Antigen binding molecule specifically binding to latent-myostatin and medical use thereof

Abstract

An antigen binding molecule specifically binding to latent-myostatin and a medical use thereof. Specifically, the present disclosure relates to a use of the antigen binding molecule specifically binding to latent-myostatin in the preparation of drugs for preventing or treating muscle atrophy, muscular dystrophy, obesity and cachexia.

Description

Antigen binding molecules that specifically bind to latent myostatin and medical uses thereof

[0001] This application claims priority to Chinese patent application CN202410891968.1 filed on July 04, 2024. TECHNICAL FIELD

[0002] The present disclosure belongs to the field of biotechnology, and more specifically, the present disclosure relates to antigen binding molecules that specifically bind to latent myostatin and medical uses thereof. BACKGROUND

[0003] The statements herein are provided only to complement what is in the prior art and necessarily do not constitute admitted prior art.

[0004] Myostatin is a protein mainly expressed and secreted by skeletal muscle, which has the function of inhibiting muscle growth, and its deletion will cause animals, including humans, to have a phenotype of excessive muscle growth. Myostatin is first synthesized in the form of a precursor (pro-myostatin) in the cell, and after hydrolysis by a precursor converting protease, it forms latent myostatin, and then after hydrolysis by BMP1 / tolloid protease, it forms a free amino-terminal propeptide and a carboxy-terminal active mature myostatin protein. Among them, latent myostatin is the main form in the blood circulation. The mature form of myostatin protein is highly similar to GDF11, but studies have found that the function of GDF11 is opposite to that of myostatin, so most antibodies targeting mature myostatin in clinical practice have failed to reach the end point of drug efficacy. Currently, there is no drug on the market targeting this target, and drugs targeting myostatin precursors (including pro-myostatin and latent myostatin) are currently in the highest clinical phase of clinical phase III, and their main indications are the treatment of spinal muscular atrophy (SMA) or obesity in combination with drugs. Therefore, there is a great demand for drugs targeting myostatin precursors in clinical practice. SUMMARY

[0005] The present disclosure provides an antigen binding molecule that specifically binds to latent myostatin, wherein the antigen binding molecule inhibits the activation of myostatin.

[0006] In some embodiments, the antigen binding molecule that specifically binds to latent myostatin as previously described comprises a heavy chain variable region and a light chain variable region, wherein:

[0007] a. the heavy chain variable region comprises 1, 2, or 3 HCDR amino acid sequences from the sequence of SEQ ID NO: 39, 34, 35, 36, 37, or 38, and / or the light chain variable region comprises 1, 2, or 3 LCDR amino acid sequences from the sequence of SEQ ID NO: 32 or 33; or

[0008] the heavy chain variable region comprises 1, 2, or 3 HCDR amino acid sequences from the sequence of SEQ ID NO: 11, and / or the light chain variable region comprises 1, 2, or 3 LCDR amino acid sequences from the sequence of SEQ ID NO: 12; or

[0009] b. the heavy chain variable region comprises 1, 2, or 3 HCDR amino acid sequences from the sequence of SEQ ID NO: 61, 59, or 60, and / or the light chain variable region comprises 1, 2, or 3 LCDR amino acid sequences from the sequence of SEQ ID NO: 56, 54, 55, 57, or 58; or

[0010] the heavy chain variable region comprises 1, 2, or 3 HCDR amino acid sequences from the sequence of SEQ ID NO: 23, and / or the light chain variable region comprises 1, 2, or 3 LCDR amino acid sequences from the sequence of SEQ ID NO: 24.

[0011] In some embodiments, the antigen binding molecule that specifically binds to latent myostatin as described in any of the preceding embodiments comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a HCDR1, a HCDR2, and a HCDR3, and the light chain variable region comprises a LCDR1, a LCDR2, and a LCDR3, wherein:

[0012] a. the HCDR1, the HCDR2, and the HCDR3 of the heavy chain variable region comprise the amino acid sequences of the HCDR1, the HCDR2, and the HCDR3, respectively, of any one of the sequences of SEQ ID NO: 39, 34, 35, 36, 37, or 38, and the LCDR1, the LCDR2, and the LCDR3 of the light chain variable region comprise the amino acid sequences of the LCDR1, the LCDR2, and the LCDR3, respectively, of any one of the sequences of SEQ ID NO: 32 or 33; or

[0013] the HCDR1, the HCDR2, and the HCDR3 of the heavy chain variable region comprise the amino acid sequences of the HCDR1, the HCDR2, and the HCDR3, respectively, of SEQ ID NO: 11, and the LCDR1, the LCDR2, and the LCDR3 of the light chain variable region comprise the amino acid sequences of the LCDR1, the LCDR2, and the LCDR3, respectively, of SEQ ID NO: 12; or

[0014] b. the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region comprise the amino acid sequences of the HCDR1, HCDR2, and HCDR3 of any one of SEQ ID NOs: 61, 59, or 60, respectively, and the LCDR1, LCDR2, and LCDR3 of the light chain variable region comprise the amino acid sequences of the LCDR1, LCDR2, and LCDR3 of any one of SEQ ID NOs: 56, 54, 55, 57, or 58, respectively; or

[0015] the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region comprise the amino acid sequences of the HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 23, respectively, and the LCDR1, LCDR2, and LCDR3 of the light chain variable region comprise the amino acid sequences of the LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 24, respectively.

[0016] In some embodiments, the antigen binding molecule that specifically binds to latent myostatin as previously described, wherein:

[0017] a. the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region comprise the amino acid sequences of the HCDR1, HCDR2, and HCDR3 of any one of SEQ ID NOs: 39, 34, 35, 36, 37, or 38, respectively, and the LCDR1, LCDR2, and LCDR3 of the light chain variable region comprise the amino acid sequences of the LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 32, respectively; or

[0018] the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region comprise the amino acid sequences of the HCDR1, HCDR2, and HCDR3 of any one of SEQ ID NOs: 39, 34, 35, 36, 37, or 38, respectively, and the LCDR1, LCDR2, and LCDR3 of the light chain variable region comprise the amino acid sequences of the LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 33, respectively; or

[0019] b. the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region comprise the amino acid sequences of the HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 61, respectively, and the LCDR1, LCDR2, and LCDR3 of the light chain variable region comprise the amino acid sequences of the LCDR1, LCDR2, and LCDR3 of any one of SEQ ID NOs: 56, 54, 55, 57, or 58, respectively; or

[0020] the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region comprise the amino acid sequences of HCDR1, HCDR2, and HCDR3, respectively, in any one of SEQ ID NOs: 59 or 60, and the LCDR1, LCDR2, and LCDR3 of the light chain variable region comprise the amino acid sequences of LCDR1, LCDR2, and LCDR3, respectively, in SEQ ID NO: 54.

[0021] In some embodiments, the antigen binding molecule that specifically binds to latent myostatin of any of the preceding embodiments, wherein:

[0022] a. the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region comprise the amino acid sequences of HCDR1, HCDR2, and HCDR3, respectively, in SEQ ID NO: 39, and the LCDR1, LCDR2, and LCDR3 of the light chain variable region comprise the amino acid sequences of LCDR1, LCDR2, and LCDR3, respectively, in SEQ ID NO: 32; or

[0023] b. the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region comprise the amino acid sequences of HCDR1, HCDR2, and HCDR3, respectively, in SEQ ID NO: 61, and the LCDR1, LCDR2, and LCDR3 of the light chain variable region comprise the amino acid sequences of LCDR1, LCDR2, and LCDR3, respectively, in SEQ ID NO: 56.

[0024] In some embodiments, the antigen binding molecule that specifically binds to latent myostatin according to any of the preceding embodiments, the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region and the LCDR1, LCDR2, and LCDR3 of the light chain variable region are defined according to a numbering scheme selected from Kabat, IMGT, Chothia, AbM, and Contact. In some embodiments, the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region and the LCDR1, LCDR2, and LCDR3 of the light chain variable region are defined according to the Kabat numbering scheme. In some embodiments, the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region and the LCDR1, LCDR2, and LCDR3 of the light chain variable region are defined according to the IMGT numbering scheme. In some embodiments, the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region and the LCDR1, LCDR2, and LCDR3 of the light chain variable region are defined according to the Chothia numbering scheme. In some embodiments, the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region and the LCDR1, LCDR2, and LCDR3 of the light chain variable region are defined according to the AbM numbering scheme. In some embodiments, the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region and the LCDR1, LCDR2, and LCDR3 of the light chain variable region are defined according to the Contact numbering scheme.

[0025] In some embodiments, the antigen binding molecule that specifically binds to latent myostatin according to any of the preceding embodiments, wherein:

[0026] a. the HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 5, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 75, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 7, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 8, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 9, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 10;

[0027] wherein:

[0028] HCDR2 is: NINPSX1GVSNYX2X3KFX4X5 SEQ ID NO: 75

[0029] wherein X1is selected from S, N, or Q, preferably S; X2is selected from A or N, preferably A; X3is selected from Q or E, preferably Q; X4is selected from Q or K, preferably Q; and X5is selected from G or I, preferably G;

[0030] or

[0031] b. the HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 17, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 76, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 19, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 77, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 78, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 22, wherein:

[0032] HCDR2 is: TISSGGTYSYYX6DSVKG SEQ ID NO: 76

[0033] LCDR1 is: KASQX7VGANVA SEQ ID NO: 77

[0034] LCDR2 is: SASYRX8R SEQ ID NO: 78

[0035] wherein X6 is selected from L or A, preferably L; X7 is selected from N, I or D, preferably N; and X8 is selected from F or Y, preferably F.

[0036] In some embodiments, the antigen binding molecule that specifically binds to latent myostatin of any of the preceding embodiments, wherein:

[0037] a. the HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 5, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 31, 29, 30, or 6, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 7, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 8, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 9, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 10; or

[0038] b. the HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 17, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 18 or 53, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 19, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 51, 52, or 20, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 21 or 50, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 22.

[0039] In some embodiments, the antigen binding molecule that specifically binds to latent myostatin of any of the preceding embodiments, wherein:

[0040] a. the HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:5, the HCDR2 comprises the amino acid sequence of SEQ ID NO:31, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:7, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO:8, the LCDR2 comprises the amino acid sequence of SEQ ID NO:9, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 10; or

[0041] b-1. the HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 17, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 18, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 19, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO:51, the LCDR2 comprises the amino acid sequence of SEQ ID NO:21 or 50, and the LCDR3 comprises the amino acid sequence of SEQ ID NO:22; or

[0042] b-2. the HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 17, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 18, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 19, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO:20, the LCDR2 comprises the amino acid sequence of SEQ ID NO:21 or 50, and the LCDR3 comprises the amino acid sequence of SEQ ID NO:22; or

[0043] b-3. the HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 17, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 18, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 19, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO:52, the LCDR2 comprises the amino acid sequence of SEQ ID NO:50, and the LCDR3 comprises the amino acid sequence of SEQ ID NO:22; or

[0044] b-4. the HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 17, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 53, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 19, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 20, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 21, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 22.

[0045] In some embodiments, the antigen binding molecule that specifically binds to latent myostatin of any of the preceding embodiments, wherein:

[0046] a. the HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 5, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 31, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 7, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 8, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 9, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 10; or

[0047] b. the HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 17, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 18, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 19, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 51, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 21, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 22.

[0048] In some embodiments, the antigen binding molecule that specifically binds to latent myostatin of any of the preceding embodiments, the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region and the LCDR1, LCDR2, and LCDR3 of the light chain variable region are defined according to the Rabat numbering convention.

[0049] In some embodiments, the antigen binding molecule that specifically binds to latent myostatin as in any of the preceding embodiments is an antibody. In some embodiments, the antigen binding molecule that specifically binds to latent myostatin is a monoclonal antibody, a monospecific antibody, a bispecific antibody, or a multispecific antibody. In some embodiments, the antigen binding molecule that specifically binds to latent myostatin is a monoclonal antibody. In some embodiments, the antigen binding molecule that specifically binds to latent myostatin is a monospecific antibody.

[0050] In some embodiments, the antigen binding molecule that specifically binds to latent myostatin as in any of the preceding embodiments is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antigen binding molecule that specifically binds to latent myostatin is a humanized antibody.

[0051] In some embodiments, the antigen binding molecule that specifically binds to latent myostatin as in any of the preceding embodiments is an antigen binding fragment. In some embodiments, the antigen binding fragment is an antibody fragment. In some embodiments, the antigen binding fragment is selected from a Fab, a Fab', a F(ab')2, a Fd, a Fv, a scFv, a dsFv, or a dAb. In some embodiments, the antigen binding fragment is selected from a Fab, a Fab', a F(ab')2, a Fv, a scFv, or a dsFv.

[0052] In some embodiments, the antigen binding molecule that specifically binds to latent myostatin as in any of the preceding embodiments comprises a framework region (FR) of a human antibody.

[0053] In some embodiments, the antigen binding molecule that specifically binds to latent myostatin as in any of the preceding embodiments, wherein the heavy chain variable region comprises a FR1, a FR2, a FR3 derived from IGHV1-46*01, and a FR4 derived from SEQ ID NO: 28, and which is either unsubstituted or comprises one or more amino acid substitutions selected from the group consisting of 1E, 28T, 48I, 67A, 69L, 71V, and 73I; and / or the light chain variable region comprises a FR1, a FR2, a FR3 derived from IGKV1-16*01, and a FR4 derived from SEQ ID NO: 27, and which is either unsubstituted or comprises one or more amino acid substitutions selected from the group consisting of 36Y and 46A. In some embodiments, the antigen binding molecule that specifically binds to latent myostatin as in any of the preceding embodiments, wherein the HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 5, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 31, 6, 29, or 30, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 7, and the FRs of the heavy chain variable region are unsubstituted or comprise one or more amino acid substitutions selected from the group consisting of 1E, 28T, 48I, 67A, 69L, 71V, and 73I; and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 8, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 9, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 10, and the FRs of the light chain variable region are unsubstituted or comprise one or more amino acid substitutions selected from the group consisting of 36Y and 46A. In some embodiments, the antigen binding molecule that specifically binds to latent myostatin as in any of the preceding embodiments, wherein the HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 5, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 31, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 7, and the FRs of the heavy chain variable region are unsubstituted or comprise one or more amino acid substitutions selected from the group consisting of 1E, 28T, 69L, 71V, and 73I; and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 8, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 9, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 10, and the FRs of the light chain variable region are unsubstituted or comprise the amino acid substitution of 36Y. In some embodiments, the above variable regions and CDRs are defined according to the Kabat numbering convention.

[0054] In some embodiments, the antigen binding molecule that specifically binds to latent myostatin as in any of the preceding embodiments, wherein the heavy chain variable region comprises a FR1, a FR2, a FR3 derived from IGHV3-21*01, and a FR4 derived from SEQ ID NO: 28, and which is either unsubstituted or comprises one or more amino acid substitutions selected from the group consisting of 30S and 49A; and / or the light chain variable region comprises a FR1, a FR2, a FR3 derived from IGKV1-16*01, and a FR4 derived from SEQ ID NO: 27, and which is either unsubstituted or comprises one or more amino acid substitutions selected from the group consisting of 36Y and 46A. In some embodiments, the antigen binding molecule that specifically binds to latent myostatin as in any of the preceding embodiments, wherein the HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 17, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 18 or 53, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 19, and the FRs of the heavy chain variable region are unsubstituted or comprise one or more amino acid substitutions selected from the group consisting of 30S and 49A; and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 51, 20, or 52, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 21 or 50, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 22, and the FRs of the light chain variable region are unsubstituted or comprise one or more amino acid substitutions selected from the group consisting of 36Y and 46A. In some embodiments, the antigen binding molecule that specifically binds to latent myostatin as in any of the preceding embodiments, wherein the HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 17, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 18, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 19, and the FRs of the heavy chain variable region are unsubstituted or comprise one or more amino acid substitutions selected from the group consisting of 30S and 49A; and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 51, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 21, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 22, and the FRs of the light chain variable region are unsubstituted or comprise one or more amino acid substitutions selected from the group consisting of 36Y and 46A. In some embodiments, the above variable regions and CDRs are defined according to the Kabat numbering convention.

[0055] In some embodiments, the antigen binding molecule that specifically binds to latent myostatin as in any of the preceding embodiments, wherein:

[0056] a. the heavy chain variable region comprises SEQ ID NO: 39, 34, 35, 36, 37, or 38, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto, and the light chain variable region comprises SEQ ID NO: 32 or 33, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto; or

[0057] the heavy chain variable region comprises SEQ ID NO: 11, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto, and the light chain variable region comprises SEQ ID NO: 12, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto; or

[0058] b. the heavy chain variable region comprises SEQ ID NO: 61, 59, or 60, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto, and the light chain variable region comprises SEQ ID NO: 56, 54, 55, 57, or 58, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto; or

[0059] the heavy chain variable region comprises SEQ ID NO: 23, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto, and the light chain variable region comprises SEQ ID NO: 24, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto.

[0060] In some embodiments, the antigen binding molecule that specifically binds to latent myostatin as set forth in any of the preceding embodiments, wherein:

[0061] a. the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 39, 34, 35, 36, 37, or 38, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto, and the light chain variable region comprises SEQ ID NO: 32, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto; or

[0062] the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 39, 34, 35, 36, 37, or 38, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto, and the light chain variable region comprises SEQ ID NO: 33, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto; or

[0063] b. the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 61, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto, and the light chain variable region comprises SEQ ID NO: 56, 54, 55, 57, or 58, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto; or

[0064] the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 59 or 60, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto, and the light chain variable region comprises SEQ ID NO: 54, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto.

[0065] In some embodiments, the antigen binding molecule that specifically binds to latent myostatin as described in any of the preceding embodiments, wherein:

[0066] a. the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 39, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 32; or

[0067] b. the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 61, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 56.

[0068] In some embodiments, the antigen binding molecule that specifically binds to latent myostatin as in any of the preceding embodiments further comprises a heavy chain constant region and a light chain constant region. In some embodiments, the heavy chain constant region of the antigen binding molecule that specifically binds to latent myostatin as in any of the preceding embodiments is a heavy chain constant region derived from human IgGl, IgG2, IgG3, or IgG4.

[0069] In some embodiments, the antigen binding molecule that specifically binds to latent myostatin as described in any of the preceding embodiments, the Fc region in the heavy chain constant region is modified to ablate Fc effector functions of the antibody and / or to prolong half-life. In some embodiments, the antigen binding molecule that specifically binds to latent myostatin as described in any of the preceding embodiments, the Fc region in the heavy chain constant region is modified to ablate Fc effector functions of the antibody. In some embodiments, the antigen binding molecule that specifically binds to latent myostatin as described in any of the preceding embodiments, the Fc region in the heavy chain constant region comprises 234A, 235A, and 237A substitutions. In some embodiments, the antigen binding molecule that specifically binds to latent myostatin as described in any of the preceding embodiments, the Fc region in the heavy chain constant region is modified to prolong half-life. In some embodiments, the antigen binding molecule that specifically binds to latent myostatin as described in any of the preceding embodiments, the Fc region in the heavy chain constant region comprises 428L and 434S substitutions. In some embodiments, the antigen binding molecule that specifically binds to latent myostatin as described in any of the preceding embodiments, the Fc region in the heavy chain constant region comprises 234A, 235A, 237A, 428L, and 434S substitutions. In some embodiments, the antigen binding molecule that specifically binds to latent myostatin as described in any of the preceding embodiments comprises a heavy chain constant region of human IgGl with one or more amino acid substitutions selected from 234A, 235A, 237A, 428L, and 434S. In some embodiments, the antigen binding molecule that specifically binds to latent myostatin as described in any of the preceding embodiments comprises a heavy chain constant region of human IgGl with 234A, 235A, and 237A substitutions. In some embodiments, the antigen binding molecule that specifically binds to latent myostatin as described in any of the preceding embodiments comprises a heavy chain constant region of human IgGl with 428L and 434S substitutions. In some embodiments, the antigen binding molecule that specifically binds to latent myostatin as described in any of the preceding embodiments comprises a heavy chain constant region of human IgGl with 234A, 235A, 237A, 428L, and 434S substitutions. In some embodiments, the antigen binding molecule that specifically binds to latent myostatin as described in any of the preceding embodiments comprises a heavy chain constant region of human IgGl with L234A, L235A, G237A, M428L, N434S substitutions. In some embodiments, the substitution sites are denoted in EU numbering.

[0070] In some embodiments, the light chain constant region of the antigen binding molecule that specifically binds to latent myostatin as described in any of the preceding embodiments is derived from a human kappa or lambda light chain constant region.

[0071] In some embodiments, the antigen binding molecule that specifically binds to latent myostatin as described in any of the preceding clauses comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 48 or 13, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto, and the light chain constant region comprises SEQ ID NO: 14, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto. In some embodiments, the antigen binding molecule that specifically binds to latent myostatin as described in any of the preceding clauses comprises the amino acid sequence of SEQ ID NO: 48 or 13, the light chain constant region comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, the antigen binding molecule that specifically binds to latent myostatin as described in any of the preceding clauses comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 48 and a light chain constant region comprising the amino acid sequence of SEQ ID NO: 14.

[0072] In some embodiments, the antigen binding molecule that specifically binds to latent myostatin as described in any of the preceding clauses, wherein:

[0073] a. the heavy chain comprises SEQ ID NO: 49, 45, 40, 41, 42, 43, or 44, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto, and the light chain comprises SEQ ID NO: 46 or 47, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto; or

[0074] the heavy chain comprises SEQ ID NO: 15, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto, and the light chain comprises SEQ ID NO: 16, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto; or

[0075] b. the heavy chain comprises SEQ ID NO: 70, 64, 62, or 63, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto, and the light chain comprises SEQ ID NO: 67, 65, 66, 68, or 69, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto; or

[0076] the heavy chain comprises SEQ ID NO: 25, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto, and the light chain comprises SEQ ID NO: 26, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto.

[0077] In some embodiments, the antigen binding molecule that specifically binds to latent myostatin as set forth in any of the preceding embodiments, wherein:

[0078] a-1. the heavy chain comprises SEQ ID NO: 49, 45, 40, 41, 42, 43, or 44, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto, and the light chain comprises SEQ ID NO: 46, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto; or

[0079] a-2. the heavy chain comprises SEQ ID NO: 45, 40, 41, 42, 43, or 44, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto, and the light chain comprises SEQ ID NO: 47, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto; or

[0080] b-1. the heavy chain comprises the amino acid sequence of SEQ ID NO: 70 or 64, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto, and the light chain comprises SEQ ID NO: 67, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto; or

[0081] b-2. the heavy chain comprises the amino acid sequence of SEQ ID NO: 64, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto, and the light chain comprises SEQ ID NO: 65, 66, 68, or 69, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto; or

[0082] b-3. the heavy chain comprises the amino acid sequence of SEQ ID NO: 62 or 63, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto, and the light chain comprises SEQ ID NO: 65, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto.

[0083] In some embodiments, the antigen binding molecule that specifically binds to latent myostatin as in any of the preceding embodiments, wherein:

[0084] a. the heavy chain comprises the amino acid sequence of SEQ ID NO: 49 or 45, and the light chain comprises the amino acid sequence of SEQ ID NO: 46; or

[0085] b. the heavy chain comprises the amino acid sequence of SEQ ID NO: 70 or 64, and the light chain comprises the amino acid sequence of SEQ ID NO: 67.

[0086] In some embodiments, the antigen binding molecule that specifically binds to latent myostatin as in any of the preceding embodiments, comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 49, and the light chain comprises the amino acid sequence of SEQ ID NO: 46.

[0087] In some embodiments, the antigen binding molecule that specifically binds to latent myostatin as in any of the preceding embodiments comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 70, and the light chain comprises the amino acid sequence of SEQ ID NO: 67.

[0088] In some embodiments, the antigen binding molecule that specifically binds to latent myostatin as in any of the preceding embodiments competes for binding to human latent myostatin or an epitope thereof with the antigen binding molecule that specifically binds to latent myostatin as in any of the preceding embodiments.

[0089] In some embodiments, the antigen binding molecule that specifically binds to latent myostatin as provided herein specifically binds to human latent myostatin and does not bind to human mature myostatin, human GDF11, human latent GDF11, human activin-A, human latent activin-A, human GDF15, human BMP9, human BMP10, and / or human TGFp 1.

[0090] In some embodiments, the antigen binding molecule that specifically binds to latent myostatin as provided herein is capable of inhibiting proteases from hydrolyzing latent myostatin to mature myostatin; in some embodiments, the proteases are the BMP1 / Tolloid protease family.

[0091] In some embodiments, the antigen binding molecule that specifically binds to latent myostatin as provided herein is capable of cross-binding to human, mouse, cynomolgus latent myostatin.

[0092] In some embodiments, the antigen binding molecule that specifically binds to latent myostatin as provided herein is capable of binding to human pro-myostatin.

[0093] In some embodiments, the antigen binding molecule that specifically binds to latent myostatin as provided herein binds to human latent myostatin with a KD value of less than 3 nM (e.g., less than 2 nM, less than 1 nM, less than 0.6 nM, less than 0.1 nM, less than 0.07 nM), as measured by Biacore.

[0094] In some embodiments, the antigen binding molecule that specifically binds to latent myostatin as provided herein inhibits latent myostatin activation with an IC 50 value of less than 7 nM (e.g., less than 3.0 nM, less than 2.8 nM, less than 2.5 nM, less than 2.0 nM, less than 1.7 nM).

[0095] In another aspect, the present disclosure provides a pharmaceutical composition comprising the antigen binding molecule that specifically binds to latent myostatin as described in any of the above, or the antigen binding molecule as described in any of the above, or the multispecific antibody as described in any of the above, and one or more pharmaceutically acceptable carriers, diluents or excipients. In some embodiments, the pharmaceutical composition further comprises at least one second therapeutic agent.

[0096] In some embodiments, the pharmaceutical composition contains 0.01-99.99% of the antigen binding molecule that specifically binds to latent myostatin as described in any of the above, based on the total weight of the composition. In some embodiments, the pharmaceutical composition contains 0.1-99.9% of the antigen binding molecule that specifically binds to latent myostatin as described in any of the above, based on the total weight of the composition. In some embodiments, the pharmaceutical composition contains 0.5-99.5% of the antigen binding molecule that specifically binds to latent myostatin as described in any of the above, based on the total weight of the composition. In some embodiments, the pharmaceutical composition contains 1-99% of the antigen binding molecule that specifically binds to latent myostatin as described in any of the above, based on the total weight of the composition. In some embodiments, the pharmaceutical composition contains 2-98% of the antigen binding molecule that specifically binds to latent myostatin as described in any of the above, based on the total weight of the composition.

[0097] In some embodiments, the pharmaceutical composition contains 0.01-99.99% of the pharmaceutically acceptable carrier, diluent or excipient, based on the total weight of the composition. In some embodiments, the pharmaceutical composition contains 0.1-99.9% of the pharmaceutically acceptable carrier, diluent or excipient, based on the total weight of the composition. In some embodiments, the pharmaceutical composition contains 0.5-99.5% of the pharmaceutically acceptable carrier, diluent or excipient, based on the total weight of the composition. In some embodiments, the pharmaceutical composition contains 1-99% of the pharmaceutically acceptable carrier, diluent or excipient, based on the total weight of the composition. In some embodiments, the pharmaceutical composition contains 2-98% of the pharmaceutically acceptable carrier, diluent or excipient, based on the total weight of the composition.

[0098] In some embodiments, the pharmaceutical composition is administered subcutaneously, intravenously, intradermally, intraperitoneally or intramuscularly.

[0099] In another aspect, the present disclosure provides an isolated nucleic acid encoding the antigen binding molecule that specifically binds to latent myostatin as described in any of the above.

[0100] In another aspect, the present disclosure provides a vector comprising the isolated nucleic acid as described in any of the above.

[0101] In another aspect, the present disclosure provides a host cell comprising the isolated nucleic acid as described in any of the above.

[0102] In another aspect, the present disclosure provides a method of reducing myostatin receptor activation in a cell present in a culture medium comprising latent myostatin, the method comprising delivering to the culture medium an antigen binding molecule that specifically binds to latent myostatin as described in any of the preceding aspects, in an amount effective to inhibit proteolytic activation of the latent myostatin.

[0103] In another aspect, the present disclosure provides a method for producing an antigen binding molecule that specifically binds to latent myostatin as described in any of the preceding aspects, the method comprising culturing a host cell as described in any of the preceding aspects in a culture medium to form and accumulate the antigen binding molecule that specifically binds to latent myostatin as described in any of the preceding aspects, and the step of recovering the antigen binding molecule from the culture.

[0104] In another aspect, the present disclosure provides a method of treating, preventing, or ameliorating a disease or disorder, the method comprising administering to a subject in need thereof an antigen binding molecule that specifically binds to latent myostatin as described in any of the preceding aspects, or a pharmaceutical composition as described in any of the preceding aspects.

[0105] In another aspect, the present disclosure provides use in the manufacture of a medicament for treating, preventing, or ameliorating a disease or disorder, comprising administering to a subject in need thereof a therapeutically effective amount or a prophylactically effective amount of an antigen binding molecule that specifically binds to latent myostatin as described in any of the preceding aspects, or a pharmaceutical composition as described in any of the preceding aspects.

[0106] In another aspect, the present disclosure provides an antigen binding molecule that specifically binds to latent myostatin as described in any of the preceding aspects, or a pharmaceutical composition as described in any of the preceding aspects, for use as a medicament. In some embodiments, the medicament is for treating, preventing, or ameliorating a disease or disorder.

[0107] In some embodiments, the disease or disorder is a myostatin-associated disease or disorder.

[0108] In some embodiments, the disease or disorder as described in any of the preceding aspects is a disease or disorder mediated by or associated with expression of myostatin.

[0109] In some embodiments, the disease or disorder is selected from muscle atrophy (e.g., spinal muscular atrophy (SMA)), muscular dystrophy (e.g., facioscapulohumeral muscular dystrophy (FSHMD)), obesity, and cachexia. BRIEF DESCRIPTION OF DRAWINGS

[0110] Figure 1A shows binding activity of chimeric antibodies to human latent myostatin.

[0111] Figure 1B shows the binding activity of chimeric antibodies to human mature myostatin.

[0112] Figures 1C to 1H show the binding activity of humanized antibodies to human latent myostatin.

[0113] Figure 2 shows the potency of humanized antibodies to inhibit latent myostatin activation.

[0114] Figure 3A shows the gastrocnemius muscle weight (g) of mice administered humanized antibodies for 21 days.

[0115] Figure 3B shows the gastrocnemius muscle mass / body mass (mg / g) of mice administered humanized antibodies for 21 days.

[0116] Figure 3C shows the lean body mass change value (g) of mice administered humanized antibodies for 21 days.

[0117] Figure 4 shows the gastrocnemius muscle mass / body mass (mg / g) of mice administered humanized antibodies for 14 days. DETAILED DESCRIPTION

[0118] Terms (definitions)

[0119] For the purposes of the present disclosure, certain technical and scientific terms are described below. Unless specifically defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0120] As used in the specification and claims, the singular forms "a," "an" and "the" include plural references unless the context clearly dictates otherwise.

[0121] Unless the context clearly indicates otherwise, throughout the patent specification and claims, the words "comprise", "have" and "include" and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense.

[0122] The term "and / or" means both "and" and "or". For example, the phrase "A, B, and / or C" is intended to cover the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0123] The three letter codes and one letter codes for amino acids used in the present disclosure are as described in J. Biol. Chem., 243, p 3558 (1968).

[0124] The term "latent-myostatin," also known as latent GDF8, has the amino acid sequence of GenBank: ABI48514.1 for the human latent-myostatin protein. In some embodiments, the human latent-myostatin has the amino acid sequence of SEQ ID NO: 1.

[0125] Amino acid sequences for latent-myostatin molecules from non-human species (e.g., mouse, rat, monkey, rabbit, dog, pig, etc.) are available from public resources, e.g., Cynomolgus monkey latent-myostatin protein, GenBank: AAL17640.1; mouse latent-myostatin protein, GenBank: AAC53167.1. Although specific database accession numbers are given herein, the skilled artisan understands that the latent-myostatin referred to herein encompasses corresponding sequences reported in other databases or literature. As used herein, the term "latent-myostatin" refers to the inactive precursor of myostatin that comprises a disulfide-linked homodimer, each molecule of which comprises an amino-terminal prodomain non-covalently bound to a carboxy-terminal mature myostatin domain. In some embodiments, "latent-myostatin" is produced from pro-myostatin that has been cleaved by a proprotein convertase, but not by a protease from the BMP / tolloid family. In other embodiments, "latent-myostatin" can be produced by combining a prodomain and a carboxy-terminal mature myostatin domain in vitro and allowing them to fold properly. See, e.g., Sengle et al., J. Biol. Chem., 286(7):5087-5099, 2011. Exemplary latent-myostatin sequences, variants thereof, and methods of producing latent-myostatin are well known in the art and are described in more detail herein.

[0126] The term "pro-myostatin," also known as "proGDF8," refers to the inactive precursor of myostatin that comprises a disulfide-linked homodimer, each molecule of which comprises an amino-terminal prodomain covalently bound to a carboxy-terminal mature myostatin domain. In some embodiments, "pro-myostatin" has not been cleaved by a proprotein convertase or a protease from the BMP / tolloid family. Exemplary pro-myostatin sequences, variants thereof, and methods of producing pro-myostatin are well known in the art and are described in more detail herein. In some embodiments, the human pro-myostatin has the amino acid sequence of SEQ ID NO: 2.

[0127] The term "mature-myostatin" refers to the mature, biologically active form of myostatin. In some embodiments, mature-myostatin is capable of myostatin receptor binding and / or activation. Activation and release of mature-myostatin from its pro-myostatin form in vivo is accomplished by several separate protease cleavage events. First, "pro-myostatin" is cleaved by a proprotein convertase, resulting in "latent-myostatin," in which mature-myostatin is shielded from binding to its receptor by a portion of the prodomain. Activation and release of mature-myostatin is accomplished after cleavage of latent-myostatin by an additional protease from the BMP / tolloid family, such as BMP1. As used herein, the term "mature-myostatin" can refer to both full-length mature-myostatin and fragments of full-length mature-myostatin that retain biological activity. Exemplary mature-myostatin sequences, variants thereof, and methods of generating mature-myostatin are well known in the art and are described in more detail herein. In some embodiments, human mature-myostatin has the amino acid sequence of SEQ ID NO: 73.

[0128] The term "proprotein convertase cleavage site" refers to the site in which pro-myostatin is cleaved by a proprotein convertase. In some embodiments, the proprotein convertase cleavage site is a conserved RXXR site between the prodomain and the biologically active domain or mature-myostatin.

[0129] The term "BMP / tolloid protease family cleavage site" refers to the site in which latent-myostatin is cleaved by a member of the BMP / tolloid protease family. In some embodiments, the member of the BMP / tolloid protease family is BMP1.

[0130] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine, a number of which are derived post-translationally in ribosomes. Amino acid analogs refer to compounds which have the same basic chemical structure as the naturally occurring amino acids, i.e., an alpha-carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, such as homo-serine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but otherwise function in a manner similar to the naturally occurring amino acids. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

[0131] The term "amino acid mutation" includes amino acid substitutions (also referred to as amino acid replacements), deletions, insertions, and modifications. Any combination of substitutions, deletions, insertions and modifications can be made to arrive at the final construct, as long as the final construct possesses the desired properties, such as reduced or no binding to an Fc receptor. Amino acid sequence deletions and insertions include deletions and insertions at the amino- and / or carboxy-terminus of a polypeptide chain. A particular amino acid mutation can be an amino acid substitution. In some embodiments, the amino acid mutation is a non-conservative amino acid substitution, i.e., replacing one amino acid with another amino acid having different structural and / or chemical properties. Amino acid substitutions include substitutions by non-naturally occurring amino acids or by derivatives of the 20 natural amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5- hydroxylysine). Amino acid mutations can be generated using genetic or chemical methods known in the art. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, and the like. Methods other than genetic engineering to alter the side chain groups of amino acids, such as chemical modifications, are also expected to be useful. Various names can be used herein to refer to the same amino acid mutation. In this regard, a particular amino acid residue at a position can be denoted in the manner of position + amino acid residue, e.g., 102S, to indicate that the amino acid residue at position 102 is S. C102S indicates that the amino acid residue at position 102 is mutated from C to S. When a claim recites a residue at a particular position in the manner of "position + amino acid residue," the original residue at that position is not a limitation on the scope of the claim.

[0132] The term "antibody" is used in the broadest sense, and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies and antibody fragments and antigen-binding fragments (or antigen-binding portions), as long as they exhibit the desired antigen-binding activity.

[0133] The term "antigen-binding molecule" is used in the broadest sense, and encompasses various molecules that specifically bind to an antigen, including but not limited to antibodies, other polypeptides with antigen-binding activity, and antibody fusion proteins that are fusion of both, as well as any molecules comprising the above antibodies, polypeptides, antibody fusion proteins, as long as they exhibit the desired antigen-binding activity. Exemplarily, an antigen-binding molecule herein is a monoclonal antibody, a monospecific antibody, a polyclonal antibody, a multispecific antibody (e.g., a bispecific antibody, a trispecific antibody).

[0134] The term "antigen binding fragment" encompasses full length antibodies, Fabs, modified Fabs, Fab', Fab'-SH, modified Fab', F(ab')2, Fv, dsFv, Fab-Fv, Fab-dsFv, Fd, single domain antibodies (sdAb, e.g., VH or VL or VHH), single chain Fab (scFab), single chain antibodies (e.g., scFv, sc(Fv)2), diabodies, linear antibodies, bivalent or trivalent or tetravalent antibodies, Bis-scFv, diabody, tribody, triabody, tetrabody, and epitope binding fragments of any of the above (see, e.g., Holliger and Hudson, 2005, Nature Biotech. 23(9): 1126-1136; Adair and Lawson, 2005, Drug Design Reviews-Online 2(3), 209-217). Methods of producing and making these antigen binding fragments are well known in the art (see, e.g., Verma et al., 1998, Journal of Immunological Methods, 216, 165-181).

[0135] The term "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, dsFv, Fab, Fab', Fab'-SH, F(ab')2, Fd, single domain antibodies (sdAb, e.g., VH, VL or VHH), single chain Fab (scFab), diabodies, linear antibodies, single chain antibodies (e.g., scFv, sc(Fv)2); and multispecific antibodies formed from antibody fragments.

[0136] The term "native antibody" refers to an immunoglobulin molecule naturally occurring. For example, a native IgG antibody is a heterotetrameric glycoprotein of about 150,000 daltons, composed of two light chains and two heavy chains that are disulfide-bonded. From N- to C-terminus, each heavy chain has a variable region (VH, also called a variable heavy domain, a heavy chain variable region), followed by three constant domains (CH1, CH2 and CH3). Similarly, from N- to C-terminus, each light chain has a variable region (VL, also called a variable light domain, or a light chain variable domain), followed by a constant light domain (CL).

[0137] The terms "full-length antibody," "intact antibody," and "whole antibody" are used herein interchangeably to refer to an antibody with a structure substantially similar to a native antibody structure or a heavy chain having an Fc region as defined herein. A native intact antibody light chain includes a light chain variable region, VL, at the amino terminus of the light chain, and a light chain constant region, CL, including kappa and lambda chains; a heavy chain includes a variable region, VH, at the amino terminus of the heavy chain, and a constant region at the carboxy terminus, where CH3 is closest to the carboxy terminus of the polypeptide, the heavy chain can be of any isotype including IgG (including IgG1, IgG2, IgG3, and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM, and IgE.

[0138] The term "variable region" or "variable domain" of an antibody refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. In this document, by the term "variable region" or "variable domain" of an antibody, we mean the variable region of the antibody heavy chain (VH) and the variable region of the antibody light chain (VL). Each of the VH and VL comprises four conserved framework regions (FRs) and three complementarity determining regions (CDRs). The term "complementarity determining region" or "CDR" refers to the regions within the variable domain that are primarily responsible for binding to an antigen; the "framework" or "FR" refers to the variable domain residues other than the CDR residues. The VH comprises three CDR regions: HCDR1, HCDR2, and HCDR3; the VL comprises three CDR regions: LCDR1, LCDR2, and LCDR3. Each of the VH and VL is composed of three CDRs and four FRs, arranged from the amino-terminus (also referred to as the N-terminus) to the carboxy-terminus (also referred to as the C-terminus) in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0139] The boundaries of the CDRs can be determined by various known schemes, such as the "Kabat" numbering convention, the "Chothia" numbering convention, the "ABM" numbering convention, the "contact" numbering convention, and the ImMunoGenTics (IMGT) numbering convention, etc.; the correspondence between the various numbering systems is well known to those skilled in the art, and exemplary is shown in Table 1 below.

[0140] Table 1. Relationship between CDR numbering systems

[0141] Unless otherwise specified, the variable regions and CDRs in the embodiments of the present disclosure are subject to the "Kabat" numbering convention. Although the Kabat numbering convention is used to define the amino acid residues in specific embodiments, the corresponding technical solutions of other numbering systems are deemed to be equivalent technical solutions.

[0142] The term "Fc region" or "fragment crystallizable region" is used to define a C-terminal region of an antibody heavy chain, including native and engineered Fc regions. In some embodiments, the Fc region comprises two subunits, which are identical or different. Suitable Fc regions for use in the antibodies described herein include Fc regions of human IgGl, IgG2 (IgG2A, IgG2B), IgG3, and IgG4. In some embodiments, the boundaries of the Fc region can also vary, e.g., the C-terminal lysine (residue 447 according to EU numbering system) of the Fc region can be absent, or the C-terminal lysine and arginine (residues 446 and 447 according to EU numbering system) of the Fc region can be absent. Unless otherwise specified, numbering of the Fc region is according to the EU numbering system, also referred to as the EU index.

[0143] The Fc region can be suitably obtained by partially digesting an IgG monoclonal antibody or the like with a proteolytic enzyme such as pepsin, followed by eluting the fraction adsorbed to a protein A or protein G column. As the proteolytic enzyme, an enzyme that can digest a full-length antibody in a limited manner to produce Fab, F(ab')2, by suitably setting the reaction conditions of the enzyme such as pH, is used without particular limitation, and for example, pepsin, papain, or the like can be exemplified.

[0144] In the present disclosure, the term "Fc region" or "Fc domain" refers to an antibody region comprising at least a CH2 domain and a CH3 domain. In the present disclosure, the term "CH2 region" or "CH2 domain" is intended to refer to the CH2 region of an immunoglobulin. Thus, for example, the CH2 region of a human IgGl antibody corresponds to amino acids 231-340 according to the EU numbering system (according to the IMGT website). However, the CH2 region can also be of any other antibody isotype as described in the present disclosure.

[0145] In the present disclosure, the term "CH3 region", "CH3 domain" or "CH3 domain" is intended to refer to the CH3 region of an immunoglobulin. Thus, for example, the CH3 region of a human IgGl antibody corresponds to amino acids 341-447 according to the EU numbering system (according to the IMGT website). However, the CH3 region can also be of any other antibody isotype as described in the present disclosure.

[0146] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different, distinct source or species.

[0147] The term "humanized" antibody is an antibody that retains the reactivity of a non-human antibody, while having lower immunogenicity in humans. For example, this can be achieved by retaining the non-human CDR regions and replacing the remainder of the antibody with its human counterpart (i.e., the constant region as well as the framework region portion of the variable region).

[0148] The terms "human antibody," "fully human antibody," "completely human antibody" are used interchangeably herein to mean an antibody having variable and constant regions that are of human sequence. The terms encompass antibodies that are derived from human genes but have had sequences changed, e.g., to reduce possible immunogenicity, to increase affinity, to eliminate a cysteine or glycosylation site that can cause undesirable folding, etc. The terms encompass these antibodies that are recombinantly produced in non-human cells that can impart non-human cell-like glycosylation. The terms also encompass antibodies that have been produced in transgenic mice that contain some or all human immunoglobulin heavy and light chain loci. The definition of human antibody expressly excludes humanized antibodies that contain non-human antigen binding residues.

[0149] The term "affinity" refers to the overall strength of the noncovalent interactions between individual binding sites of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, binding "affinity" refers to intrinsic binding affinity, which reflects the 1 : 1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its ligand Y can be generally represented by the dissociation constant (KD). Affinity can be measured by routine methods known in the art, including those described herein.

[0150] The term "kassoc" or "ka" as used herein refers to the association rate of a particular antibody-antigen interaction, and the term "kdis" or "kd" refers to the dissociation rate of a particular antibody-antigen interaction. The term "KD" refers to the dissociation constant, which is obtained from the ratio of kd to ka (i.e., kd / ka) and is expressed as a molar concentration (M). KDvalues for antibodies can be determined using methods well known in the art. For example, affinity in solution can be measured using a biosensor system, such as a Biacore® system (e.g., Biacore), or by a solution equilibrium titration method (SET).

[0151] The term "surface plasmon resonance" refers to an optical phenomenon that detects alterations in protein concentrations within a biosensor matrix in real-time, for example, using the BIAcore™ system (Biacore LifeSciences division of GE Healthcare, Piscataway, NJ).

[0152] The term "effector function" refers to those biological activities attributable to an antibody Fc region (a native sequence Fc region or a mutant Fc region having an altered amino acid sequence) and which vary with the antibody isotype. Examples of antibody effector functions include but are not limited to: Clq binding and complement dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, down regulation of cell surface receptors (e.g., B cell receptor), and B cell activation.

[0153] The term "monoclonal antibody" refers to a population of substantially homogeneous antibodies, i.e., the antibody molecules comprising the population are identical in their amino acid sequences, except for possible naturally occurring mutations that can be present in minor amounts. In contrast, polyclonal antibody preparations typically include a variety of different antibodies that are specific for different epitopes, often due to their different amino acid sequences in their variable domains. "Monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. In some embodiments, the antibodies provided by the present disclosure are monoclonal antibodies.

[0154] The term "antigen" refers to a molecule or portion of a molecule capable of being bound by an antigen binding protein, including, for example, an antibody. An antigen can have one or more epitopes capable of interacting with different antigen binding proteins, e.g., antibodies.

[0155] The term "epitope" refers to a region (area or region) on an antigen to which an antibody or antigen-binding fragment thereof specifically binds. An epitope can be formed by contiguous amino acids (linear epitope) or comprise non-contiguous amino acids (conformational epitope), e.g., brought into spatial proximity due to folding of the antigen (i.e., tertiary folding of the antigen by virtue of its protein nature). The difference between a conformational and a linear epitope is that the binding of an antibody to a conformational epitope is lost in the presence of a denaturing solvent. An epitope comprises at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial conformation. Screening for antibodies that bind to a particular epitope (i.e., those that bind to the same epitope) can be performed using routine methods in the art, such as, but not limited to, alanine scanning, peptide mapping, peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of the antigen (see Prot. Sci. 9 (2000) 487-496), and cross-blocking.

[0156] The terms "capable of specifically binding," "specifically binding," or "binds" mean that an antibody is capable of binding to a certain antigen or epitope of that antigen with a higher affinity than to other antigens or epitopes. Typically, an antibody binds to an antigen or an epitope within an antigen with an equilibrium dissociation constant (KD) of about 1 x 10 -7 M or less (e.g., about 1 x 10 -8 M, 1 x 10 -9 M, 1 x 10 -10 M, 1 x 10 -11 M or less). In some embodiments, the KD of an antibody binding to an antigen is 10% or less (e.g., 1%) of the KD of the antibody binding to a non-specific antigen (e.g., BSA, casein). KD can be measured using known methods, such as by surface plasmon resonance, e.g., using a Biacore® system. measured by surface plasmon resonance assays. However, an antibody that specifically binds to an antigen or epitope within an antigen can have cross-reactivity to other related antigens, e.g., cross-reactivity to a corresponding antigen from another species (homologous), such as a human or a monkey, e.g., a Macaca fascicularis (cynomolgus, cyno), a Pan troglodytes (chimpanzee, chimp), or a Callithrix jacchus (common marmoset, marmoset).

[0157] The term "does not bind" means that an antibody is not capable of binding to an antigen or epitope thereof in the manner of specific binding described above. For example, an antibody does not bind to an antigen or epitope thereof with a dissociation constant (KD) of about 1 x 10 -6 M or greater.

[0158] The terms "antigen binding molecule that specifically binds to latent myostatin," "anti-latent myostatin antibody," and "latent myostatin binding antibody" refer to an antibody that is capable of binding to latent myostatin or an epitope thereof with sufficient affinity.

[0159] The terms "antibody-dependent cellular cytotoxicity," "antibody-dependent cell-mediated cytotoxicity," or "ADCC" are mechanisms of inducing cell death that rely on the interaction of antibody-coated target cells with effector cells having lytic activity, such as natural killer cells (NK), monocytes, macrophages, and neutrophils, via Fcy receptors (FcyRs) expressed on the effector cells. For example, NK cells express FcyRIIIa, while monocytes express FcyRI, FcyRII, and FcyRIIIa. The ADCC activity of the antibodies provided herein can be assessed using an in vitro assay using cells expressing an antigen as target cells and NK cells as effector cells. Cell lysis is detected based on the release of a label (e.g., a radioactive substrate, a fluorescent dye, or a native intracellular protein) from the lysed cells.

[0160] The term "antibody-dependent cellular phagocytosis" (ADCP) refers to a mechanism of eliminating antibody-coated target cells through internalization by phagocytic cells, such as macrophages or dendritic cells.

[0161] The term "complement-dependent cytotoxicity" or "CDC" refers to a mechanism of inducing cell death in which the Fc effector domain of a target-bound antibody binds and activates complement component Clq, which in turn activates the complement cascade, leading to target cell death. Activation of complement can also result in deposition of complement components on the surface of target cells, which promote CDC by binding complement receptors (e.g., CR3) on leukocytes.

[0162] The term "nucleic acid" is used interchangeably herein with the term "polynucleotide" and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, that have similar binding properties to the reference nucleic acid, and are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs). An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location. An isolated nucleic acid encoding a polypeptide or fusion protein refers to one or more nucleic acid molecules encoding a polypeptide or fusion protein, including such one or more nucleic acid molecules in a single vector or separate vectors, and such one or more nucleic acid molecules present at one or more locations in a host cell. Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and

[0163] The terms "polypeptide" and "protein" are used interchangeably herein.

[0164] The term "sequence identity" refers to the extent to which the amino acid / nucleic acid of two sequences are identical at equivalent positions (percent); wherein, when the two sequences are optimally aligned, gaps are introduced if necessary, to achieve maximum percent sequence identity, and no conservative substitutions are part of the sequence identity. To determine percent sequence identity, alignment can be achieved using techniques known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0165] The term "vector" means a polynucleotide molecule capable of transporting another polynucleotide to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, e.g., a adenoviral associated virus vector (AAV or AAV2), wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. The term "expression vector" or "expression construct" refers to a vector that can be transformed into a host cell, and which contains nucleic acid sequences that direct and / or control the expression of one or more heterologous coding regions to which they are operably linked, along with the host cell. Expression constructs can include, but are not limited to, sequences that affect or control transcription, translation, and, where introns are present, RNA splicing of coding regions operably linked thereto.

[0166] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. The progeny can not be completely identical to the parent cell since there can be mutations that occur during passage due to error-prone replication. Mutant progeny that have the same function or biological activity as screened or selected for in the original transformant are included herein. Host cells include prokaryotic and eukaryotic host cells, with eukaryotic host cells including, but not limited to, mammalian cells, insect cell lines, plant cells, and fungal cells. Mammalian host cells include human, mouse, rat, canine, monkey, porcine, goat, bovine, equine, and hamster cells, including but not limited to Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, and HEK-293 cells.Fungal cells include yeast and filamentous fungal cells, including, for example, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillus sp., Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum, Physcomitrella patens, and Neurospora crassa.

[0167] "Optional" or "optionally" means that the subsequently described event or circumstance can or can not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0168] The term "pharmaceutical composition" denotes a mixture of one or more antigen binding molecules described herein that specifically bind to latent myostatin with other chemical components, such as physiologically / pharmaceutically acceptable carriers and excipients.

[0169] The term "pharmaceutically acceptable carrier" refers to a component of a pharmaceutical formulation that is different from the active ingredients and is not toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0170] The term "subject" or "individual" includes both human and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, and reptiles. The terms "patient" or "subject" are used interchangeably herein, unless otherwise indicated. In certain embodiments, the individual or subject is a human.

[0171] "Administering" or "administration," when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, means the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid.

[0172] The term "sample" refers to a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present within a subject. Exemplary samples are biological fluids such as blood, serum and serosal fluids, plasma, lymphatic fluid, urine, saliva, cyst fluid, lacrimal fluid, fecal matter, sputum, mucosal secretions of secretory tissues and organs, vaginal secretions, ascites, pleural, pericardial, peritoneal, abdominal and other body cavity fluids, fluids collected by bronchial lavage, synovial fluid, liquid solutions in contact with a subject or biological source, such as cell and organ culture media (including cell or organ conditioned media), lavage fluids, and the like, tissue biopsy samples, fine needle aspirations, surgically removed tissues, organ cultures, or cell cultures.

[0173] "Treatment" and "treating" (and grammatical variations thereof) refer to clinical intervention with the intent to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or reoccurrence of disease, alleviation of symptoms, diminishment / masking of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, the antibodies of the disclosure are used to delay development of a disease or to slow the progression of a disease.

[0174] The terms "recurrence," "relapse," "relapsed" refer to the return of a cancer or disease after a clinical assessment of the disappearance of the disease. Diagnosis of a distant metastasis or local recurrence can be considered a relapse.

[0175] The terms "refractory" or "resistant" refer to a cancer or disease that is unresponsive to treatment.

[0176] An "effective amount" is generally an amount that is sufficient to reduce the severity and / or frequency of symptoms, eliminate such symptoms and / or their underlying cause(s), prevent symptoms and / or their underlying cause(s) from occurring, or ameliorate or improve impairment(s) resulting from or associated with a disease state (e.g., a lung disease). In some embodiments, an effective amount is a therapeutically effective amount or a prophylactically effective amount. A "therapeutically effective amount" is an amount that is sufficient to treat a disease state or symptoms, particularly conditions associated with such disease state, or otherwise prevent, hinder, retard, or reverse the progression of such disease state or any other undesirable symptoms associated with such disease in any manner. A "prophylactically effective amount" is an amount that will have the intended prophylactic effect, such as preventing or delaying the onset (or recurrence) of the disease state, or reducing the likelihood of the onset (or recurrence) of the disease state or associated symptoms, when administered to a subject. The full therapeutic or prophylactic effect does not necessarily occur by administration of one dose, and can occur only after administration of a series of doses. Thus, a therapeutically or prophylactically effective amount can be administered in one or more administrations. A "therapeutically effective amount" and a "prophylactically effective amount" can vary depending on factors such as the disease state of the individual, the age, sex, and weight of the individual, and the ability of the therapeutic agent or combination of therapeutic agents to elicit a desired response in the individual. Exemplary indicators of an effective therapeutic agent or combination of therapeutic agents include, for example, improved health status of the patient.

[0177] Antigen binding molecules of the disclosure that specifically bind to latent myostatin

[0178] The present disclosure provides antigen binding molecules that specifically bind to latent myostatin, which have a number of advantageous properties, such as antigen binding activity, ability to inhibit activation of latent myostatin, good therapeutic activity, safety, pharmacokinetic properties, and drugability (e.g., solubility, viscosity, purity, and stability).

[0179] Exemplary antigen binding molecules that specifically bind to latent myostatin

[0180] For example, antigen binding molecules of the disclosure that specifically bind to latent myostatin, comprise a heavy chain variable region and a light chain variable region, wherein:

[0181] a. the amino acid sequence of HCDR1 of the heavy chain variable region is set forth in SEQ ID NO: 5, the amino acid sequence of HCDR2 is set forth in SEQ ID NO: 31, and the amino acid sequence of HCDR3 is set forth in SEQ ID NO: 7, and the amino acid sequence of LCDR1 of the light chain variable region is set forth in SEQ ID NO: 8, the amino acid sequence of LCDR2 is set forth in SEQ ID NO: 9, and the amino acid sequence of LCDR3 is set forth in SEQ ID NO: 10; or

[0182] b. The amino acid sequences of the heavy chain variable region HCDR1 are shown in SEQ ID NO: 17, HCDR2 are shown in SEQ ID NO: 18, and HCDR3 are shown in SEQ ID NO: 19; the amino acid sequences of the light chain variable region LCDR1 are shown in SEQ ID NO: 51, LCDR2 are shown in SEQ ID NO: 21, and LCDR3 are shown in SEQ ID NO: 22.

[0183] For example, the antigen-binding molecule that specifically binds to latent myostatin disclosed herein comprises a heavy chain variable region and a light chain variable region, wherein:

[0184] a. The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 39, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 32; or

[0185] b. The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 61, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 56.

[0186] For example, the antigen-binding molecule that specifically binds to latent myostatin disclosed herein comprises a heavy chain and a light chain, wherein:

[0187] a. The amino acid sequence of the heavy chain is as shown in SEQ ID NO: 49 or 45, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 46; or

[0188] b. The amino acid sequence of the heavy chain is as shown in SEQ ID NO: 70 or 64, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 67.

[0189] For example, the antigen-binding molecule that specifically binds to latent myostatin disclosed herein comprises a heavy chain and a light chain, wherein:

[0190] The amino acid sequence of the heavy chain consists of amino acid residues 1-448 of SEQ ID NO: 49, and the amino acid sequence of the light chain consists of amino acid residues 1-214 of SEQ ID NO: 46; or

[0191] The amino acid sequence of the heavy chain consists of amino acid residues 1-450 in SEQ ID NO: 70, and the amino acid sequence of the light chain consists of amino acid residues 1-214 in SEQ ID NO: 67.

[0192] antibody structure

[0193] In some embodiments, the antibodies provided herein are full-length antibodies.

[0194] In some embodiments, the antibodies provided herein are antibody fragments.

[0195] In some embodiments, the antibody fragment is a Fab, Fab', Fab'-SH or F(ab')2 fragment, particularly a Fab fragment. "Fab" is a monovalent fragment consisting of the VL, VH, CL and CH1 domains. "Fab fragment" can be produced by papain digestion of an antibody. "Fab"' contains the VL, CL, and VH and CH1, and also contains the region between the CH1 and CH2 domains, so that a disulfide bond can be formed between the two heavy chains in the two Fab' fragments, to form an F(ab')2 molecule. "Fab'-SH" is an Fab' fragment in which the cysteine residue of the constant region has a free thiol group. "F(ab')2" comprises a bivalent fragment of an antibody comprising two Fab fragments that are linked by a disulfide bond in the hinge region.

[0196] In other embodiments, the antibody fragment is a diabody, triabody or tetrabody. Diabodies are antibody fragments that comprise two antigen binding sites, in the same polypeptide chain (VH-VL), connected by a short linker that is not too long to allow pairing between the two domains on the same chain, but is too short to allow pairing between the two domains on another chain, thus forcing the domains to pair with complementary domains on another chain, resulting in two antigen binding sites, which can be the same or different.

[0197] In other embodiments, the antibody fragment is a single chain Fab fragment. "Single chain Fab fragment" or "scFab" is a polypeptide consisting of VH, CH1, VL, CL and a linker, wherein the antibody domains and the linker have one of the following orders in N-terminal to C-terminal direction: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1 or d) VL-CH1-linker-VH-CL. In some embodiments, the linker is a polypeptide of at least 30 amino acids. In other embodiments, the linker is a polypeptide of between 32 and 50 amino acids. The single chain Fab fragments are stabilized via the natural disulfide bond between CL and CH1. Additionally, these single chain Fab molecules can be further stabilized by the creation of an interchain disulfide bond by the insertion of a cysteine residue, for example at position 44 in the heavy chain variable region and at position 100 in the light chain variable region, according to Kabat numbering.

[0198] In other embodiments, the antibody fragment is a Fv fragment consisting of the VH and VL domains of a single arm of an antibody.

[0199] In other embodiments, the antibody fragment is a single-chain variable fragment (scFv). An "scFv" is a fusion protein comprising at least one antibody fragment comprising a light chain variable region and at least one antibody fragment comprising a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region are connected in tandem by a short, flexible peptide linker, is capable of being expressed as a single-chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless specifically indicated otherwise, scFv herein can have the VL and VH variable regions in either order, e.g., an scFv can comprise VL-linker-VH or can comprise VH-linker-VL, with respect to the N- and C-termini of the polypeptide.

[0200] In other embodiments, the antibody fragment is a dsFv, which is obtained by linking polypeptides in which one amino acid residue in each of the VH and VL is substituted with a cysteine residue, via a disulfide bond between the cysteine residues. The amino acid residue to be substituted with the cysteine residue can be selected based on the three-dimensional structure prediction of the antibody according to known methods (Protein Engineering. 7: 697 (1994)).

[0201] In other embodiments, the antibody fragment is a single-domain antibody (dAb). A single-domain antibody is an antibody fragment comprising all or a portion of the variable domain of the heavy chain or all or a portion of the variable domain of the light chain of an antibody.

[0202] In some embodiments, an antibody provided herein is a chimeric antibody. In some embodiments, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In some embodiments, a chimeric antibody is a "class-switched" antibody, in which the class or subclass has been changed from that of the parent antibody.

[0203] In some embodiments, an antibody is a humanized antibody. Typically, a non-human antibody is humanized by replacing some or all of the amino acids in the variable region of the antibody with corresponding amino acids from a human antibody, while retaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody comprises one or more variable regions in which the CDRs or portions thereof are derived from a non-human antibody, and the FRs or portions thereof are derived from a human antibody. Optionally, a humanized antibody will also comprise a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody can be replaced with corresponding residues from a non-human antibody, e.g., the antibody from which the CDR sequences are derived.

[0204] Humanized antibodies and methods of making them are reviewed, e.g., in Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008), and are further described, e.g., in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing specificity determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing "resurfacing"); Dall'Acqua et al., Methods 36:43-60 (2005) (describing "FR shuffling"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer 83:252-260 (2000) (describing the "guided selection" approach to FR shuffling).

[0205] Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best-fit" method (see, e.g., Sims et al., J. Immunol. 151:2296 (1993)); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light chain or heavy chain variable regions (see, e.g., Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al., J. Immunol., 151:2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions generated using a design strategy (see, e.g., U.S. Patent No. 6,187,287; and U.S. Patent No. 6,582,804).

[0206] Variants of antigen binding molecules that specifically bind to latent myostatin

[0207] In some embodiments, amino acid sequence variants of the antigen binding molecules that specifically bind to latent myostatin provided herein are encompassed. For example, it can be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into, and / or substitutions of, residues within the amino acid sequences of the antigen binding molecules that specifically bind to latent myostatin. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen binding properties.

[0208] Substitution, insertion, and deletion variants

[0209] In certain embodiments, antibody variants with one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include the CDRs and FRs. Conservative substitutions are shown in Table 2 under the heading of "preferred substitutions." More substantial changes are provided in Table 2 under the heading of "exemplary substitutions," and as further described below in reference to amino acid side chain classes. Amino acid substitutions can be introduced into an antibody of interest and the products screened for a desired activity, e.g., retained / improved antigen binding, decreased immunogenicity, or improved ADCC or CDC.

[0210] Table 2. Substitution of amino acids

[0211] According to common practice, amino acids can be grouped as follows:

[0212] (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, He;

[0213] (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;

[0214] (3) acidic: Asp, Glu;

[0215] (4) basic: His, Lys, Arg;

[0216] (5) residues that influence chain orientation: Gly, Pro;

[0217] (6) aromatic: Trp, Tyr, Phe.

[0218] Non-conservative substitutions will require replacing a member of one of these classes with a member from another class.

[0219] One type of substitutional variant involves substituting one or more amino acids in a CDR of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further study will have some alterations (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody and / or will have substantially retained certain biological properties of the parent antibody. An exemplary substitutional variant is an affinity matured antibody, which can be conveniently generated, e.g., using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more CDR residues are mutated, and the variant antibodies displayed on phage and screened for a particular biological activity (e.g., binding affinity). Changes (e.g., substitutions) can be made to a CDR, e.g., to improve antibody affinity. Such changes can be made, e.g., to CDR "hotspot" residues, i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process, and / or residues that contact antigen, while the resulting variant VHand / or VLis tested for binding affinity. In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). Secondary libraries are then created. The libraries are then screened to identify any antibody variants having the desired affinity. Another method of introducing diversity involves CDR-directed approaches, in which several CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues involved in antigen binding can be specifically identified, e.g., using alanine scanning mutagenesis or modeling. In particular, HCDR3 and LCDR3 are often targeted.

[0220] In some embodiments, substitutions, insertions, or deletions can occur within one or more CDRs so long as such changes do not substantially reduce the ability of the antibody to bind antigen. For example, conservative alterations (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity can be made to a CDR. Such alterations can be outside of antigen contacting residues in a CDR. In some embodiments of the variant VHand VLsequences provided above, each CDR is unaltered, or contains no more than 1, 2, or 3 amino acid substitutions.

[0221] One method for identifying residues or regions of an antibody that can serve as targets for mutagenesis is called "alanine scanning mutagenesis" In this approach, one or more residues or groups of residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) are identified In an antibody and replaced by alanine or a neutral or negatively charged amino acid such as polyalanine or alanine, to determine whether the interaction of the antibody with antigen is affected. Further substitutions can be introduced at the amino acid positions which display functional sensitivity to the initial substitutions. In addition, contact points between an antibody and antigen can be identified by studying crystals structures of antigen-antibody complexes. These contact residues and neighboring residues can be targeted or eliminated as candidates for substitution. Variants can be screened to determine whether they contain the desired properties.

[0222] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme or a polypeptide which increases the serum half-life of the antibody.

[0223] Recombinant methods

[0224] Antigen binding molecules that specifically bind to myostatin can be produced using recombinant methods. For these methods, one or more isolated nucleic acids encoding an antigen binding molecule that specifically binds to myostatin are provided.

[0225] In some embodiments, the disclosure provides an isolated nucleic acid encoding an antigen binding molecule that specifically binds to myostatin as previously described. Such a nucleic acid can give rise to an independent polypeptide chain as previously described. In another aspect, the disclosure provides one or more vectors (e.g., expression vectors) comprising such a nucleic acid. In another aspect, the disclosure provides a host cell comprising such a nucleic acid. In some embodiments, a method of making a polypeptide or fusion protein is provided, wherein the method comprises, culturing a host cell comprising a nucleic acid encoding the polypeptide or fusion protein, as provided above, under conditions suitable for expression, and optionally recovering the antigen binding molecule that specifically binds to myostatin from the host cell (or host cell culture medium).

[0226] To produce an antigen binding molecule that specifically binds to myostatin recombinantly, the nucleic acid encoding the protein is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acids can be readily isolated and sequenced using conventional procedures, or produced by recombinant methods or obtained through chemical synthesis.

[0227] Suitable host cells for cloning or expression of vector encoding antigen binding molecules that specifically bind to latent myostatin include prokaryotic or eukaryotic cells described herein. For example, production in bacteria is possible, particularly when glycosylation and Fc effector function are not needed. After expression, the antigen binding molecules can be isolated from bacterial cell paste in a soluble fraction and can be further purified.

[0228] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are also suitable cloning or expression hosts for vectors encoding fusion proteins, including fungal and yeast strains. Suitable host cells for expression of fusion proteins can also be derived from multicellular organisms (invertebrates and vertebrates); examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains have been identified which can be used in combination with insect cells, particularly for transfection of Spodoptera frugiperda cells; plant cell cultures also can be utilized as hosts, e.g., US5959177, US6040498, US6420548, US7125978, and US6417429; vertebrate cells can also be used as hosts, e.g., mammalian cell lines adapted to grow in suspension, e.g., the COS cell line which is a transformed monkey kidney cell line (COS-7); human embryonic kidney line (293 or 293T cells); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells); monkey kidney cells (CVl); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells; MRC 5 cells; and FS4 cells. Other suitable mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells, and myeloma cell lines, such as Y0, NS0 and Sp2 / 0. For a review of certain mammalian host cell lines suitable for production of antibodies, see, e.g., Yazaki, P. and Wu, A.M., Methods in Molecular Biology, Vol. 248, Lo, B.K.C. (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268.

[0229] Assay

[0230] The antigen binding molecules provided herein that specifically bind to latent myostatin can be identified, screened, or characterized for their physical / chemical characteristics and / or biological activities by a variety of assays known in the art. In one aspect, the antigen binding molecules of the disclosure that specifically bind to latent myostatin are tested for activity, e.g., by known methods such as ELISA, Western blotting, and the like.

[0231] Therapeutic methods and routes of administration

[0232] Any of the antigen binding molecules provided herein that specifically bind to latent myostatin can be used in a therapeutic method. In yet another aspect, the antigen binding molecules provided herein that specifically bind to latent myostatin are used in the manufacture or preparation of a medicament. In some embodiments, the disease is a disease or disorder associated with myostatin. In some embodiments, wherein the disease is selected from cachexia, muscle atrophy (e.g., spinal muscular atrophy (SMA)), muscular dystrophy (e.g., facioscapulohumeral muscular dystrophy (FSHMD)), and obesity.

[0233] In yet another aspect, a pharmaceutical composition comprising the antigen binding molecules that specifically bind to latent myostatin is provided, e.g., for any of the above pharmaceutical uses or therapeutic methods. In some embodiments, the pharmaceutical composition comprises any of the antigen binding molecules provided herein that specifically bind to latent myostatin and a pharmaceutically acceptable carrier. In other embodiments, the pharmaceutical composition further comprises at least one additional therapeutic agent.

[0234] The antigen binding molecules of the disclosure that specifically bind to latent myostatin can be used alone or in combination with other agents for therapy. For example, the antibodies of the disclosure can be co-administered with at least one additional therapeutic agent.

[0235] The antigen binding molecules of the disclosure that specifically bind to latent myostatin (and any additional therapeutic agents) can be administered by any suitable means, including parenterally, intrapulmonary, and intranasally, and, if local treatment is required, intralesionally. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any appropriate route, for example, by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short term or long term. A variety of dosing schedules are contemplated herein, including but not limited to, single or multiple administrations at multiple time points, bolus administration, and pulse infusion.

[0236] The antigen binding molecules of the disclosure that specifically bind to latent myostatin will be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the part of the body to be treated, the method of administration, the scheduling of the method of administration, and other factors known to medical practitioners. The antigen binding molecules of the disclosure that specifically bind to latent myostatin can be formulated with or without one or more other agents for preventing or treating the disorder in question. Effective amounts of such other agents depend on the amounts of the antigen binding molecules of the disclosure that specifically bind to latent myostatin present in the composition, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as described herein or in dosages and treatments differing from the above-mentioned, empirically determined, by determining the dosage through a design experiment, or by using other methods known to determine the dosage.

[0237] For the prevention or treatment of disease, the appropriate dosage of the antigen binding molecules of the disclosure that specifically bind to latent myostatin, when used alone or in combination with one or more other additional therapeutic agents, will depend on the type of disease to be treated, the type of treatment, the severity and course of the disease, whether the antigen binding molecules of the disclosure that specifically bind to latent myostatin is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antigen binding molecules of the disclosure that specifically bind to latent myostatin, and the judgment of the treating physician. The antigen binding molecules of the disclosure that specifically bind to latent myostatin is appropriately administered to the patient at one time or over a series of treatments.

[0238] Articles of manufacture

[0239] In another aspect of the disclosure, an article of manufacture (e.g., a kit) is provided that comprises materials useful for treating, preventing and / or diagnosing the conditions described above. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The containers can be formed from a variety of materials such as glass or plastic. The container holds a composition which is by itself or in combination with another composition effective for treating, preventing and / or diagnosing the condition and can have a sterile access port (for example the container can be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an antigen binding molecule of the disclosure that specifically binds to latent myostatin. The label or package insert indicates that the composition is used for treating the condition of choice. In addition, the article of manufacture can further comprise: (a) a first container wherein is deposited the composition comprising an antigen binding molecule of the disclosure that specifically binds to latent myostatin; and (b) a second container wherein is deposited a composition comprising an additional therapeutic agent. The article of manufacture in embodiments of the disclosure can further include a package insert indicating that the compositions can be used to treat the particular condition. Alternatively, or additionally, the article of manufacture can further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer. From a commercial and user standpoint, it can further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0240] Examples

[0241] The following examples and experimental examples further describe the disclosure and are not to be construed as limiting the scope of the disclosure. The examples or experimental examples of the disclosure do not include detailed descriptions of conventional methods, experimental methods not specified for specific conditions, are generally carried out according to the conventional conditions, because such methods are well known to those having ordinary skill in the art and are described in many publications, such as Cold Spring Harbor Laboratory published “Molecular Cloning” (Green M R, Sambrook J. Molecular cloning [J]. A Laboratory Manual 4th, 2012.), or Springer protocols “Antibody engineering: methods and protocols” (Antibody engineering: methods and protocols [M]. Humana Press, 2018.); or according to the conditions recommended by the raw material or commodity manufacturers; certain reagent materials not specified for specific sources are obtained from the market.

[0242] Example 1. Preparation and screening of hybridoma antibodies

[0243] This example prepared antibodies targeting human myostatin precursor (pro-myostatin and latent myostatin) by hybridoma technology. Human pro-myostatin (SEQ ID NO: 2) protein was used as antigen, Gold Adjuvant (Sigma Cat No. T2684) and Thermo Alum (Thermo Cat No. 77161) as adjuvant, the mixture of antigen and different adjuvants were used to immunize mice alternately. After the primary and booster immunization, the mice with high serum antibody titer were selected to collect spleen B cells, which were fused with myeloma cells to prepare hybridoma cells.

[0244] > Human Strep-latent myostatin antigen

[0245] Note: single underline is strep tag sequence, double underline is linker sequence between tag and antigen.

[0246] Human pro-myostatin antigen was obtained by introducing 3 point mutations to avoid enzyme digestion on the basis of human latent myostatin:

[0247] > Human Strep-pro-myostatin antigen

[0248] Note: single underline is strep tag sequence, double underline is linker sequence between tag and antigen, and wavy underline part is mutation site.

[0249] According to the growth density of hybridoma cells, the culture supernatant of hybridoma was taken for detection, and through ELISA screening, the antibody which could specifically bind to human pro-myostatin and human latent myostatin, could also bind to cynomolgus monkey and mouse latent myostatin, but could not bind to mature myostatin, and could inhibit the enzyme digestion of latent myostatin by BMP1 was screened. Several monoclonal hybridoma cell strains meeting the above conditions and having strong binding capacity were screened. The logarithmic growth period hybridoma cells were collected respectively, and the RNA was extracted by NucleoZol (MN) (according to the kit instruction steps), and then the RNA was reverse transcribed (PrimeScript TMcDNA was generated using Superscript III Reverse Transcriptase (Invitrogen, cat# 18080044) and PrimeScript Reverse Transcriptase (Takara, cat# 2680A). The cDNA was amplified by PCR using Mouse Ig-Primer Set (Novagen, TB326 Rev. B 0503) and the PCR products were sequenced. The nucleotide sequences were translated into the amino acid sequences of the antibody variable regions.

[0250] >Mouse latent myostatin-Strep antigen

[0251] Note: underlined is the strep tag sequence, double underlined is the linker sequence between the tag and the antigen.

[0252] >Chimpanzee latent myostatin-Strep antigen

[0253] Note: underlined is the strep tag sequence, double underlined is the linker sequence between the tag and the antigen.

[0254] >Human mature myostatin amino acid sequence (ProSpec Bio)

[0255] The heavy chain and light chain complementarity determining region (CDR) sequences of the mouse antibody 057 are shown in Table 3 below:

[0256] Table 3. CDR sequences of the mouse anti-latent myostatin antibody

[0257] Note: the CDRs in the table are determined according to the Kabat numbering system.

[0258] The variable region sequences are as follows:

[0259] >057 mouse antibody heavy chain variable region

[0260] >057 mouse antibody light chain variable region

[0261] Note: the primary structure of the above sequences is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, underlined is the CDR sequence determined according to the Kabat numbering system, and the non-underlined part is the FR sequence.

[0262] The heavy chain variable region and the light chain variable region of the murine anti-latency myostatin antibody 057 were recombined with human heavy chain constant region hlgGl (SEQ ID NO: 13) and human kappa light chain constant region CL (SEQ ID NO: 14), respectively, to obtain the full-length chimeric antibody Chi-057.

[0263] >hlgGl

[0264] >CL

[0265] >Chi-057 heavy chain

[0266] >Chi-057 light chain

[0267] Note: single underline indicates CDR; italic indicates constant region; wherein the CDR is determined according to the Kabat rule.

[0268] Example 2. Preparation and screening of Single B-derived antibodies

[0269] In this example, antibodies targeting human myostatin precursor were prepared by Single B technology. Human latent myostatin (SEQ ID NO: 1) was used as antigen, Gold Adjuvant (Sigma Cat No. T2684) and Thermo Alum (Thermo Cat No. 77161) as adjuvant, and the mixture of antigen and different adjuvants were used to immunize mice alternately. After the primary and booster immunization, mice with high serum antibody titer were selected, and the spleen B cells were isolated, and the B cells capable of binding to latent myostatin were sorted by flow cytometry and sequenced. The preferred single B cell CDR and variable region amino acid sequences are as follows:

[0270] Table 4. CDR sequences of murine anti-latency myostatin antibody

[0271] Note: CDR is determined according to the Kabat numbering system.

[0272] The variable region sequence is as follows:

[0273] >207 murine heavy chain variable region

[0274] >207 murine light chain variable region

[0275] Note: The primary structure of the above sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, the underlined part is the CDR sequence determined according to the Kabat numbering system, and the non-underlined part is the FR sequence.

[0276] The heavy chain variable region and the light chain variable region of the mouse anti-latent myostatin antibody were recombined with human heavy chain constant region hlgG1 (SEQ ID NO: 13) and human kappa light chain constant region CL (SEQ ID NO: 14) respectively to obtain the full-length chimeric antibody Chi-207.

[0277] >Chi-207 heavy chain

[0278] >Chi-207 light chain

[0279] Note: Single underline indicates CDR; italic indicates constant region; CDR is determined according to Kabat rule.

[0280] Example 3. Humanization of anti-latent myostatin antibody 057

[0281] At https: / / www.ncbi.nlm.nih.gov / igblast / , the murine 057 antibody light chain variable region was compared to human IgG germline sequences for homology, and IGKV1-16*01 was selected as the light chain framework region template, the murine 057 antibody light chain CDRs were grafted into the IGKV1-16*01 framework region, and FGQGTKVEIK (SEQ ID NO: 27) was added after LCDR3 as a fourth framework region to obtain the CDR-grafted light chain variable region sequence. Similarly, the murine 057 heavy chain variable region was compared to human IgG germline sequences for homology, and IGHV1-46*01 was selected as the heavy chain framework region template, the murine 057 heavy chain CDRs were grafted into the IGHV1-46*01 framework region, and WGQGTLVTVSS (SEQ ID NO: 28) was added after HCDR3 as a fourth framework region to obtain the CDR-grafted heavy chain variable region sequence. Optionally, the amino acid residues at positions 36 and / or 46 of the light chain variable region of the humanized antibody were subjected to back-mutation (e.g., the amino acid residues at specific positions of the framework region play an important role in maintaining the VH / VL conformation, and mutating the amino acid residues at these positions of the CDR-grafted antibody to the amino acid residues at the corresponding positions of the murine antibody is referred to as back-mutation) or other mutations (e.g., substituting specific amino acid residues in the CDR / FR to achieve the purpose of improving the hydrophilicity and physicochemical properties of the antibody and reducing immunogenicity); and / or the amino acid residues at positions 1, 28, 48, 54, 60, 61, 64, 65, 67, 69, 71, and / or 73 of the heavy chain variable region of the humanized antibody were subjected to back-mutation or other mutations.

[0282] Table 5-1. 057 humanized antibody design

[0283] Note: F36Y means mutating position 36 F to Y, and the rest of the mutations are similar. The position of the amino acid residue is determined according to the Kabat rule.

[0284] The CDRs of the humanized antibody of 057 are as follows:

[0285] Table 5-2. CDRs of 057 humanized antibody

[0286] The variable region sequences of the 057 humanized antibody are as follows:

[0287] >hu057VL1

[0288] >hu057VL2

[0289] >hu057VH1

[0290] >hu057VH2

[0291] >hu057VH3

[0292] >hu057VH4

[0293] >hu057VH5

[0294] >hu057VH6

[0295] Note: The primary structure of the above sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4; the underlined part is the CDR sequence determined according to the Kabat numbering system; the double underlined and bolded part is the point mutation of amino acid.

[0296] The heavy chain variable region and the light chain variable region of the 057 humanized antibody are respectively recombined with human heavy chain constant region hlgG1 (SEQ ID NO: 13) and human kappa light chain constant region CL (SEQ ID NO: 14) to obtain the humanized antibody as shown in Table 5-3 below.

[0297] Table 5-3. Humanized antibody of 057

[0298] Note: "Hu057L1H1" in the table represents a humanized antibody with the heavy chain variable region of hu057VH1 (SEQ ID NO: 34), the light chain variable region of hu057VL1 (SEQ ID NO: 32), and the heavy chain constant region as shown in SEQ ID NO: 13 and the light chain constant region as shown in SEQ ID NO: 14, and the others are inferred in the same way.

[0299] Exemplarily, the full-length sequence of the humanized antibody is as follows:

[0300] >Hu057L1H1, Hu057L2H1 antibody heavy chain sequence

[0301] >Hu057L1H2, Hu057L2H2 antibody heavy chain sequence

[0302] >Hu057L1H3, Hu057L2H3 antibody heavy chain sequence

[0303] >Hu057L1H4, Hu057L2H4 antibody heavy chain sequence

[0304] Heavy chain sequence of Hu057L1H5, Hu057L2H5 antibody

[0305] Heavy chain sequence of Hu057L1H6, Hu057L2H6 antibody

[0306] Light chain sequence of Hu057L1H1, Hu057L1H2, Hu057L1H3, Hu057L1H4, Hu057L1H5, Hu057L1H6 antibody

[0307] Light chain sequence of Hu057L2H1, Hu057L2H2, Hu057L2H3, Hu057L2H4, Hu057L2H5, Hu057L2H6 antibody

[0308] To eliminate the Fc effector function of the antibody and prolong the half-life, L234A / L235A / G237A mutation sites and M428L / N434S were introduced into Hu57L1H6, respectively, to obtain molecule Hu57L1H6-AAA / LS (the mutation sites are denoted by EU numbering). The heavy chain constant region is as follows:

[0309] >hlgGl:L234A / L235A / G237A / M428L / N434S

[0310] The full-length sequence of the humanized antibody is as follows:

[0311] Heavy chain sequence of Hu057L1H6-AAA / LS antibody

[0312] Light chain sequence of Hu057L1H6-AAA / LS antibody

[0313] Note: In the above sequences, the underlined part is the CDR sequence determined according to the Kabat numbering system, and the italic part is the constant region of the antibody.

[0314] Example 4. Humanization of anti-latent myostatin antibody 207

[0315] At https: / / www.ncbi.nlm.nih.gov / igblast / , the murine 207 antibody light chain variable region was compared to human IgG germline sequences for homology, IGKV1-16*01 was selected as the light chain framework region template, the murine 207 antibody light chain CDRs were grafted into the IGKV1-16*01 framework region, and FGQGTKVEIK (SEQ ID NO: 27) was added after LCDR3 as a fourth framework region to obtain the CDR-grafted light chain variable region sequence. Similarly, the murine 207 antibody heavy chain variable region was compared to human IgG germline sequences for homology, IGHV3-21*01 was selected as the heavy chain framework region template, the murine 207 antibody heavy chain CDRs were grafted into the IGHV3-21*01 framework region, and WGQGTLVTVSS (SEQ ID NO: 28) was added after HCDR3 as a fourth framework region to obtain the CDR-grafted heavy chain variable region sequence. Optionally, the amino acid residues at positions 28, 36, 46, and / or 55 of the light chain variable region of the humanized antibody are back-mutated or otherwise mutated; and / or the amino acid residues at positions 30, 49, and / or 60 of the heavy chain variable region of the humanized antibody are back-mutated or otherwise mutated.

[0316] Table 6-1. 207 humanized antibody design

[0317] Note: F36Y means mutating F at position 36 to Y, and so on. The position of the amino acid residue is determined according to the Kabat rule.

[0318] The CDRs of the humanized antibodies of 207 are as follows:

[0319] Table 6-2. CDRs of 207 humanized antibodies

[0320] The variable region sequences of the humanized antibodies of 207 are as follows:

[0321] > hu207VL1

[0322] > hu207VL2

[0323] > hu207VL3

[0324] > hu207VL4

[0325] > hu207VL5

[0326] > hu207VH1

[0327] >hu207VH2

[0328] >hu207VH3

[0329] Note: The primary structure of the above sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4; the underlined part is the CDR sequence determined according to the Kabat numbering system; the double underlined and bolded part is the point mutation of amino acid.

[0330] The heavy chain variable region and the light chain variable region of the 207 humanized antibody are respectively recombined with the heavy chain constant region hlgG1 (SEQ ID NO: 13) and the human kappa light chain constant region CL (SEQ ID NO: 14) to obtain the humanized antibody as shown in Table 6-3 below.

[0331] Table 6-3. Humanized antibody of 207

[0332] Note: "Hu207L1H1" in the table represents a humanized antibody with the heavy chain variable region of hu207VH1 (SEQ ID NO: 59), the light chain variable region of hu207VL1 (SEQ ID NO: 54), and the heavy chain constant region as shown in SEQ ID NO: 13 and the light chain constant region as shown in SEQ ID NO: 14, and the others are inferred in the same manner.

[0333] Exemplarily, the full-length sequence of the humanized antibody is as follows:

[0334] >Hu207L1H1 antibody heavy chain sequence

[0335] >Hu207L1H2 antibody heavy chain sequence

[0336] >Hu207L1H3, Hu207L2H3, Hu207L3H3, Hu207L4H3, Hu207L5H3 antibody heavy chain sequence

[0337] >Hu207L1H1, Hu207L1H2, Hu207L1H3 antibody light chain sequence

[0338] >Hu207L2H3 antibody light chain sequence

[0339] >Hu207L3H3 antibody light chain sequence

[0340] Light chain sequence of Hu207L4H3 antibody

[0341] Light chain sequence of Hu207L5H3 antibody

[0342] To eliminate the Fc effector function of the antibody and prolong the half-life, L234A / L235A / G237A mutation sites and M428L / N434S were introduced into Hu207L3H3 respectively, and the molecule Hu207L3H3-AAA / LS was obtained.

[0343] The full-length sequence of the humanized antibody is as follows:

[0344] Heavy chain sequence of Hu207L3H3-AAA / LS antibody

[0345] Light chain sequence of Hu207L3H3-AAA / LS antibody

[0346] Note: The underlined part of the above sequence is the CDR sequence determined according to the Kabat numbering system, and the italic part is the constant region of the antibody.

[0347] The positive control molecule used in the present disclosure is Apitegromab.

[0348] Apitegromab sequence source: WO2017049011A1

[0349] The light and heavy chain amino acid sequences of Apitegromab are as follows:

[0350] > Apitegromab heavy chain

[0351] > Apitegromab light chain

[0352] Note: Single underline: variable region; double underline: CDR; dotted underline: constant region.

[0353] Test example

[0354] Test example 1. ELISA detection of the binding ability of anti-latent myostatin antibody

[0355] Human latent myostatin (SEQ ID NO: 1) or human mature myostatin (SEQ ID NO: 73) recombinant proteins were diluted to 1 pg / mL and 0.5 pg / ml, respectively, with PBS, and a 96-well plate (Corning, CLS3590-100EA) was coated with the solution at 4°C overnight. After washing the plate, 1% BSA was added for blocking at room temperature for 1 hour. After washing the plate, gradient-diluted anti-latent myostatin antibodies were added and incubated at room temperature for 1 hour. After washing the plate, HRP-goat anti-human Fc secondary antibody (Goat Anti-Human IgG Antibody, Fc-specific, HRP-conjugated, Sigma) was added and incubated at room temperature for 0.5 hours. After washing the plate, TMB color developing substrate (KPL, 52-00-03) was added and incubated at room temperature for 5-10 minutes, 1M H2SO4 was added to stop the reaction, and OD450 was read using a VERSAmax plate reader (Molecular Devices). The results are shown in FIGS. 1A-1H.

[0356] The experimental results show that the chimeric antibodies and humanized antibodies of the present disclosure have strong binding to human latent myostatin protein and do not cross-bind human mature myostatin.

[0357] Test Example 2. Biacore detection of affinity of anti-latent myostatin antibodies to latent myostatin

[0358] The affinity of the anti-latent myostatin antibodies of the present disclosure to human latent myostatin was tested using Biacore. An Anti-His antibody biosensor chip (Cytiva, 10276998) was used to capture the latent myostatin (Acro Biosystems) antigen by affinity, and then the antibody was flowed over the surface of the chip, and the binding and dissociation curves were obtained by real-time detection of the reaction signal using the instrument. After each experimental cycle was completed, the biosensor chip was washed and regenerated using 10 mM Glycine-HCl pH 1.5 (Cytiva, BR-1003-54). The data fitting model used was a 1:1 Model. The binding and dissociation of each antibody are shown in Tables 7-1 to 7-4.

[0359] Table 7-1. Affinity of chimeric antibodies to human latent myostatin

[0360] Table 7-2. Affinity of humanized antibodies to human latent myostatin

[0361] Table 7-3. Affinity of humanized antibodies to human latent myostatin

[0362] Table 7-4 Affinity of humanized antibodies to human latent myostatin

[0363] The experimental results show that the affinity of the anti-latent myostatin antibodies involved in the present disclosure to human latent myostatin protein is higher than that of the control antibody Apitegromab.

[0364] Test Example 3. Detection of the potency of anti-latent myostatin antibodies to inhibit the activation of latent myostatin

[0365] The inhibitory effect of anti-latent myostatin antibodies on the activation of latent myostatin by BMP-1 was detected by a reporter gene method. HEK-Blue TM TGF-β cells (InvivoGen) stably expressing the corresponding receptor of myostatin and a reporter gene of the downstream signaling pathway were used, and the specific method was as follows:

[0366] In a 96-well plate (Costar, 3903), 30 nM human latent myostatin (SEQ ID NO: 1), 300 ng / mL BMP1 (R&D Systems, 1927-ZN-010) and a gradient-diluted antibody solution were mixed in equal volumes and placed in a 37°C incubator (BMP1 can cleave latent myostatin by protease activity to produce mature myostatin), and the reaction was carried out for 24 hours. At the same time, HEK-Blue TM TGF-β cells were resuspended in DMEM medium (Meilunbio, PWL003) containing 10% FBS (Gibco, 10099-141, heat-inactivated pretreatment) and 100 μg / mL Bleomycin (InvivoGen, ant-zn-1), and seeded in a 96-well plate at a density of 50,000 cells / 100 μL per well, and incubated at 37°C, 5% CO2 for 24 hours to allow them to adhere fully. The 96-well cell culture plate was removed, the supernatant was discarded, and the above enzyme-cleaved 24-hour antibody / BMP1 / myostatin mixture solution was immediately transferred to the corresponding cell culture plate at 120 μL per well, and the incubation was continued at 37°C, 5% CO2 for 24 hours to detect the active myostatin produced by enzyme cleavage in the solution by a reporter gene. After incubation, the culture plate was removed and the cell supernatant was collected, 100 μL of QUANTI-Blue TM Solution detection solution (InvivoGen, rep-qbs2) was added to each well, and incubated at 37°C for 4 hours. The OD620 was read by a plate reader, and the data was processed and plotted by GraphPad Prism software (the abscissa is the antibody concentration, and the ordinate is the inhibition rate), and the IC50 Results are shown in Figure 2, Table 8-1, Table 8-2.

[0367] Table 8-1 Potency of chimeric antibody Chi-057 to inhibit latent myostatin activation

[0368] Table 8-2 Potency of chimeric antibody Chi-207 to inhibit latent myostatin activation

[0369] The results of the experiments show that both the chimeric antibodies and the humanized antibodies according to the present disclosure are able to inhibit latent myostatin activation.

[0370] Test Example 4. Detection of binding specificity of anti-latent myostatin antibodies

[0371] Human latent myostatin (SEQ ID NO: 1), human mature myostatin (SEQ ID NO: 73), human GDF11 (R&D, C799), human latent GDF11 (SEQ ID NO: 74), human activin-A (Acro, ACA-H421b), human latent activin-A (Acro, ACA-H424x), human GDF15 (Acro, GD5-H5149), human BMP9 (Acro, GD2-H5211), human BMP10 (MCE, HY-P700019AF), human TGFpi (Acro, TG1-H4212) were diluted to 0.5 pg / mL or 1 pg / mL with PBS, coated 96-well plates (Corning, CLS3590-100EA) and incubated at 4°C overnight. After washing the plates, 1% BSA was added for blocking at 37°C for 1 hour. After washing the plates, gradient-diluted anti-latent myostatin antibodies were added and incubated at 37°C for 1 hour. After washing the plates, HRP-goat anti-human Fc secondary antibody (Goat Anti-Human IgG Antibody (Fc-specific, HRP-conjugated) - (Sigma)) was added and incubated at 37°C for 1 hour. After washing the plates, TMB color developing substrate (KPL, 52-00-03) was added and incubated at room temperature for 5-10 minutes, 1M H2SO4 was added to stop the reaction, and OD450 was read using a VERSAmax plater reader (Molecular Devices). The results are shown in Table 9.

[0372] Table 9. Detection of binding specificity of humanized antibodies

[0373] “-” means that no binding signal was detected with the addition of 100 nM antibody. The results show that the humanized antibodies according to the disclosure do not bind to mature myostatin. Also, the antibodies according to the disclosure do not bind to latent GDF11 / GDF11 and other TGF beta family proteins.

[0374] > Human latent GDF11-Strep

[0375] Note: single underline is the strep tag sequence, double underline is the linker sequence between the tag and the antigen.

[0376] Test Example 5. Promoting effect of humanized antibodies on muscle growth of SCID mice

[0377] Male CB17-SCID male mice (5 weeks old) were ordered from Vantianlihua Experimental Animal Co., Ltd. (Animal Production License No.: SCXK(Zhejiang)2024-0001). Before the experiment, the body composition of the animals was detected using a dual-energy X-ray machine, and the animals were randomly divided into groups according to the body weight, lean percent of the experimental animals, with 8 animals in each group. Drug administration started on the day of grouping (Day 0), and the animals were injected via the tail vein, once a week, with a drug administration volume of 10 mL / kg, for three weeks. The body weight of the animals was detected twice a week, and at the end of the experiment, the gastrocnemius muscles on both sides of the hind limbs of the animals were stripped and weighed. The body composition of the animals was detected using an X-ray machine before the experiment, during the experiment, and before the end of the experiment. The data of the gastrocnemius muscle weight of the animals were statistically analyzed using GraphPad Prism 10. The results are shown in FIGS. 3A-3C.

[0378] Table 10. Drug administration groups for SCID mouse pharmacodynamic model

[0379] Note: “*” means P value < 0.05, “**” means P value < 0.01, “***” means P value < 0.001, “****” means P value < 0.0001, and ns means no significant difference.

[0380] The results show that the humanized antibody Hu057L1H6-AAA / LS according to the disclosure can significantly increase the gastrocnemius muscle mass and lean body mass of CB17-SCID mice at a dose of 3 mg / kg, and can significantly increase the ratio of gastrocnemius muscle to body weight at a dose of 1 mg / kg and 3 mg / kg, and has a dose-dependent effect, compared with the isotype control. Compared with the control antibody Apitegromab, the increase in gastrocnemius muscle weight, gastrocnemius muscle / body weight, and lean body mass of the mice is more significant at the same dose of 3 mg / kg.

[0381] Test Example 6. Growth promoting effect of humanized antibody on muscle of SCID mice

[0382] Male CB17-SCID male mice (5 weeks old) were ordered from Vantianlihua Experimental Animal Co., Ltd. (Animal Production License No.: SCXK(Zhejiang)2019-0001). The animals were randomly grouped according to body weight before the start of the experiment, with 8 animals in each group. Drug administration began on the grouping day (Day 0), and was performed via tail vein injection, once a week, with a drug administration volume of 10 mL / kg, for two weeks. The animal body weight was detected twice a week, and at the end of the experiment, the gastrocnemius muscle on both sides of the hind limbs of the animals was stripped, and the weight of the gastrocnemius muscle was measured. The gastrocnemius muscle weight data was statistically analyzed using GraphPad Prism 10. The results are shown in Figure 4.

[0383] Note: “*” indicates P value < 0.05, “**” indicates P value < 0.01, “***” indicates P value < 0.001, “****” indicates P value < 0.0001, and ns indicates no significant difference.

[0384] The results show that the humanized antibody Hu207L3H3-AAA / LS according to the present disclosure can significantly increase the ratio of gastrocnemius muscle weight to body weight of CB17-SCID mice at a dose of 1 mg / kg, compared with the isotype control, indicating that the efficacy of Hu207L3H3-AAA / LS is better than that of the control antibody Apitegromab.

[0385] Although the above-mentioned invention has been described in detail with the aid of the accompanying drawings and examples for the purpose of clarity, the description and examples should not be interpreted in a limiting manner with respect to the scope of the present disclosure. The disclosures of all patents and scientific literature referred to herein are expressly incorporated herein by reference in their entirety.

Claims

1. An antigen binding molecule that specifically binds to latent myostatin, comprising a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, and a light chain variable region comprising LCDR1, LCDR2, and LCDR3, wherein: a). the HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 5, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 75, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 7, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 8, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 9, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 10; or b). the HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 17, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 76, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 19, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 77, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 78, and the LCDR3 comprises the amino acid sequence of SEQ ID NO:

22.

2. The antigen binding molecule that specifically binds to latent myostatin of claim 1, wherein: a). the HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 5, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 31, 29, 30, or 6, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 7, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 8, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 9, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 10; or b). the HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 17, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 18 or 53, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 19, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 51, 52, or 20, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 21 or 50, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 22; preferably, a). the HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 5, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 31, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 7, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 8, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 9, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 10; or b-1). the HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 17, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 18, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 19, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 51, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 21 or 50, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 22; or b-2). the HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 17, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 18, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 19, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 20, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 21 or 50, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 22; or b-3). the HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 17, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 18, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 19, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 52, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 50, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 22; or b-4). the HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 17, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 53, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 19, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 20, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 21, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 22; or More preferably, a). the HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 5, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 31, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 7, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 8, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 9, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 10; or b). the HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 17, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 18, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 19, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 51, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 21, and the LCDR3 comprises the amino acid sequence of SEQ ID NO:

22.

3. The antigen binding molecule specifically binding to latent myostatin according to claim 1 or 2, wherein the antigen binding molecule specifically binding to latent myostatin is an antibody or an antigen binding fragment thereof; Preferably, the antigen binding molecule specifically binding to latent myostatin is a murine antibody, a chimeric antibody, a humanized antibody, a fully human antibody or an antigen binding fragment thereof; More preferably, the antigen binding molecule specifically binding to latent myostatin is a humanized antibody or an antigen binding fragment selected from the group consisting of Fab, Fab', F(ab')2, Fv, scFv and dsFv.

4. The antigen binding molecule specifically binding to latent myostatin according to any one of claims 1 to 3, wherein: a). the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 39, 34, 35, 36, 37 or 38, or an amino acid sequence having at least 80% sequence identity thereto, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 32 or 33, or an amino acid sequence having at least 80% sequence identity thereto; or the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 11, or an amino acid sequence having at least 80% sequence identity thereto, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence having at least 80% sequence identity thereto; or b). the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 61, 59 or 60, or an amino acid sequence having at least 80% sequence identity thereto, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 56, 54, 55, 57 or 58, or an amino acid sequence having at least 80% sequence identity thereto; or the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 23, or an amino acid sequence having at least 80% sequence identity thereto, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 24, or an amino acid sequence having at least 80% sequence identity thereto; Preferably, a). the heavy chain variable region comprises SEQ ID NO: 39, 34, 35, 36, 37 or 38, or an amino acid sequence having at least 80% sequence identity thereto, and the light chain variable region comprises SEQ ID NO: 32, or an amino acid sequence having at least 80% sequence identity thereto; or the heavy chain variable region comprises SEQ ID NO: 39, 34, 35, 36, 37 or 38, or an amino acid sequence having at least 80% sequence identity thereto, and the light chain variable region comprises SEQ ID NO: 33, or an amino acid sequence having at least 80% sequence identity thereto; or b). the heavy chain variable region comprises SEQ ID NO: 61, or an amino acid sequence having at least 80% sequence identity thereto, and the light chain variable region comprises SEQ ID NO: 56, 54, 55, 57 or 58, or an amino acid sequence having at least 80% sequence identity thereto; or the heavy chain variable region comprises SEQ ID NO: 59 or 60, or an amino acid sequence having at least 80% sequence identity thereto, and the light chain variable region comprises SEQ ID NO: 54, or an amino acid sequence having at least 80% sequence identity thereto; More preferably, a). the heavy chain variable region comprises SEQ ID NO: 39, or an amino acid sequence having at least 80% sequence identity thereto, and the light chain variable region comprises SEQ ID NO: 32, or an amino acid sequence having at least 80% sequence identity thereto; or b). the heavy chain variable region comprises SEQ ID NO: 61, or an amino acid sequence having at least 80% sequence identity thereto, and the light chain variable region comprises SEQ ID NO: 56, or an amino acid sequence having at least 80% sequence identity thereto.

5. The antigen binding molecule specifically binding to latent myostatin of any one of claims 1 to 4, wherein the antigen binding molecule specifically binding to latent myostatin further comprises a heavy chain constant region and a light chain constant region; Preferably, the heavy chain constant region is derived from human IgGl, IgG2, IgG3, IgG4, and the light chain constant region is derived from human kappa or lambda; More preferably, the heavy chain constant region comprises SEQ ID NO: 48 or 13, or an amino acid sequence having at least 80% sequence identity thereto, and the light chain constant region comprises SEQ ID NO: 14, or an amino acid sequence having at least 80% sequence identity thereto.

6. The antigen binding molecule specifically binding to latent myostatin of claim 5, wherein the antigen binding molecule specifically binding to latent myostatin comprises a heavy chain and a light chain, wherein: a). the heavy chain comprises SEQ ID NO: 49, 45, 40, 41, 42, 43, or 44, or an amino acid sequence having at least 80% sequence identity thereto, and the light chain comprises SEQ ID NO: 46 or 47, or an amino acid sequence having at least 80% sequence identity thereto; or the heavy chain comprises SEQ ID NO: 15, or an amino acid sequence having at least 80% sequence identity thereto, and the light chain comprises SEQ ID NO: 16, or an amino acid sequence having at least 80% sequence identity thereto; or b). the heavy chain comprises SEQ ID NO: 70, 64, 62, or 63, or an amino acid sequence having at least 80% sequence identity thereto, and the light chain comprises SEQ ID NO: 67, 65, 66, 68, or 69, or an amino acid sequence having at least 80% sequence identity thereto; or the heavy chain comprises SEQ ID NO: 25, or an amino acid sequence having at least 80% sequence identity thereto, and the light chain comprises SEQ ID NO: 26, or an amino acid sequence having at least 80% sequence identity thereto; Preferably, a-1). the heavy chain comprises SEQ ID NO: 49, 45, 40, 41, 42, 43, or 44, or an amino acid sequence having at least 80% sequence identity thereto, and the light chain comprises the amino acid sequence of SEQ ID NO: 46; or a-2). the heavy chain comprises SEQ ID NO: 45, 40, 41, 42, 43, or 44, or an amino acid sequence having at least 80% sequence identity thereto, and the light chain comprises SEQ ID NO: 47, or an amino acid sequence having at least 80% sequence identity thereto; or b-1). the heavy chain comprises SEQ ID NO: 70 or 64, or an amino acid sequence having at least 80% sequence identity thereto, and the light chain comprises SEQ ID NO: 67, or an amino acid sequence having at least 80% sequence identity thereto; or b-2). the heavy chain comprises SEQ ID NO: 64, or an amino acid sequence having at least 80% sequence identity thereto, and the light chain comprises SEQ ID NO: 65, 66, 68, or 69, or an amino acid sequence having at least 80% sequence identity thereto; or b-3). the heavy chain comprises SEQ ID NO: 62 or 63, or an amino acid sequence having at least 80% sequence identity thereto, and the light chain comprises SEQ ID NO: 65, or an amino acid sequence having at least 80% sequence identity thereto; More preferably, a). the heavy chain comprises SEQ ID NO: 49 or 45, or an amino acid sequence having at least 80% sequence identity thereto, and the light chain comprises SEQ ID NO: 46, or an amino acid sequence having at least 80% sequence identity thereto; or b). the heavy chain comprises SEQ ID NO: 70 or 64, or an amino acid sequence having at least 80% sequence identity thereto, and the light chain comprises SEQ ID NO: 67, or an amino acid sequence having at least 80% sequence identity thereto.

7. A pharmaceutical composition comprising the antigen binding molecule that specifically binds to latent myostatin as claimed in any one of claims 1 to 6, and one or more pharmaceutically acceptable carriers, diluents or excipients; preferably, the pharmaceutical composition further comprises at least one second therapeutic agent.

8. An isolated nucleic acid encoding the antigen binding molecule that specifically binds to latent myostatin as claimed in any one of claims 1 to 6.

9. A host cell comprising the isolated nucleic acid as claimed in claim 8.

10. A method for producing the antigen binding molecule that specifically binds to latent myostatin as claimed in any one of claims 1 to 6, the method comprising the steps of culturing the host cell of claim 9 in a culture medium to form and accumulate the antigen binding molecule that specifically binds to latent myostatin as claimed in any one of claims 1 to 6, and recovering the antigen binding molecule that specifically binds to latent myostatin from the culture.

11. A method of treating, preventing or ameliorating a disease or disorder, the method comprising administering to a subject in need thereof a therapeutically effective amount or a prophylactically effective amount of the antigen binding molecule that specifically binds to latent myostatin as claimed in any one of claims 1 to 6, or the pharmaceutical composition according to claim 7; preferably, the disease or disorder is a disease or disorder associated with myostatin; more preferably, the disease or disorder is selected from muscle atrophy, muscular dystrophy, obesity and cachexia.

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