WO2025045894A1 - Methods and kits for diagnosing cause of nephrotic syndrome and guiding therapy - Google Patents

Abstract

The present invention describes the identification of a soluble glomerular permeability factor, anti-Vasorin (or anti-VASN) autoantibodies synthesized by immune system cells, which opens up new perspectives for pathophysiological understanding, monitoring, and therapy of nephrotic syndrome. Clinical applications can include strategies for preventing the action of autoantibodies against said podocyte protein, inhibiting the production of antibodies against this protein, or eliminating these autoantibodies. Although the exact role of anti-VASN autoantibodies and VASN in nephrotic syndrome is still not well understood, the presence of circulating autoantibodies against VASN is highly specific to nephrotic syndrome. Up to now, no anti-VASN autoantibodies have been described in healthy individuals. Thus, the present invention relates to methods and kits for determining whether a subject has or is at risk of having a nephrotic syndrome associated with anti-VASN auto-antibodies or not.

Description

METHODS AND KITS FOR DIAGNOSING CAUSE OF NEPHROTIC SYNDROME AND GUIDING THERAPY

FIELD OF THE INVENTION:

The present invention is in the field of medicine, in particular nephrology.

BACKGROUND OF THE INVENTION:

Nephrotic syndrome (NS) is defined by abundant proteinuria (characterized by proteinuria to creatininuria ratio greater than 0.2 g/mmol or greater than 2 g/g on a urine sample or by a protein output greater than 50 mg/kg/d on a urine sample) associated with hypoalbuminemia of less than 30 g/1. A spot protein/creatinine ratio greater than 200 mg/mmol in children and protein/creatinine ratio>300-350mg/mmol in adults is consistent with NS, as is a 24-hour urine collection that reveals a total protein greater than 3 to 3.5 g/24 hour in adults. A measurement of 40mg/m2/hour or greater (lg/m2/24hour) is indicative of proteinuria consistent with NS in children. Clinical signs include painless, declining soft white edema and the detection of proteinuria. In children, nephrotic syndrome is associated in 90% of cases with a disease known as idiopathic nephrotic syndrome (INS) (or nephrosis or pure nephrotic syndrome in children). In adults, INS is also known as minimal change nephrotic syndrome (MCNS) or minimal change disease (MCD), depending on the histological findings on the kidney biopsy that is routinely performed. Occasionally, glomerular lesions, particularly podocyte lesions, are evident, and the nephrotic syndrome is due to focal segmental glomerulosclerosis (FSGS), which is resistant to immunosuppressants, progresses to severe renal failure and frequently recurs on kidney transplants. Symptomatic treatment includes fluid intake management and other therapies depending on the risks and complications (antihypertensive, anticoagulant, anti- infectives). Specific treatments rely primarily on glucocorticosteroids and other broad-spectrum immunosuppressants, with considerable deleterious side effects, especially in growing children but also in adults. Therefore, there is a much-needed medical need for corticosteroid-sparing agents and cause-specific treatments.

SUMMARY OF THE INVENTION:

The claims define the present invention. In particular, the present invention relates to methods and kits for determining whether a subject has or is at risk of having a nephrotic syndrome associated with anti-VASN auto-antibodies or not. The present invention also relates to methods for treating nephrotic syndromes associated with anti-VASN autoantibodies. DETAILED DESCRIPTION OF THE INVENTION:

The present invention describes the identification of a soluble glomerular permeability factor, anti-Vasorin (or anti-VASN) autoantibodies synthesized by immune system cells, which opens up new perspectives for pathophysiological understanding, monitoring, and therapy of nephrotic syndrome. Clinical applications can include strategies for preventing the action of autoantibodies against said podocyte protein, inhibiting the production of antibodies against this protein, or eliminating these autoantibodies. Although the exact role of anti-VASN autoantibodies and VASN in nephrotic syndrome is still not well understood, the presence of circulating autoantibodies against VASN is highly specific to nephrotic syndrome. Up to now, no anti-VASN autoantibodies have been described in healthy individuals.

Thus, the first object of the present invention relates to a method of diagnosing the occurrence of nephrotic syndrome (NS) or focal segmental glomerulosclerosis (FSGS) in a subject comprising the steps of i) detecting the presence of anti-VASN autoantibodies in a sample from the subject and ii) diagnosing the occurrence of NS or FSGS if anti-VASN autoantibodies are detected in the said sample.

As used herein, the term “subject” or “patient” refers to any mammal, such as a rodent, a feline, a canine, and a primate. Notably, in the present invention, the subject is a human. In some embodiments, the subject is a human who is susceptible to having a nephrotic syndrome (NS) or focal segmental glomerulosclerosis (FSGS).

As used herein, the term “nephrotic syndrome” (NS) refers to a rare disease defined by massive proteinuria (>3g/day, or >lg of urine protein per square meter of body-surface area per day in children) and hypoalbuminemia (< 30g/l) and result from loss of integrity of the glomerular filtration barrier. The leading causes are genetic and immune. More particularly the method of the present invention is particularly suitable for diagnosing the occurrence of an idiopathic nephrotic syndrome. As used herein, the term "idiopathic nephrotic syndrome" (INS) has its general meaning in the art and represents 80% of the causes of nephrotic syndrome in children and 25% in adults. Minimal change disease (MCD) is one of the most common causes of INS in children. It accounts for 70% to 90% of children that present with nephrotic syndrome who are older than one year old as opposed to 10-15% of adults who present with nephrotic syndrome. INS is histologically characterized by the absence of lesions on light microscopy (in that case a so called MCD) and the lack of immunoglobulin or complement deposit with sometimes additional focal segmental glomerulosclerosis (FSGS) lesions. In fact, several authors believe that MCD and primary FSGS are the same disease, the second being a more advanced stage of the first, where glomerular lesions can be seen by light microscopy (Maas RJ, Deegens JK, Smeets B. et al. Minimal change disease and idiopathic FSGS: manifestations of the same disease. Nat Rev Nephrol 2016; 12: 768-776). Membranous nephropathy (MN), also known as membranous glomerulopathy, is one of the many glomerular diseases causing nephrotic syndrome. It is characterized by proteinuria, presenting with peripheral edema and frothy urine. The etiology can be primary or secondary.

In particular embodiments, regarding all the methods of the invention, the nephrotic syndrome is an idiopathic nephrotic syndrome. In particular embodiments, regarding all the methods of the invention, the nephrotic syndrome is a nephrotic syndrome with minimal change disease (NS with MCD) or a nephrotic syndrome with focal segmental glomerulosclerosis (NS with FSGS). In particular embodiments, regarding all the methods of the invention, the nephrotic syndrome is a nephrotic syndrome with membranous nephropathy (NS with MN).

A further object of the present invention relates to an in vitro or ex vivo method of determining whether a subject has or is at risk of having a nephrotic syndrome (NS) in a samplefrom the subject wherein the presence of anti-VASN autoantibodies indicates that the subject has or is at risk of having a NS.

More particularly the method of the present invention is particularly suitable for diagnosing the occurrence of NS with MCD, with FSGS or with MN. More particularly the method of the present invention is particularly suitable for diagnosing the occurrence of an antibody -mediated NS with MCD, with FSGS or with MN, and in particular, anti-VASN-mediated NS with MCD, with FSGS or with MN.

As used herein, the term "risk", in the context of the present invention, relates to the probability that an event will occur over a specific period and can mean a subject's "absolute" risk or "relative" risk. Absolute risk can be measured with reference to either actual observation postmeasurement for the relevant time cohort or with reference to index values developed from statistically valid historical cohorts that have been followed for the relevant time period. Relative risk refers to the ratio of a subject's absolute risks compared to the absolute risks of low-risk cohorts or an average population risk, which can vary by how clinical risk factors are assessed. Odds ratios, the proportion of positive events to negative events for a given test result, are also commonly used (odds are according to the formula p/(l-p) where p is the probability of an event and (1- p) is the probability of no event) to no- conversion. "Risk evaluation," or "evaluation of risk" in the context of the present invention, encompasses predicting the probability, odds, or likelihood that an event or disease state may occur, the rate of occurrence of the event or conversion from one disease state to another. Risk evaluation can also comprise the prediction of future clinical parameters, traditional laboratory risk factor values, or other indices of relapse, either in absolute or relative terms in reference to a previously measured population. The methods of the present invention may be used to make continuous or categorical measurements of the risk of conversion, thus diagnosing and defining the risk spectrum of a category of subjects defined as being at risk of conversion. In the categorical scenario, the invention can be used to discriminate between normal and other subject cohorts at higher risk. In some embodiments, the present invention may be used so as to discriminate those at risk from normal.

In some embodiments, the method of diagnosing described herein is applied to a subject who presents symptoms of NS without having undergone routine screening to rule out all possible causes for NS. The methods described herein can be part of the routine set of tests performed on a subject who presents symptoms of NS such as painless, declining soft white oedema and the detection of abundant proteinuria (i.e. proteinuria to creatininuria ratio greater than 0.2 g/mmol or greater than 2 g/g on a urine sample, or by a protein output greater than 50 mg/kg/d on a urine sample). The method of the present invention can be carried out in addition to other diagnostic tools that include, for instance, a biopsy.

As used herein, the term “sample” refers to any sample obtained from the subject for the purpose of performing the method of the present invention. In some embodiments, the sample is a bodily fluid (e.g. a blood sample) or a tissue. In some embodiments, the sample is a tissue sample. The term “tissue sample” includes sections of tissues such as biopsy or autopsy samples, fixed or and frozen sections taken for histological purposes. In some embodiments, the tissue sample may result from a biopsy performed in the patient's kidney. Typically, the tissue sample is suspected of containing glomerular deposits. As used herein, the term “blood sample” means any blood sample derived from the subject. Collections of blood samples can be performed by methods well known to those skilled in the art. In some embodiments, the blood sample is a serum or plasma sample.

In a particular embodiment, the sample has been previously obtained from the subject. As used herein, the term “Vasorin” or “VASN” refers to a typical type I membrane protein containing tandem arrays of a characteristic leucine-rich repeat motif, an epidermal growth factor-like motif, and a fibronectin type Ill-like motif at the extracellular domain. VASN is an evolutionarily conserved single-pass type I transmembrane protein, with high similarity at the DNA (95%) and protein (83%) levels between rodent and human homologs, suggesting highly conserved functions. An exemplary amino acid sequence is shown as SEQ ID NO: 1. Vasorin OS=Homo sapiens OX=9606

MCSRVPLLLPLLLLLALGPGVQGCPSGCQCSQPQTVFCTARQGTTVPRDVPPDTVGLYVF

ENGITMLDAGSFAGLPGLQLLDLSQNQIASLPSGVFQPLANLSNLDLTANRLHEITNETF

RGLRRLERLYLGKNRIRHIQPGAFDTLDRLLELKLQDNELRALPPLRLPRLLLLDLSHNS

LLALEPGILDTANVEALRLAGLGLQQLDEGLFSRLRNLHDLDVSDNQLERVPPVIRGLRG

LTRLRLAGNTRIAQLRPEDLAGLAALQELDVSNLSLQALPGDLSGLFPRLRLLAAARNPF

NCVCPLSWFGPWVRESHVTLASPEETRCHFPPKNAGRLLLELDYADFGCPATTTTATVPT

TRPWREPTALSSSLAPTWLSPTEPATEAPSPPSTAPPTVGPVPQPQDCPPSTCLNGGTC

HLGTRHHLACLCPEGFTGLYCESQMGQGTRPSPTPVTPRPPRSLTLGIEPVSPTSLRVGL

QRYLQGSSVQLRSLRLTYRNLSGPDKRLVTLRLPASLAEYTVTQLRPNATYSVCVMPLGP

GRVPEGEEACGEAHTPPAVHSNHAPVTQAREGNLPLLIAPALAAVLLAALAAVGAAYCVR

RGRAMAAAAQDKGQVGPGAGPLELEGVKVPLEPGPKATEGGGEALPSGSECEVPLMGFPG PGLQSPLHAKPYI

As used herein, the term "antibodies" or “immunoglobulins” has its general meaning in the art and relates to proteins of the immunoglobulin superfamily. The antibodies are characterized by a structural domain, i.e., the immunoglobulin domain, having a characteristic immunoglobulin (Ig) fold. The term encompasses secretory immunoglobulins. Immunoglobulins generally comprise several chains, typically two identical heavy chains and two identical light chains linked via disulfide bonds. These chains are primarily composed of immunoglobulin domains, including the VL domain (light chain variable domain), the CL domain (light chain constant domain), the VH domain (heavy chain variable domain) and the CH domains (heavy chain constant domains), CHI, optionally a hinge region, CH2, CH3, and optionally CH4. There are five main heavy chain classes (or isotypes) that determine the functional activity of an antibody molecule: mu (p) for IgM, delta (5) for IgD, gamma (y) for IgG, alpha (a) for IgA and epsilon (a) for IgE. In the context of the invention, the immunoglobulin may be an IgM, IgD, IgG, IgA or IgE. Preferably, the immunoglobulin is an IgG. As well-known by the skilled person, the IgG isotype encompasses four subclasses: IgGl, lgG2, lgG3 and lgG4. In the context of the invention, the immunoglobulin may be of any IgG subclass. Preferably, the immunoglobulin is an IgGl As used herein, the term “anti-VASN autoantibodies” refers to the antibodies produced by the subject's immune system and directed against the subject's VASN own protein.

In some embodiments, the method of the present invention comprises i) determining the level of anti-VASN autoantibodies in a sample from the subject, ii) comparing the level determined at step i) with a predetermined reference value and iii) concluding that the subject has or is at risk of having a NS when the level determined at step i) is higher than the predetermined reference value.

Typically, the predetermined reference value is a threshold or cutoff value. Typically, a "threshold value" or "cutoff value" can be determined experimentally, empirically, or theoretically. A threshold value can also be arbitrarily selected based on the existing experimental and/or clinical conditions, as would be recognized by a person of ordinary skill in the art. For example, retrospective measurement in properly banked historical subject samples may be used in establishing the predetermined reference value. The threshold value has to be determined to obtain the optimal sensitivity and specificity according to the function of the test and the benefit/risk balance (clinical consequences of false positive and false negative). Typically, the optimal sensitivity and specificity (and the threshold value) can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data. For example, after determining the level of anti-VASN autoantibodies in a group of reference, one can use algorithmic analysis to statistically treat the levels determined in samples to be tested and thus obtain a classification standard having significance for sample classification. The full name of the ROC curve is the receiver operator characteristic curve, also known as the receiver operation characteristic curve. It is mainly used for clinical and biochemical diagnostic tests. The ROC curve is a comprehensive indicator that reflects the continuous variables of true positive rate (sensitivity) and false positive rate (1-specificity). It reveals the relationship between sensitivity and specificity with the image composition method. A series of different cutoff values (thresholds or critical values, boundary values between normal and abnormal diagnostic test results) are set as continuous variables to calculate a series of sensitivity and specificity values. Then sensitivity is used as the vertical coordinate, and specificity is used as the horizontal coordinate to draw a curve. The higher the area under the curve (AUC), the higher the accuracy of diagnosis. On the ROC curve, the point closest to the far upper left of the coordinate diagram is a critical point with high sensitivity and specificity values. The AUC value of the ROC curve is between 1.0 and 0.5. When AUC>0.5, the diagnostic result improves as AUC approaches 1. When AUC is between 0.5 and 0.7, the accuracy is low. When AUC is between 0.7 and 0.9, the accuracy is moderate. When AUC is higher than 0.9, the accuracy is high. This algorithmic method is preferably done with a computer. Existing software or systems in the art may be used to draw the ROC curve, such as MedCalc 9.2.0.1 medical statistical software, SPSS 9.0, ROCPOWER.SAS, DESIGNROC.FOR, MULTIREADER POWER. SAS, CREATE-ROC.SAS, GB STAT VIO.O (Dynamic Microsystems, Inc. Silver Spring, Md., USA), etc.

In some embodiments, the predetermined reference value is the level of anti-VASN autoantibodies determined in a population of healthy individuals. Typically, it is concluded that the patient suffers from NS when the level of anti-VASN autoantibodies is at least 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 100 fold higher than the level determined in a population of healthy individuals.

Accordingly, high levels of anti-VASN autoantibodies indicate that the subject is at high risk of having NS. Conversely, low levels of anti-VASN autoantibodies indicate that the subject is at low risk of having NS. In particular embodiment, high levels of anti-VASN autoantibodies indicate that the subject is at high risk of having anti-VASN antibodies-mediated NS with MCD, or with FSGS or with MN, and low levels of anti-VASN autoantibodies indicate that the subject is at low risk of having anti-VASN antibodies-mediated NS with MCD, or with FSGS or with MN.

As used herein, the term “high” refers to a measure that is significantly greater than normal, greater than a standard, such as a predetermined reference value or a subgroup measure, or that is relatively greater than another subgroup measure. For example, high levels of anti-VASN autoantibodies refers to a level of anti-VASN autoantibodies greater than a normal anti-VASN autoantibodies level. A normal anti-VASN autoantibodies level may be determined according to any method available to one skilled in the art. A high level of anti-VASN autoantibodies may also refer to a level equal to or greater than a predetermined reference value, such as a predetermined cutoff. A high level of anti-VASN autoantibodies may also refer to a level of anti-VASN autoantibodies wherein a high anti-VASN autoantibodies subgroup has relatively greater levels of anti-VASN autoantibodies than another subgroup. For example, without limitation, according to the present specification, two distinct patient subgroups can be created by dividing samples around a mathematically determined point, such as, without limitation, a median, thus creating a subgroup whose measure is high (i.e., higher than the median) and another subgroup whose measure is low. In some cases, a “high” level may comprise a range of levels that is very high and a range of levels that is “moderately high”, where moderately high is a level that is greater than normal but less than “very high”.

As used herein, the term “low” refers to a level that is less than normal or less than a standard, such as a predetermined reference value or a subgroup measure that is relatively less than another subgroup level. For example, a low level of anti-VASN autoantibodies means a level of anti-VASN autoantibodies that is less than a normal level in a particular set of samples of patients. A normal level of anti-VASN autoantibodies measurement may be determined according to any method available to one skilled in the art. A low level of anti-VASN autoantibodies may also mean a level less than a predetermined reference value, such as a predetermined cutoff. A low level of anti-VASN autoantibodies may also mean a level wherein a low-level anti-VASN autoantibodies subgroup is relatively lower than another subgroup. For example, without limitation, according to the present specification, two distinct patient subgroups can be created by dividing samples around a mathematically determined point, such as, without limitation, a median, thus creating a group whose measure is low (i.e., less than the median) with respect to another group whose measurement is high (i.e., greater than the median).

A further object of the present invention relates to a method of predicting the risk of relapse in a subject suffering from NS i) comprising determining the level of anti-VASN autoantibodies in a sample from the subject ii) comparing the level determined at step i) with a predetermined reference value and iii) concluding that the subject is at risk of relapse when the level determined at step i) is higher than the predetermined reference value.

As used herein, the term "relapse" refers to the return of signs and symptoms of a disease after a subject has enjoyed a remission after treatment. Thus, if initially, the target disease is alleviated or healed, or the progression of the disease is halted or slowed down and subsequently, when the disease or one or more characteristics of the disease resume (e.g., proteinuria), the subject is referred to as being "relapsed." Typically, the treatment is immunosuppressive.

Typically, high levels of anti-VASN autoantibodies indicate that the subject is at high risk of relapse, whereas low levels of anti-VASN autoantibodies indicate that the subject is at low risk of relapse. A further object of the present invention relates to a method of determining whether a subject suffering from NS achieves a response with treatment comprising i) determining the level of anti-VASN autoantibodies in a sample from the subject before the treatment, ii) determining the level of anti-VASN autoantibodies in a sample from the subject after treatment initiation, iii) comparing the level determined at step i) with the level determined at step ii) and concluding that the subject achieves a response when the level determined at step ii) is lower than the level determined at step i).

In some embodiments, when it is concluded that the treatment exhibits no effect anymore or is not efficient, the treatment is adapted or modified.

Thus, the invention of the method can also be used to monitor the end of treatment.

The method is thus particularly suitable for discriminating responders from non-responders. As used herein, the term “responder” in the context of the present disclosure refers to a subject that will achieve a response, i.e., a subject who is under remission and, more particularly, a subject who does not suffer from proteinuria. A non-responder subject includes subjects for whom the disease does not show reduction or improvement after the treatment (e.g., the proteinuria remains stable or decreases).

A further object of the present invention relates to a method of determining whether a subject suffering from NS achieves remission comprising i) determining the level of anti-VASN autoantibodies in a sample from the subject before the treatment, ii) determining the level of anti-VASN autoantibodies in a sample from the subject after the treatment completion, iii) comparing the level determined at step ii) with the level determined at step i) and concluding that the subject achieves remission when the level determined at step ii) is lower than the level determined at step i).

In a particular embodiment, it is concluded that the subject achieves remission when the level of anti-VASN autoantibodies determined in step ii) is lower than the level determined at step i) and is similar to a basal level (i.e., the level determined in a population of healthy individuals). In particular embodiments, when it is concluded that the subject achieves remission, the treatment is reduced or interrupted.

As used herein, the term “remission” refers to a decrease in or diseappearance of the symptoms of NS, i.e the subject’ NS is alleviated or healed. Complete remission can be defined as the absence of proteinuria for at least 3 days, and sustained remission is defined as the absence of relapse over at least 12 months. In particular embodiment regarding all the methods of the invention, the subject suffers from an NS with MCD or with FSGS, more parti culary from an antibody-mediated NS with MCD or with FSGS, and more particularly from VASN autoantibodies-mediated NS with MCD or with FSGS. In particular embodiment regarding all the methods of the invention, the subject suffers from an NS with MN, more particularly from an antibody-mediated NS with MN, and more particularly from VASN autoantibodies-mediated NS with MN.

According to the present invention, the treatment consists of any method or drug suitable for treating NS, in particular through interference with anti-VASN actions.

In some embodiments, the treatment is immunosuppressive. As used herein, the term “immunosuppressive treatment” refers to any substance capable of producing an immunosuppressive effect, e.g., the prevention or diminution of the immune response and, in particular, the prevention or diminution of the production of Ig. Immunosuppressive drugs include, without limitation, thiopurine drugs such as azathioprine (AZA) and metabolites thereof; nucleoside triphosphate inhibitors such as mycophenolic acid (Cellcept) and its derivative (Myfortic); derivatives thereof; prodrugs thereof; and combinations thereof. Other examples include but are not limited to 6-mercaptopurine ("6-MP"), cyclophosphamide, mycophenolate, prednisolone, sirolimus, dexamethasone, rapamycin, FK506, mizoribine, azathioprine and tacrolimus.

In some embodiments, the immunosuppressive drug is a calcineurin inhibitor. As used herein, the term “calcineurin inhibitor” has its general meaning in the art and refers to substances that block calcineurin (i.e., calcium/calmodulin-regulated protein phosphatase involved in intracellular signaling) dephosphorylation of appropriate substrates, by targeting calcineurin phosphatase (PP2B, PP3), a cellular enzyme that is involved in gene regulation. A calcineurin inhibitor of the present invention is typically an immunophilin-binding compound having calcineurin inhibitory activity. Immunophilin-binding calcineurin inhibitors form calcineurin- inhibiting complexes with immunophilins, e.g., cyclophilin and macrophilin. Examples of cyclophilin-binding calcineurin inhibitors are cyclosporines or cyclosporine derivatives (hereinafter cyclosporines), and examples of macrophilin-binding calcineurin inhibitors are ascomycin (FR 520) and ascomycin derivatives (hereinafter ascomycins). A wide range of ascomycin derivatives are known, either naturally occurring among fungal species or are obtainable by manipulating fermentation procedures or by chemical derivatization. Ascomycin- type macrolides include ascomycin, tacrolimus (FK506), sirolimus, and pimecrolimus. Cyclosporine, originally extracted from the soil fungus Potypaciadium infilatum, has a cyclic 11 -amino acid structure and includes, e.g., Cyclosporines A through I, such as Cyclosporine A, B, C, D, and G. Voclosporin is a next-generation calcineurin inhibitor that is a more potent and less toxic semi -synthetic derivative of cyclosporine A. In some embodiments, the calcineurin inhibitor of the present invention is the trans-version of voclosporin, trans-ISA247 (Cas number 368455-04-3), which is described in, for example, US Patent Publication No.: 2006/0217309, which is hereby incorporated herein by reference. Further compositions of voclosporin are described, for example, in U.S. Pat. No. 7,060,672, which is hereby incorporated herein by reference. Tacrolimus (FK506) is another calcineurin inhibitor which is also a fungal product but has a macrolide lactone structure. Sirolimus (rapamycin) is a microbial product isolated from the actinomycete Streptomyces hygroscopicus. Sirolimus binds to an immunophilin (FK- binding protein 12, FKBP12), forming a complex that inhibits the mammalian target of the rapamycin (mTOR) pathway by directly binding the mTOR Complexl (mTORCl). Pimecrolimus is also a calcineurin inhibitor. Calcineurin inhibitors such as cyclosporine A, voclosporin, ascomycin, tacrolimus, pimecrolimus, an analogue thereof, or a pharmaceutically acceptable salt thereof, can be utilized in a mixed micellar composition of the present disclosure.

In some embodiments, the immunosuppressive drug is a corticosteroid. As used, the term “corticosteroids” has its general meaning in the art and refers to a class of active ingredients having a hydrogenated cyclopentoperhydrophenanthrene ring system endowed with antiinflammatory activity. Corticosteroid drugs typically include cortisone, cortisol, hydrocortisone (113,17-dihydroxy, 21-(phosphonooxy)-pregn-4-ene, 3,20-dione disodium), dihydroxy corti sone, dexamethasone (21 -(acetyloxy)-9-fluoro-l P, 17-dihydroxy- 16a-m- ethylpregna-l,4-diene-3, 20-dione), and highly derivatized steroid drugs such as beconase (beclomethasone dipropionate, which is 9-chloro-l l-P, 17,21, trihydroxy- 16P-methylpregna- 1,4 diene-3, 20-dione 17,21 -dipropionate). Other examples of corticosteroids include flunisolide, prednisone, prednisolone, methylprednisolone, triamcinolone, deflazacort and betamethasone, cortisone, hydrocortisone, methylprednisolone, prednisone, prednisolone, beclomethasone dipropionate, budesonide, dexamethasone sodium phosphate, flunisolide, fluticasone propionate, fluocinonide, betamethasone valerate, desonide, desoximetasone, fluocinolone, triamcinolone, triamcinolone acetonide, clobetasol propionate, and dexamethasone. A decrease in the level after the treatment compared to the level before the treatment of at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, 100% typically indicated that the subject achieves a response. More preferably, a level that returns to the basal level (i.e., the level determined in a population of healthy individuals) indicates that the subject has achieved a response. More preferably, a level that returns to the basal level after treatment completion (i.e., the level determined in a population of healthy individuals) indicates that the subject achieves remission.

The detection and quantification of anti-VASN autoantibodies in the sample can be detected by any method known in the art.

Typically, the detection and quantification are performed by Enzyme-linked immunosorbent assay, also called ELISA, enzyme immunoassay, or EIA, a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample. A known amount of antigen (VASN or antigenic fragment thereof) is immobilized on solid support (e.g., a polystyrene microtiter plate) either non-specifically (via adsorption to the surface) or specifically (via capture by another antibody specific to the same antigen, in a "sandwich" ELISA). Then, the sample, suspected of containing anti-VASN autoantibodies, is washed over the surface so that the auto-antibodies can bind to the immobilized antigen. The surface is washed to remove any unbound protein, and a detection antibody is applied to the surface. The detection antibody should be an anti-human Ig antibody. The detection antibody can be covalently linked to an enzyme or can be detected by a secondary antibody linked to an enzyme through bio-conjugation. Enzymes which can be used to detectably label the antibodies of the present invention include but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta- V- steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose- VI- phosphate dehydrogenase, glucoamylase and acetylcholinesterase. The plate is typically washed with a mild detergent solution between each step to remove any proteins or antibodies that are not specifically bound. After the final wash step, the plate is developed by adding an enzymatic substrate to produce a visible signal, which indicates the quantity of antigen in the sample. In some embodiments, a competitive ELISA is used. Purified anti-VASN antibodies that are not derived from the subject are coated on the solid phase of multi -wells. Serum sample recombined VASN (the antigen) or fragments thereof and horseradish peroxidase-labeled with anti-VASN antibodies (conjugated) are added to coated wells and form competitive combination. After incubation, if the auto-antibody level against VASN content is high in the sample, a complex of VASN-auto-antibodies-anti-VASN labeled with HRP will form. Wash wells will remove the complex and incubate with TMB (3, 3', 5, 5'- tetramethylbenzidene) color development substrate for localization of horseradish peroxidase-conjugated antibodies in the wells. Subsequently, there will be no color change or little color change. If there are no auto- anti-VASN autoantibodies in the serum sample, there will be much color change. Such a competitive ELSA test is specific, sensitive, reproducible, and easy to operate. In some embodiments, the detection antibody is labeled with a fluorescent compound. When the fluorescently labeled antibody is exposed to light of the proper wavelength, its presence can then be detected due to fluorescence. Among the most commonly used fluorescent labeling compounds are CY dyes, fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, and fluorescamine. Other fluorophores known to those skilled in the art can also be used, for example, those available from Life Technologies (Invitrogen; Molecular Probes (Eugene, Oreg.)) and including the ALEXA FLUOR® series of dyes (for example, as described in U.S. Pat. Nos. 5,696,157, 6, 130, 101 and 6,716,979), the BODIPY series of dyes (dipyrrometheneboron difluoride dyes, for example as described in U.S. Pat. Nos. 4,774,339, 5,187,288, 5,248,782, 5,274,113, 5,338,854, 5,451,663 and 5,433,896), Cascade Blue (an amine-reactive derivative of the sulfonated pyrene described in U.S. Pat. No. 5,132,432) and Marina Blue (U.S. Pat. No. 5,830,912). In some embodiments, the detection antibody can also be detectably labelled using fluorescence-emitting metals such as¹⁵²Eu, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentaacetic acid (DTP A) or ethylenediaminetetraacetic acid (EDTA).

In some embodiments, the detection antibody is detectably labeled by coupling it to a chemiluminescent compound (chemiluminescent immunoassay, CLIA). The presence of the chemiluminescent antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of useful chemiluminescent labeling compounds are luminol, luciferin, isoluminol, theromatic acridinium ester, imidazole, acridinium salt, and oxalate ester. In some embodiments, an automated assay systems is used and include, e.g., the BIO-FLASH™, the BEST 2000™, the DS2™, the ELx50 WASHER, the ELx800 WASHER, the ELx800 READER, and the Autoblot S20™ (INOVA Diagnostics, Inc., San Diego, CA). In some embodiments, the immunoassays comprise beads coated with native or recombinant VASN protein as described. Commonly used are beads that are dyed to establish a unique identity. Detection is performed by flow cytometry. Autoantibody detection using multiplex technologies. Other types of bead-based immunoassays are well known in the art, e.g., laser bead immunoassays and related magnetic bead assays (also known as magnetic particle chemiluminescence immunoassay, MP-CLIA) (Fritzler, Marvin J; Fritzler, Mark L, Expert Opinion on Medical Diagnostics, 2009, pp. 3: 81-89). In some embodiments, the method of the present invention involves the use of multiplex technology. Multiplex technology is the collective term for a variety of techniques that can assess multiple antibody specificities simultaneously on small volumes of blood samples. The advantage of multiplex technology is that it is able to provide very rapid test times and a very high throughput of samples. Thus, in some embodiment, the assay is a chemiluminescence immunoassay, particularly a magnetic particle chemiluminescence immunoassay (MP-CLIA).

One such technique is the addressable laser bead immunoassay (ALBIA), which is commercially available on Luminex™ based platforms. For instance, ALBIA is a semi- quantitative homogenous fluorescence-based microparticle immunoassay that can be used to simultaneously detect several autoantibodies (e.g., up to 10 autoantibodies). Each antigen (e.g., VASN) is covalently coupled to a set of distinct uniform size color-coded microspheres. The blood sample is then incubated with microspheres in a filter membrane-bottomed microplate. The beads are washed and then incubated for 3an anti-human Ig conjugated to a fluorescent label (e.g., phycoerythrin). After washing again, the beads are analyzed on a system in which separate lasers identify antigens by bead color and quantify the antibody by measuring the fluorescence of the fluorescent label. Said quantification thus indicates the level of the autoantibodies.

In some embodiments, a dot blot or a line blot is used to carry out the method of the present invention. In some embodiments, a test strip that has been coated with one or more bands of the VASN antigen purified, preferably to at least 80, 90, 95, or 99 % purity, prior to the coating procedure. If two or more antigens are used, they are preferably spatially separated. Preferably, the width of the bands is at least 30, more preferably 40, 50, 60, 70, or 80 % of the width of the test strip. The test strip may comprise one or more control bands for confirming that it has been contacted with sample sufficiently long and under sufficient conditions, particularly human serum, antibody conjugate, or both. In some embodiments, a flow path in a lateral flow immunoassay device is used. For example, the VASN antigen can be attached or immobilized on a porous membrane, such as a PVDF membrane (e.g., an Immobilon™ membrane), a nitrocellulose membrane, polyethylene membrane, nylon membrane, or a similar type of membrane.

In some embodiments, the level of anti-VASN autoantibodies in tissue samples (e.g. kidney biopsy) is determined by immunohistochemistry (IHC) or immunofluorescence (IF). Immunohistochemistry typically includes the following steps: i) fixing said tissue sample with formalin, ii) embedding said tissue sample in paraffin; alternatively, tissues that are fixed after snap-freezing, embedded in OCT compound prior to cryostat sectioning are suitable; iii) cutting said tissue sample into sections for staining, iv) incubating said sections with the binding partner specific for detecting anti-VASN autoantibodies, v) rinsing said sections, vi) incubating said section with a biotinylated secondary antibody and vii) revealing the antigen-antibody complex with avidin-biotin-peroxidase complex. Accordingly, the tissue sample is first incubated with the binding partners. After washing, the labeled antibodies that are bound to the marker of interest are revealed by the appropriate technique, depending on the kind of label is borne by the labeled antibody, e.g. radioactive, fluorescent or enzyme label. Multiple labelling can be performed simultaneously. Alternatively, the method of the present invention may use a secondary antibody coupled to an amplification system (to intensify the staining signal) and enzymatic molecules. Such coupled secondary antibodies are commercially available, e.g. from Dako, EnVision system or any suitable supplier. Counterstaining may be used, e.g. H&E, DAPI, Hoechst. Other staining methods may be accomplished using any suitable method or system as would be apparent to one of skill in the art, including automated, semi-automated or manual systems. For example, one or more labels can be attached to the antibody, thereby permitting detection of the target protein (i.e. the immune marker). Exemplary labels include radioactive isotopes, fluorophores, ligands, chemiluminescent agents, enzymes, and combinations thereof. In some embodiments, the label is a quantum dot. Non-limiting examples of labels that can be conjugated to primary and/or secondary affinity ligands include fluorescent dyes or metals (e.g. fluorescein, rhodamine, phycoerythrin, fluorescamine), chromophoric dyes (e.g. rhodopsin), chemiluminescent compounds (e.g. luminal, imidazole) and bioluminescent proteins (e.g. luciferin, luciferase), haptens (e.g. biotin). A variety of other useful fluorescers and chromophores are described in Stryer L (1968) Science 162:526-533 and Brand L and Gohlke J R (1972) Annu. Rev. Biochem. 41 :843-868. Affinity ligands can also be labeled with enzymes (e.g. horseradish peroxidase, alkaline phosphatase, beta-lactamase), radioisotopes (e.g. 3H, 14C, 32P, 35S or 1251) and particles (e.g. gold). The different types of labels can be conjugated to an affinity ligand using various chemistries, e.g. the amine reaction or the thiol reaction. However, other reactive groups than amines and thiols can be used, e.g. aldehydes, carboxylic acids and glutamine. Various enzymatic staining methods are known in the art of detecting a protein of interest. For example, enzymatic interactions can be visualized using different enzymes such as peroxidase, alkaline phosphatase, or different chromogens such as DAB, AEC or Fast Red. In other examples, the antibody can be conjugated to peptides or proteins that can be detected via a labeled binding partner or antibody. In an indirect IHC assay, a secondary antibody or second binding partner is necessary to detect the binding of the first binding partner, as it is not labeled. The resulting stained specimens are each imaged using a system for viewing the detectable signal and acquiring an image, such as a digital image of the staining. Methods for image acquisition are well known to one of skill in the art. For example, once the sample has been stained, any optical or non-optical imaging device can be used to detect the stain or biomarker label, such as, for example, upright or inverted optical microscopes, scanning confocal microscopes, cameras, scanning or tunneling electron microscopes, scanning probe microscopes and imaging infrared detectors. In some examples, the image can be captured digitally. The obtained images can then be used for quantitatively or semi-quantitatively determining the amount of the immune marker in the sample. Various automated sample processing, scanning and analysis systems suitable for use with immunohistochemistry are available in the art. Such systems can include automated staining and microscopic scanning, computerized image analysis, serial section comparison (to control for variation in the orientation and size of a sample), digital report generation, and archiving and tracking of samples (such as slides on which tissue sections are placed). Cellular imaging systems are commercially available that combine conventional light microscopes with digital image processing systems to perform quantitative analysis on cells and tissues, including immunostained samples. See, e.g., the CAS-200 system (Becton, Dickinson & Co.). In particular, detection can be made manually or by image processing techniques involving computer processors and software. Using such software, for example, the images can be configured, calibrated, standardized and/or validated based on factors including, for example, stain quality or stain intensity, using procedures known to one of skill in the art (see, e.g., published U.S. Patent Publication No. US20100136549). The image can be quantitatively or semi-quantitatively analyzed and scored based on the staining intensity of the sample. Quantitative or semi-quantitative histochemistry or immunofluorescence refers to method of scanning and scoring samples that have undergone histochemistry or immunofluorescence to identify and quantitate the presence of the specified biomarker (i.e. the protein marker). Quantitative or semi-quantitative methods can employ imaging software to detect staining densities or amount of staining or methods of detecting staining by the human eye, where a trained operator ranks results numerically. For example, images can be quantitatively analyzed using pixel count algorithms (e.g., Aperio Spectrum Software, Automated QUantitatative Analysis platform (AQUA® platform), and other standard methods that measure or quantitate or semi-quantitate the degree of staining; see, e.g., U.S. Pat. No. 8,023,714; U.S. Pat. No. 7,257,268; U.S. Pat. No. 7,219,016; U.S. Pat. No. 7,646,905; published U.S. Patent Publication No. US20100136549 and 20110111435; Camp et al. (2002) Nature Medicine, 8: 1323-1327; Bacus et al. (1997) Analyt Quant Cytol Histol, 19:316-328). A ratio of strong positive stain (such as a brown stain or fluorescence signal) to the sum of total stained area can be calculated and scored. The amount of the detected biomarker is quantified and given as a percentage of positive pixels and/or a score. For example, the amount can be quantified as a percentage of positive pixels. In some examples, the amount is quantified as the percentage of area stained, e.g., the percentage of positive pixels. For example, a sample can have at least or about at least or about 0, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more positive pixels as compared to the total staining area. In some embodiments, a score is given to the sample that is a numerical representation of the intensity or amount of the histochemical staining or of the specific antibody-associated fluorescence signal of the sample and represents the amount of target biomarker (e.g., the protein marker) present in the sample. Optical density or percentage area values can be given a scaled score, for example, on an integer scale.

A further object of the present invention relates to a kit or device for identifying the presence or the level of anti-VASN autoantibodies in a sample from a subject comprising: at least a VASN protein or fragments thereof; and at least one solid support wherein the VASN protein or fragments thereof is deposited on the support.

In other words, the present invention relates to use of a kit or device for identifying the presence or the level of anti-VASN autoantibodies in a sample from a subject comprising: at least a VASN protein or fragments thereof; and at least one solid support wherein the VASN protein or fragments thereof is deposited on the support.

In other words, the present invention relates to use of a kit or device according to the invention for perfoming the methods of the invention. In some embodiments, the VASN protein or fragments thereof that are deposited on the solid support are immobilized on the support. In some embodiments, the solid support is selected from the group comprising a bead, preferably a paramagnetic particle, a dipstick, a latex bead, a microsphere, a multi-well plate, a test strip, a microtiter plate, a blot (e.g., line blot and dot blot), a glass surface, a slide, a biochip, and a membrane. In some embodiments, the devices or kits described herein can further comprise a second labeled VASN protein or a fragment thereof that produces a detectable signal; a detection antibody, wherein the detection antibody is specific for the anti- VASN autoantibodies in the sample of the subject, and the detection antibody produces a detectable signal; or a nephelometer cuvette. In some embodiment, the detection antibody is specific to the species of the subject (i.e. specific to humans, rodents, pigs or primates). In some embodiments, the device performs an immunoassay wherein an antibodyprotein complex is formed, such as a serological immunoassay or a nephelometric immunoassay. In some embodiments, provided herein are kits comprising devices described herein and a detection antibody, wherein the detection antibody is specific for the anti-VASN autoantibodies in the subject's sample and produces a detectable signal. In some embodiments, the kit can include a second labeled VASN protein or a fragment thereof which produces a detectable signal. In some embodiments, the kits described herein further comprise standards of known amounts of the VASN or fragments thereof. In some embodiments, the kits described herein further comprise reference values of standards of known amounts of the VASN or fragments thereof to generated a standard titration curve. In some embodiments, the kits described herein further comprise reference values of the levels of anti-VASN antibodies.

The reference values are average levels of anti-VASN antibodies in samples from a population of healthy individuals. Reference values can be provided as numerical values or as standards of known amounts or titers of anti-VASN antibodies presented in pg/ml-pg/ml. In some embodiments, the kits described herein further comprise at least one sample collection container for sample collection. Collection devices and containers include but are not limited to syringes, lancets, BD VACUTAINER® blood collection tubes. In some embodiments, the kits described herein further comprise instructions for using the kit and interpretation of results.

In one embodiment, detection of auto-antibodies comprises identifying and detecting elevated amount of the mRNA that codes for the antibodies. Many methods of detecting, identifying and determining mRNA are well known in the art, e. g., Northern blots, RT-PCR, digital droplet PCR, and RNA sequencing. For example, the nucleic acid contained in the samples (e.g., cell or tissue prepared from the patient) is first extracted according to standard methods, for example, using lytic enzymes or chemical solutions or extracted by nucleic-acid- binding resins following the manufacturer's instructions. In one embodiment, the mRNA can be determined in biological fluids or isolated leukocyte fractions or tissues from the subject. Real-time PCR is an amplification technique that can be used to determine levels of mRNA expression. (See, e.g., Gibson et al., Genome Research 6:995-1001, 1996; Heid et al., Genome Research 6:986-994, 1996). Real-time PCR evaluates the level of PCR product accumulation during amplification. This technique permits quantitative evaluation of mRNA levels in multiple samples. For mRNA levels, mRNA is extracted from a biological sample, e.g. a blood sample, and cDNA is prepared using standard techniques. Real-time PCR can be performed, for example, using a Perkin Elmer/ Applied Biosystems (Foster City, Calif.) 7700 Prism instrument. Matching primers and fluorescent probes can be designed for genes of interest using, for example, the primer express program provided by Perkin Elmer/ Applied Biosystems (Foster City, Calif.). Optimal concentrations of primers and probes can be initially determined by those of ordinary skill in the art, and control (for example, beta-actin) primers and probes can be obtained commercially from, for example, Perkin Elmer/ Applied Biosystems (Foster City, Calif.). To quantify the amount of the specific nucleic acid of interest in a sample, a standard curve is generated using a control. Standard curves can be generated using the Ct values determined in the real-time PCR, which are related to the initial concentration of the nucleic acid of interest used in the assay. Standard dilutions ranging from 10-106 copies of the gene of interest are generally sufficient. In addition, a standard curve is generated for the control sequence. This permits standardization of the initial content of the nucleic acid of interest in a tissue sample to the amount of control for comparison purposes. Digital droplet PCR (ddPCR) is based on forming a water-in-oil emulsion, with each droplet having as little as one copy of the nucleic acid sample (based on dilution and statistical probability), along with the PCR reagents. Each droplet serves as a self-contained micro-reactor for PCR amplification.

Other methods of Amplification include ligase chain reaction (LCR), transcription-mediated amplification (TMA), strand displacement amplification (SDA) and nucleic acid sequencebased amplification (NASBA). Typically, the nucleic acid probes include one or more labels, for example to permit detection of a target nucleic acid molecule using the disclosed probes. In various applications, such as in situ hybridization procedures, a nucleic acid probe includes a label (e.g., a detectable label). A “detectable label” is a molecule or material that can be used to produce a detectable signal that indicates the presence or concentration of the probe (particularly the bound or hybridized probe) in a sample. Thus, a labeled nucleic acid molecule provides an indicator of the presence or concentration of a target nucleic acid sequence (e.g., genomic target nucleic acid sequence) (to which the labeled uniquely specific nucleic acid molecule is bound or hybridized) in a sample. A label associated with one or more nucleic acid molecules (such as a probe generated by the disclosed methods) can be detected either directly or indirectly. A label can be detected by any known or yet-to-be-discovered mechanism, including absorption, emission and/ or scattering of a photon (including radio frequency, microwave frequency, infrared frequency, visible frequency and ultra-violet frequency photons). Detectable labels include colored, fluorescent, phosphorescent and luminescent molecules and materials, catalysts (such as enzymes) that convert one substance into another substance to provide a detectable difference (such as by converting a colorless substance into a colored substance or vice versa, or by producing a precipitate or increasing sample turbidity), haptens that can be detected by antibody binding interactions, and paramagnetic and magnetic molecules or materials.

Particular examples of detectable labels include fluorescent molecules (or fluorochromes). Numerous fluorochromes are known to those of skill in the art and can be selected, for example, from Life Technologies (formerly Invitrogen), e.g., see, The Handbook — A Guide to Fluorescent Probes and Labeling Technologies). Examples of particular fluorophores that can be attached (for example, chemically conjugated) to a nucleic acid molecule (such as a uniquely specific binding region) are provided in U.S. Pat. No. 5,866, 366 to Nazarenko et al., such as 4-acetamido-4'-isothiocyanatostilbene-2,2' disulfonic acid, acridine and derivatives such as acridine and acridine isothiocyanate, 5-(2'-aminoethyl) aminonaphthalene- 1 -sulfonic acid (EDANS), 4-amino -N- [3 vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (Lucifer Yellow VS), N-(4-anilino-l-naphthyl)mal eimide, antllranilamide, Brilliant Yellow, coumarin and derivatives such as coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7- amino-4-trifluoromethylcouluarin (Coumarin 151); cyanosine; 4',6-diarninidino-2- phenylindole (DAPI); 5',5"dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red); 7 - diethylamino -3 - (4'-isothiocyanatophenyl)-4-methylcoumarin; diethylenetriamine pentaacetate; 4,4'-diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid; 4,4'- diisothiocyanatostilbene-2,2'-disulforlic acid; 5-[dimethylamino] naphthalene- 1 -sulfonyl chloride (DNS, dansyl chloride); 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL); 4- dimethylaminophenylazophenyl-4'-isothiocyanate (DABITC); eosin and derivatives such as eosin and eosin isothiocyanate; erythrosin and derivatives such as erythrosin B and erythrosin isothiocyanate; ethidium; fluorescein and derivatives such as 5-carboxyfluorescein (FAM), 5- (4,6diclllorotriazin-2-yDarninofluorescein (DTAF), 2'7'dimethoxy-4'5'-dichloro-6- carboxyfluorescein (JOE), fluorescein, fluorescein isothiocyanate (FITC), and QFITC Q(RITC); 2',7'-difluorofluorescein (OREGON GREEN®); fluorescamine; IR144; IR1446; Malachite Green isothiocyanate; 4-methylumbelliferone; ortho cresolphthalein; nitrotyrosine; pararosaniline; Phenol Red; B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives such as pyrene, pyrene butyrate and succinimidyl 1 -pyrene butyrate; Reactive Red 4 (Cibacron Brilliant Red 3B-A); rhodamine and derivatives such as 6-carboxy-X-rhodamine (ROX), 6- carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, rhodamine green, sulforhodamine B, sulforhodamine 101 and sulfonyl chloride derivative of sulforhodamine 101 (Texas Red); N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine; tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acid and terbium chelate derivatives. Other suitable fluorophores include thiol -reactive europium chelates which emit at approximately 617 mn (Heyduk and Heyduk, Analyt. Biochem. 248:216-27, 1997; J. Biol. Chem. 274:3315-22, 1999), as well as GFP, LissamineTM, diethylaminocoumarin, fluorescein chlorotriazinyl, naphthofluorescein, 4,7-dichlororhodamine and xanthene (as described in U.S. Pat. No. 5,800,996 to Lee et al.) and derivatives thereof. Other fluorophores known to those skilled in the art can also be used, for example, those available from Life Technologies (Invitrogen; Molecular Probes (Eugene, Oreg.)) and including the ALEXA FLUOR® series of dyes (for example, as described in U.S. Pat. Nos. 5,696,157, 6, 130, 101 and 6,716,979), the BODIPY series of dyes (dipyrrometheneboron difluoride dyes, for example as described in U.S. Pat. Nos. 4,774,339, 5,187,288, 5,248,782, 5,274,113, 5,338,854, 5,451,663 and 5,433,896), Cascade Blue (an amine reactive derivative of the sulfonated pyrene described in U.S. Pat. No. 5,132,432) and Marina Blue (U.S. Pat. No. 5,830,912).

In addition to the fluorochromes described above, a fluorescent label can be a fluorescent nanoparticle, such as a semiconductor nanocrystal, e.g., a QUANTUM DOT™ (obtained, for example, from Life Technologies (QuantumDot Corp, Invitrogen Nanocrystal Technologies, Eugene, Oreg.); see also, U.S. Pat. Nos. 6,815,064; 6,682,596; and 6,649, 138). Semiconductor nanocrystals are microscopic particles having size-dependent optical and/or electrical properties. When semiconductor nanocrystals are illuminated with a primary energy source, a secondary emission of energy occurs at a frequency corresponding to the handgap of the semiconductor material used in the semiconductor nanocrystal. This emission can be detected as colored light of a specific wavelength or fluorescence. Semiconductor nanocrystals with different spectral characteristics are described in, e.g., U.S. Pat. No. 6,602,671. Semiconductor nanocrystals that can be coupled to a variety of biological molecules (including dNTPs and/or nucleic acids) or substrates by techniques described in, for example, Bruchez et al., Science 281 :20132016, 1998; Chan et al., Science 281 :2016-2018, 1998; and U.S. Pat. No. 6,274,323. Formation of semiconductor nanocrystals of various compositions are disclosed in, e.g., U.S. Pat. Nos. 6,927, 069; 6,914,256; 6,855,202; 6,709,929; 6,689,338; 6,500,622; 6,306,736; 6,225,198; 6,207,392; 6,114,038; 6,048,616; 5,990,479; 5,690,807; 5,571,018; 5,505,928; 5,262,357 and in U.S. Patent Publication No. 2003/0165951 as well as PCT Publication No. 99/26299 (published May 27, 1999). Separate populations of semiconductor nanocrystals can be produced that are identifiable based on their different spectral characteristics. For example, semiconductor nanocrystals can be produced that emit light of different colors based on their composition, size or size and composition. For example, quantum dots that emit light at different wavelengths based on size (565 mn, 655 mn, 705 mn, or 800 mn emission wavelengths), which are suitable as fluorescent labels in the probes disclosed herein, are available from Life Technologies (Carlsbad, Calif.).

Additional labels include, for example, radioisotopes (such as 3 H), metal chelates such as DOTA and DPTA chelates of radioactive or paramagnetic metal ions like Gd3+, and liposomes. Detectable labels that can be used with nucleic acid molecules also include enzymes, for example, horseradish peroxidase, alkaline phosphatase, acid phosphatase, glucose oxidase, beta-galactosidase, beta-glucuronidase, or beta-lactamase.

Alternatively, an enzyme can be used in a metallographic detection scheme. For example, silver in situ hybridization (SISH) procedures involve metallographic detection schemes for the identification and localization of a hybridized genomic target nucleic acid sequence. Metallographic detection methods include using an enzyme, such as alkaline phosphatase, in combination with a water-soluble metal ion and a redox-inactive substrate of the enzyme. The substrate is converted to a redox-active agent by the enzyme, and the redox active agent reduces the metal ion, causing it to form a detectable precipitate. (See, for example, U.S. Patent Application Publication No. 2005/0100976, PCT Publication No. 2005/ 003777 and U.S. Patent Application Publication No. 2004/ 0265922). Metallographic detection methods also include using an oxido-reductase enzyme (such as horseradish peroxidase) along with a water soluble metal ion, an oxidizing agent and a reducing agent, again to form a detectable precipitate. (See, for example, U.S. Pat. No. 6,670,113).

Probes made using the disclosed methods can be used for nucleic acid detection, such as ISH procedures (for example, fluorescence in situ hybridization (FISH), chromogenic in situ hybridization (CISH) and silver in situ hybridization (SISH)) or comparative genomic hybridization (CGH). In situ hybridization (ISH) involves contacting a sample containing target nucleic acid sequence (e.g., genomic target nucleic acid sequence) in the context of a metaphase or interphase chromosome preparation (such as a cell or tissue sample mounted on a slide) with a labeled probe specifically hybridizable or specific for the target nucleic acid sequence (e.g., genomic target nucleic acid sequence). The slides are optionally pretreated, e.g., to remove paraffin or other materials that can interfere with uniform hybridization. The sample and the probe are both treated, for example by heating to denature the double stranded nucleic acids. The probe (formulated in a suitable hybridization buffer) and the sample are combined under conditions and for sufficient time to permit hybridization to occur (typically to reach equilibrium). The chromosome preparation is washed to remove the excess probe, and detection of specific labeling of the chromosome target is performed using standard techniques.

For example, a biotinylated probe can be detected using fluorescein-labeled avidin or avidinalkaline phosphatase. For fluorochrome detection, the fluorochrome can be detected directly, or the samples can be incubated, for example, with fluorescein isothiocyanate (FITC)- conjugated avidin. Amplification of the FITC signal can be effected, if necessary, by incubation with biotin-conjugated goat antiavidin antibodies, washing and a second incubation with FITC- conjugated avidin. For detection by enzyme activity, samples can be incubated, for example, with streptavidin, washed, incubated with biotin-conjugated alkaline phosphatase, washed again and pre-equilibrated (e.g., in alkaline phosphatase (AP) buffer). For a general description of in situ hybridization procedures, see, e.g., U.S. Pat. No. 4,888,278.

Numerous procedures for FISH, CISH, and SISH are known in the art. For example, procedures for performing FISH are described in U.S. Pat. Nos. 5,447,841; 5,472,842; and 5,427,932; and for example, in Pirlkel et al., Proc. Natl. Acad. Sci. 83:2934-2938, 1986; Pinkel et al., Proc. Natl. Acad. Sci. 85:9138-9142, 1988; and Lichter et al., Proc. Natl. Acad. Sci. 85:9664-9668, 1988. CISH is described in, e.g., Tanner et al., Am. .1. Pathol. 157: 1467-1472, 2000 and U.S. Pat. No. 6,942,970. Additional detection methods are provided in U.S. Pat. No. 6,280,929.

In some embodiments, all the methods of the present invention are performed in vitro or ex vivo (i.e method perfomed on a sample previously obtained, none step of the methods of the present invention are practiced in the animal or human body).

The diagnostic methods of the present invention are particularly suitable for guiding therapy.

In particular, removing the anti-VASN autoantibodies is expected to be of therapeutic value. Thus, when the subject is diagnosed as having a nephrotic syndrome associated with anti-VASN autoantibodies, the therapy can consist of lowering anti-VASN autoantibodies levels in body fluids and kidneys via targeting of Ig-producing cells (B-lymphocytes, plasmocytes) using B cell depleting agent and/or an agent targeting long-lived plasma cells to treat subjects with NS.

Thus, a further object of the present invention relates to a method of treating a NS associated with anti-VASN autoantibodies in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a B-cell depleting agent and/or an agent targeting long-lived plasma cells.

In a particular embodiment, the subject has been diagnosed as having a NS according to the method of the invention.

Thus, in particular embodiment, the present invention relates to a method of treating a NS associated with anti-VASN autoantibodies in a patient in need thereof comprising the steps of i) diagnosed the patients as having NS according to the invention and ii) administering to the patient a therapeutically effective amount of a B-cell depleting and/or an agent targeting long- lived plasma cells.

In particular embodiment, the NS is a NS with MCD or with FSGS. In particular embodiment, the NS is a NS with MN.

As used herein, the term “B cell depleting agent” refers to any agent capable of triggering B cells' lymphodepletion. In some embodiments, the B cell-depleting agent is an antibody having specificity for CD20. Examples of antibodies having specificity for CD20 include: “C2B8,” which is now called “Rituximab” (“RITUXAN®”) (U.S. Pat. No. 5,736,137, expressly incorporated herein by reference), a chimeric pan-B antibody targeting CD20; the yttrium-[90]- labeled 2B8 murine antibody designated “Y2B8” or “Ibritumomab Tiuxetan” ZEVALIN® (U.S. Pat. No. 5,736,137, expressly incorporated herein by reference), a murine IgGl kappa mAb covalently linked to MX-DTPA for chelating to yttrium-[90]; murine IgG2a “BI,” also called “Tositumomab,” optionally labeled with radioactive 1311 to generate the “1311-B1” antibody (iodine 131 tositumomab, BEXXAR™) (U.S. Pat. No. 5,595,721, expressly incorporated herein by reference); murine monoclonal antibody “1F5” (Press et al. Blood 69 (2):584-591 (1987) and variants thereof including “framework patched” or humanized 1F5 (W003/002607, Leung, S.; ATCC deposit HB-96450); murine 2H7 and chimeric 2H7 antibody (U.S. Pat. No. 5,677,180, expressly incorporated herein by reference); humanized 2H7, also known as ocrelizumab (PRO-70769); Ofatumumab (Arzerra), a fully human IgGl against a novel epitope on CD20 huMax-CD20 (Genmab, Denmark; W02004/035607 (U.S. Ser. No. 10/687,799, expressly incorporated herein by reference)); AME-133 (ocaratuzumab; Applied Molecular Evolution), a a fully-humanized and optimized IgGl mAb against CD20; A20 antibody or variants thereof such as chimeric or humanized A20 antibody (cA20, hA20, respectively) (U.S. Ser. No. 10/366,709, expressly incorporated herein by reference, Immunomedics); and monoclonal antibodies L27, G28-2, 93-1B3, B-CI or NU-B2 available from the International Leukocyte Typing Workshop (Valentine et al, In: Leukocyte Typing III (McMichael, Ed., p. 440, Oxford University Press (1987)). Further, suitable antibodies include, e.g., antibody GA101 (obinutuzumab), a third-generation humanized anti-CD20-antibody of Biogen Idec/Genentech/Roche. Moreover, BLX-301 of Biolex Therapeutics, a humanized anti- CD20 with optimized glycosylation or Veltuzumab (hA20), a 2nd-generation humanized antibody specific for CD20 of Immunomedics or DXL625, derivatives of Veltuzumab, such as the bispecific hexavalent antibodies of IBC Pharmaceuticals (Immunomedics) which are comprised of a divalent anti-CD20 IgG of Veltuzumab and a pair of stabilized dimers of Fab derived from milatuzumab, an anti-CD20 mAb enhanced with InNexus' Dynamic Cross Linking technology, of Inexus Biotechnology both are humanized anti-CD20 antibodies are suitable. Further suitable antibodies are BM-ca (a humanized antibody specific for CD20 (Int J. Oncol. 2011 February; 38(2):335-44)), C2H7 (a chimeric antibody specific for CD20 (Mol Immunol. 2008 May; 45(10):2861-8)), PRO131921 (a third generation antibody specific for CD20 developed by Genentech), Reditux (a biosimilar version of rituximab developed by Dr Reddy's), PBO-326 (a biosimilar version of rituximab developed by Probiomed), a biosimilar version of rituximab developed by Zenotech, TL-011 (a biosimilar version of rituximab developed by Teva), CMAB304 (a biosimilar version of rituximab developed by Shanghai CP Guojian), GP-2013 (a biosimilar version of rituximab developed by Sandoz (Novartis)), SAIT- 101 (a biosimilar version of rituximab developed by Samsung BioLogics), a biosimilar version of rituximab developed by Intas Biopharmaceuticals, CT-P10), a biosimilar version of rituximab developed by Celltrion), a biosimilar version of rituximab developed by Biocad, Ublituximab (LFB-R603, a transgenically produced mAb targeting CD20 developed by GTC Biotherapeutics (LFB Biotechnologies)), PF-05280586 (presumed to be a biosimilar version of rituximab developed by Pfizer), Lymphomun (Bi-20, a trifunctional anti-CD20 and anti-CD3 antibody, developed by Trion Pharma), a biosimilar version of rituximab developed by Natco Pharma, a biosimilar version of rituximab developed by iBio, a biosimilar version of rituximab developed by Gedeon Richter/Stada, a biosimilar version of rituximab developed by Curaxys, a biosimilar version of rituximab developed by Coherus Biosciences/Daiichi Sankyo, a biosimilar version of rituximab developed by BioXpress, BT-D004 (a biosimilar version of rituximab developed by Protheon), AP-052 (a biosimilar version of rituximab developed by Aprogen), a biosimilar version of ofatumumab developed by BioXpress, MG- 1106 (a biosimilar version of rituximab developed by Green Cross), IBI-301 (a humanized monoclonal antibody against CD20 developed by Innovent Biologies), BVX-20 (a humanized mAb against the CD20 developed by Vaccinex), 20-C2-2b (a bispecific mAb-IFNalpha that targets CD20 and human leukocyte antigen-DR (HLA-DR) developed by Immunomedics), MEDI-552 (developed by Medlmmune/AstraZeneca), the anti-CD20/streptavidin conjugates developed by NeoRx (now Poniard Pharmaceuticals), the 2nd generation anti-CD20 human antibodies developed by Favrille (now MMRGlobal), TRU-015, an antibody specific for CD20 fragment developed by Trubion/Emergent BioSolutions, as well as other precloinical approaches by various companies and entities. In some embodiments, the B cell-depleting agent is an antibody having specificity for CD19. Examples of antibodies having specificity for CD19 include: “ZB012”, which is now called “Obexelimab”, an anti-CD19 antibody with increased FcyRllb binding that suppresses B-cell activation (U.S. Pat. No. 8,524,867, expressly incorporated herein by reference). In some embodiments, the B cell-depleting agent is an antibody having specificity for CD22, such as Epratuzumab, a humanized monoclonal antibody targeting CD22 antigen (US Pat. No. 5,789,554). In some embodiments, the B cell-depleting agent is an agent blocking the binding of B-lymphocyte stimulator (BlyS) and/or a proliferation-inducing ligand (APRIL). Agent blocking the binding of BlyS and/or APRIL include atacicept (CAS Number 845264-92-8), blisibimod (CAS Number 1236126-45-6) and antibodies having specificity for BlyS and/or APRIL. Examples of antibodies having specificity for BlyS and/or APRIL include Belimumab (“BENLYSTA®”) and Tabaluman (disclosed in WO2015123782, expressly incorporated herein by reference). All aforementioned publications, references, patents, and patent applications are incorporated by reference in their entireties. All antibodies disclosed therein may be used within the present invention.

As used herein, the term “agent targeting long-lived plasma cells” refers to any agent capable of triggering Long-lived plasma cells (LLPCs) decrease. Long-lived plasma cells (LLPCs) are considered ultimately differentiated, bone marrow (BM) resident cells secreting high-affinity antibodies that disseminate through the body. The migration of plasma cells to BM where they become LLPCs is largely controlled by an interaction between the chemokine ligand CXCL12 and its receptor CXCR4. Example of agent targeting LLPCs includes AMD3100 (CAS Number 155148-31-5), an antagonist of CXCR4 blocking the CXCL12/CXCR4 interaction; Antithymocyte globulin (ATG); proteasome inhibitors such as bortezombib (CAS Number 179324-69-7), carfilzomib (CAS Number 868540-17-4), ixazomib (CAS Number 1072833-77- 2), peptide boronic acid analogs MLN9708 and CEP- 18770, peptide epoxyketones carfilzomib and PR-047, and NPI-0052, a beta-lactone compound as disclosed in Dick LR, Fleming PE. Building on bortezomib: second-generation proteasome inhibitors as anti-cancer therapy. Drug Discov Today. 2010 Mar;15(5-6):243-9.

Thus, when the subject is diagnosed as having a nephrotic syndrome associated with anti-VASN autoantibodies, the therapy can consist of an antibody-depleting strategy, typically including plasma exchange, plasmapheresis, or immunoadsorption.

Accordingly, a further object of the present invention relates to a method of treating a subject suffering from a NS by removing anti-VASN autoantibodies from body fluids from the subject comprising the steps of a) providing the extracellular body fluid (e.g., blood), which has been obtained from a subject, b) contacting the extracellular body fluid with biocompatible solid support capable of capturing the anti-VASN autoantibodies, and c) reinfusing the extracellular body fluid from step b) into the subject.

In particular embodiment, the subject has been diagnosed as having NS associated with anti- VASN autoantibodies according to the method of the invention.

In particular embodiment, the NS is a NS with MCD or with FSGS. In a particular embodiment, the NS is a NS with MN.

The removal of the anti-VASN autoantibodies is performed by any well-known method in the art and can typically involve plasma exchange or plasmapheresis. Two methods are typically used in plasmapheresis to membrane filtration and extracorporeal centrifugation. In extracorporeal immunoadsorption, circulating antibodies are extracorporeally removed using an immunoadsorbent column specific for the endogenous antibody. Blood from the patient is withdrawn either continuously or discontinuously, separated into its cellular components and plasma, and the plasma is perfused through the immunoadsorbent material in order to remove the antibody. The treated plasma and cellular components of the blood are then reinfused into the patient, either separately or simultaneously.

In some embodiments, an amount of Protein A or Protein G is immobilized in the solid support. Protein A or Protein G (for example, obtained from Miltenyi Biotec, Germany) are components of the cell wall of the bacterium Staphylococcus and have the capacity to bind non-selective immunoglobulins of the IgG class because of their high affinity to the Fc portion of the IgG antibodies.

Specific removal of circulating antibodies by extracorporeal immunoadsorption employing an immobilized antigen has been described by various investigators. See generally Kohler et al., (201 1) J Clin Apher. (6):347-55; Muller et al., (2012) Dermatology.;224(3):224-7; Koziolek et al., (2012) J Neuroinflammation. 9(l):80; Bontadi et al., (2012) J Clin Apher. doi: 10.1002/jca.21229; Westermann et al., (2012) J Dermatol. 39(2): 168-71. Moreover, this approach has been successfully commercialized as a viable system to specifically remove circulating antibodies, as exemplified by immunoadsorption columns sold under the trademarks Prosorba®, Immunosorba®, sold by Fresenius, St. Wendel, Germany, and Selesorb® sold by Kaneka, Wiesbaden, Germany.

In some embodiments, an amount of a recombinant VASN polypeptide is immobilized in the solid support. In said embodiments, the immunoadsorption is more specific so that only Ig specific to VASN are captured on the solid support; the other Ig are eluted in the extracellular body fluid.

In general, in any of these immunoadsorbent methods and compositions, the body fluids are obtained, handled, and re-infused under aseptic conditions using methods and systems that are well-known to a person skilled in the art. For example, blood is withdrawn via a needle that is introduced into, for example, a peripheral vein connected via a suitable tube to the container containing the biocompatible solid support and re-infused into the patient via an inlet tube connected to a needle inserted into another vein. In situations where large volumes are to be withdrawn from the subject, blood may be drawn, for instance, from the vena subclavia. Optionally, an anticoagulation substance such as sodium citrate, heparin, or dextran can be added to the blood when withdrawn from the body to prevent coagulation of the blood. Dextran reduces the blood viscosity and, in combination with the addition of saline, ensures an increased distance between the blood cells and the blood platelets. Such anticoagulants may be added in quantities sufficient for non-coagulation of the blood. Before reinfusion of the treated blood into the subject, the anti coagulation effect of, e.g. heparin may be reduced with the appropriate amount of heparinase, protamine, and/or vitamin K, etc. Suitable columns and perfusion systems for extracorporeal adsorption are commercially available, for example, from Fresenius, St. Wendel, Germany. Contact temperatures in the range of 35°C to about 40°C are typically used. The contact time will typically be in the range of about 1 to about 6 hours. The unbound portion of the blood or plasma is then collected for reintroduction into the patient, or it can be reintroduced directly on a continuous basis. The subject's anti-VASN autoantibodies titer may be monitored by immunoassay before and /or after the procedure to monitor the efficiency of the procedure.

To reduce the risk of embolism, precautions can be taken to avoid adsorption medium particles entering the patient upon reinfusion. Accordingly, a particle capture device is typically employed downstream of the adsorption medium container to remove any residual particles from the remainder of the body fluid before it is returned to the patient. The particle capture device may be a filter or mesh having openings of a size that retain any particulate material of the adsorption medium while letting the non-adsorbed entities of the body fluid pass through. The extracorporeal blood perfusion may be performed continuously, or alternatively, discrete volumes of blood may be removed from the patient, treated as described above, and the treated plasma and cellular components of the blood returned to the patient after the treatment is complete.

A wide variety of materials will be suitable as biocompatible solid supports for use in any of these immunoadsorbent methods and compositions, and ideally, the support matrix will be mechanically strong, sufficiently hydrophilic to avoid non-specific binding of proteins, stable and compatible with blood and other aqueous solutions. Suitable biocompatible matrix materials include, for example, synthetic and natural polymers, polysaccharides, polyamides, glass beads, particulate silica, porous glass, silica, resins, synthetic matrixes including acrylamide derivatives, methacrylamide derivatives or polystyrene derivatives, etc., in various forms including beads, fibrous form, sheets or hollow fibers. Exemplary polymers include natural and synthetic polysaccharides and other carbohydrate-based polymers, including agar, alginate, carrageenan, guar gum, gum arabic, gum ghatti, gum tragacanth, karaya gum, locust bean gum, xanthan gum, agaroses, celluloses, pectins, mucins, dextrans, starches, heparins, chitosans, hydroxy starches, hydroxypropyl starches, carboxymethyl starches, hydroxyethyl celluloses, hydroxypropyl celluloses, and carboxymethyl celluloses. Synthetic organic polymers and monomers resulting in polymers, including acrylic polymers, polyamides, polyimides, polyesters, polyethers, polymeric vinyl compounds, polyalkenes, and substituted derivatives thereof, as well as copolymers comprising more than one such polymer functionality, and substituted derivatives thereof; and mixtures thereof.

In any of these extracorporeal methods and compositions, the VASN polypeptides are typically covalently coupled to the biocompatible solid support, and standard methods for coupling proteins such as the VASN polypeptides are well known to those of skill in the art (see. e.g., Affinity Chromatography, Principles and Methods (Pharmacia-LKB), Dean, P.G., et al., eds., 1985, Affinity Chromatography: A practical approach, IRL Press, Oxford, and Scouten, W.H., 1981, Affinity Chromatography, Wiley Interscience, New York), "Immobilized Affinity Ligand Techniques" by Hermanson et al., Academic Press, Inc., San Diego, 1992). The biocompatible solid support may be derivatized (activated) to form a reactive substance that can react with one or more functional chemical groups within the VASN polypeptide, thereby forming a chemical covalent bond to coupling the VASN polypeptide to the biocompatible solid support. Thus, materials comprising hydroxyl, amino, amide, carboxyl, or thiol groups may be activated or derivatized using various activating chemicals, e.g., chemicals such as cyanogen bromide, divinyl sulfone, epichlorohydrin, bisepoxyranes, dibromopropanol, glutaric dialdehyde, carbodiimides, anhydrides, hydrazines, periodates, benzoquinones, triazines, tosylates, tresylates, and/or diazonium ions, etc. Specific exemplary activated biocompatible solid supports for use in any of these methods and compositions include for example, CNBr- Sepharose, celluloses, such as CNBr-activated Sepharose 4B (Amersham) or Epoxy-activated agarose (Sigma). Biocompatible spacers (like, for example, NHS-activated Sepharose 4 Fast Flow) or without (like, for example, CNBr-activated Sepharose 4B) may be employed and are commercially available, and methods for coupling such materials to VASN polypeptides are well known in the art and can be optimized by routine experimentation based on the manufacturer's directions.

In some embodiments, all the methods of the present invention are performed in vitro or ex vivo.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES: Figure 1: Detection of high titers of anti-VASN antibodies in children with nephrotic syndrome. About 1/3 of patients with nephrotic syndrome displayed high titers of anti-VASN autoantibodies in serum, whereas none of the 41 controls did. SDNS = steroid-dependent nephrotic syndrome; MDNS = multi -drug dependent nephrotic syndrome; MRNS = multi-drug resistant nephrotic syndrome.

EXAMPLE:

We showed that:

The VASN mRNA expression is high in laser-capture microdissected normal human glomeruli but altered in diseases with marked podocyte injuries, such as FSGS and ANCA-associated vasculitis.

A mouse model with a reporter system confirms a high selective abundance of the Vasn gene expression in normal glomeruli with a podocyte selective pattern a human podocyte cell line was transfected with a vector to express an HA-tagged full- length human VASN protein. The localization of the protein was confirmed at the plasma membrane.

These data indicate that VASN is a constitutive protein of the podocyte. We hypothesized that this type I transmembrane protein might be a target of auto-immune reactions that would, in turn, trigger podocyte dysfunction and proteinuria. The HA-VASN protein was thus affinity purified from such transfected human cells and used to set up an ELISA to detect anti-VASN antibodies in biofluids. We then screened sera from a cohort of 216 SDNS (steroid-dependent nephrotic syndrome) or MDNS (multi-drug dependent nephrotic syndrome) or MRNS (multidrug resistant nephrotic syndrome) children showing significant titers of antibodies against this protein (Figure 1).

REFERENCES:

Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

Claims

CLAIMS:

1. An in vitro or ex vivo method of diagnosing the occurrence of nephrotic syndrome (NS) in a subject comprising the steps of i) detecting the presence of anti-VASN autoantibodies in a sample from the subject and ii) diagnosing the occurrence of NS if anti-VASN autoantibodies are detected in the said sample.

2. An in vitro or ex vivo method of determining whether a subject has or is at risk of having a nephrotic syndrome (NS) in a sample from the subject wherein the presence of anti- VASN autoantibodies indicates that the subject has or is at risk of having a NS

3. The in vitro or ex vivo method according to claim 2 comprising the steps: i) determining the level of anti-VASN autoantibodies in a sample from the subject, ii) comparing the level determined at step i) with a predetermined reference value and iii) concluding that the subject has or is at risk of having a NS when the level determined at step i) is higher than the predetermined reference value.

4. An in vitro or ex vivo method of predicting the risk of relapse in a subject suffering from NS comprising i) determining the level of anti-VASN autoantibodies in a sample from the subject, ii) comparing the level determined at step i) with a predetermined reference value and iii) concluding that the subject is at risk of relapse when the level determined at step i) is higher than the predetermined reference value.

5. An in vitro or ex vivo method of determining whether a subject suffering from NS achieves a response with treatment comprising i) determining the level of anti-VASN autoantibodies in a sample from the subject before the treatment, ii) determining the level of anti-VASN autoantibodies in a sample from the subject after treatment initiation, iii) comparing the level determined at step i) with the level determined at step ii) and concluding that the subject achieves a response when the level determined at step ii) is lower than the level determined at step i).

6. An in vitro or ex vivo method of determining whether a subject suffering from NS achieves remission comprising i) determining the level of anti-VASN autoantibodies in a sample from the subject before the treatment, ii) determining the level of anti-VASN autoantibodies in a sample from the subject after the treatment completion, iii) comparing the level determined at step ii) with the level determined at step i) and concluding that the subject achieves disease remission when the level determined at step ii) is lower than the level determined at step i).

7. The in vitro or ex vivo method according to claim 6, wherein it is concluded that the subject achieves disease remission when the level of anti-VASN autoantibodies determined in step ii) is is lower than the level determined at step i) and is similar to a basal level (i.e., the level determined in a population of healthy individuals)

8. The in vitro or ex vivo method of claim 5 to7, wherein the treatment is immunosuppressive.

9. The in vitro or ex vivo method of claim 1 to 8, wherein the sample is a bodily fluid (e.g. a blood sample) or a tissue (such as kidney tissue).

10. A method of treating a NS associated with anti-VASN autoantibodies in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a B-cell depleting agent and/or an agent targeting long-lived plasma cells.

11. A method of treating a subject suffering from a NS by removing anti-VASN autoantibodies from body fluids from the subject comprising the steps of a) providing the extracellular body fluid (e.g., blood) which has been obtained from a subject, b) contacting the extracellular body fluid with biocompatible solid support capable of capturing the anti-VASN autoantibodies, and c) reinfusing the extracellular body fluid from step b) into the subject.

12. The method of claim 1 to 11, wherein the nephrotic syndrome is an idiopathic nephrotic syndrome.

13. The method of claim 1 to 12, wherein the NS is NS with minimal change disease (MCD) or with focal segmental glomerulosclerosis (FSGS).

14. The method of claim 1 to 12, wherein the NS is NS with membranous nephropathy (MN).

15. A kit or device for identifying the presence or the level of anti-VASN autoantibodies in a sample from a subject comprising at least a VASN protein or fragments thereof; and at least one solid support wherein the VASN protein or fragments thereof is deposited on the support.