An acvr2a antibody and uses thereof

Abstract

The application discloses an ACVR2A antibody and application thereof, and relates to the technical field of biological medicine. The antibody provided by the application has the advantages of stronger affinity to ACVR2A and higher selectivity to ACVR2A, and provides diversified treatment options for different clinical requirements and expands the application potential of ACVR2A targeted treatment.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an ACVR2A antibody and its applications. Background Technology

[0002] ACVR2A (Activin Receptor Type IIA) is a key type II transmembrane serine / threonine kinase receptor of the TGF-β superfamily. Antibodies against it are core therapeutic targets in areas such as muscle atrophy, pulmonary hypertension, and metabolic diseases. Current technologies mainly focus on fusion proteins, with specific antagonistic antibodies being an important research and development direction.

[0003] For example, CN118176018A confirmed that the activin signaling pathway can be antagonized by targeting ACVR2A with antibodies, and preliminarily verified its potential efficacy in promoting muscle growth and improving anemia.

[0004] However, this technical solution still has the following significant limitations: First, all the disclosed antibodies are humanized products, and their variable regions still retain non-human sequences. There are no reports of fully human anti-ACVR2A antibodies, so there is still a potential risk of immunogenicity during long-term or repeated administration. Second, the number of disclosed antibody sequences is limited, and there is still considerable room for optimization in terms of target affinity and specificity. In particular, the binding selectivity between the antibody and the highly homologous ACVR2B has not been systematically compared, nor has it been revealed whether there are novel antibody variants with stronger affinity or better specificity. This limits its application potential in scenarios requiring high-precision targeted therapy. Summary of the Invention

[0005] (a) Technical problems to be solved Therefore, one of the main objectives of this invention is to provide an ACVR2A antibody and its applications. The antibody provided by this invention has a stronger affinity for ACVR2A and higher selectivity for ACVR2A, providing diversified treatment options for different clinical needs and expanding the application potential of ACVR2A targeted therapy.

[0006] (II) Technical Solution To achieve the above objectives, the present invention provides an ACVR2A antibody or an antigen-binding fragment thereof, comprising: A heavy chain variable region (VH), the heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 region contains an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) identity with the selected VH CDR1 amino acid sequence, the VH CDR2 region contains an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) identity with the selected VH CDR2 amino acid sequence, and the VH CDR3 region contains an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) identity with the selected VH CDR3 amino acid sequence; and The light chain variable region (VL) comprises CDR 1, 2, and 3, wherein the VL CDR1 region comprises an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) identity with the selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) identity with the selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) identity with the selected VL CDR3 amino acid sequence; The selected VH CDR 1, 2, and 3 amino acid sequences and the selected VL CDR 1, 2, and 3 amino acid sequences are one of the following: (1) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 1, 2, and 3, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 4, 5, and 6, respectively; (2) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 11, 12, and 13, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 14, 15, and 16, respectively; (3) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 21, 22, and 23, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 24, 25, and 26, respectively; (4) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 31, 32, and 33, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 34, 35, and 36, respectively; (5) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 41, 42, and 43, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 44, 45, and 46, respectively.

[0007] In one embodiment, the VH comprises CDRs 1, 2, and 3 having the amino acid sequences shown in SEQ ID NO: 31, 32, and 33, respectively, and the VL comprises CDRs 1, 2, and 3 having the amino acid sequences shown in SEQ ID NO: 34, 35, and 36, respectively.

[0008] In one embodiment, the VH comprises CDRs 1, 2, and 3 having the amino acid sequences shown in SEQ ID NO: 41, 42, and 43, respectively, and the VL comprises CDRs 1, 2, and 3 having the amino acid sequences shown in SEQ ID NO: 44, 45, and 46, respectively.

[0009] In one embodiment, the antibody or antigen-binding fragment specifically binds to ACVR2A.

[0010] In one embodiment, the antibody or its antigen-binding fragment may also bind to ACVR2B.

[0011] In one embodiment, the VH comprises CDRs 1, 2, and 3 having the amino acid sequences shown in SEQ ID NO: 31, 32, and 33, respectively, and the VL comprises CDRs 1, 2, and 3 having the amino acid sequences shown in SEQ ID NO: 44, 45, and 46, respectively.

[0012] In one embodiment, the antibody or antigen-binding fragment is a humanized antibody or its antigen-binding fragment.

[0013] In one embodiment, the antibody or antigen-binding fragment is a single-chain variable fragment (scFV).

[0014] In one embodiment, the antibody or its antigen-binding fragment comprises a heavy chain framework region and / or a light chain framework region.

[0015] In one embodiment, at least a portion of the heavy chain framework region and / or light chain framework region is derived from mouse antibodies, human antibodies, primate antibodies, bovine antibodies, equine antibodies, dairy bovine antibodies, porcine antibodies, sheep antibodies, goat antibodies, canine antibodies, feline antibodies, rabbit antibodies, camel antibodies, donkey antibodies, deer antibodies, mink antibodies, chicken antibodies, duck antibodies, goose antibodies, turkey antibodies, fighting rooster antibodies, and / or their mutants.

[0016] In one embodiment, at least a portion of the heavy chain framework region and / or light chain framework region is derived from a human antibody or a mutant thereof.

[0017] In one embodiment, the antibody or its antigen-binding fragment comprises: The heavy chain variable region (VH) and the light chain variable region (VL), wherein the heavy chain variable region (VH) comprises an amino acid sequence having at least 90% (e.g., 90%, 95%, 96%, 97%, 98%, or 99%) identity with a selected VH sequence, and the light chain variable region (VL) comprises an amino acid sequence having at least 90% (e.g., 90%, 95%, 96%, 97%, 98%, or 99%) identity with a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following: (1) The selected VH sequence is SEQ ID NO: 7, and the selected VL sequence is SEQ ID NO: 8; (2) The selected VH sequence is SEQ ID NO: 17, and the selected VL sequence is SEQ ID NO: 18; (3) The selected VH sequence is SEQ ID NO: 27, and the selected VL sequence is SEQ ID NO: 28; (4) The selected VH sequence is SEQ ID NO: 37, and the selected VL sequence is SEQ ID NO: 38; (5) The selected VH sequence is SEQ ID NO: 47, and the selected VL sequence is SEQ ID NO: 48.

[0018] In one embodiment, the antibody or its antigen-binding fragment has: As shown in SEQ ID NO: 37, the heavy chain variable region is shown in SEQ ID NO: 38, and the light chain variable region is shown in SEQ ID NO: 38; And / or the heavy chain variable region as shown in SEQ ID NO: 47 and the light chain variable region as shown in SEQ ID NO: 48.

[0019] In one embodiment, the antibody or antigen-binding fragment specifically binds to ACVR2A.

[0020] In one embodiment, the antibody or its antigen-binding fragment may also bind to ACVR2B.

[0021] In one embodiment, the antibody or its antigen-binding fragment has: The heavy chain variable region is shown in SEQ ID NO: 37 and the light chain variable region is shown in SEQ ID NO: 38.

[0022] In one embodiment, the ACVR2A antibody or its antigen-binding fragment further comprises a heavy chain constant region and a light chain constant region.

[0023] In one embodiment, at least a portion of at least one of the heavy chain constant region and the light chain constant region is derived from mouse antibodies, human antibodies, primate antibodies, bovine antibodies, equine antibodies, dairy bovine antibodies, porcine antibodies, sheep antibodies, goat antibodies, canine antibodies, feline antibodies, rabbit antibodies, camel antibodies, donkey antibodies, deer antibodies, mink antibodies, chicken antibodies, duck antibodies, goose antibodies, turkey antibodies, fighting rooster antibodies, and / or their mutants.

[0024] In one embodiment, the heavy chain constant region includes a heavy chain constant region selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and / or IgD.

[0025] In one embodiment, the light chain constant region includes κ-type and / or λ-type light chain constant regions; In one embodiment, both the light chain constant region and the heavy chain constant region are derived from human antibodies or their mutants.

[0026] In one embodiment, the antibody includes a full-length monoclonal antibody, a chimeric antibody, a humanized antibody, Fv, scFv, Fab, Fab', Fab'-SH and / or F(ab')2; In one embodiment, the antigen-binding fragment of the antibody includes an F(ab')2 fragment, a Fab' fragment, a Fab fragment, an F(ab)2 fragment, an Fv fragment, a scFv fragment, and / or a scFv-Fc fusion protein.

[0027] In one embodiment, the antibody or antigen-binding fragment comprises an amino acid sequence having at least 90% (e.g., 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity with any of the following combinations of heavy and light chains: The heavy chain shown in SEQ ID NO: 39 and the light chain shown in SEQ ID NO: 40; And / or the heavy chain shown in SEQ ID NO: 49 and the light chain shown in SEQ ID NO: 50.

[0028] In one embodiment, the antibody or antigen-binding fragment specifically binds to ACVR2A.

[0029] In one embodiment, the antibody or its antigen-binding fragment may also bind to ACVR2B.

[0030] In one embodiment, the antibody or its antigen-binding fragment has: Heavy chains as shown in SEQ ID NO: 39 and light chains as shown in SEQ ID NO: 40.

[0031] In another aspect, the present invention provides a nucleic acid encoding the aforementioned antibody or its antigen-binding fragment.

[0032] In one embodiment, the nucleic acid is cDNA.

[0033] In another aspect, the present invention also provides a carrier comprising the above-mentioned nucleic acid.

[0034] In one embodiment, the carrier encodes VL and VH regions, which together are combined with ACVR2A.

[0035] In another aspect, the present invention also provides a vector pair, wherein each vector contains one of the above-described nucleic acids, wherein the vector pair together encodes a VL region and a VH region, the VL region and the VH region together binding ACVR2A.

[0036] In another aspect, the present invention also provides a cell that expresses the aforementioned nucleic acid, vector and / or vector pair.

[0037] In another aspect, the present invention provides a method for generating the above-mentioned antibody or its antigen-binding fragment, comprising: The cells were cultured under conditions sufficient to induce the production of antibodies or antigen-binding fragments; and Collect antibodies or antigen-binding fragments produced by the cells.

[0038] In another aspect, the present invention also provides an antibody or an antigen-binding fragment thereof, obtained by the above method.

[0039] In another aspect, the present invention provides a pharmaceutical composition comprising: (1) A therapeutically effective amount of the above-mentioned antibodies or their antigen-binding fragments, nucleic acids, vectors, vector pairs and / or cells; (2) Pharmaceutically or immunologically acceptable carriers or excipients.

[0040] In one embodiment, the pharmaceutical composition further includes a GLP-1 agonist.

[0041] In one embodiment, the GLP-1 agonist comprises semaglutide.

[0042] In another aspect, the present invention provides the use of the above-mentioned antibody or its antigen-binding fragment in the preparation of a medicament for the treatment of metabolic diseases in combination with a GLP-1 agonist.

[0043] In one embodiment, the GLP-1 agonist comprises semaglutide.

[0044] In one embodiment, the antibody or its antigen-binding fragment is administered first, followed by the administration of smegglutinin.

[0045] In one embodiment, smegglutinin is administered first, followed by the aforementioned antibody or its antigen-binding fragment.

[0046] In one embodiment, the antibody described above or its antigen-binding fragment and smegglutinin are administered simultaneously.

[0047] In one embodiment, the dosage of the smegglutinin is 100-140 μg / kg.

[0048] In one embodiment, the dosage of the smegglutinin is 120 μg / kg.

[0049] In one embodiment, the dosage of the antibody or its antigen-binding fragment is 10-50 mg / kg.

[0050] In one embodiment, the dosage of the antibody or its antigen-binding fragment is 20 mg / kg.

[0051] In another aspect, the present invention provides a pharmaceutical preparation comprising the above-described pharmaceutical composition.

[0052] In another aspect, the present invention also provides a pharmaceutical product comprising the above-described pharmaceutical preparation.

[0053] In one embodiment, the pharmaceutical product is a vial or box.

[0054] In another aspect, the present invention also provides the use of the above-mentioned antibodies or their antigen-binding fragments, nucleic acids, carriers, carrier pairs, cells, pharmaceutical compositions, pharmaceutical preparations and / or pharmaceutical products in the preparation of medicaments for the prevention and / or treatment of metabolic diseases and / or muscle-related diseases.

[0055] In one embodiment, the metabolic disease includes obesity or fatty liver.

[0056] In one embodiment, the muscle-related disease includes muscular dystrophy or cachexia.

[0057] In another aspect, the present invention also provides a detection reagent comprising the antibody or its antigen-binding fragment described above.

[0058] In another aspect, the present invention also provides a detection kit comprising the detection reagents described above.

[0059] In another aspect, the present invention also provides the application of the above-mentioned detection reagents and / or detection kits in the detection of ACVR2A.

[0060] (III) Beneficial Effects This invention provides an ACVR2A antibody and its application. Compared with the prior art, it has the following advantages: 1. The fully human anti-ACVR2A antibody provided maintains or enhances functional activity while minimizing the risk of immune rejection or adverse reactions caused by non-human sequences, thus significantly improving the safety of clinical applications.

[0061] 2. The provided antibody has a strong affinity for ACVR2A and high selectivity, which expands the application potential of ACVR2A targeted therapy.

[0062] 3. The efficacy of ACVR2A antibody as an inhibitor of muscle signaling pathway was verified at the functional level, and its action spectrum was further refined, providing key functional evidence for the development of differentiated drugs for different muscle-related diseases (such as muscular dystrophy, cachexia, etc.).

[0063] 4. The inhibitory spectrum of ACVR2A antibody in lipid metabolism-related signaling pathways was revealed, confirming that it not only has ACVR2A targeting, but also exhibits differentiated inhibitory characteristics in ACVR2B pathways activated by different ligands, providing an important basis for precision treatment strategies for metabolic diseases (such as obesity, fatty liver, etc.).

[0064] 5. Pharmacokinetic experiments have confirmed from an in vivo dynamic perspective that the provided antibody has a significant advantage in long-term effect, laying a key pharmacokinetic foundation for its subsequent development as a long-acting weight loss and muscle gain antibody drug. Attached Figure Description

[0065] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0066] Figure 1 This is a graph showing the results of the detection of specific binding between the antibody and the human ACVR2A recombinant protein; Figure 2 This is a graph showing the results of the detection of specific binding between the antibody and the murine ACVR2A recombinant protein; Figure 3 This is a graph showing the results of the detection of specific binding between the antibody and the human ACVR2B recombinant protein; Figure 4 This is a graph showing the results of the detection of specific binding between the antibody and the murine ACVR2B recombinant protein; Figure 5 This is a graph showing the results of the detection of specific binding of the antibody to the HEK293 cell line overexpressing ACVR2A; Figure 6 This is a graph showing the results of the detection of specific binding of the antibody to the HEK293 cell line overexpressing ACVR2B; Figure 7 This is a graph showing the inhibitory activity of the antibody against the GDF8-induced ACVR2A-ALK4-pSMAD2 / 3 signaling pathway. Figure 8 This is a graph showing the inhibitory activity of the antibody against the Activin A-induced ACVR2A-ALK4-pSMAD2 / 3 signaling pathway. Figure 9 This is a graph showing the inhibitory activity of the antibody against the GDF8-induced ACVR2B-ALK4-pSMAD2 / 3 signaling pathway. Figure 10 This is a graph showing the inhibitory activity of the antibody against the Activin A-induced ACVR2B-ALK4-pSMAD2 / 3 signaling pathway. Figure 11 This is a graph showing the inhibitory activity of the antibody against the Activin B-induced ACVR2A-ALK7-pSMAD2 / 3 signaling pathway. Figure 12 This is a graph showing the inhibitory activity of the antibody against the Activin E-induced ACVR2A-ALK7-pSMAD2 / 3 signaling pathway. Figure 13 This is a graph showing the inhibitory activity of the antibody against the Activin B-induced ACVR2B-ALK7-pSMAD2 / 3 signaling pathway. Figure 14 This is a graph showing the inhibitory activity of the antibody against the Activin E-induced ACVR2B-ALK7-pSMAD2 / 3 signaling pathway. Figure 15 This is a graph showing the pharmacokinetic results of the antibody in C57 mice; Figure 16 This is a graph showing changes in mouse weight and body composition; Figure 17 This is a graph showing the changes in mouse body weight and grip strength. Detailed Implementation

[0067] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0068] Terms and Definitions As used herein, the term "antibody" generally refers to an antibody that recognizes one or more antigenic epitopes, including but not limited to monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), heavy-chain-only antibodies, triple-chain antibodies, single-chain Fv (scFv), nanobodies, etc., and also includes antibody fragments, provided they exhibit the desired biological activity (Miller et al. (2003) Jour. of Immunology 170: 4854 4861). Antibodies can be mouse, human, humanized, chimeric, or derived from other species. Antibodies can refer to full-length heavy-chain, full-length light-chain, or intact immunoglobulin molecules; or the immunologically active portion of any of these polypeptides, i.e., a molecule or portion thereof containing an antigen-binding site that specifically binds to a target antigen of interest, such targets including but not limited to cancer cells or cells that produce autoantibodies associated with autoimmune diseases.

[0069] Within the variable region, certain areas exhibit a higher degree of variation in amino acid composition and sequence, termed "hypervariable region (HVR)." The hypervariable region is the site where antigens and antibodies bind, and is therefore also called the complementarity-determining region (CDR). Both the heavy chain and light chain variable regions contain three CDR regions. For example, these typically include amino acid residues near 23-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable region, and near 31-35B (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable region (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)); and / or amino acid residues from “high-variable rings” (e.g., near 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable region, and near 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable region (Chothia and Lesk). J. Mol. Biol. 196:901-917 (1987)).

[0070] As used in this article, the term "ACVR2A antibody" refers to an antibody that can bind to ACVR2A.

[0071] As used herein, the term “antigen-binding fragment” is equivalent to “antibody fragment” or “antigen-binding antibody fragment” and can include a portion of a complete antibody, typically a binding region or variable region. This includes, but is not limited to: Fv, scFv, Fab, Fab’, Fab’-SH, F(ab’)2, scFv-Fc fragments, or bispecific antibodies (BsAbs), linear antibodies, or any fragment that should be able to increase its half-life through chemical modification or incorporation into liposomes, such as the addition of poly(alkylene) glycols, like polyethylene glycol (“PEGylated”) (a PEGylated fragment referred to as Fv-PEG, scFv-PEG, Fab-PEG, F(ab’)2-PEG, or Fab’-PEG) (“PEG” stands for polyethylene glycol).

[0072] As used herein, the term "affinity" refers to the strength of binding between an antibody or protein and an epitope. The affinity of an antibody is given by the dissociation constant KD, defined as [Ab] × [Ag] / [Ab-Ag], where [Ab-Ag] is the molar concentration of the antibody-antigen complex, [Ab] is the molar concentration of unbound antibody, and [Ag] is the molar concentration of unbound antigen. The affinity constant KA is defined by 1 / KD. Preferred methods for determining protein affinity can be found in Harlow et al., “Antibodies: A Laboratory Manual,” Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1988); Coligan et al. (eds.), “Current Protocols in Immunology,” Greene Publishing Assoc and Wiley Interscience, New York (1992, 1993); and Muller, Meth. Enzymol., Vol. 92: pp. 589–601 (1983). A preferred standard method well known in the art for determining protein affinity is the use of surface plasmon resonance (SPR) screening (such as by analysis with the BIAcore™ SPR analysis device).

[0073] As used herein, the terms “homology,” “identity,” or “similarity” are used to describe or compare the degree of nucleotide similarity between two or more nucleotide sequences. The percentage of “sequence homology” between a first and a second sequence can be calculated by dividing the number of nucleotides in the first sequence that are identical to those at the corresponding positions by the number of nucleotides in the second sequence. This is calculated by subtracting the total number of nucleotides in the first sequence from the number of nucleotides in the second sequence and then multiplying by 100%, where each deletion, insertion, substitution, or addition of a nucleotide in the second sequence—relative to the first sequence—is considered a difference at a single nucleotide (position). Alternatively, the degree of sequence identity between two or more nucleotide sequences can be calculated using standard settings and known computer algorithms for sequence alignment, such as NCBIBlast v2.0. Other techniques, computer algorithms, and settings used to determine the degree of sequence identity include, for example, those in WO 04 / 037999, EP 0967284, EP 1085089, WO00 / 55318, WO 00 / 78972, WO 98 / 49185, and GB 2357768-A.

[0074] For peptides, the terms "(substantial) homology," "identity," or "similarity" are used to describe or compare the degree of amino acid similarity between two or more peptides or their designated sequences at optimal alignment and comparison (where appropriate insertions or deletions of nucleotides are made). The percentage of homology between two sequences varies with the number of identical positions shared by these sequences at optimal alignment (i.e., homology % = number of identical positions / total number of positions × 100), where optimal alignment is determined taking into account the number of vacancies introduced to achieve optimal alignment of the two sequences and the length of each vacancy. Sequence comparison and identity percentage determination between two sequences can be performed using mathematical algorithms, as described in the non-limiting examples below.

[0075] As used herein, the terms "vector" and "recombinant expression vector" are used interchangeably, referring to bacterial plasmids, bacteriophages, yeast plasmids, animal cell viruses, mammalian cell viruses, or other vectors well known in the art. In short, any plasmid and vector can be used as long as it can replicate and remain stable within the host. An important characteristic of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and translational control elements.

[0076] Methods well known to those skilled in the art can be used to construct expression vectors containing the ACVR2A antibody coding sequence and suitable transcription / translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, and in vivo recombination techniques. The DNA sequence can be efficiently ligated to an appropriate promoter in the expression vector to guide mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

[0077] In addition, the expression vector preferably contains one or more selective marker genes to provide phenotypic traits for selecting host cells for transformation, such as dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for Escherichia coli.

[0078] Vectors containing the appropriate DNA sequence and suitable promoters or control sequences can be used to transform suitable host cells to enable them to express proteins or peptides. Host cells can be prokaryotic cells, such as bacterial cells; lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as animal cells. Representative examples include: *Escherichia coli*, *Streptomyces*, *Agrobacterium*; fungal cells such as yeast; and animal cells.

[0079] The polynucleotides disclosed in this invention, when expressed in higher eukaryotic cells, will enhance transcription when an enhancer sequence is inserted into the vector. Enhancers are cis-acting factors of DNA, typically approximately 10 to 300 base pairs, that act on the promoter to enhance gene transcription. Those skilled in the art will understand how to select appropriate vectors, promoters, enhancers, and host cells.

[0080] As used herein, the term "pharmaceutical composition" refers to a composition comprising an ACVR2A antibody or an antigen-binding fragment thereof formulated with one or more pharmaceutically acceptable carriers.

[0081] The formulation of the pharmaceutical composition can be tailored to the application. In particular, pharmaceutical compositions can be formulated using methods known in the art to provide rapid, continuous, or delayed release of the active ingredient upon administration to mammals. For example, the formulation can be selected from any of the following: liquids and solutions, aerosols, sprays, extracts, elixirs, ointments, fluid extracts, emulsions, suspensions, decoctions, infusions, tablets, suppositories, injections, alcoholic preparations, capsules, creams, lozenges, tinctures, pastes, pills, and soft or hard gelatin capsules.

[0082] As used herein, the term "pharmaceuticalally acceptable" refers to a substance that is suitable for use in humans and / or animals without excessive adverse effects (such as toxicity, irritation, and allergic reactions), i.e., a reasonable benefit / risk ratio.

[0083] As used herein, the term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" refers to a carrier used for the administration of therapeutic agents, encompassing a variety of excipients and diluents. This term refers to pharmaceutical carriers that are not essential active ingredients themselves and do not cause excessive toxicity upon administration. Suitable carriers are well known to those skilled in the art, and a thorough discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991).

[0084] Pharmaceutically acceptable carriers in a composition include any and all solvents, dispersion media, preservatives, antioxidants, coatings, isotonic and absorption-delaying agents, surfactants, fillers, disintegrants, binders, diluents, lubricants, flow aids, pH adjusters, buffers, enhancers, wetting agents, solubilizers, surfactants, antioxidants, etc., compatible with drug administration. The use of such media and agents for pharmaceutically active substances is well known in the art. The composition may contain other active compounds that provide complementary, additional, or enhanced therapeutic functions. Solid carriers or excipients, such as lactose, starch, or talc, or liquid carriers, such as water, fatty oils, or liquid paraffin, are possible. Other examples of carriers include culture media, such as DMEM or RPMI; and cryogenic storage media containing components that scavenge free radicals, provide pH buffering, osmotic / osmotic support, energy substrates, and ion concentrations to balance intracellular states at low temperatures; and mixtures of organic solvents with water.

[0085] The active substance in the product disclosed in this invention accounts for 0.001-99.9 wt% of the total weight of the composition, with the remainder being pharmaceutically acceptable carriers and other additives.

[0086] The pharmaceutical compositions of the present invention can be administered using any known method. One of a variety of methods known to those skilled in the art can be used to administer the substance, compound, or agent to a subject using the terms "give" or "apply".

[0087] For example, compounds or agents can be administered intranasally (e.g., by inhalation), intrathecally (into the spinal canal or subarachnoid space), intraarterially, intradermally, intramuscularly, intraperitoneally, intravenously, subcutaneously, ocularly, sublingually, orally (by ingestion), intracerebrally, and transdermally (by absorption, e.g., through a skin catheter). Compounds or agents can also be suitably introduced via rechargeable or biodegradable polymeric devices or other devices (e.g., patches and pumps or formulations) that provide prolonged, slowed, or controlled release of the compound or agent. Administration can also be performed, for example, once, multiple times, and / or over one or more prolonged periods.

[0088] As used herein, the term “therapeutic effective dose” refers to a dose sufficient to treat a disease with a reasonable benefit / risk ratio suitable for medical treatment, and the effective dose level includes subject type and severity, age, sex, drug activity, drug sensitivity, time of administration, route of administration and excretion rate, duration of treatment, factors including concomitant drugs, and other factors known in the medical field.

[0089] As used in this article, “containing,” “having,” or “including” includes “containing,” “mainly composed of,” “substantially composed of,” and “composed of”; “mainly composed of,” “substantially composed of,” and “composed of” are subordinate concepts of “containing,” “having,” or “including.”

[0090] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the reagents, methods and equipment used are conventional reagents, methods and equipment in this technical field.

[0091] Example 1: Screening of ACVR2A antibodies: 1. Immunization and Cell Preparation ACVR2A antigen immunization was performed using the HUGO-eKO fully human antibody mouse platform. The endogenous antibody gene in HUGO-eKO fully human antibody mice has been replaced by a human antibody gene library, and the mouse ACVR2A gene has been knocked out, enabling the direct production of fully human antibodies. 50 μg of human ACVR2AFC fusion recombinant protein (KaiKa Bio ARA-HM22A) was emulsified with complete Freund's adjuvant and administered subcutaneously to 6-8 week old HUGO-eKO mice. After three booster immunizations (each with a protein dose of 25 μg, emulsified with incomplete Freund's adjuvant), mouse spleens were harvested, ground, and passed through a 70 μm cell sieve to prepare a single-cell suspension of spleen cells. The total B cell population was isolated from the spleen cell suspension using a commercially available mouse B cell isolation kit (STEMCELL #19844).

[0092] 2. Antigen-specific B cell labeling To specifically identify B cells capable of binding the target antigen, the prepared B cells were incubated with the fluorescently labeled ACVR2A probe at 4°C in the dark for 30 minutes. After incubation, the cells were washed twice with PBS containing 2% fetal bovine serum to remove unbound probe.

[0093] 3. Flow cytometry single-cell sorting Sorting was performed using a flow cytometer (e.g., SONY MA900). Based on the negative control (B cells not incubated with the fluorescently labeled ACVR2A probe), only individual B cells with significantly higher fluorescence signal intensity than the negative control group were collected. Using a single-cell sorting mode, each antigen-binding positive B cell was directly sorted into a single well of a 96-well PCR plate, with cell lysis buffer and RNase inhibitor pre-filled in the well.

[0094] Example 2: Recombinant expression of ACVR2A antibody: Reverse transcription and PCR amplification: The sorted single-cell lysates were subjected to reverse transcription. Using a universal primer set targeting the variable regions of the mouse antibody heavy chain (IgH) and light chain (Igκ) genes, cDNA fragments of the antibody variable regions were amplified by nested PCR.

[0095] Gene cloning and sequencing: The purified PCR product was cloned into a sequencing vector, transformed into competent cells, and clones were selected for Sanger sequencing. Then, the VH and VL genes were inserted into the mammalian expression vector pcDNA3.4 containing the constant region (heavy chain) and constant region (light chain) of mouse IgG1, respectively. CHO cells were co-transfected, expressed, and purified to obtain complete recombinant monoclonal antibodies.

[0096] The amino acid sequences of the recombinant antibodies (AB09, AB83, A0004, A0019, A0040) are shown below: AB09HCDR1 (SEQ ID NO: 1): SYSVH; AB09HCDR2 (SEQ ID NO: 2): SISSSSTYTYYADSVKG; AB09HCDR3 (SEQ ID NO: 3): GRGGVLRSFDWILFDY; AB09LCDR1 (SEQ ID NO:4):RASQSVSSWLA; AB09LCDR2 (SEQ ID NO: 5): RASSLEN; AB09LCDR3 (SEQ ID NO: 6): QQYNSYWT; AB09VH (SEQ ID NO: 7): EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSVHWVRQTPGKGLEWVSSISSSSTYTYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAIYYCARGRGGVLRSFDWILFDYWGQGTLVTVSS; AB09VL (SEQ ID NO: 8): DIQMTQSPSTLSSASVGDRVTITCRASQSVSSWLAWYQQKPGIAPKLLIYRASSLENGVPSRFSGSASGTEFTLTISSLQPDDFATYYCQQYNSYWTFGQGTKVEIK; AB09H(SEQ ID NO:9):EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSVHWVRQTPGKGLEWVSSISSSSTYTYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAIYYCARGRGGVLRSFDWILFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK; AB09L(SEQ ID NO:10):DIQMTQSPSTLSASVGDRVTITCRASQSVSSWLAWYQQKPGIAPKLLIYRASSLENGVPSRFSGSASGTEFTLTISSLQPDDFATYYCQQYNSYWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC; AB83HCDR1(SEQ ID NO:11):STSYYWG; AB83HCDR2(SEQ ID NO:12):SIHYSGSSHYNPSLKS; AB83HCDR3(SEQ ID NO:13):GEFLLRYFEDV; AB83LCDR1(SEQ ID NO:14):RASQSVSSYLA; AB83LCDR2(SEQ ID NO:15):DASNRAT; AB83LCDR3(SEQ ID NO:16):QQRTNWPPT; AB83VH(SEQ ID NO:17):QVQLQESGPGLVKPSETLSLTCSVSGGSIISTSYYWGWIRQPPGKGLEWIGSIHYSGSSHYNPSLKSRVTIYVDTSKNQFSLKLNSVTAADTAVYYCARGEFLLRYFEDVWGQGTTVTVSS; AB83VL(SEQ ID NO:18):EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQVPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRTNWPPTFGQGTKVEIK; AB83H(SEQ ID NO:19):QVQLQESGPGLVKPSETLSLTCSVSGGSIISTSYYWGWIRQPPGKGLEWIGSIHYSGSSHYNPSLKSRVTIYVDTSKNQFSLKLNSVTAADTAVYYCARGEFLLRYFEDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK; AB83L(SEQ ID NO:20):EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQVPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRTNWPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC; A0004HCDR1(SEQ ID NO:21):STNYYWG; A0004HCDR2(SEQ ID NO:22):SIHYSGNTHYNPSLKS; A0004HCDR3(SEQ ID NO:23):GEFLLRYFEDV; A0004LCDR1(SEQ ID NO:24):RASQSVSSYLA; A0004LCDR2(SEQ ID NO:25):DASNRAT; A0004LCDR3(SEQ ID NO:26):QQRTNWPPT; A0004VH(SEQ ID NO:27):QVQLQESGPGLVKPSETLSLTCSVSGGSISSTNYYWGWIRQPPGKGLEWIGSIHYSGNTHYNPSLKSRVTIYVDTSKNQFSLKLNSVTAADTAVYYCARGEFLLRYFEDVWGQGTTVTVSS; A0004VL(SEQ ID NO:28):EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQVPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRTNWPPTFGQGTKVEIK; A0004H(SEQ ID NO:29):QVQLQESGPGLVKPSETLSLTCSVSGGSISSTNYYWGWIRQPPGKGLEWIGSIHYSGNTHYNPSLKSRVTIYVDTSKNQFSLKLNSVTAADTAVYYCARGEFLLRYFEDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK; A0004L(SEQ ID NO:30):EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQVPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRTNWPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC; A0019HCDR1(SEQ ID NO:31):SYAMH; A0019HCDR2(SEQ ID NO:32):AISTNGGSTYYADSVKG; A0019HCDR3(SEQ ID NO:33):GAEGSFMDV; A0019LCDR1(SEQ ID NO:34):RSSQSLLHSNGYNYLG; A0019LCDR2(SEQ ID NO:35):LGSNRAS; A0019LCDR3(SEQ ID NO:36):MQTLQTPRT; A0019VH(SEQ ID NO:37):EVQLVESGGGLVQPGGSLRLSCLASGVTFRSYAMHWVRQAPGKGLEYISAISTNGGSTYYADSVKGRFTISRDNSKNTLYLQMSSLRDEDTAVYYCVKGAEGSFMDVWGQGTTVTVSS; A0019VL(SEQ ID NO:38):NIVMTQSPLSLPVTPGEPASMSCRSSQSLLHSNGYNYLGWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQTLQTPRTFGGGTEVEIK; A0019H(SEQ ID NO:39):EVQLVESGGGLVQPGGSLRLSCLASGVTFRSYAMHWVRQAPGKGLEYISAISTNGGSTYYADSVKGRFTISRDNSKNTLYLQMSSLRDEDTAVYYCVKGAEGSFMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK; A0019L(SEQ ID NO:40):NIVMTQSPLSLPVTPGEPASMSCRSSQSLLHSNGYNYLGWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQTLQTPRTFGGGTEVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC; A0040HCDR1(SEQ ID NO:41):SYSMH; A0040HCDR2(SEQ ID NO:42):SISSSITYTYYADSVKG; A0040HCDR3(SEQ ID NO:43):GRGGVLRSFDWILFDY; A0040LCDR1(SEQ ID NO:44):RASQSVSSWLA; A0040LCDR2(SEQ ID NO:45):RTSSLES; A0040LCDR3(SEQ ID NO:46):QQYNSYWT; A0040VH(SEQ ID NO:47):EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMHWVRQTPGKGLEWVSSISSSITYTYYADSVKGRFTISRDNAKNALYLQMNSLRAEDTAIYYCARGRGGVLRSFDWILFDYWGQGTLVTVSS; A0040VL(SEQ ID NO:48):DIQMTQSPSTLSASVGDRVTITCRASQSVSSWLAWYQQKPGIAPKLLIYRTSSLESGVPSRFSGSASGTEFTLTISSLQPDDFATYYCQQYNSYWTFGQGTKVEIK; A0040H (SEQ ID NO: 49): EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMHWVRQTPGKGLEWVSSISSSITYTYYADSVKGRFTISRDNAKNALYLQMNSLRAEDTAIYYCARGRGGVLRSFDWILFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK; A0040L (SEQ ID NO: 50): DIQMTQSPSTLSASVGDRVTITCRASQSVSSWLAWYQQKPGIAPKLLIYRTSSLESGVPSRFSGSASGTEFTLTISSLQPDDFATYYCQQYNSYWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0097] Example 3 Verification of the target binding specificity of the ACVR2A antibody: 1. Binding of the antibody to human and murine ACVR2A and ACVR2B recombinant proteins: The binding affinity of the test antibodies to human and murine ACVR2A and ACVR2B recombinant proteins was detected by enzyme-linked immunosorbent assay (ELISA). Human ACVR2A, human ACVR2B, murine ACVR2A, and murine ACVR2B recombinant proteins were diluted to 2 μg / mL with coating buffer (0.01 M PBS, pH 7.4), and 100 μL was added to each well of a 96-well ELISA plate. The plates were incubated overnight at 4°C. The coating buffer was discarded the next day, and the plates were washed three times with PBST containing 0.05% Tween-20. Then, 300 μL of PBST containing 1% bovine serum albumin was added to each well as blocking buffer, and the plates were blocked at 37°C for 2 hours. After blocking, the blocking buffer was discarded, and the plates were washed three times with PBST. The test antibodies (AB09 (Ab09), AB83 (Ab83), A0004, A0019, A0040, positive control antibody LAE-102, Bimagrumab (B), and negative control antibody Human IgG1 Isotype Control (IgG1)) were serially diluted 4-fold from 200 nM (12 dilutions in total) using blocking buffer. 100 μL of each diluted antibody was added to a well coated with a different antigen and incubated at 37°C for 1 hour. The primary antibody reaction solution was discarded, and the sample was washed 5 times with PBST. Then, 100 μL of horseradish peroxidase-labeled species-specific secondary antibody (1:5000) diluted with blocking buffer was added to each well, and the sample was incubated at 37°C for 1 hour. After 5 washes, 100 μL of TMB chromogenic buffer was added to each well, and the sample was incubated at room temperature in the dark for 10-15 minutes. Finally, 50 μL of 2M H2SO4 was added to each well to terminate the reaction. Immediately use an ELISA reader to read the absorbance value (OD) at a wavelength of 450 nm. 450 (The logarithm of antibody concentration is plotted on the x-axis, OD...) 450 Plot the combination curve with the values ​​on the ordinate and calculate the half-maximal effective concentration (EC50). 50 The cross-binding activity of the test antibody with ACVR2A and ACVR2B proteins from different sources was evaluated.

[0098] The results are as follows Figures 1-4 As shown in the ELISA results, all five recombinant antibodies expressed in Example 2 strongly bound to human and murine ACVR2A proteins, confirming their cross-species conserved binding characteristics to the ACVR2A target and supporting subsequent efficacy and safety evaluations in murine animal models. Among them, except for A0019, the other four antibodies showed only weak binding or no binding to ACVR2B, demonstrating excellent ACVR2A / ACVR2B selectivity, laying the foundation for developing highly specific ACVR2A-targeting antibodies. The dual-target binding characteristic of A0019 suggests its potential applicability to specific therapeutic scenarios requiring simultaneous regulation of the ACVR2A / ACVR2B pathways.

[0099] Example 4: Affinity of ACVR2A antibody to recombinant human ACVR2A and ACVR2B proteins: Affinity was determined using surface plasmon resonance (SPR) technology. His-tagged recombinant human ACVR2A and ACVR2B proteins were captured using anti-tag antibodies, or the test antibodies were directly conjugated to the chip surface. The test antibodies or antigens were serially diluted to five different concentrations, injected sequentially, and binding and dissociation signals were recorded. A 1:1 Langmuir binding model was used to fit the sensor map, and the binding rate constant, dissociation rate constant, and equilibrium dissociation constant were calculated. The binding specificity and family selectivity were evaluated by comparing the KD values ​​of the antibodies with ACVR2A and ACVR2B.

[0100] The affinity results of the five recombinant antibodies expressed in Example 2 for human ACVR2A are shown in Table 1, and the affinity results for human ACVR2B are shown in Table 2.

[0101] Table 1. Affinity of antibody to human ACVR2A Table 2. Affinity of antibody to human ACVR2B As shown in Table 1, the KD values ​​of A0019 and A0040 are 5.81E-10M and 9.78E-10M respectively, which are within the range of 10. -10 The M level was significantly higher than that of the known positive control LAE102; the KD values ​​of the other three antibodies (AB09, AB83, and A0004) ranged from 1.19E-09M to 2.64E-09M, falling within the range of 10. -9 M level.

[0102] As shown in Table 2, the KD values ​​of A0019 and A0040 are 4.72E-07M and 7.67E-07M, respectively. The KD values ​​of the other measurable ligands (AB09, AB83, and A0004) are between 1.37E-06M and 5.35E-06M, and their affinity is significantly weaker than that for ACVR2A (by 2 to 3 orders of magnitude). This indicates that the five recombinant antibodies expressed in Example 2 have high selectivity for ACVR2A.

[0103] Example 4: Binding of ACVR2A antibody to HEK293 cell lines overexpressing ACVR2A or ACVR2B: The binding activity of anti-ACVR2A antibody to HEK293 cell lines overexpressing the target antigen was detected by flow cytometry. Commercially available reporter gene cell lines were used: the ACVR2A reporter gene cell line (catalog number: RD01152, GenScript) was used as the ACVR2B knockout background and overexpressed ACVR2A; the ACVR2B reporter gene cell line (catalog number: RD01151, GenScript) was used as the ACVR2A knockout background and overexpressed ACVR2B. The test antibody was incubated with the above cell lines, washed, and then fluorescently labeled secondary antibody was added. The fluorescence signal intensity on the cell surface was detected by flow cytometry, and the antibody binding ability was evaluated using mean fluorescence intensity (MFI).

[0104] The results are as follows Figures 5-6 As shown, the recombinant antibodies expressed in Example 2 all exhibited significant binding signals on the surface of the HEK293 cell line overexpressing ACVR2A and knocking out ACVR2B. For the HEK293 cell line overexpressing ACVR2B and knocking out ACVR2A, AB83, AB09, A0040, and A0004 showed weak binding activity, while A0019 showed strong binding activity.

[0105] This confirms that the anti-ACVR2A antibodies of the present invention can effectively recognize the ACVR2A protein located on the cell membrane surface in its native conformation, which lays a key biological foundation for its biological function in vivo.

[0106] A0019 has been identified as a broad-spectrum candidate molecule. It exhibits strong binding activity to both ACVR2A and ACVR2B, potentially making it suitable for a wider range of therapeutic applications requiring simultaneous inhibition of two receptor signaling pathways.

[0107] The weak cross-binding properties of other antibodies (such as A0040) provide a basis for their development as drug prodrugs requiring moderate selectivity or specific pharmacokinetic properties. In summary, the antibodies of this invention are not only a set of tools for binding ACVR2A, but also a molecular library containing high specificity, broad spectrum, and intermediate selectivity, which can provide core candidate molecules for the development of diagnostic reagents or differentiated therapeutic drugs based on different disease mechanisms and therapeutic goals.

[0108] Example 5: Evaluation of the inhibitory activity of ACVR2A antibody against the ActRII-ALK4-pSMAD2 / 3 muscle signaling pathway: Commercially available reporter gene cell lines were used to determine antibody functional activity. The ACVR2A reporter gene cell line (catalog number: RD01152, GenScript) was an ACVR2B knockout background cell line overexpressing ACVR2A and SMAD-binding elements-driven luciferase reporter gene; the ACVR2B reporter gene cell line (catalog number: RD01151, GenScript) was an ACVR2A knockout background cell line overexpressing ACVR2B and SMAD-binding elements-driven luciferase reporter gene. Target gene knockout efficiency was verified by Sanger sequencing. Cells were pre-incubated with serially diluted test antibodies, followed by stimulation with ligand GDF8 or ActivinA. After 6 hours of incubation, luciferase substrate was added, and the luminescence signal was detected. The inhibition rate of the antibody against the ligand-induced signal was calculated, and the half-maximal inhibitory concentration (IC50) was fitted. 50 The study evaluated the specific antagonistic activity of the antibody against the ACVR2A and ACVR2B muscle signaling mimicry pathways.

[0109] The results are as follows Figures 7-10 As shown, the recombinant antibody expressed in Example 2 can strongly antagonize the ACVR2A-mediated muscle growth inhibition pathway. Functional experiments confirmed that all five antibodies can effectively inhibit GDF8 and Activin A signaling transmitted via the ACVR2A receptor, thus demonstrating their therapeutic potential to reverse muscle atrophy and promote muscle growth at the molecular level.

[0110] This study revealed the functional differentiation of antibodies across a dual ACVR2A / ACVR2B inhibitory spectrum. A0040 was identified as a dual-target inhibitory antibody, exhibiting weak inhibitory activity against the ACVR2B pathway as well. This characteristic may be suitable for specific pathological scenarios requiring simultaneous and moderate modulation of two homologous receptors. The remaining four antibodies were identified as selective inhibitors of ACVR2A, with their activity highly concentrated in the ACVR2A pathway. This provides clear candidate molecules for developing highly selective therapeutics aimed at precisely modulating ACVR2A signaling while avoiding interference with ACVR2B physiological function.

[0111] This experiment functionally validated the efficacy of ACVR2A antibody as an inhibitor of muscle signaling pathways and further refined its action spectrum, providing key functional evidence for the development of differentiated drugs for different muscle-related diseases (such as muscular dystrophy, cachexia, etc.).

[0112] Example 6: Evaluation of the inhibitory activity of ACVR2A antibody against the ActRII-ALK7-pSMAD2 / 3 adipocyte signaling pathway: Commercially available reporter gene cell lines were used to determine antibody functional activity. The ACVR2A reporter gene cell line (catalog number: RD01152, GenScript) was an ACVR2B knockout background cell line overexpressing ACVR2A and SMAD binding elements-driven luciferase reporter gene; the ACVR2B reporter gene cell line (catalog number: RD01151, GenScript) was an ACVR2A knockout background cell line overexpressing ACVR2B and SMAD binding elements-driven luciferase reporter gene. Cells were pre-incubated with serially diluted test antibodies, followed by stimulation with ligands Activin B or Activin E, respectively. After 6 hours of incubation, luciferase substrate was added, and luminescence signals were detected to evaluate the antagonistic efficacy of the antibodies against the ACVR2A-ALK7 pathway, while simultaneously assessing their cross-inhibition effect on the ACVR2B-ALK7 pathway.

[0113] The results are as follows Figures 11-14 As shown, the recombinant antibody expressed in Example 2 strongly inhibited the ACVR2A-ALK7-pSMAD2 / 3 adipocyte signaling pathway induced by Activin B and Activin E, demonstrating its therapeutic potential in regulating lipid metabolism-related diseases.

[0114] All five antibodies showed no significant inhibitory effect on the Activin B-induced ACVR2B-ALK7-pSMAD2 / 3 adipocyte signaling pathway, while Activin E showed a weak inhibitory effect on the Activin E-induced ACVR2B-ALK7-pSMAD2 / 3 adipocyte signaling pathway.

[0115] The inhibitory effects of antibodies on the ACVR2B-ALK7 pathway exhibited ligand-dependent differentiation. None of the antibodies significantly affected the Activin B-activated ACVR2B pathway. Against the Activin E-activated ACVR2B pathway, all five antibodies showed controllable weak inhibitory effects. This characteristic indicates that, in addition to their highly effective inhibitory function on the ACVR2A target pathway, the antibodies can also moderately regulate the ligand-dependent bypass signaling pathway (ACVR2B pathway). This "primarily targeted inhibition, supplemented by accompanying regulation" mode of action can synergistically enhance energy metabolism and muscle protein synthesis pathways, providing a multi-target synergistic therapeutic approach for metabolic syndrome accompanied by obesity and muscle atrophy.

[0116] This experiment systematically revealed the inhibitory spectrum of ACVR2A antibodies in lipid metabolism-related signaling pathways, confirming that they not only have ACVR2A targeting but also exhibit differentiated inhibitory properties in the ACVR2B pathway activated by different ligands. This provides an important basis for precision treatment strategies for metabolic diseases such as obesity and fatty liver.

[0117] Example 7: Evaluation of the in vivo pharmacokinetic properties of ACVR2A antibody: Male C57BL / 6 mice aged 6-8 weeks were randomly divided into 7 groups of 4 mice each. Each group received a single subcutaneous injection of the test antibodies (AB83, AB09, A0004, A0019, A0040) and the positive control antibody LAE102, at a dose of 20 mg / kg. Blood samples were collected before administration and at 0.5 h, 4 h, and on days 1, 3, 5, 7, 10, 14, and 21 after administration. Serum total antibody concentration (anti-human IgG detection) was measured using ELISA. Pharmacokinetic parameters, including half-life (t1 / 2), peak concentration (Cmax), and area under the curve (AUC), were calculated using a non-compartmental model. 0-t The pharmacokinetic characteristics of each antibody in mice were evaluated.

[0118] The results are as follows Figure 15 As shown, after two batches of single subcutaneous administration (20 mg / kg), each antibody showed different trends in total antibody concentration (detected by Anti-hIgG ELISA) over time in mice.

[0119] The first batch of experiments (G1-G3 groups): The pharmacokinetic characteristics of AB83, AB09, and the positive control LAE102 were compared. Total antibody concentration (pK) parameters showed that AB83 and AB09 were superior to LAE102 in all parameters: half-life (t1 / 2) was 94.57 h and 103.71 h, respectively, both longer than LAE102's 43.53 h; peak concentration (Cmax) was 382.41 μg / mL and 259.92 μg / mL, respectively, both higher than LAE102's 236.66 μg / mL; the area under the curve (AUC) was also higher. 0-t The figures are 84587.29 h respectively. μg / mL and 59131.48 h μg / mL, all higher than LAE102's 40269.07 h. μg / mL ( Figure 15 (A in the middle).

[0120] The second batch of experiments (G4-G7 groups): The pharmacokinetic characteristics of A0004, A0019, and A0040 were compared with the positive control LAE102. The results showed that the pharmacokinetic parameters of the three candidate molecules were significantly better than those of LAE102: the half-lives of A0004, A0019, and A0040 were 97.43 h, 74.72 h, and 69.11 h, respectively, all longer than LAE102's 43.16 h; the peak concentrations were 263.17 μg / mL, 232.28 μg / mL, and 219.61 μg / mL, respectively, all higher than LAE102's 145.10 μg / mL; and the areas under the curve (AUC) were 72340.95 h. μg / mL, 66750.12 h μg / mL, 58756.33 h μg / mL, all higher than LAE102's 19860.33 h μg / mL ( Figure 15 (B in the middle).

[0121] Based on the combined results of two batches of experiments, the five antibodies described in this invention (AB83, AB09, A0004, A0019, and A0040) comprehensively outperformed the positive control LAE102 in key pharmacokinetic indicators (half-life, peak concentration, and in vivo exposure), demonstrating significant overall pharmacokinetic advantages. The longer half-life and higher drug exposure suggest the potential for longer dosing intervals and more sustained efficacy in clinical applications, laying a solid pharmacokinetic foundation for the subsequent development of long-acting antibody drugs.

[0122] Example 8: Evaluation of the synergistic effect of ACVR2A antibody combined with smegglutinin in a DIO mouse model: I. Laboratory Animals and Grouping: Animal model: DIO C57BL / 6 male mice (diet-induced obesity model).

[0123] Grouping method: There are 8 groups in total (G1~G8), with 8 animals in each group.

[0124] Householding conditions: Standard SPF environment, free access to food and water.

[0125] II. Dosing regimen: Route of administration: All test substances were administered by subcutaneous injection (SC).

[0126] Group dosing details: G1: PBS (phosphate-buffered saline, control group) G2: Sema (semaglutide), 120 μg / kg, once daily (QD). G3: LAE102 + Sema, LAE102 20 mg / kg (once a week × 4 times) + Sema 120 μg / kg (QD) G4: AB09 + Sema, AB09 20 mg / kg (once a week × 4 times) + Sema 120 μg / kg (QD) G5: AB83 + Sema, AB83 20 mg / kg (once a week × 4 times) + Sema 120 μg / kg (QD) G6: A0004 + Sema, A0004 20 mg / kg (once a week × 4 times) + Sema 120 μg / kg (QD) G7: A0019 + Sema, A0019 20 mg / kg (once a week × 4 times) + Sema 120 μg / kg (QD) G8: A0040 + Sema, A0040 20 mg / kg (once a week × 4 times) + Sema 120 μg / kg (QD) III. Experimental Results: The results are as follows Figure 16 As shown: Weight loss: All groups using the "ACVR2A antibody + semaglutide" combination showed better weight loss than the semaglutide monotherapy group (G2). In the early stage of administration (D0-D25), the A0040 combination group showed the most significant weight loss; in the later stage of administration (D26-D30), the AB83 combination group showed the most significant weight loss. Figure 16 (A in the middle).

[0127] Muscle mass changes (MRI detection): Compared with the positive control antibody LAE102 combination group (G3), the combination of AB09, A0004, and A0040 with smegglutinin all showed a more significant increase in muscle mass. Figure 16 (B in the middle).

[0128] Changes in body fat mass (MRI scan): All groups using the combination of "ACVR2A antibody + semaglutide" showed better fat reduction effects than the semaglutide-only group (G2). Among them, the combination of A0040 and semaglutide showed the most significant fat reduction effect. Figure 16 (C in the middle).

[0129] The combination of ACVR2A antibody and the GLP-1 agonist semaglutide exhibited a significant synergistic effect. This combination strategy not only enhanced the weight loss and fat reduction effects of semaglutide, but more importantly, reversed the muscle loss that might result from using semaglutide alone, achieving the ideal goal of improving body composition through "fat loss and muscle gain".

[0130] A0040 showed the best performance in the early stages of weight loss and fat reduction, while AB83 showed the best performance in the later stages of weight loss. AB09, A0004, and A0040 were all superior to the positive control LAE102 in terms of muscle gain. The ACVR2A antibody described in this invention exhibited a synergistic effect superior to the positive control LAE102 in improving body composition, further verifying its functional advantages.

[0131] In vivo pharmacodynamic experiments have demonstrated the feasibility and remarkable value of combining ACVR2A antibody with existing mainstream weight-loss drugs (GLP-1 agonists) at the disease model level, providing an effective solution to the clinical challenge of "weight loss while losing muscle" in current weight-loss therapies.

[0132] Example 9: Study on the improvement of body weight and muscle function by ACVR2A antibody in CB-17 SCID mice: I. Laboratory Animals and Grouping: Animal model: CB-17 SCID male mice (severe combined immunodeficiency mice).

[0133] Grouping method: 5 groups in total (G1~G5), 8 animals in each group.

[0134] Householding conditions: Standard SPF environment, free access to food and water.

[0135] II. Dosing regimen: 1. Route of administration: All test substances were administered via subcutaneous injection (SC).

[0136] 2. Details of group dosing: G1: PBS (phosphate-buffered saline, control group), equal volume subcutaneously injected, administered weekly for 4 weeks; G2: LAE-102, Dosage: 20 μg / kg; Single dose: 26 mg; Dosage regimen: Weekly administration for 4 weeks; G3: Bimagrumab, Dosage: 20 μg / kg; Single dose: 26 mg; Dosage regimen: Weekly administration for 4 weeks; G4: AB83, Dosage: 20 μg / kg; Single dose: 26 mg; Dosage regimen: Weekly administration for 4 weeks; G5: AB09, Dosage: 20 μg / kg; Single dose: 26 mg; Dosage regimen: Weekly administration for 4 weeks.

[0137] The results are as follows Figure 17As shown, weight changes: In the CB-17 SCID mouse model, after continuous administration of antibodies (20 mg / kg) for four weeks, the weight gain effects of the antibodies AB83 and AB09 of this invention were comparable to those of the positive control antibody LAE-102, both of which could induce a certain degree of weight gain. Figure 17 (A in the middle).

[0138] Muscle function improvement (grip strength test): Both antibodies AB83 and AB09 significantly improved grip strength in CB-17 SCID mice. Among them, AB09 showed the most significant improvement in grip strength, with a statistically highly significant difference compared to the PBS control group, and was superior to LAE-102. Figure 17 (B in the middle).

[0139] The antibody was significantly superior to the positive control LAE-102 in improving muscle function. Although both had similar effects in promoting weight gain, the screening antibody (especially AB09) was able to more effectively translate the weight gain effect into a substantial improvement in muscle function, which is more in line with the core clinical need for treating diseases such as muscle atrophy or sarcopenia.

[0140] AB09 was identified as a preferred candidate molecule for functional improvement, demonstrating the strongest and most significant improvement in grip strength testing. This proves its outstanding advantages in enhancing muscle contractility and improving motor function, making it an ideal candidate molecule for developing innovative therapies aimed at "functional muscle strength enhancement".

[0141] Both antibodies AB83 and AB09 have the potential to improve muscle function, indicating that the anti-ACVR2A antibodies of the present invention are a series of molecules with common advantages in terms of functional gain.

[0142] In summary, in vivo functional studies further confirm the significant advantages of ACVR2A antibodies (especially AB09) in improving muscle function, providing key preclinical functional evidence for their therapeutic applications in sarcopenia, cachexia, and muscular atrophy, and highlighting their potential superiority over the existing positive control (LAE-102).

[0143] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0144] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An ACVR2A antibody or its antigen-binding fragment, characterized in that, The ACVR2A antibody or its antigen-binding fragment comprises VH and VL, wherein the amino acid sequences of CDR 1, 2, and 3 in VH are shown in SEQ ID NO: 41, 42, and 43, respectively, and the amino acid sequences of CDR 1, 2, and 3 in VL are shown in SEQ ID NO: 44, 45, and 46, respectively.

2. The ACVR2A antibody or its antigen-binding fragment according to claim 1, characterized in that, The VH amino acid sequence is shown in SEQ ID NO: 47, and the VL amino acid sequence is shown in SEQ ID NO:

48.

3. A nucleic acid, characterized in that, Encodes the ACVR2A antibody or its antigen-binding fragment as described in claim 1 or 2.

4. A carrier, characterized in that, It includes the nucleic acid as described in claim 3.

5. A cell, characterized in that, Express the nucleic acid as described in claim 3 or the vector as described in claim 4.

6. A pharmaceutical composition for treating obesity, characterized in that, include: (1) A therapeutically effective amount of the ACVR2A antibody or its antigen-binding fragment as described in claim 1 or 2, the nucleic acid as described in claim 3, the vector as described in claim 4, or the cell as described in claim 5; (2) Pharmaceutically or immunologically acceptable carriers or excipients.

7. The pharmaceutical composition according to claim 6, characterized in that, The pharmaceutical composition also includes a GLP-1 agonist.

8. A pharmaceutical preparation for treating obesity, characterized in that, Includes the pharmaceutical composition according to claim 6 or 7.

9. A pharmaceutical product for treating obesity, characterized in that, Includes the pharmaceutical preparation described in claim 8.

10. The use of the ACVR2A antibody of claim 1 or 2 or its antigen-binding fragment, the nucleic acid of claim 3, the vector of claim 4, the cell of claim 5, the pharmaceutical composition of claim 6 or 7, the pharmaceutical formulation of claim 8, or the pharmaceutical product of claim 9 in the preparation of a medicament for the prevention and / or treatment of obesity.

11. A detection reagent, characterized in that, Includes the ACVR2A antibody or its antigen-binding fragment as described in claim 1 or 2.

12. A test kit, characterized in that, Includes the detection reagent as described in claim 11.

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