Composition for treatment of pulmonary hypertension

Abstract

To provide a composition for treatment of pulmonary hypertension having a novel mechanism of action.SOLUTION: A composition for treatment of pulmonary hypertension contains an inhibitory substance which inhibits the function of CD49b.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a composition for treating pulmonary hypertension.

Background Art

[0002] Pulmonary hypertension (PH) shows symptoms of elevated blood pressure in the pulmonary artery. Pulmonary hypertension is classified into groups 1 to 5 according to its cause, and is diverse from primary ones such as group 1 pulmonary arterial hypertension to those associated with various underlying diseases. The pathogenesis of pulmonary hypertension has not yet been identified (Non-Patent Document 1).

[0003] For pulmonary hypertension, it has been reported that a therapeutic agent containing an inhibitor compound of interleukin 5 receptor (IL-5R) is effective (Patent Document 1).

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Non-Patent Documents

[0005]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0006] For pulmonary hypertension caused by various causes, it is preferable that there are options of therapeutic compositions having various mechanisms of action.

[0007] One aspect of the present invention aims to realize a composition for treating pulmonary hypertension having a novel mechanism of action.

Means for Solving the Problems

[0008] In order to solve the above problems, a composition for treating pulmonary hypertension according to one aspect of the present invention contains an inhibitor that inhibits the function of CD49b.

Effects of the Invention

[0009] According to one aspect of the present invention, a composition for treating pulmonary hypertension having a novel mechanism of action can be provided.

Brief Description of the Drawings

[0010]

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Mode for Carrying Out the Invention

[0011] Hereinafter, an example of an embodiment of the present invention will be described in detail, but the present invention is not limited thereto.

[0012] Unless otherwise specified in this specification, "A to B" representing a numerical range means "A or more and B or less".

[0013] In this specification, "gene" is intended to mean a polymer of nucleotides and is used synonymously with "polynucleotide", "nucleic acid" or "nucleic acid molecule". A gene may exist in the form of DNA (for example, cDNA or genomic DNA) or in the form of RNA (for example, mRNA). DNA or RNA may be double-stranded or single-stranded. Single-stranded DNA or RNA may be a coding strand (sense strand) or a non-coding strand (antisense strand). A gene may be chemically synthesized. Also, when "polynucleotide" is described in this specification, it may be DNA or RNA.

[0014] In this specification, "protein" is used synonymously with "peptide" or "polypeptide".

[0015] In this specification, the notations of bases and amino acids appropriately use the one-letter notation or three-letter notation defined by IUPAC and IUB.

[0016] [1. Summary of the Invention] Pulmonary hypertension is caused by an increase in pulmonary artery pressure due to, for example, constriction of the pulmonary artery. According to the treatment guidelines for pulmonary hypertension, when it is confirmed by right heart catheterization that the mean pulmonary artery pressure is 25 mmHg or higher, it is diagnosed as pulmonary hypertension. When pulmonary hypertension becomes chronic, for example, vascular stenosis associated with remodeling of the pulmonary peripheral blood vessels progresses. The chronic high pulmonary artery pressure increases the right ventricular load, leading to right ventricular failure. As a result, clinical symptoms such as systemic congestion, shortness of breath, easy fatigue, reduced work capacity, fainting attacks, ascites or cyanosis are caused.

[0017] Examples of treatment methods for pulmonary hypertension include administration of drugs such as prostaglandin I2 derivative preparations or calcium antagonists, vasodilator therapy with nitric oxide (NO) gas, oxygen therapy, prevention measures for heart failure, anticoagulant therapy, and surgeries such as lung transplantation.

[0018] In the study of pulmonary hypertension, a plurality of useful animal models are known. Examples of animal models include an interleukin-33 (IL-33) administration model to rodents (Japanese Patent Laid-Open No. 2015-50941), a gene-modified model in which a specific molecule is knocked out or expressed in rodents (Medoff et al., Am J.Resp. Cell Mol.Biol., 2009; 41: 397-406). Such animal models are used for pathological analysis of pulmonary hypertension and exploration of treatment methods.

[0019] The present inventors have first clarified that the integrin complex (CD49b / CD29) expressed on type 2 innate lymphocytes plays an important role in the pathological mechanism of pulmonary hypertension. And the present inventors have found that by inhibiting cell adhesion via the integrin complex (CD49b / CD29), the progression of the pathological condition in pulmonary hypertension can be reduced or prevented, and pulmonary hypertension can be effectively treated.

[0020] [2. Composition for the Treatment of Pulmonary Hypertension] (2-1. Specific Inhibition of CD49b) A composition for treating pulmonary hypertension according to one aspect of the present invention contains an inhibitor that inhibits the function of CD49b.

[0021] Pulmonary hypertension may be primary or secondary as long as it shows a pathological condition with increased pulmonary artery pressure, and may be complicated with other diseases. Further, as for secondary pulmonary hypertension, it may be either the case where there is an underlying disease or the case where it is diagnosed simultaneously with the underlying disease.

[0022] As for primary pulmonary hypertension, pulmonary arterial hypertension (PAH) is cited as a central disease. Pulmonary arterial hypertension is caused by various factors and is classified into idiopathic pulmonary arterial hypertension (ideopathic PAH), heritable pulmonary arterial hypertension (heritable PAH), drug / toxicant-induced pulmonary arterial hypertension, and pulmonary arterial hypertension associated with connective tissue disease, HIV infection, congenital heart disease, etc.

[0023] As for secondary pulmonary hypertension, pulmonary hypertension associated with left-sided heart diseases such as left ventricular failure, valvular disease, and congenital heart disease; pulmonary hypertension associated with chronic obstructive pulmonary disease (COPD), interstitial pneumonia, emphysema, sleep disordered breathing, alveolar hypoventilation disorder, and growth disorder; and pulmonary hypertension associated with chronic thromboembolic / embolic hypertension, chronic hemolytic anemia, myeloproliferative diseases, sarcoidosis, pulmonary Langerhans cell histiocytosis, lymphangioleiomyomatosis, vasculitis, glycogenosis, Gaucher disease, thyroid diseases, and chronic renal insufficiency, etc. are cited.

[0024] As used herein, "treatment" means performing an act that brings a therapeutic effect to a subject in need of treatment or potentially in need of treatment. The therapeutic effect includes a preventive effect and may be, for example, the following effects; (1) Preventing the onset of one or more symptoms related to a disease or reducing the risk as compared with the case where no drug is administered, (2) Preventing or reducing the recurrence of one or more symptoms related to the disease as compared to the case where the agent is not administered. (3) Preventing or reducing the occurrence of signs of one or more symptoms related to the disease as compared to the case where the agent is not administered. (4) Reducing the severity of one or more symptoms related to the disease as compared to the case where the agent is not administered. (5) Preventing the increase or progression of the severity of one or more symptoms related to the disease as compared to the case where the agent is not administered. (6) Reducing the rate of increase or progression of the severity of one or more symptoms related to the disease as compared to the case where the agent is not administered.

[0025] The therapeutic composition according to one aspect of the present invention may contain, as an active ingredient, other active ingredients other than an inhibitor that inhibits the function of CD49b. Such other active ingredients are preferably those that can provide a more effective therapeutic effect when combined with the inhibitor than when the inhibitor is used alone. The other active ingredients contained in the therapeutic composition according to one aspect of the present invention may be one kind or a plurality of kinds.

[0026] In addition, the therapeutic composition according to one aspect of the present invention may contain a pharmaceutically acceptable carrier, diluent, excipient, or the like. Pharmaceutically acceptable carriers, diluents, excipients, etc. are well known in the pharmaceutical field and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (edited by A.R. Gennaro, 1985). The selection of a pharmaceutically acceptable carrier, diluent, or excipient can be easily selected by those skilled in the art according to the administration route of the agent and standard pharmaceutical practices. Further, the therapeutic composition according to one aspect of the present invention may further contain a binder, lubricant, suspending agent, coating agent, solubilizing agent, or the like.

[0027] The therapeutic composition according to one aspect of the present invention is administered by any administration route effective in treatment. The therapeutic composition according to one aspect of the present invention can be administered, for example, orally, intramuscularly, intravenously, subcutaneously, intraperitoneally or transdermally to a subject to be treated. The dosage form may be, for example, an injection, a capsule, a tablet or a granule.

[0028] The subject of treatment with the therapeutic composition according to one aspect of the present invention is not particularly limited as long as it is a subject in need of treatment or potentially in need of treatment. The subject of treatment with the therapeutic composition is, for example, a mammal, such as a human, a mouse, a guinea pig, a hamster, a rat, a mouse, a rabbit, a pig, a sheep, a goat, a cow, a horse, a cat, a dog, a marmoset, a monkey and a chimpanzee.

[0029] The method of administering the therapeutic composition according to one aspect of the present invention is not particularly limited and can be carried out using any method used in the art. The formulation of the therapeutic composition according to one aspect of the present invention can be appropriately set by those skilled in the art according to the severity of the disease, the active ingredient or the administration route.

[0030] CD49b is one of the α subunits of the integrin complex and is known as the α2 subunit. CD49b may also be referred to as GPIa, ITGA2, HPA-5 or VLAA2.

[0031] As the α subunit of the integrin complex, 18 types such as α1, α10 and α11 in addition to CD49b (α2) are known, and their expression sites and the like are different. The present inventors discovered that by administering IL-33 to mice, the expression level of CD49b (α2) increases in type 2 natural lymphocytes in lung tissue, but the expression levels of other α subunits (for example, α1, α10, α11) do not increase.

[0032] The integrin complex promotes the accumulation of type 2 innate lymphoid cells in the pulmonary artery by means of their cell adhesion function. Specifically, the integrin complex expressed in the extracellular region of type 2 innate lymphoid cells interacts with the extracellular matrix such as collagen, fibronectin, and laminin, and also interacts with adhesion molecules such as ICAM (IntraCellular Adhesion Molecule) and VCAM (Vascular Cell Adhesion Molecule) on vascular endothelial cells. Administration of IL-33 is thought to promote the accumulation of the extracellular matrix such as collagen around the pulmonary artery and thus promote the accumulation of type 2 innate lymphoid cells.

[0033] Also, as described above, the IL-33 administration model in which IL-33 is administered to mice is known to be useful for pathological analysis of pulmonary hypertension. Type 2 innate lymphoid cells are known to cause pulmonary artery thickening, etc. by promoting the migration of IL-5R-expressing cells such as eosinophils around the pulmonary artery by secreting IL-5 (Patent Document 1).

[0034] In type 2 innate lymphoid cells accumulated in such an IL-33 administration model, CD49b is specifically expressed as the α subunit of the integrin complex. This indicates that by specifically inhibiting CD49b, it is possible to treat pulmonary hypertension by specifically targeting CD49b-positive type 2 innate lymphoid cells, which are considered to be the cause of inducing pulmonary hypertension. On the other hand, CD49b-negative type 2 innate lymphoid cells are involved in parasite infection defense or tissue repair, etc. The finding that targeting CD49b suggests that side effects are less likely to occur because it does not affect such CD49b-negative type 2 innate lymphoid cells.

[0035] That is, the present inventors newly found that by specifically inhibiting CD49b, it is possible to expect a therapeutic effect on pulmonary hypertension while reducing side effects. And it was demonstrated that inhibition of CD49b can reduce pulmonary artery vascular hypertrophy in the IL-33 administration model. In addition, it was also found that inhibition of CD49b reduces the accumulation of type 2 innate lymphocytes in lung tissue, but has a smaller effect on eosinophils in lung tissue and type 2 innate lymphocytes in bone marrow than inhibition of CD29, which is the β subunit of the integrin complex.

[0036] Thus, according to one aspect of the present invention, a novel therapeutic composition for pulmonary hypertension targeting CD49b of the integrin complex (CD49b / CD29) can be provided. The therapeutic composition according to one aspect of the present invention contains an inhibitor that inhibits the function of CD49b, thereby inhibiting the cell adhesion of type 2 innate lymphocytes via the integrin complex (CD49b / CD29). Thereby, the proliferation of vascular smooth muscle cells in the pulmonary artery and pulmonary vascular remodeling can be reduced, and pulmonary hypertension can be effectively treated.

[0037] According to a therapeutic composition or the like having such a novel mechanism of action, the treatment options for pulmonary hypertension increase, so that effective treatment can be provided to more patients. In addition, it becomes possible to treat pulmonary hypertension with mild side effects. Such effects also contribute to the achievement of, for example, Goal 3 of the Sustainable Development Goals (SDGs) advocated by the United Nations, "Ensure healthy lives and promote well-being for all."

[0038] (2-2. Inhibitor of CD49b) The inhibitor that inhibits the function of CD49b is not particularly limited, and may be, for example, an inhibitor that inhibits the function of promoting cell adhesion by the integrin complex. More specifically, it may be an inhibitor that inhibits the function of promoting the cell adhesion activity of type 2 innate lymphocytes in lung tissue by CD49b.

[0039] It is considered that the progenitor cells of type 2 natural lymphocytes present in the bone marrow mature and migrate to the lung tissue via the blood when they are under the long-term influence of IL-33. The present inventors newly constructed a hypothesis that type 2 natural lymphocytes that have migrated to the lung tissue accumulate in the pulmonary artery due to cell adhesion activity via the integrin complex and secrete IL-5 to cause an inflammatory reaction, and conducted intensive studies. Inhibition of cell adhesion activity by CD49b reduces the accumulation of such type 2 natural lymphocytes in the lung tissue, thus reducing the inflammatory reaction in the pulmonary artery, making it difficult for pulmonary artery thickening to occur, and also expecting improvement of pulmonary artery thickening.

[0040] Examples of the inhibitor that inhibits the function of CD49b include an antibody or an antibody fragment that binds to the extracellular region of CD49b. In order to inhibit the function of CD49b, which is the function of the integrin complex containing CD49b, it is preferable that it is an antibody or an antibody fragment that binds to the extracellular region of CD49b. The inhibition mechanism is not particularly limited, but is considered to be due to the following reasons, for example.

[0041] Both the α subunit and the β subunit of the integrin complex interact with each other in the extracellular region to form a complex. An antibody or an antibody fragment that binds to the extracellular region of CD49b can inhibit the cell adhesion function of the integrin complex by inhibiting such interaction.

[0042] In addition, the integrin complex binds to a ligand such as the extracellular matrix in the extracellular region. An antibody or an antibody fragment that binds to the extracellular region of CD49b can inhibit the cell adhesion function of the integrin complex by inhibiting the binding between the integrin complex and the ligand.

[0043] In addition, it is known that the integrin complex transitions from a bent form, which is an inactive state where a ligand cannot bind, to an extended form, which is an active state where a ligand can bind, in the extracellular region upon activation. An antibody or antibody fragment that binds to the extracellular region of CD49b can inhibit the cell adhesion function of the integrin complex by physically inhibiting such a transition to the active state.

[0044] The antibody or antibody fragment may be, for example, one that specifically binds to the extracellular region of human CD49b. "Specifically binds" means binding only to the target and not to other substances. "Specifically binds" also includes preferentially binding only to the target.

[0045] Further, the antibody or antibody fragment may be one that specifically binds to the extracellular region of CD49b derived from multiple types of mammals including humans. Thus, the antibody or antibody fragment that binds to the extracellular region of CD49b may have cross-reactivity.

[0046] Cross-reactivity is a general term for the property that an antibody or antibody fragment has a significant binding affinity for two or more antigens having similar structures. Here, antigens having similar structures include proteins with high homology.

[0047] Examples of mammals included in the range of cross-reactivity of the antibody or antibody fragment include humans, mice, guinea pigs, hamsters, rats, mice, rabbits, pigs, sheep, goats, cows, horses, cats, dogs, marmosets, monkeys, and chimpanzees. Preferably, they are humans, mice, rats, or monkeys, and more preferably, humans or mice.

[0048] The extracellular region of CD49b to which the antibody or antibody fragment binds may be, for example, a region comprising at least the 30th to 1132nd amino acid residues of the amino acid sequence of human CD49b shown in SEQ ID NO: 1. Further, the extracellular region may be a region comprising at least the 27th to 1129th amino acid residues of the amino acid sequence of mouse CD49b shown in SEQ ID NO: 2.

[0049] Examples of antibodies that inhibit the function of CD49b include, for example, the anti-mouse CD49b antibody identified by the clone name HMα. The anti-mouse CD49b antibody is a hamster IgG antibody and is a monoclonal antibody. The anti-mouse CD49b antibody is commercially available and can be easily obtained, for example, from eBioscience (Cat#16-0491-85) and abcam (Cat#ab253088), etc.

[0050] The antibody according to one aspect of the present invention may be either a monoclonal antibody or a polyclonal antibody, but is preferably a monoclonal antibody that binds to a single epitope. The monoclonal antibody may be a monoclonal antibody produced from a hybridoma or a recombinant antibody produced by genetic recombination technology.

[0051] The antibody according to one aspect of the present invention may be an antibody comprising at least a part of the constant region of a human antibody in order to reduce immunogenicity in humans. A part of the constant region may be, for example, the Fc region (Fragment crystallizable region).

[0052] Examples of antibodies comprising at least a part of the constant region of a human antibody include recombinant antibodies such as humanized chimeric antibodies (hereinafter, "chimeric antibodies"), humanized antibodies, and human antibodies. Humanized antibodies are also referred to as humanized CDR (Complementarity Determining Region) grafted antibodies.

[0053] Examples of chimeric antibodies include antibodies consisting of the variable region (V region) of an antibody from an animal other than human and the constant region (C region) of a human antibody. The animal species for the C region is not particularly limited as long as it is an animal capable of producing hybridomas such as mice, rats, hamsters, or rabbits.

[0054] For example, a humanized chimeric antibody can be prepared by inserting cDNA encoding the V region of an antibody from an animal other than human that binds to human CD49b into an expression vector having a gene encoding the C region of a human antibody, and introducing the resulting expression vector into animal cells for expression. The C region of the humanized chimeric antibody is not particularly limited as long as it is human immunoglobulin (hIg), but those of the hIgG class are preferred.

[0055] A humanized antibody is an antibody in which the CDR sequences of an antibody from an animal other than human are transplanted to appropriate positions in the V region of a human antibody. A humanized antibody can be realized by obtaining cDNA encoding a V region in which the CDRs of an antibody from an animal other than human that specifically binds to CD49b are transplanted into the framework (FR) of the V region of an arbitrary human antibody. The cDNA may be inserted into an expression vector having a gene encoding the C region of a human antibody, and the resulting expression vector may be introduced into animal cells to express the humanized antibody.

[0056] The amino acid sequence of the FR of the V region of a human antibody is not particularly limited as long as it is an amino acid sequence derived from a human antibody. The C region of a human antibody introduced into a humanized antibody is not particularly limited as long as it is hIg, but those of the hIgG class are preferred.

[0057] The antibody fragment according to one aspect of the present invention is a fragment of each of the above antibodies, and is not particularly limited as long as it binds to CD49b and inhibits the function of CD49b. Examples of the types of antibody fragments include Fab, Fab’, F(ab’)2, scFv, diabody, dsFv, and peptides containing CDRs.

[0058] Fab is an antibody fragment with an antigen-binding activity having a molecular weight of about 50,000 among the fragments obtained by treating IgG with papain (a proteolytic enzyme). The Fab of the anti-CD49b antibody can be prepared, for example, by treating the anti-CD49b antibody with papain or by inserting the cDNA encoding the Fab of the antibody into an expression vector, introducing the obtained vector into prokaryotic or eukaryotic cells, and expressing it.

[0059] F(ab’)2 is an antibody fragment with an antigen-binding activity having a molecular weight of about 100,000 among the fragments obtained by treating IgG with pepsin (a proteolytic enzyme). The F(ab’)2 of the anti-CD49b antibody can be prepared, for example, by treating the anti-CD49b antibody with pepsin or by binding Fab’ (described later) with a thioether bond or a disulfide bond.

[0060] Fab’ is an antibody fragment with an antigen-binding activity having a molecular weight of about 50,000 obtained by cleaving the disulfide bond in the hinge region of F(ab’)2. The Fab’ of the anti-CD49b antibody can be obtained, for example, by treating the F(ab’)2 of the anti-CD49b antibody with dithiothreitol. Alternatively, it can be prepared by inserting the cDNA encoding the Fab’ of the anti-CD49b antibody into an expression vector, introducing the obtained vector into prokaryotic or eukaryotic cells, and expressing it.

[0061] scFv is an antibody fragment with an antigen-binding activity obtained by linking one heavy chain variable region (VH) and one light chain variable region (VL) using an appropriate peptide linker. The scFv of the anti-CD49b antibody can be realized, for example, by obtaining the cDNA encoding VH and VL of the anti-CD49b antibody. DNA encoding scFv is constructed from the obtained cDNA, inserted into an expression vector, and introduced into prokaryotes or eukaryotes for expression to produce scFV.

[0062] A diabody is an antibody fragment in which scFv dimerizes and is an antibody fragment having bivalent antigen-binding activity. The diabody of the anti-CD49b antibody can be realized by obtaining the cDNA encoding the VH and VL of the anti-CD49b antibody. A DNA encoding the diabody is constructed from the obtained cDNA, inserted into an expression vector, and the diabody can be produced by introducing this expression vector into a prokaryote or eukaryote for expression.

[0063] dsFv is an antibody fragment in which a polypeptide with one amino acid residue in each of VH and VL replaced by a cysteine residue is bound via a disulfide bond between the cysteine residues. The dsFv of the anti-CD49b antibody can be realized by obtaining the cDNA encoding the VH and VL of the anti-CD49b antibody. A DNA encoding the dsFv is constructed from the obtained cDNA, inserted into an expression vector, and the dsFv can be produced by introducing this expression vector into a prokaryote or eukaryote for expression.

[0064] A peptide containing a CDR is a peptide containing at least one region or more of the CDRs of VH or VL. A peptide containing the CDRs of the anti-CD49b antibody can be produced by constructing a DNA encoding the CDRs of VH and VL of the anti-CD49b antibody, inserting the DNA into an expression vector, and introducing this expression vector into a prokaryote or eukaryote for expression. Also, a peptide containing the CDRs of the anti-CD49b antibody can be produced by a chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarbonyl method) or the Boc method (t-butyloxycarbonyl method). Preferably, a peptide containing six CDR regions derived from the anti-CD49b antibody can be mentioned.

[0065] In addition, the inhibitor that inhibits the function of CD49b is not limited to an antibody or an antibody fragment. The inhibitor may be, for example, a substance that binds to the extracellular region of CD49b (e.g., a peptide containing a binding sequence other than the antibody CDR, a low molecular weight compound, an aptamer, etc.), a substance that reduces the expression of the gene encoding CD49b (e.g., an antisense oligonucleotide, siRNA, shRNA, etc.), a substance that inhibits the binding of an integrin complex containing CD49b to a ligand such as the extracellular matrix (e.g., an integrin complex antagonist), and the like.

[0066] Examples of the gene encoding CD49b include, but are not limited to, the human Itga2 gene consisting of the nucleotide sequence shown in SEQ ID NO: 3, the mouse Itga2 gene consisting of the nucleotide sequence shown in SEQ ID NO: 4, etc. Information on the nucleotide sequences of Itga2 genes derived from various organisms can be obtained from known databases such as GenBank.

[0067] [3. Method for treating pulmonary hypertension] The method for treating pulmonary hypertension according to one aspect of the present invention is a method including administration of an inhibitor that inhibits the function of CD49b. The inhibitor that inhibits the function of CD49b is effective for the treatment of pulmonary hypertension by reducing vascular hypertrophy in the pulmonary artery as described above.

[0068] Administration of the inhibitor that inhibits the function of CD49b is preferably performed by administration of a composition for treating pulmonary hypertension containing the inhibitor. Regarding specific administration methods and administration targets, the description in [1. Composition for treating pulmonary hypertension] is applicable, and thus the description is omitted here.

[0069] [4. Summary] The composition for treating pulmonary hypertension according to Aspect 1 of the present invention contains an inhibitor that inhibits the function of CD49b.

[0070] The composition for treating pulmonary hypertension according to Aspect 2 of the present invention may be such that, in the above Aspect 1, the inhibitor is an antibody that binds to the extracellular region of CD49b or a fragment of the antibody.

[0071] In the composition for treating pulmonary hypertension according to Aspect 3 of the present invention, in the above Aspect 1 or 2, the function of CD49b may be a function of promoting the cell adhesion activity of type 2 natural lymphocytes derived from bone marrow.

[0072] In the composition for treating pulmonary hypertension according to Aspect 4 of the present invention, in the above Aspect 2, the antibody may include at least a part of the constant region of a human antibody.

[0073] Moreover, the method for treating pulmonary hypertension according to Aspect 5 of the present invention is a method including administration of an inhibitor that inhibits the function of CD49b.

[0074] In the method for treating pulmonary hypertension according to Aspect 6 of the present invention, in the above Aspect 5, the inhibitor may be an antibody that binds to the extracellular region of CD49b or a fragment of the antibody.

[0075] In the method for treating pulmonary hypertension according to Aspect 7 of the present invention, in the above Aspect 5 or 6, the function of CD49b may be a function of promoting the cell adhesion activity of type 2 natural lymphocytes derived from bone marrow.

[0076] In the composition for treating pulmonary hypertension according to Aspect 8 of the present invention, in the above Aspect 6, the antibody may include at least a part of the constant region of a human antibody.

[0077] [5. Supplementary matters] The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope shown in the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention. [Examples]

[0078] One embodiment of the present invention will be described below. Note that the present invention is not limited to the examples shown below.

[0079] [Mechanism of action of CD49b in pulmonary hypertension] Regarding CD49b expressed in group 2 innate lymphoid cells (ILC2), its involvement in pulmonary artery hypertrophy in pulmonary hypertension was investigated.

[0080] As a model of pulmonary hypertension (pulmonary arterial hypertension), an IL-33-administered mouse model was used. This model can be evaluated from Grade 1 to 3 out of the vascular pathological classifications Grade 1 to 4 of pulmonary arterial hypertension. It has a relatively short induction period of about 3 weeks for pulmonary arterial hypertension and is an excellent model in that no special device is required.

[0081] Figure 1 shows the types of mice and the administration schedule of IL-33 to the mice. As shown in Figure 1, 400 ng of recombinant mouse IL-33 (rmIL-33, 3626-ML, R&D systems) dissolved in 100 μL of PBS was administered intraperitoneally to the mice 3 times at 1-week intervals. On the 21st day after the start of administration, the lung tissues of the mice were analyzed.

[0082] Male mice aged 7 - 10 weeks were used. Mice expressing the fluorescent protein tdTomato and Cre in IL-5-producing cells (IL-5 / tdTomato / Cre) were mated with CD49b conditional KO mice (CD49b flox / flox, Itga2 flox(F) / f ) to generate mice in which CD49b is specifically deficient in IL-5-producing cells (IL5 + / tm Itga2 f / f , hereinafter referred to as "deficient mice"). Symmetrically, mice in which CD49b is not specifically deficient in IL-5-producing cells before mating (IL5 + / tm Itga2 + / + , hereinafter referred to as "control mice") were used.

[0083] The IL-5 / tdTomato / Cre mouse is Strain 030926 from the Jackson Laboratory and is reported in the reference (Nussbaum J C, et al. Nature 2013: 502(7470): 245-8.). Also, Itga2 flox(F) / f The mouse is Strain 018921 from the Jackson Laboratory. Itga2 is a gene that encodes CD49b in mice.

[0084] Figure 2 shows the results of evaluating the expression levels of each subunit of the integrin complex in wild-type mice (C57BL / 6J) administered IL-33 according to the schedule in Figure 1. Also, in Figure 2, the results are shown when administration was performed over a short period of 3 days with the administration interval of IL-33 being 1 day instead of 1 week. In Figure 2 and the like, "Lin" indicates the Lineage marker, and in this example, B220, CD3ε, CD4, CD8α, CD11b, CD11c, CD19, FcεRIα, Gr1, NK1.1, and Ter119 are used as the Lineage marker.

[0085] As shown in Figure 2, administration of IL-33 increased the number of CD25-positive cells in ILC2 of the lung tissue (hereinafter, "lung ILC2"). Also, CD29 (β1) had an increased expression level in lung ILC2 due to administration of IL-33. Also, CD49b (α2) was not expressed in the steady-state lung ILC2 without IL-33 administration, but its expression was confirmed in lung ILC2 by administration of IL-33. On the other hand, neither CD49a (α1), α10 nor α11 was expressed in lung ILC2. Thus, among the α subunits of the integrin complex in lung ILC2, only CD49b showed specific expression by administration of IL-33.

[0086] Note that when IL-33 was administered three times over a short period of three days instead of three weeks, no increase in the expression of CD29 was observed, nor was the expression of CD49b observed. Therefore, CD49b is considered to be involved in the pathogenesis of pulmonary hypertension only when it is affected by IL-33 in the long term in the IL-33 administration model. In the following examples, IL-33 was administered long term according to the administration schedule shown in Fig. 1.

[0087] Fig. 3 shows the amounts of pulmonary ILC2 and eosinophils in immune cells in the lung tissue of mice administered with IL-33. As shown in Fig. 3, the amount of pulmonary ILC2 increased by IL-33 administration was decreased in the deficient mice compared to the control mice. Also, the amount of eosinophils increased by IL-33 administration was decreased in the deficient mice compared to the control mice.

[0088] Fig. 4 also shows the amounts of ILC2 progenitor cells in the bone marrow (hereinafter, "bone marrow ILC2") and ILC2 in the blood (hereinafter, "blood ILC2") in mice administered with IL-33. As shown in Fig. 4, both the amounts of bone marrow ILC2 and blood ILC2 increased by IL-33 administration were decreased in the deficient mice compared to the control mice.

[0089] Fig. 5 shows a graph summarizing the results of Figs. 3 and 4. The increase rates of ILC2 in the lung, bone marrow and blood, and eosinophils in the lung tissue by IL-33 administration were all significantly decreased in the deficient mice compared to the control mice.

[0090] Fig. 6 shows the results of evaluating the pathological conditions of hypertrophied blood vessels in the pulmonary arteries of control mice and deficient mice administered with IL-33. The left figure of Fig. 6 shows the stained image by EVG (Elastica van Gieson) staining of the lung tissue section, and the right figure shows the ratio of each pathological classification by tissue evaluation in a graph. As shown in Fig. 6, when administered with IL-33, vascular hypertrophy was reduced in the deficient mice compared to the control mice. Also, in the deficient mice, the ratio of hypertrophy evaluated as particularly severe Grade 3 was greatly decreased in the pathological classification of pulmonary hypertension.

[0091] From the above results, the following hypothesis can be considered. That is, if bone marrow ILC2 is affected by IL-33 in the long term, it migrates to the lungs via the blood, induces vascular hypertrophy, and causes pulmonary hypertension. At this time, the integrin complex having CD49b as the α subunit, which is expressed on the cell surface of lung ILC2, promotes cell adhesion, and lung ILC2 accumulates around the pulmonary artery, causing chronic inflammation. Then, it is considered that eosinophils and the like accumulate due to the secretion of IL-5 by lung ILC2 and the like, promoting the inflammatory reaction and progressing pulmonary artery hypertrophy.

[0092] From the above hypothesis, it is considered that pulmonary hypertension can be treated by inhibiting the function of CD29 and / or CD49b that promotes cell adhesion in lung ILC2.

[0093] [Reduction of Pulmonary Artery Hypertrophy by CD29 or CD49b Inhibition] The effect of CD29 or CD49b inhibition on pulmonary artery hypertrophy was examined. For CD29 inhibition, an anti-CD29 antibody (Clone: HMβ1-1, Biolegend, Cat#102210) was used, and Biolegend's Cat#400933 was used as an isotype control. For CD49b inhibition, an anti-CD49b antibody (Clone: HMα2, eBioscience, Cat#16-0491-85) was used, and eBioscience's Cat#16-4888-85 was used as an isotype control.

[0094] The mice were male, 7 to 10 weeks old, and C57BL / 6J or IL-5 / Venus knock-in mice (Il5 + / v mice) were used. The knock-in mice are reported in the reference (Ikutani M, et al. J Immunol 2012: 188(2): 703-13.).

[0095] In Fig. 7, C57BL / 6J or Il5 + / vShows the administration schedule of IL-33 and anti-CD29 antibody or anti-CD49b antibody to mice. As shown in Figure 7, 400 ng of recombinant mouse IL-33 (rmIL-33, 3626-ML, R&D systems) dissolved in 100 μL of PBS was added to Il5 + / v administered to the peritoneal cavity of mice three times at one-week intervals. On the 21st day after the start of administration, the lung tissue of the mice was analyzed. This is the same administration schedule as in Figure 1.

[0096] In addition, the anti-CD29 antibody or anti-CD49b antibody was dissolved in 100 μL of PBS and intraperitoneally administered five times at 2 days before, 1 day before, on the same day, 7 days after, and 14 days after the first administration of IL-33. The dose of each administration was 50 μg. Also, as a control, an isotype control antibody was administered according to the same schedule as the inhibitory antibody.

[0097] Figure 8 shows the amounts of ILC2 and eosinophils in Il5 + / v mice administered with anti-CD29 antibody. In the IL-33 administration model, ILC2 in the lung tissue and blood decreased due to the administration of anti-CD29 antibody. Also, a decrease was observed in eosinophils in the lung tissue due to the administration of anti-CD29 antibody. On the other hand, the administration of anti-CD29 antibody did not show a significant effect on the amount of bone marrow ILC2.

[0098] Figure 9 shows the results of evaluating the pathological condition of hypertrophied blood vessels in the pulmonary artery of Il5 + / v mice administered with IL-33. The left figure in Figure 9 shows the stained image by EVG staining of the lung tissue section, and the right figure shows the ratio of each pathological classification by tissue evaluation in a graph.

[0099] As shown in Figure 9, in the IL-33 administration model, the hypertrophy of blood vessels was reduced by the administration of anti-CD29 antibody. Also, due to the administration of anti-CD29 antibody, in the pathological classification of blood vessels, especially the ratio of hypertrophy evaluated as severe Grade 3 was significantly decreased. That is, it was shown that the inhibition of CD29 reduces IL-33-induced blood vessel hypertrophy.

[0100] Figure 10 shows the amount of ILC2 and eosinophils in C57BL / 6J mice administered anti-CD49b antibody. In the IL-33 administration model, the amount of ILC2 in the lung tissue and blood was reduced by administration of anti-CD49b antibody. On the other hand, administration of anti-CD49b antibody did not significantly affect the amount of eosinophils in the lung tissue or the amount of ILC2 in the bone marrow.

[0101] This result indicates that CD49b inhibition has less effect on pulmonary ILC2s or blood ILC2s migrating to the lungs than CD29 inhibition, suggesting that specific CD49b inhibition may reduce side effects associated with administration compared to CD29 inhibition.

[0102] FIG. 11 shows the results of the analysis of Il5 mice treated with IL-33. + / v The left panel of Fig. 11 shows the results of evaluating the pathology of vascular thickening in the pulmonary artery of mice. The left panel of Fig. 11 shows the stained images of lung tissue sections stained with EVG, and the right panel shows a graph of the percentage of each pathology classification based on tissue evaluation.

[0103] As shown in Figure 11, in the IL-33 administration model, administration of anti-CD49b antibody reduced vascular thickening. In addition, administration of anti-CD49b antibody reduced the proportion of thickening evaluated as Grade 3, which is particularly severe, in the vascular pathology classification. In other words, it was shown that inhibition of CD49b also reduces IL-33-induced vascular thickening, similar to inhibition of CD29. [Industrial Applicability]

[0104] The present invention can be used to treat pulmonary hypertension.

Claims

**Claim 1** A composition for treating pulmonary hypertension, comprising an inhibitor that inhibits the function of CD49b. **Claim 2** The composition for treating pulmonary hypertension according to claim 1, wherein the inhibitor is an antibody or a fragment of the antibody that binds to the extracellular region of CD49b. **Claim 3** The composition for treating pulmonary hypertension according to claim 1 or 2, wherein the function of CD49b is a function of promoting the cell adhesion activity of type 2 natural lymphocytes derived from bone marrow. **Claim 4** The composition for treating pulmonary hypertension according to claim 2, wherein the antibody comprises at least a part of the constant region of a human antibody.

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