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 detectable radioactive or chemiluminescent label.

Membranous Nephropathy (MN) 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.

It is difficult to tailor a therapy depending on the individual patient’s needs. If a patient belongs to the first group with spontaneous remission, any medication, usually the administration of immunosuppressive drugs, will see him exposed to the considerable side effects of such treatment even though no such treatment was necessary as could be concluded with hindsight. By contrast, a patient belonging to group 3 may benefit from the administration of strong immunosuppressive drugs at an early stage of the treatment. Therefore, it has been an accepted clinical strategy to wait and watch how the clinical disease activity will develop under supportive therapy only and to consider, if required, more stringent therapy options during the follow-up time.

It would be desirable to predict as early as possible how the course of the disease will develop and in which of these three clinical categories a patient will ultimately fall. For example, it may be possible to subject a group 3 (moderate proteinuria) patient to treatment with mild immunosuppressive drugs only at an early stage and watch whether he responds positively to such treatment without developing clinical disease activity, thus potentially saving the patient from exposure to harsh immunosuppressive treatment with severe side effects. A group 2 patient, if identified as such at an early stage, could immediately be subjected to a moderate or even harsh immunosuppressive treatment as an optimal counter measure against the development of end stage renal disease.

The discovery of autoantibodies to pLA2r has paved the way to a diagnosis of the disease based on serology (Beck, L, Bonegio, R. G., Lambeau, G., Beck, D. M., Powell, D. W., Cummins, T. D., Klein J. B., Salant, D. J. (2009) M-Type Phospholipase A2 Receptor as Target Antigen in Idiopathic Membranous Nephropathy, N. Engl. J. Med. 361(1), 11-21). Autoantibodies to THSD7A may also be detected (US10107810), but the prevalence is lower.

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.

While it is tempting to assume that 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. For example, the autoantibody can be present in healthy subjects and patients in remission without any clinical symptoms (US8,507,215).

Various reasons have been provided to account for this lack of correlation. For example 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.

A recent study revealed that those patients with low antibody levels and moderate proteinuria have a higher frequency of spontaneous remission than patients with high antibody levels (Mahmud, M., Pinnschmidt, H. O., Reinhard, L. et al. (2018) Phospholipase A2 receptor 1 antibody levels at the time of diagnosis determine renal outcome in patients with membranous nephropathy, J. Am. Soc. Nephrol., 29, 377).

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.

Timmermans et al. determined the PLA2R autoantibody levels using ELISA at the time of the biopsy and suggest that level of the autoantibodies at the time of the renal biopsy predict the disease course (Timmermans, S. A., Abdul Hamid, M. A., Cohen Tervaert, J. W., Damoiseaux, J. G. and van Paassen, P. (2015) Anti-PLA2R Antibodies as a Prognostic Factor in PLA2R-Related Membranous Nephropathy, Am J Nephrol 42: 70-77). At baseline, /. e. before administration of immunosuppressive treatment, neither the PLA2R autoantibody level nor the rate of proteinuria correlated with later emergence of a relapse.

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. (Hofstra J. M., Beck, L. H. Jr., Beck, D. M., Wetzels, J. F. and Salant, D. J. (2011) Anti-phospholipase A ₂ receptor antibodies correlate with clinical status in idiopathic membranous nephropathy, Clin J Am Soc Nephrol 6: 1286-1291). 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.

Qin et al. detected the PLA2R autoantibody in nearly all Chinese patients who suffered from MN. A single sample was used from each patient and no samples from patients in remission were used (Qin, W., Beck, L. H., Zeng, C., Chen, Z., Li, S., Zuo, K., Salant, D. J. and Liu Z. (2011) Anti-Phospholipase A2 Receptor Antibody in Membranous Nephropathy, J Am Soc Nephrol 22: 1137-1143). They found that a group of 11 patients were apparently negative. However, when retesting the patients using less diluted serum and a higher concentration of detecting antibody, a low titer of anti-PLA2R antibody was detectable in 10 of them. In a preferred embodiment, 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. 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).

The problem underlying the present invention is solved by the subject-matter of the attached independent and dependent claims.

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.

In a 1 ^st aspect, 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.

In a preferred embodiment, the absolute concentration of the autoantibody is detected.

In a preferred embodiment, it is detected whether the concentration of the autoantibody has changed, preferably increased. In a preferred embodiment, 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.

In a preferred embodiment, 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.

In a preferred embodiment, the autoantibody is detected using chemiluminescence or radioactivity.

In a preferred embodiment, 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.

In a preferred embodiment, 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.

In a preferred embodiment, the autoantibody is an autoantibody to the CysR epitope of human PLA2R.

In a preferred embodiment, the autoantibody is an autoantibody binding to full length human PLA2R.

In a preferred embodiment, a recombinant and/or purified human PLA2R or a variant thereof is used.

In a preferred embodiment, the method is for the prognosis of an active MN, preferably in a patient in remission.

In a 2 ^nd aspect, 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. In a 3 ^rd aspect, 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.

In a preferred embodiment, 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.

In a 4 ^th aspect, 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.

In a 5 ^th aspect, 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. In a preferred embodiment, 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.

In a preferred embodiment, 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. In another preferred embodiment, 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. In another preferred embodiment, 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. Instead of the border value “14 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. In another preferred embodiment, 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.

As no international reference serum exists for PLA2R autoantibodies, a variety of assays are reported in the state of the art, the results of which cannot be directly compared. However, the ELISA by EUROIMMUN Medizinische Labordiagnostika AG (Lubeck, Germany) is the most frequently used assay and is commercially available (“Anti-PLA2R-ELISA”, Product number EA 1254-9601 G), hence all absolute concentration values referred to throughout this document will be based on relative units (RU, “RE” in German publications) as determined using this assay according to the manufacturer’s instructions. The assay is straightforward to use and may be used to carry out the invention or to determine whether an embodiment is within the scope of protection of the present invention. If the 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.

In a preferred embodiment, the term “PLA2R autoantibody”, as used herein also with exchangeable terms such as “autoantibody to PLA2R” or “autoantibody binding to PLA2R”, refers to a mammalian, preferably human autoantibody to PLA2R, wherein PLA2R is represented by the amino acid sequence in NP_001007268. Throughout this application, 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.

In a preferred embodiment, 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.

In a preferred embodiment, 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.

In a preferred embodiment, 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. For example, in the latter, more preferred embodiment, this means that a change of concentration from 2.5 RU/ml in a first sample to 3.5 RU/ml in a second sample obtained at a later time point, can be reliably detected as such. In a preferred embodiment, 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.

If the diagnostically useful carrier is a Western blot membrane or Western blotting is used, 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.

Depending on the experimental settings, for example 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. As a rule, 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. Also, the purer the PLA2R antigen used, the higher the specificity.

According to the present invention, a bead associated with pLA2R or a variant thereof is provided, wherein the pl_A2r or variant thereof carries a chemiluminescent or radioactive label, preferably chemiluminescent label. According to the present invention, a method is provided, 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.

In a preferred embodiment, 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. For example, 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.

In a preferred embodiment, 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.

Various PLA2R domains comprising a PLA2R autoantibody epitope have been identified that are targeted by PLA2R autoantibodies, more specifically CysR, CTLD1, CTLD7 and CTLD8. In a preferred embodiment, the PLA2R autoantibody detected is an autoantibody to CysR. In a preferred embodiment, 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.

When used to carry out the teachings of the present invention, 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. In a preferred embodiment, a lysate comprising mammalian, preferably human kidney cells is used. In a preferred embodiment, 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. In another preferred embodiment, 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. The person skilled in the art is familiar with methods for engineering nucleic acids and polypeptides encoded (for example, described in Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989), Molecular Cloning, CSH or in Brown T. A. (1986), Gene Cloning - an introduction, Chapman & Hall) and for producing and purifying native or recombinant polypeptides (for example Handbooks ..Strategies for Protein Purification", ..Antibody Purification", ..Purifying Challenging Proteins" (2009/2010), published by GE Healthcare Life Sciences, and in Burgess, R. R., Deutscher, M. P. (2009), Guide to Protein Purification). In a preferred embodiment, 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.

The teachings of the present invention may not only be carried out using 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.

In a preferred embodiment, 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. Such 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.

The term "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. In a preferred embodiment, 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. Bioinformatics, 23, 2947- 2948) is used using default settings. When designing variants, 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. Am. Soc. Nephrol., 26, 291-301. In a preferred embodiment, 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.

In a preferred embodiment, the variant is a linear, non-folded polypeptide, which is optionally denatured.

In a preferred embodiment, the 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. The person skilled in the art is familiar with methods to modify polypeptides. Any modification is designed such that it does not abolish the biological activity of the variant. Moreover, 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. In a preferred embodiment, such biological activity is the ability to bind specifically to a PLA2R autoantibody, as found in a patient suffering from MN. For example, 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.

In a preferred embodiment, 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. In a preferred embodiment, 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. In another preferred embodiment, 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. In a preferred embodiment, 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. Alternatively, 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.

In a preferred embodiment, 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. In a preferred embodiment, 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.

In a preferred embodiment, the PLA2R autoantibody is detected using chemiluminescence. For this purpose, 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. Such 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. Analytica chimica 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. Preferably, 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. Clin Chem 29: 1474-1479 (1983) and luminol or a chemiluminescent derivative thereof. Such small organic compounds may be coupled to polypeptides such as a secondary antibody. In the case of luminol, the trigger solution comprises H ₂ 0 ₂ at a high pH. In the case of 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.

According to the present invention, a kit is provided 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 method or reagent or a means or a polypeptide or composition or the inventive carrier for determining a treatment regimen for an MN patient or a subject likely to develop MN, preferably deciding, more preferably at an early stage, whether and at which time point immunosuppressive drugs should be administered and/or whether mild or strong immunosuppressive drugs are in order.

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.

According to the present invention, a method is provided for confirming the reliability of a antibody detection assay and 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. Alternatively, the solution may be a negative control not comprising the antibody to check the background.

In a preferred embodiment, 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. After the use of the medical device with a patient, 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. Alternatively, from a batch comprising a large number devices, 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:

SEQ ID N01 (HA-tagged human PLA2R):

MLLSPSLLLLLLLGAPRGCAEGVAAALTPERLLEWQDKGIFVIQSESLKKCIQAGKS VLTLENC KQANKH MLWKWVSNHGLFNIGGSGCLGLNFSAPEQPLSLYECDSTLVSLRWRCNRKMITG PLQYSVQVAHDNTVVA SRKYIHKWISYGSGGGDICEYLHKDLHTIKGNTHGMPCMFPFQYN H Q WH HECTREGRED D LL WC ATT S R Y ERDEKWGFCPDPTSAEVGCDTIWEKDLNSHICYQF NLLSSLSWSEAHSSCQMQGGTLLSITDETEENFIR EHMSSKTVEVWMGLNQLDEHAGWQW SDGTPLNYLNWSPEVNFEPFVEDHCGTFSSFMPSAWRSRDCESTL PYICKKYLNHIDHEIVE KDAWKYYATHCEPGWNPYNRNCYKLQKEEKTWHEALRSCQADNSALIDITSLA EVEFLVTLL GDENASETWIGLSSNKIPVSFEWSNDSSVIFTNWHTLEPHIFPNRSQLCVSAEQSEGHWK V KNCEERLFYICKKAGHVLSDAESGCQEGWERHGGFCYKIDTVLRSFDQASSGYYCPPALV TI TNRFEQAF ITSLISSVVKMKDSYFWIALQDQNDTGEYTWKPVGQKPEPVQYTHWNTHQPRY SGGCVAMRGRHPLGRWE VKHCRHFKAMSLCKQPVENQEKAEYEERWPFHPCYLDWESEP GLASCFKVFHSEKVLM KRTWREAEAFCE EFGAHLASFAHIEEENFVNELLHSKFNWTEERQF WIGFNKRNPLNAGSWEWSDRTPVVSSFLDNTYFGED ARNCAVYKANKTLLPLHCGSKREWI CKIPRDVKPKIPFWYQYDVPWLFYQDAEYLFHTFASEWLNFEFVC SWLHSDLLTIHSAHEQE FIHSKIKALSKYGASWWIGLQEERANDEFRWRDGTPVIYQNWDTGRERTVNNQ SQRCGFIS SITGLWGSEECSVSMPSICKRKKVWLIEKKKDTPKQHGTCPKGWLYFNYKCLLLNIPKDP SS WKNWTHAQHFCAEEGGTLVAIESEVEQAFITMNLFGQTTSVWIGLQNDDYETWLNGKPVV Y SNWSPFDII NIPSHNTTEVQKHIPLCALLSSNPNFHFTGKWYFEDCGKEGYGFVCEKMQDTS GHGVNTSDMYPMPNTLE YGNRTYKIINANMTWYAAIKTCLMHKAQLVSITDQYHQSFLTVVL NRLGYAHWIGLFTTDNGLNFDWSDG TKSSFTFWKDEESSLLGDCVFADSNGRWHSTACES FLQGAICHVPPETRQSEHPELCSETSIPWIKFKSN CYSFSTVLDSMSFEAAHEFCKKEGSNLL TIKDEAENAFLLEELFAFGSSVQMVWLNAQFDGNNETIKWFD GTPTDQSNWGIRKPDTDYFK PHHCVALRIPEGLWQLSPCQEKKGFICKMEADIHTAEALPEKGPSHSIIP LAVVLTLIVIVAICTL

SFCIYKHNGGFFRRLAGFRNPYYPATNFSTVYLEENILISDLEKSDQYPYDVPDYA SEQ ID N02 (CysR domain): kgifviqseslkkciqagksvltlenckqankhmlwkwvsnhglfniggsgclglnfsap eqplslyecdstlvslrwrcnrkmitgpl qysvqvahdntvvasrkyihkwisygsgggdicey

The present invention is further illustrated by the following non-limiting examples from which further features, embodiments, aspects and advantages of the present invention may be taken.

Example: Comparison of an assay detecting the concentration of the PLA2R autoantibody at concentrations below 14 RU/ml compared to the conventional ELISA assay

Western blotting 1. Protein preparation

In the Western blot analyses we used HA-tagged recombinant full-length human PLA2R1 protein (SEQ ID N01) obtained from whole cell lysates of HEK293 cells transfected using the Lipofectamine® system (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s protocol. Differences in cell transfection rates and hence variable amounts of expressed proteins were tested in order to optimize the amount of protein loaded on the SDS- PAGE gel. For this, an amount of protein was loaded, that produced as few unspecific cell lysate bands as possible when tested with an MN patient serum with high PLA2R antibodies. At the same time, when the same protein amount was tested with an MN patient serum with low PLA2R antibodies a promptly visible PLA2R signal was produced.

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.

2. SDS-PAGE using the Mini-Protean Electrophoresis System by Biorad

Gels and chamber were used according to the manufacturer’s instructions.

Summary of procedure:

• 4-15% Mini-PROTEAN ^® TGX™ Precast Protein Gels, 15-well, 15 mI_ were used for the experiments

• 1X SDS TG running buffer was used

• The PLA2R1 -protein Master Mix (MM) was prepared as given in Table 1.

• MM was boiled for 10 min at 97°C and 800 rpm in a Thermoshaker

• MM was cooled down to room temperature and centrifuged down shortly

• Gels were loaded in an alternating manner: 10 pL MM // 3 pl_ protein ladder

• gel-running parameters: 10 min at 80 V; ca. 50 min 160 V until blue dye front reached the lower end of the gel, but was still visible within the gel

Table 1 : Recipe for Master Mix

* The amount used varies depending on the batch of recombinant fu l-length PLA2R positive cell lysate. Here 2.2 pg/lane were loaded. 3. Western blot

3.1. Protein transfer with the Trans-Blot Turbo Transfer System by Biorad The protein transfer was performed according to the manufacturer’s instructions.

3.2. Blocking and antibody staining

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. Notably, during the blocking step, 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. In general the following setup was used (Figure 1): 6 membrane strips were incubated in MN patient serum of interest, one strip into a positive control and one strip into a negative control. The samples were incubated for a minimum of 18-20 hours at 4°C and 15 rpm on a vertical shaker. After incubation with human MN serum the membrane strips were washed 4x with 10-15 ml_ washing buffer for 5 minutes each on a vertical shaker at 15 rpm. The membrane stripes were then transferred to Petri dishes with secondary antibody (5 ml_ of 1:24.000 dilution in blocking buffer) and incubated for 1.5 hours at room temperature on a vertical shaker at 15 rpm. Then the membrane strips were washed 4x with 10-15 ml_ washing buffer for 5 min each on a vertical shaker at 15 rpm.

3.3. Development

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. For MN patient sera not giving any detectable signal, but having low background, the development step was repeated after a 2 min wash in washing buffer with Super Signal West Femto (Biorad) until the same endpoints.

4. Used chemicals and devices:

4.1. Chemicals

4.2. Buffer Recipes

All buffers were prepared in Aqua B. Braun distilled water.

Lysis-buffer (home made):

50 mM Tris (Base) pH 7.4 150 mM NaCI 1 M EDTA 1% (v/v) Triton X- 100 1x Proteaseinhibor

5x Laemmli, non-reducing (home made):

300 mM Tris HCI pH 6.8 50% (v/v) Glycerin 10% (w/v) SDS A trace of Bromophenolblau

SDS-PAGE running buffer:

1x TG SDS buffer 1x transfer buffer (home made):

25 mM Tris (Base)

192 mM Glycine 20% (v/v) Methanol Blocking buffer:

3.5% (w/v) skimmed milk powder in PBS 0.1% (v/v) Tween-20

4.3. Secondary Antibodies

4.4. Consumables 4.5. Devices

Patients:

Serum samples from eight patients suffering from a relapse following therapy-induced remission were taken at various time points before the relapse.

Results:

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.

More specifically, in the case of patient 1, PLA2R antibodies could be detected using Western blotting fifteen months earlier than using ELISA prior to a relapse. In the case of patient 2 and 3, detection of the antibody could be performed nine or three months earlier, respectively.

A group of five patients after a therapeutically induced remission who did not go through a relapse was also monitored, but no increase in PLA2R autoantibody levels could be detected, showing that the increase is associated with an increased risk of a relapse.

In the case of all 3 patients proteinuria was observed as part of a relapse, which followed the detection and concentration increase of PLA2R autoantibodies. Comparison of ELISA and Western blotting shows that the latter method may be used to resolve increases in concentration well below 14 RU/ml if optimized for this purpose. For example, in patient 2, the Western blot shows the absence of autoantibody 63 months after the MN diagnosis, but a clear band 9 months later and a noticeably stronger band (approximately at least 100% stronger) another 3 months later. At the same time, there is hardly a change in the PLA2r autoantibody levels as detected by ELISA. In fact, the ELISA in indicates a small decrease in the concentration between the first two samples, while Western blotting shows that the concentration actually increases.