control cells obtained as above are treated by suspending human lung carcinoma cells in physiological saline, by transferring the cell suspension obtained into a cylindrical plastic diffusion chamber having an internal volume of approximately 10 ml and provided with a filter membrane having pores of approximately 0.5 nm, by inclusion of the chamber in the peritoneum of an adult rat, by feeding the animal in the usual way for 4 weeks and by harvesting the cells human multiplied, or approximately 107 cells / ml. The production of hACTH is only 480 ng / ml of cell suspension, that of hMSH of 2.3 ng / ml that of hLPH of 250 ng / ml.

Example 6:

Human leukemic lymphoblastoid cells of the JBL line are implanted in the allantoic cavities of preincubated embryo eggs at 37 ° C. for 5 d, and have been given, as in Example 1, the capacity for producing hACTH of the cells. human lung carcinoma. After incubating the eggs at this temperature for another 1 week, multiply human lymphoblastoid cells are collected. These cells are treated as in Example 2 to induce the production of hACTH. The production of hACTH is 520 ng / ml of cell suspension.

In a control experiment, where human lung carcinoma cells are implanted in the allantoic cavities of embryonated eggs, no cell multiplication is observed.

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Claims (9)

652,745 1. Process for the production of the human adrenocorticotropic hormone, hACTH, characterized in that it consists in causing human lymphoblastoid cells, capable of producing this hormone, to multiply in a nutritive liquid of the organism of an animal to warm blood, by transplanting these cells into the animal's body itself, or into a device receiving the animal's nutrient fluid, and then letting the multiplied cells release the hormone. 2. Method according to claim 1, characterized in that the hormone produced is accompanied by the melanocyte stimulating hormone, hMSH, and / or lipotropin, hLPH. 2 3. Method according to one of claims 1 or 2, characterized in that induces the release of hACTH in human lymphoblastoid cells multiplied by means of one or more substances: Vasopressin, atropine methylnitrate, insulin, glucagon, a bacterial endotoxin, serotonin, propanolol. 4. Method according to one of claims 1 to 3, characterized in that the warm-blooded animal is: chicken, pigeon, dog, cat, monkey, goat, pig, cow, horse, rabbit, guinea pig, rat, hamster , mouse or naked mouse. 5. Method for preparing human lymphoblastoid cells used in the method according to one of claims 1 to 4, characterized in that human lymphoblastoid cells are hybridoma cells which are obtained by cell fusion from a lymphoblastoid line human with human cells capable of producing hACTH. 6. Method according to claim 5, characterized in that the hybridoma cells are obtained by cell fusion of a human lymphoblastoid line with human cells of chromophobic adenoma. 7. Method according to claim 5, characterized in that said hybridoma cells are obtained by cell fusion of a human lymphoblastoid line with human cells of pulmonary carcinoma. 8. Method according to claim 5, characterized in that said lymphoblastoid line is a human leukemic lymphoblastoid line. 9. Method according to claim 5, characterized in that the human lymphoblastoid line is one of the Namalwa, Ball-1, Tall-1, Nall-1 and JBL lines.