Drug for preventing or treating pulmonary hypertension

Abstract

The present disclosure addresses the problem of providing a drug for preventing or treating pulmonary hypertension, the drug having a mechanism for correcting the transformation of intestinal flora associated with PH.　This drug for preventing or treating pulmonary hypertension comprises an antibody or the like or an antigen-binding fragment thereof, the antibody or the like including: a heavy chain variable region that has a CDR1 containing the amino acid sequence represented by SEQ ID NO: 1, a CDR2 containing the amino acid sequence represented by SEQ ID NO: 2, and a CDR3 containing the amino acid sequence represented by SEQ ID NO: 3; and a light chain variable region that has a CDR1 containing the amino acid sequence represented by SEQ ID NO: 4, a CDR2 containing the amino acid sequence represented by SEQ ID NO: 5, and a CDR3 containing the amino acid sequence represented by SEQ ID NO: 6.

Description

Preventive or therapeutic drugs for pulmonary hypertension

[0001] The present disclosure relates to drugs for the prevention or treatment of pulmonary hypertension (PH).

[0002] Pulmonary hypertension is a group of progressive diseases with a poor prognosis that cause cardiac and pulmonary dysfunction due to elevated pulmonary arterial blood pressure. According to the Nice Classification, pulmonary hypertension is classified into Group 1: pulmonary arterial hypertension (PAH), Group 1': pulmonary veno-occlusive disease (PVOD) and / or pulmonary capillary hemangiomatosis (PCH), Group 1'': persistent pulmonary hypertension of the newborn, Group 2: pulmonary hypertension associated with left heart disease, Group 3: pulmonary hypertension associated with lung disease and / or hypoxemia, Group 4: chronic thromboembolic pulmonary hypertension (CTEPH), and Group 5: pulmonary hypertension associated with an unknown multifactorial mechanism.

[0003] Currently, in Japan, drugs such as endothelin receptor antagonists, phosphodiesterase 5 inhibitors, soluble guanylate cyclase stimulators, and prostaglandin I2 are used to treat PH, and although the overall prognosis is improving, the prognosis for severe and drug-resistant patients remains quite poor.In addition to genetic predisposition, inflammation and second-hit drugs are thought to be important factors in the onset and worsening of PH, but many details remain unknown.

[0004] Previously, Nakaoka et al., the present inventors, conducted metagenomic analysis of fecal DNA from PH patients and healthy individuals and demonstrated that PH patients experience a change in the intestinal microbiota (dysbiosis) characterized by ectopic colonization of oral bacteria such as Streptococcus and an increase in Ruminococcus gnavus (R. gnavus), and that this change in the intestinal microbiota promotes the pathology of PH (Patent Document 1, etc.). Additionally, Shinzo et al., the present inventors, reported an antibody that can bind to Clostridium difficile and is effective in treating inflammatory bowel disease (Patent Document 2, etc.). However, the effect of the antibody reported by Shinzo et al. on the pathology of PH is unknown.

[0005] International Publication No. 2022 / 1167088 International Publication No. 2023 / 277142

[0006] An objective of the present disclosure is to provide a preventive or therapeutic agent for PH that works by correcting alterations in the intestinal flora associated with PH.

[0007] The present inventors conducted extensive research to solve the above-mentioned problems and found that antibodies having specific CDRs can bind to oral bacteria (Streptococcus mutans, Streptococcus pasteurianus, Streptococcus sobrinus, and Streptococcus salivarius) that ectopically colonize the intestines of PH patients and bacteria (Rothia aeria and Ruminococcus gnavus) that increase in the intestinal flora of PH patients, thereby inhibiting the proliferation of these bacteria. Furthermore, the inventors found that oral administration of these antibodies to PH model rats corrects alterations in the intestinal flora and improves the pathological condition of PH. Furthermore, the inventors found that one of the mechanisms by which these antibodies improve the pathological condition of PH is an increase in butyrate-producing bacteria in the intestinal flora, resulting in an increase in the butyrate concentration in the intestinal flora. The present disclosure was completed through further research based on these findings.

[0008] That is, one embodiment of the present disclosure provides the following aspects of the invention: Item 1. A drug for preventing or treating PH, comprising at least one of antibodies and / or antigen-binding fragments thereof selected from the following (A) to (C): (A) an antibody having a heavy chain variable region having CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3; and a light chain variable region having CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6. (B) an antibody having a heavy chain variable region having CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 13, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 14, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 15; and a light chain variable region having CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 16, CDR2 comprising the amino acid sequence R A N , and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 18. (C) An antibody having a heavy chain variable region having CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 23, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 24, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 25; and a light chain variable region having CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 26, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 27, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 28. Item 2. The prophylactic or therapeutic agent for PH according to Item 1, wherein the antibody is an IgA antibody. Item 3. The prophylactic or therapeutic agent for PH according to Item 1 or 2, wherein the antibody is a humanized antibody or a chimeric antibody. Item 4. The prophylactic or therapeutic agent for PH according to any of Items 1 to 3, which is administered orally or enterally. Item 5. The prophylactic or therapeutic agent for PH according to Item 1 or 2, which is used in the prevention or treatment of pulmonary arterial hypertension. Item 6. Use of at least one of the antibodies and / or antigen-binding fragments thereof set forth in (A) to (C) for the manufacture of a prophylactic or therapeutic agent for PH. Item 7. Item 7. A method for preventing or treating PH, comprising administering an effective amount of at least one of the antibodies and / or antigen-binding fragments thereof selected from (A) to (C) above to a person in need of such prevention or treatment. Item 8. At least one of the antibodies and / or antigen-binding fragments thereof selected from (A) to (C) above, for use in a treatment for the prevention or treatment of PH.

[0009] According to one embodiment of the present disclosure, administration of an antibody having specific CDRs corrects alterations in the intestinal flora associated with PH, thereby making it possible to prevent or treat PH.

[0010] Figure 1 shows changes in the intestinal microbiota in PH model rats. Figure 1A shows the procedure for generating PH model rats. The upper panel shows hypoxia challenge, the middle panel shows monocrotaline challenge, and the lower panel shows the procedure for generating PH model rats by Sugen5416 / hypoxia / normoxia challenge (SuHx). Figure 1B shows the beta diversity of the intestinal microbiota in each model rat. Figure 1C shows a bar plot showing the family-level composition of intestinal bacteria in the intestinal microbiota of each model rat. Figure 1B shows that antibiotic administration to PH model rats improves PH pathology. Figure 1A shows the results of evaluating PH pathology (pulmonary arterial media thickening, right ventricular systolic pressure, and right ventricular hypertrophy index) in hypoxia challenge (Hx) model rats given antibiotic cocktail water (Abx) or water (vehicle) ad libitum, and in unstimulated SD rats given water ad libitum (Control). (B) MCT model rats were given antibiotic cocktail water (Abx) or water (vehicle) ad libitum in their drinking water, and the pathology of PH (pulmonary arterial medial thickening, right ventricular systolic pressure, and right ventricular hypertrophy index) was evaluated. (C) SuHx model rats were given antibiotic cocktail water (Abx) or water (vehicle) ad libitum in their drinking water, and the pathology of PH (pulmonary arterial medial thickening, right ventricular systolic pressure, and right ventricular hypertrophy index) was evaluated. Statistics are mean ± standard deviation: ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05. PH model rats (MCT model rats) were given butyrate, propionate, or water (vehicle) ad libitum in their drinking water, and the right ventricular systolic pressure and right ventricular hypertrophy index were measured. (A) Experimental procedure. Figure 1 shows the changes in the intestinal microbiota in PH patients. Figure 2 shows the alpha diversity of the intestinal microbiota in PH patients (PH, 89 cases) and healthy controls (HV, 82 cases) in relation to Faith's PD and Shannon index. Figure 3 shows the beta diversity (unweighted unifrac PCoA) of the intestinal microbiota in PH patients and healthy controls. ****P<0.0001, *P<0.05. Figure 4 shows the changes in the intestinal microbiota in PH patients. Figure 5 shows the changes in the intestinal microbiota in PH patients. Figure 6 shows the changes in the intestinal microbiota in PH patients. Figure 7 shows the changes in the intestinal microbiota in PH patients. Figure 8 shows the changes in the intestinal microbiota in PH patients and healthy controls. Figure 9 shows the changes in the intestinal microbiota in PH patients. Figure 10 shows the changes in the intestinal microbiota in PH patients and healthy controls.(B) Fecal concentrations of acetate, propionate, and butyrate are shown in PH patients and healthy controls. (C) Fecal IgA concentrations are shown in PH patients and healthy controls. Statistics are mean ± standard deviation: ****P<0.0001, **P<0.01, *P<0.05. This figure shows that in PH patients, Ruminococcus gnavus coexists with multiple oral bacteria in the intestinal network, which is associated with poor prognosis. (A) Network diagram of bacteria correlated with Ruminococcus gnavus, created using the results of a compositional analysis of the intestinal microbiota of PH patients. (B) Kaplan-Meier analysis of event-free survival in PH patients classified by the abundance of Ruminococcus gnavus and Streptococcus, Rothia, Fusobacterium, or Veillonella in the intestine. In (B), clinical events were defined as death, lung transplantation, and hospitalization due to right heart failure. Figure 1 shows changes in PH pathology in gnotobiotic PH model rats. (A) A diagram showing the method for generating gnotobiotic PH model rats. (B) Analysis of the intestinal microbiota composition in F344 rats transplanted with feces from PH patients and healthy controls. (C) Measurement of right ventricular systolic pressure and right ventricular hypertrophy index in F344 rats transplanted with feces from PH patients (MCT-PH), healthy controls (MCT-HV), germ-free F344 rats (MCT-GF), and specific-pathogen-free F344 rats (MCT-SPF). Statistics are mean ± standard deviation: **P<0.01, *P<0.05. (A) A volcano plot showing the comparison of RNA expression in the lungs of F344 rats transplanted with feces from PH patients and healthy controls. B shows the results of Gene Ontology Term analysis of RNA expression in the lung. C shows a bar plot of the differences in expression of individual genes related to B. This figure shows that Ruminococcus gnavus worsens PH through cooperation with Streptococcus pasteurianus.Figure 1 shows the protocol for an experiment in which Ruminococcus gnavus and Streptococcus pasteurianus were administered to germ-free rats. Figure 2 shows the results of measurements of right ventricular systolic pressure and right ventricular hypertrophy index. Figure 3 shows the results of an experiment in which vancomycin was administered to fecal transplanted rats from PH patients. Figure 1 shows the experimental procedure. Figure 2 shows the results of measurements of right ventricular systolic pressure and right ventricular hypertrophy index in PH patient fecal transplanted rats administered vancomycin (PH-FMT-VCM) and in PH patient fecal transplanted rats administered water (vehicle) (PH-FMT). Statistics are mean ± standard deviation: **P<0.01, *P<0.05. The relative abundance of Streptococcus pasteurianus, Ruminococcus gnavus, Eggerthella lenta, Klebsiella pneumoniae, Escherichia coli, and Fusobacterium ulcerans in the intestinal microbiota of healthy donor fecal-transplanted F344 rats (HV), vancomycin-treated PH patient fecal-transplanted rats (PH-FMT-VCM), and water (vehicle)-treated PH patient fecal-transplanted rats (PH-FMT). Statistics are means ± standard deviations. The binding of various IgA antibodies to Streptococcus mutans, Streptococcus pasteurianus, Streptococcus sobrinus, Streptococcus salivarius, Rothia aeria, and Ruminococcus gnavus was measured. NC indicates the negative control (no IgA antibody added). Figure 1 shows the results of measuring the binding of rW27 antibody to Streptococcus pasteurianus and Rothia aeria. The number of bacteria in the absence of rW27 antibody (-) is set to 100%, and the percentage of the number of bacteria in the presence of rW27 antibody (+) is shown as the growth rate (%). MCT model rats were administered with W27 antibody, W37 antibody, or PBS (vehicle), and the pathological condition of PH was evaluated. Figure 2 shows the experimental protocol. Figure 3 shows the results of measuring the mean arterial count, right ventricular systolic pressure, and right ventricular hypertrophy index.(C) shows the results of a family-level analysis of the composition of the intestinal microbiota in MCT model rats administered with W27 antibody or PBS (vehicle). (D) shows the results of a family-level analysis of the composition of the intestinal microbiota in MCT model rats administered with W37 antibody or PBS (vehicle). (A) shows the experimental protocol. (B) shows the results of measuring right ventricular systolic pressure and right ventricular hypertrophy index. (C) shows the results of analyzing the composition of the intestinal microbiota and determining the relative abundance of Streptococcus bacteria. (D) shows the results of measuring fecal butyric acid concentration. Statistics are mean ± standard deviation: **P<0.01, *P<0.05. (A) shows the results of evaluating the alteration of the intestinal microbiota in PH model gnotobiotic rats administered with W27 antibody. (B) Principal component analysis (PCA) results of the intestinal microbiota. (C) Partial least squares discriminant analysis (PLS-DA) results of the intestinal microbiota. (D) Volcano plot comparing the bacteria constituting the intestinal microbiota between the W27 antibody-treated group and the vehicle group. PH model gnotobiotic rats were administered rW27 antibody or PGSI1A antibody, and PH pathology was evaluated. (A) Experimental protocol. (B) Right ventricular systolic pressure and right ventricular hypertrophy index were measured. (C) Analysis of the intestinal microbiota composition, dividing the bacteria into those promoting PH pathology and those suppressing PH pathology, and calculating the relative frequency. Statistics are mean ± standard deviation: ***P<0.001, **P<0.01, *P<0.05. PH model gnotobiotic rats were administered rW27 antibody or PGSI1A antibody, and PH pathology was evaluated. Figure A shows the experimental protocol. Figure B shows the results of a principal component analysis (PCA) of the intestinal microbiota. Figure C shows the results of a partial least squares discriminant analysis (PLS-DA) of the intestinal microbiota. Figure D shows a violin plot showing the relative abundance (Log10 values ​​of relative frequencies) of bacteria of the genera Fusobacterium, Eggherthella, Enterococcus, Coprococcus, and Anaerostipes.rW27 antibody or 3H12LJ antibody (control antibody) was administered to gnotobiotic rats as a model for PH, and the pathological condition of PH was evaluated. (A) A diagram shows the experimental protocol. (B) Measurement results of right ventricular systolic pressure and right ventricular hypertrophy index. (C) Analysis of the composition of the intestinal microbiota to determine the relative abundance of bacteria that promote PH pathology (Streptococcus spp. and Ruminococcus gnavus) and bacteria that suppress PH pathology (Subdolignulum spp.). Statistics are mean ± standard deviation: **P<0.01, *P<0.05. rW27 antibody or 3H12LJ antibody (control antibody) was administered to gnotobiotic rats as a model for PH, and the pathological condition of PH was evaluated. (A) A diagram shows the experimental protocol. (B) Principal component analysis (PCA) results of the intestinal microbiota. C shows the results of partial least squares discriminant analysis (PLS-DA) of the intestinal microbiota. D shows a volcano plot comparing the bacteria that make up the intestinal microbiota between the rW27 antibody-administered group and the 3H12LJ antibody-administered group. E shows the results of measuring fecal butyric acid concentrations. The percentage of bacteria that bind to the rW27 antibody was measured by FACS using stool samples from PH patients and healthy individuals.

[0011] 1. Definitions Unless otherwise specified, terms used herein have the meanings that are commonly understood by those skilled in the art of medicine, pharmacology, molecular biology, microbiology, organic chemistry, etc. When a term defined herein does not have the same meaning as commonly understood, the description in this specification takes precedence.

[0012] In the present disclosure, "pulmonary hypertension (PH)" refers to a group of progressive diseases with poor prognosis that cause cardiac and pulmonary dysfunction due to elevated pulmonary artery blood pressure, specifically a condition in which the mean pulmonary artery pressure (PAP) measured at rest using right heart catheterization is 20 mmHg or higher. In one embodiment of the present disclosure, a diagnostic criterion for PH may be a mean pulmonary artery pressure of 25 mmHg or higher. According to the Nice Classification, PH is classified into the following groups: Group 1: pulmonary arterial hypertension (PAH); Group 1': pulmonary veno-occlusive disease (PVOD) and / or pulmonary capillary hemangiomatosis (PCH); Group 1'': persistent pulmonary hypertension of the newborn; Group 2: pulmonary hypertension associated with left heart disease; Group 3: pulmonary hypertension associated with lung disease and / or hypoxemia; Group 4: chronic thromboembolic pulmonary hypertension; and Group 5: pulmonary hypertension associated with an unknown multifactorial mechanism.

[0013] In this disclosure, "prevention" refers to inhibiting the onset of or prolonging the time until onset of a disease or condition, and "treatment" refers to alleviating, ameliorating, or slowing the rate of progression of a disease or condition.

[0014] In the present disclosure, an "antibody" refers to a molecule that binds to a specific antigen. Antibodies include heavy and light chains. The heavy chain includes a heavy chain variable region and a heavy chain constant region. The light chain includes a light chain variable region and a light chain constant region. The heavy and light chain variable regions each contain, from the N-terminus, CDR1, CDR2, and CDR3 as complementarity determining regions (CDRs) that recognize antigens. In the heavy and light chain variable regions, the CDRs are structurally supported by a region called the framework, except for CDR2. Antibodies may have two heavy chains and two light chains, or may be composed of one heavy chain and one light chain (sometimes referred to as a single-chain antibody). Antibodies are classified into isotypes, such as IgA, IgG, IgE, IgM, and IgY, which are further classified into subclasses, such as IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4.

[0015] In the present disclosure, the term "antigen-binding fragment" of an antibody refers to an antibody fragment that retains the ability to bind to an antigen. Antigen-binding fragments of antibodies include a Fab fragment consisting of a light chain variable region, a light chain constant region, a heavy chain variable region, and a CH1 domain that is part of the heavy chain variable region; a Fab' fragment consisting of a light chain variable region, a light chain constant region, a heavy chain variable region, a CH1 domain that is part of the heavy chain variable region, and a hinge region; a F(ab')2 fragment in which two Fab' fragments are linked by a disulfide bridge at the hinge region; and an Fv fragment consisting of a heavy chain variable region and a light chain variable region.

[0016] In the present disclosure, a "humanized antibody" is an antibody in which a non-human CDR sequence has been grafted onto the framework region of a human antibody, and is composed of a non-human antibody CDR, a human antibody framework region, and a human antibody constant region.

[0017] In the present disclosure, a "chimeric antibody" is an antibody composed of a variable region of one species and a constant region of another species. Chimeric antibodies include antibodies composed of variable regions of non-human origin and constant regions of human origin.

[0018] In this disclosure, amino acid residues in amino acid sequences are represented by single letter codes. Furthermore, in this disclosure, unless otherwise specified, the left side of an amino acid sequence represents the N-terminus and the right side represents the C-terminus.

[0019] In the present disclosure, the "sequence identity" of an amino acid sequence refers to the degree of agreement between the amino acid sequences of two or more comparable amino acid sequences. Sequence identity can be determined using the sequence analysis tool FASTA with default parameters. Alternatively, it can be determined using the BLAST algorithm by Karlin and Altschul (Karlin S, Altschul SF. "Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes," Proc Natl Acad Sci USA. 87:2264-2268 (1990); Karlin S, Altschul SF. "Applications and statistics for multiple high-scoring segments in molecular sequences," Proc Natl Acad Sci USA. 90:5873-7 (1993)). A program called BLASTX has been developed based on the BLAST algorithm. Specific techniques for these analysis methods are publicly known, and can be found on the National Center of Biotechnology Information (NCBI) website (http: / / www.ncbi.nlm.nih.gov / ).

[0020] In the present disclosure, a "conservative substitution" in an amino acid sequence refers to the substitution of an amino acid residue with an amino acid residue having a similar side chain. For example, substitutions between amino acid residues having basic side chains such as lysine, arginine, and histidine constitute a conservative substitution. Conservative substitutions also include substitutions between amino acid residues having acidic side chains such as aspartic acid and glutamic acid; amino acid residues having uncharged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; amino acid residues having nonpolar side chains such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; amino acid residues having β-branched side chains such as threonine, valine, and isoleucine; and amino acid residues having aromatic side chains such as tyrosine, phenylalanine, tryptophan, and histidine.

[0021] 2. Prophylactic or therapeutic drug for PH In one embodiment of the present disclosure, a prophylactic or therapeutic drug for PH is provided, comprising at least one antibody and / or antigen-binding fragment thereof selected from the following (A) to (C). The prophylactic or therapeutic drug for PH of the present disclosure is described in detail below.

[0022] [Antibodies and Antigen-Binding Fragments Thereof] In one embodiment of the preventive or therapeutic drug for PH disclosed herein, any of the monoclonal antibodies (A) to (C) below is used as an active ingredient for preventing or treating PH. (A) An antibody having a heavy chain variable region having CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3; and a light chain variable region having CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6 (hereinafter, this may be referred to as "antibody (A)"). (B) An antibody having a heavy chain variable region having CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 13, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 14, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 15; and a light chain variable region having CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 16, CDR2 comprising the amino acid sequence R A N , and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 18 (hereinafter, this may be referred to as "antibody (B)"). (C) An antibody having a heavy chain variable region having CDR1 comprising the amino acid sequence shown in SEQ ID NO: 23, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 24, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 25; and a light chain variable region having CDR1 comprising the amino acid sequence shown in SEQ ID NO: 26, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 27, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 28 (hereinafter, sometimes referred to as "antibody (C)").

[0023] Antibody (A) is an antibody having the same CDRs as the mouse W27 antibody and rW27 antibody used in the Examples described later. Antibody (B) is an antibody having the same CDRs as the mouse PGSI1A antibody used in the Examples described later. Antibody (C) is an antibody having the same CDRs as the mouse W37 antibody used in the Examples described later. Antibodies (A) to (C) have all been discovered as monoclonal antibodies that bind to Clostridium difficile.

[0024] A preferred embodiment of antibodies (A) to (C) is a humanized antibody. Humanized antibodies (A) to (C) may be composed of CDRs containing a predetermined amino acid sequence, a framework region derived from a human antibody, and a constant region derived from a human antibody. Known framework regions and constant regions derived from human antibodies can be used for humanized antibodies. Humanized antibodies (A) to (C) can be produced by grafting CDRs containing a predetermined amino acid sequence onto the framework region of a human antibody, and can be produced according to known techniques (e.g., Jones et al., Nature, Vol. 321, pp. 522-525 (1986); Riechman et al., Nature, Vol. 332, pp. 323-327 (1988); Verhoeyen et al., Science, Vol. 239, pp. 1534-1536 (1988)). In the humanized antibodies (A) to (C), the framework regions and constant regions derived from the human antibody may contain mutations to the extent that their antigen-binding properties are not lost. Such mutations may be, but are not limited to, substitutions, deletions, insertions, etc. For example, conservative substitutions may be employed. Furthermore, by analyzing the binding mode of the antibody and antigen in detail using three-dimensional structural analysis or the like, various mutations can be introduced into the humanized antibodies (A) to (C) to the extent that their antigen-binding properties are not lost.

[0025] Another embodiment of antibodies (A) to (C) is a chimeric antibody. The chimeric antibodies (A) to (C) may be composed of a heavy chain consisting of a heavy chain variable region having the amino acid sequences of each of the CDRs 1 to 3 and a human antibody heavy chain constant region, and a light chain consisting of a light chain variable region having the amino acid sequences of each of the CDRs 1 to 3 and a human antibody light chain constant region. In a chimeric antibody, known human antibody heavy chain constant regions and human antibody light chain constant regions can be used. Chimeric antibodies (A) to (C) can be prepared by replacing the heavy and light chain variable regions of a human antibody with heavy and light chain variable regions having the amino acid sequences of CDRs 1 to 3, according to known techniques (e.g., Morrison et al., Proc. Natl. Acad. Sci., Vol. 81, pp. 6851-6855 (1984); Neuberger et al., Nature, Vol. 312, pp. 604-608 (1984); Takeda et al., Nature, Vol. 314, pp. 452-454 (1985)). Chimeric antibodies (A) to (C) may contain mutations in regions other than the CDRs, as long as the antigen-binding properties are not lost. Such mutations may include, but are not limited to, substitutions, deletions, insertions, and the like. For example, conservative substitutions may be used. Furthermore, by analyzing the binding mode between the antibody and the antigen in detail using three-dimensional structural analysis, etc., various mutations can be introduced into the chimeric antibodies (A) to (C) as long as their antigen-binding properties are not lost.

[0026] In the chimeric IgA antibody of antibody (A), the amino acid sequence of the heavy chain variable region having CDRs comprising each of the amino acid sequences shown in SEQ ID NOs: 1 to 3 includes the amino acid sequence shown in SEQ ID NO: 7; or an amino acid sequence comprising each of the amino acid sequences shown in SEQ ID NOs: 1 to 3 and having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 7. In addition, in the chimeric IgA antibody of antibody (A), the amino acid sequence of the light chain variable region having CDRs comprising each of the amino acid sequences shown in SEQ ID NOs: 4 to 6 includes the amino acid sequence shown in SEQ ID NO: 8 or 9; or an amino acid sequence comprising each of the amino acid sequences shown in SEQ ID NOs: 4 to 6 and having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 8 or 9.

[0027] In the chimeric antibody of antibody (B), the amino acid sequence of the heavy chain variable region having CDRs comprising any of the amino acid sequences set forth in SEQ ID NOs: 13 to 15 includes the amino acid sequence of SEQ ID NO: 19; or an amino acid sequence comprising any of the amino acid sequences set forth in SEQ ID NOs: 13 to 15 and having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 19. In addition, in the chimeric antibody of antibody (B), the amino acid sequence of the light chain variable region having CDR1 comprising the amino acid sequence of SEQ ID NO: 16, CDR2 comprising the amino acid sequence RAN, and CDR3 comprising the amino acid sequence of SEQ ID NO: 18 includes the amino acid sequence of SEQ ID NO: 20; or an amino acid sequence comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 16, CDR2 comprising the amino acid sequence RAN, and CDR3 comprising the amino acid sequence of SEQ ID NO: 18, and having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 20.

[0028] In the chimeric antibody of antibody (C), the amino acid sequence of the heavy chain variable region having CDRs comprising each of the amino acid sequences set forth in SEQ ID NOs: 23 to 25 includes the amino acid sequence of SEQ ID NO: 29; or an amino acid sequence comprising each of the amino acid sequences set forth in SEQ ID NOs: 23 to 25 and having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 29. In addition, in the chimeric antibody of antibody (C), the amino acid sequence of the light chain variable region having CDRs comprising each of the amino acid sequences set forth in SEQ ID NOs: 26 to 28 includes the amino acid sequence of SEQ ID NO: 30; or an amino acid sequence comprising each of the amino acid sequences set forth in SEQ ID NOs: 26 to 28 and having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 30.

[0029] Another embodiment of antibodies (A) to (C) is a murine antibody. The murine antibodies (A) to (C) may have mutations in regions other than the CDRs, as long as their antigen-binding properties are not lost. Such mutations may be, but are not limited to, substitutions, deletions, insertions, and the like. For example, conservative substitutions can be employed. Furthermore, by analyzing the binding mode between the antibody and the antigen in detail using three-dimensional structural analysis or the like, various mutations can be introduced into the murine antibodies (A) to (C), as long as their antigen-binding properties are not lost.

[0030] Examples of the amino acid sequence of the heavy chain of the murine antibody of antibody (A) include the amino acid sequence shown in SEQ ID NO: 10; or an amino acid sequence comprising each of the amino acid sequences shown in SEQ ID NOs: 1 to 3 and having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 10. Examples of the amino acid sequence of the light chain of the murine antibody of antibody (A) include the amino acid sequence shown in SEQ ID NO: 11 or 12; or an amino acid sequence comprising each of the amino acid sequences shown in SEQ ID NOs: 4 to 6 and having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 11 or 12. DNA encoding a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 10 includes one comprising the nucleotide sequence shown in SEQ ID NO: 33. DNA encoding a light chain comprising the amino acid sequence shown in SEQ ID NO: 11 includes one comprising the nucleotide sequence shown in SEQ ID NO: 34. DNA encoding a light chain comprising the amino acid sequence shown in SEQ ID NO: 12 includes one comprising the nucleotide sequence shown in SEQ ID NO: 35.

[0031] Examples of the amino acid sequence of the heavy chain of the murine antibody of antibody (B) include the amino acid sequence shown in SEQ ID NO: 21; or an amino acid sequence comprising any of the amino acid sequences shown in SEQ ID NOs: 13 to 15, and having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 21. Examples of the amino acid sequence of the light chain of the murine antibody of antibody (B) include the amino acid sequence shown in SEQ ID NO: 22; or an amino acid sequence comprising CDR1 comprising the amino acid sequence shown in SEQ ID NO: 16, CDR2 comprising the amino acid sequence RAN, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 18, and having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 22. DNA encoding a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 21 includes DNA comprising the nucleotide sequence shown in SEQ ID NO: 36. DNA encoding a light chain comprising the amino acid sequence shown in SEQ ID NO: 22 includes DNA comprising the nucleotide sequence shown in SEQ ID NO: 37.

[0032] Examples of the amino acid sequence of the heavy chain of the murine antibody of antibody (C) include the amino acid sequence shown in SEQ ID NO: 31; or an amino acid sequence comprising each of the amino acid sequences shown in SEQ ID NOs: 23 to 25 and having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 31. Examples of the amino acid sequence of the light chain of the murine antibody of antibody (C) include the amino acid sequence shown in SEQ ID NO: 32; or an amino acid sequence comprising each of the amino acid sequences shown in SEQ ID NOs: 26 to 28 and having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 32. DNA encoding a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 31 includes one comprising the nucleotide sequence shown in SEQ ID NO: 38. Furthermore, DNA encoding a light chain comprising the amino acid sequence shown in SEQ ID NO: 32 includes one comprising the nucleotide sequence shown in SEQ ID NO: 39.

[0033] Examples of the amino acid sequences of the CDRs of antibodies (A) to (C) and the amino acid sequences of the variable regions of antibodies (A) to (C) are shown in Tables 1 to 3.

[0034] The isotype and subtype of the antibodies (A) to (C) are not particularly limited and may be, for example, IgA (IgA1, IgA2), IgG (IgG1, IgG2, IgG3, IgG4), IgM, IgD, IgE, etc., with IgA being a preferred example.

[0035] When antibodies (A) to (C) are IgA, they may be monomers or multimers. When antibodies (A) to (C) are IgA, a preferred example is a dimer. When antibodies (A) to (C) are IgA multimers, they contain a J chain. The J chain is a peptide containing an amino acid sequence that does not have an antigen recognition site, and is a peptide having an amino acid sequence different from both the heavy chain and the light chain described above. Specific examples of such amino acid sequences include, for example, the amino acid sequence shown under ACCESSION No. AAA38673; VERSION AAA38673.1 GI:196379, available on the National Center of Biotechnology Information (NCBI) website (http: / / www.ncbi.nlm.nih.gov / ), if derived from a mouse, and the amino acid sequence shown under ACCESSION No. NP_653247; VERSION NP_653247.1 GI:21489959, also available on the NCBI website, if derived from a human. Such J chains are each disulfide-bonded to the heavy chains of the monoclonal IgA antibody monomers.

[0036] Furthermore, in the preventive or therapeutic drugs for PH disclosed herein, antigen-binding fragments of antibodies (A) to (C) can also be used as active ingredients for preventing or treating PH. The type of antigen-binding fragment of antibodies (A) to (C) is not particularly limited and may be, for example, any of Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, etc.

[0037] Antibodies (A) to (C) and antigen-binding fragments thereof may be conjugated antibodies and conjugated antigen-binding fragments bound to various compounds such as polyethylene glycol, as necessary. Furthermore, antibodies (A) to (C) and antigen-binding fragments thereof may have modified sugar chains or be fused with other proteins, as necessary.

[0038] In the preventive or therapeutic agent for PH of the present disclosure, one type may be selected and used from among antibodies (A) to (C) and their antigen-binding fragments, or two or more types may be used in combination.

[0039] Antibodies (A) to (C) and their antigen-binding fragments can be produced using known genetic engineering techniques, etc. For example, antibodies (A) to (C) and their antigen-binding fragments can be produced according to the methods described in WO 2014 / 142084, WO 2023 / 277142, WO 2023 / 277166, WO 2024 / 237217, etc.

[0040] The prophylactic or therapeutic agent for PH of the present disclosure is provided in a desired dosage form by combining the antibody and / or antigen-binding fragment thereof with pharmaceutically acceptable carriers, additives, etc. Examples of pharmaceutically acceptable carriers or additives include sterile water, physiological saline, stabilizers, excipients, antioxidants, buffers, preservatives, surfactants, chelating agents, binders, etc.

[0041] The dosage form of the preventive or therapeutic agent for PH of the present disclosure is not particularly limited, provided that it can be administered orally or enterally, and examples thereof include liquids, capsules, tablets, pills, powders, granules, fine granules, film-coated agents, pellets, troches, sublingual agents, chewable agents, buccal agents, pastes, syrups, suspensions, elixirs, and emulsions. The content of the antibody and / or antigen-binding fragment thereof in the preventive or therapeutic agent for PH of the present disclosure may be appropriately determined depending on the dosage, dosage form, etc.

[0042] [Indications] Animals to which the PH preventive or therapeutic agent of the present disclosure is applicable include, but are not limited to, mammals such as humans, mice, rats, guinea pigs, rabbits, hamsters, dogs, cats, weasels, cows, pigs, etc. In a preferred embodiment, the PH preventive or therapeutic agent of the present disclosure is used for the prevention or treatment of PH in humans.

[0043] The preventive or therapeutic agent for PH of the present disclosure can correct alterations in the intestinal flora associated with PH and improve the pathological condition of PH. The clinical classification of PH to be prevented or treated by the preventive or therapeutic agent for PH of the present disclosure is not particularly limited, and PH may be classified into any of Groups 1, 1', 1'', 2, 3, 4, and 5 of the Nice classification. In one embodiment, the preventive or therapeutic agent for PH of the present disclosure can be used for the prevention or treatment of Group 1 (PAH) of the Nice classification.

[0044] Furthermore, the greater the abundance of bacteria to which the antibody and / or its antigen-binding fragment binds in the stool or gastrointestinal contents of a subject seeking prevention or treatment of PH, the more likely it is that the antibody and / or its antigen-binding fragment will be effective in preventing or treating PH. Therefore, in one embodiment of the present disclosure, the amount of bacteria to which the antibody and / or its antigen-binding fragment binds in the stool or gastrointestinal contents is tested before administration to a subject seeking prevention or treatment of PH, and subjects with high abundance of such bacteria can be selected for prevention or treatment. The abundance of bacteria to which the antibody and / or its antigen-binding fragment binds in the stool or gastrointestinal contents can be determined, for example, by recovering bacteria from the stool or gastrointestinal contents and analyzing them using a fluorescence-activated cell sorter (FACS) with labeled antibodies and / or antigen-binding fragments. The abundance of the bacteria in the stool or gastrointestinal contents can be determined, for example, by comparing the stool or gastrointestinal contents with those of healthy subjects or those of PH patients for whom the efficacy of the antibody and / or its antigen-binding fragment has been confirmed. Specifically, if the amount of the bacteria present in the stool or gastrointestinal contents of a test subject (a person for whom PH prevention or treatment is sought) is higher than that in the stool or gastrointestinal contents of a healthy person, the test subject can be determined to be more likely to benefit from the effectiveness of the antibody and / or its antigen-binding fragment in preventing or treating PH. Furthermore, if the amount of the bacteria present in the stool or gastrointestinal contents of a test subject (a person for whom PH prevention or treatment is sought) is equal to or greater than that in the stool or gastrointestinal contents of a PH patient for whom the efficacy of the antibody and / or its antigen-binding fragment has been confirmed, the test subject can be determined to be more likely to benefit from the effectiveness of the antibody and / or its antigen-binding fragment in preventing or treating PH.

[0045] Furthermore, the preventive or therapeutic agent for PH of the present disclosure may be administered in combination with other therapeutic agents for PH such as endothelin receptor antagonists, phosphodiesterase 5 inhibitors, soluble guanylate cyclase stimulators, prostaglandin I2, and sotatercept, and may also be administered to individuals who have shown resistance to these therapeutic agents for PH.

[0046] [Dosage and Administration] The route of administration of the preventive or therapeutic agent for PH of the present disclosure may be any route as long as the antibody and / or antigen-binding fragment thereof is delivered to the intestine, and examples thereof include oral administration and enteral administration. Enteral administration includes not only administration via the anus, but also administration via a tube or the like inserted into the digestive tract from outside the body, such as a gastrostomy tube. A preferred example of the route of administration of the preventive or therapeutic agent for PH of the present disclosure is oral administration.

[0047] The dosage of the preventive or therapeutic agent for PH disclosed herein may be appropriately determined to be an amount effective for preventing or treating PH depending on the age of the subject, the severity of the PH condition, the type of active ingredient used, and the like. For example, the dosage of the antibody and / or antigen-binding fragment thereof may be set to about 0.001 to 100 mg / kg / day.

[0048] The administration interval of the preventive or therapeutic agent for PH disclosed herein may be set appropriately within a range that exhibits a preventive or therapeutic effect for PH, and examples include daily, every other day, weekly, every other week, every 2 to 3 weeks, monthly, every other month, or every 2 to 3 months.

[0049] The present disclosure will be explained in more detail below by showing examples, but it should not be construed as being limited to these examples.

[0050] 1. Test Materials and Test Methods 1-1. Antibodies (1) W27 Antibody The W27 antibody is a monoclonal multimeric IgA antibody (including a J chain) produced by a mouse hybridoma. It was prepared using the method described in WO 2014 / 142084. The amino acid sequences of the W27 antibody are as follows: Heavy chain CDR1: SEQ ID NO: 1 Heavy chain CDR2: SEQ ID NO: 2 Heavy chain CDR3: SEQ ID NO: 3 Light chain CDR1: SEQ ID NO: 4 Light chain CDR2: SEQ ID NO: 5 Light chain CDR3: SEQ ID NO: 6 Heavy chain variable region: SEQ ID NO: 7 Light chain variable region: SEQ ID NO: 8 Full-length heavy chain sequence: SEQ ID NO: 10 Full-length light chain sequence: SEQ ID NO: 11

[0051] (2) rW27 antibody The rW27 antibody is a recombinant multimeric IgA antibody (including a J chain) of the W27 antibody. When creating a recombinant W27 antibody, a mutation was introduced into a portion of the light chain variable region. The rW27 antibody was produced by transfecting CHO cells with a heavy chain expression vector incorporating a nucleotide sequence encoding the heavy chain, a light chain expression vector incorporating a nucleotide sequence encoding the light chain, and a J chain expression vector incorporating a nucleotide sequence encoding the J chain. Specific production conditions were as described in WO 2023 / 277142. The amino acid sequence of the CDRs of the rW27 antibody is identical to that of the W27 antibody. The amino acid sequence of the rW27 antibody is as follows: Heavy chain CDR1: SEQ ID NO: 1 Heavy chain CDR2: SEQ ID NO: 2 Heavy chain CDR3: SEQ ID NO: 3 Light chain CDR1: SEQ ID NO: 4 Light chain CDR2: SEQ ID NO: 5 Light chain CDR3: SEQ ID NO: 6 Heavy chain variable region: SEQ ID NO: 7 Light chain variable region: SEQ ID NO: 9 Full-length heavy chain sequence: SEQ ID NO: 10 Full-length light chain sequence: SEQ ID NO: 12

[0052] (3) PGSI1A Antibody The PGSI1A antibody is a monoclonal multimeric IgA antibody (including J chain) obtained from a hybridoma formed by fusing mouse small intestinal lamina propria cells with mouse myeloma cells. The PGSI1A antibody is antibody SNK0004A described in WO 2024 / 237217 and was prepared by the method described in WO 2024 / 237217. The amino acid sequences of the PGSI1A antibody are as follows: Heavy chain CDR1: SEQ ID NO: 13 Heavy chain CDR2: SEQ ID NO: 14 Heavy chain CDR3: SEQ ID NO: 15 Light chain CDR1: SEQ ID NO: 16 Light chain CDR2: amino acid sequence: RAN Light chain CDR3: SEQ ID NO: 18 Heavy chain variable region: SEQ ID NO: 19 Light chain variable region: SEQ ID NO: 20 Full-length heavy chain sequence: SEQ ID NO: 21 Full-length light chain sequence: SEQ ID NO: 22

[0053] (4) W37 antibody The W37 antibody is a monoclonal multimeric IgA antibody produced by a mouse hybridoma. The W37 antibody is antibody SNK0003A described in WO 2023 / 277166 and was prepared by the method described in WO 2023 / 277166. The amino acid sequences of the W37 antibody are as follows: Heavy chain CDR1: SEQ ID NO: 23 Heavy chain CDR2: SEQ ID NO: 24 Heavy chain CDR3: SEQ ID NO: 25 Light chain CDR1: SEQ ID NO: 26 Light chain CDR2: SEQ ID NO: 27 Light chain CDR3: SEQ ID NO: 28 Heavy chain variable region: SEQ ID NO: 29 Light chain variable region: SEQ ID NO: 30 Full-length heavy chain sequence: SEQ ID NO: 31 Full-length light chain sequence: SEQ ID NO: 32

[0054] (5) 3H12LJ antibody The 3H12LJ antibody is a recombinant IgA multimeric antibody produced by incorporating the gene sequence of a mouse IgA antibody that exhibits weak binding to Escherichia coli into CHO cells. In this test, the 3H12LJ antibody was used as a control.

[0055] 1-2. Clinical Research With approval from the ethics committee, fecal and blood samples were collected from 89 PH patients and 82 healthy subjects. The PH patients included 40 with idiopathic / hereditary pulmonary arterial hypertension, 19 with collagen vascular disease-related pulmonary hypertension, 10 with pulmonary hypertension associated with portal hypertension, 8 with chronic thromboembolic pulmonary hypertension, 7 with pulmonary hypertension associated with congenital shunt disease, and 5 with other conditions. The healthy subjects were age- and sex-matched. Table 4 shows the background characteristics of PH patients and healthy subjects. Feces were collected from the subjects, transported to the laboratory at 4°C as quickly as possible, and stored at -80°C until analysis.

[0056]

[0057] All experiments were conducted under the guidelines of the National Cerebral and Cardiovascular Center Animal Committee and approved by the National Cerebral and Cardiovascular Center Institutional Review Board. All rats were housed at 24±1°C with a 12-hour light / 12-hour dark cycle and were provided with standard mouse chow and water.

[0058] (1) Gnotobiotic Experiments: Germ-free male F344 rats were purchased from Japan SLC Co., Ltd. and housed in a sterile isolator environment. 11-week-old rats were administered feces from healthy individuals or patients with PH, and 15-week-old rats were subcutaneously administered monocrotaline (MCT) to generate gnotobiotic PH model rats. Specifically, feces from four healthy individuals were mixed in an anaerobic chamber and diluted with anaerobic transport medium (1000 mL containing 20 g of Lablemco powder, 1 g of L-cysteine, 0.45 g of KH2PO4, 0.9 g of NaCl, 0.45 g of (NH4)2SO4, 0.045 g of CaCl2, 0.045 g of MgSO4, 400 mL of glycerol, and 600 mL of distilled water) to prepare a healthy individual feces dilution. Similarly, feces from four patients were mixed in an anaerobic chamber and diluted with anaerobic transport medium to prepare a patient feces dilution. Next, 11-week-old rats were orally administered diluted feces from healthy donors (final 10-fold dilution) or diluted feces from patients (final 10-fold dilution) in separate isolators to create fecal transplant gnotobiotic rats that replicated the intestinal flora of healthy donors and patients. Four weeks after fecal transplantation (at 15 weeks of age), the rats were subcutaneously injected with 60 mg / kg of monocrotaline (Sigma Aldrich) and then housed in an isolator for three weeks to create monocrotaline-challenged gnotobiotic rats with PH. Table 5 shows basic information for the PH patients and healthy donors used in fecal transplants.

[0059]

[0060] (2) Gnotobiotic experiments using single or dual bacterial transplants. Ruminococcus gnavus (JCM6515) and Streptococcus pasteurianus (isolated from rat feces transplanted with patient feces) were cultured in trypticase soy broth under anaerobic conditions at 37°C. One or two strains of Ruminococcus gnavus or Streptococcus pasteurianus were orally administered to 11-week-old germ-free rats. At 15 weeks of age, monocrotaline was administered subcutaneously to induce PH. Right ventricular systolic pressure and right ventricular hypertrophy index were measured 21 days after monocrotaline administration.

[0061] (3) Experiments in an SPF (Specific Pathogen Free) Environment Experiments in an SPF environment were performed using Sprague-Dawley (SD) rats purchased from Charles River Japan, Inc. Male rats aged 6 to 8 weeks were used.

[0062] (4) Short-chain fatty acid administration experiment Monocrotaline was administered subcutaneously at 60 mg / kg to 6-week-old male SD rats. From the day after monocrotaline administration, the rats were allowed to drink water, water containing 100 mM butyrate, or water containing 100 mM propionate ad libitum, and then right ventricular systolic pressure and right ventricular hypertrophy index were measured at 9 weeks of age. The rats were kept in a normal oxygen concentration environment (normoxa) throughout the entire experiment.

[0063] (5) IgA antibody administration experiment (rats housed in an SPF environment). Six-week-old male F344 rats housed in an SPF environment were subcutaneously administered 60 mg / kg of monocrotaline to induce PH. The day after monocrotaline administration, rats were divided into vehicle, W27 antibody, and W37 antibody groups. PBS was administered to the vehicle group, 500 μg of W27 antibody diluted in PBS to the W27 antibody group, and 500 μg of W37 antibody diluted in PBS to the W37 antibody group. These were administered by gavage every other day for 3 weeks. At 9 weeks of age, right ventricular systolic pressure and right ventricular hypertrophy index were measured, and feces were collected.

[0064] (6) IgA antibody administration experiment (gnotobiotic rat experiment: PBS vs. W27 antibody). 11-week-old germ-free rats were orally administered feces from PH patients and, at 15 weeks of age, administered monocrotaline subcutaneously at 60 mg / kg to create gnotobiotic PH model rats. The day after monocrotaline administration, rats were divided into a vehicle group and a W27 antibody group. PBS was administered to the vehicle group, and 500 μg of W27 antibody diluted in PBS was administered to the W27 antibody group via gavage every other day for 3 weeks. At 18 weeks of age, right ventricular systolic pressure and right ventricular hypertrophy index were measured, and feces were collected.

[0065] (7) IgA antibody administration experiment (gnotobiotic rat experiment: PBS vs. PSGI1A antibody vs. rW27 antibody). Patient-derived feces were orally administered to 11-week-old germ-free rats, and monocrotaline (60 mg / kg) was administered subcutaneously to 15-week-old rats to generate gnotobiotic PH model rats. The day after monocrotaline administration, rats were divided into vehicle, PSGI1A antibody, and rW27 antibody groups. PBS was administered to the vehicle group, 500 μg of PSGI1A antibody diluted in PBS to the PSGI1A antibody group, and 500 μg of rW27 antibody diluted in PBS to the rW27 antibody group. The vehicle group received PBS, the PSGI1A antibody group received 500 μg of rW27 antibody diluted in PBS, and the rW27 antibody group received 500 μg of rW27 antibody diluted in PBS via gavage every other day for 3 weeks. At 18 weeks of age, right ventricular systolic pressure and right ventricular hypertrophy index were measured, and feces were collected.

[0066] (8) IgA antibody administration experiment (gnotobiotic rat experiment: 3H12LJ antibody vs. rW27 antibody). Patient-derived feces were orally administered to 11-week-old germ-free rats, and 60 mg / kg of monocrotaline was subcutaneously administered to 15-week-old rats to create gnotobiotic PH model rats. The day after monocrotaline administration, the rats were divided into 3H12LJ and rW27 antibody groups. The 3H12LJ group received 200 μg of 3H12LJ antibody diluted in PBS, and the rW27 group received 200 μg of rW27 antibody diluted in PBS via gavage every other day for 3 weeks. At 18 weeks of age, right ventricular systolic pressure and right ventricular hypertrophy index were measured, and feces were collected.

[0067] 1-4. Measurement of Right Ventricular Systolic Pressure. Rats were sedated and analgesic using inhalation anesthesia with isoflurane (Pfizer). During the procedure, rat body temperature was maintained at 37–38°C using a thermostatically controlled heat pad linked to a rectal temperature monitor. After tracheotomy, rats were ventilated with a rat ventilator (Harvard apparatus) at a tidal volume of 10 μL / g and 70 breaths / min. Right ventricular pressure (RVP) was measured using a polyethylene tube inserted into the right external jugular vein and advanced to the right ventricle. The RVP signal was detected by a pressure transducer (MLT0670; AD Instruments), relayed by a pressure amplifier (ML117; AD Instruments), continuously sampled by a Power Lab system (AD Instruments, Colorado Springs, CO), and recorded on a computer using Chart software (AD Instruments). Heart rate was calculated based on the peak right ventricular systole.

[0068] 1-5. Measurement of right ventricular hypertrophy index (Fulton's index) After measuring right ventricular systolic pressure, the rats were euthanized by administering an overdose of anesthetic and the hearts were removed. The atria were removed, and the right ventricle was separated from the left ventricle and septum. After blotting, the weights of the right ventricle and the left ventricle plus septum were measured, and the weight ratio of the two (right ventricle / left ventricle weight ratio) was calculated as the right ventricular hypertrophy index to evaluate right ventricular hypertrophy.

[0069] To measure fecal short-chain fatty acid concentrations, 100 mg of feces was placed in a 2.0 mL tube with zirconia beads and suspended in MilliQ. The sample was heated at 85°C for 15 minutes, vortexed at 5 m / s for 45 seconds using a FastPrep 24 5G (MP Biomedicals, CA, USA), centrifuged at 15,350 × g for 10 minutes, and the supernatant was filtered through a 0.2 μm filter. The organic acid concentrations in the filtrate were measured by high-performance liquid chromatography (Prominence, SHIMADZU, Kyoto, Japan), a detector (CDD-10A, SHIMADZU, Kyoto, Japan), two tandem columns (Shim-pack SCR-102(H), 300 mm × 8 mm ID, SHIMADZU, Kyoto, Japan), and a guard column (Shim-pack SCR-102(H), 50 mm × 6 mm ID, SHIMADZU, Kyoto, Japan) using a post-column reaction. The mobile phase (5 mM p-toluenesulfonic acid) and reaction solution (5 mM p-toluenesulfonic acid, 100 μM EDTA, 20 mM Bis-Tris) were used.

[0070] 1-7. RNA-seq Analysis. The quality of RNA and library preparation was assured using TapeStation (Agilent). For RNA-sequencing analysis of rat lung, 100 ng of total RNA was used for ribosomal RNA removal, followed by library preparation using the TruSeq Stranded mRNA Sample Preparation Kit (Illumina). For RNA-sequencing analysis of human or rat peripheral blood mononuclear cells (PBMCs), 100 pg of total RNA was used, and library preparation was performed using the SMART-seq v4 Ultra Low Input RNA Kit (Takara Clontech). More than 25 million reads with 75 bp paired-end reads were obtained per sample. Quality control of sequencing data was performed using FastQC. Trimmed and filtered reads were aligned to the rat genome version rn6 reference genome for rats using the rat genome (Rn6) and to the human genome (hg38) reference genome for humans using Hisat2. Genes specifically expressed in lung tissue were defined as those showing a 2-fold or less change in expression level with a false discovery rate (FDR) of <0.05. Genes specifically expressed in PBMCs were defined as those showing a 1.5-fold or greater change in expression level in humans and a 2-fold or greater change in expression level in rats with a P <0.05.

[0071] 1-8. Metagenomic Analysis (1) 16S Analysis DNA was extracted from stool samples using the NecleoSpin DNA stool (Macherey-Nagel). 16S Metagenomic Sequencing Library Preparation (Illumina) was used to prepare libraries according to the manufacturer's protocol using a primer set targeting 16S ribosomal RNA V1-V2 (27Fmod: 5'-AGRGTTTGATCMTGGCTCAG-3' (SEQ ID NO: 1) and 338R: 5'-TGCTGCCTCCCGTAGGAGT-3' (SEQ ID NO: 2)). The 251-nt amplicon was sequenced using the MiSeq System (Illumina). The resulting paired-end reads were merged using PEAR (sco.h-its.org / exelixis / web / software / pear / ) and trimmed using BBtrim (bbmap.sourceforge.net). Further, up to 20,000 reads per sample were randomly extracted using random_sequence_sample.pl (ualberta.ca / ~stothard / software.html). The processed sequences were clustered into operational taxonomic units (OTUs) based on 99% identity using UCLUST version 1.2.33q. Each OTU was taxonomically classified using the Silvav138 database, and bioinformatic microbiota analysis was performed using QIIME version 2.

[0072] (2) Shotgun Analysis. Whole-genome sequencing was performed on a DNBSEQ-G400 (MGI Tech) system, generating paired-end reads of 150 bases each. The main steps of the quality check (QC) process were (i) trimming of low-quality bases, (ii) removal of duplicate reads, and (iii) identification and masking of human reads. Duplicate reads were marked using PRINSEQ-lite53 (version 0.20.4). Raw reads were trimmed using Trimomatic (version 0.39) to clip Illumina adapters and remove low-quality bases at both ends. Reads less than 60 bp in length after trimming were discarded. Next, duplicates were removed from duplicates of the same sequence, leaving only the longest read. As the final QC step, the quality-filtered reads were aligned to the human reference genome (hg38) using bowtie2 (version 2.3.5) and BMTagger (version 3.101). Only reads that failed paired-end alignment with either tool were retained. For phylogenetic annotation and abundance quantification, filtered paired-end reads were aligned to a reference genome dataset using bowtie2. For multiply mapped reads, only the best alignment was selected by alignment score. The number of reads mapped to each genome was divided by the genome length. The values ​​for each genome were summed for each sample to calculate the relative abundance of species-level clades.

[0073] The filtered paired-end reads were de novo assembled into contigs using MEGAHIT (version 1.2.9). Open reading frames (ORFs) on the contigs were predicted using MetageneMark (version 3.38). The ORF catalog was then annotated using the Kyoto Encyclopedia of Genes and Genomes (KEGG) protein database (https: / / www.kegg.jp). Annotation was performed using the KEGG gene database for prokaryotes and MGENES, a database of KEGG genes for metagenomic samples. The deduced amino acid sequences translated from the ORF catalog were aligned with the KEGG protein database using BLASTP (version v0.9.32.133) in DIAMOND. To quantify ORF abundance, the filtered paired-end reads were mapped to the assembled contigs using bowtie2 with default parameters. To avoid gene size bias, ORF abundance was defined as the region depth of each ORF in the ORF catalog according to the mapping results. After sample QC, bacterial abundance data were QC and normalized, followed by normalization of microbial gene ortholog abundance data.

[0074] 1-9. Bacterial Culture (In Vitro) Streptococcus salivarius (JCM5707), Streptococcus pasteurianus (isolated from feces of gnotobiotic rats receiving patient fecal transplants at the National Cerebral and Cardiovascular Center), Streptococcus mutans (JCM5705), and Rothia aeria (JCM11412) were cultured in Brain Heart Infusion (BHI) medium (BD). Ruminococcus gnavus (JCM6515) was cultured in Trypticase soy broth (TSB). Each bacterium was cultured from a glycerol stock in 10 ml of the respective medium overnight at 37°C. Aerobic culture was performed by shaking, and anaerobic culture was performed by static culture. Agar plates for colony counts were prepared by adding agar pellets (final concentration 1.5%, Nacalai Tesque) to each medium.

[0075] 1-10. IgA antibody bacterial binding test After measuring the number of bacteria cultured by the above method, if the number of bacteria was 1 x 10 8The bacteria were suspended in 50 mM sodium carbonate solution at a concentration of 5000 cells / 50 μl to obtain a bacterial suspension. 50 μl of the bacterial suspension was added to each well of an ELISA plate and incubated overnight at 4°C for bacterial immobilization. The ELISA plate was washed using a plate washer (Vaccu-Pette / 96 multiwell pipetter, Sigma-Aldrich). The bacterial-immobilized ELISA plate was washed three times with PBS, and 150 μl of PBS containing 1% serum albumin (BSA) (Wako) was added to each well. Blocking was performed by incubation overnight at 4°C. A dilution series of each IgA antibody was prepared in the wells of a 96-well plate. The IgA antibody dilution series consisted of serial dilutions of 1x, 1 / 3x, 1 / 10x, 1 / 30x, 1 / 100x, 1 / 300x, 1 / 1,000x, and 1 / 3,000x using PBS containing 1% BSA. After blocking, the PBS containing 1% BSA was removed from the ELISA plate, and 50 μl of serially diluted antibodies were added to each well and incubated at room temperature for 1 hour. The ELISA plate was then washed three times with PBS containing 0.05% Tween 20 (Chem Cruz). The secondary antibodies, AP (Alkaline phosphatase)-conjugated anti-goat mouse IgA (final concentration 0.5 μg / ml, Southern Biotech) or AP-conjugated anti-goat human IgA (final concentration 0.5 μg / ml, Southern Biotech), were added and incubated at room temperature for 1 hour. The substrate reaction solution for color development was prepared by dissolving magnesium chloride (final concentration 2 mM) and phosphatase substrate (final concentration 1 mg / ml, Sigma) in a solution containing sodium bicarbonate (final concentration 6.5 mM) and sodium carbonate (final concentration 18.5 mM). The plate reacted with the secondary antibody was washed again with PBS containing 0.05% Tween 20, and 50 μl of the substrate reaction solution was added to each well. OD 405 nm was measured using a Tristar2 LB942 (BERTHOLD TECHNOLOGIES) and compared with a negative control without IgA antibody to evaluate the binding of IgA antibody to bacteria.

[0076] 1-11. Bacterial Growth Inhibition Test by IgA Antibody Streptococcus pasteurianus was cultured overnight at 37°C in an aerobic chamber using TSB medium. Rothia aeria was cultured for 2 days at 30°C in an aerobic chamber using TSB medium. Streptococcus pasteurianus was centrifuged at 3,000g for 5 minutes at room temperature, washed with PBS, and diluted to 60 cells / μl. Rothia aeria was centrifuged at 5,000g for 5 minutes at room temperature and diluted to 2,000 cells / μl. Five μl of each bacterial dilution was added to separate tubes. IgA antibody solutions were prepared by adding IgA antibody to PBS containing 0.1% BSA at a concentration of 1 μg / ml. Five μl of the IgA antibody solution or PBS containing 0.1% BSA (control) was added to each bacterial dilution. Streptococcus pasteurianus was incubated at 37°C for 1 hour. Rothia aeria was incubated at 30°C for 30 minutes. After incubation, Streptococcus pasteurianus was cultured for an additional 6 hours at 37°C. Rothia aeria was cultured for an additional 20 hours at 30°C. The number of bacteria was then calculated by plating. Streptococcus pasteurianus or Rothia aeria was cultured on TSB agar medium under each condition, and the colony-forming units (CFU) were calculated.

[0077] 1-12. Measurement of the percentage of rW27 antibody-bound enterobacteria in stool samples. After measuring the bacterial counts of stool samples from healthy individuals and PH patients, PBS was added to the bacteria in the human stool samples to obtain a 6×10 7 The cells were dispensed at 6 × 10 cells / tube. After centrifugation at 8,000 g for 5 minutes at 4°C, the supernatant was removed. 100 μl of PBS containing 20% ​​normal rat serum (Wako) was added to the cells from which the supernatant was removed, and the mixture was incubated on ice for 20 minutes. This inhibited nonspecific binding of the antibody. Fluorescence activated cell sorter (FACS) buffer was then added to the cell suspension, which was centrifuged at 8,000 g for 5 minutes at 4°C, and the supernatant was removed. The number of bacteria was 6 × 10 615 μg of biotinylated mouse monoclonal IgA antibody was added to each cell and incubated on ice for 20 minutes. Next, 20 μl of PE (Phycoerythrin) / Cyanine7-Streptavidin (final concentration 10 μg / ml, BioLegend) was added to the cells and incubated on ice for 20 minutes, protected from light. FACS buffer was added to the reaction mixture, which was then centrifuged at 8,000 g for 5 minutes at 4°C. The supernatant was removed. Next, FACS buffer and thiazole orange (TO, final concentration 42 nM, BD) were added to the bacteria and incubated on ice for 10 minutes. The percentage of antibody-bound bacteria was then analyzed using a SONY Cell Sorter SH800 (SONY). FACS buffer was prepared by adding 10% fetal bovine serum (FBS, Nichirei) and EDTA (final concentration 5 μM, Nacalai Tesque) to PBS and sterilizing the mixture through a 0.22 μm filter.

[0078] 1-13. Statistics All data are expressed as mean ± standard error or standard deviation. Significant differences between multiple groups were tested using one-way ANOVA, followed by post-hoc testing. Tests between two groups were analyzed using the Student t-test or Mann-Whitney test. Event-free survival curves were derived using the Kaplan-Meier method and compared using the log-rank test. Network analysis was performed using Spearman rank correlation. A P value of less than 0.05 was considered statistically significant.

[0079] 2. Reference Test Results 2-1. PH Model Rats Show Alterations in the Intestinal Microbiota There are three representative PH disease models using rats (Figure 1A). Alterations in the intestinal microbiota were confirmed in these rat models.

[0080] Six-week-old male SD rats were continuously housed in a hypoxic chamber with 10% oxygen for 3 weeks to produce hypoxia-induced PH rats (Fig. 1A). Six-week-old male SD rats were subcutaneously administered monocrotaline at 60 mg / kg and housed in a normoxia environment (normoxia) for 3 weeks to produce monocrotaline-induced PH rats (Fig. 1A). Six-week-old male SD rats were subcutaneously administered the VEGFR2 inhibitor Sugen5416 at 20 mg / kg, housed in a hypoxic chamber with 10% oxygen for 3 weeks, and then housed in a normoxia environment (normoxia) for 2 weeks to produce Sugen5416 / hypoxia-induced PH rats (Fig. 1A).

[0081] Analysis of the gut microbiota of each PH model rat revealed altered β-diversity in the Hx, MCT, and SuHx model rats (Fig. 1B). However, the rat gut microbiota differed significantly from that of humans, with oral bacteria such as Streptococcus rarely detected in the rats, while bacteria belonging to the Mulibaculaceae family were commonly increased and Lachnospiraceae were commonly decreased (Fig. 1C).

[0082] 2-2. Antibiotic Administration Improves PH Pathology in PH Model Rats. Hx, MCT, and SuHx rat models were administered water containing 1 g / L ampicillin, 1 g / L neomycin, 1 g / L metronidazole, and 0.5 g / L vancomycin (antibiotic cocktail water) or water, and PH pathology (pulmonary arterial medial thickening, right ventricular systolic pressure, and right / left ventricular mass ratio) was evaluated. In Hx rats, PH pathology was evaluated after a 3-week housing period in a hypoxic chamber with 10% oxygen and free access to antibiotic cocktail water or water. In MCT rats, PH pathology was evaluated after a 3-week housing period in a normoxic environment (normoxia) following monocrotaline administration and free access to antibiotic cocktail water. In SuHx rats, PH pathology was evaluated after a 2-week housing period in a normoxic environment (normoxia) following hypoxia and free access to antibiotic cocktail water or water. In addition, male SD rats without PH were kept as controls with free access to water. The results showed that pulmonary arterial media thickening, right ventricular systolic pressure, and right / left ventricular mass ratio were significantly reduced in both PH model rats treated with the antibiotic cocktail compared with those treated with water (Fig. 2A–C).

[0083] 2-3. Butyrate administration improves PH pathology in PH model rats. Monocrotaline was administered subcutaneously at 60 mg / kg to 6-week-old male SD rats. From the day after monocrotaline administration, the rats were allowed to drink water, water containing 100 mM butyrate, or water containing 100 mM propionate ad libitum. Right ventricular systolic pressure and right ventricular hypertrophy index were measured at 9 weeks of age (Fig. 3A). Propionate administration did not decrease right ventricular systolic pressure or right ventricular hypertrophy index, but butyrate administration did decrease right ventricular systolic pressure and right ventricular hypertrophy index (Fig. 3B).

[0084] 2-4. PH patients exhibit altered gut microbiota characterized by ectopic colonization of oral bacteria. Based on the above results, we hypothesized that gut microbiota may promote PH pathology. Therefore, we analyzed the gut microbiota of PH patients. Analysis of the gut microbiota of PH patients (PH, 89 cases) and healthy controls (HV, 82 cases) revealed significantly reduced Faith PD and Shannon index, indicators of alpha diversity, in the gut microbiota of PH patients compared with healthy controls (Figure 4A). Furthermore, the gut microbiota of PH patients exhibited significantly altered beta diversity (unweighted unifrac PCoA) compared with healthy controls (Figure 4B).

[0085] Furthermore, the volcano plot analysis of gut microbiota composition revealed that oral bacteria such as Streptococcus, Rothia, and Actinomyces were increased in the intestine of PH patients, while short-chain fatty acid (SCFA)-producing bacteria such as Subdolignulum, Ruminococcus, and Coprococcus were decreased (Figure 5A). Furthermore, the concentrations of acetate, propionate, and butyrate in the stool samples of PH patients were significantly decreased compared with those of healthy controls (Figure 5B), suggesting a deviated gut environment from a healthy state. These results confirmed that the gut microbiota of PH patients is altered by colonization with oral bacteria. Furthermore, fecal IgA concentrations were elevated in PH patients using ELISA (Figure 5C), suggesting that ineffective IgA antibodies are produced in the intestinal tract of PH patients.

[0086] 2-5. In the intestines of PH patients, Ruminococcus gnavus coexists in a network with multiple oral bacteria, which is associated with poor prognosis. Compositional analysis of the intestinal microbiota of PH patients revealed that the relative frequency of Ruminococcus gnavus was higher in patients with a higher oral resident bacterial score. Furthermore, network analysis of bacteria correlated with Ruminococcus gnavus (Spearman correlation coefficient >0.2) revealed that Ruminococcus gnavus coexists in a network with Streptococcus, Veillonella, Rothia, and Fusobacterium (all core oral bacteria) in the intestine (Figure 6A). Furthermore, we found that PH patients who had Ruminococcus gnavus coexist with Streptococcus, Rothia, Fusobacterium, or Veillonella in the intestine had a significantly poorer prognosis (Log-rank test; Streptococcus P = 0.007, Rothia P = 0.004, Veillonella P = 0.023) (Fig. 6B). These results suggest that Ruminococcus gnavus coexists with multiple oral bacteria in the intestinal tract, forming a network, which is associated with a poor prognosis.

[0087] 2-6. Reconstitution of the gut microbiota of PH patients in PH model rats promotes PH pathology and enhances lung inflammatory signaling and responses to xenobiotics. Next, we evaluated the contribution of the altered gut microbiota present in PH patients to the pathogenesis of PH pathology. Specifically, we administered diluted feces from PH patients or healthy controls to 11-week-old germ-free F344 rats, administered monocrotaline at 15 weeks of age, and maintained them for an additional 3 weeks to generate gnotobiotic PH model rats (PH patient fecal-transplanted F344 rats or healthy control fecal-transplanted F344 rats) (Figure 6A). We also generated germ-free F344 rats by administering monocrotaline at 15 weeks of age without administering the diluted fecal solution and maintaining them for an additional 3 weeks. In addition, 11-week-old germ-free F344 rats were raised in an SPF environment at the National Cerebral and Cardiovascular Center without administering the fecal diluent, and at 15 weeks of age, monocrotaline was administered and the rats were then raised in an SPF environment for an additional 3 weeks to create SPF-reared F344 rats.

[0088] Analysis of the intestinal microbiota composition in F344 rats transplanted with feces from PH patients and healthy donors revealed significant differences in the composition of the intestinal microbiota between the two groups. The F344 rats transplanted with feces from PH patients were colonized with a high proportion of oral bacteria, Streptococcus, while the F344 rats transplanted with feces from healthy donors were colonized with a high proportion of Faecalibacterium, a typical butyrate-producing bacterium (Fig. 7B).

[0089] We also measured right ventricular systolic pressure and right / left ventricular mass ratio in each rat. Both right ventricular systolic pressure and right / left ventricular mass ratio were lower in germ-free F344 rats compared with SPF F344 rats, indicating a significant suppression of monocrotaline-induced PH (Fig. 7C). Healthy donor fecal-transplanted F344 rats also showed similar right ventricular systolic pressure and right / left ventricular mass ratio to germ-free F344 rats, indicating a significant suppression of monocrotaline-induced PH (Fig. 7C). Furthermore, patient fecal-transplanted F344 rats showed significantly elevated right ventricular systolic pressure compared with healthy donor fecal-transplanted F344 rats (Fig. 7C). These results confirm that reconstitution of the gut microbiota of PH patients in PH model rats can facilitate the development of PH pathology.

[0090] RNA sequencing analysis of the lungs of PH patient fecal-transplanted F344 rats and healthy control rats revealed increased expression of Egr1, Cma1, Mcp1, Tnf, and Gzmb in the lungs of PH patient fecal-transplanted F344 rats (Fig. 8A). Gene Ontology Term analysis revealed that genes related to defense responses against Gram-positive bacteria, genes promoting inflammatory responses, and responses to xenobiotic stimuli were upregulated in the PH patient fecal-transplanted F344 rats compared with healthy control rats (Fig. 8B). In particular, genes related to macrophage chemotaxis (e.g., C5ar1, Cxcl17), lymphocyte chemotaxis (e.g., Ccl9, Ccl6), protein processing (e.g., Cma1, Gzmb), IL-6 response (e.g., Mcpt1, Prnp), and xenobiotic stimulation (e.g., Cebpa, Egr1) were upregulated (Fig. 8C). On the other hand, in healthy donor fecal transplanted F344 rats, genes related to angiogenesis and vasculogenesis were upregulated (Fig. 8B), and in particular, the expression of genes involved in angiogenesis (Tmem100, Sox17, etc.) was upregulated (Fig. 8C).

[0091] 2-7. Ruminococcus gnavus synergizes with Streptococcus pasteurianus to worsen PH. Next, we administered Ruminococcus gnavus and Streptococcus pasteurianus to germ-free rats (Figure 9A). Specifically, 11-week-old germ-free F344 rats were inoculated with Ruminococcus gnavus and S. pasteurianus, either alone or in combination, and allowed to colonize the intestines for 4 weeks. At 15 weeks of age, monocrotaline was administered subcutaneously, and right ventricular systolic pressure and right ventricular hypertrophy index were measured 21 days after monocrotaline administration. Inoculation with Ruminococcus gnavus or Streptococcus pasteurianus did not result in a significant increase in right ventricular systolic pressure or right ventricular hypertrophy index, whereas inoculation with a combination of Ruminococcus gnavus and Streptococcus pasteurianus resulted in a significant additive increase in right ventricular systolic pressure and right ventricular hypertrophy index (Fig. 9B).

[0092] 2-8. The reconstitution of the intestinal flora of PH patients in PH model rats accelerated PH pathology, but vancomycin treatment reduced the number of Gram-positive cocci, resulting in a significant increase in vancomycin-resistant bacteria. We investigated the effects of vancomycin administration on PH pathology in gnotobiotic PH model rats (Fig. 10A). Specifically, 11-week-old germ-free F344 rats were administered diluted feces from PH patients or healthy controls, and then allowed free access to water containing 0.5 g / L vancomycin or water for 4 weeks. At 15 weeks of age, monocrotaline 60 mg / kg was administered subcutaneously. After 3 weeks of normal oxygenation, right ventricular systolic pressure and right ventricular hypertrophy index were measured.

[0093] The vancomycin group showed significantly lower right ventricular systolic pressure and right ventricular hypertrophy index than the water group (Figure 10B). Furthermore, a shotgun analysis of the gut microbiota at the species level revealed that Streptococcus pasteurianus, Ruminococcus gnavus, and Eggerthella lenta were eradicated in the vancomycin group. However, vancomycin-resistant bacteria such as Klebsiella pneumoniae, Escherichia coli, and Fusobacterium ulcerans were abnormally increased (Figure 11). These results suggest that treatments aimed at eliminating Gram-positive bacteria from the intestinal tract may be effective in addressing the altered gut microbiota of PH patients, which includes an increase in oral bacteria and Gram-positive bacteria such as Ruminococcus gnavus. However, antibiotic treatment is considered inappropriate because it leads to an abnormal increase in resistant bacteria.

[0094] 3. Test Results 3-1. Screening for Antibodies Showing Binding and Growth-Inhibitory Effects on Bacteria Involved in PH Pathogenesis We investigated the effectiveness of IgA antibodies in PH treatment by controlling intestinal microbiota alterations. First, we screened for IgA antibodies that bind to Streptococcus mutans, Streptococcus pasteurianus, Streptococcus sobrinus, Streptococcus salivarius, Rothia aeria, and Ruminococcus gnavus, bacteria thought to be closely involved in PH pathogenesis among bacteria that increase in PH patients. The results confirmed that the rW27, W37, and PGSI1A antibodies exhibited high binding to these bacteria (Figure 12). Furthermore, when growth inhibition tests were performed on Streptococcus pasteurianus and Rothia aeria using the rW27 antibody, the rW27 antibody demonstrated significant growth-inhibitory effects on all bacteria (Figure 13).

[0095] 3-2. W27 and W37 antibodies correct alterations in the intestinal microbiota and improve PH pathology in monocrotaline-challenged (MCT)-induced PH rat model. The effects of W27 and W37 antibody administration on PH pathology were examined using monocrotaline-challenged (MCT)-induced PH rat model. Specifically, 6-week-old male F344 rats housed in an SPF environment were subcutaneously administered 60 mg / kg of monocrotaline to induce PH. The day after monocrotaline administration, 500 μg of W27 or 500 μg of W37 antibody were administered by gavage every other day for 3 weeks (Figure 14A). At 9 weeks of age, mean arterial pressure, right ventricular systolic pressure, and right ventricular hypertrophy index were measured, and fecal samples were collected for intestinal microbiota analysis.

[0096] Administration of W27 or W37 antibodies reduced right ventricular systolic pressure and improved PH pathology (Figure 14B). Analysis of the intestinal microbiota revealed that administration of W27 antibody reduced the number of bacteria in the Muribaculaceae family, which are thought to promote PH pathology in rats, and increased the number of bacteria in the Lactobacillaceae family (Figure 14C). Administration of W37 antibody also reduced the number of bacteria in the Muribaculaceae family and increased the number of bacteria in the Ruminococcaceae family, similar to the results of W27 antibody (Figure 14D). These results confirmed that administration of W27 and W37 antibodies can correct alterations in the intestinal microbiota in PH and improve PH pathology.

[0097] 3-3. W27 antibody corrects alterations in the intestinal microbiota and ameliorates PH pathology in gnotobiotic rats as a model for PH. The effects of W27 antibody administration on PH pathology were examined using gnotobiotic rats as a model for PH. Specifically, germ-free F344 rats were administered a diluted fecal solution from PH patients at 11 weeks of age, monocrotaline at 60 mg / kg at 15 weeks of age, and W27 antibody at 500 μg was administered by gavage every other day for 3 weeks starting the day after monocrotaline administration (Figure 15A). At 18 weeks of age, right ventricular systolic pressure and right ventricular hypertrophy index were measured, and the collected fecal samples were used to analyze the intestinal microbiota.

[0098] Administration of the W27 antibody resulted in a decrease in right ventricular systolic pressure and right ventricular hypertrophy index, indicating an improvement in PH pathology (Figure 15B). Analysis of the intestinal microbiota revealed a significant decrease in Streptococcus species, which are thought to promote PH pathology in humans, after administration of the W27 antibody (Figure 15C). Furthermore, administration of the W27 antibody increased fecal butyrate concentrations (Figure 15D). Principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) of the intestinal microbiota also confirmed significant changes in the intestinal microbiota in the W27 antibody-treated group compared to the vehicle group (Figures 16B and 16C). Furthermore, the volcano plot analysis of gut microbiota composition revealed that the W27 antibody-treated group had reduced Eggerthella, Clostridium innocuum, Citrobacter, and Streptococcus species compared to the vehicle group, while the number of butyrate-producing bacteria, such as Eubacterium nodatum, Oscilospira, Ruminococcaceae UCD-005, Erysipelotrichaceae, Anaerofilum, and Roseburia, was increased (Figure 16D). Considering that butyrate administration improved PH pathology in PH model rats as shown in Figure 3, W27 antibody administration may have corrected alterations in the gut microbiota, resulting in increased butyrate concentrations in the improved gut microbiota, which may be one of the reasons for the improvement of PH pathology by W27 antibody. These results confirm that W27 antibody administration can correct alterations in the gut microbiota and improve PH pathology.

[0099] 3-4. rW27 and PGSI1A antibodies correct alterations in the intestinal microbiota and improve PH pathology in gnotobiotic rats. The effects of rW27 and PGSI1A antibody administration on PH pathology were examined using gnotobiotic rats. Specifically, germ-free F344 rats were administered a diluted fecal solution from PH patients at 11 weeks of age, monocrotaline at 60 mg / kg at 15 weeks of age, and 500 μg of rW27 or PGSI1A antibody were administered by gavage every other day for 3 weeks, starting the day after monocrotaline administration (Figure 17A). At 18 weeks of age, right ventricular systolic pressure and right ventricular hypertrophy index were measured, and the collected fecal samples were used to analyze the intestinal microbiota.

[0100] The results showed that administration of rW27 and PGSI1A antibodies reduced right ventricular systolic pressure and right ventricular hypertrophy index, improving PH pathology (Figure 17B). Furthermore, analysis of the intestinal microbiota revealed that administration of rW27 and PGSI1A antibodies reduced the number of Enterococcus, a Gram-positive bacterium thought to promote PH pathology in humans, as well as the number of Enterobacteriaceae and Fusobacterium bacteria that cause intestinal inflammation, such as in inflammatory bowel disease (Figure 17C). Furthermore, administration of rW27 and PGSI1A antibodies increased the number of butyrate-producing bacteria, such as Butyricoccus, Subdolignulum, Coprococcus, and Faecalicacterium, which are thought to suppress PH pathology (Figure 17C). Principal component analysis (PCA) analysis of the gut microbiota showed significant changes in the rW27 antibody-treated group compared to the vehicle group (Figure 18B). Partial least squares discriminant analysis (PLS-DA) showed significant changes in the gut microbiota in the rW27 antibody-treated and PGSI1A antibody-treated groups compared to the vehicle group (Figure 18C). Furthermore, the rW27 antibody-treated group showed significant decreases in Fusobacterium, Eggherthella, and Enterococcus, and increases in Coprococcus and Anaerostipes (all butyrate-producing bacteria), compared to the vehicle group (Figure 18D). Furthermore, the PGSI1A antibody-treated group showed significant decreases in Fusobacterium and Enterococcus, and increases in Anaerostipes, compared to the vehicle group (Figure 18D).

[0101] Furthermore, we investigated the effects of rW27 and 3H12LJ antibody administration on PH pathology using gnotobiotic rats. Specifically, germ-free F344 rats were administered a diluted fecal solution from PH patients at 11 weeks of age, monocrotaline at 60 mg / kg at 15 weeks of age, and 200 μg of rW27 or 3H12LJ antibody were administered by gavage every other day for 3 weeks, starting the day after monocrotaline administration (Fig. 19A). At 18 weeks of age, right ventricular systolic pressure and right ventricular hypertrophy index were measured, and the collected fecal samples were used to analyze the intestinal microbiota.

[0102] Treatment with the rW27 antibody significantly reduced right ventricular systolic pressure and right ventricular hypertrophy index, improving PH pathology, whereas treatment with the 3H12LJ antibody did not (Figure 19B). Analysis of the intestinal microbiota revealed that treatment with the rW27 antibody reduced the numbers of Streptococcus and Ruminococcus gnavus bacteria, which are associated with the promotion of PH pathology, and increased the numbers of Subdolignulum bacteria, which are associated with the suppression of PH pathology, compared with treatment with the 3H12LJ antibody (Figure 19C). Principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) of the intestinal microbiota revealed significant changes in the intestinal microbiota in the rW27 antibody-treated group compared with the 3H12LJ antibody-treated group (Figures 20B and 20C). Furthermore, the results of the volcano plot analysis of the gut microbiota composition revealed that the rW27 antibody-treated group had decreased levels of Turicibacter, Fusobacterium, and Streptococcus compared with the 3H12LJ antibody-treated group, while butyrate-producing bacteria such as Eubacterium, Faecalibacterium, Anaerofilum, and Roseburia were increased (Fig. 20D). Corresponding to these changes in the gut microbiota, the butyrate concentration in the gut microbiota was increased in the rW27 antibody-treated group compared with the 3H12LJ antibody-treated group (Fig. 20E).

[0103] These results confirmed that administration of rW27 and PGSI1A antibodies can correct alterations in the intestinal microbiota in PH and improve the pathology of PH. In particular, it was suggested that one of the mechanisms by which rW27 and PGSI1A antibodies improve PH pathology is through an increase in butyrate-producing bacteria and the resulting increase in butyrate concentration in the intestinal microbiota.

[0104] 3-5. The percentage of bacteria bound by rW27 antibody was higher in stool samples from PH patients than in healthy controls. The percentage of bacteria bound by rW27 antibody was measured using FACS in stool samples from PH patients and healthy controls. The results showed that the percentage of bacteria bound by rW27 antibody was higher in stool samples from PH patients (Figure 21).

[0105] These results demonstrate that the proportion of bacteria to which antibodies (A) to (C) described in the present disclosure bind is high in the intestinal flora of PH patients, and that reducing or eliminating these bacteria from the intestinal flora is effective in ameliorating PH pathology. Furthermore, it is expected that antibodies (A) to (C) described in the present disclosure will be even more effective in ameliorating PH pathology in PH patients whose stool samples show a high proportion of bacteria to which antibodies (A) to (C) described in the present disclosure bind.

[0106] The present disclosure is not limited in any way to the description of the embodiments and examples of the invention. Various modifications within the scope of the claims and within the scope that can be easily conceived by a person skilled in the art are also included in the present invention. The contents of the documents and the like shown in this specification are hereby incorporated by reference in their entirety.

Claims

1. A drug for the prevention or treatment of pulmonary hypertension, comprising at least one of the antibodies and / or antigen-binding fragments thereof selected from the following (A) to (C): (A) an antibody having a heavy chain variable region having CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3; and a light chain variable region having CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

6. (B) an antibody having a heavy chain variable region having CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 13, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 14, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 15; and a light chain variable region having CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 16, CDR2 comprising the amino acid sequence RAN, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

18. (C) An antibody having a heavy chain variable region having CDR1 comprising the amino acid sequence shown in SEQ ID NO: 23, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 24, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 25; and a light chain variable region having CDR1 comprising the amino acid sequence shown in SEQ ID NO: 26, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 27, and CDR3 comprising the amino acid sequence shown in SEQ ID NO:

28.

2. The preventive or therapeutic agent for pulmonary hypertension according to claim 1, wherein the antibody is an IgA antibody.

3. The preventive or therapeutic agent for pulmonary hypertension according to claim 1 or 2, wherein the antibody is a humanized antibody or a chimeric antibody.

4. The preventive or therapeutic drug for pulmonary hypertension according to claim 1 or 2, which is administered orally or enterally.

5. A prophylactic or therapeutic drug for pulmonary hypertension according to claim 1 or 2, which is used for the prevention or treatment of pulmonary arterial hypertension.

6. Use of at least one of the antibodies and / or antigen-binding fragments thereof selected from the following (A) to (C) for the prevention or treatment of pulmonary hypertension: (A) An antibody having a heavy chain variable region having CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3; and a light chain variable region having CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

6. (B) An antibody having a heavy chain variable region having CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 13, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 14, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 15; and a light chain variable region having CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 16, CDR2 comprising the amino acid sequence R A N , and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

18. (C) An antibody having a heavy chain variable region having CDR1 comprising the amino acid sequence shown in SEQ ID NO: 23, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 24, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 25; and a light chain variable region having CDR1 comprising the amino acid sequence shown in SEQ ID NO: 26, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 27, and CDR3 comprising the amino acid sequence shown in SEQ ID NO:

28.

7. A method for preventing or treating pulmonary hypertension, comprising administering to a person in need thereof an effective amount of at least one of the antibodies and / or antigen-binding fragments thereof selected from the following (A) to (C): (A) an antibody having a heavy chain variable region having CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3; and a light chain variable region having CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

6. (B) an antibody having a heavy chain variable region having CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 13, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 14, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 15; and a light chain variable region having CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 16, CDR2 comprising the amino acid sequence R A N , and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

18. (C) An antibody having a heavy chain variable region having CDR1 comprising the amino acid sequence shown in SEQ ID NO: 23, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 24, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 25; and a light chain variable region having CDR1 comprising the amino acid sequence shown in SEQ ID NO: 26, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 27, and CDR3 comprising the amino acid sequence shown in SEQ ID NO:

28.

8. At least one of antibodies and / or antigen-binding fragments thereof selected from the following (A) to (C), for use in a treatment for the prevention or treatment of pulmonary hypertension: (A) an antibody having a heavy chain variable region having CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3; and a light chain variable region having CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

6. (B) an antibody having a heavy chain variable region having CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 13, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 14, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 15; and a light chain variable region having CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 16, CDR2 comprising the amino acid sequence R A N , and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

18. (C) An antibody having a heavy chain variable region having CDR1 comprising the amino acid sequence shown in SEQ ID NO: 23, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 24, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 25; and a light chain variable region having CDR1 comprising the amino acid sequence shown in SEQ ID NO: 26, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 27, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 28.

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