D-dimer antibodies and uses thereof

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 the amino acid sequence shown in SEQ ID NO: 1 or a variant thereof; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 2 or a variant thereof; compared with the amino acid sequence shown in SEQ ID NO: 1, the variant of SEQ ID NO: 1 comprises a mutation at the following sites: at least one of positions 28, 52, 57, and 59; compared with the amino acid sequence shown in SEQ ID NO: 2, the variant of SEQ ID NO: 2 comprises a mutation at positions 30 and / or 98. The antibody of the present invention can specifically bind to D-dimer and can be used for the qualitative or quantitative detection of D-dimer.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to the Chinese patent application with application number 202211678586.8 filed with the China Patent Office on December 26, 2022, entitled “D-dimer antibodies and their uses”, the entire contents of which are incorporated by reference into this application. Technical Field

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

[0004] Dimers are products of fibrin formed by the sequential action of thrombin, coagulation factor XIIIa, and plasmin, and are primarily metabolized through the kidneys and reticuloendothelial system. D-dimer is a fibrin product. Elevated D-dimer levels reflect dual activation of the coagulation and fibrinolytic systems in vivo and serve as a molecular marker of hypercoagulability and hyperfibrinolysis. Inflammation, trauma, surgery, renal failure, liver injury, myocardial infarction, cerebral infarction, pulmonary embolism, arteriovenous thrombosis, tumors, disseminated intravascular coagulation, infection, and tissue necrosis can all lead to elevated D-dimer levels. Therefore, D-dimers have extensive clinical applications. Elevated D-dimer levels can monitor the course of various diseases, such as deep vein thrombosis (DVT), disseminated intravascular coagulation (DIC), myocardial infarction, severe hepatitis, and pulmonary embolism (PE). They also have a role in monitoring potential complications in preeclampsia and pregnant women with high-risk pregnancy. Furthermore, changes in D-dimer levels can be used as an indicator to monitor thrombolytic therapy and guide the dosage of thrombolytic drugs. Therefore, D-dimer testing has important clinical significance for the early diagnosis of thrombotic diseases, monitoring of the disease course, and monitoring of thrombolytic drug treatment.

[0005] Currently, D-dimer detection methods include three batch tests, latex agglutination (LATEX) method, ELISA method, immunofiltration colloidal gold colorimetric method, etc. These methods all require antibodies against D-dimer. Therefore, it is urgent to develop an antibody that can effectively bind to D-dimer. Summary of the Invention

[0006] The present invention aims to provide an antibody for D-dimer, and a reagent or kit for detecting D-dimer.

[0007] In the first aspect of the present invention, 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 the 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 a mutation at the following positions: at least one of positions 28, 52, 57 and 59; the light chain variable region comprises the 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 a mutation at positions 30 and / or 98.

[0008] In the second aspect of the present invention, the present invention provides an antibody, which includes a heavy chain and / or a light chain, wherein the heavy chain includes the heavy chain variable region defined in the antibody described in the first aspect; and the light chain includes the light chain variable region defined in the antibody described in the first aspect.

[0009] In the third aspect of the present invention, the present invention provides an antibody, which includes HCDRs and LCDRs, wherein the HCDRs include or are HCDRs consistent with the HCDRs of the heavy chain variable region defined by the antibody described in the first aspect or the second aspect, and the LCDRs include or are LCDRs consistent with the LCDRs of the light chain variable region defined by the antibody described in the first aspect or the second aspect.

[0010] In the fourth aspect of the present invention, the present invention provides a nucleic acid molecule encoding the antibody according to the first aspect, the second aspect or the third aspect.

[0011] In the fifth aspect of the present invention, the present invention provides a vector, which includes the nucleic acid molecule described in the fourth aspect.

[0012] In the sixth aspect of the present invention, the present invention provides a cell or host, which 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.

[0013] In the seventh aspect of the present invention, the present invention provides a method for preparing the antibody of the first aspect, the second aspect or the third aspect, the method comprising culturing the cell or host of the sixth aspect.

[0014] In the eighth aspect of the present invention, the present invention provides a conjugate, which comprises: the antibody described in the first aspect, the second aspect or the third aspect and a conjugated portion conjugated thereto.

[0015] In the ninth aspect of the present invention, the present invention provides a reagent or a kit, which comprises: the antibody described in the first aspect, the second aspect or the third aspect or the conjugate described in the eighth aspect.

[0016] In the tenth aspect of the present invention, the present invention proposes the use of the antibody described in the first, second or third aspect, the conjugate described in the eighth aspect, or the reagent or kit described in the ninth aspect in detecting D-dimer and preparing a product for diagnosing D-dimer-related diseases.

[0017] In the eleventh aspect of the present invention, the present invention proposes a method for detecting D-dimer, which comprises: using 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 to contact with the sample to be detected to form an immune complex.

[0018] In the twelfth aspect of the present invention, the present invention provides a method for screening D-dimer antibodies, comprising: a) designing primers for amino acid substitution at 1, 2, 3, 4, 5 or 6 sites defined in X1, X2, X3, X4, X5 and X6 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, and constructing a mutation library using the primers described in a); c) screening D-dimer antibodies from the mutation library.

[0019] In the thirteenth aspect of the present invention, the present invention provides a mutation library, which includes variants of SEQ ID NO: 1 and variants of SEQ ID NO: 2 defined in the antibody of the first aspect, the second aspect or the third aspect.

[0020] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. DETAILED DESCRIPTION

[0021] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0022] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of such features. Furthermore, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0023] The endpoints of the ranges and any values disclosed herein are not limited to the precise ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0024] To facilitate understanding of the present invention, certain technical and scientific terms are defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. The abbreviations for amino acid residues are the standard three-letter and / or one-letter codes used in the art to designate one of the 20 commonly used L-amino acids.

[0025] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention, but not excluding other contents.

[0026] As used herein, the terms "optionally," "optional," "optionally," "optional," or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0027] 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 the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences is determined by conventional methods, for example, see Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN program (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Foundation, Washington, DC). 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. 10:106); the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 10:116; the similarity search method of Pearson et al. (1990) Proc. Natl. Acad. Sci. 10:117; the similarity search method of Pearson et al. (1990 ... .70:173-187 (1997); and BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J. Mol. Biol. 215:403-410). Computer programs that utilize these algorithms are also available and include, but are not limited to, ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al., Meth. Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLAST Altschul et al., supra, FASTA, and TFASTA, available in the Genetics Computing Group (GCG) package, Version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0028] Under the premise of not substantially affecting the activity of the antibody (retaining at least 90% of the activity), those skilled in the art can replace, add and / or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more) amino acids in the sequence of the present invention to obtain variants of the antibody sequence. They are all considered to be included in the scope of protection of the present invention. For example, amino acids with similar properties are replaced in the variable region. The variant sequence of the present invention may have at least 90%, 95%, 96%, 97%, 98% or 99% identity (or homology) with the reference sequence. The sequence identity of the present invention can be measured using sequence analysis software. For example, the computer program BLAST, especially BLASTP or TBLASTN, using default parameters. The amino acid sequences described in the present invention are all shown in an N-terminal to C-terminal manner.

[0029] It should be noted that in the claims and description herein, the variants of the antibody sequences 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, and the positions or numbers of the mutation sites defined in the description 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 an amino acid before amino acid position 28 (e.g., position 10, position 27), and those skilled in the art will understand that I28R should be adjusted to I29R; or, a variant of SEQ ID NO: 1 is obtained by deleting two amino acids before amino acid position 28 (e.g., position 10, position 27), and those skilled in the art will understand that I28R should be adjusted to I26R.

[0030] As used herein, the term "at least 80% homology" refers to at least 80%, and may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% homology to each reference sequence. The term "at least 90% homology" refers to at least 90%, and may be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% homology to each reference sequence.

[0031] As used herein, the term "variant" or "mutant" may refer to any naturally occurring or engineered molecule comprising one or more nucleotide or amino acid mutations.

[0032] In this article, the term "vector" generally refers to a nucleic acid molecule that can be inserted into a suitable host and self-replicates, and the inserted nucleic acid molecule is transferred into and / or between cells or hosts. The vector may include a vector primarily used for inserting DNA or RNA into a cell, a vector primarily used for replicating DNA or RNA, and a vector primarily used for expression of the transcription and / or translation of DNA or RNA. The vector also includes a vector with a variety of the 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. Typically, the vector can produce a desired expression product by cultivating a suitable cell or host that contains the vector.

[0033] In this article, the term "cell" generally refers to a cell that has been modified or reorganized using genetic engineering techniques or cell fusion techniques to modify or reorganize the genetic material of a host cell to obtain a cell with a unique trait of stable inheritance. Wherein, the term "host cell" refers to a prokaryotic cell or eukaryotic cell into which a recombinant vector can be introduced. The terms "transformed" or "transfected" as used herein refer to the introduction of a nucleic acid (e.g., 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.

[0034] The present invention provides an antibody, a nucleic acid molecule, a vector, a cell or host, a method for preparing the antibody, a conjugate, a reagent or kit and its use, a method for detecting D-dimer, a method for screening D-dimer antibodies and a mutation library, each of which is described in detail below.

[0035] Antibody

[0036] In a first aspect, the present invention provides an antibody. According to an embodiment of the present invention, the antibody comprises: a heavy chain variable region and / or a light chain variable region; wherein the heavy chain variable region comprises the amino acid sequence 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 a mutation at at least one of positions 28, 52, 57, and 59; and the light chain variable region comprises the amino acid sequence 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 a mutation at positions 30 and / or 98.

[0037] Herein, the term "antibody" is used in the broadest sense and may include full-length monoclonal antibodies, multispecific antibodies, chimeric antibodies or functional fragments without limitation to specific structures, as long as they exhibit the desired antigen-binding activity.

[0038] As used herein, the terms "full-length antibody," "full-length monoclonal antibody," or "full-length monoclonal antibody" are all 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). They typically include a light chain with a lighter molecular weight and a heavy chain with a heavier molecular weight, with the heavy chain (H chain) and light chain (L chain) linked by disulfide bonds to form an antibody molecule. The amino acid sequence at the amino terminus (N-terminus) of the peptide chain varies greatly and is called the variable region (V region); the carboxyl terminus (C-terminus) is relatively stable and varies little, and is called the constant region (C region). The V regions of the L chain and H chain are called VL and VH, respectively.

[0039] In this article, the terms "polyantibody" and "multispecific antibody" are synonymous, both referring to antibodies that can recognize multiple antigenic epitopes, such as antibodies that can recognize two antigenic epitopes (bispecific antibodies, abbreviated as bispecific antibodies), antibodies that recognize three antigenic epitopes, or antibodies that recognize four antigenic epitopes. This is understood in a broad sense, and the specific structure is not limited, 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.

[0040] As used herein, the term "functional fragment" refers to a fragment comprising part or all of an antibody that lacks at least some of the amino acids present in the full-length chain but is still capable of specifically binding to an antigen. For example, the fragment may comprise part or all of an antibody CDR. Such fragments are biologically active because they bind to an antigen and can compete with other antigen-binding molecules (including intact antibodies) for binding to a given epitope. Such fragments include 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 at least one of the minimum recognition units. Such fragments can be produced by recombinant nucleic acid technology, or can be produced by enzymatic or chemical cleavage of antigen-binding molecules (including intact antibodies).

[0041] According to an embodiment of the present invention, the above-mentioned antibody may further include at least one of the following additional technical features:

[0042] 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 in at least one of the following sites: I28R, S52D, K57N / S / A and D59L / I.

[0043] In an optional embodiment of the present invention, the mutation at position 28 is I28R.

[0044] In an optional embodiment of the present invention, the mutation at position 52 is S52D.

[0045] In an optional embodiment of the present invention, the mutation at position 57 is K57N.

[0046] In an optional embodiment of the present invention, the mutation at position 57 is K57S.

[0047] In an optional embodiment of the present invention, the mutation at position 57 is K57A.

[0048] In an optional embodiment of the present invention, the mutation at position 59 is D59L.

[0049] In an optional embodiment of the present invention, the mutation at position 59 is D59I.

[0050] It should be noted that the above-mentioned position numbers are obtained by sequentially numbering the amino acid sequence shown in SEQ ID NO: 1 from the N-terminus to the C-terminus. For example, position 28 refers to the 28th position from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1; "I28R" means that the isoleucine at position 28 of the amino acid sequence shown in SEQ ID NO: 1 is replaced by arginine; "S52D" means that the serine at position 52 of the amino acid sequence shown in SEQ ID NO: 1 is replaced by aspartic acid; and "D59L / I" means that the aspartic acid at position 52 of the amino acid sequence shown in SEQ ID NO: 1 can be replaced by leucine or isoleucine.

[0051] 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:

[0052]

[0053]

[0054] 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 of I30K and / or H98S / A / T.

[0055] In an optional embodiment of the present invention, the mutation at position 30 is I30K.

[0056] In an optional embodiment of the present invention, the mutation at position 98 is H98S.

[0057] In an optional embodiment of the present invention, the mutation at position 98 is H98A.

[0058] In an optional embodiment of the present invention, the mutation at position 98 is H98T.

[0059] It should be noted that the above-mentioned position numbers are 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 30 refers to the 30th position from the N-terminus of the amino acid sequence shown in SEQ ID NO: 2; the "I30K" refers to the replacement of isoleucine at position 30 of the amino acid sequence shown in SEQ ID NO: 2 by lysine; the "H98S" refers to the replacement of histidine at position 98 of the amino acid sequence shown in SEQ ID NO: 2 by serine; and the "H98S / A / T" refers to the replacement of histidine at position 98 of the amino acid sequence shown in SEQ ID NO: 2 by serine, alanine, or threonine.

[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 mutations at the following sites:

[0061] mutation site VL variant 1 H98T VL variant 2 H98A VL variant 3 H98S VL variant 4 I30K VL variant 5 I30K,H98A VL variant 6 I30K,H98S VL variant 7 I30K,H98T

[0062] According to an embodiment of the present invention, the antibody includes the heavy chain variable region and light chain variable region shown in the following table:

[0063]

[0064]

[0065]

[0066] In a second aspect, 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 a heavy chain variable region defined in the antibody of the first aspect; and the light chain comprises a light chain variable region defined in the antibody of the first aspect.

[0067] According to an embodiment of the present invention, the antibody includes the heavy chain and light chain shown in the following table:

[0068]

[0069]

[0070] Those skilled in the art will appreciate that the features and advantages described above for the antibodies described in the first aspect (such as the features of the heavy chain variable region and / or light chain variable region defined in the first aspect) are also applicable to the antibodies described in the second aspect and will not be repeated here.

[0071] In a third aspect, 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 identical to the HCDRs of the heavy chain variable region defined by the antibody described in the first or second aspect, and the LCDRs comprise or are identical to the LCDRs of the light chain variable region defined by the antibody described in the first or second aspect.

[0072] As used herein, the terms "complementarity determining region," "CDR," or "CDRs" refer to the hypervariable regions of the heavy and light chains of immunoglobulins, and include one or more, or even all, of the amino acid residues that contribute primarily to the binding affinity of an antibody to its recognized antigen or epitope. In specific embodiments of the present disclosure, CDRs refer to the hypervariable regions of the heavy and light chains of the antibody.

[0073] As used herein, the heavy chain complementary determining regions (heavy chain variable region CDRs) are referred to as "HCDRs" or "HCDRs," which include HCDR1, HCDR2, and HCDR3; the light chain complementary determining regions (light chain variable region CDRs) are referred to as "LCDRs" or "LCDRs," 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 in Kabat et al., U.S. Patent No. 20 ... Exemplary defined CDRs are listed below in Table 1. Given the variable region amino acid sequence of an antibody, one skilled in the art can routinely determine which residues comprise a particular CDR.

[0074] Table 1: CDR Definition 1

[0075]

[0076]

[0077] 1 The numbering of all CDR definitions in Table 1 is according to the Kabat numbering system (see below).

[0078] 2 "AbM" as used in Table 1 with a lowercase "b" refers to CDRs defined by Oxford Molecular's "AbM" antibody modeling software.

[0079] It should be noted that the polypeptide sequences in the sequence listing and Table 2 of the present invention are not numbered according to the Kabat numbering system. However, those skilled in the art are fully capable of converting the sequence numbers in the sequence listing into Kabat numbers.

[0080] According to an embodiment of the present invention, the HCDRs and / or LCDRs are defined by the Kabat, Chothia, AbM, Contact or IMGT system.

[0081] According to an embodiment of the present invention, the HCDR1, HCDR2 and HCDR3 include or are the amino acid sequences of positions 26 to 35, 50 to 65, and 95 to 102 of the heavy chain variable region according to Kabat numbering, respectively; the LCDR1, LCDR2 and LCDR3 include or are the amino acid sequences of positions 24 to 34, 50 to 56, and 89 to 97 of the light chain variable region according to Kabat numbering, respectively.

[0082] It should be noted that CDRs defined by other methods not limited to those in Table 1 also fall within the scope of protection of the present disclosure.

[0083] According to an embodiment of the present invention, the HCDRs and LCDRs include or are the amino acid sequences shown below:

[0084] HCDR 1: GYX1FTDYSMH, wherein X1 is I or R;

[0085] HCDR 2: VIX2TYSGX3TX4YNQKFKG, wherein X2 is S or D, X3 is K, N, S or A, and X4 is D, L or I;

[0086] HCDR 3:MDDYDGDYFFDY;

[0087] LCDR1:RSSQSLX5HSNGNTYLH, where X5 is I or K;

[0088] LCDR 2: KVSNRFS;

[0089] LCDR 3: SQSRX6VPLT, where X6 is H, S, A, or T.

[0090] According to an embodiment of the present invention, X1 is 1.

[0091] According to an embodiment of the present invention, X1 is R.

[0092] According to an embodiment of the present invention, X2 is S.

[0093] According to an embodiment of the present invention, X2 is D.

[0094] According to an embodiment of the present invention, X3 is K.

[0095] According to an embodiment of the present invention, X3 is N.

[0096] According to an embodiment of the present invention, X3 is S.

[0097] According to an embodiment of the present invention, X3 is A.

[0098] According to an embodiment of the present invention, X4 is D.

[0099] According to an embodiment of the present invention, X4 is L.

[0100] According to an embodiment of the present invention, X4 is 1.

[0101] According to an embodiment of the present invention, X5 is 1.

[0102] According to an embodiment of the present invention, X5 is K.

[0103] According to an embodiment of the present invention, X6 is H.

[0104] According to an embodiment of the present invention, X6 is S.

[0105] According to an embodiment of the present invention, X6 is A.

[0106] According to an embodiment of the present invention, X6 is T.

[0107] 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:

[0108]

[0109]

[0110]

[0111] 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, wherein the heavy chain framework region comprises HFR1, HFR2, HFR3 and HFR4; and the light chain framework region comprises LFR1, LFR2, LFR3 and LFR4.

[0112] According to an embodiment of the present invention, at least a portion of at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4 is derived from at least one of mouse antibodies, human antibodies, primate antibodies, bovine antibodies, equine antibodies, dairy cow antibodies, porcine antibodies, sheep antibodies, goat antibodies, dog antibodies, cat antibodies, rabbit antibodies, camel antibodies, donkey antibodies, deer antibodies, mink antibodies, chicken antibodies, duck antibodies, goose antibodies, turkey antibodies, fighting cock antibodies or mutants thereof.

[0113] Herein, the "framework region" or "FR" region includes the heavy chain framework region and the light chain framework region, and refers to the region other than the CDR in the antibody heavy chain variable region (which can be represented as VH) and the light chain variable region (which can be represented as VL); wherein the heavy chain framework region is represented by "HFR" and can be further subdivided into adjacent regions separated by CDRs, including HFR1, HFR2, HFR3 and HFR4 framework regions; the light chain framework region is represented by "LFR" and can be further subdivided into adjacent regions separated by CDRs, including LFR1, LFR2, LFR3 and LFR4 framework regions.

[0114] As used herein, the heavy chain variable region is obtained by connecting the following numbered CDRs and FRs in the following combinations: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by connecting the following numbered CDRs and FRs in the following combinations: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0115] In an optional embodiment of the present invention, the HFR1 comprises the amino acid sequence shown in SEQ ID NO: 36 or an amino acid sequence having at least 80% homology thereto;

[0116] The HFR2 comprises the amino acid sequence shown in SEQ ID NO: 37 or an amino acid sequence having at least 80% homology thereto;

[0117] The HFR3 comprises the amino acid sequence shown in SEQ ID NO: 38 or an amino acid sequence having at least 80% homology thereto;

[0118] The HFR4 comprises the amino acid sequence shown in SEQ ID NO: 39 or an amino acid sequence having at least 80% homology thereto;

[0119] The LFR1 comprises the amino acid sequence shown in SEQ ID NO: 40 or an amino acid sequence having at least 80% homology thereto;

[0120] The LFR2 comprises the amino acid sequence shown in SEQ ID NO: 41 or an amino acid sequence having at least 80% homology thereto;

[0121] The LFR3 comprises the amino acid sequence shown in SEQ ID NO: 42 or an amino acid sequence having at least 80% homology thereto;

[0122] The LFR4 comprises the amino acid sequence shown in SEQ ID NO: 43 or an amino acid sequence having at least 80% homology thereto.

[0123] According to an embodiment of the present invention, the antibody comprises the heavy chain variable region and / or light chain variable region described in the first aspect or the second aspect.

[0124] Those skilled in the art will appreciate that the features and advantages described above for the antibodies described in the first and second aspects (e.g., features such as the heavy chain variable region and / or light chain variable region defined in the first aspect, and features such as the heavy chain and / or light chain defined in the second aspect) are also applicable to the antibodies described in the third aspect and will not be elaborated here.

[0125] According to an embodiment of the present invention, the antibodies described in the first, second and third aspects above may further include at least one of the following technical features:

[0126] According to an embodiment of the present invention, the antibody further comprises a constant region; wherein the constant region comprises at least one of a heavy chain constant region and a light chain constant region.

[0127] According to an embodiment of the present invention, at least a portion of at least one of the heavy chain constant region and the light chain constant region is derived from at least one of a mouse antibody, a human antibody, a primate antibody, a bovine antibody, a horse antibody, a dairy bovine antibody, a porcine antibody, a sheep antibody, a goat antibody, a dog antibody, a cat antibody, a rabbit antibody, a camel antibody, a donkey antibody, a deer antibody, a mink antibody, a chicken antibody, a duck antibody, a goose antibody, a turkey antibody, a fighting cock antibody, or a mutant thereof.

[0128] According to an embodiment of the present invention, the heavy chain constant region includes a heavy chain constant region selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; or, the light chain constant region includes a light chain constant region selected from κ type or λ type.

[0129] In an optional embodiment of the present invention, the light chain constant region and the heavy chain constant region are both derived from murine antibodies or mutants thereof.

[0130] 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.

[0131] In an optional embodiment of the present invention, the heavy chain constant region includes or is a heavy chain constant region as shown in SEQ ID NO:3 or an amino acid sequence with at least 80% identity thereto; or, the light chain constant region includes or is a light chain constant region as shown in SEQ ID NO:4 or an amino acid sequence with at least 80% identity thereto.

[0132] According to an embodiment of the present invention, the antibody comprises the heavy chain and / or light chain described in the second aspect.

[0133] 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 minimum recognition units.

[0134] Herein, the terms "single domain antibody", "nanoantibody" and "VHH antibody" are used interchangeably and were originally described as antigen-binding immunoglobulin (variable) domains of "heavy chain antibodies" (i.e., "antibodies lacking light chains") (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: "Naturally occurring antibodies devoid of light chains"; Nature 363, 446-448 (1993)), comprising a heavy chain variable region (VH) and conventional CH2 and CH3 regions, which specifically bind to antigen proteins (e.g., D-dimers) through the heavy chain variable region.

[0135] As used herein, the term "Fab antibody" or "Fab fragment" generally refers to an antibody or fragment containing only the Fab molecule, which is composed of the VH and CH1 of the heavy chain and a complete light chain, with the light chain and heavy chain connected by a disulfide bond.

[0136] As used herein, the term "F(ab')2 antibody" or "F(ab')2 fragment" has two antigen-binding F(ab') portions linked together by a disulfide bond.

[0137] As used herein, the term "Fv antibody" or "Fv fragment" generally refers to an antibody or fragment consisting solely of a light chain variable region (VL) and a heavy chain variable region (VH) linked by non-covalent bonds. It is the smallest functional fragment of an antibody that retains a complete antigen-binding site.

[0138] As used herein, the terms "single-chain antibody" and "scFv fragment" refer to antibodies or fragments formed by connecting the heavy chain variable region and the light chain variable region of an antibody via a short peptide.

[0139] In this article, the terms "minimum recognition unit" and "MRU" both refer to antibodies or fragments consisting of only one CDR, which has a very small molecular weight of only about 1% of the complete antibody.

[0140] Nucleic acid molecule, vector, cell or host, and method for preparing antibody

[0141] In the process of preparing or obtaining the antibodies described in the first, second or third aspects, nucleic acid molecules expressing these antibodies can be connected to different vectors and then expressed in different cells to obtain the corresponding antibodies.

[0142] In a fourth aspect, 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 aspect, the second aspect, or the third aspect. The nucleic acid molecule according to an embodiment of the present invention can encode the above-mentioned antibody.

[0143] According to an embodiment of the present invention, the nucleic acid molecule includes DNA or RNA.

[0144] It should be noted that, for nucleic acid molecules mentioned herein, those skilled in the art will understand that they actually include any one or both of the complementary double strands. For convenience, although only one strand is provided in most cases herein, the other strand complementary thereto is also disclosed. In addition, the molecular sequences in the present invention include DNA or RNA forms, and disclosure of one of them implies that the other is also disclosed.

[0145] In the fifth aspect of the present invention, the present invention proposes a vector. According to an embodiment of the present invention, the vector includes the nucleic acid molecule described in the fourth aspect. When the above-mentioned 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. Of course, these control elements can come directly from the vector itself, or they can be exogenous, that is, not from the vector itself. Of course, the nucleic acid molecule and the control elements can be operably connected. In this article, "operably connected" means that the exogenous gene is connected to the vector so that the control elements in the vector, such as transcription control sequences and translation control sequences, can play their expected function of regulating the transcription and translation of the exogenous gene. Commonly used vectors can be, for example, plasmids, phages, etc. After the vectors according to some specific embodiments of the present invention are introduced into suitable recipient cells, the expression of the aforementioned antibodies can be effectively achieved under the mediation of the regulatory system, thereby achieving large-scale in vitro acquisition of antibodies.

[0146] In some specific embodiments of the present invention, the vector is a eukaryotic expression vector, a prokaryotic expression vector, a virus or a bacteriophage.

[0147] In an optional embodiment of the present invention, the expression vector is a plasmid expression vector.

[0148] In its sixth aspect, the present invention provides a cell or host. According to embodiments 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, second, or third aspect. Under suitable conditions, the cell or host can effectively express the aforementioned antibody within the cell.

[0149] According to an embodiment of the present invention, the cell is obtained by introducing the vector described in the fifth aspect into the cell.

[0150] It should be noted that the cells of the present invention are not particularly limited and may be prokaryotic cells, eukaryotic cells, or bacteriophages. The prokaryotic cells may be Escherichia coli, Bacillus subtilis, Streptomyces, or Proteus mirabilis. The eukaryotic cells include fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Trichoderma, insect cells such as S. frugiperda, plant cells such as tobacco, and mammalian cells such as BHK cells, CHO cells, COS cells, and myeloma cells.

[0151] In an optional embodiment of the present invention, the cells are mammalian cells, including BHK cells, CHO cells, NSO cells or COS cells, and do not include animal germ cells, fertilized eggs or embryonic stem cells.

[0152] It should be noted that the "suitable conditions" described in the present invention refer to conditions suitable for the expression of the antibodies of the present invention. It will be readily understood by those skilled in the art that 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 status, suitable cell density, suitable cell culture environment, and suitable cell culture time. "Suitable conditions" are not particularly limited, and those skilled in the art can optimize the most suitable conditions for the expression of the antibodies according to the specific environment of the laboratory.

[0153] In a seventh aspect, the present invention provides a method for preparing the antibody of the first, second, or third aspect. According to embodiments of the present invention, the method comprises culturing the cell or host of the sixth aspect. The methods according to some specific embodiments of the present invention can effectively obtain the antibody in large quantities.

[0154] Based on the amino acid sequence of the antibody disclosed herein, those skilled in the art can easily conceive of using genetic engineering technology or other technologies (chemical synthesis, recombinant expression) to prepare the antibody. For example, the antibody can be isolated and purified from the culture product of recombinant cells that can recombinantly express the antibody described in any of the above items. This is easy to achieve for those skilled in the art. Based on this, no matter what technology is used to prepare the antibody disclosed herein, it falls within the scope of protection of the present disclosure.

[0155] Those skilled in the art will appreciate that the features and advantages described above for the antibodies described in the first, second and third aspects are also applicable to the nucleic acid molecule, vector, cell or host, and method for preparing the antibody, and will not be elaborated here.

[0156] Conjugate, reagent or kit and use thereof

[0157] The antibody can be used in combination with any detection reagent or therapeutic preparation, for example, in combination with diagnostic nuclides, nanomaterials, etc., to detect the target site through the radioactivity of the nuclide, and then obtain information about the target site; it can also be used in combination with therapeutic nuclides, using the radioactivity of the nuclide to specifically kill target cells, tissues, etc.

[0158] In an eighth aspect, the present invention provides a conjugate. According to embodiments of the present invention, the conjugate comprises: the antibody described in the first, second, or third aspects, and a conjugated moiety conjugated thereto. The conjugate according to embodiments 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 associated with abnormal D-dimer levels.

[0159] According to an embodiment of the present invention, the conjugate may further include at least one of the following additional technical features:

[0160] According to an embodiment of the present invention, the coupling moiety is selected from a carrier. Exemplarily, the carrier includes a purification tag or marker.

[0161] Herein, the carrier can be a substance that can be suspended or dispersed in a liquid phase (e.g., a solid phase carrier such as particles or magnetic beads), or a solid phase that can contain or carry a liquid phase (e.g., a support such as a plate, a membrane, a test tube, and a container such as a well plate, a microfluidic channel, a glass capillary, a nanocolumn, a monolithic column, etc.); it can also be a labeling carrier for labeling an antibody, such as an enzyme (e.g., peroxidase, alkaline phosphatase, luciferase, β-galactosidase), an affinity substance (e.g., one of streptavidin and biotin, one of mutually complementary sense and antisense nucleic acids), 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'-bipyridine)ruthenium, luminol), a radioactive isotope (e.g., 3 H. 14 C. 32 P. 35 S. 125 I) and gold colloid, etc.

[0162] In some specific embodiments of the present invention, the purification tag or label comprises at least one selected from colloidal gold, a radioactive label, a luminescent substance, a colored substance, an enzyme, biotin / avidin, and a spin label.

[0163] In some specific embodiments of the present invention, the purification tag or label comprises at least one selected from a fluorescent label, a chromophore label, and an electron-dense label.

[0164] In some specific embodiments of the present invention, the purification tag or label comprises one or more selected from radioisotopes, fluorophores, rhodamine and its derivatives, luciferase, luciferin, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, carbohydrate oxidase, glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase.

[0165] In some specific embodiments of the present invention, the coupling part includes at least one selected from magnetic microspheres, plastic microspheres, plastic particles, microporous plates, glass, capillaries, nylon and nitrocellulose membranes.

[0166] In the ninth aspect of the present invention, the present invention provides a reagent or kit. According to an embodiment of the present invention, the reagent or kit comprises: the antibody described in the first aspect, the second aspect or the third aspect or the conjugate described in the eighth aspect. As mentioned above, the antibodies in some specific embodiments or examples of the present invention can effectively bind to D-dimer, and therefore, the reagent or kit comprising the antibody can effectively perform qualitative or quantitative detection of D-dimer. The reagent or kit provided by the present invention can be used, for example, for detection involving the use of the specific binding properties of D-dimer and its antibody, such as immunoblotting and immunoprecipitation. As mentioned above, the antibodies in some specific embodiments or examples of the present invention have higher binding activity with D-dimer, and therefore the reagent or kit comprising the antibody has higher detection sensitivity.

[0167] These kits may include any one or more of the following: processing fluid, anti-D-dimer antibodies, D-dimer quality control, anti-IgG antibodies, instructions or literature, etc. Anti-D-dimer antibodies can be used in various diagnostic tests, such as in vitro or in vivo detection of various diseases, drugs, or other proteins. For example, they can be used to test for related diseases by testing serum or blood in subjects.

[0168] In the tenth aspect of the present invention, the present invention proposes a use of the antibody described in the first, second or third aspect, the conjugate described in the eighth aspect or the reagent or kit described in the ninth aspect in detecting D-dimer or preparing a product for diagnosing D-dimer-related diseases.

[0169] Herein, 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 Henoch-Schonlein purpura.

[0170] As used herein, the term "thrombotic disease" refers to a disease caused by thrombus, including but not limited to arteriovenous thrombosis (eg, deep vein thrombosis (DVT)), pulmonary embolism, and the like.

[0171] In an eleventh aspect, the present invention provides a method for detecting D-dimer. According to an embodiment of the present invention, the method comprises: contacting the antibody described in the first, second, or third aspect, the conjugate described in the eighth aspect, or the reagent or kit described in the ninth aspect with a sample to be detected to form an immune complex.

[0172] It should be noted that the above-mentioned "samples to be tested" can be samples to be tested from patients, such as serum samples; or they can be non-patient samples that may contain D-dimers. For example, in scientific research, the above-mentioned method is only used to detect the presence or content of D-dimers in the sample, and does not involve disease diagnosis.

[0173] According to an embodiment of the present invention, whether the sample to be tested contains D-dimer or the content of the D-dimer is determined based on the signal of the immune complex.

[0174] According to an embodiment of the present invention, the immune complex further includes a second antibody, which binds to the antibody.

[0175] According to an embodiment of the present invention, the immune complex further includes a second antibody, and the second antibody binds to D-dimer.

[0176] According to an embodiment of the present invention, the signal comprises a fluorescence signal.

[0177] In the twelfth aspect of the present invention, a method for screening for D-dimer antibodies is provided. According to an embodiment of the present invention, the method comprises: a) designing primers for amino acid substitution at one, two, three, four, five, or six of the positions X1, X2, X3, X4, X5, and X6 defined in the antibody described in the second aspect; b) constructing a mutation library using the primers described in a) 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; and c) screening for D-dimer antibodies from the mutation library.

[0178] According to an embodiment of the present invention, the mutation library is a single-point saturation mutation library.

[0179] According to an embodiment of the present invention, the D-dimer antibody includes or is the antibody described in the first aspect, the second aspect or the third aspect.

[0180] In the thirteenth aspect of the present invention, the present invention provides a mutation library, which includes variants of SEQ ID NO: 1 and variants of SEQ ID NO: 2 defined in the antibody of the first aspect, the second aspect or the third aspect.

[0181] According to an embodiment of the present invention, the mutation library is constructed using the primers in the method described in the twelfth aspect.

[0182] In the fourteenth aspect of the present invention, the present invention provides an antibody as described in the first, second or third aspect, the conjugate as described in the eighth aspect or the reagent or kit as described in the ninth aspect, for detecting D-dimer or for diagnosing D-dimer-related diseases.

[0183] In the fifteenth aspect of the present invention, the present invention proposes a use of the antibody described in the first, second or third aspect, the conjugate described in the eighth aspect or the reagent or kit described in the ninth aspect in the preparation of a product for detecting D-dimer or for diagnosing D-dimer-related diseases.

[0184] The amino acid sequences or nucleotide sequences involved herein are shown in Table 2, wherein the mutation sites of the heavy chain variable region or HCDRs are based on 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 based on the amino acid sequence shown in SEQ ID NO: 2 or the LCDRs in SEQ ID NO: 2. WT indicates no mutation.

[0185] Table 2: Amino acid sequences

[0186]

[0187]

[0188]

[0189]

[0190]

[0191]

[0192]

[0193]

[0194] Below, the scheme of the present invention will be explained in conjunction with embodiment.It will be understood by those skilled in the art 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.In the embodiment, if specific technology or conditions are not indicated, the technology or conditions described in the literature in this area or the product instructions are used.The reagents or instruments used are not indicated by the manufacturer, and are all conventional products that can be obtained by commercial purchase.

[0195] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled 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 preparations or unit doses herein, some methods and materials are now described. Unless otherwise indicated, the techniques employed or contemplated herein are standard methods. Materials, methods, and examples are illustrative and non-limiting only.

[0196] Practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a skilled artisan. The technique is fully explained in the literature, for example, in Molecular Cloning: A Laboratory Manual, 2nd ed. (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); and PCR: The Polymerase Chain Reaction. Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (J.E. Colligan et al., eds., 2011), each of which is expressly incorporated herein by reference.

[0197] 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 in our laboratory, gel recovery kits and plasmid extraction kits were commercially available, and primer synthesis and gene sequencing were performed by an outsourced company. The sequence of the D-dimer monoclonal antibody (hereinafter referred to as WT antibody) was derived from mouse hybridoma cell sequencing.

[0198] Example 1: Construction and screening of mutation library

[0199] 1. Construction of wild-type (WT) D-dimer antibody (abbreviated as WT antibody) template plasmid

[0200] (1) WT antibody gene synthesis:

[0201] The nucleotide sequences of the WT antibody's VH and VL were codon-optimized in E. coli, and the antibody gene sequences were then outsourced to a company for gene synthesis. The VH and VL amino acid sequences of the WT antibody are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0202] (2) WT antibody gene fragment amplification:

[0203] 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, and then purified using a gel recovery kit to obtain the antibody gene fragments.

[0204] (3) Enzymatic digestion and ligation of WT antibody gene fragments:

[0205] The antibody gene fragment and V01 vector plasmid obtained in step (2) were simultaneously digested with restriction endonucleases and then purified using a gel recovery kit to obtain the antibody gene fragment with sticky ends and the V01 vector. Subsequently, the antibody gene fragment and the V01 vector were ligated with T4 DNA ligase at 22°C for 4 hours. The ligation reaction product was recovered and purified, and the DNA concentration was determined. Finally, 100 ng of the plasmid was transformed into 100 ul of TG1 competent Escherichia coli cells to obtain a bacterial solution. The entire bacterial solution was then spread on an ampicillin-resistant plate and cultured at 37°C overnight.

[0206] (4) Extraction and sequencing verification of WT template plasmid

[0207] Select 10 monoclonal colonies cultured overnight in step (3), perform colony PCR and gel electrophoresis detection using Taq DNA polymerase, select bacteria with the antibody gene sequence correctly inserted for culture and amplification, use a plasmid extraction kit to obtain the WT template plasmid, and send it to a sequencing company for gene sequencing verification.

[0208] 2. Construction of single-point mutation library

[0209] 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.

[0210] (1) Primer design and synthesis

[0211] By using the degenerate base codons, 71 pairs of upstream and downstream primers for single-point saturation mutagenesis of the entire CDR region of VH and VL (71 amino acid sites) were designed and handed over to an outsourcing company for primer synthesis.

[0212] (2) Single-point saturation mutation plasmid PCR amplification

[0213] The single-point saturation mutation plasmid was amplified by PCR using the primers obtained in step (1). The reaction system was configured according to Table 3, and the PCR reaction conditions in Table 4 were used to amplify and prepare the single-point saturation mutation library plasmid. Finally, the WT template plasmid obtained in step 1 was digested with restriction endonucleases at 37°C for 1 hour to obtain 71 mutation library plasmids.

[0214] Table 3: PCR amplification reaction system

[0215]

[0216]

[0217] Table 4: PCR reaction conditions

[0218] Step 1 Step 2 Step 3 Step 4 Step 5 Step 6 temperature 95℃ 95℃ 55-60℃ 72℃ 72℃ 4℃ time 5min 30s 30s 2min 5min ∞

[0219] Note: Step 2 to Step 4 are repeated 22 times.

[0220] (3) Single-site saturation mutagenesis plasmid transformation:

[0221] Take the plasmids of the 71 mutant libraries obtained in step (2), take 10 μl of the reaction product and transform it into 100 μl of TG1 Escherichia coli competent cells to obtain bacterial liquid, then spread all the bacterial liquid on a plate containing ampicillin resistance and culture at 37°C overnight.

[0222] 3. Screening of single-point mutation libraries

[0223] (1) Antibody expression from mutant library

[0224] 500 μl of culture medium was added to a 96-well culture plate in advance. For each single-point mutation library, 92 monoclonal colonies transformed with the single-point saturation mutation plasmid cultured overnight in step 2-(3) were selected, and WT, negative control (i.e., colonies without VH\VL genes inserted), and blank control (i.e., only culture medium without colonies) colonies were set. After culturing at 37°C for 5-6 hours, the bacterial solution was transferred to a new 96-well culture plate. Then, after culturing at 37°C for 1-2 hours, induction medium was finally added, and the antibodies were expressed by culturing at 37°C overnight to obtain antibody expression supernatants of 71 mutation libraries.

[0225] (2) Mutation library screening and sequencing

[0226] Commercially available D-dimer protein was added to 71 ELISA plates at a concentration of 0.75 μg / ml and 100 μl / well, and the plates were coated overnight at 4°C. The next day, the plates were blocked with 1-2% skim milk powder. The antibody expression supernatants of the 71 mutant libraries obtained in step (1) were added to the ELISA plates at a concentration of 100 μl / well, and WT, negative control (i.e., colonies without VH\VL genes) and blank control (i.e., only culture medium without colonies) were set up. The plates were incubated at room temperature for 2 hours, and conventional ELISA detection methods were used for subsequent plate washing, color development, and reading. Finally, the data results were sorted and analyzed, and the clones with improved results were sent for sequencing. Finally, the sequencing results were analyzed, and the mutation sites of 11 unique mutation candidate clones (see Table 5) were selected for the construction of a combinatorial mutation 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 the WT; when the Ratio value is greater than 1, it means that the affinity has been improved).

[0227] Table 5: Screening results and mutation sites of candidate clones

[0228]

[0229]

[0230] Among them, WT in this article represents no mutation relative to the wild-type sequence (same as Table 6 and Table 7).

[0231] 4. Construction of combinatorial mutation library

[0232] (1) Library primer design and synthesis:

[0233] Based on the mutation sites on the VH and VL of the D-dimer antibody obtained in step 3-(2), primers for amplification of the combinatorial mutation library are designed and handed over to an outsourcing company for primer synthesis.

[0234] (2) Fragment amplification and ligation

[0235] The antibody mutant fragments were amplified according to the PCR system in Table 3 and the PCR reaction conditions in Table 4, and then the antibody mutant fragments were recovered from the gel. The antibody mutant fragments were spliced into a complete antibody fragment (heavy chain variable region or light chain variable region) using the Overlap PCR method.

[0236] Finally, the antibody fragment was inserted into the V01 vector using enzyme digestion and ligation to form a complete antibody expression plasmid (for specific steps, see step 1-(3) "WT antibody gene fragment digestion and ligation"). 100 ng of the plasmid was transformed into 100 μl of TG1 competent E. coli cells, and the entire bacterial solution was spread onto an ampicillin-resistant plate and cultured at 37°C overnight.

[0237] 5. Screening of combinatorial mutation libraries

[0238] 47 combined mutant antibodies were randomly selected for supernatant expression, ELISA screening, and positive clone sequencing analysis. The Ratio values are shown in Table 6.

[0239] Table 6: Information of candidate clones for combined mutation

[0240]

[0241]

[0242] Example 2: Expression of mutant D-dimer antibodies

[0243] In this example, the mutant D-dimer antibody obtained by screening in Example 1 was expressed, and the specific experimental procedures were as follows:

[0244] 1. Construction of eukaryotic recombinant expression plasmid

[0245] pcDNA TM 3.4 The vector was a recombinant antibody eukaryotic expression vector constructed using pcDNA3.4A, which had multiple cloning restriction sites such as HindIII, BamHI, and EcoRI introduced into the vector. The vector was named pcDNA3.4A expression vector, hereinafter referred to as 3.4A expression vector. Based on the variable region gene sequences of the 47 candidate clones obtained by screening the combinatorial mutation library (see Table 6, and for specific amino acid sequences, see Table 2), VL and VH gene-specific amplification primers and constant region overlap primers (the amino acid sequences of the heavy and light chain constant regions are shown in Table 2, where the N-terminus of the heavy chain constant region is connected to the C-terminus of the VH, representing the heavy chain, and the N-terminus of the light chain constant region is connected to the C-terminus of the VL, representing the light chain) of the corresponding antibody sequences were designed. The primers at both ends contained HindIII and EcoRI restriction sites and protective bases, respectively. A 0.73 KB light chain gene fragment and a 1.40 kb heavy chain gene fragment were amplified by PCR.

[0246] The heavy and light chain gene fragments were digested with HindIII / EcoRI, and the 3.4A vector was digested with HindIII / EcoRI. The digested antibody light and heavy chain gene fragments and vector were purified and recovered. The gene fragments encoding the antibody light and heavy chains were then ligated into the 3.4A expression vector and transformed into DH5α E. coli competent cells. After colonies grew, single colonies were picked for PCR identification of positive clones. Positive clones were selected for sequencing to confirm the correctness of the sequence. Correctly sequenced clones were selected for plasmid extraction and set aside.

[0247] 2. Sample preparation of recombinant antibodies

[0248] Resuscitate HEK293 cells in advance and subculture them into 200 ml system to make the cell density reach (3-5)×10 6 cells / ml, cell viability>95%; wash the cells by centrifugation, resolubilize with culture medium, and adjust the cell density to 2.9×10 6 cells / ml, as the cell diluent. Prepare plasmid DNA and transfection reagent diluents separately using culture medium. Add the transfection reagent diluent to the plasmid DNA diluent, mix thoroughly, and let stand at room temperature for 15 minutes. Slowly add the mixture to the cell diluent over 1 minute, mix thoroughly, and then take samples and count them. Record and observe the viability of the cells after transfection. Place the cells in a 35°C constant temperature incubator at 120 rpm and 8% CO2. After 13 days, centrifuge and collect samples. Affinity purification using a protein A affinity chromatography column yielded 47 antibodies. The amino acid sequences of the heavy chain variable regions, light chain variable regions, heavy chains, and light chains of the 47 antibodies are shown in Table 7.

[0249] Table 7: Amino acid sequences of heavy chain variable regions, light chain variable regions, heavy chains and light chains of 47 antibodies

[0250]

[0251]

[0252] Example 3: Affinity Analysis

[0253] The wild-type antibody (the amino acid sequence of the heavy chain is shown in SEQ ID NO: 115, and the amino acid sequence of the light chain is shown in SEQ ID NO: 107), the 11 single-site mutated candidate clone antibodies obtained in step 3 of Example 1, and the 47 combined mutant antibodies obtained in step 2 of Example 2 were subjected to affinity detection analysis. The specific steps are: the binding and dissociation curves of the D-dimer and the mutant D-dimer antibody are tested on the Biacore8K+ device, and the instrument automatically fits to obtain the affinity constant, association rate, and dissociation rate. In the affinity test results, KD represents the equilibrium dissociation constant, i.e., the affinity constant. The smaller the KD value, the higher the affinity; ka represents the association rate; and kd represents the dissociation rate. The results show that the affinity of the mutant antibody to the D-dimer is better than that of the wild-type antibody to the D-dimer. The affinity test results of the exemplary displayed antibodies are shown in Table 8 below:

[0254] Table 8 Antibody affinity test results

[0255]

[0256]

[0257] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0258] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are exemplary and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. An anti-D-dimer antibody, characterized in that The antibody comprises 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 comprises the heavy chain variable region defined in the antibody of claim 1 ; the light chain comprises the light chain variable region defined in the antibody of claim 1 .

3. An anti-D-dimer antibody comprising a heavy chain and a light chain as shown in the following table:

4. An anti-D-dimer antibody comprising HCDRs and LCDRs, characterized in that: The HCDRs are consistent with the HCDRs of the heavy chain variable region defined by the antibody according to claim 1, and the LCDRs are consistent with the LCDRs of the light chain variable region defined by the antibody according to claim 1; 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: HCDR 1: GYX1FTDYSMH, wherein X1 is I or R; HCDR 2: VIX2TYSGX3TX4YNQKFKG, wherein X2 is S or D, X3 is K, N, S or A, and X4 is D, L or I; HCDR 3:MDDYDGDYFFDY; LCDR1:RSSQSLX5HSNGNTYLH, where X5 is I or K; LCDR 2: KVSNRFS; LCDR 3: SQSRX6VPLT, where X6 is H, S, A, or T; The antibody comprises HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 as shown in the following table:

6. The antibody according to claim 5, characterized in that further including HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, LFR4; Among them, the HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4 are derived from mouse antibodies, primate antibodies, bovine antibodies, equine antibodies, porcine antibodies, sheep antibodies, goat antibodies, dog antibodies, cat 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, characterized in that The HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4 are derived from human antibodies, cow antibodies, turkey antibodies, or fighting cock antibodies.

8. The antibody according to claim 6, characterized in that The HFR1 comprises the amino acid sequence shown in SEQ ID NO: 36 or an amino acid sequence having at least 80% homology thereto; The HFR2 comprises the amino acid sequence shown in SEQ ID NO: 37 or an amino acid sequence having at least 80% homology thereto; The HFR3 comprises the amino acid sequence shown in SEQ ID NO: 38 or an amino acid sequence having at least 80% homology thereto; The HFR4 comprises the amino acid sequence shown in SEQ ID NO: 39 or an amino acid sequence having at least 80% homology thereto; The LFR1 comprises the amino acid sequence shown in SEQ ID NO: 40 or an amino acid sequence having at least 80% homology thereto; The LFR2 comprises the amino acid sequence shown in SEQ ID NO: 41 or an amino acid sequence having at least 80% homology thereto; The LFR3 comprises the amino acid sequence shown in SEQ ID NO: 42 or an amino acid sequence having at least 80% homology thereto; The LFR4 comprises the amino acid sequence shown in SEQ ID NO: 43 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 includes a heavy chain constant region and a light chain constant region.

10. The antibody according to claim 9, characterized in that The heavy chain constant region and the light chain constant region are derived from mouse antibodies, primate antibodies, bovine antibodies, equine antibodies, porcine antibodies, sheep antibodies, goat antibodies, dog antibodies, cat antibodies, rabbit antibodies, camel antibodies, donkey antibodies, deer antibodies, mink antibodies, chicken antibodies, duck antibodies, or goose antibodies.

11. The antibody according to claim 10, characterized in that The heavy chain constant region and the light chain constant region are derived from human antibodies, cow antibodies, turkey antibodies, or fighting cock antibodies.

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

13. The antibody according to claim 9, characterized in that The light chain constant region and the heavy chain constant region are both derived from mouse antibodies.

14. The antibody according to claim 9, characterized in that 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 thereto; or The light chain constant region includes or is the light chain constant region shown in SEQ ID NO: 4 or an amino acid sequence with at least 80% identity thereto.

15. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the antibody according to any one of claims 1 to 14.

16. A carrier, characterized in that Comprising the nucleic acid molecule of claim 15.

17. A cell or host, characterized in that include: The nucleic acid molecule according to claim 15 or the vector according to claim 16; or Expressing the antibody according to any one of claims 1 to 14.

18. A method for preparing the antibody according to any one of claims 1 to 14, characterized in that: The method comprises culturing the cell or host of claim 17.

19. A conjugate, characterized in that include: The antibody according to any one of claims 1 to 14 and the conjugated portion thereof; The coupling moiety is selected from at least one of a purification tag or marker, a magnetic microsphere, a plastic microsphere, a plastic microparticle, a microplate, glass, a capillary, nylon, and a nitrocellulose membrane; The purification tag or 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, luciferin, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, carbohydrate oxidase, glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase.

20. A reagent or kit, characterized in that include: The antibody according to any one of claims 1 to 14 or the conjugate according to claim 19.

21. Use of the antibody according to any one of claims 1 to 14, the conjugate according to claim 19, or the reagent or kit according to claim 20 in the preparation of a product for detecting D-dimer or in detecting D-dimer for non-disease diagnosis purposes.

22. Use of the antibody according to any one of claims 1 to 14, the conjugate according to claim 19, or the reagent or kit according to claim 20 in the preparation of a product for detecting D-dimer, characterized in that: include: The antibody according to any one of claims 1 to 14, the conjugate according to claim 19, or the reagent or kit according to claim 20 is brought into contact with a sample to be detected to form an immune complex.

23. The use according to claim 22, characterized in that Based on the signal of the immune complex, it is determined whether the sample to be tested contains D-dimer or the content of the D-dimer.

24. The use according to claim 22, characterized in that The immune complex further includes a second antibody that binds to the antibody.

25. The use according to claim 22, characterized in that The immune complex further includes a second antibody that binds to the D-dimer.

26. The use according to claim 23, characterized in that The signal comprises a fluorescent signal.

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