EP4010703A1 - Procédé et réactifs pour le diagnostic de mn - Google Patents

Procédé et réactifs pour le diagnostic de mn

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Publication number
EP4010703A1
EP4010703A1 EP20749917.9A EP20749917A EP4010703A1 EP 4010703 A1 EP4010703 A1 EP 4010703A1 EP 20749917 A EP20749917 A EP 20749917A EP 4010703 A1 EP4010703 A1 EP 4010703A1
Authority
EP
European Patent Office
Prior art keywords
autoantibody
pla2r
concentration
sample
patient
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP20749917.9A
Other languages
German (de)
English (en)
Inventor
Rolf Stahl
Elion HOXHA
Janine EISENMANN
Linda REINHARD
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Euroimmun Medizinische Labordiagnostika AG
Original Assignee
Euroimmun Medizinische Labordiagnostika AG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Euroimmun Medizinische Labordiagnostika AG filed Critical Euroimmun Medizinische Labordiagnostika AG
Publication of EP4010703A1 publication Critical patent/EP4010703A1/fr
Pending legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/34—Genitourinary disorders
    • G01N2800/347—Renal failures; Glomerular diseases; Tubulointerstitial diseases, e.g. nephritic syndrome, glomerulonephritis; Renovascular diseases, e.g. renal artery occlusion, nephropathy

Definitions

  • the present invention relates to a method comprising the step detecting an autoantibody to PLA2R in a sample comprising antibodies from a patient at a concentration range including 0.5 to 14 RU/ml, wherein the sample is contacted with a polypeptide comprising PLA2R or a variant thereof under conditions that allow for the formation of a complex comprising the autoantibody and PLA2r, wherein the complex is immobilized and detected; a diagnostically useful carrier comprising recombinant and/or purified human PLA2R or a variant thereof, wherein the carrier is capable of capturing an autoantibody to PLA2R for detection at a concentration below 14 RU/ml, and wherein the carrier selected from the group comprising a Western Blot membrane and a bead, preferably a magnetic bead; and a kit comprising the diagnostically useful carrier according to claim 13 and a means for detecting an autoantibody present in a sample at a concentration range below 14 RU/ml, which is a secondary antibody labeled with a
  • Membranous Nephropathy is an autoimmune disease with a prevalence of 1 to 2/100.000 persons/year and the most common cause of nephrotic syndrome in Caucasian adults.
  • the initial clinical symptoms are edema due to increased renal protein loss, defined as pathologic proteinuria, which is induced by the damage of the renal glomerular filtration barrier.
  • the clinical course of the disease is variable and ranges from spontaneous remission of proteinuria to end-stage renal disease. Patients with spontaneous remission (about 20-25% of patients) usually have an excellent clinical outcome. On the other end of the disease spectrum, about 20% of patients experience end-stage renal disease over a time course of ten years, often despite immunosuppressive therapy. The third group of patients presents with persisting, in most cases more moderate levels of proteinuria and stable renal function.
  • a commercial ELISA for the semi-quantitative determination of pl_A2R autoantibody levels is available from EUROIMMUN Medizinische Labordiagnostika AG (“Anti-PLA2R ELISA IgG Test instruction”, EA 1254-9601 G). According to the test manual, the results should be interpreted such that a concentration of ⁇ 14 RU/ml represents a negative result, a concentration between 14 and 20 RU/ml is borderline, and a result of 20 or more RU/ml represents a positive result, and that a titer increase, decrease or disappearance as detected by the present ELISA assay precedes a change in the clinical status. It is contemplated that concentrations of more than 1500 RU/ml are determined.
  • the test manual teaches detecting PLA2R in patients suffering from MN or those having undergone a kidney transplantation as part of monitoring the outcome of their therapy, but not in healthy subjects or patients without clinically active disease, for example after remission.
  • a commercial immunofluorescence assay for detecting a PLA2R autoantibody is also available (Anti-Phospholipase A2 receptor (PLA2R) II FT, for example FA 1254-1003-40, EUROIMMUN Medizinische Labordiagnostika AG), but immunofluorescence is neither a high-throughput method, nor can it be used to resolve small concentration increases. Well trained scientists, medical doctors or technicians are required to carry out the assay, since the cytoplasm is stained, as is the case in the presence of anti-nuclear antibodies or antibodies against mitochondria.
  • the autoantibody is the causative agent of the disease and, hence, antibody levels and disease activity correlate, it has been shown that this is not the case.
  • the autoantibody can be present in healthy subjects and patients in remission without any clinical symptoms (US8,507,215).
  • the antibody concentration in the serum does not necessarily correlate to the antibody concentration in the environment of the podocyte, where the PLA2R targets are located.
  • pl_A2R autoantibodies In addition to spontaneous remission, therapy-induced remission may be observed in many patients following the administration of immunosuppressive drugs. The result is a decreased level of pl_A2R autoantibodies, which are well below the cut-off or not detectable using ELISA in many patients. It is unclear whether the PLA2R autoantibodies are absent in such cases or simply below the detection limit.
  • US8,507,215 discloses the detection of an autoantibody to PLA2R in samples from patients suffering from MN, but not determining the concentration below 14 RU/ml.
  • US2018/0203020 relates to the prognosis of a relapse after a remission in MN. It is disclosed that the appearance of antibodies to epitopes CTLD1 and CTLD7 indicates that a relapse is coming up, while the detection of an autoantibody to epitope CysR only is associated with stable and mild disease. It is disclosed that a limited number of patients went into relapse and epitopes changed, but it is unclear which assay at which time was used to detect this and it is not disclosed that the concentration was monitored, let alone at which concentration range.
  • Hofstra et al. determined the PLA2R autoantibody levels using ELISA and IFT at baseline, /.e. before treatment based on immunosuppressive drugs (Hofstra, J. M., Debiec, H., Short, C. D., Pelle, T., Kleta, R., Mathieson, P. W., Ronco, P., Brenchley, P. E., Wetzels, J. F. (2012) J. Am. Soc. Nephrol 23, 1735-1743). They found that patients having autoantibody levels between 41-175 U/ml are far more likely to undergo spontaneous remission than those having between 176-610 U/ml, let alone those having more than 610 U/ml.
  • Hofstra, J. M., Beck et al. used Western blotting to detect an autoantibody to PLA2R at various stages of the disease, including remission and relapse.
  • Only one sample was measured at each stage, and signals were not quantified using standardized units. The course of the titer was not monitored at each stage of the disease.
  • the authors conclude that the detection and measurement of the autoantibody may be important for monitoring disease activity and treatment efficacy, but to not suggest predicting an increase in disease activity. In most patients that went on to relapse, no autoantibody could be detected during remission.
  • the assay according to the present invention a sample diluted 1 :5 to 1 :500, preferably 1 : 10 to 1 :300, preferably 1 : 15 to 1 :250 is used.
  • a sample diluted 1 :5 to 1 :500 preferably 1 : 10 to 1 :300, preferably 1 : 15 to 1 :250 is used.
  • the person skilled in the art is familiar with optimizing such assays using parameters such as antigen, dilution and buffer composition.
  • Hofstra et al. used Western blotting to detect an autoantibody to PLA2R in samples from patients suffering from active MN during the clinical baseline phase (Hofstra, J. M., Debiec, H., Short, C. D., Pelle, T., Kleta, R., Mathieson, P. W., Ronco, P., Brenchley P. E. and Wetzels, J. F. (2012) Antiphospholipase A2 receptor antibody titer and subclass in idiopathic membranous nephropathy, J Am Soc Nephrol 23: 1735-1743).
  • a problem underlying the present invention is to predict clinical MN disease activity in a subject at an early stage, in particular at an earlier stage than would be possible based on results from the conventional ELISA assay, in particular a subject having no active disease.
  • Another problem underlying the present invention is to determine the time point at which to administer a mild immunosuppressant drug in order to avoid the onset of clinical disease activity.
  • the problem underlying the present invention is solved by a method comprising the step detecting an autoantibody to PLA2R in a sample comprising antibodies from a patient at a concentration below 14 RU/ml, wherein the sample is contacted with a polypeptide comprising PLA2R or a variant thereof under conditions that allow for the formation of a complex comprising the autoantibody and PLA2r, wherein the complex is immobilized and detected.
  • the absolute concentration of the autoantibody is detected.
  • the concentration range wherein the PLA2R autoantibody is detected or its concentration determined or a change of its concentration is detected, comprises or is 0.5 to 14 RU/ml, preferably 1 to 10 RU/ml, more preferably 2 to 8 RU/ml, most preferably below 6 RU/ml.
  • the antibody is detected in two or more samples from the same patient, wherein the first of the samples was obtained at least two weeks, preferably at least four weeks before the second sample was obtained.
  • the autoantibody is detected using chemiluminescence or radioactivity.
  • the method is carried out using a diagnostically useful carrier selected from the group comprising a microtiter plate, a bead and a Western Blot membrane.
  • the method and the experimental setting is chosen such that a change of concentration of at least 3 RU/ml units, preferably at least 2 RU/ml units, more preferably at least 1 RU/ml units can be resolved.
  • the autoantibody is an autoantibody to the CysR epitope of human PLA2R.
  • the autoantibody is an autoantibody binding to full length human PLA2R.
  • a recombinant and/or purified human PLA2R or a variant thereof is used.
  • the method is for the prognosis of an active MN, preferably in a patient in remission.
  • the problem underlying the present invention is solved by a diagnostically useful carrier comprising recombinant and/or purified human PLA2R or a variant thereof, wherein the carrier is capable of capturing an autoantibody to PLA2R for detection at a concentration below 14 RU/ml, and wherein the carrier is selected from the group comprising a Western Blot membrane and a bead, preferably a magnetic bead.
  • the problem underlying the present invention is solved by a kit comprising the diagnostically useful carrier according to claim 13 and a means for detecting an autoantibody present in a sample at a concentration range below 14 RU/ml, which is a secondary antibody labeled with a detectable radioactive or chemiluminescent label.
  • the autoantibody is detected using a magnetic bead coupled to human PLA2R or CysR or a variant thereof and a secondary antibody labeled with a chemiluminescent label, preferably a label emitting a chemiluminscence signal when contacted with a chemiluminescence trigger solution.
  • the problem is solved by a use of the method, diagnostically useful carrier or kit according to the present invention for prognosing active MN, preferably a relapse in a patient in remission of MN, preferably at an early stage.
  • the problem underlying the present invention is solved by a use of a human PLA2R or a variant thereof, a secondary antibody labeled with a chemiluminescent label, preferably a label emitting a chemiluminescence signal when contacted with a chemiluminescence trigger solution, and a diagnostically useful carrier selected from the group comprising a microtiter plate, a bead and a Western Blot membrane for manufacturing a kit for prognosing active MN, preferably a relapse in a patient in remission of MN, preferably at an early stage, wherein an autoantibody to PLA2R is detected at a concentration below 14 RU/ml.
  • the present invention is based on the inventors’ surprising finding that at low autoantibody concentration, more precisely below 14 RU/ml, previously considered as indicating that a patient is PLA2R autoantibody-negative, or an increase of PLA2R autoantibody levels precedes and indicates clinical disease activity.
  • the present invention is based on the inventors’ surprising finding that detecting an increase in pl_A2R autoantibody concentrations using a method from the group comprising chemiluminescence and radioactivity may indicate future clinical disease activity at an early stage, more specifically before the conventional ELISA assay would detect the presence of these autoantibodies.
  • the present invention is based on the inventors’ surprising finding that, among the PLA2R autoantibodies, an autoantibody to CysR is the most sensitive autoantibody.
  • the present invention is based on the inventors’ surprising finding that based on chemiluminescence or radioactivity rather than ELISA or immunofluorescence detection, diagnostically relevant changes in the concentration of PLA2R autoantibody may be resolved.
  • the present invention relates to detecting an autoantibody to PLA2R.
  • the term “detecting”, as used herein, refers to determining the presence or absence or the absolute concentration of the PLA2R autoantibody, more preferably monitoring the concentration, more preferably detecting an increase, at a concentration range comprising a concentration below 14 RU/ml, preferably only at a concentration below 14 RU/ml. This may mean that concentration changes between those below 14 RU/ml may be detected in addition to those at a concentration of more than and including 14 RU/ml, for example concentrations of up to or more than to 100, 200, 300, 400, 500, 600, 700, 800, 1000, 1250 or 1500 RU/ml.
  • the PLA2R autoantibody concentration below 14 RU/ml includes the range between 0 and 14 RU/ml, preferably 0.01 to 14 RU/ml, preferably 0.1 to 14 RU/ml, preferably 0.25 to 14 RU/ml, preferably 0.4 to 14 RU/ml, preferably 0.5 to 14 RU/ml, preferably 1 to 12 RU/ml, preferably 1 to 10 RU/ml, more preferably 2 to 8 RU/ml.
  • the concentration below 14 RU/ml is between 0.01 to 6 RU/ml, preferably 0.05 to 6 RU/ml, preferably 0.1 to 6 RU/ml, preferably 0.2 to 6 RU/ml.
  • the concentration is between 0 and 0.6 RU/ml, preferably 0.01 and 6 RU/ml, more preferably between 0.05 and 6 RU/ml, more preferably between 0.1 and 6 RU/ml.
  • the limit according to the present invention may be defined by the cut-off of an ELISA, preferably the detection limit of an ELISA with regard to a PLA2R autoantibody, preferably the detection limit below which concentration changes cannot be detected reliably. The person skilled in the art is aware how the detection limit of a method can be determined.
  • the concentration is any concentration detectable using chemiluminescence and/or radioactivity or a concentration range in which concentration changes are detectable by chemiluminescence and/or radioactivity, but not ELISA.
  • concentration range for another type of assay needs to be defined to practice the invention, then a sample having the concentration of interest is confirmed as having this concentration by this ELISA, and the concentration of antibodies in this sample may then be determined by the other assay to yield a value in the relevant unit for such assay.
  • PLA2R autoantibody refers to a mammalian, preferably human autoantibody to PLA2R, wherein PLA2R is represented by the amino acid sequence in NP_001007268.
  • any data base codes cited refers to the Uniprot or other data base, more specifically the version available on the earliest priority date or filing date of this application.
  • the term “idiopathic MN” or simply “MN” is used to describe MN that is not caused by any known secondary etiology such as hepatitis B or lupus, but is associated with PLA2R autoantibodies.
  • a change of concentration preferably an increase is detected, wherein the change, preferably increase, is at least 0.1, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4 or 5 RU/ml or, in relative terms, an increase by 10, 20, 30, 40, 50, 75, 100 or 150% or more, preferably 50% or more, of the previous value measured, as measured densitometrically in a Western Blot or in relative detection units in the case of radioactivity or chemiluminescence detectors.
  • the method and the experimental setting is chosen such that a change of concentration of at least 3 RU/ml units, preferably at least 2 RU/ml units, more preferably at least 1 RU/ml units can be resolved, wherein preferably the change is below 14 RU/ml.
  • the PLA2R autoantibody concentration is monitored by determining it at least every 1, 2, 3, 4, 8, 12, 24 or 36 weeks, preferably at least every four weeks.
  • the method, diagnostically useful carrier or kit according to the present invention may be used for prognosing active MN, preferably a relapse in a patient in remission of MN, at an early stage, optionally before an ELISA-based assay could be used for the same purpose, for example at least 1 , 2, 3, 4, 5 or 6 months, preferably 3 months before the ELISA assay would yield a positive result.
  • the diagnostically useful carrier is a Western blot membrane or Western blotting
  • a lysate of human cells preferably kidney cells or purified mammalian, preferably PLA2R or a variant thereof may be used to capture a PLA2R antibody. It is preferred that purified PLA2R is used for increased sensitivity, specificity, reproducibility and standardization. The amount of PLA2R in cells and its immunoreactivity may vary and may be inferior to purified PLA2R.
  • the amount of PLA2R as a capturing agent, the developing time and the degree of purity of the PLA2R, the sensitivity and specificity of the diagnostically useful carrier in the assay may be adjusted in routine optimization experiments familiar to the person skilled in the art.
  • the more PLA2R is used and the longer the developing time, for example exposure to a chemiluminescent trigger solution or exposure of a radioactively labeled agent to a detection screen the higher the sensitivity.
  • the purer the PLA2R antigen used the higher the specificity.
  • a bead associated with pLA2R or a variant thereof wherein the pl_A2r or variant thereof carries a chemiluminescent or radioactive label, preferably chemiluminescent label.
  • a method comprising the steps contacting a sample with a carrier, which is preferably a bead associated with pLA2r or a variant thereof is provided, wherein the pl_A2r or variant thereof carries a chemiluminescent or radioactive label, under conditions compatible with formation of a complex comprising the bead-associated pl_A2R and an autoantibody to PLA2R, separating the bead from the sample, optionally washing the sample and detecting the complex using chemiluminescence or radioactivity detection.
  • the diagnostically useful carrier is optimized and/or configured such that it is capable of being used to detect the autoantibody at the concentration ranges and the resolution and detection limits specified herein, which includes, for example, detecting a PLA2R autoantibody at a concentration below 14 RU/ml, optionally in addition to concentration ranges above and including 14 RU/ml, detecting a change of concentration, preferably an increase by 50% or more or by 1 RU/ml or more.
  • the carrier may be optimized by calibrating it using calibrators in the concentration range contemplated and adding the right amount and type of PLA2R-based antigen to the carrier. For example, native, full-length PLA2R antibody comprising the CysR region and the CysR region itself make sensitive antigens.
  • At least two calibrators preferably three, four, five or more calibrators, each with a different, known concentration of calibrator antibody, should be used.
  • a calibrator in the range of 0.25 to 4 preferably 0.5 to 3, more preferably 1 to 3 RU/ml and a calibrator in the range of 10 to 20, preferably 12 to 16 could be used.
  • the person skilled in the art is familiar with setting up calibration curves. This will allow the person skilled in the art to calibrate a range of approximately 0.5 to 14.
  • the sample is a sample from a mammalian, preferably human patient and comprises antibodies, typically a mixture comprising both autoantibodies and other antibodies.
  • the patient may be a Caucasian patient.
  • the sample may be blood, plasma, serum or CSF and is preferably serum.
  • PLA2R domains comprising a PLA2R autoantibody epitope have been identified that are targeted by PLA2R autoantibodies, more specifically CysR, CTLD1, CTLD7 and CTLD8.
  • the PLA2R autoantibody detected is an autoantibody to CysR.
  • CysR is represented by SEQ ID N02.
  • the PLA2R autoantibodies may be distinguished by using various constructs comprising different sets of epitopes as described by Hohxa, E., Zahner, G., Reinhard, L, and Stahl, R. K. PLA2R epitope recognitions patterns and clinical outcome in patients with membranous nephropathy, Kidney International, in press.
  • PLA2R or variants thereof may be provided in any form and at any degree of purification, from liquid samples, tissues or cells comprising said polypeptide in an endogenous form, more preferably cells overexpressing the polypeptide, crude or enriched lysates of such cells, to purified and/or isolated polypeptide which is optionally essentially pure.
  • a lysate comprising mammalian, preferably human kidney cells is used.
  • the polypeptide is a native polypeptide, wherein the term “native polypeptide”, as used herein, refers to a folded polypeptide, more preferably to a folded polypeptide purified from tissues or cells, more preferably from mammalian cells or tissues, optionally from non- recombinant tissues or cells.
  • the polypeptide is a recombinant protein, wherein the term “recombinant”, as used herein, refers to a polypeptide produced using genetic engineering approaches at any stage of the production process, for example by fusing a nucleic acid encoding the polypeptide to a strong promoter for overexpression in cells or tissues or by engineering the sequence of the polypeptide itself.
  • a polypeptide is pure if at least 60, 70, 80, 90, 95 or 99 percent of the polypeptide in the respective sample consists of said polypeptide as judged by SDS polyacrylamide gel electrophoresis followed by Coomassie blue staining and visual inspection.
  • polypeptides in particular a polypeptide comprising the native sequence of PLA2R referred to in this application explicitly, for example by function, name, sequence or accession number, or implicitly, but also using variants of such polypeptides or nucleic acids.
  • the term “variant”, as used herein, may refer to at least one fragment of the full length sequence referred to, more specifically one or more amino acid or nucleic acid sequences which are, relative to the full-length sequence, truncated at one or both termini by one or more amino acids.
  • a fragment comprises or encodes for a peptide having at least 6, 7, 8, 10, 12, 15, 20, 25, 50, 75, 100, 150 or 200 successive amino acids of the original sequence or a variant thereof.
  • the total length of the variant may be at least 6, 7, 8, 9, 10, 11, 12, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 750, 1000 or more amino acids.
  • variant relates not only to at least one fragment, but also to a polypeptide or a fragment thereof comprising amino acid sequences that are at least 40, 50, 60, 70, 75, 80, 85, 90, 92, 94, 95, 96, 97, 98 or 99 % identical to the reference amino acid sequence referred to or the fragment thereof, wherein amino acids other than those essential for the biological activity, for example the ability of an antigen to bind to an (auto)antibody, or the fold or structure of the polypeptide are deleted or substituted and/or one or more such essential amino acids are replaced in a conservative manner and/or amino acids are added such that the biological activity of the polypeptide is preserved.
  • the state of the art comprises various methods that may be used to align two given nucleic acid or amino acid sequences and to calculate the degree of identity, see for example Arthur Lesk (2008), Introduction to bioinformatics, Oxford University Press, 2008, 3 rd edition.
  • the ClustalW software (Larkin, M. A., Blackshields, G., Brown, N. P., Chenna, R., McGettigan, P. A., McWilliam, H., Valentin, F., Wallace, I. M., Wilm, A., Lopez, R., Thompson, J. D., Gibson, T. J., Higgins, D. G. (2007).
  • Clustal W and Clustal X version 2.0 Clustal W and Clustal X version 2.0.
  • Bioinformatics, 23, 2947- 2948 is used using default settings.
  • the location of epitopes reactive with PLA2R autoantibodies must be considered, which is published in Fresquet, M., Jowitt, T. A., Gummadova, J., et al. (2015) Identification of a major epitope recognized by PLA2R autoantibodies in primary membranous nephropathy, J. Am. Nephrol., 26:302-313 and in Kao, L., Lam, V., Waldman, M. et al. (2015) Identification of the immunodominant epitope region in phospholipase A2 receptor-mediating autoantibody binding in idiopathic membranous nephropathy. J.
  • the variant may be flanked C-terminally or N-terminally by amino acids or amino acid sequences derived from phospholipase-A2-receptor or from any other proteins which do not prevent sterical access to the phospholipase-A2-receptor binding epitope comprised by the variant, for example linkers and/or folded domains.
  • Behnert et al. have studies and mapped the epitopes of the receptor (Behnert, A., Fritzler, M. J., Teng, B., Zhang, M., Bollig, F., Haller, H., Skoberne, A., Mahler, M., and Schiffer, M. (2013) (PLOS, 8 (4) e61669), and their results, in particular the epitopes in Table 1 and Fig. 2, may be used to guide the design of variants. Additional guidance may be found in US2019183969 AA.
  • the variant is a linear, non-folded polypeptide, which is optionally denatured.
  • polypeptide and variants thereof may, in addition, comprise chemical modifications, for example isotopic labels or covalent modifications such as glycosylation, phosphorylation, acetylation, decarboxylation, citrullination, methylation, hydroxylation and the like.
  • chemical modifications for example isotopic labels or covalent modifications such as glycosylation, phosphorylation, acetylation, decarboxylation, citrullination, methylation, hydroxylation and the like.
  • Any modification is designed such that it does not abolish the biological activity of the variant.
  • variants may also be generated by fusion with other known polypeptides or variants thereof and comprise active portions or domains, preferably having a sequence identity of at least 70, 75, 80, 85, 90, 92, 94, 95, 96, 97, 98 or 99 % when aligned with the active portion of the reference sequence, wherein the term "active portion”, as used herein, refers to an amino acid sequence, which is less than the full length amino acid sequence or, in the case of a nucleic acid sequence, codes for less than the full length amino acid sequence, respectively, and/or is a variant of the natural sequence, but retains at least some of the biological activity.
  • SEQ ID N01 represents an example of a fusion protein according to the present invention.
  • the variant of the polypeptide has biological activity.
  • biological activity is the ability to bind specifically to a PLA2R autoantibody, as found in a patient suffering from MN.
  • whether or not a variant of PLA2R has such biological activity may be checked by determining whether or not the variant of interest binds to an autoantibody from a sample of a patient which autoantibody binds to wild type PLA2R, preferably as determined by ELISA using the commercial assay.
  • the sample is derived from a subject showing no active clinical disease, which means, in a more preferred embodiment, that the 24 h urinary protein excretion, as determined by dipstick measurement, is less than 3.5 g/day, preferably less than 3 g/day, less than 2.5 g/day, less than 2 g/day, less than 1.75 g/day, less than 1.5 g/day, less than 1 g/day, less than 0.5 g/day, most preferably less than 3.5 g/day.
  • the sample is derived from a subject in spontaneous or therapeutically-induced remission, as evidenced by clinical picture, in particular a marked reduction in proteinuria and/or edema.
  • the sample is from a subject at the time of kidney biopsy.
  • the sample may be from a patient at risk of or suspected of developing MN.
  • the method according to the invention is carried out when the patient is in spontaneous or therapeutically-induced remission, which is preferably the case when he no longer shows the active disease.
  • the method is carried out using a sample from a healthy subject or to screen a healthy subject to determine whether he is at risk of developing MN.
  • the methods or products according to the present invention are used to predict the onset of active MN disease, preferably a relapse, more than three months, preferably 4, 5, 6, 8, 10, 12, 24, 36, 48 or 60 months before the onset.
  • the methods or products according to the present invention are used to predict the onset of active MN disease, preferably a relapse, 1, 2, 4, 6, 8, 10, 12, 18, 24, 36, 48 or 60 months after the remission of MN.
  • the remission may be spontaneously or therapeutically induced.
  • the PLA2R autoantibody is detected using chemiluminescence.
  • the autoantibody captured by PLA2R or a variant thereof on a diagnostically useful carrier, particularly a bead is contacted with a ligand binding specifically to the autoantibody, which ligand is labeled with a detectable chemiluminescent label.
  • a ligand may be a secondary antibody, for example one binding to the constant domain of the Ig class of the autoantibody to be detected such as IgG or for example PLA2R or a variant thereof binding to the variable domain of the autoantibody to be detected.
  • the chemiluminescent ligand may be a chemiluminescent enzyme, preferably selected from the group comprising Luciferase, Peroxidase, alkaline phosphatase and b-galactosidase or a variant thereof, which may turn over a chemiluminescent substrate without being consumed itself (Kricka, L. J. (2003). Clinical applications of chemiluminescence. Analyticazia acta, 500(1): 279-286).
  • the chemiluminescent ligand may be a small organic compound having no enzymatic activity, which emits a chemiluminescence signal upon being degraded when contacted with a chemiluminescence trigger solution which comprises inorganic and/or non- enzymatic organic compounds that are required for emitting the signal.
  • the small organic compound having no enzymatic activity is selected from the group comprising acridinium esters (Weeks, I., Beheshti, I., McCapra, F., Campbell, A. K., Woodhead, J. S. (1983) Acridinium esters as high specific activity labels in immunoassay.
  • the trigger solution comprises H 2 0 2 at a high pH.
  • the trigger solution comprises H 2 0 2 at a high pH.
  • an acridinium ester a mixture of H 2 0 2 and sodium hydroxide is frequently used.
  • the small organic compound is consumed upon emission of the chemiluminescence signal, also referred to as flash, which typically lasts for 1-5 seconds.
  • Chemiluminescence is particularly preferred as the detection method, if a bead, more preferably a magnetic bead, is used as the diagnostically useful carrier.
  • a kit which may comprise one or more, preferably all from the group comprising the diagnostically carrier according to the present invention, a ligand binding to the PLA2R autoantibody, preferably a secondary antibody, which ligand comprises a detectable radioactive or chemiluminescent label, one or more calibrator solutions, each preferably comprising an antibody to PLA2R at a known concentration, a sample dilution buffer, a washing solution and a chemiluminescence trigger solution and a chemilumescent substrate.
  • the kit may comprise instructions detailing how to carry out the inventive assay.
  • the present invention provides a use of a reagent or a means or a polypeptide or composition for detecting or binding specifically to a PLA2R autoantibody, capable of detecting a PLA2R autoantibody at a concentration below 14 RU/ml, which use is for the manufacture of a kit for diagnosing MN, preferably active clinical disease such as a relapse, at an early stage or for determining the treatment regimen for an MN patient or a subject likely to develop MN.
  • a method for confirming the reliability of a antibody detection assay may involve detecting a PLA2R autoantibody in a solution at a concentration below 14 RU/ml, which may not be a sample from a patient, but is known to comprise the autoantibody or another PLA2R antibody of the same class as the autoantibody, preferably at a known concentration.
  • the solution may be a negative control not comprising the antibody to check the background.
  • any method or use according to the present invention may be intended for testing in vitro the efficiency of a medical device designed to remove a PLA2R autoantibody from a patient’s blood, wherein the testing is performed on a liquid other than patient’s blood.
  • its capacity to remove autoantibody may be checked by running a solution comprising a PLA2R antibody through the device, followed by use of the method according to the present invention to confirm that less or no antibody is in the solution that has been passed through the device, i.e. showing that the device has still the capacity to remove antibody from the solution.
  • a small number of devices may be tested for confirming or testing the quality of the entire batch in the sense of quality control, wherein the sample or solution may comprise a known concentration of PLA2R antibody.
  • the sensitivity of the inventive method based on the detection using chemiluminescence and radioactivity may help detect a deteriorating efficiency or binding activity of the device at an earlier stage.
  • Figs. 1 to 3 depict the prognosis of a relapse of MN using a sensitive detecting method based on serological determination of PLA2R autoantibodies in three patients after a therapeutically- induced remission, more specifically Western blot, compared to conventional ELISA.
  • the present application comprises a range of sequences, more specifically:
  • SFCIYKHNGGFFRRLAGFRNPYYPATNFSTVYLEENILISDLEKSDQYPYDVPDYA SEQ ID N02 (CysR domain): kgifviqseslkkciqagksvltlenckqankhmlwkwvsnhglfniggsgclglnfsapeqplslyecdstlvslrwrcnrkmitgpl qysvqvahdntvvasrkyihkwisygsgggdicey
  • the cell lysate containing HA-tagged recombinant full-length human PLA2R1 was prepared from approximately 5 million transfected HEK293 cells, which were lysed in 800 pL lysis-buffer (50 mM Tris pH 7.4, 150 mM NaCI, 1 mM EDTA, 1% (v/v) Triton X-100, 1x protease inhibitor), sonicated 3 x 10 sec and incubated for 1 hour at 4°C at 12 rpm on a lab rotator. The cell debris was removed by centrifugation for 15 min at 4°C at 14000 g (HERAEUS Fresco 21). The supernatant was collected and the protein concentration was measured with a DC-Assay (Bio-Rad Laboratories GmbH) following the procedure provided by the manufactures. The cell lysate was divided into aliquots and stored at -20°C.
  • the PLA2R1 -protein Master Mix (MM) was prepared as given in Table 1.
  • Blocking buffer was prepared in a sterile bottle in order to avoid even small amounts of bacterial or fungal contaminations, since due to the long incubation time of the following immunoblotting steps they could have a negative effect on the sensitivity of the experiment. 3.5% (w/v) skimmed milk powder was dissolved in PBS + 0.1% (v/v) Tween-20 and left stirring for 1 hour at room temperature. Then the solution was filtered through a sterile 100 pm cell strainer to remove little milk particles. The final blocking buffer was stored at 4°C until use (maximum 2 days).
  • the PVDF membrane was blocked with 7 ml_ blocking buffer for 1-2 hours at room temperature on a vertical shaker at 17 rpm in a container.
  • the speed and volume ensured an optimal floating without bumping on the edges of the container, which would lead to false signals on the membrane brinks and increase the background.
  • very small milk particles were found to preferentially bind to the “unspecific-dot-trap” stripe described above. Therefore, the “unspecific-dot-trap” strip was cut away after the blocking step.
  • the membrane pieces were first numbered with a pencil and then cut within the marker lane using disinfected scissors, resulting in 8 membrane strips which can be analyzed using different MN patient sera.
  • Each strip was quickly dipped into washing buffer (PBS with 0.1% Tween 20) in order to remove excessed milk and then transferred into the MN patient serum (10 mL of 1:100 diluted in 0.05% (w/v) skimmed milk powder in PBS + 0.1% (v/v) Tween-20; if the serum was strongly hemolytic, a 1:200 dilution was used) of interest, which were each propounded in 10 cm Petri dishes.
  • washing buffer PBS with 0.1% Tween 20
  • a piece of parafilm (12x10 cm) was placed onto a clean plastic lid in order to generate a hydrophobic area.
  • ECL-Clarity was pre-mixed immediately before use.
  • the lower edge of the membrane strip was quickly dipped onto a paper tower to remove access liquid and then placed on the parafilm area.
  • the resulting re-assembled whole PVDF membrane was covered with 800 pl_ ECL-Clarity and incubated for 5 min in the dark.
  • the individual membrane strips were picked up with tweezers, quickly dipped on a paper towel to remove access liquid and then arranged without air bubbles between the sheets of a transparent plastic bag in order to keep it wet during the imaging process in a Luminescent Detection Imager 600.
  • the final exposure time was dependent on the serum of interest. Precisely, end points of detection were either a well-defined PLA2R-specific band, a development time of up to 15 minutes or a high background signal, whichever presented first.
  • end points of detection were either a well-defined PLA2R-specific band, a development time of up to 15 minutes or a high background signal, whichever presented first.
  • the development step was repeated after a 2 min wash in washing buffer with Super Signal West Femto (Biorad) until the same endpoints.
  • Serum samples from eight patients suffering from a relapse following therapy-induced remission were taken at various time points before the relapse.
  • Figs. 1 to 3 depict the prognosis of a relapse of an MN using a sensitive detecting method based on serological determination of PLA2R autoantibody in three patients after a therapeutically-induced remission, more specifically Western blot, compared to conventional ELISA.
  • PLA2R antibodies could be detected using Western blotting fifteen months earlier than using ELISA prior to a relapse.
  • detection of the antibody could be performed nine or three months earlier, respectively.

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Abstract

La présente invention concerne un procédé comprenant l'étape consistant à détecter un auto-anticorps dirigé contre PLA2R dans un échantillon comprenant des anticorps d'un patient à une plage de concentration comprenant 0,5 à 14 UR/ml, l'échantillon étant mis en contact avec un polypeptide comprenant PLA2R ou une variante de celui-ci dans des conditions qui permettent la formation d'un complexe comprenant l'auto-anticorps et PLA2r, le complexe étant immobilisé et détecté ; un support utile au diagnostic comprenant un PLA2R humain recombinant et/ou purifié ou un variant de celui-ci, le support pouvant capturer un auto-anticorps dirigé contre PLA2R pour une détection à une concentration inférieure à 14 UR/ml, et le support étant choisi dans le groupe comprenant une membrane de transfert Western et une bille, de préférence une bille magnétique ; et un kit comprenant le support utile au diagnostic selon la revendication 13 et un moyen de détection d'un auto-anticorps présent dans un échantillon à une plage de concentration inférieure à 14 UR/ml, qui est un anticorps secondaire marqué à l'aide d'un marqueur radioactif ou chimioluminescent détectable.
EP20749917.9A 2019-08-06 2020-08-06 Procédé et réactifs pour le diagnostic de mn Pending EP4010703A1 (fr)

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EP4166945A1 (fr) 2021-10-14 2023-04-19 EUROIMMUN Medizinische Labordiagnostika AG Nouveaux biomarqueurs de la glomérulonéphrite membranaire
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SEITZ-POLSKI BARBARA ET AL: "Supplementary Information : Cross-reactivity of anti-PLA2R1 autoantibodies to rabbit and mouse PLA2R1 antigens and development of two novel ELISAs with different diagnostic performances in idiopathic membranous nephropathy", BIOCHIMIE, vol. 118, 19 August 2015 (2015-08-19), FR, pages 1 - 12, XP093366380, ISSN: 0300-9084, Retrieved from the Internet <URL:https://ars.els-cdn.com/content/image/1-s2.0-S0300908415002540-mmc1.pdf> DOI: 10.1016/j.biochi.2015.08.007 *

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