FR2482858A1 - Non-cytotoxic immunosuppressive peptide fraction - obtained by purification of lymphoid chalone fraction fa - Google Patents

Abstract

New prepn. (I) has non-species-specific immunosuppressive activity. Also new are (a) a 'further purified' fraction and (b) a 'highly purified product', the characteristics of which are also specified below. (I) is characterised by certain of its chromatographic properties, and by the fact that it contains a "highly purified product" having the following properties: (a) substantially free from biologically inactive components, and separable from biologically active components by electrophoresis on a thin layer of cellulose in pyridine/acetic acid/acetone/water 1:2:8:40 (by volume) at pH 4.4 and 500V; (b) molecular weight below 5000; (c) wholly or partly peptide in nature; (d) detectable neither with ninhydrin- collidine nor fluorescamine in thin layer; (e) N-terminal blocked with a pyroglutamyl residue; (f) C-terminal either lysine or arginine; (g) inactivated by pronase, trypsin, pyroglutamate aminopeptidase, carboxypeptidases A and B; (h) not inactivated by leucine aminopeptidase, chymotrypsin, DNA-ase or 10A-ase;(i)specific,reversibleaffinityforIgG;(j)no selective affinity for IgM; (k) inhibits in vitro and in-vivo the formation of IgM- and IgG-carrying cells; (l) non-cytotoxic to lymphocyte cultures; (m) inhibits allogenic transplant reactions; (n) does not markedly influence spleen function in the mouse; (o) substantially free from inhibitory action on independent T antigens; (p) does not inhibit in vitro incorporation of tritiated-thymidine by mitogen-stimulated spleen lymphocyte cultures. The "further purified fraction" is characterised by its elution time under specified high pressure chromatography conditions. The new prepn. and prods. have considerable immunosuppressive activity and can be used as comparative standards for evaluation of the immunosuppressive action of investigative substances.

Description

PRODUCT OF PEPTIDE NATURE COMPRISING SUCH PROPERTIES
The invention relates to a highly purified product endowed with inimunosuppressive properties that are not specific to the species.

It can be defined as consisting of one or more substances, and more particularly as being characterized in that it is essentially free of constituents which are
both biologically inactive in the tests below
defined and separable biologically constituents
active, by cellulose thin-layer electrophoresis
of a solution of this product in a pyridine /
acetic acid / acetone / water in volume proportions
respectively 1/2/8/40, pH 4.4 under a voltage of
500 V - it has a molecular weight of less than 5,000 - it is of peptide nature (total or partial); it is not detectable by the ninhydrin collidine reagent,
nor by fluorescamine, at a dose of 5 nanomoles
thin layer of 20 cm - it contains an amino acid N-terininal blocked by a
pyroglutamyl group - it contains a C-terminal acid consisting of lysine
or arginine; - it is inactivated by the action of the following enzymes
pronase, trypsin, pyroglutamate-aminopeptidase, carboxy
peptidases A and B - it is not inactivated by leucine-amino-peptidase nor
chymotrypsin - it is not inactivated by DNase treatment and
RNases - it has a reversible specific affinity for
IgG (immobilized IgG binding at neutral pH, elution
at acid pH) - it has no selective affinity for IgM
(no binding to immobilized IgM at neutral pH) - it inhibits cell formation in vitro and in vivo
carriers of immunoglobulins IgM (19S) and IgG (75) di
against antigens, such as red blood cells
sheep in primary and secondary responses; - it is not cytotoxic with regard to lympho cultures
cytes; it has inhibitory properties of the reaction of
grafts of allogeneic origin, including cells
allogeneic rat, against the host - it does not significantly affect the ability of cells to
mouse spleen, previously incubated in his presence and
administered to mice that have themselves been pre-treated.

subjected to vital irradiation, to form
splenic colonies in the spleens of these mice - it is substantially devoid of inhibitory effect with respect to
"independent T antigens", such as trinitr-o-
phenyl lipopolysaccharides; - it does not inhibit the incorporation n vits of the
3H-thymidine in stimulated lymphocyte cultures
by mitogens, such as PHA and LPS.

The product according to the invention can be prepared from the active fraction called 11FAC ", as defined in the article by M. LENFANT et al, published in" Molecular Immunology, vol. 17, pp. 119-126) (Pergamon
Press Ltd, 1980) "and entitled" The Purification of an Immunosuppressive Factor Extracted from Bovine Spleen III "(Purification of an Immunosuppressive Factor Extracted from Beef Spleen III) and in particular using the electrophoresis technique, such as it was defined above.

 It relates in particular to a product as defined above, characterized in that all its components either do not migrate or migrate to the anode in the aforesaid electrophoretic system.

It also relates more particularly to products having all the above-mentioned characteristics and consisting essentially of - a biologically active substance, substantially homo
gene with respect to the aforementioned electrophoresis technique,
when it is repeated at least once
at pH 4.4 or at pH 6.5, the substance
migrating to the anode and being characterized at pH 6.5 by
a mobility at pH 6.5, of the order of -0.5 to -0.8, especially
from -0.54 to -0.79 vis-à-vis the mobility considered
equal to -1 of aspartic acid subjected to elec
trophoresis under the same conditions; - a biologically active substance, substantially homo
gene with respect to the aforementioned electrophoresis technique,
when it is repeated at least once
at pH 4.4 or at pH 6.5, the substance
migrating to the cathode and being characterized by a
mobility, at pH 6.5, of the order of +0.8 to +1.1, especially
from +0.83 to +1.07 for the mobility considered
equal to -1 of aspartic acid subjected to electropho
resists under the same conditions; - a biologically active substance, substantially homo
gene with respect to the aforementioned electrophoresis technique,
when it is repeated at least once
at pH 4.4 or pH 6.5, the substance
not substantially migrating under the conditions above
defined electrophoresis - a biologically active substance, substantially homo
gene with respect to the aforementioned electrophoresis technique,
when it is repeated at least once
at pH 4.4 or at pH 6.5, the substance
being characterized by a migration mobility towards
the cathode is zero or not more than 0.1
with respect to the mobilities of urea and aspar acid
respectively 0 and -1 in systems
consisting of respectively electrophoresis
on cellulose thin film solutions of the said pro
in mixtures (volume proportions):
- formic acid / acetic acid / water (1/4/45)> pH 1.9;
pyridine / acetic acid / acetone / water (1/2/8/40), pH 4.4;
pyridine / acetic acid / water (20/1/180), pH 6.5.

 Other features of the invention will still be apparent from the following description of preferred embodiments for obtaining the product according to the invention and the conditions under which the structural analyzes and the biological tests were carried out. the results allow a characterization of the product according to the invention.

Obtaining products according to the invention.

 A FAC fraction, itself obtained according to the technique already described in the above-mentioned article, is subjected to thin-layer chromatography of cellulose "MN 300" in the butanol / acetic acid / water system (60/15/25). The active fractions are located at Rf respectively between 0.2 and 0.32; 0.45 and 0.53 0.73 had 0.83. These are collected and in turn subjected to thin-layer electrophoresis of cellulose marketed under the designation "MN cell-300 uv 254" in the buffer pyridine / acetic acid / acetone / water (1/2/8/40) -pH 4.4, under a voltage of 500 V.

 The active fractions split into several sub-fractions including two active substances that migrate to the anode and flne which does not migrate. The two active substances which appear to slightly absorb the ultraviolet light could not be revealed either by the ninhydrin reagent collidine or by the fluorescamine, discounted of their low abundance. The substances contained in the two fractions that migrated to the anode were combined. They form the product according to the invention, called "SDIP" in what follows (abbreviation of the English expression "Spleen Derived Immunosuppressive Peptide").

In general, it was also used to characterize or identify the active fractions in the tests of counting the number (average value) of hemolysis ranges for 106 cells according to
JERNE (JERNE and NORDLIN, Science 140, 405, 1963) or
CUNNINGHAM (CUNNINGHAM and SZENBERG, Immunology, 14, 599, 1968) applied to mice aged 8-12 weeks of strain DBA / 2 x C57B1 / 6 or strain Balb C.

 In each case, the mice were sensitized with 0.5 ml of a sheep red blood cell (GRM) suspension (4 x 108 cells), and the antibody-producing cells were enumerated four days later. Depending on the case, the animals received one or two injections of 0.2 to 0.5 ml of a physiological C1Na solution containing the substance to be studied (or physiological solution only for the controls) 24 or 24 + 1 hours before their sacrifice.

 In many cases, it is by reference to the results obtained using these tests that the impact on the biological activity of the test substances of various chemical or biological agents has been determined.

Chemical properties.

It was thus found that the SDIP was inactivated by pronase, trypsin; that it was not inactivated by leucine amino peptidase, DNase
RNases, and more particularly-that: - treatment with pyroglutamate aminopeptidase (buffer
0.05 M phosphate, mercaptoethanol -0.01 M, 0.001 M EDTA,
2 hours, 370C) induces the total loss of biological activity
gique; the terminal NH2 group is thus blocked by
pyroglutamyl cycle. This result is confirmed by the
presence in the mass spectrum of the derived product, a
significant fragmentation peak at m / e 84.feature
of this group; the treatments with carboxypeptidases A and B (tam
0.2N ethyl morpholine acetate, pH 8.5, 2 hours,
370C) induce a loss of activity. This indicates that
the molecule carries a free terminal 'COOH' group and that
the terminal amino acid is either lysine or argi
nine.

Biological properties.

10) Study of the affinity of SDIP with respect to IgG and IgM
immobilized.

a) Fix IgG and IgM of laEin on support
("SEPHAROSE 4B").

 "Activated" SEPHAROSE is prepared from 30 ml of support by treatment at 40C in 120 ml of water (pH 10.2) with 60 mg of BrCN. The support is then washed with 2 liters of borate buffer (0.2 M, pH 8.4) and the coupling is carried out in this buffer for 24 hours with 23 mg of IgG. After filtration-washing with 500 ml of PBS buffer (0.1 M phosphate buffer, 8.5 g NaCl, pH 7), the support is stored at 40 ° C. under azide.

 The immunoglobulin fraction was fixed at 0.3 mg / ml gel.

 Under the same conditions, rabbit IgM is fixed on the same support.

 b) Chromatography of the active fraction on a column of "IG SEPHAROSE".

 The gel (10 ml) is equilibrated with pyridinium acetate buffer (0.02 M, pH 7). 20 doses (ie 20 times the dose which produces a 50% reduction in the number of hemolysis plaques in the YERNE test) of active fraction are applied in this column buffer. The column is eluted successively with 20 ml of the pyridinium acetate buffer (Fraction-1), then 20 ml of acetic acid (0.20 M, pH 2.8) (Fraction 2).

 Both fractions were tested after freeze-drying, redissolved in physiological saline, and administered to mice for their study according to the JERNE test. Fraction (1) (product eluted with pyridinium acetate) shows no reduction in control cultures, the number of hemolysis ranges in the YERNE test. On the other hand, fraction (2) (product eluted with acetic acid, pH 2.8) induces a strong reduction in the number of plaques of hemolysis by 10 6 cells, which shows the reversibility of the binding of the active fractions to immobilized IgGs. On the contrary, no binding of the active fractions according to the invention was found on IgM immobilized at neutral pH, under the conditions indicated above, in connection with IgG.

20) SDIP activity on production cell formation
IgM.

 a) In vitro activity.

SDIP has been implemented in a variant of the MISHELL and DUTTON in vitro test (Science, 153, 10041005 (1966)). This test consists of inducing the formation of IgM-bearing cells during culture of mouse spleen lymphocytes (6 × 10 6 cells / ml). The cultivation is carried out for five days at 3700 in an air atmosphere
C02 at 5%. The culture medium consists of RPMI 1640, supplemented with 1% antibiotics, 10% fetal calf serum, 1% horse, 1% glutamine, 105N mercaptoethanol.

The numbers of IgM-bearing cells are revealed by counting hemolysis plaques or PFCs (abbreviation of the corresponding English expression "plaque forming cells") in the CUNNINGHAM test in separate cultures after distinct durations, expressed in days.

The results obtained were as follows
a) The SDIP is active at the end of the lymphocyte differentiation period (fourth day of culture);
b) -The dose response shows a maximum inhibitory effect (90%> for doses ranging from 5 to 0.5 pg / ml.

A 50% inhibition of the response is observed for doses of the order of 10 -4 pg (assays performed at the amino acid analyzer).

 b) In vivo activity.

 Doses of 0.2 ng of SDIP were administered intraperitoneally to Balb C mice that had previously been sensitized by GRAM injection; at different times from this awareness.

Four days later, the animals were sacrificed and the number of hemolysis plaques per 10 cells under the conditions of the CUNNINGHAM test was reduced.

The results obtained show that the
SDIP is not active when it is administered before at the same time as the GRMs (or during the corresponding period). On the other hand, a 90% inhibition is observed, when the SDIP was administered three days after the sensitization, that is to say in the final phase of differentiation of the lymphocytes.

30) Action of SDIP on lymphocytes.

 SDIP is not cytotoxic with regard to lymphocyte cultures for which it does not modify the growth conditions.

 In addition, it does not inhibit 3H-thymidine incorporation into PHA or LPS mitogen stimulated lymphocyte cultures.

 Balb C mouse spleen cells were cultured for 48 hours in the presence of a mitogen; radioactive thymidine was added during the last 6 hours of culture; SDIP was added either at the time of culture, or 24 hours later. No activity on proliferation of B lymphocytes (LPS stimulated) or T lymphocytes (PHA stimulated) was detected.

40) Inhibition of graft versus host reaction under
the action of the SDIP.

The effects of SDIP have been studied on the splenomegaly that develops in Fl mice (C57BL / 6x
DBA / 2) irradiated at 400 R, in which a graft-versus-host reaction was induced by spleen cell grafting of the C57BL / 6 line. SDIP has been shown to be an inhibitor of this reaction either in vivo if administered to recipients simultaneously with the transplant, or in vitro when cells are incubated with this fraction prior to transplantation (at pico doses). gramme./ml). The spleen cells subjected to this treatment showed no decrease in their ability to form splenic colonies (CFUs) in the spleen of lethally irradiated C57BL / 6 mice. The SDIP thus has no inhibitory or cytotoxic effect on hemopoletic precursors. .

50) Inactivity of SDIP vis-à-vis an antigen T indpen
TNP-LPS.

 No effect of SDI could be noted on the formation of antibodies against the trinitophenyl radical, in cultures of three or four days in the presence of the antigen.

 The substantially homogeneous biologically active substances which can be obtained under the conditions which have been defined above from the SDIP have the same biological properties in the various tests which have been explained in the foregoing.

 The products of the invention therefore constitute biological reagents which, because of their considerable activity (at a dose of μg), can be used as a reference standard for assessing the immunosuppressive activities of different studied substances.

 In addition, they can be combined with pharmaceutical vehicles suitable for all modes of administration, for the preparation of pharmaceutical compositions, especially injectable solutions, suitable for the prevention or treatment of diseases involving uncontrolled production of drugs. antibodies, such as autoimmune diseases, certain forms of rheumatism, etc. Because of their total absence of cytotoxicity, including with regard to hemopoletic precursors, their use may be indicated in cases of prevention of graft-versus-host secondary reaction, as a result of grafting, including bone marrow transplants.

 The invention therefore relates to all pharmaceutical compositions containing an effective dose of the products of the invention, in particular, but not exclusively, those in the form of injectable sterile solutions.

Claims (9)

 1 - Highly purified product and endowed with non-species specific immunosuppressive properties, consisting of one or more substances, characterized in that: - it is essentially free of constituents which are  both-biologically inactive in the tests  after defined and separable biologic constituents  by thin layer electrophoresis  lulose a solution of this product in a mixture pyri  dine / acetic acid / acetone / water in  respectively 1/2/8/40, pH 4.4, under a  voltage of 500 V; - it has a molecular weight of less than 5,000 - it is of peptide nature, total or partial - it is not detectable by the reagent ninhrssrine cilidin,  nor by fluorescamine, at a dose of 5 nanomoles  thin layer of 20 cm; - it contains a N-terminal amino acid blocked by a  pyroglutamyl group; it contains a C-terminal acid constituted by lysine  or arginine - it is inactivated by the following enzymes  pronase, trypsin, pyroglutamate-aminopeptidase, carboxy  peptidases A and B; it is not inactivated by leucine amino peptidase nor  by chymotrypsin; - is not inactivated by DNase treatment and  RNases; - it has a specific reversible affinity for  IgG (immobilized IgG binding at neutral pH, elution  at acidic pH); it has no selective affinity for IgMs  (no binding to immobilized IgM at neutral pH) - it inhibits cell formation in vitro and in vivo  immunoglobulin IgG (19S) and IgG (7S) di  against antigens, such as blood cells  sheep red, in primary and secondary responses - it is not cytotoxic to lympho cultures  cytes; it has inhibitory properties of the reaction of  grafts of allogeneic origin, including cells  allogeneic rat, against the host; - it does not appreciably affect the cell capacity of  mouse spleen, previously incubated in his presence and  administered to mice that have themselves been pre-treated  subjected to lethal irradiation, to form  splenic colonies in the spleens of these mice - it is substantially devoid of inhibitory effect with respect to  "T independent" antigens, such as trinitro  phenyl lipopolysaccharides; - it does not inhibit the incorporation in glass of the  3H-thymidine in stimulated lymphocyte cultures  by mitogens, such as PHA and LPS.  2 - Product according to claim 1, characterized in that all its constituents either do not migrate or migrate to the anode in the aforesaid electrophoretic system.  3 - Product according to claim 1, characterized in that it consists essentially of a biologically active substance, substantially homogeneous with respect to the aforementioned electrophoresis technique, when it is repeated at least one additional time, or at pH 4 , 4, ie at pH 6.5, said substance migrating towards the anode and being characterized at pH 6.5 by a mobility at pH 6.5, of the order of -0.5 to -0.8 , especially -0.54 to -0.79 vis-à-vis the mobility considered equal to -1 of aspartic acid electrophoresed under the same conditions.  4 - Product according to claim 1, characterized in that it consists essentially of a biologically active substance, substantially homogeneous with respect to the aforementioned electrophoresis technique, when it is repeated at least one additional time, or at pH 4.4, or pH 6.5, said. substance migrating towards the cathode and being characterized by a mobility at pH 6.5, of the order of +0.8 to +1.1, in particular of +0.83 to +1.07 with respect to the mobility considered equal to -1 of aspartic acid subjected to electrophoresis under the same conditions.  5 - Product according to claim 1, characterized in that it consists essentially of a biologically active substance, substantially homogeneous with respect to the aforementioned electrophoresis technique, when it is repeated at least one additional time, or to pH 4.4, ie at pH 6.5, said substance not substantially migrating under the above-defined conditions of the electrophoresis.  6-- Product according to claim 1, characterized by a migration mobility to the cathode of zero or not substantially greater than 0.1 vis-à-vis the mobilities of urea and aspartic acid respectively equal to 0 and - 1 in systems of respectively subjecting to cellulose thin-layer electrophoresis solutions of said product in mixtures (volume proportions) - formic acid / acetic acid / water (1/445), pH 1.9; pyridine / acetic acid / acetone / water (1/2/8/40), pH 4.4; pyridine / acetic acid / water (20/1/180), pH 6.5.  7 - biological reagent containing the product according to any one of claims i to 6.  8 - Composition containing the product according to any one of claims 1 to 6 associated with a pharmaceutical vehicle.  9 - Composition according to claim 8, characterized in that it is injectable.