Antibodies recognizing carboxylation sites of matrix Gla protein and their applications

Abstract

The present invention discloses an antibody for recognizing the carboxylation site of matrix Gla protein and its application, wherein the antibody comprises at least one of the following CDR sequences: CDR sequences of heavy chain variable region: SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3; CDR sequences of light chain variable region: SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6. In addition, the present invention also discloses an enzyme-immunoassay kit prepared by using the antibody, which has high sensitivity.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and specifically to an antibody for recognizing the carboxylation site of a matrix Gla protein and its application, and more specifically to an antibody or antigen binding fragment, a fusion protein, a nucleic acid molecule, an expression vector, a recombinant cell, a hybridoma cell, a conjugate, a composition, a reagent or a kit or their use, a method for preparing an antibody or antigen fragment, and a method for detecting a carboxylated matrix Gla protein or a cell expressing a carboxylated matrix Gla protein. Background Art

[0002] Matrix Gla protein (MGP) is a vitamin K-dependent protein composed of five γ-carboxylated glutamic acid residues, with a molecular weight of 10.6 kDa. It is present in organs such as bones, cartilage, heart, kidneys and lungs, and is mainly distributed in cartilage, bone marrow and arterial walls. MGP is synthesized by chondrocytes in cartilage and smooth muscle cells in arterial walls, and has the function of inhibiting abnormal deposition of calcium ions in cartilage, vascular walls and other soft tissues.

[0003] MGP includes the inactivated form, i.e., uncarboxylated MGP (ucMGP), and the biologically active carboxylated MGP (cMGP). The precursor of MGP synthesized after transcription contains five glutamic acid residues and needs to be carboxylated with γ-glutamyl under the action of vitamin K to become biologically active (ucMGP-cMGP). Therefore, vitamin K is an important factor affecting its activity.

[0004] MGP is the most powerful natural calcification inhibitor in the human body. When blood vessels calcify, vascular smooth muscle continuously produces MGP to inhibit the progression of calcification, but only cMGP has an inhibitory effect. First, it has a high binding affinity for newly formed hydroxyapatite crystals, thereby eliminating their accumulation in the arterial wall; second, after binding to calcium and phosphate crystals, it upregulates arterial macrophages to promote the phagocytosis and apoptosis of MGP-hydroxyapatite complexes; finally, MGP directly inhibits the binding of bone morphogenetic protein-2 (BMP-2) to its receptor, and then downregulates its function, which has an important inhibitory effect on vascular calcification, renal calcification, cartilage calcification, etc. Lead and calcium are both divalent cations with similar absorption and metabolic pathways, so MGP is also related to lead metabolism. If the activity of MGP changes, or its activation process is obstructed, MGP cannot be carboxylated normally, and it cannot normally inhibit calcium and phosphate deposition, causing damage to tissues and organs such as blood vessels, bones, heart, and kidneys.

[0005] In healthy blood vessels and calcified blood vessels, the existence form of MGP is different. ucMGP exists in calcified blood vessels, while cMGP exists in intact blood vessel walls. The content of MGP in normal human blood vessel walls is extremely low, and is distributed in the smooth muscle cells, the elastic layer of the middle layer, and the extracellular matrix of the outer membrane. Its distribution is uneven, most of which is in the side wall of the cavity, and the content is lower towards the center of the middle layer. Since matrix Gla protein exists in two forms (i.e., carboxylated matrix Gla protein and uncarboxylated matrix Gla protein), it is difficult to screen antibodies with good specificity that can distinguish between the two forms, which makes it difficult to determine the positive serum of clinical carboxylated matrix Gla protein. Most of the diagnostic reagents for carboxylated matrix Gla protein in the industry rely on imports and are expensive.

[0006] Currently, there is an urgent need to develop an antibody with strong specificity and high sensitivity for recognizing carboxylated matrix Gla protein. Summary of the invention

[0007] The present invention aims to solve at least one of the technical problems existing in the prior art to a certain extent. To this end, the present invention provides an antibody or antigen binding fragment that can recognize carboxylated matrix Gla protein.

[0008] In the first aspect of the present invention, the present invention proposes an antibody or antigen-binding fragment. According to an embodiment of the present invention, the antibody or antigen-binding fragment comprises a CDR sequence selected from at least one of the following: a CDR sequence of a heavy chain variable region: SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3; a CDR sequence of a light chain variable region: SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6. The antibody or antigen-binding fragment according to an embodiment of the present invention has a carboxylated matrix Gla protein binding affinity, especially can specifically bind to carboxylated matrix Gla protein, and can be used to detect carboxylated matrix Gla protein and detect cells expressing carboxylated matrix Gla protein.

[0009] According to some embodiments of the present invention, the antibody or antigen-binding fragment comprises HCDR1-3 and LCDR1-3; wherein the HCDR1 has an amino acid sequence as shown in SEQ ID NO:1; the HCDR2 has an amino acid sequence as shown in SEQ ID NO:2; the HCDR3 has an amino acid sequence as shown in SEQ ID NO:3; the LCDR1 has an amino acid sequence as shown in SEQ ID NO:4; the LCDR2 has an amino acid sequence as shown in SEQ ID NO:5; and the LCDR3 has an amino acid sequence as shown in SEQ ID NO:6.

[0010] According to some embodiments of the invention, the antibody or antigen-binding fragment comprises a heavy chain framework region and / or a light chain framework region.

[0011] According to some embodiments of the present invention, at least a portion of the heavy chain framework region and / or light chain framework region is derived from at least one of a mouse antibody, a primate antibody, a bovine antibody, a horse antibody, a dairy cow antibody, a porcine antibody, a sheep antibody, a goat antibody, a dog antibody, a cat antibody, a rabbit antibody, a camel antibody, a donkey antibody, a deer antibody, a mink antibody, a chicken antibody, a duck antibody, a goose antibody, a turkey antibody, a fighting cock antibody, or a mutant thereof.

[0012] According to some embodiments of the present invention, at least a portion of the heavy chain framework region and / or the light chain framework region is derived from at least one of a murine antibody or a mutant thereof and a human antibody or a mutant thereof.

[0013] According to some embodiments of the present invention, the antibody or antigen-binding fragment comprises: a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:7; and a light chain variable region having an amino acid sequence as shown in SEQ ID NO:8.

[0014] According to some embodiments of the present invention, it further comprises a heavy chain constant region and / or a light chain constant region.

[0015] According to some embodiments of the present invention, at least a portion of at least one of the heavy chain constant region and the light chain constant region is from at least one of a mouse antibody, a primate antibody, a bovine antibody, a horse antibody, a dairy cow antibody, a porcine antibody, a sheep antibody, a goat antibody, a dog antibody, a cat antibody, a rabbit antibody, a camel antibody, a donkey antibody, a deer antibody, a mink antibody, a chicken antibody, a duck antibody, a goose antibody, a turkey antibody, a fighting cock antibody or a mutant thereof.

[0016] According to some embodiments of the invention, the heavy chain constant region comprises a heavy chain constant region selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD.

[0017] According to some embodiments of the invention, the light chain constant region comprises a light chain constant region selected from a kappa-type or a lambda-type.

[0018] According to some embodiments of the present invention, the light chain constant region and the heavy chain constant region are both derived from at least one of a murine antibody or a mutant thereof and a human antibody or a mutant thereof.

[0019] According to some embodiments of the present invention, the N-terminus of the heavy chain constant region is connected to the C-terminus of the heavy chain variable region; and / or the N-terminus of the light chain constant region is connected to the C-terminus of the light chain variable region.

[0020] According to some embodiments of the present invention, the heavy chain constant region has the amino acid sequence shown in SEQ ID NO:9; and / or the light chain constant region has the amino acid sequence shown in SEQ ID NO:10.

[0021] According to some embodiments of the present invention, the antibody includes at least one of a polyclonal antibody, a full-length monoclonal antibody, a F(ab')2 antibody, a Fab' antibody, a Fab antibody, a F(ab)2 antibody, a Fv antibody, a single-chain antibody and a minimum recognition unit.

[0022] According to some embodiments of the invention, the antigen binding fragment comprises at least one of a F(ab')2 fragment, a Fab' fragment, a Fab fragment, a F(ab)2 fragment, a Fv fragment, a scFv fragment, a scFv-Fc fusion protein, a scFv-Fv fusion protein and a minimum recognition unit.

[0023] According to some embodiments of the present invention, the antibody or antigen-binding fragment comprises: a heavy chain having an amino acid sequence as shown in SEQ ID NO:11; and a light chain having an amino acid sequence as shown in SEQ ID NO:12.

[0024] In the second aspect of the present invention, the present invention proposes a fusion protein. According to an embodiment of the present invention, the fusion protein comprises the antibody or antigen binding fragment described in the first aspect; and a biologically active protein or a fragment thereof. The fusion protein of the present invention has a carboxylated matrix GLA protein binding affinity and can be used to detect carboxylated matrix GLA protein, detect cells expressing carboxylated matrix GLA protein, etc.

[0025] According to some embodiments of the present invention, the biologically active protein or fragment thereof comprises at least one selected from a protein tag, albumin or a fragment thereof, and an Fc fragment.

[0026] In the third aspect of the present invention, the present invention provides a nucleic acid molecule. According to an embodiment of the present invention, the nucleic acid molecule encodes the antibody or antigen-binding fragment described in the first aspect, and the fusion protein described in the second aspect. The nucleic acid molecule of the present invention can encode the antibody or antigen-binding fragment of the first aspect, and the fusion protein described in the second aspect.

[0027] According to some embodiments of the invention, the nucleic acid molecule is DNA.

[0028] In a fourth aspect of the present invention, the present invention provides an expression vector. According to an embodiment of the present invention, the expression vector carries the nucleic acid molecule described in the third aspect. Thus, the expression vector of the present invention can effectively achieve the expression of the antibody or antigen-binding fragment of the first aspect and the fusion protein of the second aspect, thereby achieving the in vitro acquisition of a large amount of the antibody or antigen-binding fragment or the fusion protein.

[0029] According to some embodiments of the present invention, the expression vector comprises a vector selected from a eukaryotic expression vector or a prokaryotic expression vector.

[0030] In the fifth aspect of the present invention, the present invention provides a recombinant cell. According to an embodiment of the present invention, the recombinant cell includes carrying the nucleic acid molecule described in the third aspect or the expression vector described in the fourth aspect; or expressing the antibody or antigen-binding fragment described in the first aspect, or the fusion protein described in the second aspect. The recombinant cell can effectively express the aforementioned antibody or antigen-binding fragment, or fusion protein in the cell under suitable conditions.

[0031] According to some embodiments of the present invention, the recombinant cell is obtained by introducing the expression vector described in the fourth aspect into a host cell.

[0032] In a sixth aspect of the present invention, the present invention provides a method for preparing an antibody or antigenic fragment. According to an embodiment of the present invention, the method comprises culturing the recombinant cell described in the fifth aspect. Thus, the method of the present invention can be used to produce the aforementioned antibody or antigenic fragment.

[0033] In the seventh aspect of the present invention, the present invention provides a conjugate. According to an embodiment of the present invention, the conjugate comprises the antibody or antigen-binding fragment described in the first aspect, the fusion protein described in the second aspect; and a coupling part, wherein the coupling part is connected to the antibody or antigen-binding fragment, or the fusion protein. The conjugate of the present invention has a carboxylated matrix Gla protein binding affinity and can be used to detect carboxylated matrix Gla protein and detect cells expressing carboxylated matrix Gla protein.

[0034] According to some embodiments of the present invention, the coupling moiety comprises at least one selected from a carrier, a drug, a toxin, a cytokine, a protein tag, a modifier and a chemotherapeutic agent.

[0035] According to some embodiments of the invention, the carrier comprises a fluorescent dye.

[0036] According to some embodiments of the present invention, the fluorescent dye includes any one or more of FITC, PE, Cy5, PI, 7 AAD, APC, AlexaFluor, eFluor, PE Cy7, APC Cy7, PerCP, PerCPcy5.5, PB, BV series, QDot series and BUV series dyes.

[0037] In the eighth aspect of the present invention, the present invention proposes a composition. According to an embodiment of the present invention, the composition comprises the antibody or antigen binding fragment described in the first aspect, the fusion protein described in the second aspect, the nucleic acid molecule described in the third aspect, the expression vector described in the fourth aspect, the recombinant cell described in the fifth aspect, the antibody or antigen binding fragment prepared according to the method described in the sixth aspect, or the conjugate described in the seventh aspect. The composition of the present invention has a carboxylated matrix Gla protein binding affinity, especially can specifically bind to the carboxylated matrix Gla protein, and can be used to detect the carboxylated matrix Gla protein, or detect cells expressing the carboxylated matrix Gla protein.

[0038] In the ninth aspect of the present invention, the present invention proposes a reagent or a kit. According to an embodiment of the present invention, the reagent or the kit comprises the antibody or antigen binding fragment described in the first aspect, the fusion protein described in the second aspect, the nucleic acid molecule described in the third aspect, the expression vector described in the fourth aspect, the recombinant cell described in the fifth aspect, the antibody or antigen binding fragment prepared according to the method described in the sixth aspect, or the conjugate described in the seventh aspect. The kit of the present invention has a carboxylated matrix Gla protein binding affinity, and can effectively detect carboxylated matrix Gla protein and detect cells expressing carboxylated matrix Gla protein.

[0039] In the tenth aspect of the present invention, the present invention proposes a use of the antibody or antigen-binding fragment described in the first aspect, the fusion protein described in the second aspect, the nucleic acid molecule described in the third aspect, the expression vector described in the fourth aspect, the recombinant cell described in the fifth aspect, the antibody or antigen-binding fragment prepared according to the method described in the sixth aspect, the conjugate described in the seventh aspect, or the composition described in the eighth aspect in a kit, wherein the kit is used to detect carboxylated matrix Gla protein and cells expressing carboxylated matrix Gla protein.

[0040] In the eleventh aspect of the present invention, the present invention proposes a method for detecting carboxylated matrix Gla protein or detecting cells expressing carboxylated matrix Gla protein. According to an embodiment of the present invention, the method comprises: using the antibody or antigen binding fragment described in the first aspect, the fusion protein described in the second aspect, the antibody or antigen binding fragment prepared according to the method described in the sixth aspect, the conjugate described in the seventh aspect, the composition described in the eighth aspect, or the reagent or kit described in the ninth aspect to contact with the sample to be detected to form an immune complex. As can be seen from the above, the aforementioned antibody or antigen binding fragment, fusion protein, conjugate, reagent or kit all have carboxylated matrix Gla protein binding affinity, thus, the above-mentioned antibody or antigen binding fragment, fusion protein, conjugate, reagent or kit can effectively detect carboxylated matrix Gla protein or detect cells expressing carboxylated matrix Gla protein.

[0041] According to some embodiments of the present invention, based on the signal of the immune complex, it is determined whether the sample to be tested contains carboxylated matrix Gla protein or whether it contains cells expressing carboxylated matrix Gla protein, or the content of the carboxylated matrix Gla protein or cells expressing carboxylated matrix Gla protein in the sample to be tested is determined.

[0042] According to some embodiments of the present invention, the signal of the immune complex is detected by at least one of ELISA, WB and IHC.

[0043] According to some embodiments of the invention, the signal comprises a fluorescent signal.

[0044] The present invention provides a monoclonal antibody that recognizes the carboxylation site of the matrix Gla protein, which has the characteristics of high affinity and specificity, and also provides an in vitro diagnostic test kit based on the above-mentioned antibody. Based on this, the antibody or antigen-binding fragment, fusion protein, nucleic acid molecule, expression vector, recombinant cell, hybridoma cell, conjugate, composition, reagent or test kit of the present invention, etc., play a role in the auxiliary diagnosis of related diseases, and the enzyme-immune test kit prepared based on the antibody (such as M018 antibody) greatly improves the accuracy and convenience of diagnosis. Therefore, the present invention can solve the problem of relative shortage of antibody raw materials and test kits for carboxylation matrix Gla protein detection projects in the current market.

[0045] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0047] Figure 1 This is a gel map of subtype identification of the antibody expressed by the hybridoma cell line M018 and cloning of the antibody variable region provided in Example 7 of the present invention. The lane sequence is shown in Table 5. DETAILED DESCRIPTION

[0048] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0049] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, the meaning of "plurality" is two or more.

[0050] Detailed description of the invention

[0051] Definitions and general terms

[0052] In order to make the present invention more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present invention belongs.

[0053] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention but not excluding other contents.

[0054] As used herein, the terms "optionally", "optional" or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0055] Herein, the term "fragment" refers to a target protein or polypeptide, and a target protein or polypeptide having an N-terminal (N-terminus) or C-terminal (C-terminus) truncation, and / or internal deletion.

[0056] In order to make the present invention more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs. The abbreviations for amino acid residues are standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

[0057] In this article, the term "antibody" is used in the broadest sense, which can include full-length monoclonal antibodies, multispecific antibodies, and chimeric antibodies, and the specific structure is not limited as long as they show the desired biological activity. It usually includes a light chain with a lighter molecular weight and a heavy chain with a heavier molecular weight, and the heavy chain (H chain) and the light chain (L chain) are connected by a disulfide bond to form an antibody molecule. Among them, the amino acid sequence of the amino terminal (N terminal) of the peptide chain varies greatly, which is called the variable region (V region); the carboxyl terminal (C terminal) is relatively stable and varies little, which is called the constant region (C region). The V regions of the L chain and the H chain are called VL and VH, respectively.

[0058] In this article, the heavy chain complementary determining region (heavy chain variable region CDR) is represented by "HCDRs" or "HCDR", which includes HCDR1 (also known as CDR-H1), HCDR2 (also known as CDR-H2) and HCDR3 (also known as CDR-H3); the light chain complementary determining region (light chain variable region CDR) is represented by "LCDRs" or "LCDR", which includes LCDR1 (also known as CDR-L1), LCDR2 (also known as CDR-L2) and LCDR3 (also known as CDR-L3). Commonly used CDR definition schemes in the art include: Kabat definition, Chothia definition, IMGT definition, Contact definition and AbM definition.

[0059] In this article, the term "antibody variable region" refers to a domain in the heavy and light chains of antibodies. In nature, antibody variable regions are encoded by V, D (only heavy chain is applicable), and J fragments in immunoglobulin (heavy and light chain) genes through gene recombination splicing. The amino acid sequence of the variable region has a high degree of diversity between different antibodies (the amino acid sequences in other regions of the antibody are relatively highly identical), and is responsible for identifying and combining with specific antigenic determinants. In the antibody variable region, the framework region and CDR region (comlementarity determining region) can be further subdivided. A typical antibody variable region has 3 framework regions and 3 CDR regions (they are interspersed with each other). The framework region mainly plays the role of protein domain framework formation, while the CDR region mainly plays the role of antigen-antibody specific recognition and combination.

[0060] As used herein, the term "antigen-binding fragment" or "antibody fragment" is a fragment comprising part or all of an antibody, which lacks at least some of the amino acids present in the full-length chain but is still capable of specifically binding to an antigen, for example, the fragment may comprise part or all of an antibody CDR. Such fragments are biologically active because they bind to an antigen and can compete with other antigen-binding molecules (including intact antibodies) for binding to a given epitope. Such fragments are selected from Fab, F(ab)2, Fab', F(ab')2, Fv, scFv or single domain antibodies. Such fragments may be produced by recombinant nucleic acid technology, or may be produced by enzymatic or chemical cleavage of antigen-binding molecules (including intact antibodies).

[0061] It should be noted that, the antibodies or antigen-binding fragments, CDRs, light chain variable regions or heavy chain variable regions, etc. described in the present invention, under the premise of not substantially affecting the antibody activity (retaining at least 80%, 90%, or 95% activity), those skilled in the art can replace, 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 sequence of the present invention to obtain variants of the sequence of the antibody or its functional fragment. For example, amino acids with conservative modifications are replaced in the variable region. The variant sequence of the present invention may have at least 80% identity (or homology) with the reference sequence, and the sequence identity of the present invention can be measured using sequence analysis software. They are all deemed to be included in the scope of protection of the present invention.

[0062] In this document, the amino acid sequences described in the present invention are all shown from N-terminus to C-terminus.

[0063] The antibodies or antigen-binding fragments described in the present invention are usually prepared by biosynthetic methods. According to the nucleotide sequence described in the present invention, those skilled in the art can easily prepare the encoding nucleic acid of the present invention by various known methods. These methods are, for example, but not limited to: PCR, DNA artificial synthesis, etc., and specific methods can be found in J. Sambrook, "Molecular Cloning Laboratory Guide". As an embodiment of the present invention, the encoding nucleic acid sequence of the present invention can be constructed by synthesizing nucleotide sequences in segments and then performing overlap extension PCR. Among them, the antibodies in the present invention are numbered and defined using the IMGT numbering system. However, those of ordinary skill in the art are fully capable of converting the sequence IMGT numbering of the sequence table into "HCDRs" and / or "LCDRs" under other numbering systems (including but not limited to AbM, Kabat, Chothia), and the CDRs determined based on the heavy chain variable region and light chain variable region disclosed in this application using other rules disclosed in the art also belong to the scope of protection of the present disclosure.

[0064] As used herein, the "IMGT definition" refers to the new standardized numbering system introduced by Lefranc et al. for all protein sequences of the immunoglobulin superfamily, see Lefranc, et al., The Immunologist, 7, 132-136 (1999). The "Kabat definition" refers to the definition system described by Kabat et al., U. S. Pat. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). The "Chothia definition" refers to Chothia et al., J Mol Biol 196:901-917 (1987).

[0065] As used herein, the term "conservatively modified amino acids" refers to amino acid modifications that do not significantly affect or alter the binding properties of an antibody comprising the amino acid sequence, including amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies of the invention by standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are substitutions in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been identified in the art. These families include amino acids with basic side chains (such as lysine, arginine, histidine), amino acids with acidic side chains (such as aspartic acid, glutamic acid), amino acids with uncharged polar side chains (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with non-polar side chains (such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with beta-branched side chains (such as threonine, valine, isoleucine), and amino acids with aromatic side chains (such as tyrosine, phenylalanine, tryptophan, histidine).

[0066] As used herein, the term "identity" is used to describe an amino acid sequence or a nucleic acid sequence relative to a reference sequence, and the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences is determined by conventional methods, for example, see Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN program (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research There are many algorithms 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. 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. 85:24 ... .70:173-187 (1997); and BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J. Mol. Biol. 215:403-410). Computer programs that utilize these algorithms are also available, and include, but are not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al., Meth. Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLAST Altschul et al., supra, FASTA, and TFASTA, available in the Genetics Computing Group (GCG) package, Version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0067] As used herein, the term "at least 80% identity" refers to at least 80%, and may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity to each reference sequence.

[0068] As used herein, the term "at least 90% identity" refers to at least 90%, and may be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity to each reference sequence.

[0069] In this article, the term "fusion protein" refers to a novel protein formed by the fusion of at least two proteins or polypeptides, and the above fusion operation can usually be achieved through genetic engineering and other technologies, for example, the expression product of two genes recombined by DNA recombination technology. In this article, it refers to the antibody or antigen-binding fragment of the carboxylated matrix GLA protein and the biologically active protein or fragment thereof in a fusion state.

[0070] In this article, the term "expression vector" generally refers to a nucleic acid molecule that can be inserted into a suitable host and replicates itself, the nucleic acid molecule comprises a nucleotide sequence that can express a target protein, and the nucleic acid molecule can be transferred into a host cell and / or between host cells. The expression vector may include a vector that is mainly used to insert DNA or RNA into a cell, a vector that is mainly used to replicate DNA or RNA, and a vector that is mainly used for the expression of transcription and / or translation of DNA or RNA. The expression vector also includes vectors with a variety of the above functions. The expression vector can be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the expression vector can produce a desired expression product by culturing a suitable host cell containing the expression vector.

[0071] In this article, the term "recombinant cell" generally refers to the use of genetic engineering technology or cell fusion technology to modify or reorganize the genetic material of the recipient cell (or host cell) to obtain a cell with a unique trait of stable inheritance. Among them, the term "host cell" refers to a prokaryotic cell or eukaryotic cell that can be introduced into a recombinant expression vector. The terms "transformed" or "transfected" used herein refer to the introduction of nucleic acids (such as expression vectors) into cells by various techniques known in the art. Suitable host cells can be transformed or transfected with the DNA sequence of the present invention and can be used for the expression and / or secretion of target proteins. Examples of suitable host cells that can be used in the present invention include immortalized hybridoma cells, NS / 0 myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, Cap cells (cells derived from human amniotic fluid) and CoS cells.

[0072] As used herein, the term "composition" refers to any composition comprising the active ingredient of the present application, including but not limited to pharmaceutical compositions. The term "pharmaceutical composition" generally refers to a unit dosage form and can be prepared by any of the methods well known in the pharmaceutical field. All methods include the step of combining the active ingredient with a carrier that constitutes one or more accessory ingredients. Typically, the composition is prepared by uniformly and fully combining the active antibody or antigen-binding fragment with a liquid carrier, a finely divided solid carrier, or both.

[0073] In this article, the term "pharmaceutically acceptable excipient" may include any solvent, solid excipient, diluent or other liquid excipient, etc., suitable for a specific target dosage form. Except for any conventional excipients that are incompatible with the antibody or antigen-binding fragment of the present invention, such as any adverse biological effects produced or interactions with any other components of the pharmaceutically acceptable composition in a harmful manner, their use is also within the scope of the present invention.

[0074] Detailed description of the antibody for recognizing the carboxylation site of matrix Gla protein and its application

[0075] The present invention provides an antibody or antigen binding fragment, a fusion protein, a nucleic acid molecule, an expression vector, a recombinant cell, a hybridoma cell, a conjugate, a composition, a reagent or a kit or their uses, a method for preparing an antibody or antigen fragment, and a method for detecting carboxylated matrix Gla protein or detecting a cell expressing carboxylated matrix Gla protein, which will be described in detail below.

[0076] Antibodies or antigen-binding fragments

[0077] In the first aspect of the present invention, the present invention proposes an antibody or antigen-binding fragment. According to an embodiment of the present invention, the antibody or antigen-binding fragment comprises a CDR sequence selected from at least one of the following: a CDR sequence of a heavy chain variable region: SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3; a CDR sequence of a light chain variable region: SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6. The antibody or antigen-binding fragment according to an embodiment of the present invention has a carboxylated matrix Gla protein binding affinity, especially can specifically bind to carboxylated matrix Gla protein, and can be used to detect carboxylated matrix Gla protein and detect cells expressing carboxylated matrix Gla protein.

[0078] According to some embodiments of the present invention, the antibody or antigen-binding fragment comprises HCDR1-3 and LCDR1-3; wherein the HCDR1 has an amino acid sequence as shown in SEQ ID NO:1; the HCDR2 has an amino acid sequence as shown in SEQ ID NO:2; the HCDR3 has an amino acid sequence as shown in SEQ ID NO:3; the LCDR1 has an amino acid sequence as shown in SEQ ID NO:4; the LCDR2 has an amino acid sequence as shown in SEQ ID NO:5; and the LCDR3 has an amino acid sequence as shown in SEQ ID NO:6.

[0079] In an optional embodiment of the present invention, "the amino acid sequence as shown in SEQ ID NO:1" refers to an amino acid sequence that is completely identical to the amino acid sequence shown in SEQ ID NO:1, and an amino acid sequence that has one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO:1. "Conservative amino acid substitution" refers to the replacement of an amino acid with another amino acid residue that is biologically, chemically or structurally similar. Biologically similar means that the replacement does not destroy the biological activity of antibodies that recognize the carboxylation site of the matrix Gla protein or the carboxylated matrix Gla protein antigen. Structurally similar means that the amino acids have side chains of similar length, such as alanine, glycine or serine, or have side chains of similar size. Chemical similarity means that the amino acids have the same charge or are hydrophilic or hydrophobic. For example, hydrophobic residues isoleucine, valine, leucine or methionine are substituted for each other. Or polar amino acids are used, such as arginine replacing lysine, glutamic acid replacing aspartic acid, glutamine replacing asparagine, serine replacing threonine, etc. The same as “the amino acid sequence shown in SEQ ID NO:2”, “the amino acid sequence shown in SEQ ID NO:3”, “the amino acid sequence shown in SEQ ID NO:4”, “the amino acid sequence shown in SEQ ID NO:5”, “the amino acid sequence shown in SEQ ID NO:6”, “the amino acid sequence shown in SEQ ID NO:7”, “the amino acid sequence shown in SEQ ID NO:8”, “the amino acid sequence shown in SEQ ID NO:9”, “the amino acid sequence shown in SEQ ID NO:10”, “the amino acid sequence shown in SEQ ID NO:11”, or “the amino acid sequence shown in SEQ ID NO:12”.

[0080] According to an embodiment of the present invention, the HCDR1 has an amino acid sequence as shown in SEQ ID NO: 1 or an amino acid sequence having 1 to 2 amino acid differences therefrom.

[0081] According to an embodiment of the present invention, the HCDR2 has an amino acid sequence as shown in SEQ ID NO: 2 or an amino acid sequence having 1 to 2 amino acid differences therefrom.

[0082] According to an embodiment of the present invention, the HCDR3 has an amino acid sequence as shown in SEQ ID NO: 3 or an amino acid sequence having 1 to 2 amino acid differences therefrom.

[0083] According to an embodiment of the present invention, the LCDR1 has an amino acid sequence as shown in SEQ ID NO: 4 or an amino acid sequence having 1 to 2 amino acid differences therefrom.

[0084] According to an embodiment of the present invention, the LCDR2 has an amino acid sequence as shown in SEQ ID NO: 5 or an amino acid sequence having 1 to 2 amino acid differences therefrom.

[0085] According to an embodiment of the present invention, the LCDR3 has an amino acid sequence as shown in SEQ ID NO: 6 or an amino acid sequence having 1 to 2 amino acid differences therefrom.

[0086] As used herein, the term "or an amino acid sequence having 1 to 2 amino acid differences therefrom" refers to an amino acid sequence having 1 or 2 amino acid differences compared to the aforementioned amino acid sequence, and the difference does not significantly affect or change the binding properties of the antibody comprising the amino acid sequence, and the modification includes amino acid substitution, addition and deletion.

[0087] According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof specifically recognizes carboxylated matrix GLA protein, and in particular specifically recognizes the protein shown in SEQ ID NO:14.

[0088] According to some embodiments of the invention, the antibody or antigen-binding fragment comprises a heavy chain framework region and / or a light chain framework region.

[0089] According to some embodiments of the present invention, at least a portion of the heavy chain framework region and / or light chain framework region is derived from at least one of a mouse antibody, a primate antibody, a bovine antibody, a horse antibody, a dairy cow antibody, a porcine antibody, a sheep antibody, a goat antibody, a dog antibody, a cat antibody, a rabbit antibody, a camel antibody, a donkey antibody, a deer antibody, a mink antibody, a chicken antibody, a duck antibody, a goose antibody, a turkey antibody, a fighting cock antibody, or a mutant thereof.

[0090] According to some embodiments of the present invention, at least a portion of the heavy chain framework region and / or the light chain framework region is derived from at least one of a murine antibody or a mutant thereof and a human antibody or a mutant thereof.

[0091] According to some embodiments of the present invention, the antibody or antigen-binding fragment comprises: a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:7; and a light chain variable region having an amino acid sequence as shown in SEQ ID NO:8.

[0092] According to some embodiments of the present invention, it further comprises a heavy chain constant region and / or a light chain constant region.

[0093] According to some embodiments of the present invention, at least a portion of at least one of the heavy chain constant region and the light chain constant region is from at least one of a mouse antibody, a primate antibody, a bovine antibody, a horse antibody, a dairy cow antibody, a porcine antibody, a sheep antibody, a goat antibody, a dog antibody, a cat antibody, a rabbit antibody, a camel antibody, a donkey antibody, a deer antibody, a mink antibody, a chicken antibody, a duck antibody, a goose antibody, a turkey antibody, a fighting cock antibody or a mutant thereof.

[0094] According to some embodiments of the invention, the heavy chain constant region comprises a heavy chain constant region selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD.

[0095] According to some embodiments of the invention, the light chain constant region comprises a light chain constant region selected from a kappa-type or a lambda-type.

[0096] According to some embodiments of the present invention, the light chain constant region and the heavy chain constant region are both derived from at least one of a murine antibody or a mutant thereof and a human antibody or a mutant thereof.

[0097] According to some embodiments of the present invention, the N-terminus of the heavy chain constant region is connected to the C-terminus of the heavy chain variable region; and / or the N-terminus of the light chain constant region is connected to the C-terminus of the light chain variable region.

[0098] According to some embodiments of the present invention, the heavy chain constant region has the amino acid sequence shown in SEQ ID NO:9; and / or the light chain constant region has the amino acid sequence shown in SEQ ID NO:10.

[0099] According to some embodiments of the present invention, the antibody includes at least one of a polyclonal antibody, a full-length monoclonal antibody, a F(ab')2 antibody, a Fab' antibody, a Fab antibody, a F(ab)2 antibody, a Fv antibody, a single-chain antibody and a minimum recognition unit.

[0100] According to some embodiments of the invention, the antigen binding fragment comprises at least one of a F(ab')2 fragment, a Fab' fragment, a Fab fragment, a F(ab)2 fragment, a Fv fragment, a scFv fragment, a scFv-Fc fusion protein, a scFv-Fv fusion protein and a minimum recognition unit.

[0101] As used herein, the terms "full-length antibody", "full-length monoclonal antibody" or "full-length monoclonal antibody" are composed of at least two identical light chains and at least two identical heavy chains connected by interchain disulfide bonds, such as immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD) or immunoglobulin E (IgE).

[0102] In this article, the terms "polyantibody" and "multispecific antibody" are synonymous, both referring to antibodies that can recognize multiple antigenic epitopes, such as antibodies that can recognize two antigenic epitopes (bispecific antibodies, referred to as bispecific antibodies), antibodies that recognize three antigenic epitopes, or antibodies that recognize four antigenic epitopes. They are understood in a broad sense, and the specific structure is not limited, as long as multiple antigenic epitopes can be recognized. In the present invention, at least one of the multiple antigenic epitopes is derived from the carboxylated matrix GLA protein.

[0103] In this article, the terms "single domain antibody", "nanoantibody" and "VHH antibody" are used interchangeably, which were originally described as antigen-binding immunoglobulin (variable) domains of "heavy chain antibodies" (i.e., "antibodies lacking light chains") (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: "Naturally occurring antibodies devoid of light chains"; Nature 363, 446-448 (1993)), comprising a heavy chain variable region (VH) and conventional CH2 and CH3 regions, which specifically bind to antigen proteins (e.g., carboxylated matrix GLA protein) through the heavy chain variable region.

[0104] As used herein, the term "Fab antibody" or "Fab fragment" generally refers to an antibody or fragment containing only the Fab molecule, which is composed of the VH and CH1 of the heavy chain and a complete light chain, with the light chain and the heavy chain connected by a disulfide bond.

[0105] As used herein, the term "F(ab')2 antibody" or "F(ab')2 fragment" has two antigen-binding F(ab') portions linked together by a disulfide bond.

[0106] As used herein, the term "Fv antibody" or "Fv fragment" generally refers to an antibody or fragment consisting only of a light chain variable region (VL) and a heavy chain variable region (VH) connected by non-covalent bonds, and is the smallest functional fragment of an antibody that retains a complete antigen binding site.

[0107] As used herein, the terms "single-chain antibody" and "scFv fragment" refer to antibodies or fragments formed by connecting the heavy chain variable region and the light chain variable region of an antibody via a short peptide.

[0108] In this article, the terms "minimum recognition unit" and "MRU" both refer to antibodies or fragments consisting of only one CDR, and their molecular weight is very small, accounting for only about 1% of the complete antibody.

[0109] According to some embodiments of the present invention, the antibody or antigen-binding fragment comprises: a heavy chain having an amino acid sequence as shown in SEQ ID NO:11; and a light chain having an amino acid sequence as shown in SEQ ID NO:12.

[0110] Fusion Protein

[0111] In the second aspect of the present invention, the present invention provides a fusion protein. According to an embodiment of the present invention, the fusion protein comprises the antibody or antigen-binding fragment described in the first aspect; and a biologically active protein or a fragment thereof. The fusion protein of the present invention has a carboxylated matrix GLA protein binding affinity and can be used to detect carboxylated matrix GLA protein and detect cells expressing carboxylated matrix GLA protein.

[0112] According to some embodiments of the present invention, the biologically active protein or fragment thereof comprises at least one selected from a protein tag, albumin or a fragment thereof, and an Fc fragment.

[0113] In this article, "protein tag" generally refers to a polypeptide or protein fused with a target protein (antibody or antigen-binding fragment), which can be used for the expression, detection, disappearance or purification of the target protein, etc., including but not limited to His tag (also known as His-Tag, sequence HHHHHH), Flag tag (also known as Flag-Tag, sequence DYKDDDDK), GST tag (also known as GST-Tag, glutathione sulfhydryl transferase tag), MBP tag (also known as MBP-Tag, maltose binding protein tag), SUMO tag and C-Myc tag, etc.

[0114] Herein, "Fc fragment" generally refers to the Fc region from IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM, including CH2, CH3 region and optionally hinge region. Preferably, IgG, IgA1, IgA2, IgD, IgE or IgM is derived from mouse, human, primate or alpaca.

[0115] According to some embodiments of the invention, the albumin or a fragment thereof is serum albumin or a fragment thereof.

[0116] According to some embodiments of the invention, the albumin or fragment thereof is human serum albumin.

[0117] Nucleic acid molecule, expression vector, recombinant cell, hybridoma cell and method for preparing antibody or antigen fragment

[0118] In the process of preparing or obtaining these antibodies or antigen-binding fragments thereof, nucleic acid molecules expressing these antibodies or antigen-binding fragments thereof can be connected to different vectors and then expressed in different cells to obtain the corresponding antibodies or antigen-binding fragments thereof.

[0119] To this end, in the third aspect of the present invention, the present invention provides a nucleic acid molecule. According to an embodiment of the present invention, the nucleic acid molecule encodes the antibody or antigen-binding fragment described in the first aspect, and the fusion protein described in the second aspect. The provided nucleic acid molecule can be obtained by techniques known to those skilled in the art. Moreover, it can be species-optimized and more easily expressed in mammalian cells.

[0120] According to some embodiments of the invention, the nucleic acid molecule is DNA.

[0121] It should be noted that, for the nucleic acid molecules mentioned herein, those skilled in the art will understand that they actually include any one or both of the complementary double strands. For convenience, in this specification and claims, although only one strand is provided in most cases, the other strand complementary thereto is actually disclosed. In addition, the nucleic acid sequence in the present invention includes a DNA form or an RNA form, and disclosing one of them means that the other is also disclosed.

[0122] In the fourth aspect of the present invention, the present invention proposes an expression vector. According to an embodiment of the present invention, the expression vector carries the nucleic acid molecule described in the third aspect. When the above-mentioned nucleic acid molecule is connected to the expression vector, the nucleic acid molecule can be directly or indirectly connected to the control element on the expression vector, as long as these control elements can control the translation and expression of the nucleic acid molecule. Of course, these control elements can come directly from the expression vector itself, or they can be exogenous, that is, not from the expression vector itself. Of course, the nucleic acid molecule can be operably connected to the control element.

[0123] Herein, "operably linked" means connecting the foreign gene to the expression vector so that the control elements in the expression vector, such as transcription control sequences and translation control sequences, can play their intended functions of regulating the transcription and translation of the foreign gene. Commonly used expression vectors can be, for example, plasmids, bacteriophages, and the like.

[0124] According to some specific embodiments of the present invention, after the expression vector is introduced into a suitable recipient cell, the expression of the aforementioned antibody or antigen-binding fragment, or the aforementioned fusion protein can be effectively achieved under the mediation of a regulatory system, thereby achieving large-scale in vitro acquisition of the antibody or antigen-binding fragment, or the fusion protein.

[0125] According to an embodiment of the present invention, the expression vector comprises a vector selected from a eukaryotic expression vector or a prokaryotic expression vector.

[0126] In an optional embodiment of the present invention, the expression vector is a plasmid expression vector, a viral expression vector, such as a lentiviral expression vector.

[0127] In the fifth aspect of the present invention, the present invention provides a recombinant cell. According to an embodiment of the present invention, the recombinant cell includes carrying the nucleic acid molecule described in the third aspect or the expression vector described in the fourth aspect; or expressing the antibody or antigen-binding fragment described in the first aspect, or the fusion protein described in the second aspect. The recombinant cell can effectively express the aforementioned antibody or antigen-binding fragment, or fusion protein in the cell under suitable conditions.

[0128] It should be noted that "suitable conditions" refer to conditions suitable for the expression of the antibody or antigen-binding fragment or fusion protein of the present invention. It is easy for those skilled in the art to understand that conditions suitable for the expression of the antibody or antigen-binding fragment or fusion protein include but are not limited to suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy host cell state, suitable host cell density, suitable cell culture environment, and suitable cell culture time. "Suitable conditions" are not particularly limited, and those skilled in the art can optimize the most suitable conditions for the expression of the above-mentioned antibody or antigen-binding fragment or fusion protein according to the specific environment of the laboratory.

[0129] According to some embodiments of the present invention, the recombinant cell is obtained by introducing the expression vector described in the fourth aspect into a host cell.

[0130] It should be noted that the recombinant cells of the present invention are not particularly limited and may be prokaryotic cells, eukaryotic cells or bacteriophages. The prokaryotic cells may be Escherichia coli, Bacillus subtilis, Streptomyces or Proteus mirabilis, etc. The eukaryotic cells include fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Trichoderma, insect cells such as armyworms, plant cells such as tobacco, mammalian cells such as BHK cells, CHO cells, COS cells, myeloma cells, etc. In some embodiments, the recombinant cells of the present invention are preferably mammalian cells, including BHK cells, CHO cells, NSO cells or COS cells, and do not include animal germ cells, fertilized eggs or embryonic stem cells.

[0131] According to an embodiment of the present invention, the recombinant cell is a eukaryotic cell, preferably a mammalian cell.

[0132] In the sixth aspect of the present invention, the present invention provides a hybridoma cell. According to an embodiment of the present invention, the hybridoma cell can produce the monoclonal antibody in the antibody described in the first aspect. The hybridoma cell of the present invention can produce the monoclonal antibody in the antibody that recognizes the carboxylation site of the matrix Gla protein.

[0133] In the classic monoclonal antibody preparation method, first of all, a suitable antigen is required to be prepared and used to immunize animals. In order to prepare antibodies that recognize the carboxylation site of the matrix Gla protein, the suitable antigen must contain the matrix Gla protein carboxylation site. This antigen can be obtained by separation and purification from natural human tissue or blood, chemical synthesis, or a combination of these methods, and used for animal immunization and antibody screening detection.

[0134] In the classic monoclonal antibody preparation method, animals are first immunized with carboxylated matrix Gla protein antigens, and blood is collected at intervals to verify whether the animals have produced antibody responses to the carboxylated matrix Gla protein antigens. B cells are then isolated from the spleen of animals with antibody responses and fused with immortal myeloma cells in vitro to obtain hybridoma cells. These hybridoma cells are then diluted to the limit in a culture plate and regrown (monoclonal hybridoma cell lines), and the culture supernatant of these hybridoma cell lines is taken to detect whether it contains specific antibodies against the antigen. Based on the antibody yield, quality and cell line growth characteristics, the best monoclonal antibody production cell line can be selected for subsequent monoclonal antibody production.

[0135] Other methods can also be used to obtain monoclonal antibodies. For example, spleen cells from the above-mentioned animals can be isolated and then incubated with labeled antigens (such as fluorescein-labeled carboxylated matrix Gla protein). Since antibody-producing B cells usually have antibody molecules presented on the cell membrane surface, these cells will be bound (stained) by the labeled antigen and can be sorted by a fluorescent flow cytometer. The mRNA of these sorted B cells can be isolated, and a library of antibody variable region cDNA can be obtained by in vitro reverse transcription and specific PCR reactions. 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 a host cell suitable for this expression plasmid. These host cells (or phages) can be isolated and purified (cloned) by different methods (such as the cell limiting dilution culture method described above, or the phage plaque plating method). These cell lines or phage clones can be used to produce antibodies and analyze and identify the antibodies.

[0136] According to a preferred embodiment of the present invention, the animal immunization in the present invention adopts multiple subcutaneous injections at multiple points, which improves the titer of the final animal serum and increases the positive clone rate.

[0137] According to a preferred embodiment of the present invention, the addition of HAT screening reagent is postponed from the day of fusion to the second day, so that the cell positive rate is significantly improved.

[0138] In the seventh aspect of the present invention, the present invention provides a method for preparing an antibody or antigenic fragment. According to an embodiment of the present invention, the method comprises culturing the recombinant cell described in the fifth aspect or the hybridoma cell described in the sixth aspect. Thus, the method of the present invention can be used to produce the aforementioned antibody or antigenic fragment.

[0139] Conjugates, compositions, and reagents or kits

[0140] In the eighth aspect of the present invention, the present invention provides a conjugate. According to an embodiment of the present invention, the conjugate comprises the antibody or antigen-binding fragment described in the first aspect, the fusion protein described in the second aspect; and a coupling part, wherein the coupling part is connected to the antibody or antigen-binding fragment, or the fusion protein. The conjugate of the present invention has a carboxylated matrix Gla protein binding affinity and can be used to detect carboxylated matrix Gla protein and detect cells expressing carboxylated matrix Gla protein.

[0141] According to some embodiments of the present invention, the coupling moiety comprises at least one selected from a carrier, a drug, a toxin, a cytokine, a protein tag, a modifier and a chemotherapeutic agent.

[0142] Herein, the carrier can be a substance that can be suspended or dispersed in a liquid phase (e.g., solid phase carriers such as particles and magnetic beads), or a solid phase that can contain or carry a liquid phase (e.g., supports such as plates, membranes, test tubes, and containers such as well plates, microfluidics, glass capillaries, nanocolumns, and monolithic columns); it can also be a labeling carrier for labeling antibodies or antigen-binding fragments, or fusion proteins, such as enzymes (e.g., peroxidase, alkaline phosphatase, luciferin, β-galactosidase), affinity substances (e.g., one of streptavidin and biotin, one of mutually complementary positive and antisense nucleic acids), fluorescent dyes (e.g., fluorescein, fluorescein isothiocyanate, rhodamine, green fluorescent protein, red fluorescent protein), luminescent substances (e.g., luciferin, aequorin, acridinium esters, tris(2,2'bipyridine)ruthenium, luminol), radioactive isotopes (e.g., 3 H. 14 C. 32 P. 35 S.125 I) and gold colloid, etc.

[0143] According to an embodiment of the present invention, the carrier includes a fluorescent dye.

[0144] According to some embodiments of the present invention, the fluorescent dye includes any one or more of FITC, PE, Cy5, PI, 7AAD, APC, AlexaFluor, eFluor, PE Cy7, APCCy7, PerCP, PerCPcy5.5, PB, BV series, QDot series and BUV series dyes.

[0145] According to an embodiment of the present invention, the drug is a small molecule drug that can be combined with an antibody or an antigen-binding fragment, or a fusion protein.

[0146] According to an embodiment of the present invention, the protein tag includes but is not limited to a His tag, a Flag tag, a GST tag, an MBP tag, a SUMO tag, a C-Myc tag, and the like.

[0147] According to the embodiments of the present invention, the modifier should be understood in a broad sense, and may refer to a substance used to modify a protein. For example, it may be polyethylene glycol or a derivative thereof.

[0148] It should be noted that the binding method of the coupling part and the antibody or antigen binding fragment or fusion protein can be a method known in the art. For example, physical adsorption, covalent binding, methods using affinity substances (e.g., biotin, streptavidin) and ion binding methods can be cited.

[0149] In the ninth aspect of the present invention, the present invention proposes a composition. According to an embodiment of the present invention, the composition comprises the antibody or antigen binding fragment described in the first aspect, the fusion protein described in the second aspect, the nucleic acid molecule described in the third aspect, the expression vector described in the fourth aspect, the recombinant cell described in the fifth aspect, the hybridoma cell described in the sixth aspect, the antibody or antigen binding fragment prepared according to the method described in the seventh aspect, or the conjugate described in the eighth aspect. The composition of the present invention has a carboxylated matrix Gla protein binding affinity, especially can specifically bind to the carboxylated matrix Gla protein, and can be used to detect the carboxylated matrix Gla protein, or detect cells expressing the carboxylated matrix Gla protein.

[0150] In the tenth aspect of the present invention, the present invention proposes a reagent or a kit. According to an embodiment of the present invention, the reagent or the kit comprises the antibody or antigen binding fragment described in the first aspect, the fusion protein described in the second aspect, the nucleic acid molecule described in the third aspect, the expression vector described in the fourth aspect, the recombinant cell described in the fifth aspect, the hybridoma cell described in the sixth aspect, the antibody or antigen binding fragment prepared according to the method described in the seventh aspect, or the conjugate described in the eighth aspect. The kit of the present invention has a carboxylated matrix Gla protein binding affinity, and can effectively detect carboxylated matrix Gla protein and detect cells expressing carboxylated matrix Gla protein.

[0151] As mentioned above, the antibodies or antigen-binding fragments of some specific embodiments of the present invention can effectively bind to carboxylated matrix Gla protein, so the reagents or kits containing the antibodies or antigen-binding fragments can effectively detect carboxylated matrix Gla protein qualitatively or quantitatively. The reagents or kits provided by the present invention can be used, for example, for immunoblotting, immunoprecipitation, etc., which involve the use of carboxylated matrix Gla protein and antibody specific binding to detect reagents or kits. These kits may contain any one or more of the following: antagonists, anti-carboxylated matrix Gla protein antibodies or drug reference materials; protein purification columns; immunoglobulin affinity purification buffers; cell assay diluents; instructions or literature, etc. Anti-carboxylated matrix Gla protein antibodies can be used for different types of diagnostic tests, for example, the presence of various drugs, toxins or other proteins can be detected in vitro or in vivo, for example, by testing the serum or blood of the subject to test the content of carboxylated matrix Gla protein in the blood or serum, etc., and scientific research can also be carried out, using the kit to detect carboxylated matrix Gla protein (especially human carboxylated matrix Gla protein) in the sample to be tested. For the above-mentioned application scenarios, the binding molecules are also applicable and will not be described again here.

[0152] According to some specific embodiments of the present invention, the kit may also include conventionally used for detecting carboxylated matrix Gla protein, such as coating solution.

[0153] Purpose and inspection method

[0154] In the eleventh aspect of the present invention, the present invention proposes a use of the antibody or antigen-binding fragment described in the first aspect, the fusion protein described in the second aspect, the nucleic acid molecule described in the third aspect, the expression vector described in the fourth aspect, the recombinant cell described in the fifth aspect, the hybridoma cell described in the sixth aspect, the antibody or antigen-binding fragment prepared according to the method described in the seventh aspect, the conjugate described in the eighth aspect, or the composition described in the ninth aspect in preparing a kit, wherein the kit is used to detect carboxylated matrix Gla protein and cells expressing carboxylated matrix Gla protein.

[0155] In the twelfth aspect of the present invention, the present invention proposes a method for detecting carboxylated matrix Gla protein or detecting cells expressing carboxylated matrix Gla protein. According to an embodiment of the present invention, the method comprises: using the antibody or antigen binding fragment described in the first aspect, the fusion protein described in the second aspect, the antibody or antigen binding fragment prepared according to the method described in the seventh aspect, the conjugate described in the eighth aspect, the composition described in the ninth aspect, or the reagent or kit described in the tenth aspect to contact with the sample to be detected to form an immune complex. As can be seen from the above, the aforementioned antibody or antigen binding fragment, fusion protein, conjugate, reagent or kit all have carboxylated matrix Gla protein binding affinity, thus, the above-mentioned antibody or antigen binding fragment, fusion protein, conjugate, reagent or kit can effectively detect carboxylated matrix Gla protein or detect cells expressing carboxylated matrix Gla protein.

[0156] According to some embodiments of the present invention, based on the signal of the immune complex, it is determined whether the sample to be tested contains carboxylated matrix Gla protein or whether it contains cells expressing carboxylated matrix Gla protein, or the content of the carboxylated matrix Gla protein or cells expressing carboxylated matrix Gla protein in the sample to be tested is determined.

[0157] According to some embodiments of the present invention, the signal of the immune complex is detected by at least one of ELISA, WB and IHC.

[0158] According to some embodiments of the invention, the signal comprises a fluorescent signal.

[0159] The amino acid sequences involved in this article are shown in Table A:

[0160] Table A: Amino acid sequences

[0161]

[0162]

[0163] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

[0164] Practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a skilled artisan. This technique is fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (PCR: The Polymerase Chain Reaction) (Academic Press, Inc., 1987). Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 2011), each of which is expressly incorporated herein by reference.

[0165] In the embodiments of the present invention, the nucleotide sequence used to prepare the expression vector can be obtained according to its amino acid sequence using conventional methods or conventional software (such as the online program Vectorbuilder (website: https: / / www.vectorbuilder.cn / tool / codon-optimization.html), GeneOptimizer online program, etc.).

[0166] Example 1: Synthesis of carboxylated matrix Gla protein peptides, non-carboxylated matrix Gla protein peptides and matrix Gla protein full-length antigens

[0167] The full-length amino acid sequence encoding the matrix Gla protein is shown in SEQ ID NO: 13. The carboxylated matrix Gla protein and the non-carboxylated matrix Gla protein peptides were synthesized using conventional methods in the art, and the specific peptide sequences are as follows:

[0168] name sequence MGP-BSA (Non-carboxylated Matrix Gla Protein Peptide) QERIRERSKPVHELNREACD cMGP-KLH (carboxylated matrix Gla protein peptide) QE(c)RIR E(c)RSKPVH E(c)LNR E(c)ACD cMGP-BSA (carboxylated matrix Gla protein peptide) QE(c)RIR E(c)RSKPVH E(c)LNR E(c)ACD

[0169] Note: In the above table, "E(c)" indicates that the glutamic acid has a carboxyl modification; "BSA" indicates that the C segment of the carboxylated matrix Gla protein or the non-carboxylated matrix Gla protein peptide is linked to bovine serum albumin, which is synthesized by Shanghai Botai Biotechnology Co., Ltd.; "KLH" indicates that the C segment of the carboxylated matrix Gla protein is linked to hemocyanin, which is synthesized by Shanghai Synthetic Botai Biotechnology Co., Ltd.

[0170] Example 2: Identification of carboxylated matrix Gla protein

[0171] First, the cMGP-BSA protein prepared in Example 1 was coated on an ELISA plate at 1 μg / ml, and a purified antibody that recognizes the carboxylation site of the carboxylation matrix Gla protein was used as the primary antibody of the indirect ELISA method, and an HRP-labeled goat anti-rat antibody was used as the secondary antibody. TMB was used as the color developing solution, and then 1M H2SO4 was used to terminate the reaction. The OD value of the ELISA plate was measured by an ELISA instrument. 450 The readings showed that the antigenic activity of carboxylated matrix Gla protein was higher.

[0172] sample <![CDATA[OD 450 ]]> Carboxylated matrix Gla protein concentration (1 μg / ml) 0.813 blank 0.063

[0173] Example 3: Immunization of BALB / c mice with carboxylated matrix Gla protein

[0174] BALB / c mice aged 6 to 8 weeks were selected for the following immunization procedure:

[0175] During the first immunization, 25 μg of carboxylated matrix Gla protein was mixed with an equal amount of Freund's complete adjuvant and injected subcutaneously at multiple points after emulsification; 14 days after the first immunization, 12.5 μg of the cMGP-KLH protein in Example 1 was mixed with Freund's incomplete adjuvant and emulsified for booster immunization. 14 days after the second immunization, 12.5 μg of cMGP-KLH protein was mixed with Freund's incomplete adjuvant and emulsified for booster immunization. 14 days after the third immunization, blood was collected and serum was separated, and 1 μg / mL cMGP-BSA protein was used to coat ELISA plates for indirect ELISA tests to determine serum titers.

[0176] The results showed that the titer of the prepared mouse antiserum was 1:72900. The typical immune mouse serum test results are shown in Table 2.

[0177] Table 2: Results of serum tests on immunized mice

[0178] Serum dilution multiple <![CDATA[OD 450 ]]> 100 1.975 300 1.893 900 1.529 2700 1.231 8100 0.896 24300 0.711 72900 0.549 Background 0.081

[0179] Example 4: Cell fusion

[0180] In Example 3, cell fusion was performed 3 days after the booster immunization of mice. After removing the eyeballs of the mice to collect blood, the mice were killed by dislocation, placed in a 70% alcohol bottle for 2 minutes, and then fixed on a foam board in a biosafety cabinet. The abdominal skin was untied to find the spleen, which was removed with forceps and gently ground into a 200-mesh stainless steel filter. The cells were gently rinsed with DMEM culture medium (Thermo, 11965092), and then centrifuged at 200×g for 10 minutes at room temperature. The supernatant was discarded and set aside. When preparing feeder cells, the mice were killed by dislocation, placed in a 70% alcohol bottle for 2 minutes, and then fixed on a foam board in a biosafety cabinet. The abdominal skin was untied, and PBS was drawn with a syringe and gently injected into the peritoneum. The liquid containing the feeder cells was washed out from the other side, and then centrifuged at 200×g for 10 minutes at room temperature. The supernatant was discarded and set aside. 2.0×10 7 FO myeloma cells and 2.0×10 8 The spleen cells were mixed, centrifuged at 200 × g for 10 minutes, the supernatant was discarded, and the mixture was gently shaken to mix. In a 37°C water bath, 1 ml of a 50% PEG-1450 (Merk, P1458) aqueous solution was added dropwise within 90 seconds, and then 20 mL of DMEM culture medium was added dropwise. The mixture was centrifuged at 200 × g for 10 minutes, the supernatant was discarded, and the mixture was washed repeatedly. The mixture was centrifuged at 200 × g for 10 minutes, the supernatant was discarded, and hybridoma cells were obtained. The cells were plated into 10 96-well culture plates with 150 μL per well. Add 10,000 cells / well of feeder layer cells to the above 10 96-well cell culture plates, 100 μL per well, mark the culture plates, and place them in a cell culture incubator at 37°C containing 5% CO2. Add HAT selection medium (Merk, H0262) on the second day, and then select and culture with HAT for 1 to 2 days. A large number of tumor cells will die, and the tumor cells will disappear after 3 to 4 days. Hybrid cells will form small colonies. After 7 to 10 days of HAT selection culture medium, HT culture medium (Merk, H0137) should be used, and then maintained for another 2 weeks, and then DMEM culture medium with 20% FBS (ExCell, FSP500) should be used for continued culture. During the above selection culture period, when the hybridoma cells cover 1 / 10 of the bottom of the well, specific antibodies can be detected to screen out the desired hybridoma cell lines. During the selection culture period, half of the culture medium is generally replaced every 2 to 3 days.

[0181] Example 5: Screening and subcloning of positive hybridoma cell lines

[0182] First, the optimal coating amount of carboxylated matrix Gla protein as antigen was determined by orthogonal experiment. 0.5, 1.0, 2.0, 4.0 μg of cMGP-BSA protein in Example 1 was coated on a 96-well plate, and 6 wells of each concentration were set as 3 positive and 3 negative. Square titration was performed with positive serum of mice immunized with carboxylated matrix Gla protein cMGP-KLH at different dilution multiples, and negative serum of unimmunized mice was used as negative control. 96-well ELISA plates were coated with 0.5 μg purified carboxylated matrix Gla protein per well, overnight at 4°C; washed twice with PBST; 200 μL of 1% BSA in PBS was added to each well, blocked at room temperature for 2 hours, and then placed on trifold paper and patted dry; sample addition: 0.1 mL of the sample to be tested was added to the reaction well, incubated at 37°C for 1 hour, and then washed, and blank wells (without sample), negative control wells and positive control wells were made at the same time. Add 0.1 mL of newly diluted antibody to each reaction well, incubate at 37°C for 1 hour, and then wash 3 times. Add enzyme-labeled secondary antibody: Add 0.1 mL of newly diluted enzyme-labeled antibody to each reaction well. Incubate at 37°C for 1 hour, and wash 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 1M H2SO4 to each reaction well. Measure the OD value to determine the result, and use an enzyme reader to measure the absorbance value (A450) at 450 nm. A value that is 2.1 times or more of the negative control OD value is considered positive (calculated after adjusting the blank control well to zero). Select the hybridoma cell monoclonal clone that resists the carboxylated matrix Gla protein.

[0183] According to the above method, the positive hybridoma cells obtained by screening are subcloned, and the original wells are diluted with HAT selection medium by limiting dilution method and then redistributed into 96-well culture plates, and then the morphology and number of cells are observed. The cells are adjusted to 3-10 cells / mL. Take the cell culture plate with the feeder cell layer prepared the day before, and add 100 μL of diluted cells to each well. Culture statically in a 37°C, 5% CO2 incubator. Change the medium on the 7th day, and change the medium once every 2-3 days thereafter. Cell clones can be seen on the 8th to 9th day, and the antibody activity is detected in time. The cells in the positive wells are transferred to a 24-well plate for expansion and culture. Each clone should be frozen as soon as possible, and finally the hybridoma cell line with clone number M018 is selected for antibody production.

[0184] Example 6: Preparation of monoclonal antibodies in large quantities and determination of antibody titer

[0185] (1) Large-scale preparation of monoclonal antibodies

[0186] 8-week-old BALB / C mice were intraperitoneally injected with 0.5 mL of Freund's incomplete adjuvant. Two weeks later, 1 × 10 6The hybridoma cells of clone number M018 in Example 5 can produce ascites 7 to 10 days after inoculation. The health status and ascites signs of the animals are closely observed. When the ascites is as much as possible and the mice are close to death, the mice are killed and the ascites is sucked into a test tube with a dropper. 5 to 10 ml of ascites can be obtained from one mouse. The ascites can also be extracted with a syringe and can be collected repeatedly several times. The ascites obtained is centrifuged at 3000×g for 10 minutes, the upper layer of fat and the bottom precipitate are discarded, the supernatant is collected and distributed at -20°C, and after the ascites supernatant is thawed and equilibrated to room temperature, 1 / 10 volume of 1M Tris-HCl pH8.0 is added to adjust the sample pH value to 8.0. The protein G affinity column was balanced with 20 column volumes of 100mM Tris-HCl at pH = 8.0, the ascites supernatant after adjusting the pH to 8.0 was loaded onto the column, and then washed with 20 column volumes of 100mM Tris-HCl at pH = 8.0, and finally eluted with 100mM Glycine-HCL pH 2.5 (i.e., carboxylated matrix Gla protein antibody M018, which can be referred to as antibody M018 or M018). The antibody eluate was added to a concentrator tube (Millipore, UFC801008, 10K) and centrifuged at room temperature at 3000 × g for 20 minutes in a centrifuge (Xiangyi, L550). The solution was centrifuged in batches until the volume of the solution reached 1 ml / concentrator tube (2 tubes), 4 mL of 10mM PBS pH = 7.4 buffer was added, and the solution was centrifuged at room temperature at 3000 × g for 20 minutes. Repeat centrifugation 3 times to make the antibody buffer 10mM PBS PH = 7.4, add 10mM PBS PH = 7.4 to a total volume of 10mL. Finally, 2mL / tube of concentrated antibody solution was dispensed into centrifuge tubes and stored at -80°C. The antibody concentration was determined using a BCA kit (Solabo, PC0020), and the concentration of the purified monoclonal antibody was 3.7mg / mL.

[0187] (2) Antibody titer determination

[0188] The titer of the carboxylated matrix Gla protein antibody M018 was detected by the indirect ELISA method. The cMGP-BSA protein was diluted with PBS and coated in a 96-well ELISA plate at 0.2 μg / mL and 100 μL / well. After overnight at 4°C, the ELISA plate was washed twice with PBST solution, 300 μL / well each time, and patted dry after washing. The ELISA plate was blocked with 1% BSA in PBS solution, 200 μL / well, at room temperature for 2 hours, and patted dry after blocking; the carboxylated matrix Gla protein antibody M018 diluted to 20 ng / mL with PTB was added, and the reaction was carried out in a 37°C incubator for 1 hour. The ELISA plate was washed twice with PBST solution, 300 μL / well each time. Patted dry after washing. The HRP-labeled goat anti-mouse antibody diluted 5000 times with PTB was added, and the reaction was carried out in a 37°C incubator for 1 hour. The ELISA plate was washed twice with PBST solution, 300 μL / well each time. Patted dry after washing. TMB substrate solution was added, 100 μL / well, reacted at room temperature for 10 minutes, and 100 μL / well of 1M H2SO4 was added to terminate the reaction. The absorbance (A450) was measured at 450 nm using an ELISA reader. The results are shown in Table 3, and it can be seen that the titer of the purified antibody is 1:729000.

[0189] Table 3

[0190] Antibody dilution factor <![CDATA[OD 450 ]]> 1000 2.376 3000 1.951 9000 1.483 27000 1.291 81000 0.872 243000 0.418 729000 0.254 Background 0.061

[0191] Example 7: Sequence analysis of monoclonal antibodies

[0192] (1) Monoclonal antibody subtype identification

[0193] Hybridoma cell line M018 was cultured in a 10 cm diameter cell culture dish (37°C, 5% CO2) using DMEM medium (GIBCO, #C11995500BT) supplemented with 10% serum. After 7 days of culture, the cells were transferred to a 15 ml centrifuge tube and 4 × 10 6 After centrifugation at 200×g for 5 minutes, the supernatant was discarded, and the centrifuge tube was inverted to drain the liquid in the tube. The cells in the tube were synthesized into cDNA using the reverse transcription kit (Qiagen, 74134) of QIAGEN.

[0194] The antibody subtype is determined by PCR using antibody subtype-specific primers. The above-synthesized cDNA is used as a PCR reaction template. PCR reaction solution system: TAKARA Ex Taq (5U / μL, TAKARA, RR001B), 0.25μL; 10×Ex Taq Buffer, 5μL; dNTP mixture (2.5mM each), 4μL; template cDNA, 1μL; upstream primer (100μM), 1μL; downstream primer (100μM), 1μL; add double distilled water to a total volume of 50μL. PCR reaction temperature program: 94°C for 5 minutes for pre-denaturation, temperature cycle 30 times (94°C for 1 minute, 57°C for 1 minute, 72°C for 1 minute), and 72°C for 10 minutes for extension. After the reaction, 10μL of each PCR product was loaded on a 1% agarose gel for electrophoresis. The electrophoresis pattern is shown as follows. Figure 1 The order of samples added to each lane is shown in Table 5. The subtype of the antibody can be inferred from the PCR product results (Table 4). For specific results, see Figure 1 The monoclonal antibody M018 obtained in the present invention has a heavy chain of IgG1 and a light chain of kappa.

[0195] Table 4

[0196]

[0197] Where S=C or G, M=A or C, R=A or G, and W=A or T.

[0198] Table 5

[0199] Lanes PCR primers Antibody subtype specificity 1 DNA molecular weight standard (full gold, DNA MarkerII, #BM411) N / A 2 PCR products of primers F-VH and R-VH 1 IgG1 heavy chain 3 PCR products of primers F-VH and R-VH 2A IgG2A heavy chain 4 PCR products of primers F-VH and R-VH 2B IgG2B heavy chain 5 PCR products of primers F-VH and R-VH 3 IgG3 heavy chain 6 PCR products of primers F-VH and R-VK IgKappa light chain 7 PCR products of primers F-VL and R-VL1 IgLambda1 light chain 8 PCR products of primers F-VL and R-VL2 IgLambda2 light chain

[0200] (2) Sequencing of the variable region (V region) of the hybridoma cell line M018

[0201] The fragment obtained after PCR amplification of the V region of the antibody of the cell line M018 in step (1) of this embodiment was cut from the agarose gel and extracted using a DNA extraction kit (Qiagen, 74134). The extracted DNA fragment was linked to the pEASY-T1 cloning vector and transformed into Trans1-T1 competent cells (Transgen, CT101-1). The transformed bacterial colonies were picked into LB culture medium and DNA sequencing was performed after overnight culture. The light chain V region nucleic acid sequence of the antibody (M018) for recognizing the carboxylation site of the matrix Gla protein provided by the present invention is shown in SEQ ID NO:8, and the nucleic acid sequence of the heavy chain V region is shown in SEQ ID NO:7.

[0202] Example 8: Identification of the specificity of antibody M018 in detecting carboxylated matrix Gla protein

[0203] The specific ability of antibody M018 to detect free carboxylated matrix Gla protein was evaluated by ELISA. In this experiment, the cMGP-BSA protein in Example 1 and the MGP-BSA protein in Example 1 were diluted with PBS as antigens and coated in a 96-well ELISA plate at a coating concentration of 1 μg / mL and 0.5 ug / ml, and a volume of 100 μL / well. After overnight coating at 4°C, the ELISA plate was washed twice with PBST solution, 300 μL / well each time. After washing, pat dry, and the ELISA plate was blocked with PBS solution containing 1% BSA, 200 μL / well, at room temperature for 2 hours. Pat dry, add the antibody M018 in Example 6 (diluted to 10 ng / mL with PTB) to the above blocked ELISA plate, 100 μL / well, and react in a 37°C incubator for 1 hour. Pat dry, wash the plate 3 times with PBST, 300 μL / well each time. Pat dry, add HRP-labeled goat anti-mouse antibody diluted 5000 times with PTB to the ELISA plate, and react in a 37°C incubator 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, at room temperature for 10 minutes, then add 100 μL / well of 1M H2SO4 to terminate the reaction. Use an ELISA reader to measure the absorbance (A450) at 450nm. The experimental results are shown in Table 6, which show that antibody M018 has good specificity for detecting carboxylated matrix Gla protein.

[0204] Table 6

[0205] Antigen concentration (ng / ml) Non-carboxylated matrix Gla protein peptide Carboxylated Matrix Gla Protein Peptide 1000 0.058 1.096 500 0.049 0.689 Background 0.054 0.067

[0206] Example 9: Application of the ELISA kit prepared using antibody M018 in detecting carboxylated matrix Gla protein in human serum

[0207] (1) Horseradish peroxidase (HRP) labeling of antibody M018 and identification of the labeled product

[0208] 0.5 mL of the antibody M018 (20 nmol, 3 mg) in Example 6 was added to a dialysis bag (10 KDa, 1 cm width) and dialyzed in 2 L of 10 mM PBS solution (PH 7.4) at 4° C. overnight. 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 for 2 hours at room temperature, ready for coupling with activated HRP.

[0209] At the same time, use an analytical balance to accurately weigh 1 mg of HRP, add 0.2 mL of ultrapure water to dissolve, and make the HRP concentration 5 mg / mL. Add 40 μl of 0.1M NaIO4 to the above HRP solution, place it on a horizontal shaker, and react (activate) at room temperature in the dark for 20 minutes. Add the activated HRP solution to a dialysis bag (10KDa, width 1 cm) and dialyze it in 2L 1mM sodium acetate buffer (PH4.4) at 4°C overnight. Carefully aspirate the dialyzed HRP solution and transfer it to a new 1.5 mL centrifuge tube, add 1 / 10 volume of 0.2M carbonate buffer to raise the pH of the activated HRP solution to 9.0-9.5.

[0210] Mix the above antibodies and HRP (for coupling reaction), place on a horizontal shaker, and react at room temperature in the dark for 4 hours. After the coupling reaction is completed, add 10μl (pre-cooled ultrapure water) freshly prepared NaBH4, and terminate the reaction at 4℃ in the dark overnight. Move the antibody solution after the coupling reaction is terminated to a dialysis bag (10KDa, width 1cm), stir and dialyze at room temperature for 2 hours. Finally, transfer the solution to a brown centrifuge tube and store at 4℃.

[0211] (2) Detection of carboxylated matrix Gla protein in human serum using an ELISA kit prepared using antibody M018

[0212] In this experiment, matrix Gla protein antibody (Abcam, ab273657) was diluted to 1 μg / mL with PBS, and then 100 μL / well was coated in a 96-well ELISA plate (overnight at 4°C). The plate was washed twice with 300 μL / well PBST. Pat dry, block the ELISA plate with PBS solution containing 1% BSA, 200 μL / well, at room temperature for 2 hours. Pat dry, add 1000-fold diluted HRP-labeled antibody M018 (diluted with PTB) solution 25 μl / well to the ELISA plate, and then add different concentrations of human serum samples (serum of patients with clinically confirmed bone-related diseases was selected as the sample to be tested, and normal human serum was used as the negative sample), 75 μL / well, and reacted in a 37°C incubator for 45 minutes. Wash the plate 3 times with PBST, 300 μL / well each time. Pat dry, add TMB substrate solution, 100 μL / well, and react at room temperature for 10 minutes. Then, 100 μL / well of 1M H2SO4 was added to terminate the reaction, and the absorbance (A450) was measured at 450 nm using an ELISA reader. The results are shown in Table 7, indicating that the ELISA kit prepared using the antibody M018 of the present invention can efficiently detect carboxylated matrix Gla protein in human serum.

[0213] Table 7

[0214] Patient serum samples <![CDATA[OD 450 ]]> <![CDATA[OD 450(空白) ]]> Patient 1 0.935 0.059 Patient 2 0.496 0.039 Patient 3 0.768 0.042 Patient 4 0.268 0.065 Patient 5 0.934 0.045

[0215] In summary, the present invention provides a monoclonal antibody that recognizes the carboxylation site of the matrix Gla protein, which has the characteristics of high affinity and specificity, and also provides an in vitro diagnostic test kit based on the above antibody. The present invention solves the problem of the relative shortage of antibody raw materials and test kits for the detection of carboxylated matrix Gla protein in the current market.

[0216] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0217] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. An antibody or antigen-binding fragment thereof that recognizes the carboxylation site of a matrix Gla protein, characterized in that: The antibody or antigen-binding fragment comprises HCDR1-3 and LCDR1-3; wherein, The HCDR1 has an amino acid sequence as shown in SEQ ID NO: 1; The HCDR2 has an amino acid sequence as shown in SEQ ID NO: 2; The HCDR3 has an amino acid sequence as shown in SEQ ID NO: 3; The LCDR1 has an amino acid sequence as shown in SEQ ID NO:4; The LCDR2 has an amino acid sequence as shown in SEQ ID NO: 5; and The LCDR3 has an amino acid sequence as shown in SEQ ID NO:

6.

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

3. The antibody or antigen-binding fragment thereof according to claim 2, characterized in that: The heavy chain framework region and / or light chain framework region are from at least one of mouse antibody, primate antibody, bovine antibody, horse antibody, porcine antibody, sheep antibody, goat antibody, dog antibody, cat antibody, rabbit antibody, donkey antibody, deer antibody, mink antibody, chicken antibody, duck antibody, goose antibody, turkey antibody, fighting cock antibody or their mutants.

4. The antibody or antigen-binding fragment thereof according to claim 2, characterized in that: At least a portion of the heavy chain framework region and / or the light chain framework region is derived from at least one of a murine antibody or a mutant thereof and a human antibody or a mutant thereof.

5. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibody or antigen-binding fragment comprises: A heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 7; and The light chain variable region has an amino acid sequence as shown in SEQ ID NO:

8.

6. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: Further comprising a heavy chain constant region and / or a light chain constant region.

7. The antibody or antigen-binding fragment thereof according to claim 6, characterized in that: At least one of the heavy chain constant region and the light chain constant region is from at least one of a mouse antibody, a primate antibody, a bovine antibody, a horse antibody, a porcine antibody, a sheep antibody, a goat antibody, a dog antibody, a cat antibody, a rabbit antibody, a donkey antibody, a deer antibody, a mink antibody, a chicken antibody, a duck antibody, a goose antibody, a turkey antibody, a fighting cock antibody or a mutant thereof.

8. The antibody or antigen-binding fragment thereof according to claim 6, characterized in that: The heavy chain constant region comprises a heavy chain constant region selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; or The light chain constant region comprises a light chain constant region selected from a kappa-type or a lambda-type.

9. The antibody or antigen-binding fragment thereof according to claim 6, characterized in that: The light chain constant region and the heavy chain constant region are both derived from at least one of a murine antibody or a mutant thereof and a human antibody or a mutant thereof.

10. The antibody or antigen-binding fragment thereof according to claim 6, characterized in that: The N-terminus of the heavy chain constant region is connected to the C-terminus of the heavy chain variable region; and / or The N-terminus of the light chain constant region is connected to the C-terminus of the light chain variable region.

11. The antibody or antigen-binding fragment thereof according to claim 6, characterized in that: The heavy chain constant region has the amino acid sequence shown in SEQ ID NO: 9; and / or The light chain constant region has the amino acid sequence shown in SEQ ID NO:

10.

12. The antibody or antigen-binding fragment thereof according to claim 6, characterized in that: The antibody comprises at least one of a full-length monoclonal antibody, a F(ab')2 antibody, a Fab' antibody, a Fab antibody, a F(ab)2 antibody, a Fv antibody, or a single-chain antibody; or The antigen-binding fragment includes at least one of a F(ab')2 fragment, a Fab' fragment, a Fab fragment, a F(ab)2 fragment, a Fv fragment, a scFv fragment, a scFv-Fc fusion protein, or a scFv-Fv fusion protein.

13. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibody or antigen-binding fragment comprises: A heavy chain having an amino acid sequence as shown in SEQ ID NO: 11; and The light chain has an amino acid sequence as shown in SEQ ID NO:

12.

14. A fusion protein, characterized in that: include: The antibody or antigen-binding fragment thereof according to any one of claims 1 to 13; and Protein labeling.

15. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, or the fusion protein according to claim 14.

16. The nucleic acid molecule according to claim 15, characterized in that The nucleic acid molecule is DNA.

17. An expression vector, characterized in that: Carrying the nucleic acid molecule of claim 15 or 16.

18. The expression vector according to claim 17, characterized in that The expression vector includes one selected from a eukaryotic expression vector or a prokaryotic expression vector.

19. A recombinant cell, characterized in that: include: Carrying the nucleic acid molecule of claim 15 or 16, or the expression vector of claim 17 or 18; or Expressing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, or the fusion protein according to claim 14.

20. The recombinant cell according to claim 19, characterized in that The recombinant cell is obtained by introducing the expression vector according to claim 17 or 18 into a host cell.

21. A method for preparing an antibody or an antigen fragment, characterized in that: Comprising culturing the recombinant cell of claim 19 or 20.

22. A composition, characterized in that include: The antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, the fusion protein according to claim 14, the nucleic acid molecule according to claim 15 or 16, the expression vector according to claim 17 or 18, the recombinant cell according to claim 19 or 20, or the antibody or antigen-binding fragment prepared by the method according to claim 21.

23. A reagent or a kit, characterized in that: include: The antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, the fusion protein according to claim 14, the nucleic acid molecule according to claim 15 or 16, the expression vector according to claim 17 or 18, the recombinant cell according to claim 19 or 20, or the antibody or antigen-binding fragment prepared by the method according to claim 21.

24. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, the fusion protein according to claim 14, the nucleic acid molecule according to claim 15 or 16, the expression vector according to claim 17 or 18, the recombinant cell according to claim 19 or 20, the antibody or antigen-binding fragment prepared according to the method according to claim 21, or the composition according to claim 22 in preparing a kit for detecting carboxylated matrix Gla protein or detecting cells expressing carboxylated matrix Gla protein.

25. A method for detecting carboxylated matrix Gla protein or detecting cells expressing carboxylated matrix Gla protein for purposes other than disease diagnosis and treatment, characterized in that: include: The antibody or antigen-binding fragment according to any one of claims 1 to 13, the fusion protein according to claim 14, the antibody or antigen-binding fragment prepared according to the method of claim 21, the composition according to claim 22, or the reagent or kit according to claim 23 are contacted with the sample to be detected to form an immune complex.

26. The method according to claim 25, characterized in that Based on the signal of the immune complex, determine whether the sample to be tested contains carboxylated matrix Gla protein or contains cells expressing carboxylated matrix Gla protein, or determine the content of the carboxylated matrix Gla protein or cells expressing carboxylated matrix Gla protein in the sample to be tested.

27. The method according to claim 26, characterized in that The signal of the immune complex is detected by at least one of ELISA, Western blot and IHC.

28. The method according to claim 26, characterized in that The signal comprises a fluorescent signal.

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