JP2006062977A - Therapeutic agent for atopic dermatitis - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a therapeutic and/or a prophylactic agent efficacious for atopic dermatitis. <P>SOLUTION: The prophylactic and/or the therapeutic agent for the atopic dermatitis contain an antibody as an active ingredient, wherein the antibody comprises a monoclonal antibody against I-J molecule and is obtained by a method for selecting an antibody which does not injure an inactive-type immunocyte when added, but injures an activated immunocyte. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a preventive and / or therapeutic agent for atopic dermatitis comprising a monoclonal antibody against IJ molecule as an active ingredient.

Atopic dermatitis is a typical allergic disease in which the number of patients is currently increasing. The symptoms of atopic dermatitis are eczema with strong itching, and if it becomes a serious patient, it will interfere with daily life. The pain experienced by people in such a state is immeasurable. Although research has been conducted on treatment methods for atopic dermatitis in various directions, most of the treatment methods used are administration of symptomatic steroids, antiallergic agents, antihistamines and the like. In such treatment, there are many cases in which recurrence repeats even if symptoms improve temporarily and remit. In addition, since steroids have problems of side effects, there is a limit to long-term repeated use, and patients tend to avoid steroids.
Therefore, there has been a demand for the development of more selective and less side-effect therapeutic agents based on the onset and pathogenesis.

On the other hand, the present inventors have found that among antibodies to IJ molecules that are MHC class I-like molecules, there are antibodies that damage activated lymphocytes and lymphoma cells in a complement-independent manner. One of them was named RE2. (Refer nonpatent literature 1). Cell death caused by lymphoma cells and the like is cell death not caused by other anti-MHC class I antibodies, and is a new type of cell death that is not apoptosis. It has been reported that antibodies are effective in treating lymphoma, and that they are effective in treating hepatitis by suppressing the symptoms of artificially induced hepatitis (see Non-Patent Document 2). Moreover, it discovered that the epitope in the MHC class I molecule | numerator of RE2 antibody exists in (alpha) 2 domain (refer nonpatent literature 2). In addition, the class of the RE2 antibody was IgG _1.

However, it is not known at all that the antibody has an excellent therapeutic effect on atopic dermatitis.
J.Exp.Med.181, 2007-2015, 1995 J.Exp.Med.198, 497-503, 2003

  An object of the present invention is to provide a preventive and / or therapeutic agent for atopic dermatitis having high efficacy and few side effects.

  Under such circumstances, the present inventors have conducted various studies on a wide variety of antibodies. Among the antibodies against the I-J molecules described above, there are antibodies that damage only activated lymphocytes and lymphoma cells. The present inventors have found that the present invention has a therapeutic effect on atopic dermatitis.

  That is, the present invention relates to the prevention and / or prevention of atopic dermatitis, which comprises, as an active ingredient, an antibody obtained by a method for selecting an antibody that is a monoclonal antibody against an IJ molecule and that only damages activated immune cells. Alternatively, a therapeutic agent is provided.

  ADVANTAGE OF THE INVENTION According to this invention, the chemical | medical agent with the high preventive treatment effect with respect to atopic dermatitis and high safety | security can be provided.

  The monoclonal antibody of the present invention is a monoclonal antibody against the IJ molecule, which is obtained by a method of selecting an antibody that is administered to activated immune cells and inactivated immune cells and that only damages activated immune cells. It is what

Here, the IJ molecule is known as a molecule on the cell surface of a suppressor (suppressor) T cell, and the gene encoding this is not only mapped to the MHC region, but also in the process of T cell differentiation. The molecule is also determined passively by its environment (Nature 1985 Aug22-28; 316 (6030): 741-3). I-J molecule is defined by the MHC allotype, MHC allotypes is k (C3H mice, etc.), then ^{I-J k} molecule, b polymorphic so on (C57BL6 mice) if ^{I-J b} molecule such a molecule, for example, to obtain the I-J ^k molecules can be obtained by allotypes immunoprecipitate T cell clone lysates from k anti I-J ^k antibody, I-J ^b molecule Can be obtained by immunoprecipitation of a T cell clone lysate derived from the allotype b with an anti- ^IJk antibody. (Proc Natl Acad Sci USA. 1985 May; 82 (9): 2905-9.) (Int Immunol. 1989; 1 (1): 50-8.).

The monoclonal antibody of the present invention comprises 1) immunizing an animal with an IJ molecule, 2) preparing antibody-producing cells from the animal, and fusing them with myeloma cells to produce a hybridoma;
3) Collecting antibodies from each hybridoma, examining the cytotoxicity of these antibodies against activated immune cells and inactivated immune cells, and exhibiting cytotoxicity specifically to activated immune cells. It can be obtained by selecting the recognized antibodies.

  1) As a method for immunization, a known method can be used. For example, an IJ molecule can be used alone or in a mammal in the presence or absence of adjuvant. Intraperitoneal administration is preferable from the viewpoint of immune efficiency. In this case, administration is preferably performed every two weeks for several months. In addition, the dose of the immunizing antigen molecule is appropriately determined according to the administration route, the type of mammal, etc., but when administered intraperitoneally to a mouse, the protein amount of the normally administered IJ molecule is 1 mg for the first time. The second and subsequent times are preferably about 0.3 mg / animal. The carrier is not particularly limited as long as it does not itself have a harmful effect on the host and enhances antigenicity, and examples thereof include cellulose, polymerized amino acid, albumin, keyhole limpet hemocyanin and the like. Adjuvants include Freund's complete adjuvant, Freund's incomplete adjuvant, Ribi (MPL), Ribi (TDM), Ribi (MPL + TDM), pertussis vaccine (Bordetella pertussis vaccine), muramyl dipeptide, aluminum adjuvant (ALUM), and combinations thereof Can be illustrated. The immunized animal is not particularly limited, and mammals used for the production of known monoclonal antibodies can be used, but are selected in consideration of compatibility with bone marrow cells used for cell fusion. In general, mice, hamsters, rats and the like are preferably used. In addition, the age at the time of immunization of these rodents is preferably 8 to 10 weeks from the viewpoint of immune effect.

2) A method for preparing antibody-producing cells from the animal is performed by a known method.
Next, the obtained immune cell bone marrow cells are fused. As the bone marrow cells used here, various known cell lines for which selection means have already been established, such as NS1-Ag4 / 1 (Europian J. Immunol., 6: 511-519 (1976)), P3-U1 (Current Topic in microbiology and Immunology, 81: 1-7 (1978)) and the like can be used.

  The above fusion reaction between immune cells and myeloma cells is performed according to a known method, that is, according to the method of Oi and Herzenberg (Selected Methods in Cellular Immunology, p351-371, WHFreeman & Co., USA (1980)). It is carried out in a normal nutrient medium in the presence of a promoter. As the fusion promoter, commonly used polyethylene glycol (PEG), Sendai virus (HVJ), and the like can be used, and an auxiliary such as dimethyl sulfoxide can be added and used as desired. The usage ratio of immune cells to myeloma cells is not different from that of the usual method.

  As the medium for the fusion, any of various normal nutrient media used for this type of cell culture can be used. Representative examples thereof include RPMI-1640 medium, Dulbecco's MEM medium, and the like, and these mediums include, for example, fetal bovine serum (FCS), essential amino acid mixture, sodium pyruvate, sodium mercaptoethanol, etc. Can also be added.

  Fusion is performed by, for example, mixing a predetermined amount of the above immune cells and bone marrow cells in the above medium without serum and centrifuging, removing the supernatant, and then adding PEG preheated to about 37 ° C. to the medium. It is performed by adding and adding to the cell sediment a mixture added to a concentration of about 30-60% (W / V). This is reacted at 37 ° C. for several minutes, an appropriate medium is sequentially added, and the mixture is allowed to stand at room temperature for about 10 minutes and centrifuged. Then, the desired hybridoma is prepared by removing the supernatant.

The obtained hybridoma is separated by culturing in an ordinary selection medium such as HAT medium, and further culturing the culture in HT medium (medium containing hypoxanthine and thymidine) for several weeks.
The hybridoma thus obtained is monocloned according to a known method such as a normal limiting dilution method.

Next, the antigen binding property of the antibody produced by this hybridoma is confirmed. The confirmation is performed by reacting the hybridoma supernatant with IJ-positive lymphocyte cells, further reacting with an anti-immunoglobulin antibody labeled with FITC or the like, and confirming the staining with a flow cytometer.
The hybridoma thus obtained can be subcultured in a normal medium and stored stably in liquid nitrogen for a long period of time.

  Suitable hybridomas include, for example, a hybridoma (RE2 2004.01.14) deposited at the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center under the deposit number FERM P-19701.

3) A monoclonal antibody is collected from the hybridoma medium thus obtained. Collection is performed by culturing the hybridoma according to a conventional method and separating it from the culture supernatant, or by administering the hybridoma to a mammal compatible with the hybridoma, allowing it to proliferate, and separating it from the ascites of the animal. However, a method of separating from the culture supernatant is preferable.
Next, the obtained antibody is purified. Purification methods include known methods (for example, various column chromatography using an anion exchanger, hydroxyapatite, protein A or G immobilized column, protamine immobilized column, etc., ammonium sulfate fractionation method, PEG fractionation, etc. Column chromatography using a protein G-immobilized column is preferred, although it can be easily achieved by applying a method, an ethanol fractionation method and a hypotonic buffer precipitation method).

Furthermore, the antibody thus obtained is administered to activated immune cells and non-activated immune cells in equal amounts respectively and cultured for a certain period of time. Thereafter, the ratio of dead cells in activated immune cells and Measure the rate of dead cells in inactivated immune cells. In this case, non-stimulated peripheral lymphocytes are preferred as non-activated immune cells. Activated immune cells can be obtained by activating corresponding peripheral lymphocytes, for example, culturing T cells in the presence of IL2, or culturing B cells in the presence of LPS. Can be obtained. Dead cells can be confirmed by staining with blue when trypan blue is added.
The monoclonal antibody of the present invention can be obtained by selecting an antibody in which the rate of dead cells in activated immune cells is significantly higher than the rate of dead cells in non-activated immune cells.

  The monoclonal antibody of the present invention thus obtained reacts specifically with the IJ molecule, and does not damage normal cells but damages activated immune cells such as activated lymphocytes and lymphoma cells. Have Among these, the RE2 antibody produced from hybridoma RE2 2004.01.14 (FERM P-19701) is particularly preferable from the viewpoint of the therapeutic effect on atopic dermatitis.

In addition, the monoclonal antibody used in the preventive and / or therapeutic agent for atopic dermatitis of the present invention retains the active region of the antibody and can crosslink the antigen, thereby preventing and / or treating the atopic dermatitis. As long as it exhibits, a part of the antibody molecule can be used. That is, the monoclonal antibody of the present invention includes F (ab ′) ₂ which is a fragment thereof.

  Further, the monoclonal antibody of the present invention can be used by retaining its variable region (Fab region, Fc region) as an animal such as a mouse, and replacing other constant regions with those derived from humans (chimeric antibody), Alternatively, only the complementarity determining region (CDR) can be left as it is, and all other regions can be replaced with those derived from humans (humanized antibody). Conversion to such a chimeric antibody or humanized antibody can be performed according to a known method.

  The monoclonal antibody of the present invention, as shown in the Examples below, is an action that significantly reduces serum IgE levels and significantly improves symptoms such as eczema, redness, and ulcers when administered to mice with atopic dermatitis. Have Therefore, a preparation containing an effective amount of this antibody is useful as a medicament for the prevention and / or treatment of atopic dermatitis.

  The preparation form of the prophylactic and / or therapeutic agent for atopic dermatitis of the present invention may be either an oral administration preparation or a parenteral administration preparation, but a parenteral administration preparation is preferred, particularly for subcutaneous administration and intravenous administration. An injection is preferred.

  Such a preparation can be prepared by a conventional method. For example, in the case of injections, the monoclonal antibody of the present invention usually contains an osmotic pressure adjusting agent, a pH adjusting agent, a surfactant, a thickener and the like as necessary. The excipient used may be added and dissolved in distilled water for injection. Moreover, it is good also as a dissolution type preparation at the time of use. In the case of tablets, it may be prepared by adding usually used formulation additives such as excipients, binders, lubricants, disintegrants, and coating agents to the monoclonal antibody as necessary.

  The dose of the preventive and / or therapeutic agent for atopic dermatitis of the present invention is determined depending on the symptoms and dosage form, but as a monoclonal antibody, 0.25 to 0.5 mg per day is divided into once or several times. May be administered.

  EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, the technical scope of this invention is not limited to these illustrations.

Example 1 Production of antibodies (1) NP-40 lysate of immunized mouse T cell clone MS-S2 was immunoprecipitated with anti-IJ antibody (JK10-23) and 1-2 × 10 ⁹ MS -Immunoprecipitation product separated and purified from S2 was injected into SD rats as an immunogen. This immunization was repeated 4 times every 2 weeks.

(2) Production of hybridoma Three days after the last booster-stimulated immunization, splenocytes were excised and 3 × 10 ⁸ cells and mouse myeloma cell line P3-U1 cells 3 × 10 ^{8 were obtained.} And cell fusion using polyethylene glycol (PEG).

(3) Antibody antigen reaction It was confirmed that the antibody was reacted with IJ-positive lymphocyte cells and further reacted with an anti-immunoglobulin antibody labeled with FITC as a secondary antibody and stained with a flow cytometer. Next, it was reacted with IJ-negative lymphoid cells, and further reacted with an anti-immunoglobulin antibody labeled with FITC as a secondary antibody, and it was confirmed that it was not stained with a flow cytometer.

(4) Cloning of antibody mAb-producing hybridoma The antibody-producing hybridoma collected in this manner was subjected to limiting dilution to obtain a single clone. Five clones were obtained as hybridomas capable of producing antibodies and proliferating.

(5) Collection of antibody The monoclonal antibody used in the present invention from the hybridoma was collected by a method in which the hybridoma was cultured and separated from the culture supernatant. Further purification was performed using a protein G-Sepharose column (Pharmacia LKB, Biotecnology AB).

(6) Selection of cytotoxic antibody The antibody thus obtained was added at a dilution of 1: 100 to the target cell suspension adjusted to 1 × 10 ⁷ / ml, and incubated at 37 ° C. for 1 hour. Trypan blue was added to count the number of viable cells. Then,% injury activity was calculated using the following formula 1.
(Formula 1) (% injury activity) = (A−B) / A × 100
However, A = (number of viable cells after incubation with culture medium for 1 hour)
B = (number of viable cells after adding antibody and incubating for 1 hour)
One type of hybridoma was obtained as a hybridoma producing an antibody having a% injury activity of 80% or more. This hybridoma was deposited with the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center. The accession number is FERM P-19701. The antibody produced by this hybridoma was named RE2.

Example 2 Detection of molecules recognized by RE2 The cell surface of lymphocytes was labeled with ¹²⁵ I by the iodogen method, and membrane protein was solubilized with 1% NP-40. NP40 lysate and RE2 antibody were reacted at 4 ° C. for 12 hours, and then protein G-Sepharose beads were added for immunoprecipitation. The immunoprecipitate was dissolved in sodium lauryl sulfate (SDS) sample buffer and analyzed by SDS polyacrylamide gel electrophoresis according to the method of Laemmli.
Then, it was revealed that RE2 recognizes 44K and 60K molecules on stationary lymphocytes and recognizes 44K, 60K, and 88K molecules on activated lymphocytes.

Test Example 1 Results of Atopic Dermatitis Inflammation Treatment Experiment NC / Nga mice, the most well-known 12-week-old atopic dermatitis spontaneously mouse, are treated with 2 mg every other day for 2 weeks. The clinical score of atopic dermatitis (J. All. Clin. Immunol., 85; 927-933, Int. Immunol., 9, in the group administered and the group administered rat immunoglobulin (Ig) as a control) 461-466), IgE values, and pathological findings of the skin were compared.

  That is, the degree of Pruritus, Erythema, Edema / papulation, Excoriation and Scaling / dryness (dandruff, dryness) in the skin was scored as 1, 2, or 3 points, respectively. The total score was taken as the clinical score. Pruritus (pruritus) was observed on video, and the one with a high degree was scored high. About Erythema (red spot), 1 point was a light pink color, 2 points were red, and 3 points were red. Edema / papulation (edema) was scored 1 point for the root, 2 points for the whole, and 3 points for the whole area. For Excoriation, we scored 1 point if we weren't looking for it, 2 points if it was at a glance, and 3 points if it was too scratched. Scaling / dryness (dandruff, dryness) was scored with a high score.

  The clinical score began to decrease almost simultaneously with the start of administration of the RE2 antibody (FIG. 1), and almost healed especially with eczema, redness, and ulcers characteristic of atopic dermatitis. The serum IgE value was 15.0 μg / mL in the control mouse group, but decreased to about 11.5 μg / mL after 2 weeks of administration of the RE2 antibody (FIG. 2). Comparing the dorsal skin tissue immediately after completion of administration for 2 weeks, severe lichenification was observed in the control mouse group, whereas it was rather atrophied in the RE2 administration group (FIG. 3). It was considered that the therapeutic effect was obtained by damaging activated lymphocytes and mouse cells with the RE2 antibody.

It is the figure which showed the time-dependent change of the clinical score of atopic dermatitis. It is the figure which showed the serum IgE value after administering RE2 antibody compared with control. It is the figure which showed the mode of the skin which atopic dermatitis healed by administering RE2 antibody.

Claims (3)

  A monoclonal antibody against an I-J molecule, which is obtained by administering to activated immune cells and inactivated immune cells, and selecting an antibody that damages only activated immune cells. A preventive and / or therapeutic agent for atopic dermatitis.   The preventive and / or therapeutic agent according to claim 1, wherein the immune cells are lymphocytes and / or lymphoma cells.   The prevention according to claim 1 or 2, wherein the antibody is a monoclonal antibody RE2 produced by a hybridoma (RE2 2004.01.14) deposited as FERM P-19701 at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology. And / or therapeutic agent.

Images