FR2694767A1 - Monoclonal antibody against interleukin-6 receptor - for treating and preventing interleukin-6 related diseases, e.g. multiple myeloma, and for receptor assays - Google Patents

Abstract

A monoclonal antibody (MA6) directed against interleukin-6 receptor ( IL-6R) recognises an epitope which is the same as that recognised by one of the IgG1 monoclonal antibodies B-F19, B-R6 or B-N12 produced by hybridomas CNCM I-1256, 1255 and 1257 respectively. Also claimed are the hybridomas I-1256, 1255 and 1257, and a diagnostic reagent contg. the above Ab. Pref., these contain 0.5-5 (esp. 1) mg MAb/ml, MAb are pref. formulated with other MAbs to produce polymeric complexes between soluble IL-6R and the antibodies. Such complexes are eliminated move quickly than those formed from a single antibody. USE - MAb inhibit or suppress growth of IL-6 dependent cells so can be used to treat or prevent IL-6 associated diseases, specifically multiple myeloma; myeloid leukaemia; Castleman disease; systemic lupus erythematosus; kidney cancer and inflammatory arthropathy MAb can be coupled to a toxin, radioactive cpd. or other therapeutic agent. The MAb can also be used to detect or quantify (e.g. by ELISA) IL-6R, including its soluble forms and epitopes.

Description

ANTI-IL6R MONOCLONAL ANTIBODIES, AND THEIR
APPLICATIONS.

 The invention relates to novel monoclonal antibodies which recognize the human interleukin-6 receptor (IL-6R) and which can inhibit the interaction of IL-6 with IL-6R.

 The invention further relates to the use of said monoclonal antibodies for diagnosis and for obtaining drugs.

Interleukin-6, which was also initially called human B-cell stimulation factor 2 [KAWANO et al., Nature, 332, 83-85, (1988)], or hybridoma growth factor [BAZIN et al.
LEMIEUX, J. Immunology, 139, 78-87, (1987)] is one of the mediators of cellular immunity.

IL-6 is known to have a broad spectrum of biological functions. The effects of IL-6 on T cells [GORMAN et al., PNAS, 84, 7629-7633, (1987)], plasmacytomas [VAN DAMME et al., J. Exp. Med. 165, 914-919, (1987)], hepatocytes [ANDUS et al., FEBS
Lett. 221, 18-22, (1987)] and fibroblasts [KOHASE et al., Cell, 45, 659-666, (1986)], are described in the literature, as well as a large number of examples of the physiopathological role. IL-6, a review of which was recently published by HIRANO [International J. Cell Cloning, 9, 166-184, (1991); and Clin. Immun. Immunopath., 62, No. 1,
S60-S65, (1992)].

The IL-6 receptor present on cell membranes, which specifically binds IL-6 (gp80) has been described by TAGA et al. [J. Exp. Med., 166, 967-981, (1987)], Once IL-6 is attached to the receptor
IL-6R, gp80 associates with a molecule called gp130, which does not itself fix interleukin-6, but which transmits a signal [TAGA et al., Cell, 58, 573-581, (1989) ]. The IL-6 receptor in soluble form bound to IL-6 is also capable of inducing a signal transmitted by membrane gp130. Accordingly, it appears that IL-GR plays an important role in the pathophysiology of IL-6, and that its antagonists have an important role to play in the treatment of various diseases in which IL-6 occurs.

 In addition, for the purpose of diagnosis, it is desirable to have diagnostic reagents for measuring the concentration of the IL-GR receptor in soluble form.

 The present invention aims at the preparation of monoclonal antibodies recognizing the IL-GR receptor, and capable of inhibiting or suppressing the growth of IL-6 dependent cells, and / or of being used as reagents diagnosis.

 This objective has been achieved by the inventors by isolating new hybridoma cell lines producing IL-GR monoclonal antibodies.

The subject of the present invention is monoclonal antibodies directed against the IL-GR receptor, which antibodies are characterized in that they recognize an epitope chosen from the group consisting of:
The epitope recognized by the monoclonal antibody of isotype IgG1 called B-F19, produced by the hybridoma line deposited on August 12, 1992 with the CNCM

(NATIONAL COLLECTION OF CULTURES OF MICROORGANISMS, held by INSTITUT PASTEUR, 28, rue du Docteur Roux, 75724 PARIS CEDEX 15 (FRANCE), under the number of Dépôt I1256
the epitope recognized by the monoclonal antibody of IgGl isotype called B-R6, produced by the hybridoma line deposited on August 12, 1992 with the
CNCM, under the number of Deposit I-1255
The epitope recognized by the monoclonal antibody of IgG1 isotype called B-N12, produced by the hybridoma line deposited on August 12, 1992 with the
CNCM, under the number of Deposit I-1257.

The invention particularly encompasses class switching variants of BF-19 B antibodies.
R6 and B-N12, such as, for example, variants belonging to the IgG3, IgG1, IgG2b, IgG2a and other immunoglobulin subclasses of such variants can be obtained, for example, by the method described by US Pat.
COCO MARTIN et al. [J. Immunol.Methods. 145, p.1118, (1991)]
Monoclonal antibodies BF-19, B-R6 and B-N12 recognize three different epitopes of IL-6R, and further possess the following characteristics:
monoclonal antibody B-F19 competes with IL-6 for IL-6R binding to human cell lines, and inhibits the proliferation of IL-6 dependent cell lines.
Inventors have shown that this antibody recognizes monocytes and granulocytes.

 the monoclonal antibody B-R6 competes only partially with IL-6 for the IL-GR receptor, but nevertheless inhibits the proliferation of human IL-6 dependent cell lines. In this respect, it is 100 times more effective than the monoclonal antibody B-F19.

 The monoclonal antibody B-N12 does not compete with IL-6 at the IL-6R receptor, and furthermore, does not inhibit the proliferation of human IL-6 dependent cell lines.

 The monoclonal antibodies according to the invention can be used for the treatment of diseases in which IL-6 is involved, such as, for example, multiple myeloma, myeloid leukemia, Castleman's disease, systemic lupus erythematosus, renal carcinomas, inflammatory arthropathies, etc ...

 The monoclonal antibodies according to the invention can be used pure or coupled with toxins, or radioactive substances or any other therapeutic agent. They can also be encapsulated in liposomes.

 Pharmaceutical preparations comprising monoclonal antibodies B-F19, B-R6 and / or B-N12 may be in liquid form or in freeze-dried form. To stabilize these pharmaceutical preparations, proteins, sugars, sugar alcohols, amino acids can be used; for buffering, inorganic salts, preferably sodium phosphate in physiological saline (PBS, pH 7.4); different agents can also be used to increase their viscosity.

 The antibodies according to the invention are used at concentrations of between 0.5 and 5 mg / ml, preferably 1 mg / ml when used for therapeutic purposes. In general, therapeutic preparations containing the antibodies according to the invention are administered systemically, although local administration is not excluded.

 The monoclonal antibodies according to the invention can be used not only in therapy, but also for a prophylactic purpose.

 The antibodies according to the invention can be administered in isolation; preferentially, they will be administered together, or combined with other monoclonal antibodies to create polymeric complexes between the different monoclonal antibodies and the soluble IL-6R receptor. These complexes are then more rapidly removed than a monomeric complex comprising a single monoclonal antibody and the IL-6 receptor.

 Monoclonal antibodies B-F19, B-R6 and B-N12 can also be used as diagnostic reagents to identify IL-6R, or an epitope thereof, on the cell surface or in biological fluids. For such uses, the monoclonal antibodies can be coupled to markers, fluorescent, biotinylated, radioactive or other. It is also possible to use the monoclonal antibodies according to the invention in ELISA or RIA tests in order to assay the IL-GR receptor in biological fluids. The monoclonal antibodies according to the invention can also be used to identify and purify the IL-GR receptor, or epitopes of said receptor, from preparations of macromolecules capable of containing said receptor or its epitopes, such as, for example, cell lysates and fractions, peptide or oligosaccharide preparations resulting for example from digestion enzymatic of a cell fraction, or else obtained by chemical synthesis.

 The monoclonal antibodies according to the invention also allow the production of chimeric antibodies whose constant domain is of human origin (human immunoglobulin), and the variable part or preferably the hypervariable part is of murine origin (murine immunoglobulin). For the treatment of diseases in which IL-6 occurs, these chimeric antibodies can be used either pure, or coupled with toxins, radioactive substances, other drug substances, or encapsulated in liposomes.

 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 the monoclonal antibodies according to the invention.

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

I. PREPARATION OF MONOCLONAL ANTIBODIES
Example 1 - Immunization. fusion, clonaae and monoclonal antibody harvest B-F19, B-R6 and B-N12
An immunization protocol derived from that described by MATTHEW and SANDROCK [J. Immunol. Methods, 100, 73-82, (1987)].

Balb / c female mice were made tolerant to the cell line used for IL-GR transfection, as the mice were injected intraperitoneally with 10 x 10 6 CHO cells [TTPUCK, J. .Exp.Med. 108, 945, (1958)], which injection was followed 10 minutes, 24 hours and 48 hours later by a further intraperitoneal injection of 1 mg of cyclophosphamide in solution in saline buffer. This protocol was repeated two weeks apart four times. Two weeks after the last treatment, the mice underwent an intraperitoneal injection of 10 x 106 cells 46-20-5 (cells
CHO transfected with IL-GR). This protocol was repeated three times, at two week intervals, after which the fourth injection was made by intravenous injection, and the splenocytes were extracted four days later and fused. The fusion was carried out as follows: the splenocytes were fused with murine myeloma cells called X63Ag8653 (the splenocyte / myeloma cell ratio is 5: 1), in the presence of polyethylene glycol [KEARNEY et al., J. of
Immunol., 123, 1548, (1978)] The cell line
X63Ag8653 is deposited at the EUROPEAN COLLECTICN DE
CULTURES OF ANIMAL CELLS (ECACC), PHLS Center for Applied Microbiology and Research, Porton Down,
Salisbury, Wilshire, SP4 OJG, UK, under 12 deposit number
ECACC 850 114 20.

 The fused cell suspension was washed once, and cultured on selective medium (RPMI 1640, 10% heat inactivated horse serum, 4 mM glutamine, 13.6 mg / l hypoxanthine, 0.17 mg 1 μl of aminopterin, and 10 μg / ml of insulin).

 Ten days after fusion, supernatants from cultures in which hybridoma growth was observed were tested for the production of anti-IL-GR monoclonal antibodies.

 For this purpose, 75 μl of supernatant from each hybridoma culture were tested by flow cytometry on the CHO (IL-GR negative) and 46-20-5 (IL-6R positive) cell lines. Hybridomas producing antibodies recognizing the 46-20-5 line were retained and cloned four times, using the limit dilution method (seed density 0.2 cells per culture well). The three selected monoclonal antibodies were tested on different human cell lines, and only reacted with U266 lines [NILSSON et al., Clin. Exp.Immunol. 7: 447.

(1970)], RPMI8226 [MOORE et al., Proc.Soc.Exp.Biol.Med.

125, 1246-1250. (1967)], U937 [SUNDSTROM and NILSSON,
Int.J.Cancer., 17, 567-577, (1976)], and KG1 [KOEFFLER and GOLDE, Science, 200, 1153-1154, (1978)], all of which are known to possess an IL-6R receptor. Sandwich-type Elisa assays using the soluble IL-6R receptor and the various possible combinations of
B-F19, B-R6 and B-N12 have shown that these three monoclonal antibodies actually recognize said IL-GR receptor. In addition, monoclonal antibody B-F19 also labels monocytes and granulocytes; preincubation with human serum has no influence on this labeling, which shows that it is independent of the fragment
FcR.

EXAMPLE 2 In Vivo Production and Durability of B-F19, B-R6 and B-N12 Monoclonal Antibodies
Monoclonal antibodies are produced in large amounts in vivo by intraperitoneal injection of hybridoma cells B-F19, B-R6 and B-N12 in Balb / c mice. One week before injection of hybridoma cells, the mice receive an intraperitoneal injection of 0.5 ml of incomplete Freund's adjuvant. The ascites fluid is recovered 8 to 14 days after injection of the cells.

The monoclonal antibodies are then precipitated from the ascites fluid by addition of ammonium sulfate (45%), buffered to pH 7.7 by 0.02 mM Tris and attached to a Sepharose Q column. at the column are washed with 1% of
Tween 20 in 0.02 mM Tris, pH 7.7 and then eluted from the column with 0.02 mM Tris, 0.35 M NaCl, pH 7.7.

II. - BIOLOGICAL ACTIVITY OF ANTIBODIES B-F19
B-R6 AND B-N12
Example 3 - Inhibition of proliferation, induced by IL-6. of the IL-6 dependent human cell line XG1, because the antibodies B-F19, B-R6 and B-N12.

 The human cell line XG1 is a myeloma line, whose proliferation and properties depend on IL-6, and which has been described by KLEIN [BLOOD, 74, 749, (1989)]. The XG1 line is cultured in RPMI1640 medium in the presence of 10% fetal calf serum and beta mercaptoethanol, in the presence of 10 μg (= 1 unit) of IL-6 per culture well for three days. Cultures are performed in the presence of different concentrations of the B-F19, B-R6 and B-N12 antibodies.

The cells are then cultured for 16 hours in the presence of thymidine H3 which is incorporated in the neosynthesized DNA, which allows the evaluation of the cell growth, then they are collected, and the radioactivity is measured in a beta counter. The measurements are made in parallel, on cells cultured in the presence of different concentrations of antibodies B-F19, B-R6 and B-N12, on cells grown without antibodies in the presence of IL-6, and on cells cultured in vitro. the absence of IL-G. The average radioactivity measured on XG1 cells cultured in the absence of IL-6 is 6000 cpm. The results obtained in the absence of antibodies and in the presence of increasing concentrations of antibodies B-F19, B-R6 and B -N12 are shown in Table I below
TABLE I
RADIOACTIVITY (CPM)

<tb><SEP> B-F19 <SEP> B-R6 <SEP> B-N12
<tb> 0 <SEP> 44800 <SEP> 43500 <SEP> 48200
<tb> 1 <SEP> pg <SEP> 38300 <SEQ> 43300 <SEP> 39200
<tb> 10 <SEP> pg <SEP> 39900 <SEP> 41000 <SEP> 42300
<tb> 100 <SEP> pg <SEP> 40500 <SEP> 36400 <SEP> 40700
<tb> 1 <SEP> ng <SEP> 38900 <SEP> 26400 <SEP> 40700
<tb> 10 <SEP> ng <SEP> 36200 <SEP> 8700 <SEP> 38700
<tb> 100 <SEP> ng <SEP> 25000 <SEP> 2900 <SEP> 42400
<tb> 1 <SEP> Fg <SEP> 10600 <SEP> 1900 <SEP> 38900
<tb> 10 <SEP> fg <SEP> 4800 <SEP> 1200 <SEP> 37900
<Tb>
These results clearly show that cell growth is lower in the case of cells cultured in the presence of monoclonal antibodies
B-F19, and B-R6 than in that of the control cells cultured in the presence of IL-6 without antibodies. These two antibodies therefore inhibit the IL-6 dependent growth of the XG1 cell line. On the other hand, the B-N12 antibody has no effect on the growth of the XG1 cell line in the presence of IL-G.

Example 4 - Study of the comnetition between B-F19, B-R6.

B-N12 and IL-6 turn the IL-6R receiver.

 U266 cells are preincubated for 15 minutes in the presence of 100 ng IL-G, after which the monoclonal antibody to be tested is added. After 30 minutes of incubation, the cells are washed twice, and incubated with fluorescein-labeled goat anti-mouse serum for 30 minutes, washed again and analyzed by flow cytometry.

 Table II below represents the percentage of cells labeled with each of the different monoclonal antibodies used in decreasing concentrations. The values indicated in parentheses correspond to the percentage of cells labeled with the monoclonal antibody at the same concentration, without preincubation in the presence of IL-6.

TABLE II

<tb><SEP> B-F19 <SEP> B-R6 <SEP> B-N12
<tb> 1 <SEP> Rg <SEP> (73) <SEP> 59 <SEP> (71) <SEP> 69 <SEP> (70) <SEP> 73
<tb> 500 <SEP> ng <SEP> (66) <SEP> 64 <SEP> (74) <SEP> 71 <SEP> (68) <SEP> 71
<tb> 200 <SEP> ng <SEP> (76) <SEP> 59 <SEP> (80) <SE> 81 <SEP> (74) <SEP> 84
<tb> 100 <SEP> ng <SEP> (63) <SEP> 40 <SEP> (73) <SEP> 69 <SEP> (71) <SEP> 58
<tb> 40 <SEP> ng <SEP> (76) <SEP> 31 <SEP> (76) <SEP> 57 <SEP> (70) <SEP> 76
<tb> 20 <SEP> ng <SEP> (79) <SEP> 10 <SEP> (79) <SEP> 68 <SEP> (60) <SEP> 71
<tb> 10 <SEP> ng <SEP> (63) <SE> 63 <SEP> (59) <SEP> 66
<tb> 5 <SEP> ng <SEP> (42) <SEP> 47 <SEP> (33) <SEP> 55
<tb> 2.5 <SEP> ng <SEP> (28) <SEP> 29 <SEP> (15) <SEP> 27
<tb> 1 <SEP> ng <SEP> (10) <SEP> 8 <SEP>(<SEP> 5) <SEP> 8
<Tb>
It is observed that the binding of the monoclonal antibodies B-R6 and B-N12 to the IL-6R receptor is not inhibited by the presence of IL-6. This indicates that B-R6 and
B-N12 bind to the IL-6R receptor independently of IL-6. In contrast, B-F19 antibody competes with IL-6 at the IL-6R receptor, and binding of B-F19 antibody to the receptor
IL-6R is decreased at all concentrations by the presence of IL-G.

In another experiment, the U266 cells are incubated in the presence of three different concentrations of the monoclonal antibodies B-F19, B-R6 and
B-N12, for 30 minutes, then in the presence of 15 ng of biotinylated IL-6 for 30 minutes, after which cells are labeled with strepavidin phycoerythrin (PE) and counted by flow cytometry.

 In the presence of biotinylated IL-6 and in the absence of monoclonal antibodies, 48% of U266 cells are labeled with IL-6; this percentage is reduced to 10% when the cells are further incubated in the presence of 25 ng unlabeled IL-6; at 7% if preincubation takes place in the presence of 50 ng of unlabeled IL-6 and at 4% for preincubation in the presence of 100 ng of unlabeled IL-6.

 Table III below shows the percentage of cells labeled with biotinylated IL-6 in the presence of increasing amounts of the monoclonal antibodies according to the invention.

TABLE III

<tb> Concentra- <SEP> B-F19 <SEP> B-R6 <SEP> B-N12 <SEP>
<tb> tion <SEP> mAb
<tb> 50 <SEP> ng <SEP> 5% <SEP> 13% <SEP> 55%
<tb> 100 <SEP> ng <SEP> 8% <SEP> 21% <SEP> 55%
<tb> 500 <SEP> ng <SEP> 3% <SEP> 15% <SEP> 57%
<Tb>
The binding of IL-6 is completely blocked at all the concentrations tested, by the antibody
B-F19. In the presence of the B-N12 antibody, on the other hand, no inhibition of the IL-6 binding is observed, whereas the B-R6 antibody causes only a partial inhibition, independent of the dose of antibody used. .

In another series of experiments, U266 cells were preincubated with antibodies
B-Fl9, B-R6 at different concentrations for 15 minutes, after which the cells were incubated with 100 ng of biotinylated IL-6. After washing, the cells were labeled for flow cytometry by incubation with avidin PE.

In the absence of antibodies, the percentage of cells labeled with 100 ng of biotinylated IL-6 is 55%. The percentage of labeled cells, in the presence of 100 ng of biotinylated IL-6, and after preincubation with antibodies B-F19 or B-R6 is indicated in the Table
IV below.

TABLE IV

<tb> Antibody <SEP> B-F19 <SEP> B-R6
<tb> 2.5 <SEP> ng <SEP> 52 <SEP>% <SEP> 51 <SEP>%
<tb> 5 <SEP> ng <SEP> 45 <SEP>% <SEP> 41 <SEP>%
<tb> 10 <SEP> nQ <SEP> 29 <SEP>% <SEP> 27 <SEP>%
<tb> 20 <SEP> ng <SEP> 16 <SEP>% <SEP> 22 <SEP>%
<tb> 40 <SEP> ng <SEP> 10 <SEP>% <SEP> 19 <SEP>%
<tb> 80 <SEP> ng <SEP> 7 <SEP>% <SEP> 20 <SEP>%
<tb> 150 <SEP> ng <SEP> 5 <SEP>% <SEP> 19 <SEP>%
<tb> 300 <SEP> ng <SEP> 4 <SEP>% <SEP> 17 <SEP>%
<tb> 600 <SEP> ng <SEP> 4 <SEP>% <SEP> 16 <SEP>%
<tb> 1.25 <SEP> Rg <SEP> 3 <SEP>% <SEP><SEP> 16 <SEP>%
<tb> 2.5 <SEP> Rg <SEP> 3 <SEP>% <SEP> 18 <SEP>%
<Tb>
These results show that there is competition between B-F19 and IL-6 for the IL-GR receptor, with no binding of biotinylated IL-6 in the presence of antibody concentrations greater than 80 ng whereas in the case of antibody B-R6, it is not able to completely block the binding of IL-6 to its receptor (33% of maximal binding) even at a concentration of 2.5 Rg.

It is clear from these three experiments that as far as IL-GR receptor binding is concerned, there is only a marginal interaction between
IL-6 and the monoclonal antibody B-R6, whereas on the contrary the monoclonal antibody B-F19 competes with IL-6 for the IL-GR receptor, and inhibits IL-6 proliferation by this mechanism. dependent on the XG1 cell line. For these reasons, it is assumed that the mechanism of inhibition of B-R6 is different from that of B-F19, and all the more so since the inhibitory activity of B-R6 is 100 times greater than that of B-F19 .

EXAMPLE 5 - EDITOR recognition study between the monoclonal antibodies B-F19 B-R6 and B-N12
For this experiment, U266 cells were incubated for 30 minutes with biotinylated antibodies at a concentration of 250 ng per culture well and unlabeled antibodies at concentrations of 1 Ag, 500 ng, 250 ng, and 125 ng different combinations. After two washes, the cells are prepared for flow cytometry analyzes after incubation with streptavidin PE.

 The results, expressed as a percentage of labeled cells are shown in the following Table V.

TABLE V

<tb> mAb <SEP> labeled <SEP> B-F19 <SEP> B-R6 <SEP> B-N12
<tb> mAb
<tb> no <SEP> marked
<tb><SEP> none <SEP> 61 <SEP> 42 <SEP> 73
<tb> B-F19 <SEP> 1 <SEP> Rg <SEP> 3 <SEP> 40 <SEP> 85
<tb><SEP> 500 <SEP> ng <SEP> 41 <SEP> 80
<tb><SEP> 250 <SEP> ng <SEP> 35 <SEP> 90
<tb><SEP> 125 <SEP> ng <SEP> 48 <SEP> 94
<tb> B-R6 <SEP> 1 <SEP> Wg <SEP> 57 <SEP> 5 <SEP> 91
<tb><SEP> 500 <SEP> ng <SEP> 38 <SEP> 73
<tb><SEP> 250 <SEP> ng <SEP> 43 <SEP> 87
<tb><SEP> 125 <SEP> ng <SEP> 68 <SEP> 88
<tb> B-N12 <SEP> 1 <SEP> Rg <SEP> 54 <SEP> 39 <SEP> 8
<tb><SEP> 500 <SEP> ng <SEP> 68 <SEP> 37
<tb><SEP> 250 <SEP> ng <SEP> 60 <SEP> 43
<tb><SEP> 125 <SEP> ng <SEP> 65 <SEP> 38
<Tb>
These results show that none of the antibodies considered competes with another antibody than itself, and therefore that they recognize different epitopes.

Example 6 Scatchard Analvse of the B-R6 and B-N12 Monoclonal Antibody Binding with IL-6R
For each experiment, 40 Wg of monoclonal antibody is incubated in the presence of 125 I-labeled sodium salt (0.5 mCi) in 180 ml of PBS buffer.

Then 10 g of chloramine T (0.4 mg / ml) is added, and the reaction is stopped after one minute by addition of 10 Al sodium bisulfite (0.5 Rg / ml). The monoclonal antibodies labeled in this way are separated by chromatography on a Sephadex G-25 column of free iodine. 2.5 x 106 cells per well, line 46-20-5, or U266 are incubated with different concentrations of 125 I-labeled 125 I-labeled B-R6 or B-N12 antibodies, and one excess (500-fold). ) of unlabeled B-R6 and B-N12 antibodies. The incubation is carried out for 120 minutes at 4 ° C. in a total volume of 1 ml of 1% albumin PBS buffer. After three washes, the radioactivity is measured in a gamma counter. The measurements made allow the calculation of the specific constants of the binding reaction.
- maximum binding: Bmax,
free ligand ratio on bound ligand: B / F,
- dissociation constant: Kd,
- number of fixation sites / cell: n.

These constants are shown for each antibody and each cell line in Table VI below.
TABLE VI

<tb><SEP> 46-20-5 <SEP> U266
<tb> B-R6 <SEP> Brnax <SEP> (cpm) <SEP> 29200 <SEP> 31500
<tb><SEP> B / F <SEP> 0.0396 <SEP> 0.032
<tb><SEP> Bmax <SEP> (M) <SEP> 23.9x10 <SEP> 12 <SEP> 25.7x1012 <SEP>
<tb><SEP> Kd <SEP> (M) <SEP> 0.6 <SEP> x10-9 <SEP> 0.8x10-9 <SEP>
<tb><SEP> n <SEP> 9540 <SEP> 6160
<tb> B-N12 <SEP> Bmax <SEP> (cpm) <SEP> 38000 <SEP> 34000
<tb><SEP> B / F <SEP> 0.0282 <SEP> 0.02
<tb><SEP> Bmax <SEP> (M) <SEP> 30.6x10-12 <SEP> 27.3x10-12 <SEP>
<tb><SEP> Kd <SEP> (M) <SEP> 1.08x10-9 <SEP> 1.36x10-9 <SEP>
<tb><SEP> n <SEP> 12200 <SEP> 6550
<Tb>
EXAMPLE 7 Measurement of Soluble ILI-GR by Sandwich ELISA
The operative protocol is the following
Incubation of the microtiter plates with the B-N12 antibody (0.5 μg / well in 100 μl of PBS buffer) overnight at 4 ° C.

 Saturation with PBS buffer at 5% albumin for 90 minutes at room temperature.

Distribution in the wells of the microtiter plate of variable amounts of culture supernatant of the 46-20-5 cell line, containing the IL-GR receptor in soluble form, and adjustment to 100 l by PBS buffer; incubation for 2 hours at 37 ° C .;
- Distribution of biotinylated B-R6 antibodies in the wells of the microtiter plate (0.5 Rg in 100 l of buffer per well, in the presence of 0.005% Tween), incubation for 2 hours at room temperature
- Distribution in the wells of a solution of streptavidin labeled with peroxidase, then its substrate (O-phenylenediamine dihydrochloride) and measurement of the optical density at 405 nm.

The results are reported in the Table
VII next
TABLE VII

<tb> SURNAGEANTS <SEP> 46-20-5 <SEP> D0405 <SEP>
<tb><SEP> 100 <SEP> 1.537
<tb><SEP> 50 <SEP> l <SEP><SEP> 1,247
<tb><SEP> 25 <SEP> l <SEP> 1,073
<tb><SEP> 12 <SEP> l <SEP> 0.819
<tb><SEP> 6 <SEP> l <SEP> 0.558
<tb><SEP> 3 <SEP> til <SEP> 0.396
<tb><SEP> Noise <SEP> of <SEP> Background <SEP> 0.099
<Tb>
These results show that even on 3 l of supernatant, the presence of the IL-6R receptor can be easily detected.

Claims (10)

 1) Monoclonal antibody directed against the IL-GR receptor, characterized in that it recognizes an epitope chosen from the group consisting of:  the epitope recognized by the monoclonal antibody of IgGl isotype called B-F19, produced by the hybridoma line deposited on August 12, 1992 with the C.N.C.M., under the number of Deposit I1256  the epitope recognized by the monoclonal antibody of IgGl isotype called B-R6, produced by the hybridoma line deposited on August 12, 1992 with the C.N.C.M. under the number of Deposit 1-1255  the epitope recognized by the monoclonal antibody of IgG1 isotype called B-N12, produced by the hybridoma line deposited on August 12, 1992 with the C.N.C.M., under the number of Deposit I-1257.  2) Use of an antibody according to claim 1, for obtaining drugs.  3) Use according to claim 2, characterized in that said drugs are intended for the treatment or prophylaxis of diseases involving IL-6.  4) Use according to claim 3, characterized in that said drugs are intended for the treatment or prophylaxis of multiple myeloma, myeloid leukemia, Castleman's disease, systemic lupus erythematosus, renal carcinoma, and inflammatory arthropathies. , and other diseases IL-6 dependent.  5) Pharmaceutical compositions, characterized in that they comprise, as active ingredient, at least one antibody according to claim 1.  6) Pharmaceutical compositions according to claim 5, characterized in that said antibody is present at a concentration of between 0.5 and 5 mg / ml, preferably 1 mg / ml.  7) Use of an antibody according to claim 1 for the detection of the IL-6R receptor, or an epitope thereof.  8) Use of an antibody according to claim 1 for the determination of the soluble IL-6R receptor.  9) Diagnostic reagents, characterized in that they comprise at least one antibody according to claim 1.  10) Hybridoma, characterized in that it belongs to one of the following lines:  - line producing antibody B-F19, filed August 12, 1992 with the C.N.C.M., under the number of Depot 11256  antibody-producing line B-R6, deposited on August 12, 1992 with C.N.C.M. under the number of Depot I-1255.  - line producing antibody B-N12, filed August 12, 1992 with the C.N.C.M., under the number Deposit I-1257.