Anti-ALK1 Antibodies and Methods of Use Thereof

Abstract

Antibodies that bind to activin A receptor-like type 1 (ALK1) are provided. Treatments and uses that include administering such antibodies are also provided. [Figure 1] TIFF2025504500000008.tif117169

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 302,408, filed January 24, 2022, and U.S. Provisional Patent Application No. 63 / 335,444, filed April 27, 2022, the disclosures of which are hereby incorporated by reference in their entireties.

[0002] Antibodies that bind to activin A receptor-like type 1 (ALK1) are provided. Treatments and uses that include administering such antibodies are also provided. [Background technology]

[0003] The following discussion is presented to aid the reader in understanding the present disclosure and is not admitted to state or constitute prior art.

[0004] Activin receptor-like kinase-1 or ALK1 is a type I cell surface receptor for the transforming growth factor-β (TGF-β) family of proteins. The role of ALK1 in endothelial cell biology and in angiogenesis has been thoroughly studied. However, a possible role for ALK1 in cardiovascular homeostasis has recently been suggested. Indeed, it has now been demonstrated that ALK1 plays an important role in cardiovascular remodeling due to its ability to regulate cell proliferation and migration, as well as to modulate extracellular matrix (ECM) protein expression in several cell types.

[0005] ALK1 is not only expressed in endothelial cells, but also in smooth muscle cells, myofibroblasts, hepatic stellate cells, chondrocytes, monocytes, myoblasts, macrophages or fibroblasts, but its role in these cells has not been deeply analyzed. Due to the function of ALK1 in these cells, this receptor plays a role in several cardiovascular diseases, such as atherosclerosis. Cardiovascular diseases in general, and atherosclerosis in particular, are the leading causes of death worldwide. Summary of the Invention [Problem to be solved by the invention]

[0006] Thus, there remains a need for therapies that target ALK1, such as antibodies that bind to ALK1, for the treatment of atherosclerosis and other diseases and disorders. [Means for solving the problem]

[0007] The present disclosure provides antibodies that bind to ALK1, as well as methods / uses of the antibodies of the disclosure for treating various conditions (eg, atherosclerosis).

[0008] In one aspect, the disclosure provides an anti-ALK1 antibody comprising complementarity determining regions (CDRs) of a heavy chain variable sequence and a light chain variable sequence selected from: a heavy chain variable sequence of SEQ ID NO: 2 and a light chain variable sequence of SEQ ID NO: 3; a heavy chain variable sequence of SEQ ID NO: 4 and a light chain variable sequence of SEQ ID NO: 5; a heavy chain variable sequence of SEQ ID NO: 6 and a light chain variable sequence of SEQ ID NO: 7; a heavy chain variable sequence of SEQ ID NO: 10 and a light chain variable sequence of SEQ ID NO: 11; and a heavy chain variable sequence of SEQ ID NO: 12 and a light chain variable sequence of SEQ ID NO: 13.

[0009] The antibody may further comprise variable heavy and variable light chain sequences selected from: a heavy chain variable sequence of SEQ ID NO:2 and a light chain variable sequence of SEQ ID NO:3; a heavy chain variable sequence of SEQ ID NO:4 and a light chain variable sequence of SEQ ID NO:5; a heavy chain variable sequence of SEQ ID NO:6 and a light chain variable sequence of SEQ ID NO:7; a heavy chain variable sequence of SEQ ID NO:10 and a light chain variable sequence of SEQ ID NO:11; and a heavy chain variable sequence of SEQ ID NO:12 and a light chain variable sequence of SEQ ID NO:13.

[0010] In some embodiments, the heavy chain variable sequence comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:2, 4, 6, 10, or 12, and the light chain variable sequence comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:3, 5, 7, 11, or 13.

[0011] In some embodiments, the heavy chain variable sequence comprises 1-10 amino acid substitutions, insertions or deletions in SEQ ID NO: 2, 4, 6, 10, or 12, and the light chain variable sequence comprises 1-10 amino acid substitutions, insertions or deletions in SEQ ID NO: 3, 5, 7, 11, or 13.

[0012] In another aspect, the disclosure provides an anti-ALK1 antibody comprising a heavy chain comprising a CDRH1 comprising SSYWN (SEQ ID NO: 26), a CDRH2 comprising EVNHSGSTNYNPSLKS (SEQ ID NO: 27), and a CDRH3 comprising SPRSGRIVGAVFDY (SEQ ID NO: 28); and a light chain comprising a CDRL1 comprising GGNNIGPKSVH (SEQ ID NO: 29), a CDRL2 comprising DDSDRPS (SEQ ID NO: 30), and a CDRL2 comprising QVWDSSNDHVV (SEQ ID NO: 31).

[0013] In another aspect, the disclosure provides an anti-ALK1 antibody comprising a heavy chain comprising a CDRH1 comprising SRSYYWG (SEQ ID NO: 32), a CDRH2 comprising NIYYSGSAFYNPSLKS (SEQ ID NO: 33), and a CDRH3 comprising WDNWDVGAFDI (SEQ ID NO: 34); and a light chain comprising a CDRL1 comprising TGTSSDDVGGYNYVS (SEQ ID NO: 35), a CDRL2 comprising EVSKRPS (SEQ ID NO: 36), and a CDRL2 comprising TSFAGSNNWV (SEQ ID NO: 37).

[0014] In some embodiments, the antibody of the present disclosure binds to human ALK1, mouse ALK1, cynomolgus monkey ALK1, or combinations thereof.In some embodiments, the antibody of the present disclosure does not bind to ALK5.In some embodiments, the antibody of the present disclosure does not block BMP9 signal transduction.

[0015] The present disclosure also provides a pharmaceutical composition comprising an anti-ALK1 antibody according to any one of the above aspects or embodiments and a pharma- ceutical acceptable carrier. In some embodiments, the pharmaceutical composition may be formulated for injection or infusion.

[0016] In another aspect, the disclosure provides a method for treating or preventing a disease or disorder associated with ALK1, comprising administering to a subject having a disease or disorder associated with ALK1 an antibody or pharmaceutical composition according to any one of the preceding aspects or embodiments.

[0017] In some embodiments, the disease is atherosclerosis. In some embodiments, the risk of developing atherosclerosis is reduced, and the subject has not yet developed atherosclerotic plaque. In some embodiments, further atherosclerotic plaque formation is reduced or inhibited. In some embodiments, existing atherosclerotic plaques are destroyed. In some embodiments, the size or number of existing atherosclerotic plaques is reduced.

[0018] In some embodiments, the anti-ALK1 antibody or pharmaceutical composition is administered intravenously, subcutaneously, intramuscularly, or intraperitoneally.

[0019] In some embodiments, the subject is a human.

[0020] In another aspect, the present disclosure provides a method for blocking the interaction of ALK1 and low density lipoprotein (LDL), comprising administering to a subject an antibody or pharmaceutical composition according to any one of the above aspects or embodiments.In some embodiments, the subject has atherosclerosis or is at risk of developing atherosclerosis.In some embodiments, BMP9 signal transduction is not inhibited.In some embodiments, the subject is a human.

[0021] In another aspect, the disclosure provides the use of any of the antibodies disclosed herein for the manufacture of a medicament for treating a disease associated with ALK1, such as atherosclerosis.

[0022] In another aspect, the disclosure provides the use of any of the antibodies disclosed herein for the manufacture of a medicament for blocking the interaction of ALK1 and low density lipoprotein (LDL).

[0023] In another aspect, the disclosure provides anti-ALK1 antibodies for use in treating ALK1 associated diseases, such as atherosclerosis.

[0024] In another aspect, the disclosure provides anti-ALK1 antibodies for use in blocking the interaction of ALK1 and low density lipoprotein (LDL).

[0025] The foregoing general description and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the present disclosure as claimed. Other objects, advantages, aspects and iterations of the present disclosure will be presented in more detail below. [Brief description of the drawings]

[0026] [Figure 1]FIG. 1 shows the results of an ALK1 binding assay to confirm binding of the disclosed antibodies to various mammalian orthologs of ALK1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] Antibodies that bind to activin A receptor-like type 1 (ALK1) are provided. Antibody heavy and light chains capable of forming antibodies that bind to ALK1 are also provided. Additionally, antibodies, heavy and light chains comprising one or more particular complementarity determining regions (CDRs) are provided. Polynucleotides encoding antibodies that bind to ALK1 are provided. Polynucleotides encoding antibody heavy or light chains are also provided. Methods of making and / or purifying antibodies against ALK1 are provided. Treatments using the antibodies are provided. Such methods include, but are not limited to, methods of treating atherosclerosis.

[0028] I. Definition It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0029] Scientific and technical terms used herein have the meanings commonly understood by those of ordinary skill in the art unless otherwise defined. Unless otherwise specified, materials and / or methodologies known to those of ordinary skill in the art may be utilized in the practice of the methods described herein, based on the guidance provided herein.

[0030] As used herein, the singular terms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Reference to a singular object does not mean "one and only one" unless expressly stated to the contrary, but rather means "one or more."

[0031] As used herein, when "about" is used in conjunction with a numerical value, it means not only the numerical value explicitly stated, but also plus or minus 10% of that numerical value. For example, "about 10" should be understood as both "10" and "from 9 to 11."

[0032] Also as used herein, "and / or" refers to and includes not only the absence of a combination when interpreted as an alternative ("or"), but also refers to and includes any and all possible combinations of one or more of the associated listed items.

[0033] As used herein, a phrase of the form "A / B" or of the form "A and / or B" means (A), (B), or (A and B), and a phrase of the form "at least one of A, B, and C" means (A), (B), (C), (A and B), (A and C), (B and C), or (A, B, and C).

[0034] As used herein, the phrase "therapeutically effective amount" in relation to an anti-ALK1 antibody refers to the dose of the antibody that produces the specific pharmacological effect that the drug is administered to a subject in need of such treatment. For example, a therapeutically effective amount can be effective for reducing, reversing, or eliminating the signs and symptoms of tumor accumulation or atherosclerosis. It is emphasized that a therapeutically effective amount of an anti-ALK1 antibody is not necessarily effective for treating a disease or condition (e.g., atherosclerosis) of each individual subject, even if such a dose is considered to be a therapeutically effective amount by those skilled in the art. Those skilled in the art can adjust the considered therapeutically effective amount according to standard practice as necessary to treat a specific subject. A therapeutically effective amount can vary, for example, according to the age and weight of the subject, and / or the overall health of the subject, and / or the severity of the subject's disease or condition (e.g., atherosclerosis).

[0035] The terms "treat," "treatment," or "treating," as used herein with respect to an ALK1-associated disease or disorder (e.g., atherosclerosis), refer to reducing, reversing, or eliminating a pathophysiological feature of the disease or condition (e.g., plaque formation in atherosclerosis), reducing, reversing, or eliminating a sign or symptom of the disease or disorder, and / or otherwise improving quality of life in a subject. For example, in the case of atherosclerosis, treatment may involve improving blood flow, lowering blood pressure, inhibiting the deposition of cholesterol plaque in / on the endothelial lining of blood vessels, destroying existing plaque, or a combination of any or all of the foregoing.

[0036] The terms "prevent," "preventing," or "prevention," as used herein with respect to a disease or disorder associated with ALK1 (e.g., atherosclerosis), refer to impeding or reducing the risk of the onset of the disease or disorder. For example, preventing atherosclerosis entails preventing the formation of atherosclerosis in individuals predisposed to atherosclerosis or reducing the risk of atherosclerosis in such individuals.

[0037] The terms "individual," "subject," and "patient" are used interchangeably herein and refer to any individual mammalian subject, e.g., a cow, a dog, a cat, a horse, or a human. In certain embodiments, the subject, individual, or patient is a human.

[0038] The term "specifically binds" to an antigen or epitope is a term well understood in the art, and methods for determining such specific binding are also well known in the art. A molecule is said to "specifically bind" or "preferentially bind" if it reacts or associates with a particular cell or substance more frequently, rapidly, for a longer duration, and / or with a higher affinity than with alternative cells or substances. An antibody "specifically binds" or "preferentially binds" to a target if it binds to the target with higher affinity, avidity, more readily, and / or for a longer duration than it binds to other substances. For example, an antibody that specifically or preferentially binds to an ALK1 epitope is an antibody that binds to this epitope with higher affinity, avidity, more readily, and / or for a longer duration than it binds to other ALK1 epitopes or non-ALK1 epitopes. For example, it is understood by reading this definition that an antibody (or moiety or epitope) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. Thus, "specific binding" or "preferential binding" does not necessarily require (but may include) exclusive binding. Generally, but not necessarily, reference to binding means preferential binding. "Specificity" refers to the ability of a binding protein to selectively bind to an antigen.

[0039] The term "antibody" is used herein in the broadest sense and encompasses various antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific (e.g., bispecific T cell inducing antibodies) and trispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.

[0040] The term "CDR" refers to a complementarity determining region defined by at least one mode of identification known to those skilled in the art.

[0041] The term "heavy chain variable region" as used herein refers to a region comprising at least three heavy chain CDRs. In some embodiments, a heavy chain variable region comprises three CDRs and at least FR2 and FR3. In some embodiments, a heavy chain variable region comprises at least heavy chain HCDR1, framework (FR) 2, HCDR2, FR3, and HCDR3. In some embodiments, a heavy chain variable region also comprises at least a portion of FR1 and / or at least a portion of FR4.

[0042] The term "heavy chain constant region" as used herein refers to a region that contains at least three heavy chain constant domains, C H 1. C H 2, and C H 3. Of course, non-function-altering deletions and modifications within the domain are encompassed within the scope of the term "heavy chain constant region" unless otherwise specified. Non-limiting exemplary heavy chain constant regions include gamma, delta, and alpha. Non-limiting exemplary heavy chain constant regions also include epsilon and mu. Each heavy chain constant region corresponds to an antibody isotype. For example, an antibody comprising a gamma constant region is an IgG antibody, an antibody comprising a delta constant region is an IgD antibody, an antibody comprising an alpha constant region is an IgA antibody, an antibody comprising a mu constant region is an IgM antibody, and an antibody comprising an epsilon constant region is an IgE antibody. Certain isotypes can be further subdivided into subclasses. For example, IgG antibodies include, but are not limited to, IgG1 (containing a gamma 1 constant region), IgG2 (containing a gamma 2 constant region), IgG3 (containing a gamma 3 constant region), and IgG4 (containing a gamma 4 constant region) antibodies; IgA antibodies include, but are not limited to, IgA1 (containing an alpha 1 constant region) and IgA2 (containing an alpha 2 constant region) antibodies; and IgM antibodies include, but are not limited to, IgM1 and IgM2.

[0043] The term "heavy chain," as used herein, refers to a polypeptide comprising at least a heavy chain variable region, with or without a leader sequence. In some embodiments, a heavy chain comprises at least a portion of a heavy chain constant region. The term "full-length heavy chain," as used herein, refers to a polypeptide comprising a heavy chain variable region and a heavy chain constant region, with or without a leader sequence.

[0044] The term "light chain variable region" as used herein refers to a region comprising at least three light chain CDRs. In some embodiments, the light chain variable region comprises three CDRs and at least FR2 and FR3. In some embodiments, the light chain variable region comprises at least a light chain LCDR1, a framework (FR) 2, an LCDR2, a FR3, and an LCDR3. For example, the light chain variable region may comprise a light chain CDR1, a framework (FR) 2, a CDR2, a FR3, and a CDR3. In some embodiments, the light chain variable region also comprises at least a portion of FR1 and / or at least a portion of FR4.

[0045] The term "light chain constant region" as used herein refers to a region that includes the light chain constant domain, CL. Non-limiting exemplary light chain constant regions include λ and κ. Of course, unless otherwise specified, non-functional altering deletions and modifications within the domain are included within the scope of the term "light chain constant region."

[0046] The term "light chain" as used herein refers to a polypeptide comprising at least a light chain variable region, with or without a leader sequence. In some embodiments, a light chain comprises at least a portion of a light chain constant region. The term "full-length light chain" as used herein refers to a polypeptide comprising a light chain variable region and a light chain constant region, with or without a leader sequence.

[0047] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). The affinity of a molecule X for its partner Y is generally measured by the dissociation constant (KD Affinity can be expressed by common methods known in the art (e.g., ELISA K, including those described herein). D , KinExA, biolayer interferometry (BLI), and / or surface plasmon resonance devices (such as BIAcore® devices).

[0048] "K D The term "as used herein refers to the equilibrium dissociation constant of an antibody-antigen interaction.

[0049] "Chimeric antibody" as used herein refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, and at least a portion of the remainder of the heavy and / or light chain is derived from a different source or species. In some embodiments, a chimeric antibody refers to an antibody that comprises at least one variable region from a first species (e.g., mouse, rat, cynomolgus monkey, etc.) and at least one constant region from a second species (e.g., human, cynomolgus monkey, etc.). In some embodiments, a chimeric antibody comprises at least one mouse variable region and at least one human constant region. In some embodiments, a chimeric antibody comprises at least one cynomolgus monkey variable region and at least one human constant region. In some embodiments, all of the variable regions of the chimeric antibody are derived from a first species, and all of the constant regions of the chimeric antibody are derived from a second species. The chimeric construct may be a functional fragment as described above.

[0050] "Humanized antibody" as used herein refers to an antibody in which at least one amino acid in the framework region of a non-human variable region has been replaced by the corresponding amino acid from a human variable region. In some embodiments, a humanized antibody comprises at least one human constant region or a fragment thereof. In some embodiments, a humanized antibody is an antibody fragment, such as a Fab, scFv, (Fab')2, etc. The term humanized also refers to chimeric immunoglobulins, immunoglobulin chain forms of non-human (e.g., murine) antibodies, or fragments thereof that contain minimal sequences of non-human immunoglobulins (e.g., Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies). Humanized antibodies can encompass human immunoglobulins (recipient antibody) in which residues from the complementary determining region (CDR) of the recipient have been replaced by residues from the CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit that has the desired specificity, affinity, and capacity. In some cases, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may contain residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences, but are included to further refine and optimize antibody performance. Generally, a humanized antibody may comprise substantially all of at least one, and typically two, variable domains, with all or substantially all of the CDR regions corresponding to the CDR regions of a non-human immunoglobulin, and all or substantially all of the FR regions being of a human immunoglobulin consensus sequence. In some embodiments, the humanized antibody may also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Other forms of humanized antibodies have one or more CDRs (CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and / or CDR H3) that are altered relative to the original antibody, also referred to as one or more CDRs "derived from" one or more CDRs of the original antibody.As will be understood, a humanized sequence can be identified by its primary sequence and does not necessarily indicate the process by which the antibody was created.

[0051] "CDR-grafted antibody", as used herein, refers to a humanized antibody in which one or more complementarity determining regions (CDRs) of a first (non-human) species have been grafted onto the framework regions (FRs) of a second (human) species.

[0052] "Human antibodies", as used herein, encompass antibodies produced in humans, antibodies produced in non-human animals that contain human immunoglobulin genes, such as XenoMouse® mice, and antibodies whose antibody repertoires have been selected using in vitro methods such as phage display based on human immunoglobulin sequences (Vaughan et al., 1996, Nature Biotechnology, 14:309-314; Sheets et al., 1998, Proc. Natl. Acad. Sci. (USA) 95:6157-6162; Hoogenboom and Winter, 1991, J. Mol. Biol., 227:381; Marks et al., 1991, J. Mol. Biol., 222:581). The term "human antibodies" refers to the genus of sequences that are human sequences. Thus, the term does not designate the process by which the antibody was created, but rather the genus of relevant sequences.

[0053] Amino acid substitutions can include, but are not limited to, the replacement of one amino acid with another amino acid in a polypeptide. Exemplary conservative substitutions are shown in Table 1. Amino acid substitutions can be introduced into the antibody of interest and the products can be screened for the desired activity, such as retained / improved binding to the antigen, reduced immunogenicity, or improved ADCC or CDC.

[0054] [Table 1]

[0055] Amino acids can be divided into groups according to common side chain properties: (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0056] Non-conservative substitutions involve exchanging a member of one of these classes for a member of another class.

[0057] The term "vector" is used to describe a cloned polynucleotide that can be propagated in a host cell or a polynucleotide that can be engineered to contain a polynucleotide. A vector may contain one or more of the following elements: an origin of replication, one or more regulatory sequences (such as, for example, a promoter and / or enhancer) that regulate the expression of a polypeptide of interest, and / or one or more selectable marker genes (such as, for example, antibiotic resistance genes and genes that can be used for colorimetric assays, such as, for example, β-galactosidase). The term "expression vector" refers to a vector used to express a polypeptide of interest in a host cell.

[0058] "Host cell" refers to a cell that can be or has been the recipient of a vector or isolated polynucleotide. A host cell may be a prokaryotic or eukaryotic cell. Exemplary eukaryotic cells include mammalian cells, such as primate or non-primate animal cells; fungal cells, such as yeast; plant cells; and insect cells. Non-limiting exemplary mammalian cells include, but are not limited to, NSO cells, PER.C6® cells (Crucell), and 293 and CHO cells and their derivatives, such as 293-6E cells and DG44 cells, respectively.

[0059] The terms "pharmaceutical formulation" and "pharmaceutical composition" refer to a preparation in which the biological activity of the active ingredient(s) is effective and which does not contain additional components that are unacceptably toxic to the subject to which the formulation is administered. Such formulations may be sterile.

[0060] "Pharmaceutically acceptable carrier" refers to a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, formulation aid, or carrier that is conventional in the art for use with therapeutic agents that together constitute a "pharmaceutical composition" for administration to a subject. A pharmaceutically acceptable carrier is non-toxic to a recipient at the dosage and concentration used and is compatible with the other ingredients of the formulation. A pharmaceutically acceptable carrier is one that is suitable for the formulation used.

[0061] A "sterile" preparation is aseptic or essentially free of living microorganisms and their spores.

[0062] II. ALK1 and ALK1-Related Disorders Activin A receptor-like type 1 (ALK1; Entrez:94, Ensembl:ENSG00000139567, UniProt:P37023) is a transmembrane serine / threonine receptor kinase of the transforming growth factor-beta receptor family expressed in endothelial cells. Ligands that fall into the transforming growth factor-β (TGFβ) superfamily signal through transmembrane receptor complexes to control a myriad of processes important in development and disease, including hypocotyl patterning, cardiac and vascular development, epithelial-mesenchymal transition, and fibrosis. Among signaling receptors, Activin A receptor-like type 1 (ACVRL1, encoding ALK1) has a high degree of cell type specificity and is predominantly expressed in arterial endothelial cells (ECs). The amino acid sequence of human ALK1 is: MTLGSPRKGLLMLLMALVTQGDPVKPSRGPLVTCTCESPHCKGPTCRGAWCTVVLVREEGRHPQEHRGCGNLHRELCRGRPTEFVNHYCCDSHLCNHNVSLVLEATQPPSEQPGTDGQLALILGPVLA LLALVALGVLGLWHVRRRQEKQRGLHSELGESSLILKASEQGDSMLGDLLDSDCTTGSGSGLPFLVQRTVARQVALVECVGKGRYGEVWRGLWHGESVAVKIFSSRDEQSWFRETEIYNTVLLRHDNIL GFIASDMTSRNSSTQLWLITHYHEHGSLYDFLQRQTLEPHLALRLAVSAACGLAHLHVEIFGTQGKPAIAHRDFKSRNVLVKSNLQCCIADLGLAVMHSQGSDYLDGNNPRVGTKRYMAPEVLDEQIRTDCFESYKWTDIWAFGLVLWEIARRTIVNGIVEDYRPPFYDVVPNDPSFEDMKKVVCVDQQTPTIPNRLAADPVLSGLAQMMRECWYPNPSARLTALRIKKTLQKISNSPEKPKVIQ (SEQ ID NO: 1).

[0063] Defects in ALK1 signaling can lead to hereditary hemorrhagic telangiectasia (HHT), an autosomal dominant vascular disorder characterized by direct arterial and venous connections, or arteriovenous malformations (AVMs). In addition, it is now understood that ALK1 signaling may also play a role in other vascular-related diseases, such as pulmonary arterial hypertension, cancer, and atherosclerosis. Furthermore, ALK1 has been implicated in various aspects of sprouting angiogenesis, including tip / stalk cell selection, migration, and proliferation, and recent studies suggest a role for ALK1 in transmitting flow-based signals that govern directional endothelial cell migration in patent perfused blood vessels.

[0064] Studies in mice have demonstrated strong Alk1 expression in arterial bends and bifurcations, sites of flow obstruction that are prone to atherosclerosis. Increased ALK1 expression has also been demonstrated in the endothelium, neointima, and media of human coronary atherosclerotic lesions. It is now understood that ALK1 is directly linked to atherosclerosis by mechanisms that are apparently unrelated to its role in HHT.

[0065] Briefly, the apically localized extracellular domain of ALK1 can bind circulating ApoB100-containing low-density lipoprotein (LDL) with relatively low affinity and mediate transcytosis of LDL into the subendothelial space, initiating atherosclerotic lesions. This activity does not require BMP9 / BMP10, endoglin, BMPRII, or ALK1 kinase activity, and BMP9 / BMP10 cannot compete for LDL binding, suggesting that these ligands bind to distinct ALK1 residues. Thus, targeting the LDL / ALK1 interaction may be a feasible approach to develop new drugs to prevent atherosclerosis.

[0066] As provided in more detail below, the antibodies of the disclosure can be used to treat any of the diseases and disorders associated with ALK1, such as atherosclerosis, as well as other diseases or disorders included herein (e.g., HHT, AVM, pulmonary arterial hypertension, cancer, etc.).

[0067] III. Anti-ALK1 antibody The present disclosure provides antibodies that bind to ALK1 (i.e., "anti-ALK1 antibodies"). The antibodies of the present disclosure can selectively bind to ALK1 and can be used to treat diseases or disorders associated with ALK1, including, but not limited to, atherosclerosis, HHT, AVM, pulmonary arterial hypertension, and cancer.

[0068] ALK1 may play an important role in the accumulation of LDL cholesterol in blood vessels. The antibodies of the present disclosure (e.g., BVO-A-9F7-A10 and BVO-B-1H7-E3) can block LDL accumulation by interfering with the "LDL-ALK1 pathway" in blood vessel walls, which facilitates the transport of LDL from blood to tissues (also called LDL transcytosis). This in turn helps prevent or slow the clogging of arteries that leads to heart disease. The conformation-selective ALK1-binding monoclonal antibodies disclosed herein (e.g., BVO-A-9F7-A10 and BVO-B-1H7-E3) efficiently block LDL transcytosis to the endothelium, but do not block BMP9 signaling, resulting in a dramatic reduction in atherogenesis. The anti-ALK1 antibodies of the present disclosure can bind to human ALK1, mouse ALK1, cynomolgus monkey ALK1, or a combination thereof.

[0069] The anti-ALK1 antibodies described herein can be obtained by any means, including in vitro sources (e.g., hybridomas or recombinantly antibody-producing cell lines) and in vivo sources (e.g., rodents, rabbits, humans, etc.). The antibodies of the present disclosure can be human, humanized (partially or fully), or chimeric. Human, partially humanized, fully humanized, and chimeric antibodies can be produced by methods known in the art, such as, for example, the use of transgenic animals (e.g., mice) in which one or more endogenous immunoglobulin genes have been replaced by one or more human immunoglobulin genes. Examples of transgenic mice in which endogenous antibody genes have been effectively replaced by human antibody genes include, but are not limited to, the HUMAB-MOUSE™, the Kirin TC MOUSE™, and the KM-MOUSE™ (see, e.g., Lonberg, Nat. Biotechnol., 23(9):1117-25 (2005), and Lonberg, Handb. Exp. Pharmacol., 181: 69-97 (2008)).

[0070] Anti-Alk1 antibody disclosed herein is generally monoclonal, recombinant, or both.Monoclonal antibody (mAb) can be obtained by methods known in the art, for example, by fusing antibody-producing cells with immortalized cells to obtain hybridomas, and / or by using combinatorial antibody library technology to generate mAb from the mRNA extracted from bone marrow, B cells, and / or spleen cells of immunized animals, and / or by isolating monoclonal antibody from the serum from subjects immunized with ALK1 antigen. Recombinant antibodies can be obtained by methods known in the art, for example, using phage display techniques, yeast surface display techniques (Chao et al., Nat. Protoc., 1(2): 755-68 (2006)), mammalian cell surface display techniques (Beerli et al., PNAS, 105(38): 14336-41 (2008)), and / or by expressing or co-expressing antibody polypeptides. Other techniques for making antibodies are known in the art and can be used to obtain the antibodies used in the methods described herein.

[0071] In general, antibodies consist of four polypeptides: two identical copies of a heavy (H) chain polypeptide and two copies of a light (L) chain polypeptide. In general, each heavy chain contains one N-terminal variable (V H ) region and three C-terminal constant (C H 1. C H 2 and C H 3) region, and each light chain contains one N-terminal variable (V L ) region and one C-terminal constant (C L The variable regions of each pair of light and heavy chains form the antigen-binding site of an antibody.

[0072] The term "antibody fragment," as used herein, refers to one or more portions of an ALK1-binding antibody that exhibits the ability to bind to ALK1. Examples of binding fragments include: (i) Fab fragments (V L , V H , CL and C H1 (ii) F(ab')2 fragment (a bivalent fragment consisting of two Fab fragments linked by a disulfide bridge in the hinge region); (iii) Fd fragment (a V H and C H (iv) Fv fragments (V of a single arm of an antibody) L and V H domain), (v) dAb fragments (V H domain); and (vi) an isolated complementarity determining region (CDR), e.g., H CDR3. Other examples include single chain Fv (scFv) constructs. See, e.g., Bird et al., Science, 242:423-26 (1988); Huston et al., Proc. Natl. Acad. Sci. USA, 85:5879-83 (1988). Other examples include (i) fusions of ALK1-binding domain polypeptides (e.g., heavy chain variable region, light chain variable region, or heavy chain variable region and light chain variable region fused via a linker peptide) with immunoglobulin hinge region polypeptides; (ii) immunoglobulin heavy chain CDR3. H2 (iii) a fusion of the constant region and hinge region of an immunoglobulin heavy chain C H3 Constant region and C H2 Included are ALK1 binding domain immunoglobulin fusion proteins that include fusions of a constant region, in which the hinge region may be modified, for example by replacing one or more cysteine ​​residues with serine residues, to prevent dimer formation.

[0073] The antibody of the present disclosure may belong to a class of antibodies selected from IgG, IgM, IgA, IgE, and IgD. More specifically, the antibody of the present disclosure may be IgG1, IgG2, IgG3, or IgG4. In some embodiments, the antibody of the present disclosure may comprise all or part of the constant region, framework region, or combinations thereof of an IgG, IgM, IgA, IgE, or IgD antibody. For example, the disclosed antibody may comprise an IgG1 immunoglobulin structure, which may be modified such that the IgG1 structure is replaced (or "switched" with) another IgG class immunoglobulin, or the corresponding structure of an IgM, IgA, IgE, or IgD immunoglobulin. This type of modification or switching can be performed to enhance the neutralizing function of the peptide, for example, antibody-dependent cellular cytotoxicity (ADCC) and complement fixation (CDC). In some embodiments, the anti-ALK1 antibody may be a mammalian antibody, a human antibody, a humanized antibody, or a chimeric antibody.

[0074] In some embodiments, the anti-ALK1 antibody of the present disclosure may include one or more mutations that make the antibody more suitable for therapeutic situations. Such mutations, alterations, or modifications may include, for example, changes in the Fc region to increase the peptide's ability to mediate cytotoxic functions, such as antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and / or complement fixation (CDC). Numerous mutations in the Fc domain that enhance binding to Fc receptors have been reported, such as S239D / A330L / I332E, F243L, and G236A. Additionally or alternatively, mutations in the Fc region that increase circulating half-life can be incorporated into the structure. For example, mutations to engineer the pH-dependent interaction of the Fc domain with FcRn to increase affinity at pH 6.0 while retaining minimal binding at pH 7.4 can increase half-life under physiological conditions and improve efficacy. In some embodiments, the antibodies of the disclosure may be conjugated to polyethylene glycol (PEG) and / or albumin, which can increase the half-life and reduce potential immunogenicity of the antibody.

[0075] Exemplary anti-ALK1 antibody heavy and light chain variable amino acid sequences are disclosed below in Table 2. Nucleic acid sequences encoding the heavy and light chain variable sequences of Table 2 are shown in Table 3.

[0076] [Table 2]

[0077] [Table 3-1]

[0078] [Table 3-2]

[0079] [Table 3-3]

[0080] The present disclosure provides antibodies that include the same CDR sequences and / or the same framework region sequences and / or the same variable region sequences as one or more of the sequences disclosed in Table 2. For example, in some embodiments, an anti-ALK1 antibody of the present disclosure may include a heavy chain variable region that includes the CDRs of SEQ ID NO:2 and a light chain variable region that includes the CDRs of SEQ ID NO:3. In some embodiments, an anti-ALK1 antibody of the present disclosure may include a heavy chain variable region that includes the CDRs of SEQ ID NO:4 and a light chain variable region that includes the CDRs of SEQ ID NO:5. In some embodiments, an anti-ALK1 antibody of the present disclosure may include a heavy chain variable region that includes the CDRs of SEQ ID NO:6 and a light chain variable region that includes the CDRs of SEQ ID NO:7. In some embodiments, an anti-ALK1 antibody of the present disclosure may include a heavy chain variable region that includes the CDRs of SEQ ID NO:10 and a light chain variable region that includes the CDRs of SEQ ID NO:11. In some embodiments, an anti-ALK1 antibody of the present disclosure may include a heavy chain variable region that includes the CDRs of SEQ ID NO:12 and a light chain variable region that includes the CDRs of SEQ ID NO:13.

[0081] In some embodiments, the anti-ALK1 antibody may comprise a heavy chain comprising CDRH1 comprising SSYWN (SEQ ID NO: 26), CDRH2 comprising EVNHSGSTNYNPSLKS (SEQ ID NO: 27), and CDRH3 comprising SPRSGRIVGAVFDY (SEQ ID NO: 28); and a light chain comprising CDRL1 comprising GGNNIGPKSVH (SEQ ID NO: 29), CDRL2 comprising DDSDRPS (SEQ ID NO: 30), and CDRL2 comprising QVWDSSNDHVV (SEQ ID NO: 31). In some embodiments, the anti-ALK1 antibody is BVO-A-9F7-A10.

[0082] In some embodiments, the anti-ALK1 antibody may comprise a heavy chain comprising CDRH1 comprising SRSYYWG (SEQ ID NO: 32), CDRH2 comprising NIYYSGSAFYNPSLKS (SEQ ID NO: 33), and CDRH3 comprising WDNWDVGAFDI (SEQ ID NO: 34); and a light chain comprising CDRL1 comprising TGTSSDDVGGYNYVS (SEQ ID NO: 35), CDRL2 comprising EVSKRPS (SEQ ID NO: 36), and CDRL2 comprising TSFAGSNNWV (SEQ ID NO: 37). In some embodiments, the anti-ALK1 antibody is BVO-B-1H7-E3.

[0083] In some embodiments, the anti-ALK1 antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 2, 4, 6, 10, or 12. In some embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but an anti-ALK1 antibody comprising the sequence retains the ability to bind to ALK1. In some embodiments, a total of 1-10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) in SEQ ID NO: 2, 4, 6, 10, or 12 are substituted, inserted, and / or deleted. In some embodiments, the substitutions, insertions, or deletions are in regions outside the CDRs (i.e., in the FRs).Anti-ALK1 antibodies may comprise a VH sequence of SEQ ID NO: 2, 4, 6, 10, or 12, including post-translational modifications of that sequence.

[0084] In some embodiments, the anti-ALK1 antibody comprises a light chain variable domain (VL) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 3, 5, 7, 11, or 13. In some embodiments, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but the anti-ALK1 antibody comprising the sequence retains the ability to bind to ALK1. In some embodiments, a total of 1-10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) of SEQ ID NO: 3, 5, 7, 11, or 13 are substituted, inserted, and / or deleted. In some embodiments, the substitutions, insertions, or deletions are in regions outside the CDRs (i.e., within the FRs).Anti-ALK1 antibodies may comprise a VL sequence of SEQ ID NO: 3, 5, 7, 11, or 13, including post-translational modifications of that sequence.

[0085] As mentioned above, the antibody of the present disclosure can bind to various mammalian ALK1 proteins, including human, mouse and cynomolgus monkey.For example, BVO-B-1H7-E3 binds to human, mouse and cynomolgus monkey ALK1, and BVO-A-9E7-A10 binds to human and cynomolgus monkey ALK1.In contrast, BVN-B-2F11-E4 (control) only shows weak binding to human ALK1.

[0086] In addition to binding to ALK1, the antibodies of the present disclosure may also have other beneficial properties. For example, the antibodies of the present disclosure cannot block BMP9 (bone morphogenetic protein-9) signaling and are capable of phosphorylating SMAD1 / 5. BMP9 plays a role in inducing and maintaining the ability of fetal basal forebrain cholinergic neurons (BFCN) to respond to a neurotransmitter called acetylcholine, and also induces the differentiation of mesenchymal stem cells (MSCs) into osteoblast lineages via the SMAD signaling pathway. Of note for the purposes of the present disclosure, BMP9 is a ligand for ALK1. Endoglin, a type I membrane glycoprotein that forms the TGF-β receptor complex, is a co-receptor for ALK1 binding to GDF2 / BMP-9. Thus, the ability of certain disclosed antibodies to bind to ALK1 without blocking BMP9 signaling is unique. In some embodiments, the antibodies of the present disclosure bind to ALK1 and do not block BMP9 signaling.

[0087] Another feature of certain disclosed antibodies is their ability to bind to ALK1 with little or no cross-reactivity to ALK5.ALK5 is a TGF-β receptor, just like ALK1, and both of these receptors share similar structures and sequences.Therefore, selective binding to ALK1 without cross-reactivity to ALK5 is a desirable feature of the disclosed antibodies, as it minimizes the possibility of antibody off-target effects.In some embodiments, the disclosed antibodies do not bind to or cross-react with ALK5.Neither BVO-B-1H7-E3 nor BVO-A-9E7-A10 bind to ALK5.

[0088] Any of the antibodies disclosed herein can be used to treat and / or prevent ALK1-related diseases and disorders, such as atherosclerosis.The optimal dosage and administration route can vary based on the condition of the subject, including, for example, the administration route and dosage form, the age and weight of the subject, and / or the type and severity of disease, and can be determined by those skilled in the art.As will be discussed in more detail below, the antibody can be formulated into a pharmaceutical composition suitable for administration to the subject by any intended administration route.

[0089] IV. Pharmaceutical Compositions Pharmaceutical compositions are provided for use in the treatment or prevention of ALK1-associated diseases and disorders, including atherosclerosis, that comprise an anti-ALK1 antibody as described herein as an active ingredient.

[0090] Pharmaceutical compositions of the anti-ALK1 antibodies of the present disclosure can be prepared as formulations according to standard methods (see, for example, Remington's Pharmaceutical Science, Mark Publishing Company, Easton, USA). Pharmaceutical compositions generally include carriers and / or additives in addition to the antibody. For example, in some embodiments, the pharmaceutical composition comprises one or more surfactants (e.g., PEG and Tween), excipients, antioxidants (e.g., ascorbic acid), colorants, flavoring agents, preservatives, stabilizers, buffers (e.g., phosphoric acid, citric acid, and other organic acids), chelating agents (e.g., EDTA), suspending agents, isotonizing agents, binders, disintegrants, lubricants, flow enhancers, corrective agents, light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, carmellose calcium, carmellose sodium, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl acetal diethylaminoacetate, polyvinylpyrrolidone, gelatin, medium chain triglycerides, polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethyl cellulose, corn starch, and inorganic salts. In some embodiments, the pharmaceutical composition comprises one or more other low molecular weight polypeptides, proteins such as serum albumin, gelatin, and immunoglobulins, and amino acids such as glycine, glutamine, asparagine, arginine, and lysine.

[0091] Anti-ALK1 antibody can be prepared as an aqueous solution for injection, in which case it can be dissolved in an isotonic solution containing, for example, saline, dextrose, or other excipients or tonifiers (i.e., tonicity agents). Tonifiers can include, for example, D-sorbitol, D-mannose, D-mannitol, and sodium chloride. In addition, suitable solubilizers can be used simultaneously, for example, alcohol (e.g., ethanol), polyhydric alcohols (e.g., propylene glycol and PEG), and non-ionic surfactants (polysorbate 80 and HCO-50).

[0092] The pharmaceutical composition of the present disclosure can be formulated for any suitable administration route, including intravenous, subcutaneous, intraperitoneal, intramuscular, or oral administration.In a typical embodiment, the anti-ALK1 antibody is formulated for intravenous, subcutaneous, intraperitoneal, or intramuscular administration, such as, for example, liquid, suspension, emulsion, liposome preparation, etc. More specifically, the anti-ALK1 antibody of the present disclosure can be formulated for intravenous, subcutaneous, or intramuscular administration.

[0093] Pharmaceutically acceptable carriers for various dosage forms and administration routes are known in the art.For example, solvents, solubilizers, suspending agents, isotonicity agents, buffers, and emollients for liquid preparations are known.In some embodiments, the pharmaceutical composition comprises one or more additional components, such as one or more preservatives, antioxidants, coloring agents, sweeteners / flavoring agents, adsorbents, wetting agents, etc.

[0094] The present disclosure provides pharmaceutical compositions for use in the treatment or prevention of ALK1-associated diseases and disorders, such as atherosclerosis, in a subject, comprising an anti-ALK1 antibody disclosed herein as an active ingredient.

[0095] Any of the pharmaceutical compositions disclosed herein can be used to treat and / or prevent ALK1-related diseases and disorders, such as atherosclerosis. Optimal dosages and routes of administration may vary.

[0096] V. Treatment and Prevention The present disclosure provides a method for treating or preventing ALK1-related diseases and disorders, such as atherosclerosis. Further, the present disclosure provides a method for blocking the interaction of ALK1 and low-density lipoprotein (LDL). Further, the present disclosure provides a method for blocking the binding of ALK1 to LDL. Further, the present disclosure provides the use of the anti-ALK1 antibody of the present disclosure for treating or preventing ALK1-related diseases and disorders, such as atherosclerosis. Further, the present disclosure provides the use of the anti-ALK1 antibody of the present disclosure for blocking the interaction of ALK1 and low-density lipoprotein (LDL). Further, the present disclosure provides the use of the anti-ALK1 antibody of the present disclosure for blocking the binding of ALK1 to LDL.

[0097] As described above, ALK1 may play an important role in the accumulation of LDL cholesterol in blood vessels. The antibodies of the present disclosure (e.g., BVO-A-9F7-A10 and BVO-B-1H7-E3) inhibit LDL cholesterol in blood vessel walls, which facilitates the transport of LDL from blood to tissues (also called LDL transcytosis). By interfering with the "LDL-ALK1 pathway", LDL accumulation can be blocked. This in turn helps prevent or slow down the clogging of arteries that leads to heart disease. The conformation-selective ALK1-binding monoclonal antibodies disclosed herein (e.g., BVO-A-9F7-A10 and BVO-B-1H7-E3) effectively block LDL transcytosis into endothelium but do not block BMP9 signaling, resulting in a dramatic reduction in atheroma formation. Thus, the antibodies disclosed herein are particularly well suited for treating and / or preventing atherosclerosis.

[0098] Atherosclerosis is the buildup of fats, cholesterol, and other substances in the artery walls. This buildup is called atherosclerosis. Atherosclerosis can narrow the artery, thereby blocking blood flow. Atherosclerosis can also rupture, resulting in blood clots. Although atherosclerosis is often considered a heart problem, it can affect arteries anywhere in the body. Mild atherosclerosis usually does not cause any symptoms, but moderate to severe atherosclerosis can cause chest pain or pressure (angina), high blood pressure, and kidney failure, depending on the location of the atherosclerosis formation. Atherosclerosis can also lead to the development of comorbidities such as transient ischemic attacks (TIA), stroke, peripheral arterial disease, and heart failure.

[0099] Generally, the treatments disclosed herein include administering to a subject a therapeutically effective amount of an anti-ALK1 antibody (e.g., BVO-A-9F7-A10 or BVO-B-1H7-E3). In particular, the disclosure provides a method of treating or preventing atherosclerosis, comprising administering to a subject having atherosclerosis an anti-ALK1 antibody disclosed herein (e.g., BVO-A-9F7-A10 or BVO-B-1H7-E3). In some embodiments, the antibody can reduce the risk of developing atherosclerosis in a subject who has not yet developed atherosclerosis. In some embodiments, the antibody can inhibit or reduce further atherosclerosis formation. In some embodiments, the antibody can destroy existing atherosclerosis or reduce the size or number of existing atherosclerosis.

[0100] An effective amount of the disclosed anti-ALK1 antibody can be administered in one or multiple administrations, applications or dosages. Such delivery depends on a number of variables, including the duration of use of the individual dosage units, the bioavailability of the therapeutic agent, the route of administration, and the like. However, it is understood that the specific dosage level of the disclosed anti-ALK1 antibody for any particular subject can depend on a variety of factors, including the activity of the specific compound used, the subject's age, weight, general health, sex, and diet, the time of administration, the rate of excretion, the drug combination, and the severity and administration form of the particular disorder being treated. Therapeutic and prophylactic dosages are generally titrated to optimize safety and efficacy. Dosages can be determined by a physician and adjusted as necessary to suit the observed therapeutic effect. Generally, the relationship between dosage and effect from in vitro and / or in vivo tests can provide useful guidance initially regarding the proper dosage for administration to patients. In general, it is desired to administer an effective amount of the compound to achieve serum levels commensurate with the concentration found to be effective in vitro.

[0101] The administration of anti-ALK1 antibody (e.g., BVO-A-9F7-A10 or BVO-B-1H7-E3) can be intravenous, subcutaneous, intramuscular, intradermal, or intraperitoneal. In general, the most common route of administration is injection or infusion of the antibody. Administration can include a single dose, or repeated doses over a period of treatment.

[0102] In some embodiments of the methods and uses of the disclosure, the anti-ALK1 antibody is administered daily, once every two days, twice a week, three times a week, four times a week, five times a week, six times a week, once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, once every ten weeks, once every eleven weeks, once every twelve weeks, twice a year, once a year, and / or as needed.

[0103] In some embodiments of the methods and uses of the present disclosure, the duration of treatment or prevention is about 1 day, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 13 weeks, about 14 weeks, about 15 weeks, about 16 weeks, about 17 weeks, about 18 weeks, about 19 weeks, about 20 weeks, about 24 weeks, about 30 weeks, about 36 weeks, about 40 weeks, about 48 weeks, about 50 weeks, about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, or as needed.

[0104] The disclosure provides the use of an anti-ALK1 antibody in the manufacture of a medicament for the treatment or prevention of ALK1-associated diseases and disorders, such as atherosclerosis. EXAMPLES

[0105] The examples discussed below are intended to be purely illustrative of the present invention and should not be construed as limiting the present invention in any way. The examples do not represent that the experiments below are all or the only experiments performed.

[0106] Example 1 Isolation of ALK1-binding antibodies OmniRats were immunized using the Gene Gun method, i.e., DNA was loaded onto gold particles and administered into the animals' skin using a Gene Gun (Helios). In other words, for immunization, instead of injecting ALK1-transfected / expressing cells, plasmid DNA was used directly, thereby inducing the expression of ALK1 in skin cells / DCs of Omni rats.

[0107] Polyclonal sera from immunized animals were tested for activity. B cells of interest were isolated and hybridomas were generated. After hybridoma isolation and subcloning, the supernatants were used to reassess the monoclonal antibodies.

[0108] Example 2 ALK1 binding assessment The various isolated antibodies were evaluated for their ability to bind to ALK1 from mouse (murine), human, and cynomolgus monkey. Flow cytometry was used to measure the fluorescence intensity generated by a fluorescently labeled secondary antibody that detects the primary anti-ALK1 antibody binding to a specific cell-associated ALK1 molecule. More precisely, hybridoma supernatants containing the primary antibodies were tested by flow cytometry for binding to transiently transfected cells expressing human, cynomolgus monkey, and mouse ALK1. Signals were generated by using a PE-labeled goat anti-rat secondary antibody. As a negative control, human, cynomolgus monkey, and mouse ALK1 transfected cells were incubated with the secondary antibody alone (without supernatant), in addition to testing the supernatant of untransfected cells. Exemplary results are shown in the figure (Figure 1).

[0109] As can be seen in the figure (Figure 1), BVO-A-9F7-A10 and BVO-B-1H7-E3 showed strong binding to human and cynomolgus ALK1 but low cross-reactivity to the mouse orthologues.

[0110] It should be noted that the expression of mouse ALK1 seems to be much weaker compared to cynomolgus ALK and human ALK. Moreover, since this was done by binding of anti-tag antibodies to the N-terminal tag, it was found that in many cases the signal obtained does not necessarily reflect the "real" cell surface expression level of a given protein. In many cases, the "real" expression is higher than that indicated by the signal obtained from the tag, possibly due to the anti-tag antibody not being fully accessible to the tag sequence, resulting in a lower signal. For example, it has been found that this phenomenon even depends on the type of tag used (such as myc, FLAG or His), suggesting lower / higher expression levels in some than others, even with the same antigen, which cannot be confirmed in parallel studies with specific (anti-target) antibodies. As shown in the diagram (Figure 1), the specific anti-ALK1 antibody gives a much higher signal than the expression control generated with the anti-tag antibody.

[0111] However, the low signal for mouse ALK1 does not allow us to infer that the antibody in question has very low anti-mouse cross-reactivity, which may be due to the much lower cell surface expression of mouse ALK1 compared to cynomolgus monkeys and humans, as indicated by the much lower expression controls.

[0112] In any event, the antibodies of the present disclosure, particularly BVO-A-9F7-A10 and BVO-B-1H7-E3, demonstrated strong and specific binding affinity to multiple mammalian ALK1 orthologs.

[0113] Example 3 ALK5 binding assessment Flow cytometry was used to measure the fluorescence intensity resulting from a fluorescently labeled secondary antibody that detects anti-ALK1 primary antibody binding to specific cell-associated ALK5 molecules. More precisely, hybridoma supernatants containing primary antibodies were tested by flow cytometry for binding to transiently transfected cells expressing human and mouse ALK5. Signals were generated by using a PE-labeled goat anti-rat secondary antibody. As a negative control, in addition to testing the supernatant of untransfected cells, human and mouse ALK5 transfected cells were incubated with the secondary antibody alone (without supernatant).

[0114] Example 4 BMP9 signaling assessment Eighty-nine anti-ALK1 antibodies were evaluated to determine whether the antibodies inhibited BMP9 binding and SMAD1 / 5 signaling. Of the 89 antibodies evaluated, a total of 12 did not block BMP9 signaling.

[0115] [Table 4]

[0116] The antibodies evaluated in the above assay are listed in the table above. Of these antibodies, BVN-A-2B2, BVN-A-2C7, BVN-A-2D10, BVN-B-2F11, BVN-B-5B5, BVO-A-1D5, BVO-A-9C4, BVO-A-9C5, BVO-A-9F7, BVO-B-1H7, BVO-B-5D2, and BWG-3B3 did not block BMP9 signaling.

[0117] Example 5 Atherosclerosis Experiment BVO-A-9F7-A10 has been shown to block LDL uptake, but not BMP9 signaling, in cultured human and mouse endothelial cells. Furthermore, in preliminary experiments, treatment of mice overnight with anti-ALK antibodies reduced LDL uptake in the blood vessel wall in vivo. Similar results are expected to be achieved with BVO-B-1H7-E3.

[0118] The aim of the following predictive study is to investigate whether long-term treatment with an ALK1 antibody reduces atherosclerosis in a mouse model.

[0119] Experimental Design: Eight- to ten-week-old mice lacking the LDL receptor are fed a cholesterol-rich high-fat diet (HFD) for 14 weeks to induce experimental atherosclerosis.

[0120] Treat mice with control or anti-ALK antibodies (e.g., BVO-A-9F7-A10 or BVO-B-1H7-E3; 400-500 micrograms per mouse x 3 times per week) for a duration of 14 weeks.

[0121] At 14 weeks, the following analyses will be performed: 1. Plasma lipids (cholesterol, TG) and inflammatory cell populations by FAC 2. Plasma collection for antibody levels (PK assessment) 3. Assessment of the extent of atherosclerosis in three anatomical locations: 1. aorta (en face imaging of neutral lipid accumulation), 2. transverse section of aortic root, and 3. brachiocephalic artery. 4. Analysis of lesion composition including ApoB 100 immunochemistry, macrophages (F4 / 80 or CD68 staining), lesion necrosis and fibrous capsule (from H&E sections).

[0122] The results indicate that the antibodies of the disclosure (e.g., BVO-A-9F7-A10 and BVO-B-1H7-E3) are suitable for treating or preventing atherosclerosis.

[0123] All patents and publications mentioned in this specification are indicative of the level of those skilled in the art to which this disclosure pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[0124] Moreover, it will be readily apparent to one skilled in the art that the present disclosure is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. Modifications and other uses will occur to those skilled in the art. These modifications are encompassed within the spirit defined by the disclosure and the claims, in which non-limiting embodiments of the disclosure have been described.

[0125] The present disclosure may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. Therefore, the above-described embodiments should be considered in all respects as illustrative and not limiting of the present disclosure. The scope of the present disclosure is therefore indicated by the appended claims, not by the above description, and all changes that come within the meaning and range of equivalence of the claims are therefore intended to be embraced within their scope.

Claims

1. a. a heavy chain variable sequence of SEQ ID NO:2 and a light chain variable sequence of SEQ ID NO:3; b. a heavy chain variable sequence of SEQ ID NO:4 and a light chain variable sequence of SEQ ID NO:5; c. a heavy chain variable sequence of SEQ ID NO:6 and a light chain variable sequence of SEQ ID NO:7; d. a heavy chain variable sequence of SEQ ID NO: 10 and a light chain variable sequence of SEQ ID NO: 11; and e. A heavy chain variable sequence of SEQ ID NO: 12 and a light chain variable sequence of SEQ ID NO: 13 An anti-ALK1 antibody comprising a heavy chain variable sequence and a light chain variable sequence complementarity determining region (CDR) selected from:

2. a. a heavy chain variable sequence of SEQ ID NO:2 and a light chain variable sequence of SEQ ID NO:3; b. a heavy chain variable sequence of SEQ ID NO:4 and a light chain variable sequence of SEQ ID NO:5; c. a heavy chain variable sequence of SEQ ID NO:6 and a light chain variable sequence of SEQ ID NO:7; d. a heavy chain variable sequence of SEQ ID NO: 10 and a light chain variable sequence of SEQ ID NO: 11; and e. A heavy chain variable sequence of SEQ ID NO: 12 and a light chain variable sequence of SEQ ID NO: 13 The anti-ALK1 antibody of claim 1, further comprising a variable heavy chain sequence and a variable light chain sequence selected from:

3. 2. The anti-ALK1 antibody of claim 1, wherein the heavy chain variable sequence comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:2, 4, 6, 10, or 12, and the light chain variable sequence comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:3, 5, 7, 11, or 13.

4. 2. The anti-ALK1 antibody of claim 1, wherein the heavy chain variable sequence comprises 1 to 10 amino acid substitutions, insertions or deletions in SEQ ID NO: 2, 4, 6, 10, or 12, and the light chain variable sequence comprises 1 to 10 amino acid substitutions, insertions or deletions in SEQ ID NO: 3, 5, 7, 11, or 13.

5. a heavy chain comprising a CDRH1 comprising SSYWN (SEQ ID NO:26), a CDRH2 comprising EVNHSGSTNYNPSLKS (SEQ ID NO:27), and a CDRH3 comprising SPRSGRIVGAVFDY (SEQ ID NO:28); and a light chain comprising a CDRL1 comprising GGNNIGPKS VH (SEQ ID NO:29), a CDRL2 comprising DDSDRPS (SEQ ID NO:30), and a CDRL2 comprising QVWDSSNDHVV (SEQ ID NO:31). An anti-ALK1 antibody comprising:

6. a heavy chain comprising a CDRH1 comprising SRSYYWG (SEQ ID NO:32), a CDRH2 comprising NIYYSGSAFYNPSLKS (SEQ ID NO:33), and a CDRH3 comprising WDNWDVGAFDI (SEQ ID NO:34); and a light chain comprising a CDRL1 comprising TGTSSDVGGYNYVS (SEQ ID NO:35), a CDRL2 comprising EVSKRPS (SEQ ID NO:36), and a CDRL2 comprising TSFAGSNNWV (SEQ ID NO:37). An anti-ALK1 antibody comprising:

7. A method for treating or preventing a disease or disorder associated with ALK1, comprising administering to a subject having a disease or disorder associated with ALK1 an antibody described in any one of claims 1 to 6.

8. 8. The method of claim 7, wherein the disease is atherosclerosis.

9. The method of claim 7 or 8, wherein the subject is a human.

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