Determination method of type VI collagen alpha-6

Abstract

Disclosed herein are immunoassay methods for detecting and / or monitoring a skin disease in a patient and / or determining the severity of a skin disease in a patient. The methods include detecting and quantifying the level of a biomarker comprising the N-terminal sequence of type VI collagen alpha-6 in a blood-based sample (e.g., whole blood, plasma or serum) from a patient. Antibodies suitable for use in the methods and immunoassay kits suitable for implementing the methods are also disclosed.

Description

Technical Field

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

[0002] Remodeling of the extracellular matrix (ECM) of the skin is a continuous process necessary for maintaining tissue homeostasis. The skin can be divided into three layers: epidermis, dermis, and subcutaneous tissue, all of which have different tissue structures and functions (1, 2). The dermis provides tensile strength, elasticity, and toughness to the skin. In addition, the dermis is also the layer where fibroblasts play a key role in ECM synthesis and maintenance of tissue structure (3, 4). The ECM of the dermis can be divided into papillary and reticular dermal ECM and is composed of matricellular proteins (COMP, SPARC, thrombospondin-1, periostin, tenascin C and 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 fibrous proteins (elastin, EMLIN-1 and EMLIN-2, LTBP-4, fibrillin-4 and -5) (2). Dysregulation of papillary and reticular dermal ECM remodeling is a key event in the pathology of skin disorders, including atopic dermatitis and psoriasis (1, 2, 5).

[0003] In general, the importance of type VI collagen in maintaining homeostasis in skin tissue has been described in the literature. Type VI collagen is characterized by beaded fibrillar collagen found in the papillary and reticular dermal ECM, forming a microfibril network (6). Six different chains of type VI collagen (α1, α2, α3, α4, α5, α6) have been identified and expressed across connective tissue (7). In vitro experiments have demonstrated how important the type VI collagen α1 chain is for ECM assembly in human dermal fibroblasts, and have shown that loss of type VI collagen leads to loss of fibroblast motility (8). In skin pathology, the α3 chain measured by the serum biomarker PRO-C6 has previously been associated with the progression of systemic sclerosis (9). The role of the type VI collagen α6 chain gene (COL6α6) in patients with atopic dermatitis (AD) has been studied using whole exome sequencing, transcriptomics, immunohistochemical staining, and mRNA analysis (10-12). In one study, using immunohistochemical staining of skin samples, it was shown that the expression of COL6α6 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 compared with healthy controls (10). In addition, mRNA analysis showed that the total mRNA expression of COL6α6 was increased in skin samples (including both the dermis and epidermis) from AD patients compared with healthy controls, and COL6α6 mRNA expression was inhibited in human keratinocytes exposed to the inflammatory cytokines IL-4 and IL-13 (10). Summary of the invention

[0004] Applicants have now developed and validated a competitive ELISA for detecting and quantifying the level of a biomarker in a blood-based sample from a patient, the biomarker comprising the N-terminal amino acid sequence DSGPEYADVV of type VI collagen α6 chain (COL6α6), and have demonstrated the use of the immunoassay for detecting, monitoring and / or determining the severity of various skin diseases. Therefore, the immunoassay methods, kits and monoclonal antibodies disclosed herein can be used to analyze blood-based samples from patients in order to conveniently detect, monitor and / or evaluate such diseases.

[0005] Therefore, in a first aspect, the present invention provides an immunoassay method comprising:

[0006] i) contacting a patient sample selected from blood, serum or plasma with a monoclonal antibody that specifically binds to the N-terminal amino acid sequence DSGPEYADVV (SEQ ID NO: 1) (also referred to herein as "C6A6" and / or "target sequence");

[0007] ii) detecting and determining the amount of binding between the monoclonal antibody and the peptide in the sample.

[0008] In a preferred embodiment, the method is an immunoassay for detecting and / or monitoring a skin disease in a patient and / or determining the severity of a skin disease in a patient, the method further comprising:

[0009] iii) correlating said bound amount to a value associated with a normal healthy subject, and / or to a value associated with a known disease severity, and / or to a value obtained from said patient at a previous time point, and / or to a predetermined cut-off value.

[0010] As used herein, the term "N-terminus" refers to the N-terminal peptide sequence at the end of a polypeptide (ie, at the N-terminus of a polypeptide), and should not be construed as meaning in its general orientation.

[0011] As used herein, the terms "peptide" and "polypeptide" are used synonymously.

[0012] 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-extended version of the target sequence.

[0013] 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.

[0014] In a preferred embodiment, monoclonal antibodies are produced against a synthetic peptide having an N-terminal amino acid sequence DSGPEYADVV (SEQ ID NO: 1). For example, monoclonal antibodies can be produced by: (a) immunizing a rodent (or other suitable mammal) with a synthetic peptide comprising the N-terminal sequence DSGPEYADVV (SEQ ID NO: 1), which may optionally be linked at its C-terminus to an immunogenic carrier protein (e.g., keyhole limpet hemocyanin); (b) isolating and cloning individual antibody-producing cells; and (c) assaying the resulting monoclonal antibodies to ensure that they have the desired specificity.

[0015] As used herein, the term "monoclonal antibody" refers to intact antibodies and fragments thereof that retain the binding specificity of intact antibodies, 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, intact antibodies typically have a "Y-shaped" structure of two pairs of identical polypeptide chains, each pair consisting of a "light chain" and a "heavy chain". The N-terminal region of each light chain and heavy chain contains a variable region, while the C-terminal portion of each heavy chain and light chain constitutes a constant region. The variable region contains three complementary 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 contain at least a sufficient portion of the CDR and the rest of the variable region to retain the binding specificity.

[0016] In the present invention, monoclonal antibodies comprising any constant region known in the art can be used. In the case of mouse antibodies and human antibodies, constant light chains are classified as kappa light chains and lambda light chains. Heavy chain constant chains are classified as μ, δ, γ, α or ε, and the isotype of the antibody is defined as IgM, IgD, IgG, IgA and IgE, respectively. The IgG isotype has several subclasses, including but not limited to IgG1, IgG2, IgG3 and IgG4 of the human race, and IgG1, IgG2a, IgG2b, IgG2c and IgG3 of mice. The monoclonal antibody can preferably be an IgG isotype, including any one of the IgG subclasses (e.g., IgG1, IgG2, IgG3 or IgG4 of human antibodies).

[0017] The CDR of the antibody can be determined using methods known in the art, such as the methods described by Kabat et al. Antibodies can be produced from B cell clones, as described in the Examples. The isotype of the antibody can be determined by ELISA with specificity for IgM, IgG or IgA isotypes (human or mouse) or subclasses (human or mouse). The amino acid sequence of the antibody produced can be determined using standard techniques. For example, RNA can be isolated from cells and used to produce cDNA by reverse transcription. Then cDNA is subjected to PCR using primers for amplifying the heavy and light chains of the antibody. For example, primers with specificity for the leader sequences of all VH (variable heavy chain) sequences can be used together with primers that bind to sequences in the isotype constant region that have been previously determined. Primers that bind to the 3' end of a κ chain or a λ chain and primers that anneal to the Vκ or Vλ leader sequence can be used to amplify the light chain. Full-length heavy and light chains can be generated and sequenced.

[0018] In certain exemplary embodiments, the monoclonal antibody may preferably comprise one or more complementarity determining regions (CDRs) selected from the group consisting of:

[0019] CDR-L1:KASQNVGTDVV(SEQ ID NO:4)

[0020] CDR-L2: SASYRYS (SEQ ID NO: 5)

[0021] CDR-L3:QHYDNYPLT(SEQ ID NO:6)

[0022] CDR-H1: SYAMS (SEQ ID NO: 7)

[0023] CDR-H2:SVTSGGTHYLDSVKG(SEQ ID NO:8)

[0024] CDR-H3: GVSFAY (SEQ ID NO: 9)

[0025] Preferably, the monoclonal antibody comprises at least 2, 3, 4, 5 or 6 of the CDR sequences listed above.

[0026] Preferably, the monoclonal antibody has a light chain variable region comprising the CDR sequence:

[0027] CDR-L1:KASQNVGTDVV(SEQ ID NO:4)

[0028] CDR-L2: SASYRYS (SEQ ID NO: 5), and

[0029] CDR-L3: QHYDNYPLT (SEQ ID NO: 6).

[0030] Preferably, the monoclonal antibody has a light chain comprising a framework sequence between the CDRs, wherein the framework sequence is substantially identical or substantially similar to the framework sequence between the CDRs in the following light chain sequence (wherein the CDRs are shown in bold and underlined and the framework sequence is shown in italics).

[0031]

[0032] Preferably, the monoclonal antibody has a heavy chain variable region comprising the CDR sequence:

[0033] CDR-H1:SYAMS (SEQ ID NO:7)

[0034] CDR-H2: SVTSGGTHYLDSVKG (SEQ ID NO: 8), and

[0035] CDR-H3: GVSFAY (SEQ ID NO: 9).

[0036] Preferably, the monoclonal antibody has a heavy chain comprising a framework sequence between the CDRs, wherein the framework sequence is substantially identical or substantially similar to the framework sequence between the CDRs in the following heavy chain sequence (wherein the CDRs are shown in bold and underlined and the framework sequence is shown in italics).

[0037]

[0038] Preferably, the monoclonal antibody comprises the light chain variable region sequence:

[0039]

[0040] (CDRs are shown in bold and underlined; framework sequences are shown in italics)

[0041] and / or, heavy chain variable region sequence:

[0042]

[0043] (CDRs are shown in bold and underlined; framework sequences are shown in italics)

[0044] As used herein, if the framework amino acid sequence between the CDRs of an antibody has at least 70%, 80%, 90% or at least 95% similarity or identity with the framework amino acid sequence between the CDRs of another antibody, they are "substantially identical" or "substantially similar". Similar or identical amino acids can be continuous or non-continuous. The framework sequence may contain one or more amino acid substitutions, insertions and / or deletions. Amino acid substitutions can be conservative, meaning that the substituted amino acid has similar chemical properties as the original amino acid. A skilled person will understand which amino acids have similar chemical properties. For example, the following groups of amino acids have similar chemical properties, such as size, charge and polarity: Group 1 Ala, Ser, Thr, Pro, Gly; Group 2 Asp, Asn, Glu, Gln; Group 3 His, Arg, Lys; Group 4 Met, Leu, Ile, Val, Cys; Group 5 Phe, Thy, Trp.

[0045] Amino acid sequences can be compared using programs such as the CLUSTAL program. The program compares amino acid sequences and finds the best alignment by appropriately inserting spaces in either sequence. Amino acid identity or similarity (identity plus conservation of amino acid type) can be calculated for optimal alignment. Programs like BLASTx will align the longest segments of similar sequences and assign values ​​to fits. Therefore, a comparison in which several similar regions are found can be obtained, each with a different score. Two types of analysis are considered in the present invention. Identity or similarity is preferably calculated over the entire length of the framework sequence.

[0046] In preferred embodiments, the patient sample is serum or plasma.

[0047] In a preferred embodiment, the skin disease is atopic dermatitis, melanoma, psoriasis, hidradenitis suppurativa, or systemic lupus erythematosus.

[0048] In a preferred embodiment, the immunoassay is a competitive assay or a sandwich assay. The immunoassay may, for example, be a radioimmunoassay or an enzyme-linked immunosorbent assay (ELISA). Such assays are techniques known to those skilled in the art.

[0049] As used herein, the term "bound amount" refers to the quantification of the binding between an antibody and a peptide in a patient sample. The quantification can be determined, for example, by comparing the measured values ​​of the binding in the patient sample with a calibration curve generated using a standard sample containing a known concentration of the antibody-specific binding peptide, thereby determining the amount of the antibody-specific binding peptide in the patient sample. In the examples listed below, an ELISA method is used, wherein the binding amount in the patient sample and when the calibration curve is generated is measured using spectrophotometric analysis. However, any suitable analytical method can be used.

[0050] As used herein, the term "predetermined cutoff value" refers to a statistically determined binding amount that indicates a high likelihood that a patient has a disease or a specific severity thereof, wherein a measured value of the target peptide in a patient sample that is equal to or above the statistical cutoff value corresponds to a probability of at least 70% for the presence of the disease or a specific severity thereof, preferably a probability of at least 75%, more preferably a probability of at least 80%, more preferably a probability of at least 85%, more preferably a probability of at least 90%, and most preferably a probability of at least 95%.

[0051] As used herein, the term "value associated with normal healthy subjects" refers to the standardized binding amount determined by the above method for samples of subjects considered to be healthy (i.e., without disease); and the term "value associated with a known disease severity" refers to the standardized binding amount determined by the above method for samples of patients known to have a disease of a known severity.

[0052] In a second aspect, the present invention provides a method of treating a skin disease in a patient in need thereof, the method comprising:

[0053] (a) performing an immunoassay according to the first aspect of the present invention on a blood, serum or plasma sample from a patient to detect whether the patient suffers from a skin disease and / or determine the severity of the patient's skin disease; and

[0054] (b) if it is determined in step (a) that the patient suffers from the skin disease or a certain severity thereof, administering a drug for treating the skin disease to the patient.

[0055] The drug can be any drug suitable for treating the skin disease. The drug can, for example, include or consist of one or more topical drugs, one or more systemic drugs, or a combination thereof. Topical drugs can, for example, be formulated as creams, foams, gels, lotions, or ointments for administration to the skin area in need of treatment. Systemic drugs can, for example, be formulated for enteral or parenteral administration.

[0056] For example, where the skin disease is atopic dermatitis, suitable topical medications 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); and suitable systemic medications 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).

[0057] When the skin disease is melanoma, suitable drugs can be, for example, selected from chemotherapeutic agents (e.g., dacarbazine, temozolomide, 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 agonists, CD40 agonists, anti-PD-1 antibodies and PD-L1 antibodies) and adoptive cell transfer.

[0058] When the skin disease is psoriasis, suitable topical drugs can, for example, be selected from moisturizers, topical corticosteroids (e.g., hydrocortisone), vitamin D analogs (e.g., paricalcitol), and topical immunosuppressants, such as topical calcineurin inhibitors (e.g., tacrolimus and pimecrolimus); and suitable systemic drugs can be selected from systemic immunosuppressants (e.g., cyclosporine and methotrexate), fumarate esters (e.g., dimethyl fumarate), retinoids, and biologics (e.g., monoclonal antibodies, such as ixekizumab, secukinumab, brodalumab, guselkumab, certolizumab pegol, and ustekinumab).

[0059] When the skin disease is hidradenitis suppurativa, suitable drugs can be selected, for example, from topical antibiotics (e.g., topical clindamycin), topical retinoids (e.g., isotretinoin), oral antibiotics (e.g., rifampicin, clindamycin, tetracycline, and minocycline), corticosteroids administered by intralesional injection, antiandrogens (e.g., spironolactone, flutamide, cyproterone acetate, ethinyl estradiol, finasteride, dutasteride, and metformin), and TNF inhibitors (e.g., etanercept) and anti-TNF-α monoclonal antibodies (e.g., infliximab and adalimumab) administered by intravenous or subcutaneous injection or infusion.

[0060] In case the skin disease is systemic lupus erythematosus, suitable drugs may, for example, be selected from nonsteroidal anti-inflammatory drugs (NSAIDs), corticosteroids, antimalarials (hydroxychloroquine), BLyS-specific inhibitors (e.g., belimumab), and immunosuppressants (e.g., prednisone, mycophenolic acid, tacrolimus, methotrexate, and azathioprine).

[0061] In certain embodiments, step (a) of the method may include performing an immunoassay according to the first aspect of the invention on a blood, serum or plasma sample from a patient to determine the severity of the patient's skin disease, and step (b) of the method may include administering a drug for treating the skin disease to the patient only when the patient is determined to have a specific severity of the skin disease in step (a). For example, the method may include administering a topical medication if the severity of the disease is at or below a specific level, and administering a systemic medication if the severity of the disease is above the level. In a specific embodiment, the skin disease is atopic dermatitis, and step (b) includes administering a topical medication to the patient if it is determined in step (a) that the patient suffers from mild or moderate atopic dermatitis, and administering a systemic medication to the patient if it is determined in step (a) that the patient suffers from severe atopic dermatitis.

[0062] 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:

[0063] - Streptavidin-coated well plates;

[0064] - biotinylated peptide DSGPEYADVV-L-biotin (SEQ ID NO: 14), wherein L is an optional linker;

[0065] - Secondary antibodies for sandwich immunoassays;

[0066] - a calibration protein comprising the N-terminal amino acid sequence DSGPEYADVV (SEQ ID NO: 1);

[0067] - Antibody biotinylation kit;

[0068] -Antibody HRP labeling kit;

[0069] -Antibody radiolabeling kit; and

[0070] -Assay Visualization Kit.

[0071] 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).

[0072] 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).

[0073] 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).

[0074] The immunoassay kit according to the third aspect of the invention is particularly suitable for carrying out the method according to the first aspect of the invention. Thus, 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 of the first aspect.

[0075] 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).

[0076] 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).

[0077] 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).

[0078] 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).

[0079] The monoclonal antibodies according to the fourth aspect of the invention are particularly suitable for use in the method according to the first aspect of the invention. Therefore, further preferred embodiments and features of the monoclonal antibodies according to the fourth aspect will be apparent from the above discussion of preferred monoclonal antibodies for use in the method according to the first aspect. BRIEF DESCRIPTION OF THE DRAWINGS

[0080] Figure 1 : A) Overview of the primary structure of the type VI collagen α chain, showing the domains of all six α chains. As shown, the antibodies used for the C6A6 assay target the C6A6 target sequence at the N-terminus of the type VI collagen α-6 chain (COL6α6). B) Sequence alignment of the N-terminal sequence of COL6α6 in human, mouse, cattle and rat species, in which the C6A6 target sequence is highlighted (black box). Sequences were aligned using Uniprot. C) Specificity of the C6A6 assay. Reactivity to a standard peptide (DSGPEYADVV (SEQ ID NO: 1)), a truncated peptide (SGPEYADVV (SEQ ID NO: 3)), an extended peptide (QDSGPEYADVV (SEQ ID NO: 2)) and a nonsense standard peptide (YRDDLKKLLE (SEQ ID NO: 17)) is shown. No background signal was detected when coated with a nonsense coating peptide (YRDDLKKLLE-biotin (SEQ ID NO: 16)). Signals are shown as relative luminescence per second (RLU) versus standard peptide.

[0081] Figure 2 : Results from cohort 1. Serum levels of C6A6 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 (n=20). Mean values ​​with 95% confidence intervals (CIs) are presented. Data were analyzed using ANCOVA corrected for age and sex. Data are depicted as mean ± 95% CI.

[0082] Figure 3 : Results from cohort 2. Serum levels of C6A6 in healthy donors (n=22) and patients with atopic dermatitis (n=158). Means with 95% confidence intervals. Data were analyzed using ANCOVA corrected for age. Data are depicted as mean ± 95% CI.

[0083] Figure 4: Results from cohort 2. Serum levels of C6A6 were divided into disease severity and treatment. A) Levels of C6A6 in patients with mild and moderate AD (SCORAD; 0-50) versus patients with severe AD (SCORAD; <50). B) Levels of C6A6 in untreated AD patients versus AD patients treated with immunosuppressants. C) Levels of C6A6 in untreated patients with mild and moderate AD versus patients treated with immunosuppressants in mild and moderate AD. D) Levels of C6A6 in untreated severe AD versus severe AD patients treated with immunosuppressants. Means with 95% confidence intervals. Data were analyzed using ANCOVA corrected for age. Data are depicted as mean ± 95% CI. DETAILED DESCRIPTION

[0084] Example

[0085] The embodiments disclosed herein are described in the following examples, which are intended to help understand the present disclosure and should not be construed as limiting the scope of the present disclosure defined in the appended claims in any way. The following examples are presented to provide a complete disclosure and description of how to make and use the described embodiments for those of ordinary skill in the art, and are not intended to limit the scope of the present disclosure, nor are they intended to represent that the following experiments are all or only experiments performed. Efforts have been made to ensure the accuracy of the numbers (e.g., amount, temperature, etc.) used, but some experimental errors and deviations should be considered. Unless otherwise stated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is degrees Celsius, and pressure is atmospheric pressure or near atmospheric pressure.

[0086] Materials and methods

[0087] Unless otherwise stated, all reagents used were high quality chemicals from Merck (Whitehouse Station, NJ, USA) and Sigma (St. Louis MO, USA). All synthetic peptides used for antibody production and assay validation were purchased from Genscript (Piscataway, NJ, US) (Table 1).

[0088] Table 1. Synthetic peptide sequences used for monoclonal antibody production, assay development, and validation.

[0089]

[0090]

[0091] *Keyhole limpet hemocyanin.

[0092] Monoclonal Antibody Development, Production and Characterization

[0093] The amino acid sequence 20'↓DSGPEYADVV'30 (SEQ ID NO: 1) in human type VI collagen α6 chain (COL6α6) (also referred to herein as "C6A6" and / or "target sequence") was used to generate monoclonal antibodies (mAbs), which form the N-terminus of type VI collagen α6 chain after cleavage and removal of the signal peptide. Immunization was initiated by subcutaneous injection of 200 μl of emulsified antigen and 100 μg of immunogenic peptide (DSGPEYADVV-GG C-KLH (SEQ ID NO: 15)) in Balb / C mice aged 4 to 6 weeks using Stimmune (Thermo Fisher). Immunization was repeated every two weeks until a stable serum antibody titer level was reached. Mice with the highest serum titer were selected for fusion and rested for one month. Subsequently, mice were boosted intravenously with 50 μg of immunogenic peptide in 100 μl of 0.9% NaCl solution 3 days before spleen isolation for cell fusion. To produce hybridoma cells, mouse spleen cells were fused with SP2 / 0 myeloma cells as described by Gefter et al. (13). Subsequently, clones were plated into 96-well microtiter plates for further growth, and limiting dilution methods were applied to promote monoclonal growth. Indirect ELISA performed on streptavidin-coated plates was used to screen supernatant reactivity. DSGPEYADVV-K-biotin (SEQ ID NO: 18) was used as a screening peptide, while the standard peptide DSGPEYADVV (SEQ ID NO: 1) was used to further test the specificity of the clones. Supernatants were collected from hybridoma cells and purified using HiTrap affinity columns (GE Healthcare Life Science, Little Chalfront, Buckinghamshire, UK) according to the manufacturer's instructions, and antibody isotypes were determined using a rapid ELISA mouse monoclonal antibody typing kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol.

[0094] Native reactivity was assessed using human serum, citrate plasma, heparin plasma, EDTA plasma, and rat serum purchased from a commercial supplier (Valley Biomedical, Winchester, VA). mAbs were selected to specifically recognize a standard peptide (DSGPEYADVV (SEQ ID NO: 1)) and not to recognize an extended sequence or truncated sequence of one amino acid (QDSGPEYADVV (SEQ ID NO: 2) and SGPEYADVV (SEQ ID NO: 3), respectively).

[0095] The isotype, sequence and CDR of the monoclonal antibody selected for production and assay development were determined. The sequence of the chain is as follows (CDRs are underlined and in bold; N-terminal signal peptide and C-terminal constant region are in italics):

[0096] Heavy chain sequence (mouse IgG1 isotype)

[0097]

[0098] Light chain sequence (mouse kappa isotype)

[0099]

[0100] C6A6 Assay Development

[0101] The development of a competitive chemiluminescent immunoassay (CLIA) targeting the C6A6 target sequence included several preliminary optimization experiments, in which reagents, concentrations, incubation times, and incubation temperatures were analyzed by several tests. The final C6A6 competitive ELISA procedure was as follows: 96-well streptavidin-coated white microplates (Greiner Bio-One, Kremsmünster, Austria) were coated with 3 ng / mL biotinylated synthetic peptide (DSGPEYADVV-K-biotin (SEQ ID NO: 18)) dissolved in assay buffer (10 mM phosphate-buffered saline (PBS), 1% bovine serum albumin, 0.1% Tween-20, 0.36% Bronidox, 4 g / L NaCl, adjusted to pH 7.4 at 20°C) and incubated in the dark at 20°C with constant shaking (300 rpm) for 30 min. Next, 20 μ L / hole standard peptide (100ng / mL) and sample are added to the appropriate wells, and then 100 μ L / hole HRP-labeled antibodies (generated as described above) diluted in the assay buffer are added to a concentration of 200 ng / mL, and incubated for 1 hour in the dark at 20 ° C with continuous shaking (300rpm). After each incubation step, the wells are washed five times with standard wash buffer (20mM Tris, 50mMNaCl, pH7.2). Chemiluminescent substrate (Roche, BM chemiluminescent ELISA substrate (POD), Basel, Switzerland) working solution is mixed 15 minutes before use, and added to the plate at 100 μ L / hole, and incubated for 3 minutes in the dark at 20 ° C with continuous shaking (300rpm). On a microplate photometer reader (SpectraMax M5, Molecular Devices, CA, USA), relative light units of all wavelengths are measured within 5 minutes. The standard curve was drawn using a 4-parameter logistic curve fit Y=(AD) / (1+(x / C)^B)+D, where R>0.9. The data were analyzed using SoftMax Pro version 7.0.3 software.

[0102] Technology Assessment

[0103] Linearity was assessed using two-fold dilutions of four human serum and two rat serum samples. Linearity was calculated as the percent recovery of the undiluted sample. Antibody specificity was calculated as the percent signal inhibition of the two-fold diluted standard peptide (DSGPEYADVV (SEQ ID NO: 1)), extended peptide (QDSGPEYADVV (SEQ ID NO: 2)), truncated peptide (SGPEYADVV (SEQ ID NO: 3)) and nonsense peptide (YRDDLKKLLE (SEQ ID NO: 17)). Ten independent runs were performed with five quality controls and two kit controls to determine intra- and inter-assay variability in duplicate assays. The accuracy of the assay was measured in healthy human serum samples spiked with standard peptides and serum samples with known high COL6α6 concentrations and calculated as the percent recovery of the measured value and the expected concentration of the peptide or serum sample with high COL6α6 plus the concentration of the analyte in serum. The interferences were analyzed by adding low / high levels of hemoglobin (2.50 / 5 mg / mL), lipidemia / lipids (1.50 / 5 mg / mL) and biotin (3 / 9 ng / mL) to serum samples of known concentrations. The percent recovery was calculated using normal serum samples as reference. The normal reference levels of hemoglobin, lipidemia / lipids and biotin were 0-10 mg / dl (0-0.00161 mmol / L), <150 mg / dl (<1.6935 mmol / L) and 0.221-3.004 ng / ml, respectively. The interference was calculated as the percent recovery of the analyte not spiked into the 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), which was determined by 10 independent runs using standard peptides. The measured values ​​below the LLMR or above the ULMR were assigned the values ​​of LLMR / ULMR, respectively. The IC50 (half maximal inhibitory concentration) was determined from the standard curve. Analyte stability was examined by temperature testing and repeated freeze-thaw cycles of serum samples. Temperature testing included different time points and temperatures where C6A6 target sequence levels were measured in three human serum samples after 0, 2, 4, 24, and 48 hours of incubation at 4°C or 20°C. Recovery was estimated using the 0 hour sample as a reference. In addition, the effect of four repeated freeze / thaw cycles of three serum samples was evaluated, where freeze / thaw recoveries were calculated using the zero cycle sample as a reference. Each sample was assayed in duplicate.

[0104] Biological Assessment and Patient Demographics of the C6A6 Assay

[0105] The biological utility of the C6A6 assay (as described above) was evaluated in serum samples from two cross-sectional studies. The first cohort (cohort 1) was obtained from the commercial supplier 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 BioIVT and Lee Biosolutions.

[0106] Cohort 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 (n = 20) and matched healthy donors without symptoms or chronic diseases (n = 24). Cohort 2 included 158 patients with atopic dermatitis and 22 age-, sex-, and ethnicity-matched healthy controls. Serum samples were collected from January 2012 and June 2018 (8). Patients met the criteria defined by Hannifin and Rajka in 1980. The diagnosis of AD was confirmed by a senior physician according to the criteria. Severity was assessed by SCORAD, a commonly used severity index for AD (range, 0-103; high scores indicate severe disease). Patients were divided into disease severity groups based on SCORAD, i.e., mild (SCORAD1<25), moderate (SCORAD range 25-50), and severe disease (SCORAD1<50) (2, 3). Of the 158 patients, 47 patients were treated with topical immunosuppressants. The therapeutic agent was a topical calcineurin inhibitor. Age and gender were available only for healthy donor subjects.

[0107] Samples from both cohorts were collected after informed consent and approval by the local ethics committee and in compliance with the Helsinki Declaration of 1975. Serum samples were obtained and stored at −80°C.

[0108] Ethical Statement

[0109] All animals were handled according to animal welfare guidelines. Monoclonal antibody production in mice was approved by the Danish national agency (Animal Experimentation Inspectorate) under approval number 2013-15-2934-00956.

[0110] Statistical analysis

[0111] The patient characteristics of the two cohorts are expressed as numbers (frequency) and percentages of categorical variables and mean values ​​(standard deviation) or mean values ​​(range) of continuous variables. The statistical differences of categorical variables were evaluated using the Kruskal-Wallis test (nonparametric) of cohort 1 and the Mann-Whitney t test in cohort 2. For cohort 1, the differences between the patient groups were calculated using the ANCOVA analysis adjusted for age and gender. For cohort 2, the differences between the patient groups aged 4-90 years were calculated using the ANCOVA analysis adjusted for age. The charts are shown as mean ± 95% CI. For all statistical analyses performed, P values ​​less than 0.05 were considered significant. Statistical analysis and charts were performed using GraphPad Prism version 9 (GraphPadSoftware, Inc., La Jolla, CA) and MedCalc version 19.3 (MedCalc Software, Ostend, Belgium).

[0112] result

[0113] Specificity, accuracy, and precision of the C6A6 assay

[0114] The C6A6 assay uses a protein targeting the α6 chain of type VI collagen ( Figure 1 A) of the monoclonal antibody (mAb) with the N-terminal target sequence DSGPEYADVV (SEQ ID NO: 1). The human target sequence was aligned with the corresponding mouse, rat, and bovine sequences using UNIPROT. The mouse and rat sequences were 100% aligned with the human sequence, while the bovine sequence had one mismatch at position 1 ( Figure 1 B). The hybridomas producing the best mAbs were screened for their reactivity to standard peptides and native materials. Clone NBH-306-118E7-2C11-1F10-2C10 was selected for assay development and assayed as IgG1 subtype. To assess the specificity of the C6A6 assay, mAbs were tested against extended peptides, truncated peptides, nonsense standard peptides, and nonsense coating agents and showed no reactivity to those peptides ( Figure 1 C). Technical validation was performed to evaluate the novel C6A6 assay, and the technical results are summarized in Table 2. Overall, the C6A6 assay showed low inter- and intra-assay variability, analyte recoveries of 93% and 81% over 24 hours at 4°C / 20°C, respectively, good analyte recovery of 97% over five freeze-thaw cycles, and no interference from hemoglobin, lipemia, or biotin. Linearity was observed for human serum samples from 1:3 to 1:16 dilutions.

[0115] Table 2: Summary of technical parameters of the C6A6 assay

[0116]

[0117]

[0118] *Percentages are reported as averages

[0119] C6A6 is elevated in patients with skin diseases compared to healthy donors

[0120] Cohort 1 included patients with atopic dermatitis (mean age: 52.3 years, 10% males), melanoma (mean age: 55.6 years, 10% males), psoriasis (mean age: 50.7 years, 10% males), hidradenitis suppurativa (mean age: 49.5 years, 0% males), systemic sclerosis (mean age: 54.4 years, 5.6% males), systemic lupus erythematosus (mean age: 50.5 years, 16.7% males), urticaria (mean age: 43.6 years, 20% males), and vitiligo (mean age: 56.9 years, 25% males). The patient demographic information is shown in Table 3. Compared with healthy donors, C6A6 target sequence levels 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). Figure 2 No significant differences were found between patients with systemic sclerosis, urticaria, and vitiligo and healthy controls (p = 0.304-1.000, respectively).

[0121]

[0122] C6A6 is associated with disease severity and is suppressed by immunosuppressant treatment

[0123] Cohort 2 included 158 patients with atopic dermatitis (mean age: 30.0, 52% male) and 22 healthy donors (mean age: 29.6, 50% male), and the patient demographic information is shown in Table 4. Patients with atopic dermatitis had significantly higher serum C6A6 target sequence levels compared with healthy controls (p<0.0001). Among the 158 AD patients, 53 (33.3%) patients had mild disease, 72 (45.3%) had moderate disease, and 33 (21.4%) had severe disease. We evaluated whether C6A6 target sequence levels were associated with disease severity by comparing patients with severe AD (SCORAD>50) with patients with mild and moderate AD (SCORAD0-50). C6A6 levels were significantly higher in patients with severe AD compared with mild and moderate AD (p=0.046, Figure 4A). Of the 158 patients, 47 received topical immunosuppressant therapy. The C6A6 levels of the treated patients were significantly lower than those of the untreated patients (p = 0.0136, Figure 4 B). In addition, the C6A6 levels of mild and moderate AD patients treated with immunosuppressants were significantly lower than those of untreated patients (p = 0.035, Figure 4 C), while in patients with severe AD, there was no significant difference between those who received immunosuppressive therapy and those who did not (p = 0.544, Figure 4 D).

[0124] Table 4: Patient demographic information of cohort 2. Categorical variables are expressed as numbers (percentages) and continuous variables are expressed as means (standard deviations). BMI, body mass index

[0125]

[0126] discuss

[0127] Applicants developed and characterized a competitive ELISA in this study for detecting biomarkers comprising the N-terminus of COL6α6 in blood-based samples using a monoclonal antibody targeting the N-terminal amino acid sequence DSGPEYADVV (SEQ ID NO: 1) (also referred to herein as "C6A6" and / or "target sequence"). The main results of the study 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 was detectable in human, mouse and rat sera; 3) Elevated levels of C6A6 were present in the sera of patients diagnosed with atopic dermatitis, melanoma, psoriasis, hidradenitis suppurativa and systemic lupus erythematosus compared to healthy donors; 5) Elevated serum C6A6 levels were associated with severe atopic dermatitis, and serum levels were reduced by immunosuppressant therapy, especially in patients with mild and moderate atopic dermatitis. To the best of the applicant's knowledge, this is the first study to show that levels of this target sequence can be measured non-invasively in the serum of patients with dermatological conditions and that levels correlate with disease severity and are reduced in atopic dermatitis patients treated with topical immunosuppressants.

[0128] The C6A6 assay was characterized as a technically robust and accurate assay by showing acceptable dilution recovery, interference, and stability testing. Inter- and intravariance values ​​were accepted with values ​​of 5% and 12%, respectively. The assay was further characterized as being specific for the N-terminal target sequence of COL6α6 exposed after signal peptide cleavage.

[0129] In this study, the applicants found that serum C6A6 levels were suppressed in patients with mild and moderate atopic dermatitis when treated with topical calcineurin inhibitors (TCIs). TCIs, including pimecrolimus and tacrolimus, are widely used as first-line immunosuppressant topical treatments for atopic dermatitis and psoriasis (3). This suggests that serum levels of the target sequence correlate with treatment response. In contrast, no suppression was seen in patients with severe atopic dermatitis, which may indicate that this group of patients requires other types of treatment in addition to TCIs.

[0130] In conclusion, this data supports the use of the C6A6 biomarker in both the diagnostic and prognostic setting for patients with dermatological conditions, particularly atopic dermatitis.

[0131] In summary, the C6A6 assay showed high specificity for an N-terminal target sequence whose levels were elevated in patients with dermatological conditions. The assay was able to distinguish AD patients from healthy donors, showing high discrimination. C6A6 levels were also upregulated in patients with severe AD, suggesting that severe patients may experience more fibroblast activity and general tissue remodeling. In addition, it was found that the levels of the biomarker were reduced in patients treated with topical immunosuppressants (calcineurin inhibitors), suggesting that this biomarker is associated with treatment response.

[0132] In this specification, unless expressly stated otherwise, the word "or" is used to indicate an operator that returns a true value when either or both of the conditions are met, rather than an "exclusive or" operator that requires only one of the conditions to be met. The word "comprises" is used to mean "comprising or consisting of". All prior teachings acknowledged above are incorporated herein by reference. The acknowledgement of any prior published document herein is not to be taken as an acknowledgment or representation that the teachings therein were common general knowledge as of the date of the present application in Australia or elsewhere.

[0133] References

[0134] 1. Simon D, Aeberhard C, Erdemoglu Y, Simon HU. Th17 cells and tissue remodeling in atopic and contact dermatitis. Allergy [Internet] Allergy; 2014 [cited May 6, 2022]; 69: 125-31. Available from: https: / / pubmed.ncbi.nlm.nih.gov / 24372156 /

[0135] 2.Dengjel J,Bruckner-Tuderman L, A. Skin proteomics–analysis of the extracellular matrix in health and disease. Expert Rev Proteomics Taylor & Francis; 2020;17:377-91.

[0136] 3. Lynch MD, Watt FM. Fibroblast heterogeneity: implications for human disease. J Clin Invest [Internet] J Clin Invest; 2018 [cited May 6, 2022]; 128: 26-35. Available from: https: / / pubmed.ncbi.nlm.nih.gov / 29293096 /

[0137] 4. Cescon M, Gattazzo F, Chen P, Bonaldo P. Collagen VI at a glance. J Cell Sci 2015;128:3525-31.

[0138] 5. Wagner MFMG, Theodoro TR, Filho CDASM, Oyafuso LKM, Pinhal MAS. Extracellular matrix alterations in the skin of patients affected by psoriasis. BMC Mol Cell Biol [Internet] BioMed Central; 2021 [cited May 6, 2022]; 22. Available from: / pmc / articles / PMC8555298 /

[0139] 6. Holm Nielsen S, Mortensen J, Willumsen N, Rasmussen D, Mogensen D, DiSabatino A, et al. A Fragment of Collagen Type VI alpha-3 chain is Elevated in Serum from Patients with Gastrointestinal Disorders. Sci Rep Sci Rep; 2020; 10: 5910.

[0140] 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 May 6, 2022]; 54:345-50. Available from: https: / / pubmed.ncbi.nlm.nih.gov / 23869615 /

[0141] 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 /

[0142] 9. Dobrota R, Jordan S, Juhl P, Maurer B, Wildi L, Bay-Jensen AC, et al. Circulating collagen neo-epitopes and their role in the prediction of fibrosis in patients with systemic sclerosis: a multicentre cohort study. Lancet Rheumatol [Internet] Elsevier Ltd; 2021; 3: e175-84. Available from: http: / / dx.doi.org / 10.1016 / S2665-9913(20)30385-4

[0143] 10. Jung HJ, Heo WI, Park KY, Lee MK, Ahn JY, Park MY, et al. The Role of Collagen VIα6 Chain Gene in Atopic Dermatitis. Ann Dermatol [Internet] Ann Dermatol; 2022 [cited May 4, 2022]; 34:46-54. Available from: https: / / pubmed.ncbi.nlm.nih.gov / 35221595 /

[0144] 11. Ghosh D, Ding L, Sivaprasad U, Geh E, Myers JB, Bernstein JA et al. Multiple Transcriptome Data Analysis Reveals Biologically Relevant Atopic Dermatitis Signature Genes and Pathways. PLoS One [Internet] PLoS One; 2015 [cited May 10, 2022]; 10. Available from: https: / / pubmed.ncbi.nlm.nih.gov / 26717000 /

[0145] 12. Heo W Il, Park KY, Jin T, Lee MK, Kim MJ, Choi EH, et al. Identification of novel candidate variants including COL6Α6 polymorphisms in early-onset atopic dermatitis using whole-exome sequencing. BMC Med Genet [Internet] BMC Med Genet; 2017 [cited May 10, 2022]; 18. Available from: https: / / pubmed.ncbi.nlm.nih.gov / 28125976 /

[0146] 13. Gefter ML, Margulies DH, Scharff 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. An immunoassay method for detecting and / or monitoring a skin disease in a patient and / or determining the severity of a skin disease in a patient, the method comprising: i) contacting a patient sample selected from blood, serum or plasma with a monoclonal antibody that specifically binds to the N-terminal amino acid sequence DSGPEYADVV (SEQ ID NO: 1); ii) detecting and determining the amount of binding between the monoclonal antibody and the peptide in the sample; as well as iii) correlating said bound amount to a value associated with a normal healthy subject, and / or to a value associated with a known disease severity, and / or to a value obtained from said patient at a previous time point, and / or to a predetermined cut-off value.

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

5. A method according to any one of the preceding claims, wherein: The skin disease is atopic dermatitis, melanoma, psoriasis, hidradenitis suppurativa or systemic lupus erythematosus.

6. A method according to any one of the preceding claims, wherein: The immunoassay is a competition assay or a sandwich assay.

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

8. 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), wherein L is an optional linker; - Secondary antibodies for sandwich immunoassays; - a calibration protein 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.

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

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

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