CC16-HNE assay

Abstract

This invention discloses an immunoassay method for detecting HNE-producing fragments of club cell secreted protein 16 (CC16) in patient samples, which can be used to detect and / or monitor chronic obstructive pulmonary disease (COPD) or idiopathic pulmonary fibrosis (IPF) or a specific level of severity of these diseases in patients. Also disclosed are a monoclonal antibody and an assay kit for use with this immunoassay method.

Description

Technical Field

[0001] The present invention relates to an immunoassay method for detecting HNE-generated fragments of club cell secretory protein 16 (CC16) in a subject sample, and to its use for detecting and / or monitoring a specific level of severity of chronic obstructive pulmonary disease (COPD) or idiopathic pulmonary fibrosis (IPF) or those diseases in a subject. The present invention also relates to monoclonal antibodies and assay kits for use in the immunoassay method.

Background Art

[0002] Chronic lung diseases are common and serious health concerns worldwide [1]. Chronic obstructive pulmonary disease (COPD) is a common lung disease, and it is estimated that 212,800,000 people worldwide suffered from it in 2019 [2]. In contrast, idiopathic pulmonary fibrosis (IPF) is considered a rare disease, and it is estimated that 0.3 - 4.51 people per 10,000 people worldwide suffered from it as of 2021, which has a harmful effect on the health-related quality of life [3]. The pathological mechanisms that progress both diseases are essentially different, but there are believed to be some commonalities [4]. Overall, IPF is characterized by an increased accumulation of extracellular matrix (ECM) proteins due to lung injury, while COPD is mainly characterized by the breakdown of inflammation and an increased degradation of ECM, resulting in the loss of tissue integrity.

[0003] As described above, IPF is characterized by lung tissue injury, epithelial damage, and pathological changes in airway morphology

[19] . Pulmonary hypertension (PH) is a coexisting disease that affects IPF patients and is classified as part of PH group 3

[20] . IPF-PH is associated with a relatively high risk of death, but it is difficult to develop new treatment methods because the disease findings are currently limited

[20] . In addition, the diagnosis of PH is discouraged because its gold standard is right heart catheterization, which is an invasive procedure

[21] .

[0004] Crab cells are abundant in the epithelium of the lower airways and secrete surfactants and various other proteins.[5][6] Crab cells are the main producers of crab cell secretory protein 16 (CC16), also known as CC10, secretoglobin, or uteroglobin. The exact biological function of CC16 is not yet fully understood, but it is believed to have anti-inflammatory properties and protect the airways from inflammation and oxidative stress.[7] CC16 has been proposed as a biomarker for lung epithelial damage, and increased circulating levels are believed to reflect crab cell damage and / or increased epithelial permeability.

[0005] Studies in mice have shown that CC16 knockout significantly increased the induction of ECM remodeling and inflammation after exposure to tobacco smoke [8]. CC16 secretion has been shown to be elevated in serum and bronchoalveolar lavage fluid in IPF [9]. In contrast, CC16 levels were decreased in serum from COPD patients compared to non-smoking controls, and lower levels were associated with more severe disease

[10] ,

[11] . This is consistent with studies showing that CC16 staining is highest in airway epithelium from healthy non-smokers, decreases in healthy smokers, and is low / absent in COPD tissue [8]. Furthermore, COPD severity has been suggested to alter CC16 expression, with lower CC16 staining in severe COPD compared to moderate COPD, as determined by the Global Organization for Staging Obstructive Pulmonary Disease (GOLD) [8]. Interestingly, one study showed that low levels of serum CC16 were associated with accelerated pulmonary function decline (FEV1)

[12] . This may suggest that the effects of CC16 are opposite in patients with COPD and IPF, and that this may be one reason why COPD is more strongly associated with inflammation than IPF. Clinical trials combining immunosuppressants and anti-inflammatory drugs for IPF were terminated early because they showed adverse effects on patients and increased rates of death and hospitalization

[13] . This suggests that some degree of inflammation may serve a protective purpose in IPF. [Overview of the Initiative]

[0006] The present inventors have (1) identified neoepitopes that can be used as biomarkers for CC16 degradation by human neutrophil elastase; (2) developed and validated competitive enzyme-linked immunosorbent assays (ELISAs) specific to the identified neoepitopes; and (3) evaluated and demonstrated the biological relevance of these assays in detecting the presence and severity of chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF).

[0007] Therefore, in a first embodiment, the present invention provides an immunoassay method comprising contacting a sample of a patient with a monoclonal antibody that specifically binds to an HNE-producing neo-epitope of an HNE-producing fragment of club cell secreted protein 16 (CC16), and detecting the binding between the monoclonal antibody and a peptide in the sample and determining the amount of binding, wherein the HNE-producing neo-epitope consists of the N-terminal or C-terminal sequence of an HNE-producing fragment located at the end of the HNE-producing fragment cleaved by HNE.

[0008] In a preferred embodiment, the immunoassay method includes the following i) and ii): i) Contacting the patient's sample with a monoclonal antibody that specifically binds to the N-terminal amino acid sequence VDTLPQKPRE (SEQ ID NO: 1), or a monoclonal antibody that specifically binds to the N-terminal amino acid sequence AMELFSPDQD (SEQ ID NO: 2) (in this specification, these sequences are also referred to as the "target sequence" or "CC16-HNE target sequence"); and ii) To detect the binding between the monoclonal antibody and the peptide in the sample and to determine the amount of binding. Most preferably, step (i) above includes contacting the patient's sample with a monoclonal antibody that specifically binds to the N-terminal amino acid sequence VDTLPQKPRE (SEQ ID NO: 1).

[0009] In a preferred embodiment, the immunoassay method is an immunoassay method for detecting and / or monitoring chronic obstructive pulmonary disease (COPD) or idiopathic pulmonary fibrosis (IPF), or a specific level of severity of these diseases, in a patient, further comprising correlating the binding amount with a value associated with a normal healthy person, and / or a value associated with a known disease severity, and / or a value obtained from the patient at a past point in time, and / or a predetermined cutoff value. Most preferably, the method is a method for detecting and / or monitoring COPD or a specific level of severity of said disease in a patient; or a method for detecting and / or monitoring a specific level of severity of IPF in a patient; or a method for detecting and / or monitoring IPF with pulmonary hypertension (PH) in a patient.

[0010] If the method described above is a method for detecting a specific level of severity, it may, for example, be a method for detecting very severe (GOLD stage 4) COPD or an IPF patient with an increased risk of death. In a preferred embodiment, the patient's sample is a human biological fluid sample. Preferably, the sample is a blood material sample such as blood (whole blood), plasma, or serum.

[0011] If the monoclonal antibody is a monoclonal antibody that specifically binds to the N-terminal amino acid sequence VDTLPQKPRE (SEQ ID NO: 1), then preferably the monoclonal antibody does not specifically bind to a peptide having the N-terminal amino acid sequence LVDTLPQKPRE (SEQ ID NO: 3) (i.e., an elongated version of the target sequence extended by adding a leucine residue to the N-terminal), and / or does not specifically bind to a peptide having the N-terminal amino acid sequence DTLPQKPRE (SEQ ID NO: 4) (i.e., a truncated version of the target sequence truncated by removing the first valine residue). Preferably, the ratio of the affinity of the antibody to the target sequence to the affinity of the antibody to the extension of the target sequence is at least 10:1, more preferably at least 20:1, at least 30:1, at least 40:1, at least 50:1, or 100:1. Preferably, the ratio of the affinity of the antibody to the target sequence to the affinity of the antibody to the truncated target sequence is at least 10:1, more preferably at least 20:1, at least 30:1, at least 40:1, at least 50:1, or 100:1.

[0012] If the monoclonal antibody is a monoclonal antibody that specifically binds to the N-terminal amino acid sequence AMELFSPDQD (SEQ ID NO: 2), then the monoclonal antibody preferably has an N-terminal amino acid sequence It does not specifically bind to peptides having AAMELFSPDQD (SEQ ID NO: 5) (i.e., elongated versions of the target sequence, extended by adding an additional alanine residue to the N-terminal), and / or does not specifically bind to peptides having the N-terminal amino acid sequence MELFSPDQD (SEQ ID NO: 6) (i.e., truncated versions of the target sequence, truncated by removing the first alanine residue). Preferably, the ratio of the affinity of the antibody to the target sequence to the affinity of the antibody to the extension of the target sequence is at least 10:1, more preferably at least 20:1, at least 30:1, at least 40:1, at least 50:1, or 100:1. Preferably, the ratio of the affinity of the antibody to the target sequence to the affinity of the antibody to the truncated target sequence is at least 10:1, more preferably at least 20:1, at least 30:1, at least 40:1, at least 50:1, or 100:1.

[0013] If the monoclonal antibody is a monoclonal antibody that specifically binds to the N-terminal amino acid sequence VDTLPQKPRE (SEQ ID NO: 1), the monoclonal antibody may be produced, for example, by counteracting a synthetic peptide having the N-terminal amino acid sequence VDTLPQKPRE (SEQ ID NO: 1); or if the monoclonal antibody is a monoclonal antibody that specifically binds to the N-terminal amino acid sequence AMELFSPDQD (SEQ ID NO: 2), the monoclonal antibody may be produced, for example, by counteracting a synthetic peptide having the N-terminal amino acid sequence AMELFSPDQD (SEQ ID NO: 2). For example, monoclonal antibodies can be produced by: (a) immunizing rodents (or other suitable mammals) with a synthetic peptide comprising the N-terminal amino acid sequence VDTLPQKPRE (SEQ ID NO: 1) or AMELFSPDQD (SEQ ID NO: 2), wherein an immunogenic carrier protein (such as keyhole limpet hemocyanin ("KLH")) may be optionally linked to its C-terminal; (b) isolating and cloning single antibody-producing cells; and (c) assaying the resulting monoclonal antibodies to confirm that they have the desired specificity. Exemplary procedures for developing, producing, and characterizing suitable monoclonal antibodies are described in the Examples section below.

[0014] In certain exemplary embodiments, if the monoclonal antibody is a monoclonal antibody that specifically binds to the N-terminal amino acid sequence VDTLPQKPRE, the monoclonal antibody may preferably include one or more complementarity-determining regions (CDRs) selected from the following: CDR-L1: KSSQSLFNSGTQKNYLA (Sequence ID 7) CDR-L2: WASTRES (Sequence ID 8) CDR-L3: KQSYNLLT (Sequence ID 9) CDR-H1: DYSMH (Sequence ID 10) CDR-H2: WINTETGEPTYADDFKG (Sequence ID 11) CDR-H3: MITVAMDY (Sequence ID 12) Preferably, the monoclonal antibody contains at least two, three, four, five, or six of the CDR sequences listed above.

[0015] Preferably, the monoclonal antibody has a light chain variable region containing the following CDR sequence: CDR-L1: KSSQSLFNSGTQKNYLA (Sequence ID 7) CDR-L2: WASTRES (Sequence ID 8), and, CDR-L3: KQSYNLLT (Sequence ID 9). Preferably, the monoclonal antibody has a light chain comprising a framework sequence between CDRs, and the framework sequence is substantially identical or substantially similar to the framework sequence between CDRs in the following light chain sequence (CDRs are indicated in bold and underlined, and framework sequences are indicated in italics).

[0016]

Chemical formula

[0017] Preferably, the monoclonal antibody has a heavy chain variable region comprising the following CDR sequences. CDR-H1: DYSMH (SEQ ID NO: 10), CDR-H2: WINTETGEPTYADDFKG (SEQ ID NO: 11), and CDR-H3: MITVAMDY (SEQ ID NO: 12). Preferably, the monoclonal antibody has a heavy chain comprising a framework sequence between CDRs, and the framework sequence is substantially identical or substantially similar to the framework sequence between CDRs in the following heavy chain sequence (CDRs are indicated in bold and underlined, and framework sequences are indicated in italics).

[0018]

Chemical formula

[0019] Preferably, the monoclonal antibody has a light chain variable region sequence:

[0020]

Chemical formula

[0021] (CDRs are in bold and underlined; framework sequences are in italics) And / or, a heavy chain variable region sequence:

[0022] <l

Chemical formula

[0023] (CDR is in bold and underlined; framework array is in italics) Includes.

[0024] In this specification, the amino acid sequences of the framework present between the CDRs of an antibody are considered "substantially identical" or "substantially similar" to the amino acid sequences of the framework present between the CDRs of another antibody if they have at least 70%, 80%, 90%, or at least 95% similarity or identity with the amino acid sequences of the framework present between the CDRs of that other antibody. Similar or identical amino acids may be consecutive or discontinuous. A framework sequence may contain one or more amino acid substitutions, insertions, and / or deletions. Amino acid substitutions may be conservative substitutions, 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 in terms of size, charge, polarity, etc.: 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; Group 5 Phe, Thy, Trp.

[0025] Programs like the CLUSTAL program can be used to compare amino acid sequences. This program compares amino acid sequences and finds the optimal alignment by appropriately inserting spaces within either sequence. For optimal alignment, it is possible to calculate amino acid identity or similarity (in addition to identity, conservation of amino acid types). Programs like BLASTx align the longest segments of similar sequences and assign values ​​to the matching sites. In this way, a comparison can be obtained, and several similar regions with different scores can be discovered. In this invention, it is conceivable to use these two types of analysis. Identity or similarity is preferably calculated over the entire length of the framework sequence.

[0026] In preferred embodiments, the immunoassay is a competitive assay or a sandwich assay. The immunoassay may be, for example, a radioimmunoassay or an enzyme-linked immunosorbent assay (ELISA). Such assays are known to those skilled in the art.

[0027] As used herein, the term “N-terminal” refers to the N-terminal peptide sequence at the tip of a polypeptide, i.e., the peptide sequence at the N-end of the polypeptide, and should not be interpreted as referring to its general direction. As used herein, the term “C-terminal” refers to the C-terminal peptide sequence at the tip of a polypeptide, i.e., the peptide sequence at the C-end of the polypeptide, and should not be interpreted as referring to its general direction. As used herein, the terms “peptide” and “polypeptide” are used synonymously.

[0028] As used herein, the term “monoclonal antibody” refers to both the whole antibody and its fragments that retain the binding specificity of the whole antibody, such as Fab fragments, F(ab')2 fragments, single-chain Fv fragments, or other such fragments known to those skilled in the art. As is well known, a whole antibody typically has a “Y-shaped” structure in which two identical polypeptide chain groups are paired, with each paired element consisting of one “light” chain and one “heavy” chain. The N-terminal regions of the light and heavy chains each contain a variable region, and the C-terminal portions of the heavy and light chains each constitute a constant region. The variable region contains three complementarity-determining regions (CDRs), which are primarily responsible for antigen recognition. The constant region allows the antibody to recruit cells and molecules of the immune system. Antibody fragments that retain binding specificity include at least the CDRs and a portion of the remainder of the variable region sufficient to retain binding specificity.

[0029] In the present invention, any monoclonal antibody containing any constant region known in the art can be used. In the case of mouse antibodies and human antibodies, the constant light chain is classified as either a kappa or lambda light chain. The constant heavy chain is classified as mu, delta, gamma, alpha, or epsilon, and the antibody isotype is defined as IgM, IgD, IgG, IgA, and IgE, respectively. In the case of humans, the IgG isotype has several subclasses, including IgG1, IgG2, IgG3, and IgG4, and in the case of mice, it has several subclasses, including IgG1, IgG2a, IgG2b, IgG2c, and IgG3, but is not limited thereto. The monoclonal antibody may preferably belong to an IgG isotype containing any one of the IgG subclasses.

[0030] The CDR of an antibody can be determined using methods known in the art, such as those described by Kabat et al. The antibody can be generated from B cell clones. The isotype of the antibody can be determined by ELISA specific to the IgM, IgG, or IgA isotype or subclass. The amino acid sequence of the generated antibody can be determined using standard methods. For example, RNA can be isolated from cells and used to generate cDNA by reverse transcription. The cDNA can then be PCR-treated using primers that amplify the heavy and light chains of the antibody. For example, primers specific to the leader sequence for all VH (variable heavy chain) sequences can be used together with primers that bind to sequences located in the constant region of a predetermined isotype. The light chain can be amplified using primers that bind to the 3' end of the kappa or lambda chain, together with primers that anneal to the V-kappa or V-lambda leader sequence. The full-length heavy and light chains can be generated and sequenced.

[0031] As used herein, the term “bound amount” refers to the quantification of binding between the antibody and the peptide in the patient sample. This quantification may be determined, for example, by comparing the measured binding amount in the patient sample with a calibration curve prepared using measured binding amounts in a standard sample containing the peptide to which the antibody specifically binds at a known concentration, in order to determine the amount of peptide to which the antibody specifically binds in the patient sample. Any analytical method suitable for measuring the bound amount can be used. For example, ELISA can be used, in which spectrophotometric analysis is used to measure both the bound amount in the patient sample and the bound amount when a calibration curve is prepared.

[0032] As used herein, the term “predetermined cutoff value” means a statistically determined binding amount that indicates a high probability that a patient has a disease (e.g., COPD or IPF) or that the disease is of a particular severity, and in that sense, if the measured value of the target peptide in the patient sample is greater than or equal to the statistical cutoff value, then the measured value corresponds to a probability of at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95% that the disease is present or of a particular severity.

[0033] As used herein, the term “values ​​associated with normal healthy individuals” means the standardized binding amount determined by the method described above for samples obtained from subjects who are considered healthy, i.e., disease-free (i.e., not COPD or IPF); and the term “values ​​associated with known disease severity” means the standardized binding amount determined by the method described above for samples obtained from patients who are known to have a disease of known severity (i.e., COPD or IPF).

[0034] In a second embodiment, the present invention provides a method for treating chronic obstructive pulmonary disease (COPD) or idiopathic pulmonary fibrosis (IPF) in patients requiring treatment, comprising the following steps: (a) With respect to the sample obtained from the patient, according to the first aspect of the present invention Performing immunoassays to detect whether a person has COPD or IPF, or whether the severity of these conditions is at a specific level; and (b) If it is determined in step (a) that the patient has COPD or IPF or that the severity of either is at a certain level, the patient is given therapy to treat COPD or IPF.

[0035] The aforementioned therapy may be any therapy appropriate for treating the target disease (COPD or IPF). The therapy may include, for example, one or more medications, one or more lifestyle modifications, one or more surgeries, or a combination thereof, or consist of such therapies. The medication may be prescribed for topical administration (e.g., as a cream, foam, gel, lotion, ointment, or patch), for transgastrointestinal administration (e.g., orally, rectally, or sublingually), or for parenteral administration (e.g., via injection or inhalation). The surgery may be curative, prophylactic, palliative, and / or reconstructive.

[0036] For example, an appropriate treatment for COPD may include one or more of the following: cessation of smoking; lung rehabilitation (exercise training, nutritional adjustment, occupational therapy, educational and psychosocial counseling); influenza and pneumonia vaccination; oxygen therapy (supplementary oxygen); and bronchodilators belonging to either long-acting or short-acting types, such as beta-2-adrenergic agonists and / or anticholinergic agonists, for example, salbutamol, terbutaline, salmeterol, formoterol, indacaterol, ipratropium, tiotropium, acridi Phytochloride, umeclidinium bromide, glycopyrronium, etc.; corticosteroids (inhaled and / or oral); phosphodiesterase-4 inhibitors (PED4 inhibitors), e.g., roflumilast; antibiotics, e.g., amoxicillin, doxycycline, azithromycin, fluoroquinolones, erythromycin; methylxanthines, e.g., theophylline; mucopolysaccharide hydrolases, e.g., erdosteine, caspocysteine; and, in rare cases, surgeries such as lung decompression surgery, bronchoscopic lung decompression surgery, lung cystectomy, or lung transplantation.

[0037] Appropriate treatment for IPF may include one or more of the following: oxygen therapy (supplementary oxygen); lung rehabilitation (exercise training, nutritional adjustment, occupational therapy, educational and psychosocial counseling); influenza and pneumonia vaccinations; pirfenidone; nintedanib; lung transplant surgery; and palliative use of opioids.

[0038] In a third aspect, the present invention provides a monoclonal antibody that specifically binds to the N-terminal amino acid sequence VDTLPQKPRE (SEQ ID NO: 1) or to the N-terminal amino acid sequence AMELFSPDQD (SEQ ID NO: 2). The antibody according to the third embodiment of the invention is particularly suitable for use when carrying out the immunoassay method according to the first embodiment of the invention. Accordingly, preferred embodiments and features of the antibody according to the third embodiment are evident from the above discussion concerning preferred embodiments of the method according to the first embodiment.

[0039] In a fourth aspect, the present invention provides a monoclonal antibody according to a third aspect of the present invention, and an immunoassay kit comprising at least one of the following: - Streptoavidin-coated well plates - Biotinylated peptide VDTLPQKPRE-L-biotin (SEQ ID NO: 17), where L is an arbitrary linking group, or biotinylated peptide AMELFSPDQD-L-biotin (SEQ ID NO: 18), where L is an arbitrary linking group. - Second antibody used in sandwich immunoassays - Calibration protein containing the N-terminal amino acid sequence VDTLPQKPRE (SEQ ID NO: 1), or calibration protein containing the N-terminal amino acid sequence AMELFSPDQD (SEQ ID NO: 2) - Antibody biotinylation kit - Antibody HRP labeling kit - Antibody radiolabeling kit. The immunoassay kit according to the fourth embodiment of the invention is particularly suitable for use when carrying out the immunoassay method according to the first embodiment of the invention. Therefore, more preferred embodiments and features of the immunoassay kit according to the fourth embodiment are evident from the above discussion of preferred embodiments of the method according to the first embodiment. [Brief explanation of the drawing]

[0040] figure [Figure 1] Figure 1: Alignment of partial sequences of human, rat, and mouse club cell secreted protein 16 (CC16). Arrows point to one of the cleavage sites by human neutrophil elastase (HNE) present in CC16. The gray-highlighted areas point to portions of the human sequence (and corresponding portions of the aligned rat and mouse sequences) identified by mass spectrometry and selected for the development of monoclonal antibodies used in the CC16-HNE assay. [Figure 2] Figure 2: Specificity of the CC16-HNE assay. The CC16-HNE assay revealed that it is a specific neoepitope that reacts with the selective peptide but does not bind to any non-selective peptide. [Figure 3] Figure 3: Clinical evaluation of the CC16-HNE assay. Serum levels of CC16-HNE are shown in healthy subjects (n=20), COPD subjects (n=58), and IPF subjects (n=99) at baseline. Data are shown as median and interquartile range, along with individual data points. Data were analyzed by multiple comparison tests using Kruskar-Wallis and Dunn; **p<0.01;****p<0.0001.

[0041] [Figure 4]Figure 4: Characterization of CC16-HNE in COPD. The association between CC16-HNE serum levels and clinical parameters in COPD patients was investigated. A) CC16-HNE serum levels in comparison to females and males. B) Scatter plot of CC16-HNE levels and age. C) CC16-HNE serum levels shown by GOLD stage (2=moderate, 3=severe, 4=very severe). D) Scatter plot of CC16-HNE levels and predicted FVC%. E) Scatter plot of CC16-HNE levels and predicted FEV%. Group comparisons were performed using the Mann-Whitney test (A) or the Kruskal-Wallis and Dunn multiple comparison test (C), and the data are shown as box plots indicating the interquartile range. Correlation was evaluated by Spearman's rank correlation coefficient. FVC = Forced vital capacity; FEV1 = Forced expiratory volume in one second; GOLD = International Staging of Chronic Obstructive Pulmonary Disease (1 = Mild, 2 = Moderate, 3 = Severe, 4 = Very Severe); ns = Non-significant; SD = Standard deviation; *p<0.05.

[0042] [Figure 5] Figure 5: Survival prediction and characterization of CC16-HNE in IPF. A) Serum CC16-HNE levels were evaluated in IPF subjects (n=99) at baseline and divided into two groups based on the median. Patients with low CC16-HNE had a significantly higher all-cause mortality rate compared to IPF patients with high CC16-HNE, with a hazard ratio of 1.79 (95% CI 1.03-3.13; p=0.04). B) Serum CC16-HNE levels in comparison between females and males. C) Scatter plot of CC16-HNE levels and age in IPF patients. D) Scatter plot of CC16-HNE levels and FVC (% predicted value) in IPF patients. Group comparisons were performed using the Mann-Whitney test, and data are shown as box plots indicating the interquartile range. Correlation was evaluated using Spearman's rank correlation coefficient. FVC = Forced vital capacity; ns = Non-significant.

[0043] [Figure 6]Figure 6: Direct comparison of CC16-HNE assay and CC16 assay. A) Serum levels of CC16-HNE in healthy subjects (n=20), COPD subjects (n=58), and IPF subjects (n=99). B) CC16-HNE ROC curve for separation of healthy subjects and COPD subjects. C) CC16-HNE ROC curve for separation of healthy subjects and IPF subjects. D) Serum levels of CC16 in healthy subjects, COPD subjects, and IPF subjects. E) CC16 ROC curve for separation of healthy subjects and COPD subjects. F) CC16 ROC curve for separation of healthy subjects and IPF subjects. Data are shown as median and interquartile range along with individual data points and analyzed by multiple comparison tests using Kruskal-Wallis and Dunn. AUC = Area under the curve; ns = Non-significant; ROC = Receiver operating characteristics; **p < 0.01; ****p < 0.0001.

[0044] [Figure 7] Figure 7: CC16-HNE levels were measured in serum samples collected from subjects with idiopathic pulmonary fibrosis (IPF) with or without pulmonary hypertension (PH) (IPF + PH, n=13) or without (IPF - PH, n=86). Biomarker levels were log-transformed and analyzed using a linear regression model adjusted for age and sex. Data are presented as geometric mean evaluation values ​​± inversely transformed 95% confidence intervals (CI). ** indicates a significant difference between PH-negative and PH-positive IPF subjects (p-value < 0.01). [Examples]

[0045] The embodiments disclosed herein are described in the following examples. These examples are provided to aid in understanding the disclosure and should not be construed in any way as limiting the scope of the disclosure specified in the claims below. The examples below are provided to a person skilled in the art to provide 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 mean that the experiments described below are all or only experiments performed. Efforts have been made to ensure accuracy of the numerical values ​​used (e.g., quantity, temperature, etc.), but some experimental error and deviation should be taken into consideration. Unless otherwise indicated, parts are parts by weight, molecular weight is weight-average molecular weight, temperature is in Celsius, and pressure is atmospheric pressure or near atmospheric pressure.

[0046] Materials and methods Unless otherwise specified, all reagents used were high-quality chemicals from Merck (Whitehouse Station, NJ, USA) and Sigma (St. Louis, MO, USA). All synthetic peptides (Table 1) were purchased from GenScript (Piscataway, NJ, US).

[0047] In vitro cleavage and target identification In vitro cleavage of recombinant CC16 (uteroglobin SCGB1A1) (cat. no. MBS691837, MyBioSource, San Diego, CA, US) was performed using native human neutrophil elastase (cat. no. ab91099, Abcam, Cambridge, UK) in a 10:1 ratio in cleavage buffer (50 mM Tris-HCl, 150 mM NaCl, pH 7.5) in low-binding Eppendorf tubes at 37°C for 3 hours / 24 hours. Cleavage was confirmed by silver staining using a SilverXpress kit (in vitrogen) according to the manufacturer's instructions, and the samples were stored at -80°C. Subsequently, the cleaved samples were prepared for mass spectrometry. Briefly, the samples were passed through a 30K molecular weight cutoff (MWCO) filter (30kD filter obtained from Pall, 30K, P / N OD030C34). The sample was then desalted using a Vydac UltraMicro Spin C18 column (cat. no. 74-7206, Harvard Apparatus). The sample bound to the column material was rinsed with a 2% acetonitrile solution in 0.2% formic acid, and then eluted with a 50% acetonitrile solution in 0.1% formic acid. The sample was transferred to a vial, dried using SpeedVac, stored at -20°C until reduced with water, and then analyzed.

[0048] Peptides were identified from raw data using Mascot 2.2 (Thermo Fisher Scientific) within Proteome Discoverer software 1.4. Peptides with a probability score of p<0.05 were selected for further analysis. CC16 peptides identified in CC16 / HNE samples and not overlapping with peptides in HNE-free control samples were considered to be HNE-induced CC16 degradation fragments. The 10 amino acids at the terminal C- or N-terminal resulting from HNE cleavage of each peptide are thought to contain an HNE-generated neoepitope, and each neoepitope is expected to contain the first 6 amino acids at the terminal C- or N-terminal resulting from HNE cleavage of the peptide (because an antibody specific to one of the HNE-generated neoepitopes is expected to depend on binding to these first 6 amino acids). Therefore, each of the six amino acid sequences was analyzed for its uniqueness by protein blasting using the NPS@: Network Protein Sequencing Analysis tool

[14] , and then its homology was analyzed using the UniProt sequence sorting tool

[15] .

[0049] Development, production, and characterization of monoclonal antibodies N-terminal amino acid sequence 65↓66 A monoclonal antibody (mAb) specific to VDTLPQKPRE (SEQ ID NO: 1) was produced using the following procedure (identified and selected after HNE cleavage and fragment analysis of CC16, following the procedure described above). Five female 6-7 week old Balb / C mice were immunized by subcutaneous injection of 200 μl of emulsified antigen containing 100 μg of antigenic peptide (VDTLPQKPRE-GGC-“KLH” (SEQ ID NO: 19)) using a Thermo Fisher stimulant. Immunization was repeated every two weeks until stable serum antibody titer levels were achieved. Mice with the highest serum titer were selected for cell fusion and kept in recuperation for one month. Subsequently, these selected mice were boost-immunized by intravenous administration of 100 μl of 0.9% NaCl solution containing 50 μg of immunogenic peptide three days before spleen removal for cell fusion. Mouse splenocytes were fused with SP2 / 0 myeloma cells as described by Gefter et al.

[16] to create hybridoma cells. To further promote the proliferation of fused cells, they were cultured in each well of a 96-well microtiter plate using the limiting dilution method to stimulate monoclonal proliferation.

[0050] The reactivity of the supernatant obtained from fusion cell clones was screened using an indirect ELISA performed on streptavidin-coated 96-well microtiter plates to identify the best mAb-producing clones. The assay used a biotin-labeled screening peptide (VDTLPQKPRE-K-Biotin (SEQ ID NO: 10)), and antibody specificity was tested using a selection peptide (VDTLPQKPRE (SEQ ID NO: 1)) and non-selective peptides (extension [LVDTLPQKPRE (SEQ ID NO: 3)], truncation [DTLPQKPRE (SEQ ID NO: 4)], and nonsense [QMAGLDEKSG (SEQ ID NO: 21)]) (see Table 1). The best clones were selected based on their specificity to the selected peptide and their non-reactivity to the non-selected peptide. The supernatant was collected, and the mAbs were purified using a HiTrap affinity column (GE Healthcare Life Sciences, Little Chalfront, Buckinghamshire, UK). Subsequently, the mAbs were labeled with horseradish peroxidase (HRP) using a Roche peroxidase labeling kit (cat no. 11829696001, Merck). Finally, the antibody isotype was determined using the Rapid ELISA mouse mAb isotyping kit (Invitrogen, Carlsbad, CA, USA). All procedures were performed according to the manufacturer's instructions.

[0051] [Table 1]

[0052] CC16-HNE assay development The development of the competitive ELISA using the HRP-labeled mAb described above (hereinafter referred to as the "CC16-HNE assay") involved several preliminary optimization experiments, in which the reactivity to selective and non-selective peptides (Table 1) and human serum was tested, as well as the reagents, concentrations, incubation time, and incubation temperature were analyzed. The final CC16-HNE assay procedure was as follows: A 96-well streptavidin-coated microplate (cat. no. 11940279, Roche Diagnostics, Hvidovre, Denmark) was coated with 100 μL / well of a screening peptide (VDTLPQKPRE-K-biotin (SEQ ID NO: 20)) diluted to 15 ng / mL in coating buffer (25 mM phosphate-buffered saline (PBS) with 1% bovine serum albumin, 0.1% Tween-20, 0.36% Bronidox (BTB), 4 g / L NaCl added, pH 7.4), and incubated at 20°C for 30 minutes. Next, 20 μL / well of the selected peptide (diluted 2-fold from 1000 ng / mL), assay control, and the sample of interest were added to the appropriate wells, and the measurement was repeated twice. Then, 100 μL / well of HRP-labeled mAb diluted to 180 ng / mL in assay buffer (25 mM PBS-BTB, 4 g / L NaCl, 5% Liquid II, pH 7.4) was added, and the plate was incubated at 4°C for 20 hours. 100 μL / well of the colorimetric substrate 3,3',5,5'-tetramethylbenzidine (TMB)One (cat. no. 4380H, Chem-En-Tech, Taastrup, Denmark) was added, incubated for 15 minutes, and the reaction was stopped with 100 μL / well of 0.1% sulfuric acid. Absorbance was measured at a wavelength of 450 nm, using a microplate reader (VersaMax, molecular device, Sunnyvale, CA, USA) with 650 nm as the reference wavelength. All incubation steps were performed in the dark with shaking at 300 rpm, after which the wells were washed five times with washing buffer (20 nM Tris, 50 mM NaCl, pH 7.2). A 10-point calibration curve was plotted using a 4-parameter logistic fit model of selected peptide dilutions. The data were analyzed using SoftMax Pro version 7.1.0 software (molecular devices).

[0053] Technical evaluation Antibody specificity was evaluated using synthetic peptides and assessed as the percentage of signal inhibition of 2-fold diluted selected and non-selected peptides (Table 1) (Table 1). Nonspecific binding was tested using a nonsense screening peptide (biotin-QAAFSQYKKV (SEQ ID NO: 23)). IC50 (median inhibitory concentration) was determined from 10 independent runs of a 10-point calibration curve. The technical lower limit of detection (LLOD) was calculated as the mean signal obtained from 60 measurements of the blank sample (i.e., assay buffer) plus 3x standard deviation (SD). The upper limit of quantification (ULOQ) was determined from 10 independent runs of the calibration curve and was determined as the highest concentration of the selected peptide in which the percentage recovery was determined to be within 100 ± 20% of the nominal concentration and CV < 20%. The lower limit of quantification (LLOQ) for serum was determined by evaluating four human serum samples covering the lower region of the calibration curve through three independent runs (a total of 15 measurements per sample). The mean concentration and CV% for each sample were plotted, and the LLOQ, defined as the lowest analyte concentration when the CV% of the 95% confidence interval was equal to 20%, was determined using a regression model. Linearity of the assay was evaluated using 2-fold dilutions of the four human serum samples quantified through three independent runs, and calculated as the percentage recovery of the undiluted samples. Intra- and inter-assay variation was determined by performing 10 independent runs, each involving two measurements for 10 quality control samples (8 human serums and 2 assay controls). Assay stability was evaluated by storing the assay reagents at 20°C for 24 hours and at 37°C for 24, 72, or 168 hours. Ten quality control samples were quantified by three independent runs using stressed assay reagents, and the percentage recovery was calculated using the average concentration obtained in tests for intra-assay and inter-assay variability as a reference.

[0054] The accuracy of the assay was measured in three human serum samples that were spiked with a 2-fold dilution of a standard peptide, and in three human serum samples that were spiked with a 2-fold dilution of a human serum sample containing a high concentration of the analyte. The percentage recovery for the spiked samples was calculated using the theoretical amount of analyte present in the sample as a reference. Analytical interference was evaluated by adding low / high concentrations of hemoglobin (2.5 / 5.0 mg / mL), lipids (1.5 / 5.0 mg / mL), and biotin (5.0 / 40 ng / mL) to human serum samples of known concentrations, and calculating the percentage recovery rate in the spiked samples using the non-spiked samples as a reference. The stability of the analytes was determined in human serum samples stressed by a freeze-thaw cycle up to 5 cycles, or by storage at 4°C or 20°C for 2, 4, 24, or 48 hours. Recovery rates were calculated using unstressed samples as a reference. All tests involved repeated measurements of the sample unless otherwise specified.

[0055] Clinical evaluation of the CC16-HNE assay The clinical relevance of the novel CC16-HNE assay (described above) was evaluated in serum samples from COPD, IPF, and healthy subjects. Serum samples were measured twice, and values ​​outside the measurement range were appropriately assigned LLOQ or ULOQ values. Healthy subjects were obtained from a commercial vendor (Discovery Life Sciences, Los Osos, CA). COPD subjects were from a previously described observational study

[17] . Inclusion criteria for COPD subjects were diagnosed with COPD by a senior physician and predicted FEV1 < 80%, while exclusion criteria were acute exacerbation prior to blood collection and hospitalization for 4 weeks. IPF subjects had a common illness and were part of a previously described large study

[18] . IPF subjects were aged > 18 years and diagnosed with IPF, but those with language or intellectual impairments that prevented questionnaire completion were excluded. Clinically relevant parameters were collected at baseline for both disease groups. In addition, mortality data was recorded for IPF subjects over a five-year period.

[0056] To perform clinical evaluations, CC16-HNE levels (i.e., levels of the peptide detected and measured by the CC16-HNE assay) were measured in serum collected at baseline from healthy subjects (n=20), COPD subjects (n=58), and IPF subjects (n=99) at the study baseline. To directly compare with a commercial non-neopeptide assay for CC16, CC16 levels (i.e., levels of CC16 detected and measured by the commercial assay) and CC16-HNE levels were directly compared in serum collected at follow-up visits from healthy subjects (n=20), COPD subjects (n=20), and IPF subjects (n=19). The commercial human CC16 ELISA (cat. no. RD191022200, biovendor, Brno, Czech Republic) was performed according to the manufacturer's instructions, with two measurements repeated on each sample. To further clinically evaluate the findings, CC16-HNE levels were also measured in serum samples from IPF subjects diagnosed with and without PH (n=13 and n=86).

[0057] ethical statement All animals were handled in accordance with animal welfare guidelines. The production of monoclonal antibodies in mice was approved by the Danish National Agency (Animal Experiment Inspectorate) under approval number 2013-15-2934-00956. The collection and recovery of human serum followed international ethical guidelines for the handling of human samples and subject information. All participants signed informed consent, and the study was approved by the local ethics committee. Samples were collected in accordance with the 1975 Declaration of Helsinki after obtaining informed consent and approval from the local ethics committee.

[0058] statistical analysis Serum levels of CC16-HNE and CC16 were analyzed using the Kruskar-Wallis test and Dunn multiple comparison test. Isolation of the disease group from healthy subjects (diagnostic accuracy) was further evaluated by assessing the area under the curve (AUC) using receiver operating characteristics (ROC). Correlation was assessed using Spearman's rank correlation coefficient, and statistical significance between sexes was assessed using the Mann-Whitney U test. Interquartile ranges (IQR) and 95% confidence intervals (95% CI) of biomarker levels are displayed as appropriate. A p-value of less than 0.05 was considered statistically significant. Statistical analysis and graphing were performed using GraphPad Prism version 9.5.0 (GraphPad Software, Inc. La Jolla, CA).

[0059] result Identification of the CC16-HNE neoepitope and development of mAbs. From the peptides identified by MS analysis, two peptides with unique amino acid sequences released in vitro from HNE-mediated cleavage of CC16 were selected as potential candidates: 44↓45 AMELFSPDQD (Sequence ID 2), and 65↓66VDTLPQKPRE (Sequence ID 1). Due to the relatively high degree of species homology between humans, rats, and mice, the N-terminal amino acid sequence 65↓66 VDTLPQKPRE (SEQ ID NO: 1) was selected for antibody development. The human, rat, and mouse sequences were 100% identical at the positions of the first six amino acids closest to the cleavage site, while the rat and mouse sequences differed from the human sequence at amino acids at positions 7, 8, and 10 (Figure 1).

[0060] As described above, mAbs were generated by immunizing mice with an immunogenic peptide (VDTLPQKPRE-GGC-“KLH” (SEQ ID NO: 19)) containing the selected N-terminal amino acid sequence. The mAb with the best selectivity for the selected peptide (VDTLPQKPRE (SEQ ID NO: 1)), the best native reactivity, and stability was selected for assay development. The isotype, sequence, and CDRs of this monoclonal antibody were determined. The chain sequence is as follows (CDRs are underlined and bold; the N-terminal signal peptide and C-terminal constant region are italicized): Heavy chain sequence (mouse IgG2b isotype)

[0061] [ka]

[0062] Light chain sequence (mouse kappa isotype)

[0063] [ka]

[0064] Technical evaluation and characterization of the CC16-HNE assay. An assay using the selected mAb was developed on a competing ELISA platform and named CC16-HNE, as described above. To evaluate the specificity of the assay, reactivity to the selected and non-selected peptides was assessed (see Table 1 for details of the peptides). The CC16-HNE assay was found to detect only selective peptides and not non-selective peptides, demonstrating that this assay is specific to HNE-generating CC16 neoepitopes (Figure 2).

[0065] A summary of the technical evaluation of the CC16-HNE assay can be found in Table 2. Briefly, the measurement range (LLOQ-ULOQ) was determined to be 13.4–1000 ng / mL, and the IC50 was 36.9 ng / mL. Intra-assay and inter-assay variability were 7.1% and 11.6%, respectively, and linearity was acceptable from undiluted to 2-fold and 4-fold dilutions of human serum (recovery rate 104.3–106.5%). The assay remained stable even after long-term storage of the kit reagents at 37°C for up to 7 days (recovery rate 100.8–119.2%). The analytes in human serum remained stable even after subjecting samples to up to five freeze-thaw cycles (recovery rate 98.5–113.5%), and remained stable even after subsequent long-term storage of samples at 20°C for up to 48 hours (recovery rate 87.2–107.5%). Hemoglobin and lipids did not interfere with the measurement of CC16-HNE levels, although biotin did not interfere at low concentrations but showed slight interference at high concentrations, resulting in an analyte recovery rate slightly outside the acceptable limit of 120% (recovery rate 121.7%). The CC16-HNE assay demonstrated good accuracy, with recovery rates of 87.3–104.1% for human serum spiked with the selected peptide or another serum sample.

[0066] [Table 2]

[0067] Clinical evaluation of the CC16-HNE assay Population statistics for the initial patient cohort used to evaluate the clinical utility of CC16-HNE are summarized in Table 3. Serum CC16-HNE levels were determined in healthy individuals, COPD subjects, and IPF subjects, and significantly elevated levels were found in both COPD (median 64.2 [IQR 51.1-92.2] ng / mL, p=0.0047) and IPF (median 85.5 [IQR 66.6-108.5] ng / mL, p<0.0001) subjects compared to healthy subjects (median 43.16 [IQR 33.0-55.5] ng / mL) (Figure 3). Furthermore, a statistically significant difference in CC16-HNE levels (p = 0.0161) was found between COPD patients and IPF patients.

[0068] In the COPD cohort, CC16-HNE levels were not associated with age, sex, or forced vital capacity (FVC), but were significantly inversely correlated with FEV1 (r=-0.366, p=0.0048) (Figures 4A-B and 4D-E). Patients with very severe COPD (GOLD stage 4, median 79.9 [IQR 56.0-130.5] ng / mL) showed a slight but statistically significant increase in CC16-HNE compared to patients with moderate COPD (GOLD stage 2, median 51.0 [IQR 39.4-76.4] ng / mL; p=0.049) (Figure 4C).

[0069] In the IPF cohort, serum CC16-HNE levels were a precursor to mortality. Patients with CC16-HNE levels below the median at baseline had a significantly increased risk of death within 5 years compared to patients with levels above the median (hazard ratio 1.79 [95% CI 1.03-3.13]; p=0.04) (Figure 5A). In these patients, CC16-HNE levels at blood collection were not associated with sex (males: median 84.96 [IQR 66.6-107.2] ng / mL; females: median 91.7 [IQR 65.4-112.0] ng / mL) (Figure 5B), age (Figure 5C), or FVC (Figure 5C).

[0070] [Table 3]

[0071] In cohorts of IPF patients with PH (n=13) and IPF patients without PH (n=86), baseline CC16-HNE serum levels were found to be lower in patients with PH (PH-positive, adjusted mean 58.4 [95%CI 45.4-75.2] ng / mL) compared to patients without PH (PH-negative, adjusted mean 87.7 [95%CI 77.5-99.1] ng / mL; p = 0.004) (Figure 7).

[0072] Comparison of the potential diagnostic capabilities of the CC16-HNE assay and the CC16 assay. The performance of the CC16-HNE assay was directly compared to that of a commercial assay for intact CC16 using a direct comparison method that measured healthy subjects, COPD subjects, and IPF subjects. CC16-HNE serum levels were significantly elevated in both COPD subjects (median 87.3 [IQR 52.3-104.9] ng / mL; p=0.0018) and IPF subjects (median 82.46 [IQR 57.4-124.9] ng / mL; p=0.0001) compared to healthy subjects (median 43.2 [IQR 33.0-55.5] ng / mL) (Figure 6A). Regarding serum CC16 levels, there was no statistically significant difference between healthy individuals (median 7.78 [IQR 6.14-9.48] ng / mL) and COPD individuals (median 12.34 [IQR 8.18-17.83] ng / mL; p = 0.092). However, for IPF individuals (median 43.51 [IQR 32.7-52.85]; p<0.0001), the levels were statistically significant compared to healthy subjects (Figure 6D). Therefore, the separation between healthy subjects and COPD subjects was greater with CC16-HNE (AUC 0.80 [95% CI 0.64-0.95]; p = 0.0014) (Figure 6B) than with CC16 (AUC 0.74 [95% CI 0.59-0.90]; p = 0.0087) (Figure 6E). The separation between healthy subjects and IPF subjects was similar and highly significant for both CC16-HNE (AUC 0.90 [95% CI 0.80-1.00]; p<0.0001) (Figure 6C) and CC16 (AUC 1.00 [95% CI 1.00-1.00]; p<0.0001) (Figure 6F).

[0073] Essay In this study, the inventors developed and characterized a competitive ELISA, the "CC16-HNE assay," which can quantify the level of CC16 neoepitopes (in serum or other biological fluids) generated by cleaving CC16 using HNE. The inventors demonstrated that this assay is specific, accurate, precise, and stable for the targeted neoepitope. CC16 has long been proposed as a biomarker for COPD and IPF. Now, the inventors present data demonstrating that the CC16-HNE assay developed by the inventors (which measures the level of HNE-generating neoepitope of CC16) is superior to commercially available CC16 assays (which measure the level of intact CC16) in separating COPD subjects from healthy subjects. Furthermore, the CC16-HNE assay showed good separation between IPF subjects and healthy subjects, and the baseline levels in IPF subjects were a precursor to mortality. The CC16-HNE assay was also able to differentiate between IPF subjects with and without PH.

[0074] The CC16-HNE assay measures the proteolytic activity of HNE on CC16, quantifying a specific process associated with lung inflammation. While CC16 is believed to be released in response to inflammation or airway epithelial damage, HNE is released from migrating neutrophils as part of the response to inflammation. Therefore, CC16-HNE levels reflect lung inflammation by uniquely combining and evaluating two highly relevant processes, providing additional information compared to standard CC16 assays, and can be readily quantified in serum by the novel CC16-HNE assay. Furthermore, CC16 has been highlighted for its essential role in regulating respiratory diseases and its potential as an early-stage screening marker for lung diseases due to its abundance.[7] In this study, the inventors showed that evaluating two disease-related processes in combination improved the diagnostic value for COPD compared to evaluating intact CC16 alone. In addition, they showed that CC16-HNE neoepitope levels were not affected by age or sex, and that serological levels reflected the inflammatory state in the subjects' lungs.

[0075] In conclusion, the inventors developed a novel method for quantifying lung inflammation in serum by measuring the unique fragments of CC16 generated by HNE. The CC16-HNE assay was robust. Furthermore, the CC16-HNE assay was found to be a biomarker assay with prognostic and diagnostic capabilities in subjects with COPD and IPF.

[0076] In this specification, unless otherwise specified, the word "or" is used to mean an operator that returns true if one or both of the stated conditions are met, in contrast to the "exclusive OR" operator, which requires that only one of several conditions be met. The word "comprising" is used to mean "including, or consisting of." All prior teachings acknowledged above are incorporated into this specification by reference thereto. Any recognition of prior publications in this specification should not be taken as an admission or statement that the teachings in such publications were common knowledge in Australia or other countries at the time of this specification.

[0077] References [1] WW Labaki and MK Han, "Chronic respiratory diseases: A global survey." The Lancet Respiratory, vol. 8, pp. 531-533, 2020, doi: 10.1016 / S2213-2600(19)30356-X. [2] S. Safiri et al., "The burden of chronic obstructive pulmonary disease and its risk factors in 204 countries and territories, 1990-2019: Results from the 2019 Global Study on Disease Burden," BMJ, vol. 378, Jul. 2022, doi: 10.1136 / BMJ-2021-069679. [3] TM Mayher et al., "Global incidence and prevalence of idiopathic pulmonary fibrosis," Respir Res, vol. 22, no. 1, p. 197, Jul. 2021, doi: 10.1186 / s12931-021-01791-z. [4] B. Beguet, S. Serri, L.M. Fabri, and A. Marchioni, "COPD, pulmonary fibrosis, and ILASS in older smokers: Paradoxes regarding remarkably different responses to major risk factors." Int J Mol Sci, vol. 22, no. 17, Sep. 2021, doi: 10.3390 / IJMS22179292. [5] JE Boayers, AW Ambergen, and FBJM Sunnissen, “Number and proliferation of club cells in normal human airway epithelium.” https: / / doi.org / 10.1164 / ajrccm.159.5.9806044, vol. 159, no. 5 I, pp. 1585-1591, Dec. 2012, doi: 10.1164 / AJRCCM.159.5.9806044.

[0078] [6] AP Wong, J. Keating, and Waddell, "Airway regeneration: The role of club cell secretory proteins and the cells that express them," Cytotherapy, vol. 11, no. 6, pp. 676-687, 2009, doi: 10.3109 / 14653240903313974. [7] S. al-Muntasili, Y. Zhu, Y. Han, X. Wang, PR. Somanas, and D. Zhan, "Club cell secretory protein CC16: Potential for application in the diagnosis and treatment of lung diseases," J Clin Med, vol. 9, no. 12, pp. 1-16, Dec. 2020, doi: 10.3390 / JCM9124039. [8] ME Laucho-Contreras et al., "Expected role of club cell secreted protein-16 (CC16) in the progression of COPD," Eur Respir J, vol. 45, pp. 1544-1556, 2015, doi: 10.1183 / 09031936.00010515. [9] I. Buendía-Rolda et al., “Increased CC16 expression in patients with idiopathic pulmonary fibrosis.” 2016, doi: 10.1371 / journal.pone.0168552.

[10] A. Bernardo, FX Merchandise, S. Deperchine, R. Lowellis, and Y. Sybil, “Crab cell protein and bronchoalveolar lavage in serum,” Eur Rasplr J, vol. 5, pp. 1231-1238, 1992.

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[12] S. Guerra et al., "The relationship between circulating CC16 concentration, pulmonary function, and the progression of chronic obstructive pulmonary disease throughout life: A prospective study." Elsevier, Accessed: Feb. 21, 2023. [Online]. Available: https: / / www.sciencedirect.com / science / article / pii / S2213260015001964.

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Claims

1. Immunoassay methods comprising the following i) and ii); i) Contacting the patient's sample with a monoclonal antibody that specifically binds to the N-terminal amino acid sequence VDTLPQKPRE (SEQ ID NO: 1); and, ii) Detect the binding between the monoclonal antibody and the peptide in the sample, and determine the amount of binding.

2. The method is an immunoassay method for detecting and / or monitoring chronic obstructive pulmonary disease, or idiopathic pulmonary fibrosis, or a specific level of severity of these diseases in a patient, and further comprises iii) below, according to claim 1; iii) Correlating the binding amount with a value associated with a normal healthy person, and / or a value associated with a known disease severity, and / or a value obtained from the patient at a past point in time, and / or a predetermined cutoff value.

3. The immunoassay method according to claim 2, wherein the method is an immunoassay method for detecting and / or monitoring chronic obstructive pulmonary disease or a specific level of severity of said disease in a patient.

4. The immunoassay method according to claim 2, wherein the method is an immunoassay method for detecting and / or monitoring a specific level of severity of idiopathic pulmonary fibrosis in a patient; or an immunoassay method for detecting and / or monitoring idiopathic pulmonary fibrosis accompanied by pulmonary hypertension in a patient.

5. The immunoassay method according to any one of the claims, wherein the sample of the patient is selected from blood, plasma, or serum.

6. The immunoassay method according to any one of the claims, wherein the monoclonal antibody does not specifically bind to the peptide having the N-terminal amino acid sequence LVDTLPQKPRE (SEQ ID NO: 3).

7. The immunoassay method according to any one of the claims, wherein the monoclonal antibody does not specifically bind to the peptide having the N-terminal amino acid sequence DTLPQKPRE (SEQ ID NO: 4).

8. The immunoassay method according to any one of the claims, wherein the monoclonal antibody is produced in opposition to a synthetic peptide having the N-terminal amino acid sequence VDTLPQKPRE (SEQ ID NO: 1).

9. The immunoassay method according to any one of the claims, wherein the immunoassay is a competitive assay or a sandwich assay.

10. The immunoassay method according to any one of the claims, wherein the immunoassay is a radioimmunoassay or an enzyme-linked immunosorbent assay.

11. A monoclonal antibody that specifically binds to the N-terminal amino acid sequence VDTLPQKPRE (SEQ ID NO: 1).

12. The monoclonal antibody according to claim 11, wherein the monoclonal antibody does not specifically bind to a peptide having the N-terminal amino acid sequence LVDTLPQKPRE (SEQ ID NO: 3).

13. The monoclonal antibody according to claim 11 or claim 12, wherein the monoclonal antibody does not specifically bind to a peptide having the N-terminal amino acid sequence DTLPQKPRE (SEQ ID NO: 4).

14. The immunoassay method according to any one of claims 11 to 13, wherein the monoclonal antibody is produced in opposition to a synthetic peptide having the N-terminal amino acid sequence VDTLPQKPRE (SEQ ID NO: 1).

15. An immunoassay kit comprising a monoclonal antibody according to any one of claims 11 to 14, and at least one of the following: - Streptoavidin-coated well plates - Biotinylated peptide VDTLPQKPRE-L-biotin (SEQ ID NO: 17), where L is an arbitrary linking group. - Second antibody used in sandwich immunoassays - Calibration protein containing N-terminal amino acid sequence VDTLPQKPRE (SEQ ID NO: 1) - Antibody biotinylation kit - Antibody HRP labeling kit - Antibody radiolabeling kit.

Images