Patent Application
20230340092
A sequence listing is electronically submitted in text format in compliance with 37 C.F.R. § 1.821(c) and is incorporated by reference herein. The ASCII text file is named D7697CSEQ, was created on Dec. 21, 2022 and is 9 KB in size.
The present invention relates to a method for detecting the short isoform of Collagen type XVIII, and the use of said method in evaluating haemophilic diseases.
Recurrent haemarthroses due to vascular ruptures is a major complication in haemophilia, contributing to progressive joint damage, which leads to haemophilic (HF) arthropathy. The medical need in the HF field to reduce bleeding incidents requires measurement of the annual bleeding rate (ABR) in haemophiliacs. Although a crude measure, the ABR is associated with HF arthropathy (1) but is also a key parameter in clinical trials, ensuring quantifiable benefits to patients (2-4). Endothelial cell impairment and matrix quality may be associated with joint bleeds and later the development of HF arthropathy.
Vascular rupture is associated with the quality and turnover of the basement membrane (BM) located directly underneath the endothelial cells. Extracellular matrix turnover is a central pathological feature in many diseases due to epithelial or endothelial cell damage. While the endothelial cell function is debated, no quantifiable methods are available for specifically quantifying the damage to the vascular endothelium, which, subsequent to bleeding, results in exposure of the BM underlying the endothelial cells.
Quantifying the BM proteins specific to the endothelial cells may therefore have particular relevance to endothelial cell stability and rupture in haemophiliacs.
Collagen IV, XV ad XVIII represent the most well-known collagens of the vascular BMs, responsible for maintaining vessel wall structure and integrity of the membrane (FIG. A) (5-7).
Type XVIII collagen exists in three isoforms: short, intermediate, and long, localized in various basement membrane zones (4-6) (
Mutations in type XVIII collagen have also been lined to the autosomal recessive disorder noblock syndrome (KS). KS is characterized by various eye defects leading to blindness at a young age (11, 12). Morever, col18a/−/− knock-out mice showed delayed regression of blood vessels in the vitreous along the surface of the retina, impaired angiogenesis of retinal vessels and altered iris BM structure (8, 13-16). Thus, collagen XVIII is essential for controlling blood vessel formation in the eye, and possibly an important component in the BM zones of the entire vascular system (17).
The present Applicant is unaware of any biomarkers and/or antibodies specific to the short isotype of type XVIII collagen. The majority of commercially available antibodies recognize the C-terminal endostatin end of type XVIII collagen hence it is not possible to differentiate between the three isotypes using those antibodies.
Thus, there is a nedd for antibodies and/or biomarkers measuring the specific short isotype of type XVIII collagen and excluding the two other isoforms to quantify vascular specific basement membrane turnover in terms of detection of endothelial type XVIII collagen content.
The Applicant has now developed an assay for detecting the short isoform of collagen type XVIII, and has used that assay to assess the clinical relevance of turnover of collagen type XVIII in patients diagnosed with HF arthropathy.
In a first aspect, the invention relates to an antibody specifically reactive with short isoform collagen type XVIII, wherein said antibody does not react with intermediate isoform collagen type XVIII or with long isoform collagen type XVIII. Preferably, the antibody is specifically reactive with an N-terminal epitope of short isoform collagen type XVIII. Preferably, the Np534minal epitope is an epitope that is exposed after cleavage and removal of the N-terminal signal peptide of short isoform collagen type XVIII. Preferably, the antibody is specifically reactive with an N-terminal epitope comprised in the N-terminal amino acid sequence of H2N-EPERISEEVG . . . (SEQ ID NO: 1). Preferably, the antibody is specifically reactive with an N-terminal epitope comprising the N-terminal amino acid sequence H2N-EPERIS . . . (SEQ ID NO: 2).
Preferably, the antibody does not specifically recognise or bind an N-extended elongated version of said N-terminal amino acid sequence which is H2N-AEPERISEEVG . . . (SEQ ID NO: 3) and/or does not specifically recognise or bind an N-truncated version of said N-terminal amino acid sequence which is H2N-PERISEEVG . . . (SEQ ID NO: 4).
The antibody may be a monoclonal or polyclonal antibody. Preferably, the antibody is a monoclaonal antibody.
In a second aspect, the invention relates to a method of immunoassay for detecting or quantitating in a sample short isoform collagen type XVIII, wherein said method comprises contacting a sample comprising said short isoform collagen type XVIII with an antibody as described supra, and determining the amount of binding of said antibody.
The present invention may be directed to a method of detecting short isoform collagen type XVIII in a human patient, said method comprising:
Preferably, the method comprises detecting or quantitating an N-terminal epitope of short isoform collagen type XVIII. The N-terminal epitope is preferably comprised in the N-terminal amino acid sequence H2N-EPERISEEVG . . . (SEQ ID NO: 1). Preferably, the N-terminal epitope comprises the N-terminal amino acid sequence H2N-EPERIS . . . (SEQ ID NO: 2).
Preferably, the sample is a biofluid. The biofluid may be, but is not limited to, serum, plasma, urine, cerebrospinal fluid, or amniotic fluid.
The immunoassay may be a competition assay or a sandwich assay. The immunoassay may be a radioimmunoassay or an enzyme-linked immunosorbent assay.
The method may further comprise correlating the quantity of said short isoform collagen type XVIII determined by said method with standard haemophilic disease samples of known disease severity to evaluate the severity of a haemophilic disease.
Alternatively, or in addition to, the method may comprise comparing the quantity of said short isoform collagen type XVIII determined by said method with standard values associated with healthy subjects to evaluate the presence and/or severity of a haemophilic disease. In this regard “standard values associated with healthy subjects” means standardised quantities of short isoform collagen type XVIII determined by the method described supra for subjects considered to be healthy, i.e., without a haemophilic disease. The standardisation will depend on the height, weight, gender, etc. of the healthy subject.
Alternatively, or in addition to, the method may further comprise quantifying the amount of collagen type XVIII in at least two samples obtained from a subject at a first time point and at at least one subsequent time point, wherein an increase in the quantity of collagen type XVIII from the first time point to the at least one subsequent time point is indicative of a deterioration in a haemophilic disease from the first time point to the at least one subsequent time point, or wherein a decrease in the quantity of collagen type XVIII from the first time point to the at least one subsequent time point is indicative of an improvement in a haemophilic disease from the first time point to the at least one subsequent time point.
The haemophilic disease may be haemophilic arthropathy. The method described supra may also be used to evaluate Knoblock syndrome.
In another aspect, the present invention is directed to a method for evaluating the efficacy of a drug for treating a haemophilic disease. The method comprises using the method as described above to quantify the amount of collagen type XVIII in at least two biological samples obtained from a subject at a first time point and at at least one subsequent time point during a period of administration of the drug to the subject. A reduction in the quantity of collagen type XVIII from the first time point to the at least one subsequent timpe point during the period of administration of the drug is indicative of an efficacious drug for treating a haemophilic disease.
In a final aspect, the invention relates to an assay kit for determining the quantity of short isoform collagen type XVIII, comprising an antibody as described supra and at least one of:
As used herein, the term “N-terminal epitope” refers to an N-terminal peptide sequence at 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 “competitive ELISA” refers to a competitive enzyme-linked immunosorbent assay 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 “short isoform of collagen type XVIII” refers to the isoform of collagen type XVIII generated by promoter 1 wherein the N-terminal non-collagenous region includes the thrombospondin-1 like domain (TSP-1), but does not contain the Domain of the Unknown Function (DUF) or the Frizzled Domain (FZ). The term “intermediate isoform of collagen type XVIII” refers to the alternatively spliced isoform of collagen type XVIII generated by promoter 2 wherein the N-terminal non-collagenous region includes the TsP-1 and DUF, but does not comprise the FZ. The term “long isoform of collagen type SVIII” refers to the alternatively spliced isoform of collagen type XVIII generated by promoter 2 wherein the N-terminal non-collagenous region includes the TSP-1, the DUF and the FZ.
As used herein, the term “COL-18N” is used as shorthand to describe the herein disclosed specific assay for the N-terminal sequence EPERISEEVG (SEQ ID NO: 1) of the short isoform of collagen type XVIII.
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, temperatures, 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.
Serum was collected from 35 male HF patients aged 26 and over. This cut-off age was chosen as collagen turnover wears off at the closure of the growth plate at the age of approximately 25 years (21). The patients had a treatment history of either on-demand medication upon bleeding episodes or intake of a low dosage of prophylaxis of 5-19 IU/kg recombinant FVIII, 203 times/week. Patients had varying degrees of HF arthropathy defined by the World Federation of Haemophilia Physical Examination Score (Gilbert Score) and by radiologic evaluation according to the Pettersson score. The patients' average ABR was 18.1 ranging from 2-46. Exclusion criteria were bleeding disorders other than haemophilia, human immunodeficiency virus infection, chronic obstructive pulmonary disease, medical history of joint disease or liver fibrosis, and treatment with anti-inflammatory biologics or steroids. Study participants were enrolled at the Department of Haematology, Peking Union Medical College Hospital, Beijing, China. The study was approved by the Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Ethics Review Board, with the serial number S-720. Signed informed consent was obtained from all subjects.
A peptide corresponding to the first 10 amino acids of the N-terminal epitope of the short isoform of human type XVIII collagen α1 chain (excluding signal peptide, 34′EPERISEEVG′43) was used to generate monoclonal neo-epitope specific antibodies. Beijing Administration Office of Laboratory Animal and animal ethics committee of NOrdic Bioscience approved the animal work. Generation of monoclonal antibodies was initiated by subcutaneous immunization of 6-8 week old Balb/C mice using 200 μL emulsified Freund's complete adjuvant with 60 μg peptide conjugated to keyhole limpet hemocyanin (KLH). Consecutive immunizations were performed at 2-week intervals in Freund's incomplete adjuvant, until stable titer levels were reached. The mouse was boosted intravenously with 50 μg imunogen in 100 μL 0.9% sodium chloride solution and three days later the spleen cells were fused with SP2/0 myeloma cells (LGC Standards AB, Boras, Sweden) (21). They hybridomas were grown in 96-well plates and monoclonal growth was ensured by limited dilution. Clones were screened against the specific epitope (EPERISEEVG; SEQ ID NO: 1), elongated peptide (AEPERISEEVG; SEQ ID NO; 3) and truncated peptide (PERISEEVG; SEQ ID NO: 4). The mAb producing clone, NB632-13H11/G5, was selected based on reactivity to above-mentioned peptides, and antibody purified using Protein G columns (GE Healthcare, Hilleroed, Denmark).
The competitive COI-18N ELISA was performed as follows. A 96-well streptavidin-coated plate (Roche cat.: 19940279) was coated with 100 μl/well 1.25 ng/mL biotinylated synthetic peptide EPERISEEVG-K-Biotin (SEQ ID NO: 7) dissolved in coating buffer (20 mM Na2HPO4, 3.7 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, 0.1% Tween20, 1% BSA, pH 7.4) and incubated 30 min at 20° C. The plate was washed five times with washing buffer (20 mM Tris, 50 mM NaCl, pH 7.2). 20 μL of the standard peptide (EPERISEEVG; SEQ ID NO: 6) or samples diluted in incubation buffer (20 mM Na2HPO4, 3.7 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, 0.1% Tween20, 1% BSA, 5% Liquid II pH 7.4) were added to appropriate wells, followed by 100 μL/well monoclonal antibody NB632-13H11/G5, and incubated for 1 hr at 20° C. After washing, 100 μl rabbit-anti-mouse antibody (Jackson, 315-935-045) was added 1:3000 dissolved in coating buffer and incubated 1 hr at 20° C., 300 rpm. After final five times was, the wells were incubated with 100 μL tetramethylbenzidine (TMB) (Kem-En-Tec cat. 4380H) at 20° C., 300 rpm in the dark for 15 min., followed by the addition of 100 μL/well-stopping solution (1% H2SO4). The colorimetric reaction was measured at 450 nm with 650 nm as reference, and a calibration curve was plotted using a 4-parametric mathematical fit model.
Technical assay validation was performed according to international guidelines. The lower limit of detection (LLOD) was calculated as mean+3× standard deviation (SD) determined from 21 zero samples (i.e., the assay buffer). The upper limit of detection (ULOD) was determined as the mean−3×SD of 10 measurements of standard A (1000 ng/ml). The lower limit of quantification (LLOQ) was determined by the lowest possible concentration with an imprecision of less than 30%. The intra- and inter-assay variations were calculated as the mean of the variation of seven human samples by 10 independent runs in duplicates. Dilution recovery was determined in a 2-fold dilution of two human serum and three human citrate plasma, calculated as percentage recovery of diluted matrices compared to undiluted ones. Spiking recovery was assessed in human serum and citrate plasma spiked with standard peptide at concentrations covering the entire measure range or by combining two samples of similar concentration in order to double the concentration. Spiking recovery was calculated as the measured amount percentage recovery of the theoretical amount. Interference by hemoglobin, lipemia, biotin, and human antibodies against most antigens by human anti-mouse antibody (HAMA) was determined by adding two-fold dilutions to a serum sample of known concentration. Concentrations started at 0.500 mmol/l hemoglobin, 0.56 mmol/l lipemia, 160 μg/l biotin and 2010 ng/ml HAMA. Recovery percentage was calculated with the normal serum sample as a reference value. Analyte stability was determined for two healthy human serum samples and one healthy citrate plasma sample for four freeze-thaw cycles and calculated as the percentage recovery of the first freeze-thaw cycle. Same samples were tested at 2 hrs, 4 hrs and 24 hrs at 4° C. and 20° C. against non-stressed analytes. Finally, antibody specificity was assessed by a sanity check testing reactivity towards standard (EPERISEEVG; SEQ ID NO: 1), elongated (AEPERISEEVG; SEQ ID NO: 3), truncated (PERISEEVG; SEQ ID NO: 4) and de-selection peptides EPQIDEKKK; SEQ ID NO: 8) and CPERALERR (SEQ ID NO: 9)).
Correlations between serum COL-18N concentration and ABR were analyzed using Spearman rank correlation coefficient with GraphPad Prism v6 (GraphPad Software, La Jolla, CA, USA). Differences were considered statistically significant if p<0.05.
A novel competitive ELISA using a monoclonal antibody to detect COL-18N in human serum and plasma (citrate, EDT, heparin) samples was developed and evaluated.
The main findings were:
A competitive COL-18N ELISA that can assess endothelial BM degradation was developed. The technical performance of the ELISA is summarized in table 1, providing a measurement range from 4.8-671 ng/ml, intra- and inter-variability at 7% and 13% respectively, dilution and spike recovery within 100±20%, and analytic stability with no immunoassay interference. The normal concentration of COL-18N in serum (16.6 ng/ml), plasma citrate (12.5 ng/ml), EDTA plasma (13.2 ng/ml), and heparin plasma (15.8 ng/ml) was consistent regardless of matrices.
The NB632-13H11/G5 antibody specially recognized the first 10 amino acids of N-terminus type XVIII collagen α1 chain short isoform (selection) (
The technical evaluation of the competitive COI-18N ELISA revealed a stable sensitive assay with high specificity towards the N-terminus of vascular form of type XVIII collagen including high accuracy and precision of the assay.
COL-18N correlates to annual bleeding rate in HF patients. The haemorrhagic disorder haemophilia manifests clinically by repeated haemarthrosis resulting in unavoidable arthropathy in the absence of adequate treatment. One cardinal feature of medicinal intervention in haemophilia is lowering of ABR, albeit objective quantifiable parameters with high resolution are lacking.
The bleeding severity of haemophilia is generally inversely proportional to the degree of FVIII/IX activity in the plasma, although substantial variability in bleeding tendencies is well-known. Reduced spontaneous bleeding and lower requirements of factor concentrates are reported in a subset of 10-15% of severe HF patients (23,24). Also, development of inhibitors in non-severe HF patients may heighten the bleeding phenotype considerably (25).
Bleeding phenotype may be further compromised by large discrepancies amongst the FVIII assays caused by standardization of the assays (26) and may even be influenced by the type of FVIII concentrates used during therapy (27,28). Other assays, like thrombin generation, correlates to the bleeding phenotype in HF patients (29), but is inconsistent in HF patients with FVIII inhibitors despite the occurrence of thrombin generation (30).
In haemophilia, consequent to i) endothelial cell damage ii) bleeding and iii) delayed clotting and wound healing, the endothelial remodelling contributing to clinical symptoms of haemophilia and pathophysiological disease representation may be affected. It has now been found using the herein described assay that vascular endothelial type XVIII collagen correlates with ABR in HF patients (
In summary, the data combined suggests that the technically robust COL-18N biomarker can be related to pathologies involving vascular BM degradation and remodelling, which affects degradation of the short isoform of type XVIII collagen. In addition, the data enables the COL-18N biomarker to evaluate ABR for optimal treatment and monitoring of patients to prevent the development of arthropathy.
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 ‘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. No acknowledgement of any prior published document herein should be taken to be an admission or representation that the teaching thereof was common general knowledge in Australia or elsewhere at the date hereof.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1704182.3 | Mar 2017 | GB | national |
This is a continuation application under 35 U.S.C. § 120 of application U.S. Ser. No. 16/494,441, filed Sep. 16, 2019, now abandoned, which is a national stage application under 35 U.S.C. § 371 of international application PCT/EP2018/056320, filed Mar. 14, 2018, now abandoned, which claims priority to European Application No. 1704182.3, filed Mar. 16, 2017, now abandoned.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16494441 | Sep 2019 | US |
| Child | 18145767 | US |
