Assays for evaluating cancer

Abstract

Described herein is an immunoassay method for detecting and / or monitoring cancer in a patient, comprising contacting a biological fluid sample from the patient with a monoclonal antibody that specifically binds to an epitope at the C-terminus of type XXVIII collagen, and detecting and determining the amount of binding between the monoclonal antibody and a peptide in the sample.

Description

Technical Field

[0001] The present invention relates to an immunoassay for detecting and / or monitoring cancer in a patient. The cancer can be, in particular, lung cancer, breast cancer, colorectal cancer, or pancreatic cancer. The immunoassay can, in certain embodiments, be for detecting the stage of the patient's cancer or for determining the prognosis of the patient's cancer.

Background Art

[0002] Collagen type XXVIII has been poorly described in the literature, but studies involving its physical role are gradually emerging. It is mainly localized in the peripheral nerves and dorsal root ganglia, but is also found in the skin. 1、2 Collagen type XXVIII is a beaded collagen that is structurally similar to collagen type VI and has two von Willebrand factor A domains adjacent to a 528-amino acid collagenous domain. 3 Collagen type XXVIII was found at very low levels in normal lung tissue, but was overexpressed in bleomycin-induced lung injury, which may indicate that cells expressing collagen type XXVIII may be involved in the tissue repair process. Collagen type XXVIII has also been previously seen to be upregulated in mouse hepatocellular carcinoma. 4 5

Summary of the Invention

[0003] Here, the present inventors have determined that the formation of collagen type XXVIII is upregulated in cancers, particularly lung cancer, breast cancer, colorectal cancer, or pancreatic cancer, and have developed a competitive ELISA using a monoclonal antibody targeting the C-terminus of collagen type XXVIII, which can be used to detect and / or monitor cancer and to determine the stage and / or prognosis of cancer.

[0004] Therefore, the present invention relates to an immunoassay method for detecting and / or monitoring cancer in a patient, (i) A step of contacting a patient-derived biological fluid sample with a monoclonal antibody that specifically binds to the C-terminal epitope of type XXVIII collagen, (ii) A step of detecting and determining the amount of binding between the monoclonal antibody and the peptide in the sample, (iii) A step of correlating the amount of binding of the monoclonal antibody determined in step (ii) with a value associated with a normal healthy subject and / or a value associated with the known severity of cancer and / or a value obtained from the patient at a previous point in time and / or a predetermined cutoff value. This provides a method that includes [something].

[0005] An immunoassay may be, but is not limited to, a competitive assay or a sandwich assay. An 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.

[0006] In certain embodiments, cancer may be lung cancer, breast cancer, colorectal cancer, ovarian cancer, and / or pancreatic cancer. In particular, cancer may be lung cancer, breast cancer, colorectal cancer, and / or pancreatic cancer.

[0007] In certain embodiments, this method may be a method for detecting the severity of a patient's cancer. For example, this method may be a method for detecting the stage of a patient's cancer and / or a method for determining the prognosis of a patient's cancer (e.g., determining the patient's possible survival time or survival probability).

[0008] In certain embodiments, the patient may be, for example, a patient who has undergone cancer treatment, and the method may include a step of monitoring the patient's cancer.

[0009] The patient's biological fluid sample may be, but is not limited to, blood, serum, plasma, urine, or supernatant derived from cell or tissue cultures. Preferably, the biological fluid is serum or plasma, most preferably serum.

[0010] As used herein, the term “monoclonal antibody” refers to the whole antibody and its fragments that retain the binding specificity of the whole antibody, such as the Fab fragment, F(ab')2 fragment, single-chain Fv fragment, or other such fragments known to those skilled in the art. As is well known, the whole antibody has a “Y-type” structure, which is typically two identical pairs of polypeptide chains, each pair consisting of one “light” chain and one “heavy” chain. The N-terminal regions of each light and heavy chain contain a variable region, and the C-terminal regions of each heavy and light chain constitute a constant region. The variable region contains three complementarity-determining regions (CDRs), which primarily contribute to antigen recognition. The constant region allows the antibody to recruit cells and molecules of the immune system. An antibody fragment that retains binding specificity includes at least the CDRs and the remainder of the variable region sufficient to retain the binding specificity.

[0011] The method of the present invention may use a monoclonal antibody containing any of the constant regions known in the art. The light chain of the human constant region is classified into κ light chains and λ light chains. The heavy chain constant region is classified into μ, δ, γ, α, or ε, defining the antibody isotypes as IgM, IgD, IgG, IgA, and IgE, respectively. The IgG isotype has several subclasses, including, but is not limited to, IgGl, IgG2, IgG3, and IgG4. The monoclonal antibody may preferably be an IgG isotype containing any one of IgGl, IgG2, IgG3, or IgG4.

[0012] 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 prepared from B cell clones as described in the examples. The isotype of the antibody can be determined by ELISA specific to the isotype of human IgM, IgG, or IgA, or to a subclass of human IgG1, IgG2, IgG3, or IgG4. The amino acid sequence of the prepared antibody can be determined using standard techniques. For example, RNA can be isolated from cells and used to prepare 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 sequences of all VH (variable heavy chain) sequences can be used together with primers that bind to sequences located in the constant region of the previously determined isotype. The light chain can be amplified using primers that bind to the 3' end of the κ or λ chain, along with primers that anneal to the leader sequences of Vκ or Vλ. Full-length heavy and light chains can be prepared and sequenced.

[0013] In some embodiments of the method according to the present invention, a biological fluid sample is contacted with a monoclonal antibody that specifically binds to the C-terminal amino acid sequence QETCIQG (SEQ ID NO: 1) (also known herein as "PRO-C28"). Preferably, the monoclonal antibody does not recognize or specifically bind to the extended version of the C-terminal amino acid sequence, which is QETCIQGA (SEQ ID NO: 2). Preferably, the monoclonal antibody does not recognize or specifically bind to the truncated version of the C-terminal amino acid sequence, which is QETCIQ (SEQ ID NO: 3).

[0014] Preferably, the ratio of the affinity of the antibody to the C-terminal amino acid sequence QETCIQG (SEQ ID NO: 1) to the affinity of the antibody to the extended C-terminal amino acid sequence QETCIQGA (SEQ ID NO: 2) is at least 10:1, more preferably at least 50:1, at least 100:1, at least 500:1, at least 1,000:1, at least 10,000:1, at least 100,000:1, or at least 1,000,000:1.

[0015] Preferably, the ratio of the affinity of the antibody to the C-terminal amino acid sequence QETCIQG (SEQ ID NO: 1) to the affinity of the antibody to the truncated C-terminal amino acid sequence QETCIQ (SEQ ID NO: 3) is at least 10:1, more preferably at least 50:1, at least 100:1, at least 500:1, at least 1,000:1, at least 10,000:1, at least 100,000:1, or at least 1,000,000:1.

[0016] As used herein, the term “C-terminus” refers to the end of a polypeptide, i.e., the C-terminal peptide sequence at the C-terminus of the polypeptide, and should not be interpreted as having a general directional meaning.

[0017] Monoclonal antibodies that specifically bind to the C-terminal amino acid sequence QETCIQG (SEQ ID NO: 1) can be produced via any suitable technique known in the art. For example, monoclonal antibodies can be produced against synthetic peptides having the amino acid sequence QETCIQG (SEQ ID NO: 1) by, for example, immunizing rodents (or other suitable mammals) with a synthetic peptide consisting of the sequence QETCIQG (SEQ ID NO: 1) that can optionally bind to an immunogenic carrier protein (such as keyhole limpet hemocyanin), isolating and cloning cells that produce a single antibody, and assaying the resulting monoclonal antibodies to ensure they retain their desired specificity. An exemplary protocol for producing monoclonal antibodies that specifically bind to the C-terminal amino acid sequence QETCIQG (SEQ ID NO: 1) is described below.

[0018] In some embodiments of the method according to the present invention, the amount of binding of a monoclonal antibody specific to the C-terminal epitope of type XXVIII collagen is correlated with a value associated with a normal, healthy subject and / or a value associated with the severity of a known cancer and / or a value obtained from a patient at a previous point in time.

[0019] Where used herein, the terms “values ​​associated with normal, healthy subjects and / or values ​​associated with known cancer severity” mean the standardized amount determined by the method described above in healthy subjects, i.e., subjects considered to be cancer-free, and / or the standardized amount determined by the method described above in subjects known to have cancer with known severity. Thus, for example, if the method is for detecting lung cancer, breast cancer, or colorectal cancer, the amount of monoclonal antibody binding may correlate with the standardized amount determined by the method in healthy subjects and / or the standardized amount determined by the method in subjects known to have the above cancer with known severity.

[0020] In some embodiments of the method according to the present invention, the amount of binding of a monoclonal antibody specific to the C-terminal epitope of type XXVIII collagen is correlated with one or more predetermined cutoff values.

[0021] As used herein, “cutoff value” means the amount of binding that is statistically determined to represent a high likelihood of cancer in a patient, or a high likelihood of cancer at a particular stage or other severity level. For example, the cutoff value may be selected such that the measured value of biomarker binding in a patient sample that is greater than or equal to a statistical cutoff value corresponds to at least 70%, preferably at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% probability of the presence or likelihood of cancer or a particular stage or other severity level of cancer in the patient.

[0022] A predetermined cutoff value for the amount of monoclonal antibody binding to the C-terminal epitope of type XXVIII collagen may be, for example, at least 50 ng / mL, more preferably at least 60 ng / mL, at least 70 ng / mL, at least 80 ng / mL, at least 90 ng / mL, or at least 100 ng / mL. In this regard, through the use of statistical analysis, it has been found that measured amounts of monoclonal antibody binding to the C-terminal epitope of type XXVIII collagen that are above the above cutoff value (for example, measured amounts of 60 ng / mL or more when a cutoff value of at least 60 ng / mL is used) can represent various cancers. Furthermore, it has been found that higher measured amounts of monoclonal antibody binding are statistically correlated with later-stage cancer and a worse prognosis. Therefore, when this method is used to detect the stage of a patient's cancer, the cutoff values ​​for each stage of cancer may be used, with higher cutoff values ​​being used for later stages. Similarly, if the method is for determining the prognosis of a patient's cancer, a cutoff value corresponding to a specific prognosis (e.g., possible survival time or probability of survival) may be used. By using such a statistical cutoff value, it is possible to utilize the method of the present invention to provide a diagnosis with a high level of confidence. Applying such a statistical cutoff value is particularly preferable because it results in a standalone diagnostic assay, i.e., completely eliminates the need for direct comparison with healthy individuals and / or patients with known cancer severity to reach a diagnostic conclusion. This may also be particularly preferable when using the assay to evaluate patients who already have medical signs or symptoms that generally indicate cancer (determined, for example, by physical examination and / or diagnosis by a medical professional), because it acts as a rapid and definitive tool to demonstrate the initial diagnosis, thus potentially eliminating the need for more invasive procedures and allowing for earlier initiation of an appropriate treatment regimen. This may also avoid the need for prolonged hospitalization. In the case of certain cancers, an earlier diagnosis may allow the disease to be detected at an earlier stage, thus improving the overall survival probability.

Brief Description of the Drawings

[0023] [Figure 1] Specificity of the antibody. Reactivity was tested using a standard peptide (QETCIQG (SEQ ID NO: 1)), an extended peptide (QETCIQGA (SEQ ID NO: 2)), a nonsense peptide (GLRPGSEYTV (SEQ ID NO: 4)), and a nonsense cotter (GLRPGSEYTV-K-biotin (SEQ ID NO: 5)). [Figure 2] Levels of PRO-C28 in sera of healthy controls and cancer patients. Levels of PRO-C28 were significantly higher in pooled samples from cancer patients (Asterand, P = 0.002) and lung cancer samples (Proteogenex, P < 0.0001) (Figure 2A). Statistical differences were evaluated using ANOVA and Dunnett's multiple comparison test. PRO-C28 was able to significantly distinguish between healthy individuals and cohorts of pooled cancer patients (Figure 2B) and lung cancer patients (Figure 2C) (P = 0.0007, P < 0.0001, respectively). [Figure 3] Levels of PRO-C28 in sera from healthy controls and patients with various solid tumors defined by organ. [Figure 4] Levels of PRO-C28 in sera from various cancer patients grouped by cancer stage. The lines are fitted from linear regression analysis. [Figure 5] Overall survival curves by the Kaplan Meier method for pancreatic cancer patients who experienced chemotherapy. These patients were divided and grouped into tertiles (Q1, Q2, and Q3) by pretreatment levels of PRO-C28.

Modes for Carrying Out the Invention

[0024] Examples The embodiments disclosed herein are described in the following examples, which are provided to aid in understanding the disclosure and should not be construed as limiting in any way the scope of the disclosure as defined in the following claims. The following examples are provided to a person skilled in the art to a complete disclosure and description of how to make and use the embodiments described and are not intended to limit the scope of the disclosure, and the inventors do not intend to represent that the following experiments are all or only experiments in which experiments were performed. Although attempts have been made to ensure accuracy with respect to the numbers used (e.g., quantity, temperature, etc.), some experimental errors and deviations have been taken into account. Unless otherwise stated, parts are parts by weight, molecular weight is weight-average molecular weight, temperature is Celsius temperature, and pressure is atmospheric pressure or near atmospheric pressure.

[0025] In the following embodiments, the following materials and methods were used.

[0026] material and method All reagents used in this experiment were of high quality from companies such as Sigma Aldrich (St. Louis, MO, USA) and Merck (Whitehouse Station, NJ, USA). Synthetic peptides used in the development of immunotherapy and assays were purchased from Genscript (New Jersey, USA).

[0027] Production of monoclonal antibodies targeting PRO-C28 A monoclonal antibody targeting the C-terminus of type XXVIII collagen was produced by generating an antibody against the 7-amino acid sequence QETCIQG (SEQ ID NO: 1) ("PRO-C28") found at the C-terminus of type XXVIII collagen. This 7-amino acid sequence was selected over longer sequences, such as the 10-amino acid C-terminal sequence KECQETCIQG (SEQ ID NO: 6), to reduce the number of cysteine ​​residues and thereby avoid the formation of cis-cis crosslinks in the immunogenic peptide previously used to produce the antibody.

[0028] The protocol used for producing monoclonal antibodies targeting PRO-C28 was as follows:

[0029] Immunotherapy of 6-7 week old female Balb / C mice (body weight 14-18g) was initiated by subcutaneous injection of 200 μL of emulsified antigen solution containing 100 μg of immunogenic peptide KLH-CGG-QETCIQG (SEQ ID NO: 7) (where "KHL" represents keyhole limpet hemocyanin and CGG is a binding linker) using Stimune Immunogenic Adjuvant (SPECOL) (Cat #7925000, Invitrogen). This immunization was repeated every two weeks until a stable level of serum antibody titer was reached. Mice with the highest serum titer and best inhibition were selected for fusion and rested for at least three weeks after the last immunization. Subsequently, the mice were intravenously boosted with 100 μL of 0.9% NaCl solution containing 100 μg of immunogenic peptide three days prior to spleen isolation for cell fusion. To produce hybridoma cells, mouse spleen cells were fused with SP2 / 0 myeloma cells as described by Gefter et al. These hybridoma cells were cloned in culture dishes using a semi-solid medium method. These clones were then placed on 96-well microtiter plates for further proliferation, and monoclonal growth was promoted using limiting dilution. Indirect ELISA performed on streptavidin-coated plates was used to screen the reactivity of the supernatant. Biotin-QETCIQG (SEQ ID NO: 8) was used as a screening peptide, and the standard peptide (QETCIQG (SEQ ID NO: 1)), the extended peptide (QETCIQGA (SEQ ID NO: 2)), the nonsense peptide (GLRPGSEYTV (SEQ ID NO: 4)), and the nonsense coater (GLRPGSEYTV-K-biotin (SEQ ID NO: 5)) were used to further test the specificity of the clones. The supernatant was collected from hybridoma cells and purified using a HiTrap affinity column (GE Healthcare Life Science, Little Chalfront, Buckinghamshire, UK) according to the manufacturer's instructions. All animals were treated in accordance with animal welfare guidelines.

[0030] Clone selection and characterization Hybridomas producing the best antibody in terms of reactivity to a standard peptide (QETCIQG (SEQ ID NO: 1)) were screened in the competitive ELISA described below, and clones showing the highest reactivity in producing a monoclonal antibody targeting PRO-C28 were selected. Antibody specificity was tested using the standard peptide (QETCIQG (SEQ ID NO: 1)), an extended peptide (QETCIQGA (SEQ ID NO: 2)), a nonsense peptide (GLRPGSEYTV (SEQ ID NO: 4)), and a nonsense coater (GLRPGSEYTV-K-biotin (SEQ ID NO: 5)). The isotype of the monoclonal antibody was determined using the Clonotyping System-HRP kit, cat. 5300-05 (Southern Biotech, Birmingham, AL, USA).

[0031] PRO-C28 ELISA Roche cat.11940279 96-well streptavidin-coated ELISA plates were coated with 100 μL / well of biotinylated peptide biotin-QETCIQG (SEQ ID NO: 8) dissolved in assay buffer (25 mM TBS-BTE + 2 g / l NaCl, pH 8). The plates were incubated at 20°C for 30 minutes with shaking in a dark room, and then washed five times with wash buffer (20 mM Tris, 50 mM NaCl, pH 7.2). Subsequently, 20 μl of peptide standard or sample was added to the appropriate wells, followed by 100 μl of purified antibody solution (PRO-C28-specific monoclonal antibody dissolved in assay buffer). The plates were incubated at 20°C for 1 hour with shaking, and then washed five times with wash buffer. Next, 100 μL of secondary antibody solution (horseradish peroxidase (HRP)-labeled anti-mouse antibody dissolved in the same assay buffer used for the PRO-C28-specific monoclonal antibody) was added to each well and incubated at 20°C for 1 hour with shaking, followed by five washes with wash buffer. Finally, 100 μL of tetramethylbenzinidine (TMB) (Kem-En-Tec cat.:438OH) was added to each well, and the plate was incubated in a dark room at 20°C for 15 minutes. To stop the reaction, 100 μL of stop solution (1% H2SO4) was added, and the plate was analyzed using an ELISA reader at 450 nm with 650 nm as a reference (Molecular Devices, SpectraMax M, CA, USA). Calibration curves were plotted using a 4-parametric mathematically fitted model.

[0032] Technical evaluation of PRO-C28 ELISA Linearity was assessed using 2-fold dilutions of human serum, human urine, and human plasma samples treated with EDTA, heparin, or citrate (four samples from each sample type). Linearity was calculated as the percentage of the undiluted sample recovery.

[0033] Intra-assay and inter-assay variability was determined by 10 independent runs of five quality controls (QCs) and two kit controls with dual determination.

[0034] The accuracy of the assay was measured using healthy human serum samples spiked with standard peptides and calculated as the percentage of serum recovery in the buffer.

[0035] The lower limit of the measurement range (LLMR) and the upper limit of the measurement range (ULMR) were calculated based on 10 individual standard curves derived from intra-assay and inter-assay variability.

[0036] Biological validation of PRO-C28 as a cancer biomarker PRO-C28 levels were measured in serum samples from two cohorts of healthy controls (obtained from Lee Biosolutions, USA and Valley BioMedical, USA), from a cohort of patients with various cancer types (obtained from Asterand, USA, including samples from patients with various adenocarcinomas, invasive breast cancer, malignant melanoma of the skin, and small cell carcinoma / squamous cell carcinoma of the lung), and from a cohort of lung cancer patients (obtained from ProteoGenex, USA). PRO-C28 levels were measured blindly using the PRO-C28 ELISA protocol described above. All serum samples were collected after informed consent and approval by the local ethics committee, and all serum samples from cancer patients were collected before resection. Patient demographics are shown in Tables 1 and 2. The mean age of one patient in the healthy cohort (sample from Lee Biosolutions) was significantly different from the mean age of patients in the two cancer cohorts, but there was no correlation between age and PRO-C28 levels in any of the cohorts.

[0037] [Table 1]

[0038] [Table 2]

[0039] Biological validation of PRO-C28 as a prognostic marker for pancreatic cancer PRO-C28 was measured in pre-treated serum samples from a cohort of 701 patients with stage I–IV pancreatic cancer (PC). All PC patients were from the Danish BIOPAC (biomarker for patients with pancreatic cancer) trial (NCT03311776). Patients were recruited from six Danish hospitals from December 2008 to September 2017. PC patients had histologically confirmed tumors and were treated with various types of chemotherapy in accordance with national guidelines (www.gicancer.dk). This trial was conducted in accordance with the recommendations of the Danish Regional Committee of Health Research Ethics. The BIOPAC protocol was approved by the Danish Regional Committee of Health Research Ethics (VEK ref. KA-20060113) and the Data Protection Agency (j.nr. 2006-41-6848). All subjects provided informed consent in accordance with the Helsinki Declaration (version 8). Serum samples were obtained at the time of diagnosis or prior to surgery. Samples were processed according to nationally approved standard procedures for blood (www.herlevhospital.dk / biopac.dk).

[0040] result Clone selection and characterization Hybridomas producing the best antibody in terms of reactivity and selectivity to standard peptides were screened, and based on reactivity, clone NBH218#65 8C11-2F10-1H7 was selected and used for the production of a monoclonal antibody targeting PRO-C28 for use in the technical and biological evaluation of PRO-C28 ELISA. This monoclonal antibody was isotype: IgG2b,k. No reactivity was found to extended peptides, nonsense peptides, or nonsense coaters (Figure 1).

[0041] Technical evaluation of PRO-C28 ELISA A series of technical validations were performed to evaluate the PRO-C28 ELISA assay. A summary of the validation data is shown in Table 3.

[0042] [Table 3]

[0043] Biological evaluation of PRO-C28 as a cancer biomarker As shown in Figure 2A, PRO-C28 levels were significantly higher in the cohort of patients with mixed cancers and the cohort of patients with lung cancer compared to the cohort of healthy controls (Lee Biosolutions cohort). On average, PRO-C28 levels were assessed as 49.77 ng / ml (30.37) in the healthy controls (Lee Biosolutions cohort), 78.32 ng / ml (55.92) in the cohort of patients with mixed cancers, and 140.8 ng / ml (34.33) in the cohort of patients with lung cancer (control vs. mixed cancer P=0.002, control vs. lung cancer P<0.0001). PRO-C28 significantly differentiated between healthy individuals and those with mixed cancer (Figure 2B, AUC=0.68, P=0.0007), and between healthy individuals and those with lung cancer (Figure 2C, AUC=0.98, P<0.0001). Therefore, PRO-C28 can be used to differentiate between healthy patients and patients with cancer of various etiologies with high accuracy and significance.

[0044] The results, categorized by cancer type, are shown in Figure 3 and Table 4. Patients with lung cancer and breast cancer had significantly higher levels of PRO-C28 compared to healthy controls (Valley BioMedical cohort) when evaluated using either the Dunn multiple comparison test or the Mann-Whitney test. Colorectal cancer also showed significantly higher levels using the Mann-Whitney test, and ovarian and pancreatic cancers showed a similar tendency towards higher levels.

[0045] [Table 4]

[0046] To evaluate the association between disease stage and PRO-C28, cancer patients were similarly grouped based on whether they had stage 1 (n=15), stage 2 (n=35), stage 3 (n=40), or stage 4 (n=22) cancer, with 13 patients for whom stage information was unavailable being excluded. The PRO-C28 levels of patients in each of these groups are shown in Figure 4 and statistically summarized in Table 5. As shown, PRO-C28 levels correlated with disease stage, further suggesting that PRO-C28 levels were associated with tumor volume, and therefore possibly prognosis as well.

[0047] [Table 5]

[0048] Biological evaluation of PRO-C28 as a prognostic marker in pancreatic cancer We compared the overall survival (OS) curves of a cohort of pancreatic cancer patients using Kaplan-Meier curves and log-rank p-values. Patients here were divided into ternary groups (Q1, Q2, and Q3) based on their PRO-C28 pretreatment level. A p-value of p<0.05 was considered statistically significant. The univariate Cox proportional hazards regression model calculated the hazard ratios (HR) in 95% confidence intervals (CI) for OS per ternary group of the PRO-C28 biomarker: Q2 and Q3 compared to Q1.

[0049] As shown in Table 5 and Figure 5, when evaluating the association between PRO-C28 pretreatment levels and OS, "high" PRO-C28 levels (Q3) and "moderate" PRO-C28 levels (Q2) predicted shorter OS compared to "low" PRO-C28 levels (Q1). Here, patients in Q2 and Q3 had a 53% (HR=1.53) and 24% (HR=1.24) higher risk of death compared to patients in Q1 (HR=1.0), respectively. Similarly, the Kaplan-Meier curves for each quartile differed significantly (log rank p-value < 0.0001), and the median OS time decreased from Q1 to Q2 to Q3. These data suggest that PRO-C28 has prognostic value in PC patients.

[0050] [Table 6]

[0051] In this specification, unless otherwise explicitly stated, the term "or" is used to mean an operator that returns a true value if any or both of the multiple listed conditions are met, as opposed to the operator "exclusive or," which requires that only one of the conditions be met. The term "comprising" is used to mean "including" rather than "consisting of." All prior teachings recognized above are incorporated by reference in this specification. The acceptance of any previously published documents in this specification should not be construed as an acceptance or representation that such teachings were common knowledge in Australia or elsewhere as of the date of this specification.

[0052] References 1. Gebauer, JM, Kobbe, B., Paulsson, M. & Wagener, R. Structure, evolution and expression of collagen XXVIII: Lessons from the zebrafish. Matrix Biol. 49, 106-119 (2016). 2. Veit, G. et al. Collagen XXVIII, a novel von Willebrand factor A domain-containing protein with many imperfections in the collagenous domain. J. Biol. Chem. 281, 3494-3504 (2006). 3. Annis, DS, Mosher, DF & Roberts, DD NIH Public Access. 27, 339-351 (2009). 4. Schiller, H. B. et al. Time- and compartment-resolved proteome profiling of the extracellular niche in lung injury and repair. Mol. Syst. Biol. 11, 819-819 (2015). 5. Lai KKY, Shang S, Lohia N, Booth GC, Masse DJ, Fausto N, et al. (2011) Extracellular Matrix Dynamics in Hepatocarcinogenesis: a Comparative Proteomics Study of PDGFC Transgenic and Pten Null Mouse Models. PLoS Genet 7(6): e1002147. https: / / doi.org / 10.1371 / journal.pgen.1002147

Claims

1. An immunoassay method for detecting and / or monitoring cancer in a patient, (i) A step of contacting a patient-derived biological fluid sample with a monoclonal antibody that specifically binds to the C-terminal epitope of type XXVIII collagen, wherein the monoclonal antibody specifically binds to the C-terminal amino acid sequence QETCIQG (SEQ ID NO: 1), and the monoclonal antibody does not recognize or specifically bind to the extended version of the C-terminal amino acid sequence, which is QETCIQGA (SEQ ID NO: 2), (ii) A step of detecting and determining the amount of binding between the monoclonal antibody and the peptide in the sample, (iii) A step of correlating the amount of binding of the monoclonal antibody determined in step (ii) with a value associated with a normal healthy subject and / or a value associated with the known severity of cancer and / or a value obtained from the patient at a previous point in time and / or a predetermined cutoff value, Methods that include...

2. The method according to claim 1, wherein the cancer is lung cancer, breast cancer, colorectal cancer, or pancreatic cancer.

3. The method according to claim 2, which is a method for detecting the stage of cancer in a patient or for determining the prognosis of cancer in a patient.

4. The method according to any one of claims 1 to 3, wherein the monoclonal antibody does not recognize or specifically bind to the truncated version of the C-terminal amino acid sequence which is QETCIQ (SEQ ID NO: 3).

5. The method according to any one of claims 1 to 4, wherein the monoclonal antibody is produced against a synthetic peptide having the amino acid sequence QETCIQG (SEQ ID NO: 1).

6. The method according to any one of claims 1 to 5, wherein the biological fluid is blood, serum, plasma, urine, or supernatant derived from a cell culture or tissue culture.

7. The method according to any one of claims 1 to 6, wherein the immunoassay is a competitive assay or a sandwich assay.

8. The method according to any one of claims 1 to 7, wherein the immunoassay is a radioimmunoassay or an enzyme-linked immunosorbent assay.

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