GB2066822A - Process for preparing mouse interferon - Google Patents

Abstract

A process for producing a large amount of mouse interferon of high activity which process involves the transplanting of established mouse tumor cells to the body of a warm- blooded animal other than a mouse or inoculating the cells in a culture medium charged in a filter- membrane-interposed diffusion chamber which is designed and fitted up to or in the animal body so that its nutrient body fluid feeds the cells, multiplying the said cells, exposing the multiplied cells to the action of interferon inducer in vitro or in vivo, and then purifying and separating the induced mouse interferon.

Description

SPECIFICATION Process for preparing mouse interferon The present invention relates to a process for preparing mouse interferon.

As reported in "Science", vol. 177, pp. 797-799 (1972), it is well documented that normal mouse cells induce mouse interferon of relatively high activity, while mouse tumor cells induce mouse interferon of very low activity. Normal mouse cells are, however, not suitable for large-scale production of mouse interferon because they multiply much more slowly than mouse tumor cells.

We have now found that although a large amount of highly active mouse interferon was not obtainable by inoculating and culturing established mouse tumor cells in a culture medium in vitro, or by transplanting and multiplying the cells in the body of a mouse to use its nutrient body fluids, such interferon can easily be obtained by transplanting said cells into the body of a warm-blooded animal other than a mouse or by using the body fluids of such an animal as a nutrient for cells inoculated in a culture medium charged in a suitably designed diffusion chamber.

According to the present invention, there is provided a process for preparing mouse interferon, which process comprises (i) either transplanting mouse interferon-producing established mouse tumor cells into the body of another, different warm-blooded animal, or inoculating the cells in a culture medium which is contained within a diffusion chamber which is connected to or placed within the animal body and which is designed to enable the said cells to grow by feeding on the nutrient body fluids of the animal, (ii) multiplying the transplanted cells in the said animal body or in the diffusion chamber, (iii) exposing the multiplied cells to the action of an interferon inducer in vitro or in vivo to induce mouse interferon, and (iv) purifying and separating the induced mouse interferon.

The process according to the present invention differs from conventional processes in which the cells are multiplied in vitro, and has the advantage that it requires no, or little, nutrient medium supplemented with expensive serum. In addition, the maintenance and control of the conditions during the multiplication of the established mouse tumor cell are relatively easy, and mouse interferon of relatively high activity can be easily obtained.

More particularly, in the process according to the present invention, established mouse tumor cells can be easily multiplied in the body of a warm-blooded animal other than a mouse utilizing the body fluids of the animal as a nutrient either by transplanting said cells therein, or by embedding in or connecting to the animal body a diffusion chamber charged with a culture medium suspended with said cells, while the animal is fed in the usual way.

Compared with the conventional processes in which established mouse tumor cells are multiplied in vitro, the process according to the invention in which the cells are multiplied utilizing nutrient body fluids from the said warm-blooded animal body has further advantages. For example, the multiplication of the cells is steadier, the multiplication rate is higher, and the yield of the induced interferon per cell is much higher.

Any established mouse tumor cell may be used as far as it can multiply when transplanted in the body of the said other warm-blooded animal. Examples of suitable cell types are L 5178 Y cells, SM 36 cells, L1210 cells, FAC-C cells, T 3 cells, M 1 cells, OUIMS-2 cells, JTC II cells, ELD cells and Sarcoma 180 cells, as reported in "Protein, Nucleic Acid and Enzyme", vol. 20, no. 6, pp. 616-643 (1975).

Particularly, mouse lymphoblastoid cell lines such as L 5178 Y cells L 1210 cells, OUMS-2 cells and JTC-1 1 cells are preferable because of their high multiplicaction rates and the attainment of high activities of the induced interferon.

Any warm-blooded animal can be used in the invention as far as the transplanted established mouse tumor cells can multiply therein. Examples of suitable such animals are chickens and pigeons, and mammals such as dogs, cats, monkeys, goats, pigs, bovines, rabbits, horses, guinea pigs, rats and hamsters.

Since transplantation of mouse tumor cells into the bodies of the above-mentioned animals tends to cause undesirable immunoreactions, animals in the most immature state, namely, egg, foetus, embryo, or new-born or infant animal, should be chosen to depress the immunoreactions as much as possible.

Prior to transpiantation of the cell, the animal may be pretreated to depress immunoreactions, for example either by irradiating it with 200-600 rem of X-rays or Y-rays, or by treating the animal with antiserum or an immunosuppressive agent.

Instead of transplanting and multiplying established mouse tumor cells in the animal body, the cells can be inoculated and multiplied in a conventional type diffusion chamber which is embedded, for example intraperitoneally, in the body of the said warm blooded animal, the chamber being designed to enable the said cells to grow by using the body fluids of the animal as nutrients.

The diffusion chambers can be of various shapes and sizes and have filter membranes, for example membrane filters, ultra-filters and hollow fibres, which prevent leakage from the cells but allow ingress of nutrient body fluids. Filter membranes having pores of a size of from 1 0-7 to 1 0-5 m are preferable.

Alternatively, the established mouse tumor cells can be multiplied in a system in which the animal's nutrient body fluids are circulated through a diffusion chamber which is connected to a certain part of the animal body and placed, for example on the surface of the animal body, to allow the animal to feed the cells with its nutrient body fluid.

Furthermore, if the diffusion chamber is designed so that it can be disconnected periodically from the animal body, cells can be multiplied during the whole life of the animal without having to unnecessarily sacrifice the animal. This leads to an increased yield of multiplied cells per animal.

Furthermore, when a diffusion chamber is used, not only can the multiplied mouse tumor cells be easily harvested, but also it is not necessary to pretreat the various warm-blooded animals in order to depress their immunoreactions because there is less probability of causing immunoreactions in this aspect of the present process.

As to the induction of mouse interferon, any method can be employed so long as it will induce interferon in the multiplied living mouse cells. Thus, for example, the multiplied cells can be exposed in vivo to the action of an interferon inducer wherein they developed, or exposed in vitro to the action of interferon inducer after their isolation.

Particularly, when mouse tumor cells are multiplied in a diffusion chamber, the exposure of the multiplied cells to the action of interferon inducer in or out of the chamber induces mouse interferon.

Any known interferon inducer can be used so long as it will induce mouse interferon. Examples of suitable interferon inducers are viruses, bacteria, protozoa, rickettsia, nucleic acids, endotoxins and polysaccharides.

The induced mouse interferon can be readily purified, separated and collected by conventional purification and separation techniques, for example salting out, dialysis, filtration, centrifugation, concentration and freeze-drying.

If more highly purified mouse interferon preparations are desirable, mouse interferon of the highest purity will be obtainable by employing conventional techniques, for example adsorption on and desorption from an ion exchanger, gel-filtration, affinity-chromatography, isoelectric point fractionation and electrophoresis, in combination with the above mentioned techniques.

In the present invention, the interferon activity was determined by the plaque reduction method described by R.H. Wagner, "Biological Studies of Interferon. Virology", vol. 13, pp. 323-337 (1961), in which mouse L cells were challenged to vesicular stomatitis virus.

The hemagglutination titer was assayed according to the method described by J.E. Salk, "Journal of Immunology", vol. 49, pp. 87-98 (1944).

The present invention is illustrated by the following Examples.

EXAMPLE 1 New-born hamsters were pre-injected with anti-serum prepared from rabbit in accordance with known methods and then established mouse tumor cells of L 120 cells were subcutaneously transplanted into the hamsters. The hamsters were fed in the usual way for 3 weeks.

The resultant subcutaneous massive tumors were removed, cut finely and dissociated in a physiological saline solution containing trypsin at 40C. The cells were washed with an Eagle's minimal essential medium supplemented with 5 v/v% of fetal bovine serum at pH 7.2 and 370C, and were resuspended in the same medium to give a concentration of about 1 x 10' cells per ml. Then, to the suspension was added partially-purified mouse interferon in a proportion of about 50 units per ml. The mixture was incubated at 370C for about 6 hours.

Then, Newcastle disease virus was added to the mixture in a proportion of about 300 of hemagglutination units per ml and incubated for an additional 20 hours to induce mouse interferon.

The incubated culture was centrifuged at about 1,000 x g and about 40C to remove precipitates such as cell residues. The resulting supernatant was first dialyzed against 0.1 M hydrochloric acidpotassium chloride buffer at pH 2.0 and 40C for 70 hours, and then dialyzed against a physiological saline solution buffered at pH 7.2 with 0.01 M phosphate buffer, for 12 hours. The dialyzed mixture was carefully filtered with a membrane filter and conncentrated to obtain an interferon solution.

The resulting solution was used in the following experiments in the form of a partially-purified interferon solution.

As a control experiment, tumor cells which had been similarly multiplied by transplanting L 1210 cells into a mouse were treated as in the case of the hamsters described above to induce interferon. The obtained interferon was partially purified.

The activities of the interferon preparations obtained in accordance with the present invention and by the control experiment before and after the partial purification are shown in Table 1.

The results shown in Table 1 show that extremely high-titred mouse interferon is induced in the mouse tumor cells which are multiplied in hamsters, and the partial purification of the induced mouse interferon using hamsters results in an activity which is 100 times or more higher than that of the control preparation.

TABLE 1

Present Invention Control Animal Hamster Mouse Activity of induced interferon (units /ml) 27,000 600 Activity of partially purified interferon (units /animal) 18,000,000 110,000 EXAMPLE 2 New-born hamsters were pre-injected with antiserum to depress their immunoreactions and were intraperitoneally transplanted with established mouse tumor cells of JTC-1 1 cells. The hamsters were fed in the usual way for 4 weeks.

The cells were harvested from about 7 ml of the ascites per hamster and treated similarly as described in Example 1 to obtain a partially-purified and concentrated mouse interferon.

The interferon activity of the concentrate was about 1,700,000 units per hamster.

EXAMPLE 3 Adult hamsters were irradiated with about 300 rem of y-rays to depress their immunoreactions and intraperitoneally transplanted with established mouse tumor cells of OUMS-2 cells. The hamsters were fed in the usual way for 3 weeks.

The multiplied cells were collected from the resultant massive tumors and mouse interferon was induced, partially purified and concentrated in a manner similar to that described in Example 1. Then, the mouse interferon-containing concentrate was freeze-dried into powder.

The interferon activity of the powder was about 9.400,000 units per hamster.

EXAMPLE 4 Rats were irradiated with about 400 rem of X- rays to depress their immunoreactions, and then intraperitoneally transplanted with established mouse tumor cells of L 5178 Y cells. The rats were then fed in the usual way for 2 weeks.

Two days after intraperitoneally injecting about 5,000 hemagglutination units of Newcastle disease virus whose activity was almost inactivated, the rats were sacrificed to harvest the ascites. The ascites was treated in a manner similar to that described in Example 1 to provide a partially-purified interferon solution with an interferon activity of about 300,000 units per rat.

EXAMPLE 5 Established mouse tumor cells of OUMS-2 cells were suspended in physiological saline solution and placed in 20 ml plastic cylindrical diffusion chambers having interposed membrane filters of pore sizes of about 0.5 y. Two chambers were intraperitoneally embedded in each dog.

The dogs were fed in the usual way, and about 4 weeks later the chambers were removed from the dogs.

The cell concentration of the chambers was about 4 x 109 cells per ml which was about 102 to 103 times or more higher than that attained in vitro on a nutrient medium in a CO2 incubator.

The cells were treated, and the induced mouse interferon was partially purified and concentrated, in a manner similar to that described in Example 1. Then, the mouse interferon-containing concentrate was freeze-dried into powder.

The interferon activity of the powder was about 50,000,000 units per dog.

EXAMPLE 6 Established mouse tumor cells of JTC-1 1 cells were transplanted in the allantoic cavities of embryonated eggs which had been pre-incubated at 370C for 5 days, and then were incubated for an additional 5 days.

The eggs were opened and the multiplied mouse tumor cells were harvested. Then, the cells were treated and the induced mouse interferon was partially purified and concentrated, in a manner similar to that described in Example 1.

The interferon activity of the concentrate was about 700,000 units per 10 embryonated eggs.

Claims (7)

1. A process for preparing mouse interferon, which process comprises (i) either transplanting mouse interferon-producing established mouse tumor cells into the body of another, different warmblooded animal, or inoculating the cells in a culture medium which is contained within a diffusion chamber which is connected to or placed within the animal body and which is designed to enable the said cells to grow by feeding on the nutrient body fluids of the animal, (ii) multiplying the transplanted cells in the said animal body or in the diffusion chamber, (iii) exposing the multiplied cells to the action of an interferon inducer in vitro or in vivo to induce mouse interferon, and (iv) purifying and separating the induced mouse interferon.

2. A process according to Claim 1, in which the mouse interferon-producing established mouse tumor cells are mouse lymphoblaseoid cell lines.

3. A process according to Claim 1, in which the mouse jinterferon-producing established mouse tumor cells are L 51 78 Y cells, SM 36 cells, L 1210 cells, FAC-C cells, T3 cells, M 1 cells, JTC-1 1 cells, ELD cells, OUMS-2 cells or Sarcoma 180 cells.

4. A process according to any one of Claims 1 to 3, in which the said warm-blooded animal is a mammal.

5. A process according to any one of claims 1 to 4, in which the diffusion chambers have filter membranes which have pores of a size of from 10-7 to 10-5 m.

6. A process for preparing mouse interferon according to Claim 1 substantially as described in any one of the foregoing Examples.

7. Mouse interferon when prepared by a process as claimed in any one of the preceding claims.