CH652748A5 - PRODUCTION OF HUMAN PARATHYROIDIAN HORMONE. - Google Patents

Description

The present invention relates to a process according to the preamble of claim 1, namely a process for the production of parathyroid hormone, human, hereinafter abbreviated as HPTh.

HPTh is a hormone secreted by the parathyroid glands, which regulates the synthesis of activated vitamin D3, stimulates the release of calcium from the bone and leads to an increase in the level of calcium in the blood. To date, there is no industrial scale production process for HPTh at low cost.

The present invention is based on the unexpected finding that certain human lymphoblastoids capable of producing HPTh are suitable for the industrial production of this hormone, because of their very high multiplication power and their very high production of HPTh per cell.

The new process is characterized according to the invention by the characterizing part of claim 1.

The method according to the invention, while leading to a higher production of HPTh, requires little or no nutrient medium containing expensive serum for cell multiplication, and it makes it possible to maintain, more easily than in the case of tissue culture in vitro, the culture medium during cell multiplication. In particular, all human lymphoblastoids capable of producing HPTh can be easily multiplied, by the use of nutritive body fluid, originating from a warm-blooded animal, by transplantation of the cells in question into the body of the animal, or by suspending them in a diffusion chamber equipped to receive the nutritive body fluid, the animal being fed in the usual way. This process also gives a more stable and higher cell multiplication and a higher production of HPTh per cell.

The invention further relates to a method according to the preamble of claim 7, characterized by the characterizing part of this claim.

All human lymphoblastoids are suitable as they produce HPTh and multiply rapidly in the body of a warm-blooded animal. The preferred human lymphoblastoids are those which have genetic sites controlling the production of hPTh of human cells, producing by nature this hormone, like normal or transformed parathyroid cells, or human cells which produce ectopic hPTh like the cells of carcinoma of the lung, of ovarian tumors, of carcinoma of the kidney or of the liver, the said sites being introduced by means of cell fusion, obtained using polyethylene glycol, or by a technique of genetic recombination using DNA ligase, nuclease and DNA polymerase; other human lymphoblastoids producing ec-topical hpth are also suitable. As the transplantation into the animal body of these human lymphoblastoids results in the formation of massive tumors which can be easily disintegrated, and since these tumors are hardly contaminated by the cells of the animal host, the harvesting of the cells of multiplied human lymphoblastoids living is easy.

Suitable as usable animals all those in which cells can multiply, for example poultry like chicken and pigeon, mammals like cat, dog, monkey, goat, pig, cow, horse, rat, guinea pig, hamster, mouse or naked mouse . As this cell transplantation causes an undesired immunoreaction, it is desirable to use newborn or young animals, or at the youngest stage possible, such as egg, embryo or fetus. In order to reduce immunotherapy as much as possible, the animal can be treated, before the transplantation of the cells, by x-ray or x-ray irradiation, of approximately 200 to 600 rems, or by injection of antiserum or immunosuppressive agent prepared according to a conventional method. As the naked mouse, used as a warm-blooded animal, exhibits the weakest immunoreaction, even in adulthood, it can advantageously be used without pretreatment, to transplant and rapidly multiply therein human lymphoblastoid lines capable of produce HPTh.

Stabilized cell multiplication and increased production of hPT can be achieved by repeated transplantation, using combinations of different warm-blooded animals; these objectives can be achieved by first implanting the cells in the hamster, where they multiply, then by re-implanting them in the naked mouse. In addition, successive transplantation can be performed in animals of the same class or division, as well as in those of the same species or the same genus.

With regard to the location of the implantation of human lymphoblastoids, all the sites of the animal are suitable, provided that these cells can multiply there, for example the allanotic cavity, or the intraperitoneal, intravenous or sub- cutaneous.

Besides this direct transplantation of cells into the body of the animal, there is the possibility of easily multiplying the conventional lines of human lymphoblastoids, capable of producing HPTh, by using the nutritive body fluid originating from the animal, thanks to the inclusion, for example intraperitoneally, in the body of the animal of a conventional diffusion chamber, of appropriate size and shape, provided with a porous filtering membrane, an ultrafilter or hollow fibers of a diameter of pore in the range of 10 ~ 7 to 10 ~ 5 m, which prevents contamination of the chamber

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diffusion by the host cells while allowing the supply of nutritious body fluid from the animal to the cells. In addition, since the diffusion chamber can be designed, if necessary, to be placed on the animal host and allow the body fluid of the latter to circulate in the chamber, the walls of the latter may be provided with side windows, transparent, allowing observation of the cell suspension, as well as replacement and exchange with a cool chamber; cell multiplication is thus increased to an even higher level, related to the lifespan of the animal, and production per animal is further increased without sacrifice of the host. When this diffusion chamber is used, as the multiplied human lymphoblastoids can be easily harvested, and there is no manifestation of immunoreaction, due to the absence of direct contact between human cells and those of the The animal host can be used as host, in accordance with the invention, without pretreatment intended to reduce the immunoreaction, any warm-blooded animal.

The feeding of the animal host, to which human cells have been implanted, can easily be carried out by conventional method,

even after cell transplantation, and it doesn't require any special care.

Maximum cell multiplication is reached approximately 1 to 20 weeks, usually 1 to 5 weeks, after implantation of the cells.

107-1012 or more human lymphoblastoids can be obtained per host. In other words, the number of human cells implanted in the animal host increases 102 to 107 times or more, or is approximately 101 to 106 times or more that which is obtained with a method of tissue culture in in vitro using a nutrient medium; these human lymphoblastoids are therefore advantageously usable for the production of hPTh.

With regard to the process by which human lymphoblastoids, multiplied by one of the procedures described above, can release HPTh, any means can be used to release HPTh from these cells. For example, human lymphoblastoids, obtained by multiplication in ascites, in suspension and harvest from this ascites, or by extraction of massive tumor formed under the skin, and harvest after disintegration of the tumor, are suspended to reach a concentration 104 to 108 cells / ml in a nutritive medium, preheats to a temperature of the order of 20 to 40 ° C, then incubated at this temperature for 1 to 100 h approximately to give HPTh.

The increase in the production of HPTh can be carried out by the presence of one or more of the following compounds: amino acids like lysine, arginine, glycine, leucine and cysteine, mineral salts like chloride of sodium, potassium or calcium, and magnesium sulfate, and hormones like dopamine, isoproterenol, epinephrine and norepinephrine.

The HPTh thus obtained can be easily collected using purification and separation techniques using conventional procedures such as salting out, dialysis, filtration, centrifugation, concentration and lyophilization. When an even purer product is desired, a higher purity preparation can be obtained by combining the techniques mentioned above with other conventional procedures such as adsorption and desorption with ion exchange, gel filtration, chromatography. by affinity, fractionation at the isoelectric point and electrophoresis.

The preparation of HPTh thus obtained can be advantageously used, alone or in combination with one or more agents, for injection, external, internal or diagnostic administration, for the prevention or treatment of human diseases.

During the present description, the production of HPTh is assayed by the bioassay method described by J.A. Parsons et al., "Endocrinology", vol. 92, pp. 454-462 (1973); it is expressed by weight relative to the standard preparation of HPTh, 1300 USP units / mg being allocated to this standard available from the National Institute of Health (USA).

The invention is illustrated below by several embodiments.

Example 1:

Disaggregated human parathyroid tumor cells, obtained by extraction from a patient with parathyroid tumor, followed by hashing, and a line of Namalwa human leukemia lymphoblastoids are suspended together in a container with 140 mM saline NaCl, 54 mM KCl, 1 mM NaH2PO4 and 2 mM CaCl2, so as to obtain respective cell concentrations of the order of 104 cells / ml. The cell suspension, cooled with ice, is mixed with a fresh preparation of the same saline solution, containing Sendai virus previously inactivated by ultraviolet irradiation; the whole is transferred 5 min after mixing in an incubator at 37 ° C, and is stirred there for 30 min to achieve cell fusion, introducing the ability to produce hPTh from human parathyroid tumor cells in the lymphoblastoid line human leukemia. After cloning according to a conventional method of the hybridoma cell strain capable of producing hPT, it is implanted intraperitoneally in adult naked mice, which are then fed in the usual way for 5 weeks. The resulting massive tumors, weighing about 15 g each, are extracted, broken up by chopping, and subjected to the action of trypsin. After washing with Earle 199 medium (pH 7.2), supplemented with 10% by volume / volume of fetal calf serum, the cells are resuspended, at a concentration of 105 cells / ml, in a fresh preparation of same medium which contains 30 mM L-arginine and 20 mM CaCl2, and incubated at 37 ° C for about 40 h to obtain HPTh. The cells are then subjected to ultrasound, and the amount of HPTh present in the supernatant is assayed. The production of HPTh is approximately 830 ng / ml of cell suspension. The control cells, obtained by in vitro culture of human parathyroid tumor cells in Earle 199 medium (pH 7.2), supplemented with 10% by volume / volume of fetal calf serum, are treated as above to produce HPTh . This production is only close to 4 ng / ml of cell suspension.

Example 2:

Disaggregated human kidney carcinoma cells, obtained by extraction from a patient suffering from kidney carcinoma, followed by hashing, and a JBL human leukemic lymphoblastoid line are fused as in Example 1; the ability to produce hPTH from human kidney carcinoma cells was thus introduced into the human leukemic lymphoblastoid line. After cloning, according to a conventional method, of the source of hybridoma cells capable of producing HPTh, it is implanted subcutaneously in newborn hamsters, having previously received by injection of the antiserum prepared from 'a rabbit by the conventional method, to reduce their immunoreaction, then the animals are fed in the usual way for 3 weeks. The resulting massive tumors, formed under the skin and weighing about 10 g each, are extracted and disaggregated by chopping and suspended in physiological saline containing collagenase. After washing with Earle's medium (pH 7.4) added with 5% volume / volume of human serum, the cells are resuspended at a concentration of the order of 106 cells / ml in a fresh preparation of the same medium, containing 20 mM CaCl2 and 20 mM dopamine; then incubated at 37 ° C for 20 h to produce HPTh. The production of this hormone is of the order of 1.3 ng / ml of cell suspension.

The control cells, obtained as in Example 1 by in vitro culture of a fused line of human leukemic lymphoblastoids JBL, are treated as above. The production of HPTh is only 16 ng / ml of cell suspension.

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Example 3:

In newborn rats, a line of human leukemic lymphobastoids Ball-1 is implanted intravenously, into which the ability to produce hPTh from human ovarian tumor cell cells has been introduced as in Example 1, then the rats are fed in the usual way for 4 weeks. The resulting massive tumors, about 30 g each, are extracted and treated as in Example 1 to produce HPTh. The production is close to 900 ng / ml of cell suspension. The control experiment is carried out as in Example 1, by in vitro culture of the human leukemic lymphoblastoid line fused Ball-1 and treatment of the cells as above. The production of HPTh is only 10 ng / ml of cell suspension.

Example 4:

After having undergone an irradiation of approximately 400 rems of x-rays for the reduction of the immunoreaction, adult mice receive subcutaneously implants of a line of human leukemic lymphoblastoids Nall-1, in which the The ability to produce HPTh from human lung carcinoma cells was introduced as in Example 1, then the animals were fed in the usual way for 3 weeks. The resulting massive tumors, formed under the skin and weighing about 15 g each, are extracted and treated as in Example 2 to produce HPTh. The production is of the order of 1.2 ng / ml of cell suspension.

The control cells, obtained as in Example 1 by in vitro culture of a fused line of human leukemic lymphoblastoids Nall-1, are treated as above. The production of HPTh is only about 20 ng / ml of cell suspension approximately.

Example 5:

A line of human leukénmiques lymphoblastoïdes Tall-1, in which the aptitude to produce of the HPTh of the cells of human parathyroid tumor was introduced as in example 1, is suspended in physiological serum, then the whole is transferred to a cylindrical plastic diffusion chamber, the internal volume of which is of the order of 10 ml, provided with a filter membrane having a pore size with a diameter close to 0.5 (i. After inclusion by intraperitoneal route from this chamber 5 in an adult rat, the latter is fed in the usual manner for 4 weeks, then the chamber is removed. The density of human lymphoblastoids in the chamber, reached during the above operation, is approximately 6 x 108 cells / ml, which is approximately 102 times greater or even more than that obtained with an in vitro culture using a CO 2 incubator. The human lymphoblastoids thus obtained are treated as in example 2 for pro mute hpth. The production is of the order of 1.1 μg / ml of cell suspension.

Control cells, obtained by suspending human parathyroid tumor cells in physiological serum, transferring the resulting cell suspension into a chamber, intraperitoneally including this chamber in an adult rat, feeding the latter so usual for 4 weeks and harvest of multiplied human lymphoblastoids (cell density about 8 x 10 6 cells / ml), are treated as above. The production of HPTh is only about 3 ng / ml of cell suspension.

Example 6:

A JBL human leukemic lymphoblastoid line, in which the ability to produce hPTh from human lung carcinoma cells was introduced as in Example 1, is implanted in the allantoic cavity of embryonated eggs, previously placed incubate at 37 ° C for 5 d. After incubating the embryonated eggs at this temperature for an additional 1 week, the multiplied human lymphoblastoids thus obtained are treated as in Example 1 to produce HPTh.

The production is approximately 700 ng / ml of cell suspension. During the control experiment, in which human lung carcinoma cells are implanted in the allanic cavity of embryonated eggs, no cell multiplication is observed.

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Claims (10)

652748 1. Process for the production of human parathyroid hormone HPTh, by multiplication of human lymphoblastoids, capable of producing this hormone and release thereof by multiplied cells, characterized in that an animal is used for cell multiplication warm-blooded. 2. Method according to claim I, characterized in that the cell multiplication is carried out by implantation of human cells in the body of a warm-blooded animal. 2 3. Method according to claim 1, characterized in that the cell multiplication is carried out using a device in which the nutritive body fluid of a warm-blooded animal is supplied to human cells. 4. Method according to claim 3, characterized in that the device is a diffusion chamber equipped so that the cells of the animal host do not contaminate it. 5. Method according to one of claims 1 to 4, characterized in that the multiplied human lymphoblastoids are released to release HPTh, in the presence of one or more of the following products: glycine, leucine, lysine, arginine, cysteine, chloride sodium, potassium or calcium, magnesium sulfate, dopamine, isoproterenol, epinephrine and norepinephrine. 6. Method according to one of claims 1 to 5, characterized in that the warm-blooded animal is a poultry, in particular chicken or pigeon, or a mammal, in particular dog, cat, monkey, goat, pig, cow, horse, rat, guinea pig, hamster, mouse or naked mouse. 7. A method of preparing human lymphoblastoids used in the method according to one of claims 1 to 6, characterized in that human lymphoblastoids capable of producing HPTh are hybridoma cells which are obtained by cell fusion of a human lymphoblastoid line with human cells capable of producing HPTh. 8. Method according to claim 7, characterized in that the human cells capable of producing HPTh are cells of parathyroid tumor, renal tumor, ovarian tumor or carcinoma of the lung. 9. Method according to one of claims 7 or 8, characterized in that the line of human lymphoblastoids is a line of human leukemic lymphoblastoids. 10. Method according to claim 9, characterized in that the leukemic, human lymphoblastoids are from the line of Namalva, Ball-1, Nall-1, Tall-1 or JBL.