WO2025220732A1 - Médicament pour la prévention ou le traitement de l'hypertension pulmonaire - Google Patents

Médicament pour la prévention ou le traitement de l'hypertension pulmonaire

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Publication number
WO2025220732A1
WO2025220732A1 PCT/JP2025/015144 JP2025015144W WO2025220732A1 WO 2025220732 A1 WO2025220732 A1 WO 2025220732A1 JP 2025015144 W JP2025015144 W JP 2025015144W WO 2025220732 A1 WO2025220732 A1 WO 2025220732A1
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amino acid
seq
acid sequence
sequence shown
antibody
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English (en)
Japanese (ja)
Inventor
良和 中岡
遼太郎 浅野
礼子 新藏
竜太郎 玉野
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University of Tokyo NUC
National Cerebral and Cardiovascular Center
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University of Tokyo NUC
National Cerebral and Cardiovascular Center
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from bacteria

Definitions

  • This disclosure relates to drugs for the prevention or treatment of pulmonary hypertension (PH).
  • PH pulmonary hypertension
  • Pulmonary hypertension is a group of progressive diseases with a poor prognosis that cause cardiac and pulmonary dysfunction due to elevated blood pressure in the pulmonary arteries. According to the Nice Classification, it 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.
  • PAH pulmonary arterial hypertension
  • PVOD pulmonary veno-occlusive disease
  • PCH pulmonary capillary hemangiomatosis
  • Group 1'' persistent pulmonary hypertension of the newborn
  • Group 2 pulmonary hypertension associated with left heart disease
  • Group 3 pulmonary
  • drugs such as endothelin receptor antagonists, phosphodiesterase 5 inhibitors, soluble guanylate cyclase stimulators, and prostaglandin I2 are used to treat PH in Japan, and while the overall prognosis is improving, the prognosis for severe and drug-resistant patients remains quite poor. Furthermore, 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.
  • Nakaoka et al. the inventors of the present invention, conducted metagenomic analysis of fecal DNA from PH patients and healthy individuals, and found that PH patients experience a change in the intestinal flora (dysbiosis) characterized by ectopic colonization of oral bacteria such as Streptococcus in the intestine and an increase in Ruminococcus gnavus (R. gnavus), and that this change in the intestinal flora promotes the pathology of PH (Patent Document 1, etc.). Furthermore, Shinzo et al., the inventors of the present invention, have 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.
  • the objective of this disclosure is to provide a preventive or therapeutic drug for PH that works by correcting the alterations in the intestinal flora associated with PH.
  • the inventors have conducted extensive research to solve the above-mentioned problems and have found that antibodies with specific CDRs can bind to oral bacteria that ectopically colonize the intestines of PH patients (Streptococcus mutans, Streptococcus pasteurianus, Streptococcus sobrinus, and Streptococcus salivarius), as well as bacteria that increase in the intestinal flora of PH patients (Rothia aeria and Ruminococcus gnavus), and inhibit the proliferation of these bacteria. Furthermore, the inventors have found that orally administering these antibodies to PH model rats corrects alterations in the intestinal flora and improves the pathological condition of PH.
  • a drug for preventing or treating PH comprising at least one of the following antibodies and/or antigen-binding fragments thereof (A) to (C):
  • Item 3 The preventive 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 preventive or therapeutic drug for PH according to any one of Items 1 to 3, which is administered orally or enterally.
  • Item 5. The prophylactic or therapeutic drug for PH according to Item 1 or 2, which is used for the prophylaxis or treatment of pulmonary arterial hypertension.
  • Item 6. Use of at least one of the antibodies and/or antigen-binding fragments thereof according to (A) to (C) above for the manufacture of a drug for the prevention or treatment of PH.
  • 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 the treatment for the prevention or treatment of PH.
  • administration of an antibody having specific CDRs can correct alterations in the intestinal flora associated with PH, making it possible to prevent or treat PH.
  • FIG. 1 Figures showing changes in the intestinal microbiota in PH model rats.
  • A is a diagram showing the procedure for producing PH model rats; the upper panel shows hypoxia challenge, the middle panel shows monocrotaline challenge, and the lower panel shows the procedure for producing PH model rats by Sugen5416/hypoxia/normoxia challenge (SuHx).
  • B is a diagram showing the beta diversity of the intestinal microbiota of each model rat.
  • (C) is a bar plot showing the family-level composition of intestinal bacteria in the intestinal microbiota of each model rat. This figure shows that antibiotic administration to PH model rats improves PH pathology.
  • Hx Hypoxic (Hx) model rats were given antibiotic cocktail water (Abx) or water (vehicle) ad libitum, and unstimulated SD rats were given water ad libitum (control). PH pathology (pulmonary artery media thickening, right ventricular systolic pressure, and right ventricular hypertrophy index) was evaluated.
  • B MCT model rats were given antibiotic cocktail water (Abx) or water (vehicle) ad libitum, and PH pathology (pulmonary artery media thickening, right ventricular systolic pressure, and right ventricular hypertrophy index) was evaluated.
  • FIG. B shows the measurement results of the right ventricular systolic pressure and right ventricular hypertrophy index.
  • A shows the alpha diversity of the intestinal microbiota in PH patients (PH, 89 cases) and healthy subjects (HV, 82 cases) in relation to Faith's PD and Shannon index.
  • B shows the beta diversity (unweighted unifrac PCoA) of the intestinal microbiota in PH patients and healthy subjects. ****P ⁇ 0.0001, *P ⁇ 0.05.
  • Figure 1 shows alterations in the intestinal microbiota of PH patients.
  • A is a volcano plot comparing the bacteria (at the genus level) that make up the intestinal microbiota of PH patients and healthy subjects (HV).
  • B shows the concentrations of acetate, propionate, and butyrate in the feces of PH patients and healthy subjects.
  • C shows the IgA concentrations in the feces of PH patients and healthy subjects.
  • Statistics are mean ⁇ standard deviation: ****P ⁇ 0.0001, **P ⁇ 0.01, *P ⁇ 0.05. This figure shows that in PH patients, Ruminococcus gnavus coexists in the intestinal tract, forming a network with multiple oral bacteria, and this is associated with poor prognosis.
  • A is a network diagram of bacteria correlated with Ruminococcus gnavus, created using the results of a composition analysis of the intestinal microbiota of PH patients.
  • B is a figure showing the results of a Kaplan-Meier analysis of event-free survival in PH patients, classified according to the abundance of Ruminococcus gnavus and Streptococcus, Rothia, Fusobacterium, or Veillonella in the intestine.
  • clinical events were defined as death, lung transplantation, and hospitalization due to right heart failure.
  • This figure shows changes in PH pathology in gnotobiotic PH model rats.
  • A is a diagram showing the method for producing gnotobiotic PH model rats.
  • B is the result of analyzing the composition of the intestinal flora in F344 rats transplanted with feces from PH patients and F344 rats transplanted with feces from healthy individuals.
  • C is the result of measuring right ventricular systolic pressure and right ventricular hypertrophy index in F344 rats transplanted with feces from PH patients (MCT-PH), F344 rats transplanted with feces from healthy individuals (MCT-HV), F344 rats reared in a germ-free environment (MCT-GF), and F344 rats reared in an SPF environment (MCT-SPF).
  • Statistics are mean ⁇ standard deviation: **P ⁇ 0.01, *P ⁇ 0.05.
  • A shows a comparison of RNA expression in the lungs using a volcano plot.
  • B shows the results of a Gene Ontology Term analysis of RNA expression in the lungs.
  • C shows a bar plot of the differences in expression of individual genes related to B.
  • Ruminococcus gnavus worsens PH through a cooperative action with Streptococcus pasteurianus.
  • A is a diagram showing the protocol for an experiment in which Ruminococcus gnavus and Streptococcus pasteurianus were administered to germ-free rats.
  • B is the results of measuring right ventricular systolic pressure and right ventricular hypertrophy index.
  • A is a diagram showing the experimental procedure.
  • B is the results of measuring right ventricular systolic pressure and right ventricular hypertrophy index in PH patient fecal transplant rats administered vancomycin (PH-FMT-VCM) and PH patient fecal transplant rats administered water (vehicle) (PH-FMT).
  • Statistical values are mean ⁇ standard deviation: **P ⁇ 0.01, *P ⁇ 0.05.
  • NC indicates the negative control (no IgA antibody added).
  • NC indicates the negative control (no IgA antibody added).
  • the number of bacteria in the absence of the rW27 antibody (-) is set to 100%, and the percentage of the number of bacteria in the presence of the rW27 antibody (+) is shown as the growth rate (%).
  • MCT model rats were administered with W27 antibody, W37 antibody, or PBS (vehicle), and the results of evaluating the pathology of PH are shown.
  • A A diagram showing the experimental protocol.
  • B Measurement results of mean arterial number, right ventricular systolic pressure, and right ventricular hypertrophy index are shown.
  • C Family-level analysis of the composition of the intestinal microbiota in MCT model rats administered with W27 antibody or PBS (vehicle).
  • D Family-level analysis of the composition of the intestinal microbiota in MCT model rats administered with W37 antibody or PBS (vehicle). W27 antibody was administered to gnotobiotic rats as a model for PH, and the pathological condition of PH was evaluated.
  • A shows the experimental protocol.
  • a diagram shows the experimental protocol.
  • B shows the results of a principal component analysis (PCA) of the intestinal microbiota.
  • C shows the results of a partial least squares discriminant analysis (PLS-DA) of the intestinal microbiota.
  • FIG. 1 A volcano plot comparing the W27 antibody-administered group and the vehicle group for the bacteria that make up the intestinal microbiota.
  • rW27 antibody or PGSI1A antibody was administered to gnotobiotic rats with a PH model, and the results of evaluating PH pathology are shown.
  • A is a diagram showing the experimental protocol.
  • B is the results of measuring right ventricular systolic pressure and right ventricular hypertrophy index.
  • C is the results of analyzing the composition of the intestinal microbiota, dividing bacteria into those that promote PH pathology and those that suppress PH pathology, and calculating the relative frequency of presence.
  • Statistics are mean ⁇ standard deviation: ***P ⁇ 0.001, **P ⁇ 0.01, *P ⁇ 0.05.
  • rW27 antibody or PGSI1A antibody was administered to PH model gnotobiotic rats, and the results of evaluating the changes in the intestinal microbiota are shown.
  • A A diagram showing the experimental protocol.
  • B shows the results of principal component analysis (PCA) of the intestinal microbiota.
  • C shows the results of partial least squares discriminant analysis (PLS-DA) of the intestinal microbiota.
  • D A violin plot showing the relative abundance (Log10 value of relative frequency) of bacteria of the genera Fusobacterium, Eggherthella, Enterococcus, Coprococcus, and Anaerostipes.
  • rW27 antibody or 3H12LJ antibody (control antibody) was administered to gnotobiotic rats with a PH model, and the pathological state of PH was evaluated.
  • 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 to determine the relative abundance of bacteria that promote PH pathology (Streptococcus and Ruminococcus gnavus) and bacteria that suppress PH pathology (Subdolignulum).
  • Statistics are mean ⁇ standard deviation: **P ⁇ 0.01, *P ⁇ 0.05.
  • FIG. 1 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 volcano plot comparing the bacteria that make up the intestinal microbiota between the rW27 antibody-administered group and the 3H12LJ antibody-administered group.
  • Figure E shows the results of measuring fecal butyric acid concentration. The percentage of bacteria bound to the rW27 antibody was measured by FACS using stool samples from PH patients and healthy individuals.
  • pulmonary hypertension refers to a group of progressive diseases with poor prognosis that cause cardiac and pulmonary dysfunction due to elevated blood pressure in the pulmonary arteries, and specifically refers to a condition in which the mean pulmonary artery pressure (PAP) measured at rest using right heart catheterization is 20 mmHg or higher.
  • the diagnostic criterion for PH may be a mean pulmonary artery pressure of 25 mmHg or higher.
  • PH 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, and Group 5: Pulmonary hypertension associated with an unspecified multifactorial mechanism.
  • PAH Pulmonary Arterial Hypertension
  • PVOD Pulmonary veno-occlusive disease
  • PCH pulmonary capillary hemangiomatosis
  • 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
  • prevention refers to inhibiting the onset of a disease or symptom or prolonging the period until onset
  • treatment refers to alleviating, mitigating, or slowing the rate of progression of a disease or symptom.
  • an "antibody” is 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.
  • CDRs complementarity determining 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.
  • an "antigen-binding fragment" of an antibody refers to a fragment of an antibody that retains the ability to bind to an antigen.
  • Antigen-binding fragments of an antibody 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; an 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.
  • 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.
  • a "chimeric antibody” is an antibody composed of a variable region from one species and a constant region from another species.
  • Chimeric antibodies include antibodies composed of a variable region derived from a non-human source and a constant region derived from a human source.
  • amino acid residues in amino acid sequences are indicated by single-letter abbreviations. Furthermore, in this disclosure, unless otherwise specified, the left side of an amino acid sequence indicates the N-terminus and the right side indicates the C-terminus.
  • sequence identity of amino acid sequences is the degree of amino acid sequence agreement between 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)).
  • BLASTX based on this BLAST algorithm
  • a "conservative substitution" in an amino acid sequence refers to the replacement of an amino acid residue with an amino acid residue having a similar side chain.
  • a substitution between amino acid residues having basic side chains such as lysine, arginine, and histidine constitutes 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 ⁇ -branched side chains such as threonine, valine, and isoleucine; and amino acid residues having aromatic side chains such as tyrosine, phenylalanine, tryptophan, and histidine.
  • 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, tyros
  • prophylactic or therapeutic drug for PH comprising at least one antibody and/or antigen-binding fragment thereof selected from (A) to (C) described below.
  • the prophylactic or therapeutic drug for PH of the present disclosure is described in detail below.
  • 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 shown in SEQ ID NO: 1, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3; and a light chain variable region having CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6 (hereinafter, sometimes referred to as "antibody (A)").
  • Antibody (A) is an antibody that has the same CDRs as the mouse W27 antibody and rW27 antibody used in the Examples described below.
  • Antibody (B) is an antibody that has the same CDRs as the mouse PGSI1A antibody used in the Examples described below.
  • Antibody (C) is an antibody that has the same CDRs as the mouse W37 antibody used in the Examples described below.
  • Antibodies (A) to (C) have all been discovered as monoclonal antibodies that bind to Clostridium difficile.
  • a preferred embodiment of antibodies (A) to (C) is a humanized antibody.
  • Humanized antibodies (A) to (C) may be composed of CDRs containing a specific amino acid sequence, a framework region derived from a human antibody, and a constant region derived from a human antibody. In humanized antibodies, known framework regions and constant regions derived from a human antibody can be used.
  • Humanized antibodies (A) to (C) can be produced by grafting each CDR 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.
  • humanized antibodies (A) to (C) the framework region and constant region 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 used for substitutions. 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 humanized antibodies (A) to (C) without losing their antigen-binding properties.
  • Chimeric antibodies (A) to (C) may be composed of a heavy chain consisting of a heavy chain variable region having the amino acid sequence of each of the CDRs 1 to 3 and a heavy chain constant region of a human antibody, and a light chain consisting of a light chain variable region having the amino acid sequence of each of the CDRs 1 to 3 and a light chain constant region of a human antibody.
  • chimeric antibodies known human antibody heavy chain constant regions and human antibody light chain constant regions can be used.
  • Chimeric antibodies of antibodies (A) to (C) can be produced by replacing the heavy chain variable region and light chain variable region of a human antibody with heavy chain variable region and light chain variable region having the amino acid sequences of each of CDRs 1 to 3, and can be produced 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)).
  • 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 of antibodies (A) to (C) may contain mutations in regions other than the CDRs, as long as their antigen-binding properties are not lost.
  • the mutations may be, but are not limited to, substitutions, deletions, insertions, etc. For example, conservative substitutions can be used for substitutions.
  • various mutations can be introduced into the chimeric antibodies (A) to (C) as long as the antigen-binding properties are not lost.
  • the amino acid sequence of the heavy chain variable region having CDRs containing 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 containing 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: 7.
  • the amino acid sequence of the light chain variable region having CDRs containing 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 containing 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: 8 or 9.
  • the amino acid sequence of the heavy chain variable region having CDRs containing each of the amino acid sequences set forth in SEQ ID NOs: 13 to 15 includes the amino acid sequence set forth in SEQ ID NO: 19; or an amino acid sequence containing each 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 with the amino acid sequence set forth in SEQ ID NO: 19.
  • the amino acid sequence of the light chain variable region having CDR1 containing the amino acid sequence set forth in SEQ ID NO: 16, CDR2 containing the amino acid sequence RAN, and CDR3 containing the amino acid sequence set forth in SEQ ID NO: 18 includes the amino acid sequence set forth in SEQ ID NO: 20; or an amino acid sequence containing CDR1 containing the amino acid sequence set forth in SEQ ID NO: 16, CDR2 containing the amino acid sequence RAN, and CDR3 containing the amino acid sequence set forth 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 set forth in SEQ ID NO: 20.
  • the amino acid sequence of the heavy chain variable region having CDRs containing each of the amino acid sequences shown in SEQ ID NOs: 23 to 25 includes the amino acid sequence shown in SEQ ID NO: 29; or an amino acid sequence containing 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: 29.
  • the amino acid sequence of the light chain variable region having CDRs containing each of the amino acid sequences shown in SEQ ID NOs: 26 to 28 includes the amino acid sequence shown in SEQ ID NO: 30; or an amino acid sequence containing 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: 30.
  • mouse 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, etc. For example, conservative substitutions can 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 mouse antibodies (A) to (C), as long as their antigen-binding properties are not lost.
  • the amino acid sequence of the heavy chain of the mouse antibody of antibody (A) can be, for example, 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.
  • the amino acid sequence of the light chain of the mouse antibody of antibody (A) can be, for example, 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 can be 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 can be 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 can be one comprising the nucleotide sequence shown in SEQ ID NO: 35.
  • the amino acid sequence of the heavy chain of the mouse antibody of antibody (B) can be, for example, 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.
  • the amino acid sequence of the light chain of the mouse antibody of antibody (B) can be, for example, 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 can be one 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 can be one comprising the nucleotide sequence shown in SEQ ID NO: 37.
  • the amino acid sequence of the heavy chain of the mouse antibody of antibody (C) includes, for example, 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.
  • the amino acid sequence of the light chain of the mouse antibody of antibody (C) includes, for example, 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.
  • An example of a DNA encoding a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 31 is one comprising the nucleotide sequence shown in SEQ ID NO: 38.
  • An example of a DNA encoding a light chain comprising the amino acid sequence shown in SEQ ID NO: 32 is one comprising the nucleotide sequence shown in SEQ ID NO: 39.
  • 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.
  • the isotype and subtype of 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.
  • antibodies (A) to (C) When antibodies (A) to (C) are IgA, they may be monomers or polymers. A preferred example of antibodies (A) to (C) that are IgA is a dimer. When antibodies (A) to (C) are IgA polymers, they contain a J chain.
  • a J chain is a peptide containing an amino acid sequence that does not have an antigen recognition site, and is a peptide with an amino acid sequence that is different from both the heavy chain and the light chain described above. Specific examples of such amino acid sequences include, for mouse origin, the amino acid sequence shown in ACCESSION No.
  • AAA38673 VERSION AAA38673.1 GI:196379, which is listed on the National Center of Biotechnology Information (NCBI) website (http://www.ncbi.nlm.nih.gov/), and for human origin, the amino acid sequence shown in ACCESSION No. NP_653247; VERSION NP_653247.1 GI:21489959, which is also listed on the NCBI website.
  • Such J chains are each disulfide-bonded to the heavy chains of the monoclonal IgA antibody monomer.
  • 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.
  • Antibodies (A) to (C) and their antigen-binding fragments may be conjugated antibodies and conjugated antigen-binding fragments bound to various compounds such as polyethylene glycol, as needed. Furthermore, antibodies (A) to (C) and their antigen-binding fragments may have modified sugar chains or be fused to other proteins, as needed.
  • 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.
  • Antibodies (A) to (C) and their antigen-binding fragments can be produced using known genetic engineering techniques, etc.
  • 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.
  • the preventive 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.
  • pharmaceutically acceptable carriers or additives include sterile water, physiological saline, stabilizers, excipients, antioxidants, buffers, preservatives, surfactants, chelating agents, binders, etc.
  • the dosage form of the preventive or therapeutic drug for PH disclosed herein is not particularly limited, provided that it can be administered orally or enterally. Examples include liquids, capsules, tablets, pills, powders, granules, fine granules, film-coated formulations, pellets, troches, sublingual tablets, chewable tablets, buccal tablets, pastes, syrups, suspensions, elixirs, and emulsions.
  • the content of the antibody and/or antigen-binding fragment thereof in the preventive or therapeutic drug for PH disclosed herein may be set appropriately depending on the dosage, dosage form, etc.
  • the animals to which the PH preventive or therapeutic agent of the present disclosure is applicable are not particularly limited, and examples include mammals such as humans, mice, rats, guinea pigs, rabbits, hamsters, dogs, cats, weasels, cows, pigs, etc.
  • the PH preventive or therapeutic agent of the present disclosure is used for the prevention or treatment of PH in humans.
  • the PH preventive or therapeutic agent disclosed herein can correct alterations in the intestinal flora associated with PH and improve the pathological condition of PH.
  • the clinical classification of PH that is the target of prevention or treatment for PH in the PH preventive or therapeutic agent disclosed herein is not particularly limited, and may be classified as any of Groups 1, 1', 1'', 2, 3, 4, and 5 of the Nice classification.
  • the PH preventive or therapeutic agent disclosed herein can be used to prevent or treat Group 1 (PAH) in the Nice classification.
  • the greater the amount of bacteria to which the antibody and/or its antigen-binding fragment binds in the stool or gastrointestinal contents 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 preventive or therapeutic drug for PH disclosed herein, the amount of bacteria to which the antibody and/or its antigen-binding fragment binds in the stool or gastrointestinal contents can be tested before administration to a person for whom prevention or treatment of PH is sought, and those with a high amount of such bacteria can be selected as targets for prevention or treatment.
  • the amount 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 collecting bacteria from the stool or gastrointestinal contents and analyzing them using a fluorescence activated cell sorter (FACS) with the labeled antibody and/or its antigen-binding fragment. Furthermore, the amount of the bacteria present in the stool or gastrointestinal contents can be determined by comparing, for example, the stool or gastrointestinal contents of a healthy individual, or 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.
  • FACS fluorescence activated cell sorter
  • test subject a person for whom PH prevention or treatment is sought
  • the test subject can be determined to be more likely to benefit from the efficacy of the antibody and/or its antigen-binding fragment in preventing or treating PH.
  • test subject a person for whom PH prevention or treatment is sought
  • amount of the bacteria present in the stool or gastrointestinal contents of a test subject is equal to or greater than that 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 efficacy of the antibody and/or its antigen-binding fragment in preventing or treating PH.
  • the PH preventive or therapeutic agent disclosed herein may be administered in combination with other PH therapeutic agents 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 PH therapeutic agents.
  • PH therapeutic agents such as endothelin receptor antagonists, phosphodiesterase 5 inhibitors, soluble guanylate cyclase stimulators, prostaglandin I2, and sotatercept
  • 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 capable of being 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 ex vivo into the digestive tract, 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.
  • the dosage of the disclosed preventive or therapeutic agent for PH can be appropriately determined to be an amount effective for preventing or treating PH depending on the age of the recipient, the severity of the PH condition, the type of active ingredient used, etc.
  • the dosage of the antibody and/or antigen-binding fragment thereof can be set to approximately 0.001 to 100 mg/kg/day.
  • the administration interval for the PH preventive or therapeutic agent disclosed herein may be set appropriately within a range that achieves the preventive or therapeutic effect for PH, and may include, for example, daily, every other day, weekly, every other week, every 2-3 weeks, monthly, every other month, or every 2-3 months.
  • W27 antibody is a monoclonal multimeric IgA antibody (containing a J chain) produced by a mouse hybridoma. It was prepared according to the method described in WO 2014/142084.
  • the amino acid sequence of the W27 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: 8 Heavy chain full length sequence: SEQ ID NO: 10 Light chain full length sequence: SEQ ID NO: 11
  • the rW27 antibody is a recombinant multimeric IgA antibody (including a J chain) of the 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 Heavy chain full length sequence: SEQ ID NO: 10 Light chain full length sequence: SEQ ID NO: 12
  • the PGSI1A antibody is a monoclonal polymeric IgA antibody (containing a J chain) obtained from a hybridoma formed by fusing mouse small intestinal lamina intestinal cells with mouse myeloma cells.
  • the PGSI1A antibody is antibody SNK0004A, which is described in WO 2024/237217, and was prepared by the method described in WO 2024/237217.
  • the amino acid sequence of the PGSI1A antibody is 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 Heavy chain full length sequence: SEQ ID NO: 21 Light chain full length sequence: SEQ ID NO: 22
  • 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 sequence of the W37 antibody is 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 Heavy chain full length sequence: SEQ ID NO: 31 Light chain full length sequence: SEQ ID NO: 32
  • 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.
  • 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.
  • 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 PH patients, and 15-week-old rats were administered monocrotaline (MCT) subcutaneously to create PH model gnotobiotic rats.
  • MCT monocrotaline
  • 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 diluted healthy feces solution.
  • 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
  • mice were orally administered the diluted healthy feces solution (final 10-fold dilution) or the diluted patient feces solution (final 10-fold dilution) in separate isolators to generate fecal transplant gnotobiotic rats reproducing the gut flora of healthy individuals and patients.
  • the rats were subcutaneously injected with 60 mg/kg of monocrotaline (Sigma Aldrich) and then housed in an isolator for three weeks to generate gnotobiotic rats with monocrotaline-induced PH.
  • Table 5 shows the basic information of the PH patients and healthy controls used in fecal transplantation.
  • Ruminococcus gnavus JCM6515
  • Streptococcus pasteurianus isolated from rat feces transplanted with patient feces
  • 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.
  • monocrotaline was administered subcutaneously to induce PH.
  • Right ventricular systolic pressure and right ventricular hypertrophy index were measured 21 days after monocrotaline administration.
  • IgA antibody administration experiment (rats reared in an SPF environment) Six-week-old male F344 rats housed in a special-purpose environment were subcutaneously administered 60 mg/kg of monocrotaline to induce PH. The day after monocrotaline administration, rats were divided into three groups: vehicle, W27 antibody, and W37 antibody. The vehicle group received PBS, the W27 antibody group received 500 ⁇ g of W27 antibody diluted in PBS, and the W37 antibody group received 500 ⁇ g of W37 antibody diluted in PBS via gavage every other day for three weeks. At 9 weeks of age, right ventricular systolic pressure and right ventricular hypertrophy index were measured, and feces were collected.
  • IgA antibody administration experiment (gnotobiotic rat experiment: PBS vs. W27 antibody)
  • PH model gnotobiotic rats were generated by orally administering feces from PH patients to 11-week-old germ-free rats and subcutaneously administering 60 mg/kg of monocrotaline at 15 weeks of age.
  • rats were divided into a vehicle group and a W27 antibody group.
  • the vehicle group received PBS
  • the W27 antibody group received 500 ⁇ g of W27 antibody diluted in PBS via gavage every other day for 3 weeks.
  • right ventricular systolic pressure and right ventricular hypertrophy index were measured, and feces were collected.
  • IgA antibody administration experiment (gnotobiotic rat experiment: PBS vs. PSGI1A antibody vs. rW27 antibody)
  • PH model gnotobiotic rats were prepared by orally administering patient-derived feces to 11-week-old germ-free rats and subcutaneously administering 60 mg/kg of monocrotaline to 15-week-old rats.
  • rats were divided into vehicle, PSGI1A antibody, and rW27 antibody groups.
  • the vehicle group received PBS
  • the PSGI1A antibody group received 500 ⁇ g of PSGI1A antibody diluted in PBS
  • the rW27 antibody group received 500 ⁇ g of rW27 antibody diluted in PBS via gavage every other day for 3 weeks.
  • right ventricular systolic pressure and right ventricular hypertrophy index were measured, and feces were collected.
  • IgA antibody administration experiment (gnotobiotic rat experiment: 3H12LJ antibody vs. rW27 antibody)
  • PH model gnotobiotic rats were prepared by orally administering patient-derived feces to 11-week-old germ-free rats and subcutaneously administering 60 mg/kg of monocrotaline to 15-week-old rats.
  • rats were divided into two groups: the 3H12LJ antibody group and the rW27 antibody group.
  • the 3H12LJ antibody group received 200 ⁇ g of 3H12LJ antibody diluted in PBS
  • the rW27 antibody group received 200 ⁇ g of rW27 antibody diluted in PBS via gavage every other day for 3 weeks.
  • right ventricular systolic pressure and right ventricular hypertrophy index were measured, and feces were collected.
  • 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 ⁇ L/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.
  • 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 ratio of the two weights (right ventricle/left ventricle weight ratio) was calculated as the right ventricular hypertrophy index to evaluate right ventricular hypertrophy.
  • fecal short-chain fatty acid concentrations 100 mg 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 ⁇ m 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 ⁇ M EDTA, 20 mM Bis-Tris) were used.
  • 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.
  • Sequencing data quality control 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.
  • FDR false discovery rate
  • Metagenomic analysis (1) DNA was extracted from the 16S analysis stool using the NecleoSpin DNA stool (Macherey-Nagel). 16S Metagenomic Sequencing Library Preparation (Illumina) was used with 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)) according to the manufacturer's protocol. 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.
  • OTUs operational taxonomic units
  • 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.
  • 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.
  • 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).
  • ORFs Open reading frames
  • 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.
  • BLASTP version v0.9.32.133 was used to align the deduced amino acid sequences translated from the ORF catalog with the KEGG protein database in DIAMOND.
  • 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. Ortholog abundance data of microbial genes were then also normalized.
  • IgA Antibody Bacterial Binding Test After counting the number of bacteria cultured as described above, the bacteria were suspended in 50 mM sodium carbonate solution to a concentration of 1 x 108 bacteria/50 ⁇ l to obtain a bacterial suspension. 50 ⁇ l of the bacterial suspension was added to each well of an ELISA plate and allowed to stand overnight at 4°C to immobilize the bacteria. The ELISA plate was washed using a plate washer (Vaccu-Pette/96 multiwell pipetter, Sigma-Aldrich). The ELISA plate with immobilized bacteria was washed three times with PBS, and 150 ⁇ l of PBS containing 1% serum albumin (BSA) (Wako) was added to each well.
  • BSA serum albumin
  • Blocking was performed by allowing the plate to stand overnight at 4°C.
  • a dilution series of each IgA antibody was prepared in the wells of a 96-well plate.
  • the IgA antibody was serially diluted in 1% BSA-containing PBS at 1x, 1/3x, 1/10x, 1/30x, 1/100x, 1/300x, 1/1,000x, and 1/3,000x.
  • the 1% BSA-containing PBS was removed from the ELISA plate, and 50 ⁇ l of serially diluted antibody was added to each well and incubated at room temperature for 1 hour.
  • the ELISA plate was then washed three times with 0.05% Tween 20 (Chem Cruz)-containing PBS.
  • AP Alkaline phosphatase
  • AP-conjugated anti-goat mouse IgA final concentration 0.5 ⁇ g/ml, Southern Biotech
  • AP-conjugated anti-goat human IgA final concentration 0.5 ⁇ g/ml, Southern Biotech
  • 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 was washed again with PBS containing 0.05% Tween 20, and 50 ⁇ l of the substrate reaction solution was added to each well.
  • OD 405 nm was measured using a Tristar2 LB942 (Berthold Technologies) to evaluate the binding of IgA antibodies to bacteria, compared with a negative control without IgA antibodies.
  • 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/ ⁇ l.
  • Rothia aeria was centrifuged at 5,000g for 5 minutes at room temperature and diluted to 2,000 cells/ ⁇ l. Five ⁇ l 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 ⁇ g/ml. Five ⁇ l of the IgA antibody solution or PBS containing 0.1% BSA (control) was added to each bacterial dilution. Streptococcus pasteurianus was incubated for 1 hour at 37°C. 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 counted by plating. Streptococcus pasteurianus or Rothia aeria was cultured on TSB agar medium under each condition, and colony-forming units (CFU) were calculated.
  • CFU colony-forming units
  • Fluorescence activated cell sorter (FACS) buffer was then added to the bacterial suspension, which was then centrifuged at 8,000 g for 5 minutes at 4°C, and the supernatant was removed. 15 ⁇ g of biotinylated mouse monoclonal IgA antibody was added to 6 ⁇ 10 6 bacteria and incubated on ice for 20 minutes. Next, 20 ⁇ l of PE (Phycoerythrin)/Cyanine7-Streptavidin (final concentration 10 ⁇ g/ml, BioLegend) was added to the bacterial 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.
  • FACS buffer was prepared by adding 10% fetal bovine serum (FBS, Nichirei) and EDTA (final concentration 5 ⁇ M, Nacalai Tesque) to PBS and sterilizing the mixture through a 0.22 ⁇ m filter.
  • FBS fetal bovine serum
  • EDTA final concentration 5 ⁇ M, Nacalai Tesque
  • Fig. 1A Six-week-old male SD rats were continuously housed in a hypoxic chamber with 10% oxygen for 3 weeks to create a hypoxia-induced PH model rat (Hx) (Fig. 1A). Six-week-old male SD rats were subcutaneously administered monocrotaline at 60 mg/kg and housed in a normoxic environment (normoxia) for 3 weeks to create a monocrotaline-induced PH model rat (MCT) (Fig. 1A).
  • Hx hypoxia-induced PH model rat
  • MCT monocrotaline-induced PH model rat
  • 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.
  • PH pathology pulmonary arterial medial thickening, right ventricular systolic pressure, and right/left ventricular mass ratio
  • 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.
  • 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.
  • 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).
  • PH patients exhibit altered gut microbiota characterized by ectopic colonization of oral bacteria. Based on the above findings, we hypothesized that the 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 alpha diversity (Faith PD and Shannon index) 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).
  • the results of the volcano plot analysis of the intestinal microbiota composition revealed that oral bacteria such as Streptococcus, Rothia, and Actinomyces were increased in the intestines 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 of PH patients were significantly reduced compared to those of healthy controls (Figure 5B), suggesting that the intestinal environment deviates from a healthy state. These results confirmed that the intestinal microbiota of PH patients is altered by the colonization of oral bacteria. Furthermore, the fecal IgA concentration of PH patients was measured using ELISA, and found to be increased in PH patients ( Figure 5C), suggesting that ineffective IgA antibodies are produced in the intestinal tract of PH patients.
  • SCFA short-chain fatty acid
  • Ruminococcus gnavus coexists in a network with multiple oral bacteria, which is associated with poor prognosis.
  • Analysis of the gut microbiota of PH patients revealed that the relative frequency of Ruminococcus gnavus was higher in PH patients with a higher oral bacterial score.
  • network analysis of bacteria correlated with Ruminococcus gnavus revealed that Ruminococcus gnavus coexists in a network with Streptococcus, Veillonella, Rothia, and Fusobacterium (all core oral bacteria) in the intestine (Fig. 6A).
  • 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.
  • 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.
  • Right ventricular systolic pressure and right/left ventricular mass ratio were also measured in each rat. Both right ventricular systolic pressure and right/left ventricular mass ratio were lower in F344 rats housed in a germ-free environment compared to F344 rats housed in an SPF environment, indicating significant suppression of monocrotaline-induced PH (Figure 7C). Healthy donor fecal transplanted F344 rats also exhibited similar right ventricular systolic pressure and right/left ventricular mass ratio to F344 rats housed in a germ-free environment, indicating significant suppression of monocrotaline-induced PH (Figure 7C).
  • genes related to macrophage chemotaxis (C5ar1, Cxcl17, etc.), lymphocyte chemotaxis (Ccl9, Ccl6, etc.), protein processing (Cma1, Gzmb, etc.), IL-6 response (Mcpt1, Prnp, etc.), and xenobiotic stimulation (Cebpa, Egr1, etc.) were found to be upregulated (Fig. 8C).
  • genes related to macrophage chemotaxis C5ar1, Cxcl17, etc.
  • lymphocyte chemotaxis Ccl9, Ccl6, etc.
  • protein processing Cma1, Gzmb, etc.
  • Mcpt1, Prnp, etc. IL-6 response
  • xenobiotic stimulation (Cebpa, Egr1, etc.) were found to be upregulated (Fig. 8C).
  • genes related to angiogenesis and vasculogenesis were upregulated (Fig. 8B), and in particular, expression of angiogenesis genes
  • Ruminococcus gnavus synergizes with Streptococcus pasteurianus to worsen PH.
  • Ruminococcus gnavus and Streptococcus pasteurianus to germ-free rats (Fig. 9A).
  • 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.
  • monocrotaline was administered subcutaneously, and right ventricular systolic pressure and right ventricular hypertrophy index were measured 21 days after monocrotaline administration.
  • W27 and W37 antibodies correct alterations in the intestinal microbiota and improve PH pathology in monocrotaline-challenged (MCT)-induced PH model rats.
  • the effects of W27 and W37 antibody administration on PH pathology were examined using monocrotaline-challenged (MCT)-induced PH model rats.
  • MCT monocrotaline-challenged
  • 6-week-old male F344 rats housed in an SPF environment were subcutaneously administered 60 mg/kg of monocrotaline to induce PH.
  • 500 ⁇ g of W27 or 500 ⁇ g of W37 antibody were administered via gavage every other day for 3 weeks (Figure 14A).
  • mean arterial pressure, right ventricular systolic pressure, and right ventricular hypertrophy index were measured, and fecal samples were collected for intestinal microbiota analysis.
  • W27 antibody corrects altered intestinal microbiota and improves PH pathology in gnotobiotic rats.
  • the results of the volcano plot analysis of the intestinal microbiota composition showed that the W27 antibody-treated group had decreased Eggerthella, Clostridium innocuum, Citrobacter, and Streptococcus compared to the vehicle group, while there was an increase in butyrate-producing bacteria such as Eubacterium nodatum, Oscilospira, Ruminococcaceae UCD-005, Erysipelotrichaceae, Anaerofilum, and Roseburia (Figure 16D).
  • butyrate-producing bacteria such as Eubacterium nodatum, Oscilospira, Ruminococcaceae UCD-005, Erysipelotrichaceae, Anaerofilum, and Roseburia
  • 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, starting the day after monocrotaline administration, rW27 or PGSI1A antibody (500 ⁇ g) was administered by gavage every other day for 3 weeks (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.
  • rW27 and PGSI1A antibodies increased the number of butyricoccus, Subdolignulum, Coprococcus, and Faecalicacterium bacteria, which are butyrate-producing bacteria thought to suppress PH pathology ( Figure 17C).
  • principal component analysis (PCA) analysis of the intestinal microbiota showed significant changes in the rW27 antibody-administered group compared to the vehicle group ( Figure 18B).
  • Partial least squares discriminant analysis (PLS-DA) showed significant changes in the intestinal microbiota in the rW27 antibody-administered and PGSI1A antibody-administered groups compared to the vehicle group ( Figure 18C).
  • the rW27 antibody-administered group showed significant decreases in Fusobacterium, Eggherthella, and Enterococcus in the intestinal microbiota compared to the vehicle group, while Coprococcus and Anaerostipes (all butyrate-producing bacteria) increased (Figure 18D). Furthermore, the PGSI1A antibody-administered group showed significant decreases in Fusobacterium and Enterococcus, and an increase in Anaerostipes compared to the vehicle group ( Figure 18D).
  • rW27 and 3H12LJ antibodies were administered a diluted solution of feces from PH patients at 11 weeks of age, monocrotaline at 60 mg/kg at 15 weeks of age, and, starting the day after monocrotaline administration, rW27 antibody 200 ⁇ g or 3H12LJ antibody 200 ⁇ g were administered by gavage once every two days for three weeks (Figure 19A).
  • Figure 19A right ventricular systolic pressure and right ventricular hypertrophy index were measured, and the collected stool samples were used for intestinal microbiota analysis.

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Abstract

La présente divulgation aborde le problème de la fourniture d'un médicament destiné à la prévention ou au traitement de l'hypertension pulmonaire, le médicament ayant un mécanisme permettant de corriger la transformation de la flore intestinale associée à l'hypertension pulmonaire.　Ce médicament permettant de prévenir ou de traiter l'hypertension pulmonaire comprend un anticorps ou similaire ou un fragment de liaison à l'antigène de celui-ci, l'anticorps ou similaire comprenant : une région variable de chaîne lourde qui présente une CDR1 contenant la séquence d'acides aminés représentée par SEQ ID NO : 1, une CDR2 contenant la séquence d'acides aminés représentée par SEQ ID NO : 2, et une CDR3 contenant la séquence d'acides aminés représentée par SEQ ID NO : 3 ; et une région variable de chaîne légère qui présente une CDR1 contenant la séquence d'acides aminés représentée par SEQ ID NO : 4, une CDR2 contenant la séquence d'acides aminés représentée par SEQ ID NO : 5, et une CDR3 contenant la séquence d'acides aminés représentée par SEQ ID NO : 6.
PCT/JP2025/015144 2024-04-17 2025-04-17 Médicament pour la prévention ou le traitement de l'hypertension pulmonaire Pending WO2025220732A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014142084A1 (fr) * 2013-03-11 2014-09-18 学校法人関西文理総合学園 PROCÉDÉ DE PRODUCTION D'UN ANTICORPS IgA MONOCLONAL
WO2021167088A1 (fr) * 2020-02-21 2021-08-26 良和 中岡 Composition pour soulager l'hypertension pulmonaire, procédé de prédiction du pronostic de l'hypertension pulmonaire, procédé d'aide à la détermination de la gravité de l'hypertension pulmonaire, et procédé d'aide au diagnostic de l'hypertension pulmonaire
WO2023277166A1 (fr) * 2021-07-02 2023-01-05 国立大学法人 東京大学 Composition contenant un anticorps monoclonal pour supprimer des bactéries clostridium difficile
WO2023277142A1 (fr) * 2021-07-01 2023-01-05 国立大学法人 東京大学 Anticorps se liant à clostridium difficile

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014142084A1 (fr) * 2013-03-11 2014-09-18 学校法人関西文理総合学園 PROCÉDÉ DE PRODUCTION D'UN ANTICORPS IgA MONOCLONAL
WO2021167088A1 (fr) * 2020-02-21 2021-08-26 良和 中岡 Composition pour soulager l'hypertension pulmonaire, procédé de prédiction du pronostic de l'hypertension pulmonaire, procédé d'aide à la détermination de la gravité de l'hypertension pulmonaire, et procédé d'aide au diagnostic de l'hypertension pulmonaire
WO2023277142A1 (fr) * 2021-07-01 2023-01-05 国立大学法人 東京大学 Anticorps se liant à clostridium difficile
WO2023277166A1 (fr) * 2021-07-02 2023-01-05 国立大学法人 東京大学 Composition contenant un anticorps monoclonal pour supprimer des bactéries clostridium difficile

Non-Patent Citations (2)

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Title
CHEN YI-HANG, YUAN WEN, MENG LIU-KUN, ZHONG JIU-CHANG, LIU XIAO-YAN: "The Role and Mechanism of Gut Microbiota in Pulmonary Arterial Hypertension", NUTRIENTS, vol. 14, no. 20, 1 January 2022 (2022-01-01), CH , pages 1 - 15, XP093363770, ISSN: 2072-6643, DOI: 10.3390/nu14204278 *
重城喬行, 慢性血栓塞栓性肺高血圧症（CTEPH）の病態と治療, 呼吸器内科, 2022, vol. 41, no. 6, pp. 567-574, ISSN 1884-2887, (Respiratory medicine), non-official translation (JUJO, Takayuki, Pathogenesis and treatment of chronic thromboembolic pulmonary hypertension (CTEPH)) *

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