Anti-ACVR2a antibody or antigen-binding fragment thereof and use

Abstract

The present invention provides an anti-ACVR2A antibody or an antigen-binding fragment thereof, and also provides a polynucleotide encoding the antibody, a vector and a host cell for expressing the antibody, a pharmaceutical composition comprising the antibody, and a method and pharmaceutical use of the antibody for treating an ACVR2A-related disease.

Description

An anti-ACVR2A antibody or its antigen-binding fragment and its uses Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to anti-ACVR2A antibodies or their antigen-binding fragments, pharmaceutical compositions, and uses. Background Technology

[0002] Activin type 2 receptors (ActRII), including two distinct receptors ACVR2A (ActRIIA) and ACVR2B (ActRIIB), belong to the TGF-β receptor family. They regulate transforming growth factor β ligand signaling and participate in many physiological processes, including cell proliferation, differentiation, apoptosis, metabolism, and immune responses. Both ACVR2A and ACVR2B are transmembrane receptors for activins, possessing a cysteine-rich extracellular ligand-binding domain, a transmembrane domain, and an intracellular domain with serine / threonine kinase activity. By binding to ligands such as activins, they activate downstream signaling pathways such as Smad2 / 3 and MAPK, thereby regulating skeletal muscle mass, lipid metabolism, and embryonic development. ACVR2A and ACVR2B play crucial roles in muscle and lipid metabolism and have become potential targets for treating related diseases, necessitating improved targeted antibodies to meet clinical treatment needs. Summary of the Invention

[0003] On the one hand, the present invention provides an anti-ACVR2A antibody or its antigen-binding fragment thereof, which can bind to ACVR2A protein with high affinity and specifically, effectively inhibiting or blocking the binding of ACVR2A to ACVR2A ligands such as activin A, activin B, GDF8 and GDF11.

[0004] On one hand, the present invention provides an anti-ACVR2A antibody or its antigen-binding fragment thereof, wherein the anti-ACVR2A antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises HCDR1, HCDR2, and HCDR3 regions, and the VL comprises LCDR1, LCDR2, and LCDR3 regions, wherein the HCDR1, HCDR2, and HCDR3 regions sequentially have the same sequence as HCDR1, HCDR2, and HCDR3 of VH as shown in SEQ ID NO.1, or have a sequence with up to 5, 4, 3, 2, or 1 mutation compared to each CDR in HCDR1-3 of VH as shown in SEQ ID NO.1, and / or the LCDR1, LCDR2, and LCDR3 regions sequentially have the same sequence as LCDR1, LCDR2, and LCDR3 of VL as shown in SEQ ID NO.2 ... VH as shown in SEQ ID NO.1, and / or the LCDR1, LCDR2, and LCDR3 regions sequentially have the same sequence as Compared to the sequence shown in NO.2, each CDR in VL's LCDR1-3 has at most 5, 4, 3, 2 or 1 mutations;

[0005] The mutation is selected from insertions, deletions and / or substitutions that do not affect function, and the substitution is preferably a substitution of a conserved amino acid.

[0006] The HCDR1 has the same sequence as HCDR1 of VH as shown in SEQ ID NO.1, or the HCDR1 has a sequence with up to 5, 4, 3, 2 or 1 mutations compared to HCDR1 of VH as shown in SEQ ID NO.1; the HCDR2 has the same sequence as HCDR2 of VH as shown in SEQ ID NO.1, or the HCDR2 has a sequence with up to 5, 4, 3, 2 or 1 mutations compared to HCDR2 of VH as shown in SEQ ID NO.1; and the HCDR3 has the same sequence as HCDR3 of VH as shown in SEQ ID NO.1, or the HCDR3 has a sequence with up to 5, 4, 3, 2 or 1 mutations compared to HCDR3 of VH as shown in SEQ ID NO.1.

[0007] The LCDR1 has the same sequence as the LCDR1 of VL as shown in SEQ ID NO.2, or the LCDR1 has a sequence with up to 5, 4, 3, 2 or 1 mutations compared to the LCDR1 of VL as shown in SEQ ID NO.2; the LCDR2 has the same sequence as the LCDR2 of VL as shown in SEQ ID NO.2, or the LCDR2 has a sequence with up to 5, 4, 3, 2 or 1 mutations compared to the LCDR2 of VL as shown in SEQ ID NO.2; and the LCDR3 has the same sequence as the LCDR3 of VL as shown in SEQ ID NO.2, or the LCDR3 has a sequence with up to 5, 4, 3, 2 or 1 mutations compared to the LCDR3 of VL as shown in SEQ ID NO.2.

[0008] In some embodiments, the HCDR1, HCDR2, and HCDR3 regions have the same sequence as HCDR1, HCDR2, and HCDR3 of VH as shown in sequence a, and the LCDR1, LCDR2, and LCDR3 regions have the same sequence as LCDR1, LCDR2, and LCDR3 of VL as shown in sequence b.

[0009] The sequence a is:

[0010] EVQLVQSGAEVKKPGSSVKVSCKAX1GGTFX2SX3X4ISWVRQAPGQGLEWMGX5IIPIFGX6ANYAQKFQGRVTITADESTSTAYME LSSLRSEDTAVYYCAX7AVAWHLX8WFDPWGQGTLVTVSS; X1 is selected from S or G, X2 is selected from S or G, X3 is selected from Y, R or S, X4 is selected from A, R or K, X5 is selected from G or R, X6 is selected from T or G, X7 is selected from I or R, X8 is selected from N, A or R;

[0011] The sequence b is:

[0012] DIVMTQSPDSLAVSLGERATINCKSSQSVLX9NSNX 10 KNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQ AEDVAVYYCQQYX 11 X 12 TPPTFGQGTRLEIK, X9 is selected from Y, D, or S, X 10 Selected from N or Y, X 11 Selected from Y or L, X 12 Choose from S or A.

[0013] The HCDR1 region has the same sequence as HCDR1 of VH as shown in sequence a, the HCDR2 region has the same sequence as HCDR2 of VH as shown in sequence a, and the HCDR3 region has the same sequence as HCDR3 of VH as shown in sequence a.

[0014] The LCDR1 region has the same sequence as LCDR1 of VL as shown in sequence b, the LCDR2 region has the same sequence as LCDR2 of VL as shown in sequence b, and the LCDR3 region has the same sequence as LCDR3 of VL as shown in sequence b.

[0015] In some embodiments, X1 is selected from S or G, X2 is selected from S or G, X3 is selected from Y, R or S, X4 is selected from A, R or K, X5 is selected from G or R, X6 is selected from T or G, X7 is selected from I or R, X8 is selected from N, A or R, and X9 to X 12 Choose any one of the combinations shown in the table below:

[0016] In some implementations, X1 to X8 are selected from any of the combinations shown in the table below:

[0017] And X9 is selected from Y, D, or S, X 10 Selected from N or Y, X 11 Selected from Y or L, X 12 Choose from S or A.

[0018] In some preferred embodiments, the HCDR1, HCDR2, and HCDR3 regions have the same sequence as HCDR1, HCDR2, and HCDR3 of VH as shown in any of SEQ ID NO. 1, 5-20, and the LCDR1, LCDR2, and LCDR3 regions have the same sequence as LCDR1, LCDR2, and LCDR3 of VL as shown in any of SEQ ID NO. 2, 21-27.

[0019] In some preferred embodiments, the VH has the same sequence HCDR1, HCDR2 and HCDR3 as the VH shown in any of SEQ ID NO.1, 5-17, and the VL has the same sequence LCDR1, LCDR2 and LCDR3 as the VL shown in SEQ ID NO.2.

[0020] In some preferred embodiments, the VH has the same sequence HCDR1, HCDR2 and HCDR3 as the VH shown in any of SEQ ID NO. 12, 18, 19 and 20, and the VL has the same sequence LCDR1, LCDR2 and LCDR3 as the VL shown in SEQ ID NO. 21.

[0021] In some preferred embodiments, the VH has the same sequence HCDR1, HCDR2 and HCDR3 as the VH shown in SEQ ID NO.12, and the VL has the same sequence LCDR1, LCDR2 and LCDR3 as the VL shown in any of SEQ ID NO.22-27.

[0022] In some preferred embodiments, the HCDR1 of the VH is the same as the HCDR1 of the VH shown in any of SEQ ID NO.1, 5-17, the HCDR2 of the VH is the same as the HCDR2 of the VH shown in any of SEQ ID NO.1, 5-17, the HCDR3 of the VH is the same as the HCDR3 of the VH shown in any of SEQ ID NO.1, 5-17, the LCDR1 of the VL is the same as the LCDR1 of the VL shown in SEQ ID NO.2, the LCDR2 of the VL is the same as the LCDR2 of the VL shown in SEQ ID NO.2, and the LCDR3 of the VL is the same as the LCDR3 of the VL shown in SEQ ID NO.2.

[0023] In some preferred embodiments, the HCDR1 of the VH is the same as the HCDR1 of the VH shown in any of SEQ ID NO. 12, 18, 19, 20; the HCDR2 of the VH is the same as the HCDR2 of the VH shown in any of SEQ ID NO. 12, 18, 19, 20; the HCDR3 of the VH is the same as the HCDR3 of the VH shown in any of SEQ ID NO. 12, 18, 19, 20; the LCDR1 of the VL is the same as the LCDR1 of the VL shown in SEQ ID NO. 21; the LCDR2 of the VL is the same as the LCDR2 of the VL shown in SEQ ID NO. 21; and the LCDR3 of the VL is the same as the LCDR3 of the VL shown in SEQ ID NO. 21.

[0024] In some preferred embodiments, the HCDR1 of the VH is the same as the HCDR1 of the VH shown in SEQ ID NO. 12, the HCDR2 of the VH is the same as the HCDR2 of the VH shown in SEQ ID NO. 12, the HCDR3 of the VH is the same as the HCDR3 of the VH shown in SEQ ID NO. 12, the LCDR1 of the VL is the same as the LCDR1 of the VL shown in any of SEQ ID NO. 22-27, the LCDR2 of the VL is the same as the LCDR2 of the VL shown in any of SEQ ID NO. 22-27, and the LCDR3 of the VL is the same as the LCDR3 of the VL shown in any of SEQ ID NO. 22-27.

[0025] In some preferred embodiments, the HCDR1-3 of VH and the HCDR1-3 of VL are selected from the HCDR1-3 and LCDR1-3 of any of the antibodies shown in Table A.

[0026] In some implementations, the HCDR1, HCDR2, and HCDR3 of the VH, and the LCDR1, LCDR2, and LCDR3 of the VL are defined according to IMGT, Kabat, Chothia, AbM, Contact, or any combination thereof.

[0027] In some embodiments, the VH has an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in any of SEQ ID NO. 1, 5-20, and / or the VL has an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in any of SEQ ID NO. 2, 21-27.

[0028] In some embodiments, the VH has an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in any of SEQ ID NO. 1, 5-17, and / or the VL has an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO. 2.

[0029] In some embodiments, the VH has a sequence with up to 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mutation compared to any of the sequences shown in SEQ ID NO. 1, 5-17, and / or the VL has a sequence with up to 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mutation compared to the sequence shown in SEQ ID NO. 2; the mutations are preferably substitutions of conserved amino acids.

[0030] In some embodiments, the VH has an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in any of SEQ ID NO. 12, 18, 19, 20, and / or the VL has an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO. 21.

[0031] In some embodiments, the VH has a sequence with up to 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mutation compared to any of the sequences shown in SEQ ID NO. 12, 18, 19, 20, and / or the VL has a sequence with up to 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mutation compared to the sequence shown in SEQ ID NO. 21; the mutations are preferably substitutions of conserved amino acids.

[0032] In some embodiments, the VH has an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in any of SEQ ID NO. 12, and / or the VL has an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in any of SEQ ID NO. 22-27.

[0033] In some embodiments, the VH has a sequence with up to 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mutation compared to the sequence shown in SEQ ID NO. 12, and / or the VL has a sequence with up to 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mutation compared to the sequence shown in any of SEQ ID NO. 22-27; the mutations are preferably substitutions of conserved amino acids.

[0034] In some preferred embodiments, the VH and VL respectively comprise or are sequences selected from the group consisting of: SEQ ID NO.1 and 2; SEQ ID NO.5 and 2; SEQ ID NO.6 and 2; SEQ ID NO.7 and 2; SEQ ID NO.8 and 2; SEQ ID NO.9 and 2; SEQ ID NO.10 and 2; SEQ ID NO.11 and 2; SEQ ID NO.12 and 2; SEQ ID NO.13 and 2; SEQ ID NO.14 and 2; SEQ ID NO.15 and 2; SEQ ID NO.16 and 2; SEQ ID NO.17 and 2; SEQ ID NO.12 and 21; SEQ ID NO.18 and 21; SEQ ID NO.19 and 21; SEQ ID NO.12 and 22; SEQ ID NO.12 and 23; SEQ ID NO.12 and 24; SEQ ID NO.12 and 25; SEQ ID NO.12 and 26; SEQ ID NO.12 and 27; or, SEQ ID NO.20 and 21.

[0035] In some embodiments, the anti-ACVR2A antibody provided by the present invention further comprises a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region is selected from the constant regions of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE or variants thereof, preferably from, for example, the constant regions of human IgG1, IgG2, IgG3, and IgG4 or variants thereof; the light chain constant region is selected from the κ and λ chain constant regions or variants thereof, preferably from the human κ and λ chain constant regions or variants thereof. The variant has altered effector functions mediated by the Fc region (e.g., ADCC and / or CDC and / or ADCP activities, affinity for FcγRIIIa and / or C1q), but does not alter the function of the antibody variable region. In some embodiments, the variant Fc region has at least one amino acid substitution compared to its derived wild-type Fc region, for example, about 1-10 amino acid substitutions, or about 1-5 amino acid substitutions, or about 1-3 amino acid substitutions in the wild-type Fc region. The variant Fc region may have at least about 80%, about 81%, about 82%, about 83%, about 84%, 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%, about 99% homology with the wild-type Fc region from which it is derived. In some embodiments, the antibody or its antigen-binding fragment comprises a constant region variant of human IgG1, which has one or more of the following amino acid substitutions compared to the wild-type sequence from which it is derived: L234A, L235A, P329G, D265S, M252Y, S254T, T256E, M428L, N434S (according to the position in the EU numbering system); preferably one or any combination of the following amino acid substitutions: (1) L234A / (1) L235A (LALA); (2) L234A / L235A / P329G (LALAPG); (3) L234A / L235A / D265S (LALADS); (4) M252Y / S254T / T256E (YTE); (5) M428L / N434S (LS); More preferably, the amino acid substitutions include any one of LALA, LALAPG, LALADS and any one of YTE and LS.

[0036] In some embodiments, the heavy chain constant region comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of the sequences shown in SEQ ID NO. 28-31; or, the heavy chain constant region comprises a sequence that has undergone a mutation of up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids compared to any of the sequences shown in SEQ ID NO. 28-31; the amino acid mutation is preferably an amino acid substitution, such as a conservative substitution of amino acids, and optionally, the amino acid substitution includes any one of LALA, LALAPG, LALADS and any one of YTE and LS.

[0037] In some embodiments, the light chain constant region comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO. 4 or 32; or, the light chain constant region comprises a sequence that has undergone a mutation of up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids compared to the sequence shown in SEQ ID NO. 4 or 32; the amino acid mutation is preferably an amino acid substitution, such as a conserved substitution of amino acids.

[0038] In some embodiments, the heavy chain constant region contains an LALA mutation and contains an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO. 33; or, the heavy chain constant region contains an LALA mutation and contains a sequence that has undergone at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid mutations compared to the sequence shown in SEQ ID NO. 33.

[0039] In some embodiments, the heavy chain constant region contains LALA and YTE mutations and contains an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO. 34; or, the heavy chain constant region contains LALA and YTE mutations and contains a sequence that has undergone at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid mutations compared to the sequence shown in SEQ ID NO. 34.

[0040] In some embodiments, the heavy chain constant region contains LALA and LS mutations and contains an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO. 35; or, the heavy chain constant region contains LALA and LS mutations and contains a sequence that has undergone at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid mutations compared to the sequence shown in SEQ ID NO. 35.

[0041] In some embodiments, the heavy chain constant region contains the LALAPG mutation and contains an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO. 3; or, the heavy chain constant region contains the LALAPG mutation and contains a sequence that has undergone at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid mutations compared to the sequence shown in SEQ ID NO. 3.

[0042] In some embodiments, the heavy chain constant region contains LALAPG and LS mutations and contains an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO. 36; or, the heavy chain constant region contains LALAPG and LS mutations and contains a sequence that has undergone at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid mutations compared to the sequence shown in SEQ ID NO. 36.

[0043] In some embodiments, the heavy chain constant region contains LALAPG and YTE mutations and contains an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO. 37; or, the heavy chain constant region contains LALAPG and YTE mutations and contains a sequence that has undergone at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid mutations compared to the sequence shown in SEQ ID NO. 37.

[0044] In some embodiments, the heavy chain constant region contains LALADS and LS mutations and contains an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO. 38; or, the heavy chain constant region contains LALADS and LS mutations and contains a sequence that has undergone at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid mutations compared to the sequence shown in SEQ ID NO. 38.

[0045] In some embodiments, the heavy chain constant region contains LALADS and YTE mutations and contains an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO. 39; or, the heavy chain constant region contains LALADS and YTE mutations and contains a sequence that has undergone at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid mutations compared to the sequence shown in SEQ ID NO. 39.

[0046] In some preferred embodiments, the heavy chain constant region and the light chain constant region comprise sequences as shown in Table 2, or amino acid sequences that are at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences shown in Table 2.

[0047] In some embodiments, the heavy chain constant region lacks a C-terminal lysine. In some embodiments, the N-terminal amino acid (e.g., glutamine or glutamate) of the antibody or its antigen-binding fragment may be cyclized to pyroglutamic acid or a pyroglutamate salt.

[0048] In some embodiments, the heavy chain constant region comprises a sequence selected from any one of SEQ ID NO.3, 28-31, 33-39, preferably any one of SEQ ID NO.3, 33-39; and / or, the light chain constant region comprises a sequence selected from SEQ ID NO.4 or 32, preferably SEQ ID NO.4.

[0049] In some embodiments, the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of the sequences shown in SEQ ID NO. 40-47, or the heavy chain comprises a sequence having undergone at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid mutations compared to any of the sequences shown in SEQ ID NO. 40-47; the light chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO. 48, or the light chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of the sequences ... The sequence shown in NO.48 is a sequence with a maximum of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid mutations compared to the sequence shown in NO.48.

[0050] In some embodiments, the antibody or its antigen-binding fragment may include post-translational modifications (e.g., C-terminal lysine cleavage in the heavy chain, N-terminal glutamine or glutamate cyclization in the heavy or light chain to pyroglutamic acid or pyroglutamate salt), which may occur during recombinant expression in host cells (e.g., CHO cells) or during purification / storage.

[0051] In some embodiments, the anti-ACVR2A antibody provided by the present invention is selected from the antibodies shown in Tables 4 and 7. In some preferred embodiments, the anti-ACVR2A antibody provided by the present invention is of type IgG1.

[0052] On the one hand, the present invention provides an anti-ACVR2A antibody or its antigen-binding fragment thereof, which binds to the same ACVR2A epitope or competitively binds to ACVR2A with the aforementioned anti-ACVR2A antibody or its antigen-binding fragment.

[0053] In some embodiments, the anti-ACVR2A antibody or its antigen-binding fragment provided by the present invention has at least one of the following functions:

[0054] (1) When binding to ACVR2A (e.g., human ACVR2A), the antibody or its antigen-binding fragment has a KD of less than about 100 nM, less than about 90 nM, less than about 80 nM, less than about 70 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, less than about 10 nM, or less than about 1 nM;

[0055] (2) When used with ACVR2A (e.g., human ACVR2A), the KD of the antibody or its antigen-binding fragment is substantially the same as or better than that of the reference ACVR2A antibody;

[0056] (3) When binding to ACVR2A (e.g., human ACVR2A), the antibody or its antigen-binding fragment inhibits or blocks the binding of ACVR2A to its ligand;

[0057] (4) When the binding of ACVR2A (e.g., human ACVR2A) to its ligand is inhibited or blocked, the inhibitory or blocking effect of the antibody or its antigen-binding fragment is substantially the same as or better than that of the reference ACVR2A antibody;

[0058] (5) Combining ACVR2A from different species, for example, combining human ACVR2A and ACVR2A from at least one other mammal selected from mice, rats and monkeys (e.g., cynomolgus monkeys);

[0059] (6) As an antagonistic antibody against ACVR2A;

[0060] (7) Specifically binds to ACVR2A but does not bind or barely binds to ACVR2B;

[0061] (8) Reduce the binding of ACVR2A to at least one of its ligands, reduce the activity of ACVR2A, or reduce ACVR2A-mediated signal transduction, for example, by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95%.

[0062] KD can be determined using methods known to those skilled in the art, such as biolayer interferometry (BLI), surface plasmon resonance (SPR), or, for example, the method described in Example 4.

[0063] The ACVR2A ligand includes activins (such as activin A, activin B, activin AB, activin C, activin AC, and activin E), growth / differentiation factors (GDFs, such as GDF1, GDF3, GDF5, GDF6, GDF7, GDF8, GDF10, and GDF11), and bone morphogenetic proteins (BMPs, such as BMP2, BMP4, BMP6, BMP7, BMP8a, BMP8b, BMP9, and BMP10). In some preferred embodiments, the ACVR2A ligand includes activin A and GDF8. In some preferred embodiments, the anti-ACVR2A antibody or its antigen-binding fragment provided by the present invention binds to ACVR2A and reduces the binding level of activin A to ACVR2A, for example, completely preventing activin A from binding to ACVR2A.

[0064] The reference ACVR2A antibody can be prepared according to patents CN102753578B and CN118176018A. In some specific embodiments, the reference ACVR2A antibody is Bimagrumab or LAE102.

[0065] In some embodiments, the anti-ACVR2A antibody provided by the present invention is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody. In one specific embodiment, the anti-ACVR2A antibody provided by the present invention is a fully human antibody. In some embodiments, the anti-ACVR2A antibody is a monoclonal antibody.

[0066] In some embodiments, the antigen-binding fragment of the present invention is selected from: Fab, F(ab')2, Fab', Fd, Fv, dsFv, scFv and biantibodies.

[0067] On one hand, the present invention provides a conjugate comprising the anti-ACVR2A antibody of the present invention or its antigen-binding fragment. In one embodiment, the conjugate further comprises an effector molecule. In another embodiment, the conjugate further comprises a linker connecting the antibody or antigen-binding fragment to the effector molecule.

[0068] In some embodiments, the effector molecule is selected from antitumor agents, drugs, toxins, bioactive proteins (e.g., enzymes), other antibodies or antibody fragments, synthetic or naturally occurring polymers, nucleic acids and fragments thereof such as DNA, RNA and fragments thereof, radionuclides (e.g., radioiodides), radioisotopes, chelated metals, nanoparticles and reporter groups (e.g., fluorescent compounds), or compounds detectable by NMR or ESR spectroscopy.

[0069] On the one hand, the present invention provides a bispecific antibody comprising the anti-ACVR2A antibody of the present invention or its antigen-binding fragment.

[0070] In some embodiments, the bispecific antibody comprises a first antigen-binding domain targeting ACVR2A and at least one second antigen-binding domain targeting other targets, wherein the first antigen-binding domain comprises the aforementioned anti-ACVR2A antibody or its antigen-binding fragment. In some embodiments, each antigen-binding domain of the bispecific antibody retains its original binding specificity.

[0071] On one hand, the present invention provides a polynucleotide encoding the anti-ACVR2A antibody of the present invention or its antigen-binding fragment, conjugate, or bispecific antibody. The polynucleotide of the present invention may be, for example, DNA or RNA, and may or may not contain intron sequences. In a preferred embodiment, the polynucleotide is a cDNA molecule. The polynucleotide of the present invention can be prepared or obtained by known methods based on information of the amino acid sequence of the present invention, for example by automated DNA synthesis and / or recombinant DNA technology.

[0072] As is well known in the art, multiple codons can encode the same amino acid. Therefore, nucleic acids encoding protein sequences include those with codon degeneracy. The amino acid sequences described in this invention can be encoded by a variety of nucleic acids. The genetic code is universal and well-known. Nucleic acids encoding any of the amino acid sequences described in this invention can be readily conceived based on common knowledge in the art and can be optimized for production. Although the number of possible nucleic acid sequences encoding a given amino acid is large, given a standard table of the genetic code and with the aid of a calculator, those skilled in the art can readily generate every possible combination of nucleic acid sequences encoding a given amino acid.

[0073] On one hand, the present invention provides a vector comprising the polynucleotides of the present invention. The vector is capable of delivering and preferably expressing constructs of one or more target genes or sequences in host cells. The vector includes eukaryotic expression vectors, prokaryotic expression vectors, viral vectors such as bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.

[0074] On one hand, the present invention provides a host cell containing the polynucleotide or vector of the present invention. The host cell is capable of producing or secreting the complete antibody of the present invention or its antigen-binding fragment, conjugate, or bispecific antibody. The host cell comprises prokaryotic cells, fungal cells, or mammalian cells, such as CHO cells, NSO cells, or other mammalian cells, *Escherichia coli* or other prokaryotic cells, yeast cells, or other fungal cells.

[0075] On one hand, the present invention provides a pharmaceutical composition comprising the anti-ACVR2A antibody of the present invention or its antigen-binding fragment, conjugate, bispecific antibody, polynucleotide, expression vector or host cell, and one or more pharmaceutically acceptable vectors.

[0076] In some preferred embodiments, the pharmaceutical composition contains a therapeutically effective amount of the aforementioned anti-ACVR2A antibody or its antigen-binding fragment, the aforementioned conjugate, the aforementioned bispecific antibody or the aforementioned nucleic acid molecule, and one or more pharmaceutically acceptable carriers, diluents, buffers or excipients.

[0077] In some embodiments, the pharmaceutical composition may contain any number of excipients. Excipients that may be used include carriers, surfactants, thickeners or emulsifiers, solid binders, dispersants or suspending agents, solubilizers, colorants, flavoring agents, coating agents, disintegrants, lubricants, sweeteners, preservatives, isotonic agents, or combinations thereof. The selection and use of appropriate excipients is taught in Gennaro, ed., Remington: The Science and Practice of Pharmacy, 20th edition (Lippincott Williams & Wilkins 2003), the disclosure of which is incorporated herein by reference.

[0078] In some embodiments, the pharmaceutical composition further comprises one or more other therapeutic agents; the therapeutic agents are preferably antitumor agents, or therapeutic agents for treating obesity, weight management, reducing weight, reducing fat mass, increasing muscle mass and / or increasing lean body mass, more preferably GLP-1 receptor agonists.

[0079] On one hand, the present invention provides a kit comprising the aforementioned anti-ACVR2A antibody or its antigen-binding fragment, the aforementioned conjugate, the aforementioned bispecific antibody, or the aforementioned pharmaceutical composition.

[0080] On one hand, the present invention provides a method for preventing and / or treating a subject with an ACVR2A-related disease or condition, comprising administering to a subject in need a therapeutic or preventatively effective amount of the anti-ACVR2A antibody of the present invention or its antigen-binding fragment, conjugate, bispecific antibody, polynucleotide, expression vector, host cell, or pharmaceutical composition.

[0081] On the one hand, the present invention provides the use of the aforementioned anti-ACVR2A antibody or its antigen-binding fragment, the aforementioned conjugate, the aforementioned bispecific antibody, the aforementioned polynucleotide, the aforementioned expression vector, the aforementioned host cell or the aforementioned pharmaceutical composition in the preparation of a medicament for the prevention and / or treatment of a subject with an ACVR2A-related disease or condition.

[0082] In some embodiments, the ACVR2A-related disease or condition is a disease or condition caused by abnormal ACVR2A expression. In some embodiments, the ACVR2A-related disease or condition is a disease or condition caused by ACVR2A overexpression. In some embodiments, the ACVR2A-related disease or condition is a ligand-mediated disease or condition that binds to ACVR2A. In some embodiments, the ACVR2A-related disease or condition is an activin A-mediated disease or condition. In some preferred embodiments, the ACVR2A-related disease is selected from musculoskeletal diseases or conditions.

[0083] In some specific implementations, the diseases or conditions associated with ACVR2A are selected from muscular atrophy, spinal muscular atrophy, cachexia (e.g., cachexia caused by cancer, chronic renal failure, or chronic obstructive pulmonary disease), Duchenne muscular dystrophy, Becker muscular dystrophy, myotonic dystrophy, facioscapulohumeral muscular dystrophy, Emery-Dreifuss muscular dystrophy, oculopharyngeal muscular dystrophy, scapulopihumeral muscular dystrophy, limb girdle muscular dystrophy, Fukuyama muscular dystrophy, congenital muscular dystrophy or hereditary distal myopathy, osteoporosis, fractures, short stature, dwarfism, sarcopenia, muscle injury, muscle wasting, glucocorticoid-induced myopathy, obesity, diabetes, arthritis, multiple sclerosis, Parkinson's disease, osteoarthritis, osteopenia, and metabolic syndrome (including but not limited to diabetes, obesity, malnutrition, organ atrophy, chronic obstructive pulmonary disease, and anorexia).

[0084] In some implementations, the drug is used to improve muscle strength, muscle power, muscle mass, and / or muscle function.

[0085] On one hand, the present invention provides a method for preventing, alleviating and / or treating obesity or obesity-related diseases or conditions in a subject, comprising co-administering to a subject in need (a) the aforementioned anti-ACVR2A antibody or its antigen-binding fragment and (b) a GLP-1 receptor agonist or a pharmaceutically acceptable salt thereof.

[0086] On the one hand, this article provides the use of (a) the aforementioned anti-ACVR2A antibody or its antigen-binding fragment and (b) a GLP-1 receptor agonist or a pharmaceutically acceptable salt thereof in the preparation of a pharmaceutical composition for the prevention, relief and / or treatment of obesity or obesity-related diseases or conditions in a subject.

[0087] In any of the embodiments of the foregoing aspects, the GLP-1 receptor agonist is selected from GLP-1 (7-37) or analogues thereof. In some embodiments, the GLP-1 (7-37) analogue has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid mutations (amino acid mutations include substitutions, insertions, and / or deletions) compared to its derived natural GLP-1 (7-37). In some embodiments, the GLP-1 (7-37) analogue has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity compared to its derived natural GLP-1 (7-37). In some embodiments, the GLP-1 (7-37) analogue has side-chain modifications of amino acid residues compared to its derived natural GLP-1 (7-37), including but not limited to fatty acid chain modifications, PEG chain modifications, etc. GLP-1(7-37) or its analogues may optionally be amidated.

[0088] In any of the foregoing embodiments, specific examples of GLP-1 receptor agonists are disclosed in patent documents such as WO2006097537, WO2005000892, WO1998008871, WO2005077042, CN1381272A, CN101273134B, and CN110386975B, which are incorporated herein by reference in their entirety.

[0089] In any of the embodiments described above, the GLP-1 receptor agonist is selected from exenatide, liraglutide, lixilatide, abiglutide, dulaglutide, semaglutide, benaglutide, supaglutide, and tasglutide. The structural formula of semaglutide is as follows:

[0090] In any of the embodiments of the foregoing, the pharmaceutical composition is used for weight management, weight reduction, fat reduction, muscle mass increase and / or lean body mass increase.

[0091] In any of the embodiments of the foregoing aspect, each of (a) and (b) is present in a preventive or therapeutically effective amount.

[0092] In any of the embodiments of the foregoing aspects, co-administration can be performed simultaneously and / or sequentially in any order, such as: (a) and (b) administered simultaneously; (b) administered before (a); (a) administered before (b). When (a) and (b) are administered co-administered, they can be administered in two separate administrations on the same day, for example, it may be permissible to administer one drug a few minutes, tens of minutes, or hours after administering the other drug, with the interval between (a) and (b) including but not limited to 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, and 2 hours. Preferably, the co-administration involves a period of time during which both drugs exert their biological activity simultaneously.

[0093] In any of the foregoing embodiments, each of (a) and (b) is preferably administered subcutaneously. For example, (a) and (b) may be administered via two separate injections or via a single injection.

[0094] In any of the embodiments of the foregoing, the dose (preferably unit dose) ratio, weight ratio, or molar ratio of (a) and (b) is between 30:0.14 and 30:0.08.

[0095] In any of the embodiments of the foregoing aspect, (a) is present in a dose range of 100-1000 mg (preferably unit dose).

[0096] In any of the embodiments of the foregoing, (b) is present in a dose range of 0.1-10 mg (preferably unit dose).

[0097] In any of the preceding implementations, (a) is applied weekly, bi-weekly, tri-weekly, monthly, bi-monthly, tri-monthly, quadri-monthly, six-monthly, or semi-annually.

[0098] In any of the foregoing embodiments, (b) is applied once daily, every three days, or weekly.

[0099] In any of the embodiments of the foregoing aspect, (b) is present at a dose (preferably a unit dose) of 0.25 mg, 0.5 mg, 1.0 mg, 1.7 mg, or 2.4 mg, and / or, (b) is administered once weekly. In some embodiments, (b) is administered once weekly at a dose not exceeding 2.4 mg (preferably a unit dose), for example, once weekly at a dose of 0.5 mg (preferably a unit dose), once weekly at a dose of 1.0 mg (preferably a unit dose), once weekly at a dose of 1.7 mg (preferably a unit dose), or once weekly at a dose of 2.4 mg (preferably a unit dose).

[0100] On one hand, the present invention provides the use of the aforementioned anti-ACVR2A antibody or its antigen-binding fragment or conjugate in the detection of ACVR2A or the preparation of reagents for detecting ACVR2A. The detection of ACVR2A can be performed in vivo or in vitro and can be for non-disease diagnostic purposes. Detection of ACVR2A can be the detection of its presence or level in a sample.

[0101] The present invention also provides a method for preparing the anti-ACVR2A antibody or its antigen-binding fragment of the present invention.

[0102] The anti-ACVR2A antibody or its antigen-binding fragment described in this invention can be obtained using conventional techniques, such as the Cold Spring Harbor Laboratory Antibody Techniques Guide, Chapters 5-8 and 15. For example, mice can be immunized with human ACVR2A or its fragments, and the resulting antibody can be renatured, purified, and sequenced using conventional methods. The antigen-binding fragment can also be prepared using conventional methods. The antibody or antigen-binding fragment described in this invention uses genetic engineering methods to add one or more human FR regions to a non-human CDR region. Human FR germline sequences can be obtained from the ImMunoGeneTics (IMGT) website by comparing with the IMGT Human Antibody Variable Region Germplasm Database and MOE software, or from the Journal of Immunoglobulins, 2001 ISBN012441351. Alternatively, for example, cDNA sequences encoding the heavy and light chains can be cloned into an expression vector. The recombinant immunoglobulin expression vector can be stably transfected into CHO cells. Stable clones are obtained by expressing antibodies that specifically bind to human ACVR2A. Positive clones are scaled up in serum-free medium in a bioreactor to produce antibodies.

[0103] The anti-ACVR2A antibody or its antigen-binding fragment of the present invention can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant protein can be isolated and purified using various separation methods utilizing its physical, chemical, and other properties. These methods are well known to those skilled in the art. Typically, host cells transformed with the present invention are cultured under conditions suitable for antibody expression, and then purified using conventional immunoglobulin purification steps, such as protein A-Sepharose affinity chromatography, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis, and other conventional separation and purification methods, or combinations thereof, to obtain the anti-ACVR2A antibody or its antigen-binding fragment of the present invention. The antibody can be concentrated by filtration using conventional methods.

[0104] As a preferred embodiment of the preparation method of the anti-ACVR2A antibody or its antigen-binding fragment according to the present invention, the method for separating and purifying the anti-ACVR2A antibody or its antigen-binding fragment is protein A affinity chromatography, cation exchange, or anion exchange.

[0105] The resulting monoclonal or bispecific antibodies can be identified using conventional methods. For example, the binding specificity of an antibody can be determined by immunoprecipitation or in vitro binding assays, such as enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA). The binding affinity of an antibody can be determined, for example, by Scatchard analysis as described by Munson et al., Anal. Biochem., 107:220 (1980), or by surface plasmon resonance (SPR).

[0106] For clarity, this article defines the general terminology used in the description of compounds.

[0107] Unless otherwise stated, the following terms and phrases used herein are intended to have the following meanings. A particular term or phrase should not be considered uncertain or unclear unless specifically defined, but should be understood in its ordinary sense. When a trade name appears herein, it is intended to refer to the corresponding product or its active ingredient. The term "pharmaceutically acceptable" as used herein refers to compounds, materials, compositions, and / or dosage forms that, within the bounds of reliable medical judgment, are suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0108] The terms “ACVR2A (Activin A Receptor type 2A),” “Activin Receptor 2A,” and “ActRIIA” are used interchangeably to refer to any ACVR2A molecule and its functional homologs known to those skilled in the art, and unless otherwise stated, ACVR2A includes, but is not limited to, those derived from humans, rodents, mice, rats, primates, monkeys, and guinea pigs. The term also refers to fragments or variants of natural ACVR2A that retain at least one in vivo or in vitro activity of natural ACVR2A, such as activin-binding activity. The term includes both the full-length, unprocessed precursor form of ACVR2A and the mature form resulting from post-translational cleavage of the signal peptide. As used herein, ACVR2A also refers to a specific polypeptide expressed in cells via naturally occurring DNA sequence variations in the ACVR2A gene, such as single nucleotide polymorphisms of the ACVR2A gene. Examples of amino acid sequences corresponding to human ACVR2A registered with Genbank accessions NP_001265508.1, NP_001265509.1, or NP_001607.1 are provided. Other examples of the amino acid sequence of ACVR2A can be obtained from, for example, GenBank, UniProt, and OMIM.

[0109] The term "activin A" is used herein to refer to the activin A polypeptide as understood in the art. Activin A is a member of the TGFβ superfamily. Activin A is also known as the activin β-A chain or the inhibin β-A chain. Under physiological conditions, activin A can form a homodimer consisting of two β subunits of inhibin linked by disulfide bridges, called the βA / βA homodimer. The activin A protein is encoded by the INHBA gene. The full-length amino acid sequence of activin A and / or the nucleic acid encoding it can be found in public databases such as GenBank, UniProt, and SwissProt, e.g., GenBank accession number NM_002192.3.

[0110] The term “GDF8” (also known as “growth and differentiation factor-8” and “myosin)” is used herein to refer to the GDF8 polypeptide as understood in the art. GDF8 is a member of the TGF-β superfamily, negatively regulating muscle growth, primarily binding to ACVR2B and to a lesser extent to ACVR2A. The full-length amino acid sequence of GDF8 and / or the nucleic acid encoding it can be found in public databases such as GenBank, UniProt, and SwissProt, e.g., GenBank accession number NM_005259.3.

[0111] The term "antibody" refers to an immunoglobulin molecule that has the ability to specifically bind to a particular antigen. As used herein, the term "antibody" includes complete antibodies and any antigen-binding fragment (i.e., the "antigen-binding part") or its single chain. A complete antibody is a glycoprotein consisting of two heavy (H) chains and two light (L) chains linked together by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated as VH) and a heavy chain constant region (abbreviated as CH). The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated as VL) and a light chain constant region (abbreviated as CL). The light chain constant region consists of one domain: CL. The VH and VL regions can be further subdivided into hypervariable regions (called complementarity-determining regions (CDRs)) separated by more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant regions of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. Antibodies may contain additional modifications, such as non-naturally occurring amino acids, mutations in the Fc region, and mutations at glycosylation sites. Antibodies also include post-translational modified antibodies, fusion proteins containing antigenic determinants of the antibody, and immunoglobulin molecules containing any other modifications to antigen recognition sites, provided that these antibodies exhibit the desired biological activity.

[0112] The heavy chain of an immunoglobulin molecule can be divided into three functional regions: the Fd region, the hinge region, and the Fc region (crystallizable fragment). The Fd region contains the VH and CH1 domains and binds to the light chain to form the Fab (antigen-binding fragment). The Fc region contains the CH2 and CH3 domains, which are responsible for immunoglobulin effector functions, including, for example, complement binding and binding to homologous Fc receptors on effector cells. The hinge region, found in the IgG, IgA, and IgD immunoglobulin classes, acts as a flexible spacer, allowing the Fab portion to move freely in space relative to the Fc region. The hinge domain is structurally diverse, differing in sequence and length between immunoglobulin classes and subclasses.

[0113] The determination or definition of a CDR can be accomplished by resolving the structure of the antibody and / or the structure of the antibody-ligand complex, thereby enabling the definitive depiction of the CDR and the identification of residues containing the antibody binding site. This can be achieved using any of a variety of techniques known to those skilled in the art, such as X-ray crystallography. Various analytical methods (including combinations of methods) can be used to identify CDRs, including but not limited to Kabat definition, Chothia definition, AbM definition, IMGT definition, Contact definition, and conformational definition. All of these techniques are well-known in the art; see, for example, Kabat, E.A. et al. (1991), Sequences of Proteins of Immunological Interest, 5th ed., U.S. Department of Health and Human Services, NIH Publication No. 91-3242; Chothia, et al. (1989), Nature 342:877; Chothia, C. et al. (1987), J. Mol. Biol. 196:901-917; Allazikani et al. (1997), J. Molec. Biol. 273:927-948; and Almagro, J. Mol. Recognit. 17:132-143 (2004). The boundaries of a given CDR can vary depending on the method used for identification. Therefore, unless otherwise stated, the term "CDR" for a given antibody or its region (such as a variable region), and individual CDRs (e.g., HCDR1, HCDR2) of an antibody or its region, should be understood to encompass complementarity-determining regions as defined above by any known method described herein. In some cases, schemes for identifying a particular CDR or multiple CDRs (such as CDRs defined by the IMGT, Kabat, Chothia, or Contact methods) are described. In other cases, a specific amino acid sequence of the CDR is given. It should be noted that CDR regions can also be defined by combinations of various numbering systems (e.g., a combination of Kabat and Chothia or a combination of Kabat and IMGT). Therefore, once a variable region (e.g., VH or VL) is given, those skilled in the art will understand that CDRs within that region can be defined by different numbering systems or combinations thereof.

[0114] CDRs can also be identified using software programs, including but not limited to AbRSA (http: / / cao.labshare.cn / AbRSA / cdrs.php), abYsis (www.abysis.org / abysis / sequence_input / key_annotation / key_annotation.cgi), and IMGT (http: / / www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi#results). The positions of amino acid residues included in an exemplary defined CDR are listed in the table below: Note 1: Definitions vary slightly in different documents, especially the Chothia definition scheme; Note 2: Except for the Contact definition which uses the Chothia or Martin numbering scheme, other definition schemes are compatible with various numbering schemes; Note 3: When using Kabat numbering, the end of Chothia HCDR1 varies between H32 and H34 depending on the length of the ring (this is because the Kabat numbering scheme places the insertion at H35A and H35B).

[0115] The term “antigen-binding fragment” (or simply “antibody portion”) refers to one or more fragments of an antibody that specifically bind to an antigen (e.g., ACVR2A). Antigen-binding functionality of antibodies has been demonstrated to be achieved through fragments of full-length antibodies. Examples of binding fragments covered by the term “antigen-binding fragment” of an antibody include: (i) Fab fragments, monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments, bivalent fragments containing two Fab fragments linked by disulfide bonds in a hinge region; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of VL and VH domains in a single arm of an antibody; (v) dAb fragments consisting of a VH domain (Ward et al., (1989) Nature 341: 544-546); (vi) separated complementarity-determining regions (CDRs); and (vii) nanobodies, heavy-chain variable regions containing a single variable domain and two constant domains. Furthermore, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be linked together via adapters using recombination methods, thus forming a single protein chain in which the VL and VH regions pair to form a monovalent molecule (called a single-chain Fv (scFv); see, for example, Bird et al., (1988) Science 242:423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also included within the scope of the term "antigen-binding fragment" in the term antibody. These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and the fragment screening performed for use is the same as that for intact antibodies.

[0116] As used herein, the term "monoclonal antibody" refers to a formulation of an antibody molecule having a single molecular composition. Monoclonal antibody compositions exhibit single binding specificity and affinity for a specific epitope.

[0117] The term "mouse antibody" refers to a monoclonal antibody against human ACVR2A prepared in accordance with the knowledge and skills of the art. Preparation involves injecting the ACVR2A antigen into a test subject (rat or mouse), followed by isolating a hybridoma expressing an antibody with the desired sequence or functional characteristics. The aforementioned mouse anti-ACVR2A antibody or its antigen-binding fragment may further include a light chain constant region of a mouse κ, λ chain, or a variant thereof, or further include a heavy chain constant region of mouse IgG1, IgG2, IgG3, or a variant thereof.

[0118] The term "chimeric antibody" refers to an antibody formed by fusing the variable region of a heterologous (e.g., murine) antibody with the constant region of a parental antibody (e.g., human antibody). Chimeric antibodies can mitigate the immune response induced by heterologous antibodies. For example, to create a human-mouse chimeric antibody, a hybridoma that secretes murine-specific monoclonal antibodies must first be established. Then, the variable region gene is cloned from the murine hybridoma cells. Next, the constant region gene of the human antibody is cloned as needed. The murine variable region gene and the human constant region gene are then linked to form a chimeric gene, which is inserted into an expression vector. Finally, the chimeric antibody molecule is expressed in a eukaryotic or prokaryotic system.

[0119] The term "humanized antibody," also known as a CDR-grafted antibody, refers to an antibody generated by grafting a mouse CDR sequence into a human antibody variable region framework, i.e., a human germline antibody framework sequence of different types. This can overcome the heterologous response induced by chimeric antibodies carrying a large amount of mouse protein components. Such framework sequences can be obtained from public DNA databases that include germline antibody gene sequences or from publicly available references. For example, germline DNA sequences of human heavy chain and light chain variable region genes can be obtained from the VBase human germline sequence database and from Kabat, E.A. et al., 1991, Sequences of Proteins of Immunological Interest, 5th edition. To avoid a decrease in activity along with a decrease in immunogenicity, minimal reverse or reversion mutations can be performed on the aforementioned human antibody variable region framework sequences to maintain activity.

[0120] The terms "fully human antibody" and "fully human antibody" are used interchangeably, generally referring to antibodies whose entire composition (including the variable and constant regions) is encoded by genes of human origin. Fully human antibodies can significantly reduce the immune side effects caused by heterologous antibody components. Methods for obtaining fully human antibodies in this field include phage display technology, transgenic mouse technology, and ribosome display technology.

[0121] The term "bispecific" refers to the ability of an antibody and / or antigen-binding molecule to specifically bind to two different antigenic determinants. Typically, a bispecific antibody contains two antigen-binding sites, each specific to a different antigenic determinant. In some embodiments, the bispecific antibody or antigen-binding molecule can bind to two antigenic determinants simultaneously, particularly two antigenic determinants expressed on two different cells.

[0122] The term "variants" of the heavy chain constant region and light chain constant region refers to human-derived heavy chain constant regions or light chain constant regions that do not alter the structure and function of the antibody variable region, as disclosed in the prior art. Exemplary variants include IgG1, IgG2, IgG3, or IgG4 heavy chain constant region variants that involve site-specific modifications and amino acid substitutions in the heavy chain constant region. Specific substitutions include known prior art mutations such as YTE mutations, L234A and / or L235A mutations, S228P mutations, mutations that yield a knock-in-hole structure (resulting in a knock-Fc and hole-Fc combination in the antibody heavy chain), and L234A / L235A / P329G (LALA-PG) mutations. These mutations have been shown to impart novel properties to antibodies without altering the function of the antibody variable region.

[0123] The amino acid sequences of the constant region, κ chain, and λ chain of wild-type human IgG are summarized in the table below:

[0124] The term "mutation" in amino acid terminology encompasses the substitution (replacement, substitution), deletion, insertion, and modification of amino acids. Any combination of substitution, deletion, insertion, and modification can be performed to obtain the final construct, provided that the final construct possesses the desired characteristics. Amino acid mutations can be generated using any genetic engineering or chemical method well known in the art, such as site-directed mutagenesis, PCR, gene synthesis, chemical modification altering amino acid side chain groups, etc.

[0125] As used herein, the terms “substitution,” “replacement,” or “alternation” for amino acids are used interchangeably and refer to the substitution of an amino acid residue by another amino acid residue. Preferably, the term “substitution” refers to the substitution of an amino acid residue by another amino acid residue selected from the standard 20 naturally occurring amino acid residues, rare naturally occurring amino acid residues (e.g., hydroxyproline, hydroxylysine, allohydroxylysine, 6-N-methyllysine, N-ethylglycine, N-methylglycine, N-ethylasparagine, alloleucine, N-methylisoleucine, N-methylvaline, pyroglutamine, GABA, ornithine, ortholeucine, orthovaline), and non-naturally occurring amino acid residues that are typically synthesized (e.g., cyclohexylalanine). Preferably, the term “alternation” refers to the substitution of an amino acid residue by another amino acid residue selected from the standard 20 naturally occurring amino acid residues (G, P, A, V, L, I, M, C, F, Y, W, H, K, R, Q, N, E, D, S, and T). In this document, amino acid substitutions are referred to as follows: for example, replacing proline at position 329 of the Fc region with glycine is represented as P329G. Substitutions can be conservative or non-conservative. Conservative substitutions refer to the mutual substitution of amino acids belonging to the same class or having similar characteristics (e.g., charge, side chain size, hydrophobicity, hydrophilicity, main chain conformation, and rigidity). Examples of conservative substitutions include basic amino acids (arginine, lysine, and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine, asparagine, and threonine), hydrophobic amino acids (methionine, leucine, isoleucine, cysteine, and valine), aromatic amino acids (phenylalanine, tryptophan, and tyrosine), and small amino acids (glycine, alanine, and serine).

[0126] The term "conjugate" refers to the linkage between an antibody or its antigen-binding fragment and an effector molecule such as a chemotherapeutic agent, toxin, immunotherapeutic agent, bioactive protein, or imaging probe. The linkage can be covalent or non-covalent, such as through electrostatic forces. Linkage can be achieved chemically or through recombination. Various linkers known in the art can be used to form conjugates.

[0127] In one embodiment, the linker is chemical, wherein a reaction between the antibody or its antigen-binding fragment and the effector molecule produces a covalent bond formed between the two molecules to form a single molecule. Peptides typically contain multiple functional groups; such as carboxylic acid (COOH), free amine (-NH2), or thiol (-SH) groups, which can be used to react with suitable functional groups on the antibody to result in the binding of the chemical moiety. In the case where both the antibody and the effector molecule are peptides, the linker can be attached via their side groups to the constituent amino acids (e.g., via disulfide bonds to cysteine) or to the α-carbon amino and carboxyl groups of the terminal amino acid.

[0128] In one implementation, the linking is recombinant, and the conjugate can be provided as a fusion protein, possibly from the expression of a polynucleotide encoding the conjugate. As used herein, a "fusion protein" refers to a protein produced by linking two or more genes or gene segments that originally encode separate proteins (including peptides and polypeptides). Translation of the fusion gene produces a single protein having functional properties derived from each of the original proteins.

[0129] The term "affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise stated, as used herein, "binding affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its partner Y is typically expressed by the dissociation constant (KD). Affinity can be measured using conventional methods known in the art, including those described herein.

[0130] The term "isotype" refers to an antibody class (e.g., IgM or IgG1) encoded by a gene in the heavy chain constant region.

[0131] As used herein, the terms “comprising,” “including,” and “having” are used interchangeably to indicate the inclusiveness of a scheme, meaning that the scheme may contain elements other than those listed. It should also be understood that the use of “comprising,” “including,” and “having” in this document also provides for schemes “consisting of…”.

[0132] The terms “about” and “approximately” mean within 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of a given value or range.

[0133] As used herein, the terms “substantially non-binding” or “non-binding” mean that the antibody or binder of this disclosure does not exhibit detectable binding to a given target, for example, reactivity with the given target not exceeding 30%, not exceeding 20%, not exceeding 10%, or not exceeding 9%, 8%, 7%, 6%, 5%, or 3%.

[0134] The term "antibody" in this article includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), monovalent antibodies, multivalent antibodies, intact antibodies, antigen-binding fragments, naked antibodies, conjugated antibodies, humanized antibodies, or fully human antibodies.

[0135] The term "conservative amino acid" in this document generally refers to amino acids that belong to the same class or have similar characteristics (e.g., charge, side chain size, hydrophobicity, hydrophilicity, main chain conformation, and rigidity). For example, the amino acids in each of the following groups belong to each other's conserved amino acid residues, and substitutions of amino acid residues within a group constitute substitutions of conserved amino acids:

[0136] 1) Alanine (A), Serine (S), Threonine (T);

[0137] 2) Aspartic acid (D), glutamic acid (E);

[0138] 3) Asparagine (N), glutamine (Q);

[0139] 4) Arginine (R), Lysine (K), Histidine (H);

[0140] 5) Isoleucine (I), leucine (L), methionine (M), valine (V); and

[0141] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0142] The terms "identity," "sequence consistency," and "homology" used herein are interchangeable and are calculated as follows: To determine the percentage of "identity" between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., vacancies may be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment, or non-homologous sequences may be discarded for comparison purposes). The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. The molecules are identical at that position when a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence.

[0143] The term "epitope" refers to a region or site of an antigen that specifically binds to an antibody, such as a region or site containing contact residues that interact with the antibody. Therefore, the term "epitope" refers to a portion of a molecule that can be recognized and bound by an antibody at one or more antibody-antigen binding regions. Typically, an epitope is defined in the context of molecular interactions between an antibody or its antigen-binding fragment and its corresponding antigen. Epitopes are typically composed of surface groups of molecules such as amino acids or sugar side chains and have specific three-dimensional structural features and specific charge features. In some embodiments, an epitope may be a protein epitope. Protein epitopes can be linear or conformational. In a linear epitope, all interaction sites between the protein and the interacting molecule (such as an antibody) are linear along the primary amino acid sequence of the protein. A "non-linear epitope" or "conformational epitope" is a non-neighboring polypeptide (or amino acid) contained within an antigen protein to which an antibody specific to that epitope binds. As used herein, the term "antigen epitope" is defined as a portion of an antigen that can specifically bind to an antibody, as determined by any method known in the art (e.g., by conventional immunoassay). Alternatively, antibody generation and characterization during the discovery process may reveal information about the desired epitope. Based on this information, antibodies that bind to the same epitope can then be competitively screened. This can be achieved by conducting competitive and cross-competitive studies to find antibodies that compete or cross-competitively bind to ACVR2A, such as antibodies that compete to bind to the antigen.

[0144] When the term "competition" is used in cases where antigen-binding proteins (e.g., neutralizing antigen-binding proteins or neutralizing antibodies) compete for the same epitope, it refers to competition between antigen-binding proteins, which is determined by an assay in which the antigen-binding protein to be detected (e.g., an antibody or an immunologically functional fragment thereof) prevents or inhibits (e.g., reduces) the specific binding of a reference antigen-binding protein (e.g., a ligand or a reference antibody) to a common antigen (e.g., ACVR2A antigen or a fragment thereof). Numerous types of competitive binding assays can be used to determine whether one antigen-binding protein competes with another. These assays include: solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see, for example, Stahli et al., 1983, Methods in Enzymology 9: 242-253); solid-phase direct biotin-avidin EIA (see, for example, Kirkland et al., 1986, J. Immunol. 137: 3614-3619), solid-phase direct labeling assay, and solid-phase direct labeling sandwich assay (see, for example, Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor). Press); solid-phase direct labeling of RIA with I-125 label (see, for example, Morel et al., 1988, Molec. Immunol. 25: 7-15); solid-phase direct biotin-avidin EIA (see, for example, Cheung et al., 1990, Virology 176: 546-552); and directly labeled RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32: 77-82). The assay typically involves using a solid surface or cell to bind purified antigen loaded with either an unlabeled detection antigen-binding protein or a labeled reference antigen-binding protein. Competitive inhibition is measured by measuring the amount of label bound to the solid surface or cell in the presence of the detection antigen-binding protein. Typically, the detection antigen-binding protein is present in excess. Antigen-binding proteins identified by competitive assays (competitive antigen-binding proteins) include: antigen-binding proteins that bind to the same epitope as a reference antigen-binding protein; and antigen-binding proteins that bind to a neighboring epitope sufficiently close to the binding epitope of the reference antigen-binding protein, wherein the two epitopes spatially prevent each other from binding. Typically, when a competing antigen-binding protein is present in excess, it will inhibit (e.g., reduce) at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or 75% or more of the specific binding of the reference antigen-binding protein to the common antigen. In some cases, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.

[0145] The term "antagonist" refers to the inhibition of receptor signaling to suppress a biological response associated with receptor activation. Antagonistic antibodies are used in the broadest sense to include antibodies that partially or completely block, inhibit, or neutralize the biological activity of an epitope, peptide, or cell to which they specifically bind. Methods for identifying antagonistic antibodies may include contacting a peptide or cell to which a candidate antagonistic antibody specifically binds with the candidate antagonistic antibody and measuring detectable changes in one or more biological activities typically associated with the peptide or cell.

[0146] The term "IC" 50 "IC" refers to the half-maximal inhibitory concentration, which measures the efficacy of a substance (e.g., an antibody) in inhibiting a specific biological or biochemical reaction. 50 The smaller the value, the stronger the indicator effect.

[0147] The term "EC" 50 "EC" refers to the half-maximal effective concentration, which measures the efficacy of a substance (e.g., an antibody) in inducing a specific biological or biochemical reaction. 50 The smaller the value, the stronger the indicator effect.

[0148] The terms “ka” and “kon” are used interchangeably in this document and refer to the binding rate constant of a specific antibody-antigen interaction. The terms “kd” and “koff” are used interchangeably in this document and refer to the dissociation rate constant of a specific antibody-antigen interaction. As used herein, the term “KD” refers to the equilibrium dissociation constant, which is calculated from the ratio of kd to ka (i.e., kd / ka) and expressed as a molar concentration (M).

[0149] The term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, for example, non-human primates, rodents, rabbits, pigs, dogs, cats, chickens, amphibians, and reptiles, although mammals such as non-human primates and rodents are preferred.

[0150] The term "therapeutic effective amount" refers to the amount of the anti-ACVR2A antibody or its antigen-binding fragment of the present invention sufficient to prevent or improve symptoms associated with a disease or condition (e.g., fatty liver, diabetes, obesity, dyslipidemia, elevated glucose levels, elevated insulin levels, or diabetic nephropathy) and / or reduce the severity of the disease or condition. Therapeutic effective amount should be understood in the context of the condition being treated, where the actual effective amount can be readily identified by those skilled in the art.

[0151] The heavy chain variable region and light chain variable region (CDR) in this invention can be determined according to the definitions of Kabat, Chothia, IMGT, AbM, Contact, or combinations thereof. Table A below provides the amino acid sequences of the heavy chain / light chain CDRs of exemplary anti-ACVR2A antibodies of this invention identified by abYsis. Antibodies having the same heavy chain and light chain CDR1, CDR2, and CDR3 regions as the anti-ACVR2A antibodies of this invention are also within the scope of this invention.

[0152] Table A

[0153] As used in this article, the positive control antibody Bimagrumab can be prepared independently based on the information recorded in WHO-INN proposed list 108, 2012, vol 26, No 4, pages 407-408.

[0154] As used in this article, the positive control antibody LAE102 can be prepared independently based on the ACVR2A antibody LA01 described in patent document WO2023 / 030503. The VH and VL amino acid sequences of LA01 are shown in SEQ ID NO.12 and 38 as described in WO2023 / 030503.

[0155] Other features and advantages of the present invention will become apparent from the following detailed description and examples, and should not be construed as limiting. All references, Genbank entries, patents, and published patent applications cited throughout this application are expressly incorporated herein by reference. Attached Figure Description

[0156] Figure 1 shows the ELISA curve of Ab-01 combined with ACVR2A.

[0157] Figure 2 shows the blocking effect of Ab-01 in reporter gene cell lines.

[0158] Figures 3A to 3D show the blocking effect of the mutant antibody in reporter gene cell lines, and Figure 3E shows the binding of the antibody to ACVR2B expressed in the cells.

[0159] Figures 4 and 5 show the blocking effect of Ab-09 and its Fc mutant modified antibodies in reporter gene cell lines.

[0160] Figure 6 shows the binding of the Fc mutant modified antibody to its respective FcγR and C1q proteins.

[0161] Figures 7 and 8 show the in vivo efficacy results of Example 8.1, where Figure 7 shows the effect of the test substance on body weight and Figure 8 shows the effect of the test substance on lean body mass (W1 and W4 represent week 1 and week 4, respectively).

[0162] Figure 9 shows the in vivo efficacy results of Example 8.2, where Figure 9A shows the effect of the test substance on body weight, Figure 9B shows the effect of the test substance on lean body mass, and Figure 9C shows the effect of the test substance on fat mass. Specific Implementation

[0163] Example 1: Preparation of fully human anti-ACVR2A monoclonal antibody

[0164] Lead antibody molecules specifically targeting ACVR2A were screened using a self-made fully human natural library. Multiple rounds of liquid chromatography panning were performed using biotinylated ACVR2A or ACVR2B proteins, and multiple rounds of cell panning were performed using ACVR2A-positive or ACVR2B-positive cell lines. Phage-positive clones specifically binding to the target antigen were obtained and enriched. Positive clones were infecting TG1 bacterial culture. The fully infected culture was serially diluted and plated on agar plates containing the relevant antibodies and cultured overnight. Single clones were picked and cultured. The cultured bacterial culture was centrifuged, and the expression supernatant was used for ELISA, FACS, and other experiments to verify the positive antibody clones. The bacterial cultures corresponding to the positive clones were then cultured, plasmids extracted, PCR performed, and single-clone Sanger sequencing was conducted. The sequencing results of positive clones are shown in Table 1.

[0165] To express the full-length antibody, nucleic acid molecules encoding the light and heavy chains of the candidate antibody were synthesized based on sequencing results and inserted into the eukaryotic expression vector pcDNA3.4 to construct antibody heavy chain expression vectors and antibody light chain expression vectors, respectively. These vectors were then co-transfected into ExpiCHO-S cells, and transient antibody expression was performed according to the supplier's ExpiCHO-S expression system culture method. Cell supernatants were collected, and the antibody was purified using Protein A according to standard methods. Antibody purity was detected by SDS-PAGE and SEC-HPLC. The variable region amino acid sequence of the candidate antibody Ab-01 is shown in Table 1, and the constant region amino acid sequence is shown in Table 2. The full-length sequence of the Ab-1 heavy chain was obtained by directly splicing VH1 with the heavy chain constant region sequence, and the full-length sequence of the Ab-1 light chain was obtained by directly splicing VL1 with the light chain constant region.

[0166] Table 1

[0167] Table 2

[0168] Example 2: Validation of the in vitro activity of anti-ACVR2A antibody

[0169] 2.1 Cross-reaction between species

[0170] The binding affinity of antibodies to ACVR2A protein from different species was determined by enzyme-linked immunosorbent assay (ELISA). In short: Human or mouse ACVR2A protein was coated at a concentration of 2 μg / mL, 100 μL / well, and the coated ELISA plate was incubated overnight at 4°C. The plate was washed with 0.05% PBST, and then 150 μL of blocking buffer (PBS containing 1% BSA) was added to each well, and the plate was incubated at 37°C for 1 h. The blocking buffer was discarded, and 100 μL of candidate antibody was added to each well, with an initial concentration of 68 nM, serially diluted 4-fold. The remaining two wells were treated with dilution buffer (PBS) as a blank control, and the plate was incubated at 37°C for 1 h. The plate was washed, and 100 μL of diluted goat anti-human IgG, F(ab')2 enzyme-labeled secondary antibody (Jackson, 109-035-097) was added to each well and incubated at 37°C. Remove the ELISA plate, wash it, add 100 μL of chromogenic reagent (TMB), and incubate at 25°C for 2-3 min. Stop the reaction by adding 100 μL of 1M H₂SO₄. Read the OD450 value using the ELISA reader and calculate the EC50 value. The results showed that antibody Ab-01 exhibited good cross-binding activity against both human and mouse ACVR2A protein. Figure 1 shows the ELISA curves of the reaction between antibody Ab-01 and ACVR2A protein.

[0171] 2.2 Cell-level blockade

[0172] The blocking effects of antibodies on receptors and ligands at the cellular level were determined using fluorescent reporter gene cell lines specifically expressing ACVR2A and ACVR2B. The ACVR2A fluorescent reporter gene cell line (Genomeditech, catalog number GM-C28074) had the ACVR2B gene knocked out, thus the ligand protein could only bind to ACVR2A to activate downstream signaling pathways. Cells were resuspended and diluted in DMEM medium containing 10% FBS, and seeded at 100 μL / well in 96-well plates to ensure each well contained 2 × 10⁶ cells / well. 4Cells were cultured overnight at 37°C and 5% CO2. The next day, the cell supernatant was discarded, and the antibody was diluted to 500 nM, then serially diluted 3-fold. 50 μL of the diluted antibody was added to each well of the cells seeded the previous day, and the cells were incubated at 37°C and 5% CO2 for 1 hour to allow antibody binding. Then, ligand activator A was diluted to 2 ng / ml, and 50 μL was added to each well of the co-incubated cells. Incubation was continued at 37°C and 5% CO2 for 6 hours. After incubation, an equal volume of luciferase substrate was added to each well at room temperature for 3-5 minutes, and the fluorescence value was detected and read. The assay method for the ACVR2B fluorescent reporter gene cell line (Genomeditech, catalog number GM-C26076) was the same as that for ACVR2A. Figure 2 shows the blocking effect of the antibody on ACVR2A, ACVR2B and ligands. It can be seen that the antibody Ab-01 can specifically block the interaction between ACVR2A and activin A, but has little effect on the binding of ACVR2B to activin A.

[0173] Example 3 Antibody Engineering

[0174] Ab-01 was used as the parent antibody for affinity maturation modification to obtain antibody molecules with further improved affinity and biological activity. Specifically, single-point saturation mutations were performed on the amino acid sequence of the variable region of Ab-01. Mutation hotspots that could specifically bind to the target antigen were screened by ELISA and combined to obtain the mutant sequences of the parent antibody. The mutant sequences of the heavy chain variable region and the light chain variable region are listed in Tables 3.1 and 3.2, respectively (the underlined parts are the CDR regions defined by Kabat as identified by abYsis). The hotspot combination methods are listed in Table 3.3 (the mutation positions of the VH region are based on the SEQ ID NO.1 number; the mutation positions of the VL region are based on the SEQ ID NO.2 number; the position numbers are all counted starting from the N-terminus of VH or VL).

[0175] Table 3.1 Ab-01 heavy chain variable region mutation sequence

[0176] Table 3.2 Ab-01 light chain variable region mutation sequence

[0177] Table 3.3 Mutation Hotspot Combinations

[0178] Following the method described in Example 2.2, the ACVR2A fluorescent reporter gene cell line was used to evaluate the blocking effect of the mutant antibodies on the binding of ACVR2A to activin A. A total of 23 antibody molecules were obtained, and their sequences are shown in Table 4. The full-length heavy chain sequences of each mutant antibody were obtained by directly splicing VH2 to VH17 with the heavy chain constant region sequence shown in SEQ ID NO. 3, and the full-length light chain sequences of each mutant antibody were obtained by directly splicing VL2 to VL8 with the light chain constant region shown in SEQ ID NO. 4. The blocking effect of each antibody molecule on the binding of ACVR2A to activin A is shown in Figures 3A to 3C.

[0179] Table 4. Amino acid sequences of the variable region of mutant antibody molecules.

[0180] Following the method described in Example 2.2, the partial mutant antibody in Table 4 was used to assess the blocking effect of ACVR2B binding to activin A using the ACVR2B fluorescent reporter gene cell line. The results are shown in Figure 3D (Bimagrumab was the positive control). Additionally, the binding of the antibody to ACVR2B at the cellular level was determined using a cell line expressing only ACVR2B (Genomeditech, catalog number GM-C34942). Briefly, the cells were resuspended and diluted to 2 × 10⁻⁶ cells using FACS buffer (PBS solution containing 5% FBS). 6 Cells were seeded at a concentration of 50 μL / well in a 96-well U-plate. The antibody was diluted to 200 nM with FACS buffer, then serially diluted 4-fold for 7 spots. 50 μL of the diluted antibody was added to each well, gently mixed, and incubated on ice for 90 min. The cells were centrifuged at 3000 rpm for 3 min to remove the supernatant, and washed twice. PE anti-human IgG secondary antibody (R&D, F0157) diluted with FACS buffer was added to each well, gently mixed, and incubated at room temperature in the dark for 30 min. The cells were centrifuged at 3000 rpm for 3 min to remove the supernatant, and washed twice. 100 μL of FACS buffer was added to each well to resuspend the cells, and the cells were analyzed. The results are shown in Figure 3E (Bimagrumab was the positive control). It is evident that the mutant antibody Ab-09 neither binds to ACRV2B nor affects its interaction with its ligand; this antibody specifically blocks ACRV2A.

[0181] Example 4: Surface Plasmon Resonance (SPR) Affinity Test

[0182] Bimagrumab-Fab represents the Fab fragment of Bimagrumab, LAE102-Fab represents the Fab fragment of LAE102, and Ab-09-Fab represents the Fab fragment of Ab-09. The binding affinity of each Fab fragment to human ACVR2A was detected using a Biacore T200 (Cytiva) sensor. In summary, the affinity assay was performed at 25°C using a Protein A sensor chip with a pH 7.4 HBS-EP+ buffer. Human ACVR2A (catalog number ARA-HM22A) was coupled to the Protein A chip, and the Fab fragments were serially diluted. Fab fragments at different concentrations were injected into the chip surface at a flow rate of 30 μL / min, with binding and dissociation times of 60 s and 360 s, respectively. The chip was regenerated using 10 mM glycine hydrochloride solution (pH 1.5). The binding curves of the Fab fragments at different concentrations were used to calculate the kinetic parameters ka, kd, and KD. The results are shown in Table 5, which demonstrates that the antibody of the present invention binds to ACVR2A with better affinity.

[0183] Table 5

[0184] Example 5 Antibody Modification

[0185] The Fc region of the antibody is further modified to remove antibody effector function and / or prolong antibody half-life. That is, the heavy chain constant region of the aforementioned antibodies of the present invention is replaced with any of the heavy chain constant region sequences in Table 6 to obtain a series of modified antibodies. Table 7 exemplarily lists the full-length heavy and light chain sequences of Ab-09 and its Fc-mutated modified antibodies.

[0186] Table 6. Amino acid sequence of the heavy chain constant region of the antibody.

[0187] Table 7. Full-length heavy and light chain sequences of Ab-09 and its modified antibodies.

[0188] The inhibitory effects of Ab-09 and its Fc mutant antibodies on human ACVR2A at the cellular level were determined. HEK293 cells, a system with ACVR2B knockout and overexpression of the luciferase reporter gene, were used as assay cells. The cells were resuspended and diluted in DMEM medium containing 5% FBS, and seeded at 100 μL / well in 96-well plates to ensure each well contained 2 × 10⁶ cells / well. 4Cells were cultured overnight at 37°C and 5% CO2. The next day, the cell supernatant was discarded, and the antibody was diluted to 600 nM, then serially diluted 3-fold. 50 μL of the diluted antibody was added to each well of the previously seeded cells and incubated at 37°C and 5% CO2 for 1 hour to allow antibody binding. Then, ligand activator A was diluted to 20 ng / ml and 50 μL was added to each well of the co-incubated cells. Incubation was continued at 37°C and 5% CO2 for 6 hours. After incubation, an equal volume of luciferase substrate was added to each well at room temperature for 3-5 minutes, and the fluorescence value was detected and read. The results are shown in Figures 4 and 5. It can be seen that the mutation in the Fc region does not significantly change the antigen-binding activity of the antibody, and Ab-09 and its Fc-mutated modified antibodies can block ACVR2A binding to the ligand more effectively than Bimagrumab or LAE102. Further, following the method described in Example 2.2, the blocking effect of the modified antibodies against each Fc mutation was evaluated using the ACVR2B fluorescent reporter gene cell line (Genomeditech, catalog number GM-C26076) as the detection cell. The results showed that the modified antibodies against each Fc mutation were similar to Ab-09 and had no effect on ACVR2B.

[0189] Example 6: Fc Effect Functional Test

[0190] 6.1 Detection of ADCC and ADCP effects of antibodies

[0191] The binding of antibodies to various human FcγR proteins (CD16a, CD32a, or CD32b / c) was detected using Fortebio. In short, the antibody to be tested was immobilized onto a Protein A sensor, and the FcγR protein to be tested was diluted into several concentration gradients. The sensor was subjected to binding and dissociation reactions with the FcγR protein at each concentration. After each binding and dissociation cycle, the sensor was regenerated with buffer before starting the next cycle. After the experiment, the binding pattern was fitted using software. Figure 6 shows that Ab-09 and Ab-09.LALAPG_LS did not bind to CD16a, CD32a, or CD32b / c, respectively, with Guselkumab used as a positive control antibody. This demonstrates that the Fc mutation eliminated the ADCC and ADCP effects of the antibody.

[0192] 6.2 Detection method for antibody CDC effect

[0193] The binding of antibodies to human C1q protein was detected using Fortebio. The test antibody was immobilized onto a FAB2G sensor and blocked with an IgG4 negative antibody. C1q protein was distributed in several concentration gradients, and the sensor was subjected to binding and dissociation reactions with each concentration of C1q protein. After each binding and dissociation cycle, the sensor was regenerated with buffer before starting the next cycle. After the experiment, the binding pattern was fitted using software. Figure 6 shows that Ab-09 and Ab-09.LALAPG_LS did not bind to C1q, with Guselkumab used as a positive control antibody. This indicates that the Fc mutation eliminated the CDC effect of the antibody.

[0194] Example 7: Detection of antibody affinity kinetics for human FcRn

[0195] The affinity constant (KD) between FcRn (FCGRT & B2M dimer) receptor protein and the test antibody was detected using Fortebio. Biotinylated FcRn receptor protein was diluted to 2.5 μg / ml with diluent (0.05% PBST, pH = 6.0) and immobilized onto an SA sensor at a height of 1.0 nm. The test antibody was diluted to 100 nM with diluent (0.05% PBST, pH = 6.0), followed by a 2-fold serial dilution to prepare seven concentration points. The immobilized SA sensor was equilibrated in diluent (0.05% PBST, pH = 6.0) for 60 s, then bound to the test antibody at each concentration gradient for 60 s, followed by dissociation for 180 s. After each binding and dissociation cycle, the sensor was regenerated using diluent before starting the next cycle. After the experiment, the binding pattern was fitted using software to obtain the affinity constant (KD), binding rate constant (kon), and dissociation rate constant (kd) between the test antibody and the FcRn receptor protein. The results are shown in Tables 8.1 and 8.2. It can be seen that the Fc region mutations YTE or LS can enhance the binding of the antibody to FcRn, thereby prolonging the antibody's half-life.

[0196] Table 8.1

[0197] Table 8.2

[0198] Example 8: In vivo efficacy verification

[0199] 8.1 Validation of muscle-building effect in DIO mice

[0200] DIO mice were treated with mouse IgG-type Ab-09 and LAE102 antibodies. Specifically, the Fab domain of Ab-09 was linked to the mouse IgG2a-LALAPG Fc domain to obtain mouse IgG-format Ab-09, named mAb-09. Similarly, the Fab domain of LAE102 was linked to the mouse IgG2a-LALAPG Fc domain to obtain mouse IgG-format LAE102, named mLAE102.

[0201] High-fat diet was induced in 16-week-old (natural age 21 weeks) DIO mice (Jicui Yaokang) and they were randomly divided into groups of 10 mice each according to their body weight. All DIO mice were continuously fed an irradiated high-fat diet (Research Diets, D12492), and one group of C57BL / 6 mice on a normal diet was set up as a control group. Mice in each group received the following interventions: subcutaneous injection of 30 mg / kg of the test substance once a week, and / or subcutaneous injection of 0.14 mg / kg of semaglutide daily, as detailed in Table 9. Body weight was monitored three times a week during the experiment, and body composition analysis was performed weekly using a body composition analyzer (Suzhou Numai, QMR06-060H-PRO). In addition, food intake was monitored on days 1-3, 10-12, and 17-19 after the first administration. The experiment ended on day 28 after administration. The results are shown in Figures 7 and 8. It can be seen that mAb-09 promotes lean body mass (muscle) growth in DIO mice and works synergistically with Semigalutide for weight loss, especially in reversing the muscle loss caused by Semigalutide.

[0202] Table 9 Note: qw means once a week; qd means once a day; sc means subcutaneous injection; vehicle was administered to the Normal and Model groups.

[0203] 8.2 Combined administration of semaglutide to DIO mice

[0204] High-fat diet induction was used to induce 16-week-old (natural age 21 weeks) DIO mice (Jicui Yaokang) into randomized groups of 7 mice per group based on body weight. All DIO mice were continuously fed an irradiated, high-fat diet (Research Diets, D12492), with one group of C57BL / 6 mice on a normal diet serving as a control group. Each group of mice received the following interventions: subcutaneous injection of 30 mg / kg of the test substance once weekly, and / or subcutaneous injection of 0.08 mg / kg of Semaglutide daily, as detailed in Table 10. Body weight was monitored three times weekly during the experiment, and body composition analysis was performed weekly using a body composition analyzer (Suzhou Numai, QMR06-060H-PRO). The results are shown in Figure 9. It can be seen that at the other dosage, the antibody of this invention also showed a synergistic effect with Semaglutide, achieving better fat / weight reduction compared to Semaglutide alone. Furthermore, the antibody of this invention can reverse the muscle loss caused by Semaglutide.

[0205] Table 10 Note: qw means once a week; qd means once a day; sc means subcutaneous injection; vehicle was administered to the Normal and Model groups.

[0206] Although the invention has been described through one or more embodiments, it should be understood that the invention is not limited to these embodiments, and the specification is intended to cover all alternatives, modifications, and variations falling within the spirit and broad scope of the appended claims. All references cited in this invention are incorporated herein by reference in their entirety.

Claims

1. An anti-ACVR2A antibody or its antigen-binding fragment, characterized in that, The anti-ACVR2A antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises HCDR1, HCDR2, and HCDR3 regions, and the VL comprises LCDR1, LCDR2, and LCDR3 regions, wherein: The HCDR1, HCDR2, and HCDR3 regions have the same sequences as HCDR1, HCDR2, and HCDR3 of VH as shown in SEQ ID NO.1, or sequences with up to 5, 4, 3, 2, or 1 mutations compared to each CDR in HCDR1-3 of VH as shown in SEQ ID NO.1; and the LCDR1, LCDR2, and LCDR3 regions have the same sequences as LCDR1, LCDR2, and LCDR3 of VL as shown in SEQ ID NO.2, or sequences with up to 5, 4, 3, 2, or 1 mutations compared to each CDR in LCDR1-3 of VL as shown in SEQ ID NO.

2. The mutation is selected from insertions, deletions, and / or substitutions that do not affect function, or the mutation is a substitution of a conserved amino acid; or, The HCDR1, HCDR2, and HCDR3 regions have the same sequence as HCDR1, HCDR2, and HCDR3 of VH as shown in sequence a, and the LCDR1, LCDR2, and LCDR3 regions have the same sequence as LCDR1, LCDR2, and LCDR3 of VL as shown in sequence b. The sequence a is: EVQLVQSGAEVKKPGSSVKVSCKAX1GGTFX2SX3X4ISWVRQAPGQGLEWMGX5IIPIFGX6ANYAQKFQGRVTITADESTSTAY MELSSLRSEDTAVYYCAX7AVAWHLX8WFDPWGQGTLVTVSS; X1 is selected from S or G, X2 is selected from S or G, X3 is selected from Y, R or S, X4 is selected from A, R or K, X5 is selected from G or R, X6 is selected from T or G, X7 is selected from I or R, X8 is selected from N, A or R; The sequence b is: DIVMTQSPDSLAVSLGERATINCKSSQSVLX9NSNX 10 KNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISS LQAEDVAVYYCQQYX 11 X 12 TPPTFGQGTRLEIK, X9 is selected from Y, D, or S, X 10 Selected from N or Y, X 11 Selected from Y or L, X 12 Selected from S or A; Alternatively, the HCDR1, HCDR2, and HCDR3 regions may have the same sequence as HCDR1, HCDR2, and HCDR3 of VH as shown in any of SEQ ID NO.1, 5-20, and the LCDR1, LCDR2, and LCDR3 regions may have the same sequence as LCDR1, LCDR2, and LCDR3 of VL as shown in any of SEQ ID NO.2, 21-27.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The VH and VL have the same sequences as HCDR1-3 and LCDR1-3 selected from the group consisting of VH and VL: SEQ ID NO.1 and 2; SEQ ID NO.5 and 2; SEQ ID NO.6 and 2; SEQ ID NO.7 and 2; SEQ ID NO.8 and 2; SEQ ID NO.9 and 2; SEQ ID NO.10 and 2; SEQ ID NO.11 and 2; SEQ ID NO.12 and 2; SEQ ID NO.13 and 2; SEQ ID NO.14 and 2; SEQ ID NO.15 and 2; SEQ ID NO.16 and 2; SEQ ID NO.17 and 2; SEQ ID NO.12 and 21; SEQ ID NO.18 and 21; SEQ ID NO.19 and 21; SEQ ID NO.12 and 22; SEQ ID NO.12 and 23; SEQ ID NO.12 and 24; SEQ ID NO.12 and 25; SEQ ID NO.12 and 26; SEQ ID NO.12 and 27; or, SEQ ID NO.20 and 21.

3. The antibody or its antigen-binding fragment according to claim 1 or 2, characterized in that, The HCDR1-3 and LCDR1-3 are defined according to IMGT, Kabat, Chothia, AbM, Contact or any combination thereof.

4. The antibody or its antigen-binding fragment according to any one of claims 1-3, characterized in that, The VH has an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in any of SEQ ID NO. 1, 5-20, and / or the VL has an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in any of SEQ ID NO. 2, 21-27.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1-4, characterized in that, The VH has an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in any of SEQ ID NO. 1, 5-17, and / or the VL has an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO. 2; The VH has an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in any of SEQ ID NO. 12, 18-20, and / or the VL has an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO. 21; or, The VH has an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO. 12, and / or the VL has an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in any of SEQ ID NO. 22-27; Alternatively, VH and VL may each comprise or be sequences selected from the group consisting of: SEQ ID NO.1 and 2; SEQ ID NO.5 and 2; SEQ ID NO.6 and 2; SEQ ID NO.7 and 2; SEQ ID NO.8 and 2; SEQ ID NO.9 and 2; SEQ ID NO.10 and 2; SEQ ID NO.11 and 2; SEQ ID NO.12 and 2; SEQ ID NO.13 and 2; SEQ ID NO.14 and 2; SEQ ID NO.15 and 2; SEQ ID NO.16 and 2; SEQ ID NO.17 and 2; SEQ ID NO.12 and 21; SEQ ID NO.18 and 21; SEQ ID NO.19 and 21; SEQ ID NO.12 and 22; SEQ ID NO.12 and 23; SEQ ID NO.12 and 24; SEQ ID NO.12 and 25; SEQ ID NO.12 and 26; SEQ ID NO.12 and 27; or SEQ ID NO.20 and 21.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1-5, characterized in that, The anti-ACVR2A antibody further comprises a heavy chain constant region and a light chain constant region, and / or, the heavy chain constant region is selected from the human IgG1, IgG2, IgG3, IgG4 constant regions or variants thereof, and the light chain constant region is selected from the human κ and λ chain constant regions or variants thereof.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1-6, characterized in that, The anti-ACVR2A antibody is selected from Table 1, Table 4, or Table 7; or... The antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of the sequences shown in SEQ ID NO. 40-47, or the heavy chain comprises a sequence having undergone at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid mutations compared to any of the sequences shown in SEQ ID NO. 40-47; and the light chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO. 48, or the light chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of the sequences ... The sequence shown in NO.48 is a sequence with a maximum of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid mutations compared to the sequence shown in NO.

48.

8. An anti-ACVR2A antibody or an antigen-binding fragment thereof, which binds to the same ACVR2A epitope or competitively binds to ACVR2A with the antibody or antigen-binding fragment thereof as described in any one of claims 1-7.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1-8, characterized in that, The antibody or its antigen-binding fragment has at least one of the following functions: (1) When bound to ACVR2A, the antibody or its antigen-binding fragment has a KD of less than about 100 nM, less than about 90 nM, less than about 80 nM, less than about 70 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, less than about 10 nM, or less than about 1 nM; (2) When bound to ACVR2A, the KD of the antibody or its antigen-binding fragment is substantially the same as or better than that of the reference ACVR2A antibody; (3) When bound to ACVR2A, the antibody or its antigen-binding fragment inhibits or blocks the binding of ACVR2A to the ACVR2A ligand; (4) When the binding of ACVR2A to ACVR2A ligand is inhibited or blocked, the inhibitory or blocking effect of the antibody or its antigen-binding fragment is basically the same as or better than that of the reference ACVR2A antibody; (5) Combined with ACVR2A from different species; (6) As an antagonistic antibody against ACVR2A; (7) Specifically binds to ACVR2A but does not bind or barely binds to ACVR2B; (8) Reduce the binding of ACVR2A to at least one of its ligands, reduce the activity of ACVR2A, or reduce ACVR2A-mediated signal transduction.

10. A conjugate or bispecific antibody comprising the anti-ACVR2A antibody or its antigen-binding fragment as described in any one of claims 1-9.

11. A polynucleotide encoding an anti-ACVR2A antibody or an antigen-binding fragment thereof as described in any one of claims 1-9, or a conjugate or bispecific antibody as described in claim 10.

12. A vector comprising the polynucleotide of claim 11.

13. A host cell comprising the polynucleotide of claim 11 or the vector of claim 12.

14. A pharmaceutical composition comprising an anti-ACVR2A antibody or an antigen-binding fragment thereof as described in any one of claims 1-9, a conjugate or bispecific antibody as described in claim 10, a polynucleotide as described in claim 11, a carrier as described in claim 12 or a host cell as described in claim 13, and one or more pharmaceutically acceptable carriers.

15. A kit comprising the anti-ACVR2A antibody or its antigen-binding fragment as described in any one of claims 1-9, or the conjugate or bispecific antibody as described in claim 10, or the pharmaceutical composition as described in claim 14.

16. Use in the preparation of a medicament of any anti-ACVR2A antibody or antigen-binding fragment thereof according to any one of claims 1-9, a conjugate or bispecific antibody according to claim 10, a polynucleotide according to claim 11, a carrier according to claim 12, a host cell according to claim 13, or a pharmaceutical composition according to claim 14, for the prevention and / or treatment of a subject with an ACVR2A-related disease or condition.

17. Use of the anti-ACVR2A antibody of any one of claims 1-9 or an antigen-binding fragment thereof and a GLP-1 receptor agonist or a pharmaceutically acceptable salt thereof in the preparation of a pharmaceutical composition for the prevention, relief and / or treatment of obesity or obesity-related diseases or conditions in a subject.

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