WO2023171714A1 - Pharmaceutical composition and dendritic cell - Google Patents

Abstract

[Problem] To provide a novel therapeutic agent, for bronchial asthma, that focuses on CD109. [Solution] This pharmaceutical composition contains, as an active component, a drug that improves bronchial hypersensitivity and/or eosinophilic airway inflammation caused by CD109.

Description

Pharmaceutical composition and dendritic cells Cross-reference of related applications

This international application claims priority based on Japanese Patent Application No. 2022-036500 filed with the Japan Patent Office on March 9, 2022, and is based on Japanese Patent Application No. 2022-036500. The entire contents are incorporated by reference into this international application.

The present disclosure relates to pharmaceutical compositions and dendritic cells.

The prevalence of bronchial asthma is approximately 10% and is still on the rise. Bronchial asthma is defined by airway hyperresponsiveness and eosinophilic airway inflammation (Non-Patent Documents 1-4). In the pathology of bronchial asthma, innate immunity and acquired immunity interact, and Th2 type immune responses are activated. In a pathway mediated by innate immunity, type 2 innate lymphocytes (ILC2s) strongly induce eosinophil inflammation. On the other hand, in the pathway mediated by acquired immunity, dendritic cells (DCs) play a central role in inducing Th2 responses (Non-Patent Documents 5, 6). To date, biological preparations (antibody preparations) targeting IL-5, IL-4R (IL-4 and IL-13), IgE, etc. have been developed mainly for severe asthma patients. These biologics target functional molecules located downstream of innate and adaptive immunity. However, on the other hand, there are various phenotypes of bronchial asthma, and each phenotype has different important functional molecules. Therefore, it is necessary to select patients according to the characteristics of each biological drug, but this is not easy and the expected effects are often not achieved. In other words, even with existing drugs, the problem of treating severe asthma that is difficult to control remains unsolved. Therefore, we aim to directly control ILC2s and DCs, which are located upstream of the immune cascade in the pathology of bronchial asthma, and discover new therapeutic targets common to asthma patients of various phenotypes, as well as new treatments targeting them. It was hoped that the law would be developed.

Here, CD109 is a GPI-anchored glycoprotein present on the cell surface, and belongs to the α2 macroglobulin superfamily based on its amino acid sequence (Non-Patent Document 7). CD109 suppresses and controls signals mediated by TGF-β by binding to the TGF-β receptor (Non-Patent Document 8). CD109 is a molecule that has not received much attention so far, and its action and mechanism of action have not been fully elucidated. For example, the relationship between the CD109 molecule and allergic inflammation was unknown. In view of this background, there are expectations for the discovery of unknown functions and the possibility of CD109 as a therapeutic target.

The inventors conducted intensive studies on the significance of the CD109 molecule in allergic inflammation, particularly bronchial asthma, using CD109 gene-modified mice.
One aspect of the present disclosure is to provide a novel therapeutic agent for bronchial asthma that focuses on CD109.

One aspect of the present disclosure is a pharmaceutical composition containing as an active ingredient a drug that improves airway hyperresponsiveness and/or eosinophilic airway inflammation caused by CD109.
In one aspect of the disclosure, CD109 is induced on dendritic cells cCD2, B cells, or type 2 innate lymphocytes.

In one aspect of the present disclosure, improvement of airway hyperresponsiveness and/or eosinophilic airway inflammation is due to suppression of production of IL-4, IL-5, IL-13, and/or IL-33.
In one aspect of the present disclosure, the improvement of airway hyperresponsiveness and/or eosinophilic airway inflammation is due to activation of RUNX3 and/or phosphorylation of Smad2/3.

In one aspect of the disclosure, the agent is an anti-CD109 antibody.
In one aspect of the disclosure, the anti-CD109 antibody is an anti-mouse monoclonal antibody.
One aspect of the present disclosure is dendritic cell cDC2s derived from a subject deficient in CD109, which is used to improve airway hyperresponsiveness and/or eosinophilic airway inflammation.

In one aspect of the disclosure, dendritic cells are used for transfer.
In one aspect of the disclosure, the dendritic cells are anti-inflammatory dendritic cells.
In one aspect of the present disclosure, the improvement of airway hyperresponsiveness and/or eosinophilic airway inflammation is caused by suppressing the production of IL-6, IL-13, IL-17A, TNF, and/or IFN-γ. do.

In one aspect of the present disclosure, the improvement of airway hyperresponsiveness and/or eosinophilic airway inflammation is due to activation of RUNX3 and/or phosphorylation of Smad2/3.
In one aspect of the present disclosure, the dendritic cells have CCL24, CCL26, IL-4, IL-5, IL-13, and/or IL- 33 and increased expression of IL-18, IL-12, and/or CXCL9.

The present disclosure reveals the importance of CD109 for asthma and provides new asthma treatment strategies that include drugs targeting CD109. In particular, the present disclosure contributes to the realization of treatments for airway hyperresponsiveness and eosinophilic airway inflammation. Furthermore, the present disclosure contributes to the realization of treatments for airway hyperresponsiveness and eosinophilic airway inflammation that are superior to existing drugs.

(A) Shows the experimental timeline of Example 1. Wild type (WT) mice and CD109 ^−/− mice were intranasally administered with tick antigen (HDM). (B) WT mice sensitized with HDM (WT HDM), CD109 ^−/− mice sensitized with HDM (CD109 ^−/− HDM), and WT mice administered with PBS as a control group (WT PBS), Pulmonary airway resistance (R _L ) and pulmonary dynamic compliance (C _dyn ) of CD109 ^−/ − mice administered PBS (CD109 ^−/− PBS) are shown (n=5-7 each). The horizontal axis of each graph indicates the concentration (mg/ml) of methacholine administered. (C) Differences in cell numbers in BAL of WT HDM, CD109 ^−/− HDM, WT PBS, and CD109 ^−/− PBS (n=5-7 each) are shown. The vertical axis of the graph on the left shows the total cell number in BAL, and the vertical axis of the graph on the right shows the number of eosinophils (Eos) in BAL. (D) Section photographs and quantitative results of lung histological examination of hematoxylin and eosin staining and PAS staining in WT HDM and CD109 ^−/− HDM are shown (n=6 each). The magnification was 100 times, and the scale bar was 50 μm. (E) Cytokine (IL-4, IL-5, IL-13, IL-25, IL-33, TSLP) levels in lung homogenates in WT HDM, CD109 − ^/− HDM, WT PBS, and CD109 ^−/− PBS (each n=6). The vertical axis of each graph shows the relative proportion of each cytokine standardized with GAPDH. In each figure, each data is shown as a representative example of at least 5 independent experiments. The error bars of the bar graphs in each figure represent the mean value ± SEM. Moreover, the P value was calculated by Student's t test and one-way analysis of variance after Turkey's multiple comparison test. * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001. (A) WT mice were sensitized with ovalbumin (OVA) + alum adjuvant salt (Alum) on days 1 and 8, and challenged with OVA for 3 consecutive days on days 18 to 20. Subsequently, CD109 expression in lung immune cells was analyzed on day 21. As a control, WT mice administered with PBS were used. Representative flow cytometry (FACS) histograms and mean fluorescence intensity (MFI) of CD109 are shown (n=3-4 each). (B) WT mice were sensitized as shown in FIG. 1(A), and surface cell markers in CD109+ and CD109− lung DCs were analyzed. The gating strategy for CD109+ and CD109- lung DCs is shown. (C) Representative FACS histograms and mean fluorescence intensity in CD109+ and CD109− lung DCs are shown (n=6 each). In each figure, * indicates P<0.05, and *** indicates P<0.001. (A) Shows the experimental timeline of Example 3. WT mice and CD109 ^−/− mice were sensitized with OVA+Alum on days 1 and 8, and intranasally challenged with OVA for 3 consecutive days on days 18 to 20. Subsequently, lung DCs subsets were isolated. Sorted lung DCs subsets were co-cultured with naïve CD4+ T cells isolated from OT-II mice in the presence of OVA _323-339 peptide. (B) Identification results of lung DCs subsets (cDC1s and cDC2s) are shown, and representative FACS plots for each subset are shown. (C) Cytokines (IL-2, IL-6, IL-10, IL-13, IL-17A, TNF, IFN-γ) in the supernatant of co-cultured cDC2s from WT mice and CD109 ^−/− mice. (each n=9). In each figure, each data is shown as a representative example of two independent experiments. The error bars of the bar graphs in each figure represent the mean value ± SEM. Moreover, the P value was calculated by Student's t test. * indicates P<0.05, *** indicates P<0.001. (A) Shows the experimental timeline of Example 4. Cultured BMDCs derived from WT mice and CD109 ^−/− mice were stimulated with tick antigen (HDM) for 6 hours and administered intravenously to WT mice. Then, BMDCs cultured and stimulated with HDM in the same manner were intranasally administered on the 8th and 13th day. (B) Mice in which BMDCs were collected and cultured from WT mice and BMDCs sensitized with HDM were administered (WT BMDCs HDM), BMDCs were collected and cultured from CD109 ^−/− mice, and BMDCs sensitized with HDM were administered. BMDCs were collected and cultured from mice administered with PBS (WT BMDCs PBS), and WT mice as a control group (CD109 ^−/− BMDCs HDM), and BMDCs were collected and cultured from CD109 ^−/− mice and treated with PBS. The lung airway resistance (R _L ) and lung dynamic compliance (C _dyn ) of mice administered with (CD109 ^−/− BMDCs PBS) are shown (n=6-7 each). The horizontal axis of each graph indicates the concentration (mg/ml) of methacholine administered. (C) Shows the difference in cell numbers in BAL of WT BMDCs HDM, CD109 ^−/− BMDCs HDM, WT BMDCs PBS, and CD109 ^−/− BMDCs PBS (n=7 each). The vertical axis of the graph on the left shows the total cell number in BAL, and the vertical axis of the graph on the right shows the number of eosinophils (Eos) in BAL. (D) Section photographs and quantitative results of lung histological examination of hematoxylin and eosin staining and PAS staining in WT BMDCs HDM and CD109 ^−/− BMDCs HDM are shown (n=7 for each). The magnification was 100 times, and the scale bar was 50 μm. In each figure, each data is shown as a representative example of two independent experiments. The error bars of the bar graphs in each figure represent the mean value ± SEM. Moreover, the P value was calculated by Student's t test and one-way analysis of variance after Turkey's multiple comparison test. * indicates P<0.05, ** indicates P<0.01. WT and CD109 ^−/− mice were immunized as shown in FIG. 1(A), and lung cDC2s were isolated as shown in FIG. 3(B). (A) Heat plot showing gene expression changes in lung cDC2s from WT and CD109 ^−/− mice (n=3 each). (B) Representative FACS histograms and mean fluorescence intensity (MFI) of RUNX3 in lung cDC2s from WT and CD109 ^−/− mice (n=3 each) are shown. (C) Representative FACS histograms and mean fluorescence intensity (MFI) of phosphorylated Smad2/3 in lung cDC2s from WT and CD109 ^−/− mice (n=5 each) are shown. In addition, the error bar of the bar graph in each figure is expressed as the average value ± SEM. Moreover, the P value was calculated by Student's t test. * indicates P<0.05, ** indicates P<0.01. (A) Shows the experimental timeline of Example 6. WT mice were immunized as shown in Figure 1A, and anti-CD109 monoclonal antibody and IgG isotype were intranasally administered the day before HDM challenge. (B) Pulmonary airway resistance of WT mice administered with anti-CD109 monoclonal antibody (HDM Anti-CD109 Ab), WT mice administered with IgG isotype (HDM Isotype IgG), and WT mice administered with PBS as a control group (PBS). (R _L ) and pulmonary dynamic compliance (C _dyn ) (n=6-7 each). The horizontal axis of each graph indicates the concentration (mg/ml) of methacholine administered. (C) Shows the difference in cell numbers in BAL (each n=6-7) of HDM Anti-CD109 Ab, HDM Isotype IgG, and PBS. The vertical axis of the graph on the left shows the total cell number in BAL, and the vertical axis of the graph on the right shows the number of eosinophils (Eos) in BAL. (D) Section photographs and quantitative results of lung histological examination of hematoxylin and eosin staining and PAS staining for HDM Anti-CD109 Ab and HDM Isotype IgG are shown (n=6 each). The magnification was 100 times, and the scale bar was 50 μm. In each figure, each data is shown as a representative example of two independent experiments. The error bars of the bar graphs in each figure represent the mean value ± SEM. Moreover, the P value was calculated by Student's t test and one-way analysis of variance after Turkey's multiple comparison test. * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001. (A) Shows the experimental timeline of Example 7. WT mice and CD109 ^−/− mice were sensitized with OVA+Alum on days 1 and 8, and intranasally challenged with OVA for 3 consecutive days on days 18 to 20. (B) WT mice sensitized with OVA (WT OVA), CD109 ^−/− mice sensitized with OVA (CD109 ^−/− OVA), and WT mice administered with PBS as a control group (WT PBS), Pulmonary airway resistance (R _L ) and pulmonary dynamic compliance (C _dyn ) of CD109 ^−/ − mice administered PBS (CD109 ^−/− PBS) are shown (n=6 each). The horizontal axis of each graph indicates the concentration (mg/ml) of methacholine administered. (C) Shows the difference in cell numbers in BAL of WT OVA, CD109 ^−/− OVA, WT PBS, and CD109 ^−/− PBS (n=6 each). The vertical axis of the graph on the left shows the total cell number in BAL, and the vertical axis of the graph on the right shows the number of eosinophils (Eos) in BAL. (D) Section photographs and quantitative results of lung histological examination of hematoxylin and eosin staining and PAS staining in WT OVA, CD109 ^−/− OVA are shown (n=6 each). The magnification was 100 times, and the scale bar was 50 μm. In each figure, each data is shown as a representative example of at least two independent experiments. The error bars of the bar graphs in each figure represent the mean value ± SEM. Moreover, the P value was calculated by Student's t test and one-way analysis of variance after Turkey's multiple comparison test. * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001. WT and CD109 ^−/− mice were sensitized as shown in Figure 7. Representative FACS of phosphorylated Smad2/3 in lung cDC2s isolated from WT and CD109 ^−/− mice (n=6 each). Histograms and mean fluorescence intensity (MFI) are shown. (A) WT mice administered with IgG isotype (HDM Isotype IgG), WT mice administered with anti-IL-33 monoclonal antibody (HDM Anti-IL-33 Ab), anti-thymic stromal lymphopoietic factor (TSLP) antibody (HDM Anti-TSLP Ab), WT mice administered dexamethasone (HDM Dex), WT mice administered anti-CD109 monoclonal antibody (HDM Anti-CD109 Ab), and WT mice administered PBS as a control group. Differences in cell numbers in BAL (each n=4-5) of mice (PBS) are shown. The vertical axis of the graph on the left shows the total number of cells in the BAL, and the vertical axis of the graph on the right shows the number of eosinophils in the BAL. (B) Pulmonary airway resistance (R _L ) and pulmonary dynamic compliance ( C _dyn ) (each n=4-5). The horizontal axis of each graph indicates the concentration (mg/ml) of methacholine administered. The error bars of the bar graphs in each figure represent the mean value ± SEM. Moreover, the P value was calculated by Student's t test and one-way analysis of variance after Turkey's multiple comparison test. * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001.

One aspect of the present disclosure relates to a pharmaceutical composition containing as an active ingredient a drug that improves airway hyperresponsiveness and/or eosinophilic airway inflammation caused by CD109.
Airway hyperresponsiveness and eosinophilic airway inflammation are characterized as the underlying pathology of bronchial asthma. Airway hyperresponsiveness is a condition in which the airways and trachea are more sensitive than in a normal person, making bronchoconstriction more likely. Airway hyperresponsiveness is characterized by increased pulmonary airway resistance and decreased pulmonary dynamic compliance. In other words, the state of airway hyperresponsiveness can be evaluated by measuring these parameters. With regard to pulmonary airway resistance, when air moves in and out of the lungs, the viscosity that acts between the air and the lung wall acts to block the flow of air, resulting in a constant flow with high resistance. requires a lot of pressure. In other words, high pulmonary airway resistance means that it is difficult for air to flow. In addition, lung dynamic compliance indicates changes in lung volume due to constant pressure changes. A large lung dynamic compliance indicates a large change in lung volume in response to a unit pressure change, indicating that the lungs are easy to inflate.

Additionally, eosinophilic airway inflammation is a condition in which the number of eosinophils in the body increases, causing inflammation in the airways and trachea. Eosinophilic airway inflammation can be evaluated by measuring changes in the number of eosinophils in vivo. For example, this evaluation can be performed by performing bronchoalveolar lavage (BAL), which involves injecting and collecting physiological saline into a part of the lung, and measuring the number of eosinophils in the collected BAL. can.

In addition, histological changes associated with airway hyperresponsiveness and eosinophilic airway inflammation include increased inflammation around the airways, PAS-positive cells, bronchial wall thickness, and/or peribronchial cell infiltration.
In the present disclosure, airway hyperresponsiveness and eosinophilic airway inflammation are regulated by CD109.

CD109 is a glycosylphosphatidylinositol (GPI)-anchored glycoprotein and a cell surface antigen belonging to the α2-macroglobulin/C3, C4, C5 family of thioester-containing proteins (Lin, M. et al. Blood 99, 1683-1691, (2002). and Mii, S. et al. Pathol Int 69, 249-259, (2019).). The main function of CD109 is to bind to the transforming growth factor-β (TGF-β) receptor, TGF-β, activin receptor-like kinase 1 (ALK1), and 78-kDa glucose-related protein (GRP78). It is known to suppress signals mediated by TGF-β (Bizet, A.A. et al. Biochim Biophys Acta 1813, 742-753, (2011)., Li, C. et al. Biochem J 473, 537-547, (2016)., Vorstenbosch, J. et al. J
Invest Dermatol 137, 641-649, d (2017). , and Tsai, Y. L. et al. Proceedings of the National Academy of Sciences of the United States of America 115, E4245-e4254, (2018). ).

As described in the Examples of this application, the present inventors found that the presence of CD109 controls airway hyperresponsiveness and/or eosinophilic airway inflammation. It is believed that airway inflammation can be improved by treatments targeting CD109. In the present disclosure, "a drug that improves airway hyperresponsiveness and/or eosinophilic airway inflammation caused by CD109" refers to a drug that can weaken or eliminate airway hyperresponsiveness and/or eosinophilic airway inflammation caused by CD109. means. Typically, it is a CD109 inhibitor. Examples of CD109 inhibitors include siRNA molecules such as CD109 dsRNA or shRNA, CD109 antisense, anti-CD109 antibodies, or aptamers for CD109. Preferably it is an anti-CD109 antibody.

Here, in the present disclosure, the term "antibody" refers to the ability to specifically bind to a target such as a carbohydrate, polynucleotide, lipid, or antibody through at least one antigen recognition site located in the variable region of an immunoglobulin molecule. It is an immunoglobulin molecule. In this disclosure, antibodies include whole polyclonal or monoclonal antibodies as well as fragments thereof. It is known that the variable regions (particularly CDRs) of antibodies confer binding properties, and it is widely known to those skilled in the art that even antibody fragments that are not complete antibodies can utilize their binding properties. It is being In the present disclosure, a "fragment" or "antigen-binding fragment" of an antibody is a protein or peptide that includes a part (partial fragment) of an antibody, and is a protein or peptide that has an effect (immunoreactivity/binding ability) on the antigen of the antibody. refers to a protein or peptide that retains Such immunoreactive fragments include, for example, F(ab') ₂ , Fab', Fab, Fab ₃ , single chain Fv (hereinafter referred to as "scFv"), (tandem) bispecific single chain Fv ( sc(Fv) ₂ ), single chain triple body, nanobody, divalent VHH, pentavalent VHH, minibody, (double chain) diabody, tandem diabody, bispecific tribody, bispecific bibody, dual affinity body Targeting molecule (DART), triabody (or tribody), tetrabody (or [sc(Fv) ₂ ] ₂ , or (scFv-SA) ₄ ), disulfide-bonded Fv (hereinafter referred to as "dsFv"), compact IgG, Heavy chain antibodies or polymers thereof can be mentioned. In the present disclosure, immunoreactive fragments may be monospecific, bispecific, trispecific, or multispecific. Fragments of antibodies may also include fragments that are at least about 10 amino acids, at least about 25 amino acids, at least about 50 amino acids, at least about 75 amino acids, or at least about 100 amino acids in length.

Antibodies include any class of antibody, such as IgG, IgA, or IgM (or subclasses thereof), and need not be of any particular class. Depending on the antibody amino acid sequence of the constant domain of the heavy chain, immunoglobulins are divided into different classes. There are five major immunoglobulin classes: IgA, IgD, IgE, IgG, and IgM, and some of these are further subdivided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. sell. The corresponding heavy chain constant domains of different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional structures of different classes of immunoglobulins are well known. Preferably, the antibody may be an IgG antibody, especially an IgG1 or IgG2 antibody, and may also be a human IgG antibody.

The antibodies in the present disclosure can be monoclonal antibodies or polyclonal antibodies, and are preferably monoclonal antibodies. In this disclosure, "monoclonal antibody" refers to an antibody obtained from a cell population that produces substantially homogeneous antibodies. That is, the individual antibodies contained in the cell population are identical except for some possible natural mutants. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to typical polyclonal antibodies, which include different antigens that target different determinants (epitopes), each monoclonal antibody targets a single determinant on the antigen. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibody-producing cells, and is not to be construed as requiring production of the antibody by any particular method. It should be noted that monoclonal antibodies used in accordance with the present disclosure may be prepared by recombinant DNA methods, such as those described in US Pat. No. 4,816,567. Monoclonal antibodies used in accordance with the present disclosure may also be used, for example, by McCafferty. et al. Nature, 348:552-554 (1990). Monoclonal antibodies used in accordance with the present disclosure may also be used in accordance with the present disclosure. KOHLER&C. MILSTEIN. It may be prepared with reference to the hybridoma method described in Nature, 256:495-497 (1975).

The antibodies in this disclosure can be chimeric antibodies, humanized antibodies, fully human antibodies, or animalized antibodies adapted to any non-human animal to be treated. Examples of animals other than humans to be treated include mice, rats, hamsters, guinea pigs, rabbits, dogs, monkeys, sheep, goats, camels, chickens, ducks, etc., and preferably mice and rats. Or a rabbit. When the antibody is a human chimeric antibody, see, for example, Morrison, S. et al. L. Proc. Natl. Acad. Sci. The antibody can be produced with reference to USA, 81, 6851-6855 (1984). When the antibody of the present disclosure is a humanized antibody, for example, L. Rieohmannet al. Nature, 332, 323-7 (1988); Kettleborough, C. A. et al. Protein Eng, 4, 773-783 (1991); and Clark M. Immunol. The antibody can be produced with reference to Today, 21, 397-402 (2000).

Therefore, the "drug that improves airway hyperresponsiveness and/or eosinophilic airway inflammation caused by CD109" in the present disclosure may be an anti-CD109 monoclonal antibody, and specifically, an anti-CD109 mouse monoclonal antibody. Good too. Anti-CD109 mouse monoclonal antibodies can be prepared by, for example, producing hybridomas by cell fusion of antibody-producing cells obtained from non-human animals immunized with antigens and myeloma cells, and then producing the desired antibody (i.e., anti-CD109 mouse monoclonal antibodies) from the resulting hybridomas. ) can be obtained by selecting hybridomas that produce antibodies and allowing the selected hybridomas to produce antibodies.

More specifically, first, an antigen (immunogen) is administered to a non-human animal. Non-human animals include, for example, mice, rats, hamsters, guinea pigs, rabbits, dogs, monkeys, sheep, goats, camels, chickens, etc., and are preferably mice, rats, or rabbits.

The immunogen is not particularly limited as long as it can produce the anti-CD109 antibody of the present disclosure in non-human animals, and may be polypeptides or peptides including proteins, protein fragments, fusion proteins, etc. . As an example, the immunogen may include an epitope of CD109. Furthermore, the antigen may be administered alone or together with an adjuvant, carrier, or diluent. Any adjuvant can be used as the adjuvant, such as complete Freund's adjuvant, incomplete Freund's adjuvant, or aluminum hydroxide.

The route of administration of the immunogen (immunization route) includes any route such as intraperitoneal administration, intravenous administration, nasal administration, subcutaneous administration, intradermal administration, transpulmonary administration, and intrarectal administration. The number of immunizations (antigen administration) to non-human animals may be single or multiple times (e.g., 2, 3, 4, or 5 or more times), but it is preferable to immunize the non-human animal multiple times. . In addition, when immunizing multiple times, the administration may be repeated, for example, once every 1 to 5 or 6 weeks. Furthermore, when immunizing multiple times, the administration route may be the same, or two or more administration routes may be combined. Further, the amount of immunogen administered in one immunization can be appropriately determined depending on the non-human animal used, and may be, for example, 1-1000 μg per non-human animal.

Next, antibody-producing cells are collected from the non-human animal. Examples of antibody-producing cells include splenocytes, lymph node cells, peripheral lymphocytes, and the like. The time to collect antibody-producing cells can be set arbitrarily, and may be, for example, 1 to 7 days after the final administration of the immunogen.

Then, the antibody-producing cells and myeloma cells are fused to create a hybridoma. Examples of cell fusion methods include a method using polyethylene glycol (PEG), an electric pulse method, and the like. Myeloma cells that can be used for cell fusion include, for example, P3-X63Ag8-U1 (P3-U1), SP2/0-Ag14 (SP2/0), P3-X63-Ag8653 (653), P3-X63-Ag8 ( X63), P3/NS1/1-Ag4-1 (NS1), and the like.

Thereafter, screening for monoclonal antibody-producing hybridomas can be performed using known methods such as ELISA, Western blotting, and flow cytometry.
In the present disclosure, the antibody may be obtained by screening for a specific antibody using the Dot plot method, which is a general protein quantitative method, and extracting and purifying the protein from a clone with the lowest reaction threshold. Note that the Dot plot method is a method in which proteins are immobilized on a membrane such as a nitrocellulose membrane without being separated by electrophoresis, and the amount of protein is specifically quantified using an enzyme-labeled antibody. may also be used to screen the binding ability of antibodies.

By culturing the target antigen-specific monoclonal antibody-producing hybridoma thus obtained, a target antigen-specific monoclonal antibody (ie, anti-CD109 mouse monoclonal antibody) can be prepared in large quantities.

In one aspect of the present disclosure, the anti-CD109 mouse monoclonal antibody may be a monoclonal antibody directed against the amino acid sequence "RKYQPNIDVQESIH" (SEQ ID NO: 1), which is one of the epitopes of CD109.

In some embodiments, the antibody includes one or more constant regions. The constant region can be a heavy chain constant region and/or a light chain constant region. The antibody may comprise a constant region that has at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% identity to a human constant region. good. The antibody may include an Fc region, eg, a human Fc region. The antibody may comprise an Fc region that has at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% identity to a human Fc region. good.

Antibodies used in this disclosure specifically bind to target molecules. The binding affinity of the antibody used in the present disclosure to the antigen is less than 1×10 ⁻⁵ M, less than 5×10 ⁻⁵ M, less than 1×10 ⁻⁶ M, less than 5×10 ⁻⁷ M, 1× Less than 10 ^-7 M, less than 5 x 10 ^-8 M, less than 1 x 10 ^-8 M, less than 5 x 10 ^-9 M, less than 1 x 10 ^-9 M, less than 5 x 10 ^-10 M, 1 x 10 ^- Affinities having dissociation constants (ie, Kd) of less than ¹⁰ M, less than 5×10 ⁻¹¹ M, or less than 1×10 ⁻¹¹ M are included. The dissociation constant is also 1×10 ⁻¹⁵ M or more, 5×10 ⁻¹⁵ M or more, 1×10 ⁻¹⁴ M or more, 5×10 ⁻¹⁴ M or more, 1×10 ⁻¹³ M or more, 5×10 ⁻¹³ M or more, 1 x 10 ^-12 M or more, 5 x 10 ^-12 M or more, 1 x 10 ^-11 M or more, 5 x 10 ^-11 M or more, 1 x 10 ^-10 M or more, or 5 x 10 ^-10 M It may be more than that. Methods for determining affinity are known in the art. For example, binding affinity may be determined using a BIAcore biosensor, KinExA biosensor, scintillation proximity assay, ELISA, ORIGEN immunoassay (IGEN), fluorescence quenching, fluorescence transfer, and/or yeast display. . Affinity may be screened using appropriate bioassays.

The antibodies described in this disclosure may be modified; examples of such modifications include functionally equivalent antibodies and enhanced or attenuated activities that do not significantly affect the properties of the antibody. Examples include mutants with Modification of antibodies is a routine procedure in the art and need not be described in detail in this disclosure. Examples of modifications include conservative substitutions of amino acid residues, insertions, additions, or deletions of one or more amino acids that do not significantly impair functional activity, or the use of chemical analogs.

A conservative substitution is the replacement of an amino acid residue with an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains include: amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid). ), amino acids with uncharged polar side chains (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (eg, threonine, valine, isoleucine), and amino acids with aromatic side chains (eg, tyrosine, phenylalanine, tryptophan, histidine).

Insertions or additions of amino acid sequences include additions of amino acids to the amino and/or carboxyl termini, and insertions of single or multiple amino acid residues within the sequence. Examples of terminal insertions include antibodies with an N-terminal methionyl residue or antibodies fused to epitope tags. Other insertional variants of antibody molecules include fusions of enzymes or antibodies to the N-terminus or C-terminus of the antibody that increase the serum half-life of the antibody.

Sites where substitutional mutagenesis is most effective include CDRs, but changes in FRs are also contemplated.
Non-conservative substitutions are made by exchanging a member of one of these classes with another. More conservative substitutions involve exchanging one member of a class with another member of the same class. Amino acid substitutions in the variable region can alter binding affinity and/or specificity. For example, no more than 1-5 conservative amino acid substitutions are made within a CDR domain.

The antibodies described in this disclosure also include glycosylated and non-glycosylated antibodies, as well as antibodies with other post-translational modifications, such as glycosylation on different sugars, acetylation, and phosphorylation. Antibodies are glycosylated at conserved positions in their constant regions.

In the present disclosure, the control of airway hyperresponsiveness and/or eosinophilic airway inflammation by CD109 may involve the production of cytokines secreted by Th2 cells (Th2 cytokines). That is, in the present disclosure, improvement of airway hyperresponsiveness and/or eosinophilic airway inflammation may be due to suppression of production of cytokines secreted by Th2 cells (Th2 cytokines). Examples of Th2 cytokines include IL-4, IL-5, IL-13, IL-33, and the like.

Furthermore, in the present disclosure, improvement of airway hyperresponsiveness and/or eosinophilic airway inflammation may be due to activation of RUNX3. RUNX3 is a protein belonging to the Runt family of transcription factors. RUNX3 is known to be conjugated with Smad2/3 and involved in TGF-β. Therefore, in the present disclosure, improvement of airway hyperresponsiveness and/or eosinophilic airway inflammation may be due to phosphorylation of Smad2/3.

Therefore, the pharmaceutical composition of the present disclosure may have an effect of activating RUNX3 and an effect of phosphorylating Smad2/3.
Furthermore, the pharmaceutical composition of the present disclosure may contain pharmaceutically acceptable additives as necessary. A "pharmaceutically acceptable excipient" is one that, when combined with an active ingredient, allows the active ingredient to remain biologically active, is non-reactive with the subject's immune system when delivered, and Contains any material that is non-toxic to the body. Examples include, but are not limited to, all standard pharmaceutical carriers such as phosphate buffered saline, water, emulsions such as oil/water emulsions, and wetting agents of various types. A preferred diluent for nebulized or parenteral administration is phosphate buffered saline or saline (0.9%).

Targets for administration include mammals such as humans, rats, mice, cows, horses, dogs, cats, pigs, and sheep, and preferably humans.
The pharmaceutical composition of the present disclosure may be in any oral or parenteral formulation as long as it can be administered to a patient. Examples of compositions for parenteral administration include injections, nasal drops, and the like. Preferably, it is a nasal spray. The dosage form of the pharmaceutical composition of the present disclosure can include, for example, a liquid preparation.

Effective doses and schedules for administering the pharmaceutical compositions of the present disclosure can be determined empirically, and methods for such determination are within the common general knowledge in the art. Those skilled in the art will appreciate that the dose of the pharmaceutical composition of the present disclosure to be administered will vary depending on, for example, the mammal receiving the pharmaceutical composition of the present disclosure, the route of administration, the specific type of drug used, etc. You will understand that. A typical daily dosage of a pharmaceutical composition of the present disclosure may range, for example, from about 1 μg/kg body weight to 100 mg/kg body weight or more per day. Generally, any of the following dosages may be used: at least about 50 mg/kg body weight; at least about 10 mg/kg body weight; at least about 3 mg/kg body weight; at least about 1 mg/kg body weight; at least about 750 μg/kg body weight; At least about 500 μg/kg body weight; At least about 250 μg/kg body weight; At least about 100 μg/kg body weight; At least about 50 μg/kg body weight; At least about 10 μg/kg body weight; At least about 1 μg/kg body weight, or more. amount is administered.

Further, one aspect of the present disclosure relates to dendritic cells derived from a subject deficient in CD109, which are used to improve airway hyperresponsiveness and/or eosinophilic airway inflammation. Dendritic cells of the present disclosure may be derived from various tissues (eg, lung, bone marrow, etc.). Furthermore, in the present disclosure, the subject may be a human, a rat, a mouse, or other mammal.

Here, it is known that dendritic cells are classified into DC1s and DC2s as standard subsets. These are also described as classical dendritic cells (ie, cDC1s and cDC2s). CD109 is selectively induced in dendritic cells (cDC2s) by allergen exposure. Note that CD109 is selectively induced in type 2 innate lymphocytes and B cells in addition to cDC2s by exposure to allergens.

Furthermore, as a new classification of the cDC2s subset, in recent years, it has been classified into cDCs2a and cDCs2b based on the expression pattern of transcription factors. cDCs2a are a subset of the anti-inflammatory system with high expression of RUNX3 and T-bet, and cDCs2b are a subset of the inflammatory protective system with high expression of RORγt. Since cDC2s derived from subjects deficient in CD109 showed an expression pattern similar to that of cDCs2a, cDC2s derived from subjects deficient in CD109 were considered to be a subset of the anti-inflammatory system.

In addition, as for the genetic profile, cDC2s derived from subjects deficient in CD109 have decreased expression of eotaxin and Th2-related cytokines, and Th1 Increased expression of relevant cytokines is obtained. Examples of eotaxin include CCL24 and CCL26. Examples of Th2 include IL-4, IL-5, IL-13, and IL-33. Th1-related cytokines include IL-18, IL-12, and CXCL9.

It is also known that dendritic cells initiate adaptive responses and induce T cell activation and differentiation. In the present disclosure, for example, cytokines are induced by co-expressing cDC2s with naive T cells. Examples of such cytokines include IL-6, IL-13, IL-17A, TNF, and IFN-γ. In addition, cDC2s derived from a subject deficient in CD109 exhibit reduced expression of the cytokine, for example, compared to cDC2s derived from a subject not deficient in CD109.

The dendritic cells of the present disclosure may be used for various purposes. For example, dendritic cells (eg, cDC2s) may be used for transfer. Furthermore, dendritic cells (eg, cDC2s) may be included in the drug as an active ingredient.

Hereinafter, examples will be shown to explain the present disclosure in more detail, but the present disclosure is not limited thereto. Furthermore, all documents cited throughout this application are incorporated into this application as is by reference.

<Experimental method>
(1) Mice used in the experiment Female C57BL/6 mice and BALB/c mice (8-10 weeks old) were purchased from Japan SLC Co., Ltd. (Shizuoka, Japan), and OVA-specific T cell receptor (TCR) ) Transgenic mice (OT-II) were purchased from Kumamoto University Animal Resources Development Research Institute (Kumamoto, Japan). CD109 ^−/− mice lacking CD109 were generated according to a previous report (Mii, S. et al. Am J Pathol 181, 1180-1189, (2012).). CD109 ^−/− mice and OT-II mice were bred in the inventor's laboratory at Hamamatsu University School of Medicine according to protocols approved by the Institutional Animal Care and Use Committee (29-045).

(2) Creation of bronchial asthma model mice and measurement of airway hyperresponsiveness Mice were given 50 μg of tick antigen HDM (Dermatophagoides. Pteronyssinus, Greer Laboratories, XPB82D3A2.5) on the first day of the experiment, and on the 8th and 13th days of the experiment. Sensitization was performed intranasally (i.n.) with 10 μg of HDM.

In Example 6 described below, anti-mouse CD109 monoclonal antibody (in., 5 μg/mouse, anti-CD109 antibody, clone 4A13, Abmart) or IgG isotype (FUJIFILM, 140-09511) was administered the day before HDM challenge. Mice were treated. The anti-mouse CD109 monoclonal antibody was designed and prepared by Abmart as a monoclonal antibody against the amino acid sequence "RKYQPNIDVQESIH" (SEQ ID NO: 1), which is an epitope of CD109. In the OVA model, mice were immunized intraperitoneally with OVA (50 μg, Sigma-Aldrch, St. Louis, MO) and Alum (2 mg, Thermo Fischer Scientific, IL) on experimental days 1 and 8. The mice were challenged intranasally with 50 μg of OVA on days 19 to 21 of the experiment. Two days after HDM challenge and one day after OVA challenge, 300 μl of ketamine (10 mg/ml) and xylazine (1 mg/ml) were administered i.p. p. Mice were anesthetized by injection. Airway resistance and pulmonary dynamic compliance have been previously reported (Galle-Treger, L. et al. Nature communications 7, (2016)., Suzuki, Y. et al. The Journal of allergy and clinical i mmunology 137, 1382-1389 (2016)., and Suzuki, Y. et al. Autophagy, 1-13 (2022)), the mice were mechanically ventilated using a Fine Point RC system (Buxco Research Systems). did. Mice were treated with aerosolized PBS (baseline), increasing concentrations of methacholine (2.5 mg/ml, 5.0 mg/ml, 10 mg/ml, 20 mg/ml, 40 mg/ml; Sigma- Aldrich, A2251) were successively challenged. R _L and C _dyn values were recorded for 3 minutes after each methacholine challenge.

(3) Collection of bronchoalveolar lavage (BAL) fluid and histological examination of the lungs After measuring airway hyperresponsiveness, a test was performed as previously reported (Galle-Treger, L. et al. Nature communications 7, (2016)., Suzuki, Y. .et
al. The Journal of allergy and clinical immunology 137, 1382-1389, (2016). , and Suzuki, Y. et al. BAL cells were obtained according to Autophagy, 1-13, (2022)). In addition, lung tissue sections and lung lysates were obtained and quantified. In some experiments, lung tissue and lung lysates were preserved in RNA stabilization reagent (Qiagen, Valencia, CA, USA). Specifically, after measuring airway hyperresponsiveness, a cannula was inserted into the trachea, the lungs were washed three times with 1 ml of PBS, and BAL cells were collected. Transcardial perfusion of the lungs was performed with cold PBS, and the lungs were subsequently fixed in 4% paraformaldehyde buffered in PBS for histology. After fixation, the lungs were embedded in paraffin, cut into 4 μm sections, and stained with hematoxylin and eosin (HE) and periodic acid Schiff (PAS).

Inflammation scores were assigned in a blinded manner according to a previous report (Tong, J. et al. J Exp Med 203, 1173-1184, (2006).). The score of peribronchial inflammation by HE staining was determined as follows. 0: normal, 1: a small number of cells, 2: aggregation of inflammatory cells at a depth of 1 cell layer, 3: aggregation of inflammatory cells at a depth of 2 to 4 cell layers, 4: aggregation of inflammatory cells at a depth of 4 or more cells. Layer-depth collection of inflammatory cells. PAS staining was performed by examining at least 20 consecutive areas. A numerical score of the abundance of PAS-positive goblet cells in each airway was counted and expressed as a percentage of the total number of epithelial cells in that airway.

(4) Identification of lung DCs subsets Lung DCs were defined as deletion of classical lineage markers (CD3ε, NK1.1, CD19, CD45R) and CD45+CD64-F4/80-IA/IE+CD11c+ cells. . Next, we identified lung DCs subsets based on the expression of XCR1 and CD172a (cDC1s; XCR1+CD172a- and cDC2s; 2014). , Brown, C. C. et al. Cell 179, 846-863, (2019)., Nutt, S. L. & Chopin, M. Immunity 52, 942-956, (2020)., and Sakurai, S. et al. al. Allergology international:official journal of the Japanese Society
of Allergology 70, 351-359, (2021). ).

(5) Preparation of lung DCs subset Lungs were digested with collagenase (Roche, Basel, Switzerland) and DNase (Roche) using GentleMACSTM dissociating agent (Miltenyi Biotec) to prepare a single cell suspension. Cells were incubated with anti-mouse CD11c coated magnetic beads (Miltenyi Biotec) and positively sorted by magnetic cell sorting (MACS; Miltenyi Biotec). The following antibodies were used to classify DCs. Peridinin-chlorophyll-protein complex-Cy5.5 (PerCP-Cy5.5) labeled lineage markers (CD3e (clone 17A2, BioLegend), NK1.1 (clone PK136, BioLegend), CD19 (clone 6D5, BioLegend) , CD45R (clone RA-3-6B2)), PE-Cy7 labeled CD64 (clone X54-5/7.1, BioLegend), allophycocyamine-Cy7 (APC-Cy7) labeled F4/80 (clone BM8, BioLegend), Alexa Flour 700-labeled anti-IA/IE (clone M5/114.15.2, BioLegend), brilliant violet 510-labeled anti-CD45 (clone 30-F11, BioLegend), FITC labeled Anti-CD11c (clone N418, BioLegend), PE-labeled XCR1 (clone ZET, BioLegend), and APC-labeled CD172a (clone P84, BioLegend). DCs were sorted using a MoFlo Astrios EQ (Beckman Coulter, Brea, CA). Sorted DCs subsets were typically >95% positive for the surface marker of interest.

(6) In vitro co-culture of lung DCs subset and CD4+ T cells Naive CD4+ T cells were obtained from OT-II mouse spleen using CD4+T Cell Isolation Kit, Mouse (Miltenyi Biotec, Auburn, CA), and purified CD4+T Cells (2x10 /ml) were co-cultured with lung DCs subset ( ^2x10 /ml) for 7 days in the presence of 10 μg/m ^OVA _323-339 peptide (ISQV-HAAHAEINEAGR, Invitrogen, Carlsbad, CA). .

(7) Gene expression analysis using Nanostring nCounter technology Differences in the abundance of transcripts between lung DCs subsets purified from HDM-sensitized CD109 ^−/− mice or WT mice were analyzed using Nanostring nCounter technology (Immunology Panel). was used for analysis. Heat plots were also generated using nSolver software.

(8) Cytokine measurement The levels of IL2, IL6, IL10, IL13, IL17A, TNF and IFN-γ in the DCs-T cell co-culture supernatant were measured using a cytometry bead array kit (BD Biosciences, San Jose, CA). Measurements were made according to the manufacturer's instructions.

(9) Bone marrow-derived DCs and bone marrow-derived DCs transfer experiment Bone Marrow (BM) cells were collected from femurs and tibias. BM cells were grown at 4×10 ⁵ cells in RPMI1640 supplemented with 10% fetal calf serum (FCS), 1000 U/ml recombinant mouse GM-CSF (R&D), and 400 U/ml recombinant mouse IL-4 (R&D). /ml. Cells were collected on the 8th day of culture and incubated with HDM (20 μg/ml) for 6 hours. BM-derived DCSs (BMDCs) (2.0×10 ⁵ ) were intravenously transferred into C57BL/6 mice (day 1), and BMDCs (2.0×10 ⁵ ) were transferred on days 8 and 13. was administered intratracheally, and analysis was performed on the 15th day.

(10) Flow cytometry analysis and intracellular staining of BAL fluid BAL cells were treated with surface phycoerythrin (PE)-labeled anti-Singlec-F (clone E50-2440, BD Pharmingen, San Diego), APC-labeled anti-Ly-6G/Ly- 6C (clone RB6-8C5, BioLegend), PE-Cy (PE-Cy7) labeled anti-CD45 (clone 30-F11, BioLegend), APC-Cy7 labeled anti-CD11c (clone N418, BioLegend), PerCP-Cy5. The cells were stained with 5-labeled anti-CD3e (clone 17A2, BioLegend), FITC-labeled anti-CD19 (clone MB19-1, BioLegend), and Pacific blue-labeled anti-CD11b (clone M1/70, BioLegend).

The following antibodies were used to analyze CD109 expression in lung cells. FITC-labeled anti-EpiCAM (clone G8.8, BioLegend), APC-labeled anti-CD31 (clone 390, BioLegend), PECy7-labeled anti-CD45 (clone 30-F11, BioLegend), BV510-labeled anti-CD45 (clone 30-F11, BioLegend), FITC-labeled anti-CD11c (clone N418, BioLegend), APC-labeled anti-IA/IE (clone M5/114.15.2, BioLegend), APC-Cy7-labeled anti-F4/80 (clone BM8, BioLegend) ), Pacific blue labeled anti-CD11b (clone M1/70, BioLegend), PE-Cy7 labeled anti-CD103 (clone 2E7, BioLegend), FITC-labeled anti-CD19 (clone MB19-1, BioLegend), PerCP-Cy5.5 - labeled anti-CD3 (clone 17A2, BioLegend), APC-labeled anti-CD4 (clone RM4-5, BioLegend), BV510-labeled anti-CD45 (clone 30-F11, BioLegend), PerCP-Cy5.5-labeled anti-ST2 (clone DIH9, BioLegend), PECy7-labeled anti-CD127 (clone A7R34, BioLegend), BV510-labeled anti-CD45 (clone 30-F11, BioLegend), FITC-streptavidin (BioLegend), biotinylated anti-mouse strain (CD3e (145) -2C11; BioLegend), CD45R (RA3-6B2; BioLegend), Ly-6G/Ly-6C (RB6-8C5; BioLegend), CD11c (N418; BioLegend), CD11b (M1/70; BioLegend) ) , Ter119 (TER-119; BioLegend), NK1.1 (PK136; BioLegend), TCR-β (H57-597; BioLegend), TCR-γδ (BioLegend), and FCER1A/FCεRIα (MAR-1; BioL egend Next, the expression intensity of CD109 was evaluated using PE-labeled anti-CD109 (clone 496920, R&D).

The following antibodies were used on the cell surfaces of CD109+DCs and CD109-DCs. PE-Cy7 labeled anti-CD40 (clone 3/23, BioLegend), APC-labeled anti-CD86 (clone GL-1, BioLegend), Pacific blue-labeled anti-CD80 (clone 16-10A1, BioLegend), Biotin-labeled anti-ICOS Ligand (clone HK5.3, BioLegend), brilliant violet 421-labeled anti-streptavidin (BioLegend), PECy7-labeled anti-PD-L2 (clone TY25, BioLegend), and APC-labeled anti-PD-L1 (clone 10F.9G2, BioLegend Inc.).

For intracellular staining, PE-labeled anti-Runx3 (clone R3-5G4, BD Bioscience), BDCSytofix/Cytoperm kit (BD Bioscience), Alexa Fluor 647-labeled anti-phosphorylated Smad2 (pS465/pS467)/Smad3 (pS 423/pS425 ) (Clone O72-670, BD
Bioscience Inc.), BDCSytofix ^™ FixationBuffer and BDPhosflow ^™ PermBuffer III were used according to the manufacturer's instructions.

Analytical flow cytometry was performed using Gallios (Beckman Coulter, Brea, Calif.). The resulting data were analyzed using FlowJo version 8.6 software (TreeStar, Ashland, OR).

(11) Reverse Transcription Polymerase Chain Reaction (RT-PCR) Analysis The expression of IL4, IL5, IL13, IL25, IL33, and TSLP in lung lysates was measured by reverse transcription polymerase chain reaction (RT-PCR) assay. Total RNA was extracted using the RNAeasy mini kit (Qiagen, Valencia, CA) and then cDNA was prepared from the RNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Carlsbad, CA). did. RT-PCR was performed using StepOnePlus (Applied Biosystems, Carlsbad, CA) and ΔΔCt method (Suzuki, Y. et al. American journal of respiratory cell and mol ecular biology 46, 773-780, (2012). ) was used for data analysis. The expression of each gene was normalized with the expression of the housekeeping gene GAPDH.

The specific primer pairs for RT-PCR are as follows.
-IL-4 (forward 5'-GGTCTCAACCCCAGCTAGT-3' (SEQ ID NO: 2) and reverse 5'-GCCGATGATCTCTCTCCAAGTGAT-3' (SEQ ID NO: 3))
-IL-5 (forward 5'-CTCTGTTGACAAGCAATGAGACG-3' (SEQ ID NO: 4) and reverse 5'-TCTTCAGTATGTCTAGCCCCTG-3' (SEQ ID NO: 5))
-IL-13 (forward 5'-CCTGGCTCTTGCTTGCCTT-3' (SEQ ID NO: 6) and reverse 5'-GGTCTTGTGTGATGTTGCTCA-3' (SEQ ID NO: 7))
-IL-25 (forward 5'-ACAGGGACTTGAATCGGGTC-3' (SEQ ID NO: 8) and reverse 5'-TGGTAAAGTGGGAGAGAGTTG-3' (SEQ ID NO: 9))
-IL-33 (forward 5'-TCCAAACTCCAAGATTTCCCCG-3' (SEQ ID NO: 10) and reverse 5'-CATGCAGTAGACATGGCAGAA-3' (SEQ ID NO: 11))
・TSLP (forward 5'-ACGGATGGGGCTAACTTACAA-3' (SEQ ID NO: 12) and reverse 5'-AGTCCTCGATTTGCTCGAACT-3' (SEQ ID NO: 13))
- GAPDH (forward 5'-AACTTTGGCATTGTGGAAGG-3' (SEQ ID NO: 14) and reverse 5'-GGATGCAGGGATGATGTTCT-3' (SEQ ID NO: 15)).
The ΔΔCt method was used for data analysis.

(12) Reagents RPMI supplemented with 3mM L-glutamine (Sigma, St. Louis, MO), 1% penicillin-streptomycin (Gibco BRL, Tokyo, Japan), and 10% heat-inactivated FCS (Gibco BRL) Cells were cultured in 1640 medium (Gibco BRL).

(13) Statistical analysis For comparisons between groups, Student's t-test and one-way analysis of variance after Turkey's multiple comparison test were used. When P<0.05, it was considered that there was a statistically significant difference. All data were expressed as mean ± SEM. Statistical analyzes were performed using GraphPad Prism version 6 (GraphPad Software, Inc., San Diego, Calif.).

<Example 1: Improvement of airway hyperresponsiveness and eosinophilic airway inflammation due to CD109 deficiency>
To confirm the influence of CD109 on the development of airway hyperresponsiveness and eosinophilic airway inflammation, CD109 ^−/− mice and wild-type (WT) mice were sensitized and challenged with HDM (Figure 1A ). Two days after the final HDM challenge, pulmonary airway resistance (R _L ) and pulmonary dynamic compliance (C _dyn ) were directly measured to assess lung function. HDM-challenged CD109 ^−/− mice exhibited well-reduced airway hyperresponsiveness, lower pulmonary airway resistance, and higher pulmonary dynamic compliance compared to HDM-challenged WT mice (FIG. 1B). HDM-challenged CD109 ^−/− mice also showed lower eosinophil counts in bronchoalveolar lavage (BAL) compared to HDM-challenged WT mice (FIG. 1C). Additionally, histological analysis showed that in HDM-challenged CD109 ^−/− mice, there was a decrease in the number of PAS+ cells, as well as a decrease in bronchial wall thickness and peribronchial cell infiltration compared to HDM-challenged WT mice. confirmed (Fig. 1D). Consistent with these results, expression of IL-4, IL-5, IL-13, and IL-33 was significantly reduced in HDM-challenged CD109 ^−/− mice compared with HDM-challenged WT mice. A significant decrease was confirmed (Fig. 1E).

<Example 2: Induction of allergen sensitization in WT mice by CD109 expression>
We investigated the types of CD109-expressing immune cells in the lungs of an asthma model. At steady state, no immune cells in the lung expressed CD109. In OVA-challenged mice, CD109 expression was induced in cDC2s, B cells, and type 2 innate lymphocytes (ILC2s), but in the lungs, CD109 expression was not induced in cDC1s (FIG. 2A). In addition, as shown in Figure 2B, CD109+ DCs and CD109-DCs were sorted based on cell surface markers, and phenotypically, CD109 expression in lung DCs was compared to cases where CD109 was not expressed. The expression of PD-L1, ICOS-L, and CD80 was increased, but the expression of CD86 was decreased (Fig. 2C).

Example 3: Improved induction of cytokine production when lung cDC2s from CD109 ^−/− mice are co-cultured with naive T cells
Since CD109 is induced only in lung cDC2s and not in lung cDC1s, we investigated whether lung cDC2s from CD109 ^−/− mice alter their function with respect to Th responses. Previous studies have shown different functions of lung DCs subsets in immune responses (Murphy, T.L. et al. Annual review of immunology 34, 93-119, (2016)., Guilliams, M. et al. . Nature Reviews IMMUNOLOGY 14,571-578, (2014). BROWN, C.C.Et Al. Cell 179,846-863, (2019). (2019). 52,942 -956, (2020)., Suzuki, Y. et al. American journal of respiratory cell and molecular biology 46, 773-780, (2012)., and Furuhashi, K. et al. American journal of respiratory cell and molecular biology 46, 165-172, (2012).). In particular, cDCs2 has been reported to favorably induce Th2 localization in the steady state (Furuhashi, K. et al. American journal of respiratory cell and
Molecular biology 46, 165-172, (2012). , Gao, Y. et al. Immunity 39, 722-732, (2013). , Kumamoto, Y. et al. Immunity 39, 733-743, (2013). , and Williams, J. W. et al. Nature communications 4, (2013). ). Lung cDC2s were purified from OVA-challenged CD109 ^−/− mice and WT mice (FIGS. 3A and 3B). Subsequently, lung cDC2s were co-cultured with naive CD4+ T cells isolated from OT-II mice in the presence of OVA _323-339 peptide. Large amounts of IL-2, IL-6, IL-13, IL-17A, TNF, and IFN-γ were detected in co-culture with lung cDC2s from WT mice. Comparing lung cDC2s from CD109 ^−/− mice with lung cDC2s from WT mice, the concentration of IL-13 in lung cDC2s from CD109 ^−/− mice was significantly lower than that in lung cDC2s from WT mice. (Fig. 3C), and IL-6, IL-17A, TNF, and IFN-γ were also significantly low.

These results indicate that in this co-culture system, the ability of lung cDC2s from CD109 ^−/− mice to induce IL-13, IL-17A, and IFN-γ is weaker than that of lung cDC2s from WT mice. It shows.

<Example 4: Induction of asthmatic phenotype by CD109 expression in DC2s>
Lung cDC2s from CD109 ^−/− mice had improved induction of several cytokines, including IL-13, when co-cultured with naïve T cells, indicating that the absence of CD109 in DCs was associated with airway hyperresponsiveness and We investigated whether this is a direct factor in attenuating eosinophilic airway inflammation. WT mice were immunized intravenously and then challenged intrabronchically with HDM-loaded bone marrow-derived DCs (BMDCs) from CD109 ^−/− mice or WT mice (FIG. 4A). In this model, when HDM-loaded BMDCs from WT mice were used, they showed increased airway hyperresponsiveness and eosinophilic airway inflammation, characterizing the pathological features of asthma (Fig. 4B, Fig. 4C, Fig. 4D). However, airway hyperresponsiveness and the number of eosinophils in the BAL in mice challenged with HDM-loaded BMDCs from CD109 ^−/− mice were significantly lower compared to those challenged with HDM-loaded BMDCs from WT mice ( Figure 4B, Figure 4C). Furthermore, histological analysis showed that the number of PAS+ cells and the thickness of the bronchial wall were significantly lower in mice challenged with HDM-loaded BMDCs from CD109 ^−/− mice than in those challenged with HDM-loaded BMDCs from WT mice. The skin was thinner, and the number of inflammatory cells was also reduced (Figure 4D). These data suggested that expression of CD109 in DCs is required to appropriately induce airway hyperresponsiveness and eosinophilic airway inflammation in this model.

<Example 5: Gene expression analysis in lung DCs from CD109 ^−/− mice>
Since there is a functional difference in cytokine production induced in lung cDC2s from CD109 ^−/− mice and WT mice, we used Nanostring technology to identify genes in lung cDC2s from CD109 ^−/− mice and WT mice. The expression profile was investigated. In lung cDC2s from CD109 ^−/− mice, eotaxin (CCL24 and CCL26) and Th2-related cytokines (IL-4, IL-5, IL-13, and IL- 33) and increased expression of Th1-related cytokines (IL-18, IL-12, and CXCL9) (Figure 5A). Regarding transcription factors, lung cDC2s from CD109 ^−/− mice showed increased expression of RUNX3 and decreased expression of RORγt compared to lung cDC2s from WT mice.

Recent studies have shown different phenotypic classifications of cDC2s (cDCs2a: anti-inflammatory phenotype, and cDCs2b: pro-inflammatory phenotype) based on transcription factors (Brown, C.C. et al. Cell 179, 846-863, (2019)., and Nutt, S. L. & Chopin, M. Immunity 52, 942-956, (2020).). RUNK3 is one of the transcription factors characteristic of the anti-inflammatory phenotype of cDCs2a. Consistent with the array data, significantly increased expression of RUNX3 protein was observed in lung cDC2s from CD109 ^−/− mice compared to lung cDC2s from WT mice (FIG. 5B). Furthermore, CD109 is known to negatively regulate TGF-β signaling, and RUNX3 is known to bind to intracellular signaling factors Smad2 and Smad3 (Mii, S. et al. al. Pathol Int 69, 249-259, (2019).). Therefore, when we investigated phosphorylated Smad2/3, we found that phosphorylated Smad2/3 was also significantly increased in lung cDC2s from CD109 ^−/− mice compared to lung cDC2s from WT mice. (Fig. 5C, Fig. 5D).

<Example 6: Effect of anti-CD109 monoclonal antibody on improving airway hyperresponsiveness and eosinophilic airway inflammation in HDM-challenged mice>
To investigate whether blockade of CD109 could be a novel therapeutic strategy for asthma, we investigated whether anti-CD109 monoclonal antibodies (mAbs) ameliorate airway hyperresponsiveness and eosinophilic airway inflammation in HDM-challenged mice. investigated. WT mice were sensitized with HDM, and anti-CD109 mAb or IgG isotype was intranasally administered one day before HDM challenge (Figure 6A). As shown in Figure 6B, a significant phenomenon of airway hyperresponsiveness was observed in WT mice administered with anti-CD109 mAb compared to WT mice administered with IgG isotype. There was also a significant decrease in total cells and a significant decrease in the number of eosinophils in the BAL (Figure 6C). Furthermore, in WT mice administered with anti-CD109 mAb, there was a decrease in the number of infiltrating inflammatory cells, a decrease in the number of PAS+ cells, and a decrease in the thickness of the bronchial wall (FIG. 6D).
This suggested that anti-CD109 mAb may be useful as a novel therapeutic agent for asthma.

<Example 7: Effect of CD109 on airway hyperresponsiveness and eosinophilic airway inflammation in OVA-challenged mice>
To confirm the influence of CD109 on the development of airway hyperresponsiveness and eosinophilic airway inflammation, CD109 ^−/− mice and wild-type (WT) mice were exposed and sensitized using OVA (Figure 7A ). One day after the final OVA challenge, pulmonary airway resistance (R _L ) and pulmonary dynamic compliance (C _dyn ) were directly measured to assess lung function. OVA-challenged CD109 ^−/− mice had well-reduced airway hyperresponsiveness and exhibited lower lung airway resistance and higher pulmonary dynamic compliance compared to HDM-challenged WT mice (FIG. 7B). OVA-challenged CD109 ^−/− mice also showed lower eosinophil counts in the bronchoalveolar lavage (BAL) compared to OVA-challenged WT mice (FIG. 7C). Histological analysis also showed that in OVA-challenged CD109 ^−/− mice, there was a decrease in the number of PAS+ cells, as well as a decrease in bronchial wall thickness and peribronchial cell infiltration compared to OVA-challenged WT mice. This was confirmed (Fig. 7D). Consistent with these results, expression of IL-4, IL-5, IL-13, and IL-33 was significantly reduced in HDM-challenged CD109 ^−/− mice compared with HDM-challenged WT mice. A significant decrease was confirmed (Fig. 7E).

<Example 8: Effect on Smad2/3 by lung DCs from CD109 ^−/− mice in OVA-challenged mice>
Furthermore, when we investigated phosphorylated Smad2/3, we found that lung cDC2s from OVA-challenged CD109 ^−/− mice had lower levels of phosphorylated Smad2/3 compared to lung cDC2s from OVA-challenged WT mice. It was shown that there was a significant increase (Fig. 8).

<Example 9: Comparison with existing drugs>
To examine the utility of anti-CD109 monoclonal antibodies (mAbs) in the treatment of airway hyperresponsiveness and eosinophilic airway inflammation, we compared anti-CD109 mAbs with existing asthma treatments. Existing asthma treatment drugs include anti-IL-33 antibody (R&D Co., clone 396118, catalog number MAB555), anti-thymic stromal lymphopoietic factor (TSLP) antibody (R&D Co., clone 152614, catalog number AF3626), and Dexamethasone (Dex) (Sigma-Aldric, catalog number D4902-25MG), a type of steroid, was used.

WT mice were sensitized with HDM according to the same schedule and method as in Example 6 above, and anti-CD109 mAb (100 μg/mouse, n=5), anti-IL-33 antibody (6 μg/mouse, n=5), anti- TSLP antibody (20 μg/mouse, n = 5), Dex (20 μg/mouse, n = 5), or IgG isotype (100 μg/mouse, n = 4) was intranasally administered one day before HDM challenge (see Figure 6A). . The dosage of anti-CD109 mAb is previously reported (Song
G. et al. Ann Rheum Dis, 78, 1632-1641 (2019)). In addition, the doses of anti-IL-33 antibody, anti-TSLP antibody, and Dex were set based on a previous report (Suzuki Y. et al. Autophagy, 18, 2216-2228 (2022)).

As shown in Figure 9A, a significant decrease in the total cell number and a significant decrease in the number of eosinophils in the BAL was observed in WT mice treated with anti-CD109 mAb compared to WT mice treated with IgG isotype. . Furthermore, the reduction in total cell count and eosinophil count in BAL by anti-CD109 mAb was at least the same level as in comparison with existing therapeutic agents. Additionally, as shown in Figure 9B, a significant decrease in airway hyperresponsiveness was observed in WT mice administered with anti-CD109 mAb, with lower pulmonary airway resistance compared to existing therapeutic agents and equivalent to existing therapeutic agents. showed higher pulmonary dynamic compliance.

In clinical use, steroids tend to have side effects, which is a problem, and antibody preparations are thought to have fewer side effects. And, as mentioned above, anti-CD109 mAb was found to sufficiently suppress eosinophils and significantly improve airway hyperresponsiveness compared to existing therapeutic agents including steroids and other antibody preparations.
Therefore, it was suggested that anti-CD109 mAb may be more useful as a therapeutic agent for asthma than existing therapeutic agents.

WIPO standard ST. The sequence listing written in 25 format is shown in the table below.

 

Claims (12)

1. A pharmaceutical composition containing as an active ingredient a drug that improves airway hyperresponsiveness and/or eosinophilic airway inflammation caused by CD109.
2. The pharmaceutical composition according to claim 1, wherein the CD109 is induced into dendritic cells cCD2, B cells, or type 2 innate lymphocytes.
3. Claim 1 or Claim 2, wherein the improvement in airway hyperresponsiveness and/or eosinophilic airway inflammation is caused by suppression of production of IL-4, IL-5, IL-13, and/or IL-33. Pharmaceutical compositions as described.
4. The medicament according to any one of claims 1 to 3, wherein the improvement of airway hyperresponsiveness and/or eosinophilic airway inflammation is caused by activation of RUNX3 and/or phosphorylation of Smad2/3. Composition.
5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the drug is an anti-CD109 antibody.
6. The pharmaceutical composition according to claim 5, wherein the anti-CD109 antibody is an anti-mouse monoclonal antibody.
7. Dendritic cells cDC2s derived from a subject deficient in CD109, which are used to improve airway hyperresponsiveness and/or eosinophilic airway inflammation.
8. The dendritic cell according to claim 7, wherein the dendritic cell is used for transfer.
9. The dendritic cell according to claim 7 or 8, wherein the dendritic cell is an anti-inflammatory dendritic cell.
10. The improvement of airway hyperresponsiveness and/or eosinophilic airway inflammation is caused by suppressing the production of IL-6, IL-13, IL-17A, TNF, and/or IFN-γ. The dendritic cell according to any one of Item 9.
11. The tree according to any one of claims 7 to 10, wherein the improvement of airway hyperresponsiveness and/or eosinophilic airway inflammation is caused by activation of RUNX3 and/or phosphorylation of Smad2/3. shaped cells.
12. The expression of CCL24, CCL26, IL-4, IL-5, IL-13, and/or IL-33 is reduced in the dendritic cells compared to dendritic cells derived from the subject that are not deficient in CD109. The dendritic cell according to any one of claims 7 to 11, which has a genetic profile showing increased expression of IL-18, IL-12, and/or CXCL9.