EP0814830A1 - Lymphoblastoid natural interferon alpha production - Google Patents

Abstract

An extract of human fetal thymus which, when added to lymphoblastoid cell cultures, enables the cells to remain in a differentiated state and allows the cells to remain responsive to stimuli enabling the lymphoblastoid cells to produce interferon alpha.

Description

LYMPHOBLASTOID NATURAL INTERFERON ALPHA PRODUCTION

Bac ground of the Invention Interferon is found in many vertebrates and seems to be one of the body's main lines of non-specific defense against viral infection. Interferon is a term generically comprehending a group of vertebrate glycoproteins and proteins which are known to have various biological activities, such as antiviral, antiproliferative and immunomodulatory activity. Interferons are all polypeptides consisting of approximately 150 amino acid residues, and has proved tentative successes with its use against hepatitis, herpes simplex, the common cold and even warts. However, one problem with interferon is the burdensome cost which has resulted from the difficulty of getting significant quantities of human interferon by conventional methods.

Interferons have generally been named in terms of the species of animal cells producing the substance (e.g., human, murine, bovine, etc.), the type of cell involved (e.g., leukocyte, lymphoblastoid, fibroblast) and, occasionally, the type of inducing material responsible for the interferon production (e.g. , virus, immune). Interferon has been loosely classified by some researchers according to the induction mode as either Type I or Type II, with the former classification comprehending viral and nucleic acid induced interferon, and the latter class including the material produced as a lymphokine through induction by antigens and mitogens. More recently, an orderly nomenclature system for interferon has been devised which classifies interferons into types on the basis of antigenic specificities. Under this newer classification system, the designations alpha, beta and gamma have been used to correspond to previous designations of leukocyte, fibroblast and Type II (immune) interferons, respectively. Alpha and beta interferons are usually acid-stable and correspond to what have been called Type I interferons. Gamma interferons are usually acid-labile and correspond to what have been called Type II interferons.

The use of interferon for the treatment of disease in man and animals has been the subject of intense research efforts in many laboratories, both in industry and in educational institutions around the world. In some of the earliest research activities interferon was shown to have antiviral properties and the most successful clinical therapeutical applications to date have been in the treatment of virus-related disease states. More recently it has been found that exogenous interferon is effective for the regression or remission of some metastatic disease states. The literature is replete with reports of research and development efforts directed to defining activities and potential therapeutic uses of interferon. Most of the reports described activities of interferon in vitro or its effects in vivo following parenteral, particularly intramuscular and intradermal administration. There have been some reports of successful topical and intranasal usages.

However, interferon has seldom been administered intravenously because of substantial adverse effects attributable to "contaminants" in crude and even highly purified isolates. While the advent of recombinant DNA technology has allowed production of pure interferon species, intravenous administration of such pure compositions are not without adverse effects and drawbacks. There are many problems associated with current interferon supplies. All commercial brands and forms of natural interferon alpha available on the market are, at present, derived from live leukocytes (white blood cells) the supply of which is contingent on donors. That is, natural interferon alpha production of any given producer is limited by the number of donors at hand. Moreover, quite apart from the prohibitive cost of leukocyte derived natural interferon alpha, a growing proportion of the users and their families are becoming increasingly uneasy about both the hygienic and the genetic adequacy of the donors.

An object of the present invention is to provide a compound which can be used for maintaining a differentiated cell line, specifically, of lymphoblastoid cells capable of producing natural human interferon- alpha.

Summary of the Invention This invention relates to a novel compound and its method of use to maintain a differentiated, responsive lymphoblastoid cell line. Specifically, the novel compound is an extract of human fetal thymus.

In the human organism, the T- lymphocytes are the source of interferon alpha. Lymphoblastoid cells are the "cradle cells" for T- lymphocytes, which are one of the many existing forms of leukocytes. The lymphoblastoid cells are responsible for the interferon alpha endowment of the T- lymphocytes.

The production, in general, is based on a method originally devised by Cantell in Finland. Essentially his technique consists in making the leukocytes yield their interferon contents by stimulating them with the otherwise innocuous Sendai virus. Researchers have been addressing the perspective of growing lymphoblastoid cell cultures in an ideal auxostatic medium with a sterile laboratory environment and subject these to Cantell's method in order to incite them to issue interferon alpha rather than isolating interferon alpha from donated which blood cells.

The colossal advantages of such a production technique were obvious. First of all, production costs would shrink dramatically. Second, the production would become unrestricted, that is, limited only by space and equipment available. Third, the production would become perfectly predictable, meaning that under standardized, ideal laboratory conditions, the possibility of contamination could be safely ruled out and the mutation index would become statistically negligible. Fourth, such production would become completely independent of donors. Except for the initial starter culture, whose cells would originate from a carefully screened and selected batch of donated lymphoblastoid tissues, no donors would be required to produce natural interferon alpha. That is, the life expectancy of such a cell culture transplanted into an artificial, ideal medium is quite long and can be easily replaced at foreseen intervals.

However, a long-standing problem of the aforementioned technique is caused by the cells, upon having been transplanted into an ideal artificial environment, become deprived of their functional requirements. In other words, a cell, after being placed into a persistently perfect medium has, in a manner of speaking, no functional purpose. This lack of functionality will eventually influence its morphology. After the fourth or fifth mitotic generations, the cells in such culture will begin to lose their differentiation and will revert to one of the four primary cell forms. They will cease to behave like differentiated, specialized cells and for all sensible intents, will become free migrant cells, living without practical purposes. All research endeavors of producing natural interferon alpha from lymphoblastoid cell cultures have experienced this problem.

After a few generations, the lymphoblastoid cells began to lose their differentiation and, once their morphological change was complete, they did not react at all to any stimulus. All the attempts of hampering the loss of cellular differentiation failed. Many methods were tried, from enzyme or chemical influences to rhythmic changes of the medium, without success.

The method presented in this invention consists in the addition of a complex of natural substances to the auxostatic medium that contains the lymphoblastoid cell culture to maintain its differentiated state. Specifically, this substance is an extract made of human fetal thymuses. Human fetal cells are used rather than established human adult cells to allow for a much more vigorous mitosis rate and substantially more economic interferon production. Under the influence of this technique, the lymphoblatoid cells do not lose their differentiation, but remain receptive to the stimulation by the Cantell method. Thus, these cultures do yield unrestricted volumes of natural interferon alpha that is completely independent of donors. Such production is perfectly predictable, whose volume is only limited by the available space and technological equipment.

Detailed Description of the Preferred Embodiment The following definition of "interferon" has been accepted by an international committee assembled to devise a system for the orderly nomenclature of interferons: to qualify as an interferon, a factor must be a protein which exerts non¬ specific, antiviral activity at least in homologous cells through cellular metabolic process involving synthesis of both RNA and protein. "Interferon" as used herein in describing the present invention shall be deemed to have that definition and shall contemplate proteins, including glycoproteins, regardless of their cell source.

The interferon can be derived from human or animal cell culture, and in accordance with the preferred embodiments is alpha or beta interferon. Proteins having activities similar to natural occurring interferons but with modified amino acid sequences are also contemplated as useful in accordance with this invention. The clinical agents of choice for use in the present invention are human leukocyte interferon and lymphoblastoid interferon, mass produced by procedures involving lymphoblastoid cell cultures, induction of interferon production, via Cantell's method and isolation of interferon from culture media. The fetal thymuses extract is added to the lymphoblastoid cell cultures. The source material is supplied after careful clinical screening of the thymus donors. The thymus cells are extracted under surgical sterility regime, then flashfrozen, and ground up to a 00 level, frozen powder, in a special grinder capable of operating at extremely low temperatures. The powder is then extended on large flat containers in a one millimeter layer and thawed out at room temperature under a vacuum. After the thawing, the dehydrated cells are exposed to an acid gas flow which breaks down their membranes. Then, the open lymphoblastoid (thymus) cells are subjected to ultracentrification with the purpose of separating the DNA contained in their cell nucleuses. The thus obtained substance is then diluted in a physiological, sterile saline solution and added to the auxostatic medium that contains and nourishes the lymphoblastoid cell culture growing in that medium.

The extract made of human fetal thymuses is added to the adult lymphoblastoid cell culture at a rate of 2 milliliters per

100 liter culture medium evey 48 hours, continuously, during the cell culture's in vitro growth period. This is done in order to prevent the cell culture from loss of differentiation. The cells can successfully be used subsequently in the Cantell interferon isolation process. The cells are immortal and continue mitotic divisions as long as the aforementioned fetal thymus extract is added.

These cells can be subsequently used to produce interferon. Human alpha-interferon can be prepared through the following procedures, commonly referred to as the Cantell procedure. The process begins with packs of human leukocytes. The buffy coats in these packs are pooled into centrifuge bottles, and then are diluted with 0.83% ammonium chloride. The mixture is incubated for 15 minutes with intermittent shaking, and is then centrifuged for 20 minutes at 2000 rp . The supernatant is discarded, and the cell pellets are resuspended with a minimal volume of sterile pharmaceutical grade arabic gum (C₅H ₁₀O₅). The mixture is then diluted with ammonium chloride and centrifuged. The supernatant is again discarded, and the remaining cell pellets are resuspended with a minimal volume of a tissue culture medium such as Minimal Essential Medium (MEM), available from KC Biological. The cell concentration is determined with a Coulter counter. Interferon induction takes place in glass or plastic bottles. The induction medium contains MEM, 75mM Hepes (available from Calbiochem), 75mM Tricine (available from Sigma Chemical Co.), human gamma serum (18mg/ml), and gentamycin sulfate (from M. A. Bioproducts; 50mcg/ml). The cells are added to the induction vessels at a final concentration of about 5 to 10 million cells per milliliter. The induction vessel is incubated in a 37 degree(s) C. water bath, and alpha-interferon is added as a primer. After two hours, Sendai virus is added to the induction mixture. This causes alpha interferon to be produced in the supernatant by the leukocytes. After a 12- 18 hour incubation time, the induction mixture is centrifuged. The cells are discarded, and the supernatant is then purified. The crude interferon is chilled to 10 degree(s) C. or below in an ice bath. Five molar potassium thiocyanate is added to obtain a final concentration of 0.5M. This solution is stirred for 15 minutes, and then its pH is lowered to 3.3 by adding hydrochloric acid. The mixture is then centrifuged at 2800 rpm for 30 minutes, and the supernatant is discarded.

The pellets are then resuspended in 95% ethanol and are stirred for 15 minutes. This suspension is centrifuged at 2800 rpm for 20 minutes, and the pellets are discarded. The mixture is stirred for 10 minutes, and then centrifuged at 280 rpm for 20 minutes. The pellets are discarded. The pH of the supernatant is then adjusted to 8 with sodium hydroxide. This solution is stirred for 10 minutes, followed by centrifugation at 2800 rpm for 20 minutes. The supernatant is discarded, and the pellets are resuspended with 0.5M potassium thiocyanate in a 0.1M sodium phosphate buffer. This suspension is stirred at 4 degree(s) C. Next, the suspension is centrifuged at 2800 rpm for 20 minutes, and the pellets are discarded. The pH of the supernatant is adjusted to 5.3 with hydrochloric acid. After stirring for 10 minutes and centrifugation, the pH of the supernatant is adjusted to 2.8 with hydrochloric acid, followed by further stirring for 20 minutes. This mixture is centrifuged at 2800 rpm, and the resulting pellet is purified human alpha- interferon.

The pellet is resuspended with 0.5M potassium thiocyanate in 0. 1M sodium phosphate buffer, having a pH of about 8. It is then dialyzed against PBS at 4 degree(s) C, with two changes of PBS. This mixture is then centrifuged and the precipitate is discarded. The remaining purified alpha interferon is sterilized by filtration through a 0.2 micron filter. Other procedures are, of course, available for making interferons. For example, U.S. Patent No. 4,376,821 and 4,460,685 disclose methods of making human gamma- interferon, U.S. Pat. No. 4,276,282 discloses methods of making lymphoblastoid interferon. A method of making bovine fibroblast (beta) interferon is disclosed in applicant's U.S. Patent No.

4,462,985. Interferon Preparations are commercially available from Hoffmann-LaRoche, Burroughs-Wellcome and Schering- Plough. The fetal thymus extract could be used with these methods as well.

Claims

I claim:

1. A lymphoblastoid cell culture comprising: lymphoblastoid cells in a differentiated state and capable of producing interferon in response to interferon producing stimuli; and a compound that prolongs said differentiated state of said lymphoblastoid cells wherein said compound is an extract of mammalian fetal thymuses.

2. The cell culture of claim 1 wherein said extract of mammalian fetal thymus is an extract of human fetal thymus.

3. The cell culture of claim 1 wherein said stimuli is an innocuous virus.

4. The cell culture of claim 3 wherein said innocuous virus is a Sendai virus.

5. The cell culture of claim 1 wherein said interferon is interferon alpha.

6. The cell culture of claim 5 wherein said' interferon- producing stimuli is an interferon alpha-producing stimuli.

7. A method of maintaining a differentiated responsive lymphoblastoid cell line which produces interferon comprising: adding to lymphoblastoid cells an extract of mammalian fetal thymuses; and introducing to the lymphoblastoid cells an interferon producing stimuli.

8. The method of claim 7 wherein said extract of mammalian fetal thymuses is an extract of human fetal thymuses.

9. The method of claim 7 wherein said interferon- producing stimuli is an innocuous virus.

10. The method of claim 8 wherein said innocuous virus is a Sendai virus.

1 1. The method of claim 7 wherein said interferon- producing stimuli is interferon alpha-producing stimuli.

12. A method of maintaining a differentiated responsive lymphoblastoid cell line comprising: introducing to said lymphoblastoid cells an extract of human fetal thymuses.

13. A lymphoblastoid cell culture comprising: lymphoblastoid cells in a differentiated state which maintain their differentiated state upon introduction of an extract of mammalian fetal thymuses.

14. The cell culture of claim 13 wherein said extract of mammalian fetal thymuses is an extract of human fetal thymuses.