METHOD OF ENHANCING THE PRODUCTION OF HUMAN IFN-GAMA

Abstract: Method for stimulating the production of human IFN-//c in a culture of cells. In this method, an effective amount of a diterpene compound is added to a culture of IFN-//c producing cells.

Description: Method of Enhancing the Production of Human IF -^ Government Support Work described herein, and in parent application Serial No. 154,310, has been partially supported by grants and contracts from the U. S. Department of Health and Human Services (HHS) or its predecessor organizations. Technical Fields This invention is in the fields of cell biology and immunology. Background Art Interferon is a potent antiviral glycoprotein re- • leased by animal cells following viral infection and also after treatment of such cells with certain non- viral inducers. It constitutes one of the major de¬ fense mechanisms against viral infections in mammals, including humans. In addition to its antiviral func¬ tion, interferon has been found to have immunoregulatory activities, to affect various cellular functions includ¬ ing cell division, and to have value as an anticancer drug. Much research and development effort has been applied to the large scale production of human inter- feron. During the course of these efforts, it was learned that there are at least three distinct species of human interferon. "σ " or "Le" interferon is the major interferon species produced in cells of lyrophoi origin, whereas n j6 u or "F" interferon is the predominant species obtained after induction of non-lymphoid cells. In addition, there is a third interferon species, which has variously been referred to as V", "immune" or "type II" interferon, that is preferentially produced in cultures of lymphocytes, particularly in cultures enriched for T cells. Although it is not certain that T lymphocytes are the only source of this interferon species, it has also been suggested that this species be referred to as "T-type" interferon. " See Wietzerbin, J. et al. , "Immune Interferon Induced by Phytohemagglu- tinin in Nude Mouse Spleen Cells", Infect. Immun. 21> 966-72 (1978). In view of this, the terms '^-type inter¬ feron," " ^interferon" or simply IFN-^wiϊl be used throughout this description, and are meant to include "immune," "Type II" or "T-type" interferon. Each of the three species of interferon fit the general definition for interferon in that they are in¬ duced proteins that, on incubation with competent cells, produce a characteristic antiviral state. Nevertheless, these three interferon species have distinct properties including their antigenic characteristics and their physicochernical properties. See Vilcek, J. et al., "Synthesis and Properties of Various Human Interferons, " Microbiology - 1980, A er. Soc. for Microbiology, Washington, D. C. , pp. 204-207 (1980). IFN-^ is produced by lymphocytes in response to mitogens, or by sensitized T lymphocytes in response to specific antigens. A characteristic property of IFN-iT is its lack of stability at pH 2. This property is often used as the major criterion for . distinguishing IFN-?' from other species. An additional distinguish¬ ing property is based upon immunological analysis even though antiserum against human IFN- ^ jT is not yet avail¬ able. Antisera to <_ and _< interferon are available and they neither neutralize nor bind IFN-<ΪΓ. Thus, IFN-^ 1 is presently defined as being anti- genically "non- cr T, non-^c3". Although it ' has been suspected that IFN-<?" might be more efficient in its immunoregulatory, cell growth inhibitory and tumor growth inhibitory activities than other interferon species, very little definitive infor¬ mation has been available about IFN-^" because good pro¬ duction, and purification methods have not been discov¬ ered. One of the most widely employed methods for produc¬ ing human I N-^" to date has been the induction of human lymphocyte cells in cell culture with the plant lectin, phytohemagglutinin. See Wheelock, E. F. , "Interferon- Like Virus Inhibitor Induced in Human Leukocytes by Phytohemagglutinin," Science 149, 310-11 (1965) Al¬ though widely used, the production with phytohemagglu- tinin-induced lymphocyte cells has invariably produced relatively low yields of IFN-^I Disclosure of the Invention This invention relates to the production of IF - < ^ and is based upon the discovery that the addition of • certain diterpene compounds to cultures of white blood cells (leukocyte_s) can provide significant stimulation of human IFN-^T production. Stimulation of IFN-^ pro ¬ duction has been achieved with and without the addition of a known inducer, such as phytohemagglutinin. In preferred embodiments, a diterpene compound and IFN-^ 1 inducer are added to cultures of white blood cells to achieve the desired stimulation of production. The addition of a diterpen ' compound to stimulate IFN-_^ production increases the level of RNA for IF -^JT present in the cultures. This increased level of mRNA for I " FN-<_ facilitates the isolation of such mRNA, which can then be employed in subsequent creation and cloning of a recombinant DNA molecule containing the genetic sequence for lFτn-_ l Thus, this invention provides a method for stimu¬ lating IFN-^ production thereby increasing the likeli¬ hood that significant quantities can be produced under commercially practical conditions. - The inven- tion further provides a method for increasing the pro¬ duction in cell cultures of mRNA for IF -^ " which is use¬ ful in the isolation of this mRNA so that it can be employed in production of l N~?" by recombinant DNA procedures or other biological procedures requiring isolated mRNA. Best Mode of Carrying Out the Invention . Initially,- " a culture of cells capable of elabo¬ rating human lFN-_^"in a cell culture medium is formed. Such cells are typically sensitized lymphocytes, which can be induced by specific antigens, or unsensitized lymphocytes, which can be induced by mitogens. Of course, any other cell type capable of elaborating human IFJJ-^ would be suitable for use with this in¬ vention. In practice, the white blood cell cut (leukocytes) can be employed. This cut can be obtained from the buffy coat of a unit of blood or by cytopheresis. For example, a purified lymphocyte fraction- can be' employed, which can be obtained by blood centrifugation, column adsorption, or other art-recognized techniques. Never¬ theless, in some experiments described subsequently, unprocessed platelet pheresis residues or unprocessed buffy coat layers were more efficient interferon pro¬ ducers than their purified counterparts. The ability to omit a cell cut purification step simplifies the overall methodology, and appears to have the additional benefit of more efficient IFN-^ production in such cases. Cell culture media are employed in culturing the cells. Generally, these are synthetic formulations designed to provide sources of amino acids, vitamins, glucose, etc., to the growing cells. Typical culture media include RPMI 1640 medium, Eagle's medium, the Dulbecco-Vogt modification of Eagle's medium, and Ham's medium. Serum supplement may be added to the medium to provide certain proteins, and suitable examples of serum supplements include human plasma proteins, calf serum and fetal calf serum. Some celis suitable for producing IFN -2" can be grown in suspension cultures, such as various lymphoid lines. If, however, the- cells attach to a solid, surface, culture dishes, prescription bottles, roller tubes, roller bottle roller bottles, multi-tray units, or microcarriers can be employed. In general, art-recognized techniques are em¬ ployed to culture the white blood cells. Thus, those skilled in the art will- recognize, or be able to ascer- tain using routine experimentation, many equivalents to the methods for obtaining and culturing cells which have been specifically mentioned above. Human IFN-^can be induced in these cultures by adding an inducer for this interferon species. Mito- gens, including plant lectins such as phytohemagglu¬ tinin (PHA) and concanavalin A (CON A) , are known interferon inducers. See Friedman, R. and Cooper, H. , "Induction of Interferon in Lymphocytes by Mitogens," Proc. Soc. Exptl. Biol. Med. 125, 901-5 " (1967) and Wheelock, Ξ. F. (1965) , cited above. In addition, certain bacterial toxins, such as staphylococcal enterotoxin A have more recently been found to induce human IFN-^" from lymphocyte cells. See Langford, M. P.. et al., "Large-Scale Production and Physicochemical Characterization of Human Interferon," Infect. Immun. 26, No. 1, 36^41 (1979). Additionally, recent work indicates that certain enzymes, such as galactose oxi¬ dase, also -induce human IFN -_T production from lympho¬ cytes. See Dianzani, F. et al., "Enzymatic Induction of Interferon Production by Galactose Oxidase Treatment of Human Lymphoid Cells," Infect. Irnmun. 26, No. 3, 879-82 (1979) . It is also known that human IFN-^ can be induced in cultures of lymphocytes from sensitized donors by specific antigens, such as tuberculin. See Vilcek, J. et al. , "Syn¬ thesis and Properties of Various Human Interferons," Microbiology - 1980, A er. Soc. for ^Microbiology, 5 Washington, D. C. , pp. 204-7 (1980). Protein A is another recognized IFN-^ inducer. See Ratliff, L. R. , McCool, R. E. and Cataiona, W. S., "Interferon Induc¬ tion and Augmentation of Natural-Killer Activity by Staphylococcus Protein A," Cell. Immun. 57, 1-12 (1981) . Still yet another inducer is antilymphocyte serum, including monoclonal antibodies. See Falcoff, E. et al. , "Synthesis of Interferon in Human Lymphocytes Stimulated in vitro by Antilymphocytic Seriim," Eur. J. Clin. Biol. Res. 17, 20-6 (1972) . Other inducers of human IFN-r^are, of course, suitable for use with the' techniques of this invention. Although such known IFN-^JT inducers are ' usually employed in addition to a diterpene compound, this is not always necessary or desirable. As will be apparent from the experimental results presented subsequently, a significant stimulation of IFN-^production can some- times be achieved without the presence of a separate inducer. Thus, where a known inducer other than the diterpene compound is employed, the diterpene compound can be considered to "enhance" IFN-^" interferon produc¬ tion. Where the diterpene compound, is used alone, it might itself be considered- as an "inducer" of IFN-^ In both cases, the diterpene compound can be considered to "stimulate" IEN- < jT production. In the production of interferons, priming the cultures with relatively small amounts of interferon, as well as superinduction techniques, have often been • shown to increase production. It is believed that such priming and superinduction techniques may also be " beneficially employed with the enhanced production of IFN- "described herein. - 8 - The agents found to stimulate human IFN- productio are certain diterpene compounds, referred to herein as n 7P interferon production stimulating" diterpene compound Diterpene compounds, particularly esters, have been iso¬ lated from plant products, such as croton oil, and the diterpene moiety is derived from the tetracyclic hydro¬ carbon tigliane. See Hecker, E. , "Structure Activity Relationships in Diterpene Esters Irritant and Cocar- cinogenic to Mouse Skin," Carcinogenesis 2_, Mechanisms 10 of Tumor Promotion and Carcinogenesis, ed. Slaga, T. J. , Sivak, A. and Boutwell, R. K. , Raven Press, N. Y. (1978), the teachings of which are hereby incorporated by refer¬ ence. One class of suitable diterpene compounds is known 15 as phorbol esters, which have structural formulas rep¬ resented by: ACTIVE FORM wherein R, , 2 , ^ and R. are individually selected from hydrogen, alkyl, "alkoxy, carboxy., 'keto, aryl and alkaryl groups, and at least one of R_. and forms an ester functionality at the 12 or 13 ring position. R, and/or R~ are derived from carboxyliσ acids to form the ester bonds, and these carboxylic acids can be saturated or unsaturated hydrocarbons and also may include residues of cyclic compounds (e.g., benzene) in their chain. See, for example, Kupchan, S. M. , Sweeney, J. G. , Baxter, R. L. , Tatsuki, M. , Zimmerly, V. A. and Sickles, B. R. , J. Am. Chem. Soc. 97:3 (1975) Some specific examples of phorbol ester compounds and their abbreviations are illustrated in Table I, which follows. TABIE I.. Name ' Abbrev. ■ Z_ - ^2 - R -4- Phorbol (PHR) ' H H H, OH H Phorbol 12, 13-diacetate (PDA) CH3CO- CH 3 CO- H, OH H Phorbol 13 , 20-diacetate (P13,20DA) H CH 3 CO- CH 3 CO- H Phorbol 12, 13-dibenzoate (PDBz) C H c CO- C,H c CO- D 0 6 5 H, OH H Phorbol 12, 13-dibutyrate (PDB) C 3 H 7 CO- ' C 3 H_CO- H, OH H Phorbol 12, 13-didecanoate (PDD) • C 9 H-'CO-- C 9 ,H 19 C °- ; H, OH H Phorbol (4-0-methyl) 12- (MPMA) C 13 H 27 CO- CH 3 CO- H, OH CH myristate-13-acetate 3- Phorbol 12-monomyristate (PM) C 13 H 27 CO" H H,OH H Phorbol 12-myristate-l3- (PMA or ' CH^CO- acetate TPA) C 13 H 27 CO ~ H, OH H Phorbol (20-oxo-20-deoxy) (PMAAL) CH 3 'CO- C 13 'H 27 C ° " =0 H 12-myristate-13 acetate 4 alphα-Phorbol (4 -PHR) ' . .-• • H: . H ' H, OH H 4 alpha-Phorbol 12,13- (4 -PDD) didecanoate C 9 H 19 CO" C'gH.-^gCO- H,OH H Some of these phorbol ester compounds have been shown to be tumor-promoting. See Van Du.uren, B. L. , "Tumor-Promoting and Co-Carcinogenic Agents in Chemi¬ cal Carcinogenesis," Chemical Carcinogens, ACS Mono- graph 173,- Ed. Searle, C. E. (1976). At least one of these tumor-promoting phorbol ester compounds, phor¬ bol 12-myristate-13-acetate (PMA), ' also sometimes named 12-0-tetradecanoylphorbol-13-acetate (TPA) , has been found particularly effective for stimulating IFN-^ production from induced white blood cells. Thus, TPA is a preferred phorbol ester for purposes of stimulat¬ ing IFN- " production. Another plant-derived diterpene ester, mezerein, has been found to be an effective agent for stimu- lating IFN--? 1 production. Mezerein has been found to elicit many of the same cellular responses as TPA, but is much less effective as a tumor promoter, and in fact has significant antileukemiσ activity. See ' Mufson, R. A. et al., "Effects of 12-0-Tetradecanoyl- phorbol-13-acetate and Mezerein on Epidermal Ornithine Decarboxylase Activity, Isoproteronol-stimulated Levels of Cyclic Adenosine 3 * :5 '-Monophosphate, and Induction of Mouse Skin Tumors in Vivo, " Can. Res. 39, 4791-5 (1979) . It has a structural formula closely related to phorbol esters -' , as can be seen from the following structural formula for mezerein, which is taken from Kupchan, S. M. and Baxter, R. L. , :i Mezerein: Antileukemic Principle Isolated from Daphne mezereum L," Science 187, 652 (1974): Although TPA was added prior to the inducer in many of the experiments described below, the diterpene compound can also be added at approximately the same time as inducer is added, and possibly subsequently. In fact, as described herein, a separate inducer may be entirely omitted in some cases. The stimulated IF -^ production is " achieved according to- this invention because of the presence in the cultures of an increased level of mRNA for inter¬ feron. " This increased level of mRNA for IFN-^ is be- lieved to result because of increased expression of the gene for IFN-^ and/or increased stability for IFN--" mRNA. Because of this, the techniques described herein can be employed in the isolation of mRNA for IF -^ , the subsequent cloning of the genetic sequence for IFN-^ 1 , and the subsequent production of IF -^ by recombinant DNA technology. Such a method involves: formation of a culture of IFN-^-producing cells, pref- erably lymphocytes; the addition of a diterpene com¬ pound to stimulate cellular elaboration of IF -^; " optionally, the addition of an IFN-^ inducer, such as PHA; incubation. f the cells under conditions condu¬ cive to IF -^ production; extraction from the culture of RNA; ■ purification of the RNA to produce a fraction enriched in mRNA for IFN-^; preparation of complementary DNA- for IFN-^ , employing the enzyme, reverse trans- criptase, or other art-recognized techniques; incor¬ poration of DNA for IFN-^ 1 in a. cloning vehicle, such as a . bacterial plasmid; and, insertion of the DNA for IFN-^ into a microorganism. The invention can be further specifically illus¬ trated by the following examples. Example 1 Enhancement of PHA- nduced- /interferon . Production by TPA The following procedure was employed, unless different conditions are specifically recited. Lymphocytes from ' reshly collected heparinized venous blood were collected as follows. Leukocyte- rich plasma was centrifuged on Ficoll-Hypaque gradients. Cells collected from the interface enriched for lymphocytes were employed for the seeding of cultures. Multiple-well plastic tissue culture plates were seeded with 2 x 10 mononuclear cells/ml of RPMI 1640-medium. Culture fluids were collected after different periods of incubation with the inducer at 37° C in 5% CO„. Interferon titrationε were done by a microassay based on inhibition of vesicular stomatitis virus in human GM-258 cells with trisomy 21. See Havell, E. A. et al., J. Gen. Virol. 38, 51 (1978). ~~ By employing these general techniques, a numbe of plant lectins were screened for their induction capability for ^ interferon employing human lympho¬ cyte cell cultures. TPA was added at a concentratio of 5 ng/ml to the lymphocyte cultures three hours prior to addition of the appropriate lectins. The results were: ,u- - 15 - -Lectin Co ncentra tion- Source of • No TPA TPA j No TPA TPA Lectins Interferon, Units/ml P aseolufi (PHA) vulgaris 560 ' 15,360 •rr 0 2,560 Dolichos (DBA) .- biflor ε < 40 - 120 < 40 120 Solanura tuberosum- < 40 80 ^ 40 80 Sop ora japonica < " 40 160 < " 40 ' 80 Madura • pomif era " 40 960 <40 120 Pisum sativum <40 ' 3,840 40 5,120 Ulex (anti-HF) europeus <40 240 <40. 16.0 Ulex (anti-HC) europeus < 40 120 N.D • Arachis ypogaea < O 120 ^40 80 Glycine wa <40 ~40 <A0 40 Canavalia (COΓA) " ensif ormis 2JJ0' 10;240 < 40 960 Tr it I um (WGA) vulgaris 240 • 960 < 40 240 Ricinus (RCA-120) . Toxic " oxic comrounis _.y coper si con esculentum <40 40 40 < 40" Phytolacca [P M) 5, ,120 amencana 1,280 5,12b 960 Listeria (LPS) jnonocy to enes <T40 80 <40 40- These results illustrate that TPA was effectiv in enhancing- ?" interferon production for most of the lectins tested . Under these conditions , the enhancement of interferon production provided by the addition of TPA was several fold in most instances , and ranged up to more than 100-fold. Example 2 Effect of Cell Density on TPA-Enhanced PHA-Induced γ Interferon Production - Effective cell density was studied employing the procedures of Example 1 with pretreatment for 3 hours with 5 ng/ml TPA followed by the addition of 5 jug/ml phytohemagglutinin (PHA) in lymphocyte cultures maintained at 37° C in a humidified CO- incubator for 48 hours. The results were: Cells x 10 6 /ml Interferon, Units/ml 1.0 2,560 1.5 5,120 2.0 7,680 2.5 10,240 3.0 10,240. The results indicate that interferon enhancement increased with cell density up to about 2.5 x 10^ ells/ml, at which point production leveled off. Example 3 Effect of Pre-Treatment Time For TPA-Enhanced, PHA-Induced ^ Interferon Productiόri The effect of different times of pre-treatment was also studied employing the procedures of Example 1 and TPA at a concentration of 5 ng/ml and PHA at a concentration of 5 ;αg/ml. The results were: Hours of TPA Pre— rea ment Interferon, Units/ml 0 960 -1 1,760 -2 1,600 -3 1,280 ' . -4 1,920 -5 1,120 -16 960 -24 800 No TPA 400. The data indicate that it is preferable to pre- treat for at least 1 hour and that pre-treatment beyond 5 hours seems -to result in some loss of enhancement under these conditions. Example 4 Effect of Incubation Time on TPA-Enhanced, • PHA-Induced y - Interferon Productio The effect of incubation time was studied empl ing the procedures of Example 1 and using cultures of human lymphocytes with 5 ng/ml ' of TPA added 2 hours prior to the addition of 5 of PHA. The results .were: Hours of Interferon, Units/ml- Incubation No TPA TPA 48 200 2,300 ' 60 280 5,120 72 800 15,360 96 1,420 30,720 114 3,200 35,840 144 1,280 32,000 168 1,280 30,720. . IF -^ production with and" without TPA addition increased up to about 100 hours of incuba- tion, after which time a levelling off in interferon yields seemed to appear. Example 5 Effect of- PHA Concentration on TPA-Enhanced, FH -Iπduced γ Interferon Production The effect of PHA concentration was studied in lymphocyte cultures employing the procedures of Example 1 without any TPA and with 5 ng/ml TPA added 3 hours prior to addition of the appropriate con¬ centration of PHA. The results were: Interferon, Units/ml P PHHAA,, qg//mll N Noo TTPPAA TPA 0 <40 40 1 <40 3,840 2 400 3,840 4 560 7,680 6 _ 8 80000 " 7,680 8 280 7,680 10 240 5,120 15 320 4,480 " 20 320 3,840 3 3 55 1 10000 3,840 50 40 1,280. Under these conditions, enhancement by TPA in ¬ creased with increasing PHA concentration up to about 8 pg/ l, after which a decrease in enhancement was noted. Example 6 Effects of Varying TPA and PHA Concentrations on TPA-Enhanced, PHA-Induced W Interferon Production The combined effects of varying TPA and PHA con centrations on the production of human IFN-,^ from human lymphocyte cultures was studied employ¬ ing the procedures of Example 1 and wherein TPA was added for 3 hours prior to PHA addition. The results were: TPA at -3 hr _0 0.5 5.0 50 ng/ml eron, Units/πil 0 <40 3,840 3,200 40 0.5 <40 7,680 • 4,400 80 1.5 <40 5,600 2,400 240 .2.5 <:40 3,200 3,840 480 5.0 <40 12,800 12,800 5,120 7.5 <40 15,360 10,240 3,680 10 ^40 10,240 12,800 2,560 20 <40 25, 600 25,600 7,680 35 <40 7,680 17,920 5,760 50 <40 7,680 . 6,400 1,600. These results indicate that 5-20 ng/ml is the opt¬ imal range of TPA concentration for enhancing IFN-^T production with three different PHA concentra¬ tions under these conditions. EXAMPLE 7 TPA-Enhancement of Mitogen-Induced " ^Interferon Production The enhancement by TPA of * interferon produc¬ tion induced by various mitogens was studied employ¬ ing the procedures of Example 1, except that various mitogens were substituted for the inducers of Example 1. In cultures receiving combined treatment, TPA at a concentration of 5 ng/ml was added 3 hours before the mitogen (added in con¬ ' s centrations listed below) ♦ The results were: IFN. titer in Treatment units/ml PHA (5 μg/ml) <40 TPA/PHA 1,760 SEA (1 ' (0.02 μg/ml) 160 TPA/SEA 640 SEA (0.10 μg/ml) 160 TPA/SEA 640 PHA (5 μg/ml) 960 TPA/PHA 5,120 Gal. Oxid. (10 units/ml) 320 TPA/Gal. Oxid. 1,280 PHA (5 μg/ml) 40 TPA/PHA 1,280 Protein A (10 μg/ml) 250 TPA/Protein A 800 (1) Purified staphylococcal enterotoxin A (2) From Staphylococcus aureus EXAMPLE 8 Enhancement of PHA- nduced IF -jT Production by Various Phorbol Esters The enhancement of PHA-induced IFN-^ production by various phorbol esters was studied employing the procedures of Example 1 except that the phorbol ester noted below was substituted for TPA and plateletpheresis residues (6 x 10 cells/ml) were employed in place of fresh blood. PHA in a concentration of 5 ug/ l was added'3 hours after treatment of cell cultures with the phorbol ester. -Interferon titer was done with diploi fibroblasts employing EMC virus and the results are ex¬ pressed in laboratory units. The results were: INTERFERON TITER (UNITS/ML) Phorbol ester concentration (ng/ml) Experiment Treatment- 5 500 Without With Without With PHA PHA PHA PHA None <40 <40 Phorbol <40 <-J0 <40 <40 4α-Phorbol <40 <40 <40 <40 P12-M13-A (TPA) 120 3,840 <40 4,320 4-0-MeP12-M13-A <40 <40 <40 1,600 P12-M13-A20-al <40 110 <40 2,350 P12-M <40 <40 <40 - 2,560 P12,13-diD <40 290 <40 2,350 4α-P12,13-diD <40 <40- <40 <40 P12,13-diB <40 1,920 <40 3,000 P12,13-diBz <40 <40 <40 4,300 P12,13-diA <40 <40 -240 3,680 Pl3,20-diA <40 <40 <40 <40 None <40 160 Phorbol <40 160 <40 160 4α-Phorbol <40 150 <40 160 P12-M13-A (TPA) <40 5,120 <40' 2,560 EXAMPLE 9 Comparative Enhancement by TPA ' and MZN on PHA-Induced IFN-^ Production The comparative enhancement by TPA and Mezerein (MZN) on PHA-Induced FN-? production was studied era- ploying the procedures of Example 8, except as noted. PHA in a concentration of 5 g/ml- was added 3 hours after treatment of cell cultures with TPA or MZN. Un¬ processed plateletpheresis residues were tested, as well as mononuclear cells isolated from these residue-s. Interferon titer values are in laboratory units/ml of medium collected 48 hours after addition of PHA to the cultures. \ The results were: INTERFERON TITER Ficoll-hypaque isolated Unprocessed Treatment mononuclear cells pl telet-residue 2 x 10 6 ce His/ml 6 κ 10 cells/ml 2 x 10° cells/ml 6 x 10° cells/ml None <40^ . <40 <40 <40 P1IΛ (5 vg/ml) 320 1,280 640 <40 TPΛ (5 ng/ml) <40 <40 2,560 10,240 TPΛ/PIIΛ- 240 3,840 2,560 20,480 MZN (5 ng/ml) <40 .160 5,120 10,240 MZN/PHΛ 320 5,120 15,360 7,680 MZN (50 ng/ml) <40 160 5,120 40,960 MZN/PHA 640 5,120 10,240 40,960 EXAMPLE 10 TPA-Enhancement of OKT3- and PHA-Induced IF -^ Interferon Production and Effect of Different White Blood Cells and Cell Concentrations The effects of TPA on-IFN-^ production induced with PHA or OKT3 monoclonal antibody, or without a separate inducer, were studied employing different white blood cell concentrations of both isolated mononuclear cells and unprocessed plateletpheresis residues. In general, the procedures of Example 8 were employed, except as noted. 0KT3 is a monoclonal antibody against T cells. See Van Wauwe, J. P., DeMey, J. R. , Goossens, J. G. , "OKT3: A Monoclonal Anti-Human T Lymphocyte Antibody With Potent Mitogenic Properties," J. Immun. 124, 2708 r 13 (1980). 5 ng/ml of TPA was added to the cultures 3 hours prior ' to addition of 20 ng/ml of OKT3. The results were: Ficoll-hypaήue Isolated Mononuclear Cells Unprocessed Platelet-Residue Treatment lxlO 6 2xl0 6 3xl0 6 6xl0 6 lxlO 6 2xl0 6 3xl0 6 6xl0 6 cells/ml cells/ml cells./ml cells/ml cells/ml cells/nil cells/ml ——ce——ll—s/—m NONE <40 C40 :40 <40 40 <40 " 0 <40 TPA .40 <40 <40 ^40 <40 240 480 3840 O T-3 160 240 120 160 960 2560 2560 320 TPA/OKT-3 40 80 160 640 320 2560 10240 ' 10240 PHA 40 40 • 160 . ■ 80 , , 160 240 320 80 TPA/PHA 20 160 320 1920 480 1920 5120 5120 EXAMPLE 11 . - Preparation of Biologically Active Human IFN-^ mRNA Production an -Extraction of IF -?mRNA ' Induction of IFN--?' in human lymphocytes by PHA in the presence of TPA was carried out as described in Example 8. Briefly, mononuclear cells were isolated from lymphocyte-rich "platelet residues" by centrifuga¬ tion at 400 x g for 30 min on a Ficoll-hypaque gradient 6 (Pharmacia) . Cultures containing 6 x 10 mononuclear cells per ml .in serum-free RPMI.1640 medium (Gibco) were treated wit 5 ng/ml TPA (Consolidated Midland Corp.) for 2 hours; PHA (Burroughs Wellcome) was then added at the concentration of 5 ug/ml. After incuba- tion for the appropriate time at 37°G, cells were har- • vested by scraping with the- aid of a rubber policeman in a phosphate-buffered saline (PBS) . In a typical 9 experiment, 4 x 10 cells were pelleted by centrifu¬ gation at 800 x g for 5 min and resuspended in about 4 m! of PBS. Cells were then dispersed into 80 ml ice- cold RSB (10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 1.5 mM MgCl-) containing 10 M ribonucleoside-vanadyl com¬ plexes in a 150 ml corex tube. NP-40 was added to 0.3% (v/v) and, after thorough mixing, nuclei were removed by centrifugation at 3000 rpm for 5 min in a Sorval GSA rotor. The supernatant was poured off into a corex tube which contained 4 ml of 10% NaDodSO. , 4 ml of 2 M Tris-HCl, pH 9.0, and 2 ml of 0.25 M EDTA, and immediately extracted by phenol. The extraction was repeated twice and RNA was then precipitated by ethanol. Oligo(dT) -cellulose column chromatography ■ I ' was carried out according to the method of Taniguchi et al. See Taniguchi, T. , Sa ai, M. , F.uji-Kuriyama, Y. , Muramatsu, M. Kobayashi, S. and Sudo, T. , Proc. Japan Acad. Sci. 55 (B) , 464-9 (1979). Fractionation of the mRNAs by Sucrose Gradient Cen¬ trifugation Poly (A) -containing mRNA was heated at 70°C for 2 min and fractionated on a 5 - 25% linear sucrose gra¬ dient in 50 mM Tris-HCl, pH 7.5, 0.2 M NaCl, and 1 mM EDTA by centrifugation at 26,000 rpm for 19 hours . at 4°C in a Hitachi PRS 40 Ti rotor. . Riboso al RNA was sedimented in a parallel tube as size marker. Twenty-one fractions were collected and RNA in each fraction was precipitated by ethanol. Translation o IFN-zT mRNA in X. laevis oocytes Microinjection of mRNA into )£• laevis oocytes was carried out as described by.Pang et al. See Pang R.H.L. , Hayes, T.G. and Vilcek, J. (1980) Proc7 Nat. Acad. Sci. U.S.A. 77, 5341-3545. In the case of total poly (A) -RNA, RNA was dissolved in a solution of 10 M Tris-HCl, pH .7.5, 88 mM Na Cl (oocyte injection buffer) at the concentration of 0.5 or 1 mg per ml. Groups of 10 oocytes were incubated in 0.1 ml of Barth's medium for 24 hours at 24°C. The oocytes were then homogenized and the extract was assayed for IFN activity. Assay of IFN-^ IFN activity was assayed by inhibition of the cytopathic effect of vesicular stomatitis virus (VSV) or encephalomyocarditis (EMC) virus in human GM-258 - 30 - cells using the methods of Yip et al. See Yip, Y.K., Pang, R.H.L., Urban, C. and Vilcek, J. (1981) Proc. Nat. Acad.- Sci. ' . USA £0, 000-000. Since no reference standard for human IFN-^was available, all results are expressed in "laboratory units. " Effect of TPA on the yield of PHA-induced IFN-^ and its mRNA . IFN yields obtained from cultures of mononuclear cells induced with PHA alone or by the combined treat¬ ment with TPA and PHA were as follows: Interferon Titer - Platelet One-ml Culture Bulk Culture Residue 1to. PHA TPA/PHA TPA/PHA 1 80 ( 24) •5,120 ( 24) . 5,120 (40) 120 ( 48) 1,280 ( 48) . 40 (144) 2,560 (144) 2 1,280 ( 72) 1,920 ( 72) 1,280 (24) • 3 1,280 (114) 5,120 (114) ' 2,560 (24) 4 1,920 (138) 640 (138) 2,560 (24) 5 40 ( 90) 3,840 ( 90) 2,560 (24) 6 <40 ( 90) 20,480 ( 90) 5,120 (24) The one-ml cultures were done in 24-well microplates and the bulk cultures were done in 100 to 120 ml volumes in 140 cm diameter petri dishes. The num¬ bers in parentheses denote time of harvesting in hours after the addition of PHA. TPA was added at -2 hours. IFN yields obtained after stimulation with PHA were quite- variable, with cells from some donors pro¬ ducing over 1,000 units/ml, but with some other units producing much lower or undetectable amounts of IFN. In contrast, cultures induced by the combined treatment with TPA and" PHA produced relatively more uniform IFN yields, due to a marked enhancement of IFN production in cells which responded poorly to induction with PHA in the absence of TPA. Culture volume did not appear to greatly affect IFN production as similar yields were obtained in 1 ml microcultures and in bulk cultures of 100 to 120 ml. The same concentration of 6 x 10 mononuclear cells per ml was employed in both types of cultures. Although the kinetics of IFN production proved to be somewhat variable, the results showed that high IFN yields were reached by 24 hours after stimulation. Therefore, in experiments aimed at the extraction of IFN- ' mRNA, cells were harvested between 16 and 36 hours after induction. Most extractions were carried ou t from cells harvested at 24 hours after stimulation. To ascertain the suitability of the TPA/PHA stimulation for the extraction ' of IFN- < " mRNA, the following experiment was performed. Mononuclear cells g (about 5 x 10 total) obtained by Ficoll-hypaque centrifugation from one donor's platelet residue were divided into two halves. One half of the cells was first incubated with 500 units/ml of human IFN-∑g (fibroblast IFN) for 2 hours and then stimulated with PHA (5 ^ ug/ml) . Such "priming" with human IFN-4 or/3 has been shown to cause enhancement of PHA-stimulated IFN-<j7° production. See Wiranowska-Stewart, M. ; Lin, L. S.; Braude, I. A. and Stewart, W. E. , II; "Production, Partial Purification and Characterization of Human and Murine In¬ terferon— ype II," Molecular Immunology, Vo1. 17, pages 625-633 (1980) . The other half of the cells was incubated for 2 hours-with TPA and then stimulated with PHA. At 16 hours axter the addition of PHA, cells were harvested from both, groups of cultures, and mRNA was isolated by phenol extraction and oligo(dT)-cellulose column chroma- tography. Activity of the mRNA preparations isolated from the two groups of cultures was compared by inject¬ ing them at the same concentrations into X. laevis oocyte and assaying oocyte extracts for interferon activity. The mRNA extracted from PHA-induced cells primed with IFN-J5 produced no detectable IFN activity on oocyte in¬ jection ( <16 units/ml). In contrast, mRNA from TPA/PHA-induced cells produced clearly demonstrable IFN activity (maximum 196 units/ml) . On the basis of this experiment, all subsequent mRNA extractions were done from cells induced by the combined TPA/PHA treatment. RNA was extracted from TPA/PHA-induced cells de¬ rived from seven residues (representing-a total of 3.4 ' x 10 cells) . Cells from each residue were cultured and extracted separately. A total of 40 mg of RNA was obtained and pooled. Of this RNA, 33 mg was processed by oligo (dT)-cellulose column chromatography. Elution of the oligo (dT) -cellulose column yielded 900 ug of RNA which could be further purified by sucrose density gradient centrifugation or other.method. Antigenic Properties of IFN produced in Oocytes One of the important characteristics of IFN-7" is its lack of neutralization by antisera prepared against human IFN-& or IFN- . To ascertain that IFN synthesized in oocytes injected with RNA ' from TPA/PHA-induced mononuclear cells is indeed IFN-,? 7 , its affinity was • examined to a serum with high ϊieutralizing activity against IFN-3 and^5. Oocyte-derived IFN-7 " was ex¬ tract of X. laevis oocytes injected with sucrose gradient centrlfugation-purified fraction of mRNA isolated from cultured mononuclear cells stimulated with TPA/PHA. Control oocyte•extract was from σocytes injected with buffer. Anti-IFN • { <(Lan JS ) was anti- serum produced by immunization of a rabbit with human IFN-G_, containing neutralizing antibodies against IFN-øC and IFN-jό (but not IFN-^ . IFN preparations were incubated with an excess of this antiserum or with a control serum (no antibody) for 1 hour at 37°C and then assayed for IFN activity. The results were: IFN ' titer IFN preparation No With anti-IFN antibody (.α + β) Oocyte-derived IFN-γ 384 512 Oocyte-derived IFN-$ 512 <4 Control IFN-α 1024 <4 Control IFN- 128 <4 Control IFN-γ 512 768 Control oocyte extract <4 <4 This oocyte-derived.interferon preparation was not neutralized, indicating that the activity was due to neither IFN-oc nor IFN-jό and, therefore, it ' was likely .. to be IF -<^. A control preparation of oocyte-derived IFN- J 5 produced in the same batch of oocytes by in¬ jection with mRNA extracted from poly(I) "poly(C)-induced human fibroblasts was neutralized by this " antiserum. Control human IFN-<2. and IFN--χ3 preparations (reference standards G-023-901-527 and G-023-902-527, respectively) . were both completely neutralized by the antiserum while a control preparation of TPA/PHA induced IFN- was not. Industrial ' Applicability The invention described herein is useful in the production of human IFN-^ , which in turn is useful as an antiviral agent, immunoregulatory agent, agent for affecting cell division,- and anticancer drug. ' The enhancement gained is also useful in the production of IFN-2^ by recombinant DNA techniques or other bio¬ chemical techniques employing mRNA for IE -^". Equivalents Those skilled in the art will recognize, or be able to ascertain using no more than routine experi¬ mentation, many equivalents to the specific embodi¬ ments of the invention described herein. Such equiva- lents are intended to be encompassed by the following claims.

Claims: Claims 1. In the production of human IFN-^ from cells: The improvement of stimulating IFN-^" production by contacting said cells with an effective production- stimulating amount of at least one IFN--^" stimulating diterpene compound. 2. The improvement of Claim 1 wherein said diterpene compound comprises a phorbol ester. 3. The improvement of Claim 2 wherein said diterpene compound comprises mezerein. 4. A method 'of producing human IFN-^ , comprising: a) ' forming a cell culture of human IFN-^-producing cells in a suitable cell culture medium; b) introducing an IF -^ stimulating compound into said culture in an effective production-stimulating amount; and, c) maintaining said cell culture under conditions conducive to IF - T 1 production. 5. The method of Claim 4 additionally including the step of introducing a human IF -_^ inducer into said cell culture in an effective IFN- < jT-inducing amount. 6. A method of Claim 5 wherein said IFN-^ inducer comprises a mi ogen. 7. A method of Claim 6 wherein said IFN-^" inducer comprises a plant lectin. 5 8. A method of Claim 7 wherein said plant lectin comprises phytohemagglutinin. 9. A method of Claim 8 wherein said plant lectin comprises concanavalin A. 10. A method of Claim 5 wherein said IFN-<5" inducer 10 comprises a bacterial toxin. 11. A method of Claim 5 wherein said IF -^ inducer comprises an enzyme. 12. A method of Claim 5 wherein said IF -^ inducer comprises protein A. 15 13. A method of Claim 5 wherein said IFN-^ inducer comprises antilymphocyte antiserum. 14. - A method of Claim 13 wherein said -antiiymphocyte antiserum comprises a monoclonal antibody. 15. A method of Claims 4, 5, 6 or 14 wherein said 20 IFN-^* producing cells comprise unprocessed cyto- pheresis residues or unprocessed buffy coat cells. 16. In a method for producing mRNA for IFN-*? 7 in a culture of white blood cells including contacting said cells with an IFN-,? 7' inducer: The improvement of contacting said cells with an effective amount of a diterpene compound to thereby raise the level of mRNA for IFN-.?" 7 in said culture. 17. A method of producing IFN-^, comprising: a) forming a culture of cells; b) adding a diterpene compound to said culture in an effective amount for enhancing cellular elaboration of IFN-.?"; c) optionally adding an IFN-^ inducer to said culture; d) incubating said cells under conditions conducive to IFN-c^" production; e) extracting RNA from said cul¬ ture; f) purifying said RNA to produce a fraction enriched in mRNA for IFN-^; g) preparing DNA complementary to said mRNA for IFN-<£"; h) incorporating said DNA into a recombinant DNA vector; and, i) inserting said vector into a microorganism. 18. A method of Claim 17 wherein said diterpene com¬ pound comprises a phorbol ester. 19. A method of Claim 18 wherein said phorbol ester comprises 12-0-tetradecanoylphorbol-13-acetate. 20. A method of Claim ' 17 wherein said diterpene com¬ pound comprises mezerein. ■

Inventors: VILCEK J, YIP Y
Applicants: UNIV NEW YORK, NEW YORK UNIV
Application year: 1981
Main classification: C12P21
IPC codes: A61K38, C07C69, C07H21, C07K14, C12N15, C12P19, C12P21