Anti-d-dimer recombinant antibodies, methods and uses thereof

Abstract

The present application relates to anti-D-dimer recombinant antibodies that specifically bind with high binding affinity to fibrin and fibrinogen degradation products (FDPs) such as D-dimer, fragment DD and fragment D and do 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 said recombinant antibodies.

Description

[0001] describe Technical Field

[0002] This application relates to recombinant anti-D-dimer antibodies that specifically bind with high binding affinity to fibrin and fibrinogen degradation products (FDPs) such as D-dimer, fragment DD, and fragment D, but do not bind to fragment E and fibrinogen. The invention also relates to methods and assays for detecting D-dimers and FDP fragments in samples using said recombinant antibodies.

[0003] background

[0004] Fibrinogen is a circulating soluble protein found in blood plasma. When treated with thrombin (factor IIa), it forms fibrin polymers during the coagulation cascade. Fibrinogen consists of three chains: α, β, and γ, which form a large dumbbell-shaped structure with two terminal D domains and a central E domain. Fibrin polymers are produced by the interleaved assembly of fibrinogen monomers. Fibrin polymers can be further cross-linked by forming isopeptide bonds, covalently linking the fibrin chains to factor XIII (FXIII).

[0005] During fibrinolysis, cross-linked fibrin polymers (products of coagulation) are degraded by serine proteases called plasminase, yielding a heterogeneous mixture of degradation products, the smallest of which is the D-dimer. Figure 1 Other fibrin (fibrinogen) degradation products (FDPs) are fragments X, Y, D, and E. The D-dimer consists of two cross-linked D domains and one E domain. The D and E domains can be separated by urea extraction to obtain fragments DD and E. Fragment DD is unique because the isopeptide bond between the two D domains stabilizes the dimerization.

[0006] The detection of fibrin (fibrinogen) degradation products (FDPs) is used to diagnose venous thromboembolism (VTE), such as deep vein thrombosis (DVT) and pulmonary embolism (PE). When FDPs are present in large quantities, they 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 stability.

[0007] D-dimer and fragment DD have attracted diagnostic interest due to the presence of cross-linked D-domains, indicating coagulation events such as deep vein thrombosis (DVT) or premature rupture of membranes (PE). Many D-dimer assays are currently available; however, these assays vary due to the use of different monoclonal antibodies recognizing different epitopes, different assay formats, calibration standards and ranges, and different instruments. 1ELISA and agglutination-based assays are the most common, and the tests can be qualitative or quantitative. Agglutination assays come in many forms, and in the case of automated latex assays, beads conjugated with anti-D-dimer antibodies agglutinate in the presence of patient plasma, and turbidimetric detection is used to characterize agglutination. In some devices, bispecific antibodies binding to both D-dimer and erythrocytes are used to induce erythrocyte agglutination, thus providing qualitative results. 2 .

[0008] D-dimer testing is one of the most frequently required coagulation tests, most commonly used to rule out venous thromboembolism (VTE). VTE occurs when a blood clot (DVT) forms in the deep veins of the limbs or groin, and VTE can potentially travel to the lungs (PE). 3 The International Society of Thrombosis and Haemostasis has recognized the role of D-dimer testing in the diagnosis of disseminated intravascular coagulation (DIC). However, circulating D-dimer can be present in coronary artery disease, cancer, trauma, pregnancy, infectious diseases, inflammatory diseases, advanced age, and many other conditions and disease states. 4 When the D-dimer test is used in the diagnostic cascade for DVT or PE, further testing for DVT or PE can be excluded. This highlights the importance of performing sensitive tests so that patients with VTE are not unduly excluded. Generally, ELISA is more sensitive than latex agglutination assays; however, the value of automation and reproducibility makes latex assays easier to perform in many clinical laboratories. The specificity of the D-dimer test for a specific diagnosis depends on the pre-test probability. 5 Furthermore, the D-dimer test may be sensitive to interference from heterophilic antibodies. 6 Or, it may be sensitive 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 and needs to be considered as part of a larger body of evidence during the diagnostic process.

[0009] Many anti-D-dimer monoclonal antibodies have been produced, either while the hybridoma is in the supernatant or when it is injected into the abdomen of mice to induce ascites. Although hybridomas are generally a robust and efficient way to produce monoclonal antibodies (mAbs), this technology has limitations. First, hybridomas can become depleted and cease producing antibodies, potentially resulting in the loss of hybridomas as a source of production, even if a cell bank is created. Second, hybridomas can be lost due to cryostat malfunctions or other accidents. Finally, the fact that hybridomas produce antibodies in their natural form makes sequencing and recombinant expression necessary for further mAb engineering.

[0010] Therefore, there is a need for anti-D-dimer antibodies that can be easily and repeatedly generated and provide high specificity against D-dimer for use in thrombosis detection assays.

[0011] This invention provides recombinant antibodies and antigen-binding fragments (Fab or F(ab')2) that can specifically detect FDP and have several advantages over existing monoclonal antibodies (produced via hybridoma).

[0012] First, compared to proteins produced by hybridoma cell lines that are generally considered unstable, recombinant proteins offer improved control and reproducibility. 8,9 Second, greater batch-to-batch consistency can be achieved by using stable cell lines with controlled biochemical and physical process parameters. 10 Third, animal-free technology allows production without the use of any animals, eliminating welfare and ethical concerns. Fourth, the recombinant antibodies of this invention can be engineered to include additional functional domains for purposes such as purification or solubility. Fifth, isotypes (IgG1, IgG2a, IgG3) can be selected, if desired, to produce higher yields or more stable proteins. Finally, the ability to engineer recombinant proteins provides an additional advantage in reducing the likelihood of human anti-mouse antibody interference (HAMA). 7 Case studies have shown that HAMA causes unclear readings, which can negatively impact patient diagnosis.

[0013] Therefore, the present invention relates to recombinant antibodies capable of detecting D-dimer, fragment DD, and fragment D, and clinically effective in diagnosing DVT, PE, and other acute disease states.

[0014] Overview

[0015] The first aspect of the present invention relates to an anti-D-dimer recombinant antibody that specifically binds to fibrin and fibrinogen degradation product (FDP) D-dimer, fragment DD and fragment D, but does not bind to fragment E and fibrinogen.

[0016] In one embodiment, the recombinant antibody comprises a light chain containing complementarity-determining regions L-CDR1, L-CDR2, and L-CDR3, each of the complementarity-determining regions comprising a sequence of at least five consecutive amino acids selected from the amino acid sequence of SEQ ID NO:18. In a 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, or 50 to 56, or 89 to 97 of SEQ ID NO:18. In a 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.

[0017] In one embodiment, the light chain comprises the amino acid sequence of SEQ ID NO:18.

[0018] In another embodiment, the recombinant antibody comprises a heavy chain including complementarity-determining regions H-CDR1, H-CDR2, and H-CDR3, each of the complementarity-determining regions comprising a sequence of at least five consecutive amino acids selected from the amino acid sequence of SEQ ID NO:19, SEQ ID NO:20, 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, SEQ ID NO:20, SEQ ID NO:21, or SEQ ID NO:22 include at least one of amino acids 31 to 35, 50 to 65, or 95 to 102 of SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, or SEQ ID NO:22. In a preferred embodiment, 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.

[0019] In some embodiments, the recombinant antibody of the present invention may have heavy chains 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.

[0020] In some embodiments, the recombinant antibody of the present invention may have heavy chains 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.

[0021] 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.

[0022] In one embodiment, the recombinant antibody of the present invention may comprise:

[0023] The light chain variable region comprises 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

[0024] The heavy chain variable region comprises 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).

[0025] In another embodiment, the recombinant antibody of the present invention may comprise:

[0026] The light chain variable region contains 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 listed in SEQ ID NO:18, and

[0027] The heavy chain variable region contains at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% of the same amino acid sequence as the sequence selected from the group consisting of SEQ ID NO: 19, 20, 21, and 22.

[0028] In yet another embodiment, the recombinant antibody of the present invention may comprise:

[0029] The light chain variable region contains the amino acid sequence listed in SEQ ID NO:18, and

[0030] The heavy chain variable region contains an amino acid sequence selected from the group consisting of SEQ ID NO:19, 20, 21 and 22.

[0031] In some embodiments, the recombinant antibody of the present invention may comprise:

[0032] The light chain variable region contains the amino acid sequence listed in SEQ ID NO:18, and

[0033] The heavy chain variable region contains an amino acid sequence selected from the group consisting of SEQ ID NO:19 and 20.

[0034] In another embodiment, the recombinant antibody of the present invention is a monoclonal antibody or an antibody fragment. In some embodiments, the antibody fragment is selected from variable fragments (Fv), single-chain Fv (scFv), bispecific antibodies (sc(Fv)2), single-chain antibodies, single-domain antibodies, Fab fragments, F(ab')2 fragments, Fab' fragments, disulfide-linked Fv (dsFv), chemically conjugated Fv (ccFv), biantibodies, anti-idiotype (anti-Id) antibodies, affibody, nanobody, and unibody. In another embodiment, the antibody fragment is an antigen-binding fragment selected from Fab fragments and F(ab')2 fragments.

[0035] In one embodiment, the recombinant antibody of the present invention comprises a constant region of mouse IgG1 or mouse IgG2a.

[0036] In another embodiment, the recombinant antibody of the present invention further comprises an affinity tag. The affinity tag may be selected from the group consisting of SEQ ID NO:23, SEQ ID NO:24, or SEQ ID NO:25.

[0037] In another embodiment, the light chain of the recombinant antibody of the present invention comprises the amino acid sequence of SEQ ID NO:17. In yet another embodiment, the heavy chain of the recombinant antibody of the present 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.

[0038] In another embodiment, the light chain of the recombinant antibody of the present invention 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.

[0039] 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.

[0040] In one embodiment, the recombinant antibody of the present invention is bound to a solid support.

[0041] In another embodiment, the binding affinity of the recombinant antibody of the present invention to fragment DD is at least (about) 3 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 at least (about) 10 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) 20 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) 30 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) 40 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) 50 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) 60 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 the recombinant antibody to fragment D. In yet 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 the recombinant antibody to fragment D.

[0042] In another embodiment, the binding affinity of the recombinant antibody of the present invention to fragment DD can be at least about 20 times and about 100 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 can be at least about 40 times and about 100 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 can be at least about 60 times and about 100 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 can be at least about 70 times and about 100 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 can be at least about 70 times and about 90 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 can be at least about 80 times and about 90 times stronger than the binding affinity of the recombinant antibody to fragment D.

[0043] In another embodiment, the binding affinity of the recombinant antibody of the present invention to fragment DD is at least one order of magnitude 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 two orders of magnitude 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 one order of magnitude stronger but less than three orders of magnitude stronger than the binding affinity of the recombinant antibody to fragment D.

[0044] As used herein, the term “binding affinity” refers to the strength of the interaction between the epitope of an antigen and the antigen-binding site of an antibody, as measured by biolayer interferometry on an OctetRed96e system from Sartorius (formerly FortéBio) at approximately 23°C and 1 atmosphere.

[0045] Advantageously, compared to commercially available anti-D-dimer antibodies, the recombinant antibodies / antibodies of the present invention exhibit improved binding to fibrin degradation products. Furthermore, the recombinant antibodies / antibodies of the present invention do not show binding affinity for fibrinogen or fragment E.

[0046] In another aspect, the present invention relates to cells comprising the recombinant antibody of the present invention. The present invention also relates to a nucleic acid comprising a nucleotide sequence encoding the recombinant antibody of the present invention, a promoter operatively linked to the nucleotide sequence, and a selectable marker. The present invention further relates to cells comprising said nucleic acid.

[0047] The present invention also relates to compositions comprising the 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 particles, beads, membranes, surfaces, peptide chips, microtiter plates, or the solid phase of a chromatographic column. Preferably, the solid support is latex particles.

[0048] In another aspect, the present invention relates to a kit for detecting the presence of D-dimer, fragment DD and / or fragment D in a sample, the kit comprising at least one recombinant antibody according to the invention and a solid support, wherein the at least one recombinant antibody is covalently or non-covalently bound to the solid support.

[0049] In another aspect, the present invention relates to a method for detecting the presence of D-dimer, fragment DD, and / or fragment D in a sample, the method comprising:

[0050] -Contact the sample with at least one recombinant antibody of the present invention under conditions sufficient to form an antibody / antigen complex for a sustained period of time, and

[0051] - Detect the antibody / antigen complex.

[0052] In another aspect, the present invention relates to a method for measuring the binding affinity of D-dimer, fragment DD, and / or fragment D in a sample, the method comprising:

[0053] -Contact the sample with at least one recombinant antibody of the present invention under conditions sufficient to form an antibody / antigen complex for a sustained period of time, and

[0054] - Determine the binding affinity between the antibody and D-dimer, fragment DD, and / or fragment D in the sample.

[0055] In another aspect, the present invention relates to a method for measuring the concentration of D-dimer, fragment DD, and / or fragment D in a sample, the method comprising:

[0056] -Contact the sample with at least one recombinant antibody according to the invention under conditions sufficient to form an antibody / antigen complex and for a sustained period of time, and

[0057] - Measure the concentration of D-dimer, fragment DD and / or fragment D in the sample. Brief description of the attached diagram

[0059] Figure 1This is a schematic diagram of fibrin monomers assembling to form fibrinogen. Thrombin acts on fibrin or fibrinogen to release fibrin peptides from the E domains of the α and β chains, causing fibrin to polymerize into fibrinogen through crosslinking. FXIIIa crosslinks the α-α and γ-γ chains by forming isopeptide bonds to stabilize protoplasm. Serine protease plasmin digests fibrin, producing fragment X (280 kDa), fragment Y (150 kDa), fragment D (94 kDa), and fragment E (50 kDa). Due to crosslinking by FXIIIa, the digestion of crosslinked fibrin produces 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 to obtain fragment DD (190 kDa) and fragment E (50 kDa).

[0060] Figure 2 The SDS-PAGE results of the recombinant antibodies of the present invention under reducing (R) and non-reducing conditions (N or NR) are shown, except for antibodies #4 and #6, for which CE-SDS results are shown instead.

[0061] Figure 3 Representative light scattering data for antibodies #9 and #12 are shown. The A280 tracer peaks intersect with the measured molar mass across each peak, and the flat molar mass data for each peak indicates a monodisperse sample. #9: Predicted mass = 48.8 kDa, measured mass = 48.3 ± 1% kDa, Mw / Mn = 1.001. #12: Predicted mass = 103 kDa, measured mass = 105 ± 4% kDa, Mw / Mn = 1.000. All recombinant anti-D-dimer antibodies showed monodisperse peaks (data not shown).

[0062] Figure 4 The purified FDP used in the combined study is shown.

[0063] Figure 5 This graph shows a comparison of the binding affinity of recombinant antibody #12 with fragments DD, D, E, and fibrinogen. When using the Octet BLI platform, both fragment DD (A) from fibrinogen and fragment D (B) from fibrinogen showed binding to antibody #12. Fragments E (C) and fibrinogen (D) did not show binding to antibody #12.

[0064] Figure 6This is a graph showing the good correlation between the presence of D-dimer in plasma samples and various antibodies on the HemosILDDHS500 and Q SMART systems of the ACL TOP coagulation analyzer. A) shows the linear correlation using antibodies derived from known 8D3 hybridomas, B) shows the linear correlation in the case of IgG1 antibody (#6), C) shows the linear correlation in the case of IgG2a (#5), and D) shows the linear correlation of F(ab')2 in IgG2a#5 digested with pepsin.

[0065] Figure 7 The following are shown: A) Linear correlations between #12 and D-dimer analysis using the Grifols Q SMART system and analysis of plasma samples with different D-dimer presence using the HemosIL DDHS550 and the ACL TOP coagulation analyzer; and B) Linear correlations when analyzing D-dimer presence in plasma samples using #12 or hybridoma 8D3 mAb on the QSMART system.

[0066] definition

[0067] The following description is intended only to illustrate various embodiments of the present disclosure. Therefore, 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 may be made without departing from the spirit or scope of the subject matter presented herein, and it should be understood that such equivalent embodiments will be included herein.

[0068] As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural indicators unless the content clearly indicates otherwise.

[0069] Throughout this specification, unless the context otherwise requires, the word “comprise” or variations such as “comprises” and “comprising” shall be understood to imply inclusion of the stated element or integer, or group of elements or integers, but not to exclude any other element or integer, or group of elements or integers.

[0070] 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 pertains. Exemplary methods and materials are described below, although similar or equivalent methods and materials may also be used and will be apparent to those skilled in the art. All publications and other references mentioned herein are incorporated herein by reference in their entirety. In the event of conflict, this specification (including definitions) shall prevail. Materials, methods, and examples are illustrative only and not intended to be limiting.

[0071] Unless otherwise expressly stated, each embodiment in this specification will be applicable to every other embodiment with appropriate modifications.

[0072] Unless otherwise indicated, the following terms shall be understood to have the following meanings:

[0073] As used herein, the term "nucleic acid" refers to any material including DNA or RNA. Nucleic acids can be prepared synthetically or from living cells.

[0074] As used herein, the term "polynucleotide" refers to a polymer chain of nucleotides. This term includes DNA molecules (e.g., cDNA or genomic DNA or synthetic DNA) and RNA molecules (e.g., mRNA or synthetic RNA), as well as DNA or RNA analogs containing non-natural nucleotide analogs, non-natural nucleoside bonds, or both. Nucleic acids can be in any topological conformation. For example, nucleic acids can be single-stranded, double-stranded, triple-stranded, quadruple-stranded, partially double-stranded, branched, hairpin-shaped, circular, or padlock-shaped conformations.

[0075] As used herein, the term "protein" refers to a large biomolecule or macromolecule consisting of a chain of one or more amino acid residues. Many proteins are enzymes that catalyze biochemical reactions and are essential for metabolism. Proteins also have structural or mechanical functions, such as actin and myosin in muscle, and proteins in the cytoskeleton that form the scaffold system that maintains cell shape. Other proteins are important in cell signaling, immune responses, cell adhesion, and the cell cycle. However, proteins can be entirely artificial or recombinant, meaning they are not naturally occurring in biological systems.

[0076] As used herein, the term "peptide" refers to naturally occurring and non-natural proteins, as well as their fragments, mutants, derivatives, and analogs. Peptides can be monomeric or polymeric. Peptides can contain many different domains (peptides), each of which has one or more different activities.

[0077] As used herein, the term “recombinant” refers to a biomolecule, such as a gene or protein, that (1) has been removed from its naturally occurring environment, (2) does not associate with all or part of the polynucleotide found in nature with the gene, (3) is operatively linked with a polynucleotide not found in nature with it, or (4) is not found in nature. The term “recombinant” may also refer to cloned DNA isolates, chemically synthesized polynucleotide analogs, or polynucleotide analogs biosynthesized from heterologous systems, as well as proteins and / or mRNA encoded by such nucleic acids.

[0078] As used herein, the term "antigen" refers to a biomolecule that specifically binds to a corresponding antibody. Antibodies from different repertoires bind to specific antigenic structures through interactions of their variable regions.

[0079] As used herein, the term "fusion protein" refers to a protein that contains two or more amino acid sequences that are not found in naturally occurring proteins. Fusion proteins can 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 usually in-frame, without a stop codon between them, and are typically translated from mRNA as part of the fusion protein.

[0080] 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. Therefore, the term "antibody fragment" as used herein includes, but is not limited to, variable fragments (Fv), single-chain Fv (scFv), bispecific antibodies (sc(Fv)2), single-chain antibodies, single-domain antibodies, Fab fragments, F(ab')2 fragments, Fab' fragments, disulfide-linked Fv (dsFv), chemically conjugated Fv (ccFv), biantibodies, and anti-idiotypic (anti-Id) antibodies, as well as functionally active epitope-binding fragments of any of the above. In some embodiments, antibodies also include affinities, nanobodies, and monoantibodies. In some embodiments, specific antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules containing antigen-binding sites. 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.

[0081] As used herein, the term "antigen-binding fragment (Fab)" refers to an antibody fragment containing one constant domain and one variable domain for each of the heavy and light chains. The variable domain contains the antigen-binding site. Typically, an antibody comprises a fragment crystallizable region (Fc) and two antigen-binding fragments (Fab). The Fab fragments can be separated from the Fc region to obtain two Fab fragments, which are also referred to as F(ab')2 fragments or antigen-binding dimer fragments.

[0082] In natural antibodies, two heavy chains are linked together 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). Five major heavy chain classes (or isotypes) determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE. Each chain contains different sequence domains. The light chain contains two domains: a variable domain (VL) and a constant domain (CL). The heavy chain contains four domains: a variable domain (VH) and three constant domains (CH1, CH2, and CH3, collectively referred to as CH). The variable regions of the light chain (VL) and heavy chain (VH) determine the binding recognition and specificity to the antigen. The constant regions of the light chain (CL) and heavy chain (CH) confer important biological properties such as antibody chain association, secretion, transplacental movement, complement binding, and binding to the Fc receptor (FcR). The Fv fragment is the N-terminal portion of the Fab fragment of an immunoglobulin and consists of variable portions of a light chain and a heavy chain. Antibody specificity arises from structural complementarity between the antibody binding site and the antigenic determinant. The antibody binding site is primarily composed of residues from the hypervariable region or complementarity-determining region (CDR). Occasionally, residues from the non-hypervariable region or frame region (FR) influence the overall domain structure and thus the binding site. The complementarity-determining region, or CDR, is the amino acid sequence that collectively defines the binding affinity and specificity of the native Fv region of the immunoglobulin binding site. The light and heavy chains of immunoglobulins each have three CDRs, designated L-CDR1, L-CDR2, L-CDR3 and H-CDR1, H-CDR2, H-CDR3, respectively. Therefore, the antigen-binding site typically contains six CDRs, comprising a set of CDRs from each of the heavy chain V region and the light chain V region. The frame region (FR) is the amino acid sequence inserted between the CDRs.

[0083] CDRs can be identified according to the Kabat definition, the Chothia definition, the sum of both the Kabat and Chothia definitions, the AbM definition, the contact definition, the IMGT unique numbering definition, and / or the conformation definition, or any CDR identification method well known in the art. Antibody CDRs can be identified as hypervariable regions originally defined by Kabat et al. See, for example, Kabat et al., 1992, Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, NIH, Washington DC. The location of a CDR can also be identified as a structural loop structure originally described by Chothia and others (see, for example, Chothia et al., Nature 342:877-883, 1989). Other methods for identifying CDRs include the “AbM definition,” a compromise between Kabat and Chothia, derived using Oxford Molecular’s ​​AbM antibody modeling software (now Accelrys0); the “contact definition” of CDRs based on observed antigen contact, described in MacCallum et al., J. Mol. Biol., 262:732-745, 1996; or the “IMGT unique number,” which relies on the high conservation of the variable region’s structure (see Lefranc, M.-P. Nucl. Acids Res., 33, D593-D597, 2005). In another method, referred to here as the “conformation definition” of CDRs, the position of the CDR can be identified as the residue that makes an enthalpy contribution to antigen binding. See, for example, Makabe et al., Journal of Biological Chemistry, 283:1156-1166, 2008. Given that predictions or experiments have shown that a particular residue or group of residues, or even the entire CDR, does not significantly affect antigen binding, and that other CDR boundary definitions may not strictly follow one of the methods described above, but will still overlap with at least a portion of the Kabat CDR, although they may be shortened or lengthened. As used herein, a CDR can refer to a CDR defined by any method (including combinations of methods) known in the art. The methods used herein can utilize CDRs defined according to any of these methods. For any particular embodiment containing more than one CDR, each CDR can be defined according to any of the Kabat definition, Chothia definition, extended definition, AbM definition, contact definition, IMGT unique numbering definition, or conformational definition.

[0084] Exemplary databases of antibody sequences are described below and can be accessed via: the Abysis website www.bioinf.org.uk / abs (maintained by A.C. Martin of the Department of Biochemistry & Molecular Biology, University College London, London, England), and the VBASE2 website www.vbase2.org, as described in Retter et al., Nucl. Acids Res., 33 (Database Special Issue): D671-D674 (2005). Preferably, sequences are analyzed using the Abysis database, which integrates sequence data from Kabat, IMGT, and the Protein Database (PDB) with structural data from the PDB. Unless otherwise indicated, all CDRs listed herein are derived from the Abysis database website according to the indicated protocol.

[0085] As used herein, the term "monoclonal antibody" refers to an antibody composition having a homogeneous group of antibodies that bind to the same epitope. This term is not limited to the type or source of the antibody, nor is it intended to be limited to its preparation method. Therefore, the term includes antibodies obtained from mouse hybridomas, as well as human monoclonal antibodies obtained using human hybridomas rather than mouse hybridomas.

[0086] As used herein, the term "epitope" refers to the portion of an antigen that specifically binds to an antibody. Therefore, the term "epitope" includes any protein determinant capable of specifically binding to immunoglobulins or T-cell receptors.

[0087] As used herein, the term "recombinant antibody" refers to an antibody or fragment thereof that is not naturally occurring and can associate with polypeptides or fragments thereof that are not found in nature. Recombinant antibodies can be produced using any recombinant technique known to those skilled in the art.

[0088] As used herein, in the context of two or more nucleic acid or polypeptide sequences, the term "identical" or "identity percentage" refers to two or more sequences or subsequences that are identical or have a specific percentage of identical nucleotide or amino acid residues when compared and aligned to obtain maximum correspondence. To determine the identity percentage, sequences are aligned for optimal comparison purposes (e.g., vacancies may be introduced into the sequence of a first amino acid or nucleic acid sequence to achieve optimal alignment with a second amino acid or nucleic acid sequence). Amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, the molecules are identical at that position. The identity percentage 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 of the same length after vacancies are appropriately introduced within the sequences (e.g., excluding additional sequences extending beyond the compared sequences). For sequence comparisons between two sequences, the “corresponding” CDR refers to the CDR at the same position in both sequences (e.g., CDR-H1 for each sequence).

[0089] The determination of the percentage of identity or similarity between two sequences can be accomplished using mathematical algorithms. A preferred, non-limiting example of a mathematical algorithm for comparing two sequences is the algorithm in Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, as modified in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877. Such algorithms are incorporated into the NBLAST and XBLAST procedures in Altschul et al., 1990, J. Mol. Biol. 215:403-410. BLAST nucleotide searches can be performed using the NBLAST procedure with a score of 100 and a word length of 12 to obtain nucleotide sequences homologous to the nucleic acid encoding the protein of interest. BLAST protein searches can be performed using the XBLAST procedure with a score of 50 and a word length of 3 to obtain amino acid sequences homologous to the protein of interest. To obtain vacancy-containing alignments for comparison purposes, Gapped BLAST, as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402, can be used. When using BLAST and GappedBLAST, the default parameters of the corresponding programs (e.g., XBLAST and NBLAST) can be used. Another preferred, non-limiting example of a mathematical algorithm for sequence comparison is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When using the ALIGN program to compare amino acid sequences, the PAM120 weighted residue table, vacancy length penalty of 12, and vacancy penalty of 4 can be used.

[0090] A peptide is "immunoreactive" to an antibody when the peptide binds to the antibody due to the antibody recognizing a specific epitope contained in the peptide. Immunoreactivity can be determined by antibody binding, more particularly by the kinetics of antibody binding, and / or by competition in binding using a known peptide containing the epitope targeted by the antibody as a competitor. Techniques for determining whether a peptide is immunoreactive to an antibody are known in the art.

[0091] As used herein, the term "sample" refers to any biological material obtained from a subject or patient. In one aspect, a sample may include blood, peritoneal fluid, CSF, saliva, or urine. In other aspects, 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). A sample may also include biopsy or tissue samples, including neural tissue. In yet another aspect, a sample may include intact cells and / or cell lysates.

[0092] As used herein, the terms "diagnostic" or "diagnosed" refer to patients identified as having a pathological condition or being susceptible to disease. The sensitivity and specificity of diagnostic methods differ. The "sensitivity" of a diagnostic test is the percentage of diseased individuals who test positive (the "true positive" percentage). Diseased individuals who are not detected by the test are "false negatives." Subjects who are not diseased and test negative in the test are called "true negatives." The "specificity" of a diagnostic test is 1 minus the false positive rate, where the "false positive" rate is defined as the proportion of subjects who are not diseased and test positive. While a particular diagnostic method may not provide a definitive diagnosis of the condition, it is qualified if it provides useful indications that aid in diagnosis.

[0093] The terms “patient” or “individual” are used interchangeably herein and refer to a mammalian subject to treatment, with human patients being preferred. In some cases, the methods of the present invention can be used for the development of laboratory animals, veterinary applications, and animal models of disease, including but not limited to rodents (including mice, rats, and hamsters) and primates.

[0094] Detailed Explanation

[0095] I. Recombinant Antibody

[0096] This invention relates to a recombinant anti-D-dimer antibody that specifically binds to fibrin and fibrinogen degradation product (FDP) D-dimer, fragment DD and fragment D, but does not bind to fragment E and fibrinogen.

[0097] 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, namely a variable domain (VL) and a constant domain (CL). The heavy chain of the recombinant antibody of the present invention may comprise four domains, namely a variable domain (VH) and three constant domains (CH1, CH2, and CH3, collectively referred to as CH).

[0098] In another embodiment, the recombinant antibody of the present invention is a monoclonal antibody or an antibody fragment. In a preferred embodiment, the antibody fragment is selected from variable fragments (Fv), single-chain Fv (scFv), bispecific antibodies (sc(Fv)2), single-chain antibodies, single-domain antibodies, Fab fragments, F(ab')2 fragments, Fab' fragments, disulfide-linked Fv (dsFv), chemically conjugated Fv (ccFv), biantibodies, anti-idiotype (anti-Id) antibodies, affinity molecules, nanobodies, and monoantibodies.

[0099] In one embodiment described herein, the recombinant antibody comprises an Fc region and two Fab fragments. In other embodiments described herein, the recombinant antibody is an antigen-binding fragment and does not contain an Fc region. In other embodiments described herein, the recombinant antibody consists of one Fab fragment. In other embodiments described herein, the recombinant antibody consists of two Fab fragments (F(ab)2).

[0100] In one embodiment described herein, the recombinant antibody may be 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.

[0101] 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 yet another preferred embodiment, the recombinant antibody is of the IgG2a class.

[0102] In the most preferred embodiment, the recombinant antibody of the present invention comprises a constant region of mouse IgG1 or mouse IgG2a.

[0103] A. Light chain

[0104] In one embodiment described herein, the recombinant antibody comprises a light chain containing a complementarity-determining region (CDR). The CDR corresponds to a sequence identified according to any CDR definition method known to those skilled in the art. In some preferred embodiments, the CDR region corresponds to a sequence identified according to Kabat. In some preferred embodiments, the CDR region corresponds to a sequence identified according to Chothia. In another embodiment, the CDR can be any of the following: Kabat definition, Chothia definition, AbM definition, extended definition, contact definition, IMGT unique number definition and / or conformation definition, combined CDR, or a combination thereof.

[0105] In one embodiment described herein, the recombinant antibody comprises a light chain containing complementarity-determining regions L-CDR1, L-CDR2, and L-CDR3, each of said complementarity-determining regions comprising a (different) sequence of at least five consecutive amino acids selected from the amino acid sequence of SEQ ID NO:18. In a preferred embodiment, said 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, or 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 NO:31 to 33 represent L-CDR1, L-CDR2, and L-CDR3 as defined by Kabat in SEQ ID NO:18, respectively.

[0106] 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 with the amino acid sequence consisting of SEQ ID NO:18.

[0107] In another embodiment described herein, the recombinant antibody comprises a light chain containing 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.

[0108] B. Heavy chain

[0109] In one embodiment described herein, the recombinant antibody comprises a heavy chain containing a complementarity-determining region (CDR). The CDR corresponds to a sequence identified according to any CDR definition method known to those skilled in the art. In some preferred embodiments, the CDR region corresponds to a sequence identified according to Kabat. In some preferred embodiments, the CDR region corresponds to a sequence identified according to Chothia. In another embodiment, the CDR can be any of the following: Kabat definition, Chothia definition, AbM definition, extended definition, contact definition, IMGT unique number definition and / or conformation definition, combined CDR, or a combination thereof.

[0110] In one embodiment described herein, the recombinant antibody comprises a heavy chain including 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, SEQ ID NO:20, 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, SEQ ID NO:20, SEQ ID NO:21, or SEQ ID NO:22 include at least one of amino acids 31 to 35, 50 to 65, or 95 to 102 of SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, or SEQ ID NO:22. In a preferred embodiment, H-CDR1, H-CDR2, and H-CDR3 of the heavy chain may respectively comprise the amino acid sequences of SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36. In this embodiment, SEQ ID NO:34 and 36 represent H-CDR1 and H-CDR3 as determined by Kabat, as defined in SEQ ID NO:19 to 22, respectively, and SEQ ID NO:35 represents H-CDR2 as determined by Chothia, as defined in SEQ ID NO:19 to 22.

[0111] In another embodiment, the heavy chain may include complementary determination regions H-CDR1, H-CDR2, and H-CDR3 as defined by SEQ ID NO:34, 37, and 36, respectively. In this embodiment, SEQ ID NO:34, 37, and 36 represent H-CDR1, H-CDR2, and H-CDR3 as defined by Kabat in SEQ ID NO:19 to 20, respectively.

[0112] In yet another embodiment, the heavy chain may include complementary determination regions H-CDR1, H-CDR2, and H-CDR3 as defined by SEQ ID NO:34, 38, and 36, respectively. In this embodiment, SEQ ID NO:34, 38, and 36 represent H-CDR1, H-CDR2, and H-CDR3 as defined by Kabat in SEQ ID NO:21 to 22, respectively.

[0113] In another embodiment described herein, the variable region of the heavy chain of the recombinant antibody of the present 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.

[0114] In another embodiment described herein, the recombinant antibody comprises a heavy chain containing an amino acid sequence or a fragment thereof 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. 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 with 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.

[0115] C. Affinity Label

[0116] The recombinant antibody according to the invention may optionally contain an affinity tag. The affinity tag can be used for purification. Exemplary affinity tags include multihistidine, glutathione S-transferase (GST), chitosan-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, biotinylate carboxyl carrier protein, green fluorescent protein tag, HaloTag, Nus tag, and thioredoxin tag; however, 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. Exemplary affinity tags are multihistidine tags, which typically comprise an amino acid sequence containing between 4 and 10 consecutive histidines. Preferred affinity tags are multihistidine tags containing between 6 and 10 consecutive histidines. Exemplary affinity tags correspond to SEQ ID NO:23, SEQ ID NO:24, or SEQ ID NO:25.

[0117] The recombinant antibodies of the present invention may optionally contain an affinity tag, and may optionally be purified using said affinity tag. Several methods for purifying recombinant antibodies are available in the prior art, and these methods are well known to those skilled in the art. Exemplary purification methods for recombinant antibodies, with or without affinity tags, include immobilized metal affinity chromatography (IMAC), protein A / G affinity, exchange chromatography (IEX or IEC), hydrophobic interaction chromatography (HIC), and / or additional use of tags and affinity chromatography techniques other than IMAC or protein A / G. The purification methods and tags used should not be considered limiting.

[0118] In a preferred embodiment, the recombinant antibody of the present invention further comprises an affinity tag. The affinity tag may be selected from the group consisting of SEQ ID NO:23, SEQ ID NO:24, or SEQ ID NO:25.

[0119] D. Exemplary recombinant antibodies

[0120] In one embodiment described herein, the recombinant antibody comprises the amino acid sequence of SEQ ID NO:17.

[0121] 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.

[0122] In the preferred embodiments 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.

[0123] 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.

[0124] 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 with the amino acid sequence composed 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 with the amino acid sequence composed of SEQ ID NO:12.

[0125] In one embodiment, the recombinant antibody of the present invention is bound to a solid support.

[0126] In another embodiment, the binding affinity of the recombinant antibody of the present invention to fragment DD is at least 3 times stronger than the binding affinity of the recombinant antibody to fragment D.

[0127] II. Nucleic Acids, Cloned Cells, and Expression Cells

[0128] This invention also relates to nucleic acids comprising a nucleotide sequence encoding a recombinant antibody described herein. The nucleic acid may be DNA or RNA. The DNA comprising the nucleotide sequence encoding the recombinant antibody described herein typically contains a promoter operatively linked to that nucleotide sequence. The promoter is preferably capable of driving constitutive or inducible expression of the nucleotide sequence in cells of interest. The nucleic acid may also contain selectivity markers that can be used to select cells containing the nucleic acid of interest. Useful selectivity markers are well known to those skilled in the art. The exact nucleotide sequence of the nucleic acid is not particularly limited, as long as the nucleotide sequence encodes the recombinant antibody described herein. Codons may be selected, for example, to match the codon preferences of the expression cells of interest (e.g., mammalian cells, such as human cells) and / or for convenience during cloning. The DNA may be a plasmid, for example, which may contain an origin of replication (e.g., for replication of the plasmid in prokaryotic cells).

[0129] In one embodiment described herein, the nucleic acid comprises a nucleotide sequence encoding the recombinant antibody of the present invention, a promoter operatively linked to the nucleotide sequence, and a selectability marker.

[0130] Several aspects of the invention also relate to cells containing nucleic acids comprising nucleotide sequences encoding recombinant antibodies as described herein. The cells can be expression cells or cloned cells. Nucleic acids are typically cloned in *Escherichia coli* (E. coli), although other cloned cells can be used.

[0131] If the cell is an expression cell, the nucleic acid is optionally a chromosomal nucleic acid, i.e., in which the nucleotide sequence is integrated into the chromosome, although the nucleic acid may exist in the expression cell, for example, as extrachromosomal DNA or as a vector (such as a plasmid, granule, bacteriophage, etc.). The form of the vector should not be considered limiting.

[0132] In one embodiment described herein, the cell is typically an expression cell. The nature of the expression cell is not particularly limited. Mammalian expression cells may allow for favorable folding, post-translational modification, and / or secretion of recombinant or oligomeric recombinant antibodies, although other eukaryotic or prokaryotic cells may also be used as expression cells. Exemplary expression cells include TunaCHO, ExpiCHO, Expi293, BHK, NSO, Sp2 / O, COS, C127, HEK, HT-1080, PER.C6, HeLa, and Jurkat cells. The cell may also be selected for vector integration (more preferably for plasmid DNA integration).

[0133] The recombinant antibodies of this invention can be generated by an appropriate transfection strategy into mammalian cells using a nucleic acid containing a nucleotide sequence encoding the recombinant antibody. Those skilled in the art are aware of various techniques (liposome transfection, electroporation, etc.) that can be used to transfect nucleic acids into selected cell lines. Therefore, the choice of mammalian cell lines and transfection strategies should not be considered limiting. Cell lines can be further selected for plasmid DNA integration.

[0134] In a preferred embodiment described herein, the cells contain the recombinant antibody of the present invention.

[0135] III. Compositions and methods related to the determination

[0136] Several aspects of the present invention relate to compositions comprising recombinant antibodies as described herein.

[0137] In one embodiment described herein, the composition comprises the recombinant antibody and solid support of the present invention.

[0138] In other embodiments, the composition comprises the recombinant antibody of the present 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-covalent binding" refers to specific binding, such as specific binding between an antibody and its antigen, between a ligand and its receptor, or between an enzyme and its substrate, exemplified for example by the interaction between streptavidin-binding proteins and streptavidin, or between an antibody and its antigen.

[0139] In other embodiments, the composition comprises the recombinant antibody of the present invention and a solid support, wherein the recombinant antibody binds directly or indirectly to the solid support. As used herein, the term “direct” binding refers to the direct conjugation of a molecule to a solid support, such as the gold-thiol interaction that binds the cysteine ​​thiol of the recombinant antibody to a gold surface. As used herein, the term “indirect” binding includes the specific binding of the recombinant antibody to another molecule that binds directly to the solid support; for example, the recombinant antibody may bind to an antibody that binds directly to the solid support, thereby indirectly binding the recombinant antibody to the solid support. The term “indirect” binding is not related to the number of molecules between the recombinant antibody and the solid support, provided that (a) each interaction between the daisy chains of the molecules is a specific or covalent interaction, and (b) the terminal molecules of the daisy chains bind directly to the solid support.

[0140] Solid supports can include particles, beads, membranes, surfaces, peptide chips, microtiter plates, or the solid phase of a chromatographic column. For example, a solid support can be latex beads.

[0141] The composition may comprise more than one bead or particle, each of said more than one bead or particle being directly or indirectly bound to at least one recombinant antibody as described herein. The composition may comprise more than one bead or particle, each of said more than one bead or particle being covalently or non-covalently bound to at least one recombinant antibody as described herein.

[0142] Several aspects of the implementation scheme relate to kits for detecting the presence of D-dimer, fragment DD, and / or fragment D in a sample, said kits comprising recombinant antibodies and solid supports or compositions as described herein.

[0143] The compositions and kits described herein can be used for assays or for compositions generated during assays. Several aspects of the invention relate to diagnostic medical devices comprising compositions as described herein.

[0144] Several aspects of this invention relate to assays. Assays can be used to measure the relative binding affinity (e.g., relative to one or more control samples or standards) of the recombinant antibody of the invention with D-dimer, fragment DD, and / or fragment D in a sample. Assays can also be used to measure the relative binding affinity (e.g., relative to one or more control samples or standards) of the recombinant antibody of the invention with any fibrin (fibrinogen) degradation product.

[0145] Assays are typically characterized by a solid support that allows measurement (such as by turbidimetry, nephelometry, UV / Vis / IR spectroscopy (e.g., absorption, emission), fluorescence or phosphorescence spectroscopy, or surface plasmon resonance) or facilitates the separation of components directly or indirectly bound to a solid support from components not directly or indirectly bound to a solid support, or both. For example, an assay may include a composition comprising particles or beads that allow measurement by turbidimetry or nephelometry (e.g., in coagulation assays) and / or facilitate the mechanical separation of components directly or indirectly bound to the particles or beads.

[0146] Other exemplary assays that may include the recombinant antibodies or compositions of the present invention include, but are not limited to, ELISA, viscoelasticity tests such as Sonoclot, gel technology, fluorescence assays, and other point-of-care tests using any of these technologies.

[0147] Several aspects of the present invention relate to methods for detecting the presence of D-dimer, fragment DD, and / or fragment D in a sample.

[0148] In one embodiment described herein, a method for detecting the presence of D-dimer, fragment DD, and / or fragment D in a sample includes the following steps:

[0149] a) Contacting the sample with at least one recombinant antibody described herein under conditions sufficient to form an antibody / antigen complex and for a sustained period of time, and

[0150] b) Detect the antibody / antigen complex.

[0151] Several aspects of the present invention relate to methods for measuring the binding affinity of D-dimers, fragment DD, and / or fragment D in a sample.

[0152] In one embodiment described herein, a method for measuring the binding affinity of D-dimer, fragment DD, and / or fragment D in a sample includes the following steps:

[0153] a) Contacting the sample with at least one recombinant antibody described herein under conditions sufficient to form an antibody / antigen complex and for a sustained period of time, and

[0154] b) Determine the binding affinity between the antibody and D-dimer, fragment DD, and / or fragment D in the sample.

[0155] Several aspects of the present invention relate to methods for measuring the concentrations of D-dimer, fragment DD, and / or fragment D in a sample.

[0156] In one embodiment described herein, a method for measuring the amount of D-dimer, fragment DD, and / or fragment D in a sample includes the following steps:

[0157] a) Contacting the sample with at least one recombinant antibody described herein under conditions sufficient to form an antibody / antigen complex and for a sustained period of time, and

[0158] b) Measure the concentration of D-dimer, fragment DD and / or fragment D in the sample.

[0159] example

[0160] Example 1: Immunization strategy and selection of monoclonal antibodies

[0161] Female Balb / c mice were primed subcutaneously with 50 μg of purified D-dimer in 0.2 mL of saline mixed with 0.1 M Freund's complete adjuvant. On days 2 and 4 prior to fusion, mice were boosted intraperitoneally 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 fragments D and D-dimer from fibrinogen or non-crosslinked fibrin. Clones producing antibodies specific to D-dimer, but not fibrinogen, were used to generate ascites in the original primated Balb / c mice. The IgG fraction of the monoclonal antibody was purified from the ascites by affinity chromatography on a protein A agarose gel. 11,12 .

[0162] Example 2: De novo MS / MS sequencing of selected monoclonal antibodies

[0163] The monoclonal antibody selected in Example 1 was further subjected to de novo MS / MS sequencing.

[0164] In short, the purified antibodies are first confirmed by whole-chain mass spectrometry. Each mAb is then reduced to separate the heavy chain (HC) and light chain (LC), which are individually digested by a series of enzymes (trypsin, chymotrypsin, etc.). Bottom-up MS / MS data are collected, providing information about the digested peptides and about b and y ions, further identifying individual amino acids within the peptides for assembly. The information is then processed to determine the amino acid sequences of the heavy and light chains of the monoclonal antibody.

[0165] The sequences of a total of 16 antibodies were determined. The corresponding sequence of each antibody is disclosed below.

[0166] #1 SEQ ID NO:17 SEQ ID NO:1 #2 SEQ ID NO:17 SEQ ID NO:2 #3 SEQ ID NO:17 SEQ ID NO:3 #4 SEQ ID NO:17 SEQ ID NO:4 #5 SEQ ID NO:17 SEQ ID NO:5 #6 SEQ ID NO:17 SEQ ID NO:6 #7 SEQ ID NO:17 SEQ ID NO:7 #8 SEQ ID NO:17 SEQ ID NO:8 #9 SEQ ID NO:17 SEQ ID NO:9 #10 SEQ ID NO:17 SEQ ID NO:10 #11 SEQ ID NO:17 SEQ ID NO:11 #12 SEQ ID NO:17 SEQ ID NO:12 #13 SEQ ID NO:17 SEQ ID NO:13 #14 SEQ ID NO:17 SEQ ID NO:14 #15 SEQ ID NO:17 SEQ ID NO:15 #16 SEQ ID NO:17 SEQ ID NO:16

[0167] Example 3: Transient expression and purification of recombinant antibodies

[0168] Single transfection with a polycistronic vector containing both HC and LC, or double transfection with a vector containing either HC or LC, can be performed to generate the desired antibody or antigen-binding fragment. Tricistronic vectors utilizing the internal ribosome entry site (IRES) sequence from encephalomyocarditis virus (ECMV) can also be used.

[0169] A group of vectors with only LC, only HC, LC-IRES-HC, and HC-IRES-LC were compared, and the vector with the highest desired antibody production was selected. The highest antibody production was LC-IRES-HC. Furthermore, more than one signal peptide was used to produce the desired antibody (sequence listing). For tricistronic vectors, the IL2 signal peptide was used for both LC and HC.

[0170] During stable cell line development, ExpiCHO cells were transfected with a tricistronic vector, and selection was performed using both G418 and neomycin. Two rounds of limiting dilution clones were used to isolate clonal cell lines. The second round of limiting dilution clones were imaged to support clonality. During stable cell line development, cell viability and desired antibody productivity were assessed.

[0171] Purification of the recombinant antibodies of interest was performed via protein A or protein G purification for full-length antibodies. For antibodies lacking the Fc region (Fab and F(ab')2 forms), a C-terminal fused His tag was added to HC, allowing for IMAC purification and subsequent SEC purification.

[0172] Example 4: Characterization of the generated antibodies

[0173] SDS-PAGE

[0174] The production of each recombinant antibody was verified by SDS-PAGE under both reducing and non-reducing conditions (except for antibody #4 (protein-free) and antibody #6 (characterized by CE-SDS)). Figure 2 ).

[0175] In all cases, intact antibodies were observed under non-reducing conditions, and the corresponding HC and LC were observed under reducing conditions. Low production was observed for antibodies #1-3, while no production was shown for antibody #4.

[0176] SEC-MALS

[0177] To evaluate the assembly and aggregation of the anti-D-dimer recombinant antibody of the present invention, size exclusion multi-angle light scattering (SEC-MALS) was used. SEC-MALS provided molecular weight and characterized polydispersity (Mw / Mn). During antibody production, the purified protein was found to be monodisperse with a 1%–2% variation. Figure 3 Instance data for antibody #9 and antibody #12 are provided.

[0178] For anti-D-dimer antibodies, NHS and EDAC chemistry were used to conjugate the protein to latex beads.

[0179] Biological layer interferometry (BLI)

[0180] 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 further purified by SEC (Superdex 200 Increase 10 / 300 GL) if visible contaminants were present. Several of the fragments mentioned were generated in-house because the protein amounts were too small for SEC purification from contaminants, or they were not commercially available. To generate 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 plasminogen ether (HTI). The temperature was set to 37 °C with shaking at 800 RPM for 7 minutes. Immediately add 1000 KIU / mL aprotinin (Sigma) and incubate the sample at -20°C until ready for injection onto an SEC column (Superdex 200Increase 10 / 300GL). Maintain the collected fractions at low temperature throughout the purification process. Then, run the selected peak fractions on SDS-PAGE and pool and concentrate according to the MW corresponding to fibrinogen fragments X, Y, and D. The purified human plasma protein SDS-PAGE gel is shown on... Figure 4 As can be seen in the text.

[0181] FDP was prepared and run on 4%–20% TGX standard unstained gels to check quality and purity, followed by characterization studies.

[0182] After purification, a series of biolayer interferometry (BLI) studies were conducted to determine the binding affinity of the recombinant antibody of the present invention.

[0183] The binding affinity of recombinant antibody #12 to fragment DD from fibrinogen, fragment D from fibrinogen, fragment E, and fibrinogen was tested (at room temperature and room pressure). Figure 5 The results showed binding to both fragment DD from fibrinogen and fragment D from fibrinogen; however, the binding to fragment DD was two orders of magnitude stronger than the binding to fragment D. These binding properties, particularly the difference in binding strength between antibody #12 and fragments DD and D, have never been reported with hybridoma monoclonal antibodies known in the art. 12 .

[0184] Example 5: Determination of latex bead aggregation.

[0185] Turbidimetric measurements were performed to evaluate the function of the recombinant antibody of the present invention conjugated with latex beads through standard procedures (such as EDAC and / or NHS chemistry).

[0186] In all cases, a linear correlation was found between the known D-dimer assay using HemosIL DDHS500 and the assay developed using antibodies #6 and #5, indicating that D-dimer in plasma is readily detectable using the antibodies of this invention during POC assays. Results obtained with antibodies #6 and #5 ( Figure 6 B and Figure 6 C) and known anti-D-dimers in the prior art ( Figure 6 A) Comparable. However, F(ab')2 showed a better correlation for pepsin digestion of IgG2a#5. Figure 6 D).

[0187] exist Figure 7 In the study, a linear agreement was observed between antibody #12 and DDHS500 ACL. Figure 7 A), and on the Q SMART platform, antibody #12 showed a 1:1 consistency with existing anti-D-dimer antibodies.

[0188] Therefore, the present invention provides recombinant anti-D-dimer antibodies whose function has been evaluated by both conventional biophysical assays (BLI) and assays mounted on latex. The results surprisingly show that these anti-D-dimer antibodies differ from antibodies known in the art due to their specificity, their binding strength to fragments DD and D, and the fact that they can be readily produced in mammalian cells, thus offering several advantages for the development of latex assays compared to antibodies derived from hybridoma cell lines, as previously discussed.

[0189] Example 6: Epitope Binning Study

[0190] Using a BLI Octet Red96e system from Sartorius, antibody #12 was compared with other commercially available anti-D-dimer antibodies in binning and binding studies against purified fibrin and fibrinogen fragments. Experiments were performed at room temperature and chamber pressure. Binning studies were performed by loading antibody #12, binding it to fragment DD, and then introducing other commercially available mAbs. Responses were measured as nm shifts in the interference mode and were proportional to the number of molecules bound to the biosensor surface. In the presence of antibody #12, the selected mAbs showed binding to fragment DD (see Table 1, in nm shifts after addition), indicating that these antibodies recognize different epitopes than antibody #12.

[0191]

[0192] Table 1

[0193] Binding studies of the same set of mAbs with fibrinogen, D-dimer, and purified fibrin and fibrinogen fragments were performed using a BLI Octet Red96e system from Sartorius (see Table 2). KD was measured by loading biotinylated antibody samples onto the streptavidin BLI tip and introducing a series of dilutions of the prepared fragments. KD: ++++(10 -11 -10 -12 M); +++(10 -10 M); ++(10 -9 M); +(≥10 -8 The experiment was conducted at room temperature and room pressure.

[0194] Grifols antibody #12 showed no binding to fibrinogen or fragment E derived from fibrinogen, weak binding to fibrinogen fragments X, Y, and D; moderate binding to fragments X and D; and sub-nanomolar 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 fragment D; and compared to antibody #12, DD225 showed even weaker binding to fibrinogen fragments X and D.

[0195]

[0196] Table 2

[0197] sequence list

[0198] The sequences associated with the recombinant antibodies of this invention are summarized in Table 3 below.

[0199]

[0200]

[0201]

[0202]

[0203] References

[0204] 1. Adam, SS, Key, NS & Greenberg, CSD-Dimer antigen: Current concepts and future prospects. Blood 113, 2878–2887 (2009).

[0205] 2. Thachil, J., Lippi, G. & Favaloro, EJD-Dimer Testing: Laboratory Aspects and Current Issues. Methods Mol. Biol. 1646, 91–104 (2017).

[0206] 3. Weitz, JI, Fredenburgh, JC & Eikelboom, JWA Test in Context: D-Dimer. J. Am. Coll. Cardiol. 70, 2411–2420 (2017).

[0207] 4. Riley, RS, Gilbert, AR, Dalton, JB, Pai, S. & McPherson, RAWidely used types and clinical applications of D-Dimer assay. Lab Med. 47, 90–102 (2016).

[0208] 5. Goodacre, S., Sampson, FC, Sutton, AJ, Mason, S. & Morris, F. Variation in the diagnostic performance of D-Dimer for suspected deep vein thrombosis. QJM98, 513–27 (2005).

[0209] 6.Lippi,G.,Ippolito,L.,Tondelli,M.T.&Favaloro,E.J.Interference fromheterophilic antibodies in D-Dimer assessment.A case report.BloodCoagul.Fibrinolysis 25,277–9(2014).

[0210] 7.Robier,C.,Edler,E.,Klescher,D.&Neubauer,M.False-positive D-Dimerresult in a latex-enhanced immunoassay caused by interfering human anti-mouseantibodies.Clin.Chem.Lab.Med.52,e253–e255(2014).

[0211] 8.Coco-Martin,J.M.,Oberink,J.W.,Brunink,F.,Van der Velden-de Groot,T.A.&Beuvery,E.C.Instability of a hybridoma cell line in a homogeneouscontinuous perfusion culture system.Hybridoma 11,653–65(1992).

[0212] 9.Castillo,F.J.et al.Hybridoma stability.Dev.Biol.Stand.83,55–64(1994).

[0213] 10.Gupta,S.K.&Shukla,P.Glycosylation control technologies forrecombinant therapeutic proteins.Appl.Microbiol.Biotechnol.102,10457–10468(2018).

[0214] 11. Holvoet, P., Lijnen, HR & Collen, DA monoclonal antibody preventing binding of tissue-type plasminogen activator to fibrin: useful to monitor fibrinogen breakdown during t-PA infusion. Blood 67, 1482–7 (1986).

[0215] 12. P Holveot, JM Stassen, Y Hashimoto, D Spriggs, P Devos, DCBindingproperties of monoclonal antibodies against human fragment D-dim.... pdf. Thombosis Haemost. 61, 307–313 (1989). sequence list <110> Hofmeister Roche Ltd. <120> Anti-D-dimer recombinant antibodies, their methods and uses <130> X-23318 <150> US 62 / 979253 <151> 2020-02-20 <160> 38 <170> PatentIn version 3.5 <210> 1 <211> 441 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptides <400> 1 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Val Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Gln Thr Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Trp Ile His Trp Leu Lys Gln Gly Pro His Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Leu Asp Pro Asp Asp Ser Glu Thr His Tyr Leu Glu Lys Phe 50 55 60 Gln Gly Lys Glu Leu Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Asn Trp Asp Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Ala Ser Ala Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro 115 120 125 Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly 130 135 140 Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn 145 150 155 160 Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr 180 185 190 Trp Pro Ser Gln Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser 195 200 205 Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro 210 215 220 Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro 225 230 235 240 Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys 245 250 255 Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp 260 265 270 Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu 275 280 285 Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met 290 295 300 His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser 305 310 315 320 Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly 325 330 335 Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln 340 345 350 Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe 355 360 365 Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu 370 375 380 Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe 385 390 395 400 Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn 405 410 415 Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr 420 425 430 Glu Lys Ser Leu Ser His Ser Pro Gly 435 440 <210> 2 <211> 441 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 2 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Val Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Gln Thr Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Trp Ile His Trp Leu Lys Gln Gly Pro His Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Leu Asp Pro Asp Asp Ser Glu Thr His Tyr Leu Glu Lys Phe 50 55 60 Gln Gly Lys Glu Leu Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Asn Trp Asp Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Ala Ser Ala Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro 115 120 125 Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly 130 135 140 Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn 145 150 155 160 Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr 180 185 190 Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser 195 200 205 Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro 210 215 220 Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro 225 230 235 240 Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys 245 250 255 Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp 260 265 270 Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu 275 280 285 Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met 290 295 300 His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser 305 310 315 320 Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly 325 330 335 Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln 340 345 350 Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe 355 360 365 Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu 370 375 380 Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe 385 390 395 400 Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn 405 410 415 Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr 420 425 430 Glu Lys Ser Leu Ser His Ser Pro Gly 435 440 <210> 3 <211> 441 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 3 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Val Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Gln Thr Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Trp Ile His Trp Leu Lys Gln Gly Pro His Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Leu Asp Pro Asp Asp Ser Glu Thr His Tyr Leu Glu Lys Phe 50 55 60 Gln Gly Lys Glu Leu Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Asn Trp Asp Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Ala Ser Ala Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro 115 120 125 Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly 130 135 140 Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn 145 150 155 160 Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr 180 185 190 Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser 195 200 205 Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro 210 215 220 Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro 225 230 235 240 Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys 245 250 255 Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp 260 265 270 Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu 275 280 285 Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met 290 295 300 His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser 305 310 315 320 Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly 325 330 335 Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln 340 345 350 Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe 355 360 365 Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu 370 375 380 Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe 385 390 395 400 Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn 405 410 415 Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr 420 425 430 Glu Lys Ser Leu Ser His Ser Pro Gly 435 440 <210> 4 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 4 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Val Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Trp Ile His Trp Leu Lys Gln Gly Pro His Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Leu Asp Pro Asp Asp Ser Glu Thr His Tyr Leu Glu Lys Phe 50 55 60 Gln Gly Lys Glu Leu Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Asn Trp Asp Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ala Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro 115 120 125 Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu Gly 130 135 140 Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr Trp Asn 145 150 155 160 Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser Ser Thr 180 185 190 Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala Ser Ser 195 200 205 Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr Ile Lys Pro 210 215 220 Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu 245 250 255 Ser Pro Ile Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro 260 265 270 Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala 275 280 285 Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val 290 295 300 Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe 305 310 315 320 Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr 325 330 335 Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr Val Leu 340 345 350 Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu Thr Cys 355 360 365 Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn 370 375 380 Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys Lys 405 410 415 Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu Gly 420 425 430 Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly 435 440 445 <210> 5 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 5 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Val Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Trp Ile His Trp Leu Lys Gln Gly Pro His Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro Asp Asp Ser Glu Thr His Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Leu Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Asn Trp Asp Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ala Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro 115 120 125 Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu Gly 130 135 140 Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr Trp Asn 145 150 155 160 Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser Ser Thr 180 185 190 Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala Ser Ser 195 200 205 Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr Ile Lys Pro 210 215 220 Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu 245 250 255 Ser Pro Ile Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro 260 265 270 Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala 275 280 285 Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val 290 295 300 Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe 305 310 315 320 Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr 325 330 335 Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr Val Leu 340 345 350 Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu Thr Cys 355 360 365 Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn 370 375 380 Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys Lys 405 410 415 Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu Gly 420 425 430 Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly 435 440 445 <210> 6 <211> 441 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 6 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Val Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Trp Ile His Trp Leu Lys Gln Gly Pro His Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro Asp Asp Ser Glu Thr His Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Leu Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Asn Trp Asp Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ala Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro 115 120 125 Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly 130 135 140 Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn 145 150 155 160 Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr 180 185 190 Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser 195 200 205 Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro 210 215 220 Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro 225 230 235 240 Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys 245 250 255 Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp 260 265 270 Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu 275 280 285 Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met 290 295 300 His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser 305 310 315 320 Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly 325 330 335 Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln 340 345 350 Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe 355 360 365 Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu 370 375 380 Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe 385 390 395 400 Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn 405 410 415 Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr 420 425 430 Glu Lys Ser Leu Ser His Ser Pro Gly 435 440 <210> 7 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 7 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Val Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Gln Thr Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Trp Ile His Trp Leu Lys Gln Gly Pro His Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro Asp Asp Ser Glu Thr His Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Leu Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Asn Trp Asp Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Ala Ser Ala Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro 115 120 125 Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu Gly 130 135 140 Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr Trp Asn 145 150 155 160 Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser Ser Thr 180 185 190 Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala Ser Ser 195 200 205 Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr Ile Lys Pro 210 215 220 Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu 245 250 255 Ser Pro Ile Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro 260 265 270 Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala 275 280 285 Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val 290 295 300 Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe 305 310 315 320 Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr 325 330 335 Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr Val Leu 340 345 350 Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu Thr Cys 355 360 365 Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn 370 375 380 Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys Lys 405 410 415 Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu Gly 420 425 430 Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly 435 440 445 <210> 8 <211> 441 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 8 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Val Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Gln Thr Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Trp Ile His Trp Leu Lys Gln Gly Pro His Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro Asp Asp Ser Glu Thr His Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Leu Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Asn Trp Asp Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Ala Ser Ala Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro 115 120 125 Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly 130 135 140 Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn 145 150 155 160 Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr 180 185 190 Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser 195 200 205 Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro 210 215 220 Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro 225 230 235 240 Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys 245 250 255 Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp 260 265 270 Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu 275 280 285 Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met 290 295 300 His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser 305 310 315 320 Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly 325 330 335 Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln 340 345 350 Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe 355 360 365 Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu 370 375 380 Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe 385 390 395 400 Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn 405 410 415 Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr 420 425 430 Glu Lys Ser Leu Ser His Ser Pro Gly 435 440 <210> 9 <211> 223 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 9 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Val Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Trp Ile His Trp Leu Lys Gln Gly Pro His Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro Asp Asp Ser Glu Thr His Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Leu Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Asn Trp Asp Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ala Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro 115 120 125 Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu Gly 130 135 140 Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr Trp Asn 145 150 155 160 Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser Ser Thr 180 185 190 Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala Ser Ser 195 200 205 Thr Lys Val Asp Lys Lys Ile His His His His His His His His 210 215 220 <210> 10 <211> 231 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 10 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Val Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Trp Ile His Trp Leu Lys Gln Gly Pro His Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro Asp Asp Ser Glu Thr His Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Leu Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Asn Trp Asp Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ala Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro 115 120 125 Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu Gly 130 135 140 Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr Trp Asn 145 150 155 160 Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser Ser Thr 180 185 190 Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala Ser Ser 195 200 205 Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr Ile Lys His 210 215 220 His His His His His His His 225 230 <210> 11 <211> 243 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 11 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Val Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Trp Ile His Trp Leu Lys Gln Gly Pro His Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro Asp Asp Ser Glu Thr His Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Leu Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Asn Trp Asp Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ala Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro 115 120 125 Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu Gly 130 135 140 Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr Trp Asn 145 150 155 160 Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser Ser Thr 180 185 190 Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala Ser Ser 195 200 205 Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr Ile Lys Pro 210 215 220 Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu His His His His His 225 230 235 240 His His His <210> 12 <211> 250 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 12 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Val Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Trp Ile His Trp Leu Lys Gln Gly Pro His Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro Asp Asp Ser Glu Thr His Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Leu Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Asn Trp Asp Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ala Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro 115 120 125 Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu Gly 130 135 140 Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr Trp Asn 145 150 155 160 Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser Ser Thr 180 185 190 Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala Ser Ser 195 200 205 Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr Ile Lys Pro 210 215 220 Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe His His His His His His His His 245 250 <210> 13 <211> 264 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 13 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Val Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Trp Ile His Trp Leu Lys Gln Gly Pro His Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro Asp Asp Ser Glu Thr His Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Leu Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Asn Trp Asp Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ala Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro 115 120 125 Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu Gly 130 135 140 Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr Trp Asn 145 150 155 160 Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser Ser Thr 180 185 190 Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala Ser Ser 195 200 205 Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr Ile Lys Pro 210 215 220 Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu 245 250 255 His His His His His His His His 260 <210> 14 <211> 222 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 14 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Val Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Trp Ile His Trp Leu Lys Gln Gly Pro His Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro Asp Asp Ser Glu Thr His Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Leu Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Asn Trp Asp Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ala Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro 115 120 125 Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly 130 135 140 Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn 145 150 155 160 Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr 180 185 190 Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser 195 200 205 Thr Lys Val Asp Lys Lys Ile His His His His His His Lys 210 215 220 <210> 15 <211> 239 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 15 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Val Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Trp Ile His Trp Leu Lys Gln Gly Pro His Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro Asp Asp Ser Glu Thr His Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Leu Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Asn Trp Asp Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ala Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro 115 120 125 Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly 130 135 140 Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn 145 150 155 160 Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr 180 185 190 Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser 195 200 205 Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro 210 215 220 Cys Ile Cys Thr Val Pro Glu Val His His His His His His Lys 225 230 235 <210> 16 <211> 243 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 16 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Val Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Trp Ile His Trp Leu Lys Gln Gly Pro His Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro Asp Asp Ser Glu Thr His Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Leu Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Asn Trp Asp Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ala Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro 115 120 125 Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly 130 135 140 Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn 145 150 155 160 Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr 180 185 190 Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser 195 200 205 Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro 210 215 220 Cys Ile Cys Thr Val Pro Glu Val His His His His His His Gly Ser 225 230 235 240 Gly Gly Lys <210> 17 <211> 220 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 17 Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ala Met Ser Val Gly 1 5 10 15 Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Ser Ser Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Ser Pro Lys Leu Leu Val Tyr Phe Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ile Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln 85 90 95 His Tyr Arg Thr Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 100 105 110 Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser 115 120 125 Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn 130 135 140 Phe Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu 145 150 155 160 Arg Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp 165 170 175 Ser Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr 180 185 190 Glu Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr 195 200 205 Ser Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys 210 215 220 <210> 18 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 18 Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ala Met Ser Val Gly 1 5 10 15 Gln Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Ser Ser Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Ser Pro Lys Leu Leu Val Tyr Phe Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ile Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln 85 90 95 His Tyr Arg Thr Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 19 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 19 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Val Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Trp Ile His Trp Leu Lys Gln Gly Pro His Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro Asp Asp Ser Glu Thr His Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Leu Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Asn Trp Asp Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ala 115 <210> 20 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 20 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Val Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Gln Thr Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Trp Ile His Trp Leu Lys Gln Gly Pro His Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro Asp Asp Ser Glu Thr His Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Leu Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Asn Trp Asp Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Ala Ser Ala 115 <210> 21 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 21 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Val Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Trp Ile His Trp Leu Lys Gln Gly Pro His Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Leu Asp Pro Asp Asp Ser Glu Thr His Tyr Leu Glu Lys Phe 50 55 60 Gln Gly Lys Glu Leu Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Asn Trp Asp Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ala 115 <210> 22 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 22 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Val Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Gln Thr Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Trp Ile His Trp Leu Lys Gln Gly Pro His Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Leu Asp Pro Asp Asp Ser Glu Thr His Tyr Leu Glu Lys Phe 50 55 60 Gln Gly Lys Glu Leu Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Asn Trp Asp Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Ala Ser Ala 115 <210> twenty three <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptides <400> twenty three His His His His His His 1 5 <210> twenty four <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptides <400> twenty four His His His His His His His Lys 1 5 <210> 25 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptides <400> 25 His His His His His Gly Ser Gly Gly Lys 1 5 10 <210> 26 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptides <400> 26 Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly 1 5 10 15 Ala Val Phe Val Ser Pro Ser Ala Ser 20 25 <210> 27 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptides <400> 27 Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu 1 5 10 15 Val Thr Asn Ser 20 <210> 28 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptides <400> 28 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly 20 <210> 29 <211> twenty four <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptides <400> 29 Met Asp Pro Lys Gly Ser Leu Ser Trp Arg Ile Leu Leu Phe Leu Ser 1 5 10 15 Leu Ala Phe Glu Leu Ser Tyr Gly 20 <210> 30 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptides <400> 30 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly 20 <210> 31 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptides <400> 31 Lys Ser Ser Gln Ser Leu Leu Asn Ser Ser Ser Gln Lys Asn Tyr Leu 1 5 10 15 Ala <210> 32 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptides <400> 32 Phe Ala Ser Thr Arg Glu Ser 1 5 <210> 33 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptides <400> 33 Gln Gln His Tyr Arg Thr Pro Trp Thr 1 5 <210> 34 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptides <400> 34 Ser Tyr Trp Ile His 1 5 <210> 35 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptides <400> 35 Asp Pro Asp Asp Ser Glu 1 5 <210> 36 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptides <400> 36 Thr Asn Trp Asp Ala Trp Phe Ala Tyr 1 5 <210> 37 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptides <400> 37 Arg Ile Asp Pro Asp Asp Ser Glu Thr His Tyr Asn Gln Lys Phe Lys 1 5 10 15 Asp <210> 38 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptides <400> 38 Arg Leu Asp Pro Asp Asp Ser Glu Thr His Tyr Leu Glu Lys Phe Gln 1 5 10 15 Gly

Claims

1. A recombinant antibody against D-dimer, characterized in that it specifically binds to fibrin and fibrinogen degradation product (FDP) D-dimer, fragment DD, and fragment D, and does not bind to fragment E and fibrinogen, wherein the recombinant antibody comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprising: CDR1 consisting of the amino acid sequence of SEQ ID NO: 31, CDR2 consisting of the amino acid sequence of SEQ ID NO: 32, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 33, as defined by Kabat; the heavy chain variable region comprising: CDR1 consisting of the amino acid sequence of SEQ ID NO: 34, CDR2 consisting of the amino acid sequence of SEQ ID NO: 37, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 36, as defined by Kabat.

2. The recombinant antibody according to claim 1, further comprising: The light chain variable region comprises an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 18, and The heavy chain variable region contains an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 19 or 20.

3. The recombinant antibody according to claim 1 further comprises: The light chain variable region comprises the amino acid sequence of SEQ ID NO: 18, and The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 19 or 20.

4. The recombinant antibody according to claim 1, wherein the recombinant antibody is a monoclonal antibody or an antibody fragment.

5. The recombinant antibody according to claim 4, wherein the antibody fragment is selected from variable fragments (Fv), single-chain Fv (scFv), single-chain antibodies, Fab fragments, F(ab')2 fragments, Fab' fragments, disulfide-linked Fv (dsFv), chemically conjugated Fv (ccFv), and biantibodies.

6. The recombinant antibody according to claim 4, wherein the antibody fragment is an antigen-binding fragment selected from the Fab fragment and the F(ab')2 fragment.

7. The recombinant antibody according to claim 1, wherein the recombinant antibody comprises a constant region of mouse IgG1 or mouse IgG2a.

8. The recombinant antibody according to claim 1 further comprises an affinity tag.

9. The recombinant antibody according to claim 8, 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.

10. The recombinant antibody according to claim 1, wherein the light chain of the recombinant antibody comprises the amino acid sequence of SEQ ID NO:

17.

11. The recombinant antibody according to claim 1, wherein the heavy chain of the recombinant antibody comprises the amino acid sequence of SEQ ID NO:

12.

12. The recombinant antibody according to claim 1, 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 SEQ ID NO:

12.

13. The recombinant antibody according to claim 1, wherein the binding affinity of the recombinant antibody to fragment DD is at least 3 times stronger than the binding affinity of the recombinant antibody to fragment D, or The binding affinity of the recombinant antibody to fragment DD is at least one order of magnitude stronger than the binding affinity of the recombinant antibody to fragment D.

14. A cell comprising a recombinant antibody according to any one of the preceding claims.

15. A nucleic acid encoding a nucleotide sequence of a recombinant antibody according to any one of claims 1 to 13.

16. A vector comprising the nucleic acid according to claim 15.

17. A cell comprising the nucleic acid of claim 15.

18. A composition comprising a recombinant antibody according to any one of claims 1 to 13 and a solid support, wherein the recombinant antibody is covalently or non-covalently bound to the solid support.

19. The composition of claim 18, wherein the solid support comprises a solid phase of particles, beads, membranes, surfaces, polypeptide chips, microtiter plates, or chromatographic columns.

20. The composition of claim 18, wherein the solid support is latex particles.

21. A kit for detecting the presence of D-dimer, fragment DD, and / or fragment D in a sample, the kit comprising at least one recombinant antibody according to any one of claims 1 to 13 and a solid support, wherein the at least one recombinant antibody is covalently or non-covalently bound to the solid support.

22. Use of at least one recombinant antibody according to any one of claims 1 to 13 in the preparation of a reagent for use in a method for detecting the presence of D-dimer, fragment DD, and / or fragment D in a sample, said method comprising: The sample is contacted with at least one recombinant antibody according to any one of claims 1 to 13 under conditions sufficient to form an antibody / antigen complex and for a sustained period of time. The antibody / antigen complex was detected.

23. Use of at least one recombinant antibody according to any one of claims 1 to 13 in a reagent for preparing a method for measuring the binding affinity of D-dimer, fragment DD, and / or fragment D in a sample, said method comprising: The sample is contacted with at least one recombinant antibody according to any one of claims 1 to 13 under conditions sufficient to form an antibody / antigen complex and for a sustained period of time. Determine the binding affinity between the antibody and the D-dimer, fragment DD, and / or fragment D in the sample.

24. Use of at least one recombinant antibody according to any one of claims 1 to 13 in the preparation of a reagent for use in a method for measuring the concentration of D-dimer, fragment DD, and / or fragment D in a sample, said method comprising: The sample is contacted with at least one recombinant antibody according to any one of claims 1 to 13 under conditions sufficient to form an antibody / antigen complex and for a sustained period of time. The concentrations of D-dimer, fragment DD, and / or fragment D in the sample were measured.

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