GB2118560A

GB2118560A - Process for producing human Immune Response Suppressor

Info

Publication number: GB2118560A
Application number: GB08303870A
Authority: GB
Inventors: Kaname Sugimoto
Original assignee: Hayashibara Seibutsu Kagaku Kenkyujo KK; Hayashibara Biochemical Laboratories Co Ltd
Current assignee: Hayashibara Seibutsu Kagaku Kenkyujo KK
Priority date: 1982-02-12
Filing date: 1983-02-11
Publication date: 1983-11-02
Also published as: JPS58138395A; IT1167074B; GB8303870D0; GB2118560B; FR2521588B1; KR840003419A; FR2521588A1; KR900005860B1; CH661745A5; JPH0214038B2; IT8347689A0

Abstract

A process for producing human immune Response Suppressor (hIRS) which comprises in vivo multiplication of human cells capable of producing hIRS, and in vitro production of hIRS with the human cells thus obtained.

Description

SPECIFICATION Process for producing human Immune Re sponse Suppressor The present invention relates to a process for producing a biologically-active substance, more particularly, it relates to a process for producing human Immune Response Suppressor (referred to hereinafter as hlRS) possess ing promising pharmacological applications.

hlRS designates Immune Response Sup pressor which can be obtained from viable human cells.

As described by Carl Waltenbaugh, "Biology of the Lymphokines", pp. 422-427 (1 979), published by Academic Press, Inc.

(New York), Immune Response Suppressor (IRS) is a proteinaceous substance found in body fluid, which inhibits non-species-specifically immune responses, independent of the type of antigen.

Accordingly, IRS is considered to be an effective prophylactic or therapeutic agent for hypersensitiveness, for example, allergy, anaphylactic-shock or hyper-autoimmunization, or rejection of tissue or organ transplants.

Although the non-species-specificity of IRS suggests that it may be possible to use IRS of non-human origin in the treatment of human diseases, the use of hlRS produced from viable human cells is safe and efficacious because of its lower antigenicity.

No industrial-scale process for producing readily usable hlRS for the prevention or treatment of human diseases has been, established to date.

There exists, therefore, a need for a lowcost process for producing hlRS which is feasible on an industrial-scale.

The present invention is based on the finding that production of hlRS using human cells, obtained by multiplying in vivo human cells capable of producing hlRS while utilizing a non-human warm-blooded animal, is substantially higher than that attained with human cells multiplied in vitro.

According to the present inventon, there is provided a process for the production of hlRS which process comprises transplanting human cells capable of producing hlRS to a nonhuman warm-blooded animal, allowing the human cells to utilize the nutrient body fluid of the animal for their multiplication, and allowing the multiplied human cells to release hlRS, or, placing human cells capable of producing hlRS in a device whereby the nutrient body fluid of a non-human warm-blooded animal is supplied to the cells, allowing the human cells to utilize the body fluid for their multiplication, and allowing the multiplied human cells to release hlRS.

In a preferred embodiment, the invention provides a process for the production of hlRS which process comprises transplanting human cells capable of producing hlRS to a nonhuman warm-blooded animal, feeding the ani mal so as to allow the human cells to utilize the nutrient body fluid of the animal for their multiplication, extracting and disaggregating the resultant tumor, formed in the animal, to obtain the multiplied human cells, culturing the human cells in an in vitro nutrient medium in the presence or absence of an IRS inducer for a period sufficient to accumulate a significant amunt of hlRS, and harvesting the accumulated hlRS from the cuiture, or, placing a suspension of human cells capable of producing hlRS in a conventional diffusion chamber by which the nutrient body fluid of a non-human warm-blooded animal is supplied to the human cells, embedding or placing the chamber in or on the non-human warmblooded animal, feeding the animal to allow the human cells to utilize the body fluid for their multiplication, collecting the multiplied human cells from the chamber, culturing the human cells in an in vitro nutrient medium in the presence or absence of an IRS inducer for a period sufficient to accumulate a significant amount of hlRS, and harvesting the accumulated hlRS from the culture.

Unlike conventional processes using in vitro tissue culture, the present process using the in vivo procedure requires no, or at least substantially less, nutrient medium containing expensive serum, and the procedure renders the maintenance of the culture during the cell multiplication stage much easier, in addition to producing a much higher-titered hlRS.

More particularly, in the process using a non-human warm-blooded animal as a host, according to the invention, certain human cells can be easily multiplied while utilizing the nutrient body fluid supplied from the animal by transplanting them to the animal, or, alternatively, placing them in a conventional diffusion chamber devised to receive the body fluid, and embedding or placing the chamber in or on the animal, and feeding the animal in the usual way.

In addition, the present process can be distinguished from previous in vitro cell multiplication methods using tissue culture because one obtains much more stable and rapid cell multiplication, higher cell production, and substantially higher levels of hlRS production per cell.

The human cells which can be used in the invention are those which produce a substantial amount of hlRS, and which are capable of being multiplied in a non-human warmblooded animal upon being transplanted into such an animal. For example, normal human cells originating from periferal blood, from the spleen, or the tonsils, those obtained by transformation of the normal human cells using any carcinogenic virus or radiation; human cells originating from tonsil tumors, liver carcinomas, spleen tumors and lung carcinomas; and established cell lines of the above human cells, are all capable of being used in the invention. Known human cell lines, such as Ad-L, HL-4, L 84-ly, NPC-501, and HB-7, as described in The Tissue Culture, Vol. 6, pp.

527-546 (1980), can be used according to the present invention.

Instead of using the above-mentioned human cells, a human lymphoblastoid line may also be used. These cells are much more readily subjected to sub-culture, and are capable of having human gene(s) coding hlRS production introduced into them by means of cell fusion using polyethylene glycol or Sendai virus, or by gene recombinant technique using nuclease, ligase and DNA polymerase. This results in a great increase in the cell multiplication rate and/or hlRS production per cell, of as much as about 2-10 times greater than that which can be attained in the abovedescribed normal or tumor cells.

Transplantation of such a human lymphob- lastoid line to the animal tends to form massive tumor(s) which are hardly contaminated with the host animal cells. In addition, the subsequent disaggregation of the extracted tumor is very easy and, therefore, the harvest of the viable human cells can be carried out easily.

Known lymphoblastoid lines of human leukaemic or lymphoma origin, such as Nam alwa, BALL-1, NALL-1, TALL-1 and JBL cells, are especially suitable for use in the present invention.

The non-human warm-blooded animal usable in the invention maybe any such animal in which the human cells are multipliable.

Examples of suitable animals are birds such as chickens, or pigeons; and mammals, such as dogs, cats, monkeys, goats, pigs, horses, rabbits, cows, guinea pigs, rats, nude rats, hamsters, mice or nude mice.

Since transplantation of such human cells to the animal results in undesirable immunoreactions, the use of a non-human warmblooded animal in the youngest stage possible, for example, in the form of an egg, embryo, or foetus, or as a newborn or infant animal, is desirable in order to reduce the incidence of immunoreactions as much as possible.

Pior to the transplantation, the animal may be treated further with X-ray or y-ray irradiation of about 200-600 rem, or by means of an injection of antiserum or immunosuppressant in order to reduce immunoreactions to the lowest possible level.

The use of an immunodeficient laboratory animal, such as a nude mouse or a nude rat, as the host animal, results in less undesirable immunoreaction even when it is an adult animal, and such animals can be readily transplanted with any of the above-mentioned human cells without a pretreatment to suppress or reduce immunoreactions, since in such cases immunoreaction is less likely.

One may obtain both stabilization of cell multiplication and increase in hlRS production by repeated transplantation using a combination of different non-human warm-blooded animals. For example, both objectives may be attained by first transplanting the human cells to a hamster and multiplying them therein, followed by retransplanting the multi pied human cells to a nude mouse. In this case, the repeated transplantation may be carried out with non-human warm-blooded animals of the same class or order, as well as with those of the same species or genus.

The cells can be transplanted to any site of the host animal as long as the human cells multiply at that site. Examples of suitable sites are, in the allantoic cavity, or intravenously, intraperitoneally, or subcutaneously.

Instead of transplanting the human cells to the animal, any of the human cells described hereinbefore can be multiplied easily by placing them in a conventional diffusion chamber of various shapes and sizes, equipped with, for example, a membrane filter, ultra-filter or hollow fiber of a nominal pore size of about 10-1 to 10-5m, which prevents ingress of host cells into the chamber, but supplies the human cells with the nutrient body fluid from the host. The chamber is embedded, for example, intraperitoneally in the animal and the human cells are allowed to multiply therein by utilizing the nutrient body fluid supplied from the animal.

The diffusion chamber can also be designed if desired, to enable the culture contained in the chamber to be observed during the cell multiplication through transparent side window(s) provided on the chamber wall(s), and/or the chamber can be continually replaced with a fresh one, so as to both continue the cell multiplication over the period of the hosts' life-span and to greatly increase the rate of production of cells per animal without sacrificing the host animal.

Since the use of such diffusion chamber results in a lower incidence of undesirable immunoreactions on account of the absence of direct contact of the human cells with the animal cells, any non-human warm-blooded animal may be readily used as the host without pretreatment, and the multiplied viable human cells can be harvested easily therefrom.

The feeding of the animal can be carried out easily in the usual way, and no special care is required even after the transplantation.

The period required to obtain maximum cell multiplication is generally from 1 to 20 weeks. When the human cells are of human tumor or lymphoblastoid origin, the maximum cell multiplication may be generally obtained within about 1 to 5 weeks. The number of the human cells thus obtained may be about 107 to 1012 per animal or higher. In particular, according to the present process, the transplanted human cells increase by about 102 to 107 times or higher, which is about 10 to 106 times or higher than that obtained by in vitro multiplication procedure using nutrient medium. Therefore, it is preferable to multiply the human cells by the present process in order to produce hlRS.

Cell generation of the human cells thus multiplied may be regulated by placing them in vitro culture for a period of about 1 to 4 days, prior to the induction step described hereinafter.

Any method by which the multiplied human cells are induced to produce hlRs can be employed in the invention. For example, the multiplied human cells, obtained by harvesting from ascite suspension, or from the extraction and disaggregation of the resultant massive tumor(s), for example, the subcutaneous tumors, are suspended in a nutrient medium, prewarmed to a temperature in the range of about 20 to 40"C, to give a cell density of about 104 to 108 cells per ml, and incubated therein to obtain hlRS. The human cells may also be exposed to an IRS inducer to further increase the quntity of IRS produced.

The IRS inducer used in the present process may be one or more substances which can induce hlRS production in the human cells obtained by the present process; examples of such substances are mitogens, such as phytohaemagglutinin, concanavalin A, pokeweed mitogen, lipopolysaccharide, endotoxin, polysaccharide or bacteria; virus; or nucleic acids.

Stabilization of the hlRS in the culture and enhancement of hlRS production may be achieved by adding to the culture an hlRS stabilizer or enhancer.

The hlRS in the culture can be easily collected by purification and separation methods using conventional procedures, such as concentration, salting-out, dialysis, filtration, centrifugation, and/or lyophilization. If a more highly purified hlRS preparation is required, a preparation of the utmost purity can be obtained by the above described procedures in combination with other conventional procedures, such as adsorption and desorption with ion exchange, gel filtration, electrophoresis, affinity-chromatography and/or isoelectric point fractionation.

The present hlRS can be used advantageously alone or in combination with one or more agents, such as a vitamin, a hormone, or a carcinostatic agent, for internal administration or injection for the prevention or treatment of human diseases.

The following Examples illustrate the present invention. In this specification, the titers of hlRS were determined by the method, with slight modification, as reported by Robert R.

Rich and Carl W. Pierce, Journal of Immunology, Vol. 112, pp. 1360-1368 (1974), and expressed by the amount which suppresses the response of plaque-forming cells. According to the method, one ml of a cell suspension containing 1 X 107 spleen cells from a C 57BL/6 mouse was placed in each of a number of Petri dishes, and 0.1 ml aliquots of hlRS preparations having serial dilutions were added. The mixtures in the dishes were then incubated for a set period, after which the plaque formed during the incubation was counted.

The hlRS titer corresponds to the dilution at which 50%-plaque reduction is obtained.

EXAMPLE 1 Adult nude mice were subcutaneously transplanted with a human adenoid line, Ad-L, and fed in the usual way for three weeks.

Such cell transplantation and subsequent feeding of the animals resulted in the formation of massive subcutaneous tumors, about 1 2 g each, which were then extracted, minced, and disaggregated by suspension in a physiological saline solution containing trypsin.

The human cells were then washed with serum-free RPMI 1640 medium (pH7.2),-and suspended in a fresh medium of the same composition to give a cell density of about 1 X 106 cells per ml. To the cell suspension was added concanavalin A, (1y g per ml of cell suspension), and the mixture was incubated at 37"C for 20 hours to induce hlRS production.

The culture was then centrifuged at 8,000 rpm for 30 minutes, and the hlRS titer in the supernatant was determined according to the hereinbefore described hlRS assay method.

The hlRS production in the culture was about 2,400 units per 0.1 ml of culture.

The control experiment was carried out in the following manner. The human adenoid line, Ad-L, was cultivated in vitro in Eagle's medium (pH7.2), supplemented with 1 v/v % foetal calf serum and 20 v/v % meat extract, and the multiplied cells were then treated similarly as above to iduce hlRS production.

The hlRS production in the resultant culture was only about 90 units per 0.1 ml of culture.

EXAMPLE 2 Disaggregated human spleen tumor cells, which had been obtained by extracting and mincing tumor tissue from a patient, and a human lymphoblastoid line, Namalwa, were suspended together in a salt solution, consisting of 140 mM NaCI, 54 mM KC1, 1 mM NaH2PO4 and 2 mM CaCI2, to give respective cell densities of about 103 cells per ml. To the cell suspension was added under ice-cold conditions a fresh salt solution of the same composition, containing in addition UV-irradiation preinactivated Sendai virus, and the mixture was then transferred to a 37"C-incubator about 5 minutes after the addition. The cell suspension was stirred therein for about 30 minutes to effect cell fusion, whereby the ability of the human spleen tumor cells to produce hlRS had been introduced into the human lymphoblastoid line.

Adult nude mice were transplanted intraperitoneally with the resultant hybrid cells, and fed in the usual way for 5 weeks.

The resultant massive tumors, about 1 5 g each, were extracted, and treated to the same method of induction of hlRS production, as set out in Example 1, except that the concanavalin A was replaced with phytohaemagglutinin at a concentration of about 10 y g per ml.

The hlRS production was about 12,800 units per 0.1 ml of culture.

The control, wherein the hybridized cells were cultivated in vitro for their multiplication, and then treated similarly as described in EXAMPLE 1 to induce hlRS production, led to only about 400 units of hlRS production per 0.1 ml of culture.

EXAMPLE 3 After injecting antiserum, prepared from rabbit in the usual way, into newborn hamsters to reduce their immunoreactions, the newborn hamsters were subcutaneously transplanted with a hybridized human lymphoblastoid line, JBL, wherein the ability to produce hlRS was introduced in a manner similar to that described in EXAMPLE 2, and fed in usual way for three weeks.

The resultant massive tumors, about 1 8 g each formed subcutaneously, were extracted, and treated in a manner similar to that described in EXAMPLE 1 so as to induce hlRS production, except that the RPMI 1 640 medium was replaced with Eagle's medium (pH 7.2) supplemented with 20 v/v % meat extract.

The hlRS production was about 8,500 units per 0.1 ml of culture.

The control, wherein the hybridized JBL cells were cultivated in vitro for their multiplication, and then treated similarly as described in EXAMPLE 1 to induce hlRS production, led to only about 240 units of hlRS production per 0.1 ml of culture.

EXAMPLE 4 Newborn rats were transplanted intravenously with a hyridized human lymphoblastoid line, BALL-1, wherein the ability of producing hlRS was introduced in a manner similar to that described in EXAMPLE 2, and the newborn rats were fed in the usual way for four weeks.

The resultant massive tumors, about 35 g each, were extracted, and treated similarly as described in EXAMPLE 2 to induce hlRS production.

The hlRS production was about 9,400 units per 0.1 ml of culture.

The control, wherein the hybridized BALL-1 cells were cultivated in vitro for their multiplication, and then treated similarly as described in EXAMPLE 1 to induce hlRS production, led to only about 350 units of hlRS production per 0.1 ml of culture.

EXAMPLE 5 After irradiating adult rats with y-ray, about 400 rem,so as to reduce their immunoreaction, the adult rats were transplanted subcutaneously with a cell line of human lymph node origin, HL-4, and then fed in the usual way for four weeks.

The resultant massive tumors, about 1 5 g each, formed subcutaneously, were extracted, and treated similarly as described in EXAMPLE 3 to induce hlRS production.

The hlRS production was about 3,600 units per 0.1 ml culture.

The control, wherein the human cell line was cultivated in vitro for its multiplication, and then allowed to release hlRS, led to only about 30 units of hlRS production per 0.1 ml of culture.

EXAMPLE 6 A human lymphoblastoid line, Ad-L, was suspended with physiological saline solution in a plastic cylindrical diffusion chamber (inside volume of about 10 ml), equipped with a membrane filter having a nominal pore size of about 0.5y. The chamber was then embedded intraperitoneally into an adult rat, and the adult rat was fed in the usual way for four weeks. After completion of the feeding, the chamber was removed, and the human cells were collected.

The human cell density in the chamber was as high as about 109 cells per ml, which was about 102 times higher than that attained by an in vitro tissue culture method using a CO2 incubator.

The human cells were then treated in a manner similar to that described in EXAMPLE 3 to induce hlRS production.

The hlRS production was about 4,200 units per 0.1 ml of culture.

EXAMPLE 7 A hybridized human lymphoblastoid line, BALL-1, into which the ability to produce hlRS had been introduced in a manner similar to that described in EXAMPLE 4, was transplanted to the allantoic cavities of embryonated eggs which had been preincubated at 37"C for five days, and the eggs were then further incubated at this temperature for an additional one week.

The human cells were collected from the eggs, and treated similarly as described in EXAMPLE 1 to induce hlRS production.

The hlRS production was about 3,200 units per 0.1 ml of culture.

Claims

1. A process for producing hlRS, which process comprises transplanting human cells capable of producing hlRS to a non-human warm-blooded animal, allowing the human cells to utilize the nutrient body fluid of the animal for their multiplication, and allowing the multiplied human cells to release hlRS, or, placing human cells capable of producing hlRS in a device whereby the nutrient body fluid of a non-human warm-blooded animal is supplied to the cells, allowing the human cells to utilize the body fluid for their multiplication, and allowing the multiplied human cells to release hlRS.

2. A process according to Claim 1, which process comprises transplanting human cells capable of producing hlRS to a non-human warm-blooded animal, feeding the animal so as to allow the human cells to utilize the nutrient body fluid of the animal for their multiplication, extracting and disaggregating the resultant tumor, formed in the animal, to obtain the multipled human cells, culturing the human cells in an in vitro nutrient medium in the presence or absence of an IRS inducer for a period sufficient to accumulate a significant amount of hlRS, and harvesting the accumulated hlRS from the culture, or, placing a suspension of human cells capable of producing hlRS in a conventional diffusion chamber by which the nutrient body fluid of a non-human warm-blooded animal is supplied to the human cells, embedding or placing the chamber in or on the non-human warmblooded animal, feeding the animal to allow the human cells to utilize the body fluid for their multiplication, collecting the multiplied human cells from the chamber, culturing the human cells in an in vitro nutrient medium in the presence or absence of an IRS inducer for a period sufficient to accumulate a significant amount of hlRS, and harvesting the accumulated hlRS from the culture.

3. A process according to Claim 1 or 2, wherein the human cells capable of producing hlRS are normal human cells selected from human periferal blood, spleen cells, and tonsil cells.

4. A process according to Claim 1 or 2, wherein the human cells are human tumor cells selected from human tonsil tumor cells, liver carcinoma cells, spleen tumor cells, lung carcinoma cells, and cells obtained by transformation of the human cells described in Claim 3.

5. A process according to Claim 1 or 2, wherein the human cells are hybrid cells into which the ability to produce hlRS has been introduced.

6. A process according to Claim 5, wherein the human cells are a human lymphoblastoid cell line hybridized with any of the human cells described in Claims 3 and 4 so as to introduce into the former the ability of the latter to produce hlRS.

7. A process according to Claim 6, wherein the human lymphoblastoid line is a member selected from Namalwa, BALL-1, NALL-1, TALL-1 and JBL cells.

8. A process according to any one of the preceding Claims, wherein the non-human warm-blooded animal is a mammal or a bird.

9. A process according to any one of Claim 2 to 8, wherein the IRS inducer is one or more of a mitogen, a virus, or a nucleic acid.

10. In the process for producing hlRS which comprises multiplying human cells capable of producing hlRS, and culturing the multiplied human cells in the presence or absence of an IRS inducer for a period sufficient to accumulate a significant amount of hlRS, the improvement whereby extreme enhancements of cell multiplication rate and hlRS production are both attained, comprising transplanting human cells capable of producing hlRS to a non-human warm-blooded animal, feeding the animal to allow the human cells to utilize the nutrient body fluid of the animal for their multiplication, extracting and disaggregating the resultant tumor, formed in the animal, to obtain the multiplied human cells, culturing the human cells in an in vitro nutrient medium in the presence of or absence of an IRS inducer for a period sufficient to accumulate a significant amount of hlRS, and harvesting the accumulated hlRS from the culture, or, placing a suspension of human cells capable of producing hlRS in a conventional diffusion chamber by which the nutrient body fluid is supplied to the human cells, embedding or placing the chamber in or on a non-human warm-blooded animal, feeding the animal to allow the human cells to utilize the body fluid for their multiplication, collecting the multiplied human cells from the chamber, culturing the human cells in an in vitro nutrient medium in the presence or absence of an IR-S inducer for a period sufficient to accumulate a significant amount of hlRS, and harvesting the accumulated hlRS from the culture.

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

1 2. hlRS whenever prepared by a process as claimed in any one of the preceding Claims.