Assay of type XIX collagen

Abstract

The present invention relates to monoclonal antibodies that target collagen XIX, and immunoassays and kits that use said antibodies. The assays of the present invention can be used for the diagnosis and monitoring of cancer.

Description

Technical Field

[0001] The present invention relates to a monoclonal antibody targeting type XIX collagen, as well as an immunoassay and a kit using the antibody.

Background Art

[0002] Introduction Type XIX collagen is a collagen related to the basement membrane, and its property evaluation has not advanced. This collagen shows regulatory changes during the progression of breast cancer, and the NC1(XIX) domain shows anti-tumor signal transduction properties. However, little is known about the potential of type XIX collagen as a biomarker in cancer.

[0003] Lung cancer is the most frequently diagnosed cancer and a major cause of cancer death (Non-Patent Document 1). Non-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancer cases, and adenocarcinoma (AC) and squamous cell carcinoma (SCC) are the most common subtypes (Non-Patent Documents 2 and 3). Since many cases of lung cancer are diagnosed at an advanced stage, the overall 5-year survival rate is a serious 19% (Non-Patent Document 4). However, in the case of patients diagnosed at a limited stage where most patients can benefit from surgical resection, the 5-year survival rate is 56% (Non-Patent Documents 2 and 4). Therefore, early detection is one of the major ways to improve the survival rate of lung cancer patients.

[0004] The tumor microenvironment is intricately associated with the conventional features that define cancer progression (Non-Patent Document 5). One of the major components of the tumor microenvironment is the extracellular matrix (ECM), the acellular part of the tissue, which actually affects all of these features (Non-Patent Document 6). The most major protein in the ECM is collagen, and there are 28 different types (Non-Patent Document 7). Type XIX collagen is a minor collagen in which three α1(XIX) chains form a homotrimer of 400 kDa. Each chain contains five collagenous triple helix domains and six non-collagenous domains. Based on its primary sequence, type XIX collagen belongs to the family of Fibril-Associated Collagen with Interrupted Triple helices, which mediates the interaction between fibrillar collagen and other ECM components (Non-Patent Documents 8 to 10).

[0005] The expression of type XIX collagen is widely observed in developing mice, but is more restricted in adults, with most of it accumulating in brain tissue (Non-Patent Document 11). In adults, its expression has been found in the brain, skeletal muscle, spleen, prostate, kidney, liver, placenta, colon, skin, and breast tissue (Non-Patent Documents 12, 13). Although the function of type XIX collagen is not fully understood, it is actually thought to play a role in development. Type XIX collagen is involved in the differentiation of embryonic muscle and the development of the esophagus (Non-Patent Documents 10, 14, 15). Type XIX collagen is also involved in synapse formation in the hippocampus and axon formation in spinal cord neurons (Non-Patent Documents 16, 17). Overexpression of type XIX collagen in the muscles of patients with amyotrophic lateral sclerosis (ALS) has also been reported, which is associated with a worse prognosis (Non-Patent Documents 18, 19).

[0006] Overall, the tissue localization of type XIX collagen protein is mostly associated with blood vessels, nerves, muscles, and the basement membrane zone (BMZ) of some epithelia (Non-Patent Document 12). Interestingly, protein staining of type XIX collagen in the epithelial BMZ of breast cancer is partially lost in localized tubular carcinoma and completely absent in invasive carcinoma. Since this disappearance occurs earlier than that of type IV collagen and laminin, it has been suggested that the decrease in type XIX collagen levels is an early result of BMZ remodeling in pre-invasive tumors (Non-Patent Document 13).

[0007] Similar to the release of matricains from the NC1 domains of type IV, XV, and XVIII collagens, the C-terminal NC1 domain of type XIX collagen may be cleaved and released. The resulting peptide can suppress melanoma growth and angiogenesis in vivo and invasion in vitro (Non-Patent Document 20). The NC1 domain is cleaved by plasmin protease, interacts with αvβ3 integrin to inhibit the FAK / PI3K / Akt / mTOR signaling pathway, and inhibits GSK3β phosphorylation (Non-Patent Documents 21, 22). Interestingly, the NC1 domain induces the formation of inhibitory nerve terminals, interacting with α5β1, a different integrin receptor, in the process (Non-Patent Document 23).

Prior Art Documents

Non-Patent Documents

[0008]

Non-Patent Document 1

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Non-Patent Document 26

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Non-Patent Document 37

Non-Patent Document 38

Summary of the Invention

Means for Solving the Problems

[0009] The inventors have developed a monoclonal antibody that specifically recognizes and binds to the C-terminus of type XIX collagen, particularly the α1 chain; and an immunoassay, specifically, an enzyme-linked immunosorbent assay (ELISA) for detecting type XIX collagen in a biological fluid sample. The inventors have determined that type XIX collagen can be used as a biomarker for cancer detection. Specifically, PRO-C19 is particularly excellent in discriminating between NSCLC and healthy controls and can be used as a biomarker for early detection of NSCLC.

[0010] Accordingly, in a first aspect, the present invention relates to a monoclonal antibody that specifically recognizes and binds to the C-terminus of the α1 chain of type XIX collagen (also referred to herein as the target peptide), and the C-terminus has the amino acid sequence SHAHRTGGN (SEQ ID NO: 1) (also referred to herein as the target sequence).

[0011] Preferably, the monoclonal antibody is a monoclonal antibody raised against a synthetic peptide having a C-terminal amino acid sequence SHAHQRTGGN (SEQ ID NO: 1). The synthetic peptide used to raise the antibody may be a synthetic peptide linked to a transport protein at its N-terminus. Exemplary transport proteins include, but are not limited to, proteins such as keyhole limpet hemocyanin (KLH). The synthetic peptide may be linked to the transport protein via any suitable linkage, and the peptide may contain one or more additional amino acid residues at the N-terminus of the peptide. The monoclonal antibody may be raised by any suitable technique known to those skilled in the art, such as immunizing a mouse or other mammal, isolating spleen cells from the immunized mammal and fusing them with hybridoma cells, and then culturing the resulting hybridoma cells to ensure monoclonal growth, but is not limited thereto.

[0012] In a preferred embodiment, the monoclonal antibody does not specifically recognize or specifically bind to a peptide having a C-terminal amino acid sequence SHAHQRTGGNX (SEQ ID NO: 2), where X represents any amino acid. Thus, the monoclonal antibody preferably does not specifically recognize or specifically bind to an extended variant of the target peptide in which the target amino acid sequence is extended by one or more amino acids at the C-terminus. Preferably, the monoclonal antibody does not specifically recognize or specifically bind to a peptide having a C-terminal amino acid sequence SHAHQRTGGNA (SEQ ID NO: 3).

[0013] In a preferred embodiment, the monoclonal antibody does not specifically recognize or specifically bind to a peptide having a C-terminal amino acid sequence SHAHQRTGG (SEQ ID NO: 4). Thus, the monoclonal antibody preferably does not specifically recognize or specifically bind to a truncated variant of the target peptide in which the target amino acid sequence is truncated by one or more amino acids at the C-terminus.

[0014] In a preferred embodiment, the monoclonal antibody does not specifically recognize or specifically bind to a peptide having the C-terminal amino acid sequence GVAPGIGPGG (SEQ ID NO: 5). Thus, the monoclonal antibody preferably does not specifically recognize or specifically bind to a nonsense standard peptide.

[0015] In a second aspect, the present invention relates to a method for an immunoassay for detecting type XIX collagen in a human biological fluid sample, the method comprising contacting a human biological fluid sample with a monoclonal antibody according to the first aspect of the present invention and detecting a binding between the monoclonal antibody and a peptide in the sample.

[0016] Preferably, the detection is quantitative. Thus, the method may include detecting and determining the amount of binding between the monoclonal antibody and a peptide in the sample.

[0017] Preferably, the immunoassay is a competitive immunoassay.

[0018] Preferably, the immunoassay is an enzyme-linked immunosorbent assay (ELISA). Preferably, the ELISA is a competitive ELISA.

[0019] The human biological fluid sample may be, by way of example, blood, serum, plasma, or urine. Preferably, the sample is serum or plasma.

[0020] The human biological fluid sample may be a sample from a human patient having medical signs or symptoms indicative of cancer. Preferably, the biological fluid sample is a sample from a human patient having medical signs or symptoms indicative of cancer of the pancreas, colon, kidney, stomach, ovary, breast, bladder, lung, head and neck, prostate, or liver, or melanoma, preferably cancer of the breast, lung, or ovary, more particularly lung cancer, especially non-small cell lung cancer (NSCLC).

[0021] The method may be an immunoassay method for diagnosing, and / or monitoring, and / or evaluating the possibility of cancer in a patient. The method includes contacting a biological fluid sample obtained from the patient with a monoclonal antibody, detecting and determining the amount of binding between the monoclonal antibody and a peptide in the sample, and correlating the amount of binding with a value associated with a normal healthy subject, and / or a value associated with a known disease severity, and / or a value obtained from the patient at a previous time point. Preferably, the cancer is cancer of the pancreas, large intestine, kidney, stomach, ovary, breast, bladder, lung, head and neck, prostate, or liver, or melanoma, more preferably cancer of the breast, lung, or ovary, specifically lung cancer, particularly non-small cell lung cancer (NSCLC).

[0022] In some embodiments of the method according to the second aspect, the amount of binding of a monoclonal antibody specific for an epitope of a type XIX collagen peptide having the C-terminal amino acid sequence SHAHQRTGGN (SEQ ID NO: 1) correlates with one or more predetermined cut-off values.

[0023] As used herein, "cut-off value" means an amount of binding that is statistically determined to indicate a high likelihood of being a subject having cancer. A measured value of biomarker binding in a patient sample that is above the statistical cut-off value may correspond to a probability of at least 70%, preferably at least 80%, preferably at least 85%, more preferably at least 90%, most preferably at least 95% of the presence or possibility of cancer. The "cut-off value" can be calculated by comparing the results obtained from patients diagnosed with cancer and healthy controls.

[0024] The predetermined cut-off value for the binding amount of the monoclonal antibody specific to the C-terminal amino acid sequence SHAHQRTGGN (SEQ ID NO: 1) may be in the range of 50.0 to 200.0 ng / mL. Preferably, the predetermined cut-off value for the binding amount of the monoclonal antibody specific to the C-terminal amino acid sequence SHAHQRTGGN (SEQ ID NO: 1) is in the range of 75.0 to 150.0 ng / mL, more preferably in the range of 90.0 to 120.0 ng / mL, and most preferably at least 118.9 ng / mL. In this regard, through the use of statistical analysis, if the measured amount of binding of the monoclonal antibody specific to the C-terminal amino acid sequence SHAHQRTGGN (SEQ ID NO: 1) is within the range of 50.0 to 200.0 ng / mL, specifically at least 118.9 ng / mL or more, it has been found that there may be cases where a patient has cancer, specifically lung cancer, such as NSCLC. Due to the existence of a statistical cut-off value within the range of 50.0 to 200.0 ng / mL, specifically at least 118.9 ng / mL, it is possible to predict cancer diagnosis with high reliability using the method of the present invention. Specifically, there may be cases where a patient with NSCLC is determined with a value within the range of 50.0 to 200.0 ng / mL, specifically at least 118.9 ng / mL or more. Applying such a statistical cut-off value is particularly advantageous as it results in an independent diagnostic assay; that is, any need for direct comparison with healthy individuals and / or patients known to have cancer to reach a diagnosis conclusion is thereby eliminated. By making a definitive prediction easily, the patient may be treated earlier, which in turn may improve the overall prognosis of survival and / or reduce the risk of hospitalization.

[0025] In patients diagnosed with lung cancer, particularly NSCLC, an index for the cancer stage can be provided using a predetermined cut-off value for the binding amount of a monoclonal antibody specific to the C-terminal amino acid sequence SHAHQRTGGN (SEQ ID NO: 1). The predetermined cut-off value for the binding amount of the monoclonal antibody specific to the C-terminal amino acid sequence SHAHQRTGGN (SEQ ID NO: 1) can be in the range of 40.0 to 75.0 ng / mL, preferably at least 55.6 ng / mL. In this regard, through the use of statistical analysis, it has been found that if the measured amount of binding of the monoclonal antibody specific to the C-terminal amino acid sequence SHAHQRTGGN (SEQ ID NO: 1) is in the range of 40.0 to 75.0 ng / mL, preferably at least 55.6 ng / mL or more, there may be cases where patients having advanced cancer in stage III or stage IV NSCLC are identified. With a statistical cut-off value in the range of 40.0 to 75.0 ng / mL, particularly at least 55.6 ng / mL, it is possible to predict the cancer stage and progression with high reliability using the method of the present invention. Specifically, there may be cases where patients with NSCLC at least in stage III are identified with values in the range of 40.0 to 75.0 ng / mL, preferably at least 55.6 ng / mL or more. The fact that a definitive prediction can be made simply can be useful for monitoring the progression of cancer in patients and the effectiveness of treatment. The use of the cut-off value may help to identify whether the treatment regime is working and whether the cancer is progressing, and as a result, alternative treatments can be sought earlier, which may in turn improve the overall outlook for survival.

[0026] In a third aspect, the present invention a monoclonal antibody according to the first aspect of the present invention and: - a streptavidin-coated well plate; - an N-terminal biotinylated peptide having the C-terminal amino acid sequence SHAHQRTGGN (SEQ ID NO: 1); and - a calibration peptide having the C-terminal amino acid sequence SHAHQRTGGN (SEQ ID NO: 1) at least one of; relates to an assay kit comprising

[0027] The kit may be for use in the diagnosis or prediction of cancer risk, preferably in combination with a method according to a second aspect of the invention. Preferably, the cancer is pancreatic, colorectal, renal, gastric, ovarian, breast, bladder, lung, head and neck, prostate, or liver cancer, or melanoma, more preferably breast, lung, or ovarian cancer, particularly lung cancer, especially non-small cell lung cancer (NSCLC). Definitions

[0028] As used herein, the terms "peptide" and "polypeptide" are used synonymously.

[0029] As used herein, the term "monoclonal antibody" refers to both the whole antibody and fragments thereof that retain the binding specificity of the whole antibody, such as Fab fragments, Fv fragments, or other such fragments known to those skilled in the art. Antibodies that retain the same binding specificity may contain the same complementarity determining regions (CDRs). The CDRs of an antibody can be determined using methods known to those skilled in the art, such as those described by Kabat et al. [Non-Patent Document 38].

[0030] Antibodies can be generated from B cell clones as described in the examples. The isotype of an antibody can be determined by ELISA specific for human IgM, IgG, or IgA isotypes, or human IgG1, IgG2, IgG3, or IgG4 subclasses. Other suitable methods can be used to identify the isotype.

[0031] 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. This cDNA is then subjected to PCR using primers that amplify the heavy and light chains of the antibody. For example, primers specific for the leader sequence can be used for all VH (variable heavy chain) sequences, together with primers that bind to sequences located in the constant region of a pre-determined isotype. The light chain can be amplified by using primers that bind to the 3' end of the kappa or lambda chain, together with primers that anneal to the V kappa or V lambda leader sequence. Full-length heavy and light chains can be generated and sequenced.

[0032] As used herein, the term "C-terminus" refers to the end of the polypeptide, i.e., the C-terminus of the polypeptide, and is not to be construed in its ordinary directional sense.

[0033] Similarly, the term "N-terminus" refers to the end of the polypeptide, i.e., the N-terminus of the polypeptide, and is not to be construed in its ordinary directional sense. As used herein, the term "competitive immunoassay" refers to an immunoassay in which, with respect to binding to an antibody, a target peptide (if any) present in a sample competes with a known amount of a peptide target (e.g., one that is bound to a solid substrate or labeled), which is a technique known to those of skill in the art.

[0034] As used herein, the term "ELISA" (enzyme-linked immunosorbent assay) refers to an immunoassay in which a target peptide (if any) present in a sample is detected using an antibody conjugated to an enzyme, e.g., horseradish peroxidase or alkaline phosphatase. The activity of the enzyme is then evaluated by incubation with a substrate that produces a measurable product. Thereby, the presence and / or amount of the target peptide in the sample can be detected and / or quantified. ELISA is a technique known to those of skill in the art.

[0035] As used herein, the term "binding amount" refers to the quantification of the binding between a monoclonal antibody and a target peptide, and the quantification is determined by comparing the measured value of the target peptide in a biological fluid sample with a calibration curve, which is generated using standard samples of known concentrations of the target peptide. In the specific assay disclosed herein for measuring a target peptide having the C-terminal amino acid sequence SHAHQRTGGN (SEQ ID NO: 1) in a biological fluid, the calibration curve is generated using standard samples of known concentrations of a calibration peptide having the C-terminal amino acid sequence SHAHQRTGGN (SEQ ID NO: 1, and in particular, which may consist of the amino acid sequence SHAHQRTGGN (SEQ ID NO: 1)). By comparing the value measured in the biological fluid sample with the calibration curve, the actual amount of the target peptide in the sample is determined.

[0036] As used herein, the term "PRO-C19" means type XIX collagen having the C-terminal amino acid sequence SHAHQRTGGN (SEQ ID NO: 1).

[0037] The present invention is demonstrated in the following examples with reference to the following figures.

Brief Description of the Drawings

[0038]

Figure 1

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Mode for Carrying Out the Invention

[0039] Method ELISA protocol for PRO-C19: 10-amino acid peptide found at the C-terminus of type XIX collagen (UniProtKB: Q14993) 1133 SHAHQRTGGN 1142(SEQ ID NO:1) was purchased from Genscript (Piscataway, New Jersey, USA) and used for immunization. The production of monoclonal antibodies has been described elsewhere (Non-Patent Document 24). Several optimizations were performed on ELISA, including the selection of assay buffer, incubation time, and temperature, as well as the concentrations of antibodies and peptides. The final PRO-C19 protocol was executed as follows: A 96-well streptavidin-coated ELISA plate was coated with 100 μl / well of 2.5 ng / ml biotinylated SHAHRTGGN peptide dissolved in assay buffer (25 mM TBS, 1% BSA (w / v), 0.1% Tween-20 (w / v), 2 g / l NaCl, pH 8.0) and incubated at 20 °C for 30 minutes with shaking at 300 RPM. After washing 5 times with wash buffer (25 mM Tris, 50 mM NaCl, pH 7.2), 20 μl / well of the sample was added in two portions, followed by 100 μl / well of 60 ng / ml HRP-labeled monoclonal antibody in assay buffer, and incubated at 20 °C for 1 hour with shaking at 300 RPM. After the second washing cycle, 100 μl / well of TMB was added and incubated for 15 minutes at 20 °C with shaking at 300 RPM in the dark. The reaction was stopped by adding 1% H2SO4, 100 μl / well. Absorbance was measured at 450 nm with reference to 650 nm. To create a standard curve, 20 μl / well of a two-fold dilution series of 500 ng / ml SHAHQRTGGN (SEQ ID NO:1) peptide was added to appropriate wells, and the curve was created using four-parameter mathematical fitting. Each plate included five quality control samples, including one human serum, one horse serum, one bovine cartilage extract, and two peptide samples in assay buffer, to monitor intra-assay and inter-assay variability.

[0040] Technical validity of ELISA for PRO-C19: The specificity of the antibody was tested by signal inhibition by 2-fold dilution of not only the standard peptide (SHAHQRTGGN - SEQ ID NO: 1), the extended peptide (SHAHQRTGGNA - SEQ ID NO: 3), the shortened peptide (SHAHQRTGG - SEQ ID NO: 4), but also the nonsense standard peptide (GVAPGIGPGG - SEQ ID NO: 5), and the nonsense coter peptide (Biotin-GVAPGIGPGG). Linearity or parallelism was tested by calculating the recovery rate against the dilution factor with human serum samples as a 2-fold dilution series. Accuracy was tested by spiking the standard peptide into human serum samples and calculating the recovery rate of the peptide in the spiked samples. The effects of common interferents including hemoglobin, lipid, and biotin were evaluated with human serum spiked with either high or low concentrations of interferents (hemoglobin low = 2.5 mg / ml, high = 5 mg / ml; lipid low = 1.5 mg / ml, high = 5 mg / ml; biotin low = 3 ng / ml, high = 9 ng / ml). Assay interference was calculated as the recovery rate of the spiked sample against the non-spiked sample. Assay variability was tested by 10 independent runs using 10 runs of quality control samples in duplicate measurements. Five of the quality control samples were human serum, one was horse serum, one was bovine cartilage extract, and three were standard peptides in assay buffer at various concentrations. Intra-assay variability was calculated as the average coefficient of variation (CV%) of each duplicate measurement in 10 runs. Inter-assay variability was calculated as the total CV% over 10 runs. The lower and upper limits of the measurement range (LLMR and ULMR, respectively) were measured over 10 independent runs and indicate the boundaries of the linear range of the standard curve. The stability of the analyte was measured against three human serum samples incubated at 4 or 20 °C for 2, 4, 24, or 48 hours. Stability was calculated as the recovery rate of the incubation sample against the control sample stored at -20 °C. Freeze-thaw stability was evaluated by freeze-thawing human serum samples up to 4 cycles. Stability was calculated as the recovery rate of the thawed sample against the sample that had undergone 1 freeze-thaw cycle. The lower limit of detection was calculated as the average concentration of 21 blank samples containing assay buffer and adding 3 standard deviations.The upper detection limit was calculated as the average concentration of the standard peptide corresponding to the highest concentration of the standard curve over 10 independent runs, minus three standard deviations.

[0041] Patient samples: The first cohort was obtained in part from the vendor Asterand Bioscience (Detroit, Michigan, USA). This included sera from 75 cancer patients with breast (n = 12), colon (n = 7), stomach (n = 9), melanoma (n = 6), NSCLC (n = 11), ovary (n = 8), pancreas (n = 2), prostate (n = 13), and small cell lung cancer (SCLC) (n = 7), along with 38 healthy controls obtained from the vendor Valley Biomedical (Winchester, Virginia, USA).

[0042] The second and third cohorts were obtained from the vendor Proteogenex (Los Angeles, California, USA). The second cohort included 40 NSCLC patients, 20 of stage III and 20 of stage IV. This also included 24 healthy controls obtained from Valley Biomedical. The third cohort included 34 NSCLC patients, 10 of stage I, 10 of stage II, 9 of stage III, and 5 of stage IV. This also included 30 healthy controls obtained from Proteogenex and Valley Biomedical.

[0043] The fourth cohort included 20 cancer patients each with pancreas, colon, kidney, stomach, ovary, breast, bladder, lung, melanoma, head and neck, and prostate cancer. This also included 3 liver cancer patients and 33 healthy controls. All cancer samples were obtained from Proteogenex, and healthy controls were obtained from BioIVT (Westbury, New York, USA).

[0044] In accordance with the operator, sample collection was approved by the Institutional Review Board or Independent Ethical Committee, and patients provided informed consent. All investigations were conducted in accordance with the Helsinki Declaration.

[0045] Statistics: PRO-C19 levels were log-transformed and tested for normality using the D’Agostino-Pearson omnibus test. Comparisons of PRO-C19 levels between healthy subjects and NSCLC, and between NSCLC subtypes, were performed using unpaired two-sided t-tests. Comparisons of PRO-C19 levels across several groups were performed using ordinary one-way ANOVA corrected for multiple comparisons using Dunnett’s test. Differences in age between groups were evaluated using unpaired two-sided t-tests. Differences in sex and ethnicity were evaluated using Fisher’s exact test. Correlations between PRO-C19 levels and BMI, age, smoking, and sampling date were evaluated using linear regression. Diagnostic accuracy was tested using the area under the receiver operating characteristic curve (AUROC). Sensitivity and specificity were determined at the optimal cut-off value estimated according to the Youden Index. p-values less than 0.05 were considered significant. Asterisks indicate the following significance levels: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. When performing multiple comparison tests, multiplicity-adjusted p-values are reported. Statistical analysis and graphs were performed using GraphPad Prism (version 8.2 for Windows, GraphPad Software, San Diego, California, USA, www.graphpad.com) and MedCalc (MedCalc Statistical Software version 18.11.6 (MedCalc Software bvba, Ostend, Belgium; https: / / www.medcalc.org; 2019)).

[0046] Results The specificity of the PRO-C19 assay was evaluated by the proficiency of the peptide competing for binding to the monoclonal antibody. The peptides tested included a standard peptide (SHAHQRTGGN - SEQ ID NO: 1), an extended peptide (SHAHQRTGGNA - SEQ ID NO: 3), a shortened peptide (SHAHQRTGG - SEQ ID NO: 4), a nonsense peptide (GVAPGIGPGG - SEQ ID NO: 5), and a nonsense-coated peptide (Biotin-GVAPGIGPGG). Only the standard peptide inhibited the signal in a dose-dependent manner (Figure 1 shows the specificity of the PRO-C19 assay). The nonsense-coated peptide did not generate a detectable signal. This indicates in all cases that the assay is specific for the SHAHQRTGGN (SEQ ID NO: 1) epitope of type XIX collagen.

[0047] The technical validity of the PRO-C19 assay is summarized in Table 1. The linearity and parallelism of dilution were within the acceptable range when the serum sample was diluted 1:4, and thereafter, the average dilution recovery rate was 101.7% (Figure 2). The matrix precision in serum was within the acceptable range, and using the standard peptide spiked into human serum samples at a final dilution of 1:4, the average spike recovery rate was 118.6%. No effects of common interferents were observed, including hemoglobin, lipids, and biotin. The inter-assay variation was 10.9% and the intra-assay variation was 6.6%. The measurement range was determined to be 3.31 - 214.3 ng / ml, and the limit of detection was 1.23 - 443.5 ng / ml. The stability of the analyte was within the acceptable range up to 24 hours at 4°C and up to 4 hours at 20°C. The freeze-thaw stability was within the acceptable range for more than 4 freeze-thaw cycles.

[0048] To investigate the utility of PRO-C19 in the context of cancer, PRO-C19 was evaluated in a cohort consisting of various cancer types including 12 breast cancer samples, 7 colon cancers, 9 gastric cancers, 6 melanomas, 11 NSCLC, 8 ovarian cancers, 2 pancreatic cancers, 13 prostate cancers, 7 SCLCs, as well as 38 healthy controls (Table 2). No significant associations were seen between PRO-C19 levels and age, BMI, or smoking history in the cancer groups. PRO-C19 levels were significantly elevated in cancers of NSCLC (p<0.0001), SCLC (p=0.0081), breast (p=0.0005), and ovary (p<0.0001) (Figure 3). In the colon and pancreatic cancer groups, although not significant, the mean PRO-C19 levels were higher compared to healthy controls and lower in gastric cancer. With a cut-off value of 63.3 ng / ml, PRO-C19 has the potential to discriminate between healthy individuals and NSCLC with an AUROC of 0.995, corresponding sensitivity of 100%, and specificity of 94.74% (Table 5). PRO-C19 can also potentially discriminate between healthy individuals and SCLC at a cut-off value of 54.3 ng / ml with an AUROC of 0.808, corresponding sensitivity of 71.4%, and specificity of 84.2%. PRO-C19 can also potentially discriminate between healthy individuals and breast cancer at a cut-off value of 41.85 ng / ml with an AUROC of 0.814, corresponding sensitivity of 75%, and specificity of 78.9%. Finally, PRO-C19 can also potentially discriminate between healthy individuals and ovarian cancer at a cut-off value of 60.31 ng / ml with an AUROC of 0.839, corresponding sensitivity of 75%, and specificity of 92.1%. Overall, the circulating blood levels of type XIX collagen appear to be elevated in several different cancer types. Next, the role of PRO-C19 in NSCLC was investigated.

[0049] PRO-C19 was evaluated in a cohort of NSCLC patients including 20 stage III and 20 stage IV patients, as well as 24 healthy controls (Table 3). The control group was significantly younger, had a lower proportion of males, and a lower proportion of whites compared to the NSCLC group. Within the NSCLC group itself, no significant associations were found between PRO-C19 levels and sample collection date, gender, age, BMI, smoking history, tumor grade, or histological subtype (AC and SCC). Mean PRO-C19 levels were significantly elevated up to 3.5-fold compared to controls in the NSCLC group (p<0.0001) (Figure 4). At a cutoff value of 55.6 ng / ml, PRO-C19 had the potential to discriminate between healthy individuals and NSCLC with an AUROC of 0.980, corresponding to a sensitivity of 97.5% and a specificity of 91.67% (Table 5). When separated into stages III and IV, PRO-C19 levels in each stage were also significantly elevated compared to healthy controls (p<0.0001) (Figure 5). These results confirm that PRO-C19 levels are elevated in the circulating blood of NSCLC patients.

[0050] Next, the use of PRO-C19 in even earlier stages of NSCLC was investigated. For this purpose, PRO-C19 was evaluated in a separate cohort of NSCLC patients including 10 stage I, 10 stage II, 9 stage III, 5 stage IV, as well as 30 healthy controls (Table 4). The control group was significantly younger and had a lower proportion of white people compared to the NSCLC group. There was no significant difference in gender between the two groups. Within the NSCLC group, no significant associations were found between the sample collection date, gender, age, BMI, smoking history, tumor grade, or histological subtype (AC or SCC). The mean PRO-C19 levels were significantly increased up to two-fold in NSCLC compared to controls (Figure 6). At a cut-off value of 118.9 ng / ml, PRO-C19 has the potential to discriminate between healthy individuals and NSCLC with an AUROC of 0.823, corresponding sensitivity of 82.4%, and specificity of 76.7% (Table 5). When comparing individual stages to healthy controls, PRO-C19 was also significantly increased in stage II (p = 0.0011), stage III (p = 0.0012), and stage IV (p = 0.0041) compared to healthy controls (Figure 7). There was a tendency for the mean PRO-C19 levels to be higher with increasing stage. To evaluate PRO-C19 as an early detection marker for NSCLC, the diagnostic accuracy for stage I+II was evaluated. PRO-C19 has the potential to discriminate between healthy individuals and stage I+II NSCLC with an AUROC of 0.762 at a cut-off value of 118.9, corresponding sensitivity of 70.0%, and specificity of 76.7% (Table 5). At a higher specificity of 96.7%, the sensitivity decreased to 35%.

[0051] Finally, PRO-C19 was evaluated in a separate cancer cohort including 20 pancreatic cancer patients, colorectal cancer (CRC), kidney cancer, stomach cancer, ovarian cancer, breast cancer, bladder cancer, lung cancer, melanoma, head and neck (H&N) cancer, prostate cancer, and finally 3 liver cancer patients, as well as 33 healthy controls (Table 6). No significant differences in age or gender were found between cancer patients and healthy controls.

[0052] However, the cancer samples were only from white patients, while the healthy controls were a mix of white, black, and Hispanic ethnicities. No significant associations were found between PRO-C19 levels and sample collection date, gender, age, or BMI. The mean PRO-C19 level was significantly elevated in all cancers compared to controls (Figure 8). PRO-C19 generally performed well in differentiating between healthy and cancer individuals across all cancer types. This is summarized in Table 7.

[0053] Discussion This study demonstrated the technical validity of an ELISA that measures the C-terminus of type XIX collagen, named PRO-C19. PRO-C19 was specific to the intended epitope and technically robust. PRO-C19 was evaluated in a panel of serum samples from healthy individuals and cancer patients to demonstrate its biological relevance and potential as a biomarker. PRO-C19 levels were significantly elevated in several types of cancer, providing evidence of its superiority in differentiating between NSCLC and healthy individuals and showing moderate diagnostic accuracy in the early stages of NSCLC.

[0054] In adults, as exemplified by type XIX collagen, which accounts for 10-6% of the dry weight of umbilical cord tissue, the expression of type XIX collagen may be very limited (Non-Patent Document 25). However, separate studies quantifying type XIX collagen in different tissue extracts and biological fluids have shown that it can be detected in circulating blood (Non-Patent Document 26). From this data, type XIX collagen is released into the circulating blood of healthy adults in moderate amounts, and circulating type XIX collagen levels are significantly increased in some cancer types. Type XIX collagen has previously been associated with the progression of breast cancer. In this context, as the BMZ around breast tumors is disrupted during cancer progression, the staining property of type XIX collagen protein is also lost (Non-Patent Document 13). The expression of type XIX collagen is generally strongly associated with the BMZ, and disruption of the BMZ in breast epithelium and blood vessels may result in the release of type XIX collagen into the circulating blood. From this data, an increase in circulating type XIX collagen levels is also associated with breast cancer. For example, there may be clear differences in the organization of the BMZ among different tumor types. The epithelial BMZ is disrupted around invasive breast cancers, while it remains intact around invasive glands in epithelial malignancies of the colon, prostate, and lung (Non-Patent Documents 13, 27). The limitation of this approach to quantifying type XIX collagen is that, although the tissue where the tumor is found is likely to be a factor, the origin tissue cannot be determined.

[0055] Type XIX collagen is also associated with neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) and Parkinson's disease. In the peripheral blood of Parkinson's disease patients, the expression of type XIX collagen is decreased (Non-Patent Document 35). In contrast, in ALS, type XIX collagen increases with disease progression and increases the risk of death (Non-Patent Documents 18, 36, 37). Therefore, the PRO-C19 assay may also be used for the detection and diagnosis of neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) and Parkinson's disease.

[0056] The presence of type XIX collagen related to lung cancer has not been demonstrated so far. While it has been observed in moderate amounts in the lungs of mouse embryos, only trace amounts are found in adults, which may suggest a developmental role of type XIX collagen in the lungs (Non-Patent Document 11). Such expression patterns are seen in some proteins and pathways important for cancer progression, and indeed, some aspects of the developmental process, including EMT28-30, are reactivated during tumor formation. Thus, the role in development may also suggest a role in cancer.

[0057] Antitumor properties have been attributed to type XIX collagen. Interestingly, the NC1 domain, once cleaved, can inhibit invasion and angiogenesis in melanoma (Non-Patent Document 22). This has been demonstrated in an in vivo mouse model where the NC1(XIX) peptide inhibited tumor growth and angiogenesis by inhibiting MMP-14 and VEGF (Non-Patent Document 20). Subsequently, it was discovered that NC1(XIX) signaling is likely mediated by αvβ3 integrin (Non-Patent Document 31). In a separate study, it was demonstrated that the NC1(XIX) peptide promotes the formation of inhibitory nerve terminals through α5β1 integrin (Non-Patent Document 23). Since these integrin receptors are expressed by epithelial and endothelial lung cells and play a role in NSCLC, it may be interesting to see the effect of the NC1(XIX) peptide in lung cancer (Non-Patent Documents 32-34).

[0058] The PRO-C19 assay is not specific for neoepitopes generated during plasmin cleavage and subsequent release of the NC1 domain. However, it can quantify any fragment containing the C-terminal epitope. In the absence of knowledge about how type XIX collagen is cleaved or otherwise processed, PRO-C19 may, on the hypothesis, be able to measure a large and diverse population of type XIX collagen fragments all containing the C-terminal epitope. Further investigation is needed into how type XIX collagen is processed and whether any of these fragments can be quantified in circulating blood. A separate assay specific for the neoepitopes generated during plasmin cleavage may be useful in this regard.

[0059] With regard to diagnostic accuracy, PRO-C19 did not perform as well in stages I and II of NSCLC patients. Considering the small sample size, the relatively wide confidence intervals included AUC values ranging from 0.62 corresponding to poor diagnostic ability to 0.87 corresponding to good diagnostic ability. Follow-up studies are needed to confirm and specify the validity of the diagnostic accuracy of PRO-C19. In addition, at a high specificity of over 95% where diagnostic tests are usually most appropriate, the sensitivity of PRO-C19 in early NSCLC decreased to 35%. In future studies, it would also be desirable to examine combining PRO-C19 with other NSCLC biomarkers to improve overall accuracy. Furthermore, in future studies on early detection, it may also be possible to evaluate PRO-C19 in high-risk individuals prior to a definitive NSCLC diagnosis.

[0060] This study has several major limitations. That is, considering the exact nature of the research objectives, bias can be introduced by the use of so-called "samples of convenience" and post hoc analysis. Numerically, this bias is demonstrated by differences in sample size, age, gender, and ethnicity of the compared groups. Also, since the clinical data of the study participants are limited, there may be additional hidden biases. Therefore, the results and conclusions of this study are only our first attempt to explore the biology of type XIX collagen in cancer.

[0061] In conclusion, an ELISA targeting the C-terminus of type XIX collagen, named PRO-C19, was developed and its validity was confirmed. PRO-C19 was used to quantify type XIX collagen in the sera of cancer patients, and type XIX collagen was significantly elevated in all cancer types investigated compared to healthy controls. Subsequently, PRO-C19 was evaluated in two separate NSCLC cohorts, and in this case as well, PRO-C19 was significantly elevated and showed moderate diagnostic accuracy in early NSCLC. PRO-C19 and type XIX collagen show potential as cancer biomarkers.

[0062]

Table 1

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Table 3

Table 4

Table 5

Table 6

Table 7

Claims

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

2. The monoclonal antibody according to claim 1, which is a monoclonal antibody raised against a synthetic peptide having the amino acid sequence SHAHQRTGGN (SEQ ID NO: 1) at the C-terminus.

3. The monoclonal antibody according to claim 1 or 2, which does not specifically bind to a peptide having the amino acid sequence SHAHQRTGGNX (SEQ ID NO: 2) at the C-terminus, wherein X represents any amino acid.

4. The monoclonal antibody according to any one of claims 1 to 3, which does not specifically bind to a peptide having the amino acid sequence SHAHQRTGGNA (SEQ ID NO: 3) at the C-terminus.

5. The monoclonal antibody according to any one of claims 1 to 4, which does not specifically bind to a peptide having the amino acid sequence SHAHQRTGG (SEQ ID NO: 4) at the C-terminus.

6. The monoclonal antibody according to any one of claims 1 to 5, which does not specifically bind to a peptide having the amino acid sequence GVAPGIGPGGG (SEQ ID NO: 5) at the C-terminus.

7. A method for immunoassay for detecting type XIX collagen in a human biological fluid sample, comprising contacting a fluid sample obtained from a human patient with a monoclonal antibody that specifically binds to the C-terminal amino acid sequence SHAHQRTGGN (SEQ ID NO: 1), and detecting the binding between the monoclonal antibody and the peptide in the sample.

8. The method according to claim 7, wherein the detection is quantitative.

9. The method according to claim 7 or 8, wherein the immunoassay is a competitive immunoassay.

10. The method according to any one of claims 7 to 9, wherein the monoclonal antibody is a monoclonal antibody raised against a synthetic peptide having the amino acid sequence SHAHQRTGGN (SEQ ID NO: 1) at the C-terminus.

11. The method according to any one of claims 7 to 10, wherein the antibody does not specifically bind to a peptide having the amino acid sequence SHAHQRTGGNX (SEQ ID NO: 2) at the C-terminus, wherein X represents any amino acid.

12. The method according to any one of claims 7 to 11, wherein the antibody does not specifically bind to a peptide having the amino acid sequence SHAHQRTGGNA (SEQ ID NO: 3) at the C-terminus.

13. The method according to any one of claims 7 to 12, wherein the antibody does not specifically bind to a peptide having the amino acid sequence SHAHQRTGG (SEQ ID NO: 4) at the C-terminus.

14. The method according to any one of claims 7 to 13, wherein the antibody does not specifically bind to a peptide having the amino acid sequence GVAPGIGPGG (SEQ ID NO: 5) at the C-terminus.

15. The method according to any one of claims 7 to 14, wherein the fluid sample is from a human patient having medical signs or symptoms indicative of cancer.

16. An immunoassay method for diagnosing and / or evaluating the likelihood of cancer in a patient, comprising: contacting a biological fluid sample obtained from the patient with a monoclonal antibody; detecting and determining the amount of binding between the monoclonal antibody and a peptide in the sample; correlating the amount of binding with a value associated with a normal healthy subject and / or a value associated with a known disease severity and / or a value obtained from the patient at a previous time point; The method according to any one of claims 7 to 15, wherein an increase in the amount of binding compared to a healthy subject indicates that the patient may have cancer.

17. The method according to claim 15 or claim 16, wherein the cancer is breast, lung, colon, head and neck, kidney, liver, pancreas, prostate, stomach, bladder, or ovarian cancer, or melanoma.

18. The method according to claim 16, wherein a binding amount within the range of 50.0 to 200.0 ng / mL may confirm that the patient may have cancer.

19. A monoclonal antibody that specifically recognizes and specifically binds to a peptide having the amino acid sequence SHAHQRTGGN (SEQ ID NO: 1) at the C-terminus, and: - a streptavidin-coated well plate; - an N-terminal biotinylated peptide having the amino acid sequence SHAHQRTGGN (SEQ ID NO: 1) at the C-terminus; and - a calibration peptide having the amino acid sequence SHAHQRTGGN (SEQ ID NO: 1) at the C-terminus at least one of, An assay kit comprising.

20. The assay kit according to claim 19, wherein the monoclonal antibody is a monoclonal antibody raised against a synthetic peptide having the amino acid sequence SHAHQRTGGN (SEQ ID NO: 1) at the C-terminus.

21. The assay kit according to claim 19 or claim 20, wherein the monoclonal antibody does not specifically bind to the C-terminal amino acid sequence SHAHQRTGGNX (SEQ ID NO: 2), where X represents any amino acid.

22. The assay kit according to any one of claims 19 to 21, wherein the monoclonal antibody does not specifically bind to the C-terminal amino acid sequence SHAHQRTGGNA (SEQ ID NO: 3).

23. The assay kit according to any one of claims 19 to 22, wherein the monoclonal antibody does not specifically bind to the C-terminal amino acid sequence SHAHQRTGG (SEQ ID NO: 4).

24. The assay kit according to any one of claims 19 to 23, wherein the monoclonal antibody does not specifically bind to the C-terminal amino acid sequence GVAPGIGPGGG (SEQ ID NO: 5).

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