Anti-ACVR1 antibodies and their use in the treatment of trauma-induced ectopic ossification

Abstract

This invention provides monoclonal antibodies that bind to the activin AI receptor (ACVR1) protein, their antigen-binding fragments, and methods for using them. In various embodiments of the invention, the antibody is a fully human antibody that binds to ACVR1. In some embodiments, the antibody and its antigen-binding fragment are useful for inhibiting ACVR1-mediated bone morphogenetic protein (BMP) signaling, and thus provide a means for treating or preventing diseases, disorders, or conditions associated with ACVR1.

Description

[Technical Field]

[0001] Cross-reference of related applications This application was filed on October 26, 2023, as a PCT international patent application claiming the benefit of priority from U.S. Provisional Application No. 63 / 381,245, filed on October 27, 2022, which is incorporated in its entirety by reference herein.

[0002] Reference to sequence listings This application includes a sequence listing, which is submitted electronically in XML format and is incorporated herein by reference in its entirety. The XML file, created on 26 October 2022, is named Sequence-Listing-40848-0115USP1.xml and has a size of 87,923 bytes.

[0003] The present invention relates to antibodies and antigen-binding fragments of antibodies that specifically bind to activin A receptor type 1 (ACVR1) and / or ACVR1 mutant proteins, as well as treatment and diagnostic methods using these antibodies. [Background technology]

[0004] Activin A receptor type 1 (ACVR1; ActR1; or activin receptor-like kinase 2; also known as ALK2) is a single-pass transmembrane receptor and a member of the type I bone morphogenetic protein (BMP) receptor superfamily of TGF-β receptors. Upon ligand binding, ACVR1, along with type II receptors, initiates a downstream signaling cascade, leading to the activation of receptor-specific R-SMAD proteins (SMAD1, SMAD5, or SMAD8), which subsequently associate with SMAD4, resulting in gene transcriptional regulation (Massague 1998, Massaque et al. 2005).

[0005] Heterotopic ossification (HO) is a common complication associated with post-traumatic healing in clinical conditions such as fractures, spinal cord injuries, traumatic brain injuries, blast injuries, severe burns, and extensive surgeries including hip arthroplasty, acetabular surgery, and elbow joint surgery. In addition to trauma-induced heterotopic ossification, this pathological phenomenon is also seen in rare genetic disorders associated with certain mutations in the ACVR1 gene.

[0006] Mutations in the ACVR1 gene, which encodes the BMP type I receptor ALK2 (also known as the ACVR1 protein), can cause fibrosis ossificans progressive (FOP), a rare disorder resulting in progressive ectopic ossification of soft tissues with severe impairment of body movement due to extraosseous ponsions. The ACVR1 mutations causing FOP lead to dysregulation of SMAD-dependent downstream signaling, conferring the ability of the mutated receptor to respond to the non-standard ligand activin A, thereby inducing ectopic ossification. Acquisition of functional mutations in the gene encoding ACVR1 results in debilitating disorders of extraosseous (ectopic) ossification in humans, such as FOP. For example, a typical FOP patient may have the amino acid arginine substituted for the amino acid histidine at position 206 of the ACVR1 protein. This causes a change in the protein's glycine-serine activating domain, which converts the Acvr1:activin A:Acvr2 non-signaling complex into a signaling complex. The result of activin's new function is that fibroadipose progenitor cells (FAPs) initiate endochondral ossification. Atypical mutations involving other residues may act similarly, preventing the ACVR1 protein from escaping its active conformation even in the absence of BMPs. Mutations in the ACVR1 gene may also be associated with diffuse pontine glioma (DIPG).

[0007] The hepatic expression of the important iron regulator hepcidin is regulated by the bone morphogenetic protein (BMP) / SMAD pathway. BMP signaling requires a ligand (e.g., BMP7, BMP6, or BMP2), a type I receptor (e.g., ACVR1), a type II receptor (e.g., ACVR2 or BMPR2), and a co-receptor hemoduvelin (HJV) to phosphorylate SMAD proteins. BMP6-mediated activation of ACVR1 directly activates the transcription of Hamp, the gene encoding hepcidin. Hepcidin is a negative regulator of iron levels by causing the internal translocation of ferroportin (slc40a1), the only known iron transporter. Inhibition of the BMP6-ACVR1 signaling cascade leads to decreased Hamp transcription, resulting in reduced circulating hepcidin levels. The reduction in circulating hepcidin levels increases ferroportin levels, which in turn allows for increased iron uptake from the small intestine, thereby increasing circulating iron levels.

[0008] Monoclonal antibodies against ACVR1 are described by Katagiri et al., Patent Documents 1, 2, and 3, and by Idone et al., Patent Documents 4 and 5. [Prior art documents] [Patent Documents]

[0009] [Patent Document 1] U.S. Patent No. 10428148 [Patent Document 2] U.S. Patent Application Publication No. 20180118835 [Patent Document 3] International Publication No. 2019172165 [Patent Document 4] U.S. Patent Application Publication No. 20210253716 [Patent Document 5] International Publication No. 2021163170 [Overview of the project] [Problems that the invention aims to solve]

[0010] High-affinity, fully human antibodies that specifically bind to the ACVR1 protein, its antigen-binding fragments, or their mutants, and inhibit ACVR1-mediated osteomorphonectomy (BMP) signaling, are important for the prevention and treatment of conditions such as ectopic ossification, osteodysplasia, anemia, or diffuse pontine glioma. [Means for solving the problem]

[0011] The present invention provides an antibody and its antigen-binding fragment that specifically binds to the activin A receptor type 1 (ACVR1) protein and inhibits ACVR1-mediated BMP signaling. In certain embodiments, the anti-ACVR1 antibody is a fully human antibody that binds to ACVR1 with high affinity and blocks ACVR1 or destabilizes its activating conformation. The antibody of the present invention is particularly useful for inactivating or reducing the activity of the ACVR1 protein. In certain embodiments, the antibody is useful for preventing, treating, or reversing at least one symptom or sign of ACVR1-related disease or disorder in a subject. In certain embodiments, the antibody is administered prophylactically or therapeutically to a subject who has or is at risk of having ACVR1-related disease or disorder. In certain embodiments, the antibody is used for the prevention and treatment of certain cancers, including ectopic ossification, dysplasia, anemia, or brain tumors, when administered to a subject requiring prevention and treatment.

[0012] In some embodiments, the antibodies of the present invention bind to the ACVR1 protein and / or its mutants. Furthermore, the antibodies disclosed herein bind to the ACVR1 protein or its mutants with high affinity. Examples of ACVR1 proteins used in the present invention include proteins derived from mammals such as humans or mice. For example, the full-length amino acid sequence of human ACVR1 is available by referring to UniProtKB accession number Q04771 (SEQ ID NO: 61).

[0013] The ACVR1 protein may, for example, contain a signal peptide located at positions 1-20 of the ACVR1 protein with accession number Q04771 (SEQ ID NO: 61). The mature ACVR1 protein may, for example, contain amino acids 21-509 of accession number Q04771 (SEQ ID NO: 61). The ACVR1 protein may, for example, contain an extracellular domain located at amino acids 21-123 of accession number Q04771 (SEQ ID NO: 61). The ACVR1 protein may, for example, contain a transmembrane domain located at amino acids 124-146 of accession number Q04771 (SEQ ID NO: 61). The ACVR1 protein may, for example, contain a protein kinase domain located at positions 208-502 of accession number Q04771 (SEQ ID NO: 61). The ACVR protein may, for example, contain a glycosylation at amino acid position 102 containing N-linked (GlcNAc...)asparagine of accession number Q04771 (SEQ ID NO: 61). ACVR proteins can contain modified residues, such as phosphoserine, at position 501 of accession number Q04771 (sequence number 61).

[0014] Mutations in the ACVR1 gene can cause a variety of diseases, including FOP. The ACVR1 protein may be a mutant ACVR1 protein with amino acid substitutions found in various cases of familial and sporadic FOP. Human ACVR1 proteins include L196P (a mutation substituting leucine at position 196 with proline), delP197_F198insL (a mutation deleting proline at position 197 and phenylalanine at position 198 and inserting leucine), R202I (a mutation substituting arginine at position 202 with isoleucine), R206H (a mutation substituting arginine at position 206 with histidine), Q207E (a mutation substituting glutamine at position 207 with glutamic acid), R258S (a mutation substituting arginine at position 258 with serine), and R258G (2 This may include, but is not limited to, various mutations such as G325A (a mutation that replaces arginine at position 58 with glycine), G328E (a mutation that replaces glycine at position 328 with glutamate), G328R (a mutation that replaces glycine at position 328 with arginine), G328W (a mutation that replaces glycine at position 328 with tryptophan), G356D (a mutation that replaces glycine at position 356 with aspartate), and R375P (a mutation that replaces arginine at position 375 with proline).

[0015] As another example, the full-length amino acid sequence of the mouse ACVR1 protein is available by referring to accession number P37172 (sequence number 62).

[0016] The antibodies of the present invention may be full-length (e.g., IgG1 or IgG4 antibodies) or may consist only of antigen-binding moieties (e.g., Fab, F(ab')2, or scFv fragments), and may be modified to affect their functionality, for example, to increase persistence in the host or to eliminate residual effector function (Reddy et al., 2000, J.Immunol. 164: pp. 1925-1933). In certain embodiments, the antibodies are bispecific.

[0017] In a first embodiment, the present invention provides an isolated recombinant monoclonal antibody or an antigen-binding fragment thereof that specifically binds to the ACVR1 protein. In some embodiments, the antibody is a fully human monoclonal antibody.

[0018] The exemplary anti-ACVR1 antibodies of the invention include the amino acid sequences and nucleic acid sequences listed in Tables 1, 2, and 3 herein. Table 1 lists the amino acid sequence identifiers for the heavy chain variable region (HCVR), light chain variable region (LCVR), heavy chain complementarity-determining region (HCDR) (HCDR1, HCDR2, and HCDR3), and light chain complementarity-determining region (LCDR) (LCDR1, LCDR2, and LCDR3) of the exemplary antibodies. Table 2 lists the nucleic acid sequence identifiers for the HCVR, LCVR, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the exemplary antibodies. Table 3 lists the amino acid sequences and nucleic acid sequences of the heavy and light chains of the exemplary antibodies.

[0019] The present invention provides an HCVR comprising an amino acid sequence selected from any of the HCVR amino acid sequences listed in Table 1, or an antibody or antigen-binding fragment thereof comprising a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0020] The present invention also provides an antibody or antigen-binding fragment thereof comprising an LCVR containing an amino acid sequence selected from any of the LCVR amino acid sequences listed in Table 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0021] The present invention also provides an antibody or antigen-binding fragment thereof comprising an HCVR and LCVR amino acid sequence pair (HCVR / LCVR) containing one of the HCVR amino acid sequences listed in Table 1 paired with one of the LCVR amino acid sequences listed in Table 1. According to a particular embodiment, the present invention provides an antibody or antigen-binding fragment thereof comprising an HCVR / LCVR amino acid sequence pair contained within one of the exemplary anti-ACVR1 antibodies listed in Table 1. The present invention provides an antibody or antigen-binding fragment thereof comprising a heavy chain variable region (HCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 30, and 46, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0022] The present invention also provides an antibody or antigen-binding fragment of an antibody comprising a light chain variable region (LCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 22, 38, and 54, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0023] The present invention also provides an antibody or an antigen-binding fragment thereof comprising an HCVR and LCVR (HCVR / LCVR) sequence pair selected from the group consisting of SEQ ID NOs: 14 / 22, 30 / 38, and 46 / 54.

[0024] The present invention also provides an antibody or antigen-binding fragment of an antibody comprising a heavy chain CDR3 (HCDR3) domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 36, and 52, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; and a light chain CDR3 (LCDR3) domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 28, 44, and 60, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0025] In certain embodiments, the antibody or the antigen-binding portion of the antibody comprises an HCDR3 / LCDR3 amino acid sequence pair selected from the group consisting of SEQ ID NOs: 20 / 28, 36 / 44, and 52 / 60.

[0026] The present invention also provides an antibody or fragment thereof comprising: a heavy chain CDR1 (HCDR1) domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 32, and 48, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; a heavy chain CDR2 (HCDR2) domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 34, and 50, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; a light chain CDR1 (LCDR1) domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 24, 40, and 56, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; and a light chain CDR2 (LCDR2) domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 26, 42, and 58, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0027] Certain non-limiting exemplary antibody and antigen-binding fragments of the present invention each contain an HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 16-18-20-24-26-28 (e.g., REGN 5166); 32-34-36-40-42-44 (REGN 5167); and 48-50-52-56-58-60 (e.g., REGN 5168).

[0028] In related embodiments, the present invention comprises an antibody or antigen-binding fragment of an antibody that specifically binds to activin A receptor type 1 (ACVR1) and / or ACVR1 mutant protein, wherein the antibody or fragment comprises heavy and light chain CDR domains contained within a heavy and light chain variable region (HCVR / LCVR) sequence selected from the group consisting of SEQ ID NOs: 14 / 22, 30 / 38, and 46 / 54.

[0029] The present invention also provides an antibody comprising HCVR and LCVR, or an antigen-binding fragment thereof, wherein the HCVR comprises an amino acid sequence listed in Table 1 having 12 or fewer amino acid substitutions, and / or the LCVR comprises an amino acid sequence listed in Table 1 having 10 or fewer amino acid substitutions. For example, the present invention provides an antibody comprising HCVR and LCVR, or an antigen-binding fragment thereof, wherein the HCVR comprises an amino acid sequence listed in Table 1, and the amino acid sequence has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid substitutions. In another example, the present invention provides an antibody comprising HCVR and LCVR, or an antigen-binding fragment thereof, wherein the LCVR comprises an amino acid sequence listed in Table 1, and the amino acid sequence has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. In one embodiment, the present invention provides an anti-ACVR1 antibody comprising HCVR and LCVR or an antigen-binding fragment thereof, wherein the HCVR comprises an amino acid sequence listed in Table 1, the amino acid sequence having at least one amino acid substitution, and / or the LCVR comprises an amino acid sequence listed in Table 1, the amino acid sequence having at least one amino acid substitution.

[0030] The present invention also provides a heavy chain CDR1 (HCDR1) comprising an amino acid sequence selected from any of the HCDR1 amino acid sequences listed in Table 1, or an antibody or antigen-binding fragment thereof comprising a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0031] The present invention also provides a heavy chain CDR2 (HCDR2) comprising an amino acid sequence selected from any of the HCDR2 amino acid sequences listed in Table 1, or an antibody or antigen-binding fragment thereof comprising a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0032] The present invention also provides a heavy chain CDR3 (HCDR3) comprising an amino acid sequence selected from any of the HCDR3 amino acid sequences listed in Table 1, or an antibody or antigen-binding fragment thereof comprising a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0033] The present invention also provides an antibody or antigen-binding fragment thereof comprising a light chain CDR1 (LCDR1) containing an amino acid sequence selected from any of the LCDR1 amino acid sequences listed in Table 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0034] The present invention also provides an antibody or antigen-binding fragment thereof, comprising a light chain CDR2 (LCDR2) containing an amino acid sequence selected from any of the LCDR2 amino acid sequences listed in Table 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0035] The present invention also provides an antibody or antigen-binding fragment thereof, comprising a light chain CDR3 (LCDR3) containing an amino acid sequence selected from any of the LCDR3 amino acid sequences listed in Table 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0036] The present invention also provides an antibody or antigen-binding fragment thereof comprising an HCDR3 and LCDR3 amino acid sequence pair (HCDR3 / LCDR3) which includes one of the HCDR3 amino acid sequences listed in Table 1 paired with one of the LCDR3 amino acid sequences listed in Table 1.

[0037] According to certain embodiments, the present invention provides an antibody or antigen-binding fragment thereof comprising an HCDR3 / LCDR3 amino acid sequence pair contained within one of the exemplary anti-ACVR1 antibodies listed in Table 1. In certain embodiments, the HCDR3 / LCDR3 amino acid sequence pair is selected from the group consisting of SEQ ID NOs: 20 / 28 (e.g., REGN 5166), 36 / 44 (e.g., REGN 5167), and 52 / 60 (e.g., REGN 5168).

[0038] The present invention also provides antibodies comprising HCVR and LCVR, or antigen-binding fragments thereof, wherein the HCVR comprises HCDR1 comprising an amino acid sequence having one amino acid different from the amino acid sequence listed in Table 1, HCDR2 comprising an amino acid sequence having one amino acid different from the amino acid sequence listed in Table 1, and HCDR3 comprising an amino acid sequence having one amino acid different from the amino acid sequence listed in Table 1. In certain embodiments, the present invention provides antibodies comprising HCVR and LCVR, or antigen-binding fragments thereof, wherein the LCVR comprises LCDR1 comprising an amino acid sequence having one amino acid different from the amino acid sequence listed in Table 1, LCDR2 comprising an amino acid sequence having one amino acid different from the amino acid sequence listed in Table 1, and LCDR3 comprising an amino acid sequence having one amino acid different from the amino acid sequence listed in Table 1.

[0039] For example, the present invention provides an antibody comprising HCVR and LCVR, or an antigen-binding fragment thereof, wherein the HCVR comprises HCDR1 comprising the amino acid sequence of SEQ ID NO: 16, 32, or 48, or an amino acid sequence that differs from SEQ ID NO: 16, 32, or 48 by one amino acid; HCDR2 comprising the amino acid sequence of SEQ ID NO: 18, 34, or 50, or an amino acid sequence that differs from SEQ ID NO: 18, 34, or 50 by one amino acid; and HCDR3 comprising the amino acid sequence of SEQ ID NO: 20, 36, or 52, or an amino acid sequence that differs from SEQ ID NO: 20, 36, or 52 by one amino acid. In another exemplary embodiment, the present invention provides an antibody comprising HCVR and LCVR, or an antigen-binding fragment thereof, wherein the LCVR comprises LCDR1 comprising the amino acid sequence of SEQ ID NO: 24, 40, or 56 or an amino acid sequence that differs from SEQ ID NO: 24, 40, or 56 by one amino acid; LCDR2 comprising the amino acid sequence of SEQ ID NO: 26, 42, or 58 or an amino acid sequence that differs from SEQ ID NO: 26, 42, or 58 by one amino acid; and LCDR3 comprising the amino acid sequence of SEQ ID NO: 28, 44, or 60 or an amino acid sequence that differs from SEQ ID NO: 28, 44, or 60 by one amino acid.

[0040] The present invention also provides an antibody or antigen-binding fragment thereof containing six CDR sets (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within any of the exemplary antibodies listed in Table 1. In certain embodiments, the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence set is selected from the group consisting of SEQ ID NOs: 16-18-20-24-26-28 (e.g., REGN 5166); 32-34-36-40-42-44 (REGN 5167); and 48-50-52-56-58-60 (e.g., REGN 5168).

[0041] In related embodiments, the present invention provides an antibody or antigen-binding fragment thereof comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within an HCVR / LCVR amino acid sequence pair as defined by any of the exemplary antibodies listed in Table 1. For example, the present invention comprises an antibody or antigen-binding fragment thereof comprising a set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences contained within an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 14 / 22, 30 / 38, and 46 / 54.

[0042] Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs within the designated HCVR and / or LCVR amino acid sequences disclosed herein. Exemplary conventions that can be used to identify CDR boundaries include, for example, the Kabat definition, the Chothia definition, and the AbM definition. Generally, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of structural loop regions, and the AbM definition is a hybrid of the Kabat and Chothia approaches. See, for example, Kabat, "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J.Mol.Biol.273:927-948 (1997); and Martin et al., Proc.Natl.Acad.Sci.USA 86:9268-9272 (1989). Publicly available databases can also be used to identify CDR sequences within antibodies.

[0043] In certain embodiments, the present invention comprises an antibody or an antigen-binding fragment thereof that specifically binds to ACVR1, wherein the antibody or antigen-binding fragment comprises three heavy chain complementarity-determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable region (HCVR) and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within a light chain variable region (LCVR), wherein the HCVR is (i) an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 30, and 46; (ii) an amino acid sequence having at least 90% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 30, and 46; (iii) selected from the group consisting of SEQ ID NOs: 14, 30, and 46 (iv) an amino acid sequence having at least 95% identity with the amino acid sequence to be performed; or an amino acid sequence selected from the group consisting of SEQ ID NOs. 14, 30, and 46 having 12 or fewer amino acid substitutions, wherein LCVR includes (a) an amino acid sequence selected from the group consisting of SEQ ID NOs. 22, 38, and 54; (b) an amino acid sequence having at least 90% identity with the amino acid sequence selected from the group consisting of SEQ ID NOs. 22, 38, and 54; (c) an amino acid sequence having at least 95% identity with the amino acid sequence selected from the group consisting of SEQ ID NOs. 22, 38, and 54; or (d) an amino acid sequence selected from the group consisting of SEQ ID NOs. 22, 38, and 54 having 10 or fewer amino acid substitutions.

[0044] In certain preferred embodiments, the present invention includes an antibody or its antigen-binding fragment that specifically binds to ACVR1 in an antagonist manner, i.e., reduces or inhibits the binding and / or activity of ACVR1.

[0045] In some embodiments, the anti-ACVR1 antibody or its antigen-binding fragment of the present invention reduces trauma-induced ectopic ossification (HO). In some cases, the anti-ACVR1 antibody or its antigen-binding fragment of the present invention can reduce the recurrence of trauma-induced ectopic ossification (HO) after surgical resection.

[0046] Anti-ACVR1 antibodies or their antigen-binding fragments are provided for use in the treatment, prevention, or recovery of at least one symptom or sign of ACVR1-related disease or disorder, where ACVR1-related disease or disorder is selected from the group consisting of heterotopic ossification, trauma-induced heterotopic ossification, heterotopic ossification, osteodysplasia, anemia, and diffuse pontine glioma. In some cases, ACVR1-related disease or disorder is not progressive ossifying fibrosis. Anti-ACVR1 antibodies or their antigen-binding fragments are provided for use in reducing the recurrence of trauma-induced heterotopic ossification (HO) after surgical excision.

[0047] The present invention comprises an anti-ACVR1 antibody having a modified glycosylation pattern. In some embodiments, modifications to remove undesirable glycosylation sites may be useful, or the antibody may lack a fucose moiety present on the oligosaccharide chain to enhance antibody-dependent cell-mediated cytotoxicity (ADCC) function, for example (see Shield et al. (2002) JBC 277:26733). In other applications, galactosylation modifications can be made to modify complement-dependent cell-mediated cytotoxicity (CDC).

[0048] In certain embodiments, the present invention provides an antibody exhibiting pH-dependent binding to ACVR1 and its antigen-binding fragment. For example, the present invention includes an antibody and its antigen-binding fragment that binds ACVR1 with higher affinity at neutral pH than at acidic pH (i.e., binding is reduced at acidic pH).

[0049] The present invention also provides antibodies or antigen-binding fragments thereof comprising CDRs of HCVR and LCVR, and antibodies and antigen-binding fragments thereof that compete for specific binding to ACVR1, wherein HCVR and LCVR each have an amino acid sequence selected from the HCVR and LCVR sequences listed in Table 1.

[0050] The present invention also provides a reference antibody or its antigen-binding fragment comprising the CDR of HCVR and the CDR of LCVR, and an antibody or its antigen-binding fragment that cross-compete for binding to ACVR1, wherein HCVR and LCVR each have an amino acid sequence selected from the HCVR and LCVR sequences listed in Table 1.

[0051] The present invention also provides an antibody and its antigen-binding fragment that bind to the same epitope as a reference antibody or its antigen-binding fragment, which comprises three CDRs of HCVR and three CDRs of LCVR, where HCVR and LCVR each have an amino acid sequence selected from the HCVR and LCVR sequences listed in Table 1.

[0052] The present invention also provides an isolated antibody and its antigen-binding fragment that inhibits ligand-induced signaling by BMP7, activin A, or other TGF beta-family ligands that form a signaling complex with the activin type II receptor. In some embodiments, the antibody or its antigen-binding fragment prevents ACVR1 from forming a signaling complex with the activin type II receptor. The present invention provides an isolated and its antigen-binding fragment that can bind to the same epitope on ACVR1 as BMP7, activin A, or the activin type II receptor, or to an epitope on ACVR1 that is different from BMP7, activin A, or the activin type II receptor.

[0053] In certain embodiments, the antibody or antigen-binding fragment of the present invention is bispecific, comprising a first binding specificity to a first epitope of ACVR1 and a second binding specificity to a second epitope of ACVR1, where the first and second epitopes are distinct and non-overlapping.

[0054] In a particular embodiment, the present invention has the following features: (a) It must be a fully human monoclonal antibody; (b) Dissociation constant (K) less than 15 nM, less than 10 nM, less than 5 nM, less than 3 nM, less than 2 nM, less than 1 nM, less than 0.5 nM, less than 0.3 nM, less than 0.2 nM, or less than 0.1 nM when measured in a surface plasmon resonance assay. D ) Binds to the human ACVR1 extracellular domain (SEQ ID NO: 64) fused to the mFc at 37°C; (c) K levels less than 50 nM, less than 10 nM, less than 5 nM, less than 3 nM, less than 2 nM, less than 1 nM, and less than 0.5 nM when measured in a surface plasmon resonance assay. D It binds to the human ACVR1 extracellular domain (e.g., SEQ ID NO: 63) fused to the myc-myc-hexahis tag at 37°C; (d) K levels less than 50 nM, less than 10 nM, less than 5 nM, less than 3 nM, less than 2 nM, less than 1 nM, and less than 0.5 nM when measured in a surface plasmon resonance assay. D To bind to the mouse ACVR1 extracellular domain (e.g., SEQ ID NO: 65) fused to the myc-myc-hexahis tag at 37°C; (e) K less than 10 nM, less than 5 nM, less than 3 nM, less than 2 nM, less than 1 nM, less than 0.5 nM, less than 0.2 nM, or less than 0.1 nM D To bind to the mouse ACVR1 extracellular domain fused to mFc at 37°C; (f) Binding to cells expressing human ACVR1 protein or human ACVR(R206H) protein; (g) IC values ​​less than 10 nM, less than 5 nM, less than 3 nM, less than 2 nM, or less than 1 nM when measured in a cell-based bioassay. 50 This involves inhibiting the activation of cells expressing human ACVR1(R206H) by human activin A; (h) ICs less than 10 nM, less than 5 nM, less than 3 nM, less than 2 nM, or less than 1 nM when measured in a cell-based bioassay 50 This involves inhibiting the activation of cells expressing human ACVR1(R206H) by human BMP7; and (i) The HCVR includes an amino acid sequence selected from the group consisting of HCVR sequences listed in Table 1, and the LCVR includes an amino acid sequence selected from the group consisting of LCVR sequences listed in Table 1. The present invention provides an isolated antibody or its antigen-binding fragment having one or more of the following characteristics.

[0055] In a second aspect, the present invention provides nucleic acid molecules encoding an anti-ACVR1 antibody or a portion thereof. For example, the present invention provides nucleic acid molecules encoding any of the HCVR amino acid sequences listed in Table 1; in a particular embodiment, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in Table 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0056] The present invention also provides nucleic acid molecules encoding any of the LCVR amino acid sequences listed in Table 1; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCVR nucleic acid sequences listed in Table 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0057] The present invention also provides nucleic acid molecules encoding any of the HCDR1 amino acid sequences listed in Table 1; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR1 nucleic acid sequences listed in Table 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0058] The present invention also provides nucleic acid molecules encoding any of the HCDR2 amino acid sequences listed in Table 1; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR2 nucleic acid sequences listed in Table 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0059] The present invention also provides nucleic acid molecules encoding any of the HCDR3 amino acid sequences listed in Table 1; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR3 nucleic acid sequences listed in Table 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0060] The present invention also provides nucleic acid molecules encoding any of the LCDR1 amino acid sequences listed in Table 1; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR1 nucleic acid sequences listed in Table 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0061] The present invention also provides nucleic acid molecules encoding any of the LCDR2 amino acid sequences listed in Table 1; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR2 nucleic acid sequences listed in Table 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0062] The present invention also provides nucleic acid molecules encoding any of the LCDR3 amino acid sequences listed in Table 1; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR3 nucleic acid sequences listed in Table 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0063] The present invention also provides a nucleic acid molecule encoding HCVR, wherein HCVR comprises three CDR sets (i.e., HCDR1-HCDR2-HCDR3), where the HCDR1-HCDR2-HCDR3 amino acid sequence set is defined by one of the exemplary antibodies listed in Table 1.

[0064] The present invention also provides a nucleic acid molecule encoding LCVR, wherein LCVR comprises three CDR sets (i.e., LCDR1-LCDR2-LCDR3), where the LCDR1-LCDR2-LCDR3 amino acid sequence set is defined by one of the exemplary antibodies listed in Table 1.

[0065] The present invention also provides nucleic acid molecules encoding both HCVR and LCVR, where HCVR comprises an amino acid sequence of any of the HCVR amino acid sequences listed in Table 1, and where LCVR comprises an amino acid sequence of any of the LCVR amino acid sequences listed in Table 1. In certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in Table 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto, and a polynucleotide sequence selected from any of the LCVR nucleic acid sequences listed in Table 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments of this aspect of the present invention, the nucleic acid molecule encodes both HCVR and LCVR, where both HCVR and LCVR are derived from the same anti-ACVR1 antibody listed in Table 1.

[0066] In related embodiments, the present invention provides recombinant expression vectors capable of expressing polypeptides comprising the weight and / or light chain variable regions of an antibody. For example, the present invention includes recombinant expression vectors comprising any of the nucleic acid molecules described above, i.e., nucleic acid molecules encoding any of the HCVR, LCVR, and / or CDR sequences listed in Table 2. In certain embodiments, the present invention provides expression vectors comprising (a) a nucleic acid molecule comprising a nucleic acid sequence encoding the HCVR of an antibody conjugating ACVR1 comprising an amino acid sequence selected from the group consisting of sequences listed in Table 1; and / or (b) a nucleic acid molecule comprising a nucleic acid sequence encoding the LCVR of an antibody conjugating ACVR1 comprising an amino acid sequence selected from the group consisting of sequences listed in Table 1. Also within the scope of the present invention are host cells into which such vectors have been introduced, and methods for producing an antibody or a portion thereof by culturing the host cells under conditions that enable the production of an antibody or antibody fragment, and recovering the antibody and antibody fragment thus produced. In certain embodiments, the host cells include mammalian cells or prokaryotic cells. In certain embodiments, the host cells are Chinese hamster ovary (CHO) cells or Escherichia coli (E. coli) cells. In certain embodiments, the present invention provides a method for producing the antibody or its antigen-binding fragment, the method comprising: introducing an expression vector into host cells containing a nucleic acid sequence encoding the HCVR and / or LCVR of the antibody or its antigen-binding fragment, operably linked to a promoter; culturing the host cells under conditions suitable for the expression of the nucleic acid sequence; and isolating the antibody or its antigen-binding fragment from the culture medium and / or host cells. The isolated antibody or its antigen-binding fragment is purified using any of the methods known in the prior art.

[0067] In a third aspect, the present invention provides a pharmaceutical composition comprising at least one recombinant monoclonal antibody or its antigen-binding fragment that specifically binds a therapeutically effective amount of ACVR1, and a pharmaceutically acceptable carrier. In a related aspect, the present invention features a composition which is a combination of an anti-ACVR1 antibody and a second therapeutic agent. In one embodiment, the second therapeutic agent is any agent which is suitably combined with the anti-ACVR1 antibody.

[0068] Examples of agents suitably combined with the anti-ACVR1 antibody include, but are not limited to, other agents that conjugate and / or activate ACVR1 activity (including other antibodies or their antigen-binding fragments) and / or agents that do not directly conjugate ACVR1 but nevertheless treat or restore at least one symptom or sign of ACVR1-related disease or disorder (disclosed elsewhere herein). Further combination therapies and co-formulations comprising the anti-ACVR1 antibody of the present invention are disclosed elsewhere herein.

[0069] In a fourth aspect, the present invention provides a therapeutic method for treating a disease or disorder associated with ACVR1 in a subject using the anti-ACVR1 antibody or antigen-binding portion of the antibody of the present invention, wherein the therapeutic method comprises administering a pharmaceutical composition comprising a therapeutically effective amount of the antibody or antigen-binding fragment of the antibody to a subject in need of treatment. The disorder to be treated is any disease or condition (e.g., anemia, ectopic ossification, dysplasia, or diffuse pontine glioma) that is improved, restored, inhibited, or prevented by enhancement of ACVR1 activity. In certain embodiments, the present invention provides a method for preventing or treating an ACVR1-related disease or disorder, comprising administering a therapeutically effective amount of the anti-ACVR1 antibody or antigen-binding fragment of the present invention to a subject in need of treatment. In some embodiments, the antibody or antigen-binding fragment is administered prophylactically or therapeutically to a subject having or at risk of having an ACVR1-related disease or disorder. In certain embodiments, the antibody or antigen-binding fragment of the present invention is administered to a subject in need of treatment in combination with a second therapeutic agent.

[0070] The second therapeutic agent is selected from the group consisting of anti-activin A antibodies or their antigen-binding fragments, anti-BMP7 antibodies or their antigen-binding fragments, anti-ACVR2 antibodies or their antigen-binding fragments, anti-inflammatory drugs, steroids, bisphosphonates, muscle relaxants, or retinoic acid receptor (RAR) gamma agonists, lifestyle modifications, nutritional supplements, and any other drugs or therapies known in the art. In certain embodiments, the second therapeutic agent is a drug that helps to counteract or reduce any possible side effects associated with the antibody or its antigen-binding fragment of the present invention, if such side effects occur. The antibody or its fragment is administered subcutaneously, intravenously, intradermally, intraperitoneally, or orally, intramuscularly, or intraventricularly. The antibody or its fragment is administered in doses of about 0.1 mg / kg to about 100 mg / kg of the subject's body weight. In certain embodiments, the antibody of the present invention is administered in one or more doses containing 10 mg to 600 mg.

[0071] The present invention also includes the use of the anti-ACVR1 antibody or its antigen-binding fragment in the manufacture of a pharmaceutical for the treatment of a disease or disorder for which the activation of ACVR1 binding and / or activity is beneficial.

[0072] Other embodiments will become apparent from the detailed description that follows. [Brief explanation of the drawing]

[0073] [Figure 1A] This matrix shows the results of an antibody cross-competition assay in which a first anti-ACVR1 antibody was applied to hACVR1.mmh captured on a biosensor chip coated with His antibody, followed by immersion in a solution of the second anti-ACVR1 antibody (50 μg / mL). Binding responses indicated by white boxes show no competition for hACVR1 binding, suggesting separate binding regions. [Figure 1B]The results are shown in Figure 1A, which is a schematic diagram of the antibody cross-competition assay format. The first anti-ACVR1 antibody is applied to hACVR1.mmh captured on a biosensor chip coated with His antibody, and then immersed in a solution of the second anti-ACVR1 antibody. [Figure 2] This figure shows bar graphs of total ectopic bone mass measured by micro-CT at 5 and 9 weeks after trauma in C57BL / 6 mice conjugated with either the anti-ACVR1 antibody REGN 5168 or the isotype control antibody REGN 1945. The anti-ACVR1 antibody REGN 5168 significantly reduced post-traumatic ectopic ossification (HO) compared to the isotype control. [Figure 3] This figure shows a bar graph of serum hepcidin levels in prophylactic drug administration studies conducted in C57BL / 6 mice conferred with either the anti-ACVR1 antibody REGN 5168 or the isotype control antibody REGN 1945. Serum hepcidin levels were significantly reduced in mice conferred with the anti-ACVR1 antibody REGN 5168 compared to the control group. [Figure 4] This figure shows bar graphs of total ectopic bone mass measured by micro-CT at 3, 6, 9, and 12 weeks after trauma in No MAHA transgenic mice conjugated with anti-ACVR1 antibodies REGN 5166, REGN 5168, or the isotype control antibody REGN 1945. Anti-ACVR1 antibodies REGN 5166 and REGN 5168 significantly reduced post-traumatic ectopic ossification (HO) compared to the control, respectively. [Figure 5] This figure shows a bar graph of serum iron levels in prophylactic drug studies conducted in No MAHA transgenic mice conferred with anti-ACVR1 antibodies REGN 5166 or REGN 5168, or isotype control antibody REGN 1945. Serum iron levels were significantly increased in mice conferred with anti-ACVR1 antibody REGN 5168 compared to isotype controls. [Figure 6]This figure shows bar graphs of total ectopic bone mass measured by micro-CT at 3, 6, and 9 weeks after trauma in C57BL / 6 mice conferred with either the anti-ACVR1 antibody REGN 5168 or the isotype control antibody REGN 1945 in a delayed-dose study in which treatment was initiated 3 weeks after trauma. The anti-ACVR1 antibody REGN 5168 significantly reduced post-traumatic ectopic ossification (HO) at 6 and 9 weeks after trauma compared to the isotype control. [Figure 7] This figure shows bar graphs of total ectopic bone volume measured by micro-CT in No MAHA transgenic mice after HO resection surgery 7 weeks after trauma in an Achilles tendon rupture and burn tHO mouse model, and after the start of treatment. The arrows indicate HO resection surgery and the start of treatment at week 7. Inhibition of ACVR1 with the neutralizing antibody REGN 5168 significantly inhibited HO recurrence at weeks 12, 15, and 18 post-resection in No MAHA mice compared to mice treated with isotype controls. [Modes for carrying out the invention]

[0074] Before describing the methods of the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, and therefore such methods and conditions are diverse. Furthermore, since the scope of the present invention is limited only by the accompanying claims, it should be understood that the terms used herein are intended to describe only specific embodiments and are not intended to be restrictive.

[0075] Unless otherwise noted, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art in which the present invention pertains. Any methods and materials similar to or equivalent to those described herein may be used in the practice or experimentation of the present invention, but preferred methods and materials are described herein. All publications, patents, and patent applications referenced herein are incorporated herein by reference in their entirety.

[0076] definition ACVR1, also known as ALK2, refers to the activin A receptor type 1 (also known as activin-like kinase 2). ACVR1 is a single-pass type I membrane protein. The full-length amino acid sequence of human ACVR1 is available by referring to UniProtKB accession number Q04771 (SEQ ID NO: 61) as having 509aa residues. The protein has an extracellular domain at amino acid residues 21-123, a transmembrane domain at amino acid positions 124-146, and a cytoplasmic domain at positions 147-509. Upon ligand binding, ACVR1 forms a receptor complex consisting of two type II and two type I transmembrane serine / threonine kinases. The type II receptor phosphorylates and activates the type I receptor. After autophosphorylation, the type I receptor binds and activates SMAD transcription factors. ACVR1 is the receptor for activin.

[0077] The amino acid sequence of the full-length human ACVR1 protein is exemplified by the amino acid sequence provided as accession number Q04771 (sequence number 61) in UniProtKB / Swiss-Prot. The full-length amino acid sequence of the mouse ACVR1 protein is available by referring to accession number P37172 (sequence number 62).

[0078] The term "ACVR1" encompasses recombinant ACVR1 protein or fragments thereof. The term also encompasses ACVR1 protein or fragments coupled to, for example, a histidine tag, a PADRE tag, mouse or human Fc, or a signal sequence (e.g., SEQ ID NOs. 63-65).

[0079] The term "ACVR1" can include an ACVR1 protein containing a mutation or a fragment thereof. For example, the mutation can be based on the corresponding amino acid sequence of human ACVR1 UniProtKB accession number Q04771 (SEQ ID NO: 61) or a fragment thereof. For example, the ACVR1 protein or a fragment thereof can include mutations including, but not limited to, L196P, delP197_F198insL, R202I, R206H, Q207E, R258S, R258G, G325A, G328E, G328R, G328W, G356D, and R375P of SEQ ID NO: 61.

[0080] As used herein, the term "antibody" is intended to refer to an immunoglobulin molecule (i.e., a "complete antibody molecule") composed of four polypeptide chains of two heavy (H) chains and two light (L) chains covalently linked by disulfide bonds, as well as multimers thereof (e.g., IgM) or antigen-binding fragments thereof. Each heavy chain is composed of a heavy chain variable region ("HCVR" or "V H ") and a heavy chain constant region (domains C H 1, C H 2 and C H 3). Each light chain is composed of a light chain variable region ("LCVR" or "V L ") and a light chain constant region (C L ). The V H and V L regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). The V H and V LEach consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In certain embodiments of the present invention, the FRs of the antibody (or its antigen-binding fragment) are either identical to the human germline sequence or are naturally or artificially modified. The amino acid consensus sequence is defined based on a side-by-side analysis of two or more CDRs.

[0081] Substitution of one or more CDR residues or omission of one or more CDRs is also possible. Antibodies that can omit one or two CDRs for binding have been documented in the scientific literature. Padlan et al. (1995 FASEB J.9:133-139) analyzed the contact region between antibodies and their antigens based on publicly available crystal structures and concluded that only about one-fifth to one-third of the CDR residues actually contact the antigen. Padlan also found many antibodies that do not have amino acids in which one or two CDRs contact the antigen (see also Vajdos et al. 2002 J Mol Biol 320:415-428).

[0082] CDR residues that do not come into contact with the antigen can be identified by molecular modeling and / or empirically from regions of Kabat CDRs unrelated to Chothia CDRs, based on previous studies (e.g., residues H60-H65 in CDRH2 are often unnecessary). If a CDR or its residue(s) is omitted, it is usually replaced by an amino acid occupying the corresponding position in another human antibody sequence or a consensus of such a sequence. The position of the substitution within the CDR and the amino acid to be substituted can also be selected empirically. Empirical substitutions may be conserved or non-conserved.

[0083] The fully human anti-ACVR1 monoclonal antibodies disclosed herein may contain one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR region of the heavy and light chain variable domains when compared to the corresponding germline sequence. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein with germline sequences available, for example, from publicly available antibody sequence databases. The present invention comprises antibodies derived from any of the amino acid sequences disclosed herein, and antigen-binding fragments thereof, wherein one or more amino acids in one or more frameworks and / or CDR regions are mutated to a corresponding residue(or more) in the germline sequence from which the antibody is derived, or to a corresponding residue(or more) in another human germline sequence, or to a conserved amino acid substitution of a corresponding germline residue(or more) (such sequence changes are collectively referred to herein as “germline mutations”). Those skilled in the art can readily produce a number of antibodies and antigen-binding fragments containing one or more individual germline mutations or combinations thereof starting from the heavy and light chain variable domain sequences disclosed herein. In certain embodiments, V H and / or V LAll of the framework and / or CDR residues within the domain are mutated back to residues found in the original germline sequence from which the antibody originates. In other embodiments, only certain residues are mutated back to the original germline sequence, for example, only mutated residues found within the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or only mutated residues found within CDR1, CDR2, or CDR3. In other embodiments, one or more of the framework and / or CDR residues are mutated to the corresponding residue(s) in a different germline sequence (i.e., a germline sequence different from the germline sequence from which the antibody originally originates). Furthermore, the antibody of the present invention may contain any combination of two or more germline mutations within the framework and / or CDR region, for example, where certain individual residues are mutated to corresponding residues in a particular germline sequence, while certain other residues different from the original germline sequence are maintained or mutated to corresponding residues in a different germline sequence. Antibodies and antigen-binding fragments containing one or more germline mutations, once obtained, can be readily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonistic biological properties, or reduced immunogenicity. Antibodies and antigen-binding fragments obtained in this general manner are encompassed within the present invention.

[0084] The present invention also includes a fully human anti-ACVR1 monoclonal antibody comprising a variant of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, the present invention includes an anti-ACVR1 antibody having an HCVR, LCVR, and / or CDR amino acid sequence having, for example, 10 or fewer, 8 or fewer, 6 or fewer, or 4 or fewer conservative amino acid substitutions to any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein.

[0085] The terms “human antibody” or “fully human antibody,” as used herein, are intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human mAbs of the present invention may include, for example, amino acid residues not encoded by human germline immunoglobulin sequences in the CDR, particularly CDR3 (e.g., mutations introduced by random or site-directed mutagenesis in vitro, or somatic mutations in vivo). However, the terms “human antibody” or “fully human antibody,” as used herein, are not intended to include mAbs in which a germline-derived CDR sequence from another mammalian species (e.g., mouse) is grafted onto a human FR sequence. The terms encompass antibodies recombinantly produced in or in non-human mammals. The terms are not intended to include antibodies isolated from or generated in human subjects.

[0086] As used herein, the term "recombinant" refers to an antibody or antigen-binding fragment of the present invention created, expressed, isolated, or obtained by a technique or method known in the art, such as recombinant DNA technology including DNA splicing and transgenic expression. The term also refers to an antibody expressed in a non-human mammal (including transgenic non-human mammals, e.g., transgenic mice) or cell (e.g., CHO cells) expression system, or isolated from a recombinant combinatorial human antibody library.

[0087] Terms such as "specifically bind to" or "specifically bind to" mean that the antibody or its antigen-binding fragment forms a complex with an antigen that is relatively stable under physiological conditions. Specific binding is at least about 1 × 10⁻⁶. -8 It can be characterized by an equilibrium dissociation constant of M or less (for example, a smaller K). D(This exhibits a stronger binding.) Methods for determining whether two molecules specifically bind are well known in the art, including, for example, equilibrium dialysis and surface plasmon resonance. As described herein, surface plasmon resonance, for example BIACORE®, has been used to identify antibodies that specifically bind to ACVR1. Furthermore, polyspecific antibodies that bind to one domain in ACVR1 and one or more further antigens, or bispecific antibodies that bind to two different regions of ACVR1, are also considered "specifically binding" antibodies as used herein.

[0088] The term "high affinity" antibody is defined as having at least 10 when measured by surface plasmon resonance, e.g., BIACORE® or solution affinity ELISA. -8 M; preferably 10 -9 M;comfort10 -10 M, more preferably 10 -11 M's K D This refers to an mAb that has binding affinity to ACVR1, which is represented as follows.

[0089] The terms "slow off rate," "Koff," or "kd" refer to a 1 × 10⁻¹⁰ saturation rate, as determined by surface plasmon resonance, e.g., BIACORE®. -3 s -1 Or less, preferably 1 × 10 -4 s -1 This refers to an antibody that dissociates from ACVR1 with a rate constant of or less than that.

[0090] The terms “antigen-binding portion” and “antigen-binding fragment” of an antibody, as used herein, include any naturally occurring, enzymatically obtainable, synthesized, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. The terms “antigen-binding fragment” or “antibody fragment” of an antibody, as used herein, refer to one or more fragments of an antibody that retain the ability to bind to the ACVR1 protein.

[0091] In certain embodiments, the antibody or antibody fragment of the present invention is conjugated to a ligand or therapeutic moiety ("immunoconjugate"), a second anti-ACVR1 antibody, or any other therapeutic moiety useful for treating an ACVR1-related disease or disorder.

[0092] As used herein, "isolated antibody" is intended to mean an antibody that substantially does not contain other antibodies (Abs) with different antigen specificities (for example, an isolated antibody that specifically binds ACVR1 or a fragment thereof substantially does not contain Abs that specifically bind antigens other than ACVR1).

[0093] When used herein, "inactivating antibody" or "antagonist antibody" (or "antibody that reduces or inhibits ACVR1 activity" or "antibody that destabilizes the activating conformation") is intended to refer to an antibody whose binding to ACVR1 results in the inactivation of at least one biological activity of ACVR1. For example, the antibodies of the present invention may reduce anemia when administered to subjects in need of treatment.

[0094] As used herein, the term "surface plasmon resonance" refers to an optical phenomenon that enables real-time analysis of biomolecular interactions by detecting changes in protein concentration within a biosensor matrix, for example, using the BIACORE® system (Pharmacia Biosensor AB, Uppsala, Sweden and Piskataway, NJ).

[0095] "K D When used herein, the term "equilibrium dissociation constant" is intended to refer to the equilibrium dissociation constant of a particular antibody-antigen interaction.

[0096] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site in a variable region of an antibody molecule, known as a paratope. A single antigen can have more than one epitope. Therefore, different antibodies can bind to different regions on an antigen and may have different biological effects. The term "epitope" also refers to a site on an antigen to which B and / or T cells respond. The term "epitope" also refers to a region of an antigen to which an antibody binds. Epitopes are defined as structural or functional. Functional epitopes are generally a subset of structural epitopes and have residues that directly contribute to the affinity of the interaction. Epitopes are also three-dimensional, i.e., composed of non-linear amino acids. In certain embodiments, epitopes may include determinants that are chemically active surface groups of molecules, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in certain embodiments, may have specific three-dimensional structural features and / or specific charge features.

[0097] The term "cross-competition," as used herein, means that an antibody or its antigen-binding fragment binds to an antigen and inhibits or blocks the binding of another antibody or its antigen-binding fragment. The term also encompasses competition between two antibodies in both orientations, i.e., a first antibody binding to a second antibody and blocking its binding, and vice versa. In certain embodiments, the first and second antibodies may bind to the same epitope. Alternatively, the first and second antibodies may bind to different but overlapping epitopes such that the binding of one antibody inhibits or blocks the binding of the second antibody, for example, via steric hindrance. Cross-competition between antibodies is measured by methods known in the art, for example, by a real-time label-free biolayer interference assay. Cross-competition between two antibodies is represented as the binding of the second antibody being smaller than the background signal resulting from self-self binding (where the first and second antibodies are the same antibody). Cross-competition between the two antibodies is expressed, for example, as the binding percentage of the second antibody being smaller than the baseline self-background binding (where the first and second antibodies are the same antibody).

[0098] The terms “substantially identical” or “substantially identical” refer to nucleic acids or fragments thereof, as discussed below, and indicate that nucleotide sequence identity exists in at least about 90%, more preferably at least about 95%, 96%, 97%, 98%, or 99% of the nucleotide bases when optimally aligned with another nucleic acid (or its complementary strand) using appropriate nucleotide insertions or deletions, as measured by any well-known algorithm of sequence identity such as FASTA, BLAST, or GAP. A nucleic acid molecule having substantial identity with respect to a reference nucleic acid molecule may, in certain cases, encode a polypeptide that is identical to, or substantially similar to, the polypeptide encoded by the reference nucleic acid molecule.

[0099] When applied to polypeptides, the terms “substantial similarity” or “substantially identical” mean that two peptide sequences share at least 90% sequence identity, more preferably at least 95%, 98%, or 99% sequence identity, when optimally aligned by a programmed GAP or BESTFIT using default gap weights. Preferably, non-identical residue positions are distinguished by conserved amino acid substitutions. A “conservative amino acid substitution” is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functional properties of the protein. If two or more amino acid sequences differ from each other by conservative substitutions, the percentage or degree of similarity is adjusted up to compensate for the conservative nature of the substitutions. Means for making this adjustment are well known to those skilled in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24: pp. 307–331, incorporated herein by reference. Examples of amino acid groups with side chains having similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conserved amino acid substituents are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood matrix, as disclosed by Gonnet et al. (1992) Science 256:1443, p. 45. A "moderately conservative" substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0100] Sequence similarity of polypeptides is typically measured using sequence analysis software. Protein analysis software matches similar sequences using similarity measurements assigned to various substitutions, deletions, and other modifications, including conserved amino acid substitutions. For example, GCG software includes programs such as GAP and BESTFIT, which, when used with default parameters, can determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species, or between wild-type proteins and their mutaines. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using FASTA, a program in GCG version 6.1, with default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignment and sequence identity percentage of the best overlap region between the query sequence and the search sequence (Pearson (2000) above). Another preferred algorithm for comparing the sequences of the present invention with a database containing numerous sequences from different organisms is the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. See, for example, Altschul et al. (1990) J.Mol.Biol.215:403-410 and (1997) Nucleic Acids Res.25:3389-3402. These are incorporated herein by reference, respectively.

[0101] The phrase "therapeutably effective dose" refers to the amount administered that produces the desired effect. The exact amount depends on the purpose of the treatment and can be determined by those skilled in the art using known techniques (see, for example, Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

[0102] As used herein, the term “Subject” means an animal, preferably a mammal, more preferably a human, that requires recovery, prevention, and / or treatment of ACVR1-related disease or disorder, such as anemia or heterotopic ossification. The term includes human subjects that have or are at risk of having such disease or disorder.

[0103] As used herein, the terms “to treat,” “to treat,” or “to treat” mean a reduction or recovery of the severity of at least one symptom or sign of ACVR1-related disease or disorder resulting from the administration of a therapeutic agent, such as the antibody of the present invention, to a subject in need of treatment. The terms include inhibition of disease progression or worsening of symptoms / signs. The terms also include a prospective prognosis of the disease, i.e., the subject may be disease-free or disease-reduced upon administration of a therapeutic agent, such as the antibody of the present invention. The therapeutic agent is administered to the subject in a therapeutic dose.

[0104] The terms “prevent,” “prevent,” or “prevent” refer to the inhibition of findings of ACVR1-related disease or disorder, or any symptoms or signs of such disease or disorder, upon administration of the antibody of the present invention.

[0105] The term "heterotopic ossification" (HO) refers to the formation of benign, mature bone elements in extraosseous areas, including soft tissues and joints.

[0106] Antigen-binding fragments of antibodies Unless otherwise specified, the term “antibody” as used herein is understood to encompass an antibody molecule containing two immunoglobulin heavy chains and two immunoglobulin light chains (i.e., a “complete antibody molecule”) and its antigen-binding fragment. Terms such as “antigen-binding portion” of an antibody, “antigen-binding fragment” of an antibody, as used herein, include any naturally occurring, enzymatically obtainable, synthesized, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen and forms a complex. The terms “antigen-binding fragment” or “antibody fragment” as used herein refer to one or more fragments of an antibody that possess the ability to specifically bind to the ACVR1 protein, its fragments, and / or their mutants. Antibody fragments may include Fab fragments, F(ab')2 fragments, Fv fragments, dAb fragments, fragments containing CDRs, or isolated CDRs. In certain embodiments, the term “antigen-binding fragment” refers to a polypeptide fragment of a multispecific antigen-binding molecule. Antibody antigen-binding fragments are obtained from a complete antibody molecule using any suitable standard technique, such as, for example, proteolytic digestion or recombinant gene manipulation techniques, including the manipulation and expression of DNA encoding antibody variable and (optionally) constant domains. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. The DNA can be sequenced and manipulated using chemical or molecular biological techniques to, for example, position one or more variable and / or constant domains in appropriate positions, or to modify, add, or delete amino acids by introducing codons, for example, to create cysteine ​​residues.

[0107] Non-limiting examples of antigen-binding fragments include (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv(scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues that mimic the hypervariable region of an antibody (e.g., isolated complementarity-determining regions (CDRs) such as the CDR3 peptide), or restrictive FR3-CDR3-FR4 peptides. Domain-specific antibodies, single-domain antibodies, domain deletion antibodies, chimeric antibodies, CDR graft antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and other manipulated molecules such as shark variable IgNAR domains are also included within the expression “antigen-binding fragment” as used herein.

[0108] Antigen-binding fragments of antibodies typically contain at least one variable domain. The variable domain can have any size or amino acid composition and generally contains at least one CDR adjacent to or in-frame with one or more framework sequences. L Domain and associated V H In an antigen-binding fragment having a domain, V H and V L Domains are positioned relative to each other in any appropriate arrangement. For example, a variable region may be a dimer, V H -V H , V H -V L or V L -V L It may contain dimers. Alternatively, the antigen-binding fragment of the antibody may contain monomer V H or V L It can contain a domain.

[0109] In certain embodiments, the antigen-binding fragment of the antibody may contain at least one variable domain covalently bound to at least one constant domain. Non-limiting and exemplary arrangements of variable and constant domains found within the antigen-binding fragment of the antibody of the present invention are shown below: (i)V H -C H 1;(ii)V H -C H 2; (iii)V H -C H 3;(iv)V H -C H 1-C H 2;(v)V H -C H 1-C H 2-C H 3;(vi)V H -C H 2-C H 3;(vii)V H -C L ;(viii)V L -C H 1;(ix)V L -C H 2;(x)V L -CH3;(xi)V L -C H 1-C H 2;(xii)V L -C H 1-C H 2-C H 3;(xiii)V L -CH2-CH3; and (xiv)V L -C LExamples include: In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains are linked directly to each other or by a complete or partial hinge region or linker region. The hinge region consists of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, which result in a flexible or semi-flexible linkage between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, the antigen-binding fragment of the antibody of the present invention has homodimers or heterodimers (or other macromers) of any of the configurations of variable and constant domains listed above, linked non-covalently to each other and / or one or more monomers V H or V L It can be included along with the domain (for example, by disulfide bonds).

[0110] Similar to complete antibody molecules, antigen-binding fragments are monospecific or polyspecific (e.g., bispecific). A polyspecific antigen-binding fragment of an antibody typically comprises at least two distinct variable domains, each capable of specifically binding to a different antigen or to a different epitope on the same antigen. Any polyspecific antibody form, including the exemplary bispecific antibody forms disclosed herein, can be adapted for use in the context of the antibody antigen-binding fragments of the present invention using routine techniques available in the art.

[0111] Manufacturing of human antibodies Methods for generating human antibodies in transgenic mice are known in the art. Any such known method can be used in the context of the present invention to produce human antibodies that specifically bind to ACVR1.

[0112] Human antibodies against ACVR1 can be isolated from full-length human IgG synthetic naive libraries using an in vitro yeast selection system and related methods (Rouha H, et al. MAbs. 2015; 7(1): pp. 243-254). As previously described, approximately 1 × 10⁶ antibodies in the diversity. 10 The antibody libraries are designed and propagated (Rouha et al. 2015, Xu et al., Protein Eng Des Sel. 2013; 26(10): pp. 663-670). ACVR1-conjugated antibodies are enriched by incubating biotinylated ACVR1-Fc and Myc-His monomers ACVR1 at various concentrations together with antibody-expressing yeast cells, followed by several consecutive selection rounds, for example, by magnetic bead selection using fluorescent streptavidin or extravidin secondary reagents (Miltenyi Biotec) or flow cytometry on a FACSAria II cell sorter (BD Biosciences). Antibodies cross-reactive to off-target proteins ALK1, ALK3, and ALK6 are actively depleted from the selection output. After the final round of enrichment, the yeast cells can be seeded on agar plates, analyzed by DNA sequencing, and grown for IgG production. The heavy chains from the naive output are used to produce a light chain diversification library, which is then used for further selection rounds. In particular, heavy chains are extracted from the naive selection round output, for example, 1 × 10⁶. 6 By converting it into a light chain library consisting of individual unique light chains, for example, approximately 1 × 10⁶ in total diversity, 8 Numerous new libraries can be created. Antibody optimization is completed in three phases. Heavy chain optimization via diversification of complementarity-determining regions (CDRs) HCDR1 and HCDR2 is performed either by mutagenic PCR-based diversification of the entire heavy chain variable region or by diversification of the light chain LCDR1 and LCDR2 segments. The HCDR1 and HCDR2 regions are, for example, approximately 1 × 10⁶. 8Lead variants are diversified using a pre-existing library of HCDR1 and HCDR2 variants with individual diversities. The lead variants are further diversified through diversification of the DNA oligonucleotide sequences of HCDR3 or LCDR3. The diversified antibody populations are selected for enhanced binding to the target protein while avoiding undesirable cross-reactivity. The methods used for selection in the diversified populations may be similar to, or identical to, the methods used to isolate the original lead IgG (Xu et al., 2013).

[0113] Alternatively, antibodies against the ACVR1 protein can be generated using an immunogen comprising any one of the following: In certain embodiments, the antibodies of the present invention are obtained from mice immunized with the full-length native ACVR1 protein (e.g., UniProtKB / Swiss-Prot accession number Q04771) (SEQ ID NO: 61), or with DNA encoding the protein or a fragment thereof. Alternatively, the protein or a fragment thereof may be produced and modified using standard biochemical techniques and used as an immunogen.

[0114] In some embodiments, the immunogen is a recombinant ACVR1 protein or a fragment thereof expressed in E. coli or in any other eukaryotic or mammalian cell such as Chinese hamster ovary (CHO) cells (e.g., SEQ ID NOs. 63-65).

[0115] High-affinity chimeric antibodies against ACVR1, possessing human variable regions and mouse constant regions, are initially isolated using VELOCIMMUNE® technology (see, for example, U.S. Patent No. 6,596,541, Regeneron Pharmaceuticals, VELOCIMMUNE®) or any other known method for generating monoclonal antibodies. VELOCIMMUNE® technology is involved in generating transgenic mice having a genome containing human heavy and light chain variable regions operably ligated to an endogenous mouse constant region locus, so that the mice produce antibodies containing human variable regions and mouse constant regions in response to antigen stimulation. The DNA encoding the weight and light chain variable regions of the antibody is isolated and operably ligated to the DNA encoding the human heavy and light chain constant regions. The DNA is then expressed in cells capable of expressing fully human antibodies.

[0116] Generally, VELOCIMMUNE® mice are loaded with the target antigen, and lymphoid cells (such as B cells) are recovered from the antibody-expressing mice. These lymphoid cells are fused with myeloma cell lines to produce immortal hybridoma cell lines, which are then screened and selected to identify hybridoma cell lines that produce antibodies specific to the target antigen. DNA encoding the variable regions of the heavy and light chains is isolated and ligated to the desired isotype constant regions of the heavy and light chains. Such antibody proteins are produced in cells such as CHO cells. Alternatively, antigen-specific chimeric antibodies or DNA encoding the variable domains of the light and heavy chains are isolated directly from antigen-specific lymphocytes.

[0117] First, a high-affinity chimeric antibody having a human variable region and a mouse constant region is isolated. As seen in the experimental sections below, the antibody is characterized and selected for desirable properties, including affinity, selectivity, and epitope. The mouse constant region is replaced with the desired human constant region to produce the fully human antibody of the present invention, e.g., wild-type or modified IgG1 or IgG4. The selected constant region may vary depending on the specific use, while the high-affinity antigen-binding and target specificity features reside in the variable region.

[0118] biological equivalent The anti-ACVR1 antibodies and antibody fragments of the present invention include proteins having amino acid sequences that are modified from the amino acid sequence of the described antibody, but that retain the ability to bind to the ACVR1 protein. Such variant antibodies and antibody fragments include the addition, deletion, or substitution of one or more amino acids compared to the parent sequence, but exhibit biological activity that is essentially equivalent to the biological activity of the described antibody. Similarly, the antibody-coding DNA sequences of the present invention include sequences that encode antibodies or antibody fragments that are essentially biologically equivalent to the antibodies or antibody fragments of the present invention, but include the addition, deletion, or substitution of one or more nucleotides compared to the disclosed sequence.

[0119] Two antigen-binding proteins, or antibodies, are considered bioequivalent or substitutes if, for example, they are administered under similar experimental conditions at the same molar dose, either as a single or multiple dose, and their absorption rates and extents do not show a significant difference. Some antibodies are considered equivalent or substitutes if their absorption rates are equivalent, but their absorption extents are not. However, they are considered bioequivalent if such a difference in absorption rates is intentional, reflected in the labeling, and is not considered medically important to the specific drug studied, for example, for achieving effective drug concentrations in the body with respect to chronic use.

[0120] In one embodiment, two antigen-binding proteins are bioequivalent if there is no clinically significant difference between them in terms of safety, purity, or potency.

[0121] In one embodiment, the two antigen-binding proteins are bioequivalent if the patient can switch between the reference product and the biological product once or more, without expecting an increased risk of adverse effects, including clinically significant changes in immunogenicity or decreased efficacy, compared to continuous therapy without switching between the reference product and the biological product.

[0122] In one embodiment, two antigen-binding proteins are physically equivalent insofar as such mechanisms are known, provided that they both act by a common mechanism of action (one or more) under the conditions (one or more) of use.

[0123] Bioequivalence is demonstrated by in vivo and / or in vitro methods. Measurements of bioequivalence include, for example, (a) in vivo tests in humans or other mammals in which the concentration of the antibody or its metabolites is measured as a function of time in blood, plasma, serum, or other biological fluids; (b) in vitro tests that correlate with and reasonably predict human in vivo bioavailability data; (c) in vivo tests in humans or other mammals in which the appropriate acute pharmacological effect of the antibody (or its target) is measured as a function of time; and (d) well-controlled clinical trials to establish the safety, efficacy, or bioavailability or bioequivalence of the antibody.

[0124] Bioequivalent variants of the antibody of the present invention are constructed, for example, by making various substitutions of residues or sequences, or by deleting terminal or internal residues or sequences that are not required for biological activity. For example, cysteine ​​residues that are not essential for biological activity can be deleted to prevent the formation of unnecessary or inaccurate intramolecular disulfide crosslinks during regeneration, or they can be replaced with other amino acids. In other circumstances, bioequivalent antibodies may include antibody variants that involve amino acid changes that modify the glycosylation characteristics of the antibody, such as mutations that eliminate or remove glycosylation.

[0125] Anti-ACVR1 antibody containing Fc variant According to a particular embodiment of the present invention, for example, an anti-ACVR1 antibody is provided comprising an Fc domain containing one or more mutations that enhance or reduce antibody binding to the FcRn receptor at acidic pH compared to neutral pH. For example, the present invention provides an Fc domain containing C H 2 or C HThe antibody contains an anti-ACVR1 antibody with mutations in three regions, where the mutation(s) increase the affinity of the Fc domain to FcRn in an acidic environment (e.g., in endosomes with a pH in the range of approximately 5.5 to 6.0). Such mutations may result in an increased serum half-life of the antibody when administered to animals. Non-restrictive examples of such Fc modifications include, for example, modifications at position 250 (e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., L / Y / F / W or T), 254 (e.g., S or T), and 256 (e.g., S / R / Q / E / D or T); or modifications at 428 and / or 433 (e.g., H / L / R / S / P / Q or K) and / or 434 (e.g., A, W, H, F or Y [N434A, N434W, N434H, N434F or N434Y]); or modifications at position 250 and / or 428; or modifications at position 307 or 308 (e.g., 308F, V308F), and 434. In one embodiment, the modifications include 428L (e.g., M428L) and 434S (e.g., N434S) modifications; 428L, 259I (e.g., V259I), and 308F (e.g., V308F) modifications; 433K (e.g., H433K) and 434 (e.g., 434Y) modifications; 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modifications; 250Q and 428L modifications (e.g., T250Q and M428L); and 307 and / or 308 modifications (e.g., 308F or 308P). In yet another embodiment, the modifications include 265A (e.g., D265A) and / or 297A (e.g., N297A) modifications.

[0126] For example, the present invention includes one or more pairs or groups of mutations selected from the group consisting of 250Q and 248L (e.g., T250Q and M248L); 252Y, 254T and 256E (e.g., M252Y, S254T and T256E); 428L and 434S (e.g., M428L and N434S); 257I and 311I (e.g., P257I and Q311I); 257I and 434H (e.g., P257I and N434H); 376V and 434H (e.g., D376V and N434H); 307A, 380A and 434A (e.g., T307A, E380A and N434A); and 433K and 434F (e.g., H433K and N434F), and includes an anti-ACVR1 antibody comprising an Fc domain containing one or more of such pairs or groups of mutations. All contemplated combinations of the foregoing Fc domain mutations and other mutations within the antibody variable domains disclosed herein are intended to be within the scope of the present invention.

[0127] The present invention also includes an anti-ACVR1 antibody comprising a chimeric heavy chain constant (C H ) region, wherein the chimeric C H region comprises segments derived from the C H regions of more than one immunoglobulin isotype. For example, the antibodies of the present invention can comprise a chimeric C H region comprising a part or all of the C H 3 domain of a human IgG1, human IgG2 or human IgG4 molecule combined with a part or all of the C H 2 domain of a human IgG1, human IgG2 or human IgG4 molecule. According to certain embodiments, the antibodies of the present invention comprise a chimeric C HThe region includes. For example, the chimeric hinge may include an "upper hinge" amino acid sequence (amino acid residues from positions 216 to 227 according to EU numbering) derived from the human IgG1, human IgG2, or human IgG4 hinge region, combined with a "lower hinge" sequence (amino acid residues from positions 228 to 236 according to EU numbering) derived from the human IgG1, human IgG2, or human IgG4 hinge region. According to a particular embodiment, the chimeric hinge region includes amino acid residues derived from the human IgG1 or human IgG4 upper hinge and amino acid residues derived from the human IgG2 lower hinge. Chimeric C as described herein H Antibodies containing the region may, in certain embodiments, exhibit modified Fc effector function without adversely affecting the therapeutic or pharmacokinetic properties of the antibody. (See, for example, U.S. Patent Application Publication 2014 / 0243504, the disclosure of which is incorporated herein by reference in its entirety.)

[0128] Biological characteristics of antibodies In general, the antibodies of the present invention function by binding to the ACVR1 protein and reducing its activity. For example, the present invention reduces K to less than 50 nM when measured by surface plasmon resonance using an assay format such as that specified in Example 3 herein. D The product includes an antibody that binds to the human ACVR1 protein (e.g., at 25°C or 37°C) and an antigen-binding fragment of the antibody.

[0129] In certain embodiments, the antibody or its antigen-binding fragment is measured by surface plasmon resonance using an assay format such as that specified in Example 3 herein, or a substantially similar assay, and has a K content of less than approximately 50 nM, less than approximately 40 nM, less than approximately 30 nM, less than approximately 20 nM, less than approximately 10 nM, less than approximately 5 nM, less than approximately 2.5 nM, less than approximately 1 nM, less than approximately 0.5 nM, less than approximately 0.3 nM, less than approximately 0.2 nM, and less than approximately 0.1 nM. DThe human ACVR1 protein is then bound. In certain embodiments, the present invention provides an isolated anti-ACVR1 antibody, which is a fully human monoclonal antibody, or an antigen-binding fragment thereof.

[0130] In certain embodiments, the antibody or its antigen-binding fragment, when measured in a surface plasmon resonance assay using an assay format such as that specified in Example 3 herein, or a substantially similar assay, has a dissociation constant (K) of less than 15 nM, less than 10 nM, less than 5 nM, less than 3 nM, less than 2 nM, less than 1 nM, less than 0.5 nM, less than 0.3 nM, less than 0.2 nM, or less than 0.1 nM. D It binds to the human ACVR1 extracellular domain (SEQ ID NO: 64) fused to the mFc at 37°C.

[0131] In certain embodiments, the antibody or its antigen-binding fragment is measured in a surface plasmon resonance assay using an assay format such as that specified in Example 3 herein, or a substantially similar assay, and has a K content of less than 50 nM, less than 10 nM, less than 5 nM, less than 3 nM, less than 2 nM, less than 1 nM, or less than 0.5 nM. D At 37°C, it binds to the human ACVR1 extracellular domain fused to the myc-myc-hexahis tag (mmh) (e.g., SEQ ID NO: 63).

[0132] In certain embodiments, the antibody or its antigen-binding fragment is conjugated to human and mouse ACVR1 using an assay format such as that specified in Example 3 herein, or a substantially similar assay.

[0133] In certain embodiments, the antibody or its antigen-binding fragment is measured in a surface plasmon resonance assay using an assay format such as that specified in Example 3 herein, or a substantially similar assay, and has a K content of less than 50 nM, less than 10 nM, less than 5 nM, less than 3 nM, less than 2 nM, less than 1 nM, or less than 0.5 nM. DBinds to the mouse ACVR1 extracellular domain (e.g., SEQ ID NO: 65) fused to myc-myc-hexahis tag (mmh) at 37°C.

[0134] In certain embodiments, the antibody or antigen-binding fragment thereof has a K less than 10 nM, less than 5 nM, less than 3 nM, less than 2 nM, less than 1 nM, less than 0.5 nM, less than 0.2 nM, or less than 0.1 nM when measured in a surface plasmon resonance assay using an assay format as defined in Example 3 herein, or a substantially similar assay. D Binds to the mouse ACVR1 extracellular domain fused to mFc at 37°C.

[0135] The invention also encompasses an antibody or antigen-binding fragment thereof that binds to a cell expressing human ACVR1 protein or human ACVR(R206H) protein using an assay format as defined in Example 4 herein, or a substantially similar assay.

[0136] In certain embodiments, the antibody or antigen-binding fragment thereof has an IC less than 10 nM, less than 5 nM, less than 3 nM, or less than 1 nM when measured in a cell-based bioassay using an assay format as defined in Example 5 herein, or a substantially similar assay. 50 Inhibits the activation of cells expressing human ACVR1(R206H) by human activin A.

[0137] In certain embodiments, the antibody or antigen-binding fragment thereof has an IC less than 10 nM, less than 5 nM, less than 3 nM, less than 2 nM, or less than 1 nM when measured in a cell-based bioassay using an assay format as defined in Example 5 herein, or a substantially similar assay. 50 Inhibits the activation of cells expressing human ACVR1(R206H) by human BMP7.

[0138] The present invention also includes an antibody or its antigen-binding fragment that reduces serum hepcidin.

[0139] In certain embodiments, the antibody or its antigen-binding fragment increases serum iron concentration.

[0140] In certain embodiments, the antibody or its antigen-binding fragment inhibits wild-type ACVR1 signaling.

[0141] In certain embodiments, the anti-ACVR antibody or its antigen-binding fragment according to the present invention significantly reduces post-traumatic ectopic ossification (HO).

[0142] In certain embodiments, the antibody or its antigen-binding fragment specifically binds human ACVR1, the fragment thereof, or mutants thereof, and includes an HCVR comprising an amino acid sequence selected from the group consisting of HCVR sequences listed in Table 1, and an LCVR comprising an amino acid sequence selected from the group consisting of LCVR sequences listed in Table 1.

[0143] In one embodiment, the present invention provides an isolated recombinant antibody or its antigen-binding fragment that specifically binds to the ACVR1 protein and inhibits ACVR1-mediated bone morphogenetic protein (BMP) signaling, wherein the antibody or fragment exhibits one or more of the following features:

[0144] In certain embodiments, the present invention has the following characteristics: (a) being a fully human monoclonal antibody; (b) having a dissociation constant (K) less than 15 nM, less than 10 nM, less than 5 nM, less than 3 nM, less than 2 nM, less than 1 nM, less than 0.5 nM, less than 0.3 nM, less than 0.2 nM, or less than 0.1 nM when measured in a surface plasmon resonance assay. D (c) To bind to the human ACVR1 extracellular domain (SEQ ID NO: 64) fused to mFc at 37°C; (c) K levels less than 50 nM, less than 10 nM, less than 5 nM, less than 3 nM, less than 2 nM, less than 1 nM, and less than 0.5 nM when measured in a surface plasmon resonance assay. D(d) Binding to the human ACVR1 extracellular domain fused to the myc-myc-hexahis tag at 37°C (e.g., SEQ ID NO: 63); (f) K levels less than 50 nM, less than 10 nM, less than 5 nM, less than 3 nM, less than 2 nM, less than 1 nM, and less than 0.5 nM when measured in a surface plasmon resonance assay. D (e) K at 37°C to bind to the mouse ACVR1 extracellular domain fused to the myc-myc-hexahis tag (e.g., SEQ ID NO: 65); (e) K at concentrations of less than 10 nM, less than 5 nM, less than 3 nM, less than 2 nM, less than 1 nM, less than 0.5 nM, less than 0.2 nM, or less than 0.1 nM D (f) To bind to the mouse ACVR1 extracellular domain fused to mFc at 37°C; (g) To bind to cells expressing human ACVR1 protein or human ACVR(R206H) protein; (g) To achieve IC levels of less than 10 nM, less than 5 nM, less than 3 nM, less than 2 nM, or less than 1 nM when measured in a cell-based bioassay. 50 (h) inhibiting the activation of cells expressing human ACVR1(R206H) by human activin A; (h) IC2012 < 10 nM, < 5 nM, < 3 nM, < 2 nM, or < 1 nM, or less than that, as measured in a cell-based bioassay. 50 The present invention provides an antibody or antigen-binding fragment having one or more of the following properties: (i) inhibiting the activation of cells expressing human ACVR1(R206H) by human BMP7; and (i) comprising an HCVR containing an amino acid sequence selected from the group consisting of HCVR sequences listed in Table 1, and an LCVR containing an amino acid sequence selected from the group consisting of LCVR sequences listed in Table 1.

[0145] The antibodies of the present invention may possess one or more of the biological characteristics described above, or any combination thereof. Other biological characteristics of the antibodies of the invention will be apparent to those skilled in the art from the summary of this disclosure, including the actual examples provided herein.

[0146] Epitope mapping and related technologies The present invention comprises an anti-ACVR1 antibody that interacts with one or more amino acids found within one or more regions of the ACVR1 protein molecule. The epitope to which the antibody binds consists of a single adjacent sequence of three or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids located within any of the aforementioned domains of the ACVR1 protein molecule (e.g., a linear epitope in a domain). Alternatively, the epitope consists of a plurality of non-adjacent amino acids (or amino acid sequences) (e.g., a structural epitope) located within one or both of the aforementioned domains of the protein molecule.

[0147] Various techniques known to those skilled in the art can be used to determine whether an antibody "interacts with one or more amino acids" within a polypeptide or protein. Exemplary techniques include routine cross-blocking assays, such as those described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, NY). Other methods include alanine scanning mutation analysis, peptide blot analysis (Reineke (2004) Methods Mol. Biol. 248: pp. 443-463), peptide cleavage analysis crystallographic studies, and NMR analysis. Furthermore, methods such as antigen epitope excision, epitope extraction, and chemical modification can also be used (Tomer (2000) Prot. Sci. 9: pp. 487-496). Another method that can be used to identify amino acids within polypeptides with which antibodies interact is hydrogen / deuterium exchange detected by mass spectrometry. In general terms, hydrogen / deuterium exchange methods involve deuterium labeling of the protein of interest, followed by the binding of an antibody to the deuterium-labeled protein. Next, the protein / antibody complex is transferred to water, and the exchangeable protons within the amino acids protected by the antibody complex undergo reverse exchange from deuterium to hydrogen at a slower rate than the exchangeable protons within the amino acids that are not part of the interface. As a result, the amino acids that form part of the protein / antibody interface retain deuterium and can therefore exhibit a relatively higher mass compared to amino acids not included in the interface. After antibody dissociation, the target protein is subjected to protease cleavage and mass spectrometry, thereby revealing the deuterium-labeled residues corresponding to the specific amino acids with which the antibody interacts. See, for example, Ehring (1999) Analytical Biochemistry 267:252-259; Engen and Smith (2001) Anal. Chem. 73:256A-265A.

[0148] The term "epitope" refers to a site on an antigen to which B and / or T cells respond. B cell epitopes can be formed from both adjacent amino acids or non-adjacent amino acids juxtaposed by tertiary folding of proteins. Epitopes formed from adjacent amino acids are usually retained upon exposure to denaturing solvents, while epitopes formed by tertiary folding are usually lost upon treatment with denaturing solvents. Epitopes typically contain at least 3, more commonly at least 5 or 8-10, amino acids in a specific spatial conformation.

[0149] Modification-Assisted Profiling (MAP), also known as Antigen Structure-Based Antibody Profiling (ASAP), is a method for classifying a large number of monoclonal antibodies (mAbs) against the same antigen according to the similarity of the binding profiles of each antibody to chemically or enzymatically modified antigen surfaces (see US2004 / 0101920, which is incorporated herein by reference in its entirety). Each category may reflect unique epitopes that are either distinctly different from or partially overlapping with the epitopes represented by other categories. This technique enables rapid filtering of genetically identical antibodies, allowing characterization to focus on genetically distinct antibodies. When applied to hybridoma screening, MAP can facilitate the identification of rare hybridoma clones that produce mAbs with desired characteristics. Using MAP, the antibodies of the present invention can be sorted into groups of antibodies that bind to different epitopes.

[0150] In certain embodiments, the present invention comprises an anti-ACVR1 antibody and its antigen-binding fragment that interacts with one or more epitopes found within the extracellular domain of ACVR1. The epitope(s) consist of one or more adjacent sequences of three or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids located within the extracellular domain of ACVR1. Alternatively, the epitope consists of multiple disproportionate amino acids (or amino acid sequences) located within the ACVR1 protein.

[0151] The present invention encompasses anti-ACVR1 antibodies that bind to the same epitope, or a portion of the epitope, as any of the specific exemplary antibodies listed in Table 1. Similarly, the present invention encompasses anti-ACVR1 antibodies that compete with any of the specific exemplary antibodies listed in Table 1 for binding to the ACVR1 protein or a fragment thereof. For example, the present invention encompasses anti-ACVR1 antibodies that cross-compete with one or more of the antibodies listed in Table 1 for binding to the ACV protein.

[0152] By using routine methods known in the art, it is easily possible to determine whether an antibody binds to the same epitope as the reference anti-ACVR1 antibody, or whether it competes for binding to it. For example, to determine whether a test antibody binds to the same epitope as the reference anti-ACVR1 antibody of the present invention, the reference antibody is bound to the ACVR1 protein or peptide under saturated conditions. The ability of the test antibody to bind to the ACVR1 protein molecule is then evaluated. If the test antibody is able to bind to ACVR1 after saturated binding with the reference anti-ACVR1 antibody, it can be concluded that the test antibody binds to a different epitope than the reference anti-ACVR1 antibody. On the other hand, if the test antibody is unable to bind to the ACVR1 protein after saturated binding with the reference anti-ACVR1 antibody, the test antibody may bind to the same epitope to which the reference anti-ACVR1 antibody of the present invention binds.

[0153] To determine whether an antibody competes for binding with the reference anti-ACVR1 antibody, the binding methodology described above is carried out in two directions: In the first direction, the reference antibody is bound to the ACVR1 protein under saturated conditions, and then the binding of the test antibody to the ACVR1 molecule is evaluated. In the second direction, the test antibody is bound to the ACVR1 molecule under saturated conditions, and then the binding of the reference antibody to the ACVR1 molecule is evaluated. In both directions, if only the first (saturated) antibody is able to bind to the ACVR1 molecule, it is concluded that the test antibody and the reference antibody compete for binding to ACVR1. As will be understood by those skilled in the art, an antibody that competes for binding with a reference antibody cannot necessarily bind to the same epitope as the reference antibody, and can sterically block the binding of the reference antibody by binding to an overlapping or adjacent epitope.

[0154] Two antibodies bind to the same or overlapping epitopes if each competitively inhibits (blocks) the binding of the other antibody to the antigen. That is, one, five, ten, twenty, or 100-fold excess of one antibody inhibits the binding of the other by at least 50%, but preferably 75%, 90%, or even 99%, when measured in a competitive binding assay (see, for example, Junghans et al., Cancer Res. 1990 50: pp. 1495-1502). Alternatively, the two antibodies have the same epitope if substantially all amino acid mutations in the antigen that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other. The two antibodies have overlapping epitopes if several amino acid mutations that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other.

[0155] Subsequently, further routine experiments (e.g., peptide mutation and binding analysis) can be performed to confirm whether the observed lack of binding of the test antibody is actually due to binding to the same epitope as the reference antibody, or whether stereoblocking (or another phenomenon) is involved in the observed lack of binding. These types of experiments can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art.

[0156] Immunoconjugate The present invention encompasses human anti-ACVR1 monoclonal antibodies or their antigen-binding fragments ("immunoconjugates") conjugated to a therapeutic site for treating ACVR1-related diseases or disorders (e.g., ectopic ossification, anemia, or ectopic ossification). As used herein, the term "immunoconjugate" refers to an antibody chemically or biologically conjugated to a radiopharmaceutical, cytokine, interferon, target or reporter moiety, enzyme, peptide or protein, or therapeutic agent. The antibody is conjugated to the radiopharmaceutical, cytokine, interferon, target or reporter moiety, enzyme, peptide or therapeutic agent at any position along the molecule, as long as the antibody is able to conjugate its target. Examples of immunoconjugates include antibody-drug conjugates and antibody-toxin fusion proteins. In one embodiment, the drug is a second, distinct antibody against the ACVR1 protein. The type of therapeutic moiety conjugated to the anti-ACVR1 antibody takes into account the condition to be treated and the desired therapeutic effect to be achieved. Examples of agents suitable for forming immunoconjugates are known in the art; see, for example, WO05 / 103081, incorporated herein by reference.

[0157] multispecific antibody The antibodies of the present invention are monospecific, bispecific, or polyspecific. Polyspecific antibodies may contain antigen-binding domains specific to different epitopes of a single target polypeptide, or to more than one target polypeptide. See, for example, Tutt et al., 1991, J.Immunol. 147:60-69; Kufer et al., 2004, Trends Biotechnol. 22:238-244. These are incorporated herein by reference, respectively.

[0158] Any of the multispecific antigen-binding molecules of the present invention, or variants thereof, are constructed using standard molecular biological techniques (e.g., recombinant DNA and protein expression techniques) as are known to those skilled in the art.

[0159] In some embodiments, ACVR1-specific antibodies are produced in a bispecificity form ("bispecificity") in which variable regions that bind to distinct domains of the ACVR1 protein are linked together to confer bidomain specificity within a single binding molecule. A well-designed bispecificity may enhance the overall inhibitory efficacy of the ACVR1 protein by increasing both specificity and binding avidity. Variable regions that have specificity for individual domains (e.g., segments of the N-terminal domain), or that can bind to different regions within a single domain, are paired on a structural scaffold that allows each region to bind to separate epitopes or to different regions within a single domain simultaneously. An example of bispecificity involves a binder-derived heavy chain variable region (V) that has specificity for one domain. H ) is a series of binder-derived light chain variable regions (V) that have specificity for the second domain. L ) rearrange it, and that V H Without destroying the original singularity for the original V H Non-homogeneous V that can be paired with it L Identify the partner. In this way, a single V L segment (e.g., V) L 1) Two different V H Domain (for example, VH 1 and V H 2) Combined with two bonds "arms", it forms a bispecificity (V H 1-V L 1 and V H 2-V L 1) can be generated. A single V L The use of segments reduces the complexity of the system, thereby simplifying and increasing the efficiency of the cloning, expression, and purification processes used to generate bispecificity (see, for example, US2011 / 0195454 and US2010 / 0331527).

[0160] Alternatively, antibodies that bind to more than one domain and a second target, such as, but not limited to, a second different anti-ACVR1 antibody, can be prepared in a bispecific form using the techniques described herein or other techniques known to those skilled in the art. Antibody variable regions that bind to distinct regions can be ligated together with variable regions that bind to relevant sites on, for example, the extracellular domain of ACVR1, thereby conferring biantigen specificity within a single binding molecule. A well-designed bispecificity of this property performs a dual function. Variable regions with specificity to the extracellular domain are paired on a structural scaffold that allows each variable region to bind to a different antigen, in combination with variable regions that have specificity to the area outside the extracellular domain.

[0161] An exemplary bispecific antibody form that can be used in the context of the present invention is the first immunoglobulin (Ig) C H 3 domains and 2nd Ig C H This involves the use of three domains, where the first and second Ig C H The three domains differ from each other by at least one amino acid, where the difference of at least one amino acid reduces the binding of the bispecific antibody to protein A compared to a bispecific antibody lacking the amino acid difference. In one embodiment, the first Ig C H The 3 domains bind to protein A, and the second Ig C HThe 3 domains contain mutations that reduce or eliminate protein A binding, such as the H95R modification (according to IMGT exon numbering; H435R according to EU numbering). Second C H 3 may further include Y96F modification (by IMGT; Y436F by EU). Second C H Further modifications found within 3 include, for IgG1 antibodies, D16E, L18M, N44S, K52N, V57M, and V82I (according to IMGT; D356E, L358M, N384S, K392N, V397M, and V422I); for IgG2 antibodies, N44S, K52N, and V82I (according to IMGT; N384S, K392N, and V422I); and for IgG4 antibodies, Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (according to IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I). The variations in the bispecific antibody forms described above are intended to be within the scope of the present invention.

[0162] Other exemplary bispecificity forms that can be used in the context of the present invention include, for example, scFv-based or diabody bispecificity forms, IgG-scFv fusions, dual variable domain (DVD)-Ig, Quadroma, knob-in-hole, common light chain (e.g., common light chain with knob-in-hole), CrossMab, CrossFab, (SEED)body, leucine zipper, Duobody, IgG1 / IgG2, dual-acting Fab(DAF)-IgG, and Mab 2Bispecific forms include, but are not limited to, Klein et al., 2012, mAbs 4:6, pp. 1-11, and the references cited therein, for an overview of the aforementioned forms. Bispecific antibodies can also be constructed using peptide / nucleic acid conjugations, for example, using non-natural amino acids with orthogonal chemical reactivity to generate site-specific antibody-oligonucleotide conjugates, which then self-assemble into multimeric complexes with a predetermined composition, valency, and geometry. (See, e.g., Kazane et al., J.Am.Chem.Soc. [Epub: December 4, 2012]).

[0163] Therapeutic administration and formulations The present invention provides therapeutic compositions comprising the anti-ACVR1 antibody or its antigen-binding fragment. The therapeutic compositions according to the present invention are administered together with suitable pharmaceutically acceptable carriers, excipients, and other agents incorporated into the formulation to provide improved transfer, delivery, tolerance, etc. Numerous suitable formulations can be found in formulation collections known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)-containing vesicles (e.g., LIPOFECTIN®), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsion carbowaxes (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowaxes. See also Powell et al., "Compendium of excipients for parenteral formulations," PDA (1998), J Pharm Sci Technol 52: pp. 238-311.

[0164] The antibody dose varies depending on the age and size of the recipient, the target disease, the condition, and the route of administration. When the antibody of the present invention is used to treat or prevent a disease or disorder in adult patients, it is usually preferable to administer it as a single dose of about 0.1 to about 100 mg / kg body weight. The frequency and duration of treatment can be adjusted depending on the severity of the condition. In certain embodiments, the antibody of the present invention or its antigen-binding fragment can be administered as an initial dose of at least about 0.1 mg to about 800 mg, about 1 to about 600 mg, about 5 to about 500 mg, or about 10 to about 400 mg. In certain embodiments, a second or more subsequent doses of the antibody or its antigen-binding fragment may be administered following an initial dose, in an amount approximately equal to or less than the initial dose, wherein the subsequent doses are separated by at least 1 to 3 days; at least 1 week; at least 2 weeks; at least 3 weeks; at least 4 weeks; at least 5 weeks; at least 6 weeks; at least 7 weeks; at least 8 weeks; at least 9 weeks; at least 10 weeks; at least 12 weeks; or at least 14 weeks.

[0165] Various delivery systems are known and can be used to administer the pharmaceutical compositions of the present invention, for example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, and receptor-mediated endocytosis (see, e.g., Wu et al. (1987) J. Biol. Chem. 262: pp. 4429-4432). Methods of delivery include, but are not limited to, intradermal, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, intraventricular, and oral routes. The compositions are administered by any convenient route, for example, by infusion or bolus injection, or by absorption through the epithelium or mucosal lining (e.g., oral mucosa, rectal and intestinal mucosa), and are administered together with other bioactive agents. Administration is systemic or topical. The pharmaceutical compositions can also be delivered in vesicles, particularly liposomes (see, e.g., Langer (1990) Science 249: pp. 1527-1533).

[0166] The use of nanoparticles for delivering the antibodies of the present invention is also intended herein. Antibody-conjugated nanoparticles are used for both therapeutic and diagnostic applications. Antibody-conjugated nanoparticles, as well as methods for their manufacture and use, are described in detail by reference to Arruebo, M. et al. 2009 ("Antibody-conjugated nanoparticles for biomedical applications," J. Nanomat. Volume 2009, Article ID 439389, p. 24, doi:10.1155 / 2009 / 439389). Nanoparticles may be developed and conjugated to antibodies contained in pharmaceutical compositions to target cells. Nanoparticles for drug delivery are also described, for example, in US8257740 or US8246995, which are both incorporated herein in their entirety.

[0167] In certain circumstances, pharmaceutical compositions can be delivered using a controlled-release system. In one embodiment, a pump is used. In another embodiment, a polymer material can be used. In yet another embodiment, the controlled-release system can be positioned close to the target of the composition, and therefore only a fraction of the total systemic dose is required.

[0168] Injectable preparations can be administered in various forms, including intravenous, subcutaneous, intracranial, intraperitoneal, and intramuscular injections, as well as intravenous infusions. These injectable preparations are manufactured by known and publicly available methods.

[0169] The pharmaceutical compositions of the present invention can be delivered subcutaneously or intravenously using standard needles and syringes. Furthermore, with respect to subcutaneous delivery, pen delivery devices are readily applicable to delivering the pharmaceutical compositions of the present invention. Such pen delivery devices are reusable or disposable. Reusable pen delivery devices generally utilize replaceable cartridges containing the pharmaceutical composition. Once all of the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge is easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. Disposable pen delivery devices do not have replaceable cartridges. Rather, disposable pen delivery devices are pre-filled with the pharmaceutical composition held in a reservoir within the device. Once the pharmaceutical composition is depleted from the reservoir, the entire device is discarded.

[0170] In the treatment of certain diseases or conditions (e.g., DIPG), it is necessary to overcome the blood-brain barrier. In certain embodiments, the blood-brain barrier is overcome by using one or more approaches disclosed in the art, for example, Parodi et al., 2019, Pharmaceuticals 11:245, incorporated herein by reference.

[0171] Preferably, the oral or parenteral pharmaceutical compositions described above are manufactured in a unit dosing form suitable for administering the active ingredient. Examples of such unit dosing forms include tablets, pills, capsules, injections (ampoules), and suppositories. The amount of antibody contained is generally about 5 to about 500 mg per unit dosing form; in particular, it is preferable that the antibody is contained in an amount of about 5 to about 300 mg in the injection form and about 10 to about 300 mg in the other dosing forms.

[0172] Therapeutic uses of antibodies The antibody or antigen-binding fragment of the present invention is useful for treating and / or preventing diseases, disorders, or conditions associated with ACVR1, and / or for improving at least one symptom associated with such disease, disorder, or condition. In certain embodiments, the antibody or antigen-binding fragment of the present invention is administered in a therapeutic dose to a patient having a disease, disorder, or condition associated with ACVR1 or a mutant ACVR protein.

[0173] In certain embodiments, the antibodies or antigen-binding fragments of the present invention are useful in treating or preventing at least one symptom or sign of an ACVR1-related or ACVR1 mutant protein-related disease or disorder selected from the group consisting of ectopic ossification, trauma-induced ectopic ossification, ectopic ossification, osteodysplasia, anemia, and diffuse pontine glioma. In some cases, the ACVR1-related or ACVR1 mutant protein-related disease or disorder is not fibrosis ossificans progressive (FOP). In some cases, the ACVR1 mutant protein-related disease or disorder is FOP.

[0174] It is also intended herein that one or more antibodies of the present invention may be used prophylactically in subjects at risk of developing ACVR1-related disease or disorder.

[0175] In one embodiment of the present invention, the antibody is used to manufacture a pharmaceutical composition or pharmaceutical for treating a patient suffering from one of the diseases, disorders, or conditions disclosed herein. In another embodiment of the present invention, the antibody is used as an adjunct therapy to any other agent or therapy known to those skilled in the art that is useful for treating or reversing one of the diseases, disorders, or conditions disclosed herein.

[0176] Combination therapy Combination therapies may include the antibody of the present invention and any further therapeutic agents that can be suitably combined with the antibody of the present invention or with a biologically active fragment of the antibody of the present invention. The antibody of the present invention can be synergistically combined with one or more drugs or therapies used to treat ACVR1-related or ACVR1 mutant protein-related diseases or disorders. In some embodiments, the antibody of the present invention can be combined with a second therapeutic agent to restore one or more symptoms of the above-mentioned diseases or conditions.

[0177] Depending on the disease, disorder, or condition, the antibodies of the present invention are used in combination with one or more further therapeutic agents.

[0178] Examples of further therapeutic agents for heterotopic ossification that can be administered in combination with anti-ACVR1 antibodies include, but are not limited to, anti-activin A inhibitors or their antigen-binding fragments, anti-ACVR2 antibodies or their antigen-binding fragments, anti-inflammatory drugs, steroids, bisphosphonates, muscle relaxants, and retinoic acid receptor (RAR) gamma agonists.

[0179] Activin belongs to the transforming growth factor-beta (TGF-β) superfamily and exerts a wide range of biological effects on cell proliferation, differentiation, metabolism, homeostasis, and apoptosis, as well as immune responses and tissue repair. Activin A is a disulfide-linked homodimer (two β-A chains) that binds to and activates heteromeric complexes of type I (Act RI-A and Act RI-B) and type II (Act RII-A and Act RII-B) serine-threonine kinase receptors. Activin A can act as a ligand for ACVR1 protein or ACVR1 mutant protein.

[0180] Examples of anti-inflammatory drugs include aspirin, diclofenac, indomethacin, ibuprofen, ketoprofen, naproxen, piroxicam, rofecoxib, celecoxib, azathioprine, penicillamine, methotrexate, sulfasalazine, leflunomide, infliximab, and etanercept. Examples of steroids include prednisolone, beclomethasone, betamethasone, fluticasone, dexamethasone, and hydrocortisone. Examples of bisphosphonates include alendronate, simadronate, clodronate, etidronate, ibandronate, incadronate, minodronate, neridronate, olpadronate, pamidronate, pyridronate, risedronate, tildronate, and zoledronate. Examples of muscle relaxants include cyclobenzaprine, metaxalone, and baclofen. An example of a retinoic acid receptor gamma agonist is parobalotene. Examples of further treatments for anemia include recombinant erythropoietin (EPO) and iron supplements. Examples of further treatments for diffuse pontine gliomas include radiotherapy or experimental chemotherapy.

[0181] As used herein, the term "in combination with" means that a further therapeutically active component(s) is administered before, concurrently with, or after the administration of the anti-ACVR1 antibody of the present invention. The term "in combination with" also includes the sequential or concurrent administration of the anti-ACVR1 antibody and the second therapeutic agent.

[0182] Further therapeutically active components(s) are administered to the subject before administration of the anti-ACVR1 antibody of the present invention. For example, if the first component is administered one week, 72 hours, 60 hours, 48 ​​hours, 36 hours, 24 hours, 12 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 30 minutes, or less than 30 minutes before administration of the second component, the first component is considered to have been administered "before" the second component. In other embodiments, further therapeutically active components(s) are administered to the subject after administration of the anti-ACVR1 antibody of the present invention. For example, if the first component is administered 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours or more after the administration of the second component, the first component is considered to have been administered "after" the second component. In yet another embodiment, further therapeutically active components(s) are administered to the subject simultaneously with the administration of the anti-ACVR1 antibody of the present invention. For the purposes of the present invention, "simultaneous" administration includes, for example, the administration of the anti-ACVR1 antibody and further therapeutically active components to the subject in single-dose forms or in separate dosing forms, administered to the subject within approximately 30 minutes or less from each other. When administered in separate dosage forms, each dosage form may be administered via the same route (e.g., the anti-ACVR1 antibody and the further therapeutically active component may both be administered intravenously); or each dosage form may be administered via different routes (e.g., the anti-ACVR1 antibody may be administered intravenously, and the further therapeutically active component may be administered orally). In either case, administering components in single doses, in separate dosage forms via the same route, or in separate dosage forms via different routes, is all considered “concurrent administration” for the purposes of this disclosure. For the purposes of this disclosure, administration of anti-ACVR1 antibody “before,” “at the same time as,” or “after” administration of further therapeutically active component (these terms are as defined herein above) is considered administration of anti-ACVR1 antibody “in combination with” further therapeutically active component.

[0183] The present invention encompasses pharmaceutical compositions in which the anti-ACVR1 antibody of the present invention is co-formulated with one or more further therapeutically active components as described elsewhere herein.

[0184] Diagnostic applications of antibodies The antibodies of the present invention are used, for example, for diagnostic purposes to detect and / or measure the ACVR1 protein in a sample. Some embodiments intend the use of one or more antibodies of the present invention in assays for detecting ACVR1-related or ACVR mutant protein-related diseases or disorders. An exemplary diagnostic assay relating to ACVR1 may, for example, involve contacting a sample obtained from a patient with the anti-ACVR1 antibody of the present invention, where the anti-ACVR1 antibody is labeled with a detectable label or reporter molecule, or used as a capture ligand to selectively isolate ACVR1 from the patient sample. Alternatively, an unlabeled anti-ACVR1 antibody can be used in diagnostic applications in combination with a secondary antibody that is itself detectably labeled. The detectable label or reporter molecule is 3 H, 14 C, 32 P, 35 S, or 125 ACVR1 may be a radioactive isotope such as 1; a fluorescent or chemiluminescent moiety such as fluorescein isothiocyanate or rhodamine; or an enzyme such as alkaline phosphatase, β-galactosidase, horseradish peroxidase, or luciferase. Specific exemplary assays that can be used to detect or measure ACVR1 in a sample include enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), and fluorescence-activated cell sorting (FACS).

[0185] Samples that can be used in the ACVR1 diagnostic assay according to the present invention include any tissue or liquid sample obtainable from a patient that contains either ACVR1 protein or a fragment thereof in an amount detectable under normal or pathological conditions. Generally, a baseline or standard level of ACVR1 is first established by measuring the level of ACVR1 protein in a specific sample obtained from a healthy patient (e.g., a patient without a disease associated with ACVR1). Subsequently, this baseline level of ACVR1 can be compared to the level of ACVR1 measured in a sample obtained from an individual suspected of having an ACVR1-related disease or symptoms associated with such a condition.

[0186] Antibodies specific to the ACVR1 protein may or may not contain further labeling or sites, or they may contain labeling or sites at the N-terminus or C-terminus. In one embodiment, the labeling or site is biotin. In a binding assay, the position of the label (if present) can determine the orientation of the peptide relative to the surface to which it is bound. For example, if the surface is coated with avidin, a peptide containing N-terminal biotin will be oriented so that the C-terminus of the peptide is distal to the surface. [Examples]

[0187] The following examples are presented to provide a complete disclosure and explanation of the methods and compositions of the present invention and how to use them, and are not intended to limit the scope of what the inventors consider to be the present invention. Efforts have been made to ensure accuracy with respect to the numbers used (e.g., quantity, temperature, etc.), but some experimental errors and deviations should be explained. Unless otherwise noted, parts are parts by weight, molecular weight is average molecular weight, temperature is Celsius, room temperature is approximately 25°C, and pressure is atmospheric pressure or near atmospheric pressure. [Examples]

[0188] Production of human antibodies against activin A receptor 1 (ACVR1) Human antibodies against ACVR1 (human and mouse cross-reactive) were isolated from a full-length human IgG synthetic naive library using an in vitro yeast selection system and related methods (Rouha H, et al. MAbs. 2015; 7(1): 243-254). As previously described, approximately 1 × 10⁶ antibodies in the diversity. 10 Several antibody libraries were designed and propagated (Rouha et al. 2015, Xu et al., Protein Eng Des Sel. 2013; 26(10): pp. 663-670). ACVR1-binding antibodies were enriched by incubating biotinylated ACVR1-Fc and Myc-His monomers ACVR1 at various concentrations with antibody-expressing yeast cells, followed by several consecutive selection rounds using magnetic bead selection with fluorescent streptavidin or extraavidin secondary reagents (Miltenyi Biotec) or flow cytometry on a FACSAria II cell sorter (BD Biosciences). Antibodies cross-reactive to off-target proteins ALK1, ALK3, and ALK6 were actively depleted from the selection output. After the final round of enrichment, yeast cells were seeded on agar plates, analyzed by DNA sequencing, and grown for IgG production. The heavy chains from the naive output were used to produce a light chain diversification library, which was then used for further selection rounds. In particular, heavy chains were extracted from the naive selection round output and 1 × 10⁶ 6 Converted into a light chain library consisting of individual unique light chains, approximately 1 × 10⁶ in total diversity. 8 Numerous new libraries were created. Antibody optimization was completed in three phases. Heavy chain optimization via diversification of complementarity-determining regions (CDRs) HCDR1 and HCDR2 was performed either by mutagenic PCR-based diversification of the entire heavy chain variable region or by diversification of the light chain LCDR1 and LCDR2 segments. The HCDR1 and HCDR2 regions were approximately 1 × 10⁶ 8Diversification was carried out using pre-existing libraries of HCDR1 and HCDR2 variants with a diversity of 1 × 10⁶. Mutagenic PCR-based and pre-existing libraries containing HCDR1 and HCDR2 variants each had approximately 1 × 10⁶ differences. 7 pieces and 1 × 10 5 There were approximately 1 × 10¹⁶ read variants. The read variants were further diversified through diversification of the DNA oligonucleotide sequences of HCDR3 or LCDR3. The oligonucleotide libraries for HCDR3 and LCDR3 were approximately 1 × 10¹⁶ each. 4 pieces and 1 × 10 3 The diversified antibody lineages exhibited individual diversity. The diversified populations were selected for enhanced binding to target proteins while avoiding undesirable cross-reactivity. The methods used for selection in the diversified populations were similar to, or identical to, the methods used to isolate the original lead IgGs (Xu et al., 2013).

[0189] The exemplary antibodies produced as disclosed above were referred to as REGN 5166, REGN 5167, and REGN 5168. ACVR1 Fabs were generated from the corresponding ACVR1 Mabs.

[0190] The biological properties of the exemplary antibodies produced according to the method of this example are described in detail in the examples below. [Examples]

[0191] Amino acid and nucleotide sequences of the heavy and light chain variable regions Table 1 lists the amino acid sequence identifiers of the heavy and light chain variable regions and CDRs of the selected anti-ACVR1 antibodies of the present invention.

[0192] [Table 1]

[0193] Table 1 shows that the amino acid sequence of LCDR2 SEQ ID NO: 26 is GAS; LCDR2 SEQ ID NO: 42 is GAS; and LCDR2 SEQ ID NO: 58 is KAS.

[0194] The corresponding nucleic acid (DNA) sequence identifiers are listed in Table 2.

[0195] [Table 2]

[0196] Table 2 shows that the DNA sequence of LCDR2 Sequence ID No. 25 is GGCGCATCC; LCDR2 Sequence ID No. 41 is GGTGCATCC; and LCDR2 Sequence ID No. 57 is AAAGCCTCC.

[0197] The antibodies referred to herein typically have a fully human variable region, but may have a human or mouse constant region. As will be understood by those skilled in the art, antibodies having a particular Fc isotype can be converted to antibodies having a different Fc isotype (for example, an antibody having mouse IgG1 Fc can be converted to an antibody having human IgG4), but in any case, the variable domain (including the CDR) indicated by the numerical identifier shown in Table 1 or 2 remains the same, and it was expected that the antigen-binding properties would be identical or substantially similar regardless of the nature of the Fc domain. In certain embodiments, a selected antibody having mouse IgG1 Fc was converted to an antibody having human IgG4 Fc. In one embodiment, the IgG4 Fc domain contained two or more amino acid changes, as disclosed in US20100331527. In one embodiment, the human IgG4 Fc contained a serine-to-proline mutation (S108P) in the hinge region to promote dimerization. Unless otherwise noted, all antibodies used in the following examples contained the human IgG4 isotype.

[0198] Table 3 lists the nucleic acid (DNA) and amino acid (PEP) sequence identifiers of the heavy and light chains (HC and LC) of the selected anti-ACVR1 antibodies of the present invention.

[0199] [Table 3] [Examples]

[0200] Antibody binding to ACVR1 as measured by surface plasmon resonance The Biacore binding kinetics of anti-ACVR1 monoclonal antibodies bound to the ACVR1 reagent were measured at 37°C. The reagent / mAb antibody clones tested are shown in Table 4.

[0201] [Table 4]

[0202] Experimental Procedure Equilibrium dissociation constant (K) for ACVR1 binding to purified anti-ACVR1 monoclonal antibody DThe binding was determined using a real-time surface plasmon resonance (SPR)-based Biacore T200 biosensor. All binding studies were performed at 37°C in 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% v / v surfactant Tween-20, pH 7.4 (HBS-EP) running buffer. The Biacore CM5 sensor surface was first derivatized by amine coupling with a monoclonal mouse anti-human Fc antibody (REGN2567) to capture an anti-ACVR1 monoclonal antibody. Various concentrations of ACVR1 reagent, human ACVR1 extracellular domain expressed with a C-terminal myc-myc-hexahistidine tag (hACVR1-mmh; REGN3111, SEQ ID NO: 63), mouse ACVR1 extracellular domain expressed with a C-terminal myc-myc-hexahistidine tag (mACVR1-mmh; REGN3407, SEQ ID NO: 65), human ACVR1 extracellular domain expressed with a C-terminal mouse IgG2a Fc tag (hACVR1-mFc; REGN3112, SEQ ID NO: 64), and mouse ACVR1 extracellular domain expressed with a C-terminal mouse IgG2a Fc tag (mACVR1-mFc, R&D system) were injected at a flow rate of 50 μL / min for 1.5 minutes in a series of 3-fold dilutions prepared in HBS-EP running buffer at concentrations ranging from 0.41 nM to 100 nM. The dissociation of various ACVR1 reagents bound to anti-ACVR1 monoclonal antibodies was monitored for 20 minutes in HBS-EP running buffer. At the end of each cycle, the capture surface of the anti-ACVR1 monoclonal antibody was regenerated using a 12-second injection of 20 mM H3PO4. Binding rate (ka) and dissociation rate (kd) were determined by fitting real-time binding sensorgrams to a 1:1 binding model with mass transport constraints using Scrubber 2.0c curve fitting software. Binding-dissociation equilibrium constant (KD) and dissociation half-life (t) were determined. 1 / 2 The following was calculated from the motion velocity:

number

[0203] Table 5 to Table 8 show the binding kinetic parameters of the anti-ACVR1 monoclonal antibody of the present invention to various ACVR1 reagents at 37°C.

[0204] [Table 5]

[0205] [Table 6]

[0206] [Table 7]

[0207] [Table 8]

[0208] At 37°C, the anti-ACVR1 monoclonal antibody bound to hACVR1-mmh at a K D value ranging from 227 pM to 1.21 nM, as shown in Table 5.

[0209] At 37°C, the anti-ACVR1 monoclonal antibody bound to hACVR1-mFc at a K D value ranging from 47.8 pM to 191 pM, as shown in Table 6.

[0210] At 37°C, the anti-ACVR1 monoclonal antibody bound to mACVR1-mmh at a K D value ranging from 226 pM to 1.24 nM, as shown in Table 7.

[0211] At 37°C, the anti-ACVR1 monoclonal antibody bound to mACVR1-mFc at a K D value ranging from 17.9 pM to 36.2 pM, as shown in Table 8.

[0212] To measure the binding constant of ACVR1 Fab, hACVR1.mmh was captured with myc antibody (REGN 642) immobilized on a CM5 chip. Various concentrations of ACVR1 Fab were injected into hACVR1.mmh at 37°C. Binding rate constants and equilibrium dissociation rate constants were calculated by fitting the data using a 1:1 Langmuir binding model (Scrubber 2.0c). Table 9 shows the binding constants of ACVR1 Fab to human ACVR1 (data for REGN 5168 Fab are not shown).

[0213] [Table 9]

[0214] All three ACVR1 Fabs bound to monomeric human ACVR1 with similar binding kinetics (less than 2.5-fold difference) to their respective mAbs.

[0215] To measure the binding constant of ACVR1 Fab, mACVR1.mmh was captured with myc antibody (REGN 642) immobilized on a CM5 chip. Various concentrations of ACVR1 Fab were injected into mACVR1.mmh at 37°C. The binding rate constant and equilibrium dissociation rate constant were calculated by fitting the data using a 1:1 Langmuir binding model (Scrubber 2.0c). The binding constants of ACVR1 Fab to mouse ACVR1 are shown in Table 10 (data for REGN 5168 Fab are not shown).

[0216] [Table 10]

[0217] All three ACVR1 Fabs bound to monomeric mouse ACVR1 with similar binding kinetics (less than 2.5-fold difference) to their respective mAbs. [Examples]

[0218] Antibody binding to ACVR1 family members as determined by surface plasmon resonance spectroscopy The cross-reactivity of ACVR1 mAbs to a panel of osteomorphonectomy protein type 1 receptors, including human and mouse ACVR1, BMPR1A, BMPR1B, and ACVRL1, was tested using a Biacore T200 as described below. To measure the specificity of ACVR1 mAbs, they were captured with anti-human Fab immobilized on a CM5 chip. 100 nM or 10 nM dimerized human or mouse ACVR1, ACVRL1, BMPR1A, or BMPR1B were injected at 30 μL / min for 2 minutes (double repeated injection), and the experiment was performed at 25°C. Binding of various receptors to the captured mAbs was determined using Scrubber 2.0c software. The results are shown in Table 11.

[0219] [Table 11]

[0220] Results: None of the anti-ACVR1 mAbs bound to any other human or mouse receptors, ACVRL1, BMPR1A, or BMPR1B. As shown in Table 11, these mAbs, REGN 5166, REGN 5167, and REGN 5168, were specific to ACVR1. The anti-ACVR1 mAbs REGN 5166, REGN 5167, and REGN 5168 bound to human and mouse ACVR1, as shown in Table 11. [Examples]

[0221] Cross-competition analysis of anti-ACVR1 antibodies A cross-competition assay was performed to evaluate the ability of a panel of three antibodies (REGN 5166, REGN 5167, and REGN 5168) to compete with each other for binding to human ACVR1. An isotype-matched antibody that does not bind to ACVR1 was used as a negative control.

[0222] As shown in Figure 1B, hACVR1.mmh was captured on a His antibody Octet® biosensor and then saturated by immersing it for 4 minutes in a well containing 50 μg / mL of ACVR1 mAb (referred to as the first mAb). Next, the first mAb-saturated biosensor was immersed for 3 minutes in wells containing various 50 μg / mL ACVR1 mAbs (referred to as the second mAbs). The binding of the second mAbs to the complex of hACVR1.mmh and the first mAb was determined, and the observed wavelength shift (nm) was reported in Figure 1A. The shaded cells represented bidirectional competition, whereas the white cells represented no competition.

[0223] These data indicated that REGN 5166 and REGN 5167 recognize partially overlapping epitopes (or that binding of one of them to ACVR1 sterically inhibits the binding of the other), and that it is highly likely that REGN 5168 and REGN 5167 bind to overlapping epitopes. No cross-competition was seen between REGN 5168 and REGN 5166, indicating that the epitope recognized by REGN 5168 is distinct from the epitope recognized by REGN 5166.

Example

[0224] Cell Binding by Flow Cytometry Using HEK293 / hACVR1-R206H Cells To evaluate cell binding by the anti-hACVR1 antibodies of the present invention, HEK293 cells (human fetal kidney, ATCC) were engineered to stably overexpress full-length human ACVR1 (amino acids 1-509 of accession number Q04771, R206H) and a BMP response element (BRE-Luc) fused to a firefly luciferase reporter. A single clone of the cell line was isolated and the resulting cell line was designated HEK293 / BRE-luc / hACVR1-R206H-clone H2. Hereinafter, it was referred to as HEK293 / hACVR1-R206H.

[0225] To evaluate the binding of the anti-ACVR1 antibody of the present invention to ACVR1 receptors expressed on the cell surface, 70 nM of the antibody was incubated in PBS (calcium and magnesium-free) (flow buffer) containing 2% FBS at 4°C for 30 minutes at a rate of 5 × 10⁶ 5 Cells were incubated with the primary antibody. After incubation with the primary antibody, cells were stained with 3.2 μg / mL of Alexa Fluor®-647 conjugate secondary antibody (Jackson ImmunoResearch Laboratories Inc., anti-human #109-607-003) at 4°C for 25 minutes. Cells were fixed using BD CytoFix® (Becton Dickinson, #554655) and analyzed with an IQue® Flow Cytometer (IntelliCyt®). Unstained and secondary antibody-only controls were also examined. Results were analyzed using ForeCyt® (IntelliCyt®) software to determine the geometric mean of fluorescence in viable cells, and the binding ratio was calculated by normalizing the geometric mean of the examination conditions by the corresponding geometric mean of unstained cells. The results are shown in Table 12.

[0226] [Table 12]

[0227] As shown in Table 12, all three antibodies of the present invention bound to HEK293 / hACVR1-R206H cells with binding ratios ranging from 1868 to 2247 times. The antibodies of the present invention bound to HEK293 parent cells with binding ratios ranging from 27 to 34 times. Samples of the human IgG control antibody and secondary antibody alone showed binding ratios ranging from 1 to 3 times. [Examples]

[0228] Functional inhibition of hACVR1 signaling in cell-based bioassays using hBMP7-activated HEK293 / BRE-luc / hACVR1-R206H-clonal H2 cells Activin A receptor type I, ACVR1 (also known as ActRI, ACVR1A, or Alk2), is a single-pass transmembrane receptor and a member of the type I BMP receptor superfamily of TGF-β receptors. Upon ligand binding, ACVR1 interacts with type II receptors, triggering signaling (Massague 1998, Massague et al. 2005).

[0229] To evaluate the anti-ACVR1 antibody-mediated inhibition of ACVR1, a bioassay was established using HEK293 cells (human fetal kidney, ATCC) stably overexpressing full-length human ACVR1 (amino acids 1-509 of accession number Q04771, R206H) and a BMP response element (BRE-Luc) fused to a firefly luciferase reporter. HEK293 cells endogenously express receptors and other components of the BMP signaling mechanism. A single clone of the cell lineage was isolated, and the resulting cell lineage was named HEK293 / BRE-luc / hACVR1-R206H-clone H2. Hereafter, it will be referred to as HEK293 / BRE-luc / hACVR1-R206H.

[0230] For the bioassay, HEK293 / BRE-luc / hACVR1-R206H cells were seeded at 10,000 cells / well in assay buffer (DMEM High glucose + 10% FBS + Pen / Strep / L-glutamine) in a 96-well plate and incubated at 37°C in 5% CO2. After 5 hours, anti-hACVR1 antibody or human IgG control antibody was serially diluted 1:4 in assay buffer from 300 nM to 73.2 pM (plus a sample containing buffer alone without the test molecule) and added to the cells, and incubated at 25°C for 30 minutes. After 30 minutes, 2 nM human bone morphogenetic protein 7 (hBMP7, R&D System 354-BP / C) was added to the cells. To obtain ligand-mediated dose-dependent activation, hBMP7 was serially diluted 1:3 from 100 nM to 1.7 pM in assay buffer (plus a sample containing buffer alone without the test molecule) and added to cells alone. After incubation overnight at 37°C in 5% CO2, luciferase activity was measured using OneGlo® reagent (Promega, #E6031) and an Envision plate reader (Perkin Elmer). The results were analyzed using nonlinear regression (4-parameter logistic regression) with Prism software (GraphPad) and EC 50 and IC 50 The value was obtained. The percentage of inhibition was calculated using the RLU value with the following equation:

number

[0231] In this equation, "RLU ベースライン " is the luminescence value from cells treated with 2nM hBMP7 that do not contain antibodies, and is "RLU". 実験 "RLU" is the lowest luminescence value recorded across the antibody concentration range tested. 背景 " is the luminescence value from cells that do not have any ligand or antibody. The results are shown in Table 13.

[0232] [Table 13]

[0233] Summary of results and conclusions: The three anti-human ACVR1 antibodies of the present invention were tested for their ability to inhibit BMP-7-mediated activation in HEK293 / BRE-luc / ACVR1-R206H cells. As shown in Table 13, each antibody of the present invention showed 109-111% inhibition of 2 nM hBMP7 and IC51. 50 The values ​​ranged from 980 pM to 1.3 nM. The IgG control antibody showed 39.5% inhibition against 2 nM hBMP7, and no concentration-dependent activity was observed within the test range, therefore IC was positive. 50 The value could not be determined. Based on the dose response of hBMP7, BMP7 activates HEK293 / BRE-luc / hACVR1-R206H cells and EC 50 The value was determined to be 1.22 nM.

[0234] References: Massague J, TGF-beta Signal Transduction, Annu.Rev.Biochem.1998.67:753~91, PMID:9759503 Massague J, Seoane J, Wotton D, Smad transcription factors, Genes Dev.2005, 19:2783~2810, PMID:16322555 [Examples]

[0235] Research on trauma-induced heterotopic ossification in burn and Achilles tendon rupture mouse models The anti-ACVR1 antibody of this disclosure was evaluated using a trauma-induced ectopic ossification model mouse. C57Bl / 6 (WT) and transgenic No MAHA mice (8-10 weeks old) were obtained from suppliers or Velocigene®. "No MAHA" was a special mouse species engineered to minimize the anti-human antibody immune response. Mice were acclimatized for 2 days at the Regeneron Animal Facility before the start of the experiment. Mice were anesthetized using inhaled isoflurane according to the protocol. Buprenorphine ER was administered once preoperatively (sustained release over 72 hours), with a second dose at 72 hours. The dorsal hair was trimmed using an automatic clipper, and the surgical site was sterile-prepared by alternating application of povidone-iodine and alcohol three times. A custom-made rectangular block (2 × 3 cm) was preheated in a 60°C water bath. The block was placed on the dorsal side of the mouse and held in place for 30 seconds to create a partial-layer thermal burn covering 30% of the total body surface area. The block was placed at the midpoint of the distance between the base of the cervical vertebrae and the lumbosacral vertebrae. The same procedure was performed on dummy animals, but instead the molds were placed in a 30°C water bath. After burns, each mouse was resuscitated with 1 ml of warm saline solution administered intraperitoneally (IP) and 0.5 ml of saline solution administered subcutaneously (SC).

[0236] All mice (excluding dummy animals) were subjected to Achilles tendon transection on one hind limb, which, simply put, was surgical severance of the Achilles tendon. The skin at the Achilles tendon transection site was prepared for sterile surgery (three repetitions of rubbing with 10% povidone-iodine followed by wiping with 70% alcohol). A 1 cm skin incision was made along the outer surface using sterile iris scissors.

[0237] Achilles tendon. The Achilles tendon was sterilely exposed from the distal part of the gastrocnemius muscle to its insertion point on the calcaneus. The tendon was sharply divided at its midpoint, and the skin incision was closed with non-absorbable sutures such as nylon. The sutures were removed or tissue adhesive was used after 10–14 days. Mice were weighed, and the wounds were examined daily for signs of infection.

[0238] The Achilles tendon rupture and burn treatment were performed on the same day, with one procedure following the other, and the order was not important.

[0239] MicroCT (microcomputed tomography) scans were performed using the Quantum GX microCT Imaging System (Revvity Inc.), with scan parameters of 18-second scan time, voxel size of 90 μm, and FOV of 45 mm.

[0240] For prophylactic / delayed medication studies, total ectopic bone mass was measured as follows: Total HO = [Volume of the calcaneus (HO + native skeleton) - calcaneus] 体側 ] + HO 非付着 (HO 踵 +HO 腓腹 )

[0241] Prophylactic drug administration study in C57BL / 6 mice To confirm that ACVR1 antibodies can inhibit WT ACVR1 in vivo under HO conditions, the anti-ACVR1 antibody REGN 5168 was tested in WT mice in a trauma-induced HO (tHO) burn tendon rupture model. A prophylactic drug study was conducted in C57BL / 6 mice. Mice were subcutaneously administered 25 mg / kg of anti-ACVR1 antibody REGN 5168 (n=9) or REGN 1945 (n=9, isotype control) once a week for a total of 13 doses starting 12 weeks after trauma (Achilles tendon rupture and burn). MicroCT (Quantum GX, Revvity) scans of the hind limbs were obtained 5 and 9 weeks after trauma and used to quantify ectopic bone mass. The results are shown in Table 14 and Figure 2.

[0242] [Table 14]

[0243] Results: Inhibition of ACVR1 by REGN 5168 significantly reduced total ectopic osteogenesis in wild-type mice 5 and 9 weeks after trauma in a prophylactic treatment compared to isotype controls for Achilles tendon rupture and burns, as shown in Table 14 and Figure 2. These results confirm that antibody-mediated inhibition of WT ACVR1 blocks tHO.

[0244] Measurement of serum hepcidin and iron. Serum hepcidin was measured using a mouse hepcidin ELISA kit (Intrinsic Biosciences) from serum obtained from end-of-life blood collected at the time of euthanasia, according to the manufacturer's protocol. Serum iron was measured using a QuantiChrom Iron Assay Kit (BioAssay Systems, DIFE-250) from serum obtained from end-of-life blood collected at the time of euthanasia, according to the manufacturer's protocol.

[0245] Results: Serum hepcidin levels were significantly reduced in WT mice treated prophylactically with REGN 5168 compared to isotype-controlled WT mice, as shown in Figure 3. Mean serum iron levels increased in WT mice treated prophylactically with REGN 5168 compared to isotype-controlled WT mice, but the trend in data was not statistically significant in this experiment (data not shown). These data demonstrate that the same ACVR1 antibody that inhibits wild-type ACVR1 in vivo can extend its physiological effects to systems other than HO. ACVR1 antibodies may be considered as a treatment option for trauma-induced HO and in other conditions where increasing iron levels is desirable.

[0246] No MAHA mouse prophylactic drug administration study A prophylactic drug administration study was conducted in No MAHA transgenic mice. Mice were subcutaneously administered either 25 mg / kg of REGN 1945 isotype control or the anti-ACVR1 antibody REGN 5168 or REGN 5166 once a week (n=9 in each group). The REGN 5166 group received eight antibody doses, the first two of which were 12.5 mg / kg. Mice treated with REGN 1945 or REGN 5168 received a total of 12 doses. MicroCT (Quantum GX, Revvity) scans of the hind limbs were obtained 3, 6, 9, and 12 weeks after trauma and used to quantify ectopic bone mass. The results are shown in Table 15 and Figure 4.

[0247] [Table 15]

[0248] Results: Inhibition of ACVR1 with REGN 5168 or REGN 5166 significantly reduced total ectopic osteogenesis in No MAHA transgenic mice at 3, 6, 9, and 12 weeks after trauma, compared to isotype controls in Achilles tendon rupture and burns, as shown in Table 15 and Figure 4.

[0249] Serum iron in No MAHA mice was measured using the QuantiChrom® Iron Assay Kit (BioAssay Systems, DIFE-250) in serum obtained from end-of-life blood collected at the time of euthanasia, according to the manufacturer's protocol. Results: Serum iron was significantly increased in No MAHA mice conferred with REGN 5168 compared to mice conferred with the isotype control REGN 1945, as shown in Figure 5, but not in mice conferred with REGN 5166.

[0250] Delayed drug delivery study in C57BL / 6 mice A delayed-dose study was conducted in C57BL / 6 mice. For the delayed-dose study, mice were subjected to Achilles tendon transection and burns, and heterotopic bone was allowed to develop until week 3. At that point, they were divided into either REGN 1945 (n=10) or REGN 5168 (n=8) treatment groups. Mice were subcutaneously treated once a week at 25 mg / kg for 8 weeks from the start of treatment, at which point treatment was discontinued. HO bone mass was measured using microCT scans of the hind limbs at weeks 3, 6, and 9. The results are shown in Table 16 and Figure 6.

[0251] [Table 16]

[0252] Results: Inhibition of ACVR1 by REGN 5168 significantly reduced total ectopic osteogenesis in wild-type mice 6 and 9 weeks after trauma in delayed treatment studies compared to isotype controls in Achilles tendon rupture and burns, as shown in Table 16 and Figure 6. [Examples]

[0253] Ectopic bone resection study mouse model Surgical resection studies were conducted in No MAHA transgenic mice.

[0254] In the excision study, mice were allowed to develop heterotopic bone 7 weeks after trauma (Achilles tendon rupture and burns). At that point, the heterotopic bone was excised, and the mice were divided into groups receiving either REGN 1945 or REGN 5168.

[0255] Mice were anesthetized using inhaled isoflurane or ketamine and xylazine. After the onset of anesthesia, buprenorphine ER was administered, the skin at the site of the ectopic bone was shaved, and the exposed skin was prepared for sterile surgery (three repetitions of rubbing with 10% povidone-iodine followed by wiping with 70% alcohol). A small incision (approximately 1 cm) was made at the site of the ectopic bone lesion to expose the lesion. Each ectopic bone lesion was excised with as little surrounding muscle and soft tissue as possible. Hemostasis was achieved by blood pressure control for 1-2 minutes or cauterization as needed. The incision was closed intermittently with (5-0 Vicryl) absorbable sutures, wound clips, or tissue adhesive.

[0256] Mice were administered either REGN 1945 or REGN 5168 at a dose of 25 mg / kg once a week for a total of 11 doses. Micro-CT scans of the hind limbs were used to monitor the progression of HO, obtained at 3 and 6 weeks after trauma, 7 weeks before and 7 weeks after resection, and 9, 12, 15, and 18 weeks after trauma.

[0257] Ectopic bone regrowth was evaluated by in vivo microCT imaging. MicroCT scans were performed using a Quantum GX (Perkin Elmer) scanner, with scan parameters of 18-second scan time, voxel size of 90 μm, and FOV of 45 mm.

[0258] For the resection study, total ectopic bone mass was defined as follows:

[0259] Total HO = [Volume of the calcaneus (HO + native skeleton)] + HO 非付着 (HO 踵 +HO 腓腹 )

[0260] The results are shown in Table 17 and Figure 7.

[0261] [Table 17]

[0262] Results: Inhibition of ACVR1 by the neutralizing antibody REGN 5168 in Achilles tendon rupture and burn tHO mouse models significantly inhibited HO recurrence at 12, 15, and 18 weeks after trauma (5, 8, and 11 weeks after excision) in No MAHA mice, as shown in Table 17 and Figure 7.

[0263] The present invention is not limited in scope by the specific embodiments described herein. In fact, various modifications of the present invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the accompanying claims.

[0264] array Sequence ID 1 caggtgcagc tggtggagtc tgggggaggc gtagtccagc ctgggaggtc cctgagactc 60 tcctgtgcag cgtctggatt caccttcagt gcgtattgga tgcactgggt ccgccaggct 120 ccaggcaagg ggctggagtg ggtggcagtt atatcgtatg atggaagtaa taaatactat 180 gcagactccg tgaagggccg attcaccatc tccagagacc attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggcggtgt actactgcgc aaaaggggac 300 gcctacgact cctggaggat actactcggc ggagattact acggcatgga tgtttggggc 360 cagggaacaa ctgtcaccgt ctcctcagcc tccaccaagg gcccatcggt cttccccctg 420 gcgccctgct ccaggagcac ctccgagagc acagccgccc tgggctgcct ggtcaaggac 480 tacttccccg aaccggtgac ggtgtcgtgg aactcaggcg ccctgaccag cggcgtgcac 540 accttcccgg ctgtcctaca gtcctcagga ctctactccc tcagcagcgt ggtgaccgtg 600 ccctccagca gcttgggcac gaagacctac acctgcaacg tagcacaa gcccagcaac accaaggtgg acaagagagt tgagtccaaa tatggtcccc catgcccacc ctgcccagca cctgagttcc tggggggacc atcagtcttc ctgttccccc caaaacccaa ggacactctc 780 atgatctccc ggacccctga ggtcacgtgc gtggtggtgg acgtgagcca ggagacccc 840 gaggtccagt tcaactggta cgtggatggc gtggaggtgc ataatgccaa gacaaagccg cgggaggagc agttcaacag cacgtaccgt gtggtcagcg tcctcaccgt cctgcaccag 960 1020. gactggctga acggcaagga gtacaagtgc aaggtctcca acaaaggcct cccgtcctcc atcgagaaaa ccatctccaa agccaaaggg cagccccgag agccacaggt gtacaccctg cccccatccc aggaggat gaccaagac caggtcagcc tgacctgcct ggtcaaaggc ttctacccca gcgacatcgc cgtggagtgg gagagcaatg ggcagccgga gaacaactac 1200 aagaccacgc ctcccgtgct ggactccgac ggctccttct tcctctacag caggctcacc 1260 gtggacaaga gcaggtggca ggaggggaat gtcttctcat gctccgtgat gcatgaggct 1320 ctgcacaacc actacacaca gaagtccctc tccctgtctc tgggtaaatg a 1371 Sequence ID 2 QVQLVESGGG VVQPGRSLRL SCAASGFTFS AYWMHWVRQA PGKGLEWVAV ISYDGSNKYY 60 ADSVKGRFTI SRDHSKNTLY LQMNSLRAED TAVYYCAKGD AYDSWRILLG GDYYGMDVWG 120 QGTTVTVSSA STKGPSVFPL APCSRSTSES TAALGCLVKD YFPEPVTVSW NSGALTSGVH 180 TFPAVLQSSG LYSLSSVVTV PSSSLGTKTY TCNVDHKPSN TKVDKRVESK YGPPCPPCPA 240 PEFLGGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSQEDP EVQFNWYVDG VEVHNAKTKP 300 REEQFNSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKGLPSS IEKTISKAKG QPREPQVYTL 360 PPSQEEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSRLT 420 VDKSRWQEGN VFSCSVMHEA LHNHYTQKSL SLSLGK 456 Sequence ID 3 gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60 gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60 ctctcctgca gggccagtca gagtgttagc agcagctact tagcctggta ccagcagaaa 120 ctctcctgca gggccagtca gagtgttagc agcagctact tagcctggta ccagcagaaa 120 cctggccagg ctcccaggcc cctcatctat ggcgcatcca gcagggccac tggcatccca 180 cctggccagg ctcccaggcc cctcatctat ggcgcatcca gcagggccac tggcatccca 180 gacagattca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240 gacagattca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240 cctgaagatt ttgcagtgta ttactgtcag cagggggggg acgcccctcc ttacactttc 300 cctgaagatt ttgcagtgta ttactgtcag cagggggggg acgcccctcc ttacactttc 300 ggcggaggga ccaaggttga gatcaaacga actgtggctg caccatctgt cttcatcttc 360 ggcggaggga ccaaggttga gatcaaacga actgtggctg caccatctgt cttcatcttc 360 ccgccatctg atgagcagtt gaaatctgga actgcctctg ttgtgtgcct gctgaataac 420 ccgccatctg atgagcagtt gaaatctgga actgcctctg ttgtgtgcct gctgaataac 420 ttctatccca gagaggccaa agtacagtgg aaggtggata acgccctcca atcgggtaac 480 ttctatccca gagaggccaa agtacagtgg aaggtggata acgccctcca atcgggtaac 480 tcccaggaga gtgtcacaga gcaggacagc aaggacagca cctacagcct cagcagcacc 540 tcccaggaga gtgtcacaga gcaggacagc aaggacagca cctacagcct cagcagcacc 540 ctgacgctga gcaaagcaga ctacgagaaa cacaaagtct acgcctgcga agtcacccat 600 ctgacgctga gcaaagcaga ctacgagaaa cacaaagtct acgcctgcga agtcacccat 600 cagggcctga gctcgcccgt cacaaagagc ttcaacaggg gagagtgtta g 651 cagggcctga gctcgcccgt cacaaagagc ttcaacaggg gagagtgtta g 651 配列番号4 Accession No. 4 EIVLTQSPGT LSLSPGERAT LSCRASQSVS SSYLAWYQQK PGQAPRPLIY GASSRATGIP 60 EIVLTQSPGT LSLSPGERAT LSCRASQSVS SSYLAWYQQK PGQAPRPLIY GASSRATGIP 60 DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QGGDAPPYTF GGGTKVEIKR TVAAPSVFIF 120 PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN SQESVTEQDS KDSTYSLSST 180 LTLSKADYEK HKVYACEVTH QGLSSPVTKS FNRGEC 216 Sequence number 5 cagctgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60 acctgcactg tctctggtgg ctccatcacg agtagtagtt actactgggc gtggatccgc 120 cagcccccag ggaaggggct ggagtggatt gggaagatct attatagtgg gagcacccat 180 tacaacccgt ccctcaagag tcgagtcacc atatccgtag acacgtccaa gaaccagttc 240 tccctgaagt tgagttctgt gaccgccgca gacacggcgg tgtactactg cgccagagtt 300 ggaggacacg gctacggaga ctccgcaggg ttagccttcg atatctgggg tcagggtaca 360 atggtcaccg tctcctcagc ctccaccaag ggcccatcgg tcttccccct ggcgccctgc 420 tccaggagca cctccgagag cacagccgcc ctgggctgcc tggtcaagga ctacttcccc 480 gaaccggtga cggtgtcgtg gaactcaggc gccctgacca gcggcgtgca caccttcccg 540 gctgtcctac agtcctcagg actctactcc ctcagcagcg tggtgaccgt gccctccagc 600 agcttgggca cgaagaccta cacctgcaac gtagatcaca agcccagcaa caccaaggtg 660 gacaagagag ttgagtccaa atatggtccc ccatgcccac cctgcccagc acctgagttc 720 ctggggggac catcagtctt cctgttcccc ccaaaaccca aggacactct catgatctcc 780 cggacccctg aggtcacgtg cgtggtggtg gacgtgagcc aggaagaccc cgaggtccag 840 ttcaactggt acgtggatgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag 900 cagttcaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg 960 aacggcaagg agtacaagtg caaggtctcc aacaaaggcc tcccgtcctc catcgagaaa 1020 accatctcca aagccaaagg gcagccccga gagccacagg tgtacaccct gcccccatcc 1080 caggaggaga tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctacccc 1140 agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg 1200 cctcccgtgc tggactccga cggctccttc ttcctctaca gcaggctcac cgtggacaag 1260 agcaggtggc aggaggggaa tgtcttctca tgctccgtga tgcatgaggc tctgcacaac 1320 cactacacac agaagtccct ctccctgtct ctgggtaaat ga 1362 Sequence number 6 QLQLQESGPG LVKPSETLSL TCTVSGGSIT SSSYYWAWIR QPPGKGLEWI GKIYYSGSTH 60 YNPSLKSRVT ISVDTSKNQF SLKLSSVTAA DTAVYYCARV GGHGYGDSAG LAFDIWGQGT 120 MVTVSSASTK GPSVFPLAPC SRSTSESTAA LGCLVKDYFP EPVTVSWNSG ALTSGVHTFP 180 AVLQSSGLYS LSSVVTVPSS SLGTKTYTCN VDHKPSNTKV DKRVESKYGP PCPPCPAPEF 240 LGGPSVFLFP PKPKDTLMIS RTPEVTCVVV DVSQEDPEVQ FNWYVDGVEV HNAKTKPREE 300 QFNSTYRVVS VLTVLHQDWL NGKEYKCKVS NKGLPSSIEK TISKAKGQPR EPQVYTLPPS 360 QEEMTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF FLYSRLTVDK 420 SRWQEGNVFS CSVMHEALHN HYTQKSLSLS LGK 453 Sequence number 7 gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60 ctctcctgca gggccagtca gagtgttagc agcgccttct tagcctggta ccagcagaaa 120 cctggccagg ctcccaggct cctcatctat ggtgcatcct acaggcacac tggcatccca 180 gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240 cctgaagatt ttgcagtgta ttactgtcag cactacggag ttggtcctag gactttcggc 300 ggagggacca aggttgagat caaacgaact gtggctgcac catctgtctt catcttcccg 360 ccatctgatg agcagttgaa atctggaact gcctctgttg tgtgcctgct gaataacttc 420 tatcccagag aggccaaagt acagtggaag gtggataacg ccctccaatc gggtaactcc 480 caggagagtg tcacagagca ggacagcaag gacagcacct acagcctcag cagcaccctg 540 acgctgagca aagcagacta cgagaaacac aaagtctacg cctgcgaagt cacccatcag 600 S ggcctgagct cgcccgtcac aaagagcttc aacaggggag agtgttag 648 SEQ ID NO:8 EIVLTQSPGT LSLSPGERAT LSCRASQSVS SAFLAWYQQK PGQAPRLLIY GASYRHTGIP 60 DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ HYGVGPRTFG GGTKVEIKRT VAAPSVFIFP 120 PSDEQLKSGT ASVVCLLNNF YPREAKVQWK VDNALQSGNS QESVTEQDSK DSTYSLSSTL 180 It should be noted that there may be some inaccuracies in the translation as the original text seems to be a nucleotide sequence or some other biological sequence data which requires more context for a more precise translation. The above is a literal translation based on the rules.TLSKADYEKH KVYACEVTHQ GLSSPVTKSF NRGEC 215 Sequence number 9 caggtgcagc tggtgcagtc tggggctgag gtgaaggagc ctggggcctc agtgaaggtt 60 tcctgcaagg catctggata caccttcgcg gagtactata tgcactgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggagcg attaacccta gtggtagtca tacaagctac 180 gcacagaagt tccagggcag agtcaccatg accagggaca cgtccacgag cacagtctac 240 atggagctga gcagcctgag atctgaggac acggcggtgt actactgcgc tagaagtatt 300 agtggcaaac gaggtggcga ttactacggc atggatgttt ggggccaggg aacaactgtc 360 accgtctcct cagcctccac caagggccca tcggtcttcc ccctggcgcc ctgctccagg 420 agcacctccg agagcacagc cgccctgggc tgcctggtca aggactactt ccccgaaccg 480 gtgacggtgt cgtggaactc aggcgccctg accagcggcg tgcacacctt cccggctgtc 540 [[ID=2又]]ctacagtcct caggactcta ctccctcagc agcgtggtga ccgtgccctc cagcagcttg 600 ggcacgaaga cctacacctg caacgtagat cacaagccca gcaacaccaa ggtggacaag 660 (注:你提供的原文中“[[ID=2又]]”可能有误,我按原样翻译了。)agagttgagt ccaaatatgg tcccccatgc ccaccctgcc cagcacctga gttcctgggg 720 ggaccatcag tcttcctgtt ccccccaaaa cccaaggaca ctctcatgat ctcccggacc 780 cctgaggtca cgtgcgtggt ggtggacgtg agccaggaag accccgaggt ccagttcaac 840 tggtacgtgg atggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagttc 900 aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaacggc 960 aaggagtaca agtgcaaggt ctccaacaaa ggcctcccgt cctccatcga gaaaaccatc 1020 tccaaagcca aagggcagcc ccgagagcca caggtgtaca ccctgccccc atcccaggag 1080 gagatgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta ccccagcgac 1140 atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc 1200 gtgctggact ccgacggctc cttcttcctc tacagcaggc tcaccgtgga caagagcagg 1260 tggcaggagg ggaatgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 1320 acacagaagt ccctctccct gtctctgggt aaatga 1356 SEQ ID NO: 10 QVQLVQSGAE VKEPGASVKV SCKASGYTFA EYYMHWVRQA PGQGLEWMGA INPSGSHTSY 60 AQKFQGRVTM TRDTSTSTVY MELSSLRSED TAVYYCARSI SGKRGGDYYG MDVWGQGTTV 120 TVSSASTKGP SVFPLAPCSR STSESTAALG CLVKDYFPEP VTVSWNSGAL TSGVHTFPAV 180 LQSSGLYSLS SVVTVPSSSL GTKTYTCNVD HKPSNTKVDK RVESKYGPPC PCPPAPEFLG 240 GPSVFLFPPK PKDTLMISRT PEVTCVVVDV SQEDPEVQFN WYVDGVEVHN AKTKPREEQF 300 NSTYRVVSVL TVLHQDWLNG KEYKCKVSNK GLPSSIEKTI SKAKGQPREP QVYTLPSQE 360 EMTKNQVSLT CLVKGFYPSD ​​IAVEWESNGQ PENNYKTTPP VLDSDGSFFL YSRLTVDKSR 420 WQEGNVFSCS VMHEALHNHY TQKSLSLSLG K 451 Sequence ID 11 gacatccaga tgacccagtc tccttccacc ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgcc gggccagtca gagtattagt agctggttgg cctggtatca gcagaaacca 120 gggaaagccc ctaagctcct gatctataaa gcctccagct tggaaagtgg ggtcccatca 180 aggttcagcg gcagtggatc tgggacagaa ttcactctca ccatcagcag cctgcagcct 240 gatgattttg caacttatta ctgccagcag tacagcatct tccctttcac ttttggcgga 300 gggaccaagg ttgagatcaa acgaactgtg gctgcaccat ctgtcttcat cttcccgcca 360 tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 420 cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 480 gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 540 ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600 ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gttag 645 SEQ ID NO: 12 DIQMTQSPST LSASVGDRVT ITCRASQSIS SWLAWYQQKP GKAPKLLIYK ASSLESGVPS 60 RFSGSGSGTE FTLTISSLQP DDFATYYCQQ YSIFPFTFGG GTKVEIKRTV AAPSVFIFPP 120 SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT 180 LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC 214 SEQ ID NO: 13 caggtgcagc tggtggagtc tgggggaggc gtagtccagc ctgggaggtc cctgagactc 60 tcctgtgcag cgtctggatt caccttcagt gcgtattgga tgcactgggt ccgccaggct 120 ccaggcaagg ggctggagtg ggtggcagtt atatcgtatg atggaagtaa taaatactat 180 gcagactccg tgaagggccg attcaccatc tccagagacc attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggcggtgt actactgcgc aaaaggggac 300 gcctacgact cctggaggat actactcggc ggagattact acggcatgga tgtttggggc 360 cagggaacaa ctgtcaccgt ctcctca 387 Sequence ID 14 QVQLVESGGG VVQPGRSLRL SCAASGFTFS AYWMHWVRQA PGKGLEWVAV ISYDGSNKYY 60 ADSVKGRFTI SRDHSKNTLY LQMNSLRAED TAVYYCAKGD AYDSWRILLG GDYYGMDVWG 120 QGTTVTVSS 129 Sequence ID 15 ggattcacct tcagtgcgta ttgg 24 Sequence ID 16 GFTFSAYW 8 Sequence ID 17 atatcgtatg atggaagtaa taaa 24 Sequence ID 18 ISYDGSNK 8 Sequence ID 19 gcaaaagggg acgcctacga ctcctggagg atactactcg gcggagatta ctacggcatg 60 gatgtt 66 Sequence ID 20 AKGDAYDSWR ILLGGDYYGM DV 22 Sequence ID 21 gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60 ctctcctgca gggccagtca gagtgttagc agcagctact tagcctggta ccagcagaaa 120 cctggccagg ctcccaggcc cctcatctat ggcgcatcca gcagggccac tggcatccca 180 gacagattca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240 cctgaagatt ttgcagtgta ttactgtcag cagggggggg acgcccctcc ttacactttc 300 ggcggaggga ccaaggttga gatcaaa 327 Sequence ID 22 EIVLTQSPGT LSLSPGERAT LSCRASQSVS SSYLAWYQQK PGQAPRPLIY GASSRATGIP 60 DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QGGDAPPYTF GGGTKVEIK 109 Sequence ID 23 cagagtgtta gcagcagcta c 21 Sequence ID 24 QSVSSSY 7 Sequence ID 25 GGCGCATCC 9 Sequence ID 26 GAS 3 Sequence ID 27 cagcaggggg gggacgcccc tccttacact 30 Sequence ID 28 QQGGDAPPYT 10 Sequence ID 29 cagctgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60 acctgcactg tctctggtgg ctccatcacg agtagtagtt actactggggc gtggatccgc 120 cagcccccag ggaaggggct ggagtggatt gggaagatct attatagtgg gagcacccat 180 tacaacccgt ccctcaagag tcgagtcacc atatccgtag acacgtccaa gaaccagttc 240 tccctgaagt tgagttctgt gaccgccgca gacacggcgg tgtactactg cgccagagtt 300 ggaggacacg gctacggaga ctccgcaggg ttagccttcg atatctgggg tcagggtaca 360 atggtcaccg tctcctca 378 Sequence ID 30 QLQLQESGPG LVKPSETLSL TCTVSGGSIT SSSYYWAWIR QPPGKGLEWI GKIYYSGSTH 60 YNPSLKSRVT ISVDTSKNQF SLKLSSVTAA DTAVYYCARV GGHGYGDSAG LAFDIWGQGT 120 MVTVSS 126 Sequence ID 31 ggtggctcca tcacgagtag tagttactac 30 Sequence ID 32 GGSITSSSYY 10 Sequence ID 33 atctattata gtgggagcac c 21 Sequence ID 34 IYYSGST 7 Sequence ID 35 gccagagttg gaggacacgg ctacggagac tccgcagggt tagccttcga tatc 54 Sequence ID 36 ARVGGHGYGD SAGLAFDI 18 Sequence ID 37 gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60 ctctcctgca gggccagtca gagtgttagc agcgccttct tagcctggta ccagcagaaa 120 cctggccagg ctcccaggct cctcatctat ggtgcatcct acaggcacac tggcatccca 180 gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240 cctgaagatt ttgcagtgta ttactgtcag cactacggag ttggtcctag gactttcggc 300 ggagggacca aggttgagat caaa 324 Sequence ID 38 EIVLTQSPGT LSLSPGERAT LSCRASQSVS SAFLAWYQQK PGQAPRLLIY GASYRHTGIP 60 DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ HYGVGPRTFG GGTKVEIK 108 Sequence ID 39 cagagtgtta gcagcgcctt c 21 Sequence ID 40 QSVSSAF 7 Sequence ID 41 GGTGCATCC 9 Sequence ID 42 GAS 3 Sequence ID 43 cagcactacg gagttggtcc taggact 27 Sequence ID 44 QHYGVGPRT 9 Sequence ID 45 caggtgcagc tggtgcagtc tggggctgag gtgaaggagc ctggggcctc agtgaaggtt 60 tcctgcaagg catctggata caccttcgcg gagtactata tgcactgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggagcg attaacccta gtggtagtca tacaagctac 180 gcacagaagt tccagggcag agtcaccatg accagggaca cgtccacgag cacagtctac 240 atggagctga gcagcctgag atctgaggac acggcggtgt actactgcgc tagaagtatt 300 agtggcaaac gaggtggcga ttactacggc atggatgttt ggggccaggg aacaactgtc 360 accgtctcct ca 372 Sequence ID 46 QVQLVQSGAE VKEPGASVKV SCKASGYTFA EYYMHWVRQA PGQGLEWMGA INPSGSHTSY 60 AQKFQGRVTM TRDTSTSTVY MELSSLRSED TAVYYCARSI SGKRGGDYYG MDVWGQGTTV 120 TVSS 124 Sequence ID 47 ggatacacct tcgcggagta ctat 24 Sequence ID 48 GYTFAEYY 8 Sequence ID 49 attaacccta gtggtagtca taca 24 Sequence ID 50 INPSGSHT 8 Sequence ID 51 gctagaagta ttagtggcaa acgaggtggc gattactacg gcatggatgt t 51 Sequence ID 52 ARSISGKRGG DYYGMDV 17 Sequence ID 53 gacatccaga tgacccagtc tccttccacc ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgcc gggccagtca gagtattagt agctggttgg cctggtatca gcagaaacca 120 gggaaagccc ctaagctcct gatctataaa gcctccagct tggaaagtgg ggtcccatca 180 aggttcagcg gcagtggatc tgggacagaa ttcactctca ccatcagcag cctgcagcct 240 gatgattttg caacttatta ctgccagcag tacagcatct tccctttcac ttttggcgga 300 gggaccaagg ttgagatcaa a 321 Sequence ID 54 DIQMTQSPST LSASVGDRVT ITCRASQSIS SWLAWYQQKP GKAPKLLIYK ASSLESGVPS 60 RFSGSGSGTE FTLTISSLQP DDFATYYCQQ YSIFPFTFGG GTKVEIK 107 Sequence ID 55 cagagtatta gtagctgg 18 Sequence ID 56 QSISSW 6 Sequence ID 57 AAAGCCTCC 9 Sequence ID 58 KAS 3 Sequence ID 59 cagcagtaca gcatcttccc tttcact 27 Sequence ID 60 QQYSIFPFT 9 Sequence ID 61 MVDGVMILPV LIMIALPSPS MEDEKPKVNP KLYMCVCEGL SCGNEDHCEG QQCFSSLSIN 60 DGFHVYQKGC FQVYEQGKMT CKTPPSPGQA VECCQGDWCN RNITAQLPTK GKSFPGTQNF 120 HLEVGLIILS VVFAVCLLAC LLGVALRKFK RRNQERLNPR DVEYGTIEGL ITTNVGDSTL 180 ADLLDHSCTS GSGSGLPFLV QRTVARQITL LECVGKGRYG EVWRGSWQGE NVAVKIFSSR 240 DEKSWFRETE LYNTVMLRHE NILGFIASDM TSRHSSTQLW LITHYHEMGS LYDYLQLTTL 300 DTVSCLRIVL SIASGLAHLH IEIFGTQGKP AIAHRDLKSK NILVKKNGQC CIADLGLAVM 360 HSQSTNQLDV GNNPRVGTKR YMAPEVLDET IQVDCFDSYK RVDIWAFGLV LWEVARRMVS 420 NGIVEDYKPP FYDVVPNDPS FEDMRKVVCV DQQRPNIPNR WFSDPTLTSL AKLMKECWYQ 480 NPSARLTALR IKKTLTKIDN SLDKLKTDC 509 Sequence ID 62 MVDGVMILPV LMMMAFPSPS VEDEKPKVNQ KLYMCVCEGL SCGNEDHCEG QQCFSSLSIN 60 DGFHVYQKGC FQVYEQGKMT CKTPPSPGQA VECCQGDWCN RNITAQLPTK GKSFPGTQNF 120 HLEVGLIILS VVFAVCLLAC ILGVALRKFK RRNQERLNPR DVEYGTIEGL ITTNVGDSTL 180 AELLDHSCTS GSGSGLPFLV QRTVARQITL LECVGKGRYG EVWRGSWQGE NVAVKIFSSR 240 DEKSWFRETE LYNTVMLRHE NILGFIASDM TSRHSSTQLW LITHYHEMGS LYDYLQLTTL 300 DTVSCLRIVL SIASGLAHLH IEIFGTQGKS AIAHRDLKSK NILVKKNGQC CIADLGLAVM 360 HSQSTNQLDV GNNPRVGTKR YMAPEVLDET IQVDCFDSYK RVDIWAFGLV LWEVARRMVS 420 NGIVEDYKPP FYDVVPNDPS FEDMRKVVCV DQQRPNIPNR WFSDPTLTSL AKLMKECWYQ 480 NPSARLTALR IKKTLTKIDN SLDKLKTDC 509 Sequence ID 63 MEDEKPKVNP KLYMCVCEGL SCGNEDHCEG QQCFSSLSIN DGFHVYQKGC FQVYEQGKMT 60 CKTPPSPGQA VECCQGDWCN RNITAQLPTK GKSFPGTQNF HLEEQKLISE EDLGGEQKLI 120 SEEDLHHHHH H 131 Sequence ID 64 MEDEKPKVNP KLYMCVCEGL SCGNEDHCEG QQCFSSLSIN DGFHVYQKGC FQVYEQGKMT 60 CKTPPSPGQA VECCQGDWCN RNITAQLPTK GKSFPGTQNF HLEEPRGPTI KPCPPCKCPA 120 PNLLGGPSVF IFPPKIKDVL MISLSPIVTC VVVDVSEDDP DVQISWFVNN VEVHTAQTQT 180 HREDYNSTLR VVSALPIQHQ DWMSGKEFKC KVNNKDLPAP IERTISKPKG SVRAPQVYVL 240 PPPEEEMTKK QVTLTCMVTD FMPEDIYVEW TNNGKTELNY KNTEPVLDSD GSYFMYSKLR 300 VEKKNWVERN SYSCSVVHEG LHNHHTTKSF SRTPGK 336 Sequence ID 65 VEDEKPKVNQ KLYMCVCEGL SCGNEDHCEG QQCFSSLSIN DGFHVYQKGC FQVYEQGKMT 60 CKTPPSPGQA VECCQGDWCN RNITAQLPTK GKSFPGTQNF HLEEQKLISE EDLGGEQKLI 120 SEEDLHHHHH H 131

Claims

1. An isolated antibody or its antigen-binding fragment that specifically binds to the activin A receptor type 1 (ACVR1) protein and / or its mutant, and which interacts with one or more amino acids contained within the extracellular domain of ACVR1 (amino acids 21-123 of SEQ ID NO: 61) and binds to cells expressing the full-length ACVR1 protein and / or its mutant.

2. The isolated antibody or antigen-binding fragment according to claim 1, wherein the full-length ACVR1 protein or its mutant is the full-length human ACVR protein or its mutant.

3. The fully-length human ACVR1 protein comprises amino acids 21 to 509 of SEQ ID NO: 61, wherein the isolated antibody or its antigen-binding fragment is as described in claim 2.

4. The isolated antibody or its antigen-binding fragment according to claim 1 or 2, wherein the mutant ACVR1 protein comprises a mutation selected from the group consisting of ACVR1 L196P, delP197_F198insL, R202I, R206H, Q207E, R258S, R258G, G325A, G328E, G328R, G328W, G356D, and R375P of SEQ ID NO:

61.

5. The isolated antibody or antigen-binding fragment according to claim 4, which binds to the ACVR1(R206H) protein and inhibits ACVR1(R206H)-mediated bone morphogenetic protein (BMP) signaling.

6. An isolated antibody or its antigen-binding fragment that specifically binds to the ACVR1 protein, wherein (i) it binds to cells expressing human ACVR1; and / or (ii) it binds to ACVR1 and inhibits ACVR1-mediated bone morphogenetic protein (BMP) signaling.

7. The antibody or its antigen-binding fragment according to any one of claims 1 to 6, wherein the antibody is a fully human monoclonal antibody.

8. The antibody must (a) be a fully human monoclonal antibody; and (b) have a dissociation constant (K) less than 15 nM, less than 10 nM, less than 5 nM, less than 3 nM, less than 2 nM, less than 1 nM, less than 0.5 nM, less than 0.3 nM, less than 0.2 nM, or less than 0.1 nM as measured by surface plasmon resonance assay. Ｄ (c) To bind to the human ACVR1 extracellular domain (SEQ ID NO: 64) fused to mFc at 37°C; (c) K levels less than 50 nM, less than 10 nM, less than 5 nM, less than 3 nM, less than 2 nM, less than 1 nM, and less than 0.5 nM when measured in a surface plasmon resonance assay. Ｄ (d) binding to the human ACVR1 extracellular domain fused to the myc-myc-hexahis tag at 37°C (e.g., SEQ ID NO: 63); (d) K levels less than 50 nM, less than 10 nM, less than 5 nM, less than 3 nM, less than 2 nM, less than 1 nM, and less than 0.5 nM when measured in a surface plasmon resonance assay. Ｄ (e) binding to the mouse ACVR1 extracellular domain fused to the myc-myc-hexahis tag at 37°C (e.g., SEQ ID NO: 65); (e) K < 10 nM, < 5 nM, < 3 nM, < 2 nM, < 1 nM, < 0.5 nM, < 0.2 nM, or < 0.1 nM Ｄ (f) To bind to the mouse ACVR1 extracellular domain fused to mFc at 37°C; (g) To bind to cells expressing human ACVR1 protein or human ACVR(R206H) protein; (g) To achieve IC250 or less than 10 nM, 5 nM, 3 nM, 2 nM, or 1 nM as measured in a cell-based bioassay. ５０ (h) inhibiting the activation of cells expressing human ACVR1 (R206H) by human activin A; (h) IC2012 < 10 nM, < 5 nM, < 3 nM, < 2 nM, or < 1 nM as measured in a cell-based bioassay. ５０ The antibody or antigen-binding fragment according to any one of claims 1 to 6, wherein it inhibits the activation of cells expressing human ACVR1 (R206H) by human BMP7; and has one or more properties selected from the group consisting of (i) an HCVR comprising an amino acid sequence selected from the group consisting of HCVR sequences listed in Table 1 and an LCVR comprising an amino acid sequence selected from the group consisting of LCVR sequences listed in Table 1.

9. The antibody or antigen-binding fragment according to any one of claims 1 to 8, comprising three heavy chain complementarity-determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable region (HCVR); and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within a light chain variable region (LCVR), wherein the HCVR has an amino acid sequence selected from the group consisting of HCVR sequences listed in Table 1.

10. The antibody or antigen-binding fragment thereof according to claim 9, comprising an LCVR having an amino acid sequence selected from the group consisting of LCVR sequences listed in Table 1.

11. (a) HCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 32, and 48; (b) HCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 34, and 50; (c) HCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 36, and 52; (d) An LCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 24, 40, and 56; (e) An LCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 26, 42, and 58; Furthermore (f) LCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 28, 44, and 60 The antibody or antigen-binding fragment thereof according to claim 9 or 10, comprising one or more of the group consisting of the following.

12. The antibody or antigen-binding fragment according to claim 11, comprising an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 14 / 22, 30 / 38, and 46 / 54.

13. The antibody or antigen-binding fragment thereof according to claim 12, comprising a CDR selected from the group consisting of (a) SEQ ID NOs: 16, 18, 20, 24, 26, and 28; (b) SEQ ID NOs: 32, 34, 36, 40, 42, and 44; and (c) SEQ ID NOs: 48, 50, 52, 56, 58, and 60.

14. The antibody or antigen-binding fragment thereof according to claim 13, comprising an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 30 / 38 and 46 / 54.

15. An antibody or antigen-binding fragment that binds to ACVR1, wherein the antibody or antigen-binding fragment comprises three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within HCVR and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within LCVR; HCVR is, (i) an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 30, and 46; (ii) an amino acid sequence having at least 90% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 30, and 46; (iii) an amino acid sequence having at least 95% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 30, and 46; or (iv) Amino acid sequences selected from the group consisting of SEQ ID NOs. 14, 30, and 46, having 12 or fewer amino acid substitutions. Includes, LCVR is, (a) an amino acid sequence selected from the group consisting of SEQ ID NOs: 22, 38, and 54; (b) an amino acid sequence having at least 90% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 22, 38, and 54; (c) an amino acid sequence having at least 95% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 22, 38, and 54; or (d) Amino acid sequences selected from the group consisting of SEQ ID NOs. 22, 38, and 54, which have 10 or fewer amino acid substitutions. An antibody or its antigen-binding fragment, including an antibody.

16. The antibody or antigen-binding fragment thereof according to claim 15, comprising an HCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 30, and 46.

17. The antibody or antigen-binding fragment thereof according to claim 15, comprising an LCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 22, 38, and 54.

18. The antibody or antigen-binding fragment according to any one of claims 15 to 17, comprising three CDRs contained in an HCVR selected from the group consisting of SEQ ID NOs: 14, 30, and 46; and three CDRs contained in an LCVR selected from the group consisting of SEQ ID NOs: 22, 38, and 54.

19. An antibody or antigen-binding fragment according to any one of claims 15 to 18, comprising an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 14 / 22, 30 / 38, and 46 / 54.

20. (a) HCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 32, and 48; (b) HCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 34, and 50; (c) HCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 36, and 52; (d) An LCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 24, 40, and 56; (e) an LCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 26, 42, and 58; and (f) LCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 28, 44, and 60 The antibody or antigen-binding fragment thereof according to claim 15, comprising:

21. The antibody or antigen-binding fragment thereof according to claim 20, comprising a CDR selected from the group consisting of (a) SEQ ID NOs: 16, 18, 20, 24, 26, and 28; (b) SEQ ID NOs: 32, 34, 36, 40, 42, and 44; and (c) SEQ ID NOs: 48, 50, 52, 56, 58, and 60.

22. The antibody or antigen-binding fragment according to claim 21, comprising an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 14 / 22, 30 / 38, and 46 / 54.

23. An isolated monoclonal antibody or antigen-binding fragment thereof that inhibits ACVR-mediated and / or ACVR1(R206H)-mediated osteomorphogenetic protein (BMP) signaling, comprising three CDRs of HCVR and three CDRs of LCVR, wherein the HCVR has an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 30, and 46, and the LCVR has an amino acid sequence selected from the group consisting of SEQ ID NOs: 22, 38, and 54.

24. An antibody or antigen-binding fragment thereof that competes with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 23 for binding to ACVR1.

25. An antibody or antigen-binding fragment that binds to the same epitope as the antibody or antigen-binding fragment described in any one of claims 1 to 23.

26. A pharmaceutical composition comprising an isolated antibody bound to ACVR1 according to any one of claims 1 to 25 or an antigen-binding fragment thereof, and a pharmaceutically acceptable carrier or diluent.

27. An isolated polynucleotide molecule comprising a polynucleotide sequence encoding the HCVR of an antibody according to any one of claims 1 to 25.

28. An isolated polynucleotide molecule comprising a polynucleotide sequence encoding the LCVR of an antibody according to any one of claims 1 to 25.

29. A vector comprising the polynucleotide sequence described in claim 27 and / or the polynucleotide sequence described in claim 27.

30. A host cell expressing the vector described in claim 29.

31. A method for producing an anti-ACVR1 antibody or an antigen-binding fragment thereof, comprising growing a host cell according to claim 30 under conditions that enable the production of the antibody or fragment, and recovering the antibody or fragment thus produced.

32. The method according to claim 31, further comprising formulating an antibody or an antigen-binding fragment thereof as a pharmaceutical composition comprising an acceptable carrier.

33. A method for treating, preventing, reversing, or reducing the recurrence of at least one symptom or sign of ACVR1-related disease or disorder, comprising administering to a subject in need of treatment a therapeutically effective amount of a pharmaceutical composition comprising an antibody or antigen-binding fragment according to any one of claims 1 to 25.

34. The method according to claim 33, wherein the ACVR1-related disease or disorder is selected from the group consisting of heterotopic ossification, trauma-induced heterotopic ossification, heterotopic ossification, osteodysplasia, anemia, and diffuse pontine glioma.

35. The method according to claim 33 or 34, wherein the pharmaceutical composition is administered prophylactically or therapeutically to a subject in need of prevention or treatment.

36. The method according to any one of claims 33 to 35, wherein the pharmaceutical composition is administered in combination with a second therapeutic agent.

37. The method according to claim 36, wherein the second therapeutic agent is selected from the group consisting of an anti-activin A inhibitor, an anti-BMP7 antibody or its antigen-binding fragment, an anti-ACVR2 antibody or its antigen-binding fragment, an anti-inflammatory agent, a steroid, a bisphosphonate, a muscle relaxant, and a retinoic acid receptor (RAR) gamma agonist, lifestyle modifications, and nutritional supplements.

38. The method according to any one of claims 33 to 37, wherein the pharmaceutical composition is administered subcutaneously, intravenously, intradermally, intraperitoneally, intramuscularly, or intraventricularly.

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