JPH07242566A - Immunosuppressant - Google Patents

Abstract

(57) [Summary] [Structure] An immunosuppressant comprising an antibody reactive to human nucleolin as an active ingredient. [Effect] The immunosuppressive agent of the present invention is a drug having efficacy against various immune diseases and is extremely useful industrially.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an immunosuppressive agent containing an antibody as an active ingredient. More specifically, it relates to an immunosuppressive agent containing, as an active ingredient, an antibody that specifically reacts with human nucleolin.

[0002]

2. Description of the Related Art In recent years, with the progress of organ transplantation technology, various inhibitors for immune rejection that occur during transplantation have been developed, and the demand for them has increased with the increase in the number of transplants. However, the current situation is that a complete immunosuppressant has not been developed. Specifically, it is known that cyclosporin A, a steroid drug and the like, which are prominent as inhibitors of immune rejection during organ transplantation, show a high immunosuppressive effect on humoral immune response.
It has a weakness that it cannot suppress a part of cell rejection. Therefore, various antibody preparations having the ability to suppress this cellular rejection have been developed. One of them is a mouse monoclonal antibody (OKT-3) against CD3 which is a surface antigen of T cells which are responsible for immune reaction. ^{(1) , (2), (3)} . A similar antibody preparation is BMA031, a mouse monoclonal antibody that recognizes T cell receptors ^{(4), (5)} . Also, a polyclonal antibody preparation against human lymphocytes has been developed. ^{(6), (7)} .

The immune system, which is a defense mechanism in the body, is mainly classified into three stages: recognition of antigen, activation of immunocompetent cells, and cytotoxicity. The first step, the recognition of the antigen, is mainly in charge of two types of lymphocytes, one is the native form of the antigen by the membrane-bound immunoglobulin expressed on the surface of the B cell which B cell is in charge of. Recognition.
The other is the T cell receptor on the T cell for the antigen presented to the MHC molecule on the cell surface, which is phagocytosed by the antigen presenting cell and fragmented (peptide) by the cellular immune mechanism mainly responsible for the T cell. Is the recognition of. The recognition of this antigen causes the next step, activation of immunocompetent cells, simply by the binding of the antigen to membrane-bound immunoglobulin or the complex of MHC molecule + antigen between the antigen-presenting cell and the T cell. Activation of the responsible cell does not occur by signal transduction only by the binding of the body to the T cell receptor, and at that time, L of CD2, CD4, CD8, CD28, gp39,
FA-3, MHC ClassII, MHC ClassI,
It is known that transmission of a secondary signal to each cell by binding with B7 and CD40 is essential. or,
Regarding the mechanism of cytotoxicity, the antigen presented on MHC Class I was used as the MH expressing CD8 on the cell surface.
C Class I-restricted T cells (cytotoxic T cells) recognize and secrete granules containing perforin and cause cytotoxicity. While searching for antibodies that recognize the surface antigens of mononuclear cells that play an important role in these immune responses and investigating their immunosuppressive effects, nucleolin (its own involvement in the immune system is known. It has been found that an antibody that recognizes (not yet) has an immunosuppressive effect.

[0004] The immunosuppressive effects of the above various anti-adhesion molecule antibodies have been reported. However, anti-nucleolin antibodies are known as autoantibodies of autoimmune disease SLE, and the immunosuppressive effects of these antibodies have been reported. Not yet.
Here, the nucleolin itself known to date is briefly described. This protein, which is also called C23, is known as a non-histone protein that is mainly expressed in the nucleus during the growth phase of eukaryotic cells. It is suggested that the shuttle protein ⁽⁸⁾ between cytoplasm and nucleus binds to rRNA and participates in the regulation of rRNA transcription. It was discovered that it binds to cytotoxic protein Granzyme A ⁽⁹⁾ and FK506 binding protein ⁽¹⁰⁾ in cells, but its cell distribution is not only intracellular, but also activated T cells and human hepatocytes. Stock HepG
Although 2 cells ^{(1 1)} there is a low affinity for the LDL has been expressed on the surface ⁽¹¹⁾ reports such have been made, the one is not sure to play a What specific features. The amino acid primary sequence predicted from the cloned nucleolin cDNA is as shown in SEQ ID NO: 1 ⁽¹²⁾ . From this amino acid primary sequence, nucleolin is characteristic and highly hydrophilic overall, with a high charge near the N-terminus, a nuclear translocation signal near the center, and RN from the center to the C-terminus.
It has an A-binding domain and a region bound to glycine near the C-terminus ⁽¹³⁾ . Although it is a nucleolin with many personalities in this way, there are many points that its function has not yet been elucidated.

[0005]

An object of the present invention is to develop and provide a novel immunosuppressive agent containing an antibody as an active ingredient.

[0006]

Means for Solving the Problems To solve the above problems, the present inventors have screened and examined various antibodies from the viewpoint of immunosuppression, and as a result, surprisingly, antibodies against human nucleolin have immunosuppressive activity. The present invention has been completed and the present invention has been completed. 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, an antibody reactive with human nucleolin. Then, examples of the antibody having reactivity with human nucleolin include a monoclonal antibody, a polyclonal antibody, an F (ab) ₂ fragment and the like. As the monoclonal antibody-producing hybridoma, the hybridoma cell line K28 (FERM
BP-4577) is used.

The present invention will be described in detail below. As the antibody that can be used in the present invention, all of the following can be used as long as they retain or do not impair the antigen specificity to human nucleolin. A: 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 modified or produced by the gene recombination technique shown below for the purpose of humanization, improvement of affinity, elimination or enhancement of complementary activity, etc. Insert the variable region ⁽¹⁴⁾ or hypervariable region ⁽¹⁵⁾ of the antibody of A into the constant region or constant region + framework of various immunoglobulins. • Binds the variable or hypervariable regions of antibody A to molecules other than immunoglobulins ⁽¹⁶⁾ .

-It has two specificities of having the variable region of the antibody of A and the variable region of the antibody different from the antibody (bisp
esific) antibody ⁽¹⁷⁾ .・ Screen for those with higher affinity for the antigen than those expressing the antibody fragment on the phage surface
⁽¹⁸⁾ . -A product having a high affinity by causing a DNA mutation by using the PCR method or the like.

The purpose of removing complement activity from the C: A antibody
With enzyme treatment (eg pepsin treatment) ⁽¹⁹⁾And Plasmin
Reason⁽²⁰⁾Fc part is removed) or chemical modification
(For example, β-propiolactone treatment^{(twenty one)}, Disulfide
Reductive alkylation of bonds^{(twenty two)}Inactivates the Fc part
Modified). D: Human immunoglobulin producing transgenic animal
object^{(twenty three)}Made using.

E: Human antibody-producing cells introduced into an immunodeficient animal, and produced from the cells. F: Polyclonal antibody prepared by a conventional method Since the present invention suppresses not only the antigen-presenting reaction but also the cytotoxic reaction, a rejection reaction that occurs at the time of organ transplantation between allogeneic species and xenogeneic species or at the time of cell transplantation of bone marrow cells or the like It can be used as an inhibitor. It can also be used as a therapeutic drug for autoimmune diseases that recognize self-antigens and cause cytotoxicity against it, but in that case, antibodies other than humans are considered to be capable of producing antibodies against the antibodies by long-term administration. It is desirable to make humanized.

The present invention can be used in the form of injection, preferably intravenous administration, and the dose and the number of doses can be determined in consideration of the medical condition of the target disease patient, but it is 10 μg to 1000 mg / adult. Can be administered up to the day. The present invention can be prepared as a liquid preparation or a lyophilized preparation, and if necessary, contains pharmaceutically acceptable excipients, diluents, stabilizers, tonicity agents, and buffers as additives. I can do things. Preferred additives include sugars such as maltose, surfactants such as polysorbate, proteins such as human serum albumin, amino acids such as glycine, and salts such as sodium chloride. [Example of antibody preparation] Preparation of antibody (1) Acquisition of antibody-producing hybridoma Female Balb / c mouse 8 week old B cell line (CCRF-SB ATCC No. C
2 × 10 ⁷ lymphocytes activated with CL-120) were immunized by intravenous injection into the tail. The spleen of this immunized mouse was removed and the lymphocytes prepared from this spleen and mouse myeloma (p3X63 6.5.3 A
TCC No. CRL-1580) was subjected to cell fusion using polyethylene glycol 1500 (Bellinger). 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 using the following method was washed with phosphate buffer (PBS), and the blood cell fraction was suspended in 0.1% FCS (fetal calf serum) / PBS and overlaid with Ficoll. 1
After centrifugation at 500 rpm for 30 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 and centrifuged at 1500 rpm, and the leukocyte-enriched fraction (buffy coat) was taken 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 lymphocytes prepared from this spleen and p3X636.5.3 were cell-fused with polyethylene glycol 1500. The fused cells were seeded in a 96-well plate, 37 ° C, 5% CO ₂ HAT (hypoxanthine, aminopterin, thymidine Sigma) + 10% F
The cells were cultured in a CS / DMEM (Dulbecco's modified Eagle medium) medium for 10 to 20 days.

Human lymphocytes prepared by a conventional method were suspended in a suspension A (2% FCS / PBS containing 0.1% NaN ₃ ) at 1 × 1.
0 ⁶ adjusted to / ml, which was transferred to a 96-well plate at 100 [mu] l / well. 100 μl / well of the above-mentioned fused cell culture supernatant was added to this plate, and the mixture was allowed to stand at 4 ° C. for 45 minutes. Then, the lymphocytes were washed with suspension A, and goat anti-mouse IgG labeled with alkaline phosphatase
(TAGO) was reacted with the lymphocytes for 30 minutes at 4 ° C. After the reaction, the lymphocytes were washed again with suspension A, and a color-developing substrate (KPL PHOSPHATASE SUBSTRATE SYSTEM FOR EIA; Funakoshi Co., Ltd.) was added for color development.

The fused cells corresponding to the culture supernatant showing a positive reaction were limited-diluted and plated in a 96-well plate at 1 cell / well, and the positive strain was reselected by the above-mentioned enzyme-linked immunosorbent assay. A plurality of positive hybridoma strains were obtained by repeating the above series of limiting dilution operations. One of these strains was named K28. This hybridoma cell line K28
Has made an international deposit under the Budapest Treaty with the Institute of Biotechnology, Institute of Biotechnology, and the deposit number is FE.
RM BP-4577. (2) Preparation of monoclonal antibody K28N Antibody-secreting hybridoma K28 was serum-free cultured. This culture supernatant was passed through a protein A disk column, and the adsorbed IgG fraction was eluted with a buffer solution. Specifically, the following method was used.

Hybridoma K28 with 1 liter ER
The cells were cultured at 37 ° C. for 6 days in DF medium (Kyokuto Pharmaceutical Co., Ltd.) (+ insulin + transferrin, etc.). This culture solution was centrifuged to collect the culture supernatant. This culture supernatant is used as protein A
The immunoglobulin fraction was adsorbed through a column (Fuji Filter Industry). Apply this column to 1.5M Glycine 3MNa
It was washed with Cl pH 8.9 and the immunoglobulin fraction was eluted with 0.1 M Glycine pH 2.5 to prepare 30 mg of monoclonal antibody. The monoclonal antibody obtained from the K28 hybridoma was designated as K28N. (3) Preparation of K28N F (ab) ₂ F (ab) ₂ is basically prepared by Immuno Pure F (ab) ₂ pre.
It conformed to the operation method of the paration kit (PIERCE). Specifically, the following method was used.

After the K28N antibody was adjusted to a concentration of 200 mg / ml, the buffer was replaced with 20 mM sodium acetate (pH 3.5) by dialysis, and then the antibody concentration was adjusted to 10 mg / ml. Add 0.25 ml of immobilized pepsin slurry to digestion buffer (dig
Estion buffer) and washed 3 times, and suspended in 0.5 ml of digestion buffer. 1.0 ml antibody sample was mixed with 0.5 ml immobilized pepsin. After incubating at 37 ° C for 8 hours in a constant temperature water bath equipped with a shaker, the immobilized pepsin was removed and F (a
b) ₂ , Fc fragment and undigested IgG were recovered. Pro
The F (ab) ₂ fraction was purified by a tein A affinity column, dialyzed and replaced with PBS. Purified F (ab) ₂
The fraction was replaced with PBS by dialysis. 2. Analysis of antibody (1) Analysis of antigen distribution of antibody K28N by automatic fluorescence analyzer Mononuclear cells were prepared from human peripheral blood according to a conventional method. Antibody K28N and commercially available CD2, CD4, CD8, CD1
4. Fluorescent secondary staining was performed using the CD56 and MHC Class II antibodies. The fluorescence-stained mononuclear cells were analyzed using flow cytometry FACScan (Becton Dickinson) to examine the distribution of antibody-recognizing antigens. Specifically, the following method was used.

A mononuclear cell fraction was prepared from peripheral blood by a density gradient centrifugation method using Ficol (Pharmacia). The T cells were suspended in 0.1% NaN ₃ 2% FCS / PBS, human immunoglobulin was added, and the mixture was allowed to stand on ice for 30 minutes. Next, this was washed three times with 0.1% NaN ₃ 2% FCS / PBS, antibody K28N was added, and the mixture was allowed to stand at room temperature for 30 minutes, followed by FITC (fluorescein isothiocyanate: TAGO) or PE.
(Picoerythrin: TAGO) Labeled anti-mouse Ig
G antibody was added, and this was added to 0.1% NaN ₃ 2% FCS / PBS.
It was washed 3 times.

Each commercially available PE or FITC-labeled anti-human CD2 antibody (anti-Leu 5b Becton Dickinson), anti-human CD4 antibody (anti-Leu 3a Becton Dickinson), anti-human CD8 antibody (anti-Leu 5b) Leu 2a Becton Dickinson), anti-human CD14 antibody (anti-Leu-M)
3 Becton Dickinson), anti-human CD19 antibody (an
ti-Leu12 Becton Dickinson), anti-human CD56
Antibodies (anti-Leu19 Becton Dickinson), anti-human M
HC Class II antibody (anti-HLA DR Becton Dickinson) was added, and the mixture was allowed to stand at room temperature for 30 minutes. This was washed 3 times with 0.1% NaN ₃ 2% FCS / PBS.

The thus-obtained fluorescently labeled mononuclear cells are subjected to dot blot analysis using FACScan to obtain commercially available CD2, CD4 and CD.
8, CD14, CD19, CD56, MHC Class
The difference in the cell recognition group between the II antibody and the obtained antibody was tested.
As a result, it was found that the antigen recognized by the antibody K28N was expressed in the antigen-presenting cells including B cells in the inactive state, and was expressed in the above-mentioned antigen-presenting cells and T cells in the active state. (2) Identification of antigen recognized by K28N A. Preparation of antibody column using K28N Preparation of antibody column was performed using Hi Trap ^™ Affinity columns.
(Pharmacia) was used. Specifically, the following method was used.

After adjusting the concentration of K28N antibody to 10 mg / ml,
Coupling buffer (0.2M NaHCO3, 0.5M NaCl pH 8.
Replaced with 3). After equilibrating 30 ml of 1 mM HCl through a HiTrap column, inject 5 ml of antibody solution and seal with parafilm.
It was allowed to stand at 25 ° C for 20 minutes. Wash buffer A (0.
5M ethanolamine, 0.5M NaCl, pH8.3) 10 ml 3 times,
Washing buffer B (0.1M acetic acid, 0.5M NaCl, pH 4.0) 10ml
Is injected 3 times, 10 ml of washing buffer A is further injected 3 times,
It was left as it was for 1 hour to block unreacted active groups. Inject 10 ml of washing buffer B 3 times, 10 ml of washing buffer A 3 times, 3 times of 10 ml of washing buffer B 3 times, and finally 10 ml of PBS to adjust the pH to immobilize the column. finished. B. Purification of antigen that binds to K28N antibody using K28N antibody column After freeze-thawing 5 × 10 ⁸ SKW6.4 cells, 9 times volume of suspension buffer (1 mM CaCl ₂ , 1 mM NaHCO ₃ , 0.8 mg / ml) EDTA, p-APMSF
26 μg / ml, Leupeptin 10 μg / ml). This suspension was homogenized with a microhomogenizer (OMEGAERECTRIC), and the homogenate was centrifuged at 2000 xg for 15 minutes to recover the pellet. This pellet was suspended in 4.5 volumes of the above buffer solution. Furthermore, the operations from homogenization to pellet collection by centrifugation were repeated twice more.

The resulting pellet was suspended in a buffer solution to 13 ml, and this suspension was added from the bottom at 41% (W / W) sucrose solution 5 ml, 37
% (W / W) sucrose solution (7 ml) and 31% (W / W) sucrose solution (12.5 ml) were layered on a discontinuous gradient phase and centrifuged at 25,000 rpm for 2 hours. A 31% interface fraction was collected, suspended in 37 ml of 0.3 M sucrose solution, and further centrifuged at 25,000 rpm for 30 minutes. After centrifugation, the pellet was suspended in 10 ml of PBS and the cell membrane fraction was collected.

Regarding the obtained cell membrane fraction sample, S
When DS-PAGE was performed and Western blotting using K28N antibody was performed, bands of about 50K and about 100K that were specifically stained with K28N antibody were detected. Next, the whole cell fraction of SKW6.4 cells (cell lysate)
It was applied to the antibody column described in 1. After performing SDS-PAGE on the antibody column-adsorbed fraction and confirming that a similar band was detected by Western blotting using K28N antibody, SDS-PAGE was performed again on the antibody column-adsorbed fraction. The gel was stained with Ponceau (Sigma). The band portion having the antibody-binding property confirmed by the Western blotting was cut out from the gel, extracted, and then digested with lysyl endopeptidase (Wako Pure Chemical Industries, Ltd.). This digested peptide fragment was isolated by reverse phase HPLC and used as a protein sequencer PPSQ.
The amino acid sequence was determined using -10 (Shimadzu).
363 of the human nucleolin amino acid sequence shown in SEQ ID NO: 1
It matched the sequence from the residue to the 370th residue (ALELTGLK), the sequence from the 573rd residue to the 577th residue (TLFVK), and the sequence from the 637th residue to the 643rd residue (VTLDWAK).

Based on the molecular weight and the partial amino acid sequence of the antigen, it was determined that the K28N antibody is an antibody that specifically binds to human nucleolin. 3. Effect of K28N on B cell line B cell line SKW having K28N antibody recognition antigen on the cell surface
The effect of K28N on the spontaneous growth of 6.4 was observed. The method is shown below in advance the final concentration of K28N each 0ng / ml,
It was adjusted to 3.85 ng / ml, 38.5 ng / ml and 385 ng / ml. 10% F
1 × 10 ⁵ SKW6.4 / ml using CS / RPMI medium
200 μl each was rolled up in a 96-well plate and left in a 5% CO ₂ incubator for 4 days. Meanwhile 24
The cell concentration was measured after 48 hours and 96 hours.

The results are shown in FIG. As shown in this figure, K28N had little effect on the growth of SKW6.4. [Test Example] Immunosuppressive effect of anti-human nucleolin antibody (1) Immunosuppressive effect of anti-human nucleolin antibody in in vitro immunosuppressive ability test using human peripheral blood Lymphocyte mixed culture test In this experiment, monoclonal antibody K28N and its F (ab)
_A lymphocyte mixed culture test was performed on the _two fragments.

In the immune reaction, lymphocytes stimulated by antigen-presenting cells are activated and their proliferation is promoted.
This was reproduced in an in vitro system by a lymphocyte mixed culture test, which is used to determine the effects of various immunosuppressive substances ⁽²⁴⁾ . That is, it is examined whether or not the addition of the test sample suppresses the lymphocyte proliferation that occurs when lymphocytes having different haplotypes (hereinafter referred to as human A and human B for convenience) are mixed. Lymphocytes prepared in advance from human A peripheral blood are deproliferated with mitomycin C (Kyowa Hakko Co., Ltd.). This non-proliferated lymphocyte
(Stimulator) and lymphocytes (Responder) prepared from peripheral blood of human B to which antibody has been added are mixed, and 4 days later, ³ H-labeled thymidine is added to measure proliferation of human B lymphocytes. Specifically, the following method was used.

Lymphocytes were separated and prepared from the peripheral blood of human A and human B having different haplotypes by density gradient centrifugation using Ficoll. Next, human A lymphocytes
1 x 10 ⁷ RP containing serum prepared from 10% human B
The suspension was suspended in 5 ml of MI (Gibco), 250 μg of mitomycin C was added to this lymphocyte suspension, and the mixture was incubated at 37 ° C. for 30 minutes in the presence of 5% CO ₂ . Then, after centrifugation at 1500 rpm for 5 minutes, the precipitate is suspended in 1% FCS / PBS and further centrifuged (washing operation). After repeating this washing operation 4 times, 10%
It was suspended in human B serum / RPMI at 5 × 10 ⁵ / ml and used as a stimulator. In addition, human B lymphocytes 5 × 1
0 such that the ⁵ / ml were suspended in 10% B Mr. serum / RPMI, Re
sponder.

100 μl each of Stimolator and Responder
Mixed on a U-bottom 96-well plate. At this time, 1 hour before premixing, K28N antibody or F (ab) ₂ of K28N antibody was mixed.
Put the fragment on the Responder side. The mixed lymphocytes were cultured at 37 ° C. for 4 days in the presence of 5% CO ₂ . After that, 0.25 μCi / well of ³ H thymidine was added to this plate, and the temperature was 37 ° C.
It was allowed to stand for 16 to 24 hours in the presence of 5% CO ₂ .

A cell harvester (manufactured by Wallac: a device for crushing cultured cells on a culture plate to recover DNA) was used to recover DNA containing ³ H thymidine from the cells. The β dose of incorporated ³ H thymidine was measured with a liquid scintillation counter (Wallac).
The result is shown in FIG. As shown in this figure, it was found that the K28N antibody and its F (ab) ₂ fragment exhibited a suppressive effect. B. Target cytotoxicity test In this experiment, a target cytotoxicity test was performed on the monoclonal antibody K28N.

The cell damage caused by lymphocytes that occurs in the final stage of the above-mentioned series of immune reactions is closely related to the immune reaction occurring during transplantation, the immune reaction to virus-infected cells, autoimmune disease and the like. This cytotoxic effect in vivo
Since it cannot be tested in humans at o, the effect of K28N antibody on cytotoxicity was confirmed by the following cytotoxicity test ⁽²⁵⁾ , which is widely used as an in vitro test method against this. To explain the outline of the cytotoxicity test, first, mononuclear cells are obtained from human peripheral blood. Next, by culturing the mononuclear cells in the presence of a human B cell line (allostimulated cells) that has been non-proliferated with mitomycin C, cytotoxic T cells are produced from the mononuclear cells. Separately, the same human B that was subjected to the previous mitomycin C treatment
Create cells (target cells) that have incorporated ⁵¹ Cr into their cell lines. Then, the target cells and the cytotoxic T cells are cultured together, and the damage of the B cell line caused by the cytotoxic T cells is measured using the released amount of radioactive chromium as an index. When investigating the immunosuppressive effect of a test sample, cytotoxic T cells should be prepared before mixing them with target cells.
A pretreatment of adding a test siple to the cell side is performed, and it is examined to what extent the pretreatment suppresses the release of radioactive chromium. Specifically, it carried out as follows.

The blood cell fraction obtained by centrifuging human peripheral blood at 1500 rpm for 10 minutes was suspended in PBS and layered on Ficoll. Then, the mixture was centrifuged at 1500 rpm for 30 minutes, and Buffy coat was collected as a lymphocyte fraction. After washing twice with 2% FCS / PBS, the cells were suspended in 10% RPMI to obtain human peripheral blood-derived mononuclear cells. Next, the human B cell line SKW6.4 was suspended in 10% FCS,
Add mitomycin C to a final concentration of 50 μg / ml,
After standing at 37 ° C. for 30 minutes, the cells were washed 4 times with PBS to prepare allostimulated cells.

Mixing the mononuclear cells and allostimulatory cells described above
The cells were cultured at 37 ° C, 5% CO _{2 for} 6 days. After culturing, perform the same procedure as the procedure for obtaining mononuclear cells described above, and obtain the resulting cell fraction at 10%.
The cells were suspended in human serum RPMI to obtain cytotoxic T cells. Separately, the B cell lines SKW 6.4 was suspended in 10% FCS / RPMI, this ⁵¹ Cr (NEN) is added to 37 ℃ 5% CO ₂ 1 hour after incubation, washed 5 times with 1% FCS / PBS ⁵¹ Target cells incorporating Cr were prepared.

Mixing cytotoxic T cells and target cells,
It was cultured in 10% human serum / RPMI for 4 hours at 37 ° C. and 5% CO ₂ . As a control, mononuclear cells that had not been subjected to allostimulation (that is, no culture treatment in the presence of allostimulated cells) were used. The supernatant after culturing was collected by a supernatant collection system (Dainippon Pharmaceutical Co., Ltd.), and the labeled substance in the collected supernatant was measured by a gamma counter (AUTO-GAMMA 5650 Packard). K for watching immunosuppressive reaction
The 28N antibody was added to the cytotoxic cells 1 hour before mixing the target cells and the cytotoxic cells.

The results are shown in FIG. As shown in this figure, it was found that the K28N antibody has an effect of suppressing cell damage. C. Inhibition test on lymphocyte proliferation stimulated by mitogen In this experiment, an inhibition test on lymphocyte proliferation stimulated by mitogen was conducted for the monoclonal antibody K28N. LPS and PHA were used as mitogens. Specifically:

The blood cell fraction obtained by centrifuging human peripheral blood at 1500 rpm for 10 minutes was suspended in PBS and layered on Ficoll. Then, the mixture was centrifuged at 1500 rpm for 30 minutes, and Buffy coat was collected as a lymphocyte fraction. This was washed twice with 2% FCS / PBS and then suspended in 10% RPMI to obtain human peripheral blood-derived mononuclear cells. This was spread on a U-bottom 96-well plate at 1 × 10 ⁵ cells / well / 200 μl.

PHA was added to the above plate at a final concentration of 5 μg / m 2.
l or LPS was added to a final concentration of 5 μg / ml. At the same time, add K28N antibody to a final concentration of 500 ng / ml to 4 ng / ml.
Add serial dilution until 37 ℃, 5% CO _{2 for} 3 days
Cultured in the presence. Add 0.25 μCi / well of ³ H thymidine to the plate after culturing, and further 5% CO 2 at 37 ℃ for 16-24 hours
_It was allowed to stand in the presence of ₂ .

Using a cell harvester (wallac),
DNA incorporating ³ H thymidine was recovered from the cells. The β dose of ³ H thymidine was measured with a liquid scintillation counter (Wallac). The result is shown in FIG. As shown in this figure, the K28N antibody did not show an inhibitory effect on the growth stimulation by PHA, but showed an inhibitory effect on the growth stimulation by LPS. (2) In vitro using mouse or rat spleen cells
Immunosuppression test of o. Mixed lymphocyte culture test using mouse spleen cells In this test, the monoclonal antibody K28N was used, and basically the above (1) A. The test was carried out according to the human mixed lymphocyte culture test method described in. Specifically:

C3H mice with different haplotypes and B
Mononuclear cells were prepared from ALB / C mouse spleen cells according to a conventional method. Next, 1 x 10 ⁷ cells prepared from Balb / C
Suspend in 5 ml of 10% FCS / RPMI (Gibco), add 250 μg of mitomycin C to this suspension, and leave at 37 ℃ for 30 minutes.
Incubated in the presence of 5% CO ₂ . Then 1500 rpm
After centrifugation for 5 minutes, the precipitate is suspended in 1% FCS / PBS and then centrifuged (washing operation). This washing operation was repeated 4 times, and then suspended in 10% FCS / RPMI at 5 × 10 ⁵ / ml to give a stimulator. Further, cells prepared from C3H were suspended in 10% FCS / RPMI at 5 × 10 ⁵ / ml to prepare a responder.

100 μl each of Stimulator and Responder U
Mixed on bottom 96-well plate. At this time, the K28N antibody is placed on the Responder side 1 hour before the premixing.
The mixed lymphocytes were cultured at 37 ° C. for 4 days in the presence of 5% CO ₂ . Then, add 0.25μ of ³ H thymidine to this plate.
Ci / well was added and the mixture was allowed to stand at 37 ° C. for 16 to 24 hours in the presence of 5% CO ₂ .

Using a cell harvester (wallac),
DNA incorporating ³ H thymidine was recovered from the cells. The β dose of incorporated ³ H thymidine was measured with a liquid scintillation counter (Wallac). The result is shown in FIG. As shown in this figure, the K28N antibody, which is a monoclonal antibody against human nucleolin,
It was also cross-reactive with mouse lymphocytes and had a suppressive effect. B. Mixed lymphocyte culture test using rat spleen cells In this test, a monoclonal antibody K28N and a rabbit anti-rat nucleolin polyclonal antibody were used. The test was carried out according to the human mixed lymphocyte culture test method described in. Specifically:

Haplotype-different Lewis rats and ACI
Mononuclear cells were prepared from rat spleen cells by a conventional method. Next, 1 × 10 ⁷ cells prepared from ACI rats were added to 10
Suspend in 1 ml of FCS / RPMI (Gibco), add 50 μg of mitomycin C to this suspension and add 5% at 37 ℃ for 30 minutes.
Incubated in the presence of CO ₂ . Then 1500 rpm 5
After centrifugation for 5 minutes, the precipitate is suspended in 1% FCS / PBS and then centrifuged (washing operation). This washing operation was repeated 4 times, and then suspended in 10% FCS / RPMI at 4 × 10 ⁶ / ml to obtain a stimulator. In addition, cells prepared from Lewis rats were suspended in 10% FCS / RPMI at 4 × 10 ⁶ / ml to prepare Responders. Stimulator and Responder 10 each
0 μl was mixed on a U-bottom 96-well plate. At this time, 1 hour before premixing, K28N antibody (final concentration 10 μg / m
l), rabbit anti-rat nucleolin polyclonal antibody
⁽²⁶⁾ Responde (final concentration 10%) or mouse anti-rat CD3 antibody G4.18 (Seikagaku Corporation) (final concentration 10 μg / ml)
Put it on the r side. 5% of this mixed lymphocyte at 37 ℃ for 4 days
Cultured in the presence of CO ₂ . Then, 0.25 μCi / well of ³ H thymidine was added to this plate, and the plate was allowed to stand at 37 ° C. for 21 hours in the presence of 5% CO ₂ .

Using a cell harvester (wallac),
DNA incorporating ³ H thymidine was recovered from the cells. The β dose of incorporated ³ H thymidine was measured with a liquid scintillation counter (Wallac). The result is shown in FIG. As shown in this figure, not only the K28N monoclonal antibody but also the rabbit anti-rat nucleolin polyclonal antibody showed a remarkable immunosuppressive effect. (3) In vivo immunosuppressive effect in pathological model animals A. Experiment with Type II Collagen-induced Arthritis Model Mouse In this experiment, the inhibitory effect in the indicated arthritis model was tested using the K28N antibody. This model is a system for inducing arthritis by immunizing mice with a mixture of complete Freund's adjuvant (hereinafter referred to as CFA) and type II collagen.
Widely used as a model for rheumatism. An antibody was administered to this model animal to examine its immunosuppressive effect.
The method is shown below.

CFA (DIFCO LABORATORY) and type II collagen (Sigma) were mixed to prepare an emulsion in ice, and 100 μl was subcutaneously injected into the ridge of 24 DBA1 mice (6 weeks old male: Charles River Japan). Make a sensitization. Furthermore, booster immunization is performed on the 25th and 40th days after the first sensitization to induce edema of limbs. The prophylactic administration group, the therapeutic administration group, and the non-administration group are divided into groups of 8 animals each, and the administration group for which the prophylactic effect is observed is K28N at 5-6 day intervals from 1 day before the first sensitization.
Was intravenously administered at 300 μg / animal. In addition, K28N was intravenously administered at 300 μg / mouse from the 20th day after the first sensitization at an interval of 5 to 6 days to the administration group for which the therapeutic effect was observed.

Mice in which the development of edema was observed in any of the four limbs of each mouse were counted as the affected individuals, and those in which no macroscopic edema was observed were counted as the unaffected individuals.
The result is shown in FIG. 7. As shown in this figure, the antibody K28
N showed an inhibitory effect on type II collagen-induced arthritis. B. Experiments with MRL lpr / lpr mice In this experiment, K28N antibody was used to test the inhibitory effect in a systemic autoimmune disease model (MRL lpr / lpr mice). MRL lpr / lpr mice spontaneously develop systemic autoimmune disease with symptoms such as nephritis, vasculitis, lymphoid hypertrophy, Sjogren's syndrome, joint lesions, and elevated IgG1, IgG2a, and SLE (systemic lupus erythematosus). It is widely used as a model animal for squirrels and scrotum. Since there is a time-dependent increase in serum urea nitrogen concentration due to lupus nephritis, which is a condition of SLE, and a decrease in the survival rate associated with the development of autoimmune disease, the urea nitrogen concentration in serum and the survival rate are used as indicators. The immunosuppressive effect of antibody administration was examined.
The method is shown below.

The urea nitrogen concentration in the serum of MRL lpr / lpr mice was measured using a urea nitrogen measurement kit (urea-nitrogen B test Wako (Wako Pure Chemical Industries)), and the antibody K28N administration group and non-administration group were evenly distributed. Ten animals were divided into groups. To the mice in the administration group, administration of 200 μg / animal of the antibody was started at 1-week intervals from before the appearance of lymph node hypertrophy (80 days old). Serum urea nitrogen concentration and survival rate of the administration group and the non-administration group were measured and the administration effect was observed over time.

The results are shown in FIGS. As shown in the figure, the antibody K28N increased the concentration of urea nitride in serum over time.
(Fig. 8) and decrease in survival rate due to autoimmune disease development
The suppression effect was shown for (Fig. 9). C. Experiment Using GVHD Model Mouse In this experiment, K28N antibody was administered to a GVHD model mouse that was developed by injecting mouse bone marrow cells with different haplotypes into an X-irradiated mouse, and the immunosuppressive effect was examined.
Specifically, the method is shown below.

C3H mice (Charles River) were irradiated with X-ray irradiation equipment MBR1520R (Hitachi) 10 Gy of X-rays to kill bone marrow cells, and grouped into 8 bone marrow cell-transfected groups and 3 bone marrow cell-untransfected groups. Was done. Furthermore, 8 mice in the bone marrow cell transfer group were divided into 4 groups in the antibody-treated group and 4 groups in the untreated group. In the bone marrow cell transfer group, 9.0 × 10 ⁶ bone marrow cells prepared from C57 / Black (Charles River) were intravenously injected.
In addition, four bone marrow cells treated with antibody were treated with K28N antibody (5 μg / ml) for 1.5 hours before the bone marrow cells prepared from C57 / Black were transferred, and further transferred after this bone marrow cell transfer. K3N antibody 300μg / animal / in tail vein of C3H
It was administered for 7 days.

The results are shown in FIG. G as shown
K28N showed an inhibitory effect on the decrease in survival rate due to the onset of VHD.

[0048]

【Example】

Example 1 Solution-1 anti-human nucleolin monoclonal antibody (K28N) 10m
g, Japanese Pharmacopoeia 1.0 mg, sodium chloride 16 mg, 2 m
l Aseptically prepare a solution consisting of PBS, fill an ampoule,
It was used as an injection solution.

Example 2 Solution-2 F of anti-human nucleolin monoclonal antibody (K28N)
(Ab) _{2 A} solution consisting of 10 mg, JP polysorbate 1.0 mg, sodium chloride 16 mg, and 2 ml PBS was aseptically prepared and filled in an ampoule to give an injection solution. Example 3 Solution-3 Anti-Human Nucleolin Polyclonal Antibody 100 mg, Japanese Pharmacy Polysorbate 1.0 mg, Sodium Chloride 16 mg, 2 ml PBS
Was prepared aseptically, filled in ampoules, and used as an injection solution.

Example 4 Lyophilizer-1 Anti-human nucleolin monoclonal antibody (K28N) 10m
g, Japanese Pharmacopoeia 1.0 mg, sodium chloride 16 mg, 2 m
l A lyophilizate prepared by aseptically preparing a solution consisting of PBS. It is dissolved in distilled water for injection and used as an injection solution. Example 5 Lyophilizer-2 F of anti-human nucleolin monoclonal antibody (K28N)
(Ab) ₂ A lyophilizate prepared by aseptically preparing a solution consisting of 10 mg, JP-A polysorbate 1.0 mg, sodium chloride 16 mg, and 2 ml PBS. It is dissolved in distilled water for injection and used as an injection solution.

Example 6 Freeze-Drying Agent-3 Anti-human Nucleolin Polyclonal Antibody 100 mg, Japanese Polysorbate 1.0 mg, Sodium Chloride 16 mg, 2 ml PBS
A lyophilizate prepared by aseptically preparing a solution consisting of. It is dissolved in distilled water for injection and used as an injection solution.

[0052]

The present invention is to provide a novel immunosuppressive agent. The immunosuppressive agent of the present invention containing an antibody reactive to human nucleolin as an active ingredient is an immunosuppressive agent effective against various immune diseases and is extremely useful industrially. References (1) Von Wauwe JP, Demey JR, Goosens JG J Immunol 1980; 124: 2708 (2) Ellenhorn JDI, Woodle ES, Ghobrial I., T
histlethwaite JR, Bluestone JA Transplantation 1990; 50: 608 (3) OKT-3 TNF-a IL2 r-IFN expression in serum (4) Smely S., Weschka M., Hillebrand G., Dendorfe
r U., Krombach F., Kurrle R., Land W., Hammer C., Transplant proc 1990; 22: 1785 (5) Hayasaka Y., Takahasi K., Yagisawa T., Oota K. Transplantation Now 1992; 5 : 125 (6) Aarbulin (dried anti-human lymphocyte equine immunoglobulin) Green Cross Catalog (7) Presmuin (dried anti-human lymphocyte equine immunoglobulin) Hoechst Japan Catalog (8) RABorer, CFLehner, HM Eppenberger, and E.
A. Nigg Cell 1989, 56,379-390 (9) Mark S. Pasternack, Kevin J. Bleier, and Th
omas N. McInerney The Journal of Biological Chemistry 286,22,14703
~ 14708, 1991 (10) Youb Jiu Jin and Steven J. Burakoff Proc. Natl. Acad. Sci. USA 1993, 90, 7769 ~ 7773 (11) Serivastava M., Mcbride OW, Fleming PJ,
Pollard HB and BurnsA.LJ Biol. Chem. 265, 14922-14931 (1990) (12) Srivastava M., Fleming P. J., Pollard HB &
Burns AL FEBS Lett, 1989 250, 99-105 (13) Clay F. Semenkovich, Richard E. Ostland, Jr,
Mark OJOlson, andjoseph W. Yang Biochemistry 1990,29,9708-9713 (14) Bouliane GL, Hozumi N. & Shulman MJ, Natu
re 1984 312,643-646 (15) Riechman, L., Clark, M .., Waldman, H. & Winter, G.Na
ture 1988 332,323-327 (16) Kreitman RJ, Chaudhary VK, Waldman T., W
illingham MC, Fitz Gerald DJ & Pastan I. Proc. Natl. Acad. Sci. USA 1990 87 8291-8295 (17) Bator JM & Reading CL, In Therapeutic Mo
noclonal Antibodies. 1990 (18) McCafferty J., Griffiths AD, Winter G. & C
hiswell DJ, Nature 1990 348, 552-554 (19) Barandun S., Castel V., Makura MF, Morell
A., Plan R. & SkvarilF. Vox Sang. 1975 28 157-175 (20) Koblet H., Barandun S. & Diggelman H., Vox
Sang. 1967 13,93-102 (21) Stephan W., Vox Sang.1975 28 422-437 (22) Morell A., Vox Sang. 1986 51 (suppl 2) 44-49 (23) Brugman M., Proc .Natl.Acad.Sci .USA 1989 8
6,6709-6713 (24) Yoshihisa Ito All about Cellular Immune Function Testing 173 (25) Junichi Yada, Michio Fujiwara New Lymphocyte Function Retrieval Method Chugai Medical 189 (26) Kiyoji Kata Clinical Immunity 1992 24 (4), 573-580

[0053]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 707 Sequence type: Amino acid Sequence type: Protein Topology: Linear Sequence feature: Human nucleolin amino acid primary structure Sequence Met Val Lys Leu Ala Lys Ala Gly Lys Asn Gln Gly Asp Pro Lys Lys 5 10 15 Met Ala Pro Pro Pro Lys Glu Val Glu Glu Asp Ser Glu Asp Glu Glu 20 25 30 Met Ser Glu Asp Glu Glu Asp Asp Ser Ser Gly Glu Glu Val Val Ile 35 40 45 Pro Gln Lys Lys Gly Lys Lys Ala Ala Ala Thr Ser Ala Lys Lys Val 50 55 60 Val Val Ser Pro Thr Lys Lys Val Ala Val Ala Thr Pro Ala Lys Lys 65 70 75 80 Ala Ala Val Thr Pro Gly Lys Lys Ala Ala Ala Thr Pro Ala Lys Lys 85 90 95 Thr Val Thr Pro Ala Lys Ala Val Thr Thr Pro Gly Lys Lys Gly Ala 100 105 110 Thr Pro Gly Lys Ala Leu Val Ala Thr Pro Gly Lys Lys Gly Ala Ala 115 120 125 Ile Pro Ala Lys Gly Ala Lys Asn Gly Lys Asn Ala Lys Lys Glu Asp 130 135 140 Ser Asp Glu Glu Glu Asp Asp Asp Ser Glu Glu Asp Glu Glu Asp Asp 145 150 155 160 Glu Asp Glu Asp Glu Asp Glu Asp Glu Ile Glu Pro Ala Ala Met Lys 165 170 175 Ala Ala Ala Ala Ala Pro Ala Ser Glu Asp Glu Asp Asp Glu Asp Asp 180 185 190 Glu Asp Asp Glu Asp Asp Asp Asp Asp Glu Glu Asp Asp Ser Glu Glu 195 200 205 Glu Ala Met Glu Thr Thr Pro Ala Lys Gly Lys Lys Ala Ala Lys Val 210 215 220 Val Pro Val Lys Ala Lys Asn Val Ala Glu Asp Glu Asp Glu Glu Glu 225 230 235 240 Asp Asp Glu Asp Glu Asp Asp Asp Asp Asp Glu Asp Asp Asp Asp Glu Asp Asp 245 250 255 Asp Asp Glu Asp Asp Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Pro 260 265 270 Val Lys Glu Ala Pro Gly Lys Arg Lys Lys Glu Met Ala Lys Gln Lys 275 280 285 Ala Ala Pro Glu Ala Lys Lys Gln Lys Val Glu Gly Thr Glu Pro Thr 290 295 300 Thr Ala Phe Asn Leu Phe Val Gly Asn Leu Asn Phe Asn Lys Ser Ala 305 310 315 320 Pro Glu Leu Lys Thr Gly Ile Ser Asp Val Phe Ala Lys Asn Asp Leu 325 330 335 Ala Val Val Asp Val Arg Ile Gly Met Thr Arg Lys Phe Gly Tyr Val 340 345 350 Asp Phe Glu Ser Ala Glu Asp Leu Glu Lys Ala Leu Glu Leu Thr Gly 355 360 365 Leu Lys Val Phe Gly Asn Glu Ile Lys Leu Glu Lys Pro Lys Gly Lys 370 375 380 Asp Ser Lys Lys Glu Arg Asp Ala Arg Thr Leu Leu Ala Lys Asn Leu 385 390 395 400 Pro Tyr Lys Val Thr Gln Asp Glu Leu Lys Glu Val Phe Glu Asp Ala 405 410 415 Ala Glu Ile Arg Leu Val Ser Lys Asp Gly Lys Ser Lys Gly Ile Ala 420 425 430 Tyr Ile Glu Phe Lys Thr Glu Ala Asp Ala Glu Lys Thr Phe Glu Glu 435 440 445 Lys Gln Gly Thr Glu Ile Asp Gly Arg Ser Ile Ser Leu Tyr Tyr Thr 450 455 460 Gly Glu Lys Gly Gln Asn Gln Asp Tyr Arg Gly Gly Lys Asn Ser Thr 465 470 475 480 Trp Ser Gly Glu Ser Lys Thr Leu Val Leu Ser Asn Leu Ser Tyr Ser 485 490 495 Ala Thr Glu Glu Thr Leu Gln Glu Val Phe Glu Lys Ala Thr Phe Ile 500 505 510 Lys Val Pro Gln Asn Gln Asn Gly Lys Ser Lys Gly Tyr Ala Phe Ile 515 520 525 Glu Phe Ala Ser Phe Glu Asp Ala Lys Glu Ala Leu Asn Ser Cys Asn 530 535 540 Lys Arg Glu Ile Glu Gly Arg Ala Ile Arg Leu Glu Leu Gln Gly Pro 545 550 555 560 Arg Gly Ser Pro Asn Ala Arg Ser Gln Pro Ser Lys Thr Leu Phe Val 565 570 575 Lys Gly Leu Ser Glu Asp Thr Thr Glu Glu Thr Leu Lys Glu Ser Phe 580 585 590 Asp Gly Ser Val Arg Ala Arg Ile Val Thr Asp Arg Glu Thr Gly Ser 595 600 605 Ser Lys Gly Phe Gly Phe Val Asp Phe Asn Ser Glu Glu Asp Ala Lys 610 615 620 Glu Ala Met Glu Asp Gly Glu Ile Asp Gly Asn Lys Val Thr Leu Asp 625 630 635 640 Trp Ala Lys Pro Lys Gly Glu Gly Gly Phe Gly Gly Arg Gly Gly Gly 645 650 655 Arg Gly Gly Phe Gly Gly Arg Gly Gly Gly Arg Gly Gly Arg Gly Gly 660 665 670 Phe Gly Gly Arg Gly Arg Gly Gly Phe Gly Gly Arg Gly Gly Phe Arg 675 680 685 Gly Gly Arg Gly Gly Gly Gly Asp His Lys Pro Gln Gly Lys Lys Thr 690 695 700 Lys Phe Glu 705

[Brief description of drawings]

FIG. 1 is a diagram showing the influence of antibodies on B cell lines.

FIG. 2 shows the inhibitory effect of antibodies in a mixed human lymphocyte culture test.

FIG. 3 shows the inhibitory effect of antibodies in human target cytotoxicity tests.

FIG. 4 shows the inhibitory effect of antibodies on mitogen-stimulated proliferation.

FIG. 5 shows the inhibitory effect of antibodies in a mouse lymphocyte mixed culture test.

FIG. 6 shows the inhibitory effect of antibodies in a rat lymphocyte mixed culture test.

FIG. 7 is a view showing the inhibitory effect of antibodies in type II collagen-induced arthritis model mice.

FIG. 8 shows the inhibitory effect (blood urea nitrogen concentration) of an antibody on MRLlpr / lpr.

FIG. 9: Inhibitory effect of antibody on MRLlpr / lpr (survival rate)
FIG.

FIG. 10 shows the inhibitory effect of antibodies in GVHD model mice.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location (C12P 21/08 C12R 1:91)

Claims (6)

[Claims] 1. An immunosuppressant comprising an antibody reactive to human nucleolin as an active ingredient. 2. The human nucleolin-reactive antibody is a monoclonal antibody.
The immunosuppressant described. 3. The antibody having reactivity with human nucleolin is a polyclonal antibody.
The immunosuppressant described. 4. The immunosuppressant according to claim 1, wherein the antibody reactive with human nucleolin is an F (ab) ₂ fragment. 5. An antibody having reactivity with human nucleolin is a hybridoma cell line K28 (FERM BP-45).
The immunosuppressive agent according to claim 2, which is a monoclonal antibody produced by 77). 6. The immunosuppressive agent according to claim 4, wherein the F (ab) ₂ fragment is derived from a monoclonal antibody produced by the hybridoma cell line K28 (FERMBP-4577).