PRO-C5 assay

Abstract

Disclosed is an immunoassay method for detecting and / or monitoring cancer in a patient, comprising measuring the level of the N-terminal amino acid sequence TAALGDIMGH (SEQ ID NO: 1) of the C-terminal propeptide cleaved from the α2 chain of type V collagen ("PRO-C5") in a patient sample.

Description

[Technical Field]

[0001] The present invention relates to immunoassay methods suitable for detecting and / or monitoring cancer in a subject. [Background technology]

[0002] Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease, with many patients presenting with metastatic disease, and only 20% of patients are suitable for surgery. [1-4] Patients with PDAC also suffer from a high degree of treatment resistance. Therefore, palliative care, including chemotherapy, is often the standard of care. [2] One explanation for the reduced therapeutic response is the fact that the PDAC tumor microenvironment is highly fibrotic. Cancer fibrosis causes increased interstitial pressure, reducing drug delivery. [5,6] Additionally, tumor fibrosis inhibits T-cell activity and migration, thereby reducing the efficacy of immunotherapy. [7-11] Therefore, there is a strong need for novel strategies to overcome cancer fibrosis in the treatment of PDAC patients, and therefore for identifying patients with high fibrotic activity.

[0003] Cancer fibrosis is characterized by increased activity of cancer-associated fibroblasts (CAFs), which leads to abnormal extracellular matrix (ECM) remodeling.

[12] CAF-mediated ECM remodeling results in the degradation of existing collagen fibers and their replacement with new, more densely packed collagen fibers, forming a stromal barrier surrounding cancer cells. [12,13] Type I collagen is the most abundant CAF-derived fibrillar collagen and is the major component of collagen fibers that play an important role during cancer progression. [14-19] Although the pathogenic phenotype of CAFs was initially thought to result from their ability to produce large amounts of collagen, recent studies focusing on type I collagen have highlighted the importance of collagen quality and fibrous structure as a central component in their tumorigenic potential. [14,20]Interestingly, type V collagen, a minor fibrillar collagen, has been suggested to be an important regulator of type I collagen structure.

[0004] Type V collagen has multiple roles in the healthy ECM and can bind to different ECM proteins such as other collagens, TGF-β, elastin, and metalloproteinases, thereby regulating cell behavior.

[21] The most abundant isoform of type V collagen consists of two α1 chains and one α2 chain, which form a heterotrimer. In healthy tissues, types I and V collagen are closely related, and the two collagens copolymerize to form heterotypic fibrils. [22-24] The binding of type V collagen to type I collagen is crucial for structural integrity. A good example is the Ehlers-Danlos syndrome, characterized by abnormal collagen fibrils, caused by a deficiency of type V collagen.

[25] Another study showed that type V collagen-deficient mice (COL5a1- / -) died at day 10.5 and lacked type I collagen fibrils. Furthermore, heterozygous type V collagen mice (COL5a1+ / -) survived but showed a 50% reduction in collagen content and fibril density.

[22] Thus, the binding between type V and type I collagen results in homeostatic fibrillar and tissue structure. Compared to type I collagen, type V collagen is expressed at relatively low levels in healthy tissues, and its role in cancer progression is not fully understood.

[25] Type V collagen has not only been shown to be upregulated in several cancers, such as colorectal cancer (CRC), gastric cancer, and breast cancer, but has also been shown to be associated with cell proliferation, invasion, metastasis, and angiogenesis. [24-34] . Summary of the Invention

[0005] Applicant explored the potential of measuring the level of the N-terminal amino acid sequence TAALGDIMGH (SEQ ID NO: 1) of the C-terminal propeptide cleaved from the α2 chain of type V collagen (also referred to herein as "PRO-C5," "target sequence," or "PRO-C5 target sequence") in the serum of cancer patients as a biomarker. Applicant demonstrated that PRO-C5 levels were significantly elevated in discovery and validation cohorts of PDAC patients, as well as in cohorts of patients with various other types of cancer, thus demonstrating the utility of PRO-C5 as a biomarker for cancer.

[0006] Thus, in a first aspect, the present invention provides an immunoassay method as described below. 1. An immunoassay method for detecting and / or monitoring cancer in a subject, the method comprising: i) contacting a patient's sample with a monoclonal antibody that specifically binds to the N-terminal amino acid sequence TAALGDIMGH (SEQ ID NO: 1); ii) detecting binding between the monoclonal antibody and the peptide in the sample and determining the amount of binding; and iii) correlating the amount of binding with values ​​associated with normal healthy individuals and / or with values ​​associated with known disease severity and / or with values ​​obtained from the subject at previous time points and / or with predetermined cut-off values.

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

[0008] As used herein, the term "monoclonal antibody" refers to both whole antibodies and fragments thereof that retain the binding specificity of the whole antibody, such as Fab fragments, F(ab')2 fragments, single-chain Fv fragments, or other such fragments known to those skilled in the art. As is well known, whole antibodies typically have a "Y-shaped" structure consisting of two identical paired polypeptide chains, each of which is composed of one "light" chain and one "heavy" chain. The N-terminal regions of each of the light and heavy chains comprise the variable region, while the C-terminal portions of each of the heavy and light chains constitute the constant region. The variable regions contain three complementarity-determining regions (CDRs), which are primarily responsible for antigen recognition. The constant region enables the antibody to recruit cells and molecules of the immune system. Antibody fragments that retain binding specificity contain at least the CDRs and a sufficient portion of the remainder of the variable region to retain binding specificity.

[0009] The present invention can use monoclonal antibodies containing any constant region known in the art. In mouse and human antibodies, constant light chains are classified as kappa or lambda light chains. Constant heavy chains are classified as mu, delta, gamma, alpha, or epsilon, defining the antibody isotype as IgM, IgD, IgG, IgA, or IgE, respectively. The IgG isotype has several subclasses, including IgG1, IgG2, IgG3, and IgG4 in humans, and several subclasses, including IgG1, IgG2a, IgG2b, IgG2c, and IgG3 in mice, but is not limited to these. The monoclonal antibody preferably belongs to the IgG isotype, including any one of the IgG subclasses.

[0010] The CDRs of an antibody can be determined using methods known in the art, such as those described by Kabat et al. Antibodies can be generated from B cell clones. The isotype of the antibody can be determined by ELISA specific for IgM, IgG, or IgA isotype or subclass. The amino acid sequence of the generated antibody can be determined using standard techniques. For example, RNA can be isolated from cells and used to generate cDNA by reverse transcription. The cDNA can then be subjected to PCR using primers that amplify the heavy and light chains of the antibody. For example, primers specific to the leader sequences of all VH (variable heavy) sequences can be used together with primers that bind to sequences located in the constant region of a predetermined isotype. The light chain can be amplified using a primer that binds to the 3' end of the kappa or lambda chain together with a primer that anneals to the leader sequence of Vkappa or Vlamda. Full-length heavy and light chains can be generated and sequenced.

[0011] As used herein, the term "amount bound" refers to the quantification of binding between an antibody and a peptide in a patient sample. This quantification may be determined, for example, by comparing the measured binding in the patient sample to a calibration curve generated using measured binding in standard samples containing known concentrations of the peptide to which the antibody specifically binds, to determine the amount of peptide in the patient sample to which the antibody specifically binds. Any analytical method suitable for measuring the amount of binding can be used. For example, an ELISA method can be used, which uses spectrophotometric analysis to measure both the amount of binding in the patient sample and the amount of binding when generating the calibration curve.

[0012] As used herein, the term "predetermined cutoff value" refers to a statistically determined amount of binding that indicates a high likelihood that a subject has a disease (i.e., cancer) or a particular severity of the disease, and in this sense, when a measured value of the target peptide in a subject's sample is equal to or greater than the statistical cutoff value, the measured value corresponds to at least a 70% probability, preferably at least a 75% probability, more preferably at least an 80% probability, more preferably at least an 85% probability, more preferably at least a 90% probability, and most preferably at least a 95% probability of the presence of the disease or of the disease being of a particular severity.

[0013] As used herein, the term "value associated with a normal healthy individual" refers to a normalized binding amount determined by the above method for a sample obtained from a subject who is considered healthy, i.e., disease-free (i.e., cancer-free); and the term "value associated with known disease severity" refers to a normalized binding amount determined by the above method for a sample obtained from a subject known to have a disease of known severity (i.e., cancer).

[0014] In a preferred embodiment, the method may be an immunoassay method for detecting and / or monitoring bladder cancer, breast cancer, colon cancer, head and neck cancer, renal cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer or gastric cancer, in particular, the method may be an immunoassay method for detecting and / or monitoring pancreatic ductal adenocarcinoma.

[0015] In certain embodiments, the method may be an immunoassay method for detecting a particular level of cancer severity in a subject. In some embodiments, the method may be a method for detecting a stage of cancer in a subject. In some embodiments, the method may be a method for detecting a level of cancer severity that is associated with prognosis or average overall survival.

[0016] In a preferred embodiment, the monoclonal antibody does not specifically bind to a peptide having the N-terminal amino acid sequence LTAALGDIMGH (SEQ ID NO: 2) (i.e., an extension of the PRO-C5 target sequence that is extended relative to the PRO-C5 target sequence by the addition of a lysine residue at its N-terminus). Preferably, the ratio of the affinity of the antibody for the PRO-C5 target sequence to the extension of the target sequence is at least 10:1, more preferably at least 20:1, or at least 30:1.

[0017] In a preferred embodiment, the monoclonal antibody does not specifically bind to a peptide having the N-terminal amino acid sequence AALGDIMGH (SEQ ID NO: 3) (i.e., a truncation of the PRO-C5 target sequence that is truncated relative to the PRO-C5 target sequence by removing the N-terminal threonine). Preferably, the ratio of the affinity of the antibody for the PRO-C5 target sequence to the affinity of the antibody for the truncated target sequence is at least 10:1, more preferably at least 20:1, or at least 30:1.

[0018] In a preferred embodiment, the monoclonal antibody is raised against a synthetic peptide having the N-terminal amino acid sequence TAALGDIMGH (SEQ ID NO: 1). For example, a monoclonal antibody can be raised by: (a) immunizing a rodent (or other suitable mammal) with a synthetic peptide comprising the N-terminal amino acid sequence TAALGDIMGH (SEQ ID NO: 1), optionally linked to an immunogenic carrier protein (such as keyhole limpet hemocyanin) at its C-terminus; (b) isolating and cloning a single antibody-producing cell; and (c) assaying the resulting monoclonal antibodies to determine that they have the desired specificity.

[0019] In a preferred embodiment, the patient sample is selected from blood, serum, or plasma. In a preferred embodiment, the immunoassay is a competitive assay or a sandwich assay. The immunoassay may be, for example, a radioimmunoassay or an enzyme-linked immunosorbent assay (ELISA). Such assays are techniques known to those skilled in the art.

[0020] In a second aspect, the present invention provides a method of treating cancer in a patient in need thereof, the method comprising the steps of: (a) performing an immunoassay for detecting cancer according to the first aspect of the invention on a sample obtained from the patient; and (b) if in step (a) it is determined that the patient has cancer, administering to the patient a therapy to treat the cancer.

[0021] Preferred embodiments of the method according to the second aspect will be apparent from the above discussion of preferred embodiments of the method according to the first aspect. For example, step (a) may comprise performing an immunoassay for detecting, inter alia, bladder cancer, breast cancer, colon cancer, head and neck cancer, kidney cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer or gastric cancer; and / or for detecting cancer of a particular severity.

[0022] The therapy may be any therapy suitable for treating the target cancer. The therapy may include or consist of, for example, one or more surgeries, one or more radiation therapies, one or more medications (e.g., one or more chemotherapy, one or more immunotherapy, and / or one or more hormone therapy), or a combination thereof. The medication may be formulated for local or systemic administration. Topical medications may be formulated, for example, as creams, foams, gels, lotions, or ointments. Systemic medications may be formulated, for example, for administration via the gastrointestinal tract or parenterally. The surgery may be curative surgery, preventative surgery, debulking surgery, palliative surgery, and / or reconstructive surgery.

[0023] For example, if the cancer is bladder cancer, suitable therapies include, for example, one or more of the following: transurethral resection of bladder cancer (TURBT) with or without intravesical chemotherapy or intravesical immunotherapy; radical cystectomy with neoadjuvant chemotherapy, or transurethral resection with chemoradiotherapy, or partial cystectomy with neoadjuvant chemotherapy; cisplatin-based chemotherapy, optionally followed by radical cystectomy or chemoradiotherapy; carboplatin-based chemotherapy; immune checkpoint inhibitors; radical cystectomy; and palliative radiation therapy.

[0024] Where the cancer is breast cancer, appropriate therapies 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 the production of estrogen (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 radiation therapy.

[0025] If the cancer is colon cancer, suitable therapies include, for example, one or more of the following: endoscopic mucosal resection or endoscopic submucosal dissection; partial colectomy (or proctocolectomy of the rectal region); chemotherapy agents such as capecitabine, fluorouracil, irinotecan, oxaliplatin, or UTF; antiangiogenic agents such as bevacizumab; epidermal growth factor receptor inhibitors such as aflibercept, cetuximab, and panitumumab; radiation therapy; immune checkpoint inhibitors; and monoclonal antibodies such as pembrolizumab and dostarlimab.

[0026] Where the cancer is head and neck cancer, suitable therapies include, for example, one or more of the following: surgery, including, but not limited to, laser surgery; radiation therapy, including, but not limited to, three-dimensional conformal radiation therapy, intensity-modulated radiation therapy, 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 Gendicine; and immune checkpoint inhibitors.

[0027] If the cancer is renal cancer, appropriate therapies include, for example, one or more of the following: surgical removal of all or part of the kidney; freezing or hyperthermia of the cancer; biological therapy 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 radiation therapy.

[0028] Where the cancer is lung cancer, suitable therapies include, for example, one or more of the following: surgery, such as performing a lobectomy, segmentectomy (wedge resection), or pneumonectomy (lung resection); radiation therapy, specific examples of which include radiation therapy in combination with chemotherapy, radiation therapy given after surgery, brachytherapy (localized radiation therapy) applied directly to the inside of the airways, prophylactic whole-brain irradiation, stereotactic radiation therapy, and palliative radiation therapy. chemotherapy using one or more agents such as cisplatin / carboplatin, etoposide, gemcitabine, paclitaxel, docetaxel, vinorelbine, topotecan, irinotecan, and pemetrexed; epidermal growth factor receptor (EGR) 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.

[0029] If the cancer is melanoma, suitable therapies include, for example, one or more of the following: surgical removal of the cancer and, optionally, lymph nodes in the area of ​​the cancer; interferon treatment; chemotherapy, for example, using agents such as dacarbazine; small molecule targeted therapy, for example, using BRAF inhibitors (such as vemurafenib and dabrafenib), MEK inhibitors (trametinib), C-kit inhibitors, and / or NRAS inhibitors; cytokines (e.g., IL-2 and / or IFN-α), immune checkpoint inhibitors, and / or anti-cancer drugs. immunotherapy using checkpoint inhibitors (e.g., anti-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 leratolimab, and / or PD-L1 antibodies, etc.) and / or adoptive cell transfer (e.g., using pre-stimulated, modified T cells or dendritic cells); and radiation therapy.

[0030] Where the cancer is ovarian cancer, suitable therapies include, for example, one or more of the following: removal of one ovary (unilateral oophorectomy) or both ovaries (bilateral oophorectomy), optionally with removal of the fallopian tube (salpingectomy), uterus (hysterectomy), and / or retina (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; radiation therapy; hormone therapy; immunotherapy, such as the antibody drug bevacizumab.

[0031] For example, if the cancer is pancreatic cancer such as PDAC, appropriate therapies include, for example, one or more of the following: surgical resection, such as cancer resection, Whipple procedure, total pancreatectomy, or distal pancreatectomy; radiation therapy, including, but not limited to, radiation therapy combined with chemotherapy, postoperative radiation therapy, brachytherapy (localized radiation therapy), stereotactic radiation therapy, and palliative radiation therapy; chemotherapy using one or more agents such as gemcitabine, 5-FU, erlotinib, FOLFIRINOX, nab-paclitaxel, or combinations thereof; somatostatin analogs; lanreotide; targeted therapy using everolimus or sunitinib; nuclear medicine therapy using radiolabeled peptides or hormones such as iobenguane; and procedures such as radiofrequency ablation (RFA), cryoablation, or transcatheter arterial embolization.

[0032] Where the cancer is prostate cancer, suitable therapies include, for example, one or more of the following: radiation therapy; chemotherapy using chemotherapeutic agents such as docetaxel, cabazitaxel, docetaxel, thalidomide, and combinations thereof; immunotherapy such as the monoclonal antibody bevacizumab; hormonal therapy such as abiraterone and enzalutamide; external beam radiation therapy; particle beam radiation therapy; high intensity focused ultrasound; cryotherapy; and surgical procedures such as radical prostatectomy.

[0033] When the cancer is gastric cancer, suitable therapies include, for example, one or more of the following: 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 combinations thereof; targeted therapy using epidermal growth factor receptor 2 inhibitors such as trastuzumab; and radiation therapy. [Brief explanation of the drawings]

[0034] figure [Figure 1] Figure 1: Type V collagen regulation of type I collagen structure. Collagen fibers are composed of collagen fibrils, which are formed by the self-assembly of collagen microfibrils. (A) Type V collagen is incorporated into the structure of type I collagen microfibrils and regulates the structural integrity of type I collagen through homeostatic functions. (B) Deficiency of type V collagen (low COLV) leads to abnormal type I collagen fibrils and the well-known Ehlers-Danlos syndrome. Increased type V collagen expression (high COLV) causes type I collagen fibrils to become more condensed and linear, generating the dense fibers characteristic of cancer fibrosis. COL I: Type I collagen. COL V: Type V collagen.

[0035] [Figure 2] Figure 2: PRO-C5 is elevated in serum from patients within the PDAC discovery cohort. (A) Individual serum PRO-C5 levels in PDAC patients (n = 33), chronic pancreatitis patients (n = 12), and healthy controls (n = 20). (B) Individual serum PRO-C5 levels in patients with PDAC stages I, II, III, and IV. The association between PRO-C5 levels and PDAC stage was analyzed using least squares regression analysis.

[0036] [Figure 3]Figure 3: High levels of serum PRO-C5 are associated with poor overall survival (OS) in the discovery cohort. Kaplan-Meier survival plot showing the association between overall survival and PRO-C5 levels. Patients were stratified into quartiles; Q1 includes patients with the lowest levels of PRO-C5, and Q4 includes patients with the highest levels of PRO-C5. Hazard ratios (HRs), 95% confidence intervals (CIs), and log-rank tests are shown.

[0037] [Figure 4] Figure 4: High serum PRO-C5 levels are associated with poor overall survival (OS) in the validation cohort. (A) Individual serum PRO-C5 levels in patients with PDAC stages I, II, III, and IV. The association between PRO-C5 levels and PDAC stage was analyzed using least squares regression analysis. (B) Kaplan-Meier survival plot showing the association between overall survival and PRO-C5 levels. Patients were stratified into those with low (Q1-Q3) and high (Q4) PRO-C5 serum levels. Hazard ratios (HRs), 95% confidence intervals (CIs), and log-rank tests are shown.

[0038] [Figure 5] Figure 5: High serum levels of PRO-C5 are associated with poor OS in PDAC stages II, III, and IV within the validation cohort. Kaplan-Meier survival plots showing the association of PRO-C5 levels with overall survival in stage II (A), stage III (B), and stage IV (C). In all plots, patients were stratified into low (Q1-Q3) and high (Q4) levels of PRO-C5 serum. Hazard ratios (HRs), 95% confidence intervals (CIs), and log-rank tests are shown.

[0039] [Figure 6]Figure 6: PRO-C5 is elevated in serum from patients with different types of cancer. Individual serum PRO-C5 levels in patients with various stages of cancer, including bladder cancer (n = 20), breast cancer (n = 20), colorectal cancer (n = 20), head and neck cancer (n = 20), renal cancer (n = 20), lung cancer (n = 20), malignant melanoma (n = 20), ovarian cancer (n = 20), PDAC (n = 20), prostate cancer (n = 20), or gastric cancer (n = 20), are shown compared with healthy controls (n = 33). Ns: not significant; *p<0.05, ***p<0.001, ****p<0.0001.

[0040] [Figure 7] Figure 7: Specificity of the PRO-C5 assay. Reactivity to standard peptides, extended peptides, and nonsense peptides is shown. [Example]

[0041] The embodiments disclosed herein are described in the following examples. These examples are presented to aid in understanding the present disclosure and should not be construed in any way to limit the scope of the disclosure as defined in the claims that follow. The examples set forth below are presented so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the described embodiments, and are not intended to limit the scope of the disclosure, nor are they intended to imply that the experiments described below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

[0042] Materials and Methods patient statistics Cohort 1 (PDAC discovery cohort) consisted of 19 age- and sex-matched healthy controls, 12 pancreatitis patients, and 33 PDAC patients (stages I-IV). Cohort 2 (PDAC validation cohort) consisted of 800 PDAC patients (stages I-VI). Serum samples from patients with pancreatitis and PDAC in cohorts 1 and 2 were obtained from the Danish BIOPAC study "Biomarkers in Patients with Pancreatic Cancer (BIOPAC) - Can They Provide New Information About the Disease and Improve Patient Diagnosis and Prognosis?" (ClinicalTrials.gov ID: NCT03311776). This study has been described elsewhere. [35-37] This study was conducted in accordance with the Danish Regional Committee for Health Research Ethics. The BIOPAC protocol was approved by the Danish Regional Committee for Health Research Ethics (VEK ref. KA-20060113; and the retrospective protocol VEK H-17039022) and the Data Protection Authority (J.nr. 2006-41-6848, 2012-58-0004, HGH-2015-027; I-Suite J.nr. 03960; and PACTIUS P-2020-834). All subjects signed informed consent in accordance with the 8th edition of the Declaration of Helsinki. Serum samples and clinical data were collected prospectively. Subjects were followed until September 2022 or death, whichever occurred first. Serum samples were blinded to clinical characteristics. Clinical data included age, sex, stage (American Joint Commission, eighth edition), number of metastatic sites, liver metastases, body mass index (BMI), tobacco consumption, alcohol consumption, carbohydrate antigen 19-9 (CA19-9), performance status (PS), Charlson age comorbidity index (CACI), and overall survival (OS).

[0043] Cohort 3 consisted of 33 age- and sex-matched healthy controls and 220 patients with different types of cancer: bladder cancer (n=20), breast cancer (n=20), colorectal cancer (n=20), head and neck cancer (n=20), renal cancer (n=20), lung cancer (n=20), malignant melanoma (n=20), ovarian cancer (n=20), PDAC (n=20), prostate cancer (n=20), and gastric cancer (n=20). Cancer samples from Cohort 3 were obtained from a commercial vendor, Proteogenex (CA, USA). Healthy controls for Cohorts 1 and 3 were obtained from Valley Biomedical (VA, USA). An appropriate institutional review board / independent ethics committee approved sample collection, and all subjects provided informed consent. Patient demographics for Cohorts 1 and 2 are shown in Table 1, and patient demographics for Cohort 3 are shown in Table 2.

[0044] [Table 1-1]

[0045] [Table 1-2]

[0046] [Table 1-3]

[0047] [Table 2]

[0048] Monoclonal antibody development The PRO-C5 monoclonal antibody, i.e., a monoclonal antibody specific for the PRO-C5 target sequence (TAALGDIMGH (SEQ ID NO: 1)) located at the N-terminal beginning of the C-terminal propeptide cleaved from the α2 chain of type V collagen, was generated as previously described.

[39] . Briefly, six 4- to 6-week-old Balb / C mice were immunized by subcutaneous injection into the abdominal region of 200 μL of emulsified antigen containing 50 μg of the immunogenic peptide (TAALGDIMGH-GGC-OVA (SEQ ID NO: 4)) in Freund's incomplete adjuvant. Immunizations were performed every two weeks until a stable titer level was achieved. Serum antibody titers were measured at each blood draw, and mice with the highest antibody titers and the best serum and urine reactivity were selected for cell fusion. The selected mice were boosted intravenously with 100 μL of 0.9% sodium chloride solution containing 50 μg of the immunogenic peptide 3 days before harvesting the spleen for cell fusion.

[0049] To generate hybridoma cells, mouse splenocytes were fused with mouse SP2 / 0 myeloma cells as described by Gefter et al. Hybridoma cells were cloned using limiting dilution and plated into 96-well microtiter plates for further expansion. Monoclonal growth was promoted using standard limiting dilution methods. Supernatants were screened using indirect ELISA, which used the biotinylated peptide TAALGDIMGH-GGC-Biotin (SEQ ID NO: 5) as a capture peptide on streptavidin-coated microtiter plates.

[0050] Native reactivity and peptide binding of the monoclonal antibodies in human and rat serum and urine was assessed using a preliminary ELISA using 10 ng / mL biotinylated peptide coaters on streptavidin-coated microtiter plates and supernatants obtained from growing monoclonal hybridoma cells. The specificity of the clones was tested against a standard peptide (TAALGDIMGH (SEQ ID NO: 1)) and a nonsense peptide. Monoclonal antibody isotyping was performed using the Clonotyping System-HRP kit, cat. 5300-05 (Southern Biotech, Birmingham, AL). Selected clones were purified and dialyzed using a protein G column according to the manufacturer's instructions (GE Healthcare Life Sciences, Little Chalfont, Buckinghamshire, UK).

[0051] Antibody specificity The specificity of the PRO-C5 monoclonal antibody was calculated as the percentage of signal inhibition by two-fold dilutions of the standard peptide (TAALGDIMGH (SEQ ID NO: 1)), the extended peptide (LTAALGDIMGH (SEQ ID NO: 2)), and the nonsense peptide.

[0052] Assessment of monoclonal PRO-C5 levels in human serum Serum levels of PRO-C5 were measured using an ELISA-based PRO-C5 assay according to the manufacturer's instructions (Nordic Biosciences A / S, Denmark). Details of the assay methodology have been previously described. [38,39] .

[0053] Briefly, 96-well streptavidin plates (Roche Diagnostics, Basel, Switzerland) were coated with 3 ng of biotinylated synthetic peptide (TAALGDIMGH-GGC-Biotin (SEQ ID NO: 5)) diluted in coater buffer (25 mM PBS, 1% BSA, 0.1% TWEEN®-20, pH 7.4, and 0.36% bronidox) and incubated at 20°C for 30 min. Twenty microliters of peptide calibrator (TAALGDIMGH (SEQ ID NO: 1)) or sample was added to the appropriate wells, followed by 100 μL of a dilution of 15 ng of a conjugate of PRO-C5 monoclonal antibody and horseradish peroxidase (HRP) (Innovabioscience, Babraham, Cambridge, UK) in the same buffer used for coating and incubation at 20°C for 1 hour. Finally, 100 μL of tetramethylbenzidine (TMB) (Tem-En-Tec cat. 438 OH, Taastrup, Denmark) was added, and the plate was incubated for a further 15 minutes at 20°C in the dark. All incubation steps were performed with shaking at 300 rpm. After each incubation step, the plate was washed five times with wash buffer (20 mM Tris, 50 mM NaCl, pH 7.2). The TMB reaction was stopped by adding 100 μL of stop solution (1% HCl) and measured at 450 nm with a reference at 650 nm. A calibration curve was plotted using a four-parameter mathematical fitting model with a starting concentration of 200 ng of the standard peptide (TAALGDIMGH (SEQ ID NO: 1)) followed by two-fold dilutions.

[0054] statistics Biomarker results are reported in REMARK (reporting recommendations for tumor marker prognostic study ) reported in accordance with guidelines

[40] .

[0055] The Kruskal-Wallis multiple comparison test was used to test for differences in PRO-C5 serum levels between healthy controls, patients with pancreatitis, and patients with PDAC (Cohort 1), as well as between healthy controls and patients with 11 different cancer diagnoses (Cohort 3). Least-squares regression analysis was used to assess the association between PRO-C5 serum levels and PDAC stage in Cohort 1 and Cohort 2. Kaplan-Meier curves were used to assess the association between high and intermediate PRO-C5 serum levels and OS.

[0056] In Cohort 1, patients were stratified into quartiles according to PRO-C5 serum levels (Q1, Q2, Q3, and Q4). In Cohort 2, patients were stratified into two groups: one group including the Q1-Q3 quantiles and the other group including the Q4 quantile. When investigating the association between PRO-C5 serum levels and OS in specific stages of PDAC, patients were also stratified into two groups (Q1-Q3 vs. Q4). In Cohort 1, hazard ratios (HRs) with 95% confidence intervals (CIs) for short overall survival were calculated by level of PRO-C5 biomarker (Q4 or Q3 or Q2 vs. Q1) using univariate Cox proportional-hazards regression models.

[0057] In cohort 2, univariate Cox proportional-hazards regression models were used to calculate hazard ratios (HRs) with 95% confidence intervals (CIs) for short overall survival by PRO-C5 biomarker level (all categories and >Q1-Q3 vs. ≤Q4) and the following clinical covariates: age (all categories), sex (female vs. male), number of metastatic sites (≥1 vs. 0), liver metastases (yes vs. no), BMI (all categories), stage (all categories), diabetes (yes vs. no), smoking (yes vs. no), alcohol intake (below Danish Health Authority recommendations [DHAR] vs. above recommendations), CA19-9 (>median vs. ≤median [median = 483 U / mL]), PS (1 + 2 + 3 vs. 0), and CACI (≥4 vs. <4).

[0058] In addition, in cohort 2, the independent prognostic potential of the PRO-C5 biomarker for predicting mortality risk was assessed using a multivariate Cox proportional hazards regression model including PRO-C5 (category-by-category and >Q1-Q3 vs. ≤Q4), age, metastatic site (≥1 vs. 0), liver metastasis (yes vs. no), stage (category-by-category), CA19-9 (>median vs. ≤median [median = 483 U / mL]), PS (1 + 2 + 3 vs. 0), and CACI (≥4 vs. <4). When the model was applied to patients of individual stages, only age, CA19-9 (>median vs. ≤median [median = 483 U / mL]), PS (1 + 2 + 3 vs. 0), and CACI (≥ 4 vs. < 4) were included for stage II and III patients. For stage IV patients, age, CA19-9 (>median vs. ≤median [median = 483 U / mL]), PS (1 + 2 + 3 vs. 0), CACI (≥ 4 vs. < 4), metastatic site (≥ 1 vs. 0), and liver metastasis (yes vs. no) were included. P values ​​< 0.05 were considered statistically significant. Graphs and statistical analyses were performed using GraphPad Prism version 9 (GraphPad Software, Inc., La Jolla, CA) and MedCalc version 19.3 (Medcalc Software).

[0059] result antibody specificity To evaluate the specificity of the PRO-C5 monoclonal antibody and assay, the antibody was tested against the standard peptide, the extended peptide, and the nonsense standard peptide. No reactivity was observed against the extended peptide or the nonsense standard peptide, demonstrating experimentally the specificity of the antibody and assay for the PRO-C5 target sequence (Figure 7).

[0060] PRO-C5 is elevated in PDAC patients and associated with poor overall survival (OS) - Discovery cohort. The PDAC discovery cohort (Cohort 1) included 19 healthy controls, 12 pancreatitis patients, and 33 PDAC patients (stages I-IV) (Table 1). Serum PRO-C5 levels were significantly elevated in PDAC patients compared with healthy controls (PDAC: 1071.3 ng / mL vs. healthy controls: 549.5 ng / mL, p<0.001). PRO-C5 levels were not significantly elevated in pancreatitis patients (pancreatitis: 786.4 ng / mL) compared with healthy controls, nor were they significantly elevated in PDAC patients compared with pancreatitis. However, there was a trend toward increased PRO-C5 levels from healthy controls to pancreatitis and then to PDAC (Figure 2A). When PDAC patients were stratified according to disease stage (stages I-IV), there was a significant correlation (least squares regression: p=0.0005) between serum PRO-C5 levels and disease stage (Figure 2B).

[0061] To investigate the association between PRO-C5 serum levels and OS, the prognostic potential of PRO-C5 was evaluated using Kaplan-Meier curves and univariate Cox proportional hazards models. Patients were stratified into quartiles: Q1 included patients with the lowest PRO-C5 levels, and Q4 included patients with the highest PRO-C5 levels. Patients in Q1 had a median OS of 28.0 months, patients in Q2 had a median OS of 7.1 months, patients in Q3 had a median OS of 15.5 months, and patients in Q4 had a median OS of 3.8 months (log-rank, p = 0.0005). Thus, the difference in median OS between Q4 and Q1 was more than 2 years (24.2 months) (Figure 3). In support of this, a univariate Cox proportional-hazards model showed that the risk of death in patients in Q4 was increased by 940% compared with patients in Q1 (Q4 vs. Q1: HR 95% CI: 10.4 (2.9-37.2), p=0.0003).

[0062] PRO-C5 is elevated in PDAC patients and associated with poor overall survival (OS) - Validation cohort. To validate the prognostic potential of PRO-C5 in the PDAC discovery cohort, we measured serum PRO-C5 levels from the PDAC validation cohort (BIOPAC, Cohort 2) (Table 1), which included 800 PDAC patients (stages I-IV). Overall, the findings from the discovery cohort were confirmed. When patients were stratified by stage (stages I-IV), there was a significant correlation (least squares regression: p = 0.0008) between serum PRO-C5 levels and disease stage (Figure 4A). To evaluate the association between serum PRO-C5 levels and OS in the PDAC validation cohort, patients were stratified into two groups based on the results from the discovery cohort: one group included patients with relatively low serum PRO-C5 levels (Q1-Q3), and the other group included patients with the highest serum PRO-C5 levels (Q4). Patients with relatively low PRO-C5 levels had a median OS of 10.1 months, whereas patients with the highest PRO-C5 levels had a median OS of 6.4 months (log-rank, p<0.0001) (Figure 4B). Furthermore, univariate Cox proportional hazards modeling showed that the risk of death in patients with the highest PRO-C5 levels was increased by 50% compared with patients with relatively low PRO-C5 levels (high PRO-C5 vs. low PRO-C5: HR 95% CI: 1.5 (1.3-1.8), p<0.0001) (Figure 4B and Table 3).

[0063] To assess whether the association between OS and PRO-C5 was independent of clinical covariates, a multivariate Cox proportional hazards model including age, number of metastatic sites, liver metastasis, stage, CA19-9, PS, and CACI was performed. The model showed that the prognostic value of PRO-C5 remained statistically significant even when adjusting for clinical covariates (HR 95% CI: 1.4 (1.2-1.6), p = 0.0002) (Table 3). This indicates that PRO-C5 is a risk factor independent of other common risk factors.

[0064] [Table 3-1]

[0065] [Table 3-2]

[0066] [Table 3-3]

[0067] Abbreviations: BMI, body mass index; CACI, Charlson age comorbidity index; CA19-9, carcinoembryonic antigen 19-9; DHAR, Danish Health Authority recommendation; No, number; and PS, performance status. Hazard ratio (HR), 95% confidence interval (CI). *p-value indicates significance.

[0068] We further evaluated the association between high serum PRO-C5 levels and OS in patients stratified by specific stages of PDAC. Patients with stage I disease were not analyzed due to the low number (n = 15).

[0069] In stage II patients (n=117), patients with high serum PRO-C5 levels had a median OS of 8.5 months compared with 24.3 months for patients with low serum PRO-C5 levels (log-rank, p=0.0041) (Figure 5A). Thus, the difference in median OS between low and high PRO-C5 was more than 1 year (15.8 months). Furthermore, univariate Cox proportional hazards modeling showed that patients with stage II PDAC and high PRO-C5 levels had a 100% increased risk of death compared with patients with low PRO-C5 levels (high PRO-C5 vs. low PRO-C5: HR 95% CI: 2.0 (1.2-3.3), p = 0.0049) (Figure 5A). Furthermore, a multivariate Cox proportional-hazards model including age, CA19-9, PS, and CACI showed that the association between high levels of PRO-C5 and OS was independent of clinical covariates in patients with PDAC stage II (high PRO-C5 vs. low PRO-C5: HR 95% CI: 2.0 (1.2-3.4), p = 0.0100) (Table 4).

[0070] Among patients with stage III PDAC (n = 227), patients with low serum PRO-C5 levels had a median OS of 13.1 months compared with 9.5 months for patients with high serum PRO-C5 levels (log-rank, p = 0.0191) (Figure 5B). Univariate and multivariate Cox proportional hazards models confirmed that patients with stage III PDAC and high PRO-C5 levels had a 50% increased risk of death compared with patients with low PRO-C5 levels (high PRO-C5 vs. low PRO-C5: HR 95% CI: 1.5 (1.1-2.1), p = 0.0191) (Figure 5B). Furthermore, a multivariate Cox proportional-hazards model including age, CA19-9, PS, and CACI showed that the association between high levels of PRO-C5 and OS was independent of clinical covariates in patients with PDAC stage III (high PRO-C5 vs. low PRO-C5: HR 95% CI: 1.5 (1.1-2.1), p = 0.0177) (Table 4).

[0071] [Table 4-1]

[0072] [Table 4-2]

[0073] Abbreviations: CACI, Charlson Age Comorbidity Index; CA19-9, Carcinoembryonic Antigen 19-9; No, number; and PS, performance status. Hazard ratio (HR), 95% confidence interval (CI). *p-value indicates significance.

[0074] In patients with PDAC stage IV (n = 435), patients with low serum PRO-C5 levels had a median OS of 6.3 months compared with 4.8 months for patients with high serum PRO-C5 levels (log-rank, p = 0.0012) ( Figure 5C ). Univariate Cox proportional hazards model showed that patients with stage III PDAC and high PRO-C5 levels had a 40% increased risk of death compared with patients with low PRO-C5 levels (high PRO-C5 vs. low PRO-C5: HR 95% CI: 1.4 (1.1-1.7), p = 0.0012) (Figure 5C). Furthermore, a multivariate Cox proportional hazards model including age, CA19-9, PS, CACI, number of metastatic sites, and liver metastasis showed that the association between high PRO-C5 levels and OS was independent of clinical covariates in patients with stage IV PDAC (high PRO-C5 vs. low PRO-C5: HR 95% CI: 1.3 (1.1-1.6), p = 0.0085) (Table 4).

[0075] PRO-C5 biomarker is increased in sera from patients with different types of cancer Next, we explored the potential of PRO-C5 as a biomarker in other cancer types. We measured serum PRO-C5 levels from patients with various stages of bladder cancer (n = 20), breast cancer (n = 20), colorectal cancer (n = 20), head and neck cancer (n = 20), renal cancer (n = 20), lung cancer (n = 20), malignant melanoma (n = 20), ovarian cancer (n = 20), PDAC (n = 20), prostate cancer (n = 20), or gastric cancer (n = 20) and compared them with healthy controls (n = 33) (Table 2). PRO-C5 levels were elevated in all cancer diagnoses compared with healthy controls. The elevations were statistically significant (p range, <0.05-0.0001) except for prostate and gastric cancer (Figure 6), which also showed clear elevations of PRO-C5 levels. This suggests that PRO-C5 has potential as a biomarker for diseases other than PDAC.

[0076] [Essay] The potential biomarker offered by measuring the α2 chain of type V collagen propeptide (PRO-C5) was evaluated in serum from cancer patients, particularly PDAC. In a discovery cohort including PDAC patients, pancreatitis patients, and healthy controls, PRO-C5 levels were elevated in PDAC patients compared with pancreatitis patients and healthy controls. Furthermore, high levels of PRO-C5 were associated with later stages of PDAC, and PDAC patients with high serum PRO-C5 levels had an increased risk of death compared with patients with low levels. PRO-C5 has previously been known to be associated with fibrosis in liver disease.

[63] This is the first time that PRO-C5 has been found to be associated with cancer.

[0077] These initial results were validated in the BIOPAC cohort. In the BIOPAC cohort, high PRO-C5 was associated with poor OS in stage II, III, and IV disease, independent of other risk factors, including CA19-9. Interestingly, the largest relative difference in OS was observed in stage II patients. In stage II patients, patients with low PRO-C5 had a median OS >1 year longer than patients with high PRO-C5. In contrast, CA19-9 was also independently associated with OS in patients with stage III and IV disease, but not in stage II disease. Beyond PDAC, the association of PRO-C5 was observed in other solid tumor types, with distinctly elevated PRO-C5 levels in numerous cancer types. While upregulation of the α2 chain gene expression has been observed in numerous cancer diagnoses, [24-34] ,No research has been conducted on PRO-C5 to date.

[0078] Similar to PRO-C5, several other biomarkers arising from the fibrotic compartment of cancer have shown significant potential as prognostic and predictive biomarkers across various cancer diagnoses, including PDAC. [36、37、41-48]As an example, PRO-C3, which measures the formation of type III collagen, has been shown to be prognostic for OS in PDAC patients. [36、49、50] Furthermore, measuring type III collagen turnover has been shown to predict response to the investigational antifibrotic drug PEGPH20, significantly better than tissue biopsies.

[46] Like type I collagen, type III collagen is also a major fibrillar collagen and is present in both healthy and diseased tissues.

[51] In contrast to type I and type III collagens, which form fibrillar masses, type V collagen is thought to be a more minor collagen, supporting the major fibrillar collagens in the fibrillar assembly. In recent years, fibrillar types V and XI collagens, as well as the FACIT collagens types XIX, XX, and XXII, have gained increasing interest as more disease-specific biomarkers and / or targets. Biomarkers measuring types XI, XIX, XX, and XXII collagens have also been found to be upregulated in various cancers. [37、52-54] Furthermore, types XI, XX, and XXII collagens can all predict prognosis for short OS in PDAC patients. [37、52、53] These minor collagens are expressed at high levels primarily during embryonic development and cancer progression, and their upregulation may be more pathological than the relative upregulation of the already abundant major collagens.

[0079] Interestingly, despite the fact that the major type I collagen is important for cancer progression and has a close interaction with type V collagen, biomarkers measuring serum type I collagen formation were not elevated in different cancer types within the same cohort compared to controls (cohort 3) where PRO-C5 was measured here.

[52] This supports the hypothesis that changes in cancer fibrosis may involve more than just type I collagen. This may be because type I collagen is the most abundant protein in the body and therefore less sensitive as a serological biomarker than the less abundant type V collagen, which also supports the important role of type V collagen in cancer fibrosis.

[0080] Furthermore, it should be noted that not all biomarkers derived from type V collagen are suitable for measuring cancer. It has been found that the levels of C5M (a biomarker derived from type V collagen but unrelated to PRO-C5) do not vary between healthy controls and patients with bladder cancer, breast cancer, colon cancer, liver cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, or gastric cancer (data not shown). Therefore, the PRO-C5 assay is specific and can distinguish cancer patients from healthy controls.

[0081] Cancer fibrosis is recognized as a process that stiffens tissue and results in the production of more linearly aligned collagen fibers by CAFs. [36、55-57] Cancer cells are thought to use these linearized collagen fibers to metastasize.

[58] One study showed that when the collagen V / collagen I ratio was increased in vitro, fibrils became smaller. [59、60] However, what this means exactly in terms of biological function remains unclear. Recently, Chen et al. showed that pancreatic cancer cells produce a unique type I collagen homotrimer (α1 / α1 / α1) compared to the type I collagen heterotrimer (α1 / α2 / α1) produced by fibroblasts.

[14] Furthermore, they showed that deletion of type I collagen homotrimers increased survival in a PDAC mouse model, as well as T cell infiltration and efficacy against anti-PD-1 immunotherapy.

[14] Studies have also shown that type V collagen can exist as both a homotrimer and a heteropolymer, e.g., α1(V)2α2(V), α1(V)3, or α1(V)α2(V)α3(V). [25、61、62] The impact of this has yet to be determined. Overall, Applicant hypothesizes from these data that increased type V collagen expression leads to the linearization of more type I collagen fibers, promoting cancer progression and metastasis, as depicted in Figure 1. Because PDAC is a fatal disease, it is important to treat patients as soon as possible, before they reach the later stages of PDAC. The PRO-C5 assay could be used to guide treatment already in stage 2 PDAC. Furthermore, with the increasing recognition of cancer fibrosis, interest in anti-cancer fibrosis compounds continues to grow. Therefore, the PRO-C5 assay may have potential for monitoring drug efficacy and detecting susceptible individuals.

[0082] In conclusion, PRO-C5 was shown to be elevated in serum from cancer patients. High levels of PRO-C5 were independently associated with shorter OS in PDAC, particularly in stage II PDAC. This highlights the importance and complexity of cancer fibrosis. PRO-C5 may have potential applications in multiple areas, including drug discovery, patient stratification, and drug efficacy.

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Claims

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

2. 10. The method of claim 1, wherein the method is an immunoassay method for detecting and / or monitoring bladder cancer, breast cancer, colon cancer, head and neck cancer, renal cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, or gastric cancer.

3. 10. The method of claim 1, wherein the method is an immunoassay method for detecting and / or monitoring pancreatic ductal adenocarcinoma.

4. 10. The method of any preceding claim, wherein the method is an immunoassay method for detecting cancer of a particular severity in a subject.

5. 5. The method of claim 4, wherein the method is for detecting the stage of cancer in a subject.

6. 6. The method of claim 4 or 5, wherein the method is a method for predicting a level of severity associated with probable or average overall survival.

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

8. 10. The method of any preceding claim, wherein the monoclonal antibody does not specifically bind to a peptide having the N-terminal amino acid sequence AALGDIMGH (SEQ ID NO: 3).

9. 10. A method according to any preceding claim, wherein the monoclonal antibody is raised against a synthetic peptide having the N-terminal amino acid sequence TAALGDIMGH (SEQ ID NO: 1).

10. 10. The method of any preceding claim, wherein the patient sample is selected from blood, serum or plasma.

11. 10. The method of any preceding claim, wherein the immunoassay is a competitive assay or a sandwich assay.

12. 10. The method of any preceding claim, wherein the immunoassay is a radioimmunoassay or an enzyme-linked immunosorbent assay.

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