Methods for obtaining soluble calprotectin, polypeptides, polypeptide oligomers, uses of the polypeptides or polypeptide oligomers, measurement methods using the polypeptides in analytical assays, polypeptides for use in methods for diagnosing inflammatory diseases, and kits

Abstract

The present invention relates to polypeptides comprising a first strand comprising an amino acid sequence having at least 80% sequence identity with SEQ ID No. 1, a second strand comprising an amino acid sequence having at least 80% sequence identity with SEQ ID No. 2, and a linker connecting the first and second strands. The present invention is further directed to methods of using these polypeptides as calibrators and standards in diagnostic methods.

Description

Technical Field

[0001] The present invention relates to a polypeptide comprising a first chain having an amino acid sequence with at least 80% sequence identity to SEQ ID NO:1, a second chain having an amino acid sequence with at least 80% sequence identity to SEQ ID NO:2, and a linker connecting the first chain and the second chain. Furthermore, the present invention also targets methods of using these polypeptides as calibration substances and reference substances in diagnostic methods.

Background Art

[0002] Calprotectin (CP) is a cytoplasmic protein expressed in various myeloid cell types such as neutrophils, monocytes, and macrophages. In neutrophils, calprotectin is constitutively expressed and can account for approximately 40% of the total cytoplasmic protein, while in epithelial cells and keratinocytes, the expression of calprotectin can be induced.

[0003] Calprotectin consists of two polypeptide chains, Mrp8 (alias: S100A8, calgranulin A) and Mrp14 (alias: S100A9, calgranulin B), and forms a stable dimer. In the presence of about 150 mM Ca 2+ two Mrp8 / Mrp14 heterodimers form a heterotetramer and have an important function in nutritional immunity as a metal ion sequestration complex for divalent cations such as Zn 2+ to starve pathogens during the progression of inflammation (Non-Patent Document 1).

[0004] Since it is released at the site of inflammation, calprotectin is regarded as a warning substance and is frequently used as a biomarker for monitoring the inflammatory process. For example, fecal calprotectin is currently the gold standard for the diagnosis and monitoring of inflammatory bowel diseases (IBD) such as Crohn's disease (CD) and ulcerative colitis (UC) (Non-Patent Document 2).

[0005] Furthermore, serum CP has been validated as a biomarker for monitoring various (chronic) inflammatory diseases, such as rheumatoid arthritis (Non-Patent Literature 3).

[0006] To develop reliable immunoassays for measuring calprotectin, it is crucial to use highly purified calprotectin antigens that are as close as possible to natural (wild-type) calprotectin. This avoids assay problems caused by differences in antibody reactivity between calibrators, standards (controls), and samples. Currently, calprotectin is primarily used as a fecal biomarker to differentiate organic irritable bowel syndrome (IBD) from non-organic irritable bowel syndrome (IBS), but there are no generally accepted and / or effective standards for calibrating blood and fecal analyses.

[0007] Comparisons of methods have shown that there are clear differences in calibration between fecal calprotectin assays from different manufacturers (Non-Patent Literature 4).

[0008] State-of-the-art methods for obtaining pure calprotectin include bacterial expression of Mrp8 and Mrp14 in insoluble inclusion bodies involving a sophisticated refolding process (Non-Patent Literature 5), or purification of calprotectin from granulocytes isolated from human blood (Non-Patent Literature 6).

[0009] However, both methods have several drawbacks. Refolding of Mrp8 and Mrp14 obtained from inclusion bodies may result in homodimers or other refolding products (Non-Patent Literature 7). On the other hand, purification of calprotectin from blood donors has low yields, is laborious and costly, and does not allow for site-directed mutagenesis of calprotectin.

[0010] Therefore, in this field, there is a demand for artificial calprotectins that are highly similar to natural (wild-type) calprotectins and can function as reliable calibrators and standards for immunoassays and other assays. [Prior art documents] [Non-patent literature]

[0011] [Non-Patent Document 1] Zygiel EM, Nolan EM. Transition Metal Sequestration by the Host-Defense Protein Calprotectin (2018). Annu. Rev. Biochem. 87: 621-43 [Non-Patent Document 2] Konikoff MR, Denson LA. Role of Fecal Calprotectin as a Biomarker of Intestinal Inflammation in Inflammatory Bowel Disease (2006). Inflamm. Bowel Dis. 12 (6): 524-34 [Non-Patent Document 3] Austermann J et al. S100 proteins in rheumatic diseases (2018). Nat. Rev. Rheumatol. 14: 528-541; Ometto F et al. Calprotectin in rheumatic diseases (2017). Exp. Biol. Med. 242: 859-873 [Non-Patent Document 4] De Sloovere MW et al. Analytical and Diagnostic Performance of Two Automated Fecal Calprotectin Immunoassays for Detection of Inflammatory Bowel Disease (2017). Clin. Chem. Lab. Med. 55 (9): 1435-1446 [Non-Patent Document 5] Hadley and Nolan, Methods in Molecular Biology, 2019 [Non-Patent Document 6] Nilsen T, Haugen SH. Extraction, isolation, and concentration of calprotectin antigen (S100A8 / S100A9) from granulocytes (2018). Health Sci. Rep. 1 : e35 [Non-Patent Document 7] Vogl et al. Biophysical Characterization of S100A8 and S100A9 in Absence and Presence of Bivalent Cations (2006). Biochim.Biophys. Acta 1763 (11 ): 1298-12306 [Overview of the project] [Problems that the invention aims to solve]

[0012] Therefore, the challenge to be addressed is to provide recombinant calprotectin that retains the important properties of naturally occurring calprotectin. [Means for solving the problem]

[0013] This problem is solved by a method for expressing soluble calprotectin, comprising expressing calprotectin from a vector comprising a first strand containing a nucleotide sequence having at least 80% sequence homology with SEQ ID NO:11, a second strand containing a nucleotide sequence having at least 80% sequence homology with SEQ ID NO:12, and a linker connecting the first strand and the second strand.

[0014] Furthermore, the above problem is also solved by the following polypeptide. a) A first strand having an amino acid sequence having at least 80% sequence identity with SEQ ID NO:1, b) A second strand having an amino acid sequence having at least 80% sequence identity with SEQ ID NO:2, c) A linker connecting the first strand and the second strand, where the linker is a peptide linker containing a protease recognition sequence.

[0015] The present invention also targets a method for measuring S100A8, S100A9, S100A8 / A9, or an oligomer thereof in a sample using the polypeptide of the present invention in an analytical assay, the method comprising the following steps. a) Measuring various amounts of the polypeptide using the analytical assay; b) Establishing a calibration curve using the analytical results obtained in step a); c) Measuring the sample; d) Comparing the analytical results of the sample with the calibration curve of step b); and e) Quantifying the concentration of S100A8, S100A9, S100A8 / A9, or an oligomer thereof in the sample.

[0016] The present invention also targets the polypeptide of the present invention for use in a method for diagnosing acute or chronic inflammatory diseases in a subject, the method comprising a) Providing a biological sample from the subject, b) By using the polypeptide of the present invention and its oligomers as calibration reference substances, quantifying the amount of S100A8, S100A9, S100A8 / A9, or their oligomers in the biological sample of step a), and c) comparing the amount of S100A8, S100A9, S100A8 / A9, or their oligomers determined in step b) with reference data from subjects already known to be suffering from an acute or chronic inflammatory disease.

[0017] The present invention relates to a) the polypeptide of the present invention and / or its oligomers; b) a test container; c) a buffer solution; and d) a first binding reagent, and the present invention also targets a kit containing the same.

Brief Description of the Drawings

[0018] [Figure 1] Size-exclusion chromatography of the fusion protein of the present invention in buffer A (20 mM HEPES pH 7.5, 100 mM NaCl, 2.5% glycerol, 1 mM DTT) on HiPrep Sephacryl S200 16 / 60 (GE Healthcare) is shown. The purified fusion protein is monodisperse and elutes from the column as a monomer (26.8 kDa). [Figure 2] 3C digest of the fusion protein. 15 μg of purified rCAL (Mrp14-Mrp8 fusion) monomer was digested with 150 ng (++) or 50 ng (+) of purified 3C protease at room temperature for 3 hours. Next, samples were prepared for SDS-PAGE using LDS buffer (×4) and denatured at 95 °C for 12 minutes. It was revealed by SDS-PAGE that the fusion protein was efficiently cleaved into S100A8 chain and S100A9 chain by 3C protease. [Figure 3]When 2 mM CaCl2 was added to buffer A, the amount of recombinant fusion polypeptide eluted from Example 1 changed significantly, suggesting dimerization. This is equivalent to the calcium-dependent heterotetramerization of S100A8 / S100A9 in endogenous calprotectin. [Figure 4] The binding of the monoclonal antibody 27E10 to calprotectin requires heterodimerization of S100A8 and S100A9 (Hessian PA, Fisher L. The heterodimeric complex of MRP-8 (S100A8) and MRP-14 (S100A9). Antibody recognition, epitope definition and the implications for structure (2001). Eur. J. Biochem. 268: 353-363). The fusion protein showed high affinity for 27E10mAB in BLI (biolayer interferometry) measurements, suggesting that the 3D structure of the fusion polypeptide of the present invention is similar to the heterodimer surface structure of the endogenous calprotectin heterotetramer. [Figure 5] Sandwich ELISA measurement employing a dilution series of recombinant fusion polypeptide (SEQID NO:3) using monoclonal capture and detection antibodies. The capture antibody is linked to HRP, which yields a concentration-dependent indication in OD450 after TMB addition. The linear correlation between the spiked fusion polypeptide concentration (from 0.5 pg / mL to 10 pg / mL) and the OD450 signal suggests that the fusion polypeptide (SEQID NO:3) is recognized by the monoclonal calprotectin antibody in a concentration-dependent manner. [Figure 6]This is a turbidimetric method employing a dilution series with a polyclonal antibody against the recombinant fusion polypeptide (SEQID NO:3). PETIA (particle-enhanced turbidimetric immunoassay) also shows an absorption signal correlated with the spiked fusion polypeptide concentration (from 0 to 21.7 μg / mL). Therefore, the polyclonal antibody against S100A8 / S100A9 readily recognizes the fusion polypeptide (SEQID NO:3) of the present invention, which supports the structural similarity of its counterpart to endogenous calprotectin. [Figure 7] Figure 6 shows a comparison of results obtained from 23 serum human samples by immunoturbidimetric analysis. For the first formulation, the immunoassay was calibrated with purified endogenous calprotectin (S100A8 / A9 heterotetramer), and for the second formulation, it was performed using the recombinant fusion polypeptide of the present invention (SEQID NO:3). The two result sets were then correlated and displayed as a passing bar block plot. [Figure 8] This study compares results obtained from four different dilutions of the recombinant fusion polypeptide (SEQID NO:3) of the present invention with results obtained from 23 human serum samples using ELISA (Figure 5) employing the monoclonal antibody described in Example 5 versus immunoturbidimetry (Figure 6) employing the polyclonal antibody described in Example 5. Both assays were calibrated with endogenous calprotectin. The two result sets were then correlated and displayed as passing-bubble plots. [Figure 9]This figure shows site-directed mutagenesis of the SEQID NO:3 fusion protein and the results. The mutation that impairs the tetramerization of S100A8 / S100A9 (European Patent Application Publication No. 3248015), namely E78A of the S100A9 polypeptide chain, was introduced into the fusion protein. This E78A mutant remained monomeric even in the presence of CaCl2. In S200 size exclusion chromatography using buffer A + 2mMCaCl2, even when the non-mutant fusion protein eluted to a volume indicating dimerization (approximately 63 mL), the elution profile of the fusion protein with the E78A mutation (approximately 56 mL) did not change compared to buffer A without calcium. [Modes for carrying out the invention]

[0019] The present invention relates to a method for expressing soluble calprotectin, which includes expressing calprotectin from a vector comprising: a first chain containing a nucleotide sequence having at least 80% sequence homology with SEQ ID NO:11; a second chain containing a nucleotide sequence having at least 80% sequence homology with SEQ ID NO:12; and a linker connecting the first chain and the second chain.

[0020] In a preferred embodiment, the linker connecting the first and second strands on the vector has a length of 18 to 180 nucleotides, 18 to 120 nucleotides, 18 to 90 nucleotides, or 18 to 60 nucleotides, most preferably 21 to 30 nucleotides. A linker of this length ensures sufficient proximity between the two strands to enable correct folding.

[0021] As used herein, the term "S100A8" refers to a polypeptide having SEQID NO:1. Human S100A8 encoded by SEQ ID NO:11 is also known in the art as Mrp8 or cargranulin A.

[0022] As used herein, the term "S100A9" refers to a polypeptide having SEQ ID NO:2. Human S100A9 encoded by SEQ ID NO:12 is also known in the art as Mrp14 or cargranulin B.

[0023] Heterodimers of S100A8 and S100A9 spontaneously form as soon as these two molecules come into contact with each other. Metal ions, especially Ca 2+ In the presence of divalent metal ions such as , two S100A8 / S100A9 heterodimers may form a heterotetramer. Here, the term "S100A8 / S100A9 oligomer" refers to oligomers containing one or more S100A8 and S100A9 monomers, including heterodimers, heterotetramers, and polymers. In particular, "S100A8 / S100A9 oligomer" refers to a heterotetramer containing endogenous "calprotectin," i.e., two units each of S100A8 and S100A9.

[0024] Prior to the present invention, recombinant calprotectin was obtained by expressing each subunit separately in inclusion bodies, then purifying, refolding, and dimerizing the subunits to form naturally occurring heterodimers and heterotetramers. This process is not only time-consuming but also prone to errors, as it often produces homodimers of low analytical value.

[0025] The inventors have surprisingly found that by linking two subunits and expressing them as a single fusion protein, a correctly folded heterodimer at the time of expression is directly obtained, and therefore subsequent purification becomes very easy. Accordingly, in preferred embodiments, the method of the present invention does not involve the extracellular refolding (re-folding) step of calprotectin. In other words, the method of the present invention ensures that calprotectin is already correctly folded at the time of expression.

[0026] The heterodimers obtained by the method of the present invention subsequently associate into heterotetramers in the presence of divalent metal ions, similar to natural (wild-type) calprotectin heterodimers. The soluble calprotectin obtained by the method of the present invention is structurally essentially identical to natural calprotectin and is therefore capable of binding to monoclonal antibodies specific to the S100A8 / S100A9 heterodimer, particularly the monoclonal antibody mAb27E10.

[0027] Monoclonal antibodies containing clone ID mAb27E10 are commercially available, for example, from Abcam Ltd. (ab17050) in the UK or Santa Cruz Biotechnology Ltd. (sC-33714) in Texas, USA. mAb27E10 is further described in Hessian and Fisher, Eur. J. Biochem. 268, 353-363 (2001).

[0028] The present invention further, a) A first chain having an amino acid sequence with at least 80% sequence identity with SEQ ID NO:1, b) A second chain containing an amino acid sequence having at least 80% sequence identity with SEQ ID NO:2, and c) Regarding a polypeptide including a linker connecting the first chain and the second chain, The linker is a peptide linker that has a protease recognition sequence.

[0029] The polypeptide of the present invention comprises two chains having amino acid sequences having at least 80% sequence identity to SEQ ID NO:1 and 2, respectively. In the context of the present invention, the term “sequence having at least 80% sequence identity” consists of sequences having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, and most preferably 100%, sequence identity to each respective sequence. In all cases, identity is identity across the entire length of the corresponding amino acid sequence.

[0030] SEQ ID NO:1 corresponds to human S100A8, and SEQID NO:2 corresponds to human S100A9. Thus, the polypeptide of the present invention has an amino acid sequence similar to or identical to the two monomers that constitute human calprotectin. Here, the first chain is assumed to be one of the S100A8 isoforms known in the art, in particular the isoform having 93 amino acids (SEQID NO:1). Similarly, the first chain may correspond to an S100A8 isoform having 101, 116, or 117 amino acids.

[0031] The first chain of the polypeptide of the present invention is preferably at least 80, 85, or 90 amino acids long. In a particularly preferred embodiment, the first chain is exactly 93, 101, 116, or 117 amino acids long.

[0032] The second chain of the polypeptide of the present invention is preferably at least 90, 100, or 110 amino acids long. In a particularly preferred embodiment, the first chain is exactly 114 amino acids long.

[0033] In the polypeptide of the present invention, the two chains are linked by a peptide linker. Such a linker has the advantage of being easily adaptable to and integrated into the polypeptide of the present invention.

[0034] Preferably, the peptide linker has a length of 1 to 20 amino acids. In a particularly preferred embodiment, the peptide linker has a length of 5 to 10 amino acids. Peptide linkers having this length are particularly suitable for linking two chains of the polypeptide of the present invention in a manner that allows for an interaction between the two chains, which is quite similar to the dimer interaction between S100A8 and S100A9 in endogenous calprotectin.

[0035] The peptide linker has a protease recognition sequence, that is, an amino acid sequence that can be recognized and cleaved by a protease. This has the advantage that the first and second chains of the polypeptide of the present invention can be separated from each other by the addition of their respective proteases. Thus, the polypeptide according to this embodiment can be expressed and assembled, and then cleaved into two chains corresponding to Mrp8 and Mrp14, making it possible to restore a physiological state in which Mrp8 and Mrp14 are not permanently bound.

[0036] Many examples of protease recognition sequences are known in the art. For example, the protein recognition sequence may be LEVLFQGP for rhinovirus 3C protease ("Prescission" protease), ENLYFQG for TEV protease, LVPRGS for thrombin, IEDGR for factor Xa, or DDDDK for enteropeptidase.

[0037] The linker can connect either the N-terminus or C-terminus of the first chain to the second chain. In a preferred embodiment, the first chain is linked to the linker and the second chain at its N-terminus. This arrangement is advantageous because it facilitates the soluble expression of the polypeptide in E. coli.

[0038] In one aspect of the present invention, the polypeptides of the present invention can oligomerize, particularly dimerize, each other in the presence of a metal ion. Preferably, the metal ion is a divalent metal ion, most preferably Ca 2+ When oligomerized, the polypeptide of the present invention can form a structure very similar to that of naturally occurring calprotectin heterotetramers. This is useful for testing and calibrating immunoassays aimed at detecting endogenous calprotectin.

[0039] In another aspect of the present invention, the polypeptide of the present invention can form structures that are recognized by a binding reagent that recognizes S100A8 monomers, S100A9 monomers, S100A8 / S100A9 dimers, and / or their oligomers, which contain endogenous calprotectin.

[0040] The conjugate reagent may be any S100A8 monomer, S100A9 monomer, S100A8 / S100A9 dimer, and / or their oligomers, in particular any that recognize calprotectin. In particular, the conjugate reagent may be an antibody or a fragment thereof, a sybody, nanobody, monobody, CAMELIDHC antibody, affibody, TandAb, bicyclic peptide, DARPin, avimer, Ankyrin repeat sequence, BiTE, DART, anticalin, or nucleotide sequence.

[0041] The polypeptides of the present invention may further include tags, markers, recognition sequences, or signals known in the art to enable the purification, identification, localization, and recognition of the polypeptides of the present invention. In one embodiment, the polypeptide of the present invention includes a His-tag to enable efficient purification of the polypeptide by chromatography. Other tags or markers that may be used with the polypeptides of the present invention are FLAG-tags, HA-tags, Strep-tags, or GFP, which are known to all those skilled in the art.

[0042] In preferred embodiments, the polypeptides of the present invention have at least 80% sequence identity with SEQID NO: 7, 8, 9, 10, 18, or 19. In preferred embodiments, the polypeptides of the present invention have 85%, 90%, or 95% sequence identity with SEQID NO: 7, 8, 9, 10, 18, or 19. In particularly preferred embodiments, the polypeptides of the present invention have 100% sequence identity with SEQID NO: 7, 8, 9, 10, 18, or 19. SEQ ID NO: 7 to 10 include a His-tag linked to the first or second chain by a short amino acid sequence having the protease sequence of rhinovirus SC protease, and the His-tag can be cleaved from the polypeptide portion having the two chains and linker by the addition of rhinovirus 3C protease. Therefore, polypeptides having SEQID NO: 7 to 10 can be efficiently purified, and the His-tag can then be discarded. Furthermore, the linker between the two strands used in SEQID NO:7 to 10 has a recognition sequence for TEV protease, so the two strands can be dissociated by adding TEV protease.

[0043] In other embodiments shown in SEQ ID NO:3 and 4, the polypeptide of the present invention consists of the same protease recognition sequence between the His-tag and the two strands, and thus the removal of the His-tag and cleavage between the two strands are possible in a single step.

[0044] In further embodiments, the polypeptide chains of the present invention have one or more specific mutations compared to naturally occurring (non-artificial) S100A8 and S100A9. These mutations may affect the oligomerization of the chain and / or recognition by specific binders.

[0045] For example, the polypeptides of the present invention may have mutations, such as those described in European Patent Application Publication No. 3248015, that prevent oligomerization between the heterodimers of S100A8 and S100A9. These polypeptides can be advantageously used as calibrators or standards because they provide a clearly defined structure that is not affected by external conditions. For example, these polypeptides do not heterotetramerize in the presence of calcium ions and / or other substances present in assay buffers or biological samples.

[0046] The present invention also covers polynucleotide sequences that encode the polypeptide of the present invention. In one embodiment, the polynucleotide sequence is a polynucleotide sequence that hybridizes with the polynucleotide sequence of SEQID NO:5 under high stringency conditions. In another embodiment, the polynucleotide sequence is a polynucleotide sequence that hybridizes with the polynucleotide sequence of SEQID NO:6 under high stringency conditions. SEQ ID NO:5 encodes the amino acid sequence of SEQID NO:3, and SEQ ID NO:6 encodes the amino acid sequence of SEQID NO:4.

[0047] As used herein, the terms “hybridization” or “hybridize” encompass any process by which a strand of nucleic acid molecules binds to a complementary strand through base pairing (J. Coombs (1994) Dictionary of Biotechnology, Stockton Press, New York). Hybridization and the strength of hybridization (i.e., the strength of the bond between nucleic acid molecules) are influenced by factors such as the degree of complementarity between nucleic acid molecules, the stringency of the conditions involved, the Tm of the hybrid formed, and the G:C ratio within the nucleic acid molecule.

[0048] As used herein, the term "Tm" is used in relation to "melting temperature." The melting temperature is the temperature at which half of a population of double-stranded nucleic acid molecules dissociates into single strands. Formulas for calculating the Tm of nucleic acid molecules are well known in the art. A simple estimate of the Tm value can be calculated by the following formula, as shown in standard literature: When the nucleic acid molecule is in an aqueous solution of 1 M NaCl, Tm = 81.5 + 0.41 (%G + C) (see, e.g., Anderson and Young, Quantitative Filter Hybridization, in Nucleic Acid Hybridization (1985)). Other literature includes more sophisticated calculations that take into account structural properties as well as sequence properties for calculating Tm. Stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6.

[0049] Specifically, the term "stringency conditions" refers to conditions under which 100 or more consecutive nucleotides, 150 or more consecutive nucleotides, 200 or more consecutive nucleotides, or 250 or more consecutive nucleotides, which are fragments of complementary nucleic acid molecules (DNA, RNA, ssDNA, or ssRNA) or identical to such molecules, hybridize with a specific nucleic acid molecule (DNA; RNA, ssDNA, or ssRNA) in a 50°C solution of 7% sodium dodecyl sulfate (SDS), 0.5M NaPO4, and 1 mM EDTA, accompanied by washing with 2×SSC using 0.1% SDS, at 50°C or 65°C (preferably at 65°C). Preferably, the hybridization conditions are equivalent to those for hybridization in a 50°C solution of 7% sodium dodecyl sulfate (SDS), 0.5M NaPO4, and 1mM EDTA, accompanied by washing with 0.1×SSC using 0.1% SDS at 50°C or 65°C (preferably at 65°C). More preferably, the hybridization conditions are equivalent to those for hybridization in a 50°C solution of 7% sodium dodecyl sulfate (SDS), 0.5M NaPO4, and 1mM EDTA, accompanied by washing with 0.1×SSC using 0.1% SDS at 50°C or 65°C (preferably at 65°C). Preferably, the complementary nucleotide hybridizes with the nucleic acid fragment or whole. Alternatively, preferred hybridization conditions include hybridization in 1×SSC at 65°C or 1×SSC at 42°C and 50% formamide, followed by washing in 0.3×SSC at 65°C or hybridization in 4×SSC at 50°C, or hybridization in 6×SSC and 50% formamide at 40°C, followed by washing in 2×SSC at 50°C. Even more preferred hybridization conditions are 0.1%SDS, 0.1×SSD, and 65°C.

[0050] The polynucleotide sequence of the present invention may further have a promoter, a nuclease recognition site, a marker gene, an enhancer, and other genetic elements useful for transformation using the polynucleotide sequence and for the expression of the polynucleotide sequence.

[0051] The polypeptides of the present invention may form oligomers. Therefore, in one embodiment, the present invention relates to polypeptide oligomers comprising two or more polypeptides of the present invention. In a preferred embodiment, the polypeptide oligomer comprises just two polypeptides of the present invention. Such a polypeptide oligomer corresponds to an endogenous calprotectin heterotetramer consisting of two S100A8 and two S100A9 monomers. In a particularly preferred embodiment, the polypeptide oligomer comprises a divalent metal ion, preferably Ca 2+ It is formed in the presence of.

[0052] The polypeptides or oligomers of the present invention can be used for immunization of animals. In this specification, “immunization” means the administration of a substance to a subject or animal for the purpose of inducing an immune response in the subject or animal. The immune response may include the production of antibodies.

[0053] The animal may be any animal useful for clinical research or industrial application, such as a mouse, rat, rabbit, goat, dog, hen, shark, camelid, pig, or monkey. When immunizing an animal with the polypeptide of the present invention, the polypeptide can be prepared by any method known in the art, for example, by adding further immunogenic compounds.

[0054] During the immunization process, the polypeptide of the present invention can substitute for endogenous calprotectin in order to obtain antibodies against calprotectin.

[0055] The polypeptides or oligomers of the present invention can also be used as epitopes for in vitro selection or affinity purification of S100A8 monomers, S100A9 monomers, S100A8 / S100A9 dimers, and / or their oligomers, particularly binding reagents that recognize endogenous calprotectin. Since the polypeptides of the present invention can be easily obtained in large quantities, their use in in vitro selection and purification methods allows for cost-effective and reproducible selection and purification of appropriate binding reagents.

[0056] In another embodiment, the polypeptide of the present invention can be used as a pharmaceutical. In one embodiment, the pharmaceutical may be useful for treating or preventing diseases requiring modulation of the immune response or reduction / inhibition of inflammatory processes.

[0057] In another preferred embodiment, the polypeptide of the present invention may be used as a calibration reference material. Hereinafter, a calibration reference material is understood to mean a substance used as a (internationally recognized) (highest-grade) primary reference material for traceability and / or interchangeability of any type of analytical assay, or as a standard when establishing a novel analytical assay. Thus, a calibration reference material can be used to test the sensitivity and specificity of a novel binder or measurement system. Similarly, a calibration reference material can be used to calibrate an analytical assay. Therefore, the polypeptide of the present invention can be used in methods for establishing novel assays, particularly immunoassays, and can be used not only for testing binders but also for calibration of assays. In these embodiments, the polypeptide of the present invention exhibits all the essential properties of natural calprotectin, such as epitope presentation and binding properties, but can be used as a substitute for standard heterodimer or standard heterotetramer calprotectin as a calibration reference material because it is more readily available than calprotectins conventionally used as calibration reference materials.

[0058] The present invention also relates to a method for measuring S100A8, S100A9, S100A8 / A9, or their oligomers, particularly calprotectin, in a sample using the polypeptide of the present invention in an analytical assay, and includes the following steps. a) A step of measuring various amounts of polypeptide using an analytical assay; b) A step to establish a calibration curve using the analytical results obtained in step a); c) Step of measuring the sample; d) A step of comparing the analysis results of the sample with the calibration curve from step b); and, e) A step of quantifying the concentration of S100A8, S100A9, S100A8 / A9, or their oligomers in the sample.

[0059] Those skilled in the art will know how to calibrate analytical assays. In short, analytical assays are used to measure various amounts of the polypeptide of the present invention to obtain analytical results, which are then used to establish a calibration curve. In other words, the polypeptide of the present invention or its oligomers are used as calibration reference materials for analytical assays. Due to its monodispersity and homogeneity, calibration using the polypeptide of the present invention is rapid and highly reliable.

[0060] In the method of the present invention, the sample is then measured using the same analytical test, and the analytical results of these measurements are compared with a calibration curve. From this comparison, the concentrations of s100A8, S100A9, S100A8 / A9, or their oligomers in the sample can be quantified.

[0061] The polypeptide or oligomer of the present invention can also be used in a method for diagnosing acute or chronic inflammatory diseases in subjects, and this method is a) A step of providing a biological sample from the subject, b) A step of quantifying the amount of S100A8, S100A9, S100A8 / A9, or their oligomers, particularly calprotectin, in the biological sample from step a) using the polypeptide oligomer of the present invention as a calibration reference material; and, c) The step includes comparing the amount of S100A8, S100A9, S100A8 / A9, or their oligomers determined in step b) with reference data from subjects known to already have acute or chronic inflammatory diseases.

[0062] Those skilled in the art are familiar with various types of acute or chronic inflammatory diseases, such as allergies, asthma, autoimmune diseases, celiac disease, glomerulonephritis, hepatitis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, reperfusion injury, transplant rejection, infectious colitis, necrotizing enterocolitis, cystic fibrosis, rheumatoid arthritis, juvenile idiopathic arthritis, ankylosing spondylitis, joint swelling, psoriasis, psoriatic arthritis, Behçet's disease, gingivitis, tonsillitis, appendicitis, lupus, fever of unknown origin, sepsis, heart disease, myocardial infarction, multiple sclerosis, and cancers such as colorectal cancer. In preferred embodiments, the inflammatory disease is selected from the group consisting of inflammatory bowel disease, particularly ulcerative colitis or Crohn's disease, and inflammatory arthritis, particularly rheumatoid arthritis, juvenile idiopathic arthritis or ankylosing spondylitis.

[0063] In one aspect of the present invention, a significant increase in the amount of S100A8, S100A9, S100A8 / A9, or their oligomers, particularly calprotectin, compared to reference data, suggests that the subject is suffering from an acute or chronic inflammatory disease.

[0064] By using the polypeptide or oligomer of the present invention, inflammatory diseases can be easily and reliably diagnosed, and their treatment methods can be monitored.

[0065] In the method of the present invention, the sample may be any biological sample used in the art, particularly blood, serum, plasma, synovial fluid, saliva, urine, tears, sweat, gingival crevicular fluid, feces, gastrointestinal lavage fluid, bronchial lavage fluid, cell culture supernatant, or tissue extract. In preferred embodiments, the sample is a fecal sample (stool sample) or a blood sample.

[0066] The analytical assay may be any analytical assay known in the art, such as an immunoassay, biochemical assay, biophysical assay, or physical assay. In a preferred embodiment, the analytical assay is an immunoassay based on a method that requires recognition of the analyte by a high-affinity binding reagent, such as an enzyme-linked immunosorbent assay (ELISA), a lateral flow immunoassay (LFIA), or a particle-enhanced immunoturbidimetry assay (PETIA).

[0067] In the method of the present invention, the analytical results of the sample may be based on optical readout, absorption, UV / VIS spectroscopy, light scattering, turbidimetry, nephelometry, light scattering, reflectance measurement, fluorescence, luminescence, chemiluminescence, surface plasmon resonance, amperometry, magnetic measurement, voltammetry, potentiometry, conductometry, coulometry, polarography, gravimetric measurement, or cantilever. In a preferred embodiment, the analytical results are based on an immunoassay measured by absorption, UV / VIS spectroscopy, light scattering, turbidimetry, nephelometry, reflectance measurement, fluorescence, luminescence, or chemiluminescence. In the most preferred embodiment, the analytical results are based on an immunoassay measured by absorption spectroscopy, light scattering, or chemiluminescence.

[0068] The present invention a) polypeptides and / or oligomers thereof of the present invention; b) Test container; c) Buffer; and d) Kits comprising a first binding reagent, preferably immobilized on a solid support, that is specific to S100A8, S100A9, S100A8 / A9, or their oligomers, particularly calprotectin.

[0069] In one embodiment, the first binding reagent is labeled with a substance that enables the quantification of the polypeptide and S100A8, S100A9, S100A8 / A9, or their oligomers, or is bound to such a substance.

[0070] The test container is used to carry out the analytical assay. For example, the test container may be a test cartridge, membrane, tube, titer plate, or container. Those skilled in the art know how to select a suitable buffer solution. For example, buffers may include phosphates, maleates, chloroacetates, formates, benzoates, pyridines, piperazines, propions, 3-N-morpholinopropanesulfonic acid (MOPS), 1,3-bistris-hydroxymethyl)methylaminopropane (Bis-TRIS), tris-(hydroxymethyl)aminomethane (TRIS), tris-(hydroxymethyl)aminomethanemaleate (TRIS-maleate), and 2-(-tris-(hydroxymethyl)methylamino)ethane Examples include sulfonic acid (TES), 1,4-piperazine bis-ethanesulfonic acid (PIPES), 4-morpholinoethanesulfonic acid (MES), N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), N-(2-acetamide)iminodiacetic acid (ADA), N-(2-acetamide)-2-aminoethanesulfonic acid (ACES), and others known to those skilled in the art. The buffer may further contain salts, antimicrobial agents, detergents, chelating agents, chaotropic agents, and / or antifoaming agents.

[0071] The kit of the present invention further comprises a first binder specific to S100A8, S100A9, S100A8 / S100A9, or their oligomers, and optionally a second binder. In a preferred embodiment, the first / second binder is specific to a heterotetramer containing S100A8 and S100A9, i.e., calprotectin. In another preferred embodiment, the first / second binder is specific to a heterodimer containing S100A8 and S100A9. However, the present invention also envisions the first / second binder being specific to the S100A8 monomer or the S100A9 monomer.

[0072] Due to the sequence of the polypeptide of the present invention, binders specific to S100A8, S100A9, S100A8 / S100A9, or their oligomers, also recognize the polypeptide of the present invention.

[0073] In a preferred embodiment, the first binder is immobilized on a solid carrier. This allows the kit to be used in ELISA, LFIA, or PETIA.

[0074] According to the present invention, the first / second binding reagent may be labeled with a substance that enables the quantification of the polypeptide and S100A8, S100A9, S100A8 / A9, or their oligomers, or may be bound to a substance that enables the quantification. Those skilled in the art will know which substances or labels can be used for the quantification of the analyte. For example, the binding agent can be labeled with latex, fluorescent, (para)magnetic, colored cellulose, iron, gold, or silica (nano) particles; fluorophores (fluorescent dye molecules) or fluorescent dyes; quantum dots; enzymes such as horseradish peroxidase (HRP), alkaline phosphate (AP), glucose oxidase, glucose 6-phosphate dehydrogenase, maleate dehydrogenase, NADH dehydrogenase, acetylcholinesterase; chemiluminescent reactants such as luciferase and luminol and its derivatives; fluorescent proteins such as green fluorescent protein (GFP), red fluorescent protein, yellow fluorescent protein, and radioisotopes.

[0075] If necessary, this kit may include means for purifying biological samples.

[0076] In this way, the kit of the present invention enables the quantitative measurement of calprotectin in a sample. Since the polypeptides of the present invention specifically form heterodimers or heterotetramers, their use in the kit avoids false positives and thus enables reliable quantitative determination of calprotectin in a sample. [Examples]

[0077] Example 1: Preparation of the polypeptide of the present invention SEQID NO:5 was cloned into the IPTG-inducible T7 transcription pET21 vector (Novagen). Expression strains of E. coli BL21 were transformed with the corresponding plasmid. Protein expression induction by IPTG was performed according to a standard protocol to obtain a soluble polypeptide containing SEQID NO:3. More specifically, each expression strain was grown in LB medium containing 100 pg / mL carbenicillin at 37°C and 200 rpm until OD600 = 0.5. Subsequently, the cell culture was cooled to 18°C, and protein expression induction was started at OD600 ≈ 0.8 by adding 0.2 mM IPTG. After expression at 18°C ​​and 200 rpm for 14-16 hours, the E. coli expression strains were collected by centrifugation at 7,000 × g and washed in buffer A (20 mM HEPES pH 7.5, 100 mM NaCl, 2.5% glycerol, 1 mM DTT).

[0078] The induced expression strain was resuspended in lysation buffer (buffer A + 20 mM imidazole), and then the cells were disrupted by sonication (product name "Branson Sonifier"). Soluble proteins were separated from the cell debris by centrifugation at 20,000 × g. The supernatant was applied to NiNTA resin, and the purified fusion protein was eluted using an imidazole concentration gradient. By applying to a Q Sepharose column, the fusion protein was separated from nucleic acid contaminants using a NaCl concentration gradient. A final purification step using an S200 size exclusion column (Figure 1) yielded pure, monodisperse fusion protein in buffer A (Figure 2, lane 3).

[0079] Example 2: Proteolytic cleavage of fusion polypeptide into monomers In this type of fusion protein (SEQID NO:3), the 3C protease (registered trademark "PreScission") recognition sequences that link the His-tag and S100A9, and that link S100A9 and S100A8, allow for the proteolytic degradation of a single fusion polypeptide to form three polypeptides. These polypeptides correspond to the free His-tag, S100A8, and S100A9, each possessing their respective residual amino acids from the 3C recognition sequences (Figure 2, lane 4).

[0080] Example 3: Ca 2+ Dimerization of recombinant fusion polypeptides in the presence of ions This recombinant fusion polypeptide shares characteristics with endogenous calprotectin, which is a heterodimer of S100A8 and S100A9. Size exclusion chromatography with buffer A containing 2mMCaCl2 showed a significant shift in the elution amount of the fusion protein, indicating dimerization. This corresponds to the calcium-dependent heterotetramerization of the two heterodimers of S100A8 and S100A9 (Figure 3). The recombinant polypeptide monomer (corresponding to the S100A8 / S100A9 dimer) eluted in approximately 62 mL, while the recombinant polypeptide dimer (corresponding to the S100A8 / A9 tetramer) eluted more rapidly in approximately 58 mL.

[0081] Example 4: Immunological properties of the recombinant fusion polypeptide of the present invention The aforementioned fusion protein (SEQID NO:3) exhibits immunological properties similar to endogenous calprotectin. Monoclonal antibody 27E10 (Hessian PA, Fisher L. The heterodimeric complex of MRP-8 (S100A8) and MRP-14 (S100A9). Antibody recognition, epitope definition and the implications for structure (2001). Eur. J. Biochem. 268: 353-363) was immobilized on a Biacore chip sensor to recognize the fusion protein (SEQID NO:3). The fusion protein's response was indicated by a concentration-dependent response unit during interaction with the fusion protein (SEQ ID NO:3) using surface plasmon resonance (Figure 4).

[0082] Example 5: Immunoassay based on the recombinant fusion polypeptide of the present invention ELISA using monoclonal capture antibodies was performed in the presence of CaCl2 with various concentrations of the fusion protein (SEQID NO:3). A color change based on TMB (3,3',5,5'-tetramethylbenzidine) was induced using an HRP-binding detection antibody. After adding an acidic stop solution, the OD at 450 nm was measured and showed a linear correlation with the spiked concentration of the purified fusion protein (SEQID NO:3) previously evaluated by A280 absorption measurement (Figure 5). Similarly, immunoturbidimetric analysis using polyclonal antibodies also correlated with various concentrations of the fusion protein (SEQID NO:3) (Figure 6), confirming that the fusion protein (SEQID NO:3) exhibits essentially the same properties as those described for endogenous calprotectin (S100A8 / S100A9), and therefore can be used interchangeably with S100A8 / S100A9 purified from endogenous human sources such as fecal samples or blood-derived granulocytes.

[0083] Example 6: Interoperability of calibration substances comprising either endogenous calprotectin or the recombinant fusion polypeptide of the present invention. This study compares the results obtained by turbidimetry of 23 human serum samples using the recombinant fusion polypeptide (SEQID NO:3) of the present invention as a calibrator, with the results obtained by the same immunoassay using purified endogenous calprotectin (S100A8 / A9 heterotetramer) as a calibrator. When the results were plotted against each other, a perfect correlation was observed regardless of the calibrator used (slope = 1.009, R). 2 The result obtained was (=0.999) (Figure 7).

[0084] Example 7: The fusion polypeptide of the present invention exhibits the same immunological properties with respect to polyclonal and monoclonal antibodies. Four different dilutions of the recombinant fusion polypeptide and 23 human serum samples were analyzed comparatively using an ELISA employing the monoclonal antibody described in Example 5 and a turbidimetric immunoassay employing the polyclonal antibody also described in Example 5. In this experiment, endogenous calprotectin was used as a calibrator in both assays. The two result sets were interrelated and displayed as a passing-bub block plot (Figure 8). No substantially quantitative differences were observed between the results obtained with the polyclonal antibody and the monoclonal antibody, for both the fusion polypeptide of the present invention (SEQID NO:3) and the human serum samples.

[0085] Example 8: Prevention of dimerization or oligomerization of the fusion polypeptide (SEQID NO:3) of the present invention by introduction of point mutations. The E78A mutation was introduced into the S100A9 chain of the fusion protein (SEQID NO:3) (see European Patent Application Publication No. 3248015) using site-directed mutagenesis. In endogenous calprotectin, this mutation suppresses heterodimerization (Leukert N et al. Calcium-dependent tetramer formation of S100A8 and S100A9 is essential for biological activity (2006). J. Mol. Biol. 359: 961-972). Similarly, introduction of this mutation into the fusion protein (SEQ ID NO:3) inhibited CaCl2-dependent dimerization. S200 size exclusion chromatography of the mutant and non-mutant fusion proteins (SEQID NO:3) in buffer A containing 2 mM CaCl2 was performed as described in Examples 1 and 3 (Figure 9).

Claims

1. A method for obtaining soluble calprotectin comprising the steps of: expressing calprotectin from a vector comprising a first strand encoding an amino acid sequence having at least 90% sequence identity to SEQ ID NO:1, a second strand encoding an amino acid sequence having at least 90% sequence identity to SEQ ID NO:2, and a linker connecting the first strand and the second strand, The method, wherein the linker encodes a peptide linker having a protease recognition sequence.

2. The method of claim 1, wherein the soluble calprotectin is capable of binding to monoclonal antibody mAb27E10.

3. The method of claim 1 or 2, which does not include refolding the calprotectin extracellularly.

4. The method of claim 1 , wherein the linker has a length between 18 and 180 nucleotides.

5. The method of claim 1 , wherein the linker comprises a nucleotide sequence encoding a protease recognition sequence.

6. a) a first strand comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:1; b) a second strand comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:2; c) a linker connecting the first chain and the second chain, A polypeptide, wherein the linker is a peptide linker having a protease recognition sequence.

7. 7. The polypeptide of claim 6, wherein the protease recognition sequence is a rhinovirus 3C protease recognition sequence, a TEV protease recognition sequence, a thrombin recognition sequence, a factor Xa recognition sequence, or an enteropeptidase recognition sequence.

8. 8. The polypeptide of claim 6 or 7, which has the ability to oligomerize in the presence of metal ions.

9. The polypeptide of claim 6 , which has the ability to form a dimer in the presence of a divalent metal ion.

10. A polypeptide described in any one of claims 6 to 9, having the ability to form a structure recognized by an antibody that recognizes S100A8 monomers, S100A9 monomers, S100A8 / S100A9 dimers, and / or oligomers thereof.

11. 7. The polypeptide of claim 6, comprising an amino acid sequence having at least 80% sequence identity with the polypeptide of SEQ ID NO:

3.

12. The polypeptide of claim 6, comprising an amino acid sequence having at least 80% sequence identity to a polypeptide of SEQ ID NO: 4, 7 to 10, 18, or 19.

13. A polypeptide oligomer comprising two or more of the polypeptides according to any one of claims 6 to 12.

14. Use of a polypeptide according to any one of claims 6 to 12, or a polypeptide obtainable by a method according to any one of claims 1 to 5, or a polypeptide oligomer according to claim 13, for the immunization of animals (excluding humans).

15. Use of a polypeptide according to any one of claims 6 to 12, or a polypeptide obtainable by a method according to any one of claims 1 to 5, or a polypeptide oligomer according to claim 13, as an epitope for in vitro selection of antibodies recognizing S100A8 monomers, S100A9 monomers, S100A8 / S100A9 dimers and / or oligomers thereof.

16. Use of a polypeptide according to any one of claims 6 to 12, or a polypeptide obtainable by a method according to any one of claims 1 to 5, or a polypeptide oligomer according to claim 13, for affinity purification of antibodies recognizing S100A8 monomers, S100A9 monomers, S100A8 / S100A9 dimers and / or oligomers thereof.

17. A polypeptide according to any one of claims 6 to 12 for use as a medicament.

18. Use of a polypeptide according to any one of claims 6 to 12, or a polypeptide obtainable by a method according to any one of claims 1 to 5, or a polypeptide oligomer according to claim 13, as a calibration standard.

19. 13. A method for measuring S100A8, S100A9, S100A8 / A9 dimers or oligomers thereof in a sample using a polypeptide according to any one of claims 6 to 12 in an analytical assay comprising the steps of: a) measuring different amounts of said polypeptide using said analytical assay; b) establishing a calibration curve using the analytical results obtained in step a); c) measuring the sample using said analytical assay; d) comparing the analytical results of the sample with the calibration curve of step b); e) quantifying the concentration of S100A8, S100A9, S100A8 / A9 dimers, or oligomers thereof in the sample.

20. 20. The method of claim 19, wherein the sample is blood, serum, plasma, synovial fluid, saliva, urine, tears, sweat, gingival crevicular fluid, feces, gastrointestinal lavage fluid, bronchial lavage fluid, cell culture supernatant, or a tissue extract.

21. 21. The method of claim 19 or 20, wherein the analytical assay is an immunoassay, a biochemical assay, a biophysical assay, or a physical assay.

22. 22. The method of any one of claims 19 to 21, wherein the analysis of the sample is based on optical readout, UV / VIS spectroscopy, turbidimetry, nephelometry, light scattering, reflectometry, fluorescence, luminescence, chemiluminescence, surface plasmon resonance, amperometry, magnetometry, voltammetry, potentiometry, conductometry, coulometry, polarography, gravimetry, or cantilever.

23. 1. A method for diagnosing an acute or chronic inflammatory disease in a subject, comprising: a) providing a biological sample from said subject; b) quantifying S100A8, S100A9, or S100A8 / A9 dimers or oligomers thereof in the biological sample of step a) by using a polypeptide according to any one of claims 6 to 12 as a calibration standard; and c) comparing the amounts of S100A8, S100A9 and S100A8 / A9 dimer determined in step b) with reference data from subjects already known to suffer from an acute or chronic inflammatory disease, A polypeptide according to any one of claims 6 to 12.

24. a) a polypeptide according to any one of claims 6 to 12 or a polypeptide oligomer according to claim 13; b) a test vessel; and c) a buffer solution; and d) a first antibody specific for S100A8, S100A9, S100A8 / A9 dimer, or an oligomer thereof.

25. the first antibody is labeled with or bound to a substance that allows for the quantification of the polypeptide and S100A8, S100A9, S100A8 / A9 dimer, or oligomers thereof; 25. The kit of claim 24, wherein the substance enabling quantification is selected from latex particles, fluorescent particles, (para)magnetic particles, colored cellulose particles, iron particles, gold particles, silica particles, fluorophores (fluorescent dye molecules), fluorescent dyes, quantum dots, enzymes, chemiluminescent reactants, fluorescent proteins, or radioisotopes.

26. further comprising a second antibody specific for S100A8, S100A9, S100A8 / A9 dimer, or oligomers thereof; the first antibody is immobilized on a solid support; The kit of claim 25, wherein the second antibody is labeled with or bound to the substance, and the kit is capable of quantifying the polypeptide and S100A8, S100A9, S100A8 / A9 dimer, or oligomers thereof.

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