JPH0733680A - Immunosuppressor - Google Patents

Abstract

PURPOSE:To obtain an immunosuppressor containing three kinds of monoclonal antibodies as the active components. CONSTITUTION:This immunosuppressor contains three kinds of monoclonal antibodies respectively having a specific activity on a human T-cell surface antigen, CD2, CD4 or CD8 as the active components. Since a mixture of three kinds of antibodies, i.e., CD2 antibody, CD4 antibody and CD8 antibody is used, a remarkable immunosuppressive effect is exhibited in comparison with the case of one of the antibodies alone or combination of two antibodies. Further, even in the case one of the antibodies unfavorably exhibits a T-cell growth activity when used by itself, this unfavorable activity can be suppressed by a synergistic effect. In addition, this immunosuppressor is free from a side effect caused by an activity on cells not taking part in an immunoreaction unlike a polyclonal antibody preparation. The mixture ratio of three kinds of monoclonal antibodies is determined so that the respective components may be within a range of 10 to 60wt.% based on 100wt.% whole mixture.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an immunosuppressive agent containing a plurality of monoclonal antibodies as active ingredients. More specifically, human T cell surface antigens CD2, CD4, and CD
The present invention relates to an immunosuppressive agent containing, as an active ingredient, three types of monoclonal antibodies that specifically react with 8 respectively.

[0002]

2. Description of the Related Art In recent years, with the progress of organ transplantation technology, various inhibitors for immune rejection that occur at the time of transplantation have been developed, and the demand for them has increased with the increase in the number of transplants. The current situation is that a complete immunosuppressive drug has not been developed. Specifically, cyclosporin A, which is well-known as an inhibitor of immune rejection during organ transplantation, and steroids are known to show a high immunosuppressive effect on humoral immune response. It has a weakness that some cannot be suppressed.

Also, various antibody preparations having the ability to suppress cellular rejection have been developed. Mouse monoclonal antibody (OKT-3) against CD3, which is the surface antigen of T cells that are responsible for immune reaction, is one of them, but it has side effects such as extreme fever chills to activate T cells. ^{(1), (2), (3)} . In addition, a mouse monoclonal antibody BMA03 that recognizes a T cell receptor with the same antibody preparation
1 ^{(4) and (5)} do not have side effects such as T cell activation, but their specific activity is low, so that they have to be administered in large amounts, and as a result, intravenous drip is inevitable. On the other hand, although polyclonal antibody preparations have been developed, they have side effects such as erythrocyte and thrombocytopenia due to their reactivity with cells other than immunocompetent cells ^{(6), (7)} .

The immune system, which is a defense mechanism in the body, is mainly classified into three stages: antigen recognition, activation of immunocompetent cells, and cytotoxicity. In the first step, two types of lymphocytes are mainly responsible for antigen recognition. One is humoral immunity, which is in charge of B cells, and it is due to the membrane-bound immunoglobulin expressed on the surface of B cells. Recognition of the native form of the antigen. The other is the cellular immune system that is mainly responsible for T cells. The T cell receptor on the T cell for the antigen that is phagocytosed by the antigen-presenting cells and fragmented (peptide-ized) and presented to the MHC molecule on the cell surface. It is the recognition of the body. The recognition of this antigen causes the next step, activation of immunocompetent cells, which is a signal only by the binding between the MHC molecule + antigen complex and the T cell receptor between the antigen presenting cell and the T cell. T cell activation is not caused by transduction, but at that time, LFA of CD2, CD4, and CD8
-3, It is known that transmission of a secondary signal to T cells by binding to MHC Class I and MHC Class II is essential. Regarding the mechanism of cytotoxicity, MH
The antigen presented on C Class I is transferred to the CD8 on the cell surface.
Is recognized by MHC Class I-restricted T cells (cytotoxic T cells) that express, and secretes granules containing perforin and causes cytotoxicity. Surface antigens CD2, CD4, CD8 on T cells that are deeply involved in these immune responses
Is a cell adhesion molecule belonging to the immunoglobulin superfamily, and is known to be involved in T cell activation. The function is shown below. CD2 is
It was distributed on the surface of all T cells and was initially sheep red blood cells (SRB
C) Receptor identified to cause E rosette formation and having a molecular weight of 50K consisting of 327 amino acid residues
Is a membrane-bound glycoprotein of LFA-3 as its ligand
(CD58) is known ⁽⁸⁾ . CD2 binds to LFA-3 expressed on antigen-presenting cells, strengthens the binding between T cells and antigen-presenting cells without intervening antigen, and also carries out signal transduction into T cell itself ^{( 9)} .

CD4 is a single-chain, membrane-bound protein with a molecular weight of 59K that is known to be distributed on the surface of a subset of T cells and monocytes and some B cell lines. As its ligand, there is a constant region of major histocompatibility complex MHC Class II, which enhances the binding of MHC Class II and T cell receptor as its CD4 function, and CD4 itself also performs signal transduction through tylosin kinase p56lck. It is known that ⁽¹⁰⁾ .

CD8 is distributed on the surface of a subset of T cells and thymocytes and natural killer cells, and α
Constitutes a heterodimer ⁽¹¹⁾ consisting of a chain and a β chain,
MHC Class I ⁽¹²⁾ is known as its ligand. As its function, it is known to enhance the binding between MHC Class I and T cell receptor, and perform signal transduction via CD56 itself via the tylosin kinase p56lck, which is in contact with the cell ^{(10). )} .

A monoclonal antibody reactive with the human T cell surface antigen CD2 (hereinafter referred to as anti-CD2 antibody),
The immunosuppressive action of a monoclonal antibody reactive with human T cell surface antigen CD4 (hereinafter referred to as anti-CD4 antibody) and a monoclonal antibody reactive with human T cell surface antigen CD8 (hereinafter referred to as anti-CD8 antibody) Has been pointed out. Specifically, anti-CD2 antibody shows an immunosuppressive effect on xenogeneic organ transplantation ⁽¹³⁾ ,
Anti-CD4 antibody shows immunosuppressive effect on allogeneic organ transplantation, and anti-CD8 antibody causes GVH in bone marrow transplantation
It is already known that it has an immunosuppressive effect on D ⁽¹⁴⁾ . In addition, as an example of the immunosuppressive effect of the combination, anti-CD4 antibody, anti-CD8 antibody and anti-I
Examples of three types of L2 receptor antibody combinations and two types of anti-CD4 antibody and anti-CD8 antibody ⁽¹⁶⁾ combinations are known.

[0008] However, there is no known case of an immunosuppressant comprising a combination of three kinds of antibodies, anti-CD2 antibody, anti-CD4 antibody and anti-CD8 antibody.

[0009]

DISCLOSURE OF THE INVENTION An object of the present invention is to eliminate the above-mentioned drawbacks relating to conventional immunosuppressive agents containing an antibody as an active ingredient, which have no cross-reactivity with cells other than T cells, and
It is to develop and provide an immunosuppressant that does not activate cells.

[0010]

Means for Solving the Problems In order to solve the above-mentioned problems, the inventors of the present invention investigated various monoclonal antibodies against human T cell surface antigens, and as a result, anti-CD2
When three kinds of antibodies, ie, an antibody, an anti-CD4 antibody and an anti-CD8 antibody, are used in combination, the mixture is used alone or
Surprisingly, as compared with the combination of two or conventional immunosuppressive antibody preparations, it is possible to exert an excellent synergistic effect that is immunocompetent cell-specific, has less T cell activation, and has a significantly high immunosuppressive effect. Found and completed the present invention.

The present invention relates to the provision of a novel immunosuppressive agent. That is, the present invention is an immunosuppressive agent containing, as an active ingredient, three types of monoclonal antibodies that specifically react with human T cell surface antigens CD2, CD4, and CD8, respectively. As the monoclonal antibody that can be used in the present invention, all of the following can be used as long as they retain or lose the respective antigen specificity to CD2, CD4, and CD8.

A: Monoclonal antibody produced by an antibody-producing hybridoma prepared using B cells derived from various animals including mice by a conventional method B: The antibody of A is genetically modified as exemplified below. Those that have been modified or produced using the technology for the purpose of humanization, improvement of affinity, removal or enhancement of complement activity, etc.

The variable region or hypervariable region of the antibody A is inserted into the constant region or constant region portion of various immunoglobulins or the portion consisting of the constant region and the framework. ^{(19), (20).} The variable region or hypervariable region of the antibody of A is bound to a molecule other than immunoglobulin. ⁽²¹⁾ A bispecific antibody having a variable region of the antibody A and a variable region of the antibody different from the antibody is created. ⁽²²⁾ -Screen for those with a higher affinity for the antigen than those expressing the antibody fragment on the surface of the phage.
⁽²³⁾ -A product that has a DNA mutation and is modified to have a high affinity by using the PCR method or the like. C: A antibody is treated with an enzyme for the purpose of removing complement activity, etc.
(For example, the Fc portion is removed by pepsin treatment ⁽²⁵⁾ or plasmin treatment ⁽²⁶⁾ ) or chemical modification (for example, β-
Modified by propiolactone treatment ⁽²⁷⁾ and reductive alkylation of disulfide bond ⁽²⁸⁾ to inactivate the Fc portion.

D: Prepared using a human immunoglobulin producing transgenic animal ⁽²⁴⁾ . E: Human antibody-producing cells introduced into an immunodeficient animal and produced from the cells. The present invention not only suppresses the antigen-presenting reaction but also suppresses the cytotoxic reaction. Therefore, not only as a rejection inhibitor that occurs during allogeneic and xenogeneic organ transplantation or cell transplantation of bone marrow cells, but also self-antigen It can also be used as a general therapeutic agent for autoimmune diseases that recognizes and causes cytotoxicity against it. In that case, since it is considered that non-human antibodies can form antibodies against the antibodies by long-term administration, humanization is preferable.

The mixing ratio of each of the three types of monoclonal antibodies in the immunosuppressive agent of the present invention is 10 to 60%.
The ratio is within the range of and becomes 100% as a whole. The present invention can be used in the form of injections, preferably intravenous administration, and the dose and the number of administrations can be determined in consideration of the medical condition of the target disease patient.
It can be administered up to 100 μg / day.

The present invention can be prepared as a solution formulation or a lyophilized formulation, and if necessary, a pharmaceutically acceptable excipient, diluent, stabilizer, isotonicity agent, buffer is added. It can be included as a thing. Examples of preferable additives include sugars such as maltose, surfactants such as polysorbate, amino acids such as glycine, proteins such as human serum albumin, and salts such as sodium chloride.

[0017]

The anti-CD2 antibody and anti-CD4 antibody of the present invention,
An immunosuppressive agent consisting of a combination of three kinds of anti-CD8 antibodies has a remarkable immunosuppressive effect as compared with the case where they are used alone or in combination of two, and in particular, any one of the antibodies alone is not preferable as T cell proliferative activity. When it has a property, it is suppressed by a synergistic effect. Therefore, this immunosuppressive drug not only exerts an excellent immunosuppressive effect as compared with conventional immunosuppressive drugs, but even if the antibody used has T cell proliferating activity, it is suppressed by mixing and side effects based on this activity are exerted. It is very useful industrially, because it has the extraordinary effect of suppressing the irritation, and there is no fear of side effects due to reactivity to cells other than immunocompetent cells as in the polyclonal antibody preparation. (Preparation example) Preparation of antibody (1) Screening and cloning of antibody Female Balb / c mouse 8 weeks old B cell line (CCRF-SB ATCC No.
CCL-120) activated T cells (2 × 10 ⁷⁾ were immunized by intravenous injection into the tail. The spleen of this immunized mouse was removed and the lymphocytes prepared with this spleen and mouse myeloma p3X63 6.5.3.
(ATCC No.CRL-1580) with polyethylene glycol 1500
(Bellinger) was used to perform cell fusion. The fused cells were cloned into antibody secretion positive strains using the limiting dilution method. The ELISA method was used to determine the positive strain. Specifically, human peripheral blood was washed with PBS and the blood cell fraction was adjusted to 0.1% F.
The cells were suspended in CS / PBS and overlaid with Ficoll. 30 at 1500 rpm
After centrifugation for minutes, the buffy coat was collected as a mononuclear cell fraction.
This mononuclear cell fraction was passed through an IgG column to remove B cells. The T cells thus obtained and CCRF-SB were mixed and cultured at 37 ° C. for 6 days to activate the T cells. The cells were layered on Ficol, centrifuged at 1500 rpm, buffy coat was removed, and washed 3 times with PBS to prepare an immunogen. This immunogen was injected into the tail vein of Balb / c mice. The spleen of this mouse was removed and the lymphocytes prepared from this spleen and p3X63 6.5.
3 was cell-fused with polyethylene glycol 1500. Spread the fused cells in a 96-well plate at 37 ° C, 5% CO ₂ HA
The cells were cultured in T (hypoxanthine, aminopterin, thymidine, Sigma) + 10% FCS RPMI medium for 10 to 20 days.
The culture supernatant was assayed using an enzyme immuno-antibody method against T cells, and the resulting positive strain was subjected to limiting dilution and seeded at 1 / well into a 96-well plate. By repeating this limiting dilution operation, a human T cell recognizing antibody-secreting hybridoma was cloned. (2) Analysis of antibody by automatic fluorescence analyzer T cells were prepared from peripheral blood according to a conventional method. The T cells were subjected to fluorescent secondary staining using the above antibody and commercially available CD2, CD4 and CD8 antibodies. The fluorescently stained T cells were analyzed by flow cytometry FACSCAN (Becton Dickinson) to identify the antibody. The anti-human CD2, CD4, and CD8 antibodies thus obtained were used as the following in vitro evaluation system samples. Specifically, a mononuclear cell fraction was prepared by separating lymphocytes from peripheral blood by density gradient centrifugation using Ficol (Pharmacia). The T cells were suspended in 0.1% NaN ₃ 2% FCS / PBS, human immunoglobulin was added, and the mixture was left on ice for 30 minutes.
After washing three times with 0.1% NaN ₃ 2% FCS / PBS, FITC-labeled anti-mouse IgG antibody was added, and this was added to 0.1% NaN ₃ 2% FCS.
/ After washing 3 times with PBS, each commercially available anti-human CD2 antibody labeled with picoerythrin (anti-Leu5b Becton Dickinson) anti-human CD4 antibody (anti-Leu3a Becton Dickinson) anti-human CD8 antibody (anti-Leu2a Becton Deckonson) Each) was left for 30 minutes at room temperature. This was washed 3 times with 0.1% NaN ₃ 2% FCS / PBS.
The fluorescently labeled T cells thus obtained were subjected to FACSca
The difference in the T cell recognition group between each commercially available CD2, CD4, and CD8 antibody and the above monoclonal antibody was assayed by dot blot analysis using n. FIG. 1 shows a dot plot pattern by double staining using each T cell antibody and commercially available CD2, CD4, and CD8 antibodies. The antibody obtained from FIG. 1 was found to be an antibody that competes with each commercially available CD2, CD4, and CD8 antibody or recognizes the same T cell group. (3) Purification of antibody Each anti-human lymphocyte antibody-secreting hybridoma was subjected to serum-free culture. This culture supernatant was passed through a protein A disk column, and the adsorbed IgG fraction was eluted with a buffer solution. Specifically, the hybridoma was cultured at 37 ° C. for 6 days in ERDF medium ((Kyokuto Pharmaceutical Co., Ltd.) + insulin + transferrin etc.). This culture solution was centrifuged to collect the culture supernatant. This culture supernatant was passed through a protein A column (Fuji Filter Industry) to adsorb the immunoglobulin fraction. This column
Washed with 1.5M Glycine 3M NaCl pH8.9, 0.1M Glyc
The immunoglobulin fraction was eluted with ine pH 2.5. (Test Example) Immunosuppression test (1) Lymphocyte mixed culture test In the above-mentioned immune reaction, lymphocytes stimulated by antigen-presenting cells are activated and their proliferation is promoted. This is an in vitro system, which is a mixed lymphocyte culture test, and can be used to determine the effects of various immunosuppressive substances ⁽¹⁷⁾ . Specifically, humans with different haplotypes
Lymphocytes (hereinafter referred to as human A and human B for convenience) are mixed to promote lymphocyte proliferation, which is suppressed by the above antibody. Lymphocytes prepared from the peripheral blood of human A are deproliferated with mitomycin C in advance. Lymphocytes (Responder) prepared from peripheral blood of human B to which the non-proliferated lymphocytes (Stimulator) and antibody were added
4 days later, tritium-labeled thymidine is added and the proliferation of human B lymphocytes is measured. The method is shown below. 1) Preparation of lymphocytes From peripheral blood of human A and human B with different haplotypes from Fico
Lymphocytes were separated by density gradient centrifugation using l. 2) Preparation of Stimulator 1 × 10 ⁷ human A lymphocytes were suspended in 5 ml of RPMI medium (Gibco) containing 10% serum prepared from human B.
To this lymphocyte suspension, 250 μg of mitomycin C was added and incubated at 37 ° C. for 30 minutes in the presence of 5% CO ₂ . 1500r
After centrifuging at 5 pm for 5 minutes, the precipitate was suspended in 1% FCS PBS, further centrifuged, and the washing operation was repeated 4 times.
Suspended in RPMI. 3) Preparation of Responder Suspended in ⁵ % 10 ⁵ / ml human B lymphocytes in 10% B serum RPMI 4) Mixing lymphocytes 100 μl each of Stimulator and Responder were mixed on a U-bottom 96-well plate. At this time, place the antibody on the Responder side one hour before mixing in advance. The mixed lymphocytes were cultured at 37 ° C. for 4 days in the presence of 5% CO ₂ . 5) Incorporation of thymidine 0.25 μCi / well of ³ H thymidine was added to this plate and left at 37 ° C. for 16 to 24 hours in the presence of 5% CO ₂ . 6) Measurement of label uptake amount DNA containing ³ H thymidine was recovered by a cell harvester (Wallac Co.). Β of this ³ H thymidine
The dose was measured with a liquid scintillation counter (Wallac).

Results Mouse anti-human CD2 monoclonal antibody obtained by us
(62D4), mouse anti-human CD4 monoclonal antibody (a2
0-4), mouse anti-human CD8 monoclonal antibody (h32-1)
FIG. 2 shows the thymidine uptake (inhibitory effect) in the lymphocyte mixed culture test of the sample alone and the sample mixed 1: 1: 1. Each sample was prepared so that the final concentration of the total antibody was 700 ng / ml. Mouse anti-human CD3 monoclonal antibody (OKT-3) has higher T cell proliferation activity than control (uptake of thymidine 14000cp
m), each anti-CD2 antibody, anti-CD4 antibody, and anti-CD8 antibody showed high immunosuppressive ability.
(Thymidine incorporation 1000-1200 cpm). Further anti-CD2
The antibody, the anti-CD4 antibody, and the anti-CD8 antibody mixed at 1: 1: 1 showed higher immunosuppressive activity (260 cpm) even though the final concentration of the mixed total was the same as the single antibody.
From this, it seems that a synergistic effect due to the combination of the anti-CD2, CD4 and CD8 antibodies is shown.

Next, another mouse anti-human CD4 monoclonal antibody (k35-37), mouse anti-human CD8 monoclonal antibody (a86-2) and the above-mentioned anti-human CD2 monoclonal antibody (62D4) which were obtained by us were used. The immunosuppressive effect of each antibody alone and in combination is shown in FIG. When each antibody is used alone, anti-CD2 antibody, anti-C
D4 antibody shows an immunosuppressive effect as described above, and anti-C
Although the D8 antibody alone is a combination showing T cell proliferative activity, even such a combination showed a high immunosuppressive effect when mixed with three kinds of anti-CD2 antibody, anti-CD4 antibody and anti-CD8 antibody. And this effect is
It was higher than any of the two combinations of D2, CD4, and CD8 antibodies.

Furthermore, the mouse anti-human CD2 monoclonal antibody (h82-35, 62D 4) and mouse anti-human CD obtained by us
4 monoclonal antibody (k35-37, N93-18) and mouse anti-human CD8 monoclonal antibody (h32-1, N49-4) were used alone and as anti-CD2 antibody, anti-CD4 antibody and anti-CD, respectively.
3 kinds of 8 antibodies (anti-CD2 antibody, anti-CD4 antibody,
The immunosuppressive effect of anti-CD8 antibody = 1: 1: 1) is shown in FIG. In any combination of these antibodies, a synergistic effect of mixing three kinds of anti-CD2, CD4 and CD8 antibodies was shown.

Further, commercially available mouse anti-human CD2 monoclonal antibody (IOT-11: IMMUNOTECH), mouse anti-human CD
4 monoclonal antibody (A40: PHARMINGEN), mouse anti-human CD8 monoclonal antibody (G-42-8: PHARMINGEN)
FIG. 5 shows the immunosuppressive effects of the above alone and the mixture of three types. Also in this case, a synergistic effect of mixing three kinds was confirmed.

Anti-CD2 antibody (62D4), CD4 (k35-37)
The concentration dependence of the immunosuppressive activity of the CD8 antibody (h32-1) is shown in FIG. Anti-CD2, CD4, CD8 antibodies alone and 3 types mixed at a final concentration of 2.8 ng / ml-350 ng / ml
As a result of examining the immunosuppressive effect by (anti-CD2: CD4: CD8 antibody = 1: 1: 1), a synergistic effect was observed by mixing three kinds of anti-CD2, CD4, and CD8 antibodies at any antibody concentration.

From these facts, not only anti-CD2, CD4, and CD8 antibodies alone have sufficient immunosuppressive activity in mixed lymphocyte culture tests, but also anti-CD2 and CD8
4. It was confirmed that when the CD8 antibody alone had a low immunosuppressive activity or had T cell proliferating activity, a synergistic effect could be obtained by mixing three types. In addition, Responder lymphocytes and Stmul in these assays
As for ater lymphocytes, different human ones are used for each assay (for example, if the data in FIG. 2 is human A and human B, it is called human C and human D in FIG. 3). Like), any of them have a synergistic effect,
It can be said that this effect is observed regardless of individual differences in humans. (2) Target cytotoxicity test Cytotoxicity by lymphocytes that occurs at the final stage of the above-mentioned immune reaction is closely related to immune reaction during transplantation, immune reaction against virus-infected cells, autoimmune disease, etc. There is. Since this cytotoxic effect cannot be measured in vivo in humans, this in vitro measurement method such as the cytotoxicity test is widely used.
⁽¹⁸⁾ . Specifically, cytotoxic T cells were prepared by co-culturing a human B cell line that had been non-proliferated with mitomycin C as a target and mononuclear cells prepared from human peripheral blood. A fresh human B cell line (target cell) into which ⁵¹ Cr has been incorporated is co-cultured with the above cytotoxic T cells, and the amount of chromium in the culture supernatant released from the damaged B cell line is measured. The method is shown below. 1) Preparation of mononuclear cells from human peripheral blood Human peripheral blood was centrifuged at 1500 rpm for 10 minutes, and the blood cell fraction was suspended in PBS and overlaid with Ficoll. This was centrifuged at 1500 rpm for 30 minutes, Buffy coat was collected as a lymphocyte fraction, and 2% FCS /
After washing twice with PBS, suspend in 10% RPMI. 2) Preparation of allostimulatory cells (target cells) Suspend B cell line SKW.6.4 to be target cells with 10% FCS,
Add mitomycin C to a final concentration of 50 μg / ml,
After leaving it at 37 ℃ for 30 minutes, wash it 4 times with PBS. 3) Allostimulation of mononuclear cells (Preparation of cytotoxic T cells) The mononuclear cells prepared in 1) above were mixed with the cells of 2) above and incubated at 37 ° C for 5 days in 5% CO ₂ . The cells were prepared by the same procedure as in 1) above and suspended in 10% human serum RPMI. As a control, a cell in which the cells of 2) above were not mixed was used. 4) Uptake of ⁵¹ Cr into target cells B cell line SKW.6.4 was suspended in 10% FCS RPMI, and ⁵¹ Cr was added thereto.
(NEN) was added, and the mixture was incubated at 37 ° C., 5% CO _{2 for} 1 hour, and then washed 5 times with 1% FCS / PBS. 5) Assay Mix the cells from 3) and 4) above and mix with 10% human serum.
It was incubated at 37 ° C., 5% CO _{2 for} 4 hours in RPMI. The culture supernatant was collected by a supernatant collection system (Dainippon Pharmaceutical Co., Ltd.). Gamma counter
(AUTO-GAMMA5650 Packard). The antibody was added to the cytotoxic cells 1 hour before mixing the target cells (4) above and the cytotoxic cells (3) above.

Results Anti-CD2 antibody (h82-35, 62D4), anti-CD4 antibody (k35-3
7, N93-18), CD8 antibody (h32-1) alone and a mixture of three anti-CD2, CD4 and CD8 antibodies (anti-CD
2: CD4: CD8 antibody = 1: 1: 1) at a final concentration of 200 ng /
FIG. 7 shows the immunosuppressive effect when added so that the amount becomes ml. Each of them showed a higher immunosuppressive effect when the three kinds were mixed than when the anti-CD2, CD4, and CD8 antibodies were used alone, and a synergistic effect due to the mixing was recognized. Next, using the same antibody, two kinds (1: 1) and three kinds (1: 1 :) of CD2, CD4, and CD8 antibodies were mixed respectively.
The immunosuppressive effect in case 1) is shown in FIGS. 8 and 9. The antibody was added so that the final concentration of the total antibody was 350 ng / ml in FIG. 8 and 200 ng / ml in FIG. As a result, the immunosuppressive effect of some of the two types was rather decreased, but the synergistic immunosuppressive effect was exhibited in all of the three types. Similarly anti-C
D2 antibody (62D4), CD4 (k35-37, N93-18), CD8
Antibodies (N49-4, h32-1) alone and anti-CD2,
Mixed CD4 and CD8 antibodies (anti-CD2: CD4: C
The immunosuppressive effect when D8 antibody = 1: 1: 1) was added to a final concentration of 200 ng / ml is shown in FIG.

In each of these assays, cytotoxic T cells were prepared from different humans.
Mixture of 3 types of D2, CD4, and CD8 antibodies (anti-CD2: CD
The synergistic effect of the combination of 4: CD8 antibody = 1: 1: 1) was confirmed. In addition, a commercially available mouse anti-human CD2 monoclonal antibody (IOT-11: IMMUNOTECH), mouse anti-human CD4 monoclonal antibody (A40: PHARMINGEN),
Mouse anti-human CD8 monoclonal antibody (G-42-8: PHARM
FIG. 11 shows the immunosuppressive effect of INGEN) alone or in combination with three types. Also in this case, a synergistic effect of mixing three kinds was confirmed. Anti-CD2 antibody (62D4), anti-CD4 antibody (k35-37), anti-CD8 antibody (h32-1), OKT-3 (mouse anti-human CD3 monoclonal antibody Ortho Pharmaceutical Co., Ltd.) alone and anti-CD2, CD4,
FIG. 12 shows the concentration dependence of the cytotoxic activity-suppressing effect when the final concentration of each CD3 antibody mixture (1: 1: 1) was 3 ng / ml to 700 ng / ml. Anti-C
Mixture of 3 types of D2, CD4, and CD8 antibodies, final concentration 10ng / ml
As described above, each showed a higher inhibitory effect than the antibody alone. Furthermore, OKT-3 shows about 36 %% of lysis at 700 ng / ml, while about 350% / 33% about 3% in case of mixing three kinds.
Of% of lysis.

Anti-CD2 antibody (62D4), anti-CD4 antibody (k3
5-37), anti-CD8 antibody (h32-1) was added to anti-CD2, CD4, C
The D8 antibody mixing ratio was 1: 0: 0, 0: 1: 0, 0: 0:
1, 7: 5: 3, 2: 1: 1, 1: 2: 1, 1: 1:
2, when added at a ratio of 1: 1: 1 and a total antibody concentration of 700 ng / ml, and OKT-3 (anti-human CD3 monoclonal antibody Ortho Pharmaceutical Co., Ltd.) 700 ng / ml
FIG. 13 shows the cytotoxic activity suppressing effect of each antibody when added.

A mixture of three kinds of anti-CD2, CD4 and CD8 antibodies shows a higher immunosuppressive effect than any of the antibodies alone,
In addition, the mixing ratio is anti-CD2: CD4: CD8 antibody = 1:
The highest immunosuppressive synergistic effect was observed in this assay at 1: 2. This may be due to the presence of CD8-positive cells in many of the cytotoxic T cells.

[0028]

【Example】

Example 1 Solution-1 2 mg each of anti-CD2 antibody, anti-CD4 antibody and anti-CD8 antibody, 1 mg of polysorbate, 16 mg of sodium chloride, 2 ml
A solution consisting of PBS was aseptically prepared and filled in an ampule to give an injection solution.

Example 2 Liquid formulation-2 Anti-CD2 antibody, anti-CD4 antibody and anti-CD8 antibody were prepared, respectively.
A solution consisting of 1.5 mg, 1.5 mg, 3 mg, polysorbate 1 mg, sodium chloride 16 mg, and 2 ml PBS was aseptically prepared and filled with Alpur to prepare an injection solution. Example 3 Solution-3 Anti-CD2 antibody, anti-CD4 antibody, anti-CD8 antibody
A solution consisting of 1.5 mg, 3 mg, 1.5 mg, polysorbate 1 mg, sodium chloride 16 mg, and 2 ml PBS was aseptically prepared and filled with Alpur to obtain an injection solution.

Example 4 Solution-4 A solution consisting of 3 mg, 1.5 mg and 1.5 mg of anti-CD2 antibody, anti-CD4 antibody and anti-CD8 antibody, polysorbate 1 mg, sodium chloride 16 mg and 2 ml PBS was prepared aseptically. An ampoule was filled and used as an injection solution. Example 5 Solution-5 A solution consisting of anti-CD2 antibody, anti-CD4 antibody and anti-CD8 antibody in an amount of 2 mg, 1 mg, 3 mg, polysorbate 1 mg, sodium chloride 16 mg and 2 ml PBS was aseptically prepared, filled in an ampoule and used as an injection solution. did.

Example 6 Solution-6 A solution consisting of 3 mg, 1 mg and 2 mg of anti-CD2 antibody, anti-CD4 antibody and anti-CD8 antibody respectively, 1 mg of polysorbate, 16 mg of sodium chloride and 2 ml PBS was aseptically prepared and filled with alpur, It was used as an injection solution. Example 7 Lyophilizer-11 Anti-human T lymphocytes (CD2, CD4, C in 1 vial)
D8) Monoclonal antibody 6 mg (anti-CD2 antibody, anti-CD
4 antibody, anti-CD8 antibody (2 mg each) and polysorbate 1 mg as additive, sodium chloride 16 mg, 2 ml P
A solution consisting of BS is dissolved aseptically in a freeze-dried preparation and distilled water for injection, and then used as an injection solution.

Example 8 Lyophilization agent-2 1 Anti-human T lymphocytes (CD2, CD4, C) in a vial.
D8) Monoclonal antibody 6 mg (anti-CD2 antibody, anti-CD
4 antibody, anti-CD8 antibody 1.5mg, 1.5mg, 3m respectively
g) and 1 mg of polysorbate as an additive, 16 mg of sodium chloride, 2 ml of a solution of 2 ml PBS are dissolved in an aseptically prepared lyophilizer and distilled water for injection, and then used as an injection solution.

Example 9 Lyophilizer-31 Anti-human T lymphocytes (CD2, CD4, C) in one vial
D8) Monoclonal antibody 6 mg (anti-CD2 antibody, anti-CD
4 antibody, anti-CD8 antibody 1.5mg, 3mg, 1.5m respectively
g) and 1 mg of polysorbate as an additive, 16 mg of sodium chloride, 2 ml of a solution of 2 ml PBS are dissolved in an aseptically prepared lyophilizer and distilled water for injection, and then used as an injection solution.

Example 10 Lyophilization agent-41 Anti-human T lymphocytes (CD2, CD4, C) in one vial
D8) Monoclonal antibody 6 mg (anti-CD2 antibody, anti-CD
4 antibody, anti-CD8 antibody 3mg, 1.5mg, 1.5m respectively
g) and 1 mg of polysorbate as an additive, 16 mg of sodium chloride, 2 ml of a solution of 2 ml PBS are dissolved in an aseptically prepared lyophilizer and distilled water for injection, and then used as an injection solution.

Example 11 Lyophilization agent-5 Anti-human T lymphocytes (CD2, CD4, C) in one vial
D8) Monoclonal antibody 6 mg (anti-CD2 antibody, anti-CD
4 antibody, anti-CD8 antibody 3mg, 1.5mg, 1.5m respectively
g) and 1 mg of polysorbate as an additive, 16 mg of sodium chloride, 2 ml of a solution of 2 ml PBS are dissolved in an aseptically prepared lyophilizer and distilled water for injection, and then used as an injection solution.

Example 12 Freeze-Drying Agent-6 Anti-human T lymphocytes (CD2, CD4, C) in one vial
D8) Monoclonal antibody 6 mg (anti-CD2 antibody, anti-CD
4 antibody, anti-CD8 antibody (3 mg, 1 mg, 2 mg, respectively) and polysorbate 1 mg as an additive, sodium chloride
A solution consisting of 16 mg and 2 ml PBS is dissolved in an aseptically prepared lyophilizer and distilled water for injection, and then used as an injection solution. [References] (1) Von Wauwe JP, Demey JR, Goosens JG J Immunol, 124 , 2708 (1980) (2) Ellenhorn JDI, Woodle ES, Ghobrial I, Th
istlethwaite JR, Bluestone JA Transplantation, 50 , 608 (1990) (3) Gaston RS: Deierhoi MH et al., Kidney Int. Jan.
1991, 39 (1) p141-8 (4) Smely S., Weschka M., Hillebrand G., Dendorfe
r U., Krombach F., Kurrle R., Land W., Hammer C Transplant proc 22 , 1785 (1990) (5) Hayasaka Y., Takahasi K., Yagisawa T., Oota
K. Transplantation Now, 5 , 125 (1992) (6) Aarbulin (dried anti-human lymphocyte equine immunoglobulin) Midori cross catalog (7) Presmuin (dried anti-human lymphocyte equine immunoglobulin) Hoechst Japan catalog (8) Peterson A. , Seed B. Nature. 329 , 842 (1987) (9) Chang. H., Moingeon, P. et al. J. Exp. Med., 169 , 2073 (1989) (10) Veillette A., Bookman MA, Horak EM et al Nature, 338 , 257 (1989) (11) Jay G., Palladino MA, Khoury G. et al Proc Natl Acad Sci USA 79 , 2654 (1982) (12) Potter TA, Rajian TV, DickII RF, Bluest
onc JA Nature, 337 , 73 (1989) (13) Chavin KD, Lau HT, Bromberg JS Transplantation, 54 , 286 (1992) (14) Champlin R., Gajewski J., Feig S., Giorji J.,
Lyddane N., Lee K., Schmidt I., Winston D., Ho W.,
Reichert T. Transplantation proceedings, 21 , 2947 (1989) (15) I. Smyej, 8th International Congress of immun
ology (1992) (16) Cobbold SP, Marin G., Waldman H. Eur J Immunol, 20 , 2747 (1990) (17) Itoh Yoshihisa All about Cellular Immune Function Tests 171 (1985) (18) Junichi Yada, Michio Fujiwara New Lymphocyte Function Search Method Chugai Medical Co., Ltd. 189 (1987) (19) Bouliane GL, Hozumi N., Shulman MJ Nature, 312 , 643 (1984) (20) Riechman L., Clark M., Waldman H., Winter G.,
Nature, 332 , 323 (1988) (21) Kreitman RJ, Chaudhary VK, Waldman T., Wi
llingham MC, FitzGerald DJ, Pastan I., Proc Natl
Acad Sci USA 87 , 8291 (1990) (22) Bator JM, Reading CL, In Therapeutic Monoc
lonal Antibodies (1990) (23) McCafferty J., Griffiths AD, Winter G., Chi
swell DJ, Nature, 348 , 552 (1990) (24) Brugman M., Proc Natl Acad Sci USA 86 , 6709 (19
89) (25) Barandun S., Castel V., Makura MF, Morell
A., Plan R., Skvaril F., Vox. Sang., 28 , 157 (1975) (26) Koblet H., Barandun S., Diggelman H., Vox. San
g., 13 , 93 (1967) (27) Stephan W., Vox. Sang., 28 , 422 (1975) (28) Morell A., Vox. Sang., 51 (suppl 2), 44 (1986)

[Brief description of drawings]

FIG. 1 Staining pattern of lymphocytes with various antibodies and commercially available anti-CD2, CD4, and CD8 antibodies by an automatic fluorescence analyzer.

FIG. 2 is a view showing the results of a mixed lymphocyte culture test. Cont: 10% FCS medium (control) A: 62D4 (anti-CD2 antibody) B: k35-37 (anti-CD4
Antibody) C: h32-1 (anti-CD8 antibody) OKT-3: anti-CD3 antibody A + B + C: mixture of A, B, and C A: B: C = 1:
1: 1 Each antibody alone or mixed final concentration is 700ng / ml

FIG. 3 shows the results of mixed lymphocyte culture test. T (R): Responder T cell mT (S): Stimulator T cell non-proliferated with mitomycin C A: 62D4 (anti-CD2 antibody) B: k35-37 (anti-CD4
Antibody) C: a86-2 (anti-CD8 antibody) A + B: A, B mixture A: B = 1: 1 A + C: A, C mixture A: C = 1: 1 B + C: B, C mixture B: C = 1: 1 A + B + C: Mixture of A, B and C A: B: C = 1: 1:
1 Each antibody alone or mixed final concentration is 700ng / ml

FIG. 4 shows the results of mixed lymphocyte culture test. A: h82-35 (anti-CD2 antibody) B: 62D4 (anti-CD2 antibody) C: k35-37 (anti-CD4 antibody) D: N93-18 (anti-CD4 antibody)
Antibody) E: h32-1 (anti-CD8 antibody) F: N49-4 (anti-CD8
Antibody) OKT-3: anti-CD3 antibody (ortho) The mixing ratio of each anti-CD2, CD4, CD8 antibody is 1:
1: 1 Each antibody alone or mixed final concentration is 200ng / ml

FIG. 5 shows the results of mixed lymphocyte culture test. Anti-CD2: Anti-CD2 antibody (IOT-11: IMMUNOTECH) Anti-CD4: Anti-CD4 antibody (A-40: PHARMINGEN) Anti-CD8: Anti-CD8 antibody (C-42-8: PHARMINGEN) Mix: Anti-CD2 antibody, Mixed anti-CD4 and anti-CD8 antibodies Anti-CD2: Anti-CD4: Anti-CD8 = 1: 1: 1 Each antibody alone or mixed at a final concentration of 350 ng / ml

FIG. 6 shows the results of mixed lymphocyte culture test. anti-CD2: 62D4 anti-CD4: k35-37 anti-CD8: h32-1, Mix: anti-CD2 + anti-CD4, anti-CD8 mixed anti-CD2 antibody, anti-CD4 antibody, anti-CD8 antibody mixed ratio Anti- CD2:
Anti-CD4: Anti-CD8 = 1: 1: 1

FIG. 7 shows the results of cytotoxicity tests. A: h82-35 (anti-CD2 antibody) B: 62D4 (anti-CD2 antibody) C: k35-37 (anti-CD4 antibody) D: N93-18 (anti-CD4 antibody)
Antibody) E: h32-1 (anti-CD8 antibody) Each antibody alone or mixed at a final concentration of 200 ng / ml Anti-CD2 antibody, anti-CD4 antibody, anti-CD8 antibody mixing ratio is 1: 1: 1

FIG. 8 shows results of cytotoxicity test. A: 62D4 (anti-CD2 antibody) B: k35-37 (anti-CD4 antibody) C: h32-1 (anti-CD8 antibody) Each antibody alone or in a mixed concentration of 350 ng / ml anti-CD2 antibody, anti-CD4 antibody, anti-CD8 antibody The mixing ratio is
1: 1 in case of mixing 2 kinds, 1: 1: 1 in case of mixing 3 kinds

FIG. 9 shows the results of cytotoxicity test. A: h82-35 (anti-CD2 antibody) B: 62D4 (anti-CD2 antibody) C: k35-37 (anti-CD4 antibody) D: N93-18 (anti-CD4 antibody)
Antibody) E: h32-1 (anti-CD8 antibody) Each antibody alone or mixed at a final concentration of 200 ng / ml. Anti-CD2 antibody, anti-CD4 antibody, anti-CD8 antibody mixing ratio is
1: 1 in case of mixing 2 kinds, 1: 1: 1 in case of mixing 3 kinds

FIG. 10 shows the results of cytotoxicity tests. A: 62D4 (anti-CD2 antibody) B: k35-37 (anti-CD4
Antibody: C: N93-18 (anti-CD4 antibody) D: N49-4 (anti-CD8
Antibody) E: h32-1 (anti-CD8 antibody) Each antibody alone or mixed at a final concentration of 200 ng / ml Anti-CD2 antibody, anti-CD4 antibody, anti-CD8 antibody mixing ratio is 1: 1: 1

FIG. 11 shows the results of a cytotoxicity test. Anti-CD2: Anti-CD2 antibody (IOT-11: IMMUNOTECH) Anti-CD4: Anti-CD4 antibody (A-40: PHARMINGEN) Anti-CD8: Anti-CD8 antibody (C-42-8: PHARMINGEN) Mix: Anti-CD2 antibody, Mixed anti-CD4 and anti-CD8 antibodies Anti-CD2: Anti-CD4: Anti-CD8 = 1: 1: 1 Each antibody alone or mixed at a final concentration of 350 ng / ml

FIG. 12 shows the results of cytotoxicity test. anti-CD2: 62D4 anti-CD4: k35-37 anti-CD8: h32-1,
OKT-3: (anti-CD3 antibody: ORTHO) Mix: anti-CD2 antibody, anti-CD4 antibody, anti-CD8 antibody mixed ratio is Anti-CD2: Anti-CD4: Anti-CD8 = 1: 1: 1

FIG. 13 shows the results of a cytotoxicity test. anti-CD2: 62D4 anti-CD4: k35-37 anti-CD8: h32-1,
OKT-3: (anti-CD3 antibody: ORTHO) The mixing ratio of anti-CD2 antibody, anti-CD4 antibody, and anti-CD8 antibody is 1.7: 5: 3, 2.2: 1: 1, 3.1: 2: 1 respectively. 1: 1: 2
5.1: 1: 1 Each antibody alone or mixed final concentration is 700ng / ml

Claims (2)

[Claims] 1. An immunosuppressant containing the following three monoclonal antibodies (a), (b) and (c) as active ingredients. (a) Monoclonal antibody reactive with human T cell surface antigen CD2 (b) Monoclonal antibody reactive with human T cell surface antigen CD4 (c) Monoclonal antibody reactive with human T cell surface antigen CD8 2. The immunosuppressant according to claim 1, wherein the monoclonal antibody is derived from mouse.