Antibody against activin receptor ii and use thereof

Abstract

The present disclosure provides an antibody against activin receptor II and a use thereof. The antibody can be used for treating and / or preventing activin receptor II-related diseases or disorders, including obesity, diabetes, muscle atrophy and / or cancer cachexia, etc.

Description

Antibodies against activin receptor II and their uses

[0001] This application claims priority to Chinese Patent Application No. 2025100765834, filed January 17, 2025, entitled "Antibody against activin receptor II and its use thereof". The contents of that application are incorporated herein by reference. Technical Field

[0002] This disclosure relates to the field of biomedicine, and more specifically, to an antibody targeting activin receptor IIA / B and its use. Background Technology

[0003] Obesity is a global health problem. According to the World Health Organization (WHO), there are currently 650 million obese people worldwide, and this number is projected to reach 1.12 billion by 2030. Obesity not only affects an individual's quality of life but is also closely related to various chronic diseases such as cardiovascular disease and diabetes.

[0004] GLP-1 receptor agonists (GLP-1RAs) are currently important drugs for the treatment of diabetes and weight loss. Several GLP-1RA drugs are available globally, and sales are experiencing rapid growth. However, while GLP-1RAs promote weight loss, they also lead to muscle loss. Multiple clinical and retrospective studies have shown that nearly 40% of the weight lost by GLP-1RA participants is lean body mass, primarily composed of muscle mass. Muscle loss increases the risk of cardiovascular disease, osteoporosis, and other illnesses. Therefore, maintaining muscle mass while losing fat, thereby promoting metabolic health, is key to achieving high-quality weight management.

[0005] Activin receptor type II (ActRII) is an important member of the transforming growth factor β (TGF-β) receptor superfamily, comprising two forms: activin receptor IIA (ACVR2A) and activin receptor IIB (ACVR2B). They play roles in various physiological processes, including muscle growth, fat distribution, and skeletal development. Both ACVR2A and ACVR2B have approximately 500 amino acid residues and are single-transmembrane serine / threonine kinases, including a ligand-binding cys-rich extracellular domain, a transmembrane domain, and an intracellular domain. Myostatin, activin A, and GDF-11 are negative regulators of skeletal muscle growth, acting through the activin receptor II signaling pathway to inhibit muscle protein synthesis and myocyte differentiation and proliferation. Blocking this signaling pathway holds promise for inhibiting muscle atrophy and promoting muscle mass increase, potentially helping obese patients improve body composition and metabolism while losing weight.

[0006] Patent document WO2010125003A1 discloses Bimagrumab, a monoclonal antibody targeting ActRII, which binds to human ActRIIB with extremely high affinity and to human ActRIIA with relatively low affinity, and is formulated for intravenous administration. Bimagrumab has shown preliminary clinical potential for fat loss and muscle gain, and its combination regimen with GLP-1RA is currently undergoing clinical trials. However, there is still a need to develop antibodies targeting ActRII with better efficacy and safety. Summary of the Invention

[0007] To address the aforementioned issues, this disclosure provides a Bimagrumab mutant that, compared to the parent Bimagrumab, exhibits a stronger affinity for ActRIIA / B, a more potent muscle-building and fat-reducing effect, and lower immunogenicity.

[0008] In a first aspect, this disclosure provides an antibody or antigen-binding fragment thereof targeting activin receptor II, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 of the heavy chain variable region shown in SEQ ID NO: 33; and / or the light chain variable region comprising LCDR1, LCDR2 and LCDR3 of the light chain variable region shown in SEQ ID NO: 44.

[0009] In some embodiments, the HCDR1 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 1: GYTFX1SSYIN (SEQ ID NO: 1), wherein X1 is selected from Y, W and F.

[0010] The HCDR2 contains, or is composed of, the amino acid sequence shown in SEQ ID NO: 5: TINPVSGX2TSYAQKFQG (SEQ ID NO: 5), where X2 is Q or N.

[0011] The HCDR3 contains, or is composed of, the amino acid sequence shown in SEQ ID NO: 8: X3GWFDY (SEQ ID NO: 8), wherein X3 is selected from T, S, A and R.

[0012] The LCDR1 contains, or is composed of, the amino acid sequence shown in SEQ ID NO: 13;

[0013] The LCDR2 contains, or is composed of, the amino acid sequence shown in SEQ ID NO: 14;

[0014] The LCDR3 contains, or is composed of, the amino acid sequence shown in SEQ ID NO: 15.

[0015] In some embodiments, the HCDR1 comprises the amino acid sequence shown in any one of SEQ ID NO: 2-4.

[0016] In some embodiments, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 6 or 7.

[0017] In some embodiments, the HCDR3 comprises the amino acid sequence shown in any one of SEQ ID NO: 9-12.

[0018] In some embodiments, the heavy chain variable region comprises HCDR1, which contains the amino acid sequence shown in SEQ ID NO: 2; HCDR2, which contains the amino acid sequence shown in SEQ ID NO: 6; and HCDR3, which contains the amino acid sequence shown in SEQ ID NO: 9.

[0019] In some embodiments, the heavy chain variable region further comprises HFR1, which contains the amino acid sequence shown in SEQ ID NO: 16: QVQLVQSGAEVKKPGASVKVSCKAS (SEQ ID NO: 16).

[0020] In some embodiments, the heavy chain variable region further comprises HFR2, which contains the amino acid sequence shown in SEQ ID NO: 17: WVRQAPGQGLEWX4G (SEQ ID NO: 17), wherein X4 is selected from M, I, and L.

[0021] In some embodiments, the heavy chain variable region further comprises HFR3, which comprises the amino acid sequence shown in SEQ ID NO: 18: RVTMTRDTSISTAYMELSX5LRSX6DTAVYYCAR (SEQ ID NO: 18), wherein X5 is selected from R, S and T, and X6 is D or E.

[0022] In some embodiments, the heavy chain variable region further comprises HFR4, which contains the amino acid sequence shown in SEQ ID NO: 19: WGQGTLVTVSS (SEQ ID NO: 19).

[0023] In some embodiments, the heavy chain variable region further comprises: HFR1, which comprises the amino acid sequence shown in SEQ ID NO: 16; HFR2, which comprises the amino acid sequence shown in any one of SEQ ID NO: 20-22; HFR3, which comprises the amino acid sequence shown in any one of SEQ ID NO: 23-28; and HFR4, which comprises the amino acid sequence shown in SEQ ID NO: 19.

[0024] In some embodiments, the light chain variable region further comprises LFR1, which contains the amino acid sequence shown in SEQ ID NO: 29.

[0025] In some embodiments, the light chain variable region further comprises LFR2, which contains the amino acid sequence shown in SEQ ID NO: 30.

[0026] In some embodiments, the light chain variable region further comprises LFR3, which contains the amino acid sequence shown in SEQ ID NO: 31.

[0027] In some embodiments, the light chain variable region further comprises LFR4, which contains the amino acid sequence shown in SEQ ID NO: 32.

[0028] In some embodiments, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 33 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or higher sequence identity with SEQ ID NO: 33.

[0029] In some embodiments, the heavy chain variable region comprises an amino acid sequence shown in any one of SEQ ID NO: 34-43 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or higher sequence identity with any one of SEQ ID NO: 34-43.

[0030] In some embodiments, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 35 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or higher sequence identity with SEQ ID NO: 35, provided that it contains the T30Y, M48I, S57Q, R85S, D89E, and G99T mutations. In some embodiments, the heavy chain variable region comprises an amino acid sequence having about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with SEQ ID NO: 35, provided that it contains the T30Y, M48I, S57Q, R85S, D89E, and G99T mutations.

[0031] In some embodiments, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 44 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or higher sequence identity with SEQ ID NO: 44.

[0032] In some embodiments, the antibody or antigen-binding fragment includes a full-length antibody, Fab, Fab', Fv fragment, F(ab')2, scFv, or di-scFv.

[0033] In some embodiments, the heavy chain of the antibody or its antigen-binding fragment further comprises a constant region or a variant thereof; preferably, the heavy chain comprises a constant region of human IgG1, IgG2, IgG3 or IgG4 or a variant thereof; more preferably, the heavy chain further comprises the amino acid sequence shown in any one of SEQ ID NO: 45-53.

[0034] In some embodiments, the light chain of the antibody or its antigen-binding fragment further comprises a constant region or a variant thereof; preferably, the light chain comprises a constant region of human Igλ or a variant thereof; more preferably, the light chain further comprises the amino acid sequence described in SEQ ID NO: 64.

[0035] In some embodiments, the heavy chain of the antibody or its antigen-binding fragment comprises the amino acid sequence shown in any one of SEQ ID NO: 54-63, 88-89 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or higher sequence identity with any one of SEQ ID NO: 54-63, 88-89.

[0036] In some embodiments, the heavy chain of the antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 57 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or higher sequence identity with SEQ ID NO: 57, provided that it contains the T30Y, M48I, S57Q, R85S, D89E, and G99T mutations. In some embodiments, the heavy chain of the antibody or its antigen-binding fragment comprises an amino acid sequence having about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with SEQ ID NO: 57, provided that it contains the T30Y, M48I, S57Q, R85S, D89E, and G99T mutations.

[0037] In some embodiments, the light chain of the antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 65 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or higher sequence identity with SEQ ID NO: 65.

[0038] In some embodiments, the antibody or its antigen-binding fragment is targeted at ActRIIA and / or ActRIIB.

[0039] In a second aspect, this disclosure provides a fusion protein comprising the antibody or its antigen-binding fragment described in the first aspect of this disclosure.

[0040] Thirdly, this disclosure provides bispecific or multispecific antibodies comprising the antibodies or antigen-binding fragments thereof described in the first aspect of this disclosure.

[0041] Fourthly, this disclosure provides a pharmaceutical combination comprising an antibody against activin receptor II as described in the first aspect of this disclosure or an antigen-binding fragment thereof, or a fusion protein as described in the second aspect of this disclosure, or a bispecific or multispecific antibody as described in the third aspect of this disclosure, and a GLP-1R agonist.

[0042] In some embodiments, the GLP-1R agonist comprises human GLP-1 or a variant thereof, or a functional fragment of human GLP-1 or a variant thereof.

[0043] In some embodiments, the GLP-1R agonist comprises exenatide or a variant thereof, or a functional fragment of exenatide or a variant thereof.

[0044] In some embodiments, the GLP-1R agonist comprises: GLP-1(7-37), GLP-1(7-36)-NH2, liraglutide, abiglutide, lixisenatide, dulaglutide, smegglutide, exendin-4, exendin-3, polyethylene glycol loxenatide, telpopotide, GMA102, PB119, ibennatide, mazdutide, supaglutide, cotadutide, Danuglipron, HM-15211, efinopegdutide, etc.

[0045] In some embodiments, the GLP-1R agonist comprises the amino acid sequence shown in any one of SEQ ID NO: 66-75, 85-86.

[0046] Fifthly, this disclosure also provides a composition comprising the antibody or antigen-binding fragment thereof described in the first aspect of this disclosure, the fusion protein described in the second aspect of this disclosure, the bispecific or multispecific antibody described in the third aspect of this disclosure, the pharmaceutical combination described in the fourth aspect of this disclosure, and a pharmaceutically acceptable carrier.

[0047] In a sixth aspect, this disclosure provides nucleic acids encoding the antibodies or antigen-binding fragments thereof described in the first aspect of this disclosure, the fusion proteins described in the second aspect of this disclosure, and the bispecific or multispecific antibodies described in the third aspect of this disclosure.

[0048] In a seventh aspect, this disclosure provides a vector comprising the nucleic acid described in the sixth aspect of this disclosure.

[0049] Eighthly, this disclosure provides recombinant cells comprising the nucleic acids described in the sixth aspect of this disclosure or the vectors described in the seventh aspect of this disclosure.

[0050] In a ninth aspect, this disclosure provides a method for preparing an antibody against activin receptor II or an antigen-binding fragment thereof, the method comprising culturing the recombinant cells described in the eighth aspect of this disclosure and obtaining the antibody against activin receptor II or an antigen-binding fragment thereof from the culture.

[0051] In a tenth aspect, this disclosure provides a method for treating and / or preventing activin receptor II-related diseases or conditions, comprising administering to a patient in need an antibody or antigen-binding fragment thereof as described in the first aspect of this disclosure, a fusion protein as described in the second aspect of this disclosure, a bispecific or multispecific antibody as described in the third aspect of this disclosure, a pharmaceutical combination as described in the fourth aspect of this disclosure, a composition as described in the fifth aspect of this disclosure, a nucleic acid as described in the sixth aspect of this disclosure, a vector as described in the seventh aspect of this disclosure, or a recombinant cell as described in the eighth aspect of this disclosure.

[0052] In some embodiments, the activin receptor II-related diseases or conditions include obesity, diabetes, musculoskeletal disorders, and / or cancer cachexia.

[0053] In some implementations, the musculoskeletal disease is muscle atrophy.

[0054] The eleventh aspect of this disclosure provides the use of the antibodies or antigen-binding fragments thereof described in the first aspect of this disclosure, the fusion proteins described in the second aspect of this disclosure, the bispecific or multispecific antibodies described in the third aspect of this disclosure, the pharmaceutical combinations described in the fourth aspect of this disclosure, the compositions described in the fifth aspect of this disclosure, the nucleic acids described in the sixth aspect of this disclosure, the vectors described in the seventh aspect of this disclosure, or the recombinant cells described in the eighth aspect of this disclosure in the preparation of medicaments for the treatment and / or prevention of activin receptor II-related diseases or conditions.

[0055] In some embodiments, the activin receptor II-related diseases or conditions include obesity, diabetes, musculoskeletal disorders (e.g., muscle atrophy), and / or cancer cachexia.

[0056] The twelfth aspect of this disclosure provides a method for non-therapeutic fat reduction and / or muscle gain in humans, comprising administering to a subject in need an antibody or antigen-binding fragment thereof as described in the first aspect of this disclosure, a fusion protein as described in the second aspect of this disclosure, a bispecific or multispecific antibody as described in the third aspect of this disclosure, a pharmaceutical combination as described in the fourth aspect of this disclosure, a composition as described in the fifth aspect of this disclosure, a nucleic acid as described in the sixth aspect of this disclosure, a vector as described in the seventh aspect of this disclosure, or a recombinant cell as described in the eighth aspect of this disclosure.

[0057] The twelfth aspect of this disclosure provides a drug delivery device, including a drug infusion module and optionally a drug monitoring module.

[0058] In some embodiments, the drug infusion module contains the antibody or antigen-binding fragment thereof described in the first aspect of this disclosure, the fusion protein described in the second aspect of this disclosure, the bispecific or multispecific antibody described in the third aspect of this disclosure, the drug combination described in the fourth aspect of this disclosure, the composition described in the fifth aspect of this disclosure, the nucleic acid described in the sixth aspect of this disclosure, the vector described in the seventh aspect of this disclosure, or the recombinant cell described in the eighth aspect of this disclosure.

[0059] Thirteenth aspect of this disclosure provides a kit comprising at least a first kit and a second kit, wherein the first kit comprises an antibody or antigen-binding fragment thereof as described in the first aspect of this disclosure, a fusion protein as described in the second aspect of this disclosure, a bispecific or multispecific antibody as described in the third aspect of this disclosure, a drug combination as described in the fourth aspect of this disclosure, a composition as described in the fifth aspect of this disclosure, a nucleic acid as described in the sixth aspect of this disclosure, a vector as described in the seventh aspect of this disclosure, or a recombinant cell as described in the eighth aspect of this disclosure.

[0060] In some embodiments, the second kit contains a GLP-1R agonist.

[0061] In some embodiments, the GLP-1R agonist comprises the amino acid sequence shown in any one of SEQ ID NO: 66-75, 85-86.

[0062] In some embodiments, the GLP-1R agonist comprises the amino acid sequence shown in SEQ ID NO: 85. Attached Figure Description

[0063] Figure 1 shows the inhibitory effects of Bima-AU003 and Bimagrumab on (A)Activin A / (B)GDF-8 / (C)GDF-11-induced SMAD2 / 3 activation in the HEK293-SBE reporter gene system.

[0064] Figure 2 shows the following in vivo efficacy studies in DIO mice: (A) percentage change in body weight relative to D0 for each group; (B) percentage of body weight in fat mass at the endpoint of the experiment; (C) rate of change in fat mass in groups G2-G5 relative to group G1 at the endpoint of the experiment; (D) percentage of body weight in lean mass at the endpoint of the experiment; (E) rate of change in lean mass in groups G2-G5 relative to group G1 at the endpoint of the experiment; and (F) changes in blood concentrations of Bimagrumab and Bima-AU003 in groups G4 and G5. In Figure 2 (C) and (E), %change = (mean Gm - mean G1) / (mean G1) * 100, where Gm represents each administered group.

[0065] Figure 3 shows the following in the in vivo efficacy study of CB-17SCID mice: (A) percentage change in body weight relative to D0 for each group, (B) fat mass of each group at the endpoint of the experiment, (C) rate of change of fat mass and fat mass / body weight percentage in groups G2-G5 relative to G1 at the endpoint of the experiment, (D) lean body mass of each group at the endpoint of the experiment, and (E) rate of change of lean body mass and lean body mass / body weight percentage in groups G2-G5 relative to G1 at the endpoint of the experiment. In Figure 3 (C) and (E), %change = (mean Gm - mean G1) / (mean G1) * 100, where Gm represents each administered group.

[0066] Figure 4 shows the inhibitory effects of Bima-AU003-FcAG-LS, Bima-AU003-FcAG, and Bimagrumab on (A)GDF-8 / (B)GDF-11-induced SMAD2 / 3 activation in the HEK293-SBE reporter gene system. Detailed Implementation

[0067] Terminology Definition

[0068] Unless otherwise indicated or defined, all terms used herein have their ordinary meaning as will be understood by those skilled in the art. References include, for example, standard manuals such as Sambrook et al., “Molecular Cloning: A Laboratory Manual” (2nd edition), Volumes 1–3, Cold Spring Harbor Laboratory Press (1989); Lewin, “Genes IV”, Oxford University Press, New York (1990); and Roitt et al., “Immunology” (2nd edition), Gower Medical Publishing, London, New York (1989), and general prior art cited herein; furthermore, unless otherwise stated, all methods, steps, techniques, and operations not specifically detailed herein can and have been performed in a manner known per se as will be understood by those skilled in the art. Also refer to, for example, standard manuals, the aforementioned general prior art, and other references cited therein.

[0069] As used herein, the terms “activin receptor IIA / B” or “activin receptor type 2 A / B” or “ActRIIA / B” or “ACVR2A / ACVR2B” refer to any variant, functional fragment, allotype, or species homologue of human ActRIIA / B. Exemplary human ActRIIA / B can be found in the proteins disclosed in Uniprot ID NO: P27037 (ACVR2A) and Uniprot ID NO: Q13705 (ACVR2B).

[0070] As used herein, “antibody” refers to immunoglobulins and immunoglobulin fragments, whether natural or partially or wholly synthetic (e.g., recombinant), including any fragment that retains the binding specificity of the full-length immunoglobulin molecule, containing at least a portion of the variable region of the immunoglobulin molecule. Therefore, an antibody includes any protein having a binding domain homologous or substantially homologous to an immunoglobulin antigen-binding domain (antibody binding site). Antibodies include antibody fragments, such as anti-ActRIIA / B antibody fragments. As used herein, the term antibody therefore includes synthetic antibodies, recombinant antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, intracellular antibodies, and antibody fragments, such as, but not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, disulfide-linked Fv (dsFv), Fd fragments, Fd' fragments, single-chain Fv (scFv), single-chain Fab (scFab), biantibodies, anti-idiotypic (anti-Id) antibodies, or antigen-binding fragments of any of the above antibodies. The antibodies described herein include members of any immunoglobulin type (e.g., IgG, IgM, IgD, IgE, IgA, and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass (e.g., IgG2a and IgG2b).

[0071] As used herein, an “antibody fragment” or “antigen-binding fragment” refers to any portion of a full-length antibody that is less than full-length but contains at least a portion of the antibody’s variable region (e.g., one or more CDRs and / or one or more antigen-binding sites) that binds to an antigen, and thus retains binding specificity as well as at least a portion of the full-length antibody’s specific binding capacity. Therefore, an antigen-binding fragment refers to an antibody fragment containing an antigen-binding portion that binds to the same antigen as an antibody fragment derived from the antibody fragment. Antibody fragments include antibody derivatives produced by enzymatic treatment of a full-length antibody, as well as synthetically produced derivatives, such as recombinant derivatives. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, single-chain Fv (scFv), Fv, dsFv, biantibodies, Fd and Fd' fragments, and other fragments, including modified fragments (see, for example, Methods in Molecular Biology, Vol 207: Recombinant Antibodies for Cancer Therapy Methods and Protocols (2003); Chapter 1; p 3-25, Kipriyanov). The fragment may comprise multiple chains linked together, for example by disulfide bonds and / or by peptide linkers. Antibody fragments generally contain at least or about 50 amino acids, and typically at least or about 200 amino acids. Antigen-binding fragments include any antibody fragment that, upon insertion into an antibody framework (e.g., by replacing the corresponding region), acquires immune-specific binding (i.e., exhibits at least or at least about 10 amino acids). 7 -10 8 M -1 Antibodies against the Ka antigen.

[0072] As used herein, “monoclonal antibody” refers to a population of identical antibodies, meaning that each individual antibody molecule in a population of monoclonal antibodies is identical to the others. This characteristic contrasts with that of a polyclonal population of antibodies, which contains antibodies with a variety of different sequences. Monoclonal antibodies can be prepared by a number of well-known methods (Smith et al. (2004) J. Clin. Pathol. 57, 912-917; and Nelson et al., J Clin Pathol (2000), 53, 111-117). For example, monoclonal antibodies can be prepared from immortalized B cells, for instance, by fusing with myeloma cells to generate hybridoma cell lines or by infecting B cells with a virus such as EBV. Recombinant techniques can also be used to prepare antibodies in vitro from a clonal population of host cells by transforming host cells with plasmids carrying artificial sequences of nucleotides encoding the antibody.

[0073] As used herein, “conventional antibody” refers to an antibody containing two heavy chains (which may be labeled H and H') and two light chains (which may be labeled L and L') and two antigen-binding sites, wherein each heavy chain may be a full-length immunoglobulin heavy chain or any functional region thereof that retains antigen-binding ability (e.g., heavy chains include, but are not limited to, V). H Chain, V H -C H 1 chain and V H -C H 1-C H 2-C H 3 chains), and each light chain can be a full-length light chain or any functional area (e.g., light chains include, but are not limited to, V). L Chain and V L -C L Each heavy chain (H and H') is paired with a light chain (L and L', respectively).

[0074] As used in this article, a full-length antibody is a antibody with two full-length heavy chains (e.g., V). H -C H 1-C H 2-C H 3 or V H -C H 1-C H 2-C H 3-C H 4) and two full-length light chains (V L -C L Antibodies with the hinge region, such as antibodies naturally produced by B cells through antibody secretion and synthetically produced antibodies with the same structural domain.

[0075] As used in this article, dsFv refers to a stable V H -V L Fv of engineered intermolecular disulfide bonds.

[0076] As used herein, the Fab fragment is an antibody fragment obtained by digesting a full-length immunoglobulin with papain, or a fragment with the same structure synthesized, for example, through recombinant methods. The Fab fragment contains a light chain (containing V... L and C L ) and another chain, the other chain containing the variable structural domain (V) of the heavy chain. H ) and a constant region structural domain of the heavy chain (C H 1).

[0077] As used herein, the F(ab')2 fragment is an antibody fragment resulting from the digestion of immunoglobulins with pepsin at pH 4.0–4.5, or a fragment with the same structure synthesized, for example, by a recombinant method. The F(ab')2 fragment essentially comprises two Fab fragments, each heavy chain containing several additional amino acids, including cysteine ​​residues that form the disulfide bond connecting the two fragments.

[0078] As used in this article, the Fab' fragment is a fragment that contains half of the F(ab')2 fragment (one heavy chain and one light chain).

[0079] As used herein, scFv fragments refer to variable light chains (V) covalently linked in any order via peptide linkers. L ) and variable heavy chain (V H The antibody fragment has a linker length that allows the two variable domains to bridge with minimal interference. An exemplary linker is (Gly-Ser) with some Glu or Lys residues dispersed to increase solubility. n Residues.

[0080] The term "chimeric antibody" refers to an antibody in which the variable region sequence is derived from one species and the constant region sequence is derived from another species, such as an antibody in which the variable region sequence is derived from a mouse antibody and the constant region sequence is derived from a human antibody.

[0081] "Humanized" antibodies refer to non-human (e.g., mouse) antibody forms that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) containing a minimal sequence derived from a non-human immunoglobulin. Preferably, the humanized antibody is a human immunoglobulin (recipient antibody) in which residues of the complementarity-determining region (CDR) of the recipient antibody are replaced by CDR residues from a non-human species (donor antibody) with the desired specificity, affinity, and capability, such as mouse, rat, or rabbit.

[0082] Furthermore, in humanization, amino acid residues in the CDR1, CDR2, and / or CDR3 regions of VH and / or VL may be mutated to improve one or more binding properties (e.g., affinity) of the antibody. Mutations can be introduced, for example, through PCR-mediated mutations, and their effects on antibody binding or other functional properties can be assessed using the in vitro or in vivo assays described herein. Typically, conserved mutations are introduced. Such mutations can be amino acid substitutions, additions, or deletions. Additionally, mutations within the CDRs typically do not exceed one or two. Therefore, the humanized antibodies described in this disclosure also cover antibodies containing one or two amino acid mutations within the CDRs.

[0083] As used herein, a variable domain or variable region is a specific Ig domain of the antibody heavy or light chain, containing a variable amino acid sequence that varies between different antibodies. Each light chain and each heavy chain has one variable region domain V. L and V H Variable domains provide antigen specificity and are therefore responsible for antigen recognition. Each variable region contains a CDR and a frame region (FR), the CDR being part of the antigen-binding site domain.

[0084] As used herein, "antigen-binding domain" and "antigen-binding site" are used synonymously to refer to the domain within an antibody that recognizes and physically interacts with the corresponding antigen. Natural, conventional full-length antibody molecules have two conventional antigen-binding sites, each containing a variable region portion of the heavy chain and a variable region portion of the light chain. The conventional antigen-binding site contains a loop connecting antiparallel β-chains within the variable region domain. The antigen-binding site may contain other portions of the variable region domain. Each conventional antigen-binding site contains three hypervariable regions from the heavy chain and three hypervariable regions from the light chain. The hypervariable regions are also called complementarity-determining regions (CDRs).

[0085] As used herein, the terms “hypervariant region,” “HV,” “complementarity-determining region,” “CDR,” and “antibody CDR” can be used interchangeably to refer to one of the multiple portions within each variable region that together form the antigen-binding site of the antibody. Each variable region domain contains three CDRs, named CDR1, CDR2, and CDR3. For example, the light chain variable region domain contains three CDRs, named VL CDR1, VL CDR2, and VL CDR3; the heavy chain variable region domain contains three CDRs, named VH CDR1, VH CDR2, and VH CDR3. The three CDRs in the variable region are discontinuous along the linear amino acid sequence but are close together in the folded polypeptide. The CDRs are located within the loop of the parallel chain connecting the β-sheet of the variable region.

[0086] It should be understood that combinations of CDRs defined using different numbering rules based on this document are still within the scope of protection of this invention. Those skilled in the art know and can identify CDRs based on Kabat, Chothia, or IMGT numbering (see, for example, Kabat, E.A. et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242 and Chothia, C. et al. (1987) J.Mol.Biol. 196:901-917). Alternative methods for numbering the amino acid residues of CDRs are also known in the art. For example, AbM CDRs represent a compromise between the Kabat hypervariable region and the Chothia structural loop, and are used in Oxford Molecular's AbM antibody modeling software; for example, IMGT CDRs are defined according to the unique numbering system of IMGT (International ImMunoGeneTics Information System). The residue descriptions of CDRs from each method are as follows:

[0087] However, it should be noted that, as is known in the art, the total number of amino acid residues in each CDR may differ and may not correspond to the total number of amino acid residues indicated by the Kabat number (i.e., one or more positions according to the Kabat number may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed by the Kabat number). This means that, in general, the Kabat number may or may not correspond to the actual number of amino acid residues in the actual sequence.

[0088] As used in this article, the frame region (FR) is a domain located within the antibody variable region domain within the β-sheet; in terms of amino acid sequence, the FR region is relatively more conserved than the hypervariable region.

[0089] As used in this article, the "constant region" domain is a domain in the antibody heavy or light chain that contains a more conserved amino acid sequence than the variable region domain. In a typical full-length antibody molecule, each light chain has a single light chain constant region (C0). L ) structural domains, and each heavy chain contains one or more heavy chain constant regions (C H ) structural domain, including C H 1. C H 2. C H 3 and CH 4. Full-length IgA, IgD, and IgG isotypes include C. H 1. C H 2. C H 3 and hinge region, while IgE and IgM contain C H 1. C H 2. C H 3 and C H 4. C H 1 and C L The structural domain extends the Fab arm of the antibody molecule, thus facilitating interaction with antigens and the rotation of the antibody arm. The antibody constant region can serve effector functions, such as, but not limited to, clearing antigens, pathogens, and toxins specifically bound to the antibody, for example, through interactions with various cells, biomolecules, and tissues.

[0090] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of its constant region. This term includes native sequence Fc regions and variant Fc regions. A native immunoglobulin "Fc domain" contains two or three constant domains: a CH2 domain, a CH3 domain, and optionally a CH4 domain. For example, in native antibodies, the immunoglobulin Fc domain contains the second and third constant domains (CH2 and CH3 domains) of two heavy chains derived from IgG, IgA, and IgD antibodies; or it contains the second, third, and fourth constant domains (CH2, CH3, and CH4 domains) of two heavy chains derived from IgM and IgE antibodies. Unless otherwise stated herein, the amino acid residues in the Fc region or heavy chain constant region are numbered according to the EU numbering system (also known as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interes, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991. In this document, the term "Fc region" does not include the heavy chain variable region VH and light chain variable region VL of immunoglobulins, nor the heavy chain constant region CH1 and light chain constant region CL, but in some cases may include the hinge region at the N-terminus of the heavy chain constant region. In some embodiments, the Fc region of this disclosure is derived from IgG1, IgG2, IgG3, or IgG4.

[0091] As used herein, the terms “specific binding” and “immune-specific binding” for antibodies or their antigen-binding fragments are used interchangeably and refer to the ability of an antibody or antigen-binding fragment to form one or more non-covalent bonds with the same antigen through a non-covalent interaction between the antibody and the antigen’s antibody-binding site. Typically, antibodies that immune-specifically bind (or specifically bind) antigens have a binding rate of approximately 1 × 10⁻⁶. 7 M -1 Or 1x 10 8 M - 1 Or a larger affinity constant Ka (or 1 x 10⁻⁶) -7 M or 1×10 -8 M or a lower dissociation constant (K) d The affinity constant can be determined by standard kinetic methods of antibody reactions, such as immunoassay, surface plasmon resonance (SPR) (Rich and Myszka (2000) Curr. Opin. Biotechnol 11:54; Englebienne (1998) Analyst. 123:1599), isothermal titration calorimetry (ITC), or other kinetic interaction assays known in the art (see, for example, Paul, ed., Fundamental Immunology, 2nd ed., Raven Press, New York, pages 332-336 (1989)). Instruments and methods for real-time detection and monitoring of binding rates are known and commercially available (see, BiaCore 2000, Biacore AB, Upsala, Sweden and GE Healthcare Life Sciences; Malmqvist (2000) Biochem. Soc. Trans. 27:335).

[0092] As used herein, a polypeptide refers to two or more amino acids covalently linked together. The terms "polypeptide" and "protein" are used interchangeably herein.

[0093] "Isolated protein," "isolated polypeptide," or "isolated antibody" means that the protein, polypeptide, or antibody (1) is not associated with its natural associated component in its native state, (2) does not contain other proteins from the same species, (3) is expressed by cells from a different species, or (4) does not occur naturally. Therefore, a chemically synthesized polypeptide or a polypeptide synthesized in a cellular system of a different natural source cell will be "isolated" from its natural associated component. Isolation can also render a protein substantially free of its natural associated component, i.e., using protein purification techniques well known in the art.

[0094] In peptides or proteins, suitable conserved amino acid substitutions are known to those skilled in the art and can generally be performed without altering the biological activity of the resulting molecule. Typically, those skilled in the art recognize that single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter its biological activity (see, for example, Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub.co., p. 224).

[0095] As used herein, the term "GLP-1" generally refers to glucagon-like peptide-1. Natural GLP-1 molecules are processed in vivo, cleaving the first six amino acids; therefore, it is customary in the art to designate the first N-terminal amino acid of the GLP-1 amino acid sequence as position 7 and the last C-terminal amino acid as position 37. The processed peptide can be further modified in vivo by removing the C-terminal glycine residue and replacing it with an amide group. GLP-1 typically exists in two biologically active forms: GLP-1(7-37)OH and GLP-1(7-36)NH2. The "GLP-1" described in this application includes natural, synthetic, or modified GLP-1 proteins, as well as complete GLP-1 proteins or functional fragments thereof, and GLP-1 proteins in different biologically active forms. For example, wild-type human GLP-1 may contain the amino acid sequence shown in SEQ ID NO:66, with the N-terminal amino acid residue H designated as position 7. Therefore, the term "K26" in this application generally refers to the 26th amino acid position counting from the 7th H position at the N-terminus of the GLP-1 protein, which is amino acid K in the amino acid sequence shown in SEQ ID NO:66; the term "W31" generally refers to the 31st amino acid position counting from the 7th H position at the N-terminus of the GLP-1 protein, which is amino acid W in the amino acid sequence shown in SEQ ID NO:66. In this application, when describing amino acid mutations and / or substitutions, the numbering of amino acid residues for GLP-1 peptide variants is different from the numbering of amino acid residues in the Fc region, which is numbered according to EU encoding.

[0096] The term "GLP-1R" typically refers to the glucagon-like peptide-1 receptor. GLP-1R binding to GLP-1 activates a signaling cascade, leading to activation of adenylate cyclase and an increase in intracellular cAMP levels. The GLP-1R described in this application may include the full-length molecule, variants, fragments, or synthetic compounds of natural GLP-1R, as long as they retain the activity of GLP-1R. For example, the GLP-1R described in this application may contain the amino acid sequence shown in NCBI database accession number NP_002053.3.

[0097] The term “GLP-1R agonist” generally refers to substances that bind to and activate the GLP-1 receptor, including but not limited to GLP-1 and its active variants.

[0098] As used herein, the term "at least partial activity of human GLP-1" generally refers to having one or more activities of the human GLP-1 protein, or having at least 20% (e.g., at least 25%, 30%, 35%, 40%, 45%, or 50% or more) of the activity of the human GLP-1 protein. The human GLP-1 in "at least partial activity of human GLP-1" can be wild-type, such as the amino acid sequence shown in SEQ ID NO: 66. The human GLP-1 in "at least partial activity of human GLP-1" can be a GLP-1 variant modified from wild-type human GLP-1, such as the amino acid sequence shown in SEQ ID NO: 68. For example, the human GLP-1 peptide variant of this application can have at least partial activity of human GLP-1 with the amino acid sequence shown in SEQ ID NO: 66. In other cases, the human GLP-1 peptide variant of this application can have at least partial activity of human GLP-1 with the amino acid sequence shown in SEQ ID NO: 68.

[0099] The activity is not required to be at the same level as human GLP-1 protein; it can be higher, similar to, or lower than that of human GLP-1 protein. In some cases, "at least part of the activity of human GLP-1" can refer to one or more selected from the group consisting of: activity binding to the GLP-1 receptor, activity activating the GLP-1 receptor, activity activating adenylate cyclase, activity promoting an increase in intracellular cyclic adenosine monophosphate (cAMP) levels, activity positively regulating intracellular Ca2+ levels, activity stimulating insulin secretion, activity increasing liver glycogen storage, activity delaying gastric emptying, activity inhibiting gastric motility, activity reducing appetite, activity inhibiting β-cell apoptosis, activity inhibiting postprandial glucagon secretion, activity alleviating hypoglycemia, and activity reducing weight. For example, it can be detected by measuring the ability to bind to the GLP-1 receptor and the expression level of cAMP. For example, it can be detected by detecting the activation level of the cAMP / PKA signaling pathway using a luciferase assay. The “at least partial activity of human GLP-1” can be at least partial activity of a fusion protein containing human GLP-1 (e.g., a fusion protein with the Fc region).

[0100] As used herein, the terms “polynucleotide” and “nucleic acid molecule” refer to oligomers or polymers containing at least two linked nucleotides or nucleotide derivatives, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) which are typically linked together by phosphodiester bonds.

[0101] As used herein, isolated nucleic acid molecules are nucleic acid molecules isolated from other nucleic acid molecules present in natural sources of nucleic acid molecules. “Isolated” nucleic acid molecules, such as cDNA molecules, may be substantially free of other cellular material or culture medium when prepared by recombinant technology, or substantially free of chemical precursors or other chemical components when chemically synthesized. Exemplary isolated nucleic acid molecules provided herein include isolated nucleic acid molecules encoding provided antibody or antigen-binding fragments.

[0102] Sequence “identity” has a generally accepted meaning in the art, and the percentage of sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated using publicly available techniques. Sequence identity can be measured along the full length of a polynucleotide or polypeptide or along a region of that molecule. (See, for example: 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., M Stockton Press, New York, 1991). Although there are many methods for measuring the identity between two polynucleotides or polypeptides, the term “identity” is well known to those skilled in the art (Carrillo, H. & Lipman, D., SIAM J Applied Math 48:1073 (1988)).

[0103] As used herein, “operably linked” in relation to a nucleic acid sequence, region, element, or domain indicates that the nucleic acid regions are functionally related to each other. For example, a promoter can be operably linked to a nucleic acid encoding a polypeptide, thereby regulating or mediating the transcription of that nucleic acid.

[0104] As used herein, “expression” refers to the process by which a polypeptide is produced through the transcription and translation of polynucleotides. The expression level of a polypeptide can be evaluated using any method known in the art, including, for example, methods for determining the amount of polypeptide produced from host cells. Such methods may include, but are not limited to, quantifying polypeptides in cell lysates by ELISA, Coomassie blue staining following gel electrophoresis, Lowry protein assays, and Bradford protein assays.

[0105] As used herein, a “host cell” is a cell used to receive, maintain, replicate, and amplify a vector. Host cells can also be used to express the polypeptide encoded by the vector. When a host cell divides, the nucleic acids contained in the vector replicate, thereby amplifying the nucleic acids. Host cells can be eukaryotic or prokaryotic cells. Suitable host cells include, but are not limited to, CHO cells, various COS cells, HeLa cells, and HEK cells such as HEK 293 cells.

[0106] As used herein, a "vector" is a reproducible nucleic acid from which one or more heterologous proteins can be expressed when the vector is transformed into a suitable host cell. Vectors include those into which nucleic acids encoding polypeptides or fragments thereof can typically be introduced via restriction enzyme digestion and ligation. Vectors also include those containing nucleic acids encoding polypeptides. Vectors are used to introduce nucleic acids encoding polypeptides into host cells for amplification of nucleic acids or for expression / display of the polypeptide encoded by the nucleic acid. Vectors are typically kept free but can be designed to integrate genes or portions thereof into the chromosome of the genome. Vectors for artificial chromosomes, such as yeast artificial vectors and mammalian artificial chromosomes, are also considered. The selection and use of such vectors are well known to those skilled in the art.

[0107] As used in this article, vectors also include "viral vectors." Viral vectors are engineered viruses that are operatively linked to foreign genes to transfer (as a medium or shuttle) the foreign genes into cells.

[0108] As used herein, "expression vector" includes a vector capable of expressing DNA operatively linked to regulatory sequences, such as promoter regions, that influence the expression of such DNA fragments. These additional fragments may include promoter and terminator sequences and optionally include one or more origins of replication, one or more selection markers, enhancers, polyadenylation signals, etc. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both. Therefore, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, bacteriophage, recombinant virus, or other vector, which, when introduced into a suitable host cell, results in the expression of clonal DNA. Suitable expression vectors are well known to those skilled in the art and include reproducible expression vectors in eukaryotic and / or prokaryotic cells, as well as expression vectors that remain free or are integrated into the host cell genome.

[0109] As used herein, “treatment” for an individual suffering from a disease or disease condition means that the individual’s symptoms are partially or completely relieved, or remain unchanged after treatment. Therefore, treatment includes prevention, treatment, and / or cure. Prevention refers to preventing underlying disease and / or preventing the worsening of symptoms or the development of disease. Treatment also includes any antibodies or antigen-binding fragments thereof provided, and any pharmaceutical use of the compositions provided herein.

[0110] As used in this article, “therapeutic effect” refers to the effect resulting from treatment of an individual, which alters, usually improves or enhances the symptoms of a disease or condition, or cures a disease or condition.

[0111] As used herein, "therapeutic effective amount" or "therapeutic effective dose" refers to an amount of substance, compound, material, or composition containing a compound that, when applied to a subject, is at least sufficient to produce a therapeutic effect. Therefore, it is the amount necessary to prevent, cure, improve, block, or partially block the symptoms of a disease or condition.

[0112] As used herein, the term “and / or” refers to any one of the options or two or more of the options.

[0113] As used herein, the terms “comprising” or “including” mean to include the stated elements, integers, or steps, but do not exclude any other elements, integers, or steps. In this document, when the terms “comprising” or “including” are used, unless otherwise specified, they also cover combinations of the stated elements, integers, or steps.

[0114] The term "administration" generally refers to a method of administering a dose of a compound or pharmaceutical composition to a subject (e.g., a patient). Administration can be performed by any suitable means, including parenteral, intrapulmonary, and intranasal administration, as well as intralesional administration (if necessary for local treatment). Parenteral administration includes, for example, intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In this application, the term "about" generally refers to a variation within a range of 0.5% to 10% above or below a specified value, such as variations within the 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. Unless otherwise specified, all values ​​mentioned in this application are considered modified by "about". If there is any doubt, or if the error range for a particular value or parameter is not generally understood in the field, then “about” means ±5% of that value or parameter.

[0115] The term "pharmaceutical composition" refers to a composition which is present in a form that allows the biological activity of the active ingredient contained therein to be effective, and which does not contain any additional ingredients that would have unacceptable toxicity to a subject administering the composition.

[0116] When used in this article, "treatment" means to slow down, interrupt, block, alleviate, stop, reduce, or reverse the progression or severity of existing symptoms, conditions, illnesses, or diseases.

[0117] The term "therapeutic agent" as used herein encompasses any active substance or agent that is effective in preventing or treating a disease to be treated.

[0118] The terms "drug combination" or "combination product" refer to non-fixed combination products or fixed combination products, including but not limited to pillboxes and pharmaceutical compositions. The term "non-fixed combination" means that the active ingredients (e.g., (i) antibodies or antigen-binding fragments thereof disclosed herein, and (ii) other therapeutic agents) are administered to a patient simultaneously, without a specific time limit, or sequentially at the same or different time intervals, in separate entities, wherein such administration to the patient provides a preventive or therapeutically effective level. The term "fixed combination" means that two or more active ingredients are administered to a patient simultaneously in the form of a single entity. Preferably, the dosage and / or time interval of the two or more active ingredients are selected so that the combined use of the components produces an effect greater than that achievable by using any one ingredient alone in treating a disease or condition. The components may be in separate formulations, and their formulations may be the same or different.

[0119] The term "lean mass," also known as "lean body mass," "lean weight," or "fat-free body mass," is composed of the weight of body cells, extracellular water, and the solid portion after fat removal. Its main components are bones and muscles. Unless otherwise specified, the lean body mass referred to in this article can be measured using a Bruker Minispec LF50 instrument.

[0120] The term "fat mass," also known as "liposome weight," refers to the weight of fat stored in the body. Unless otherwise specified, lean body mass as used in this article can be measured using a Bruker minispec LF50 instrument.

[0121] Invention Details

[0122] I. Antibodies targeting activin receptor II

[0123] This disclosure relates to an antibody or antigen-binding fragment thereof targeting activin receptor II, said antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, said heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 of the heavy chain variable region shown in SEQ ID NO: 33; and / or said light chain variable region comprising LCDR1, LCDR2 and LCDR3 of the light chain variable region shown in SEQ ID NO: 44.

[0124] In some embodiments, the HCDR1 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 1:

[0125] GYTFX1SSYIN(SEQ ID NO: 1), where X1 is selected from Y, W and F.

[0126] In some embodiments, the HCDR1 comprises, or is composed of, the amino acid sequence shown in any one of SEQ ID NO: 2-4.

[0127] In some embodiments, the HCDR2 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 5:

[0128] TINPVSGX2TSYAQKFQG(SEQ ID NO: 5), where X2 is Q or N.

[0129] In some embodiments, the HCDR2 comprises, or is composed of, the amino acid sequence shown in SEQ ID NO: 6 or 7.

[0130] In some embodiments, the HCDR3 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 8.

[0131] X3GWFDY (SEQ ID NO: 8), where X3 is selected from T, S, A and R.

[0132] In some embodiments, the HCDR3 comprises, or is composed of, the amino acid sequence shown in any one of SEQ ID NO: 9-12.

[0133] In some embodiments, the heavy chain variable region of the antibody or its antigen-binding fragment comprises: HCDR1, which comprises or is composed of the amino acid sequence shown in SEQ ID NO: 1; HCDR2, which comprises or is composed of the amino acid sequence shown in SEQ ID NO: 5; and HCDR3, which comprises or is composed of the amino acid sequence shown in SEQ ID NO: 8.

[0134] In some embodiments, the heavy chain variable region of the antibody or its antigen-binding fragment comprises: HCDR1, which comprises or consists of the amino acid sequence shown in any one of SEQ ID NO: 2-4; HCDR2, which comprises or consists of the amino acid sequence shown in SEQ ID NO: 6 or 7; and HCDR3, which comprises or consists of the amino acid sequence shown in any one of SEQ ID NO: 9-12.

[0135] In some embodiments, the heavy chain variable region of the antibody or its antigen-binding fragment comprises: HCDR1, which comprises or is composed of the amino acid sequence shown in SEQ ID NO: 2; HCDR2, which comprises or is composed of the amino acid sequence shown in SEQ ID NO: 6; and HCDR3, which comprises or is composed of the amino acid sequence shown in SEQ ID NO: 9.

[0136] In some embodiments, the antibody or its antigen-binding fragment further comprises a light chain variable region comprising: LCDR1, wherein the LCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO: 13; LCDR2, wherein the LCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO: 14; and LCDR3, wherein the LCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO: 15.

[0137] In some embodiments, the antibody or antigen-binding fragment thereof described in this disclosure comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, which comprises or is composed of the amino acid sequence shown in SEQ ID NO: 1; HCDR2, which comprises or is composed of the amino acid sequence shown in SEQ ID NO: 5; and HCDR3, which comprises or is composed of the amino acid sequence shown in SEQ ID NO: 8; the light chain variable region comprises: LCDR1, which comprises or is composed of the amino acid sequence shown in SEQ ID NO: 13; LCDR2, which comprises or is composed of the amino acid sequence shown in SEQ ID NO: 14; and LCDR3, which comprises or is composed of the amino acid sequence shown in SEQ ID NO: 15.

[0138] In some embodiments, the antibody or antigen-binding fragment thereof described in this disclosure comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: HCDR1, wherein HCDR1 comprises or consists of the amino acid sequence shown in any one of SEQ ID NO: 2-4; HCDR2, wherein HCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 6 or 7; and HCDR3, wherein HCDR3 comprises or consists of the amino acid sequence shown in any one of SEQ ID NO: 9-12; wherein the light chain variable region comprises: LCDR1, wherein LCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 13; LCDR2, wherein LCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 14; and LCDR3, wherein LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 15.

[0139] In some embodiments, the antibody or antigen-binding fragment thereof described in this disclosure comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: HCDR1, wherein HCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO: 2; HCDR2, wherein HCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO: 6; and HCDR3, wherein HCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO: 9; the light chain variable region comprises: LCDR1, wherein LCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO: 13; LCDR2, wherein LCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO: 14; and LCDR3, wherein LCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO: 15.

[0140] In some embodiments, the antibody or antigen-binding fragment thereof described in this disclosure comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 2, HCDR2 shown in SEQ ID NO: 6, and HCDR3 shown in SEQ ID NO: 9; and the light chain variable region comprises LCDR1 shown in SEQ ID NO: 13, LCDR2 shown in SEQ ID NO: 14, and LCDR3 shown in SEQ ID NO: 15.

[0141] In other embodiments, the heavy chain variable region of the antibody or its antigen-binding fragment described in this disclosure further includes a framework region, such as HFR1, HFR2, HFR3, and HFR4.

[0142] In some embodiments, the heavy chain variable region further comprises: HFR1, which comprises or is composed of the amino acid sequence shown in SEQ ID NO: 16 (QVQLVQSGAEVKKPGASVKVSCKAS); HFR2, which comprises or is composed of the amino acid sequence shown in SEQ ID NO: 17 (WVRQAPGQGLEWX4G), wherein X4 is selected from M, I, and L; HFR3, which comprises or is composed of the amino acid sequence shown in SEQ ID NO: 18 (RVTMTRDTSISTAYMELSX5LRSX6DTAVYYCAR), wherein X5 is selected from R, S, and T, and X6 is D or E; and HFR4, which comprises or is composed of the amino acid sequence shown in SEQ ID NO: 19 (WGQGTLVTVSS).

[0143] In some embodiments, the heavy chain variable region further comprises: HFR1, which comprises or is composed of the amino acid sequence shown in SEQ ID NO: 16; HFR2, which comprises or is composed of the amino acid sequence shown in any one of SEQ ID NO: 20-22; HFR3, which comprises or is composed of the amino acid sequence shown in any one of SEQ ID NO: 23-28; and HFR4, which comprises or is composed of the amino acid sequence shown in SEQ ID NO: 19.

[0144] In some embodiments, the heavy chain variable region further comprises: HFR1, which comprises or is composed of the amino acid sequence shown in SEQ ID NO: 16; HFR2, which comprises or is composed of the amino acid sequence shown in SEQ ID NO: 21; HFR3, which comprises or is composed of the amino acid sequence shown in SEQ ID NO: 23; and HFR4, which comprises or is composed of the amino acid sequence shown in SEQ ID NO: 19.

[0145] In other embodiments, the light chain variable region of the antibody or its antigen-binding fragment described in this disclosure further includes a framework region, such as LFR1, LFR2, LFR3, and LFR4.

[0146] In some embodiments, the light chain variable region further comprises: LFR1, which comprises or is composed of the amino acid sequence shown in SEQ ID NO: 29; LFR2, which comprises or is composed of the amino acid sequence shown in SEQ ID NO: 30; LFR3, which comprises or is composed of the amino acid sequence shown in SEQ ID NO: 31; and LFR4, which comprises or is composed of the amino acid sequence shown in SEQ ID NO: 32.

[0147] In some embodiments, the heavy chain variable region of the antibody or its antigen-binding fragment described in this disclosure comprises, or is composed of, the amino acid sequence shown in SEQ ID NO: 33:

[0148] QVQLVQSGAEVKKPGASVKVSCKASGYTFX1SSYINWVRQAPGQGLEWX4GTINPVSGX2TSYAQKFQGRVTMTRDTSISTAYMELSX5LRSX6DTAVYYCARX3GWFDYWGQGTLVTVSS (SEQ ID NO: 33), wherein X1 is selected from Y, W or F, X2 is Q or N, X3 is selected from T, S, A or R, X4 is I or L, X5 is S or T, and X6 is E.

[0149] In some embodiments, the heavy chain variable region of the antibody or its antigen-binding fragment described in this disclosure comprises, or is composed of, the amino acid sequence shown in any one of SEQ ID NO: 34-43.

[0150] In some embodiments, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 35 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or higher sequence identity with SEQ ID NO: 35, provided that it contains the T30Y, M48I, S57Q, R85S, D89E, and G99T mutations. In some embodiments, the heavy chain variable region comprises an amino acid sequence having about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with SEQ ID NO: 35, provided that it contains the T30Y, M48I, S57Q, R85S, D89E, and G99T mutations.

[0151] In some embodiments, the light chain variable region of the antibody or its antigen-binding fragment described in this disclosure comprises, or is composed of, the amino acid sequence shown in SEQ ID NO: 44 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or higher sequence identity with SEQ ID NO: 44.

[0152] In some embodiments, the heavy chain of the antibody or antigen-binding fragment thereof described in this disclosure further comprises a constant region or a variant thereof, such as a constant region derived from mouse or human. In some embodiments, the heavy chain comprises a constant region of human IgG1, IgG2, IgG3, or IgG4 or a variant thereof; for example, the heavy chain comprises a constant region, which may comprise or consist of the amino acid sequence described in SEQ ID NO: 45.

[0153] In some embodiments, the Fc region of the heavy chain of the antibody or its antigen-binding fragment may further contain mutations that reduce or eliminate effector function. The effector function may be antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC), etc. Specifically, the mutations that reduce or eliminate effector function weaken or eliminate the binding between immunoglobulins and FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb, and / or C1q.

[0154] The mutation that weakens or eliminates the effector function can be any known mutation or combination of mutations in the prior art, for example, Esohe EI, J Immunol 2000; 164:4178-4184; Hutchins, JT, Proc. Natl. Acad. Sci. USA 1995, 92, 11980–11984; Xu D., Cell Immunol. 2000, 200, 16–26; Hezareh, M., J. Virol. 2001, 75, 12161–12168; Schlothauer, T., Protein Eng. Des. Sel. 2016, 29, 457–466; Chu, SY, Mol. Immunol. 2008, 45, 3926–3933; Sazinsky, SL, Proc. Natl. Acad. Sci. USA 2008, 105, 20167–20172; Oganesyan, V., Acta Crystallogr.Sect.D Biol.Crystallogr.2008, 64Pt 6, 700–704; An, Z., MAbs 2009, 1, 572–579; Moore, GL, Methods 2019, 154, 38–50; Schlothauer, T., Protein Eng.Des.Sel.2016, 29, 457–466; Strohl, W., US20150337053; Engelberts, PJ, EBioMedicine 2020, 52, 102625, etc.

[0155] In some embodiments, the Fc region may further include mutations selected from the following that reduce or eliminate effector function: D265A, D270A, N297A, N297Q, N297G, N297D, K322A, P329A, P331G, D265A / P331G, L235A / G237A / E318A, L234A / L235A, S228P / L235E, G236R / L328R, S29 8G / T299A, L234F / L235E / P331S, H268Q / V309L / A330S / P331S, E233P / L234V / L235A / G236del / S267K, L234A / L235A / P329G, L234F / L235E / D265A, or V234A / G237A / P238S / H268A / V309L / A330S / P331S. Preferably, the Fc region may further contain mutations selected from the following that reduce or eliminate effector function: L234A / L235A (hereinafter abbreviated as AA) or D265A / P331G (hereinafter abbreviated as AG).

[0156] In some embodiments, the heavy chain of the antibody or its antigen-binding fragment contains a constant region comprising, or being composed of, the amino acid sequence shown in SEQ ID NO: 46 or 47.

[0157] In some embodiments, the Fc region of the heavy chain of the antibody or its antigen-binding fragment may also contain a mutation that modulates its ability to bind to FcRn. For example, the Fc region variant improves binding to FcRn under acidic pH conditions without affecting binding to FcRn under neutral pH conditions, thereby achieving a longer half-life compared to the parent.

[0158] The mutation regulating the binding affinity to FcRn and / or prolonging the half-life can be any known mutation or combination of mutations in the prior art, such as those described in Dall'Acqua W, J. Immunol. 2002; 169:5171–5180; Dall'Acqua W, J. Biol. Chem. 2006b; 281:23514–23524; Hinton PR, J. Immunol. 2006; 176:346–356; Petkova, J. Exp. Med. 2006; 203:275–280; Yeung, J. Immunol. 2009; 182:7663–7671; Zalevsky, Nat. Biotechnol. 2010; 28:157–159; Monnet, MAbs. 2014; 6:422–436, etc.

[0159] In some embodiments, the Fc region may contain mutations selected from the following that regulate the binding affinity to FcRn and / or prolong the half-life: M252Y / S254T / T256E, T250Q / M428L, N434A, M428L / N434S, N315D / A330V / N361D / A378V / N434Y, E294D / T307P / N434Y, V259I / N315D / N434Y, T307A / N315D / A330V / E382V / N389T / N434Y, or L234F / L235E / D265A. Preferably, the immunoglobulin Fc region variant may include mutations selected from the following that regulate the binding ability to FcRn and / or prolong the half-life: M252Y / S254T / T256E (hereinafter abbreviated as YTE) or M428L / N434S (hereinafter abbreviated as LS).

[0160] In some embodiments, the antibody or its heavy chain contains a constant region comprising, or being composed of, the amino acid sequence shown in SEQ ID NO: 48 or 49.

[0161] In some embodiments, the heavy chain of the antibody or its antigen-binding fragment includes a constant region, and the Fc region of the constant region may simultaneously contain mutations that reduce or eliminate effector function, as well as mutations that regulate its binding ability to FcRn. In some embodiments, the Fc region of the constant region simultaneously contains AA / LS, AA / YTE, AG / LS, or AG / YTE mutations. In some embodiments, the heavy chain of the antibody or its antigen-binding fragment includes a constant region, and the constant region contains, or is composed of, the amino acid sequence shown in any one of SEQ ID NO: 50-53.

[0162] In some embodiments, the heavy chain of the antibody or its antigen-binding fragment described in this disclosure comprises, or is composed of, the amino acid sequence shown in any one of SEQ ID NO: 54-63, 88-89.

[0163] In some embodiments, the heavy chain of the antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 57 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or higher sequence identity with SEQ ID NO: 57, provided that it contains, or is composed of, the T30Y, M48I, S57Q, R85S, D89E, and G99T mutations. In some embodiments, the heavy chain of the antibody or its antigen-binding fragment comprises an amino acid sequence having about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with SEQ ID NO: 57, provided that it contains, or is composed of, the T30Y, M48I, S57Q, R85S, D89E, and G99T mutations.

[0164] In some embodiments, the light chain of the antibody or antigen-binding fragment thereof described in this disclosure further comprises a constant region or a variant thereof, for example, a constant region derived from mouse or human. In some embodiments, the light chain comprises a constant region of human Igλ or a variant thereof; for example, the light chain comprises a constant region, which may comprise or consist of the amino acid sequence described in SEQ ID NO: 64.

[0165] In some embodiments, the light chain of the antibody or antigen-binding fragment thereof described in this disclosure comprises, or is composed of, the amino acid sequence shown in SEQ ID NO: 65. In some embodiments, the light chain of the antibody or antigen-binding fragment thereof described in this disclosure is derived from Bimagrumab.

[0166] In some embodiments, the antibody or antigen-binding fragment thereof disclosed herein targets ActRIIA and / or ActRIIB.

[0167] In some embodiments, the antibody or antigen-binding fragment thereof disclosed herein has at least one of the following characteristics:

[0168] (1) Capable of blocking the activation of signaling pathways induced by Activin A, GDF-8 and / or GDF-11, for example, the antibody or its antigen-binding fragment blocks the IC50 activation of the above signaling pathways. 50The value is not higher than that of Bimagrumab, or even lower than that of Bimagrumab; for example, the IC50 value of the antibody or its antigen-binding fragment blocking the activation of the Activin A and / or GDF-8-induced signaling pathway is ≤1000pM, preferably ≤500pM, more preferably ≤300pM, and even more preferably ≤200pM; the IC50 value of the antibody or its antigen-binding fragment blocking the activation of the GDF-11-induced signaling pathway is ≤100μg / ml, preferably ≤75μg / ml, more preferably ≤50μg / ml, and even more preferably ≤25μg / ml;

[0169] (2) The equilibrium dissociation constant (KD) with human ActRIIA and / or ActRIIB is not higher than that with Bimagrumab, and is even lower than that with Bimagrumab. The KD value can be detected using biomembrane interference technology. For example, the KD value of the antibody or its antigen-binding fragment with human ActRIIA is ≤1000 pM, preferably ≤750 pM, more preferably ≤500 pM, and even more preferably ≤250 pM; or, for example, the KD value of the antibody or its antigen-binding fragment with human ActRIIB is ≤100 pM, preferably ≤50 pM, more preferably ≤20 pM, and even more preferably ≤10 pM; and

[0170] (3) Able to increase lean body mass and / or lean body mass / body weight percentage in mammals, and / or able to decrease fat mass and / or fat mass / body weight percentage in mammals; the aforementioned mammals may be mice, rats, rabbits, cynomolgus monkeys and humans, etc.

[0171] II. Drug Combinations and Compositions

[0172] This disclosure also relates to a pharmaceutical combination comprising an antibody against activin receptor II or an antigen-binding fragment thereof as described in the first aspect, and a GLP-1R agonist.

[0173] In some embodiments, the GLP-1R agonist comprises human GLP-1 or a variant thereof, or a functional fragment of human GLP-1 or a variant thereof, or exenatide or a variant thereof, or a functional fragment of exenatide or a variant thereof.

[0174] The GLP-1R agonist has at least some of the activity of human GLP-1. For example, in some cases, the GLP-1R agonist may have one or more activities selected from the group consisting of: GLP-1 receptor binding activity, GLP-1 receptor activation activity, adenylate cyclase activation activity, activity promoting the increase of intracellular cyclic adenosine monophosphate (cAMP) levels, and positive regulation of intracellular Ca2+. +The activity of the GLP-1R agonist includes: stimulation of insulin secretion, increase of liver glycogen storage, delay of gastric emptying, inhibition of gastric motility, reduction of appetite, inhibition of β-cell apoptosis, inhibition of postprandial glucagon secretion, relief of hypoglycemia, and weight loss. For example, the activity of the GLP-1R agonist can be detected by measuring its ability to bind to the GLP-1 receptor and the level of cAMP expression. The activity of the GLP-1R agonist can be at least 1% (e.g., at least 5%, at least 10%, at least 20%, at least 25%, 30%, 35%, 40%, 45%, or 50% or higher) of human GLP-1 activity.

[0175] In some embodiments, the GLP-1R agonist comprises human GLP-1 (SEQ ID NO: 66).

[0176] In some embodiments, the GLP-1R agonist comprises a human GLP-1 variant, such as:

[0177] Arg 34 -GLP-1(7-37); Glu 30 -GLP-1(7-37); Lys 22 -GLP-1(7-37); Gly 8,36 Glu 22 -GLP-1(7-37); Val 8 Glu 22 Gly 36 -GLP-1(7-37); Gly 8,36 Glu 22 Lys 33 Asn 34 -GLP-1(7-37); Val 8 Glu 22 Lys 33 Asn 34 Gly 36 -GLP-1(7-37); Gly 8,36 Glu 22 Pro 37 -GLP-1(7-37); Val 8 Glu 22 Gly 36 Pro 37 -GLP-1(7-37); Gly 8,36 Glu 22 Lys 33 Asn 34 Pro 37 -GLP-1(7-37); Val8 ,Glu 22 ,Lys 33 ,Asn 34 ,Gly 36 ,Pro 37 -GLP-1(7-37);Aib 8 ,Lys 37 -GLP-1(7-37);Aib 8 ,Lys 36 -GLP-1(7-37);Aib 8,22 ,Gly 36 -GLP-1(7-37);Aib 8 ,Glu 22 ,Gly 36 -GLP-1(7-37);Aib 8 ,Arg 34 -GLP-1(7-37);Aib 8 ,Tyr 16 ,Glu 22 ,Gly 36 -GLP-1(7-37);Aib 8 ,Lys 18,33 ,Glu 22,23,30 ,Val 25 ,Arg 26 ,Leu 27 ,Asn 34 ,Gly 36 -GLP-1(7-37);Aib 8 ,Lys 18,33 ,Glu 22,23,30 ,Leu 27 ,Gly 36 -GLP-1(7-37);Aib 8,22 ,Ile 9 ,Gly 36 -GLP-1(7-37);Aib 8,22 ,Glu 15 ,Gly 36 -GLP-1(7-37);Gly 8,36 ,Glu 22 -GLP-1(7-36);Val 8 ,Glu 22 ,Gly 36 -GLP-1(7-36);Val 8 ,Glu 22 ,Asn 34 ,Gly 36 -GLP-1(7-36);Gly8,36 Glu 22 Asn 34 -GLP-1(7-36);

[0178] In some embodiments, the GLP-1R agonist comprises exenatide (Exendin-4, Ex-4) or a variant thereof, such as: Exendin-4 (SEQ ID NO: 67); Leu 14 -Ex-4; Leu 14 Phe 25 -Ex-4; Leu 14 Ala 19 Phe 25 -Ex-4; Leu 14 Lys 17,20 Ala 19 Glu 21 Phe 25 ,Gln 28 -Ex-4; Leu 14 Lys 17,20 Ala 19 Glu 21 ,Gln 28 -Ex-4; Phe 4 Leu 14 Lys 17,20 Ala 19 Glu 21 ,Gln 28 -Ex-4; Val 11 ,Ile 13 Leu 14 Ala 16 Lys 21 Phe 25 -Ex-4; Ex-4-Lys 40 .

[0179] The variant description includes "AA" m "", means that in wild-type human GLP-1 (SEQ ID NO: 66) or exenatide (SEQ ID NO: 67), the original amino acid residue is mutated to AA at position m, where m is an integer (m starts from 7 for human GLP-1; m starts from 1 for exenatide), and AA is an abbreviation for any amino acid residue.

[0180] In some embodiments, the GLP-1R agonist described herein may comprise: GLP-1(7-37), GLP-1(7-36)-NH2, liraglutide, abiglutide, lixisenatide, dulaglutide, smegglutide, exendin-4, exendin-3, polyethylene glycol loxenatide, telpopotide, GMA102, PB119, ibennatide, mazdutide, supaglutide, cotadutide, Danuglipron, HM-15211, efinopegdutide, etc.

[0181] In some embodiments, the GLP-1R agonist comprises a human GLP-1 variant having, for example, the amino acid sequence shown in SEQ ID NO: 68.

[0182] In some embodiments, the human GLP-1 variant is selected from the GLP-1 variant sequence disclosed in patent application WO2022143516, the entire text of which is incorporated herein by reference.

[0183] In some embodiments, the GLP-1R agonist comprises a human GLP-1 variant, which may further include amino acid substitutions at one or more positions of Y19, K26, and W31 compared to the amino acid sequence shown in SEQ ID NO: 68.

[0184] In some embodiments, the GLP-1R agonist comprises a human GLP-1 variant, which may further include amino acid substitutions at positions Y19, K26, and W31 compared to the amino acid sequence shown in SEQ ID NO: 68. For example, the amino acid substitutions at positions Y19, K26, and W31 are selected from combinations of the following mutations:

[0185] a) Y19A, K26R, and W31Y;

[0186] b) Y19F, K26R, and W31Y;

[0187] c) Y19L, K26R, and W31Y;

[0188] d)Y19T, K26R, and W31Y;

[0189] e)Y19I, K26R and W31Y;

[0190] f)Y19V, K26R and W31Y; and

[0191] g)Y19S, K26R and W31Y.

[0192] In some embodiments, the GLP-1R agonist comprises the amino acid sequence shown in any one of SEQ ID NO: 69-75.

[0193] In some embodiments, the GLP-1R agonist further comprises an immunoglobulin Fc region.

[0194] In some embodiments, the immunoglobulin Fc region is an Fc region derived from IgG; preferably, the immunoglobulin Fc region is an Fc region derived from IgG1, IgG2, IgG3, or IgG4. For example, the immunoglobulin Fc region contains an amino acid sequence as shown in SEQ ID NO: 76.

[0195] In some embodiments, the immunoglobulin Fc region contains an amino acid mutation that, compared to the unmutated immunoglobulin Fc region, selectively enhances the binding affinity of the immunoglobulin Fc region to the Fc receptor; in some preferred embodiments, the amino acid mutation selectively enhances the binding affinity of the immunoglobulin Fc region to the neonatal Fc receptor (FcRn). In some embodiments, enhanced binding affinity selectivity refers to increased binding affinity under acidic conditions compared to the unmutated state.

[0196] In some implementations, the aforementioned immunoglobulin Fc region may contain amino acid mutations at positions M252, S254, and / or T256, according to the Kabat EU index number.

[0197] In some implementations, the immunoglobulin Fc region may contain amino acid mutations at positions M252, S254, and T256, according to the Kabat EU index number.

[0198] In this disclosure, the Fc region may be an Fc region derived from IgG1, IgG2, IgG3, or IgG4, and may contain amino acid mutations of M252Y, S254T, and T256E. For example, the Fc region may be an Fc region derived from IgG1, and may contain amino acid mutations of M252Y, S254T, and T256E. For example, the Fc region may be an Fc region derived from IgG2, and may contain amino acid mutations of M252Y, S254T, and T256E. For example, the Fc region may be an Fc region derived from IgG3, and may contain amino acid mutations of M252Y, S254T, and T256E. For example, the Fc region may be an Fc region derived from IgG4, and may contain amino acid mutations of M252Y, S254T, and T256E.

[0199] In some embodiments, the immunoglobulin Fc region contains an amino acid sequence as shown in SEQ ID NO: 77.

[0200] In some embodiments, the GLP-1R agonist optionally includes a linker, preferably a peptide linker, between the human GLP-1 variant and the immunoglobulin Fc region. For example, the peptide linker has a length of 2-20 amino acids.

[0201] In some embodiments, the peptide linker may comprise an amino acid sequence as shown in any one of SEQ ID NO: 78-84.

[0202] In some embodiments, the human GLP-1 variant is located at the N-terminus of the peptide linker, and the immunoglobulin Fc is located at the C-terminus of the peptide linker; in other embodiments, the human GLP-1 variant is located at the C-terminus of the peptide linker, and the immunoglobulin Fc is located at the N-terminus of the peptide linker.

[0203] In some embodiments, the GLP-1R agonist comprises the amino acid sequence shown in SEQ ID NO: 85 or 86.

[0204] This disclosure also provides a composition, for example, preferably, a pharmaceutical composition, comprising an antibody or antigen-binding fragment thereof of this disclosure formulated with a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition further comprises a GLP-1R agonist as a second active substance, the GLP-1R agonist referring to the definition in Part II above.

[0205] As used herein, "pharmaceutically acceptable carriers" include any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, buffers, stabilizers, isotonic agents, and absorption delay agents. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., the antibody molecule, may be encapsulated in a material to protect it from acids and other natural conditions that could inactivate it.

[0206] The amount of active ingredient that can be combined with a carrier material to prepare a single-dose formulation varies depending on the target population and the specific route of administration. Generally, the amount of active ingredient that can be combined with a carrier material to prepare a single-dose formulation is the amount of the composition that produces the therapeutic effect. Typically, this amount, expressed as 100%, ranges from about 0.01% to about 99% of the active ingredient, for example, from about 0.1% to about 70%, or from about 1% to about 30% of the active ingredient, combined with a pharmaceutically acceptable carrier.

[0207] The actual dose level of the active ingredient in the pharmaceutical compositions disclosed herein may be varied to obtain an amount of active ingredient that is effective in achieving the desired therapeutic response in a particular patient, composition, and route of administration, without toxicity to the patient. The selected dose level depends on a variety of pharmacokinetic factors, including the activity of the specific composition of this disclosure or its esters, salts, or amides, the route of administration, the time of administration, the excretion rate of the specific compound applied, the duration of treatment, other drugs, compounds, and / or materials used in combination with the specific composition applied, the age, sex, weight, condition, general health status, and medical history of the patient receiving treatment, and similar factors known in the medical field.

[0208] The compositions disclosed herein can be administered via one or more routes of administration using one or more methods known in the art. Those skilled in the art will understand that the route and / or manner of administration varies depending on the desired outcome. Preferred routes of administration for the antibodies disclosed herein include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other parenteral routes, such as injection or infusion. As used herein, the phrase "parenteral administration" refers to a mode of administration other than enteral and local administration, typically by injection, including but not limited to intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, spinal, epidural, and intrasternal injections and infusions.

[0209] In some embodiments, this disclosure also provides a kit containing the drug combination, for example, the kit containing, within the same package:

[0210] - A first container containing a pharmaceutical composition comprising molecules of the present disclosure;

[0211] - Optionally, it also includes a second container containing a pharmaceutical composition comprising one or more other therapeutic agents (in some embodiments, the two or more other therapeutic agents are in the same container or in separate containers).

[0212] In some implementations, other therapeutic agents encompass any therapeutic agent used to treat the diseases described in this disclosure.

[0213] III. Nucleic acids, and the vectors and host cells containing them.

[0214] This disclosure provides nucleic acids encoding any of the antibodies or antigen-binding fragments thereof described above. This disclosure also covers nucleic acids that hybridize with the nucleic acids described above under stringent conditions, nucleic acids having one or more substitutions (e.g., conserved substitutions), deletions, or insertions compared to the nucleic acids described above, or nucleic acid sequences having at least 80%, at least 85%, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity compared to the nucleic acids described above.

[0215] For example, the nucleic acid disclosed herein comprises a nucleic acid encoding an amino acid sequence selected from any one of SEQ ID NO:1-89, or a nucleic acid encoding an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from any one of SEQ ID NO:1-89.

[0216] As will be apparent to those skilled in the art, due to codon degeneracy, the amino acid sequence of each antibody or its antigen-binding fragment can be encoded by multiple nucleic acid sequences. The nucleic acid sequence encoding the molecule disclosed herein can be generated using methods well known in the art, such as de novo solid-phase DNA synthesis or by PCR amplification.

[0217] In one embodiment, the nucleic acid encoding the antibody or antigen-binding fragment thereof disclosed herein may be in the same vector or in different vectors. In yet another embodiment, the nucleic acids encoding the strands of the antibody or antigen-binding fragment thereof disclosed herein may be introduced into the same or different host cells for expression. Therefore, in some embodiments, a method for producing the antibody or antigen-binding fragment thereof disclosed herein includes the steps of: culturing host cells containing nucleic acids encoding the strands of the molecule under conditions suitable for expression of the strands of the molecule to produce the antibody or antigen-binding fragment thereof disclosed herein.

[0218] On the other hand, this disclosure provides vectors comprising the aforementioned nucleic acids. The term "vector" generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which transfers inserted nucleic acid molecules into host cells and / or between host cells. The vectors may include vectors primarily for inserting DNA or RNA into cells, vectors primarily for replicating DNA or RNA, and expression vectors primarily for transcription and / or translation of DNA or RNA. The vectors also include vectors having a variety of the aforementioned functions. The vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, by culturing suitable host cells containing the vector, the vector can produce the desired expression product.

[0219] In a preferred embodiment, the vector is an expression vector. This expression vector typically contains at least one nucleic acid of this disclosure, operably linked to one or more suitable expression regulatory elements (e.g., promoters, enhancers, terminators, etc.). Selection of these elements and their sequences for expression in a particular host is common knowledge to those skilled in the art. Specific examples of regulatory elements and other elements useful or necessary for the expression of the fusion proteins of this disclosure include, for example, promoters, enhancers, terminators, integrators, selection markers, leader sequences, and reporter genes. Those skilled in the art will readily understand that vectors commonly used in the art to which this disclosure pertains can be applied to this disclosure.

[0220] In one embodiment, this disclosure provides a host cell comprising the nucleic acid or the vector.

[0221] The term "host cell" refers to a cell into which exogenous polynucleotides have been introduced, including the progeny of such cells. Host cells include "transformers" and "transformed cells," which include primary transformed cells and their derived progeny, regardless of the number of passages. Progeny may not be identical to the parent cells in terms of nucleic acid content and may contain mutations. Mutant progeny with the same function or biological activity screened or selected from the initially transformed cells are included herein. Host cells are any type of cell system that can be used to produce the antibody molecules disclosed herein, including eukaryotic cells, such as mammalian cells (e.g., CHO cells or HEK293 cells), insect cells, yeast cells; and prokaryotic cells, such as E. coli cells. Host cells include cultured cells, as well as cells within transgenic animals, transgenic plants, or cultured plant or animal tissues.

[0222] IV. Treatment Methods and Applications

[0223] This disclosure also provides a method for treating and / or preventing activin receptor II-related diseases or conditions, comprising administering to a patient in need one or more of the disclosed antibodies or antigen-binding fragments thereof, fusion proteins, bispecific or multispecific antibodies, nucleic acids, vectors, recombinant cells, pharmaceutical compositions or combinations of pharmaceuticals.

[0224] In some embodiments, the activin receptor II-related disease or condition refers to an indication associated with abnormal activation of ActRIIA or ActRIIB. In some embodiments, the activin receptor II-related disease or condition includes obesity, diabetes, muscular dystrophy, and / or cancer cachexia.

[0225] In some embodiments, this disclosure also provides a method for treating and / or preventing obesity, diabetes, muscle atrophy, and / or cancer cachexia, comprising administering to a patient in need one or more of the antibodies of this disclosure or their antigen-binding fragments, fusion proteins, bispecific or multispecific antibodies, nucleic acids, vectors, recombinant cells, drug combinations, and pharmaceutical compositions.

[0226] In some embodiments, this disclosure also provides a method for treating and / or preventing obesity, diabetes, muscle atrophy, and / or cancer cachexia, comprising administering to a patient in need one of the antibodies or antigen-binding fragments thereof of this disclosure, fusion proteins, bispecific or multispecific antibodies, nucleic acids, vectors, recombinant cells, and pharmaceutical compositions, as well as the GLP-1R agonist described in this disclosure.

[0227] In other embodiments, this disclosure also provides the use of the said antibody or its antigen-binding fragment, fusion protein, bispecific or multispecific antibody, nucleic acid, vector, recombinant cell, pharmaceutical combination, and pharmaceutical composition in the preparation of a medicament for the treatment and / or prevention of the related diseases or conditions mentioned herein, such as activin receptor II-related diseases or conditions. In some embodiments, the activin receptor II-related diseases or conditions include obesity, diabetes, muscular dystrophy, and / or cancer cachexia.

[0228] In some embodiments, this disclosure also provides the use of the antibody or its antigen-binding fragment, nucleic acid, carrier, and / or pharmaceutical composition in combination with the GLP-1R agonist described herein in the preparation of a medicament for the treatment and / or prevention of the related diseases or conditions mentioned herein. For example, the medicament is used to treat and / or prevent obesity, diabetes, muscular dystrophy, and / or cancer cachexia.

[0229] In other embodiments, antibodies or antigen-binding fragments thereof, nucleic acids, vectors, pharmaceutical compositions or combinations thereof are further provided for the treatment and / or prevention of the related diseases or conditions mentioned herein, such as activin receptor II-related diseases or conditions. In some embodiments, the activin receptor II-related diseases or conditions include obesity, diabetes, muscular dystrophy, and / or cancer cachexia.

[0230] In some embodiments, this disclosure also provides the antibody or its antigen-binding fragment, fusion protein, bispecific or multispecific antibody, nucleic acid, vector, recombinant cell and / or pharmaceutical composition, which can be used in combination with the GLP-1R agonist described herein for the treatment and / or prevention of the related diseases or conditions mentioned herein. In some embodiments, the diseases or conditions include obesity, diabetes, muscular dystrophy and / or cancer cachexia.

[0231] Example

[0232] Example 1: Antibody Acquisition and Preparation

[0233] Using AuraQuest and AuraPicasso platforms from Xunming Biotechnology (https: / / www.bigmarker.com / ciiwebinars / Next-Gen-Antibody-Engineering-for-GPCRs-by-integrating-Autonomous-Yeast-Display-and-AI or https: / / acrobiosystems.com.cn / S29-San-Diego-Antibody-Workshop.html), with Bimagrumab (containing VH as shown in SEQ ID NO: 87 and VL as shown in SEQ ID NO: 44) as the parent culture, yeast was auto-evolved for rapid mutation. Simultaneously, a key site mutation was predicted using an AI model, the mutant molecules were expressed in yeast, and dominant clones were screened by flow cytometry. After sequencing, the AI ​​model was optimized, and after multiple iterations, the variant AU003 was obtained, which has the following VH sequence:

[0234] The Bimagrumab heavy chain sequence bimaHC was synthesized, and its VH sequence was point-mutated according to AU003. This mutation was then cloned into a conventional mammalian expression vector to obtain the antibody heavy chain plasmid. The heavy chain bimaHC-AU003 has the amino acid sequence shown in SEQ ID NO: 57. The Bimagrumab light chain sequence bimaLC was synthesized and cloned into a conventional mammalian expression vector to obtain the antibody light chain plasmid. The light chain bimaLC has the amino acid sequence shown in SEQ ID NO: 65. The sequence of the final construct was verified by DNA sequencing.

[0235] A mixture of antibody heavy chain and antibody light chain plasmids was transfected into HEK293 cells for transient antibody expression. The recombinant expression plasmid was diluted with Freestyle293 medium and added to the required PEI (Polyethylenimine) solution for transformation. Each plasmid / PEI mixture was added to the HEK293 cell suspension, and the cells were incubated at 37°C, 5% CO2, and 130 rpm on a shaker. After four hours, EX-CELL293 medium (Merck, catalog number: 14571C-1000ML) and 2 mM glutamine were added, and the cells were incubated on a shaker at 130 rpm. After 24 hours, 3.8 mM VPA (sodium valproate, Sigma, catalog number: P4543-100g) was added. After 72 hours, 4 g / L glucose and 6 g / L peptone D (bottled TC YEASTOLATE UF, Gibco, catalog number: 2557875) were added. After culturing for 5-6 days, the supernatant of transient expression culture was collected and purified to obtain the target protein bima-AU003 by Protein A affinity chromatography.

[0236] Example 2: Characterization of antibody bioactivity in vitro

[0237] 2.1 Inhibition of ligand-induced SMAD2 / 3 activation

[0238] HEK293 cells (a HEK293 cell line expressing firefly luciferase reporter gene under the control of SMAD binding element (SBE), constructed by introducing the SBE-driven reporter gene sequence into the HEK293 genome using a lentiviral system) were resuspended in 2.5% FBS+DMEM, 25,000 cells / 50 μL per well, and incubated overnight at 37°C. Activin A (ACRO biosystems, Cat:#ACA-h421b, Lot:#RC20dyP1-233EW1-19J), GDF-8 (Abcam, Cat:#AB256090, Lot:#1038430-1), and GDF-11 (R&D, Cat:1958-GD, Lot:#MMW2623081) were prepared using DMEM (final concentrations of 10 ng / mL, 100 ng / mL, and 10 ng / mL, respectively), and 25 μL / well was added to each well of a 96-well plate. Bimagrumab or Bima-AU003 samples were prepared using DMEM (final concentration of 25 μg / mL), and 25 μL / well was added to each well of the 96-well plate. The plates were incubated at 37°C for 6 h or 20 h, and then 50 μL of substrate (One-Lite Luciferase Assay) was added to each well. The system (catalog number DD1203, Novizan) was used to detect luciferase activity. The results, shown in Figure 1, indicate that Bima-AU003 and Bimagrumab have similar effects in blocking Actvin A / GDF8 / GDF11-induced signaling pathway activation.

[0239] Using a similar method as described above, the blocking effects of Bima-AU003-FcAG (replacing the AA mutation in Bima-AU003 with the AG mutation, having the heavy chain shown in SEQ ID NO: 59 and the light chain shown in SEQ ID NO: 65) and Bima-AU003-FcAG-LS (adding the LS mutation to the AG mutation, having the heavy chain shown in SEQ ID NO: 89 and the light chain shown in SEQ ID NO: 65) on the GDF8 / GDF11-induced signaling pathway were tested. The results are shown in Figures 4A-4B. The blocking effects of Bima-AU003-FcAG and Bima-AU003-FcAG-LC on the signaling pathway were slightly better than those of Bimagrumab.

[0240] 2.2 Affinity to ActRIIA / B

[0241] The binding kinetics of bima-AU003 to human ACVR2A or ACVR2B were detected using biolayer interferometry (BLI) and compared with bimagrumab. Bimagrumab or bima-AU003 was directly immobilized onto the AHC sensor, and then the receptor proteins human ACVR2A (e.g., cat#10257-H08H, lot#LC17OC2514) were diluted to 5 concentrations (25, 12.5, 6.25, 3.13, 1.56 nM) or ACVR2B (e.g., cat#10229-H08H, lot#LC17DE0418) were diluted to 4 concentrations (50, 25, 12.5, 3.13 nM) to bind to the immobilized proteins. The equilibrium dissociation constant (KD), binding rate (ka), and dissociation rate (kdis) were calculated using Data Analysis HT 12.0.2.59 software. The results are shown in Table 1. Bima-AU003 has a higher affinity for human ActRIIA and ActRIIB than Bimagrumab.

[0242] Table 1. Affinity of Bima-AU003 and Bimagrumab with Human ACVR2A and ACVR2B

[0243] Example 3: Characterization of antibody bioactivity in vivo

[0244] 3.1 In vivo efficacy study in DIO mice

[0245] Twenty 25-week-old male DIO (diet-induced obesity) mice (purchased from Jicui Pharmaceutical) were randomly divided into 5 groups of 4 mice each based on their pre-fasting body weight. The groups were: G1 PBS, G2 Duyakrwy-C12-Fc4 (32.5 nmol / kg, sc), G3 Bimagrumab (141 nmol / kg, sc), G4 Bimagrumab + Duyakrwy-C12-Fc4 (141 + 32.5 nmol / kg, sc), and G5 Bima-AU003 + Duyakrwy-C12-Fc4 (141 + 32.5 nmol / kg, sc). Bimagrumab and Bima-AU003 were administered subcutaneously once weekly (sc), while Duyakrwy-C12-Fc4 was administered subcutaneously twice weekly (sc). The day of administration was designated D0, and the experimental endpoint was D28. Mouse body weight was measured twice weekly. On D28, body fat mass (fat mass and lean mass) was measured using small nuclear magnetic resonance imaging (MRI). Serum was collected at fixed time points to detect the blood concentrations of Bimagrumab or Bima-AU003. Anti-drug antibodies (ADA) were detected using ELISA on D0 and D21. The results are shown in Figures 2A-2F. Compared to Bimagrumab, Bima-AU003 exhibited a stronger muscle-building and fat-reducing effect in this model and was more stable in mice. Furthermore, in the ADA detection results, 2 out of 4 mice in group G4 tested positive for ADA, while no mice in group G5 tested positive for ADA. This indicates that Bima-AU003 has lower immunogenicity in this model compared to Bimagrumab.

[0246] Duyakrwy-C12-Fc4 is a long-acting GLP-1R agonist with the amino acid sequence shown in SEQ ID NO: 85.

[0247] 3.2 In vivo efficacy study in CB-17SCID mice

[0248] Twenty-five 10-week-old female CB17 / ICR-Prkdcscid / Crl (SCID) immunodeficient mice (purchased from Vital River) were randomly divided into five groups of five mice each, based on their pre-fasting body weight. The groups were: G1 PBS, G2 Bimagrumab (5 mg / kg), G3 Bimagrumab (20 mg / kg), G4 Bima-AU003 (5 mg / kg), and G5 Bima-AU003 (20 mg / kg). Bimagrumab and bima-AU003 were administered subcutaneously (sc) once weekly. The day of administration was designated D0, and the experimental endpoint was D25. Mouse body weight was measured on D0, D3, D7, D15, D18, and D21, and body fat mass (fat mass and lean mass) was measured on the endpoint D25. The results are shown in Figures 3A-3E. Compared with Bimagrumab, Bima-AU003 exhibited a stronger fat-reducing and muscle-building effect in this model.

[0249] Sequence information:

[0250] SEQ ID NO: 1(HCDR1-X1), where X1 represents any amino acid

[0251] SEQ ID NO: 2(HCDR1 X1=Y)

[0252] SEQ ID NO: 3 (HCDR1 X1 = W)

[0253] SEQ ID NO: 4 (HCDR1 X1 = F)

[0254] SEQ ID NO: 5(HCDR2-X2), where X2 represents any amino acid

[0255] SEQ ID NO: 6 (HCDR2 X2 = Q)

[0256] SEQ ID NO: 7(HCDR2 X2=N)

[0257] SEQ ID NO: 8(HCDR3-X3), where X3 represents any amino acid

[0258] SEQ ID NO: 9(HCDR3 X3=T)

[0259] SEQ ID NO: 10 (HCDR3 X3 = S)

[0260] SEQ ID NO: 11 (HCDR3 X3 = A)

[0261] SEQ ID NO: 12(HCDR3 X3=R)

[0262] SEQ ID NO: 13(LCDR1)

[0263] SEQ ID NO: 14(LCDR2)

[0264] SEQ ID NO: 15 (LCDR3)

[0265] SEQ ID NO: 16(HFR1)

[0266] SEQ ID NO: 17(HFR2-X4), where X4 represents any amino acid

[0267] SEQ ID NO: 18(HFR3-X5 X6), where X5 and X6 represent any amino acid.

[0268] SEQ ID NO: 19(HFR4)

[0269] SEQ ID NO: 20(HFR2 X4=M)

[0270] SEQ ID NO: 21(HFR2 X4=I)

[0271] SEQ ID NO: 22(HFR2 X4=L)

[0272] SEQ ID NO: 23(HFR3 X5=S X6=E)

[0273] SEQ ID NO: 24(HFR3 X5=R X6=E)

[0274] SEQ ID NO: 25(HFR3 X5=T X6=E)

[0275] SEQ ID NO:26(HFR3 X5=S X6=D)

[0276] SEQ ID NO:27(HFR3 X5=R X6=D)

[0277] SEQ ID NO:28(HFR3 X5=T X6=D)

[0278] SEQ ID NO: 29(LFR1)

[0279] SEQ ID NO: 30(LFR2)

[0280] SEQ ID NO: 31(LFR3)

[0281] SEQ ID NO: 32(LFR4)

[0282] SEQ ID NO:33(VH

[0283] SEQ ID NO:34(VH T30Y S57Q G99T)

[0284] SEQ ID NO:35(VH T30Y M48I S57Q R85SD89E G99T)

[0285] SEQ ID NO:36(VH T30W S57Q G99T)

[0286] SEQ ID NO:37(VH T30W M48I S57Q R85SD89E G99T)

[0287] SEQ ID NO:38(VH T30Y S57N G99T)

[0288] SEQ ID NO:39(VH T30Y M48I S57N R85SD89E G99T)

[0289] SEQ ID NO:40(VH T30Y S57Q G99S)

[0290] SEQ ID NO:41(VH T30Y M48I S57Q R85SD89E G99S)

[0291] SEQ ID NO:42(VH T30Y S57Q G99A)

[0292] SEQ ID NO:43(VH T30Y M48I S57Q R85SD89E G99A)

[0293] SEQ ID NO:44(VL)

[0294] SEQ ID NO:45(CH IgG1)

[0295] SEQ ID NO:46(CH IgG1 AA)

[0296] SEQ ID NO:47(CH IgG1 AG)

[0297] SEQ ID NO:48(CH IgG1 LS)

[0298] SEQ ID NO:49(CH IgG1 YTE)

[0299] SEQ ID NO:50(CH IgG1 AALS)

[0300] SEQ ID NO:51(CH IgG1 AAYTE)

[0301] SEQ ID NO:52(CH IgG1 AG LS)

[0302] SEQ ID NO: 53(CH IgG1 AG YTE)

[0303] SEQ ID NO: 54(HC T30Y S57Q G99T IgG1)

[0304] SEQ ID NO:55(HC T30Y M48I S57Q R85S D89E G99T IgG1)

[0305] SEQ ID NO: 56(HC T30Y S57Q G99T IgG1 AA)

[0306] SEQ ID NO:57(HC T30Y M48I S57Q R85S D89E G99T IgG1 AA)

[0307] SEQ ID NO:58(HC T30Y S57Q G99T IgG1 AG)

[0308] SEQ ID NO: 59(HC T30Y M48I S57Q R85S D89E G99T IgG1 AG)

[0309] SEQ ID NO: 60(HC T30Y S57Q G99T IgG1 LS)

[0310] SEQ ID NO:61(HC T30Y M48I S57Q R85S D89E G99T IgG1 LS)

[0311] SEQ ID NO: 62(HC T30Y S57Q G99T IgG1 YTE)

[0312] SEQ ID NO: 63(HC T30Y M48I S57Q R85S D89E G99T IgG1 YTE)

[0313] SEQ ID NO: 64(CL)

[0314] SEQ ID NO: 65(LC)

[0315] SEQ ID NO:66(GLP-1)

[0316] SEQ ID NO:67(Exendin-4)

[0317] SEQ ID NO:68(GLP-1-Dula)

[0318] SEQ ID NO:69(GLP-1-Dula Y19AK26R W31Y)

[0319] SEQ ID NO:70(GLP-1-Dula Y19F K26R W31Y)

[0320] SEQ ID NO:71(GLP-1-Dula Y19L K26R W31Y)

[0321] SEQ ID NO:72(GLP-1-Dula Y19T K26R W31Y)

[0322] SEQ ID NO:73(GLP-1-Dula Y19I K26R W31Y)

[0323] SEQ ID NO:74(GLP-1-Dula Y19V K26R W31Y)

[0324] SEQ ID NO:75(GLP-1-Dula Y19S K26R W31Y)

[0325] SEQ ID NO:76(IgG4 Fc)

[0326] SEQ ID NO:77(IgG4 Fc YTE)

[0327] SEQ ID NO:78

[0328] SEQ ID NO:79

[0329] SEQ ID NO:80

[0330] SEQ ID NO:81

[0331] SEQ ID NO:82

[0332] SEQ ID NO:83

[0333] SEQ ID NO:84

[0334] SEQ ID NO:85(Duyakrwy-C12-Fc4)

[0335] SEQ ID NO:86(Duyakrwy-C12-Fc4 YTE)

[0336] SEQ ID NO:87(Bimagrumab VH)

[0337] SEQ ID NO:88(HC T30Y M48I S57Q R85S D89E G99T IgG1 AALS)

[0338] SEQ ID NO:89(HC T30Y M48I S57Q R85S D89E G99T IgG1 AG LS)

Claims

1. An antibody or antigen-binding fragment thereof against Activin Receptor II, comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 in the heavy chain variable region set forth in SEQ ID NO: 33; and / or the light chain variable region comprising LCDR1, LCDR2 and LCDR3 in the light chain variable region set forth in SEQ ID NO:

44.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein, the HCDR1 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 2-4. the HCDR2 comprises an amino acid sequence set forth in SEQ ID NO: 6 or 7. the HCDR3 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 9-12. the heavy chain variable region comprises HCDR1 comprising an amino acid sequence set forth in SEQ ID NO: 2; HCDR2 comprising an amino acid sequence set forth in SEQ ID NO: 6; and HCDR3 comprising an amino acid sequence set forth in SEQ ID NO:

9.

3. The antibody or antigen-binding fragment thereof of claim 2, wherein, ​ 4. The antibody or antigen-binding fragment thereof of claim 2 or 3, wherein, ​ 5. The antibody or antigen-binding fragment thereof of any one of claims 2-4, wherein, ​ 6. The antibody or antigen-binding fragment thereof of any one of claims 2-5, wherein, ​ 7. The antibody or antigen-binding fragment thereof of any one of claims 2-6, the heavy chain variable region further comprising: HFR1 comprising the amino acid sequence set forth in SEQ ID NO: 16: QVQLVQSGAEVKKPGASVKVSCKAS, (SEQ ID NO: 16); HFR2 comprising the amino acid sequence set forth in SEQ ID NO: 17: WVRQAPGQGLEWX4G (SEQ ID NO: 17), wherein X4 is selected from M, I, and L; HFR3 comprising the amino acid sequence set forth in SEQ ID NO: 18: RVTMTRDTSISTAYMELSX5LRSX6DTAVYYCAR (SEQ ID NO: 18), wherein X5 is selected from R, S, and T, and X6 is D or E; and HFR4 comprising the amino acid sequence set forth in SEQ ID NO: 19: WGQGTLVTVSS (SEQ ID NO: 19).

8. The antibody or antigen-binding fragment thereof of any one of claims 2-7, the heavy chain variable region further comprising: HFR1 comprising the amino acid sequence set forth in SEQ ID NO: 16; HFR2 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 20-22; HFR3 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 23-28; and HFR4 comprising the amino acid sequence set forth in SEQ ID NO:

19.

9. The antibody or antigen-binding fragment thereof of any one of claims 2-8, the light chain variable region further comprising: LFR1 comprising the amino acid sequence set forth in SEQ ID NO: 29; LFR2 comprising the amino acid sequence set forth in SEQ ID NO: 30; LFR3 comprising the amino acid sequence set forth in SEQ ID NO: 31; and LFR4 comprising the amino acid sequence set forth in SEQ ID NO:

32.

10. The antibody or antigen-binding fragment thereof of any one of claims 1-9, the heavy chain variable region comprising the amino acid sequence set forth in any one of SEQ ID NOs: 33-43 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or greater sequence identity to any one of SEQ ID NOs: 33-43.

11. The antibody or antigen-binding fragment thereof of any one of claims 1-10, the light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 44 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or greater sequence identity to SEQ ID NO:

44.

12. The antibody or antigen-binding fragment thereof of any one of claims 1-11, which comprises a full-length antibody, a Fab, a Fab’, a Fv fragment, a F(ab’)2, a scFv, or a di-scFv.

13. The antibody or antigen-binding fragment thereof of any one of claims 1-12, wherein the heavy chain of the antibody or antigen-binding fragment thereof further comprises a constant region or a variant thereof; preferably, the heavy chain comprises a constant region of human IgGl, IgG2, IgG3, or IgG4 or a variant thereof; more preferably, the heavy chain further comprises an amino acid sequence set forth in any one of SEQ ID NOs: 45-53.

14. The antibody or antigen-binding fragment thereof of any one of claims 1-13, wherein the light chain of the antibody or antigen-binding fragment thereof further comprises a constant region or a variant thereof; preferably, the light chain comprises a constant region of human IgA or a variant thereof; more preferably, the light chain further comprises an amino acid sequence set forth in SEQ ID NO:

64.

15. The antibody or antigen-binding fragment thereof of any one of claims 1-14, wherein the heavy chain of the antibody or antigen-binding fragment thereof comprises an amino acid sequence set forth in any one of SEQ ID NOs: 54-63 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or more sequence identity to any one of SEQ ID NOs: 54-63.

16. The antibody or antigen-binding fragment thereof of any one of claims 1-15, wherein the light chain of the antibody or antigen-binding fragment thereof comprises an amino acid sequence set forth in SEQ ID NO: 65 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or more sequence identity to SEQ ID NO:

65.

17. The antibody or antigen-binding fragment thereof of any one of claims 1-16, which is directed against ActRIIA and / or ActRIIB.

18. A fusion protein comprising the antibody or antigen-binding fragment thereof of any one of claims 1-17.

19. A bispecific or multispecific antibody comprising the antibody or antigen-binding fragment thereof of any one of claims 1-17.

20. A pharmaceutical combination comprising the antibody or antigen-binding fragment thereof directed against Activin Receptor II of any one of claims 1-17, the fusion protein of claim 18, or the bispecific or multispecific antibody of claim 19, and a GLP-1R agonist.

21. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof directed against Activin Receptor II of any one of claims 1-17, the fusion protein of claim 18, or the bispecific or multispecific antibody of claim 19, the pharmaceutical combination of claim 20, and a pharmaceutically acceptable carrier.

22. A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1-17 or the fusion protein of claim 18 or the bispecific or multispecific antibody of claim 19; preferably, the nucleic acid is DNA or RNA.

23. A vector comprising the nucleic acid molecule of claim 22.

24. A recombinant cell comprising the nucleic acid molecule of claim 22 and / or the vector of claim 23.

25. A method of producing an antibody or antigen-binding fragment thereof against Activin Receptor II, characterized in that, The antibody or antigen-binding fragment thereof against Activin Receptor II is obtained from the culture comprising culturing the recombinant cell of claim 24.

26. A method of treating and / or preventing an Activin Receptor II related disease or disorder, comprising administering to a patient in need thereof the antibody or antigen-binding fragment thereof of any one of claims 1-17 and / or the fusion protein of claim 18 and / or the bispecific or multispecific antibody of claim 19 and / or the pharmaceutical combination of claim 20 and / or the pharmaceutical composition of claim 21 and / or the nucleic acid molecule of claim 22 and / or the vector of claim 23 and / or the recombinant cell of claim 24.

27. The method of claim 26, wherein the Activin Receptor II related disease or disorder comprises obesity, diabetes, musculoskeletal disease and / or tumor cachexia; preferably, the musculoskeletal disease is muscle atrophy.

28. A method of human fat reduction and / or muscle augmentation for non-therapeutic purposes, comprising administering to a subject in need thereof the antibody or antigen-binding fragment thereof of any one of claims 1-17 and / or the fusion protein of claim 18 and / or the bispecific or multispecific antibody of claim 19 and / or the pharmaceutical combination of claim 20 and / or the pharmaceutical composition of claim 21 and / or the nucleic acid molecule of claim 22 and / or the vector of claim 23 and / or the recombinant cell of claim 24.

29. A drug delivery device characterized in that, A drug infusion module and optionally a drug monitoring module, the drug infusion module containing the antibody or antigen-binding fragment thereof of any one of claims 1-17 and / or the fusion protein of claim 18 and / or the bispecific or multispecific antibody of claim 19 and / or the pharmaceutical combination of claim 20 and / or the pharmaceutical composition of claim 21 and / or the nucleic acid molecule of claim 22 and / or the vector of claim 23 and / or the recombinant cell of claim 24.

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