Immunoassay method for detecting N-terminal peptide of proteolytic cleavage product of cartilage interlayer protein-1

Abstract

Described herein is a monoclonal antibody that binds to the N-terminal sequence of cartilage interlayer protein-1 (SLNPDTGLWE (SEQ ID NO: 1), also referred to herein as' CILP-M ', resulting from cleavage of MMP-1, MMP-8, and MMP-12. Also disclosed are immunoassay methods and kits for detecting CILP-M in a sample of a patient, which methods and kits can be used to detect and monitor joint disease and assess the likelihood of a patient to respond to a drug treatment.

Description

Technical Field

[0001] The present invention relates to a monoclonal antibody that binds to the N-terminal sequence of cartilage intermediate layer protein-1 produced by cleavage of MMP-1, MMP-8 and MMP-12. The present invention also relates to an immunoassay method for detecting the N-terminal sequence of cartilage intermediate layer protein-1 in a sample (such as whole blood, plasma or serum) from a patient, and an immunoassay kit suitable for performing the method. Background Art

[0002] Articular cartilage is a heterogeneous tissue in which cells are organized into a matrix. The matrix is ​​formed by chondrocytes, the main components of which are fibrillar collagens, such as type I and type III collagen, and large aggrecan (1). To form the cartilage matrix, other non-collagenous extracellular matrix proteins are also present, such as cartilage intermediate layer protein (CILP) (2). CILP-1 is a glycoprotein that was first identified in articular cartilage and is thought to play a role in the cartilage scaffold (2). CILP-1 is secreted by articular chondrocytes (2), which are responsible for maintaining cartilage homeostasis through low-turnover remodeling of matrix proteins (3). The hallmark of rheumatic joint disease is altered cartilage turnover, resulting in an imbalance in cartilage components. During cartilage erosion, CILP-1 is secreted from articular chondrocytes and deposited into the cartilage extracellular matrix (2). CILP-1 has been shown to be cleaved in vitro by various human proteases, resulting in the production of multiple fragments (4). It has been shown that the synthesis of CILP is increased in early osteoarthritis (OA) cartilage (5) and is associated with musculoskeletal disorders (6, 7). In another study, CILP-1 was highly expressed in the intervertebral disc, and its expression was increased in lumbar disc disease (8). The immune response to CILP is also thought to contribute to the pathogenesis of inflammatory joint destruction present in rheumatoid arthritis (RA) and OA (9). In addition, CILP mRNA expression has been observed in the synovial tissue of patients with OA (10), indicating that its expression is not limited to articular cartilage. These findings suggest that CILP protein may play a role in cartilage structure and rheumatic diseases. Summary of the invention

[0003] Applicants have now discovered that CILP-1 is degraded by MMP-1, MMP-8, and MMP-12 to produce a new epitope having an N-terminal sequence SLNPDTGLWE (SEQ ID NO: 1) (referred to herein as "CILP-M" and / or "target sequence"). Applicants have also discovered that CILP-M is produced and released into the body's circulation, and have developed an immunoassay targeting CILP-M. In addition, applicants have evaluated the biological value of CILP-M in joint-related diseases (RA, AS, and OA) in two exploratory patient studies, and have studied the prognostic potential of CILP-M in anti-TNF-α therapy, and have shown that the measured levels of CILP-M in serum samples reflect disease activity.

[0004] Therefore, in a first aspect, the present invention provides a monoclonal antibody that specifically binds to the N-terminal amino acid sequence SLNPDTGLWE (SEQ ID NO: 1).

[0005] 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.

[0006] As used herein, the terms "peptide" and "polypeptide" are used synonymously.

[0007] 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 portion of the CDRs and the remaining variable region sufficient to retain the binding specificity.

[0008] 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 in the human case, and IgG1, IgG2a, IgG2b, IgG2c and IgG3 in the mouse case. The monoclonal antibody can preferably be an IgG isotype, including any one of the IgG subclasses (e.g., IgG1, IgG2, IgG3 or IgG4 in the case of human antibodies).

[0009] 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.

[0010] In a preferred embodiment, the monoclonal antibody does not specifically bind to a peptide having the N-terminal amino acid sequence WSLNPDTGLWE (SEQ ID NO: 2).

[0011] In a preferred embodiment, the monoclonal antibody does not specifically bind to a peptide having the N-terminal amino acid sequence LNPDTGLWE (SEQ ID NO: 3).

[0012] In a preferred embodiment, the monoclonal antibody does not specifically bind to a peptide having the N-terminal amino acid sequence NPDTGLWE (SEQ ID NO: 4).

[0013] In a preferred embodiment, monoclonal antibodies are generated against a synthetic peptide having an N-terminal amino acid sequence SLNPDTGLWE (SEQ ID NO: 1). For example, monoclonal antibodies can be generated by: (a) immunizing a rodent (or other suitable mammal) with a synthetic peptide comprising the N-terminal sequence SLNPDTGLWE (SEQ ID NO: 1), which may optionally be linked to an immunogenic carrier protein (e.g., keyhole limpet hemocyanin) at its C-terminus; (b) isolating and cloning individual antibody-producing cells; and (c) assaying the resulting monoclonal antibodies to ensure that they have the desired specificity.

[0014] In certain exemplary embodiments, the monoclonal antibody may preferably comprise one or more complementarity determining regions (CDRs) selected from the group consisting of:

[0015] CDR-L1:KASQNVGTDVV (SEQ ID NO:5)

[0016] CDR-L2:SASYRYS (SEQ ID NO:6)

[0017] CDR-L3:QHYDNYPLT (SEQ ID NO:7)

[0018] CDR-H1: NYYIH (SEQ ID NO: 8)

[0019] CDR-H2:WISPGIVDTEYNEKFKN (SEQ ID NO:9)

[0020] CDR-H3:RSAGSYGDFDY (SEQ ID NO:10)

[0021] Preferably, the monoclonal antibody comprises at least 2, 3, 4, 5 or 6 of the above CDR sequences.

[0022] Preferably, the monoclonal antibody has a light chain variable region comprising the CDR sequence:

[0023] CDR-L1:KASQNVGTDVV (SEQ ID NO:5)

[0024] CDR-L2: SASYRYS (SEQ ID NO:6) and

[0025] CDR-L3: QHYDNYPLT (SEQ ID NO:7).

[0026] 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).

[0027]

[0028] Preferably, the monoclonal antibody has a heavy chain variable region comprising the CDR sequence:

[0029] CDR-H1: NYYIH (SEQ ID NO: 8)

[0030] CDR-H2: WISPGIVDTEYNEKFKN (SEQ ID NO:9) and

[0031] CDR-H3: RSAGSYGDFDY (SEQ ID NO:10).

[0032] 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).

[0033]

[0034] Preferably, the monoclonal antibody comprises:

[0035] Light chain variable region sequence:

[0036]

[0037] (CDRs are shown in bold and underlined; framework sequences are shown in italics)

[0038] and / or, heavy chain variable region sequence:

[0039]

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

[0041] 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.

[0042] 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.

[0043] In a second aspect, the present invention provides an immunoassay method comprising:

[0044] i) contacting a patient sample with a monoclonal antibody according to the first aspect of the invention;

[0045] ii) detecting and determining the amount of binding between the monoclonal antibody and the peptide in the sample.

[0046] In a preferred embodiment, the method is a method for detecting and / or monitoring a disease in a patient and / or assessing the likelihood of a patient responding to treatment with a drug to treat the disease, the method further comprising:

[0047] iii) correlating said bound amount to a value associated with a normal healthy subject, and / or a value associated with a known disease severity, and / or a value obtained from said patient at a previous time point, and / or a predetermined cut-off value.

[0048] In a preferred embodiment, the disease is a joint disease. Preferably, the joint disease is rheumatoid arthritis, ankylosing spondylitis or osteoarthritis.

[0049] In a preferred embodiment, the treatment of the disease being treated with the drug is an anti-TNF-α treatment.

[0050] In a preferred embodiment, the patient sample is selected from blood, serum or plasma. Preferably, the sample is serum or plasma.

[0051] 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.

[0052] As used herein, the term "binding amount" refers to the quantification of the binding between the antibody and the peptide in the patient sample. The quantification can be determined, for example, by comparing the measured value of the binding in the patient sample with the calibration curve generated by the measured value of the binding in the standard sample containing the known concentration of the peptide of the antibody specific binding, thereby determining the amount of the peptide specifically bound by the antibody in the patient sample. In the embodiments listed below, the binding amount when the ELISA method (wherein, spectrophotometric analysis is used) is measured in the patient sample and the calibration curve is generated. However, any suitable analytical method can be used.

[0053] 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 the 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%.

[0054] As used herein, the term "value associated with normal healthy subjects" refers to the standardized binding amount determined by the above method for samples from subjects who are 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 from patients known to have a disease of a known severity, such as a known likelihood of response to treatment.

[0055] In a third aspect, the present invention provides a method of treating a joint disease in a patient in need thereof, the method comprising:

[0056] (a) performing an immunoassay according to the second aspect of the invention on a sample from a patient to detect whether the patient suffers from a joint disease and / or to predict whether the patient will respond to treatment for a joint disease; and

[0057] (b) if the patient is determined to be suffering from the joint disease in step (a), or if the patient is determined to be likely to respond to treatment for the joint disease, administering to the patient a drug for treating the joint disease.

[0058] The drug may be any drug suitable for treating the joint disease. The drug may, for example, include or consist of one or more local drugs, one or more systemic drugs, or a combination thereof. The local drug may, for example, be formulated as a cream, foam, gel, lotion or ointment for administration to one or more areas of the joint in need of treatment. The systemic drug may, for example, be formulated for enteral or parenteral administration.

[0059] For example, where the joint disease is osteoarthritis, suitable topical medications may be selected from nonsteroidal anti-inflammatory drugs (e.g., diclofenac or ketoprofen) or topical capsaicin; suitable systemic medications may be selected from analgesics (e.g., acetaminophen or opioids); anti-TNF-α therapy (e.g., etanercept, infliximab, golimumab, adalimumab or apremilast); glucocorticoids (e.g., hydrocortisone or triamcinolone) or platelet-rich plasma.

[0060] In the case where the joint disease is rheumatoid arthritis, suitable drugs can be, for example, topical drugs, which can be selected from non-steroidal anti-inflammatory drugs (such as diclofenac or ketoprofen) or topical capsaicin; suitable systemic drugs can be selected from disease-modifying antirheumatic drugs (such as methotrexate, sulfasalazine, leflunomide, hydroxychloroquine); anti-TNF-α therapy (such as etanercept, infliximab, golimumab, adalimumab or apremilast); antibody therapy (such as rituximab or tocilizumab); analgesics (such as acetaminophen or opioids); and glucocorticoids (such as hydrocortisone or triamcinolone).

[0061] Where the joint disease is ankylosing spondylitis, suitable drugs may, for example, be selected from anti-inflammatory drugs (e.g. ibuprofen, phenylbutazone, diclofenac, indomethacin, naproxen or COX-2 inhibitors), disease-modifying antirheumatic drugs (e.g. methotrexate, sulfasalazine, leflunomide, hydroxychloroquine), anti-interleukin-6 inhibitors (e.g. tocilizumab or rituximab); interleukin-17A inhibitors (e.g. secukinumab and ixekizumab); janus kinase inhibitors (e.g. tofacitinib); and anti-TNF-α therapy (e.g. etanercept, infliximab, golimumab, adalimumab or apremilast).

[0062] In certain embodiments, step (a) of the method may comprise performing an immunoassay according to the first aspect of the invention on a sample from a patient to determine whether the patient is likely to respond to treatment for the joint disease, and step (b) of the method may comprise administering to the patient a drug for treating the joint disease only if it is determined in step (a) that the patient is likely to respond to treatment.

[0063] For example, the method may include administering a drug if the likelihood of responding to treatment is equal to or above a certain level. In a specific embodiment, the joint disease is ankylosing spondylitis, and step (b) includes administering anti-TNF-α therapy to the patient if the level of CILP-M in the patient sample is determined in step (a) to be associated with a likelihood of responding to anti-TNF-α therapy, and administering anti-TNF-α therapy if it is determined that the patient is likely to respond to treatment.

[0064] In a fourth aspect, the present invention provides an immunoassay kit comprising a monoclonal antibody according to the first aspect of the present invention and at least one of the following;

[0065] - Streptavidin-coated well plates;

[0066] - biotinylated peptide SLNPDTGLWE-L-biotin (SEQ ID NO: 15), wherein L is an optional linker;

[0067] - Secondary antibodies for sandwich immunoassays;

[0068] - a calibration protein comprising the N-terminal amino acid sequence SLNPDTGLWE (SEQ ID NO: 1);

[0069] - Antibody biotinylation kit;

[0070] -Antibody HRP labeling kit;

[0071] -Antibody radiolabeling kit; and

[0072] -Assay Visualization Kit.

[0073] The immunoassay kit according to the fourth aspect of the invention is particularly suitable for carrying out the method according to the second aspect of the invention. Hence, further preferred embodiments and features of the immunoassay kit according to the fourth aspect will be apparent from the above discussion of preferred embodiments of the method according to the second aspect. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1 : Alignment and specificity of the CILP-M assay.A) Sequence alignment of the targeting sequences of CILP-M in human (SEQ ID NO:21) and mouse (SEQ ID NO:22), bovine (SEQ ID NO:23) and rat (SEQ ID NO:24). The target sequence is highlighted and protease cleavage is marked with an arrow. B) Specificity of the CILP-M assay. Reactivity to standard peptide (SLNPDTGLWE) (SEQ ID NO:1), truncated peptide (NPDTGLWE) (SEQ ID NO:4), extended peptide (WSLNPDTGLWE) (SEQ ID NO:2) and nonsense standard peptide and coater (DSGPEYADVV) (SEQ ID NO:16). Signals are shown as relative luminescence per second (RLU) changes with standard peptide.

[0075] Figure 2 : In vitro cleavage of human articular cartilage by enzymes. CILP-M levels were measured in human articular cartilage cleaved by MMP1, MMP2, MMP3, MMP8, MMP9, MMP10, MMP12, MMP13, ADAMTS4, and ADAMTS5 using a CILP-M competitive chemiluminescent immunoassay.

[0076] Figure 3 : CILP-M levels and associated ROC curves in the discovery and validation cohorts. A) CILP-M levels in the discovery cohort included healthy donors (n=13), patients with rheumatoid arthritis (RA, n=18), patients with ankylosing spondylitis (AS, n=14), and patients with osteoarthritis (OA, n=8). B) ROC curve analysis evaluated the ability of CILP-M to distinguish healthy controls and RA / OA / AS, respectively, in the discovery cohort. C) CILP-M levels in the validation cohort included healthy donors (n=105), patients with RA (n=23), and patients with AS (n=89). D) ROC curve analysis evaluated the ability of CILP-M to distinguish healthy controls and RA / AS, respectively, in the validation cohort. Data were analyzed using ANCOVA test (adjusted for age and sex in the validation cohort) and receiver operating characteristic area under the curve (AUROC) analysis.

[0077] Figure 4 : CILP-M levels stratified into responders (n=26) and non-responders to anti-TNF-α therapy (n＝9) Response to anti-TNF-α treatment in AS patients at baseline (week 0) and after 12 weeks. Data are shown as Tukey box plots, and a logistic regression model was used to predict the response to anti-TNF-α based on a 50% reduction in the BASDAI index after 3 months of treatment. DETAILED DESCRIPTION

[0078] The embodiments disclosed herein are described in the following examples, which are set forth to help understand the present disclosure and should not be construed as limiting the scope of the present disclosure as 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 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 used (e.g., amounts, temperatures, etc.), 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.

[0079] Materials and methods

[0080] 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).

[0081] Table 1. Sequences of synthetic peptides used for monoclonal antibody production, assay development and validation

[0082] Peptide type sequence Immunogenic peptides SLNPDTGLWE-GGC-KLH*(SEQ ID NO:17) Select peptide SLNPDTGLWE (SEQ ID NO: 1) Extended select peptide WSLNPDTGLWE (SEQ ID NO: 2) Truncated select peptides NPDTGLWE (SEQ ID NO:4) Nonsense coating and standard peptides DSGPEYADVV (SEQ ID NO: 16)

[0083] *Keyhole limpet hemocyanin

[0084] Monoclonal Antibody Development, Production and Characterization

[0085] Amino acid sequence 700 '↓SLNPDTGLWE' 710(SEQ ID NO:1) was used to generate monoclonal antibodies (mAb). Immunization was initiated by subcutaneous injection of 200 μl of emulsified antigen and 100 μg of immunogenic peptide (SLNPDTGLWE-GGC-KLH) in 4- to 6-week-old Balb / C mice 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 separation of spleen 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. SLNPDTGLWE-K-biotin (SEQ ID NO: 18) was used as a screening peptide, while the standard peptide SLNPDTGLWE (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.

[0086] 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 (SLNPDTGLWE (SEQ ID NO: 1)) and not to recognize an extended or truncated sequence of one amino acid (WSLNPDTGLWE (SEQ ID NO: 2) and NPDTGLWE (SEQ ID NO: 14), respectively).

[0087] CILP-M Assay Development

[0088] The development of competitive chemiluminescent immunoassay (CLIA) included preliminary optimization experiments, in which reagents, concentrations, incubation times and incubation temperatures were analyzed by several tests. The CILP-M competitive ELISA procedure was as follows: 96-well streptavidin-coated white microplates (Greiner Bio-One, Kremsmünster, Austria) were coated with 2.5 ng / mL biotinylated synthetic peptide (SLNPDTGLWE-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 at 20°C in the dark with constant shaking (300 rpm) for 30 minutes. Next, the standard peptide (100ng / mL) and sample of 20 μ L / holes are added to appropriate holes, then the HRP-labeled antibody diluted to the concentration of 100ng / mL in the assay buffer of 100 μ L / holes is added, and incubated for 1 hour at 20 ℃ in the dark under continuous shaking (300rpm). After each incubation step, the holes are washed five times with standard wash buffer (20mM Tris, 50mM NaCl, pH 7.2). Chemiluminescent substrate (Roche, BM chemiluminescent ELISA substrate (POD), Basel, Switzerland) working solution is mixed before use for 15 minutes, and 100 μ L / holes are added to the plate, and incubated for 3 minutes at 20 ℃ in the dark under continuous shaking (300rpm). On microplate photometer reader (SpectraMax M5, Molecular Devices, CA, USA), the relative light units of all wavelengths are measured in 5 minutes. The standard curve was drawn using a 4-parameter logistic curve fit Y = (AD) / (1+(x / C)^B)+D with R>0.9. The data were analyzed using SoftMax Pro version 7.0.3 software.

[0089] Technology Assessment

[0090] The linearity of the immunoassay was assessed using two-fold dilutions of four human serum samples. Linearity was calculated as the percent recovery of undiluted samples. Antibody specificity was calculated as the percent signal inhibition of two-fold diluted standard peptides (SLNPDTGLWE) (SEQ ID NO: 11), extended peptides (WSLNPDTGLWE) (SEQ ID NO: 2), truncated peptides (LNPDTGLWE) (SEQ ID NO: 3) and nonsense peptides (DSGPEYADVV) (SEQ ID NO: 16). Five quality controls and two kit controls were run in duplicate assays to determine intra- and inter-assay variation. The accuracy of the assay was measured in healthy human serum samples spiked with standard peptides and serum samples with known high CILP-M concentrations, and was calculated as the percent recovery of the measured values ​​and the expected concentration of the peptide or serum sample with high CILP-M plus the concentration of the analyte in serum. Analytical interferences were performed by spiking serum samples of known concentrations with low / high levels of hemoglobin (2.50 / 5 mg / mL), lipids / lipids (1.50 / 5 mg / mL), and biotin (3 / 9 ng / mL). Percent recovery was calculated using normal serum samples as reference. Normal reference levels for hemoglobin, lipids / lipids, and biotin are 0-10 mg / dl (0-0.00161 mmol / L), <150 mg / dl (<1.6935 mmol / L), and 0.221-3.004 ng / ml, respectively. Interference was calculated as the percent recovery of the analyte in unspiked 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 from 10 independent runs using standard peptides. Measurements below the LLMR or above the ULMR were assigned the value of LLMR / ULMR, respectively. IC was determined from the standard curve 50 (half maximal inhibitory concentration). Analyte stability was checked by temperature testing and repeated freeze-thaw cycles of serum samples. The temperature testing included different time points and temperatures, where CILP-M 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. 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.

[0091] In vitro cleavage

[0092] Articular cartilage biopsy samples of OA patients undergoing knee replacement surgery were obtained from Gentofte Hospital Denmark. The collection and retrieval of human cartilage complied with international ethical guidelines for handling human samples and patient information. All participants signed informed consent, and the study was approved by the local ethics committee. Various enzymes known to cleave human articular cartilage were tested (4). The enzymes used were MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-10, MMP-12, MMP-13, ADAMTS4 and ADAMTS5. Cartilage cleavage was performed as follows; the crushed cartilage sample (30 mg) was incubated with 1 μg of each enzyme with 250 μL digestion buffer as described previously (4). Digestion was repeated for 24 h. The reaction was terminated by adding 5 mM EDTA. The cleavage products were measured using CILP-M ELISA.

[0093] Biological Evaluation of CILP-M ELISA

[0094] The biological utility of the CILP-M ELISA was evaluated in serum samples from the discovery cohort and validation cohort. The discovery cohort was obtained from the commercial supplier Proteogenex (Culver City, CA). The discovery cohort included a group of healthy donors (n = 13), patients diagnosed with RA (n = 18), patients diagnosed with AS (n = 14), and patients diagnosed with OA (n = 8). The validation cohort was collected at the University of Alberta, Canada, and included serum samples from patients diagnosed with RA (n = 23), patients diagnosed with AS (n = 89), and the results were compared with age-matched healthy donors (n = 105) from the commercial supplier BioIVT (Westerbury, NY, USA). The Bath AS Disease Activity Index (BASDAI) and the modified Stoke AS Spine Score (mSASSS) were monitored for each AS patient in the validation cohort. Samples from both cohorts were collected in accordance with the 1975 Declaration of Helsinki after informed consent and approval by the local ethics committee. Serum samples were obtained and stored at −80 °C until use.

[0095] Ethical Statement

[0096] All animals were handled according to the guidelines for animal care. The production of monoclonal antibodies in mice was approved by the Danish National Agency (Animal Experimentation Inspectorate) under approval number 2013-15-2934-00956.

[0097] Statistical analysis

[0098] For all statistical analyses performed, p values ​​below 0.05 were considered significant. Baseline characteristics were described as the number (frequency) and percentage of categorical variables, and the mean (± SD) of continuous variables. The Kruskal-Wallis rank test was used to examine baseline differences between participant groups. A one-way ANCOVA analysis was performed for both studies at baseline. An analysis of the area under the receiver operating characteristic curve (AUROC) was performed to study the discrimination accuracy of CILP-M at baseline between patients with AS or RA and healthy controls. The analysis of the validation cohort was adjusted for age and gender. A logistic regression model was used to predict the response of patients with AS to anti-TNF-α treatment (etanercept, adalimumab, infliximab, or golimumab), based on a 50% reduction in the BASDAI index after 3 months of treatment in the validation study (patients were biologically primitive before treatment).

[0099] Statistical analyses and graphs were performed using GraphPad Prism version 9 (GraphPad Software, Inc., La Jolla, CA) and R studio version 4.2.1 (R Foundation for Statistical Computing, Vienna, Austria. URL https: / / www.R-project.org).

[0100] result

[0101] Technical evaluation and characterization of CILP-M

[0102] The monoclonal antibody clone NB326#76 13B5-1C6-2C4 showed the best native reactivity, peptide affinity and assay stability and was selected for assay development. The isotype, sequence and CDR of this monoclonal antibody were determined. The sequence of the chain is as follows (CDRs are underlined and bold; N-terminal signal peptide and C-terminal constant region are italicized):

[0103] Heavy chain sequence (mouse IgG1 isotype)

[0104]

[0105]

[0106] Light chain sequence (mouse kappa isotype)

[0107]

[0108] A summary of the technical evaluation of the CILP-M assay can be found in Table 2. In brief, the measurement range (LLOQ-ULOQ) was determined to be 0.54-30.00 ng / mL. The inter- and intra-assay variations were 10.3% and 7.2%, respectively, and linearity was approved in human serum from undiluted to 2-fold dilution. The stability of the analyte was acceptable during long-term storage of human serum samples at 4°C and 20°C (95% and 92%, respectively) and three freeze-thaw cycles (92-119%). Hemoglobin, lipemia, and biotin did not interfere with the measurement of CILP-M in human serum.

[0109] Table 2. Summary of CILP-M technical parameters

[0110]

[0111] The human target sequence of CILP-M (SEQ ID NO: 21) was aligned with the corresponding mouse (SEQ ID NO: 22), bovine (SEQ ID NO: 23), and rat (SEQ ID NO: 24) sequences using UNIPROT. The corresponding target sequences of mouse and bovine were 100% aligned with the human sequence, while the rat sequence had a mismatch at position 5 ( Figure 1 A). To evaluate the specificity of the CILP-M assay, mAbs were tested against extended peptides, truncated peptides, nonsense standard peptides, and nonsense coatings and showed no reactivity to these peptides ( Figure 1 B).

[0112] To investigate the enzymes responsible for CILP-M production, human articular cartilage was lysed using a panel of enzymes. It was observed that CILP-M was produced primarily by MMP-1, MMP-8, and MMP-12 ( Figure 2 ).

[0113] Baseline demographic and clinical characteristics

[0114] Tables 3 and 4 show the demographic characteristics of the two cohorts. In the discovery cohort, significant differences were found in age between the groups (p<0.0001). The significant difference was driven by OA patients, who were on average 30 years older than healthy donors, RA, and AS patients in this cohort. There was no difference in gender distribution between the groups. In the validation cohort, there were also significant differences in age between the groups (p<0.0001), with RA patients on average 15 years older than healthy donors and AS patients. In addition, there were more males in the RA and AS groups compared with healthy donors (p<0.0001).

[0115] Table 3. Patient demographics of the discovery cohort

[0116]

[0117] Categorical variables are written as numbers (percentages), while continuous variables are means (standard deviations). The Kruskal-Wallis rank test was used to compare differences between groups. Abbreviations: BMI, body mass index.

[0118] Table 4. Patient Demographics of the Validation Cohort

[0119]

[0120] Categorical variables are written as numbers (percentages), whereas continuous variables are means (standard deviations). The Kruskal-Wallis rank test was used to compare differences between the groups. Abbreviations: CRP, C-reactive protein; ESR, erythrocyte sedimentation rate; BASDAI, Bath Ankylosing Spondylitis Disease Activity Index; mSASSS, modified Stoke Ankylosing Spondylitis Spine Score

[0121] CILP-M is upregulated in rheumatic diseases in the discovery and validation cohorts

[0122] The levels of CILP-M were measured in two independent cohorts. The discovery cohort consisted of healthy donors and patients diagnosed with RA, AS, and OA. Here, RA, AS, and OA patients showed significantly higher levels of CILP-M compared to healthy donors (p=0.001, p=0.0007, p=0.006, respectively). Figure 3 A). No differences were found between the patient groups. The diagnostic efficacy (AUROC) of CILP-M was 0.966 (95% CI = 0.910-1.000, p < 0.0001) for patients with RA compared with healthy donors, 0.962 (95% CI = 0.896-1.000, p < 0.0001) for patients with AS compared with healthy donors, and 0.971 (95% CI = 0.911-1.000, p = 0.0004) for patients with OA compared with healthy donors. Figure 3 As shown in B.

[0123] The validation cohort included healthy donors and patients diagnosed with RA or AS. Here, patients with RA and AS demonstrated higher CILP-M levels in patients with rheumatic diseases (p < 0.0001 for both, Figure 3 C). The diagnostic efficacy (AUROC) of CILP-M was 0.936 (95% CI = 0.894-0.978, p < 0.0001) for patients with RA compared with healthy donors, and 0.955 (95% CI = 0.926-0.984, p < 0.0001) for patients with AS compared with healthy donors. Figure 3 As shown in D.

[0124] In the validation cohort, 67 patients with AS were treated with anti-TNF-α therapy, and 35 of the 67 patients had BASDAI measurements before and after treatment. In order to investigate the response to treatment based on a 50% reduction in BASDAI, CILP-M levels were measured in the 35 patients for whom BASDAI information was available. It was found that the likelihood of responding to anti-TNF-α treatment showed a higher trend for a unit increase in CILP-M at baseline (OR = 3.28, 95% CI = [0.85; 12.64], p value = 0.07, Figure 4 ).

[0125] discuss

[0126] Applicants developed and characterized a competitive ELISA for detecting CILP-M using monoclonal antibodies targeting MMP-generated CILP-1 neoepitopes. The main findings of the examples are: 1) a technically robust and specific assay for CILP-M neoepitopes generated primarily by MMP1, MMP8, and MMP12 was developed; 2) CILP-M is measurable in human serum, and levels of CILP-M are increased in patients with RA, AS, and OA, as shown in the discovery and validation cohorts; and 3) CILP-M shows the ability to discriminate between responders and non-responders to anti-TNF-α therapy.

[0127] The CILP-M assay was characterized as a technically robust and accurate assay by showing acceptable dilution recovery, interference, and stability tests. Inter- and intra-variances of 10.3% and 7.2%, respectively, were received. The epitope CILP-M target was previously discovered by mass spectrometry of human articular cartilage (4), which is primarily produced by ADAMTS5. In order to confirm which protease produces the CILP-M cleavage site, a series of proteases were tested. Compared with previous findings, our in vitro cleavage showed that the CILP-M epitope is produced by MMP-1, MMP-8, and MMP-12. Destruction and fusion of articular cartilage are key features in rheumatic diseases (11-13). When joint damage occurs and cartilage degradation occurs, serine proteases and MMPs are highly expressed in the extracellular matrix (13). ADAMTS4 and ADAMTS5 are considered to be the main aggrecan degrading enzymes in cartilage (3), while MMPs can degrade all components of the ECM (14). MMP-1 and MMP-8 are the two most classical collagenases that are most important for collagen degradation within cartilage (14). MMP-1 is produced primarily by synovial cells at the margins of joints and plays a major role in OA as it controls the process of collagen degradation (15). High levels of CILP-1 have been found in synovial tissue (10), which can be further cleaved by MMP-1. MMP-8 is expressed at sites of inflammation and has been associated with a variety of inflammatory conditions (13, 16), while MMP-12 is secreted by inflammatory macrophages (17), and increased MMP-12 expression in macrophages has been found to exacerbate the development of inflammatory arthritis in rabbits (18).

[0128] To the best of the applicant's knowledge, this is the first study to study the cleavage products of CILP-1 in rheumatic diseases and the first study to detect the epitope of CILP-1 in blood-based samples. The study found that compared with healthy donors, CILP-M levels were increased in patients with RA, AS and OA. In previous studies, CILP-1 was associated with cartilage degenerative diseases, particularly OA (6, 7). Studies have also shown that anti-CILP antibodies are found in patients with OA and RA, indicating that autoimmune responses to CILP are associated with the pathogenesis of OA and RA (19). To the best of the applicant's knowledge, this is the first study to study the role of CILP-1 in patients with AS. The present findings are consistent with previous studies that have shown that patients with AS have higher extracellular matrix remodeling in cartilage (20, 21). However, CILP-1 is also associated with pulmonary hypertension and cardiac fibrosis, suggesting that it is not limited to articular cartilage (22, 23).

[0129] As better tools are needed to monitor the response to treatment in patients with AS, we also investigated whether CILP-M could predict response to anti-TNF-α, and we observed that patients who responded to anti-TNF-α treatment had higher levels of CILP-M before treatment.

[0130] In conclusion, a novel neo-epitope biomarker, CILP-M, was developed to measure MMP-1-, MMP-8-, and MMP-12-mediated CILP-1 fragments and validated for use in human serum samples. CILP-M was upregulated in patients with RA and AS in two independent cohorts. Based on these results, CILP-M may be used to assess cartilage remodeling in joint degenerative diseases.

[0131] 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 acknowledgment of any prior published document herein is not to be taken as an acknowledgment or representation that the teachings therein were common general knowledge in Australia or elsewhere as of the date of the application.

[0132] References

[0133] 1. Sophia Fox AJ, Bedi A, Rodeo SA. The Basic Science of Articular Cartilage: Structure, Composition, and Function. Sports Health [Internet] SAGE Publications; 2009 [cited March 9, 2022]; 1: 461. Available from: / pmc / articles / PMC3445147 /

[0134] 2. Lorenzo P, Bayliss MT, D. A Novel Cartilage Protein (CILP) Present in the Mid-zone of Human Articular Cartilage Increases with Age*. 1998 [cited July 19, 2022]; available from: http: / / www.jbc.org

[0135] 3. Goldring MB, Marcu KB. Cartilage homeostasis in health and rheumatic diseases. Arthritis Res Ther [Internet] BioMed Central; 2009 [cited July 19, 2022]; 11: 224. Available from: / pmc / articles / PMC2714092 /

[0136] 4. Zhen EY, Brittain IJ, Laska DA, Mitchell PG, Sumer EU, Karsdal MA, et al. Characterization of metalloprotease cleavage products of human articular cartilage. Arthritis Rheum [Internet] Arthritis Rheum; 2008 [cited 2022 Jul 19]; 58: 2420-31. Available from: https: / / pubmed.ncbi.nlm.nih.gov / 18668564 /

[0137] 5.Lorenzo P,Bayliss MT, D. Altered patterns and synthesis of extracellular matrix macromolecules in early osteoarthritis. Matrix Biol 2004; 23: 381-91.

[0138] 6. Valdes AM, Hart DJ, Jones KA, Surdulescu G, Swarbrick P, Doyle CV, et al. Association study of candidate genes for the prevalence and progression of knee osteoarthritis. Arthritis Rheum [Internet] John Wiley & Sons, Ltd; 2004 [cited 19 Jul 2022]; 50: 2497-507. Available from: https: / / onlinelibrary.wiley.com / doi / full / 10.1002 / art.20443

[0139] 7. Valdes AM, Van Oene M, Hart DJ, Surdulescu GL, Loughlin J, Doherty M, et al. Reproducible genetic associations between candidate genes and clinical knee osteoarthritis in men and women. Arthritis Rheum [Internet] John Wiley & Sons, Ltd; 2006 [cited 2022 Jul 19]; 54: 533-9. Available from: https: / / onlinelibrary.wiley.com / doi / full / 10.1002 / art.21621

[0140] 8. Seki S, Kawaguchi Y, Chiba K, Mikami Y, Kizawa H, Oya T, et al. A functional SNP in CILP, encoding cartilage intermediate layer protein, is associated with susceptibility to lumbar disc disease. Nat Genet [Internet] Nature Publishing Group; 2005 [cited 2022 Jul 19]; 37: 607-12. Available from: https: / / u-toyama.elsevierpure.com / en / publications / a-functional-snp-in-cilp-encoding-cartilage-intermediate-layer-pr

[0141] 9. Tsuruha JI, Masuko-Hongo K, Kato T, Sakata M, Nakamura H, Nishioka K, et al. Implication of Cartilage Intermediate Layer Protein in Cartilage Destruction in Subsets of Patients With Osteoarthritis and Rheumatoid Arthritis. ARTHRITIS Rheum 2001; 44: 838-45.

[0142] 10. Kerna I, Kisand K, Tamm A, Tamm A. Cartilage intermediate layer protein is produced in synovial membrane of osteoarthritic joint and upregulated inosteoarthritis associated fibrosis. Bone Abstr [Internet] BioScientifica; 2013 [cited 2022 Jul 19]; 1. Available from: http: / / www.bone-abstracts.org / ba / 0001 / ba0001pp15

[0143] 11. Park DR, Kim J, Kim GM, Lee H, Kim M, Hwang D, et al. Osteoclast-associated receptor blockade prevents articular cartilage destruction via chondrocyte apoptosis regulation. Nat Commun 2020 111 [Internet] Nature Publishing Group; 2020 [cited 2022 Jul 19]; 11: 1-11. Available from: https: / / www.nature.com / articles / s41467-020-18208-y

[0144] 12. Bleil J, Sieper J, Maier R, Schlichting U, Hempfing A, Syrbe U, et al. Cartilage in facet joints of patients with ankylosing spondylitis (AS) shows signs of cartilage degeneration rather than chondrocyte hypertrophy: Implications for joint remodeling in AS. Arthritis Res Ther [Internet] BioMedCentral Ltd.; 2015 [cited May 31, 2021]; 17. Available from: / pmc / articles / PMC4506408 /

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[0147] 15. A, Przybyszewski M, Graczyk M, Bartuzi Z. The role of extracellular matrixmetalloproteinases and their inhibitors in allergic diseases. Postedermatologiii Alergol 2012; 29: 384-9.

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[0149] 17. Chen YE. MMP-12, An Old Enzyme Plays a New Role in the Pathogenesis of Rheumatoid Arthritis? Am J Pathol [Internet] American Society for Investigative Pathology; 2004 [cited 2022 Aug 3]; 165:1069. Available from: / pmc / articles / PMC1618647 /

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Claims

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

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

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

4. The monoclonal antibody according to any one of claims 1 to 3, wherein The monoclonal antibody does not specifically bind to a peptide having the N-terminal amino acid sequence NPDTGLWE (SEQ ID NO: 4).

5. The monoclonal antibody 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 SLNPDTGLWE (SEQ ID NO: 1).

6. An immunoassay method comprising: i) contacting the patient sample with a monoclonal antibody that specifically binds to the N-terminal amino acid sequence SLNPDTGLWE (SEQ ID NO: 1); ii) detecting and determining the amount of binding between the monoclonal antibody and the peptide in the sample.

7. The method according to claim 6, wherein: The method is a method for detecting and / or monitoring a disease in a patient and / or assessing the likelihood that the patient will respond to treatment with a drug to treat the disease, the method further comprising: iii) correlating said bound amount to a value associated with a normal healthy subject, and / or a value associated with a known disease severity, and / or a value obtained from said patient at a previous time point, and / or a predetermined cut-off value.

8. The method according to claim 7, wherein: The disease is a joint disease.

9. The method according to claim 8, wherein: The joint disease is rheumatoid arthritis, ankylosing spondylitis or osteoarthritis.

10. The method according to any one of claims 7 to 9, wherein: The treatment of the disease using drugs is anti-TNF-α therapy.

11. The method according to any one of claims 6 to 10, wherein: The monoclonal antibody does not specifically bind to a peptide having the N-terminal amino acid sequence WSLNPDTGLWE (SEQ ID NO: 2).

12. The method according to any one of claims 6 to 11, wherein: The monoclonal antibody does not specifically bind to a peptide having the N-terminal amino acid sequence LNPDTGLWE (SEQ ID NO: 3).

13. The method according to any one of claims 6 to 12, wherein: The monoclonal antibody does not specifically bind to a peptide having the N-terminal amino acid sequence NPDTGLWE (SEQ ID NO: 4).

14. The method according to any one of claims 6 to 13, wherein: The monoclonal antibody was raised against a synthetic peptide having the N-terminal amino acid sequence SLNPDTGLWE (SEQ ID NO: 1).

15. The method according to any one of claims 6 to 14, wherein: The patient sample is selected from blood, serum or plasma.

16. The method according to any one of claims 6 to 15, wherein: The immunoassay is a competition assay or a sandwich assay.

17. The method according to any one of claims 6 to 16, wherein: The immunoassay is a radioimmunoassay or an enzyme-linked immunosorbent assay.

18. An immunoassay kit comprising a monoclonal antibody that specifically binds to the N-terminal amino acid sequence SLNPDTGLWE (SEQ ID NO: 1), and at least one of the following: - Streptavidin-coated well plates; - Biotinylated peptide SLNPDTGLWE-L-biotin (SEQ ID NO: 15), wherein L is an optional linker; - Secondary antibodies for sandwich immunoassays; - a calibration protein comprising the N-terminal amino acid sequence SLNPDTGLWE (SEQ ID NO: 1); - Antibody biotinylation kit; -Antibody HRP labeling kit; -Antibody radiolabeling kit; and -Assay Visualization Kit.

19. The immunoassay kit according to claim 18, wherein The monoclonal antibody does not specifically bind to a peptide having the N-terminal amino acid sequence WSLNPDTGLWE (SEQ ID NO: 2).

20. The immunoassay kit according to claim 18 or 19, wherein The monoclonal antibody does not specifically bind to a peptide having the N-terminal amino acid sequence LNPDTGLWE (SEQ ID NO: 3).

21. The immunoassay kit according to any one of claims 18 to 20, wherein The monoclonal antibody does not specifically bind to a peptide having the N-terminal amino acid sequence NPDTGLWE (SEQ ID NO: 4).

22. The immunoassay kit according to any one of claims 18 to 21, wherein The monoclonal antibody was raised against a synthetic peptide having the N-terminal amino acid sequence SLNPDTGLWE (SEQ ID NO: 1).

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