Soluble forms of low affinity Fc gamma receptors, process for their identification and dosage, a corresponding dosage kit, and applications

Receptors, characterized by the fact that they consist of the product obtained by affinity chromatography on a column coupled with 3 G8 antibodies or lectins or polyclonal anti-receptor FcR antibodies of a biological fluid of human origin, then by gel permeation. The spectrum of said product, electrophoresis acrylamide gel in reducing condition, comprising a major band corresponding to a molecular mass of between 72000 and 76000 daltons, and a number of minor bands. According to its purified form, the receptor consists of a glycoprotein with a molecular mass of between 72000 and 76000 daltons, recognized by ELISA and Western Blotting by the monoclonal anti-Leu 11b antibody. Application of said receptors to diagnosis and to follow-up treatment of diseases involving Fc receptors (infectious diseases, diseases of the autoimmune system, rejection of transplants, cancer and myeloma and AIDS), as well as to the study of human polymorphisms.

The present invention relates to soluble forms of low affinity receptors 
for the Fc fragment of IgG molecules; the present invention also relates 
to a method for the identification and assay of soluble forms of low 
affinity Fc gamma receptors, especially human soluble Fc receptors; it 
also relates to a kit of the reagents necessary for carrying out this 
method; it relates finally to the applications of this method for the 
diagnosis of pathological conditions in which the above-mentioned 
receptors are involved, such as infectious diseases, autoimmune diseases, 
transplant rejections, human complex diseases, cancers, myelomas and 
acquired immune deficiency syndrome (AIDS), for the therapeutic monitoring 
of the course of these conditions, as well as for the study of human 
polymorphisms. 
At the surface of many cells, in particular the cells of the immune system 
(macrophages, B lymphocytes, polymorphonuclear leukocytes, NK cells, and 
the like), there are receptors which permit a relationship between 
antibodies or immunoglobulins and these cells are involved in immunity. 
These receptors have an affinity for the Fc fragment of antibodies (FcR). 
This Fc fragment corresponds to the portion of immunoglobulins not endowed 
with antibody activity. The importance of these receptors is considerable, 
since their mediation makes possible the recognition of foreign antigens 
(viruses, tumor cells and the like) and the initiation of a cytotoxic 
reaction, by T lymphocytes or humoral, with secretion of specific 
antibodies by B lymphocytes; phagocytosis, that is to say the extraction 
from tissues and the blood of particles recognized as foreign by the cells 
of the reticuloendothelial system (monocytes, macrophages); cell 
interactions between different cells involved in immunity; the 
transplacental transfer of antibodies from mother to fetus; and the 
clearance of immune complexes whose pathogenic role in many conditions is 
known. 
The existence of a secretion of these receptors for the Fc fragment (FcR) 
by mouse T cell cultures activated by a specific activator has been 
demonstrated. These Fc receptor molecules, secreted in vitro under very 
special conditions of activation and culture (in particular, in medium 
devoid of all serum, which has a deleterious effect on the cells in 
culture), still possess the capacity to bind immunoglobulins through their 
Fc fragment, and they have hence been christened "immunoglobulin binding 
factor" (IBF). The existence of this type of IBF has been demonstrated for 
three classes of immunoglobulins, IgG, IgA and IgE, namely the factors 
IgGBF, IgABF and IgEBF. 
It has, moreover, been demonstrated that these IBF molecules are capable, 
when they are in contact with B lymphocytes in culture, or even myeloma 
cells in vitro, of completely inhibiting the activation of these cells 
and, as a result, the secretion of immunoglobulins by these B or myeloma 
cells. The molecules in question are hence secreted by cells belonging to 
the immune system under artificial conditions of in vitro culture, but are 
nevertheless endowed with a functional capacity to bind antibodies and to 
bring about a substantial suppression of the activation of the B 
lymphocyte system (lymphokine). 
The existence of an Fc receptor for mouse IgG immunoglobulins (FcR) in 
mouse serum has been demonstrated. These molecules have been defined both 
by their capacity to be recognized by a monoclonal antibody specific for 
mouse Fc gamma receptors (monoclonal antibody 2.4G2) and their functional 
capacity to be purified from immunoglobulin Fc fragments. This discovery 
is extremely important, since this lymphokine is secreted at a level which 
increases with age (level zero at birth, appearing at around the fifth day 
in newborn mice and becoming systematically detectable at around the 
seventh day), that this level increases in proportion to the presence of 
infections or, more generally, that the immune system is stimulated (mice 
kept in a microbe-free environment having strictly zero levels throughout 
their life), this level being, moreover, determined in a relative manner 
by genetic factors within a population of homozygotic mice. 
In addition, the existence has now been discovered, in particular in human 
serum, of a lymphokine having the capacity to bind to the Fc fragments of 
immunoglobulin, namely the circulating soluble serum form of low affinity 
Fc gamma receptors, also designated by the abbreviation Fc-gamma RLo, or 
alternatively "CD16". 
These receptors are present in biological fluids (in particular serum) in 
polymerized form, as could be observed by molecular weight measurements 
which, for example, yielded a value above approximately 700,000 for the 
glycoprotein which has been shown to have a molecular weight in the 
monomer state of 72,000 to 76,000. 
The subject of the present invention is a low affinity, soluble Fc .gamma. 
R type III receptor (or CD16), consisting of a glycoprotein of molecular 
mass 72,000-76,000 daltons which is recognized in ELISA and Western 
Blotting by the monoclonal antibody anti-Leu 11b. 
The subject of the invention is also a low affinity, soluble Fc .gamma. R 
type III receptor (or CD16), which consists of the product obtained by 
affinity chromatography, on a column coupled to 3G8 antibodies or to 
lectins (for example Lens Culinaris Agglutinin (LCA), wheatgerm agglutinin 
or concanavalin) or to anti-FcR receptor polyclonal antibodies, of a 
biological fluid of human origin, followed by gel filtration, the spectrum 
of the said product, in acrylamide gel electrophoresis under reducing 
conditions, containing a major band corresponding to a molecular mass of 
between 72,000 and 76,000 daltons and a plurality of minor bands, of which 
the main ones correspond to molecular masses, respectively, of: 
between 64,000 and 68,000 daltons 
between 51,000 and 55,000 daltons 
between 42,000 and 46,000 daltons 
between 33,000 and 37,000 daltons. 
The invention also relates to an Fc .gamma. R type III receptor essentially 
comprising the fraction of molecular mass 33,000-37,000 daltons, as 
appears in acrylamide gel electrophoresis in the presence of a reducing 
agent and a detergent agent such as sodium dodecyl sulfate (SDS). 
The biological fluids to which the present invention relates are, inter 
alia, serum and plasma fluids, cephalo-rachidian fluids, urines and 
ascitic fluids. 
A feature of all these receptors which may be mentioned is that, in the Dot 
Blot technique, they recognize a rabbit anti-FcR receptor polyclonal 
antibody. 
The invention finally relates to each of the fractions of the Fc .gamma. R 
type III receptor, as are defined above, as well as to all possible 
combinations of these fractions. 
The present invention also relates to a method for the identification, 
detection or assay of these lymphokines having the capacity to bind to the 
Fc fragments of immunoglobulin, in particular the human serum soluble Fc 
receptor.

The method according to the present invention for the identification or 
assay of soluble forms of the low affinity Fc .gamma. R type III receptor 
(or CD16) consists in: 
(a) binding, to a solid phase, a first antibody, which is a monoclonal or 
polyclonal antibody or alternatively a fraction of a monoclonal or 
polyclonal antibody (for example a Fab fragment), directed against a 
conformational epitope of the Fc .gamma. receptor to be identified or 
assayed; 
(b) washing the said solid phase to remove the said first antibody which is 
not coupled; 
(c) incubating the sample containing the Fc .gamma. receptor to be assayed 
in the presence of the solid phase coated with the said first antibody; 
(d) washing to remove the material not specifically bound to the said first 
antibody; 
(e) incubating, in the presence of the resulting solid phase, a second 
antibody, which is a monoclonal or polyclonal antibody, or alternatively a 
fraction of a monoclonal or polyclonal antibody (for example a Fab 
fragment), and which is an anti-Fc receptor recognizing the same category 
of Fc receptors as the first antibody, but by a completely different 
epitope; 
(f) washing to remove the said second antibody not specifically bound; 
(g) incubating, in the presence of the resulting solid phase, a third 
antibody, which is an antibody capable of specifically recognizing the 
said second antibody; 
(h) washing to remove the third antibody not specifically bound; and 
(i) assaying the third antibody bound, and deducing therefrom the quantity 
of Fc receptor initially present in the sample. 
In the stage (a), the monoclonal antibody 3G8 is used, in particular, as 
the first antibody. In the stage (e), a mouse IgM consisting of the 
monoclonal antibody anti-Leu 11b is used, in particular, as the second 
antibody. In the stage (g), a polyclonal antibody, namely a goat 
anti-mouse IgM antibody, is used as the third antibody. 
According to an especially preferred embodiment of the method according to 
the invention, in the stage (g), a third antibody labeled with an enzyme 
is employed and, in the stage (i), a colorimetric substrate for the said 
enzyme is added and, after the colorimetric reaction has been stopped, for 
example by adding aqueous sulfuric acid solution, the colorimetric change 
is read, from which the quantity of Fc receptor sought is deduced. In 
effect, the colorimetric change is proportional to the quantity of the 
third antibody and, as a result, proportional to the quantity of second 
antibody, and hence of Fc receptor initially present in the sample. 
Preferably, the enzyme is peroxidase, in particular horseradish peroxidase, 
and the colorimetric substrate for peroxidase is ortho-phenylenediamine, 
in the presence of hydrogen peroxide, the colorimetric reading being 
performed at 492 nm. 
The incubation of the stage (a), for the binding of the first antibody to 
the solid phase, is performed, for example, at a temperature of the order 
of 4.degree. C., for a period of time ranging from 8 to 12 hours. 
As regards the incubations of the stages (c), (e) and (g), these are 
performed, in particular, at room temperature, over a period of time 
ranging from 1 to 4 hours. 
FIG. 7 of the attached drawing explains the assay according to the 
invention. The lymphokine sought, which is assayed specifically by this 
method, contains a first site of capture by the first antibody (3G8), and 
a 10 second site of detection by the sequence .alpha.Leu 11b-goat 
anti-mouse IgM antibody and peroxidase. 
The subject of the present invention is also the kit of the reagents 
necessary for carrying out this method, this kit comprising: 
a solid support, in particular a microtitration plate, provided with a 
first antibody which is a monoclonal or polyclonal antibody, or 
alternatively a fraction of a monoclonal or polyclonal antibody, and which 
is directed against a conformational epitope of the Fc .gamma. receptor to 
be assayed; 
a second antibody, which is a monoclonal or polyclonal antibody, or 
alternatively a fraction of a monoclonal or polyclonal antibody, and which 
is an anti-Fc receptor recognizing the same category of Fc receptors as 
the first antibody, but by a completely different epitope; 
a third antibody, which is an antibody capable of specifically recognizing 
the second antibody; and 
a system for assay of said third antibody. 
These three antibodies, as well as a system for assay of the third 
antibody, have been defined above. 
As a detector of background, that is to say as a negative control, 
xenogenic sera (fetal calf, horse, goat) are used. 
As a standard enabling provision to be made for a qualitative positive 
control of the method, a positive known serum is used, and, in order to be 
able to quantify the method, an extract of human polymorphonuclear 
leukocytes are used. These polymorphonuclear leukocytes are purified 
according to classical purification techniques, are counted and are then 
lysed with a mild detergent which does not destroy the conformational 
structures of the polymorphonuclear cell receptors, which would no longer 
be able to be recognized subsequently during the test with the 3G8 
antibody, and, knowing the number of Fc receptors per polymorphonuclear 
cell, the number of polymorphonuclear cells at the start and the different 
dilutions at which these polymorphonuclear cells are tested, it is 
possible, by a simple relationship, to determine the correspondence 
between the soluble Fc receptors in the sera tested and a theoretical 
number of Fc receptors of polymorphonuclear cells detected in the cell 
lysate. A lysate, purified according to classical techniques, of human 
polymorphonuclear cells, lysed exclusively with an aqueous solution of 30 
mM octylthio beta-d-glucoside+5 mM DFP at 4.degree. C. 
The applications of this method are essentially diagnostic applications and 
applications for monitoring the therapeutic use of these lymphokines. The 
latter are, in effect, substances, the variations of which are linked to 
the fine activation of the B lymphocyte system and/or tho macrophage 
system and whose variation in level enables the state of stimulation of 
this system to be determined. This is of major importance in infectious 
diseases, autoimmune diseases, transplant rejections, cancers and 
myelomas, and acquired immune deficiency syndrome (AIDS). Moreover, after 
injection of the lymphokine (human Fc receptor), it is possible, by means 
of this test, to assay the variations in the serum levels and, as a 
result, to manage more effectively and with less toxicity the therapeutic 
capabilities of this lymphokine in human medicine. In effect, such a 
lymphokine is capable, like its in vitro homolog, of specifically 
inhibiting the secretion of an immunoglobulin class and hence of halting 
an effector mechanism in some pathological conditions, such as the 
abovementioned diseases, without thereby, as a result of its 
specificity--in effect, a receptor for IgG will bring about the inhibition 
of only IgG secretion--bringing about an inhibition of the secretion of 
the other immunoglobulin classes, hence enabling a resultant 
immunosuppression of the patient to be avoided. 
Likewise, by blocking the Fc fragments of immunoglobulins already engaged 
in an adverse antigen-antibody reaction (as in the case of antiplatelet 
autoantibodies in idiopathic thrombocytopenic purpura, for example), this 
lymphokine may prevent the relationship between these immune complexes and 
the cells of the reticuloendothelial system which would normally have 
phagocytosed these antigens (platelets in the case of idiopathic 
thrombocytopenic purpura), leading to the pathogenic effect 
(thrombocytopenia in the chosen example). 
The invention also relates to a medicinal product containing an Fc .gamma. 
R type III receptor or at least one fraction which is a constituent 
thereof, as are defined above. 
A particular embodiment of the assay method of the invention will be 
described below. 
(1) Preparation of the Microtitration Plates 
The microtitration plates used are 96-well U-bottomed poly(vinyl) chloride 
plates (manufactured by Societe "Dynatech"). 
The monoclonal antibody 3G8 is introduced, on the basis of 3 .mu.g per 
well, in 100 .mu.l of carbonate buffer at pH 9.6. The incubation lasts 8 
to 12 hours at a temperature of 4.degree. C. 
(2) Washing 
The plates are washed 6 times with PBS (Phosphate Buffered Saline) 
containing 0.1% v/v of Tween 20. This washing solution, which will be used 
again in subsequent stages of this assay, will now be designated more 
simply by the term "PBS Tween". 
(3) Deposition of the Sample to be Assayed 
100 .mu.l of the sample to be assayed, pure or diluted in PBS Tween which 
has been left for 4 hours at approximately 22.degree. C. (room 
temperature), are deposited in each well. 
(4) Washing 
This washing is performed in the same manner as in the stage (2). 
(5) Deposition of Anti-Leu-11b Antibody 
80 ng of anti-Leu-11b antibody (source: Beckton-Dickinson), diluted in 100 
.mu.l of PBS Tween, are deposited in each well. Incubation is performed 
for 2 hours at approximately 22.degree. C. 
(6) Washing 
This washing is performed in the same manner as in the stage (2). 
(7) Deposition of Goat Anti-mouse IgM Antibody Conjugated to Horseradish 
Peroxidase 
The abovementioned antibody (source: Jackson Immuno-Research Laboratories 
Inc.), diluted to 1/5,000 in PBS Tween producing in total 100 .mu.l per 
well, is introduced. Incubation is performed for 1 hour at 22.degree. C. 
(8) Washing 
This washing is performed in the same manner as in the stage (2). 
(9) Deposition of the Colorimetric Substrate for Peroxidase 
150 .mu.l per well of citrate phosphate buffer containing 4 mg/ml of 
ortho-phenylenediamine and 0.8 .mu.l/ml of hydrogen peroxide are 
deposited. 
The colorimetric reaction is stopped by depositing 75 .mu.l per well of 10% 
aqueous sulfuric acid solution, and the result is read at 492 nm in an 
ELISA reading apparatus. 
By means of the assay method according to the present invention, a molecule 
corresponding to a soluble serum form of the low affinity receptor for the 
Fc fragment of IgG has been demonstrated in human blood. 
The characterization of the Fc .gamma. R type III receptors of the 
invention will now be described. 
A normal human serum (68 ml) is passed, in a first stage, through a column 
of Sepharose coupled to 3G8 monoclonal antibodies (2 mg of 3G8 antibody 
per ml of Sepharose; total column volume: 1 ml), at room temperature. This 
column is then washed using 150 ml of a 0.1% strength solution of "Tween 
20" in phosphate buffered saline solution, and the material adsorbed is 
then eluted using 1/2M acetic acid solution, the fragments having a volume 
of 0.5 ml. These fractions are immediately neutralized using 3M sodium 
bicarbonate solution. The serum, before and after affinity chromatography, 
as well as the elution fractions, are tested for their content of soluble 
low affinity type III Fc receptor. The fractions which show immunological 
activity are then combined, and 0.2 ml of this pool of fractions is 
subjected to gel filtration on a column of volume 25 ml of "Superose 6" 
(Pharmacia); this "Superose 6" column is equilibrated in 50 mM ammonium 
bicarbonate solution at pH 8. The fractions having a volume of 0.5 ml are 
collected and tested using a direct ELISA assay method. This ELISA method 
consists in testing the elution fractions without passing through the 
stage of capture using the monoclonal antibody 3G8. These fractions are 
affixed directly to a solid support of the 96-well PVC plate type, and are 
then, after washing, reacted with the monoclonal antibody anti-Leu 11b, 
followed, after 2 hours, by a second anti-mouse IgM antibody labeled with 
peroxidase. 
FIG. 1 of the attached drawing shows the results of this second stage of 
the purification, hence corresponding to the gel filtration. Two curves 
are seen in this figure. The elution fraction numbers, as well as the time 
elapsed (expressed in minutes) are plotted as abscissae, and the optical 
density as seen on the reading of the wells in the direct ELISA method is 
plotted as ordinates. The curve in the form of a continuous line 
corresponds to the elution chromatogram of this gel filtration, and gives 
only the quantity of proteins without giving their characteristics. The 
second curve (.smallcircle.--.smallcircle.) represents the 
immunoreactivity of each of the elution fractions, in terms of purified 
soluble Fc receptor. It is hence observed that the peak between the 
fractions 20 and 25 bears virtually the whole of the purified soluble Fc 
type III receptor type immunoreactivity. 
The fractions 20 and 22, which are the richest in soluble Fc receptor type 
immunoreactivity since the optical density rises to around 2 units, were 
lyophilized, and they were subjected to acrylamide gel electrophoresis in 
the presence of SDS and a reducing agent. These fractions are shown in 
FIG. 2, and show the existence of a characteristic principal major band 
whose molecular weight is 72,000 to 76,000 daltons, but also bands of 
somewhat minor importance at around 66,000, 3,000, 43,000 and 35,000 
daltons. 
These bands are then transferred by horizontal flow onto a nitrocellulose 
filter, according to the technique known as "Western Blotting"; this 
cellulose nitrate paper is then reacted with the monoclonal antibody 
anti-Leu 11b and, after washing, this cellulose nitrate filter is reacted 
with anti-mouse IgM polyclonal antibody labeled with alkaline phosphatase. 
This is shown in FIG. 3. It is found that only one of these bands actually 
reacts in the Western Blotting technique, and is hence still recognized by 
the anti-Leu 11b antibody; it is the major band at 72,000-76,000 daltons. 
FIGS. 4 and 5 illustrate the results of adsorption experiments on columns 
of lectin. The experiment was as follows: 500 microliters of normal human 
serum were deposited twice in succession, at room temperature, in an 
equivalent volume of agarose coupled to different lectins. In FIG. 4, the 
lectin in question is that known as Lens Culinaris Agglutinin (or LCA). 
After the column eluant had been collected, that is to say after 
everything not retained by the column had been collected, the Fc receptor 
reactivity was measured using the indirect ELISA technique as described 
above. The immunoreactivity of the sera before passage through these 
lectins (solid column) and of the effluent, that is to say of the 
substances nor retained in the column, was tested, and an almost 90% 
adsorption of the material according to the invention, representing the Fc 
receptor immunoreactivity, on these columns is observed. This proves that 
this material is glycoprotein in nature, and contains glucose and/or 
mannose residues. 
The material adsorbed on this column was then eluted, that is to say 
detached; this is shown in FIG. 5; this was carried out by applying on the 
column a solution which enters into competition with the Fc receptor, 
namely a solution which contains the sugar specific for this lectin. In 
this instance, the elution solution is a 0.5M .alpha.-methyl mannoside 
solution. In this way, it was possible to elute the Fc receptors from this 
lectin column; this is shown in FIG. 5. The immunoreactivity, as obtained 
using the classical sandwich ELISA technique, of the different elution 
fractions, which are numbered as abscissae, are shown in this figure, and 
the ordinates indicate the reactivity in ELISA in terms of optical 
density. It is seen that a classical elution curve, with a peak around the 
second and third fraction, is indeed obtained. This confirms once more 
that the Fc receptor according to the invention, as it circulates in the 
serum, is indeed a glycoprotein. 
FIG. 6 illustrates the results of a so-called "Dot Blot" experiment. The 
Dot Blot technique is fully known and referred to in the literature. 
According to this technique, a cellulose nitrate filter is brought into 
contact with a solution containing the antigens to be assayed. These 
antigens are bound to this cellulose nitrate paper by the application of a 
vacuum on the other side of this porous filter. As a result of the action 
of the vacuum, the antigen molecules become bound to the cellulose nitrate 
paper. This paper is then reacted with different antibodies and enables 
colorimetric reactions to be observed by means of the use of 
enzyme-labeled antibodies. 
In this case, there were three samples, which were tested in parallel: 
on the bottom line, the sample is a cell extract, as also described above. 
This cell extract is tested at four dilutions, from the left to the right 
of the figure, and shows a classical dilution curve with an 
immunoreactivity corresponding to the colorimetric reaction observed on 
the nitrocellulose filter which decreases progressively in proportion to 
the dilution. 
On the middle line, in place of the cell extract, the sample is a serum 
considered to be positive from the results of the sandwich ELISA technique 
according to the invention. In this case also, it is found that the test 
shows a decrease in the optical density obtained, proportional to the 
dilution of serum which also goes from the left to the right of the 
figure, hence showing that the reaction in question is equally as specific 
as that of the sandwich ELISA technique. 
On the top line, a serum was tested which was considered to be negative 
from the results of the sandwich ELISA technique, that is to say, when it 
is reacted in a first stage with capture by 3G8 on a PVC plate and in the 
second instance with the system comprising anti-Leu 11b antibody and 
peroxidase-labeled anti-mouse IgM polyclonal antibody, which usually gave 
a negative result, the presence was observed, in this negative serum, of 
material antigenically recognized as an Fc receptor. The only difference 
here, compared with the sandwich ELISA technique, is, in the first place, 
that there is no capture stage, and hence no need for this antigen to be 
recognized by the 3G8 monoclonal antibody, and in the second place, that 
the antibodies used for visualizing the Fc receptor are not the Leu 11b 
antibody and anti-mouse IgM antibody but, directly, a rabbit anti-Fc 
receptor polyclonal antibody, manufactured in the laboratory by 
immunization of a rabbit with the purified Fc receptor according to the 
technique described in relation to FIG. 1, and a peroxidase-labeled rabbit 
anti-immunoglobulin antibody as second antibody. 
This shows that some sera considered to be negative by the use of the 
3G8/anti-Leu 11b system, are, however, soluble Fc type III receptor 
carriers. This suggests that an antigen polymorphism exists, some 
individuals having soluble plasma Fc receptors which are recognized by 
3G8/anti-Leu 11b, others having soluble serum Fc receptors which are not 
recognized by the G8/anti-Leu 11b system. 
By means of the assay and purification methods according to the invention, 
a molecule is made available which can be used in man in the treatment of 
different conditions: 
1--Monoclonal secretions of immunoglobulins, such as multiple myeloma or 
Kahler's disease. 
2--Immune complex disease or diseases accompanied by the presence of tissue 
or serum immune complexes, such as lupus erythematosus and other 
autoimmune diseases. 
3--Lymphomas and leukemias, in particular Burkitt's lymphoma. 
4--Bone marrow or organ (liver, kidney, heart, for example) transplant 
rejection, for curative or preventive purposes. 
5--Acquired immune deficiencies and in particular during infection by HIV 
virus.