Patent Application
20230052142
The contents of the electronic sequence D7755CIPSEQ.xml with a size of 17 kb and created on Aug. 1, 2022 is hereby incorporated by reference in its entirety.
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).
The basement membrane (BM) is a specialized extracellular matrix (ECM), which functions as a scaffold for epithelial and endothelial cells, and acts as a barrier between tissues (1,2). Two of the main BM proteins are collagen type IV and laminin, which together form a distinct network linked together by nidogen and heparin sulphate proteoglycans (2-5). Collagen type IV has six different α-chains, α1-6, which form the heterotrimers expressed in the mammalian BMs(6). The α3 chain of collagen type IV (COL4α3), has been described to have restricted distribution across BMs and is generally found in the lungs and kidneys (7). The structural role of COL4α3 is illustrated by clinical manifestations of Alport's syndrome, Goodpasture's syndrome, and several autoimmune disease targeting the lungs and kidneys' BM. These diseases are characterized by damage to COL4α3 by either mutations or immune attacks which cause leakage of the BM (8-11). The BM serves as a barrier for cell invasion. Breaching of the BM and loss of BM integrity are associated with an invasive cancer phenotype (12). Cancer biomarkers associated with breach and disorganization of the BM are therefore needed.
Tumstatin (TUM) is a 28-kDa fragment of COL4α3 that binds to endothelial cells via the αvβ3 integrin (13). It is a matrikine generated by matrix metalloproteinase-9 (MMP-9), and it is known to keep pathological angiogenesis and tumour growth in check (13-15). MMP-9 is needed to cleave tumstatin from COL4α3 so that tumstatin can function as a protective matrikine. Lack of MMP-9 accelerates tumour growth in MMP-9 knockout mice. High levels of COL4α3 mRNA were associated with a poor prognosis in patients with lung cancer (15,16). Several studies have speculated that matrikines may be potential biomarkers with therapeutical potential (10, 17-19).
Luo et al. (20) developed a sandwich ELISA for the quantification of COL4α3/tumstatin in human serum and tissue extracts. However, no significant difference was found in patients with lung carcinoma without metastatic disease compared to healthy controls, with the only relevant finding being a decreased level of COL4α3/tumstatin in patients with metastatic lung carcinoma compared to patients without metastatic lung carcinoma. Thus, whilst the ELISA of Luo may be able to quantify COL4α3/tumstatin in human serum and tissue extracts, the diagnostic utility of that assay is shown to be somewhat limited.
The present inventors have now developed a tumstatin assay that demonstrates excellent diagnostic utility.
Thus, in a first aspect the present invention relates to 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 a monoclonal antibody specifically reactive with said N-terminus amino acid sequence PGLKGKRGDS (SEQ ID NO:1), and determining the amount of binding between said monoclonal antibody and said N-terminus amino acid sequence.
Preferably, 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. In this regard “N-extended elongated version of said N-terminus amino acid sequence” means one or more amino acids extending beyond the N-terminus of the sequence H2N-PGLKGKRGDS (SEQ ID NO: 1). For example, if the N-terminal amino acid sequence H2N-PGLKGKRGDS (SEQ ID NO: 1) was elongated by a leucine residue then the corresponding “N-extended elongated version” would be H2N-LPGLKGKRGDS(SEQ ID NO: 2). Similarly, “N-truncated shortened version of said N-terminus amino acid sequence” means one or more amino acids removed from the N-terminus of the sequence H2N-PGLKGKRGDS(SEQ ID NO: 1). For example, if the N-terminal amino acid sequence H2N-PGLKGKRGDS (SEQ ID NO: 1) was shortened by one amino acid residue then the corresponding “N-truncated shortened version” would be H2N-GLKGKRGDS (SEQ ID NO: 3).
Monoclonal antibodies that specifically bind to the N-terminus amino acid sequence PGLKGKRGDS (SEQ ID NO: 1) can be generated via any suitable techniques known in the art. For example, the monoclonal antibody may be raised against a synthetic peptide having the amino acid sequence PGLKGKRGDS (SEQ ID NO: 1), such as for example by: immunizing a rodent (or other suitable mammal) with a synthetic peptide consisting of the sequence PGLKGKRGDS (SEQ ID NO: 1), which optionally may linked to an immunogenic carrier protein (such as keyhole limpet hemocyanin), isolating and cloning a single antibody producing cell, and assaying the resulting monoclonal antibodies to ensure that they have the desired specificity. An exemplary protocol for producing a monoclonal antibody that that specifically bind to the N-terminus amino acid sequence PGLKGKRGDS (SEQ ID NO: 1) is described infra.
Preferably, the monoclonal antibody or fragment thereof may preferably comprise one or more complementarity-determining regions (CDRs) selected from:
Preferably the antibody or fragment thereof comprises at least 2, 3, 4, 5 or 6 of the above listed CDR sequences.
Preferably the monoclonal antibody or fragment thereof has a light chain variable region comprising the CDR sequences
Preferably the monoclonal antibody or fragment thereof has a light chain that comprises framework sequences between the CDRs, wherein said framework sequences are substantially identical or substantially similar to the framework sequences between the CDRs in the light chain sequence below (in which the CDRs are shown in bold and underlined, and the framework sequences are shown in italics):
ISSQNLVYSNGDTYLE
WYLLKPGQSPKLLIY
KVSNRFS
GVPDRFSGSGSG
TDFTLKISRVEAEDLGIYYC
FQGSHYPYT.
Preferably the monoclonal antibody or fragment thereof has a heavy chain variable region comprising the CDR sequences
Preferably the monoclonal antibody or fragment thereof has a heavy chain that comprises framework sequences between the CDRs, wherein said framework sequences are substantially identical or substantially similar to the framework sequences between the CDRs in the heavy chain sequence below (in which the CDRs are shown in bold and underlined, and the framework sequences are shown in italics):
NYWMH
WILQRPGQGLEWIG
EIDPKNGHSNYNEKFKN
KASLTVDIYSSTAY
MQLSSLRSEDSAVYYCTR
DDYYGAL
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.
The framework sequences may contain one or more amino acid substitutions, insertions and/or deletions. Amino acid substitutions may be conservative, by which it is meant the substituted amino acid has similar chemical properties to the original amino acid. A skilled person would 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.
A program such as the CLUSTAL program to can be used to compare amino acid sequences. This program compares amino acid sequences and finds the optimal alignment by inserting spaces in either sequence as appropriate. It is possible to calculate amino acid identity or similarity (identity plus conservation of amino acid type) for an optimal alignment. A program like BLASTx will align the longest stretch of similar sequences and assign a value to the fit. It is thus possible to obtain a comparison where several regions of similarity are found, each having 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 sequences.
In certain preferred embodiments, the monoclonal antibody or fragment thereof may comprise the light chain variable region sequence:
DVLLTQSPVSLPVSLGDPASISC
ISSQNLVYSNGDTYLE
WYLLKPGQSPK
LLIY
KVSNRFS
GVPDRFSGSGSGTDFTLKISRVEAEDLGIYYC
FQGSHYP
YT
FGGGTKLEIK (CDRs bold and underlined; Framework
QAQLQQPGAELVKPGASVKLSCKTYGYTFT
NYWMH
WILQRPGQGLEWIG
E
IDPKNGHSNYNEKFKN
K
ASLTVDIYSSTAYMQLSSLRSEDSAVYYCTR
DD
YYGAL
WGQGTLVTVSA (CDRs bold and underlined;
In a second aspect, the present invention relates to a method of immunoassay for detecting lung cancer in a patient, the method comprising contacting a patient biofluid sample with a monoclonal antibody specifically reactive with an N-terminus amino acid sequence PGLKGKRGDS (SEQ ID NO: 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. The lung cancer may be, but is not limited to, non-small cell lung cancer (NSCLC).
The predetermined cut-off value may be at least 2.00 ng/mL, preferably at least 2.30 ng/mL, more preferably at least 2.60 ng/mL, and most preferably at least 3.00 ng/mL. In this regard, through the combined use of various statistical analyses it has been found that a measured amount of binding between the monoclonal antibody (described above) and the N-terminus biomarker of at least 2.00 ng/mL or greater may be determinative of the presence of lung cancer, such as NSCLC. By having a statistical cutoff value of at least 2.00 ng/mL, preferably at least 2.30 ng/mL, more preferably at least 2.60 ng/mL, and most preferably at least 3.00 ng/mL, it is possible to utilise the method of the invention to diagnose lung cancer with a high level of confidence. Or, in other words, applying the statistical cutoff value to the method of the invention is particularly advantageous as it results in a standalone diagnostic assay; i.e. it removes the need for any direct comparisons with healthy individuals and/or patients with known disease severity in order to arrive at a diagnostic conclusion. This may also be particularly advantageous when utilising the assay to evaluate patients that already have medical signs or symptoms that are generally indicative of lung cancer (e.g. as determined by a physical examination and/or consultation with a medical professional) as it may act as a quick and definitive tool for corroborating the initial prognosis and thus potentially remove the need for more invasive procedures, such as endoscopy and/or biopsy, and expedite the commencement of a suitable treatment regimen. In the particular case of lung cancer, an expedited conclusive diagnosis may result in the disease being detected at an earlier stage, which may in turn improve overall chances of survival.
Preferably, the monoclonal antibody used in the above method 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.
If the patient is determined to have lung cancer as the amount of binding detected is determinative of the presence of lung cancer, then the method may further comprise the step of treating the patient. This may involve administering to the patient suitable treatment for lung cancer. Treatments for lung cancer include surgery, immunotherapy such as pembrolizumab or atezolizumab, cryotherapy, radiofrequenct ablation, photodynamic therapy, radiotherapy, chemotherapy, chemoradiotherapy, EGFR inhibitors such as gefitinib, afatanib, erlotinib, dacomitinib, osmertinib; ALK inhibitors such as alectinib, crizotinib, ceritinib, and brigatinib; and nintedanib with or without docetaxel.
In a third aspect, the present invention relates to a method of immunoassay for detecting chronic kidney disease (CKD) in a patient, the method comprising contacting a patient biofluid sample with a monoclonal antibody specifically reactive with an N-terminus amino acid sequence PGLKGKRGDS (SEQ ID NO: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 CKD severity and/or iii) values obtained from said patient at a previous time point and/or iv) a predetermined cut-off value.
The CKD may be, but is not limited to, CKD resulting from systemic lupus erythematosus (SLE), lupus nephritis (LN) or diabetes.
The predetermined cut-off value may be at least 2.00 ng/mL, preferably at least 2.30 ng/mL, more preferably at least 2.60 ng/mL, and most preferably at least 3.00 ng/m L.
Preferably, 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.
If the patient is determined to have chronic kidney disease as the amount of binding detected is determinative of the presence of chronic kidney disease, then the method may further comprise the step of treating the patient. This may involve administering to the patient suitable treatment for chronic kidney disease. Treatments for chronic kidney disease include dialysis, diuretics such as furosemide and kidney transplantation. Treatment can also involve controlling associated conditions such as high blood pressure and high cholesterol by administering medicaments including angiotensin-converting enzyme (ACE) inhibitors such as ramipril, enalapril and lisinopril, statins such as atorvastatin and simvastatin.
In a fourth aspect, the present invention relates to 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 a monoclonal antibody specifically reactive with an N-terminus amino acid sequence PGLKGKRGDS (SEQ ID NO: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 SLE or LN severity and/or iii) values obtained from said patient at a previous time point and/or iv) a predetermined cut-off value.
The predetermined cut-off value may be at least 2.00 ng/mL, preferably at least 2.30 ng/mL, more preferably at least 2.60 ng/mL, and most preferably at least 3.00 ng/m L.
Preferably, 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.
If the patient is determined to have systemic lupus erythematosus (SLE) or lupus nephritis (LN) as the amount of binding detected is determinative of the presence of SLE or LN, then the method may further comprise the step of treating the patient. This may involve administering to the patient suitable treatment for SLE or LN. Treatments for SLE include belimumab, rituximab, hydroxychloroquine, methotrexate, immunosuppressants including immunosuppressive, steroid-sparing drugs such as azathioprine, mycophenolate mofetil, calcineurin inhibitors (ciclosporin and tacrolimus); corticosteroids such as methylprednisolone and prednisolone, Treatments for LN include medicaments known to control blood pressure such as angiotensin-converting enzyme (ACE) inhibitors and angiotensin II receptor blockers (ARBs); immunosuppressive medicines such as cyclophosphamide, azathioprine and mycophenolate (MMF); steroids such as prednisolone; Cyclosporine; Tacrolimus; Cyclophosphamide; Azathioprine (Imuran); Mycophenolate (CellCept); Rituximab (Rituxan); Belimumab (Benlysta); dialysis; and kidney transplant.
In all of the above described methods according to any of the first to fourth aspects of the invention, the patient biofluid sample may be, but is not limited to, blood, urine, synovial fluid, serum or plasma. In certain preferred embodiments the biofluid sample may be urine or serum. In methods of immunoassay for detecting chronic kidney disease (CKD), systemic lupus erythematosus (SLE) or lupus nephritis (LN), it may in particular be preferred that the biofluid sample is urine.
In a fifth aspect, the present invention relates to a monoclonal antibody specifically reactive with an N-terminus amino acid sequence PGLKGKRGDS (SEQ ID NO:1).
Preferably, 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.
In a sixth aspect, the present invention relates to 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:
Preferably, 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. The monoclonal antibody described supra and/or included in the assay kit may be raised against a synthetic peptide having the amino acid sequence PGLKGKRGDS (SEQ ID NO:1).
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.
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.
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.
The CDR of an antibody can be determined using methods known in the art such as that described by Kabat et al19. 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.
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.
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.
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.
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.
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).
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.
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).
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.
In the following examples, the following materials and methods were employed.
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).
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 2nd 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.
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).
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% H2SO4) 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.
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.
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.
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.
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.
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 FEV1% of predicted value and FEV1/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 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.
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).
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).
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.
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.
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 (
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%).
1Percentages are reported as mean,
2Average recovery after salt interference.
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
In cohort 2, TUM was measured in samples from healthy controls, and patients with NSCLC as shown in
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.
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
TUM was also measured in a rat model of diabetic kidney disease, the results of which are shown in
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.
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.
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.
doi:10.1016/S1535-6108(03)00133-8.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1721387.7 | Dec 2017 | GB | national |
This is a continuation-in-part application under 35 U.S.C. § 120 of pending application U.S. Ser. No. 16/956,369, filed Jun. 19, 2020, which is a national stage application under 35 U.S.C. § 371 of International Application PCT/EP2018/085855, filed Dec. 19, 2018, now abandoned, which claimed priority to European Application No: 1721387.7, filed Dec. 20, 2017, now abandoned.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16956369 | Jun 2020 | US |
| Child | 17933113 | US |
