Single-domain antibody of human thromboregulatory protein and application of single-domain antibody

Abstract

The invention relates to the field of biological medicines, and discloses a single-domain antibody of human Thrombomodulin (TM) and an application of the single-domain antibody of the human Thrombomodulin (TM). Specifically, the invention provides a single-domain antibody combined with thromboregulatory protein and a pairing application of the thromboregulatory protein single-domain antibody. Furthermore, the single-domain antibody disclosed by the invention is suitable for constructing various human thromboregulatory protein quantitative detection methods based on an antigen-antibody recognition principle, such as ELISA (Enzyme-Linked Immunosorbent Assay), fluorescence, chemiluminescence and the like. The single-domain antibody is obtained through camel immunization, immune library construction, phage panning and the like, and compared with a conventional monoclonal antibody, the single-domain antibody has the advantages of being small in molecular weight, capable of recognizing hidden epitopes and special structures, resistant to high temperature, acid and alkali, easy to express and produce, high in expression quantity, good in water solubility and the like.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to an anti-human thrombomodulin single domain antibody and applications thereof. Background Art

[0002] Thrombomodulin (TM) is a transmembrane protein widely present on the surface of vascular endothelium. Mature thrombomodulin contains 557 amino acids (19-575aa, P07204), including 5 major domains, from N-terminus to C-terminus, which are C-type lectin-like domain, 6 epidermal growth factor-like repeat structures, serine / threonine-rich domain, transmembrane domain and cytoplasmic tail. TM can form a complex with thrombin with high affinity. This complex not only exerts anticoagulant effect by activating protein C, but also exerts thromboprotective effect by activating thrombin activated fibrinolysis inhibitor (TAFI) (activated TAFI (TAFIa) inhibits plasmin-mediated fibrinolysis). Studies have shown that the anticoagulant or antifibrinolytic effect of TM is related to the concentration of TM: low concentration is conducive to TAFI activation, and high concentration is conducive to protein C activation.

[0003] TM can bind to thrombin with high affinity. After binding, thrombin cannot activate platelets or cleave fibrinogen. It plays a very important role in maintaining the non-coagulation state of blood in blood vessels. When endothelial cell function is impaired, TM falls off the surface of vascular endothelial cells and becomes soluble TM (soluble thrombomodulin, sTM). sTM is one of the key indicators of the new four clinical thrombosis tests and can be used as a marker of the degree of damage to vascular endothelial cell function. The normal value is about 3-50ng / mL or 3.8~13.3TU / mL.

[0004] Clinically, elevated sTM levels can indicate vascular endothelial damage and are often used in combination with other indicators (such as D-dimer) to assess thrombotic risk. The TM antibodies currently used in enzyme immunoassay (EIA) and enzyme linked immunosorbent assay (ELISA) for sTM detection are not ideal in terms of detection limit, stability and other indicators. Moreover, monoclonal antibodies have strict storage conditions, are prone to deterioration, have high production costs, are expensive, and are difficult to transform and modify. In addition, different domains of TM have different effects such as anti-inflammatory, anti-coagulation, and anti-fibrinolysis, and related antibodies have important value in mechanism research and potential drugability. Scientific research institutions, medical institutions, and third-party testing institutions are in urgent need of TM antibodies that are both high-quality and low-priced. Solving these factors and existing problems is of great significance to the in vitro diagnostic research of TM and related research on multiple functional domains of TM.

[0005] Single-domain antibody (SdAb), derived from heavy chain antibody (HcAb) naturally lacking light chain in camels or sharks, was first discovered in camel serum by Hamers-Casterman team in 1993. Single-domain antibody is a single variable domain of heavy chain of heavy chain antibody (VHH), which is the smallest active antigen-binding fragment, with a molecular weight of only 10% of traditional antibodies and a diameter of 2.5nm. Compared with traditional monoclonal antibodies, VHH antibodies contain larger CDR1 and CDR3 regions, which are more convenient for identifying tiny hidden epitopes and special structures. In addition, VHH antibodies also have the advantages of strong affinity, low immunogenicity, diversified administration methods, high water solubility, strong stability, easy production, easy coupling and modification, and low production cost, and have broad application prospects in diagnostic antibodies and therapeutic antibodies. Summary of the invention

[0006] Based on the above background, the present invention provides a single domain antibody capable of specifically binding to human thrombomodulin and its application.

[0007] In the first aspect of the present invention, an anti-thrombomodulin single domain antibody is provided, wherein the complementary determining region (CDR) region of the VHH chain in the single domain antibody is as follows: CDR1 shown in SEQ ID NO: 1, CDR2 shown in SEQ ID NO: 2, and CDR3 shown in SEQ ID NO: 3.

[0008] In another preferred embodiment, the CDR1, CDR2 and CDR3 are separated by the framework regions FR1, FR2, FR3 and FR4 of the VHH chain;

[0009] In another preferred embodiment, the VHH chain further comprises a framework region FR, and the framework region FR is as follows: FR1 shown in SEQ ID NO: 4, FR2 shown in SEQ ID NO: 5, FR3 shown in SEQ ID NO: 6, and FR4 shown in SEQ ID NO: 7;

[0010] In another preferred embodiment, the CDR region of the single-domain antibody VHH chain comprises an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence similarity to SEQ ID NO: 1-3;

[0011] In another preferred embodiment, the amino acid sequence of the CDR region of the single-domain antibody VHH chain comprises one or more amino acid substitutions compared with SEQ ID NO: 1-3, preferably conservative amino acid substitutions;

[0012] In another preferred embodiment, any one of the above amino acid sequences further comprises a derivative sequence which is optionally subjected to addition, deletion, modification and / or substitution of at least one (such as 1-3, preferably 1-2, more preferably 1) amino acid and can retain the ability to specifically bind to thrombomodulin;

[0013] In another preferred embodiment, the anti-thrombomodulin single domain antibody has an amino acid sequence as shown in SEQ ID NO: 8;

[0014] In another preferred embodiment, the single domain antibody is capable of specifically binding to thrombomodulin;

[0015] In another preferred embodiment, the thrombomodulin is human thrombomodulin.

[0016] The second aspect of the present invention provides an anti-thrombomodulin antibody, which is an antibody against thrombomodulin and has the anti-thrombomodulin single domain antibody described in the first aspect of the present invention;

[0017] In another preferred embodiment, the anti-thrombomodulin antibody comprises one or more anti-thrombomodulin single domain antibodies;

[0018] In another preferred embodiment, the anti-thrombomodulin antibody includes a monomer, a bivalent body (bivalent antibody), a tetravalent body (tetravalent antibody), and / or a multivalent body (multivalent antibody);

[0019] In another preferred embodiment, the anti-thrombomodulin antibody comprises one or more VHH chains having an amino acid sequence as shown in SEQ ID NO: 8;

[0020] In another preferred embodiment, the anti-human thrombomodulin antibody has an amino acid sequence as shown in SEQ ID NO: 8;

[0021] In another preferred embodiment, the antibody can specifically bind to thrombomodulin.

[0022] The third aspect of the present invention provides a polynucleotide encoding a protein selected from the group consisting of: the anti-human thrombomodulin single domain antibody described in the first aspect of the present invention or the anti-human thrombomodulin antibody described in the second aspect of the present invention;

[0023] In another preferred embodiment, the polynucleotide is in a combined form;

[0024] In another preferred embodiment, the polynucleotide sequence comprises one or more sequences shown in SEQ ID NO: 9;

[0025] In another preferred embodiment, the polynucleotide includes DNA or RNA.

[0026] The fourth aspect of the present invention provides an expression vector, wherein the expression vector contains the polynucleotide according to the third aspect of the present invention;

[0027] In another preferred embodiment, the expression vector is selected from the following group: DNA, RNA, viral vector, plasmid, transposon, other gene transfer system, or a combination thereof;

[0028] In another preferred embodiment, the expression vector includes a viral vector, such as a lentivirus, adenovirus, AAV virus, or retrovirus.

[0029] The fifth aspect of the present invention provides a host cell, the host cell contains the expression vector according to the fourth aspect of the present invention, or the polynucleotide according to the third aspect of the present invention is integrated into its genome;

[0030] In another preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell;

[0031] In another preferred embodiment, the host cell is selected from the group consisting of Escherichia coli, yeast cells, mammalian cells, bacteriophages, or a combination thereof;

[0032] In another preferred embodiment, the prokaryotic cell is selected from the group consisting of Escherichia coli, Bacillus subtilis, lactic acid bacteria, Streptomyces, Proteus mirabilis, or a combination thereof;

[0033] In another preferred embodiment, the eukaryotic cell is selected from the group consisting of Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Trichoderma, or a combination thereof;

[0034] In another preferred embodiment, the host cell is Escherichia coli.

[0035] The sixth aspect of the present invention provides a method for producing an anti-thrombomodulin single domain antibody, comprising the steps of:

[0036] (1) culturing the host cell according to the fifth aspect of the present invention under conditions suitable for producing the single domain antibody, thereby obtaining a culture containing the anti-thrombomodulin single domain antibody;

[0037] (2) isolating or recovering the anti-thrombomodulin single domain antibody from the culture;

[0038] (3) Optionally, purifying and / or modifying the anti-thrombomodulin single domain antibody obtained in step (b).

[0039] The seventh aspect of the present invention provides the use of the anti-human thrombomodulin single domain antibody as described in the first aspect of the present invention or the anti-human thrombomodulin antibody as described in the second aspect of the present invention for preparing a reagent, a detection plate or a kit;

[0040] Wherein, the reagent, detection plate or kit is used for: detecting human thrombomodulin in a sample;

[0041] In another preferred embodiment, the detection includes enzyme immunoassay (EIA), colloidal gold, enzyme-linked immunosorbent assay (ELISA), lateral flow chromatography, flow cytometry, and cell immunofluorescence detection.

[0042] The eighth aspect of the present invention provides a method for detecting human thrombomodulin in a sample, the method comprising the steps of:

[0043] (1) contacting a sample with the anti-human thrombomodulin single domain antibody described in the first aspect of the present invention or the anti-human thrombomodulin antibody described in the second aspect of the present invention;

[0044] (2) Detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of human thrombomodulin in the sample.

[0045] In another preferred embodiment, the method is a non-diagnostic and non-therapeutic method.

[0046] The ninth aspect of the present invention provides a human thrombomodulin detection reagent, the detection reagent comprising:

[0047] (1) the anti-human thrombomodulin single domain antibody according to the first aspect of the present invention, or the anti-human thrombomodulin antibody according to the second aspect of the present invention; and

[0048] (2) A carrier that is acceptable for testing;

[0049] In another preferred embodiment, the test-acceptable carrier is a non-toxic, inert aqueous carrier medium;

[0050] In another preferred embodiment, the detection reagent is one or more reagents selected from the following group: enzymes, isotope tracers, contrast agents, flow detection reagents, cell immunofluorescence detection reagents, nanomagnetic particles and imaging agents;

[0051] In another preferred embodiment, the dosage form of the detection reagent is liquid or powder (such as aqueous solution, injection, lyophilized powder, tablet).

[0052] The tenth aspect of the present invention provides a kit for detecting human thrombomodulin, the kit comprising the detection reagent described in the ninth aspect of the present invention, and instructions;

[0053] In another preferred embodiment, the instructions state that the kit is used for non-invasively detecting the expression of human thrombomodulin in a test subject.

[0054] It should be understood that within the scope of the present invention, the above-mentioned various technical features of the present invention and the various technical features specifically described below (such as embodiments) can be arbitrarily combined to form new or preferred technical solutions. Due to space limitations, they will not be described one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 This is the nucleic acid electrophoresis diagram: a 700bp band in the first round of PCR and a 400bp band in the second round of PCR.

[0056] Figure 2 This is a plate scan: a plate containing bacterial culture solutions with 10-fold dilution differences for library capacity calculation.

[0057] Figure 3 The nucleic acid electrophoresis diagram shows 24 randomly selected monoclonal colonies used to detect the insertion rate of VHH fragments.

[0058] Figure 4 Plate scan: streaking plate for phage display biopanning.

[0059] Figure 5 The SDS-PAGE protein electrophoresis diagram of the antibody of the present invention.

[0060] Figure 6 This is a specific analysis of the antibodies of the present invention.

[0061] Figure 7 This is an analysis of the binding activity of the antibody of the present invention.

[0062] Figure 8This is a thermal stability analysis of the antibodies of the present invention.

[0063] Fig. 9 A double antibody sandwich ELISA was constructed based on the antibody of the present invention to quantitatively detect thrombomodulin molecules. DETAILED DESCRIPTION

[0064] After in-depth research and a large number of selections, the inventors successfully obtained a class of anti-thrombomodulin single-domain antibodies. Specifically, the present invention subcutaneously injected the prepared high-purity thrombomodulin antigen fused with an Fc tag into a camel to enhance immunogenicity, and obtained an immune library containing single-domain antibody gene fragments by genetic engineering. By coating the thrombomodulin antigen molecule fused with a His tag on an ELISA plate to display its epitope, the single-domain antibody immune library was further screened using phage display technology, thereby obtaining a thrombomodulin-specific single-domain antibody gene. Finally, this gene was transferred into an Escherichia coli expression strain to obtain a single-domain antibody of thrombomodulin. The experimental results show that the thrombomodulin single-domain antibody obtained by the present invention can effectively bind to human thrombomodulin.

[0065] the term

[0066] In order to better understand the present invention, the following terms are defined.

[0067] The singular terms also include the plural form, active tense and past tense of the terms unless otherwise specified.

[0068] The term "about" includes values ​​within a standard deviation of the stated value unless otherwise indicated.

[0069] Throughout the specification and claims, the words "comprises", "including", "having" and the like should be understood to have an inclusive rather than an exclusive meaning; that is, the meaning of "including but not limited to", unless otherwise indicated.

[0070] As used herein, the terms "antibody of the present invention", "single domain antibody of the present invention", "thrombomodulin single domain antibody", "anti-thrombomodulin single domain antibody", "anti-human thrombomodulin single domain antibody", and "TM single domain antibody" have the same meaning and interchangeability does not change the meaning. They all refer to single domain antibodies that specifically recognize and have binding activity to thrombomodulin (including recombinant human thrombomodulin or thrombomodulin derived from human plasma).

[0071] In this article, the terms "single domain antibody", "VHH antibody" and "nanoantibody" have the same meaning and can be interchanged without changing the meaning. They all refer to single domain antibodies consisting of only one heavy chain variable region, which are constructed by cloning the variable structural region of camel heavy chain antibodies. They are the smallest active antigen-binding fragment with complete functions.

[0072] Herein, the terms "variable region" and "complementarity determining region (CDR)" are used interchangeably.

[0073] In a preferred embodiment of the present invention, the heavy chain of the antibody includes the above-mentioned heavy chain variable region and heavy chain constant region.

[0074] In the present invention, the terms "antibodies of the present invention", "proteins of the present invention", or "polypeptides of the present invention" are used interchangeably, and all refer to polypeptides that specifically bind to thrombomodulin, such as proteins or polypeptides having a heavy chain variable region. They may or may not contain an initial methionine.

[0075] The present invention also provides other proteins or fusion expression products having the antibodies of the present invention. Specifically, the present invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a heavy chain containing a variable region, as long as the variable region is identical to or at least 90% homologous to the heavy chain variable region of the antibodies of the present invention, preferably at least 95% homologous.

[0076] Generally, the antigen binding properties of an antibody can be described by three specific regions located in the variable region of the heavy chain, called the variable region (CDR). This segment is divided into four framework regions (FR). The amino acid sequences of the four FRs are relatively conservative and do not directly participate in the binding reaction. These CDRs form a ring structure, and the β-folds formed by the FRs in between are close to each other in spatial structure. The CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen binding site of the antibody. The amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR region.

[0077] The variable regions of the heavy chains of the antibodies of the present invention are of particular interest because they are at least partially involved in binding to antigen. Therefore, the present invention includes molecules having antibody heavy chain variable regions with CDRs, as long as their CDRs have more than 90% (preferably more than 95%, and most preferably more than 98%) homology with the CDRs identified herein.

[0078] The present invention includes not only complete antibodies, but also fragments of antibodies with immunological activity or fusion proteins formed by antibodies and other sequences. Therefore, the present invention also includes fragments, derivatives and analogs of the antibodies.

[0079] As used herein, the terms "fragment", "derivative" and "analog" refer to polypeptides that substantially retain the same biological function or activity as the antibodies of the present invention. The polypeptide fragments, derivatives or analogs of the present invention may be:

[0080] (1) A polypeptide in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code;

[0081] (2) polypeptides having a substitution group in one or more amino acid residues;

[0082] (3) a polypeptide formed by the fusion of a mature polypeptide with another compound (e.g., a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol);

[0083] (4) A polypeptide formed by fusion of an additional amino acid sequence to the polypeptide sequence (such as a leader sequence or a secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or a fusion protein formed with a 6His tag).

[0084] Based on the teachings herein, these fragments, derivatives and analogs are well within the scope known to those skilled in the art.

[0085] The antibody of the present invention refers to a polypeptide having thrombomodulin binding activity and including the above-mentioned CDR region. The term also includes variant forms of polypeptides having the same function as the antibody of the present invention and including the above-mentioned CDR region. These variant forms include (but are not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acid deletions, insertions and / or substitutions, and addition of one or several (usually within 20, preferably within 10, and more preferably within 5) amino acids at the C-terminus and / or N-terminus. For example, in the art, when amino acids with similar or similar properties are substituted, the function of the protein is usually not changed. For another example, adding one or several amino acids at the C-terminus and / or N-terminus usually does not change the function of the protein. The term also includes active fragments and active derivatives of the antibodies of the present invention.

[0086] Variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of the antibody of the present invention under high or low stringency conditions, and polypeptides or proteins obtained using antiserum against the antibody of the present invention.

[0087] The present invention also provides other polypeptides, such as fusion proteins comprising single domain antibodies or fragments thereof. In addition to almost full-length polypeptides, the present invention also includes fragments of single domain antibodies of the present invention. Typically, the fragment has at least about 50 consecutive amino acids of the antibody of the present invention, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids.

[0088] In the present invention, "conservative variants of the antibodies of the present invention" refer to polypeptides formed by replacing at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids with amino acids having similar or similar properties compared to the amino acid sequence of the antibodies of the present invention. These conservative variant polypeptides are preferably generated by amino acid substitution according to Table 1.

[0089] Table 1

[0090]

[0091]

[0092] The present invention also provides a polynucleotide molecule encoding the above-mentioned antibody or its fragment or its fusion protein. The polynucleotide of the present invention can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand.

[0093] The polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence encoding only a mature polypeptide; a coding sequence of a mature polypeptide and various additional coding sequences; a coding sequence of a mature polypeptide (and optional additional coding sequences) and non-coding sequences.

[0094] The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, or may include additional coding and / or non-coding sequences.

[0095] The present invention also relates to polynucleotides that hybridize to the above-mentioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that hybridize to the polynucleotides of the present invention under stringent conditions. In the present invention, "stringent conditions" refers to:

[0096] (1) Hybridization and elution at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C;

[0097] (2) Denaturing agents are added during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, etc.;

[0098] (3) Hybridization occurs only when the identity between the two sequences is at least 90%, preferably 95% or more. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0099] The full-length nucleotide sequence of the antibody of the present invention or its fragment can usually be obtained by PCR amplification, recombination or artificial synthesis. A feasible method is to synthesize the relevant sequence by artificial synthesis, especially when the fragment length is short. Usually, a fragment with a very long sequence can be obtained by synthesizing multiple small fragments first and then connecting them. In addition, the coding sequence of the heavy chain and the expression tag (such as 6His) can be fused together to form a fusion protein.

[0100] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the proliferated host cells by conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in the present invention include biomolecules in isolated form.

[0101] At present, the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) can be obtained completely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention by chemical synthesis.

[0102] The sequences of the present invention are shown in Table 2:

[0103] Table 2

[0104]

[0105] The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.

[0106] Host cells can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells such as CHO, COS7, 293 cells, etc.

[0107] Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl2 method, the steps used are well known in the art. Another method is to use MgCl2. If necessary, transformation can also be carried out using electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be selected: calcium phosphate coprecipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0108] The obtained transformant can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used in the culture can be selected from various conventional culture media. Culture is carried out under conditions suitable for the growth of the host cells. After the host cells grow to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

[0109] The recombinant polypeptide in the above method can be expressed in the cell, on the cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be separated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods.

[0110] The antibodies of the invention may be used alone or in combination or conjugated to a detectable marker (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety, or any combination of these.

[0111] Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing a detectable product.

[0112] Therapeutic agents that can be combined or coupled to the antibodies of the present invention include, but are not limited to: (1) radionuclides; (2) biological toxins; (3) cytokines such as IL-2, etc.; (4) gold nanoparticles / nanorods; (5) viral particles; (6) liposomes; (7) nanomagnetic particles; (8) drug-activated enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); (9) therapeutic agents (e.g., cisplatin) or any form of nanoparticles, etc.

[0113] Detection Methods

[0114] The present invention also relates to a method for detecting thrombomodulin. The method generally comprises the following steps: obtaining a cell and / or tissue sample; dissolving the sample in a medium; and detecting the level of thrombomodulin in the dissolved sample.

[0115] In the detection method of the present invention, the sample used is not particularly limited, and a representative example is a sample containing cells in a cell storage solution.

[0116] Reagent test kit

[0117] The present invention also provides a kit containing the antibody (or fragment thereof) or the detection plate of the present invention. In a preferred embodiment of the present invention, the kit further includes a container, instructions for use, a buffer, and the like.

[0118] The present invention also provides a detection kit for detecting the level of thrombomodulin, which includes an antibody that recognizes thrombomodulin, a lysis medium for dissolving the sample, and common reagents and buffers required for detection, such as various buffers, detection labels, detection substrates, etc. The detection kit can be an in vitro diagnostic device.

[0119] application

[0120] As described above, the single domain antibody of the present invention has a wide range of biological and clinical application values, and its application involves multiple fields such as diagnosis and treatment of diseases related to thrombomodulin, basic medical research, biological research, etc. A preferred application is for clinical diagnosis of thrombomodulin.

[0121] The main advantages of the present invention include:

[0122] (1) The single domain antibody of the present invention can effectively bind to thrombomodulin and does not react with D-dimer and fibrinogen.

[0123] (2) The single domain antibody of the present invention has high purity, good specificity, better binding activity than commercially available monoclonal antibodies, and stronger thermal stability, providing a new option for the detection of thrombomodulin and the diagnosis of diseases with abnormal thrombomodulin levels.

[0124] (3) The single domain antibody of the present invention is suitable for constructing a variety of quantitative detection methods for human thrombomodulin based on the principle of antigen-antibody recognition, including ELISA, fluorescence, chemiluminescence, electrochemiluminescence, time-resolved fluorescence, etc.

[0125] The present invention will be further described below in conjunction with specific examples. It should be understood that these specific examples are only further detailed descriptions of the present invention and are not intended to limit the scope of the present invention in any form. The experimental methods in the following examples where specific conditions are not specified are usually based on conventional parameters, such as the conditions described in (Sambrook and Russell et al., Molecular Cloning: A Laboratory Manual (Molecular Cloning-A Laboratory Manual) (3rd edition) (2001) CSHL Press), or according to the conditions recommended by the manufacturer. Percentages and parts are calculated by weight unless otherwise stated.

[0126] Example 1: Preparation of thrombomodulin antigen for immunization

[0127] (1) The sequences of human thrombomodulin and human IgG1 protein Fc fragment were queried from the UniProt database and the HuFc-TM recombinant plasmid was designed and constructed.

[0128] (2) The constructed HuFc-TM recombinant plasmid was transferred into Escherichia coli DH5α by chemical transformation and plated.

[0129] (3) On the second day, a single clone was picked from the ampicillin-resistant LB plate and inoculated into LB medium (containing ampicillin) for activation and cultured at 37°C overnight.

[0130] (4) Take the activated bacteria and culture them in a shake flask at 37°C overnight.

[0131] (5) Use a transfection-grade plasmid extraction kit to extract the plasmid, perform agarose gel electrophoresis on the extracted plasmid to verify the band size, and perform sequencing at the same time.

[0132] (6) HEK293F cells were passaged to 2E6, and the cell viability was greater than 90%.

[0133] (7) Add plasmid DNA to F17 medium, mix well, and filter to sterilize.

[0134] (8) PEI was added to F17 medium, mixed and then mixed with the filter-sterilized plasmid. After standing, the mixture was slowly added to HEK293F cells.

[0135] (9) On the second day after transfection, transfection enhancer was added, and on the fifth day after transfection, cell culture supernatant was collected for purification.

[0136] Example 2: Construction and screening of thrombomodulin single domain antibody library

[0137] Construction and validation of VHH antibody library:

[0138] (1) The thrombomodulin antigen HuFc-TM prepared by the HEK293F expression system was mixed with an equal volume of Freund's adjuvant, and the mixture of thrombomodulin antigen and Freund's adjuvant was injected subcutaneously at multiple points in a Bactrian camel every week.

[0139] (2) After 7 immunizations, peripheral blood was collected from camels, and the mononuclear cells (PBMCs) were isolated and total RNA was extracted from them, which was then reverse transcribed into cDNA.

[0140] (3) Obtaining the gene encoding the single-domain antibody VHH fragment by two rounds of PCR amplification, such as Figure 1 shown.

[0141] (4) The VHH fragment was double-digested with restriction endonucleases Pst I and Not I and then recombined and ligated.

[0142] (5) The library was constructed by electroporation into TG1 Escherichia coli cells.

[0143] (6) The quality of the constructed library was evaluated by testing the library capacity and the accuracy of VHH fragment insertion. Figure 2 As shown, the library capacity is 2.4×10 9 CFU (colony forming units). 24 monoclonal colonies were randomly selected for colony PCR detection, such as Figure 3 As shown, the library insertion accuracy was 100%.

[0144] Thrombomodulin-positive clone enrichment and screening:

[0145] (1) Use 100 mM NaHCO3, pH 8.4 to dilute the thrombomodulin antigen (MedChemExpress: HY-P70724A) to 50 ug / mL (and use 100 mM NaHCO3 as a control). Coat the diluted thrombomodulin and the control on NUNC high-binding ELISA plates (coating volume is 100 uL / well) and place in a refrigerator at 2-8°C for overnight coating.

[0146] (2) Wash the coated ELISA plate overnight with sterile 1xPBST, then add 100uL of filter-sterilized 0.1% BSA and block at room temperature for 2h.

[0147] (3) After washing again to remove excess blocking solution, 100 μL of phage library was added to the thrombomodulin well and the control well, and allowed to bind at room temperature for 1 h.

[0148] (4) The thrombomodulin and control wells were washed with 1xPBST to remove non-specific phages.

[0149] (5) Add triethylamine solution to the thrombomodulin and control wells to dissociate specific phages, followed by neutralization by adding an equal volume of Tris-HCl.

[0150] (6) Finally, the neutralized phages were infected with TG1 cells that had been cultured to the logarithmic growth phase for amplification and enrichment and used for the next round of screening.

[0151] (7) Figure 4 As shown, three rounds of screening were performed, and 380 monoclonal colonies were picked from the second round of screening plates.

[0152] (8) 380 monoclonal colonies were inoculated into TB medium (containing ampicillin) in a sterile deep-well plate, cultured in a shaking incubator at 37°C until the logarithmic growth phase, and IPTG was added to induce expression.

[0153] (9) E. coli cells were lysed by osmotic shock to obtain periplasmic proteins, and positive clones were identified by periplasmic extract ELISA (PE-ELISA). When the OD value of the target well was more than 3 times that of the control well (Ratio + / - > 3), it was defined as a positive clone.

[0154] (10) The positive clones were inoculated into LB medium (containing ampicillin) for amplification and sent for sequencing.

[0155] (11) After obtaining the differential sequences, the differentially positive clones were inoculated into small shakes, amplified overnight, and stored in glycerol culture medium. The plasmids were then extracted for subsequent transformation.

[0156] Example 3: Expression of Thrombomodulin Single Domain Antibody in Escherichia coli WK6 Cells

[0157] (1) The pMECS-Nb recombinant plasmid of the extracted positive clone was added into Escherichia coli WK6 cells, and the exogenous plasmid was introduced into WK6 cells by electroporation.

[0158] (2) After culturing at 37°C in a shaking incubator for 1 hour, the culture was spread on an LB plate containing ampicillin and glucose and cultured at 37°C overnight.

[0159] (3) On the second day, a single clone was picked and inoculated into LB medium (containing ampicillin) for activation and cultured at 37°C overnight.

[0160] (4) On the second day, the activated bacteria were cultured in shake flasks and induced to express by IPTG.

[0161] (5) The supernatant containing the thrombomodulin single domain antibody was obtained by swelling the WK6 Escherichia coli cells with high concentration sucrose, osmotic pressure lysis with low concentration double volume sucrose, and high-speed centrifugation.

[0162] (6) Thrombomodulin single domain antibody was obtained after Ni column affinity purification, imidazole gradient elution and ultrafiltration concentration.

[0163] (7) The molecular weight and purity of the thrombomodulin single domain antibody were detected by SDS-PAGE protein electrophoresis.

[0164] like Figure 5 As shown, gel electrophoresis of the thrombomodulin single domain antibody Nb14 showed a molecular weight of approximately 15 kDa and good purity.

[0165] Example 4: Specificity, binding activity and thermal stability of thrombomodulin single domain antibodies

[0166] Specificity of Thrombomodulin Single Domain Antibody:

[0167] (1) 2ug / mL thrombomodulin antigen (MedChemExpress), human D-dimer antigen (Medix Biochemica), and human fibrinogen antigen (Sangon Biotech) diluted in 100mM NaHCO3 were coated on NUNC high-binding ELISA plates and placed in a refrigerator at 2-8°C for overnight coating.

[0168] (2) After washing 4 times with 1xPBST, 1% skim milk powder was added for blocking at room temperature for 2 h.

[0169] (3) After washing, add 2 ug / mL of thrombomodulin single domain antibody and place in a 37°C incubator for 1 h.

[0170] (4) After washing, add mouse anti-HA antibody and place in a 37°C incubator for 1 h.

[0171] (5) After washing, add HRP-labeled rabbit anti-mouse IgG and place in a 37°C incubator for 1 h.

[0172] (6) After washing and patting dry, add 100uL TMB substrate to each well and react at room temperature in the dark for 5-10 min or until the color is fully developed. Then add 50uL 2M H2SO4 to stop the color development.

[0173] like Figure 6 As shown, the antibody of the present invention can specifically recognize thrombomodulin molecules and does not react with D-dimer and fibrinogen.

[0174] Binding activity of thrombomodulin single domain antibody:

[0175] (1) Coat 2ug / mL TM diluted with 100mM NaHCO3 onto NUNC high-binding ELISA plates and place in a 2-8℃ refrigerator overnight.

[0176] (2) After washing 4 times with 1xPBST, 1% skim milk powder was added for blocking at room temperature for 2 h.

[0177] (3) After washing, serially diluted TM single domain antibody Nb14 and control mouse monoclonal antibody (Abcam: Anti-Thrombomodulin antibody [PBS-01]) were added, with the dilution solution being used as the zero concentration, and the cells were placed in a 37°C incubator for 1 h.

[0178] (4) After washing, the antibody of the present invention is added to the mouse anti-HA antibody (the monoclonal antibody is added to an equal amount of diluent) and placed in a 37° C. incubator for 1 hour.

[0179] (5) After washing, add HRP-labeled rabbit anti-mouse IgG and place in a 37°C incubator for 1 h.

[0180] (6) After washing and patting dry, add 100uL TMB substrate to each well and react at room temperature in the dark for 5-10 min or until the color is fully developed. Then add 50uL 2M H2SO4 to stop the color development.

[0181] like Figure 7 As shown, compared with the commercially available thrombomodulin monoclonal antibody, the antibody of the present invention has better binding activity.

[0182] Thermal stability of thrombomodulin single domain antibody:

[0183] (1) Coat 2ug / mL TM diluted with 100mM NaHCO3 onto NUNC high-binding ELISA plates and place in a 2-8℃ refrigerator overnight.

[0184] (2) After washing 4 times with 1xPBST, 1% skim milk powder was added for blocking at room temperature for 2 h.

[0185] (3) After washing, 2ug / mL TM single domain antibody Nb14 and 2ug / mL control mouse monoclonal antibody that had been heated in a water bath at different temperatures for 5 min and in a water bath at the same temperature for different times were added and placed in a 37°C incubator for 1 h.

[0186] (4) After washing, the antibody of the present invention is added to the mouse anti-HA antibody (the monoclonal antibody is added to an equal amount of diluent) and placed in a 37° C. incubator for 1 hour.

[0187] (5) After washing, add HRP-labeled rabbit anti-mouse IgG and place in a 37°C incubator for 1 h.

[0188] (6) After washing and patting dry, add 100uL TMB substrate to each well and react at room temperature in the dark for 5-10 min or until the color is fully developed. Then add 50uL 2M H2SO4 to stop the color development.

[0189] like Figure 8 As shown, compared with the commercially available thrombomodulin monoclonal antibody, the antibody of the present invention has better thermal stability.

[0190] Example 5: Construction of a double antibody sandwich method based on thrombomodulin single domain antibody

[0191] (1) 5 ug / mL of thrombomodulin single domain antibody Nb1 diluted with 100 mM NaHCO3 was coated on NUNC high binding ELISA plate and placed in a refrigerator at 2-8°C for overnight coating.

[0192] (2) After washing 4 times with 1xPBST, 1% casein was added for blocking at room temperature for 2 h.

[0193] (3) After washing, commercially available thrombomodulin antigen was diluted 2-fold from 1000 to 31.25 ng / mL, and the dilution was used as the zero concentration. The antigen diluted to the target concentration was added to the corresponding wells and placed in a 37°C incubator for 1 h.

[0194] (4) After washing, 100 uL of 2 ug / mL biotinylated thrombomodulin single domain antibody Nb14 was added to each well and placed in a 37°C incubator for 1 h.

[0195] (5) After washing, HRP-labeled streptavidin was added at a volume ratio of 1:2000 and placed in a 37°C incubator for 1 h.

[0196] (6) After washing and patting dry, add 100uL TMB substrate to each well and react at room temperature in the dark for 5-10 min or until the color is fully developed. Then add 50uL 2M H2SO4 to stop the color development.

[0197] like Fig. 9 As shown, the double antibody sandwich ELISA constructed based on the antibody of the present invention can be used to quantitatively detect thrombomodulin molecules.

[0198] Finally, it should be noted that the technical solution of the present invention is described in detail above in conjunction with the preferred embodiments, but it is not intended to limit the present invention. It should be understood by those skilled in the art that the present invention may be modified, deformed, improved, etc. without departing from the spirit and scope of the present invention, and any modification, deformation, improvement, etc. are within the protection scope of the present invention. The protection scope of the present invention is defined by the attached claims and their equivalents.

Claims

1. An anti-thrombomodulin single domain antibody, characterized in that: The complementary determining regions CDRs of the VHH chain in the single domain antibody are as follows: CDR1 shown in SEQ ID NO: 1, CDR2 shown in SEQ ID NO: 2, and CDR3 shown in SEQ ID NO:

3.

2. The anti-thrombomodulin single domain antibody according to claim 1, characterized in that The VHH chain of the single-domain antibody also includes a framework region FR, and the framework region FR is as follows: FR1 shown in SEQ ID NO:4, FR2 shown in SEQ ID NO:5, FR3 shown in SEQ ID NO:6, and FR4 shown in SEQ ID NO:

7.

3. An anti-thrombomodulin antibody, characterized in that: The antibody comprises one or more anti-thrombomodulin single domain antibodies as claimed in claim 1.

4. A polynucleotide, characterized in that The polynucleotide encodes a protein selected from the group consisting of the anti-thrombomodulin single domain antibody of claim 1 and the anti-thrombomodulin antibody of claim 3.

5. An expression vector, characterized in that: The expression vector contains the polynucleotide according to claim 4.

6. A host cell, characterized in that The host cell contains the expression vector of claim 5, or the polynucleotide of claim 4 is integrated into its genome.

7. A method for producing an anti-thrombomodulin single domain antibody, characterized in that: The method comprises the following steps: (1) culturing the host cell of claim 6 under conditions suitable for producing single-domain antibodies, thereby obtaining a culture containing the anti-thrombomodulin single-domain antibody; (2) isolating or recovering the anti-thrombomodulin single-domain antibody from the culture; and (3) optionally, purifying and / or modifying the anti-thrombomodulin single-domain antibody obtained in (2).

8. A method for detecting thrombomodulin in a sample, characterized in that: The method comprises the steps of: (1) contacting a sample with the anti-thrombomodulin single domain antibody of claim 1 and the anti-thrombomodulin antibody of claim 3; and (2) detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of thrombomodulin in the sample.

9. A thrombomodulin detection reagent, characterized in that: The detection reagent comprises: the anti-thrombomodulin single domain antibody according to claim 1, the anti-thrombomodulin antibody according to claim 3; and a carrier acceptable in detection.

10. A kit for detecting thrombomodulin, characterized in that: The kit contains the detection reagent according to claim 9 and instructions.

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