GB2093038A

GB2093038A - Process for the production of human placental lactogen

Info

Publication number: GB2093038A
Application number: GB8137708A
Authority: GB
Original assignee: Hayashibara Seibutsu Kagaku Kenkyujo KK; Hayashibara Biochemical Laboratories Co Ltd
Current assignee: Hayashibara Seibutsu Kagaku Kenkyujo KK
Priority date: 1980-12-19
Filing date: 1981-12-15
Publication date: 1982-08-25
Also published as: FR2496466B1; IT1148017B; JPS57102819A; JPS6045851B2; KR860001575B1; IT8149901A0; FR2496466A1; GB2093038B; CH652746A5; KR830007088A

Abstract

A process for the production of human placental lactogen (hPL) comprising in vivo multiplication of human lymphoblastoid cells capable of producing said hormone, using a non-human warm-blooded animal, and in vitro production of hPL with the multiplied human lymphoblastoid cells.

Description

SPECIFICATION Process for the production of human placental lactogen The present invention relates to a process for the production of human placental lactogen (or human chorionic somatomammotropin, abbreviated hPL hereinafter).

hPL is a hormone, secreted by the human syncytiotrophoblast cells of the chorionic villi, which has growth-stimulating and anti-insulin activities.

No process for the mass production of lowcost hPL has been established so far.

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

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

The process according to the invention provides a higher hPL yield, 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 hPL can be multiplied easily while utilising the nutrient body fluid supplied form the non-human warm-blooded animal body by transplanting said cells to the animal body, or suspending said cells in a conventional 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 higher hPL production per cell.

As to the human lymphoblastoid cells which can be used in the present invention, any human lymphoblastoid cells can be used as long as they produce hPL and multiply rapidly in the non-human warm-blooded animal body.

Preferable human lymphoblastoid cells are those introduced with the hPL production governing genetic sites of human cells which inherently produce hPL such as syncytiotrophoblast cells of the chorionic villi, or chorionic epithelioma cells, or those which produce ectopic hPL such as lung carcinoma cells, by means of cell fusion using polyethylene glycol or Sendai virus, or genetic recombination techniques using DNA ligase, nuclease and DNA polymerase; and human lymphoblastoid cells which produce ectopic hPL. Since the use of such human lymphoblastoid cells results in the formation of easily disaggregatable massive tumors when the cells are transplanted to the animal body, and the massive tumors are hardly contaminated with the host animal cells, the multiplied live human lymphoblastoid cells can be easily harvested.

Any non-human warm-blooded animal can be used to perform the process of the present 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, rats, guinea pigs, hamsters, mice and nude mice. Since transplantation of the human lymphoblastoid cells to the non-human warmblooded animal gives rise to undesirable immunoreactions, the use of a newborn or infant animal, or those in the youngest possible stage, for example, in the form of 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 V-ray irradiation, at about 200-600 rem, or with an injection of antiserum or an immunosuppressive agent prepared according to conventional methods. Since nude mice, exhibit weaker immunoreactions, even as adults; thus, any human lymphoblastoid lines can be transplanted in nude mice without pretreatment to suppress immunoreactions, and then multiplied rapidly.

Both stabilised cell multiplication and enhancement of hPL production can be carried out by repeated transplantation using combination(s) of different non-human warmblooded animals: the objectives are attainable first by implanting said cells to hamsters and multiplying the cells therein, and then 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 of the same species or genus.

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

As well as direct cell transplantation of the human lymphoblastoid cells to the animal, it is also possible to multiply easily any conventional human lymphoblastoid lines capable of producing hPL 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 having a pore size of about 10-7 to 10-5m in diameter which prevents contamination with the host animal cells into the chamber and allows the animal to supply its body fluid to the chamber.The diffusion chamber can be designed, if necessary, so it can be placed, on the host animal in such a manner that the nutrient body fluid from the animal body can circulate into the chamber, and 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.When such a diffusion chamber is used, since no immunoreaction arises owing to the absence of direct contact of the human lymphobiastoid cells with the host animal cells, and the multiplied human cells can be harvested easily, any nonhuman warm-blooded animals can be used as the host in the invention without any pretreatment to reduce immunoreactions.

Feeding of the 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 can be attained within 1-20 weeks, generally 1-5 weeks, after the cell transplantation.

According to the invention, 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 transplanted to the animal body increases about 102 to 107 times or more, or about 101 to 106 times or more than that attained by in vitro tissue culture using a nutrient medium. The cells are therefore suitable for use in the production of hPL.

As regards the method by which the human lymphoblastoid cells are allowed to release hPL, any method can be employed as long as the human lymphoblastoid cells release hPL thereby. For example, the human lymphoblastoid cells, obtained by multiplying in ascite in suspension and harvesting from said ascite, or by extracting massive tumor formed subcutaneously and harvesting after the disaggregation of the massive tumor, are suspended to give a cell concentration of about 104 to 108 cells per ml in a nutrient medium, prewarmed at a temperature of about 20-40"C, and then incubated at this temperature for 1-50 hours to produce hPL.Enhancement of hPL production may be carried out by the presence of one or more of amino acids such as leucine, lysine, arginine and cysteine; inorganic salts such as sodium chloride, potassium chloride, calcium chloride and magnesium sulfate; and hormones such as luteinising hormone releasing hormone. Furthermore, during the hPL production human chorionic gonadotropin (abbreviated hCG hereinafter) can be produced simultaneously according to the invention.

The hPL thus obtained can be collected easily by purification and separation techniques using conventional procedures such as salting-out, dialysis, filtration, centrifugation, concentration and lyophilisation. If a more highly purified hPL 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, affinity chromatography, gel filtration, iso-electric point fractionation and electophoresis.

The hPL 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 diesases.

The following Examples illustrate the present invention.

In this specification, hPL procuetion was determined by radio-immunoassay method as described in P. Bech et al, J. Clin. Endocrinol., Vol. 25, pp. 1457-1462 (1965), and expressed by weight in terms of the standard hPL preparation available from the National Institutes of Health (USA). Also, hGC production was determined by a radio-immunoassay method as described in A.R. Midgley Jr., Endocrinology, Vol. 79, pp. 10-18 (1966), and expressed in International Units (IU).

EXAMPLE 1 Disaggregated human chorionic epithelioma cells, extracted from a patient suffering from chorionic epithelioma and minced, and a human leukemic lymphoblastoid 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. The ice-chilled cell suspension was mixed with a fresh preparation of the same salt solution containing UV-irradiation pre-inactivated Sendai virus, transferred into a 37"C incubator five minutes after mixing, and stirred therein for 30 minuted to effect cell fusion, introducing the hPL productibility of the human chorionic epithelioma cells into the human leukemic lymphoblastoid line. After cloning by conventional methods the hybridoma cell strain capable of producing hPL, the hybridoma cell strain was implanted intraperitoneally 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 contain ing trypsin. After washing the cells with Earle's 199 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 30 mM L-arginine, and then incubated at 35"C for 20 hours to produce hPL.Thereafter, the cells were ultra-sonicated, and the hPL in the resulting supernatant was determined. The hPL production was about 340 yg per ml cell suspension.

The simultaneous hCG production was about 230 IU per ml cell suspension.

Control cells were obtained by implanting the human chorionic epithelioma cells in nude mice, feeding the animals in 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 described above to induce hPL. The hPL production was only about 3 jug per ml cell suspension.

EXAMPLE 2 Disaggregated human lung carcinoma cells, obtained from a patient suffering from lung carcinoma and mincing, and a human leukemic lymphoblastoid line JBL were fused in a manner similar to that described in Example 1, thereby introducing the hPL productability of the human lung carcinoma cells into the human leukemic lymphoblastoid line. After cloning using conventional methods the hybridoma cell strain capable of producing hPL, the hybridoma cell strain was implanted subcutaneously in newborn hamsters which had been preinjected with an antiserum-pre- pared from rabbits by conventional methods-to reduce their immunoreaction, and the animals were 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 suspending 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 20 mM L-lysine and 10 mM magnesium sulfate, and then incubated at 37"C for 15 hours to produce hPL. The hPL production was about 1 50 jug per ml cell suspension.

Control cells were obtained by implanting the human lung carcinoma cells in 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 described above. The hPL production was only about 0.9 'Lg per ml cell suspension.

EXAMPLE 3 Newborn rats were implanted intravenously with a human leukemic lymphoblastoid line BALL-1 into which the hPL productibility of the human chorionic epithelioma cells had 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 resulting massive tumors, about 30 g each, were extracted and disaggregted. After washing the cells with RPMI 1640 medium (pH 7.4), supplemented with 10 v/v% foetal calf serum, the cells were resuspended to give a cell concentration of abut 107 cells per ml in a fresh preparation of the same medium which contained 30 mM L-arginine, and then incubated at 30"C for 40 hours to produce hPL.

The hPL production was about 410 9 per ml cell suspension.

The simultaneous hCG production was about 870 IU per ml cell suspension.

Control cells were obtained by implanting the human chorionic epithelioma cells in 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 hPL production was only about 2 pg per ml cell suspension.

EXAMPLE 4 After about 400 rem X-ray irradiation of adult mice to reduce their immunoreaction, the mrce were implanted subcutaneously with a human leukemic lymphoblastoid line NALL1 into which the hPL productibility of the human lung carcinoma cells had been introduced in a manner similar to that described in Example 2, and then 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 obtained cells were treated in a manner similar to that described in Example 2 to produce hPL. The hPL production was about 210 jug per ml cell suspension.

Control cells were obtained by implanting the human lung carcinoma 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 described above. The hPL production was only about 1 y9 per ml cell suspension.

EXAMPLE 5 A human leukemic lymphoblastoid line TALL-1 into which the hPL productibility of the human chorionic epithelioma cells had been introduced in a manner similar to that described in Example 1 was suspended in physiological saline solution, and then trans ferred 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 in diameter. 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 lymphoblastoid cells thus obtained were treated similarly as described in Example 3 to produce hPL. The hPL production was about 380 ,ug per ml cell suspension.

The simultaneous hCG production was about 750 IU per ml cell suspension.

Control cells were obtained by suspending the human chorionic epithelioma in physiological saline solution, transferring the resulting cell suspension into the diffusion chamber, embedding the chamber intraperitoneally in a rat, feeding the animal in the usual way for four weeks, and harvesting the multiplied human lymphoblastoid cells (cell density, about 107 cells per ml). The control cells were treated similarly as described above. The hPL production was only about 1 ,ug per ml cell suspension.

EXAMPLE 6 A human leukemic lymphoblastoid line JBL into which the hPL productibility of human syncytiotrophoblast cells from chorionic villi 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 37"C for five days, and the eggs were incubated at this temperature for an additional one week. After harvesting the multiplied human lymphoblas- toid cells, the cells were treated similarly as described in Example 1 to produce hPL. The hPL production was about 1 90 ,ug per ml cell suspension.

The simultaneous hCG production was about 210 IU per ml cell suspension.

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

Claims

1. A process for the production of human placental lactogen (hPL), which process comprises multiplying human lymphoblastoid cells capable of producing said hormone by transplanting said cells to a non-human warmblooded animal body, and allowing the multiplied human lymphoblastoid cells to release said hormone; or multiplying human lymphob lastoid cells capable of producing said hor mone by allowing said cells to multiply within a device in which the nutrient body fluid of a - non-human warm-blooded animal is supplied to said cells, and allowing the multiplied human lymphoblastoid cells to release said hor mone.

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

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

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

5. 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.

6. A process according to Claim 2, wherein the human lymphoblastoid line is a human leukemic lymphoblastoid line.

7. A process according to Claim 2, wherein the human lymphoblastoid line is Namalwa, NALL-1, BALL-1, TALL-1 or JBL.

8. A process according to any one of the preceding Claims, wherein the multiplied human lymphoblastoid cells are allowed to release hPL in the presence of one or more of leucine, lysine, arginine, cysteine, sodium chloride, potassium chloride, calcium chloride, magnesium sulfate and luteinizing hormone releasing hormone.

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

10. A process according to any one of the preceding Claims, wherein human chorionic gonadotropin is also produced.

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

1 2. Human placental lactogen whenever prepared by a process as claimed in any one of the preceding claims.