Anti-dkk1 antibody and use thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to an anti-DKK1 antibody and application thereof, which comprises a light chain variable region VL and a heavy chain variable region VH, the light chain variable region VL comprises LCDR 1-3 with an amino acid sequence being at least 95%, at least 98%, at least 99% or at least 100% identical to SEQ ID NO: 3-5, and the heavy chain variable region VH comprises HCDR 1-3 with an amino acid sequence being at least 95%, at least 98%, at least 99% or at least 100% identical to SEQ ID NO: 6-8. The anti-DKK1 antibody of the application has high affinity and high specificity, and is suitable for detection of DKK1 in human blood. The application fills the blank of insufficient supply of antibody raw materials in the current DKK1 detection field, and provides an important detection tool and means for early diagnosis and treatment prognosis evaluation of diseases such as osteoporosis, multiple myeloma bone disease (MBD) and degenerative arthritis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically, it relates to an anti-DKK1 antibody and its application. Background Technology

[0002] Dickkopf-related protein 1 (DKK1) is a secreted glycoprotein that plays a crucial regulatory role in various physiological and pathological processes, including embryonic development, bone formation, and tumorigenesis. In bone tissue, DKK1 is a significant negative regulator of bone formation, reducing bone matrix synthesis and mineralization by inhibiting osteoblast differentiation and function. Simultaneously, DKK1 can also act on osteoclasts, regulating bone resorption. In some bone metabolic diseases, aberrant expression of DKK1 leads to altered bone density and abnormal bone structure. During embryonic development, DKK1 participates in body axis formation and organogenesis by regulating the Wnt signaling pathway and can also guide cell differentiation into specific cell types.

[0003] Although some research has been conducted on DKK1-related antibodies, such as CN119097708B, WO2024015463A1, and WO2023143444A1, these studies mainly focus on pharmaceutical applications such as improving osteoporosis and anti-tumor treatments. Dedicated detection antibodies for DKK1 remain scarce, making it difficult to meet the needs for accurate identification and quantitative analysis. Currently, there is an urgent need to develop a highly specific and sensitive anti-DKK1 antibody for the development of DKK1 reagent kits. Summary of the Invention

[0004] The purpose of this invention is to overcome the problems existing in the prior art and provide an anti-DKK1 antibody and its applications. This invention provides an antibody or its antigen-binding fragment that specifically binds to DKK1, which can be in the form of a monoclonal antibody or a human chimeric antibody. The antibody provided by this invention has the characteristics of high affinity and specificity.

[0005] The objective of this invention and the technical problem it solves are achieved by the following technical solutions.

[0006] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0007] As used herein, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this disclosure, "a plurality of" means at least two, such as two, three, etc., unless otherwise expressly and specifically defined.

[0008] As used herein, the terms “comprising” or “including” are open-ended expressions, meaning they include the contents specified in this invention but do not exclude other aspects.

[0009] As used herein, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0010] In one aspect, this disclosure provides an antibody or antigen-binding fragment thereof that specifically binds to DKK1, comprising a light chain variable region VL and a heavy chain variable region VH, wherein the light chain variable region VL comprises LCDR1-3 having an amino acid sequence as shown in SEQ ID NO: 3-5 or having at least 95%, at least 98%, at least 99%, or at least 100% identity with it, and the heavy chain variable region VH comprises HCDR1-3 having an amino acid sequence as shown in SEQ ID NO: 6-8 or having at least 95%, at least 98%, at least 99%, or at least 100% identity with it.

[0011] In some embodiments of the antibodies or antigen-binding fragments thereof that specifically bind to DKK1 disclosed herein, the amino acid sequence of the VL is as shown in SEQ ID NO: 1 or has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with it, and the amino acid sequence of the VH is as shown in SEQ ID NO: 2 or has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with it.

[0012] In some embodiments of the antibody or antigen-binding fragment thereof that specifically binds to DKK1 disclosed herein, the amino acid sequence of the VL comprises LCDR1, LCDR2, and LCDR3 sequences having one or more conserved amino acid substitutions, deletions, or insertions thereof, or any combination thereof, as shown in SEQ ID NO: 1; and the amino acid sequence of the VH comprises HCDR1, HCDR2, and HCDR3 sequences having one or more conserved amino acid substitutions, deletions, or insertions thereof, or any combination thereof, as shown in SEQ ID NO: 2.

[0013] As used herein, the term "antibody" refers to an immunoglobulin molecule that has the ability to specifically bind to a particular antigen. Antibodies typically contain variable and constant regions in each of their heavy and light chains. The variable regions of the antibody heavy and light chains contain binding domains that interact with the antigen. The constant regions of the antibody mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system such as C1q (the first component in the classical complement activation pathway). Therefore, most antibodies have a heavy chain variable region (VH) and a light chain variable region (VL) that together form the antibody portion that binds to the antigen.

[0014] As used herein, the term "antibody analogue" refers to a derivative obtained by biological or chemical methods, based on the antibody structure, through the deletion, addition, or modification of chemical groups (such as amino acids). These derivatives still contain structures similar to the antibody variable region (or the CDR region within the antibody variable region) and can undergo reactions similar to antigen-antibody binding through these structures.

[0015] As used herein, the terms "binding" or "specific binding" refer to a non-random binding reaction between two molecules, such as an antibody and its target antigen. In some embodiments, an antibody that specifically binds to an antigen refers to an antibody that binds to the antigen with an affinity corresponding to a KD of less than about 10⁻⁵ M, for example, less than about 10⁻⁶ M, 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M, or 10⁻¹⁰ M or smaller. As used herein, "KD" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, used to describe the binding affinity between the antibody and the antigen. The smaller the KD, the higher the binding affinity between the antibody and the antigen.

[0016] As used herein, the term "antibody variable region" refers to a domain within the heavy and light chains of an antibody. It includes the light chain variable region (VL) and the heavy chain variable region (VH). In nature, antibody variable regions are encoded by the V, D (only applicable to the heavy chain), and J segments from immunoglobulin (heavy and light chain) genes through genetic recombination. The amino acid sequences of variable regions are highly diverse between different antibodies (while the amino acid sequences of other regions of the antibody are relatively highly similar), and they are responsible for recognizing and binding to specific antigenic determinants. Within the antibody variable region, both the VL and VH domains contain framework regions (FRs) and CDRs (completional radii) from the amino terminus to the carboxyl terminus. A typical antibody variable region has three framework regions and three CDRs, interleaved: FR1, CDR1, FR2, CDR2, FR3, and CDR3. The framework regions (FRs) primarily function as the protein domain framework, while the CDR regions primarily function for antigen-antibody specific recognition and binding. The CDR1, CDR2, and CDR3 of the VL domain are also referred to as LCDR1, LCDR2, and LCDR3, respectively, in this paper; the CDR1, CDR2, and CDR3 of the VH domain are also referred to as HCDR1, HCDR2, and HCDR3, respectively, in this paper.

[0017] The amino acid sequence of each VL and VH domain is consistent with any conventional definition of CDR. Conventional definitions include the Kabat definition (Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991)), the Chothia definition (Chothia and Lesk, J. Mol. Biol. 196:901-917, 1987; Chothia et al., Nature 342:878-883, 1989); the complex of Chothia and Kabat CDRs, where CDR-H1 is a complex of Chothia and Kabat CDRs; the AbM definition used by OxfordMolecular's antibody modeling software; and the CONTACT definition by Martin et al. (bioinfo.org.uk / abs). Kabat provides a widely used numbering convention (Kabat numbering system) in which corresponding residues between different heavy chains or between different light chains are assigned the same number. This disclosure may use CDRs defined according to any of these numbering systems, but preferred embodiments use CDRs defined by Kabat or Chothia.

[0018] As used herein, the term “anti-DKK1 antibody (or antibody analog)” refers to a single (or monoclonal) form of an antibody (or antibody analog) containing a single structure (such as a single amino acid sequence).

[0019] As used herein, the term “identity” is used to describe the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences relative to a reference sequence, determined by conventional methods, see, for example, Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN procedure (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Center)). Foundation, Washington, DC). Numerous algorithms exist for aligning sequences and determining sequence identity, including: the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48: 443; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2: 482; the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85: 2444; the Smith-Waterman algorithm (Meth. Mol. Biol. 70: 173-187 (1997); and the BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J. Mol. Biol. 215: 403-410). Computer programs utilizing these algorithms are also available, including but not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul...). See, Meth.Enzym., 266:460-480 (1996); or GAP, BESTFIT, BLAST Altschul, etc., above, FASTA, and TFASTA, available in Genetics Computing Group (GCG) package, version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0020] Without substantially affecting antibody activity (retaining at least 95% activity), those skilled in the art can substitute, add, and / or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) amino acids in the sequences of this invention to obtain variants of the antibody or its functional fragments. These are all considered to be included within the scope of protection of this invention. For example, amino acids with similar properties can be substituted in the variable region. The variant sequences of this invention can have at least 80% identity (or homology) with the reference sequence. Sequence identity described in this invention can be measured using sequence analysis software, such as the computer program BLAST using default parameters, especially BLASTP or TBLASTN. The amino acid sequences described in this invention are shown from the N-terminus to the C-terminus.

[0021] As used herein, the term "conserved amino acid substitution" refers to the substitution of an amino acid by another amino acid with biologically, chemically, or structurally similar residues. Biological similarity means that the substitution does not impair the biological activity of the DKK1 antibody or the DKK1 antigen. Structural similarity means that the amino acid has a side chain of similar length, such as alanine, glycine, or serine, or a side chain of similar size. Chemical similarity means that the amino acids have the same charge or are both hydrophilic or hydrophobic. For example, hydrophobic residues such as isoleucine, valine, leucine, or methionine can be substituted for each other. Alternatively, polar amino acids can be used, such as arginine replacing lysine, glutamic acid replacing aspartic acid, glutamine replacing asparagine, serine replacing threonine, etc.

[0022] Therefore, the antibody described in this invention can also be directly synthesized from the antibody variable region amino acid sequence provided by this invention, and can be further chemically modified to derive different antibody analogs.

[0023] In some embodiments of the antibodies or antigen-binding fragments thereof that specifically bind to DKK1 disclosed herein, they further comprise a light chain constant region CL and / or a heavy chain constant region CH, wherein the light chain constant region CL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 13, and the heavy chain constant region CH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 14.

[0024] As used herein, the term "heavy chain constant region" includes an amino acid sequence derived from the immunoglobulin heavy chain. A polypeptide containing a heavy chain constant region comprises at least one of the following: a CH1 domain, a hinge (e.g., an upper hinge region, a middle hinge region, and / or a lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, an antigen-binding polypeptide for use in this disclosure may comprise: a polypeptide chain containing a CH1 domain; a polypeptide chain containing at least a portion of a CH1 domain, a hinge domain, and a CH2 domain; a polypeptide chain containing both a CH1 domain and a CH3 domain; a polypeptide chain containing at least a portion of a CH1 domain, a hinge domain, and a CH3 domain; or a polypeptide chain containing at least a portion of a CH1 domain, a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, the polypeptide of this disclosure comprises a polypeptide chain containing a CH3 domain. Furthermore, antibodies for use in this disclosure may lack at least a portion of the CH2 domain (e.g., all or part of the CH2 domain). As described above, those skilled in the art will understand that the heavy chain constant region can be modified to differ from naturally occurring immunoglobulin molecules in its amino acid sequence.

[0025] Based on the amino acid sequence of the constant region of the antibody heavy chain, immunoglobulin molecules can be classified into five classes (isotypes): IgA, IgD, IgE, IgG, and IgM, and can be further divided into different subtypes, such as IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, etc. Based on the amino acid sequence of the light chain, the antibody light chain can be divided into lambda (λ) chains and kappa (κ) chains. The antibodies disclosed herein can be any of the above classes or subtypes.

[0026] In some embodiments of the antibodies or antigen-binding fragments thereof that specifically bind to DKK1 disclosed herein, the antibodies have an isotype selected from IgG. In some embodiments, the antibodies have a subtype selected from IgG1.

[0027] As used herein, the term "antigen-binding fragment" is also known as "antibody fragment." Antibody fragments typically refer to antigen-binding antibody fragments, which can include a portion of a complete antibody. They are generally antigen-binding regions or variable regions. Examples of antibody fragments include Fab, Fab', F(ab')2, Fd, Fv, scFv, dAb, Fab / c, complementarity-determining regions, single-chain antibodies, double-chain antibodies, or single-domain antibody molecules. A "Fab fragment" consists of a light chain, a heavy chain (CH1), and a variable region. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule. The Fab' fragment having one or more cysteine ​​residues at the C-terminus of the CH1 domain of the Fab fragment; the F(ab')2 fragment being a divalent fragment consisting of two Fab' fragments linked by disulfide bonds in the hinge region; the Fd fragment having VH and CH1 domains; the Fv fragment having VL and VH domains in one arm of the antibody; the dAb fragment consisting of either a VH or VL domain; a separate CDR region; and any of the above fragments in a modified form that retains antigen-binding activity.

[0028] In some embodiments of the antibodies or antigen-binding fragments thereof that specifically bind to DKK1 disclosed herein, the antigen-binding fragments are selected from double-stranded antibodies.

[0029] In some embodiments of the antibodies or antigen-binding fragments thereof that specifically bind to DKK1 disclosed herein, the antibodies are murine monoclonal antibodies or humanized chimeric antibodies.

[0030] In some embodiments of the antibodies or antigen-binding fragments thereof that specifically bind to DKK1 disclosed herein, the antibody is a monoclonal antibody that further comprises a light chain and a heavy chain, the light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 13, and the heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 14.

[0031] As used herein, the term "monoclonal antibody" refers to an antibody derived from a substantially homogeneous population of antibodies. That is, each antibody that makes up the population is identical, except for a small number of mutations that may be naturally present. Monoclonal antibodies are highly specific and target a single antigen. The term "monoclonal antibody" as used herein is not limited to antibodies produced by hybridoma technology, nor should it be construed as requiring antibodies to be produced by any particular method.

[0032] In some embodiments of the antibodies or antigen-binding fragments thereof that specifically bind to DKK1 disclosed herein, the antibody is a humanized chimeric antibody that further comprises a light chain and a heavy chain, the light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 15, and the heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 16.

[0033] As used herein, the term "chimeric antibody" refers to an antibody in which a portion of the heavy chain and / or light chain originates from one source or species, while the remainder of the heavy chain and / or light chain originates from a different source or species. "Humanized chimeric antibody" refers to an antibody obtained by transfecting host cells with a plasmid constructed by inserting the nucleic acid sequences of the light chain V region and heavy chain V region of a monoclonal antibody into pGmab-K (containing the constant region of the human antibody kappa chain) and pGmab-H (containing the constant region of the human antibody IgG1 chain), respectively, using molecular cloning methods. The term "humanized chimeric antibody" in this document is not limited to antibodies produced using the techniques described herein, nor should it be construed as requiring antibodies produced by any specific method.

[0034] In another aspect, the present invention provides a nucleic acid comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof that specifically binds to DKK1 as disclosed herein.

[0035] In another aspect, this disclosure provides a carrier containing the nucleic acid disclosed herein.

[0036] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is attached. One type of vector is the "plasmid," which refers to a circular double-stranded DNA loop capable of attaching an additional DNA fragment. Another type of vector is a viral vector, in which an additional DNA fragment can be attached to a viral genome. Some vectors are capable of autonomous replication in the host cell to which they are introduced (e.g., bacterial vectors with bacterial origins of replication and free mammalian vectors). Other vectors (e.g., non-free mammalian vectors) can integrate into the host cell's genome after introduction and thus replicate along with the host genome. Furthermore, some vectors are capable of directing the expression of genes operatively attached to them. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In some implementations, the vector includes, but is not limited to: (1) plasmids; (2) phage particles; (3) granules; (4) artificial chromosomes, such as yeast artificial chromosomes, bacterial artificial chromosomes or artificial chromosomes derived from P1; (5) bacteriophages, such as λ phage or M13 phage; (6) animal viruses, such as retroviruses, adenoviruses, adeno-associated viruses, sporocyst viruses, poxviruses, and baculoviruses.

[0037] When ligating the nucleic acid disclosed in this invention to a vector, the nucleic acid can be directly or indirectly linked to control elements on the vector, as long as these control elements can control the translation and expression of the nucleic acid. These control elements can be directly derived from the vector itself or be exogenous, i.e., not derived from the vector itself. Of course, the polynucleotides can be operatively linked to the control elements. In this document, "operatively linked" means linking the exogenous gene to the vector so that the control elements within the vector, such as transcriptional control sequences and translational control sequences, can perform their intended functions of regulating the transcription and translation of the exogenous gene. Of course, the polynucleotides used to encode the antibody heavy and light chains can be inserted independently into different vectors, but commonly they are inserted into the same vector.

[0038] In another aspect, this disclosure provides a host cell containing the nucleic acid or vector disclosed herein.

[0039] As used herein, the term "host cell" refers to a cell into which an expression vector has been introduced. The expression vector can be introduced into a host cell to construct recombinant cells, which are then used to express the antibodies or antigen-binding fragments provided by this invention. The corresponding antibodies can be obtained by culturing these recombinant cells. In some embodiments, the host cell includes, for example, CHO cells, such as CHOS cells and CHO-K1 cells, or HEK293 cells, such as HEK293A, HEK293T, and HEK293F.

[0040] In some embodiments of the host cell disclosed herein, the host cell is a eukaryotic cell.

[0041] In some embodiments of the host cell disclosed herein, the host cell is a mammalian cell, including but not limited to 293F cells and CHO cells.

[0042] In another respect, this disclosure provides hybridoma cells that can produce antibodies or antigen-binding fragments that specifically bind to DKK1 as disclosed in this invention.

[0043] As used herein, the term "hybridoma cell" refers to a cell formed by fusing myeloma cells and B lymphocytes during the preparation of monoclonal antibodies. In classic monoclonal antibody preparation methods, a suitable antigen is first required, which is then used to immunize animals. To prepare antibodies (or antibody analogs) against DKK1, a suitable antigen must contain DKK1. This antigen can be isolated and purified from natural human tissue or blood, or obtained through artificial recombinant protein expression in prokaryotic or eukaryotic cells followed by purification. The antigen is used for animal immunization and antibody screening.

[0044] In classic monoclonal antibody preparation methods, animals are first immunized with the DKK1 antigen, and blood is collected at intervals to verify whether the animals have developed an antibody response to the DKK1 antigen. Then, B cells are isolated from the spleens of animals with antibody responses and fused in vitro with immortalized myeloma cells to obtain hybridoma cells. These hybridoma cells are then extremely diluted in culture plates and regrow (monoclonal hybridoma cell lines), and the culture supernatant of these hybridoma cell lines is collected to detect whether they contain specific antibodies against the antigen. Based on antibody yield, quality, and cell line growth characteristics, the optimal monoclonal antibody production cell line can be selected for subsequent monoclonal antibody production.

[0045] Other methods can also be used to obtain monoclonal antibodies. For example, spleen cells from the aforementioned animals can be isolated and incubated with labeled antigens (such as fluorescein-labeled DKK1). Since antibody-producing B cells typically have antibody molecules present on their cell membranes, these cells bind to the labeled antigens (staining) and can then be sorted using a fluorescence flow cytometry sorter. The mRNA from these sorted B cells can be isolated and used to obtain a library of antibody variable region cDNA through in vitro reverse transcription and specific PCR. This cDNA library can be inserted into an expression plasmid (such as an antibody expression plasmid suitable for expression in mammalian cells, or a phage expression plasmid suitable for expression in bacterial cells) and expressed in host cells suitable for that plasmid. These host cells (or phages) can be isolated and purified (cloned) using various methods (such as the previously described cell limiting dilution culture method, or the phage plating method). These cell lines or phage clones can be used to produce antibodies and to analyze and identify the antibodies.

[0046] In another aspect, this disclosure provides a method for preparing the antibody or its antigen-binding fragment disclosed in this invention, comprising: culturing the host cell or hybridoma cell disclosed in this invention under conditions that cause the antibody or its antigen-binding fragment to be expressed.

[0047] In another aspect, this disclosure provides antibody markers comprising: (i) an antibody that specifically binds to DKK1 as disclosed in this invention or an antigen-binding fragment thereof, nucleic acid, vector, host cell or hybridoma cell; and (ii) a fluorescent marker.

[0048] In another aspect, this disclosure provides antibody-conjugates comprising the antibody or antigen-binding fragment thereof that specifically binds to DKK1 as disclosed in this invention.

[0049] In the disclosure of this invention, suitable substances for fluorescent labeling include, but are not limited to, horseradish peroxidase (HRP), alkaline phosphatase (ALP), acridinium ester, and ruthenium tripyridine.

[0050] In another aspect, the present invention provides a detection reagent comprising an antibody or antigen-binding fragment thereof that specifically binds to DKK1 as disclosed in the present invention.

[0051] In some embodiments of the detection reagents disclosed herein, an antibody or antigen-binding fragment of DKK1, after fluorescent labeling, serves as a flow cytometry detection reagent component that specifically recognizes DKK1.

[0052] In another aspect, this disclosure provides the use of the antibodies or antigen-binding fragments thereof that specifically bind to DKK1 disclosed in this invention, or the detection reagents, in the preparation of reagents or products for detecting the DKK1 content in a sample, reagents or products for diagnosing DKK1-related diseases, or medicaments for the prevention and / or treatment of DKK1-related diseases.

[0053] In some embodiments of the uses disclosed herein, the DKK1-related diseases are selected from: osteoporosis, multiple myeloma bone disease (MBD), and osteoarthritis.

[0054] In another aspect, this disclosure provides the use of the antibodies or antigen-binding fragments thereof that specifically bind to DKK1 disclosed in this invention, nucleic acids, vectors, host cells, hybridoma cells, compositions and / or antibody-conjugates in the preparation of any of the products shown below: (1) Products tested for DKK1; (2) Products for the prevention and / or treatment of DKK1-related diseases.

[0055] The anti-DKK1 antibody of the present invention can be used to detect DKK1 in human serum, or substances containing DKK1, as well as substances that can specifically bind to DKK1 (such as anti-DKK1 antibodies). The detection principle is mainly based on the specific recognition and binding of the DKK1 antibody of the present invention to DKK1, and the detection is performed using chemical labeling technology (such as labeled antibodies, or labeled specific secondary antibodies) or physical detection technology (such as light scattering technology, plasma resonance technology, etc.).

[0056] This invention provides monoclonal antibody M925 and / or humanized chimeric antibody hM925 against DKK1, which exhibit high affinity and specificity. It also provides a flow cytometry detection method based on these antibodies. This invention addresses the relative shortage of antibody raw materials for DKK1 detection in the current market and can serve as an auxiliary diagnostic tool for related cardiovascular diseases, filling a gap in the domestic market.

[0057] By employing the above technical solution, the present invention has at least the following advantages: This invention provides a monoclonal antibody against DKK1, and a modified human chimeric antibody hM925 that avoids the HAMA reaction in natural samples. The antibody or its antigen-binding fragment provided by this invention exhibits high affinity and specificity. The product of this invention solves the problems of poor specificity and low sensitivity of antibody raw materials for DKK1 detection projects currently on the market. The antibody disclosed in this invention has high sensitivity and specificity, providing an important detection tool and method for the early diagnosis and prognostic assessment of various skeletal diseases, such as osteoporosis, multiple myeloma bone disease (MBD), and osteoarthritis.

[0058] In antibody screening, this invention uses eukaryotic cell-expressed antigens and compares antibody specificity detection. After multiple rounds of screening, M925 was found to be an antibody with good specificity for DKK1. Multiple subcutaneous injections at multiple sites during animal immunization improved the final serum titer and increased the positive clone rate. Delaying the addition of the HAT screening reagent from the day of fusion to the second day significantly improved the cell positivity rate.

[0059] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the preferred embodiments of the present invention are described in detail below. Attached Figure Description

[0060] Figure 1 Construct a map for the pCDNA3.1-DKK1 plasmid; Figure 2 To determine the purity of the DKK1 target protein using an SDS-PAGE assay; Figure 3 A plasmid image of pGmab-K-hM925, a humanized chimeric antibody expression plasmid constructed using antibody M925 according to an embodiment of the present invention; Figure 4 A plasmid image of pGmab-H-hM925, a humanized chimeric antibody expression plasmid constructed using antibody M925 according to an embodiment of the present invention; Figure 5 shows a comparison of the concentrations of DKK1 in human blood using the magnetic microparticle chemiluminescence detection method provided by the embodiments of the present invention, specifically antibodies hM925 and M925. Detailed Implementation

[0061] To make the technical means, creative features, achieved objectives, and effects of this invention readily understandable, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0062] The nucleic acids encoding the heavy and / or light chains of the antibodies of this invention are within the scope of this invention. Based on the amino acid sequences of the heavy and / or light chains, those skilled in the art can easily obtain the corresponding nucleic acid sequences, as shown in Table 1. It should be noted that the CDR sequences listed in Table 1 below were obtained from the IMGT database. Those skilled in the art should understand that CDR sequences obtained from different databases may differ, but these variations should all be included within the scope of protection of this invention.

[0063] Table 1. Sequence information corresponding to different numbers

[0064] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0065] Example 1: Construction of DKK1 recombinant protein expression plasmid

[0066] The nucleic acid sequence of DKK1 is based on the NCBI Gene ID: NM_012242.4 (DKK1) sequence. During the synthesis of the DNA sequence, a protein purification tag sequence (10×His) was ligated to the 3' end of the full-length DKK1 sequence (1Met-266His). EcoRI and XbaI sites were designed upstream and downstream of the DNA sequence, respectively. The designed DNA sequence was synthesized by a third-party gene synthesis company and constructed into the multiple cloning site of the pUC57 plasmid. The constructed plasmid was named pUC57-DKK1. Using the NEButsmart endonuclease system, 2 μg of pUC57-DKK1 and pCDNA3.1 plasmid were added to 2 U EcoRI and 2 U XbaI endonucleases, respectively. After digestion at 37°C for 2 hours, the positive bands were separated and recovered using 1% agarose gel, and the concentration of the recovered DNA fragments was detected using A260. 2 nmol of each of the double-digested fragments of pUC57-DKK1 and pCDNA3.1 plasmid were added, and 1 U T4 ligase was added. The volume was adjusted to 20 μL with T4 ligase buffer and ultrapure water. After ligation at 16°C overnight, the cells were transformed into DH5α competent cells and plated on LB plates. Single colonies were picked and sequenced using FCMV and SV40 universal primers. The sequenced gene sequences were found to be correct. The constructed plasmid was named pCDNA3.1-DKK1, and its structure is as follows: Figure 1As shown.

[0067] Example 2: Expression and Identification of Recombinant DKK1 Protein

[0068] First, the expression plasmid pCDNA3.1-DKK1 was transfected into 293F cells. The transfection reagent used for transfecting suspension-cultured 293F cells was Free Style MAX (Invitrogen, 16447100), and the transfection procedure was performed according to the manufacturer's instructions.

[0069] Subsequently, the transfected 293F cells were transferred to a 125 mL Erlenmeyer flask and cultured in 30 mL of FreeStyle293 medium at a cell density of 1 × 10⁶ cells / mL. Cells were cultured in a CO₂ incubator (37 °C, 5% CO₂) at a shaker speed of 130 rpm. The culture supernatant was collected after 7 days of cell culture, and the concentration of recombinant DKK1 protein (expressed and secreted by 293F cells) in the supernatant was measured using a sandwich ELISA method.

[0070] Specifically, in the sandwich ELISA, the human DKK1 ELISA kit (Novus, #DKK100B) was used to detect the transfection supernatant. To determine the activity concentration of DKK1, a DKK1 control antigen (R&D, #5439-DK) of known concentration (detected by the A280 method) was used to create a concentration standard curve. The detection experiment was performed according to the kit instructions. Sample addition: The DKK1 transfection supernatant and control antigen were diluted with the sample diluent provided with the kit at different ratios or concentrations. 0.1 mL of the diluted sample was added to each well and incubated at 37 °C for 1 hour, followed by washing. Blank wells (without sample), negative control wells, and positive control wells were also prepared. 0.1 mL of freshly diluted antibody was added to each well and incubated at 37 °C for 1 hour, followed by washing 3 times. Adding enzyme-labeled antibody: 0.1 mL of enzyme-labeled antibody solution was added to each well. The wells were incubated at 37 °C for 1 hour and washed 3 times. Add substrate solution for color development: Add 0.1 mL of TMB substrate solution to each reaction well and let stand at room temperature for 10 minutes. Add 0.1 mL of stop solution to each reaction well. Measure OD value to determine the results. Use a microplate reader to measure the absorbance at 450 nm (A450). The results are shown in Table 2.

[0071] Finally, the ELISA CALC was used to calculate the logistic curve of the control protein's four parameters as a standard curve, where R... 2 =0.990. The calculated concentration of DKK1 protein in the supernatant of the test cells was approximately 4.1 μg / mL, indicating that the target protein was significantly expressed in the supernatant of 293F cells transfected with pCDNA3.1-DKK1.

[0072] Table 2 Absorbance values ​​corresponding to different protein concentrations

[0073] Example 3: Production and purification of DKK1 recombinant protein

[0074] Cell culture stage: The expression plasmid pCDNA3.1-DKK1 was transfected into 293F cells. The transfection reagent used for transfecting suspension-cultured 293F cells was Free Style MAX (Invitrogen, 16447100), and the transfection procedure was performed according to the manufacturer's instructions.

[0075] Subsequently, the transfected 293F cells were transferred to a 1L Erlenmeyer flask and cultured in 220 mL of FreeStyle 293 medium at a cell density of 1×10⁻⁶ cells / mL. 6 Cells / mL. Cells were cultured in a CO2 incubator (37 °C, 5% CO2) at 130 rpm on a shaker. After 7 days of culture, the cell culture medium was removed, centrifuged at 200 g for 5 minutes (to separate cells and coarse cell debris), and the supernatant was transferred to a new bottle and centrifuged again at 3000 g for 15 minutes (to separate fine cell debris). The separated supernatant was filtered through a 0.22 µM filter cup for sterilization and stored at 4 °C.

[0076] Protein purification stage: After equilibrating the sterilized cell supernatant to room temperature, purification was performed using a Ni column. Ni column packing material (medium), GE Healthcare, catalog number: #17-3712-02. A 30 mL column was packed, resulting in a final packing volume of 10 mL. 200 mL of cell supernatant was loaded at a flow rate of 1.0 mL / min, followed by elution with different concentrations of imidazole elution buffer (50 mM Tris-HCl, pH 7.5, 10-500 mM imidazole concentration gradient). The DKK1 recombinant protein eluted in 100 mM and 250 mM imidazole elution buffers. Samples from each elution step were taken, and the purity of the DKK1 target protein was determined by SDS-PAGE. The results are shown below. Figure 2 The samples added to lanes 1-9 were: supernatant, flow-through buffer, marker, washing buffer, 10mM imidazole elution buffer, 30mM imidazole elution buffer, 100mM imidazole elution buffer, 250mM imidazole elution buffer, and 500mM imidazole elution buffer, respectively. The purified DKK1 protein was mainly found in lanes 7 and 8 (i.e., in 100mM and 250mM imidazole elution buffers). Figure 2The results showed that the purified DKK1 recombinant protein was mainly found in lanes 7 and 8, with a molecular weight of approximately 40 kDa. The molecular weight was larger than the theoretical value (25.8 kDa) due to glycosylation. The purity was estimated to be 90%.

[0077] The eluent containing recombinant DKK1 protein (Lane 7 and Lane 8) was concentrated using 10 kDa ultrafiltration tubes (Millipore, UFC900396) and dialyzed overnight using a 10 kDa dialysis bag with 50 mM pH 7.5 Tris buffer. The recombinant protein concentration after dialyzing was determined using the A280 method. A typical total protein concentration was 0.82 mg / mL. Purified DKK1 was stored at -80 °C.

[0078] Example 4: Immunizing BALB / c mice with recombinant DKK1 protein

[0079] BALB / c mice aged 6-8 weeks were immunized according to the following procedure: For the initial immunization, 50 µg of DKK1 protein was mixed with an equal volume of Freund's complete adjuvant, emulsified, and injected subcutaneously at multiple sites. Fourteen days after the initial immunization, 25 µg of DKK1 protein was mixed with Freund's incomplete adjuvant, emulsified, and used as a booster immunization. Fourteen days after the second immunization, 25 µg of DKK1 protein was mixed with Freund's incomplete adjuvant, emulsified, and used as a booster immunization. Fourteen days after the third immunization, blood was collected and serum was separated. ELISA plates were coated with DKK1 protein, and an indirect ELISA assay was performed to determine the serum titer. The results showed that the prepared mouse antiserum titer was 1:24300. Typical serum test results from immunized mice are shown in Table 3.

[0080] Table 3. Detection of mouse antiserum titer

[0081] Example 5: Cell Fusion

[0082] In Example 5, after the third immunization, mice were given a booster immunization. The specific steps were as follows: 25 µg of DKK1 protein was mixed with PBS to a volume of 200 μL and injected intraperitoneally. Cell fusion was performed 3 days after the booster immunization. After removing the mouse eyeballs to collect blood, the mice were euthanized by dislocation and placed in a bottle of 70% alcohol for 2 minutes. Then, the mice were fixed to a foam board in a biosafety cabinet, the abdominal skin was opened to locate the spleen, which was removed with forceps, and the cells were gently crushed in a 200-mesh stainless steel filter. The cells were gently washed with DMEM medium (Thermo, 11965092), and then centrifuged at 200 g for 10 minutes at room temperature. The supernatant was discarded and the cells were used for further processing.

[0083] When preparing feeder cells, mice were euthanized by dislocation, placed in a bottle of 70% alcohol for 2 minutes, and then fixed on a foam board in a biosafety cabinet. The abdominal skin was opened, and PBS was gently injected subperitoneally with a syringe. The liquid containing feeder cells was washed out from the other side. The mixture was then centrifuged at 200 g for 10 minutes at room temperature, and the supernatant was discarded for later use. Mix 2.0 × 10⁷ FO myeloma cells with 1.0 × 10⁸ spleen cells, centrifuge at 200 g for 10 minutes, discard the supernatant, gently vortex to mix, and in a 37°C water bath, add 1 mL of 50% PEG-1450 (Merk, P1458) aqueous solution over 90 seconds, then add 20 mL of DMEM medium, centrifuge at 200 g for 10 minutes, discard the supernatant, repeat the washing once, centrifuge at 200 g for 10 minutes, discard the supernatant, and obtain hybridoma cells. Seed the cells into 10 96-well culture plates, 150 μL per well. Add 10,000 feeder cells / well to 10 wells of the above-mentioned 96-well cell culture plates, 100 μL per well. After labeling the culture plates, incubate them in a cell culture incubator at 37°C with 5% CO2. On the second day, add HAT selection medium (Merk, H0262) and continue HAT selection culture for 1-2 days. A large number of tumor cells will die, and after 3-4 days, the tumor cells will disappear, and hybrid cells will form small colonies. Maintain the HAT selection culture medium for 7-10 days, then switch to HT culture medium (Merk, H0137) and maintain for another 2 weeks. Then, continue culturing in DMEM medium with 20% FBS (ExCell, FSP500). During the above selection culture, when the hybridoma cells cover 1 / 10 of the well area, you can start detecting specific antibodies and screening for the desired hybridoma cell lines. During the selection culture, generally change half of the culture medium every 2-3 days.

[0084] Example 6: Screening and subclonal culture of positive hybridoma cell lines

[0085] First, the optimal coating amount of DKK1 protein as antigen was determined using the square titration method. 0.5, 1.0, 2.0, and 4.0 µg of DKK1 protein were coated onto 96-well plates, with each concentration represented by 6 wells (3 positive and 3 negative). Square titration was performed using DKK1 protein diluted with different concentrations from mouse positive serum, with unimmunized mouse negative serum serving as a negative control. 0.5 μg of purified DKK1 protein was coated onto each well of a 96-well ELISA plate and incubated overnight at 4°C. The plates were washed twice with PBST. 200 µL of 1% BSA in PBS was added to each well, and the plates were blocked at room temperature for 2 hours, then patted dry on folded paper. Sample loading: 0.1 mL of the test sample was added to each well, and the plates were incubated at 37°C for 1 hour, followed by washing. Blank wells (without sample), negative control wells, and positive control wells were also prepared. 0.1 mL of freshly diluted antibody was added to each well, and the plates were incubated at 37°C for 1 hour, followed by washing three times. Add enzyme-labeled secondary antibody: Add 0.1 mL of freshly diluted enzyme-labeled antibody (Solepro, #SE131) to each reaction well. Incubate at 37 ℃ for 1 hour, washing 3 times. Add substrate solution for color development: Add 0.1 mL of TMB substrate solution to each reaction well and incubate at room temperature for 10 minutes. Add 0.1 mL of 1M H2SO4 to each reaction well. Measure OD value to determine the result: Measure the absorbance at 450 nm (A450) using a microplate reader. A positive result is defined as OD value greater than 2.1 times that of the negative control (calculated after zeroing the blank control well). Select single clones of hybridoma cells anti-DKK1.

[0086] Following the above method, the selected positive hybridoma cells were subcloned. The original wells were diluted with HAT selective medium using a limiting dilution method and then re-distributed into 96-well culture plates. Cell morphology and quantity were then observed. The cell density was adjusted to 3-10 cells / mL. 100µL of diluted cells was added to each well of a cell culture plate containing a feeder cell layer prepared the previous day. The plates were incubated statically at 37 ℃ with 5% CO2. The medium was changed on day 7, and thereafter every 2-3 days. Cell clone formation was observed on days 8-9, and antibody activity was promptly detected. Cells from the positive wells were transferred to 24-well plates for further culture. The binding activity of antibodies to DKK1 protein in the culture supernatant of different cell lines was compared using an indirect ELISA method. The cell line M925, with the highest OD value (OD450=2.153), was selected for later experiments. Cells were cryopreserved as soon as possible, and the hybridoma cell line with clone number M925 was ultimately selected for antibody production.

[0087] Example 7: Large-scale preparation of monoclonal antibodies and determination of antibody titer

[0088] (1) Large-scale preparation of monoclonal antibodies

[0089] Hybridoma cells were cultured in DMEM complete medium with 10% serum, with 10⁶ cells per 10 cm diameter cell culture dish, 10 mL of medium per dish, for a total of 20 dishes. Cell supernatant was collected 7–10 days after seeding, yielding 7–10 mL of supernatant per dish. The obtained cell supernatant was centrifuged at 3000 g for 10 minutes, the bottom cell pellet was discarded, and the supernatant was aliquoted and stored at -20°C. After thawing and equilibration to room temperature, 1 / 10 volume of 1 M Tris–HCl pH 8.0 was added to adjust the sample pH to 8.0. Protein G affinity columns were equilibrated with 20 column volumes of 100 mM pH 8.0 Tris–HCl. The ascites supernatant adjusted to pH 8.0 was loaded onto the column, followed by washing with 20 column volumes of 100 mM pH 8.0 Tris–HCl, and finally eluting the antibody with 100 mM Mlycine–HCl pH 2.5. The antibody eluent was added to a concentration tube (Millipore, UFC801008, 10K) and centrifuged at 3000×g for 20 minutes at room temperature using a centrifuge (Xiangyi, L550). The mixture was then centrifuged in batches to a final volume of 1 mL per concentration tube (2 tubes). 4 mL of 10 mM PBS pH 7.4 buffer was added, and the mixture was centrifuged again at 3000×g for 20 minutes at room temperature. This centrifugation was repeated three times to bring the antibody buffer to a final volume of 10 mM PBS pH 7.4. 10 mM PBS pH 7.4 was then added to bring the total volume to 10 mL. Finally, 2 mL of the concentrated antibody solution was aliquoted into centrifuge tubes and stored at -80°C. The antibody concentration was determined using the A280 method, and the purified monoclonal antibody M925 concentration was 0.41 mg / mL.

[0090] (2) Antibody titer determination

[0091] The titer of anti-DKK1 antibody M925 was detected using an indirect ELISA method. DKK1 protein was diluted with PBS and coated into 96-well microplates at a concentration of 0.2 µg / mL (100 µL / well). After incubation at 4°C overnight, the plates were washed twice with PBST solution (300 µL / well each time) and then blotted dry. The plates were then blocked with 200 µL / well of 1% BSA in PBS solution at room temperature for 2 hours and blotted dry. Different dilutions of anti-DKK1 antibody M925 (PTB) were added, and the plates were incubated at 37°C for 1 hour. The plates were then washed twice with PBST solution (300 µL / well each time) and blotted dry. Finally, HRP-labeled goat anti-mouse antibody (5000-fold diluted with PTB) was added, and the plates were incubated at 37°C for 1 hour. The plates were then washed twice with PBST solution (300 µL / well each time) and blotted dry. Add 100 µL of TMB substrate solution to each well and react at room temperature for 10 minutes. Terminate the reaction by adding 100 µL of 1M H₂SO₄ to each well. Measure the absorbance at 450 nm using a microplate reader (A450). The results are shown in Table 4, indicating that the purified antibody titer is 1:243000.

[0092] Table 4 Antibody titer determination

[0093] Example 7: Sequence Analysis of Monoclonal Antibodies

[0094] (1) Identification of monoclonal antibody subtypes

[0095] Hybridoma cell line M925 was cultured in DMEM medium (GIBCO, #C11995500BT) supplemented with 10% serum in 10 cm diameter cell culture dishes (37℃, 5% CO2). After 7 days of culture, the cells were transferred to 15 mL centrifuge tubes, counted using a hemocytometer, and 5 × 10⁶ cells were collected. The cells were centrifuged at 200 g for 5 minutes, the supernatant was discarded, and the centrifuge tubes were inverted and drained. cDNA was synthesized from the cells using a reverse transcription kit from Qiagen (Qiagen, 74134).

[0096] Antibody subtypes were determined by PCR using antibody subtype-specific primers. The synthesized cDNA was used as a template for the PCR reaction. Primer sequence information used for PCR is shown in Table 5.

[0097] Table 5 PCR primer sequence information

[0098] Note: In the table, S = C or G, M = A or C, R = A or G, and W = A or T.

[0099] PCR reaction solution system: TAKARA Ex Taq (5 U / µL, TAKARA, RR001B), 0.25 μL; 10×ExTaq Buffer, 5µL; dNTP mixture (2.5 mM each), 4µL; template cDNA, 1µL; upstream primer (100 µM), 1µL; downstream primer (100 µM), 1µL; add double-distilled water to a total volume of 50 µL.

[0100] PCR reaction temperature program: 94℃ for 5 minutes pre-denaturation, 30 temperature cycles (94℃ for 1 minute, 57℃ for 1 minute, 72℃ for 1 minute), 72℃ for 10 minutes extension.

[0101] After the reaction, 10 µL of each PCR product was loaded onto a 1% agarose gel for electrophoresis. Analysis of the electrophoresis pattern showed positive bands of 650 bp for the IgG1 heavy chain and the kappa light chain, respectively. The antibody subtype can be deduced based on the PCR product results. The monoclonal antibody M925 obtained in this invention has an IgG1 heavy chain and a kappa light chain.

[0102] (2) Sequencing of the variable region (V region) of the M925 hybridoma cell line antibody

[0103] The V region fragment of the antibody from cell line M925, obtained after PCR amplification (see above), was cleaved from an agarose gel and extracted using a DNA extraction kit (Qiagen, 74134). The extracted DNA fragment was ligated into the pEASY-T1 cloning vector and transformed into Trans1-T1 competent cells (Transgen, CT101-1). The transformed bacterial colonies were picked into LB medium, cultured overnight, and then subjected to DNA sequencing. The light chain V region nucleic acid sequence of the anti-DKK1 antibody (M925) provided by this invention is shown in SEQ ID NO. 9, and the heavy chain V region nucleic acid sequence is shown in SEQ ID NO. 10.

[0104] Based on the above, the amino acid sequences of the light chain variable region (VL) and heavy chain variable region (VH) of the obtained monoclonal antibody M925 are shown in Table 6. The CDR sequence of the antibody from the IMGT database is shown in Table 7. The sequences of the light chain constant region and heavy chain constant region of the antibody are shown in Table 8. The complete heavy chain and light chain sequences of the antibody are shown in Table 9.

[0105] Table 6. VL and VH sequences of monoclonal antibody M925

[0106] Table 7. CDR sequence of monoclonal antibody M925

[0107] Table 8. Light chain constant region and heavy chain constant region of monoclonal antibody M925

[0108] Table 9. Light and heavy chains of monoclonal antibody M925

[0109] Example 8: Modification, expression, and purification of human chimeric antibody M925

[0110] (1) Construction of pGmab-K-hM925 / pGmab-H-hM925 plasmid

[0111] The previously obtained nucleic acid sequences of the light chain V region of antibody M925 (as shown in SEQ ID NO. 9) and the heavy chain V region (as shown in SEQ ID NO. 10) were used to synthesize the designed DNA sequences by a third-party gene synthesis company and constructed into the pUC57 plasmid. The constructed plasmids were named pUC57-M925-VL and pUC57-M925-VH. Using the NEB Cutsmart endonuclease system, 2 μg of pUC57-M925-VL or pUC57-M925-VH and pGmab-K (containing the constant region of the human antibody kappa chain) or pGmab-H (containing the constant region of the human antibody IgG1 chain) plasmids were added to 2 U of XbaI and 2 U of... After digestion with BamHI restriction enzyme at 37℃ for 2 hours, the positive bands were separated and recovered using 1% agarose gel electrophoresis. The concentration of the recovered DNA fragments was detected using an A260 assay. 2 nmol each of the insert fragment (M925-VL or M925-VH) and the expression plasmid double-digested fragment (pGmab-K or pGmab-H) were added, and 1 U of T4 ligase was added. The mixture was then diluted to 20 μL with T4 ligase buffer and ultrapure water, and ligated overnight at 16℃. The resulting cells were then transformed into DH5α competent cells and plated on LB agar plates. Single colonies were picked and sequenced using FCMV and SV40 universal primers. The sequencing confirmed the correct gene sequences. The constructed expression plasmids were named pGmab-K-hM925 and pGmab-H-hM925, respectively. (See plasmid diagram for details.) Figure 3 and Figure 4 .

[0112] The amino acid sequences of the light chain variable region (VL) and heavy chain variable region (VH) of the hM925 antibody are shown in Table 10. The CDR sequences of the antibody according to the Kabat system are shown in Table 11. The heavy chain and light chain sequences of the antibody are shown in Table 12.

[0113] Table 10. Amino acid sequences of the light chain variable region (VL) and heavy chain variable region (VH) of the hM925 antibody.

[0114] Table 11 CDR sequence of hM925 antibody

[0115] Table 12 Light and Heavy Chains of hM925 Antibody

[0116] (2) Expression and purification of human chimeric antibody hM925

[0117] First, the two plasmids pGmab-K-hM925 / pGmab-H-hM925 constructed in the above process were mixed at a 1:1 ratio and transfected into ExpiCHO cells. The transfection reagent used for transfecting suspension-cultured ExpiCHO cells was FreeStyleMAX (Invitrogen, 16447100), and the transfection procedure was performed according to the manufacturer's instructions. Subsequently, the transfected ExpiCHO cells were transferred to a 125 mL Erlenmeyer flask and cultured in 30 mL of FreeStyle CHO medium at a cell density of 1 × 10⁶ cells / mL. The cells were cultured in a CO₂ incubator (37 ℃, 5% CO₂) at a shaker speed of 130 rpm. Finally, the culture supernatant was collected after 7 days of cell culture, and the activity concentration of the anti-DKK1 humanized antibody (named hM925) in the supernatant (expressed and secreted by ExpiCHO cells) was detected using an indirect ELISA method. In this experiment, DKK1 protein was diluted with PBS as an antigen and coated onto 96-well microplates at a concentration of 1 µg / mL, with a volume of 100 µL / well. After overnight coating at 4°C, the plates were washed twice with PBST solution, 300 µL / well each time. After washing, the plates were blotted dry and blocked with PBS solution containing 1% BSA, 200 µL / well, at room temperature for 2 hours. After blotting dry, 100 µL / well of cell supernatant expressing hM925, diluted with PTB at different ratios, was added to the blocked plates and incubated at 37°C for 1 hour. After blotting dry, the plates were washed three times with PBST, 300 µL / well each time. After blotting dry, HRP-labeled goat anti-human antibody diluted 5000-fold with PTB was added to the plates and incubated at 37°C for 1 hour. After washing three times with PBST, 300 µL / well was added to each well. Pat dry, add 100 µL of TMB substrate solution to each well of the microplate, incubate at room temperature for 10 minutes, then add 100 µL of 1M H2SO4 to terminate the reaction. Measure the absorbance at 450 nm (A450) using a microplate reader. Results are shown in Table 13. The activity and titer of the anti-DKK1 humanized antibody hM925 in the cell supernatant were detected, and the titer of hM925 in the cell supernatant was 72900.

[0118] Table 13. Titer of anti-DKK1 humanized antibody hM925 in cell supernatant

[0119] Centrifuge the cell supernatant at 3000 ×g for 10 minutes, then add 1 / 10 volume of 1 M Tris–HCl pH 8.0 to adjust the sample pH to 8.0. Equilibrate the protein G affinity column with 20 column volumes of 100 mM pH 8.0 Tris–HCl. Load the ascites supernatant adjusted to pH 8.0 onto the column, then wash with 20 column volumes of 100 mM pH 8.0 Tris–HCl. Finally, elute the antibody with 100 mM Glycine–HCl pH 2.5. Add the antibody eluent to a concentration tube (Millipore, UFC801008, 10K) and centrifuge at 3000 ×g for 20 minutes at room temperature using a centrifuge (Xiangyi, L550). Centrifuge in batches to a final volume of 1 mL / concentration tube (2 tubes), add 4 mL of 10 mM PBS pH 7.4 buffer, and continue centrifuging at 3000 ×g for 20 minutes at room temperature. Repeat centrifugation three times to adjust the antibody buffer to 10 mM PBS pH 7.4, then add 10 mM PBS pH 7.4 to a total volume of 10 mL. Finally, aliquot the concentrated antibody solution into centrifuge tubes (2 mL / tube) and store at -80°C. The antibody concentration was determined using a BCA kit (Solepro, PC0020), and the purified human chimeric antibody hM925 concentration was 3.2 mg / mL. For the activity titer assay of the purified anti-DKK1 humanized antibody hM925, DKK1 protein was diluted with PBS and coated onto 96-well microplates at a concentration of 1 µg / mL (100 µL / well). After overnight coating at 4°C, the microplates were washed twice with PBST solution (300 µL / well each time). After washing, the plates were patted dry and blocked with PBS solution containing 1% BSA (200 µL / well) at room temperature for 2 hours. Pat dry, add different concentrations of hM925 antibody diluted with PTB to the blocked ELISA plate, 100 µL / well, and incubate at 37 °C for 1 hour. Pat dry, wash the plate 3 times with PBST, 300 µL / well each time. Pat dry, add HRP-labeled goat anti-human antibody diluted 5000 times with PTB to the ELISA plate, and incubate at 37 °C for 1 hour. Wash the plate 3 times with PBST, 300 µL / well. Pat dry, add TMB substrate solution to the ELISA plate, 100 µL / well, incubate at room temperature for 10 minutes, then add 100 µL / well of 1MH2SO4 to stop the reaction. Measure the absorbance at 450 nm using an ELISA reader (A450). The results are shown in Table 14. The titer of hM925 in the cell supernatant was detected to be 7,290,000.

[0120] Table 14. Titer assay of affinity-purified humanized antibody hM925

[0121] Example 9: Identification of the sensitivity and specificity of antibody hM925 in detecting DKK1

[0122] (1) Identification of the sensitivity of antibodies hM925 and M925 in detecting DKK1

[0123] The sensitivity of antibody hM925 in detecting DKK1 was evaluated using an indirect ELISA method. The difference in sensitivity between hM925 and the control antibody (MCE, #HY-P86848) was compared. In this experiment, DKK1 protein was diluted with PBS as an antigen and coated onto 96-well microplates at a concentration of 1 µg / mL, with a volume of 100 µL / well. After overnight coating at 4°C, the plates were washed twice with PBST solution, 300 µL / well each time. After washing and drying, the plates were blocked with PBS solution containing 1% BSA, 200 µL / well, at room temperature for 2 hours. After drying, different concentrations of antibody M925, hM925, and control antibody diluted with PTB were added to the blocked plates, 100 µL / well, and incubated at 37°C for 1 hour. After drying, the plates were washed three times with PBST, 300 µL / well each time. Pat dry, add HRP-labeled goat anti-human antibody diluted 5000-fold with PTB to the microplate, and incubate at 37°C for 1 hour. Wash the plate 3 times with PBST, 300 µL / well. Pat dry, add TMB substrate solution, 100 µL / well, incubate at room temperature for 10 minutes, then add 100 µL / well of 1M H2SO4 to stop the reaction. Measure the absorbance at 450 nm (A450) using a microplate reader.

[0124] The experimental results are shown in Table 16. Antibody M925 and hM925 showed better sensitivity in detecting DKK1 protein than the control antibody. Furthermore, hM925 showed higher sensitivity for DKK1 protein detection. The results analysis showed that the DKK1 protein standard curve R... 2 =0.972, IC 50 =25 ng / mL. The standard curve range of this method is 0-3000 ng / mL, and the limit of detection is 100 ng / mL for the control antibody, 50 ng / mL for the M925 antibody, and 25 ng / mL for the hM925 antibody. The results are shown in Table 15.

[0125] Table 15. Identification of the sensitivity of antibody M925 and hM925 to DKK1 protein assay.

[0126] (2) Identification of the specificity of antibodies hM925 and M925 in detecting DKK1

[0127] The ability of antibodies hM925 and M925 to detect the specificity of DKK1 was evaluated using an indirect ELISA method. In this experiment, cells (293F, as the negative control) were cultured for 3 passages in DMEM medium with 10% serum. Cell lysis buffer (Beyotime, #P0013) was then used as the negative control. DKK1 293F overexpression cell lysis buffer (NOVUS, #NBL1-09901) was used as the positive detection protein. Both cell lysis buffers were diluted to 1 μg / mL with carbonated buffer at pH 9.5 and transferred to 96-well cell culture plates at a volume of 100 µL / well for overnight coating. The plates were washed twice with PBST solution, 300 µL / well each time. After washing, the plates were blotted dry, and the 96-well plates were blocked with PBS solution containing 1% BSA, 200 µL / well, and blotted dry at room temperature for 2 hours. Add 100 µL / well of different concentrations of antibodies hM925 and M925 diluted with PTB, and control antibody (RayBiotech, #130-10141-100), and incubate at 37 °C for 1 hour. Pat dry, wash 3 wells three times with PBST, 300 µL / well each time. Pat dry, add HRP-labeled secondary antibody diluted 5000 times with PTB to each well, and incubate at 37 °C for 1 hour. Wash 3 wells three times with PBST, 300 µL / well. Pat dry, add 100 µL / well of TMB substrate solution, incubate at room temperature for 10 minutes, then add 100 µL / well of 1M H2SO4 to stop the reaction. Measure the absorbance at 450 nm (A450) using a microplate reader. The experimental results are shown in Table 16, indicating that antibody hM925 has better specificity for detecting cell lysates overexpressing DKK1 protein compared to antibody M925 and the control antibody.

[0128] Table 16 Identification of the specificity of antibody M925 and hM925 in detecting DKK1.

[0129] Example 10: Comparison of the application of antibodies hM925 and M925 in the detection of DKK1 in human blood using a magnetic microparticle chemiluminescence detection method.

[0130] (1) Alkaline phosphatase (ALP) labeling of antibodies hM925 and M925

[0131] Antibodies hM925 and M925 (20 nmol, 3 mg) were diluted to 0.5 mL and added to a dialysis bag (10 kDa, 1 cm wide). The bag was dialyzed overnight at 4°C in 2 L of 10 mM PBS solution (pH 7.4). The next day, the dialysis bag containing the antibody solution was placed in 1 L of 10 mM carbonate buffer (pH 9.5) and dialyzed at room temperature for 2 hours with stirring, in preparation for coupling with EDC (carbodiimide)-activated ALP.

[0132] Meanwhile, accurately weigh 0.1 mg of ALP using an analytical balance and add it to the dialyzed antibody solution. Add ALP while stirring to prevent powder from adhering to the walls. Place the solution in a refrigerator or freezer at 4°C and continue stirring for 12–18 hours. After binding is complete, centrifuge the conjugate at 3000×g for 20 minutes to remove any small amount of precipitate. Transfer the conjugate to a dialysis bag and dialyze overnight in a beaker containing 10 mM PBS (pH 7.4). Pass the overnight dialyzed labeled antibody through a dextran G-25 column to separate the free ALP. Collect the labeled fluorescent antibody and store at 4°C.

[0133] (2) Components of the magnetic microparticle chemiluminescence detection reagent constructed using ALP-labeled antibodies hM925 and M925

[0134] In this experiment, the magnetic particle chemiluminescence detection includes reagent R1 or reagent R2, magnetic separation reagent, a series of calibrator solutions, and chemiluminescence substrate solution.

[0135] Among them, reagent R1 includes: R1 antibody: Alkaline phosphatase (ALP)-labeled anti-DKK1 monoclonal antibody M925, at a concentration of 2 μg / ml; Buffer solution: includes Tris (12.0 g / L); sodium azide (1.98 g / L); sodium chloride (5.9 g / L); 1.0 mL / L 1M magnesium chloride solution; 1.0 mL 0.1M zinc chloride solution; 10 g / L fish skin gelatin; 5 g / L bovine serum albumin; 30 g / L newborn calf serum; the remainder is deionized water. The pH of the buffer solution for reagent R1 is 8.0.

[0136] Among them, reagent R2 includes: R2 antibody: alkaline phosphatase-labeled anti-DKK1 protein antibody hM925, at a concentration of 2 μg / ml; Buffer solution: The buffer solution includes Tris (12.0 g / L), sodium azide (1.98 g / L), sodium chloride (5.9 g / L), 1.0 mL / L 1M magnesium chloride solution, 1.0 mL 0.1M zinc chloride solution, 10 g / L fish skin gelatin, 5 g / L bovine serum albumin, 30 g / L newborn calf serum, and the remainder is deionized water. The pH of the buffer solution for reagent R2 is 8.0.

[0137] The magnetic separation reagents include: Magnetic microparticles: Magnetic microparticles coated with anti-DKK1 monoclonal antibody (antibodies-online, #ABIN524880) at a concentration of 1 mg / ml; Buffer solution: comprising Tris (11.08 g / L); sodium azide (1.917 g / L); sodium chloride (5.56 g / L); 1 M magnesium chloride solution (1.0 mL / L); 0.1 M zinc chloride solution (1.0 mL / L); bovine serum albumin (4.81 g / L); premium horse serum (4.91 g / L); and deionized water as the remaining component. The buffer solution for the magnetic separation reagent has a pH of 8.0.

[0138] The calibration solution series includes: DKK1 recombinant protein antigen; Buffer solutions include: Tris (9.1 g / L); sodium chloride (12.9 g / L); tetracycline hydrochloride (0.01 g / L); neomycin sulfate (0.01 g / L); sodium azide (2.0 g / L); casein (10.0 g / L); and Tween 20 (3.1 g / L). The pH of the buffer solution series is 7.6. The calibrator series includes calibrators containing different concentrations (0 pg / mL, 50 pg / mL, 100 pg / mL, 250 pg / mL, 500 pg / mL, and 1000 pg / mL) of DKK1 recombinant protein antigen.

[0139] The chemiluminescent substrate solution is prepared by diluting a 0.2 M Tris-HCl buffer solution with a pH of 9.3 with a molar concentration of 0.2 M and containing 0.25 mg / mL of dioxane compound (APCL).

[0140] (3) Application of the magnetic microparticle chemiluminescence detection reagent constructed from antibodies hM925 and M925 in the detection of DKK1 in human blood

[0141] 50 μL of the calibrator series (DKK1 recombinant protein antigen concentrations of 0 pg / mL, 50 pg / mL, 100 pg / mL, 250 pg / mL, 500 pg / mL, and 1000 pg / mL, respectively) were added sequentially to the reaction tube along with 50 μL of reagent R1 or 50 μL of reagent R2 from Example 1, and mixed and incubated at 37°C for 15 minutes. The above reagent series were then combined with 50 μL of magnetic separation reagent and incubated at 37°C for 5 minutes. Wash three times with washing solution to remove unbound antibodies and impurities; Each of the above reagent series was then added to 150 μL of the luminescent substrate solution. After ALP catalyzed the luminescence of the substrate, the relative luminescence intensity (RLU) was measured using a chemiluminescence detector. The results are shown in Table 17 below. Table 17 Relative Luminescence Values ​​(RLU) of DKK1 Standard Samples

[0142] 5) Fit the values ​​in Table 17 to obtain the standard curve of DKK1 protein concentration-luminescence value.

[0143] 6) Add 50 μL of the test sample (blood samples of different disease types, of which negative samples are physical examination samples, and positive samples are heart failure and septic myocarditis samples, respectively) to a reaction tube with 50 μL of reagent R1 or 50 μL of reagent R2, and mix and incubate at 37°C for 15 minutes. 7) After combining the above reagents with 50 μL of magnetic separation reagent, incubate at 37°C for another 5 minutes; 8) Wash three times with washing buffer (0.1 M TrisHCl buffer, pH 8, containing 0.02% Tween 20 and 15 w / w sodium chloride) to remove unbound antibodies and impurities; 9) Add 150 μL of luminescent substrate solution to each of the above reagent series. After ALP catalyzes the luminescence of the substrate, measure the relative luminescence intensity (RLU) using a chemiluminescence detector. 10) Calculate the DKK1 protein concentration corresponding to the RLU of the sample based on the DKK1 recombinant protein concentration-luminescence value standard curve from step 1. The results are shown in Table 18, and the plotting results can be found in [reference needed]. Figure 5 .

[0144] Table 18 Comparison of antibody hM925 and M925 in detecting DKK1 concentration in human blood using magnetic microparticle chemiluminescence detection method.

[0145] As shown in Table 18, both hM925 and M925 antibodies can be clearly detected in human blood using magnetic particle chemiluminescence detection, and the specificity of hM925 antibody is significantly higher than that of M925 antibody. Therefore, it can be concluded that hM925 antibody has a significant advantage in specifically recognizing DKK1 protein in human blood using magnetic particle chemiluminescence detection.

[0146] This invention provides monoclonal antibodies M925 and / or hM925 against DKK1, which exhibit high affinity and specificity. It also provides a method for detecting DKK1 in human peripheral blood serum using a magnetic microparticle chemiluminescence method based on the aforementioned hM925 antibody. This invention addresses the current shortage of antibody raw materials for DKK1 detection in the market.

[0147] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0148] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the methods and techniques disclosed above without departing from the scope of the present invention to create equivalent embodiments. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. An anti-DKK1 antibody comprising a light chain variable region VL and a heavy chain variable region VH, wherein the light chain variable region VL comprises LCDR 1-3 having an amino acid sequence having at least 95%, at least 98%, at least 99%, or at least 100% identity as shown in SEQ ID NO: 3-5, and the heavy chain variable region VH comprises HCDR 1-3 having an amino acid sequence having at least 95%, at least 98%, at least 99%, or at least 100% identity as shown in SEQ ID NO: 6-8.

2. The anti-DKK1 antibody according to claim 1, wherein the amino acid sequence of VL is as shown in SEQ ID NO: 1 or has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with it, and the amino acid sequence of VH is as shown in SEQ ID NO: 2 or has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with it.

3. The anti-DKK1 antibody according to claim 2, wherein the amino acid sequence of the VL comprises LCDR1, LCDR2, and LCDR3 sequences having one or more conserved amino acid substitutions, deletions, or insertions thereof as shown in SEQ ID NO:1 or any combination thereof; and the amino acid sequence of the VH comprises HCDR1, HCDR2, and HCDR3 sequences having one or more conserved amino acid substitutions, deletions, or insertions thereof as shown in SEQ ID NO:2 or any combination thereof.

4. The anti-DKK1 antibody according to any one of claims 1-3, wherein the antibody is a murine monoclonal antibody or a humanized chimeric antibody.

5. The anti-DKK1 antibody according to claim 4, wherein the antibody light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 13, and the antibody heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:

14.

6. The anti-DKK1 antibody according to claim 5, further comprising a light chain and a heavy chain, the light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 15, and the heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:

16.

7. A nucleic acid comprising encoding an anti-DKK1 antibody according to any one of claims 1-6.

8. A detection reagent comprising an anti-DKK1 antibody according to any one of claims 1-6.

9. The detection reagent according to claim 8, wherein the anti-DKK1 antibody, after being labeled with alkaline phosphatase (ALP), is used as a component of the magnetic microparticle chemiluminescent detection reagent that specifically recognizes DKK1.

10. Claim 1 The use of any one of the anti-DKK1 antibodies in claim 6, or any one of the detection reagents in claims 8-9, in the preparation of reagents or products for detecting the DKK1 content in a sample, reagents or products for diagnosing DKK1-related diseases, or medicaments for the prevention and / or treatment of DKK1-related diseases; preferably, the DKK1-related diseases are selected from: osteoporosis, multiple myeloma bone disease (MBD), and osteoarthritis.

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