Activin receptor ii antibody and use

Abstract

The present application relates to an activin receptor II antibody and a use. The activin receptor II antibody comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3. A mutation relative to CDRs of a parental antibody includes one or more selected from the following: a heavy chain CDR2 V54A mutation, a heavy chain CDR2 G56L mutation, a heavy chain CDR2 T58K mutation, a heavy chain CDR3 G99S mutation, a heavy chain CDR3 G99T mutation, a light chain CDR1 G24H mutation, a light chain CDR1 D28W mutation, a light chain CDR1 Y32F mutation, a light chain CDR2 P57E mutation, a light chain CDR3 S97A mutation, and a light chain CDR3 Y99N mutation. A balanced activin receptor II dual-blocking antibody that exhibits higher affinity for ActRIIA while not significantly reducing affinity for ActRIIB is obtained by screening.

Description

An activin receptor II antibody and its application Technical Field

[0001] This application relates to the field of biomedicine, specifically to an activin receptor II antibody and its applications. Background Technology

[0002] Myostatin and activin, members of the transforming growth factor β (TGFβ) superfamily, promote muscle protein degradation, inhibit protein production, and affect the differentiation and proliferation of myocytes and stellate cells by activating the receptor complex of activin receptor II (ActRII, including ActRIIA and ActRIIB) and activin receptor-like kinase, thereby causing muscle loss.

[0003] ActRII blockers (such as antibody blockers) have shown significant increases in muscle mass in clinical practice. Bimagrumab, a monoclonal antibody, blocks the ActRIIA / B signaling pathway by binding to both ActRIIA and ActRIIB, with significantly higher affinity for ActRIIB than for ActRIIA. Compared to blocking only ActRIIA or ActRIIB, simultaneous blocking of both receptors maximizes the muscle-building effect of ActRII blockers. Developing a blocking antibody with higher affinity for ActRIIA without significantly reducing affinity for ActRIIB would provide a more balanced blockade of both receptors, potentially leading to better muscle-building effects from ActRII blockers.

[0004] Invention Overview

[0005] This application provides an activin receptor II antibody and its application. The inventors screened and obtained a balanced activin receptor II dual-blocking antibody with higher affinity for ActRIIA while not significantly reducing affinity for ActRIIB. The balanced activin receptor II dual-blocking antibody exhibits superior muscle-building and / or fat-loss effects compared to Bimagrumab.

[0006] A first aspect of this application provides an activin receptor II antibody or an antigen-binding fragment thereof, comprising heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3, wherein one or more CDRs of said heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 are mutated relative to their parent antibody CDRs, and the mutations relative to the parent antibody CDRs include one or more selected from the following:

[0007] Heavy chain CDR2 V54A mutation, heavy chain CDR2 G56L mutation, heavy chain CDR2 T58K mutation, heavy chain CDR3 G99S mutation, heavy chain CDR3 G99T mutation, light chain CDR1 G24H mutation, light chain CDR1 D28W mutation, light chain CDR1 Y32F mutation, light chain CDR2 P57E mutation, light chain CDR3 S97A mutation, and light chain CDR3 Y99N mutation;

[0008] The heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2 and light chain CDR3 of the parent antibody are shown in the amino acid sequences of SEQ ID NO:1, 5, 21, 24, 37 and 41, respectively.

[0009] The heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 of the activin receptor II antibody or its antigen-binding fragment and its parent antibody are divided according to the Kabat rule.

[0010] In some embodiments, the activin receptor II antibody or its antigen-binding fragment comprises a heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 selected from the following:

[0011] Heavy chain CDR1, as shown in the amino acid sequence of SEQ ID NO:1;

[0012] Heavy chain CDR2, as shown by the amino acid sequence selected from SEQ ID NO:7 and SEQ ID NO:10;

[0013] Heavy chain CDR3, as shown by the amino acid sequences selected from SEQ ID NO:22 and SEQ ID NO:23;

[0014] The light chain CDR1, as shown by the amino acid sequences selected from SEQ ID NO:25, SEQ ID NO:26 and SEQ ID NO:27;

[0015] The light chain CDR2 is shown in the amino acid sequence of SEQ ID NO:38;

[0016] The light chain CDR3 is shown in the amino acid sequences selected from SEQ ID NO:41, SEQ ID NO:42 and SEQ ID NO:43.

[0017] In some embodiments, the above-mentioned activin receptor II antibody or its antigen-binding fragment comprises:

[0018] Heavy chain CDR1 as shown in SEQ ID NO:1, heavy chain CDR2 as shown in SEQ ID NO:7, heavy chain CDR3 as shown in SEQ ID NO:22, light chain CDR1 as shown in SEQ ID NO:27, light chain CDR2 as shown in SEQ ID NO:38, and light chain CDR3 as shown in SEQ ID NO:43;

[0019] Or, as shown in SEQ ID NO:1, heavy chain CDR1; as shown in SEQ ID NO:7, heavy chain CDR2; as shown in SEQ ID NO:22, heavy chain CDR3; as shown in SEQ ID NO:25, light chain CDR1; as shown in SEQ ID NO:38, light chain CDR2; and light chain CDR3 as shown in SEQ ID NO:42.

[0020] Or, as shown in SEQ ID NO:1, heavy chain CDR1; as shown in SEQ ID NO:10, heavy chain CDR2; as shown in SEQ ID NO:23, heavy chain CDR3; as shown in SEQ ID NO:27, light chain CDR1; as shown in SEQ ID NO:38, light chain CDR2; and light chain CDR3 as shown in SEQ ID NO:41.

[0021] Or, as shown in SEQ ID NO:1, heavy chain CDR1; as shown in SEQ ID NO:7, heavy chain CDR2; as shown in SEQ ID NO:22, heavy chain CDR3; as shown in SEQ ID NO:25, light chain CDR1; as shown in SEQ ID NO:38, light chain CDR2; and light chain CDR3 as shown in SEQ ID NO:43.

[0022] Alternatively, heavy chain CDR1 as shown in SEQ ID NO:1, heavy chain CDR2 as shown in SEQ ID NO:7, heavy chain CDR3 as shown in SEQ ID NO:22, light chain CDR1 as shown in SEQ ID NO:26, light chain CDR2 as shown in SEQ ID NO:38, and light chain CDR3 as shown in SEQ ID NO:43.

[0023] In some embodiments, the activin receptor II antibody or its antigen-binding fragment comprises heavy chain framework regions FR1, FR2, FR3, FR4 and light chain framework regions FR1, FR2, FR3, FR4, wherein:

[0024] The heavy chain frame region FR1 is shown in SEQ ID NO:44;

[0025] And / or, the heavy chain frame region FR2 is as shown in SEQ ID NO:45;

[0026] And / or, the heavy chain frame region FR3 is as shown in SEQ ID NO:46;

[0027] And / or, the heavy chain frame region FR4 is as shown in SEQ ID NO:47;

[0028] And / or, the light chain frame region FR1 is as shown in SEQ ID NO:48;

[0029] And / or, the light chain frame region FR2 is as shown in SEQ ID NO:49;

[0030] And / or, the light chain framework region FR3 is as shown in SEQ ID NO:50;

[0031] And / or, the light chain frame region FR4 is as shown in SEQ ID NO:51.

[0032] In some embodiments, the above-mentioned activin receptor II antibody or its antigen-binding fragment comprises:

[0033] The heavy chain variable region having at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:54, and the light chain variable region having at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:59;

[0034] Alternatively, a heavy chain variable region having at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:54, and a light chain variable region having at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:56;

[0035] Alternatively, a heavy chain variable region having at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:55, and a light chain variable region having at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:57;

[0036] Alternatively, a heavy chain variable region having at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:54, and a light chain variable region having at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:58;

[0037] Alternatively, a heavy chain variable region having at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:54, and a light chain variable region having at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:60.

[0038] In some embodiments, the above-mentioned activin receptor II antibody or its antigen-binding fragment:

[0039] Its heavy chain variable region is shown in SEQ ID NO:54, and its light chain variable region is shown in SEQ ID NO:59;

[0040] Alternatively, its heavy chain variable region is shown in SEQ ID NO:54, and its light chain variable region is shown in SEQ ID NO:56;

[0041] Alternatively, its heavy chain variable region is shown in SEQ ID NO:55, and its light chain variable region is shown in SEQ ID NO:57;

[0042] Alternatively, its heavy chain variable region is shown in SEQ ID NO:54, and its light chain variable region is shown in SEQ ID NO:58;

[0043] Alternatively, its heavy chain variable region is shown as SEQ ID NO:54, and its light chain variable region is shown as SEQ ID NO:60.

[0044] In some embodiments, the activin receptor II antibody described above includes a heavy chain constant region and a light chain constant region, wherein:

[0045] The amino acid sequence of the heavy chain constant region is shown as SEQ ID NO:52 or as shown as SEQ ID NO:71;

[0046] And / or, the amino acid sequence of the constant region of the light chain is as shown in SEQ ID NO:53.

[0047] In some embodiments, the above-mentioned activin receptor II antibody comprises:

[0048] Heavy chain sequences as shown in SEQ ID NO:63 and light chain sequences as shown in SEQ ID NO:69;

[0049] Or, as shown in SEQ ID NO:63, the heavy chain sequence, and as shown in SEQ ID NO:66, the light chain sequence;

[0050] Or, as shown in SEQ ID NO:64, the heavy chain sequence, and as shown in SEQ ID NO:67, the light chain sequence;

[0051] Or, as shown in SEQ ID NO:65, the heavy chain sequence, and as shown in SEQ ID NO:68, the light chain sequence;

[0052] Alternatively, the heavy chain sequence as shown in SEQ ID NO:63, and the light chain sequence as shown in SEQ ID NO:70.

[0053] In some implementations, the aforementioned activin receptor II antibody is an scFv antibody, a Fab antibody, a monospecific antibody, a bispecific antibody, or a trispecific antibody.

[0054] A second aspect of this application also provides an isolated polynucleotide sequence encoding any of the aforementioned activin receptor II antibodies or their antigen-binding fragments.

[0055] A third aspect of this application also provides a vector comprising one or more of the aforementioned isolated polynucleotide sequences.

[0056] A fourth aspect of this application also provides an isolated host cell comprising one or more activin receptor II antibodies or their antigen-binding fragments, or one or more isolated polynucleotide sequences, or one or more vectors.

[0057] A fifth aspect of this application also provides a pharmaceutical composition comprising any one of the aforementioned activin receptor II antibodies or their antigen-binding fragments, or any one or more of the aforementioned isolated polynucleotide sequences, or one or more of the aforementioned vectors, or one or more of the aforementioned isolated host cells.

[0058] In some embodiments, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

[0059] A sixth aspect of this application also provides the use of any of the above-mentioned activin receptor II antibodies or their antigen-binding fragments, or any of the above-mentioned isolated polynucleotide sequences, or any of the above-mentioned vectors, or any of the above-mentioned isolated host cells, or any of the above-mentioned pharmaceutical compositions in the preparation of a medicament; optionally, the medicament is a fat-reducing and / or muscle-building drug.

[0060] A seventh aspect of this application also provides a method for using any of the above-mentioned activin receptor II antibodies or antigen-binding fragments thereof, or any of the above-mentioned isolated polynucleotide sequences, or any of the above-mentioned vectors, or any of the above-mentioned isolated host cells, or any of the above-mentioned pharmaceutical compositions for fat reduction and / or muscle gain.

[0061] An eighth aspect of this application also provides a method for fat reduction and / or muscle gain, comprising the steps of: administering to a patient with a need for fat reduction and / or muscle gain an effective amount of any of the above-mentioned activin receptor II antibodies or their antigen-binding fragments, or any of the above-mentioned isolated polynucleotide sequences, or any of the above-mentioned vectors, or any of the above-mentioned isolated host cells, or any of the above-mentioned pharmaceutical compositions.

[0062] The optional embodiments of this application have been described in detail above; however, this application is not limited thereto. Within the scope of the technical concept of this application, various simple modifications can be made to the technical solution of this application, including combining various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in this application and are all within the protection scope of this application. The aspects and embodiments of this application described herein include aspects and embodiments described as "comprising," "forming," and "substantially consisting of." Attached Figure Description

[0063] Figures 1a-1c show the antagonistic activity curves of five representative antibodies against Activin A activation in different cells in Example 2, where: Figure 1a is on HEK293-ActRIIA cells; Figure 1b is on HEK293-ActRIIB cells; and Figure 1c is on 293-TGFβ-Res cells.

[0064] Figures 2a-2c show the antagonistic activity curves of five representative antibodies against Myostatin activation in different cells in Example 2, where: Figure 2a is on HEK293-ActRIIA cells; Figure 2b is on HEK293-ActRIIB cells; and Figure 2c is on 293-TGFβ-Res cells.

[0065] Figure 3 shows the mouse weight data of each group on day 25, the experimental endpoint in Example 4.

[0066] Figure 4 shows the percentage change in body weight of mice in each group on day 25, the experimental endpoint of Example 4, compared to day 0.

[0067] Figure 5a shows the percentage difference in gastrocnemius muscle weight between the groups of mice in Example 4 and the solvent group; Figure 5b shows the percentage difference in quadriceps muscle weight between the groups of mice and the solvent group; Figure 5c shows the percentage difference in triceps muscle weight between the groups of mice and the solvent group.

[0068] Invention Details

[0069] the term

[0070] For the purposes of this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa. Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which the described technology pertains. All technical and patent disclosures referenced in this application are incorporated herein by reference in their entirety.

[0071] In this application, the term "antibody" has its conventional meaning in the art and is used in the broadest sense. In the biosciences, analysis of the amino acid sequences of different antibody heavy and light chains reveals that the amino acid sequences near the N-terminus of both heavy and light chains vary considerably, while the amino acid sequences in other parts remain relatively constant. Therefore, the regions in the antibody light and heavy chains with significant amino acid sequence variation near the N-terminus are called variable regions (V), and the regions with relatively stable amino acid sequences near the C-terminus are called constant regions (C). The V regions of the heavy and light chains are abbreviated as VH and VL, respectively, and the C regions of the heavy and light chains are abbreviated as CH and CL, respectively. Within the antibody variable regions, a small subset of amino acid residues exhibits particularly strong variation; these regions, where the composition and sequence of amino acid residues are more prone to variation, are called hypervariable regions (HVR). There are three hypervariable regions in the V regions of both the L and H chains. Because these regions can form precise complementarity with the antigenic determinants in their spatial structure, they are also called complementarity determining regions (CDR). In antibodies, common CDR (Cellular Recognition Domain) partitioning rules include Kabat, AbM, Chothia, Contact, and IMGT, which are well-known to those skilled in the art. When applying websites that execute these rules, simply inputting the VH and VL sequences and selecting the corresponding rule will yield CDR sequences based on different rules. Those skilled in the art should understand that the scope of protection of this application covers combinations of CDR sequences obtained through analysis using different rules. The six CDR regions of an antibody collectively determine its recognition ability and specificity against the corresponding antigen. Those skilled in the art should understand that when this application defines the amino acid sequences of the six CDR regions, the antibody's recognition ability and specificity against the corresponding antigen are predictable.

[0072] In this application, the term "antigen-binding fragment" has the conventional meaning in the art, referring to a key site on an antibody that can specifically recognize and bind to an antigen, such as the VH and / or VL regions.

[0073] In this application, the term "separated" generally refers to substances obtained artificially from their natural state. If a substance or component is found in nature as a "separated" substance, it may be due to a change in its natural environment, the separation of the substance from its natural environment, or both. For example, a certain unseparated polynucleotide or polypeptide may naturally exist in the body of a living animal, and a high-purity identical polynucleotide or polypeptide separated from this natural state is called a separated substance. The term "separated" does not exclude substances obtained artificially from their natural state and then synthesized, nor does it exclude the presence of other impurities that do not affect the substance's activity.

[0074] In this application, the terms "isolated nucleic acid molecule" and "isolated polynucleotide sequence" generally refer to an isolated form of nucleotide, deoxyribonucleotide, or ribonucleotide of any length, which may be isolated from its natural environment or synthetically produced analogues.

[0075] In this application, the term "vector" generally refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a protein is inserted, thereby enabling the protein to be expressed. Vectors can transform, transduce, or transfect host cells, allowing the genetic material they carry to be expressed within the host cells. Examples of vectors include: plasmids; phage particles; Cos plasmids; artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses used as vectors include retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, vectors may contain a replication initiation site. The vector may also include components that help it enter the cell, such as viral particles, liposomes (like LNP), or protein coats, but not only these substances.

[0076] In this application, the term "affinity" refers to the strength of the interaction between an antibody and an antigen at a single antigenic site; the stronger the interaction, the greater the affinity.

[0077] In this application, the term "affinity maturation" refers to the nucleotide changes in the variable regions of genes during antibody production due to somatic hypermutation, which occurs with each cell division. This process results in subtle differences between each progeny B cell and its parent DNA in the variable regions of the antibody amino acid chain. In mutated progeny cells, some antibodies produce antibodies with decreased affinity for the antigen compared to those produced by the parent, while others may exhibit increased affinity. Those B cells expressing antibodies with stronger affinity receive stronger survival signals from the weaker antibodies during interactions with other parts of the immune system, which eventually disappear due to apoptosis. This process, which gradually increases the binding affinity of produced antibodies, is the affinity maturation process.

[0078] In this application, the term "ka" refers to the binding rate constant of a specific antibody-antigen interaction, the term "kd" refers to the dissociation rate constant of a specific antibody-antigen interaction, and the term "KD" refers to the dissociation equilibrium constant, which is derived from the ratio of kd to ka (i.e., kd / ka) and expressed as a molar concentration (M). The KD value of an antibody can be determined using methods well-established in the art; in this application, it is based on the Biacore affinity assay.

[0079] In this application, the term "parent antibody" refers to a Bimagrumab monoclonal antibody, the heavy chain variable region of which is shown in SEQ ID NO:61, the light chain variable region of which is shown in SEQ ID NO:62, the heavy chain constant region of which is shown in SEQ ID NO:52, and the light chain constant region of which is shown in SEQ ID NO:53.

[0080] In this application, the terms "comprising" or "including" generally mean including the explicitly specified features, but do not exclude other elements.

[0081] In this application, the term "about" generally refers to a range of fluctuations acceptable to a person skilled in the art above or below a specified value, such as a variation within ±0.5% to 10%, for example, a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value.

[0082] It should be understood that this application includes the various aspects, embodiments, and combinations of said aspects and / or embodiments described herein. The above description and the following embodiments are intended to illustrate, not limit, the scope of this application. Within the scope of the technical concept of this application, various simple modifications can be made to the technical solutions of this application, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in this application and are all within the protection scope of this application.

[0083] Unless otherwise stated, the practice of this application will employ conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology.

[0084] It should be understood that this application includes the various aspects, embodiments, and combinations of said aspects and / or embodiments described herein. The above description and the following examples are intended to illustrate, not limit, the scope of this application. Other aspects, improvements, and modifications within the scope of this application will be apparent to those skilled in the art to which this application pertains. Therefore, those skilled in the art should recognize that the scope of this application also includes the improvements and modifications to the said aspects and embodiments.

[0085] Example 1: Affinity maturation experiment of Bimagrumab

[0086] Bimagrumab affinity maturation protocol is based on single-point saturation mutagenesis and high-throughput mammalian cell system expression, and the specific process is as follows:

[0087] Using Bimagrumab antibody as the parent antibody (its heavy chain variable region is shown in SEQ ID NO:61, heavy chain constant region in SEQ ID NO:52, light chain variable region in SEQ ID NO:62, and light chain constant region in SEQ ID NO:53), the CDR region of the Bimagrumab antibody was confirmed using the Kabat definition protocol (heavy chain CDR1-CDR3 are shown in SEQ ID NO:1, 5, and 21, respectively, and light chain CDR1-CDR3 are shown in SEQ ID NO:24, 37, and 41, respectively). For each amino acid site in the CDR region, primers containing 17 specific amino acid mutations (20 natural amino acids excluding methionine, cysteine, and the original amino acid at that site) were designed and synthesized. After PCR amplification, DNA encoding 17 antibody sequences with single-point mutations at that site was obtained. The obtained DNA was inserted into a vector to construct a single-point saturated mutant plasmid library for that site, which was then transfected into competent cells and plated on agar plates for culture. Each agar plate corresponds to a specific amino acid site, and the cell cloning theory on the plate covers all 17 mutant plasmids for that site. Eighty-eight single clones were randomly selected from each plate and cultured in the corresponding 96-well plates. Mutant plasmids were then obtained through high-throughput plasmid extraction, yielding 88 single-clone mutant plasmids for each amino acid site. Simultaneously, the DNA encoding the maternal antibody was inserted into a vector to construct plasmids, following the same procedures as with the aforementioned single-site saturated mutant plasmid library.

[0088] High-throughput mammalian cell expression was performed using HEK293 cells to obtain expression supernatants of corresponding monoclonal mutant plasmids and maternal antibody plasmids. The expression supernatants were screened using an ELISA method, described below: Low concentrations of goat anti-human IgG (Fc) (Jacksonimmunoresearch, catalog number 109-005-008, 1:5000 dilution) were coated onto ELISA plates and incubated overnight at 4°C. After washing, the plates were blocked with casein blocking buffer at 25°C for 1 hour. After washing again, the expression supernatant of the antibody to be tested was added, and the plates were incubated at 37°C for 2 hours. After washing the plate, add biotin-labeled extracellular fragment of ActRIIA (Kaikai Biotechnology, ACV-HM001) and incubate at 25°C for 5 minutes; add PBS and incubate at 25°C for 30 minutes to dissociate; after washing the plate, add HRP-labeled streptavidin (Sangon Biotech, D111054-0001) and incubate at 25°C for 45 minutes; after washing the plate, add TMB chromogenic buffer (Beyotime Biotechnology, P0209-500ml) and incubate at 25°C in the dark for 5 minutes; finally, add TMB chromogenic stop buffer (Beyotime Biotechnology, P0215-500ml) to stop the reaction and read the OD. 450 value.

[0089] OD 450The value was significantly higher than that of the maternal antibody OD. 450 Sample of values ​​(i.e., OD) 450 样品 / OD 450 母本抗体 Gene sequencing was performed on the plasmids corresponding to >1) to identify mutation hotspots that could potentially enhance antibody affinity. Data on some representative mutation hotspots are shown in Table 1.

[0090] Table 1. Expression supernatant OD of antibodies with different amino acid mutations during affinity maturation. 450 With maternal antibody OD 450 ratio

[0091] By combining different amino acid mutations in the heavy chain CDRs and light chain CDRs shown in Tables 2 and 3, antibody sequences containing mutations at one or more amino acid sites were designed. The mutation site locations of the heavy chain CDRs and light chain CDRs are described according to the corresponding amino acid sequence in the parent antibody heavy chain and light chain, respectively. For example, the heavy chain CDR2V54A mutation indicates that the mutation site is located at the 54th amino acid from the N-terminus to the C-terminus of the parent antibody heavy chain, changing from V to A, and it belongs to the CDR2 region of the heavy chain according to Kabat rules; the light chain CDR1 G24H mutation indicates that the mutation site is located at the 24th amino acid from the N-terminus to the C-terminus of the parent antibody light chain, changing from G to H, and it belongs to the CDR1 region of the light chain according to Kabat rules. The specific CDR sequences in the new antibodies are shown in Table 4. The heavy chain FR1, FR2, FR3, FR4 and light chain FR1, FR2, FR3, FR4 of the mutant antibodies are identical to those of Bimagrumab, as shown in SEQ ID NO:44-51, respectively. The light chain constant region of the mutant antibodies is identical to that of Bimagrumab, as shown in SEQ ID NO:53. The heavy chain constant region of the mutant antibodies is divided into two versions. JMTA11-1 to JMTA11-10, JMTA11-30 to JMTA11-36, and JMTA11-39 to JMTA11-45 use the heavy chain constant region shown in SEQ ID NO:71. The remaining antibodies use the heavy chain constant region identical to that of Bimagrumab, as shown in SEQ ID NO:52. The heavy chain constant regions shown in SEQ ID NO:52 and SEQ ID NO:71 differ by only one amino acid (counting the 97th amino acid from the heavy chain constant region). This site, K or R, is a natural variation and does not affect antibody function. The sequences of each mutant antibody, as described above (e.g., JMTA11-28, JMTA11-29, JMTA11-36, JMTA11-37, and JMTA11-38), are shown in Tables 5 and 6, respectively, for their heavy chain variable region, light chain variable region, full-length heavy chain, and full-length light chain. The corresponding antibodies were expressed using CHO cells and then purified. As described in Examples 2 and 3, the bioactivity and antigen-binding activity of the purified antibodies were tested.

[0092] Table 2. Heavy chain CDR mutations or combinations thereof

[0093] Table 3. Light chain CDR mutations or combinations thereof

[0094] Table 4. Specific CDR sequences and their numbers in mutant antibodies

[0095] Table 5. Variable region sequence of mutant antibodies

[0096] Table 6. Heavy chain and light chain sequences of mutant antibodies

[0097] Example 2: Detection of antibody antagonistic activity using reporter gene assay

[0098] The antagonistic activity of antibodies against the downstream ActRIIA / B signaling pathway activated by ActivinA or Myostatin was detected using the reporter gene assay.

[0099] ActRII was naturally expressed in 293-TGFβ-Res cells (nearshore organism, XCC03-1), and a luciferase reporter gene plasmid responding to SMAD2 / 3 signaling pathway activation was stably transfected. Activin A or Myostatin-induced SMAD2 / 3 activation upregulated luciferase expression. Luciferase activity was measured to determine the activation effect of Activin A or Myostatin and the antagonistic activity of antibodies against the downstream ActRIIA / B signaling pathway activated by Activin A or Myostatin.

[0100] To specifically determine the blocking effect of antibodies on ActRIIA or ActRIIB, the inventors further stably transfected 293-TGFβ-Res cells with plasmids carrying ActRIIA (SinoBiological, Cat#HG10257-UT) or plasmids carrying ActRIIB (SinoBiological, Cat#HG10229-UT), obtaining 293-TGFβ-Res cells that stably and highly express ActRIIA or ActRIIB (hereinafter referred to as HEK293-ActRIIA and HEK293-ActRIIB in this application). The method for determining the antagonistic activity of antibodies in three cell types—293-TGFβ-Res cells, HEK293-ActRIIA cells, and HEK293-ActRIIB cells—is described as follows: First, 40 μL of diluted antibody in cell culture medium was added to a 96-well cell culture plate (Corning). Then, 40 μL of diluted ActivinA (Kaikai Biotechnology; 30 ng / mL for 293-TGFβ-Res cells; 7.5 ng / mL for HEK293-ActRIIA / HEK293-ActRIIB cells) or Myostatin (R&D Systems; 30 ng / mL) was added. Finally, 40 μL of each of the three cell suspensions (cell count 10) was added. 4 After ( / well), incubate overnight in a 37°C / 5% CO2 incubator. The next day, use One-Glo TM The (Promega) kit detects luciferase levels in cells.

[0101] Representative results of the antagonistic activity of antibodies against the activation of downstream ActRIIA / B signaling pathways by Activin A or Myostatin are shown in Tables 7 and 8, Figures 1a-1c and 2a-2c. Among these, antibodies with optimized sequences such as JMTA11-28, JMTA11-29, and JMTA11-37 showed significantly increased antagonistic activity against Activin A or Myostatin activation in HEK293-ActRIIA cells compared to the parent antibody Bimagrumab (IC50). 50 The value decreased significantly, but the antagonistic activity of the antibody on HEK293-ActRIIB cells was not weakened (IC50 value decreased significantly). 50 (The value did not increase significantly); on 293-TGFβ-Res cells that naturally express the ActRII receptor, the antagonistic activity of these antibodies was also superior to that of the parent antibody Bimagrumab. This suggests that these antibodies will have a better effect in blocking the activation of ActivinA or Myostatin, and potentially have a better muscle-promoting effect.

[0102] Table 7. Representative results of the antagonistic activity of antibodies against Activin A activation in different cell types.

[0103] Table 8. Representative results of the antagonistic activity of antibodies against Myostatin activation in different cell types.

[0104] Example 3: Antibody Affinity Assay

[0105] Affinity determination of antibodies based on the Biacore affinity assay: The equilibrium dissociation constant (KD) of antibodies with human ActRIIA was determined by surface plasmon resonance at 25°C to assess their affinity for human ActRIIA, thereby screening for antibodies with significantly enhanced affinity for ActRIIA. Furthermore, the equilibrium dissociation constant (KD) of antibodies with ActRIIA was accurately determined by measuring their affinity at concentrations of 3.125–200 nM human ActRIIA. The method is described as follows: In the Biacore 8K system, the antibody to be tested was diluted to 2 μg / mL with running buffer (HBS-EP), and coupled to a Protein A chip (Cytiva / 29127556) at a flow rate of 10 μL / min, with a capture volume of approximately 350–650 RU. At a flow rate of 30 μL / min, the kinetics and affinity data of different concentrations of ActRIIA bound to the antibody were detected. The binding time was set at 100 s and the dissociation time at 120 s. Representative affinity data of some test antibodies at a concentration of 200 nM ActRIIA are shown in Table 9; representative affinity data of some test antibodies under multiple concentrations of ActRIIA are shown in Table 10.

[0106] Table 9. Affinity data of antibodies at a concentration of 200 nM ActRIIA

[0107] Table 10. Affinity data of antibodies under multiple concentrations of ActRIIA.

[0108] In addition, the affinities of some antibodies with human ActRIIA, human ActRIIB, mouse ActRIIA, and mouse ActRIIB were determined using the Biacore affinity assay at room temperature (25°C). The methods for chip preparation and analytical assay are as follows:

[0109] Chip preparation: The antibody was diluted to 10 μg / mL with a fixation reagent (10 mM sodium acetate, pH 4.5). The surface of the CM5 chip was activated for 420 s with 400 mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 100 mM N-hydroxysuccinimide (NHS) at a flow rate of 10 μL / min. 10 μg / mL of antibody was injected into the experimental channels at a flow rate of 10 μL / min, with a fixation volume of approximately 1000 RU. Finally, the chip was blocked with 1 M ethanolamine at a flow rate of 10 μL / min for 420 s. No antibody was injected into the reference channels; other procedures were the same as for the experimental channels.

[0110] Analytical assay: The antigen was serially diluted 2-fold using the running reagent. The diluted antigen was then injected sequentially into the experimental and reference channels at a flow rate of 30 μL / min, with binding for 90 s and dissociation for 210 s. Both binding and dissociation steps were performed using the running reagent. After each concentration analysis, the chip was regenerated with glycine-hydrochloric acid at pH 1.5 at a flow rate of 20 μL / min for 30 s to wash away any undissociated analytes.

[0111] Table 11 shows representative affinity data for some antibodies with human ActRIIA, human ActRIIB, mouse ActRIIA, and mouse ActRIIB. Several sequence-optimized antibodies showed a 5-15 fold increase in affinity for ActRIIA. For example, JMTA11-28, JMTA11-37, and JMTA11-38 showed approximately a 7-fold increase in affinity for ActRIIA without significantly weakening affinity for ActRIIB, achieving comparable affinity between the antibodies and both ActRIIA and ActRIIB.

[0112] Table 11. Affinity data of antibodies with human ActRIIA, human ActRIIB, mouse ActRIIA, and mouse ActRIIB

[0113] Example 4: Evaluation of the muscle-building efficacy of the antibody in mice

[0114] This study evaluated the effect of antibodies on muscle weight in mice. The method was as follows: Male CB17-SCID mice (n=8 per group) aged 12-14 weeks were randomly assigned to groups based on body weight. Grouping was defined as day 0. Subsequently, on days 0, 7, 14, and 21, mice were injected via tail vein with a solvent, 4 mg / kg or 20 mg / kg of Bimagrumab, or 4 mg / kg of JMTA11-37 or JMTA11-38, respectively. Body weight was monitored twice weekly. On day 25, mice were weighed, and following animal welfare requirements, they were euthanized. The gastrocnemius, triceps, and quadriceps muscles were dissected and weighed.

[0115] Figures 3 and 4 show the total body weight (mean ± SEM) and the percentage change in body weight relative to day 0 (mean ± SEM), respectively. Figures 5a-5c show the percentage change in weight of each muscle tissue (mean ± SEM). Statistical analysis was performed using the one-way ANOVA test. In these experiments, differences between the treatment group and the solvent group were indicated by asterisks: * indicates 0.01 < p < 0.05, ** indicates 0.001 < p < 0.01, *** indicates 0.0001 < p < 0.001, and **** indicates p < 0.0001; when p > 0.05, there was no statistically significant difference, and when p < 0.05, the difference was significant. In these experiments, the differences between the treatment group and the 4 mg / kg bimagrumab group were indicated by #: # indicates 0.01 < p < 0.05, ## indicates 0.001 < p < 0.01, ### indicates 0.0001 < p < 0.001, and #### indicates p < 0.0001. When p > 0.05, there was no statistically significant difference, and when p < 0.05, the difference was significant.

[0116] As shown in Figure 3, the 4 mg / kg or 20 mg / kg Bimagrumab groups, the 4 mg / kg JMTA11-37 group, and the 4 mg / kg JMTA11-38 group all significantly increased the absolute body weight compared to the solvent group; the 4 mg / kg JMTA11-37 group significantly increased the absolute body weight of mice compared to the same dose of bimagrumab. As shown in Figure 4, the 4 mg / kg or 20 mg / kg Bimagrumab groups, the 4 mg / kg JMTA11-37 group, and the 4 mg / kg JMTA11-38 group all significantly increased the percentage change in body weight compared to the solvent group; the 20 mg / kg Bimagrumab group and the 4 mg / kg JMTA11-37 group significantly increased the percentage change in body weight compared to the 4 mg / kg bimagrumab group. Since the mice involved in Figures 3 and 4 were non-obese mice, and their adipose tissue was examined with no significant increase in adipose tissue weight, the increase in body weight was due to an increase in muscle mass.

[0117] As shown in Figures 5a-5c, compared to the solvent group, the 4 mg / kg or 20 mg / kg Bimagrumab group, the 4 mg / kg JMTA11-37 group, and the 4 mg / kg JMTA11-38 group all significantly increased the weight of the gastrocnemius and quadriceps muscles in mice, while the 4 mg / kg JMTA11-37 group significantly increased the weight of the triceps muscles in mice. Compared to the 4 mg / kg Bimagrumab group, the 4 mg / kg JMTA11-37 group significantly increased the weight of the gastrocnemius and quadriceps muscles in mice, while the 4 mg / kg JMTA11-38 group significantly increased the weight of the quadriceps muscles in mice.

[0118] Example 5: Assessment of ActRII antibody for fat loss and muscle gain

[0119] This study evaluated the fat reduction and muscle gain effects of antibodies in diet-induced obese (DIO) mice. Male C57BL / 6 mice were induced to be obese (50-55g) on ​​a high-fat diet (60% of calories from fat) and then randomly assigned to groups based on body weight. The mice were administered solvent (intravenous injection; once weekly), Bimagrumab (10mg / kg; intravenous injection; once weekly), or JMTA11-37 (10mg / kg; intravenous injection; once weekly), respectively. Mouse body weight and food intake were monitored daily. After 2 weeks, the animals were euthanized according to animal welfare requirements. The left tibialis anterior, soleus, gastrocnemius, and extensor digitorum longus muscles were dissected and separated; the left triceps and biceps muscles were separated; and epididymal adipose tissue (eWAT) and inguinal adipose tissue (iWAT) were dissected and weighed.

[0120] Data are presented as mean ± standard deviation, with fat and muscle weights in mg. In these experiments, differences between the treatment and solvent groups were indicated by asterisks, and a p-value < 0.05 was considered statistically significant: * indicates 0.01 < p < 0.05, ** indicates 0.001 < p < 0.01, and *** indicates 0.0001 < p < 0.001.

[0121] The results are shown in Tables 12 and 13. Compared with the solvent group, the JMTA11-37 group increased muscle mass, such as in the tibialis anterior, extensor digitorum longus, and gastrocnemius muscles. Compared with the Bimagrumab group, the JMTA11-37 group showed a more significant increase in muscle mass, such as in the gastrocnemius muscle. Compared with the solvent group, the JMTA11-37 group reduced fat mass, such as inguinal adipose tissue. Compared with the Bimagrumab group, the JMTA11-37 group also showed a more significant reduction in inguinal adipose tissue mass.

[0122] Table 12. Muscle weight data of different parts of mice

[0123] Table 13. Weight data of adipose tissue in different parts of mice

[0124] Example 6: Pharmacokinetic Study of ActRII Antibody Administered Subcutaneously to Cynomolgus Monkeys via Single Dosage

[0125] Different groups of cynomolgus monkeys were given JMTA11-37 via single subcutaneous and intravenous injections. The blood drug concentration in the animals was measured at different time points before and after administration. The pharmacokinetic characteristics of JMTA11-37 in cynomolgus monkeys within the set dose range were investigated. The absolute bioavailability of subcutaneous injection was also investigated.

[0126] method

[0127] This experiment used 24 cynomolgus monkeys, divided into 4 groups of 6 animals each (half male and half female). The groups were designated as low-, medium-, and high-dose subcutaneous injection groups and an intravenous injection group. The day of administration was D1. Blood biochemistry and hematology tests were performed before administration (D-2), and animals with normal test results were included in the groups. Pharmacokinetic samples were collected from animals receiving subcutaneous injection at time points: before administration (0h) and at 1h, 8h, 24h (D2), 48h (D3), 72h (D4), 120h (D6), 168h (D8), 240h (D11), 336h (D15), 408h (D18), 504h (D22), 576h (D25), and 672h (D29). Animals in the intravenous injection group... Pharmacokinetic samples were collected before administration (0h) and at 0.25h, 1h, 8h, 24h (D2), 48h (D3), 72h (D4), 120h (D6), 168h (D8), 240h (D11), 336h (D15), 408h (D18), 504h (D22), 576h (D25), and 672h (D29) after administration. Administration began at time zero of the blood collection point. Blood drug concentrations were measured using a validated ELISA method. The main pharmacokinetic parameters of the drug were calculated using a non-compartmental (NCA) model with PhoenixWinNonlin 8.4.0 software: half-life (T1 / 2), maximum blood concentration (Cmax), time to peak concentration (Tmax), area under the curve (AUC0~t and AUC0~∞), apparent volume of distribution (Vz), clearance (Cl), and mean residence time (MRT0~t and MRT0~∞).

[0128] Table 14. Dosing regimen design

[0129] Table 15. Pharmacokinetic parameters of JMTA11-37 after subcutaneous and intravenous injection in cynomolgus monkeys. Note: The parameter Tmax is a range value.

[0130] result:

[0131] Absolute bioavailability of subcutaneous injection

[0132] In this study, the mean AUC0~t of the subcutaneous injection medium-dose group (4mg / kg) of JMTA11-37 was 3635h*μg / mL;

[0133] The mean AUC0–t of the intravenous JMTA11-37 dose group (4 mg / kg) was 5623 h*μg / mL. Therefore, at the 4 mg / kg dose level, the AUC0–t value of JMTA11-37 administered subcutaneously to cynomolgus monkeys was [not specified]. 0～tThe absolute bioavailability was 64.64%, which was significantly better than the 42% bioavailability of the monoclonal antibody Bimagrumab targeting the same target in humans (Bioavailability of Bimagramab from Pharmacokinetics and Pharmacodynamics of Bimagrumab (BYM338). Clinical pharmacokinetics vol.62,1(2023):141-155.).

[0134] Apart from the sequences listed in Tables 4, 5, and 6, the remaining sequences involved in this invention are as follows:

[0135] SEQ ID NO:44 Heavy chain frame region FR1

[0136] SEQ ID NO:45 Heavy chain frame region FR2

[0137] SEQ ID NO:46 Heavy chain frame region FR3

[0138] SEQ ID NO:47 Heavy chain frame region FR4

[0139] SEQ ID NO:48 Light chain frame region FR1

[0140] SEQ ID NO:49 Light chain framework region FR2

[0141] SEQ ID NO:50 Light chain frame region FR3

[0142] SEQ ID NO:51 Light chain framework region FR4

[0143] SEQ ID NO:52 Heavy chain constant region

[0144] SEQ ID NO:53 Light chain constant region

[0145] SEQ ID NO:61 Heavy chain variable region of Bimagrumab

[0146] SEQ ID NO:62 Light chain variable region of Bimagrumab

[0147] SEQ ID NO:71 Heavy chain constant region

Claims

1. An activin receptor II antibody or an antigen-binding fragment thereof, comprising heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3, wherein one or more CDRs of said heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 are mutated relative to their parent antibody CDRs, and the mutation relative to the parent antibody CDRs comprises or is selected from one or more of the following: Heavy chain CDR2 V54A mutation, heavy chain CDR2 G56L mutation, heavy chain CDR2 T58K mutation, heavy chain CDR3 G99S mutation, heavy chain CDR3 G99T mutation, light chain CDR1 G24H mutation, light chain CDR1 D28W mutation, light chain CDR1 Y32F mutation, light chain CDR2 P57E mutation, light chain CDR3 S97A mutation, and light chain CDR3 Y99N mutation; The heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 of the parent antibody are shown in the amino acid sequences of SEQ ID NO: 1, 5, 21, 24, 37, and 41, respectively; and The heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 of the activin receptor II antibody or its antigen-binding fragment and its parent antibody are divided according to the Kabat rule.

2. The activin receptor II antibody or its antigen-binding fragment according to claim 1, wherein the activin receptor II antibody or its antigen-binding fragment comprises heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 selected from the following: Heavy chain CDR1, as shown in the amino acid sequence of SEQ ID NO:1; Heavy chain CDR2, as shown by the amino acid sequence selected from SEQ ID NO:7 and SEQ ID NO:10; Heavy chain CDR3, as shown by the amino acid sequences selected from SEQ ID NO:22 and SEQ ID NO:23; The light chain CDR1, as shown by the amino acid sequences selected from SEQ ID NO:25, SEQ ID NO:26 and SEQ ID NO:27; The light chain CDR1, as shown in the amino acid sequence of SEQ ID NO:38; The light chain CDR1 is shown in the amino acid sequences selected from SEQ ID NO:41, SEQ ID NO:42 and SEQ ID NO:

43.

3. The activin receptor II antibody or its antigen-binding fragment according to claim 2, comprising: Heavy chain CDR1 as shown in SEQ ID NO:1, heavy chain CDR2 as shown in SEQ ID NO:7, heavy chain CDR3 as shown in SEQ ID NO:22, light chain CDR1 as shown in SEQ ID NO:27, light chain CDR2 as shown in SEQ ID NO:38, and light chain CDR3 as shown in SEQ ID NO:43; Or, as shown in SEQ ID NO:1, heavy chain CDR1; as shown in SEQ ID NO:7, heavy chain CDR2; as shown in SEQ ID NO:22, heavy chain CDR3; as shown in SEQ ID NO:25, light chain CDR1; as shown in SEQ ID NO:38, light chain CDR2; and light chain CDR3 as shown in SEQ ID NO:

42. Or, as shown in SEQ ID NO:1, heavy chain CDR1; as shown in SEQ ID NO:10, heavy chain CDR2; as shown in SEQ ID NO:23, heavy chain CDR3; as shown in SEQ ID NO:27, light chain CDR1; as shown in SEQ ID NO:38, light chain CDR2; and light chain CDR3 as shown in SEQ ID NO:

41. Or, as shown in SEQ ID NO:1, heavy chain CDR1; as shown in SEQ ID NO:7, heavy chain CDR2; as shown in SEQ ID NO:22, heavy chain CDR3; as shown in SEQ ID NO:25, light chain CDR1; as shown in SEQ ID NO:38, light chain CDR2; and light chain CDR3 as shown in SEQ ID NO:

43. Alternatively, heavy chain CDR1 as shown in SEQ ID NO:1, heavy chain CDR2 as shown in SEQ ID NO:7, heavy chain CDR3 as shown in SEQ ID NO:22, light chain CDR1 as shown in SEQ ID NO:26, light chain CDR2 as shown in SEQ ID NO:38, and light chain CDR3 as shown in SEQ ID NO:

43.

4. The activin receptor II antibody or its antigen-binding fragment according to claim 1, comprising heavy chain framework regions FR1, FR2, FR3, FR4 and light chain framework regions FR1, FR2, FR3, FR4, wherein: The heavy chain frame region FR1 is shown in SEQ ID NO:44; And / or, the heavy chain frame region FR2 is as shown in SEQ ID NO:45; And / or, the heavy chain frame region FR3 is as shown in SEQ ID NO:46; And / or, the heavy chain frame region FR4 is as shown in SEQ ID NO:47; And / or, the light chain frame region FR1 is as shown in SEQ ID NO:48; And / or, the light chain frame region FR2 is as shown in SEQ ID NO:49; And / or, the light chain framework region FR3 is as shown in SEQ ID NO:50; And / or, the light chain frame region FR4 is as shown in SEQ ID NO:

51.

5. The activin receptor II antibody or its antigen-binding fragment according to claim 4, comprising: The heavy chain variable region having at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:54, and the light chain variable region having at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:59; Alternatively, a heavy chain variable region having at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:54, and a light chain variable region having at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:56; Alternatively, a heavy chain variable region having at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:55, and a light chain variable region having at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:57; Alternatively, a heavy chain variable region having at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:54, and a light chain variable region having at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:58; Alternatively, a heavy chain variable region having at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:54, and a light chain variable region having at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:

60.

6. The activin receptor II antibody or its antigen-binding fragment according to claim 5, Its heavy chain variable region is shown in SEQ ID NO:54, and its light chain variable region is shown in SEQ ID NO:59; Alternatively, its heavy chain variable region is shown in SEQ ID NO:54, and its light chain variable region is shown in SEQ ID NO:56; Alternatively, its heavy chain variable region is shown in SEQ ID NO:55, and its light chain variable region is shown in SEQ ID NO:57; Alternatively, its heavy chain variable region is shown in SEQ ID NO:54, and its light chain variable region is shown in SEQ ID NO:58; Alternatively, its heavy chain variable region is shown as SEQ ID NO:54, and its light chain variable region is shown as SEQ ID NO:

60.

7. The activin receptor II antibody or its antigen-binding fragment according to claim 1, comprising a heavy chain constant region and a light chain constant region, wherein: The amino acid sequence of the heavy chain constant region is shown in SEQ ID NO:52 or SEQ ID NO:71; And / or, the amino acid sequence of the constant region of the light chain is as shown in SEQ ID NO:

53.

8. The activin receptor II antibody or its antigen-binding fragment according to claim 7, comprising: Heavy chain sequences as shown in SEQ ID NO:63 and light chain sequences as shown in SEQ ID NO:69; Or, as shown in SEQ ID NO:63, the heavy chain sequence, and as shown in SEQ ID NO:66, the light chain sequence; Or, as shown in SEQ ID NO:64, the heavy chain sequence, and as shown in SEQ ID NO:67, the light chain sequence; Or, as shown in SEQ ID NO:65, the heavy chain sequence, and as shown in SEQ ID NO:68, the light chain sequence; Alternatively, the heavy chain sequence as shown in SEQ ID NO:63, and the light chain sequence as shown in SEQ ID NO:

70.

9. The activin receptor II antibody according to claim 8, wherein the antibody is an scFv antibody, a Fab antibody, a monospecific antibody, a bispecific antibody, or a trispecific antibody.

10. An isolated polynucleotide sequence encoding an activin receptor II antibody or an antigen-binding fragment thereof as described in any one of claims 1-9.

11. A vector comprising one or more isolated polynucleotide sequences as described in claim 10.

12. An isolated host cell comprising one or more activin receptor II antibodies or antigen-binding fragments thereof as described in any one of claims 1-9, or one or more isolated polynucleotide sequences as described in claim 10, or one or more vectors as described in claim 11.

13. A pharmaceutical composition comprising one or more activin receptor II antibodies or antigen-binding fragments thereof as described in any one of claims 1-9, or one or more isolated polynucleotide sequences as described in claim 10, or one or more vectors as described in claim 11, or one or more isolated host cells as described in claim 17.

14. The pharmaceutical composition of claim 13, further comprising pharmaceutically acceptable excipients.

15. Use of the activin receptor II antibody or its antigen-binding fragment as described in any one of claims 1-9, or the isolated polynucleotide sequence as described in claim 10, or the vector as described in claim 11, or the isolated host cell as described in claim 12, or the pharmaceutical composition as described in claim 13 or 14 in the preparation of a medicament; optionally, the medicament is a fat-reducing and / or muscle-building drug.

16. The activin receptor II antibody or its antigen-binding fragment according to any one of claims 1-9, or the isolated polynucleotide sequence according to claim 10, or the vector according to claim 11, or the isolated host cell according to claim 12, or the pharmaceutical composition according to claim 13 or 14, for use in a method of fat reduction and / or muscle gain.

17. A method for fat loss and / or muscle gain, comprising the following steps: Administer to a patient with fat loss and / or muscle gain an effective amount of the activin receptor II antibody or its antigen-binding fragment as described in any one of claims 1-9, or the isolated polynucleotide sequence as described in claim 10, or the vector as described in claim 11, or the isolated host cell as described in claim 12, or the pharmaceutical composition as described in claim 13 or 14.

18. A method for preparing an antibody, comprising culturing the isolated host cell of claim 12 and recovering the antibody from the cell culture.

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