Combination therapy of grem1 antagonists and activin receptor antagonists for the treatment of PAH

Abstract

The present disclosure provides herein a novel gremlin 1 (GREM1) antagonist. The present disclosure also provides therapies for treating pulmonary arterial hypertension (PAH), including monotherapy comprising the GREM1 antagonist, and combination therapy comprising the GREM1 antagonist and an activin receptor antagonist and optionally one or more therapeutic agents for treating PAH.

Description

Technical Field

[0001] This disclosure generally relates to a novel combination therapy for the treatment of pulmonary arterial hypertension (PAH) comprising a GREM1 antagonist and an activin receptor antagonist. Background Technology

[0002] Pulmonary arterial hypertension (PAH) is a chronic and progressive disease characterized by high blood pressure in the blood vessels (pulmonary arteries) that supply the lungs. PAH is a serious condition that can damage the right side of the heart. The walls of the pulmonary arteries thicken and harden, and become unable to dilate to allow blood to flow. Reduced blood flow makes it more difficult for the right side of the heart to pump blood through the arteries, which, if left untreated, can lead to heart failure and eventually death. See, for example, Montani, D., Günther, S., Dorfmüller, P. et al., Pulmonary arterial hypertension. Orphanet J Rare Dis 8, 97 (2013).

[0003] Several therapeutic agents have been developed for the treatment (e.g., medical administration) of PAH, including (but not limited to) prostaglandins (e.g., trepoprostenil, epoprostenol, iloprost), endothelin receptor antagonists (e.g., bosentan, ambrisentan), and phosphodiesterase type 5 inhibitors (e.g., sildenafil, tadalafil).

[0004] Sildenafil has been used in Europe since 2005 for patients with PAH class II to III, and is licensed in Canada and the United States for patients with PAH class II to IV. Class I, II, and IV are four categories proposed by the New York Heart Association (NYHA) to classify patients into functional levels based on the degree of respiratory limitation during physical activity. See, for example, Montani, D., Günther, S., Dorfmüller, P. et al., Pulmonary arterial hypertension. Orphanet J Rare Dis 8, 97 (2013).

[0005] Despite treatment advancements, PAH remains incurable, and many patients develop right ventricular failure. Therefore, novel therapies for PAH with improved efficacy and reduced side effects are needed. Summary of the Invention

[0006] Among other things, this disclosure provides a method for treating pulmonary arterial hypertension (PAH) in a subject in need. In some embodiments, the method includes administering a combination of a GREM1 antagonist and an activin receptor antagonist to the subject to treat the subject's PAH.

[0007] In one aspect, this disclosure provides a method for treating PAH in a subject with an inadequate or unsatisfactory response to an activin receptor antagonist, the method comprising administering a GREM1 antagonist to the subject, optionally in combination with an activin receptor antagonist, and further optionally in combination with at least one additional therapeutic agent.

[0008] In another aspect, this disclosure provides an anti-GREM1 antibody or its antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises QVQLVQSGAEVKKPGASVKVSCKASGYX1FTSSGIGWVX2QAX3GQGLEWX4GEIYPRSGNTYNNEKFKGRX5TX6TX7DX8STSTX9YMELRSLRSDDTAVYX 10 CX 11 The amino acid sequence shown in REAYSHHYYAMDYWGQGTTVTVSS (SEQ ID NO: 13), and / or the light chain variable region contains DX. 12 VMTQTPLSLX 13 VTPGQPASISCRSSQSLLHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRX 14 The amino acid sequence shown in SEQ ID NO: 17 is as follows: X1 is S or T; X2 is K or R; X3 is S or P; X4 is I or M; X5 is A or V; X6 is L or M; X7 is A or T; X8 is K or T; X9 is V or A; X10 is F or Y; X11 is V or A; X12 is V or I; X13 is P or S; X14 is L or F.

[0009] In another aspect, this disclosure provides a pharmaceutical composition comprising the anti-GREM1 antibody or its antigen-binding fragment provided herein.

[0010] In another aspect, this disclosure provides a kit comprising (a) a first composition comprising the anti-GREM1 antibody or an antigen-binding fragment thereof provided herein, and (b) a second composition comprising an activin receptor antagonist.

[0011] In another aspect, this disclosure provides a method for treating PAH in a subject in need, comprising administering to the subject a therapeutically effective amount of the antiGREM1 antibody or antigen-binding fragment thereof provided herein, or a pharmaceutical composition or kit provided herein.

[0012] It should be understood that the foregoing general description and the following detailed description are exemplary and explanatory only, and do not limit the invention. Furthermore, the accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and, together with the description, serve to explain the principles of the invention. Attached Figure Description

[0013] Figure 1 This demonstrates the binding affinity of the humanized 36F5 antibody provided in this paper for human GREM1.

[0014] Figure 2 This demonstrates the binding affinity of the humanized 36F5 antibody provided in this paper for human GREM1.

[0015] Figure 3 The humanized 36F5 antibody presented in this paper demonstrates a higher affinity for binding to human GREM1 compared to the chimeric 36F5.

[0016] Figure 4 This study demonstrates the activity of the humanized 36F5 antibody provided in blocking the binding of human GREM1 to BMP2.

[0017] Figure 5 This study demonstrates the activity of the humanized 36F5 antibody provided in blocking the binding of human GREM1 to BMP4.

[0018] Figure 6 This study demonstrates the activity of the humanized 36F5 antibody provided in blocking the binding of human GREM1 to BMP7.

[0019] Figure 7 Compared with the solvent group, the sildenafil treatment group, the 36F5-HdLa treatment group, the ActRIIA-mFc treatment group, and the 36F5-HdLa+ActRIIA-mFc treatment group all showed a decrease in right ventricular systolic pressure (RVSP), and the combination group of 36F5-HdLag+ActRIIA-mFc was more effective than individual treatment with 36F5-HdLa or ActRIIA-mFc alone.

[0020] Figure 8Compared with the solvent group, the sildenafil treatment group, the 36F5-HdLa treatment group, the ActRIIA-mFc treatment group, and the 36F5-HdLa+ActRIIA-mFc treatment group all showed a decrease in the Right Ventricular Hypertrophy Index (RVHI). Furthermore, the combination group of 36F5-HdLa+ActRIIA-mFc was more effective than 36F5-HdLa, ActRIIA-mFc, or sildenafil alone.

[0021] Figure 9 Compared with the solvent group, the percentage of pulmonary artery wall thickness (PAWT%) was reduced in the sildenafil treatment group, the 36F5-HdLa treatment group, the ActRIIA-mFc treatment group, and the 36F5-HdLa+ActRIIA-mFc treatment group. Furthermore, the combination group of 36F5-HdLa+ActRIIA-mFc was more effective than 36F5-HdLa, ActRIIA-mFc, or sildenafil alone.

[0022] The same reference numerals will be used throughout the accompanying drawings to refer to the same or similar parts. Detailed Implementation

[0023] The following description of this disclosure is intended only to illustrate various embodiments of this disclosure. Therefore, the specific modifications discussed should not be construed as limiting the scope of this disclosure. It will be apparent to those skilled in the art that various equivalents, variations, and modifications can be made without departing from the scope of this disclosure, and it should be understood that such equivalent embodiments are included herein. All references cited herein, including publications, patents, and patent applications, are incorporated herein by reference in their entirety.

[0024] In this application, unless otherwise specifically stated, the use of the singular includes the plural. In this application, unless otherwise stated, the use of “or” means “and / or”. Furthermore, the use of the term “including” and other forms (such as “includes” and “included”) is not restrictive. Additionally, unless otherwise specifically stated, terms such as “element” or “component” cover elements and components that include one unit as well as elements and components that include more than one subunit. Furthermore, the section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0025] definition As used herein, unless otherwise indicated herein or clearly contradicted by the context, the terms “a / an,” “the,” and similar terms used in the context of this invention (especially in the context of the claims) shall be interpreted to cover both the singular and the plural.

[0026] As used herein, terms such as “comprises,” “comprised,” “comprising,” “contains,” “containing,” etc., are intended to be inclusive or open-ended and do not exclude other unlisted elements or methodological steps.

[0027] In this document, the reference to "about" a value or parameter includes (and describes) the implementation of said value or parameter itself. For example, a description of "about X" includes a description of "X". Numerical ranges include the numbers that define the range. Generally, the term "about" refers to the greater of a specified value of a variable and all values ​​of the variable within the experimental error of the specified value (e.g., within the 95% confidence interval of the mean) or within 10% of the specified value. When the term "about" is used in the context of a time period (year, month, week, day, etc.), the term "about" means the time period plus or minus the next subordinate time period (e.g., about 1 year means 11-13 months; about 6 months means 6 months plus or minus 1 week; about 1 week means 6-8 days; etc.) or within 10% of the specified value.

[0028] As used herein, the term "antagonist" in relation to proteins (e.g., GREM1, activin receptors of ActRIIA, for example) refers to any molecule that partially or completely prevents, blocks, inhibits, neutralizes, or reduces the biological activity or function of a protein (e.g., GREM1, activin receptors of ActRIIA, for example), acting directly on the protein or indirectly by acting on another molecule that interacts with the protein (e.g., a protein binding partner, such as a protein ligand). Suitable antagonists may include (but are not limited to) antibodies, antisense oligonucleotides, peptides, and small molecules.

[0029] As used herein, the term "antibody" includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multivalent antibody, bivalent antibody, monovalent antibody, multispecific antibody, or bispecific antibody that binds to a specific antigen, or any polypeptide that mimics an antibody in its ability to bind to a specific antigen. A natural, complete antibody comprises two heavy (H) chains and two light (L) chains. Mammalian heavy chains are classified as α, δ, ε, γ, and μ, each consisting of a variable region (VH) and first, second, and third constant regions (CH1, CH2, and CH3, respectively); mammalian light chains are classified as λ or κ, each consisting of a variable region (VL) and a constant region. Antibodies have a "Y" shape, where the stem of the Y consists of the second and third constant regions of two heavy chains bonded together by disulfide bonds. Each arm of the Y comprises the variable region and first constant region of a single heavy chain, which binds to the variable and constant regions of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding. The variable regions in both chains typically contain three highly variable loops, called complementarity-determining regions (CDRs) (light chain CDRs include LCDR1, LCDR2, and LCDR3; heavy chain CDRs include HCDR1, HCDR2, and HCDR3). The CDR boundaries of the antibody and antigen-binding domains disclosed in this paper can be defined or identified using the conventions of Kabat, IMGT, AbM, Chothia, or Al-Lazikani (Al-Lazikani, B., Chothia, C., Lesk, AM, ...). J. Mol. Biol. , 273(4), 927 (1997); Chothia, C. et al., J Mol Biol. December 5; 186(3):651-63 (1985); Chothia, C. and Lesk, AM, J.Mol.Biol. , 196,901 (1987); NR Whitelegg et al., Protein Engineering , v13(12), 819-824 (2000); Chothia, C. et al., Nature . December 21–28; 342(6252): 877–83 (1989); Kabat EA et al., National Institutes of Health Bethesda, Md. (1991); Marie-Paule Lefranc et al. Developmental and Comparative Immunology , 27: 55-77 (2003); Marie-Paule Lefranc et al., Immunome Research , 1(3), (2005); Marie-Paule Lefranc, Molecular Biology of B cells (Second Edition)(Chapter 26, 481-514, (2015)). Three CDRs are inserted between side extensions called framework regions (FRs), which are more conserved than the CDRs and form a scaffold supporting the hypervariable loop. The constant regions of the heavy and light chains do not participate in antigen binding but exhibit various effector functions. Antibodies are classified based on the amino acid sequence of their heavy chain constant regions. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Several major antibody classes are divided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain). In some embodiments, the antibodies provided herein encompass any of their antigen-binding fragments.

[0030] As used herein, the term "antigen-binding fragment" refers to a fragment formed from an antibody fragment containing one or more CDRs (e.g., an antibody fragment), or any other part bound to an antigen but not containing the complete structure of a natural antibody (e.g., an antibody moiety). Examples of antigen-binding fragments / migrations include (but are not limited to) bifunctional antibodies, Fab, Fab', F(ab')2, Fd, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized bifunctional antibodies (ds bifunctional antibodies), single-chain antibody molecules (scFv), scFv dimers (bivalent bifunctional antibodies), multispecific antibodies, camelified single-domain antibodies, nanobodies, domain antibodies, and bivalent domain antibodies. Antigen-binding fragments / migrations are capable of binding to the same antigen to which the parent antibody is bound. In some embodiments, the antigen-binding fragment / migration may contain one or more CDRs from a specific parent antibody.

[0031] The term "Fab" in antibody refers to the monovalent antigen-binding fragment of the antibody, which consists of a single light chain (both the variable and constant regions) bound by disulfide bonds to the variable region and the first constant region of a single heavy chain. Fab can be obtained by papain digestion of the antibody at residues near the N-terminus of the disulfide bonds between the heavy chains in the hinge region.

[0032] "Fab" refers to a Fab fragment that includes a portion of the hinge region, which can be obtained by pepsin digestion of the antibody at residues near the C-terminus of the disulfide bonds between the heavy chains in the hinge region. Therefore, it differs from Fab in that it contains a small number of residues (including one or more cysteine ​​residues) in the hinge region.

[0033] “F(ab')2” refers to a Fab' dimer that contains two light chains and a portion of two heavy chains.

[0034] The term "Fv" in antibody refers to the smallest fragment of the antibody that carries the complete antigen-binding site. An Fv fragment consists of a variable region of a single light chain that binds to a variable region of a single heavy chain. "dsFv" refers to an Fv fragment stabilized by disulfide bonds, where the variable region of the single light chain is linked to the variable region of the single heavy chain by a disulfide bond.

[0035] "Single-chain Fv antibody" or "scFv" refers to an engineered antibody composed of a light chain variable region and a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region are directly linked to each other or linked to each other through peptide linker sequences (Huston JS et al., Proc Natl Acad Sci USA , 85:5879(1988)). A “scFv dimer” refers to a single chain containing two heavy chain variable regions and two light chain variable regions, as well as a linker. In some embodiments, the “scFv dimer” is a bivalent bifunctional antibody or a bivalent scFv (BsFv) comprising a dimerized VH-VL (linked via a peptide linker) and another VH-VL moiety, such that the VH of one moiety coordinates to the VL of the other moiety, forming two binding sites that can target the same antigen (or epitope) or different antigens (or epitopes). In other embodiments, the “scFv dimer” is a bispecific bifunctional antibody comprising associated VH1-VL2 (linked via a peptide linker) and VL1-VH2 (also linked via a peptide linker), such that VH1 coordinates to VL1 and VH2 coordinates to VL2, with each coordination pair having a different antigen specificity.

[0036] "Single-chain Fv-Fc antibody" or "scFv-Fc" refers to an engineered antibody composed of scFvs linked to the Fc region of the antibody.

[0037] "Camelized single-domain antibodies," "heavy-chain antibodies," or "nanobodies" refer to antibodies containing two VH domains and lacking a light chain (Riechmann L. and Muyldermans S.). J Immunol Methods December 10; 231(1-2):25-38 (1999); Muyldermans S., J Biotechnol. June;74(4):277-302 (2001); WO94 / 04678; WO94 / 25591; US ​​Patent No. 6,005,079). Heavy chain antibodies were originally derived from camelids (camels, dromedaries, and llamas). Although they do not contain light chains, camelidized antibodies possess a true antigen-binding lineage (Hamers-Casterman C. et al., NatureJune 3; 363(6428):446-8 (1993); Nguyen VK. et al., “Heavy-chain antibodies in Camelidae; a case of evolutionary innovation”, Immunogenetics April; 54(1):39-47 (2002); Nguyen VK et al., Immunology May; 109(1):93-101 (2003). The variable domain (VHH domain) of heavy chain antibodies represents the smallest known antigen-binding unit generated by adaptive immune responses (Koch-Nolte F. et al., FASEB J. November;21(13):3490-8. Electronic version June 15, 2007 (2007)). “Bifunctional antibodies” comprise small antibody fragments having two antigen-binding sites, wherein said fragments contain a VH domain (VH-VL or VL-VH) linked to a VL domain in a single polypeptide chain (see, for example, Holliger P. et al., Proc Natl Acad Sci USA July 15; 90(14):6444-8 (1993); EP404097; WO93 / 11161). Two domains on the same strand cannot pair because the linker is too short, so the domain is forced to pair with a complementary domain on another strand, thus creating two antigen-binding sites. The antigen-binding sites can target the same or different antigens (or epitopes).

[0038] "Domain-specific antibodies" refer to antibody fragments containing only heavy chain variable regions or light chain variable regions. In some embodiments, two or more VH domains are covalently linked by peptide linkers to form bivalent or multivalent domain antibodies. The two VH domains of a bivalent domain antibody can target the same or different antigens.

[0039] In some implementations, “(dsFv)2” comprises three peptide chains: two VH moieties are linked by peptide linkers and are bound to two VL moieties by disulfide bridging bonds.

[0040] In some embodiments, the “bispecific ds bifunctional antibody” comprises VH1-VL2 (linked via peptide linkers) and VL1-VH2 (also linked via peptide linkers) bound by a disulfide bridge between VH1 and VL1.

[0041] In some embodiments, the “bispecific dsFv” or “dsFv-dsFv” comprises three peptide chains: VH1-VH2 moieties, wherein the heavy chain is bound via a peptide linker (e.g., a long, flexible linker) and paired with the VL1 and VL2 moieties respectively via disulfide bridges. Each disulfide-paired heavy and light chain has a different antigen specificity.

[0042] As used herein, the term "humanized" means that the antibody or antigen-binding fragment includes a CDR derived from a non-human animal, a FR region derived from a human, and, where appropriate, a constant region derived from a human. In some embodiments, amino acid residues of the variable region framework of the humanized gremlin antibody are substituted for sequence optimization. In some embodiments, the variable region framework sequence of the humanized gremlin antibody chain has at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with the corresponding human variable region framework sequence.

[0043] As used herein, “anti-GREM1 antibody” refers to an antibody that can specifically bind to GREM1 (e.g., human GREM1 or non-human GREM1) with sufficient specificity and / or affinity, for example, to provide therapeutic use.

[0044] As used in this article, “affinity” refers to the strength of the non-covalent interaction between an immunoglobulin molecule (i.e., an antibody) or a fragment thereof and an antigen.

[0045] As used herein, the terms "specific binding" or "specifically binds" refer to a non-random binding reaction between two molecules (e.g., between an antibody and an antigen). In some embodiments, the antibody or antigen binding fragments provided herein are in the form of ≤10 -6 M (e.g., ≤5×10) -7 M, ≤2×10 -7 M, ≤10 -7 M, ≤5×10 -8 M, ≤2×10 -8 M, ≤10 -8 M, ≤5×10 -9 M, ≤4×10 -9 M, ≤3×10 -9 M, ≤2×10 -9 M or ≤10 -9 The binding affinity (KD) of grimlin1 (M) specifically binds to human and / or non-human grimlin1. As used in this article, KD refers to the ratio of dissociation rate to association rate (k...). off / k onThe KD value can be determined using any conventional method known in the art, including (but not limited to) surface plasmon resonance methods, micro-thermophoresis methods, HPLC-MS methods, and flow cytometry (e.g., FACS). In some embodiments, the KD value can be appropriately determined using flow cytometry. Various immunoassay modalities can be used to select antibodies that have a specific immunoreactivity with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies that have a specific immunoreactivity with a protein (for a description of immunoassay modalities and conditions that can be used to determine specific immunoreactivity, see, for example, Harlow and Lane). Using Antibodies, A Laboratory Manual (1998)). Typically, the signal generated by the specific or selective binding reaction is at least twice the background signal and more typically, at least 10 to 100 times the background signal.

[0046] The "sequence identity percentage (%)" for an amino acid sequence (or nucleic acid sequence) is defined as the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to those in a reference sequence after sequence alignment and, where necessary, the introduction of vacancies to achieve maximum correspondence. Alignment can be performed, for example, using publicly available tools such as BLASTN, BLASTp (available on the National Center for Biotechnology Information (NCBI) website, and see also Altschul SF et al.), to determine the amino acid (or nucleic acid) sequence identity percentage. J. Mol. Biol. , 215:403-410 (1990); Stephen F. et al., Nucleic Acids Res. , 25:3389-3402 (1997)), ClustalW2 (available on the European Institute for Bioinformatics website, see also Higgins DG et al.), Methods in Enzymology , 266:383-402 (1996); Larkin MA et al., Bioinformatics (Oxford, England)(see, 23(21): 2947-8(2007)), and ALIGN or Megalign (DNASTAR) software. Those skilled in the art can use the default parameters provided by the tools, or can, for example, customize parameters suitable for alignment by selecting an appropriate algorithm. In some embodiments, the positions of different residues may differ due to conserved amino acid substitutions. A “conserved amino acid substitution” is an amino acid substitution in which an amino acid residue is replaced by another amino acid residue with a side chain (R group) having similar chemical properties (e.g., charge or hydrophobicity). Generally, conserved amino acid substitutions do not substantially alter the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conserved substitutions, the similarity percentage or similarity can be increased to correct for the conservatism of the substitution. Methods for making such adjustments are well known to those skilled in the art. See, for example, Pearson (1994). Methods Mol. Biol. 24:307-331, which are incorporated into this paper by reference.

[0047] As used herein, a “homologue sequence” is a polynucleotide sequence (or its complementary strand) or amino acid sequence that, when optionally aligned, has at least 80% (e.g., at least 85%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with another sequence.

[0048] As used herein, the terms “treatment,” “treat,” or “treating” in relation to a condition refer to the management, elimination, reduction, or improvement of the condition and / or its associated symptoms. While not excluded, treating a condition does not necessarily require the complete elimination of the condition or its associated symptoms. The term “treatment” as used herein can include “preventive treatment,” which is applied prior to the development of any symptoms or manifestations of the condition to reduce the likelihood of the occurrence or recurrence of the condition in individuals who are not affected but are at risk or susceptible to recurrence, or to reduce the likelihood of recurrence of a previously controlled condition. Within the meaning of this invention, “treatment” also includes prevention of recurrence or a preventive phase, as well as treatment of acute or chronic signs, symptoms, and / or functional impairments. Treatment may target symptoms, for example, to suppress symptoms. Treatment may be effective for short, moderate, or long-term periods, or may be a long-term treatment, such as in the case of maintenance therapy.

[0049] The term "therapeutic effective amount" or "effective amount" means the amount of a drug agent suitable for any treatment, alone or in combination with other doses, that produces a desired local or systemic therapeutic effect with a reasonable benefit / risk ratio. In the case of treating a specific disease, the desired local or systemic therapeutic effect is preferably related to inhibiting the course of the disease. This includes slowing disease progression and, in particular, interrupting or reversing disease progression. When administered for disease prevention, the amount is sufficient to avoid or delay the onset of the disease. Therapeutic effective amount or effective amount is not necessarily curative or preventative of the occurrence of the disease or symptom. The effective amount of the drug agent described herein will depend on the symptom to be treated; the severity of the disease; the patient's individual parameters, including age, physiological condition, body size, and weight; the duration of treatment; the type of concomitant therapy (if present); the specific route of administration; and similar factors. Therefore, the dosage of the drug agent described herein may depend on a variety of such parameters. Higher doses (or more effective doses achieved through different, more local routes of administration) may be used when the initial dose is insufficient to elicit a response in the patient. In some embodiments, the therapeutic effective amount of the drug agent will depend on factors such as its therapeutic index, solubility, etc.

[0050] As used herein, the “combination” administration of a drug with one or more other drugs includes simultaneous (parallel) administration and continuous administration in any order and in any form. It should be understood that, for example, the “combination” administration of drug A with drug B, or the “combination” use of drug A with drug B, can cover at least three situations, such as: 1) drug A and drug B are formulated as a single molecule or a single dosage form before or at the time of administration; 2) drug A and drug B are formulated as two separate dosage forms and provided (e.g., manufactured or sold) with instructions for the combined administration of drug A and drug B; and 3) drug A and drug B are formulated as two separate dosage forms and provided (e.g., manufactured or sold) separately with instructions for the following: i) when drug A is provided (e.g., manufactured or sold), drug A is administered in combination with drug B, or ii) when drug B is provided (e.g., manufactured or sold), drug B is administered in combination with drug A. In other words, the combined use of drug A and drug B does not necessarily mean that drug A and drug B must be provided (e.g., manufactured or sold) together.

[0051] As used herein, the term “pharmaceuticalally acceptable” indicates that a substance or composition is chemically and / or toxicologically compatible with other components constituting the formulation and / or the object to be treated therewith.

[0052] Treatment 1. Treatment of PAH with a combination of GREM1 antagonists and activin receptor antagonists In another aspect, this disclosure provides a method for treating pulmonary arterial hypertension (PAH) in a subject in need, comprising administering to the subject a combination of a GREM1 antagonist (e.g., in an effective amount) and an activin receptor antagonist (e.g., in an effective amount or a sub-effective amount).

[0053] In some embodiments, activin receptor antagonists and GREM1 antagonists exhibit at least an additive effect in treating PAH. As used herein, the term "additive" effect refers to an effect achieved by combining one agent with another that is not less than the sum of the effects expected from the individual agents. In some embodiments, the additive effect is equal to the sum of the effects of each agent when used independently.

[0054] In some implementations, the methods presented herein exhibit a synergistic effect in treating PAH. As used herein, a "synergistic" effect refers to an effect achieved by combining one drug with another that is greater than the simple sum of the effects achieved by administering the individual drugs alone. Synergistic effects are not merely additive. For example, if drug A produces a 2-unit effect and drug B produces a 2-unit effect, the additive effect is equal to a 4-unit effect; the synergistic effect is greater than 4 units, and its upper limit is unpredictable.

[0055] In some implementations, activin receptor antagonists and GREM1 antagonists exhibit at least an additive effect in reducing the right ventricular hypertrophy index (RVHI). RVHI, calculated as the ratio of right ventricular weight to the sum of left ventricular weight and interventricular septal weight, reflects the severity of right ventricular hypertrophy. The severity of right ventricular hypertrophy reflects the progression of PAH, a syndrome of right heart failure. In early PAH, the right ventricle tends to adapt to afterload by increasing contractility, while the size of the right ventricle increases very little or not at all. However, as the disease progresses, this adaptive response may become insufficient, leading to right ventricular dilation and eventually right ventricular failure. Right-sided heart failure is a leading cause of death in PAH (Grzegorz). Kopeć et al., Sotatercept as a next-generation therapy for pulmonary arterialhypertension: insights from the STELLAR trial. Cardiovascular Research(2023)119, e155-e157). Therefore, regular monitoring of right ventricular structure and function (e.g., right ventricular hypertrophy) is crucial for assessing the progression and prognosis of PAH. Thus, RVHI is an important measure for evaluating the effectiveness of PAH treatment. A decrease in RVHI indicates improved treatment efficacy for PAH, demonstrating a positive response to treatment intervention.

[0056] In some implementations, the activator receptor antagonist is administered in an effective amount.

[0057] In some implementations, the activator receptor antagonist is administered at a sub-effective amount. As used herein, the term "sub-effective amount" means an amount or dose of the active agent that is less than the effective amount required to achieve the desired therapeutic effect when administered alone.

[0058] For example, the sub-effective amount is a reduction of approximately 20%, 30%, 40%, 50%, 60%, or 70% relative to the effective amount. In some embodiments, the sub-effective amount of the activin receptor antagonist is approximately 0.1 mg / kg, approximately 0.2 mg / kg, approximately 0.3 mg / kg, approximately 0.4 mg / kg, approximately 0.5 mg / kg, or approximately 0.6 mg / kg.

[0059] Compared to the usual or effective dose when applied alone, the sub-effective dose of active agent is expected to produce lower therapeutic efficacy due to the reduced amount.

[0060] On the other hand, reduced doses may also produce fewer adverse effects or lower toxicity. In some embodiments, sub-effective doses of activin receptor antagonists reduce side effects compared to those observed when administered at effective doses. In some embodiments, side effects include (but are not limited to) nosebleeds, dizziness, telangiectasia, increased heme levels, thrombocytopenia, and elevated blood pressure.

[0061] Without being bound by any theory, it is believed that combining the GREM1 antagonist presented in this article with an activin receptor antagonist (where the activin receptor antagonist is administered at a sub-effective dose) can enhance the therapeutic efficacy of the activin receptor antagonist to a level comparable to that achieved with an effective dose, while reducing adverse effects or toxicities typically associated with effective doses.

[0062] In some implementations, the therapeutic efficacy of the activin receptor antagonist is generally maintained at a level comparable to that observed when administered at an effective dose. As used herein, the term "comparable to" regarding therapeutic efficacy means a level not less than 70%, 80%, or 90% of a reference level.

[0063] In some implementations, the GREM1 antagonist is administered before, simultaneously with, or after the activin receptor antagonist.

[0064] In some implementations, administration is performed orally, nasally, intravenously, subcutaneously, sublingually, or intramuscularly.

[0065] i) GREM1 antagonists In some embodiments, the GREM1 antagonists provided herein comprise an anti-GREM1 antibody or an antigen-binding fragment thereof. In some embodiments, the anti-GREM1 antibody or its antigen-binding fragment comprises the heavy chains HCDR1, HCDR2, and HCDR3 and / or the light chains LCDR1, LCDR2, and LCDR3 of any one of the anti-GREM1 antibodies (e.g., 14E3, 69H5, 22F1, 56C11, 36F5, 42B9, 67G11) described in PCT patent application PCT / CN2022 / 072297, which is incorporated herein by reference. In some embodiments, the anti-GREM1 antibody or its antigen-binding fragment provided herein comprises heavy chains HCDR1, HCDR2, and HCDR3 and / or light chains LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises an amino acid sequence containing SSGIG (SEQ ID NO: 1) or a homologous sequence having at least 80% sequence identity with it; HCDR2 comprises an amino acid sequence containing EIYPRSGNTYNNEKFKG (SEQ ID NO: 2) or a homologous sequence having at least 80% sequence identity with it; HCDR3 comprises an amino acid sequence containing EAYSHHYYAMDY (SEQ ID NO: 3) or a homologous sequence having at least 80% sequence identity with it; LCDR1 comprises an amino acid sequence containing RSSQSLLHSNGNTYLE (SEQ ID NO: 4) or a homologous sequence having at least 80% sequence identity with it; and LCDR2 comprises an amino acid sequence containing KVSNRFS (SEQ ID NO: 4). 5) an amino acid sequence or a homologous sequence having at least 80% sequence identity with it, and LCDR3 contains an amino acid sequence containing FQGSHVPFT (SEQ ID NO: 6) or a homologous sequence having at least 80% sequence identity with it.In some embodiments, the anti-GREM1 antibody or its antigen-binding fragment comprises heavy chains HCDR1, HCDR2, and HCDR3 and / or light chains LCDR1, LCDR2, and LCDR3, wherein: HCDR1 comprises an amino acid sequence containing TYGMA (SEQ ID NO: 25) or a homologous sequence having at least 80% sequence identity with it; HCDR2 comprises an amino acid sequence containing WINTLSGEPTYADDFKG (SEQ ID NO: 26) or a homologous sequence having at least 80% sequence identity with it; HCDR3 comprises an amino acid sequence containing EPMDY (SEQ ID NO: 27) or a homologous sequence having at least 80% sequence identity with it; LCDR1 comprises an amino acid sequence containing KSSQSLLDSDGKTYLS (SEQ ID NO: 28) or a homologous sequence having at least 80% sequence identity with it; LCDR2 comprises an amino acid sequence containing LVSKLDS (SEQ ID NO: 29) or a homologous sequence having at least 80% sequence identity with it; and LCDR3 comprises an amino acid sequence containing WQGAHFPLT (SEQ ID NO: 25) or a homologous sequence having at least 80% sequence identity with it. The amino acid sequence of NO: 30 or a homologous sequence that has at least 80% sequence identity with it.

[0066] It is known that CDRs are responsible for antigen binding; however, it has been found that not all six CDRs are necessarily indispensable or immutable. In other words, one, two, or three CDRs in an anti-GREM1 antibody can be replaced, altered, or modified, but the specific binding affinity to GREM1 is largely preserved.

[0067] In some embodiments, the anti-GREM1 antibody or its antigen-binding fragment comprises the heavy chain CDR3 sequence of EPMDY (SEQ ID NO: 27) or EAYSHHYYAMDY (SEQ ID NO: 3). The heavy chain CDR3 region is located at the center of the antigen-binding site and is therefore considered to have the most contact with the antigen and provide the most free energy for the antibody's affinity for the antigen. It is also believed that the heavy chain CDR3 is the most diverse CDR in terms of length, amino acid composition, and conformation among antigen-binding sites to date, due to a variety of diverse mechanisms (Tonegawa S). Nature 302:575-81). The diversity of heavy chain CDR3 is sufficient to produce most antibody specificity (Xu JL, Davis MM). Immunity . 13:37-45) and the desired antigen-binding affinity (Schier R et al., J Mol Biol. 263:551-67).

[0068] In some embodiments, the anti-GREM1 antibody or its antigen-binding fragment comprises all or a portion of the heavy chain variable domain and / or all or a portion of the light chain variable domain. In one embodiment, the anti-GREM1 antibody is a single-domain antibody comprising all or a portion of the heavy chain variable domain provided herein. More information on such single-domain antibodies is available in the art (see, for example, U.S. Patent No. 6,248,516).

[0069] In some embodiments, the anti-GREM1 antibody or its antigen-binding fragment provided herein comprises a heavy chain variable region and a light chain variable region. In some embodiments, the heavy chain variable region comprises QVQLVQSGAEVKKPGASVKVSCKASGYX1FTSSGIGWVX2QAX3GQGLEWX4GEIYPRSGNTYNNEKFKGRX5TX6TX7DX8STSTX9YMELRSLRSDDTAVYX 10 CX 11 The amino acid sequence shown in REAYSHHYYAMDYWGQGTTVTVSS (SEQ ID NO: 13) is as follows: X1 is S or T; X2 is K or R; X3 is S or P; X4 is I or M; X5 is A or V; X6 is L or M; X7 is A or T; X8 is K or T; X9 is V or A; X10 is F or Y; X11 is V or A. In some embodiments, X1 is S; X7 is A; X8 is K; X11 is V. In some embodiments, X1 is S; X3 is P; X5 is V; X7 is A; X8 is K; X10 is F; X11 is V. In some embodiments, X1 is S; X2 is K; X4 is I; X6 is L; X7 is A; X8 is K; X9 is V; X11 is V. In some embodiments, the heavy chain variable region comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12.

[0070] In some implementations, the light chain variable region includes DX 12 VMTQTPLSLX 13 VTPGQPASISCRSSQSLLHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRX 14The amino acid sequence shown in SEQ ID NO: 17 is SGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPFTFGQGTKLEIK, where X12 is V or I; X13 is P or S; and X14 is L or F. In some embodiments, X12 is V. In some embodiments, the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16.

[0071] In some embodiments, the heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 7, and / or the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 14.

[0072] In some embodiments, the heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 7, and / or the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 15.

[0073] In some embodiments, the heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 7, and / or the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 16.

[0074] In some embodiments, the heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 8, and / or the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 14.

[0075] In some embodiments, the heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 8, and / or the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 15.

[0076] In some embodiments, the heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 8, and / or the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 16.

[0077] In some embodiments, the heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 9, and / or the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 14.

[0078] In some embodiments, the heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 9, and / or the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 15.

[0079] In some embodiments, the heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 9, and / or the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 16.

[0080] In some embodiments, the heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 10, and / or the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 14.

[0081] In some embodiments, the heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 10, and / or the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 15.

[0082] In some embodiments, the heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 10, and / or the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 16.

[0083] In some embodiments, the heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 11, and / or the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 14.

[0084] In some embodiments, the heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 11, and / or the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 15.

[0085] In some embodiments, the heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 11, and / or the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 16.

[0086] In some embodiments, the heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 12, and / or the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 14.

[0087] In some embodiments, the heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 12, and / or the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 15.

[0088] In some embodiments, the heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 12, and / or the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 16.

[0089] In some embodiments, the anti-GREM1 antibody or its antigen-binding fragment provided herein comprises the heavy chain variable regions (e.g., Hu14E3-Ha VH, Hu14E3-Hb VH, Hu14E3-Hc VH, Hu22F1-Ha VH, Hu22F1-Hb VH, Hu22F1-Hc VH, Hu22F1-Hd VH, Hu56C11-H0 VH, Hu56C11-HaVH, Hu56C11-Hb VH, Hu56C11-Hc VH) and light chain variable regions (e.g., Hu14E3-La VL, Hu14E3-Lb VL, Hu22F1-La VL, Hu22F1-Lb VL, Hu56C11-L0 VL, Hu56C11-La) described in PCT patent application PCT / CN2022 / 072297. The application (VL, Hu56C11-LbVL) is incorporated herein by reference.

[0090] In some embodiments, the heavy chain variable region is selected from the group consisting of Hu14E3-Ha VH, Hu14E3-Hb VH and Hu14E3-Hc VH as described in PCT patent application PCT / CN2022 / 072297, and the light chain variable region is selected from the group consisting of Hu14E3-La VL and Hu14E3-Lb VL as described in PCT patent application PCT / CN2022 / 072297, which is incorporated herein by reference.

[0091] In some embodiments, the heavy chain variable region is selected from the group consisting of Hu22F1-Ha VH, Hu22F1-Hb VH, Hu22F1-Hc VH and Hu22F1-Hd VH as described in PCT patent application PCT / CN2022 / 072297, and the light chain variable region is selected from the group consisting of Hu22F1-La VL and Hu22F1-Lb VL as described in PCT patent application PCT / CN2022 / 072297, which is incorporated herein by reference.

[0092] In some embodiments, the heavy chain variable region is selected from the group consisting of Hu56C11-H0 VH, Hu56C11-HaVH, Hu56C11-Hb VH and Hu56C11-Hc VH as described in PCT patent application PCT / CN2022 / 072297, and the light chain variable region is selected from the group consisting of Hu56C11-L0 VL, Hu56C11-La VL and Hu56C11-Lb VL as described in PCT patent application PCT / CN2022 / 072297, which is incorporated herein by reference.

[0093] In some embodiments, the heavy chain variable region contains the amino acid sequence shown in QVQLVQSGSELKKPGASVKVSCKASGYTFTTYGMAWMRQAPGQGLEWMGWINTLSGEPTYADDFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCAREPMDYWGQGTMVTVSS (SEQ ID NO: 31), and / or the light chain variable region contains the amino acid sequence shown in DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLSWLQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGAHFPLTFGQGTKLEIK (SEQ ID NO: 32).

[0094] In some embodiments, the anti-GREM1 antibody or its antigen-binding fragment provided herein further comprises one or more amino acid residue substitutions or modifications, while still retaining specific binding specificity or affinity for hGREM1. In some embodiments, at least one substitution or modification is located in one or more CDR sequences, and / or in one or more non-CDR regions of the VH or VL sequence.

[0095] In some embodiments, the anti-GREM1 antibody or its antigen-binding fragment further comprises an immunoglobulin constant region, optionally a constant region of human IgG. In some embodiments, the constant region comprises a constant region of human IgG1, IgG2, IgG3, or IgG4, and optionally comprises a heavy chain constant region containing the sequence of SEQ ID NO: 18 and / or a light chain constant region containing the sequence of SEQ ID NO: 19.

[0096] In some embodiments, an anti-GREM1 antibody or its antigen-binding fragment is linked to one or more conjugate moieties. In some embodiments, the conjugate moieties comprise scavenging modulators, therapeutic agents (e.g., chemotherapeutic agents), toxins, radioisotopes, detectable labels (e.g., lanthanides, luminescent labels, fluorescent labels, biotin / avidin, or enzyme-substrate labels), pharmacokinetic modulators, DNA alkylating agents, topoisomerase inhibitors, tubulin binders, and other anticancer drugs (e.g., androgen receptor inhibitors), such as those described in PCT patent application PCT / CN2022 / 072297, which is incorporated herein by reference.

[0097] ii) Activin receptor antagonists As used herein, the term "activin receptor" refers to an activin-binding receptor having a single transmembrane domain and a specific hydrophilic, Cys-rich ligand-binding domain. Two types of activin receptors exist: type I (ActRI) and type II (ActRII) serine-threonine kinase receptors (Williams Textbook of Endocrinology (13th Edition), 2016). In some embodiments, the activin receptor is ActRI, ActRIIA, or ActRIIB. In some embodiments, the activin receptor is a type IIA activin receptor (ActRIIA). In some embodiments, the activin receptor antagonist comprises an antibody against an ActRIIA ligand or an antigen-binding fragment thereof, an inhibitory ActRIIA ligand mimic peptide, an inhibitory nucleic acid targeting ActRIIA ligand RNA or DNA, a polynucleotide encoding said inhibitory nucleic acid, a compound inhibiting the interaction between the ActRIIA ligand and ActRIIA, and a compound inhibiting the activity of the ActRIIA ligand.

[0098] In some embodiments, the activin receptor antagonist comprises a ligand trapping agent capable of capturing ActRIIA ligands such as activin, growth and differentiation factor (GDF) and / or bone morphogenetic protein (BMP).

[0099] In some embodiments, the ligand trapping agent comprises an extracellular domain of ActRIIA. In some embodiments, the ligand trapping agent comprises an extracellular domain of human ActRIIA. As used herein, the term "extracellular domain of human ActRIIA" includes the extracellular domain of human ActRIIA, its analogs, and derivatives. As used herein, the term "analog" refers to a molecule having a structure similar to the extracellular domain of human ActRIIA but with a slightly different amino acid sequence. Analogs generally retain the same or similar biological activity as the original protein domain. They may be naturally occurring variants or artificially produced through genetic engineering or chemical synthesis. As used herein, the term "derivative" refers to a molecule based on the structure of the extracellular domain of human ActRIIA but modified in some way. Modifications may include altering the amino acid sequence, adding chemical groups, or changing the three-dimensional structure of the protein. Depending on the nature of the modification, derivatives may have altered biological activity compared to the original protein domain. They are typically produced through chemical synthesis or protein engineering techniques to improve certain properties, such as stability, solubility, or binding affinity.

[0100] In some embodiments, the extracellular domain of human ActRIIA comprises the amino acid sequence at positions 21 to 135 of human ActRIIA. In some embodiments, human ActRIIA comprises the amino acid sequence of SEQ ID NO: 24. In some embodiments, the extracellular domain of human ActRIIA comprises the amino acid sequence of SEQ ID NO: 21 or a homologous sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with it.

[0101] In some embodiments, the ligand trapping agent further comprises the Fc domain of human IgG. In some embodiments, the extracellular domain of human ActRIIA is fused with the Fc domain of human IgG (e.g., IgG1, IgG2, IgG3, or IgG4). As used herein, the term "Fc domain" includes Fc domains, their analogs, and derivatives. As used herein, the term "analyte" refers to a molecule having a structure similar to an Fc domain but with a slightly different amino acid sequence. Analytes typically retain the same or similar biological activity as the original protein domain. They may be naturally occurring variants or artificially produced through genetic engineering or chemical synthesis. As used herein, the term "derivative" refers to a molecule based on the structure of an Fc domain but modified in some way. Modifications may include altering the amino acid sequence, adding chemical groups, or changing the three-dimensional structure of the protein. Depending on the nature of the modification, derivatives may have altered biological activity compared to the original protein domain. They are typically produced through chemical synthesis or protein engineering techniques to improve certain properties, such as stability, solubility, or binding affinity.

[0102] In some embodiments, the extracellular domain of human ActRIIA is fused with the Fc domain of human IgG1. In some embodiments, the Fc domain of human IgG1 comprises the amino acid sequence of SEQ ID NO: 22 or a homologous sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with it.

[0103] In some embodiments, the ligand trapping agent comprises the amino acid sequence of SEQ ID NO: 23 or a homologous sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with it. In some embodiments, the ligand trapping agent comprises sotatercept. The amino acid sequence of sotatercept is shown in SEQ ID NO: 23.

[0104] 2. Treatment of PAH with anti-GREM1 antibody or its antigen-binding fragment In another aspect, this disclosure provides a method for treating PAH in subjects with inadequate or unsatisfactory response to activin receptor antagonists, comprising administering a GREM1 antagonist to the subject.

[0105] Subjects exhibiting "insufficient or unsatisfactory response" to a drug may refer to subjects whose disease or symptoms continue to progress despite drug administration. This can be attributed to various reasons, such as inability to tolerate an effective dose due to, for example, severe side effects, differences in drug metabolism or response among subjects, the presence of comorbidities, or the severity of underlying conditions. In one case, the subject may experience side effects from the drug, necessitating a reduction in the drug dosage, which in turn reduces the therapeutic effect. In some cases, the subject may no longer tolerate the side effects of the drug, thus requiring discontinuation of such medication. In some embodiments, the activin receptor antagonist is combined with... ii) Activin receptor antagonists The activator receptor antagonist described in and incorporated herein is the same as that described in [the original text]. In some embodiments, the activator receptor antagonist is sotexip. It has been observed that patients receiving sotexip sometimes cannot tolerate its side effects. In these cases, a reduced dose of sotexip may be administered, such as a sub-effective dose, which may result in a diminished therapeutic effect. In addition, some patients may become intolerant to sotexip or experience disease progression even at reduced doses. These patients fall within the scope of consideration for “subjects with inadequate or unsatisfactory response to activator receptor antagonists”.

[0106] While not wishing to be bound by any theoretical framework, it is believed that GREM1 antagonists are suitable for the aforementioned subjects, possibly by reducing GREM1-mediated inhibition of the BMP signaling pathway, thereby restoring the balance between pro-proliferative and anti-proliferative signaling in the pulmonary artery. This has become a key mechanism for the "next-generation therapy" of PAH. See, for example, Grzegorz. Kopeć et al., Sotatercept as a next-generation therapy for pulmonary arterial hypertension: insights from the STELLAR trial. Cardiovascular Research (2023) 119, e155-e157.

[0107] In some embodiments, the GREM1 antagonist may be provided as a monotherapy. In some embodiments, the GREM1 antagonist may be combined with other standard care therapies for PAH, such as those in Grzegorz. Kopećet al., Sotatercept as a next-generation therapy for pulmonary arterialhypertension: insights from the STELLAR trial. Cardiovascular Research The conventional PAH therapy described in (2023)119, e155-e157, the entire contents of which are incorporated herein by reference.

[0108] In some implementations, the GREM1 antagonist contains " i) Anti-GREM1 antibody The anti-GREM1 antibody or its antigen-binding fragment described in the document is incorporated herein by reference.

[0109] iii) Additional treatments In some embodiments, the GREM1 antagonist is administered in combination with at least one additional therapeutic agent. In some embodiments, the therapeutic agent is selected from the group consisting of: drugs targeting the endothelin pathway (e.g., endothelin receptor antagonists), drugs targeting the nitric oxide pathway (e.g., phosphodiesterase type 5 inhibitors, soluble guanylate cyclase activators), drugs targeting the prostacyclin pathway (e.g., prostacyclin analogs, prostacyclin receptor agonists), drugs targeting the vasodilatory pathway (e.g., Getagozumab monoclonal antibody, therapeutic vaccine ETR-002 peptide, Pemziviptadil (vasoactive intestinal peptide analog), endothelial progenitor cells (eNOS gene)), diuretics, anticoagulants, cardiac glycosides, vasodilators, prostacyclin analogs, calcium channel blockers, endothelial antagonists, lipid-lowering agents, phosphodiesterase inhibitors, thromboxane inhibitors, and endopeptidase inhibitors.

[0110] Diuretics that may be used in combination therapies disclosed herein include, but are not limited to, chlorthalidone, indapamide, bendro-flumethiazid, metolazone, cyclopenthiazide, polythiazide, mefruside, ximapide, chlorothiazide, and hydrochlorothiazide.

[0111] In some implementations, anticoagulants such as warfarin may be suitable for patients with pulmonary hypertension who have an increased risk of thrombosis and thromboembolism.

[0112] In some embodiments, the vasodilator is selected from the group consisting of prostacyclin, eprostacyclin, treprocycline and nitric oxide (NO).

[0113] In some embodiments, the prostacyclin analogue is selected from the group consisting of ilomedin, treprostacyclin, and eprostadil.

[0114] Examples of calcium channel blockers that can be used include (but are not limited to) diltiazem, felodipine, amlodipine, and nifedipine.

[0115] For lipid-lowering agents, HMG CoA reductase inhibitors, such as simvastatin, pravastatin, atorvastatin, lovastatin, itavastatin, fluvastatin, pitavastatin, rosuvastatin, ZD-4522, and cerivastatin, are suitable options.

[0116] In some implementations, phosphodiesterase inhibitors, especially phosphodiesterase V inhibitors, such as tadalafil, sildenafil, and vardenafil, may also be used in combination therapy.

[0117] 3. Pulmonary hypertension (PAH) The World Health Organization (WHO) classifies pulmonary hypertension (PH) into five groups based on similarity in pathophysiology, clinical presentation, and treatment options: Group 1 (PAH), Group 2 (pulmonary hypertension due to left-sided heart disease), Group 3 (pulmonary hypertension due to lung disease and / or hypoxia), Group 4 (chronic thromboembolic pulmonary hypertension (CTEPH)), and Group 5 (pulmonary hypertension with unclear or multifactorial etiologies). See, for example, Simonneau G et al., Haemodynamic definitions and updated clinical classification of pulmonary hypertension. Eur Respir J2019; 53: 1801913, the entire contents of which are incorporated herein by reference. PAH is one of five subgroups of pulmonary arterial hypertension (PH). As used herein, “pulmonary arterial hypertension” (“PH”) refers to hypertension of the lungs from any cause. As used herein, “hypertension” or “high blood pressure” refers to hypertension of the arteries throughout the body.

[0118] In some implementations, the subject is a person who has or has been diagnosed with Group 1 (PAH).

[0119] As used herein, the term “pulmonary hypertension” or “PAH” refers to a lung condition characterized by a persistently elevated pulmonary artery pressure, such as a mean pulmonary artery pressure (mPAP) equal to or greater than 20 mm Hg (or greater than 25 mm Hg), which can be measured in a resting subject using routine techniques such as right heart catheterization. See, for example, Bradley A. Maron, Revised Definition of Pulmonary Hypertension and Approach to Management: A Clinical Primer. J Am Heart Assoc. 2023;12:e029024. However, an abnormally elevated mPAP alone is insufficient to define pulmonary vascular disease, as an abnormally elevated mPAP can be caused by increased cardiac output or increased pulmonary artery wedge pressure. Therefore, following the recommendations of the Working Group of the 6th World Symposium on Pulmonary Hypertension (WSPH), pulmonary vascular resistance ≥3 Wood Units is included in the definition of all forms of precapillary hypertension, provided that mPAP > 20 mmHg. See, for example, Simonneau G et al., Haemodynamic definitions and updated clinical classification of pulmonary hypertension. Eur Respir J 2019;53: 1801913.

[0120] PAH is also typically characterized by proliferative remodeling and progressive luminal narrowing of the pulmonary arterioles. The resulting increase in pulmonary artery pressure strains the heart, ultimately leading to right ventricular failure and death. See, for example, Marius M. Hoeper, Phase 3 Trial of Sotatercept for Treatment of Pulmonary Arterial Hypertension. N Engl J Med 2023; 388:1478-1490. PAH can also be characterized by pulmonary arteries that are narrower, thicker, and / or stiffer than healthy pulmonary arteries. For example, healthy pulmonary arteries are flexible, allowing blood to flow easily through them; mild PAH is characterized by thick, stiff arterial walls that restrict blood flow and increase resistance; moderate PAH is characterized by increasingly restricted blood flow as the artery narrows; and severe PAH (e.g., severe PAH with plexiform lesions) is characterized by advanced vascular lesions (abnormal growth) and thrombosis (blood clots). See, for example, from November 28, 2023. https: / / phassociation.org / types-pulmonary- hypertension-groups / Search results for About Pulmonary Hypertension, Pulmonary Hypertension Association.

[0121] Pulmonary artery stiffness can be measured in vivo using invasive or non-invasive methods, based on measurements such as pressure and inner or outer diameter or cross-sectional area. See, for example, Tian L et al., In vivo and in vitro measurements of pulmonary arterial stiffness: A brief review. Pulm Circ. October 2012;2(4):505-17, the entire contents of which are incorporated herein by reference. In some embodiments, pulmonary artery stiffness may be indicated by the percentage of pulmonary artery wall thickness (PAWT%) of each arteriole, which can be measured by non-invasive imaging methods combined with image processing using software such as ImageJ.

[0122] Symptoms and / or complications of PAH that characterize these conditions and aid in diagnosis are well known in the art, such as tinnitus, vertigo, dizziness, headache, or syncope. Tests used to diagnose PAH include (but are not limited to) blood pressure measurements. A family history of PAH can also help determine whether a person is likely to have or currently has PAH.

[0123] WHO also provides a clinical classification of PAH, as shown in Table 1 (Simonneau G et al., Haemodynamic definitions and updated clinical classification of pulmonary hypertension). Eur Respir J 2019; 53: 1801913 (the entire contents of which are incorporated herein by reference). Table 1: Clinical Classification of PAH The severity of PAH can also be represented, for example, by the WHO functional class of patients with PAH. The WHO functional classification is based on the NYHA system and assesses activity tolerance, monitors disease progression, and treatment response. It includes four classes: Class I (pulmonary hypertension that does not restrict physical activity; normal physical activity does not cause excessive dyspnea or fatigue, chest pain, or near-syncope), Class II (pulmonary hypertension that causes mild restriction of physical activity; the patient is comfortable at rest; normal physical activity causes excessive dyspnea or fatigue, chest pain, or near-syncope), Class III (pulmonary hypertension that causes significant restriction of physical activity; the patient is comfortable at rest; less than normal physical activity causes excessive dyspnea or fatigue, chest pain, or near-syncope), and Class IV (pulmonary hypertension that results in an inability to perform any physical activity without symptoms; the patient presents with signs of right heart failure; dyspnea and / or fatigue are present even at rest; any physical activity increases discomfort).

[0124] In one implementation, the subject is undergoing treatment for PAH, assessing for PAH, or has a risk of PAH, such as PAH ([WHO] Functional Class I, [WHO] Functional Class II, [WHO] Functional Class III, or [WHO] Functional Class IV).

[0125] In some implementations, the object includes humans and non-human animals. The term "non-human animal" covers all vertebrate species, including (but not limited to) mammals and non-mammals, examples of which include non-human primates, mice, rats, cats, rabbits, sheep, dogs, cattle, chickens, amphibians, and reptiles. Unless otherwise specified, the terms "patient" and "object" are used interchangeably throughout this document.

[0126] In some implementations, the object is a person.

[0127] Anti-GREM1 antibody, polynucleotide and recombinant method In another respect, this disclosure provides " i) Anti-GREM1 antibody The anti-GREM1 antibody or its antigen-binding fragment described in [the document] is incorporated herein. In another aspect, this disclosure provides isolated polynucleotides encoding the anti-GREM1 antibody or its antigen-binding fragment provided herein. The DNA encoding the monoclonal antibody can be isolated and sequenced using routine procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antibody). The encoding DNA can also be obtained by synthetic methods.

[0128] Recombinant techniques known in the art can be used to insert isolated polynucleotides encoding the anti-GREM1 antibody disclosed herein or its antigen-binding fragment into a vector for further cloning (DNA amplification) or expression. Many vectors are available. Vector components generally include (but are not limited to) one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0129] This disclosure provides vectors (e.g., expression vectors) containing a nucleic acid sequence encoding an anti-GREM1 antibody or an antigen-binding fragment thereof, as provided herein, at least one promoter operatively linked to the nucleic acid sequence, and at least one selection marker. Examples of vectors include (but are not limited to) retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses, multifocal papillomaviruses (e.g., SV40), λ phages and M13 phages, plasmids pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, and pGEMEX. , pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p1 5TV-L, pPro18, pTD, pRS10, pLexA, pACT2.2, pCMV-SCRIPT.RTM., pCDM8, pCDNA1.1 / amp, pcDNA3.1, pRc / RSV, PCR 2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos, etc.

[0130] A vector containing a multinucleotide sequence encoding the anti-GREM1 antibody or its antigen-binding fragment disclosed herein can be introduced into a host cell for cloning or gene expression. Suitable host cells for cloning or expressing the DNA in the vectors described herein are prokaryotic, yeast, or higher eukaryotic cells as described above. Suitable prokaryotes include eubacteria (…). eubacteria ), such as Gram-negative or Gram-positive organisms, such as Enterobacteriaceae ( Enterobacteriaceae ), for example, Escherichia coli ( Escherichia ), such as E. coli ( E. coli Enterobacteriaceae ( Enterobacter Erwinia spp. Erwinia Klebsiella spp. Klebsiella Proteus spp. Proteus Salmonella ( ) Salmonella ), such as Salmonella typhimurium ( Salmonella typhimurium Serratia spp. Serratia ), such as Serratia marcescens ( Serratia marcescans ); and Shigella spp. ( Shigella ); and Bacillus ( Bacilli ), such as Bacillus subtilis ( B. subtilis ) and Bacillus licheniformis ( B. licheniformis ); Pseudomonas spp. ( Pseudomonas ), such as Pseudomonas aeruginosa ( P. aeruginosa ); and Streptomyces ( Streptomyces ).

[0131] This disclosure also provides a method for expressing the anti-GREM1 antibody or its antigen-binding fragment disclosed herein, comprising culturing host cells under conditions of an expression vector. Host cells used to generate the anti-GREM1 antibody or its antigen-binding fragment provided herein can be cultured in a variety of culture media. Commercially available culture media, such as Ham's F10 (Sigma), Minimal Essential Medium (MEM) (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM) (Sigma), are suitable for culturing host cells. Alternatively, any of the culture media described below can be used as the culture medium for host cells: Ham et al., Meth. Enz. 58:44 (1979); Barnes et al., Anal. Biochem. (1980) 102:255; U.S. Patent Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90 / 03430; WO 87 / 00195; or U.S. Patent Reexamination Case 30,985. Any of these culture media may be supplemented as needed with hormones and / or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium, and phosphate), buffers (e.g., HEPES), nucleotides (e.g., adenosine and thymidine), and antibiotics (e.g., GENTAMYCIN). TM The ingredients include drugs, trace elements (defined as inorganic compounds typically present in a final concentration in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations known to those skilled in the art. Culture conditions (e.g., temperature, pH, etc.) are those previously used with the host cells selected for expression and will be apparent to those skilled in the art.

[0132] Pharmaceutical Composition In another aspect, this disclosure provides pharmaceutical compositions comprising a GREM1 antagonist and one or more pharmaceutically acceptable carriers. In some embodiments, the GREM1 antagonist is capable of blocking the binding of GREM1 to BMPs (e.g., BMP2, BMP4, and / or BMP7). In some embodiments, the GREM1 antagonist is an anti-GREM1 antibody or an antigen-binding fragment thereof disclosed herein.

[0133] In some embodiments, the pharmaceutical composition further comprises a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from the above-mentioned " ii) Activin receptor antagonists "Chapter or " iii) Additional treatments The therapeutic agents described in the previous chapter are incorporated into this article.

[0134] The GREM1 antagonist and second therapeutic agent described above can each be administered in the form of any suitable pharmaceutical composition. The term "pharmaceutical composition" refers to a formulation comprising a therapeutically effective agent (e.g., the GREM1 antagonist and second therapeutic agent described above), preferably together with a pharmaceutically acceptable carrier, diluent, and / or excipient. The pharmaceutical composition can be used to treat, prevent, or reduce the severity of a disease or condition by administering the pharmaceutical composition to a subject.

[0135] Pharmaceutically acceptable carriers used in the pharmaceutical compositions disclosed herein may include, for example, pharmaceutically acceptable liquid, gel, or solid carriers, aqueous solvents, non-aqueous solvents, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending / dispersing agents, multivalent chelating agents or chelating agents, diluents, adjuvants, excipients or non-toxic excipients, other components known in the art, or various combinations thereof.

[0136] Pharmaceutical compositions are typically provided in a homogeneous dosage form and can be prepared in a manner known in the art. Pharmaceutical compositions may be, for example, in liquid dosage forms, such as solutions or suspensions; or in solid dosage forms, such as tablets and capsules. The pharmaceutical compositions described herein are generally administered in a therapeutically effective and pharmaceutically acceptable formulation.

[0137] Reagent test kit In another aspect, this disclosure provides a kit for treating PAH in individuals in need.

[0138] In some embodiments, the kit comprises a composition containing the GREM1 antagonist described herein.

[0139] In some embodiments, the kit comprises a first composition containing a GREM1 antagonist described herein and a second composition containing a second therapeutic agent (e.g., an activin receptor antagonist). The second therapeutic agent is selected from the above-mentioned " ii) Activator receptor antagonists "Chapter or " iii) Additional treatments The therapeutic agents described in the section are incorporated herein. In some embodiments, the first and second compositions are located in separate containers. In some embodiments, the first and second compositions are located in a single container.

[0140] In some implementations, the kit includes a product information leaflet containing instructions on the use of the GREM1 antagonist in combination with a second therapeutic agent, such as an activator receptor antagonist.

[0141] As used herein, the term "instructions for use of a drug" refers to the instructions included in the commercial packaging of a drug that contain information such as indications, dosage, usage, administration, contraindications, other drugs in combination with the packaged product, and / or warnings regarding the use of such drugs.

[0142] The kit may further include other materials desired from a commercial and user perspective, including additional buffers, diluents, filters, needles, and syringes.

[0143] In another aspect, this disclosure provides a method for treating PAH in a subject in need, comprising administering to the subject a therapeutically effective amount of an anti-GREM1 antibody or an antigen-binding fragment thereof as described herein. In some embodiments, the anti-GREM1 antibody or an antigen-binding fragment thereof is administered in combination with a therapeutically effective amount of at least one additional therapeutic agent, said at least one additional therapeutic agent as described above. iii) Additional treatments The therapeutic agents described in the previous chapter are incorporated into this article.

[0144] In another aspect, this disclosure provides the use of a combination of a GREM1 antagonist and an activin receptor antagonist in the preparation of a medicament for treating pulmonary arterial hypertension (PAH) in subjects of need.

[0145] In another aspect, this disclosure provides the use of a GREM1 antagonist in the preparation of a medicament for treating pulmonary arterial hypertension (PAH) in subjects of need, wherein the medicament is used in combination with an activin receptor antagonist.

[0146] In another aspect, this disclosure provides the use of a GREM1 antagonist in the preparation of a medicament for treating pulmonary arterial hypertension (PAH) in a subject in need, wherein the treatment comprises administering a combination of a GREM1 antagonist and an activin receptor antagonist to the subject, thereby treating the subject's PAH.

[0147] In another aspect, this disclosure provides the use of a GREM1 antagonist in the preparation of a medicament for treating PAH in subjects with inadequate or unsatisfactory response to an activin receptor antagonist, wherein the treatment comprises administering the GREM1 antagonist to the subject, optionally in combination with an activin receptor antagonist, and further optionally in combination with at least one additional therapeutic agent.

[0148] Example While this disclosure has been specifically shown and described with reference to particular embodiments, some of which are preferred embodiments, those skilled in the art will understand that various changes in form and detail may be made to this disclosure without departing from the spirit and scope of the disclosure herein.

[0149] Example 1: Humanization of gremlin1 (GREM1) antibody This embodiment illustrates the generation and characterization of a humanized antibody against clone 36F5. The generation and characterization of mouse hybridoma 36F5 have been described in PCT patent application PCT / CN2022 / 072297, which is incorporated herein by reference.

[0150] The humanized antibody for 36F5 was designed according to the following scheme: three-dimensional structural simulation and humanization via CDR transplantation.

[0151] The first step in antibody humanization was to simulate the three-dimensional structure of the variable domains of 36F5. The sequences of each variable domain (Vk and Vh) of the mouse antibody were BLAST-aligned in the Protein Data Bank (http: / / www.rcsb.org / ) database to identify the antibody sequences with the highest homology and known high-resolution structures. The selected structural template for modeling 36F5 exhibited the best similarity to the target antibody. We modified each residue of the structure to conform to the target sequence. While preserving the main chain conformation, certain side chain conformations were adjusted. At positions where the parent structure and the simulated structure had the same residues, the side chain conformation remained unchanged; if some residues differed between the template structure and the modeled structure, the side chain conformation was mutated and optimized based on the template structure and packaging considerations.

[0152] We also simulated the structure of the 36F5 graft from the CDR to guide the design of reverse mutations and the assessment of the developability and stability of humanized antibodies. Structural simulations were performed in a similar manner.

[0153] Humanization was achieved through CDR transplantation. After BLAST alignment of the mu36F5 sequence in the IMGT human immunoglobulin gene database, CDR transplantation was performed using the human germline framework sequence IGHV / 7-4 targeting the heavy chain and IGKV / 2-30 targeting the light chain, respectively, resulting in humanized 36F5 without reversion mutations. To further maintain the activity of the humanized 36F5, the framework sequence of the humanized antibody was aligned with that of the mouse antibody. These different residues were double-checked in the mouse antibody structural model; if any of these residues were located at a position that could interact with and affect the CDR residues, they were reverted to mouse residues. Six humanized heavy chain variants with different reversion mutations were identified as Hu_36F5_H0 (SEQ ID NO: 7), Hu_36F5_Ha (SEQ ID NO: 8), Hu_36F5_Hb (SEQ ID NO: 9), Hu_36F5_Hc (SEQ ID NO: 10), Hu_36F5_Hd (SEQ ID NO: 11), and Hu_36F5_He (SEQ ID NO: 12), and three light chain variants with different reversion mutations were identified as Hu_36F5_L0 (SEQ ID NO: 14), hu_36F5_La (SEQ ID NO: 15), and hu_36F5_Lb (SEQ ID NO: 12). 16), and the cDNA of these humanized heavy and light chain variable regions fused with the constant regions of hIgG1 and hKappa was inserted into a mammalian vector. Subsequently, each humanized heavy chain was co-expressed with a humanized light chain to obtain 18 forms of humanized antibodies, including Hu_36F5_H0L0, Hu_36F5_H0La, Hu_36F5_H0Lb, Hu_36F5_HaL0, Hu_36F5_HaLa, Hu_36F5_HaLb, and Hu... The expression and purification procedures for _36F5_HbL0, Hu_36F5_HbLa, Hu_36F5_HbLb, Hu_36F5_HcL0, Hu_36F5_HcLa, Hu_36F5_HcLb, Hu_36F5_HdL0, Hu_36F5_HdLa, Hu_36F5_HdLb, Hu_36F5_HeL0, Hu_36F5_HeLa, and Hu_36F5_HeLb are the same as those for chimeric antibodies.

[0154] The expression and purification of the recombinant antibody protein were performed as follows: ExpiCHO cells were seeded at 5–6 × 10^6 cells / ml in ExpiCHO expression medium. Subsequently, ExpiCHO cells were transfected using an ExpiCHO transfection kit with equal volumes of heavy and light chain vector DNA at a final concentration of 1.0 μg / ml. The transfected cells were cultured in shake flasks at 125 rpm in an incubator at 37°C and 8% CO2. ExpiCHO feed was added 18–22 hours post-transfection. Cell culture was collected on day 10. Collected cell culture medium (HCCF) was obtained by centrifugation. The HCCF was then loaded onto an rProtein A column (GE Healthcare) and washed with PBS. The final IgG antibody was eluted with a solution containing 20 mM citrate at pH 3.2. Finally, the eluted antibody protein was neutralized and stored at -80°C for long-term use. The obtained antibodies were analyzed using SDS-PAGE and size exclusion chromatography (TSKgel G3000SWXL, TOSOH) to determine purity levels.

[0155] Example 2: Binding affinity of the humanized antibody provided in this paper to human GREM1 This embodiment illustrates the binding affinity of the humanized antibody provided herein to human GREM1.

[0156] Transparent polystyrene plates (BEAVER) were coated overnight at 4°C with 100 μl / well of high-pH coating buffer containing 0.5 μg / ml human gremlin1 (ACRO). The plates were then washed once with PBS + 0.1% Tween 20 (Sigma) using an automated plate washer. A blocking solution consisting of PBS + 1% BSA + 1% normal goat serum + 0.5% Tween 20 (Sigma) was added to each well and incubated at room temperature for 2 hours. Subsequently, 100 μl of antibody dilution buffer containing PBS + 1% BSA + 1% normal goat serum + 0.01% Tween 20, starting at 2 μg / ml (13.33 nM) and then serially diluted 4-fold, was added to each well and the plates were incubated at room temperature for 1 hour. The plate was then washed three times with 200 μl PBS + 0.1% Tween 20, followed by the addition of 100 µl / well of 1:10000 mouse anti-human IgG-HRP (abcam) and incubation at room temperature for 1 hour. It was then washed three times with PBS + 0.1% Tween 20. Finally, 100 μl / well of TMB (InnoReagents) was added to each well, and 50 μl of stop solution was added to each well after 2 minutes. The plate was read at 450 nm using a Multiscan FC microplate reader (Thermo Scientific). EC50 values ​​are also displayed. Figure 1 middle.

[0157] like Figure 1 As shown, the humanized 36F5 antibodies Hu36F5-HdLa, Hu36F5-HcLa, Hu36F5-HbLa, Hu36F5-HdL0, Hu36F5-HbL0, and Hu36F5-HcL0 showed similar binding to human Gremlin1 and were superior to Hu36F5-HaL0, Hu36F5-H0L0, Hu36F5-H0La, and Hu36F5-HaLa. Hu36F5-HdLa was selected for a second screening. Figure 2 As shown, compared with Hu36F5-HdLa, the humanized 36F5 antibodies Hu36F5-HdLb, Hu36F5-HeLa and Hu36F5-HeLb showed similar binding to human Gremlin1.

[0158] Example 3: Binding affinity of the humanized antibody provided in this paper to human GREM1 This embodiment illustrates the binding affinity of the humanized antibody provided herein to human GREM1.

[0159] Human gremlin protein was diluted with kinetic buffer (PBS pH 7.4, 0.1% BSA + 0.002% Tween-20) to obtain a concentration of 2 μg / ml in the loading column of a 96-well half-zone microplate (Greiner Bio-one), 100 μl per well. 0 nM was used as a reference control. The antibody to be tested was diluted with ForteBio kinetic buffer in the association column of the plate to concentrations of 100 nM, 50 nM, and 25 nM, 100 μl per well. The HIS1K sensor was placed in the first baseline column for 60 s to obtain the first baseline, followed by 90 s in the loading column to capture Gremlin-his protein. The sensor was then placed in the second baseline column for 60 s to obtain the second baseline. It was then placed in the association column for 90 s to allow complete association of Gremlin / Gremlin Ab to obtain Kon factor data. The sensor was placed in the dissociation column for 60 seconds to obtain Koff factor data. The data was then analyzed using ForteBio (Octet96) to obtain affinity under global conditions.

[0160] like Figure 3 As shown, 36F5-HeLb exhibits better affinity than chimeric antibodies.

[0161] Example 4: BMP2 / 4 / 7 blocking assay of the humanized antibody provided in this paper This example illustrates the results of the BMP2 / 4 / 7 blocking assay of the humanized antibody provided herein.

[0162] As previously described in the art, the plate was coated overnight with recombinant human BMP2 / 4 / 7 (0.5 μg / ml). The plate was then washed once with PBS + 0.1% Tween 20 (Sigma) on an automated plate washer. 100 μl of a blocking solution consisting of PBS + 1% BSA + 1% normal goat serum + 0.5% Tween 20 (Sigma) was added to each well and incubated at room temperature for 2 hours. The plate was then washed three (3) times. Subsequently, 55 μl of serially diluted chimeric antibody and 55 μl of dilution buffer containing 0.5 μg / ml human Gremlin1-his in PBS + 1% BSA + 1% normal goat serum + 0.01% Tween 20 were mixed separately and incubated at room temperature for 1 hour. Then, 100 μl of this complex was added to the coated plate and incubated at room temperature for another hour. The plate was then washed three times, and 100 μl of dilution buffer containing anti-his HRP (GenScript) was added. The plate was then developed with TMB solution and the reaction was stopped by adding a stop solution. After washing three times with washing buffer, the plate was read at 450 nm using a microplate reader.

[0163] like Figure 4 , Figure 5 and Figure 6 As shown, consistent with the binding results, Hu36F5-HdLa, Hu36F5-HdLb, Hu36F5-HeLa and Hu36F5-HeLb exhibited similar effective activity in blocking the binding of GREM1 to BMP2, BMP4 and BMP7.

[0164] Example 5: Efficacy of anti-Gremlin1 antibody in a rat model of pulmonary arterial hypertension (PAH) induced by lily alkaloid (MCT). In this embodiment, the anti-pulmonary hypertension modulatory effects of the anti-Gremlin 1 antibodies (e.g., Hu36F5-HdLa, Hu36F5-HdLb, Hu36F5-HeLa, and Hu36F5-HeLb) and ActRIIA-mFc provided herein were comprehensively evaluated by measuring right ventricular systolic pressure (RVSP), right ventricular hypertrophy index (RVHI), and pulmonary artery wall thickness percentage (PAWT%).

[0165] ActRIIA-mFc: ActRIIA (Aa21-135) of mouse Fc linked by a TGGG linker is a ligand trapping agent containing the extracellular domain of human ActRIIA having the amino acid sequence shown in SEQ ID NO: 21. The amino acid sequence of the mouse Fc is shown in SEQ ID NO: 20.

[0166] Sildenafil: A compound with the following structural formula: , Sildenafil is an inhibitor of cGMP-specific PDE-5 in the smooth muscle of pulmonary blood vessels, where PDE-5 is responsible for cGMP degradation. Therefore, sildenafil increases cGMP in pulmonary vascular smooth muscle cells, inducing relaxation, and is commonly used to improve motor function in adults with PAH and children aged 1 year and older. In this study, sildenafil was used as a positive control. In an MCT-induced PAH model, sildenafil showed significant differences in RVSP, RVHI, and PAWT indices.

[0167] In this study, rats were subcutaneously injected with a single dose of MCT (60 mg / kg) to induce PAH. MCT-induced PAH rat models are frequently used to study the pathophysiological processes in PAH and to identify novel treatment strategies. See, for example, Nogueira-Ferreira, R. et al., Exploring the monocrotaline animal model for the study of pulmonary arterial hypertension: A network approach. Pulm. Pharmacol. Ther. 2015, 35, 8-16; Jasenovec, T. et al., Monocrotaline-Induced Pulmonary Arterial Hypertension and Bosentan Treatment in Rats: Focus on Plasma and Erythrocyte Parameters. Pharmaceuticals December 27, 2022 (15 / 12 / 27)

[0168] Based on their body weight on day 0, MCT-induced PAH rats were randomly divided into 5 groups of 10 animals each. Following a single injection of MCT, rats were administered solvent, sildenafil, 36F5-HdLa, ActRIIA-mFc, or 36F5-HdLa plus ActRIIA-mFc at the doses specified in Table 2. On day 22 after MCT modeling, rats were anesthetized with sodium pentobarbital, and RVSP was measured by inserting a venous catheter into the right jugular vein, followed by access to the right ventricle via the right atrium. Rats were euthanized by exsanguination under deep anesthesia. Bilateral thoracotomy or cervical dislocation was performed as adjuncts to ensure death. Left lung tissue was collected and immediately subjected to H&E staining in 10% NBF to determine PAWT% (arterial diameter range 50–100 µm). Heart samples were excised and weighed. The right ventricular wall was then dissected, and the remaining left ventricular wall and interventricular septum were weighed. RVHI was calculated as the ratio of right ventricular weight to (left ventricular weight + interventricular septum weight). Data were analyzed using GraphPad Prism 7, and each column represents the mean ± SEM. p < 0.05 was considered statistically significant.

[0169] Table 2. Overview of the effects of test samples on RVSP / RVHI / PAWT in the PAH model result: Right ventricular systolic pressure (RVSP): such as Figure 7 As shown, RVSP decreased in all treatment groups compared to the solvent group. The 36F5-HdLa-10 mg / kg group showed a trend towards decreased RVSP, and the combination of 36F5-HdLa-10 mg / kg + ActRIIA-mFc-2.5 mg / kg showed a statistically significant difference compared to the solvent control group. Furthermore, it was more effective than either 36F5-HdLa-10 mg / kg or ActRIIA-mFc-2.5 mg / kg alone, indicating a synergistic effect of the combination therapy.

[0170] Right ventricular hypertrophy index (RVHI): such as Figure 8 As shown, RVHI was reduced in all treatment groups compared to the solvent group. The 36F5-HdLa-10 mg / kg group showed a significant reduction in RVHI, and the 36F5-HdLa-10 mg / kg + ActRIIA-mFc-2.5 mg / kg group showed a statistically significant difference compared to the solvent control group, and was more effective than treatment with 36F5-HdLa-10 mg / kg, ActRIIA-mFc-2.5 mg / kg or sildenafil 25 mg / kg alone, indicating a synergistic effect of combination therapy.

[0171] Pulmonary artery wall thickness percentage (PAWT%): as follows Figure 9 As shown, PAWT% decreased in all treatment groups compared to the solvent group. Specifically, the 36F5-HdLa-10 mg / kg group showed a significant decrease in PAWT%, and the 36F5-HdLa-10 mg / kg + ActRIIA-mFc-2.5 mg / kg group showed a statistically significant difference compared to the solvent control group, and was more effective than treatment with 36F5-HdLa-10 mg / kg, ActRIIA-mFc-2.5 mg / kg, or sildenafil 25 mg / kg alone, indicating a synergistic effect of combination therapy.

[0172] The results showed that ActRIIA-mFc-2.5 mg / kg and its combination with 36F5-HdLa-10 mg / kg significantly reduced RVSP, RVHI, and PAWT%, and the combination therapy of 36F5-HdLa-10 mg / kg and ActRIIA-mFc-2.5 mg / kg was slightly better than the monotherapy groups of 36F5-HdLa-10 mg / kg and ActRIIA-mFc-2.5 mg / kg. These results provide evidence that 36F5, as a monotherapy or in combination with active agents such as ActRIIA-mFc, is effective in treating PAH. Combination therapy showed greater therapeutic activity than any single agent in controlling and improving the progression of PAH.

[0173] Table 3. Sequences mentioned or used in this disclosure.

Claims

1. A method for treating pulmonary arterial hypertension (PAH) in a subject of need, the method comprising: PAH in the subject was treated by administering a combination of a GREM1 antagonist and an activin receptor antagonist.

2. The method of claim 1, wherein the activin receptor antagonist and the GREM1 antagonist exhibit at least an additive effect in treating PAH.

3. The method of claim 2, wherein the activin receptor antagonist and the GREM1 antagonist exhibit at least an additive effect in reducing the right ventricular hypertrophy index (RVHI).

4. The method according to any one of claims 1 to 3, wherein the activator receptor antagonist is administered in an effective amount.

5. The method according to any one of claims 1 to 3, wherein the activator receptor antagonist is administered in a sub-effective amount.

6. The method according to claim 5, wherein the sub-effective amount is about 0.1 mg / kg, about 0.2 mg / kg, about 0.3 mg / kg, about 0.4 mg / kg, about 0.5 mg / kg, or about 0.6 mg / kg.

7. The method of claim 5 or 6, wherein the side effects of the activin receptor antagonist are reduced relative to those observed when administered in an effective amount.

8. The method according to any one of claims 5 to 7, wherein the therapeutic efficacy of the activin receptor antagonist is maintained substantially at a level comparable to that observed when applied in an effective amount.

9. The method according to any one of the preceding claims, wherein the administration of the activator receptor antagonist is performed before, simultaneously with, or after the administration of the GREM1 antagonist.

10. A method of treating PAH in a subject with an inadequate or unsatisfactory response to an activin receptor antagonist, comprising administering to the subject a GREM1 antagonist, optionally in combination with an activin receptor antagonist, and further optionally in combination with at least one additional therapeutic agent.

11. The method of claim 10, wherein the additional therapeutic agent is selected from the group consisting of: drugs targeting the endothelin pathway (e.g., endothelin receptor antagonists), drugs targeting the nitric oxide pathway (e.g., phosphodiesterase type 5 inhibitors, soluble guanylate cyclase activators), drugs targeting the prostacyclin pathway (e.g., prostacyclin analogs, prostacyclin receptor agonists), drugs targeting the vasodilatory pathway (e.g., guitarazine monoclonal antibody, therapeutic vaccine ETR-002 peptide, pegvetilide (vasoactive intestinal peptide analog), endothelial progenitor cells (eNOS gene)), diuretics, anticoagulants, cardiac glycosides, vasodilators, prostacyclin analogs, calcium channel blockers, endothelial antagonists, lipid-lowering agents, phosphodiesterase inhibitors, thromboxane inhibitors, and endopeptidase inhibitors.

12. The method according to any one of the preceding claims, wherein the GREM1 antagonist comprises an anti-GREM1 antibody or an antigen-binding fragment thereof.

13. The method of claim 12, wherein the anti-GREM1 antibody or its antigen-binding fragment comprises heavy chains HCDR1, HCDR2, and HCDR3 and / or light chains LCDR1, LCDR2, and LCDR3, wherein: (1) The HCDR1 contains an amino acid sequence containing SSGIG (SEQ ID NO: 1) or a homologous sequence having at least 80% sequence identity with it; The HCDR2 contains an amino acid sequence containing EIYPRSGNTYNNEKFKG (SEQ ID NO: 2) or a homologous sequence having at least 80% sequence identity with it; The HCDR3 contains an amino acid sequence containing EAYSHHYYAMDY (SEQ ID NO: 3) or a homologous sequence having at least 80% sequence identity with it; The LCDR1 contains an amino acid sequence containing RSSQSLLHSNGNTYLE (SEQ ID NO: 4) or a homologous sequence having at least 80% sequence identity with it; The LCDR2 contains an amino acid sequence containing KVSNRFS (SEQ ID NO: 5) or a homologous sequence having at least 80% sequence identity with it; and The LCDR3 contains an amino acid sequence containing FQGSHVPFT (SEQ ID NO: 6) or a homologous sequence having at least 80% sequence identity with it; or (2) The HCDR1 contains an amino acid sequence containing TYGMA (SEQ ID NO: 25) or a homologous sequence having at least 80% sequence identity with it; The HCDR2 contains an amino acid sequence containing WINTLSGEPTYADDFKG (SEQ ID NO: 26) or a homologous sequence having at least 80% sequence identity with it; The HCDR3 contains an amino acid sequence containing EPMDY (SEQ ID NO: 27) or a homologous sequence having at least 80% sequence identity with it; The LCDR1 contains an amino acid sequence containing KSSQSLLDSDGKTYLS (SEQ ID NO: 28) or a homologous sequence having at least 80% sequence identity with it; The LCDR2 comprises an amino acid sequence containing LVSKLDS (SEQ ID NO: 29) or a homologous sequence having at least 80% sequence identity with it; and The LCDR3 contains an amino acid sequence containing WQGAHFPLT (SEQ ID NO: 30) or a homologous sequence having at least 80% sequence identity with it.

14. The method of claim 13, wherein the anti-GREM1 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein (1) The heavy chain variable region includes QVQLVQSGAEVKKPGASVKVSCKASGYX1FTSSGIGWVX2QAX3GQGLEWX4GEIYPRSGNTYNNEKFKGRX5TX6TX7DX8STSTX9YMELRSLRSDDTAVYX 10 CX 11 The amino acid sequence shown in REAYSHHYYAMDYWGQGTTVTVSS (SEQ ID NO: 13), and / or The light chain variable region contains DX 12 VMTQTPLSLX 13 VTPGQPASISCRSSQSLLHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRX 14 The amino acid sequence shown in SEQ ID NO: 17 is as follows: X1 is S or T; X2 is K or R; X3 is S or P; X4 is I or M; X5 is A or V; X6 is L or M; X7 is A or T; X8 is K or T; X9 is V or A; X10 is F or Y; X11 is V or A; X12 is V or I; X13 is P or S; X14 is L or F; or (2) The heavy chain variable region comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 31, SEQ ID NO: 33 and SEQ ID NO: 34, and / or The light chain variable region comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 32 and SEQ ID NO:

35.

15. The method according to claim 14, wherein X1 is S; X7 is A; X8 is K; and X11 is V.

16. The method according to claim 14, wherein X1 is S; X3 is P; X5 is V; X7 is A; X8 is K; X10 is F; and X11 is V.

17. The method according to claim 14, wherein X1 is S; X2 is K; X4 is I; X6 is L; X7 is A; X8 is K; X9 is V; and X11 is V.

18. The method according to any one of claims 14 to 17, wherein X12 is V.

19. The method according to any one of claims 13 to 18, wherein The heavy chain variable region comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12; and / or The light chain variable region comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO:

16.

20. The method of claim 19, wherein The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 7, and / or the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 14; or The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 7, and / or the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 15; or The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 7, and / or the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 16; or The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 8, and / or the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 14; or The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 8, and / or the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 15; or The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 8, and / or the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 16; or The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 9, and / or the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 14; or The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 9, and / or the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 15; or The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 9, and / or the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 16; or The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 10, and / or the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 14; or The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 10, and / or the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 15; or The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 10, and / or the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 16; or The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 11, and / or the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 14; or The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 11, and / or the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 15; or The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 11, and / or the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 16; or The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 12, and / or the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 14; or The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 12, and / or the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 15; or The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 12, and / or the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 16; or The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 31, and / or the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 32; or The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 31, and / or the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 35; or The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 33, and / or the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 32; or The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 33, and / or the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 35; or The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 34, and / or the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 32; or The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 34, and / or the light chain variable region contains the amino acid sequence shown in SEQ ID NO:

35.

21. The method according to any one of the preceding claims, wherein the activin receptor antagonist comprises an antibody against an ActRIIA ligand or an antigen-binding fragment thereof, an inhibitory ActRIIA ligand mimic peptide, an inhibitory nucleic acid targeting ActRIIA ligand RNA or DNA, a polynucleotide encoding the inhibitory nucleic acid, a compound that inhibits the interaction between the ActRIIA ligand and ActRIIA, and a compound that inhibits the activity of the ActRIIA ligand.

22. The method according to any one of the preceding claims, wherein the activin receptor antagonist comprises a ligand trapping agent capable of trapping at least one ActRIIA ligand, optionally the at least one ActRIIA ligand comprising activin.

23. The method of claim 22, wherein the ligand trapping agent comprises an extracellular domain of human ActRIIA.

24. The method of claim 23, wherein the ligand trapping agent further comprises an Fc domain of human IgG (e.g., IgG1, IgG2, IgG3, or IgG4).

25. The method according to any one of claims 22 to 24, wherein the ligand trapping agent comprises sotexip.

26. The method according to any one of the preceding claims, wherein the object is a person.

27. The method according to any one of the preceding claims, wherein the administration is by oral, nasal, intravenous, subcutaneous, sublingual, or intramuscular administration.

28. An anti-GREM1 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region as defined in any one of claims 14 to 20.

29. An isolated polynucleotide encoding the anti-GREM1 antibody or its antigen-binding fragment as described in claim 28.

30. A carrier comprising the isolated polynucleotide according to claim 29.

31. A host cell comprising the vector according to claim 30.

32. A method for expressing the anti-GREM1 antibody or an antigen-binding fragment thereof according to claim 28, comprising culturing the host cells according to claim 31 under conditions of expressing the vector according to claim 30.

33. A pharmaceutical composition comprising the anti-GREM1 antibody or an antigen-binding fragment thereof as claimed in claim 28 and a pharmaceutically acceptable carrier.

34. The pharmaceutical composition of claim 33, further comprising an activin receptor antagonist as defined in any one of claims 21 to 25.

35. A kit comprising (a) a first composition comprising an anti-GREM1 antibody or an antigen-binding fragment thereof as claimed in claim 28, and (b) a second composition comprising an activin receptor antagonist as defined in any one of claims 21 to 25.

36. The kit of claim 35, wherein the first composition and the second composition are located in separate containers.

37. The kit of claim 35, wherein the first composition and the second composition are located in a container.

38. A kit comprising a composition containing the anti-GREM1 antibody or an antigen-binding fragment thereof as claimed in claim 28.

39. The kit according to any one of claims 35 to 38, further comprising a pharmaceutical instruction manual containing instructions on the use of the anti-GREM1 antibody or its antigen-binding fragment in combination with an activin receptor antagonist as defined in any one of claims 21 to 25.

40. A method for treating PAH in a subject in need, comprising administering to the subject a therapeutically effective amount of the antiGREM1 antibody of claim 28 or an antigen-binding fragment thereof, or a pharmaceutical composition of claim 33 or 34, or a kit according to any one of claims 35 to 39.

41. The method of claim 40, wherein the anti-GREM1 antibody or its antigen-binding fragment is administered in combination with an activin receptor antagonist.

42. The method according to any one of claims 1 to 9 and 40 to 41, wherein the anti-GREM1 antibody or its antigen-binding fragment is further administered in combination with a therapeutically effective amount of at least one additional therapeutic agent.

43. The method of claim 42, wherein the therapeutic agent is selected from the group consisting of: drugs targeting the endothelin pathway (e.g., endothelin receptor antagonists), drugs targeting the nitric oxide pathway (e.g., phosphodiesterase type 5 inhibitors, soluble guanylate cyclase activators), drugs targeting the prostacyclin pathway (e.g., prostacyclin analogs, prostacyclin receptor agonists), drugs targeting the vasodilatory pathway (e.g., guitarazine monoclonal antibody, therapeutic vaccine ETR-002 peptide, pegvetidinil (vasoactive intestinal peptide analog), endothelial progenitor cells (eNOS gene)), diuretics, anticoagulants, cardiac glycosides, vasodilators, prostacyclin analogs, calcium channel blockers, endothelial antagonists, lipid-lowering agents, phosphodiesterase inhibitors, thromboxane inhibitors, and endopeptidase inhibitors.

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