Recombinant fusion protein for the treatment of pulmonary hypertension

Abstract

This invention provides a recombinant fusion protein comprising an extracellular domain of human activin receptor type 2A fused to the constant region of an immunoglobulin heavy chain, optionally by a linker, wherein the extracellular domain of human ACVR2A includes an Asn(N) to Ala(A) mutation at a site corresponding to position 24 of the sequence described in SEQ ID NO: 1 to remove a glycosylation site.

Description

[Technical Field]

[0001] This application relates to a recombinant fusion protein comprising the extracellular domain of human activin receptor type 2A (ACVR2A) fused to the constant region of an immunoglobulin heavy chain, wherein the extracellular domain of human ACVR2A comprises a mutation at a site corresponding to position 24 of the amino acid sequence of SEQ ID NO: 1, which removes a glycosylation site. This application also relates to the use of the recombinant fusion protein of this disclosure in the treatment of pulmonary hypertension and related complications. [Background technology]

[0002] Pulmonary hypertension (PH) is a chronic and progressive disease characterized by elevated pressure in the pulmonary arteries, which, if left untreated, can lead to right heart failure and ultimately death. PH can be idiopathic or associated with a variety of underlying conditions, including connective tissue disorders, congenital heart disease, and chronic lung disease. Symptoms of PH include shortness of breath, fatigue, chest pain, and syncope.

[0003] PH is diagnosed through a combination of physical examination, imaging tests such as echocardiography, right heart catheterization, and blood tests to assess oxygen levels and other indicators of disease severity. Treatment for PH depends on the underlying cause but often includes medications that dilate the pulmonary arteries and reduce pressure; supplemental oxygen; and lifestyle modifications.

[0004] Sotatercept is a fusion protein used in the treatment of pulmonary hypertension (PH) that contains the extracellular domain of wild-type activin receptor 2A (ACVR2A) fused with the Fc domain of an immunoglobulin. It captures activin and growth and differentiation factors involved in pulmonary hypertension, thereby inhibiting signaling pathways that lead to abnormal proliferation and constriction of pulmonary blood vessels, reducing the load on the right side of the heart and improving blood flow to the lungs.

[0005] Clinical trials have shown that treatment with sotatercept can significantly improve exercise capacity, reduce pulmonary vascular resistance, and improve hemodynamic parameters in patients with PAH. Further clinical trials are currently underway to evaluate the long-term safety and efficacy of sotatercept in PAH and other pulmonary vascular disorders.

[0006] WO2021262718A1 discloses a composition comprising an ACVR2A-Fc fusion protein (e.g., sotatercept) and a method for treating or preventing PH and PH-related complications using the same.

[0007] No reference or identification of any document in this application constitutes an admission that such document is available as prior art to this disclosure. [Overview of the project]

[0008] The inventors of the present invention have designed and prepared a recombinant fusion protein containing the extracellular domain of human activin receptor type 2A (ACVR2A) fused to the constant region of an immunoglobulin heavy chain, which contains a deglycosylation mutation in the extracellular domain of human ACVR2A at the site corresponding to position 24 of SEQ ID NO: 1, and is superior to sotatercept in the treatment or alleviation of pulmonary hypertension and related complications. In particular, administration of the recombinant fusion protein of the present invention can further improve animal survival compared to sotatercept in a monocrotaline-induced pulmonary arterial hypertension model.

[0009] Accordingly, in a first embodiment, the disclosure provides a recombinant fusion protein comprising i) an extracellular domain of activin receptor type 2A (ACVR2A), ii) an extracellular domain linked to a constant region of an immunoglobulin heavy chain, wherein the extracellular domain of ACVR2A may include a mutation from asparagine (Asn,N) to alanine (Ala,A) at a site corresponding to position 24 of the amino acid sequence of SEQ ID NO: 1, and is capable of binding to activin or growth / differentiation factors with particularly high affinity / potency. The extracellular domain of ACVR2A may be capable of binding to activin and growth / differentiation factors with particularly high affinity or potency. In certain embodiments, the extracellular domain of ACVR2A may be capable of binding to activin and growth / differentiation factors with high affinity or potency. The extracellular domain of ACVR2A may include asparagine (Asn,N) or alanine (Ala,A) at a site corresponding to position 47 of the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the extracellular domain of ACVR2A may contain asparagine (Asn,N) at the site corresponding to position 47 of the amino acid sequence of SEQ ID NO: 1.

[0010] Activin receptor type 2A may be human activin receptor type 2A. The extracellular domain of human activin receptor type 2A may contain a mutation from asparagine (Asn,N) to alanine (Ala,A) at the site corresponding to position 24 of the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the extracellular domain of ACVR2A may contain asparagine (Asn,N) at the site corresponding to position 47 of the amino acid sequence of SEQ ID NO: 1. The extracellular domain of human activin receptor type 2A may contain an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to SEQ ID NO: 1 (X1=A,X2=N). In certain embodiments, the extracellular domain of human activin receptor type 2A may include an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 1 (X1=A, X2=N). In certain embodiments, the extracellular domain of human activin receptor type 2A may include the amino acid sequence of SEQ ID NO: 1 (X1=A, X2=N).

[0011] The immunoglobulin heavy chain constant region may be a human immunoglobulin heavy chain constant region, for example, a human IgG1 or IgG4 heavy chain constant region. The immunoglobulin heavy chain constant region may also be the Fc domain of the immunoglobulin heavy chain constant region. In certain embodiments, the immunoglobulin heavy chain constant region may be the Fc domain of a human IgG1 heavy chain constant region, for example, containing the amino acid sequence of SEQ ID NO: 2.

[0012] The recombinant fusion protein of this disclosure may include the extracellular domain and the constant immunoglobulin heavy chain region of human ACVR2A from the N-terminus to the C-terminus.

[0013] The extracellular domain of activin receptor type 2A may be fused to the constant region of the immunoglobulin heavy chain via a linker. The linker may be a peptide of about 5 to 30 amino acid residues. In certain embodiments, the linker may be a peptide of about 5 to 20 amino acid residues. In certain embodiments, the linker may be a GS linker containing, for example, the amino acid sequence of SEQ ID NO: 3. The recombinant fusion protein of the Disclosure may comprise the extracellular domain of human activin receptor type 2A fused via a linker to the Fc domain of the constant region of the human immunoglobulin heavy chain, wherein the extracellular domain of human activin receptor type 2A may comprise a mutation from asparagine (Asn, N) to alanine (Ala, A) at the site corresponding to position 24 of the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the recombinant fusion protein of the Disclosure may comprise the extracellular domain of human activin receptor type 2A, the linker, and the Fc domain of the constant region of the human immunoglobulin heavy chain from the N-terminus to the C-terminus. The extracellular domain of the human activin receptor type 2A may contain asparagine (Asn,N) at the site corresponding to position 47 of the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the recombinant fusion protein of the Disclosure may contain an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 4 (X1=A, X2=N). In certain embodiments, the recombinant fusion protein of the Disclosure may contain the amino acid sequence of SEQ ID NO: 4 (X1=A, X2=N).

[0014] The Disclosure also provides dimers comprising the recombinant fusion proteins of the Disclosure. In certain embodiments, the Disclosure provides homodimers of the recombinant fusion proteins of the Disclosure comprising two recombinant fusion proteins of the Disclosure linked, for example, via one or more disulfide bonds.

[0015] The recombinant fusion proteins or dimers (including homodimers) of this disclosure may bind to one or more ligands selected from the group consisting of, for example, human-derived activin A, activin B, BMP-10, GDF-8, and GDF11, and may also block the interaction between activin A, activin B, BMP-10, GDF-8, or GDF11 and human activin receptor type 2A.

[0016] In a second embodiment, the Disclosure provides a nucleic acid molecule encoding a recombinant fusion protein of the Disclosure, an expression vector which may contain the nucleic acid molecule, and a host cell which may contain the expression vector or which may incorporate the nucleic acid molecule into its genome. A method for preparing the recombinant fusion protein or homodimer of the Disclosure using a host cell is also provided, which may comprise the steps of (i) expressing the recombinant fusion protein or homodimer in a host cell and (ii) isolating the recombinant fusion protein or homodimer from the host cell or its cell culture medium.

[0017] This disclosure provides compositions which may comprise recombinant fusion proteins, dimers (including homodimers), nucleic acid molecules, expression vectors, or host cells. In certain embodiments, the compositions are pharmaceutical compositions which may further comprise pharmaceutically acceptable carriers.

[0018] This disclosure also provides a kit which may include the recombinant fusion protein or dimer (including homodimer) of this disclosure and an injection device. The recombinant fusion protein or dimer (including homodimer) of this disclosure may be lyophilized or soluble. In certain embodiments, the recombinant fusion protein or dimer (including homodimer) is lyophilized.

[0019] In a third aspect, the present disclosure provides a method for treating or alleviating pulmonary hypertension (PH) and / or related complications in a subject that requires it, comprising the step of administering a therapeutically effective amount of the pharmaceutical composition of the present disclosure to the subject. Related complications may include, but are not limited to, smooth muscle and / or endothelial cell proliferation in the pulmonary artery, angiogenesis in the pulmonary artery, dyspnea, chest pain, pulmonary vascular remodeling, right ventricular hypertrophy, and pulmonary fibrosis. In certain embodiments, the subject is human.

[0020] The present disclosure also provides the use of the pharmaceutical composition of the present disclosure in the treatment or alleviation of pulmonary hypertension and / or related complications and in the preparation of a medicament for the treatment or alleviation of pulmonary hypertension and / or related complications.

[0021] Other features and advantages of the present disclosure will become apparent from the following detailed description and examples, which should not be considered limiting. The contents of all references, GenBank entries, patents, and patent application publications cited throughout this application are hereby expressly incorporated by reference into this specification.

[0022] Therefore, the object of the present application is that none of the products known from before, the manufacturing processes of such products, or the methods of using such products are included within the present application. Therefore, the applicant reserves the rights and discloses herein a disclaimer of any products, processes, or methods known from before. Furthermore, it should be noted that the present application does not intend to include within the scope of the present application any products, the manufacturing processes of such products, or the methods of using such products that do not meet the written description and enablement requirements of the USPTO (35 U.S.C. § 112, paragraph 1) or the EPO (Article 83 of the EPC); therefore, the present applicant reserves the rights and discloses a disclaimer of any products, manufacturing processes, or methods of using products described previously. In the implementation of the present application, it may be advantageous to comply with Article 53(c) of the EPC, Rules 28(b) and (c) of the EPC. All rights to expressly abandon any embodiments that are the subject of any registered patents of the applicant in the line of the present application, or in any other line, or in any application filed previously by any third party are expressly reserved. Nothing in this specification shall be construed as a promise.

[0023] In the present disclosure, particularly in the claims and / or paragraphs, terms such as "comprises", "comprised", "comprising" and the like may have the meaning given to them in United States patent law; for example, they can mean "includes", "included", "including" and the like; and it should be noted that terms such as "consisting essentially of" and "consists essentially of" have the meaning given to them in United States patent law, for example, they allow elements not expressly recited but exclude elements found in the prior art or elements that affect the basic or novel characteristics of the present application. Description of the Drawings

Brief Description of the Drawings

[0024] The following detailed description is provided as an example, but is not intended to limit this application to the specific embodiments described, and will be best understood in conjunction with the accompanying drawings.

[0025] [Figure 1] The SEC-HPLC results for sotatercept analog (A), ACVR2A-Fc (B), ACVR2A-24A-Fc (IMM72, C), ACVR2A-47A-Fc (D), and ACVR2A-24A / 47A-Fc (E) are shown.

[0026] [Figure 2] This shows an SDS-PAGE gel image of ACVR2A-Fc fusion proteins, including sotatercept analog (lane 1), ACVR2A-Fc (lane 2), ACVR2A-24A-Fc (IMM72, lane 3), ACVR2A-47A-Fc (lane 4), and ACVR2A-24A / 47A-Fc (lane 5).

[0027] [Figure 3] The BLI binding curves for ACVR2A-Fc(A), ACVR2A-24A-Fc(IMM72, B), ACVR2A-47A-Fc(C), and ACVR2A-24A / 47A-Fc(D) to activin A are shown.

[0028] [Figure 4] The BLI binding curves for sotatercept analog (A) and IMM72 (B) to activin A are shown.

[0029] [Figure 5] This shows the binding ability of the ACVR2A-Fc fusion protein to activin A, as measured by ELISA.

[0030] [Figure 6] The KD values ​​(A) and shifts (B) in the BLI test, which measures the binding affinity of the ACVR2A-Fc fusion protein to five ligands, are shown.

[0031] [Figure 7] The ACVR2A-Fc fusion protein exhibits the ability to inhibit the binding or interaction of ACVR2A with activin A(A), activin B(B), or BMP-10(C) in cell-based assays.

[0032] [Figure 8-1] The ACVR2A-Fc fusion protein exhibits the ability to inhibit the binding or interaction of ACVR2A with activin A(A), activin B(B), or GDF-11(C) in cell-based assays. [Figure 8-2] The ACVR2A-Fc fusion protein exhibits the ability to inhibit the binding or interaction of ACVR2A with BMP-10(D) or GDF-8(E) in cell-based assays.

[0033] [Figure 9] This shows the survival rates of rats treated with monocrotaline and ACVR2A-Fc fusion protein.

[0034] [Figure 10] The PAAT / PET ratio (A) and RVH index (B) of rats treated with monocrotaline and ACVR2A-Fc fusion protein are shown. Detailed description of this application

[0035] To ensure that this disclosure is easily understood, certain terms are defined first. Additional definitions are provided throughout the detailed description.

[0036] The term "ACVR2A" refers to activin receptor type 2A. The term "ACVR2A" includes variants, isoforms, homologs, orthologs, and paralogs. A "variant" or "mutant" of ACVR2A or its extracellular domain refers to an ACVR2A protein or ACVR2A extracellular domain that contains one or more mutations compared to the wild-type ACVR2A protein or wild-type ACVR2A extracellular domain. For example, an ACVR2A variant may include a mutation in the extracellular domain corresponding to the change from asparagine (Asn,N) to alanine (Ala,A) at position 24 of the amino acid sequence of SEQ ID NO: 1.

[0037] The term "immunoglobulin," also known as antibody, refers to molecules produced by white blood cells that help the body defend against infections and cancer. There are five types of immunoglobulins: IgG, IgM, IgA, IgD, and IgE. Whole or full-length antibodies contain at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Mammalian Ig heavy chains have five types, denoted by Greek letters: α, δ, ε, γ, and μ. These chains are found in IgA, IgD, IgE, IgG, and IgM antibodies, respectively. Each heavy chain has a heavy chain variable region (V in this specification). H (Abbreviated as) and heavy chain steady region (C H ) includes. The heavy chain constant region is identical in all antibodies of the same isotype, but differs in antibodies of different isotypes. The heavy chain γ, α, and δ consist of three tandem Ig domains - C H1 , C H2 , C H3 - It has a constant region composed of and a hinge region for adding flexibility. Heavy chains μ and ε have a constant region composed of four immunoglobulin domains. The constant region of the antibody's heavy chain may mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0038] The term "Fc domain" of an immunoglobulin or antibody is the tail region of the antibody that interacts with several proteins of the Fc receptor and the complement system and activates the immune system. The IgG, IgA, and IgD Fc domains are composed of two identical fragments derived from the second and third constant domains (C H2 and C H3 ) of the heavy chain of the antibody, while the IgM and IgE Fc regions contain three heavy chain constant domains (C H domains 2-4).

[0039] The term "EC 50 " is also known as the half-maximal effective concentration and refers to the concentration of a molecule, such as a recombinant fusion protein or dimer of the present disclosure, that induces an intermediate response between baseline and maximum after a specific exposure time.

[0040] The term "IC 50 " is also known as the half-maximal inhibitory concentration and refers to the concentration of a molecule, such as a recombinant fusion protein or dimer of the present disclosure, that inhibits a specific biological or biochemical function by 50% compared to the case where the molecule is absent.

[0041] The term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles, including mammals and non-mammals, but mammals such as non-human primates, sheep, dogs, cats, cows, and horses are preferred.

[0042] As used herein, “sequence identity” refers to the percentage of nucleotide / amino acid residues in a target sequence that are identical to those in a reference sequence after the sequences have been aligned, gaps introduced where necessary, and the maximum sequence identity percentage between the sequences has been achieved. Pairwise and multiple sequence alignment can be achieved in various ways known to those skilled in the art, for example, by using publicly available computer software such as ClustalOmega, T-coffee, Kalign, and MAFFT, for the purpose of determining percentage sequence identity between two or more amino acid or nucleic acid sequences. When using such software, default parameters for, for example, gap penalties and extension penalties are preferably used.

[0043] The term "therapeutic effective dose" means an amount of a molecule, such as a recombinant fusion protein or dimer of the Disclosure, sufficient to prevent or improve symptoms associated with a disease or condition (e.g., pulmonary hypertension) and / or reduce the severity of that disease or condition. The therapeutic effective dose is understood in the context of the condition being treated, where the actual effective amount is readily apparent to those skilled in the art.

[0044] In this specification, the term "high affinity / high activity" means that the recombinant fusion protein of this disclosure has binding affinity, activity, or activity equivalent to or higher than that of sotatercept or wild-type ACVR2A.

[0045] Pulmonary hypertension (PH) is a type of hypertension that affects the arteries on the right side of the heart and in the lungs. Pulmonary arterial hypertension (PAH) is a form of PH characterized by pulmonary vascular remodeling, which is caused by increased proliferation or decreased apoptosis of vascular endothelial and smooth muscle cells, leading to narrowing or damage of the pulmonary blood vessels.

[0046] Osteogenesis imperfecta receptor type 2 (BMPR2) is a member of the transforming growth factor β (TGF-β) superfamily and plays a crucial role in maintaining pulmonary artery endothelial cell integrity; however, mutations in this protein can lead to PAH. Sotatercept acts as a ligand trap for activin and other members of the TGF-β superfamily, including osteomorphogenetic proteins, restoring balance between pro- and anti-proliferative signaling pathways.

[0047] The inventors of this disclosure have designed and prepared a recombinant fusion protein comprising the extracellular domain of human activin receptor type 2A (ACVR2A) fused to the constant region of an immunoglobulin heavy chain, wherein the glycosylation site in the extracellular domain of human activin receptor 2A located at the site corresponding to position 24 of the amino acid sequence of SEQ ID NO: 1 differs from that of sotatercept. In particular, the recombinant fusion protein of this disclosure lacks the glycosylation site in the extracellular domain of human activin receptor type 2A located at the site corresponding to position 24 of the amino acid sequence of SEQ ID NO: 1. Due to the deglycosylation mutation at a specific site, the recombinant fusion protein of this disclosure exhibits a higher response than sotatercept analogs in activin A, activin B, BMP-10, GDF-11, or GDF-8 binding affinity assays. That is, the response at equilibrium is higher for the recombinant fusion protein of this disclosure, i.e., it appeared that the recombinant fusion protein of this disclosure could bind to more ligand molecules compared to sotatercept analogs (see Examples 3 and 5). The recombinant fusion protein of this disclosure also demonstrates significantly higher binding affinity to activin A than sotatercept analogs and ACVR2A(WT)-Fc protein in ELISA (see Example 4). Furthermore, the recombinant fusion protein of this disclosure has a higher ability to inhibit ACVR2A-activin A interaction than ACVR2A(WT)-Fc protein and sotatercept analogs, and inhibits IC50 when inhibiting ACVR2A-activin B / GDF11 / BMP-10 interaction. 50 This indicates a low value (see Example 6).

[0048] Most importantly, the recombinant fusion protein of this disclosure is superior to sotatercept in treating or alleviating pulmonary hypertension and related complications. Specifically, administration of the recombinant fusion protein of this disclosure is more effective than sotatercept in alleviating right ventricular hypertrophy in monocrotaline-induced pulmonary arterial hypertension models, and therefore improves animal survival.

[0049] In the field of protein engineering, it is well known that deglycosylation mutations can alter the biological activity of proteins in unpredictable ways.

[0050] Wang et al. disclosed that N-linked glycosylation of specific amino acid residues in the PD-L1 molecule plays a crucial role in PD-L1's binding affinity to PD-1 and its immunosuppressive function in vivo. Deglycosylation promotes the degradation of PD-L1 and inhibits its binding to PD-1, and removal of N-linked glycosylation in the PD-L1 molecule in fixed tissue samples improved antibody binding.

[0051] Tansky et al. showed that complete removal of N-linked glycosylation of NK1R in vitro was achieved. max We revealed that it significantly reduced [a certain factor], but did not affect its binding affinity to substance P. Furthermore, deglycosylation did not significantly affect NK1R's ability to activate the MAP kinase family (p42 / p44, JNK, and p38), but it did affect SP-induced IL-8 secretion.

[0052] The extracellular domain of activin receptor type 2A contains at least two putative N-linked glycosylation sites corresponding to positions 24 and 47 of the amino acid sequence of Sequence ID No. 1. Deglycosylation at these two sites unexpectedly resulted in homogeneity and activity of different molecules.

[0053] In particular, the inventors of this disclosure constructed three recombinant fusion protein dimers, namely ACVR2A-24A-Fc (or IMM72), ACVR2A-47A-Fc, and ACVR2A-24A / 47A-Fc, each containing a variant extracellular domain of the activin receptor type 2A. IMM72, ACVR2A-47A-Fc, and ACVR2A-24A / 47A-Fc are otherwise identical, except that IMM72 contains the N24A deglycosylation mutation, ACVR2A-47A-Fc contains the N47A deglycosylation mutation, and ACVR2A-24A / 47A-Fc contains both the N24A and N47A deglycosylation mutations, and the amino acid residue numbering of these fusion proteins is based on the numbering of Sequence ID No. 1. Surprisingly, as the following examples demonstrate, the protein homogeneity (demonstrated by SEC-HPLC and SDS-PAGE data) and the biological activity of IMM72 (shown by Grato affinity and ELISA binding data) are significantly better than those of ACVR2A-47A-Fc or ACVR2A-24A / 47A-Fc.

[0054] Even more surprisingly, the protein homogeneity and in vivo efficacy of IMM72, which has an N-to-A mutation at the site corresponding to position 24 of Sequence ID No. 1, are even better than those of sotatercept, which utilizes the wild-type ACVR2A extracellular domain.

[0055] The recombinant fusion protein of this disclosure comprises i) an extracellular domain of activin receptor type 2A, ii) an optional linker, and iii) a constant region of an immunoglobulin heavy chain.

[0056] Activin receptor type 2A may be human activin receptor type 2A. The extracellular domain of human activin receptor type 2A may contain a mutation that corresponds to a change from asparagine (Asn,N) to alanine (Ala,A) at position 24 of the amino acid sequence of SEQ ID NO: 1. The extracellular domain of ACVR2A may contain asparagine (Asn,N) at the site corresponding to position 47 of the amino acid sequence of SEQ ID NO: 1. The extracellular domain of human activin receptor type 2A may contain an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to SEQ ID NO: 1 (X1=A,X2=N). In certain embodiments, the extracellular domain of human activin receptor type 2A may include an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 1 (X1=A, X2=N). In certain embodiments, the extracellular domain of human activin receptor type 2A may include the amino acid sequence of SEQ ID NO: 1 (X1=A, X2=N).

[0057] N-linked glycosylation occurs at the consensus sequence Asn-X-Ser / Thr, where glycan is linked to the amino group of asparagine and X represents any amino acid except proline. Any change to the consensus sequence can result in deglycosylation. In other words, a mutation may be applied to any one of the amino acid residues located at the sites corresponding to positions 24 through 26 of SEQ ID NO: 1, as long as the mutation can result in deglycosylation. For example, asparagine (Asn,N) at the site corresponding to position 24 of SEQ ID NO: 1 may be deleted or replaced with another amino acid to remove the glycosylation site. The amino acid residue at the site corresponding to position 25 of SEQ ID NO: 1 may be deleted or replaced with proline (Pro,P). Alternatively, serine (Ser,S) or threonine (Thr,T) at the site corresponding to position 26 of SEQ ID NO: 1 may be deleted or replaced with an amino acid residue other than Ser or Thr.

[0058] The extracellular domain of activin receptor type 2A may include one or more conservative modifications. In the art, it is understood that certain conservative sequence modifications can be added, for example, that do not remove the protein's ability to bind to a ligand. The extracellular domain of activin receptor type 2A of the present disclosure may bind with or without conservative modifications to one or more ligands selected from the group consisting of activin A, activin B, and GDF11. The extracellular domain of activin receptor type 2A of the present disclosure may further bind with or without conservative modifications to one or more ligands selected from the group consisting of BMP10, GDF8, and BMP6.

[0059] As used herein, the term “conservative sequence modification” is intended to mean an amino acid modification that does not significantly affect or alter the binding properties of the ACVR2A extracellular domain, which includes an amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the ACVR2A extracellular domain of this disclosure by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. A conservative amino acid substitution is the substitution of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, one or more amino acid residues in the ACVR2A extracellular domain of this disclosure can be substituted with other amino acid residues of the same side chain family, and the altered extracellular domain can be tested using the functional assays described herein for residual function, such as binding ability to activin A, activin B, GDF11, BMP10, GDF8, and / or BMP6.

[0060] The immunoglobulin heavy chain constant region is used primarily to stabilize the structure of the recombinant fusion protein of the disclosure and / or to extend the serum half-life of the recombinant fusion protein of the disclosure. The full-length heavy chain constant region or a fragment thereof, such as an Fc domain, may be used to the extent that it can stabilize the structure of the recombinant fusion protein of the disclosure and / or to extend the serum half-life.

[0061] The immunoglobulin heavy chain constant region may be a human immunoglobulin heavy chain constant region, for example, a human IgG1 or IgG4 heavy chain constant region. The immunoglobulin heavy chain constant region may also be the Fc domain of the immunoglobulin heavy chain constant region. In certain embodiments, the immunoglobulin heavy chain constant region may be the Fc domain of a human IgG1 heavy chain constant region, for example, containing the amino acid sequence of SEQ ID NO: 2.

[0062] The extracellular domain of the activin receptor type 2A may be fused to the constant region of the immunoglobulin heavy chain via a linker.

[0063] The linker may consist of amino acids linked together by peptide bonds, preferably 5 to 30 amino acids linked by peptide bonds, where the amino acids are selected from 20 naturally occurring amino acids. As will be understood by those skilled in the art, one or more of these amino acids may be glycosylated. In one embodiment, the 5 to 30 amino acids may be selected from glycine, alanine, proline, asparagine, glutamine, serine, and lysine. In one embodiment, the linker is largely composed of sterically unhinged amino acids such as glycine and alanine. Exemplary linkers are polyglycine, particularly poly(Gly-Ala), and polyalanine. In certain embodiments, the linker may be a peptide of about 5 to 20 amino acid residues. In certain embodiments, the linker may be a GS linker containing, for example, the amino acid sequence of SEQ ID NO: 3.

[0064] The linker may also be a non-peptide linker. For example, alkyl linkers such as -NH-, -(CH2)sC(O)- can be used, where s = 2 to 20. These alkyl linkers can also be lower alkyl (e.g., C 1-6 ) can be substituted with any non-sterically hindered group, such as lower acyls, halogens (e.g., CI, Br), CN, NH2, phenyl, etc.

[0065] The selection of linker peptides may alter the biological activity of the protein. The length, flexibility, composition, and context of the linker peptides may have a significant impact on the fusion protein activity. For example, Silacci et al. disclose that longer linker lengths correspond to higher activity of IL-17A against peptide inhibitors. Conversely, however, Guo et al. have shown that the catalytic efficiency of the fusion protein enzyme gradually decreases with increasing GS repeats.

[0066] In another embodiment, the Disclosure provides nucleic acid molecules that encode recombinant fusion proteins or dimers (including homodimers) of the Disclosure.

[0067] Nucleic acid molecules can exist in whole cells, in cell lysates, or in partially purified or substantially pure forms. When nucleic acids are purified by standard techniques from other cellular components or other contaminants, such as other cellular nucleic acids or proteins, they are “isolated” or “substantially purified.” The nucleic acids of this disclosure may be, for example, DNA or RNA, and may or may not contain intron sequences. In a preferred embodiment, the nucleic acid is a cDNA molecule.

[0068] The nucleic acid molecules of this disclosure can be obtained using standard molecular biology techniques. For example, the nucleic acid molecules may be chemically synthesized. Alternatively, the nucleic acid molecules may be obtained by introducing mutations into a nucleic acid molecule encoding the extracellular domain of wild-type activin receptor type 2A fused to the constant region of an immunoglobulin heavy chain, for example, via site-directed mutagenesis or PCR-mediated mutagenesis. The DNA fragment encoding the ACVR2A extracellular domain may be functionally linked to the DNA fragment encoding the constant region of an immunoglobulin heavy chain. As used in this context, the term “functionally linked” is intended to mean that the two DNA fragments are linked in such a way that the amino acid sequences encoded by the two DNA fragments remain in frame.

[0069] The recombinant fusion protein or dimer of the present disclosure may be produced by (i) inserting a nucleotide sequence encoding the recombinant fusion protein into an expression vector functionally linked to a regulatory sequence that controls transcription or translation; (ii) transduction or transfection of host cells using the expression vector; and (iii) expressing the recombinant fusion protein of the present disclosure and forming the dimer of the present disclosure.

[0070] The term “regulatory sequence” is intended to include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of nucleic acid molecules in this disclosure.

[0071] The expression vector can encode a signal peptide that promotes the secretion of recombinant fusion proteins from host cells. The nucleic acid molecule of the Disclosure can be cloned into a vector such that the signal peptide is in-frame ligated to the amino terminus of the recombinant fusion protein of the Disclosure.

[0072] The expression vectors disclosed herein may carry additional sequences, such as sequences that regulate vector replication within host cells (e.g., origins of replication) and selection marker genes. Selection marker genes facilitate the selection of host cells into which the vector has been introduced. Typically, for example, selection marker genes confer resistance to drugs such as G418, hygromycin, or methotrexate to host cells into which the vector has been introduced. Preferred selection marker genes include the dihydrofolate reductase (DHFR) gene (for use in DHFR-host cells with methotrexate selection / amplification) and the neo gene (for G418 selection).

[0073] Expression vectors can be transfected into host cells using standard techniques. The various forms of the term "transfection" are intended to encompass a wide range of techniques commonly used to introduce foreign DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and similar methods. While it is theoretically possible to express the recombinant fusion proteins of this disclosure in either prokaryotic or eukaryotic host cells, expression of recombinant fusion proteins in eukaryotic cells, most preferably mammalian host cells, is most preferred because such eukaryotic cells, and especially mammalian cells, are more likely to associate and secrete molecules with better folded immunological activity than prokaryotic cells.

[0074] Expression vectors that can be used in this application include, but are not limited to, plasmids, viral vectors, yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), transformation-competent artificial chromosomes (TACs), mammalian artificial chromosomes (MACs), and human artificial episomal chromosomes (HAECs).

[0075] In another embodiment, the Disclosure provides a pharmaceutical composition which may comprise a recombinant fusion protein or dimer, nucleic acid molecule, expression vector, or host cell of the Disclosure, formulated with a pharmaceutically acceptable carrier. The pharmaceutical composition may optionally contain one or more additional pharmaceutically active ingredients, such as additional pharmaceutically active ingredients effective in treating or alleviating pulmonary hypertension and related complications. The pharmaceutical composition of the Disclosure may be used in combination with additional pharmaceutically active ingredients effective in treating or alleviating pulmonary hypertension and related complications.

[0076] A pharmaceutical composition may contain any number of excipients. Excipients that can be used include carriers, surfactants, thickeners or emulsifiers, solid binders, dispersion or suspension aids, solubilizers, colorants, fragrances, coatings, disintegrants, lubricants, sweeteners, preservatives, isotonic agents, and combinations thereof.

[0077] Preferably, the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active ingredient may be coated with a material to protect it from the action of acids and other natural conditions that may inactivate it. As used herein, the term “parenteral administration” means, but is not limited to, methods of administration other than enteral and topical administration, usually by injection, and includes intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions. Alternatively, the pharmaceutical compositions of this disclosure may be administered via parenteral routes, e.g., topical, epidermal, or mucosal administration routes, e.g., nasal, oral, transvaginal, transrectal, sublingual, or topical.

[0078] The pharmaceutical composition may be in the form of a sterile aqueous solution or dispersion. It may also be formulated into a microemulsion, liposome, or other ordered structure suitable for high drug concentrations.

[0079] The pharmaceutical composition may be a sustained-release formulation including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable and biocompatible polymers such as ethylene vinyl acetate, polyacid anhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used.

[0080] The administration of the pharmaceutical compositions of this disclosure may be determined, for example, by a surgeon, for example, a physician, in accordance with the subject's, for example, sex, age, medical history and similar factors.

[0081] The therapeutically effective dose of the pharmaceutical composition of this disclosure may result in a reduction in the severity of disease symptoms or an increase in the frequency and duration of periods without disease symptoms. For example, in the treatment of subjects with pulmonary hypertension and related complications, the therapeutically effective dose may increase the PAAT / PET ratio by at least about 20%, more preferably at least 40%, even more preferably at least 60%, and even more preferably at least 80%, compared to untreated subjects, or alternatively, reduce the index of right ventricular hypertrophy by at least about 20%, more preferably at least 40%, even more preferably at least 60%, and even more preferably at least 80%. Usage and Method

[0082] The pharmaceutical compositions of this disclosure may be used to treat or alleviate pulmonary hypertension and related complications.

[0083] This disclosure provides a method of combination therapy in which the pharmaceutical composition of this disclosure is administered in combination with one or more additional agents effective in treating or alleviating pulmonary hypertension and related complications.

[0084] In one embodiment, the Disclosure provides a method for treating pulmonary arterial hypertension (PAH) in a subject requiring such treatment, comprising the step of administering to the subject a therapeutically effective dose of the pharmaceutical composition of the Disclosure. Administration of the pharmaceutical composition may result in changes in one or more of the following hemodynamic or functional parameters: a decrease in pulmonary vascular resistance (PVR); an increase in 6-minute walk distance (6MWD); a decrease in N-terminal pro-B natriuretic peptide (NT-proBNP) levels; prevention or reduction of progression of pulmonary hypertension functional class as recognized by the World Health Organization (WHO); promotion or increase of regression of pulmonary hypertension functional class as recognized by the WHO; improvement of right ventricular function; improvement of pulmonary artery pressure; and / or improvement of mean right atrial pressure.

[0085] In another embodiment, the Disclosure provides a method for treating, preventing, or reducing the rate of progression and / or severity of one or more complications of pulmonary arterial hypertension in a subject requiring such treatment, comprising administering a therapeutically effective amount of the pharmaceutical composition of the Disclosure to such subject. In a particular embodiment, the one or more complications of pulmonary arterial hypertension are selected from the group consisting of smooth muscle and / or endothelial cell proliferation in the pulmonary arteries, angiogenesis in the pulmonary arteries, dyspnea, chest pain, pulmonary vascular remodeling, right ventricular hypertrophy, and pulmonary fibrosis.

[0086] In another embodiment, the Disclosure provides a method for treating or preventing cardiopulmonary remodeling associated with pulmonary arterial hypertension in a subject requiring such treatment, comprising administering a therapeutically effective amount of the pharmaceutical composition of the Disclosure to the subject requiring such treatment. The administration of the pharmaceutical composition may slow down and / or reverse cardiac remodeling. In some embodiments, cardiopulmonary remodeling is ventricular remodeling. In some embodiments, ventricular remodeling is right ventricular remodeling. In some embodiments, cardiopulmonary remodeling is ventricular dilation.

[0087] In certain embodiments, administration of the pharmaceutical compositions of the present disclosure may increase the PAAT / PET ratio, decrease the index of right ventricular hypertrophy, and / or improve patient survival.

[0088] Various aspects and embodiments of this disclosure will be discussed with reference to the drawings and the following examples. Other aspects and embodiments will be obvious to those skilled in the art. All references described herein are incorporated herein by reference in their entirety. While this application has been described with reference to exemplary embodiments, many changes and modifications will be obvious to those skilled in the art in view of this disclosure. Accordingly, the exemplary embodiments of this disclosure are provided for illustrative purposes only and should not be construed as limiting. Various modifications can be made to the embodiments without departing from the spirit and scope of this application. [Examples]

[0089] IMM72, also known as ACVR2A-24A-Fc, is a homodimer of an exemplary recombinant fusion protein of this disclosure containing the amino acid sequence of SEQ ID NO: 4 (X1=A, X2=N), which contains an N-to-A mutation at position 24 of SEQ ID NO: 1.

[0090] ACVR2A-47A-Fc is a homodimer of a recombinant fusion protein containing the amino acid sequence of SEQ ID NO: 4 (X1=N, X2=A); this recombinant fusion protein contains an N-to-A mutation at position 47 of SEQ ID NO: 1.

[0091] ACVR2A-24A / 47A-Fc is a homodimer of a recombinant fusion protein containing the amino acid sequence of SEQ ID NO: 4 (X1=A, X2=A), and this recombinant fusion protein contains an N-to-A mutation at position 24 of SEQ ID NO: 1 and an N-to-A mutation at position 47 of SEQ ID NO: 1.

[0092] ACVR2A-Fc, also known as ACVR2A(WT)-Fc, is a homodimer of a recombinant fusion protein containing the amino acid sequence of SEQ ID NO: 4 (X1=N, X2=N), in which the wild-type extracellular domain of human ACVR2A (SEQ ID NO: 1, X1=N, X2=N) is fused to the Fc domain of the human IgG1 heavy chain constant region.

[0093] Sotatercept analogs are homodimers of recombinant fusion proteins containing the extracellular domain of wild-type activin receptor type 2A fused to the Fc domain of the human IgG heavy chain constant region, where the recombinant fusion protein contains the amino acid sequence of SEQ ID NO: 5. Example 1 Generation and purification of recombinant ACVR2A-Fc fusion protein

[0094] DNA fragments encoding recombinant ACVR2A-Fc fusion proteins, including ACVR2A-24A-Fc, ACVR2A-47A-Fc, ACVR2A-24A / 47A-Fc, ACVR2A-Fc, and sotatercept analogs, were synthesized, digested with HindIII and NotI, and ligated to pMac-Fc vectors digested with the same two restriction enzymes to construct expression vectors.

[0095] CHO-S cells were cultured in TransFx-C CHO transient transfection medium (Cat#SH30942.02, Hyclone). Transient transfection was performed by transfection of CHO-S cells with an expression vector using polyethyleneimine transfection reagent (Polysciences, Cat#24765) according to the manufacturer's instructions. After 8 to 10 days, the culture supernatant of the transfected cells was collected and loaded onto a protein A Sepharose column (Bestchrom, Cat#AA0273). The column was washed with wash buffer (20 mM PB + 140 mM NaCl, pH 7.4 ± 0.1), and the recombinant fusion protein was then eluted with elution buffer (25 mM NaAc + 100 mM NaCl, pH 3.5 ± 0.1). The recovered fraction was neutralized to pH 5.2 ± 0.2 with 2 M Tris. Example 2 Characterization of recombinant ACVR2A-Fc fusion protein

[0096] The recombinant ACVR2A-Fc fusion protein prepared in Example 1 was measured for protein size and structure using size exclusion-high performance liquid chromatography (SEC HPLC). Briefly, the SEC column (Xbridge BEH 200 Å, 3.5 μm, 186007640, Waters) was mounted on an HPLC system (ARC-HPLC, Waters) and washed with ultrapure water at 0.5 ml / min for 30 minutes. Next, five column volumes (CV) of 50 mM PBS-300 mM NaCl (pH 7.3) were flowed at a flow rate of 0.5 ml / min to equilibrate the columns until the baseline flattened. 50 μg of each fusion protein was then extracted using an autosampler and injected into the system, and the absorbance was read at 280 nm. The data were analyzed using Empower to determine the peak areas, which were later normalized to calculate the percentage of each component relative to the total components.

[0097] The results are shown in Figure 1. The main peak of the IMM72 molecule is symmetrical with a narrow peak width (Figure 1(C)), which suggests that the IMM72 molecule is likely homogeneous and pure, while the ACVR2A-47A-Fc and ACVR2A-24A / 47A-Fc proteins show multiple peaks (Figure 1(D and E)), which means they may be structurally unstable.

[0098] The ACVR2A-Fc fusion proteins were further subjected to reduced SDS-PAGE. Briefly, 10 μl of each fusion protein, diluted in 1 mg / ml ultrapure water, was mixed with 2.5 μl of 5× protein loading dye (Cat#: C508320-0001, Sangon Biotech) and incubated at 80°C for 10 minutes. A 12% precast polyacrylamide gel (Cat#: C661105-0001, Sangon Biotech) was inserted into the electrophoresis chamber, and SDS-PAGE buffer was then added. The fusion protein samples, along with the loading dye, were loaded into the gel wells. Electrophoresis was performed at a voltage of 120 V for 80 minutes. The gel was then transferred to an automated staining machine (eStain® L1, GenScript) for staining and destaining, with the program set as follows: equilibrium, 1 cycle, 0.5 minutes; staining, 1 cycle, 0.5 minutes; and destaining, 3 cycles, 4 minutes per cycle. The gel was removed from the staining machine and placed on a white plate for imaging.

[0099] The results are shown in Figure 2. The main band in lane 3 containing the IMM72 molecule is clear and narrow, indicating that the IMM72 molecule is more homogeneous than the ACVR2A-Fc, ACVR2A-47A-Fc, and ACVR2A-24A / 47A-Fc proteins. Example 3 Binding affinity of recombinant ACVR2A-Fc fusion protein to activin A

[0100] The binding affinity of recombinant ACVR2A-Fc fusion protein to activin A (Cat#:ACV-HM001,Kactus) was measured by bio-layer interferometry (BLI) using a label-free biomolecular detector (Access Medical Systems, Gator bio). The assay used Q buffer (10 mM pH 7.4 PBS + 0.02% Tween® + 0.2% BSA) and an anti-human-Fc probe (Cat# 20-5036, Gator bio). The detector operated in kinetics assay mode, with each cycle including loading, association, dissociation, and regeneration (detailed program settings are shown in Table 1). The probe was regenerated using regeneration buffer (pH 2.0), and the detector was set to 25°C. Table 1. Program settings for label-free biomolecular detectors [Table 1]

[0101] The data was analyzed using an interaction model (global, Rmax not linked per sensor), K D The value was determined and reported.

[0102] The results are shown in Table 2 and Figure 3. IMM72 has a higher dissociation rate constant than the ACVR2A-Fc protein, but ACVR2A-47A-Fc and ACVR2A-24A / 47A-Fc showed little to no activin A binding affinity. Table 2. Binding affinity data for ACVR2A-Fc fusion proteins [Table 2]

[0103] Following the protocol described above, the binding affinity of IMM72 and sotatercept analogs to activin A was measured. The results are shown in Table 3 and Figure 4.

[0104] The two proteins mentioned above showed similar binding affinity. However, the reaction at equilibrium was higher for the IMM72 molecule; that is, it appeared that more activin A molecules could bind to the IMM72 protein compared to the sotatercept analog. Table 3. Binding affinity data for IMM72 and sotatercept analogs. [Table 3] Example 4 Binding ability of recombinant ACVR2A-Fc fusion protein to activin A

[0105] Recombinant fusion proteins were tested for their binding ability to activin A.

[0106] Briefly, ELISA plates were coated with 100 μl of 0.5 μg / mL activin-A protein (Cat#: ACV-HM001, Kactusbio) in 1×PBS overnight at 2–8°C, and blocked with 150 μl of PBS containing 3% skim milk for 2 hours at room temperature. Then, 100 μl of serially diluted recombinant fusion protein, starting at 10 μg / mL and diluted 3-fold, was added to the plates and incubated at 37°C for 1 hour. The plates were washed with 300 μl of wash buffer, and 100 μl of peroxidase cojugate AffiniPure F(ab')2 fragment goat anti-human IgG, Fc-γ fragment specific (diluted 1:20000 in PBS containing 3% skim milk, Cat#: 109-036-098, Jackson Immuno) was added and incubated at 37°C for 1 hour. The plate was washed with 300 μl of washing buffer, 100 μl of TMB (Cat#: 51200050, KPL) was added, and the plate was kept in the dark. The reaction was terminated by adding 50 μl of 2 M H2SO4 to the plate, and the absorbance was read at 450 nm. The data was analyzed using GraphPad Prism, and the OD450 values ​​were log 10 [Protein concentration] was plotted against this.

[0107] The results are shown in Figure 5. IMM72, which has an N-to-A mutation at position 24 of SEQ ID NO: 1, showed significantly higher binding affinity to activin A than sotatercept analogs and ACVR2A-Fc protein, while the fusion protein, which has an N-to-A mutation at position 47 of SEQ ID NO: 2, almost completely lost its binding affinity to activin A. Example 5. Binding affinity of recombinant ACVR2A-Fc fusion protein to ACVR2A ligand

[0108] The binding affinity of recombinant ACVR2A-Fc fusion proteins, including ACVR2A-24A-Fc protein (IMM72), sotatercept analogues, and ACVR2A-Fc protein, to recombinant human activin A (Cat#:ACV-HM001,Kactus), recombinant activin B protein (Cat#659-AB-025,R&D systems), recombinant human BMP-10 protein (Cat#2926-BP-025,R&D systems), recombinant GDF-11 protein (Cat#1958-GD-010,R&D systems), and recombinant human GDF-8 (Cat#cyt-418,ProSpec) was measured by biolayer interferometry (BLI) using a label-free biomolecular detector (Access Medical Systems, Gator bio). Table 4. Program settings for label-free biomolecular detectors [Table 4] Table 5. Binding affinity data for ACVR2A-Fc fusion proteins [Table 5]

[0109] The assay uses Q buffer (10 mM pH 7.4 PBS + 0.02% Tween® + 0.2% BSA) and an anti-human Fc probe (Cat# 160003, Gator). The detector operates in kinetics assay mode, and each cycle includes loading, association, dissociation, and regeneration (detailed program settings are shown in Table 4). The probe is regenerated using regeneration buffer (pH 2.0), and the detector is set to 25°C.

[0110] The data was analyzed using an interaction model (global, Rmax not linked per sensor), K D The value was determined and reported.

[0111] The results are shown in Table 5 and Figure 6. The three recombinant fusion proteins showed similar binding affinity (K) to each ligand (A). D This indicates that the reaction at equilibrium was higher for the IMM72 molecule, meaning that more ligand molecules could bind to the IMM72 protein compared to the sotatercept analog and ACVR2A-Fc protein (B). Example 6. Ability of recombinant ACVR2A-Fc fusion protein to block ACVR2A-ligand interactions

[0112] Recombinant ACVR2A-Fc fusion proteins, including ACVR2A-24A-Fc protein (IMM72), sotatercept analogues, and ACVR2A-Fc protein, were tested for their ability to inhibit the binding / interaction of ACVR2A with its ligands, including activin A, GDF11, and BMP10.

[0113] In short, 50 μl of serially diluted IMM72, sotatercept analog, or ACVR2A-Fc protein, starting at 120 μg / mL and then diluting 2-fold, was added to a 96-well plate. Subsequently, 50 μl of recombinant human activin A protein (Cat#ACV-HM001, KACTUS) at 1.0 μg / mL was added, and the mixture was incubated at room temperature for 45 minutes. MPC-11 cells (Cat#CBP60951, Co-bioer) had a cell density of 1 × 10⁶ in DMEM. 5 The cells were suspended in 50 μl / mL, and 50 μl of such a cell suspension was added to each well of the plate. The plate was incubated in a 37°C, 5% CO2 incubator for 3 days. Then, 10 μl of CCK-8 solution from Cell Counting Kit-8 (Cat#C0040, Beyotime) and 40 μl of DMEM were added, mixed homogeneously, and incubated in the incubator for 4 hours. The absorbance of the plate was read at 450 nm using a microplate reader (Tecan, sunrise).

[0114] Plate wells containing MPC-11 cells and CCK-8 solution were used as positive controls, while plate wells containing MPC-11 cells, ligand (activin A), and CCK-8 solution were used as blank controls. The inhibition rate, calculated as 1 - (OD450 test - OD450 blank) / (OD450 positive - OD450 blank) × 100%, was used to measure the reduction in MPC-11 cell death and, consequently, the inhibitory effect of recombinant ACVR2A-Fc fusion protein on ACVR2A-activin A binding / interaction.

[0115] Following the protocol described above, the inhibitory effect of recombinant ACVR2A-Fc fusion protein on the binding / interaction of ACVR2A with other ligands was measured using recombinant human GDF-11 protein (Cat#1958-GD-010, R&D systems) and recombinant human BMP-10 protein (Cat#2926-BP-025, R&D systems).

[0116] The results are shown in Figure 7. The IMM72 protein demonstrated a higher ability to inhibit ACVR2A-activin A-mediated MPC-11 cell death than the ACVR2A(WT)-Fc protein and sotatercept analog (A). The inhibitory effect of the IMM72 protein on the binding / interaction of ACVR2A to human GDF-11 protein (B) or recombinant human BMP-10 protein (C) was approximately equivalent to that of the ACVR2A(WT)-Fc protein or sotatercept analog.

[0117] The recombinant ACVR2A-Fc fusion protein was also tested for its ability to block the binding / interaction of ACVR2A with its ligands, including activin A, activin B, GDF11, BMP10, and GDF8, using 293T-pCAGA12-Luc cells prepared by transfection of 293T cells (Cat#GNHu43, National Collection of Authenticated Cell Cultures) with the pCAGA12-TA-Luc (Cat#D4026-1μg, Beyotime) plasmid. ACVR2A's interaction with its ligands on the cell surface may induce luciferase expression.

[0118] In short, 293T-pCAGA12-Luc cells have a cell density of 5 × 10⁶ in DMEM. 5 The cells were suspended in 1 / mL, and 40 μl of such cell suspension was added to each well of a 96-well opaque flat-bottom plate. 40 μL of serially diluted IMM72, sotatercept analog, or ACVR2A-Fc protein, starting at 90 μg / mL and diluted 3-fold, was added to the plate and incubated in a CO2 incubator for 45 minutes. Next, 40 μL of 100 ng / mL recombinant human activin A protein (Cat#ACV-HM001,KACTUS) in DMEM was added to the plate and incubated overnight. Finally, 70 μl of substrate solution from Steady-Lumi® II Firefly Luciferase Assay Kit (Cat#RG059M,Beyotime) was added to the plate and incubated in an incubator for 10 minutes. The plates were read using a microplate reader (Molecular Devices, SpectraMax M3) to obtain relative fluorescence units (RFU).

[0119] Plate wells containing 293T-pCAGA12-Luc cells, ligand (activin A), and substrate solutions from the Steady-Lumi® II Firefly Luciferase Assay Kit were used as positive controls, while plate wells containing 293T-pCAGA12-Luc cells and substrate solutions from the Steady-Lumi® II Firefly Luciferase Assay Kit were used as blank controls. The inhibition rate, calculated as 1-(RFU test-RFU blank) / (RFU positive-RFU blank) × 100%, was used to measure the reduction in fluorescence signal from 293T-pCAGA12-Luc cells, and consequently, the inhibitory effect of recombinant ACVR2A-Fc fusion protein on ACVR2A-activin A binding / interaction.

[0120] Following the protocol described above, the inhibitory effect of ACVR2A-Fc fusion protein on the binding / interaction of ACVR2A with other ligands was measured using recombinant human activin B protein (Cat#659-AB-025, R&D systems), recombinant human GDF-11 protein (Cat#1958-GD-010, R&D systems), recombinant human BMP-10 protein (Cat#2926-BP-025, R&D systems), and recombinant human GDF-8 protein (Cat#cyt-418, ProSpec).

[0121] The results are shown in Figures 8-1 and 8-2. The IMM72 protein exhibited a much higher ability to inhibit the ACVR2A-activin A interaction than the ACVR2A(WT)-Fc protein and sotatercept analog (A), and a lower IC50 than the ACVR2A-Fc(WT) protein and sotatercept analog in blocking the ACVR2A-activin B interaction (B), ACVR2A-GDF11 interaction (C), and ACVR2A-BMP-10 interaction (D). 50The values ​​were shown. The inhibitory effect of the IMM72 protein on the binding and interaction of ACVR2A with the GDF-8 protein is approximately equivalent to that of the sotatercept analog (E). Example 7. In vivo efficacy of recombinant ACVR2A-Fc fusion protein

[0122] Recombinant ACVR2A-Fc fusion proteins were tested for their in vivo efficacy in treating or alleviating pulmonary hypertension in a monocothaline-induced pulmonary arterial hypertension model.

[0123] Briefly, 40 male Sprague-Dawley (SD) rats were subcutaneously injected with either monocrotaline (MCT) or vehicle control (PBS) at a dose of 60 mg / kg body weight to induce pulmonary hypertension. After 7 days, the MCT-treated rats were randomly assigned to three groups based on body weight and began receiving intraperitoneal (IP) injections of IMM72, sotatercept analog, or vehicle control (PBS) according to the scheme in Table 6, with this day designated as day 0. The experiment concluded on day 23. Table 6. Scheme for animal experiments [Table 6]

[0124] The rats were observed daily for their overall appearance (including fur) and activity level, and their weight was recorded once a week.

[0125] On day 23, 8, 4, and 5 rats survived in groups 2, 3, and 4, respectively. These rats were measured using a VisualSonics Vevo2100 imaging system for pulmonary artery acceleration time (PAAT), pulmonary artery ejection time (PET), right ventricular systolic pressure (PVSP), percentage change in right ventricular area (RVFAC), tricuspid annular systolic displacement (TAPSE), and tricuspid regurgitation peak velocity (TRpeak). They were then sacrificed, and their hearts and lungs were recovered for further investigation. Cardiac and lung tissue from rats that died earlier was also recovered. The weights of the right ventricle (RV), left ventricle (LV), and interventricular septum (S) were measured, and the right ventricular hypertrophy index (RVHI) was calculated as RV / (LV+S) × 100%. Lung tissue was subjected to HE or Masson staining to reveal minor vascular dysfunction.

[0126] The results are shown in Figures 9 and 10.

[0127] Rats in groups 2 through 4 injected with monocrotaline gained weight more slowly than those in group 1, and, according to lung tissue staining results, all deaths in the three monocrotaline groups were due to pulmonary abnormalities such as pulmonary congestion and / or respiratory failure.

[0128] As shown in Figure 9, administration of IMM72 or sotatercept analogs reduced or delayed animal mortality compared to vehicle control, and the survival rate of the IMM72-treated group was slightly higher than that of the sotatercept-treated group.

[0129] Furthermore, as shown in Figure 10, administration of IMM72 or sotatercept analogs slightly increased the PAAT / PET ratio (indicating lower pulmonary artery pressure) and mitigated right ventricular hypertrophy compared to vehicle control (A) (B). The p-value for the t-test between Group 2 and Group 4 regarding the right ventricular hypertrophy index was 0.0858, suggesting a trend toward statistical significance.

[0130] The arrangement of this application is described below. [Table 7] JPEG2026519452000008.jpg191135

[0131] While the above description relates to one or more embodiments of this application, it should be understood that the application is not limited to those embodiments, and the description is intended to encompass all alternatives, modifications, and equivalents that may fall within the spirit and scope of the appended claims. All references cited herein are further incorporated by reference in their entirety. References 1.McLaughlin, VV et al.,(2009). Journal of the American College of Cardiology, 53(17), 1573-1619. 2.Chin, KM et al.,(2021). The Lancet Respiratory Medicine, 9(2), 146-158 3.Ying-Nai Wang, Heng-Huan Lee, Jennifer L. Hsu, Dihua Yu & Mien-Chie Hung(2020) Journal of Biomedical Science, 27, 77 4.Morris F. Tansky et al.,(2007)PNAS, 104(25), 10691-10696 5.Michela Silacci et al.,(2014)J Biol Chem. 289(20), 14392-14398 6. Huili Guo et al.,(2017)Mol Biosyst. 13(3), 598-606 7.Yuelin Kong et al.,(2016)Enzyme Microb Technol.82, 105-109. 8.Marc Humbert et al.,(2021)N Engl J Med 384, 1204-1215 9. Marius M. Hoeper et al., (2023) N Engl J Med 388, 1478-1490

Claims

1. Recombinant fusion protein, i) The extracellular domain of human activin receptor type 2A (ACVR2A), and ii) Constant region of immunoglobulin heavy chain Equipped with, The extracellular domain of the human ACVR2A is linked to the constant region of the immunoglobulin heavy chain, The extracellular domain of the human ACVR2A contains a mutation from Asn(N) to Ala(A) at the site corresponding to position 24 of Sequence ID No.

1. The recombinant fusion protein is capable of binding to activin and / or growth and differentiation factors. Recombinant fusion protein.

2. The recombinant fusion protein according to claim 1, comprising the extracellular domain of human ACVR2A and the constant immunoglobulin heavy chain region from the N-terminus to the C-terminus.

3. The recombinant fusion protein according to claim 1, wherein the extracellular domain of human ACVR2A comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 1, and the amino acid residues at positions 24 and 47 in SEQ ID NO: 1 are A and N, respectively.

4. The recombinant fusion protein according to claim 3, wherein the extracellular domain of human ACVR2A comprises the amino acid sequence of SEQ ID NO: 1, and the amino acid residues at positions 24 and 47 of SEQ ID NO: 1 are A and N, respectively.

5. The recombinant fusion protein according to claim 1, wherein the immunoglobulin heavy chain constant region is a human IgG1 or IgG4 heavy chain constant region.

6. The recombinant fusion protein according to claim 5, wherein the immunoglobulin heavy chain constant region is the Fc domain of the heavy chain constant region of human IgG1 or IgG4.

7. The recombinant fusion protein according to claim 6, wherein the immunoglobulin heavy chain constant region comprises the amino acid sequence of SEQ ID NO:

2.

8. The recombinant fusion protein according to claim 1, wherein the extracellular domain of human ACVR2A is linked to the constant region of the immunoglobulin heavy chain via a linker.

9. The recombinant fusion protein according to claim 8, wherein the linker is a GS linker.

10. The recombinant fusion protein according to claim 9, wherein the linker comprises the amino acid sequence of SEQ ID NO:

3.

11. The recombinant fusion protein according to claim 1, comprising the amino acid sequence of SEQ ID NO: 4, wherein the amino acid residues at positions 24 and 47 of SEQ ID NO: 1 are A and N, respectively.

12. A homodimer of a recombinant fusion protein according to any one of claims 1 to 11, wherein the homodimer comprises two recombinant fusion proteins linked by one or more disulfide bonds.

13. A nucleic acid molecule encoding a recombinant fusion protein according to any one of claims 1 to 11.

14. An expression vector comprising the nucleic acid molecule described in claim 13.

15. A host cell comprising the expression vector described in claim 14 or having the nucleic acid molecule described in claim 13 incorporated into its genome.

16. A pharmaceutical composition comprising a recombinant fusion protein according to any one of claims 1 to 11, a homodimer according to claim 12, a nucleic acid molecule according to claim 13, an expression vector according to claim 14 or a host cell according to claim 15, and at least one pharmaceutically acceptable carrier.

17. Use of the pharmaceutical composition according to claim 16 in the preparation of a pharmaceutical for the treatment of pulmonary hypertension and related complications.

Images