JP6944698B2 - Drugs for treating type I allergic diseases - Google Patents

Description

The present invention relates to a specific therapeutic agent for a type I allergic disease.

In type I allergic diseases, IgE binds to the high-affinity IgE receptor (FcεR1) on mast cells and basophils, and the allergen binds to and cross-links the IgE, resulting in histamine from these cells. Releases chemical mediators such as serotonin. These chemical messengers cause vasodilation and increased vascular permeability, resulting in various allergic symptoms. Known typical diseases include urticaria, food allergy, hay fever, allergic rhinitis, bronchial asthma, atopic dermatitis, and anaphylactic shock.

Omalizumab, an anti-IgE antibody, is commercially available as a therapeutic agent for intractable asthma, which is a type I allergic disease. Omalizumab is a humanized anti-IgE monoclonal antibody that reduces the amount of high-affinity receptor (FcεR1) and IgE on the surface of mast cells by removing free IgE from the blood, thereby reducing the amount of mast cell activity. It is said to suppress the conversion (Non-Patent Document 1).

Zolea (registered trademark) package insert Int. Arch Allergy Immunol 85: 47-54 (1988) Int. Arch Allergy Immunol 128: 24-32 (2002)

Omalizumab is an anti-IgE antibody that recognizes the IgE molecule Fcε3, which is the binding site for the IgE receptor (FcεR1) on the surface of mast cells. Therefore, omalizumab recognizes the binding site of IgE FcεR1 and cannot bind to IgE already attached to mast cells. Therefore, by removing free IgE in the blood, the amount of IgE that binds to mast cells is reduced, and by suppressing the activation of mast cells, the type I allergic reaction is indirectly attenuated. The mechanism of action. In addition, since omalizumab is an IgG and has a molecular weight of about 150,000, it is pointed out that it may bind to IgE molecules on mast cells and induce anaphylactic shock by cross-linking formation. The side effects of anaphylactic shock have already been reported in the literature.

Therefore, it is desired to develop a new therapeutic agent for type I allergic disease by a mechanism of action different from that of the conventional anti-IgE antibody (omalizumab).

Therefore, the present inventor has produced various anti-IgE antibodies and investigated their actions. As a result, the monoclonal anti-IgE antibody (HMK-12) produced by the hybridoma is an IgE receptor on mast cells (HMK-12), similarly to the above-mentioned omalizumab. We have found that it is an anti-IgE antibody that recognizes Fcε3 of IgE molecule, which is a binding site of FcεR1), and reported it earlier (Non-Patent Documents 2 and 3). Further investigation revealed that another monoclonal anti-IgE antibody different from this had an IgE-Fcε2 whose IgE recognition site was completely different from the IgE-Fcε3 (recognition site of omalizumab and HMK-12) to which FcεRI of IgE binds. We have succeeded in producing a Fab fragment of an anti-IgE antibody. By using this new anti-IgE antibody Fab fragment, it binds directly to IgE bound to mast cells and directly inhibits type I allergic reaction through cross-linking by allergens, thus strongly suppressing allergic symptoms. do. Further, since it is a Fab fragment, it does not form a crosslink by itself, has a small molecular weight of about 50,000, and has good safety, and completed the present invention.

That is, the present invention provides the following [1] to [12].

[1] A therapeutic agent for type I allergic disease containing a Fab fragment of a monoclonal anti-IgE antibody that recognizes Fcε2 of IgE and binds to the site as an active ingredient.
[2] The therapeutic agent for type I allergic disease according to [1], wherein the Fab fragment recognizes IgE Fcε2 on mast cells and directly binds to IgE Fcε2 on mast cells.
[3] The agent for treating type I allergic disease according to [1] or [2], wherein the anti-IgE antibody is a monoclonal anti-IgE antibody produced by hybridoma 6HD5.
[4] Use of a Fab fragment of a monoclonal anti-IgE antibody that recognizes Fcε2 of IgE and binds to the site for the production of a therapeutic agent for a type I allergic disease.
[5] The use according to [4], wherein the Fab fragment recognizes IgE Fcε2 on mast cells and directly binds to IgE Fcε2 on mast cells.
[6] The use according to [4] or [5], wherein the anti-IgE antibody is a monoclonal anti-IgE antibody produced by hybridoma 6HD5.
[7] A Fab fragment of a monoclonal anti-IgE antibody that recognizes Fcε2 of IgE and binds to the site for treating a type I allergic disease.
[8] The Fab fragment according to [7], wherein the Fab fragment recognizes IgE Fcε2 on mast cells and directly binds to IgE Fcε2 on mast cells.
[9] The Fab fragment according to [7] or [8], wherein the anti-IgE antibody is a monoclonal anti-IgE antibody produced by hybridoma 6HD5.
[10] A method for treating a type I allergic disease, which comprises administering an effective amount of a Fab fragment of a monoclonal anti-IgE antibody that recognizes Fcε2 of IgE and binds to the site.
[11] The method according to [10], wherein the Fab fragment recognizes IgE Fcε2 on mast cells and directly binds to IgE Fcε2 on mast cells.
[12] The method according to [10] or [11], wherein the anti-IgE antibody is a monoclonal anti-IgE antibody produced by hybridoma 6HD5.

The Fab fragment (Fab-6HD5) of the anti-IgE antibody of the present invention recognizes Fcε2 of IgE, directly binds to Fcε2 of IgE bound to mast cells, and causes a type I allergic reaction through cross-linking formation by an allergen. It is useful as a therapeutic drug for type I allergic disease because it directly inhibits type I allergic symptoms compared to the conventional therapeutic drug (omalizumab) by removing blood-free IgE. .. Further, since the present invention is a Fab fragment, the molecular weight is small and the risk of side effects is low.

It has an effect on β-hexosaminidase release. The flow cytometry result is shown. The flow cytometry result is shown. The results of Western blotting are shown. The results of Western blotting are shown.

The active ingredient of the therapeutic agent for type I allergic disease of the present invention is a Fab fragment of a monoclonal anti-IgE antibody that recognizes Fcε2 of IgE and binds to the site.
Omalizumab, an anti-IgE antibody conventionally used as a therapeutic agent for type I allergic diseases, is an antibody that recognizes Fcε3 (a binding site of IgE to the IgE receptor (FcεR1) on the surface of mast cells) of IgE molecules. By removing free IgE in the blood, the amount of IgE bound to mast cells is reduced to indirectly inhibit the type I allergic reaction, and it does not act directly on IgE bound to mast cells. .. On the other hand, since the Fab fragment of the monoclonal anti-IgE antibody used in the present invention recognizes Fcε2 instead of Fcε3, it directly binds to IgE bound to mast cells and is type I via allergen-induced cross-linking. Since it directly inhibits the allergic reaction, it can strongly and continuously suppress type I allergic symptoms.

Examples of such a monoclonal anti-IgE antibody include a monoclonal anti-IgE antibody produced by hybridoma 6HD5 (Non-Patent Documents 2 and 3).

Hybridoma 6HD5 is prepared by immunizing a non-human animal (SD rat) with IgE of an anti-dinitrophenol (DNP) IgE monoclonal antibody (B53: derived from BALB / c mouse). For example, it is a hybridoma obtained by cell fusion of an antibody-producing cell obtained by immunizing IgE of a mouse-derived monoclonal anti-DNPIgE (B53) with a rat and a myeloma cell (P3U1).

As the sensitizing antigen, anti-DNPIgE (B53) is used. As the non-human animal immunized with the sensitizing antigen, for example, a rat is used.

To immunize an animal with a sensitizing antigen, a known method is used. For example, as a general method, the sensitizing antigen is injected intraperitoneally or subcutaneously into a mammal. Specifically, the sensitizing antigen is diluted with PBS (Phosphate-Buffered Saline), physiological saline or the like to an appropriate amount, suspended, and if desired, a normal adjuvant, for example, Freund's complete adjuvant, is mixed in an appropriate amount, and after emulsification, Administer to mammals several times every 4 to 21 days. In addition, a suitable carrier can be used at the time of immunization with a sensitizing antigen.

After immunizing the mammal in this way and confirming that the desired antibody level is elevated in the serum, immune cells are collected from the mammal and subjected to cell fusion. Preferred immune cells include splenocytes in particular.

Mammalian myeloma cells are used as parent cells to be fused with the immune cells. The myeloma cells are derived from various known cell lines such as P3 (P3x63Ag8.653) (Kearney et al., J Immunol 1979; 123: 1548-1550), NS-1 (Kohler. G. and Milstein, C Eur). J Immunol 1976; 6: 511-519), SP2 / 0 (Shulman, M. et al., Nature 1978; 276: 269-270) and the like are preferably used.

The cell fusion of the immune cell and the myeloma cell is basically a known method, for example, the method of Kohler. G. and Milstein, C., Methods Enzymol 1981; 73: 3-46). It can be done according to the above.

More specifically, the cell fusion is carried out in a normal culture medium, for example, in the presence of a cell fusion promoter. As the fusion accelerator, for example, polyethylene glycol (PEG), Sendai virus (HVJ) and the like are used, and if desired, an auxiliary agent such as dimethyl sulfoxide can also be used in order to increase the fusion efficiency.

The usage ratio of immune cells and myeloma cells can be set arbitrarily. For example, it is preferable to increase the number of immune cells to 1 to 10 times that of myeloma cells. As the culture medium used for the cell fusion, for example, RPMI1640 culture medium suitable for the growth of the myeloma cell line, MEM culture medium, and other ordinary culture mediums used for this type of cell culture can be used, and further. , Fetal bovine serum (FBS) and other serum supplements can also be used in combination.

For cell fusion, a predetermined amount of the immune cells and myeloma cells are well mixed in the culture solution, and a polyethylene glycol solution (for example, an average molecular weight of about 1000 to 6000) preheated to about 37 ° C. is usually 30 to 60%. By adding at a concentration of (w / v) and mixing, the desired fusion cells (hybridoma) are formed. Subsequently, an appropriate culture solution is sequentially added, and the operation of centrifuging to remove the supernatant is repeated to remove a cell fusion agent or the like that is unfavorable for the growth of hybridomas.

The hybridoma thus obtained is selected by culturing in a usual selective culture medium, for example, a HAT culture medium (a culture medium containing hypoxanthine, aminopterin and thymidine). The culture in the above HAT culture medium is continued for a sufficient time (usually several days to several weeks) for cells other than the target hybridoma (non-fused cells) to die. The usual limiting dilution method is then performed to screen and single clone hybridomas that produce the antibody of interest.

For hybridoma screening, monoclonal anti-IgE antibodies can be selected, for example, by ELISA using IgE used as a sensitizing antigen.

The hybridoma producing the monoclonal antibody thus produced can be subcultured in a normal culture medium and can be stored for a long period of time in liquid nitrogen.

To obtain a monoclonal antibody from the hybridoma, the hybridoma is cultured according to a usual method and obtained as a culture supernatant thereof, or the hybridoma is administered to a mammal compatible with the hybridoma to proliferate and used as ascites. The method of obtaining is adopted. The former method is suitable for obtaining high-purity antibody, while the latter method is suitable for mass production of antibody.

The anti-IgE antibody of the present invention is an experimental system of rat anti-mouse IgE, but it can also be chimeric or humanized. Further, IgG is preferable as the anti-IgE antibody.

The hybridoma 6HD5 that produces the anti-IgE antibody of the present invention can be produced, for example, according to the description of Non-Patent Documents 2 and 3.

The Fab fragment of the anti-IgE antibody can be obtained, for example, by treating the anti-IgE antibody with a protease according to the description of Non-Patent Document 3 and then removing the Fc fragment using a protein A column. Papain can be used as the protease.

The Fab fragment of the anti-IgE antibody of the present invention recognizes Fcε2, not Fcε3, and directly binds to IgE bound to mast cells, as shown in Examples below, and I through cross-linking formation by an allergen. Directly inhibits type allergic reactions. Moreover, the anti-allergic action of the Fab fragment of the anti-IgE antibody of the present invention is strong and persistent. Furthermore, since it is a Fab fragment, its molecular weight is small and the risk of side effects is low. Therefore, the Fab fragment of the anti-IgE antibody of the present invention is useful as a therapeutic agent for various type I allergic diseases.

Examples of the type I allergic disease targeted by the therapeutic agent for the type I allergic disease of the present invention include urticaria, food allergy, pollinosis, allergic rhinitis, bronchial asthma, atopic dermatitis, anaphylactic shock and the like.

Examples of the administration form of the therapeutic agent for type I allergic disease of the present invention include eye drops, injections, oral preparations (tablets, granules, powders, capsules), ointments, creams and the like. Of these, injections are particularly preferred. In the form of these pharmaceutical compositions, they can be formulated with a pharmaceutically acceptable carrier. Such carriers include, for example, lactose, glucose, D-mannitol, starch, crystalline cellulose, calcium carbonate, kaolin, starch, gelatin, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, polyvinylpyrrolidone, ethanol, carboxymethyl cellulose, carboxymethyl cellulose calcium. Salt, magnesium stearate, talc, acetyl cellulose, sucrose, titanium oxide, benzoic acid, paraoxybenzoic acid ester, sodium dehydroacetate, gum arabic, tragant, methyl cellulose, egg yolk, surfactant, sucrose, monosyrup, citric acid, distillation There are water, ethanol, glycerin, propylene glycol, macrogol, sodium phosphate-sodium hydrogen phosphate, sodium dihydrogen phosphate, sodium phosphate, glucose, sodium chloride, phenol, thimerosal, paraoxybenzoic acid ester, sodium hydrogen sulfite, etc. It is used by mixing with the compound (1) of the present invention according to the form of.

Further, the content of the active ingredient of the present invention in the pharmaceutical composition of the present invention varies greatly depending on the form of the preparation and is not particularly limited, but is usually 0.01 to 0.01 to the total amount of the composition. It is 20% by mass, preferably 0.1 to 10% by mass.

The dose of the therapeutic agent for type I allergic disease of the present invention varies depending on the symptom, age, and administration method of the patient to be administered, but is preferably 0.01 to 100 mg / kg / week to 3 weeks.

Next, the present invention will be described in more detail with reference to examples.

Example 1 (Manufacturing of 6HD5)
After immunizing SD rats with anti-DNPIgE (B53: derived from Balb / c mice) together with FCA, 6HD5 was prepared by cloning a monoclonal anti-IgE antibody of Rat anti-mouse IgE by a hybridoma method (Materials and Methods; Int). Arch Allergy Immunol 85: 47-54 (1988)).

Example 2 (Production of Fab Fragment)
A rat IgG anti-mouse IgE antibody was papainized to prepare a Fab fragment having a molecular weight of about 50,000 (marerials and Methods; Int. Arch Allergy Immunol 128: 24-32 (2002). The obtained Fab fragment of the anti-IgE antibody was prepared. It is called Fab-6HD5.

Example 3 (PCA reaction)
The in vivo passive anaphylactic reaction (PCA) of Fab-6HD5 was examined. That is, female SD rats were used. 100 ng / 0.1 mL of anti-DNPIgE (SPE-7) or anti-TNPIgE (142a) was intradermally administered to several parts of the shaved skin of 2 animals in each group. Twenty-four hours later, Fab-6HD5, 6HD5, Fab-HMK-12, HMK-12, rat IgG and anti-κ antibody were injected into the same site. Two hours after the second injection, 1 mL of 0.5% Evans blue dye saline and 1 mg / mL of DNP-BSA or TNP-BSA were administered intravenously. After 30 minutes, the diameter of the blue part was measured.
As a result, as shown in Table 1, Fab-6HD5 suppressed the PCA reaction at the lowest concentration of 1.25 μg / mL, and the other antibody group 6HD5 was 2.5 μg / mL, Fab-HMK-12. , HMK-12 suppressed the PCA reaction at a concentration of 10 μg / mL. The control Rat IgG did not suppress the PCA response. From this result, it was shown that Fab-6HD5 having a molecular weight of 40,000 has a stronger inhibitory power than 6HD5 having a molecular weight of 180,000 divalent, and has a stronger inhibitory power than other anti-IgE antibody HMK-12. This may be due to the different binding sites of 6HD5 and HMK-12 for IgE molecules.
Experiments using allotype and Anti-TNP IgE (100 ng, 141a) having different antigen specificities were conducted with the aim of obtaining similar inhibition of PCA with IgE having other specificities. Similarly, Fab-6HD5 strongly suppressed the PCA reaction, but the positive controls Fab-anti-κ and Rat IgG did not block the PCA reaction. The fact that Fab-anti-κ does not block the PCA response indicates that this suppression does not involve a light chain.

Example 4 (sustained effect of Fab-6HD5 on PCA reaction)
The duration of the PCA inhibitory effect of Fab-6HD5 was examined by time course study.
After skin sensitization of anti-DNP IgE on Day 0, Fab-6HD5 on Day 1 is intradermally administered to the site where IgE was sensitized in a concentration-dependent manner. Two hours later on the same day, the group induced by antigen + Evans blue (day 1 group: Day 1) and the group induced by Day 10 (day 10 group: Day 10) were examined.
As a result, as shown in Table 2, in the Day 1 group, the PCA reaction was suppressed at a concentration of 0.63 μg / mL of Fab-6HD5, and in the Day 10 group, the PCA reaction was suppressed at a concentration of 0.32 μg / mL. This result indicates that Fab-6HD5 can sustain the inhibition of type I allergic reaction in the PCA reaction for at least 10 days. The results of the Day 10 group show that pre-administration of Fab-6HD5 can prevent type I allergic reactions. That is, preventive treatment can be expected in which an allergic reaction can be avoided by administering Fab-6HD5 before being exposed to a known allergen such as pollinosis. In addition, since the PCA reaction is suppressed 2 hours after the antibody administration in the Day 1 group, it can be expected to be effective against anaphylactic shock, which is an acute type I allergic disease.

Example 5 (Action on β-hexosaminidase release)
Fab-6HD5 suppressed the release of β-hexosaminidase from RBL-2H3 cells.
After culturing RBL-2H3 cells overnight, IgE (SPE-7) concentration 1 μg / mL is cultured for 16 hours and fixed to the cells. Then, Fab-6HD5 was added as an inhibitor in a concentration-dependent manner (0,1.25, 2.5, 5.0, 10 μg / mL) to suppress the release of β-hexosaminidase after the addition of the antigen (DNP-BSA). Was measured.
As shown in FIG. 1, it was shown that Fab-6HD5 suppressed the release of β-hexosaminidase in a concentration-dependent manner. Baniyash et al. Are antibodies in which Fab'fragment anti-IgE treated with various pepsin recognizes the binding site of FcεR1 on RBL / 2H3 cells of IgE, and suppresses the release of serotonin from cells in the coexistence of this antibody and IgE. It is reported that (Eur. J. Immunol 1984 14: 799-807). However, it is the first report that the release of chemical mediators was suppressed after IgE was adhered to RBL / 2H3 cells, indicating that Fab-6HD5 is not a competitive inhibition of IgE-FcεR1.

Example 6 (flow cytometry)
The following experiment was performed using flow cytometry to investigate the binding site of Fab-6HD5 to IgE.
RBL / 2H3 is used with FITC-SPE-7 (anti-DNP IgE ^b : IgE.λ, κ) and FITC-142a (anti-TNP IgE ^a : IgE, κ) and various antibody groups (anti-λ, anti-κ). , Fab-6HD5, Fab-HMK-12, Rat IgG) + PE-rabbit IgG anti-rat IgG (secondary antibody) for double staining, antibody group against IgE molecule on RBL / 2H3 The degree of binding was observed by co-expression.
As shown in FIGS. 2 and 3, the positive controls SPE-7 ⁺ and λ ⁺ cells were 84.67% positive, and the SPE-7 ⁺ and Fab-6HD5 ⁺ cells were 91.1% positive. On the contrary, SPE-7 ⁺ and Fab-HMK-12 ⁺ were 64.7%. The negative control SPE-7 ⁺ rat IgG ⁺ was 3.95%. Similar results were also obtained in experiments using 142a (anti-TNP IgE ^{a (IgE, κ)), that is, 93.4% of positive controls 142a +} , κ ⁺ positive cells, 142a +, Fab-. 6DH5 + cells were 82.2%, while 142a ⁺ and Fab-HMK-12 ⁺ cells were 43.0%. Negative controls 142a + and Rat IgG + were 0.64%.
In addition, FITC-IgE (SPE-7,141a) and various antibody groups (anti-λ, anti-κ, Fab-6HD5, Fab-HMK-12, Rat IgG) were incubated for 1 hour in advance, and then cultured with RBL / 2H3. Then, we tried to inhibit the competition between IgE and various antibodies. As shown in As shower Fig 1b, the positive controls SPE-7 + and λ + cells were 92.3% positive (1). SPE-7 + and Fab-6HD5 + cells were 90.2% positive. On the contrary, SPE-7 + and Fab-HMK-12 + strongly inhibited it to be 4.05%. The negative control SPE-7 ⁺ rat IgG ⁺ was 3.95%.
Similar results were obtained in experiments using 142a (anti-TNP IgEa (IgE, κ), that is, 88.8% of the positive controls 142a +, κ + positive cells, 142a +, 97. Fab-6DH5 + cells. Although it was 7%, 142a + and Fab-HMK-12 + cells were strongly inhibited and 1.09%. Negative controls 142a + and Rat IgG + were 0.58%.
The above results indicate that Fab-6HD5 binds to IgE that is attached to the mast cells, while Fab-HMK-12 recognizes the vicinity of the binding site of FcεR1 and IgE on the mast cells. Therefore, in FIG. 2, IgE + and Fab-HMK-12 + are stained with dim at 64.7% and 43.0%, and in FIG. 3, IgE + HMK-12 + cells because HMK-12 inhibits the binding of IgE to FcεRI. Was found to be almost undetectable. It is considered that the binding of FITC-IgE to mast cells was blocked by Fab-HMK-12, and HMK-12 + IgE + cells were not detected.

Example 7 (Western blot)
In order to search the binding site of Fab-6HD5 for IgE in more detail, an IgE-producing hybridoma was cloned to prepare a fragment IgE (CH1-4) of the Fc portion of IgE. Western blotting using Fab-6HD5 showed that 6HD5 did not react with CH1, CH3 and CH4, but only strongly with CH2 (FIGS. 4 and 5). As a result, it was found that Fab-6HD5 binds to IgECε2 and is different from the binding site of IgE to FcεR1 (IgEε3), which is the recognition site of omalizumab.

(Fab-6HD5), which is a monomer and has a molecular weight of about 50,000, strongly suppresses type I allergic reaction in vivo and in vitro by directly binding to IgECε2 of IgE (FcεRI-Bund IgE) on mast cells. It has been found. Until now, all the allergy-suppressing mechanisms of anti-IgE antibodies have been due to inhibition of binding between IgE and FcRI.
Fab-6HD5 of the present invention is an antibody that strongly blocks type I allergic reaction by a completely new mechanism, that is, by directly binding to IgECε2 on mast cells.

Claims (3)

A therapeutic agent for type I allergic diseases containing a Fab fragment of a monoclonal anti-IgE antibody that recognizes IgE Fcε2 and binds to the site as an active ingredient. The Fab fragment, I allergic diseases by claim 1, wherein recognizing the Fcε2 of IgE on mast cells directly bonded. The agent for treating type I allergic disease according to claim 1 or 2, wherein the anti-IgE antibody is a monoclonal anti-IgE antibody produced by hybridoma 6HD5.

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