METHOD OF ENHANCING INTERFERON PRODUCTION

Abstract: Method for enhancing the production of interferon from normal human diploid fibroblast cells. In this method, a distinct interferon-production phase is established in which the temperature is initially elevated for a brief period followed by a reduction in temperature for the balance of the interferon-production phase.

Description: Method of Enhancing Interferόn Production Description Support Work described herein was supported by grants or contracts from the National Science Foundation, the National Institutes of Health, and the Lewis and Rosa Strauss Memorial Foundation. Technical Field This invention is in the field of cell biology. Background Art Interferon can be defined as an induced protein, probably a glycoprotein, that produces a characteristic antiviral state upon incubation with competent cells. Interferon is released by animal cells following viral infection and also after treatment of such cells with certain non-viral inducers. It constitutes one of the major defense mechanisms against viral infections in mammals, including humans. In addition, interferpn has been found to have immunoregulatory activities, to af- feet various cellular functions, including cell divi¬ sion, and to have potential value as an anticancer drug. It is known that there are at -least three distinct species of human interferon. Each of these has been named from their major source. Thus, Le_ interferon is the major interferon species produced in cells of lym- phoid origin, such as leukocytes; F_ interferon is the predominant interferon species obtained from non- lymphoid cells, such as fibroblasts; and T-type or OMPI immune interferon is preferentially produced in cul¬ tures of lymphocytes, particularly such cultures en¬ riched for T cells. To date, it has been difficult to obtain adequate amounts of any species of interferon to properly evalu¬ ate its clinical potential. The species of interferon most widely used in clinical trials to date is the Le_ interferon produced by the leukocyte/New Castle Disease virus (NDV) system. See Strander, H. and Cantell, K. in "The Production and Use of Interferon for the Treat¬ ment and Prevention of Human Virus Infections," C. Weymouth, Ed., Tissue Culture Association, Rockville, Md., USA, pp. 4-11, 1974. The leukocyte/NDV system has certain drawbacks, however, which include the limited supply of leukocytes and the risk of viral contamina¬ tion, such as with Hepatitis virus. Because of this, much research effort has been directed to producing other species of interferon, particularly F_ interferon. Production of F_ interferon employing human fibro- blasts has certain advantages. Because the cells can be grown in cell cultures, for example, scale-up is possible. Additionally, cell stocks can be continu¬ ously monitored to insure the absence of pathogens and a normal karyotype. Also, fibroblasts can be induced to produce interferon employing non-viral inducers. Such research has involved the testing of many different types of fibroblast cells and cell lines. Additionally, much effort has been directed towards varying the experimental culture conditions including the number of cells employed, the ingredients in the culture medium, the temperature employed for incubation and production, and other such parameters of the growth - 3 - processes. Techniques such as priming interferon-pro- ducing cells with small amounts of interferon and su- perinduction employing antimetabolites have evolved from such research. While priming, superinduction, and other recently discovered techniques have improved in¬ terferon production considerably, much greater in¬ creases in productivity are still required. Disclosure of the Invention This invention relates to a method of enhancing interferon production, particularly interferon produc¬ tion by fibroblast cells. In its most general sense, this invention relates to the establishment of a dis¬ tinct interferon-production phase in which the tempera¬ ture is initially elevated for a short period of time and then subsequently reduced to a selected interferon- production temperature capable of sustaining interferon production for prolonged periods. The invention can be practiced by forming a cell culture of inter eron-pro- ducing cells and introducing an interferon-inducer into the culture in an amount sufficient to induce inter¬ feron production. In a particularly preferred embodi¬ ment, the fibroblast cells are cultured on microcar- riers suspended in cell culture medium. One method by which the distinct production phase can be established is by introducing one or more antimetabolites to effec¬ tively block interferon production during the induction period and subsequently removing the antimetabolites to initiate the distinct interferon-production phase. This interferon-production phase is initiated at a first temperature, such as 37° C, but after a short pe¬ riod, the temperature is reduced to a lower tempera¬ ture, such as 30° C. The initial elevation in tempera- 'BURE ture, in combination with the subsequent reduced tem¬ perature, allows interferon production to be sustained at high rates and for prolonged periods compared to the rates and periods if a uniform temperature is employed during production. Often, the rate of interferon pro¬ duction is increased during the period of elevated tem¬ perature. More surprising, however, is the fact that the rate of interferon production is elevated compared to the rate which would exist if production was carried out at a uniform temperature. Additionally, interferon production is usually prolonged when the temperature shift is employed. As a result, interferon productiv¬ ity is improved and total interferon yield is increased significantly by a simple temperature shift during the distinct production phase. Brief Decription of the Drawing Figure 1 is a plot graphically illustrating the growth characteristics of a 5-liter FS-4 fibroblast cell culture at a microcarrier concentration of 5 grams per liter; Figure 2 is a plot graphically illustrating the effect of time of exposure to the antimetabolite actinomycin-D on interferon yield from FS-4 cells under one set of experimental conditions; and, Figures 3, 4 and 5 are each plots graphically il¬ lustrating comparative kinetic data for interferon pro¬ duction both with and without a temperature shift dur¬ ing the production phase. Best Mode for Carrying out the Invention For the work described hsi'ein, interferon activity was -assayed by determining the 50% level of cell pro¬ tection for samples and standard solutions using FS-4 cells challenged with Vesicular Stomatitis Virus (VSV) , Indiana strain. See, Havell, E. A. ' and Vilcek, J., "Production of High-Titered Interferon in Cultures of Human Diploid Cells," Antimicrobial Agents and Chemo¬ therapy, 2 ^ , pp. 476-84, 1972. Interferon titers are determined by including internal standards calibrated against International Standard GO23-901-527 (obtained from The National Institutes ' of Health, Bethesda, Md.) in each assay. The cells employed in the experimental work described herein were human diploid foreskin cells des¬ ignated as FS-4 cells. These are known to be good pro¬ ducers of F_-interferon and probably also produce some L interferon. Other normal human diploid cell strains, which might be employed, include embryonic lung fibro- blasts, such as WI-38, MRC-5, RS-48, and IMR-90, fore¬ skin fibroblasts, such as FS-49; and even human non- fibroblast cell strains, including epithelial and kid¬ ney cells. In addition, it is believed that certain . established cell lines would be suitable, including such lines as HT-1376 (bladder carcinoma); MG-63 (osteosarcoma-derived) ; 108 (SV40 transformed WI-38) ; etc. Generally, it is believed that any mammalian cell strain or line which produces interferon can be emp¬ loyed. The preferred culturing techniques involve the use of microcarrier cultures. Microcarrier cultures offer many potential advantages including ease of innovative cellular manipulation and potential high density cell growth in very limited volumes. A speci- fie example of microcarriers found suitable are those cell culture microcarriers sold under the trademark "Superbead" by Flow Laboratories, McLean, Virginia. An extensive description of such microcarriers and their use in cell cultures is presented in U. S. Patent No. 4,189,534, issued to David W. Levine et al., the teach¬ ings of which are hereby incorporated ' y reference. -BUREA Such microcarrier cultures employ standard cell culture media, such as Dulbecco's Modified Eagle Medium (DMEM), RPMI-1640 medium, Weymouth's 199, etc. Serum supplement is usually added, but it was found that the growth of FS-4 cells in microcarrier culture required less serum than generally employed with other culturing systems, such as roller bottles. It was found that good growth of FS-4 cells could be routinely achieved if careful attention was paid to the preparation of the inoculum and to control of the cell density at inoculation, despite previous findings that normal human cells were sometimes more difficult to grow on microcarriers than most cell lines. One ex¬ ample of a technique which was found to be consistently successful employed, an inoculum grown in Corning 490 plastic roller bottles using DMEM supplemented with 10% fetal bovine serum (FBS). Confluent FS-4 cells in rol¬ ler bottles, not older than 4 weeks, were split 1:2 and grown in roller bottles and then harvested. These cells were then used to inoculate microcarrier cul¬ tures. In order to obtain uniform growth of cells on the microcarriers, it was found that the cells should be well dispersed and that it was desirable to exceed a certain minimum requirement of cells per surface area. As an example, it was found preferable, at a microcar¬ rier concentration of 5 grams/liter, to have an inocu¬ lum density of 3 x 10^ cells/ml or more. Also, as pre¬ viously mentioned, the FBS concentration was reduced from 10% to 5% in order to promote good attachment of cells to the microcarriers during the stationary growth phase and during the interferon induction and produc¬ tion phases. Figure 1 illustrates a typical growth curve of the FS-4 cells under such conditions, as more completely described in Example 1, below. Although microcarrier cultures were employed in work described herein, and are preferred, any other ex¬ isting cell culturing technique can be employed. Thus, petri dishes, prescription bottles, roller tubes, rol- ler bottles, etc., can be employed for anchorage- dependent cells and suspension cultures can be employed for those cells capable of growing in direct suspen¬ sions. Priming, which is the addition of a small amount of interferon prior to induction, was found to be use¬ ful with the FS-4 cells grown in microcarrier cul¬ tures. In general, low amounts of interferon, such as 50 units/ml medium, are employed. In addition to en- hanciηg production, priming appeared to add consistency to the results achieved. Priming is normally carried out by adding the small amount of interferon prior to adding an interferon inducer to the cell culture. Priming has been described in the literature, and has been found to help in some cases, but not all. See, for example, Havell, E. A. and Vilcek, J., cited above; and Myers, M. W. and Friedman, R. M. , "Potentiation of Human Interferon Production by Superinduction," J. Nat. Cancer Inst., 47, No. 4, 757-63, (1971). Induction of the FS-4 cells was achieved using the known interferon inducer, polyinosinic-polycytidylic acid (poly I-poly C). Other synthetic inducers, such as Poly I, Poly I-Poly C-Polylysine and Poly I-Poly C- DEAE Dextran could also be employed. Additionally, viral inducers including Sendai Virus, New Castle Disease Virus (NDV) and others, could be employed, live or inactivated (such as by irradiation with UV light). These inducers are all known to those skilled in the art. See, for example, Havell and Vilcek, cited above. -SU EA OMPI Super induction techniques were employed in the work descr ibed . Generally , superinduction involves the addition of antimetabolites to an induced cell culture to prevent interferon production until the antimetabo- lite is removed . This also separates the process into distinct interferon-induction and interferon-production phases . Specif ically , a superinduction technique employing both cycloheximide and actinomycin-D was " employed . Whereas previous work had used 50 jig/ml of cyclohex¬ imide , studies undertaken in conjunction with this work indicated that lower amounts produced better yields . As a result , the concentration for cycloheximide was lowered and 10 ;ug/ml is the specif ic concentration which was used in much of the work . Similarly , the effect of actinomycin-D concentra¬ tion was studied from 0 to 2 jύg/ml , and there were no s ignif icant differences found for concentrations rang¬ ing between from about 0.25 and 2 .0 g/ml . However , lowering the concentration below 0. 125 jαg/ml resulted in a two-fold drop in interferon yield . Thus , the con¬ centration of 1 jug/ml was chosen. The time of exposure to the antimetabolites was also studied . As a result, it was determined that the microcarrier cultures with FS-4 cells had optimum in¬ terferon yields if at least a 5 hour exposure to cyclo¬ heximide was employed . Studies also indicated that the time of exposure to actinomycin-D was a s ignif icant factor in interferon yield . In studies wherein cyclo- heximide and poly I-poly C were added to cultures s i¬ multaneously and actinomycin— D was added four hours later, it was determined that signif icant interferon yields could be obtained only after ten minutes of exposure to actinomycin-D and that ' increasing exposure time beyond one hour, e.g., up to two hours, resulted in further gains in yield. Figure 2 is a plot of ac¬ tual data obtained in experiments conducted according to Example 1, below, except that the time of exposure to actinomycin-D was varied. The results indicate that the interferon yield appeared to be increased up to an exposure time of about three hours with actinomycin-D. In addition to cycloheximide and actinomycin-D, other antimetabolites are suitable. Examples include inhibitors of RNA synthesis such as alpha-amanatin and camptothecin as well as inhibitors of protein synthesis such as puromycin, trichoder in, oxytetracycline, and p-fluorophenylanine are also suitable. See Atherton, K. T. and Burke, D. C. , "The Effects of Some Different Metabolic Inhibitors on Interferon Superinduction," J. gen Virol. , ATI , 229-37, 1978. The crux of this invention, however, is the tem¬ perature switch made during the distinct production phase. Production is started at an initial tempera¬ ture, which is elevated in terms of the interferon pro¬ duction temperature at which production will be sus¬ tained. As an example, it was found with the FS—4 mi¬ crocarrier cultures that an initial temperature of 37° C for one hour in the production phase followed by sus¬ tained interferon production at 30° C produced out¬ standing results. As mentioned previously, the initial period at an elevated temperature often produces an in¬ creased rate of interferon production at the elevated temperature and, in addition produces an increased rate at the lower production temperature, and usually a pro¬ longed period of production, as well. In general, the initial temperature and period should be sufficient to produce the increased rate at the later and lower production temperature. Prefer¬ ably, the initial temperature is at least about 3° C above the final production temperature. In terms of time, studies were made involving the use of an elevated temperature for 1, 2, 3 and 4 hours. The results indicated that one hour was sufficient and that ultimate production fell off if this period were extended. Of course, these temperatures and times will vary with the specific cells and other culture parameters involved. The reason that the ultimate production can be so significantly enhanced by using a temperature shift in the production phase is not understood. One possible explanation for the improvement in yields resulting from the temperature manipulation is that the inter¬ feron mRNA is somehow stabilized by lowering the tem¬ perature and that the initial period at 37° C is re- quired in order to provide the high initial production rate which is then sustained or decreases gradually over a long period of time. Two possible ways in which interferon RNA could be stabilized are: (1) a direct stabilizing effect on interferon mRNA of lowering its decay rate, i.e " . , increasing its half-life; or (2) indi¬ rect stabilization by interfering with the ability of a repressor protein to inactivate interferon mRNA. The latter possibility could involve interference at one or more levels of repressor protein activity, including production of repressor protein mRNA, - 11 - translation of repressor protein mRNA, or interaction of the repressor protein with interferon mRNA. The fact that increased yields can be achieved even after four hours at the elevated temperature would tend to rule out the first and second possibilities, since four hours would seem to be sufficient time for the repressor protein to be produced. It tends to support the third possibility, i.e., that the repressor protein is less effective in shutting off or inactivating interferon mRNA at the lower temperature. The invention can be further and more specifically described by the following examples. EXAMPLE 1 Human diploid foreskin cells (FS-4) were em- ployed. The cells were obtained at approximately the 18th population doubling and were actually used in the experiments between the 30th and 40th population doubl¬ ings. Stock cultures of these cells were prepared in plastic roller bottles containing Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bo¬ vine serum. Antibiotics used were penecillin (100 units/ml) and streptomycin (100 jug/ml). These stock cultures were maintained in a walk-in incubator at 37° C until needed. The roller bottle cultures of FS-4 cells were split 1:2 two days prior to inoculation of microcar¬ riers. Forty-eight hours after the 1:2 split, the cells were dispersed with a solution of trypsin-EDTA (Q.1%/0.02%) and seeded onto microcarriers suspended in - 12 - DMEM plus 5% fetal bovine serum at a concentration of 3-4 x 10^ cells ml. Prior to use, the microcarriers were sterilized in glass bottles by autoclaving. Microcarrier concentration was 5 grams/liter, the culture size was 5 liters, and cultures were formed in 14-liter fermenters. The cultures had a pH of about 7.3 and a temperature was maintained at 37° C. After three days of incubation, 50% of the growth medium was removed and replaced with fresh growth medium. After five days, the cells had reached post exponential growth and had a density of about 1 x 10^ cells/ml. The post exponential growth cells were primed with interferon. This was done by removing the cells from the growth medium, washing the cells with DMEM, without serum, and then incubating the cells for 16 hours in DMEM containing 0.5% human plasma protein (HPP) and 50 units interferon/ml. - Subsequently, the cells were induced by first withdrawing the priming medium, washing two times with DMEM and then replacing the medium with DMEM containing 50 jug/ml poly I—poly C plus 10 jg/ml cyclohexamide. The temperature during the entire induction period was 34° C. After four hours of incubation, actinomycin-D was added to give a final concentration of 1 g/ml. After two additional hours of incubation, the medium was removed and the cells were washed two times with DMEM " URE Production medium comprising DMEM plus 0.5% HPP was warmed to 37° C and added to the cells. The cul¬ ture was incubated at 37° C for one hour after which the temperature was lowered to 37° C. Culture fluids were collected after 24 and 48 hours of production. Culture fluids were clarified by centrifugation at 2,000 rpm for ten minutes at 5° C and samples were then removed for assay. The interferon assay is one described by Havell and Vilcek, ibid. In this procedure, samples were assayed in duplicate in CoStar 96-well culture dishes. Growth medium (100 ul) was added to each well and two¬ fold dilutions of each sample were made in duplicate. Each well was seeded with 50 x 10 3 FS-4 cells in 100 ul growth medium and dishes were incubated for 20-24 hours in a humidified incubator at 37° C supplied with 10% C0 2 - Cells were then challenged with 1,000 PFU per well of VSV (Indiana strain). Several wells in each dish served as cell and virus controls. Dishes were incubated at 37° C and scored microscopically after 48-72 hours. The highest dilution of the samples show¬ ing a 50% destruction of cells was considered the end point. An internal standard calibrated against the In¬ ternational Standard GO23-901-527 was included with each assay. Cells in microcarrier cultures were enumerated by counting nuclei using a modification of the method of Sanford et al. as described by vanWezel. See Sanford, K. K. et al., "The Measurement of Proliferation in Tissue Cultures of Enumeration of Cell Nuclei," J. OMPI Nat. Cancer Inst. , 11, 773-95, 1951 and vanWezel, A. L. , "Microcarrier Cultures of Animal Cells," in Tissue Culture: Methods and Applications, pp 372-7, Ed. ' Kruse and Patterson, Academic Press, Inc., New York, 1973. Roller bottle cell counts were made by dispersion with a trypsin-EDTA solution, followed by counting with a hemacytometer. The results of two runs were: Interferon Production (Units/10 6 Cells - 24 hours) Run First 24 Hours Second 24 Hours 1 24 , 000 24 , 000 2 65 , 000 45 , 000. Figure 3 presents a plot including the data from a run according to this Example as well as comparative runs using the same experimental conditions except that the comparative runs were done with uniform production temperatures of 30 ° C and 37 ° C. Figure 4 presents similar comparative data for similar experiments wherein the exposure time to actinomycin-D was one hour and the elevated production temperature was maintained for 3 hours . Figure 5 presents data from similar com¬ parative experiments in which exposure to actinomycin-D was one hour and the elevated temperature during pro- duction was maintained for two hours , and the cultures were sampled on a daily basis . ' BURE - 15 - EXAMPLE 2 A series of experiments were conducted to show the effect of various temperature shifts during the produc¬ tion phase. The procedures and materials of Example 1 5 were employed except for the following differences: (1) The incubation with actinomycin-D was reduced from two hours to one hour; (2) production was initially started at 37°C, and after two hours the temperature was shifted as indicated for the balance of the production 0 period. The cells were primed with 50 units per ml of interferon for sixteen hours at 37°C prior to induction. The results of these temperature studies are as follows: Temperature Conditions (° C) Interferon Yield Induction Production (IU/ml) 34 37 30,000 34 37—>34 46,000 34 37—>30 54,000 34 37—>25 22,000. Since cell density was 1 x 10 6 cells/ml, the yield fig¬ ures also could be stated as units per 10° cells. EXAMPLE 3 Studies were made to determine the effect of the time of temperature shift during production phase em- • ploying the conditions and materials of Example 2. The results were : 16 - Temperature Conditions (° C) Interferon Yield Induction Production ( IU/ l ) 34 37 15,800 34 37—->30(1 hr) 43,400 34 37— 30(2 hr) 37,500 34 37—>30(3 hr) 28,100 34 37— 30(4 " hr) 36,300 34 30 12,300. Industrial Applicability The invention described herein is useful in the production of interferon, especially interferon from normal human diploid fibroblast cells. Interferon, in turn, is useful as an antiviral agent, immunoregulatory agent, agent for effecting cell division, and antican- cer drug. Equivalents Those skilled in the art will recognize, or be able to ascertain using no more than routine experimen¬ tation, many equivalents to the specific embodiments of the invention described herein. Additionally, it should be noted that it is believed that the tempera¬ ture switch described herein could also be useful in the production of other induced cell products, such as enzymes and hormones. Such equivalents are intended to be encompassed by the following claims.

Claims: Claims 1. In the production of interferon including induc¬ tion of cultured cells to stimulate interferon production followed by maintenance of said induced cells at an interferon-production temperature se¬ lected to sustain interferon production: The improvement of establishing a dis¬ tinct interferon-production phase in which the temperature is initially el- evated above the selected interferon- production temperature to a level and for a period sufficient to provide an increased sustained interferon produc¬ tion rate after said elevated tempera- ture is reduced to said selected interferon-production temperature. 2. The improvement of Claim 1 wherein said distinct interferon production phase is established by add¬ ing an antimetabolite to the cell culture during the induction phase and subsequently removing said antimetabolite to thereby establish said distinct production phase. 3. The improvement of Claim 2 wherein said cells com¬ prise fibroblast cells. 4. The improvement of Claims 1, 2 or 3 wherein said cells are cultured on microcarriers suspended in cell culture medium. 5. The improvement of Claim 4 wherein said elevated temperature is at least 3° C above the selected interferon-production temperature. 6. The improvement of Claim 5 wherein said elevated temperature is maintained for less than 4 hours after initiation of said distinct interferon pro¬ duction phase. 7. A method of producing interferon, comprising: (a) forming a cell culture of inter- feron—producing cells; (b) introducing an interferon-inducer into said cell culture in an amount suffi- cient to induce interferon production by said cells; (c) introducing an antimetabolite into said cell culture to effectively block the interferon production during the induction period; (d) removing said antimetabolite from said cell culture to initiate a distinct interferon production phase at a first tem¬ perature; and, (e) after a short period of interferon production at said first temperature, re- ducing the temperature of said cell culture to a temperature sufficient to sustain in¬ terferon production. 8. A method of Claim 7 wherein said interferon in¬ ducer comprises a non-viral inducer. 9. A method of Claim 8 wherein said interferon pro¬ ducing cells comprise fibroblast cells. 10. A method of Claim 9 wherein said first temperature is at least 3° C higher than the reduced tempera- ture. 11. A method of Claim 10 wherein said elevated tem¬ perature is maintained for a period of less than 4 hours before reducing the temperature. 12. A method of Claim 7, 9 or 11 wherein said cells are cultured on microcarriers suspended in cell culture medium. 13. A method of Claim 12 wherein said interferon in¬ ducer comprises polyinosinic-polycytidylic acid. 14. A method of Claim 13 wherein said antimetabolite includes actinomycin-D. 15. A method of Claim 14 wherein the induced cells are exposed to actinomycin-D for a period of at least one hour. 16. A method of Claim 15 wherein said interferon pro- duction phase is continued for at least 48 hours. 17. A method of producing interferon in a culture of interferon-producing cells, comprising: (a) forming a suspension of cell cul¬ ture microcarriers in cell culture medium; "BU E OMPI - 20 - (b) inoculating interferon-producing fibroblast cells into said suspension of microcarriers to thereby form a cell cul¬ ture of interferon-producing fibroblast cells; (c) maintaining said cell culture un¬ der conditions whereby said cells grow to a high cell density; (d) introducing an interferon inducer into said cell culture in an amount suffi¬ cient to induce interferon production by said fibroblast cells; (e) introducing an antimetabolite into said cell culture to effectively block in¬ terferon production during an induction pe¬ riod; (f) removing said antimetabolite from said cell culture to initiate a distinct interferon-production phase at a first tem¬ perature; and, (g) after a short period of interferon production, reducing the temperature of said cell culture to a temperature suffi¬ cient to sustain interferon production for a prolonged period. 18. In the production of cell by-products induced in cultured cells involving distinct induction and production phases, the improvement of initiating the production, at an elevated temperature and subsequently reducing the temperature for the balance of the production. 19. The improvement of Claim 18 wherein said cell by-product comprises a hormone. 20. The improvement of Claim 18 wherein said cell ID by-product comprises an enzyme. OMPI

Inventors: FLEISCHAKER R, GIARD D
Applicants: MASSACHUSETTS INST TECHNOLOGY, MASSACHUSETTS INST
Application year: 1981
Main classification: C12P21
IPC codes: C07K14, C12N9, C12P21