GB2091741A - Process for the Production of Human Adrenocorticotropic Hormone - Google Patents

Abstract

Processes for the production of human adrenocorticotropic hormone (hACTH), comprising in vivo multiplication of human lymphoblastoid cells capable of producing said hormone, using a non- human warm-blooded animal, and exposing the multiplied human lymphoblastoid cells to an adrenocorticotropic hormone inducer. Simultaneous production of other human hormones such as human melanocyte-stimulating hormone and human lipotropic hormone also occurs.

Description

SPECIFICATION Process for the Production of Human Adrenocorticotropic Hormone The present invention relates to processes for the production of human adrenocorticotropic hormone (abbreviated as hACTH hereinafter).

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

We have investigated processes for the mass production of hACTH and have unexpectedly found that human lymphoblastoid cells capable of producing said hormone are suitable for the mass production of hACTH due to their very high multiplication rates and hACTH production per cell.

According to the present invention there is provided a process for the production of hACTH, which process comprises multiplying human lymphoblastoid cells capable of producing hACTH by transplanting said cells to a non-human warmblooded animal body, or alternatively by allowing said cells to multiply within a device in which the nutrient body fluid of a non-human warmblooded animal is supplied to said cells, and allowing the human lymphoblastoid cells multiplied by either of the above multiplication procedures to release hACTH.

The process according to the invention, besides realising a higher hACTH production, requires much less nutrient medium containing expensive serum for the cell multiplication or no such medium, and renders much easier the maintenance of the culture medium during the cell multiplication than in in vitro tissue culture.

Particularly, any human lymphoblastoid cells capable of producing hACTH can be multiplied easily while utilising the nutrient body fluid supplied from the non-human warm-blooded animal body by transplanting said cells to the animal body, or suspending said cells in a conventional diffusion chamber arranged to receive the nutrient body fluid of the animal, and feeding the animal in the usual way. Also, in the present process one obtains more stable and increased cell multiplication, and higher hACTH production per cell.

As regards the human lymphoblastoid cells which can be used in the present invention, any human lymphoblastoid cells can be used as long as they produce hACTH and multiply in the nonhuman warm-blooded animal body. Preferably human lymphoblastoid cells are those which have had incorporated therein hACTH production governing genetic sites of human cells capable of producing said hormone, for example, human cells which inherently produce hACTH such as cells from the anterior lobe of the pituitary gland, pituitary tumor cells or chromophobe adenoma cells, or other human cells which produce ectopic hACTH such as pancreas carcinoma cells from Langerhans island or lung carcinoma cells, by means of cell fusion using polyethylene glycol or Sendai virus, or by genetic recombination techniques using DNA ligase, nuclease and DNA polymerase; and human lymphoblastoid cells which produce ectopic hACTH.

Since the use of the human lymphoblastoid cells results in the formation of easily disaggregatable massive tumors when the cells are transplanted to the non-human warm-blooded animal body, and the massive tumors are hardly contaminated with the host animal cells, the multiplied live human lymphoblastoid cells can be harvested easily.

Any warm-blooded animal body can be used to perform the process of the present invention so long as the human lymphoblastoid cells multiply therein. Examples of suitable animals are poultry such as chickens or pigeons, and mammals such as dogs, cats, monkeys, goats, pigs, cows, horses, rabbits, guinea pigs, rats, hamsters, mice and nude mice. Since transplantation of the human lymphoblastoid cells to the animal gives rise to undesirable immunoreactions, the use of a newborn or infant animal, or those in the youngest possible stage, for example, an egg, embryo or foetus, is desirable.In order to reduce the incidence of immunoreactions as much as possible, prior to the cell transplantation the animal may be treated with X-ray or y-ray irradiation at about 200-600 rem, or by injection of antiserum or immnuosuppressive agent prepared according to conventional methods. Nude mice, even in adult form, are found to exhibit weaker immunoreactions; thus any human lymphoblastoid lines can be transplanted therein, and multiplied rapidly without pretreatment to suppress immunoreactions.

Stabilised cell multiplication and enhancement of hACTH production can be both carried out by repeated transplantation using combination(s) of different non-human warm-blooded animals; the objectives are attainable first by implanting the human lymphoblastoid cells in hamsters and multiplying the cells therein, then by reimplanting the cells in nude mice. The repeated transplantation may be carried out with animals of the same class or division as well as those in the same species or genus.

The human cells to be multiplied can be implanted in any site on the animal as long as the human lymphoblastoid cells multiply at that site; for example, in the allantoic cavity, or intraveneously, intraperitoneally, or subcutaneously.

As well as direct cell transplantation to the animal body, it is also possible to multiply any conventional human lymphoblastoid lines capable of producing hACTH by using the nutrient body fluid supplied from a non-human warm-blooded animal body by embedding, for example, intraperitoneally, in said animal body a conventional diffusion chamber, of various shapes and sizes, and equipped with a porous membrane filter, ultra filter or hollow fiber having a pore size of about 10-7 to 1 0~5mien diameter which prevents contamination with the host animal cells into the chamber.Since the diffusion chamber can be designed, if necessary, so that it can be placed, for example, on the host animal in such a manner as to allow the body fluid to circulate from the animal body into the chamber, so as to enable observation of the cell suspension in the chamber through transparent side window(s) equipped on the chamber wall(s), and so as to enable replacement and exchange with a fresh chamber.

By such a method cell production per host thereby increases to an even higher level over the period of the animal life without any sacrifice of the host animal. Furthermore, when such a diffusion chamber is used, since immunoreactions do not arise owing to the absence of direct contact of the human cells with the host animal cells, various non-human warm-blooded animals can be used as the host without pretreatment to reduce immunoreactions and the multiplied human kymphoblastoid cells can be harvested easily.

Feeding of the host animal implanted with the human lymphoblastoid cells can be carried out easily by conventional methods even after the cell transplantation, and no special care is required.

Maximum cell multiplication is generally attained within 1-20 weeks after the cell transplantation. According to the present method, the number of the human lymphoblastoid cells obtained per host ranges from about 107 to 1012 or more. In other words, the number of the human lymphoblastoid cells implanted in the animal body increases about 102 to 107 times or more, or about 101 to 106 times at least over the level attained by in vitro tissue culture methods using nutrient medium; the human lymphoblastoid cells are thus suitable for the hACTH production.

Any suitable method can be used for hACTH induction as long as the human lymphoblastoid cells obtained according to the invention release said hormone as a result of the method of induction. For example, the human lymphoblastoid cells, obtained by multiplying in ascite in suspension and harvesting from said ascite, or by extracting the massive tumor formed subcutaneously and harvesting after the disaggregation of the massive tumor, are suspended to give a cell concentration of about 104 to 1 oa cells per ml in a nutrient medium, pre- warmed at a temperature of about 20--400C, and then subjected to an adrenocorticotropic hormone inducer at this temperature for about 1 to 50 hours to induce said hormone.

As regards the adrenocorticotropic hormone inducers usable in the invention, any inducers can be used as long as the human lymphoblastoid cells are induced to release said hormone thereby.

Preferable adrenocorticotropic hormone inducers are vasopressin, atropine methyl nitrate, insulin, glucagon, bacterial endotoxin, serotonin and propranolol.

We have found that after the hACTH induction a large amount of human melanocyte-stimulating hormone (abbreviated hMSH hereinafter) and human lipotropin (hLPH) are produced simultaneously.

The hACTH, hMSH and hLPH can be collected easily by purification and separation techniques using conventional procedures such as saltingout, dialysis, filtration, centrifugation, concentration and lyophiiisation. If a more highly purified preparation is desirable, a preparation of the highest purity can be obtained by the above mentioned techniques in combination with other conventional procedures 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 injection, or for external, internal or diagnostical administration in the prevention and treatment of human diseases such as pituitary insufficiency.

The following Examples illustrate the present invention.

In this specification, hACTH production was determined by a radio-immunoassay method as described in S. Matsukura et al., J. Lab. Clin.

Med., Vol, 77, pp. 490--500 (1971), and is expressed as a weight per ml cell suspension.

Also, hMSH and hLPH production was determined by a radio-immunoassay method as described in K. Abe eft at J. Clin, Invest., Viol, 46.

pp. 1906-1916 (1967), first, the total amount of the hormones was determined with human p- MSH standard, then the weight ratio of the hormones was calculated with the amounts of human p-MSH and jB-LPH isolated by gel filtration.

Example 1 Human lung carcinoma cells, extracted from a patient suffering from lung carcinoma and minced, and a human leukemic lymphoblastoid line Namalwa were suspended together in a vessei with a salt solution, containing 140 mM NaCI, 54 mM KCI, 1 mM NaH2PO4 and 2 mM CaCI2, to give a respective cell concentration of about 104 cells per ml. The ice-chilled cell suspension was mixed with a fresh preparation of the same salt solution containing UV-irradiation preinactivated Sendai virus, transferred into a 370C incubator five minutes after the mixing, and stirred therein for 30 minutes to effect cell fusion, thereby introducing the hACTH productibility of the human lung carcinoma cells into the human leukemic lymphoblastoid line. After cloning the hybridoma cell strain capable of producing hACTH using conventional methods, the hybridoma cell strain was implanted in adult nude mice which were then fed in the usual way for five weeks. The resulting massive tumors, formed subcutaneously and about 1 5 g each, were extracted and disaggregated by mincing and suspending in a physiological saline solution containing trypsin.

After washing the cells with Earle's 1 99 medium (pH 7.2), supplemented with 10 v/v % foetal calf serum, the cells were resuspended to give a cell concentration of about 106 cells per ml in a fresh preparation of the same medium which contained 0.1 jug glucagon per ml as the inducer, and then incubated at 350C for 20 hours to induce hormones. Thereafter, the cells were ultrasonicated,and the hACTH and hLPH in the supernatant were determined.

The hACTH production was 250 ,ug per ml cell suspension; hMSH, 580 ug per ml; and hLPH, 40 ,a9 per ml.

Control cells were obtained by implanting the human lung carcinoma cells in adult nude mice, feeding the animals in the usual way for five weeks, extracting the resulting massive tumors, formed subcutaneously and about 5 g each, and disaggregating the massive tumors. The control cells were treated similarly as above to induce hormones. The hACTH production was only 600 ng per ml cell suspension; hMSH, 1.2 FLg per ml; and hLPH, 350 ng per ml.

Example 2 Human chromophobe adenoma cells, extracted from a patient suffering from chromophobe adenoma of the pituitary gland and minced, and a human leukemic lymphoblastoid line JBL were fused in a manner similar to that described in Example 1, introducing the hACTH productibility of the human chromophobe adenoma cells into the human leukemic lymphoblastoid line. After cloning the hybridoma cell strain capable of producing hACTH using conventional methods, the hybridoma cell strain was implanted subcutaneously in newborn hamsters which had been preinjected to reduce their immunoreaction with an antiserum prepared from rabbits by conventional methods; the hamsters were then fed in the usual way for three weeks.The resulting massive tumors, formed subcutaneously and about 10 g each, were extracted and disaggregated by mincing and suspended in a physiological saline solution containing collagenase. After washing the cells with Eagle's minimal essential medium (pH 7.2), supplemented with 5 v/v % human serum, the cells were resuspended to give a cell concentration of about 105 cells per ml in a fresh preparation of the same medium which contained 3 juU vasopressin per ml as the inducer, and incubated at 370C for 15 hours to induce hormones. The human hormone production was determined as described in Example 1. The hACTH production was 380 fig per ml cell suspension; hMSH, 740 y9 per ml; and hLPH, 510g per ml.

Control cells were obtained by implanting the human chromophobe adenoma cells in newborn hamsters, feeding the animals in the usual way for three weeks, extracting the resulting massive tumors, formed subcutaneously and about 3 g each, and disaggregating the massive tumors. The control cells were treated similarly as above to induce hormones. The hACTH production was only 750 ng per ml cell suspension; hMSH, 560 ng per ml; and hLPH, 430 ng per ml.

Example 3 Newborn rats were implanted intraveneously with human leukemic lymphoblastoid line BALL-2 into which the hACTH productibility of the human lung carcinoma cells had been introduced in a manner similar to that described in Example 1, and then fed in the usual way for four weeks. The resulting massive tumors, about 30 g each, were extracted and disaggregated. After washing the cells with RPMI 1 640 medium (pH 7.4), supplemented with 10 v/v % foetal calf serum, the cells were resuspended to give a cell concentration of about 107 cells per ml in a fresh preparation of the same medium which contained 6 mM serotonin as the inducer, and then incubated at 300C for about 40 hours to induce hormones. The hACTH production was 730 ft9 per ml cell suspension; and hMSH, 940 g per ml.

Control cells were obtained by implanting the human lung carcinoma cells in newborn rats, feeding the animals in the usual way for four weeks, extracting the resulting massive tumors, about 5 g each, and disaggregating the massive tumors. The control cells were treated similarly as above. The hACTH production was only 650 ng per ml cell suspension; and hMSH 1.8 ,ag per ml.

Example 4 After about 400 rem X-ray irradiation of adult mice to reduce their immunoreaction, the mice were implanted subcutaneously with a human leukemic lymphoblastoid line NALL-1 into which the hACTH productibility of the human chromophobe adenoma cells had been introduced in a manner similar to that described in Example 2, and fed in the usual way for three weeks. The resulting massive tumors, formed subcutaneously and about 1 5 g each, were extracted and disaggregated. The cells were treated similarly as described in Example 2 to induce hormones. The hACTH production was 480 g per ml cell suspension; and hLPH, 350 y9 per ml.

Control cells were obtained by implanting the human chromphobe adenoma cells in mice, feeding the animals in the usual way for three weeks, extracting the resulting massive tumors, about 5 g each, and disaggregating the massive tumors. The control cells were treated similarly as above. The hACTH production was only 520 ng per ml cell suspension; and hLPH, 790 ng per ml.

Example 5 A human leukemic lymphoblastoid line TALL-1 into which the hACTH productibility of the human lung carcinoma cells had been introduced in a manner similar to that described in Example 1 was suspended in physiological saline solution, transferred into a plastic cylindrical diffusion chamber (inner volume: about 10 ml) and equipped with a membrane filter having a pore size of about 0.5 y. After intraperitoneal embedding of the chamber into an adult rat, the rat was fed in the usual way for four weeks, and the chamber was removed. The human cell density in the chamber attained by the above operation was about 7 x 108 cells per ml which was about 102 times higher or more than that attained by in vitro cultivation using a CO2 incubator.The human cells thus obtained were treated similarly as described in Example 3 to induce hormones. The hACTH production was 280 yg per ml cell suspension; hMSH, 770 g per ml; and hLPH, 160 ug per ml.

Control cells were obtained by suspending the human lung carcinoma cells in physiological saline solution, transferring the resulting cell suspension into a plastic cylindrical diffusion chamber (inner volume: about 10 ml) and equipped with a membrane filter having a pore size of about 0.5 FL. The chamber was embedded intraperitoneally into an adult rat, the rat was fed in the usual way for four weeks, and the multiplied human cells (about 107 cells per ml) were harvested. The control cells were treated similarly as described above. The hACTH production was only 480 ng per ml cell suspension; hMSH, 2.3 jug per ml; and hLPH, 250 ng per ml.

Example 6 A human leukemic lymphoblastoid line JBL into which the hACTH productibility of the human lung carcinoma cells had been introduced in a manner similar to that described in Example 1 was implanted in the allantoic cavities of embryonated eggs which had been preincubated at 370C for five days. After incubation of the eggs at this temperature for a further week, the multiplied human lymphoblastoid cells were harvested. The human lymphoblastoid cells were treated similarly as described in Example 2 to induce hACTH. The hACTH production was 520 ,a9 per ml cell suspension.

In a control experiment in which the human lung carcinoma cells were implanted in the allantoic cavities of embryonated eggs, no cell multiplication was noted.

Claims (10)

Claims

1. A process for the production of human adrenocorticotropic hormone (hACTH), which process comprises multiplying human lymphoblastoid cells capable of producing said hormone by transplanting said cells to a nonhuman warm-blooded animal body, and allowing the multiplied human lymphoplastoid cells to release said hormone; or multiplying human lymphoblastoid cells capable of producing said hormone by allowing said cells to multiply within a device in which the nutrient body fluid of a nonhuman warm-blooded animal is supplied to said cells, and allowing the multiplied human lymphoblastoid cells to release said hormone.

2. A process according to Claim 1, wherein the human lymphoblastoid cells are hybridoma cells obtained by means of cell fusion of a human lymphoblastoid line with human cells capable of producing hACTH.

3. A process according to Claim 2, wherein the hybridoma cells are obtained by cell fusion of a human lymphoblastoid line with human chromophobe adenoma cells.

4. A process according to Claim 2, wherein the hybridoma cells are obtained by cell fusion of a human lymphoblastoid line with human lung carcinoma cells.

5. A process according to any one of Claims 2 to 4, wherein the human lymphoblastoid line is a human leukemic lymphoblastoid line.

6. A process according to any one of Claims 2 or 4, wherein the human lymphoblastoid line is Namalwa, BALL-1, TALL-1, NALL-1, or JBL.

7. A process according to any one of the preceding claims, wherein the multiplied human lymphoblastoid cells are induced to release hACTH with one or more inducers selected from vasopressin, atropine methyi nitrate, insulin, glucagon, bacterial endotoxin, serotonin and propranolol.

8. A process according to any one of the preceding claims, wherein the non-human warmblooded animal is a chicken, pigeon, dog, cat, monkey, goat, pig, cow, horse, rabbit, guinea pig, rat, hamster, mouse or nude mouse,

9. A process according to any one of the preceding claims, wherein a culture product containing hACTH is produced, and this product also contains human melanocyte-stimulating hormone and human lipotropic hormone.

10. Human adrenocorticotropic hormone whenever prepared by a process as claimed in any one of the preceding claims.