C3F assay

Abstract

This document discloses an immunoassay method for detecting fibroblast-activating protein (FAP)-producing fragments of type III collagen in patient samples, and its use for detecting and / or monitoring cancer or arthritis or a specific level of their severity in patients. It also describes monoclonal antibodies and assay kits for use with the immunoassay method.

Description

[Technical Field]

[0001] The present invention relates to an immunoassay method for detecting fibroblast-activating protein (FAP)-producing fragments of type III collagen in patient samples, and to the use thereof for detecting and / or monitoring cancer or arthritis or a specific level of their severity in patients. The present invention also relates to monoclonal antibodies and assay kits for use in said immunoassay method. [Background technology]

[0002] In the United States alone, it is estimated that there will be more than 230,000 new cases of lung cancer and more than 130,000 deaths from lung cancer in 2022, indicating that lung cancer remains one of the leading causes of cancer-related deaths. [1] . The tumor microenvironment (TME) is well-established as crucial for tumor progression and patient survival, and therefore, interest in TMEs for the investigation and treatment of various cancers is increasing. [2-5] TME includes the extracellular matrix (ECM), tumor cells, immune cells, and stromal cells. [6] . Cancer-associated fibroblasts (CAFs) are a major component of tumor stroma. [6,7] CAFs are involved in cancer progression, immunosuppression, and ECM reconstitution. [8-10] CAFs affect ECM remodeling by excessive synthesis of collagen and other ECM components, and by secreting metalloproteinases (MMPs). [10,11] .

[0003] Fibroblast-activating protein (FAP) is a type II transmembrane serine protease that is expressed almost exclusively in the interstitium under pathological conditions including arthritis, fibrosis, and cancer, and is one of the major biological markers of CAF. [3,12,13] . Several studies have investigated the diagnostic and prognostic value of FAP. [2,14-17]The results of these studies were somewhat inconclusive regarding the general prognostic value of FAP, but FAP has shown potential as a prognostic marker for outcomes in lung, gastric, and colorectal cancers. [16-18] FAP has also been shown to be involved in ECM remodeling and fibrosis.

[19] . For example, excessive ECM degradation by MMP-mediated cleavage during tumor progression generates protein fragments that are released into the circulation. These fragments can be non-invasively evaluated as a liquid biopsy and quantified as disease biomarkers. [20,21] Since FAP has been shown to cleave ECM components including type I and type III collagens, fragments derived from these cleavages can be expected to appear in the circulation. [12,22] . FAP belongs to the dipeptidyl peptidase 4 (DPP4) protein family and, like other enzymes in the DPP4 family, FAP has dipeptidyl peptidase activity. Furthermore, FAP also has endopeptidase activity, which distinguishes FAP from other DPP4 proteins and leads to potential unique cleavage targets. [3,13] . Zhang et al. used terminal amine isotope labeling of substrates (TAILS) on mouse embryonic fibroblast substrates to identify the natural substrates of human FAP. Using this degradomics approach, Zhang et al. identified multiple potential FAP cleavage sites in various ECM-related proteins containing 1069 AGPSGAPGPA 1078 (SEQ ID NO: 20).

[22] . SUMMARY OF THE INVENTION

[0004] The inventors have now found that the amino acid residues P 1069 and A 1070We developed an enzyme-linked immunosorbent assay (ELISA) targeting a fragment of the alpha-1 chain of human type III collagen, which is produced by cleavage of the alpha-1 chain between and . This fragment may serve as a biomarker for FAP activity in the extracellular matrix (ECM). We further measured the levels of this biomarker in a lung cancer cohort and compared the levels in patients with non-small cell lung cancer (including adenocarcinoma and squamous cell carcinoma) with those in healthy individuals. We also measured the levels of this biomarker in patients with spondyloarthritis. We demonstrated that the levels of this biomarker were elevated in patients with cancer and elevated in patients with arthritis.

[0005] Therefore, in a first aspect, the present invention provides an immunoassay method comprising contacting a sample of a patient with a monoclonal antibody that specifically binds to a fibroblast-activating protein (FAP)-producing neoepitope of the alpha-1 chain of human type III collagen FAP-producing fragment, and detecting the binding between the monoclonal antibody and a peptide in the sample and determining the amount of binding, wherein the FAP-producing neoepitope consists of an N-terminal sequence or a C-terminal sequence of the FAP-producing fragment located at the end of the FAP-producing fragment cleaved by FAP. In a preferred embodiment, the immunoassay method includes the following steps: i) Contacting the patient's sample with a monoclonal antibody that specifically binds to the N-terminal amino acid sequence AGPAGAPGPA (SEQ ID NO: 1) (the sequence is also referred to herein as the “target sequence” and / or “C3F”); and ii) Detect the binding between the monoclonal antibody and the peptide in the sample, and determine the amount of binding.

[0006] In a preferred embodiment, the immunoassay method is an immunoassay method for detecting and / or monitoring cancer or a specific level of cancer severity in a patient, the method further comprising correlating the binding amount with a value associated with a normal healthy person and / or a value associated with a known disease severity and / or a value obtained from the patient at a past point in time and / or a predetermined cutoff value. In a preferred embodiment, the cancer is lung cancer. In a preferred embodiment, the lung cancer is non-small cell lung cancer (NSCLC).

[0007] In another preferred embodiment, the immunoassay method is an immunoassay method for detecting and / or monitoring arthritis or a specific level of severity of arthritis in a patient, the method further comprising correlating the binding amount with a value associated with a normal healthy person and / or a value associated with a known disease severity and / or a value obtained from the patient at a past point in time and / or a predetermined cutoff value. In a preferred embodiment, the arthritis is spondyloarthritis. In a preferred embodiment, the patient sample is a human biological fluid sample. Preferably, the sample is a blood material sample such as blood (whole blood), plasma, or serum.

[0008] In a preferred embodiment, the monoclonal antibody does not specifically bind to the peptide having the N-terminal amino acid sequence PAGPAGAPGPA (SEQ ID NO: 2) (i.e., the extended version of the target sequence, which is extended by adding a proline residue to the N-terminal position). Preferably, the ratio of the antibody's affinity to the target sequence to the antibody's affinity to the extended version of the target sequence is at least 10:1, more preferably at least 20:1, at least 30:1, at least 40:1, at least 50:1, or at least 100:1. In a preferred embodiment, the monoclonal antibody does not specifically bind to the peptide having the N-terminal amino acid sequence GPAGAPGPA (SEQ ID NO: 3) (i.e., the truncated target sequence, which is truncated by removing the first alanine residue at the N-terminal). Preferably, the ratio of the antibody's affinity to the target sequence to the antibody's affinity to the truncated target sequence is at least 10:1, more preferably at least 20:1, at least 30:1, at least 40:1, at least 50:1, or at least 100:1.

[0009] In a preferred embodiment, the monoclonal antibody is produced by immunizing a synthetic peptide having the N-terminal amino acid sequence AGPAGAPGPA (SEQ ID NO: 1). For example, the monoclonal antibody may be produced by: (a) immunizing a rodent (or other suitable mammal) with a synthetic peptide containing the N-terminal sequence AGPAGAPGPA (SEQ ID NO: 1), which may optionally be linked at its C-terminal position to an immunogenic carrier protein (such as keyhole limpet hemocyanin ("KLH")); (b) isolating and cloning a single antibody-producing cell; and (c) assaying the resulting monoclonal antibody to confirm that it has the desired specificity. Exemplary protocols for the development, production, and characterization of suitable monoclonal antibodies are described in the following Examples section. In preferred embodiments, the immunoassay is a competitive assay or a sandwich assay. The immunoassay may be, for example, a radioimmunoassay or an enzyme-linked immunosorbent assay (ELISA). Such assays are known to those skilled in the art.

[0010] As used herein, the term “N-terminal” refers to the N-terminal peptide sequence at the tip of a polypeptide, i.e., the peptide sequence at the N-end of the polypeptide, and should not be interpreted as referring to its general direction. As used herein, the term “C-terminal” refers to the C-terminal peptide sequence at the tip of a polypeptide, i.e., the peptide sequence at the C-end of the polypeptide, and should not be interpreted as referring to its general direction. As used herein, the terms “peptide” and “polypeptide” are used synonymously.

[0011] As used herein, the term “monoclonal antibody” refers to both the whole antibody and its fragments that retain the binding specificity of the whole antibody, such as Fab fragments, F(ab')2 fragments, single-chain Fv fragments, or other such fragments known to those skilled in the art. As is well known, a whole antibody typically has a “Y-shaped” structure in which two identical polypeptide chain groups are paired, with each paired element consisting of one “light” chain and one “heavy” chain. The N-terminal regions of the light and heavy chains each contain a variable region, and the C-terminal portions of the heavy and light chains each constitute a constant region. The variable region contains three complementarity-determining regions (CDRs), which are primarily responsible for antigen recognition. The constant region allows the antibody to recruit cells and molecules of the immune system. Antibody fragments that retain binding specificity include the CDRs and a portion of the remainder of the variable region sufficient to retain binding specificity.

[0012] In the present invention, any monoclonal antibody containing a constant region known in the art can be used. In the case of mouse antibodies and human antibodies, the constant light chain is classified into a kappa light chain or a lambda light chain. The constant heavy chain is classified into mu, delta, gamma, alpha or epsilon, and each of IgM, IgD, IgG, IgA and IgE is defined as an isotype of the antibody. The IgG isotype has several subclasses including IgG1, IgG2, IgG3 and IgG4 in the case of humans, and has several subclasses including IgG1, IgG2a, IgG2b, IgG2c and IgG3 in the case of mice, but is not limited thereto. The monoclonal antibody may preferably belong to an IgG isotype containing any one of the IgG subclasses.

[0013] The CDRs of the antibody can be determined using methods known in the art as described by Kabat et al. The antibody can be produced from B cell clones. The isotype of the antibody can be determined by ELISA specific for the IgM, IgG or IgA isotype, or subclass. The amino acid sequence of the produced antibody can be determined using standard techniques. For example, RNA can be isolated from cells and used to generate cDNA by reverse transcription. The cDNA is then PCR processed using primers that amplify the heavy and light chains of the antibody. For example, a primer specific for the leader sequence for all VH (variable heavy chain) sequences can be used together with a primer that binds to a sequence located in the constant region of a pre-determined isotype. The light chain can be amplified using a primer that binds to the 3' end of the kappa or lambda chain together with a primer that anneals to the leader sequence of V kappa or V lambda. The full-length heavy and light chains can be produced and sequenced.

[0014] In certain exemplary embodiments, the monoclonal antibody may preferably comprise one or more complementarity determining regions (CDRs) selected from the following: CDR-L1: KSSQSLVYSDGKTYMN (SEQ ID NO: 4) CDR-L2: LVSKLDS (SEQ ID NO: 5) CDR-L3: WQGTHSPLT (SEQ ID NO: 6) CDR-H1: SYWMN (SEQ ID NO: 7) CDR-H2: QIYPGTGDTNYNGKFKG (SEQ ID NO: 8) CDR-H3: ITTAYYFDY (SEQ ID NO: 9). Preferably, the monoclonal antibody comprises at least two, three, four, five or six of the CDR sequences listed above.

[0015] Preferably, the monoclonal antibody has a light chain variable region comprising the following CDR sequences: CDR-L1: KSSQSLVYSDGKTYMN (SEQ ID NO: 4) CDR-L2: LVSKLDS (SEQ ID NO: 5), and CDR-L3: WQGTHSPLT (SEQ ID NO: 6). Preferably, the monoclonal antibody has a light chain comprising the framework sequences between the CDRs, and the framework sequences are substantially identical or substantially similar to the framework sequences between the CDRs in the following light chain sequence (the CDRs are shown in bold and underlined, and the framework sequences are shown in italics).

[0016]

Chemical Structure

[0017] <值="0000116">Preferably, the monoclonal antibody has a heavy chain variable region comprising the following CDR sequences: CDR-H1: SYWMN (SEQ ID NO: 7) CDR-H2: QIYPGTGDTNYNGKFKG (SEQ ID NO: 8), and CDR-H3: ITTAYYFDY (SEQ ID NO: 9). Preferably, the monoclonal antibody has a heavy chain containing a framework sequence between CDRs, the framework sequence being substantially identical or substantially similar to the framework sequence between CDRs in the heavy chain sequence below (CDRs are shown in bold and underline, and framework sequences are shown in italics).

[0018] [ka]

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

[0020] [ka]

[0021] (CDR is in bold and underlined; framework array is in italics) and / or heavy chain variable region sequence:

[0022] [ka]

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

[0024] As used herein, the amino acid sequences of the framework between the CDRs of an antibody are "substantially identical" or "substantially similar" to the amino acid sequences of the framework between the CDRs of another antibody if they have at least 70%, 80%, 90%, or at least 95% similarity or identity to the amino acid sequences of the framework between the CDRs of another antibody. Similar or identical amino acids may be consecutive or discontinuous. A framework sequence may contain one or more amino acid substitutions, insertions, and / or deletions. Amino acid substitutions may be conservative substitutions, meaning that the substituted amino acid has similar chemical properties to the original amino acid. Those skilled in the art will understand which amino acids share similar chemical properties. For example, the following groups of amino acids share similar chemical properties in terms of size, charge, polarity, etc.: Group 1 Ala, Ser, Thr, Pro, Gly; Group 2 Asp, Asn, Glu, Gln; Group 3 His, Arg, Lys; Group 4 Met, Leu, Ile, Val, Cys; Group 5 Phe, Thy, Trp.

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

[0026] As used herein, the term “bound amount” refers to the quantification of binding between the antibody and the peptide in the patient sample. This quantification may be determined, for example, by comparing the measured binding amount in the patient sample with a calibration curve prepared using measured binding amounts in a standard sample containing the peptide to which the antibody specifically binds at a known concentration, in order to determine the amount of peptide to which the antibody specifically binds in the patient sample. Any analytical method suitable for measuring the bound amount can be used. For example, ELISA can be used, in which spectrophotometric analysis is used to measure both the bound amount in the patient sample and the bound amount when a calibration curve is prepared.

[0027] As used herein, the term “predetermined cutoff value” means a statistically determined binding amount that indicates a high likelihood that a patient has a disease (e.g., cancer or arthritis) or that the disease is of a particular severity, and in that sense, if the measured value of the target peptide in the patient sample is greater than or equal to the statistical cutoff value, then the measured value corresponds to a probability of at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95% that the disease is present or of a particular severity.

[0028] As used herein, the term “value associated with normal healthy individuals” means the standardized binding amount determined by the method described above for samples obtained from subjects considered to be healthy, i.e., disease-free (i.e., not cancerous or arthritis); and the term “value associated with known disease severity” means the standardized binding amount determined by the method described above for samples obtained from patients known to have a disease of known severity (i.e., cancer or arthritis).

[0029] In a second embodiment, the present invention provides a method relating to cancer in patients requiring treatment, comprising the following steps: (a) Performing an immunoassay on a sample obtained from a patient in accordance with a first aspect of the present invention to detect whether it is cancerous or whether its severity is at a specific level; and (b) If it is determined in step (a) that the patient has cancer or that the severity of the cancer is at the specific level, the patient is given a treatment to treat the cancer.

[0030] The aforementioned therapy may be any therapy appropriate for the treatment of the target cancer. The therapy may include, for example, one or more surgeries, one or more radiation therapies, one or more drugs (e.g., one or more chemotherapy, one or more immunotherapies, and / or one or more hormone therapies), or a combination thereof, or consist of such therapies. The medication may be prescribed for topical or systemic administration. Topical medications may be prescribed in dosage forms such as creams, foams, gels, lotions, or ointments. Systemic medications may be prescribed in dosage forms such as enteral or parenteral administration. The surgery may be curative, prophylactic, debulking, palliative, and / or reconstructive surgery.

[0031] For example, if the cancer is lung cancer (e.g., NSCLC), appropriate therapy may include, for example, one or more of the following therapies: surgery such as lobectomy, segmentectomy (wedge resection), or pneumonectomy (lung resection); and radiotherapy. Specific examples include, but are not limited to, radiotherapy combined with chemotherapy, radiotherapy performed after surgery, brachytherapy (localized radiotherapy), prophylactic whole-brain irradiation, stereotactic radiotherapy, and palliative radiotherapy. ; for example, chemotherapy using one or more agents such as cisplatin, carboplatin, etoposide, gemcitabine, paclitaxel, docetaxel, vinorelbine, topotecan, irinotecan, and pemetrexed; epidermal growth factor receptor (EGFR) inhibitors such as erlotinib, gefitinib, afatinib, dacomitinib, or osimertinib; targeted therapy using one or more drugs such as crizotinib, and For example, immunotherapy using one or more monoclonal antibodies such as anti-PD-L1 monoclonal antibodies like atezolizumab, nivolumab, or pembrolizumab, monoclonal antibodies targeting cytotoxic T cell-associated protein 4 (CTLA-4) such as ipilimumab, and / or monoclonal antibodies targeting vascular endothelial growth factor such as bevacizumab, nesitumumab, mobocertinib, or cetuximab.

[0032] In a third embodiment, the present invention provides a method relating to arthritis in a patient requiring treatment, comprising the following steps: (a) Perform an immunoassay on a sample obtained from a patient in accordance with a first aspect of the present invention to detect whether it is arthritis and whether its severity is at a specific level; and (b) If it is determined in step (a) that the patient has arthritis or that the severity of the arthritis is at the specific level, the patient shall be given therapy to treat the arthritis.

[0033] Any therapy may be used as long as it is appropriate for treating the arthritis in question. Therapy may include, for example, one or more surgeries, one or more physiotherapy treatments, one or more medications, or a combination thereof, or consist of such therapies. Drugs may be prescribed for topical or systemic administration. Topical drugs may be prescribed in the form of, for example, creams, foams, gels, lotions, or ointments. Systemic drugs may be prescribed in the form of, for example, enteral or parenteral administration. For example, if the arthritis is spondyloarthritis, appropriate therapy may include, for example, one or more immunosuppressants, such as one or more tumor necrosis factor (TNF) inhibitors.

[0034] In a fourth embodiment, the present invention provides a monoclonal antibody that specifically binds to the N-terminal amino acid sequence AGPAGAPGPA (SEQ ID NO: 1). The antibody according to the fourth embodiment of the invention is particularly suitable for use when carrying out the immunoassay method according to the first embodiment of the invention. Therefore, preferred embodiments and features of the antibody according to the third embodiment are evident from the above discussion concerning preferred embodiments of the method according to the first embodiment.

[0035] In a fifth aspect, the present invention provides a monoclonal antibody according to the fourth aspect of the invention, and an immunoassay kit comprising at least one of the following: - Streptoavidin-coated well plates -Biotinylated peptide AGPAGAPGPA-L-biotin (SEQ ID NO: 14), where L is an optional linking group. - Second antibody used in sandwich immunoassay Calibration protein containing the N-terminal amino acid sequence AGPAGAPGPA (SEQ ID NO: 1) - Antibody biotinylation kit - Antibody HRP labeling kit - Antibody radiolabeling kit. The immunoassay kit according to the invention of the fifth aspect is particularly suitable for use in carrying out the immunoassay method according to the invention of the first aspect. Therefore, more preferred embodiments and features of the immunoassay kit according to the fifth aspect are apparent from the above considerations regarding the preferred embodiments of the method according to the first aspect.

Brief Description of the Drawings

[0036] [Figure 1] Figure 1: Specificity of C3F ELISA. (A) Specificity of C3F antibody and assay for 10 - amino acid selected peptide (AGPAGAPGPA (SEQ ID NO: 1)), 30 - amino acid selected peptide (AGPAGAPGPAGSRGAPGPQGPRGDKGETGE (SEQ ID NO: 19)), truncated peptide (GPAGAPGPA (SEQ ID NO: 3)), and extended peptide (PAGPAGAPGPA (SEQ ID NO: 2)). (B) Specificity of C3F ELISA for FAP - cleaved type III collagen shown by C3F measurement of collagen III solution after incubation with FAP for 24 hours. [Figure 2] Figure 2: Specificity of C3F ELISA for FAP - cleaved type III collagen shown by (A) C3F measurement and (B) C3M measurement of collagen III solution after incubation with FAP or MMP9 for 24 hours. [Figure 3] Figure 3: C3F and C3M levels in serum of patients with lung cancer. Quantification, aggregation (A - B) of C3F (A and C) and C3M (B and D) in serum from healthy subjects (n = 26) and patients with lung cancer (n = 40), and classification into patients with adenocarcinoma (n = 26) or squamous cell carcinoma (n = 14) (C - D). Biomarker levels between groups are compared using Mann - Whitney test (A - B) and Kruskal - Wallis test followed by Dunn's multiple comparison test (C - D). ns indicates p > 0.05, * indicates 0.01 < p < 0.05, *** indicates 0.001 < p < 0.0001, and **** indicates p < 0.0001. [Figure 4]Figure 4: Diagnostic accuracy of the C3F and C3M assays. AUROC for separation of healthy individuals from patients with adenocarcinoma (A and C), and for separation of patients with adenocarcinoma from patients with squamous cell carcinoma (B and D), based on serum levels of C3F (A - B) or C3M (C - D).

[0037] [Figure 5] Figure 5: Correlation between C3F and C3M. C3F and C3M values obtained from patients with adenocarcinoma (triangles) or squamous cell carcinoma (circles). The correlation between C3F and C3M was tested using Spearman correlation analysis. Spearman's r and p values are shown in the figure. [Figure 6] Figure 6: Serum C3F levels in patients with lung cancer. (A) Quantification of C3F in sera from healthy individuals (n = 42) and patients with NSCLC (n = 109). Biomarker levels between groups were compared using the Mann - Whitney test. (B) AUROC for separation of healthy individuals from patients with NSCLC, based on serum levels of C3F. ns indicates p > 0.05, * indicates 0.01 < p < 0.05, *** indicates 0.001 < p < 0.0001, and **** indicates p < 0.0001. [Figure 7] Figure 7: Correlation between C3F and C3M. C3F and C3M values obtained from patients with NSCLC (n = 32). The correlation between C3F and C3M was tested using Spearman correlation analysis. Spearman's r and p values are shown in the figure. [Figure 8] Figure 8: Serum C3F levels in patients with spondyloarthritis. (A) Quantification of C3F in sera from healthy individuals (n = 19) and patients with spondyloarthritis (SpA) (n = 17). Biomarker levels between groups were compared using the Mann - Whitney test. (B) AUROC for separation of healthy individuals from patients with SpA, based on serum levels of C3F. ns indicates p > 0.05, * indicates 0.01 < p < 0.05, *** indicates 0.001 < p < 0.0001, and **** indicates p < 0.0001.

Example

[0038] The embodiments disclosed herein are described in the following examples. These examples are provided to aid in understanding the disclosure and should not be construed in any way as limiting the scope of the disclosure specified in the claims below. The examples below are provided to a person skilled in the art to provide a complete disclosure and description of how to make and use the described embodiments and are not intended to limit the scope of the disclosure, nor are they intended to mean that the experiments described below are all or only experiments performed. Efforts have been made to ensure accuracy of the numerical values ​​used (e.g., quantity, temperature, etc.), but some experimental error and deviation should be taken into consideration. Unless otherwise indicated, parts are parts by weight, molecular weight is weight-average molecular weight, temperature is in Celsius, and pressure is atmospheric pressure or near atmospheric pressure.

[0039] Materials and methods Development, production, and characterization of monoclonal antibodies N-terminal amino acid sequence 1070 AGPAGAPGPA 1079 (Sequence ID 1)(Amino acid residue P of the alpha-1 chain of type III collagen (UniprotKB: P02461)) 1069 and A 1070 A monoclonal antibody (mAb) specific to the N-terminal of the C-terminal fragment of the alpha-1 chain of type III collagen (which is produced when the interstitial space between these cells is cleaved by fibroblast-activating protein (FAP)) was produced using the following procedure.

[0040] To use as a target peptide, AGPAGAPGPA (SEQ ID NO: 1), a 10-amino acid peptide, was purchased from GenScript (Piscataway, NJ, USA). The immunogenic peptide (AGPAGAPGPA-GGC-"KLH" (SEQ ID NO: 15)) was generated by covalently crosslinking the target peptide to a keyhole limpet hemocyanin ("KLH") carrier protein using sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, SMCC (Thermo Scientific, Waltham, MA, USA, cat.no.22322). To ensure correct linkage of the carrier protein to the target peptide, glycine and cysteine ​​residues were added to the C-terminal end of the target peptide as linking groups. Five female Balb / C mice, 6-7 weeks old, were subcutaneously immunized with 200 μl of emulsified antigen containing 100 μg of immunogenic peptide using the Sigma Adjuvant System (Sigma). Immunization was repeated every two weeks until a stable serum antibody titer level was reached. The mouse with the highest serum titer was selected for cell fusion and kept in recuperation for one month. Three days before spleen removal for cell fusion, the selected mouse was boosted by intravenous administration of 100 μl of 0.9% NaCl solution containing 50 μg of immunogenic peptide. To create hybridoma cells, mouse splenocytes were fused with SP2 / 0 myeloma cells as described by Gefter et al. To further promote the proliferation of the fused cells, they were cultured in each well of a 96-well microtiter plate using limiting dilution to promote monoclonal proliferation.

[0041] The reactivity of the supernatant obtained from fusion cell clones was screened using an indirect ELISA performed on streptavidin-coated 96-well microtiter plates to identify the best mAb-producing clones. The assay used a biotin-labeled screening peptide (AGPAGAPGPA-K-biotin (SEQ ID NO: 16)), and antibody specificity was tested using a selective peptide (AGPAGAPGPA (SEQ ID NO: 1)) and non-selective peptides (extended peptide (PAGPAGAPGPA (SEQ ID NO: 2)) and truncated peptide (GPAGAPGPA (SEQ ID NO: 3))). The best clones were selected based on their specificity to the selected peptide and their non-reactivity to the non-selected peptide. The supernatant was collected, and the mAbs were purified using a HiTrap affinity column (GE Healthcare Life Sciences, Little Chalfront, Buckinghamshire, UK). Finally, antibody isotypes were determined using the SBA Clonetyping System-HRP kit (Southern Biotech, Birmingham, AL, USA), and the monoclonal antibody sequences and CDRs were determined. All procedures were performed according to the manufacturer's instructions.

[0042] The chain sequence of the monoclonal antibody is as follows (CDR is underlined and bold, N-terminal signal peptide and C-terminal constant region are italicized): Heavy chain sequence (mouse IgG2b isotype)

[0043] [ka]

[0044] Light chain sequence (mouse kappa isotype)

[0045] [ka]

[0046] C3F ELISA Protocol The development of a competitive ELISA (also referred to herein as the "C3F assay") targeting the N-terminal amino acid sequence AGPAGAPGPA (SEQ ID NO: 1) (also referred to herein as "C3F") using the aforementioned mAb involved several preliminary optimization experiments, which included various parameters such as assay buffer, incubation time and temperature, and antibody and peptide concentrations.

[0047] The final C3F assay procedure was as follows: A 96-well streptavidin-coated ELISA plate was coated with 100 μL / well of biotinylated peptide (AGPAGAPGPA-K-biotin (SEQ ID NO: 16)) dissolved at 3.5 ng / mL in assay buffer (25 mM PBS, 1% BSA (w / v), 0.018% Bronidox (v / v), 0.1% Tween-20 (w / v), 2 g / L NaCl, pH 7.4), and incubated in the dark at 20°C for 30 minutes with shaking (300 rpm). After washing five times with washing buffer (25 mM Tris, 50 mM NaCl, pH 7.2), 20 μL / well of pre-diluted sample (in the initial version of the assay procedure, the ratio was 1:4, but in the final version of the assay procedure, it was pre-diluted to 1:2) was added twice in duplicate, followed by the addition of 12 ng / mL monoclonal antibody in 100 μL / well of assay buffer, and incubation was performed in the dark at 4°C for 20 hours with shaking (300 rpm). After another washing cycle (five times with washing buffer), 100 μL / well of goat anti-mouse IgG II antibody, HRP (Thermo Fisher, cat#31437), diluted to a final dilution of 1:5000, was added to the assay buffer, and incubation was performed in the dark at 20°C for 1 hour with shaking (300 rpm). After the final washing cycle, 100 μL / well of 3,3',5,5'-tetramethylbenzidine (TMB) was added, and the mixture was incubated in the dark at 20°C for 15 minutes with shaking (300 rpm). The reaction was stopped by adding 100 μL / well of 1% H2SO4. Absorbance was measured at 450 nm, with 650 nm as the reference. A standard curve was created by adding a 30-amino acid standard peptide (AGPAGAPGPAGSRGAPGPQGPRGDKGETGE (SEQ ID NO: 19)) with an intensity of 800 nM, diluted twofold in series, at 20 μL / well, and the curve was fitted using a four-parameter logistic (4PL) model.

[0048] Specificity of C3F ELISA Selected peptides with a length of 10 amino acids ( 1070 AGPAGAPGPA 1079The specificity of the antibody against (SEQ ID NO: 1) is determined by binding to the selected peptide and the peptide extension ( 1069 PAGPAGAPGPA 1079 (Sequence No. 2)) and trimmed items ( 1071 GPAGAPGPA 1079 The binding was evaluated by comparing it with that to (SEQ ID NO: 3). Furthermore, recombinant type III collagen (Abcam, cat#ab7535) was incubated with FAP (Bio-Techne, cat#3715-SE) or 4-aminophenylmercury acetate (Sigma-Aldrich, cat#A9563) activated MMP-9 (Bio-Techne, cat#911-MP) for 24 hours, followed by measurement using C3F and C3M (an established ELISA for measuring MMP-9-cleaved type III collagen (Nordic BioScience, cat#1200AG01)), which showed specificity for FAP-cleaved type III collagen.

[0049] Patient samples - First cohort C3F and C3M levels were measured in a lung cancer cohort including serum from 26 healthy individuals, 26 patients with adenocarcinoma, and 14 patients with squamous cell carcinoma. Cancer serum samples were purchased from Proteogenex (Los Angeles, CA, USA), and healthy control samples were obtained from BioIVT (Westbury, NY, USA). Details of the cohort are shown in Table 1.

[0050] [Table 1]

[0051] Patient samples - second cohort C3F levels were measured in a second lung cancer cohort, including serum from 42 healthy individuals and 109 patients with NSCLC. C3M levels were measured in serum from 32 of these patients with NSCLC. Cancer serum samples were purchased from Proteogenex (Los Angeles, CA, USA), and healthy control samples were obtained from BioIVT (Westbury, NY, USA). Details of the cohort are shown in Table 2.

[0052] [Table 2]

[0053] Patient samples - Third cohort C3F levels were also measured in a spondyloarthritis cohort, which included serum from 19 healthy individuals and 17 patients with spondyloarthritis. Details of the cohort are shown in Table 3.

[0054] [Table 3]

[0055] statistics The comparison of both C3F levels and C3M levels in patients within the first cohort (patients with adenocarcinoma and patients with squamous cell carcinoma, and healthy control subjects) was performed by conducting the Kruskal-Wallis test, followed by Dunn's multiple comparison test. The comparison of C3F levels in patients within the second cohort (patients with NSCLC and healthy control subjects), and the comparison of C3F levels in patients within the third cohort (patients with spondyloarthritis and healthy control subjects) were performed by conducting the Mann-Whitney test. The diagnostic accuracy of the C3F assay for discriminating healthy subjects from both lung cancer subtypes (adenocarcinoma and squamous cell carcinoma), and likewise for discriminating patients with different subtypes from each other, for discriminating healthy subjects from patients with NSCLC, and for discriminating healthy subjects from spondyloarthritis, was evaluated by the area under the receiver operating characteristic curve (AUROC). To compare with the diagnostic potential of C3F, the same analysis was performed for C3M measurements. The correlation between C3M measurements and C3F measurements was performed using Spearman correlation analysis. Significance was examined with a p-value < 0.05 as follows: *0.01 < p < 0.05; **0.001 < p < 0.01; ***0.0001 < p < 0.001; ****p < 0.0001. Statistical analysis was performed using GraphPad Prism (version 9.5.0 for Windows or version 10.1.2 for Windows, GraphPad Software, San Diego, California, USA, www.graphpad.com).

[0056] Results specificity The specificity of the assay was evaluated by the ability of different peptides to compete for binding to monoclonal antibodies in a competitive ELISA format. The peptide groups were amino acid residues P 1069 and A 1070The assay included standard peptides consisting of 10 amino acids and 30 amino acids corresponding to the N-terminal of the C-terminal fragment generated from FAP cleavage of the alpha-1 chain of type III collagen between the two molecules (AGPAGAPGPA (SEQ ID NO: 1) and AGPAGAPGPAGSRGAPGPQGPRGDKGETGE (SEQ ID NO: 19)), an elongated peptide (PAGPAGAPGPA (SEQ ID NO: 2)), and a truncated peptide (GPAGAPGPA (SEQ ID NO: 3)) (Figure 1A). No cross-reactivity was observed with either the elongated or truncated peptides, demonstrating that the C3F assay and antibody recognize the target N-terminal sequence and are specific to it, but do not recognize the sequence when it is located within a peptide (and therefore lacks a free N-terminal amine group), thus demonstrating the neoepitope specificity of the antibody and assay.

[0057] To detect FAP-cleaved type III collagen and to confirm the assay and antibody capabilities supporting the specificity of the ELISA, C3F levels were measured in a solution of recombinant type III collagen after 24 hours of incubation with or without FAP (using this assay). In the initial tests, a signal was detected from a solution containing type III collagen but without FAP, but there was a clear amplification of the signal in a solution containing both FAP and type III collagen, indicating that FAP mediates the formation of type III collagen-derived fragments and that these fragments can be detected and quantified by C3F ELISA (Figure 1B). Further tests further confirmed the specificity of the C3F assay for FAP-generated fragments of type III collagen, with no signal detected from a solution containing type III collagen but without FAP (Figure 2A). For comparison, the results of measuring C3M levels in a solution of recombinant type III collagen after 24 hours of incubation with or without MMP9 (using the C3M assay) are also shown (Figure 2B).

[0058] C3F in serum from patients with lung cancer (first cohort) C3F levels were significantly elevated in serum from lung cancer patients compared to healthy individuals (Figure 3A), suggesting the presence of FAP-cleaved type III collagen fragments in circulation and their increase during lung cancer progression. Using a C3M assay (an established ELISA for measuring MMP-9-cleaved type III collagen), C3M levels in the same serum samples were also significantly elevated in serum from lung cancer patients compared to healthy individuals (Figure 3B).

[0059] When patients with lung cancer were divided into those with adenocarcinoma (n=26) and those with squamous cell carcinoma (n=14), C3F levels were significantly higher in patients with squamous cell carcinoma compared to those with adenocarcinoma (Figure 3C). C3F demonstrated better diagnostic accuracy for the isolation of adenocarcinoma from squamous cell carcinoma (AUROC=0.77, p=0.006) (Figure 4B) than for isolation from healthy individuals (AUROC=0.66, p=0.048) (Figure 4A). In contrast to C3F, C3M could not significantly distinguish patients with adenocarcinoma from those with squamous cell carcinoma (Figure 3D). Furthermore, C3M showed good diagnostic accuracy in the isolation of patients with adenocarcinoma from healthy individuals (AUROC=0.92, p<0.0001) (Figure 4C), and poor diagnostic accuracy in the isolation of patients with adenocarcinoma from patients with squamous cell carcinoma (AUROC=0.56, p=0.53) (Figure 4D), which was the opposite of what was observed for C3F. The C3F and C3M levels showed only moderate overall correlation (Spearman's r=0.53, p=0.0004) (Figure 5), further supporting the idea that C3F reflects something different from C3M. In summary, these results suggest that the circulating levels of FAP-cleaved type III collagen fragments (measured by the C3F assay) reflect a different biological process than the circulating levels of MMP-9-cleaved type III collagen fragments (measured by the C3M assay), and therefore offer additional value and diagnostic potential.

[0060] C3F in serum from patients with lung cancer (second cohort) C3F levels were significantly elevated in serum from patients with NSCLC compared to healthy individuals (p<0.0001) (Figure 6A), and could be significantly differentiated between healthy individuals and patients with NSCLC (AUROC=0.78, p<0.0001) (Figure 6B). This reaffirmed and confirmed that FAP-cleaved type III collagen fragments are present in circulation and increase during the progression of lung cancer. Although this cohort included a larger number of patients and healthy controls, there was no difference in C3F levels between patients with adenocarcinoma and those with squamous cell carcinoma. C3M levels were measured in a subset of the same serum samples using the C3M assay. C3F and C3M levels were uncorrelated (Spearman's r=0.17, p=0.35) (Figure 7), further supporting the idea that C3F reflects something different from C3M. In summary, these results further suggest that the circulating levels of FAP-cleaved type III collagen fragments (measured by the C3F assay) reflect a different biological process than the circulating levels of MMP-9-cleaved type III collagen fragments (measured by the C3M assay), and therefore offer additional value and diagnostic potential.

[0061] C3F in serum from patients with spondyloarthritis (third cohort) C3F levels were significantly elevated in serum from patients with spondyloarthritis compared to healthy individuals (p<0.0015) (Figure 8A), and could be significantly distinguished between healthy individuals and patients with NSCLC (AUROC=0.78, p<0.0048) (Figure 8B), suggesting that circulating FAP-cleaved type III collagen fragments are increased in patients with spondyloarthritis.

[0062] Essay The inventors have developed a competitive ELISA to measure the AGPAGAPGPA(SEQ ID NO: 1)N terminal sequence, and the level of the AGPAGAPGPA(SEQ ID NO: 1)N terminal sequence reflects FAP activity via FAP cleavage of type III collagen. The immunoassay can detect and quantify the level of this biomarker in blood material samples from patients with lung cancer. The biomarker can be distinguished from another type III collagen degradation marker, C3M (MMP-9 mediated cleavage of type III collagen), and has shown different diagnostic potential than C3M. Therefore, using the immunoassay methods, kits, and monoclonal antibodies disclosed herein, patient-derived blood material samples can be analyzed to detect, monitor, and / or evaluate cancer or arthritis.

[0063] In this specification, unless otherwise specified, the word "or" is used to mean an operator that returns true if one or both of the stated conditions are met, in contrast to the "exclusive OR" operator, which requires that only one of the conditions be met. The word "comprising" is used to mean "including, or consisting of." All prior teachings acknowledged above are incorporated into this specification by reference thereto. Any recognition of prior publications in this specification should not be taken as an admission or statement that the teachings in such publications were common knowledge in Australia or other countries at the time of this specification.

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Claims

1. An immunoassay method including the following steps; i) Contacting the patient's sample with a monoclonal antibody that specifically binds to the N-terminal amino acid sequence AGPAGAPGPA (SEQ ID NO: 1); and, ii) To detect the binding between the monoclonal antibody and the peptide in the sample and to determine the amount of binding.

2. The immunoassay method is an immunoassay method for detecting and / or monitoring cancer or a specific level of severity of said cancer in a patient, the method further comprising the following steps, according to claim 1; iii) Correlating the binding amount with a value associated with a normal healthy person, and / or a value associated with a known disease severity, and / or a value obtained from the patient at a past point in time, and / or a predetermined cutoff value.

3. The immunoassay method according to claim 2, wherein the cancer is lung cancer.

4. The immunoassay method according to claim 3, wherein the lung cancer is non-small cell lung cancer.

5. The immunoassay method described above is a method for detecting and / or monitoring arthritis or a specific level of severity of said arthritis in a patient, the method further comprising the following steps, according to claim 1; iii) Correlating the binding amount with a value associated with a normal healthy person, and / or a value associated with a known disease severity, and / or a value obtained from the patient at a past point in time, and / or a predetermined cutoff value.

6. The immunoassay method according to claim 5, wherein the arthritis is spondyloarthritis.

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

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

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

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

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

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

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

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

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

16. The monoclonal antibody according to any one of claims 13 to 15, wherein the monoclonal antibody is produced in opposition to a synthetic peptide having the N-terminal amino acid sequence AGPAGAPGPA (SEQ ID NO: 1).

17. An immunoassay kit comprising a monoclonal antibody according to any one of claims 13 to 16, and at least one of the following: - Streptoavidin-coated well plates - Biotinylated peptide AGPAGAPGPA-L-biotin (SEQ ID NO: 14), where L is an arbitrary linking group. - Second antibody used in sandwich immunoassays - Calibration protein containing the N-terminal amino acid sequence AGPAGAPGPA (SEQ ID NO: 1) - Antibody biotinylation kit - Antibody HRP labeling kit - Antibody radiolabeling kit.

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