Collagen XX assay

Abstract

Described herein is an immunoassay for detecting and / or monitoring cancer in a patient, comprising contacting a patient sample with a monoclonal antibody that specifically binds to a C-terminal amino acid sequence corresponding to the C-terminus of type XX collagen. Also described are the monoclonal antibody and an immunoassay kit for use in performing the immunoassay.

Description

[Technical field]

[0001] The present invention relates to immunoassays, particularly immunoassays for detecting and / or monitoring cancer in a patient, and to monoclonal antibodies and immunoassay kits for use in performing such immunoassays. [Background technology]

[0002] Cancer is a major global health problem, and the burden of cancer incidence and mortality continues to increase worldwide [1]. Part of this problem is the lack of valid biomarker tools, especially those that can detect cancer earlier and predict response to treatment. Moreover, traditional biomarkers usually involve invasive measures such as tissue biopsy [2]. A promising alternative to these approaches is liquid biopsy. In recent years, more attention has been paid to the tumor microenvironment around cancer cells, which is the most important site to look for non-invasive biomarkers.

[0003] In particular, the extracellular matrix, defined as the noncellular portion of tissues, is increasingly recognized as a key part of cancer development [3]. The major ECM proteins are collagens, which are important for tumor stiffness, tumor immunity, and cancer metastasis [4]. In cancer, the dynamic balance between ECM formation and degradation is disrupted. In the tumor microenvironment, cells influence collagen remodeling and collagens reciprocally influence cell behavior [4, 5]. There are detailed reports on the cancer-associated expression patterns, localization, and functions of collagens, such as type IV, which are ubiquitous in basement membranes and act as a barrier against invasive tumor cells, but which tumor cells can degrade, thereby facilitating metastasis [6, 7]. However, most cancer studies interested in collagens have focused on abundant and well-characterized collagens, such as collagen types I, III, or IV, leaving many of the less well-characterized collagens unexplored.

[0004] Type XX collagen is one such underexplored collagen. Based on its structural features, type XX collagen is part of the fibril-associated collagens with interrupted helices (FACIT) family, which is thought to associate with and regulate the organization and interactions of fibrillar collagens [8]. Structural features of type XX collagen include multiple fibronectin type III repeats, a von Willebrand factor A domain, a thrombospondin-like domain, and a collagenous triple-helical domain (GXY) with interspersed non-collagenous domains [8]. Within the FACIT family, types XII and XIV are most closely related to type XX collagen; they are the only FACITs that also contain a fibronectin domain [8].

[0005] Very little is known about the expression, localization, and function of type XX collagen. Type XX collagen was originally cloned from chicken embryos, and its expression is almost exclusively restricted to corneal epithelium, but is detectable in fetal skin, lung, sternocostal cartilage, and tendons [8]. Several papers have pointed out parallels between cancer and development [9, 10]. RNA expression data from the Human Protein Atlas (https: / / www.proteinatlas.org / ENSG00000101203-COL20A1 / tissue) also suggest strong expression of COL20A1 in human brain, with weaker expression in testis and spleen tissues. A role in the brain is also confirmed by data from The Cancer Genome Atlas (TCGA) Initiative, while COL20A1 levels are relatively high in glioma tissues (https: / / www.proteinatlas.org / ENSG00000101203-COL20A1 / pathology). Thus, if we look carefully at the expression level, type XX collagen may play a role in brain function and brain tumors, since type XX collagen does not appear to be abundant in other tissues.

[0006] Despite the apparent rarity of XX collagen expression, COL20A1 has been described in the cancer literature. Reports have usually described COL20A1 at the DNA or RNA level, and usually in broad and generalized screens. Consistent with the cDNA microarray-based brain association described above, COL20A1 was increased in so-called brain tumor-initiating cells compared to normal glioma cell lines and normal brain astrocytes

[12] . In another study, using a biopsy-derived glioma cell model, downregulation of COL20A1 RNA was observed after treatment with a histone deacetylase inhibitor

[13] . In other cancers, reports of XX collagen are rare, but COL20A1 was included in a 16-gene signature associated with breast cancer recurrence, metastasis, and poor survival in a Chinese population

[11] . COL20A1 has also been reported to be upregulated in early-stage prostate tumorigenesis

[14] . In particular, these screens have not been followed up with any functional testing of type XX collagen, and no insight into its function and distribution has been currently provided. Summary of the Invention

[0007] Applicants have now developed an enzyme-linked immunosorbent assay (ELISA) to quantify the presence of a biomarker for type XX collagen in blood, referred to herein as "PRO-C20". The assay was optimized, validated, and used to measure circulating type XX collagen levels in the serum of cancer patients and healthy controls. The assay was found to be robust, specific for type XX collagen, and sensitive to detect levels in both healthy controls and diseased (cancer) patients. The data show that PRO-C20 levels are significantly higher in the serum of cancer patients compared to healthy controls. The data also show that higher levels of PRO-C20 are associated with poor overall survival in cancer patients.

[0008] Thus, in a first aspect, the present invention provides an immunoassay method comprising the steps of: (i) contacting the patient sample with a monoclonal antibody that specifically binds to the C-terminal amino acid sequence QGASTQGLWE (SEQ ID NO:1) (which C-terminal amino acid sequence is referred to herein as "PRO-C20" and / or the "target sequence"; a peptide consisting of or containing said C-terminal amino acid sequence is also referred to herein as "PRO-C20" and / or the "target peptide"); and (ii) detecting and measuring the amount of binding between the monoclonal antibody and the peptide in the sample.

[0009] The method is preferably an immunoassay for detecting and / or monitoring disease in a patient and / or for assessing the severity of disease in a patient. The method preferably further comprises the step (iii) of: (iii) correlating the amount of binding of the monoclonal antibody as measured in step (ii) to a value associated with normal healthy subjects, and / or to a value associated with a known disease severity, and / or to a value obtained from the patient at an earlier time point and / or to a predefined cut-off value.

[0010] In a preferred embodiment, the disease is cancer.The cancer may be, for example, bladder cancer, breast cancer, colorectal cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer or gastric cancer.The disease may be, in particular, pancreatic ductal adenocarcinoma (PDAC).

[0011] As mentioned above, in certain embodiments, the method may be a method of assessing the severity of a patient's disease, such as cancer, etc. For example, the method may be a method of determining the prognosis of cancer in a patient, such as determining the possible survival time and / or survival probability of a patient.

[0012] In a preferred embodiment, the monoclonal antibody does not specifically bind to an extended version of the target sequence QGASTQGLWES (SEQ ID NO:2) (i.e., a version of the PRO-C20 target sequence extended by the addition of a serine residue at its C-terminus). Preferably, the ratio of the affinity of the antibody for the PRO-C20 target sequence relative to the affinity of the antibody for the extended target sequence is at least 10:1, and more preferably at least 20:1, or at least 30:1.

[0013] In a preferred embodiment, the monoclonal antibody does not specifically bind to a truncated version of the target sequence QGASTQGLW (SEQ ID NO:3) (i.e., a version of the PRO-C2 target sequence truncated by removing the last glutamic acid residue). Preferably, the ratio of the affinity of the antibody to the PRO-C20 target sequence relative to the affinity of the antibody to the truncated target sequence is at least 10:1, and more preferably at least 20:1, or at least 30:1.

[0014] Preferably, the monoclonal antibody is a monoclonal antibody raised against a synthetic peptide having the C-terminal amino acid sequence QGASTQGLWE (SEQ ID NO:1).

[0015] The sample is preferably a biological fluid, which may be, but is not limited to, blood, serum, plasma, urine or cell or tissue culture supernatant. Preferably, the biological fluid is blood, serum or plasma.

[0016] The immunoassay may be, but is not limited to, 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.

[0017] The term "amount of binding" herein refers to the quantification of the binding between the monoclonal antibody and the peptide in the patient sample. The quantification may be evaluated, for example, by comparing the measured value of binding in the patient sample with a calibration curve made using the measured value of binding in a standard sample containing a known concentration of the peptide to which the antibody specifically binds, thereby determining the amount of the peptide to which the antibody specifically binds in the patient sample. In the examples described later in this specification, an ELISA method is used, in which spectroscopic analysis is used both to measure the amount of binding in the patient sample and to measure the amount of binding in the calibration curve. However, any suitable analytical method can be used.

[0018] The term "predetermined cut-off value" as used herein refers to the amount of binding that is statistically measured as indicating a high probability or a specific severity of disease in a patient, and when the measured value of the target peptide in a patient sample is the same as or higher than the statistical cut-off value, it corresponds to 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 for the presence or specific severity of the disease.For example, in a PDAC patient, when PRO-C20 level is 2.59nM, in certain embodiments, it can be used as a predetermined cut-off value that indicates the severity of cancer, such that the patient has a high probability (for example, about 75%) of dying within the next 6 months and / or a very high probability (for example, at least 95%) of dying within about the next 2 years.

[0019] As used herein, the term "value associated with a normal healthy subject" refers to a standardized amount of binding measured by the above method for samples obtained from subjects considered healthy (i.e., disease-free); and the term "value associated with a known disease severity" refers to a standardized amount of binding measured by the above method for samples obtained from known patients with a disease of known severity.

[0020] The term "C-terminus" as used herein refers to a C-terminal peptide sequence present at the end of a polypeptide, i.e., the C-terminus of the polypeptide, and should not be construed as referring to the general orientation thereof.

[0021] As used herein, the terms "peptide" and "polypeptide" are used synonymously.

[0022] The term "monoclonal antibody" herein refers to both complete antibodies and fragments thereof, where the fragments retain the binding specificity of the complete 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, complete antibodies typically have a "Y-shaped" structure of two identical pairs of polypeptide chains, each pair consisting of one "light" chain and one "heavy" chain. The N-terminal regions of each light chain and each heavy chain comprise the variable region, while the C-terminal portions of each light chain and each heavy chain form the constant region. The variable regions comprise three complementarity determining regions (CDRs), which are primarily responsible for antigen recognition. The constant regions allow the antibody to recruit cells and molecules of the immune system. An antibody fragment that retains the binding specificity comprises at least the CDRs and a sufficient remaining portion of the variable region to retain the binding specificity.

[0023] The method of the present invention can utilize monoclonal antibodies containing any constant region known in the art. Human light chain constant regions are classified as kappa light chains and lambda light chains. Heavy chain constant regions are classified as mu, delta, gamma, alpha, or epsilon, which define the antibody isotypes IgM, IgD, IgG, IgA, and IgE, respectively. There are multiple subclasses of the IgG isotype, including, but not limited to, IgG1, IgG2, IgG3, and IgG4. The monoclonal antibody may be of the IgG isotype, preferably including any one of IgG1, IgG2, IgG3, or IgG4.

[0024] The CDRs of the antibody can be determined using methods known in the art, such as those reported by Kabat et al. The antibody can be generated from a B cell clone as described in the Examples. The isotype of the antibody can be determined by ELISA specific for human IgM, IgG or IgA isotype, or human IgG1, IgG2, IgG3 or IgG4 subclasses. The amino acid sequence of the generated antibody can be determined using standard techniques. For example, RNA can be isolated from the cells and used to generate cDNA by reverse transcription. This cDNA is then subjected to PCR using primers that amplify the heavy and light chains of the antibody. For example, a primer specific to the leader sequence of the entire VH (variable heavy) sequence can be used together with a primer that binds to a sequence located in the constant region of the previously determined isotype. The light chain can be amplified using a primer that binds to the 3' end of the kappa or lambda chain together with a primer that anneals to the leader sequence of Vkappa or Vlambda. Full-length heavy and light chains can be generated and sequenced.

[0025] The monoclonal antibody or fragment thereof preferably comprises: CDR-H1: DYSMH (SEQ ID NO: 11) CDR-H2: WINTETGEPTYADGFKG (SEQ ID NO: 12) CDR-H3:GPY CDR-L1: RSSQSIVHNNGKIYLE (SEQ ID NO: 13) CDR-L2: KVSNRFS (SEQ ID NO: 14) CDR-L3: FQGSHVPYT (SEQ ID NO: 15) It may comprise one or more complementarity determining regions (CDRs) selected from the following: Preferably, the antibody or fragment thereof comprises at least two, three, four, five, or six of the CDR sequences shown in the above list.

[0026] Preferably, the monoclonal antibody or fragment thereof has a light chain variable region comprising the following CDR sequences: CDR-L1: RSSQSIVHNNGKIYLE (SEQ ID NO: 13) CDR-L2: KVSNRFS (SEQ ID NO: 14) and CDR-L3: FQGSHVPYT (SEQ ID NO: 15)

[0027] Preferably, the monoclonal antibody or fragment thereof has a light chain comprising framework sequences between the CDRs that are substantially identical to or substantially similar to the framework sequences between the CDRs of the light chain sequence set forth below (in which the CDRs are shown in bold and underlined; and the framework sequences are shown in italics): [ka]

[0028] Preferably, the monoclonal antibody or fragment thereof has a heavy chain variable region comprising the following CDR sequences: CDR-H1: DYSMH (SEQ ID NO: 11) CDR-H2: WINTETGEPTYADGFKG (SEQ ID NO: 12) and CDR-H3:GPY

[0029] Preferably, the monoclonal antibody or fragment thereof has a heavy chain comprising framework sequences between the CDRs that are substantially identical to or substantially similar to the framework sequences between the CDRs of the heavy chain sequence set forth below (in which the CDRs are shown in bold and underlined; and the framework sequences are shown in italics): [ka]

[0030] As used herein, two antibodies are considered substantially identical or substantially similar if the framework amino acid sequence between the CDRs of the antibody has at least 70%, 80%, 90%, or at least 95% similarity or identity to the framework amino acid sequence present between the CDRs of another antibody. The similar or identical amino acids may be contiguous or non-contiguous.

[0031] The framework sequence may contain one or more amino acid substitutions, insertions and / or deletions. The amino acid substitutions may be conservative, meaning that the substituted amino acid has similar chemical properties to the original amino acid. Those skilled in the art will understand which amino acids share similar chemical properties. For example, the following groups of amino acids share similar chemical properties (such as size, charge and polarity): Group 1: Ala, Ser, Thr, Pro, Gly; Group 2: Asp, Asn, Glu, Gln; Group 3: His, Arg, Lys; Group 4: Met, Leu, Ile, Val, Cys; Group 5: Phe Thy Trp.

[0032] Programs such as the CLUSTAL program can be used for amino acid sequence comparison. This program compares amino acid sequences and finds the optimal alignment by inserting spaces into either sequence as necessary. This program can calculate amino acid identity or similarity (identity and conservation of amino acid types) to obtain the optimal alignment. Programs such as BLASTx align the longest lengths of similar sequences and determine a value for the match. In this way a comparison can be obtained which finds multiple regions of similarity each with a different score. Both types of analysis are contemplated in the present invention. Identity or similarity is preferably calculated over the entire length of the framework sequence.

[0033] In certain preferred embodiments, the monoclonal antibody or fragment thereof comprises: The light chain variable region sequence: [ka] and / or The heavy chain variable region sequence: [ka] (CDRs are bold and underlined; framework sequences are italicized).

[0034] In a second aspect, the present invention provides a monoclonal antibody which specifically binds to the C-terminal amino acid sequence QGASTQGLWE (SEQ ID NO:1), which is suitable for use in an immunoassay according to the first aspect of the invention; and preferred and other optional embodiments of the monoclonal antibody according to the second aspect of the invention will be apparent from the above discussion of the monoclonal antibodies for use in the first aspect of the invention and its preferred and other optional embodiments.

[0035] In a third aspect, the present application relates to an immunoassay kit, the kit comprising: A monoclonal antibody according to the second aspect, and · Streptavidin-coated well plates; the biotinylated peptide Biotin-L-QGASTQGLWE (SEQ ID NO:4); where L is an optional linker; · Secondary antibodies for use in sandwich immunoassays; A calibration protein comprising the C-terminal amino acid sequence QGASTQGLWE (SEQ ID NO:1); · Antibody biotinylation kit; · Antibody HRP labeling kit; · Antibody radiolabeling kits; and Assay Visualization Kit Contains at least one of the following: [Brief description of the drawings]

[0036] [Figure 1] Specificity test of the PRO-C20 assay. Inhibition curves for standard peptide (RHLEGRGEPGAVGQMGSPGQQGASTQGLWE (SEQ ID NO:6)), extended peptide (QGASTQGLWES (SEQ ID NO:2)), truncated peptide (QGASTQGLW (SEQ ID NO:3)), and nonsense standard peptide (SHAHQRTGGN (SEQ ID NO:8)) and nonsense coater peptide (biotin-SHAHQRTGGN (SEQ ID NO:9)). Peptides were serially diluted 2-fold to assess their ability to compete for antibody binding. Signal (B) is the negligible background absorbance (B0); it corresponds to the assay buffer and is expressed as a function of peptide concentration on a logarithmic scale. Error bars indicate the standard deviation of duplicate measurements. [Diagram 2]PRO-C20 was measured in serum from multiple cancer patient groups. Quantification of PRO-C20 in serum from healthy controls (n=33) and from patients with bladder (n=20), breast (n=20), colorectal (n=20), head and neck (n=20), kidney (n=20), liver (n=3), lung (n=20), melanoma (n=20), ovarian (n=19), pancreatic (n=20), prostate (n=20) and gastric cancer (n=20). PRO-C20 levels are represented as Tukey-type box plots with data point variation. Horizontal bars represent median values; upper and lower hinges of the box represent the first and third quartiles (25th and 75th percentiles); whiskers in either the positive or negative direction extend from the hinge to the maximum and minimum values, but are less than 1.5*IQR (where IQR is the interquartile range between the first and third quartiles). Samples that measured lower than the lower limit of measurement range (LLMR) when measured in the validation of PRO-C20 were given the LLMR value. Differences in PRO-C20 levels between cancer-affected groups and healthy controls were assessed by ordinary ANOVA, with multiple comparisons of controls performed using Dunnett's test. **** represents p-value less than 0.0001. *** less than 0.001. ** less than 0.01. * less than 0.05. [Diagram 3] Quantification of PRO-C20 in serum of healthy controls (n=20) and PDAC patients (n=36). [Figure 4] Kaplan–Meier curves assessing the association between high PRO-C20 levels (>2.59 nM) and overall survival in PDAC patients.

[0037] Working Example The following examples illustrate embodiments of the present disclosure, but are presented to aid in the understanding of the disclosure and should not be construed as limiting the scope of the disclosure as defined in the claims that follow. The following examples are provided to fully disclose and describe to one of ordinary skill in the art how to make and use the described embodiments, and are not intended to limit the scope of the disclosure, nor are they intended to represent that the experiments described below represent 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 variation will be expected. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in °C, and pressure is at or near atmospheric pressure.

[0038] Materials and Methods Generation of monoclonal antibodies targeting PRO-C20 A 10 amino acid target peptide corresponding to the C-terminus of type XX collagen (UniprotKB:Q9P218) 1275 QGASTQGLWE 1284 (SEQ ID NO:1) was purchased from Genscript (Piscataway, NJ, USA) and used for immunization.

[0039] More specifically, the immunogenic peptide (KLH-CGG-QGASTQGLWE (SEQ ID NO: 7)) was generated by covalently cross-linking the target peptide to keyhole limpet hemocyanin (KLH) carrier protein using sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, SMCC (Thermo Scientific, Waltham, MA, USA; Cat. No.: 22322). Glycine and cysteine ​​residues were added to the N-terminus to ensure correct linkage to the carrier protein. 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). Successive immunizations were performed at 2-week intervals until stable serum titer levels were achieved. The mice with the highest titers were rested for 4 weeks and then boosted intravenously with 100 μg of immunogenic peptide in 100 μL of 0.9% NaCl solution. Hybridoma cells were generated by fusing spleen cells with SP2 / 0 myeloma cells as previously reported (Gefter, Margulies and Scharff, 1977). The resulting hybridoma cells were then cultured in 96-well microtiter plates and expanded to monoclonal size using standard limiting dilution techniques.

[0040] Monoclonal antibodies were purified using a protein G column (GE Healthcare Life Sciences, Little Chalfont, UK; Catalogue No. 17-0404-01) according to the manufacturer's instructions.

[0041] The best antibody clones for the biomarkers were selected based on a preliminary competitive ELISA for reactivity to the selected peptide (target peptide, QGASTQGLWE (SEQ ID NO: 1)); but not to the extended peptide (QGASTQGLWES (SEQ ID NO: 2)), truncated peptide (QGASTQGLW (SEQ ID NO: 3)), and nonsense KLH conjugate peptide (IRQCPDRTYG-GGC-KLH (SEQ ID NO: 10)).

[0042] Antibodies produced by the best performing clones were sequenced to determine the CDRs. The sequences of the chains are shown below (CDRs are bold and underlined; constant regions are in italics): Heavy chain: amino acid sequence (511aa) [ka]

[0043] PRO-C20 ELISA Protocol Multiple optimizations were performed on the ELISA, including the selection of assay buffer, incubation time and temperature, and antibody and peptide concentrations. The final PRO-C20 assay protocol was performed as follows: Streptavidin-coated 96-well ELISA plates were coated with 100 μL / well of 1.25 ng / mL of biotinylated target peptide (Biotin-QGASTQGLWE (SEQ ID NO:5)), which was dissolved in assay buffer (25 mM TBS, 1% BSA (w / v), 0.1% Tween-20 (w / v), 2 g / L NaCl, pH 8.0); the plate was incubated at 20° C. for 30 minutes with shaking at 300 RPM. After washing five times with wash buffer (25 mM Tris, 50 mM NaCl, pH 7.2), samples were added in duplicate at 20 μL / well, followed by 100 μL / well of 50 ng / mL HRP-labeled monoclonal antibody in assay buffer and incubated for 1 hour at 20° C. with shaking at 300 RPM. After the second wash cycle, TMB was added at 100 μL / well and incubated for 15 minutes at 20° C. in the dark with shaking at 300 RPM. The reaction was stopped by adding 100 μL / well of 1% H2SO4. The absorbance was measured at 450 nm with a reference at 650 nm. To generate a standard curve, 50ng / mL of the standard peptide (RHLEGRGEPGAVGQMGSPGQQGASTQGLWE (SEQ ID NO:6)) was added in serial 2-fold dilutions at 20μL / well to the appropriate wells; the curve was fitted using a 4-parameter logistic regression model. Five quality control samples were used on each plate to monitor intra- and inter-assay variability, including one human serum, one horse serum, one human plasma, and two peptide-in-assay buffer samples.

[0044] Technical validation of the PRO-C20 ELISA Antibody specificity was evaluated by signal inhibition using serial two-fold dilutions of the standard peptide (RHLEGRGEPGAVGQMGSPGQQGASTQGLWE (SEQ ID NO: 6)), the extended peptide (QGASTQGLWES (SEQ ID NO: 2)), the truncated peptide (QGASTQGLW (SEQ ID NO: 3)), as well as the nonsense standard peptide (SHAHQRTGGN (SEQ ID NO: 8)) and the nonsense coater peptide (biotin-SHAHQRTGGN (SEQ ID NO: 9)).

[0045] Linearity or parallelism was assessed by serially diluting the human serum samples two-fold and calculating the % recovery for the dilution. Precision was assessed by spiking known amounts of standard peptides into the human serum samples and calculating the % recovery of the expected concentration of the unspiked sample and the measured concentration of the spiked sample relative to the known amount spiked. Similarly, precision was assessed by spiking one human serum sample into another in different ratios (e.g., 50:50 or 25:75) and calculating the % recovery for the sum of the values ​​quantified individually.

[0046] The effect of common interfering substances, including hemoglobin, lipids, and biotin, was assessed by spiking human serum samples with known amounts of interfering substances (hemoglobin, low = 2.5 mg / mL, high = 5 mg / mL; lipids, low = 1.5 mg / mL, high = 5 mg / mL; biotin, low = 5 ng / mL, high = 100 ng / mL) and calculating the % recovery relative to unspiked samples.

[0047] Assay variation was tested by performing the assay ten independent times in duplicate with ten quality control samples. Five of the quality control samples were human serum, one horse serum, one human plasma, and three were standard peptides in assay buffer at various concentrations. Intra-assay variation was calculated as the average coefficient of variation (CV%) between duplicate measurements for each of the ten runs. Inter-assay variation was calculated as the overall CV% for all ten runs. The lower and upper limits of the measurement range (LLMR and ULMR) were determined as concentrations that represent the limits of the linear range of the assay. The lower detection limit was calculated as the interpolated mean concentration of 21 blank samples containing only assay buffer, plus three standard deviations. The upper detection limit was calculated as the interpolated mean concentration of the standard peptide corresponding to the highest concentration on the standard curve minus three standard deviations.

[0048] Test article stability was assessed for three human serum samples incubated at either 4° C. or 20° C. for 2, 4, 24, or 48 hours; this was performed by calculating the % recovery of the incubated samples relative to the corresponding control samples kept at −20° C. Freeze / thaw stability was assessed by repeatedly freezing / thawing the human serum samples for up to four cycles and calculating the % recovery of these multiple cycle samples relative to the corresponding control samples that underwent a single freeze / thaw cycle.

[0049] Patient Samples – Cohort 1 The cohort included 222 cancer samples and 33 healthy samples. It included 20 patients with pancreatic, colorectal, renal, gastric, breast, bladder, lung, melanoma, head and neck, and prostate cancer, 19 patients with ovarian cancer, 3 patients with liver cancer, and 33 age-matched healthy controls. All cancer samples were obtained from Proteogenex (Los Angeles, CA, USA), and those of the healthy controls were obtained from BioIVT (Westbury, NY, USA). A summary of the cohort characteristics is shown in Table 1.

[0050] [Table 1] TIFF2024525130000008.tif193159

[0051] Statistics – Cohort 1 Comparison of PRO-C20 levels between groups was performed using conventional one-way analysis of variance (ANOVA), followed by pairwise comparisons to the control group using Dunnett's test. Diagnostic accuracy was assessed by area under the receiver operating characteristic (AUROC) curve. p values ​​less than 0.05 were considered significant. Asterisks in the figures represent the following significance levels: * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001.

[0052] Statistical analyses and graphing were performed with GraphPad Prism (version 9.1.0 for Windows, GraphPad Software; San Diego, CA, USA; www.graphpad.com ) and R version 4.0.4 (R Core Team (2021), R Foundation for Statistical Computin, Vienna, Austria; https: / / www.R-project.org ).

[0053] Patient samples – Cohort 2 The cohort included 20 healthy donors and 36 patients with pancreatic ductal adenocarcinoma (PDAC). All patients were from the Danish BIOPAC study "BIOmarkers in patients with Pancreatic Cancer" (NCT03311776). Patients were recruited between December 2008 and September 2017 in six Danish hospitals. PC patients had histologically confirmed tumors. PDAC patients had been treated with different types of chemotherapy according to national guidelines (www.gicancer.dk). The study was performed according to the recommendations of the Danish Regional Committee for Health-related Research Ethics. The BIOPAC protocol was approved by the Danish Regional Committee for Health-related Research Ethics (VEK ref. KA-20060113) and the Data Protection Authority (j.nr.2006-41-6848). Blood (serum) samples were obtained at the time of diagnosis or before surgery. All subjects provided written informed consent in accordance with the Declaration of Helsinki. A summary of cohort characteristics is shown in Table 2.

[0054] [Table 2]

[0055] Statistics – Cohort 2 The Wilcoxon test was used to compare PRO-C20 levels between healthy and PDAC samples. PRO-C20 diagnostic accuracy was evaluated using the area under the receiver operating characteristic curve (AUC). In addition, the sensitivity, specificity, positive predictive value, and negative predictive value of the PRO-C20 cutoff value at which Youden's index was maximized were measured. The 75th percentile of PRO-C20 levels in PDAC samples (i.e., a PRO-C20 level of 2.59 nM) was used as the cutoff point to define the group with high PRO-C20 levels. The association between high PRO-C20 levels and overall survival was evaluated using Kaplan-Meier curves and log-rank tests. In addition, the association of high PRO-C20 levels with the presence of metastases and adjustment for age and sex was also evaluated using multivariate Cox regression analysis. The following significance levels are indicated by asterisks: **** p<0.0001.

[0056] result Development of PRO-C20 ELISA Optimization of the ELISA protocol included optimal time and temperature of incubation, choice of assay buffer and concentration of kit components. Settings were selected for maximum sensitivity in human serum while meeting the technical requirements outlined below. The format chosen for the ELISA was a competitive ELISA, where the specificity of the assay was evaluated by the ability of different peptides to compete for binding to the monoclonal antibody. The set of peptides included a standard peptide corresponding to the C-terminus of collagen XX (RHLEGRGEPGAVGQMGSPGQQGASTQGLWE (SEQ ID NO:6)); an extended peptide corresponding to a C-terminal epitope with an extra amino acid (QGASTQGLWES (SEQ ID NO:2)); a truncated peptide with one less amino acid (QGASTQGLW (SEQ ID NO:3)); a nonsense standard peptide corresponding to an unrelated epitope (SHAHQRTGGN (SEQ ID NO:8)); and finally a nonsense coater peptide (biotin-SHAHQRTGGN (SEQ ID NO:9)).

[0057] Only the standard peptide inhibited the signal in a meaningful dose-dependent manner; on the other hand, the extended and truncated forms showed minimal competition even at high concentrations (Figure 1). A 10 amino acid long variant of the standard peptide (QGASTQGLWE (SEQ ID NO: 1)) was also tested and showed exactly the same behavior as the 30 amino acid variant (data not shown). The nonsense coater peptide did not give a detectable signal, as expected. This setup provides confirmation that the monoclonal antibody is specific for the desired epitope at the C-terminus of type XX collagen.

[0058] Other aspects of the technical validation are outlined in Table 3.

[0059] [Table 3]

[0060] Linearity or parallelism of dilution was acceptable from undiluted to 1:2 dilution. At 1:4 dilution, recovery of human serum samples fell below the acceptance limit of 80% analyte recovery. Precision testing using spike recovery assays revealed good recovery of the standard peptide in human serum, with recovery of 101%. This was also the case for matrix-in-matrix spikes, where recovery of 95% was achieved when analytes from human serum samples were spiked into other human serum samples. No interference from common interfering substances was observed, with recovery within 15% even at the highest concentration of biotin. Assay variability was excellent, with both inter- and intraassay variability of approximately 6%. Analyte stability was assessed for up to 48 hours at either 4°C or 20°C; recovery was 15%. Stability after four freeze / thaw cycles was also good, with recovery of 90%.

[0061] PRO-C20 in serum of cancer patients (cohort 1) PRO-C20 levels were significantly elevated in all cancers compared to healthy controls (Figure 2). PRO-C20 proved to be an excellent discriminator between healthy and cancer, as evident from the AUROC values ​​(Table 4).

[0062] [Table 4]

[0063] PRO-C20 was particularly good at discriminating between lung cancer and healthy controls (AUROC of 0.92). Overall, these results suggest that circulating levels of PRO-C20, and thus type XX collagen, are elevated in multiple different cancer types.

[0064] PRO-C20 in serum of PDAC patients (cohort 2) Quantification of PRO-C20 in serum from healthy controls (n=20) and PDAC patients (n=36) detected elevated levels of PRO-C20 in PDAC compared to healthy controls (Figure 3). PRO-C20 had good diagnostic accuracy as suggested by the AUC, sensitivity, and specificity values ​​(Table 5). In PDAC patients, high PRO-C20 levels (>2.59 nM) were associated with poor overall survival (Figure 4). High PRO-C20 levels were also an independent predictor of survival after adjustment for age, sex, and the presence of metastases (Table 6).

[0065] [Table 5]

[0066] [Table 6]

[0067] conclusion In this study, we successfully developed, optimized and validated an ELISA to quantify the presence of type XX collagen in blood. The PRO-C20 ELISA was technically robust, highly accurate and sensitive. It was possible to assess circulating type XX collagen levels in the serum of cancer patients and healthy controls, and PRO-C20 levels were significantly higher in all cancers tested compared to healthy controls. In the serum of patients with pancreatic ductal adenocarcinoma (PDAC), it was further found that high levels of PRO-C20 correlated with poor overall survival.

[0068] In this specification, unless expressly stated otherwise, the term "or" is used to mean an operator that returns a value of true if either or both of the conditions it refers to 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 or consisting of". All of the prior knowledge set forth above is incorporated herein by reference. Acknowledgment of any previously published document cited herein should not be taken as an admission or representation that the contents thereof constitute the common general knowledge whether in Australia or elsewhere at the time of filing. References [Table 7] TIFF2024525130000015.tif217159TIFF2024525130000016.tif226159TIFF2024525130000017.tif67159

Claims

1. An immunoassay method for detecting and / or monitoring cancer in a patient, the method comprising: (i) contacting a sample from the patient with a monoclonal antibody that specifically binds to the C-terminal amino acid sequence QGASTQGLWE; and (ii) detecting and measuring the amount of binding between the monoclonal antibody and the peptide in the sample. A method.

2. The method further comprises: (iii) correlating the amount of binding of the monoclonal antibody as measured in step (ii) with a value related to a normal healthy subject and / or a value related to a known disease severity and / or a value obtained from the patient at a previous time point, and / or a predefined cut-off value. The method according to claim 1, further comprising the step of

3. The method according to claim 1 or 2, wherein the cancer is bladder cancer, breast cancer, colorectal cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, or gastric cancer.

4. The method according to claim 1, wherein the monoclonal antibody does not specifically bind to an extended form of the C-terminal amino acid sequence QGASTQGLWE with an additional amino acid E at the C-terminus.

5. The method according to claim 1 or 4, wherein the monoclonal antibody does not specifically bind to a shortened form of the C-terminal amino acid sequence QGASTQGLWE lacking the last amino acid E.

6. The method according to claim 1, wherein the monoclonal antibody is raised against a synthetic peptide having the C-terminal amino acid sequence QGASTQGLWE.

7. The method according to claim 1, wherein the sample is a biological fluid sample selected from blood, serum, or plasma.

8. The method according to claim 1, wherein the immunoassay is a competitive assay or a sandwich assay.

9. The method according to claim 1 or 8, wherein the immunoassay is a radioimmunoassay or an enzyme immunoassay.

10. An immunoassay kit comprising a monoclonal antibody that specifically binds to the C-terminal amino acid sequence QGASTQGLWE, the immunoassay kit comprising: - a streptavidin-coated well plate; - a biotinylated peptide, Biotin-L-QGASTQGLWE, where L is an optional linker; - a secondary antibody for use in a sandwich immunoassay; - a calibration protein comprising the C-terminal amino acid sequence QGASTQGLWE; - an antibody biotinylation kit; - an antibody HRP-labeling kit; - an antibody radiolabeling kit; and - An assay visualization kit containing at least one type of immunological assay kit.

11. The immunological assay kit according to claim 10, wherein the monoclonal antibody does not specifically bind to an extended form of the C-terminal amino acid sequence that is QGASTQGLWE.

12. The immunological assay kit according to claim 10 or 11, wherein the monoclonal antibody does not specifically bind to a shortened form of the C-terminal amino acid sequence that is QGASTQGLW.

13. The immunological assay kit according to claim 10, wherein the monoclonal antibody is prepared against a synthetic peptide having the C-terminal amino acid sequence QGASTQGLWE.

14. A monoclonal antibody that specifically binds to the C-terminal amino acid sequence QGASTQGLWE.

15. The monoclonal antibody according to claim 14, which does not specifically bind to an extended form of the C-terminal amino acid sequence that is QGASTQGLWE.

16. The monoclonal antibody according to claim 14 or 15, which does not specifically bind to a shortened form of the C-terminal amino acid sequence that is QGASTQGLW.

17. The monoclonal antibody according to claim 14, which is prepared against a synthetic peptide having the C-terminal amino acid sequence QGASTQGLWE.

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