Collagen VI alpha-6 assay

Abstract

An immunoassay method for detecting and / or monitoring a skin disorder and / or determining the severity of a skin disorder in a patient is disclosed. The method comprises detecting and quantifying the level of a biomarker comprising the N-terminal sequence of type VI collagen alpha-6 in a sample derived from the patient's blood, such as whole blood, plasma, or serum. Antibodies suitable for use in the method and immunoassay kits suitable for carrying out the method are also disclosed.

Description

[Technical Field]

[0001] The present invention relates to an immunoassay method for detecting and / or monitoring and / or determining the severity of a skin disease in a patient. The method comprises detecting and quantifying the level of a biomarker comprising the N-terminal sequence of type VI collagen alpha-6 in a patient's blood-derived sample, such as whole blood, plasma, or serum. The present invention also relates to antibodies suitable for use in the method and immunoassay kits suitable for carrying out the method. The skin disease may be, in particular, a skin disease selected from atopic dermatitis, melanoma, psoriasis, hidradenitis suppurativa, systemic sclerosis, and systemic lupus erythematosus. [Background technology]

[0002] Skin extracellular matrix (ECM) remodeling is a continuous process necessary for maintaining tissue homeostasis. Skin is divided into three layers: the epidermis, dermis, and subcutaneous tissue, each with distinct tissue structures and functions (References 1, 2). The dermis is the layer where fibroblasts play a key role in synthesizing the ECM and maintaining tissue structure, as well as providing tensile strength, elasticity, and resilience to the skin (References 3, 4). The dermal ECM is classified into papillary and reticular dermal ECMs, which are composed of matricellular proteins (COMP, SPARC, thrombospondin-1, periostin, tenascin C, and tenascin X), proteoglycans (decorin, versican, biglycan, fibromodulin, and lumican), collagens (I, III, V, VI, XII, XIV, and XV), fibrillin microfibrils (fibrillin-1 and fibrillin-2), and elastic fiber proteins (elastin, EMLIN-1, EMLIN-2, LTBP-4, fibulin-4, and fibulin-5) (Reference 2). Dysregulation of papillary and reticular dermal ECM remodeling is a key event in dermatological pathologies, including atopic dermatitis and psoriasis (References 1, 2, 5).

[0003] The importance of type VI collagen in maintaining skin tissue homeostasis has been documented in the literature. Type VI collagen is characterized as a beaded filament collagen present in the papillary and reticular dermal ECM and is thought to form a microfibril network (Ref. 6). Six distinct chains of type VI collagen (α1, α2, α3, α4, α5, and α6) have been identified and are expressed throughout connective tissue (Ref. 7). In vitro experiments have demonstrated that the α1 chain of type VI collagen is crucial for ECM assembly in human dermal fibroblasts, and loss of type VI collagen has been shown to result in loss of fibroblast motility (Ref. 8). In skin pathology, the α3 chain, as measured by the serum biomarker PRO-C6, has been associated with the progression of systemic sclerosis (Ref. 9). The role of the type VI collagen α6 chain gene (COL6α6) in patients with atopic dermatitis (AD) has been investigated using whole-exome sequencing, transcriptome analysis, immunohistochemical staining, and mRNA analysis (References 10-12). A study comparing COL6α6 expression with that of healthy controls using immunohistochemical staining of skin samples found that COL6α6 expression was decreased in the epidermis and increased in the dermis of AD patients, decreased in the epidermis and dermis of psoriasis patients, increased in the epidermis and decreased in the dermis of papular urticaria patients, and decreased in the epidermis of pityriasis rosea patients (Reference 10). Furthermore, mRNA analysis showed that the total expression of COL6α6 mRNA in skin samples (including both the dermis and epidermis) obtained from AD patients was increased compared to healthy controls, and that the expression of COL6α6 mRNA in human keratinocytes exposed to the inflammatory cytokines IL-4 and IL-13 was suppressed (Reference 10). Summary of the Invention

[0004] Applicant has now developed and validated a competitive ELISA for detecting and quantifying the level of a biomarker containing the N-terminal sequence DSGPEYADVV of type VI collagen α6 chain (COL6α6) in samples derived from patient blood. Applicant has demonstrated the use of this immunoassay to detect and / or monitor and / or determine the severity of various skin diseases. Thus, the immunoassay methods, kits, and monoclonal antibodies disclosed herein can be used to analyze samples derived from patient blood for the purpose of conveniently detecting, monitoring, and / or evaluating such diseases.

[0005] Thus, in a first aspect, the present invention provides an immunoassay method comprising the steps of: i) contacting a patient sample selected from blood, serum, and plasma with a monoclonal antibody that specifically binds to the N-terminal amino acid sequence DSGPEYADVV (SEQ ID NO: 1) (sometimes referred to herein as "C6A6" and / or the "target sequence"); and ii) detecting binding between the monoclonal antibody and the peptide in the sample and determining the amount of binding.

[0006] In a preferred embodiment, the method is an immunoassay method for detecting and / or monitoring and / or determining the severity of a skin disorder in a subject, further comprising the steps of: iii) correlating the determined amount of binding with values associated with normal healthy individuals and / or values associated with known disease severity and / or values obtained from the subject at previous time points and / or predetermined cut-off values.

[0007] The term "N-terminus" as used herein refers to the N-terminal amino acid sequence at the extremity of a polypeptide, i.e., the amino acid sequence at the N-end of a polypeptide, and should not be construed as implying a general orientation thereof. As used herein, the terms "peptide" and "polypeptide" are used interchangeably.

[0008] In a preferred embodiment, the monoclonal antibody does not specifically bind to a peptide having the N-terminal amino acid sequence QDSGPEYADVV (SEQ ID NO: 2) (ie, an N-extension of the target sequence). In a preferred embodiment, the monoclonal antibody does not specifically bind to a peptide having the N-terminal amino acid sequence SGPEYADVV (SEQ ID NO: 3) (ie, an N-truncated version of the target sequence).

[0009] In a preferred embodiment, the monoclonal antibody is raised against a synthetic peptide having the N-terminal amino acid sequence DSGPEYADVV (SEQ ID NO: 1). For example, monoclonal antibodies can be raised by (a) immunizing a rodent (or other suitable mammal) with a synthetic peptide comprising the N-terminal amino acid sequence DSGPEYADVV (SEQ ID NO: 1), optionally linked to an immunogenic carrier protein (such as keyhole limpet hemocyanin) at its C-terminus; (b) isolating and cloning a single antibody-producing cell; and (c) assaying the resulting monoclonal antibodies to determine that they have the desired specificity.

[0010] As used herein, the term "monoclonal antibody" refers to both whole antibodies and fragments thereof 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, whole antibodies typically have a "Y-shaped" structure consisting of two identical paired polypeptide chains, each of which is composed of one "light" chain and one "heavy" chain. The N-terminal regions of each of the light and heavy chains comprise the variable region, while the C-terminal portions of each of the heavy and light chains constitute the constant region. The variable regions contain three complementarity-determining regions (CDRs), which are primarily responsible for antigen recognition. The constant region enables the antibody to recruit cells and molecules of the immune system. Antibody fragments that retain binding specificity contain at least the CDRs and a sufficient portion of the remainder of the variable region to retain binding specificity.

[0011] Monoclonal antibodies containing any constant region known in the art can be used in the present invention. In mouse and human antibodies, constant light chains are classified as kappa and lambda light chains. Constant heavy chains are classified as mu, delta, gamma, alpha, or epsilon, defining the antibody isotype as IgM, IgD, IgG, IgA, or IgE, respectively. The IgG isotype has several subclasses, including IgG1, IgG2, IgG3, and IgG4 in humans, and several subclasses, including IgG1, IgG2a, IgG2b, IgG2c, and IgG3 in mice, but is not limited to these. The monoclonal antibody preferably belongs to the IgG isotype, including any one of the IgG subclasses (e.g., IgG1, IgG2, IgG3, or IgG4 in the case of human antibodies).

[0012] The CDRs of an antibody can be determined using methods known in the art, such as those described by Kabat et al. Antibodies can be generated from B cell clones as described in those examples. The isotype of an antibody can be determined by ELISA specific for the IgM, IgG, or IgA isotype (human or mouse) or subclass (human or mouse). The amino acid sequence of the generated 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 can then be 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 primers that bind to sequences located in the constant region of a predetermined isotype. The light chain can be amplified using a primer that binds to the 3' end of the kappa or lambda chain together with a primer that anneals to the leader sequence of Vkappa or Vlamda. Full-length heavy and light chains can be generated and sequenced.

[0013] In certain exemplary embodiments, the monoclonal antibody may preferably comprise one or more complementarity determining regions (CDRs) selected from the following: CDR-L1: KASQNVGTDVV (SEQ ID NO: 4) CDR-L2: SASYRYS (SEQ ID NO: 5) CDR-L3: QHYDNYPLT (SEQ ID NO: 6) CDR-H1: SYAMS (SEQ ID NO: 7) CDR-H2: SVTSGGTHYLDSVKG (SEQ ID NO: 8) CDR-H3: GVSFAY (SEQ ID NO: 9)

[0014] Preferably, the monoclonal antibody comprises at least two, three, four, five, or six of the above CDR sequences.

[0015] Preferably, the monoclonal antibody has a light chain variable region comprising the following CDR sequences: CDR-L1: KASQNVGTDVV (SEQ ID NO: 4) CDR-L2: SASYRYS (SEQ ID NO: 5) and CDR-L3: QHYDNYPLT (SEQ ID NO: 6)

[0016] Preferably, the monoclonal antibody has a light chain comprising inter-CDR framework sequences that are substantially identical to or substantially similar to the inter-CDR framework sequences in the light chain sequence below (CDRs are shown in bold and underlined, framework sequences are shown in italics):

[0017] [ka]

[0018] Preferably, the monoclonal antibody has a heavy chain variable region comprising the following CDR sequences: CDR-H1: SYAMS (SEQ ID NO: 7) CDR-H2: SVTSGGTHYLDSVKG (SEQ ID NO: 8) and CDR-H3: GVSFAY (SEQ ID NO: 9)

[0019] Preferably, the monoclonal antibody has a heavy chain comprising inter-CDR framework sequences that are substantially identical to or substantially similar to the inter-CDR framework sequences of the heavy chain sequence below (CDRs are shown in bold and underlined, framework sequences are shown in italics):

[0020] [ka]

[0021] Preferably, the monoclonal antibody comprises the light chain variable region sequence:

[0022] [ka]

[0023] (CDRs are in bold and underlined, framework sequences are in italics) and / or heavy chain variable region sequence:

[0024] [ka]

[0025] (CDRs are in bold and underlined, framework sequences are in italics) may include:

[0026] As used herein, the amino acid sequence of a framework present between the CDRs of an antibody is "substantially identical" or "substantially similar" to the amino acid sequence of a framework present between the CDRs of another antibody if it has at least 70%, 80%, 90%, or at least 95% similarity or identity to the amino acid sequence of the framework present between the CDRs of the other antibody. The similar or identical amino acids may be contiguous or non-contiguous.

[0027] The framework sequence may contain one or more amino acid substitutions, insertions, and / or deletions. The 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 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.

[0028] Programs such as the CLUSTAL program can be used to compare amino acid sequences. This program compares amino acid sequences and finds the optimal alignment by inserting spaces in any appropriate sequence. 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 values to matching positions. In this way, a comparison can be made, and several regions of similarity, each with a different score, are found. It is contemplated that these two types of analysis can be used in the present invention. Identity or similarity is preferably calculated over the entire length of the framework sequence.

[0029] In a preferred embodiment, the patient sample is serum or plasma. In preferred embodiments, the skin disease is atopic dermatitis, melanoma, psoriasis, hidradenitis suppurativa, or systemic lupus erythematosus. In a preferred embodiment, 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 techniques known to those skilled in the art.

[0030] As used herein, the term "amount bound" refers to the quantification of binding between an antibody and a peptide in a patient sample. This quantification may be determined, for example, by comparing measurements of binding in a patient sample to a calibration curve generated using measurements of binding in standard samples containing known concentrations of the peptide to which the antibody specifically binds, to determine the amount of peptide in the patient sample to which the antibody specifically binds. In the examples provided below, an ELISA method is used, which utilizes spectrophotometric analysis to measure both the amount of binding in the patient sample and the amount of binding when generating the calibration curve. However, any suitable analytical method may be used.

[0031] As used herein, the term "predetermined cutoff value" refers to a statistically determined binding amount that indicates a high probability of disease in a patient or a particular severity of the disease, in the sense that the measured value of the target peptide in a patient sample is equal to or greater than the statistical cutoff value, corresponding to at least a 70% probability, 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.

[0032] As used herein, the term "value associated with a normal healthy individual" refers to a standardized amount determined by the above method for a sample obtained from a subject who is considered healthy, i.e., disease-free; and the term "value associated with a known disease severity" refers to a standardized amount determined by the above method for a sample obtained from a patient known to have a disease of known severity.

[0033] In a second aspect, the present invention provides a method for treating a skin disorder in a patient in need thereof, the method comprising the steps of: (a) performing an immunoassay method according to the first aspect of the invention on a sample of blood, serum or plasma obtained from a patient to detect whether the patient has a skin disease and / or to determine the severity of the patient's skin disease; and (b) if it is determined in step (a) that the patient has a skin disease or that the skin disease is of a particular severity, administering to the patient a drug for treating the skin disease.

[0034] The pharmaceutical agent may be any agent suitable for treating the target skin condition. The pharmaceutical agent may, for example, include or consist of one or more topical pharmaceutical agents, one or more systemic pharmaceutical agents, or a combination thereof. Topical pharmaceutical agents may be formulated, for example, as a cream, foam, gel, lotion, or ointment, for application to one or more areas of the skin requiring treatment. Systemic pharmaceutical agents may, for example, be formulated for administration via the gastrointestinal tract or parenterally.

[0035] For example, if the skin disease is atopic dermatitis, applicable topical agents may be selected from moisturizers, topical corticosteroids (e.g., hydrocortisone), topical immunosuppressants such as topical calcineurin inhibitors (e.g., tacrolimus and pimecrolimus), and PDE-4 inhibitors (e.g., crisaborole); applicable systemic agents may be selected from systemic immunosuppressants (e.g., cyclosporine, methotrexate, interferon gamma-1b, mycophenolate mofetil, and azathioprine), biologics (e.g., monoclonal antibodies such as dupilumab and tralokinumab), and JAK inhibitors (e.g., abrocitinib and upadacitinib).

[0036] When the skin disease is melanoma, the applicable drug may be selected from, for example, chemotherapeutic agents (e.g., dacarbazine, temozolamide, cisplatin, carboplatin, and paclitaxel), BRAF inhibitors (e.g., vemurafenib and dabrafenib), MEK inhibitors (e.g., trametinib), C-Kit inhibitors, NRAS inhibitors, cytokines (e.g., IL-2 and IFN-α), immune checkpoint inhibitors (e.g., anti-CTLA-4 monoclonal antibodies, TLR antagonists, CD40 antagonists, anti-PD-1 antibodies, and PD-L1 antibodies), and adoptive cell transfer.

[0037] When the skin disease is psoriasis, applicable topical agents may be selected from, for example, moisturizers, topical corticosteroids (e.g., hydrocortisone), vitamin D analogues (e.g., paricalcitol), and topical immunosuppressants such as, for example, topical calcineurin inhibitors (e.g., tacrolimus and pimecrolimus); applicable systemic agents may be selected from systemic immunosuppressants (e.g., cyclosporine and methotrexate), fumarates (e.g., dimethyl fumarate), retinoids, and biologics (e.g., monoclonal antibodies such as ixekizumab, secukinumab, brodalumab, guselkumab, certolizumab, and ustekinumab).

[0038] When the skin disease is hidradenitis suppurativa, applicable medications may be selected from, for example, topical antibiotics (e.g., topical clindamycin), topical retinoids (e.g., isotretinoin), oral antibiotics (e.g., rifampicin, clindamycin, tetracycline, and minocycline), intralesional corticosteroids, antiandrogens (e.g., spironolactone, flutamide, cyproterone acetate, ethinyl estradiol, finasteride, dutasteride, and metformin), and intravenous, subcutaneous, or infusion-based TNF inhibitors (e.g., etanercept) and anti-TNF-alpha monoclonal antibodies (e.g., infliximab and adalimumab).

[0039] When the skin disease is systemic lupus erythematosus, applicable drugs may be selected from, for example, nonsteroidal anti-inflammatory drugs (NSAIDs), corticosteroids, antimalarials (e.g., hydroxychloroquine), BLyS-specific inhibitors (e.g., belimumab), and immunosuppressants (e.g., prednisone, mycophenolate, tacrolimus, methotrexate, and azathioprine).

[0040] In a particular embodiment, step (a) of the method may comprise performing an immunoassay according to the first aspect of the present invention on a blood, serum or plasma sample obtained from the patient to determine the severity of the patient's skin disease, and step (b) of the method may comprise administering to the patient a drug for treating the skin disease only if the patient is determined to have a skin disease of a particular severity in step (a). For example, the method may administer a topical agent if the severity of the disease is at or below a certain level, but administer a systemic agent if the severity of the disease exceeds a certain level. In particular, in one embodiment, the skin disease is atopic dermatitis, and step (b) may include administering a topical medication to the patient if step (a) determines that the patient has mild or moderate atopic dermatitis, and administering a systemic medication to the patient if step (a) determines that the patient has severe atopic dermatitis.

[0041] In a third aspect, the present invention provides an immunoassay kit comprising a monoclonal antibody that specifically binds to the N-terminal amino acid sequence DSGPEYADVV (SEQ ID NO: 1), and at least one of the following: - streptavidin-coated well plates; - biotinylated peptide DSGPEYADVV-L-biotin (SEQ ID NO: 14), where L is an optional linking group; - Secondary antibodies used in sandwich immunoassays; - a calibration peptide comprising the N-terminal amino acid sequence DSGPEYADVV (SEQ ID NO: 1); - Antibody biotinylation kit; - Antibody HRP labeling kit; - antibody radiolabeling kit; and - Assay visualization kit.

[0042] In a preferred embodiment, the monoclonal antibody does not specifically bind to a peptide having the N-terminal amino acid sequence QDSGPEYADVV (SEQ ID NO: 2). In a preferred embodiment, the monoclonal antibody does not specifically bind to a peptide having the N-terminal amino acid sequence SGPEYADVV (SEQ ID NO: 3). In a preferred embodiment, the monoclonal antibody is raised against a synthetic peptide having the N-terminal amino acid sequence DSGPEYADVV (SEQ ID NO: 1).

[0043] The immunoassay kit according to the third aspect of the invention is particularly suitable for use in carrying out the method according to the first aspect of the invention. Accordingly, further preferred embodiments and features of the immunoassay kit according to the third aspect will be apparent from the above discussion of preferred embodiments of the method according to the first aspect.

[0044] In a fourth aspect, the present invention provides a monoclonal antibody that specifically binds to the N-terminal amino acid sequence DSGPEYADVV (SEQ ID NO: 1). In a preferred embodiment, the monoclonal antibody does not specifically bind to a peptide having the N-terminal amino acid sequence QDSGPEYADVV (SEQ ID NO: 2). In a preferred embodiment, the monoclonal antibody does not specifically bind to a peptide having the N-terminal amino acid sequence SGPEYADVV (SEQ ID NO: 3). In a preferred embodiment, the monoclonal antibody is raised against a synthetic peptide having the N-terminal amino acid sequence DSGPEYADVV (SEQ ID NO: 1). [Brief explanation of the drawings]

[0045] [Figure 1]Figure 1: A) Overview of the primary structure of type VI collagen alpha chain, showing all six domains of the alpha chain. As shown, the antibody used in the C6A6 assay targets the C6A6 target sequence at the N-terminus of type VI collagen alpha 6 chain (COL6α6). B) Sequence alignment of the N-terminal sequences of COL6α6 from human, mouse, bovine, and rat species, with the C6A6 target sequence highlighted in a black box. Sequences were aligned using Uniprot. C) Specificity of the C6A6 assay, demonstrating reactivity with the standard peptide (DSGPEYADVV (SEQ ID NO: 1)), truncated peptide (SGPEYADVV (SEQ ID NO: 3)), extended peptide (QDSGPEYADVV (SEQ ID NO: 2)), and nonsense standard peptide (YRDDLKKLLE (SEQ ID NO: 17)). No background signal was detected when coated with the nonsense coating peptide (YRDDLKKLLE-biotin (SEQ ID NO: 16)). The signal is expressed in relative light units per second (RLU) as a function of the standard peptide.

[0046] [Figure 2] Figure 2: Results from Population 1. Serum C6A6 levels were assessed in healthy donors (n = 20) and patients with atopic dermatitis (n = 20), melanoma (n = 20), psoriasis (n = 20), hidradenitis suppurativa (n = 6), systemic sclerosis (n = 14), systemic lupus erythematosus (n = 12), urticaria (n = 19), and vitiligo vulgaris (n = 20). Means and 95% confidence intervals (CI). Data were analyzed using ANCOVA adjusted for age and sex. Data are plotted as mean ± 95% CI. [Figure 3] Figure 3: Results from population 2. Serum C6A6 levels in healthy donors (n=22) and patients with atopic dermatitis (n=158). Mean and 95% confidence interval. Data were analyzed using ANCOVA adjusted for age. Data are plotted as mean ± 95% CI.

[0047] [Figure 4-1] Figure 4: Results from population 2. Serum C6A6 levels were stratified by disease severity and treatment. A) C6A6 levels in patients with mild and moderate AD (SCORAD; 0-50) compared with patients with severe AD (SCORAD; <50). B) C6A6 levels in untreated AD patients compared with immunosuppressant-treated AD patients. [Figure 4-2] Figure 4: Results from population 2. Serum C6A6 levels stratified by disease severity and treatment. C) C6A6 levels in untreated patients with mild and moderate AD versus patients with mild and moderate AD treated with immunosuppressants. D) C6A6 levels in untreated patients with severe AD versus patients with severe AD treated with immunosuppressants. Mean and 95% confidence interval. Data were analyzed using ANCOVA adjusted for age. Data are plotted as mean ± 95% CI. [Example]

[0048] The embodiments disclosed herein are described in the following examples. These examples are presented to aid in understanding the present disclosure and should not be construed in any way to limit the scope of the disclosure as defined in the claims that follow. The examples set forth below are presented so as 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 disclosure, nor are they intended to imply that the experiments described below 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 indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

[0049] Materials and Methods All reagents used in the experiments were high quality chemicals from Merck (Whitehouse Station, NJ, USA) and Sigma (St. Louis MO, USA) unless otherwise stated. All synthetic peptides used for antibody production and assay validation were purchased from GenScript (Pitcataway, NJ, US) (Table 1).

[0050] [Table 1]

[0051] Monoclonal antibody development, production and characterization The amino acid sequence 20'↓DSGPEYADVV'30 (SEQ ID NO: 1) (also referred to herein as "C6A6" and / or the "target sequence") within the α6 chain of human type VI collagen (COL6α6) was used to generate monoclonal antibodies (mAbs), which form the N-terminus of the α6 chain of type VI collagen after the signal peptide is cleaved and removed. Immunization was initiated by subcutaneous injection of 200 μl of emulsified antigen containing 100 μg of the immunogenic peptide (DSGPEYADVV-GGC-KLH (SEQ ID NO: 15)) into 4-6 week-old Balb / C mice using Stimune (Thermo Fisher Scientific). Immunizations were repeated every 2 weeks until serum antibody titers stabilized. Mice with the highest serum antibody titers were selected for cell fusion and allowed to rest for 1 month. Three days before isolating the spleen for cell fusion, the mice were boosted with 100 μl of 0.9% NaCl solution containing 50 μg of the immunogenic peptide intravenously. To generate hybridoma cells, mouse splenocytes were fused with mouse SP2 / 0 myeloma cells as described by Gefter et al. (13). Clones were then cultured in 96-well microtiter plates and subjected to limiting dilution to promote monoclonal proliferation. Supernatants were screened for reactivity by indirect ELISA on streptavidin-coated plates. DSGPEYADVV-K-biotin (SEQ ID NO: 18) was used as the screening peptide, and the standard peptide DSGPEYADVV (SEQ ID NO: 1) was used to further test the specificity of the clones. Supernatants were collected from the hybridoma cells and purified using a HiTrap affinity column (GE Healthcare Life Sciences, Little Chalfront, Buckinghamshire, UK) according to the manufacturer's instructions, and the antibody isotype was determined using a Rapid ELISA Mouse Monoclonal Antibody Isotyping Kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.

[0052] Native reactivity was assessed using human serum, citrated plasma, heparinized plasma, EDTA plasma, and rat serum purchased from a commercial supplier (Valley Biomedical, Winchester, VA). mAbs were selected that specifically recognized the standard peptide (DSGPEYADVV (SEQ ID NO: 1)) but not the one-amino acid-extended sequence (QDSGPEYADVV (SEQ ID NO: 2)) or the one-amino acid-truncated sequence (SGPEYADVV (SEQ ID NO: 3)).

[0053] The isotype, sequence, and CDRs of the monoclonal antibodies selected for production and assay development were determined. The sequences of the peptide chains are as follows (CDRs are underlined and bold; N-terminal signal peptide and C-terminal constant region are italic):

[0054] Heavy chain sequence (mouse IgG1 isotype)

[0055] [ka]

[0056] Light chain sequence (mouse kappa isotype)

[0057] [ka]

[0058] C6A6 assay development The development of a competitive chemiluminescent immunoassay (CLIA) targeting the C6A6 target peptide involved several preliminary optimization experiments in which several tests were performed to analyze the reagents, concentrations, incubation times, and incubation temperatures. The final C6A6 competitive ELISA procedure was as follows: A 96-well streptavidin-coated white microplate (Greiner Bio-One, Kremsmunster, Austria) was coated with 3 ng / mL biotinylated synthetic peptide (DSGPEYADVV-K-biotin (SEQ ID NO: 18)) in assay buffer (10 mM phosphate-buffered saline (PBS), 1% bovine serum albumin, 0.1% Tween-20, 0.36% bronidox, 4 g / L NaCl, pH 7.4 at 20°C) and incubated for 30 min at 20°C with shaking (300 rpm) in the dark. Next, 20 μl / well of selected peptide (100 ng / mL) and sample were added to the appropriate wells, and 100 μl / well of HRP-labeled antibody (prepared as described above) diluted to a concentration of 200 ng / mL in assay buffer was added and incubated for 1 hour at 20° C. with shaking (300 rpm) in the dark. After each incubation step, the wells were washed five times with standard wash buffer (20 mM Tris, 50 mM NaCl, pH 7.2). A working solution of chemiluminescent substrate (Roche, BM Chemiluminescence ELISA substrate (POD), Basel, Switzerland) was mixed 15 min before use, and 100 μL / well was added to the plate and incubated for 3 min at 20°C with shaking (300 rpm) in the dark. Relative light units were measured at all wavelengths within 5 min using a microplate luminometer reader (Spectramax M5, Molecular Devices, CA, USA). The standard curve was calculated using a four-parameter logistic curve fit [Y = (AD) / (1 + (x / C) ∧ B) + D, where R > 0.9. Data were analyzed using SoftMax Pro version 7.0.3 software.

[0059] Technical evaluation Linearity was assessed using two-fold dilutions of four human serum samples and two rat serum samples and was calculated as the percent recovery of the undiluted samples. Antibody specificity was calculated as the percent signal inhibition by two-fold dilutions of the standard peptide (DSGPEYADVV (SEQ ID NO: 1)), the extended peptide (QDSGPEYADVV (SEQ ID NO: 2)), the truncated peptide (SGPEYADVV (SEQ ID NO: 3)), and the nonsense peptide (YRDDLKKLLE (SEQ ID NO: 17)).

[0060] Intra- and inter-assay variation was determined by 10 independent measurements of five quality controls and two kit controls in duplicate. The accuracy of the assay was measured in healthy human serum samples spiked with the standard peptide and in serum samples containing known high concentrations of COL6α6, and was calculated as the percent recovery of the measured value and the expected concentration obtained by adding the concentration of the analyte in serum to the concentration of the standard peptide or serum sample containing high concentrations of COL6α6. Analytical interferences were assessed by spiking serum samples with known concentrations of low and high hemoglobin (2.50 / 5 mg / mL), hyperlipidemia (1.50 / 5 mg / mL), and biotin (3 / 9 ng / mL). Percent recoveries were calculated using normal serum samples as references. Normal reference levels were 1-10 mg / dL (0-0.00161 mmol / L), <150 mg / dL (<1.6935 mmol / L), and 0.221-3.004 ng / mL for hemoglobin, hyperlipidemia, and biotin, respectively. Interferences were calculated as the percent recovery of the analyte in non-spiked serum. The measurement range was defined as the range between the lower limit of the measurement range (LLMR) and the upper limit of the measurement range (ULMR), determined from 10 independent measurements using standard peptides. Measurements below the LLMR or above the UMLR were assigned a value of LLMR / UMLR, respectively. IC50 (half-maximum inhibitory concentration) was determined from the standard curve.

[0061] Analyte stability was investigated by performing temperature studies and by performing repeated freeze-thaw cycling studies on serum samples. In the temperature study, three human serum samples were incubated at 4°C or 20°C for 0, 2, 4, 24, and 48 hours, and then the levels of C6A6 target peptide in the samples were measured after varying incubation times and temperatures. Recovery rates were assessed using the 0-hour sample as a reference. Additionally, three serum samples were evaluated for the effect after four cycles of freeze-thaw cycling, and freeze-thaw recovery was calculated using the zero-cycle sample as a reference.

[0062] Biological evaluation of the C6A6 assay and patient demographics The biological utility of the C6A6 assay was evaluated using serum samples from two cross-sectional studies. The first cohort (cohort 1) was obtained from a commercial vendor, Proteogenex (Culver City, CA, USA), while the second cohort (cohort 2) was obtained from the Department of Dermatology, Bispebjerg Hospital, University of Copenhagen, Denmark. Healthy donor samples were obtained from Bio IVT and Lee Biosolutions.

[0063] Group 1 included patients with atopic dermatitis (n=20), melanoma (n=20), psoriasis (n=20), hidradenitis suppurativa (n=6), systemic sclerosis (n=18), systemic lupus erythematosus (n=12), urticaria (n=19), and vitiligo vulgaris (n=20), as well as healthy donors (n=24) who met the criteria of having no signs of disease or chronic illness. Cohort 2 included 158 patients with atopic dermatitis and 22 healthy controls matched for age, sex, and ethnicity. Serum samples were collected between January 2012 and June 2018 (Reference 8). Patients met the criteria established by Hannifin and Rajka (1980). AD diagnosis was confirmed by a senior physician according to the diagnostic criteria. Severity was assessed using the SCORAD score, a commonly used severity index for AD (range 0-103; higher scores indicate more severe disease). Patients were divided into disease severity groups based on SCORAD: mild (SCORAD < 25), moderate (SCORAD 25-50), and severe (SCORAD < 50) (References 2, 3). Of the 158 patients, 47 were receiving topical immunosuppressant therapy. Treatment was a topical calcineurin inhibitor. Age and sex were only available for healthy donors.

[0064] After informed consent and approval by the local ethical committee, samples were collected from both populations in accordance with the Declaration of Helsinki of 1975. Serum samples were obtained and stored at -80°C.

[0065] 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 Authority (Animal Experimentation Inspectorate) under approval number 2013-15-2934-00956.

[0066] statistical analysis Characteristics of patients in the two populations were expressed as number (frequency) and percentage for categorical variables and either mean (standard deviation) or mean (range) for continuous variables. Statistical differences for categorical variables were evaluated using the Kruskal-Wallis test (nonparametric) for population 1 and the Mann-Whitney t-test for population 2. For population 1, differences between patient groups were calculated using ANCOVA analysis adjusted for age and sex. For population 2, differences between patient groups ranging in age from 4 to 90 years were calculated using ANCOVA analysis adjusted for age. Graphs are presented as mean ± 95% CI. For all statistical analyses performed, a P value of less than 0.05 was considered significant. Statistical analysis and graphing were performed using GraphPad Prism version 9 (GraphPad Software, Inc., La Jolla, CA) and MedCalc version 19.3 (MedCalc Software, Ostend, Belgium).

[0067] 〔result〕 Specificity, accuracy and precision of the C6A6 assay The C6A6 assay uses a monoclonal antibody (mAb) directed against the target sequence DSGPEYADVV (SEQ ID NO: 1) at the N-terminus of type VI collagen, alpha 6 chain (Figure 1A). The human target sequence was aligned to the corresponding mouse, rat, and bovine sequences using UNIPROT; the mouse and rat sequences aligned 100% to the human sequence, whereas the bovine sequence had one mismatch at position 1 (Figure 1B). Hybridomas producing the best mAbs were screened for reactivity to standard peptides and native material. Clone NBH-306-11 8E7-2C11-1F10-2C10 was selected for assay development and determined to be of the IgG1 subtype. To assess the specificity of the C6A6 assay, the mAb was tested against extended peptides, truncated peptides, nonsense standard peptides, and nonsense coater peptides, and showed no reactivity to these peptides (Figure 1C). A technical validation was performed to evaluate the novel C6A6 assay. The technical results are summarized in Table 2. Overall, the C6A6 assay demonstrated low intra- and inter-assay variability, good analyte recovery at 4°C / 20°C for 24 hours (93% and 81%), good analyte recovery at 97% after five freeze-thaw cycles, and no interference from hemoglobin, hyperlipidemia, or biotin. Linearity with human serum samples was observed from 1:3 to 1:16 dilutions.

[0068] [Table 2]

[0069] C6A6 was elevated in patients with dermatological diseases compared with healthy donors Group 1 included patients with atopic dermatitis (mean age: 52.3 years, 10% male), melanoma (mean age: 55.6 years, 10% male), psoriasis (mean age: 50.7 years, 10% male), hidradenitis suppurativa (mean age: 49.5 years, 0% male), systemic sclerosis (mean age: 54.4 years, 5.6% male), systemic lupus erythematosus (mean age: 50.5 years, 16.7% male), urticaria (mean age: 43.6 years, 20% male), and vitiligo vulgaris (mean age: 56.9 years, 25% male). Patient demographics are shown in Table 3. Levels of the C6A6 target peptide were significantly elevated in the sera of patients with atopic dermatitis (p<0.0001), melanoma (p<0.0001), psoriasis (p<0.0001), hidradenitis suppurativa (p<0.0095), and systemic lupus erythematosus (p<0.0032) compared with healthy donors (Figure 2). No significant differences were observed between patients with systemic sclerosis, urticaria, and vitiligo and healthy controls (p=0.304-1.000, respectively).

[0070] [Table 3]

[0071] C6A6 is associated with disease severity and is suppressed by immunosuppressant treatment Group 2 included 158 patients with atopic dermatitis (mean age: 30.0 years, 52% male) and 22 healthy donors (mean age: 29.6 years, 50% male). Patient demographics are shown in Table 4. Patients with atopic dermatitis had significantly higher serum C6A6 target sequence levels than healthy controls (p<0.0001).Of 158 AD patients, 53 (33.3%) patients had mild, 72 (45.3%) patients had moderate, and 33 (21.4%) patients had severe disease. We evaluated whether C6A6 target sequence levels correlated with disease severity by comparing severe AD patients (SCORAD > 50) with mild and moderate AD patients (SCORAD 0-50). C6A6 levels in severe AD patients were significantly higher than those in mild and moderate AD patients (p = 0.046, Figure 4A). Of the 158 patients, 47 patients were receiving topical immunosuppressants. C6A6 levels in treated patients were significantly lower than those in untreated patients (p = 0.0136, Figure 4B). C6A6 levels in mild and moderate AD patients receiving immunosuppressants were also significantly lower than those in untreated mild and moderate AD patients (p = 0.035, Figure 4C). However, there was no significant difference between patients receiving immunosuppressants and those not receiving immunosuppressants in the severe AD patient group (p = 0.544, Figure 4D).

[0072] [Table 4]

[0073] [Essay] In this study, the applicant developed and characterized a competitive ELISA for detecting biomarkers containing the N-terminus of COL6α6 in blood-derived samples using a monoclonal antibody targeting the N-terminal amino acid sequence DSGPEYADVV (SEQ ID NO: 1) (sometimes referred to herein as "C6A6" and / or "target sequence"). The main findings are as follows: 1) a robust and specific assay for the target sequence DSGPEYADVV (SEQ ID NO: 1) was developed; 2) the C6A6 target sequence could be detected in human, mouse, and rat serum; 3) serum C6A6 levels were shown to be elevated in patients diagnosed with atopic dermatitis, melanoma, psoriasis, hidradenitis suppurativa, and systemic lupus erythematosus compared with healthy donors; 5) elevated serum C6A6 levels were associated with severe atopic dermatitis, and serum levels were reduced by treatment with immunosuppressants, especially in patients with mild and moderate atopic dermatitis. To the applicant's knowledge, this is the first study to demonstrate that the levels of this target sequence can be measured non-invasively using serum from patients with skin lesions, and that the levels of this target sequence are associated with disease severity in atopic dermatitis and are reduced in patients with atopic dermatitis treated with topical immunosuppressants.

[0074] The C6A6 assay was characterized as technically stable and accurate, demonstrating acceptable dilution recovery, interference, and stability tests. The intra- and inter-assay variability was acceptable, at 5% and 12%, respectively. Furthermore, the assay was characterized as specific for the N-terminal target sequence of COL6α6, which is exposed after cleavage of the signal peptide.

[0075] In this study, we discovered that serum C6A6 levels were suppressed when patients with mild to moderate atopic dermatitis were treated with topical calcineurin inhibitors (TCIs). TCIs, including pimecrolimus and tacrolimus, are widely used as first-line immunosuppressants in the topical treatment of atopic dermatitis and psoriasis (Reference 3). This finding indicates that serum target sequence levels are associated with treatment response. Conversely, no suppression was observed in patients with severe atopic dermatitis, a result that may indicate the need for types of treatment other than TCIs in this severe patient population. The findings support the use of the C6A6 biomarker in diagnosing and predicting patients with skin conditions, particularly those with atopic dermatitis.

[0076] In conclusion, the C6A6 assay demonstrated high specificity for its N-terminal target sequence, whose levels were elevated in patients with skin lesions. The assay demonstrated high discriminatory power, enabling the differentiation of AD patients from healthy donors. C6A6 levels were also elevated in patients with severe AD, suggesting that severe AD may be due to increased fibroblast activity and global tissue remodeling. Furthermore, decreased biomarker levels were found in patients treated with topical immunosuppressants (calcineurin inhibitors), indicating that this biomarker is associated with treatment response.

[0077] In this specification, unless otherwise specified, the word "or" is used to mean an operator that returns a true value if one or both of the stated conditions are met, as opposed 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 herein by reference. Any acknowledgment in this specification of a prior-published document should not be taken as an admission or representation that the teaching of that document was common general knowledge in Australia or elsewhere at the time of this specification.

[0078] [References] 1. Simon D, Aeberhard C, Erdemoglu Y, Simon HU. Th17 cells and tissue remodeling in atopic and contact dermatitis. Allergy [Internet] Allergy; 2014 [cited 2022 May 6];69:125-31. Available from: https: / / pubmed.ncbi.nlm.nih.gov / 24372156 / 2. Denguier J, Bruckner-Tudelmann L, Nyström A. Skin proteomic analysis of the extracellular matrix in healthy and disease states. Expert Rev Proteomics Taylor & Francis; 2020;17:377-91 3. Lynch MD, Watt FM. Fibroblast heterogeneity: relevance to human disease. J Clin Invest [Internet] J Clin Invest; 2018 [cited 2022 May 6];128:26-35. Available from: https: / / pubmed.ncbi.nlm.nih.gov / 29293096 / 4. Sescon M, Gattazzo F, Chen P, Bonaldo P. A general overview of collagen VI. J Cell Sci 2015;128:3525-31 5. Wagner MFMG, Theodoro TR, Filho CDASM, Oyiafuso LKM, Pinhal MAS. Alterations in the extracellular matrix in the skin of patients with psoriasis. BMC Mol Cell Biol [Internet] BioMed Central; 2021 [cited 2022 May 6];22. Available from: / pmc / articles / PMC8555298 / 6. Holm-Nielsen S, Mortensen J, Wilmsen N, Rasmussen D, Mogensen D, Di Sabatino A et al. Fragments of type VI collagen alpha-3 chain are elevated in the serum of patients with gastrointestinal disease. Sci Rep Sci Rep;2020;10:5910

[0079] 7. Fitzgerald J, Holden P, Hansen U. The expanded collagen VI family: new chains and new questions. Connect Tissue Res [Internet] Connect Tissue Res; 2013 [cited 2022 May 6];54:345-50. Available from: https: / / pubmed.ncbi.nlm.nih.gov / 23869615 / 8. GT, ZD, AP K, AH B, DA L, KM B et al. Type VI collagen regulates dermal matrix assembly and fibroblast motility. J Invest Dermatol [Internet] J Invest Dermatol; 2016 [cited 2021 Sep 8];136:74-83. Available from: https: / / pubmed.ncbi.nlm.nih.gov / 26763426 / 9. Dobrota R, Jordan S, Juhl P, Mourea B, Wildi L, Bey-Jensen AC et al. Neoepitopes of circulating collagen and their role in predicting fibrosis in patients with systemic sclerosis: a multicenter cohort study. Lancet Rheumatol [Internet] Elsevier Ltd; 2021;3:e175-84. Available from: http: / / dx.doi.org / 10.1016 / S2665-9913(20)30385-4 10. Jung HJ, Heo W Il, Park KY, Lee MK, Ahnn JY, Park MY, et al. The role of collagen VI α6 chain gene in atopic dermatitis. Ann Dermatol [Internet] Ann Dermatol; 2022 [cited 2022 May 4];34:46-54. Available from: https: / / pubmed.ncbi.nlm.nih.gov / 35221595 / 11. Ghosh D, Ding L, Shivapraisad U, Ge E, Myers JB, Bernstein JA et al. Multiplex transcriptome data analysis reveals biologically relevant atopic dermatitis signature genes and pathways. PLoS One [Internet] PLoS One; 2015 [cited 2022 Mar 10];10. Available from: https: / / pubmed.ncbi.nlm.nih.gov / 26717000 / 12. Heo W Il, Park KY, Jin T, Lee MK, Kim MJ, Choi EH, et al. Identification of novel candidate variants, including COL6A6 polymorphisms, in the early stage of atopic dermatitis using whole-exome sequencing. BMC Med Genet [Internet] BMC Med Genet; 2017 [cited 2022 Mar 10];18. Available from: https: / / pubmed.ncbi.nlm.nih.gov / 28125976 / 13. Gefter ML, Margulies DH, Scarfe MD. A simple method for polyethylene glycol-promoted hybridization of mouse myeloma cells. Somatic Cell Genet [Internet] 1977;3:231-6. Available from: http: / / www.ncbi.nlm.nih.gov / pubmed / 605383

Claims

1. 1. An immunoassay method for detecting and / or monitoring a skin disease in a subject and / or determining the severity of a skin disease in a subject, the method comprising: i) contacting a patient sample selected from blood, serum, and plasma with a monoclonal antibody that specifically binds to the N-terminal amino acid sequence DSGPEYADVV (SEQ ID NO: 1); ii) detecting binding between the monoclonal antibody and the peptide in the sample and determining the amount of binding; and iii) correlating the determined amount of binding with values associated with normal healthy individuals and / or with values associated with known disease severity and / or with values obtained from the subject at previous time points and / or with predetermined cut-off values.

2. The method of claim 1, wherein the monoclonal antibody does not specifically bind to a peptide having the N-terminal amino acid sequence QDSGPEYADVV (SEQ ID NO: 2).

3. The method of claim 1 or 2, wherein the monoclonal antibody does not specifically bind to a peptide having the N-terminal amino acid sequence SGPEYADVV (SEQ ID NO: 3).

4. 4. The method of any one of claims 1 to 3, wherein the monoclonal antibody is raised against a synthetic peptide having the N-terminal amino acid sequence DSGPEYADVV (SEQ ID NO: 1).

5. 5. The method of claim 1, wherein the skin disease is atopic dermatitis, melanoma, psoriasis, hidradenitis suppurativa, or systemic lupus erythematosus.

6. The method of claim 1 , wherein the immunoassay is a competitive assay or a sandwich assay.

7. 7. The method of claim 1, wherein the immunoassay is a radioimmunoassay or an enzyme-linked immunosorbent assay.

8. A monoclonal antibody that specifically binds to the N-terminal amino acid sequence DSGPEYADVV (SEQ ID NO: 1), and at least one of the following: - streptavidin-coated well plates; - biotinylated peptide DSGPEYADVV-L-biotin (SEQ ID NO: 14), where L is an optional linking group; - secondary antibodies used in sandwich immunoassays; a calibration peptide comprising the N-terminal amino acid sequence DSGPEYADVV (SEQ ID NO: 1); - antibody biotinylation kit; - Antibody HRP labeling kit; - antibody radiolabeling kit; and - Assay Visualization Kit 1. An immunoassay kit comprising:

9. The immunoassay kit of claim 8, wherein the monoclonal antibody does not specifically bind to a peptide having the N-terminal amino acid sequence QDSGPEYADVV (SEQ ID NO: 2).

10. The immunoassay kit according to claim 8 or 9, wherein the monoclonal antibody does not specifically bind to a peptide having the N-terminal amino acid sequence SGPEYADVV (SEQ ID NO: 3).

11. 11. The immunoassay kit of any one of claims 8 to 10, wherein the monoclonal antibody is raised against a synthetic peptide having the N-terminal amino acid sequence DSGPEYADVV (SEQ ID NO: 1).

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

13. The monoclonal antibody of claim 12, wherein the monoclonal antibody does not specifically bind to a peptide having the N-terminal amino acid sequence QDSGPEYADVV (SEQ ID NO: 2).

14. The monoclonal antibody according to claim 12 or 13, which does not specifically bind to a peptide having the N-terminal amino acid sequence SGPEYADVV (SEQ ID NO: 3).

15. 15. The monoclonal antibody of any one of claims 12 to 14, wherein the monoclonal antibody is raised against a synthetic peptide having the N-terminal amino acid sequence DSGPEYADVV (SEQ ID NO: 1).

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