EP3027652A1 - Anti-pla2r antibody and uses thereof - Google Patents

Abstract

A human Phospholipase A ₂ receptor (PLA ₂ R) binding molecule directed against a PLA ₂ R- derived peptide of formula (II) comprising the following amino sequence: wherein: -y is an integer consistingin 0 or 1, -z is an integer consisting in 0 or 1, -Nt is a peptide of 1 to 20 amino acids in length, -Ct is a peptide of 1 to 20 amino acids in length.

Description

ANTI-PLA2R ANTIBODY AND USES THEREOF

FIELD OF THE INVENTION

The invention relates to PLA₂R-binding molecules such as anti-PLA₂R antibodies. Thus the invention relates also to the identification of novel epitopes of PLA₂R, immunogenic compounds, kits and compositions, as well as methods for obtaining such antibodies.

According to one of its aspects, the invention relates to methods for detecting the presence of PLA₂R in a sample.

According to another of its aspects, the invention relates to methods for diagnosing the occurrence of a membranous nephropathy in an individual, such as an idiopathic (or PLA₂R-positive) membranous nephropathy.

BACKGROUND OF THE INVENTION

Membranous nephropathy (MN) is an organ-specific autoimmune disease which targets the kidney glomerulus, resulting in the formation of immune deposits on the outer aspect of the glomerular basement membrane (GBM) and complement mediated proteinuria). MN is the most common cause of nephrotic syndrome (defined by massive urinary protein loss) in Caucasian adults. It is a very heterogenous disease with variable outcome and sensitivity to treatment.

Membranous nephropathy may be classified as a primary MN, or as a secondary MN, and is preferably diagnosed by renal biopsy. Primary membranous nephropathy may be also referred herein as an idiopathic membranous nephropathy (iMN), or as a PLA₂R-positive membranous nephropathy. More generally, the diagnosis of idiopathic membranous nephropathy is established in the absence of secondary features, which include positivity for anti-nuclear antibodies, anti-double stranded DNA, hepatitis B antigenemia, or electron-dense deposits on renal biopsy in locations other than subepithelial. On the other hand, a secondary MN may be, non-exhaustively, linked to an infection, a tumor, a drug intoxication or an autoimmune condition such as lupus.

Membranous nephropathy may also be referred in the literature as a membranous glomerulonephritis. Because of this heterogeneity, the prognosis of membranous nephropathy remains difficult due to the lack of reliable biomarkers (Debiec & Ronco; 2011; PLA₂R autoantibodies and PLA₂R glomerular deposits in membranous nephropathy; N. Engl. J. Med.; 364(7):689-90).

Diffuse subepithelial deposits of immune complexes are characteristic membranous lesions which may be observed both in primary and in secondary MN including lupus membranous nephropathy, albeit with several distinct pathologic characteristics (Kolasinski et al; 2002; What Do We Know About Lupus Membranous Nephropathy? An Analytic Review ; Arthritis & Rheumatism ; 47(4) pp 450-455).

The M-type phospholipase A₂ receptor (PLA₂ R) was recently identified as a major target antigen in "idiopathic" MN (iMN) in adults, as shown in Beck et al. (Beck et al. ; 2009; M-type phospholipase A₂ receptor as target antigen in idiopathic membranous nephropathy ; N Engl J Med. ; 361 : 11-21).

In addition, the strong association of the PLA2R1 gene with iMN in a recent genome-wide association study suggests that genetic variants of PLA2R1 are at risk for the disease, as shown by Stanescu et al. (Stanescu et al. ; 2011; Risk HLA-DQA1 and PLA(2)R1 alleles in idiopathic membranous nephropathy; N Engl J Med ; 364(7):616-26).

Although the exact role of PLA₂R in the pathogenesis of iMN is still not yet well understood, the presence of circulating anti-PLA₂R is highly specific for iMN.

Indeed, several recent studies have shown that anti-PLA₂R antibody levels correlate with the clinical manifestations of disease activity, decreasing during a spontaneous or treatment-induced remission and reappearing during a relapse (see for review Debiec & Ronco; 2010; Antigen Identification in Membranous Nephropathy Moves toward Targeted Monitoring and New Therapy ; JASN ; vol. 21(4), 564-569).

To our knowledge, no anti-PL A₂R antibodies have been detected in healthy individuals.

Changes in circulating levels of anti-PLA₂R antibody often precede changes in proteinuria. Without wishing to be bound by any theory, it is believed that, in human idiopathic membranous nephropathy, glomerular deposits (in particular subepithelial glomerular deposits) form in situ through binding of circulating anti-PLA₂R antibodies to the PLA₂R antigen which is expressed on the surface of podocytes. Because PLA₂R antibody titers can fluctuate and even become negative, detection of PLA₂R antigen in immune glomerular deposits is more sensitive than serology.

Our recent study in French and Czech cohorts of patients with iMN (Debiec & Ronco; 2011; PLA₂R autoantibodies and PLA₂R glomerular deposits in membranous nephropathy; N. Engl. J. Med.; 364(7):689-90) has suggested that assessment of both (i) circulating PLA₂R antibodies and (ii) PLA₂R in biopsy might better categorize patients into different groups, in comparison to the sole assessment of circulating anti-PLA₂R antibodies. Indeed, this study has shown that the absence of circulating PLA₂R antibody at the time of kidney biopsy and proteinuria was probably not sufficient to rule out a diagnosis of PLA₂R-related MN. As a consequence, the diagnosis of membranous nephropathy is preferably established by renal biopsy in all cases.

Thus it is recommended that PLA₂R antigen in glomeruli should be examined in all biopsies of patients with a suspected idiopathic (or PLA₂R-positive) MN, especially if they have negative anti-PL A₂R activity in the serum Search for PLA₂R antigen in archival kidney biopsies is also important for the monitoring of patients who will benefit from a kidney transplant

Advantageously, detection of PLA₂R in kidney biopsies can discriminate between different forms of membranous nephropathy, in particular between iMN and a secondary MN such as lupus MN. Because therapeutic strategies are different for patients with iMN and lupus MN, discriminating between these two groups of patients is of utmost clinical importance.

Commercial antibodies against PLA₂R have been reported in the literature. One may, for instance, refer to the goat polyclonal antibody commercialized by LSBio (ref. LS-C108906), which is raised against an internal region of human PLA₂R and which is suitable for western blots and ELISA methods. However, that antibody is not suitable for immunohistochemistry, which may indicate that the targeted epitope is not available in situ for antibody binding.

The polyclonal rabbit anti-PLA₂R antibody commercialized by SIGMA- ALDRICH^® (ref. HPA012657) is raised against another epitope which ranges from amino- acid 395 to 530 of the receptor PLA₂R of sequence SEQ ID N°l . This polyclonal antibody is validated by the Human Protein Atlas project and is suitable for immunohistochemistry, but also for protein array and western blotting.

Another polyclonal rabbit anti-PLA₂R antibody is commercialized by ABCAM® (ref. ab99466) and is raised against the epitope which ranges from amino-acid 946 to 957 of the receptor PL A₂R of sequence SEQ ID N° 1.

There remains a need for novel antibodies which bind selectively to diagnostically relevant epitopes of PLA₂R, and which may be used notably immunohistochemical detection assays.

There remains a need for new tools for detecting glomerular deposits within biopsies, and more particularly renal biopsies.

There remains a need for methods for detecting the presence of PLA₂R in biological samples, and/or for diagnosing the occurrence of a membranous nephropathy disease.

In particular, there remains a need for improved tools which are able to discriminate between the different forms of membranous nephropathy, such as for example idiopathic membranous nephropathy and lupus membranous nephropathy.

SUMMARY OF THE INVENTION

The Phospholipase A₂ receptor (PLA₂R) is involved in the formation of immune glomerular deposits, which are characteristic to membranous nephropathy. The emergence of kidney biopsy for the diagnostic of membranous nephropathy requires to identify new tools for the detection of PLA₂R.

Thus, the invention relates to the assessment of the presence of PLA₂R for diagnostic purposes.

In particular, the invention relates to novel PLA₂R epitopes and the identification of highly specific PLA₂R binding molecules.

According to one of its aspects, the invention relates to a human Phospholipase A2 receptor (PLA₂R) binding molecule directed against a PLA₂R-derived peptide of formula (I) comprising the following amino sequence:

H₂-[Nt]_y - FXiWX₂ [Ct]_z - COOH (I)

wherein: - y is an integer consisting in 0 or 1,

- z is an integer consisting in 0 or 1,

- Nt is a peptide of 1 to 20 amino acids in length,

- Ct is a peptide of 1 to 20 amino acids in length,

- Xi and X₂ are identical or different and represent any amino acid.

According to another of its aspects, the invention relates to a PLA₂R-derived peptide in the form of an immunogenic compound comprising the PLA₂R-derived peptide of SEQ ID N°8, wherein the said peptide is covalently linked to a carrier molecule.

This invention also pertains to an immunogenic compound comprising a PLA₂R-derived peptide of formula:

H₂-[Nt]_y - ImEp - [Ct]_z - COOH

wherein:

- y is an integer consisting in 0 or 1,

- z is an integer consisting in 0 or 1,

- Nt is a peptide of 1 to 20 amino acids in length,

- Ct is a peptide of 1 to 20 amino acids in length,

- ImEp is an immunogenic epitope of PLA₂R of SEQ ID N°8.

Thus, the invention also relates to a method for generating antibodies binding to immune complexes between auto-antibodies directed against human phospholipase A₂ receptor (PLA₂R) and human PLA₂R comprising a step of providing an immunogenic compound containing a PLA₂R-derived peptide of SEQ ID N°8.

According to another of its aspects, the invention relates to a complex between (i) a human PLA₂R binding molecule and (ii) human PLA₂.

Thus the invention relates to an epitope of a human Phospholipase A₂ receptor bound by the said human PLA₂R binding molecule.

The invention also relates to a composition comprising a PLA₂R binding molecule according to the invention.

According to another of its aspects, the invention relates to a method for detecting the presence of human PLA₂R in a sample comprising the steps of:

a) providing a sample to be tested,

b) bringing into contact the sample provided at step a) with a PLA₂R binding molecule according to the invention, and c) detecting the binding of the said PLA₂R binding molecule onto the said sample, whereby the presence of human PLA₂R is detected.

Thus the invention relates to a kit for detecting human PLA₂R, the said kit comprising a PLA₂R binding molecule according to the invention and one or more reagents necessary for detecting the extent of binding between the said PLA₂R binding molecule and a sample to be tested.

Advantageously, the sample may be a biological sample taken from an individual suspected of having glomerular deposits.

Thus the invention also relates to a method for diagnosing the occurrence of a membranous nephropathy (MN) disease in an individual comprising the steps of:

(i) performing a method for detecting the presence of human PLA₂R in a sample from an individual to be tested, and

(ii) diagnosing the occurrence of a membranous nephropathy disease in the said individual if glomerular deposits comprising human PLA₂R are detected.

The invention further relates to a method for discriminating between idiopathic (or PLA₂R-positive) MN and lupus MN comprising the steps of:

a) providing a tissue sample from an individual to be tested,

b) bringing into contact the tissue sample provided at step a) with a PLA₂R binding molecule according to the invention, and

c) detecting the binding of the said PLA₂R binding molecule onto the said tissue sample, the presence of PLA₂R is indicative of idiopathic MN.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: Sequences of the ten overlapping peptides that were used in ELISA to identify the specific minimal epitope of the anti-PLA₂R antibody. The amino-acid sequence "PepC" of sequence SEQ ID N°8 ranges from amino-acid 875 to 900 of PLA₂R, of sequence SEQ ID N°l . Corresponding overlapping 10-aa long peptides in double amino-acid steps were synthesized in order to identify a specific minimal epitope.

Figure 2: Assessment by ELISA of peptide antibody reactivity in monoclonal hybridoma fluid, in complex with a HRP-conjugated secondary antibody. Absorbance (y-axis) is read at 450 nm after addition of TMB substrate. Dark grey bars and light grey bars relate to two independent ELISA measurements. Numbering of peptides (x-axis) is identical to figure 1.

Figure 3: Alanine Scanning on the immunogenic epitope of PLA₂R on hybridoma fluids and by ELISA. Panel A: Numbering of tested peptides. Panel B: Absorbance (y-axis) is read at 450 nm after addition of TMB substrate. Numbering of peptides (x-axis) is identified in panel A.

Figure 4: Assessment by Western Blot of the specificity of the PLA₂R antibody. Western blot analysis on recombinant PLA₂R in presence of recombinant PLA₂R (lane 1); after preincubation with an excess of EFRWR peptide (lane 2); after preincubation with a control peptide SWIGLQ (lane 3).

Figure 5 Location of various antigenic epitopes contained in PLA₂R. The position of the corresponding PLA₂R epitopes is indicated in the figure. The PLA₂R receptor is a type I transmembrane glycoprotein composed of a large extracellular portion that consists of an N-terminal cysteine-rich region (CRD), a fibronectin-like type II domain (FN2D), a tandem repeat of eight C-type lectin domains (CTLD), a transmembrane domain (TMD) and a short intracellular C-terminal domain (CD).

DETAILED DESCRIPTION OF THE INVENTION

In order to search for the presence of PLA₂R antigen in kidney biopsies, highly specific monoclonal antibodies that efficiently and specifically bind to PLA₂R on tissue samples embedded in paraffin sections are highly needed.

Thus, it has been determined that anti-PLA₂R autoantibodies from patients with nephropathy only recognize a reduction- sensitive, or conformation-dependent, epitope. In view of the above, it is assumed herein that not all epitopes from PLA₂R are equally relevant for obtaining highly specific monoclonal antibodies suitable for kidney biopsies.

It is emphasized that kidney biopsies from patients with membranous nephropathy may present in situ glomerular deposits formed both by the accumulation of pathogenic antigens from PLA₂R and of autoimmune anti-PLA₂R antibodies. When such glomerular deposits contain autoimmune antibodies, they may also be referred herein as immune glomerular deposits.

Without wishing to be bound by any theory, the present inventors believe that the formation of glomerular deposits may be viewed, at least in part, as a multiple-step process wherein circulating (auto)antibodies bind to a native protein. The formed PLA₂R- autoantibody complex may then be released in the subepithelial space, attach to the underlying glomerular basement membrane, resist degradation and persist as an immune deposit which would over time accumulate and be detectable on biopsies.

The PLA₂R is a known 185 kDa type I transmembrane receptor for secretory phospholipase which belongs to the mannose receptor family. It is composed of a short C- terminal cytoplasmic domain, a transmembrane domain, and an N-terminal extracellular domain. The extracellular domain comprises a cysteine-rich domain, a fibronectin type II domain, and eight C-type lectin -like domains. Thus, the folded PLA₂R transmembrane receptor is likely to possess linear, antigenic and/or accessibility motifs.

It has been determined herein that monoclonal or polyclonal antibodies directed against PLA₂R do not all possess the suitable binding properties for detecting the presence of PLA2R in biopsy tissue samples, including in paraffin-embedded biopsy tissue samples. It has been determined herein that relevant PLA₂R-derived antigenic epitopes may not be available in situ within the glomerular deposits, or may not be available when the target sample consists of paraffin-embedded biopsy tissue samples. One reason may be that the targeted epitope is a conformational epitope, and thus not accessible on the natively-folded protein. Another reason may be that the epitope is hidden after binding of anti-PLA₂R autoantibodies to the PLA₂R target. In this context, contacting the sample with a random PLA₂R binding molecule would result in the absence of binding of the said PLA₂R binding molecule to its antigenic target, and thus would result in a lack of detection signal, irrespective of whether glomerular deposits are present in the sample tested.

The lack of detection signal may thus be highly prejudicial for the reliability of a detection assay performed with such PLA₂R binding molecule on a biopsy tissue sample, and ultimately may be highly prejudicial for the patients' health as it may delay the establishment of a suitable treatment. Thus, an antibody suitable for kidney biopsies, such as paraffin-embedded kidney biopsies, should be ideally directed against a PLA₂R-derived epitope that is distinct from "pathogenic" epitopes recognized by patients' sera.

Due to the lack of structural information on the PLA₂R receptor in situ in the art, the inventors have determined herein novel relevant epitopes from PLA₂R that are exposed in tissue samples, including biopsy tissue samples, which encompasses paraffin- embedded tissue samples, for the purpose of generating antibodies directed against such epitopes that would be highly relevant for detection.

In order to find a good candidate for immunization, peptides derived from the PLA₂R sequence and containing putative linear epitopes, antigenic and/or accessibility motifs were synthesized and used as an antigen for the immunization of rabbits.

The amino acids will be referred herein according to their usual one-letter code, and as defined in the following list: A (Alanine), G (Glycine), V (Valine), L (Leucine), I (Isoleucine, M (Methionine), P (Proline), W (Tryptophane), F (Phenylalanine), M (Methionine), H (Histidine), S (Serine), T (Threonine), C (Cysteine), N (Asparagine), Q (Glutamine), Y (Tyrosine), D (Aspartic Acid), E (Glutamic Acid), K (Lysine) and R (Arginine).

The immunization of rabbits with a polypeptide derived from a 25-long fragment of PLA₂R led to the isolation of highly specific antibodies against PLA₂R.

In particular, the inventors have selected an antigenic fragment, also referred herein as peptide C (or "pep C"), which consists in the PLA₂R-derived amino acid sequence WIGLOEERA DEFRWRD GTP VI YONWD (SEQ ID N°8). The "pep C" antigenic fragment consists of the 875-900 fragment of full-length PLA₂R of sequence SEQ ID N⁰1.

Surprisingly, the inventors have found that the immunization of rabbits with the above-mentioned PLA₂R-derived peptide leads to the production of antibodies with improved specificity towards a specific epitope of PLA₂R, which will be referred herein as the minimal immunogenic epitope of PLA₂R, which PLA₂R-derived epitope is available in tissue samples containing PLA₂R deposits, which includes tissue samples wherein the PLA₂R deposits are complexed with anti-PLA₂R autoantibodies. Importantly, it has been determined herein that the said PLA₂R-derived epitope is available in paraffin-embedded tissue samples containing PLA₂R deposits. Thus, the inventors have identified a minimal immunogenic epitope of PLA₂R, which comprises, or consists in, a short peptide motif of sequence SEQ ID N°2, or FX1WX2, wherein Xi and X₂ are identical or different and each of Xi and X₂ consists of an amino acid residue. The meaning of the term "amino acid residue" is disclosed previously in the instant specification.

In particular, Xi and X₂ may represent a lysine (K), an arginine (R) or an alanine (A), and preferably mean an arginine.

According to this embodiment, the immunogenic epitope may thus comprise or consist in an epitope of sequence SEQ ID N°2 to SEQ ID N°8, of sequence SEQ ID N°15 to SEQ ID N°18, of sequence SEQ ID N°22, of sequence SEQ ID N°24 to SEQ ID N°25, or of sequence SEQ ID N°26, in particular an epitope of sequence SEQ ID N°4.

More particularly, the minimal immunogenic epitope consists in a short motif FRWR of sequence SEQ ID N°4.

According to another embodiment, the immunogenic epitope comprises, or consists in, the short peptide motif of sequence SEQ ID N°3, or X₃FXiWX₂, wherein Xi and X₂ are as defined above, and wherein X₃ may represent any amino acid residue.

In particular, Xi and X₂ may represent a lysine (K), an arginine (R) or an alanine (A), and preferably an arginine.

In particular, X₃ may represent an aspartate (D), a glutamate (E) or an alanine (A), and preferably a glutamate.

According to this embodiment, the immunogenic epitope comprises or consists of EFRWR (SEQ ID N°5).

Thus, the immunogenic epitope may comprise or consist of EERA DEFRWRD (SEQ ID N°6).

Thus, the immunogenic epitope may comprise or consist of

EERA DEFRWRDGTPVIYQNWD (SEQ ID N°7).

Thus, the immunogenic epitope may comprise or consist of WIGLQEERA DEFRWRDGTPVIYQNWD (SEQ ID N°8).

According to another embodiment, the invention pertains to a PLA₂R-derived peptide of formula:

H₂-[Nt]_y - ImEp - [Ct]_z - COOH

wherein: - y is an integer consisting in 0 or 1,

- z is an integer consisting in 0 or 1,

- Nt is a peptide of 1 to 20 amino acids in length,

- Ct is a peptide of 1 to 20 amino acids in length,

- ImEp is an immunogenic epitope of PLA₂R as defined above.

This invention also pertains to an immunogenic compound comprising a PLA₂R-derived peptide of formula:

H₂-[Nt]_y - ImEp - [Ct]_z - COOH

wherein:

- y is an integer consisting in 0 or 1,

- z is an integer consisting in 0 or 1,

- Nt is a peptide of 1 to 20 amino acids in length,

- Ct is a peptide of 1 to 20 amino acids in length,

- ImEp is an immunogenic epitope of PLA₂R as defined above.

This invention also relates to an immunogenic compound comprising the PLA₂R-derived peptide of SEQ ID N°8.

This invention also relates to an immunogenic compound of SEQ ID N°8. This invention also concerns an immunogenic compound comprising a PLA₂R-derived peptide of formula:

NH₂-[Nt]y - ImEp - [Ct]_z - COOH

wherein:

- y is an integer consisting in 0 or 1,

- z is an integer consisting in 0 or 1,

- Nt is a peptide of 1 to 20 amino acids in length,

- Ct is a peptide of 1 to 20 amino acids in length,

- ImEp is an immunogenic epitope of PLA₂R of SEQ ID N°8.

In particular, this invention pertains to an immunogenic compound covalently linked to a carrier molecule.

In some embodiments of the said immunogenic compound, the said PLA₂R- derived peptide is covalently linked to a carrier molecule, such as for example Keyhole Limpet Hemocyanin (also termed KLH). According to an exemplary embodiment, the invention also pertains to an immunogenic compound of SEQ ID N°8 covalently linked to a carrier molecule. The carrier molecule is preferably a carrier protein. The carrier protein may be selected among those which are well known by the one skilled in the art for preparing immunogenic compounds aimed at inducing an antibody response after administration to a human or to a non- human mammal.

This invention also pertains to a human Phospholipase A₂ receptor (PLA₂R) binding molecule directed against a PLA₂R-derived peptide, including against a PLA₂R- derived peptide comprised in an immunogenic compound as disclosed herein.

According to a preferred embodiment, the said human Phospholipase A₂ receptor (PLA₂R) binding molecule is an antibody, or an antibody-derived fragment.

According to other embodiments, the said PLA₂R binding molecule consists of a nucleic acid aptamer or of a peptide aptamer that specifically binds to a PLA2R-derived peptide comprising the minimal immunogenic epitope described above. Methods for selecting target-specific nucleic acid aptamers or peptide aptamers are well known in the art.

An antibody-derived fragment may be selected in the group comprising Fab, Fab', F(ab')₂, scFv Fd antibody fragments; Fv antibody fragments, isolated CDRs and diabodies, or an epitope-binding fragment thereof.

According to a more preferred embodiment, the said human Phospholipase A₂ receptor (PLA₂R) binding molecule is an antibody.

Most preferably, the human Phospholipase A₂ receptor (PLA₂R) binding molecule is a monoclonal antibody.

Thus the invention relates to a human Phospholipase A₂ receptor (PLA₂R) binding molecule directed against a PLA₂R-derived peptide of formula:

H₂-[Nt]_y - ImEp - [Ct]_z - COOH

wherein:

- y is an integer consisting in 0 or 1,

- z is an integer consisting in 0 or 1,

- Nt is a peptide of 1 to 20 amino acids in length,

- Ct is a peptide of 1 to 20 amino acids in length, - ImEp is an immunogenic epitope of PLA₂R as defined above, and in particular of sequence SEQ ID N° 2, SEQ ID N°3 or SEQ ID N°8.

A peptide of 1 to 20 amino acids in length encompasses peptides possessing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20 amino acids length. In particular, a peptide of 1 to 20 amino acids in length encompasses a peptide of 1 to 15 amino acids in length.

Advantageously, a human Phospholipase A₂ receptor (PLA₂R) binding molecule of the invention, as defined above and further herebelow, is directed against a PLA₂R-derived peptide with a minimal size of at least 10 amino acids, which includes at least 10, 11, 12, 13, 14 and 15 amino acids.

In particular, a human Phospholipase A₂ receptor (PLA₂R) binding molecule of the invention, such as an antibody, is directed against a PLA₂R-derived peptide with a minimal size of at least 12 amino acids, and preferably of at least 15 amino acids, in order to be sufficiently immunogenic.

The individual length of the N-terminal (Nt) and C-terminal (Ct) ends of the said PLA₂R-derived peptide may be equal or different.

According to a particular embodiment, the N-terminal end is of 12 amino acids in length and the C-terminal end is of 11 amino acids in length.

According to one exemplary embodiment, the PLA₂R-derived peptide is 25- amino acids long. This invention also concerns a human Phospholipase A₂ receptor (PLA₂R) binding molecule directed against a PLA₂R-derived peptide of formula (I) comprising the following amino acid sequence:

NH₂-[Nt]_y - FXiWX₂ [Ct]_z - COOH (I)

wherein:

- y is an integer consisting in 0 or 1,

- z is an integer consisting in 0 or 1,

- Nt is a peptide of 1 to 20 amino acids in length,

- Ct is a peptide of 1 to 20 amino acids in length,

- Xi and X₂ are identical or different and represent any amino acid.

The invention also relates to a human Phospholipase A2 receptor (PLA2R) binding molecule directed against a PLA₂R-derived peptide of formula (I) comprising the following amino acid sequence: H₂-[Nt]_y - X3FX1WX2 [Ct]_z - COOH (I)

wherein:

- y is an integer consisting in 0 or 1,

- z is an integer consisting in 0 or 1,

- Nt is a peptide of 1 to 20 amino acids in length,

- Ct is a peptide of 1 to 20 amino acids in length,

- Xi and X₂ are identical or different and represent either a lysine or an arginine, and preferably an arginine,

- X3 represents any amino acid.

Thus the invention relates to a PLA₂R binding molecule directed against a

PLA₂R derived peptide, wherein the said PLA₂R-derived peptide comprises the amino acid sequence X₃FXiWX₂.

In particular the invention relates to a PLA₂R binding molecule directed against a PLA₂R derived peptide, wherein the said PLA₂R-derived peptide comprises, or consists of, the amino acid sequence FRWR (SEQ ID N° 4).

According to another of its objects, the invention pertains to a PLA₂R binding molecule directed against a PLA₂R derived peptide, wherein the said PLA₂R-derived peptide comprises, or consists of, the amino acid sequence EFRWR (SEQ ID N° 5).

This invention also concerns a human Phospholipase A2 receptor (PLA₂R) binding molecule directed against a PLA₂R-derived peptide of formula (II) comprising the following amino acid sequence:

H₂-[Nt]_y - FRWR-[Ct]_z - COOH (II),

wherein:

- y is an integer consisting in 0 or 1,

- z is an integer consisting in 0 or 1,

- Nt is a peptide of 1 to 20 amino acids in length,

- Ct is a peptide of 1 to 20 amino acids in length.

According to another of its objects, the invention also relates to a human Phospholipase A2 receptor (PLA₂R) binding molecule directed against a PLA₂R-derived peptide of formula (III) comprising the following amino acid sequence:

NH₂-[Nt]_y - EFRWR-[Ct]_z - COOH (III) wherein: - y is an integer consisting in 0 or 1,

- z is an integer consisting in 0 or 1,

- Nt is a peptide of 1 to 20 amino acids in length,

- Ct is a peptide of 1 to 20 amino acids in length.

This invention also relates to a PLA₂R binding molecule directed against a

PLA₂R-derived peptide, wherein the said PLA₂R-derived peptide comprises the amino acid sequence WIGLQEERA DEFRWRDGTPVIYQNWD (SEQ ID N° 8).

According to a preferred embodiment, the invention relates to a PLA₂R binding molecule directed against a PLA₂R-derived peptide, wherein the said PLA₂R- derived peptide consists in the amino acid sequence WIGLQEERANDEFRWRDGTPVIYQNWD (SEQ ID N° 8).

The one skilled in the art may easily confirm that a PLA₂R binding molecule is directed against a PLA₂R-derived peptide as defined in the present specification, for example by performing conventional immunoassays (e.g. ELISA or RIA assays) using the PLA₂R-derived peptide as the bait molecule.

Also, the one skilled in the art may perform a "competition assay" with the ELISA screening method which is taught in example 1, the western blot method which is taught in example 3 or the immunohistochemistry method which is taught in example 3.

In some embodiments, a PLA₂R binding molecule of the invention is able to compete for binding to PLA₂R with a peptide of sequence SEQ ID N°5 (EFRWR) but not with a peptide of sequence SEQ ID N°l 1 (SWIGLQ).

In particular, the PLA₂R binding molecule is able to compete by ELISA screening method or immunohistochemistry method.

Thus a PLA₂R binding molecule of the invention is able to compete for binding to PLA₂R with a peptide of sequence SEQ ID N°5 (EFRWR) but not with a peptide of sequence SEQ ID N°l l (SWIGLQ) by ELISA screening method or immunohistochemistry method, in particular by immunohistochemistry method.

Such a "competition assay" is well known in the art. In particular, a competition assay relies on the establishment of a background signal. Thus, a positive signal may be detected when the value is statistically above the established background.

This invention also relates to a complex between (i) a human PLA₂R binding molecule as defined herein and (ii) human PLA₂. This invention notably relates to such complexes between (i) a human PLA₂R binding molecule as defined herein and (ii) human PLA₂.

The invention also relates to an epitope of a human PLA₂R bound by a PLA₂R binding molecule as defined above.

Surprisingly, the inventors have found that generating antibodies binding to the human phospholipase A₂ receptor (PLA₂R), and more specifically directed against a minimal immunogenic epitope as defined above, is particularly advantageous for targeting glomerular deposits.

According to the invention, "glomerular deposits" refer to the accumulation of deposits which are characteristic of a membranous nephropathy (MN) disease. In particular, subepithelial glomerular deposits may be indicative of an idiopathic membranous nephropathy. Such glomerular deposits are among the most dramatic findings in renal pathology and are well known in the art.

Glomerular deposits may be either PLA₂R-positive or PLA₂R-negative glomerular deposits.

For more information on the organization of such deposits, the man skilled in the Art may refer to Beck et al. (Beck et al. ; 2009; M-type phospholipase A₂ receptor as target antigen in idiopathic membranous nephropathy ; N Engl J Med. ; 361 : 11-21).

Non-exhaustively, glomerular deposits may be found in the following membranous nephropathy diseases: idiopathic membranous nephropathy, lupus membranous nephropathy and MN associated with cancer, sarcoidosis, infections such as hepatitis B, syphilis, and drug intoxication.

For the purpose of the invention, an "idiopathic membranous nephropathy" is also referred herein as a PLA₂R-positive membranous nephropathy, or as primary membranous nephropathy.

Of course the "idiopathic" terminology usually refers to a pathology for which the cause is unknown. However the recent identification of PLA₂R as a marker for membranous nephropathies, may lead to a change in the current classification (idiopathic vs. secondary MN).

In particular, membranous nephropathies may now be further classified as either PLA₂R-positive or PLA₂R-negative membranous nephropathies. Such a change should not be viewed, however as a limitation regarding the ability of antibodies and methods of the invention as tools to detect the occurrence of a membranous nephropathy in general, and of the PLA₂R epitope in glomerular deposits in particular. In particular, the PLA₂R binding molecules of the invention are able to target glomerular deposits comprising PLA₂R, including glomerular deposits comprising PLA₂R which are complexed with anti-PLA₂R auto-antibodies. Glomerular deposits which are complexed with anti-PLA₂R auto-antibodies may be also termed herein "immune glomerular deposits". In particular, immune glomerular deposits may be subepithelial immune glomerular deposits along glomerular capillary loops.

According to the invention, auto-antibodies are antibodies that are endogenously produced by the immune system of an individual and that are directed against one or more of the individual's own proteins, which auto-antibodies encompass in particular those directed against PLA₂R.

Thus, the invention relates to a PLA₂R binding molecule as defined in the present specification, which PLA₂R binding molecule binds to glomerular deposits and immune glomerular deposits.

This invention also relates to a method for generating antibodies binding to immune complexes between auto-antibodies directed against human phospholipase A₂ receptor (PLA₂R) and human PLA₂R comprising a step of providing an immunogenic compound containing a PLA₂R-derived peptide of SEQ ID N°2, in particular of SEQ ID N°4, and preferably of sequence SEQ ID N°8.

Notably, this invention relates to a method for generating antibodies as defined above, wherein the immunogenic compound is covalently linked to a carrier molecule. Most preferably, the said carrier molecule is Keyhole Limpet Hemocyanin (KLH).

This invention notably pertains to a method for generating antibodies directed against a PLA₂R-derived peptide as described herein comprising the steps of:

a) immunizing a non-human mammal with an immunogenic compound as described herein, and

b) collecting the antibodies produced by the immunized non-human mammal obtained at the end of step a).

Monoclonal antibodies may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique (Kohler, et al.,1975, Nature 256:495-497; Kozbor, et at , 1985, J. Immunol. Methods 81 :31-42; Cote, et al, 1983, Proc.Natl.Acad.Sci. 80:2026-2030; Cole, et al, 1984, MoL Cell Biol. 62: 109-120).

In some embodiments, a PLA₂R binding molecule as defined above may be labeled covalently or non-covalently to a detectable molecule. Examples of detectable molecules are well known in the Art.

In a non-limitative way, a PLA₂R binding molecule (for instance an antibody) may be linked to a detectable molecule chosen in the following list: (i) a radioactive molecule, (ii) a fluorescent molecule, (iii) a luminescent molecule, (iv) a receptor molecule that is selectively recognized by a ligand and (v) a metallic tag.

In particular one may consider a PLA₂R binding molecule linked to a naturally-occuring or radioactive heavy isotope, a luminescent molecule such as a chromophore, a luminophore, a fluorophore, a calorimetric agent, a magnetic substance, an electro-chemo-luminescent marker, a spin marker, gold or silver particles, and enzymes or substrates.

Advantageously, a PLA₂R binding molecule may be linked to fluorophores, to lanthanide complexes and metallic tags, such as Europium or Terbium, fluorescein, fluorescein isothiocyanate (FITC), dichlorotriazinylamine, rhodamine, eosine, coumarin, methyl-coumarin, pyrene, Malachite green, Cy^®-type or Alexa^®-type (Invitrogen) fluorophores such as Cy^®3, Cy^®5, Alexa Fluor^® 488, Lucifer Yellow, Cascade Blue, Texas Red, dansyle chrloride, phycoerythrin, luciferin, Green Fluorescent Protein and its variants, bore-dipyromethene (BODIPY), and others which are described in Haugland, Molecular Probes Handbook, (Eugene, Oreg.) 6th Edition; The Synthegen catalog (Houston, Tex.), Lakowicz, Principles of Fluorescence Spectroscopy, 2nd Ed., Plenum Press New York (1999).

In other embodiments, a PLA₂R binding molecule is not labeled with a detectable molecule. According to that embodiment, binding of the PLA₂R binding molecule to its target may then be detected using a secondary labelled molecule. Such a case may occur for instance when the PLA₂R binding molecule is an antibody. In a non- limitative way, such an antibody may be revealed using a secondary labelled antibody when performing a Western Blot assay or Immunohistochemistry. Methods of detection and diagnosis

A PLA₂R binding molecule of the invention is particularly aimed at detecting the presence of human phospholipase A₂ receptor (PLA₂R) in a sample.

Advantageously, a PLA₂R binding molecule of the invention is labeled with a detectable molecule, or can interact with a labeled molecule.

For instance, a sample may be a biological sample, such as a blood-derived sample or a biopsy. Advantageously, the sample is a biological sample that is susceptible to contain a human phospholipase A2 receptor, and/or glomerular deposits, such as a blood serum or a tissue sample.

A tissue sample may be a biopsy, such as a kidney biopsy, and more particularly a paraffin-embedded kidney biopsy. The preparation of a kidney biopsy is well known in the Art. One may for instance provide a paraffin embedded kidney biopsy specimen from an individual suspected of having a membranous nephropathy disease.

Advantageously, the binding of the PLA₂R binding molecule may be detected using immunofluorescence staining, in particular Antigen retrieval immunofluorescence staining, and more particularly Heat Induced Epitope Retrieval (HIER) method, as described in examples 3 or 4. Such methods are well known in the Art (see for instance Shi et al; 2011; Antigen Retrieval Immunohistochemistry Review and Future Prospects in Research and Diagnosis over Two Decades ; J Histochem Cytochem.; 59(1): 13-32).

Thus, the invention relates to a method for detecting the presence of human phospholipase A2 receptor (PLA₂R) in a sample comprising the steps of:

a) providing a sample to be tested,

b) bringing into contact the sample provided at step a) with a PLA₂R binding molecule, and

c) detecting the binding of the said PLA₂R binding molecule onto the said sample, whereby the presence of human PLA₂R is detected.

Advantageously, when the method for detecting the presence of human phospholipase A2 receptor (PLA₂R) in a sample is applied to a sample suspected of containing glomerular deposits, it can be used as a method for diagnosing the occurrence of a membranous nephropathy (MN) disease in an individual. Thus the invention relates to a method for diagnosing the occurrence of a membranous nephropathy (MN) disease in an individual to be tested, and more particularly for diagnosing the occurrence of an idiopathic membranous nephropathy disease.

According to that embodiment, the method for diagnosing the occurrence of a membranous nephropathy (MN) disease relies on the detection of glomerular deposits within the sample by a PLA₂R binding molecule of the invention.

Detection of the PLA₂R-derived immunogenic epitope of the invention within glomerular deposits is indicative of a membranous nephropathy disease.

In particular, glomerular deposits may be an immune glomerular deposit, which means that glomerular deposits may further comprise autoantibodies, such as anti- PLA₂R autoantibodies.

Advantageously, the immunogenic epitope of the invention is not recognized by anti-PLA₂R autoantibodies, which means that it is also accessible in immune glomerular deposits. Thus, the PLA₂R immunogenic epitope may be detected both in glomerular deposits and immune glomerular deposits.

Thus, the invention also relates to a method for detecting the presence of immune complexes between auto-antibodies directed against human phospholipase A2 receptor (PLA₂R) and human PLA₂R in a sample comprising the steps of:

a) providing a tissue sample from an individual to be tested,

b) bringing into contact the tissue sample provided at step a) with a PLA₂R binding molecule of the invention, and

c) detecting the binding of the said PLA₂R binding molecule onto the said tissue sample, whereby the said immune complexes are detected.

Detection of glomerular deposits comprising the PLA₂R immunogenic epitope of the invention is indicative of the occurrence of a membranous nephropathy, preferably a primary or PLA₂R-positive form of membranous nephropathy, which is also referred herein as idiopathic membranous nephropathy.

Thus the invention also relates to a method for diagnosing the occurrence of a membranous nephropathy (MN) disease in an individual to be tested comprising the steps of: (i) performing a method for detecting the presence of human phospholipase A₂ receptor (PLA₂R) in a sample as defined above from the said individual and

(ii) diagnosing the occurrence of a membranous nephropathy disease if glomerular deposits comprising human PLA₂R are detected in the said sample.

According to that embodiment, the sample may be any sample from the said individual that is suspected of containing glomerular deposits. Preferably, a sample suspected of containing glomerular deposits may be a renal biopsy, or part of a renal biopsy.

Even more advantageously, the method for diagnosing the occurrence of a membranous nephropathy (MN) disease in an individual may comprise a step of detecting the presence of anti-PLA₂R autoantibodies in a sample.

Thus the invention relates to a method for diagnosing the occurrence of a membranous nephropathy (MN) disease in an individual, comprising the step of detecting the presence of anti-PLA₂R autoantibodies in a sample from the said individual.

According to that embodiment, the sample may be the same sample as the sample where the method for detecting the presence of human phospholipase A2 receptor (PLA₂R) is performed, or a different sample.

The step of detecting the presence of anti-PLA₂R autoantibodies in a sample may be performed according to any method known in the Art, and/or as described in Debiec & Ronco (Debiec & Ronco; 2011; PLA₂R autoantibodies and PLA₂R glomerular deposits in membranous nephropathy; N. Engl. J. Med.; 364(7): 689-90) or in Hofstra & Wetzels (Hofstra & Wetzels ; 2012 ; Anti-PLA₂R antibodies in membranous nephropathy: ready for routine clinical practice? ; Neth J Med.; 70(3): 109-13).

According to a particular embodiment, the step of detecting the presence of anti-PLA₂R autoantibodies may be performed on a blood-derived sample such as the serum of an individual.

In a non-limitative way, the presence of anti-PLA₂R autoantibodies may be assessed in serum by Western blotting under non-reducing conditions, using glycoproteins extracted from normal human glomeruli after removing contaminating endogenous immunoglobulin G. It may also be assessed using an immunofluorescence assay with the use of human embryonic kidney 293 cells that ware transiently transfected with full-length complementary DNA encoding a PLA₂R1 isoform. Thus the invention relates to a method for diagnosing the occurrence of a membranous nephropathy (MN) disease in an individual comprising the steps of:

(i) performing a method for detecting the presence of human phospholipase A2 receptor (PLA₂R) in a tissue sample from the said individual and as defined above,

(ii) detecting the presence of anti-PLA₂R autoantibodies in a sample from the said individual,

(iii) diagnosing the occurrence of a membranous nephropathy disease if anti-PLA₂R autoantibodies and glomerular deposits are detected in the said individual,

wherein steps (i) and (ii) may be performed in any order,

wherein steps (i) and (ii) may be performed on the said sample or on different samples.

According to a preferred embodiment, the method for detecting the presence of human phospholipase A₂ receptor (PLA₂R) in a sample or a tissue sample is performed on a kidney biopsy, and preferably a paraffin-embedded kidney biopsy.

When the PLA₂R immunogenic epitope of the invention is detected within glomerular deposits (PLA₂R-positive deposits), it is indicative of a membranous nephropathy, and more particularly a primary ("idiopathic") membranous nephropathy.

More precisely, subepithelial PLA₂R-positive glomerular deposits may appear during iMN in a finely granular pattern of sub-epithelial deposits along glomerular capillary loops.

In contract, lupus membranous nephropathy is mostly characterized by PLA₂R-negative mesangial or subendothelial deposits.

Thus, when the PLA₂R immunogenic epitope of the invention is not detected within glomerular deposits (PLA₂R-negative deposits), it may be indicative of a secondary membranous nephropathy.

According to a particular embodiment, the method for detecting the presence of anti-PLA₂R autoantibodies in a sample is performed on serum.

When anti-PLA₂R autoantibodies are detected in a sample from an individual, it is indicative of membranous nephropathy, and more particularly a primary ("idiopathic") membranous nephropathy.

On the other hand, the absence of circulating anti-PLA₂R autoantibodies is not sufficient to rule out a membranous nephropathy, or even to discriminate between an idiopathic membranous nephropathy and a lupus membranous nephropathy, especially at an early stage of the disease.

Thus, it is not always possible to rule out the possibility of a secondary membranous nephropathy, especially when the clinical signs and the serology do not clearly fit into any standard criteria.

That is the case when a lupus membranous nephropathy is suspected. Thus, it may be difficult to assess in an individual whether a suspected membranous nephropathy may be actually due to an underlying lupus or to an idiopathic cause.

A recent study (Svobodova et al. 2012; Kidney biopsy is a sensitive tool for retrospective diagnosis of PLA₂R-related membranous nephropathy; Nephrol Dial Transplant; 0: 1-5) has shown that a PLA₂R antigen may be detected in immune deposits and within kidney biopsies for a majority of primary "idiopathic" membranous nephropathy patients. On the other hand, the same study has shown that PLA₂R-positive immune deposits were not detected in lupus MN.

Thus, the invention further relates to a method for discriminating between idiopathic MN and lupus MN comprising the steps of:

a) providing a tissue sample from an individual to be tested,

b) bringing into contact the tissue sample provided at step a) with a PLA₂R binding molecule of the invention, and

c) detecting the binding of the said PLA₂R binding molecule onto the said tissue sample, the presence of PLA₂R being indicative of idiopathic MN.

According to a particular embodiment, the invention relates to a method for discriminating between idiopathic (or PLA₂R-positive) MN and lupus MN, which may optionally comprise a step of detecting the presence of autoantibodies indicative of a lupus and/or anti-PLA₂R autoantibodies in a sample from the said individual, wherein the step of detecting the presence of autoantibodies may be performed on the tissue sample of step a) or on a different sample.

Preferably, the invention relates to a method for discriminating between idiopathic MN and lupus MN comprising a step of detecting the presence of anti-PLA₂R autoantibodies in a sample from the said individual, wherein the step of detecting the presence of anti-PLA₂R autoantibodies may be performed on the tissue sample of step a) or on a different sample. Thus the invention also relates to a method for discriminating between idiopathic MN and lupus MN comprising the steps of:

a) providing a tissue sample from an individual to be tested,

b) bringing into contact the tissue sample provided at step a) with a PLA₂R binding molecule of the invention,

c) detecting the binding of the said PLA₂R binding molecule onto the said tissue sample, the presence of PLA₂R being indicative of idiopathic MN,

d) detecting the presence of anti-PLA₂R autoantibodies in a sample from the said individual,

the presence of PLA₂R in the tissue sample and of anti-PLA₂R autoantibodies in the sample being indicative of idiopathic MN,

wherein step (d) may be performed in any order,

wherein steps (c) and (d) may be performed on the said sample or on different samples. Compositions and kits

According to one embodiment, the invention relates to a composition comprising a PLA₂R binding molecule as defined by the invention.

According to another embodiment, the invention relates to a kit for detecting human PLA₂R, the said kit comprising a PLA₂R binding molecule as defined by the invention and one or more reagents necessary for detecting the extent of binding between the said PLA₂R binding molecule and a sample to be tested.

Of course, the nature and quantity of reagents included in the kit may vary depending on the nature of the sample to be tested, and the technique that is used for detection.

According to said embodiment, a reagent necessary for detecting the extent of binding between the said PLA₂R binding molecule and a sample to be tested can be a peptide sequence of SEQ ID N°l to 26, an epitope of a human PLA₂R bound by a PLA₂R binding molecule, or a complex between (i) a human PLA₂R binding molecule and (ii) human PLA₂R.

According to another embodiment, the invention relates to a kit for diagnosing a membranous nephropathy disease, which comprises at least a PLA₂R binding molecule of the invention. It may further comprise one or more reagents necessary for detecting the presence of autoantibodies in a sample, in particular for detecting autoantibodies against PLA₂R and autoantibodies indicative of a lupus.

Kits for the detection of autoantibodies are well known in the art. In a non- limitative way, commercial autoantibody assay kits may thus include reagents for immunofluorescence, immunodiffusion, immunoblotting, or ELISA methods.

EXAMPLES

A. MATERIAL & METHODS

A.l. Immunization with PLA2R-derived antigenic peptides and purification of rabbit antibodies

We selected three antigenic peptides derived from the PLA2R sequence: peptide A (pepA) 437-467 aa, peptide B (pepB) 481-507 aa, and peptide C (pep C) 875- 900 aa from the sequence of PLA2R of sequence SEQ ID N°l, that contain linear epitopes, antigenic and/or accessibility motifs.

Peptide A is a 29-long polypeptide of sequence SEQ ID N°9.

Peptide B is a 27-long polypeptide of sequence SEQ ID N°10.

Peptide C is a 25-long polypeptide of sequence SEQ ID N°8.

These three peptides were synthesized and after coupling with KLH carrier were used, as an antigen for immunization of rabbits (two rabbits for each peptide) and development of polyclonal anti-peptide antibodies according to the standard protocols known in the Art. After the last immunization, the serum from each rabbit was tested by ELISA against the peptide used for immunization. All sera taken from rabbits were positive in this test. Next, rabbit IgG against pep C were purified on a carrier sepharose linked with peptide C by a cysteine residue according to the usual methods.

A.2. Production and cloning of hybridomas

For production of monoclonal antibodies, peptide C was chosen to immunize BALB/C mice. Immunisation, fusion and screening of the positive hybridoma and subsequent cloning of hybridomas selected at the polyclonal step were conducted according to the standard protocols known in the Art. In our laboratory, we screened all of the above samples on paraffin-embedded biopsy specimens from MN patients. Finally, we selected several hybridoma supernatants which stained PLA₂R in subepithelial deposits in glomeruli.

A.3. Analysis of sera and hybridoma fluids by peptide ELISA and alanine scanning

The immunization protocol, production and cloning of hybridomas, and analysis of sera and hybridoma fluids by peptide ELISA were done in our laboratory. Overlapping peptides (of sequences SEQ ID N° 12 to 21) defined in figure 1 with a spacer sequence GSGS and biotin residue at the N-terminus were coated on NeutrAvidin ELISA (Thermo Fisher ref 15507) plate at a concentration of l .C^g/ml. The peptide-containing plates were washed three times with washing buffer (0.05% Tween-20 in TBS), and blocked with SuperBlock blocking buffer (ThermoFisher ref 37535). Next, the plate was incubated overnight at +4°C with monoclonal antibody 12D8 immunopurified from hybridoma fluid and diluted at l^g/ml in TBS 1 : 1 SuperBlock blocking buffer. After three washing steps with washing buffer, bound antibodies were detected by incubation with goat anti-mouse IgG-URP conjugate (goat anti-mouse abeam ref ab7023) diluted 1 :4000 in TBS 1 : 1 SuperBlock blocking buffer. After washing with washing buffer, enzyme substrate TMB was added, and after 10 min the reaction was stopped with H2SO4. The absorbance was read at 450 nm.

Antibodies from patients sera were isolated according to the standard protocols. Sera were sampled before treatment at the time of renal biopsy. Those patients had circulating anti-PLA2R antibodies and biopsy-proven MN.

The critical amino-acids in the minimal epitope were identified further by alanine screening. Peptides with one amino-acid substituted by alanine (spacer sequence GSGS and biotin residue at the N-terminus, a-carboxamide at the C terminus), of sequences SEQ ID N°5 and SEQ ID N°22 to 26, were coated on NeutrAvidin plate. An ELISA with hybridoma fluids was performed according to the same protocols as previously described.

A.4. Selection of paraffin-embedded patient's kidney biopsy specimen Biopsies were obtained from a cohort of patients published in : Debiec H and Ronco P (2011), N Engl J Med 364 :689-690).

A.5. Western Blot on recombinant PLA₂R Recombinant PLA₂R (the extracellular domain of the human PLA2R isoform 1 (SEQ ID N°l, aminoacid residues 1-1397, 300 ng/track) was run on a 10% SDS-PAGE gel under non-reducing conditions, and the gel was electroblotted onto immobilion membrane using semi-dry conditions. After blocking, with SuperBlok blocking buffer, each track was cut out for incubation with: the monoclonal antibody (concentration 0.3 μg/ml), the monoclonal antibody pre-incubated with 20 μg of blocking peptide (EFRWR) or with a same concentration of control peptide (SWIGLQ). Bound antibodies were detected by incubation with goat anti-mouse Ig-HRP (abeam) conjugate diluted at 1 :5000 in TBS 1 : 1 SuperBlock blocking buffer. After washing with washing buffer, immunoglobulins were visualized with Super Signal West Pico Chemiluminescent Substrate (Pierce ref 34080)

A.6. Immunofluorescence with a labelled anti-PLA₂R antibody

Patients biopsy was fixed in solution containing ethanol (75%): formol (3%): acetic acid (5%). Fixed patients biopsy specimens were embedded in paraffin according to standard techniques. Devaxed sections (4μπι thickness) were hydrated and antigens were retrieval by Heat Induced Epitope Retrieval (HIER) at pH 6 and temperature 95°C (Diagnostic BioSystem ref K035).

HIER at pH 6 was followed by 5-min incubation with HistoReveal (Abeam ref. ab 103720). The concentration of a monoclonal antibody of the invention is set at 0.3 μg/ml and the secondary antibody is a polyclonal goat Alexa488-conjugated anti- mouse IgG antibody at a dilution of 1/500 (Molecular Probes - ref.Al 1001)

Kidney biopsies were obtained from diagnosed MN patients; adjacent sections (4 μπι) were incubated with the monoclonal antibody only (A). Adjacent sections were also pre-incubated with 20 μg of blocking peptide (B) or with 20 μg of control peptide (C) prior to incubation with the monoclonal antibody of the invention.

Example 1 : Identification of a specific minimal binding epitope from a monoclonal hybridoma fluid

To identify the specific minimal epitope, the monoclonal 12D8 hybridoma fluid was tested by ELISA with overlapping peptides. The amino-acid sequences of pepC and overlapping 10-aa long peptides in double amino-acid steps were synthesized as shown in the Material & Methods section. The absorbance at 450 nm is indicative of binding of the secondary anti-mouse IgG HRP conjugate to the primary monoclonal anti-PLA₂R antibody isolated from a mouse hybridoma. Thus, a high signal necessarily relates to the formation of a complex between the primary anti-PLA₂R of the invention and the peptide to be tested.

Results

As shown in figures 1 and 2, peptides 4-7 show significant absorbance over the background, whereas peptide 3 shows a weak signal indicative of residual binding.

Thus, it can be inferred from the above that peptides 4-7 share the same minimal epitope which binds selectively to a monoclonal anti-PLA₂R of the invention. All four peptides comprise the same FRWR minimal epitope. Thus, the ELISA screening establishes that the monoclonal anti-PLA₂R antibody of the invention recognizes specifically the minimal epitope FRWR.

In addition, the mild signal obtained from peptide 3 suggests that the glutamic acid which is part of the EFRWR motif may contribute weakly to the overall affinity of the antibody to the pep C polypeptide.

Other ELISA screenings with peptides 1, 2, 8, 9 and 10 show no signal, which indicates that the minimal epitope is absent from those constructs. Thus, it is assumed from the above that the anti-PLA₂R does not bind to any one of those regions, due to the lack of signal above the background.

Of note, patients' sera were not reactive with the tested peptides, which indicates that the monoclonal anti-PLA₂R antibody of the invention is reactive against a different epitope of PLA₂R than anti-PLA₂R auto-antibodies isolated from patients.

Within the minimal (E)FRWR binding epitope, alanine scanning (see figure 3) was further performed in order to identify the critical amino-acids involved in this interaction.

These experiments provide evidence that F and W residues in the minimal epitope are critical for antibody binding, due to complete loss of signal when the peptide is of sequence SEQ ID N°23 or SEQ ID N° 25.

On the other hand, it is shown that other residues may be mutated into an Alanine without detectable loss of affinity in an ELISA test, and when the peptide is of sequence SEQ ID N°22, SEQ ID N°24 or SEQ ID N°26. Of note, an Alanine possesses distinct steric hindrance and charge properties in comparison with amino acids such as a glutamic acid or an arginine. This data provides evidence that other amino acids are optional for binding, and may thus be mutated by any amino acid.

Example 2 : Peptide C-specific antibodies as tools for targeting glomerular deposits within kidney biopsies

Antigen-affinity purified rabbit IgG against peptide C were tested on paraffin- embedded biopsy sample from patients with membranous nephropathy.

Results

In this test only IgG from rabbit immunized with peptide C stained PLA₂R in subepithelial deposits along glomerular capillary loops in patients with MN whereas IgG antigen affinity purified from sera of rabbits immunized with peptide A or B did not react with PLA₂R in glomerular immune deposits.

Thus, it is shown that the PLA₂ receptor in glomerular immune deposits comprises epitopes which are not accessible within kidney biopsy specimens.

On the other hand, this result shows that the PLA₂R-derived peptide C (or

"pepC") comprises an epitope which can be targeted for detection of PLA₂R-enriched glomerular deposits directly from biopsies.

Example 3 ; Immunofluorescence staining of the paraffin embedded patients ' kidney biopsy specimen showing membranous nephropathy: relevance of the (E)FRWR motif for detection in situ

In addition to example 1 and example 2, this experiment shows that in situ targeting is abolished after competition with the EFRWR peptide.

Results

Specificity of monoclonal antibody binding (12D8 hybridoma fluid) to the MN patients' glomeruli was confirmed by extinction of fluorescence with an excess (20μg) of EFRWR peptide but not with a control peptide SWIGLQ (data not shown). Similarly, our monoclonal antibody recognized recombinant PLA₂R protein (SEQ ID N°l, amino-acid residues 1-1397) in Western blot analysis and this immunoreactivity (Figure 4; lane 1) was suppressed when the antibody was pre-incubated with an excess of EFRWR peptide (Figure 4; lane 2) but not with the control peptide (Figure 4; lane 3). Thus the (E)FRWR motif is shown to be a biomarker of glomerular deposits within kidney biopsy specimen. Of note, the SWIGLQ control peptide mimics the N-terminal end of the peptide C fragment; thus this experiment establishes, as a secondary control, that the (E)FRWR motif is relevant for modulating in situ staining of glomerular deposits. Example 4 ; Immunofluorescence staining of the paraffin embedded patients ' kidney biopsy specimen showing membranous nephropathy: comparison of an anti-PLAiR antibody of the invention with previously reported anti-PLAjR antibodies

Along with example 3, this experiment shows that anti-PLA₂R antibodies against the immunogenic epitope (E)FRWR provide superior specificity for glomerular deposits as compared to other commercial polyclonal antibodies.

Results

We tested our monoclonal antibody in parallel with the reference rabbit polyclonal antibody (Atlas antibody produced against aa 395-530) on thirty (30) biopsies from patients with MN. Those thirty biopsies were taken from patients with histologically- proven idiopathic MN. Both antibodies stained PLA₂R in subepithelial immune deposits along glomerular capillary loops in all biopsies (data not shown). Thus this experiment shows that the targeted epitope on PLA₂R is an excellent biomarker for membranous nephropathy diseases.

In addition we tested twenty (20) biopsies with histologically-proven lupus nephritis MN with both antibodies and all samples were negative.

Thus this experiment provides evidence that the PLA₂R antibodies of the invention are particularly suitable for kidney biopsies.

There are also commercially distributed polyclonal antibodies raised against the internal or C-terminal parts of PLA₂R. However these commercial antibodies target epitopes that do not stain PLA₂R in immune deposits (Figure 5). Altogether, these results indicate that the target epitope of our monoclonal antibody and of Atlas polyclonal antibodies but not that for other antibodies is accessible for binding in glomerular immune deposits in MN patients and can be used for diagnosis.

In conclusion, our approach (i.e. screening of hybridomas against biopsies), allowed us to select clones reactive with a specific PLA₂R epitope that is accessible and available on paraffin-embedded biopsies from MN patients. Thus, anti-PLA₂R binding antibodies of the invention are the first very well characterized monoclonal antibody that can be made commercially available for histopathologic purposes.

SEQUENCE LISTING

SEQ ID N°l

MLLSPSLLLLLLLGAPRGCAEGVAAALTPERLLEWQDKGIFVIQSESLK KCIQAGKSVLTLENCKQA KHMLWKWVS HGLFNIGGSGCLGL FSAPEQPLSL YECDSTLVSLRWRC RKMITGPLQYSVQVAHDNTVVASRKYIHKWISYGSGGGD ICEYLHKDLHTIKGNTHGMPCMFPFQYNHQWHHECTREGREDDLLWCATTSRYE RDEKWGFCPDPTSAEVGCDTIWEKDLNSHICYQF LLSSLSWSEAHSSCQMQGGT LLSITDETEE FIREHMSSKTVEVWMGLNQLDEHAGWQWSDGTPLNYLNWSPEV FEPFVEDHCGTFSSFMPSAWRSRDCESTLPYICKKYL HIDHEIVEKDAWKYYA THCEPGW PYNRNCYKLQKEEKTWHEALRSCQADNSALIDITSLAEVEFLVTLLG DENASETWIGLSSNKIPVSFEWS DSSVIFTNWHTLEPHIFP RSQLCVSAEQSEGH WKVKNCEERLFYICKKAGHVLSDAESGCQEGWERHGGFCYKIDTVLRSFDQASS GYYCPPALVTITNRFEQAFITSLISS VVKMKDS YFWIALQDQ DTGEYTWKPVGQ KPEPVQYTHWNTHQPRYSGGCVAMRGRHPLGRWEVKHCRHFKAMSLCKQPVE NQEKAEYEERWPFHPCYLDWESEPGLASCFKVFHSEKVLMKRTWREAEAFCEEF GAHLASFAHIEEE FV ELLHSKFNWTEERQFWIGF KR PLNAGSWEWSDRTPV VSSFLDNTYFGEDARNCAVYKA KTLLPLHCGSKREWICKIPRDVKPKIPFWYQY DVPWLFYQDAEYLFHTFASEWL FEFVCSWLHSDLLTIHSAHEQEFIHSKIKALSK YGASWWIGLOEERA DEFRWRDGTPVIYONWDTGRERTVNNOSORCGFISSITGL WGSEEC S VSMP SICKRKK VWLIEKKKDTPKQHGTCPKGWL YFNYKCLLLNIPKDP SSWKNWTHAQHFCAEEGGTLVAIESEVEQAFITMNLFGQTTSVWIGLQ DDYET WLNGKPVVYSNWSPFDIINIPSHNTTEVQKHIPLCALLSS P FHFTGKWYFEDCG KEG YGF VCEKMQDT S GHGVNT SDMYPMPNTLE YG RT YKIIN A MT W Y A AIKT CLMHKAQLVSITDQYHQSFLTVVL RLGYAHWIGLFTTDNGL FDWSDGTKSSF TFWKDEESSLLGDCVFADSNGRWHSTACESFLQGAICHVPPETRQSEHPELCSETS IPWIKFKSNCYSFSTVLDSMSFEAAHEFCKKEGS LLTIKDEAENAFLLEELFAFGS SVQMVWLNAQFDGN ETIKWFDGTPTDQSNWGIRKPDTDYFKPHHCVALRIPEG LWQLSPCQEKKGFICKMEADIHTAEALPEKGPSHSIIPLAVVLTLIVIVAICTLSFCI YKHNGGFFRRLAGFR PYYPATNFSTVYLEENILISDLEKSDQ

SEQ ID N°2

FXWX

SEQ ID N°3

XFXWX

SEQ ID N°4

FRWR SEQ ID N°5

EFRWR

SEQ ID N°6

EERA DEFRWRD

SEQ ID N°7

EERANDEFRWRDGTPVIYQNWD SEQ ID N°8

WIGLOEERANDEFRWRDGTPVIYONWD SEQ ID N°9

TWIGLSSNKIPVSFEWSNDSSVIFTNWHT SEQ ID N°10

AEQSEGHWKVKNCEERLFYICKKAGHV

SEQ ID N°ll

SWIGLQ

SEQ ID N°12

WIGLQEERAN

SEQ ID N°13

GLQEERANDE

SEQ ID N°14

QEERANDEFR

SEQ ID N°15

ERANDEFRWR SEQ ID N°16

ANDEFRWRDG

SEQ ID N°17 DEFRWRDGTP

SEQ ID N°18 FRWRDGTPVI

SEQ ID N°19 WRDGTPVIYQ

SEQ ID N°20 DGTPVIYQNW

SEQ ID N°21

TPVIYQNWD

SEQ ID N°22 AFRWR

SEQ ID N°23

EARWR

SEQ ID N°24

EFAWR

SEQ ID N°25 EFRAR

SEQ ID N°26

EFRWA

Claims

1. A human Phospholipase A₂ receptor (PLA₂R) binding molecule directed against a PLA₂R-derived peptide of formula (II) comprising the following amino sequence:

H₂-[Nt]_y - FRWR - [Ct]_z - COOH (II)

wherein:

- y is an integer consisting in 0 or 1,

- z is an integer consisting in 0 or 1,

- Nt is a peptide of 1 to 20 amino acids in length,

- Ct is a peptide of 1 to 20 amino acids in length.

2. The PLA₂R binding molecule according to claim 1, wherein the said binding molecule is an antibody, or an antibody- derived fragment selected in the group comprising Fab, Fab', F(ab')₂, scFv Fd antibody fragments, Fv antibody fragments, isolated CDRs and diabodies, or an epitope-binding fragment thereof.

3. The PLA₂R binding molecule according to claim 2, wherein the said PLA2R-derived peptide consists in the amino acid sequence WIGLQEERANDEFRWRD GTPVIYQNWD (SEQ ID N°8).

4. The PLA₂R binding molecule according to any one of the preceding claims, which binds to glomerular deposits and immune glomerular deposits.

5. The PLA₂R binding molecule according to any one of the preceding claims, wherein the binding molecule is directed against a PLA₂R-derived peptide with a minimal size of at least 10 amino acids.

6. A complex between (i) a human PLA₂R binding molecule according to claim 1 and (ii) human PLA₂R.

7. An epitope of a human PLA₂R bound by a PLA₂R binding molecule as defined in any one of claims 1 to 5.

8. A method for detecting the presence of human phospholipase A2 receptor (PLA₂R) in a sample comprising the steps of:

a) providing a sample to be tested,

b) bringing into contact the sample provided at step a) with a PLA₂R binding molecule as defined in any one of claims 1 to 5, and c) detecting the binding of the said PLA₂R binding molecule onto the said sample, whereby the presence of human PLA₂R is detected.

9. A method for diagnosing the occurrence of a membranous nephropathy (MN) disease in an individual to be tested comprising the steps of:

(i) performing the method according to claim 8 on a sample from the said individual and

(ii) diagnosing the occurrence of a membranous nephropathy disease if glomerular deposits comprising human PLA₂R are detected in the said sample.

10. A method for diagnosing the occurrence of a membranous nephropathy (MN) disease in an individual as defined in claim 9, comprising the step of detecting the presence of anti-PLA₂R autoantibodies in a sample from the said individual, wherein the sample is the same as in claim 9 or a different sample.

11. A method for discriminating between PLA₂R-positive MN and lupus MN comprising the steps of:

a) providing a tissue sample from an individual to be tested,

b) bringing into contact the tissue sample provided at step a) with a PLA₂R binding molecule as defined in any one of claims 1 to 5, and

c) detecting the binding of the said PLA₂R binding molecule onto the said tissue sample, the presence of PL A₂R being indicative of PLA₂R-positive MN.

12. A composition comprising a PLA₂R binding molecule according to any one of claims 1 to 5.

13. A kit for detecting human PLA₂R, the said kit comprising a PLA₂R binding molecule according to any one of claims 1 to 5 and one or more reagents necessary for detecting the extent of binding between the said PLA₂R binding molecule and a sample to be tested.

14. A method for generating antibodies binding to immune complexes between auto-antibodies directed against human phospholipase A₂ receptor (PLA₂R) and human PLA₂R comprising a step of providing an immunogenic compound containing a PLA₂R-derived peptide of SEQ ID N°8.

15. A PLA₂R-derived peptide of formula:

NH₂-[Nt]_y - ImEp - [Ct]_z - COOH

wherein: - y is an integer consisting in 0 or 1,

- z is an integer consisting in 0 or 1,

- Nt is a peptide of 1 to 20 amino acids in length,

- Ct is a peptide of 1 to 20 amino acids in length,

- ImEp is an immunogenic epitope of PLA₂R of SEQ ID N°8.

16. An immunogenic compound comprising the PLA₂R-derived peptide according to claim 15, wherein the said peptide is covalently linked to a carrier molecule.