PRO-C17 assay

Abstract

Disclosed is an immunoassay method ("PRO-C17 assay") for measuring the level of type XVII collagen ectodomains present in patient samples, which can be used to detect and / or monitor cancer in patients and / or to assess the severity of cancer. Also disclosed are monoclonal antibodies and assay kits suitable for use in carrying out the method.

Description

[Technical Field]

[0001] The present invention relates to an immunoassay method suitable for use in the detection and / or monitoring of cancer and / or assessment of its severity, and to antibodies and assay kits suitable for use in carrying out said method. [Background technology]

[0002] Cancer is the leading cause of death worldwide, accounting for nearly 10 million deaths in 2020. The use of biomarkers is crucial for early diagnosis and the selection of appropriate treatments to reduce the burden of cancer. [1] In recent years, attention has been focused on the tumor microenvironment, more specifically the extracellular matrix (ECM), which has been reported to promote tumor progression and reduce the response to cancer treatment. [2] .

[0003] Collagen is a major component of the extracellular matrix (ECM), and 28 different types of collagen have been identified to date. [3] In healthy tissues, collagen production and degradation are highly regulated to maintain tissue homeostasis. However, in cancer, excessive metabolic turnover and remodeling occur, resulting in a loss of systematic cellular behavior within the tissue and leading to unbalanced angiogenesis that can promote disease progression and tumorigenesis. [4] The protein fragments resulting from this excessive ECM remodeling are released into circulation and can be non-invasively measured by liquid biopsy, allowing them to be used as biomarkers. Extensive research and biomarker discovery have focused on the most dominant collagens of the ECM, such as types I, III, and IV, but little is known about the less abundant, highly specialized groups of collagen.

[0004] Membrane-associated collagens (MACITs), including type XIII, XVII, XXIII, and XXV collagens, are type II proteins containing large ectodomains that are not present in other collagens and can be detached and released into the pericellular matrix.[5,6] Overall, MACIT is expressed at low levels in adult tissues but is upregulated during both embryogenesis and tumorigenesis. MACIT may contribute to the maintenance of ECM stability, and there is increasing evidence that they play an essential role in the process of determining cell fate by binding to ECM components such as cell surface receptors or growth factors in both healthy and cancerous tissues. [7-9] MACIT consists of an N-terminal cytoplasmic domain composed of different collagenous domains (COL) and non-collagenous domains (NC), a transmembrane domain, and a C-terminal ectodomain.

[10] Types XIII, XXIII, and XXV collagens have similar structures, and their ectodomains are cleaved by furin proteases, while type XVII collagen has a different structure and is cleaved by ADAM9, 10, and 17. [11-13] 。

[0005] Type XVII collagen, also known as BP180 or BPAG2, consists of three identical 180 kDa α-chains and is part of the hemidesmosome, providing stable adhesion between basal keratinocytes and the basement membrane and thus playing an important role in the skin.

[14] Several blistering skin diseases are associated with both hereditary and acquired deficiencies of type XVII collagen. For example, mutations in the COL17A1 gene can cause junctional epidermolysis bullosa (JEB), while autoimmunity against the NC16A domain of shed type XVII collagen induces bullous pemphigoid (BP). [15-17] In one study, it was shown that a polyclonal antibody produced against the peptide sequence LQGMAPAAG (corresponding to amino acids 524 - 532 of the NC16A domain of type XVII collagen) preferentially reacts with the shed ectodomain of type XVII collagen, and that the sera of patients with bullous pemphigoid react with this same peptide, suggesting that the neoepitope of the shed ectodomain of type XVII collagen is located in this series of amino acids and can be the target of blister-inducing antibodies.

[24] 。

[0006] In cancer, type XVII collagen and its detached ectodomains appear to play a major role in the development and metastasis of epithelial carcinoma.

[18] Abnormal expression of the COL17A1 gene has been detected in many epithelial tumors, such as colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, or squamous cell carcinoma, and is associated with a poor prognosis. [19-22] Furthermore, recent studies on the role of type XVII collagen in cancer stem cell dormancy have revealed that COL17A1-KO colorectal cancer organoids tend to form larger colonies than wild-type organoids due to increased proliferation rates. These studies also suggest that COL17A1-KO colorectal cancer organoids contribute to dormant LGR. + p27 + By causing cell disappearance and finding that the cells were more sensitive to chemotherapy, we suggested that COL17A1 may play a role in maintaining cellular dormancy.

[23] . [Overview of the project]

[0007] The inventors developed and validated an ELISA (also referred to herein as the “PRO-C17 assay”) targeting the ectodomain of type XVII collagen, and measured the levels of circulating PRO-C17 in serum samples (i.e., the levels of type XVII collagen ectodomain detected by the PRO-C17 assay) to study the potential of PRO-C17 as a biomarker in patients with cancer. The assay was found to be technically robust, and the data showed that PRO-C17 levels were significantly elevated in all cancer types, and that colorectal cancer patients with high PRO-C17 levels had lower overall survival and an increased risk of death.

[0008] Therefore, in the first embodiment, the present invention provides an immunoassay method, which is i) Contact the patient's sample with a monoclonal antibody that specifically binds to the amino acid sequence QGMAPAAGA (SEQ ID NO: 1); ii) The method includes detecting the binding between the monoclonal antibody and the peptide in the sample and determining the amount of binding. In a preferred embodiment, the method is an immunoassay for detecting and / or monitoring cancer in a patient and / or assessing the severity of cancer in a patient, the method further comprising the following steps; 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. In a preferred embodiment, the monoclonal antibody specifically binds to the amino acid sequence LQGMAPAAGA (SEQ ID NO: 2).

[0009] In a preferred embodiment, the monoclonal antibody does not specifically bind to the amino acid sequence GMAPAAGADL (SEQ ID NO: 3) (i.e., the truncated QGMAPAAGA (SEQ ID NO: 1) sequence, which is truncated by removing the N-terminal glutamine residue). Preferably, the ratio of the antibody's affinity for the QGMAPAAGA (SEQ ID NO: 1) sequence to the truncated sequence GMAPAAGADL (SEQ ID NO: 3) 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 amino acid sequence LQGLAPLGSE (SEQ ID NO: 4). Preferably, the ratio of the antibody's affinity for the QGMAPAAGA (SEQ ID NO: 1) sequence to its affinity for the LQGLAPLGSE (SEQ ID NO: 4) 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 amino acid sequence LQGMAYTVQG (SEQ ID NO: 5). Preferably, the ratio of the affinity of the antibody to the QGMAPAAGA (SEQ ID NO: 1) sequence to the affinity of the antibody to the LQGMAYTVQG (SEQ ID NO: 5) 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.

[0010] In a preferred embodiment, the monoclonal antibody is produced by counteracting a synthetic peptide containing the amino acid sequence LQGMAPAAGA (SEQ ID NO: 2). Preferably, the synthetic peptide consists of the amino acid sequence LQGMAPAAGA (SEQ ID NO: 2). For example, the monoclonal antibody can be produced by: (a) immunizing a rodent (or other suitable mammal) with a synthetic peptide containing the amino acid sequence LQGMAPAAGA (SEQ ID NO: 2), which may optionally have an immunogenic carrier protein (such as keyhole limpet hemocyanin) linked to its C-terminal; (b) isolating and cloning a single antibody-producing cell; and (c) assaying the resulting monoclonal antibody to confirm that it has the desired specificity.

[0011] In preferred embodiments, the cancer is pancreatic cancer, colorectal cancer, kidney cancer, ovarian cancer, bladder cancer, lung cancer, melanoma, breast cancer, head and neck cancer, prostate cancer, or stomach cancer. Preferably, the cancer is colorectal cancer, kidney cancer, ovarian cancer, bladder cancer, breast cancer, or head and neck cancer. Most preferably, the cancer is colorectal cancer. As described above, in one embodiment, the method is an immunoassay for evaluating the severity of cancer in a patient. For example, the method may be a method for detecting a level of severity related to mean overall survival. 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. 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.

[0012] 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.

[0013] As used herein, the term “monoclonal antibody” refers to both the whole antibody and its fragments that retain the binding specificity of the whole antibody, such as Fab fragments, F(ab')2 fragments, single-chain Fv fragments, or other such fragments known to those skilled in the art. As is well known, a whole antibody typically has a “Y-shaped” structure in which two identical polypeptide chain groups are paired, with each paired element consisting of one “light” chain and one “heavy” chain. The N-terminal regions of the light and heavy chains each contain a variable region, and the C-terminal portions of the heavy and light chains each constitute a constant region. The variable region contains three complementarity-determining regions (CDRs), which are primarily responsible for antigen recognition. The constant region allows the antibody to recruit cells and molecules of the immune system. Antibody fragments that retain binding specificity include at least the CDRs and a portion of the remainder of the variable region sufficient to retain binding specificity.

[0014] In the present invention, any monoclonal antibody containing any constant region known in the art can be used. In the case of mouse antibodies and human antibodies, the constant light chain is classified as either a kappa light chain or a lambda light chain. The constant heavy chain is classified as mu, delta, gamma, alpha, or epsilon, defining the antibody isotypes as IgM, IgD, IgG, IgA, and IgE, respectively. The IgG isotype has several subclasses in the case of humans, including IgG1, IgG2, IgG3, and IgG4, and in the case of mice, it has several subclasses including IgG1, IgG2a, IgG2b, IgG2c, and IgG3, but is not limited thereto. The monoclonal antibody may preferably belong to an IgG isotype containing any one of the IgG subclasses.

[0015] The CDR of an antibody can be determined using methods known in the art, such as those described by Kabat et al. The antibody can be generated from B cell clones. The isotype of the antibody can be determined by ELISA specific to the IgM, IgG, or IgA isotype or subclass. The amino acid sequence of the generated antibody can be determined using standard methods. For example, RNA can be isolated from cells and used to generate cDNA by reverse transcription. The cDNA is then subjected to PCR using primers that amplify the heavy and light chains of the antibody. For example, primers specific to the leader sequence for all VH (variable heavy chain) sequences can be used together with primers that bind to sequences located in the constant region of a predetermined isotype. The light chain can be amplified using primers that bind to the 3' end of the kappa or lambda chain, together with primers that anneal to the V-kappa or V-lambda leader sequence. The full-length heavy and light chains can be generated and sequenced.

[0016] As used herein, the term "amount bound" refers to the quantification of the binding between an antibody and a peptide in a subject sample. The quantification may be determined, for example, by comparing the measured value of the binding in the subject sample to a calibration curve generated using the measured value of the binding in a standard sample containing a peptide to which the antibody specifically binds at a known concentration, in order to determine the amount of the peptide to which the antibody specifically binds in the subject sample. Any method may be used as long as it is an analytical method suitable for measuring the amount of binding. For example, an ELISA method may be used, in which spectrophotometric analysis is utilized to measure both the amount of binding in the subject sample and the amount of binding when the calibration curve was created.

[0017] As used herein, the term "predetermined cut-off value" means a statistically determined amount of binding that indicates a high likelihood that a subject has a disease (e.g., cancer), or a particular severity of the disease, and in this sense, when the measured value of the target peptide in the subject sample is above the statistical cut-off value, the measured value corresponds to a probability that the disease is present or that the disease is of a particular severity of at least 70% probability, preferably at least 75% probability, more preferably at least 80% probability, more preferably at least 85% probability, more preferably at least 90% probability, and most preferably at least 95% probability.

[0018] As used herein, the term "value related to a normal healthy subject" means a standardized amount of binding determined by the above method for a sample obtained from a subject considered to be healthy, i.e., not having a disease (i.e., not having cancer); and the term "value related to a known disease severity" means a standardized amount of binding determined by the above method for a sample obtained from a subject known to have a disease (i.e., cancer) of known severity.

[0019] In a second aspect, the present invention provides a method for treating cancer in a patient who needs treatment, the method comprising the following steps: (a) Performing an immunoassay method on a sample obtained from a patient according to the first aspect of the present invention; and (b) When it is determined in step (a) that the patient has cancer or the severity of cancer is at a specific level, administering a therapy for treating cancer to the patient. A preferred embodiment of the method according to the second aspect is apparent from the above considerations regarding the preferred embodiment of the method according to the first aspect. For example, step (a) may particularly include performing an immunoassay method for detecting bladder cancer, breast cancer, colorectal cancer, head and neck cancer, kidney cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, or gastric cancer; or for detecting a specific level of cancer severity.

[0020] The above therapy may be any therapy suitable for treating 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, etc.), or a combination thereof, or may consist of those therapies. The drugs may be formulated for local or systemic administration. The local drugs may be formulated, for example, in dosage forms such as creams, foams, gels, lotions, or ointments. The systemic drugs may be formulated, for example, in dosage forms for oral or parenteral administration. The surgery may be radical surgery, preventive surgery, debulking surgery, palliative surgery, and / or reconstructive surgery.

[0021] For example, when the cancer is bladder cancer, appropriate therapies may include, for example, one or more of the following: transurethral resection of bladder tumor (TURBT) with or without concomitant intravesical chemotherapy or intravesical immunotherapy; radical cystectomy with neoadjuvant chemotherapy, or transurethral resection with concomitant chemoradiation therapy, or partial cystectomy with neoadjuvant chemotherapy; cisplatin-based chemotherapy, optionally followed by radical cystectomy or chemoradiation therapy; carboplatin-based chemotherapy; immune checkpoint inhibitors; radical cystectomy; and palliative radiation therapy.

[0022] If the cancer is breast cancer, appropriate therapy may include, for example, one or more of the following: mastectomy, quarter-mastectomy, or breast-conserving surgery; estrogen receptor blockers (such as tamoxifen); aromatase inhibitors that block estrogen production (such as anastrozole or letrozole); CDK inhibitors; one or more chemotherapeutic agents such as a combination of cyclophosphamide, doxorubicin, and optionally a taxane (such as docetaxel), or a combination of cyclophosphamide, methotrexate, and fluorouracil; one or more monoclonal antibodies such as trastuzumab and / or pertuzumab; and radiotherapy.

[0023] If the cancer is colorectal cancer, appropriate therapy may include, for example, one or more of the following: endoscopic mucosal resection or endoscopic submucosal dissection; partial colectomy (or corectomy of the rectal region); chemotherapeutic agents such as capecitabine, fluorouracil, irinotecan, oxaliplatin, or UFT; anti-angiogenic agents such as bevacizumab; epidermal growth factor receptor inhibitors such as aflibercept, cetuximab, and panitumumab; radiotherapy; immune checkpoint inhibitors; and monoclonal antibodies such as pembrolizumab and dostallimab.

[0024] If the cancer is head and neck cancer, appropriate therapy may include, for example, one or more of the following: surgery, including laser surgery; radiotherapy, including three-dimensional conformal radiotherapy, intensity-modulated radiotherapy, particle beam therapy, and brachytherapy; one or more chemotherapeutic agents, such as paclitaxel, carboplatin, cetuximab, docetaxel, cisplatin, and fluorouracil; photodynamic therapy using Amfinex; monoclonal antibodies such as cetuximab, bevacizumab, erlotinib, pembrolizumab, or nivolumab; gene therapy such as Gendicin; and immune checkpoint inhibitors.

[0025] If the cancer is kidney cancer, appropriate treatment may include, for example, one or more of the following therapies: total or partial removal of the kidney by surgery; cryotherapy or high-temperature treatment of the cancer; biological therapies such as everolimus, torisel, nexavar, sutent, axitinib, sunitinib, pazopanib, sorafenib, cabozantinib, and / or lenvatinib; immunotherapy using interferon and / or interleukin-2; monoclonal antibodies such as nivolumab; immune checkpoint inhibitors; and radiotherapy.

[0026] If the cancer is lung cancer, appropriate treatment includes, for example, one or more of the following: surgery such as lobectomy, segmentectomy (wedge resection), or pneumonectomy (lung resection); and radiotherapy. Specific examples include radiotherapy combined with chemotherapy, radiotherapy performed after surgery, brachytherapy (localized radiotherapy) applied directly to the inside of the airway, 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; and immunotherapy using PD-L1 monoclonal antibodies such as 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.

[0027] If the cancer is melanoma, appropriate therapy may include, for example, one or more of the following: surgical removal of the cancer and optionally lymph nodes in the cancerous area; interferon therapy; chemotherapy using agents such as dacarbazine; small molecule targeted therapy using agents such as BRAF inhibitors (e.g., vemurafenib and dabrafenib), MEK inhibitors (trametinib), C-kit inhibitors, and / or NRAS inhibitors; cytokines (e.g., IL-2, and / or IFN-α), immunotherapy. Immunotherapy using CTLA-4 monoclonal antibodies such as ipilimumab or tremelimumab, Toll-like receptor (TLR) agonists, CD40 agonists, anti-PD-1 antibodies such as pembrolizumab, pidilizumab, or nivolumab, LAG-3 inhibitors such as relatrimab, and / or PD-L1 antibodies, etc.) and / or adoptive cell transplantation (e.g., using pre-stimulated and modified T cells or dendritic cells); and radiotherapy.

[0028] If the cancer is ovarian cancer, appropriate treatment may include, for example, one or more of the following: removal of one ovary (unilateral oophorectomy) or both ovaries (bilateral oophorectomy), optionally also removing the fallopian tubes (salpingectomy), uterus (hysterectomy), and / or omentum (omentectomy); debulking surgery; chemotherapy (including neoadjuvant or adjuvant chemotherapy) using agents such as paclitaxel, cisplatin, topotecan, doxorubicin, epirubicin, gemcitabine, carboplatin, docetaxel, vincristine, dactinomycin, etoposide, cyclophosphamide, oxaliplatin, or combinations thereof; radiotherapy; hormone therapy; and immunotherapy, such as the antibody drug bevacizumab.

[0029] For example, if the cancer is pancreatic cancer such as PDAC, appropriate therapy may include, for example, one or more of the following therapies: surgical resection such as cancer resection, Whipple surgery, total pancreatectomy, or distal pancreatectomy; radiotherapy, including, but not limited to, radiotherapy combined with chemotherapy, radiotherapy performed after surgery, brachytherapy (localized radiotherapy), stereotactic radiotherapy, and palliative radiotherapy; chemotherapy using one or more agents such as gemcitabine, 5-FU, erlotinib, FOLFIRINOX therapy, nab-paclitaxel, or combinations thereof; somatostatin analogs; targeted therapy using lanreotide, everolimus, or sunitinib; nuclear medicine therapy using radiolabeled peptides or hormones such as iobenguan; and techniques such as radiofrequency ablation (RFA), cryoablation, or transcatheter artery embolization.

[0030] If the cancer is prostate cancer, appropriate treatment may include, for example, one or more of the following therapies: radiotherapy; chemotherapy using chemotherapeutic agents such as docetaxel, cabazitaxel, docetaxel, thalidomide, and combinations thereof; immunotherapy such as bevacizumab, a monoclonal antibody; hormone therapy such as abiraterone and enzalutamide; external beam radiation therapy; particle beam therapy; intensive focused ultrasound; cryotherapy; and surgical procedures such as radical prostatectomy.

[0031] If the cancer is gastric cancer, appropriate treatment may include, for example, one or more of the following therapies: surgical procedures such as endoscopic mucosal resection, endoscopic submucosal dissection, or gastrectomy; chemotherapy using agents such as fluorouracil, capecitabine, BCNU, methyl-CCNU, doxorubicin, mitomycin C, cisplatin, taxotere, or a combination thereof; targeted therapy using epidermal growth factor receptor 2 inhibitors such as trastuzumab; and radiotherapy.

[0032] In a third embodiment, the present invention provides a monoclonal antibody that specifically binds to the amino acid sequence QGMAPAAGA (SEQ ID NO: 1). The antibody according to the third embodiment of the invention is particularly suitable for use when carrying out the immunoassay method according to the first embodiment of the invention. Accordingly, preferred embodiments and features of the antibody according to the third embodiment are evident from the above discussion concerning preferred embodiments of the method according to the first embodiment.

[0033] In a fourth aspect, the present invention provides a monoclonal antibody according to the third aspect of the invention, and an immunoassay kit comprising at least one of the following: - Streptoavidin-coated well plates -Biotinylated peptide QGMAPAAGA-L-biotin (SEQ ID NO: 6), where L is an optional linking group. - Second antibody used in sandwich immunoassay - Calibration protein containing amino acid sequence QGMAPAAGA (SEQ ID NO: 1) - Antibody biotinylation kit - Antibody HRP labeling kit - Antibody radiolabeling kit The immunoassay kit according to the fourth embodiment of the invention is particularly suitable for use when carrying out the immunoassay method according to the first embodiment of the invention. Therefore, more preferred embodiments and features of the immunoassay kit according to the fourth embodiment are evident from the above discussion of preferred embodiments of the method according to the first embodiment. [Brief explanation of the drawing]

[0034] [Figure 1]Figure 1: Specificity of PRO-C17 monoclonal antibodies. The reactivity of monoclonal antibodies used in competitive PRO-C17 ELISA was evaluated against a selective peptide (LQGMAPAAGA (SEQ ID NO: 2)) and two non-selective peptides (LQGMAYTVQG (SEQ ID NO: 5) and LQGLAPLGSE (SEQ ID NO: 4)). The monoclonal antibodies were also specific to the elongated peptide (RLQGMAPAAGA (SEQ ID NO: 7)) and the truncated peptide (QGMAPAAGA (SEQ ID NO: 1)). B / B0 is the ratio between the OD when the analyte is present (B) and the maximum OD when the analyte is absent (B0). [Figure 2] Figure 2: Depiction of the NC16A domain of type XVII collagen. Physiological cleavage sites are indicated by black arrows, and selected peptides are highlighted. [Figure 3] Figure 3: Specificity of the PRO-C17 monoclonal antibody against the further elongated peptide (DSMDRIEKDRLQGMAPAAGADLDKIGLHSD (SEQ ID NO: 8)) and another peptide (GMPAPAAGADL (SEQ ID NO: 9)) selected from epitope mapping. The antibody was specific to the further elongated peptide but not to the further truncated peptide. [Figure 4] Figure 4: Western blotting results for type XVII collagen in supernatant and cell lysate derived from A-431 cells using commercially available type XVII antibody (right) and PRO-C17 antibody (left). In the supernatant, only a 120 kDa fragment corresponding to the type XVII ectodomain was detected. In the cell lysate, a 180 kDa fragment corresponding to full-size type XVII collagen was detected. [Figure 5] Figure 5: Type XVII collagen levels measured in non-small cell lung cancer (NSCLC) patients compared to healthy controls using the PRO-C17 assay (left) and a commercially available type XVII collagen kit (right). Comparisons were made using parametric t-tests (ns = not statistically significant). [Figure 6]Figure 6: Correlation between type XVII collagen levels using the PRO-C17 assay and a commercially available type XVII assay. The Pearson correlation coefficient (r) was -0.007357 (p>0.05, 95% CI -0.5012 to 0.4902).

[0035] [Figure 7] Figure 7: Serum PRO-C17 levels in cancer patients. Quantification of PRO-C17 in serum from healthy controls (n=23) and patients with bladder cancer (n=19), breast cancer (n=19), colorectal cancer (n=20), head and neck cancer (n=20), renal cancer (n=20), lung cancer (n=17), melanoma (n=20), ovarian cancer (n=20), pancreatic cancer (n=20), prostate cancer (n=19), and gastric cancer (n=20). PRO-C17 levels are presented as box plots with data point jitter: horizontal bars indicate the median; the top and bottom hinges of the box indicate the first and third quartiles (25th and 75th percentiles); whiskers extend from the hinges to the maximum or minimum value but do not exceed 1.5*IQR (IQR is the interquartile range between the first and third quartiles) in either the positive or negative direction. Samples with measurement results less than LLOQ were given the LLOQ value determined in PRO-C17 validation. Differences in PRO-C17 levels between the cancer group and healthy controls were evaluated by standard ANOVA followed by Dunnett's multiple comparison t-test. **** indicates a p-value less than 0.0001, *** indicates a p-value less than 0.001, ** indicates a p-value less than 0.01, and * indicates a p-value less than 0.05. [Figure 8] Figure 8: PRO-C17 levels measured in colorectal cancer patients. PRO-C17 levels were significantly elevated in metastatic colorectal cancer patients (n=212) compared to healthy controls (n=23). The comparison was performed using a parametric t-test. **** indicates p<0.0001. [Figure 9]Figure 9: Kaplan-Meier survival plot. High levels of PRO-C17 were associated with reduced overall survival (OS) in patients with metastatic colorectal disease. The upper line represents low PRO-C17 levels (tertile 1 + tertile 2), and the lower line represents high PRO-C17 levels (tertile 3). [Figure 10] Figure 10: Hazard ratio (HR). HR was calculated using multivariate Cox proportional-hazards analysis stratified by treatment line. [Examples]

[0036] 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.

[0037] Materials and methods Antibody development for PRO-C17 A target peptide consisting of 10 amino acids corresponding to aa524-533 in the NC16A domain of type XVII collagen. 524 LQGMAPAAGA 533 (Sequence ID 2) (UniprotKB: Q9UMD9) was purchased from GenScript (Piscataway, NJ, USA) and used for immunization. More specifically, an immunogenic peptide (LQGMAPAAGA-GGC-“KLH” (SEQ ID NO: 10)) 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.

[0038] Monoclonal antibodies were generated by subcutaneous immunization of 6-week-old Balb / C mice with 200 μL of emulsified antigen containing 100 μg of immunogenic peptide mixed with Sigma Adjuvant System (Sigma, cat. No. S6322) Specol (Invitrogen, cat. No. 7925000). Immunization was performed continuously every two weeks until a stable serum titer level was reached. Mice with the highest titer were selected for cell fusion and rested for 4 weeks before being immunized intravenously with 100 μL of 0.9% NaCl solution containing 100 μg of immunogenic peptide. Then, as previously described (Gefter, Margulies, and Scharff, 1977), spleen cells were fused with SP2 / 0 myeloma cells to create hybridoma cells. The resulting hybridoma cells were cultured in 96-well microtiter plates, and monoclonal proliferation was ensured using standard limiting dilution methods.

[0039] The monoclonal antibody was purified using a protein-G column according to the manufacturer's instructions (GE Healthcare Life Sciences, Little Chalfont, UK, cat. #17-0404-01). The best antibody clone for the biomarker was selected based on preliminary competitive ELISA tests for reactivity to the target peptide (LQGMAPAAGA (SEQ ID NO: 2)), the elongated peptide (RLQGMAPAAGA (SEQ ID NO: 7)), the truncated peptide (QGMAPAAGA (SEQ ID NO: 1)), two non-selective peptides (LQGMAYTVQG (SEQ ID NO: 5) and LQGLAPLGSE (SEQ ID NO: 4)), the targeted KLH-binding peptide (LQGMAPAAGA-GGC-KLH (SEQ ID NO: 10)), and the nonsense KLH-binding peptide (DCTSTFPRV-GGC-KLH (SEQ ID NO: 11)). The supernatant was collected from the selected clones, the monoclonal antibodies (mAbs) were purified and used for assay development.

[0040] PRO-C17 ELISA Protocol The development of the competitive ELISA using the aforementioned mAbs (also referred to herein as the "PRO-C17 assay") involved several preliminary optimization experiments, in which several assay buffers, incubation times and temperatures, and antibody and peptide concentrations were tested.

[0041] The final PRO-C17 protocol was as follows: A 96-well streptavidin-coated ELISA plate was coated with 100 μL / well of biotinylated QGMAPAAGA (SEQ ID NO: 1) peptide dissolved at 2.5 ng / mL in assay buffer (50 mM PBS, 1% BSA (w / v), 0.018% Bronidox (v / v), 0.1% Tween-20 (w / v), 8 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 (1:2) was added in parallel in double succession. Subsequently, 25 ng / mL of HRP-labeled monoclonal antibody in assay buffer was added as 100 μL / well, and the mixture was incubated in the dark at 4°C for 20 hours with shaking (300 rpm). After the second washing cycle, 100 μL / well of 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 sequentially diluting 30 ng / mL of the QGMAPAAGADLDKIGLHSDSQEELWMFVRK (SEQ ID NO: 12) peptide twofold and adding it as 20 μl / well. Curve fitting was performed using a four-parameter logistic (4PL) model. Each plate contained five quality control samples, including porcine serum (1), human serum (2), donkey serum (1), and peptide in assay buffer (1), to monitor inter-assay and intra-assay variability.

[0042] Technical evaluation of PRO-C17 ELISA The specificity of the antibody and assay against the target peptide (LQGMAPAAGA (SEQ ID NO: 2)) was evaluated by including the peptide's elongated form (RLQGMAPAAGA (SEQ ID NO: 7)) and truncated form (QGMAPAAGA (SEQ ID NO: 1)), as well as two non-selective peptides (LQGMAYTVQG (SEQ ID NO: 5) and LQGLAPLGSE (SEQ ID NO: 4)). Linearity was verified by performing serial dilutions of serum samples in assay buffer and calculating the percentage recovery rate for each dilution. The accuracy of the assay was evaluated by adding (spiking) another serum sample containing a lower concentration of the analyte to a serum sample containing a high concentration of the analyte at different ratios (100:0, 75:25, 50:50, 25:75, 0:100), and calculating the percentage recovery rate relative to the analyte concentration when the samples were measured separately. Interference with the most common endogenous analytes (hemoglobin, biotin, and lipids) was evaluated by adding known amounts of interfering substances (low hemoglobin = 2.5 mg / mL, high hemoglobin = 5 mg / mL; low lipid = 1.5 mg / mL, high lipid = 5 mg / mL; low biotin = 5 ng / mL, high biotin = 100 ng / mL) to serum samples and calculating the percentage recovery rate compared to samples without the interfering substances.

[0043] Variability was determined by performing duplicate measurements on 10 independent plates containing three human serum samples, three quality control samples (QC), two peptides in assay buffer, and two kit controls (CO). Intra-assay variability was calculated as the average CV% between sample replicas within the same plate. Intra-assay variability was tolerated at CV% < 10. Inter-assay variability was calculated as the average CV% between runs of sample replicas on different plates, with an acceptable threshold of CV% < 15%. The lower and upper limits of the measurement range (LLOQ and ULOQ) were defined as the highest and lowest concentrations precisely measured within the linear range of the assay. The stability of the analytes was assessed by storing three serum samples at both 4°C and 20°C for 2, 4, 24, and 48 hours, and calculating the percentage recovery compared to the corresponding control sample stored at -20°C. Freeze-thaw stability was determined by freezing and thawing serum samples multiple times (4 cycles) and calculating the percentage recovery rate by comparing them to a control sample that was thawed only once before measurement. The performance of the PRO-C17 ELISA was compared to a commercially available type XVII ELISA kit (MyBioSource, San Diego, CA, USA) by measuring eight serum samples from patients with non-small cell lung cancer (NSCLC) and eight healthy controls.

[0044] Cell culture of A-431 cells A-431 is an epithelial cell line derived from a patient with epidermoid carcinoma that constitutively expresses and cleaves type XVII collagen. The A-431 cell line was purchased from ATCC (Rockville, MD, USA). Cells were cultured under two different conditions: keratinocyte SFM (KSFM; Divco, UK) supplemented with human recombinant epidermal growth factor (rEGF), bovine pituitary extract (BPE), and 0.4 mM CaCl2, and Dulbecco's Modified Eagle Medium (DMEM, Divco, United Kingdom) supplemented with 10% fetal bovine serum (FBS; Divco, United Kingdom).

[0045] Western blot of A-431 cells The supernatant and cell lysates of A-431 cells were electrophoresed on NuPAGE 4-12% Bis-Tris gel (Invitrogen, Carlsbad, CA, USA) under reducing conditions using NuPAGE MES SDS electrophoresis buffer (Invitrogen). Proteins from the polyacrylamide gel were transferred to iBlot nitrocellulose membranes (Life Technologies, Bengaluru, India) using the iBlot dry blotting system (Life Technologies, Carlsbad, CA, USA). The membranes were then blocked for 60 minutes with 5% skim milk (Sigma-Aldrich, St. Louis, MO, USA) in TBST (0.1%, Tris-buffered saline (TBS) containing Tween-20). Subsequently, the membranes were incubated overnight at 4°C with 0.001 mg / mL of type XVII monoclonal antibody.

[0046] The following day, the membrane was washed three times with TBST for 10 minutes each, and then immediately incubated with the second antibody (HRP-labeled) (1:5000) for 60 minutes. The membrane was washed three times with TBST for 10 minutes each and incubated with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Waltham, MA, USA) for 1-5 minutes, and the protein bands were visualized with a C-DiGit BlotScanner (LI-COR Biosciences, Lincoln, NE, USA). After development, the membrane was washed with TBST for 15 minutes. Stripping was performed by incubating the membrane with 15 mL of Restore Western Blot Stripping Buffer (Thermo Fisher Scientific, Waltham, MA, USA) for 15 minutes. The membrane was then washed three times with TBST for 10 minutes each to remove the stripping buffer, and re-blocked for 1 hour with 5% skim milk in 30 mL of TBST. Subsequently, the membrane was incubated with a 1:1000 ratio of commercially available type XVII collagen antibody (MyBioSource, San Diego, CA, USA) and processed according to the previously described procedure.

[0047] Patient sample Cohort 1 consisted of 214 cancer patients and 23 healthy controls. Cohort 1 included 19 samples from bladder, breast, and prostate cancer; 20 samples from colorectal, head and neck, kidney, melanoma, ovarian, pancreatic, and gastric cancer; 17 samples from lung cancer; and 23 age-matched healthy controls. Cancer serum samples were purchased from Proteogenics (Los Angeles, CA, USA), and healthy controls were obtained from BioIVT (Westbury, NY, USA). A summary of the characteristics of Cohort 1 can be seen in Table 1.

[0048] [Table 1]

[0049] Cohort 2 included pre-treatment serum samples from 212 patients with metastatic colorectal cancer, part of the 2011-2016 CREBB study, “Evaluation of Biomarkers in Bevacizumab Therapy for Colorectal Cancer,” which originated from four Swedish hospitals and three Danish hospitals. Serum samples were collected at baseline before the application of palliative care with chemotherapy in combination with bevacizumab. The study was conducted in accordance with the recommendations of the Regional Committee on Healthcare Research Ethics in Denmark. The CREBB protocol was approved by the Regional Committee on Healthcare Research Ethics in Denmark (Approval ID: H-3-2010-121) and the Data Protection Authority (Approval ID: 2007-58-0015 / HEH.750.24-44). The study was conducted in accordance with the principles of the Declaration of Helsinki, and all patients submitted written informed consent before enrollment. Clinical data included: age, sex, total number of drugs, site, number of metastatic sites at study selection, performance status, and information on synchronous metastatic disease and primary tumor resection. A summary of the characteristics of Cohort 2 can be seen in Table 2.

[0050] [Table 2-1]

[0051] [Table 2-2]

[0052] statistics In Cohort 1, PRO-C17 levels in different cancer patients were compared to healthy controls using a one-way ANOVA followed by multiple comparisons using t-tests. The diagnostic accuracy of the assay was evaluated by the area under the receiver operating characteristic curve (AUROC). In Cohort 2, PRO-C17 levels in patients with metastatic colorectal disease were compared to those in healthy controls using t-tests. Kaplan-Meier curves were used to compare overall survival (OS) in patients with low PRO-C17 levels (tertile 1 + tertile 2) with that of patients with high PRO-C17 levels (tertile 3), and the difference between the curves was determined using a log-rank test. Multivariate Cox regression analysis, stratified by treatment line, was performed to evaluate independent predictors of PRO-C17 after adjusting for age, sex, total number of drugs, site, concurrent metastatic disease, performance status, primary tumor resection, number of metastatic sites, and PRO-C3 level at baseline. For statistical significance, the following p-values ​​were considered: * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001. Statistical analysis was performed using GraphPad Prism (version 9.5.0 for Windows, GraphPad Software, San Diego, California, USA, www.graphpad.com) and R version 4.2.2 (R Core Team (2022), R Foundation for Statistical Computing, Vienna, Austria, https: / / www.R-project.org).

[0053] result Development of PRO-C17 ELISA Optimization of the assay protocol involved selecting the optimal incubation time (20 hours) and antibody incubation temperature (4°C), as well as the concentrations of the assay buffer (50 mM PBS, 1% BSA (w / v), 0.018% Bronidocus (v / v), 0.1% Tween-20 (w / v), 8 g / L NaCl, pH 7.4) and other kit components. The final assay parameters were selected according to those that yielded the highest sensitivity in human serum, while adhering to the technical specifications listed below.

[0054] 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 group included a standard peptide consisting of 30 amino acids corresponding to the internal epitope of the NC16A domain of type XVII collagen (LQGMAPAAGADLDKIGLHSDSQEELWMFVRK (SEQ ID NO: 12)), an elongated peptide (RLQGMAPAAGA (SEQ ID NO: 7)), a truncated peptide (QGMAPAAGA (SEQ ID NO: 1)), and two non-selective peptides (LQGMAYTVQG (SEQ ID NO: 5) and LQGLAPLGSE (SEQ ID NO: 4)) (Figure 1). Although no cross-reactivity was observed for the two non-selective peptides, both the elongated and truncated peptides were recognized by the antibody (Figure 1). Epitope mapping was performed to test antibody affinity to adjacent epitopes corresponding to peptides derived from different physiological cleavage sites in the NC16A domain, including a further elongated (DSMDRIEKDRLQGMAPAAGADLDKIGLHSD (SEQ ID NO: 8)) and further truncated (GMAPAAGADL (SEQ ID NO: 3)) product of the selected peptide (Figure 2). The antibody recognized DSMDRIEKDRLQGMAPAAGADLDKIGLHSD (SEQ ID NO: 8), but no cross-reactivity was observed for GMAPAAGADL (SEQ ID NO: 3), suggesting that the antibody is sequence-specific rather than neoepitope-specific (Figure 3).

[0055] Western blot analysis showed that type XVII collagen ectodomain (120 kDa) was present in the A-431 supernatant, while the complete type XVII fragment (180 kDa) was detected only in the cell lysate (Figure 4). Dilution linearity was acceptable from 1:2 to 1:16. Matrix-in-matrix spike testing for accuracy showed a recovery rate of 99.6%. Interference from common endogenous analytes was observed but was within an acceptable recovery range (80-120%) for all samples. Intra-assay and inter-assay variability were 4.2% and 9.3%, respectively. Analyte stability was assessed at 4°C or 20°C for up to 48 hours, with an average recovery rate of 102.3%. Stability after 4 freeze-thaw cycles showed a recovery rate of 95.2%. Table 3 summarizes the different technical verification processes.

[0056] [Table 3]

[0057] When measuring serum samples from patients with non-small cell lung cancer (NSCLC), the PRO-C17 assay showed increased levels of type XVII collagen compared to healthy individuals, although this was not statistically significant (p>0.05), while the commercially available kit showed the opposite trend (Figure 5). Furthermore, there was no correlation between type XVII collagen levels in the two assays (r=-0.007357, p=0.9784), suggesting that the two assays target different epitopes of type XVII collagen (Figure 6).

[0058] PRO-C17 in patients with multiple cancers In Cohort 1, PRO-C17 levels were significantly elevated in serum from patients with colorectal cancer, renal cancer, ovarian cancer, bladder cancer, breast cancer, and head and neck cancer compared to healthy controls (p<0.05). These levels were also visually elevated (though not statistically significant) in serum from patients with pancreatic cancer, lung cancer, melanoma, prostate cancer, and gastric cancer (Figure 7). Table 4 shows the AUROC values ​​for different cancer types.

[0059] [Table 4]

[0060] PRO-C17 was particularly effective in differentiating between colorectal cancer patients and healthy controls with an AUROC of 0.904 (Table 4). These results suggest that circulating levels of PRO-C17 are elevated in several cancer types, and that PRO-C17 may be particularly associated with colorectal cancer.

[0061] PRO-C17 in the serum of patients with metastatic colorectal cancer predicts an unfavorable outcome. In Cohort 2, we found that PRO-C17 levels were elevated in serum from patients with metastatic colorectal cancer compared to healthy individuals (p<0.0001) (Figure 8). To investigate the prognostic value of PRO-C17, patients in Cohort 2 were divided into two groups, “low” (T1+T2) and “high” (T3), according to their biomarker levels. Kaplan-Meier survival analysis showed that patients with low PRO-C17 levels had a median overall survival (OS) of 539 days, compared to 390 days for patients with high PRO-C17 levels (log-rank p=0.007) (Figure 9). We then investigated the ability of PRO-C17 to predict OS using a multivariate Cox proportional-hazards model stratified by treatment line. When PRO-C17 levels were adjusted for covariates such as age, sex, total number of drugs, synchronous metastatic disease, PRO-C3 level at baseline, tumor site, surgical status, performance status, and number of metastatic sites, high PRO-C17 levels predicted poor OS and an increased risk of 53% death (HR=1.53, 95%CI=1.02, 2.28, p=0.04) (Figure 10).

[0062] conclusion The inventors have successfully developed, optimized, and validated an ELISA (PRO-C17 assay) for quantifying type XVII collagen fragments in blood. The PRO-C17 assay is technically robust, precise, and highly sensitive. The inventors further found that PRO-C17 levels were increased in patients with cancer, particularly colorectal cancer, compared to healthy controls. Furthermore, in patients with metastatic colorectal cancer treated with bevacizumab in combination with chemotherapy, high levels of the biomarker predicted poor overall survival and an increased risk of death.

[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 for detecting and / or monitoring cancer in a patient and / or assessing the severity of cancer in a patient, wherein the immunoassay method is: i) Contacting the patient's sample with a monoclonal antibody that specifically binds to the amino acid sequence QGMAPAAGA (SEQ ID NO: 1); ii) Detecting the binding between the monoclonal antibody and the peptide in the sample and determining the amount of binding; and, iii) The method includes 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.

2. The immunoassay method according to claim 1, wherein the monoclonal antibody specifically binds to the amino acid sequence LQGMAPAAGA (SEQ ID NO: 2).

3. The immunoassay method according to any one of the above claims, wherein the monoclonal antibody does not specifically bind to the amino acid sequence GMAPAAGADL (SEQ ID NO: 3).

4. The immunoassay method according to any of the above claims, wherein the monoclonal antibody does not specifically bind to the amino acid sequence LQGLAPLGSE (SEQ ID NO: 4).

5. The immunoassay method according to any one of the above claims, wherein the monoclonal antibody does not specifically bind to the amino acid sequence LQGMAYTVQG (SEQ ID NO: 5).

6. The immunoassay method according to any one of the above claims, wherein the monoclonal antibody is produced in opposition to a synthetic peptide having the amino acid sequence LQGMAPAAGA (SEQ ID NO: 2).

7. The immunoassay method according to any one of the above claims, wherein the cancer is pancreatic cancer, colorectal cancer, kidney cancer, ovarian cancer, bladder cancer, lung cancer, melanoma, breast cancer, head and neck cancer, prostate cancer, or gastric cancer.

8. The immunoassay method according to any of the above claims, wherein the cancer is colorectal cancer.

9. The immunoassay method according to any one of the claims, wherein the immunoassay method is a method for evaluating the severity of cancer in a patient.

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

11. The immunoassay method according to any one of the above 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.

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