Antibodies Targeting SOST and Their Uses

Abstract

The present invention relates to antibodies targeting SOST, antigen-binding fragments thereof, and their uses. Specifically provided are antibodies targeting SOST or antigen-binding fragments thereof, or variants having at least 85% sequence identity with the antibody or antigen-binding fragment thereof and retaining their SOST-binding activity, wherein the antibody or antigen-binding fragment thereof can specifically bind to the SOST protein.

Description

Technical Field

[0001] The present disclosure relates to monoclonal antibodies against sclerostin, antigen-binding fragments thereof, and their uses. Background Art

[0002] Currently, drugs for treating osteoporosis include two major categories: anti-resorptive and bone anabolic. Anti-resorptive drugs include calcitonin, bisphosphonates, estrogen replacement agents, estrogen receptor modulators, and monoclonal antibodies specific for receptor activator of nuclear factor-κB ligand (RANKL). Such drugs target osteoclasts, slowing down osteoclast-mediated bone resorption. Although they can slow down further bone loss, they cannot restore the lost bone mass and bone microstructure. Bone anabolic drugs include parathyroid hormone (PTH) analogs, parathyroid hormone-related protein (PTHrP) analogs, and monoclonal antibodies specific for sclerostin (SOST). PTH and PTHrP analogs have been shown to stimulate bone formation, but they need to be administered subcutaneously every day. Their high frequency of administration, inconvenience in use, and potential safety issues limit their application. Romosozumab developed by Amgen and UCB is currently the only approved monoclonal antibody targeting sclerostin for treating osteoporosis globally. This antibody has been proven to effectively promote bone formation and inhibit bone resorption, and it is a drug for treating osteoporosis with a new mechanism of action. Therefore, it is highly valuable to develop drugs targeting the same target with better efficacy and safety.

[0003] Sclerostin is mainly secreted by osteocytes, and its binding to low-density lipoprotein receptor-related protein 5 / 6 (LRP5 / 6) can inhibit the classical Wnt signaling pathway. Studies on sclerosteosis and van Buchem disease have shown that patients with these diseases have abnormal bone mass due to the deletion or mutation of the sclerostin gene, resulting in the absence or reduced expression of sclerostin protein. Anti-sclerostin antibodies bind to sclerostin, blocking its binding to LRP5 / 6, allowing Wnt to form a heterotrimer with LRP5 / 6 and Frizzled receptors and mediate downstream signaling, promoting osteoblast activation and new bone formation. Summary of the Invention

[0004] The present invention first provides an antibody against sclerostin (SOST) or an antigen-binding fragment thereof, or a variant having at least 85% sequence identity with the antibody or its antigen-binding fragment and retaining its SOST-binding activity, wherein the antibody targets SOST. Preferably, the antibody targets the flexible loop region 2 of SOST, which contains the P1 polypeptide fragment (PNAIGRGKWWRPSGPDFR).

[0005] In one or more embodiments, the antibody comprises: three HCDRs of the heavy chain variable region shown in SEQ ID NO:1, and / or three LCDRs of the light chain variable region shown in SEQ ID NO:2.

[0006] In one or more embodiments, HCDR1 of the antibody comprises SEQ ID NO:3, or a sequence having at least 85% sequence identity therewith.

[0007] In one or more embodiments, HCDR2 of the antibody comprises SEQ ID NO:4 or a sequence having at least 85% sequence identity therewith.

[0008] In one or more embodiments, HCDR3 of the antibody comprises SEQ ID NO:5 or a sequence having at least 85% sequence identity therewith.

[0009] In one or more embodiments, LCDR1 of the antibody comprises SEQ ID NO:6 or a sequence having at least 85% sequence identity therewith.

[0010] In one or more embodiments, LCDR2 of the antibody comprises SEQ ID NO:7 or a sequence having at least 85% sequence identity therewith.

[0011] In one or more embodiments, LCDR3 of the antibody comprises SEQ ID NO:8 or a sequence having at least 85% sequence identity therewith.

[0012] In one or more embodiments, the heavy chain of the antibody comprises the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4, or a sequence having at least 85% sequence identity therewith. Alternatively or additionally, the light chain of the antibody comprises the light chain constant region of human κ chain, λ chain, or a sequence having at least 85% sequence identity therewith.

[0013] In one or more embodiments, the heavy chain of the antibody comprises the heavy chain constant region of human IgG1 (SEQ ID NO:9), or a sequence having at least 85% sequence identity therewith, and the light chain comprises the light chain constant region of human κ chain (SEQ ID NO:10), or a sequence having at least 85% sequence identity therewith.

[0014] In one or more embodiments, the heavy chain of the antibody has the sequence of SEQ ID NO:11, or has at least 80%, 90%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO:11, or alternatively or in addition, the light chain of the antibody has the sequence shown in SEQ ID NO:12, or has at least 80%, 90%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO:12.

[0015] In one or more embodiments, the antibody is a multispecific antibody, preferably a bispecific antibody.

[0016] In one or more embodiments, the antibody is a monoclonal antibody.

[0017] In one or more embodiments, the antibody is a chimeric antibody or a fully human antibody.

[0018] In one or more embodiments, the antibody targeting SOST is a whole antibody, a Fab fragment, an F(ab’)2 fragment, or a scFv fragment.

[0019] The present invention also provides polynucleotides selected from:

[0020] (1) the coding sequence of the antibody or its antigen-binding fragment according to any embodiment herein;

[0021] (2) the complementary sequence of (1).

[0022] The present invention also provides a nucleic acid construct comprising the polynucleotide according to any embodiment herein.

[0023] In one or more embodiments, the nucleic acid construct is a vector, such as an integrating vector, a cloning vector or an expression vector.

[0024] The present invention also provides a phage comprising the antibody or its antigen-binding fragment according to any embodiment herein or a library comprising such phage.

[0025] In one or more embodiments, the antibody or its antigen-binding fragment is displayed on the surface of the phage.

[0026] The present invention also provides a host cell which:

[0027] (1) expresses and / or secretes the antibody or its antigen-binding fragment according to any embodiment herein;

[0028] (2) comprises the polynucleotide described herein; and / or

[0029] (3) comprises the nucleic acid construct described herein.

[0030] In one or more embodiments, the host cell is selected from prokaryotic cells or eukaryotic cells.

[0031] In one or more embodiments, the host cell is a mammalian cell.

[0032] The present invention also provides a method for producing an antibody or an antigen-binding fragment thereof, comprising: culturing the host cell described herein under conditions suitable for producing an antibody or an antigen-binding fragment thereof, and optionally purifying the antibody or an antigen-binding fragment thereof from the culture.

[0033] The present invention also provides a pharmaceutical composition comprising the antibody or an antigen-binding fragment thereof, polynucleotide, nucleic acid construct, phage or host cell described herein, and a pharmaceutically acceptable excipient.

[0034] In one or more embodiments, the excipient is a carrier, diluent or vehicle.

[0035] In one or more embodiments, the pharmaceutical composition is used for treating osteoporosis, osteogenesis imperfecta, fractures or other metabolic bone diseases.

[0036] In one or more embodiments, the osteoporosis includes primary osteoporosis and secondary osteoporosis.

[0037] The present invention also provides the use of the antibody or an antigen-binding fragment thereof, polynucleotide, nucleic acid construct or host cell according to any one of the embodiments herein in the preparation of a drug for preventing or treating a disease.

[0038] In one or more embodiments, the disease is osteoporosis, osteogenesis imperfecta, fractures or other metabolic bone diseases.

[0039] The present invention also provides a method for treating or preventing a disease, the method comprising administering to a patient in need a therapeutically effective amount of the antibody or an antigen-binding fragment thereof or the pharmaceutical composition according to any one of the embodiments of the present invention.

[0040] The present invention also provides a kit for detecting sclerostin for evaluating the therapeutic effect of a drug, the kit comprising the antibody or an antigen-binding fragment thereof, polynucleotide, nucleic acid construct, phage or host cell according to any one of the embodiments herein.

[0041] In one or more embodiments, the kit further comprises a reagent for detecting the binding of sclerostin to the antibody or an antigen-binding fragment thereof. For example, a reagent for detecting the binding by an enzyme-linked immunosorbent assay.

[0042] In one or more embodiments, the reagent for detecting binding is a detectable label capable of being linked to an antibody or an antigen-binding fragment thereof, such as biotin. The detectable label is linked to the antibody or an antigen-binding fragment thereof or is present separately in the kit.

[0043] The present invention also provides a non-diagnostic method for detecting the presence of sclerostin in a sample, the method comprising: incubating the sample with the antibody or an antigen-binding fragment thereof according to any one of the embodiments herein, and detecting the binding of sclerostin to the antibody or an antigen-binding fragment thereof, thereby determining the presence of sclerostin in the sample. The detection is by an enzyme-linked immunosorbent assay.

[0044] The present invention also provides the use of the antibody or an antigen-binding fragment thereof according to any one of the embodiments herein in the preparation of a kit for detecting sclerostin in a sample and evaluating the therapeutic effect of a drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 . Epitope analysis of m-9 and romosozumab binding to SOST. DETAILED DESCRIPTION

[0046] Unless otherwise defined, scientific and technical terms used herein shall have the meanings commonly understood by those of ordinary skill in the art. Generally, the nomenclature and techniques used herein in connection with cell and tissue culture, molecular biology, and the chemistry and hybridization of proteins and oligonucleotides or polynucleotides are well known and commonly used in the art.

[0047] Specifically, the present invention obtains a monoclonal antibody against human SOST by immunizing mice, constructing a mouse scFv-phage immune library and screening, constructs a plasmid vector expressing the heavy and light chains of a chimeric antibody, transfects the above plasmid vector into Expi293F cells, further purifies to obtain a highly pure antigen-binding protein, and detects the antibody activity, specificity and binding epitope by Topflash and BLI methods. The results show that the antibody has high affinity, high inhibitory activity against sclerostin and a binding epitope different from that of romosozumab.

[0048] As used herein, the term "antibody" includes full-length antibodies having an immunoglobulin Fc, antibody compositions having multi-epitope specificity, multi-specific antibodies (e.g., bispecific antibodies), diabodies and single-chain molecules, and antibody fragments, particularly antigen-binding fragments, such as Fab, F(ab')2 and Fv.

[0049] The basic four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light chains (L) and two identical heavy chains (H). IgM antibodies consist of five basic heterotetrameric units and an additional polypeptide called the J chain, containing ten antigen-binding sites; while IgA antibodies contain two to five basic four-chain units, which can combine with the J chain to polymerize into multivalent assemblies. In the case of IgG, the four-chain unit is typically about 150,000 daltons. Each light chain is linked to the heavy chain by a covalent disulfide bond, and the two heavy chains are linked to each other by one or more disulfide bonds, the number of which depends on the heavy chain isotype. Each heavy and light chain also has regularly spaced intra-chain disulfide bridges. Each heavy chain has a variable domain (VH) at the N-terminus, followed by three (CH1, CH2, and CH3 for each α and γ chain) and four (CH1, CH2, CH3, and CH4 for μ and ε isotypes) constant domains (CH), as well as a hinge region (Hinge) located between the CH1 and CH2 domains. Each light chain has a variable domain (VL) at the N-terminus, followed by a constant domain (CL) at the other end. VL aligns with VH, and CL aligns with the first constant domain (CH1) of the heavy chain. Specific amino acid residues are thought to form an interface between the light and heavy chain variable domains. Pairs of VH and VL together form an antigen-binding site. For the structure and properties of different classes of antibodies, see, for example, Basic and Clinical Immunology, 8th edition, edited by Daniel P. Sties, Abba I. Terr, and Tristram G. Parsolw, Appleton & Lange, Norwalk, CT, 1994, pages 71 and chapter 6. Light chains from any vertebrate species can be grouped into one of two distinct types called κ and λ, based on the amino acid sequence of their constant domains. Immunoglobulins can be grouped into different classes or isotypes based on the amino acid sequence of their heavy chain constant domains (CH). There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, which have heavy chains called α, δ, ε, γ, and μ, respectively. Based on relatively minor differences in CH sequence and function, the γ and α classes can be further divided into subclasses, for example, humans express the following subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1, and IgA2.

[0050] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. The variable domains of the heavy and light chains can be referred to as "VH" and "VL", respectively. These domains are generally the most variable parts of the antibody (relative to other antibodies of the same type) and contain the antigen-binding site.

[0051] The term "variable" refers to the situation where certain segments in the variable domain vary widely among antibody sequences. The variable domain mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across all the amino acids spanned by the variable domain. Instead, it is concentrated in three segments called hypervariable regions (HVRs) (both in the light-chain and heavy-chain variable domains), namely HCDR1, HCDR2, HCDR3 in the heavy-chain variable region (which can be abbreviated as CDR1, CDR2, CDR3 in heavy-chain antibodies) and LCDR1, LCDR2, and LCDR3 in the light-chain variable region. The more highly conserved portions of the variable domain are called framework regions (FRs). The variable domains of native heavy and light chains each contain four FR regions (FR1, FR2, FR3, and FR4), which mostly adopt a β-sheet conformation and are connected by the three HVRs that form loops and in some cases are part of the β-sheet structure. The HVRs in each chain are held very close together by the FR regions and together with the HVRs of the other chain contribute to the formation of the antigen-binding site of the antibody. Generally, the structure of the light-chain variable region is FR1-LCDR1-FR2-LCDR2-FR3-LCDR3-FR4, and the structure of the heavy-chain variable region is FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4. The constant domain does not directly participate in the binding of the antibody to the antigen but exhibits various effector functions, such as the participation of the antibody in antibody-dependent cell-mediated cytotoxicity.

[0052] The "Fc region" (fragment crystallizable region) or "Fc domain" or "Fc" refers to the C-terminal region of the antibody heavy chain, which mediates the binding of the immunoglobulin to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells), or binding to the first component (C1q) of the classical complement system. In the IgG antibody isotype, the Fc region consists of two identical protein fragments from the CH2 and CH3 domains of the two heavy chains of the antibody. Although the boundaries of the Fc region of the immunoglobulin heavy chain can vary, the human IgG heavy-chain Fc region is generally defined as the sequence segment from the amino acid residue at position C226 or P230 of the heavy chain to the carboxyl terminus, where this numbering is according to the EU index, as in Kabat. As used herein, the Fc region can be a native sequence Fc or a variant Fc.

[0053] "Antibody fragment" includes a portion of a full antibody, preferably the antigen-binding region and / or variable region of a full antibody. The antibody fragment is preferably an antigen-binding fragment of an antibody. Examples of antibody fragments include Fab, Fab', F(ab')2 and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; scFv-Fc fragments; multispecific antibodies formed by antibody fragments; and any fragment that should be able to increase the half-life through chemical modification or incorporation into liposomes. Digestion of an antibody with papain yields two identical antigen-binding fragments called "Fab" fragments and a residual "Fc" fragment, the name of which reflects its ability to crystallize easily. The Fab fragment consists of the complete light chain and the variable domain of the heavy chain (VH) and the first constant domain of the heavy chain (CH1). Each Fab fragment is monovalent in terms of antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of an antibody yields a larger F(ab')2 fragment, which roughly corresponds to two Fab fragments linked by a disulfide bond, with different antigen-binding activities and still capable of cross-linking antigens. The Fab' fragment differs from the Fab fragment by the addition of some additional residues at the carboxyl terminus of the CH1 domain, including one or more cysteines from the antibody hinge region. The F(ab')2 antibody fragment is initially generated as a pair of Fab' fragments with hinge cysteines between the Fab' fragments. Other chemical conjugations of antibody fragments are also known. The Fc fragment contains the carboxyl-terminal portions of the two heavy chains held together by disulfide bonds. The effector function of an antibody is determined by the sequence in the Fc region, which is also the region recognized by Fc receptors (FcR) found on certain types of cells.

[0054] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., each antibody constituting the population is identical except for possible naturally occurring mutations and / or post-translational modifications (such as isomerization, amidation) that may be present in minor amounts. Monoclonal antibodies are highly specific, directed against a single antigenic site. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on an antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they are synthesized by hybridoma culture and are not contaminated with other immunoglobulins. The modifier "monoclonal" indicates the characteristic of an antibody obtained from a substantially homogeneous population of antibodies and should not be construed as requiring that the antibody be produced by any particular method. For example, monoclonal antibodies used according to the present invention can be generated by a variety of techniques, including, for example, the hybridoma method, phage display method, recombinant DNA method, and techniques for generating human or human-like antibodies from animals having part or all of the human immunoglobulin locus or genes encoding human immunoglobulin sequences, single-cell sequencing method.

[0055] Monoclonal antibodies also include "chimeric" antibodies herein, in which part of the heavy and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remaining part of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, provided that they exhibit the desired biological activity.

[0056] The "humanized" form of a non-human (e.g., murine) antibody refers to a chimeric antibody that minimally contains a sequence derived from a non-human immunoglobulin. Thus, a "humanized antibody" generally refers to a non-human antibody in which the framework regions of the variable domains are exchanged with sequences found in human antibodies. Typically, in a humanized antibody, the entire antibody (except for the CDRs) is encoded by polynucleotides of human origin or is identical to such an antibody (except for the CDRs). The CDRs (some or all of which are encoded by nucleic acids derived from a non-human organism) are transplanted into the β-sheet framework of the human antibody variable region to generate an antibody whose specificity is determined by the transplanted CDRs. Methods for producing such antibodies are well known in the art, such as using mice with a genetically engineered immune system. In the present invention, antibodies, single-domain antibodies, heavy-chain antibodies, etc. all include humanized variants of the respective antibodies.

[0057] A "human antibody" refers to an antibody having an amino acid sequence corresponding to that of an antibody produced by a human and / or produced using any of the techniques for generating human antibodies disclosed herein. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues. Human antibodies can be generated using a variety of techniques known in the art, including phage display libraries.

[0058] In the present invention, the antibody has HCDR1 shown in SEQ ID NO:3, HCDR2 shown in SEQ ID NO:4, and HCDR3 shown in SEQ ID NO:5, and / or LCDR1 shown in SEQ ID NO:6, LCDR2 shown in SEQ ID NO:7, and LCDR3 shown in SEQ ID NO:8.

[0059] The FR1, FR2, FR3, and FR4 of the antibodies described herein can be selected from (1) the FR1, FR2, FR3, and FR4 of the heavy-chain variable region shown in SEQ ID NO:1, or (2) the FR1, FR2, FR3, and FR4 of the light-chain variable region shown in SEQ ID NO:2. Preferably, the amino acid sequence of the heavy-chain variable region of the antibody described herein is as shown in SEQ ID NO:1, and the amino acid sequence of the light-chain variable region of the antibody described herein is as shown in SEQ ID NO:2.

[0060] In one or more embodiments, the antibodies described herein further include a heavy chain constant region and / or a light chain constant region. The heavy chain constant region is, for example, a heavy chain constant region derived from IgA, IgD, IgE, IgG, and IgM, or a heavy chain constant region derived from IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1, and IgA2, or a sequence having at least 85% sequence identity thereto. The light chain constant region is, for example, a light chain constant region of a κ or λ chain, or a sequence having at least 85% sequence identity thereto.

[0061] The heavy chain constant region of the antibodies described herein may be the constant region of a human heavy chain antibody, including CH1, CH2, and CH3. Preferably, the antibody constant region is derived from: the constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD, more preferably from the constant region of any one of IgG1, IgG2, IgG3, and IgG4. In one or more embodiments, the heavy chain constant region described herein is the constant region of human IgG1 Fc, as shown in SEQ ID NO:9.

[0062] The light chain constant region of the antibodies described herein may be the constant region of a human antibody light chain. In one or more embodiments, the sequence of the light chain constant region is as shown in SEQ ID NO:10.

[0063] The term "targeting" refers to specifically binding to an antigen molecule. Exemplarily, an antibody "targeting SOST" herein specifically refers to an antibody that specifically binds to SOST.

[0064] The present invention also includes derivatives and analogs of the various antibodies described above (such as single-domain antibodies, heavy chain antibodies or antigen-binding fragments thereof, multivalent single-domain antibodies, multispecific single-domain antibodies, antibodies or antigen-binding fragments thereof). "Derivatives" and "analogs" refer to polypeptides that substantially retain the same biological functions or activities of the various antibodies of the present invention. The derivatives or analogs of the present invention may be (i) polypeptides having a substitution group in one or more amino acid residues, or (ii) polypeptides formed by fusing a mature polypeptide with another compound (such as a compound that prolongs the polypeptide half-life, such as polyethylene glycol), or (iii) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (such as a leader sequence or a secretion sequence or a sequence used to purify this polypeptide or a proprotein sequence, or a fusion protein formed with a 6*His tag). According to the teachings herein, these derivatives and analogs are within the scope well-known to those skilled in the art.

[0065] Without substantially affecting the antibody activity, those skilled in the art can change one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more) amino acids of the sequences of the present invention to obtain variants of the sequences of the antibody or its functional fragments. These variants include (but are not limited to): deletion, insertion and / or substitution of one or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acids, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and / or N-terminus. In the art, when conservative substitutions are made with amino acids having similar or close properties, the function of the protein is usually not changed. For example, amino acids with similar properties are substituted in the FR and / or CDR regions of the variable region. Amino acid residues that can be conservatively substituted are well known in the art. Such substituted amino acid residues may or may not be encoded by the genetic code. Also, for example, adding one or several amino acids at the C-terminus and / or N-terminus usually does not change the function of the protein. They are all considered to be included within the scope of protection of the present invention.

[0066] Variant forms of the various antibodies described herein include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNAs that can hybridize with the coding DNAs of the various antibodies of the present invention under high or low stringency conditions, and polypeptides or proteins obtained using antisera against the various antibodies of the present invention.

[0067] In some embodiments, the sequences of the variants of the present invention may have at least 95%, 96%, 97%, 98% or 99% identity with their source sequences. The sequence identity described herein can be measured using sequence analysis software. For example, using the computer program BLAST with default parameters, especially BLASTP or TBLASTN. The present invention also includes those molecules having a heavy chain variable region of an antibody with CDRs or a light chain variable region of an antibody with CDRs, as long as their CDRs have more than 90% (preferably more than 95%, most preferably more than 98%) homology with the CDRs identified herein.

[0068] The antibodies of the present invention can be prepared by conventional methods in the art, such as the well-known hybridoma technology in the art. Alternatively, various antibodies of the present invention can be expressed in other cell lines. Suitable mammalian host cells can be transformed with the sequences encoding various antibodies of the present invention. The transformation can be carried out by any known method, for example, including packaging the polynucleotide in a virus (or viral vector) and transducing the host cell with the virus (or vector). The transformation procedures used depend on the host to be transformed. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art, including dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulating the polynucleotide in liposomes, and direct microinjection of DNA into the nucleus, etc. Mammalian cell lines that can be used as hosts for expression are well known in the art, including but not limited to various immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., HepG2), etc. Particularly preferred cell lines are selected by determining which cell lines have high expression levels and produce antibodies with substantially SOST-binding properties.

[0069] The invention also provides polynucleotides encoding the above-mentioned antibodies or their antigen-binding fragments. The polynucleotides of the present invention can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA, or synthetic DNA. The DNA can be single-stranded or double-stranded, usually in a double-stranded structure, with a sense strand and an antisense strand that are complementary to each other. Among them, the sense strand is also called the coding strand, which refers to the strand with genetic information in the DNA double strand and cannot be transcribed. It contains the same nucleotide sequence as the mRNA encoding the functional protein, with the only difference being that T in DNA is U in mRNA. The antisense strand is also called the template strand, which refers to the DNA strand used by RNA polymerase during transcription and serves as the template for transcription. RNA polymerase moves along the template strand and transcribes it into mRNA, which is then translated into a polypeptide.

[0070] Narrowly speaking, the "polynucleotide sequence encoding..." or "coding sequence of..." refers to the sequence that can directly guide transcription on the antisense strand of DNA or the sequence that can directly guide translation on mRNA. The polynucleotide can include a coding sequence (or coding region) and a non-coding sequence (or non-coding region, such as an intron). The coding sequence can be continuous or discontinuous. The discontinuous coding sequence fragments can be separated by non-coding sequences. The complete coding region obtained by sequentially connecting the coding sequence fragments is the coding sequence of the polypeptide.

[0071] The present invention also includes degenerate variants of the polynucleotide sequences encoding the fusion proteins, that is, nucleotide sequences encoding the same amino acid sequence but having different nucleotide sequences. As will be appreciated by those skilled in the art, due to the degeneracy of the genetic code, an extremely large number of nucleic acids can be made, all of which encode the antibodies or antigen-binding fragments of the present invention. Thus, given the identification of a particular amino acid sequence, those skilled in the art can make any number of different nucleic acids by simply modifying the sequence of one or more codons in a manner that does not change the amino acid sequence of the encoded protein. Accordingly, the present invention also relates to polynucleotides that hybridize to the above-described polynucleotide sequences and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the present invention under stringent conditions. In the present invention, "stringent conditions" mean: (1) hybridization and washing at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) in the presence of a denaturing agent during hybridization, such as 50% (v / v) formamide, 0.1% bovine serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the identity between the two sequences is at least 90% or more, preferably 95% or more. Moreover, the polypeptides encoded by the hybridizable polynucleotides have the same biological functions and activities as the mature polypeptides.

[0072] The full-length nucleotide sequences or fragments thereof of the various antibodies of the present invention can generally be obtained by PCR amplification, recombination or artificial synthesis. A feasible method is to synthesize the relevant sequences by artificial synthesis, especially when the fragment length is short. Generally, very long fragments can be obtained by first synthesizing multiple small fragments and then ligating them. In addition, the coding sequence of the heavy chain can be fused with an expression tag (such as 6*His) to form a fusion protein.

[0073] Once the relevant sequences are obtained, the relevant sequences can be obtained in large quantities by recombination. This is usually done by cloning them into a vector, then introducing them into cells, and then isolating the relevant sequences from the proliferated host cells by conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in the present invention include biomolecules in isolated form. At present, it is already possible to obtain the DNA sequence encoding the protein (or its fragment, or its derivative) of the present invention entirely by chemical synthesis. Then the DNA sequence can be introduced into various existing DNA molecules (or such as vectors) and cells known in the art. In addition, mutations can be introduced into the protein sequence of the present invention by chemical synthesis.

[0074] Accordingly, the present invention also relates to nucleic acid constructs, such as expression vectors and recombinant vectors, comprising the above-described suitable DNA sequences and suitable promoters or control sequences. These vectors can be used to transform suitable host cells to enable them to express proteins. Vectors generally contain sequences for plasmid maintenance and for cloning and expressing exogenous nucleotide sequences. The sequences (collectively referred to as "flanking sequences" in some embodiments) generally include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence containing donor and acceptor splice sites, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a multiple linker region for inserting nucleic acids encoding the antibody to be expressed, and optional marker elements.

[0075] Host cells can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells such as Drosophila S2 or Sf9 cells; eukaryotic cells such as CHO, COS7, 293 cells, etc.

[0076] In some embodiments, the host cells can be various functional cells well-known in the art, such as various killer cells, including but not limited to cytokine-induced killer cells (CIK), dendritic cell-stimulated cytokine-induced killer cells (DC-CIK), cytotoxic T lymphocytes (CTL), γδ T cells, natural killer cells (NK), tumor-infiltrating lymphocytes (TIL), lymphokine-activated killer cells (LAK), CD3AK cells (killer cells of anti-CD3 monoclonal antibody), and CAR-T / TCR-T cells. In some embodiments, the killer cells are T cells or NK cells. Exemplary NK cells include but are not limited to primary NK cells, NK cell lines (such as NK92), and NKT cells. In some embodiments, the NK cells are primary NK cells. Exemplary T cells include but are not limited to peripheral blood T lymphocytes, cytotoxic killer T cells (CTL), helper T cells, suppressor / regulatory T cells, γδ T cells, and T cells of a mixed cell population such as cytokine-induced killer cells (CIK), tumor-infiltrating lymphocytes (TIL), etc. In some embodiments, the T cells are peripheral blood T lymphocytes and T cells derived from TIL.

[0077] Transformation of host cells with recombinant DNA can be carried out by conventional techniques well-known to those skilled in the art. When the host is a prokaryote such as Escherichia coli, competent cells capable of taking up DNA can be harvested after the exponential growth phase and treated with the CaCl2 method, the steps of which are well-known in the art. Another method is to use MgCl2. If desired, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods can be selected: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0078] The obtained transformants can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used in the culture can be selected from various conventional culture media. The culture is carried out under conditions suitable for the growth of the host cell. When the host cell grows to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature shift or chemical induction), and the cells are cultured for a further period of time.

[0079] The polypeptide in the above method can be expressed intracellularly, or on the cell membrane, or secreted extracellularly. If desired, the recombinant protein can be separated and purified by various separation methods utilizing its physical, chemical, and other properties. These methods are well-known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting-out method), centrifugation, osmotic lysis, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations of these methods.

[0080] All aspects of the various antibodies described herein can be used for the preparation of agents for preventing or treating osteoporosis, osteogenesis imperfecta, fractures, or other metabolic bone diseases, etc.

[0081] The pharmaceutical compositions herein contain the antibodies described herein, as well as pharmaceutically acceptable excipients, including but not limited to diluents, carriers, solubilizers, emulsifiers, preservatives, and / or adjuvants. The excipients are preferably non-toxic to the recipient at the doses and concentrations employed. Such excipients include (but are not limited to): saline, buffers, glucose, water, glycerol, ethanol, and combinations thereof. In certain embodiments, the pharmaceutical composition may contain substances for improving, maintaining, or retaining, for example, the pH, osmotic pressure, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, absorption, or permeability of the composition. These substances are known in the prior art. The optimal pharmaceutical composition can be determined depending on the intended route of administration, mode of delivery, and required dose.

[0082] Pharmaceutical compositions for in vivo administration are typically provided in the form of sterile preparations. Sterilization is achieved by filtration through a sterile filtration membrane. This method can be used for sterilization before or after lyophilization and reconstitution of the composition. The pharmaceutical compositions of the present invention can be selected for parenteral delivery. Compositions for parenteral administration can be stored in lyophilized form or in solution. For example, they are prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Parenteral compositions are usually placed in containers having a sterile access port, such as intravenous solution bags or vials with stoppers pierceable by a hypodermic needle. Alternatively, the compositions can be selected for inhalation or delivery through the digestive tract (such as orally). The preparation of the pharmaceutically acceptable compositions is within the skill of the art. Other pharmaceutical compositions will be apparent to those skilled in the art, including formulations containing antibodies in sustained or controlled release delivery formulations. Techniques for formulating a variety of other sustained or controlled delivery modalities, such as liposomal carriers, bioerodible microparticles or porous beads, and depot injections, are also known to those skilled in the art.

[0083] Once formulated, the pharmaceutical compositions are stored in sterile vials in the form of solutions, suspensions, gels, emulsions, solids, crystals, or as dehydrated or lyophilized powders. The formulations can be stored in ready-to-use form or in a form that is rehydrated before administration (e.g., lyophilized). The present invention also provides kits for producing single-dose administration units. The kits of the present invention can each contain a first container with the dried protein and a second container with the aqueous formulation. In certain embodiments of the present invention, kits containing single-chamber and multi-chamber prefilled syringes (e.g., liquid syringes and lyophilized syringes) are provided.

[0084] The present invention also provides methods for preventing or treating osteoporosis, osteogenesis imperfecta, fractures, or other metabolic bone diseases by administering an antibody or its pharmaceutical composition according to any embodiment of the present invention. As used herein, the terms "subject", "individual", "subject" are used interchangeably herein and include any living organism, preferably an animal, more preferably a mammal (such as a rat, mouse, dog, cat, rabbit, etc.), and most preferably a human.

[0085] The therapeutically effective amount of the pharmaceutical composition containing the antibody of the present invention to be employed will depend, for example, on the degree and target of treatment. Those skilled in the art will appreciate that the appropriate dosage level for treatment will vary in part depending on the molecule being delivered, the indication, the route of administration, and the size (body weight, body surface area, or organ size) and / or condition (age and general health status) of the patient. In certain embodiments, the clinician can titrate the dose and change the route of administration to obtain the optimal therapeutic effect. For example, from about 10 micrograms per kilogram of body weight per day to about 50 milligrams per kilogram of body weight.

[0086] The dosing frequency will depend on the pharmacokinetic parameters of the antibody in the formulation used. Clinicians typically administer the composition until a dose is reached that achieves the desired effect. The composition can thus be administered as a single dose, or over time as two or more doses (which may or may not contain the same amount of the desired molecule), or by continuous infusion via an implant device or catheter.

[0087] The administration route of the pharmaceutical composition is according to known methods, such as oral, by intravenous, intraperitoneal, intracerebral (intraparenchymal), intraventricular, intramuscular, intraocular, intraarterial, portal vein or intralesional injection; by sustained release system or by implant device.

[0088] The present invention also provides a detection kit, which includes an antibody, polynucleotide, nucleic acid construct or host cell that recognizes the SOST protein, a lysis medium for lysing the sample, general reagents and buffers required for detection, such as various buffers, detection labels, detection substrates, etc.

[0089] The present invention is further illustrated by the following examples, but the present invention is not limited to the scope of the described examples. For the experimental methods without specific conditions noted in the following examples, they are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0090] Examples

[0091] Example 1. Screening of Monoclonal Antibodies Against Sclerostin

[0092] Monoclonal antibodies against human SOST were obtained by immunizing mice, constructing a murine scFv-phage immune library and screening. The human SOST protein antigen was purchased from Acro Biosystems (#HST-H5245). The BALB / c mice (6-8 weeks old) used for immunization were purchased from Jiangsu Jicui Yakang Biotechnology Co., Ltd. The SOST protein antigen was mixed with Freund's complete adjuvant in an equal volume ratio of 1:1 to form a stable emulsion, and each mouse was subcutaneously injected with an emulsion containing 50-100 μg of the protein antigen (day 0). Booster immunizations were performed on days 14 and 28. The SOST protein antigen was mixed with Freund's incomplete adjuvant in an equal volume ratio of 1:1 to form a stable emulsion, and each mouse was intraperitoneally injected with an emulsion containing 25-50 μg of the protein. Blood was collected on day 35 to detect the antibody titer in the mouse serum. The mouse with the highest antibody titer was selected for spleen cell extraction and cDNA synthesis.

[0093] Using mouse spleen cell cDNA as a template, a primer set that specifically binds to the V gene and J gene of mouse antibodies was used to amplify the heavy and light chains of the immune mouse antibodies. The amplified heavy and light chain DNAs were recovered separately, and scFv DNA was obtained by PCR using the heavy and light chains as templates. The scFv fragment was inserted into a phage display vector to construct a library of approximately 10 9 in size. Clones that can bind to human SOST were enriched by solid-phase or liquid-phase screening methods.

[0094] Example 2. Expression and purification of anti-sclerostin monoclonal antibody

[0095] The anti-SOST monoclonal antibody sequence obtained by screening was constructed into the form of full-length IgG1. Primers were designed and the VH and VL gene fragments of the antibody were obtained by PCR amplification. They were respectively subjected to overlap PCR with the constant region gene fragments CH1-Hinge-CH2-CH3 and CL of the heavy and light chains to obtain a fusion DNA sequence, which was inserted into the mammalian expression vector pCDNA3.1 to construct a plasmid vector expressing chimeric antibody heavy and light chains. After the sequence was verified to be correct by sequencing, the plasmid vector was extracted using an endotoxin-free plasmid extraction kit and stored at -20°C for later use. Expi293F cells were diluted to a density of approximately 3x10 6 cells / mL, and the plasmids expressing the antibody light and heavy chains were co-transfected into Expi293F cells using the PEI40000 (polysciences) transfection reagent. Four days later, the cell culture supernatant was collected. After high-speed centrifugation, the cell culture supernatant was collected and filtered through a 0.22 μm filter membrane to remove residual cell debris. The filtered cell culture supernatant was purified using a Protein A column. The Protein A column was rinsed with PBS buffer to remove miscellaneous proteins. After the A280 reading dropped to a stable baseline, the target protein was eluted with a 0.1 M acetic acid-sodium acetate solution at pH 3.2, and the target protein peak was collected and neutralized with a 1 M Tris-HCl solution at pH 8.0. After the sample was concentrated, it was further purified using a gel chromatography column ENrichTM SEC650 (Bio-red) to remove aggregates, and the monomer peak was collected. After the collected sample was detected by 4-12% SDS-PAGE gradient gel electrophoresis, it was aliquoted and stored at -80°C for later use.

[0096] Example 3. Activity detection of anti-sclerostin monoclonal antibody neutralizing sclerostin

[0097] The activity of anti-sclerostin antibody neutralizing sclerostin was detected by Topflash assay. The binding of Wnt to LRP5 / 6 and Frizzled receptors can stabilize β-catenin in the cytoplasm, causing β-catenin to translocate into the nucleus and stimulate the expression of target proteins. In the Topflash assay, a vector containing a lymphoid enhancer factor / T cell factor (LEF / TCF) element and a luciferase gene, as well as a plasmid expressing Wnt1 protein, were transfected into HEK293 cells. The binding of Wnt1 protein to LRP5 / 6 and Frizzled receptors on HEK293 cells enabled β-catenin to bind to LEF / TCF and stimulate the expression of the luciferase gene. The fluorescence generated by the hydrolysis of the luciferin substrate by luciferase can characterize the intensity of Wnt signal activation. When SOST was added, the fluorescence signal value decreased. If the anti-SOST antibody could block the binding of SOST to LRP5 / 6, the fluorescence signal would recover when the anti-SOST antibody was added simultaneously. By this method, the strength of the antibody neutralizing the inhibitory activity of SOST on Wnt signal can be detected.

[0098] HEK293 cells (1.5x10 4 cells / well) were seeded into white-bottom 96-well plates treated with poly-D-lysine. The next day, pCMV-Wnt1 plasmid (Sino Biological, #HG10721-UT), Topflash or Fopflash plasmid (internally constructed), and pGL4.74[hRluc / TK] (Promega, #E6921) were co-transfected into HEK293 cells. The plasmid dosage per well of cells was 40 ng of pCMV-Wnt1, 40 ng of Topflash plasmid, and 20 ng of pGL4.74[hRluc / TK] plasmid. 6 hours after plasmid transfection, the cell culture supernatant was discarded, and 100 μL / well of fresh culture medium containing 2 μg / mL SOST protein and 10 μg / mL anti-SOST antibody was added to the cells. Samples without protein treatment were used as blank controls. After 16 hours, the activity of luciferase was quantified using the Dual-Glo® Luciferase Assay System (Promega, #E2920). The fluorescence value of the blank sample was taken as 100%, and the other values were processed accordingly. As shown in Table 1, the candidate antibody m-9 has the activity of neutralizing the inhibitory effect of SOST on Wnt1 signal transduction, and its neutralizing activity is superior to that of the control antibody romosozumab (self-made).

[0099] Table 1. Detection of the activity of anti-SOST antibody neutralizing the inhibitory effect of SOST on Wnt1 signal transduction

[0100]

[0101] Example 4. Detection of the Affinity and Species Specificity of Anti-Sclerostin Antibody

[0102] The binding affinity of the purified m-9 antibody to human and mouse SOST was analyzed using biolayer interferometry (BLI). The antibody was diluted to 20 µg / ml with sodium acetate solution (pH 6.0). After the amino (AR2G) sensor was wetted with purified water for 10 minutes, EDC / NHS was added to the amino group on the activated sensor, and then the sodium acetate solution containing the immobilized antibody was added. The antibody was immobilized on the sensor through the reaction of carboxyl and amino groups. Further, ethanolamine was added to block the unbound amino sites. A buffer solution containing different concentrations of SOST (0.8 - 12.5 nM) (PBS containing 0.5% BSA and 0.05% Tween 20, pH = 7.4) was prepared. The sensor immobilized with the antibody to be tested was successively allowed to bind to different concentrations of SOST, and fitting was performed according to the 1:1 binding mode using Octet AnalysisStudio software to obtain the association rate constant (k a ), and the dissociation rate constant (k d ). The dissociation equilibrium constant (KD) was calculated based on the ratio of the association and dissociation rate constants. As shown in Table 2, the anti-SOST antibody m-9 can bind to human and mouse SOST proteins with high affinity.

[0103] Table 2. Affinity Detection of Anti-SOST Antibody m-9 Binding to SOST Proteins of Different Species

[0104]

[0105] Example 5. Detection of the Binding Epitope of Anti-Sclerostin Antibody m-9 to SOST

[0106] Studies have shown that the binding site of SOST to the E1 domain of LRP6 is mainly located within the flexible peptide segment P1 of the SOST protein sequence: the PNAIGRGKWWRPSGPDFR region. Biotin-labeled P1 peptide and P2 peptide (KWWRPSGPDFRCIPDRYRAQRV) were synthesized to detect the binding epitope of m-9 and compare it with romosozumab. Using biolayer interferometry (BLI) analysis, the SA probe was immersed in solutions containing P1 and P2 peptides respectively to immobilize the peptides. Then the probe was immersed in a solution containing 200 nM antibody. As Figure 1 can be seen, both m-9 and romosozumab can bind to P1 and P2 peptides, but the affinity of m-9 for binding to P1 peptide is significantly higher than that for P2 peptide, and the binding epitopes are not completely overlapped with those of romosozumab.

[0107] Partial Sequence

[0108] SEQ ID NO:1 Heavy Chain Variable Region

[0109] QVQLQQPGAELVRPGASLKLSCKASGYSFSSYWVTWVKQRPGQGLEWIGMIHPSDSETRLNQNFKDKATLTVDKSSSTAYIQLSSPTSEDSAVYYCARGGGYGNFDYWGQGTTVTVSS

[0110] SEQ ID NO:2 Light chain variable region

[0111] DIVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPFTFGSGTKLEIK

[0112] SEQ ID NO:3 HCDR1

[0113] GYSFSSYW

[0114] SEQ ID NO:4 HCDR2

[0115] IHPSDSET

[0116] SEQ ID NO:5 HCDR3

[0117] ARGGGYGNFDY

[0118] SEQ ID NO:6 LCDR1

[0119] QSVDYDGDSY

[0120] SEQ ID NO:7 LCDR2

[0121] AAS

[0122] SEQ ID NO:8 LCDR3

[0123] QQSNEDPFT

[0124] SEQ ID NO:9 Heavy chain constant region

[0125] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0126] SEQ ID NO: 10 Light chain constant region

[0127] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0128] SEQ ID NO: 11 Heavy chain

[0129] QVQLQQPGAELVRPGASLKLSCKASGYSFSSYWVTWVKQRPGQGLEWIGMIHPSDSETRLNQNFKDKATLTVDKSSSTAYIQLSSPTSEDSAVYYCARGGGYGNFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0130] SEQ ID NO:12 Light chain

[0131] DIVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPFTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Claims

1. An antibody or antigen-binding fragment thereof targeting sclerostin, wherein the HCDR1 of the antibody is as shown in SEQ ID NO: 3, the HCDR2 of the antibody is as shown in SEQ ID NO: 4, the HCDR3 of the antibody is as shown in SEQ ID NO: 5, the LCDR1 of the antibody is as shown in SEQ ID NO: 6, the LCDR2 of the antibody is as shown in SEQ ID NO: 7, and the LCDR3 of the antibody is as shown in SEQ ID NO:

8.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein The heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO: 1, and the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:

2.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that, The heavy chain variable region of the antibody contains murine or human heavy chain FR regions, and the light chain variable region of the antibody contains murine or human light chain FR regions.

4. The antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that, The antibody further comprises a heavy chain constant region and / or a light chain constant region.

5. The antibody or antigen-binding fragment thereof according to claim 4, wherein The heavy chain of the antibody contains the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4, and / or the light chain of the antibody contains the light chain constant region of human kappa chain or lambda chain.

6. The antibody or antigen-binding fragment thereof according to claim 5, wherein The heavy chain of the antibody contains the heavy chain constant region of human IgG1, and the light chain contains the light chain constant region of human kappa chain.

7. The antibody or antigen-binding fragment thereof according to claim 6, wherein, The antibody is a multispecific antibody, or a monoclonal antibody, or a chimeric antibody or a fully human antibody.

8. The antibody or antigen-binding fragment thereof according to claim 6, wherein The antibody is a bispecific antibody.

9. A polynucleotide, the sequence of which is selected from: (1) The coding sequence of the antibody or antigen-binding fragment thereof according to any one of claims 1-8, (2) The complementary sequence of (1).

10. A nucleic acid construct comprising the polynucleotide according to claim 9.

11. The nucleic acid construct according to claim 10, wherein, The nucleic acid construct is a cloning vector.

12. The nucleic acid construct according to claim 10, wherein, The nucleic acid construct is an expression vector.

13. The nucleic acid construct according to claim 10, wherein The nucleic acid construct is an integration vector.

14. A host cell, selected from: (1) A host cell expressing and / or secreting the antibody or antigen-binding fragment thereof according to any one of claims 1-8; (2) A host cell containing the polynucleotide according to claim 9; and / or (3) A host cell containing the nucleic acid construct according to any one of claims 10-13.

15. The host cell according to claim 14, wherein, The host cell is selected from prokaryotic cells or eukaryotic cells.

16. The host cell according to claim 14 or 15, characterized in that, The host cell is a mammalian cell.

17. A method for producing the antibody or antigen-binding fragment thereof according to any one of claims 1-8, comprising: Culturing the host cell according to any one of claims 14-16 under conditions suitable for producing the antibody or antigen-binding fragment thereof, and optionally purifying the antibody or antigen-binding fragment thereof from the culture.

18. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-8, the polynucleotide according to claim 9, the nucleic acid construct according to any one of claims 10-13, or the host cell according to any one of claims 14-16, and a pharmaceutically acceptable excipient.

19. Use of the antibody or antigen-binding fragment thereof according to claims 1-8, the polynucleotide according to claim 9, the nucleic acid construct according to any one of claims 10-13, or the host cell according to any one of claims 14-16 in the preparation of a medicament for preventing or treating osteoporosis.

20. A kit for detecting sclerostin, which is used to evaluate the therapeutic effect of a drug. The kit comprises the antibody or its antigen-binding fragment as claimed in claims 1-8, the polynucleotide as claimed in claim 9, the nucleic acid construct as claimed in any one of claims 10-13, or the host cell as claimed in any one of claims 14-16.

21. A non-diagnostic method for detecting the presence of decorin in a sample, the method comprising: Incubate the sample with the antibody or its antigen-binding fragment as claimed in any one of claims 1-8, and detect the binding of sclerostin to the antibody or its antigen-binding fragment, so as to determine the presence of sclerostin in the sample.

22. The method according to claim 21, wherein The detection is performed by enzyme-linked immunosorbent assay.

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