Assay for detecting cancer by using an antibody that binds to a C-terminal epitope of type IX collagen

Abstract

The present invention relates to immunoassay methods suitable for detecting and / or monitoring cancer in patients, immunoassay kits suitable for carrying out said methods, and antibodies suitable for use in said methods and kits.

Description

[Technical Field]

[0001] The present invention relates to immunoassay methods suitable for detecting and / or monitoring cancer in patients, immunoassay kits suitable for carrying out said methods, and antibodies suitable for use in said methods and kits. [Background technology]

[0002] Lung cancer is the most commonly diagnosed cancer and the leading cause of cancer death worldwide (1). Non-small cell lung cancer (NSCLC) represents approximately 85% of all lung cancer cases, with adenocarcinoma and squamous cell carcinoma being the most common subtypes (2). One of the major components of the tumor microenvironment is the extracellular matrix (ECM), the noncellular portion of tissue (3). Collagen is the most prominent ECM protein, and there are 28 different types of collagen with 46 side chains (4).

[0003] Fragments of fibril-associated collagen with interrupted triple helices (FACIT) collagen have previously been shown to be associated with cancer (5). The localization and thorough description of type IX collagen in human lung is lacking in the literature. It has been extensively studied in human articular cartilage, where it colocalizes with type II collagen (6, 7). A previous study by Chung et al. from 2017 indicated that certain single nucleotide polymorphisms (SNPs) in the COL9A1 gene are associated with an increased risk of oral cancer (8). Piotrowski et al. (2006) demonstrated a reduction in methylation of CpG islands in the COL9A1 gene in breast cancer tumor tissue samples by microarray hybridization and bisulfite sequencing (9). Summary of the Invention

[0004] Applicants have now developed an immunoassay method that targets the C-terminal amino acid sequence QRAFNKGPDP of type IX collagen alpha-1 chain (referred to herein as "PRO-C9," "target sequence," or "PRO-C9 target sequence") and demonstrated that elevated levels of PRO-C9 are present and can be detected in serum samples from patients with a number of different cancers, including non-small cell lung cancer (NSCLC), thereby demonstrating the utility of the immunoassay method as a means for detecting and / or monitoring cancer.

[0005] Thus, in a first aspect, the present invention provides a method of immunoassay comprising: i) contacting the patient sample with a monoclonal antibody that specifically binds to the C-terminal amino acid sequence QRAFNKGPDP (SEQ ID NO: 1); ii) detecting binding between the monoclonal antibody and the peptide in the sample and determining the amount of binding. The present invention provides a method comprising:

[0006] Preferably, the method is an immunoassay method for detecting and / or monitoring cancer in a patient, comprising: iii) comparing said amount of binding with a value associated with normal healthy subjects and / or a value associated with a known disease severity and / or a value obtained from said patient at a previous time point and / or a predetermined cut-off value. The method further comprises:

[0007] As used herein, the term "C-terminus" refers to a C-terminal peptide sequence at the end of a polypeptide, i.e., at the C-terminus of the polypeptide, and should not be construed as meaning in that general orientation.

[0008] As used herein, the terms "peptide" and "polypeptide" are used interchangeably.

[0009] Disclosed herein are monoclonal antibodies suitable for use in the methods of the invention that specifically bind to an epitope consisting of or within the C-terminal amino acid sequence QRAFNKGPDP-COOH (SEQ ID NO: 1).

[0010] Preferably, the monoclonal antibody does not substantially recognize or bind to an extended version of the above C-terminal amino acid sequence, QRAFNKGPDPG-COOH (SEQ ID NO: 2), i.e., a version of the PRO-C9 target sequence extended at the C-terminus by the addition of a glycine residue.

[0011] Preferably, the monoclonal antibody does not recognize or bind to a truncated version of the above C-terminal amino acid sequence having the amino acid sequence QRAFNKGPD-COOH (SEQ ID NO: 3).

[0012] As used herein, the term "monoclonal antibody" refers to both whole antibodies and fragments thereof that retain the binding specificity of the whole antibody, such as Fab fragments, F(ab')2 fragments, single-chain Fv fragments, nanobodies, or other such fragments known to those skilled in the art. As is well known, whole antibodies typically have a "Y" 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 of the light and heavy chains contain a variable region, and the C-terminal portions of each of the heavy and light chains constitute a constant region. The variable regions comprise three complementarity-determining regions (CDRs), which are primarily responsible for antigen recognition. The constant regions enable antibodies to recruit cells and molecules of the immune system. Antibody fragments that retain binding specificity comprise at least the CDRs and a sufficient residual portion of the variable region to retain the binding specificity.

[0013] In the present invention, monoclonal antibodies comprising any constant region known in the art can be used. In mouse and human antibodies, constant light chains are classified as either kappa or lambda light chains. Heavy chain constant chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody isotype as IgM, IgD, IgG, IgA, and IgE, respectively. The IgG isotype has several subclasses, including, but not limited to, IgG1, IgG2, IgG3, and IgG4 in humans, and IgG1, IgG2a, IgG2b, IgG2c, and IgG3 in mice. Monoclonal antibodies may preferably be of the IgG isotype, including any one of the IgG subclasses.

[0014] The CDRs of an antibody can be determined using methods known in the art, such as those described by Kabat et al. The antibody can be generated from a B cell clone. The antibody isotype can be determined by ELISA specific for IgM, IgG, or IgA isotype or subclass. The amino acid sequence of the generated antibody can be determined using standard techniques. For example, RNA can be isolated from cells and used to generate cDNA by reverse transcription. The cDNA 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 for all VH (variable heavy) 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 a primer that binds to the 3' end of the kappa or lambda chain together with a primer that anneals to the Vkappa or Vlamda leader sequence. Full-length heavy and light chains can be generated and sequenced.

[0015] Monoclonal antibodies that specifically bind to the C-terminal amino acid sequence QRAFNKGPDP-COOH (SEQ ID NO: 1) can be generated via any suitable technique known in the art. For example, monoclonal antibodies can be generated against a synthetic peptide having the amino acid sequence QRAFNKGPDP (SEQ ID NO: 1), such as by immunizing a rodent (or other suitable mammal) with a synthetic peptide consisting of the sequence QRAFNKGPDP (SEQ ID NO: 1), optionally linked to an immunogenic carrier protein (e.g., keyhole limpet hemocyanin), isolating and cloning single antibody-producing cells, and assaying the resulting monoclonal antibodies to ensure they have the desired specificity. An exemplary protocol for producing monoclonal antibodies that specifically bind to the C-terminal amino acid sequence QRAFNKGPDP (SEQ ID NO: 1) is described below.

[0016] Preferably, the monoclonal antibody or fragment thereof may comprise one or more complementarity determining regions (CDRs) selected from the following: [ka]

[0017] Preferably, the antibody or fragment thereof comprises at least two, three, four, five or six of the CDR sequences listed above.

[0018] Preferably, the monoclonal antibody or fragment thereof has a light chain variable region comprising the following CDR sequences: CDR-L1: KSSQSLLYSSNQMNYLA (SEQ ID NO: 4) CDR-L2: WASTRES (SEQ ID NO: 5) CDR-L3: HQYFSSRT (SEQ ID NO: 6)

[0019] 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 in the light chain sequence below (CDRs are shown in bold and underlined, and framework sequences are shown in italics): [ka]

[0020] Preferably, the monoclonal antibody or fragment thereof has a heavy chain variable region comprising the following CDR sequences: [ka]

[0021] 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 in the heavy chain sequence below (CDRs are shown in bold and underlined, and framework sequences are shown in italics): [ka]

[0022] As used herein, the framework amino acid sequences between the CDRs of an antibody are substantially identical or substantially similar to the framework amino acid sequences between the CDRs of another antibody if they have at least 70%, 80%, 90%, or at least 95% similarity or identity. Similarity or identity may be measured over the entire length of each intervening framework sequence. The similar or identical amino acids may be contiguous or non-contiguous.

[0023] The framework sequence may contain one or more amino acid substitutions, insertions, and / or deletions. The amino acid substitutions may be conservative, which means 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.

[0024] A program such as the CLUSTAL program can be used to compare amino acid sequences. This program compares amino acid sequences and finds the optimal alignment by inserting spaces into either sequence as appropriate. For optimal alignment, it is possible to calculate amino acid identity or similarity (a combination of identity and conservation of amino acid type). Programs such as BLASTx align the longest stretch of similar sequences and assign a value for fit. This makes it possible to obtain a comparison in which several regions of similarity are found, 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.

[0025] In certain preferred embodiments, the monoclonal antibody or fragment thereof comprises the light chain variable region sequence: [ka] and / or heavy chain variable region sequence: [ka] may comprise:

[0026] As used herein, the term "amount of binding" refers to the quantification of the binding between the antibody and the peptide in the patient sample.The quantification can be determined, for example, by comparing the binding value measured in the patient sample with the calibration curve generated using the binding value measured in the standard sample containing the peptide that the antibody specifically binds to at a known concentration, to determine the amount of the peptide that the antibody specifically binds to in the patient sample.In the example shown below, an ELISA method is used in which spectrophotometric analysis is used to measure the amount of binding both in the patient sample and when generating the calibration curve.However, any suitable analytical method can be used.

[0027] The term "specifically binds," as used herein, means that antibody binding is selective for the antigen and that this binding can be distinguished from unwanted or nonspecific interactions. The ability of a monoclonal antibody to bind to a specific epitope or peptide sequence can be measured through either enzyme-linked immunosorbent assay (ELISA) as described herein or other techniques familiar to those skilled in the art, such as surface plasmon resonance (SPR) technology (e.g., analyzed on a BIAcore instrument) and traditional binding assays. The degree of binding of a monoclonal antibody to an unrelated protein is less than about 10% of the binding of the monoclonal antibody to the epitope or peptide, as measured, for example, by ELISA. "Affinity" refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., the epitope-binding region of an antibody) and its binding partner (e.g., an epitope or antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antigen-binding moiety and an antigen). The affinity of a molecule for its partner can generally be represented by the dissociation constant (Kd), which is the ratio of the dissociation and association rate constants (koff and kon, respectively). Therefore, equivalent affinities may comprise different rate constants, as long as the ratio of the rate constants remains the same. The dissociation constant represents the concentration of antigen at which half of the binding sites on the antibody are occupied. A lower Kd indicates a higher binding affinity between the antibody and the antigen, while a higher Kd reflects weaker binding. Several methods are available for measuring the Kd of an antibody, including surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), and fluorescence-based assays. In certain embodiments, a monoclonal antibody that binds to an epitope or peptide has a Kd of <1 pM, <100 nM, <10 nM, <1 nM, <0.1 nM, <0.01 nM, or <0.001 nM (e.g., 10 8 M or less, e.g. 10 8 M~10 13 M, e.g., 10 9 M~10 13 It has a dissociation constant (KD) of 1 M.

[0028] As used herein, the term "predetermined cutoff value" refers to an amount of binding that is statistically determined to be indicative of a high likelihood of a disease (i.e., cancer) or a particular severity thereof in a patient, wherein a measured value of a target peptide in a patient sample that is equal to or greater than the statistical cutoff value corresponds to at least a 70% probability of the presence of said disease, preferably at least a 75% probability, more preferably at least an 80% probability, more preferably at least an 85% probability, more preferably at least a 90% probability, and most preferably at least a 95% probability.

[0029] As used herein, the term "value associated with a normal healthy subject" refers to a standardized amount of binding determined by the methods described above for samples from subjects who are considered to be healthy, i.e., disease-free (i.e., cancer), and the term "value associated with a known disease severity" refers to a standardized amount of binding determined by the methods described above for samples from patients who are known to have a disease (i.e., cancer) of a known severity.

[0030] In a preferred embodiment, the cancer is bladder cancer, breast cancer, colorectal cancer, gastric cancer, head and neck cancer, lung cancer, ovarian cancer, pancreatic cancer, kidney cancer or melanoma. In another preferred embodiment, the cancer is lung cancer, and most preferably, the lung cancer is non-small cell lung cancer.

[0031] In a preferred embodiment, the monoclonal antibody does not specifically bind to a peptide having the C-terminal amino acid sequence QRAFNKGPDPG (SEQ ID NO: 2) (i.e., a version of the PRO-C9 target sequence extended at its C-terminus by the addition of a glycine residue). Preferably, the ratio of the affinity of the antibody for the PRO-C9 target sequence to the affinity of the antibody for the extended version of the target sequence is at least 10:1, and more preferably at least 20:1 or at least 30:1.

[0032] In a preferred embodiment, the monoclonal antibody does not specifically bind to a peptide having the C-terminal amino acid sequence QRAFNKGPD (SEQ ID NO: 3) (i.e., a version of the PRO-C9 target sequence truncated by removal of the final proline residue). Preferably, the ratio of the affinity of the antibody for the PRO-C9 target sequence to the affinity of the antibody for the truncated version of the target sequence is at least 10:1, and more preferably at least 20:1 or at least 30:1.

[0033] In a preferred embodiment, monoclonal antibodies are generated against a synthetic peptide having the C-terminal amino acid sequence QRAFNKGPDP. For example, monoclonal antibodies may be generated by (a) immunizing a rodent (or other suitable mammal) with a synthetic peptide comprising the C-terminal sequence QRAFNKGPDP (SEQ ID NO: 1), optionally linked at its N-terminus to an immunogenic carrier protein (e.g., keyhole limpet hemocyanin), (b) isolating and cloning a single antibody-producing cell, and (c) assaying the resulting monoclonal antibodies to ensure they have the desired specificity.

[0034] In a preferred embodiment, the patient sample is selected from blood, serum, or plasma.

[0035] In a preferred embodiment, the immunoassay is a competitive assay or a sandwich assay. The immunoassay may be, for example, a radioimmunoassay or an enzyme-linked immunosorbent assay (ELISA). Such assays are techniques known to those skilled in the art.

[0036] In a second aspect, the present invention provides a method of treating cancer in a patient in need thereof, comprising: (a) performing an immunoassay method for detecting and / or monitoring cancer according to the first aspect of the invention on a sample from a patient to detect whether the patient has cancer; and (b) if it is determined in step (a) that the patient has said cancer, administering to the patient a therapy for the treatment of said cancer. The present invention provides a method comprising:

[0037] The treatment may be any treatment suitable for treating the cancer in question. The treatment may, for example, comprise or consist of one or more surgeries, one or more radiation treatments, one or more medications (such as one or more chemotherapy, one or more immunotherapy, and / or one or more hormone therapy), or a combination thereof. The medication may be formulated for topical or systemic administration. The topical medication may, for example, be formulated as a cream, foam, gel, lotion, or ointment for administration. The systemic medication may, for example, be formulated for enteral or parenteral administration. The surgery may be curative surgery, preventive surgery, debulking surgery, palliative surgery, and / or reconstructive surgery.

[0038] For example, if the cancer is lung cancer, suitable treatments include, for example, surgery, such as performing a lung resection, such as a lobectomy, sublobar excision (wedge resection), or removal of the entire lung (pneumonectomy); radiation therapy, examples of which include, but are not limited to, radiation therapy given in conjunction with chemotherapy, postoperative radiation therapy, brachytherapy (localized radiation therapy), prophylactic cranial irradiation, stereotactic radiation, and palliative radiation therapy; chemotherapy using, for example, one additional agent, such as cisplatin, carboplatin, etoposide, gemcitabine, paclitaxel, docetaxel, vinorelbine, topotecan, irinotecan, and pemetrexed; epidermal growth factor receptor (EGFR) inhibitors, such as erlotinib, gefitinib, afatinib, and the like. targeted therapy using one or more drugs such as crizotinib, and immunotherapy using one or more monoclonal antibodies, such as anti-PD-L1 monoclonal antibodies, for example, atezolizumab, nivolumab, or pembrolizumab, monoclonal antibodies targeting cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), for example, ipilimumab, and / or monoclonal antibodies targeting vascular endothelial growth factor, for example, bevacizumab, necitumumab, mobocertinib, or cetuximab.

[0039] Where the lung cancer is non-small cell lung cancer, suitable treatments may, for example, comprise one or more of the lung cancer treatments listed above.

[0040] In a third aspect, the present invention provides a monoclonal antibody that specifically binds to the C-terminal amino acid sequence QRAFNKGPDP (SEQ ID NO: 1).

[0041] Antibodies according to the third aspect of the invention are particularly suitable for use in carrying out the immunoassay method according to the first aspect of the invention. Preferred embodiments and features of antibodies according to the third aspect will therefore be apparent from the above discussion of preferred embodiments of the method according to the first aspect.

[0042] For example, in a preferred embodiment, a monoclonal antibody does not specifically bind to a peptide having the C-terminal amino acid sequence QRAFNKGPDPG (SEQ ID NO: 2) and / or does not specifically bind to a peptide having the C-terminal amino acid sequence QRAFNKGPD (SEQ ID NO: 3) and / or is raised against a synthetic peptide having the C-terminal amino acid sequence QRAFNKGPDP (SEQ ID NO: 1).

[0043] In a fourth aspect, the present invention provides a method for producing a monoclonal antibody according to the third aspect of the invention, below: - streptavidin-coated well plates, - the biotinylated peptide Biotin-L-QRAFNKGPDP (SEQ ID NO: 18), where L is an optional linker; - secondary antibodies for use in sandwich immunoassays, - a calibrator protein comprising the C-terminal amino acid sequence QRAFNKGPDP, - antibody biotinylation kit, - Antibody HRP labeling kit, - antibody radiolabeling kits, and - Assay Visualization Kit with at least one of An immunoassay kit is provided, comprising:

[0044] Immunoassay kits according to the fourth aspect of the invention are particularly suitable for use in carrying out immunoassay methods according to the first aspect of the invention. Further preferred embodiments and features of immunoassay kits according to the fourth aspect will therefore be apparent from the above discussion of preferred embodiments of the methods according to the first aspect. [Brief explanation of the drawings]

[0045] [Figure 1] Depiction of the three chains of type IX collagen (α1, α2, and α3), highlighting the PRO-C9 target sequence on the α1 chain. [Figure 2] Type IX collagen sequence alignment and specificity of the PRO-C9 assay. (A) Sequence alignment of the C-terminal region of the human, mouse, and rat α1 chain of type IX collagen. The PRO-C9 sequences of the mouse and rat α1 chain and the aligned C-terminal sequences are marked with boxes. (B) Specificity of the PRO-C9 assay. Reactivity to the standard peptide (QRAFNKGPDP SEQ ID NO:1), truncated peptide (QRAFNKGPD SEQ ID NO:3), extended peptide (QRAFNKGPDPG SEQ ID NO:2), and nonsense standard peptide and coater (DQAAGGLRQH SEQ ID NO:14) is depicted. Signal is shown as relative light units (RLU) per second as a function of the standard peptide. [Figure 3] Levels of PRO-C9 in healthy controls (n = 43) and patients with NSCLC (n = 40). (A) PRO-C9 is significantly elevated in patients with NSCLC compared to healthy controls (p = 0.006). Data were analyzed by age- and sex-adjusted t-test and presented as Tukey box plots. (B) ROC curve analysis was applied to evaluate the ability of PRO-C9 to distinguish between healthy controls and NSCLC patients. [Figure 4] Serum PRO-C9 levels from Cohort 1, including healthy donors (n=13), bladder (n=19), breast (n=20), colorectal (n=20), gastric (n=20), head and neck (n=20), lung (n=20), pancreatic (n=20), prostate (n=20), or kidney (n=20) cancer. Differences in PRO-C9 were assessed by Kruskal-Wallis test with Dunn's multiple comparison test to compare solid tumor types with healthy donors. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05. DETAILED DESCRIPTION OF THE INVENTION

[0046] Example Embodiments of the present disclosure are described in the following examples, which are provided to aid in the understanding of the present disclosure and should not be construed in any way to limit the scope of the present disclosure as defined in the claims that follow. The following examples are presented to provide those skilled in the art with a complete disclosure and description of how to make and use the described embodiments, and are not intended to limit the scope of the disclosure, nor are they intended to represent that the experiments described below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure.

[0047] material and method All reagents used were high-quality chemicals from Merck (Whitehouse Station, NJ, USA) and Sigma (St. Louis, MO, USA) unless otherwise stated. All synthetic peptides used for antibody production and assay validation were purchased from Genscript (Piscataway, NJ, USA) (Table 1).

[0048] [Table 1]

[0049] Monoclonal antibody development, production, and characterization Amino acid sequence 912 'QRAFNKGPDP' 921The antibody was used to generate monoclonal antibodies. Immunization was initiated in 4-6 week-old Balb / C mice by subcutaneous injection of 200 μl of emulsified antigen and 100 μg of immunogenic peptide (KLH-CGG-QRAFNKGPDP (SEQ ID NO: 15)) using Steimmune (Thermo Fisher). Immunization was repeated every 2 weeks until a stable serum antibody titer level was reached. Mice with the highest serum titers were selected for fusion and rested for 1 month. Subsequently, mice were boosted intravenously with 50 μg of immunogenic peptide in 100 μl of 0.9% NaCl solution 3 days before isolation of the spleen for cell fusion. To produce hybridoma cells, mouse spleen cells were fused with SP2 / 0 myeloma cells as described by Gefter et al. (10). Subsequently, clones were plated into 96-well microtiter plates for further expansion, and limiting dilution methods were applied to promote monoclonal expansion.

[0050] Indirect ELISA performed on streptavidin-coated plates was used to screen supernatant reactivity. Biotin-QRAFNKGPDP was used as the screening peptide, and the standard peptide QRAFNKGPDP was used to further test the specificity of the clones. Supernatants were collected from hybridoma cells and purified using HiTrap affinity columns (GE Healthcare Life Science, Little Chalfront, Buckinghamshire, UK) according to the manufacturer's instructions, and antibody isotypes were determined using a Rapid ELISA Mouse monoclonal antibody Isotyping Kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol.

[0051] Native reactivity was assessed using human serum purchased from a commercial supplier (Valley Biomedical, Winchester, VA). Monoclonal antibodies (mAbs) were selected to specifically recognize the standard peptide (QRAFNKGPDP (SEQ ID NO: 1)) and not the one amino acid extended or truncated sequences (QRAFNKGPDPG (SEQ ID NO: 2) and QRAFNKGPD (SEQ ID NO: 3), respectively).

[0052] Selected antibodies were sequenced and the CDRs were determined. The sequences of the chains are as follows (CDRs in bold; framework sequences in italics; constant regions underlined): [ka]

[0053] [ka]

[0054] Development of the PRO-C9 assay The development of the competitive chemiluminescence immunoassay (CLIA) involved several preliminary optimization experiments, in which reagents, concentrations, incubation times, and temperatures were analyzed through several tests. The PRO-C9 competitive ELISA procedure was as follows: 96-well streptavidin-coated white microplates (Greiner Bio-One, Kremsmunster, Austria) were coated with 5 ng / mL of biotinylated synthetic peptide (biotin-QRAFNKGPDP SEQ ID NO: 18) dissolved in assay buffer (10 mM phosphate-buffered saline (PBS), 1% bovine serum albumin, 0.1% Tween-20, 0.36% Bronidox, 4 g / L NaCl, pH 7.4, 20°C) and incubated for 30 minutes at 20°C with constant shaking (300 rpm) in the dark.

[0055] Next, 20 μL / well of standard peptide (100 ng / mL) and samples were added to the appropriate wells, followed by 100 μL / well of HRP-labeled antibody diluted in assay buffer to a concentration of 100 ng / mL, and incubated for 20 hours at 4°C with constant shaking (300 rpm) in the dark. After each incubation step, wells were washed with a standard wash buffer (20 mM Tris, 50 mM NaCl, pH 7.2). A chemiluminescent substrate (Roche, BM Chemiluminescence ELISA Substrate (POD), Basel, Switzerland) standard solution was mixed 15 minutes before use, and 100 μL / well was added to the plate and incubated for 3 minutes at 20°C with constant shaking (300 rpm) in the dark. Relative light units were measured at all wavelengths within 5 minutes on a microplate luminometer reader (SpectraMax M5, Molecular Devices, CA, USA).

[0056] Standard curves were plotted using a four-parameter logistic curve fit: Y = (AD) / (1 + (x / C)^B) + D (R > 0.9). Data were analyzed using SoftMax Pro version 7.0.3 software.

[0057] Technical evaluation Linearity was assessed using two-fold dilutions of four human serum samples. Linearity was calculated as the percentage of recovery of the undiluted sample. Antibody specificity was calculated as the percentage of signal inhibition by two-fold diluted standard peptide (QRAFNKGPDP (SEQ ID NO: 1)), extended peptide (QRAFNKGPDPG (SEQ ID NO: 2)), truncated peptide (QRAFNKGPD (SEQ ID NO: 3)), and nonsense peptide (DQAAGGLRQH (SEQ ID NO: 14)). Intra- and inter-assay variation was determined by 10 independent runs of five quality controls and two kit controls performed in duplicate.

[0058] The accuracy of the assay was measured in healthy human serum samples spiked with the standard peptide and serum samples with known high PRO-C9 concentrations, and calculated as the percentage recovery of the measured and expected concentrations of the peptide, or by adding the analyte concentration in serum to the serum samples with high PRO-C9 concentrations. Analytical interferences were introduced by adding low and high concentrations of hemoglobin (2.50 / 5 mg / mL), lipemia / lipids (1.50 / 5 mg / mL), and biotin (3 / 9 ng / mL) to serum samples of known concentrations. The percentage recovery was calculated using normal serum samples as a reference. Normal reference levels for hemoglobin, lipemia / lipids, and biotin were 0-10 mg / dL (0-0.00161 mmol / L), <150 mg / dL (<1.6935 mmol / L), and 0.221-3.004 ng / mL, respectively. Interference was calculated as the percentage recovery of the analyte in unspiked serum. The measurement range was defined as the range between the lower limit of quantitation (LLOQ) and the upper limit of quantitation (ULOQ), determined from 10 independent runs using the standard peptide. Measurements below or above the LLOQ were assigned a value of LLOQ / ULOQ, respectively. The IC50 (half maximal inhibitory concentration) was determined from the standard curve.

[0059] Analyte stability was investigated through temperature testing and repeated freeze-thaw cycling of serum samples. The temperature testing involved different time points and temperatures, and PRO-C9 levels were measured in three human serum samples after 0, 2, 4, 24, and 48 hours of incubation at either 4°C or 20°C. Recovery was estimated using the 0-hour sample as a reference. Additionally, the effect of four repeated freeze / thaw cycles of three serum samples was evaluated, and freeze / thaw recovery was calculated using the 0-cycle sample as a reference. Each sample was run in duplicate.

[0060] Biological evaluation of PRO-C9 The bioavailability of PRO-C9 was evaluated in serum samples from patients with NSCLC (n=40) and healthy donors (n=43) from the commercial vendor Proteogenex (Culver City, CA). Serum samples were obtained and stored at -80°C until use.

[0061] The biological utility of PRO-C9 was also evaluated in serum samples from patients with various cancers (n=219) and healthy donors (n=13) from the commercial vendor Proteogenex (Culver City, CA). Serum samples were obtained and stored at -80°C until use.

[0062] Ethical statement All animals were treated in accordance with animal welfare guidelines. Monoclonal antibody production in mice was approved by the Danish National Authority (The Animal Experiments Inspectorate) under approval number 2013-15-2934-00956. Collection and retrieval of human cartilage followed international ethical guidelines for the handling of human samples and patient information. All participants signed informed consent, and the study was approved by the local ethics committee. Samples from both cohorts were collected in accordance with the 1975 Declaration of Helsinki after informed consent and approval by the local ethics committee.

[0063] statistical analysis PRO-C9 levels were log-transformed to obtain normalized values. Comparison of PRO-C9 between healthy controls and NSCLC was performed using a t-test adjusted for age and sex. Diagnostic accuracy was tested by AUROC. A p-value below 0.05 was considered significant. Statistical analysis and graphs were performed using GraphPad Prism version 9 (GraphPad Software, Inc., La Jolla, CA) and R studio version 4.2.1 (R Foundation for Statistical Computing, Vienna, Austria, URL https: / / www.R-project.org).

[0064] result Technical evaluation and characterization of the PRO-C9 assay Monoclonal antibody clone NBH313 #46 9H11-1D9-2B6 demonstrated the best native reactivity, peptide affinity, and assay stability and was selected for assay development. A summary of the technical evaluation of the PRO-C9 assay can be found in Table 2.

[0065] [Table 2]

[0066] The measurement range (LLOQ-ULOQ) was determined to be 0.65–120 ng / mL. Inter- and intra-assay variations were 12.0% and 3.9%, respectively, and linearity was observed from undiluted to 2-fold dilutions in human serum. Analyte stability was acceptable (92.1–112.8%) for five freeze-thaw cycles. Hemoglobin, lipemia, and biotin did not interfere with the measurement of PRO-C9 in human serum. Alignment of the human sequence using UNIPROT revealed mismatches at positions 1 and 5 with the corresponding mouse and rat sequences (Figure 2A). To evaluate the specificity of the PRO-C9 assay, the mAb was tested against extended peptides, truncated peptides, nonsense standard peptides, and nonsense coagulum, and showed no reactivity to these peptides (Figure 2B).

[0067] Baseline demographics and clinical characteristics The PRO-C9 assay was measured in serum from patients with NSCLC. The patient cohort consisted of healthy controls and patients diagnosed with NSCLC. Patient demographics can be found in Table 3. Here, patients with NSCLC had significantly higher levels of PRO-C9 compared to healthy controls when corrected for age and sex (p = 0.006, Figure 3A). The diagnostic power of PRO-C9 for patients with NSCLC compared to healthy controls was AUROC = 0.890 (95% CI: 0.82-0.96 p < 0.001, Figure 3B).

[0068] [Table 3]

[0069] The PRO-C9 assay was used to analyze serum from a second cohort of patients with a range of cancers. The second patient cohort consisted of healthy controls and patients diagnosed with cancer. Patient demographics can be found in Table 4. Here, patients with bladder, breast, colorectal, gastric, head and neck, lung, ovarian, pancreatic, and kidney cancers, as well as melanoma, had significantly higher levels of PRO-C9 compared to healthy controls when adjusted for age and sex (Figure 4).

[0070] [Table 4]

[0071] Consideration We developed and characterized a competitive CLIA assay for the detection of type IX collagen using a monoclonal antibody reactive with the PRO-C9 target sequence. Key findings included: 1) successful development of a technically robust and specific assay targeting the C-terminal sequence of the α-1 chain of type IX collagen (PRO-C9); 2) PRO-C9 levels were measurable in human serum; 3) PRO-C9 levels were significantly elevated in patients with NSCLC, bladder, breast, colorectal, gastric, head and neck, lung, ovarian, pancreatic, and renal cancers, as well as melanoma, compared with healthy controls; and 4) the PRO-C9 assay demonstrated an AUROC of 0.890, indicating its potential as a diagnostic biomarker.

[0072] This is the first demonstration that PRO-C9 can be measured non-invasively in blood and with biological relevance in patients with cancer, including NSCLC.

[0073] In conclusion, a CLIA targeting the PRO-C9 target sequence was developed and validated. PRO-C9 levels were quantified in serum from patients with NSCLC and other cancers, and were significantly elevated compared with healthy controls, suggesting that PRO-C9 levels can serve as a cancer biomarker.

[0074] References Table 5 JPEG2025541757000014.jpg138170

Claims

1. 1. An immunoassay method for detecting and / or monitoring cancer in a patient, comprising: i) contacting the patient sample with a monoclonal antibody that specifically binds to the C-terminal amino acid sequence QRAFNKGPDP; ii) detecting binding between said monoclonal antibody and the peptide in said sample and determining the amount of said binding; and iii) comparing said amount of binding with a value associated with normal healthy subjects and / or with a value associated with a known disease severity and / or with a value obtained from said patient at a previous time point and / or with a predetermined cut-off value. The method comprising:

2. 10. The method of claim 1, wherein the cancer is bladder cancer, breast cancer, colorectal cancer, gastric cancer, head and neck cancer, lung cancer, ovarian cancer, pancreatic cancer, kidney cancer or melanoma.

3. 3. The method of claim 1 or claim 2, wherein the cancer is lung cancer.

4. The method of claim 2 or claim 3, wherein the lung cancer is non-small cell lung cancer.

5. The method of any one of claims 1 to 4, wherein the monoclonal antibody does not specifically bind to a peptide having the C-terminal amino acid sequence QRAFNKGPDPG.

6. The method of any one of claims 1 to 5, wherein the monoclonal antibody does not specifically bind to a peptide having the C-terminal amino acid sequence QRAFNKGPD.

7. The method of any one of claims 1 to 6, wherein the monoclonal antibody is raised against a synthetic peptide having the C-terminal amino acid sequence QRAFNKGPDP.

8. The method of any one of claims 1 to 7, wherein the patient sample is selected from blood, serum or plasma.

9. The method according to any one of claims 1 to 8, wherein the immunoassay is a competitive assay or a sandwich assay.

10. The method of any one of claims 1 to 9, wherein the immunoassay is a radioimmunoassay or an enzyme-linked immunosorbent assay.

11. A monoclonal antibody that specifically binds to the C-terminal amino acid sequence QRAFNKGPDP.

12. The monoclonal antibody of claim 11, which does not specifically bind to a peptide having the C-terminal amino acid sequence QRAFNKGPDPG.

13. The monoclonal antibody of claim 11 or 12, which does not specifically bind to a peptide having the C-terminal amino acid sequence QRAFNKGPD.

14. The monoclonal antibody of any one of claims 11 to 13, which is raised against a synthetic peptide having the C-terminal amino acid sequence QRAFNKGPDP.

15. a monoclonal antibody that specifically binds to the C-terminal amino acid sequence QRAFNKGPDP; below: - streptavidin-coated well plates, - the biotinylated peptide Biotin-L-QRAFNKGPDP (where L is an optional linker); - secondary antibodies for use in sandwich immunoassays, a calibrator protein comprising the N-terminal amino acid sequence QRAFNKGPDP, - antibody biotinylation kit, - antibody HRP labeling kit, - antibody radiolabeling kit, and - Assay Visualization Kit with at least one of An immunoassay kit comprising:

16. 16. The immunoassay kit of claim 15, wherein the monoclonal antibody does not specifically bind to a peptide having the C-terminal amino acid sequence QRAFNKGPDPG.

17. 17. The immunoassay kit of claim 15 or 16, wherein the monoclonal antibody does not specifically bind to a peptide having the C-terminal amino acid sequence QRAFNKGPD.

18. The immunoassay kit of any one of claims 15 to 17, wherein the monoclonal antibody is raised against a synthetic peptide having the C-terminal amino acid sequence QRAFNKGPDP.

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