Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2851—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the lectin superfamily, e.g. CD23, CD72
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
  • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/32—Immunoglobulins specific features characterized by aspects of specificity or valency specific for a neo-epitope on a complex, e.g. antibody-antigen or ligand-receptor
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/28—Neurological disorders
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/28—Neurological disorders
  • G01N2800/2842—Pain, e.g. neuropathic pain, psychogenic pain

Definitions

• the invention relates to PLA 2 R-binding molecules such as anti-PLA 2 R antibodies.
• PLA 2 R-binding molecules such as anti-PLA 2 R antibodies.
• the invention relates also to the identification of novel epitopes of PLA 2 R, immunogenic compounds, kits and compositions, as well as methods for obtaining such antibodies.
• the invention relates to methods for detecting the presence of PLA 2 R in a sample.
• the invention relates to methods for diagnosing the occurrence of a membranous nephropathy in an individual, such as an idiopathic (or PLA 2 R-positive) membranous nephropathy.
• MN Membranous nephropathy
• GBM glomerular basement membrane
• 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 PLA 2 R-positive membranous nephropathy.
• iMN idiopathic membranous nephropathy
• PLA 2 R-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.
• 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; PLA 2 R autoantibodies and PLA 2 R 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 A 2 receptor (PLA 2 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 A 2 receptor as target antigen in idiopathic membranous nephropathy ; N Engl J Med. ; 361 : 11-21).
• PLA 2 R antigen in glomeruli should be examined in all biopsies of patients with a suspected idiopathic (or PLA 2 R-positive) MN, especially if they have negative anti-PL A 2 R activity in the serum Search for PLA 2 R antigen in archival kidney biopsies is also important for the monitoring of patients who will benefit from a kidney transplant
• detection of PLA 2 R 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.
• polyclonal rabbit anti-PLA 2 R antibody commercialized by SIGMA- ALDRICH ® (ref. HPA012657) is raised against another epitope which ranges from amino- acid 395 to 530 of the receptor PLA 2 R 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-PLA 2 R 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 A 2 R of sequence SEQ ID N° 1.
• membranous nephropathy such as for example idiopathic membranous nephropathy and lupus membranous nephropathy.
• Phospholipase A 2 receptor (PLA 2 R) 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 PLA 2 R.
• the invention relates to the assessment of the presence of PLA 2 R for diagnostic purposes.
• the invention relates to novel PLA 2 R epitopes and the identification of highly specific PLA 2 R binding molecules.
• the invention relates to a human Phospholipase A2 receptor (PLA 2 R) binding molecule directed against a PLA 2 R-derived peptide of formula (I) comprising the following amino sequence:
• - 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 X 2 are identical or different and represent any amino acid.
• the invention relates to a PLA 2 R-derived peptide in the form of an immunogenic compound comprising the PLA 2 R-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 PLA 2 R-derived peptide of formula:
• - 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 PLA 2 R of SEQ ID N°8.
• the invention also relates to a method for generating antibodies binding to immune complexes between auto-antibodies directed against human phospholipase A 2 receptor (PLA 2 R) and human PLA 2 R comprising a step of providing an immunogenic compound containing a PLA 2 R-derived peptide of SEQ ID N°8.
• the invention relates to a complex between (i) a human PLA 2 R binding molecule and (ii) human PLA 2 .
• the invention relates to an epitope of a human Phospholipase A 2 receptor bound by the said human PLA 2 R binding molecule.
• the invention also relates to a composition comprising a PLA 2 R binding molecule according to the invention.
• the invention relates to a method for detecting the presence of human PLA 2 R in a sample comprising the steps of:
• step b) bringing into contact the sample provided at step a) with a PLA 2 R binding molecule according to the invention, and c) detecting the binding of the said PLA 2 R binding molecule onto the said sample, whereby the presence of human PLA 2 R is detected.
• the invention relates to a kit for detecting human PLA 2 R, the said kit comprising a PLA 2 R binding molecule according to the invention and one or more reagents necessary for detecting the extent of binding between the said PLA 2 R binding molecule and a sample to be tested.
• the sample may be a biological sample taken from an individual suspected of having glomerular deposits.
• the invention also relates to a method for diagnosing the occurrence of a membranous nephropathy (MN) disease in an individual comprising the steps of:
• the invention further relates to a method for discriminating between idiopathic (or PLA 2 R-positive) MN and lupus MN comprising the steps of:
• Figure 1 Sequences of the ten overlapping peptides that were used in ELISA to identify the specific minimal epitope of the anti-PLA 2 R antibody.
• the amino-acid sequence "PepC" of sequence SEQ ID N°8 ranges from amino-acid 875 to 900 of PLA 2 R, 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 PLA 2 R 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 PLA 2 R antibody.
• FIG. 5 Location of various antigenic epitopes contained in PLA 2 R. The position of the corresponding PLA 2 R epitopes is indicated in the figure.
• the PLA 2 R 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).
• CCD N-terminal cysteine-rich region
• FN2D fibronectin-like type II domain
• CLD C-type lectin domain
• TMD transmembrane domain
• CD short intracellular C-terminal domain
• kidney biopsies from patients with membranous nephropathy may present in situ glomerular deposits formed both by the accumulation of pathogenic antigens from PLA 2 R and of autoimmune anti-PLA 2 R antibodies.
• glomerular deposits contain autoimmune antibodies, they may also be referred herein as immune glomerular deposits.
• 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 PLA 2 R- 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 PLA 2 R 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 PLA 2 R transmembrane receptor is likely to possess linear, antigenic and/or accessibility motifs.
• PLA 2 R monoclonal or polyclonal antibodies directed against PLA 2 R 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 PLA 2 R-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-PLA 2 R autoantibodies to the PLA 2 R target.
• contacting the sample with a random PLA 2 R binding molecule would result in the absence of binding of the said PLA 2 R 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.
• kidney biopsies such as paraffin-embedded kidney biopsies
• an antibody suitable for kidney biopsies should be ideally directed against a PLA 2 R-derived epitope that is distinct from "pathogenic" epitopes recognized by patients' sera.
• PLA 2 R receptor Due to the lack of structural information on the PLA 2 R receptor in situ in the art, the inventors have determined herein novel relevant epitopes from PLA 2 R 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.
• peptides derived from the PLA 2 R sequence and containing putative linear epitopes, antigenic and/or accessibility motifs were synthesized and used as an antigen for the immunization of rabbits.
• 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).
• an antigenic fragment also referred herein as peptide C (or “pep C”), which consists in the PLA 2 R-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 PLA 2 R of sequence SEQ ID N 0 1.
• PLA 2 R-derived peptide leads to the production of antibodies with improved specificity towards a specific epitope of PLA 2 R, which will be referred herein as the minimal immunogenic epitope of PLA 2 R, which PLA 2 R-derived epitope is available in tissue samples containing PLA 2 R deposits, which includes tissue samples wherein the PLA 2 R deposits are complexed with anti-PLA 2 R autoantibodies.
• tissue samples containing PLA 2 R deposits which includes tissue samples wherein the PLA 2 R deposits are complexed with anti-PLA 2 R autoantibodies.
• the said PLA 2 R-derived epitope is available in paraffin-embedded tissue samples containing PLA 2 R deposits.
• a minimal immunogenic epitope of PLA 2 R which comprises, or consists in, a short peptide motif of sequence SEQ ID N°2, or FX1WX2, wherein Xi and X 2 are identical or different and each of Xi and X 2 consists of an amino acid residue.
• amino acid residue is disclosed previously in the instant specification.
• Xi and X 2 may represent a lysine (K), an arginine (R) or an alanine (A), and preferably mean an arginine.
• 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.
• the minimal immunogenic epitope consists in a short motif FRWR of sequence SEQ ID N°4.
• the immunogenic epitope comprises, or consists in, the short peptide motif of sequence SEQ ID N°3, or X 3 FXiWX 2 , wherein Xi and X 2 are as defined above, and wherein X 3 may represent any amino acid residue.
• Xi and X 2 may represent a lysine (K), an arginine (R) or an alanine (A), and preferably an arginine.
• X 3 may represent an aspartate (D), a glutamate (E) or an alanine (A), and preferably a glutamate.
• the immunogenic epitope comprises or consists of EFRWR (SEQ ID N°5).
• the immunogenic epitope may comprise or consist of EERA DEFRWRD (SEQ ID N°6).
• immunogenic epitope may comprise or consist of
• the immunogenic epitope may comprise or consist of WIGLQEERA DEFRWRDGTPVIYQNWD (SEQ ID N°8).
• the invention pertains to a PLA 2 R-derived peptide of formula:
• - 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 PLA 2 R as defined above.
• This invention also pertains to an immunogenic compound comprising a PLA 2 R-derived peptide of formula:
• - 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 PLA 2 R as defined above.
• This invention also relates to an immunogenic compound comprising the PLA 2 R-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 PLA 2 R-derived peptide of formula:
• - 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 PLA 2 R of SEQ ID N°8.
• this invention pertains to an immunogenic compound covalently linked to a carrier molecule.
• the said PLA 2 R- derived peptide is covalently linked to a carrier molecule, such as for example Keyhole Limpet Hemocyanin (also termed KLH).
• a carrier molecule such as for example Keyhole Limpet Hemocyanin (also termed KLH).
• 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 A 2 receptor (PLA 2 R) binding molecule directed against a PLA 2 R-derived peptide, including against a PLA 2 R- derived peptide comprised in an immunogenic compound as disclosed herein.
• PHA 2 R Phospholipase A 2 receptor
• the said human Phospholipase A 2 receptor (PLA 2 R) binding molecule is an antibody, or an antibody-derived fragment.
• the said PLA 2 R 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.
• the said human Phospholipase A 2 receptor (PLA 2 R) binding molecule is an antibody.
• the human Phospholipase A 2 receptor (PLA 2 R) binding molecule is a monoclonal antibody.
• PLA 2 R human Phospholipase A 2 receptor
• - 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 PLA 2 R 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.
• a peptide of 1 to 20 amino acids in length encompasses a peptide of 1 to 15 amino acids in length.
• a human Phospholipase A 2 receptor (PLA 2 R) binding molecule of the invention is directed against a PLA 2 R-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.
• a human Phospholipase A 2 receptor (PLA 2 R) binding molecule of the invention such as an antibody, is directed against a PLA 2 R-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 PLA 2 R-derived peptide may be equal or different.
• the N-terminal end is of 12 amino acids in length and the C-terminal end is of 11 amino acids in length.
• the PLA 2 R-derived peptide is 25- amino acids long.
• This invention also concerns a human Phospholipase A 2 receptor (PLA 2 R) binding molecule directed against a PLA 2 R-derived peptide of formula (I) comprising the following amino acid sequence:
• - 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 X 2 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 PLA 2 R-derived peptide of formula (I) comprising the following amino acid sequence: H 2 -[Nt] y - X3FX1WX2 [Ct] z - COOH (I)
• - 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 X 2 are identical or different and represent either a lysine or an arginine, and preferably an arginine,
• the invention relates to a PLA 2 R binding molecule directed against a
• PLA 2 R derived peptide wherein the said PLA 2 R-derived peptide comprises the amino acid sequence X 3 FXiWX 2 .
• the invention relates to a PLA 2 R binding molecule directed against a PLA 2 R derived peptide, wherein the said PLA 2 R-derived peptide comprises, or consists of, the amino acid sequence FRWR (SEQ ID N° 4).
• the invention pertains to a PLA 2 R binding molecule directed against a PLA 2 R derived peptide, wherein the said PLA 2 R-derived peptide comprises, or consists of, the amino acid sequence EFRWR (SEQ ID N° 5).
• This invention also concerns a human Phospholipase A2 receptor (PLA 2 R) binding molecule directed against a PLA 2 R-derived peptide of formula (II) comprising the following amino acid sequence:
• - 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.
• the invention also relates to a human Phospholipase A2 receptor (PLA 2 R) binding molecule directed against a PLA 2 R-derived peptide of formula (III) comprising the following amino acid sequence:
• - 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 PLA 2 R binding molecule directed against a
• PLA 2 R-derived peptide wherein the said PLA 2 R-derived peptide comprises the amino acid sequence WIGLQEERA DEFRWRDGTPVIYQNWD (SEQ ID N° 8).
• the invention relates to a PLA 2 R binding molecule directed against a PLA 2 R-derived peptide, wherein the said PLA 2 R- derived peptide consists in the amino acid sequence WIGLQEERANDEFRWRDGTPVIYQNWD (SEQ ID N° 8).
• PLA 2 R binding molecule is directed against a PLA 2 R-derived peptide as defined in the present specification, for example by performing conventional immunoassays (e.g. ELISA or RIA assays) using the PLA 2 R-derived peptide as the bait molecule.
• conventional immunoassays e.g. ELISA or RIA assays
• 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.
• a PLA 2 R binding molecule of the invention is able to compete for binding to PLA 2 R with a peptide of sequence SEQ ID N°5 (EFRWR) but not with a peptide of sequence SEQ ID N°l 1 (SWIGLQ).
• the PLA 2 R binding molecule is able to compete by ELISA screening method or immunohistochemistry method.
• a PLA 2 R binding molecule of the invention is able to compete for binding to PLA 2 R 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.
• 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 PLA 2 R binding molecule as defined herein and (ii) human PLA 2 .
• This invention notably relates to such complexes between (i) a human PLA 2 R binding molecule as defined herein and (ii) human PLA 2 .
• the invention also relates to an epitope of a human PLA 2 R bound by a PLA 2 R binding molecule as defined above.
• the inventors have found that generating antibodies binding to the human phospholipase A 2 receptor (PLA 2 R), and more specifically directed against a minimal immunogenic epitope as defined above, is particularly advantageous for targeting glomerular deposits.
• PHA 2 R human phospholipase A 2 receptor
• glomerular deposits refer to the accumulation of deposits which are characteristic of a membranous nephropathy (MN) disease.
• MN membranous nephropathy
• 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 PLA 2 R-positive or PLA 2 R-negative glomerular deposits.
• 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.
• an "idiopathic membranous nephropathy” is also referred herein as a PLA 2 R-positive membranous nephropathy, or as primary membranous nephropathy.
• membranous nephropathies may now be further classified as either PLA 2 R-positive or PLA 2 R-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 PLA 2 R epitope in glomerular deposits in particular.
• the PLA 2 R binding molecules of the invention are able to target glomerular deposits comprising PLA 2 R, including glomerular deposits comprising PLA 2 R which are complexed with anti-PLA 2 R auto-antibodies.
• Immuno glomerular deposits which are complexed with anti-PLA 2 R auto-antibodies may be also termed herein "immune glomerular deposits".
• immune glomerular deposits may be subepithelial immune glomerular deposits along glomerular capillary loops.
• 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 PLA 2 R.
• the invention relates to a PLA 2 R binding molecule as defined in the present specification, which PLA 2 R 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 A 2 receptor (PLA 2 R) and human PLA 2 R comprising a step of providing an immunogenic compound containing a PLA 2 R-derived peptide of SEQ ID N°2, in particular of SEQ ID N°4, and preferably of sequence SEQ ID N°8.
• this invention relates to a method for generating antibodies as defined above, wherein the immunogenic compound is covalently linked to a carrier molecule.
• the said carrier molecule is Keyhole Limpet Hemocyanin (KLH).
• This invention notably pertains to a method for generating antibodies directed against a PLA 2 R-derived peptide as described herein comprising the steps of:
• step 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).
• a PLA 2 R binding molecule as defined above may be labeled covalently or non-covalently to a detectable molecule.
• detectable molecules are well known in the Art.
• a PLA 2 R binding molecule 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.
• a PLA 2 R 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.
• 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.
• a PLA 2 R 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.), Lakowic
• a PLA 2 R binding molecule is not labeled with a detectable molecule. According to that embodiment, binding of the PLA 2 R binding molecule to its target may then be detected using a secondary labelled molecule. Such a case may occur for instance when the PLA 2 R 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.
• a PLA 2 R binding molecule of the invention is particularly aimed at detecting the presence of human phospholipase A 2 receptor (PLA 2 R) in a sample.
• a PLA 2 R binding molecule of the invention is labeled with a detectable molecule, or can interact with a labeled molecule.
• a sample may be a biological sample, such as a blood-derived sample or a biopsy.
• 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.
• the binding of the PLA 2 R 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.
• immunofluorescence staining in particular Antigen retrieval immunofluorescence staining, and more particularly Heat Induced Epitope Retrieval (HIER) method, as described in examples 3 or 4.
• HIER Heat Induced Epitope Retrieval
• the invention relates to a method for detecting the presence of human phospholipase A2 receptor (PLA 2 R) in a sample comprising the steps of:
• the method for detecting the presence of human phospholipase A2 receptor (PLA 2 R) in a sample when 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.
• 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.
• the method for diagnosing the occurrence of a membranous nephropathy (MN) disease relies on the detection of glomerular deposits within the sample by a PLA 2 R binding molecule of the invention.
• Detection of the PLA 2 R-derived immunogenic epitope of the invention within glomerular deposits is indicative of a membranous nephropathy disease.
• glomerular deposits may be an immune glomerular deposit, which means that glomerular deposits may further comprise autoantibodies, such as anti- PLA 2 R autoantibodies.
• the immunogenic epitope of the invention is not recognized by anti-PLA 2 R autoantibodies, which means that it is also accessible in immune glomerular deposits.
• the PLA 2 R immunogenic epitope may be detected both in glomerular deposits and immune glomerular deposits.
• the invention also relates to a method for detecting the presence of immune complexes between auto-antibodies directed against human phospholipase A2 receptor (PLA 2 R) and human PLA 2 R in a sample comprising the steps of:
• Detection of glomerular deposits comprising the PLA 2 R immunogenic epitope of the invention is indicative of the occurrence of a membranous nephropathy, preferably a primary or PLA 2 R-positive form of membranous nephropathy, which is also referred herein as idiopathic membranous nephropathy.
• 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 A 2 receptor (PLA 2 R) in a sample as defined above from the said individual and
• the sample may be any sample from the said individual that is suspected of containing glomerular deposits.
• a sample suspected of containing glomerular deposits may be a renal biopsy, or part of a renal biopsy.
• 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-PLA 2 R autoantibodies in a sample.
• 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-PLA 2 R autoantibodies in a sample from the said individual.
• MN membranous nephropathy
• the sample may be the same sample as the sample where the method for detecting the presence of human phospholipase A2 receptor (PLA 2 R) is performed, or a different sample.
• PHA 2 R human phospholipase A2 receptor
• the step of detecting the presence of anti-PLA 2 R 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; PLA 2 R autoantibodies and PLA 2 R glomerular deposits in membranous nephropathy; N. Engl. J. Med.; 364(7): 689-90) or in Hofstra & Wetzels (Hofstra & Wetzels ; 2012 ; Anti-PLA 2 R antibodies in membranous nephropathy: ready for routine clinical practice? ; Neth J Med.; 70(3): 109-13).
• the step of detecting the presence of anti-PLA 2 R autoantibodies may be performed on a blood-derived sample such as the serum of an individual.
• the presence of anti-PLA 2 R 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 PLA 2 R1 isoform.
• MN membranous nephropathy
• steps (i) and (ii) may be performed in any order
• steps (i) and (ii) may be performed on the said sample or on different samples.
• the method for detecting the presence of human phospholipase A 2 receptor (PLA 2 R) in a sample or a tissue sample is performed on a kidney biopsy, and preferably a paraffin-embedded kidney biopsy.
• PLA 2 R immunogenic epitope of the invention When the PLA 2 R immunogenic epitope of the invention is detected within glomerular deposits (PLA 2 R-positive deposits), it is indicative of a membranous nephropathy, and more particularly a primary ("idiopathic") membranous nephropathy.
• subepithelial PLA 2 R-positive glomerular deposits may appear during iMN in a finely granular pattern of sub-epithelial deposits along glomerular capillary loops.
• lupus membranous nephropathy is mostly characterized by PLA 2 R-negative mesangial or subendothelial deposits.
• PLA 2 R immunogenic epitope of the invention when not detected within glomerular deposits (PLA 2 R-negative deposits), it may be indicative of a secondary membranous nephropathy.
• the method for detecting the presence of anti-PLA 2 R autoantibodies in a sample is performed on serum.
• anti-PLA 2 R autoantibodies When anti-PLA 2 R autoantibodies are detected in a sample from an individual, it is indicative of membranous nephropathy, and more particularly a primary ("idiopathic") membranous nephropathy.
• the absence of circulating anti-PLA 2 R 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.
• the invention further relates to a method for discriminating between idiopathic MN and lupus MN comprising the steps of:
• the invention relates to a method for discriminating between idiopathic (or PLA 2 R-positive) MN and lupus MN, which may optionally comprise a step of detecting the presence of autoantibodies indicative of a lupus and/or anti-PLA 2 R 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.
• the invention relates to a method for discriminating between idiopathic MN and lupus MN comprising a step of detecting the presence of anti-PLA 2 R autoantibodies in a sample from the said individual, wherein the step of detecting the presence of anti-PLA 2 R autoantibodies may be performed on the tissue sample of step a) or on a different sample.
• the invention also relates to a method for discriminating between idiopathic MN and lupus MN comprising the steps of:
• step (d) may be performed in any order
• steps (c) and (d) may be performed on the said sample or on different samples.
• the invention relates to a composition comprising a PLA 2 R binding molecule as defined by the invention.
• the invention relates to a kit for detecting human PLA 2 R, the said kit comprising a PLA 2 R binding molecule as defined by the invention and one or more reagents necessary for detecting the extent of binding between the said PLA 2 R binding molecule and a sample to be tested.
• 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.
• a reagent necessary for detecting the extent of binding between the said PLA 2 R 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 PLA 2 R bound by a PLA 2 R binding molecule, or a complex between (i) a human PLA 2 R binding molecule and (ii) human PLA 2 R.
• the invention relates to a kit for diagnosing a membranous nephropathy disease, which comprises at least a PLA 2 R 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 PLA 2 R and autoantibodies indicative of a lupus.
• kits for the detection of autoantibodies are well known in the art.
• commercial autoantibody assay kits may thus include reagents for immunofluorescence, immunodiffusion, immunoblotting, or ELISA methods.
• peptide A 437-467 aa
• peptide B pepB
• 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.
• 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 PLA 2 R in subepithelial deposits in glomeruli.
• 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.
• 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)
• HIER Heat Induced Epitope Retrieval
• HIER at pH 6 was followed by 5-min incubation with HistoReveal (Abeam ref. ab 103720).
• concentration of a monoclonal antibody of the invention is set at 0.3 ⁇ g/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 ⁇ ) were incubated with the monoclonal antibody only (A). Adjacent sections were also pre-incubated with 20 ⁇ g of blocking peptide (B) or with 20 ⁇ g 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
• 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-PLA 2 R antibody isolated from a mouse hybridoma.
• a high signal necessarily relates to the formation of a complex between the primary anti-PLA 2 R of the invention and the peptide to be tested.
• peptides 4-7 show significant absorbance over the background, whereas peptide 3 shows a weak signal indicative of residual binding.
• peptides 4-7 share the same minimal epitope which binds selectively to a monoclonal anti-PLA 2 R of the invention. All four peptides comprise the same FRWR minimal epitope.
• the ELISA screening establishes that the monoclonal anti-PLA 2 R antibody of the invention recognizes specifically the minimal epitope FRWR.
• 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.
• 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.
• the PLA 2 receptor in glomerular immune deposits comprises epitopes which are not accessible within kidney biopsy specimens.
• pepC comprises an epitope which can be targeted for detection of PLA 2 R-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
• this experiment shows that in situ targeting is abolished after competition with the EFRWR peptide.
• the (E)FRWR motif is shown to be a biomarker of glomerular deposits within kidney biopsy specimen.
• 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
• this experiment shows that anti-PLA 2 R antibodies against the immunogenic epitope (E)FRWR provide superior specificity for glomerular deposits as compared to other commercial polyclonal antibodies.
• anti-PLA 2 R binding antibodies of the invention are the first very well characterized monoclonal antibody that can be made commercially available for histopathologic purposes.

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