The present invention provides an apoptosis inducer and a therapeutic agent for eosinophilic diseases which comprises, as an active ingredient, an antibody which reacts specifically with eosinophils and induces apoptosis of eosinophils; and a method for inducing eosinophil apoptosis using the antibody, and a method for specifically reducing or removing eosinophils in peripheral blood or tissues using the antibody.

Description of the Invention

SUMMARY OF THE INVENTION

The development of a clinically more effective treatment having lower side effects has been long awaited for the treatment of inflammatory diseases, such as chronic bronchial asthma, and eosinophil associated diseases, such as eosinophilic granuloma.

Inventors of the present invention have found that human eosinophil-specific apoptosis induced by an anti-IL-5 receptor .alpha.-chain antibody with an Fc region of the human IgG1 subclass as disclosed in WO97/10354 is mediated by antibody-dependent cellular cytotoxicity. Since apoptosis of eosinophils mediated by antibody-dependent cellular cytotoxicity does not cause the release of cytotoxic proteins, reduced side effects can be expected. In addition, the inventors of the present invention have shown that the antibody induces apoptosis of IL-5-independent activated eosinophils, suggesting that the antibody is useful in the treatment for eosinophilic diseases.

Specifically, the present invention relates to the following (1) to (20):

(1) An apoptosis inducer, comprising an antibody which reacts specifically with an eosinophil and induces apoptosis of the eosinophil as an active ingredient.

(2) The apoptosis inducer of above mentioned (1), wherein the apoptosis-inducing antibody has antibody-dependent cellular cytotoxicity.

(3) The apoptosis inducer of above mentioned (1) or (2), wherein the antibody which reacts specifically with an eosinophil is an anti-human interleukin-5 receptor .alpha.-chain monoclonal antibody.

(4) The apoptosis inducer of above mentioned (3), wherein the anti-human interleukin-5 receptor .alpha.-chain monoclonal antibody is produced by an animal cell.

(5) The apoptosis inducer of above mentioned (3), wherein the anti-human interleukin-5 receptor .alpha.-chain monoclonal antibody is produced by a transformant KM8399 (FERM BP-5648).

(6) A therapeutic agent for eosinophilic diseases, comprising an antibody which reacts specifically with an eosinophil and induces apoptosis of the eosinophil as an active ingredient.

(7) The therapeutic agent for eosinophilic diseases of above mentioned (6), wherein the apoptosis-inducing antibody has cellular cytotoxicity.

(8) The therapeutic agent for eosinophilic diseases of above mentioned (6) or (7), wherein the antibody which reacts specifically with an eosinophil is an anti-human interleukin-5 receptor .alpha.-chain monoclonal antibody.

(9) The therapeutic agent for eosinophilic diseases of above mentioned (8), wherein the anti-human interleukin-5 receptor .alpha.-chain monoclonal antibody is produced by an animal cell.

(10) The therapeutic agent for eosinophilic diseases of above mentioned (8), wherein the anti-human interleukin-5 receptor .alpha.-chain monoclonal antibody is produced by the transformant KM8399 (FERM BP-5648).

(11) A method for specifically inducing apoptosis of an eosinophil using an antibody which reacts specifically with an eosinophil and induces apoptosis of the eosinophil.

(12) The method of above mentioned (11), wherein the apoptosis-inducing antibody has cellular cytotoxicity.

(13) The method of above mentioned (11) or (12), wherein the antibody which reacts specifically with an eosinophil is an anti-human interleukin-5 receptor .alpha.-chain monoclonal antibody.

(14) The method of above mentioned (13), wherein the anti-human interleukin-5 receptor .alpha.-chain monoclonal antibody is produced by an animal cell.

(15) The method of above mentioned (13), wherein the anti-human interleukin-5 receptor .alpha.-chain monoclonal antibody is produced by the transformant KM8399 (FERM BP-5648).

(16) A method for specifically reducing or removing eosinophils in peripheral blood or in a tissue infiltrated with eosinophils using an antibody which specifically reacts to an eosinophil and induces apoptosis of the eosinophil.

(17) The method of above mentioned (16), wherein the apoptosis-inducing antibody has antibody-dependent cellular cytotoxicity.

(18) The method of above mentioned (16) or (17) for specifically reducing or removing eosinophils, wherein the antibody which reacts specifically with an eosinophil is an anti-human interleukin-5 receptor .alpha.-chain monoclonal antibody.

(19) The method of above mentioned (18), wherein the anti-human interleukin-5 receptor .alpha.-chain monoclonal antibody is produced by an animal cell.

(20) The method of above mentioned (18), wherein the anti-human interleukin-5 receptor .alpha.-chain monoclonal antibody is produced by the transformant KM8399 (FERM BP-5648).

As the antibody used for the present invention, any antibody which reacts specifically to an eosinophil and induce apoptosis of the eosinophil can be used.

Examples of antibodies which react specifically to eosinophils include antibodies against receptors expressed on the surfaces of eosinophils. Examples of antibodies against receptors expressed on the surfaces of eosinophils include anti-human interleukin-5 receptor .beta.-chain antibodies, anti-human interleukin-3 receptor antibodies, anti-human monocyte/macrophage colony-stimulating factor receptor antibodies, and anti-human interleukin-5 receptor .alpha.-chain (hereinafter, referred to as hIL-5R .alpha.) antibodies. The anti-hIL-5R .alpha. antibody is preferred.

Examples of antibodies which induce apoptosis of eosinophils include antibodies having activity to inhibit signal transduction involved in the differentiation or proliferation of eosinophils, and antibodies having cellular cytotoxicity. Antibodies having cellular cytotoxicity are preferred, in order to induce apoptosis of any eosinophil as described below.

Hence, examples of antibodies capable of reacting specifically with eosinophils and inducing apoptosis of the eosinophils include antibodies against receptors expressed on the surfaces of eosinophils, which have antibody-dependent cellular cytotoxicity, and preferably, anti-hIL-5R .alpha. antibodies which have antibody-dependent cellular cytotoxicity. Other examples of such antibodies include antibodies against receptors expressed on the surfaces of eosinophils, which are produced by animal cell lines, such as CHO cells, YB2/3.0-Ag20 cells, SP2/0-AG14 cells and NS0 cells, and preferably, anti-hIL-5R .alpha. antibodies which are also produced by animal cell lines. Further examples of such antibodies include human IgG1 type antibodies against receptors expressed on the surfaces of eosinophils, and preferably, human IgG1 type anti-hIL-5R .alpha. antibodies. An example is an anti-hIL-5R .alpha. human CDR-grafted antibody KM8399 produced by a transformant KM8399 (FERM BP-5648).

The anti-IL-5 receptor .alpha.-chain antibody can be produced by a method described in WO97/10354.

Apoptosis of eosinophils induced by the above antibody can be confirmed by the following method.

1. Isolation of Eosinophil

(1) Isolation of Granulocyte from Peripheral Blood

Peripheral blood should be first treated with an anticoagulant to isolate the granulocytes from peripheral blood. Examples of anticoagulants include heparin sodium, disodium EDTA and dipotassium EDTA. Normally, 100 units of heparin sodium is used for 20 to 30 ml of peripheral blood.

Peripheral blood is collected with a syringe containing an anticoagulant, superposed on a suitable isolation medium, and centrifuged, thereby separating leukocytes into different cell populations, such as mononuclear cells, granulocytes and monocytes [Nature, 204, 793 (1964)].

Examples of media for separating peripheral blood-derived mononuclear cells (hereinafter referred to as PBMC) from granulocytes include Lymphoprep, Polymorphoprep (NYCOMED), Ficoll (Sigma) or the like. Further, isolation can also be performed using isotonic Percoll (Pharmacia) (0.15 M NaCl) adjusted to density of 1.085 to 1.088 by a densimeter. Centrifugation using the above isolation medium is always performed at room temperature.

(2) Isolation of Eosinophils From Granulocytes

Granulocytes separated in above mentioned (1) contain neutrophils and eosinophils, or may also contain erythrocytes. Erythrocytes can be removed through hemolyzation by either one of the following methods:

The pellet of granulocytes containing erythrocytes in a centrifuge tube is suspended in ice-cooled distilled water. After 30 seconds, an ice-cooled 1/10 volume of isosmotic 10-fold concentration buffer is added to stop hemolytic reaction. Centrifugation is carried out at 4.degree. C. for 5 minutes at 400.times.g to remove the supernatant. Erythrocytes can be removed by repeating the procedure a few times.

Alternatively, the pellet of granulocytes containing erythrocytes is suspended in an ice-cooled 0.2% NaCl solution. After 15 seconds, an equivalent volume of ice-cooled 1.6% NaCl solution is added to stop the hemolytic reaction, followed by centrifugation at 4.degree. C. for 5 minutes at 300.times.g, so that erythrocytes can also be removed [Clinical Immunology, 29., (Suppl. 17), 41 1997].

Subsequent to removal of erythrocytes, neutrophils should be removed.

Neutrophils expressing CD16 antigen on their surface can be removed by performing sorting.

First, granulocytes are incubated with mouse anti-CD 16 antibodies and then sheep anti-mouse immunoglobulin antibody immobilized on the Dynabead.TM. (DYNAL) is added. Using Magnetic bead concentrator MPC-1 (DYNAL), Dynabead-bound CD 16 positive cells are captured to collect the remaining suspended cells, thereby isolating the eosinophils (Allergy, 50, 34 (1995); Eur. J. Immunol., 24, 518 (1994); J. Immunol. Methods, 122, 97 (1989)).

Neutrophils can also be separated from granulocytes by MACS.TM. system (Miltenyi) using anti-CD 16 antibody immobilization microbeads (J. Immunol. Methods, 165, 253 (1993), J. Immunol. Methods 127, 153 (1990)).

(3) Induction of Activated Eosinophil

Activated eosinophils can be obtained by culturing for a few days of above-mentioned (2) with IL-3[J. Clin. Invest., 81 1986 (1988)], or by co-culturing with PBMC for 2 days. Furthermore, blood collected from a body is centrifuged with cell isolation media of different densities, so that activated eosinophils which have densities lower than the normal levels can be obtained [Clin. Exp. Immunol., 85, 312 (1991)].

The presence of activated eosinophils can be confirmed by the expression of CD69 molecules [J. Exp. Med., 172, 701 (1990)].

(4) Method for Culturing Eosinophils

Eosinophils can be cultured in RPMI1640 media supplemented with 1% or 10% fetal calf serum (hereinafter referred to as FCS), to which any one of cytokines including IL-5, IL-3 and GM-CSF is added at a final concentration of 1 ng/ml under the air containing 5% CO.sub.2 at 37.degree. C.

2. Method for Inducing Apoptosis of Human Eosinophils by Antibodies

Inhibition of signal-transduction involved in the differentiation and proliferation of eosinophils causes normodense eosinophils to die. However, the inhibition of signal-transduction involved in differentiation and proliferation of eosinophils is not enough to cause hypodense eosinophils (activated eosinophils) to die. Hypodense eosinophils cause effector function of antibody such as complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC) or the like to die.

There are two types of cell death, necrosis and apoptosis. However, the mechanism of action is yet to be elucidated.

Apoptosis can be induced by cytotoxic activity of antibody [Cancer Imunol. Immunother, 43, 220 (1996)]. However, the cellular cytotoxicity causes not only apoptosis, but also necrosis.

Cell death of eosinophils induced by treating antibody used for the present invention having cellular cytotoxicity can be analyzed by the following.

An example of a method for detecting necrotic cells involves staining intracellular DNA with PI (Propidium Iodide) reagent; and an example of a method for detecting apoptotic cells uses annexin V. Specifically, apoptotic cells can be evaluated by measuring cell surface phosphatidylserine (hereinafter referred to as PS) with annexin V [J. Immunol. Methods, 217, 61 (1998)] as an indication as described in the following method.

PS on the cell membrane is located on the side of cytoplasm in a living cell. When apoptosis is induced, PS is exposed on the cell surface within 1 hour. Accordingly, FITC-labeled annexin V which binds to PS in a calcium-dependent manner can detect the PS exposed apoptotic cells, so that early apoptosis can be detected before the cell membrane is damaged [J. Exp. Med. 182, 1545 (1995)].

Double staining with annexin V-FITC and PI is preferred, because binding of annexin V to cell membranes may also be observed in necrotic cells. Early apoptosis can be detected by the fact that it is stained with annexin V-FITC, but not with PI.

The antibody-dependent cellular cytotoxicity (hereinafter abbreviated as ADCC) can be measured according to the method of 3 described later. Thus induction of apoptosis in the target cells can be evaluated using the annexin V method.

3. Measurement of ADCC Activity

To measure ADCC activity, effector cells and target cells are used.

Examples of effector cells include natural killer (NK) cells, large granular lymphocytes (LGL), and PBMC comprising NK and LGL, or leukocytes having Fc receptors on the cell surfaces, such as neutrophils, eosinophils and macrophages.

Effector cells can be isolated according to the method of above mentioned 1.

As the target cells, any cells which express, on the cell surfaces, antigens that antibodies to be evaluated can recognize can be used. An example of such a target cell is an eosinophil which expresses IL-5 receptor on the cell surface.

Target cells are labeled with a reagent that enables detection of cytolysis.

Examples of reagents for labeling include a radio-active substance such as sodium chromate (Na.sub.2.sup.51CrO.sub.4, hereinafter referred to as .sup.51Cr) [Immunology, 14, 181 (1968)], calcein-AM [J. Immunol. Methods, 172, 227 (1994)], Europium [J. Immunol. Methods, 184, 29 (1995)] and .sup.51Cr is preferred.

When human peripheral blood eosinophils, which are terminally differentiated cells and have low labeling efficiency, are used as target cells, the death of target cells should be detected by another method after ADCC reaction. In this situation, cell death can be detected by the method described in above mentioned 2.

4. Method for Specifically Reducing or Removing Eosinophils in Peripheral Blood or in Tissues Infiltrated with Eosinophils

Eosinophils can be specifically reduced or removed from peripheral blood or tissues infiltrated with eosinophils using an apoptosis inducer which comprises, as an active ingredient, an antibody of the present invention that specifically reacts to the eosinophils and induces apoptosis of the eosinophils. Examples of such antibodies as an active ingredient include anti-hIL-5R .alpha.-chain antibodies, or preferably anti-hIL-5R .alpha. antibodies produced by animal cells. For example, direct action of anti-hIL-5R .alpha.-chain monoclonal antibodies KM8399 on peripheral blood or tissues enables induction of eosinophil apoptosis, and reduction or removal of eosinophils in peripheral blood or tissues infiltrated with eosinophils.

5. Form of Agent

The above-described apoptosis inducer or the therapeutic agent for eosinophilic diseases comprising, as an active ingredient, an antibody which specifically reacts to eosinophils and induces apoptosis of the eosinophils, may be solely administered as an agent. Normally, the inducer or the therapeutic agent is preferably provided as pharmaceutical preparations which are produced by mixing with one or more pharmacologically acceptable carriers according to any method known in the pharmaceutical technical field.

It is preferable to use an administration which is most effective in carrying out a treatment. Examples include oral administration and parenteral administration such as intraoral, bronchial, intrarectal, subcutaneous, intramuscular, intravenous administrations and the like. In an antibody-containing pharmaceutical formulation, intravenous administration is preferrable.

Examples of dosage form include nebulae, capsules, tablets, granules, syrups, emulsions, suppositories, injection, an ointments, tapes and the like.

Examples of formulation suitable for oral administration include emulsions, syrups, capsules, tablets, powders, granules and the like.

Liquid preparations, such as emulsions and syrups, can be produced by using as an additive, water; sugar, such as sucrose, sorbitol, fructose etc.; glycol, such as polyethylene glycol, propylene glycol etc.; oil, such as sesami oil, olive oil, soybean oil etc.; antiseptic such as p-hydroxy benzoate ester etc.; flavoring, such as strawberry flavors, peppermint flavors; and the like.

Capsules, tablets, powders, granules or the like can be produced by using as an additive, excipients such as lactose, glucose, sucrose, mannitol etc.; disintegrators, such as starch, sodium alginate etc.; lubricants, such as magnesium stearate, talc etc.; binders, such as polyvinyl alcohol, hydroxypropylcellulose, gelatin etc.; surfactants, such as fatty acid ester etc.; plasticizers, such as glycerine etc; and the like.

Examples of pharmaceutical preparations suitable for parenteral administration include injectables, suppositories, nebulae and the like.

An injection is prepared by using a carrier or the like which comprises a saline solution, a glucose solution, a mixture of both or the like.

A suppository is prepared by using a carrier, such as cacao butter, hydrogenated fat, carboxylic acid and the like.

A nebula is prepared by using the antibody preparation itself or using a carrier or the like which facilitates absorption by allowing the compound to disperse as fine particles without stimulating the mouth cavity and bronchial mucous membrane of a recipient.

Examples of carriers include lactose, glycerine and the like. Preparations, such as aerosol and dry powder, can be used, depending on the properties of the antibody and the carrier to be used. In addition, these parenteral preparations can be supplemented with components illustrated as additives for oral preparations.

The applied dose and the number of administration vary depending on target therapeutic effects, medication methods, treatment period, age and body weight of the patient. Normally, 10 .mu.g/kg to 8 mg/kg is administered per day to an adult patient.

The term "eosinophil associated diseases" of the present invention refers to diseases caused by eosinophils, including allergic diseases, such as asthma bronchiale and atopic dermatitis; and hypereosinophilic syndrome (HES), such as eosinophilia (e.g., eosinophilic pneumonia and sudden eosinophilia), eosinophilic enterogastritis, eosinophilic leukemia, eosinophilic granuloma and Kimura's disease.

The apoptosis inducer and the therapeutic agent for eosinophilic diseases of the present invention can be used as the therapeutic agent for the above eosinophil associated diseases.
 

Claim 1 of 12 Claims

1. A method for specifically inducing apoptosis of an eosinophil, comprising: administering an anti-human interleukin-5 receptor .alpha.-chain monoclonal antibody with an Fc region of the human IgG1 subclass that has antibody-dependent cellular cytotoxicity to a patient in need thereof in an amount effective to induce apoptosis of an eosinophil in said patient.

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