D-Dimer Antibodies and Their Uses

Abstract

The present invention provides an antibody, comprising: a heavy chain variable region and / or a light chain variable region; wherein, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 1 or a variant thereof, and compared with the amino acid sequence shown in SEQ ID NO: 1, the variant of SEQ ID NO: 1 comprises mutations at at least one of the following positions: the 50th, 59th, 99th, and 102nd positions; the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 2 or a variant thereof, and compared with the amino acid sequence shown in SEQ ID NO: 2, the variant of SEQ ID NO: 2 comprises mutations at at least one of the following positions: the 32nd, 34th, 60th, and 61st positions. The antibody of the present invention can specifically bind to D-dimer and can be used for qualitative or quantitative detection of D-dimer.

Description

[0001] Cross - reference to related applications

[0002] This application claims the priority of a Chinese patent application with the application number 202211679637.9 and the title "D - dimer antibody and its uses" filed with the Chinese Patent Office on December 26, 2022, the entire content of which is incorporated herein by reference. Technical field

[0003] The present invention belongs to the technical field of antibodies. Specifically, the present invention relates to a D - dimer antibody and its uses. More specifically, the present invention relates to an antibody, nucleic acid molecule, vector, cell or host of D - dimer, a method for preparing an antibody, a conjugate, a reagent or a kit and its uses, a method for detecting D - dimer, a method for screening D - dimer antibodies, and a mutant library. Background art

[0004] D - dimer is the end - product of cross - linked fibrin after the action of plasmin. During the blood - clotting process, after thrombin hydrolyzes fibrinogen, fibrinopeptide A (FPA) and peptide B (FPB) are successively released, and the remaining part is soluble fibrin monomer (SFM). Under the action of transamidase, SFM is transformed into fibrin, and then blood coagulation occurs. The process is completed after a series of cross - linkings. The formed fibrin is stable in nature and generally insoluble, but can be degraded by plasmin. During the degradation of cross - linked fibrin by plasmin, several kinds of polymers are gradually generated, and D - dimer is one of its specific products, with a molecular weight of 184,000 - 202,000. Under pathological conditions, the dynamic balance between blood coagulation and fibrinolysis is disrupted, the tendency of blood coagulation is enhanced, so the fibrin degradation products increase, resulting in an increase in the content of D - dimer. An increase in the D - dimer level indicates that there is fibrin thrombosis formation and fibrinolysis in the body, so it can be used clinically as a molecular marker for hypercoagulable state and hyperfibrinolysis in the body.

[0005] The detection of D - dimer has important value for the diagnosis and treatment of various diseases. In particular, an increase in the content of D - dimer can detect the course of various diseases, such as deep vein thrombosis (DVT), disseminated intravascular coagulation (DIC), myocardial infarction, severe hepatitis, pulmonary embolism (PE), etc.; it also has a certain monitoring effect on the possible complications of pregnant women with pre - eclampsia and high - risk pregnancy. Moreover, the change in the D - dimer level can be used as an index for monitoring thrombolytic therapy and guiding the dosage of thrombolytic drugs. Thus, the detection of D - dimer has important clinical significance in aspects such as the early diagnosis of thrombotic diseases, course monitoring, and treatment monitoring of thrombolytic drugs.

[0006] Currently, the detection of D-dimer includes three batches of tests, latex agglutination method (LATEX), ELISA method, immunofiltration colloidal gold color reaction method, etc. These methods all require antibodies against D-dimer. Therefore, it is urgent to develop an antibody that can effectively bind D-dimer. Summary of the Invention

[0007] The present invention aims to provide an antibody against D-dimer, a reagent or a kit for detecting D-dimer.

[0008] In the first aspect of the present invention, an antibody is proposed, which includes: a heavy chain variable region and / or a light chain variable region; wherein, the heavy chain variable region includes an amino acid sequence shown in SEQ ID NO: 1 or a variant thereof. Compared with the amino acid sequence shown in SEQ ID NO: 1, the variant of SEQ ID NO: 1 includes mutations at at least one of the following positions: position 50, position 59, position 99, and position 102; the light chain variable region includes an amino acid sequence shown in SEQ ID NO: 2 or a variant thereof. Compared with the amino acid sequence shown in SEQ ID NO: 2, the variant of SEQ ID NO: 2 includes mutations at at least one of the following positions: position 32, position 34, position 60, and position 61.

[0009] In the second aspect of the present invention, an antibody is proposed, which includes a heavy chain and / or a light chain. The heavy chain includes the heavy chain variable region defined in the antibody described in the first aspect; the light chain includes the light chain variable region defined in the antibody described in the first aspect.

[0010] In the third aspect of the present invention, an antibody is proposed, which includes HCDRs and LCDRs. The HCDRs include or are HCDRs that are identical to the HCDRs of the heavy chain variable region defined in the antibody described in the first aspect or the second aspect, and the LCDRs include or are LCDRs that are identical to the LCDRs of the light chain variable region defined in the antibody described in the first aspect or the second aspect.

[0011] In the fourth aspect of the present invention, a nucleic acid molecule is proposed, which encodes the antibody described in the first aspect, the second aspect, or the third aspect.

[0012] In the fifth aspect of the present invention, a vector is proposed, which includes the nucleic acid molecule described in the fourth aspect.

[0013] In the sixth aspect of the present invention, a cell or a host is proposed, which includes: the nucleic acid molecule described in the fourth aspect or the vector described in the fifth aspect; or expresses the antibody described in the first aspect, the second aspect, or the third aspect.

[0014] In a seventh aspect of the present invention, there is provided a method for preparing the antibody according to the first, second or third aspect, the method comprising culturing the cell or host according to the sixth aspect.

[0015] In an eighth aspect of the present invention, there is provided a conjugate, the conjugate comprising: the antibody according to the first, second or third aspect and a conjugate moiety conjugated thereto.

[0016] In a ninth aspect of the present invention, there is provided a reagent or kit, the reagent or kit comprising: the antibody according to the first, second or third aspect or the conjugate according to the eighth aspect.

[0017] In a tenth aspect of the present invention, there is provided the use of the antibody according to the first, second or third aspect, the conjugate according to the eighth aspect or the reagent or kit according to the ninth aspect in detecting D-dimer or in preparing a product for diagnosing D-dimer related diseases.

[0018] In an eleventh aspect of the present invention, there is provided a method for detecting D-dimer, the method comprising: contacting a sample to be detected with the antibody according to the first, second or third aspect, the conjugate according to the eighth aspect or the reagent or kit according to the ninth aspect to form an immune complex.

[0019] In a twelfth aspect of the present invention, there is provided a method for screening D-dimer antibodies, the method comprising: a) designing primers for amino acid substitution at 1, 2, 3, 4, 5, 6, 7 or 8 sites among X1, X2, X3, X4, X5, X6, X7 and X8 defined in the antibody according to the second aspect; b) constructing a mutant library with the primers described in a) using the nucleic acid molecule according to the fourth aspect, the vector according to the fifth aspect or the cell according to the sixth aspect as a template; c) screening D-dimer antibodies from the mutant library.

[0020] In a thirteenth aspect of the present invention, there is provided a mutant library, the mutant library comprising variants of SEQ ID NO:1 and variants of SEQ ID NO:2 defined in the antibody according to the first, second or third aspect.

[0021] Additional aspects and advantages of the present invention will be given in part in the following description, become apparent in part from the following description, or be learned through the practice of the present invention. Detailed Description of the Invention

[0022] The embodiments of the present invention will be described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0023] It should be noted that the terms "first" and "second" are for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, features defined with "first" and "second" may explicitly or implicitly include one or more of such features. Further, in the description of the present invention, unless otherwise specified, "a plurality of" means two or more.

[0024] The endpoints and any values disclosed herein in ranges are not limited to the exact ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0025] For easier understanding of the present invention, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention pertains. The abbreviations of amino acid residues are the standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 common L-amino acids.

[0026] In this document, the term "comprising" or "including" is an open-ended expression, that is, it includes the content specified by the present invention, but does not exclude other aspects.

[0027] In this document, the terms "optionally", "optional", "option", "optionally", "optional", or "option" generally mean that the subsequent events or conditions may but do not necessarily occur, and this description includes the cases where such events or conditions occur, as well as the cases where such events or conditions do not occur.

[0028] As used herein, the terms "identity", "homology", or "similarity" are used to describe an amino acid sequence or nucleic acid sequence relative to a reference sequence, and refer to the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences determined by conventional methods, see, e.g., Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN program (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Foundation, Washington, D.C.)). There are many algorithms for aligning sequences and determining sequence identity, including, the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48:443; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85:2444; the Smith-Waterman algorithm (Meth. Mol. Biol. 70:173-187 (1997)); and the BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J. Mol. Biol. 215:403-410). Computer programs utilizing these algorithms are also available and include, but are not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al., Meth. Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLAST Altschul et al., supra, FASTA, and TFASTA, available in the Genetics Computing Group (GCG) package, version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0029] Without substantially affecting the antibody activity (retaining at least 90% of the activity), those skilled in the art can substitute, add, and / or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) amino acids to the sequence of the present invention to obtain variants of the antibody sequence. They are all considered to be included within the scope of protection of the present invention. Amino acids with similar properties are substituted in the variable region. The variant sequences of the present invention can have at least 90%, 95%, 96%, 97%, 98%, or 99% identity (or homology) with the reference sequence. The sequence identity described in the present invention can be measured using sequence analysis software. For example, using the computer program BLAST with default parameters, especially BLASTP or TBLASTN. The amino acid sequences described in the present invention are all shown in the N-terminal to C-terminal manner.

[0030] It should be noted that in the claims and the specification herein, the variants of the antibody sequence are obtained by adding and / or deleting one or more amino acids on the basis of the heavy chain, light chain, heavy chain variable region, or light chain variable region. The position or number of the mutation sites defined in the specification and claims of the present invention also need to be adjusted according to the number and position of the added and / or deleted amino acids. For example, a variant of SEQ ID NO:1 is obtained by adding one amino acid before the 50th amino acid (e.g., the 10th or 27th amino acid). Those skilled in the art can understand that Y50F should be adjusted to Y51F; alternatively, a variant of SEQ ID NO:1 is obtained by deleting two amino acids before the 50th amino acid (e.g., the 10th and 27th amino acids). Those skilled in the art can understand that Y50F should be adjusted to Y48F.

[0031] In this article, the term "at least 80% homology" means at least 80% with each reference sequence, and can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% homology. The term "at least 90% homology" means at least 90% with each reference sequence, and can be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% homology.

[0032] In this article, the term "variant" or "mutant" can refer to any naturally occurring or engineered molecule that contains one or more nucleotide or amino acid mutations.

[0033] In this text, the term "vector" generally refers to a nucleic acid molecule capable of self - replicating after being inserted into a suitable host, which transfers the inserted nucleic acid molecule into cells or hosts and / or between cells or hosts. The vector can include vectors mainly used for inserting DNA or RNA into cells, vectors mainly used for replicating DNA or RNA, and expression vectors mainly used for transcription and / or translation of DNA or RNA. The vector also includes vectors with multiple above - mentioned functions. The vector can be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable cell or host. Generally, by culturing a suitable cell or host containing the vector, the vector can produce a desired expression product.

[0034] In this text, the term "cell" generally refers to a cell obtained by modifying or recombining the genetic material of a host cell using genetic engineering techniques or cell fusion techniques, and having unique traits with stable inheritance. Among them, the term "host cell" refers to a prokaryotic cell or a eukaryotic cell that can introduce a recombinant vector. The terms "transformed" or "transfected" as used herein refer to introducing nucleic acid (such as a vector) into a cell by various techniques known in the art. Suitable host cells can be transformed or transfected with the DNA sequence of the present invention and can be used for the expression and / or secretion of a target protein. Examples of suitable host cells that can be used in the present invention include immortalized hybridoma cells, NS / 0 myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, Cap cells (cells derived from human amniotic fluid), and CoS cells.

[0035] The present invention provides an antibody, a nucleic acid molecule, a vector, a cell or a host, a method for preparing an antibody, a conjugate, a reagent or a kit and their uses, a method for detecting D - dimer, a method for screening D - dimer antibodies, and a mutant library, which will be described in detail below respectively.

[0036] Antibody

[0037] In the first aspect of the present invention, the present invention provides an antibody. According to an embodiment of the present invention, the antibody includes: a heavy - chain variable region and / or a light - chain variable region; wherein, the heavy - chain variable region includes an amino acid sequence as shown in SEQ ID NO:1 or its variant, and compared with the amino acid sequence shown in SEQ ID NO:1, the variant of SEQ ID NO:1 includes mutations at at least one of the following positions: position 50, position 59, position 99, and position 102; the light - chain variable region includes an amino acid sequence as shown in SEQ ID NO:2 or its variant, and compared with the amino acid sequence shown in SEQ ID NO:2, the variant of SEQ ID NO:2 includes mutations at at least one of the following positions: position 32, position 34, position 60, and position 61.

[0038] As used herein, the term "antibody" is used in the broadest sense and can include full-length monoclonal antibodies, multispecific antibodies, chimeric antibodies or functional fragments, without limitation as to the specific structure, provided that they exhibit the desired antigen-binding activity.

[0039] As used herein, the terms "full-length antibody", "full-length monoclonal antibody" or "full-length monoclonal antibody" are each composed of at least two identical light chains and at least two identical heavy chains linked by interchain disulfide bonds, such as immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD) or immunoglobulin E (IgE). It generally includes a light chain with a relatively low molecular weight and a heavy chain with a relatively high molecular weight, and the antibody molecule formed by the heavy chain (H chain) and the light chain (L chain) linked by disulfide bonds. Among them, the amino-terminal (N-terminal) amino acid sequence of the peptide chain varies greatly and is called the variable region (V region); the carboxyl-terminal (C-terminal) is relatively stable and varies little, and is called the constant region (C region). The V regions of the L chain and the H chain are called VL and VH, respectively.

[0040] As used herein, the terms "polyclonal antibody" and "multispecific antibody" are synonymous and both refer to antibodies that can recognize multiple antigenic epitopes, such as antibodies that can recognize two antigenic epitopes (bispecific antibodies, abbreviated as bispecific antibodies), antibodies that can recognize three antigenic epitopes or antibodies that can recognize four antigenic epitopes. It is understood in a broad sense and there is no limitation as to the specific structure, as long as it can recognize multiple antigenic epitopes. In the present invention, at least one of the multiple antigenic epitopes is derived from D-dimer.

[0041] As used herein, the term "functional fragment" is a fragment that contains a part or all of an antibody, which lacks at least some of the amino acids present in the full-length chain but still has the performance activity of specifically binding to an antigen. For example, the fragment may contain a part or all of the CDR of the antibody. Such fragments are biologically active because they bind to the antigen and can compete with other antigen-binding molecules (including intact antibodies) for binding to a given epitope. Such fragments include at least one of Fv fragments, disulfide-stabilized Fv fragments (dsFv), F(ab’)2 fragments, Fab’ fragments, Fab fragments, F(ab)2 fragments, scFv fragments, scFv-Fc fusion proteins, scFv-Fv fusion proteins, Fv-Fc fusion proteins, multispecific antibodies formed by functional fragments, single-domain antibodies, VHH nanobodies, domain antibodies, bivalent domain antibodies or minimum recognition units. Such fragments can be produced by recombinant nucleic acid techniques or can be produced by enzymatic cleavage or chemical cleavage of antigen-binding molecules (including intact antibodies).

[0042] Exemplarily, the variants of SEQ ID NO:1 include / are the mutation at position 50, the mutation at position 59, the mutation at position 99, the mutation at position 102, the mutations at positions 50 and 59, the mutations at positions 50 and 99, the mutations at positions 50 and 102, the mutations at positions 59 and 99, the mutations at positions 59 and 102, the mutations at positions 99 and 102, the mutations at positions 50, 59 and 99, the mutations at positions 50, 59 and 102, the mutations at positions 59, 99 and 102, and one of the mutations at positions 50, 59, 99 and 102.

[0043] Exemplarily, the variants of SEQ ID NO:2 include / are the mutation at position 32, the mutation at position 34, the mutation at position 60, the mutation at position 61, the mutations at positions 32 and 34, the mutations at positions 32 and 60, the mutations at positions 32 and 61, the mutations at positions 34 and 60, the mutations at positions 34 and 61, the mutations at positions 60 and 61, the mutations at positions 32, 34 and 60, the mutations at positions 32, 34 and 61, the mutations at positions 34, 60 and 61, and one of the mutations at positions 32, 34, 60 and 61.

[0044] According to an embodiment of the present invention, the above antibody may further include at least one of the following additional technical features:

[0045] According to an embodiment of the present invention, compared with the amino acid sequence shown in SEQ ID NO:1, the variants of SEQ ID NO:1 include mutations at the following sites: at least one of Y50F, A59I / R / L / F, S99G and Y102I / W.

[0046] In some alternative embodiments of the present invention, the mutation at position 50 is Y50F.

[0047] In some alternative embodiments of the present invention, the mutation at position 59 is A59I, A59R, A59L or A59F.

[0048] In some alternative embodiments of the present invention, the mutation at position 59 is A59I.

[0049] In some alternative embodiments of the present invention, the mutation at position 59 is A59R.

[0050] In some alternative embodiments of the present invention, the mutation at position 59 is A59L.

[0051] In some alternative embodiments of the present invention, the mutation at position 59 is A59F.

[0052] In some alternative embodiments of the present invention, the mutation at the 99th position is S99G.

[0053] In some alternative embodiments of the present invention, the mutation at the 102nd position is Y102I or Y102W.

[0054] In some alternative embodiments of the present invention, the mutation at the 102nd position is Y102I.

[0055] In some alternative embodiments of the present invention, the mutation at the 102nd position is Y102W.

[0056] It should be noted that the numbering of the above sites is obtained by sequentially numbering the amino acid sequence shown in SEQ ID NO: 1 from the N-terminus to the C-terminus. For example, the 50th position refers to the 50th position starting from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1; "Y50F" means that the tyrosine at the 50th position of the amino acid sequence shown in SEQ ID NO: 1 is replaced by phenylalanine; "A59I" means that the alanine at the 59th position of the amino acid sequence shown in SEQ ID NO: 1 is replaced by isoleucine;; "A59I / R / L / F" means that the alanine at the 59th position of the amino acid sequence shown in SEQ ID NO: 1 can be replaced by isoleucine, arginine, leucine or phenylalanine.

[0057] According to an embodiment of the present invention, compared with the amino acid sequence shown in SEQ ID NO: 1, the variant of SEQ ID NO: 1 includes mutations at the following sites:

[0058]

[0059]

[0060] According to an embodiment of the present invention, compared with the amino acid sequence shown in SEQ ID NO: 2, the variant of SEQ ID NO: 2 includes at least one of the following site mutations: S32E, S34T / H / Y / F, R60Q, and E61H / A.

[0061] In some alternative embodiments of the present invention, the mutation at the 32nd position is S32E.

[0062] In some alternative embodiments of the present invention, the mutation at the 34th position is S34T, S34H, S34Y or S34F.

[0063] In some alternative embodiments of the present invention, the mutation at the 34th position is S34T.

[0064] In some alternative embodiments of the present invention, the mutation at position 34 is S34H.

[0065] In some alternative embodiments of the present invention, the mutation at position 34 is S34Y.

[0066] In some alternative embodiments of the present invention, the mutation at position 34 is S34F.

[0067] In some alternative embodiments of the present invention, the mutation at position 60 is R60Q.

[0068] In some alternative embodiments of the present invention, the mutation at position 61 is E61H or E61A.

[0069] In some alternative embodiments of the present invention, the mutation at position 61 is E61H.

[0070] In some alternative embodiments of the present invention, the mutation at position 61 is E61A.

[0071] It should be noted that the numbering of the above sites is obtained by sequentially numbering the amino acid sequence shown in SEQ ID NO:2 from the N-terminus to the C-terminus. For example, position 32 refers to the 32nd position starting from the N-terminus of the amino acid sequence shown in SEQ ID NO:2; "S32E" means that serine at position 32 of the amino acid sequence shown in SEQ ID NO:2 is replaced by glutamic acid; "R60Q" means that arginine at position 60 of the amino acid sequence shown in SEQ ID NO:2 is replaced by glutamine; "E61H / A" means that glutamic acid at position 61 of the amino acid sequence shown in SEQ ID NO:2 is replaced by histidine or alanine.

[0072] According to an embodiment of the present invention, compared with the amino acid sequence shown in SEQ ID NO:2, the variant of SEQ ID NO:2 includes mutations at the following sites:

[0073]

[0074]

[0075] According to an embodiment of the present invention, the antibody includes the heavy chain variable region and the light chain variable region as shown in the following table:

[0076]

[0077]

[0078] In a second aspect of the present invention, the present invention provides an antibody. According to an embodiment of the present invention, the antibody comprises a heavy chain and / or a light chain, wherein the heavy chain comprises the heavy chain variable region defined in the antibody described in the first aspect; and the light chain comprises the light chain variable region defined in the antibody described in the first aspect.

[0079] According to an embodiment of the present invention, the antibody comprises the heavy chain and the light chain as shown in the following table:

[0080]

[0081]

[0082] Those skilled in the art can understand that the features and advantages described above for the antibody in the first aspect (such as the features of the heavy chain variable region and / or the light chain variable region defined in the first aspect) also apply to the antibody in the second aspect, and will not be repeated here.

[0083] In a third aspect of the present invention, the present invention provides an antibody. According to an embodiment of the present invention, the antibody comprises HCDRs and LCDRs, wherein the HCDRs comprise or are HCDRs identical to the HCDRs of the heavy chain variable region defined in the antibody described in the first aspect or the second aspect, and the LCDRs comprise or are LCDRs identical to the LCDRs of the light chain variable region defined in the antibody described in the first aspect or the second aspect.

[0084] As used herein, the terms "complementary determining region", "CDR" or "CDRs" refer to the highly variable regions of the heavy and light chains of an immunoglobulin, and refer to regions containing one or more or even all of the major amino acid residues that contribute to the binding affinity of the antibody for the antigen or epitope it recognizes. In the specific embodiments of the present disclosure, the CDRs refer to the highly variable regions of the heavy and light chains of the antibody.

[0085] In this text, the heavy chain complementarity determining regions (CDRs of the heavy chain variable region) are denoted by "HCDRs" or "HCDR", which include HCDR1, HCDR2, and HCDR3; the light chain complementarity determining regions (CDRs of the light chain variable region) are denoted by "LCDRs" or "LCDR", which include LCDR1, LCDR2, and LCDR3. Commonly used CDR numbering schemes in the art include: Kabat numbering, Chothia numbering, IMGT numbering, ChothiaMartin numbering, and AHoLesk numbering. CDR definition schemes include: Kabat definition, Chothia definition, IMGT definition, Contact definition, and AbM definition. As used herein, "Kabat numbering" and "Kabat definition" refer to the numbering and definition system described by Kabat et al., U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). "Chothia definition" is referred to Chothia et al., J Mol Biol 196: 901-917 (1987). Exemplary defined CDRs are listed in Table 1 below. Given the amino acid sequence of the variable region of a given antibody, those skilled in the art can routinely determine which residues contain a specific CDR.

[0086] Table 1: CDR Definitions 1

[0087] CDR Kabat <![CDATA[AbM 2 > IMGT HCDR1 31～35 26-35 26-35 HCDR2 50～65 50-58 51-56 HCDR3 95～102 95-102 93-102 LCDR1 24～34 24-34 27-32 LCDR2 50～56 50-56 50-51 LCDR3 89～97 89-97 89-97

[0088] 1 The numbering of all CDR definitions in Table 1 is based on the Kabat numbering system (see below).

[0089] 2 As used in Table 1, "AbM" with a lowercase "b" refers to the CDRs defined by the "AbM" antibody modeling software of Oxford Molecular.

[0090] It should be noted that the polypeptide sequences in the sequence listing of the present invention and in Table 2 are not numbered according to the Kabat numbering system. However, those of ordinary skill in the art are fully capable of converting the sequence numbers in the sequence listing to Kabat numbers.

[0091] According to an embodiment of the present invention, the HCDRs and / or LCDRs are defined by the Kabat, Chothia, AbM, Contact, or IMGT system.

[0092] According to an embodiment of the present invention, the HCDR1, HCDR2, and HCDR3 sequentially include or are the amino acid sequences at positions 26-35, 50-65, and 95-102 of the heavy chain variable region numbered by Kabat; the LCDR1, LCDR2, and LCDR3 sequentially include or are the amino acid sequences at positions 24-34, 50-56, and 89-97 of the light chain variable region numbered by Kabat.

[0093] It should be noted that CDRs defined by other methods not limited to those in Table 1 also fall within the protection scope of the present disclosure.

[0094] According to an embodiment of the present invention, the HCDRs and LCDRs include or are the amino acid sequences shown below:

[0095] HCDR 1: GYTFTDYNLH;

[0096] HCDR 2: X1IYPYIGETX2YNQKFKS, where X1 is Y or F, and X2 is A, I, R, L, or F;

[0097] HCDR 3: X3GDX4DPWFTY, where X3 is S or G, and X4 is Y, I, or W;

[0098] LCDR1: KSSQSLFNX5GX6QKNYLT, where X5 is S or E, and X6 is S, T, H, Y, or F;

[0099] LCDR 2: WASTX7X8S, where X7 is R or Q, and X8 is E, H, or A;

[0100] LCDR 3: QNDYSYPLT.

[0101] In some alternative embodiments of the present invention, X1 is Y.

[0102] In some alternative embodiments of the present invention, X1 is F.

[0103] In some alternative embodiments of the present invention, X2 is A.

[0104] In some alternative embodiments of the present invention, X2 is I.

[0105] In some alternative embodiments of the present invention, X2 is R.

[0106] In some alternative embodiments of the present invention, X2 is L.

[0107] In some alternative embodiments of the present invention, X2 is F.

[0108] In some alternative embodiments of the present invention, X3 is S.

[0109] In some alternative embodiments of the present invention, X3 is G.

[0110] In some alternative embodiments of the present invention, X4 is Y.

[0111] In some alternative embodiments of the present invention, X4 is I.

[0112] In some alternative embodiments of the present invention, X4 is W.

[0113] In some alternative embodiments of the present invention, X5 is S.

[0114] In some alternative embodiments of the present invention, X5 is E.

[0115] In some alternative embodiments of the present invention, X6 is S.

[0116] In some alternative embodiments of the present invention, X6 is T.

[0117] In some alternative embodiments of the present invention, X6 is H.

[0118] In some alternative embodiments of the present invention, X6 is Y.

[0119] In some alternative embodiments of the present invention, X6 is F.

[0120] In some alternative embodiments of the present invention, X7 is R.

[0121] In some alternative embodiments of the present invention, X7 is Q.

[0122] In some alternative embodiments of the present invention, X8 is E.

[0123] In some alternative embodiments of the present invention, X8 is H.

[0124] In some alternative embodiments of the present invention, X8 is A.

[0125] In an alternative embodiment of the present invention, X1 being Y, X2 being A, X3 being S, X4 being Y, X5 being S, X6 being S, X7 being R, and X8 being E do not exist simultaneously.

[0126] According to an embodiment of the present invention, the antibody comprises or is HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 as shown in the following table,

[0127]

[0128]

[0129]

[0130] According to an embodiment of the present invention, the antibody further comprises at least one of a heavy chain framework region and a light chain framework region. Among them, the heavy chain framework region includes HFR1, HFR2, HFR3, and HFR4; the light chain framework region includes LFR1, LFR2, LFR3, and LFR4.

[0131] According to an embodiment of the present invention, at least a part of at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4 is derived from at least one of a murine antibody, a human antibody, a primate antibody, a bovine antibody, a horse antibody, a dairy cow antibody, a porcine antibody, a sheep antibody, a goat antibody, a dog antibody, a cat antibody, a rabbit antibody, a camel antibody, a donkey antibody, a deer antibody, a mink antibody, a chicken antibody, a duck antibody, a goose antibody, a turkey antibody, a fighting chicken antibody, or a mutant thereof.

[0132] As used herein, the "framework region" or "FR" region includes the heavy chain framework region and the light chain framework region, and refers to the regions other than the CDRs in the heavy chain variable region (which can be denoted as VH) and the light chain variable region (which can be denoted as VL) of the antibody; among them, the heavy chain framework region is denoted as "HFR" and can be further subdivided into adjacent regions separated by CDRs, including the HFR1, HFR2, HFR3, and HFR4 framework regions; the light chain framework region is denoted as "LFR" and can be further subdivided into adjacent regions separated by CDRs, including the LFR1, LFR2, LFR3, and LFR4 framework regions.

[0133] As used herein, the heavy chain variable region is obtained by connecting the CDRs numbered as follows with the FRs in the following combined arrangement: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by connecting the CDRs numbered as follows with the FRs in the following combined arrangement: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0134] In an alternative embodiment of the present invention, HFR1 comprises the amino acid sequence shown in SEQ ID NO: 39 or an amino acid sequence having at least 80% homology thereto;

[0135] HFR2 comprises the amino acid sequence shown in SEQ ID NO: 40 or an amino acid sequence having at least 80% homology thereto;

[0136] HFR3 comprises the amino acid sequence shown in SEQ ID NO: 41 or an amino acid sequence having at least 80% homology thereto;

[0137] The HFR4 includes the amino acid sequence shown in SEQ ID NO: 42 or an amino acid sequence having at least 80% homology thereto.

[0138] The LFR1 includes the amino acid sequence shown in SEQ ID NO: 43 or an amino acid sequence having at least 80% homology thereto.

[0139] The LFR2 includes the amino acid sequence shown in SEQ ID NO: 44 or an amino acid sequence having at least 80% homology thereto.

[0140] The LFR3 includes the amino acid sequence shown in SEQ ID NO: 45 or an amino acid sequence having at least 80% homology thereto.

[0141] The LFR4 includes the amino acid sequence shown in SEQ ID NO: 46 or an amino acid sequence having at least 80% homology thereto.

[0142] According to an embodiment of the present invention, the antibody described above includes the heavy chain variable region and / or the light chain variable region described in the first aspect or the second aspect.

[0143] Those skilled in the art can understand that the features and advantages described above for the antibodies in the first aspect and the second aspect (for example, features such as the heavy chain variable region and / or the light chain variable region defined in the first aspect, and features such as the heavy chain and / or the light chain defined in the second aspect) also apply to the antibodies in this third aspect, and will not be elaborated herein.

[0144] According to an embodiment of the present invention, the antibodies described in the above first aspect, second aspect, and third aspect may further include at least one of the following technical features:

[0145] According to an embodiment of the present invention, the antibody further includes a constant region; wherein, the constant region includes at least one of a heavy chain constant region and a light chain constant region.

[0146] According to an embodiment of the present invention, at least a part of at least one of the heavy chain constant region and the light chain constant region is derived from at least one of a murine antibody, a human antibody, a primate antibody, a bovine antibody, a horse antibody, a dairy cow antibody, a pig antibody, a sheep antibody, a goat antibody, a dog antibody, a cat antibody, a rabbit antibody, a camel antibody, a donkey antibody, a deer antibody, a mink antibody, a chicken antibody, a duck antibody, a goose antibody, a turkey antibody, a fighting chicken antibody, or a mutant thereof.

[0147] According to an embodiment of the present invention, the heavy chain constant region comprises a heavy chain constant region selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; alternatively, the light chain constant region comprises a light chain constant region selected from kappa-type or lambda-type light chain constant regions.

[0148] In an alternative embodiment of the present invention, both the light chain constant region and the heavy chain constant region are from a murine antibody or a mutant thereof.

[0149] According to an embodiment of the present invention, the N-terminus of the heavy chain constant region is connected to the C-terminus of the heavy chain variable region, and the N-terminus of the light chain constant region is connected to the N-terminus of the light chain variable region.

[0150] In an alternative embodiment of the present invention, the heavy chain constant region comprises or is the heavy chain constant region shown in SEQ ID NO:3 or an amino acid sequence having at least 80% identity therewith; alternatively, the light chain constant region comprises or is the light chain constant region shown in SEQ ID NO:4 or an amino acid sequence having at least 80% identity therewith.

[0151] According to an embodiment of the present invention, the antibody comprises the heavy chain and / or light chain described in the second aspect.

[0152] According to an embodiment of the present invention, the antibody comprises at least one selected from polyclonal antibodies, full-length monoclonal antibodies, Fab antibodies, Fab' antibodies, F(ab')2 antibodies, Fv antibodies, single-chain antibodies, single-domain antibodies, and minimal recognition units.

[0153] As used herein, the terms "single-domain antibody", "nanobody" and "VHH antibody" are used interchangeably and were originally described as the antigen-binding immunoglobulin (variable) domain of "heavy-chain antibodies" (i.e., "antibodies lacking light chains") (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: "Naturally occurring antibodies devoid of light chains"; Nature 363, 446-448 (1993)), which contain a heavy-chain variable region (VH) and conventional CH2 and CH3 regions and specifically bind to antigenic proteins (such as D-dimers) through the heavy-chain variable region.

[0154] As used herein, the term "Fab antibody" or "Fab fragment" generally refers to an antibody or fragment containing only the Fab molecule, which is composed of VH and CH1 of the heavy chain and the complete light chain, and the light chain and the heavy chain are connected by a disulfide bond.

[0155] As used herein, the term "F(ab')2 antibody" or "F(ab')2 fragment" refers to two antigen-binding F(ab') portions linked together by disulfide bonds.

[0156] As used herein, the term "Fv antibody" or "Fv fragment" generally refers to an antibody or fragment consisting only of a light chain variable region (VL) and a heavy chain variable region (VH) linked by non-covalent bonds, which is the smallest functional fragment of an antibody molecule that retains the complete antigen-binding site.

[0157] As used herein, the terms "single-chain antibody", "scFv fragment" refer to an antibody or fragment formed by linking the heavy chain variable region and the light chain variable region of an antibody with a short peptide.

[0158] As used herein, the terms "minimal recognition unit" and "MRU" both refer to an antibody or fragment consisting only of one CDR, which has a very small molecular weight, accounting for only about 1% of the complete antibody.

[0159] According to an embodiment of the present invention, the antibody comprises a light chain and / or a heavy chain, and the heavy chain comprises the heavy chain variable region and the heavy chain constant region defined above; the light chain comprises the light chain variable region and the light chain constant region defined above.

[0160] Nucleic acid molecule, vector, cell or host, method for preparing an antibody

[0161] In the process of preparing or obtaining the antibody described in the first, second or third aspect, nucleic acid molecules expressing these antibodies can be used, linked to different vectors, and then expressed in different cells to obtain the corresponding antibodies.

[0162] In the fourth aspect of the present invention, the present invention provides a nucleic acid molecule. According to an embodiment of the present invention, the nucleic acid molecule encodes the antibody described in the first, second or third aspect. The nucleic acid molecule according to the embodiment of the present invention can encode and obtain the above-mentioned antibody.

[0163] According to an embodiment of the present invention, the nucleic acid molecule comprises DNA or RNA.

[0164] It should be noted that for the nucleic acid molecules mentioned herein, those skilled in the art should understand that it actually includes either any one of the complementary double strands, or both. For convenience, in this article, although only one strand is given in most cases, the other complementary strand is actually also disclosed. In addition, the molecular sequences in the present invention include DNA form or RNA form, and disclosing one means that the other is also disclosed.

[0165] In the fifth aspect of the present invention, the present invention provides a vector. According to an embodiment of the present invention, the vector comprises the nucleic acid molecule described in the fourth aspect. When the above nucleic acid molecule is connected to the vector, the nucleic acid molecule can be directly or indirectly connected to the control elements on the vector, as long as these control elements can control the translation and expression of the nucleic acid molecule, etc. Of course, these control elements can be directly from the vector itself or exogenous, that is, not from the vector itself. Of course, the nucleic acid molecule and the control element are operably linked. As used herein, "operably linked" means that an exogenous gene is linked to a vector such that the control elements within the vector, such as transcriptional control sequences and translational control sequences, etc., can perform their intended functions of regulating the transcription and translation of the exogenous gene. Commonly used vectors can be, for example, plasmids, phages, etc. After the vector according to some specific embodiments of the present invention is introduced into a suitable recipient cell, under the mediation of a regulatory system, the expression of the aforementioned antibody can be effectively achieved, and thus a large amount of the antibody can be obtained in vitro.

[0166] In some specific embodiments of the present invention, the vector is a eukaryotic expression vector, a prokaryotic expression vector, a virus or a phage.

[0167] In an alternative embodiment of the present invention, the expression vector is a plasmid expression vector.

[0168] In the sixth aspect of the present invention, the present invention provides a cell or a host. According to an embodiment of the present invention, the cell or host comprises: the nucleic acid molecule described in the fourth aspect or the vector described in the fifth aspect; or expresses the antibody described in the first aspect, the second aspect or the third aspect. Using this cell under suitable conditions, the aforementioned antibody can be effectively expressed intracellularly.

[0169] According to an embodiment of the present invention, the cell is obtained by introducing the vector described in the fifth aspect into the cell.

[0170] It should be noted that the cells of the present invention are not particularly limited and can be prokaryotic cells, eukaryotic cells or phages. The prokaryotic cells can be Escherichia coli, Bacillus subtilis, Streptomyces or Proteus mirabilis, etc. The eukaryotic cells include fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Trichoderma, etc., insect cells such as Spodoptera frugiperda, plant cells such as tobacco, and mammalian cells such as BHK cells, CHO cells, COS cells, myeloma cells, etc.

[0171] In an alternative embodiment of the present invention, the cell is a mammalian cell, including BHK cells, CHO cells, NSO cells or COS cells, and does not include animal germ cells, fertilized eggs or embryonic stem cells.

[0172] It should be noted that the "suitable conditions" described in the present invention refer to the conditions suitable for the expression of the antibodies of the present invention. It is easy for those skilled in the art to understand that the conditions suitable for the expression of the antibodies include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy cell states, suitable cell densities, suitable cell culture environments, and suitable cell culture times. The "suitable conditions" are not particularly limited, and those skilled in the art can optimize the most suitable conditions for the expression of the antibodies according to the specific environment of the laboratory.

[0173] In the seventh aspect of the present invention, the present invention provides a method for preparing the antibody according to the first aspect, the second aspect, or the third aspect. According to an embodiment of the present invention, the method includes culturing the cell or host according to the sixth aspect. The method according to some specific embodiments of the present invention can effectively obtain a large amount of the antibody.

[0174] Based on the amino acid sequence of the antibody of the present disclosure, it is easy for those skilled in the art to think of using genetic engineering techniques or other techniques (chemical synthesis, recombinant expression) to prepare the antibody. For example, the antibody can be isolated and purified from the culture product of recombinant cells capable of recombinantly expressing the antibody described in any one of the above. This is easy to achieve for those skilled in the art. Based on this, no matter what technique is used to prepare the antibody of the present disclosure, it falls within the protection scope of the present disclosure.

[0175] Those skilled in the art can understand that the features and advantages described above for the antibodies according to the first aspect, the second aspect, and the third aspect also apply to the nucleic acid molecule, the vector, the cell or host, and the method for preparing the antibody, and will not be repeated here.

[0176] Conjugates, reagents or kits and their uses

[0177] The antibody can be used in combination with any detection reagent or therapeutic agent. For example, it can be used in combination with diagnostic radionuclides, nanomaterials, etc. to detect the target site through the radioactivity of the radionuclide and obtain information about the target site. It can also be used in combination with therapeutic radionuclides to specifically kill target cells, tissues, etc. using the radioactivity of the radionuclide.

[0178] In the eighth aspect of the present invention, the present invention provides a conjugate. According to an embodiment of the present invention, the conjugate includes: the antibody according to the first aspect, the second aspect, or the third aspect, and a conjugate moiety conjugated thereto. The conjugate according to the embodiment of the present invention can specifically target and bind to D-dimer, and can be used for qualitative or quantitative detection of D-dimer, or for diagnosing diseases related to abnormal D-dimer.

[0179] According to an embodiment of the present invention, the above conjugate may further include at least one of the following additional technical features:

[0180] According to an embodiment of the present invention, the coupling moiety is selected from carriers. Exemplarily, the carrier includes a purification tag or label.

[0181] As used herein, the carrier may be a substance capable of being suspended or dispersed in a liquid phase (e.g., solid-phase carriers such as particles, magnetic beads, etc.), or a solid phase capable of accommodating or carrying a liquid phase (e.g., supports such as plates, membranes, test tubes, etc., and containers such as microtiter plates, microfluidic channels, glass capillaries, nano-columns, monolithic columns, etc.); it may also be a labeling carrier for labeling an antibody, such as an enzyme (e.g., peroxidase, alkaline phosphatase, luciferin, β-galactosidase), an affinity substance (e.g., one of streptavidin and biotin, one of nucleic acids of sense and antisense strands complementary to each other), a fluorescent substance (e.g., fluorescein, fluorescein isothiocyanate, rhodamine, green fluorescent protein, red fluorescent protein), a luminescent substance (e.g., luciferin, Aequorin, acridinium ester, tris(2,2'-bipyridyl)ruthenium, luminol), a radioisotope (e.g., 3 H, 14 C, 32 P, 35 S, 125 I), and gold colloids, etc.

[0182] In some specific embodiments of the present invention, the purification tag or label includes at least one selected from colloidal gold, radioactive label, luminescent substance, colored substance, enzyme, biotin / avidin, and spin label.

[0183] In some specific embodiments of the present invention, the purification tag or label includes at least one selected from fluorescent label, chromophore label, and electron-dense label.

[0184] In some specific embodiments of the present invention, the purification tag or label includes one or more selected from radioisotopes, fluorophores, rhodamine and its derivatives, luciferase, fluorescein, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, carbohydrate oxidase, glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase.

[0185] In some specific embodiments of the present invention, the coupling moiety includes at least one selected from magnetic microspheres, plastic microspheres, plastic particles, microtiter plates, glass, capillaries, nylon, and nitrocellulose membranes.

[0186] In a ninth aspect of the present invention, the present invention provides a reagent or a kit. According to an embodiment of the present invention, the reagent or the kit comprises: the antibody according to the first, second or third aspect, or the conjugate according to the eighth aspect. As described above, the antibodies in some specific embodiments or examples of the present invention can effectively bind to D-dimer. Therefore, the reagent or the kit containing the antibody can effectively qualitatively or quantitatively detect D-dimer. By applying the reagent or the kit provided by the present invention, it can be used, for example, in immunoblotting, immunoprecipitation and other detections that utilize the specific binding property between D-dimer and its antibody. As described above, the antibodies in some specific embodiments or examples of the present invention have a higher binding activity with D-dimer. Therefore, the reagent or the kit containing the antibody has a higher detection sensitivity.

[0187] These kits may contain any one or more of the following: treatment solution, anti-D-dimer antibody, D-dimer control product, anti-IgG antibody, instruction manual or literature, etc. The anti-D-dimer antibody can be used in different types of diagnostic tests. For example, it can detect the presence of various diseases, drugs, or other proteins in vitro or in vivo. For example, it can be used to test related diseases by detecting the serum or blood of a subject.

[0188] In a tenth aspect of the present invention, the present invention provides the use of the antibody according to the first, second or third aspect, the conjugate according to the eighth aspect, or the reagent or the kit according to the ninth aspect in detecting D-dimer or in preparing a product for diagnosing D-dimer related diseases.

[0189] In this context, the D-dimer related diseases include, but are not limited to, at least one of thrombotic diseases, disseminated intravascular coagulation, systemic lupus erythematosus, myocardial infarction, cirrhosis or hepatitis, cancer or tumor, mycoplasma pneumonia, diabetes, and anaphylactoid purpura.

[0190] In this context, the term "thrombotic disease" refers to a disease caused by a thrombus, including but not limited to arterial and venous thrombosis (e.g., deep vein thrombosis (DVT)), pulmonary embolism, etc.

[0191] In an eleventh aspect of the present invention, the present invention provides a method for detecting D-dimer. According to an embodiment of the present invention, the method comprises: contacting a sample to be detected with the antibody according to the first, second or third aspect, the conjugate according to the eighth aspect, or the reagent or the kit according to the ninth aspect to form an immune complex.

[0192] According to an embodiment of the present invention, based on the signal of the immune complex, it is determined whether the sample to be detected contains D-dimer or the content of D-dimer.

[0193] According to an embodiment of the present invention, the immune complex further comprises a second antibody that binds to the antibody.

[0194] According to an embodiment of the present invention, the immune complex further comprises a second antibody that binds to D-dimer.

[0195] According to an embodiment of the present invention, the signal includes a fluorescence signal.

[0196] In a twelfth aspect of the present invention, the present invention provides a method for screening D-dimer antibodies. According to an embodiment of the present invention, the method comprises: a) designing primers for amino acid substitution at 1, 2, 3, 4, 5, 6, 7, or 8 sites among X1, X2, X3, X4, X5, X6, X7, or X8 defined in the antibody described in the second aspect; b) using the nucleic acid molecule described in the fourth aspect, the vector described in the fifth aspect, or the cell described in the sixth aspect as a template to construct a mutant library with the primers described in a); c) screening D-dimer antibodies from the mutant library.

[0197] According to an embodiment of the present invention, the mutant library is a single-site saturation mutant library.

[0198] According to an embodiment of the present invention, the D-dimer antibody comprises or is the antibody described in the first aspect, the second aspect, or the third aspect.

[0199] In a thirteenth aspect of the present invention, the present invention provides a mutant library that includes variants of SEQ ID NO:1 and variants of SEQ ID NO:2 defined in the antibody described in the first aspect, the second aspect, or the third aspect.

[0200] According to an embodiment of the present invention, the mutant library is constructed using the primers in the method described in the twelfth aspect.

[0201] In a fourteenth aspect of the present invention, the present invention provides the antibody described in the first aspect, the second aspect, or the third aspect, the conjugate described in the eighth aspect, or the reagent or kit described in the ninth aspect for detecting D-dimer or for diagnosing D-dimer-related diseases.

[0202] In a fifteenth aspect of the present invention, the present invention provides the use of the antibody described in the first aspect, the second aspect, or the third aspect, the conjugate described in the eighth aspect, or the reagent or kit described in the ninth aspect in the preparation of products for detecting D-dimer or for diagnosing D-dimer-related diseases.

[0203] Those skilled in the art can understand that the antibodies, the described features and advantages mentioned in the foregoing for the first, second, and third aspects are equally applicable to the conjugate, reagent or kit, and their uses and methods, and will not be elaborated herein.

[0204] The amino acid sequences or nucleotide sequences involved herein are shown in Table 2. Among them, the mutation sites of the heavy chain variable region or HCDRs are referenced to the amino acid sequence shown in SEQ ID NO:1 or the HCDRs in SEQ ID NO:1, and the mutation sites of the light chain variable region or LCDRs are referenced to the amino acid sequence shown in SEQ ID NO:2 or the LCDRs in SEQ ID NO:2. WT indicates no mutation:

[0205] Table 2: Amino Acid Sequences

[0206]

[0207]

[0208]

[0209]

[0210]

[0211]

[0212]

[0213]

[0214]

[0215] The solution of the present invention will be explained below in conjunction with the examples. Those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. For those not specified in the examples regarding specific technologies or conditions, they shall be carried out according to the technologies or conditions described in the literature in the art or according to the product specifications. For the reagents or instruments not specified regarding the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0216] Unless otherwise defined, 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 doses herein, some methods and materials are now described. Unless otherwise stated, the technologies adopted or considered herein are standard methods. The materials, methods, and examples are illustrative only and not restrictive.

[0217] Unless otherwise indicated, the practice of the present disclosure will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of those of ordinary skill in the art. Such techniques are fully explained in the literature, such as "Molecular Cloning: A Laboratory Manual", Second Edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (ed. M.J. Gait, 1984); "Animal Cell Culture" (ed. R.I. Freshney, 1987); "Methods in Enzymology" (Academic Press, Inc.); "Handbook of Experimental Immunology" (ed. D.M. Weir and C.C. Blackwell); "Gene Transfer Vectors for Mammalian Cells" (ed. J.M. Miller and M.P. Calos, 1987); "Current Protocols in Molecular Biology" (ed. F.M. Ausubel et al., 1987); "PCR: The Polymerase Chain Reaction" (ed. Mullis et al., 1994); and "Current Protocols in Immunology" (ed. J.E. Coligan et al., 2011), each of which is hereby expressly incorporated by reference.

[0218] In this example, restriction endonucleases, T4 DNA ligase, and DNA polymerase were purchased from New England Biolabs, Taq DNA polymerase was purchased from TaKaRa, the V01 expression vector was constructed by our laboratory, the gel extraction kit and plasmid extraction kit were commercially available, and primer synthesis and gene sequencing were completed by an outsourcing company. The sequence of the D-dimer monoclonal antibody (hereinafter referred to as the WT antibody) was derived from the sequencing of mouse hybridoma cells.

[0219] Example 1: Construction and Screening of a Mutation Library

[0220] 1. Construction of the wild-type (WT) D-dimer antibody (abbreviated as WT antibody) template plasmid

[0221] (1) Synthesis of the WT antibody gene:

[0222] The VH (amino acid sequence shown in SEQ ID NO: 1) and VL (amino acid sequence shown in SEQ ID NO: 2) of the WT antibody sequence were optimized for E. coli codons, and then the antibody gene sequence was handed over to an outsourcing company for gene synthesis.

[0223] (2) Amplification of the WT antibody gene fragment:

[0224] The nucleotide sequences of the antibody VH and VL synthesized in step (1) were amplified by PCR using DNA polymerase, and then the antibody bands were separated by agarose gel electrophoresis. Subsequently, the antibody gene fragment was purified using a gel recovery kit.

[0225] (3) Enzyme digestion and ligation of the WT antibody gene fragment:

[0226] The antibody gene fragment obtained in step (2) and the V01 vector plasmid were simultaneously digested with restriction endonucleases for double digestion, and then the antibody gene fragment and the V01 vector with sticky ends were purified using a gel recovery kit. Subsequently, the antibody gene fragment and the V01 vector were ligated with T4 DNA ligase at 22 °C for 4 hours. Then, the ligation reaction product was recovered and purified, and the DNA concentration was measured. Finally, 100 ng of the plasmid was transformed into 100 μl of TG1 E. coli competent cells to obtain a bacterial solution. Then, the entire bacterial solution was spread on a plate containing ampicillin resistance and cultured overnight at 37 °C.

[0227] (4) Extraction and sequencing verification of the WT template plasmid

[0228] Ten monoclonal colonies cultured overnight in step (3) were selected, and colony PCR and gel electrophoresis were performed using Taq DNA polymerase. The bacteria with the correct insertion of the antibody gene sequence were selected for culture and amplification. The WT template plasmid was obtained using a plasmid extraction kit and sent to a sequencing company for gene sequencing verification.

[0229] 2. Construction of the single-point mutation library

[0230] In this part of the experiment, single-point saturation mutagenesis was performed on the entire CDR regions of VH and VL of the WT antibody obtained in step 1 to construct a single-point mutation library.

[0231] (1) Primer design and synthesis

[0232] Using degenerate base codons, 70 pairs of upstream and downstream primers for single-point saturation mutagenesis of the entire CDR regions of VH and VL (70 amino acid sites) were designed and handed over to an outsourcing company for primer synthesis.

[0233] (2) PCR amplification of single-point saturated mutation plasmid

[0234] Using the primers obtained in step (1), perform PCR amplification on the single-point saturated mutation plasmid by the PCR method. Configure the reaction system according to Table 3, and then perform the amplification preparation of the single-point saturated mutation library plasmid using the PCR reaction conditions in Table 4. Finally, digest the WT template plasmid obtained in step 1 with restriction endonuclease at 37°C for 1 hour to obtain plasmids of 70 mutation libraries.

[0235] Table 3: Reaction system for PCR amplification

[0236] WT template plasmid 50 ng DNA polymerase 1 μl DNA polymerase buffer 10 μl dNTP (2.5 mM) 4 μl Forward primer (10 μM) 1 μl Reverse primer (10 μM) 1 μl <![CDATA[ddH2O]]> Make up to 50 μl

[0237] Table 4: PCR reaction conditions

[0238] Step1 Step2 Step3 Step4 Step5 Step6 Temperature 95℃ 95℃ 55-60℃ 72℃ 72℃ 4℃ Time 5 min 30s 30s 2 min 5 min ∞

[0239] Note: 22 cycles are performed for Step2 - Step4.

[0240] (3) Transformation of single-point saturated mutation plasmid:

[0241] Take 10 μl of the reaction products of the 71 plasmids of the mutation libraries obtained in step (2), and transform each into 100 μl of TG1 Escherichia coli competent cells to obtain bacterial solutions. Then spread all the bacterial solutions on plates containing ampicillin resistance and culture overnight at 37°C.

[0242] 3. Screening of single-point mutation library

[0243] (1) Antibody expression of mutation library

[0244] Pre-add 500 μl of medium to a 96-well culture plate. For each single-point mutation library, select 92 monoclonal colonies transformed with the single-point saturated mutation plasmid cultured overnight in step 2-(3), and set up WT, negative control (i.e., colonies without inserting VH\VL genes), and blank control (i.e., only medium without colonies). Culture at 37°C for 5 - 6 hours, then transfer the bacterial solution to a new 96-well culture plate. Then, after culturing at 37°C for 1 - 2 hours, finally add induction medium and culture overnight at 37°C to express antibodies, obtaining the antibody expression supernatants of 70 mutation libraries.

[0245] (2) Screening and sequencing of mutation library

[0246] The commercially available D-dimer protein was added to 70 ELISA plates at a concentration of 1 μg / ml and a volume of 100 μl / well, and incubated overnight at 4 °C for coating. The next day, it was blocked with 1-2% skim milk powder. The antibody expression supernatants of the 70 mutant libraries obtained in step (1) were added to the ELISA plate wells at a volume of 100 μl / well, and WT, negative control (i.e., colonies without the insertion of VH\VL genes) and blank control (i.e., only medium without colonies) were set. Incubate at room temperature for 2 hours, and then perform subsequent plate washing, color development, and reading using the detection method of conventional ELISA. Finally, organize and analyze the data results, send the clones with improved performance for sequencing, and finally analyze the sequencing results to select the mutation sites of 16 unique mutant candidate clones (see Table 5) for the construction of a combinatorial mutant library. (The Ratio value in Table 5 represents the degree of affinity improvement. When the Ratio value is equal to 1, it means that the affinity of the mutant clone is the same as that of WT; when the Ratio value is greater than 1, it means that the affinity has been improved).

[0247] Table 5: Screening results and mutation sites of candidate clones

[0248]

[0249] 4. Construction of combinatorial mutant library

[0250] (1) Design and synthesis of library primers:

[0251] According to the mutation sites on VH and VL of the D-dimer antibody obtained in step 3-(2), amplification primers for the combinatorial mutant library were designed and handed over to an outsourcing company for primer synthesis.

[0252] (2) Fragment amplification and ligation

[0253] According to the PCR system in Table 3 and the PCR reaction conditions in Table 4, the antibody mutant fragments were amplified, and then the antibody mutant fragments were recovered by gel extraction. Using the method of Overlap PCR, the antibody mutant fragments were spliced into complete antibody fragments (heavy chain variable region or light chain variable region).

[0254] Finally, the antibody fragments were inserted into the V01 vector by enzymatic digestion and ligation to form a complete antibody expression plasmid (for specific steps, refer to the steps of "Enzymatic digestion and ligation of WT antibody gene fragments" in step 1-(3)). 100 ng of the plasmid was transformed into 100 μl of TG1 Escherichia coli competent cells, and the bacterial solution was spread on a plate containing ampicillin resistance and cultured overnight at 37 °C.

[0255] 5. Screening of combinatorial mutant library

[0256] Thirty-seven combinatorial mutant antibodies were randomly selected for expression in the supernatant, ELISA screening, and positive clone sequencing analysis. The specific screening results, mutation site information, and Ratio value measurements are shown in Table 6.

[0257] Table 6: Information on Combinatorial Mutant Candidate Clones

[0258]

[0259]

[0260] Example 2: Expression of Mutant D-Dimer Antibody

[0261] In this example, the antibodies screened in Example 1 were expressed. The specific experimental operations are as follows:

[0262] 1. Construction of Eukaryotic Recombinant Expression Plasmid

[0263] pcDNA TM 3.4 The pcDNA3.4 vector was the constructed eukaryotic expression vector for the recombinant antibody. This expression vector had introduced multiple cloning sites such as HindIII, BamHI, and EcoRI, and was named the pcDNA3.4A expression vector, hereinafter referred to as the 3.4A expression vector for short. According to the variable region gene sequences of the 37 candidate clones screened from the above combinatorial mutant library (see Table 6, and the specific amino acid sequences are shown in Table 2), VL and VH gene-specific amplification primers for the corresponding antibody sequences and overlap primers for the constant regions (the amino acid sequences of the heavy chain constant region and the light chain constant region are shown in Table 2. Among them, the N-terminus of the heavy chain constant region is connected to the C-terminus of VH to form the heavy chain, and the N-terminus of the light chain constant region is connected to the C-terminus of VL to form the light chain) were designed. The primers at both ends carried HindIII and EcoRI restriction sites and protective bases. A 0.73KB light chain gene fragment and a 1.40kb heavy chain gene fragment were amplified by PCR.

[0264] The heavy chain and light chain gene fragments were respectively digested with HindIII / EcoRI double enzymes, and the 3.4A vector was digested with HindIII / EcoRI double enzymes. The digested antibody light and heavy chain gene fragments and the vector were purified and recovered. Then, the gene fragments encoding the antibody light and heavy chains were respectively ligated into the 3.4A expression vector and transformed into DH5α Escherichia coli competent cells. After colonies grew, single colonies were respectively picked for PCR identification of positive clones. Positive clones were picked for sequencing to determine the correctness of the sequences. Clones with correct sequencing were selected for plasmid extraction for standby.

[0265] 2. Sample Preparation of Recombinant Antibody

[0266] Resuscitate HEK293 cells in advance, passage and culture them in a 200 ml system until the cell density reaches (3 - 5)×10 6 cells / ml and the cell viability > 95%; centrifuge and wash the cells, resuspend them with the medium, and at the same time adjust the cell density to 2.9×10 6 cells / ml as the cell diluent. Prepare plasmid DNA and transfection reagent diluents with the medium respectively. Add the transfection reagent diluent to the plasmid DNA diluent, mix well and let it stand at room temperature for 15 min; slowly add this mixture to the cell diluent within 1 min, mix well, sample and count, record and observe the viability of the cells after transfection, and place them in an incubator at 35°C for culture, with a rotation speed of 120 rmp and a CO2 content of 8%. After 13 days, centrifuge to collect the samples. Use a protein A affinity chromatography column for affinity purification to obtain 37 antibodies. Among them, the amino acid sequences of the heavy chain variable region, light chain variable region, heavy chain and light chain of the 37 antibodies are shown in Table 7.

[0267] Table 7: Amino acid sequences of the heavy chain variable region, light chain variable region, heavy chain and light chain of 37 antibodies

[0268]

[0269]

[0270] 3. Affinity analysis

[0271] Perform affinity detection and analysis on the wild-type antibody (the amino acid sequence of the heavy chain is as shown in SEQ ID NO: 165, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 166), the 16 single-site mutant candidate cloned antibodies obtained in step 3 of Example 1, and the 37 combinatorial mutant antibodies obtained in step 2 of Example 2. The specific steps are as follows: Test the binding and dissociation curves of D-dimer and mutant D-dimer antibodies on a Biacore8K+ device, and the instrument automatically fits to obtain the affinity constant, binding rate, and dissociation rate. In the affinity detection results, KD represents the equilibrium dissociation constant, that is, the affinity constant. The smaller the KD value, the higher the affinity; ka represents the binding rate; kd represents the dissociation rate. The results show that the affinity of the mutant antibodies for D-dimer is better than that of the wild-type antibody for D-dimer. An exemplary display of the antibody affinity detection results is shown in Table 8 below:

[0272] Table 8 Antibody affinity detection results

[0273]

[0274]

[0275] In the description of this specification, the description referring to terms such as "one embodiment", "some embodiments", "examples", "specific examples", or "some examples", etc. means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0276] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. An anti-D-dimer antibody, characterized in that, The antibody includes a heavy chain variable region and a light chain variable region as shown in the following table:

2. An anti-D-dimer antibody, comprising a heavy chain and a light chain, characterized in that, The heavy chain includes the heavy chain variable region defined in the antibody of claim 1; the light chain includes the light chain variable region defined in the antibody of claim 1.

3. The antibody according to claim 2, wherein The antibody includes the heavy chain and the light chain as shown in the following table:

4. An anti-D-dimer antibody, comprising HCDRs and LCDRs, characterized in that, The HCDRs are HCDRs that are identical to the HCDRs of the heavy chain variable region defined by the antibody of any one of claims 1 to 3, and the LCDRs are LCDRs that are identical to the LCDRs of the light chain variable region defined by the antibody of any one of claims 1 to 3; The HCDRs and LCDRs are defined by the Kabat, Chothia, AbM, Contact or IMGT system.

5. An anti-D-dimer antibody, comprising HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, characterized in that, The HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 are the amino acid sequences shown below: HCDR1: GYTFTDYNLH; HCDR2: X1IYPYIGETX2YNQKFKS, wherein X1 is Y or F, and X2 is A, I, R, L or F; HCDR3: X3GDX4DPWFTY, wherein X3 is S or G, and X4 is Y, I or W; LCDR1: KSSQSLFNX5GX6QKNYLT, wherein X5 is S or E, and X6 is S, T, H, Y or F; LCDR2: WASTX7X8S, wherein X7 is R or Q, and X8 is E, H or A; LCDR3: QNDYSYPLT; X1 being Y, X2 being A, X3 being S, X4 being Y, X5 being S, X6 being S, X7 being R and X8 being E do not exist simultaneously: The antibody includes HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 as shown in the following table:

6. The antibody according to claim 5, wherein The antibody further includes HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, LFR4; Wherein, the HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4 are from murine antibodies, primate antibodies, bovine antibodies, equine antibodies, porcine antibodies, ovine antibodies, caprine antibodies, canine antibodies, feline antibodies, rabbit antibodies, camel antibodies, donkey antibodies, deer antibodies, mink antibodies, chicken antibodies, duck antibodies, or goose antibodies.

7. The antibody according to claim 6, wherein The HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4 are from human antibodies, dairy cow antibodies, turkey antibodies, or gamecock antibodies.

8. The antibody according to claim 6, wherein The HFR1 includes the amino acid sequence shown in SEQ ID NO: 39 or an amino acid sequence having at least 80% homology thereto; The HFR2 includes the amino acid sequence shown in SEQ ID NO: 40 or an amino acid sequence having at least 80% homology thereto; The HFR3 includes the amino acid sequence shown in SEQ ID NO: 41 or an amino acid sequence having at least 80% homology thereto; The HFR4 comprises the amino acid sequence as shown in SEQ ID NO: 42 or an amino acid sequence having at least 80% homology thereto; The LFR1 comprises the amino acid sequence as shown in SEQ ID NO: 43 or an amino acid sequence having at least 80% homology thereto; The LFR2 comprises the amino acid sequence as shown in SEQ ID NO: 44 or an amino acid sequence having at least 80% homology thereto; The LFR3 comprises the amino acid sequence as shown in SEQ ID NO: 45 or an amino acid sequence having at least 80% homology thereto; The LFR4 comprises the amino acid sequence as shown in SEQ ID NO: 46 or an amino acid sequence having at least 80% homology thereto.

9. The antibody according to any one of claims 1 to 8, characterized in that, The antibody further comprises a constant region; Wherein, the constant region comprises a heavy chain constant region and a light chain constant region.

10. The antibody according to claim 9, wherein The heavy chain constant region and the light chain constant region are from a murine antibody, a primate antibody, a bovine antibody, a horse antibody, a porcine antibody, a sheep antibody, a goat antibody, a dog antibody, a cat antibody, a rabbit antibody, a camel antibody, a donkey antibody, a deer antibody, a mink antibody, a chicken antibody, a duck antibody, or a goose antibody.

11. The antibody according to claim 10, wherein The heavy chain constant region and the light chain constant region are from a human antibody, a dairy cow antibody, a turkey antibody, or a gamecock antibody.

12. The antibody according to claim 9, wherein The heavy chain constant region comprises a heavy chain constant region selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; or The light chain constant region comprises a light chain constant region selected from kappa type or lambda type.

13. The antibody according to claim 9, wherein Both the light chain constant region and the heavy chain constant region are from a murine antibody.

14. The antibody according to claim 9, wherein The N-terminus of the heavy chain constant region is connected to the C-terminus of the heavy chain variable region of the antibody, and the N-terminus of the light chain constant region is connected to the N-terminus of the light chain variable region of the antibody.

15. The antibody according to claim 9, wherein, The heavy chain constant region comprises or is the heavy chain constant region as shown in the amino acid sequence of SEQ ID NO: 3; or The light chain constant region comprises or is the light chain constant region as shown in the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence having at least 80% identity thereto.

16. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the antibody according to any one of claims 1 to 15.

17. A carrier, characterized in that, Comprises the nucleic acid molecule according to claim 16.

18. A cell or host, characterized in that, Comprises: The nucleic acid molecule according to claim 16 or the vector according to claim 17; or Expresses the antibody according to any one of claims 1 to 15.

19. A method for preparing the antibody according to any one of claims 1 to 15, characterized in that, The method comprises culturing the cell or host according to claim 18.

20. A conjugate, characterized in that, Comprises: The antibody according to any one of claims 1 to 15 and a conjugate part conjugated thereto; The conjugate part is selected from a purification tag or a label; The label is selected from at least one of colloidal gold, biotin / avidin, spin label, fluorescent label, chromophore label, electron-dense label, radioisotope, fluorophore, rhodamine, luciferase, fluorescein, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, carbohydrate oxidase, glucose oxidase, galactose oxidase, glucose-6-phosphate dehydrogenase, magnetic microspheres, plastic microspheres, plastic particles, microtiter plates, glass, capillary tubes, nylon, and nitrocellulose membranes.

21. A reagent or kit, characterized in that, Comprising: The antibody according to any one of claims 1 to 15 or the conjugate according to claim 20.

22. Use of the antibody according to any one of claims 1 to 15, the conjugate according to claim 20, or the reagent or kit according to claim 21 for detecting D-dimer for non-disease diagnosis purposes.

23. Use of the antibody according to any one of claims 1 to 15, the conjugate according to claim 20, or the reagent or kit according to claim 21 in the preparation of a product for detecting D-dimer.

24. A method for screening D-dimer antibodies, characterized in that, Comprising: a) Designing primers for amino acid substitution at 1, 2, 3, 4, 5, 6, 7, or 8 sites among X1, X2, X3, X4, X5, X6, X7, and X8 defined in the antibody according to claim 5; b) A mutant library constructed with the primers described in a) using the nucleic acid molecule according to claim 16, the vector according to claim 17, or the cell according to claim 18 as a template; c) Screening for D-dimer antibodies from the mutant library.

25. The method according to claim 24, characterized in that, The mutant library is a single-site saturation mutant library.

26. The method according to claim 24, characterized in that The D-dimer antibody comprises or is the antibody according to any one of claims 1 to 15.

27. A mutant library, characterized in that, The mutant library comprises variants of SEQ ID NO:1 and variants of SEQ ID NO:2 defined in the antibody according to any one of claims 1 to 15.

28. The mutant library according to claim 27, wherein, The mutant library is constructed using the primers in the method according to any one of claims 24 to 26.

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