Antibody binding to ACVR2a and / or ACVR2b, and preparation therefor and use thereof

Abstract

Provided are an antibody binding to ACVR2A and / or ACVR2B, and a preparation therefor and a use thereof. A heavy chain variable region of the antibody comprises HCDRs 1-3, and amino acid sequences of the HCDRs 1-3 are as shown in SEQ ID NOs: 13-15 or as shown in SEQ ID NOs: 19-21. Also provided are a biological material, pharmaceutical composition and use related to the antibody.

Description

Antibodies binding to acvr2a and / or acvr2b and preparation and use thereof TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to an antibody binding to ACVR2A and / or ACVR2B and preparation and use thereof. BACKGROUND

[0002] Activin type II receptors (ACVR2) include ACVR2A and ACVR2B. They are widely expressed in muscle, fat and various organ cells, are a class of transmembrane proteins, and mediate the activation of various protein signaling pathways. The ACVR2 ligands include Activin A, Activin B, Myostatin (GDF8), GDF11, etc., among which Activin A and GDF8 are recognized as negative regulators of muscle, which inhibit muscle growth under physiological conditions and avoid excessive muscle hypertrophy. The ligands such as Activin A bind to ACVR2 and activate Activin type I receptors (ACVR1), and then activate the phosphorylation of transcription factors Smad2 and Smad3, and further regulate the expression of related genes such as MyoD.

[0003] Muscle plays an important role in health, and muscle fibers can synthesize and release myokines. Muscle factors have an impact on cognition, lipid and glucose metabolism, white fat browning, bone formation, endothelial cell function, maintenance of skin structure and tumor growth, etc. Various factors can cause muscle loss, including acute diseases (severe infection, postoperative stress, etc.), chronic diseases (diabetes, cancer, etc.), malnutrition, lack of exercise, age-related natural muscle atrophy, etc. The use of drugs can also cause muscle loss, such as GLP-1RA drugs for treating diabetes and obesity. Therefore, to avoid or slow down muscle loss caused by various reasons, targeting ACVR2 antibodies can be an important treatment strategy.

[0004] In addition, the role of ACVR2 in the treatment of obesity has also been increasingly valued. Obesity has become a global health problem and a major risk factor for many metabolic diseases. In obese people, the expression level of ACVR2 ligand GDF8 is significantly increased. Studies have shown that knocking out GDF8 can increase muscle weight, reduce fat mass, and improve insulin sensitivity; inhibiting the activity of ACVR2 can, on the one hand, reduce the uptake of glucose by fat through increasing the uptake of glucose by muscle and the secretion of cytokines such as IL-6, and increase the decomposition of fat; on the other hand, by acting on adipocytes to increase the expression of heat-producing proteins such as UCP1, inducing the browning of white adipose tissue. Therefore, targeting ACVR2 or synergizing with GLP-1 drugs for the treatment of obesity can have lower side effects and better therapeutic effects. SUMMARY

[0005] The present application provides antibodies and antigen-binding fragments thereof that bind to ACVR2A and / or ACVR2B, as well as methods of making such antibodies and antigen-binding fragments thereof, pharmaceutical compositions comprising such antibodies and antigen-binding fragments thereof, or immunoconjugates thereof. The present application also relates to methods and uses of active molecules, pharmaceutical compositions comprising the antibodies and antigen-binding fragments thereof for preventing or treating diseases.

[0006] The first aspect of the present application provides an antibody or antigen-binding fragment thereof that binds to ACVR2A and / or ACVR2B, wherein the heavy chain variable region comprises HCDR1-3, the amino acid sequences of which are shown as SEQ ID NO: 13-15 or as SEQ ID NO: 19-21.

[0007] The antibody or antigen-binding fragment thereof that binds to ACVR2A and / or ACVR2B further comprises light chain variable region LCDR1-3, the amino acid sequences of which are shown as SEQ ID NO: 16-18 or as SEQ ID NO: 22-24.

[0008] The second aspect of the present application provides a bispecific antibody that binds to ACVR2A / ACVR2B, comprising the antibody or antigen-binding fragment thereof that binds to ACVR2A and / or ACVR2B of the above-mentioned first aspect.

[0009] The third aspect of the present application provides a polynucleotide encoding the antibody or antigen-binding fragment thereof that binds to ACVR2A and / or ACVR2B of the above-mentioned first aspect, or the bispecific antibody that binds to ACVR2A / ACVR2B of the second aspect.

[0010] The fourth aspect of the present application provides a vector comprising the polynucleotide of the above-mentioned third aspect.

[0011] The fifth aspect of the present application provides a host cell, which comprises the vector of the fourth aspect described above, or the genome of the host cell is integrated with the polynucleotide of the third aspect described above.

[0012] The sixth aspect of the present application provides a preparation method, which comprises the following steps: 1) culturing the host cell of the fifth aspect described above, so as to obtain a culture comprising the antibody or antigen-binding fragment thereof or bispecific antibody described above; 2) isolating or recovering the antibody or antigen-binding fragment thereof or bispecific antibody described above from the culture; 3) optionally, purifying and / or modifying the antibody or antigen-binding fragment thereof or bispecific antibody obtained in step 2).

[0013] The seventh aspect of the present application provides an immunoconjugate, which comprises the antibody or antigen-binding fragment thereof binding to ACVR2A and / or ACVR2B of the first aspect described above or the bispecific antibody binding to ACVR2A / ACVR2B of the second aspect, and a conjugated moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a carrier protein, a radionuclide, an enzyme or a combination thereof.

[0014] The eighth aspect of the present application provides a pharmaceutical composition, which comprises the antibody or antigen-binding fragment thereof binding to ACVR2A and / or ACVR2B of the first aspect described above, the bispecific antibody binding to ACVR2A / ACVR2B of the second aspect, or the immunoconjugate of the seventh aspect described above.

[0015] The ninth aspect of the present application provides the use of the antibody or antigen-binding fragment thereof binding to ACVR2A and / or ACVR2B of the first aspect described above, the bispecific antibody binding to ACVR2A / ACVR2B of the second aspect, the immunoconjugate of the seventh aspect, or the pharmaceutical composition of the eighth aspect for preventing and / or treating a disease or disorder selected from the group consisting of:

[0016] 1) a disease or disorder associated with ACVR2A, ACVR2B, or a ligand thereof;

[0017] 2) an endocrine and metabolic disease, a tumor, a respiratory disease, a skin and musculoskeletal disease, a nervous system disease.

[0018] In particular, the disease or disorder can be selected from the group consisting of obesity and its metabolic disorders, overweight, cachexia, type 2 diabetes, hip fracture, difficulty walking, muscle atrophy, inclusion body myositis, sarcopenia, myelofibrosis, osteoporosis, chronic obstructive pulmonary disease, pulmonary arterial hypertension, depression, primary myelofibrosis, anemia, chronic kidney disease-mineral and bone disorder, thalassemia, congenital pure red cell anemia, beta thalassemia, bladder cancer, head and neck tumor, multiple myeloma, myelodysplastic syndrome, small cell lung cancer, non-small cell lung cancer, pancreatic cancer, metastatic solid tumor, chronic myelomonocytic leukemia.

[0019] The application has the beneficial effect that, through animal immunization, and multiple rounds of optimization and activity screening, a monoclonal antibody with high affinity and blocking activity to ACVR2A and / or ACVR2B is constructed, which has an affinity and blocking activity to ACVR2A or ACVR2B comparable to the positive controls BYM338 and LA01. To further enhance the activity, a bispecific antibody is constructed based on the monoclonal antibody. The bispecific antibody exhibits blocking activity to ACVR2A and ACVR2B and their downstream signaling pathways at the same time, and also shows excellent effects of reducing fat and enhancing efficiency in animal models. Surprisingly, the bispecific antibody also exhibits better blocking activity to the downstream signaling pathway of ACVR2A than BYM338. Therefore, the existence of such difference is expected to have new clinical application scenarios relative to existing treatment methods, and to provide new treatment options for diseases or disorders related to ACVR2A, ACVR2B or their ligands, such as obesity, overweight, muscle atrophy, etc. BRIEF DESCRIPTION OF DRAWINGS

[0020] FIG. 1A is a detection result of the binding activity of a chimeric antibody to human ACVR2A.

[0021] FIG. 1B is a detection result of the binding activity of a chimeric antibody to human ACVR2B.

[0022] FIG. 2A is a detection result of the blocking activity of a chimeric antibody to the binding of ACVR2A to Activin A protein.

[0023] FIG. 2B is a detection result of the blocking activity of a chimeric antibody to the binding of ACVR2B to Activin A protein.

[0024] FIG. 3 is a detection result of the blocking activity of a chimeric antibody to the downstream signaling pathway of ACVR2A.

[0025] FIG. 4 is a detection result of the blocking activity of a chimeric antibody to the downstream signaling pathway of ACVR2B.

[0026] FIG. 5A is a detection result of the binding activity of 124H1A4 humanized antibody to human ACVR2A (Table 5).

[0027] Figure 5B shows the results of testing the binding activity of 124H1A4 humanized antibody to human ACVR2B (Table 5).

[0028] Figure 6A shows the results of testing the binding activity of 124H1A4 humanized antibody to human ACVR2A (Table 6).

[0029] Figure 6B shows the results of testing the binding activity of 124H1A4 humanized antibody to human ACVR2B (Table 6).

[0030] Figure 7 shows the results of testing the blocking activity of 124H1A4 humanized antibody to ACVR2A binding to Activin A protein.

[0031] Figure 8 shows the results of testing the blocking activity of 124H1A4 humanized antibody to ACVR2B binding to Activin A protein.

[0032] Figure 9 shows the results of testing the blocking activity of 124H1A4 humanized antibody to Activin A protein activating downstream signaling pathway of ACVR2A.

[0033] Figure 10 shows the results of testing the binding activity of 10E8H3 humanized antibody to human ACVR2B.

[0034] Figure 11 shows the results of testing the blocking activity of 10E8H3 humanized antibody to ACVR2B binding to Activin A protein.

[0035] Figure 12 shows the results of testing the blocking activity of 10E8H3 humanized antibody to Activin A protein activating downstream signaling pathway of ACVR2B.

[0036] Figure 13 shows the results of testing the binding epitope to ACVR2B.

[0037] Figure 14A shows the results of testing the binding activity of the bispecific antibody to human ACVR2A.

[0038] Figure 14B shows the results of testing the binding activity of the bispecific antibody to human ACVR2B.

[0039] Figure 15A shows the results of testing the blocking activity of the bispecific antibody to ACVR2A binding to Activin A protein.

[0040] Figure 15B shows the results of testing the blocking activity of the bispecific antibody to ACVR2B binding to Activin A protein.

[0041] Figure 16 shows the results of testing the blocking activity of the bispecific antibody to Activin A protein activating downstream signaling pathway of ACVR2A.

[0042] Figure 17 shows the results of testing the blocking activity of the bispecific antibody to Activin A protein activating downstream signaling pathway of ACVR2B.

[0043] Figure 18 shows the detection results of simultaneous blocking of ACVR2A and ACVR2B by bispecific antibodies to activate downstream signaling pathways mediated by Activin A protein in cells.

[0044] Figure 19 shows the results of the detection of the blocking activity of the bispecific antibody against the downstream signaling pathway of ACVR2A cells activated by GDF8 protein.

[0045] Figure 20 shows the results of the detection of the blocking activity of the bispecific antibody against the downstream signaling pathway of GDF8 protein activation in ACVR2B cells.

[0046] Figure 21A shows the detection results of bispecific antibody-ACVR2A binding epitope competition.

[0047] Figure 21B shows the detection results of bispecific antibody-ACVR2B binding epitope competition.

[0048] Figure 22A shows the results of detecting the effect of drug administration on brown adipose tissue in a mouse model.

[0049] Figure 22B shows the results of detecting the effect of drug administration on white adipose tissue in a mouse model.

[0050] Figure 22C shows the results of detecting the effect of drug administration on muscle mass in a mouse model.

[0051] Figure 23 shows the results of the bispecific antibody binding activity assay for FcRn.

[0052] Figure 24 shows the results of the detection of the rate of change in body weight of cynomolgus monkeys after double antibody treatment.

[0053] Figure 25A shows the rate of change of the effect of the double antibody on the fat content of cynomolgus monkeys.

[0054] Figure 25B shows the results of the rate of change detection of the effect of the double antibiotics on the lean meat content of cynomolgus monkeys.

[0055] Figure 26 shows the results of the rate of change of the effect of the double antibody on adiponectin in cynomolgus monkeys.

[0056] Figure 27 shows the effect of drug administration on liver weight after 2 weeks in the DIO mouse model.

[0057] Figure 28 shows the effect of drug administration on heart weight after 2 weeks in the DIO mouse model.

[0058] Figure 29 shows the effect of drug administration for 2 weeks on total muscle weight in the DIO mouse model.

[0059] Figure 30 shows the effect of drug administration for 2 weeks on total fat weight in the DIO mouse model.

[0060] Figure 31A shows the effect of drug administration for 2 weeks on serum ALT levels in the DIO mouse model.

[0061] Figure 31B shows the effect of drug administration for 2 weeks on serum AST levels in the DIO mouse model.

[0062] Figure 31C shows the effect of drug administration for 2 weeks on serum ALP levels in the DIO mouse model.

[0063] Figure 31D shows the effect of drug administration for 2 weeks on serum LDH levels in the DIO mouse model. Detailed Implementation

[0064] The following specific examples illustrate the implementation methods of this application. Those skilled in the art can understand other advantages and effects of this application from the content disclosed in this specification. This application can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of this application.

[0065] Before further describing the specific embodiments of this application, it should be understood that the scope of protection of this application is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of this application is for describing specific embodiments and not for limiting the scope of protection of this application; in the specification and claims of this application, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0066] This application first provides an antibody or antigen-binding fragment thereof that binds ACVR2A and / or ACVR2B.

[0067] Antibodies typically exist as one or more Y-shaped monomers (full-length antibodies), each consisting of four polypeptide chains: two identical heavy chains and two identical light chains. The heavy and light chains are distinguished by their molecular weight. Based on differences in the structure of small polypeptide molecules, light chains are classified as κ or λ types, and heavy chains as μ, δ, γ, α, or ε, defining the antibody class or type as IgM, IgD, IgG, IgA, and IgE, respectively.

[0068] Typically, the variable region of an antibody includes a heavy chain variable region and a light chain variable region (single-domain antibodies may only include the heavy chain variable region). The heavy chain variable region (VH) is located at the N-terminus of the antibody heavy chain (H chain), and the light chain variable region (VL) is located at the N-terminus of the antibody light chain (L chain). These variable regions include complementarity-determining regions (CDRs), which are crucial regions in the antibody molecule for specific binding to antigens. CDR regions exhibit high sequence diversity, enabling antibodies to recognize and bind to various different antigens. In the antibody variable region, there are three CDR regions each in the light and heavy chains, denoted as CDR1, CDR2, and CDR3, respectively. CDR regions directly interact with specific epitopes of the antigen, and their sequence diversity determines the antibody's specificity and affinity.

[0069] "Anti" and "binding" refer to non-random binding reactions between two molecules, such as the reaction between an antibody and the antigen it targets. Typically, antibodies bind in groups of less than approximately 10... -7 M, for example, less than approximately 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 An antibody binds to an antigen with an equilibrium dissociation constant (KD) of M or less. The term "KD" refers to the equilibrium dissociation constant of a specific antibody-antigen interaction, used to describe the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the stronger the antibody-antigen binding and the higher the affinity between the antibody and the antigen. For example, the binding affinity between the antibody and the antigen can be determined using surface plasmon resonance (SPR) in a BIACORE instrument or using ELISA to determine the relative affinity of the antibody to the antigen.

[0070] In specific embodiments of this application, the heavy chain variable region and / or light chain variable region further include amino acid mutations; preferably, the amino acid mutations are located in the FR framework region. The FR framework region refers to the region in the antibody variable region other than the three CDRs, and its main function is to stabilize the spatial conformation of the CDR region and assist in the accurate and specific binding of the antigen. Each antibody chain has four framework regions, namely FR1, FR2, FR3, and FR4. The relatively conserved amino acid sequences of the framework regions form a β-sheet structure, supporting the CDR region located at the top of the barrel structure, enabling the CDR region to bind to the antigen epitope.

[0071] In one embodiment, the heavy chain variable region comprises HCDR1-3, the amino acid sequences of which are shown in SEQ ID NO:13-15 or SEQ ID NO:19-21.

[0072] In one embodiment, the system further includes a light chain variable region LCDR1-3, the amino acid sequence of which is shown in SEQ ID NO:16-18 or SEQ ID NO:22-24.

[0073] In one implementation, the heavy chain variable region or the light chain variable region has the following characteristics:

[0074] 1) The heavy chain variable region is selected from SEQ ID NO:4, 8, 25, 28, 29, 31, 32, 34; and / or

[0075] 2) The light chain variable region is selected from SEQ ID NO: 6, 10, 26, 27, 30, 33, 35; and / or

[0076] 3) The heavy chain variable region is selected from amino acid sequences having at least 99%, 95%, 90%, 85%, or 80% identity with SEQ ID NO:4, 8, 25, 28, 29, 31, 32, and 34; and the light chain variable region is selected from amino acid sequences having at least 99%, 95%, 90%, 85%, or 80% identity with SEQ ID NO:6, 10, 26, 27, 30, 33, and 35.

[0077] In one implementation, the heavy chain variable region and / or light chain variable region of 3) above have the same function as 4, 8, 25, 28, 29, 31, 32, 34 or as 6, 10, 26, 27, 30, 33, 35.

[0078] Methods for determining sequence identity known to those skilled in the art include, but are not limited to: Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., Stockton Press, New York, 1991; and Carillo, H. and Lipman, D., SIAM J. Applied. Math., 48:1073 (1988). Preferred methods for determining identity involve obtaining the largest possible match between the tested sequences, and these methods are compiled into publicly available computer programs. Preferred computer program methods for determining identity between two sequences include, but are not limited to: the GCG package (Devereux, J. et al., 1984), BLASTP, BLASTN, and FASTA (Altschul, S., F. et al., 1990). The BLASTX program (BLAST manual, Altschul, S. et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S. et al., 1990) is also publicly available from NCBI and other sources, and the Smith Waterman algorithm can also be used for identity determination.

[0079] In one embodiment, the heavy chain and light chain variable regions comprise SEQ ID NO:8 and SEQ ID NO:10 (124H1A4-ch).

[0080] In one embodiment, the heavy chain and light chain variable regions comprise SEQ ID NO:25 and SEQ ID NO:26. (124H-Hu1-L1)

[0081] In one embodiment, the heavy chain and light chain variable regions comprise SEQ ID NO:25 and SEQ ID NO:27. (124H-Hu1-L2)

[0082] In one embodiment, the heavy chain and light chain variable regions comprise SEQ ID NO:28 and SEQ ID NO:26. (124H-Hu2-L1)

[0083] In one embodiment, the heavy chain and light chain variable regions comprise SEQ ID NO:28 and SEQ ID NO:27. (124H-Hu2-L2)

[0084] In one embodiment, the heavy chain and light chain variable regions comprise SEQ ID NO:29 and SEQ ID NO:30. (124H-HuH1-L3)

[0085] In one embodiment, the heavy chain and light chain variable regions comprise SEQ ID NO:31 and SEQ ID NO:30. (124HDF-HuH2-L3)

[0086] In one embodiment, the heavy chain and light chain variable regions comprise SEQ ID NO:4 and SEQ ID NO:6. (10E8H3-ch)

[0087] In one embodiment, the heavy chain and light chain variable regions comprise SEQ ID NO:32 and SEQ ID NO:33. (10E8H-HuGG)

[0088] In one embodiment, the heavy chain and light chain variable regions comprise SEQ ID NO:34 and SEQ ID NO:33. (10E8H-Hu1G)

[0089] In one embodiment, the heavy chain and light chain variable regions comprise SEQ ID NO:34 and SEQ ID NO:35. (10E8H-Hu1)

[0090] In one embodiment, the heavy chain and light chain variable regions comprise SEQ ID NO:32 and SEQ ID NO:35. (10E8H-HuG1)

[0091] This application also provides an antibody or antigen-binding fragment thereof that binds to ACVR2A and / or ACVR2B, which competes with the aforementioned antibody or antigen-binding fragment thereof for binding.

[0092] In one embodiment, it competes with antibodies or antigen-binding fragments thereof comprising HCDR-1-3 of SEQ ID NO:13-15 and LCDR 1-3 of SEQ ID NO:16-18 for binding to ACVR2A.

[0093] The antibody or its antigen-binding fragment provided in this application is selected from full-length antibodies, nanobodies, single-chain antibodies, biantibodies, scFv, Fv, Fd, Fab, F(ab')2 or F(ab').

[0094] Those skilled in the art will recognize that antibodies in non-complete tetrameric forms, including but not limited to Fab, Fab', F(ab') or F(ab')2, nanobodies (VHH), single-chain antibodies (scFv), BsFv, dsFv, (dsFv)2, or Fv, can also exert the effect of specifically binding antigens. Therefore, this application also includes antigen-binding fragments of antibodies.

[0095] In specific embodiments of this application, the antibody is selected from murine antibodies, chimeric antibodies, and humanized antibodies.

[0096] Specifically, murine antibodies are antibodies whose encoding genes are entirely derived from mice. Chimeric antibodies are monoclonal antibodies produced by inserting the variable regions of the light and heavy chains of murine monoclonal antibodies into a vector containing the constant regions of human antibodies using DNA recombination technology, followed by expression in mammalian cells. Chimeric antibodies can achieve a humanization rate of up to 70%, fully retaining the variable regions and parental activity of the murine monoclonal antibody, while the introduction of the human antibody constant regions reduces immunogenicity. Humanized antibodies are antibodies modified through genetic engineering, with their structure largely or entirely composed of human antibody sequences to reduce immunogenicity in humans. This modification typically involves transplanting the complementarity-determining regions (CDRs) of murine monoclonal antibodies into the framework regions (FRs) of human antibodies, while retaining the antigen-binding sites of the murine antibody. This results in modified antibodies with lower immunogenicity in humans while maintaining their original specificity and affinity.

[0097] The antibody or its antigen-binding fragment provided in this application may contain a constant region, which may contain mutated amino acids. This results in a correspondingly altered effector function through mutation.

[0098] The constant region can be human or mouse-derived. The heavy chain constant region of the antibody can be the heavy chain constant region of IgG (IgG1, IgG2, IgG3 or IgG4), IgA, IgM, IgD, or IgE. The light chain constant region of the antibody can be the Kappa light chain constant region or the Lambda light chain constant region.

[0099] For example, in some embodiments of this application, the heavy chain constant region of the antibody is the human antibody IgG1 heavy chain constant region, and the light chain constant region is the human Kappa light chain constant region. Mutations in the constant region include, for example, LALA mutations, YTE mutations, etc.

[0100] In one embodiment, the heavy chain constant region comprises an amino acid sequence as shown in SEQ ID NO: 11, and the light chain constant region comprises an amino acid sequence as shown in SEQ ID NO: 12.

[0101] This application also provides bispecific antibodies that bind ACVR2A / ACVR2B, comprising the antibodies that bind ACVR2A and / or ACVR2B as described above, or their antigen-binding fragments.

[0102] In one embodiment, the bispecific antibody comprises an antibody or its antigen-binding fragments I and II that bind to different antigenic epitopes. For example, I binds to ACVR2A and II binds to ACVR2B; or I and II bind to different epitopes of ACVR2A and / or ACVR2B.

[0103] In one implementation, I and II are optionally scFv and full-length antibody.

[0104] In one implementation, I combines ACVR2B, and II combines ACVR2A and ACVR2B.

[0105] In one embodiment, the bispecific antibody exhibits superior activity against ACVR2A. This activity includes, but is not limited to, binding affinity to ACVR2A / ACVR2B and blocking effects on its ligands such as Activin A and GDF8 proteins.

[0106] In one implementation, the bispecific antibody is at a concentration of less than 10. -9 M's KD combination with ACVR2A, at less than 5*10 -9 M's KD binds to ACVR2B, wherein the KD is determined by the SPR method.

[0107] In one embodiment, the bispecific antibody comprises a heavy chain with an amino acid sequence as shown in SEQ ID NO: 38 and a light chain with an amino acid sequence as shown in SEQ ID NO: 30.

[0108] The antibodies or antigen-binding fragments thereof provided in this application, as well as the bispecific antibodies, have any of the following characteristics:

[0109] 1) Combined with ACVR2A and / or ACVR2B;

[0110] 2) Block the binding of ACVR2A and / or ACVR2B to their ligands;

[0111] 3) Block downstream signaling pathways activated by ACVR2A and / or ACVR2B and their ligand complexes;

[0112] 4) Increase muscle mass, increase brown adipose tissue content, or decrease white adipose tissue content.

[0113] The antibodies or antigen-binding fragments thereof provided in this application, as well as bispecific antibodies, comprise monomers or dimers formed from monomers, wherein the dimers may be homologous or heterologous.

[0114] This application also provides a polynucleotide encoding the above-described antibody or its antigen-binding fragment, as well as a bispecific antibody.

[0115] Polynucleotides are polymers of nucleotides typically linked from one deoxyribose or ribose to another. The polynucleotides in this application are not limited in size and may include polynucleotides containing modifications, particularly modified nucleotides. The polynucleotides in this application may, for example, contain coding sequences and regulatory sequences, and optionally natural or artificial introns, which may have their initial codon or may have optimized codon usage, and are particularly suitable for expression in desired host cells or host organisms. In some embodiments, the polynucleotide may be RNA, DNA, or cDNA, etc. The preparation methods of the polynucleotides are prior art, and the nucleic acids of this application can be prepared or obtained in a manner known per se (e.g., by automated DNA synthesis and / or recombinant DNA technology) based on the amino acid sequence information of the protein given herein. In specific embodiments of this application, the polynucleotides include, but are not limited to, those listed in the sequence listing, such as the nucleotide sequences of chimeric antibodies SEQ ID NO: 3, 5, 7, 9.

[0116] Based on the above-mentioned polynucleotides, the polynucleotides can be inserted into a vector using existing technology to enable their expression. Therefore, this application also provides a vector containing the above-mentioned polynucleotides.

[0117] In this application, "vector" refers to a polynucleotide capable of carrying at least one polynucleotide fragment. The vector can exist in a circular or linear (linearized) form and includes vector fragments, or it can be an artificial chromosome or similar polynucleotide containing a transferable exogenous nucleic acid fragment. The vector may contain at least one expression cassette containing a regulatory sequence for the proper expression of the polynucleotide incorporated therein. The polynucleotide to be introduced into the cell (e.g., a polynucleotide encoding a target product or a selectable marker) can be inserted into the expression cassette of the vector for expression therefrom. The vector can also integrate exogenous DNA sequences into the host cell's genome, thereby achieving stable expression of the exogenous gene. It typically contains homologous arms or specific DNA sequences that can undergo homologous recombination with specific locations in the host cell's genome. The vector may be selected from DNA vectors, RNA vectors, plasmids, transposon vectors, CRISPR / Cas9 vectors, or viral vectors, etc.

[0118] Based on the above-described polynucleotides and vectors, this application may also provide a host cell containing the above-described vectors or having the above-described polynucleotides integrated into its genome.

[0119] The host cells can be selected from bacterial cells, fungal cells, insect cells, plant cells, mammalian cells, etc. Specifically, they can be selected from Escherichia coli, Streptomyces, Salmonella typhimurium, yeast, filamentous fungi, Drosophila S2 or Sf9 cells, CHO cells, COS cells, HEK293F cells, Bowes melanoma cells, NS0 cells, BHK cells, PER.C6 cells, etc.

[0120] Furthermore, the host cell is a cell in which a vector containing the aforementioned polynucleotide has been introduced. The method for introducing the vector into the host cell can be conventional, depending on the actual situation. For example, methods such as microinjection, gene gun, electroporation, virus-mediated transformation, electron bombardment, calcium phosphate precipitation, liposome transfection, and PEI can be selected.

[0121] This application also provides a method for preparing an antigen-binding protein or a chimeric antigen receptor, comprising culturing a host cell containing the aforementioned vector or a host cell whose genome has integrated exogenous polynucleotides, under conditions that allow the expression of the antigen-binding protein or chimeric antigen receptor, and recovering the antigen-binding protein or chimeric antigen receptor from the cultured host cell culture.

[0122] According to the method for preparing antibodies by genetic engineering, this application also provides a method for preparing antibodies or antigen-binding fragments thereof that bind ACVR2A and / or ACVR2B, or bispecific antibodies that bind ACVR2A / ACVR2B, wherein the above-mentioned host cells are cultured under conditions that allow the expression of the antibodies or antigen-binding fragments thereof and the bispecific antibodies, and the antibodies or antigen-binding fragments thereof and the bispecific antibodies are recovered from the cultured host cell culture.

[0123] The conditions for expression typically refer to effective transfection or transduction methods, favorable promoter conditions, stable gene copies, suitable culture conditions, and optimized post-translational modifications.

[0124] The effective transfection or transduction methods can be selected from microinjection, gene gun, electroporation, virus-mediated transformation, electron bombardment, calcium phosphate precipitation, liposome transfection, PEI, and other methods.

[0125] The recovered antibody or its antigen-binding fragment can be selected according to the actual situation, such as using Protein A affinity chromatography for purification.

[0126] This application may also provide an immunoconjugate comprising the antibody or antigen-binding fragment thereof that binds ACVR2A and / or ACVR2B, or a bispecific antibody that binds ACVR2A / ACVR2B, and a conjugation portion selected from the group consisting of: detectable markers, drugs (e.g., ADCs), toxins, cytokines, carrier proteins, radionuclides, enzymes, or combinations thereof.

[0127] Antibody-drug conjugates (ADCs) are small molecule drugs with biological activity linked to an antibody via a chemical chain. The antibody acts as a carrier to target and deliver the drug to target cells. Their general structural formula can be A-(LU)n, where: A is an antibody or its antigen-binding fragment; U is a therapeutic agent; L is a linker; and n is an integer selected from 1 to 8.

[0128] Antibody-drug conjugates include linkers that conjugate the antibody or its antigen-binding fragment to the therapeutic agent. The linkers can be cleavable linkers or non-cleavable linkers. Cleavable linkers can include acid-cleavable linkers, reducible linkers, enzyme-cleavable linkers, etc., while non-cleavable linkers are divided into two categories: thioether or maleimide hexanoyl (MC).

[0129] The therapeutic agents refer to small molecule drugs with cytotoxicity that are covalently coupled to antibodies via linkers. These therapeutic agents are the core components of ADCs and are responsible for exerting anti-tumor effects. Examples include microtubule inhibitors, DNA damaging agents, topoisomerase inhibitors, apoptosis inducers, immunomodulators, and protein degraders. Microtubule inhibitors can be, for example, maytansine derivatives (DM1, DM3, DM4), monomethylolpropionate E (MMAE), paclitaxel, and eribulin. DNA damaging agents can be, for example, calicheamicin, duocarmysin, atrazodone (PBD), camptothecins, and camptothecin derivatives such as SN-3. Topoisomerase inhibitors can be, for example, DXd (Deruxtecan), SN-38, and Exatecan (DX-895). Immunomodulators can be, for example, TLR agonists, STING agonists, and BTK inhibitors.

[0130] This application also provides a pharmaceutical composition comprising, in an effective amount of, an antibody or antigen-binding fragment thereof binding to ACVR2A and / or ACVR2B, a bispecific antibody binding to ACVR2A / ACVR2B, or the aforementioned immunoconjugate.

[0131] The effective amount refers to the amount of a pharmaceutical compound or composition that causes a measurable clinical, biological, or pharmaceutical change or response in a biomarker, cell, tissue, system, or patient.

[0132] The form of the pharmaceutical composition is not limited and can be in various forms such as solid, liquid, gel, semi-liquid, or aerosol.

[0133] In specific embodiments of this application, the pharmaceutical composition may further include pharmaceutically acceptable excipients or carriers. Pharmaceutically acceptable refers to non-toxic materials that do not interfere with the bioactivity and effectiveness of the active ingredient. Specific examples of pharmaceutically acceptable excipients or carriers include sterile water or physiological saline, stabilizers, excipients, antioxidants (ascorbic acid, etc.), buffers (phosphate, citric acid, other organic acids, etc.), preservatives, surfactants (PEG, Tween, etc.), chelating agents (EDTA, etc.), binders, etc. Furthermore, it may also contain other low-molecular-weight peptides; proteins such as serum albumin, gelatin, or immunoglobulins; amino acids such as glycine, glutamine, asparagine, arginine, and lysine; sugars or carbohydrates such as polysaccharides and monosaccharides; and sugar alcohols such as mannitol or sorbitol. When preparing aqueous solutions for injection, such as physiological saline, isotonic solutions containing glucose or other excipients like D-sorbitol, D-mannose, D-mannitol, or sodium chloride, appropriate solubilizers such as alcohols (ethanol, etc.), polyols (propylene glycol, PEG, etc.), and nonionic surfactants (Tween 80, HCO-50) can be used. Excipients or carriers can be adjusted according to the desired dosage form, and the ratio can be adjusted based on actual needs. The dosage forms of the pharmaceutical composition include gastrointestinal or parenteral dosage forms. Parenteral dosage forms include intravitreal injection, intravenous injection, intravenous infusion, subcutaneous injection, local injection, intramuscular injection, intratumoral injection, intraperitoneal injection, intracranial injection, intracavitary injection, or inhalation.

[0134] In specific embodiments of this application, the antibody or antibody-drug conjugate in the pharmaceutical composition can be a single active ingredient or can be combined with one or more other active ingredients (molecules) to form a combination formulation for treating the target disease. There is no conflict in efficacy or safety between the components. The content of each component in the combination formulation is typically a safe and effective amount, which can be adjusted based on actual usage (e.g., patient weight, type of application, disease condition, severity).

[0135] In one embodiment, the second active ingredient includes a GLP-1 analog or a GLP-1R agonist, such as Semaglutide, Dulaglutide, or Exenatide.

[0136] This application also provides the use of the above-mentioned antibodies binding ACVR2A and / or ACVR2B or their antigen-binding fragments, bispecific antibodies binding ACVR2A / ACVR2B, immunoconjugates, and pharmaceutical compositions for the prevention and / or treatment of diseases or conditions.

[0137] This application also provides the use of the above-mentioned antibodies binding ACVR2A and / or ACVR2B or their antigen-binding fragments, bispecific antibodies binding ACVR2A / ACVR2B, immunoconjugates, and pharmaceutical compositions in the preparation of medicaments for the prevention and / or treatment of diseases or conditions.

[0138] This application also provides methods for administering the above-described antibodies or antigen-binding fragments of ACVR2A and / or ACVR2B, bispecific antibodies binding ACVR2A / ACVR2B, immunoconjugates, and pharmaceutical compositions to subjects in need for the prevention and / or treatment of diseases or conditions.

[0139] The above diseases or symptoms are selected from:

[0140] 1) Diseases or conditions associated with ACVR2A, ACVR2B, or their ligands;

[0141] 2) Endocrine and metabolic diseases, tumors, respiratory diseases, skin and musculoskeletal diseases, and nervous system diseases;

[0142] Preferably, the disease or condition is selected from obesity and its metabolic disorders, overweight, cachexia, type 2 diabetes, hip fracture, dyskinesia, muscle atrophy, inclusion body myositis, sarcopenia, myelofibrosis, osteoporosis, chronic obstructive pulmonary disease, pulmonary hypertension, depression, primary myelofibrosis, anemia, chronic kidney disease-mineral and bone disorders, thalassemia, congenital simple erythrocytic aplasia, β-thalassemia, bladder cancer, head and neck tumors, multiple myeloma, myelodysplastic syndrome, small cell lung cancer, non-small cell lung cancer, pancreatic cancer, metastatic solid tumors, and chronic myelomonocytic leukemia. More specifically, muscle atrophy is spinal muscular atrophy; cachexia is cancer cachexia.

[0143] ACVR2A or ACVR2B related diseases or conditions refer to diseases or conditions involving cells or tissues in which ACVR2A or ACVR2B is expressed, overexpressed, or abnormally expressed. ACVR2A or ACVR2B ligand-related diseases or conditions refer to diseases or conditions involving certain cells or tissues in which one or more ACVR2A or ACVR2B ligands are expressed, overexpressed, or abnormally expressed. ACVR2A ligands include activins (e.g., activin A, activin B, activin C), growth / differentiation factors GDF (e.g., GDF1, GDF3, GDF5, GDF6, GDF7, GDF8), bone morphogenetic proteins (BMPs), etc.

[0144] ACVR2A (activin receptor 2A) and ACVR2B (activin receptor 2B) play crucial roles in various physiological processes, including cell growth, differentiation, and tissue repair. These two transmembrane proteins possess cysteine-rich ligand-binding domains and kinase-active catalytic domains. By binding to ligands such as activins, they activate downstream signaling pathways like Smad2 / 3 and MAPK, thereby regulating skeletal muscle mass, lipid metabolism, and embryonic development. Although their functions overlap, differences in expression and ligand affinity in specific tissues allow them to play specific roles in different physiological processes. Due to their importance in muscle and lipid metabolism, ACVR2A and ACVR2B have become potential drug targets for the treatment of muscle atrophy and metabolic diseases, and related drugs are under development. For example, the monoclonal antibody Bimagrumab (i.e., the control antibody BYM338 in this example), targeting ACVR2A and ACVR2B, has shown effects in clinical trials on improving patients' walking ability, increasing muscle mass, reducing fat, and improving metabolic indicators such as blood glucose levels. Its potential indications include obesity, cachexia, type 2 diabetes, hip fracture, metastatic non-small cell lung cancer, dywalking, muscle atrophy, inclusion body myositis, pancreatic cancer, chronic obstructive pulmonary disease, and sarcopenia. The monoclonal antibody LAE-102 (i.e., the control antibody LA01 in this example), targeting ACVR2A, has potential indications including obesity, overweight, metastatic solid tumors, and non-small cell lung cancer. Sotatercept is a marketed fusion protein that forms a ligand trap by fusing the extracellular domain of ACVR2A with the Fc terminus of an antibody, thereby inhibiting the activation of downstream signaling pathways. Its indications include depression, pulmonary hypertension, anemia, chronic kidney disease-mineral and bone disorders, primary myelofibrosis, thalassemia, congenital simple erythroblastic aplastic anemia, β-thalassemia, bladder cancer, chronic myelomonocytic leukemia, head and neck tumors, multiple myeloma, myelodysplastic syndromes, myelofibrosis, osteoporosis, and small cell lung cancer. Depression and pulmonary hypertension have already been approved for marketing.

[0145] In this application, the above-mentioned pharmaceutical composition and method can be used alone or in combination with other methods, such as other targeted drug therapy, chemotherapy drug therapy, and surgical treatment.

[0146] In one embodiment, the prevention or treatment method further includes administration of a second agent comprising a second active molecule, said active molecule being a GLP-1 analog or a GLP-1R agonist; preferably, the second active molecule comprises Semaglutide, Dulaglutide, or Exenatide.

[0147] In this application, the target of administration of the above-described pharmaceutical composition and method can be a mammal, such as, but not limited to, humans, primates, and laboratory test animals (e.g., mice, rabbits, rats, guinea pigs, hamsters). Primates are preferred; humans are more preferred.

[0148] The following will illustrate the specific implementation through examples. When numerical ranges are given in the examples, it should be understood that, unless otherwise stated in this application, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the examples, based on the knowledge of the prior art possessed by those skilled in the art and the description in this application, any prior art methods, equipment, and materials similar to or equivalent to those described, used, and materials in the examples of this application may be used to implement this application. Unless otherwise specified, the instruments, materials, and reagents used in the examples are obtained through conventional means.

[0149] Example 1: Animal Immunization and Preparation and Screening of Hybridomas

[0150] 1.1 Mouse Immunization

[0151] Balb / c mice were immunized with human ACVR2A-His (amino acid sequence as SEQ ID NO:39) and ACVR2B-His (amino acid sequence as SEQ ID NO:40) proteins (purity >95%) expressed in HEK-293F cells. Balb / c mice were initially immunized by subcutaneous injection at multiple sites with a complex of human ACVR2A-His, ACVR2B-His, and a fully Freund's adjuvanted emulsion (50 μg / mouse / 0.5 mL). Subsequently, immunizations were performed every 14 days with a complex of ACVR2A-His, ACVR2B-His, and an incompletely Freund's adjuvanted emulsion (50 μg / mouse / 0.5 mL), for a total of four immunizations. Three weeks later, mice were challenged by intraperitoneal injection of soluble human ACVR2A-His and ACVR2B-His proteins, 50 μg / mouse / 0.2 mL. Three to four days later, the spleens of the mice were harvested for fusion experiments.

[0152] 1.2 Hybridoma cell preparation and screening

[0153] Three to four days after the last immunization of mice, mouse spleen cells were PEG-fused with mouse myeloma cells SP2 / 0. The fused cells were then uniformly suspended in complete culture medium consisting of RPMI 1640-GLUMAX, 1% penicillin-streptomycin, 20% FBS (fetal bovine serum), and 1*HAT. The fused cells were then cultured at a density of 3*102.4 Cells were cultured at 200 μl / well in 60 96-well plates. After 7-12 days, the supernatant was harvested, and wells with positive hybridomas binding to human ACVR2A / B were screened by ELISA.

[0154] The method for screening hybridoma wells with positive human ACVR2A / B binding activity using ELISA is as follows: Human ACVR2A-Fc (amino acid sequence as shown in SEQ ID NO:41) and ACVR2B-Fc (amino acid sequence as shown in SEQ ID NO:42) expressed in HEK-293F cells were diluted to 1 μg / ml with PBS buffer, and 100 μl / well was added to each well of an ELISA plate and incubated overnight at 4°C. The next day, the supernatant was discarded, 5% skim milk powder was added, and the plate was blocked at 37°C for 2 hours. The plate was washed 3 times with PBST and set aside. The collected hybridoma supernatant was added sequentially to the blocked plate, 100 μl / well, and incubated at 37°C for 1 hour. The plate was washed 3 times with PBST, and HRP-labeled goat anti-mouse IgG secondary antibody was added and incubated at 37°C for 30 minutes. After washing 3 times with PBST, residual droplets were patted dry on absorbent paper, and 100 μl of TMB was added to each well. The plate was incubated at room temperature in the dark for 5 minutes. 50 μl of 2M TMB was added to each well. The substrate reaction was terminated with H2SO4 stop solution, and the OD value was read at 450 nm using a microplate reader to analyze the binding ability of the test antibody to the target antigen ACVR2A / B. Hybridoma cell lines amplified and screened in serum-containing complete medium were centrifuged and transferred to serum-free Hybridoma-SFM medium to achieve a cell density of 1–2 × 10⁶ cells / mL. 7 / ml, cultured at 8% CO2 and 37℃ for 1 week, centrifuged to obtain the culture supernatant, purified by Protein G affinity chromatography to obtain monoclonal antibody protein against human ACVR2A / B, and screened by ELISA to obtain a total of 39 hybridoma cell lines.

[0155] 1.3 Binding ability of murine antibodies to human ACVR2A-Fc and ACVR2B-Fc proteins

[0156] The binding ability of mouse antibodies to human ACVR2A and ACVR2B proteins was determined using an indirect enzyme-linked immunosorbent assay (ELISA). The specific method is as follows: ACVR2A-Fc and ACVR2B-Fc proteins were diluted to 1 μg / ml with coating buffer (50 mM carbonate coating buffer, pH 9.6) and coated overnight at 4°C; then blocked with 5% skim milk powder and incubated at 37°C for 2 hours; after washing the plate 3 times with PBST, the laboratory-prepared anti-human ACVR2A / B mouse antibody was serially diluted with 1% BSA buffer, and 100 μl / well was added to the pre-coated ACVR2A-Fc and ACVR2B-Fc plates, respectively, and incubated at 37°C for 1 hour; after washing the plate 3 times with PBST, HRP-labeled goat anti-mouse IgG secondary antibody was added, and the plate was incubated at 37°C for 30 min; after washing the plate 3 times with PBST, residual droplets were patted dry on absorbent paper, 100 μl of TMB was added to each well, and the plate was incubated at room temperature in the dark for 5 min, and then 50 μl of TMB was added to each well. The substrate reaction was terminated with 2MH2SO4 stop solution, and the OD value was read at 450 nm using an ELISA reader. The binding ability of the test antibodies to the antigens human ACVR2A-Fc and ACVR2B-Fc was analyzed. The results showed that most murine antibodies had good binding activity to the target antigen ACVR2B-Fc, while some antibodies also bound to ACVR2A-Fc. Among them, seven murine antibodies: 60B5C4B11, 10E8H3, 133A3C6, 124H1A4, 1E9H1, 11G1G8, and 15A5B4 showed ECG binding to ACVR2B-Fc. 50 Less than 0.2 nM; 12 murine antibodies: 133A3C6, 124H1A4, 18A11G10, 34C2A1, 15B11G3, 11D6E10, 10B6E3, 1E9H1, 40F6F2, 2F12E11, 21F5E12, 23B5A1B3 binding EC50 to ACVR2A-Fc 50 Less than 0.4 nM.

[0157] 1.4 Determination of the blocking activity of murine antibodies against ACVR2A-Fc and ACVR2B-Fc binding to Activin A protein

[0158] In this embodiment, the blocking activity of mouse antibodies against ACVR2A and ACVR2B binding to Activin A protein was determined by enzyme-linked immunosorbent assay (ELISA).

[0159] The control antibody BYM338 used in this experiment has a sequence derived from patent WO2010125003A1, and its heavy chain amino acid sequence is shown in SEQ ID NO: 1; the light chain amino acid sequence of BYM338 is shown in SEQ ID NO: 2.

[0160] In this experiment, ACVR2A-Fc and ACVR2B-Fc were used to coat ELISA plates at a concentration of 100 ng / well. Bio-Activin A protein was prepared by linking a biotin tag to Biotin N-hydroxysuccinimide ester reagent (Sigma). The purified mouse antibody was serially diluted 3-fold from 12 μg / ml in 11 gradients with 1% BSA-PBS, with 1% BSA-PBS serving as a blank control. 100 μl of antibody dilution buffer was mixed with Bio-Activin A protein (final concentration 20 ng / ml, 20 μl / well), and then 100 μl of the mixture was added to the ELISA-coated plate. The plate was incubated at 37°C for 1 h, washed 3 times with PBST, and HRP-labeled streptavidin secondary antibody was added. The plate was incubated at 37°C for 30 min, washed 3 times with PBST, and residual droplets were patted dry on absorbent paper. 100 μl of TMB was added to each well, and the plate was incubated at room temperature in the dark for 5 min. 50 μl of 2M TMB was added to each well. The substrate reaction was terminated with H2SO4 stop solution, and the OD value was read at 450 nm using a microplate reader. The blocking activity of the test antibodies against ACVR2A and ACVR2B binding to Activin A protein was analyzed. The obtained data were fitted and analyzed using GraphPad Prism 10 software. The results showed that all 39 antibodies had the activity of blocking the binding of ACVR2B and Activin A protein; among them, 11 antibodies had the activity of blocking the binding of both ACVR2A and Activin A protein.

[0161] Based on the combined affinity and blocking assay results, it can be determined that 10E8H3 has a good affinity for ACVR2B, and its blocking activity is close to that of BYM338 (IC50 of 10E8H3 and BYM338 is similar). 50 The values ​​are 3.586 nM and 4.865 nM respectively. 133A3C6, 124H1A4, 23B5A1B3, and 1E9H1 can simultaneously block ACVR2A and ACVR2B and have good affinity.

[0162] Example 2: Preparation and Screening of Chimeric Antibodies

[0163] 2.1 Preparation of chimeric antibodies

[0164] In this embodiment, the heavy chain variable region and light chain variable region of hybridomas 1E9H1, 10E8H3, 23B5A1B3, 124H1A4, and 133A3C6 were obtained through relevant molecular biology methods, and chimeric antibodies were further constructed.

[0165] RNA was extracted from five hybridoma cells (1E9H1, 10E8H3, 23B5A1B3, 124H1A4, and 133A3C6) using Trizol, and cDNA was obtained through reverse transcription of mRNA. Subsequently, using cDNA as a template, PCR was performed using degenerate primers for the heavy and light chains of the mouse antibody (Antibody Engineering, Volume 1, Edited by Roland Kontermann and Stefan Dübel; the sequence of the combined primers is from page 323). The obtained PCR products were sequenced and analyzed using the Kabat database to confirm that the obtained sequences were the variable region sequences of the mouse antibody.

[0166] The obtained hybridoma heavy chain variable region sequences were spliced ​​with the human IgG1 constant region (containing LALA mutation) (amino acid sequence as shown in SEQ ID NO: 11), and the light chain variable region sequences were spliced ​​with the human kappa chain constant region (amino acid sequence as shown in SEQ ID NO: 12). The heavy and light chains of each chimeric antibody were constructed into the pcDNA3.4 expression vector (Invitrogen), transfected into HEK-293F cells, and purified by Protein A to obtain each chimeric antibody. The molecular weight of each expressed antibody was determined to be around 150 kDa by SDS-PAGE electrophoresis and SEC-HPLC, and the antibody purity was >95%. The antibodies were quantified, aliquoted, and frozen at -80℃ for later use.

[0167] 2.2 ELISA method was used to determine the binding activity of each chimeric antibody to human ACVR2A-His and ACVR2B-His.

[0168] ACVR2A-His and ACVR2B-His proteins were diluted to 0.3 μg / mL using ELISA coating buffer and coated onto ELISA plates at 100 μL / well. Coating was carried out at 4°C for 16 h. The ELISA plates were washed three times with PBST (PBS containing 0.05% Tween 20), blotted dry, and then blocked at room temperature for 2 h with 200 μL / well of 2% BSA prepared in PBS. The plates were washed once with PBST and blotted dry. Each chimeric antibody was diluted with 1% BSA prepared in PBST at an initial concentration of 100 nM, and serially diluted 3-fold (10 gradients) at 100 μL / well. The plates were incubated at room temperature for 1 h, with each sample in duplicate. The plates were washed three times with PBST and blotted dry. The anti-human antibody was diluted with 1% BSA prepared in PBST at an appropriate ratio. Fc-HRP (Sigma) secondary antibody was added to ELISA plates at 100 μL / well, and incubated at room temperature for 0.5 h. The plates were washed five times with PBST and patted dry. TMB chromogenic buffer was added at 100 μL / well, and the plates were incubated until the desired color depth was reached. The incubation was terminated with 2M H2SO4 at 50 μL / well. All reaction solutions were shaken thoroughly and the OD was measured at 450 nm using a microplate reader. The data were analyzed using GraphPad Prism 10 software to calculate EC50. 50 .

[0169] The experimental results are shown in Figure 1A. Chimeric antibodies 1E9H1-ch, 23B5A1B3-ch, 124H1A4-ch, and 133A3C6-ch effectively bound to human ACVR2A-His, with 133A3C6-ch and 124H1A4-ch showing the strongest affinity for ACVR2A. As shown in Figure 1B, chimeric antibodies 1E9H1-ch, 23B5A1B3-ch, 124H1A4-ch, 133A3C6-ch, and 10E8H3-ch effectively bound to human ACVR2B-His, with affinity levels similar to the control antibody BYM338, and binding to EC... 50 See Table 1.

[0170] Table 1. Binding EC of chimeric antibodies to ACVR2A and ACVR2B 50

[0171] 2.3 Determination of the blocking activity of chimeric antibodies against ACVR2A and ACVR2B binding to Activin A protein

[0172] ACVR2A-His and ACVR2B-His proteins were diluted to 0.3 μg / mL using ELISA coating buffer, and coated onto ELISA plates at 100 μL / well, incubating overnight at 4°C. The ELISA plates were washed three times with PBST and patted dry on absorbent paper. Then, 200 μL / well of 2% BSA (prepared with PBS) was used to block the plates, and the plates were incubated at room temperature for 2 hours. The plates were washed once with PBST and patted dry. Activin A-Biotin protein (purchased from Sinocare and biotinylated) was diluted to 10 nM (final concentration 5 nM) with 1% BSA (prepared with PBST). The antibody was serially diluted 3-fold (10 dilutions total), starting at a maximum concentration of 200 nM (final maximum concentration 100 nM). The diluted antibody was then mixed with an equal volume of 10 nM Activin A-Biotin. A-Biotin protein was thoroughly mixed, then 100 μL / well was added to each ELISA plate, with two replicates per sample. The plates were incubated at room temperature for 1 h. Unbound or non-specifically bound primary antibody was washed with PBST and the plates were patted dry. Following the antibody manufacturer's instructions, SA-HRP(BD) secondary antibody was diluted with 1% BSA prepared with PBST and added to each well at 100 μL / well. The plates were incubated at room temperature for 0.5 h. The plates were washed five times with PBST and patted dry on absorbent paper. TMB chromogenic solution was added to each well, and the plates were developed to the desired color depth. 2M H2SO4 was added to each well to stop the development. The absorbance was measured at 450 nm using a multi-mode microplate reader. The data were analyzed using GraphPad Prism 10 software to calculate the IC50. 50 .

[0173] The experimental results are shown in Figure 2A. Chimeric antibodies 1E9H1-ch, 124H1-1A4-ch, and 133A3C6-ch effectively blocked the binding of Activin A to human ACVR2A-His, with stronger blocking activity than 23B5A1B3-ch and 10E8H3-ch. As shown in Figure 2B, chimeric antibodies 1E9H1-ch, 23B5A1B3-ch, 124H1A4-ch, 133A3C6-ch, and 10E8H3-ch effectively blocked the binding of Activin A to human ACVR2B-His, with 10E8H3-ch exhibiting the strongest blocking activity. The IC50 values ​​for each chimeric antibody were [not specified in the original text]. 50 See Table 2.

[0174] Table 2. IC50 of chimeric antibodies blocking the binding of Activin A to ACVR2A / 2B 50

[0175] 2.4 Detection of the blocking activity of chimeric antibodies against the ACVR2A downstream signaling pathway

[0176] To screen for antibodies that can effectively block the downstream signaling activity of ACVR2A, the inventors introduced a reporter gene (plasmid pCAGA12-TA-Luc, purchased from Beyotime Biotechnology Co., Ltd.) into A-204 (human rhabdomyosarcoma cell line), a cell line with stronger ACVR2A specificity, to obtain the A-204-4B3-Luc cell line. ACVR2A ligands such as Activin A and GDF8 can activate Luciferase expression in the A-204-4B3-Luc cell line by binding to ACVR2A. Using this system, the inventors detected the blocking effect of the antibody on Activin A activation of A-204-4B3-Luc cells.

[0177] Collect and count A-204-4B3-Luc cells in the logarithmic growth phase. Dilute the cells to 1E6 / mL with DMEM + 10% FBS and add 50 μL / well to each well of a 96-well white plate (CORNING). Dilute the target protein with DMEM + 10% FBS to a starting concentration of 10000 nM (final concentration 4000 nM). Perform eight serial dilutions (5-fold each) and add 50 μL / well to each well of the plate. Incubate for 1 hour. After 1 hour, add 25 μL / well of 15 nM Activin diluted with DMEM + 10% FBS. A (final concentration 3 nM), final plate volume 125 μL / well, after pipetting and mixing, incubated in a cell culture incubator for 20 hours; remove the plate and let it sit at room temperature for 15 min, then add 100 μL / well of thawed Bio-glo reagent (Promega) to the wells. After 7 min, read the fluorescence values ​​using a SpectraMax i3 instrument (reading mode LUM, read type Endpoint). The obtained data were fitted and analyzed using GraphPad Prism 10 software to calculate IC50. 50 .

[0178] The experimental results are shown in Figure 3. The chimeric antibody 124H1A4-ch showed stronger inhibition of Activin A stimulation of A-204-4B3-luc cells than 133A3C6-ch; the chimeric antibody 10E8H3-ch had no effect on inhibiting Activin A stimulation of A-204-4B3-luc cells. The IC50 values ​​of each chimeric antibody are shown in Figure 3. 50 See Table 3.

[0179] Table 3. IC50 of chimeric antibodies blocking Activin A activation of A-204-4B3-luc cells 50

[0180] 2.5 Detection of the blocking activity of chimeric antibodies against the ACVR2B downstream signaling pathway

[0181] The H-ACVR2B reporter cell line, purchased from Geneplus Biotechnology, is used to detect the activity of ligand-specific activation of the ACVR2B signaling pathway by knocking out human ACVR2A and overexpressing human ACVR2B. The inventors used this system to detect the blocking effect of various antibodies on the activation of the cellular signaling pathway by Activin A via ACVR2B.

[0182] Collect and count H-ACVR2B reporter cell lines in the logarithmic growth phase. Dilute cells to 1.5E5 / mL with DMEM + 10% FBS, and add 100 μL / well to a 96-well white plate. Incubate overnight for 18 h. Discard the supernatant. Dilute the antibody to be tested with DMEM + 10% FBS medium to an initial concentration of 6000 nM (final concentration 3000 nM). Perform 8 serial dilutions (4-fold) and add 50 μL / well to each cell plate. Incubate in a cell culture incubator. After 1 hour, add 50 μL / well of 0.08 nM Activin A diluted with DMEM + 10% FBS medium (final concentration 0.04 nM). Incubate in a cell culture incubator for 6 hours. Remove the plate and place it at room temperature for 15 min. Then, add 60 μL / well of thawed Bio-glo reagent to the cell wells. After 1 min, incubate in SpectraMax. Fluorescence values ​​were read in the i3 instrument (readout mode LUM, readout type Endpoint). The obtained data were fitted and analyzed using GraphPad Prism 10 software to calculate IC50. 50 .

[0183] The experimental results are shown in Figure 4 and Table 4. The chimeric antibody 10E8H3-ch effectively blocked the activation of H-ACVR2B reporter cells by Activin A, and its blocking activity was stronger than that of 124H1A4-ch. The blocking activity of 133A3C6-ch was the weakest.

[0184] Table 4. IC50 of chimeric antibodies blocking Activin A activation of H-ACVR2B reporter cells 50

[0185] Example 3: Preparation of humanized antibodies

[0186] The amino acid sequences of the light chain variable regions and heavy chain variable regions of each candidate murine antibody were analyzed, and the three antigen complementarity-determining regions (CDRs) and four frame regions (FRs) of the murine antibody were determined according to Kabat rules. Specifically, the amino acid sequences of the heavy chain complementarity-determining regions of 124H1A4 were HCDR1: DTDLH (SEQ ID NO: 13), HCDR2: RIDPANDNTKYDPRFQG (SEQ ID NO: 14), and HCDR3: ETYHGRTYVGCAY (SEQ ID NO: 15), while the amino acid sequences of the light chain complementarity-determining regions were LCDR1: RSSQIILHSNGNTYLE (SEQ ID NO: 16), LCDR2: KVSNRFS (SEQ ID NO: 17), and LCDR3: FQGSHVPYT (SEQ ID NO: 18). The amino acid sequences of the heavy chain complementarity-determining region of 10E8H3 are HCDR1: SYAMS (SEQ ID NO: 19), HCDR2: EISSGGTYTRYPDTVTG (SEQ ID NO: 20), and HCDR3: DYRHAMDY (SEQ ID NO: 21), while the amino acid sequences of the light chain complementarity-determining region are LCDR1: RASESVEYYGTSLMQ (SEQ ID NO: 22), LCDR2: AASNVES (SEQ ID NO: 23), and LCDR3: QQSRKVPRT (SEQ ID NO: 24).

[0187] Humanized templates that best matched the non-FR regions of the aforementioned murine antibodies were selected from the Germline database. The CDR regions of the murine antibodies were then transplanted into the selected humanized templates, replacing the CDR regions of the human templates. The heavy chain variable region was then recombined with the human IgG1 constant region (containing the LALA mutation), and the light chain variable region was recombined with the human kappa chain constant region. Simultaneously, based on the three-dimensional structure of the antibody, reversion mutations were performed on embedded residues, residues that directly interact with the CDR region, and residues that significantly affect the conformation of the VL and VH regions of each antibody, ultimately obtaining multiple humanized antibodies. The heavy and light chains of each humanized antibody were constructed into pcDNA3.4 expression vectors, transfected into HEK-293F cells, and purified using Protein A. The molecular weight and purity (>95%) of each antibody were confirmed by SDS-PAGE electrophoresis and SEC-HPLC.

[0188] Example 4: Screening of humanized antibodies against 124H1A4

[0189] 4.1 ELISA method was used to determine the binding ability of 124H1A4 humanized antibody to human ACVR2A and ACVR2B proteins.

[0190] The binding affinity of each humanized antibody to human ACVR2A and ACVR2B proteins was determined by ELISA, with the experimental methods described in Example 2.2. The control antibody LA01 targeting ACVR2A used in this experiment has a sequence derived from patent WO2023030503A1, with its VH amino acid sequence shown in SEQ ID NO: 36 and its VL amino acid sequence shown in SEQ ID NO: 37.

[0191] The experimental results are shown in Figures 5A, 5B, 6A, 6B and Tables 5 and 6. The six humanized antibodies of 124H1A4 all showed good binding activity to ACVR2A and ACVR2B. The affinity for ACVR2A was comparable to that of the control antibodies BYM338 and LA01, and the affinity for ACVR2B was comparable to that of BYM338. LA01 did not bind to ACVR2B.

[0192] Table 5 ECGs of 124H1A4 humanized antibody bound to ACVR2A and ACVR2B 50 -1

[0193] Table 6. ECGs of affinity between 124H1A4 humanized antibody and ACVR2A and ACVR2B 50 -2

[0194] 4.2 ELISA method was used to determine the blocking activity of 124H1A4 humanized antibody against ACVR2A and ACVR2B binding to Activin A protein.

[0195] The blocking activity of each 124H1A4 humanized antibody against the binding of human ACVR2A and ACVR2B to Activin A protein was determined by ELISA, with the relevant experimental methods referring to Example 2.3. The results of the blocking activity of the 124H1A4 humanized antibody against the binding of human ACVR2A to Activin A protein are shown in Figure 7 and Table 7. The 124H1A4 humanized antibodies can effectively block the binding of ACVR2A to Activin A protein, and the blocking activity is similar to that of the chimeric antibody and the control antibody.

[0196] Table 7. IC50 of 124H1A4 humanized antibody blocking the binding of Activin A to ACVR2A 50

[0197] The blocking activity of the 124H1A4 humanized antibody against the binding of human ACVR2B to Activin A protein is shown in Figure 8 and Table 8. The 124H1A4 humanized antibody can effectively block the binding of ACVR2B to Activin A protein, and the blocking activity is similar to that of 124H1A4-ch.

[0198] Table 8. IC50 of 124H1A4 humanized antibody blocking the binding of Activin A to ACVR2B

[0199] 4.3 Blocking effect of 124H1A4 humanized antibody on the downstream signaling pathway of Activin A protein activation of ACVR2A

[0200] Here, the blocking activity of each 124H1A4 humanized antibody against the activation of downstream pathways in the A-204-4B3-Luc cell line induced by Activin A protein binding to ACVR2A was determined by reporter gene assay. The relevant experimental methods are as described in Example 2.4.

[0201] As shown in Figure 9 and Table 9, 124H-HuH1-L3 and 124H-HuH2-L3 showed relatively strong blocking activity against the activation of downstream signaling pathways in A-204-4B3-Luc cells.

[0202] Table 9. IC50 of 124H1A4 humanized antibody blocking Activin A activation of A-204-4B3-Luc cells 50

[0203] Example 5: Screening of 10E8H3 humanized antibodies

[0204] 5.1 Binding ability of 10E8H3 humanized antibody to human ACVR2B protein

[0205] The binding affinity of each humanized antibody to human ACVR2B protein was determined by ELISA, following the experimental methods described in Example 2.2. The results are shown in Figure 10. All four humanized antibodies effectively bound ACVR2B, exhibiting affinity comparable to BYM338. The EC50 of each antibody was [missing information]. 50 See Table 10.

[0206] Table 10 ECGs of 10E8H3 humanized antibody bound to ACVR2B 50

[0207] 5.2 Blocking activity of 10E8H3 humanized antibody against ACVR2B binding to Activin A protein

[0208] The blocking activity of each 10E8H3 humanized antibody against the binding of human ACVR2B to Activin A protein was determined by ELISA, with the relevant experimental methods referring to Example 2.3. As shown in Figure 11 and Table 11, all 10E8H3 humanized antibodies could effectively block the binding of ACVR2B to Activin A, and the blocking activity was comparable to that of BYM338.

[0209] Table 11 IC50 of 10E8H3 humanized antibody blocking the binding of Activin A to ACVR2B 50

[0210] 5.3 Blocking effect of 10E8H3 humanized antibody on the downstream signaling pathway of Activin A protein activation of ACVR2B

[0211] The blocking activity of each 10E8H3 humanized antibody against the activation of downstream signal transduction of H-ACVR2B reporter cell line by Activin A binding ACVR2B was determined by reporter gene assay. The relevant experimental methods are as described in Example 2.5.

[0212] The results are shown in Figure 12 and Table 12. All four humanized 10E8H3 antibodies can effectively block ACVR2B-mediated signal transduction. Among them, 10E8H-Hu1, 10E8H-Hu1G and 10E8H3-ch are similar to 10E8H-HuGG and 10E8H-HuG1.

[0213] Table 12 IC50 of 10E8H3 humanized antibody blocking Activin A activation of H-ACVR2B reporter cells 50

[0214] Example 6: ACVR2B combined with epitope detection

[0215] This embodiment uses H-ACVR2B reporter cells overexpressing ACVR2B to detect the competition between 10E8H-Hu1 and 124H-HuH1-L3 for the ACVR2B binding epitope. H-ACVR2B reporter cells in logarithmic growth phase were digested and counted, resuspended in PBS containing 1% BSA to 1E6 cells / mL, and added to U-shaped 96-well cell culture plates at 100 μL / well. 10E8H-Hu1-APC antibody (i.e., 10E8H-Hu1 conjugated with the APC-647 fluorescent group) and 124H-HuH1-L3-FITC antibody (i.e., 124H-HuH1-L3 conjugated with the FITC fluorescent group) were diluted with PBS containing 1% BSA to 200 nM (single-drug group) or 400 nM (…). The 10E8H-Hu1-APC and 124H-HuH1-L3-FITC combination groups were prepared by mixing monoclonal antibodies of equal concentration and volume. 100 μL of each antibody dilution solution was added to the cell plate and mixed with the cells. The plate was then incubated at 4 °C for 1 h. The cells were washed three times with PBS and resuspended in 200 μL / well. The fluorescence signals of the cells were then read using a Cytoflex flow cytometer through the FITC and APC channels.

[0216] As shown in Figure 13, 10E8H-Hu1 and 124H-HuH1-L3 can simultaneously bind to the ACVR2B protein on the surface of H-ACVR2B reporter cells, and they do not compete completely.

[0217] Example 7: Preparation and Screening of Bispecific Antibodies

[0218] 7.1 Preparation of bispecific antibodies

[0219] The bispecific antibody 10E8scfv-124H1-124HuL1 is produced by linking the heavy chain (VH) and light chain (VL) of 10E8H-Hu1 together via a linker sequence, then linking the linker to the N-terminus of the heavy chain of 124H-HuH1-L3. This linker is then co-transfected with the light chain of 124H-HuH1-L3 into HEK-293F cells. After purification with Protein A, the molecular weight of the expressed protein was determined to be approximately 201 kDa by SDS-PAGE electrophoresis and SEC-HPLC, with antibody purity >95%. The antibody was quantified, aliquoted, and stored at -80°C for later use. The linker sequence is (G4S)4; the amino acid sequence of the 10E8scfv-124H1-124HuL1 heavy chain is shown in SEQ ID NO: 38, and the amino acid sequence of the light chain is shown in SEQ ID NO: 30.

[0220] 7.2 Binding ability of the bispecific antibody to human ACVR2A-His and ACVR2B-His proteins

[0221] The binding affinity of the bispecific antibody to human ACVR2A and 2B proteins was determined by ELISA, and the relevant experimental methods are as described in Example 2.2.

[0222] The experimental results are shown in Figures 14A and 14B. The bispecific antibody 10E8scfv-124H1-124HuL1 can effectively bind to ACVR2A and ACVR2B. Its affinity is similar to that of its parent antibodies 124H-HuH1-L3 and 10E8H-Hu1, as well as the control antibodies BYM338 and LA01. The EC50 values ​​of each antibody are [not specified in the original text]. 50 See Table 13.

[0223] Table 13 EC5 Affinity of each antibody to ACVR2A and ACVR2B 50

[0224] 7.3 Binding kinetics of the bispecific antibody to human ACVR2A and ACVR2B proteins

[0225] The binding constants, dissociation constants, and equilibrium dissociation constants of the bispecific antibody and each control monoclonal antibody for human ACVR2A and ACVR2B were determined using the surface plasmon resonance (SPR) method. The experimental methods are as follows:

[0226] The fusion protein was obtained using a chip covalently coupled with Protein A. The relevant operating parameters were as follows: fusion protein concentration of 2 μg / mL, contact time of 60 s, flow rate of 10 μL / min, and regeneration contact time of 30 s. The proteins ACVR2A-His or ACVR2B-His were diluted separately using HBS-EP + pH 7.4 + 150 mM NaCl buffer, with a maximum concentration of 250 nM. Nine 2-fold dilutions were performed, with a 0 concentration point set. 6 M guanidine hydrochloride solution was used as the regeneration buffer. The protein was injected onto a Biacore 8K chip with the following parameters: binding time of 300 s, dissociation time of 900 s, flow rate of 30 μL / min, and regeneration contact time of 30 s. The data were analyzed using Biacore 8K Evaluation Software.

[0227] The experimental results are shown in Table 14. The KD value of the bispecific antibody 10E8scfv-124H1-124HuL1 against ACVR2A was 5.09E-10M, which was superior to its two parent antibodies and the control antibodies BYM338 and LA01; the KD value against ACVR2B was 1.18E-9M, which was superior to its two parent antibodies and the control antibody LA01. This indicates that 10E8scfv-124H1-124HuL1 has good binding activity against both ACVR2A and ACVR2B.

[0228] Table 14. Determination of binding constant, dissociation constant, and equilibrium dissociation constant of antibodies against human ACVR2A and ACVR2B.

[0229] 7.4 The blocking effect of bispecific antibodies on the binding of ACVR2A or ACVR2B to Activin A protein

[0230] The blocking activity of the antibody against the binding of human ACVR2A or ACVR2B to Activin A protein was determined by ELISA, and the relevant experimental methods are as described in Example 2.3.

[0231] As shown in Figures 15A and 15B and Table 15, 10E8scfv-124H1-124HuL1 can effectively bind to ACVR2A and ACVR2B and block the binding of ActivinA. Its blocking activity on ACVR2A is comparable to that of the control antibodies BYM338 and LA01, and superior to that of the parent antibody 10E8H-Hu1; on ACVR2B, it is comparable to that of the control antibody BYM338, and superior to that of the parent antibody 124H-HuH1-L3.

[0232] Table 15 shows the IC50 values ​​of each antibody that block the binding of Activin A to ACVR2B or ACVR2A. 50

[0233] Example 8: Bispecific antibodies can simultaneously block ACVR2A and ACVR2B-mediated cell activation.

[0234] 8.1 Detection of the blocking activity of the bispecific antibody against the ACVR2A downstream signaling pathway activated by Activin A protein.

[0235] The blocking effect of each antibody on the activation of downstream signaling pathways in A-204-4B3-Luc cells stimulated by Activin A protein was determined by reporter gene assay. The relevant experimental methods are as described in Example 2.4.

[0236] The experimental results are shown in Figure 16 and Table 16. The activity of 10E8scfv-124H1-124HuL1 in blocking Activin A and activating downstream signaling in A-204-4B3-Luc cells is stronger than that of the control antibody BYM338 and its parent monoclonal antibody, and comparable to that of the control antibody LA01.

[0237] Table 16 IC50 of each antibody blocking the activation of downstream signaling in A-204-4B3-luc cells by Activin A. 50

[0238] 8.2 Detection of the blocking activity of the bispecific antibody against the downstream signaling pathway of Activin A protein activation of ACVR2B

[0239] The blocking activity of each antibody against the downstream signaling pathway of H-ACVR2B reporter cell line (H-ACVR2B cells) associated with Activin A protein activation was determined by reporter gene assay. The relevant experimental methods are as described in Example 2.5.

[0240] The results are shown in Figure 17 and Table 17. The bispecific antibody 10E8scfv-124H1-124HuL1 showed significantly stronger blocking activity against Activin A-activated H-ACVR2B cells than the control antibody LA01 and its parent monoclonal antibody.

[0241] Table 17 shows the IC50 values ​​of each antibody that block Activin A's activation of downstream signaling in H-ACVR2B cells. 50

[0242] 8.3 Detection of simultaneous blockade of ACVR2A and ACVR2B by bispecific antibodies against downstream signaling pathways mediated by Activin A protein activation in cells.

[0243] Activin A Reporter cells (purchased from Jimon Biotechnology) simultaneously express ACVR2A and ACVR2B receptors. Activin A, acting on these cells, can simultaneously activate downstream signaling pathways induced by both ACVR2A and ACVR2B. Here, a reporter gene assay was used to determine whether the bispecific antibody could simultaneously bind to ACVR2A and ACVR2B and inhibit Activin A activation of cells.

[0244] Collect and count Activin A Reporter cells in the logarithmic growth phase. Dilute the cells with DMEM + 10% FBS and add 75 μL / 10,000 cells / well to 96-well white plates and incubate overnight. Dilute the test antibody with DMEM + 10% FBS to an initial concentration of 4000 nM (final concentration 800 nM), serially dilute 10 times in a 6-fold series, and add 25 μL / well to the cell plate and incubate in a cell culture incubator. After 1 hour, add 25 μL / well of Activin A diluted with DMEM + 10% FBS (final concentration 5 ng / mL), gently pat to mix, and incubate in a cell culture incubator for 6 hours. Remove the plate and let it sit at room temperature for 15 min. Then add 80 μL / well of thawed Bio-glo reagent to the cell wells. After 1 min, read the fluorescence values ​​on a SpectraMax i3 instrument (reading mode LUM, read type Endpoint). The obtained data are then processed using a GraphPad Prism analyzer. 10. Software fitting analysis to calculate IC 50 .

[0245] The experimental results are shown in Figure 18 and Table 18. 10E8scfv-124H1-124HuL1 can effectively inhibit the activation of the downstream signaling pathway of Activin A Reporter cell mediated by Activin A binding to ACVR2A and ACVR2B. Its inhibitory activity is comparable to that of the control antibody BYM338 and is significantly better than that of the control antibody LA01 and various parent antibodies.

[0246] Table 18 shows the IC50 values ​​of each antibody blocking the downstream pathways of Activin A activation of ACVR2A and ACVR2B co-expressed cells. 50

[0247] Example 9: Bilateral anti-inflammatory drugs block GDF8 protein activation of ACVR2A or ACVR2B-mediated downstream signaling pathways in cells.

[0248] 9.1 Detection of the blocking activity of the bispecific antibody against the downstream signaling pathway of GDF8 protein activation in ACVR2A cells

[0249] The blocking activity of each antibody against the activation of the GDF8 protein-stimulated A-204-4B3-Luc cell pathway was determined by reporter gene assay. The relevant experimental methods were as described in Example 2.4, where the final concentration of GDF8 protein was 4 nM.

[0250] The results are shown in Figure 19 and Table 19. The bispecific antibody 10E8scfv-124H1-124HuL1 can effectively block the downstream signaling of A-204-4B3-Luc cells activated by GDF8 binding to ACVR2A. Its activity is stronger than that of the control antibody BYM338 and the parent monoclonal antibody, and comparable to that of the control antibody LA01.

[0251] Table 19 IC50 of each antibody in blocking GDF8-activated downstream signaling in A-204-4B3-luc cells 50

[0252] 9.2 Detection of the blocking activity of the double antibody against the downstream signaling pathway activated by GDF8 protein in ACVR2B cells

[0253] The antibody's blocking activity against H-ACVR2B reporter cell line activation associated with GDF8 protein activation was determined by reporter gene assay. The experimental method was the same as in Example 2.5, with the final concentration of GDF8 protein being 15 ng / mL.

[0254] The results are shown in Figure 20 and Table 20. The bispecific antibody 10E8scfv-124H1-124HuL1 showed stronger blocking activity against the downstream signaling pathway of GDF8-activated ACVR2B cells than the control antibody LA01 and each parent monoclonal antibody.

[0255] Table 20 shows the IC50 values ​​of various antibodies blocking GDF8-activated downstream signaling in H-ACVR2B reporter cell lines. 50

[0256] Example 10: Detection of binding epitope competition between bispecific antibodies and ACVR2A and ACVR2B

[0257] In this embodiment, the competitive ELISA method was used to detect the competition between 10E8scfv-124H1-124HuL1 and BYM338 for the binding epitopes of ACVR2A and ACVR2B proteins, respectively. ACVR2A-His or ACVR2B-His protein was diluted to 0.5 μg / mL using ELISA coating buffer and coated onto ELISA plates at 100 μL / well. The plates were then placed in a humidified chamber at 4°C for 16 h. The ELISA plates were washed three times with PBST and blotted dry. Then, 200 μL of 2% BSA (prepared with PBS) was added to each well, and the plates were blocked at room temperature for 2 h. The plates were washed once with PBST and blotted dry. The antibody 10E8scfv-124H1-124HuL1-Biotin (i.e., biotinylated 10E8scfv-124H1-124HuL1) was diluted to 2 nM (final concentration 1 nM) with 1% BSA (prepared with PBST). BYM338 or 10E8scfv-124H1-124HuL1 was then subjected to a 3-fold gradient. The ELISA plate was diluted 11 times, starting at a concentration of 240 nM (final concentration 120 nM). The diluted BYM338 or 10E8scfv-124H1-124HuL1 was mixed with an equal volume of 10E8scfv-124H1-124HuL1-Biotin protein, and 100 μL / well was added to each ELISA well. The plate was incubated at room temperature for 1 hour, with two replicates per sample. The ELISA plate was washed three times with PBST. Following the antibody manufacturer's instructions, SA-HRP was diluted to the appropriate concentration with antibody diluent and added to the ELISA plate at 100 μL / well. The plate was incubated at room temperature for 0.5 hours. The plate was washed five times with PBST, and the ELISA plate was patted dry on absorbent paper to remove excess liquid. TMB chromogenic solution (1:1 mixture of ELISA chromogenic solutions A and B) was added at 100 μL / well. The plate was developed to the desired color depth, and 2M... H2SO4, 50 μL / well, was added to terminate the color development. The absorbance was measured at 450 nm using a multi-functional microplate reader, and the data were analyzed using GraphPad Prism 10 software.

[0258] As shown in Figure 21A, BYM338 was unable to compete with 10E8scfv-124H1-124HuL1-biotin for binding to ACVR2A, indicating that the bispecific antibody 10E8scfv-124H1-124HuL1 does not compete with BYM338 for the ACVR2A binding epitope.

[0259] As shown in Figure 21B, BYM338 was able to compete with 10E8scfv-124H1-124HuL1-biotin for binding to ACVR2B, but its competitive ability was not as strong as that of the bispecific antibody 10E8scfv-124H1-124HuL1 itself. This indicates that there is partial competition between the bispecific antibody 10E8scfv-124H1-124HuL1 and BYM338 for the ACVR2B binding epitope.

[0260] Example 11: Fat Reduction and Muscle Gain Effects in Mice (In vivo Efficacy)

[0261] Based on the good affinity of 10E8scfv-124H1-124HuL1 for ACVR2A and ACVR2B, and its ability to effectively inhibit the biological functions of muscle negative regulators Activin A and GDF8 at the cellular level, the inventors used CB17 SCID mice as an animal model to investigate whether 10E8scfv-124H1-124HuL1 has a muscle-building and fat-reducing effect. The experimental methods are as follows:

[0262] Eighty CB17 SCID mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.), weighing 20-22g, were randomly divided into four groups of 10 mice each. G1 served as the blank control group. The other test groups included: G2 (BYM338 administration group, 20mg / kg); G3 (LA01 administration group, 20mg / kg); and G4 (10E8scfv-124H1-124HuL1 administration group, 28mg / kg). All groups received subcutaneous injections twice weekly for a total of eight administrations. After the eighth administration, whole blood was collected via cardiac puncture under anesthesia, and the animals were euthanized. The quadriceps femoris, tibialis anterior, digitorum longus, gastrocnemius, soleus, and pectoralis major muscles, as well as inguinal fat, brown fat, epididymal fat, perirenal fat, retroperitoneal fat, and mesenteric fat (including the mesentery) were dissected, weighed, and their contents recorded. The data obtained after normalizing the tissue data and mouse body weight were analyzed by fitting using GraphPad Prism 10 software.

[0263] The experimental results are shown in Figures 22A, 22B, and 22C. Compared with the blank control group, 10E8scfv-124H1-124HuL1 significantly increased the proportion of brown adipose tissue, decreased the proportion of white adipose tissue (total weight of inguinal fat, epididymal fat, perirenal fat, retroperitoneal fat, and mesenteric fat), and significantly increased total muscle mass (total weight of left and right quadriceps femoris, left and right tibialis anterior, digitorum longus, left and right gastrocnemius, soleus, and pectoralis major muscles). Compared with the control antibody group, the increased proportion of brown adipose tissue in the 10E8scfv-124H1-124HuL1 group was similar to that in the BYM338 group; the reduction in white adipose tissue by 10E8scfv-124H1-124HuL1 was lower than that of BYM338 but higher than that of LA01; the increase in total muscle mass by 10E8scfv-124H1-124HuL1 was similar to that of BYM338 but significantly higher than that of LA01. This demonstrates that the 10E8scfv-124H1-124HuL1 bispecific antibody of the present invention can effectively increase muscle and brown adipose tissue content and decrease white adipose tissue content in animals.

[0264] Example 12 Optimization of Bispecific Antibody Half-Life Extension

[0265] 12.1 Modification and Acquisition of Dual Antibody Molecules

[0266] To prolong the half-life of the 10E8scfv-124H1-124HuL1 molecule in vivo and extend the dosing interval, the inventors utilized molecular cloning technology to mutate the Fc region (EU number) of the bispecific antibody molecule into M252Y, S254T, and T256E (YTE) molecules, obtaining the 10E8scfv-124H1-124HuL1-YTE bispecific antibody molecule. The demutated heavy chain was constructed into the pcDNA3.4 expression vector, and co-transfected with its respective light chain into HEK-293F cells. After purification with Protein A, the expressed protein molecular weight was determined to be approximately 201 kDa by SDS-PAGE electrophoresis and SEC-HPLC, with antibody purity >95%. The antibody was quantified, aliquoted, and stored at -80°C for later use.

[0267] 12.2 Determination of the affinity of the bispecific antibody molecule for FcRn

[0268] The affinity of the antibody for FcRn was determined by ELISA. The experimental method is as follows:

[0269] Dilute proteins SSS67 and SSS67(no YTE) to 0.5 μg / mL using ELISA coating buffer, then add 100 μL / well to each ELISA plate and incubate overnight at 4°C. The next day, wash the ELISA plate three times with PBST at pH 7.4 and blot dry. Then add 200 μL / well of 2% BSA prepared with PBST at pH 7.4 and block at room temperature for 1–2 h. Wash the ELISA plate once with PBST at pH 7.4 and blot dry. Dilute the biotinylated FcRn-his protein in PBST containing 1% BSA at pH 7.4 in a 3-fold serial gradient, with a maximum concentration of 20000 nM, for 12 dilutions. Add 100 μL / well to each well and incubate at room temperature for 1 h. After washing the ELISA plate with PBST at pH 7.4 to remove unbound or non-specifically bound proteins, blot dry. Dilute the Streptavidin HRP secondary antibody to the appropriate concentration with PBST at pH 7.0 and 1% BSA according to the manufacturer's instructions. Add 100 μL / well to the ELISA plate and incubate at room temperature for 0.5 h. Wash the ELISA plate three times with PBST at pH 7.4 and blot dry. Add 100 μL / well of TMB chromogenic solution and develop to the desired color depth. Then, add 50 μL / well of 2M H2SO4 to stop the development. After termination, measure the absorbance at 450 nm using a multi-microplate reader and analyze the data using a GraphPad Prism 10. The binding assay at pH 6.0 is performed in the same manner as the binding assay at pH 7.4, except that the PBST at pH 7.4 is replaced with PBST at pH 6.0.

[0270] The experimental results are shown in Figure 23 and Table 21. At pH 7.4, the affinities of 10E8scfv-124H1-124HuL1-YTE and 10E8scfv-124H1-124HuL1 with FcRn are similar. At pH 6.0, the affinity of 10E8scfv-124H1-124HuL1-YTE for FcRn is approximately 3.3 times stronger than that of 10E8scfv-124H1-124HuL1. The addition of the YTE mutation results in a stronger affinity for FcRn at pH 6.0; therefore, 10E8scfv-124H1-124HuL1-YTE may have a longer half-life in vivo compared to 10E8scfv-124H1-124HuL1.

[0271] Table 21. Binding EC of each antibody to FcRn at different pH values. 50

[0272] Example 13: Fat Reduction and Muscle Building Effects in Crab-Eating Mammals; In Vivo Pharmacological Efficacy

[0273] Studies have shown significant differences in the distribution of ACVR2A and ACVR2B ligands between mice and primates. Specifically, GDF8, which is biased towards ACVR2B, is distributed much more abundantly in mice than in primates, while Activin A, which has a direct effect on fat, is expressed and distributed at much higher levels in primates than in mice. Therefore, the inventors compared the fat-reducing and muscle-building effects of 10E8scfv-124H1-124HuL1-YTE and BYM338 in cynomolgus monkeys.

[0274] Nine cynomolgus macaques were randomly divided into three groups of three based on their DEXA and body weight results. Group G1 was the solvent control group, Group G2 was the BYM338 administration group (20 mg / kg), and Group G3 was the 10E8scfv-124H1-124HuL1-YTE administration group (28 mg / kg). All groups received intravenous infusions once a week for a total of four administrations. Body fat scans were performed using DEXA before administration and on days 14, 28, and 35. Adiponectin levels were measured before administration and on day 35.

[0275] As shown in Figure 24, the body weight of the animals in the Vehicle group remained relatively stable during the experiment. Both BYM338 and SSS67 showed an increase in body weight, with BYM338 showing the largest increase on Day 28, an increase of 8.32% ± 2.95% compared to before administration (Day 0). SSS67 showed the largest increase in body weight on Day 35, an increase of 7.40% ± 6.62% compared to before administration (Day 0).

[0276] As shown in Figures 25A and 25B, the body fat index in the Vehicle group remained stable during the experiment. After administration, compared to the Vehicle group, the positive control group (BYM338) showed no significant change in total body fat content, but an increase in total body muscle content (Day 35, p<0.05). In the test substance SSS67 group, compared to the Vehicle group, the animals showed a decrease in total body fat content (Day 14, p<0.05) and a significant increase in total body muscle content (Day 14, Day 28, and Day 35, p<0.05). Compared to the positive control group (BYM338), the trends in various indicators in the test substance SSS67 group were more stable and consistent, with a more pronounced increase in muscle and decrease in body fat.

[0277] As shown in Figure 26, after drug administration, serum adiponectin levels in the Vehicle and control drug BYM338 decreased compared to pre-drug levels, while adiponectin levels in the SSS67 group increased. Day 35 data showed that the percentage changes in adiponectin levels in the Vehicle, BYM338, and SSS67 groups compared to pre-drug levels were -25.97% ± 21.52%, -32.98% ± 22.40%, and 25.42% ± 37.11%, respectively.

[0278] Example 14: The effect of weakening ACVR2B blocking on reducing adverse reactions

[0279] 14.1 Attenuating ACVR2B blocking effects can reduce liver toxicity caused by co-administration with semaglutide.

[0280] Using a high-fat diet-induced DIO (Diabetic Osteoproliferative Disorder) mouse model, this study investigated the synergistic effects of 10E8scfv-124H1-124HuL1 and semaglutide on fat reduction, muscle preservation, and toxicity in DIO obese mice. The experimental design included a normal mouse group, a blank control group, and a treatment group (n=8). In the treatment groups, 10E8scfv-124H1-124HuL1 and BYM338 were administered subcutaneously at doses of 28 mg / kg and 20 mg / kg (equimolar doses), respectively, twice weekly. Semaglutide was also administered subcutaneously once daily at 30 nmol / kg. At the experimental endpoint, all mice were euthanized, and the maximum amount of serum collected was determined. Mouse fat (epididymal fat, mesenteric fat, inguinal fat, retroperitoneal fat, brown fat), muscle (tibialis anterior, gastrocnemius, extensor digitorum longus, quadriceps femoris), liver, and heart tissue were separated and weighed. Blood biochemistry (ALT, AST, ALP, LDH) tests were performed on them.

[0281] The results of liver weight analysis are shown in Figure 27. 1) Compared with DIO and Vehicle sc, the test sample BYM338,20mpk,sc (P<0.05) and the combination of BYM338,20mpk,sc and Semaglutide,30nmol / kg,sc significantly increased liver weight (P<0.001). 10E8scfv-124H1-124HuL1 also increased liver weight, but the difference was not statistically significant. 4HuL1,28mpk,sc+Semaglutide,30nmol / kg,sc did not significantly increase liver weight, while Semaglutide,30nmol / kg,sc significantly decreased liver weight; 2) Compared with Semaglutide,30nmol / kg,sc, the test sample BYM338,20mpk,sc+Semaglutide,30nmol / kg,sc significantly increased liver weight and the increase exceeded that of DIO vehicle.

[0282] The heart weight results from the autopsy are shown in Figure 28. Compared with the DIO, Vehicle, sc group, the heart weight of the group treated with semaglutide, 30 nmol / kg, sc alone was significantly reduced. The combination of BYM338, 20 mpk, sc + semaglutide, 30 nmol / kg, sc or 10E8scfv-124H1-124HuL1, 28 mpk, sc + semaglutide, 30 nmol / kg, sc could salvage the heart weight loss induced by semaglutide. Compared with semaglutide, 30 nmol / kg, sc, the test sample BYM338, 20 mpk, sc + semaglutide, 30 nmol / kg, sc significantly increased heart weight. 10E8scfv-124H1-124HuL1, 28 mpk, sc + semaglutide, 30 nmol / kg, sc also increased heart weight, but the difference was not statistically significant.

[0283] The total muscle weight results from the dissection are shown in Figure 29. Compared with Semaglutide, 30 nmol / kg, sc, 10E8scfv-124H1-124HuL1, 28mpk, sc + Semaglutide, 30 nmol / kg, sc was more effective in reversing muscle loss than the test sample BYM338, 20mpk, sc + Semaglutide, 30 nmol / kg, sc, but the difference was not statistically significant.

[0284] The results of the total white fat weight analysis are shown in Figure 30. Compared with Semaglutide, 30 nmol / kg, sc, the test samples BYM338, 20 mpk, sc + Semaglutide, 30 nmol / kg, sc and 10E8scfv-124H1-124HuL1, 28 mpk, sc + Semaglutide, 30 nmol / kg, sc significantly reduced the total white fat weight. Among them, the test sample BYM338, 20 mpk, sc + Semaglutide, 30 nmol / kg, sc reduced the white fat weight more effectively than the test sample 10E8scfv-124H1-124HuL1, 28 mpk, sc + Semaglutide, 30 nmol / kg, sc.

[0285] The biochemical results, as shown in Figures 31A-D, indicate that compared to the model group, the combination therapy group (BYM338+semaglutide) significantly increased serum ALT, AST, ALP, and LDH levels, while the other test groups showed no significant changes in ALT, AST, ALP, and LDH levels. This suggests that the combination therapy of BYM338+semaglutide may cause liver toxicity, while 10E8scfv-124H1-124HuL1+semaglutide did not exhibit this toxicity.

[0286] 14.2 Reducing ACVR2B blocking effect can decrease adverse reactions in C57BL / 6J.

[0287] In this experiment, 72 C57BL / 6J mice, half male and half female, were randomly divided into 6 groups according to sex and body weight. The weight of male animals was 21.26-23.25g and that of female animals was 17.64-19.92g at the time of grouping. Groups 1 and 4 were vehicle control groups (administered with 12.50mL / kg PBS solution), groups 2 and 5 were 10E8scfv-124H1-124HuL1 dose groups (80mg / kg), and groups 3 and 6 were BYM338 dose groups (57mg / kg). The 10E8scfv-124H1-124HuL1 dose groups and the BYM338 dose groups were equimolar doses. The drug was administered via slow bolus injection into the tail vein twice a week for a total of 13 administrations (on D1, D4, D8, D11, D15, D18, D22, D25, D29, D32, D36, D39, and D43, respectively). Surviving animals in groups 1 to 3 were administered 5 times and dissected on D16, while surviving animals in groups 4 to 6 were administered 13 times and dissected on D44.

[0288] Under the conditions of this experiment, C57BL / 6J mice were repeatedly administered intravenous injections of 80 mg / kg of 10E8scfv-124H1-124HuL1 and 57 mg / kg of BYM338 (both at equimolar doses). All animals survived to the planned dissection, with no animals showing signs of mortality or death. 10E8scfv-124H1-124HuL1 and BYM338 had no significant effects on the general condition, food intake, or blood biochemistry of C57BL / 6J mice, and no abnormalities related to the administration of the test substances were found during gross necropsy.

[0289] During the experiment, the body weight of both male and female animals in all groups, including the solvent control group, showed a continuous increasing trend. The body weight increase trend of both male and female animals in the two test sample groups was higher than that of the solvent control group during the same period, and there was a statistical difference (P<0.05). Among them, the body weight increase of both male and female animals in the BYM338 dose group was higher than that of both male and female animals in the 10E8scfv-124H1-124HuL1 dose group.

[0290] Compared with the solvent control group, at D16, the liver weight of male animals in the 10E8scfv-124H1-124HuL1 group was significantly increased (p<0.05); the heart and liver weight (and / or organ-to-body ratio, organ-to-brain ratio) of both male and female animals in the BYM338 group were significantly increased (p<0.05), the spleen weight and organ-to-brain ratio of female animals were significantly increased (p<0.01), and the uterine weight (and organ-to-body ratio, organ-to-brain ratio) of female animals were significantly decreased (p<0.05). At day 44, in the 10E8scfv-124H1-124HuL1 group, the weight of the heart and liver (towards the visceral-brain ratio) was significantly increased in male animals (p < 0.05), and the weight of the testes (towards the visceral-body ratio and the visceral-brain ratio) was significantly decreased in male animals (p < 0.05). In the BYM338 group, the weight of the heart and liver (towards the visceral-brain ratio) was significantly increased in male animals (p < 0.05), and the weight of the testes (towards the visceral-body ratio and the visceral-brain ratio) was significantly decreased in male animals (p < 0.01), while the weight of the liver in female animals was significantly increased (p < 0.01); the weight of the uterus showed a significant decreasing trend. Compared with the equimolar dose of the 10E8scfv-124H1-124HuL1 group, the BYM338 group showed a greater increase in heart and liver weight and a greater decrease in testicular and uterine weight. Adverse drug findings related to 10E8scfv-124H1-124HuL1 mainly included increased liver weight and corresponding histopathological diffuse hepatocyte hypertrophy; adverse drug findings related to BYM338 included increased heart, liver, and spleen weight, decreased testicular and uterine weight, and corresponding histopathological examinations showing diffuse hepatocyte hypertrophy and periportal hepatocyte vacuolation, increased extramedullary hematopoiesis in the spleen's red pulp, and uterine atrophy.

[0291] Comprehensive evaluation showed that the pathological target organ after administration of the test product 10E8scfv-124H1-124HuL1 was the liver, while the pathological target organs after administration of the test product BYM338 were the liver, spleen, and uterus. Compared to BYM338, equimolar doses of 10E8scfv-124H1-124HuL1 had significantly milder adverse effects on the heart, liver, and reproductive system organs.

Claims

1. An antibody or antigen-binding fragment thereof that binds ACVR2A and / or ACVR2B, characterized in that, Its heavy chain variable region contains HCDR1-3, the amino acid sequences of which are shown in SEQ ID NO:13-15 or SEQ ID NO:19-21.

2. The binding antibody or its antigen-binding fragment as described in claim 1, characterized in that, It also includes the light chain variable region LCDR1-3, the amino acid sequence of which is shown in SEQ ID NO:16-18 or SEQ ID NO:22-24.

3. The antibody or its antigen-binding fragment as described in claim 1 or 2, characterized in that, 1) The heavy chain variable region is selected from SEQ ID NO:4, 8, 25, 28, 29, 31, 32, 34; and / or 2) The light chain variable region is selected from SEQ ID NO: 6, 10, 26, 27, 30, 33, 35; and / or 3) The heavy chain variable region is selected from amino acid sequences having at least 99%, 95%, 90%, 85%, or 80% identity with SEQ ID NO:4, 8, 25, 28, 29, 31, 32, and 34; and the light chain variable region is selected from amino acid sequences having at least 99%, 95%, 90%, 85%, or 80% identity with SEQ ID NO:6, 10, 26, 27, 30, 33, and 35. Preferably, the antibody or its antigen-binding fragment further includes a constant region.

4. An antibody or antigen-binding fragment thereof that binds ACVR2A and / or ACVR2B, characterized in that, It competes with the antibody or its antigen-binding fragment as described in any one of claims 1-3; preferably, it competes with the antibody or its antigen-binding fragment comprising HCDR1-3 of SEQ ID NO:13-15 and LCDR1-3 of SEQ ID NO:16-18 for binding to ACVR2A.

5. The antibody or its antigen-binding fragment as described in claim 1 or 2, characterized in that, The antibody or its antigen-binding fragment is selected from full-length antibodies, nanobodies, single-chain antibodies, biantibodies, scFv, Fv, Fd, Fab, F(ab')2 or F(ab'); and / or, the antibody or its antigen-binding fragment is selected from murine, chimeric or humanized sources.

6. A bispecific antibody binding ACVR2A / ACVR2B, characterized in that, The antibody or antigen-binding fragment thereof that binds ACVR2A and / or ACVR2B as described in any one of claims 1-5 is preferably linked to the antibody or antigen-binding fragment thereof that binds ACVR2A and the antibody or antigen-binding fragment thereof that binds ACVR2B by a linker sequence, and more preferably, the linker sequence is (G4S)4.

7. The bispecific antibody as described in claim 6, characterized in that, It contains a heavy chain with an amino acid sequence as shown in SEQ ID NO: 38 and a light chain with an amino acid sequence as shown in SEQ ID NO:

30.

8. The antibody or antigen-binding fragment thereof binding ACVR2A and / or ACVR2B as described in any one of claims 1-5, and the bispecific antibody binding ACVR2A / ACVR2B as described in claim 6 or 7, characterized in that, It has any of the following characteristics: increased muscle mass, increased brown or adipose tissue mass, or decreased white tissue mass.

9. A polynucleotide molecule, characterized in that, The polynucleotide molecule encodes the antibody or its antigen-binding fragment as described in any one of claims 1-5, or the bispecific antibody as described in claim 6 or 7.

10. An expression carrier, characterized in that, The expression vector contains the polynucleotide molecule as described in claim 9.

11. A host cell, characterized in that, The host cell contains the expression vector as described in claim 10 or its genome integrated with the polynucleotide as described in claim 9.

12. A method for preparing an antibody or antigen-binding fragment thereof binding to ACVR2A and / or ACVR2B, or a bispecific antibody binding to ACVR2A / ACVR2B, characterized in that, Including the following steps: 1) Culturing the host cells of claim 11 to obtain a culture containing the antibody or its antigen-binding fragment or bispecific antibody; 2) Isolate or recover the antibody or its antigen-binding fragment or bispecific antibody from the culture; 3) Optionally, purify and / or modify the antibody or its antigen-binding fragment or bispecific antibody obtained in step 2).

13. An immunoconjugate, characterized in that, The immunoconjugate contains: 1) An antibody or antigen-binding fragment thereof binding to ACVR2A and / or ACVR2B as described in any one of claims 1-5, or a bispecific antibody binding to ACVR2A / ACVR2B as described in claim 6 or 7; and 2) Selected conjugates from the following groups: detectable markers, drugs, toxins, cytokines, carrier proteins, radionuclides, enzymes, or combinations thereof.

14. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises: an antibody or antigen-binding fragment thereof that binds ACVR2A and / or ACVR2B as described in any one of claims 1-5, or a bispecific antibody that binds ACVR2A / ACVR2B as described in claim 6 or 7, or an immunoconjugate as described in claim 13.

15. The pharmaceutical composition according to claim 14, characterized in that, It also includes a second active molecule, which is a GLP-1 analog or a GLP-1R agonist; preferably, the second active molecule includes Semaglutide, Dulaglutide, or Exenatide.

16. The use of an antibody or antigen-binding fragment thereof binding ACVR2A and / or ACVR2B as described in any one of claims 1-5, or a bispecific antibody binding ACVR2A / ACVR2B as described in claim 6 or 7, or an immunoconjugate as described in claim 13, or a pharmaceutical composition as described in claim 14, for the prevention and / or treatment of a disease or condition selected from: 1) Diseases or conditions associated with ACVR2A, ACVR2B, or their ligands; 2) Endocrine and metabolic diseases, tumors, respiratory diseases, skin and musculoskeletal diseases, and nervous system diseases; Preferably, the disease or condition is selected from obesity and its metabolic disorders, overweight, cachexia, type 2 diabetes, hip fracture, dysphagia, muscle atrophy, inclusion body myositis, sarcopenia, myelofibrosis, osteoporosis, chronic obstructive pulmonary disease, pulmonary hypertension, depression, primary myelofibrosis, anemia, chronic kidney disease-mineral and bone disease, thalassemia, congenital simple erythroblastic aplasia, β-thalassemia, bladder cancer, head and neck tumors, multiple myeloma, myelodysplastic syndrome, small cell lung cancer, non-small cell lung cancer, pancreatic cancer, metastatic solid tumors, and chronic myelomonocytic leukemia.

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