WO1993012146A1

WO1993012146A1 - Trophoblast interferons and their use

Info

Publication number: WO1993012146A1
Application number: PCT/DK1992/000370
Authority: WO
Inventors: Peter Ebbesen; George Aboagye-Mathiesen; Ferenc Toth
Original assignee: Landsforeningen Til Kraeftens Bekaempelse
Priority date: 1991-12-06
Filing date: 1992-12-07
Publication date: 1993-06-24
Also published as: JPH07504161A; CA2125181A1; US5540923A; AU3255393A; EP0619824A1

Abstract

The invention relates to methods for producing, isolating and purifying trophoblast interferon proteins such as β-interferon proteins, αI-interferon proteins, αII-interferon proteins and η-interferon proteins, and to the use of the interferon proteins, e.g. for inhibiting tumoral growth or metastatic processes, preventing graft-versus-host reaction, against leukemia, against viral activity, for enhancing in vitro fertility, for preventing pregnancy or inducing abortion, as well as antibodies against the interferon proteins.

Description

TROPHOBLAST INTERFERONS AND THEIR USE

The present invention relates to trophoblast interferon proteins, their preparation, and their use, as well as antibodies against the interferon proteins.

The invention is based on the discovery that human tropho- blasts produce proteins which conform with the internatio¬ nally accepted characterizations of interferons, but which show properties indicating that they are not identical with known interferons, and on the isolation and characteriza- tion of the trophoblast-produced interferons. Thus, the interferon proteins are human interferon proteins/glycopro- teins produced by human trophoblasts, or the same or sub¬ stantially the same proteins when produced by other means, such as by recombinant techniques. The invention relates to these proteins when existing outside their natural environ¬ ment, in other words in non-naturally occurring forms, e.g. as homogeneous proteins, or in purified or pure or sub¬ stantially pure form, such as disclosed herein. The inven¬ tion also provides methods for producing these novel pro- teins by cell cultivation and protein purification, in¬ cluding methods for production in a scale which makes it possible to use the interferon proteins for prophylactic and therapeutic treatment of humans and other warm-blooded animals, and in a scale which is realistic for the estab- lishment of the amino acid sequence of the interferon proteins as well as the sequence of DNA coding for the proteins.

As described in the following, substantially pure human trophoblast interferon proteins/glycoproteins have been isolated by two-dimensional high performance liquid chroma- tography and other methods suitable for production in quan¬ tities sufficient for prophylactic or therapeutic treatment of humans or other warm blooded animals. The properties of the interferon proteins combined with the fact that present development makes it possible to produce them in large quantities will make it possible to use them, e.g., in the following applications:

- Prevention of virus, in particular human immunodefi- ciency virus and herpes simplex virus, from infected mother to noninfected fetus during pregnancy- and during labour,

- treatment of virus and non-virus infections of the placenta, such as treatment of the fetal membranes, treatment via the amnion fluid, and treatment of the fetus in cases where acute systemic maternal infec¬ tions are diagnosed,

- treatment of benign and malignant tumors (choriocarci- nomas) arising from the placenta,

- identification of the trophoblast produced interferons in amnion fluid, and maternal blood, using, e.g., antibodies according to the invention, can be used as a signal of disease,

as well as a number of other applications as explained in the following.

The human trophoblast interferons are products of particu¬ larly the isolated first and term placenta non-MHC carrying trophoblasts such as villous trophoblasts. They are com- pounds which, being interferon, per definition have anti¬ viral activity, but which in addition have been found to show ability to inhibit tumour cell growth and modulate immune responses. BACKGROUND ART

Interferons are characterised as alpha class I, alpha class II, beta and gamma interferons as determined by inactiva- tion of the antiviral effects with internationally accepted standard antibodies. Trophoblast-produced interferons are also, according to international rules, designated tropho¬ blast interferons. A number of interferons derived from trophoblasts of non-human animals such as sheep and cattle, are known.

The known trophoblast interferons have been implied to be cytokines with an autocrine influence on differentiation of the normal trophoblasts, defence against virus infections, regulation of cell division and modulation of maternal immune attacks against the foetal tissues.

Several groups have demonstrated alpha interferon in amnion fluid, placenta tissue and cord blood, (Lebon et al. 1982, DucGoiran et al. 1985, Chard et al. 1986) even from appa¬ rently uninfected term used placenta (Bocci et al, 1985) . The cell(s) of origin and chemical composition except for neutralization by anti-interferon alpha antibodies was not delineated in these studies. Immunochemistry furthermore showed human trophoblasts to harbour alpha (Howatson et al., 1988). Earlier studies also showed animal trophoblasts capable of in vitro production of interferons (Godkin et al., 1982).

It had been reported that purified human villous tropho¬ blasts isolated by negative selection (non MHC-1, A, B, C expression) may produce interferon in vitro. Term placenta trophoblasts were exposed to polyriboinosinic-polyribocyti- dylic acid (poly 1:C) which led to production of beta type interferon activity (Toth et al., 1990). DISCLOSURE OF THE INVENTION

The present invention relates to isolated and characterized human trophoblast-produced interferons, some of which are novel, and important developments related thereto, such as novel uses. The relevant interferons are not only β-inter- feron, but also α_j-interferon, α _j-interferon, and ₇-inter- feron. The invention also relates to methods for producing, isolating and purifying the novel interferons. Furthermore, the realization and understanding of the character of the human trophoblast-produced interferons makes it possible to devise a number of important uses of the interferons, not only for purposes established for known human interferons, but also for a number of purposes related to the fact that the interferons are produced by and characteristic to trophoblast cells, their environment, and their function.

The human trophoblast interferons share antiviral, anti- cellular, and immunosuppressive properties with Type 1 leukocyte interferons, but are believed to lack the cyto- toxic effects of the latter.

Generally, the production of human trophoblast interferons according to the invention is based on establishment of primary cultures of isolated human trophoblast, stimulation with chemical or virus and subsequent recovery of inter¬ ferons from the culture medium. Particular embodiments include, e.g., the following:

1) The human placenta material is chemically minced and enzymatically digested. The resulting cell suspension is density gradient centrifuged and the harvested band is exposed to negative selection with magnetic beads carrying antibodies removing cells expressing the major histocompa- tibility complex MHC-1. Residual cells after test for purity is seeded. 2) Stimulation of seeded cells with the synthetic double stranded RNA Poly I:C or the RNA Sendai virus or Newcastle Disease virus.

* 3) Immobilization of Cibacron Blue F 3GA on HEMA-BIO 1000 5 VS (see Figure 7 and Example 2) via a spacer arm, 1,4-

» diaminobutane, and immobilization of anti interferon alpha and beta on HEMA 1000 VS, thereby creating new types of very efficient adsorbents for interferon purification under High performance conditions, that is, under HPLC condi- 10 tions.

4) Tandem high-performance affinity chromatography where the interferon absorbed to HEMA-BIO 100 VST 3GA was eluted into HEMA 1000 VS-antilFN-beta and HEMA 1000 VS-anti-lFN- alpha. Interferons bound to these two columns were eluted 15 separately.

The present invention is additionally directed to a method for protecting human placenta and human fetus against in¬ fection with the human immunodeficiency virus (HIV) during pregnancy and protection of the human fetus against infec- 20 tion with a virus such as HIV and Human Herpes vira such as Herpes Simplex virus or cytomegalovirus during labour.

The results presented in the examples herein here show that trophoblast cultures infected, e.g., with Sendai virus produce a mixture of -interferon, α _j-interferon, β- 25 interferon; the interferon production increased after dif¬ ferentiation of the cytotrophoblast to syncytiotrophoblast in vitro (Toth et al, 1990). Similarly, Burke et al, (1978)

* have reported that the production of interferon and inter¬ feron sensitivity change during differentiation of mouse *. 30 embryonal carcinoma in vitro.

The trophoblast interferons (αj, an, β ) are antigenically distinct which is an indication that they are structurally different. Antiserum to human α_j -interferon could not neutralize trophoblast α -interferon component (fractions 21 to 40 from Figure 1) or trophoblast β-interferon but completely neutralized trophoblast α_jj-interferon purified by HP-IAC. The ability of polyclonal anti-human ø-inter- feron (lymphoblastoid) to neutralize both the antiviral activities of trophoblast α -interferon and trophoblast -interferon can be explained by the fact that α j-inter- feron is a component of natural mixtures of lymphoblastoid and leukocyte IFN preparations (Adolf, 1987) used to pre- pare polyclonal antibodies. The neutralization results sup¬ port the suggestion (Adolf, 1987) that human type 1 inter¬ feron is made up of three antigenically distinct proteins, <*■£-interferon, a_jj-interferon and β-interferon.

The response of different cell lines to the antiviral effects of the trophoblast interferons when tested by inhi¬ bition of plaque formation varied. The results presented in Table 4 show that the trophoblast interferons differ from one another in their ability to confer protection against VSV infection of different human and bovine cell species. Trophoblast -interferon showed a degree of species speci¬ ficity, protecting human cells but not bovine (MDBK) cells. However, the trophoblast α_j-interferon and trophoblast α_jj- interferon protect both human and bovine cells. The varia¬ tions in the relative antiviral activities of trophoblast interferons in the different cells may indicate that they differentially affect biochemical pathways induced by interferons, such as phosphorylation of the eukaryotic ini¬ tiation factor-2 (eIF-2) by the double-stranded (ds) RNA- dependent protein kinase or activation of 2'- S'-oligo- adenylate-system (Marie et al., 1990; Baglioni, 1979). That interferons differentially induce biochemical pathways which correlate with particular specific antiviral activi¬ ties in various lines of cells is indicated by studies with different interferon preparations (Rosenblum et al. , 1990) . Differences between distinct interferons or between dis¬ tinct cell types may be of importance for the therapeutic application of interferons, particularly when such diffe¬ rences occur within the same organism.

The human placenta is important from pharmacological and virological perspective, since among its numerous functions it also acts as a selective barrier to virus traffic to the foetus. The fetal-placental unit depends on the unique functional and structural characteristics of the placental syncytiotrophoblast which is in direct contact with mater¬ nal blood and forms a continuous layer that mediates the interaction between maternal and fetal components (Douglas and King, 1990) .

In the first trimester, the cytotrophoblasts are highly proliferative and invasive and undergo a series of diffe¬ rentiation to form a multinuclear syncyntiotrophoblast that displays little potential to proliferate. The balance between proliferation and differentiation determines the structure and function of the trophoblast and its "pseudo- malignant*¹ properties. The processes underlying the morpho¬ logical transformation are, however, presently not well understood.

Cytokines, such as interferons, interleukins and tumour necrosis factor may play an important role in regulating the growth and differentiation of trophoblast cells as demonstrated (Berkowitz et al.. 1988; Athanassakis et al.. 1987) to stimulate or inhibit the proliferation of malig¬ nant human trophoblast.

The effect of trophoblast interferon on the proliferation of choriocarcino a cells in vitro was investigated as described in Example 12. As appears from Figures 17 and 18, a strong inhibitory effect of tro-IFN-_/S on proliferation on to differenct choriocarcinoma cell lines can be obtained with interferon in concentrations on 100 and 1000 IU/ml. Thus, the results strongy indicate that human _/9-trophoplast interferon (tro-IFN-^) would be a potent drug in the treat¬ ment of choriocarcinoma in humans.

One aspect of the biochemical differentiation of cytotro- phoblast is demonstrated in vitro in Example 5 herein by demonstrating differential interferon production in first and third trimester trophoblast cultures and syncytio trophoblast cultures stimulated with mitogens and viruses. It was found that the first trimester trophoblast stimula¬ ted with different inducers led to enhanced production of interferons compared to the third trimester trophoblast, whereas syncytio trophoblast at term produce more inter¬ feron than the mononuclear trophoblast when stimulated with the viruses.

The trophoblast layer of the human placenta constitutes the maternal-foetal interface and acts as a barrier to the transmission of infection from mother to foetus. The abili¬ ty of trophoblast cells to produce interferons may repre¬ sent a system for the protection of the foetus from viral infection. This is shown by the fact that, in some cases, maternal infection may spread to the placenta but fail to progress to the foetus (Yamauchi et al. , 1974; Klein et al.,1976; Remington & Desmonts, 1976). In addition to the antiviral activity of interferons, there is increasing evidence that interferons are also involved in the normal physiological and regulatory processes such as cell proli¬ feration and differentiation (Zullo et al. , 1985; Fisher & Grant, 1985) . Cells of the cytotrophoblast have been shown (Bulmer et al. , 1988) to be highly proliferative, as mea¬ sured by the proliferative marker Ki67 or the presence of mitoses, but lose their proliferative activity and begin to differentiate as they migrate into ducidua during implanta¬ tion. Although the factors that control such aspects of trophoblast behaviour are not known, interferons may play a major role in the development of these differentiated states. Furthermore, the a-interferon has been shown to be involved in prolongation of allograft survival (Hirsch et al., 191 ; Mobraaten et al. ,1973) and suppression of graft-versus-host disease (Corettini et al., 1973). Such modulation of the immune response by interferon may be important in the rejection of the allogenic foetus.

As indicated above, the production and characterization of the novel interferons, as well as the fact the effective methods for producing the interferons disclosed herein, make it possible to obtain high amounts of the novel inter¬ ferons in extremely high purity, which, inter alia, can be utilized for amino acid sequencing and DNA sequencing purposes, as well as for the establishment of antibodies. Thus, the invention relates not only to the novel inter¬ ferons when produced by human trophoblasts, but also to the identical or substantially identical interferon proteins when produced by other means using, e.g, recombinant tech¬ nique.

Thus, in one aspect, the interferon protein dealt with herein is an interferon protein which is identical or sub¬ stantially identical to a human interferon protein which is produced by a human trophoblast cell and which is cha¬ racterized as

a -interferon which is produced by a term trophoblast cell or a trophoblast cell derived from a provoked vaginal deli¬ very, which trophoblast cell is a trophoblast such as a villous trophoblast which is not bound by magnetic beads carrying immobilized antibodies to the tissue types MHC-1, A, B, or C,

the β-interferon being obtainable in purified form from a filtered (0.22 μm filter) supernatant of a stimulated cul- ture of the trophoblast cells, by a purification scheme comprising either 1) High-performance dye-ligand affinity chromatography followed by applying the thus obtained interferon-containing fractions to High-performance immuno- affinity chromatography using immobilized anti-^-interferon antibodies or 2) High-performance dye-ligand chromatography followed by Reversed phase HPLC,

the /3-interferon possessing at least one of the following characteristics i-iv:

i) the β-interferon in purified form appears substantially as only one silver-stainable band showing antiviral activity with a molecular weight in the range of 22-26 kDa, in a slab SDS-PAGE, applying an amount of β- interferon of 40.000 IU, under reducing conditions using 5% 2-mercaptoethanol or under non-reducing conditions without using 2-mercaptoethanol,

ii) the antiviral activity of the ^-interferon, as measured by inhibition of vesicular stomatitis virus plaque formation in a human amniotic cell line WISH cells (ATCC, CCL 25) , is retained in a pattern which re¬ sembles the specific pattern of retainment to an extent of about 55% of the initial value of antiviral activity after 3 hours at a temperature of 37°C, and to an extent of about

45-55%, in particular about 48-52%, after 10 minutes at a temperature of 56"C,

38-47%, in particular about 41-45%, after 15 minutes at a temperature of 56°C,

0-10%, in particular about 1-5%, after 60 minutes at a temperature of 56°C,

iii) the β-interferon substantially retains its anti¬ viral activity after storage in 0.1 M glycine at pH 2 for 24 or even 48 hours, as measured by inhibition of the plaque formation in human amniotic cell line WISH caused by vesicular stomatitis virus (VSV) , Indiana strain,

iv) the β-interferon shows a high degree of hydropho¬ bicity as indicated by it requiring a concentra¬ tion of 50% of the hydrophobic eluent ethylene glycol in 0.02 M sodium phosphate buffer pH 7.2 containing 1.0 M NaCI to be eluted from the High performance dye-ligand affinity chromatography column.

The above-mentioned isolation and purification methods are, of course, not the only available methods for isolating and purifying the β-interferon protein according to the inven- tion, but they have been found to be highly useful and efficient methods for the purpose and, thus, well suited for defining the purified forms of the -interferon. The exact manner in which the isolation and purification may be performed using these methods appears from the below dis- closure and the examples, where the unique novel HEMA adsorbents are used both for the High performance dye- ligand chromatography and for the High performance immuno- affinity chromatography.

The purity obtainable by either of the above-mentioned purification schemes is normally sufficient to ensure that the β-interferon will appear substantially as only one silver-stainable band showing antiviral activity with a molecular weight in the range of 23-25 kDa, in particular at 24 kDa, determined as described above.

For most purposes, it is preferred the β-interferon protein according to the invention is in substantially pure form, such as in a purity of at least 95%, preferably at least 99%, as measured by densitometric scanning of a Commassie Blue gel at 595 nm. As appears from Example 3, a purity of at least 95% of the -interferon according to the invention, as measured by densitometric scanning of a Coomassie Blue gel at 595 nm, has been obtained by subjecting the -interferon to high performance dye-ligand affinity chromatography, and a combination of high performance dye-ligand chromatography and subsequent high performance im unoaffinity chromato¬ graphy using immobilized anti-_/9-interferon antibodies has resulted in a purity of at least 99%, as measured by densi- tometric scanning of a Coomassie Blue gel at 595 nm.

As appears from Example 3, the specific activity of the β- interferon of the invention has been found to be at least about 1.0 x 10⁸ IU/mg of protein when the interferon is in a purified form.

Whether the β-interferon of the invention is in a purified form or not, it has a high temperature stability, such as appears from characteristic ii) above (Example 3) . This temperature stability pattern is different from the tempe¬ rature stability patterns of known human fibroblast inter- ferons induced by poly(rl) .poly(rC) or virus which have been reported to have half lives in the range of 2 to 7 minutes.

One interesting characteristic of the -interferon protein according to the invention is that it is capable of protec- ting human cell lines WISH (ATCC) , GM 2504 and GM 2767 trisomy 21 fibroblasts (Cell Repository, Cambden, N. . , U.S.A.) against an infection with vesicular stomatitis virus (VSV) , Indiana strain, but is not capable of protec¬ ting bovine cell line MBDK against a vesicular stomatitis virus (VSV) , Indiana strain, infection, such as appears from Table 3 (Example 3) .

As shown in Example 2, the ø-interferon produced by the human trophoblast cells binds to Concanavalin A and hence, is a glycoprotein. When produced by recombinant DNA tech¬ nique in bacteria in analogy with the known production of known interferons, the β-interferon protein according to the invention will be obtained in unglycosylated form. The invention comprises both the glycosylated and the unglyco¬ sylated form.

In another aspect, the invention relates to an isolated interferon protein which is identical or substantially identical to a human interferon protein which is produced by a human trophoblast cell and which is characterized as

an α -interferon which is produced by a term trophoblast cell or a trophoblast cell from a provoked vaginal deli¬ very, which trophoblast cell is a trophoblast such as a villous trophoblast which is not bound by magnetic beads carrying immobilized antibodies to the tissue types MHC-1, A, B, or C,

the α -interferon being obtainable in purified form from a filtered (0.22 μm filter) supernatant of a stimulated culture of the trophoblast cells by a purification scheme comprising either 1) High-performance dye-ligand affinity chromatography followed by applying the thus obtained interferon-containing fractions to High-performance immuno- affinity chromatography using immobilized anti-α -inter- feron antibodies or 2) High-performance dye-ligand chroma¬ tography followed by Reversed phase HPLC,

the α -interferon possessing at least one of the following characteristics i-iii:

i) the α -interferon in purified form appears substan- tially as only one silver-stainable band showing antiviral activity with a molecular weight in the range of 15-20 kDa in a slab SDS-PAGE, applying an amount of α_j-interferon of about 45.000 IU, under reducing conditions using 5% 2-mercaptoethanol or under non-reducing conditions without using 2-mercap- toethanol,

ii) the α_j-interferon substantially retains its antiviral activity after storage in 0.1 M glycine at pH 2 for 24 hours measured by inhibition of the plaque formation in human amniotic cell line WISH (ATCC) caused by vesicular stomatitis virus (VSV) , Indiana strain,

iii) the α_j-interferon is bound to the High performance dye-ligand chromatography column by electrostatic forces as evidenced by its ability to be displaced from the column by the ionic salt NaCI.

What has been stated above in connection with the β-inter¬ feron according to the invention ith respect to isolation and purification, purity, and purity percentage, also applies to the α^■-■•-interferon according to the invention, the SDS band characteristic to this interferon, however, corresponding to a molecular weight of 15-17 kDa, in parti¬ cular 16 kDa, determined as described above.

One interesting characteristic of the α_j-interferon protein according to the invention is that it is capable of protec¬ ting the human cell lines WISH (ATCC) , GM 2504 and GM 2767 trisomic 21 fibroblasts (Cell Repository, Cambden, N.J., USA) against a viral infection with vesicular stomatitis virus (VSV) , Indiana strain, and which is capable of pro¬ tecting the bovine cell line MDBK against a viral infection with vesicular stomatitis virus (VSV) , Indiana strain, to a higher extent than it protects the human cell line WISH, such as described in Example 3.

As shown in Example 3, the human trophoblasts produce the α_j-interferon in unglycosylated form.

In a further aspect, the invention relates to an isolated interferon protein which is identical or substantially identical to a human interferon protein which is produced by a human trophoblast cell and which is characterized as

an α_jj-interferon which is produced by a first trimester or term trophoblast cell or a trophoblast cell derived from a provoked vaginal delivery, which trophoblast cell is a villous trophoblast which is not bound by magnetic beads carrying immobilized antibodies to the tissue types MHC-1, A, B, or C,

the α_j -interferon being obtainable in purified form from a filtered (0.22 μm filter) supernatant of a stimulated culture of the trophoblast cells by a purification scheme comprising either 1) High-performance dye-ligand affinity chromatography followed by applying the thus ₀btained interferon-containing fractions to High-performance immuno- affinity chromatography using immobilized anti-α_j -inter- feron antibodies or 2) High-performance dye-ligand chroma¬ tography followed by Reversed phase HPLC,

the α_jj-interferon possessing at least one of the following characteristics i-iii:

i) the α_jj-interferon in purified form appears substantially as only one silver-stainable band showing antiviral activity with a molecular weight in the range of 20-24 kDa, in a slab SDS-PAGE, applying an amount of α _j-interferon of about 45.000 UI, under reducing conditions using 5% 2-mercaptoetha- nol or under non-reducing conditions without using 2-mercaptoethano1, ii) the α _j-interferon substantially retains its antiviral activity after storage in 0.1 M glycine at pH 2 for 24 hours measured by inhibition of the plaque formation in human amniotic cell line WISH (ATCC) caused by vesicular stomatitis virus (VSV) , Indiana strain. What has been stated above in connection with the β-inter¬ feron according to the invention with respect to isolation and purification, purity, and purity percentages, also applies to the α^_j-interferon according to the invention, the SDS band characteristic to this interferon, however, corresponding to a molecular weight of 21-23 kDa, in parti¬ cular 22 kDa, determined as described above.

One interesting characteristic of the ct _j-interferon pro¬ tein according to the invention is that it is capable of protecting human cell lines WISH (ATCC) , GM 2504 and GM 2767 trisomy 21 fibroblasts (Cell Repository, Cambden, N.J. , U.S.A.) against an infection with vesicular stomati¬ tis virus (VSV) , Indiana strain, and not capable of protec¬ ting bovine cell line MBDK against a vesicular stomatitis virus (VSV) , Indiana strain, infection, to a higher degree than it protects the human cell lines, such as appears from Table 3 (Example 3) .

In a still further aspect, the invention relates to an isolated interferon protein which is identical or substan- tially identical to a human interferon protein which is produced by a human trophoblast cell and which is charac¬ terized as

a ₇-interferon which can be obtained from a first trimester trophoblast cell from a provoked vaginal delivery, which trophoblast cell is a villous trophoblast which dis not bound by magnetic beads carrying immobilized antibodies to the tissue types MHC-1, A, B, or C,

or which can be obtained from a first trimester trophoblast cell from a provoked vaginal delivery, which trophoblast cell is isolated from at first trimester placenta using differential trypsinization periods, the -interferon being obtainable in purified form from a stimulated culture of the trophoblast cells by the follow¬ ing purification scheme comprising High-performance dye- ligand affinity chromatography followed by binding to immobilized Concanavalin A and subsequent displacement with a sugar and then controlled pore glass affinity chromato¬ graphy. This interferon is not stable at pH 2 under the conditions described above where the above-mentioned inter¬ ferons are stable.

A component of the β-interferon fractions from the High performance dye-ligande chromatography has cytotoxic effect as determinable by standard methods, such as test for influence on the metabolic reduction of 3-(4,5-dimethyl- thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT test), and each of the interferons has antiproliferal effect as determinable by standard methods, such as assay of [³H] thymidine incorporation in vitro.

Based upon observations to the effect that a human tropho¬ blast cell derived from abortion placentas of 5 to 12 weeks of age was capable of producing antiviral activity which could not be associated with any of the established human interferon classifications, it is justified to assume that the human trophoblast cells at this particular very early stage and earlier are capable of producing a special novel type of interferon, the function of which may conceivably be the same as the function of corresponding interferons ("trophoblast proteins", TP) produced in certain non-human animals, that is, as fertility enhancers which also have antiviral activity, in particular as a compound influencing the uterine epithelium, corpus lutea and the pituitary gland to the effect of maintaining the progesteron level and/or modulate the local maternal immunologic and nonimmu- nologic response to the fetal allograft. Furthermore, the human trophoblasts are likely to be capable of producing interferon related to the known interferon beta 2 (inter- leukin 6) . Thus, this aspect of the invention relates to an interferon which is produced by a human trophoblast cell derived from abortion placentas of 5 to 12 weeks of age, which tropho¬ blast cell is a villous trophoblast which is not bound by magnetic beads carrying immobilized antibodies to the tissue types MHC-1, A, B, or C,

the interferon being obtainable in purified form from the supernatant of a stimulated culture of the trophoblast cells by the following purification schemes comprising 1) High-performance dye-ligand affinity chromatography, the interferon not pertaining to any of the interferon classes β , α_j_, α-_jj and ₇.

As explained above, apart from the -interferon activity reported in Toth et al., 1990, human trophoblast inter- ferons, whether isolated/purified or not, are believed never previously, prior to the present invention, to have been produced by culturing human trophoblast cells in vitro. According to the present invention, it has been found that by suitable stimulation, other interferons identified and characterized above are produced by human trophoblasts.

Thus, one aspect of the invention relates to a method for producing an interferon selected from ₇-interferon, a_j- interferon, and α_jj-interferon from human trophoblasts, comprising cultivating the human trophoblast, and stimula¬ ting the culture with an agent capable of inducing the production of an interferon selected from ₇-interferon, α_j-interferon, and α_jj-interferon.

However, for all practical considerations, the process aspect of the present invention is one in which the inter¬ feron or interferons produced is/are obtained and isolated from the culture. Thus, this aspect can be expressed as a method for producing an interferon or interferons selected from -interferon, α_j-interferon and α_jj-interferon and ₇- interferon from human trophoblasts, comprising cultivating the human trophoblast, stimulating the culture with an agent capable of inducing the production of an interferon selected from -interferon, α_j-interferon, α_jj-interferon and ₇-interferon, and isolating the interferon or inter¬ ferons from the culture.

Stimulating agents which have been found, according to the invention, to stimulate the production of these interferons by trophoblasts, are virus, synthetic double stranded RNA, interleukins, plant mitogens, and growth factors. Thus, e.g., a very suitable stimulating agent for producing ₇- interferon is a plant mitogen and/or a cytokine. In parti¬ cular, an effective stimulating agent for producing ₇- interferon is a combination of phytohaemagglutinin and interleukin-2, such as a combination of 50-200 U/ml, e.g. 100 U/ml, of interleukin 2 together with 3-10 μg/ml, such as 5 μg/ml, of phytohaemagglutinin.

Other stimulating agents suitable for inducing the .in vitro production of the above-mentioned interferons by human trophoblasts are Concanavalin A, lipopolysaccharide, pock wheat mitogens and 4-^-phorbol-12-_/9-myristate-13-α-acetate.

As appears from Example 5, stimulation with different virus can result in differences in the composition of the inter- ferons produced. It is also important to note that Example 5 demonstrates that first trimester trophoblasts (5 to 12 weeks) yield 5-7 timers higher interferon yields than third trimester trophoblasts.

According to a particular aspect of the invention, the interferon production by the stimulated culture of human trophoblast cells is increased when the culture is kept under an oxygen-containing atmosphere. In this aspect, the invention relates to a method for producing a human tropho¬ blast interferon, comprising cultivating human trophoblast cells under an oxygen-containing atmosphere, stimulating the culture with an agent capable of inducing the produc¬ tion of interferon, and obtaining interferon from the culture.

The oxygen-enriched atmosphere is suitably nitrogen with about 2-10%, such as 3-7%, e.g. 5%, of oxygen.

According to the invention, human trophoblasts have been found to vary with their development stage with respect to their capability of producing a particular interferon. Thus, e.g., human ₇-interferon is produced by the tropho¬ blast cells at a rather early stage of development. These cells, when obtained by normal means, such as de¬ scribed herein, will tend to attach, together with other cells and other types of sort to surfaces in the vessels in which they are cultured. According to an aspect of the invention, however, it is possible to substantially selec¬ tively loosen or free these trophoblast cells from the surface, leaving the other cell still bound to the surface, thus obtaining, in the culture medium, a substantially pure population of these trophoblast cells.

Thus, in one aspect, the invention relates to a method of producing an interferon protein according to the invention, from trophoblast cells in a culture which, in addition to the trophoblast cells, contains other cells and which grows in a culture attached to a surface, the method comprising treating the culture with a calcium binding chelator, isolating the cells thereby detached from the surface, culturing the thus isolated cells, stimulating the resul¬ ting trophoblast cell culture with an agent capable of stimulating an interferon production, and obtaining the interferon protein from the culture.

This method is especially effective and valuable when the the culture is a culture obtained from a provoked first trimester vaginal delivery, the vaginal tissue typically being placental tissue which is has been digested enzymati- cally, such as with trypsin, followed by pooling and gra¬ dient centrifugation. The temperature of the culture is preferably about 0°C during the treatment with the calcium binding chelator. The calcium binding chelator is preferab¬ ly EDTA, such as EDTA in a concentration of the about 0.02%. When the trophoblast cells have been liberated from the surface in this manner, they are suitably reseeded on a fresh culture medium.

In conformity with what is stated above in connection with the characterization and purity of the individual inter¬ ferons, the generally preferred method of the invention for isolating and purifying an interferon produced by a human trophoblast cell culture comprises subjecting supernatant from the culture to affinity chromatography and obtaining, from the eluate from the chromatography, the fractions containing interferon activity. The affinity chromatography is preferably a high performance affinity chromatography selected from high performance dye-ligand affinity chroma- tography, high performance Concanavalin A affinity chroma¬ tography, high performance immunoaffinity chromatography, and Reversed phase HPLC. Evidently, in selecting the puri¬ fication scheme, the stabilities of the interferons should be taken into consideration. Thus, e.g. , -interferon, which is not stable at pH 2, should not be purified using immunoaffinity which involves exposure to such low pH.

A particular aspect of the invention is the use of adsor¬ bents based on the 2-hydroxyethyl ethacrylate (HEMA) polymer to which a ligand has been bound, such as a dye, e.g. Cibachrpn Blue F 3GA, or antibodies, e.g. via a spacer arm such as explained in Example 2 and illustrated in Fig. 7. These adsorbents permit very high throughput and result in extremely high purifications of the interferons, such as is illustrated herein. The invention also relates to a monoclonal or polyclonal antibody which binds to an interferon as de ined above or a part thereof. A most useful type of antibody is a monoclo¬ nal antibody; however, also a polyclonal antibody may of great importance provided it shows a sufficient selectivi¬ ty, which may be obtained, e.g., by means of known absorp¬ tion methods.

The term "antibody" refers to a substance which is produced by a mammal or more precisely a cell of mammalian origin belonging to the immune system as a response to exposure to the polypeptides or carbohydrates of the invention.

The variant domain of an antibody is composed of variable and constant sequences. The variant part of the domain defines the idiotype of the antibody. This part of the antibody is responsible for the interaction with the anti¬ gen, such as the interferon, and thereby the antigen bin¬ ding.

The idiotypic structure is antigenic and can thus give rise to specific antibodies directed against the idiotypic structure. Production of such anti-idiotypic antibody can be done in mice. The antibodies raised against the idio¬ type, the anti-idiotypic antibodies, may mimic the struc¬ ture of the original antigen and therefore may function as the original antigen to raise antibodies reactive with the original antigen. This approach may be advantageous as it circumvents the problem associated with the characteriza¬ tion and synthesis of the important immunogenic parts of the antigen in question. This is most important in the case of conformational epitopes, which might otherwise be diffi- cult to identify. The present invention therefore also relates to an anti-idiotypic antibody which is directed against the site of an antibody which binds the antigen or the epitope according to the invention. The antibodies of the present invention may be produced by a method which comprises administering in an immunogenic form at least a part of the interferon of the invention or an anti-idiotypic antibody as defined above to obtain cells producing antibodies reactive with the interferon or a part thereof, and isolating the antibody containing material from the organism or the cells. The methods of producing antibodies of the invention will be explained further below.

The antibody of the invention may be used in an assay for the identification and/or purification and/or quantifica¬ tion of at least a form and/or a part of the interferon of the invention present in a sample. The identification and/or purification and/or quantification performed by the use according to the present invention may be any identifi¬ cation and/or purification and/or quantification involving the interferon of the invention. The identification and/or purification and/or quantification may be performed for both a scientific, a clinical and an industrial purpose. As will be further described below, it is especially important in clinical routine to identify or quantify antigens or epitopes of the invention.

The sample may be a specimen obtained from a living orga¬ nism such as a human or an animal. The specimen may be a sample of body fluid, such as a blood sample, or a tissue sample.

In one preferred embodiment of the invention it is prefer¬ red that the antibody used in the method of the invention is a monoclonal antibody as this generally provides a high¬ er precision and accuracy of the assay, at the same time possibly requiring less time to perform. Furthermore, a mixture of two or more different monoclonal antibodies may be employed as this may increase the detection limit and sensitivity of the test. The monoclonal antibody may be obtained by the method described below. Antibodies posses¬ sing high avidity may be selected for catching techniques.

The antibody used in the present method is preferably in substantially pure form (purified according to suitable techniques or by the methods of the invention, see below) in order to improve the precision and/or accuracy of the assays of the invention.

The determination of antibodies reactive with the inter¬ feron of the invention and being present in a sample, e.g. as defined above, may be carried out by use of a method comprising contacting the sample with the antibody of the invention and detecting the presence of bound antibody resulting from said contacting and correlating the result with a reference value.

When the antibody of the invention is to be employed in an assay for determining the presence of the interferon of the invention in a sample, it may be in the form of a diagnos¬ tic reagent or a diagnostic agent. As will be apparent to a person skilled in the art several techniques may be applied in connection with such diagnostic reagents.

Another field of the invention is a method for producing an antibody which binds to an interferon of the invention, which comprises immunizing an animal with an interferon or part thereof or an anti-idiotypic antibody or an interferon produced by cultivating cells harboring a plasmid which contains and is capable of expressing an interferon or part thereof as described above, whereby cells producing an antibody specific for the interferon or part thereof is obtained and the antibody is isolated from the animal or the cells.

The antibody is preferably a monospecific antibody. The monospecific antibody may be prepared by injecting a sui¬ table animal with a substantially pure preparation of the interferon of the invention or a part thereof followed by one or more booster injections at suitable intervals (e.g. one or two weeks to a month) up to four or five months be¬ fore the first bleeding. The established immunization sche- dule is continued, and the animals are bled about one week after each booster immunization, and antibody is isolated from the serum in a suitable manner (cf. e.g. Harboe and Ingild, Scand. J. Immun. 2 (Suppl. 1), 1973, pp. 161-164.)

For purposes not requiring a high assay specificity, the antibody may be a polyclonal antibody. Polyclonal anti¬ bodies may be obtained, e.g. as described in Harboe and Ingild, see above. More specifically, when polyclonal antibodies are to be obtained, the compound comprising an interferon of the invention or part thereof or an anti- idiotype antibody as described above is prepared and prefe¬ rably after addition of a suitable adjuvant, such as Freund's incomplete or complete adjuvant, injected into an animal. The animals are bled regularly, for instance at weekly intervals, and the blood obtained is separated into an antibody containing serum fraction, and optionally said fraction is subjected to further conventional procedures for antibody purification, and/or procedures involving use of purified compounds comprising an interferon of the invention or a part thereof or an idio-typic antibody as described above.

In another preferred embodiment, monoclonal antibodies are obtained. The monoclonal antibody may be raised against or directed substantially against an essential component of the compounds comprising an interferon of the invention or a part thereof or an anti-idiotypic antibody as described above. The monoclonal antibody may be produced by conven¬ tional techniques (e.g. as described by Kδhler and Mil¬ stein, Nature 256, 1975, p. 495) e.g. by use of a hybridoma cell line, or by clones or subclones thereof or by cells carrying genetic information from the hybridoma cell line coding for said monoclonal antibody. The monoclonal anti- body may be produced by fusing cells producing the monoclo¬ nal antibody with cells of a suitable cell line, and selec¬ ting and cloning the resulting hybridoma cells producing said monoclonal antibody. Alternatively, the monoclonal antibody may be produced by immortalizing an unfused cell line producing said monoclonal antibody, subsequently growing the cells in a suitable medium to produce said antibody, and harvesting the monoclonal antibody from the growth medium.

The immunized animal used for the preparation of antibodies of the invention is preferably selected from the group consisting of rabbit, monkey, sheep, goat, mouse, rat, pig, horse and guinea pigs. The cells producing the antibodies of the invention may be spleen cells or lymph cells, e.g. peripheric lymphocytes.

When hybridoma cells are used in the production of anti¬ bodies of the invention, these may be grown in vitro or in a body cavity of an animal. The antibody-producing cell is injected into an animal such as a mouse resulting in the formation of an ascites tumour which releases high concen¬ trations of the antibody in the ascites of the animal. Although the animals will also produce normal antibodies, these will only amount to a minor percentage of the mono¬ clonal antibodies which may be purified from ascites by standard purification procedures such as centrifugation, filtration, precipitation, chromatography or a combination thereof.

An example of a suitable manner in which the monoclonal antibody may be produced is as a result of fusing spleen cells from immunized mice (such as Balb/c mice) with myelo¬ ma cells using conventional techniques (e.g. as described by R. Dalchau, J. Kirkley, J.W. Fabre, "Monoclonal antibody to a human leukocyte-specific membrane glycoprotein pro¬ bably homologous to the leukocyte-common (L-C) antigen of the rat"_r Eur. J. Immunol. 10. 1980, pp. 737-744). The fusions obtained are screened by conventional techniques such as binding assays employing compounds comprising antigen or epitope of the invention or an anti-idiotypic antibody as described above isolated by the above-described methods.

As mentioned above, the interferon proteins according to the invention have possess a number of properties which make them valuable as therapeutic or prophylactic agents, including antiviral activity, antitumour activity and immune system modulating activity . These activities make them useful generally as antiviral and antitumour agents and agents modulating the activity of the immune system. However, the fact that the new interferons are produced by the human trophoblasts, that is, cells present in a very specialized environment, with their very specialized func¬ tions, make these interferon proteins especially valuable in connection with treatment of adverse conditions in connection with pregnancy or labour, such as indicated above.

In accordance with this, one aspect of the invention rela¬ tes to a method for inhibiting tumoral growth or metastatic processes in a warm-blooded animal such as a human, com¬ prising administering an effective amount of an interferon according to the invention or a mixture of such interferons to an animal. In particular, the interferon is an α-inter- feron, or a mixture of an a- and a ₇-interferon, according to the invention, but in accordance with what is stated above, also mixtures of α-interferons according to the invention and mixtures of one or more α-interferons accor- ding to the invention together with other interferons according to the invention are of interest in this connec¬ tion.

Another aspect of the invention is a method for preventing graft-versus-host reaction in a warm-blooded animal such as a human, comprising administering an effective amount of an interferon according to the invention or a mixture of such interferons to the animal. The preferred interferon for this purpose is α-interferon and -interferon according to the invention.

Another aspect of the invention is a method for prolonga- ting an allograft survival in a warm-blooded animal such as a human, comprising administering an effective amount of an interferon according to the invention to the animal. In this case, α-interferon and -interferon protein according to the invention will often be preferred.

A further aspect of the invention is a method for treating of leukemia, such as hairy cell leukemia or chronic myeloid leukemia, in a warm-blooded animal such as a human, com¬ prising administering an effective amount of an interferon according to the invention, in particular an α-interferon, or a mixture and α- and a ₇-interferon of the invention to the animal. The amount of the interferon administered is typically about 1000 million units over a year.

A further aspect of the invention relates to a method for treating myelomatosis in a warm-blooded animal such as a human, comprising administering an effective amount of an interferon according to the invention, in particular a α- interferon according to the invention, or a β-protein according to the invention, or a mixture of a- and ₇-inter- ferons according to the invention, to the animal. Also in this case, the amount of interferon administered is sui¬ tably about 1000 million units over a year.

A still further aspect of the invention is a method for inhibiting, controlling or preventing viral activity in a warm-blooded animal such as a human, comprising admini¬ stering to the animal an effective amount of an interferon protein as defined in claim 1 or a mixture of such inter¬ feron proteins. The viral activity may, e.g., be retroviral activity, such as HIV activity, or it may be hepatitis activity or herpes simplex virus activity. For this pur¬ pose, α- or β-interferon according to the invention, or mixtures of interferons according to the invention com¬ prising ₇-interferons according to the invention, will normally be preferred.

In accordance with what is stated above, this method is particularly valuable when used for preventing virus from a human mother infected with the virus from infecting her noninfected fetus during pregnancy or during labour. The administration may suitably be performed by injection into the amniotic fluid.

For these antiviral purposes, the interferon protein is suitably an α- or a -interferon according to the invention as characterized under d) in claim 1, administered, e.g., in an amount of about 5 x 10⁶ units 3 times a week by intravenous injection or by injection into the amniotic fluid.

Another important aspect of the invention is a method for treating infections selected from virus infections and nonvirus infections in a warm-blooded animal such as a human in cases where acute systemic maternal infections are diagnosed, comprising administering to the animal an effec¬ tive amount of an interferon protein as defined in above or a mixture of such interferon proteins. Also in this case, the administration is suitably performed by intravenous injection or injection into the amniotic fluid. The inter¬ feron protein is preferably an α- or a -interferon as described above in a dose of 100,000 to 1,000,000 units per injection.

A further aspect of the invention is a method for treating malignant tumours (choriocarcinomas) arising from the pla¬ centa in a warm-blooded animal such as a human, comprising administering to the animal an effective amount of an interferon protein according to the invention. Again, the administration is suitably performed by injection into the amniotic fluid. The interferon protein is suitably a mix¬ ture of α-interferons according to the invention or a mixture of α- and ₇-interferons or a mixture of β- and 7- interferons according to the invention administered in an amount of about 5 x 10⁶ units 3 times a week.

For the above purposes, the interferons are suitably used in the form of pharmaceutical compositions comprising an interferon or interferons in association with a stabilizer in a manner known per se. The stabilizer may, e.g., be human albumin.

Additional important aspects of the invention are set out in the following points, termed 82-105, and corresponding to claims 82-105:

82. A method for inhibiting posttransplantational graft rejection, comprising administering, to a mammal in need thereof, an a- or -interferon according to 1 in an amount sufficient to effective to inhibit proliferation of immune¬ competent lymphocytes such as T.cells or B-cells, such as an amount corresponding to a plasma concentration of about 100-1000 International Units per milliliter.

83. A method according to point 82, wherein the admini¬ stration is performed posttransplantationally.

84. A method according to point 83, wherein the admini- stration is continued life-lasting.

85. A method for inhibiting a posttransplantational graft- versus host reaction, comprising administering, to a mammal in need thereof, an α- or -interferon according to point 1 in an amount sufficient to effective to inhibit prolifera- tion of immunecompetent lymphocytes such as T.cells or B- cells, such as an amount corresponding to a plasma concen- tration of about 100-1000 International Units per milli- liter.

86. A method according to point 85, wherein the admini¬ stration of the interferon started prior to or simultane- ously with the transplantation.

87. A method according to point 86, wherein the interferon is incorporated in the transplantate.

88. A method according to any of claims 85-87 wherein the administration is continued life-lasting.

89. A method for heat-stabilizing an interferon according to claim 1, comprising adding, to the interferon, a stabi¬ lizer selected from the group consisting of ethylene gly¬ col, in particular 30-40% ethylene glycol, reducing agents, such as ME, and detergents, such as SDS.

90. A method according to point 89, wherein the stabilizer comprises a combination of a reducing agent and a deter¬ gent.

91. A method according to point 89 or 90, wherein the reducing agent is ME, and the stabilizer is SDS.

92. A method for inactivating any pathogenic components, such as virus or prions, in a preparation containing an interferon according to claim 1, in particular an α- or β- interferon according to claim 1, such as an aqueous solu¬ tion thereof, comprising adding a heat-stabilizer to the preparation and subjecting the preparation to heat treat¬ ment at a temperature and for a period sufficient to inac¬ tive the pathogenic components, the kind and amount of the heat stabilizer being sufficient to stabilize the inter¬ feron during the heat treatment. 93. A method according to point 92, wherein the heat treat¬ ment comprises heating to a temperature of at least 56°C.

94. A method according to point 92 or 93, wherein the heat stabilizer comprises a reducing agent or a detergent, or a combination thereof.

95. A heat-stabilized interferon composition, comprising an interferon according to claim 1, in particular an α- or β-interferon, together with a stabilizer selected from the group consisting of ethylene glycol, in particular 30-40% ethylene glycol, reducing agents, such as ME, and deter¬ gents, such as SDS.

96. A composition according to point 95, wherein the stabi¬ lizer comprises a combination of a reducing agent and a detergent.

97. A composition according to point 95 or 96, wherein the reducing agent is ME, and the stabilizer is SDS.

98. A method for enhancing fertility, comprising admini¬ stering an α- or -trophoblast interferon according to claim 1 to a female mammal such as a female human in an amount effect to have luteotropic effect.

99. A method according to point 98, wherein the α- or β- trophoblast interferon is administered parenterally in an amount corresponding to a plasma concentration of about 10- 50 International Units per illiliter.

100. A method for enhancing in vitro fertility, in particu¬ lar human in vitro fertility, comprising administering to a culture fluid containing the egg and sperm, an α- or β- trophoblast interferon according to claim 1 in an amount effective for enhancing cell viability. 101. A method according to point 100, wherein the a- or β - trophoblast interferon is administered to the culture fluid in an amount corresponding to a concentration of about 5- 20, in particular about 10, International Units per milli- liter.

102. A method for contraception (preventing pregnancy) , comprising administering an α- or -trophoblast interferon according to claim 1 to a female mammal such as a female human in an amount effective to inhibit trophoblast in vitro production of human chorion gonadotropin.

103. A method according to point 102, wherein the α- or β- interferon is administered parenterally in an amount corre¬ sponding to a plasma concentration of about 100-1000 Inter¬ national Units per milliliter.

104. A method for inducing abortion (rejection of the nidated fertilized egg) , comprising administering to a female mammal, in particular a female human, a ₇-tropho¬ blast interferon according to claim 1 in an amount effec¬ tive to enhance cellular expression of the major histocom- patibility complexes and enhance cellular immune responses.

105. A method according to point 104, wherein the ₇-inter¬ feron is administered parenterally in an amount correspond¬ ing to a plasma concentration of about 100 to about 1000 International Units per milliliter.

106. A method for treating preeclampsia comprising admini¬ stering alpha or beta-interferon protein according to claim 1 to a female mammal such as a female human in an amount effective to have effects on the arachidonic acid derivati¬ ves (prostacyclines and thromboxanes) .

107. A method according to point 106, wherein the alpha or beta-interferon protein according to claim 1 is admini¬ stered parenterally in an amount corresponding to a plasma concentration of about 10 International Units per milli¬ liter.

108. A method for treating moles and choriocarcinomas com¬ prising administering gamma-interferon protein according to claim 1 or a mixture of gamma, alpha and beta-interferon protein according to claim 1 to a female mammal such as a female human in an amount effective to enhance the expres¬ sion of the major histocompatibility complex and stimulate cellular immune reactivity.

109. A method according to point 108, wherein the gamma- interferon protein according to claim 1 is administered parenterally in an amount corresponding to a plasma concen¬ tration of about 100 to 1000 International Units per milli¬ liter.

110. A method for enhancing the viability of lymphoid cells to be used in transplantation of haematopoietic stem cells among mammals such as humans comprising administering alpha or beta-interferon protein according to claim 1 to the cells in suspension in an amount effective to promote cell pro1iferation.

111. A method according to point 110, wherein the alpha or beta-interferon protein according to claim 1 is admini¬ stered so as to give a concentration in the suspending medium of about 10 International Units per milliliter.

112. A method for treatment of virus, bacteria, fungi and protozoans infecting mammals such as humans in the placen¬ ta, the fetus and the chorion-amnion membranes comprising administering alpha or beta-interferon protein according to claim 1 intravenously or into the amnion cavity in an amount effective to inhibit growth of the microbes.

113. A method according to point 112, wherein the alpha or beta-interferon protein according to claim 1 is ad ini- stered so as to give a concentration in the serum or amnion fluid of about 100 to 1000 International Units per milli¬ liter.

In addition to being used alone or in admixture with each other, the interferons according to the invention may also be used in admixture with other active compounds, in parti¬ cular other interferons, depending on the condition to be treated.

FIGURES

Figure 1. HP-DIAC of crude trophoblast interferon on HEMA- BIO 1000 VS 3GA. Interferon (2.05X10⁶ IU) was applied to a column and interferon activity (D) was eluted with a 60 min linear gradient of 0.2 M- to 1.0 M-NaCl ( ) for 60 min.

The column was further eluted with increasing concentra¬ tions of ethylene glycol ( ) from 0 to 50% for 10 min and

50% for 35 min. The A2_8O i-³ shown by the continuous line.

Figure 2. Silver-stained SDS-PAGE containing purified trophoblast interferons. (a) Lane 1, standard protein markers; lane 2, trophoblast α_j-interferon and trophoblast α_jjl-interfe on purified from a crude trophoblast inter¬ feron preparation on a HEMA-BIO 1000 VS-anti-α-interferon antibody column; lanes 3 and 4, HP-IAC-purified trophoblast α_j-interferon under reducing and non-reducing conditions respectively, (b) Lane 1, standard protein markers; lanes 2 and 3, HP-DLAC-purified trophoblast β-interferon (from fraction 56 to 58 in Figure 1) under reducing and non- reducing conditions respectively.

Figure 3. Con A affinity chromatography of the trophoblast interferons. Trophoblast ø-interferon and trophoblast α_j- and α_jj-interferons were purified using tandem HPAC (high performance affinity chromatography) and the resulting fractions were injected separately onto HEMA 1000 VS-Con A column (50 mm x 4.6 mm I.D) . After washing the column with column buffer (20 mM sodium phosphate buffer, pH 7.2, containing 1 M NaCI and MnCl₂, CaCl₂ and MgCl₂) the bound interferon having interferon activity were eluted with 0.1 M α-methyl-D-mannopyranoside and 50% ethylene glycol (1 ) in the column buffer (a) and (b) . Trophoblast α_j-interferon did not bind to the column but was eluted in the flow- through fractions (c) . Figure 4. Densitometric analysis of Coomassie-stained SDS gel of the purified trohoplast -interferon preparation. The scanning analysis showed the purity to be greater than 99%.

Figure 5. RP-HPLC of HP-DLAC-purified trophoblast β-inter¬ feron on Separon SGX C-18. 25 ml of interferon sample, containing 1.47 x 10⁶ IU, was applied to a column (3 mm x 150 mm) equilibrated with 20% propan-1-ol in mobile phase A (pyridine/acetic acid pH 5.0). Bound proteins were eluted with a linear gradient ( ) of 20-70% propan-1-ol in

Buffer A. Interferon activity (D) was eluted between 40 and 43% propan-1-ol.

Figure 6. SDS-PAGE of the RP-HPLC-purified trophoblast β- interferon. Lane l is standard molecular mass markers. Lanes 2-4 are pooled fractions 22-24 (see Figure 5) with the highest interferon antiviral activity.

Figure 7. The structure of HEMA-BIO 1000 VS 3GA. Cibachron Blue 3GA (A) was attached via spacer arm, 1,2-diaminobutane (B) to HEMA-BIO (D) activated with divinyl sulphone (C) .

Figure 8. The cytotoxic effect of natural killer (NK) cells, obtained from adult donors, against cells of the NK susceptible control cell line K562. IFN-treated effector cells (open symbols) were incubated with tro-IFN 500 U/ml of complete medium at the concentration of lxlO⁶ cells/ml for 24 h. An untreated NK population was used as control (filled symbols) . The specific lyses of target cells was calculated at different effector target ratios.

Figure 9. The cytotoxic effect of natural killer (NK) cells, obtained from adult donors, against HSV-1 infected fibroblasts. IFN-treated effector cells (open symbols) were incubated with tro-IFN 500 U/ml of complete medium at the concentration of lxlO⁶ cells/ml for 24 h. An untreated NK population was used as control (filled symbols) . The speci- fie lyses of target cells was calculated at different effector target ratios.

Figure 10. The cytotoxic effect of natural killer (NK) cells, obtained from adult donors, against trophoblast cells. IFN-treated effector cells (open symbols) were incubated with tro-IFN 500 U/ml of complete medium at the concentration of lxlO⁶ cells/ml for 24 h. An untreated NK population was used as control (filled symbols) . The speci¬ fic lyses of target cells was calculated at different effector target ratios.

Figure 11. The cytotoxic effect of fetal natural killer (NK) cells, obtained from umbilical cords, against cells of the NK susceptible control cell line K562. IFN-treated effector cells (open symbols) were incubated with tro-IFN 500 U/ml of complete medium at the concentration of 1x10 - cells/ml for 24 h. An untreated NK population was used as control (filled symbols) . The specific lyses of target cells was calculated at different effector target ratios.

Figure 12. The cytotoxic effect of fetal natural killer (NK) cells, obtained from umbilical cords, against auto- logous trophoblast cells. IFN-treated effector cells (open symbols) were incubated with tro-IFN 500 U/ml of complete medium at the concentration of lxlO⁶ cells/ml for 24 h. An untreated NK population was used as control (filled sym- bols) . The specific lyses of target cells was calculated at different effector target ratios.

Figure 13. The cytotoxic effect of fetal natural killer (NK) cells, obtained from umbilical cords, against HSV-1 infected autologous trophoblast cells. IFN-treated effector cells (open symbols) were incubated with tro-IFN 500 U/ml of complete medium at the concentration of lxlO⁶ cells/ml for 24 h. An untreated NK population was used as control (filled symbols) . The specific lyses of target cells was calculated at different effector target ratios. Figure 14. The cytotoxic effect of fetal natural killer (NK) cells, obtained from umbilical cords, against tro-IFN treated HSV-1 infected autologous trophoblast cells. IFN- treated effector cells were incubated with tro-IFN 500 U/ml of complete medium at the concentration of lxlO⁶ cells/ml for. The specific lyses of target cells was calculated at different effector target ratios.

Figure 15. The expression of MHC-1 antigens in human tro¬ phoblast cultures. Trophoblast-IFN-beta treated and untrea- ted cells were removed from culture bottles every 12 h after the addition of IFN. Suspended cells were incubated with relevant monoclonal antibody and then incubated with secondary antibody. Controls were incubated with unspecific mouse IgG as primary antibody. Cells were analyzed using a FACStar Plus Flow Cytometer.

Figure 16. The expression of MHC-1 antigens in human tro¬ phoblast cultures. Trophoblast-IFN-alpha treated and untre¬ ated cells were removed from culture bottles every 12 h after the addition of IFN. Suspended cells were incubated with relevant monoclonal antibody and then incubated with secondary antibody. Controls were incubated with unspecific mouse IgG as primary antibody. Cells were analyzed using a FACStar Plus Flow Cytometer.

Figure 17. The inhibitive effect of tro-IFN-beta on the proliferation of JAR cells. JAR cells were cultured in RPMI 1640 + 10% fetal calf serum at a concentration of 5 x 10⁴ cells per well in the presence or absence of 100 and 1000 IU/ml of tro-IFN-3. At 24 h intervals cells were washed with phosphate buffer saline and culture medium were recon- stituted. ³H-Thymidine (6.7 Ci/mmol) at a concentration of 1 mCi per well was added to the cultures for 4 h. Proli¬ ferative response was evaluated from beta counts of cells harvested at the end of the inoculation period. The percent inhibition of proliferation was calculated. Figure 18. The inhibitive effect of tro-IFN-beta on the proliferation of BeWo cells. BeWo cells were cultured in RPMI 1640 + 10% fetal calf serum at a concentration of 5 x 10⁴ cells per well in the presence or absence of 100 and 1000 lU/ml of tro-IFN-^. At 24 h intervals cells were washed with phosphate buffer saline and culture medium were reconstituted. ³H-Thymidine (6.7 Ci/mmol) at a concentra¬ tion of 1 mCi per well was added to the cultures for 4 h. Proliferative response was evaluated from beta counts of cells harvested at the end of the inoculation period. The percent inhibition of proliferation was calculated accor¬ ding to the following formula:

Figure 19. The proliferation of PHA stimulated T-lymphocy¬ tes. ^τ ₀ determine the effect of tro-IFN- on the prolifera- tion of T-lymphocytes, cultures were supplemented with PHA (1 μl/ml) and different concentrations of tro-IFN- . The lymphocytes were stimulated with 10 IU/ml, 100 IU/ml and 1000 IU/ml. Cultures were pulsed at different periods (24, 48, 72 and 96 hours after stimulation) with [³H]-thymidine (1 μg/ml with a specific activity of 2 Ci/mmol, Amersham International) for 4 hours and the incorporation of radio¬ activity was assessed by scintillation counting.

Figure 20. The proliferation of PHA stimulated CD4-lympho- cytes. ^τ ₀ determine the effect of tro-IFN-^ on the pro- liferation of T-lymphocytes, cultures were supplemented with PHA (1 μl/ml) and different concentrations of tro-IFN- β . The lymphocytes were stimulated with 10 IU/ml, 100 IU/ml and 1000 IU/ml. Cultures were pulsed at different periods (24, 48, 72 and 96 hours after stimulation) with [³H]- thymidine (1 μg/ml with a specific activity of 2 Ci/mmol, Amersham International) for 4 hours and the incorporation of radioactivity was assessed by scintillation counting. Figure 21. The relative inhibitive effect of tro-IFN-beta on the proliferation of PHA stimulated CD4-lymphocytes. ^τ ₀ determine the effect of tro-IFN-^ on the proliferation of T-lymphocytes, cultures were supplemented with PHA (1 μl/ml) and different concentrations of tro-IFN-yS. The lymphocytes were stimulated with 10 IU/ml, 100 IU/ml and 1000 IU/ml. Cultures were pulsed at different periods (24, 48, 72 and 96 hours after stimulation) with [³H]-thymidine (1 μg/ml with a specific activity of 2 Ci/mmol, Amersham International) for 4 hours and the incorporation of radio¬ activity was assessed by scintillation counting.

Figure 22. The proliferation of PHA stimulated CD8-lympho- cytes. ^τ ₀ determine the effect of tro-IFN-_/3 on the pro¬ liferation of T-lymphocytes, cultures were supplemented with PHA (1 μl/ml) and different concentrations of tro-IFN- β. The lymphocytes were stimulated with 10 IU/ml, 100 IU/ml and 1000 IU/ml. Cultures were pulsed at different periods (24, 48, 72 and 96 hours after stimulation) with [³H]- thymidine (1 μg/ml with a specific activity of 2 Ci/mmol, Amersham International) for 4 hours and the incorporation of radioactivity was assessed by scintillation counting.

Figure 23. The relative inhibitive effect of tro-IFN-beta on the proliferation of PHA stimulated CD8-lymphocytes. ^τ ₀ determine the effect of tro-IFN-^ on the proliferation of T-lymphocytes, cultures were supplemented with PHA

(1 μl/ml) and different concentrations of tro-IFN-^. The lymphocytes were stimulated with 10 IU/ml, 100 IU/ml and 1000 IU/ml. Cultures were pulsed at different periods (24, 48, 72 and 96 hours after stimulation) with [³H]-thymidine (1 μg/ml with a specific activity of 2 Ci/mmol, Amersham International) for 4 hours and the incorporation of radio¬ activity was assessed by scintillation counting.

Figure 24. The relative inhibitive effect of tro-IFN-beta on the proliferation of PWM stimulated B-lymphocytes. ^τ ₀ determine the effect of tro-IFN-_/-.' on the proliferation of T-lymphocytes, cultures were supplemented with PWM (5 μg/ml) and different concentrations of tro-IFN-^. The lymphocytes were stimulated with 10 IU/ml, 100 IU/ml and 1000 IU/ml. Cultures were pulsed at different periods (24, 48, 72 and 96 hours after stimulation) with [³H]-thymidine (1 μg/ml with a specific activity of 2 Ci/mmol, Amersham International) for 4 hours and the incorporation of radio¬ activity was assessed by scintillation counting.

EXAMPLE 1

Isolation of human placental trophoblast cells, establish¬ ment of a trophoblast cell culture and induction of inter¬ feron production

Isolation and purification of human placental trophoblast cells and establishment of a trophoblast cell culture

Human placental trophoblast cells from term placenta were isolated as follows.

1) Biopsies were taken from the placenta after removing the shell-site.

2) The tissue was placed in a container with PBS contain¬ ing Fungizone®, penicillin G and streptomycin (fungi- zone-B 10.000 μg/ml, amphofericin-B 250 μg/ml, peni¬ cillin 100 IU/ml and streptomycin 100 μg/ml was added to 500 ml s-PBS (sterile PBS) ) .

3) The biopsies were cut into lxlxl cm pieces.

4) The tissue was transferred to another container and s-PBS was added. After sedimentation of the tissue, the tissue was washed repeatedly with s-PBS until the supernatant was clear.

5) Thereafter, the tissue was filtered through a double layer of gauze.

6) The thereby obtained tissue was transferred to a new container with 300 ml trypsinization liquid with a temperature of 37°C. The trypsinization liquid was prepared by adding 1.125 g of trypsin to 900 ml PBS containing 5 mM Mg⁺⁺ and adjusting the pH to 7.4 followed by sterilization. 300 μl DNAse was added per 300 ml trypsinating liquid. 7) After 30 min at 37°C under rotation, all was filtered through a double layer of gauze and the tissue was transferred to another round of 30 min of trypsiniza¬ tion. Thesupernatant was discarded.

8) The sample was filtrated through a double layer of gauze into 10 ml fetal calf serum whereby the trypsin was inactivated. The retained tissue was taken through another round of trypsinization for 30 minutes.

9) The supernatant was removed and the cells resuspended in 2x40-50 ml of s-PBS. The cell suspension was cen¬ trifugated at 300g for 7 minutes.

10) All was filtered through two layers of gauze into 10 ml fetal calf serum. The retained tissue was discard¬ ed.

11) The supernatant was centrifugated at 300g for 7 min.

12) The supernatant was removed and the cells resuspended in 25-35 ml of PBS and filtrated through a six-layer of gauze and was then centrifugated at 300g for 7 min.

13) The cells were placed on a Percoll gradient by suspen- ding the cells in 10 ml 70% Percoll which was applied underneath 20 ml 25% Percoll. Finally, 10 ml s-PBS was applied and the resulting gradient was centrifugated for 20 min at 800g at 4°C.

14) The mononuclear cells were collected from the gra- dient, resuspended in PBS and centrifugated at 300g for 7 min.

15) The supernatant was removed, the cells resuspended in PBS and centrifugated again at 300g for 7 min. 16) The supernatant was removed, the cells were resuspen¬ ded in a known volume of PBS and counted.

17) The cells were centrifugated and resuspended in 30% non heat inactivated fetal calf serum. In 925 μl of RPMI containing the cells 75 μl DMSO was added and the cells were stored in frozen condition.

First trimester trophoblast cells from a provoked vaginal delivery were isolated as described in Example 4.

The cells from the term placenta isolated as described above was purified by immunomagnetic microspheres as de¬ scribed by Douglas & King (1989) . The purification was performed as follows.

1) After thawing in RPMI, the cells were centrifugated at 300g for 7 min.

2) The supernatant was removed and the cells resuspended in RPMI at a concentration of 20xl0⁶ cells/ml.

3) 20 μl of HLA-ABC antibody was added per ml of cell suspension.

4) The cells were incubated on ice under gentle rotation for 30 min followed by 3 washes in RPMI.

5) The cells were centrifugated at 300g for 7 min and resuspended in RPMI to a concentration of 20xl0⁶ cell/ml.

6) The following Dynabeads (Douglas & King, 1989) were added:

50% CK1 pos.cells: 150 μl Dynabeads M-450 (sheep anti- mouse IgG) /ml suspension. 70% CK1 pos.cells: 100 μl Dynabeads M-450 (sheep anti- mouse IgG) /ml suspension. 90% CK1 pos.cells: 50 μl Dynabeads M-450 (sheep anti- mouse IgG)/ml suspension.

9) The cells were incubated for 30 min on ice and gently hand-shaken from time to time.

10) The cell suspension was then placed on a magnetic plate. The cells were left for a few minutes and the supernatant were removed. The Dynabeads were washed with PBS and placed on the magnetic plate again for remowal of the remaining Dynabeads.

11) PBS was added and the sample was centrifugated at 300g for 7 min. The cells were resuspended and counted.

12) The cells were examined for the presence of CK-1,

M 717 and M 821, HLA-abc, M 736, vimentin M 877 and M 725, macrophage M 718 (Dakopatts A/S, Denmark) by indirect fluorescence. The secondary rabbit to mouse FITC labelled antibody F 313 of F 201 were obtained from Dakopatts.

13) The trophoblast cell culture was established by seed¬ ing the trophoblast cells at a density of 1 x 10 - cells/ml in RPMI-1640 supplemented with 10% FCS.

Production of interferon

In order to induce a production of interferon, stimulation of the cells was needed.

The cells were cultured for 24 h in RPMI-1640 suplemented with 10% fetal calf serum, at 37°C and 5% C0₂ in air. The induction of the interferon performed either by infecting the cells with Sendai virus or by using polyriboinosinic- polyribocytidylic acid (poly(rl)-poly(rC) ) . When using Sendai virus as inducer of the interferon, the cells were infected with Sendai virus using 200 HAU/10⁶ cells for 1 h in serum-free RPMI-1640. Unadsorbed virus was removed by washing the cells twice with serumfree medium. When using polyriboinosinic-polyribocytidylic acid (poly(rl)-poly(rC) ) (Sigma Chemical Company, St. Louis, MO, USA) , the cells were incubated with 10 μg/ml for 1 hour and the culture was washed three times with serum-free medium and then further cultured in RPMI 1640 + 5% fetal calf serum.

Culture supernatants were harvested 18 h after induction. Virus-stimulated supernatants were acidified to pH 2.0 using hydrochloric acid for 48 h at 4°C and then neutral¬ ized to pH 7.2. The crude interferon was precipitated by adding 1 g ammonium sulphate per 2 ml of interferon prepa¬ ration. The precipitate was redissolved in 0.02-M sodium phosphate buffer pH 7.2 and dialyzed against two changes of 500-fold excess of the same buffer for 10 h at 4°C.

EXAMPLE 2

Purification of the various types of interferon and further examination of the purified interferon

Preparation of the columns used in the chromatographic procedures

The HEMA-BIO 1000 VS 3GA adsorbent (HEMA-BIO 1000 VS = 2- hydroxyethyl methacrylate vinyl sulphone with an exclusion limit at molecular weight lOOoA) , synthesized as described below, is a unique adsorbent for interferon purification. It does not swell in water. It is rigid and can therefore tolerate the high mobile phase flow rates typical of HPLC. As appears from the following, it results in a very high degree of purification.

Synthesis Of HEMA-BIO 1000 VS 3GA

HEMA-BIO 1000 VS 3GA (see Figure 7) was prepared by cova¬ lently attaching Cibacron Blue 3GA (Sigma Chemical Company, St. Louis, Mo, USA) via a spacer arm, 1,4-diaminobutane, to vinyl sulphonate-activated HEMA-BIO 1000 (10 μm particle size, Tessek A/S, Denmark) . 6 g of dry HEMA-BIO 1000 VS (10 μm) was suspended in 25 ml of 0.1 M NaHC0₃ - Na₂C0₃ buffer pH 11.5 for 10 min and 1,4-diaminobutane was added to a final concentration of 0.5 M. The mixture was incu¬ bated overnight at room temperature by rotation on a Rotamix (HETO, Birkerød, Denmark) . The gel was settled by centrifugation and extensively washed with water (500 ml) . The gel was then suspended in 30 ml of 0.1 M NaHC0₃ -

Na C0₃ buffer pH 9.0 containing Cibacron Blue 3GA (600 mg, 0.775 mmol) . The mixture was incubated overnight at room temperature by rotation on a Rotamix. After incubation, the gel was settled by centrifugation and the supernatant was removed. The HEMA-BIO 1000 VS 3GA was washed with water (500 ml), 1.5 M NaCI (300 ml) and 0.02 M sodium phosphate buffer pH 7.2 (500 ml). The gel was then packed in a bio¬ compatible PEEK (Poly Ether Ether Ketone) column (7.5 mm x 250 mm, Tessek A/S, Aarhus, Denmark) by the upward slurry packing technique under pressure (6 MPa) . The concentration of the Cibacron Blue 3GA immobilized on the HEMA-BIO 1000 VS 3GA was determined by spectrophotometric analysis of alkaline hydrolysates as described by Lowry et al (1981) . The binding capacity was determined by frontal analysis with human serum albumin in 0.02 M sodium phosphate buffer pH 7.2. Thus, continuous injections of human serum albumin were applied to a PEEK column (4.6 mm x 50 mm packed with HEMA-BIO 1000 VS 3GA) until protein activity was observed in the eluent. The column was washed and the bound protein was eluted with 2 M NaCI in the same buffer. The concen¬ tration of the eluted protein was then assayed.

Synthesis of HEMA-BIO 1000 vs - antibody column

Immunoadsorbents (such as anti-α-interferon antibodies and ant i.-β-interferon antibodies) and Concanavalin A (Con A) columns were prepared by adsorption-promoted enhanced co- valent immobilization of the antibodies and Con A on macro- porous HEMA-BIO 1000 VS.

0.5 g of dry HEMA-BIO 1000 VS was swollen with immobiliza¬ tion buffer (0.1-M sodium borate/0.75 M ammonium sulphate buffer pH 8.0) and 5 mg antibodies or 10 mg of Con A (Sigma Chemical Company, St. Louis, MO, USA) dissolved in 4 ml or 15 ml (for Con A) of immobilization buffer was added to the wetted gels. The immobilization was allowed to proceed under gentle rotation for 16 to 20 h at 4°C. The gels were then settled by centrifugation, the supernatants were removed, the gels were washed eight times with distilled water and residual reactive groups were blocked by incuba¬ tion with 3 ml of 0.1-M ethanolamine in 0.1-M sodium borate buffer pH 9.0 for 6 h. The gels were then packed in biocom- patible PEEK hardware (50 mm x 4.6 mm internal diameter).

Chromatoσraphic procedures

High-performance dye-ligand affinity chromatography (HP- DLAC)

Concentrated crude trophoblast interferon (2.05 x lo⁶ IU) was filtered through 0.22 μm Millipore filter and applied to HEMA-BIO 1000 VS 3GA column (250 mm x 7.5 mm I.D.) equilibrated with column buffer (0.02-M sodium phosphate buffer pH 7.2). After the column had been washed with the same buffer containing 0.2-M NaCI, proteins were eluted with a linear gradient of 0.2 to 1.0-M NaCI in 35 min at a flow rate of 1.5 ml/min. The elution from the column was monitored continuously at UV absorbance of 280 nm. The column was further washed with 1.0-M NaCI in the column buffer for 20 min until the absorbance was almost zero. Protein was then eluted from the column by increasing con¬ centration of ethylene glycol in 0.02-M sodium phosphate buffer pH 7.2 containing 1.0-M NaCI; elution was with 0 to 50% for 10 min and 50% for 35 min at a flow rate of 1.5 ml/min. Fractions of 1.5 ml were collected and assayed for interferon antiviral activity.

High-performance immunoaffinity chromatography (HP-IAC)

Crude trophoblast interferon preparations and the fractions containing interferon antiviral activity from the HEMA-BIO 1000 VS 3GA column were applied separately to HEMA-BIO 1000 VS-anti-α-interferon (polyclonal) antibody or anti-^-inter¬ feron antibody columns. In all cases, the columns were washed 10 min with PBS (phosphate buffer saline) pH 7.4 and proteins were eluted with 0.1-M glycine-HCl pH 2.4. Frac¬ tions of 0.5 ml were collected and dialyzed against PBS buffer for 16 h at 4°C to restore the pH to neutral, and then were assayed for interferon antiviral activity.

Reversed-phase high performance liquid chromatography (RP-HPCL)

The interferon-containing fractions from the HP-DLAC step were pooled together and dialysed against 0.02 M sodium phosphate buffer, pH 7.2, containing 30% glycerol for 1 h to remove ethylene glycol and also to concentrate the sample. The dialysed sample was applied to Separon SGX C-18 column (CGC glass cartridge of 3 mm x 150 mm, with a metal column holder) that has previously been equilibrated with a mobile phase consisting of 80% Solvent A (1 M pyridine/ace- tic acid pH 5.0) and 20% Solvent B (100% propan-1-ol) . The column was washed for 20 min with the same mobile phase, followed by elution of interferon activity with a linear gradient from 20 to 70% Solvent B in Solvent A. The gradi¬ ent elution was carried out in 60 minutes at a flow rate of 0.25 ml/min. Elution from the column was monitored conti- nuously by UV absorbance at 280 nm (0.32 absorbance units full-scale) and fractions of 0.5 ml were collected and then were assayed for interferon activity. Purification of the trophoblast interferons

Figure 1 illustrates the HP-DLAC of crude trophoblast interferon on HEMA-BIO 1000 VS 3GA. The trophoblast inter¬ ferons bound completely when applied in 0.02 M sodium phos- phate buffer pH 7.2 at low ionic strength (0.2 M NaCI) . Development of the column with a linear concentration gra¬ dient of sodium chloride separated 20% of the total inter¬ feron activity applied into five peaks (fractions 21 to 40 eluted at 0.6 to 0.8 M NaCI).

Further development of the column with a linear concentra¬ tion gradient of the hydrophobic solute ethylene glycol produced two interferon peaks (fractions 50 to 53 and 54 to 70) , eluted from the column at ethylene glycol concentra¬ tions of 40 to 50% and 50%. A summary of the HP-DLAC of the trophoblast interferons is presented below in Table 1.

Table 1

Purification of placental trophoblast interferons (IFN) by HP-DLAC

Table 2

Summary of a two-dimensional purification of trophoblast β-interferon.

^a % Recovery over the total ø-interferon activity in the crude preparation

-^b Total β-interferon activity in the crude preparation ^c Recovery of trophoblast -interferon over the total interferon activity.

Further purification in the form of rechromatography of the HP-DLAC-purified trophoblast -interferon by RP-HPLC on

Separon SGX C-18 is shown in Table 2 above. As appears from the table, the rechromatography resulted in a purification of β-interferon having a specific activity of 1.03xl0⁸ IU/mg of protein with a 17,517-fold purification over the crude preparation. The chromatogram is shown in Figure 5.

Determination of the protein concentration

The concentration of protein in crude trophoblast inter¬ feron preparations was determined by the dye binding assay (Bradford, 1976) using bovine serum albumin as a standard. The concentration of purified interferon was measured by absorbance at 280 nm, or derivatization with fluorescamine and injecting the samples into a fluorescence HPLC monitor and using known concentrations of BSA as a standard. Polyacrylamide gel electrophoresis

Slab SDS-PAGE was carried out according to Laemmli (1970) using a voltage of 200V and running the SDS-PAGE for 8 h or until the sample is 1 cm from the bottom. Freeze-dried interferon samples were prepared with 5% 2-mercaptoethanol or without 2-mercaptoethanol. The following amounts of the various interferons were used in order to determine the size of the various interferon molecules: 40.000 IU of β- interferon, 45.000 IU of α_j-interferon, 45.000 IU of α_j - interferon.

After electrophoresis, the gels were silver stained using Bio-rad silver staining kit (Catalogue number 161-0443) and scanned on Biomed SLR 1 D/2D scanner (Biomed Instruments Inc. California, USA) to determine the molecular mass by comparison with Bio-Rad standard protein standards. To determine the interferon antiviral activity on unstained gels, 2-mm slides of the gel were extracted at 4°C for 24 h into 0.5 ml volumes of PBS buffer pH 7.4 containing 0.1 % SDS and 0.02% NaN₃. Aliquots (100 μl) were then taken and assayed for interferon antiviral activity.

Figure 2 shows the patterns obtained by SDS-PAGE on frac¬ tions from HP-IAC performed on crude trophoblast interferon preparation and the patterns obtained by SDS-PAGE on HP- DLAC-purified trophoblast α_j-interferon (fractions 21 to 40 from Figure 1) on a HEMA-BIO 1000 VS-anti-α-interferon (polyclonal) antibody column. Antiviral activity neutrali¬ zation assays of interferons extracted from unstained gels (similar to the gel in Figure 2(a) under reducing and non- reducing conditions, showed that trophoblast α -interferon and trophoblast α_j l-interferon activities corresponded to the protein bands with molecular masses of 16 and 22 kDa respectively. No interferon activity was associated with the 67 kDa protein band. Figure 2(b) shows the SDS-PAGE of trophoblast -interferon (from fractions 56 to 58 in Figure 1) ; trophoblast β-interferon migrated as a 24-kDa protein on reducing and non-reducing SDS-PAGE (Figure 2(b) lanes 2 and 3, respectively) .

The antiviral activity of the crude trophoblast interferon preparation and the purified interferon were stable after 24 hours of incubation at pH 2 in 0.1-M glycine.

EXAMPLE 3

Examination of the interferon produced by the trophoblast cells

Glycoprotein analysis

The presence of sugar residues in the trophoblast inter¬ ferons was determined by their binding to Con A, by employ¬ ing affinity chromatography on HEMA 1000 VS-Con A.

After injection of the interferon samples, the column was washed with column buffer (20 mM sodium phosphate buffer pH 7.2, containing 1 M NaCI and MnCl₂, CaCl , and MgCl₂) • Bound interferons were then eluted with 0.1 M α-methyl-D- mannopyranoside and 50% ethylene glycol in the column buffer.

Trophoblast α_j -interferon and trophoblast β-interferon bound to Con A affinity column (se Figure 3) , whereas tro¬ phoblast αχ-interferon did not bind. This suggests that trophoblast α_jj-interferon and trophoblast β-interferon are glycoproteins whereas α_j_-interferon may not be a glycopro- tein. Examination of a cytotoxic compound secreted by tropho¬ blasts after induction with virus

When determining the biological activity of fractions 55-70 in Figure 1, it was found that apart from antiviral acti- vity, the purified interferon had cytotoxic effect. At first, it was believed that it was the impurity that gave the cytotoxic effect. Therefore, the material was further purified on an anti-^-interferon column. The cytotoxic com¬ pound did not bind to the column. Even in dilutions up to 1,000,000, the eluate showed cytotoxic activity.

In the following, the investigation is made of the cyto- toxicity of this compound, prepared by pooling the above- mentioned fractions 55-70, subjecting them to the High performance dye ligand chromatography, and subjecting the eluate from this chromatography to High performance immuno¬ affinity chromatography on the anti-β-interferon column (whereby the -interferon is very efficiently bound by the HEMA-BIO 1000 VS 3GA, while the cytotoxic compound passes through the column) .

To determine the cytotoxic drug response of the compound in question, two different target cell lines, L-929 and K-562 were used in a tetrazoliu dye reduction assay (MTT assay) . (P.R. Twentyman, Modification of cytotoxic drug resistance by non-immuno-suppressive cyclosporins. British Journal of Cancer 1988, 57, pp. 254-258) . In brief, the target cells L-929 and K-562 were grown in RPMI 10% at 37"C in 96-well microwells in 100 μl, 20000 cells/well. On day 0 the trophoblast compound was added to the well to obtain different dilutions: 1:10, 1:1000, 1:10000, 1:100000, 1:1000000. After 2 days of cultivation in 5%

C0₂, 100 μl of medium were added to each well. After 4 days of cultivation 20 μl MTT solution were added to all wells and incubated for further 4 hours. The plates were centri¬ fugated 1 minute at 300 rpm in order to pellet the cells. 150 ml were harvested from each well and lOOμl isopropanol were added. After allowing the crystals to dissolve for 5 minutes, the plates were grown on an ELISA reader using an optical density of 570 nm. Control cultures were grown in medium alone. It was found that the cells were killed by the compound in all of the dilutions mentioned.

Examination of the purity of the interferons

The purity of the -interferons purified as described in Example 2 above using HP-DLAC and RP-HPLC purification was determined by densitometric analysis of Coomassie SDS- polyacrylamide gel (see Figure 6) using Biomed SLR-1D/2D densitometer (Biomed Instruments Inc. , California, 92621)

In Figure 4 is shown the densitometric analysis of the purity of -interferon. As appears from the figure, a single peak was obtained, showing that a purity of more than 99% was obtained.

Examination of the antiviral activity of the trophoblast inter erons.

The antiviral activity of the various interferon prepa¬ rations was determined by assaying the capability of the sample to inhibit plaque formation in human amniotic cell line WISH (American Type Cell Culture, ATCC) by vesicular stomatitis virus (VSV) , Indiana strain.

The cells were seeded in 96-well microplates (40,000 cells/well) . Interferon preparations were titrated by adding 100 μl of two- or four-fold serial dilutions to each well. After incubation for 24 h at 37^QC and 5% C0₂ in air, the cells were infected with VSV (50 plaque-forming units per well) . The titrations were scored microscopically 24 h after virus inoculation. The highest dilution giving 50% reduction of the viral plaques were considered as the end point. The interferon titers were standardized by comparison with the National Institute of Health Standard for human α- interferon G-023-902-530) and ,0-interferon (G-023-902-527) . Also, interferon titers, using human trisomic 21 fibroblast cell lines GM 2504 and GM 2767 (Cell Repository, Camden, N. J. , U.S.A) and bovine (MDBK) cells (ATCC) were performed similarly using vesicular stomatitis virus as the challen¬ ge; results were expressed as the highest dilution giving 50% protection and the activity expressed as the dilution factor in units/ml.

Antigenic characterization of the interferons using anti¬ body neutralizing test

The antigenic specificities of the crude and isolated tro- phoblast interferons were determined by antibody neutra¬ lization tests. Polyclonal anti-human-α-interferon, anti- human-_/-}-interferon, anti-₇-interferon and anti-human recom¬ binant α_j l-interferon antibodies were used for the assay and for the preparation of the HPLC sorbents.

Polyclonal anti- -interferon obtained from Boehringer Mann¬ heim (produced in Balb/c mice using human α-interferon purified from Sendai virus-stimulated human lymphoblastoid cells) neutralizes human α-interferon and recombinant α - interferon and α -interferon but do not react with anti- yθ-interferon or anti-₇-interferon.

Polyclonal human anti-^-interferon antibodies, produced in horses immunized with human -interferon (fibroblast) , was obtained form Boehringer Mannheim (Germany) and do not react with human α_j-interferon, α_jj-interferon or ₇-inter- feron.

Anti-recombinant α _jl-interferon antibodies was supplied by Gϋnther R. Adolf (Dept. of Biotechnology, Dr. Boehringer- Gasse, A-l 121 Vienna, Austria) and do not react with a_j-, β- or ₇-interferon. Serial dilutions of interferon and a fixed dilution of antibody in 10-fold excess were incubated for 1 h at 37°C and residual antiviral activity was determined as described above in "Examination of the antiviral activity of the trophoblast interferons".

Antiviral specificity of trophoblast interferons on diffe¬ rent types of cells

Table 3 shows the antiviral activities of the trophoblast interferons on different human and bovine cell lines.

Table 3

Protection of cell lines from different species against VSV infection by trophoblast interferon (IFN)

Antiviral activity*

Species Cell line tro-IFN-α tro-IFN-α_jjl tro-IFN-j8

256

768

2

* Antiviral activity is expressed as the highest dilution giving 50% protection against VSV.

As appears from Table 3, the trophoblast interferons ex¬ hibited a broad spectrum of antiviral activities on the human cells tested (WISH (American Type Cell Culture) , GM 2504 and GM 2767 (both trisomy 21 fibroblasts, Cell Reposi¬ tory, Cambden, N.J., U.S.A.), but trophoblast α_j-interferon and α_jj-interferon protected bovine cell lines better than human cells (two fold and four fold, respectively, relative to human WISH cells) . More interestingly, the protection of MBDK cells by trophoblast α _j-interferon was twice that conferred by trophoblast α_j-interferon. In contrast, tro¬ phoblast β-interferon did not protect bovine MDBK cells, but did protect all the human cell lines tested.

Characterization of trophoblast interferons

The trophoblast interferons were identified by antiviral neutralization tests as described above using polyclonal antisera to human interferon aχ~, β- and ₇- and αul-inter- feron.

Table 4

Antibody neutralization of trophoblast interferons (IFN) isolated by HP-DLAC"

Residual antiviral activity after incubation with antisera (%)

* Neutralization tests were performed with a 10-fold excess of the respective antiserum. D ND = Not determined. Polyclonal antiserum to human -interferon neutralized 75% of the antiviral activity of crude trophoblast interferon samples (initially 800 IU/ml, 200 IU/ml after treatment) whereas the anti-α-interferon neutralized 25% of the acti- vity (initially 800 IU/ml, 600 IU/ml after treatment) . The results appears from Table 4 above. Fractions 21 to 40 (see Figure 1), eluted from the column by NaCI (0.6 to 0.8-M) were completely neutralized by antiserum to human interferon α_j antibodies, but not by anti-αχχl-interferon, anti-^-interferon and anti-₇-interferon. This showed that the five interferon peaks were α_j_-interferon. However, the interferon peak fractions (50 to 53) that were eluted from the column using ethylene glycol concentrations between 40 to 50% were partially neutralized by polyclonal anti-β- interferon antibodies, and to the same extent by anti-α- interferon and anti-α_j__jl-interferon antibodies. These interferon fractions were completely neutralized by a com¬ bination of anti-α χl-interferon and anti-ø-interferon antibodies, and polyclonal anti-α-interferon and anti-y9- interferon antibodies, but not polyclonal anti-α-interferon and anti-α l-interferon antibodies. This suggests that fractions 50 to 53 were a mixture of trophoblast α_jj-inter- feron and trophoblast β-interferon.

Fractions 50 to 53 (see Figure 1) were further characteri- zed by passing through HEMA-BIO 1000 VS-anti-^-interferon (polyclonal) column as described above. The antiviral acti¬ vity of the flow-through fractions and the bound interferon eluted from the column at pH 2.4 was characterized further using antiserum to human α-interferon, β-interferon and recombinant human α-_j- l-interferon as appears from Table 5. These assays showed that the antiviral activity of the flow-through fractions was not neutralized by antiserum to β-interferon but was completely neutralized by anti-αιχl- interferon and polyclonal anti-α-interferon antisera. How- ever, the bound interferon was completely neutralized by human anti-^-interferon antiserum. Table 5

Neutralization of the antiviral activity of fractions 50 to 53 after passage through an anti-_/3-interferon antibody column

Residual antiviral activity (%)

Anti-human IFN seru Flowthrough Bound

None 100 100 _jjl 0 100 α 0 100 β 100 0

The anti-IFN immune sera were used in 10-fold excess.

Determination of the thermal stability of the antiviral activity

The thermal stability of -interferon was determined at a temperature of 37°C and 56°C. The interferon samples were heated to 37°C or 56°C for various times. After heating, samples were allowed to cool to room temperature or were stored on ice for 1 hour until they were assayed for anti¬ viral activity using the method as described above.

It was found that the β-interferon retained 55% of its antiviral activity at 37°C for 3 hours. However, 50%, 75% and 97% of the antiviral activity was lost after 10 min, 15 min and 60 min respectively at 56°C. EXAMPLE 4

Production of -interferon by first trimester placental trophoblasts cell cultures

Isolation of first trimester trophoblast

First trimester trophoblasts were isolated by a modifica¬ tion of the method of Fisher (1989) as follows.

1) The tissue is transported from the hospital in PBS containing PeniciIlium/Streptomycin and Fungizone® (the same concentration as used for term trophoblast cells) .

2) The tissue was sectioned in pieces of 2-3 mm².

3) The tissue was allowed to sediment and thereafter washed repeatedly with s-PBS until the supernatant was clear. The tissue was centrifugated, collected and centrifugated again for 10 min at 300g.

4) The tissue was weighed.

5) The tissue was placed in a 100 ml bottle with a tryp- sinating liquid consisting of 0.625 g trypsin to

600 ml PBS with 5 mM Mg⁺⁺, pH 7.4, and 200 μl DNA-se (this corresponded to approximately 40 g of tissue) .

6) After 35 min at 37°C with rotation, the thus obtained sample was filtered trough 2 layers of gauze, inacti¬ vated with sterile serum and centrifugated at 300g for 7 min.

7) The tissue were weighed. 8) The tissue was washed and centrifugated at least twice and filtered through 6-8 layers of gauze.

9) The cells were resuspended in 10 ml 70% Percoll which was applied underneath 20 ml 25% Percoll. Finally, 10 ml s-PBS was applied and the resulting gradient was centrifugated for 20 min at 800g at 4°C (break off) .

10) The bands containing cells (as seen under microscope) were aspirated.

11) The cells were washed in s-PBS and counted. Then, the cells were stored frozen in 30% RPMI and 15 % DMSO at a concentration of approximately 10 millions of cells per ampoule.

12) Selective detaching of trophoblast cells using EDTA on ice was used to further purify the cells. 10⁶ cells/ml were seeded for 24 h in RPMI 1640 containing 10% fetal calf serum. First trimester trophoblasts were detached from the mixture by adding 0.02% EDTA to the cells after aspiration of the medium. The growth container containing the cells was kept on ice for 10 - 12 min. Detached trophoblast cells were collected and washed twice with RPMI containing 10% medium and were re- seeded at a density of 5 x 10⁵ cells/ml in the same medium.

Induction of the production of ₇-interferon

First trimester trophoblasts cells seeded at a density of 5 x 10⁵/ml and cultured for 24 hours in RPMI-1640 supple¬ mented by 10% fetal calf serum. After 24 h, the medium was removed and replaced with RPMI-1640 + 10% fetal calf serum containing the ₇-interferon inducer in the form of 100 U/ml of IL-2 (interleukin-2) and 5 μg/ml of phytohaemagglutinin (PHA) . The cells were cultured for 5 days and 200 μl of medium were collected every day and examined for interferon activity. On the 5th day, the supernatant was collected and centrifuged at 2000 rpm for 20 min at 4°C and stored at -70°C.

Interferon bioassay

Examination of antiviral activity of the ₇-interferon was performed as described above by assaying the capability of the ₇-interferon to inhibit the plaque formation in human amniotic cell line WISH (ATCC) by vesicular stomatitis virus (VSV) , Indiana strain. Cells were seeded in 96-well microplates (40,000 cells/well). Interferon preparations were titrated by adding 100 μl of two-fold serial dilutions to each well. After incubation for 24 h at 37°C and 5% C0₂ in air, the cells were infected with VSV (50 plaque-forming units per well) . The titrations were scored microscopically 24 h after virus inoculation. The highest dilution giving 50% reduction of the viral plaques were considered as the end point.

Antibody neutralization of the ₇-interferon activity

The antibody neutralization assay was performed as descri- bed above. Polyclonal rabbit and monoclonal mouse antibo¬ dies to human ₇-interferon were obtained from Serotec, Oxford, England, and Janssen Biochimica, Belgium respec¬ tively. These antibodies do not react with human α-inter¬ feron or -interferon. A single dilution capable of neutra- lizing 10-times the activity of the test interferon acti¬ vity was incubated with various concentrations of the tro¬ phoblast ₇-interferon preparation for 1 h at 37°C. The samples were then assayed for the residual interferon anti¬ viral activity.

In Table 6 below is shown the production of ₇-interferon in first trimester trophoblast cell cultures stimulated with 1L-2 and phytohaemagglutinin. As seen from the Table, the ₇-interferon production peaked 96 h (4 days) after the induction.

Table 6

₇-interferon production in first trimester trophoblast as a function of time

Time (h) interferon titre (U/ml)

0 0 24 80

48 80

72 160

96 640

120 640

Table 7 shows the antiviral activity of ₇-interferon as assayed using the neutralization assay and using specific human ₇-interferon antibodies. The three interferon prepa¬ rations, that is samples 1, 2 and 3 had antiviral activi- ties of 640 U/ml, 320 U/ml and 160 U/ml respectively. The antiviral activity of the samples were completely neutra¬ lized by anti-human ₇-interferon but not anti-human β- interferon or anti-human α-interferon. Therefore, the anti¬ viral activity in the supernatant of the human first tri- mester trophoblast cell culture stimulated by phytohaemag¬ glutinin and IL-2 is caused by ₇-interferon. Table 7

Trophoblast interferon (IFN) antiviral neutralization test

Residual antiviral activity (%) after incubation with

anti-human anti-human anti-human

IFN samples α-IFN -IFN ₇-IFN

tro-IFN (PHA/IL-2) 1 100 100 0 tro-IFN (PHA/IL-2) 2 100 100 0 tro-IFN (PHA/IL-2) 3 100 100 0

Effect of IL-2 and TPA on the induction of IFN-₇ produc¬ tion in first trimester trophoblast cultures

IFN-₇ production was performed as described above for induction of the production of ₇-interferon except that PHA was substituted with 5 ng/ml 12-O-tetradecanoylphorbol 13- acetate (TPA) .

Table 7a

Effect of IL-2 and TPA on the production of IFN-₇ in first trimester trophoblast cultures

Stimulant IFN-₇ titre (IU/ml)

None 0 IL-2 0

IL-2 + PHA 640 PHA 640 TPA 0

TPA + PHA 640 PHA 640

IL-2 and TPA did not influence the production of IFN-₇ in first trimester trophoblast cultures stimulated with PHA.

EXAMPLE 5

Differential interferon production in human first and third trimester trophoblast cultures stimulated with virus

Viruses

Newcastle Disease virus (NDV, Strain IB) and Sendai virus (Parainfluenza 1) were obtained from American Type Culture Collection (ATCC) and were propagated in the allantoic cavities of 10 day old embryonated eggs by inoculation of a 10~² or 10~³ dilution of infected allantoic fluids. After the eggs were incubated at 35°C for 3 days, the allantoic fluids were harvested and the infectivity was titrated in chicken erythrocytes. Isolation of first and third trimester trophoblasts

Third trimester trophoblast cells were isolated by immuno- magnetic microspheres as described above in Example 1. First trimester trophoblasts were isolated as described in Example 4. _β

Induction of trophoblast interferons

Trophoblast cells were seeded at a density of 1x10 - cells/ml in RPMI-1640 supplemented with 10% fetal calf serum. The cells were cultured for 24 hours at 37°C and 5% C0₂ in air, and were then infected with NDV (30 HAU/10⁶ cells) or Sendai virus (200 HAU/10⁶) for 1 hour in serum- free RPMI 1640. The unadsorbed virus was removed by washing the cells twice with serum-free medium and further cul- turing in RPMI 1640 + 5% fetal calf serum. Culture super- natants were harvested 18 hours after induction. Virus- stimulated supernatants were acidified to pH 2.0 for 48 hours at 4°C and then neutralized to pH 7.2.

Interferon antiviral and neutralization assay

These assays were performed as described previously in Example 3.

Results

Table 8 shows the differential interferon production in first and third trimester trophoblast cultures stimulated with Sendai and Newcastle Disease viruses. The data demon- strated that first trimester trophoblasts produced higher levels (about 5-7 times) of interferons than third trimes¬ ter trophoblasts. Furthermore, the interferon compositions varied depending on the inducer used to induce the inter¬ feron production. However, the interferon compositions were the same in both cases when induced by the same inducer in the first and third trimester trophoblast cultures. Table 8

Interferon yields and compositions of Sendai and Newcastle Disease virus-stimulated first and third trimester tropho¬ blast cultures.

Inducer IFN yield Composition* (IU/10⁶ IFN IFN

Trophoblast virus cells α (%) β (%)

First trimester Sendai

Third trimester Sendai

First trimester Newcastle

Third trimester Newcastle

*Determined by interferon antiviral neutralization assay

The experiment described above was repeated, and the re¬ sults were as follows:

Table 8a

Interferon yields and compositions of Sendai- and Newcastle Disease virus-stimulated first and third trimester tropho¬ blast and syncytiotrophoblast cultures.

IFN yield Composition* Inducer IU/10⁶ IFN IFN virus cells α (%) β (%)

Trophoblast

First trimester Sendai 42440±4220 25 75

(9.9%)

Third trimester Sendai 7400± 880 25 75

(11.9%) First trimester Newcastle 8120016340 35 65

(7.8%)

Third trimester Newcastle 16000±1480 35 65

(9.3%)

Syncytiotrophoblast

First trimester Sendai 64240+4820 25 75

(7.5%)

Third trimester Sendai 12800±1240 25 75

(9.7-%) First trimester Newcastle 86680±5560 35 65

(6.4%) Third trimester Newcastle 32860±2400 35 65

(7.3%)

Data are the mean ± s.d. and the coefficient of variation of IFN antiviral activities in parentheses; number of IFN preparations (N) is 9 and replication of assay (n) is 6 (P<0.05). *Determined by IFN neutralization test. EXAMPLE 6

Determination of the chemical stability of trophoblast interferons

Purified tro-IFN preparations dialysed against 0.02 M sodium phosphate buffer pH 7.2 were tested for their stabi¬ lity towards sodium dodecyl sulphate (SDS) , β-mercaptoetha- nol and urea. Samples were incubated in 1% SDS or 1% SDS, 1% _/9-mercaptoethanol and 5 M urea or 1% ^-mercaptoethanol and 5 M urea. Incubation times and temperature were 1 hour at 37°C or 1 minute at 100°C, respectively. After treat¬ ment, samples were diluted in assay medium (Eagle's minimal essential medium containing 5% FCS) before assaying for residual antiviral activity.

The antiviral activity of the purified tro-IFNs (α and β ) was stable at acidic conditions (pH 2.0) for 2 weeks at

4°C. Purified tro-IFN-^ at a concentration of 30 μg/ml was stable for one month in 0.02 M sodium phosphate buffer pH 7.2 containing 50% ethylene glycol at 4°C. The antiviral activities of the tro-IFNs were stabilized to different degrees by SDS under reducing and non-reducing conditions at 37°C and 100°C. As shown in Table 9 below, the antiviral activity of tro-IFN-α was stable in 1% SDS at 37°C and 100°C but less stable under reducing (1% SDS, 1% 3-mercap- toethanol and 5 M urea) conditions whereas tro-IFN-^ was more stable under reducing conditions at 37°C and 100°C.

Table 9

Chemical stability

Antiviral activity*

Treatment Incubation tro-IFN-α tro-IFN-y8

None

SDS

1% SDS + 1% -ME +

5 M urea 1% β-NE + 5 M urea

None

SDS

1% SDS + 1% β-NE + 5 M urea

1% -ME + 5 M urea

* Antiviral activity is expressed as the highest dilution giving 50% protection against VSV. β-KE = ^-mercaptoethanol

EXAMPLE 7

Leukotriene-reσulated interferon gamma production in human placental first trimester trophoblast cultures

As shown in Example 4, human first trimester trophoblast cultures when stimulated with phytohaemagglutinin (PHA) produced IFN-₇, and the IFN-₇ production was not mediated by interleukin 2 (IL-2) or 12-O-tetradecanoylphorbol 13- acetate (TPA) , which has been demonstrated for ₇-inter¬ ferons produced by T cells or T cell lines. However, first trimester trophoblast cultures produced leukotrienes, a product of the lipoxygenase pathway of arachidonic acid metabolism, which may positively regulate the IFN-₇ pro¬ duction.

IFN-₇ plays a central role in the mediation or regulation of several immunologic activities. Examples of such acti- vities are the induction and/or enhancement of both natural killer cell activity (Weigent et al., 1983) and specific T cell activity (Farrar et al., 1981) and/or enhancement of expression of class I (Wallach and Revel, 1982) and class II (Steeg. et al., 1982; Basham and Merigan, 1983) antigens of the major histocompatibility complex. Regulation of these immunologic events may be intimately related to the regulation of the production of IFN-₇.

Mitogen induction of IFN-₇ in peripheral blood lymphocytes of humans involves helper cells, suppressor cells and IFN-₇ producer cells (Torres et al., 1982; Papermester et al., 1983) . Helper cells requirement for IFN-₇ production is mediated through IL-2 by a mechanism that does not involve DNA synthesis (Johnson et al., 1982). The ability of the second messenger, cyclic GMP and a variety of substances that stimulate guanlyate cyclase activity in cells to pro¬ duce the IL-2 requirement for IFN-₇ production suggests that helper effects involve elevation of cyclic GMP acti¬ vity in IFN-₇ producing cells (Johnson et al. , 1982). Data suggests also that IL-2 increases cyclic GMP levels in treated cells (Hadden and Goffey, 1982) .

Products of the lipoxygenase pathway of arachidonic acid metabolism such as leukotriene B₄ (LTB₄) have been shown to activate guanylate cyclase activity, for example in lympho¬ cytes (Hadden and Goffey, 1982) . Johnson and Torres (1984) have shown that leukotrienes can mediate the helper cell requirements for IFN-₇. They further reported that cells that are rich sources of leukotrienes could play important roles in positive regulation of IFN-₇ production.

The production of IFN-₇ in first trimester trophoblast cul- tures di fer from the induction mechanism in T cells or T cell lines. IL-2 and TPA play a second messenger role for IFN-₇ production in T cells or T cell lines. IL-2 and TPA mobilize protein kinase C to the membranes of T cells (Farrar and Anderson, 1985) and increase cyclic GMP levels in cells and hence mediate IFN-₇ production.

Leukotrienes have been suggested to have a positive signal for regulation of IFN-₇ production (Johnson and Torres, 1984) , and therefore cells rich in leukotrienes may produce IFN-₇ when stimulated directly with PHA. The secretion of leukotrienes was therefore tested in first and third tri¬ mester trophoblast cultures. First trimester trophoblast cultures produced leukotriene B₄ (24-27 pg/ l) whereas no leukotriene B4 could be detected from cultures of third trimester trophoblast. Third trimester trophoblast cul- tures, however, did not produce IFN-₇. These results indi¬ cate that leukotrienes may mediate IFN-₇ production in first trimester trophoblast cultures.

EXAMPLE 8

Effect of trophoblast-IFN on the natural killer cell acti- vity against Herpes Simplex-1 infected cells.

In pregnancy, IFNs may accomplish a number of complex actions at the cellular level, not only in the immunologi- cal relationship between mother and foetus, but also in the defence mechanism against intrauterine infection of the foetus. IFNs are known as proteins which are induced by vertebrate cells as a response to a viral infection. They bind to receptors on the cell surface and stimulate an antiviral state, inhibit cell growth, induce cell differen¬ tiation, and stimulate the cytotoxic activity of natural killer (NK) cells.

NK cells represent a subset of lymphocytes having the abi- lity to lyse a variety of isogenic and allogenic virus- infected target and tumour cells in a MHC-non-restricted manner and in the absence of prior sensitization. The majority of peripheral blood lymphocytes mediating MHC- nonrestricted cytotoxicity express the CD56 antigen. These cells are thought to represent a first line of immunolo- gical defence against viral pathogens and tumour cells.

IFNs are known to enhance the cytotoxic activity of peri¬ pheral blood NK cells. The effect of trophoblast-IFN on the NK cell activity was therefore investigated.

Target cells

Term placentas from normal pregnancies (36 to 42 weeks of gestation) were obtained within 30 min of spontaneous vagi¬ nal delivery. Human villous trophoblasts were isolated as previously described (21) using immunomagnetic sorting. Villous tissue was dissected from the placenta, enzymati- cally dissociated and fractionated on gradient of Percoll (Pharmacia, Uppsala, Sweden) . The cells were then negative¬ ly sorted on a magnetic concentrator using immunomagnetic microspheres (Dyna-beads; Dynal A.S., Oslo, Norway) coupled to anti-HLA-ABC mAb (Dakopatts, Copenhagen, Denmark) . Fetal fibroblasts were harvested from the 45-50% Percoll band after 30 min of enzymatic treatment. Suspensions were ad¬ justed to 1.5 x 10⁶ cell/ml in RPMI (GIBCO, Grand Island, NY, USA) supplemented with 10% FCS. Cells were allowed to adhere to plastic dishes for approximately 2 h at 37°C and unattached cells were removed by washing six times with PBS. The monolayers were then exposed to 0.1% trypsin in PBS for 10 min at room temperature, and the removed fibro¬ blasts were washed twice with complete medium and then propagated in the same medium. Fibroblasts were used after 5-7 passages. The positive NK cell target control, K562 cells (human erythroleukemia cell line) , was obtained from the American Type Culture Collection, Rockville, MD, USA (ATCC) . Adherent target cells, trophoblasts and fibroblasts were plated in 96-well flat-bottomed microtiter plates (Nunc, Roskilde, Denmark) at 10⁴ cells per well in 100 μl and incubated for 24 h or 72 h before infection. Human herpes simplex virus type 1 (strain Mclntyre) was obtained from ATCC. Virus infection of target cells was done with a multiplicity of infection of 1 (units of the virus divided by the number of target cells) .

Isolation of peripheral blood lymphocyte effector cells

For the preparation of effector cells, buffy coats from healthy adult blood donors were used (Skejby Hospital,

Aarhus, Denmark) . The cells were centrifugated on a Ficoll- Hypaque gradient (Pharmacia) . The crude preparation of PBMC was washed and then used in a cellular cytotoxic assay at appropriate concentrations. For isolation of CD56⁺ NK cells with magnetic beads, the MACS separation system (Miltenyi Biotec, Sunnyvale, CA, USA) was used. First, PBMC were depleted of monocytes by allowing them to adhere to culture flask for 2 h. The non-adhering cells were then reacted with mouse antihuman CD56 mAb (Becton Dickinson, Monoclonal Center, Mountain View, CA, USA) for 30 min at 4°C. After washing, magnetic microbeads with conjugated antimouse IgG were added and incubated for 30 min. Finally, the cells were recovered using a magnetic separator. Isolated popula¬ tions were viable in excess of 95%, and their purity was higher than 96% CD56⁺, as shown by anti-CD56 staining and analysis with FACStar Plus Cytometer (Becton Dickinson) . All cell cultures were grown in RPMI-1640 containing 10% FCS, penicillin (100 U/ml) , streptomycin (100 μg/ml) , gentamycin (40 μg/ml) and 10 mM HEPES. IFN treatment of effector cells

Effector cells were incubated with tro-IFN 500 U/ml of com¬ plete medium at the concentration of lxlO⁶ cells/ml for 24 h. Thereafter the cells were used in the cytotoxic assay.

Natural killer cell cytotoxic assay

NK activity was measured using a ⁵¹Cr release assay. A 4 or 12 hour chromium release cytotoxicity assay was used. The target cell suspension was plated in a 96-well flat- bottomed tissue culture plate (NUNC, Denmark) at a cell density of 10⁴/well. After incubation for 24 h or 72 h the placental cells were infected with HSV-1 (Herpes Simplex Virus 1) or mock-infected. Cells were then washed and incubated 3 h at 37°C, and then labeled with 5 μCi of ⁵¹Cr (as NaCr0₄, Amersham, England) for 2 h in 40 μl of RPMI

1640. Cells were then washed 5 times and various numbers of effector cells were added to give different effector to target cell ratios in a final volume of 0.2 ml. The plates were centrifugated at lOOg for 2 min and incubated at 37°C in 5% C0₂. After 4 or 12 h of incubation plates were cen¬ trifugated at lOOOg for 5 min and 100 μl of supernatant was harvested from each well. The non-adherent positive NK cell target control, K562 was labeled with ⁵¹Cr 100 μCi/10⁶ cells. The cells were washed in culture media and plated in 96-well tissue culture plate (NUNC, Denmark) at 10⁴ cells/well in 100 μl of medium. Effector cells were then added. Radioactivity was counted in a gamma scintillation counter. All experiments were carried out in triplicate. Spontaneous release was less than 20% of maximum release. Maximum release was measured by lysing the ⁵¹Cr-labeled target cells with 5% Triton X-100. The percentage specific lysis was calculated as follows: Lysis ₍%₎= exp. com - spon. release cpm x 100 max. release cpm - spon. release cpm

Results

It was found that pretreatment of NK cells with tropho- blast-IFN alpha significantly enhanced the NK cell activity against the normally susceptible cell line K562 (figure 8) and against virus-infected fibroblast (figure 9) , but not against virus-infected term trophoblast (figure 10) . This suggests that tro-IFN like human leukocyte IFN enhances the cytotoxic activity of NK cells.

EXAMPLE 9.

Effect of trophoblast-IFN on the fetal natural killer cell activity against Herpes Simplex-1 infected cells.

The trophoblast cells were purified as described in example 8.

Lymphocytes.

Effector cells were isolated from cord blood and prepared by Ficoll-hypaque gradient centrifugation [Timonen 1980] . Because of erythrocyte contamination in the fetal blood, these were gradient centrifuged twice. After centrifugation the cells were purified for CD56 using monoclonal antibo¬ dies coupled to magnetic beads. The cells were incubated in 10% RPMI 1640 at the same conditions as the trophoblast. The cells were characterised by flow cytometry on FACstar plus Becton Dickinson using monoclonal antibodies against CD56, CD8, CD3 and CD19 from DAKO Denmark. K562

The K562 (flowter) was passed once weekly using 1640 RPMI 10% FCS.

Trophoblast interferon

Tro-IFN were produced by the method described by Mathiesen (Mathiesen et al. 1990) .

Interferon treatment

Effector cells were incubated with 500 IU/ml of tropho- blast-IFN beta for 24h, and target cells with lOOIU/ml.

Cytotoxic assay

The trophoblast were seeded out in flat-bottomed 96 well plates from NUNC Denmark, 10.000 cells/well in 100 μl and infected with HSV-1 using a multiplicity of infection on 1. Incubated for lhr and washed 3 times. Afterwards the cells were incubated in 51-Cr(Amersham) for 4 hours, washed 3 times in RPMI and resuspended in lOOul 10% RPMI. Effector cells were added in various dilutions thus obtaining effec- tor:target (E:T) ratios at 100:1, 50:1, 25:1 and 12:1. The total volume was 200ul. Spontaneous release was obtained from wells containg only cromium labeled trophoblast in 200ul of medium. Maximum release was assessed using lOOul of 2.5% Triton X-100. Spontaneous release was always below 20% of maximum. Because of the low number of effector cells which could be obtained the assay was run in duplicate. After 8h the plates were centrifugated at 2000g for 2 minutes, and lOOul was harvested from each well. The radio¬ activity was measured in a gamma counter (Perkin Elmer) . The cell lysis was calculated by the equation:

Lysis (%)= ex . cp - spon. release cpm _{χ 1QQ} max. release cpm - spon. release cpm Results

It was found that Tro-IFN treatment of NK-cells enhanced the cytotoxic activity against the NK susceptible K562 cell line, especially at higher effector target ratios (Figure 11) . Killing of autologous trophoblast was not seen after interferon treatment of the NK-cells (Figure 12) . This was not altered by infecting the trophoblast cells (Figure 13) . Only when both effector cells and HSV-1 in¬ fected target cells were treated with Tro-IFN a cytotoxic effect was seen, which, however, was lower than the effect on K562 cells (Figure 14) . The negative calculated specific lysis sometimes seen is due to ⁵¹Cr uptake of the NK-cells.

Discussion

It has been shown above that trophoblast interferons are able to enhance the cytotoxic activity of fetal NK cells. These results are comparable to results obtained with human leukocyte interferons. The enhancement is thought to occur at tree different levels. 1) better binding of NK cells to target cells and higher ratio of NK cells with cytotoxic abilities 2) quicker lysis 3) increase in the number of target cells one NK-Cell can lyse (Trinchieri at al. 1989) . The cytotoxic inertness of the trophoblast cell, even after HSV-1 infection and tro-IFN treatment of the NK-cells, is in line with results described by Petersen (Petersen et al. 1992) . However, it has been observed that double treatment i.e. HSV-l infected and IFN treated trophoblast and IFN- treated cord blood cells makes the trophoblast susceptible to killing. This result suggests that the fetus has an ability in fighting the infection and that tro-IFN, which is produced during pregnancy, has a special role in dealing with virus from mother to the foetus. EXAMPLE 10

Effect of tro-IFNs on PGE₂ secretion

Trophoblast cells, seeded at a density of 1 x 10⁶/ml, were incubated with 1000 IU/ml of purified tro-IFN-α or -β in (KGM) supplemented with 5% FCS at 37°C, 5% C0₂. Control cells were incubated under the same conditions without addition of IFN. Culture medium from IFN-treated and un¬ treated cultures was harvested at 12 h intervals. The con¬ centrations of PGE₂ in the culture medium was determined using PGE enzyme immunoassay kit (Cayman Chemical Company, MI, USA) according to the manufacturer's instructions.

Inhibition of PGE₂ secretion by tro-IFNs

In the absence of tro-IFNs, PGE₂ secretion by trophoblast cultures increased from 3.6 ± 0.44 pg/10⁶ cells/ml at 12 h incubation to 14.9 + 0.77 pg/10⁶ cells/ml at 72 h. 1000

IU/ml of tro-IFN-α and -β reduced the secretion of PGE in trophoblast cultures by 48.9% and 58.8%, respectively, as compared to the secretion by untreated cultures after 72 h of incubation.

Evidence has accumulated suggesting an immunomodulatory function for IFNs (Bocci, 1981; Minato et al., 1980; Metz, 1975) . At least one manner in which tro-IFN might be immu- noregulatory is by an effect on PG metabolism. Trophoblast as well as other placental cells have been demonstrated to produce arachidonic acid metabolites (Yagel et al., 1988; Papadogiannakis et al., 1985) with direct immunosuppressive effects. In particular, PGE has been implicated in the immunosuppression at the feto-placental interface (Papado¬ giannakis et al., 1985; Tawfik et al., 1986; Lala et al., 1989) . The tro-IFNs have a proven capacity to modulate PGE production in human trophoblast cultures. For example, tro- IFN-α and -β caused inhibition of PGE production by cul¬ tures trophoblast cells. This suggests that the tro-IFNs are potent local regulatory factors and that their actions on PG secretion may be one of their fundamental properties. More direct evidence derives from the observation that in¬ fection in pregnancy leads to an increased generation of prostaglandins by intrauterine tissues and can cause pre- term labour, apparently exerting the same functions as with normal for term labour (Mitchell et al., 1990). This and the high levels of IFN production by trophoblast stimulated with viruses suggest the notion that the tro-IFNs may regu- late prostaglandin production during infection in the feto- placental interface during pregnancy.

EXAMPLE 11

Human trophoblast interferons enhance MHC class I antigen expression in human trophoblast cultures

summary

The expression and regulation of major histocompatibility complex class I (MHC-I) antigens by virus-induced human trophoblast interferons (tro-IFNs) were examined in term of trophoblast cultures. Flow cytometry studies using fluores- cent monoclonal antibodies against MHC-I antigens revealed that isolated cytotrophoblasts can express MHC-I antigens. The expression of these antigens increased with stimulation of trophoblast cultures with tro-IFN-α and -β. The increa¬ sed expression of the MHC-I antigens by the tro-IFNs was dependent on the type (α or β ) and the dose of the IFN applied. Tro-IFN-^ (100 IU/ml) induced significantly higher levels of MHC-I antigens as compared to tro-IFN-α at 100 and 1000 IU/ml. The tro-IFN-enhanced expression of MHC-I antigens may be important by increasing the efficiency of local and viral antigen presentation and cytotoxicity by T-cell response and local inflammatory processes and thereby prevent virus spreading from mother to foetus. Introduction

The majority of the cells of the human trophoblast layer forming the interface between mother and foetus do not express major histocompatibility complex (MHC) antigens (Faulk and Temple, 1976; Faulk, 1983; Goodfellow et al., 1976) . Specifically, MHC antigens do not appear to be pre¬ sent on villous syncytiotrophoblasts or cytotrophoblasts in intact villous placental tissue. In contrast, some MHC class I (MHC-I) antigens have been detected in non-villous trophoblast cells (Redman et al., 1984) and early prolife¬ rating cytotrophoblasts (Butterworth et al., 1985). Fur¬ thermore, evidence that MHC antigens could be expressed in gestation is derived from studies of HLA gene regulation in trophoblast lineage cells where genes encoding MHC anti- gens appear to remain accessible and hence regulatable

(Feinman et al., 1987). The expression of MHC-I antigens by trophoblasts has been suggested to be biologically impor¬ tant since the expression of paternal MHC antigens by pla¬ centa enables it to absorb potentially harmful maternal antibodies directed against fetal histocompatibility anti¬ gen (Anderson and Berkowitz, 1985) . Furthermore, the ex¬ pression of MHC-I antigens by trophoblast cells may result in a better target for maternal cytotoxicity T cells since their action requires an autologous class I antigen signal in combination with foreign antigen (Anderson and Berko¬ witz, 1985; Zinkernagel and Doherty, 1985) . The absence or regulated expression of MHC antigens on fetal and chorionic tissues has therefor been suggested as one approach to ex¬ plain the phenomenon of the foetus to escape rejection (Feinman et al., 1987).

The expression of MHC antigens in many cell species has been demonstrated to increase in response to IFNs (Kato et al., 1991; Halloran et al., 1989; Feinman et al., 1987). For example, IFN has been shown to enhance the expression of MHC class I antigens in murine lymphoid cells. Specifi¬ cally, IFN-₇ produced by lymphocytes has been demonstrated to enhance the expression of MHC-I antigens in cultured human trophoblasts (Feinman et al. , 1987), choriocarcinoma cells (Anderson and Berkowitz, 1985; Kato et al. , 1991) and human amnion cells (Hunt and Wood, 1986) .

In the present studies, the effect of the local virus- induced tro-IFNs on the expression of MHC-I antigens at the cell surface in cultured term trophoblasts was investigated using fluorescence-activated cell sorter (FACS) . The obser¬ vations indicate that virus-induced tro-IFN-α /β enhances the expression of MHC-I antigens in normal cytotrophoblast cells in vivo.

Materials and Methods

Trophoblast culture and IFN treatment

Human term trophoblast cells were isolated from term pla- centas as described in Example 2. The purity of these cells was 99.9% as tested with FACStar Plus Flow Cytometer (Bec¬ ton Dickinson Immunocytometry Systems, Mountain View, CA) using fluorescin-conjugated monoclonal antibody to human cytokeratin (Dakopatts, Denmark) . Virus-induced tro-IFNs were produced and purified as described above. Trophoblast cells were seeded out in 25 cm² culture bottles at a den¬ sity of 4 x 10⁵ cells/cm² using 5 ml of RPMI 1640 supple¬ mented with 10% foetal calf serum (FCS) and 100 or 1000 IU/ml of purified tro-IFN-α or -β.

Antibodies specific to human MHC class I antigens (W6/32) were obtained from Dakopatts. Unspecific mouse IgG (Dako¬ patts) was used as a negative control for the flow cyto¬ metry method. FITC-labelled polyclonal rabbit anti-mouse IgG (Dakopatts) was used as secondary antibody for the double, layer technique. Analysis by flow cytometry

Tropphoblast-IFN treated and untreated cells were removed from culture bottles every 12 h after the addition of IFN by brief trypsination followed by resuspension and washing with PBS. A suspension of 5 x 10⁵ cells was incubated with 5 ml of relevant monoclonal antibody in 1 ml of PBS supple¬ mented with 1% foetal calf serum at 4°C, washed twice and then incubated with secondary antibody. Controls were incu¬ bated with unspecific mouse IgG as primary antibody. Cells were analyzed using FACStar Plus Flow Cytometer (Becton Dickinson) . Argon laser 488 nm was used for the detection of FITC fluorescence. The fluorescence intensity was dis¬ played as a fluorescence histogram. The mean fluorescence was calculated using Lysis 2 software (Becton Dickinson) .

Results

Under the present in vitro condition, human placental cyto¬ trophoblasts can express the MHC-I antigen. Addition of virus-induced tro-IFN-α and -β enhanced the expression of the MHC-I proteins (Figures 15 and 16) . The expression was, however, dependent on the type (α or β ) of tro-IFN used in the stimulation. The relative increase in the fluorescence of IFN-stimulated and unstimulated trophoblasts labeled with monoclonal antibodies W6/32 is shown in Table 10. There was a twofold increase in the fluorescence intensity at 36 hours after treatment of the trophoblast cultures with 1000 IU/ml of tro-IFN-α. Stimulation of the tropho¬ blast cultures with 100 IU/ml of tro-IFN-α showed only 2-5% increase in the expression of MHC-I antigens as compared to the untreated cultures (Figure 16) . TABLE 10

Effects of tro-IFN-α and -β on the expression of MHC-1 antigens on trophoblast

Time Sfter IFN Relative increase in flourescence after treatment (h) treatment with

The kinetics of the expression of MHC-I antigens on tro- IFN- stimulated cultures were different from cultures

20 treated with tro-IFN-α. 100 IU/ml of tro-IFN-^ showed greater than 800% increase in the expression of MHC-I at 24 h after stimulation (Figure 15) . The expression, how¬ ever, decreased and was similar to the control at 36 h after stimulation. 1000 IU/ml of tro-IFN-^ enhanced the

25 expression of the MHC-I antigens by 200% at 24 h after stimulation. The expression extended to 72 h after stimula¬ tion (Figure 15) .

Discussion

The present data suggest a local immunologic role of enhan- 30 cing the expression of MHC class I expression. Modulation of MHC-I locally by virus-induced trophoblast IFNs may be relevant to i-mmunological processes in vivo, since quanti- tative expression of MHC class I antigens affects T cell recognition, viral antigen presentation (Unanue et al., 1984; Matis et al., 1983) and susceptibility of infected cells to cellular cytotoxicity (Flyer et al., 1985). One possible in vivo role of tro-IFN is probably the transient enhancement of MHC class I expression as part of the nor¬ mally but tightly controlled maternal immune response to the foetus (Faulk and Hunt, 1989) . It has been reported that treatment of animals with high doses of IFN-7 may participate in abortion (Mattsson et al., 1991).

It is likely that IFNs and MHC class I expression play a role in patients suffering from recurrent spontaneous abortion.

Another possible role of trophoblast interferons may be enhancement of MHC class I expression on trophoblast cells during viral infections. This may increase the chances for viral proteins to occupy the antigen binding groove on MHC class I molecules, facilitating T cell response and local inflammatory processes, thereby preventing virus spreading from mother to foetus.

EXAMPLE 12

Effect of trophoblast FN-ff on the proliferation of chorio¬ carcinoma cells in vitro

The choriocarcinoma cell lines JAR (ATCC HTB 144) and BeWo (ATCC CCL 98) are malignant tumours of trophoblast cells comprising a mixed population of cellular intermediates that are in different stages of differentiation (Hoshina et al. , 1983; Bagshawe, 1969; Hertz, 1978). In contrast to the normal cytotrophoblasts, the JAR and BeWo cells divide rapidly in culture. The antiproliferative effect of the human trophoblast interferon beta on the proliferation choriocarcinoma cell lines JAR and BeWo in vitro is tested as described below.

JAR and BeWo cells were cultured in RPMI 1640 + 10% fetal calf serum at a concentration of 5 x 10⁴ cells per well in the presence or absence of 100 and 1000 IU/ml of tro-IFN-j9. At 24 h intervals cells were washed with phosphate buffer saline and culture medium were reconstituted. ³H-Thymidine (6.7 Ci/mmol) at a concentration of 1 mCi per well was added to the cultures for 4 h. Proliferative response was evaluated from beta counts of cells harvested at the end of the inoculation period. The percent inhibition of prolife¬ ration was calculated according to the following formula:

% inhibition of proliferation = 1- (CPM of sample/CPM of control) X 100

Results

Tro-IFN- inhibited the proliferation of JAR and BeWo cell lines (Figure 17 and 18 and Tables 11 and 12).

Table 11

Antiproliferative effects of tro-IFN-^ on JAR cells

Incubation time % inhibition of proliferation of (days) JAR cells after treatment with tro-

IFN-0

100 IU/ml 1000 IU/ml

10 44.3 18.1 51.4

11 39.0 Table 12

Antiproliferative effects of tro-IFN-S on BeWo cells

Incubation time % inhibition of proliferation of (days) BeWo cells after treatment with tro-IFN-^

100 IU/ml 1000 IU/ml

1 64 66 2 10 57 3 18 47

EXAMPLE 13

Immunosuppressive (antiproliferative) effects of human placental trophoblast interferon on lymphocytes in vitro

The immunosuppressive (antiproliferative) effects of human placental trophoblast interferon (tro-IFN) on mitogen sti¬ mulated human T and B lymphocyte populations were investi¬ gated in vitro.

Preparation of lymphocytes

Human peripheral blood was obtained from Aarhus University Hospital (Denmark) and mononuclear lymphocytes were iso¬ lated by gradient centrifugation with Ficoll-Paque as de¬ scribed at the WHO Workshop, 1974. The intermediate band containing peripheral blood mononuclear cells was harvested and washed in phosphate buffered saline (PBS) .

CD4⁺ and CD8⁺ lymphocytes were isolated from the resulting mixture by adding mouse anti-human monoclonal antibodies against CD4 and CD8 (Becton Dickinson) and afterwards coupling these with magnetic beads coated with sheep anti- mouse monoclonal antibodies (Dynal A/S, Norway) . The purity of the CD4⁺ and CD8⁺ lymphocyte populations was greater than 95% as tested with FACStar Plus Flow Cytometer (Becton Dickinson Immunocytometry Systems, Mountain View, CA) using FITC-conjugated monoclonal antibody to CD4 and CD8 (Becton Dickinson) . The cells were washed in RPMI 1640 and resus- pended to a final concentration of 1 x 10⁶ viable cells/ml in RPMI 1640 supplemented with 10% fetal calf serum (FCS) .

PHA-induced proliferation of crude T, CD4⁺ and CD8⁺ lympho¬ cytes

Lymphocytes (5.0 x 10⁴) were seeded in 50 μl of culture medium supplemented with different concentrations of PHA (0, 1, 5, 10 and 20 μl) in 96-well microtiter plates. Cul¬ tures were maintained at 37°C in a 5% C0₂ humid atmosphere. At different time periods (24, 48, 72 and 96 hours) after PHA-stimulation, the cultures were pulsed with [³H]-thymi- dine (1 μg/ml with a specific activity of 2 Ci/mmol, Amer¬ sham International) for 4 hours. The cells were harvested and absorbed on a filter disc. The filters were washed in 10% TCA, 5% TCA and finally dried in 99.9% ethanol, and transferred to plastic beta-vials. 2 ml of scintillation fluid was added to the vials and radioactivity was then assessed using a liquid scintillation counter.

To determine the effect of tro-IFN-θ on the proliferation of T-lymphocytes, cultures were supplemented with PHA

(1 μl/ml) and different concentrations of tro-IFN-^. Crude T-lymphocytes were stimulated with 10 IU/ml, 100 IU/ml and 1000 IU/ml and the separated lymphocytes with 100 IU/ml, 500 IU/ml and 1000 IU/ml of trophoblast interferon activi¬ ty. Cultures were pulsed at different periods (24, 48, 72 and 96 hours after stimulation) with [³H]-thymidine (1 μg/ml with a specific activity of 2 Ci/mmol, Amersham International) for 4 hours and the incorporation of radio¬ activity was assessed by scintillation counting.

Pokeweed mitogen stimulation of B-lymphocytes under in¬ fluence of tro-IFN-^

Pokeweed mitogen preferentially stimulates B-cell prolife¬ ration; however, there is a requirement for the presence of T-cells to optimize the stimulatory effect (Ling, 1976, and Hume, 1980) . Therefore, a crude lymphocyte population was used, seeded out in 10% RPMI 1640 supplemented with 5 μg/ml of pokeweed mutagen and various concentrations of tro-IFN-^ (10 IU/ml, 100 IU/ml and 1000 IU/ml). The proliferation was assessed by the uptake of [³H]-thymidine using the same pulsing hours and applying the same method as described above. Results

Table 13

Suppressive effects of tro-IFN- on PHA-stimulated

T-lymphocytes

Incubation time % suppression* of T-lymphocytes (days) after treatment with tro-IFN-9

10 IU/ml 100 IU/ml 1000 IU/ml

1 2 3 4

* % suppression as compared to untreated T-lymphocytes

Table 14

Suppressive effects of tro-IFN- on CD4⁺ T-lymphocytes

Incubation time % suppression* of CD4⁺ T-lympho¬ (days) cytes after treatment with tro-IFN- β

100 IU/ml 500 IU/ml 1000 IU/ml

1 2 3

4 5

* % suppression as compared to untreated CD4⁺ T-lymphocytes

Table 15

Suppressive effects of tro-IFN-^ on CD8⁺ T-lymphocytes

Incubation time % suppression* of CD8⁺ T-lympho- (days) cytes after treatment with tro-IFN- β

100 IU/ml 500 IU/ml 1000 IU/ml

1 2 3 4 5

* % suppression as compared to untreated CD8⁺ T-lymphocytes

Table 16

Suppressive effects of tro-IFN-^ on PWM stimulated B-lymphocytes

Incubation time % suppression* of B-lymphocytes (days) after treatment with tro-IFN-y9

10 IU/ml 100 IU/ml 1000 IU/ml

1 2

3 4

suppression as compared to untreated B-lymphocytes Tro-IFN-^ inhibited PHA-activated proliferation of human T- lymphocytes (Figure 19) . The inhibitory effect of tro-IFN upon crude T-lymphocytes started at day 1 and reached a peak at days 3 and 4 (Table 13) . Overall, the proliferation was inhibited in a dose dependent manner. The inhibition observed at 10 IU/ml was from 5% to 27%. At 100 IU/ml the effect ranged from 36% to 55%. The inhibition increased to 73% by using 1000 IU/ml (Figure 19 and Table 13) .

To examine whether the inhibition observed with respect to crude lymphocytes covered any difference between different subpopulations, T-lymphocytes were separated into CD4⁺ and CD8⁺. At 100 IU/ml of tro-IFN-S, CD4⁺- and CD8⁺-lymphocyte proliferation was suppressed by 55-59% and 37-47%, respec¬ tively, of the control values (Figures 20, 21, 23 and 23) . At 1000 IU/ml of tro-IFN-0, the suppression of the CD4⁺- and CD8⁺-lymphocyte proliferation was 62-74% and 67-82%, respectively, of the control values (Tables 14 and 15) . In both subpopulations of the T-lymphocytes, the extent of inhibition was concentration dependent.

B-lymphocyte proliferation induced by pokeweed mitogen was suppressed by tro-IFN-j9 (Figure 24) . The suppression was observed from day 2. At 10 IU/ml and 100 IU/ml, the inhibi¬ tion ranged from 20% to 56% and from 26% to 67%, respec¬ tively (Table 16) . The highest concentration (1000 IU/ml) gave a response similar to 100 IU/ml.

Discussion

Several mechanisms have been suggested to explain the survival of the fetal allograft. Local immunoregulation is one of the processes attributed to the success of this phenomenon. Decidual cells, placental cells (trophoblasts) and endometric cells have all been reported to play an immunoregulatory role at the feto-maternal tissue interface via such products as hormones (progestrone) , prostaglandins (PGs) and other suppressor molecules. The data presented here show the suppression of maternal T- and B-lymphocytes by tro-IFN. This suggests that tro-IFNs play a local role in the immunosuppression in the feto-placental interface during pregnancy and may account for the successful sur- vival of the foetus.

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Claims

1. An isolated interferon protein which is identical or substantially identical to a human interferon protein produced by a human trophoblast cell and being selected from the group consisting of:

a) a -interferon which is produced by a first trimester or term trophoblast cell or a trophoblast cell derived from a provoked vaginal delivery, which trophoblast cell is a trophoblast which is not bound by magnetic beads carrying immobilized antibodies to the tissue types MHC-l, A, B, or C,

the 0-interferon being obtainable in purified form from a filtered (0.22 μm filter) supernatant of a stimulated culture of the trophoblast cells, by a purification scheme comprising either 1) High-perfor¬ mance dye-ligand affinity chromatography followed by applying the thus obtained interferon-containing fractions to High-performance immunoaffinity chromato¬ graphy using immobilized ant i-β-interferon antibodies or 2) High-performance dye-ligand chromatography followed by Reversed phase HPLC, the β-interferon possessing at least one of the fol¬ lowing characteristics i-iv:

i) the -interferon in purified form appears substantially as only one silver-stainable band showing antiviral activity with a molecular weight in the range of 22-26 kDa, in a slab SDS-PAGE, applying an amount of β- interferon of 40.000 IU, under reducing conditions using 5% 2-mercaptoethanol or under non-reducing conditions without using 2-mercaptoethano1, ii) the antiviral activity of the β-interferon, as measured by inhibition of vesicular stomatitis virus plaque formation in cells of the human amniotic cell line WISH (ATCC, CCL 25) , is retained in a pattern which resembles the specific pattern of antiviral activity to an extent of about 55% of the initial value of antiviral activity after 3 hours at a temperature of 37°C, and to an extent of about

45-55%, in particular about 48-52%, after 10 minutes at a temperature of 56°C,

38-47%, in particular about 41-45%, after 15 minutes at a temperature of 56°C,

0-10%, in particular about 1-5%, after 60 minutes at a temperature of 56°C,

iϋ) the β-interferon substantially retains its anti¬ viral activity after storage in 0.1 M glycine at pH 2 for 24 or even 48 hours, as measured by inhibition of the plaque formation in human amniotic cell line WISH caused by vesicular stomatitis virus (VSV) , Indiana strain,

an α_j-interferon which is produced by a term trophoblast cell or a trophoblast cell from a provoked vaginal delivery, which trophoblast cell is a trophoblast which is not bound to magnetic beads carrying immobilized antibodies to the tissue types MHC-1, A, B, or C,

the α_j-interferon being obtainable in purified form from a filtered (0.22 μm filter) supernatant of a stimulated culture of the trophoblast cells by a purification scheme comprising either 1) High-perfor¬ mance dye-ligand affinity chromatography followed by applying the thus obtained interferon-containing fractions to High-performance immunoaffinity chromato¬ graphy using immobilized anti-αχ-interferon antibodies or 2) High-performance dye-ligand chromatography followed by Reversed phase HPLC,

the α-_j--interferon possessing at least one of the following characteristics i-iii:

i) the α_j-interferon in purified form appears sub¬ stantially as only one silver-stainable band showing antiviral activity with a molecular weight in the range of 15-20 kDa in a slab SDS-

PAGE, applying an amount of α_j-interferon of about 45.000 IU, under reducing conditions using 5% 2-mercaptoethanol or under non-reducing condi¬ tions without using 2-mercaptoethanol,

ii) the α_j-interferon substantially retains its antiviral activity after storage in 0.1 M glycine at pH 2 for 24 hours measured by inhibition of the plaque formation in human amniotic cell line WISH (ATCC) caused by vesicular stomatitis virus

(VSV) , Indiana strain,

iii) the α_j-interferon is bound to the High perfor¬ mance dye-ligand chromatography column by elec¬ trostatic forces as evidenced by its ability to be displaced from the column by the ionic salt NaCI.

An α _j-interferon which is produced by a first trimester or term trophoblast cell or a tropho- blast cell derived from a provoked vaginal deli¬ very, which trophoblast cell is a trophoblast which is not bound by magnetic beads carrying immobilized antibodies to the tissue types MHC-1, A, B, or C,

the α_jj-interferon being obtainable in purified form from a filtered (0.22 μm filter) supernatant of a stimulated culture of the trophoblast cells by a purification scheme comprising either 1) High-performance dye-ligand affinity chromato- graphy followed by applying the thus obtained interferon-containing fractions to High-perfor¬ mance immunoaffinity chromatography using immo¬ bilized anti-α_j -interferon antibodies or 2) High-performance dye-ligand chromatography fol- lowed by Reversed phase HPLC,

i) the α_jj-interferon in purified form appears substantially as only one silver-stainable band showing antiviral activity with a molecular weight in the range of 20-24 kDa, in a slab SDS-PAGE, applying an amount of α_j -interferon of about 45.000 UI, under reducing conditions using 5% 2-mercaptoetha- nol or under non-reducing conditions without using 2-mercaptoethanol,

ii) the α_j -interferon substantially retains its antiviral activity after storage in 0.1 M glycine at pH 2 for 24 hours measured by inhibition of the plaque formation in human amniotic cell line WISH (ATCC) caused by vesicular stomatitis virus (VSV) , Indiana strain,

ϋi) the α_jj-interferon shows a hydrophobic nature as indicated by it requiring a concentration of 35- 40% of the hydrophobic eluent ethylene glycol in 0.02 M sodium phosphate buffer pH 7.2 containing 1.0 M NaCI to be eluted from the High performance dye-ligand affinity chromatography column,

d) a ₇-interferon which can be obtained from a first trimester trophoblast cell from a provoked vagi¬ nal delivery, which trophoblast cell is a tropho¬ blast which is not bound bymagnetic beads carry- ing immobilized antibodies to the tissue types MHC-1, A, B, or C,

or which can be obtained from a first trimester tro¬ phoblast cell from a provoked vaginal delivery, which trophoblast cell is isolated from a first trimester placenta using differential trypsinization periods

the ₇-interferon being obtainable in purified form from a stimulated culture of the trophoblast cells by a purification scheme comprising High-performance dye- ligand affinity chromatography followed by binding to immobilized Concanavalin A and subsequent displacement with a sugar and then controlled pore glass affinity chromatography, or by a purification scheme comprising controlled-pore glass (mesh size, 120/200, CPG Inc., N.J.) affinity chromotography followed by binding to immobilized Concanavalin A and subsequent displacement with a sugar and then high-perfomance dye-ligand affi¬ nity chromatography or reversed-phase high-performance liquid chromatography, the ₇-interferon not _retaining its antiviral activity after storage in 0.1 M glycine at pH 2 for 24 hours measured by inhibition of the plaque formation in human amniotic cell line WISH (ATCC) caused by vesicu- lar stomatitis virus (VSV) , Indiana strain,

e) an interferon which is produced by a trophoblast cell derived from abortion placentas of 5 to 12 weeks of age, which trophoblast cell is a tropho¬ blast which is not bound by magnetic beads carry- ing immobilized antibodies to the tissue types MHC-1, A, B, or C,

or or which can be obtained from a first trimester trophoblast cell from a provoked vaginal delivery, which trophoblast cell is isolated from a first tri¬ mester placenta using differential trypsinization periods,

the interferon being obtainable in purified form from the supernatant of a stimulated culture of the tropho¬ blast cells by High-performance dye-ligand affinity chromatography,

the interferon not pertaining to any of the interferon classes β, α_j, α and ₇.

2. An interferon according to claim 1 wherein the tropho¬ blast cell from which it can be produced is a villous trophoblast.

3. An interferon protein according to claim 1, which is a glycoprotein.

4. A _/9-interferon according to claim 1 a) which in purified form appears substantially as only one silver-stainable band showing antiviral activity with a molecular weight in the range of 23-25 kDa, determined as described in claim la) .

5. A -interferon according to claim 1 a) which in purified form appears substantially as only one silver-stainable band showing antiviral activity with a molecular weight of 24 kDa, determined as described in claim la) .

6. A ^-interferon according to any of claims 1 a) , 4 and 5, the antiviral activity of said β-interferon, as measured by inhibition of vesicular stomatitis virus plaque formation in cells of the human amniotic cell line WISH (ATCC, CCL 25) , is retained in a pattern which resembles the specific pattern of antiviral activity of -interferon stored for 1 hour at 37°C to an extent of about

16% after treatment for 1 hour i a solution of 1% SDS (sodium dodecyl sulphate) at 37°C

64% after treatment for 1 hour in a solution of 1% (w/v) SDS, 1% (v/v) _/5-ME (/---mercaptoethanol) and 5 M urea at 37°C,

1.6% after treatment for 1 hour in a solution of 1% (v/v) β-KE and 5 M urea at 37°C,

0.8% after storage for 1 minute at 100°C,

16% after treatment for 1 minute in a solution of 1% (w/v) SDS 100°C,

64% after treatment for 1 minute in a solution of 1% (w/v) SDS, 1% (v/v) ø-ME and 5 M urea at 100°C

19.2% after treatment for 1 minute in a solution of 1% (v/v) jS-ME and 5 M urea at 100°C,

7. A β-interferon according to any of claims 1 a) , 4 and 5, the J-interferon showing a concentration dependent inhibi¬ tory effect on the proliferation of crude human T-lympho¬ cytes, CD4+-lymphocytes and CD8+-lymphocytes grown in cul- ture at 37°C and a 95%/5% 0 /C0 humid atmosphere, the proliferation of said lymphocytes being induced by phyto- hemagglutinin, the inhibitory effect on proliferation of T-lymphocytes being

in the range 5%-27% at a concentration of 10 IU/ml of β-interferon,

in the range 36%-55% at a concentration of 100 IU/ml of β-interferon,

about 73% at a concentration of 1000 IU/ml of ^-inter¬ feron,

the inhibitory effect on proliferation of CD4+-lympho- cytes being

in the range 55%-59% at a concentration of 100 IU/ml of β-interferon,

in the range 62%-74% at a concentration of 1000 IU/ml of -interferon,

and the inhibitory effect on proliferation of CD8+- lymphocytes being

in the range 37%-47% at a concentration of 100 IU/ml of -interferon and,

in the range 67%-82% at a concentration of 1000 IU/ml of β-interferon,

8. A 9-interferon according to any of claims 1 a) , 4 and 5, said β-interferon showing a concentration dependent inhibi- tory effect on the proliferation of crude human B-lymphocy¬ tes grown in culture at 37°C and a 95%/5% 0₂/C0 humid atmosphere, the proliferation of said B-lymphocytes being induced by poke weed mutagen, the inhibitory effect being

in the range 20%-56% at a concentration of 10 IU/ml of β-interferon and,

in the range 26%-67% at concentrations of 100 IU/ml and of 1000 IU/ml of -interferon,

9. A β-interferon according to any of claims 1 a) , 4 and 5, said -interferon substantially retaining its antiviral activity after storage in 0.1 M glycine at pH 2 for 1 week at 4°C, as measured by inhibition of the plaque formation in human amniotic cell line WISH caused by vesicular stoma¬ titis virus (VSV) , Indiana strain.

10. An α_j-interferon according to claim l b), 2, 4 and 5 which in purified form appears substantially as only one silver-stainable band showing antiviral activity with a molecular weight in the range of 15-17 kDa, determined as described in claim lb) .

11. An α_j-interferon according to claim 1 b) , which in purified form appears substantially as only one silver- stainable band showing antiviral activity with a molecular weight of 16 kDa, determined as described in claim lb) .

12. An α_jj-interferon according to claim 1 c) , which in purified form appears substantially as only one silver- stainable band showing antiviral activity with a molecular weight in the range of 21-23 kDa, determined as described in claim 1 c) .

13. An α_j -interferon according to claim 1 c) , which in purified form appears substantially as only one silver- stainable band showing antiviral activity with a molecular weight of 22 kDa, determined as described in claim 1 c) .

14. A human trophoblast cell-produced compound which will be present together with β-interferon according to claim l, such as in fractions 55-70 in Figure 1 under the conditions described in connection with Figure 1, which compound is not bound to anti-^-interferon antibody, which compound has cytotoxic effect as determinable by standard methods.

15. An interferon according to claim 1, selected from β- interferon according to claim l a), α -interferon according to claim 1 b) , α _j-interferon according to claim 1 c) , and ₇-interferon according to claim 1 d) , which has antiproli¬ ferative effect, as determinable by standard methods.

16. A β-interferon protein according to claim 1 a) , which is capable of protecting human cell lines WISH (ATCC) , GM

2504 and GM 2767 trisomy 21 fibroblasts (Cell Repository, Cambden, N.J., U.S.A.) against an infection with vesicular stomatitis virus (VSV) , Indiana strain, but is not capable of protecting bovine cell line MBDK against a vesicular stomatitis virus (VSV) , Indiana strain, infection

17. An α_j-interferon protein according to claim 1 b) , which is capable of protecting the human cell line GM 2767, trisomic 21 fibroblasts (Cell Repository, Cambden, N.J. , USA) against a viral infection with vesicular stomatitis virus (VSV) , Indiana strain, and which is capable of pro¬ tecting the bovine cell line MDBK against a viral infection with vesicular stomatitis virus (VSV) , Indiana strain, to a higher extent than it protects the human cell line.

18. An α_jj-interferon according to claim 1 c) , which is capable of protecting the human cell lines WISH (ATCC) , GM 2504 and GM 2767 trisomic 21 fibroblasts (Cell Repository, Cambden, N.J., USA) against a viral infection with vesicu¬ lar stomatitis virus (VSV) , Indiana strain, and which is capable of protecting the bovine cell line MDBK against a viral infection with vesicular stomatitis virus (VSV) , Indiana strain, to a higher extent than it protects the human cell line WISH

19. An interferon protein according to claim 1, which is in substantially pure form.

20. A β-interferon protein according to claim 1 a), which has a purity of at least 95%, as measured by densitometric scanning of a Comassie Blue gel at 595 nm.

21. A β-interferon protein according to claim l a), which has a purity of at least 99%, as measured by densitometric scanning of a Comassie Blue gel at 595 nm.

22. A -interferon protein according to claim 1 a) , which, when having been subjected to high performance dye-ligand affinity chromatography, has a purity of at least 95%, as measured by densitometric scanning of a Comassie Blue gel at 595 nm.

23. A /3-interferon protein according to claim 1 a), which, when having been subjected to high performance dye-ligand chromatography and subsequent high performance immunoaffi¬ nity chromatography using immobilized anti-α_j-interferon antibodies, has a purity of at least 99%, as measured by densitometric scanning of a Coomassie Blue gel at 595 nm.

24. A -interferon protein according to claim 1 a), which has a specific activity of at least about 1.0 x 10⁸ IU/mg of protein.

25. An α_j-interferon according to claim 1 b) , which has a purity of at least 95% as measured by densitometric scan¬ ning of a Coomassie Blue gel at 595 nm.

26. An α_j-interferon according to claim 1 b) , which has a purity of at least 99% as measured by densitometric scan¬ ning of a Coomassie Blue gel at 595 nm.

27. An α_j-interferon according to claim 1 b) , which, when having been subjected to high performance dye-ligand affi¬ nity chromatography, has a purity of at least 95% as measu¬ red by densitometric scanning of a Coomassie Blue gel at 595 nm.

28. An α_j-interferon according to claim 1 b) , which, when having been subjected to high performance dye-ligand chro¬ matography and subsequent high performance immunoaffinity chromatography using immobilized anti-α -interferon anti¬ bodies, has a purity of at least 99% as measured by densi¬ tometric scanning of a Coomassie Blue gel at 595 nm.

29. An α_jj-interferon according to claim 1 c) , which has a purity of at least 95% as measured by densitometric scan¬ ning of a Coomassie Blue gel at 595 nm.

30. An α_jj-interferon according to claim 1 c) , which has a purity of at least 99% as measured by densitometric scan- ning of a Coomassie Blue gel at 595 nm.

31. An α_j-interferon according to claim 1 c) , which, when having been subjected to high performance dye-ligand affi¬ nity chromatography, has a purity of at least 95% as measu¬ red by densitometric scanning of a Coomassie Blue gel at 595 nm.

32. An α_j-interferon according to claim 1 c) , which, when having been subjected to high performance dye-ligand chro¬ matography and subsequent high performance immunoaffinity chromatography using immobilized anti-α -interferon anti- bodies, has a purity of at least 99% as measured by densi¬ tometric scanning of a Coomassie Blue gel at 595 nm.

33. A composition containing a mixture of two or more of the interferon proteins according to claim 1 in purified form.

34. A method for producing an interferon selected from 7- interferon, α_j-interferon, and α_j__j-interferon from human trophoblasts, comprising cultivating the human trophoblast, stimulating the culture with an agent capable of inducing the production of an interferon selected from ₇-interferon, α_j-interferon, and α_j -interferon, or for producing an interferon or interferons selected from -interferon, αjo¬ interferon and α τ_-interferon and ₇-interferon from human trophoblasts, comprising cultivating the human trophoblast, stimulating the culture with an agent capable of inducing the production of an interferon selected from β-interferon, α_j-interferon, α_jj-interferon and ₇-interferon, and isola¬ ting the interferon or interferons from the culture.

35. A method according to claim 1, in which the stimulating agent is selected from virus, synthetic double stranded RNA, cytokines such as interleukins, plant mitogens, and growth factors.

36. A method according to claim 34 for producing ₇-inter¬ feron, in which the stimulating agent is a plant mitogen and/or a cytokine.

37. A method according to claim 36, in which the stimula- ting agent is a combination of phytohaemagglutinin and interleukin-2.

38. A method according to claim 37 in which the stimulating agent is a combination of 50-200 U/ml of interleukin 2 together with 3-10 μg/ml of phytohaemaglutinin.

39. A method according to claim 35, in which the stimula¬ ting agent is selected from Concanavalin A, lipopolysac- charide, pock wheat mitogens and

state-13-α-acetate

40. A method for producing a human trophoblast interferon, comprising cultivating human trophoblast cells under an oxygen-containing atmosphere, stimulating the culture with an agent capable of inducing the production of interferon, and obtaining interferon from the culture.

41. A method according to claim 40, wherein the oxygen- containing atmosphere is nitrogen with 2-10% of oxygen.

42. A method of producing an interferon protein according to claim 1 from trophoblast cells in a culture which, in addition to the trophoblast cells, contains other cells and which grows in a culture attached or unattached to a sur¬ face, comprising treating the culture with a calcium bind- ing chelator, isolating the cells thereby detached from the surface, culturing the thus isolated cells, stimulating the resulting trophoblast cell culture with an agent capa¬ ble of stimulating an interferon production, and obtaining interferon protein from the culture.

43. A method according to claim 42, wherein the interferon protein is ₇-interferon as defined in claim 1, and the culture is a culture obtained from a provoked first trimes¬ ter vaginal delivery.

44. A method according to claim 43, in which the vaginal tissue which has been subjected to a separation is placen¬ tal tissue which is treated enzymatically followed by pooling and gradient centrifugation.

45. A method according to claim 42, in which the tempera¬ ture of the culture is about 0°C during the treatment with the calcium binding chelator.

46. A method according to claim 42, in which the calcium binding chelator is EDTA.

47. A method according to claim 46 in which the concentra¬ tion of the EDTA is about 0.02%.

48. A method according to claim 42, in which the isolated cells are reseeded on a fresh culture medium.

49. A method for isolating and purifying an interferon produced by a human trophoblast cell culture, comprising subjecting supernatant from the culture to affinity chroma- tography and obtaining, from the eluate from the chromato¬ graphy, the fractions containing interferon activity.

50. A method according to claim 49, wherein the affinity chromatography is a high performance affinity chromato¬ graphy selected from high performance dye-ligand control pore glass affinity chromatography, high performance con¬ canavalin A affinity chromatography and high performance immunoaffinity chromatography.

51. A method according to claim 50 wherein the affinity chromatography is a combination of at least two of the affinity chromatography treatments to give a tandem high performance affinity chromatography.

52. A method according to claim 51 in which the affinity chromatography is a combination of high performance dye ligand chromatography and high performance immunoaffinity chromatography.

53. An antibody raised against, or directed substantially only against, an interferon according to claim 1.

54. An antibody according to claim 53 which is a monoclonal antibody.

55. An antibody according to claim 53 which is a polyclonal antibody.

56. A method of producing an antibody according to claim 53, which comprises immunizing an animal with an interferon according to claim 1 to obtain cells producing an antibody specific for the interferon and fusing the cells with cells of cell line capable of rendering the fused cells immortal, and selecting and cloning the resulting hybridoma cells producing the antibody, or immortalizing an unfused cell line producing the antibody, followed by growing the cells in a medium to produce the antibody, and harvesting the antibody from the growth medium.

57. A method of producing an antibody according to claim 53, which comprises immunizing an animal with an interferon according to claim 1, and obtaining serum containing anti¬ bodies specific for the interferon from the animal.

58. A method according to claim 57, in which the immunized animal is a mammal selected from the group consisting of rabbit, monkey, sheep, goat, mouse, rat, pig, horse, and guinea pig.

59. A method according to claim 57, in which the immunized animal is a mammal selected from the group consisting of rabbit, monkey, sheep, goat, mouse, rat, pig, horse, and guinea pig.

60. A method according to claim 56, in which the cells producing the antibody are spleen or lymph cells.

61. A method according to claim 56, in which the hybridoma cells are grown in vitro or in a body cavity of an animal such as a mouse.

62. A method for inhibiting tumoral growth or metastatic processes in a warm-blooded animal such as a human, com- prising administering an effective amount of an interferon according to claim 1 or a mixture of such interferons to an animal, the interferon preferably being selected from an α- interferon, or a mixture of an α- and a ₇-interferon.

63. A method for preventing graft-versus-host reaction in a warm-blooded animal such as a human, comprising admini¬ stering an effective amount of an interferon according to claim 1 or a mixture of such interferons to the animal.

64. A method according to claim 63, in which the interferon is selected from an α-interferon, a β-interferon, and mixtures of α-interferons according to claim 1 and -inter¬ ferons.

65. A method for prolongating an allograft survival in a warm-blooded animal such as a human, comprising admini- stering an effective amount of an interferon according to claim 1 to the animal, the interferon preferably being selected from an α-interferon, a -interferon, and mixtures of α-interferons according to claim 1 and -interferons.

66. A method for treating of leukemia, such as hairy cell leukemia or chronic yeloid leukemia, in a warm-blooded animal such as a human, comprising administering an effec¬ tive amount of an interferon according to claim 1 or a mixture of such interferons to the animal, the interferon preferably being selected from an α-interferon, a -inter- feron, and mixtures of α-interferons according to claim 1 and -interferons.

67. A method according to claim 66, in which the amount is about 1000 million units over a year.

68. A method for treating myelomatosis in a warm-blooded animal such as a human, comprising administering an effec¬ tive amount of an interferon according to claim 1 or a mixture of such interferons to the animal, the interferon preferably being selected from an α-interferon, a ₇-inter¬ feron, and mixtures of α- and ₇-interferons, as well as a -interferon, and mixtures of α- and β-interferons accord¬ ing to claim 1.

69. A method according to claim 68, in which the amount is about 1000 million units over a year.

70. A method for inhibiting, controlling or preventing viral activity in a warm-blooded animal such as a human, comprising administering to the animal an effective amount of an interferon protein as defined in claim 1 or a mixture of such interferon proteins, the interferon preferably being selected from an α-interferon, a -interferon, and mixtures of α-interferons according to claim 1.

71. A method according to claim 70, wherein the viral activity is retroviral activity.

72. A method according to claim 71, wherein the retroviral activity is HIV activity.

73. A method according to claim 72, in which the viral activity is hepatitis activity.

74. A method according to claim 70, wherein the viral activity is selected from Herpes simplex virus activity and cytomegalovirus.

75. A method according to claim 70 when used for preventing virus from a human mother infected with the virus from infecting her noninfected fetus during pregnancy or during labour.

76. A method according to claim 75, in which the admini¬ stration is performed by injection into the amniotic fluid.

77. A method according to claim 70, in which the interferon protein is selected from an α-interferon according to claim 1, a -interferon according to claim 1, and a mixture of α- and -inteferons according to claim 1.

78. A method according to claim 77, in which the admini¬ stration is performed by intravenous injection or by injec¬ tion into the amniotic fluid.

79. A method for treating infections selected from virus infections and nonvirus infections of the placenta in a warm-blooded animal such as a human in cases where acute systemic maternal infections are diagnosed, comprising administering to the animal an effective amount of an interferon protein as defined in claim 1 or a mixture of such interferon proteins.

80. A method according to claim 79, in which the admini¬ stration is performed by intravenous injection or injection into the amniotic fluid.

81. A method according to claim 70, in which the interferon protein is an α-interferon according to claim 1, a ^-inter- feron according to claim 1, and a mixture of α- and β- interferons according to claim 1, in a dose of 100,000 to 1,000,000 units per injection.

82. A method for treating malignant tumours (choriocarci- no as) arising from the placenta in a warm-blooded animal such as a human, comprising administering to the animal an effective amount of an interferon protein according to claim 1.

83. A method according to claim 82, in which the admini¬ stration is performed by injection into the amniotic fluid.

84. A method according to claim 83, in which the interferon protein is selected from a mixture of α-interferons accord- ing to claim, mixtures of α- and ₇-interferons according to claim 1, a -interferon according to claim 1, a ₇-inter¬ feron according to claim 1, and mixtures of a -interferon and a ₇-interferon according to claim 1, administered in an amount of about 5 x 10⁶ units 3 times a week.

85. A pharmaceutical composition comprising an interferon according to claim 1 in association with a stabilizer.

86. A composition according to claim 80 in which the stabi¬ lizer is human albumin

87. A method for inhibiting posttransplantational graft rejection, comprising administering, to a mammal in need thereof, an α- or ^-interferon according to 1 in an amount sufficient to effective to inhibit proliferation of immune- competent lymphocytes such as T.cells or B-cells, such as an amount corresponding to a plasma concentration of about 100-1000 International Units per milliliter.

88. A method according to claim 87, wherein the administra¬ tion is performed posttransplantationally.

89. A method according to claim 88, wherein the administra- tion is continued life-lasting.

90. A method for inhibiting a posttransplantational graft- versus host reaction, comprising administering, to a mammal in need thereof, an α- or β-interferon according to claim 1 in an amount sufficient to effective to inhibit prolifera- tion of immunecompetent lymphocytes such as T.cells or B- cells, such as an amount corresponding to a plasma concen¬ tration of about 100-1000 International Units per milli¬ liter.

91. A method according to claim 90, wherein the administra- tion of the interferon started prior to or simultaneously with the transplantation.

92. A method according to claim 91, wherein the interferon is incorporated in the transplantate.

93. A method according to any of claims 90-92 wherein the administration is continued life-lasting.

94. A method for heat-stabilizing an interferon according to claim 1, comprising adding, to the interferon, a stabi¬ lizer selected from the group consisting of ethylene gly¬ col, in particular 30-40% ethylene glycol, reducing agents, such as ME, and detergents, such as SDS.

95. A method according to claim 94, wherein the stabilizer comprises a combination of a reducing agent and a deter¬ gent.

96. A method according to claim 94 or 95, wherein the reducing agent is ME, and the stabilizer is SDS.

97. A method for inactivating any pathogenic components, such as virus or prions, in a preparation containing an interferon according to claim 1, in particular an a- or β- interferon according to claim 1, such as an aqueous solu¬ tion thereof, comprising adding a heat-stabilizer to the preparation and subjecting the preparation to heat treat¬ ment at a temperature and for a period sufficient to in¬ active the pathogenic components, the kind and amount of the heat stabilizer being sufficient to stabilize the interferon during the heat treatment.

98. A method according to claim 97, wherein the heat treat¬ ment comprises heating to a temperature of at least 56°C.

99. A method according to claim 97 or 98, wherein the heat stabilizer comprises a reducing agent or a detergent, or a combination thereof.

100. A heat-stabilized interferon composition, comprising an interferon according to claim l, in particular an α- or -interferon, together with a stabilizer selected from the group consisting of ethylene glycol, in particular 30-40% ethylene glycol, reducing agents, such as ME, and deter¬ gents, such as SDS.

101. A composition according to claim 100, wherein the sta¬ bilizer comprises a combination of a reducing agent and a detergent.

102. A composition according to claim 100 or 101, wherein the reducing agent is ME, and the stabilizer is SDS.

103. A method for enhancing fertility, comprising admini¬ stering an α- or β-trophoblast interferon according to claim 1 to a female mammal such as a female human in an amount effect to have luteotropic effect.

104. A method according to claim 103, wherein the α- or β - trophoblast interferon is administered parenterally in an amount corresponding to a plasma concentration of about 10- 50 International Units per milliliter.

105. A method for enhancing in vitro fertility, in parti¬ cular human in vitro fertility, comprising administering to a culture fluid containing the egg and sperm, an α- or β- trophoblast interferon according to claim 1 in an amount effective for enhancing cell viability.

106. A method according to claim 105, wherein the α- or β- trophoblast interferon is administered to the culture fluid in an amount corresponding to a concentration of about 5- 20, in particular about 10, International Units per milli¬ liter.

107. A method for contraception (preventing pregnancy), comprising administering an α- or -trophoblast interferon according to claim 1 to a female mammal such as a female human in an amount effective to inhibit trophoblast in vitro production of human chorion gonadotropin.

108. A method according to claim 107, wherein the α- or β- interferon is administered parenterally in an amount corre¬ sponding to a plasma concentration of about 100-1000 Inter¬ national Units per milliliter.

109. A method for inducing abortion (rejection of the nidated fertilized egg) , comprising administering to a female mammal, in particular a female human, a ₇-tropho¬ blast interferon according to claim 1 in an amount effec¬ tive to enhance cellular expression of the major histocom¬ patibility complexes and enhance cellular immune responses.

110. A method according to claim 109, wherein the 7-inter- feron is administered parenterally in an amount correspond¬ ing to a plasma concentration of about 100 to about 1000 International Units per milliliter.