Antibodies targeting SOST and uses thereof

Abstract

The present invention relates to antibodies targeting SOST, antigen binding fragments thereof, and uses thereof. In particular, provided are SOST-targeting antibodies, or antigen-binding fragments thereof, capable of specifically binding to SOST proteins, or variants having at least 85% sequence identity to said antibodies, or antigen-binding fragments thereof, and retaining SOST binding activity thereof.

Description

Technical Field

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

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

[0003] Sclerostin is mainly secreted by osteocytes, and its binding to low-density lipoprotein receptor-related protein 5\6 (LRP5\6) can inhibit classical Wnt signaling. Studies on sclerosteosis and van Buchem disease have shown that these patients have abnormal bone mass due to the absence or mutation of the sclerostin gene, which causes the absence or reduction of sclerostin protein expression. Anti-sclerostin antibodies bind to sclerostin and block its binding to LRP5 / 6, allowing Wnt to form heterotrimers 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 or an antigen-binding fragment thereof targeting sclerostin (SOST), or a variant thereof 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 SOST flexible loop structural region 2, which comprises 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, the HCDR1 of the antibody comprises SEQ ID NO: 3, or a sequence having at least 85% sequence identity thereto.

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

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

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

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

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

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

[0014] In one or more embodiments, the heavy chain of the antibody has the sequence of SEQ ID NO:11, or is at least 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:11, or or in addition, the light chain of the antibody has the sequence of SEQ ID NO:12, or is at least 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to 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, a F(ab')2 fragment, or a scFv fragment.

[0019] The present invention also provides a polynucleotide selected from: (1) A coding sequence of an antibody or antigen-binding fragment thereof as described in any embodiment of the present invention; (2) The complementary sequence of (1).

[0020] The present invention also provides a nucleic acid construct, which comprises the polynucleotide described in any embodiment herein.

[0021] In one or more embodiments, the nucleic acid construct is a vector, such as an integration vector, a cloning vector, or an expression vector.

[0022] The present invention also provides a phage comprising the antibody or antigen-binding fragment thereof described in any embodiment of the present invention or a library comprising the phage.

[0023] In one or more embodiments, the antibody or antigen-binding fragment thereof is displayed on the surface of the bacteriophage.

[0024] The present invention also provides a host cell, which: (1) expressing and / or secreting the antibody or antigen-binding fragment thereof described in any embodiment of the present invention; (2) comprising a polynucleotide as described herein; and / or (3) comprising the nucleic acid construct described herein.

[0025] In one or more embodiments, the host cell is selected from a prokaryotic cell or a eukaryotic cell.

[0026] In one or more embodiments, the host cell is a mammalian cell.

[0027] 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 the antibody or the antigen-binding fragment thereof, and optionally purifying the antibody or the antigen-binding fragment thereof from the culture.

[0028] The present invention also provides a pharmaceutical composition comprising the antibody or antigen-binding fragment polynucleotide, nucleic acid construct, phage or host cell described herein, and pharmaceutically acceptable excipients.

[0029] In one or more embodiments, the auxiliary material is a carrier, a diluent, or an excipient.

[0030] In one or more embodiments, the pharmaceutical composition is used to treat osteoporosis, osteogenesis imperfecta, bone fractures or other metabolic bone diseases.

[0031] In one or more embodiments, the osteoporosis includes primary osteoporosis and secondary osteoporosis.

[0032] The present invention also provides use of the antibody or antigen-binding fragment thereof, polynucleotide, nucleic acid construct or host cell described in any embodiment herein in the preparation of a medicament for preventing or treating a disease.

[0033] In one or more embodiments, the disease is osteoporosis, osteogenesis imperfecta, bone fractures or other metabolic bone diseases.

[0034] The present invention also provides a method for treating or preventing a disease, comprising administering a therapeutically effective amount of the antibody or antigen-binding fragment thereof or pharmaceutical composition according to any embodiment of the present invention to a patient in need thereof.

[0035] The present invention also provides a kit for detecting sclerostin, which is used to evaluate the therapeutic effect of a drug, wherein the kit comprises the antibody or antigen-binding fragment thereof, polynucleotide, nucleic acid construct, bacteriophage or host cell described in any embodiment of the present invention.

[0036] In one or more embodiments, the kit further comprises a reagent for detecting the binding of sclerostin to the antibody or its antigen-binding fragment, such as a reagent for detecting the binding by enzyme-linked immunosorbent assay.

[0037] In one or more embodiments, the reagent for detecting binding is a detectable label that can be linked to the antibody or antigen-binding fragment thereof, such as biotin. The detectable label is linked to the antibody or antigen-binding fragment thereof or exists separately in the kit.

[0038] 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 antigen-binding fragment thereof described in any embodiment of the present invention, and detecting the binding of sclerostin to the antibody or antigen-binding fragment thereof, thereby determining the presence of sclerostin in the sample. The detection is an enzyme-linked immunosorbent assay.

[0039] The present invention also provides use of the antibody or antigen-binding fragment thereof described in any embodiment of the present invention in preparing a kit for detecting sclerostin in a sample and evaluating the therapeutic effect of a drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 . Epitope analysis of m-9 and romosozumab binding to SOST. DETAILED DESCRIPTION

[0041] Unless otherwise defined, the scientific and technical terms used herein should have the meanings commonly understood by those with routine skills in the art. In general, the nomenclature and techniques used in conjunction with cell and tissue culture, molecular biology, and protein and oligonucleotide or polynucleotide chemistry and hybridization described herein are well known and commonly used in the art.

[0042] Specifically, the present invention immunizes mice, constructs a mouse scFv-phage immune library and screens it to obtain a monoclonal antibody against human SOST, constructs a plasmid vector expressing the heavy and light chains of the chimeric antibody, transfects the above plasmid vector into Expi293F cells, and further purifies to obtain a high-purity antigen-binding protein. The antibody activity, specificity and binding epitope are detected by Topflash and BLI methods. The results show that the antibody has high affinity, high inhibitory activity of sclerostin and a binding epitope different from that of romosozumab.

[0043] As used herein, the term "antibody" includes full-length antibodies having an immunoglobulin Fc, antibody compositions having multiple epitope specificities, multispecific antibodies (e.g., bispecific antibodies), diabodies and single-chain molecules, as well as antibody fragments, especially antigen-binding fragments, e.g., Fab, F(ab')2 and Fv.

[0044] The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light chains (L) and two identical heavy chains (H). IgM antibodies consist of 5 basic heterotetrameric units and an additional polypeptide called J chain, containing 10 antigen binding sites; while IgA antibodies contain 2-5 basic 4-chain units, which can be combined with J chains to form multivalent assemblies. In the case of IgG, the 4-chain unit is usually about 150,000 Daltons. Each light chain is connected to the heavy chain by one covalent disulfide bond, while the two heavy chains are connected to each other by one or more disulfide bonds, the number of which depends on the isotype of the heavy chain. Each heavy and light chain also has regularly spaced intrachain 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) and a hinge region (Hinge) between the CH1 domain and the CH2 domain. Each light chain has a variable domain (VL) at the N-terminus, followed by a constant domain (CL) at the other end. VL is arranged with VH, while CL is arranged with the first constant domain (CH1) of the heavy chain. Specific amino acid residues are considered to form an interface between the light chain 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, e.g., Basic and Clinical Immunology, 8th edition, Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw, ed., Appleton & Lange, Norwalk, CT, 1994, p. 71 and Chapter 6. Light chains from any vertebrate species can be assigned to one of two distinct types, called kappa and lambda, based on their constant domain amino acid sequences. Immunoglobulins can be assigned to different classes or isotypes based on their heavy chain constant domain (CH) amino acid sequences. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with 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.

[0045] The "variable region" or "variable domain" of an antibody refers to the amino terminal domain of the heavy or light chain of an antibody. The variable domains of the heavy and light chains may be referred to as "VH" and "VL", respectively. These domains are usually the most variable parts of an antibody (relative to other antibodies of the same type) and contain the antigen binding site.

[0046] The term "variable" refers to the situation that certain segments in the variable domain vary widely in the antibody sequence. The variable domain mediates antigen binding and defines the specificity of a particular antibody to its specific antigen. However, the variability is not evenly distributed over all the amino acids spanned by the variable domain. Instead, it is concentrated in three segments called hypervariable regions (HVRs) (in both the light chain and heavy chain variable domains), namely, HCDR1, HCDR2, HCDR3 (heavy chain antibodies can be referred to as CDR1, CDR2, CDR3) of the heavy chain variable region and LCDR1, LCDR2 and LCDR3 of the light chain variable region. The more highly conserved parts of the variable domain are called framework regions (FRs). The variable domains of natural heavy and light chains each contain four FR regions (FR1, FR2, FR3 and FR4), most of which adopt a β-folded conformation, connected by three HVRs that form a loop connection and in some cases form a part of the β-folded structure. The HVR in each chain is held together in close proximity by the FR region and contributes to the formation of the antigen binding site of the antibody together with the HVR of the other chain. Typically, 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 is not directly involved in the binding of the antibody to the antigen, but exhibits a variety of effector functions, such as the participation of the antibody in antibody-dependent cell-mediated cytotoxicity.

[0047] "Fc region" (fragment crystallizable region) or "Fc domain" or "Fc" refers to the C-terminal region of an antibody heavy chain that 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 is composed of two identical protein fragments, the CH2 domain and the CH3 domain from the two heavy chains of the antibody. Although the boundaries of the Fc region of an 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 the numbering is according to the EU index, as in Kabat. As used herein, the Fc region may be a native sequence Fc or a variant Fc.

[0048] "Antibody fragments" comprise a portion of an intact antibody, preferably the antigen binding and / or variable regions of an intact antibody. Antibody fragments are preferably antigen binding fragments of antibodies. 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 from antibody fragments; and any fragments that should be able to increase half-life by chemical modification or by incorporation into liposomes. Digestion of antibodies with papain produces two identical antigen binding fragments called "Fab" fragments, and a residual "Fc" fragment, the name of which reflects its ability to crystallize easily. Fab fragments consist of an intact light chain and heavy chain variable domain (VH) and one heavy chain first constant domain (CH1). Each Fab fragment is monovalent in terms of antigen binding, i.e., it has a single antigen binding site. Pepsin treatment of antibodies produces a larger F(ab')2 fragment, which is roughly equivalent to two Fab fragments linked by disulfide bonds, with different antigen binding activities and still able to cross-link antigen. Fab' fragments differ from Fab fragments 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. F(ab')2 antibody fragments were originally generated as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings 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 functions of antibodies are determined by sequences in the Fc region, which is also the region recognized by Fc receptors (FcR) found on certain types of cells.

[0049] Herein, the term "monoclonal antibody" refers to an antibody obtained from a group of substantially homogeneous antibodies, that is, except for possible natural mutations and / or post-translational modifications (e.g., isomerization, amidation) that may exist in small amounts, the individual antibodies constituting the group are identical. Monoclonal antibodies are highly specific and are directed to a single antigenic site. Compared with polyclonal antibody preparations (which typically include different antibodies directed to different determinants (epitopes)), each monoclonal antibody is directed to a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they are synthesized by hybridoma culture without being contaminated by other immunoglobulins. The modifier "monoclonal" indicates that the antibody is obtained from a substantially homogeneous antibody group and should not be interpreted as requiring the production of antibodies by any ad hoc method. For example, the monoclonal antibody used according to the present invention can be generated by a variety of techniques, including, for example, hybridoma method, phage display method, recombinant DNA method, and technology, single cell sequencing method for generating human or human-like antibodies from animals with part or all of the human immunoglobulin locus or genes encoding human immunoglobulin sequences.

[0050] Monoclonal antibodies herein also include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical or homologous to the corresponding sequence in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.

[0051] The "humanized" form of a non-human (e.g., mouse) antibody refers to a chimeric antibody that contains at least a sequence derived from a non-human immunoglobulin. Therefore, a "humanized antibody" generally refers to a non-human antibody in which the variable domain framework region is exchanged with a sequence found in a human antibody. Typically in a humanized antibody, the entire antibody (except for the CDR) is encoded by a polynucleotide of human origin or is identical to such an antibody (except for the CDR). The CDRs (some or all of which are encoded by nucleic acids derived from non-human organisms) are transplanted into the β-folded skeleton of the variable region of a human antibody to produce an antibody, the specificity of which is determined by the transplanted CDR. The production methods of such antibodies are well known in the art, for example, 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 each of the antibodies.

[0052] "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 disclosed herein for producing human antibodies. This definition of human antibody explicitly excludes humanized antibodies comprising non-human antigen-binding residues. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries.

[0053] 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.

[0054] FR1, FR2, FR3, and FR4 of the antibody described herein may be selected from (1) FR1, FR2, FR3, and FR4 of the heavy chain variable region shown in SEQ ID NO: 1, or (2) 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.

[0055] 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, such as 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 thereof having at least 85% sequence identity, and a light chain constant region, such as a light chain constant region of a κ or λ chain, or a sequence thereof having at least 85% sequence identity.

[0056] The heavy chain constant region of the antibody described herein can be the constant region of the heavy chain of a human heavy chain antibody, comprising 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, 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.

[0057] The light chain constant region of the antibody described herein can be the constant region of the light chain of a human antibody. In one or more embodiments, the sequence of the light chain constant region is as shown in SEQ ID NO:10.

[0058] The term "targeting" refers to specific binding to an antigen molecule. For example, the antibody "targeting SOST" herein refers to an antibody that specifically binds to SOST.

[0059] The present invention also includes derivatives and analogs of the various antibodies (e.g., 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 maintain 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 fusion of a mature polypeptide with another compound (such as a compound that prolongs the half-life of a polypeptide, such as polyethylene glycol), or (iii) polypeptides formed by fusion of an additional amino acid sequence to this polypeptide sequence (such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify the polypeptide, or a fusion protein formed with a 6*His tag). According to the teachings herein, these derivatives and analogs are within the scope known to those skilled in the art.

[0060] Without substantially affecting the activity of the antibody, a person skilled in the art may change one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more) amino acids in the sequence of the present invention to obtain variants of the antibody or its functional fragment sequence. These variants include (but are not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acid deletions, insertions and / or substitutions, and addition of one or more (usually within 20, preferably within 10, and more preferably within 5) amino acids at the C-terminus and / or N-terminus. In the art, conservative substitutions with amino acids with similar or similar properties generally do not change the function of the protein. 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. For another example, adding one or more amino acids to the C-terminus and / or N-terminus will generally not change the function of the protein. They are all considered to be included in the scope of protection of the present invention.

[0061] Variant forms of the various antibodies described herein include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA 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.

[0062] In some embodiments, the sequence of the variant of the present invention may have at least 95%, 96%, 97%, 98% or 99% identity to its source sequence. The sequence identity of the present invention can be measured using sequence analysis software. For example, the computer program BLAST, especially BLASTP or TBLASTN, using default parameters. The present invention also includes molecules having antibody heavy chain variable regions with CDRs or molecules having antibody light chain variable regions with CDRs, as long as their CDRs have more than 90% (preferably more than 95%, and most preferably more than 98%) homology with the CDRs identified herein.

[0063] The antibodies of the present invention can be prepared by conventional methods in the art, such as hybridoma technology well known in the art. Alternatively, the various antibodies of the present invention can be expressed in other cell lines. Suitable mammalian host cells can be transformed with sequences encoding various antibodies of the present invention. Transformation can be carried out by any known method, for example, including packaging the polynucleotide in a virus (or a viral vector) and transducing the host cell with the virus (or vector). The transformation procedure used depends 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, encapsulation of polynucleotides in liposomes, and direct microinjection of DNA into the nucleus. Mammalian cell lines that can be used as hosts for expression are well known in the art, including but not limited to a variety of 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 substantial SOST binding properties.

[0064] The invention also provides a polynucleotide encoding the above-mentioned antibody or its antigen-binding fragment. The polynucleotide of the present invention can be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA or artificially synthesized DNA. DNA can be single-stranded or double-stranded, usually a double-stranded structure, with a sense strand and an antisense strand that complement each other. Among them, the sense strand, also known as the sense strand, refers to the strand with genetic information in the DNA double strand, which cannot be transcribed and 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, also known as the template strand, refers to the DNA strand used by RNA polymerase during the transcription process, acting as a template for transcription. The RNA polymerase moves along the template strand and transcribes it into mRNA, which is then translated into a polypeptide by the mRNA.

[0065] In a narrow sense, "polynucleotide sequence encoding..." or "coding sequence of..." refers to a sequence that is present on the antisense strand of DNA and can directly guide transcription, or a sequence that is present on mRNA and can directly guide translation. A polynucleotide may contain a coding sequence (or coding region) and a non-coding sequence (or non-coding region, such as introns). The coding sequence may be continuous or discontinuous, and discontinuous coding sequence fragments may 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.

[0066] The present invention also includes degenerate variants of polynucleotide sequences encoding fusion proteins, i.e., nucleotide sequences encoding the same amino acid sequence but with different nucleotide sequences. As will be appreciated by those skilled in the art, due to the degeneracy of the genetic code, a very large number of nucleic acids can be produced, all of which encode the antibodies or antigen-binding fragments thereof of the present invention. Thus, in the case where a specific amino acid sequence has been identified, one skilled in the art can produce 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. Therefore, the present invention also relates to polynucleotides that hybridize with the above-mentioned polynucleotide sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that are hybridizable with the polynucleotides of the present invention under stringent conditions. In the present invention, "stringent conditions" refer to: (1) hybridization and elution at relatively low ionic strength and relatively high temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) addition of denaturants during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, preferably at least 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0067] The full-length nucleotide sequences or fragments thereof of various antibodies of the present invention can usually 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. Usually, a fragment with a very long sequence can be obtained by first synthesizing multiple small fragments and then connecting them. In addition, the coding sequence of the heavy chain and the expression tag (such as 6*His) can be fused together to form a fusion protein.

[0068] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the host cells after proliferation by conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in the present invention include biomolecules in isolated form. At present, the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) can be obtained completely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention by chemical synthesis.

[0069] Therefore, the present invention also relates to nucleic acid constructs, such as expression vectors and recombinant vectors, comprising the above-mentioned appropriate DNA sequences and appropriate promoters or control sequences. These vectors can be used to transform appropriate host cells to enable them to express proteins. The vectors usually contain sequences for plasmid maintenance and for cloning and expressing exogenous nucleotide sequences. The sequences (collectively referred to as "flanking sequences" in certain embodiments) usually include one or more of the following nucleotide sequences: promoters, one or more enhancer sequences, replication origins, transcription termination sequences, complete intron sequences containing donor and acceptor splice sites, sequences encoding leader sequences for polypeptide secretion, ribosome binding sites, polyadenylation sequences, multiple linker regions for inserting nucleic acids encoding antibodies to be expressed, and selectable marker elements.

[0070] 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.

[0071] In certain embodiments, the host cell can be various functional cells known in the art, such as various killer cells, including but not limited to cytokine-induced killer cells (CIK), cytokine-induced killer cells stimulated by dendritic cells (DC-CIK), cytotoxic T lymphocytes (CTL), γδT cells, natural killer cells (NK), tumor infiltrating lymphocytes (TIL), lymphokine-activated killer cells (LAK), CD3AK cells (killer cells of anti-CD3 monoclonal antibodies) and CAR-T / TCR-T cells. In certain 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 certain 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, inhibitory / regulatory T cells, γδT cells, and cytokine-induced killer cells (CIK), tumor infiltrating lymphocytes (TIL) and other mixed cell populations of T cells. In certain embodiments, the T cells are peripheral blood T lymphocytes and TIL-derived T cells.

[0072] Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl2 method, the steps used are well known in the art. Another method is to use MgCl2. If necessary, transformation can also be carried out by electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be selected: calcium phosphate coprecipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0073] The obtained transformant 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. Culture is carried out under conditions suitable for the growth of the host cells. After the host cells grow to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

[0074] The polypeptide in the above method can be expressed in the cell, on the cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be separated and purified by various separation methods using 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 renaturation treatment, treatment with protein precipitants (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods.

[0075] All aspects of the various antibodies described herein can be used to prepare antibodies for preventing or treating osteoporosis, osteogenesis imperfecta, bone fractures or other metabolic bone diseases.

[0076] The pharmaceutical compositions herein contain antibodies described herein, and pharmaceutically acceptable excipients, including but not limited to diluents, carriers, solubilizers, emulsifiers, preservatives and / or adjuvants. Excipients are preferably non-toxic to the recipient at the dose and concentration used. Such excipients include (but are not limited to): saline, buffer, 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, permeability, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, absorption or penetration of the composition. These substances are known in the prior art. The optimal pharmaceutical composition can be determined based on the expected route of administration, delivery method, and required dosage.

[0077] Pharmaceutical compositions for in vivo administration are usually provided in the form of sterile preparations. Sterilization is achieved by filtering through a sterile filtration membrane. When the composition is freeze-dried, this method can be used for sterilization before or after freeze-drying and reconstitution. The pharmaceutical composition of the present invention can be selected for parenteral delivery. The composition for parenteral administration can be stored in a freeze-dried form or in a solution. For example, it is prepared by conventional methods with physiological saline or an aqueous solution containing glucose and other adjuvants. Parenteral compositions are usually placed in a container with a sterile access hole, such as an intravenous solution strip or a vial with a stopper that can be pierced by a hypodermic injection needle. Alternatively, the composition can be selected for inhalation or delivery through the digestive tract (such as oral). The preparation of the pharmaceutically acceptable composition is within the technology 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. Technologies for preparing a variety of other sustained or controlled delivery methods (such as liposome carriers, bioerodible microparticles or porous beads and accumulated injections) are also known to those skilled in the art.

[0078] Once the pharmaceutical composition is formulated, it is stored in a sterile vial in the form of a solution, suspension, gel, emulsion, solid, crystal or in the form of a dehydrated or lyophilized powder. The formulation can be stored in a ready-to-use form or in a form of rehydration before administration (e.g., lyophilization). The present invention also provides a kit for producing a single-dose administration unit. The kit of the present invention can each contain a first container with a dried protein and a second container with an aqueous formulation. In certain embodiments of the present invention, a kit containing a single-chamber and multi-chamber prefilled syringe (e.g., a liquid syringe and a lyophilized syringe) is provided.

[0079] The present invention also provides a method for preventing or treating osteoporosis, osteogenesis imperfecta, bone fractures or other metabolic bone diseases by administering an antibody or pharmaceutical composition thereof according to any embodiment of the present invention. Herein, the terms "subject", "individual" and "object" are used interchangeably herein and include any organism, preferably an animal, more preferably a mammal (e.g., rat, mouse, dog, cat, rabbit, etc.), and most preferably a human.

[0080] The therapeutically effective amount of the pharmaceutical composition containing the antibody of the present invention to be used will depend on, for example, the degree of treatment and the target. It will be appreciated by those skilled in the art that the appropriate dosage level for treatment will vary depending in part on the size (body weight, body surface or organ size) and / or condition (age and general health) of the delivered molecule, indication, route of administration and the patient. In certain embodiments, the clinician can titrate the dosage and change the route of administration to obtain the best therapeutic effect. For example, about 10 micrograms / kg body weight-about 50 milligrams / kg body weight per day.

[0081] The frequency of administration will depend on the pharmacokinetic parameters of the antibody in the formulation used. The clinician typically administers the composition until a dose is reached that achieves the desired effect. The composition may therefore be administered as a single dose, or as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via an implant device or catheter.

[0082] The route of administration of the pharmaceutical composition is according to known methods, for example, orally, by injection via intravenous, intraperitoneal, intracerebral (intracerebroparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, portal vein or intralesional routes; by sustained release systems or by implant devices.

[0083] The present invention also provides a detection kit, which includes antibodies, polynucleotides, nucleic acid constructs or host cells that recognize SOST protein, a lysis medium for dissolving samples, and universal reagents and buffers required for detection, such as various buffers, detection markers, detection substrates, etc.

[0084] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0085] Example

[0086] Example 1. Screening of anti-sclerostin monoclonal antibodies

[0087] Monoclonal antibodies against human SOST were obtained by immunizing mice, constructing mouse scFv-phage immune libraries, and screening. Human SOST protein antigen was purchased from Acro Biosystems (#HST-H5245). BALB / c mice (6-8 weeks old) for immunization were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. The SOST protein antigen was mixed with Freund's complete adjuvant in an equal volume of 1:1 to form a stable emulsion, and each mouse was subcutaneously injected with an emulsion containing 50-100 μg of protein antigen (day 0). On days 14 and 28, booster immunization was performed, and the SOST protein antigen was mixed with Freund's incomplete adjuvant in an equal volume of 1:1 to form a stable emulsion, and each mouse was intraperitoneally injected with an emulsion containing 25-50 μg of protein. Blood was collected on day 35 to detect the antibody titer of mouse serum. The mouse with the highest antibody titer was selected for spleen cell extraction and cDNA synthesis.

[0088] Using mouse spleen cell cDNA as a template, primers that specifically bind to mouse antibody V and J genes were used to amplify the heavy and light chains of the immune mouse antibody. The amplified heavy and light chain DNAs were recovered, 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 10 9 The clones that can bind to human SOST are enriched by solid phase or liquid phase screening.

[0089] Example 2. Expression and purification of anti-sclerostin monoclonal antibodies

[0090] The anti-SOST monoclonal antibody sequence obtained by screening was constructed into the form of full-length IgG1. Primers were designed and PCR amplified to obtain the VH and VL gene fragments of the antibody, which were overlapped with the constant region gene fragments CH1-Hinge-CH2-CH3 and CL of the heavy chain and light chain to obtain fusion DNA sequences, which were inserted into the mammalian expression vector pCDNA3.1 to construct a plasmid vector expressing the heavy and light chains of the chimeric antibody. After sequencing to verify the correctness of the sequence, the plasmid vector was extracted using an endotoxin-free plasmid extraction kit and stored at -20°C for future use. Expi293F was diluted to a density of approximately 3x10 6 cells / mL, and the plasmids expressing the light and heavy chains of the antibody were co-transfected into Expi293F cells using PEI40000 (polysciences) transfection reagent. Four days later, the cell culture supernatant was collected, and the cell culture supernatant was collected after high-speed centrifugation, and filtered with a 0.22μm filter to remove residual cell debris. The filtered cell culture supernatant was purified using a Protein A column, and the Protein A column was rinsed with PBS buffer to remove impurities. After the A280 reading dropped to a stable baseline, the target protein was eluted with a 0.1M acetic acid-sodium acetate solution at pH3.2, and the target protein peak was collected and neutralized with a 1M Tris-HCl, pH8.0 solution. After the sample was concentrated, it was further purified using a gel chromatography column ENrichTM SEC650 (Bio-red), aggregates were removed, and the monomer peak was collected. The collected samples were detected by 4-12% SDS-PAGE gradient gel electrophoresis, and then aliquoted and stored at -80°C for later use.

[0091] Example 3. Detection of the activity of anti-sclerostin monoclonal antibodies in neutralizing sclerostin

[0092] The activity of anti-sclerostin antibodies in 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 transfer to the nucleus and stimulate the expression of target proteins. In Topflash assay, a vector containing lymphoid enhancer factor / T cell factor (LEF / TCF) elements and luciferase gene and a plasmid expressing Wnt1 protein were transfected into HEK293 cells. The binding of Wnt1 protein to LRP5 / 6 and Frizzled receptors on HEK293 cells caused β-catenin to bind to LEF / TCF and stimulate the expression of luciferase gene. The fluorescence generated by luciferase hydrolysis of luciferin substrate can characterize the intensity of Wnt signal activation. When SOST was added, the fluorescence signal value decreased. If the anti-SOST antibody can block the binding of SOST to LRP5 / 6, the fluorescence signal will be restored when the anti-SOST antibody is added at the same time. This method can be used to detect the strength of the antibody neutralizing SOST to inhibit Wnt signaling activity.

[0093] HEK293 cells (1.5x10 4 Cells / well) were seeded into a 96-well plate with a white bottom treated with poly-D-lysine. The next day, pCMV-Wnt1 plasmid (Sino Biological, #HG10721-UT), Topflash or Fopflash plasmid (constructed in-house) and pGL4.74[hRluc / TK] (Promega, #E6921) were co-transfected into HEK293 cells. The amount of plasmids per well was 40ng pCMV-Wnt1, 40ng Topflash plasmid and 20ng pGL4.74[hRluc / TK] plasmid. Six hours after plasmid transfection, the cell culture supernatant was discarded, and 100μL / well of fresh culture medium containing 2μg / mL SOST protein and 10μg / mL anti-SOST antibody was added to the cells. The sample without protein treatment was used as a blank control. 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 remaining values ​​were treated accordingly. As shown in Table 1, the candidate antibody m-9 has the activity of neutralizing SOST to inhibit Wnt1 signaling, and its neutralizing activity is better than that of the control antibody romosozumab (homemade).

[0094] Table 1. Detection of the activity of anti-SOST antibodies in neutralizing SOST to inhibit Wnt1 signaling

[0095] Example 4. Affinity and species specificity detection of anti-sclerostin antibodies

[0096] The binding affinity of the purified m-9 antibody to human and mouse SOST was analyzed using biomembrane interferometry (BLI). The antibodies were diluted to 20 µg / ml with sodium acetate solution (pH 6.0). After the amino group (AR2G) sensor was moistened with purified water for 10 minutes, the amino groups on the EDC / NHS active sensor were added, and then the sodium acetate solution containing the fixed antibody was added to fix the antibody to the sensor through the reaction between the carboxyl group and the amino group. Ethanolamine was further added to block the unbound amino sites. Buffer solutions containing different concentrations of SOST (0.8-12.5 nM) were prepared (PBS containing 0.5% BSA and 0.05% Tween20, pH=7.4), and the sensors with fixed antibodies to be tested were allowed to bind to different concentrations of SOST in turn. The binding rate constant (k) was obtained by fitting according to the 1:1 binding mode using Octet AnalysisStudio software. 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.

[0097] Table 2. Affinity test of anti-SOST antibody m-9 binding to SOST proteins of different species

[0098] Example 5. Detection of Anti-Sclerostin Antibody m-9 Binding to SOST Epitope

[0099] Studies have shown that the binding site of SOST and LRP6 E1 domain is mainly located in the flexible peptide P1 of SOST protein sequence: PNAIGRGKWWRPSGPDFR. The synthetic biotin-labeled P1 peptide and P2 peptide (KWWRPSGPDFRCIPDRYRAQRV) were used to detect the binding epitope of m-9 and compared with romosozumab. Using biomembrane interferometry (BLI) analysis, the SA probe was immersed in solutions containing P1 and P2 peptides to fix the peptides. The probe was then immersed in a solution containing 200 nM antibody. Figure 1 It can be seen that m-9 and romosozumab can bind to both P1 and P2 peptides, but the affinity of m-9 binding to P1 peptide is significantly higher than that to P2 peptide, and the binding epitope with romosozumab does not completely overlap.

[0100] Partial sequence

[0101] SEQ ID NO:1 Heavy chain variable region

[0102] QVQLQQPGAELVRPGASLKLSCKASGYSFSSYWVTWVKQRPGQGLEWIGMIHPSDSETRLNQNFKDKATLTVDKSSSTAYIQLSSPTSEDSAVYYCARGGGYGNFDYWGQGTTVTVSS

[0103] SEQ ID NO:2 Light chain variable region

[0104] DIVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPFTFGSGTKLEIK

[0105] SEQ ID NO:3 HCDR1

[0106] GYSFSSYW

[0107] SEQ ID NO:4 HCDR2

[0108] IHPSDSET

[0109] SEQ ID NO:5 HCDR3

[0110] ARGGGYGNFDY

[0111] SEQ ID NO:6 LCDR1

[0112] QSVDYDGDSY

[0113] SEQ ID NO:7 LCDR2

[0114] AAS

[0115] SEQ ID NO:8 LCDR3

[0116] QQSNEDPFT

[0117] SEQ ID NO:9 Heavy chain constant region

[0118] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0119] SEQ ID NO:10 Light chain constant region

[0120] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0121] SEQ ID NO:11 Heavy chain

[0122] QVQLQQPGAELVRPGASLKLSCKASGYSFSSYWVTWVKQRPGQGLEWIGMIHPSDSETRLNQNFKDKATLTVDKSSSTAYIQLSSPTSEDSAVYYCARGGGYGNFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0123] SEQ ID NO: 12 Light chain

[0124] DIVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPFTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Claims

1. An antibody or antigen-binding fragment thereof targeting sclerostin (SOST), in, (A) the heavy chain variable region of the antibody comprises the amino acid sequence shown in SEQ ID NO: 1; and / or the light chain variable region of the antibody comprises the amino acid sequence shown in SEQ ID NO: 2, or (B) 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, or (C) The HCDR1 of the antibody comprises the sequence shown in SEQ ID NO: 3, the HCDR2 of the antibody comprises the sequence shown in SEQ ID NO: 4, the HCDR3 of the antibody comprises the sequence shown in SEQ ID NO: 5, the LCDR1 of the antibody comprises the sequence shown in SEQ ID NO: 6, the LCDR2 of the antibody comprises the sequence shown in SEQ ID NO: 7, and the LCDR3 of the antibody comprises the sequence 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 comprises a murine or human heavy chain FR region, and the light chain variable region of the antibody comprises a murine or human light chain FR region.

3. 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.

4. The antibody or antigen-binding fragment thereof according to claim 3, wherein: The heavy chain of the antibody comprises the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4, and / or the light chain of the antibody comprises the light chain constant region of human κ chain or λ chain.

5. The antibody or antigen-binding fragment thereof according to claim 4, wherein: The heavy chain of the antibody comprises a heavy chain constant region of human IgG1, and the light chain comprises a light chain constant region of a human κ chain.

6. The antibody or antigen-binding fragment thereof according to claim 5, wherein: The antibody is a multispecific antibody, or a monoclonal antibody, or a chimeric antibody or a fully human antibody.

7. The antibody or antigen-binding fragment thereof according to claim 5, wherein: The antibody is a bispecific antibody.

8. A polynucleotide selected from: (1) a coding sequence of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, (2) The complementary sequence of (1).

9. A nucleic acid construct comprising the polynucleotide according to claim 8.

10. The nucleic acid construct according to claim 9, wherein The nucleic acid construct is a cloning vector, an expression vector or an integration vector.

11. A host cell selected from: (1) expressing and / or secreting the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7; (2) comprising the polynucleotide according to claim 8; and / or (3) Comprising the nucleic acid construct according to claim 9 or 10.

12. The host cell according to claim 11, characterized in that The host cell is selected from a prokaryotic cell or a eukaryotic cell.

13. The host cell according to claim 11 or 12, characterized in that The host cell is a mammalian cell.

14. A method for producing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, comprising: Cultivating the host cell of any one of claims 11 to 13 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.

15. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, the polynucleotide according to claim 8, the nucleic acid construct according to claim 9 or 10, or the host cell according to any one of claims 11 to 13, and a pharmaceutically acceptable excipient.

16. Use of the antibody or antigen-binding fragment thereof according to claims 1-7, the polynucleotide according to claim 8, the nucleic acid construct according to claim 9 or 10, or the host cell according to any one of claims 11-13 in the preparation of a medicament for preventing or treating a disease.

17. A kit for detecting sclerostin for evaluating the therapeutic effect of a drug, the kit comprising the antibody or antigen-binding fragment thereof according to claims 1-7, the polynucleotide according to claim 8, the nucleic acid construct according to claim 9 or 10, or the host cell according to any one of claims 11-13.

18. A non-diagnostic method for detecting the presence of sclerostin in a sample, the method comprising: The antibody or antigen-binding fragment thereof according to claims 1-7 is incubated with the sample, and the binding of sclerostin to the antibody or antigen-binding fragment thereof is detected, thereby determining the presence of sclerostin in the sample.

19. The method according to claim 18, characterized in that The detection is an enzyme-linked immunosorbent assay.

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