BE897263A - INTERPERON-BASED PREPARATION AND MANUFACTURING METHOD THEREOF - Google Patents

Description

       

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   TITLE "Interferon-based preparation and its manufacturing process" (Inventors: JARVIS Jr. Allan P. and KOSOWSKY David I.)

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The present invention relates to L: ne new composition of matter (hereinafter called "epsilon interferon" or "IFN-e" useful for example in cultures of human cells or others as an antiviral and / or control agent against proliferation, as well as methods of making such a composition and methods of treating human epithelial cells to resist viral infections.



   Interferons are materials that have antiviral properties. They are prepared using certain types of cells which have been stimulated by exposure to a virus, certain nucleic acids and antigen-mitogenic complexes. Interferons are extremely potent pharmaceuticals that hold great promise as clinical antiviral and anti-tumor agents.
There are currently three known types of human interferons: alpha interferon (IFN-a), prepared from human leukocytes or lymphoblastpid cells, beta interferon (IFN-P), prepared from fibroblasts, and l gamma interferon (IFN-y), prepared from human T lymphoeytes.

   These three interferons are secreted by the respective cells after stimulation of these by viruses, certain nucleic acids or mitogenic antigen complexes. Recent indications show that these interferons can comprise a mixture of proteins with a linked structure, and not a single protein. Human interferons can be differentiated from each other by their different level of activity on human and bovine cultures of this species. An analysis used for measuring activity can be performed as described in The Interferon System, William E. Stewart, Springer Verlr3p'Co., New York, 1978, which is a variant of the method of Ho and Enders, The Proceedings of the National Academy of Sciences, Volume 45, pages 385-389, 1959.

   IFN-a has an antiviral activity level on oxen kidney cell cultures that is 1 to 5 times its activity on

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 human fibroblasts. IFN-P has a level of antiviral activity on cultures of ox kidney cells from 1/100 to 2/1 00 times its activity on cultures of human fibroblasts. IFN-γ has an activity on ox cell cultures less than 1/100 times its activity on human fibroblast cultures.



   In the most productive process of making IFN-a from human leukocytes, about one unit of interferon is produced per 50 cells. IFN-γ is prepared from human T lymphocytes with a yield of approximately 1 unit per 1000 cells. IFN-ss prepared from human fibroblasts has an optimal yield of about 1 unit per 15 cells.



   An antiviral unit of interferon corresponds to the concentration which protects half of the cells of a culture of human fibroblasts subjected to competition from the vesicular stomatitis virus at a normalized or standard concentration (one pfu / cell).



   Antivir activity has been detected; d. ins of; media from other cells of human and animal origin. Interference with the multiplication of the yrippe virus was first detected by Issacs and Lindenman zwans of chorioallantoic membrane cultures of chicken. Another example is the Hela cell series which is a transformed cancer cell of epithelial (cervical) origin as reported by G. Gey et al., Cancer Research, Vol. 12, p. 264-165, 1952. Interferon activity has been detected in various series of transformed or neoplastic cells originally derived from human epithelial tissue, as described in Production of Interferon by Human Tumor Cell Lines, Jameson et al, Archives of Virology 62, 209-219 (1979).



  It has been observed that known preparations of interfuron humjin have antiviral activity in human cell cultures.



   The invention relates to the discovery and production of a new type of antiviral material, called "interferon E". This new material which, like the others

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 interferons, appears to be composed of at least two functionally linked proteins, can be produced for example by treatment of normal human epithelial cells by an interferon induction technique, then by incubation of the cells under conditions which favor the production of the new interferon. IFN-E, unlike the known alpha, beta and gamma substances, has a level of antiviral activity in cultures of ox kidney cells, between approximately 1/4 and 1/2 times its activity in human cultures.

   IFN-E does not exhibit significant mutual activity with antisera prepared in opposition to IFN-a, IFN-P, or IFN-y in neutralization analyzes. It is stable at pH 2. Its production by epithelial cells is eliminated by the introduction of inhibitors of "RNA" synthesis or inhibitors of protein synthesis into the culture. The antiviral activity of preparation containing IFN-E is destroyed by proteases.



   IFN-E can be prepared by any type of primary human diploid epithelial cell. Cultures of human cells of epidermis and connective, vaginal and esophageal tissues have also been used with success.



  Non-limiting examples of other types of epithelial tissue which can be used are the tissues of the nose, pharynx, bronchi, pleura and intestine.



  It has been advantageously discovered that primary epithelial cells can be induced to produce interferon-E, using techniques already used in known manner for the production of other types of interferons from leukocytes , lymphoblasts and fibroblasts. In an advantageous induction technique using Newcastle Disease Virus (Bankowski strain) or Sendai Virus, 100 to 500 epithelial cells are required for the production of one unit of IFN-E.



   As previously indicated, IFN-E is prepared by primary diploid epithelial cells of human origin. The expression the primary diploid epithelial cells

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 ", or cultures of such cells, refers herein to cells sampled from healthy human tissue or a culture of such cells prepared, for example, by the method described in U.S. Patent 4,016,036 These types of cell cultures should be distinguished from non-human cells, human cells of non-epithelial origin, and transformed or neoplastic epithelial cells.



   The new interferon can also be produced in naturally or artificially transformed eukaryotic cells containing the part of the human epithelial nucleic acid responsible for the production of IFN-E and in cells modified by recombinant DNA technology.



   It was also discovered that a prepared interferon '. from a given type of human cell, for example epithelial cells, had greater antiviral activity on that particular type of cell than on any other? types of human cells. Air15i, interferon8cpsiluil has increased the antiviral and antitumor possibilities in the treatment of epithelial tissue subjected to a tumor or viral infection, compared to its relative effectiveness with other types of human cells.



   Thus, the invention relates to a new type of antiviral substance useful for example for the protection of human cells against attack by viruses. It also relates to a method of manufacturing interferon of the type native to normal healthy human epithelial tissue.



  It also relates to a method of treating tumor growth or viral infection in a particular type of tissue by exposure to an interferon derived from the same type of tissue. It also relates to a type of interferon which is more active on human epithelial cells than on other types of human cells, for example human fibroblasts.



   Generally, normal human epithelial cells can be grown in vitro (for example,

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 according to the method described in the patent of the United States of America No. 4,016,036) in the form of a monolayer, in microcapsules by implementing the method described in the patent application of the United States of America No. 243,586 filed March 14, 1981 by Jarvis et al., Or by other methods, during the preparation of interferon E.



  The cells are then subjected to an IFN induction technique by exposure to certain viruses or nucleic acids.



  For example, the culture medium can be replaced by a medium containing a known IFN inducer of the viral or nucleic acid type. After a short incubation, for example of the order of one hour, the inducing agent is removed, and the cells are placed in a new normal culture or maintenance medium and incubate for example for 24 hours. Exposure of cells to the inducing agent triggers expression of the cell's DNA code for the production of epsilon interferon. During the subsequent incubation, the cell synthesizes and secretes IFN-E.

   At this time, the crop is harvested and, if it is. is analyzed for example by the method of Ho and Enders (Proceedings of the National Academy of Science, Volume 45, pages 385-389, 1959), it is found that it has an antiviral activity of IFN-E.



   The product which is obtained by this method does not show significant mutual reactivity with an antibody to IFN-a, IFN-P or IFN-y in neutralization experiments. It is stable at pH 2 and is protein in nature. Immunoabsorption experiments indicate that anti-IFN-p antisera react with part of the interferon produced by induced epithelial cells. The new interferon also has a different activity profile in mammalian cells than that of the known type of interferon.



  These chemical characteristics clearly show that IFN-E is an original substance.



   IFN-E is currently being satisfactorily produced by normal epithelial cells

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 epidermis, connective tissue, vaginal tissue and human esophagus, by "normal induction" (Field et al., Proceedings of the National Academy of Sciences, Vol. 58, p. 1004-1009,1967) and "superinduction" (Havell and Vilcek, Antimicrobial Agents in Chemotherapy, Vol. 2, p. 476-484, 1972) with nucleic acid, poly I'C (Miles Laboratories), with Newcastle Disease Virus ( Baron and Issacs, British Medical J., Vol. 56, p. 18-20 1962) and with the Sendai virus (paragrippe-1, Gresser, Proceedings of the
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 Society of Experimental and Biological p. 303-307, 1961).

   The results of the analyzes show that all the induction techniques and all the types of cultures of upruvial cells.



  The production levels vary with the nature of the inductor used and with the various modifications of the '. induction techniques. Newcastle disease virus works well. Other viruses that can be used are listed in the table below.

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  TABLE I
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 <tb>
 <tb> Virus <SEP> References
 <tb> Flu <SEP> A <SEP> Gress <SEP> and <SEP> Dull <SEP> (1964) <SEP>;
 <tb> Andrews <SEP> (1961)
 <tb> Paragrippe-3 <SEP> Chany <SEP> (1960)
 <tb> Measles <SEP> Petralli, <SEP> Merigan, <SEP> and <SEP> Wilbur
 <tb> (1965a) <SEP>; <SEP> DeMaeyer <SEP> and <SEP> Enders <SEP> (1961)
 <tb> Parotitis <SEP> epidermal. <SEP> Cantell <SEP> (1961) <SEP>;

    <SEP> Waddell, <SEP> Wilbur,
 <tb> and <SEP> Merigan <SEP> (1968)
 <tb> Rubella'Neva <SEP> and <SEP> Weller <SEP> (1964]
 <tb> Syncytium Respiratory <SEP> <SEP> Ray, <SEP> Gravelle, <SEP> and <SEP> Chin <SEP> (1967) <SEP>;
 <tb> Moehring <SEP> and <SEP> Forsyth <SEP> (1971)
 <tb> Virus <SEP> from <SEP> the <SEP> rage <SEP> WiKtor <SEP> and <SEP> al. <SEP> (1972)
 <tb> Stomatitis <SEP> vesicular <SEP> Marcus <SEP> and <SEP> Sekellick <SEP> (1977) <SEP>;
 <tb> Wilcek, <SEP> Yamazaki <SEP> and <SEP> Havell <SEP> (1977)
 <tb> Chikungunya <SEP> Zimmermann <SEP> and <SEP> al. <SEP> (1972)
 <tb> Sindbis <SEP> Gress <SEP> and <SEP> Enders <SEP> (1962)
 <tb> Encephalitis <SEP> equine <SEP> Luby, <SEP> S @ nders, <SEP> and <SEP> Sulkin <SEP> (1971)
 western <tb>
 <tb> Yellow fever <SEP> Wheelock <SEP> and <SEP> Sibley <SEP> (1965) <SEP>;

    <SEP>
 <tb> Wheelock <SEP> and <SEP> Edelman <SEP> (1969) <SEP>
 <tb> Poliovirus-Type <SEP> 1 <SEP> Gress, <SEP> Chany, <SEP> and <SEP> Enders <SEP> (1965)
 <tb> Poliovirus-Type <SEP> 2 <SEP> Smorodintsev <SEP> and <SEP> al. <SEP> (1970) <SEP>;
 <tb> Ho <SEP> and <SEP> Enders <SEP> (1959a, <SEP> b)
 <tb> Encephalomyocarditis <SEP> Stewart <SEP> II, <SEP> Gosser, <SEP> and <SEP> Lockart
 <tb> (1971a)
 <tb> Rhinovirus-2 <SEP> Smorodintsev <SEP> and <SEP> al. <SEP> (1971a) <SEP>;

  
 <tb> Fiala <SEP> (1972)
 <tb> Rhinovirus-12 <SEP> Gatmaitan, <SEP> Stanley, <SEP> and <SEP> Jackson
 <tb> (1973)
 <tb> Reovirus-2 <SEP> Goose, <SEP> Loh, <SEP> and <SEP> Rotnayake <SEP> (1973)
 <tb> Language <SEP> blue <SEP> from <SEP> sheep <SEP> Jameson <SEP> and <SEP> Grossberg <SEP> (1977)
 <tb> Adenovirus <SEP> Lysov <SEP> and <SEP> al. <SEP> (1971)
 <tb> Chickenpox <SEP> Vaczi, <SEP> Horvath, <SEP> and <SEP> Hadhazy <SEP> (1965)
 <tb> Cytomegalovirus <SEP> human <SEP> Vaczi, <SEP> Horvath, <SEP> and <SEP> Hadhazy <SEP> (1965) <SEP>;
 <tb> Glasgow <SEP> (1974)
 <tb> Herpes <SEP> simplex <SEP> Rasmussen <SEP> and <SEP> ale
 <tb> Vaccine <SEP> Wheelock <SEP> (1964) <SEP>;

    <SEP> Epstein,
 <tb> Stevens, <SEP> and <SEP> Merigan <SEP> (1972)
 <tb>
 

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It has also been found that interferons made from a selected type of human cells such as epithelials, fibroblasts, vascular, hepatic, renal, etc., are particularly effective as antiviral agents or antitumors for the treatment of formed human tissues of the type chosen cell.

   Thus, IFN-E is especially effective when used for the treatment of epithelial cells, and it is found that it has a more
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 low activity when used to treat other human cells. nL for origin of human fibroblasts) is more effective for the treatment of human tissues or fibroblasts and therefore has weaker anti-tumor and antiviral properties vis-à-vis cells of other types of human tissue.



   Thus, the following method can be used to protect a given tissue or type of cells against viral infection, so that the multiplication of a virus which has infected a given tissue or type of cells is stopped , or that the growth of a tumor in a given tissue is stopped.



   A culture of cells of the type considered is first formed by growth by the implementation of conventional techniques such as (in the case of epithelial cells) the Green technique described in the aforementioned patent of the United States of America No. 4 016 036. The cells of the culture are then treated so that they induce the expression of the part of its DNA code responsible for the production of interferon. The operation can be carried out for example by the IFN induction techniques described in the rest of this memo.



  The product is then harvested, for example in the medium after incubation. The product can be used as an effective antiviral or anti-tumor substance for the treatment of cells or the type of tissue in question.
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  In vitro cell cultures are protected against viral attack by the addition of 2 to 5 units of IFN-E / cm3.

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  This concentration is effective for the protection of epithelial cells having a density of 0.5105 to 2.50.10 cells / cm.



   Significant amounts of this new antiviral substance can also be made by two additional cellular techniques currently known.



  The first uses types of eukaryotic cells transformed spontaneously, virally or chemically, analogous to those used previously for the production of IFN-a from series of human lymphoblastoid cells. The second technique uses known recombinant DNA techniques, analogous to those which are currently used currently for the production of IFN-a and IFN-ss.



   According to the first method, transformed cells capable of producing IFN-E can be obtained by using one of the two techniques: in vivo or in vitro. The first technique (in vivo) involves direct isolation from fresh tissue. type of transformed cells, the second technique (in vitro) involves a selection operation in which a strain of normal human diploid cells is cultivated in the long term until a small but detectable number of the population undergoes spontaneous transformation or the initial strain is treated for a short period with a known virus or mutagen such as EMS, MMS, or MNNG so that the frequency of transformation is increased.



  Some of the cells transformed by one? from r? s two in vitro techniques contain part of the DNA code of the epithelial cells responsible for IFN-E production.



   Transformed cultures thus obtained can be induction to produce IFN-E using conventional induction techniques as described above for normal human diploid epithelial cells.



   In the second technique, mRNA synthesized in epithelial cells following the induction of IFN

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 is extracted from the cells, is optionally purified by ultracentrifugation or the like so that the mRNA fraction obtained has increased specificity, and the extract is used as a template for the synthesis of complementary DNA (cDNA). The cDNA can be prepared using the reverse transcriptase enzyme Avian Myoblastosis.

   The final product resulting from the operation, double standard complementary DNA (dscDNA) which contains the sequence coding for the synthesis of IFN-E, and a eukaryotic or bacterial vector are then treated with a restrictive enzyme which causes cleavage of DNA and gives binding sites by which the dscDNA and the vector can be attached. The vector and DNA are then mixed, annealed and linked by covalent bonds using a ligase enzyme. At this time, the recombinant vector preparation is used for the transformation of a bacterial or eukaryotic cell.

   The transformed cells thus contain DNA complementary to all or part of the RNA originally contained in the epithelial cells during their stage of production of TFN-E.
These recombinant vectors are then incubated with an appropriate type of cell which allows the operation of the vector. A cell population then appears which contains individual cells capable of synthesizing and secreting IFN-E. The cell population is then systematically analyzed to give a subpopulation of cells producing IFN-E, and this subpopulation is cultured so that it gives a series of cells capable of producing relatively large quantities of IFN- E.



   Other characteristics of this technique using recombinant DNA appear for example in the following documents:

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1. U.S. Patent No. 4,237,224 Cohen et al "Process for producing biologically functional molecular chimeras";
2. Scheller, R. et al, 1977, Clones of individual repetitive sequences from sea urchins DNA constructed with synthetic eco R1 sites, Science, Vol. 196, p. 197-200;
3. Blattner, F. et al, (1977), Charon Phages: Safer derivatives of bacteriophage lambda for DNA cloning, Science, Vol. 196, p. 161-169;
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 4. Broach, J. R., and J. D., 0,)) tion and recombination functions associated with yeast plasmid, 2 micron circle, Cell, Vol. 21, p. 501-508; and
5.

   Hamer, D. 1981, Synthesis processing and secretion of eukaryotic protcinsin thé SV40 - Monkey cell system, Recombinant DNA Abstracts, Vol. 1, p. 4.



   Other characteristics and advantages of the invention will emerge more clearly from the description which follows of nonlimiting examples given purely by way of illustration.



   'Example 1
A culture of human epidermis epithelial cells is grown from the laboratory of Howard Green, Massachusetts Institute of Technology to a density
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 5 2 cells from 1 to par in a minimal essential medium (MEM, "Gibco") containing 10% of fetal calf serum inactivated thermally (incubated at 56 ° C. for 30 minutes: HIFCS). Four equivalent cultures are exposed incubated in MEM and 2% HIFCS for 1 hour at 37 C at poly (I. C) - (9S) which is a nucleic acid of high molecular mass commercially available from PL Laboratories (Milwaukee, Wisconsin), in an amount equal to 0.1, 10 and 100 micrograms / cm3 (previous induction technique of Field et al.).

   The cultures are then washed twice with MEM and incubated in the same medium with the addition of 2% HIFCS for 24 hours.



  The medium is collected and analyzed by the Stewart II method, The Interferon System, p. 17-18. No activity is observed

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 detectable antiviral in cultures with 0 and 3 3 1 g / cm3 of poly (I. About 10 of IFN-E are detected in the samples obtained from cultures having undergone induction with 10 and 100 lig of poly (I-C).



   Example 2
The process of Example 1 is repeated, but a modified "superinduction" technique (Havell and Vilcek, supra) is used in an attempt to increase the production of IFN- #. 50 g / cm of cyclohexamide (protein synthesis inhibitor) are incorporated with poly (I-C) acid. After the one hour incubation, the cells are washed and incubated for an additional 3 hours in MEM containing 32% HIFCS and 50 g / cm of cyclohexamide. The cells are then washed again and incubated with MEM
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 3 3 container. 50 g / cm3 of cyclohexamide and 5.0 g / cm3 of actino'mycin D (inhibitor of RNA synthesis) for an additional 2 hours.

   At the end of this incubation, wash
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 two ciihç-i- at 370C in a normal environment. The culture of the medium and the analysis as indicated in Example 1 do not allow the production of any detectable amount of IFN-E in the cultures
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 g induced with 0 and 1 of poly (I-C). The culture 3 3 induced with 10 g / cm 3 of poly (I-C) gives 100 3 of IFN-E. The cultures induced by 100 g / cm3 of poly (I-C) g ensure the production of more than 330 lig / cm Example 3
A culture of epithelial cells of epidermal origin containing 1 to 2 is used. 105 cells (Example 1) for the preparation of IFN-E using the Newcastle disease virus (NDV) induction method (Baron and Issacs, supra).

   The virus used was the Odnkowski strain of NDV available from Poultry Heoith Laboratn'ies, Davis, California. Four samples of 1 cm of MEM containing 2% HIFCS and enough Nil \ / pOull are prepared, the final concentration being respectively 0, 100-200, 25-50 and 1-16 pfu virus / cell in the samples.

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  3 0.2 cm of the respective preparations of virus 3 of 1 (virus inducing agent) are then added to the cell cultures which are then shaken for 30 minutes at 5 minute intervals at 37 C. The remaining part of 0.8 NDV preparations are then added and the cultures are incubated for an additional 30 minutes. The cell cultures are then washed twice with a
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 3 normal medium, 1 medium is added to each culture, and the cultures are incubated for 24 hours.



   The medium is then harvested, acidified to pH 2 with 0.1 N HCl and stored at 4 ° C. for 5 to 6 days so that NDV becomes inactive. Then, the harvested medium is neutralized with 0.1 N NaOH and the presence of IFN- is analyzed.



  In the sample not containing NDV virus (control), all antiviral activity is absent. The sample has undergone induction with 100 to 200 virus particles per
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 3 NDV cell contains about 500 to 1000 units of.



  The culture induced with the preparation of NDV containing 25 to 50 particles of virus per cell contains approximately "3 170 to 330 units of IFN-E / cm. The culture induced with the preparation of NDV containing 1 to 16 particles of virus
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 3 per cell contains approximately 50 to 170 units of.



   As this experiment indicates, a culture of cells containing 1 to 10 cells / cm3 can give
3 approximately 500 to 1000 units of IFN-E / cm3. The yield is thus of the order of 1 unit of IFN-E produced for 100 to 500 culture cells.



   Example 4
The process of Example 3 is repeated, but the paragrippe-1 virus (Sandai) is used. at various concentrations, as an inducing agent at pt. tce of NDV virus (see Gresser, supra). Sendai virus as purchased (Flow Laboratories) contains 10,000 to 40,000 hc units-
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 l.

   magglutinous
In a culture in which the commercial preparation of virus is diluted 1 to 3 times,
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 3 1000 units / cm of A dilution of 1 to 10 gives

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TABLE II Results of interferon experiments
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 <tb>
 <tb> Type <SEP> of <SEP> cells <SEP> Method <SEP> induction <SEP> Units <SEP> IFN-e / cm <SEP> Units <SEP> IFN - # / cell
 <tb> Conjunctive <SEP> human <SEP> poly <SEP> I-C * <SEP> 33 <SEP> 3.3 <SEP> x <SEP> 10-5
 <tb> " <SEP> " <SEP> poly <SEP> I.C + <SEP> 330 <SEP> 3.3 <SEP> x <SEP> 10-4
 <tb> " <SEP> " <SEP> none <SEP> (witness) <SEP> 0 <SEP> 0
 <tb> Epidermis <SEP> human <SEP> poly <SEP> I.C * <SEP> 0 <SEP> 0
 <tb> " <SEP> " <SEP> Sendai ++ <SEP> 330 <SEP> 3.3 <SEP> x <SEP> 10-4
 <tb> "" NDV <SEP> ** <SEP> 1000 <SEP> 1,

    <SEP> 0 <SEP> x <SEP> 10 ' <SEP>
 <tb> " <SEP> " <SEP> none <SEP> (witness) <SEP> 0 <SEP> 0
 <tb>
 
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 3 * Induction of Example 1 with 100 pg / cm poly 1.



  3 + Induction method of Example 2 with 100 μg / cm 3 of poly I.



  ** Induction method of Example 3 with 1/3 dilution of NDV (1C-21 virus particles / cell) ++ Induction method of Example 4 with 1/4 dilution of the s

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 3,200 units / cm3 and a dilution of 1 to 33 No antiviral activity can be detected in the control culture from which the virus has been omitted.



   Table II above summarizes the results of the experiments of Examples 1 to 4 carried out with cultures of cells of human connective tissue and cultures of cells of human epidermis. with various induction techniques.



   Example 5
As it has conventionally been shown that interferons are by definition proteins, samples of IFN-E have been tested in order. to determine if the antiviral activity present in the samples was due to a protein-based component. The experiments are carried out by incubating samples of IFN-a (NIH standard), IFN-P (NIH standard), and IFN-E (taken from NDV-induced epithelial keratinocytes) in the presence of known proteolytic enzymes, then by nnrilyr. f of the material treated so that the remaining antiviral activity is known to be determined.

   Samples of IFN-a (16 units / cm3), IFN- (16
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 3 3 and IFN-E (32 in MEM, in the presence of either trypsin (Sigma Chemical Co., St. Louis, Missouri) or Pronase (Sigma) at concentrations of
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 3 3, 3-100 ug / cm. the end of the reaction, the activity
After proteolytic remaining in the samples is neutralized by addition of HIFCS until final concentration of 33% (volume / volume). The samples are then analyzed so that the remaining antiviral activity induced by IFN is determined by microtiter on GM-2767 cells.



  The results are as follows:

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 PROTEOLYTIC SENSITIVITY OF IFN-E
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 <tb>
 <tb> Enzyme <SEP> Concentration
 <tb> proteolytic Enzyme <SEP> <SEP> Activity <SEP> antiviral <SEP> IFN <SEP> remaining
 <tb> (g / cm) <SEP> IFN- &alpha;

    <SEP> IFN-ss <SEP> IFN- # <SEP>
 <tb> Trypsin <SEP> 100 <SEP> 0 <SEP> 0 <SEP> 0
 <tb> 33 <SEP> 0. <SEP> 0 <SEP> 0
 <tb> 10 <SEP> 1-2 <SEP> 4
 <tb> 3.3 <SEP> 8 <SEP> 8 <SEP> 16
 <tb> 0 <SEP> 16 <SEP> 1 <SEP> Li <SEP> 3-- <SEP>
 <tb> Pronase. <SEP> 10 <SEP> 0 <SEP> 0 <SEP> 0
 <tb> 3.3 <SEP> 0 <SEP> 0 <SEP> 0 <SEP>
 <tb> 0 <SEP> 16 <SEP> 16 <SEP> 32
 <tb>
 
These results show that the antiviral activity observed in the IFN-E samples is due to the presence of one or more components in the form of proteins, given the total elimination of the activity by the treatment with proteolytic enzymes. The character. IFN- # protein also appears in the fact that inhibitors of protein and RNA synthesis block the production of IFN-E.



   Example 6
This example shows the stability of IFN-E for acid pH. IFN-E is prepared by induction of human epidermis cells using NDV according to Example 3.



  When the medium is harvested, it is divided into three parts. The virus is inactivated in the first part
 EMI17.2
 as described in Example 3, by acidification to pH 2 with HCl. The second part is filtered for 5 days on a "Millipore 01310 filter (molecular weight limit 100,000, Millipore Corp., Bedford, MA) so that the virus is extracted. The third part is filtered by a" Millipore "filter PTMK01310, having a molecular weight limit of 100,000, previously impregnated for 4 hours in a 1% BSA solution.

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   In all cases, the resulting medium is analyzed so that the presence of IFN and residual virus is determined. The results are as follows:
Stability of IFN- # at acid pH
 EMI18.1
 
 <tb>
 <tb> Processing <SEP> Title <SEP> initial <SEP> Title-Final <SEP> iiter IFN <SEP> <SEP>
 <tb> of <SEP> IFN-de <SEP> virus <SEP> from <SEP> virus
 <tb> (pfu / cm3) <SEP> (pfu / m3) <SEP> (u / cm) <SEP>
 <tb> pH <SEP> 2.5 <SEP> days <SEP> 1, <SEP> 1 <SEP>. <SEP> 108 <SEP> 0 <SEP> 128
 <tb> Filtered <SEP> by
 <tb> PTHK01310 <SEP> 0 <SEP> 1 <SEP>? <SEP> S <SEP>
 <tb> Filtered <SEP> by
 <tb> PSVP01310 <SEP> 1, <SEP> 1. <SEP> 10 <SEP> 0 <SEP> 128
 <tb>
 
None of the samples showed viral activity except. elconque and all samples had the same amount of IFN-E activity.

   This result shows that the activity of IFN-E is stable at an acid pH.



   Example 7
The immunological properties of IFN- # (prepared by NDV induction of human epidermis cells as described in Example 3), of IFN-a, of IFN- (NIH) and of IFN-γ (obtained from S. Baron, University of Texas Medical School, Galveston, Texas). Titrations are carried out by neutralization of each interferon using anti-IFN-a (NIH), anti-IFN-ss (NIH and YH Tan, Calgary University, Calgary, Alberta, Canada), anti -IFN-y (S. Baron), and mixtures of these antisera. 2 fold dilutions of appropriate antisera are performed in MEM with a 98 cell microtiter plate.
 EMI18.2
 



  3 Dilutions ranging from 32 to 3 2 of each IFN preparation are added to the tanks and the plates are incubated first at 37 ° C. for 1 hour, then at 4 ° C. for 1 hour so that the antibody-IFN complex can
 EMI18.3
 form. It is then seeded with fibiohiastuu (deposit of human mutant cells at the rate A of 2. cells per tank, and, after 20 to 24 hours

  <Desc / Clms Page number 19>

    incubation at 37 ° C., they are subjected to a standard dose of vesicular stomatitis virus (1 pfu / cell).



  After 24 to 48 hours at 37 ° C., all the tanks are observed under the microscope so that the inhibition transmitted by interferon I the cytopathic effect induced by the virus is determined (CPE). The results are as follows:
Quantity of antiserum required to neutralize IFN 3 (neutralization unit per cm)
 EMI19.1
 
 <tb>
 <tb> Sample <SEP> Conc. <SEP> Anti- &alpha; a <SEP> Anti-ssa <SEP> Anti- &alpha; a
 <tb> units / cm <SEP> Anti-ssa
 <tb> IFN-a <SEP> 16 <SEP> N. <SEP> D. <SEP>> <SEP> 3 <SEP> 000 <SEP> N. <SEP> D.
 <tb>



  (NIH <SEP> Std) <SEP> 8 <SEP> N. <SEP> D. <SEP>> <SEP> 3 <SEP> 000 <SEP> N. <SEP> D.
 <tb>



  4 <SEP> 62 <SEP>> <SEP> 3 <SEP> 000 <SEP> 125
 <tb> IFN-ss <SEP> 8 <SEP>> <SEP> 3 <SEP> 000 <SEP> 375 <SEP> 750
 <tb> (NIH <SEP> std) <SEP> 4 <SEP>> <SEP> 3 <SEP> 000 <SEP> 188 <SEP> 750
 <tb> 2 <SEP>> <SEP> 3000 <SEP> 188 <SEP> 47
 <tb> IFN- # <SEP> 8 <SEP>> <SEP> 3 <SEP> 000 <SEP>> <SEP> 3 <SEP> 000 <SEP> 188
 <tb> AR-IVb <SEP> 4 <SEP>> <SEP> 3 <SEP> 000 <SEP> 375 <SEP> 188
 <tb> 2 <SEP>> <SEP> 3 <SEP> 000 <SEP> 375 <SEP> 47
 <tb> IFN-E <SEP> 16 <SEP>> <SEP> 3 <SEP> 000 <SEP>> <SEP> 3 <SEP> 000 <SEP> 375
 <tb> AOB <SEP> 8 <SEP>> <SEP> 3 <SEP> 000 <SEP>> <SEP> 3 <SEP> 000 <SEP> 375
 <tb> c <SEP> 2 <SEP>> <SEP> 3 <SEP> 000 <SEP> 1 <SEP> 500 <SEP> 94
 <tb>
 The sheep control serum against fibroblast IFN has no neutralizing activity. NOT.

   D. = not determined
 EMI19.2
 a with respective sheep b-derivatives of epidermal keratinocytes, not necessarily devoid of fibroblasts (induced by NDV) c - derivatives of epidermal keratinocytes devoid of fibroblasts (induced by NDV).



   We can see from the above tables that:

  <Desc / Clms Page number 20>

   a) IFN-a is neutralized by both the anti-a and anti-a / anti-P antisera in a mixture. As expected, you need twice as much anti-alpha / ss as anti-alpha; to neutralize the same amount of IFN-a.



  (Analogous amounts of antisera neutralize analogous amounts of IFN-a). b) IFN-ss is neutralized at. both by anti and anti-a / anti antisera. in mixture. Two times more anti-a / ss antiserum is needed than anti-antiserum to neutralize the same amount of JTN-p. c) Less than half the antiserum mixture is needed. anti-a / anti-ss compared to the anti-ss antiserum for the neutralization of the same amount of IFN-E.



  IFN-E does not react with antisera directed against IFN-y.



   This example shows that IFN-e is immunologically distinct from IFN-a and IFN-P, and IFN-y and is therefore chemically distinct from these known human interferons.



   Example 8
In addition to the serological indications presented in Example 7, IFN-E, which has undergone partial immunoprecipitation, is used in a neutralization experiment. Add to this interferon samples. anti-S IFN (polyclonal obtained from NIH or monoclonal obtained from Y. H. Tan). The mixture incubates for 1 hour at 350 ° C. and then for 1 hour at 4 ° C. so that the IFN-antibody complex can be formed, and the complex, with any excess of antibodies, is removed by incubation of the complexed and non-complexed antisera in the presence of protein "S'3pharfJse" A (Sigma) with a ratio of 30 μl to 1.5 μl of the reaction mixture.

   Uncomplexed and remaining IFN is subjected to a second stage of immunoprecipitation and the samples are analyzed so that the activity of IFN is determined as described, using the CPE inhibition analysis. The results are as follows:

  <Desc / Clms Page number 21>

 
 EMI21.1
 
 <tb>
 <tb> 0Sample <SEP> Title <SEP> 1st <SEP> precip. <SEP> Title <SEP> 2nd <SEP> precip. <SEP> Title
 <tb> IFN <SEP> original <SEP> IFN Antibody <SEP> IFN <SEP> Antibody <SEP> IFN <SEP>
 <tb> units / cm3 <SEP> with <SEP>: -after <SEP> with <SEP>:

    <SEP> after
 <tb> 1st <SEP> 20 <SEP>
 <tb> AOB-IFN-E <SEP> 1024 <SEP> Anti-ss <SEP> 256 <SEP> Anti-ss <SEP> 256
 <tb> polyclonal <SEP> polyclonal
 <tb> AOB-1FN-E <SEP> 1024 <SEP> Anti-ss <SEP> 1024 <SEP> Anti-ss <SEP> 1024
 <tb> monoclonal <SEP> monoclonal <SEP>
 <tb> AOB-IFN-E <SEP> 1024 <SEP> Anti-ss <SEP> 1024 <SEP> Anti-ss <SEP> 1024
 <tb> monoclonal <SEP> polyclonal
 <tb> AOB-IFN- # <SEP> 1024 <SEP> Anti- <SEP> 256 <SEP> Anti-ss <SEP> 256
 <tb> polyclonal <SEP> monoclonal
 <tb>
   When AOB-IFN- # having previously precipitated is used in the neutralization test, carried out exactly as indicated in example 7, the following results are obtained:

   
Amount of antiserum required to neutralize IFN (neutralization units)
 EMI21.2
 ...
 EMI21.3
 
 <tb>
 <tb> Sample <SEP> Concentration <SEP> IFN <SEP> Anti-ss <SEP> Mixture <SEP> of anti-a / ss
 <tb> u <SEP> Icm3 <SEP>
 <tb> AOB <SEP> IFN-E <SEP> 16 <SEP>> <SEP> 3 <SEP> 000 <SEP> 1 <SEP> 500
 <tb> absorbed <SEP> with <SEP> 8 <SEP>> <SEP> 3 <SEP> 000 <SEP> 375
 <tb> antiserum <SEP> 4 <SEP>> <SEP> 3 <SEP> 000 <SEP> 94
 <tb> anti-ss
 <tb> AOB <SEP> IFN-C <SEP> 16 <SEP>> <SEP> 3 <SEP> 000 <SEP> 1 <SEP> 500
 <tb> 1 <SEP> absorbed <SEP> with <SEP> 8 <SEP>> <SEP> 3 <SEP> 000 <SEP> 188 <SEP>
 <tb> anti <SEP> serum <SEP> 4 <SEP> 1 <SEP> 500 <SEP> 94
 <tb> witness
 <tb> AOB <SEP> IFN-e <SEP>> <SEP> 3 <SEP> 000 <SEP> 1 <SEP> 500 <SEP>
 <tb> no <SEP> absorbed <SEP> 4 <SEP> 375 <SEP> 94
 <tb> 1 <SEP> 12 <SEP> 24
 <tb> NIH <SEP> IFN-P <SEP> 16 <SEP> 750 <SEP> 750
 <tb>

  8 <SEP> 188 <SEP> 375
 <tb> 4 <SEP> 94 <SEP> 188 <SEP>
 <tb>
 The fibroblast IFN sheep control serum has no neutralizing activity.

  <Desc / Clms Page number 22>

 



   The results of the above tables show that part of the IFN- # preparation cannot be neutralized by the anti-ss antiserum even when two different anti-antibodies are used. However, even after precipitation pushed by anti-ss antisera, an additional part of IFN- can be precipitated
 EMI22.1
 with a mixture of anti-a / P antisera These results also show that IFN-E is immunologically and chemically distinct from other known human interferons.



   Example 9
IFN-E produced by induction of human epidermis cells with NDV as indicated in Example 3, can be purified and concentrated by affinity chromatography on a number of immobilized ligands. These include, but are not limited to, reactive red agarose, phenyl- "Sepharose" (Sigma Chemical Co., St. Louis, no.), Procion red agarose, phenyl agarose
 EMI22.2
 (BRL, Gaithersburg, Md.), Glycogel B, ..propionyl) aminodecane (CPAO) agarose and N-pyromellityl- N- (3-carboxy-aminodecane (PMAD) agarose (Pierce Chemical Co., Rockford,. Ill.).
 EMI22.3
 



  3 8 of container containing 3,900 units of IFN-E are applied in a column of 0.5 of reactive red agarose 3. The column is washed with 10m 20mM phosphate buffer (pH 7.4). IFN is then eluted with three 3 quantities equal to 2 of 50% ethylene glycol in a 20 mM phosphate buffer containing 1 M sodium chloride.



  Each fraction is collected in an equal volume of 20 mM phosphate buffer so that the ethylene glycol is diluted.



  The column is finally washed with an additional 5 cm of 20 mM phosphate buffer. Protein concentrations are measured pure absorption at 280 nm (OD-2UU).



  The results are shown in the following table:

  <Desc / Clms Page number 23>

 
 EMI23.1
 .



  1- '(U U 0) bu 1-.



  QJ bU: 1-.



  QJ In 0 1-. bu (U 0) D QJ o "ri fa 0) - ( <j r Z LL QJ Q) C o - lu 0) U - ri 4- - 1-.



  I] CL
 EMI23.2
 
 <tb>
 <tb> 4-c
 <tb> l
 <tb> - <SEP>
 <tb> 4-41 <SEP> Co <SEP> C <SEP>:) n
 <tb> - <SEP>: <SEP> CD
 <tb> - '
 <tb> E
 <tb> laughed <SEP> 0 <SEP>
 <tb> rq <SEP> -i
 <tb> r) <SEP>
 <tb> F-1 <SEP> 4D <SEP> 1-0
 <tb> C)
 <tb> O <SEP>
 <tb>
 

  <Desc / Clms Page number 24>

 
The first two ethylene glycol fractions are combined and the resulting fraction contains any IFN-E applied to the column, showing a purification of factor 6 compared to the original material.
 EMI24.1
 



  In other similar experiments, 3 to 68 IFN-E are applied to the column and IFN 3 is recovered in a final volume of 8 This represents an 8.5-fold concentration of the material.



   When IFN-E is applied to at least two ligands immobilized successively, a greater purification can be obtained. For example, apply a sample of IFN- to a CPAD column and elute as described in the previous method. The combined fractions containing IFN indicate a purification of approximately 9 times and undergo dialysis with a 20 mM phosphate buffer so '. that ethylene glycol be extracted. The sample is then applied to a column of agarose PMAD, developed in the same way. IFN-E is recovered with a yield of 70 2 100% and an additional purification of 5 times pal compared to the total protein.



   This example shows that IFN-E can be purified by various immobilized ligands to be considered alone or in combination.



   Example 10
A centrifugation is first subjected to unfractionated IFN-e at 300 g for 20 minutes. The supernatant is mixed by inversion on a Fischer Rotarack device with a glass of controlled porosity (C. P. G.)
 EMI24.2
 (Electronucleonics, Inc.) at 4 C. 0.28 g of glass 3 of controlled porosity is used for 30 of culture which floats. cm After 3 hours, the floating material is extracted and the glass beads of controlled porosity are piled up in a column (2.5 x 3.1 cm).

   The column is first washed with 4 column volumes of PBS, then 4 column volumes of NaCl 1, U M-20 mM phosphate buffer at pH 7.4. IFN- # is then eluted with 4 column volumes of 50% ethylene glycol by volume / volume-1.0 M NaCl-20 mM phosphate buffer at pH 7.4,

  <Desc / Clms Page number 25>

 
 EMI25.1
 lSl u <C riD m Q) fa f-.



  + -> ID TJ TI Q) Q) Q) Or M 'Q)' CD.



  UJ o o el CL TI QJ CD [-.



  [-.



   > QJ CD QJ CD 'o -' n tW 1 Z LL H CD TI C o - + -> m u - 4- - ri [-.



  : CL
 EMI25.2
 
 <tb>
 <tb> <: <SEP>: <SEP> t1
 <tb> pi <SEP>
 <tb> 0lu)
 <tb> U
 <tb> CD
 <tb> CD
 <tb> pu
 <tb>
 <tb> rD <SEP>
 <tb> mi <SEP> H <SEP> CO <SEP> CD <SEP> I <SEP> ura <SEP> 0 <SEP> or
 <tb> 1 <SEP>
 <tb> CN <SEP> O <SEP> rs <SEP> CO <SEP> ar <SEP> I <SEP> CN <SEP> eDCD <SEP> 1
 <tb> 1
 <tb> E
 <tb> U
 <tb> 9 <SEP>
 <tb> o <SEP> ^ 0
 <tb> i <SEP>
 <tb> ¯ <SEP>
 <tb> d, <SEP>
 <tb> o <SEP> 4D <SEP> tXt <SEP> z <SEP> Q <SEP> O <SEP> I <SEP> h <SEP> UM.
 <tb>



  D <SEP>
 <tb> o <SEP>
 <tb> E <SEP>
 <tb>
 

  <Desc / Clms Page number 26>

 
 EMI26.1
 then is collected in an equal volume of NaCl 1, phosphated 20 mM so that the final concentration of ethylene glycol is 25%. The fraction containing IFN-E is concentrated by ultrafiltration at 1.0-1.6 and is applied.
1to a column of polyacrylamide-agarose ("Ultragel" AcA54) (1.6 x 85 cm) balanced with 25% ethylene glycol (by volume / volume) -NaCl 1.0 M 20mM phosphate buffer at pH 7.4 .
 EMI26.2
 IFN-E is then eluted with a balancing buffer solution 3 3 at a rate of 6.0 cm / h. Fractions of are collected and the presence of IFN and the protein content are analyzed (Loury analysis).

   The results are given in the table above. t
Example 11
The preparations of IFN-E, IFN-a and IFN-ss, containing partially purified raw material according to Example 10 (p-IFN-E) and absorbed p-IFN-E and anti-mouse interferon, are subjected to molecular weight analysis by SOS gel electrophoresis. according to the Lamelli process.



  The samples are incubated at room temperature for 1 hour in the presence of 1.0% SOS, 0.05 M tris-HCl buffer (pH 6.8), 10% glycerol (volume / volume) and 0.001% bromophenol blue, loaded onto 12.5% polyacrylamide gels, and testing lasted approximately 16 hours. After electrophoresis, the elongated zones containing the molecular weight references are seeded. The regions containing IFN are cut into 3 mm slices and extracted by shaking
3 with 0.5 cm of PBS containing 0.5% SOS for 20 hours at room temperature. The analysis of these fractions is carried out on GM-2767 cells and the molecular mass of each activity peak is calculated by comparison with the position on the gel relative to the molecular mass references.

   The results are as follows:

  <Desc / Clms Page number 27>

 
0 Molecular mass of IFN
 EMI27.1
 
 <tb>
 <tb> M. <SEP> M. <SEP> of <SEP> maxima <SEP> of activity <SEP> (". <SEP> of activity <SEP>
 <tb> INF <SEP> retrieved)
 <tb> a <SEP> 18 <SEP> K <SEP> (27.6) <SEP> 19, <SEP> 7 <SEP> K <SEP> (44.8) <SEP> 22.5 <SEP> K <SEP> (27.6)
 <tb> ss <SEP> - <SEP> (1, <SEP> 83).

    <SEP> - <SEP> - <SEP> 22, <SEP> 0 <SEP> K <SEP> (98.2)
 <tb> # <SEP> (gross) <SEP> 17.5 <SEP> K <SEP> (41, <SEP> 0) <SEP> 20, <SEP> 0 <SEP> K <SEP> (7.4) <SEP> 22.0 <SEP> K <SEP> (51.6)
 <tb> p-IFN-E <SEP> 17, <SEP> 5 <SEP> K <SEP> (35.4) <SEP> 20, <SEP> 0 <SEP> K <SEP> (2, <SEP> 53) <SEP> 22.0 <SEP> K <SEP> (62.0)
 <tb> p-IFN-E <SEP> no <SEP> linked <SEP> 17.5 <SEP> K <SEP> (9.2) <SEP> 20.0 <SEP> K <SEP> 0 <SEP> 22.0 <SEP> K <SEP> (90.8)
 <tb> p-IFN-E <SEP> linked <SEP> 17, <SEP> 5 <SEP> K <SEP> (68.0) <SEP> 20, <SEP> 0 <SEP> K <SEP> (15, <SEP> 3) <SEP> 22.0 <SEP> K <SEP> (16.8)
 <tb>
 The three fractions obtained with the samples of IFN- # show antiviral activity on GM-2767 cells.



  The fraction of molecular weight 22, 0.13 obtained from IFN-E does not show any activity on oxen kidney cells (MDBK). The other two fractions of IFN- (17.5K,! O, OK) have an activity of approximately 25%, on MDBK cells as on GM-2767 cells.



   Example 12
The antiviral power of IFN- # (produced by NDV induction from human epidermis cells as in Example 3), IFN-a (obtained from National Institute of Health, NIH), and IFN-P (NIH ) is compared in normal human epidermis cell cultures, compared to normal human diploid fibroblasts.



  Serial dilutions of the three TFN preparations are added to the cells of a microtiter plate with reference cultures of normal human epidermis cells and normal human fibroblasts. After 18 to 24 hours of incubation at 37 ° C., the cells of each tank are subjected to the action of a standard dose of vesicular stomatitis virus (1 pfu / cell). After 36 to 96 h (when witnesses indicate 100% cell death), each cell is observed under a microscope until resistance to the virus is determined.

   The results are as follows:

  <Desc / Clms Page number 28>

 TABLE III
 EMI28.1
 
 <tb>
 <tb> - <SEP> 3 <SEP>
 <tb> Units <SEP> activity / cm <SEP> from <SEP> IFN <SEP> on <SEP>: <SEP>
 <tb> Interferon <SEP> Fibroblasts <SEP> (FS-4) <SEP> Epithelium
 <tb> IFN-e <SEP> 32-64 <SEP> 512-7048 <SEP>
 <tb> IFN-a <SEP> 128-512'64 <SEP>
 <tb> IFN-P <SEP> 32-256 <SEP> 8-32
 <tb>
 normal human diploid a-fibroblasts obtained from J. Vilcek (N. Y. U., N. Y., N. Y.).



   As the table above indicates, IFN-E produced by the DNA code of epithelial cells has a much higher potency (by a factor of the order of 16) as an antiviral agent compared to epithelial cells than compared to fibroblasts. IFN-a prepared from DNA drawn from human leukocytes and lymphoblastoid cells, has a higher potency (by a factor of up to 8) compared to fibroblasts than compared to epithelial cells, and IFN-P, produced from DNA code of fibroblasts has a greater power (up to 30x) vis-à-vis fibroblasts than vis-à-vis epithelial cells.



   Example 13
The method of Example 11 is used for the analysis of the relative antiviral activity of interferons and E on ox kidney cells (MDBK ATCC CCL22 cells), compared to human trisomal fibroblasts (3 copies of chromosome 21- deposition of human mutant cells GM-2767 47, XX, t21).

   The results of the analysis are as follows:
 EMI28.2
 
 <tb>
 <tb> 3
 <tb> Units <SEP> activity / cm <SEP> from <SEP> IFN <SEP> on
 <tb> Type <SEP> IFN <SEP> MDBK <SEP> GM-2767
 <tb> a <SEP> 512 <SEP> 512 <SEP>
 <tb> ss <SEP> 0 <SEP> 12ss <SEP>
 <tb> y <SEP> 0-128 <SEP>
 <tb> E <SEP> 8-16 <SEP> 32 <SEP>
 <tb>
 

  <Desc / Clms Page number 29>

 
 EMI29.1
 The results obtained for IFN a, sset are in accordance therewith the results already indicated. The IFN-E results show that this new interferon has an activity of approximately 2 to 4 times greater on GM-2767 cells than on ox kidney cells.



   Example 14
The new interferon also has activity against proliferation, as indicated by the inhibition of the multiplication of human lymphoblastoid cells (Daudi cells). Approximately 10,000 human lymphoblastoldps cells are coated with 200 microliters of medium
 EMI29.2
 in each tank of a microtiter plate. Various dilutions of IFN a, ss and E are then added to each tank and the number of viable cells is increased after 1, 2 and d days. The results are shown in the following table:
ANALYSIS OF DAUDI CELLS - 4 Number of viable cells / cell (x1D).
 EMI29.3
 
 <tb>
 <tb>



  Type <SEP> and <SEP> concentration IFN <SEP> <SEP> Day <SEP> 1 <SEP> Day <SEP> 2 <SEP> Day <SEP> 4
 <tb> 100u <SEP> 6, <SEP> 0 <SEP> 4.8 <SEP> 6.8
 <tb> 10 <SEP> u <SEP> 4.8 <SEP> 5.2 <SEP> 6.0
 <tb> ss <SEP> - <SEP> 100 <SEP> u <SEP> 7.2 <SEP> 9.6 <SEP> 6.4
 <tb> 10 <SEP> u <SEP> 5.2 <SEP> 9.6 <SEP> 18.4
 <tb> # <SEP> - <SEP> 100 <SEP> u <SEP> 6.0 <SEP> 5.2 <SEP> 4.8
 <tb> 10 <SEP> u <SEP> 5.6 <SEP> 5.2 <SEP> 6.0
 <tb> Witness <SEP> (without <SEP> IFN) <SEP> 8.0 <SEP> 21, <SEP> 5 <SEP> 67
 <tb>
 These results show that 10 units of INF-e can suppress the growth of Daudi cells by more than 90%.

  <Desc / Clms Page number 30>

 



   Example 15
IFN-a, P and # samples are balanced with phophate buffered saline (PBS), sodium dodecyl sulfate (SOS), and a mixture of SDS,
 EMI30.1
 p and urea at 100 C. The SOS detergent denatures proteins in a known manner by altering their conformation, and BME reduces the disulphide bonds. We see that INF-a. ss and -different very clearly by their sensitivity to these reagents.

   More specifically, INF-o. is stable in SOS alone, but is made significantly inactive in the presence of a mixture of SOS, urea and BME; IFN-P is practically inactivated in SOS alone but is stable in a mixture of SOS, urea and BME, and INF-E is relatively stable in SOS alone and in the mixture of SOS and. of BME-urea. In PBS, at 100 ° C., less than 5% of the activity of the three interferons remains.



   In analogous experiments, it is determined that INF-a. which was previously inactivated by the SOS-BME mixture, can be reactivated from its equilibrium at 100 C with a. mixture of SDS-PBS, that INF-ss which has been previously inactivated in SOS alone can be reactivated in a mixture of SDS-BME-urea-PBS, and that IFN-S which has been previously inactivated in PBS can be reactivated by setting in
 EMI30.2
 equilibrium at 100 ° C. in the presence of PBS-SOS, with or without BME-urea. These observations also show the originality of the preparation of IFN-E according to the invention.



   Example 16
This experiment concerns the comparison of the binding and elution properties of IFS-C and IFN-P on a blue "Sepharose" column (Pharmcia). The columns are prepared according to the manufacturer's instructions and loaded at room temperature with samples IFN-ss or IFN-E in PBS. The elutions are carried out with NaCl 1, OM in PBS and 50% ethlyene glycol (in volume / volume) in a 20mM sodium phosphate buffer.



  Each interferon preparation is treated in two columns
 EMI30.3
 containing different lots of blue "Sepharose", and the following results are obtained

  <Desc / Clms Page number 31>

 
 EMI31.1
 Interferon binding to blue "Sepharosis"
 EMI31.2
 
 <tb>
 <tb>:% <SEP> of activity <SEP> recovered <SEP> with
 <tb> Fraction
 <tb> IFN-P <SEP> IFN-E <SEP>
 <tb> Crossing <SEP> column
 <tb> + <SEP> 20mM <SEP> from <SEP> buffer <SEP> phosphated <SEP> 44, <SEP> 0-44, <SEP> 2 <SEP> 2.5-10, <SEP> 2
 <tb> Elution <SEP> with <SEP> NaCl <SEP> 1, <SEP> OM <SEP> 2, <SEP> 7- <SEP> 4, <SEP> 3 <SEP> 19, <SEP> 3-30
 <tb> Elution <SEP> with <SEP> ethylene <SEP> glycol
 <tb> 50% / NaCl <SEP> 1, <SEP> OM <SEP> in <SEP> 20mM <SEP> from
 <tb> buffer <SEP> phosphated <SEP> 51.9-52, <SEP> 3 <SEP> 60.0-78,

    <SEP> 2
 <tb>
 
This example shows that the interferons E and ss have different affinities to blue "Sepharose" and different behaviors during elution. The blue "Sepharosis" fixes approximately 90 to 97% of the activity of INF-E but only approximately 60% of INF-e are linked under the same conditions. In addition, while 20-30% of INF- is recovered with 1.0 M NaCl 3-4% only of the activity of IFN-E is recovered under analogous conditions.



  Finally, up to about 80% of INF-ss activity is eluted with a combination of 50% ethylene glycol and 1.0% NaCl while only about 50% of INF-E activity is recovered under similar conditions.


    

Claims (16)

CLAIMS 1. Preparation based on interferon having antiviral activity, the preparation being produced by culturing living cells containing at least part of the DNA code of a human epithelial cell so that the interferon is synthesized and by harvesting a fraction rich in the interferon of the culture, * the interferon preparation being characterized in that: i) it is stable at pH 2, ii) its antiviral activity is destroyed by proteolytic enzymes, iii) it does not exhibit of mutual reactivity towards an antibody of interferon-a or interferon-y, and iv) its antiviral activity on cells of kidneys of MDBK oxen is between 1/4 and-la 1 / 2 of its activity on  EMI32.1  human Down's syndrome fibroblasts (GM-2767). ..  2. Preparation according to claim 1, characterized in that the culture is a culture of human epithelial cells.  3. Preparation according to claim 1, characterized in that the culture contains transformed eukaryotic cells.  4. Preparation according to claim 1, characterized in that the culture contains cells in which the part of the DNA code is inserted into the cells by recombinant DNA techniques.  5. Preparation according to claim 1, characterized by an original profile of mutual reactivity with respect to a mixture of anti-a and anti-ss antisera such that twice as much of the interferon are neutralized by the mixture as by the anti-ss antiserum alone, for equal units of neutralizing activity.  <Desc / Clms Page number 33>    6. Method for manufacturing an interferon preparation, characterized in that it comprises the following steps: A. production of a living cell culture containing a portion of the DNA code of human epithelial cells; B. incubation of the cells in a medium under conditions which favor the synthesis of interferon; and C. the harvest of a fraction rich in the interferon of cells, the interferon having the characteristic:    i) to be stable at pH 2, ii) to have an antiviral activity destroyed by proteolytic enzymes,  EMI33.1  iii) not to show mutual reactivity '. vis-G-vis an antibody to interferon-a or interferon-y, and iv) to have antiviral activity on MDBK ox kidney cells between 1J4 and 1/2 of its activity on human Down's syndrome fibroblasts (GM-2767).  7. Method according to claim 6, characterized in that the culture of living cells is a culture of human epithelial cells, and before step B, a virus is used so that it induces the production of interferon by the cells .  8. Method according to claim 6, characterized in that the culture of step A contains transformed eukaryotic cells.  9. Method according to claim 6, characterized in that the culture of step A contains cells in which the part of the DNA code is inserted into the cells by techniques using "recombinant DNA".  10. Method according to claim 7, characterized in that the virus is chosen from the group which comprises Newcastle disease virus and Sendai virus.  <Desc / Clms Page number 34>    011. Method according to claim 10, characterized in that the virus is the Bankowski strain of the Newcastle disease virus.  12. The method of claim 7, characterized in that the cell culture is formed from cells sampled from human epithelial tissue selected from the group which includes the epidermis, connective tissue, vaginal tissue and esophagus.    13. A process for the treatment of living cells, intended to inhibit infection by a virus, said method being characterized in that it comprises bringing the cells to be treated into contact with an effective amount of the interferon preparation according to the invention. any of claims 1 to 5 so that infection with the virus is inhibited.  14. Method according to claim 13, characterized in that the living cells are human epithelial cells.    15. A method of treating living neoplastic cells so that the proliferation of cells is inhibited, said method being characterized in that it comprises bringing the cells to be treated into contact with an effective amount of the interferon preparation according to one any of claims 1 to 5 so that said proliferation is inhibited.  16. The method of claim 15, characterized in that the living neoplastic cells are human epithelial cells.