WO2012010677A1 - Diagnostic method for assaying anti-srp antibodies - Google Patents

Abstract

The present invention refers to a method for diagnosing an acquired myopathy in a mammal, particularly necrotizing or inflammatory myopathy, comprising a step of measuring the quantity and/or the concentration of anti-SRP antibodies in a biological sample of said mammal. The present invention also refers to a method for monitoring and/or predicting responsiveness to a treatment, and to a kit for measuring anti-SRP antibodies.

Description

Diagnostic method for assaying anti-SRP antibodies

The present invention refers to a quantitative test for measuring anti-SRP antibodies (Abs), and to methods for diagnosing an acquired myopathy, particularly necrotizing or inflammatory myopathy, and/or for monitoring and/or predicting the responsiveness to a treatment of an acquired myopathy, particularly necrotizing or inflammatory myopathy in a mammal.

Autoantibodies (aAbs) directed against signal recognition particles (SRP) are present in a minority (4-6%) of patients with inflammatory (Targoff et al., 1990; Troyanov et al., 2005) and/or acquired necrotizing myopathies (Brouwer et al., 2001; Hengstman et al, 2006; Hoogendijk et al., 2004). The myopathy associated with anti-SRP aAbs is a severe necrotizing immune-mediated disease characterized by rapidly progressive proximal muscle weakness, markedly elevated serum creatine kinase (CK) levels, and poor responsiveness to traditional immunosuppressive therapies. Growing pathological evidence suggests that anti-SRP myopathy is an immune-mediated necrotizing myopathy that differs from polymyositis, characterized by important muscle necrosis contrasting with little or no inflammatory infiltrates and HLA class I expression, and inconstantly particular patterns of complement membrane attack complex (C5b-9) deposition.

Anti-SRP aAbs are directed against components of the signal recognition particle. The SRP complex is composed of six SRP family proteins associated with a small RNA molecule. The physiological role of the SRP complex is to guide the translocation of growing polypeptides into the endoplasmic reticulum during protein synthesis. Whereas

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patient anti-SRP sera may recognise each of the six SRP subunits or the 7S RNA, the 54 kDa subunit (SRP54) - which directly binds signal peptides (Janda et al., 2010) - remains their main target and reactivity to SRP54 is always present (Okada et al., 1987; Reeves et al., 1986; Romisch et al., 2006). Functionally, it has been shown that anti- SRP aAbs purified from patients can inhibit the in vitro translocation of secretory proteins into the endoplasmic reticulum (Romisch et al., 2006). Given that the level of serum creatine kinase (CK) reflects muscle necrosis in a given patient, studying the correlation between anti-SRP aAbs titers and CK level would be helpful to better understand the pathogenesis of this form of acquired myopathy and may define a surrogate marker of disease activity.

Currently, the detection of circulating anti-SRP aAbs is based upon an indirect immunofluorescence test on HEp-2 cells that typically shows a cytoplasmic pattern (Targoff et al., 1990). Since this profile is not specific, the diagnosis of anti-SRP aAbs has to be confirmed by an immunodot assay or by protein immunoprecipitation. Yet, these latter assays are not quantitative and do not allow for reproducible titration of anti- SRP aAbs.

Moreover, the etiology of necrotizing myopathy is obscure. Particularly, it is unclear whether the anti-SRP aAbs are the cause of the disease. In general, when pathologies are associated with the presence of specific aAbs, the simple qualitative detection of these aAbs allows the diagnosis of said pathologies. However, there is a need to quantify the evolution of autoantibodies quantity or concentration overtime, in order to better understand the role of these autoantibodies in the pathogenesis of the disease, and the effect of the therapy. Thus, there remains a need to develop methods for the diagnosis of acquired myopathies, particularly necrotizing or inflammatory myopathy, in subjects suffering thereof. Such methods have to be easy to perform, with high reproducibility and sensitivity of the results. Surprisingly, the inventors have discovered that there is a correlation between anti-SRP aAbs titers and serum CK levels in patients with necrotizing myopathy. This correlation is observed even at different stages of the disease. Thus, the anti-SRP aAbs quantity or concentration is a good marker of necrotizing myopathy. According to one embodiment, the present invention encompasses a method for diagnosing an acquired myopathy, and particularly necrotizing or inflammatory myopathy, in a mammal, comprising a step of measuring the quantity and/or the concentration of anti-SRP antibodies in a biological sample of said mammal.

Preferably, said diagnostic method comprises a further step of comparing the quantity and/or the concentration of anti-SRP antibodies in the biological sample of said mammal with a reference value.

According to a second embodiment, the present invention targets a method for predicting the responsiveness to a treatment of a mammal suffering from an acquired myopathy, particularly necrotizing or inflammatory myopathy, said method comprising measuring the quantity and/or the concentration of anti-SRP antibodies in the biological sample of said mammal.

According to a third embodiment, the present invention aims a method for assessing the effectiveness of a drug to treat an acquired myopathy, preferably necrotizing or inflammatory myopathy, in a mammal suffering from said acquired myopathy, preferably necrotizing or inflammatory myopathy, comprising the following steps:

(A) measuring the quantity or concentration Dl of anti-SRP antibodies in a biological sample obtained from said mammal;

(B) after a period of therapy, measuring the quantity or concentration D2 of anti- SRP antibodies in a biological sample obtained from said mammal in the same manner as in step (A);

(C) comparing quantities Dl and D2, or concentrations Dl and D2, and

(D) if D2 <D1, deducing that the drug is effective in treating said acquired myopathy, preferably necrotizing or inflammatory myopathy.

Finally, according to a fourth embodiment, the present invention refers to measuring anti-SRP antibodies quantity or concentration, comprising:

at least one labelled SRP element; and

means for detecting anti-SRP antibodies. The methods according to the invention are specific to the anti-SRP antibodies, specifically detect anti-SRP antibodies, and allow a finer and a more sensitive and reliable diagnosis or monitoring of positive patients as compared to conventional methods. Indeed, they allow the diagnosis or monitoring of positive patients, i.e. patients afflicted with an acquired myopathy, whereas said patients have been diagnosed or monitored as negative with other conventional methods, like Dot-Blot.

In the context of the invention, the term "diagnosing" or "diagnosis", as used herein, means identifying the disease, particularly after its first clinical signs or symptoms are declared.

The term "monitoring the responsiveness to a treatment" means following the evolution of the response to said treatment. The term "predicting the responsiveness to a treatment" means anticipating the response to said treatment.

As used herein, the term "antibody" means any type of antibody, and includes any autoantibody.

As used herein, the term "SRP" means signal recognition particle. As said above, the SRP complex is composed of six SRP family proteins associated with a small RNA molecule. By "anti-SRP antibody", it is meant any antibody which targets, as antigen, one element of the SRP complex, i.e. one of the six SRP proteins or the SRP small RNA molecule. Preferably, the anti-SRP antibody used according to the invention target the 54kDa subunit (SRP54) as antigen.

The term "acquired myopathy" means any myopathy which appears on a previously healthy muscle, in opposition to monogenic myopathies such as heritable muscular dystrophies. The acquired myopathies include for instance inflammatory myopathies, necrotizing myopathy and drug myopathies.

Drug myopathies are due to myotoxic drugs, like hypocholesterolaemics, chloroquin or colchicin, anti-retroviral drugs or corticosteroids.

Inflammatory myopathies comprise polymyositis, dermatomyositis, inclusion body myositis and inflammatory myopathies associated with connective tissue diseases (overlap myositis).

Preferably, the methods according to the present invention target necrotizing myopathy. As used herein, the term "mammal" is a human or a non human mammal. Preferably, said non human mammal is a rodent, a feline, a canine, an equidae or a primate; preferably, the mammal is chosen from a horse, a mouse, a rat and a rabbit. Preferably the mammal according to the invention is a human.

As used herein, the term "quantity" refers to the quantity of anti-SRP Abs measured in the biological sample. The term "concentration" refers to the quantity of anti-SRP Abs per biological sample volume.

The terms "biological sample" as used herein 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). In a preferred embodiment, said biological liquids comprise blood, plasma, serum, saliva and exsudates. Thus, preferably, the biological sample is chosen from blood samples, plasma samples, saliva samples, exsudate samples and serum samples. Preferably, the biological sample is a blood sample, a serum sample or a plasma sample.

The methods according to the invention are ex vivo or in vitro methods, preferably in vitro methods.

The methods according to the present invention are easy to perform.

Acquired myopathy diagnostic method

The method for diagnosing an acquired myopathy, preferably necrotizing or inflammatory myopathy, in a mammal comprises a step of measuring the quantity and/or the concentration of anti-SRP antibodies in a biological sample of said mammal.

The quantity or concentration of anti-SRP Abs in the biological sample obtained from the mammal may be determined using any available technique, including immunoassays such as competition, direct reaction, array chips, or sandwich type assays. Such assays include, but are not limited to agglutination tests; enzyme-labelled and -mediated immunoassays, such as ELISAs or Luminex; biotin/avidin type assays; radio- immunoassays; or solid-phase affinity. Preferably, the quantity or concentration of anti- SRP Abs in the biological sample is measured thanks to an immunoassay.

Preferably, said diagnostic method comprises a further step of comparing the quantity and/or the concentration of anti-SRP antibodies in the biological sample of said mammal with a reference value. Said reference value is predictive of the presence or absence of the disease in said mammal. Said reference value may be an average anti- SRP Abs quantity or concentration obtained from healthy mammals, or obtained from mammals suffering from an acquired myopathy.

Preferably, the method is for diagnosing necrotizing myopathy; in this case, said reference value is preferably an average anti-SRP Abs quantity or concentration obtained from healthy mammals.

Thanks to the only quantitative detection of anti-SRP Abs, the present invention allows the diagnosis of an acquired myopathy, preferably necrotizing myopathy.

Method for monitoring and/or predicting the responsiveness to a treatment

The present invention targets a method for monitoring and/or predicting the responsiveness to a treatment of a mammal suffering from an acquired myopathy, preferably necrotizing or inflammatory myopathy, said method comprising measuring the quantity and/or the concentration of anti-SRP antibodies in the biological sample of said mammal.

Said method preferably further comprises a step of comparing the quantity and/or the concentration of anti-SRP antibodies with reference values. Said reference values are quantity and/or concentration of anti-SRP antibodies, which may be obtained from unaffected control mammals. Said reference values may also be obtained from responder and non-responder group of mammals. Responder and non-responder groups of mammals to a treatment are respectively mammals who ameliorate their condition (i.e. recover, ameliorate or stabilize) thanks to said treatment, and those who do not. Said reference values may also be obtained from said mammal in the absence of said treatment and before beginning this treatment; said reference values may also be obtained on the first day of treatment.

Necrotizing myopathy treatments may be corticosteroids; immunosupressants including azathioprine, methotrexate and mycophenolate mofetil; plasma exchange; intravenous immunoglobulins; or therapeutic antibodies including Rituximab. The method according to the invention is useful for predicting the responsiveness to one of said treatments.

Such a method is useful for screening a drug candidate able to treat an acquired myopathy, preferably necrotizing myopathy. Indeed, thanks to the responsiveness of the mammal to the treatment, it is possible to identify leading drugs able to treat the disease.

Method for assessing the effectiveness of a drug

Finally, the present invention aims a method for assessing the effectiveness of a drug to treat an acquired myopathy in a mammal suffering from said acquired myopathy, necrotizing myopathy, comprising the following steps:

(A) measuring the quantity or concentration Dl of anti-SRP Abs in a biological sample obtained from said mammal;

(B) after a period of therapy, measuring the quantity or concentration D2 of anti- SRP Abs in a biological sample obtained from said mammal in the same manner as in step (A);

(C) comparing quantities Dl and D2, or concentrations Dl and D2, and

(D) if D2 < Dl, deducing that the drug is effective in treating said acquired myopathy. Preferably, said acquired myopathy is necrotizing myopathy.

Anti-SRP Abs are thus good markers of acquired myopathies, preferably of necrotizing myopathy, and measuring their quantity or concentration in mammals, before and after treatment, is predictive of the efficacy of said treatment. Kits for measuring anti-SRP antibodies quantity or concentration

The present invention aims a kit for measuring anti-SRP antibodies quantity concentration, comprising:

at least one labelled SRP element; and

means for detecting anti-SRP antibodies.

By "SRP element", it is meant any element of the SRP complex, i.e. any one of the 6 proteins or the small RNA. Typically the SRP element may be labelled with a detectable molecule or substance, such as a fluorescent molecule, a radioactive molecule or any others labels known in the art. Labels are known in the art that generally provide (either directly or indirectly) a signal. As used herein, the term "labelled", with regard to the SRP element, is intended to encompass direct labelling of the SRP element by coupling (i.e. physically linking) a detectable substance, such as a radioactive agent or a fluorophore (e.g. fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or Indocyanine (Cy5)) to the SRP element, as well as indirect labelling of the SRP element by reactivity with a detectable substance.

The SRP element may also be labelled with a radioactive molecule by any method known to the art. For example radioactive molecules include but are not limited

123 124 111 186 188

radioactive atom for scintigraphic studies such as I , 1 , In , Re , Re

Said SRP element may also be labelled with a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine- 123, iodine-131, indium-Ill, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

Preferably, the labelled SRP element is the SRP54 subunit, labelled with fluorescent beads.

By "means for detecting anti-SRP antibodies", it is meant any means able to detect the presence of said anti-SRP antibodies. For example, such means may be secondary antibodies, like anti-IgG antibodies. For example, if the anti-SRP antibodies are human, the secondary antibodies may be mouse anti-human IgG antibodies. These secondary antibodies may also be labelled as described above, but the label being different from the label of the SRP element.

Thanks to this kit, the quantity or concentration of anti-SRP Abs in a sample can be determined. For example, the SRP element is labelled with fluorescent beads. Then the anti-SRP Abs sample is mixed with the labelled SRP element, and thereafter labelled secondary antibodies (targeting the anti-SRP antibodies) are added. Measurements of the labelled SRP element and of the labelled secondary antibodies give the quantity or concentration of SRP element/anti-SRP Ab complex.

Said SRP element may be a recombinant protein, which has been tagged. Said tagged SRP element can then bind to fluorescent beads, like Luminex beads. For example, said SRP element may be tagged with at least one histidine tag, and then can bind to fluorescent beads like Luminex beads, because of their nickel moiety. Further aspects and advantages of this invention will be disclosed in the following figures and examples, which should be regarded as illustrative and not limiting the scope of the invention.

Brief description of the Figures

Figure 1: Development of a quantitative assay for the detection of anti-SRP54 antibodies.

(A) Structure of the recombinant human SRP54 protein containing a hexa-histidine tag in C-terminal position (arrowhead). N, G and M represent the N-terminal, GTP -binding and Methionin-rich domains, respectively. The epitopes recognised by the goat and chicken anti-SRP antibodies (raised against SRP peptides) are depicted by arrows.

(B) SDS-PAGE analysis of the recombinant SRP54 protein after Coomassie blue staining.

(C) Western blot analysis of the recombinant SRP54 protein using an antibody directed to the C-terminal histidine tag and different anti-SRP54 antibodies.

(D) Fluorescent microsphere immunoassay (Luminex™) validation using serial dilutions of goat (closed circles) and chicken (open squares) anti-SRP54 antibodies. M.F.I, values are mean of triplicate determinations (error bars = SD). Inlay represents magnification of the curve for low anti-SRP54 antibody concentrations (horizontal dotted line depicts threshold for positivity of the assay = mean + 2 SD of negative control).

Figure 2: Specificity of the SRP54-coated beads.

(A) SRP54, Jo-1 and intrinsic factor-specific beads were prepared using 10 μg/ml of the corresponding hexa-histidine tagged protein and analysed by fluorescent microsphere immunoassay using a human anti-SRP54, anti-Jo-1 or anti-intrinsic factor positive serum.

(B) Dose-dependant inhibition of human anti-SRP antibodies binding to SRP54-coated beads by free human recombinant SRP54 protein. Percent inhibition is given relative to the M.F.I, value in the absence of free SRP54.

(C) Specificity of inhibition of the assay by the homologous but not heterologous proteins. Percent inhibition of four different human anti-SRP54 positive sera by 100 μg/mL of free homologous (SRP54) or heterologous (Jo-1 and intrinsic factor) proteins.

Figure 3: Titration of anti-SRP54 antibodies. The same human anti-SRP54 positive serum was used.

(A) DOT analysis of 1/150 diluted serum showing specific reactivity against SRP54 but not other antigens.

(B) Calculation of anti-SRP54 antibody titre by fluorescent microsphere immunoassay. Anti-SRP54 titres are determined by reference to the M.F.I, value given, in the same assay, by a calibrator (highly anti-SRP54 positive serum whose titre is arbitrarily set to 100 arbitrary units A.U./mL). The assay is first performed using a 1/500 screening dilution of the serum. In case the sample's M.F.I, at 1/500 dilution is higher than 80% of the calibrator's M.F.I., further dilutions are performed and the first dilution yielding a M.F.I, inferior to 80% of calibrator M.F.I, is retained for calculation.

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Figure 4: Diagnostic value of ALBIA-SRP54. Sera from anti-SRP patients were compared to that of healthy blood donors. These two groups are fully discriminated using a threshold of positivity of 60 A.U./mL (dotted line). Sera from patients with different inflammatory/autoimmune conditions including rheumatoid arthritis (RA),

+ systemic sclerosis (SS), systemic lupus erythematosus (SLE), anti-tRNA synthetase myositis or inclusion body myositis (IBM), as well as patients with polyclonal hyper- gamma globulinemia were also assayed.

Figure 5: Monitoring of anti-SRP titres in patients with necrotising myopathy.

(A) Evolution of plasmatic anti-SRP54 titres in a patient before (black squares) and after (open squares) plasma exchanges performed at days 49, 58 and 60.

(B) Evolution of serum anti-SRP54 titres (open squares, dotted line) and CK levels (black dots, plain line) in 8 patients under therapy (see Table 1 for treatment details).

Figure 6: Determination of the concentration of human recombinant SRP54 protein required for coating. Fluorescent microsphere immunoassay of two human sera (with high and low titres of anti-SRP54 antibodies) with different concentration of SRP54 protein used for coating. Uncoated beads were used as negative control.

Figure 7: ROC analysis of ALBIA-SRP54. Thirty-one anti-SRP⁺ patients are compared to 190 healthy blood donors. Sensitivity and (1 -specificity) values are given for two different thresholds of positivity of the test. Values obtained with the threshold of 35 A.U./mL (corresponding to the 99^th percentile of the control sera distribution) and with the threshold of 60 U.A./mL (yielding the best diagnostic value) are depicted by dots and specifically indicated. Inlay is a magnification of the curve around these values. Area under curve is 1. Figure 8: Distribution of anti-SRP54 IgG isotypes. Subclasses were determined by ALBIA-SRP54 using an isotype-specific secondary Ab. Results from 21 anti-SRP⁺ patients are indicated (numbers of patients are given within circles).

Figure 9: Monitoring of anti-SRP titres in comparison to total IgG concentrations in patients with necrotizing myopathy. Evolution of serum anti-SRP54 titres (black squares, plain line) and total IgG concentrations (black circles, dotted line) in 8 patients under therapy (see Table 1 for treatment details). Example: Anti-SRP auto-antibodies titers correlate with serum CK levels in patients with necrotizing myopathy

PATIENTS AND METHODS

Patients

This study called "Development of a new technique for the diagnosis and monitoring of anti-SRP antibodies" has been approved by the Ethical Review Committee of the Cochin Hospital, Paris. Thirty one patients from 2 French centres (Rouen University

Hospital and Paris Pitie-Salpetriere University Hospital) with anti-SRP antibodies were

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included in the study. Among the 31 anti-SRP patients, 8 were sampled over time for monitoring of anti-SRP titers and CK levels (at least 3 times consecutively). The date of the first sample was referred to as day 0 and this date also corresponded to the initiation of treatment (for naive patients) or restart of a modified one (for relapsing patients). Clinical characteristics, treatments and outcomes of these 8 patients are summarised in Table 1 below.

Table I Clinical characteristics and follow-up of 8 patients with anti-SRP aAbs

M: male, F: female, C: Caucasian origin, A: african origin, ICU: intensive care unit, LGMD: limb girdle muscular dystrophy, Pred: prednisone, Azat: azathioprine, RTX: rituximab, IVIg: intravenous immunoglobulins, MMF: myocophenolate mofetil, PE: plasma exchange, MTX: methotrexate.

AOF : arms outstretched forward (AOF). The arms outstretched forward (AOF) test measures the duration of time (number of seconds) during which the arms are upheld in this position (normal > 150 seconds) (Sharshar et al).

MRC psoas : muscle strength in the deltoid and psoas muscles is determined as a score of severity (0 = absence of any movement to 5 = normal strength) using the 6-point British Medical Research Council (MRC) manual muscle strength test. Controls

Control sera were collected from 190 healthy blood donors and from 140 patients with different inflammatory/autoimmune diseases, according to established classification criteria: ACR revised criteria for systemic lupus erythematosus (Tan et al., 1982) with anti-dsDNA aAbs (n=20), ARA criteria for rheumatoid arthritis (Arnett et al, 1988) with anti-CPP antibodies and/or rheumatoid factor (n=40), revised European criteria for primary Sjogren's syndrome (Vitali et al, 2002) with anti-SSA and/or anti-SSB aAbs (n=20), modified Bohan and Peter criteria for polymyositis (Bohan and Peter, 1975) with anti-tRN A- synthetase antibodies (anti-Jo- 1, n=27 ; anti-PL-7, n=l ; anti-PL- 12, n=2) and Griggs criteria for inclusion-body myositis (Griggs et al., 1995) (n=30). Another control consisted in sera from patients with polyclonal hypergammaglobulinemia (serum IgG: 23.1 ± 6.9 g/L, mean ± SD) (n=27). Serum samples were stored at -80°c until use.

Detection of auto-Abs

Indirect immunofluorescence was performed on HEp-2® cells (Reference SA2014-Ro, Immunoconcepts, Sacramento, CA, USA). Sera were tested at 1/80 screening dilution in PBS buffer, using a FITC-coupled antibody against human IgG. Samples exhibiting cytoplasmic fluorescence were further serially diluted, and those still reacting at 1/160 or over dilution were considered as positive. On these cells, the fluorescence pattern suggestive for anti-SRP is composed of an absence of nuclear staining associated to a homogeneous fluorescence of cytoplasm.

Myositis-associated aAbs were detected by Dot-blot immunoassay using BlueDOT Polymyositis / Scleroderma dot (PMS8D-24, D-Tek SA, Mons, Belgium), intended for the detection in human sera of IgG antibodies against Jol, PL-7, PL-12, Mi-2, Ku, Pm- Scl, Scl-70 and SRP-54 antigens.

Western blot analysis of recombinant SRP54 protein

Purity of the recombinant SRP54 protein was first determined by 4-10% gradient sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing conditions, followed by Coomassie Blue staining. Western blot analysis was further performed by transfer of proteins separated by non- reducing SDS-PAGE to a nitrocellulose membrane followed by incubation with anti- histidine Abs, anti-SRP54 Abs or a human anti-SRP54 positive serum. Goat polyclonal anti-histidine, chicken polyclonal anti-SRP54 and goat polyclonal anti-SRP54 Abs were from Sigma (Saint Louis, MO, USA), US Biological (Swampscott, MA, USA) and Santa Cruz Biotechnology (Santa Cruz, CA, USA), respectively. Biotinylated mouse anti-goat Abs, biotinylated goat anti-chicken IgY and biotinylated mouse anti-human IgG (clone JDC-10) were from Sigma, GeneTex (Irvine, CA, USA) and Southern Biotech (Birmingham, AL, USA), respectively. Reactivity was revealed using sreptavidin Alexa Fluor® 680 from Invitrogen (Cergy Pontoise, France) on a Li-Cor Odyssey system (Lincoln, Nebraska, USA).

Addressable Laser Bead Immunoassays for quantification of SRP54-specific antibodies (ALBIA-SRP54)

Full-length human recombinant SRP54 protein fused to a hexa-histidine tag was obtained from Diarect AG (Freiburg, Germany). LiquiChip™ Ni-NTA beads and streptavidin-R- phycoerythrin were from Qiagen (Hilden, Germany). 50 μΐ of recombinant SRP54 was coupled to 1.25x105 fluorescent LiquiChip™ Ni-NTA microspheres (total volume of 100 μΐ in PBS without calcium and magnesium, DPBS). After optimisation to maximise the signal to noise ratio, the concentration of SRP54 protein per coupling reaction was set at 10 μg/ml (Fig.6). After overnight incubation under agitation at 4°C in the dark, 900 μΐ of storage buffer (DPBS without calcium and magnesium and 0.05% sodium azide) were added and coated beads were either used immediately or stored at 4°C in the dark for future use. When indicated, hexa-histidine tagged human recombinant Jo-1 and Intrinsic Factor proteins (Diarect) were used similarly in place of SRP54.

Immediately prior to use, SRP54-coated beads were vigorously agitated for 30 s. Then, a 10 μΐ volume containing 1,250 beads was added to 150 μΐ of serum from patients or controls (diluted in DPBS plus 1% fetal bovine serum, FBS) in Multiscreen 96-well plates (Millipore, Breford, MA, USA). Plates were incubated for 2 h at room temperature in the dark on a plate shaker at 650 rpm. Blank (no serum, secondary antibody only), negative control (anti-SRP negative serum) and positive controls (highly positive human anti-SRP serum; goat anti-SRP Abs with appropriate secondary antibody) were included in every assay. Beads were collected by filtration under vacuum and washed twice with 150 μΐ DPBS containing 0, 1% Tween-20. Biotinylated mouse anti-human IgG Ab (or isotype-specific Ab) was added at 1/2,000 dilution and incubated for 1 h at room temperature under shaking. After washing, beads were incubated with 50 μΐ of streptavidin-R-phycoerythrin (Qiagen) at 1/1,000 dilution for 15 min. Finally, beads were resuspended in 100 μΐ of DPBS and Mean Fluorescence Intensity (MFI) was determined on a Bio-Plex apparatus using the Bio-Plex Manager Software 4.0 (Bio-Rad Laboratories, Hercules, CA, USA).

The analytical sensitivity of detection of ALBIA-SRP54 was determined by triplicate assay of serial dilutions of goat and chicken anti-SRP54 antibodies. For each well, after subtraction of negative control MFI value, mean MFI and standard deviation (SD) were calculated.

The analytical specificity of detection of ALBIA-SRP54 was determined by using a highly positive human anti-SRP54, anti-Jo-1 or anti-intrinsic factor serum at 1/1,000 dilution. Sera were incubated with purified SRP54, Jo-1 or intrinsic factor beads for 1 h at room temperature and MFI was determined as described above.

Specific inhibition of ALBIA-SRP54 was performed using growing concentrations of recombinant SRP54 protein, with 1/2,000 dilution of the anti-SRP54 serum used as

pre-adsorbed serum serum

100% control. Percent inhibition was calculated as [1 - (MFI / MFI )] x 100. Homologous and heterologous inhibition of ALBIA-SRP54 was further performed by pre-ab sorption of 4 anti-SRP, 4 anti-Jo-1 or 4 anti-intrinsic factor positive sera with 100 μg/ml of recombinant SRP54, Jo-1 or intrinsic factor protein.

All patients' sera were initially assayed at a 1/500 dilution. The anti-SRP titers were

serum calibrator

determined at a 1/D dilution using the following formula: [MFI / MFI ] x [titer of calibrator] x D/500. The calibrator is a highly positive human anti-SRP serum (the same throughout the study) whose titer was arbitrarily set to 100 Arbitrary Units (U.A. / mL). When sample MFI at 1/500 dilution was higher than 80% of calibrator MFI, further dilutions were performed and the first dilution yielding a MFI inferior to 80% of calibrator MFI was retained for calculation of titer.

Reproducibility of ALBIA-SRP54 was determined using three independent anti-SRP positive sera (low, medium and high titer). Intra-assay variation was determined by measurements of the same samples within the same run. Inter-assay variation (assay to assay) was determined by measurements of the same samples in separate runs. Inter- assay variation (batch to batch) was determined by measurement of the same samples in the same run using different batches of coated beads. Coefficients of variation were computed as SD/mean.

Statistical analysis

The normality of data distribution was analysed by the D'Agostino and Pearson test. The relationship between anti-SRP titers and CK levels was tested using a linear mixed model.

RESULTS

Development of a quantitative assay of anti-SRP54 aAbs (ALBIA-SRP54)

To allow the quantitative analysis of anti-SRP54 aAbs and their follow-up overtime in patients, we developed an addressable laser bead immunoassay (ALBIA). For this, we used a recombinant human SRP54 protein harbouring a hexa-histidine-tag in its C- terminal M domain (Fig. 1A). The purity of the SRP54 protein was confirmed by

Coomassie blue staining after SDS-PAGE which revealed a unique band of 60 kDa (Fig.

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IB) specifically recognised by chicken or goat anti-SRP54 Abs, and human anti-SRP serum (Fig. 1C).

The tagged recombinant SRP54 protein was coupled to fluorescent beads through interaction of histidines with the beads Nickel moiety, and further used to measure the levels of anti-SRP Abs in a Luminex™-based assay (Verkaik et al., 2008) which will be referred to as Addressable Laser Bead Immunoassay for quantification of SRP54- specific antibodies (ALBIA-SRP54). A series of parameter were optimised, including incubation times and buffers, washing steps, secondary antibodies (not shown) and the concentration of recombinant SRP54 protein for bead coating which was set to 10 μg/ml (Fig. 6). ALBIA-SRP54 could detect concentrations as low as 2 ng/mL chicken or 8 ng/mL goat anti-SRP54-specific Abs, indicating a good analytical sensitivity of the assay, and the degree of linearity covered at least 3 orders of magnitude (Fig. ID).

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Only anti-SRP54 but not irrelevant anti-Jo-1 or anti-intrinsic factor serum reacted with SRP54 beads (Fig. 2A). Similarly, beads coated with Jo-1 or intrinsic factor recombinant proteins that were produced and purified using the same process that SRP54 only revealed the corresponding aAb-positive serum (Fig. 2A). Further experiments showed that the free SRP54 protein could inhibit the binding of anti-SRP54 aAbs in a dose-dependent manner (Fig. 2B) while no inhibition was observed using an heterologous protein (Fig. 2C).

The method used for calculating the titer of anti-SRP54 aAbs is illustrated in Figure 3. The patient's serum used in this example displayed a characteristic immunofluorescence cytoplasmic pattern (data not shown) and was positive for anti-SRP54 dot blot (Fig.3 A). Using ALBIA-SRP54, this serum showed a saturating signal at the 1/500 screening dilution and a further 1/5,000 dilution was retained to perform calculation by reference to the calibrator used throughout the study whose titer was arbitrarily fixed to 100 A.U./mL (Fig. 3B).

The reproducibility of the test was next assessed by determining the level of intra- and inter-assay titer variations for sera with high, medium and low anti-SRP54 titers. The intra-assay coefficients of variation (%CV) were lower than 5% (see Table 2 below).

Table II Reproducibility ofALBIA-SRP54

Similarly, assay to assay and batch to batch variations ranged from 0.4 to 3.3%. Together, these data indicate excellent analytical sensitivity, specificity and reproducibility of ALBIA-SRP54.

Diagnostic value of ALBIA-SRP54

The diagnostic value of the test was determined by comparing serum titers from anti-

+

SRP patients to that of healthy blood donors. The values obtained with control sera did not follow a normal distribution (n=190, p<0.0001), their median was 2 A.U./mL and th

the 99 percentile was 35 A.U./mL. At the 35 A.U./mL threshold, all sera from anti-

+

SRP patients scored positive with titers ranging from 66 to 22,080 A.U./mL, whereas 189/190 (99.5%) control sera were negative (Fig. 4), yielding a specificity of the test of 99.5%) and a sensitivity of 100%. ROC analysis (Fig. 7) revealed that the best diagnostic value of ALBIA-SRP54 is obtained when the threshold of positivity is set at 60 A.U./mL. In this condition, all sera from patients score positive, all control sera are negative, the specificity of the test is 100%> with a confidence interval of [98%> - 100%>], the sensitivity is 100%) [89%> - 100%>] and the area under curve in ROC analysis is 1.

Patients with different inflammatory/autoimmune conditions including rheumatoid

+

arthritis, systemic sclerosis, lupus, anti-tRNA synthetase myositis or inclusion body myositis, as well as patients with polyclonal hyper-gamma globulinemia were all negative (Fig. 4). Hence, ALBIA-SRP54 is a test of high diagnostic value to detect and quantify anti-SRP aAbs.

IgG isotypes of anti-SRP Abs

The most frequent isotype of anti-SRP aAbs was IgGl, which was present in 78%> (18 out of 23) of sera analysed (Fig. 8). Anti-SRP IgGl were either present alone (9 out of 18, 50%)) or associated to one or two other isotypes. Presence of 2 or more IgG isotypes was found in 43% (10 out of 23) of the cases whereas the remaining 57% had a single subclass of anti-SRP54 IgG. IgG2 was never found as a single isotype but always in association.

Effect of plasma exchange on anti-SRP titers

+

Patient 6 presented an evolutive form of anti-SRP myopathy since she was not far to loose her walk (need of a walker) at time of diagnosis. We decided then to start (at day 0) a treatment associating prednisone (1 mg/kg/day), methotrexate (0.3 mg/kg/week) and intravenous immunoglobulins (2 g/kg/month). Because of the absence of obvious result on muscular strength, plasma exchanges were additionally performed on days 49, 58 and 60. An aliquot was sampled from the machine tubing in order to determine plasmatic anti-SRP titers at the beginning and end of each procedure (data not shown). As expected, anti-SRP titers always decreased following plasma exchange (ranging from 54 to 64%> aAb depletion), with a rebound observed on subsequent days since the titer augmented at the beginning of the next exchange, although not reaching the previous level. Hence, plasma exchange indeed diminishes the titers of anti-SRP54 aAbs and ALBIA-SRP54 may be useful for longitudinally monitoring the efficacy of this therapy.

Correlation between anti-SRP titers and CK levels

Eight patients were sampled over time for monitoring of anti-SRP titers and CK levels. All of them received a new or modified treatment on day 0, and were followed up from 165 to 1318 days thereafter (mean 722 days). With these treatments, 6 out the 8 patients presented a clear clinical improvement of their muscular strength (Table 1), which was manifested by a noticeable regression of their muscular deficit (patients 1, 3, and 5) or even normalization of muscle strength (patients 2, 4, and 8), whereas in patients 6 and 7, only a modest improvement was observed (Table 1). Nonetheless, the general feature is a decrease to normalisation of CK levels under therapy in all patients.

The most striking result of this study is the observation that the evolution of anti-SRP aAb titers closely correlates with that of CK levels. Indeed, titers dropped in parallel with CK decrease but also augmented in correlation with CK rises such as at day 1063 in patient 2 or at day 1301 for patient 5, for instance. Using a linear mixed model taking into account the correlations among measurements made on the same patient, anti-SRP titers appeared significantly associated with CK levels (p<0.005) while disease duration had no effect (p=0.26). Clinically, there was also a parallel amelioration of muscular strength in 6 out of the 8 patients (Table 1).

Correlation between anti-SRP titers and total IgG concentrations

As shown in Figure 9, anti-SRP aAb titers do not correlate with global IgG concentrations (see for example Patients 5, 6 and 8).

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Claims

1. Method for diagnosing an acquired myopathy in a mammal, characterized in that it comprises a step of measuring the quantity and/or the concentration of anti- SRP antibodies in a biological sample of said mammal.

2. Method according to claim 1, characterized in that it comprises a further step of comparing the quantity and/or the concentration of anti-SRP antibodies in the biological sample of said mammal with a reference value.

3. Method according to any one of claims 1 to 2, characterized in that the mammal is a human or a non human mammal, said non human mammal being preferably chosen from a rodent, a feline, an equidae, a canine and a primate.

4. Method according to any one of claims 1 to 3, characterized in that said biological sample is chosen from blood samples, plasma samples, saliva samples, exsudate samples and serum samples.

5. Method according to any one of claims 1 to 4, characterized in that the step of measuring the quantity and/or the concentration of anti-SRP antibodies is an immunoassay.

6. A method for monitoring and/or predicting the responsiveness to a treatment of a mammal suffering from an acquired myopathy, said method comprising measuring the quantity and/or the concentration of anti-SRP antibodies in the biological sample of said mammal.

7. The method according to claim 6 which further comprises a step of comparing the quantity and/or the concentration of anti-SRP antibodies with reference values obtained from responder and non-responder group of mammals.

8. The method according to any one of claims 1 to 7, in which the quantity or concentration of anti-SRP antibodies are determined by the following steps: a SRP element is labelled with fluorescent beads; then said biological sample is mixed with the labelled SRP element, and thereafter

labelled secondary antibodies targeting the anti-SRP antibodies are added;

wherein the measurement of the labelled secondary antibodies gives the quantity or concentration of SRP element/anti-SRP antibody complexes.

9. The method according to any one of the claims 6 to 8 for screening a drug candidate able to treat an acquired myopathy.

10. A method for assessing the effectiveness of a drug to treat an acquired myopathy in a mammal suffering from said acquired myopathy, comprising the following steps:

(A) measuring the quantity or concentration Dl of anti-SRP antibodies in a biological sample obtained from said mammal;

(B) after a period of therapy, measuring the quantity or concentration D2 of anti- SRP antibodies in a biological sample obtained from said mammal in the same manner as in step (A);

(C) comparing quantities Dl and D2, or concentrations Dl and D2, and

(D) if D2 <D1, deducing that the drug is effective in treating said acquired myopathy.

11. Method according to any one of claims 1 to 10, characterized in that the acquired myopathy is necrotizing myopathy.

12. Kit for measuring anti-SRP antibodies quantity or concentration, comprising:

at least one labelled SRP element; and

means for detecting anti-SRP antibodies.

13. Kit according to claim 12, characterized in that the means for detecting anti-SRP antibodies are secondary antibodies, preferably anti-IgG antibodies.