FR2669824A1 - Use of interferon alpha variants for obtaining medicaments - Google Patents

Abstract

The invention relates to the applications of interferon- alpha variants, of general formula: X1-R0 (I> or alternatively X1-X2-R0 (II> or alternatively X1-X2-X3-R0 (III> in which X1, X2 and X3 are identical or different and each represent an amino acid having a non-polar or neutral side chain, and R0 represents the amino acid sequence of the mature form of an interferon- alpha , for obtaining medicaments or diagnostic reagents, and more especially of a trophoblastin variant designated APrT. The invention also relates to a method for the purification of APrT from yeast culture medium.

Description

 The invention relates to the use of variants of interferons a, in particular class II interferons a, such as trophoblastin, which variants are obtained by genetic engineering in a eukaryotic expression system, for obtaining medicaments, and more particularly for obtaining therapeutic and diagnostic products intended to improve the fertility of farm animals.

 Trophoblastin, or oTP, was highlighted respectively by MARTAL et al. [J. Reprod.

Fert., 56, 63-73 (1979); Pro. 10th intern. Congress on
Anim. Reprod. and Al, Urbana-Champaign (USA), 11, 509 (short communication) (1984)] and GODKIN et al.

[J. Reprod. Fert., 65, 141-150 (1982)] in sheep where it is produced in abundance by the embryo, between the 12th and the 21st day of gestation; in cattle, it is produced between the 16th and the 24th day. It exists in at least 5 isoforms corresponding to genetic variants. These isoforms are described in the Application
Internationale PCT 89/08706, on behalf of the INSTITUTE
NATIONAL AGRONOMIC RESEARCH. The molecular weight of trophoblastin is 20 KDa and its isoelectric point is between 5.3 and 5.5, depending on the isoform considered. It has been proposed to classify trophoblastin as a class II interferon a.

 Trophoblastin produced by embryos, like other interferons, has various biological activities.

 Trophoblastin is involved in the mechanism of recognition of the embryo by the maternal organism.

GODKIN et al [J. Reprod. Fert., 71, 57-64 (1984)] have shown that intrauterine injection of purified OTP prolongs the secretion of luteal progesterone for a few days in recipient cyclic ewes. FINCHER et al. [J. Reprod. Fert., 76, 425-433 (1986)] found that the injection of natural OTP into the uterus delays the lytolysis induced by oxytocin or ltoestradiol by a few days and is accompanied by a reduction in secretion prostaglandin F2a. Similar observations have been made with class I interferons [PLANTE et al., Endocrinology 122, 2342-2344, (1988); STEWART et al., (1989) J. Repr.

Fer. Supp. p. 127-138].

 An antiviral activity of this protein has also been demonstrated. This activity has been described by MARTAL et al. [J.Reprod.Fert.Abs.series, 2, 3, (1988)] and POUTZER et al., [Biochem.Biophys.Res.Com., 152, 801-807, (1988) l, as well as in the PCT application 89/08706 cited above. This Application also proposes the use of trophoblastin for the prevention of rejection of organ transplants.

 These properties make it possible to envisage numerous therapeutic uses of trophoblastin. The development of such applications has however come up against the fact that trophoblastin can only be obtained from conceptus, which does not allow its production in sufficient quantity for industrial use.

However, the cloning in E. coli of complementary DNA (cDNA) from messenger RNA (mRNA) coding for ovine trophoblastin has been described in the Application
PCT 89/08706 cited above. It could therefore be envisaged to construct, from this cDNA, chimeric genes allowing the production of trophoblastin in microorganisms.

 The constructions carried out initially in E. Coli did not allow the production of trophoblastin in sufficient quantity. Cloning tests in other hosts have therefore been carried out.

 An expression system allowing the secretion of trophoblastin from yeasts was thus sought, and a construction allowing a high level of expression of the trophoblastin gene was developed.

In this construction, the region corresponding to the signal sequence of the trophoblastin cDNA was deleted and replaced by the signal sequence of the yeast prepropheromone, then the whole was cloned into the expression vector PTG 7908.

 The recombinant trophoblastin which results from the expression of the chimeric gene thus constructed contains 2 additional amino acids (alanineproline) preceding the N-terminal cysteine of natural trophoblastin. This recombinant trophoblastin is hereinafter called APrT.

The expression system thus obtained, as well as the synthesized trophoblastin synthesized, are the subject of a Patent Application, in the name of the Company
TRANSGENE, filed at the same time as this Application.

 A quantitatively sufficient expression of the trophoblastin gene after cloning in yeast could therefore only be obtained at the cost of the addition of two amino acids at the N-terminal end of the polypeptide chain. It was to be feared that this modification of the primary structure would have consequences on the tertiary structure such that the physiological activity of the recombinant protein obtained is different from that of the natural protein. Indeed, in interferons a, the cysteine residue at the N-terminal end intervenes in the tertiary structure of the molecule, by formation of a disulfide bridge cysl-cys99, and it is generally considered that the position of said residue is essential for the maintenance of an active conformation. Until now, the possibility of using in natural a interferons, interferons a whose N-terminal end has been modified, had therefore absolutely not been considered.

 Now, the inventors have carried out the study of the physiological properties of APRT, and during this study, have found that, surprisingly, and despite the two additional N-terminal amino acids which are capable of modifying the tertiary structure and to cause a conformational gene disrupting the activity of the molecule, the immunological and biological properties (antiviral, antiluteolytic, and immunosuppressive) of this recombinant interferon a were equivalent to those of the natural molecule.

It therefore appears, in the light of the observations made by the Inventors, that variants of interferons has modified by adding to their end
N-terminal, of one or more amino acids, for example a di- or a tripeptide, retain the properties of natural a interferons, in particular if they are amino acids with a non-polar or neutral side chain, such as alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, glycine, serine and threonine,
The subject of the present invention is the applications of variants of interferon a, of general formula
X1-Ro (I)
or
X1-X2-RO (Il)
or
X1-X2-X3-Ro (III)
in which X1, X2 and X3 are identical or different, and each represents an amino acid with a non-polar or neutral side chain,
and Ro represents the amino acid sequence of the mature form of an interferona,
for obtaining drugs or diagnostic reagents.

According to a preferred embodiment of the present invention, X1, X2 and X3 each represent an amino acid chosen from the group consisting of alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, glycine, serine and threonine
By interferona (IFN-a) is meant in particular an IFN-a of class I (IFN-aI) or of class II (IFN-aII).

An IFN-aI is characterized by a sequence of 166 amino acids while an IFN-aII has an extension
C-terminal of 6 amino acids compared to an IFN-aI.

 This definition obviously includes the natural allelic variants of the interferons a. For example, with regard to interferons a of human origin, at least fifteen genes are known whose coding sequences have more than 85 homologies between them.

According to a preferred embodiment of the present invention, a variant of interferona, of general formula
Ala-Pro-RO '(IV)
in which R0, is an interferon aII, is used in the applications defined above. Preferably, this variant corresponds to the formula
Ala-Pro-Ro "(V)
in which Roi 'is an IFN aII of bovine or ovine origin.

According to a particularly advantageous modality of this embodiment, an interferona variant called APrT, of general formula
Ala-Pro-Ro "'(VI)
in which Ro "'represents the amino acid sequence of the mature form of any of the trophoblastin isoforms, is used in said applications.

 In accordance with the invention, the interferona variants as defined above can be used in all applications of natural a interferons such as, for example, obtaining antiviral, immunomodulatory, anti-inflammatory, antitumour drugs.

 Advantageously, APRT is, in addition to the general applications of the interferons defined above, more specifically used for obtaining antiluteolytic drugs, as well as products intended to improve the survival of embryos during their transplantation, and for l obtaining reagents for the diagnosis of embryo viability at an early stage of their development.

 According to a preferred embodiment of the present invention, APRT is used for the treatment of herds, in order to improve their fertility.

 According to another preferred embodiment of the present invention, APRT is used for the treatment of embryos during their transplantation in the various techniques of reproduction of farm animals implying a transfer of embryos, in particular those associated with cryopreservation, in-vitro fertilization, embryonic cloning, embryonic transgenesis.

 According to yet another preferred embodiment of the present invention, APRT is used for obtaining reagents and kits allowing the diagnosis of viability of embryos at an early stage of their development.

 Such a diagnosis is based on the dosage of trophoblastin produced by the embryos. This assay can for example be carried out by immunological methods; in this case, APRT can be used as an immunogen for the production of antibodies, or as an antigen, in competitive type methods. Trophoblastin produced by embryos can also be quantified by its antiviral activity; APRT can be used as a standard for antiviral activity in such an assay.

The present invention also relates to a process for purifying APRT from yeast culture medium producing it, which process is characterized in that APrT is purified by DEAE type chromatography on an ion exchange column cationic, by a 3-step elution
- a KCl gradient from 0 to 0.135 M
- an isocratic phase at approximately 0.135 M
of KCl
- a KCl gradient of 0.135 M to 0.5 M, the APRT being collected at the end of the isocratic phase.

 APrT can also be purified by reverse phase chromatography, or by affinity chromatography, using anti-trophoblastin antibodies.

 The present invention will be better understood with the aid of the additional description which follows, which refers to examples of preparation and use of APRT, and of demonstration of its properties.

 It goes without saying, however, that these examples are given solely by way of illustration of the subject of the invention, of which they do not in any way constitute a limitation.

Example 1: PRODUCTION AND PURIFICATION OF THE
RECOMBINANT TROPHOBLASTINE (APrT)
a) Production of APRT in yeast.

The DNA sequence coding for the ovine trophoblastin precursor, as described in
PCT application 89/08706 is cloned as a fragment
EcoRI in the vector M13TG131 described in the article by
MP KIENY et al., [Gene, 26:91 (1983)]. The vector M13TG771 is thus obtained. In order to be able to isolate the fragment coding for the mature protein, that is to say, the DNA fragment without the signal sequence, a HindIII site is created at 5 ′ of the mature sequence of the protein (FIG. 1). by site-directed mutagenesis using the kit
Amersham and the oligonucleotide OTG2102, the sequence of which is as follows: GAGGATCTCAAGCTTGTTACCTAT.

 The antisense strand of trophoblastin, carried by the vector M13TG771 single strand, is represented below line I and the oligonucleotide OTG2102 is represented line II; the stars * represent the mismatches which will cause the mutations sought.

Line I: CT CCT AGA GAC CCA ACA ATG GAT A
Line II: GA GGA TCT CAA GCT TGT TAC CTA T.

** *
The vector M13TG7720 is thus obtained. The point mutations which have been introduced into the coding sequence induce the replacement of the amino acids Leucine in position -2 and Glycine in position -1 of the precursor of trophoblastin by glutamine and Alanine respectively. M13TG7720 is then digested with
EcoRI and the EcoRI DNA fragment coding for the mutated precursor of trophoblastin is inserted into the vector pTG769 (described in Patent Application EP0258118 in the name of TRANSGENE) previously digested with EcoRI. The vector pTG7901 is thus obtained.

On the other hand, to produce trophoblastin in yeast, the DNA fragment coding for trophoblastin must be put under the control of a yeast promoter. For this, use is made of the vector M13TG3841, described in the PCT Patent Application filed on April 28, 1990, the filing number of which is FR90 / 00306, and which contains in particular
- the promoter of the gene coding for factor alpha 1 (MFalphal);
- the MFalphal pre sequence coding for the signal peptide of the alpha 1 factor precursor; and
- the MFalphal pro sequence.

A SmaI restriction site is created between the second and third codon of the pro sequence of
MFalphal by site-directed mutagenesis using the kit
Amersham and the oligonucleotide OTG2072 whose sequence is as follows: TCCGCATTAGCTGCTCCCGGGAACACTACAACAGAA.

The antisense strand of the pre-pro sequences of
MFalphal, carried by the vector M13TG3841, is represented above line I and the oligonucleotide OTG2072 is represented line II; the stars * indicate the mismatches which will cause the mutations sought.

Line I AGG CGT AAT CGA CGA GGT CAG TTG TGA TGT TGT CTT
Line II TCC GCA TTA GCT GCT CCC GGG AAC ACT ACA ACA GAA
* **
SmaI
The vector M13TG3869 is thus obtained. The point mutations which have been introduced into the coding sequence induce the replacement of the amino acid Glycine by valine.

The vector pTG7901 is digested with HindIII to release the DNA fragment HindIII, coding for mature trophoblastin which is then treated with Mung nuclease
Bean. This fragment is inserted into the vector M13TG3869 previously digested with SmaI. The vector M13TG7740 is thus obtained which comprises in sequence and in phase
- the promoter of MFalphal,
- the MFalphal pre sequence followed by the first two codons of the pro sequence, ie those coding for the amino acids alanine and proline and,
- the DNA fragment coding for ovine trophoblastin.

The SphI DNA fragment from the vector
M13TG7740, comprising the MFalphal promoter, the sequence followed by the codons of alanine and proline and the DNA sequence coding for mature trophoblastin is inserted into the yeast vector pTG3828 (described in the PCT Patent Application whose the filing number is FR90 / 00306) previously digested by SphI. The plasmid pTG7908 is thus obtained.

A yeast strain of the species Saccharomyces cerevisiae of genotype MATalpha, ura3-251, -373, -328, leu2-3, -112, his3, pep4-3 is transformed by the plasmid pTG7908 by the acetate method of lithium [H. ITO et al., J. Bacteriol. (1983) 153] and the prototrophs for uracil (Ura +) are selected on a YNBG medium (14 g / l of nitrogenous bases for yeast (Yeast Nitrogen
Base), 10 g / l glucose) added with 10 g / l casamino acids.

 The Al-Pro-trophoblastin variant is produced by "Fed-Batch" in a 20 L BIOLAFFITE fermenter.

12 l of medium D kàppeli [A. FICCHTER et al., Adv.

Microbial. Physiol. (1981), 22, 123-183] concentrated 1.5 times which contains 10 g / l of glucose and of HY Case SF (sold by SHEFFIELD) are seeded with 400 ml of a preculture of a yeast clone transformed by pTG9708.

This preculture is carried out in an Erlenmeyer flask at 30 ° C. on a selective YNBG medium. Before the start of the fermentation, the DO600 of the medium thus seeded is 0.2. The fermentation takes place at 30 "C, at a pH of 4 , 5 controlled by adding a 10% ammonia solution and a partial oxygen pressure corresponding to 30% of the saturation pressure, kept constant by regulation of the agitation. When all the glucose is consumed, the DO600 is measured (OD at the start of the feeding) and the glucose feeding is started in 3 hour exponential steps, knowing that the specific growth rate (F) is 0.1 hits ~ 1 and the amount of glucose added (QS) is 0.045 g / h / DO. Fermentation is stopped when DO600 = 100, and the harvest is done by centrifugation at 5000g. 13 l of culture supernatant containing the variant are thus obtained.
Ala-trophoblastin.

b) Purification of the APRT:
The yeast culture supernatants are centrifuged then concentrated and dialyzed against 0.05M of Tris-HCl buffer (pH 8.2) in an ultrafiltration cell (AMICON), on a FILTRON membrane retaining molecules greater than 10 KDa. The isolation of APrT was carried out on a semi-preparative scale by high performance liquid chromatography (HPLC), on a balanced TSK DEAE 5PW anion exchanger column (150 x 21.5) by buffer
Tris-HCl, 0.05M, pH 8.2. The flow rate is 4ml / min. After injection of the sample, the elution is carried out with a KCl gradient of 0 to 0.135 M (Tris-HCl buffer, 0.05 M, pH 8.2) for 90 minutes, followed by an isocratic plateau phase at 0.135 M KCl for 30 min, then a second gradient, up to 0.5 M KC1 for 80 min.

The elution is followed by measuring the absorption at 280 nm.

The peak corresponding to APRT is identified using a natural anti-trophoblastin immune serum (the similarity of the antigenic properties of APRT and natural trophoblastin is demonstrated below, in Example 2), then the corresponding fractions are collected. APrT leaves at the end of the isocratic plateau phase at 0.135 M KC1
Figure 1 illustrates the elution profile obtained; the peak corresponding to the APRT is hatched.

The apparent molecular weight of APRT in polyacrylamide gel electrophoresis in the presence of
SDS is around 21 KDa. Figure 2 shows the electrophoretic profile obtained (well a: APrT; well b molecular weight markers).

Example 2: COMPARISON OF THE IMMUNOLOGICAL PROPERTIES OF THE APrT WITH THOSE OF THE TROPHOBLASTIN NATURETLv
The existence of antigenic cross-reactions between APRT and trophoblastin is evaluated by radioimmunoassay (RIA), using an anti-trophoblastin polyclonal antiserum, and a preparation of trophoblastin labeled with iodine 125, according to the protocol described in the Application. PCT 89/08706
The RIA inhibition curves obtained under these conditions are shown in Figure 3; on the abscissa is the Log of the dilution, and on the ordinate the corresponding Logit = Log (Bo / 1-Bo) (Bo represents the maximum concentration of bound trophoblastin, for a constant concentration of anti-trophoblastin antiserum)
- (g) control: purified preparation of trophoblastin; ;
Y = - 0.9572X - 0.2341
linear regression coefficient = O, 9957
- (x) culture medium for yeasts secreting APRT (dilutions from 1 to 1/100)
Y = - 0.9372X - 0.921
linear regression coefficient = O, 9996
- (X) preparation of APRT (dilutions from 1 to 1/4000) obtained as described in Example 1,
Y = - 0.9341X - 2.41
linear regression coefficient = O, 9955
These curves are parallel to each other, which shows that the antigenic properties of APRT are similar to those of natural trophoblastin.

Example 3: ANTIVIRAL ACTIVITY TEST
The antiviral activity of APRT is measured according to the protocol described by LA BONNARDIERE and LAUDE, [Infection and Immunity, 32, 28-31, (1981)], on MDBK cells (Madin Darby Bovine Kidney), a line of calf kidney cells, in the presence of the vesicular stomatitis virus.

The results obtained are compared with those obtained from a reference interferon, which is a class I porcine interferon, of a titre of 1000 IU, itself calibrated against the human reference standard.
The results of this antiviral activity test show an equivalent activity, for the supernatants of yeast culture medium (0.8 x 108 IU / mg), the purified APRT (0.55 x 108 IU / mg) , and natural trophoblastin (0.7 x 108 IU / mg).

Example 4: IN VIVO DEMONSTRATION OF THE ACTIVITY
APrT ANTILUTEOLYTIC
Animals and hormone therapy
The experiment was carried out on 30 Préalpes-du-Sud sheep. The oestrus cycles are synchronized by means of vaginal sponges impregnated with 300 mg of 17 alpha-acetoxy-9 alpha-fluoro-11ss-hydroxyprogesterone (SEARLE, INTERVET). These sponges are left in place for 14 days and on the day of withdrawal (D14), the sheep are injected, intramuscularly, 500 IU of PMSG (pregnant mare serum gonadotrophin); 48 hours later, the sheep begin a new cycle (OJ).

Placement of intrauterine catheters
The sheep are anesthetized, then, by laparotomy at the level of the white line, the operator accesses the uterus and proceeds to mark the yellow bodies with Indian ink. A sterile catheter (SILASTIC, DOW
CORNING) of 0.076 mm internal diameter, 0.165 mm external diameter and 70 cm long is equipped with a short sleeve at one of its ends, which keeps the catheter in place in the uterine horn, substantially at level of the utero-tubal junction.

The opening is closed with a needle crimped to a "catgut" wire (BRUNEAU laboratory). A purse suture is made around the insertion hole of the catheter ensuring the durable positioning of the device. A safety point made with a needle crimped with silk (BRUNEAU Laboratory) fixes the catheter to the broad ligament. The other end is closed with a linen thread and a loop is made, so that the operator, after having pierced the abdominal wall at the level of the right flank, can pull the catheter out by pulling on the loop. A SILASTIC brake was placed 30 cm from the intrauterine end to limit the exit of the catheter and serve as a stop inside the abdominal wall. A length of approximately 40 cm is available to the operator who performs the intrauterine injections. A point made around the abdominal orifice of emergence of the catheter, reinforces the solidity of the device. The catheter poses are practiced between the 9th and island days of the cycle.

Intrauterine injections
Three batches of sheep were made up: intrauterine injections start between the 10th and 12th day of the cycle, and are carried out twice a day, for 8 days. APrT is in solution in physiological saline containing 50,000 IU / ml of penicillin G and BSA at 2 '/. The volume of solution injected is 1 ml.

Lot A: It is a control lot composed of 10 ewes which receive 2 ml of solution of twice a day.
BSA (fraction V, SIGMA) at 2ç / oo in a physiological serum (0.9% NaCl) containing 50,000 IU / ml of penicillin G.

 Lot B: This group is made up of 8 ewes to which 170 Ag of PrTT were administered bi-daily.

Lot C: This group is made up of 4 ewes which receive twice a day 80 Rg of APRT
Lot D: 5 ewes receive twice a day 340 Rg of APRT.

At the end of the experiment, all the ewes undergo an exploratory laparotomy in order, on the one hand, to verify that the catheters have remained in place and, on the other hand, to check in each batch the presence or not of corpus luteum ( s) marked with
China.

Radioimmunoassay of progesterone
The blood of the animals is taken from the jugular vein using Vacutainer tubes (BECTON-DICKINSON) without anticoagulant. The concentration of serum progesterone is determined by direct radioimmunoassay without extraction, according to the protocol described by
HEYMAN et al. [J. Reprod. Fer., 70, 533-540, (1984) 1. Tritium-labeled progesterone and a specific antiprogesterone immune serum (INSTITUT PASTEUR) are used for this assay.

 In control batch A, the concentration of progesterone in the peripheral blood suddenly decreases in all ewes from the 14th day, reaching levels below 0.5 ng / ml between the 15th and 17th day post-estrus . The average duration of the cycle in this batch is 15.2 + 0.3 days.

The administration of 80 Rg of APRT per day (batch C) does not extend this duration.

 In batch B, (170 Rg / day), on the 14th day of the cycle, there is a slowing down of the fall in progesteronemia compared to batch A: 7 out of 8 ewes present at this stage progesteronemia levels higher than l ng / ml (compared to 4 out of 10 in lot A); on the 15th day of the cycle, 5 out of 8 ewes still have progesterone levels higher than 1 ng / ml (compared to 2 out of 10 in lot A).

 In this batch, luteolysis is delayed on average by 2 days compared to batch A.

In lot D, intrauterine administration of APRT at a dose of 340 Ag / day maintains luteal function well beyond the duration of the normal cycle in four out of five ewes (25, 32, 45, and 64 days respectively, in sheep ne9037, 9431, 9458, and 9053) -
The comparative average profile of progesterone secretion between the different batches, represented by the
Figure 4 clearly shows that there is a clear persistence of luteal activity in lot D.

(W) Lot A
(O) Lot B
(X) Lot C
(O) Lot D
In addition, during laparotomy surgical control, no newly formed yellow body was found in any of the 4 ewes in lot D mentioned above, which shows that the progesterone measured corresponds to the persistence of cyclic yellow bodies before the injections. of APRT.

Previous experiments showed the antiluteolytic activity of natural trophoblastin, but they did not allow to affirm that trophoblastine alone was sufficient to prevent luteolysis, and had not made it possible to determine what was the role of the different isoforms of trophoblastin. However, the experiments described above show that the APRT obtained from a single isoform is sufficient, in appropriate doses, to inhibit luteolysis, despite the two additional amino acids of the N-terminal end
Example 5: DEMONSTRATION OF IMMUNOSUPPRESSED PROPERTIES
SIVES OF THE APART
These properties have been demonstrated by three types of tests allowing the demonstration of three different immunological modes of action - antimitotic activity, evaluated by the action on the proliferation of murine or human lymphocytes; - the activity of inhibiting the cytological reaction of graft rejection, evaluated using the in vitro test of the mixed lymphocyte reaction (RLM); - the immunoregulatory activity on the population of NK killer lymphocytes, independent of the antigens of the major histocompatibility complex (MHC).

1) Action of APrT on the proliferation of murine lymphocytes activated by phytohemagglutinin
Murine lymphocytes are obtained from spleen of C3H / He or Balb / c mice after grinding with
Potter and washing twice in culture medium
RPMI 1640 at 1500 rpm for 10 min. Finally, the isolated lymphocytes are mixed in the same culture medium, supplemented with 10% fetal calf serum (SVF), at a final concentration of 5,106 cells / ml.

The culture medium is composed of 500 ml of RPMI 1640 (GIBCO) + 5 ml of penicillin G / strep (GIBCO) + 5 ml of 7.5% sodium bicarbonate (GIBCO) + 5 ml of glutamine.

 100 A1 per well of culture medium containing 6 × 105 lymphocytes at a rate of 5 fg / ml PHA (WELLCOME) are incubated with 100 Rl of a solution of AprT at 3 pLg / ml (108 IU / mg) or 100 Rl of culture medium (control) in 96-well microtest plates at 37 ° C., in an air-CO2 atmosphere (95% -5%), for 48 hours.

 Lymphocyte proliferation is evaluated by measuring the incorporation of tritiated thymidine. 25 F1 of 3H thymidine (0.04 mCi / ml) are added to each well and the cells are harvested 24 hours later and deposited on a filter (Glass Micro fiber Filters-GFM-WHATMAN). After drying, the filters are placed in tubes to which 1 ml of scintillation liquid (ECONOFLUOR) is added.

Radioactivity is measured in an ss radiation counter (BECKMAN).

 The results are illustrated in Figure 5, which shows that APRT very markedly inhibits (55%) the proliferation of murine lymphocytes treated with PHA.

A: Witness
B: APrT.

Radioactivity in cpm is plotted on the ordinate
2) Action of the APRT on. a mixed lymphocyte reaction
The mixed lymphocyte reactions are carried out by incubating per 150 l of culture medium containing 5 × 10 8 reactive C3H / He cells per ml with 5 × 10 6 stimulatory cells, isogenic or allogenic, irradiated at 1,800 Rads / ml.

 C3H / He mouse cells are used for the isogenic reaction and Balb / c mouse cells are used for the allogenic reaction. 100 A1 of APRT at 3 μLg / ml (108 IU / mg) or of culture medium (control) is added per well in 96-well microtest plates (FALCON 3072) at 37 ° C., and the cultures are left under a gaseous atmosphere. air-CO2 (95% - 5%) for 4 days.

Lymphocyte lysis by cytotoxic lymphocytes produced during the mixed lymphocyte reaction is evaluated by measuring the incorporation of tritiated thymidine. 25 A1 of 3H thymidine are added 24 h before the removal of the lymphocytes and their deposit on a filter. The radioactivity of the filters is measured in a scintillation counter
The results are shown in Figure 6
A: Control
B: Natural trophoblastin
C: APrT
Radioactivity in cpm is plotted on the ordinate.

 In bidirectional culture of lymphocytes from two strains of mice (Balb / c and C3H / He), APRT inhibits by 90% the lysis of marine lymphocytes by the cytotoxic cells (CTL) produced.

3) Action of APRT on cell lysis by NK cells
K562 cells (human erythroleukemic line) are centrifuged for 10 min at 1800 rpm and 0.5 ml of 51 Cr are deposited on the pellet. After 1 h of incubation at 37 ° C. in 5% CO 2 - 95% of air, the cells are washed three times in RPMI culture medium and resuspended in the same culture medium, at a concentration of 2 × 10 5 cells / ml.

 Three types of incubation in a microtiter plate at 37 ° C, in an air-CO2 atmosphere (95% - 5%) are carried out in parallel: - 100 A1 of labeled K562 cells + 100 l of human lymphocytes 50 to 100 times more concentrates + 100 fl of APRT at 3 μLg / mi (108 IU / mg) or of culture medium (control) make it possible to determine the radioactive proteins of the experimental medium (prot. rad. exp.); - 100 fl of labeled K562 cells + 200 fl of 4N HCl make it possible to evaluate the radioactive proteins of the total medium (total rad prot.); - 100 l of labeled K562 + 200 μl of culture medium make it possible to determine the radioactive proteins of the spontaneous medium (prot. Rad. Spont.).

 After 4 h of incubation, 100 μl of supernatant are taken from each well and the radioactivity due to the release of proteins labeled with 51 Cr in the medium is counted in cpm in a gamma radiation counter.

The results are expressed as an average percentage of cell lysis of samples and calculated according to the following formula
cpm prot. rad. exp. - cpm prot. rad. spont.

% of lysis =
cpm prot. rad. early. - cpm prot. rad. spont.

Figure 7 shows the results
A: Control
B: APrT
C: IFN alpha
D: IFN gamma
It therefore clearly appears that APRT activates cell lysis of K562 target cells by NK cells.

This activation of cell lysis by NK cells is analogous to that observed from human interferon a class 1 and lower than that of the reference human interferon r
The preceding examples show that APRT has immunological and biological properties comparable to those which had previously been shown for natural trophoblastin, which allows its use in all the applications recommended for the latter.

As is apparent from the above, the invention is in no way limited to those of its modes of implementation, embodiment and application which have just been described more explicitly; on the contrary, it embraces all the variants which may come to the mind of the technician in the matter, without departing from the framework, or the scope, of the present
Invention.

Claims (10)

CLAIMS for obtaining diagnostic drugs or reagents. and Ro represents the amino acid sequence of the mature form of an interferona, in which X1, X2 and X3 are identical or different, and each represents an amino acid with a non-polar or neutral side chain, X1-X2-X3-R (III) or X1-X2-RO (II) or X1-Ro (I)  1 ") Applications of variants of interferon a, of general formula X3 chosen from the group consisting of alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, glycine, serine and threonine and Ro represents the amino acid sequence of the mature form an interferon a, for obtaining drugs or diagnostic reagents.  2 ") Applications according to Claim 1, characterized in that a variant in which X1, X2 and  in which Ro 'is an interferon aII, is used.  Ala-Pro-RO '(IV) Claims 1 or 2, characterized in that a variant of interferon a, of general formula  3) Applications according to any one of  in which Roi 'represents an interferon aII of ovine or bovine origin, is used.  Ai-Pro-R0, '(V)  4) Application according to Claim 3, characterized in that a variant of general formula  in which Ro "'represents the amino acid sequence of the mature form of trophoblastin, is used.  Ala-Pro-R, "'(VI)  5) Applications according to Claim 4 characterized in that a variant, called APrT, of general formula VI is used to obtain anti-inflammatory, antiviral, anti-tumor, immunomodulatory or antiluteolytic drugs. Claims 1 to 5, characterized in that a variant of interferon a, of general formula I, II, III, IV, V or  6) Applications according to any one of  7) Applications according to Claim 6 characterized in that the interferon variant used is the APRT of formula VI.  8) Application of the APRT for the treatment of embryos during their transplantation.  9) Application of APRT for obtaining reagents allowing a diagnosis of viability of embryos at an early stage of their development.  - a KCl gradient of 0.135 M to 0.5 M, the APRT being collected at the end of the isocratic phase.  of KCl  - an isocratic phase at approximately 0.135 M  - a KCl gradient from 0 to 0.135 M  10) Method for purifying APRT from yeast culture medium, characterized in that the APRT is purified on a chromatography column, by elution in 3 steps