Collagen type XXII assay

Abstract

The present invention relates to immunoassays, in particular immunoassays for detecting and / or monitoring cancer in a patient, and / or monoclonal antibodies and immunoassay kits for use in performing such immunoassays.

Description

[Technical field]

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

[0002] Cancer is a major health problem worldwide, estimated to result in over 600,000 cancer deaths in 2021 in the United States alone [1]. Despite ongoing research and advances, the burden of cancer persists. One of the most pressing needs for cancer patients is good, non-invasive diagnostic and prognostic biomarkers. In contrast to traditional tissue-based approaches, blood-based biomarkers do not require tissue biopsy and are less invasive, resulting in less discomfort and complications [2], [3].

[0003] The tumor microenvironment is intricately involved in cancer development and has recently been recognized as one of the classic hallmarks of cancer [4]. The tumor microenvironment includes cancer cells and the surrounding stroma. Within the stroma are various cells of the immune system and fibroblasts. Fibroblasts contribute to the production of collagen, a key component of the extracellular matrix (ECM), the large non-cellular component of most tissues. The ECM can accumulate around tumors and form a surrounding layer of matrix that encapsulates the growth and inhibits the ability of immune cells or drugs to challenge the cancer [5]. In healthy tissues, components of the ECM are constantly produced and degraded in a fine balance. In cancer, however, this turnover balance is distorted [6], [7].

[0004] Collagens, 28 of which have been identified to date, are major components of the ECM together with laminins, proteoglycans, and glycoproteins. Collagens extend from the basement membrane (BM) on the basal side of epithelial and endothelial cells into the interstitial matrix (IM). In the basement membrane region at the interface between the BM and IM, several so-called rare collagens, such as FACIT (fibrillar associated collagens with interrupted triple helices), are present. The FACIT family of collagens has been proposed to function as cross-links, as they mediate interactions between collagen fibers and other ECM components. In this way, they help in the organization and stability of the ECM [7],[8]. Type XXII collagen (COL22) is a rare collagen and a member of the EACIT family of collagens. COL22 is known to be expressed at the sites of tissue junctions in muscle, cartilage, heart, and skin, and to be associated with the maintenance of vascular stability [9],

[10] . COL22 also has prognostic value in head and neck squamous cell carcinoma (HNSCC), where COL22A1 mRNA expression was significantly increased in cases with lymph node metastasis. Furthermore, mRNA expression levels were significantly associated with early disease recurrence, and upregulation of COL22A1 mRNA was associated with reduced disease-free survival

[11] . Individually, COL22 was identified in human ex vivo and in vivo models as an early response gene to transforming growth factor beta using both skin- and lung-derived cells (including adenocarcinoma human alveolar basal epithelial cells, A549 cells). This induction of COL22A1 was significantly increased at both the mRNA and protein levels

[12] . For several FACITs, particularly collagens XVI, XX, and XXII, it has been observed that they are more prominent during development and / or tissue repair

[13] ,

[14] ,

[15] .

[0005] The role of COL22 in cancer has been investigated by the present inventors, and some parallels are suggested to exist between cancer and development, as well as tissue repair. Summary of the Invention

[0006] The applicant has now developed an enzyme-linked immunosorbent assay (ELISA) for quantifying the presence of a biomarker of collagen XXII (herein referred to as "PRO-C22") in blood. The assay has been optimized, validated, and used to determine the levels of circulating collagen XXII in the serum of cancer patients and healthy controls. The assay has been found to be very powerful in terms of specificity for collagen XXII and in terms of sensitivity for detecting levels in both healthy controls and patients with disease (cancer). The data show that the levels of PRO-C22 are significantly higher in the serum of cancer patients compared to healthy controls.

[0007] Thus, in a first aspect, the present invention provides a method of immunoassay, the method comprising: i) contacting a sample from a patient with a monoclonal antibody that specifically binds to the C-terminal amino acid sequence AARPGNVKGP (SEQ ID No. 1) (the C-terminal amino acid sequence is referred to herein as "PRO-C22" and / or the "target sequence", and a peptide consisting of or comprising said C-terminal amino acid sequence is also referred to herein as "PRO-C22" and / or the "target sequence"); ii) detecting and determining the amount of binding between said monoclonal antibody and peptide in said sample. Includes.

[0008] The method is preferably an immunoassay method for detecting and / or monitoring disease in a patient. The method preferably comprises: iii) correlating the amount of binding of said monoclonal antibody determined in step ii) with values ​​associated with normal healthy subjects, and / or with values ​​associated with known disease severity, and / or with values ​​obtained from said patient at previous time points, and / or with predefined cut-off values. Further includes:

[0009] In a preferred embodiment, the disease is cancer, such as bladder cancer, breast cancer, colorectal cancer, head and neck cancer, kidney cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, or gastric cancer. Preferably, the cancer is pancreatic cancer.

[0010] If the patient has pancreatic cancer, the method can be used to provide a prognosis of overall survival.Thus, the method can include providing a prognosis of overall survival, and the amount of binding of the monoclonal antibody determined in step (ii) that is greater than the median (50th percentile) associated with known pancreatic cancer patients is associated with poor overall survival.An amount of binding that is less than the median (50th percentile) is associated with improved overall survival.

[0011] In a preferred embodiment, the monoclonal antibody does not specifically bind to an extended version of the target sequence (i.e. a version of the PRO-C22 target sequence extended at its C-terminus by the addition of an amino acid) which is AARPGNVKGPX (SEQ ID No. 2) (wherein X is any amino acid). Preferably, X is A and the extended version of the target sequence is AARPGNVKGPA (SEQ ID No. 3) (i.e. a version of the PRO-C22 target sequence extended at its C-terminus by the addition of an alanine). Preferably, the ratio of the affinity of said antibody for the PRO-C22 target sequence to the affinity of said antibody for the extended version of the target sequence is at least 10 to 1, more preferably at least 20 to 1 or at least 30 to 1.

[0012] In a preferred embodiment, the monoclonal antibody does not specifically bind to a truncated version of the target sequence, AARPGNVKG (SEQ ID No. 4) (i.e., a version of the PRO-C22 target sequence truncated by removal of the last proline residue). Preferably, the ratio of the affinity of said antibody for the PRO-C22 target sequence to the affinity of said antibody for the truncated version of the target sequence is at least 10 to 1, more preferably at least 20 to 1 or at least 30 to 1.

[0013] Preferably, the monoclonal antibody is a monoclonal antibody raised against a synthetic peptide having the C-terminal amino acid sequence AARPGNVKGP (SEQ ID No. 1).

[0014] If a disease is detected in a patient, the method may further include administering an appropriate treatment to the patient, for example, if the disease is cancer, a treatment known for that cancer, such as chemotherapy, radiation therapy, hormone therapy, immunotherapy, stem cell transplant, surgery, targeted therapy, or a combination thereof, may be administered.

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

[0016] The immunoassay may be, but is not limited to, a competitive assay or a sandwich assay. The immunoassay may be, for example, a radioimmunoassay or an enzyme-linked immunosorbent assay (ELISA). Such assays are techniques known to those skilled in the art.

[0017] As used herein, the term ELISA (enzyme-linked immunosorbent assay) refers to an immunoassay in which the target peptide present in a sample (if any) is detected using an antibody linked to an enzyme such as horseradish peroxidase or alkaline phosphatase. The activity of the enzyme is thus assessed by incubation with a substrate that produces a measurable product. The presence and / or amount of the target peptide in the sample can thereby be detected and / or quantified. ELISA is a technique known to those skilled in the art.

[0018] As used herein, the term "competitive ELISA" refers to a competitive enzyme-linked immunosorbent assay, in which a target peptide present in a sample (if present) competes with a known amount of the target peptide (that is, for example, bound to an immobilized substrate or labeled) for binding to an antibody, a technique known to those skilled in the art.

[0019] As used herein, the term "sandwich immunoassay" refers to the use of at least two antibodies for the detection of an antigen in a sample, and is a technique known to those skilled in the art.

[0020] As used herein, the term "amount of binding" refers to the quantification of binding between a monoclonal antibody and a peptide in a patient sample. Such quantification can be determined, for example, by comparing the binding values ​​measured in a patient sample to a standard curve generated using binding values ​​measured in standard samples containing known concentrations of the peptide to which the antibody specifically binds, thereby determining the amount of the peptide to which the antibody specifically binds in the patient sample. In the examples described below, an ELISA method is used, in which spectrophotometric analysis is used to measure the amount of binding in the patient sample and when generating the standard curve. However, any suitable method can be used.

[0021] As used herein, the term "predetermined cutoff value" refers to an amount of binding that is statistically determined to represent a high likelihood of said disease or a particular severity thereof in a patient, in that a measured value of the 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 the disease or a particular severity thereof, preferably at least a 75% probability, 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.

[0022] 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, and the term "value associated with a known disease severity or prognosis" refers to a standardized amount of binding determined by the methods described above for samples from patients known to have a disease of known severity.

[0023] 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-terminal end of a polypeptide, and should not be construed as meaning in its general orientation.

[0024] As used herein, the terms "peptide" and "polypeptide" are used synonymously.

[0025] 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, or other such fragments known to those skilled in the art. As is well known, whole antibodies usually have a "Y-shaped" structure, which is two identical pairs of polypeptide chains, each pair consisting of one "light" chain and one "heavy" chain. The N-terminal regions of each light and heavy chain comprise the variable region, while the C-terminal parts of each heavy and light chain constitute the constant region. The variable regions contain three complementarity determining regions (CDRs), which are primarily responsible for antigen recognition. The constant regions allow the antibody to recruit cells and molecules of the immune system. An antibody fragment that retains the binding specificity comprises at least the CDRs and a sufficient remaining portion of the variable region to retain said binding specificity.

[0026] In the method of the present invention, monoclonal antibodies containing any constant region known in the art can be used. Human constant light chains are classified as kappa light chains and lambda light chains. Heavy chain constant regions are classified as mu, delta, gamma, alpha, or epsilon, which define the antibody's isotype as gM, IgD, IgG, IgA, and IgE, respectively. The IgG isotype has several subclasses, including but not limited to IgG1, IgG2, IgG3, and IgG4. The monoclonal antibody can preferably be of the IgG isotype, including any of IgG1, IgG2, IgG3, or IgG4.

[0027] The CDRs of the antibody can be determined using methods known in the art, such as those described by Kabat et al.

[18] . The antibody can be generated from a clone of a B cell as described in the Examples. The isotype of the antibody can be determined by ELISA specific for human IgM, IgG, or IgA isotypes, or human IgG1, IgG2, IgG3, or IgG4 subclasses. The amino acid sequence of the generated antibody can be determined using standard techniques. For example, RNA can be isolated from the cells and used to generate cDNA by reverse transcription. 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 sequence for all VH (variable heavy) sequences can be used together with a primer that binds to a sequence located in the constant region of the previously determined isotype. The light chain can be amplified using a primer that binds to the 3' end of the kappa or lambda chain together with a primer that anneals to the Vkappa or Vlambda leader sequence. Full-length heavy and light chains can be generated and sequenced.

[0028] In a second aspect, the present invention provides a monoclonal antibody which specifically binds to the C-terminal amino acid sequence AARPGNVKGP (SEQ ID No. 1), which is suitable for use in an immunoassay according to the first aspect of the invention, and preferred and other optional embodiments of the monoclonal antibody according to the second aspect of the invention will be apparent from the above discussion of monoclonal antibodies for use in the first aspect of the invention and its preferred and other optional embodiments.

[0029] Monoclonal antibodies that specifically bind to the C-terminal amino acid sequence AARPGNVKGP (SEQ ID No. 1) may be made via any suitable technique known in the art. For example, monoclonal antibodies may be produced against a synthetic peptide having the amino acid sequence AARPGNVKGP (SEQ ID No. 1) by, for example, immunizing a rodent (or other suitable mammal) with a synthetic peptide consisting of the sequence AARPGNVKGP (SEQ ID No. 1), which may optionally be 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 that they have the desired specificity. An exemplary protocol for producing monoclonal antibodies that specifically bind to the C-terminal amino acid sequence AARPGNVKGP (SEQ ID No. 1) is described below.

[0030] Preferably, the monoclonal antibody or fragment thereof is CDR-L1: KASQDIYSSLS(SEQ ID No. 5) CDR-L2: RANRLIN (SEQ ID No. 6) and CDR-L3: LQYDDFPYM(SEQ ID No. 7) CDR-H1: TYGVH(SEQ ID No. 8) CDR-H2: AIWRGGSTDYNPAFMS (SEQ ID No. 9) and CDR-H3: RSTLFYFDY(SEQ ID No. 10) It may comprise one or more complementarity determining regions (CDRs) selected from:

[0031] Preferably, the antibody or fragment thereof comprises at least two, three, four, five, or six of the above-listed CDR sequences.

[0032] Preferably, the monoclonal antibody or fragment thereof comprises the CDR sequences CDR-L1: KASQDIYSSLS(SEQ ID No. 5) CDR-L2: RANRLIN (SEQ ID No. 6) and CDR-L3: LQYDDFPYM(SEQ ID No. 7) The light chain variable region comprises:

[0033] Preferably, the monoclonal antibody or fragment thereof has a light chain comprising framework sequences between the CDRs that are substantially identical or substantially similar to the framework sequences between the CDRs of the light chain sequence below (where the CDRs are shown in bold and underlined and the framework sequences are shown in italics): [ka]

[0034] Preferably, the monoclonal antibody or fragment thereof comprises the CDR sequences CDR-H1: TYGVH(SEQ ID No. 8) CDR-H2: AIWRGGSTDYNPAFMS (SEQ ID No. 9) and CDR-H3: RSTLFYFDY(SEQ ID No. 10) The heavy chain variable region comprises:

[0035] Preferably, the monoclonal antibody or fragment thereof has a heavy chain comprising framework sequences between the CDRs, said framework sequences being substantially identical or substantially similar to the framework sequences between the CDRs of the following heavy chain sequence (where the CDRs are indicated in bold and underlined and the framework sequences are indicated in italics): [ka]

[0036] 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. The similar or identical amino acids may be contiguous or non-contiguous.

[0037] The framework sequence may include one or more amino acid substitutions, insertions, and / or deletions. The amino acid substitutions may be conservative, meaning that the substituted amino acid has similar chemical properties as 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.

[0038] Programs such as the CLUSTAL program can be used to compare amino acid sequences. This program compares amino acid sequences by inserting spaces in either sequence as appropriate to find the optimal alignment. For optimal alignment it is possible to calculate amino acid identity or similarity (identity + conservation of amino acid type). Programs such as BLASTx align the longest stretch of similar sequences and assign a value to the match. It is thus 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.

[0039] In certain preferred embodiments, the monoclonal antibody or fragment thereof comprises the light chain variable region sequence: [ka] [ka]

[0040] In a third aspect, the present application relates to a monoclonal antibody according to the second aspect, Streptavidin-coated well plates, Biotinylated peptide Biotin-L-AARPGNVKGP (SEQ ID No. 15), where L is an optional linker; Secondary antibodies for use in sandwich immunoassays, a calibrator protein containing the C-terminal amino acid sequence AARPGNVKGP (SEQ ID No. 1); Antibody biotinylation kit, Antibody HRP Labeling Kit, Antibody radiolabeling kit, and Assay Visualization Kit At least one of The present invention relates to an immunoassay kit comprising:

[0041] The invention will now be described by way of example with reference to the following figures. [Brief description of the drawings]

[0042] [Figure 1]Figure 1 shows the specificity of the PRO-C22 ELISA. Dose-dependent inhibition of the biotinylated coating peptide (biotin-AARPGNVKGP (SEQ ID No. 15)) and the interaction of the monoclonal antibody with the assay specific for standard peptides of 30 (GPPGPPGQCDPSQCAYFASLAARPGNVKGP (SEQ ID No. 16)) and 10 (AARPGNVKGP (SEQ ID No. 1)) amino acid residues (AA), respectively. No inhibition was observed when the peptide sequence was truncated (AARPGNVKG (SEQ ID No. 4)) or extended (AARPGNVKGPA (SEQ ID No. 3)) from the C-terminus. The nonsense 30AA peptide PGMRGMPGSPGGPGSDGKPGPPGSQGESGR (SEQ ID No. 17) had no inhibitory effect and addition of the nonsense coating peptide PPGSQGESGR-biotin (SEQ ID No. 18) in combination with the assay-specific 30AA standard peptide had no effect on the interaction. Error bars represent standard PRO-C22 in cancer patient serum. [Diagram 2] FIG. 2 shows the quantification of PRO-C22 in serum of healthy controls (n=33) and bladder (n=20), breast (n=20), colorectal (n=20), head and neck (n=20), kidney (n=20), lung (n=20), melanoma (n=20), ovarian (n=20), pancreatic (n=20), prostate (n=20), and gastric cancer (n=20). PRO-C22 levels are presented individually and bars indicate mean values ​​as scatter plots. Samples measuring below the LLOQ were given the LLOQ value determined in the validation of PRO-C22. **** represents a value below 0.0001; *** is below 0.001; ** is below 0.01. [Diagram 3] Figure 3 shows PRO-C22 levels measured in serum from patients enrolled in the BIOPAC cohort. (Left) PRO-C22 levels were significantly higher in PDAC patients (n=39) compared to healthy controls (n=20). [Figure 4]FIG. 4 shows that high PRO-C22 (median: above 50th percentile) is significantly associated with poor overall survival in pancreatic cancer. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0043] Working Example The disclosed embodiments of the present invention 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 disclosure defined in the claims that follow herein below. The following examples are presented to provide those of ordinary skill 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 following experiments are complete or the most suitable experiments have been performed. An attempt has been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, 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 near atmospheric pressure.

[0044] material and method Generation of monoclonal antibodies targeting PRO-C22 A 10-amino acid peptide corresponding to the C-terminus of type XII collagen (UniprotKB:Q8NFW1) 1616 AARPGNVKGP 1626 (SEQ ID No. 1) was purchased from Genscript (Piscataway, NJ, USA) and used for immunization.

[0045] Six-week-old female Balb / C mice were subcutaneously injected with emulsified KLH-CGG conjugated immunogenic peptide (KLH-CGG-AARPGNVKGP (SEQ ID No. 19)), which was generated by covalently cross-linking the target peptide to keyhole limpet hemocyanin (KLH) carrier protein using sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, SMCC (Thermo Scientific, Waltham, MA, USA, cat. no. 22322). Glycine and cysteine ​​residues were added to the N-terminus to ensure correct linkage of the carrier protein. Immunizations using 200 μl of emulsified antigen containing 100 μg of immunogenic peptide mixed with Sigma Adjuvant System (Sigma cat. no. S6322) were performed at 2-week intervals until stable serum titer levels were achieved. The mouse with the highest titer was rested for 4 weeks and then boosted intravenously with 100 μg of immunogenic peptide in 100 μl of 0.9% NaCl solution and terminated.

[0046] Hybridoma cells were produced by fusing spleen cells with SP2 / 0 melanoma cells as previously described (Gefter, Margulies and Scharff, 1977). The resulting hybridoma cells were then cultured in 96-well microtiter plates and subcloning was performed during limiting dilution of the cells. Selected monoclonal cells were seeded in 24-well plates and then expanded in T25 flasks, then T75 flasks and finally in T150 flasks, after which the supernatant was harvested. Approximately 500 mL was harvested and purified using a protein G column according to the manufacturer's instructions (GE Healthcare Life Sciences, Little Chalfont, UK, cat. #17-0404-01).

[0047] The generated antibodies were sequenced and the CDRs were determined. Total RNA was isolated from hybridoma cells according to the technical manual of RNA-easy Isolation Reagent. Total RNA was then reverse transcribed into cDNA using isotype-specific antisense or universal primers according to the technical manual of SMARTScribe Reverse Transcriptase. VH and VL antibody fragments were amplified according to the standard operating procedure (SOP) of RACE (rapid amplification of cDNA ends) from GenScript. The amplified antibody fragments were cloned separately into standard cloning vectors. Colony PCR was performed to screen for clones with the correct size insert. Five or more colonies with the correct size insert were sequenced for each fragment. The sequences of the different clones were aligned to provide a consensus sequence of these clones.

[0048] The sequences of the chains are as follows (CDRs: bold; framework sequences: italics; constant regions: underlined): [ka]

[0049] [ka]

[0050] PRO-C22 ELISA Protocol The final PRO-C22 protocol was performed as follows: 96-well streptavidin-coated ELISA plates were coated with 100 μl / well of 20 ng / mL biotinylated AARPGNVKGP (Biotin-AARPGNVKGP (SEQ ID No. 15)) 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 five times with wash buffer (25 mM Tris, 50 mM NaCl, pH 7.2), 20 μL / well of sample was added in duplicate, followed by 100 μl / well of monoclonal antibody labeled with HRP at 35 ng / mL in assay buffer and incubated for 20 hours at 4° C. with shaking at 300 RPM. A second wash cycle was performed, followed by the addition of 100 μL / well of 3,3′,5,5′-tetramethylbenzidine (TMB) and incubation in the dark at 20° C. with shaking at 300 RPM. The reaction was stopped by adding 100 μl / well of 1% H2SO4. Absorbance was measured at 450 nm with 650 nm as reference. A standard curve was generated using 2-fold serial dilutions of 125 ng / mL GPPGPPGQCDPSQCAYFASLAARPGNVKGP (SEQ ID No. 16) standard peptide at 20 μl / well. The curve was fitted using a four-parameter logistic regression model. Each plate included two kit controls, peptide in assay buffer, and three quality control samples, including one human serum sample, one porcine serum sample, and one synovial fluid sample.

[0051] Technical validation of PRO-C22 ELISA The specificity of the antibodies was assessed by signal inhibition using two versions of the selected peptide, a 10 amino acid (AA) version AARPGNVKGP (SEQ ID No. 1) and a 30AA version (GPPGPPGQCDPSQCAYFASLAARPGNVKGP (SEQ ID No. 16)) and comparing these with a truncated version (AARPGNVKG (SEQ ID No. 4)) and an extended version (AARPGNVKGPA (SEQ ID No. 3)), testing a nonsense biotinylated peptide (PGMRGMPGSP-biotin (SEQ ID No. 22)) and a nonsense standard peptide (PGMRGMPGSPGGPGSDGKPGPPGSQGESGR (SEQ ID No. 17)).

[0052] The lower and upper limits of the measurement range (LLMR and ULMR), defined as the concentration limits of the linear range of the assay, were determined through 10 independent runs. From these runs, the inter- and intra-assay variations were determined based on 10 samples covering the measurement range of LLMR-ULMR. The 10 samples included three human serum samples, two peptides in assay buffer, in addition to two kit controls and three quality control samples. The intra-assay variation was calculated as the average coefficient of variation (CV%) within a plate. The inter-assay variation was calculated as the average CV% between plates. From the 10 runs, the upper limit of quantification (ULOQ) was determined as the highest acceptable standard point based on this recovery (RE%) and CV%. The criteria for acceptability were CV% ≤ 20% and RE% within 80-120%. The lower limit of quantification (LLOQ) was determined as the concentration at which the CV% was greater than 20% for the majority of samples, based on four human serum samples with low analyte concentrations measured in sextuplicate across five independent runs.

[0053] Linearity and parallelism were tested by serially diluting four healthy human serum samples two-fold and calculating the CV% through three independent runs. The RE% was calculated relative to the 1:2 diluted value. The acceptance criteria for CV% was ≦20% and the acceptance criteria for RE% was within 80-120%.

[0054] The accuracy was evaluated by spiking the 30AA standard peptide into three human serum samples and calculating the RE%. The acceptance criteria for the average RE% was 80-120%. The accuracy was further evaluated by mixing three sets of serum samples in different amounts (e.g., 25% serum A + 75% serum B, 50% serum A + 50% serum B, etc.) and calculating the RE% for each reference serum sample value. The RE% for each spike ratio was within 80-120%. The effect of the commonly interfering substances biotin, lipids, and hemoglobin was tested using three serum samples at different concentrations (biotin low: 5ng / mL, high: 100ng / mL; lipids low: 1.5mg / mL, high: 5mg / mL; hemoglobin: 10 concentrations ranging from 0.5mg / mL to 5.0mg / m, varying by 0.5mg / mL). Interference was evaluated by RE% and was allowed within the range of 80-120%.

[0055] Analyte stability was assessed by incubating triplicate serum sample aliquots at 4°C and 20°C for 2, 4, 24, or 48 hours. The RE% was calculated relative to a control serum sample that was thawed after analysis, and the RE% was accepted within 80-120%. Stability was further assessed through repeated freeze-thaw cycles of serum samples: up to 5 rounds of freeze-thaw cycles. Freeze-thaw stability was calculated based on the RE% for samples that underwent a single thaw prior to analysis, and was accepted if the RE% was within 80-120%. The technical stability of the assay was assessed by performing the assay in triplicate using kit reagents incubated at 20°C for 24 hours, and by calculating the RE% of 10 samples measured using reagents from frozen storage, where the acceptance criteria for RE% was within 80-120%.

[0056] Patient samples The first cohort included 223 cancer samples and 33 healthy samples. It included 20 patients with pancreatic, colorectal, renal, gastric, breast, bladder, lung, melanoma, head and neck, prostate, and ovarian cancer, respectively, and 33 age-matched healthy controls. All cancer samples were obtained from Proteogenex (Los Angeles, CA, USA) and healthy controls were obtained from BioIVT (Westbury, NY, USA). A summary of the characteristics of the first cohort can be found in Table 1.

[0057] [Table 1]

[0058] The second cohort included 20 healthy donors and 39 patients with invasive pancreatic ductal adenocarcinoma (PDAC). All patients were from the Danish BIOPAC study "BIOmarkers in patients with Pancreatic Cancer" (NCT03311776). Patients were recruited from six Danish hospitals between December 2008 and September 2017. PC patients had histologically confirmed tumors. PC patients were treated with different types of chemotherapy according to national guidelines (www.gicancer.dk). The study was performed in accordance with the recommendations of the Danish Regional Committee on Health Research Ethics. The BIOPAC protocol was approved by the Danish Regional Committee on Health Research Ethics (VEK ref. KA-20060113) and the Data Protection Authority (j.nr. 2006-41-6848). Blood samples were obtained at the time of diagnosis or prior to intervention. All subjects provided written informed consent in accordance with the Declaration of Helsinki.

[0059] [Table 2]

[0060] statistics Comparisons of PRO-C22 levels were performed using the Kruskal-Wallis test followed by Dunn's multiple comparison test, comparing each solid tumor type with a group of healthy controls. This was followed by testing the diagnostic accuracy of PRO-C22 by area under the receiver operating characteristic curve (AUROC). Using the BIOPAC cohort, the association with overall survival (OS) was evaluated using the median as the cut point to define the group with high PRO-C22 levels. Kaplan-Meier curve analysis and univariate Cox regression analysis were used to evaluate the association between PRO-C22 and OS. Asterisks represent the following levels of significance: **p<0.01; ***p<0.001; ****p<0.0001.

[0061] result Development of ELISA for PRO-C22 Throughout the ELISA development process, optimization steps were performed to find the appropriate assay buffer, incubation time, and temperature, as well as antibody and peptide concentrations, that provided the best competitive conditions for the assay. The competitive immunoassay PRO-C22 quantified the amount of COL22 in serum samples using a horseradish peroxidase-labeled mAb (HRP-mAb). The assay contained a standard peptide (GPPGPPGQCDPSQCAYFASLAARPGNVKGP (SEQ ID No. 16)), which by competing away quantified the amount of circulating COL22 in serum using the interaction between the HRP-mAb and a biotinylated version of the peptide (biotin-AARPGNVKGP (SEQ ID No. 15)). Specificity was tested using a truncated (AARPGNVKG (SEQ ID No. 4)) and extended (AARPGNVKGPA (SEQ ID No. 3)) version of the peptide, which showed no competition with the HRP-mAb. The Nansen-Coater (PGMRGMPGSP-Biotin (SEQ ID No. 22)) and the nonsense 30AA standard peptide (PGMRGMPGSPGGPGSDGKPGPPGSQGESGR (SEQ ID No. 17)) were also evaluated for their ability to disrupt competition, but they did not show any effect (Figure 1). In contrast, the 10AA sequence-selected peptide (AARPGNVKGP (SEQ ID No. 1)) and the 30AA sequence standard peptide (GPPGPPGQCDPSQCAYFASLAARPGNVKGP (SEQ ID No. 16)) successfully inhibited the interaction in a dose-dependent manner (Figure 1).

[0062] The range of linear measurement was determined to be 1.0-53.0 ng / mL, and the LLOQ and ULOQ were determined to be 1.95 and 125.0 ng / mL, respectively. The precision of the assay was determined through intra- and inter-variation studies. Both intra- and inter-variation were determined to be 3.6%. The MRD (minimum required dilution) of serum was assessed by linearity of healthy serum samples diluted 2-fold in the assay. This determined 1:2 (1+1) as the MRD, and the dilution was limited to 1:4 (1+3) for healthy serum samples. This was based on an average RE% of 103.7%.

[0063] Accuracy was determined by spiking recovery test, where the average RE% of peptides in serum was 105.1% and the average RE% of serum in serum was 103.5%. The substrates biotin, lipids, and hemoglobin were tested for interference at different concentrations. A low concentration of biotin of 5 ng / ml was found to have an average RE% of 99.1 in three different samples, and a high concentration of 100 ng / ml had an average RE% of 85.5%. A low lipid concentration of 1.5 mg / mL had an average RE% of 104.6% and a high concentration of 5 mg / mL had an average RE% of 102.3% in three serum samples. The interference cutoff for hemoglobin was determined using concentrations ranging from 0.50 mg / mL to 5.0 mg / mL of hemoglobin in steps of 0.5 mg / mL. At 3.0 mg / mL, the mean RE% of the three serum samples was 126.6%, which exceeded the acceptable range of RE% (80-120%), so the cutoff was determined to be a concentration of 2.5 mg / mL based on a mean RE% of 112.5%.

[0064] Stability was evaluated both with respect to the analyte and the kit reagent stability. The average RE% of three serum samples undergoing five freeze-thaw cycles was 103.1%. Serum samples incubated at 4°C for up to 48 hours had an RE% of 107.2% and 106.1% for samples incubated at 20°C for up to 48 hours. Kit reagents (buffer, biotinylated peptide, and HRP-mAb) were evaluated after 24 hours of incubation at 20°C. Reagent stability was evaluated by measuring the same 10 samples used within and between variations and calculating the RE%. The 10 samples measured in three individual runs showed an average RE% of 94.4%.

[0065] An overview of the technical variations is given in Table 3.

[0066] [Table 3]

[0067] PRO-C22 in the serum of cancer patients In cohort 1, PRO-C22 was significantly higher in all solid tumor types compared to controls (Figure 2). This suggests that circulating levels of PRO-C22, i.e., COL22, are higher in serum from patients suffering from various types of solid tumors. The area under the receiver operating characteristic curve (AUROC) was used to evaluate the ability of PRO-C22 to distinguish cancer patients from healthy controls (Table 4).

[0068] [Table 4]

[0069] The range of AUROC varied from 0.886-0.976, which indicates that PRO-C22 distinguishes cancer patients from healthy controls well (AUROC >0.8). PRO-C22 was validated to be elevated in pancreatic cancer patients in the second (BIOPAC) cohort (Figure 3). When assessing and correlating with overall survival (OS), high PRO-C22 (median: >50th percentile) was significantly associated with poor OS in pancreatic cancer with a hazard ratio of 3.987 (95%CI: 1.724-8.806) compared to patients with low levels. Similarly, the median OS time was 162 days in patients with high PRO-C22 and 1363 days in patients with low PRO-C22 (log-rank P-value = 0.0011).

[0070] conclusion In this study, an ELISA for quantifying the presence of collagen XXII in blood was successfully developed, optimized and validated. PRO-C22 ELISA is technically robust, accurate and sensitive. The level of circulating collagen XXII can be evaluated in the serum of cancer patients and healthy controls, where the level of PRO-C22 was significantly higher in all cancers tested compared to healthy controls. The level of PRO-C22 can be used to distinguish cancer patients and healthy controls, and the level of PRO-C22 can also be used to provide prognosis for pancreatic cancer overall survival.

[0071] In this specification, unless expressly stated otherwise, the word "or" is used in its operator sense, selecting a truth value when either or both of the stated conditions are met, as opposed to the operator "exclusive or", which requires that only one of the conditions be met. The word "comprising" is used to mean "including or consisting of". All prior art acknowledged above is hereby incorporated by reference. All published prior art in this specification should not be deemed an admission that it is the common general knowledge in Australia or anywhere else at the date of this specification.

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[12] T. Watanabe et al., “A Human Skin Model Recapitulates Systemic Sclerosis Dermal Fibrosis and Identifies COL22A1 as a TGFβ Early Response Gene that Mediates Fibroblast to Myofibroblast Transition,” Genes (Basel)., vol. 10, no. 2, p. 75, Jan. 2019, doi: 10.3390 / genes10020075.

[13] R. Bauer et al., “Inhibition of Collagen XVI Expression Reduces Glioma Cell Invasiveness,” Cell. Physiol. Biochem., vol. 27, no. 3‐4, pp. 217‐226, 2011, doi: 10.1159 / 000327947.

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Claims

1. 1. An immunoassay method for detecting and / or monitoring cancer in a patient, comprising: i) contacting a sample from a patient with a monoclonal antibody that specifically binds to the C-terminal amino acid sequence AARPGNVKGP (SEQ ID NO: 1); ii) detecting and determining the amount of binding between said monoclonal antibody and peptide in said sample; A method comprising:

2. iii) correlating the amount of binding of said monoclonal antibody determined in step ii) with values ​​associated with normal healthy subjects, and / or with values ​​associated with known disease severity, and / or with values ​​obtained from said patient at previous time points, and / or with a predetermined cut-off value; The method of claim 1 further comprising:

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

4. The method of claim 1 , wherein the cancer is pancreatic cancer.

5. 5. The method of claim 4, further comprising the step of providing a prognosis of overall survival, wherein an amount of binding of the monoclonal antibody determined in step ii) that exceeds the median associated with known pancreatic cancer patients is associated with poor overall survival.

6. The method of any one of claims 1 to 5, wherein the monoclonal antibody does not specifically bind to an extended version of the C-terminal amino acid sequence AARPGNVKGPA (SEQ ID NO: 3).

7. The method of any one of claims 1 to 5, wherein the monoclonal antibody does not specifically bind to a truncated version of the C-terminal amino acid sequence AARPGNVKG (SEQ ID NO: 4).

8. The method of any one of claims 1 to 5, wherein the monoclonal antibody is raised against a synthetic peptide having the C-terminal amino acid sequence AARPGNVKGP (SEQ ID NO: 1).

9. The method according to any one of claims 1 to 5, wherein the sample is a biological fluid sample selected from blood, serum, or plasma.

10. The method according to any one of claims 1 to 5, wherein the immunoassay is a competitive assay or a sandwich assay.

11. The method according to any one of claims 1 to 5, wherein the immunoassay is a radioimmunoassay or an enzyme-linked immunosorbent assay.

12. a monoclonal antibody that specifically binds to the C-terminal amino acid sequence AARPGNVKGP (SEQ ID NO: 1); Streptavidin-coated well plates, Biotinylated peptide, where L is an optional linker: biotin-L-AARPGNVKGP (SEQ ID NO: 15); Secondary antibodies for use in sandwich immunoassays, a calibrator protein comprising the C-terminal amino acid sequence AARPGNVKGP (SEQ ID NO: 1); antibody biotinylation kit, Antibody HRP labeling kit, Antibody radiolabeling kit, and Assay Visualization Kit and at least one of: An immunoassay kit comprising:

13. 13. The immunoassay kit of claim 12, wherein the monoclonal antibody does not specifically bind to an extended version of the C-terminal amino acid sequence of ARPGNVKGPA (SEQ ID NO: 3).

14. 13. The immunoassay kit of claim 12, wherein the monoclonal antibody does not specifically bind to a truncated version of the C-terminal amino acid sequence AARPGNVKG (SEQ ID NO: 4).

15. The kit of any one of claims 12 to 14, wherein the monoclonal antibody is raised against a synthetic peptide having the C-terminal amino acid sequence AARPGNVKGP (SEQ ID NO: 1).

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

17. 17. The monoclonal antibody of claim 16, wherein the monoclonal antibody does not specifically bind to an extended version of the C-terminal amino acid sequence ARPGNVKGPA (SEQ ID NO: 3).

18. 17. The monoclonal antibody of claim 16, wherein the monoclonal antibody does not specifically bind to a truncated version of the C-terminal amino acid sequence AARPGNVKG (SEQ ID NO: 4).

19. The monoclonal antibody of any one of claims 16 to 18, wherein the monoclonal antibody is raised against a synthetic peptide having the C-terminal amino acid sequence AARPGNVKGP (SEQ ID NO: 1).

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