Immuno-suppressive material and process for preparing same

An immuno-suppressor is described in the form of an immuno-suppressor serum and/or an IS-IgG extracted from this serum, produced by immunisation of an animal with antibody-covered cells; also described is the process for making said immuno-suppressor and its use for suppressing the humoral and/or cellular immune response.

FIELD OF THE INVENTION 
The present invention is directed to an immuno-suppressive material and 
process for the production thereof, as well as methods of use thereof. 
STATE OF THE ART 
Immuno-suppressive techniques occupy an important position in human 
medicine. The most important indications for this therapy are: organ 
transplants, immune-hematological syndrome, immuno-pathologically caused 
diseases of the liver and collagen diseases. 
The immuno-suppressives utilized heretofore in clinical work, have a 
substantial number of side effects, partly as a result of toxicological 
properties of the materials, partly as the result of a broad 
immuno-suppressive action. 
Thus, Cyclosporin which is utilized in organ transplants, while showing 
exceedingly powerful immuno-suppressive action, possesses a number of 
toxic side effects, for example, nephro-toxicity and hepato-toxicity, 
which give rise to problems in clinical use. An important role in the 
treatment of rejection crisis after organ transplants, has been played by 
anti-lymphocyte globulin (ALG). Unfortunately, the production of such 
product having assured immuno-suppressive action and minimal toxicity, 
still remains problematical. 
Next, encouraging results were shown by the action of selectively acting 
monoclonal antibodies (for example, OKT 3, T 12, anti-lymphoblast 
antibodies). Also here, further studies have failed to fulfill the desired 
goals completely. 
SUMMARY OF THE INVENTION 
The goal of the present invention is therefore to provide an 
immuno-suppressive material which has a powerful suppressive action solely 
upon a predetermined antigen (antigen-specific) is neither toxic nor 
cytotoxic and induces long acting immuno-suppression after treatment for a 
predetermined but limited length of time. 
In accordance with the present invention this problem was solved by an 
immuno-suppressive material obtained by the immunization of animals with 
cells coated with antibodies. 
This immuno-suppressive material, as provided in accordance with the 
present invention, comprises an immuno-suppressive serum and/or an 
immuno-suppressive IgG. 
Among the cells suitable for this purpose there may be mentioned blood 
cells, spleen cells, bone marrow cells and the like, among which blood 
cells are particularly preferred. 
The antibodies may be provided in the form of monopolyclonal antibodies or 
antisera, preferably polyclonal antibodies are utilized. 
The recovery or production of the immuno-suppressive material in accordance 
with the procedure of the present invention, occurs in that cells taken 
from an animal are incubated with homologous or heterologous antibodies, 
these cells are injected into an animal, after an incubation period, blood 
is extracted from the injected animal and, in ways known to the art, the 
serum (that is to say, immuno-suppressive serum, hereinafter IS-serum), is 
isolated and also IgG, that is to say, immuno-suppressive IgG, hereinafter 
IS-IgG, is separated in a manner also known to the art. 
In the separation of the IS-serum from the blood, the blood is permitted to 
coagulate for from about 3 to about 24 hours, suitably at room 
temperature. The material is then subjected to centrifugation, suitably 
for about 10 minutes at about 4,000 rpm, whereby the serum is separated as 
the supernate and can be removed. The IS-serum is then subjected to ion 
exchange chromatography, suitably on a Mono-Q or Q-Sepharose Fast Flow 
column (Trademarked product sold by Pharmacia Fine Chemicals, AB, Uppsala, 
Sweden), in accordance with the general procedure set forth in Separation 
News, 1982/1, published by Pharmacia Fine Chemicals, which is incorporated 
herein by reference and made part hereof. Elution with phosphate buffer 
(suitably 0.3M, pH 6.5) yields 3 peaks of which the first is the IgG peak. 
Yet a further purification step may be carried out utilizing a Protein A 
gel, suitably Affi-Gel Protein A Agarose (manufactured by Biorad) in 
accordance with the procedures set forth in the Affi-Gel Protein A MAPS II 
Instruction Kit Manual, published by Biorad, the disclosure of which is 
incorporated by reference and made part hereof. 
In this procedure the IgG solution is placed upon the column in the 
provided binding buffer and eluted with the provided elution buffer. The 
eluate is rebuffered on Sephadex G25. The product may, if desired, be 
further purifed to provide anti-human IgG antibodies by affinity 
chromatography utilizing Sepharose coated with human IgG in accordance 
with the procedures set forth in Affinity Chromatography Principals and 
Methods, published by Pharmacia Fine Chemicals, 1983/2, which is 
incorporated herein by reference and made a part hereof. 
The injection or processing of the cells taken from the animal, (i.e., 
blood, blood cells and the like), with homologous or heterologous 
antibodies, is carried out according to the procedures of the present 
invention, in methods and conditions well known in the art. For example, 
the incubation time is from about 30 to about 60 minutes and the 
incubation is suitably carried out at 4.degree. C., room temperature, 
(that is to say, 22.degree. C.) or even at 37.degree. C., preferably at 
room temperature. 
The cells covered with antibody obtained as a result of such incubation, 
are thereafter injected into an animal. In accordance with the preferred 
embodiment of the process of the present invention, the cells coated with 
antibodies after incubation are washed prior to injection into the animal 
Suitable media for this washing are cell culture media, such as the 
conventional phosphate buffer solution, RPMI, Hanks, and the like. 
The injection of the cells coated with antibody into the animal is carried 
out in accordance with conventional immunization methods and under 
conventional immunization conditions. Suitably, the injection may take 
place several times, suitably 3 times. It is preferred to utilize an 
incubation time of from about 3 to 24 days, wherein the animals are 
maintained at room temperature. 
The cells taken from one animal and coated with antibodies can, in 
accordance with the present invention,. be reinjected into the same animal 
or, alternatively, they can be injected into another, that is to say, an 
animal different from the donor animal, wherein in the latter case, the 
animal may belong to either the same or a different species. Examples of 
animals suitable in the present invention may be mentioned: rats, mice, 
rabbits, and the like. 
For example, in accordance with the present invention, rat IS-serum was 
produced in the highly incompatible BN.fwdarw.LEW system (as schematically 
illustrated in FIG. 1), as well as in other systems and similarly 
utilized. 
Further, rabbit IS-serum can be obtained by the immunization of rabbits 
with autologous or allogeneic blood cells covered with antibodies. 
A serum produced by use of antigen-bound autologous or heterologous 
antibodies demonstrates strong suppressive action without containing 
cytotoxic antibodies. Thus, the undesired side effects associated 
therewith are eliminated. A single treatment with such a serum leads to 
the suppression of primary and secondary antibody response. (compare for 
example, FIG. 2), as well as to the suppression of proliferative cellular 
immuno response 
It has been found that a single dose of the immuno-suppressive material of 
the present invention, together with antigen, leads also to the 
suppression of the secondary antibody response Furthermore, it has been 
shown that even repeated administration of antigen does not remove the 
suppressive effect. This is a substantial advantage in relation to the 
immuno-suppression agents generally utilized at the present time which 
require repeated administration for the maintenance of suppression. 
Experiments have further shown that the primary antibody response (see FIG. 
3), as well as the secondary antibody response (see FIG. 4), are 
suppressed even against a third party donor through a single 
administration of the immuno-suppressive material of the present 
invention. Thus, an immuno-suppressive material of the present invention 
that has been obtained by BN cells coated with antibodies, suppresses not 
only the primary but also the secondary antibody response against 
unrelated third party donors, for example, DA and WF. 
It was found that the suppression is dose dependent. According to the 
amount of IS-serum administered, there may be obtained a weak or total 
suppression of the antibody response. When lymphocytes are incubated in 
vitro with IS-serum, the Graft versus Host assay, shows a suppression of 
the proliferative T cell response. 
Experiments have shown that the IS-serum produced in accordance with the 
present invention, contains a broadly reactive immuno-suppressive factor. 
The immuno-suppressive activity can be shown to be present in the IgG 
fraction. For example, thus it was found that 0 8 mg. of IS-IgG separated 
from the IS-serum by means of ion exchange chromatography, gave rise to a 
significant suppression of the antibody response in comparison to the 
untreated control or to a control treated with "normal" IgG (see FIG. 5). 
The effect can be shown, either in donor specific (BN-LEW) as well as 
non-specific (DA-LEW) systems. 
The experimental result shown in FIG. 5, was obtained in that 0.8 mg. of 
IS-IgG, together with BN or DA blood, were injected into LEW rats, while 
in the control animals, only blood or blood in connection with 0 8 mg. IgG 
from the serum of untreated rats, was utilized 
Further studies gave the result that the immuno-suppressive factor is an 
antibody which is directed against Neo-determinants of IgG, which are 
formed through antigen binding action (anti-Neo-IgG antibodies). 
The immuno-suppressives formed in accordance with the present invention can 
be used for the suppression of humoral and/or cellular immune response and 
thus, can be successfully used in immuno-suppressive therapy as has been 
demonstrated hereinabove.

WAYS OF CARRYING OUT THE INVENTION 
The following examples serve to serve the further explanation of the 
invention under consideration. 
GENERAL PROCEDURES 
The following procedures exemplify generally accepted methods of isolation 
and purification. 
Procedure 1 
A. Immunization of Rabbits 
New Zealand white rabbits (ca. 2 to 3 kgs.) are utilized. Approximately 1 
ml. of blood is drawn from the rabbits and incubated with 1 mg. of human 
anti-rabbit erythrocyte IgG for 30-60 minutes at room temperature, the 
antibody excess is removed by washing 3 times with phosphate buffer. 1 ml. 
of this preparation is injected 3 times i.v., into the rabbit at 3 day 
intervals. 
B. Mouse IS-Serum 
(i) BALB/c mice were utilized. 0.05 to 0.1 ml. of blood was removed from 
the mouse, incubated with 0.1 mg. human anti-mouse erythrocyte IgG for 
30-60 minutes at room temperature, the excess antibodies removed by 
washing 3 times with phosphate buffer 0.1 ml. of this product was injected 
3 to 5 times i.v , in weekly intervals into the mouse. 
(ii) In an alternative procedure, anti-mouse IgG 20 antibodies are 
extracted from the sera of human patients treated with monoclonal mouse 
IgG antibodies. IgG is prepared in the usual manner from this sera and 0.1 
mg. of mouse IgG is then incubated with 0.1 mg. of human anti-mouse IgG 
for 60 minutes at room temperature and then as described above, repeatedly 
injected into the BALB/c mice. Three days after the last immunization, the 
spleen cells are removed from the mice and used for the preparation of 
monoclonal anti-human IgG antibodies by the well known procedure of Kohler 
and Milstein, Nature (1975) 256; 495. 
Procedure 2 
Preparation of IS-Serum 
Blood is taken from the animals inoculated in accordance with the methods 
of Procedure #1 above and set aside at room temperature for from about 3 
to about 24 hours. The blood is thus permitted to coagulate and then is 
centrifuged for 10 minutes at 4,000 rpm. An initial charge of 1 ml. of 
blood yields approximately 0.3 ml. of supernatant serum. 
Procedure 3 
A. IgG Separation by Ion Exchange Chromatography 
There are utilized: 
1. Mono-Q (5.times.50 mm. column) (Mono-Q is a product of Pharmacia). 
2. Phosphate Buffer A (0.025 M pH 6.7) 
3. Phosphate Buffer B (0.3 M pH 6.5) 
0.2 ml. of the IS-serum is diluted with 0.8 ml. of phosphate buffer A and 
placed on the top of the column Elution is carried out at a flow rate of 2 
ml./minute utilizing a gradient of 0/50%/100% phosphate buffer B for a 
total separation time of 15 minutes 1 ml. fractions were collected to 
yield 3 peaks. The first peak contains the IgG fraction. 
The aforesaid IgG fraction is rebuffered over on a PD10 (Sephadex G25, 
Pharmacia) column which had previously been equilibrated with 20 ml. of 
phosphat buffer, pH 7.4. 2.5 ml. of IgG solution are placed on the column 
and eluted with phosphate buffer. There are thus provided approximately 3 
ml. of rebuffered IgG solution which are further purified in accordance 
with procedure #4. 
Procedure 4 
Purification of IgG on a Protein A Column 
There is utilized the Affi-Gel Protein A MAPS II (manufactured by Biorad) 
column , utilizing the binding buffer and elution buffer provided in the 
kit. The column volume is 2 ml. 3 ml. of the IgG solution obtained in 
accordance with with Procedure 3 are mixed with 3 ml. of the binding 
buffer and placed on the column. 30 ml. of binding buffer are washed 
through the column, followed by 10 ml. of the elution buffer. The 10 ml. 
of eluate are rebuffered on a PD10 column in pH 7 phosphate buffer in 
accordance with the procedure set forth in Procedure #3. The product, 
(i.e., 10 ml. IgG solution) is treated with 2 g. of Sephadex 25 to yield a 
concentrated IgG solution of about 1 ml. This product may be utilized as 
the IgG solution in the following examples or, if desired, it may be 
further purified by affinity chromatography in accordance with Procedure 
5. 
Procedure 5 
Separation of Antibody by Affinity Chromatography 
There is utilized a Sepharose column of 0.5 ml. containing Sepharose coated 
with human IgG at a concentration of 0.5 mg. IgG/ml. of Sepharose. 0.5 ml. 
of IS-IgG solution (IgG concentration 0.5 mg./ml.) are placed on the 
column. Thereafter, 2.5 ml. of elution buffer (as utilized in Procedure 4) 
are utilized for elution. The resulting IS antibodies (2.5 ml.) are 
rebuffered on the PD10 column (in accordance with the procedure of 
Procedure #3), there is thus obtained purified IS antibody of volume 3 ml. 
which may be utilized in this form. 
EXAMPLE I 
A. Rat IS-serum was produced, as schematically illustrated in FIG. 1, in 
the highly incompatible BN-LEW system, in the following manner: 
1 ml. of BN blood was incubated at 20.degree. C. for 30 minutes with 0.2 
ml. of LEW-anti-BN serum. After the incubation, the excess antiserum was 
washed out and the cells resuspended to the original volume with phosphate 
buffer solution 1 ml. of cell suspension was injected 3 times i.v., into 
LEW rats in weekly intervals. The serum of the treated animals (IS) was 
collected one week later Only sera without cytotoxic anti-BN activity were 
used. In the experiments, IS serum and allogenic blood or leukocytes were 
injected i.v. into the recipients. The cytotoxic antibody titer was 
determined in weekly intervals. 
A sample of IS-serum produced as described above, demonstrates a strong 
suppressive action without however containing cytotoxic antibodies. This 
removes the undesired side effects associated therewith A one-time 
treatment with such a serum in the rat, leads to suppression of the 
primary and secondary antibody response (see FIG. 2) with respect to blood 
cells with which the IS-serum was produced Furthermore, it has been shown 
that repeated antigen administration does not remove the suppression once 
it has been induced. This again, shows a substantial advantage with 
respect to presently generally utilized immuno-suppressives which require 
continual administration, in order to maintain the suppression. 
B. The following experiments show that not only the primary antibody 
response, but also the secondary antibody response against a third party 
donor is suppressed by a single administration of IS-serum. 
1 ml. of DA- or WF- blood was injected into LEW rats, together with 1 ml. 
of the IS-serum produced as above. Control animals received soley DA or WF 
blood. Examination showed suppression of the primary antibody response in 
the rats treated with IS-serum. The results of these experiments are set 
forth in FIG. 3. 
As shown in FIG. 4, 3 weeks after treatment with IS-serum, the animals were 
boosted with 1 ml. of DA or WF blood (without IS-serum). A significant 
suppression of antibody response to the DA or WF cells was found. 
EXAMPLE II 
Suppression of the Antibody with Thromobocyt Transfusion 
Thrombocyte transfusion is a form of support therapy frequently utilized in 
human medicine. Patients who do not have hematological neoplasty can be 
given high dosage chemotherapy by means of thrombocyte substitution, 
whereas thrombocytopenic patients with primary bone marrow diseases, 
(i.e., acute leukemia), the thrombocyte transfusion by giving rise to a 
longer survival time, permits an effective therapy. The main problem of 
thrombocyte substitution is that generally speaking, sensitization of the 
patient against donor antigens will occur, which reduces the effectiveness 
of the transfusion. An experiment in this direction was carried out with 
LEW rats, which gave rise to the results which are set forth in FIG. 6. 
To LEW rats, there was given a 3 times administration (on days 0, 3 and 7), 
of 5.times.10.sup.6 BN leucocytes (control animals) or 5.times.10.sup.6 BN 
leucocytes, together with 0.2 ml. of the IS-serum obtained in the 
accordance with Example I. Three weeks later, both groups were boosted 
with 10.sup.9 BN thrombocytes (without IS-serum). In these experiments, 
the control LEW rats generated a strong anti-BN antibody response whereas 
with a simultaneous provision of IS-serum with the leucocytes to the LEW 
rats, the anti-thrombocyte antibody response was almost entirely 
suppressed. 
EXAMPLE III 
Suppression of Humoral Sensitization after Blood or Leucocyte Transfusion 
In clinical kidney transplants, it has been found that the preparation of 
the potential recipient by means of blood transfusions, leads to a 
substantial improvement of survival of the transplant. However, 
pretransplant blood transfusion can lead to the formation of cytotoxic 
antibodies. If these antibodies are directed against the potential donor, 
the transplant cannot be carried out because of the risk of a hyperacute 
rejection. A portion of potential receptors, the so-called highly 
immunized patients, have a high percentage of cytotoxic antibodies so that 
it is exceedingly difficult to find a suitable donor. Even where a 
transplant for these patients is possible, the survival time of the 
transplant is shorter. 
In living related kidney transplants, donor specific blood transfusions are 
often carried out. Approximately a third of the recipients develop 
anti-donor antibodies and so cannot be transplanted with the kidney of the 
prospective living donor. By avoiding the sensitization effect of the 
pretransplant blood transfusion, the positive influence of the blood 
transfusion upon the transplant survival can be considerably improved. It 
was found that the simultaneous treatment with blood transfusion and 
IS-serum, leads to a suppression of the cytotoxic and antibody response. 
This suppression remains even in further transplants. 
Experiments results in this direction are set forth in FIG. 2. The results 
were obtained in the following manner. 
LEW rats are injected on day 0 with 1 ml of the IS-serum obtained in 
accordance with Example I, together with 1 ml. of BN blood The control 
animals received only BN blood and normal LEW serum. As shown in FIG. 2, 
the groups of rats which received the IS-serum showed total suppression of 
the cytotoxic antibody response Three weeks later, both groups, that is to 
say, the control group and the group to which IS-serum was administered, 
were boosted with 1 ml of BN blood without IS-serum. The induced 
suppression remained even after the booster transfusion. 
Experiments in the rat have shown that the administration of a high dose of 
allogenous erythrocytes can lead to suppression of the antibody response. 
In order to eliminate a possible erythrocyte mediated suppression in blood 
transfusion experiments, LEW rats were transfused with BN leucocytes with 
or without administration of IS-serum (leucocytes are known to be strongly 
immunogenic and can lead to acceleration of rejection). Thus, on day 0, 
the LEW rats were injected with 5.times.10 BN leucocytes and 0.5 ml. of 
the IS-serum produced in accordance with Example I. The thus obtained 
results are shown in FIG. 7. As can be seen from FIG. 7, a single 
administration of IS-serum gave rise to a total suppression of 
antileucocyte antibody response which persisted even after repeated 
booster transfusions (without IS-serum) with cells of the same donor 
EXAMPLE IV 
Suppression of the Graft verus Host (GvH Reaction) 
The GvH reaction has a considerable influence upon the success of clinical 
bone marrow transplants. In spite of many attempts, there is no clear 
solution for this problem The experiments with rats have shown that the 
pretreatment of the lymphocyte donor or the incubation of lymphocytes with 
IS-serum, lead to a considerable reduction of the GVH reaction. The 
experiments were carried out in the following manner. 
5.times.10 LEW spleen cells were twice incubated at 22.degree. C. for 90 
minutes with 0.5 ml. of the IS-serum produced 5 in accordance with Example 
I. After each incubation, the cells were washed. LBN-rats (that is to say, 
LEW.times.BN) were injected with 5.times.10 with treated or untreated 
spleen cells subcutaneously into the footpads. Five days later, the 
popliteal lymph nodes were removed and weighed. The results obtained are 
set forth in FIG. 8. As will shown, there is demonstrated, a very strong 
suppression of the GvH reaction. 
EXAMPLE V 
Possible Prolongation of Kidney Transplant Survival Through Treatment with 
Antibody Coated Cells 
Where LEW rats are treated prior to transplantation with antibody coated BN 
cells without further immuno-suppressive therapy, a substantial increase 
in the survival time of BN kidneys is noted. This was confirmed by the 
following experiments. 
LEW rats were treated 3 times, in weekly intervals, with cells, which were 
obtained from 1 ml of BN blood which had been incubated with 0.05 ml. LEW 
anti-BN serum and subsequently washed 3 times with phosphate buffer 
solution. The control animals were pretreated with 0.05 ml. 
LEW-anti-BN-serum or untreated BN blood, or not pretreated at all. The 
results of these experiments are shown in FIG. 9. The survival time 
(X.+-.standard error) of the animals which had been pretreated with 
antibody coated cells, were shown to be in the region of 109.+-.27 days, 
in contrast to the control animals with had a survival time of 8.4.+-..4 
days. 
EXAMPLE VI 
Rabbit IS-serum was obtained by the immunization of rabbits with autologous 
or allogeneic blood cells covered by human antibodies. These experiments 
were based on the knowledge that humans can spontaneously generate 
antibodies against rabbit erythrocytes. Thus, there exists the possibility 
of coating rabbit cells with human IgG. One ml. of rabbit blood was 
incubated for 30 minutes with 1.5 mg. of human IgG antibodies. The excess 
of antibodies was washed out and the rabbits were immunized 3 times in 3 
day intervals with the antibody covered cells by intravenous 
administration. One week later, the IS-serum was collected Neither in 
autologous nor in allogenic IS-serum, could human anti-lymphocyte 
antibodies be found. 
Rabbit IS-serum was tested in vitro for suppression of the activity of 
human B and T cells. The results showed that all sera suppressed the B 
cell response by 50 to 82% (measured by the indirect plaque forming cell 
assay with pokeweed mitogen stimulation). The allogenic IS-serum was more 
strongly suppressive than the autologous IS-serum. A suppression of the 
proliferative (MLC) and cytotoxic (CML) T cell response could similarly be 
demonstrated. Again, a stronger effect of the allogenic IS-serum was 
detected.