CN112851818A

CN112851818A - Binding protein for D-dimer, application thereof and method for detecting D-dimer

Info

Publication number: CN112851818A
Application number: CN201911193168.8A
Authority: CN
Inventors: 孟媛; 崔鹏; 何志强; 钟冬梅; 叶颖; 覃婷
Original assignee: Dongguan Pengzhi Biotechnology Co Ltd
Current assignee: Dongguan Pengzhi Biotechnology Co Ltd
Priority date: 2019-11-28
Filing date: 2019-11-28
Publication date: 2021-05-28
Anticipated expiration: 2039-11-28
Also published as: CN112851818B

Abstract

The present invention discloses a binding protein for D-dimer, its application, and a method for detecting D-dimer, and relates to the technical field of antibodies. The binding protein disclosed in the present invention includes an antigen-binding domain; the antigen-binding domain includes At least one of the following complementarity determination regions: G‑Y‑X1‑F‑T‑X2‑Y‑N‑X3‑H, Y‑I‑X1‑P‑Y‑X2‑G‑E‑T‑X3‑Y ‑N‑Q and A‑R‑X1‑G‑X2‑Y‑X3‑P‑W‑F‑T; the binding protein provided by the present invention can specifically bind to D‑dimer, and has better binding activity and Affinity, which is beneficial to improve the specificity and sensitivity of detection, can be used for the detection of D-dimer and the diagnosis of diseases with abnormal D-dimer levels, and provides a variety of protein choices for the detection of D-dimer. The binding protein It can also be used to prepare a thrombus-directed thrombolytic agent, which provides a new idea and choice for the thrombolysis of thrombus.

Description

Binding protein for D-dimer, application thereof and method for detecting D-dimer

Technical Field

The invention relates to the technical field of antibodies, in particular to a binding protein aiming at a D-dimer, application thereof and a method for detecting the D-dimer.

Background

D-dimer (DD) is fibrin in blood, and is activated and hydrolyzed to produce a specific, simplest degradation product, which is a specific marker of the fibrinolytic process. The fibrinolysis system in human body mainly maintains normal permeability of vascular wall, blood flow state and tissue repair, the organism generates blood coagulation in pathological state, thrombin can act on fibrin to convert into cross-linked fibrin, and the cross-linked fibrin releases fragments of X ', Y', D ', E' and the like in the dissolving process to form compounds of DD, DD/E, YD/YD, YY/DD and the like. These fragments are further degraded to the smallest fragments DD, with a molecular weight of about 62000D, and a half-life >3h in vivo, mainly excreted via the kidneys, and DD/E complexes.

The rise of the D-dimer reflects the existence of important molecular markers of coagulation and fibrinolytic activity enhancement in vivo, represents that blood clots are formed in the vascular circulation system, and is a sensitive marker of acute thrombosis. Myocardial infarction, cerebral infarction, pulmonary embolism, venous thrombosis, surgery, tumor, disseminated intravascular coagulation, infection and tissue necrosis can all cause D-dimer to rise.

In 1972, Gaffney first proposed D-dimer detection as a "useful tool" for monitoring blood coagulation disorders. In 1983, Rylatt et al subsequently proposed the detection of D-Dimer based on monoclonal antibodies (latex agglutination method). In recent years, researchers have introduced thrombus-directed thrombolytic agents using monoclonal antibodies and protein ligation techniques. The basic principle is to utilize the affinity between antigen (D-dimer) and antibody (anti-D-dimer monoclonal antibody) to link the monoclonal antibody of the antithrombotic specific component with the thrombolytic drug to form an antibody-thrombolytic complex. The monoclonal antibody can carry a thrombolytic agent to be specifically combined with thrombus like a missile, so that the thrombolytic agent at the thrombus part is highly aggregated, and the dissolving effect on the thrombus is enhanced. Later, the anti-D-dimer monoclonal antibody is used for labeling the radioactive nuclide, the radioactive nuclide can be carried to the local part of the thrombus in the process of the specific combination of the antibody and the antigen, and the distribution of the radioactive nuclide in vivo is monitored by a radioactive nuclide detector, so that the aim of positioning and diagnosing the thrombus by utilizing the guide tracer is fulfilled.

The D-dimer is clinically used as a specific monitoring index for thrombolytic therapy of thrombotic diseases. Since only the D-dimer cross-linked fragments in the degradation products of fibrinolytic proteins reflect the thrombolytic activity after thrombus formation. Therefore, in theory, the quantitative detection of D-dimer can quantitatively reflect the thrombolytic effect of the drug, and can be used for diagnosing and screening newly formed thrombus. In thrombolytic therapy, the D-dimer content changes are generally characterized as follows: firstly, the content of D-dimer after thrombolysis obviously rises in a short period and then gradually falls, thus prompting that the treatment is effective; ② the D-dimer content continuously rises or slowly falls after thrombolysis, which indicates insufficient dose of thrombolytic drug; ③ thrombolytic therapy should be continued until the D-dimer content is reduced to the normal range. In addition, after the completion of thrombolytic therapy, changes in D-dimer should be observed periodically over a period of time to prevent recurrence of thrombus.

Tumors have also been reported to cause elevated concentrations of D-dimer in patients, and may be used as a criterion for staging, prognosis, etc. Masatoshi Oya et al found in one study that D-dimer was significantly higher in colorectal cancer patients than in benign patients, and that pre-operative D-dimer was positively correlated with pathological outcome and staging of tumors. Patients with high preoperative D-dimer levels had significantly shorter postoperative survival than patients with low levels. D-dimer assays were performed on 128 patients with malignant tumors and found that: the acute leukemia group, the malignant lymphoma group and the solid tumor group are respectively obviously higher than the control group, have obvious difference (P <0.05), and have the remission stage obviously lower than the initial stage (P < 0.05).

Research shows that in liver diseases, the content of plasma D-dimer is obviously increased and is in positive correlation with the severity of liver diseases. Wilder et al determined 59 of the plasma D-dimers from individuals with acute and chronic liver disease, 48 of which were elevated to varying degrees. The D dimer level of each hepatitis patient is obviously higher than that of the control group and has significant difference (P <0.01), and the D dimer level of the patients is sequentially in the severe hepatitis group >; liver cirrhosis group >; in the chronic hepatitis group, severe group >; acute liver group >; chronic hepatitis, which may be associated with an impaired anticoagulant system.

In addition, the document also reports the application of the D-dimer in the clinical detection of various diseases such as cerebral infarction, myocardial infarction, nephropathy, neonatal asphyxia and the like.

There are many monoclonal antibodies that can distinguish between D-dimer and monomeric D in the clinic today, and can monitor fibrin degradation products in the presence of fibrinogen. These anti-D-dimer antibodies can detect not only free D-dimer, but also the polymerized D domain of large fibrin complexes, including the hinge fibrin complex formed early in the coagulation process. Accurate determination of the D-dimer antigen allows early detection of intravascular coagulation.

The detection method of D-dimer includes three P test, LATEX agglutination method (LATEX), ELISA method, immune percolation colloidal gold color reaction method, etc., and the clinical method is most commonly: ELISA, LATEX and the immune filtration colloidal gold staining method, wherein the LATEX method has high measuring speed, but has poor result repeatability and poor sensitivity compared with the ELISA method; the ELISA method has high sensitivity, but the detection time is long. The immune filtration colloidal gold color reaction can be quantitatively detected, the sensitivity is high, the result is comparable to the ELISA result, but the specificity is not strong, and the interference of lipid particles is avoided.

The detection methods used at present are all based on specific monoclonal antibodies, that is, the preparation of monoclonal antibodies aiming at D-dimers becomes the key for improving the detection sensitivity and specificity. There are some defects in sensitivity, specificity and affinity of the monoclonal antibodies currently used for detecting D-dimer, and there is a large room for improvement, and thus there is still a strong need in the art for monoclonal antibodies directed to detecting D-dimer.

Disclosure of Invention

The invention aims to provide a binding protein of a D-dimer, application thereof and a method for detecting the D-dimer. The binding protein provided by the invention can be specifically bound with a D-dimer, has better binding activity and affinity, is favorable for improving the specificity and sensitivity of detection, can be used for detecting the D-dimer and diagnosing diseases with abnormal D-dimer level, provides multiple protein choices for detecting the D-dimer, can also be used for preparing a thrombus-oriented thrombolytic agent, and provides a new idea and choice for thrombolysis of thrombus.

Noun definitions

The term "binding protein" broadly refers to all proteins/protein fragments, in particular antibodies or functional fragments of antibodies, comprising CDR regions. The term "antibody" includes polyclonal and monoclonal antibodies, and "antibody functional fragments" include antigen-compound-binding fragments of these antibodies, including Fab, F (ab') 2, Fd, Fv, scFv, diabodies, and minimal recognition units, as well as single chain derivatives of these antibodies and fragments. The type of antibody can be selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, IgD. Furthermore, the term "antibody" includes naturally occurring antibodies as well as non-naturally occurring antibodies, including, for example, chimeric (chimeric), bifunctional (bifunctional) and humanized (humanized) antibodies, as well as related synthetic isomeric forms (isoforms). The term "antibody" is used interchangeably with "immunoglobulin".

The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. The variable domain of the heavy chain may be referred to as "VH". The variable domain of the light chain may be referred to as "VL". These domains are usually the most variable parts of an antibody and contain an antigen binding site. The light or heavy chain variable region (VL or VH) is composed of framework regions interrupted by three hypervariable regions, termed "complementarity determining regions" or "CDRs". The extent of the framework regions and CDRs has been precisely defined, for example, in Kabat (see Sequences of Proteins of Immunological Interest), E.Kabat et al, U.S. department of Health and Human Services (U.S.. department of Health and Human Services), (1983), and Chothia. The framework regions of the antibody, which constitute the combination of the essential light and heavy chains, serve to locate and align the CDRs, which are primarily responsible for binding to the antigen.

As used herein, "framework region" or "FR" region means the region of an antibody variable domain excluding those defined as CDRs. Each antibody variable domain framework can be further subdivided into adjacent regions separated by CDRs (FR1, FR2, FR3 and FR 4).

Typically, the variable domains VL/VH of the heavy and light chains are obtained by linking the CDRs and FRs numbered as follows in a combinatorial arrangement: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR 4.

As used herein, the term "purified" or "isolated" in relation to a polypeptide or nucleic acid means that the polypeptide or nucleic acid is not in its native medium or native form. Thus, the term "isolated" includes a polypeptide or nucleic acid that is removed from its original environment, e.g., from its natural environment if it is naturally occurring. For example, an isolated polypeptide is generally free of at least some proteins or other cellular components that are normally bound to or normally mixed with it or in solution. Isolated polypeptides include the naturally-produced polypeptide contained in a cell lysate, the polypeptide in purified or partially purified form, recombinant polypeptides, the polypeptide expressed or secreted by a cell, and the polypeptide in a heterologous host cell or culture. In connection with a nucleic acid, the terms "isolated" or "purified" mean that the nucleic acid is not in its natural genomic context (e.g., in a vector, as an expression cassette, linked to a promoter, or artificially introduced into a heterologous host cell).

Exemplary embodiments of the invention:

in a first aspect, embodiments of the present invention provide a binding protein for a D-dimer, the binding protein comprising an antigen binding domain; the antigen binding domain comprises at least one of the following complementarity determining regions, or a similar complementarity determining region having at least 80% sequence identity with the sequence of at least one of the complementarity determining regions:

a complementarity determining region CDR-VH1 having the amino acid sequence G-Y-X1-F-T-X2-Y-N-X3-H, wherein: x1 is T or S, X2 is D or N, X3 is L, V or I;

a complementarity determining region CDR-VH2 having the amino acid sequence Y-I-X1-P-Y-X2-G-E-T-X3-Y-N-Q, wherein: x1 is Y or D, X2 is L, V or I, X3 is A or G;

a complementarity determining region CDR-VH3 having the amino acid sequence a-R-X1-G-X2-Y-X3-P-W-F-T, wherein: x1 is S or T, X2 is N or D, X3 is N or D;

a complementarity determining region CDR-VL1 having the amino acid sequence Q-S-X1-X2-N-S-G-X3-Q-K-X4-Y, wherein: x1 is I or L, X2 is F, V or L, X3 is S or T, X4 is D or N;

a complementarity determining region CDR-VL2 having the amino acid sequence X1-a-S-X2-R-X3-S, wherein: x1 is G, F or W, X2 is S or T, X3 is D or E;

a complementarity determining region CDR-VL3 having the amino acid sequence X1-X2-D-Y-S-Y-P-X2-T, wherein: x1 is Q or K, X2 is Q or N, and X3 is I, V or L.

The binding protein provided by the embodiment of the invention contains an antigen binding domain, the antigen binding domain comprises at least one of the complementarity determining regions, the amino acid sequence of the complementarity determining region is found and disclosed for the first time, the binding protein is a new sequence, the binding protein can be endowed with the capacity of specifically binding D-dimer antigen, and the binding protein has better binding activity and affinity.

In alternative embodiments, in the complementarity determining region CDR-VH1, X2 is D; in the complementarity determining region CDR-VH2, X1 is Y; in the complementarity determining region CDR-VH3, X1 is S; in the complementarity determining region CDR-VL1, X4 is N; in the complementarity determining region CDR-VL2, X2 is T; in the complementarity determining region CDR-VL3, X1 is Q.

In an alternative embodiment, in the complementarity determining region CDR-VH1, X1 is T.

In an alternative embodiment, in the complementarity determining region CDR-VH1, X1 is S.

In an alternative embodiment, in the complementarity determining region CDR-VH1, X3 is L.

In an alternative embodiment, in the complementarity determining region CDR-VH1, X3 is V.

In an alternative embodiment, in the complementarity determining region CDR-VH1, X3 is I.

In an alternative embodiment, in the complementarity determining region CDR-VH2, X2 is L.

In an alternative embodiment, in the complementarity determining region CDR-VH2, X2 is V.

In an alternative embodiment, in the complementarity determining region CDR-VH2, X2 is I.

In an alternative embodiment, in the complementarity determining region CDR-VH2, X3 is a.

In an alternative embodiment, in the complementarity determining region CDR-VH2, X3 is G.

In an alternative embodiment, in the complementarity determining region CDR-VH3, X2 is N.

In an alternative embodiment, in the complementarity determining region CDR-VH3, X2 is D.

In an alternative embodiment, in the complementarity determining region CDR-VH3, X3 is N.

In an alternative embodiment, in the complementarity determining region CDR-VH3, X3 is D.

In an alternative embodiment, in the complementarity determining region CDR-VL1, X1 is I.

In an alternative embodiment, in the complementarity determining region CDR-VL1, X1 is L.

In an alternative embodiment, in the complementarity determining region CDR-VL1, X2 is F.

In an alternative embodiment, in the complementarity determining region CDR-VL1, X2 is V.

In an alternative embodiment, in the complementarity determining region CDR-VL1, X2 is L.

In an alternative embodiment, in the complementarity determining region CDR-VL1, X3 is S.

In alternative embodiments, in the complementarity determining region CDR-VL1, X3 is T.

In an alternative embodiment, in the complementarity determining region CDR-VL2, X1 is G.

In an alternative embodiment, in the complementarity determining region CDR-VL2, X1 is F.

In an alternative embodiment, in the complementarity determining region CDR-VL2, X1 is W.

In an alternative embodiment, in the complementarity determining region CDR-VL2, X3 is D.

In an alternative embodiment, in the complementarity determining region CDR-VL2, X3 is E.

In an alternative embodiment, in the complementarity determining region CDR-VL3, X2 is Q.

In an alternative embodiment, in the complementarity determining region CDR-VL3, X2 is N.

In an alternative embodiment, in the complementarity determining region CDR-VL3, X3 is I.

In an alternative embodiment, in the complementarity determining region CDR-VL3, X3 is V.

In an alternative embodiment, in the complementarity determining region CDR-VL3, X3 is L.

In alternative embodiments, the similar complementarity determining regions have at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequences of the complementarity determining regions described above.

In alternative embodiments, the antigen binding domain has a K with the D-dimer protein_D≤5.95×10^- ⁷Affinity of mol/L.

In alternative embodiments, the antigen binding domain has a K with the D-dimer protein_D≤5×10^-7mol/L、4×10^-7mol/L、3×10^-7mol/L、2×10^-7mol/L、1×10^-7mol/L、9×10^-8mol/L、8×10^-8mol/L、7×10^-8mol/L、6×10^-8mol/L、5×10^-8mol/L、4×10^-8mol/L、3×10^-8mol/L、2×10^-8mol/L、1×10^-8mol/L、9×10^-9mol/L、8×10^-9mol/L、7×10^-9mol/L、6×10^-9mol/L or 5X 10^-9Affinity of mol/L.

In alternative embodiments, the antigen binding domain has a size of 5.40 × 10 with the D-dimer protein^-9mol/L≤K_D≤5.95×10^-7Affinity of mol/L.

The detection of KD was performed with reference to the method in the examples of the present invention.

In an alternative embodiment, the mutation site (i.e., Xn site, n ═ 1, 2, 3, or 4) in each of the complementarity determining regions described above is selected from any one of the following combinations of mutations 1-51:

in alternative embodiments, in the complementarity determining region CDR-VH1, X2 is N; in the complementarity determining region CDR-VH2, X1 is D; in the complementarity determining region CDR-VH3, X1 is T; in the complementarity determining region CDR-VL1, X4 is D; in the complementarity determining region CDR-VL2, X2 is S; in the complementarity determining region CDR-VL3, X1 is K.

In alternative embodiments, the mutation site (i.e., Xn site, n ═ 1, 2, 3, or 4) in each of the complementarity determining regions described above is selected from any one of the following combinations of mutations 52-60:

in alternative embodiments, the binding protein includes at least 3 complementarity determining regions (e.g., 3 complementarity determining regions of a heavy chain, or3 complementarity determining regions of a light chain); alternatively, the binding protein comprises at least 6 complementarity determining regions (e.g., 3 complementarity determining regions of a heavy chain and 3 complementarity determining regions of a light chain);

in alternative embodiments, the binding protein is a whole antibody comprising a variable region and a constant region.

In alternative embodiments, the binding protein is a functional fragment of an antibody, such as any one of a nanobody, F (ab ') 2, Fab', Fab, Fv, scFv, diabody, and antibody minimal recognition unit;

functional fragments of the above antibodies typically have the same binding specificity as the antibody from which they are derived. As will be readily understood by those skilled in the art based on the teachings of the present invention, functional fragments of the above antibodies can be obtained by methods such as enzymatic digestion (including pepsin or papain) and/or by chemical reduction to cleave disulfide bonds.

Functional fragments of the above antibodies can also be obtained by recombinant genetic techniques also known to those skilled in the art or synthesized by, for example, automated peptide synthesizers, such as those sold by Applied BioSystems and the like.

In an alternative embodiment, the binding protein comprises the light chain framework regions FR-L1, FR-L2, FR-L3 and FR-L4 in the sequence shown in SEQ ID Nos. 1-4, and/or the heavy chain framework regions FR-H1, FR-H2, FR-H3 and FR-H4 in the sequence shown in SEQ ID Nos. 5-8.

In addition, based on the disclosure of the present invention, the species source of the heavy chain or light chain framework region of the binding protein may be human, so as to constitute a humanized antibody.

In alternative embodiments, the binding protein further comprises an antibody constant region.

In alternative embodiments, the antibody constant region is selected from the constant regions of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD.

In alternative embodiments, the species of the antibody constant region is from a cow, horse, dairy cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose, turkey, chicken fountains, or human.

In alternative embodiments, the antibody constant region is derived from a mouse.

In alternative embodiments, the light chain constant region sequence of the antibody constant region is set forth in SEQ ID NO. 9 and the heavy chain constant region sequence of the antibody constant region is set forth in SEQ ID NO. 10.

The sequences of SEQ ID NOS: 1-10 are shown in the following table:

in a second aspect, the present embodiments provide the use of a binding protein according to any one of the preceding embodiments in the manufacture of a reagent or kit for the diagnosis of a disease with abnormal levels of D-dimers.

It should be noted that the binding protein provided by the present invention can detect D-dimer, therefore, any disease using D-dimer as a marker can be used to prepare the diagnosis or prognosis evaluation of the disease, and all of them fall into the scope of the present invention.

In an alternative embodiment, the disorder in which the level of D-dimer is abnormal is a thrombotic disorder.

In alternative embodiments, the thrombotic disorder is selected from one or more of myocardial infarction, cerebral infarction, pulmonary embolism, renal disease, tumor, and disseminated intravascular coagulation.

In a third aspect, the embodiments of the present invention provide a reagent or a kit for diagnosing a disease with abnormal D-dimer levels, which contains the binding protein as described above.

In a fourth aspect, embodiments of the present invention provide a thrombus-directed thrombolytic agent comprising a thrombolytic drug linked to a binding protein as described above.

The thrombolytic drug may be selected according to actual needs, and the present invention is not limited to the type thereof, and any thrombolytic drug may be selected, so long as the thrombolytic drug is a thrombus-oriented thrombolytic agent formed by linking the thrombolytic drug to the binding protein provided by the present invention, and the thrombolytic drug is within the scope of the present invention.

In a fifth aspect, an embodiment of the present invention provides a method for detecting a D-dimer, including: mixing a binding protein according to any one of the preceding embodiments with a sample to be tested.

In an alternative embodiment, the above detection method is aimed at the diagnosis of non-diseases.

It should be noted that one skilled in the art can perform qualitative or quantitative detection of the D-dimer protein in the sample to be tested based on the characteristics of the immune complex formed by the antibody/antigen combination. The method for detecting an antigen or an antibody based on the formation of an immune complex upon binding of the antibody to the antigen comprises:

(1) the detection purpose is realized by a precipitation reaction, which comprises the following steps: a one-way immunodiffusion test, a two-way immunodiffusion test, an immunoturbidimetry, a countercurrent immunoelectrophoresis, an immunoblotting, and the like;

(2) the detection purpose is realized by marking an indicator for displaying the signal intensity, and the method comprises the following steps: immunofluorescence, radioimmunoassay, enzyme-linked immunoassay (e.g., double antibody sandwich, indirect or competitive assay), and chemiluminescence immunoassay.

The indicator may be selected appropriately according to different detection methods, including but not limited to the indicators described below:

(1) in the immunofluorescence method, the indicator may be a fluorescent dye, for example, a fluorescein-based dye (including isothiocyanate (FIIC), hydroxyphoton (FAM), tetrachloro-fluorescein (TET), etc. or an analog thereof), a rhodamine-based dye (including red Rhodamine (RBITC), Tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc. or an analog thereof), a Cy-series dye (including Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy3, etc. or an analog thereof), an Alexa-series dye (including Alexa Fluor350, 405, 430, 488, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or an analog thereof), a protein-based dye (including Phycoerythrin (PE), phycocyanin (APC), allophycocyanin (phycocyanin), polymetaxanthin-chlorophyll protein (precP), etc.);

(2) in radioimmunoassays, the indicator may be a radioisotope, for example: 212Bi, 131I, 111In, 90Y, 186Re, 211At, 125I, 188Re, 153Sm, 213Bi, 32P, 94mTc, 99mTc, 203Pb, 67Ga, 68Ga, 43Sc, 47Sc, 110mIn, 97Ru, 62Cu, 64Cu, 67Cu, 68Cu, 86Y, 88Y, 121Sn, 161Tb, 166Ho, 105Rh, 177Lu, 172Lu, 18F, and the like.

(3) In enzyme-linked immunoassays, the indicator may be an enzyme that catalyzes the development of a substrate (e.g., horseradish peroxidase, alkaline phosphatase, or glucose oxidase, etc.).

(4) In chemiluminescent immunoassays, the indicator may be a chemiluminescent reagent such as acridinium ester, horseradish peroxidase and luminol, alkaline phosphatase and AMPPD, electrochemiluminescent agents ruthenium and tripropylamine, and the like.

Based on the disclosure of the above binding protein, those skilled in the art can easily think of using any one or a combination of several methods or other methods to realize the quantitative or qualitative detection of the D-dimer in the sample to be detected, and it is within the scope of the present invention to use the binding protein disclosed in the present invention to detect the D-dimer.

In alternative embodiments, the binding protein is labeled with an indicator that indicates the intensity of the signal, such that complexes of the binding protein bound to the D-dimer protein are detected.

In a sixth aspect, embodiments of the invention provide an isolated nucleic acid encoding a binding protein according to any one of the preceding embodiments;

in alternative embodiments, the nucleic acid is DNA or RNA.

Based on the disclosure of the amino acid sequence of the binding protein, one skilled in the art can easily obtain the nucleic acid sequence encoding the binding protein according to the codon corresponding to the amino acid, and obtain various nucleic acid sequences encoding the binding protein according to the degeneracy of the codon, which are within the protection scope of the present invention as long as they encode the binding protein.

In a seventh aspect, the embodiments provide a vector comprising a nucleic acid according to the previous embodiments.

Wherein the nucleic acid sequence is operably linked to at least one regulatory sequence. "operably linked" means that the nucleic acid sequence is linked to the regulatory sequence in a manner that allows expression. Regulatory sequences, including promoters, enhancers and other expression control elements, are selected to direct the expression of the protein of interest in a suitable host cell.

Herein, a vector may refer to a molecule or agent comprising a nucleic acid of the invention or a fragment thereof, capable of carrying genetic information and capable of delivering the genetic information into a cell. Typical vectors include plasmids, viruses, bacteriophages, cosmids and minichromosomes. The vector may be a cloning vector (i.e., a vector for transferring genetic information into a cell, which may be propagated and in which the genetic information may be present or absent) or an expression vector (i.e., a vector which comprises the necessary genetic elements to permit expression of the genetic information of the vector in a cell). Thus, a cloning vector may contain a selectable marker, as well as an origin of replication compatible with the cell type specified by the cloning vector, while an expression vector contains the regulatory elements necessary to effect expression in a specified target cell.

The nucleic acid of the invention or fragments thereof may be inserted into a suitable vector to form a cloning or expression vector carrying the nucleic acid fragment of the invention. Such novel vectors are also part of the present invention. The vector may comprise a plasmid, phage, cosmid, minichromosome, or virus, as well as naked DNA that is transiently expressed only in a particular cell. The cloning and expression vectors of the invention are capable of autonomous replication and thus provide high copy numbers for high level expression or high level replication purposes for subsequent cloning. The expression vector may comprise a promoter for driving expression of the nucleic acid fragment of the invention, optionally a signal peptide sequence encoding for secretion or integration of the protein expression product into a membrane, and optionally a nucleic acid sequence encoding for a terminator. When the expression vector is manipulated in a production strain or cell line, the vector, when introduced into a host cell, may or may not be integrated into the genome of the host cell. Vectors typically carry a replication site, as well as a marker sequence capable of providing phenotypic selection in transformed cells.

In an eighth aspect, embodiments of the present invention provide a host cell comprising a vector according to the previous embodiments.

The expression vectors of the invention are useful for transforming host cells. Such transformed host cells are also part of the invention and may be cultured cells or cell lines for propagation of the nucleic acid fragments and vectors of the invention, or for recombinant production of the binding proteins of the invention. Host cells of the present invention include microorganisms such as bacteria (e.g., Escherichia coli, Bacillus, etc.). Host cells also include cells from multicellular organisms such as fungi, insect cells, plant cells or mammalian cells, preferably from mammals, e.g., CHO cells.

In a ninth aspect, embodiments of the invention provide a method of producing a binding protein of any one of the preceding embodiments, comprising:

the host cell of the previous embodiment is cultured, and the binding protein is isolated and purified from the culture medium or from the cultured host cell.

The production method may be, for example, transfecting a host cell with a nucleic acid vector encoding at least a portion of the binding protein, and culturing the host cell under suitable conditions such that the binding protein is expressed. The host cell may also be transfected with one or more expression vectors, which may comprise, alone or in combination, DNA encoding at least a portion of the binding protein. The bound protein may be isolated from the culture medium or cell lysate using conventional techniques for purifying proteins and peptides, including ammonium sulfate precipitation, chromatography (e.g., ion exchange, gel filtration, affinity chromatography, etc.), and/or electrophoresis.

Construction of suitable vectors containing the coding and regulatory sequences of interest can be carried out using standard ligation and restriction techniques well known in the art. The isolated plasmid, DNA sequence or synthetic oligonucleotide is cleaved, tailed and religated as desired. Any method may be used to introduce mutations into the coding sequence to produce variants of the invention, and these mutations may comprise deletions or insertions or substitutions or the like.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is a reduced SDS-PAGE of the D-dimer monoclonal antibody of example 1.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the formulations or unit dosages herein, some are now described. Unless otherwise indicated, the techniques employed or contemplated herein are standard methods. The materials, methods, and examples are illustrative only and not intended to be limiting.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the skill of the art. Such techniques are well explained in the literature, e.g. "molecular cloning: a Laboratory Manual, second edition (Sambrook et al, 1989); oligonucleotide Synthesis (oligo Synthesis) (eds. m.j. goal, 1984); animal Cell Culture (Animal Cell Culture), ed.r.i. freshney, 1987; methods in Enzymology (Methods in Enzymology), Handbook of Experimental Immunology (Handbook of Experimental Immunology) (ed. D.M.Weir and C.C.Black well), Gene Transfer Vectors for Mammalian Cells (ed. J.M.Miller and M.P.Calos) (ed. J.M.and M.P.Calos) (ed. 1987), Methods in Current Generation (Current Protocols in Molecular Biology) (ed. F.M.Ausubel.et al, 1987), PCR, Polymerase Chain Reaction (ed. PCR: The Polymerase Chain Reaction) (ed. Mullis et al, 1994), and Methods in Current Immunology (ed. J.1991).

Several aspects of the invention are described below with reference to example applications for illustration. It should be understood that numerous specific details, relationships, and methods are set forth to provide a full understanding of the invention. One of ordinary skill in the relevant art will readily recognize, however, that the invention can be practiced without one or more of the specific details or with other methods. The present invention is not limited by the illustrated ordering of activities or events, as some activities may occur in different orders and/or concurrently with other activities or events. Moreover, not all illustrated acts or events are required to implement a methodology in accordance with the present invention.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

Restriction enzyme, Prime Star DNA polymerase, was purchased from Takara in this example. MagExtractor-RNA extraction kit was purchased from TOYOBO. BD SMART^TMRACE cDNA Amplification Kit was purchased from Takara. pMD-18T vector was purchased from Takara. Plasmid extraction kits were purchased from Tiangen corporation. Primer synthesis and gene sequencing were performed by Invitrogen corporation.

This example provides a method for preparing recombinant antibodies against D-dimer

1 construction of recombinant plasmid

(1) Primer and method for producing the same

Amplification of Heavy Chain and Light Chain 5' RACE primers:

(2) antibody variable region gene cloning and sequencing

RNA is extracted from a hybridoma cell line secreting an anti-D-dimer monoclonal antibody, first strand cDNA synthesis is carried out by using an SMARTERTM RACE cDNA Amplification Kit and SMARTER II A Oligonucleotide and 5' -CDS primers in the Kit, and an obtained first strand cDNA product is used as a PCR Amplification template. The Light Chain gene was amplified with Universal Primer A Mix (UPM), Nested Universal Primer A (NUP) and mIgG CKR primers, and the Heavy Chain gene was amplified with Universal Primer A Mix (UPM), Nested Universal Primer A (NUP) and mIgG CHR primers. The primer pair of Light Chain can amplify a target band about 0.8KB, and the primer pair of Heavy Chain can amplify a target band about 1.4 KB. The product was purified and recovered by agarose gel electrophoresis, and the product was subjected to A addition reaction with rTaq DNA polymerase, inserted into pMD-18T vector, transformed into DH 5. alpha. competent cells, and after colonies were grown, the Heavy Chain and Light Chain genes were cloned, respectively, and sent to Invitrogen for sequencing.

(3) Sequence analysis of variable region Gene of anti-D-dimer monoclonal antibody

Putting the gene sequence obtained by sequencing in an IMGT antibody database for analysis, and analyzing by using VNTI11.5 software to determine that the genes amplified by the heavy Chain primer pair and the Light Chain primer pair are correct, wherein in the gene fragment amplified by the Light Chain, the VL gene sequence is 342bp, belongs to VkII gene family, and a leader peptide sequence of 57bp is arranged in front of the VL gene sequence; in the gene fragment amplified by the Heavy Chain primer pair, the VH gene sequence is 357bp, belongs to a VH1 gene family, and has a leader peptide sequence of 57bp in front.

(4) Construction of recombinant antibody expression plasmid

pcDNA^TM 3.4

vector is a constructed recombinant antibody eukaryotic expression vector, and multiple cloning enzyme cutting sites such as HindIII, BamHI, EcoRI and the like are introduced into the expression vector and named as pcDNA3.4A expression vector, and the vector is called as 3.4A expression vector for short in the following; according to the sequencing result of the antibody gene in the pMD-18T, the light chain and heavy chain gene specific primers of the anti-D-dimer antibody are designed, two ends of the primers are respectively provided with HindIII and EcoRI enzyme cutting sites and protective bases, and the primers are as follows:

a0.75 KB Light Chain gene fragment and a 1.42KB Heavy Chain gene fragment were amplified by PCR amplification. The gene fragments of the Heavy Chain and the Light Chain are subjected to double enzyme digestion by HindIII/EcoRI respectively, the 3.4A vector is subjected to double enzyme digestion by HindIII/EcoRI, the Heavy Chain gene and the Light Chain gene are respectively connected into the 3.4A expression vector after the fragments and the vector are purified and recovered, and recombinant expression plasmids of the Heavy Chain and the Light Chain are respectively obtained.

2 Stable cell line selection

(1) Transient transfection of recombinant antibody expression plasmid into CHO cells and determination of expression plasmid activity

Plasmid was diluted to 400ng/ml with ultrapure water and CHO cells were conditioned at 1.43X 10⁷cells/ml are put into a centrifuge tube, 100 mul of plasmid is mixed with 700 mul of cells, the mixture is transferred into an electric rotating cup and is electrically rotated, the sampling counting is carried out on 3 rd, 5 th and 7 th days, and the sampling detection is carried out on 7 th day.

Diluting goat anti-mouse IgG1 mu g/ml with the coating solution to coat the microplate, wherein each well is 100 mu l, and the temperature is 4 ℃ overnight; the next day, washing with the washing solution for 2 times, and patting dry; adding blocking solution (20% BSA + 80% PBS), beating to dry at 37 deg.C for 1 hr, and adding blocking solution (120 μ l per well); adding diluted cell supernatant at a concentration of 100 μ l/well at 37 deg.C for 60 min; throwing off liquid in the plate, patting dry, adding 20% mouse negative blood, sealing, and sealing at 37 ℃ for 1h, wherein each hole is 120 mu l; throwing off the liquid in the plate, patting to dry, adding diluted D-dimer antigen 100 mul per hole, 37 ℃ for 40 min; washing with washing solution for 5 times, and drying; adding HRP-labeled D-dimer monoclonal antibody into the reaction solution, wherein each well is 100 mu l, and the temperature is 37 ℃ for 30 min; adding a developing solution A (50 μ l/hole), adding a developing solution B (50 μ l/hole), and standing for 10 min; adding stop solution into the mixture, wherein the concentration of the stop solution is 50 mu l/hole; OD readings were taken at 450nm (reference 630nm) on the microplate reader. The results showed that the OD of the reaction after the cell supernatant was diluted 1000 times was still greater than 1.0, and the OD of the reaction in the wells without the cell supernatant was less than 0.1, indicating that the antibodies generated immediately after the plasmid was transformed were active against the D-dimer protein.

(2) Linearization of recombinant antibody expression plasmids

The following reagents were prepared: 50 mul Buffer, 100 mul DNA/tube, 10 mul Puv I enzyme, and sterile water to 500 mul, water bath enzyme digestion overnight at 37 ℃; sequentially extracting with equal volume of phenol/chloroform/isoamyl alcohol (lower layer) 25:24:1 and then chloroform (water phase); precipitating with 0.1 volume (water phase) of 3M sodium acetate and 2 volumes of ethanol on ice, rinsing with 70% ethanol, removing organic solvent, re-melting with appropriate amount of sterilized water after ethanol is completely volatilized, and finally measuring concentration.

(3) Stable transfection of recombinant antibody expression plasmid, pressurized screening of stable cell lines

Plasmid was diluted to 400ng/ml with ultrapure water and CHO cells were conditioned at 1.43X 10⁷cells/ml are put into a centrifuge tube, 100 mul of plasmid is mixed with 700 mul of cells, and the mixture is transferred into an electric rotating cup and is electrically rotated, and the next day is counted; 25 mu mol/L MSX 96-well pressure culture for about 25 days。

Observing the marked clone holes with cells under a microscope, and recording the confluence degree; taking culture supernatant, and sending the culture supernatant to a sample for detection; selecting cell strains with high antibody concentration and relative concentration, transferring the cell strains into 24 holes, and transferring the cell strains into 6 holes after 3 days; after 3 days, the seeds were kept and cultured in batches, and the cell density was adjusted to 0.5X 10⁶cells/ml, 2.2ml, cell density 0.3X 10⁶cell/ml, 2ml for seed preservation; and (4) 7 days, carrying out batch culture supernatant sample sending detection in 6 holes, and selecting cell strains with small antibody concentration and cell diameter to transfer TPP for seed preservation and passage.

3 recombinant antibody production

(1) Cell expanding culture

After the cells are recovered, the cells are cultured in a shaking flask with the specification of 125ml, the inoculation volume is 30ml, the culture medium is 100% Dynamis culture medium, and the cells are placed in a shaking table with the rotation speed of 120r/min, the temperature of 37 ℃ and the carbon dioxide of 8%. Culturing for 72h, inoculating and expanding culture at an inoculation density of 50 ten thousand cells/ml, wherein the expanding culture volume is calculated according to production requirements, and the culture medium is 100% Dynamis culture medium. Then the culture is expanded every 72 h. When the cell amount meets the production requirement, the production is carried out by strictly controlling the inoculation density to be about 50 ten thousand cells/ml.

(2) Shake flask production and purification

Shake flask parameters: the rotating speed is 120r/min, the temperature is 37 ℃, and the carbon dioxide is 8 percent. Feeding in a flowing mode: daily feeding was started when the culture was carried out for 72h in a shake flask, 3% of the initial culture volume was fed daily to HyCloneTM Cell BoostTM Feed 7a, and one thousandth of the initial culture volume was fed daily to Feed 7b, up to day 12 (day 12 feeding). Glucose was supplemented with 3g/L on the sixth day. Samples were collected on day 13. Affinity purification was performed using a proteinA affinity column. Mu.g of the purified antibody (i.e., the D-dimer monoclonal antibody) was subjected to reducing SDS-PAGE, and the electrophoretogram thereof was as shown in FIG. 1. Two bands were shown after reducing SDS-PAGE, 1 with 50kD of Mr (i.e., heavy chain, SEQ ID NO:14) and 28kD of Mr (i.e., light chain, SEQ ID NO: 12).

Example 2

Detection of antibody Performance

(1) Example 1 Activity assay of antibodies and mutants thereof

For further analysis, the variable region of the heavy chain of the D-dimer monoclonal antibody (WT) of example 1 is shown in SEQ ID NO 13, wherein the amino acid sequences of the complementarity determining regions of the heavy chain are as follows:

CDR-VH1：G-Y-S(X1)-F-T-N(X2)-Y-N-V(X3)-H；

CDR-VH2：Y-I-D(X1)-P-Y-L(X2)-G-E-T-A(X3)-Y-N-Q；

CDR-VH3：A-R-T(X1)-G-N(X2)-Y-D(X3)-P-W-F-T；

the light chain variable region is shown as SEQ ID NO. 11, wherein the amino acid sequences of the complementarity determining regions of the light chain are as follows:

CDR-VL1：Q-S-I(X1)-V(X2)-N-S-G-S(X3)-Q-K-D(X4)-Y；

CDR-VL2：G(X1)-A-S-S(X2)-R-D(X3)-S；

CDR-VL3：K(X1)-Q(X2)-D-Y-S-Y-P-I(X2)-T。

on the basis of the D-dimer monoclonal antibody of example 1, mutations were made in the complementarity determining regions at sites relevant to the activity of the antibody, wherein X1, X2, X3, and X4 were all mutated sites. See table 1 below.

TABLE 1 mutant sites associated with antibody Activity

And (3) detecting the binding activity:

coating solution (PBS) dilutes goat anti-mouse IgG1 mug/ml for coating a microplate, each well is 100 mug, and the temperature is kept overnight at 4 ℃; the next day, washing with washing solution (PBS) for 2 times, and patting dry; adding blocking solution (20% BSA + 80% PBS), beating to dry at 37 deg.C for 1 hr, and adding blocking solution (120 μ l per well); adding diluted D-dimer monoclonal antibody, 100 μ l/well, 37 deg.C, 60 min; throwing off liquid in the plate, patting dry, adding 20% mouse negative blood, sealing, and sealing at 37 ℃ for 1h, wherein each hole is 120 mu l; throwing off the liquid in the plate, patting to dry, adding diluted D-dimer, 100 mul per hole, 37 ℃, 40 min; washing with washing solution for 5 times, and drying;adding another HRP-labeled D-dimer monoclonal antibody into the mixed solution, wherein each well is filled with 100 mu l of the HRP-labeled D-dimer monoclonal antibody, and the temperature is 37 ℃ for 30 min; adding color development liquid A (50 μ L/well containing 2.1g/L citric acid, 12.25g/L citric acid, 0.07g/L acetanilide and 0.5g/L carbamide peroxide) and adding color development liquid B (50 μ L/well containing 1.05g/L citric acid, 0.186g/L LEDTA.2Na, 0.45g/L TMB and 0.2ml/L concentrated HCl) for 10 min; adding stop solution (containing 0.75 g/EDTA-2 Na and 10.2ml/L concentrated H)₂SO₄) 50 μ l/well; OD readings were taken at 450nm (reference 630nm) on the microplate reader. The results are shown in Table 2.

TABLE 2 Activity data of antibodies and mutants thereof

Antibody concentration (ng/ml)	125	62.5	31.25	15.625	7.8125	0
							WT	1.725	1.213	0.956	0.652	0.431	0.023
Mutation 1	1.984	1.481	1.129	0.871	0.539	0.048
							Mutation 2	1.918	1.482	1.105	0.861	0.546	0.074
Mutation 3	1.900	1.474	1.136	0.894	0.581	0.067
							Mutation 4	1.976	1.449	1.109	0.886	0.517	0.059
Mutation 5	0.845	0.567	0.315	0.243	0.201	0.046
							Mutation 6	0.224	0.031	-	-	-	-
Mutation 7	0.356	0.021	-	-	-	-

As can be seen from the data in Table 2, WT and mutations 1 to 5 had binding activity to antigen, while mutations 6 and 7 did not detect OD values at concentrations below 31.25ng/ml, indicating that the amino acid mutation patterns at the mutation sites listed in Table 1 have different expectations.

(2) Example 1 affinity assays for antibodies and mutants thereof

(a) Based on mutation 1, other sites were mutated, and the sequence of each mutation is shown in table 3 below.

TABLE 3 mutation sites related to antibody affinity

And (3) affinity detection:

using AMC sensors, purified antibodies (Table 3 in each of the mutant antibodies) with PBST from 50 u g/ml for 2 times gradient dilution, D dimer with PBST gradient dilution;

the operation flow is as follows: equilibrating in buffer 1(PBST) for 60s, immobilizing antibody in antibody solution for 300s, incubating in buffer 2(PBST) for 180s, binding in antigen solution for 420s, dissociating in buffer 2 for 1200s, regenerating the sensor with 10mM GLY solution pH 1.69 and buffer 3, and outputting the data. (KD represents the equilibrium dissociation constant, i.e.affinity; kon represents the association rate; kdis represents the dissociation rate.)

Table 4 affinity assay data

As can be seen from Table 4, the affinity of mutation 1 and mutations 1-1 to 1-50 is better, which indicates that the mutation pattern of the mutation sites in Table 5 has no negative effect on the affinity, and better affinity can be obtained after mutation.

(b) Based on WT, mutation is carried out on other sites, and the affinity of each mutant is detected, the sequence of each mutation is shown in Table 5, and the corresponding affinity data is shown in Table 6.

TABLE 5 mutations with WT as backbone

TABLE 6 results of affinity assay of mutations with WT as backbone

The data in Table 6 show that the mutant sites listed in Table 5 have less influence on the affinity of the antibody and all have better affinity.

(3) Evaluation of stability against naked antibody

Placing the antibodies in the table at 4 ℃ (refrigerator), -80 ℃ (refrigerator) and 37 ℃ (thermostat) for 21 days, taking samples at 7 days, 14 days and 21 days for state observation, and performing activity detection on the samples at 21 days, wherein the result shows that no obvious protein state change is seen in 21 days of placing the antibodies under three examination conditions, and the activity is more prone to decrease with the increase of the examination temperature, which indicates that the self-produced antibodies are stable. The following table shows the results of the 21-day evaluation of the OD enzyme immunity assay with mutation 1.

TABLE 7

Antibody concentration (ng/ml)	125	31.25	0
				Samples at 4 ℃ for 21 days	1.827	1.108	0.059
21 days samples at-80 deg.C	1.841	1.116	0.048
				At 37 deg.C for 21 daysSample (I)	1.894	1.135	0.074

After the antibody is stored for 21 days at different temperatures, the activity of the antibody provided by the embodiment of the invention is not changed greatly, which indicates that the antibody provided by the embodiment of the invention has better stability.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

SEQUENCE LISTING

<110> Dongguan City of Pengzhi Biotech Co., Ltd

<120> binding protein for D-dimer, use thereof, and method for detecting D-dimer

<160> 14

<170> PatentIn version 3.5

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Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Gln Thr Val

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Glu Val Gln Phe Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

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Gly Tyr Ile Asp Pro Tyr Leu Gly Glu Thr Ala Tyr Asn Gln Lys Phe

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Lys Ser Lys Ala Thr Leu Thr Val Asp Asp Ser Ser Ser Thr Ala Tyr

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Glu Val Gln Phe Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

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Gly Tyr Ile Asp Pro Tyr Leu Gly Glu Thr Ala Tyr Asn Gln Lys Phe

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Lys Ser Lys Ala Thr Leu Thr Val Asp Asp Ser Ser Ser Thr Ala Tyr

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Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

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Ala Arg Thr Gly Asn Tyr Asp Pro Trp Phe Thr Tyr Trp Gly Gln Gly

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Thr Leu Val Thr Val Ser Ala Ala Lys Thr Thr Pro Pro Ser Val Tyr

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Pro Leu Ala Pro Gly Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu

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Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Ser Val Thr Val Thr Trp

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Asn Ser Gly Ser Leu Ser Ser Ser Val His Thr Phe Pro Ala Leu Leu

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Gln Ser Gly Leu Tyr Thr Met Ser Ser Ser Val Thr Val Pro Ser Ser

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Thr Trp Pro Ser Gln Thr Val Thr Cys Ser Val Ala His Pro Ala Ser

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Ser Thr Thr Val Asp Lys Lys Leu Glu Pro Ser Gly Pro Ile Ser Thr

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Ile Asn Pro Cys Pro Pro Cys Lys Glu Cys His Lys Cys Pro Ala Pro

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Asn Leu Glu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Asn Ile Lys

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Asp Val Leu Met Ile Ser Leu Thr Pro Lys Val Thr Cys Val Val Val

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Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn

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Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr

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Asn Ser Thr Ile Arg Val Val Ser Ala Leu Pro Ile Gln His Gln Asp

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Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu

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Pro Ser Pro Ile Glu Arg Thr Ile Ser Lys Ile Lys Gly Leu Val Arg

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Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu Met Thr Lys

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Lys Asp Val Ser Leu Thr Cys Leu Val Val Gly Phe Asn Pro Gly Asp

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Ile Ser Val Glu Trp Thr Ser Asn Gly His Thr Glu Glu Asn Tyr Lys

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Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser

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Lys Leu Asn Met Lys Thr Ser Lys Trp Glu Lys Thr Asp Ser Phe Ser

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Cys Asn Val Arg His Glu Gly Leu Lys Asn Tyr Tyr Leu Lys Lys Thr

435 440 445

Ile Ser Arg Ser Pro Gly

450

Claims

1. A binding protein to a D-dimer, wherein said binding protein comprises an antigen binding domain; the antigen binding domain comprises at least one of the following complementarity determining regions, or a similar complementarity determining region having at least 80% sequence identity with the sequence of at least one of the complementarity determining regions:

2. The binding protein of claim 1,

in the complementarity determining region CDR-VH1, X2 is D;

in the complementarity determining region CDR-VH2, X1 is Y;

in the complementarity determining region CDR-VH3, X1 is S;

in the complementarity determining region CDR-VL1, X4 is N;

in the complementarity determining region CDR-VL2, X2 is T;

in the complementarity determining region CDR-VL3, X1 is Q;

preferably, in the complementarity determining region CDR-VH1, X1 is T;

preferably, in the complementarity determining region CDR-VH1, X1 is S;

preferably, in the complementarity determining region CDR-VH1, X3 is L;

preferably, in the complementarity determining region CDR-VH1, X3 is V;

preferably, in the complementarity determining region CDR-VH1, X3 is I;

preferably, in the complementarity determining region CDR-VH2, X2 is L;

preferably, in the complementarity determining region CDR-VH2, X2 is V;

preferably, in the complementarity determining region CDR-VH2, X2 is I;

preferably, in the complementarity determining region CDR-VH2, X3 is a;

preferably, in the complementarity determining region CDR-VH2, X3 is G;

preferably, in the complementarity determining region CDR-VH3, X2 is N;

preferably, in the complementarity determining region CDR-VH3, X2 is D;

preferably, in the complementarity determining region CDR-VH3, X3 is N;

preferably, in the complementarity determining region CDR-VH3, X3 is D;

preferably, in the complementarity determining region CDR-VL1, X1 is I;

preferably, in the complementarity determining region CDR-VL1, X1 is L;

preferably, in the complementarity determining region CDR-VL1, X2 is F;

preferably, in the complementarity determining region CDR-VL1, X2 is V;

preferably, in the complementarity determining region CDR-VL1, X2 is L;

preferably, in the complementarity determining region CDR-VL1, X3 is S;

preferably, in the complementarity determining region CDR-VL1, X3 is T;

preferably, in the complementarity determining region CDR-VL2, X1 is G;

preferably, in the complementarity determining region CDR-VL2, X1 is F;

preferably, in the complementarity determining region CDR-VL2, X1 is W;

preferably, in the complementarity determining region CDR-VL2, X3 is D;

preferably, in the complementarity determining region CDR-VL2, X3 is E;

preferably, in the complementarity determining region CDR-VL3, X2 is Q;

preferably, in the complementarity determining region CDR-VL3, X2 is N;

preferably, in the complementarity determining region CDR-VL3, X3 is I;

preferably, in the complementarity determining region CDR-VL3, X3 is V;

preferably, in the complementarity determining region CDR-VL3, X3 is L;

preferably, the antigen binding domain has a K with the D-dimer protein_D≤5.95×10^-7Affinity of mol/L;

preferably, the mutation site of each complementarity determining region of the antigen binding domain is selected from any one of the following combinations of mutations 1-51:

。

3. the binding protein of claim 1,

in the complementarity determining region CDR-VH1, X2 is N;

in the complementarity determining region CDR-VH2, X1 is D;

in the complementarity determining region CDR-VH3, X1 is T;

in the complementarity determining region CDR-VL1, X4 is D;

in the complementarity determining region CDR-VL2, X2 is S;

in the complementarity determining region CDR-VL3, X1 is K;

preferably, the mutation site of each complementarity determining region of the antigen binding domain is selected from any one of the following combinations of mutations 52-60:

。

4. the binding protein according to any one of claims 1 to 3, wherein at least 3 complementarity determining regions are included in the binding protein; alternatively, the binding protein comprises at least 6 complementarity determining regions;

preferably, the binding protein is an antibody or a functional fragment thereof;

preferably, the binding protein is selected from any one of nanobody, F (ab ') 2, Fab', Fab, Fv, scFv, diabody, and antibody minimal recognition unit;

preferably, the binding protein comprises light chain framework regions FR-L1, FR-L2, FR-L3 and FR-L4 which have the sequences shown in SEQ ID NO. 1-4 in sequence, and/or heavy chain framework regions FR-H1, FR-H2, FR-H3 and FR-H4 which have the sequences shown in SEQ ID NO. 5-8 in sequence;

preferably, the binding protein further comprises an antibody constant region;

preferably, the antibody constant region is selected from the constant regions of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD;

preferably, the species of the antibody constant region is from cattle, horses, dairy cattle, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, camels, donkeys, deer, mink, chickens, ducks, geese, turkeys, bangles, or humans;

preferably, the antibody constant region is derived from a mouse;

preferably, the light chain constant region sequence of the antibody constant region is shown as SEQ ID NO. 9, and the heavy chain constant region sequence of the antibody constant region is shown as SEQ ID NO. 10.

5. Use of a binding protein according to any one of claims 1 to 4 in the manufacture of a reagent or kit for the diagnosis of a disease with abnormal levels of D-dimer;

preferably, the disorder in which the level of D-dimer is abnormal is a thrombotic disorder;

preferably, the thrombotic disease is selected from one or more of myocardial infarction, cerebral infarction, pulmonary embolism, nephropathy, tumor and disseminated intravascular coagulation.

6. A reagent or kit for diagnosing a disease with abnormal D-dimer levels, comprising the binding protein of any one of claims 1 to 4;

7. A thrombus-directed thrombolytic agent comprising a thrombolytic drug having attached thereto a binding protein according to any one of claims 1-4.

8. A method for detecting D-dimers, comprising: mixing the binding protein of any one of claims 1-4 with a sample to be tested;

preferably, the method is a method for detecting D-dimer by precipitation reaction or a method for detecting D-dimer by labeling an indicator showing signal intensity;

preferably, the method for detecting the D-dimer by the precipitation reaction is selected from any one or more of the following methods: a one-way immunodiffusion test, a two-way immunodiffusion test, an immunoturbidimetry, a countercurrent immunoelectrophoresis, an immunoelectrophoresis, and an immunoblotting method;

preferably, the method for detecting the D-dimer by marking the indicator showing the signal intensity is selected from any one or more of the following methods: immunofluorescence, radioimmunoassay, enzyme-linked immunoassay, and chemiluminescent immunoassay;

preferably, the indicator is selected from any one of a fluorescent dye, a radioisotope, an enzyme catalyzing color development of a substrate, and a chemiluminescent reagent.

9. A host cell, comprising a vector; the vector comprises a nucleic acid encoding the binding protein of any one of claims 1 to 4.

10. A method of producing the binding protein of any one of claims 1 to 4, comprising:

culturing the host cell of claim 9, and isolating and purifying the binding protein from the culture medium or from the cultured host cell.