Tumstatin Assay

Abstract

The present invention relates to an assay for detecting Tumstatin, and its use in evaluating lung cancers, such as non-small cell lung cancer (NSCLC), chronic kidney disease (CKD), such as CKD resulting from diabetes, lupus nephritis (LN) and systemic lupus erythematosus (SLE).

Claims

1. A monoclonal antibody specifically reactive with an N-terminus amino acid sequence PGLKGKRGDS (SEQ ID NO: 1).

2. The monoclonal antibody as claimed in claim 1, wherein the monoclonal antibody does not specifically recognise or bind an N-extended elongated version of said N-terminus amino acid sequence or an N-truncated shortened version of said N-terminus amino acid sequence.

3. An immunoassay method for quantifying peptides having an N-terminus amino acid sequence PGLKGKRGDS (SEQ ID NO: 1) in a patient biofluid sample, said method comprising contacting said patient biofluid sample with the monoclonal antibody of claim 1 and determining the amount of binding between said monoclonal antibody and said N-terminus amino acid sequence.

4. The immunoassay method of claim 3, wherein the monoclonal antibody does not specifically recognise or bind an N-extended elongated version of said N-terminus amino acid sequence or an N-truncated shortened version of said N-terminus amino acid sequence.

5. The immunoassay method of claim 3, wherein the patient biofluid sample is blood, urine, synovial fluid, serum or plasma.

6. A method of immunoassay for detecting lung cancer in a patient, the method comprising contacting a patient biofluid sample with the monoclonal antibody of claim 1, determining the amount of binding between said monoclonal antibody and peptides comprising said N-terminus amino acid sequence, and correlating said amount of binding with i) values associated with normal healthy subjects and/or ii) values associated with known lung cancer severity and/or iii) values obtained from said patient at a previous time point and/or iv) a predetermined cut-off value.

7. The method as claimed in claim 6, wherein the lung cancer is non-small cell lung cancer (NSCLC).

8. The method as claimed in claim 6, wherein the predetermined cut-off value is at least 2.00 ng/mL.

9. The method of claim 6, wherein the monoclonal antibody does not specifically recognise or bind an N-extended elongated version of said N-terminus amino acid sequence or an N-truncated shortened version of said N-terminus amino acid sequence.

10. The method of claim 6, wherein the patient biofluid sample is blood, urine, synovial fluid, serum or plasma.

11. A method of immunoassay for detecting chronic kidney disease (CKD) in a patient, the method comprising contacting a patient biofluid sample with the monoclonal antibody of claim 1, determining the amount of binding between said monoclonal antibody and peptides comprising said N-terminus amino acid sequence, and correlating said amount of binding with values associated with normal healthy subjects and/or values associated with known CKD severity and/or values obtained from said patient at a previous time point and/or a predetermined cut-off value.

12. The method as claimed in claim 11, wherein the chronic kidney disease is chronic kidney disease resulting from systemic lupus erythematosus, lupus nephritis or diabetes.

13. The method of claim 11, wherein the monoclonal antibody does not specifically recognise or bind an N-extended elongated version of said N-terminus amino acid sequence or an N-truncated shortened version of said N-terminus amino acid sequence.

14. The method of claim 11, wherein the patient biofluid sample is blood, urine, synovial fluid, serum or plasma.

15. A method of immunoassay for detecting systemic lupus erythematosus (SLE) or lupus nephritis (LN) in a patient, the method comprising contacting a patient biofluid sample with the monoclonal antibody of claim 1, determining the amount of binding between said monoclonal antibody and peptides comprising said N-terminus amino acid sequence, and correlating said amount of binding with values associated with normal healthy subjects and/or values associated with known SLE or LN severity and/or values obtained from said patient at a previous time point and/or a predetermined cut-off value.

16. The method of claim 15, wherein the monoclonal antibody does not specifically recognise or bind an N-extended elongated version of said N-terminus amino acid sequence or an N-truncated shortened version of said N-terminus amino acid sequence.

17. The method of claim 15, wherein the patient biofluid sample is blood, urine, synovial fluid, serum or plasma.

18. An assay kit comprising a monoclonal antibody specifically reactive with an N-terminus amino acid sequence PGLKGKRGDS (SEQ ID NO: 1), and at least one of: a streptavidin coated well plate; a biotinylated peptide PGLKGKRGDS-L-Biotin (SEQ ID NO: 17), wherein L is an optional linker; a secondary antibody for use in a sandwich immunoassay; a calibrator peptide comprising the sequence PGLKGKRGDS (SEQ ID NO: 1); an antibody biotinylation kit; an antibody HRP labeling kit; an antibody radiolabeling kit; and an assay visualization kit.

19. The assay kit as claimed in claim 18, wherein the monoclonal antibody is raised against a synthetic peptide having the amino acid sequence PGLKGKRGDS (SEQ ID NO: 1).

20. The assay kit as claimed in claim 18, wherein the monoclonal antibody does not specifically recognise or bind an N-extended elongated version of said N-terminus amino acid sequence or an N-truncated shortened version of said N-terminus amino acid sequence.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0046] FIG. 1 is a TUM ELISA showing a typical standard curve and native reactivity against human serum and human urine. The standard peptide was 2-fold diluted starting from 20 ng/mL. The samples were run from undiluted and up to 8-fold dilution as indicated.

[0047] FIG. 2 shows assay specificity. Reactivity to the standard peptide (PGLKGKRGDS; SEQ ID NO: 1), the elongated peptide (LPGLKGKRGDS; SEQ ID NO: 2), the truncated peptide (GLKGKRGDS; SEQ ID NO: 3) and a non-sense peptide (LRSKSKKFRR; SEQ ID NO: 4) was tested in the TUM assay.

[0048] FIG. 3 shows results from Cohort 1. Serum TUM levels were assessed in healthy controls (n=8), patients with IPF (n=7), COPD (n=8) and NSCLC (n=8). Data was analyzed using a Kruskal-Wallis test adjusted for Dunn's multiple comparisons test. Data are presented as Tukey box plots. Significance levels: *: p<0.05 and **: p<0.001.

[0049] FIG. 4 shows the results from cohort 2. Serum TUM levels were assessed in healthy controls (n=20) and NSCLC (n=40). Data was analyzed using a Mann Whitney t-test. Data are presented as Tukey box plots. Significance levels: *: p<0.05.

[0050] FIGS. 5A-5B show levels of TUM in urine (ng/mg Creatinine) (FIG. 5A) and serum (ng/ml) (FIG. 5B) samples from healthy individuals and from patients with lupus nephritis (LN). **p<0.01.

[0051] FIG. 6 shows levels of TUM in serum samples (ng/ml) from healthy individuals and patients with systemic lupus erythematosus (SLE).

[0052] FIG. 7 shows levels of TUM in urine samples in a rat model of diabetic kidney disease.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

[0053] As used herein the term “N-terminus” refers to the extremity of a polypeptide, i.e. at the N-terminal end of the polypeptide, and is not to be construed as meaning in the general direction thereof.

[0054] As used herein the term “monoclonal antibody” refers to both whole antibodies and to fragments thereof that retain the binding specificity of the whole antibody, such as for example a Fab fragment, F(ab′)2 fragment, single chain Fv fragment, or other such fragments known to those skilled in the art. As is well known, whole antibodies typically have a “Y-shaped” structure of two identical pairs of polypeptide chains, each pair made up of one “light” and one “heavy” chain. The N-terminal regions of each light chain and heavy chain contain the variable region, while the C-terminal portions of each of the heavy and light chains make up the constant region. The variable region comprises three complementarity determining regions (CDRs), which are primarily responsible for antigen recognition. The constant region allows the antibody to recruit cells and molecules of the immune system. Antibody fragments retaining binding specificity comprise at least the CDRs and sufficient parts of the rest of the variable region to retain said binding specificity.

[0055] In the present invention, the monoclonal antibody may comprise any constant region known in the art. Human constant light chains are classified as kappa and lambda light chains. Heavy constant chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. The IgG isotype has several subclasses, including, but not limited to IgGI, IgG2, IgG3, and IgG4. The monoclonal antibody may preferably be of the IgG isotype, including any one of IgGI, IgG2, IgG3 or IgG4.

[0056] The CDR of an antibody can be determined using methods known in the art such as that described by Kabat et al.sup.19. Antibodies can be generated from B cell clones as described in the examples. The isotype of the antibody can be determined by ELISA specific for human IgM, IgG or IgA isotype, or human IgG1, IgG2, IgG3 or IgG4 subclasses. The amino acid sequence of the antibodies generated 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 which amplify the heavy and light chains of the antibody. For example primers specific for the leader sequence for all VH (variable heavy chain) sequences can be used together with primers that bind to a sequence located in the constant region of the isotype which has been previously determined. The light chain can be amplified using primers which bind to the 3′ end of the Kappa or Lamda chain together with primers which anneal to the V kappa or V lambda leader sequence. The full length heavy and light chains can be generated and sequenced.

[0057] 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 antibodies linked to an enzyme, such as horseradish peroxidase or alkaline phosphatase. The activity of the enzyme is then assessed by incubation with a substrate generating a measurable product. The presence and/or amount of target peptide in a sample can thereby be detected and/or quantified. ELISA is a technique known to those skilled in the art.

[0058] As used herein the term, the term “competitive ELISA” refers to a competitive enzyme-linked immunosorbent assay In a “competitive ELISA” the target peptide present in a sample (if any) competes with known amount of target of peptide (which for example is bound to a fixed substrate or is labelled) for to binding an antibody, and is a technique known to the person skilled in the art.

[0059] 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 the person skilled in the art.

[0060] As used herein the term “amount of binding” refers to the quantification of binding between antibody and biomarker, which said quantification is determined by comparing the measured values of biomarker in the biofluid samples against a calibration curve, wherein the calibration curve is produced using standard samples of known concentration of the biomarker. In the specific assay disclosed herein which measures in biofluids the N-terminus biomarker having the N-terminus amino acid sequence PGLKGKRGDS (SEQ ID NO: 1), the calibration curve is produced using standard samples of known concentration of the calibration peptide PGLKGKRGDS (SEQ ID NO: 1). The values measured in the biofluid samples are compared to the calibration curve to determine the actual quantity of biomarker in the sample. The present invention utilises spectrophotometric analysis to both produce the standard curve and measure the amount of binding in the biofluid samples; in the Examples set out below the method utilises HRP and TMB to produce a measurable colour intensity which is proportional to the amount of binding and which can be read by the spectrophotometer. Of course, any suitable analytical method could also be used.

[0061] As used herein the “cut-off value” means an amount of binding that is determined statistically to be indicative of a high likelihood of a disease (e.g. lung cancer, such as NSCLC, or chronic kidney disease, systemic lupus erythematosus, or lupus nephritis), in a patient, in that a measured value of biomarker in a patient sample that is at or above the statistical cutoff value corresponds to at least a 70% probability, preferably at least an 80% probability, preferably at least an 85% probability, more preferably at least a 90% probability, and most preferably at least a 95% probability of the presence or likelihood of a disease (e.g. lung cancer, such as NSCLC, or chronic kidney disease, systemic lupus erythematosus, or lupus nephritis).

[0062] As used herein the term “values associated with normal healthy subjects and/or values associated with known disease severity” means standardised quantities of Tumstatin determined by the method described supra for subjects considered to be healthy, i.e. without a disease (e.g. without lung cancer, such as NSCLC, or chronic kidney disease, systemic lupus erythematosus, or lupus nephritis), and/or standardised quantities of Tumstatin determined by the method described supra for subjects known to have a disease (e.g. lung cancer, such as NSCLC, or chronic kidney disease, systemic lupus erythematosus, or lupus nephritis), of a known severity.

[0063] As used herein, “TUM ELISA” refers to the specific competitive ELISA disclosed herein which quantifies in a sample the amount peptides having the N-terminus amino acid sequence PGLKGKRGDS (SEQ ID NO:1).

Examples

[0064] The presently disclosed embodiments are described in the following Examples, which are set forth to aid in the understanding of the disclosure, and should not be construed to limit in any way the scope of the disclosure as defined in the claims which follow thereafter. The following examples are put forth so as 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 present disclosure nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

[0065] In the following examples, the following materials and methods were employed.

Materials and Methods

[0066] All reagents used for the experiments were high quality standards from companies such as Sigma Aldrich (St. Louis, Mo., USA) and Merck (Whitehouse Station, N.J., USA). The synthetic peptides used for immunization and assay development were purchased from the Genscript (New Jersey, USA).

Generation of Monoclonal Antibodies

[0067] The amino acid sequence 1426′PGLKGKRGDS′1436 (SEQ ID NO: 1) is located in the α3 chain of type IV collagen. This sequence is generated towards human tumstatin, and has a mismatch in amino acid (AA) position 6 in rats and a mismatch in AA position 5 in mice. Immunization was initiated by subcutaneous injection of 200 μL emulsified antigen and 50 ug immunogenic peptide (PGLKGKRGDS-GGC-KLH; SEQ ID NO: 5) in 4-6 weeks old Balb/C mice using Freund's incomplete adjuvant. The immunizations were repeated every 2.sup.nd week until stable serum antibody titer levels were reached. The mouse with the highest serum titer was selected for fusion and rested for a month. Subsequently, the mouse was boosted intravenously with 50 μg immunogenic peptide in 100 μL 0.9% NaCl solution three days before isolation of the spleen for cell fusion. To produce hybridoma cells, the mouse spleen cells were fused with SP2/0 myeloma cells as described by Gefter et al. The hybridoma cells were cloned in culture dishes using the semi-solid medium method. The clones were then plated into 96-well microtiter plates for further growth, and the limiting dilution method was applied to promote monoclonal growth. Indirect ELISA performed on streptavidin-coated plates was used for the screening of supernatant reactivity. PGLKGKRGDS-K-Biotin (SEQ ID NO:6) was used as the screening peptide, while the standard peptide PGLKGKRGDS (SEQ ID NO:1) was used for further test of specificity of clones. Supernatant was collected from the hybridoma cells, and purified using HiTrap affinity columns GE Healthcare Life Science, Little Chalfront, Buckinghamshire, UK) according to manufacturer's instructions. The production of monoclonal antibodies performed in mice was approved by the National Authority (The Animal Experiments Inspectorate) under approval number 2013-15-2934-00956. All animals were treated according to the guidelines for animal welfare.

Clone Characterization

[0068] Native reactivity and peptide affinity for the standard peptide were assessed using human serum and human urine purchased from a commercial supplier (Valley Biomedical, VA 22602, USA). Antibody specificity was tested in a preliminary assay using truncated (GLKGKRGDS; SEQ ID NO:3) and elongated peptides (LPGLKGKRGDS; SEQ ID NO:2). The isotype of the monoclonal antibody was determined using the Clonotyping System-HRP kit, cat. 5300-05 (Southern Biotech, Birmingham, Ala., USA).

TUM ELISA

[0069] The TUM competitive ELISA procedure was as follows: 96-well streptavidin-coated ELISA plates (Roche, cat. 11940279) were coated with 10 ng/mL biotinylated peptide PGLKGKRGDS-K-Biotin (SEQ ID NO:6) dissolved in assay buffer (25 mM Tris-BTB 2 g. NaCl/L, pH 8.0, 100 μL/well) and incubated for 30 min at 20° C. in the dark with 300 rpm shaking. Plates were washed five times in washing buffer (20 mM TRIS, 50 mM NaCl, pH 7.2). Subsequently, 20 μL of standard peptide or sample were added to appropriate wells, followed by 100 μL of 7 ng/mL horseradish peroxidase (HRP) labeled monoclonal antibody solution. The plates were incubated for 1 hour at 20° C. with shaking, and subsequently washed in washing buffer. Finally, 100 μL 3,3′,5,5-tetramethylbenzinidine (TMB) (Kem-En-Tec cat. 4380H) was added, and incubated for 15 min at 20° C. To stop the enzyme reaction of TMB, 100 μL of stopping solution (1% H.sub.2SO.sub.4) was added. The plate was analyzed by an ELISA reader at 450 nm with 650 nm as reference (Molecular Devices, VersaMax, CA, USA). A standard curve was performed by serial dilution of the standard peptide and plotted using a 4-parametric mathematical fit model. Standard concentrations were 0, 0.3125, 0.625, 1.25, 2.5, 5, 10, and 20 ng/mL. Each plate included five kit controls to monitor inter-assay variation. All samples were measured within the range of the assay, and all samples below lower limit of measurement range (LLMR) were reported as the value of LLMR.

Technical Evaluation

[0070] A twofold dilution of four human serum and human urine samples was used to assess the linearity. The linearity was calculated as a percentage of recovery of the undiluted sample.

[0071] Antibody specificity was calculated as percentage of signal inhibition by 2-fold diluted standard peptide (PGLKGKRGDS; SEQ ID NO:1), elongated peptide (LPGLKGKRGDS; SEQ ID NO:2), truncated peptide (GLKGKRGDS; SEQ ID NO:3) and non-sense peptide (LRSKSKKFRR; SEQ ID NO:4). The lower limit of detection (LLOD) was estimated from 21 determinations of the lowest standard (buffer). LLOD was calculated as mean−3*standard deviation (SD). Upper limit of detection (ULOD) was determined as the mean±3*SD of 10 measurements of Standard A. The intra- and inter-assay variation was determined by 10 independent runs of five quality control (QC) and two kit controls run in double determinations. Accuracy of the assay was measured in healthy human serum/urine samples spiked with standard peptide and a serum/urine sample with a known high Tumstatin concentration, and calculated as the percentage recovery of serum/urine in buffer. Following, spiking recovery was determined by calculating the percentage recovery of spiked serum in buffer. Interference was measured in healthy human serum spiked with either biotin (low=30 ng/ml, high=90 ng/ml), hemoglobin (low=0.155 mM, high=0.310 mM), or lipids (low=4.83 mM, high=10.98 mM). The interference was calculated as the percentage recovery of the analyte in non-spiked serum.

[0072] Furthermore, a human anti-mouse antibody (HAMA) panel was used to study the interference. Five healthy human serum samples were added to the HAMA panel. These were analyzed with and without 5% Liq II in the dilution buffer. Salt interference was tested by measuring salt samples with a concentration of 8.14 g/L NaCl at pH 7.0 and 8.0. To define the standard concentration of Tumstatin, the normal range was determined by analyzing 32 healthy human serum samples in relation to age and gender of the sample donors. Lower limit of measurement range (LLMR) and upper limit of measurement range (ULMR) was calculated based on the 10 individual standard curves from the intra- and inter-assay variation. The analyte stability was determined for three healthy human serum samples which were incubated at either 4 or 20° C. for 2, 4 and 24 hours respectively. The stability of the samples was evaluated by calculating the percentage variation in proportion to the sample kept at −20° C. (0 hour sample). Furthermore, the analyte stability was determined for three healthy human serum samples, exposed to four freeze and thaw cycles. To assess the stability of the analyte, the percentage of recovery was calculated of a sample undergone only one freeze/thaw cycle.

Biological Validation of TUM as a Biomarker for Lung Cancer

[0073] TUM was measured in serum samples from two different cohorts. Both cohorts were obtained from the commercial vendor Proteogenex (Culver City, Calif., USA). Cohort 1 included patients diagnosed with IPF, COPD, non-small cell lung cancer (NSCLC) and colonoscopy-negative controls with no symptomatic or chronic disease. Patient demographics are shown in Table 1. Cohort 2 included patients diagnosed with NSCLC in cancer stage I, II, III and IV together with colonscopy-negative controls with no symptomatric or chronic disease. Patient demographics of this cohort can be found in Table 2.

[0074] Table 1 contains the patient demographics of cohort 1. Data is presented as mean (SD) unless otherwise stated. Comparison of age, gender and BMI was performed using Kruskal-Wallis adjusted for Dunn's multiple comparisons test, while comparison of FEV.sub.1% of predicted value and FEV.sub.1/FVC ratio % were calculated using the Mann-Whitney unpaired t-test. P-values below 0.05 were considered significant. Abbreviations: BMI; body mass index; IPF; idiopathic pulmonary fibrosis, COPD; chronic obstructive pulmonary disease, FEV1, forced expiratory volume in one second, FVC, forced vital capacity.

TABLE-US-00007 TABLE 1 Patient demographics of cohort 1 Healthy controls IPF COPD NSCLC (n = 8) (n = 7) (n = 8) (n = 8) p-value Age 54.88 74.13  75.38 60.50 <0.001 (7.85) (8.36) (1.69) (9.32) Male, 6 (75%) 4 (57%) 4 (50%) 7 (87.5%) 0.102 n (%) BMI 26.25 25.79  27.24 N/A 0.170 (1.27) (1.58) (1.84) FEV.sub.1% of — 64.38 61.5 — 0.634 predicted (3.42) (7.19) value FEV.sub.1/FVC — 76.00  58.38 — 0.016 ratio % (1.51) (15.20)

[0075] Table 2 contains the patient demographics of cohort 2. Data is presented as mean (SD) unless otherwise stated. Comparison of age, gender and BMI was performed using a Mann-Whitney t-test. P-values below 0.05 were considered significant. Abbreviations: BMI; body mass index.

TABLE-US-00008 TABLE 2 Patient demographics of cohort 2 Healthy controls NSCLC (n = 20) (n = 40) p-value Age 61.85 (1.95) 61.93 (2.14) 0.593 Male, n (%) 10 (50%) 20 (50%) 1.000 BMI 26.14 (2.67) 25.55 (4.23) 0.533

Statistical Analysis

[0076] Levels of TUM in serum samples was compared using Kruskal-Wallis adjusted for Dunn's multiple comparisons test (non-paramteric data). Results are presented as Mean±Standard Error of Mean (SEM).

[0077] The diagnostic power of TUM was investigated by an area under the receiver operating characteristics (AUROC) curve. Statistical analysis and graphs were performed using GraphPad Prism version 7 (GraphPad Software, Inc., CA, USA).

Biological Validation-TUM as a Biomarker for CKD, SLE and LN

[0078] TUM was measured in two different patient cohorts. Cohort 1 (18 patients) included individuals with lupus nephritis (LN) and healthy controls, with TUM levels being measured in both serum and urine samples. Cohort 2 (126 patients) included individuals with systemic lupus erythematosus (SLE) and healthy controls, with TUM levels being measured in serum samples only. The patient demographics for Cohort 1 are shown below in Table 3.

TABLE-US-00009 TABLE 3 Cohort No: 1:18 patients with lupus nephritis (serum and urine) N or mean Min-Max Gender (male) 4 Age 40 17-62 eGFR 87  17.4-165.0 Proteinuria 2.8   0-13.9 (g/day)

[0079] Additionally, TUM was measured in a rat model of diabetic kidney disease. Sprague-Dawley rats (n=8) were injected with streptozocin (STZ) in the tail vein to induce diabetes, and the rats were considered diabetic if their blood glucose was stable above 15 mmol L-1 after 48 hours. After 2 weeks, STZ-treated rats underwent ischemic reperfusion injury (IRI). Control rats (n=7) received a sham operation. Urine samples were taken from the rats at days 0, 1, 5 and 8 after the operation (IRI or sham), and the levels of TUM in the urine samples were measured.

Results

Clone Characterization

[0080] The best antibody producing hybridomas were screened for reactivity towards the standard peptide and native material in the competitive ELISA. Based on the reactivity, the clone NBH134 #102-3GF was chosen for assay developed and determined to be the IgG1 subtype. Native reactivity was observed in human serum and urine (FIG. 1), while no reactivity was found towards the elongated peptide, truncated peptide, non-sense standard peptide and non-sense coater (FIG. 2).

Technical Evaluation of the TUM ELISA Assay

[0081] A series of technical validations were performed to evaluate the TUM ELISA assay. A summary of the validation data can be found in Table 4. The measurement range (LLMR-ULMR) of the assay was determined to 0.26-9.92 ng/mL. The inter- and intra-variation was 8.04% and 10.96% respectively. Linearity of the human samples was observed from undiluted to 1:4 for human serum, and undiluted to 1:2 for human urine. Spiking recover tog standard peptide in human serum, and human serum in human serum resulted in a mean recovery of 90% and 99%, respectively. Neither hemoglobin, lipids nor biotin interfered with measurements of the TUM analyte in human serum. The stability of the analyte was acceptable during both prolonged storage of human serum samples at 4° C. and 20° C. (102.4% and 80.1%) and during freeze/thaw cycles (80.8%).

TABLE-US-00010 TABLE 4 Technical validation data of the TUM ELISA assay Technical validation test TUM IC50 1.6 ng/mL Detection range 0.26-9.92 ng/mL Intra-assay variation.sup.1 8.04% Inter-assay variation.sup.1 10.96%  Dilution recovery in human serum.sup.1   89% Dilution recovery in human urine.sup.1   98% Analyte recovery 24 h, 4° C./20° C..sup.1 102.4%/80.1%  Hemoglobin recovery, low/high.sup.1 100%/100% Lipemia recovery, low/high.sup.1 100%/100% Biotin recovery, low/high.sup.1 120%/106% Salt recovery, pH 6.0/pH 7.0/pH 8.0.sup.2   97% Spiking recovery (peptide in serum).sup.1   90% Spiking recovery (serum in serum).sup.1   99% Analyte recovery, 3 freeze/thaw cycles.sup.1 80.8% .sup.1Percentages are reported as mean, .sup.2Average recovery after salt interference.

Biological Evaluation— TUM as a Biomarker for Lung Cancer

[0082] TUM was measured in two different cohorts, cohort 1 and cohort 2. Cohort 1 consists of healthy controls and patients diagnosed with IPF, COPD and NSCLC, and the results are shown in FIG. 3. Results from cohort 1 showed that TUM was significantly elevated in serum from NSCLC compared to healthy controls, IPF patients and COPD patients (p=0.007, p=0.03 and p=0.001 respectively). No significant difference was observed between healthy controls, IPF patients and COPD patients, which indicated that TUM may play a role in NSCLC, but not fibrotic lung disorders.

[0083] In cohort 2, TUM was measured in samples from healthy controls, and patients with NSCLC as shown in FIG. 4. Here TUM was significantly upregulated in patients with NSCLC compared to healthy controls (p=0.002). There was no significant difference between the different cancer stages.

[0084] The AUROC was used to evaluate the discriminative power of TUM in relation to NSCLC and healthy controls. As shown in Table 5, TUM was able to discriminate between NSCLC patients and healthy controls in cohort 1 with an AUROC of 0.97, NSCLC patients and IPF patients with an AUROC 0.98 and NSCLC and COPD patients with an AUROC of 1.00. In cohort 2, TUM was able to identify NSCLC patients from healthy controls with an AUROC 0.73. These findings indicate that TUM levels are able to separate healthy controls from patients with NSCLC with a high diagnostic accuracy.

TABLE-US-00011 TABLE 5 Discriminative performance of TUM in healthy controls and NSCLC Cut-off value AUROC (ng/mL) Sensitivity Specificity (95% Cl) p-value Cohort 1 1.97 100 87.5 0.97 0.002 NSCLC vs. (0.89-1.05) healthy controls NSCLC vs. IPF 3.67 87.5 100 0.98 <0.0001 (0.75-1.00) NSCLC vs. 2.37 100 100 1.00 <0.0001 COPD (0.79-1.00) Cohort 2 1.27 100 44 0.73 0.003 NSCLC vs. (0.60-0.84) healthy controls NSCLC stage IV 1.27 100 60 0.87 0.001 vs. healthy (0.73-1.01) controls NSCLC: non-small cell lung cancer, IPF: idiopathic pulmonary fibrosis, COPD: chronic obstructive pulmonary disease.

Biological Validation-TUM as a Biomarker for CKD, SLE and LN

[0085] TUM was measured in two different patient cohorts; cohort 1 and cohort 2. In cohort 1, TUM was measured in serum and urine samples from healthy controls and patients with lupus nephritis (LN), and the results are shown in FIGS. 5B and 5A, respectively. In cohort 2, TUM was measured in serum samples from healthy controls and patients with systemic lupus erythematosus (SLE, and the results are shown in FIG. 6. Levels of TUM were found to be up to 2-fold upregulated in serum of patients with systemic lupus erythematosus (SLE) and lupus nephritis (LN) and 10-fold upregulated in urine of patients with LN.

[0086] TUM was also measured in a rat model of diabetic kidney disease, the results of which are shown in FIG. 7. Levels of TUM in the urine were found to be more elevated in diabetic rats (type 1 diabetes) compared to controls, and increased over time in the diabetic rats, with a peak 5 days after ischemic reperfusion injury (IRI).

Conclusions

[0087] A novel competitive ELISA using a monoclonal antibody for detecting tumstatin has been developed (herein referred to as “TUM ELISA”). The assay was technically robust and specific towards the amino acid sequence PGLKGKRGDS (SEQ ID NO:1). The TUM fragment was detectable in human serum and urine, and was found to be significantly elevated in patients with NSCLC, compared to IPF, COPD and healthy controls; significantly elevated in patients with SLE or LN, compared to healthy controls; and significantly elevated in a rat model of diabetic kidney disease.

[0088] As shown herein, the TUM ELISA has a diagnostic potential within diagnosis of lung cancers, particularly NSCLC, and can separate these patients from patients with lung fibrosis. Based on the high diagnostic accuracy, this could be a biomarker of BM remodeling in lung cancer. Likewise, it has been shown herein that the TUM ELISA has a diagnostic potential within diagnosis of systemic lupus erythematosus (SLE), lupus nephritis (LN), and chronic kidney disease, particularly resulting from diabetes, SLE or LN.

[0089] In this specification, unless expressly otherwise indicated, the word ‘or’ is used in the sense of an operator that returns a true value when either or both of the stated conditions is met, as opposed to the operator ‘exclusive or’ which requires that only one of the conditions is met. The word ‘comprising’ is used in the sense of ‘including’ rather than in to mean ‘consisting of’. All prior teachings acknowledged above are hereby incorporated by reference.

REFERENCES

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