GB2092157A

GB2092157A - Process for the production of human calcitonin

Info

Publication number: GB2092157A
Application number: GB8138589A
Authority: GB
Original assignee: Hayashibara Seibutsu Kagaku Kenkyujo KK; Hayashibara Biochemical Laboratories Co Ltd
Current assignee: Hayashibara Seibutsu Kagaku Kenkyujo KK
Priority date: 1980-12-30
Filing date: 1981-12-22
Publication date: 1982-08-11
Also published as: JPS57115195A; IT1148020B; GB2092157B; JPS5825440B2; CH652749A5; FR2497100A1; KR860001590B1; FR2497100B1; IT8149980A0; KR830007086A

Abstract

A process for the production of human calcitonin (hCT) comprising in vivo multiplication of human lymphoblastoid cells capable of producing said hormone, and in vitro hCT production with the multiplied human lymphoblastoid cells.

Description

SPECIFICATION Process for the production of human calcitonin The present invention relates to a process for the production of human calcitonin (hereinafter abbreviated as "hCT").

hCT is a hormone secreted by human parafollicular cells in the thyroid gland, and which suppresses the release of calcium from bones into the blood and also the secretion of insulin.

However, no effective process to produce a large amount of hCT at low cost is known.

We have investigated processes for the mass production of hCT and have unexpectedly found that the use of human lymphablastoid cells that are capable of producing hCT results in a high cell multiplication rate and also in a high hCT production per cell; human lymphoblastoid cells capable of producing hCT are therefore very effective for use in hCT production.

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

The process according to the invention besides giving a greater hCT production, requires much less nutrient medium containing expensive serum for 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 hCT can be multiplied easily by using the nutrient body fluid supplied from a non-human warm-blooded animal by transplanting the cells to the animal, or by suspending the cells in a diffusion chamber devised 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 a higher human hCT production per cell than that attained with the cells multiplied by in vitro tissue culture.

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 product hCT and multiply easily in the nonhuman warm-blooded animal body. Examples of suitable such human cells are human lymphoblastoid cells wherein the genetic sites coding for the production of hCT are introduced from human cells capable of inherently producing hCT such as human parafollicular cells in thyroid gland or thyroidea adenoma cells, or those from human tumor cells capable of producing ectopic hCT such as human carcinoid cells or human lung tumor cells by cell fusion techniques using agents such as polyethylene glycol or Sendai virus, or by genetic recombination techniques using enzymes such as DNA ligase nuclease and DNA polymerase, and also human lymphoblastoid cells that are capable of producing ectopic hCT.

Furthermore, since transplantation of the above-mentioned estabiished human lymphoblastoid cell lines to the animal body results in the formation of massive tumors, and said massive tumors are barely contaminated with the host animal cell and disaggregated easily, the multiplied viable human lymphoblastoid cells can be easily harvested.

Any warm-blooded non-human anirnal can be used to perform the process of the invention as 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, or nude mice.

Since such cell transplantation give use to undesirable irnmunoreactions, the use of a newborn or infant animal, or an animal in the youngest possible stage, for example, in the form of an egg, embryo, of foetus, is desirable. In order to reduce immunoreactions, the animal can be treated, prior to the cell transplantation, with irradiation of about 200 to 600 rem of X-ray or 7 > -ray, or with an injection of antiserum or an immunosuppressive agent prepared according to conventional methods. Nude mice. even in adult form, exhibit weak immunoreactions; consequently, any established human lymphoblastoid cell lines can be conveniently and easily transplanted and multiplied therein without subjecting the mice to such pretreatment.

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

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

In addition to direct cell transplantation of the human lymphoblastoid cells to the animal body, any conventional established human lymphoblastoid cell line capable of producing hCT can be multiplied by using the nutrient body fluid supplied from the animal body by embedding, for example, intraperitoneally, in the animal body a conventional diffusion chamber, of various shapes and sizes, and equipped with a porous membrane filter, ultra filter or hollow fiber with pore sizes of about 10-' to 10-5 m in diameter which prevents ingress of the host cells into the diffusion chamber but allows the animal to supply its nutrient body fluid to the human lymphoblastoid cells.

Additionally, the diffusion chamber can be designed if desired, so as to enable observation of the cell suspension in the chamber through transparent side window(s) provided on the chamber wall(s), and so as to enable replacement and exchange with a fresh chamber. In this way, cell multiplication can be increased to an even higher level over the period of the animal's life and the human lymphoblastoid cell production per animal is further augmented without sacrificing the host animal.

Furthermore, when such diffusion chamber is used, since the multiplied human lymphoblastoid cells can be harvested easily and no immunoreactions arise owing to the absence of direct contact of human lymphoblastoid cells with the host animal cells, any non-human warmblooded animal can be used as the host in the present process without the need for any pretreatment to reduce immunoreactions.

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

The period required to obtain maximum cell multiplication in the host animal is one to 20 weeks, usually one to five weeks.

According to the invention, the number of human lymphoblastoid cells obtained per host animal is about 107 to 1012 or more. In other words, the number of human lymphoblastoid cells transplanted into the animal body increases about 102 to 7times or more, corresponding to about 10 to 106 times or more than that attained by in vitro tissue culture method using nutrient medium; the human lymphobiastoid cells can therefore be advantageously used for hCT production.

As regards the method for release of hCT from the multiplied humam lymphoblastoid cells, any method can be employed as long as the human lymphoblastoid cells release hCT thereby. For example, the multiplied human lymphoblastoid cellsoótained by multiplying the cells in ascite in suspension and harvesting them from the ascite, or by extracting the massive tumor formed subcutaneously and disaggregating and collecting the resultant cellular products -- are suspended in a concentration of about 104 to 106 cells per ml in a nutrient medium, kept at a temperature of about 200 to 400C, and then incubated at this temperature for about one to 100 hours to release hCT. In order to increase hCT production from the human lymphoblastoid cells, any hCT inducing agent can be added to the above mentioned nutrient medium.Example of suitable hCT inducers are amino acids such as glycine, leucine, lysine, arginine and cysteine; inorganic salts such as sodium chloride, potassium chloride, calcium chloride and magnesium sulfate; and hormones such as dibutylcyclic AMP and prostagladin E.

These may be used alone or as mixtures of hCT inducers.

The hCT thus obtained can be collected easily by purification and separation using conventional techniques such as salting-out, dialysis, filtration, centrifugation, concentration and lyophilization. If a more highly purified hCT is desirable, a hCT preparation of the utmost purity can be obtained by the above-mentioned techniques in combination with conventional techniques such as adsorption and desorption with ion exchange, gel filtration, affinity chromatography, isolelectric point fractionation and electrophoresis.

The hCT preparation thus obtained is advantageously usable 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.

The following Examples illustrate the present invention: The hCT in the culture medium was determined by the radioimmunoassay method described in N. A. Samaan et al, J. Lad. Clin. Med. Vol. 81, pp.

671-681(1973), and expressed by a weight which is determined with reference to a doseresponse curve obtained with Internationai reference preparation of calcitonin available from the World Health Organization.

EXAMPLE 1 Minced and disaggregated human thyrophyma cells extracted from a thyrophma patient and human leukemic lymphoblastoid cell line Namalwa were suspended together in a vessel with a salt solution, containing 140 mM NaCI, 54 mM KCI, 1 mM NaH2PO4 and 2 mM Cacti2, to give a respective cell concentration of about 104 cells per ml, and the cell suspension was mixed with a fresh preparation of a salt solution having the same composition and containing UVpreinactivated Sendai virus under ice-cold conditions. Five minutes after the mixing, the mixed suspension was placed in a 370C incubator and incubated for 30 minutes with agitation to effect cell fusion and thereby introducing the abaility to produce hCT into the human lymphoblastoid cell line Namalwa.After cloning the hybridoma cell line capable of producing hCT using conventional methods, the hybriderma cell line was implanted intraperitoneally into adult nude mice which where then fed in the usual way for five weeks. The resultant massive tumors, about 1 5 grams each, were extracted, minced and then suspended in a saline solution containing trypsin to disaggregate said massive tumors. After the disaggregated cells were washed with Earle's medium 199 (pH 7.2) supplemented with 10 v/v% foetal bovine serum, the cells were resuspended in a fresh preparation of the same medium containing 20 mM CaCI2 and 30 mM L-arginine to give a cell concentration of about 105 per ml, and then incubated at 370C for 40 hours to produce hCT. At the end of the incubation period, the suspension was treated ultrasonically and the supernatent of the suspension was determined for its hCT content. The hCT production was about 4.4 y9 per ml of the suspension.

Control cells were obtained by cultivating in vitro the human thyrophyma cells in Earle's 199 medium (pH 7.2), supplemented with 10 v/v% foetal bovine serum, and incubated at 37 C.

These control cells were treated similarly as described above, to produce hCT. The hCT production was only about 10 rlg per ml of the suspension.

EXAMPLE 2 Minced and disaggregated human carcinoid cells extracted from a patient with argentaffinoma of bronchus and a human leukemic lymphoblastoid cell line JBL were treated in a manner similar to that described in Example 1 to effect cell fusion and thereby introduce the ability of producing hCT into the human lymphoblastoid cell line JBL. After cloning the hybriderma cell line capable of producing hCT by using conventional methods the hybriderma cell line was implanted subcutaneously in newborn hamsters pretreated with an antiserum prepared from rabbits by conventional methods and the newborn hamsters bearing the hybridoma cell line were fed for three weeks in the usual way.The resultant massive tumors, about 10 grams each, formed subcutaneously in the hamsters were extracted, minced and then suspended in a saline solution containing collagenase to disaggregate the massive tumors. After the disaggregated hybriderma cells were washed with Eagle's minimal essential medium (pH 7.4) containing 5 v/v% human serum the cell suspension was resuspended in a fresh preparation of the same medium which contained 10 mM CaCI2, 40 mM MgSO4 and 0.1 mM dibutylcyclic AMP to give a cell concentration of about 106 per ml and then incubated at 370C for 30 hours to release hCT.

The hCT production was about 3.7tug per ml of the suspension.

A control test was carried out similarly as described in Example 1 by cultivating in vitro the fused human lymphoblastoid cell line JBL into which the ability of producing hCT had been introduced, and allowing the multiplied said cells to release hCT. The hCT production was only about 60 rlg per ml of the suspension.

EXAMPLE 3 Newborn rats were implanted intravenously with a human leukemic lymphoblastoid line BALL-1 into which the ability of producing hCT of human thyrophyma cells has been introduced in a manner similar to that described in Example 1, and then the rats were fed in the usual way for four weeks. The resultant massive tumors, about 30 grams each, were extracted and treated similarly as described in Example 1 to release hCT. The hCT production was about 4.1 ,ug per ml of the suspension.

A control test was carried out similarly as described in Example 1 in cultivating in vitro the fused human leukemic lymphoblastoid line BALL-1 into which the ability of producing hCT had been introduced, and allowing the multiplied said cell line to release hCT. The hCT production was only about 85 w7g per ml of the suspension.

EXAMPLE4 Adult mice were irradiated with about 400 rem of X-ray to reduce their immunoreactions and were then implanted subcutaneously with a human leukemic lymphoblastoid line NALL-1 into which the ability of producing hCT of human lung tumor cells had been introduced in a manner similar to that descibed in Example 2. The mice were then fed in the usual way for three weeks.

The resultant massive tumors, formed subcutaneously, about 1 5 grams each, were extracted and treated similarly as described in Example 1 to release hCT. The hCT production was about 5.2 ,ug per ml of the suspension.

A control test was carried out similarly as described in Example 2 by cultivating in vitro the human lymphoblastoid cell line NALL-1, into which the ability of producing hCT had been introduced, and treating the multiplied cells to release hCT.

The hCT production was only about 90 q/g per ml of the suspension.

EXAMPLE 5 A human leukemic lymphoblastoid line TALL-1 into which the hCT-producing ability of thyrophyma cells had been introduced in a manner similar to that described in Example 1 was suspended in a saline solution and transferred into a cylindrical plastic diffusion chamber (inner volume: about 10 ml), and having a membrane filter with pore sizes of a diameter of about 0.5 u.

The chamber was embedded intraperitoneally in an adult rat and after feeding the rat for four weeks in the usual way, the chamber was removed. The human cell density in the chamber attained by the above operation was about 7 x los cells per ml which was about 102 times higher or more than that attained by in vitro cultivation using a CO2 incubator. The cells thus obtained were treated similarly as described in Example 2 to release hCT. The hCT production was about 3.lug per ml of the suspension.

A control test was carried out similarly as described above by suspending the human thyrophyma cells into a diffusion chamber which was then embedded intraperitoneally into an adult rat, and feeding the rat in the usual way for four weeks. The human cell density thus obtained in this test was about 6 x 102 cells per ml. The cells were treated similarly as described in Example 2 to release hCT. The hCT production was only about 1 5 77g per ml of the suspension.

EXAMPLE 6 A human leukemic lymphoblastoid line JBL into which the ability of producting hCT of the human carcinoid cells had been introduced in a manner similar to that described in Example 1 was implanted into 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 cells were harvested. The cells were treated similarly as described in Example 1 to release hCT. The hCT production was about 2.6 trg per ml of the suspension.

A control test was carried out similarly as described above by implanting the human carcinoid cells into the allantoic cavities of embryonated eggs. No multiplication of the cells was observed.

Claims

1. A process for the production of human calcitonin (hCT) which process comprises multiplying human lymphoblastoid cells capable of producing said hormone by transplanting said cells to a non-human warm-blooded animal body, and allowing the multiplied human lymphoblastoid cells to release said hormone; or multiplying human lymphoblastoid cells capable of producing said hormone by allowing the multiplied human lymphoblastoid cells to release said hormone.

2. A process according to Claim 1, wherein the human tymphoblastoid cells are those obtained by cell fusion of a human lymphoblastoid cell line with human thyrophyma cells.

3. A process according to Claim 1, wherein the human lymphoblastoid cells are those obtained by cells fusion of a human lymphoblastoid line with human carcinoid cells.

4. A process according to Claim 1, wherein the human lymphoblastoid cells are those obtained by cell fusion of a human leukemic lymphoblastoid line with human cells capable of producing hCT.

5. A process according to Claim 4, wherein the leukemic lymphoblastoid line is Namalwa, BALL-1, NALL-1 , TALL-1 ,orJBL.

6. 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, guinea pig, rat, hamster, mouse or nude mouse.

7. A process according to any one of the preceding claims, wherein the multiplied lymphoblastoid cells are allowed to release hCT in the presence of one or more of an amino acid, an inorganic salt and a hormone.

8. A process according to Claim 1, substantially as hereinbefore described in any one of the Examples.

9. Human calcitonin whenever prepared by a process as claimed in any one of the preceding claims.