Calprotectin assay

Abstract

Disclosed herein are immunoassay methods for detecting and quantifying human neutrophil elastase (HNE)-generated fragments of calprotectin in biological fluid samples, as well as monoclonal antibodies and assay kits for use in such methods. The methods can be used to detect, monitor, and / or determine the condition or severity of diseases characterized by or manifested by inflammation in patients.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to immunoassay methods for detecting and preferably quantifying calprotectin fragments generated by human neutrophil elastase (HNE) in biological fluid samples, and the use of such methods to detect, monitor, and / or determine the state or severity of diseases characterized by or manifested by inflammation, including, but not limited to, diseases driven by inflammation, in a patient. The invention also relates to monoclonal antibodies and assay kits for use in such methods. [Background technology]

[0002] background Calprotectin is expressed intracellularly, particularly in neutrophil granulocytes, where it accounts for approximately 60% of the total cytosolic material. 1 Calprotectin is a ligand for TLR receptors and is relatively protease-resistant. 2 Neutrophil granulocytes are specialized hematopoietic cells from the myeloid lineage and a subgroup of cells from the innate immune system. They serve as the first line of defense against pathogen and bacterial invasion and are therefore highly expressed in inflamed tissues. 3~5 .

[0003] Calprotectin can be measured in the stool of patients with inflammatory bowel disease (IBD) (fecal calprotectin). 1 Fecal calprotectin has been shown to be a robust sandwich ELISA biomarker for distinguishing patients with IBD from those with irritable bowel syndrome. 6~8 Because serum / plasma samples are much easier to handle than stool samples and stool consistency has been shown to affect fecal calprotectin levels, which vary widely from day to day and even between stools, many fecal calprotectin assays are being tested to see if they can also be applied to serum / plasma samples. However, calprotectin has a very short half-life in plasma (5 hours). 9Currently available data regarding serum / plasma calprotectin and its usefulness as a biomarker applicable to IBD are controversial. 10~15 .

[0004] Activation of neutrophil granulocytes generates neutrophil extracellular traps (NETs), in which the cells secrete all nuclear and cytosolic material in an attempt to capture and damage, for example, invading bacteria. 16 Calprotectin and human neutrophil elastase (HNE) are secreted into inflamed tissue as a result of NET formation. 17 As a result, tissue and extracellular matrix (ECM) and other secreted proteins are degraded by HNE, resulting in the generation of neoepitope-containing protein fragments. 18 ' 19 In addition, HNE has been shown to be associated with tissue inflammation in IBD. 20 Neoepitope-containing protein fragments from collagen degradation and formation have been demonstrated to be measured in serum from IBD patients and preclinical models and correlated with clinical disease parameters. 18 ' 21~25 .

[0005] Inflammation-induced release of calprotectin and HNE also occurs in other diseases. Chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF) are characterized by extensive inflammation and extracellular matrix (ECM) remodeling, which potentially leads to a severe decline in lung function over time. 34 ' 35 Neutrophils are highly abundant in COPD-affected lungs and cause persistent tissue damage. 36 Patients with IPF experience similar persistent damage due to fibrotic changes, and increased HNE release has been observed in bronchoalveolar lavage fluid in patients with IPF. 37 Neutrophils are cellular responders to inflammation and can release both the protease NE and the protein calprotectin. 38 .

[0006] Inflammation also predisposes to cancer development and promotes all stages of tumorigenesis. Cancer cells and surrounding stromal and inflammatory cells engage in well-orchestrated interactions to form the inflammatory tumor microenvironment (TME). 39 Immune checkpoint inhibitors, including but not limited to anti-PD-1 therapy, are a class of drugs used to treat cancers such as metastatic melanoma. However, peripheral biomarkers associated with response and resistance to anti-PD-1 therapy and other such immune checkpoint inhibitors in metastatic melanoma patients represent an unmet medical need. High neutrophil activity is also associated with the dysfunction of immune checkpoint inhibitors, due in part to the release of NETs, ​​which have been shown to protect tumor cells from cytotoxic attack. As noted above, HNE and calprotectin are major NET components. Summary of the Invention [Means for solving the problem]

[0007] The inventors theorized that HNE-generated neoepitope-containing fragments of calprotectin may be a measure of active local tissue inflammation and activated leukocytes, including neutrophils, rather than systemic inflammation or circulating leukocytes. Furthermore, the inventors developed a robust and reliable immunoassay for detecting and quantifying HNE-generated neoepitope-containing fragments of calprotectin in biological fluids, such as serum and plasma, and demonstrated the use of the immunoassay in assessing inflammation and disease activity in diseases such as IBD, COPD, IPF, metastatic melanoma, SCLC / NSCLC, rheumatoid arthritis, ankylosing spondylitis, psoriasis, psoriatic arthritis, and osteoarthritis. [Effects of the Invention]

[0008] Thus, in a first aspect, the present invention relates to an immunoassay method for detecting HNE-generated calprotectin fragments, comprising contacting a sample of a human biological fluid with a monoclonal antibody that specifically recognizes and binds to an HNE-generated neoepitope consisting of an N-terminal or C-terminal sequence of an HNE-generated calprotectin fragment, and detecting binding between the monoclonal antibody and a peptide in the sample.

[0009] Preferably, the detection is quantitative and the method further comprises determining the amount of binding between said monoclonal antibody and the peptide in the sample.

[0010] In a preferred embodiment, the method is an immunoassay method for detecting and / or monitoring the progression and / or determining the state or severity of a disease in a patient, wherein the disease is characterized or manifested by inflammation, comprising contacting a sample of biological fluid obtained from said patient with said monoclonal antibody, detecting and determining the amount of binding between the monoclonal antibody and a peptide in the sample, and correlating said amount of binding with values ​​associated with normal healthy subjects and / or values ​​associated with a known state or severity of the disease and / or values ​​obtained from said patient at a previous time point and / or a predetermined cut-off value.

[0011] The disease can be, for example, a disease driven by inflammation, such as inflammatory bowel disease (IBD), rheumatoid arthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, osteoarthritis, Sjogren's syndrome, or lupus.

[0012] In a preferred embodiment, the disease is inflammatory bowel disease (IBD). In particular, the immunoassay method can be a method for detecting and / or monitoring the progression of and / or determining the condition or severity of multiple types of inflammatory bowel disease (IBD), such as ulcerative colitis (UC) and / or Crohn's disease (CD).

[0013] In another preferred embodiment, the disease is rheumatoid arthritis, ankylosing spondylitis, psoriasis, psoriatic arthritis or osteoarthritis.

[0014] In other preferred embodiments, the disease may be chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF) or asthma.

[0015] In yet other embodiments, the disease may be cancer. The cancer may be, for example, breast cancer, prostate cancer, lung cancer, gastric cancer, colorectal cancer, pancreatic cancer, melanoma, ovarian cancer, kidney cancer, head and neck cancer, or bladder cancer. The cancer may be, for example, metastatic cancer. In certain embodiments, the cancer may be, for example, metastatic melanoma, small cell lung cancer (SCLC), or non-small cell lung cancer (NSCLC).

[0016] Where the method is a method of determining disease status or severity, the method may be for assessing, for example, whether the disease is active or in remission, or for assessing potential patient survival or progression-free survival, or for assessing potential response to a medical intervention, such as potential patient survival or progression-free survival following treatment with one or more drugs (such as one or more chemotherapeutic agents (i.e., cytotoxic agents) and / or immune checkpoint inhibitors).

[0017] The monoclonal antibody may specifically recognize and bind to an HNE-generated neoepitope consisting of the N-terminal or C-terminal sequence of any HNE-generated calprotectin fragment, such as any of the peptides listed in Table 2 below. However, in a preferred embodiment, the monoclonal antibody specifically recognizes and binds to the following HNE-generated calprotectin fragment: KLGHPDTLNQGEFKELV (also referred to herein as "NBH-222") (SEQ ID NO: 1) RKDLQNFLKKENKNEKV ("NBH-223") (SEQ ID NO: 2) RKDLQNFLKKENKNEKVI (SEQ ID NO: 3) EHIMEDLDTNADKQL (SEQ ID NO: 4) SHEKMHEGDEGPGHHHKPGLGEGTP ("NBH-224") (SEQ ID NO: 5) YRDDLKKLLET ("NBH-225") (SEQ ID NO: 6) WFKELDINTDGAV ("NBH-226") (SEQ ID NO: 7) The peptide specifically recognizes and binds to the N-terminal or C-terminal sequence of a peptide selected from the following:

[0018] In a particularly preferred embodiment, the monoclonal antibody specifically recognizes and binds to the N- or C-terminal sequence of the peptide KLGHPDTLNQGEFKELV ("NBH-222") (SEQ ID NO: 1).

[0019] In a preferred embodiment, the monoclonal antibody specifically recognizes and binds to the N-terminal sequence of the HNE-generated calprotectin fragment. Preferably, the monoclonal antibody does not specifically recognize or bind to an N-extended or N-truncated version of the N-terminal amino acid sequence. In this context, "an N-extended or N-extended version of the N-terminal amino acid sequence" means that one or more amino acids are extended beyond the N-terminus of the sequence. Similarly, "an N-truncated or N-truncated version of the N-terminal amino acid sequence" means that one or more amino acids have been removed from the N-terminus of the sequence. Thus, for example, if a monoclonal antibody specifically recognizes and binds to the N-terminal sequence of the peptide KLGHPDTLNQGEFKELV "NBH-222" (SEQ ID NO: 1), the "N-extended or N-extended version" is VKLGHPDTLNQ... (SEQ ID NO: 8) and the "N-truncated version" is LGHPDTLNQ... (SEQ ID NO: 9).

[0020] In certain other embodiments, the monoclonal antibody specifically recognizes and binds to the C-terminal sequence of an HNE-generated calprotectin fragment; preferably, the monoclonal antibody does not specifically recognize or bind to a C-extended version of the C-terminal amino acid sequence or a C-truncated version of the C-terminal amino acid sequence. In this context, "a C-extended version of the C-terminal amino acid sequence" means that one or more amino acids are extended beyond the C-terminus of the sequence. Similarly, "a C-truncated version of the C-terminal amino acid sequence" means that one or more amino acids have been removed from the C-terminus of the sequence. Thus, for example, if a monoclonal antibody specifically recognizes and binds to the C-terminal sequence of the peptide KLGHPDTLNQGEFKELV "NBH-222" (SEQ ID NO: 1), then the "C-extended version" is ...LNQGEFKELVR (SEQ ID NO: 10) and the "C-truncated version" is ...LNQGEFKEL (SEQ ID NO: 11).

[0021] The monoclonal antibody is preferably a monoclonal antibody raised against a synthetic peptide containing the N-terminal or C-terminal sequence. Thus, for example, if a monoclonal antibody specifically recognizes and binds to the N-terminal sequence of the peptide KLGHPDTLNQGEFKELV ("NBH-222") (SEQ ID NO: 1), the monoclonal antibody can be a monoclonal antibody raised against a synthetic peptide having the sequence KLGHPDTLNQ (SEQ ID NO: 12) so as to specifically recognize and bind to the N-terminal sequence of the peptide KLGHPDTLNQGEFKELV (SEQ ID NO: 1). Suitable exemplary protocols for raising monoclonal antibodies against synthetic peptides are described in the Examples below.

[0022] In one exemplary embodiment, the monoclonal antibody specifically recognizes and binds to the N-terminal sequence of the peptide KLGHPDTLNQGEFKELV "NBH-222" (SEQ ID NO: 1), and preferably does not specifically recognize or bind to an N-extended extended form of said N-terminal amino acid sequence or an N-truncated truncated form of said N-terminal amino acid sequence, and the monoclonal antibody preferably CDR-L1: KSSQSLLNSGNQKNYLA (SEQ ID NO: 13) CDR-L2: GASTRES (SEQ ID NO: 14) CDR-L3: LNDHSYPYT (SEQ ID NO: 15) CDR-H1: DHVIN (SEQ ID NO: 16) CDR-H2: EIYPGSGSTYYNEKFKG (SEQ ID NO: 17) CDR-H3: FAY (SEQ ID NO: 18) It may comprise one or more complementarity determining regions (CDRs) selected from:

[0023] Preferably, the monoclonal antibody comprises at least two, three, four, five or six of the CDR sequences listed above.

[0024] Preferably, the monoclonal antibody comprises a light chain variable region comprising the CDR sequences: CDR-L1: KSSQSLLNSGNQKNYLA (SEQ ID NO: 13) CDR-L2: GASTRES (SEQ ID NO: 14) and CDR-L3: LNDHSYPYT (SEQ ID NO: 15) It has.

[0025] Preferably, the monoclonal antibody has a light chain comprising framework sequences between the CDRs that are substantially identical to or substantially similar to the framework sequences between the CDRs in the light chain sequence below (in which the CDRs are shown in bold and underlined and the framework sequences are shown in italics): KSSQSLLNSGNQKNYLA WYQQKPGQPPKLLIY GASTRES GVPDRFTGSGSGTDFLTISSVQAEDLAVYYC LNDHSYPYT (SEQ ID NO: 19)

[0026] Preferably, the monoclonal antibody comprises a heavy chain variable region comprising the CDR sequences: CDR-H1: DHVIN (SEQ ID NO: 16) CDR-H2: EIYPGSGSTYYNEKFKG (SEQ ID NO: 17) and CDR-H3: FAY (SEQ ID NO: 18) It has.

[0027] Preferably, the monoclonal antibody has a heavy chain comprising framework sequences between the CDRs that are substantially identical to or substantially similar to the framework sequences between the CDRs in the heavy chain sequence below (in which the CDRs are shown in bold and underlined and the framework sequences are shown in italics): DHVIN WVRQRTGQGLEWIG EIYPGSGSTYYNEKFKG KATLTADKSSNTAYMQLSSLTSEDSAVYFCAW FAY (SEQ ID NO: 20)

[0028] Preferably, the monoclonal antibody comprises the light chain variable region sequence: DIVMTQSPSSLSVSAGEKVTMSC KSSQSLLNSGNQKNYLA WYQQKPGQPPKLLIY GASTRES GVPDRFTGSGSGTDFLTISSVQAEDLAVYYC LNDHSYPYT FGGGTKLEIK (SEQ ID NO: 21) (CDRs are bold and underlined; framework sequences are italicized) and / or heavy chain variable region sequence: QVQLQQSGPELVKPGASVKMSCKASGYTFT DHVIN WVRQRTGQGLEWIG EIYPGSGSTYYNEKFKG KATLTADKSSNTAYMQLSSLTSEDSAVYFCAW FAY WGQGTLVTVSA (SEQ ID NO: 22) (CDRs are bold and underlined; framework sequences are italicized) Includes:

[0029] The biological fluid sample can be any type of biological fluid, such as, for example, blood, urine, synovial fluid, serum, bronchoalveolar lavage fluid (BALF), or plasma, etc. However, in preferred embodiments, the biological fluid sample is plasma or serum.

[0030] The immunoassay may be, but is not limited to, a competitive assay or a sandwich assay. Similarly, the immunoassay may be, but is not limited to, an enzyme immunoassay (EIA) or a radioimmunoassay. Preferably, the immunoassay is a competitive assay. Preferably, the immunoassay is an enzyme-linked immunosorbent assay (ELISA), particularly a competitive ELISA.

[0031] In a second aspect, the present invention relates to a monoclonal antibody which specifically recognises and binds to an HNE-generated neoepitope consisting of an N-terminal or C-terminal sequence of an HNE-generated calprotectin fragment, the monoclonal antibody being suitable for use in an immunoassay according to the first aspect of the invention, and preferred and optional alternative embodiments of the monoclonal antibody according to the second aspect of the invention will be apparent from the above discussion of monoclonal antibodies for use in the first aspect of the invention and any of its preferred alternative embodiments.

[0032] In a third aspect, the present invention provides a method for the treatment of a cancer cell comprising administering to a patient a monoclonal antibody according to the second aspect of the invention; -Streptavidin-coated well plates; biotinylated peptides comprising said N-terminal or C-terminal sequence linked to biotin; - secondary antibodies for use in sandwich immunoassays; a calibrator peptide comprising said N-terminal or C-terminal sequence; -Antibody biotinylation kit; -Antibody HRP labeling kit; and -Antibody Radiolabeling Kit and at least one of the following:

[0033] In a preferred embodiment, the immunoassay kit comprises a monoclonal antibody according to the second aspect of the invention; -Streptavidin-coated well plates; biotinylated peptides comprising said N-terminal or C-terminal sequence linked to biotin; - a calibrator peptide containing the N-terminal or C-terminal sequence Includes one, two or all of the following:

[0034] For example, if a monoclonal antibody binds to the N-terminal sequence of the peptide KLGHPDTLNQGEFKELV ("NBH-222") (SEQ ID NO: 1), a suitable biotinylated peptide may be a peptide having the sequence KLGHPDTLNQ-L-biotin (SEQ ID NO: 23), where L is an optional linker. Similarly, if a monoclonal antibody binds to the N-terminal sequence of the peptide KLGHPDTLNQGEFKELV ("NBH-222") (SEQ ID NO: 1), a suitable calibrator peptide may be a peptide having the sequence KLGHPDTLNQ (SEQ ID NO: 12).

[0035] definition As used herein, the terms "peptide" and "polypeptide" are used interchangeably.

[0036] The term "monoclonal antibody" as used herein refers to both whole antibodies and fragments thereof that retain the binding specificity of the whole antibody, such as, for example, Fab fragments, Fv fragments, or other such fragments known to those skilled in the art. Antibodies that retain the same binding specificity may contain the same complementarity-determining regions (CDRs). The CDRs of an antibody are described by Kabat et al. 33 This can be determined using methods known in the art, such as those described by

[0037] Antibodies can be generated from B cell clones as described in the Examples. The antibody isotype can be determined by ELISA specific for human IgM, IgG, or IgA isotypes, or human IgG1, IgG2, IgG3, or IgG4 subclasses. Other suitable methods can be used to identify the isotype.

[0038] The amino acid sequence of the produced antibody can be determined using standard techniques. For example, RNA can be isolated from cells and used to generate cDNA by reverse transcription. The cDNA is then subjected to PCR using primers that amplify the heavy and light chains of the antibody. For example, primers specific to the leader sequences of all VH (variable heavy) sequences can be used together with a primer that binds to a sequence located in the constant region of a previously determined isotype. The light chain can be amplified using a primer that anneals to the Vkappa or Vlambda leader sequence and a primer that binds to the 3' end of the kappa or lambda chain. Full-length heavy and light chains can then be generated and sequenced.

[0039] As used herein, a framework amino acid sequence between the CDRs of an antibody is "substantially identical" or "substantially similar" to a framework amino acid sequence between the CDRs of another antibody if they share at least 70%, 80%, 90%, or at least 95% similarity or identity. Similar or identical amino acids may be contiguous or non-contiguous. A framework sequence may contain one or more amino acid substitutions, insertions, and / or deletions. Amino acid substitutions may be conservative, meaning that the substituted amino acid has similar chemical properties to the original amino acid. Those skilled in the art will understand which amino acids share similar chemical properties. For example, the following groups of amino acids share similar chemical properties, such as size, charge, and polarity: Group 1 Ala, Ser, Thr, Pro, Gly; Group 2 Asp, Asn, Glu, Gln; Group 3 His, Arg, Lys; Group 4 Met, Leu, Ile, Val, Cys; and Group 5 Phe, Thy, Trp.

[0040] Amino acid sequences can be compared using programs such as the CLUSTAL program. This program finds the optimal alignment by comparing amino acid sequences and inserting spaces into either sequence as needed. For optimal alignment, it is possible to calculate amino acid identity or similarity (identity plus conservation of amino acid type). Programs such as BLASTx align the longest stretch of similar sequences and assign a value to the fit. In this way, it is possible to obtain a comparison in which several similar regions are found, each with a different score. Both types of analysis are contemplated in the present invention. Identity or similarity is preferably calculated over the entire length of the framework sequence.

[0041] As used herein, the term "N-terminus" refers to the end of a polypeptide, i.e., the N-terminus of a polypeptide, and should not be interpreted in its general orientation. Similarly, the term "C-terminus" refers to the end of a polypeptide, i.e., the C-terminus of a polypeptide, and should not be interpreted in its general orientation.

[0042] As used herein, the term "competitive immunoassay" refers to an immunoassay in which a target peptide (if any) present in a sample competes with a known amount of peptide target (e.g., bound to an immobilized substrate or labeled) for binding to an antibody, a technique known to those skilled in the art.

[0043] The term "target peptide" as used herein refers to a peptide that comprises or consists of an HNE-generated neoepitope consisting of the N-terminal or C-terminal sequence of an HNE-generated calprotectin fragment, which is specifically recognized and bound by a monoclonal antibody.

[0044] As used herein, the term "sandwich immunoassay" refers to an immunoassay that uses at least two antibodies for the detection of an antigen in a sample, and is a technique known to those skilled in the art.

[0045] As used herein, the term "ELISA" (enzyme-linked immunosorbent assay) refers to an immunoassay in which a target peptide (if any) present in a sample is detected using an antibody conjugated to an enzyme such as horseradish peroxidase or alkaline phosphatase. The activity of the enzyme is then assessed by incubation with a substrate that produces a measurable product. The presence and / or amount of the target peptide in the sample can thereby be detected and / or quantified. ELISA is a technique known to those skilled in the art.

[0046] The term "amount of binding" as used herein refers to the quantification of binding between a monoclonal antibody and a target peptide, which is determined by comparing the measured value of the target peptide in a biological fluid sample with a standard curve, which is prepared using a standard sample of known concentration of the target peptide. In a specific assay disclosed below, in which a target peptide having an HNE-generated neoepitope consisting of the N-terminal sequence of the HNE-generated calprotectin fragment KLGHPDTLNQGEFKELV ("NBH-222") (SEQ ID NO: 1) is measured in a biological fluid, the standard curve is prepared using a standard sample of known concentration of the calibration peptide having the N-terminal amino acid sequence KLGHPDTLNQ (SEQ ID NO: 12) (and may particularly consist of the amino acid sequence KLGHPDTLNQ (SEQ ID NO: 12)). The value measured in the biological fluid sample is compared with the standard curve to determine the actual amount of the target peptide in the sample.

[0047] As used herein, a "predetermined cutoff value" refers to an amount of binding that is statistically determined to be indicative of a high likelihood of a disease in a patient (i.e., a disease characterized by or exhibiting inflammation, such as, for example, an inflammation-driven disease) or a particular condition or severity thereof (such as an active disease state or disease prognosis), in the sense that a measurement of the target peptide in a patient sample that is equal to or greater than the statistical cutoff value corresponds to at least a 70% probability of the presence of said disease or said particular condition or severity thereof, preferably at least a 75% probability, more preferably at least an 80% probability, more preferably at least an 85% probability, more preferably at least a 90% probability, and most preferably at least a 95% probability.

[0048] As used herein, the term "value associated with a normal, healthy subject and / or value associated with a known disease state or severity" refers to a standardized amount of a target peptide determined by immunoassay in a subject who is considered healthy, i.e., does not have a disease (i.e., a disease that is characterized by or exhibits inflammation, such as, for example, a disease that is driven by inflammation), and / or a standardized amount of a target peptide determined by immunoassay in a subject who is known to have a disease of a known state or severity (i.e., a disease that is characterized by or exhibits inflammation, such as, for example, a disease that is driven by inflammation). [Brief explanation of the drawings]

[0049] [Figure 1] Figure 1: Overview of the sequences of calprotectin proteins S100A9 and S100A8 (A and B) and their fragments generated by human neutrophil elastase (HNE). The HNE cleavage points are indicated by downward arrows (↓), some of the resulting HNE-generated fragments are highlighted in gray, and the N-terminal sequences that form all or the beginning of the N-terminal neoepitopes of the fragments designated NBH-222, NBH-223, NBH-224, NBH-225, and NBH-226 are highlighted in darker gray shading. [Figure 2]Figure 2: Relative abundance of CPa9-HNE (N-terminal neoepitope of NBH-222) in samples containing HNE-cleaved calprotectin, full-length calprotectin, or HNE tested by mass spectrometry (A); reactivity of the CPa9-HNE antibody and assay tested against selected peptides, extended peptides, truncated peptides, and nonsense peptides (B); and abundance of CPa9-HNE in samples containing HNE-cleaved calprotectin, full-length calprotectin, or HNE as detected by the CPa9-HNE antibody and assay (C). [Figure 3] Figure 3: Spearman's rho correlation of CPa9-HNE with fecal calprotectin (fecal CP) and neutrophil count (A and B). [Figure 4] Figure 4: Measured levels of CPa9-HNE in serum from patients with inflammatory bowel disease (IBD), ulcerative colitis (UC), and Crohn's disease (CD) and healthy subjects (A and C), and associated receiver operating characteristic curves (B, D, and E). Data are shown as mean and standard error of the mean (SEM). Asterisks (*) indicate significant differences: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 5] Figure 5: Correlation of CPa9-HNE and fecal calprotectin (fecal CP) with endoscopic scores for ulcerative colitis (MES) and Crohn's disease (SES-CD) (A-D). R = correlation coefficient, P = P value, SES-CD = simple endoscopic score for Crohn's disease, MES = Mayo endoscopic score. [Figure 6] Figure 6: Measured levels of CPa9-HNE and associated ROC curves in healthy subjects, UC patients in clinical remission, and UC patients with active disease (A-C); measured levels of fecal CP and associated ROC curves in UC patients in clinical remission and UC patients with active disease (D and E); and correlations of CPa9-HNE with partial Mayo score (pMayo) and Trulove and Witts score (TW-score) (F and G). [Figure 7]Figure 7: (A) Serum biomarker levels of CPa9-HNE in COPD patients and healthy controls. Data are shown as Tukey boxplots, with the lower limit of measurable range (LLMR) indicated by a dashed line. Significance was found by a two-tailed Mann-Whitney test. (B) ROC curve showing the diagnostic performance of CPa9-HNE in COPD patients. AUC: 0.9996. [Figure 8] Figure 8: (A) Serum biomarker levels of CPa9-HNE in IPF patients and healthy controls. Data are shown as Tukey's boxplots, with LLMR indicated by dashed lines. Significance was found by a two-tailed Mann-Whitney test. (B) ROC curve showing the diagnostic performance of CPa9-HNE in IPF patients. AUC: 0.9813. [Figure 9] Figure 9: Kaplan-Meier plot to assess progression-free survival and overall survival of metastatic melanoma patients treated with PD-1 inhibitors associated with CPa9-HNE at baseline by grouping (dichotomizing) at 75th percentile (Q1+Q2+Q3 vs. Q4). [Figure 10] Figure 10: Serum biomarker levels of CPa9-HNE in lung cancer patients and controls. Data are shown as scatter plots with a line at the median and the lower limit of measurable range (LLMR) indicated by a dashed line. Significance was found by first testing for normality and lognormality, followed by applying Dunnett's multiple comparison test. [Figure 11] Figure 11: Serum biomarker levels of CPa9-HNE in patients with joint disease and healthy controls. Data are shown as Tukey box plots. DETAILED DESCRIPTION OF THE INVENTION

[0050] Example The embodiments of the present disclosure described in the following examples are set forth to aid in understanding the disclosure and should not be construed in any way to limit the scope of the disclosure, as defined in the claims that follow. The following examples are presented to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the described embodiments, and are not intended to limit the scope of the disclosure, nor are they intended to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise specified, parts are parts by weight, molecular weight is weight average molecular weight, temperature is degrees Celsius, and pressure is at or near atmospheric. The following materials and methods were used in the following examples.

[0051] method In vitro cleavage of calprotectin Calprotectin fragments were generated in vitro by adding purified calprotectin, a heterodimer composed of the proteins S100A9 and S100A8, to an Eppendorf tube and adding human neutrophil elastase (HNE) at a protein:protease ratio of 100:1 (10 μg calprotectin per 0.1 μg HNE). After 24 h, the proteolytic reaction was inhibited by adding 5 mM EDTA stop buffer. Eppendorf tubes containing only protease buffer, calprotectin without HNE, or HNE without calprotectin served as experimental controls.

[0052] mass spectrometry 100 μl of the cut sample or control was desalted on a reversed-phase Vydac UltraMicro Spin C18 column (Harvard Apparatus, catalog number 74-7206) according to the manufacturer's instructions. Untargeted mass analysis was performed on a quadrupole-Orbitrap benchtop mass spectrometer QExactive (Thermo Scientific) equipped with an Easy nano-LC 1000 system (ThermoFisher Scientific). Separation was performed on a 75 μm x 25 cm Acclaim Pepmap™ RSLC C18 capillary column (ThermoFisher Scientific) packed with 2 μm particles. For desolvation, a spray voltage of +2000 V was used with a heated ion transfer setting of 275 °C. Online reversed-phase separation was performed using a flow rate of 300 nl / min and an 85-minute linear binary gradient. The gradient started at 3% solvent B for 4 min, then increased to 35% solvent B at 64 min, and then to 45% solvent B at 5 min. Finally, the organic solvent concentration was increased to 90% over 5 min and held at 90% for 7 min. At a resolution of 70,000 at 200 m / z, 1 × 10 6 MS scans (400–1200 m / z) were recorded on an Orbitrap mass spectrometer set to an automatic gain control (AGC) target and a 100 ms maximum ion injection time. (44) Following MS, the 15 most intense multiply charged ions were analyzed at a resolution of 17,500, 2 × 10 4 Data-dependent collision-induced dissociation MS / MS scans were performed with an intensity threshold of 100 m / z, an isolation width of 2 m / z, and dynamic exclusion valid for 30 seconds.

[0053] Calprotectin fragment identification Identification from discovery data was performed using the Homo sapiens proteome (UniProt proteome ID UP000005640,n20200 downloaded on December 6, 2015 with Proteome Discoverer 2.1 software (ThermoFisher Scientific)). The processing workflow consisted of the following nodes: Spectrum Selector for spectrum preprocessing (precursor mass range: 100-10,000 Da; S / N threshold: 1.5), Sequest-HT search engine (protein database: see above; enzyme: no enzyme; max. missed cleavage site: 2; peptide length range: 6-144 amino acids; precursor mass tolerance: 10 ppm; fragment mass tolerance: 0.02 Da; dynamic modification: oxidation; static modification: cysteine ​​carbamidomethylation); and Percolator for peptide validation (FDR < 0.01 based on peptide q-values). Peptide intensities were quantified using a proprietary algorithm developed in Proteome Discoverer 2.1 (ThermoFisher Scientific).

[0054] Monoclonal antibody generation and clone characterization Briefly, monoclonal antibodies targeting HNE-generated neoepitopes consisting of N- or C-terminal sequences of HNE-generated calprotectin fragments (more specifically, targeting the N-terminal neoepitope of the HNE-generated calprotectin fragment designated "NBH-222," also designated "CPa9-HNE" herein) were generated as follows.

[0055] Balb / C mice aged 4–6 weeks were subcutaneously immunized with 200 μL of emulsified antigen and 50 μg of immunogenic peptide (KLGHPDTLNQ-GGC-keyhole limpet hemocyanin (KLH) (SEQ ID NO: 24)) in Freund's incomplete adjuvant (Sigma-Aldrich). Mice were immunized at 2-week intervals until a stable serum titer level was reached. Mice with the highest serum titers were selected for monoclonal antibody generation. After resting for one month, mice were immunized intravenously with 50 μg of immunogenic peptide in 100 μL of 0.9% sodium chloride (NaCl) solution. Three days later, splenocytes were isolated for cell fusion. Briefly, splenocytes were fused with SP2 / 0 myeloma cells to generate hybridoma cells, which were then cloned in culture dishes using the semisolid medium method. Clones were seeded into 96-well microtiter plates, and limiting dilution was used to ensure monoclonal growth. Supernatants were screened for reactivity against the selected peptide (KLGHPDTLNQ (SEQ ID NO: 12)) and native material (serum and truncated material) in an indirect competitive ELISA using streptavidin-coated plates (Roche, Hvidovre, Denmark, catalog no. 11940279). Clones with the highest reactivity were purified using a protein G column according to the manufacturer's instructions (GE Healthcare Life Sciences, Little Chalfont, Buckinghamshire, UK). These clones were tested for reactivity against the selected peptide and extended, truncated, and nonsense peptides (see Table 1). Clones showing the highest selectivity for the selected peptide were selected for monoclonal antibody generation and assay development. The optimal incubation buffer, time, temperature, and ratio between biotinylated peptide and antibody were determined.

[0056] [Table 1]

[0057] Monoclonal antibodies selected for production and assay development were also sequenced to determine the CDRs and isotype. The sequences of the chains are as follows (CDRs are underlined and in bold; N-terminal signal peptide and C-terminal constant region are in italics): Heavy chain sequence (mouse IgG1 isotype) MEWRIFLFILSGTAGVHSQVQLQQSGPELVKPGASVKMSCKASGYTFT DHVIN WVRQRTGQGLEWIG EIYPGSGSTYYNEKFKG KATLTADKSSNTAYMQLSSLTSEDSAVYFCAW FAY WGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO: 25) Light chain sequence (mouse kappa isotype) MESQTQVLISLLFWVSGACGDIVMTQSPSSLSVSAGEKVTMSC KSSQSLLNSGNQKNYLA WYQQKPGQPPKLLIY GASTRES GVPDRFTGSGSGTDFLTISSVQAEDLAVYYC LNDHSYPYT FGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO: 26)

[0058] Immunoassay (ELISA) Protocol The levels of HNE-generated neoepitopes consisting of N- or C-terminal sequences of HNE-generated calprotectin fragments (more specifically, the levels of CPa9-HNE, i.e., the N-terminal neoepitope of the HNE-generated fragment of calprotectin NBH-222) in the samples were assessed by solid-phase competitive enzyme-linked immunosorbent assay performed as follows.

[0059] A 96-well plate (Roche Diagnostics, catalog number 11940279, Hvidovre, Denmark) precoated with streptavidin was coated with biotinylated antigen (KLGHPDTLNQ-K-biotin (SEQ ID NO: 27)) and incubated with the biotinylated antigen for 30 minutes at room temperature. Unbound biotinylated coating antigen was discarded, and the wells were washed with wash buffer (25 mM TRIZMA, 50 mM NaCl, 0.036% Bronidox L5, 0.1% Tween 20) using a standardized ELISA plate washer (BioTek Instruments, Microplate washer, ELx405 Select CW, Winooski, USA). To maintain protein stability and block the sample, the sample was diluted in incubation buffer containing 1% bovine serum albumin (Sigma Aldrich, catalog number a-7906, purity ≥98%). Samples and controls were added to the wells and incubated with a primary monoclonal antibody against the HNE-generated neoepitope (CPa9-HNE) for 1 hour at 20°C with agitation at 300 rpm. Unbound primary antibody and samples were discarded, and the wells were washed with wash buffer. Subsequently, an HRP-conjugated AffiniPure rabbit anti-mouse IgG secondary antibody (Jackson catalog no. 315-035-045) was added to the wells and incubated for 1 hour at 20°C. Unbound secondary antibody was discarded by washing the wells with wash buffer. Chemiluminescent substrate (Roche Diagnostic catalog no. 11582950001) was added to the wells (100 μl / well), the plate was incubated at room temperature for 3 minutes, and the plate was read. Finally, the relative light units (RLU) emitted from the plate at 440 nm and 650 nm were quantified using an ELISA reader (VersaMAX; Molecular Devices, Wokingham, Berkshire, UK). Standard curves were plotted using a four-parametric mathematical fitting model.

[0060] Immunoassay Development To test the robustness of the above assay, dilution recovery, peptides in serum, analyte stability, freeze / thaw, antibody specificity (soundness test), interferences (hemolysis, biotin, and lipids added in serum), inter-assay / intra-assay variability, and biological relevance (cleaved material, serum, and plasma) were tested.

[0061] Biological validation in IBD patients Patient Demographics Serum samples from a patient cohort consisting of a total of 29 UC and 72 CD patients were tested. Demographic data, medical history, and treatment were obtained from electronic medical records and questionnaires. Anthropometric parameters were measured at enrollment. When available, patients' endoscopic disease activity was based on the simple endoscopic score for CD (SES-CD) and the Mayo endoscopic score (MES) for UC. The MES score was used to add information about disease extension. 26 Inflammatory activity was also defined as a combination of clinical and biochemical disease activity using the Crohn's Disease Activity Index (CDAI), partial Mayo score (pMayo), and C-reactive protein (CRP). Patients were stratified based on the endoscopic score as follows: SES-CD (remission = 0–2, mild = 3–6, moderate = 7–15, severe >15) and MES (remission = 0–2, mild = 3–6, moderate = 7–15, severe >15). Clinical and biochemical activity was defined as CDAI ≥ 150 or CRP > 5 for CD and pMayo > 1 or CRP > 5 for UC. Disease severity and progression were assessed according to the Montreal classification.

[0062] statistical analysis To achieve normal distribution, logarithmic transformation of the data was applied before statistical analysis. For normally distributed data, the Student's t-test and one-way ANOVA were applied to analyze statistical differences. When normal distribution was not achieved by logarithmic transformation, the Mann-Whitney U test and Kruskal-Wallis test were applied. The false discovery rate method (FDR = 5%) was used to correct for multiple comparisons. Target peptide levels are presented as non-logarithmic transformed data with the mean and standard error of the mean (SEM).

[0063] Receiver operating characteristic (ROC) curves were calculated to evaluate the diagnostic power of the target peptides. A P value of 0.05 or less was considered statistically significant. Statistical analysis was performed using Graphpad Prism 7.03 and MedCalc. Figures were generated using GraphPad Prism version 7.03.

[0064] Biological validation in COPD and IPF patients CPa9-HNE was measured in serum from COPD patients (n=68) and healthy controls (n=36), and from IPF patients (n=16) and healthy controls (n=10).

[0065] Biological validation in patients with metastatic melanoma CPa9-HNE was measured in pretreatment serum of metastatic melanoma patients treated with anti-PD-1 therapy (pembrolizumab) (n=35). Patients were treated with pembrolizumab as standard of care at Copenhagen University Hospital, Hereu, after informed consent and approval by the Ethics Committee of the Capital Region of Denmark in accordance with the 1975 Declaration of Helsinki. Serum samples were measured blinded. The association of CPa9-HNE levels with progression-free survival (PFS) and overall survival (OS) was assessed by Kaplan-Meier and Cox regression analyses, both independently and after adjusting for PDL1 expression (≥1%), lactate dehydrogenase (LDH), BRAF mutation status, and C-reactive protein (CRP).

[0066] Biological validation in SCLC and NSCLC patients CPa9-HNE was measured in serum from small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), and healthy controls.

[0067] Biological validation in patients with joint disease CPa9-HNE was measured in serum from patients with rheumatoid arthritis (n = 15, age range: 39-47), psoriatic arthritis (n = 11, age range: 31-64), psoriasis (n = 12, age range: 27-52), ankylosing spondylitis (n = 11, age range: 35-53), juvenile osteoarthritis (n = 13, age < 51 [range: 41-50]), and elderly osteoarthritis (n = 10, age > 50) and healthy controls (n = 33). 41 of the patients were women.

[0068] result Generation and identification of calprotectin neoepitopes Untargeted mass spectrometry analysis using UniProt Proteome ID UP000005640 and subsequent identification of calprotectin fragments revealed that several neoepitope-containing calprotectin fragments were generated by HNE from both the S100A9 and S100A8 proteins (Table 2 and Figure 1). Five HNE-generated calprotectin fragments (NBH-222, NBH-223, NBH-224, NBH-225, and NBH-226) were considered candidates for assay development based on their PSMs, quality q values, and quality PEP scores. Of these, NBH-222 was selected as the calprotectin (S100A9) fragment for the development of an immunoassay targeting the HNE-generated N-terminal neoepitope (CPa9-HNE) of this fragment due to its PSM number (Table 2) and the uniqueness of its N-terminal neoepitope sequence.

[0069] [Table 2] TIFF0007730841000003.tif169159

[0070] Immunoassay development for CPa9-HNE Specificity, accuracy and precision Mass spectrometry data demonstrated that NBH-222 was present only in samples containing full-length human calprotectin plus human neutrophil elastase, but not in control samples containing only full-length human calprotectin or human neutrophil elastase (Figure 2A). A monoclonal antibody targeting CPa9-HNE (the HNE-generated N-terminal neoepitope of NBH-222) (also referred to herein as the CPa9-HNE antibody) was developed and used in the above-described ELISA protocol (also referred to hereafter as the CPa9-HNE assay). The specificity of this antibody in this immunoassay was tested against selected peptides, as well as extended, truncated, and nonsense peptides (with sequences listed in Table 1). The CPa9-HNE antibody demonstrated reactivity only with the CPa9-HNE neoepitope sequence (Figure 2B). In samples containing only HNE-cleaved calprotectin, only intact full-length human calprotectin, or only human neutrophil elastase, the CPa9-HNE antibody was able to identify the target neoepitope sequence only when human neutrophil elastase-cleaved calprotectin was present (Figure 2C). The final specifications of the CPa9-HNE assay are listed in Table 3.

[0071] [Table 3]

[0072] Patient demographics of IBD patients It was demonstrated that CPa9-HNE levels in serum from IBD patients correlated with fecal calprotectin levels (i.e., levels of intact calprotectin in the stool) and neutrophil counts in UC patients (Fig. 3A and B).

[0073] CPa9-HNE serum levels in IBD CPa9-HNE is elevated in serum of IBD compared with healthy controls We measured serum levels of CPa9-HNE in serum from UC, CD, and healthy subjects. CPa9-HNE was demonstrated to be approximately four-fold higher in serum from IBD patients compared to healthy subjects (AUC: 0.92, P < 0.0001) (Figure 4A and B). When patients were divided into CD and UC, serum CPa9-HNE was similarly elevated in UC and CD patients, and was approximately four-fold higher in CD patients (AUC: 0.92, P < 0.0001) and UC patients (AUC: 0.94, P < 0.0001) compared to healthy subjects (Figure 4C, D, and E).

[0074] CPa9-HNE is associated with endoscopic disease activity in UC and CD CPa9-HNE correlated well with endoscopic disease activity for CD (SES-CD: r = 0.057, P < 0.0001) and UC (MES: r = 0.71, P = 0.0003) (Figures 5A and 5B). Fecal CP correlated well with endoscopic disease activity for CD (SES-CD: r = 0.39, P = 0.005) but not for UC (MES: r = 0.31, P = 0.09) (Figures 5C and 5D).

[0075] CPa9-HNE is associated with clinical disease activity in UC When UC patients were divided into those in clinical remission and those with clinically active disease based on the partial Mayo score, CPa9-HNE was significantly elevated in UC patients with clinically active disease compared with those in remission (AUC: 0.88, P < 0.0001) and healthy donors (AUC: 0.93, P < 0.0001) (Figure 6A, B, and C). Comparing the performance of CPa9-HNE with fecal calprotectin demonstrated that CPa9-HNE was comparable to or slightly superior to fecal calprotectin, with increased AUC and sensitivity (Figure 6A–E). CPa9-HNE was also demonstrated to correlate with the partial Mayo score (r = 0.51, P = 0.008) and Trulove and Witts score (r = 0.64, P = 0.0005) for UC (Figure 6F and G).

[0076] CPa9-HNE is elevated in COPD patients compared with healthy controls CPa9-HNE was measured in serum samples from COPD patients and healthy controls. Figure 7A shows the present difference (P<0.0001) between healthy controls (n=36) and COPD patients (n=68). Furthermore, the diagnostic ability was calculated using a receiver operating characteristic (ROC) curve, and the area under the curve (AUC) was determined to be 0.9996 (Figure 7B). The measurement values ​​were not affected by the patient's BMI, age, or gender.

[0077] CPa9-HNE is elevated in IPF patients compared with healthy controls CPa9-HNE was measured in serum samples from IPF patients (n = 16) and healthy controls (n = 10). Figure 8A shows the difference between healthy controls and IPF patients (P < 0.0001). Furthermore, the diagnostic performance was calculated using a receiver operating characteristic curve (ROC) and the AUC was determined to be 0.9813 (Figure 8B).

[0078] High CPa9-HNE is associated with poor prognosis in metastatic melanoma The association between CPa9-HNE and survival outcomes in patients with metastatic melanoma was assessed by Kaplan-Meier analysis. Using the 75th percentile cutpoint, patients with high CPa9-HNE levels (>75th percentile) were found to have significantly worse PFS (P = 0.011) and OS (P = 0.0002) compared with patients with low CPa9-HNE levels (Figure 9). In support, univariate Cox regression identified high (>75th percentile) pretreatment CPa9-HNE as a predictor of poor PFS (HR = 3.32, 95% CI = 1.25-8.82, p = 0.016) and OS (HR = 11.31, 95% CI = 2.27-56.33, p = 0.003) compared with low CPa9-HNE (Table 4). Multivariate Cox regression revealed that high CPa9-HNE was an independent predictor of poor PFS (HR = 8.22, 95% CI = 1.30 to 52.14, p = 0.025) and OS (HR = 76.87, 95% CI = 4.73 to 1248.57, p = 0.002) when adjusted for PDL1 expression, LDH, BRAF mutation, and CRP (Table 4).

[0079] [Table 4]

[0080] CPa9-HNE is elevated in SCLC and NSCLC patients compared with healthy controls We measured CPa9-HNE in serum from patients with small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), and healthy controls. We found that lung cancer patients experienced a statistically significant increase in CPa9-HNE serum levels in both SCLC (p = 0.0435, n = 10, median = 212.1 [IQR 164.0-407.1]) and NSCLC (p = 0.0467, n = 10, median = 281.3 [IQR 186.8-385.8]) (Figure 10).

[0081] CPa9-HNE is elevated in joint disease compared to healthy donors CPa9-HNE was measured in serum from patients with rheumatoid arthritis (n = 15, age [range: 39-47]), psoriatic arthritis (n = 11, age [range: 31-64]), psoriasis (n = 12, age [range: 27-52]), ankylosing spondylitis (n = 11, age [range: 35-53]), juvenile osteoarthritis (n = 13, age < 51 [range: 41-50]), elderly osteoarthritis (n = 10, age > 50) and healthy controls (n = 33). The CPa9-HNE biomarker was significantly elevated in all joint diseases compared to healthy controls, with the greatest differences observed in patients with ankylosing spondylitis (P<0.001), psoriatic arthritis (P<0.001), juvenile osteoarthritis (P<0.001), and rheumatoid arthritis (P<0.01) compared to healthy controls (Figure 11).

[0082] Consideration In this study, we demonstrated that the CPa9-HNE assay is biologically and clinically relevant and technically robust as a serum-based assay for detecting and assessing disease activity in, among others, IBD, COPD, IPF, SCLC, NSCLC, rheumatoid arthritis, ankylosing spondylitis, psoriasis, psoriatic arthritis, and osteoarthritis. Furthermore, we demonstrated that the CPa9-HNE assay is valuable in determining the prognosis of patients with metastatic melanoma.

[0083] As shown in Table 3, CPa9-HNE containing the calprotectin fragment (NBH-222) was stable in serum for at least 48 hours at 4°C, which also means that this neoepitope-containing fragment has a much longer half-life in serum and plasma than intact calprotectin, which has been shown to be stable in stool for 7 days. 1 Nevertheless, it only has a half-life of 5 hours in plasma, which means that the assay performance is similar to that of CRP. 10~12 This may explain the poor clinical applicability of fecal calprotectin assays when applied to serum / plasma. The high stability of the CPa9-HNE fragment in serum (at least 48 hours at 4°C) can be explained by the fact that it is a degradation / metabolite of calprotectin, and the fragment is more resistant to further degradation. In addition, the CPa9-HNE assay is more sensitive than the fecal calprotectin assay because it measures in nanograms / mL instead of micrograms / grams. 27 .

[0084] Because metalloproteinases (MMPs) require zinc ions for activation, the ability of calprotectin to bind metal ions, such as calcium and zinc ions, makes it highly resistant to degradation by MMPs. HNE, a serine protease that does not require activation by metal ions, can cleave calprotectin and generate HNE-derived neoepitope-containing calprotectin fragments. 5 ' 28~30Therefore, CPa9-HNE levels may represent local tissue inflammation, as CPa9-HNE-containing fragments are generated only by activated neutrophil granulocytes and other activated leukocytes, as well as by the presence of HNE. This is in contrast to intact calprotectin, which can be released randomly into the circulation and stool by non-activated circulating leukocytes and, to some extent, by epithelial cells that also express calprotectin. 10,31、32 .

[0085] CPa9-HNE was demonstrated to be highly abundant in the serum of CD and UC patients, demonstrating its high potential as a surrogate biomarker to aid in the diagnosis of IBD. This is in agreement with fecal calprotectin, as this biomarker can reliably distinguish between IBS and IBD patients. 6~8 Furthermore, CPa9-HNE also correlated with fecal calprotectin, neutrophil granulocyte counts, and disease activity in CD and UC, suggesting that CPa9-HNE can be used to monitor disease activity and may serve as a surrogate biomarker for endoscopic evaluation of CD and UC.

[0086] The CPa9-HNE assay also revealed significant differences between patients with COPD and IPF compared with healthy controls. The CPa9-HNE assay measures the specific cleavage site on calprotectin generated by neutrophil elastase. Therefore, these results indicate that NE activity is higher in these lung diseases.

[0087] The CPa9-HNE assay was also demonstrated to predict overall survival and progression-free survival in metastatic melanoma patients treated with anti-PD-1 therapy based on baseline (pre-treatment) concentrations of serum CPa9-HNE.

[0088] Finally, CPa9-HNE was also shown to be significantly elevated in the serum of lung cancer patients (both SCLC and NSCLC) compared to healthy controls, and in the serum of patients with rheumatoid arthritis, ankylosing spondylitis, psoriasis, psoriatic arthritis, and osteoarthritis compared to healthy controls.

[0089] conclusion The CPa9-HNE ELISA, a novel serum biomarker of HNE-mediated degradation of calprotectin, demonstrated high specificity for the neoepitope in both in vitro cleavage samples and human IBD serum samples. CPa9-HNE was highly associated with CD and UC patients and demonstrated high diagnostic accuracy in distinguishing IBD patients from healthy donors. Furthermore, CPa9-HNE also correlated with endoscopic disease activity for CD and UC, SES-CD, and MES, respectively. CPa9-HNE also correlated with clinical disease activity scores (partial Mayo score and Trulove and Witts score) for UC. Thus, CPa9-HNE is a surrogate biomarker of disease activity for CD and UC, performing at least as well as or slightly better than fecal CP. Therefore, CPa9-HNE is a novel clinically relevant biomarker for diagnosing and monitoring disease activity in IBD, as well as other inflammation-driven diseases, including rheumatoid arthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, osteoarthritis, Sjögren's syndrome, and lupus. It can also be used to predict or monitor treatment efficacy in prospective studies of, for example, TNF-alpha, vedolizumab, and ustekinumab.

[0090] Furthermore, CPa9-HNE has been shown to be a clinically relevant biomarker in pulmonary diseases such as COPD and IPF, metastatic diseases such as metastatic melanoma, and lung cancers such as SCLC and NSCLC.

[0091] As used herein, unless expressly indicated otherwise, the word "or" is used in the sense of an operator that returns a true value when either or both stated conditions are met, as opposed to the operator "exclusive or," which requires that only one of the conditions be met. As used herein, the word "comprising" means "including" or "consisting of." All prior teachings accepted herein are hereby incorporated by reference.

Claims

1. An immunoassay method for detecting HNE-generated calprotectin fragments, comprising contacting a human biological fluid sample with a monoclonal antibody that specifically recognizes and binds to an HNE-generated neoepitope consisting of the N-terminal sequence of the HNE-generated calprotectin fragment, and detecting binding between the monoclonal antibody and a peptide in the sample, wherein the monoclonal antibody specifically recognizes and binds to the N-terminal or C-terminal sequence of the peptide KLGHPDTLNQGEFKELV (SEQ ID NO: 1).

2. 10. The method of claim 1, wherein the detection is quantitative, and the method further comprises determining the amount of binding between the monoclonal antibody and the peptide in the sample.

3. 3. The method of claim 2, wherein the method is an immunoassay method for detecting and / or monitoring the progression of and / or determining the state or severity of a disease in a patient, wherein the disease is characterized or manifested by inflammation, comprising contacting a biological fluid sample obtained from the patient with the monoclonal antibody, detecting and determining the amount of binding between the monoclonal antibody and a peptide in the sample, and correlating the amount of binding with values ​​associated with normal healthy subjects and / or values ​​associated with a known state or severity of the disease and / or values ​​obtained from the patient at a previous time point and / or a predetermined cut-off value.

4. 4. The method of claim 3, wherein the disease is an inflammation-driven disease.

5. 5. The method of claim 4, wherein the disease is inflammatory bowel disease (IBD), rheumatoid arthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, or osteoarthritis.

6. 4. The method of claim 3, wherein the disease is chronic obstructive pulmonary disease (COPD) or idiopathic pulmonary fibrosis (IPF).

7. The method of claim 3 , wherein the disease is cancer.

8. 8. The method of claim 7, wherein the disease is metastatic melanoma, small cell lung cancer (SCLC), or non-small cell lung cancer (NSCLC).

9. The method according to any one of claims 1 to 8, wherein the monoclonal antibody is a monoclonal antibody produced against a synthetic peptide containing the N-terminal or C-terminal sequence.

10. 10. The method of any one of claims 1 to 9, wherein the monoclonal antibody specifically recognizes and binds to the N-terminal sequence of the HNE-generated calprotectin fragment, but does not specifically recognize or bind to an N-extended version of the N-terminal amino acid sequence or an N-truncated version of the N-terminal amino acid sequence.

11. The method according to any one of claims 1 to 10, wherein the biological fluid sample is plasma or serum.

12. The method according to any one of claims 1 to 11, wherein the immunoassay is a competitive immunoassay and / or the immunoassay is an enzyme-linked immunosorbent assay (ELISA).

13. A monoclonal antibody that specifically recognizes and binds to an HNE-generated neoepitope consisting of the N-terminal or C-terminal sequence of an HNE-generated calprotectin fragment, wherein the monoclonal antibody specifically recognizes and binds to the N-terminal or C-terminal sequence of the peptide KLGHPDTLNQGEFKELV (SEQ ID NO: 1).

14. The monoclonal antibody described in Claim 13, wherein the monoclonal antibody is a monoclonal antibody produced against a synthetic peptide containing the N-terminal or C-terminal sequence.

15. A monoclonal antibody described in claim 13 or 14, which specifically recognizes and binds to the N-terminal sequence of the HNE-generated calprotectin fragment, but does not specifically recognize or bind to an N-extended extended form of the N-terminal amino acid sequence or an N-truncated shortened form of the N-terminal amino acid sequence.

16. A monoclonal antibody according to any one of claims 13 to 15, - Streptavidin coated well plate - biotinylated peptides containing said N-terminal or C-terminal sequences linked to biotin - Secondary antibodies for use in sandwich immunoassays - a calibrator peptide containing the N-terminal or C-terminal sequence - Antibody Biotinylation Kit - Antibody HRP labeling kit - Antibody radiolabeling kit and at least one of the following:

17. A monoclonal antibody according to any one of claims 13 to 15, - Streptavidin coated well plate - biotinylated peptides containing said N-terminal or C-terminal sequences linked to biotin - a calibrator peptide containing the N-terminal or C-terminal sequence 17. The assay kit of claim 16, comprising one, two or all of:

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