Recombinant anti-D-dimer antibodies, methods and uses thereof

Abstract

This application relates to an anti-D-dimer recombinant antibody that specifically binds with high binding affinity to fibrin and fibrinogen degradation products (FDPs), such as D-dimer, fragment DD, and fragment D, but does not bind to fragment E and fibrinogen. The present invention also relates to methods and assays for detecting D-dimer and FDP fragments in a sample using the recombinant antibody.

Description

[Technical Field]

[0001] This application relates to an anti-D-dimer recombinant antibody that specifically binds with high binding affinity to fibrin and fibrinogen degradation products (FDPs), such as D-dimer, fragment DD, and fragment D, but does not bind to fragment E and fibrinogen. The present invention also relates to methods and assays for detecting D-dimer and FDP fragments in a sample using the recombinant antibody. [Background technology]

[0002] Fibrinogen is a circulating soluble protein present in plasma that forms fibrin polymers when processed by the enzyme thrombin (factor IIa) during the coagulation cascade. Fibrinogen consists of three chains: alpha, beta, and gamma, which form a larger dumbbell-shaped structure with two terminal D domains and a central E domain. Fibrin polymers are generated from staggered assembly of fibrinogen monomers. Fibrin polymers can be further cross-linked by factor XIII (FXIII), which forms isopeptide bonds that covalently link the fibrin chains.

[0003] During fibrinolysis, cross-linked fibrin polymers, a product of coagulation, are degraded by the serine protease plasmin to produce a heterogeneous mixture of degradation products, the smallest of which is the D-dimer (Figure 1). Other fibrin(ogen) degradation products (FDPs) are fragments X, Y, D, and E. The D-dimer consists of two cross-linked D domains and an E domain. The D and E domains can be separated by urea extraction, resulting in fragments DD and E. Fragment DD is unique in that its dimerization is stabilized by an isopeptide bond between the two D domains.

[0004] Detection of fibrin (and fibrinogen) degradation products (FDPs) is utilized in the diagnosis of venous thromboembolism (VTE), such as deep vein thrombosis (DVT) and pulmonary embolism (PE). When present in large amounts, FDPs can interfere with the hemostatic process by binding to the platelet surface, interfering with platelet function, and forming soluble complexes with fibrin monomers, preventing polymerization and clot stabilization.

[0005] D-dimer and fragment DD are of interest for diagnostics due to the presence of a cross-linked D domain, which is indicative of a coagulation event such as DVT or PE. There are many current D-dimer assays available, which all differ due to the use of different monoclonal antibodies that recognize different epitopes, different assay formats, assay calibration standards and ranges, and different equipment. 1 ELISA and agglutination-based assays are the most common, and the tests can be either qualitative or quantitative. Agglutination tests come in many configurations; in automated latex assays, beads conjugated with anti-D-dimer antibodies agglutinate in the presence of patient plasma, and turbidimetric detection is used to characterize the agglutination. In some devices, bispecific antibodies that bind both D-dimer and red blood cells are used to cause hemagglutination and provide a qualitative result. 2 .

[0006] The D-dimer assay is one of the most commonly required coagulation tests and is most commonly used to rule out venous thromboembolism (VTE), which occurs when a blood clot forms in the deep veins of the legs or groin (DVT) and can travel to the lungs (PE). 3 The International Society of Thrombosis and Haemostasis supports the role of D-dimer testing for disseminated intravascular coagulation (DIC). However, circulating D-dimer may be present in coronary artery disease, cancer, trauma, pregnancy, infection, inflammatory disease, elderly, and many other conditions and disease states. 4D-dimer testing, when used in a diagnostic cascade for DVT or PE, can preclude further testing for DVT or PE. This emphasizes the importance of having a highly sensitive test so that patients with VTE are not inappropriately excluded. In general, the sensitivity of ELISA is higher than that of latex agglutination assays, but the value of automation and reproducibility makes latex assays more accessible to many clinical laboratories. The specificity of D-dimer testing for a particular diagnosis depends on the pre-test probability. 5 In addition, the D-dimer test detects heterophile antibodies. 6 May be sensitive to interference from human anti-mouse antibodies or to interfering human anti-mouse antibodies (if mouse anti-D-dimer monoclonal antibodies are used) 7 Like most clinical tests, it cannot exist in isolation but must be considered part of a larger body of evidence in the diagnostic process.

[0007] Many anti-D-dimer monoclonal antibodies have been generated and are produced as hybridomas, either in the supernatant or injected into the abdominal cavity of mice to produce ascites. While hybridomas are often a robust and efficient method for producing monoclonal antibodies (mAbs), the technology has limitations. First, hybridomas can become exhausted and stop producing antibodies, even if a cell bank is created, potentially resulting in the loss of the hybridoma as a production source. Second, hybridomas can be lost due to freezing failures or other accidents. Finally, hybridomas produce antibodies in their native form, requiring sequencing and recombinant expression to further engineer the mAb.

[0008] Therefore, there is a need for anti-D-dimer antibodies that can be easily and reproducibly generated and that provide high specificity for D-dimers for use in thrombus detection assays.

[0009] The present invention provides recombinant antibodies and antigen-binding fragments (Fab or F(ab')2) capable of specifically detecting FDP, which have several advantages over state-of-the-art monoclonal antibodies (produced in hybridomas).

[0010] First, hybridoma cell lines that are generally considered unstable 8,9 Second, greater lot-to-lot consistency can be achieved by using stable expression strains with controlled biochemical and physical process parameters. 10 Third, animal-free technology allows for production without the use of any animals, eliminating welfare and ethical issues. Fourth, the recombinant antibodies of the present invention can be engineered to contain other functional domains, particularly for purification or solubility. Fifth, if necessary, the isotype (IgG1, IgG2a, IgG3) can be selected to produce higher yields or stable proteins. Finally, recombinant proteins can be engineered to avoid interference by human anti-mouse antibodies (HAMA), which have been shown in case studies to cause ambiguous readings and adversely affect patient diagnosis. 7 This provides an additional benefit in reducing the likelihood of

[0011] Thus, the present invention relates to recombinant antibodies that enable assays for the detection of D-dimer, fragment DD, and fragment D, which have utility in the diagnosis of DVT, PE, and other acute disease states in the clinic. [Prior art documents] [Non-patent literature]

[0012] [Non-Patent Document 1] Kabat et al., 1992, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, NIH, Washington DC [Non-patent document 2] Chothia et al., Nature 342:877-883, 1989 [Non-patent document 3] MacCallum et al., J. Mol. Biol., 262:732-745, 1996 [Non-patent document 4] Lefranc, M.-P. Nucl. Acids Res., 33, pp. D593-D597, 2005 [Non-Patent Document 5] Makabe et al., Journal of Biological Chemistry, 283:1156-1166, 2008 [Non-patent document 6] Retter et al., Nucl. Acids Res., 33 (Database issue): pp. D671-D674 (2005) [Non-Patent Document 7] Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268 [Non-patent document 8] Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877 [Non-Patent Document 9] Altschul et al., 1990, J. Mol. Biol. 215:403-410 [Non-Patent Document 10] Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402 Summary of the Invention [Means for solving the problem]

[0013] A first aspect of the present invention relates to an anti-D-dimer recombinant antibody that specifically binds to fibrin and fibrinogen degradation products (FDPs), namely D-dimer, fragment DD, and fragment D, but does not bind to fragment E and fibrinogen.

[0014] In one embodiment, the recombinant antibody comprises a light chain comprising complementarity determining regions L-CDR1, L-CDR2, and L-CDR3, each of which comprises a sequence of at least five consecutive amino acids selected from the amino acid sequence of SEQ ID NO: 18. In one preferred embodiment, the at least five consecutive amino acids selected from the amino acid sequence of SEQ ID NO: 18 comprise at least one of amino acids 24 to 34, 50 to 56, or 89 to 97 of SEQ ID NO: 18. In one preferred embodiment, the L-CDR1, L-CDR2, and L-CDR3 of the light chain comprise the amino acid sequences of SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 33, respectively.

[0015] In one embodiment, the light chain comprises the amino acid sequence of SEQ ID NO:18.

[0016] In another embodiment, the recombinant antibody comprises a heavy chain comprising complementarity determining regions H-CDR1, H-CDR2, and H-CDR3, each of which comprises a sequence of at least five consecutive amino acids selected from the amino acid sequences of SEQ ID NO: 19, or SEQ ID NO: 20, or SEQ ID NO: 21, or SEQ ID NO: 22. In a preferred embodiment, the at least five consecutive amino acids selected from the amino acid sequences of SEQ ID NO: 19, or SEQ ID NO: 20, or SEQ ID NO: 21, or SEQ ID NO: 22 comprise at least one of amino acids 31 to 35, or 50 to 65, or 95 to 102 of SEQ ID NO: 19, or SEQ ID NO: 20, or SEQ ID NO: 21, or SEQ ID NO: 22. In one preferred embodiment, the H-CDR1, H-CDR2, and H-CDR3 of the heavy chain comprise the amino acid sequences of SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36, respectively.

[0017] In some embodiments, the recombinant antibody of the present invention may have heavy chain H-CDR1, H-CDR2, and H-CDR3 comprising the amino acid sequences of SEQ ID NO:34, SEQ ID NO:37, and SEQ ID NO:36, respectively.

[0018] In some embodiments, a recombinant antibody of the present invention may have heavy chain H-CDR1, H-CDR2, and H-CDR3 comprising the amino acid sequences of SEQ ID NO:34, SEQ ID NO:38, and SEQ ID NO:36, respectively.

[0019] In one embodiment, the heavy chain comprises the amino acid sequence of SEQ ID NO:19, or SEQ ID NO:20, or SEQ ID NO:21, or SEQ ID NO:22.

[0020] In one embodiment, the recombinant antibody of the present invention may comprise: a light chain variable region comprising the amino acid sequence of SEQ ID NO: 31 (L-CDR1); the amino acid sequence of SEQ ID NO: 32 (L-CDR2); and the amino acid sequence of SEQ ID NO: 33 (L-CDR3); and A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 34 (H-CDR1); the amino acid sequence of SEQ ID NO: 35 (H-CDR2); and the amino acid sequence of SEQ ID NO: 36 (H-CDR3).

[0021] In another embodiment, the recombinant antibody of the present invention may comprise: a light chain variable region comprising an amino acid sequence that is at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% identical to the sequence set forth in SEQ ID NO: 18; and A heavy chain variable region comprising an amino acid sequence that is at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 19, 20, 21, and 22.

[0022] In still further embodiments, the recombinant antibody of the present invention may comprise: a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 18; and A heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 20, 21, and 22.

[0023] In some embodiments, the recombinant antibody of the present invention may comprise: a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 18; and A heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 19 and 20.

[0024] In another embodiment, the recombinant antibody of the present invention is a monoclonal antibody or antibody fragment. In some embodiments, the antibody fragment is selected from a variable fragment (Fv), a single-chain Fv (scFv), a bispecific antibody (sc(Fv)2), a single-chain antibody, a single-domain antibody, a Fab fragment, a F(ab')2 fragment, a Fab' fragment, a disulfide-linked Fv (dsFv), a chemically conjugated Fv (ccFv), a diabody, an anti-idiotypic (anti-Id) antibody, an affibody, a nanobody, and a unibody. In another embodiment, the antibody fragment is an antigen-binding fragment selected from a Fab fragment and a F(ab')2 fragment.

[0025] In one embodiment, the recombinant antibody of the present invention comprises a constant region of mouse IgG1 class or mouse IgG2a class.

[0026] In another embodiment, the recombinant antibody of the present invention further comprises an affinity tag, which may be selected from the group consisting of SEQ ID NO:23, SEQ ID NO:24, or SEQ ID NO:25.

[0027] In a further embodiment, the light chain of a recombinant antibody of the invention comprises the amino acid sequence of SEQ ID NO: 17. In another embodiment, the heavy chain of a recombinant antibody of the invention comprises the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16.

[0028] In another embodiment, the light chain of a recombinant antibody of the present invention comprises the amino acid sequence of SEQ ID NO: 17, and the heavy chain of said recombinant antibody comprises the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16.

[0029] In another embodiment described herein, the recombinant antibody comprises the amino acid sequence of SEQ ID NO:17 and the amino acid sequence of SEQ ID NO:12.

[0030] In one embodiment, the recombinant antibody of the invention is bound to a solid support.

[0031] In another embodiment, the binding affinity of a recombinant antibody of the present invention to fragment DD is at least (about) three times stronger than the binding affinity of the recombinant antibody to fragment D. For example, the binding affinity of a recombinant antibody of the present invention to fragment DD is at least (about) ten times stronger than the binding affinity of the recombinant antibody to fragment D. In another embodiment, the binding affinity of a recombinant antibody of the present invention to fragment DD is at least (about) twenty times stronger than the binding affinity of the recombinant antibody to fragment D. In another embodiment, the binding affinity of a recombinant antibody of the present invention to fragment DD is at least (about) thirty times stronger than the binding affinity of the recombinant antibody to fragment D. In another embodiment, the binding affinity of a recombinant antibody of the present invention to fragment DD is at least (about) forty times stronger than the binding affinity of the recombinant antibody to fragment D. In another embodiment, the binding affinity of a recombinant antibody of the present invention to fragment DD is at least (about) fifty times stronger than the binding affinity of the recombinant antibody to fragment D. In another embodiment, the binding affinity of a recombinant antibody of the present invention to fragment DD is at least (about) sixty times stronger than the binding affinity of the recombinant antibody to fragment D. In another embodiment, the binding affinity of the recombinant antibody of the present invention to fragment DD is at least (about) 70 times stronger than the binding affinity of said recombinant antibody to fragment D. In another embodiment, the binding affinity of the recombinant antibody of the present invention to fragment DD is at least (about) 80 times stronger than the binding affinity of said recombinant antibody to fragment D.

[0032] In another embodiment, the binding affinity of a recombinant antibody of the present invention to fragment DD may be at least about 20 to about 100 times greater than that of fragment D of the recombinant antibody. For example, the binding affinity of a recombinant antibody of the present invention to fragment DD may be at least about 40 to about 100 times greater than that of fragment D of the recombinant antibody. For example, the binding affinity of a recombinant antibody of the present invention to fragment DD may be at least about 60 to about 100 times greater than that of fragment D of the recombinant antibody. For example, the binding affinity of a recombinant antibody of the present invention to fragment DD may be at least about 70 to about 100 times greater than that of fragment D of the recombinant antibody. For example, the binding affinity of a recombinant antibody of the present invention to fragment DD may be at least about 70 to about 90 times greater than that of fragment D of the recombinant antibody. For example, the binding affinity of a recombinant antibody of the present invention to fragment DD may be at least about 80 to about 90 times greater than that of fragment D of the recombinant antibody.

[0033] In another embodiment, the binding affinity of the recombinant antibody of the present invention to fragment DD is at least 10 times stronger than the binding affinity of the recombinant antibody to fragment D. For example, the binding affinity of the recombinant antibody of the present invention to fragment DD is about 100 times stronger than the binding affinity of the recombinant antibody to fragment D. In one embodiment, the binding affinity of the recombinant antibody of the present invention to fragment DD is at least 10 times, but less than 1000 times, the binding affinity of the recombinant antibody to fragment D.

[0034] As used herein, the term "binding affinity" refers to the strength of the interaction between an epitope of an antigen and the antigen-binding site of an antibody as measured by biolayer interferometry on a Sartorius (formerly ForteBio) Octet Red 96e system at about 23°C and 1 atmosphere.

[0035] Advantageously, compared to commercially available anti-D-dimer antibodies, the recombinant antibodies of the present invention exhibit improved binding to fibrin degradation products. Furthermore, the recombinant antibodies of the present invention do not exhibit binding affinity to fibrinogen or fragment E.

[0036] In a further aspect, the present invention refers to a cell comprising a recombinant antibody of the present invention. The present invention also refers to a nucleic acid comprising a nucleotide sequence encoding the recombinant antibody of the present invention, a promoter operably linked to said nucleotide sequence, and a selectable marker. The present invention also relates to a cell comprising said nucleic acid.

[0037] The present invention also refers to a composition comprising a recombinant antibody of the present invention and a solid support, wherein the recombinant antibody is covalently or non-covalently bound to the solid support. In a preferred embodiment, the solid support comprises a particle, bead, membrane, surface, polypeptide chip, microtiter plate, or the solid phase of a chromatography column. Preferably, the solid support is a latex particle.

[0038] In another aspect, the present invention refers to a kit for detecting the presence of D-dimer, fragment DD, and / or fragment D in a sample, said kit comprising at least one recombinant antibody according to the present invention and a solid support, wherein said at least one recombinant antibody is covalently or non-covalently bound to the solid support.

[0039] In a further aspect, the present invention refers to a method for detecting the presence of D-dimer, fragment DD, and / or fragment D in a sample, comprising the steps of: - contacting said sample with at least one recombinant antibody of the invention for a time and under conditions sufficient for the formation of an antibody / antigen complex; and - detecting said antibody / antigen complex.

[0040] In another aspect, the present invention refers to a method for measuring the binding affinity of D-dimer, fragment DD, and / or fragment D in a sample, comprising the steps of: - contacting said sample with at least one recombinant antibody of the invention for a time and under conditions sufficient for the formation of an antibody / antigen complex; and - determining the binding affinity between said antibody and D-dimer, fragment DD, and / or fragment D in the sample.

[0041] In a further aspect, the present invention refers to a method for determining the concentration of D-dimer, fragment DD, and / or fragment D in a sample, comprising the steps of: - contacting said sample with at least one recombinant antibody according to the invention for a time and under conditions sufficient for the formation of an antibody / antigen complex; and - measuring the concentration of D-dimer, fragment DD, and / or fragment D in the sample. [Brief explanation of the drawings]

[0042] [Figure 1] Schematic diagram of the assembly of fibrin monomers to form fibrinogen. Thrombin acts on fibrin or fibrinogen to liberate fibrinopeptides in the E domain from the α and β chains, resulting in the polymerization of fibrin into fibrinogen, which is thought to be a staggered linkage of fibrin. FXIIIa stabilizes the fibril by cross-linking the α-α and γ-γ chains through the formation of isopeptide bonds. The serine protease plasmin digests fibrin to produce fragments X (280 kDa), Y (150 kDa), D (94 kDa), and E (50 kDa). Due to cross-linking by FXIIIa, digestion of cross-linked fibrin produces various products of various sizes, the smallest of which is the D-dimer (240 kDa). The D and E domains of the D-dimer can be separated by urea extraction, resulting in the fragments DD (190 kDa) and E (50 kDa). [Figure 2] FIG. 1 shows SDS-PAGE results for recombinant antibodies of the invention under reducing (R) and non-reducing (N or NR) conditions, with the exception of antibodies #4 and #6, for which CE-SDS results are shown instead. [Figure 3]Representative light scattering data for antibodies #9 and #12 are shown. The A280 trace peaks are intersected by the measured molar mass of each peak, and flat molar mass data across each peak indicates a monodisperse sample. #9: Expected mass = 48.8 kDa, Measured mass = 48.3 ± 1% kDa, Mw / Mn = 1.001. #12: Expected mass = 103 kDa, Measured mass = 105 ± 4% kDa, Mw / Mn = 1.000. All recombinant anti-D-dimer antibodies showed monodisperse peaks (data not shown). [Figure 4] FIG. 1 shows purified FDP used in binding studies. [Figure 5] Figure 1 shows a comparison of the binding affinity of recombinant antibody #12 to fragments DD, D, E, and fibrinogen. Both fibrin-derived fragment DD (A) and fibrinogen-derived fragment D (B) show binding to antibody #12 using the Octet BLI platform. Neither fragment E (C) nor fibrinogen (D) show binding to antibody #12. [Figure 6] Graphs showing that the presence of D-dimer in plasma samples correlates well between the HemosIL DDHS500 on the ACL TOP Coagulation Analyzer and various antibodies on the Q SMART system. A) Linear correlation using the known 8D3 hybridoma-derived antibody, B) Linear correlation with an IgG1 antibody (#6), C) Linear correlation with an IgG2a (#5), and D) Linear correlation with the pepsin-digested F(ab')2 of IgG2a #5. [Figure 7] A) Linear correlation between #12 and D-dimer using the Grifols Q SMART system and the ACL TOP coagulation analyzer using the HemosIL DDHS550 for plasma samples containing various D-dimers, and B) Linear correlation when D-dimers present in plasma samples are analyzed using #12 or hybridoma 8D3 mAb on the Q SMART system. DETAILED DESCRIPTION OF THE INVENTION

[0043] definition The following description is intended merely to illustrate various embodiments of the present disclosure. As such, the specific modifications discussed are not intended to be limiting. It will be apparent to those skilled in the art that various equivalents, changes, and modifications can be made without departing from the spirit or scope of the subject matter presented herein, and it is understood that such equivalent embodiments are to be included herein.

[0044] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0045] Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer, or group of elements or integers, but not the exclusion of any other element or integer, or group of elements or integers.

[0046] 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 invention belongs. Exemplary methods and materials are described below; however, methods and materials similar or equivalent to those described herein can also be used and will be apparent to those skilled in the art. All publications and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. The materials, methods, and examples are illustrative only and are not intended to be limiting.

[0047] Each embodiment in this specification may be applied mutatis mutandis to every other embodiment unless expressly stated otherwise.

[0048] The following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0049] As used herein, the term "nucleic acid" refers to any material composed of DNA or RNA. Nucleic acids can be produced synthetically or by living cells.

[0050] As used herein, the term "polynucleotide" refers to a polymeric chain of nucleotides. The term includes DNA molecules (e.g., cDNA or genomic or synthetic DNA) and RNA molecules (e.g., mRNA or synthetic RNA), as well as analogs of DNA or RNA containing non-natural nucleotide analogs, non-natural internucleoside linkages, or both. Nucleic acids can have any topological structure. By way of example, nucleic acids can be single-stranded, double-stranded, triple-stranded, quadruplexed, partially double-stranded, branched, hairpinned, circular, or padlock-shaped.

[0051] As used herein, the term "protein" refers to a large biological molecule or macromolecule composed of one or more chains of amino acid residues. Many proteins are enzymes that catalyze biochemical reactions and are crucial to metabolism. Proteins also have structural or mechanical functions, such as actin and myosin in muscles and proteins in the cytoskeleton, which form the scaffolding system that maintains the shape of cells. Other proteins are important in cell signaling, immune response, cell adhesion, and the cell cycle. However, proteins may also be entirely artificial or recombinant, i.e., not naturally occurring in living systems.

[0052] As used herein, the term "polypeptide" refers to both naturally occurring and non-naturally occurring proteins, as well as fragments, variants, derivatives, and analogs thereof. A polypeptide can be a monomer or a polymer. A polypeptide can contain several different domains (peptides), each of which has one or more distinct activities.

[0053] As used herein, the term "recombinant" refers to a biomolecule, e.g., a gene or protein, that (1) has been removed from its naturally occurring environment, (2) whose gene is not associated with all or a portion of a polynucleotide found in nature, (3) is operably linked to a polynucleotide with which it is not naturally linked, or (4) is not found in nature. The term "recombinant" can be used in reference to cloned DNA isolates, chemically synthesized polynucleotide analogs, or polynucleotide analogs synthesized in vivo by heterologous systems, as well as proteins and / or mRNAs encoded by such nucleic acids.

[0054] As used herein, the term "antigen" refers to a biological molecule that specifically binds to a respective antibody. Antibodies from a diverse repertoire bind specific antigenic structures through their variable region interactions.

[0055] As used herein, the term "fusion protein" refers to a protein containing two or more amino acid sequences that do not coexist in a naturally occurring protein. A fusion protein may contain two or more amino acid sequences from the same or different organisms. The two or more amino acid sequences of a fusion protein are typically in-frame, do not contain a stop codon between them, and are typically translated from mRNA as part of the fusion protein.

[0056] As used herein, the term "antibody" includes polyclonal antibodies, monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies (fully or partially humanized), animal antibodies, recombinant antibodies, chimeric antibodies, and antibody fragments. Accordingly, the term "antibody fragment" as used herein includes, but is not limited to, variable fragments (Fv), single-chain Fvs (scFv), bispecific antibodies (sc(Fv)2), single-chain antibodies, single-domain antibodies, Fab fragments, F(ab')2 fragments, Fab' fragments, disulfide-linked Fvs (dsFv), chemically conjugated Fvs (ccFv), diabodies, and anti-idiotypic (anti-Id) antibodies, as well as functionally active, epitope-binding fragments of any of the above. In certain embodiments, antibodies also include affibodies, nanobodies, and unibodies. In certain embodiments, specific antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules containing an antigen-binding site. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgAi, and IgA2) or subclass.

[0057] As used herein, the term "antigen-binding fragment (Fab)" refers to an antibody fragment containing one constant domain and one variable domain of each of the heavy and light chains. The variable domains contain the antigen-binding site. Generally, an antibody contains a fragment crystallizable region (Fc) and two antigen-binding fragments (Fab). The Fab fragment can be separated from the Fc region to generate two Fab fragments, which are also known as F(ab')2 fragments or dimeric fragment antigen binding.

[0058] In natural antibodies, two heavy chains are linked to each other by disulfide bonds, and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chains: lambda (λ) and kappa (κ). There are five major heavy chain classes (or isotypes) that determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE. Each chain contains distinct sequence domains. Light chains contain two domains: a variable domain (VL) and a constant domain (CL). Heavy chains contain four domains: a variable domain (VH) and three constant domains (CH1, CH2, and CH3, collectively referred to as CH). The variable regions of both the light chain (VL) and the heavy chain (VH) determine binding recognition and specificity to antigens. The constant region domains of the light chain (CL) and heavy chain (CH) confer important biological properties, such as antibody chain assembly, secretion, transplacental mobility, complement fixation, and Fc receptor (FcR) binding. An Fv fragment is the N-terminal portion of an immunoglobulin Fab fragment and consists of one light chain variable portion and one heavy chain variable portion. Antibody specificity resides in the structural complementarity between the antibody-binding site and an antigenic determinant. An antibody-binding site is composed of residues primarily from hypervariable or complementarity-determining regions (CDRs). Occasionally, residues from non-hypervariable or framework regions (FRs) contribute to the overall domain structure and, therefore, the binding site. Complementarity-determining regions or CDRs refer to amino acid sequences that collectively define the binding affinity and specificity of the native Fv region of a native immunoglobulin-binding site. Each immunoglobulin light and heavy chain has three CDRs, designated L-CDR1, L-CDR2, and L-CDR3, and H-CDR1, H-CDR2, and H-CDR3, respectively. Thus, an antigen-binding site typically contains six CDRs, including a set of CDRs from each of the heavy and light chain V regions. Framework region (FR) refers to the amino acid sequences interposed between the CDRs.

[0059] CDRs can be identified according to the definitions of Kabat, Chothia, both Kabat and Chothia, AbM, contact, IMGT unique numbering, and / or conformational definitions, or any method of CDR determination known in the art. Antibody CDRs can be identified as hypervariable regions as originally defined by Kabat et al. See, e.g., Kabat et al., 1992, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, NIH, Washington, DC. CDR locations can also be identified as loop structures of the structure originally described by Chothia et al. (See, e.g., Chothia et al., Nature 342:877-883, 1989). Other approaches to CDR identification include the "AbM definition" (a compromise of Kabat and Chothia, derived using Oxford Molecular's AbM antibody modeling software (now Accelrys0)), the "contact definition" of CDRs based on observed antigen contacts (as set forth in MacCallum et al., J. Mol. Biol., 262:732-745, 1996), or "IMGT unique numbering," which relies on the high structural conservation of variable regions (see Lefranc, M.-P. Nucl. Acids Res., 33, D593-D597, 2005). In another approach, referred to herein as the "conformational definition" of CDRs, CDR positions can be identified as residues that generate enthalpic contributions to antigen binding. See, e.g., Makabe et al., Journal of Biological Chemistry, 283:1156-1166, 2008). Still other CDR boundary definitions do not strictly follow one of the above approaches, but still overlap with at least a portion of the Kabat CDRs, although they may be shortened or extended in light of predictions or experimental findings that particular residues or groups of residues, or even entire CDRs, do not significantly affect antigen binding.As used herein, CDR refers to a CDR defined by any approach known in the art, including a combination of approaches. The methods used herein may utilize CDRs defined according to any of these approaches. For any given embodiment containing more than one CDR, each CDR may be defined according to any one of Kabat, Chothia, extended, AbM, contact, IMGT unique numbering, or conformational definition.

[0060] Exemplary databases of antibody sequences are described and can be accessed through the "Abysis" website at www.bioinf.org.uk / abs (maintained by A.C. Martin, Department of Biochemistry & Molecular Biology, University College London, London, England) and the VBASE2 website at www.vbase2.org as described in Retter et al., Nucl. Acids Res., 33 (Database issue): D671-D674 (2005). Preferably, sequences are analyzed using the Abysis database, which integrates sequence data from Kabat, IMGT, and the Protein Data Bank (PDB) with structural data from the PDB. Unless otherwise indicated, all CDRs presented herein have been derived according to the scheme shown in accordance with the Abysis database website.

[0061] As used herein, the term "monoclonal antibody" refers to an antibody composition having a homogeneous antibody population that binds to the same epitope. The term is not limited with respect to the species or source of the antibody, nor is it intended to be limited by the method by which it is made. Thus, the term encompasses antibodies obtained from murine hybridomas, as well as human monoclonal antibodies obtained using human rather than murine hybridomas.

[0062] As used herein, the term "epitope" refers to the portion of an antigen to which an antibody specifically binds. Thus, the term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor.

[0063] As used herein, the term "recombinant antibody" refers to an antibody or fragment thereof that is not naturally occurring and may be associated with a polypeptide or fragment thereof not found in nature. Recombinant antibodies can be made by any of the recombinant techniques well known to those of skill in the art.

[0064] As used herein, the term "identical" or "percent identity" in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same when compared and aligned for maximum correspondence. To determine percent identity, the sequences are aligned for optimal comparison (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions (e.g., overlapping positions) × 100). In some embodiments, the two sequences being compared are the same length (e.g., excluding additional sequences extending beyond the sequences being compared), after introducing gaps into the sequences, if necessary. For sequence comparison between two sequences, a "corresponding" CDR refers to a CDR in the same location in both sequences (e.g., CDR-H1 in each sequence).

[0065] The determination of percent identity or percent similarity between two sequences can be accomplished using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm utilized for comparing two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm has been incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403-410. BLAST nucleotide searches can be performed using the NBLAST program, score=100, wordlength=12, to obtain nucleotide sequences homologous to a nucleic acid encoding a protein of interest. BLAST protein searches can be performed using the XBLAST program, score = 50, word length = 3, to obtain amino acid sequences homologous to the protein of interest. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402. When utilizing BLAST and Gapped BLAST, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. Another preferred, non-limiting example of a mathematical algorithm utilized for sequence comparison is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm has been incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

[0066] A polypeptide is "immunologically reactive" with an antibody if it binds to the antibody due to antibody recognition of a particular epitope contained within that polypeptide. Immunological reactivity can be determined by antibody binding, more specifically, by the kinetics of antibody binding, and / or by competition in binding using as a competitor a known polypeptide containing the epitope to which the antibody is directed. Techniques for determining whether a polypeptide is immunologically reactive with an antibody are known in the art.

[0067] As used herein, the term "sample" refers to any biological material obtained from a subject or patient. In one embodiment, a sample may include blood, ascites, CSF, saliva, or urine. In other embodiments, a sample may include whole blood, plasma, serum, B cells enriched from a blood sample, and cultured cells (e.g., B cells from a subject). Samples may also include biopsies or tissue samples, including neural tissue. In yet other embodiments, a sample may include whole cells and / or cell lysates.

[0068] As used herein, the terms "diagnostic" or "diagnosed" refer to identifying the presence or nature of a pathological condition or a patient predisposed to a disease. Diagnostic methods vary in their sensitivity and specificity. The "sensitivity" of a diagnostic assay is the percentage of diseased individuals who test positive (the "percent true positives"). Diseased individuals not detected by the assay are "false negatives." Subjects who are not diseased and test negative in the assay are called "true negatives." The "specificity" of a diagnostic assay is 1 minus the false positive rate, where the "false positive" rate is defined as the proportion of those without the disease who test positive. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it is sufficient if the method provides a positive indication that aids in diagnosis.

[0069] The terms "patient" or "individual" are used interchangeably herein and refer to a mammalian subject to be treated, with human subjects being preferred. Optionally, the methods of the present invention are used in laboratory animals, in veterinary applications, and in the development of animal models of disease, including, but not limited to, rodents, including mice, rats, and hamsters; and primates.

[0070] Detailed Description 1. Recombinant antibodies The present invention relates to an anti-D-dimer recombinant antibody that specifically binds to fibrin and fibrinogen degradation products (FDPs), D-dimer, fragment DD, and fragment D, but does not bind to fragment E and fibrinogen.

[0071] In one embodiment described herein, the recombinant antibody comprises a light chain and a heavy chain. In other embodiments described herein, the recombinant antibody comprises two light chains and two heavy chains. The light chain of the recombinant antibody of the present invention may comprise two domains: a variable domain (VL) and a constant domain (CL). The heavy chain of the recombinant antibody of the present invention may comprise four domains: a variable domain (VH) and three constant domains (CH1, CH2, and CH3, collectively referred to as CH).

[0072] In another embodiment, the recombinant antibody of the present invention is a monoclonal antibody or antibody fragment. In a preferred embodiment, the antibody fragment is selected from a variable fragment (Fv), a single-chain Fv (scFv), a bispecific antibody (sc(Fv)2), a single-chain antibody, a single-domain antibody, a Fab fragment, a F(ab')2 fragment, a Fab' fragment, a disulfide-linked Fv (dsFv), a chemically conjugated Fv (ccFv), a diabody, an anti-idiotypic (anti-Id) antibody, an affibody, a nanobody, and a unibody.

[0073] In one embodiment described herein, the recombinant antibody comprises an Fc region and two Fab fragments. In another embodiment described herein, the recombinant antibody is an antigen-binding fragment and does not comprise an Fc region. In another embodiment described herein, the recombinant antibody consists of one Fab fragment. In another embodiment described herein, the recombinant antibody consists of two Fab fragments (F(ab)2).

[0074] In one embodiment described herein, the recombinant antibody can be of any known type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), or any known class (e.g., IgG1, IgG2, IgG3, IgG4, IgAi, and IgA2), or any known subclass.

[0075] In one embodiment described herein, the recombinant antibody is of the IgG type. In a preferred embodiment, the recombinant antibody is of the IgG1, IgG2, IgG3, or IgG4 class. In another preferred embodiment, the recombinant antibody is of the IgG1 or IgG2 class. In another preferred embodiment, the recombinant antibody is of the IgG2a class.

[0076] In a most preferred embodiment, the recombinant antibody of the present invention comprises a constant region of the mouse IgG1 class or the mouse IgG2a class.

[0077] A. Light Chain In one embodiment described herein, the recombinant antibody comprises a light chain comprising complementarity determining regions (CDRs). The CDRs correspond to sequences identified according to any CDR definition approach known to those of skill in the art. In some preferred embodiments, the CDR regions correspond to sequences identified according to Kabat. In some preferred embodiments, the CDR regions correspond to sequences identified according to Chothia. In other embodiments, the CDRs can be any of Kabat, Chothia, AbM, extended, contact, IMGT unique numbering, and / or conformation-defined, combined CDRs, or a combination thereof.

[0078] In one embodiment described herein, the recombinant antibody comprises a light chain comprising complementarity determining regions L-CDR1, L-CDR2, and L-CDR3, each of which comprises a (distinct) sequence of at least five consecutive amino acids selected from the amino acid sequence of SEQ ID NO: 18. In one preferred embodiment, the at least five consecutive amino acids selected from the amino acid sequence of SEQ ID NO: 18 comprise at least one of amino acids 24 to 34, 50 to 56, or 89 to 97 of SEQ ID NO: 18. In one embodiment, the L-CDR1, L-CDR2, and L-CDR3 of the light chain comprise the amino acid sequences of SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 33, respectively. In this embodiment, SEQ ID NOs: 31 to 33 represent the Kabat-defined L-CDR1, L-CDR2, and L-CDR3 of SEQ ID NO: 18, respectively.

[0079] In another embodiment described herein, the variable region of the light chain of the recombinant antibody of the present invention comprises the amino acid sequence of SEQ ID NO: 18 or a fragment thereof. In yet another embodiment, the variable region of the light chain of the recombinant antibody may have about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology to the amino acid sequence consisting of SEQ ID NO: 18.

[0080] In another embodiment described herein, the recombinant antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 18, or a fragment thereof. In other embodiments, the light chain of the recombinant antibody may have about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology to the amino acid sequence consisting of SEQ ID NO: 17 or SEQ ID NO: 18.

[0081] B. Heavy Chain In one embodiment described herein, the recombinant antibody comprises a heavy chain comprising complementarity determining regions (CDRs). The CDRs correspond to sequences identified according to any CDR definition approach known to those of skill in the art. In some preferred embodiments, the CDR regions correspond to sequences identified according to Kabat. In some preferred embodiments, the CDR regions correspond to sequences identified according to Chothia. In other embodiments, the CDRs can be any of Kabat, Chothia, AbM, extended, contact, IMGT unique numbering, and / or conformation-defined, combined CDRs, or a combination thereof.

[0082] In one embodiment described herein, the recombinant antibody comprises a heavy chain comprising complementarity determining regions H-CDR1, H-CDR2, and H-CDR3, each of which comprises a sequence of at least five consecutive amino acids selected from the amino acid sequence of SEQ ID NO: 19, or SEQ ID NO: 20, or SEQ ID NO: 21, or SEQ ID NO: 22. In a preferred embodiment, the at least five consecutive amino acids selected from the amino acid sequence of SEQ ID NO: 19, or SEQ ID NO: 20, or SEQ ID NO: 21, or SEQ ID NO: 22 comprise at least one of amino acids 31 to 35, or 50 to 65, or 95 to 102 of SEQ ID NO: 19, or SEQ ID NO: 20, or SEQ ID NO: 21, or SEQ ID NO: 22. In one preferred embodiment, the H-CDR1, H-CDR2, and H-CDR3 of the heavy chain may comprise the amino acid sequences of SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36, respectively. In this embodiment, SEQ ID NOs: 34 and 36 represent the H-CDR1 and H-CDR3, respectively, of SEQ ID NOs: 19-22 as determined by Kabat, and SEQ ID NO: 35 represents the H-CDR2 of SEQ ID NOs: 19-22 as determined by Chothia.

[0083] In a further embodiment, the heavy chain may comprise complementarity determining regions H-CDR1, H-CDR2, and H-CDR3 as defined, respectively, by SEQ ID NOs: 34, 37, and 36. In this embodiment, SEQ ID NOs: 34, 37, and 36 represent H-CDR1, H-CDR2, and H-CDR3, respectively, as defined by Kabat in SEQ ID NOs: 19-20.

[0084] In still further embodiments, the heavy chain may comprise complementarity determining regions H-CDR1, H-CDR2, and H-CDR3 as defined, respectively, by SEQ ID NOs: 34, 38, and 36. In this embodiment, SEQ ID NOs: 34, 38, and 36 represent H-CDR1, H-CDR2, and H-CDR3, respectively, as defined by Kabat in SEQ ID NOs: 21-22.

[0085] In another embodiment described herein, the variable region of the heavy chain of the recombinant antibody of the invention comprises the amino acid sequence of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO: 22, or a fragment thereof. In other embodiments, the variable region of the heavy chain of the recombinant antibody may have about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology to the amino acid sequence consisting of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO: 22.

[0086] In another embodiment described herein, the recombinant antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, or SEQ ID NO:22, or a fragment thereof. In other embodiments, the heavy chain of the recombinant antibody may have about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology to the amino acid sequence consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, or SEQ ID NO:22.

[0087] C. Affinity Tags The recombinant antibody according to the present invention may comprise an affinity tag. Affinity tags are useful for purification. Exemplary affinity tags include polyhistidine, glutathione S-transferase (GST), chitin-binding protein, maltose-binding protein (MBP), streptavidin-binding peptide (Strep tag), isopeptide bond formation, FLAG tag, V5 tag, Myc tag, HA tag, NE tag, AviTag, calmodulin tag, polyglutamic acid, S tag, SBP tag, Softag 1, Softag 3, TC tag, VSV tag, Xpress tag, Isopeptag, SpyTag, SnoopTag, biotin carboxyl carrier protein, green fluorescent protein tag, HaloTag, Nus tag, and thioredoxin tag, but the choice of affinity tag is not particularly limited. However, the recombinant antibody may lack an affinity tag, for example, if the affinity tag is removed after use or if the recombinant antibody is purified using a strategy that does not require an affinity tag. An exemplary affinity tag is polyhistidine, which typically comprises an amino acid sequence containing between 4 and 10 consecutive histidines. A preferred affinity tag is a polyhistidine tag containing between 6 and 10 consecutive histidines. An exemplary affinity tag corresponds to SEQ ID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 25.

[0088] The recombinant antibodies of the present invention may comprise an affinity tag and may be purified using said affinity tag. Several methods of purification for recombinant antibodies are available in the state of the art and are well known to those skilled in the art. Exemplary methods of purification for recombinant antibodies, with or without an affinity tag, are immobilized metal affinity chromatography (IMAC), Protein A / G affinity, exchange chromatography (IEX or IEC), hydrophobic interaction chromatography (HIC), and / or the additional use of tag and affinity chromatography techniques beyond IMAC or Protein A / G. The purification methods and tags employed should not be considered limiting.

[0089] In a preferred embodiment, the recombinant antibody of the present invention further comprises an affinity tag, which may be selected from the group consisting of SEQ ID NO:23, SEQ ID NO:24, or SEQ ID NO:25.

[0090] D. Exemplary Recombinant Antibodies In one embodiment described herein, the recombinant antibody comprises the amino acid sequence of SEQ ID NO:17.

[0091] In one embodiment described herein, the recombinant antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16.

[0092] In a preferred embodiment described herein, the recombinant antibody comprises the amino acid sequence of SEQ ID NO:17 and an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16.

[0093] In the most preferred embodiment described herein, the recombinant antibody comprises the amino acid sequence of SEQ ID NO:17 and the amino acid sequence of SEQ ID NO:12.

[0094] In another preferred embodiment, the light chain of the recombinant antibody may have about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology to the amino acid sequence consisting of SEQ ID NO: 17, and the heavy chain of the recombinant antibody may have about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology to the amino acid sequence consisting of SEQ ID NO: 12.

[0095] In one embodiment, the recombinant antibody of the invention is bound to a solid support.

[0096] In another embodiment, the binding affinity of the recombinant antibody of the present invention to fragment DD is at least three times stronger than the binding affinity of said recombinant antibody to fragment D.

[0097] III. Nucleic Acids, Cloning, and Expression Cells The present invention also relates to a nucleic acid comprising a nucleotide sequence encoding a recombinant antibody described herein. The nucleic acid may be DNA or RNA. DNA comprising a nucleotide sequence encoding a recombinant antibody described herein typically comprises a promoter operably linked to the nucleotide sequence. The promoter is preferably capable of driving constitutive or inducible expression of the nucleotide sequence in a target expression cell. The nucleic acid may also comprise a selectable marker useful for selecting cells containing the target nucleic acid. Useful selectable markers are well known to those skilled in the art. The exact nucleotide sequence of the nucleic acid is not particularly limiting, as long as the nucleotide sequence encodes a recombinant antibody described herein. Codons may be selected, for example, to match the codon bias of the target expression cell (e.g., a mammalian cell such as a human cell) and / or for convenience during cloning. The DNA may comprise an origin of replication (e.g., for replication of plasmids in prokaryotic cells), e.g., a plasmid.

[0098] In one embodiment described herein, the nucleic acid comprises a nucleotide sequence encoding a recombinant antibody of the invention, a promoter operably linked to the nucleotide sequence, and a selectable marker.

[0099] Various aspects of the present invention also relate to cells containing nucleic acids comprising nucleotide sequences encoding recombinant antibodies as described herein. The cells can be expression cells or cloning cells. Nucleic acids are typically cloned in E. coli, although other cloning cells may be used.

[0100] If the cell is an expression cell, the nucleic acid may be a chromosomal nucleic acid, i.e., where the nucleotide sequence is integrated into the chromosome, although the nucleic acid may then be present in the expression cell, for example, as extrachromosomal DNA or as a vector, e.g., a plasmid, cosmid, phage, etc. The type of vector should not be considered limiting.

[0101] In one embodiment described herein, the cell is typically an expression cell. The nature of the expression cell is not particularly limiting. Mammalian expression cells may allow for convenient folding, post-translational modification, and / or secretion of the recombinant antibody or oligomeric recombinant antibody, although other eukaryotic or prokaryotic cells may also be used as expression cells. Exemplary expression cells include TunaCHO, ExpiCHO, Expi293, BHK, NS0, Sp2 / 0, COS, C127, HEK, HT-1080, PER.C6, HeLa, and Jurkat cells. Cells may also be selected for integration of vectors, more preferably for integration of plasmid DNA.

[0102] The recombinant antibodies of the present invention can be produced by appropriate transfection strategies of a nucleic acid containing a nucleotide sequence encoding the recombinant antibody into mammalian cells. Those skilled in the art are aware of the various techniques (lipofection, electroporation, etc.) available for transfecting nucleic acids into the cell line of choice. Therefore, the choice of mammalian cell line and transfection strategy should not be considered limiting. The cell line can further be selected for integration of the plasmid DNA.

[0103] In one preferred embodiment described herein, the cells comprise a recombinant antibody of the invention.

[0104] IV. COMPOSITIONS AND ASSAY-RELATED METHODS Various aspects of the present invention relate to compositions comprising recombinant antibodies as described herein.

[0105] In one embodiment described herein, the composition comprises a recombinant antibody of the invention and a solid support.

[0106] In another embodiment, a composition comprises a recombinant antibody of the invention and a solid support, wherein the recombinant antibody is covalently or non-covalently bound to the solid support. As used herein, the term "non-covalently bound" refers to a specific bond, such as between an antibody and its antigen, a ligand and its receptor, or an enzyme and its substrate, exemplified by the interaction between a streptavidin-binding protein and streptavidin or between an antibody and its antigen.

[0107] In other embodiments, a composition comprises a recombinant antibody of the present invention and a solid support, wherein the recombinant antibody is directly or indirectly bound to the solid support. As used herein, the term "direct" binding refers to direct conjugation of a molecule to the solid support, e.g., a gold-thiol interaction that binds a cysteine ​​thiol of the recombinant antibody to a gold surface. As used herein, the term "indirect" binding includes specific binding of a recombinant antibody to another molecule that is directly bound to the solid support, e.g., a recombinant antibody may bind an antibody that is directly bound to the solid support, thereby indirectly binding the recombinant antibody to the solid support. The term "indirect" binding is independent of the number of molecules between the recombinant antibody and the solid support, as long as (a) each interaction between the daisy chain of molecules is a specific or covalent interaction, and (b) the terminal molecule of the daisy chain is directly bound to the solid support.

[0108] The solid support may comprise a particle, bead, membrane, surface, polypeptide chip, microtiter plate, or the solid phase of a chromatography column. For example, the solid support may be a latex bead.

[0109] The composition may comprise a plurality of beads or particles, each bead or particle of said plurality of beads or particles being directly or indirectly associated with at least one recombinant antibody as described herein.The composition may comprise a plurality of beads or particles, each bead or particle of said plurality of beads or particles being covalently or non-covalently associated with at least one recombinant antibody as described herein.

[0110] Various aspects of the embodiments relate to kits for detecting the presence of D-dimer, fragment DD, and / or fragment D in a sample, the kit comprising a recombinant antibody and a solid support or composition as described herein.

[0111] The compositions and kits as described herein can be used either in assays or in compositions made during the performance of an assay.Various aspects of the present invention relate to diagnostic medical devices that include compositions as described herein.

[0112] Various aspects of the present invention relate to assays. The assay can be an assay for measuring the relative binding affinity of a recombinant antibody of the present invention to D-dimer, fragment DD, and / or fragment D in a sample (e.g., compared to one or more control samples or standards). The assay can be an assay for measuring the relative binding affinity of a recombinant antibody of the present invention to any fibrin(ogen) degradation product (e.g., compared to one or more control samples or standards).

[0113] Assays typically feature a solid support that either or both allows for measurements such as turbidimetry, nephelometry, UV / Vis / IR spectroscopy (e.g., absorbance, transmittance), fluorescence or phosphorescence spectroscopy, or surface plasmon resonance, or aids in the separation of components that directly or indirectly bind to the solid support from components that do not directly or indirectly bind to the solid support. For example, an assay may include a composition comprising particles or beads that allows for measurements by turbidimetry or nephelometry (e.g., in a clotting assay) and / or aids in the mechanical separation of components that directly or indirectly bind to the particles or beads.

[0114] Other exemplary assays that may include the recombinant antibodies or compositions of the present invention include, but are not limited to, ELISA, viscoelastic tests such as Sonoclot, gel technology, fluorescent assays, and other point-of-care tests using any of these technologies.

[0115] Various aspects of the present invention relate to methods of detecting the presence of D-dimer, fragment DD, and / or fragment D in a sample.

[0116] In one embodiment described herein, a method for detecting the presence of D-dimer, fragment DD, and / or fragment D in a sample comprises the steps of: a) contacting the sample with at least one recombinant antibody described herein for a time and under conditions sufficient for the formation of an antibody / antigen complex; and b) detecting said antibody / antigen complex.

[0117] Various aspects of the present invention relate to methods for determining the binding affinity of D-dimer, fragment DD, and / or fragment D in a sample.

[0118] In one embodiment described herein, a method for measuring the binding affinity of D-dimer, Fragment DD, and / or Fragment D in a sample comprises the steps of: a) contacting the sample with at least one recombinant antibody described herein for a time and under conditions sufficient for the formation of an antibody / antigen complex; and b) determining the binding affinity between the antibody and D-dimer, fragment DD, and / or fragment D in the sample;

[0119] Various aspects of the present invention relate to methods for determining the concentration of D-dimer, fragment DD, and / or fragment D in a sample.

[0120] In one embodiment described herein, a method for measuring the amount of D-dimer, fragment DD, and / or fragment D in a sample comprises the steps of: a) contacting the sample with at least one recombinant antibody described herein for a time and under conditions sufficient for the formation of an antibody / antigen complex; and b) measuring the concentration of D-dimer, fragment DD, and / or fragment D in said sample. [Example]

[0121] Example 1 Immunization strategies and monoclonal antibody selection Female Balb / c mice were subcutaneously primed with 50 μg purified D-dimer in 0.2 mL of saline mixed with 0.1 M complete Freund's adjuvant. Four and two days before fusion, the mice were intraperitoneally boosted with the same amount of antigen in saline. P3X63-Ag8-6.5.3 myeloma cells were fused with spleen cells from immunized Balb / c mice. Cell culture supernatants were screened for antibodies specific to purified fragment D and D-dimer from fibrinogen or noncrosslinked fibrin. Clones producing antibodies specific to D-dimer but not fibrinogen were used to produce ascites in pristine-primed Balb / c mice. The IgG fraction of monoclonal antibodies was purified from the ascites by affinity chromatography on protein A Sepharose. 11,12 .

[0122] Example 2 De novo MS / MS sequencing of selected monoclonal antibodies The monoclonal antibodies selected in Example 1 were further subjected to de novo MS / MS sequencing.

[0123] Briefly, purified antibodies were first confirmed by intact mass analysis. Each mAb was then reduced and separated into heavy chains (HC) and light chains (LC), which were then separately digested with a set of enzymes (trypsin, chymotrypsin, etc.). Bottom-up MS / MS data were collected, providing information about the digested peptides as well as b- and y-ions, which further identify individual amino acids within the peptides for assembly. The information was then processed to establish the amino acid sequences of the heavy and light chains of the monoclonal antibodies.

[0124] In total, the sequences of 16 antibodies were established, and the respective sequences for each antibody are disclosed below.

[0125] [Table 1]

[0126] Example 3 Transient expression and purification of recombinant antibodies Single transfection of a multicistronic vector containing both the HC and LC, or double transfection of vectors containing either the HC or the LC, was performed to produce the desired antibody or antigen-binding fragment. A tricistronic vector using an internal ribosome entry site (IRES) sequence from encephalomyocarditis virus (ECMV) was used.

[0127] A set of vectors with LC only, HC only, LC-IRES-HC, and HC-IRES-LC was compared, and the vector with the highest producer of the desired antibody was selected. The highest producer of antibody was LC-IRES-HC. Additionally, multiple signal peptides were used to generate the desired antibody (Sequence Listing). For the tricistronic vector, the IL2 signal peptide was utilized along with both the LC and HC.

[0128] During stable line development, ExpiCHO cells were transfected with the tricistronic vector and selected with both G418 and neomycin. Two rounds of limiting dilution cloning were used to isolate clonal cell lines. The second round of limiting dilution cloning was imaged to confirm clonality. During stable line development, cells were assessed for viability and productivity of the desired antibody.

[0129] The recombinant antibodies of interest were purified by either Protein A or Protein G for full-length antibodies. For antibodies lacking the Fc region (Fab and F(ab')2 formats), a C-terminal His tag was added to the HC, allowing for IMAC purification followed by SEC policing.

[0130] Example 4 Characterization of the produced antibodies SDS-PAGE The production of each recombinant antibody was verified by SDS-PAGE under reducing and non-reducing conditions, with the exception of antibodies #4 (no protein) and #6 (characterized by CE-SDS) (Figure 2).

[0131] In all cases, the intact antibody was visible under non-reducing conditions, and the respective HC and LC were visible under reducing conditions. Low production was observed for antibodies #1-3, while no production was demonstrated for #4.

[0132] SEC-MALS To assess the assembly and aggregation of the anti-D-dimer recombinant antibodies of the present invention, size-exclusion multi-angle light scattering (SEC-MALS) was used. SEC-MALS provides molecular weight and characterizes polydispersity (Mw / Mn). During antibody production, purified proteins were found to be monodisperse with a difference of 1-2%. Figure 3 provides example data for antibodies #9 and #12.

[0133] For anti-D-dimer antibodies, NHS and EDAC chemistry has been used to conjugate the protein to latex beads.

[0134] Biolayer Interferometry (BLI) Prior to BLI testing, human plasma-derived fragments D, DD, E, fibrinogen, fibrinogen fragment X, and fibrinogen fragment Y were characterized in-house by SDS-PAGE and, if visible contaminants were present, further purified by SEC (Superdex 200 Increase 10 / 300 GL). Some of the mentioned fragments were prepared in-house because the protein amounts were too small to remove contaminants by SEC purification or because they were not commercially available. To prepare fibrinogen fragment X, fibrinogen fragment Y, and fibrinogen fragment D, 1 mg of purified human fibrinogen (Aniara) was incubated with 2 mM CaCl2, 50 mM Tris-HCl pH 7.4, 100 mM NaCl, and 0.055 U / mL human plasmin (HTI). The temperature was set to 37°C and shaken at 800 RPM for 7 minutes. 1000 KIU / mL aprotinin (Sigma) was immediately added, and the sample was placed at -20°C until ready for injection onto an SEC column (Superdex 200 Increase 10 / 300 GL). Collected fractions were kept cold for the duration of the purification. Selected peak fractions were then run on SDS-PAGE and pooled and concentrated according to the MW corresponding to fibrinogen fragments X, Y, and D. An SDS-PAGE gel of purified human plasma proteins is shown in Figure 4.

[0135] FDPs were prepared and run on a 4-20% TGX criterion stain-free gel to check quality and purity before proceeding to characterization studies.

[0136] After purification, a series of biolayer interferometry (BLI) studies were performed to establish the binding affinity of the recombinant antibodies of the invention.

[0137] The binding affinity (at room temperature and pressure) of recombinant antibody #12 was tested for fibrin-derived fragment DD, fibrinogen-derived fragment D, fragment E, and fibrinogen (Figure 5). The results showed that there was binding to both fibrin-derived fragment DD and fibrinogen-derived fragment D, but that the binding to fragment DD was 100-fold stronger than that to fragment D. These binding characteristics, particularly the difference in binding strength of antibody #12 to fragment DD and fragment D, have not been reported for any hybridoma monoclonal antibody known in the art. 12 .

[0138] Example 5 Latex bead agglutination assay After conjugation to latex beads by standard procedures (eg, EDAC and / or NHS chemistry), nephelometric assays were performed to assess the functionality of the recombinant antibodies of the invention.

[0139] A linear correlation was observed in all cases between the known D-dimer assay, HemosIL DDHS500, and the assay developed using antibodies #6 and #5, demonstrating that D-dimer in plasma can be easily detected by the antibodies of the present invention during POC assays. The results obtained for antibodies #6 and #5 (Figures 6B and 6C) are comparable to the known state-of-the-art anti-D-dimer assay (Figure 6A). However, a better correlation was observed for the pepsin-digested F(ab')2 of IgG2a #5 (Figure 6D).

[0140] In Figure 7, we can see the correlation between antibody #12 and DDHS500 ACL (Figure 7A), which shows a linear agreement, and the correlation of antibody #12 with state-of-the-art anti-D-dimer on the Q SMART platform, which shows a 1:1 agreement.

[0141] Thus, the present invention provides recombinant anti-D-dimer antibodies whose functionality has been evaluated by both traditional biophysical assays (BLI) and implementation in latex assays. The results surprisingly show that the anti-D-dimer antibodies are different from antibodies known in the prior art due to their specificity, their binding strength to fragment DD and fragment D, and the fact that they can be easily produced in mammalian cells, thus offering several advantages for latex assay development compared to hybridoma cell line-derived antibodies as discussed previously.

[0142] Example 6 Epitope binning studies Antibody #12 was compared with other commercially available anti-D-dimer antibodies in binning and binding studies against purified fibrin and fibrinogen fragments using the Sartorius BLI Octet Red96e system. Experiments were performed at room temperature and pressure. Binning studies were performed by loading antibody #12 and allowing it to bind to fragment DD, followed by the introduction of other commercially available mAbs. Responses were measured as nm shifts in the interference pattern and are proportional to the number of molecules bound to the biosensor surface. Selected mAbs showed binding to fragment DD in the presence of antibody #12 (see Table 1, units are nm shifted after addition), suggesting that these antibodies recognize a different epitope than antibody #12.

[0143] [Table 2]

[0144] The same set of mAbs was subjected to binding studies with fibrinogen, D-dimer, and purified fibrin and fibrinogen fragments using the BLI Octet Red96e system from Sartorius (see Table 2). KD was measured by loading biotinylated antibody samples onto a streptavidin BLI chip and introducing serial dilutions of the prepared fragments. KD: ++++ (10 -11 ~10 -12 M); +++ (10 -10 M); ++(10 -9 M); + (≧10 -8 M). Experiments were conducted at room temperature and pressure.

[0145] Grifols antibody #12 showed no binding to fibrinogen or fibrin or fibrinogen-derived fragment E, but showed weak binding to fibrinogen fragments X, Y, and D; moderate binding to fragments X and D; and subnanomolar binding to D-dimer and fragment DD. In contrast, DCABY-4394 showed the strongest binding to D-dimer and fragment DD, but also strong binding to fibrinogen. 3B6 did not recognize fragment X and showed weaker binding to D-dimer and fragment DD; NB110-8376 weakly recognized fibrinogen fragments X and Y but not D; and DD225 showed weaker binding to fibrin fragments X and D than antibody #12.

[0146] [Table 3]

[0147] Sequence Listing The sequences relevant to the recombinant antibodies of the present invention are outlined in Table 3 below.

[0148] [Table 4A]

[0149] Table 4B

[0150] Table 4C

[0151] Table 4D

[0152] Table 4E

[0153] [Table 4F]

[0154] [References] TIFF0007807152000010.tif214169

Claims

1. An anti-D-dimer recombinant antibody characterized by specifically binding to fibrin and fibrinogen degradation products (FDPs), namely, D-dimer, fragment DD, and fragment D, but not to fragment E and fibrinogen, the recombinant antibody comprising: a light chain variable region comprising the amino acid sequence of SEQ ID NO: 31 (L-CDR1); the amino acid sequence of SEQ ID NO: 32 (L-CDR2); and the amino acid sequence of SEQ ID NO: 33 (L-CDR3); and An anti-D-dimer recombinant antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 34 (H-CDR1); the amino acid sequence of SEQ ID NO: 35 (H-CDR2); and the amino acid sequence of SEQ ID NO: 36 (H-CDR3).

2. The recombinant antibody of claim 1, wherein the amino acid sequence of H-CDR2 further comprises a sequence selected from the group consisting of SEQ ID NO: 37 and SEQ ID NO:

38.

3. The recombinant antibody of claim 1 or 2, comprising: a light chain variable region comprising an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% identical to SEQ ID NO: 18; and A heavy chain variable region comprising an amino acid sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 19, 20, 21, and 22.

4. The recombinant antibody according to any one of claims 1 to 3, comprising: a light chain variable region comprising the amino acid sequence of SEQ ID NO: 18; and A heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 20, 21, and 22.

5. An anti-D-dimer recombinant antibody characterized by specifically binding to fibrin and fibrinogen degradation products (FDPs), namely, D-dimer, fragment DD, and fragment D, but not to fragment E and fibrinogen, a light chain variable region comprising the amino acid sequence of SEQ ID NO: 18, and An anti-D-dimer recombinant antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:

19.

6. The recombinant antibody of any one of claims 1 to 5, which is a monoclonal antibody or antibody fragment.

7. The antibody fragment may be a variable fragment (Fv), a single-chain Fv (scFv), a bispecific antibody (sc(Fv) 2 ), single chain antibody, Fab fragment, F(ab') 2 The recombinant antibody of claim 6, selected from a fragment, a Fab' fragment, a disulfide-linked Fv (dsFv), a chemically conjugated Fv (ccFv), a diabody, and an anti-idiotypic (anti-Id) antibody.

8. The antibody fragments include Fab fragments and F(ab') 2 The recombinant antibody of claim 6, which is an antigen-binding fragment selected from the group consisting of:

9. The recombinant antibody according to any one of claims 1 to 8, comprising a constant region of mouse IgG1 class or mouse IgG2a class.

10. The recombinant antibody of any one of claims 1 to 9, further comprising an affinity tag.

11. 11. The recombinant antibody of claim 10, wherein the amino acid sequence of the affinity tag is selected from the group consisting of SEQ ID NO: 23, SEQ ID NO: 24, or SEQ ID NO:

25.

12. A recombinant antibody according to any one of claims 1 to 11, wherein the light chain of the recombinant antibody comprises the amino acid sequence of SEQ ID NO:

17.

13. 13. The recombinant antibody of any one of claims 1 to 12, wherein the heavy chain of the recombinant antibody comprises the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:

16.

14. 14. The recombinant antibody of any one of claims 1 to 13, wherein the light chain of the recombinant antibody comprises the amino acid sequence of SEQ ID NO: 17 and the heavy chain of the recombinant antibody comprises the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO:

16.

15. A recombinant antibody according to any one of claims 1 to 14, comprising the amino acid sequence of SEQ ID NO: 17 and the amino acid sequence of SEQ ID NO:

12.

16. The recombinant antibody of any one of claims 1 to 15, which is bound to a solid support.

17. the binding affinity of said recombinant antibody to fragment DD is at least three times stronger than the binding affinity of said recombinant antibody to fragment D; or the binding affinity of the recombinant antibody to fragment DD is at least 10 times stronger than the binding affinity of the recombinant antibody to fragment D; A recombinant antibody according to any one of claims 1 to 16.

18. A cell comprising a recombinant antibody according to any one of claims 1 to 17.

19. A nucleic acid comprising a nucleotide sequence encoding the recombinant antibody of any one of claims 1 to 15 or 17, a promoter operably linked to the nucleotide sequence, and a selectable marker.

20. 20. A cell comprising the nucleic acid of claim 19.

21. A composition comprising the recombinant antibody of any one of claims 1 to 15 or 17 and a solid support, wherein the recombinant antibody is covalently or non-covalently bound to the solid support.

22. 22. The composition of claim 21, wherein the solid support comprises a particle, bead, membrane, surface, polypeptide chip, microtiter plate, or the solid phase of a chromatography column.

23. 22. The composition of claim 21, wherein the solid support is a latex particle.

24. 19. A kit for detecting the presence of D-dimer, fragment DD, and / or fragment D in a sample, said kit comprising at least one recombinant antibody according to any one of claims 1 to 15 or 17, and a solid support, wherein said at least one recombinant antibody is covalently or non-covalently bound to the solid support.

25. A method for detecting the presence of D-dimer, fragment DD, and / or fragment D in a sample, comprising the steps of: contacting the sample with at least one recombinant antibody according to any one of claims 1 to 15 or 17 for a time and under conditions sufficient for the formation of an antibody / antigen complex; detecting said antibody / antigen complex.

26. A method for measuring the binding affinity of D-dimer, fragment DD, and / or fragment D in a sample, comprising the steps of: contacting the sample with at least one recombinant antibody according to any one of claims 1 to 15 or 17 for a time and under conditions sufficient for the formation of an antibody / antigen complex; Determining the binding affinity between the antibody and D-dimer, fragment DD, and / or fragment D in the sample.

27. 1. A method for determining the concentration of D-dimer, fragment DD, and / or fragment D in a sample, comprising the steps of: contacting the sample with at least one recombinant antibody according to any one of claims 1 to 15 or 17 for a time and under conditions sufficient for the formation of an antibody / antigen complex; Measuring the concentration of D-dimer, fragment DD, and / or fragment D in the sample.

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