WO2022043415A1 - Methods for detecting the presence of pemphigus-specific autoantibodies in a sample - Google Patents

Abstract

Pemphigus is a group of rare autoimmune diseases that causes blistering of the skin and mucous membranes, and includes pemphigus vulgaris (PV) and pemphigus foliaceus (PF). Patients with pemphigus have various combinations of autoantibodies to keratinocyte muscarinic acetylcholine receptor subtype M3 (CHRM3), the secretory pathway Ca 2+ /Mn 2+ -ATPase 10 isoform 1 (SPCA1), and desmocollin 3 (DSC3). However, there is still a need to characterize these autoantibodies and optimize their detection for diagnosis and disease monitoring. The inventors now developed an ALBIA for the 3 proteins and successfully detected and quantified the presence of auto-antibodies directed against each of these antigens in pemphigus patients. Furthermore, they showed that detection and quantification of anti-SPCA1 and/or anti-CHRM3 15 were also associated to a risk of relapse in the first year following the treatment using rituximab as a first-line agent. The present invention thus relates to methods for detecting the presence of said pemphigus-specific autoantibodies.

Description

METHODS FOR DETECTING THE PRESENCE OF PEMPHIGUS-SPECIFIC AUTOANTIBODIES IN A SAMPLE

FIELD OF THE INVENTION:

The present invention is in the field of medicine, in particular dermatology and immunology.

BACKGROUND OF THE INVENTION:

Pemphigus is a group of rare autoimmune diseases that causes blistering of the skin and mucous membranes, including mouth, nose, throat, eyes, and genitals. The type of disease depends on what layer in the skin the blisters form and where they are located on the body, which include pemphigus vulgaris (PV) and pemphigus foliaceus (PF). For instance, PV is the most common type of pemphigus in the United States and Europe. Flaccid blisters appear on erythematous or normal-appearing skin and mucous membranes. The sores almost always start in the mouth. The blisters of PV form within the deep layer of the epidermis, and are often painful. Blistered skin becomes so fragile that it may peel off by rubbing a finger on it. The blisters normally heal without scarring, but pigmented spots (spots where skin appears darker than the surrounding skin) may remain for a number of months. Without treatment, PV leads to significant skin erosions, resulting in high morbidity and mortality. It is known that the type of autoantibodies typically defines the type of disease. In particular, the discovery that autoantibodies targeting desmoglein (Dsg) 1 and Dsg3 cause blister formation has been potentially the most critical event in understanding pemphigus pathogenesis to date. Patients with pemphigus have various combinations of autoantibodies to keratinocyte muscarinic acetylcholine receptor subtype M3 (CHRM3), the secretory pathway Ca²⁺/Mn²⁺-ATPase isoform 1 (SPCA1), and desmocollin 3 (DSC3). However, there is still a need to characterize these autoantibodies and optimize their detection for diagnosis and disease monitoring.

SUMMARY OF THE INVENTION:

The present invention is defined by the claims. In particular, the present invention relates to methods for detecting the presence of pemphigus-specific autoantibodies in a sample.

DETAILED DESCRIPTION OF THE INVENTION:

The first object of the present invention relates to a method for detecting the presence of pemphigus-specific autoantibodies in a subject comprising the steps of a) placing a sample obtained from the subject, in a single assay receptacle, in the presence of particles belonging to either of the 3 different groups, a first group of particles being conjugated to DSC3 polypeptide, a second group of particles being conjugated to a SPCA1 polypeptide and a third group of particles being conjugated to a CHRM3 polypeptide

- b) incubating the mixture under conditions which allow the formation of immunocomplexes on particles, c) eliminating the immunoglobulins which have not bound to the particles, and d) detecting the immunocomplexes of step b) on the plurality of particles, whereby the presence or absence of anti-DSC3 autoantibodies, anti-SPCAl autoantibodies or anti-CHRM3 autoantibodies is revealed.

As used herein, the term “sample" refer to a biological sample obtained for the purpose of in vitro evaluation. Typical biological samples to be used in the method according to the invention are blood samples (e.g. whole blood sample or serum sample).

As used herein, the term “blood sample” means any blood sample derived from the subject. Collections of blood samples can be performed by methods well known to those skilled in the art. In some embodiments, the blood sample is a serum sample or a plasma sample.

As used herein, the term "antigen" refers to a substance that can cause the immune system to produce an antibody response against it, and possibly can trigger a biological reaction when an antibody binds to it under the appropriate in vivo conditions. The term antigen as used herein shall refer to a whole target molecule or a fragment of such molecule recognized by an antigen binding site. Specifically, substructures of an antigen, e.g. a polypeptide, generally referred to as "epitopes", which are immunologically relevant, may be recognized by an antibody. Thus, in some embodiments, the antigen of the present invention comprises at least one epitope. Methods for identifying and characterizing epitopes are well known in the art. Typically, said methods include but are not limited to epitope prediction algorithms and MHC associated peptidome identified by mass spectrometry (MS).

As used herein, the term “antibody”, "immunoglobulin" or “Ig” has its general meaning in the art and relates to proteins of the immunoglobulin superfamily. The immunoglobulins are characterized by a structural domain, i.e., the immunoglobulin domain, having a characteristic immunoglobulin (Ig) fold. The term encompasses secretory immunoglobulins. Immunoglobulins generally comprise several chains, typically two identical heavy chains and two identical light chains which are linked via disulfide bonds. These chains are primarily composed of immunoglobulin domains, including the VL domain (light chain variable domain), the CL domain (light chain constant domain), the VH domain (heavy chain variable domain) and the CH domains (heavy chain constant domains) CHI, optionally a hinge region, CH2, CH3, and optionally CH4. There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: mu (p) for IgM, delta (6) for IgD, gamma (y) for IgG, alpha (a) for IgA and epsilon (a) for IgE. In the context of the invention, the immunoglobulin may be an IgM, IgD, IgG, IgA or IgE. Preferably, the immunoglobulin is an IgG. As well-known from the skilled person, the IgG isotype encompasses four subclasses: the subclasses IgGl, lgG2, lgG3 and lgG4. The IgA isotype encompasses 2 subclasses: IgAl and IgA2 immunoglobulins.

As used herein, the term "autoantibody" refers to an antibody produced by a subject, where the antibody is directed against one or more 'self antigens (e.g., antigens that are native to the individual, e.g., an antigen on a cell or tissue, or an endogenous peptide or protein).

As used herein, the term "particle" has its general meaning in the art and refers to a particle from 1 nm to 1000 nm, preferably from 100 to 500 nm and even more preferably from 350 to 450 nm in size. In some embodiments, the size of the particle is about 400 nm. A particle may typically be spherical, though the shape is not limited to that of a sphere and may include other shapes like spheroid, irregular particles, cubes, irregular cubes, and disks. According to the present invention the term “particle” is interchangeable with the term “bead”.

In some embodiments, the particle of the present invention is made of an organic polymer. Organic polymers encompass, but are not limited to, polystyrene, poly(vinyl acetate), poly (methyl styrene), poly(acrylamide), poly(acrylonitrile), poly(vinyl chloride), poly(butyl acrylate), poly(acrylic acid), copolymers of styrene and Cl-C4alkyl (meth)acrylate, copolymers of styrene and acrylamide, copolymers of styrene and acrylonitrile, copolymers of styrene and vinyl acetate, copolymers of acrylamide and C1-C4 alkyl (meth)acrylates, copolymers from acrylonitrile and C1-C4 alkyl (meth)acrylate, copolymers of acrylonitrile and acrylamide, terpolymers from styrene, acrylonitrile and acrylamide, poly(methyl methacrylate), poly(ethyl methacrylate), copolymers styrene/butadiene, styrene/acrylic acid, styrene/vinylpyrrolidone and butadiene/acrylonitrile, or methoxy poly(ethylene glycol)-poly(lactide) copolymer (MPEG-PLA). Polymer particles can be crosslinked or not. For instance, organic particles include, but are not limited to, nylon (for example marketed by ATOCHEM), polyethylene powders (for example marketed by PLAST LABOR), poly-2-alanine powders, polyfluorinated powders such as polytetrafluoroethylene (for example marketed by DUPONT DE NEMOURS), acrylic copolymer powders (for example marketed by DOW CHEMICA), polystyrene powders (for example marketed by PRESPERESE), polyester powders, expanded microspheres in thermoplastic material (for example marketed by EXPANCEL), microballs of silicon resins (for example marketed by TOSHIBA), synthetic hydrophilic polymer powders such as polyacrylates (for example marketed by MATSUMOTO), acrylic polyamides (for example marketed by ORIS), insoluble polyurethanes (for example marketed by TOSHNU), porous microspheres of cellulose, micro- or particles of PTFE (polytetrafluoroethylene).

In some embodiments, the particles are selected to have a variety of properties useful for particular experimental formats. For example, particles can be selected that remain suspended in a solution of desired viscosity or to readily precipitate in a solution of desired viscosity.

In some embodiments, the particles are magnetic and coded.

Particles also can be coded for identification purposes, such as by bar codes, luminescence, fluorescence and the like. A variety of coded particles are well known to those skilled in the art, and include for example, Luminex® and Cyvera® coded particles. With regard to coded particles, each particle can include a unique code, preferably, the coded particles contain a code other than that present in the detectable tag used to detect the presence or amount of modified substrate (e.g., support-bound product portion, free product portion, or modified support-bound substrate). The code can be embedded (for example, within the interior of the particle) or otherwise attached to the particle in a manner that is stable through hybridization and analysis. The code can be provided by any detectable means, such as by holographic encoding, by a fluorescence property, color, shape, size, light emission, quantum dot emission and the like to identify particle and thus the capture probes immobilized thereto. For example, the particles may be encoded using optical, chemical, physical, or electronic tags. Examples of such coding technologies are optical bar codes fluorescent dyes, or other means. One exemplary platform utilizes mixtures of fluorescent dyes impregnated into polymer particles as the means to identify each member of a particle set to which a specific capture probe has been immobilized. Another exemplary platform uses holographic barcodes to identify cylindrical glass particles. For example, Chandler et al. (U.S. Pat. No. 5,981,180) describes a particle-based system in which different particle types are encoded by mixtures of various proportions of two or more fluorescent dyes impregnated into polymer particles. Soini (U.S. Pat. No. 5,028,545) describes a particle-based multiplexed assay system that employs time-resolved fluorescence for particle identification. Fulwyler (U.S. Pat. No. 4,499,052) describes an exemplary method for using particle distinguished by color and/or size. U.S. Patent Publication Nos. 2004-0179267, 2004- 0132205, 2004-0130786, 2004-0130761, 2004-0126875, 2004-0125424, and 2004-0075907 describe exemplary particles encoded by holographic barcodes. U.S. Pat. No. 6,916,661 describes polymeric particles (e.g., microparticles) that are associated with particles that have dyes that provide a code for the particles.

As used herein, the term “magnetic particle” encompasses any particle having at least some magnetic characteristic, e.g., ferromagnetic, paramagnetic, and superparamagnetic property. A magnetic particle can include magnetic materials such as iron, nickel, and cobalt, as well as metal oxides such as FesCU, BaFenOig, MmCh, CnCh, CoO, NiO, and CoMnP. In some embodiments, the magnetic particle contains, or fully consists of, a polymeric magnetic material. Polymeric magnetic material includes for example, material in which the magnetic material is mixed with polymeric material and magnetic material that is coated with polymeric material. Preferably the magnetic material is only one component of the microparticle whose remainder consists of a polymeric material to which the magnetically responsive material is affixed (see coded particles below). Exemplary methods for the preparation of or composition of magnetic particles are described in, e.g., U.S. Pat. Nos. 6,773,812 and 6,280,618.

As used herein, the terms “polypeptide”, “peptide” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. Polypeptides when discussed in the context of the present invention refer to the respective intact polypeptide, or any fragment or genetically engineered derivative thereof, which retains the desired biochemical function and/or conformation of the intact protein.

As used herein, the term “DSC3” has its general meaning in the art and refers to the Desmocollin-3. DSC3 is a component of intercellular desmosome junctions and is involved in the interaction of plaque proteins and intermediate filaments mediating cell-cell adhesion. The term is also known as Cadherin family member 3 or HT-CP. In some embodiments, the DSC3 polypeptide comprises or consists in the cytoplasmic domain of the DSC3

In some embodiments, the DSC3 polypeptide comprises or consists in the sequence as set forth in SEQ ID NO: !.

As used herein, the term “SPCA1” has its general meaning in the art and refers to secretory pathway Ca²⁺/Mn²⁺-ATPase isoform 1. SPCA1 is a magnesium-dependent enzyme that catalyses the hydrolysis of ATP coupled with the transport of the calcium.

In some embodiments, the SPCA1 polypeptide comprises or consists in the sequence as set forth in SEQ ID NO:2.

The term “CHRM3” has its general meaning in the art and refers to the muscarinic acetylcholine receptor M3. The muscarinic acetylcholine receptor mediates various cellular responses, including inhibition of adenylate cyclase, breakdown of phosphoinositides and modulation of potassium channels through the action of G proteins.

In some embodiments, the CHRM3 polypeptide comprises or consists in the sequence as set forth in SEQ ID NO:3.

In some embodiments, the polypeptide is attached to the surface of the particle by any conventional method well known in the art, such as described in Hermanson, Greg T. Bioconjugate techniques. Academic press, 2013. In some embodiments, l-ethyl-3-[3- dimethylaminopropyl] carbodiimide hydrochloride (EDC)- N-hydroxysulfosuccinimide (Sulfo NHS) reactions are used for conjugating the polypeptides to the particles. In some embodiments, the particle is conjugated to an avidin moiety that can create an avidin-biotin complex with the biotinylated polypeptides and the particles. Additional, appropriate crosslinking agents for use in the invention include a variety of agents that are capable of reacting with a functional group present on a surface of the particle. Reagents capable of such reactivity include homo- and hetero-bifunctional reagents, many of which are known in the art. Heterobifunctional reagents are preferred. A typical bifunctional cross-linking agent is N- succinimidyl(4-iodoacetyl) aminobenzoate (SLAB). However, other crosslinking agents, including, without limitation, dimaleimide, dithio-bis-nitrobenzoic acid (DTNB), N- succinimidyl-S-acetyl-thioacetate (SATA), N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl 4-(N-maleimidomethyl)cyclohexane-l -carboxylate (SMCC) and 6- hydrazinonicotimide (HYNIC) may also be used. For further examples of cross-linking reagents, see, e.g., S. S. Wong, "Chemistry of Protein Conjugation and Cross-Linking," CRC Press (1991), and G. T. Hermanson, "Bioconjugate Techniques," Academic Press (1995).

In some embodiments, the receptacle may be any solid container, for example a test tube, a microplate well or a reaction cuvette made of polypropylene.

In some embodiments, the elimination of the unbound reagents may be carried out by any technique known to those skilled in the art, such as e.g. washing by means of repeated centrifugation steps.

As used herein the term “immunocomplex” refers to the complex formed between the pemphigus-specific autoantibodies of the subject and their specific antigen, i.e. the polypeptide that is conjugated to the particle.

The presence and amount of the immunocomplexes may be detected by methods known in the art, including label-based and label-free detection. In some embodiments, the method of the present invention includes use of a secondary antibody that is coupled to an indicator reagent comprising a signal generating compound.

In some embodiments, the secondary antibody has specificity for a particular immunoglobulin. In some embodiments, the secondary antibody is an anti-human IgG antibody, including anti- IgGl, IgG2, IgG3 and IgG4 antibodies.

In some embodiments, the secondary antibody is an anti-human IgAl or IgA2 antibody.

In some embodiments, the antibody having specificity for a particular type immunoglobulin is a rabbit or goat antibody. In some embodiments, the antibody of the present invention is a monoclonal antibody or a polyclonal antibody.

Indicator reagents include chromogenic agents, catalysts such as enzyme conjugates, fluorescent compounds such as fluorescein and rhodamine, chemiluminescent compounds such as dioxetanes, acridiniums, phenanthridiniums, ruthenium, and luminol, radioactive elements, direct visual labels, as well as cofactors, inhibitors and magnetic particles. Examples of enzyme conjugates include alkaline phosphatase, horseradish peroxidase and beta-galactosidase. In some embodiments, the secondary antibody is conjugated to phycoerythrin.

Methods for detecting the particle identity codes, e.g., a fluorescent code, are known in the art and are described below. Examples of systems that read (detect or analyze) multiplex assay signals from Luminex beads include, e.g., the Luminex xMAP 100 and xMAP 200 instruments or the Bio-Plex 100 and Bio-Plex 200 from BioRad instruments. Another method for detecting and/or separating particle sets based on ID codes is flow cytometry. Methods of and instrumentation for flow cytometry are known in the art, and those that are known can be used in the practice of the present invention. Flow cytometry, in general, involves the passage of a suspension of the particles as a stream past a light beam and electro-optical sensors, in such a manner that only one particle at a time passes through the region. As each particle passes this region, the light beam is perturbed by the presence of the particle, and the resulting scattered and fluorescent light are detected. The optical signals are used by the instrumentation to identify the subgroup to which each particle belongs, along with the presence and amount of label, so that individual assay results are achieved. Descriptions of instrumentation and methods for flow cytometry are known in the art and include, e.g., McHugh, “Flow Microsphere Immunoassay for the Quantitative and Simultaneous Detection of Multiple Soluble Analytes,” Methods in Cell Biology 42, Part B (Academic Press, 1994); McHugh et al., “Microsphere-Based Fluorescence Immunoassays Using Flow Cytometry Instrumentation,” Clinical Flow Cytometry, Bauer, K. D., et al., eds. (Baltimore, Md., USA: Williams and Williams, 1993), pp. 535-544; Lindmo et al, “Immunometric Assay Using Mixtures of Two Particle Types of Different Affinity,” J. Immunol. Meth. 126: 183-189 (1990); McHugh, “Flow Cytometry and the Application of Microsphere-Based Fluorescence Immunoassays,” Immunochemica 5: 116 (1991); Horan et al., “Fluid Phase Particle Fluorescence Analysis: Rheumatoid Factor Specificity Evaluated by Laser Flow Cytophotometry,” Immunoassays in the Clinical Laboratory, 185-189 (Liss 1979); Wilson et al, “A New Microsphere-Based Immunofluorescence Assay Using Flow Cytometry,” J. Immunol. Meth. 107: 225-230 (1988); Fulwyler et al., “Flow Microsphere Immunoassay for the Quantitative and Simultaneous Detection of Multiple Soluble Analytes,” Meth. Cell Biol. 33: 613-629 (1990); Coulter Electronics Inc., United Kingdom Patent No. 1,561,042 (published Feb. 13, 1980); and Steinkamp et al., Review of Scientific Instruments 44(9): 1301-1310 (1973).

Typically, the detecting step thus involved the use of detector.

As used herein, the term “detector” is intended to mean a device or apparatus that converts the energy of contacted photons into an electrical response. For instance, the term can include an apparatus that produces an electric current in response to impinging photons such as in a photodiode or photomultiplier tube. A detector can also accumulate charge in response to impinging photons and can include, for example, a charge coupled device. In particular, the detector involves the use of a radiation source.

As used herein, the term “radiation source” is intended to mean an origin or generator of propagated electromagnetic energy. The term can include any illumination sources including, for example, those producing electromagnetic radiation in the ultraviolet, visible and/or infrared regions of the spectrum. A radiation source can include, for example, a lamp such as an arc lamp or quartz halogen lamp, or a laser.

As used herein, the term “laser” is intended to mean a source of radiation produced by light amplification by stimulated emission of radiation. The term can include, for example, an ion laser such as argon ion or krypton ion laser, helium neon laser, helium cadmium laser, dye laser such as a rhodamine 6G laser, YAG laser or diode laser. These and other lasers useful in the apparatus of the invention are known in the art as described, for example, in Shapiro, Practical Flow Cytometry, 3rd Ed. Wiley-Liss, New York (1995).

In some embodiments, the detector is a flow cytometer.

As used herein, the term “flow cytometer” is intended to mean a device or apparatus having a means for aligning the particles in a sample stream and a detector aligned such that the particles individually enter a zone of detection. A sample stream can include any mobile phase that passes particles in single file including, for example, a fluid stream or fluid jet.

In some embodiments, the method of the present invention comprises the steps wherein the sample is separated in 3 aliquots, and each aliquot is then placed in a single assay receptacle, wherein the first aliquot is placed in presence of a first of particles being conjugated to a DSC3 polypeptide, the second aliquot is placed in presence of particles being conjugated to a a SPCA1 polypeptide and the third aliquot is placed in presence of particles being conjugate to a CHRM3 polypeptide.

As used herein, the term “aliquot” refers to a subset of the sample.

In said embodiments, the presence or absence of anti-DSC3 autoantibodies, anti-SPCAl autoantibodies and or anti-CHRM3 autoantibodies is revealed for one sample.

In some embodiments, the method of the present invention comprises the steps of: a) placing a sample obtained from the subject, in a single assay receptacle, in the presence of a plurality of particles belonging to at least 2 different groups, a first group of particles being conjugated to a SPCA1 polypeptide and a second group of particles being conjugated to a CHRM3 polypeptide

- b) incubating the mixture under conditions which allow the formation of immunocomplexes on particles, c) eliminating the immunoglobulins which have not bound to the particles, d) incubating the mixture of step b) with at least one secondary antibody that is coupled to an indicator reagent and has specificity for a particular immunoglobulin (e.g. an anti-human IgG or anti-IgA antibody), e) eliminating the secondary antibodies not bound to the immunocomplexes of step b), and f) simultaneously detecting, by means of a detector capable of differentiating the particles mentioned above, the immunocomplexes of step d) on each particle, whereby the presence or absence of anti-SPCAl autoantibodies or anti-CHRM3 autoantibodies is revealed.

In said embodiments, the method of the present invention is particularly suitable for simultaneously detecting the presence or absence of anti-SPCAl autoantibodies (IgG and/or IgA) and anti-CHRM3 autoantibodies (IgG and/or IgA). In some embodiments, the groups of said particles differ from one another by their identity codes (e.g. fluorophores) as described above. In said embodiments, the method of the present invention thus involves the use of a multiplex technology.

As used herein, the term “multiplex technology” is the collective term for a variety of techniques which can assess multiple immunoglobulin specificities simultaneously on small volumes of sample. The advantage of multiplex technology is that it is able to provide very rapid test times and very high throughput of samples. In some embodiments, the method of the present invention involves an addressable laser bead immunoassay (ALBIA), which is commercially available on Luminex™-based platforms. For instance, ALBIA is a semi-quantitative homogenous fluorescence-based microparticle immunoassay that can be used for the simultaneous detection of several immunogobulins (e.g. up to 10 immunoglobulins). Each antigen (i.e. DSC3, SPCA1 or CHRM3) is covalently coupled to a set of distinct uniform size colour-coded particles. The sample is then incubated with the particles in the single assay receptacle or may be separated in 3 aliquots as described above and thus are contacted with group of particles. The particles are then washed and then incubated with secondary anti-human IgG conjugated to a fluorescent label (e.g. phycoerythrin). After washing again, the particles are analysed on a system in which separate lasers identified antigen by bead colour and quantified the antibody by measuring the fluorescence of the fluorescent label. Said quantification thus indicated the level of the detected autoantibodies.

In some embodiments, the method of the present invention further involves placing a fourth aliquot, in a single assay receptacle, in the presence of a fourth group of particles being conjugated to a desmoglein polypeptide.

In some embodiments, the fourth group of particles is conjugated to a DSG1 polypeptide. In some embodiments, the fourth group of particles is conjugated to a DSG3 polypeptide.

In some embodiments, the method of the present invention further involves i) placing a fourth aliquot, in a single assay receptacle, in the presence of a fourth group of particles being conjugated to a desmoglein polypeptide and ii) placing a fifth aliquot, in a single assay receptacle, in the presence of a fifth group of particles being conjugated to a desmoglein polypeptide

In some embodiments, the fourth group of particles is conjugated to a DSG1 polypeptide. In some embodiments, the fifth group of particles is conjugated to a DSG3 polypeptide.

As used herein, the term “DSG1” has its general meaning in the art and refers to the desmoglein 1. An exemplary amino acid sequence for DSG1 is represented by SEQ ID NO:4.

As used herein, the term “DSG3” has its general meaning in the art and refers to the desmoglein 3. An exemplary amino acid sequence for DSG3 is represented by SEQ ID NO:5.

In some embodiments, the method of the present invention is particularly suitable for detecting the presence or absence of anti-DSGl autoantibodies (IgG and/or IgA), anti-DSG3 autoantibodies (IgG and/or IgA), anti-DSC3 autoantibodies (IgG and/or IgA), anti-SPCAl autoantibodies (IgG and/or IgA) or anti-CHRM3 autoantibodies (IgG and/or IgA).

The method of the present invention is particularly suitable for the diagnosis of pemphigus and more particularly for the diagnosis of pemphigus vulgaris and pemphigus foliaceus. As used herein, the term “pemphigus vulgaris” or “PV” has its general meaning in the art and refers to an acquired, rare, chronic, disabling, and potentially life- threatening autoimmune vesiculobullous disorder, characterized by mucocutaneous erosions or blisters. The disease is caused by pathogenic antibodies directed against desmoglein 1 and 3, which are members of the desmosomal cadherin family. The in vivo binding of these anti-desmoglein autoantibodies (mainly IgG4 and IgGl) leads to the loss of adhesion between keratinocytes resulting in the formation of intra-epidermal blisters. These blisters eventually lead to erosions in the skin which, prior to steroid therapy, resulted in significant mortality.

As used herein, the term “pemphigus foliaceus” or “PF” has its genera meaning in the art and refers to the second most common type of pemphigus. It is an autoimmune skin disorder characterized by the loss of intercellular adhesion of keratinocytes in the upper parts of the epidermis (acantholysis), resulting in the formation of superficial blisters.

In some embodiments, the method of diagnosis described herein is applied to a subject who presents symptoms of PV or PF without having undergone the routine screening to rule out all possible causes for PV or PF. The methods described herein can be part of the routine set of tests performed on a subject who presents symptoms of PV or PF such as painful blisters that start in the mouth or skin areas, skin blisters near the surface of the skin that come and go, as well as oozing, crusting, or peeling at the blister site. The method of the present invention can be carried out in addition of other diagnostic tools such as histology.

The method of the present invention is also particularly suitable for determining whether a subject suffering from PV or PF achieves a response with a treatment.

The method is thus particularly suitable for discriminating responder from non-responder.

As used herein the term “responder” in the context of the present disclosure refers to a subject that will achieve a response, i.e. a subject who is under remission and more particularly a subject who does not suffers from blisters. As used herein the term “non-responder” refers to a subject for whom the disease does not show reduction or improvement after the treatment (e.g. the blisters remains stable or increases).

In particular, detecting the absence of the autoantibodies indicates that the patient achieve a response with the treatment.

In other word, the present invention refers to a method for monitoring the treatment of pemphigus vulgaris or pemphigus foliaceus in a subject in need thereof comprising the steps of i) detecting the presence of pemphigus-specific autoantibodies in a sample obtained from the subject according to the method of the invention, and ii) concluding that subject achieve a response to said treatment when the pemphigus-specific autoantibodies is not detected at step i) or concluding that the treatment is not efficient when the pemphigus-specific autoantibodies is detected at step i)

The method of the present invention is also particularly suitable for determining whether a subject is at risk of relapse after a treatment.

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

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

In other word, the present invention refers to a method for determining whether a subject is at risk of relapse after a treatment comprising the steps of i) detecting the presence of pemphigusspecific autoantibodies in a sample obtained from the subject according to the method of the invention, and ii) concluding that subject is at risk of relapse after a treatment when the pemphigus-specific autoantibodies is detected at step i).

In particular detection of the presence of anti-SPCAl autoantibodies and/or anti-CHRM3 autoantibodies indicates that the patient is at risk of relapse, in particular, in the first year following the treatment using a B cell depleting agent (e.g. rituximab) as a first-line agent.

Thus, the present invention refers to a method for determining whether a subject is at risk of relapse after a treatment comprising the steps of i) detecting the anti-SPCAl autoantibodies and/or anti-CHRM3 in a sample obtained from the subject according to the method of the invention, and ii) concluding that subject is at risk of relapse after a treatment when the pemphigus-specific autoantibodies is detected at step i).

In particular detection of the presence of anti-SPCAl autoantibodies and/or anti-CHRM3 autoantibodies and anti-DSC3 autoantibodies indicates that the patient is at risk of relapse, in particular, in the first year following the treatment using a B cell depleting agent (e.g. rituximab) as a first-line agent.

According to the present invention, the treatment consists in any method or drug that could be suitable for the treatment of PV or PF.

In some embodiments, the treatment consists in an antibody depleting strategy, which typically include plasma exchange, plasmapheresis or immunoadsorption.

In some embodiments, the treatment consists in administering immunoglobulins (e.g. by intravenous route).

In some embodiments, the treatment is an immunosuppressive treatment.

As used herein, the term “immunosuppressive treatment” refers to any substance capable of producing an immunosuppressive effect, e.g., the prevention or diminution of the immune response and in particular the prevention or diminution of the production of Ig. Immunosuppressive drugs include, without limitation Prednisone, Dexamethasone, Rituximab, Mycophenolate mofetil, Azathioprine, and in more refractory cases, Cyclophosphamide, and Methotrexate

In some embodiments, the immunosuppressive drug is a corticosteroid. As used, the term “corticosteroid” has its general meaning in the art and refers to class of active ingredients having a hydrogenated cyclopentoperhydrophenanthrene ring system endowed with an anti-inflammatory activity. Corticosteroid drugs typically include cortisone, cortisol, hydrocortisone (1 ip,17-dihydroxy, 21-(phosphonooxy)-pregn-4-ene, 3,20-dione disodium), dihydroxy corti sone, dexamethasone (21 -(acetyloxy)-9-fluoro- 1 P, 17-dihydroxy- 16a-m- ethylpregna-l,4-diene-3, 20-dione), and highly derivatized steroid drugs such as beconase (beclomethasone dipropionate, which is 9-chloro-l l-P, 17,21, trihydroxy- 16P-methylpregna- 1,4 di ene-3, 20-dione 17,21 -dipropionate). Other examples of corticosteroids include flunisolide, prednisone, prednisolone, methylprednisolone, triamcinolone, deflazacort and betamethasone, corticosteroids, for example, cortisone, hydrocortisone, methylprednisolone, prednisone, prednisolone, betamethesone, beclomethasone dipropionate, budesonide, dexamethasone sodium phosphate, flunisolide, fluticasone propionate, triamcinolone acetonide, betamethasone, fluocinolone, fluocinonide, betamethasone dipropionate, betamethasone valerate, desonide, desoximetasone, fluocinolone, triamcinolone, triamcinolone acetonide, clobetasol propionate, and dexamethasone.

In some embodiments, the treatment consists of administering a B cell depleting agent.

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

In some embodiments, the method of the present invention is performed in vitro or ex vivo.

A further object of the present invention relates to a kit for performing the method of the present invention. The kit comprises one or more plurality of particles as above described and means for determining the immunocomplexes. Reagents for particular types of assays can also be provided in kits of the invention. Thus, the kits can include different groups of particles each identified by a specific identity, plates that comprises the single assay receptacles (e.g. a multiwell plate), and secondary antibodies as described above. In some embodiments, the kits comprise a device such as a detector as described above. The groups of particles, the plate, and the devices are useful for performing the immunoassay of the present invention. In addition, the kits can include various diluents and buffers, labelled conjugates or other agents for the detection of the specifically immunocomplexes, and other signal-generating reagents, such as enzyme substrates, cofactors and chromogens. Other components of a kit can easily be determined by one of skill in the art.

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

FIGURES: Figure 1. Schematic representation of the ALBIA technique used to detect and quantify the auto-antibodies in pemphigus patients. The antigens are either DSC3, SPCA1 or CHRM3.

Figure 2. Anti-DSC3 auto-antibodies serum level in (A) total IgG, (B) IgAl and (C) IgA2 in pemphigus patients (n=146) compared to healthy donors (HD) (n=100), and serum level of anti- SPCA1 (D-F) and anti-CHRM3 (G-I). The positivity cut-off value, which is calculated with the mean of 100 HD + 2 (IgG anti-SPCAl and IgG anti-CHRM3) or 3 (IgG and IgA anti-DSC3, IgA anti-SPCAl and IgA anti-CHRM3) standard deviation, is indicated as horizontal dotted line and mean values are shown as solid bars.

Figure 3. Evaluation of in vitro pathogenicity of anti-DSC3 antibodies from pemphigus patients and anti-DSC3 immunized-mice. (A) Keratinocyte dissociation assay from one patient containing solely anti-DSC3 IgA which shown to be pathogenic in the absence of anti- DSG1 and anti-DSG3. (B) keratinocyte dissociation assays using sera from two patients with mucocutaneous type of PV containing anti-DSG3 IgG and anti-DSC3 IgAl (patient D), and anti-DSG3 and anti-DSC3 IgG (patient E). A decrease of the pathogenicity was observed after pre-adsorption of anti-DSC3 Abs. (C) The dissociation assay was performed with sera from two patients (F and G) who presented a cutaneous type of PV containing anti-DSGl IgG and anti-DSC3 IgAl. A decrease of serum pathogenicity was observed after pre-adsorption of anti- DSC3 Abs with recombinant DSC3. For A, B and C * For healthy donor: n=9, **The P-value is calculated in comparison to the healthy donor/negative control. (D) Mice were immunized with recombinant human DSC3 protein and antibodies were detected as of day 56. Purified IgG anti-DSC3 from immunized mice were shown to be pathogenic. DSC3, Desmocollin 3; DSG, Desmoglein; IF A, Incomplete Freund’s Adjuvant; Ig, Immunoglobulin. * The P-value is calculated in comparison to the non-immunized mice, ** For the purified IgG pre-adsorbed in anti-DSC3: n=l.

Figure 4: Evaluation of in vitro pathogenicity of anti-SPCAl and anti-CHRM3 antibodies from pemphigus patients. (A) Keratinocyte dissociation assay from two patients containing anti-SPCAl and anti-CHRM3 IgG associated with anti-DSGl and/or anti-DSG3 antibodies at baseline which showed to be pathogenic even after adsorption of anti-SPCAl or anti-CHRM3 antibodies. (B) Keratinocyte dissociation assays using sera from these two patients containing anti-SPCAl and anti-CHRM3 antibodies, but in absence of anti-DSGl/3 antibodies at the time of relapse. The in vitro pathogenicity was abolished after pre-adsorption of anti-SPCAl or anti- CHRM3 antibodies. CHRM3: Cholinergic Receptor Muscarinic 3; DSG, Desmoglein; Ig, Immunoglobulin, SPCA1 : Secretory pathway of Ca2+/Mn2+ ATPase type 1. EXAMPLE 1:

Methods

Adressable laser bead immunoassay (ALBIA) test for the quantification of anti-DSC3, anti- SPCA1 and anti-CHRM3 auto-antibodies

An ALBIA test was developed for each of these three proteins (Figure 1). It consisted of coupling human recombinant DSC3, SPCA1 or CHRM3 protein to fluorescent beads (Biorad) according to the manufacturer’s protocol.

To quantify the auto-antibodies, 1000 coated beads were incubated in Multiscreen 96-well plates with lOOpl of sera diluted at 1 :37.5 for DSC3 and at 1 :600 for SPCA1 and CHRM3 in Dulbecco’s phosphate-buffered saline (DPBS) with Ca²⁺/Mg²⁺ and 1% fetal bovine serum for 2h on a plate shaker and then washed. Then, beads were incubated for Ih in the same conditions with lOOpl of specific biotinylated mouse anti-human secondary antibodies (SoutherB iotech) at the following dilution: anti-IgG at 1 :2000, anti-IgAl at 1 : 125 and anti-IgA2 at 1 :200, and washed. Finally, beads were incubated for 15 min with 50pL of streptavidin-R-phycoerythrin (Biorad) diluted at 1 :400. The mean fluorescence intensity (MFI) was determined on a Bio- Plex apparatus using Manager software version 4.0 (Bio-Rad). Negative control (no serum, secondary antibody only) and positive control (highly positive serum) were included in every assay.

The auto-antibodies level were determined with the following formula that was previously described: (MFI^serum/MFI^{positlve control}) x 100, in which the positive control is a patient highly positive in anti-DSC3, anti-SPCAl or anti-CHRM3 auto-antibodies. These patients serve as positive controls for each antigen and are used on every 96-well plates. Their level was arbitrarily set to 100 arbitrary units (AU)/mL.

Results

The ALBIA test developed for the 3 proteins successfully detected and quantified the presence of auto-antibodies directed against each of these antigens in pemphigus patients (Figure 2). For DSC3, IgA (IgAl and/or IgA2) anti-DSC3 were detected in 23/146 (16%) patients compared to 3/100 (3%) in healthy donors (P=0.0012) and IgG anti-DSC3 were detected in 10/146 (7%) patients compared to 2/100 (2%) in healthy donors (P=0.13). Therefore, the main isotype identified was IgA, and the IgAl titers significantly decreased following the introduction of a treatment from 188+132 AU/mL at day (D) 0 to 40+35 AU/mL at D90 after treatment (P=0.0015), and the IgA2 titers from 26+26 AU/mL at DO to 0.9+1.4 AU/mL at D90 (P=0.07).

For SPCA1, IgG anti-SPCAl were detected in 32/146 (22%) patients compared to 3/100 (3%) in healthy donors (P<0.0001) and no IgA were detected. The IgG titers significantly decreased following the introduction of a treatment from 88+26 AU/mL at DO to 25+21 AU/mL at D90 (P0.0001).

For CHRM3, IgG anti-CHRM3 were detected in 45/146 (31%) patients compared to 2/100 (2%) in healthy donors (P<0.0001) and IgA anti-CHRM3 were detected in 15/146 (10%) patients compared to 3/100 (3%) in healthy donors (P=0.07). The titers significantly decreased following the introduction of a treatment from 60+27 AU/mL at DO to 31+21 AU/mL at D90 (P<0.0001).

The detection and quantification of anti-SPCAl and/or anti-CHRM3 were also associated to a risk of relapse in the first year following the treatment using rituximab as a first-line agent. Amongst patients with anti-SPCAl at DO, 5/10 (50%) patients relapsed in the first year compared to 4/36 (11%) patients who were negative in anti-SPCAl (P=0.0056). Amongst patients with anti-CHRM3 at DO, 4/12 (33%) patients relapsed in the first year compared to 5/34 (15%) patients who were negative in anti-SPCAl (P=0.16). Therefore, the presence of either of these autoantibodies was a predictor of relapse and could help monitor disease evolution.

EXAMPLE 2:

Materials and methods

Keratinocyte Dissociation Assay

HaCaT cells were cultivated with 600 000 cells per well in 24-well-plates with DMEM + GlutaMAX (Gibco) containing ImM CaC12 in a humidified atmosphere (5% CO2) at 37°C. After reaching confluency in 24 h, positive control AK23 (10 pg/mL), 50 pg of HD or PV purified IgG or flow (Ig without IgG) or fractions pre-adsorbed in anti-DSC3, anti-SPCAl or anti-CHRM3 were added and incubated for 24 hours. For adsorption, purified IgG or flow diluted at 1 :5 in DPBS were incubated twice for 2 h with 2500 DSC3-coated beads and washed. Subsequently, the cells were treated with dispase solution (2.4 U/ml; Sigma) at 37°C until monolayers were released from plate. Monolayers were stained with crystal violet (Sigma Aldrich) and subjected to mechanical stress by vigorously pipetting 7 times with a ImL pipette. Cell fragments were fixed and counted manually, and photos were taken of each well. All experiments were performed in triplicate.

Mouse immunization with human recombinant Desmocollin 3

Eight-week-old female C57BL/6 mice were purchased from Janvier, France. The protocol for animal experimentation was approved by an institutional ethics committee (Comite regional d’Ethique en Experimentation Animale, Mont Saint Aignan, France, protocol number 27457) were conducted according to the NIH Guide for the Care and Use of Laboratory Animals. Mice were immunized with subcutaneous injection of 10 pg of human rDSC3 (Antibodies online, Aachen, Germany) emulsified in Incomplete Freund's Adjuvant (IF A) (Thermo Scientific). Additional boosts were performed after 7 and 35 days using 10 pg rDSC3 plus IFA. The production of anti-DSC3 after immunization was assessed from mice serum by ALBIA, as previously described, except that anti-mouse IgG secondary Ab (Southern Biotech) was used for revelation. Pathogenicity of the newly formed IgG anti-DSC3 was evaluated with a keratinocyte dissociation essay.

Results:

Pathogenicity of anti-Dsc3 antibodies

To confirm the pathogenic activity of anti-DSC3 Abs, we evaluated the in vitro pathogenicity of sera containing anti-DSC3 Abs. First, patient A had a mucosal type of PV with suprabasilar acantholysis despite the absence of anti-DSGl and anti-DSG3 Abs. Anti-DSC3 IgA Abs from this patient’s serum induced in vitro monolayer dissociation, which was significantly lowered following pre-adsorption with recombinant DSC3 (rDSC3) (Figure 3 A).

Second, patients D and E had a mucocutaneous type of PV despite the exclusive presence of anti-DSG3 Abs (without anti-DSGl Abs). Figure 3B shows that purified anti-DSC3 IgA and IgG Abs from these sera induced monolayer dissociation. The pathogenic activity of patients’ purified anti-DSC3 IgA Abs was abolished after pre-adsorption of the IgA fraction with rDSC3 (patients D). Similarly, the in vitro pathogenicity of patients’ purified IgG containing both anti- DSG3 and anti-DSC3 Abs was decreased when removing anti-DSC3 IgG Abs (patient E). This suggests that the combination of both anti-DSG3 and anti-DSC3 Abs were necessary to induce an in vitro pathogenic effect with this serum. Third, two patients (F and G) had a cutaneous type of PV (without mucosal lesions) and suprabasilar acantholysis despite the presence of exclusive anti-DSGl Abs (without anti-DSG3 Abs). Figure 3C shows that the pathogenic activity of anti-DSC3 IgA Abs was abolished by pre-adsorption of these sera with rDSC3.

Finally, female C57BL/6 mice were immunized with human rDSC3. Anti-DSC3 IgG Abs were detected in mice sera 56 days after immunization (Figure 3D). Purified IgG from these anti- DSC3 Ab-containing sera induced a monolayer dissociation, while no pathogenic effect was observed with control sera from non-immunized mice. In addition, we performed a specific preadsorption of anti-DSC3 from the purified IgG fraction obtain from mice, and this data support its pathogenicity since the monolayer fragmentation was abolished. These experiments further suggest the role of anti-DSC3 Abs in the pathogenesis of pemphigus.

Pathogenicity of anti-SPC Al and anti-CHRM3 antibodies

To confirm the pathogenic activity of anti-SPCAl and anti-CHRM3 Abs, we evaluated the in vitro pathogenicity of sera containing these two auto-Abs. Purified IgG from the sera of two pemphigus patients containing anti-SPCAl and anti-CHRM3 Abs were tested in a keratinocyte dissociation assay before treatment (in active disease, at baseline) and at the time of relapse. Indeed, anti-SPCAl and anti-CHRM3 IgG were associated with anti-DSGl and/or anti-DSG3 antibodies at baseline, but not at the time of relapse. At baseline, purified IgG from patients’ sera induced the monolayer fragmentation and showed to be pathogenic even after the adsorption of anti-SPCAl or anti-CHRM3 Abs. This is probably explained by the presence of anti-DSGl and/or anti-DSG3 Abs (Figure 4A). Conversely, purified IgG from these two patients’ sera containing anti-SPCAl and anti-CHRM3 antibodies, but in absence of anti- DSG1/3 antibodies at the time of relapse, induced the monolayer fragmentation, which was abolished after pre-adsorption of anti-SPCAl or anti-CHRM3 antibodies (Figure 4B). These results showed the in vitro pathogenic effect of anti-SPCAl and anti-CHRM3 Abs and thus suggested the role of these two auto-Abs in the pathogenesis of pemphigus.

REFERENCES:

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

Claims

CLAIMS:

1. A method for detecting the presence of pemphigus-specific autoantibodies in a subject comprising the steps of: a) placing a sample obtained from the subject, in a single assay receptacle, in the presence of particles belonging to either of the 3 different groups, a first group of particles being conjugated to DSC3 polypeptide, a second group of particles being conjugated to a SPCA1 polypeptide and a third group of particles being conjugated to a CHRM3 polypeptide b) incubating the mixture under conditions which allow the formation of immunocomplexes on particles, c) eliminating the immunoglobulins which have not bound to the particles, and d) detecting the immunocomplexes of step b) on the plurality of particles, whereby the presence or absence of anti-DSC3 autoantibodies, anti-SPCAl autoantibodies or anti- CHRM3 autoantibodies is revealed.

2. The method of claim 1 wherein the sample is a blood sample.

3. The method of claim 1 wherein the particles are magnetic and coded.

4. The method of claim 1 wherein the DSC3 polypeptide comprises the sequence as set forth in SEQ ID NO: 1.

5. The method of claim 1 wherein the SPCA1 polypeptide comprises the sequence as set forth in SEQ ID NO:2.

6. The method of claim 1 wherein the CHRM3 polypeptide comprises the sequence as set forth in SEQ ID NO:3.

7. The method of claim 1 wherein a secondary antibody that is coupled to an indicator reagent comprising a signal generating compound is used.

8. The method of claim 7 wherein the secondary antibody is an anti-human IgG antibody, including anti-IgGl, IgG2, IgG3 and IgG4 antibodies. The method of claim 7 wherein the secondary antibody is an anti-human IgAl or IgA2 antibody. The method of claim 1 that comprises the steps wherein the sample is separated in 3 aliquots, and each aliquot is then placed in a single assay receptacle, wherein the first aliquot is placed in presence of a first of particles being conjugated to a DSC3 polypeptide, the second aliquot is placed in presence of particles being conjugated to a a SPCA1 polypeptide and the third aliquot is placed in presence of particles being conjugate to a CHRM3 polypeptide. The method of claim 1 that comprises the steps of: a) placing a sample obtained from the subject, in a single assay receptacle, in the presence of a plurality of particles belonging to at least 2 different groups, a first group of particles being conjugated to a SPCA1 polypeptide and a second group of particles being conjugated to a CHRM3 polypeptide b) incubating the mixture under conditions which allow the formation of immunocomplexes on particles, c) eliminating the immunoglobulins which have not bound to the particles, d) incubating the mixture of step b) with at least one secondary antibody that is coupled to an indicator reagent and has specificity for a particular immunoglobulin (e.g. an antihuman IgG or anti-IgA antibody), e) eliminating the secondary antibodies not bound to the immunocomplexes of step b), and f) simultaneously detecting, by means of a detector capable of differentiating the particles mentioned above, the immunocomplexes of step d) on each particle, whereby the presence or absence of anti-SPCAl autoantibodies or anti-CHRM3 autoantibodies is revealed. The method of claim 1 that involves an addressable laser bead immunoassay (ALBIA). Use of the method of claim 1 for the diagnosis of pemphigus and more particularly for the diagnosis of pemphigus vulgaris and pemphigus foliaceus.

14. Use of the method of claim 1 for determining whether a subject suffering from PV or PF achieves a response with a treatment.

15. Use of the method of claim 1 for determining whether a subject is at risk of relapse after a treatment. 16. The use of claim 15 wherein detection of the presence of anti-SPCAl autoantibodies and/or anti-CHRM3 autoantibodies indicates that the patient is at risk of relapse, in particular, in the first year following the treatment using a B cell depleting agent (e.g. rituximab) as a first-line agent.