Calprotectin-binding peptide

Abstract

The present invention relates to peptides capable of binding to calprotectin. The present invention further relates to methods for the detection and purification of calprotectin.

Description

[Technical field]

[0001] The present invention relates to synthetic peptide ligands capable of binding to calprotectin. [Background technology]

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

[0003] Calprotectin consists of two polypeptide chains, Mrp8 (synonym: S100A8, calgranulin A) and Mrp14 (synonym: S100A9, calgranulin B), which form a stable dimer. 2+ In the presence of Zn, two Mrp8 / Mrp14 heterodimers can form a heterotetramer. 2+ As a sequestering complex for divalent cations such as , it plays an important role in nutritional immunity and starves microbes during inflammatory processes (Zygiel EM, Nolan EM. Transition Metal Sequestration by the Host-Defense Protein Calprotectin (2018), Annu. Rev. Biochem. 87: 621-43).

[0004] Because it is released at sites of inflammation, calprotectin is considered an alarmin and is frequently used as a biomarker to monitor inflammatory processes. For example, fecal calprotectin is currently the gold standard for diagnosing and monitoring inflammatory bowel diseases (IBD), such as Crohn's disease (CD) and ulcerative colitis (UC) (KonikoffMR, DensonLA. Role of Fecal Calprotectin as a Biomarker of Intestinal Inflammation in Inflammatory Bowel Disease (2006). Inflamm. Bowel Dis. 12 (6):524-34).

[0005] Furthermore, serum CP has been validated as a biomarker for monitoring various (chronic) inflammatory diseases, e.g., rheumatoid arthritis (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).

[0006] Existing calprotectin assays are antibody-based and therefore face challenges generally associated with antibody affinity reagents, particularly limited shelf life, high manufacturing costs, high batch variability, and non-uniformity of immobilization.

[0007] In contrast to antibodies, synthetic peptide ligands consisting of tens to hundreds of amino acids are amenable to large-scale production, show little batch-to-batch variability, and are stable during storage, but such ligands have not yet been described for calprotectin. Summary of the Invention [Problem to be solved by the invention]

[0008] Thus, there is a need in the art to provide high affinity peptide ligands capable of binding calprotectin that can be used not only for the detection of calprotectin, but also for its purification and for medical purposes. [Means for solving the problem]

[0009] In one embodiment the invention relates to a peptide capable of binding calprotectin, said peptide comprising the sequence ΦΖΨΣΧΘΘΘΩ, Φ is L-phenylalanine, L-tryptophan, or an analog thereof; X and Z represent α-amino acids in the L-configuration, Ψ represents a non-polar aliphatic L-amino acid; Σ is L-tyrosine, L-tryptophan, L-phenylalanine, or an aromatic L-amino acid, Θ represents an α-amino acid, Ω represents a hydrophobic L-amino acid; Each occurrence of Φ, each occurrence of Z, each occurrence of X, each occurrence of Ψ, each occurrence of Σ, each occurrence of Θ, and each occurrence of Ω are selected independently of one another.

[0010] In another aspect, the present invention relates to a method for detecting calprotectin in a sample, said method comprising the steps of: a) providing a sample containing calprotectin; b) contacting said sample with a peptide according to the invention to form a complex between said peptide and said calprotectin; and c) detecting the complex formed in step b).

[0011] In another aspect, the present invention relates to a kit for detecting calprotectin in a sample, said kit comprising a peptide according to the invention.

[0012] In another aspect, the present invention relates to a method for purifying calprotectin, said method comprising purifying calprotectin using a peptide according to the invention.

[0013] In another aspect, the present invention relates to a pharmaceutical composition comprising a peptide according to the present invention.

[0014] In another embodiment, the present invention relates to the use of a peptide according to the invention for tagging a protein of interest. [Brief description of the drawings]

[0015] [Figure 1-1] Fluorescence polarization results of phage display derived calprotectin binding peptides. [Figure 1-2] Fluorescence polarization results of phage display derived calprotectin binding peptides. [Figure 1-3] Fluorescence polarization results of phage display derived calprotectin binding peptides. [Figure 1-4] Fluorescence polarization results of phage display derived calprotectin binding peptides. [Figure 1-5] Figure 1 shows the fluorescence polarization results of calprotectin-binding peptides derived from phage display. Peptide sequences selected from three different phage display libraries were enriched after biopanning with human recombinant calprotectin. These peptides were then synthesized and cross-linked between cysteines. Fluorescence polarization experiments revealed affinities for human recombinant calprotectin in the nanomolar range.

[0016] [Diagram 2]Figure 2 shows the characteristics of a calprotectin-binding peptide (peptide 3) with the sequence RSPESVAFPMFQSHWYSG. Various cross-linked isomers were synthesized, purified, and tested by fluorescence polarization. A) Isomer 1, in which adjacent cysteines are cross-linked, showed the most significant increase in fluorescence polarization, with a KD of approximately 24 ± 10 nM. B) Further characterization of the requirement for cross-linking was achieved by selectively substituting cysteines with serine. Isomers 1a and 1b, each with only one adjacent cysteine ​​cross-link, were synthesized, as well as a fully linear peptide containing only serine instead of cysteine. All three peptides showed comparable affinity for recombinant calprotectin, suggesting that peptide 3 binds calprotectin in an extended linear conformation.

[0017] FIG. 3 shows an alanine scan of the calprotectin binding peptide to reveal the amino acids involved in binding. [Figure 3-A] A) Linear peptides were synthesized substituting alanine or glycine for selected amino acids. [Figure 3-B] Slightly modified linear peptides from peptide 3 were then tested for affinity to recombinant calprotectin using B) surface plasmon resonance (SPR). [Figure 3-C1] Slightly modified linear peptides from peptide 3 were also tested for affinity to recombinant calprotectin using C) fluorescence polarization (FP). [Figure 3-C2] Slightly modified linear peptides from peptide 3 were tested for affinity to recombinant calprotectin using C) Fluorescence Polarization (FP). [Figure 3-C3] Slightly modified linear peptides from peptide 3 were tested for affinity to recombinant calprotectin using C) Fluorescence Polarization (FP). [Figure 3-C4]Slightly modified linear peptides from peptide 3 were tested for affinity to recombinant calprotectin using C) Fluorescence Polarization (FP). [Fig.3-C5] Slightly modified linear peptides from peptide 3 were tested for affinity to recombinant calprotectin using C) Fluorescence Polarization (FP). [Fig.3-C6] Slightly modified linear peptides from peptide 3 were tested for affinity to recombinant calprotectin using C) Fluorescence Polarization (FP). [Fig.3-C7] Slightly modified linear peptides from peptide 3 were tested for affinity to recombinant calprotectin using C) fluorescence polarization (FP). Most of the amino acid substitutions did not significantly change the binding affinity. Substitution of F8, M10, F11, H14, and Y16 to alanine showed a significant decrease in affinity in SPR and FP, suggesting that these amino acids are responsible for the majority of the interaction of peptide 3 with human calprotectin.

[0018] FIG. 4 shows the X-ray structure of the peptide-calprotectin complex, highlighting the binding interface between the peptide and the calprotectin tetramer. [Fig. 4-AB] A) Co-crystallization of calprotectin with the peptides revealed that two peptides 3 can bind to one calprotectin tetramer. One peptide shows contacts with two S100A8 (Mrp-8) and one S100A9 (Mrp-14). Thus, the complete calprotectin binding groove for peptide 3 requires the alignment of two S100A8 molecules that are only found in the tetramer, explaining the affinity of peptide 3 for the tetramer. B) Expanded binding groove with peptide 3 in a stick model. Amino acids important for binding affinity according to the alanine scan are underlined. [Figure 4-C] C) Valence structure of peptide 3 with interaction with calprotectin.

[0019] FIG. 5 shows the suitability of calprotectin binding peptides in ELISA. [Figure 5-ABC] A) Example of enzyme-linked immunoabsorbent assay (ELISA) setup recognizing calprotectin. Calprotectin-binding antibodies are coated on a plate to immobilize calprotectin from samples. Calprotectin detection was performed by incubation with biotinylated calprotectin-binding peptide 3 conjugated with streptavidin conjugated horse radish peroxidasa (Step-HRP) for tetramethylbenzidine (TMB) conversion. B) Comparison of biotinylation at the C-terminus and N-terminus of peptide 3 in an ELISA setup. A concentration-dependent signal of calprotectin is prominent when biotin is added to the C-terminus. C) Increased time resolution of the signal depending on calprotectin concentration in the ELISA shows that high sensitivity can be achieved when an incubation time of 60 min is used. [Figure 5-D] D) Measurement of background signal in an ELISA setup. A low background signal is obtained without peptide or calprotectin. C-terminal biotinylation of peptide no. 3 is advantageous to obtain a better signal / noise ratio.

[0020] FIG. 6 shows calprotectin binding peptides in a lateral flow immunoassay set-up. [Figure 6-A1] A) Dipstick experiment with coated calprotectin as control dots and rabbit polyclonal anti-calprotectin antibody as test dots. Gold nanoparticles are conjugated with peptide 3 and added to sample wells as well as various concentrations of calprotectin. [Figure 6-A2] The intensity of the test dots increased with increasing calprotectin concentration in the samples, suggesting the possibility of quantitative measurement of calprotectin using this setup based either on the intensity of the test dots or on the ratio of test dots to control dots. [Figure 6-B] B) Complete lateral flow setup with anti-calprotectin antibody at the test line and recombinant calprotectin at the control line. This setup is suitable for the detection of 950ng / mL calprotectin from serum or blood samples as well as from buffer samples. [Figure 6-C-D] C) Various linear calibration curves based on the T / C ratio (ratio of intensity of the test line to the control line) of buffer or serum spiked with recombinant calprotectin or with natural calprotectin from granulocytes. D) Comparison of the T / C ratios of sera from 18 rheumatoid arthritis (RA) patients and 9 healthy donors illustrates that peptide-based lateral flow based peptides may have diagnostic value for inflammatory diseases with elevated calprotectin concentrations. [Figure 6-E] E) Receiver operating curve analysis based on these data showed that the area under the curve was less than 0.858.

[0021] [Figure 7]Figure 7 shows the L-alanine, D-alanine, and β-alanine scans of peptide 3. The KD values ​​in the figure are the average of the KD values ​​obtained by kinetic and steady-state analysis in SPR. Compounds that gave signals less than 2 response units (much lower than the 40 unit change typically observed for peptide 3) at the highest concentration tested (500 nM) were assigned KD values ​​>10-5 M. The green dashed line indicates the KD of peptide 3. The SPR data correlate well with affinity measurements in a fluorescence polarization (FP)-based assay: 95% of the compounds that showed binding in SPR (KD values ​​less than 10-5 M) showed binding in FP by an increase in fluorescence anisotropy of at least 4 units at 330 nM calprotectin.

[0022] [Figure 8] FIG. 8 shows the mutation of amino acids Phe8, Met10, Phe11, His14, and Tyr16 in peptide 3, which were found to be most important for binding in the L-alanine scan. All positions were mutated to at least one aromatic amino acid (Phe, Tyr, or Trp), Glu (as a representative of charged amino acids), Gln (as a representative of polar amino acids), and Ile (as a representative of aliphatic amino acids). In addition, several mutations were examined. The KD values ​​in the figure are the average KD values ​​obtained by kinetic and steady-state analysis in SPR. Compounds that gave signals of less than 2 response units (much lower than the 40 unit change usually observed for peptide 3) at the highest concentration tested (500 nM) were assigned KD values ​​>10-5 M. The green dashed line indicates the KD of peptide 3. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] In one embodiment the invention relates to a peptide capable of binding to calprotectin, said peptide comprising the sequence ΦΖΨΣΧΘΘΘΩ, Φ is L-phenylalanine, L-tryptophan, or an analog thereof; X and Z represent α-amino acids in the L-configuration, Ψ represents a non-polar aliphatic L-amino acid; Σ is L-tyrosine, L-tryptophan, L-phenylalanine, or an aromatic L-amino acid, Θ represents an α-amino acid, Ω represents a hydrophobic L-amino acid; Each occurrence of Φ, each occurrence of Z, each occurrence of X, each occurrence of Ψ, each occurrence of Σ, each occurrence of Θ, and each occurrence of Ω are selected independently of one another.

[0024] As used herein, when a variable is defined as "x, x, or x," this phrase is considered equivalent to the variable "selected from the group consisting of x, x, and x."

[0025] As used herein, the term "peptide" refers to an amino acid chain having a maximum length of 200 amino acids.

[0026] As used herein, the term "amino acid" refers to an organic compound containing amino and carboxylate functional groups and, optionally, one or more side chains which may carry functional groups. In amino acids having a carbon chain attached to the a-carbon (e.g., lysine), the carbons are designated α, β, γ, δ, etc. In some amino acids, the amine group is attached to, for example, the α-, β-, or γ-carbon, which are referred to as α-, β-, or γ-amino acids, respectively.

[0027] Proteinogenic amino acids, also called naturally occurring amino acids, are amino acids that are biosynthetically incorporated into proteins during translation. In addition to amino acids encoded by naturally occurring base triplets, proteinogenic amino acids include selenocysteine ​​and pyrrolysine.

[0028] Non-proteinogenic amino acids are amino acids that are not coded for but can nevertheless be integrated into peptides. Those skilled in the art know which compounds fall under the definition of non-proteinogenic amino acids. Non-proteinogenic amino acids include, for example, all-S, all-E-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid (ADDA), B-alanine, 4-aminobenzoic acid, γ-aminobutyric acid, S-aminoethyl-L-cysteine, 2-aminoisobutyric acid, aminolevulinic acid, azetidine-2-carboxylic acid, canaline, canavanine, carboxyglutamic acid, chloroalanine, citrulline, cysteine, dehydroalanine, These include diaminopimelic acid, dihydroxyphenylglycine, endoracidin, homocysteine, homoserine, 4-hydroxyphenylglycine, hydroxyproline, hypusine, lanthionine, B-leucine, mimosine, norleucine, norvaline, ornithine, penicillamine, placohypaforin, pyroglutamic acid, quisqualic acid, sarcosine, theanine, tranexamic acid, trichoromic acid and 3,4-dihydroxyphenylalanine (L-DOPA).

[0029] The term "(amino acid) derivative" is defined herein as a proteinogenic or non-proteinogenic amino acid modified by the addition or substitution of individual functional groups.

[0030] Amino acids are generally classified according to the chemical nature of their side chains, i.e., branching from the parent structure of the amino acid. Aromatic and aliphatic amino acids are terms known in the art. Aromatic amino acids contain at least one aromatic ring. Aromatic amino acids include phenylalanine, tryptophan, tyrosine, and their derivatives. Aliphatic amino acids contain at least one aliphatic side chain or a side chain that exhibits properties similar to aliphatic side chains (i.e., non-polar and hydrophobic). Aliphatic amino acids include alanine, leucine, isoleucine, norleucine, proline, valine, methionine, and their derivatives. Hydrophobic amino acids include tyrosine, phenylalanine, tryptophan, and isoleucine.

[0031] The inventors have surprisingly identified the sequence ΦΖΨΣΧΘΘΘΩ, where Φ is L-phenylalanine, L-tryptophan or analogues thereof, Χ and Z represent α-amino acids in the L-configuration, Ψ represents a non-polar aliphatic L-amino acid, Σ is L-tyrosine, L-tryptophan, L-phenylalanine or an aromatic L-amino acid, Θ represents an α-amino acid and Ω represents a hydrophobic L-amino acid. Also, each occurrence of Φ, each occurrence of Z, each occurrence of Χ, each occurrence of Ψ, each occurrence of Σ, each occurrence of Θ and each occurrence of Ω are independently selected as peptides capable of binding native tetrameric calprotectin with high affinity. Advantageously, calprotectin dimers are bound with significantly lower affinity. Because the peptides of the invention bind to the interface between Mrp8 and Mrp14 that is only fully formed upon tetramerization (Figure 4), the peptides of the invention are therefore particularly suitable for recognising, detecting and purifying the naturally occurring tetrameric form of calprotectin.

[0032] In one embodiment, the peptides of the invention have an equilibrium dissociation constant K between 1 pM and 750 nM. D In a preferred embodiment, K D is less than 200 nM, more preferably less than 50 nM. The calprotectin binding ability of a given peptide can be assessed by methods known to those skilled in the art, such as surface plasmon resonance, fluorescence polarization or biolayer interferometry.

[0033] In one embodiment, Ψ represents L-methionine, L-leucine, L-isoleucine or L-norleucine. In another embodiment that may be combined with the previous embodiment, Z represents L-proline or L-alanine.

[0034] In another embodiment, the peptide of the present invention meets at least one of the following criteria: (1) Φ is L-phenylalanine or a derivative thereof; (2) Z is L-proline or a derivative thereof; (3) Ψ is L-methionine or a derivative thereof; (4) Σ is L-phenylalanine or a derivative thereof; (5) Ω is L-tyrosine or a derivative thereof.

[0035] In a preferred embodiment, the peptide satisfies condition (1) and at least one of conditions (2), (3), (4) or (5). In another embodiment, the peptide satisfies condition (2) and at least one of conditions (1), (3), (4) or (5). In another embodiment, the peptide satisfies condition (3) and at least one of conditions (1), (2), (4) or (5). In another preferred embodiment, the peptide satisfies conditions (1) and (2). In another preferred embodiment, the peptide satisfies conditions (1) and (2) and at least one of conditions (3), (4) or (5). In another preferred embodiment, the peptide satisfies conditions (1), (2) and (3) and at least one of conditions (4) or (5). In a particularly preferred embodiment, the peptide satisfies all five conditions.

[0036] In another preferred embodiment, the peptide comprises the sequence FPLFQΘXΘY, FPIFQΘXΘY, FP(Nle)FQΘXΘY, FPLFQΘXΘF, WPLFQΘXΘY, FPIFQΘXΘF, FP(Nle)FQΘXΘF, WPIFQΘXΘY, WPLFQΘXΘY, WPIFQΘXΘF, WP(Nle)FQΘXΘF, or FZMFXΘHΘY.

[0037] In particularly preferred embodiments the peptide comprises the sequence FPLFQΘXΘY, FPIFQΘXΘY, FP(Nle)FQΘXΘY, FPLFQΘXΘF or WPLFQΘXΘY, most preferably FPLFQΘXΘY.

[0038] The peptides according to the invention preferably consist of 9-30 amino acids, more preferably 15-20 amino acids, most preferably 18 amino acids. Their small size compared to antibodies facilitates chemical synthesis, provides better tissue penetration and higher resistance to cleavage and inactivation by proteases, and extends the half-life of the peptides both in vivo and in vitro. In a particularly preferred embodiment, the peptide has or consists of the sequence RCPECVAFPMFQCHWYCG or RSPESVAFPMFQSHWYSG.

[0039] In another particularly preferred embodiment, the peptide capable of binding to calprotectin is selected from the group consisting of CTQSPCPLYDSHQCSCK, VCPCPLFRAHGCSRFSCQ, CQCPWDLFSQHSLSDCCD, WCTQSPCPLYDSHQCSCK, TCPLNRTQCPLYACTTCP, GCDLAHQPCPLYKCTKCP, VCQQTASRCPVWECQRCP, ACRTCPLFTCPSCG, RCPECVAFPMFQCHWYCG, RSPESVAFPMFQSHWYSG or SCQCPWDLFSQHSLSDCCD. These peptides have been shown to have excellent binding affinity to calprotectin (Figure 1).

[0040] In the peptides of the present invention, cysteine ​​residues may be cross-linked with each other so that each peptide contains two cyclic structures. Such peptides are also called bicyclic peptides. Therefore, the cysteine-containing peptides of the present invention are preferably bicyclic peptides. Due to the presence of one or more disulfide bonds in the peptide, bicyclic peptides are constrained in their conformation, which results in a relatively small entropic cost upon binding, and thus has good binding affinity and specificity. Unlike antibodies, bicyclic peptides can cross the blood-brain barrier.

[0041] In yet another aspect, the present invention relates to a method for detecting calprotectin in a sample, the method comprising the steps of: a) providing a sample containing calprotectin; b) contacting the sample with a peptide according to the invention to form a complex between the peptide and calprotectin; and c) detecting the complex formed in step b).

[0042] In a preferred embodiment, the sample is a biological sample such as a blood sample, a serum sample, a plasma sample, a saliva sample, a urine sample or a stool sample. The method of detecting calprotectin in a biological sample is useful for monitoring inflammatory processes. To facilitate the detection process and to stabilize calprotectin in the sample, the sample may be further purified, stabilized, diluted or otherwise treated.

[0043] According to the method of the invention, the sample is then contacted with a peptide according to the invention, allowing the peptide to form a complex with calprotectin. This step of including the sample with the peptide may take any form suitable for contacting the sample with the peptide. For example, the peptide may be added directly to the sample or the sample may be added to a vessel containing the peptide. In the latter embodiment, the peptide may be immobilized on a solid support or stationary phase.

[0044] In one embodiment, the peptide is detectably labeled. The term "label" herein refers to any entity that can be bound or conjugated to a peptide to facilitate detection of the peptide. Preferred labels used according to the present invention include nanoparticles (e.g., gold nanoparticles), proteins (e.g., streptavidin), enzymes (e.g., horseradish peroxidase), dyes (e.g., luminescent or fluorescent dyes), and small molecules (e.g., biotin). In a preferred embodiment, the peptide of the present invention is labeled with gold.

[0045] In the final step according to the method of the invention, the complex comprising calprotectin and the peptide is detected. Any detection method known in the art can be used. The choice of detection method may depend on the label with which the peptide is labeled. Detection methods useful for application in the method of the invention include optical readout, absorption, UV / VIS spectroscopy, turbidimetry, nephelometry, light scattering, reflectometry, fluorescence, luminescence, chemiluminescence, surface plasmon resonance, amperometry, magnetometry, voltammetry, potentiometry, conductometry, coulometry, polarography, gravimetry and cantilevers.

[0046] In yet another aspect, the present invention relates to a kit for detecting calprotectin in a sample, the kit comprising a peptide according to the present invention. The kit according to the present invention may comprise means for carrying out a method for detecting calprotectin according to the present invention. In a preferred embodiment, the kit may be in the form of a lateral-flow immunoassay (LFI), in which the peptide is detectably labelled with nanoparticles (e.g. cellulose, polystyrol or europium, preferably gold) and coated on a peeling pad or immobilised on a membrane.

[0047] In another preferred embodiment, the kit may be in the form of a particle enhanced turbidimetric immunoassay (PETIA), in which the peptides are bound to nanoparticles, hi another preferred embodiment, the kit may be in the form of an enzyme-linked immunosorbent assay (ELISA), in which the peptides are linked, directly or indirectly, to a detection enzyme, chemiluminescent or fluorescent marker.

[0048] Kits according to the invention may also include buffers, solutions and instructions for carrying out the methods of the invention.

[0049] In another aspect, the invention relates to a method for purifying calprotectin, comprising purifying calprotectin using a peptide according to the invention. Due to their high affinity for calprotectin, the peptides according to the invention can be used for capturing and purifying calprotectin. For example, the method of the invention can be used to purify calprotectin from granulocytes or inclusion bodies. In one embodiment, the peptides according to the invention are immobilized on a stationary phase. In one embodiment, the method comprises the step of contacting calprotectin with a peptide according to the invention.

[0050] In another aspect, the present invention relates to a pharmaceutical composition comprising a peptide according to the present invention.

[0051] In another aspect, the present invention relates to the use of a peptide according to the present invention for tagging a protein of interest. As used herein, the term "tagging" refers to the covalent or non-covalent attachment of a peptide to a protein. A protein of interest can be tagged with a peptide tag linked to calprotectin and subsequently purified. EXAMPLES

[0052] Example 1: Affinity testing of phage display derived synthetic peptides against human calprotectin

[0053] Recombinantly expressed fusion calprotectin (His6-linker-S100A9-linker-S100A8) (product code: B-RACL, BUHLMANN Laboratories AG Schonenbuch, Switzerland) was immobilized on magnetic beads by random biotinylation of amino groups and addition to streptavidin- or neutravidin-coated beads. To avoid enrichment of streptavidin- or neutravidin-specific peptides, the two types of beads were used alternately. Neutravidin beads were prepared by reacting 6 mg of neutravidin (Pierce) with 10 mL of tosyl-activated magnetic beads (Dynal, M-280 from Invitrogen) according to the supplier's instructions.

[0054] Add 500 μL of calprotectin (10 μM) to 5 μL of EZ-Link in 20 mM HEPES (pH 7.5). TM Calprotectin was biotinylated by incubation with Sulfo-NHS-LC-Biotin (ThermoFisher Scientific) (10 mM, final concentration 200 μM, 20-fold molar excess), 150 mM NaCl, and 2 mM CaCl2. The reaction was incubated for 1 h at room temperature. Protein was separated from unreacted reagents using a PD-10 column (GE Healthcare). Samples were concentrated and stored at -80°C.

[0055] Biotinylated proteins were immobilized on magnetic beads by incubating the proteins with pre-washed beads in 200 μL of wash buffer (10 mM Tris, pH 7.4, 150 mM NaCl, 10 mM MgCl2, 2 mM CaCl2) for 30 min at room temperature on a rotating wheel (10 rpm). Then, 3 μL of biotin (1 mM) was added and the remaining positions on the beads were blocked by further incubation for 30 min. The beads were washed three times with 1 mL of wash buffer and resuspended in 400 μL of wash buffer containing 1% BSA and 0.1% Tween-20.

[0056] The phage display library was generated as described (Kong et al. Generation of a Large Peptide Phage Display Library by Self-Ligation of Whole-Plasmid PCR Product ACS Chem. Biol. 2020). The glycerol stock of the library was inoculated into 0.5 L of 2YT / tetracycline (100 μg / mL) culture. A sample was taken to calculate the initial phage titer. The culture was shaken (200 rpm) at 30 °C overnight. The next day, the culture was pelleted at 4500 g at 4 °C and a sample of the supernatant was taken. Phage precipitation was performed by adding 125 mL of chilled PEG / NaCl solution (20% PEG-6000 (w / v), 2.5 M NaCl) followed by incubation on ice for 30 min. The phages were then centrifuged at 6500 g for 45 min at 4 °C. The phage pellet was then resuspended in 15 mL of degassed reaction buffer (20 mM NH4HCO3, pH 8.0, 0.5 mM EDTA). Residual cells were removed by centrifugation at 4500 g for 15 min at 4° C. Phage aliquots were taken before and after precipitation to calculate the phage titer.

[0057] Cysteine ​​residues of the peptides were reduced by adding 1 mM TCEP for 30 min at 25°C. Phages were precipitated again by adding PEG / NaCl and resuspended in 18 mL of degassed reaction buffer. For each linker, 4.5 mL of phage was taken and 30 μM to 40 μM linker was added in 0.5 mL of ACN. These were incubated for 1 h at 30°C, and phage were precipitated again by adding PEG / NaCl. The phage pellet was resuspended in 5 mL of binding buffer (10 mM Tris, pH 7.4, 150 mM NaCl, 10 mM MgCl2, 2 mM CaCl2, 1% BSA, and 0.1% Tween-20) and stored at 4°C overnight.

[0058] To select phages against calprotectin, proteins were immobilized on magnetic beads as described above. 5 μg, 2.5 μg, and 1 μg of target protein were immobilized on 20 μL of streptavidin beads (first and third rounds) or 10 μL of neutravidin beads (second round), respectively. The beads were then added with each modified phage and incubated for 30 min with rotation. Unbound phages were removed by washing the beads eight times with wash buffer (containing 0.1% Tween-20) and three times with wash buffer. The beads were resuspended in 100 μL of glycine buffer (20 mM, pH 2.2) and incubated for five min to elute the phages. The solution was neutralized by adding 100 μL of Tris-Cl buffer (1 M, pH 8.0).

[0059] The eluted phages were incubated with 10 mL of E. coli TG1 cells at OD 600 = 0.4. After incubation at 37 °C for 30 min without shaking, freshly infected bacteria were plated onto 2YT / tetracycline (100 μ / mL) plates and grown overnight at 30 °C. Bacterial cells from overnight grown colonies were harvested in 2YT medium containing 20% ​​glycerol, flash frozen, and stored at -80 °C until the next round of selection.

[0060] For the next round of selection, the scale of phage production was scaled down to 25 mL per linker and the remaining solution volumes were adjusted accordingly. After the third round of selection, 24 clones per linker were sequenced by Sanger sequencing (Macrogen) and the resulting sequences were grouped based on similarity.

[0061] Solid phase peptide synthesis (SPPS) based on these sequences was performed on a MultiPep RSi parallel peptide synthesizer (Intavis Inc.) using Fmoc-chemistry with DMF as solvent and linked amide AM resin. Peptides were synthesized on a 25 μmol scale. Amino acids were coupled twice (2.5 eq.) with HATU (2.5 eq.) and NMM (4 eq.), each coupling was performed for 45 min at room temperature. After the coupling reaction, seven washing cycles with DMF were performed. The N-terminal amines remaining free after coupling were capped with acetic anhydride (5% v / v) and lutidine (6% v / v) for 30 min at room temperature. Seven washing cycles were performed again. The Fmoc group was deprotected twice with piperidine (20% v / v) in DMF for 5 min at room temperature. Seven washing cycles were performed again. The N-terminal carboxyfluorescein moiety was manually incorporated by adding 5(6)-carboxyfluorescein (3 eq.), HATU (4 eq.) and DIEA (3 eq.) for 2 × 45 min, after which the resin was washed with DMF, piperidine (20% v / v), DMF and DCM.

[0062] All cleavage of peptides was performed using a standard cleavage cocktail (90% TFA, 2.5% thioanisole, 2.5% H2O, 2.5% 1,2-ethanediol, 2.5% phenol). 5 mL of the cleavage cocktail was added to each peptide and incubated for 4 h with shaking. The peptide-containing solution was collected by vacuum filtration, after which the peptides were first purified by cold ether precipitation. 50 mL of ice-cold diethyl ether was added to the peptides and incubated at -20°C for 30 min, followed by centrifugation at 2700g for 10 min. The peptide pellet was washed once more with 35 mL of diethyl ether and centrifuged again to remove residual diethyl ether.

[0063] Preparative C18 reversed-phase column (Sunfire TMThe linear peptides were purified using an HPLC system (Prep LC 2535 HPLC, Waters) using a prep C18 OBD 10 μm, 100 Å, 19 × 250 mm, Waters) with a flow rate of 20 mL / min and an appropriate linear gradient (A: HO, 0.1% TFA; B: ACN, 0.1% TFA) in 40 min. Fractions containing the desired peptide were pooled together and lyophilized. This intermediate purification step was only performed for double-bridge peptides or when the crude mixture was too complex to directly cyclize.

[0064] The purified peptide was dissolved in 10 mL of 30% v / v ACN and 70% V / V aqueous buffer (60 mM NH4HCO3, pH 8.0) and the cyclization reagent was added to ACN (3 eq., 100 μL). The reaction mixture was incubated at 30 °C for 1 h and the reaction completion was assessed by LCMS. The reaction was stopped by adding HCOOH (200 μL) and the cyclized peptide was lyophilized. Final purification was performed using a reversed-phase C18 column (X-bridge peptide BEH C18 5 μm, 300 Å, 10 × 250 mm, Waters) at a flow rate of 6 mL / min with an appropriate linear gradient for 400 min. The fractions containing the desired peptide were lyophilized. The purity of the peptides was assessed by analyzing approximately 20 μg of peptides by RP-HPLC (1260 HPLC system, Agilent) using a C18 column (ZORBAX 300SB-C18, 5 μm, 300 Å, 4.6 × 250 mm, Agilent). The peptides were analyzed at a flow rate of 1 mL min -1 A linear gradient of 0–100% of solvent B (A: 94.9% HO, 5% ACN, and 0.1% TFA; B: 99.9% ACN and 0.1% TFA) over 15 min was performed at a flow rate of 100 Hz. Mass was measured by electrospray ionization mass spectrometry (ESI-MS) in positive ion mode using a single quadrupole liquid chromatography mass spectrometer (LCMS-2020, Shimadzu).

[0065] To measure the affinity of the selected and synthesized peptides in fluorescence polarization, calprotectin was serially diluted in 20 mM HEPES, 100 mM NaCl, 2 mM CaCl2, pH 7.5, 1 mM DTT with 0.01% v / v Tween-20. 16 μL of protein was added to 4 μL of fluorescent peptide (final concentration 20 nM) in a 96-well microtiter plate (black, half area). Fluorescence anisotropy was measured in a microwell plate reader (Infinite M200Pro, Tecan) using filters for excitation 485 nm and emission 535 nm. Dissociation constants (K d ) was calculated in Prism 5 (GraphPad) using the following formula:

[0066]

number

[0067] For measurements with calprotectin dimers, the dilution buffer did not contain CaCl2 to avoid tetramer formation. The rest of the protocol was the same.

[0068] The identified and synthesized peptides showed affinities for calprotectin in the nanomolar range (Figure 1).

[0069] Example 2: Identification of a linear binding sequence based on peptide 3 (RCPECVAFPMFQCHWYCG) sufficient to bind calprotectin

[0070] A peptide defined with a pair of cysteines bridged with a chemical linker was synthesized by protecting the two cysteines with a Dpm group and two Mmt groups instead of the previously used Trt. After linear synthesis of the peptide and before total cleavage, the resin was treated with 5 mL of TFA:TIS:DCM (1:5:94) for 8 × 2 min in a fritted syringe. The resin was washed three times with DCM and three times with DMF. 1.5 eq. of cyclization reagent and 4 eq. DIPEA in 4 mL of DMF were added and the reaction mixture was shaken for 1 h at room temperature. The reaction solution was removed and the resin was washed three times with DCM. The resin then underwent comprehensive deprotection with 90% TFA, 2.5% thioanisole, 2.5% H2O, 2.5% 1,2-ethanedithiol, 2.5% phenol for 6–8 h to ensure that all Dpm groups were removed. Ether and HPLC purification was carried out as previously described.

[0071] The linear peptide 3-biotin was synthesized according to the previous protocol, the second Lys installed in the sequence was added as Fmoc-Lys(Dde)-OH, and the last amino acid was incorporated as Boc-Arg(Pbf)-OH. After completion of the synthesis, the protecting group of Lys was removed using 2% hydrazine in DMF, and biotin was introduced at the deprotected amino group. Finally, the peptide was fully deprotected and removed from the resin.

[0072] Subsequent fluorescence polarization experiments were performed according to Example 1. Calprotectin binding affinity was compromised when non-adjacent cysteines in peptide 3 were cross-linked, suggesting that peptide 3 binds calprotectin in an elongated conformation. Consistent with this observation, linear peptides in which all cysteines are replaced by serine show affinity for calprotectin similar to isomers 1, 1a and isomer 1b (Figure 2).

[0073] Example 3: Alanine scanning and affinity measurement by surface plasmon resonance

[0074] The contribution of various amino acids within linear peptide 3 was assessed using alanine scanning. For this, linear peptides were synthesized in which individual amino acids were selectively replaced with alanine. The affinity of these linear peptides for calprotectin was measured by fluorescence polarization (FP) and surface plasmon resonance (SPR).

[0075] To complement and validate the results obtained by FP, the binding reactions and kinetics of peptides with immobilized calprotectin were analyzed by SPR. TM The assay was performed using an 8K instrument (GE Healthcare). Calprotectin (5 μg / mL) was dissolved in 10 mM acetate buffer (pH 5.0) and immobilized on a CM5 series S chip by standard amine coupling methods in running buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 2 mM CaCl2, 0.005% v / v Tween-20) at 25 °C. Typical immobilization levels were 3000 resonance units (RUs). A reference cell was treated similarly but no protein was injected. A single concentration (500 nM) of each peptide was injected in running buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 2 mM CaCl2, 0.005% v / v Tween-20 and 0.5% v / v DMSO) to measure binding levels. For determination of binding kinetics and dissociation constants, five serial dilutions (3-fold) of peptides were prepared in running buffer (containing 0.5% DMSO) and analyzed in single-cycle kinetic mode with contact and dissociation times of 90 and 120 s, respectively.

[0076] Both SPR and FP revealed that the amino acids mainly responsible for calprotectin affinity in linear peptide 3 were F8, M10, F11, H14, and Y16 (Figure 3 ).

[0077] Example 4: Co-crystallization of calprotectin with linear peptide 3

[0078] Fusion calprotectin was expressed, cleaved, and purified as described previously. For crystallization purposes, the cysteines of fusion calprotectin were replaced by serines by site-directed mutagenesis to prevent disulfide bond formation. Prior to crystallization, the His-Tag and the linker sequence between S100A8 and S100A9 were cleaved with 3C precision protease in a 1:200 molar ratio overnight at 4°C. The protein was then concentrated to a final concentration of 11 mg / mL (440 μM) in 20 mM HEPES, 100 mM NaCl, 1 mM CaCl2, pH 7.4 using a centrifuge device with a 3 kDa cutoff. Prior to crystallization, linear peptide 3 (6.2 mM, 14 equivalents) was incubated with the protein to form the complex.

[0079] Crystals of recombinant calprotectin containing peptide were grown at 18°C ​​using sitting drop vapor diffusion. Crystallization conditions were screened using a Mosquito crystal automated robot (SPT Labtech) and PACT Premier (Molecular Dimensions), resulting in the appearance of crystals between the third and fifth day. Droplets contained 200 nL of protein solution and 100 nL of precipitant solution and were equilibrated against 100 μL of precipitant solution in a 96-well intelli-plate (Hampton Research). The best crystals were grown in conditions containing 0.1 mM MIB (disodium malonate monohydrate, imidazole, boric acid), pH 6.0, and 25% w / v PEG1500 as precipitant solution. Crystals were transferred to cryosolution (25% glycerol) and flash frozen in liquid nitrogen.

[0080] Crystal data for the complex of calprotectin with peptide 3 were collected at beamline PXIII of the Swiss Light Source at the Paul Scherrer Institute (SLS, Villigen, Switzerland) at a wavelength of 1.0 Å. The raw data were processed with the program XDS. The structure was solved by molecular replacement using Phenix, with the atomic coordinates of calprotectin (PDBID: 1XK4) as a search model. The peptide was manually modeled into extra density using Coot. The structure was completed by iterative refinement in Phenix and model building in Coot, yielding a final model at 1.85 Å resolution. The calprotectin tetramer is formed by symmetry, with one copy of calprotectin (S100A and S100A9) and one copy of the peptide constituting the asymmetric unit. An initial difference electron density map showed density consistent with the presence of three Ca(II) ions, one Ni(II) ion, one Na(I) ion, and one K(I) ion. The molecular geometry was generated using PyMOL.

[0081] The structure of the peptides with calprotectin shows the exact binding epitope of peptide 3 on calprotectin. Two peptides bind to one calprotectin tetramer in an elongated fashion. The binding epitope involves one calprotectin dimer, Mrp8 / Mrp14, as well as a second dimer, Mrp8. This binding mode also indicates that peptide 3 requires the calprotectin tetramer for binding (Figure 4).

[0082] Example 5: Sandwich ELISA using calprotectin binding peptides

[0083] Wells of a 96-well Immulon 4 HBX-Extra High Binding plate (Thermo Fisher Scientific) were coated with 80 μL of a 0.5 μg / mL solution of anti-calprotectin rabbit polyclonal antibody in Na2HCO3 (0.2 M, pH 9.4) by overnight incubation at 4°C. The plate was rinsed four times with 200 μL of washing buffer (25 mM Tris, 150 mM NaCl, 2 mM CaCl2, pH 7.4, containing 0.05% v / v Tween-20) per well. All wells were then blocked with 300 μL of blocking buffer (25 mM Tris, 150 mM NaCl, pH 7.4, containing 0.05% v / v Tween-20 and 3% v / v BSA) by incubation with shaking for 1 h at room temperature. After this, the blocking buffer was removed and 80 μL of calprotectin was added in dilution buffer (washing buffer containing 1% w / v BSA) and shaken for 1 h at room temperature. The wells were washed four times with washing buffer. Peptide (linear peptide 3-biotin at a final concentration of 50 nM) was premixed with Strep-HRP (four-fold dilution, Thermo Fisher Scientific) for 30 min, added to the wells and shaken for 2 h at room temperature. The solution was removed and the wells were washed six times with washing buffer. 80 μL of TMB substrate (ready-made solution, SigmaAldrich) was added for 30 min and the reaction was stopped with 40 μL of sulfuric acid (2 M). The absorbance was read at 450 nm in a microwell plate reader (Infinite M200Pro, Tecan).

[0084] Although the ELISA results show that C-terminal conjugation with biotin and streptavidin-HRP is favored over N-terminal conjugation, in both setups an ELISA signal that depends on the calprotectin concentration can be measured. Extending the incubation time of the antibody-captured calprotectin complex with Strep-HRP biotinylated peptide to 60 min could allow the detection of calprotectin concentrations below 5 nM (Figure 5).

[0085] Example 6: Use of calprotectin-binding peptides in setting up a lateral flow assay

[0086] First, peptide-gold nanoparticles were prepared: C-terminally biotinylated linear peptide 3 was incubated with streptavidin-coated gold nanoparticles (AuNPs) at a concentration of 1 μM. Free streptavidin sites were blocked by adding excess biotin. The peptide-AuNPs were concentrated by centrifugation and resuspended in binding resuspension buffer. These peptide-gold nanoparticles were used to fabricate half-strip and lateral flow assays, respectively.

[0087] For the half-strip assay, the nitrocellulose membrane was attached to an adhesive mount with an absorbent pad attached to overlap the nitrocellulose membrane by 2 mm. 0.5 cm strips were cut with scissors. Anti-calprotectin antibody was applied as dots in the test area 1.9 cm from the bottom of the nitrocellulose membrane. Recombinant calprotectin (B-RCAL) was applied as dots in the test area 1.5 cm from the bottom of the nitrocellulose membrane. The half-strips were placed vertically into wells of a flat-bottom 96-well plate containing 100 μL of chase buffer containing peptide-AuNPs and calprotectin. The half-strips were removed after 15 min. For quantitative analysis, the intensity of the dots was measured using Image J. Curves were plotted using GraphPad Prism.

[0088] Lateral flow assays were made by assembling the membrane and pad as described above, cutting them into 0.5 cm wide strips, and assembling them into cassette housings. Samples containing calprotectin were applied to the LF strips. Blood was anticoagulated with EDTA and spiked with 9.5 μg / mL B-RCAL. Plasma was obtained by centrifuging the spiked blood at 1,500 × g and 4 °C for 15 min. Blood and plasma samples were diluted 10-fold in chase buffer. Patient samples were collected as serum fractions and diluted 10-fold with chase buffer before being added to the LF strips. To perform the LFA, 80 μL of premixed sample was added to the SAP. After the sample solution was allowed to permeate the membrane for 15 min at room temperature, the appearance of red lines at C and T positions indicated that calprotectin was detected. A negative result was indicated by the appearance of a red line only at the C line. For quantitative analysis, Quantum Blue (R) Intensity was measured using a 3rd generation reader and curves were plotted using GraphPad Prism.

[0089] The prototype lateral flow assay is able to quantitatively measure calprotectin concentrations and in the current setup works with buffer, serum, and capillary blood as matrices requiring individual calibration curves. Furthermore, an initial data set of serum from rheumatoid arthritis (RA) patients showed a significant increase in calprotectin concentrations compared to normal donors in this setup.

[0090] Example 7: Structure-activity relationship (SAR) analysis of peptide 3

[0091] Structure-activity relationship (SAR) analysis of peptide 3 was assessed by synthesizing multiple mutants of peptide 3 in which only one amino acid was changed at a time and measuring the binding affinity by surface plasmon resonance (SPR) and fluorescence polarization (FP).

[0092] In the first part, an L-alanine scan (usually called the alanine scan), a D-alanine scan, and a β-amino acid alanine scan were performed. The L-alanine scan was to provide information on the importance of the side chains of each of the 18 amino acids in peptide 3 (RSPESVAFPMFQSHWYSG). The D-alanine scan was expected to tell us whether the inverse stereocenter of the α-carbon is tolerated for the 18 amino acids. In such mutations, the amino acids have two possibilities to preserve the binding interactions. The first is to keep the orientation of the backbone atoms (amino group, carbonyl, α-carbon) the same and change the position and orientation of the side chain, and the second is to keep the side chain interactions the same but accept a large change in the backbone interactions, which also affect the neighboring amino acids. The results of the D-alanine scan mainly provide valuable information on the positions of the amino acids to which L-alanine is bound, but not on the positions where mutation to L-alanine renders the peptide inactive (because in such cases it is unclear whether the loss of activity is due to the loss of the side chain or the change of the stereocenter). A β-amino acid alanine scan was performed on peptide 3 at position 18 to determine whether a backbone change (the addition of one carbon atom) was tolerated, which would apparently affect the adjacent amino acids. As with the D-alanine scan, the results of the β-amino acid alanine scan were conclusive only if the L-amino acid mutation was active.

[0093] The results of the three "scans" are shown in Figure 7. These results indicate that the side chains of five amino acids are most important for the binding of peptide 3 (F8, M10, F11, H14, Y16). This means that these side chains are likely to contribute most of the binding energy of peptide 3 (results of the L-alanine scan). This result is in total agreement with the phage display selection data, where these positions were the most conserved in peptides rich in calprotectin binding. Surprisingly, the P9 amino acid was not included among the five amino acids whose side chains play an important role, despite the strong conservation of this position in the phage display selection (almost all phage display selected peptides contained proline at this position). L-alanine is well accepted due to its similarity to L-proline, and the side chain at this position may also contribute significantly to the binding affinity of peptide 3.

[0094] Of the 13 amino acids in peptide 3 that were tolerated with minimal loss of binding affinity upon mutation to L-alanine, there are two that showed a large loss of binding when mutated to D-alanine or β-alanine: P9 and Q13. As noted above, P9 may tolerate L-alanine due to its similarity to L-proline, but this position critically does not accept changes in the backbone or α-carbon configuration. For Q13, the results indicate that the side chain at this position is less important, but the backbone region is important, for example to keep adjacent amino acids ideally spaced and oriented. It is therefore important that both positions P9 and Q13 are occupied by amino acids that are α-amino acids and have an L-configuration.

[0095] A more extensive side chain scan at five amino acid positions that appear to have important side chain interactions based on the L-alanine scan revealed the extent to which each side chain could be varied and also revealed positions where the side chain could not be modified significantly (Figure 8). Phe8 can be modified with Trp without significant loss of binding, but cannot be substituted with other aromatic amino acids such as Tyr or His. In the case of Tyr, a steric clash between the tyrosine alcohol at the para position and calprotectin is the main culprit. Met10 could be substituted with other aliphatic amino acids (Ile, Leu, and Nle) without loss of binding. This result encourages future substitutions to avoid methionine oxidation. Phe11 tolerated both aromatic side chains of Tyr and Trp. His14 and Tyr16 tolerated a variety of different amino acids with only a slight decrease in affinity. Interestingly, mutation of Tyr16 to Phe did not result in loss of binding, suggesting that the alcohol at Tyr16 plays a minor role.

Claims

1. A peptide capable of binding to calprotectin, said peptide comprising the sequence ΦΖΨΣΧΘΘΘΩ; Φ is L-phenylalanine, L-tryptophan, or an analog thereof; X and Z represent α-amino acids in the L-configuration; Ψ represents a non-polar aliphatic L-amino acid; Σ is L-tyrosine, L-tryptophan, L-phenylalanine, or an aromatic L-amino acid, Θ represents an α-amino acid, Ω represents a hydrophobic L-amino acid; Φ at each occurrence, Z at each occurrence, X at each occurrence, Ψ at each occurrence, Σ at each occurrence, Θ at each occurrence, and Ω at each occurrence are selected independently of each other, peptides.

2. 2. The peptide of claim 1, wherein Ψ is L-methionine, L-leucine, L-isoleucine, or L-norleucine, and / or Z is L-proline or L-alanine.

3. The following conditions: Φ is L-phenylalanine or a derivative thereof; Z is L-proline or a derivative thereof; Ψ is L-methionine or a derivative thereof; Σ is L-phenylalanine or a derivative thereof, and / or Ω is L-tyrosine or a derivative thereof; The peptide according to claim 1 or 2, which satisfies at least one of the above.

4. 4. The peptide of any one of claims 1 to 3, wherein the peptide comprises the sequence FPLFQΘΘY, FPIFQΘΘY, FP(Nle)FQΘΘY, FPLFQΘΘF, WPLFQΘΘY, FPIFQΘΘΘF, FP(Nle)FQΘΘΘF, WPIFQΘΘΘY, WP(Nle)FQΘΘΘY, WPLFQΘΘF, WPIFQΘΘΘF, WP(Nle)FQΘΘΘF, or FZMFXΘHΘY.

5. The peptide according to any one of claims 1 to 4, wherein the peptide consists of 9 to 30 amino acids.

6. A peptide according to any one of claims 1 to 5 having the sequence RCPECVAFPMFQCHWYCG or RSPESVAFPMFQSHWYSG.

7. 2. The peptide of claim 1, comprising the sequence CTQSPCPLYDSHQCSCK, VCPCPLFRAHGCSRFSCQ, CQCPWDLFSQHSLSDCCD, WCTQSPCPLYDSHQCSCK, TCPLNRTQCPLYACTTCP, GCDLAHQPCPLYKCTKCP, VCQQTASRCPVWECQRCP, ACRTCPLFTCPSCG, or SCQCPWDLFSQHSLSDCCD.

8. The equilibrium dissociation constant K of the peptide to calprotectin Ｄ The peptide according to any one of claims 1 to 7, wherein the affinity of the peptide is between 1 pM and 750 nM.

9. 1. A method for detecting calprotectin in a sample, comprising the steps of: d) providing a sample containing calprotectin; e) contacting said sample with a peptide according to any one of claims 1 to 8 to form a complex between said peptide and said calprotectin; f) detecting the complex formed in step b); The method includes:

10. The method of claim 9, wherein the peptide is detectably labeled, preferably with a nanoparticle, streptavidin, biotin, a fluorescent marker, a luminescent marker or an enzyme.

11. A kit for detecting calprotectin in a sample, comprising a peptide according to any one of claims 1 to 8.

12. A method for purifying calprotectin, comprising purifying calprotectin using a peptide according to any one of claims 1 to 8.

13. The method of claim 9 , wherein the peptide is immobilized on a stationary phase.

14. A pharmaceutical composition comprising a peptide according to any one of claims 1 to 8.

15. Use of a peptide according to any one of claims 1 to 8 for tagging a protein of interest.

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