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

Abstract

The present invention relates to immunoassay methods suitable for detecting and / or monitoring cancer in a patient, immunoassay kits suitable for performing said methods and antibodies suitable for use in said methods and kits.

Description

Technical Field

[0001] The invention relates to immunoassay methods suitable for detecting and / or monitoring cancer in patients, immunoassay kits suitable for performing said methods, and antibodies suitable for said methods and kits. Background Art

[0002] Lung cancer is the most diagnosed cancer worldwide and a major cause of cancer death (1). Non-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancer cases, among which adenocarcinoma and squamous cell carcinoma are the most common subtypes (2). One of the major components of the tumor microenvironment is the extracellular matrix (ECM), the acellular part of tissues (3). Collagen is the most prominent ECM protein, among which there are 28 different types with 46 side chains (4).

[0003] Previous studies have shown that fragments of fibril-associated collagens with interrupted triple helices (FACIT) collagens are associated with cancer (5). There is a lack of localization and comprehensive description of type IX collagen in the human lung in the literature. Type IX collagen has been extensively studied in human articular cartilage, where it co-localizes with type II collagen (6, 7). A previous study by Chung et al. in 2017 showed that certain single nucleotide polymorphisms (SNPs) in the COL9A1 gene are associated with an increased risk of oral cancer (8). Piotrowski et al. demonstrated a decrease in CpG island methylation in the COL9A1 gene in breast cancer tumor tissue samples by microarray hybridization and bisulfite sequencing in 2006 (9). Summary of the Invention

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

[0005] Accordingly, in a first aspect, the present invention provides an immunoassay method, which comprises:

[0006] i) contacting a patient sample with a monoclonal antibody that specifically binds to the C-terminal amino acid sequence QRAFNKGPDP (SEQ ID No. 1);

[0007] ii) detecting and determining the amount of binding between the monoclonal antibody and the peptide in the sample.

[0008] Preferably, the method is an immunoassay method for detecting and / or monitoring cancer in a patient, and the method further comprises:

[0009] iii) correlating the binding amount 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 and / or with a predetermined cut-off value.

[0010] As used herein, the term "C-terminus" refers to the C-terminal peptide sequence at the end of a polypeptide (i.e., at the C-terminus of the polypeptide) and should not be construed in the sense of its general orientation.

[0011] As used herein, the terms "peptide" and "polypeptide" are used synonymously.

[0012] Monoclonal antibodies suitable for the methods of the invention are disclosed herein, which specifically bind to an epitope consisting of the C-terminal amino acid sequence QRAFNKGPDP-COOH (SEQ ID NO:1) or within the C-terminal amino acid sequence QRAFNKGPDP-COOH.

[0013] Preferably, the monoclonal antibody substantially does not recognize or bind to an extended form of the C-terminal amino acid sequence QRAFNKGPDPG-COOH (SEQ ID NO:2), i.e., a form of the PRO-C9 target sequence extended at the C-terminus by addition of a glycine residue.

[0014] Preferably, the monoclonal antibody does not recognize or bind to a shortened form of the C-terminal amino acid sequence having the amino acid sequence QRAFNKGPD-COOH (SEQ ID NO:3).

[0015] As used herein, the term "monoclonal antibody" refers to 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, intact antibodies typically have a "Y-shaped" structure of two pairs of identical polypeptide chains, each pair consisting of one "light chain" and one "heavy chain". The N-terminal regions of each light and heavy chain contain variable regions, while the C-terminal portions of each heavy and light chain constitute constant regions. The variable region contains three complementarity-determining regions (CDRs), which are mainly responsible for antigen recognition. The constant region allows the antibody to recruit cells and molecules of the immune system. Antibody fragments that retain binding specificity contain at least the CDRs and a sufficient portion of the remaining variable region to retain the binding specificity.

[0016] In the present invention, monoclonal antibodies can be used that comprise any constant region known in the art. In the case of murine and human antibodies, the constant light chain is classified as either a κ or λ light chain. The constant heavy chain is classified as μ, δ, γ, α, or ε, and the isotype of the antibody is defined 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 the case of humans and IgG1, IgG2a, IgG2b, IgG2c, and IgG3 in the case of mice. The monoclonal antibody can preferably be of the IgG isotype, including any one of the IgG subclasses.

[0017] The CDRs of the antibody can be determined using methods known in the art, such as those described by Kabat et al. Antibodies can be produced from B cell clones. The isotype of the antibody can be determined by ELISA specific for the IgM, IgG, or IgA isotype or subclass. The amino acid sequence of the produced 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 primer pairs that amplify the heavy and light chains of the antibody. For example, primers specific for the leader sequence of all VH (variable heavy chain) sequences can be used in conjunction with primers that bind to sequences located in the constant region of the isotype that has been previously determined. Primers that bind to the 3’ end of the κ or λ chain and primers that anneal to the Vκ or Vλ leader sequence can be used to amplify the light chain. The full-length heavy and light chains can be produced and sequenced.

[0018] Monoclonal antibodies that specifically bind to the C-terminal amino acid sequence QRAFNKGPDP-COOH (SEQ ID No.1) can be produced by any suitable technique known in the art. For example, monoclonal antibodies can be produced against a synthetic peptide having the amino acid sequence QRAFNKGPDP (SEQ ID No.1), for example, by immunizing a rodent (or other suitable mammal) with a synthetic peptide consisting of the sequence QRAFNKGPDP (SEQ ID No.1), which can optionally be linked to an immunogenic carrier protein (such as keyhole limpet hemocyanin); isolating and cloning individual antibody-producing cells; and assaying the resulting monoclonal antibodies to ensure that 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.

[0019] Preferably, the monoclonal antibody or its fragment can comprise one or more complementarity determining regions (CDRs) selected from the following:

[0020] CDR-L1: KSSQSLLYSSNQMNYLA (SEQ ID No.4)

[0021] CDR-L2: WASTRES (SEQ ID No.5)

[0022] CDR-L3: HQYFSSRT (SEQ ID No.6)

[0023] CDR-H1: IYTMN (SEQ ID No.7)

[0024] CDR-H2: RIRSKSENYATYYADSVKD (SEQ ID No.8)

[0025] CDR-H3: QGIYYDYYGAMDY (SEQ ID No.9)

[0026] Preferably, the antibody or its fragment comprises at least 2, 3, 4, 5 or 6 of the above CDR sequences.

[0027] Preferably, the monoclonal antibody or its fragment has a light chain variable region comprising the following CDR sequences:

[0028] CDR-L1: KSSQSLLYSSNQMNYLA (SEQ ID No.4)

[0029] CDR-L2: WASTRES (SEQ ID No.5)

[0030] CDR-L3: HQYFSSRT (SEQ ID No.6)

[0031] Preferably, the monoclonal antibody or its fragment has a light chain comprising framework sequences between the CDRs, wherein the framework sequences are substantially identical or substantially similar to the framework sequences between the CDRs in the following light chain sequence (wherein the CDRs are shown in bold and underlined and the framework sequences are shown in italics):

[0032]

[0033] Preferably, the monoclonal antibody or its fragment has a heavy chain variable region comprising the following CDR sequences:

[0034] CDR-H1: IYTMN (SEQ ID No.7)

[0035] CDR-H2: RIRSKSENYATYYADSVKD (SEQ ID No.8)

[0036] CDR-H3: QGIYYDYYGAMDY (SEQ ID No.9)

[0037] Preferably, the monoclonal antibody or fragment thereof has a heavy chain comprising framework sequences between the CDRs, wherein the framework sequences are substantially the same as or substantially similar to the framework sequences between the CDRs in the following heavy chain sequences (wherein the CDRs are shown in bold and underlined and the framework sequences are shown in italics):

[0038]

[0039] As used herein, if the framework amino acid sequence between the CDRs of an antibody has at least 70%, 80%, 90% or at least 95% similarity or identity to the framework amino acid sequence between the CDRs of another antibody, they are substantially the same or substantially similar. Similarity or identity can be measured over the entire length of each intervening framework sequence. Similar or identical amino acids can be contiguous or non - contiguous.

[0040] The framework sequence may contain one or more amino acid substitutions, insertions and / or deletions. Amino acid substitutions can 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 have similar chemical properties. For example, the following groups of amino acids have 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.

[0041] Programs such as the CLUSTAL program can be used to compare amino acid sequences. The program compares amino acid sequences and finds the best alignment by appropriately inserting gaps in either sequence. Amino acid identity or similarity (identity plus conservation of amino acid type) can be calculated for the best alignment. Programs like BLASTx will align the longest segments of similar sequences and assign a value for the fit. Thus, a comparison can be obtained in which several similar regions are found, each with a different score. Two types of analysis are contemplated in the present invention. Identity or similarity is preferably calculated over the entire length of the framework sequence.

[0042] In certain preferred embodiments, the monoclonal antibody or fragment thereof may comprise a light chain variable region sequence:

[0043] NIMMTQSPSSLAVSAGEKATMSC KSSQSLLYSSNQMNYLA WYQQKPGQ SPKLLIY WASTRES DVPDRFTGSGSGTDFTLTISSVQTEDLAVYYC HQYFSSRT FGGGTKLEIK(SEQ ID No.12)

[0044] (The CDRs are shown in bold and underlined; the framework sequences are shown in italics)

[0045] and / or, heavy chain variable region sequence:

[0046] EVQLVESGGGLVQPKGSLKLSCAASGFTFN IYTMN WVRQAPGKGLEWVA RIRSKSENYATYYADSVK D RFTISRDDSQSILYLQMNNLKTEDTAIYYCVR QGI YYDYYGAMDY WGQGTSVTVS (SEQ ID No.13)

[0047] (The CDRs are shown in bold and underlined; the framework sequences are shown in italics)

[0048] As used herein, the term "binding amount" refers to the quantification of the binding between an antibody and a peptide in a patient sample. The quantification can be determined, for example, by comparing the measured value of the binding in the patient sample with a calibration curve generated from the measured value of the binding in a standard sample containing a peptide specifically bound by an antibody at a known concentration, so as to determine the amount of the peptide specifically bound by the antibody in the patient sample. In the examples listed below, the ELISA method is used, wherein spectrophotometric analysis is used to measure the binding amount in the patient sample and when generating the calibration curve. However, any suitable analytical method can be used.

[0049] As used herein, the term "specifically binds" means that an antibody binds selectively to an antigen, and that binding can be distinguished from unwanted or non-specific interactions. The ability of a monoclonal antibody to bind a specific epitope or peptide sequence can be measured by the enzyme-linked immunosorbent assay (ELISA) described herein or other techniques familiar to those skilled in the art, such as surface plasmon resonance (SPR) techniques (e.g., analyzed on a BIAcore instrument) and conventional binding assays. The degree of binding of a monoclonal antibody to an irrelevant protein is less than about 10% of the degree of binding of the monoclonal antibody to the epitope or peptide, e.g., as measured by ELISA. "Affinity" refers to the strength of the sum of non-covalent 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 stated, "binding affinity" as used herein refers to the intrinsic binding affinity, reflecting the 1:1 interaction between the 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 rate constant and the association rate constant (koff and kon, respectively). Thus, equivalent affinities can include different rate constants as long as the ratio of the rate constants remains the same. The dissociation constant represents the antigen concentration 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 can be used to measure the Kd of an antibody, including surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), and fluorescence-based assays. In certain aspects, the dissociation constant (KD) of a monoclonal antibody that binds an epitope or peptide is <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., from 10 8 M - 10 13 M, e.g., from 10 9 M - 10 13 M).

[0050] As used herein, the term "predetermined cut-off value" refers to a binding amount that is statistically determined to indicate a high likelihood of disease (i.e., cancer) or its specific severity in a patient, wherein a measured value of a target peptide in a patient sample that is equal to or higher than the statistical cut-off value corresponds to at least a 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 of the presence of the disease.

[0051] As used herein, the term "value associated with a normal healthy subject" refers to the normalized binding amount determined by the above method for a subject sample that is considered healthy (i.e., not suffering from a disease (i.e., cancer)); and the term "value associated with a known disease severity" refers to the normalized binding amount determined by the above method for a patient sample known to have a disease (i.e., cancer) of a known severity.

[0052] 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 (renal) cancer, or melanoma. In another preferred embodiment, the cancer is lung cancer, and most preferably the lung cancer is non-small cell lung cancer.

[0053] 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., in the form 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 form of the target sequence is at least 10 to 1, more preferably at least 20 to 1 or at least 30 to 1.

[0054] 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., in the form of the PRO-C9 target sequence truncated by the removal of the last 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 form of the target sequence is at least 10 to 1, more preferably at least 20 to 1 or at least 30 to 1.

[0055] In a preferred embodiment, the monoclonal antibody is produced against a synthetic peptide having the C-terminal amino acid sequence QRAFNKGPDP. For example, the monoclonal antibody can be produced by: (a) immunizing a rodent (or other suitable mammal) with a synthetic peptide containing the C-terminal sequence QRAFNKGPDP (SEQ ID.No: 1), which peptide can optionally be linked at its N-terminus to an immunogenic carrier protein (such as keyhole limpet hemocyanin); (b) isolating and cloning individual antibody-producing cells; and (c) assaying the resulting monoclonal antibodies to ensure that they have the desired specificity.

[0056] In a preferred embodiment, the patient sample is selected from blood, serum, or plasma.

[0057] In a preferred embodiment, the immunoassay is a competitive assay or a sandwich assay. The immunoassay can be, for example, a radioimmunoassay or an enzyme-linked immunosorbent assay (ELISA). Such assays are techniques known to those skilled in the art.

[0058] In a second aspect, the present invention provides a method for treating cancer in a patient in need thereof, the method comprising:

[0059] (a) performing an immunoassay method for detecting and / or monitoring cancer according to the first aspect of the present invention on a sample from the patient to detect whether the patient has cancer; and

[0060] (b) if it is determined in step (a) that the patient has the cancer, administering to the patient a therapy for treating the cancer.

[0061] The therapy can be any therapy suitable for treating the cancer in question. The therapy can, for example, include one or more surgical procedures, one or more radiotherapy treatments, one or more drugs (such as one or more chemotherapy treatments, one or more immunotherapy treatments and / or one or more hormone therapy treatments) or a combination thereof, or consist of the same. The drugs can be formulated for local or systemic administration. Local drugs can, for example, be formulated as creams, foams, gels, lotions or ointments for administration. Systemic drugs can, for example, be formulated for enteral or parenteral administration. The surgical procedure can be radical surgery, prophylactic surgery, debulking surgery, palliative surgery and / or reconstructive surgery.

[0062] For example, in the case where the cancer is lung cancer, suitable therapies can, for example, include one or more of the following: surgical procedures, such as performing a pulmonary resection, such as a lobectomy, a sublobar resection (wedge resection) or a resection of the entire lung (pneumonectomy); radiotherapy treatments, examples of which include but are not limited to: radiotherapy given in combination with chemotherapy, postoperative radiotherapy, brachytherapy (local radiotherapy), prophylactic cranial radiotherapy, stereotactic radiotherapy and palliative radiotherapy; chemotherapy using, for example, one or more agents such as cisplatin, carboplatin, etoposide, gemcitabine, paclitaxel, docetaxel, vinorelbine, topotecan, irinotecan and pemetrexed; epidermal growth factor receptor (EGFR) inhibitor drugs, such as erlotinib, gefitinib, afatinib, dacomitinib or osimertinib; 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, such as atezolizumab, nivolumab or pembrolizumab, monoclonal antibodies targeting cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), such as ipilimumab, and / or monoclonal antibodies targeting vascular endothelial growth factor, such as bevacizumab, necitumumab, mobocertinib or cetuximab.

[0063] When the lung cancer is non-small cell lung cancer, suitable therapies can, for example, include one or more of the above-mentioned lung cancer therapies.

[0064] 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).

[0065] The antibody according to the third aspect of the present invention is particularly suitable for performing the immunoassay method according to the first aspect of the present invention. Therefore, from the above discussion of the preferred embodiments of the method according to the first aspect, the preferred embodiments and features of the antibody according to the third aspect will be apparent.

[0066] For example, 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); and / or does not specifically bind to a peptide having the C-terminal amino acid sequence QRAFNKGPD (SEQ ID No.3); and / or is produced against a synthetic peptide having the C-terminal amino acid sequence QRAFNKGPDP (SEQ ID No.1).

[0067] In a fourth aspect, the present invention provides an immunoassay kit comprising the monoclonal antibody according to the third aspect of the present invention, and at least one of the following:

[0068] - Streptavidin-coated microtiter plates;

[0069] - Biotinylated peptide: biotin-L-QRAFNKGPDP (SEQ ID No.18), where L is an optional linker;

[0070] - Secondary antibody for sandwich immunoassay;

[0071] - Calibration protein comprising the C-terminal amino acid sequence QRAFNKGPDP;

[0072] - Antibody biotinylation kit;

[0073] - Antibody HRP-labeling kit;

[0074] - Antibody radiolabeling kit; and

[0075] - Assay visualization kit.

[0076] The immunoassay kit according to the fourth aspect of the present invention is particularly suitable for performing the immunoassay method according to the first aspect of the present invention. Therefore, from the above discussion of the preferred embodiments of the method according to the first aspect, other preferred embodiments and features of the immunoassay kit according to the fourth aspect will be apparent. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] Figure 1:Depiction of the three chains (α1, α2, and α3) of type IX collagen, where the PRO-C9 target sequence on the α1 chain is highlighted.

[0078] Figure 2 :Sequence alignment of type IX collagen and specificity of PRO-C9 assay. (A) Sequence alignment of the C-terminal regions of the α1 chains of type IX collagen from human, mouse, and rat. The PRO-C9 sequences of the mouse and rat α1 chains and the aligned C-terminal sequences are boxed. (B) Specificity of the PRO-C9 assay. Reactivity to a standard peptide (QRAFNKGPDP, SEQ ID No.1), a truncated peptide (QRAFNKGPD, SEQ ID No.3), an extended peptide (QRAFNKGPDPG, SEQ ID No.2), a nonsense standard peptide, and a coating agent (DQAAGGLRQH, SEQ ID No.14) is described. Signals are expressed as relative light units per second (RLU) relative to the standard peptide.

[0079] Figure 3 :PRO-C9 levels in healthy controls (n = 43) and NSCLC patients (n = 40). (A) PRO-C9 was significantly elevated in NSCLC patients compared to healthy controls (p = 0.006). Data were analyzed by t-test adjusted for age and gender and presented as Tukey box plots. (B) ROC curve analysis was used to evaluate the ability of PRO-C9 to distinguish between healthy controls and NSCLC patients.

[0080] Figure 4 :PRO-C9 levels in sera from cohort 1, including healthy donors (n = 13), bladder cancer (n = 19), breast cancer (n = 20), colorectal cancer (n = 20), gastric cancer (n = 20), head and neck cancer (n = 20), lung cancer (n = 20), pancreatic cancer (n = 20), prostate cancer (n = 20), or kidney cancer (n = 20). Differences in PRO-C9 between solid tumor types and healthy donors were evaluated by Kruskal-Wallis test and Dunn's multiple comparison test. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05. Detailed Description

[0081] Examples

[0082] The presently disclosed embodiments are described in the following examples, which are intended to facilitate understanding of the disclosure and should not be construed as limiting the scope of the disclosure as defined in the appended claims in any way. The following examples are presented to provide a complete disclosure and description to those of ordinary skill in the art 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 following experiments are all or the only experiments conducted. Efforts have been made to ensure the accuracy of the numbers used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be taken into account. Unless otherwise indicated, parts are parts by weight, molecular weight is weight-average molecular weight, temperature is in degrees Celsius, and pressure is atmospheric or near atmospheric pressure.

[0083] Materials and Methods

[0084] Unless otherwise indicated, all reagents used were high-quality chemicals from Merck (Whitehouse Station, NJ, USA) and Sigma (St. Louis MO, USA). All synthetic peptides used for antibody production and assay validation were purchased from Genscript (Piscataway, NJ, US) (Table 1).

[0085] Table 1. Sequences of synthetic peptides for monoclonal antibody production, assay development, and validation

[0086] Peptide type Sequence Immunogenic peptide KLH-CGG-QRAFNKGPDP(SEQ ID NO.15) Selected peptide QRAFNKGPDP(SEQ ID No.1) Extended selected peptide QRAFNKGPDPG(SEQ ID No.2) Truncated selected peptide QRAFNKGPD(SEQ ID No.3) Nonsense-coated and standard peptides DQAAGGLRQH(SEQ ID NO.14) Biotinylated selected peptide Biotin-QRAFNKGPDP(SEQ ID No.18)

[0087] KHL = keyhole limpet hemocyanin

[0088] Monoclonal antibody development, production, and characterization

[0089] Amino acid sequence 912 ’QRAFNKGPDP’ 921 For monoclonal antibody production. Immunization was initiated by subcutaneous injection of 200 μl of emulsified antigen and 100 μg of immunogenic peptide (KLH-CGG-QRAFNKGPDP (SEQ ID No. 15)) in 4- to 6-week-old Balb / C mice using Stimmune (ThermoFisher). Immunization was repeated every two weeks until a stable serum antibody titer level was reached. Mice with the highest serum titers were selected for fusion and rested for one month. Subsequently, three days before isolating the spleen for cell fusion, the mice were boosted intravenously with 50 μg of immunogenic peptide in 100 μl of 0.9% NaCl solution. To generate hybridoma cells, the spleen cells of the mice were fused with SP2 / 0 myeloma cells as described by Gefter et al. (10). Subsequently, the clones were plated into 96-well microtiter plates for further growth, and the limiting dilution method was applied to promote monoclonal growth.

[0090] An indirect ELISA performed on streptavidin-coated plates was used to screen for supernatant reactivity. Biotin-QRAFNKGPDP was used as the screening peptide, while the standard peptide QRAFNKGPDP was used to further test the specificity of the clones. Supernatants were collected from the hybridoma cells, purified using a HiTrap affinity column (GE Healthcare Life Science, Little Chalfront, Buckinghamshire, UK) according to the manufacturer's instructions, and the antibody isotypes were determined using a Quick ELISA Mouse Monoclonal Antibody Isotyping Kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol.

[0091] Natural reactivity was evaluated using human serum purchased from a commercial vendor (Valley Biomedical, Winchester, VA). Monoclonal antibodies (mAbs) were selected to specifically recognize the standard peptide (QRAFNKGPDP (SEQ ID No.1)), but not an extended or truncated sequence of one amino acid (QRAFNKGPDPG (SEQ ID No.2) and QRAFNKGPD (SEQ ID No.3), respectively).

[0092] The selected antibodies were sequenced and the CDRs were determined.

[0093] The sequences of the chains are as follows (CDRs are shown in bold; framework sequences are shown in italics; constant regions are underlined):

[0094] Light chain: Amino acid sequence (219aa)

[0095]

[0096] CDR-L1: KSSQSLLYSSNQMNYLA (SEQ ID No.4)

[0097] CDR-L2: WASTRES (SEQ ID No.5)

[0098] CDR-L3: HQYFSSRT (SEQ ID No.6)

[0099] Heavy chain: Amino acid sequence (448aa):

[0100]

[0101] CDR-H1: IYTMN (SEQ ID No.7)

[0102] CDR-H2: RIRSKSENYATYYADSVKD (SEQ ID No.8)

[0103] CDR-H3: QGIYYDYYGAMDY (SEQ ID No.9)

[0104] PRO-C9 assay development

[0105] The development of a competitive chemiluminescent immunoassay (CLIA) included several preliminary optimization experiments, in which several test analytical reagents, concentrations, incubation times, and temperatures were analyzed. The PRO-C9 competitive ELISA procedure was as follows: A 96-well streptavidin-coated white microplate (Greiner Bio-One, Kremsmünster, Austria) was coated with 5 ng / mL 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, adjusted to pH 7.4 at 20 °C) and incubated for 30 minutes at 20 °C in the dark with constant shaking (300 rpm).

[0106] Next, 20 μL / well of the standard peptide (100 ng / mL) and samples were added to the appropriate wells, followed by the addition of 100 μL / well of the HRP-labeled antibody diluted in assay buffer to a concentration of 100 ng / mL, and incubated for 20 hours at 4 °C in the dark with continuous shaking (300 rpm). After each incubation step, the wells were washed with standard wash buffer (20 mM Tris, 50 mM NaCl, pH 7.2). The chemiluminescent substrate (Roche, BM Chemiluminescent ELISA Substrate (POD), Basel, Switzerland) working solution was mixed 15 minutes before use and added to the plate at 100 μL / well, and incubated for 3 minutes at 20 °C in the dark with constant shaking (300 rpm). Relative light units were measured at all wavelengths within 5 minutes on a microplate photometer reader (SpectraMax M5, Molecular Devices, CA, USA).

[0107] A standard curve was plotted using 4-parameter logistic curve fitting Y = (A - D) / (1 + (x / C)^B) + D, where R > 0.9. The data were analyzed using SoftMax Pro version 7.0.3 software.

[0108] Technical evaluation

[0109] Doubled dilutions of four human serum samples were used to evaluate linearity. Linearity was calculated as the percentage of recovery of the undiluted sample. Antibody specificity was calculated as the percentage of signal inhibition of the doubled 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-assay and inter-assay variability were determined by 10 independent runs of five quality controls and two kit controls run in duplicate.

[0110] The accuracy of the assay was measured for healthy human serum samples spiked with the standard peptide and serum samples with known high PRO-C9 concentrations, and was calculated as the percentage of recovery of the measured value and the expected concentration of the peptide or serum sample with high PRO-C9 plus the concentration of the analyte in the serum. Analytical interference was performed by adding low / high amounts 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 concentration. The percentage of recovery was calculated using normal serum samples as a reference. The 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 of recovery of the analyte in the unspiked serum. The measurement range was defined as the range between the lower limit of quantitation (LLOQ) and the upper limit of quantitation (ULOQ), which was determined by 10 independent runs using the standard peptide. Measured values below the LLOQ or above the ULOQ were assigned the value of LLOQ / ULOQ, respectively. The IC50 (half-maximal inhibitory concentration) was determined from the standard curve.

[0111] Analyte stability was examined by temperature testing of serum samples and repeated freeze-thaw cycles. Temperature testing included different time points and temperatures, where PRO-C9 levels were measured for three human serum samples after incubation at 4 °C or 20 °C for 0 h, 2 h, 4 h, 24 h, and 48 h. Recovery was estimated using the 0 h sample as a reference. In addition, the effect of four repeated freeze / thaw cycles on three serum samples was evaluated, where the freeze / thaw recovery was calculated using the zero-cycle sample as a reference. Each sample was run in duplicate.

[0112] Biological evaluation of PRO-C9

[0113] The biological utility of PRO-C9 was evaluated in serum samples from patients with NSCLC (n = 40) and healthy donors (n = 43), which were obtained from the commercial supplier Proteogenex (Culver City, CA). After the serum samples were obtained, they were stored at -80 °C until use.

[0114] The biological utility of PRO-C9 was also evaluated in serum samples from patients with various cancers (n = 219) and healthy donors (n = 13), which were obtained from the commercial supplier Proteogenex (Culver City, CA). After the serum samples were collected, they were stored at -80 °C until use.

[0115] Ethical statement

[0116] All animals were handled according to the guidelines of animal welfare. The production of monoclonal antibodies in mice was approved by the Danish national agency (Danish Animal Experiment Inspectorate) with the approval number 2013-15-2934-00956. The collection and retrieval of human cartilage complied with the international ethical guidelines for handling human samples and patient information. All participants signed an informed consent form, and the study was approved by the local ethics committee. Both groups of samples were collected after signing the informed consent form and being approved by the local ethics committee, and complied with the Helsinki Declaration of 1975.

[0117] Statistical analysis

[0118] The PRO-C9 levels were log-transformed to obtain normality. The comparison of PRO-C9 between healthy controls and NSCLC was performed by a t-test corrected for age and gender. The diagnostic accuracy was tested by AUROC. A p-value lower than 0.05 was considered significant. Statistical analysis and graphing 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).

[0119] Results

[0120] Technical evaluation and characterization of the PRO-C9 assay

[0121] The monoclonal antibody clone NBH313#46 9H11-1D9-2B6 showed the best native reactivity, peptide affinity, and stability for the assay and was selected for assay development. A summary of the technical evaluation of the PRO-C9 assay is shown in Table 2.

[0122] Table 2. Summary of technical parameters for PRO-C9 assay

[0123]

[0124] The measurement range (LLOQ-ULOQ) was determined to be 0.65 - 120 ng / mL. The between- and within-assay variability were 12.0% and 3.9%, respectively, and the linearity in human serum was approved from undiluted to 2-fold dilution. The stability of the analyte was acceptable for five freeze-thaw cycles (92.1 - 112.8%). Hemoglobin, lipemia, and biotin did not interfere with the measurement of PRO-C9 in human serum. The human sequence was aligned using UNIPROT, and the corresponding sequences in mice and rats had mismatches at positions 1 and 5 ( Figure 2 A). To evaluate the specificity of the PRO-C9 assay, mAbs were tested against extended peptides, truncated peptides, nonsense standard peptides, and nonsense coatings and showed no reactivity to these peptides ( Figure 2 B).

[0125] Baseline demographics and clinical characteristics

[0126] The PRO-C9 assay was measured in sera from NSCLC patients. The patient cohort consisted of healthy controls and patients diagnosed with NSCLC. Patient demographic information can be seen in Table 3. Here, when adjusted for age and gender, patients with NSCLC had significantly higher levels of PRO-C9 compared to healthy controls (p = 0.006, Figure 3 A). The diagnostic efficacy of PRO-C9 in patients with NSCLC compared to healthy controls was AUROC = 0.890 (95% CI: 0.82 - 0.96 p < 0.001, Figure 3 B).

[0127] Table 3. Demographic information of NSCLC patients

[0128]

[0129] The PRO-C9 assay was used to analyze sera from a second patient cohort with a range of cancers. The second patient cohort consisted of healthy controls and patients diagnosed with cancer. Patient demographic information can be seen in Table 4. Here, when adjusted for age and gender, patients with bladder cancer, breast cancer, colorectal cancer, gastric cancer, head and neck cancer, lung cancer, ovarian cancer, pancreatic cancer, and renal cancer, as well as melanoma, had significantly higher levels of PRO-C9 compared to healthy controls ( Figure 4 ).

[0130] Table 4. Demographic information of patients in cohort 2

[0131]

[0132] Discussion

[0133] The applicant developed and characterized a competitive CLIA assay for detecting type IX collagen using monoclonal antibodies that react with the PRO-C9 target sequence. The main findings were as follows: 1) A technically robust and specific assay targeting the C-terminal sequence (PRO-C9) of the α-1 chain of type IX collagen was successfully developed; 2) PRO-C9 levels were measurable in human serum; 3) PRO-C9 levels were significantly elevated in patients with NSCLC, bladder cancer, breast cancer, colorectal cancer, gastric cancer, head and neck cancer, lung cancer, ovarian cancer, pancreatic cancer, and kidney cancer, as well as melanoma, compared to healthy controls; and 4) The PRO-C9 assay showed an AUROC = 0.890, indicating its potential as a diagnostic biomarker.

[0134] This is the first demonstration that PRO-C9 can be measured non-invasively in blood and is biologically relevant for patients with cancer, including NSCLC.

[0135] In summary, the present invention developed and validated a CLIA targeting the PRO-C9 target sequence. Quantification of PRO-C9 levels in the sera of NSCLC and other cancer patients showed a significant elevation compared to healthy controls and can be used as a cancer biomarker.

[0136] References

[0137] 1. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin Wiley; 2018; 68: 394-424.

[0138] 2. Molina JR, Yang P, Cassivi SD, Schild SE, Adjei AA. Non-small cell lung cancer: epidemiology, risk factors, treatment, and survivorship. Mayo Clin Proc [Internet]. Mayo Clin Proc; 2008 [cited 2022 Oct 21]; 83:584-94. Available from: https: / / pubmed.ncbi.nlm.nih.gov / 18452692 /

[0139] 3. Nissen NI, Karsdal M, Willumsen N. Collagens and Cancer associated fibroblasts in the reactive stroma and its relation to Cancer biology. J Exp Clin Cancer Res [Internet]. J Exp Clin Cancer Res; 2019 [cited 2022 Oct 21]; 38. Available from: https: / / pubmed.ncbi.nlm.nih.gov / 30841909 /

[0140] 4. Ricard-Blum S. The Collagen Family. Cold Spring Harb Perspect Biol 2011; 3:1-19.

[0141] 5. Thorlacius-Ussing J, Manon-Jensen T, Sun S, Leeming DJ, Sand JM, Karsdal M, et al. Serum Type XIX Collagen is Significantly Elevated in Non-Small Cell Lung Cancer: A Preliminary Study on Biomarker Potential. Cancers (Basel) [Internet]. Cancers (Basel); 2020 [cited 2022 Oct 21]; 12:1-14. Available from: https: / / pubmed.ncbi.nlm.nih.gov / 32527017 /

[0142] 6. Diab M. The role of type IX collagen in osteoarthritis and rheumatoid arthritis. Orthop Rev [Internet]. Orthop Rev; 1993 [cited 2022 Oct 21]; 22:165-70. Available from: https: / / pubmed.ncbi.nlm.nih.gov / 8451069 /

[0143] 7. Parsons P, Gilbert SJ, Vaughan-Thomas A, Sorrell DA, Notman R, Bishop M, et al. Type IX collagen interacts with fibronectin providing an important molecular bridge in articular cartilage. J Biol Chem [Internet]. J Biol Chem; 2011 [cited 2022 Oct 21]; 286:34986-97. Available from: https: / / pubmed.ncbi.nlm.nih.gov / 21768108 /

[0144] 8. Chung CM, Lee CH, Chen MK, Lee KW, Lan CCE, Kwan AL, et al. Combined genetic biomarkers and betel quid chewing for identifying high-risk group for oral cancer occurrence. Cancer Prev Res [Internet]. American Association for Cancer Research Inc.; 2017 [cited 2022 October 21]; 10:355-61. Available from: https: / / aacrjournals.org / cancerpreventionresearch / article / 10 / 6 / 355 / 258417 / Combined-Genetic-Biomarkers-and-Betel-Quid-Chewing

[0145] 9. Piotrowski A, Benetkiewicz M, Menzel U, Díaz De T, Mantripragada K, Grigelionis G, et al. Microarray-based survey of CpG islands identifies concurrent hyper- and hypomethylation patterns in tissues derived from patients with breast cancer. Genes Chromosomes Cancer [Internet]. Genes Chromosomes Cancer; 2006 [cited 2022 October 21]; 45:656-67. Available from: https: / / pubmed.ncbi.nlm.nih.gov / 16575877 /

[0146] 10. Gefter ML, Margulies DH, Scharff MD. A simple method for polyethylene glycol-promoted hybridization of mouse myeloma cells. Somatic Cell Genet [Internet]. 1977;3:231-6. Available from: http: / / www.ncbi.nlm.nih.gov / pubmed / 605383

Claims

1. A method for an immunoassay 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 QRAFNKGPDP; ii) detecting and determining the amount of binding between the monoclonal antibody and the peptide in the sample; and iii) 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, and / or a predetermined cut-off value.

2. The method according to 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. The method according to claim 1 or 2, wherein The cancer is lung cancer.

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

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

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

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

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

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

10. The method according to any one of the preceding claims, 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 according to claim 11, wherein, The monoclonal antibody does not specifically bind to a peptide having the C-terminal amino acid sequence QRAFNKGPDPG.

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

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

15. An immunoassay kit comprising a monoclonal antibody that specifically binds to the C-terminal amino acid sequence QRAFNKGPDP, and at least one of the following: - a streptavidin-coated microtiter plate; - Biotinylated peptide: biotin-L-QRAFNKGPDP, wherein, L is an optional linker; - a secondary antibody for a sandwich immunoassay; - a calibration protein comprising the N-terminal amino acid sequence QRAFNKGPDP; - an antibody biotinylation kit; - an antibody HRP-labeling kit; - an antibody radiolabeling kit; and - an assay visualization kit.

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

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

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