Substrate for IGG degrading enzymes

Abstract

The invention relates to the field of enzymology. In this study, the inventors described a simple quantitative assay that allows to precisely measure 1 / the activity of IgG degrading enzymes like IdeS, IdeS orthologs or IdeS mutants and 2 / the neutralizing activity of pre- existing or induced anti-IgG degrading enzymes antibodies towards IgG degrading enzymes in plasma, serum or purified polyclonal or monoclonal antibodies pools. Thus, the invention relates to an IgG-degrading enzyme substrate with different characteristics.

Description

[0001] SUBSTRATE FOR IGG DEGRADING ENZYMES

[0002] FIELD OF THE INVENTION:

[0003] The invention relates to an IgG-degrading enzyme substrate with different characteristics.

[0004] BACKGROUND OF THE INVENTION:

[0005] The IgG-degrading enzyme (IdeS) or Imlifidase is a cysteine protease produced by Streptococcus pyogenes (1, 2). Together with other proteases such as the IgG endoglycosidase Endo S and the cysteine proteinase SpeB, IdeS acts as a defense mechanism against the host humoral immune response. IdeS is a 339 amino-acid long protein with a molecular weight of 37 kDa. The active site of IdeS includes Lys 84, Cys 94, His 262 and Asp 284 (2). Mutation of the Cys 94 to an Ala residue inactivates the proteinase (3). IdeS exhibits exquisite specificity for human IgG (4). It also cleaves IgG from different species, including rabbit, pig and monkeys. It however does not cleave all mouse IgG subclasses, or human IgM, IgA or IgE (4). The cleavage of IgG by IdeS proceeds in two steps. IdeS first hydrolyses one of the two heavy chains between the hinge region and the CH2 domains between Gly / Ala 236 and Gly 237. It then cleaves the second heavy chain, thus dissociating the F(ab’)2 and the Fc fragment (5). The first cleavage is rapid and characterized by a kcat of 10.1 s-1 and a catalytic efficiency of 1 402 778 M-ls-1 (Km = 7.2 pM). The second cleavage is 100-fold slower: kcat = 0.1 s-1, Km = 28 pM, catalytic efficiency of 3571 M-ls-1 (5). Removal of the Fc fragment induces a drastic acceleration of the catabolism of the antigen-binding moiety of the IgG that is not recycled upon binding to the neonatal Fc receptor anymore. In addition, the absence of the Fc fragment precludes opsonization, complement-dependent cytotoxicity or antibody-dependent cell-mediated cytotoxicity when the F(ab’)2 binds to its cognate antigen. IdeS is also able to cleave the B-cell receptor at the surface of IgG+ memory B cells both in vitro (6) and in vivo (7).

[0006] The efficacy of IdeS in vivo in removing human IgG was first demonstrated in healthy individuals (ClinicalTrials.gov Identifier: NCTO 1802697), where complete IgG removal was achieved in 2-6 hours for an IdeS dose of 0.24 mg / kg body weight; IgG returned to pre-dosing levels within 14 days or more (8). IdeS demonstrated a circulating half-life of 4.9±2.8 hours. The therapeutic potential of IdeS was demonstrated in kidney transplant patients with donor specific antibodies. IdeS administration eliminated anti-donor HLA IgG and prevented acutehumoral rejection of the grafted organs (9). Now marketed as Idefirix and recommended for the deimmunization of hyperimmunized adult patients scheduled for renal transplant.

[0007] A three-year follow-up confirmed good long-term graft survival (10). IdeS was also tested with positive results in patients with Goodpasture syndrome (11, 12). The potential value of IdeS was also investigated in a series of preclinical models of human diseases including rheumatoid arthritis (13), immune thrombocytopenic purpura (14), neuromyelitis optica (15), heparin-induced thrombocytopenia (16), as well as in the context of gene therapy in the presence of anti -AAV capsid antibodies (17).

[0008] The use of IdeS is however confronted by two major hindrances: pre-existing immunity and immunogenicity. The seminal work by Akesson et al documented the presence of anti-IdeS IgG in the serum from patients with pharyngotonsillitis, bacteremia and erysipelas (18). The presence of pre-existing anti -IdeS IgG was confirmed in cohorts of healthy individuals (8) and in patients with chronic kidney disease (7). In both situations, and as seen with patients undergoing bacterial infection (18), the levels of anti -IdeS IgG increased following administration of IdeS to the individuals. In healthy individuals and kidney disease patients, the peak levels were reached about two weeks after dosing, which is indicative of memory rather than naive immune responses. It is thus probable that pre-dosing anti-IdeS IgG result from past streptococcal infections. Of note, Lonze et al reported the testing of kidney recipients for the presence of anti -IdeS IgE antibodies (19), however the presence of such antibodies and their prevalence in the studied population are not described.

[0009] The neutralizing activity of anti-IdeS antibodies towards IdeS has been poorly investigated. Akesson et al detected IdeS neutralizing antibodies in the serum from patients with bacterial infections (18). In one patient, IdeS-neutralizing activity was contained in IgG fraction of the serum. In contrast, Johansson et al (14) found that anti-IdeS antibodies in serum from 19 healthy donors do not inactivate the IgG-cleaving activity of IdeS when used at a concentration that completely cleaves IgG in human blood in vitro (i.e., 20 pg IdeS / ml when indicated). The discrepancy in the results may be due to the type of assay used and to the concentration of IdeS used in vitro, and particularly the ratio of IdeS versus serum or plasma IgG used in the different experiments. Besides, IdeS-mediated IgG degradation was followed by SDS-PAGE which is not the most sensitive technique.

[0010] Taken together, these observations raise the question of the biological relevance of anti-IdeS antibodies with respect the efficiency of therapeutic IdeS. Indeed, it remains unclear whether the levels of pre-existing anti-IdeS IgG in patients may reduce or block the therapeutic efficacy of IdeS. More critically, the development of a strong and rapid anti-IdeS memory IgGresponse may preclude redosing of the patients with IdeS (17). The existence of a threshold in anti-IdeS IgG titers above which therapeutic IdeS loses efficacy has to our knowledge not been investigated.

[0011] SUMMARY OF THE INVENTION:

[0012] In this study, the inventors described a simple quantitative assay that allows to precisely measure 1 / the activity of IgG degrading enzymes like IdeS, IdeS orthologs or IdeS mutants and 2 / the neutralizing activity of pre-existing or induced anti-IgG degrading enzymes antibodies towards IgG degrading enzymes in plasma, serum or purified polyclonal or monoclonal antibodies pools. They describe a novel synthetic FRET substrate that is specifically cleaved by IgG degrading enzymes like IdeS. Using a cohort of 7 healthy donors and 6 patients with acquired hemophilia, they confirm the presence of anti-IdeS IgG in the plasma of all tested individuals, and validate a neutralization assay that allows to determine the neutralizing potential of anti-IdeS antbodies in plasma. The neutralization assay shall be applicable using all biological samples, and will allow to determine the threshold of circulating levels of anti-IgG degrading enzymes antibodies (like anti-IdeS antibodies) above which IgG degrading enzymes lose their proteolytic activity.

[0013] Thus, the present invention relates to an IgG-degrading enzyme substrate with different characteristics.

[0014] Particularly, the invention is described by its claims.

[0015] DETAILED DESCRIPTION OF THE INVENTION:

[0016] The substrate of the invention

[0017] A first aspect of the invention relates to an IgG-degrading enzyme substrate comprising or consisting of two different monomers fused to a detecting agent, wherein

[0018] a. each monomer consists of the hinge-CH2-CH3 proteins of an immunoglobulin; b. each monomer is fused to one detecting agent at its N-Terminal or C-terminal part;

[0019] c. the CH3 domain of the monomer comprises at least one mutation; and d. each monomer is link to one detecting agent thanks to a linker and the hinge region or fragment thereof wherein the hinge region comprises the peptidic sequence GPSVF (SEQ ID NO: 1).As used herein, the term IgG-degrading enzyme denotes an IgG protease which is a cysteine protease, a thiol protease or a serine protease. Particularly, the protease may be of the endo-type that hydrolyzes peptide bonds internally in polypeptide chains (endoprotease or endopeptidase). Examples of endopeptidases include, for example, IdeS, IdeZ, IgdE, IdeMC, trypsin, chymotrypsin, papain and pepsin. Examples of proteases that may be used in the invention include, for example and without limitation, cysteine proteases from Streptococcus pyogenes, Streptococcus equi, Mycoplasma canis, S. agalactiae, S. pseudoporcinus or Pseudomonas putida. More particularly, the protease is the IdeS from Streptococcus pyogenes.

[0020] In preferred embodiment, the IgG-degrading enzyme is IdeS, and more particularly IdeS from Streptococcus pyogenes.

[0021] As used herein the terms “CH2” or “CH3” correspond to the two constant domains of the Fc part of an antibody.

[0022] As used herein, the term “hinge region” denotes a stretch of heavy chains between the Fab and Fc portions of an antibody. Its unique structure and position provide segmental flexibility, which is essential for normal functioning of antibodies.

[0023] In one embodiment, the hinge region of the IgG-degrading enzyme substrate of the invention is a hinge cleaved by an IgG-degrading enzyme like an IgG protease like the endopeptidase IdeS.

[0024] In one embodiment, the hinge region of the IgG-degrading enzyme substrate of the invention is a hinge of an antibody of any species and particularly from human, monkey, sheep, rabbit or bat and which is cleaved by an IgG-degrading enzyme like the endopeptidase IdeS.

[0025] In a particular embodiment, the hinge region is the hinge region of human IgG.

[0026] In another particular embodiment, the hinge region is a hinge region from a human IgGl, IgG2, IgG3 or IgG4 antibody.

[0027] In another particular embodiment, the hinge region comprises the peptidic sequence as set for SEQ ID NO: 1 : GPSVF.

[0028] In another particular embodiment, the sequence as set for SEQ ID NO: 2: APELLG is inserted between the linker and the sequence of SEQ ID NO: 1.

[0029] In another particular embodiment, the hinge region has the sequence as set for SEQ ID NO: 3 : APELLGGPSVF. Particularly, the IgG-degrading enzyme will cleave the hinge before the G of the sequence GPSVF (SEQ ID NO: 1).In another particular embodiment, the hinge region has the sequence as set for SEQ ID NO: 4 : PAPEFLGGPSVF, SEQ ID NO:5: PPPELLGGPSVF or SEQ ID NO:6 PAPPVAGGPSVF.

[0030] In one embodiment, the linker is a peptide.

[0031] In a particular embodiment, the linker of the IgG-degrading enzyme substrate of the invention has the following sequence ((G)nS)x wherein n is between 1 and 10 and x between 1 and 5.

[0032] In a particular embodiment, the linker has the sequence as set for in SEQ ID NO: 7 :GGGS.

[0033] In one embodiment, the linker of the IgG protease substrate of the invention has the following sequence ((G)nR)x wherein n is between 1 and 10 and x between 1 and 5.

[0034] In a particular embodiment, the linker has the sequence as set for in SEQ ID NO: 8: GGGR.

[0035] A combination of peptidic sequences SEQ ID NO: 7 and 8 can also be used to generate the linker of the invention. For example the linker can have the sequence as set for in SEQ ID NO: 9: GGGSGGGSGGGSGGGR.

[0036] As used herein, the term “detecting agent” refers to any molecule, molecular complex, particle, or system capable of directly or indirectly producing, modulating, or enabling generation of a detectable signal in a proximity-dependent signaling assay ((such as Forster Resonance Energy Transfer (FRET) assay for example), wherein the signal is dependent upon the spatial proximity of the detecting agent to one or more other assay components. As used herein, a detecting agent includes, but is not limited to, fluorophores, chromophores, luminophores, enzymes, enzyme substrates, enzyme cofactors, quenchers, nanoparticles, energy donors, energy acceptors, redox-active moieties, signal amplification elements, and combinations thereof, which are configured such that detectable signal generation, transfer, enhancement, suppression, or modulation occurs when the detecting agent is brought into spatial proximity with a corresponding interaction partner.

[0037] In particular embodiment, the detecting agent comprises at least a donor fluorophore and an acceptor fluorophore positioned such that energy transfer occurs when the construct is intact, and the detecting agent is configured to produce a detectable change in fluorescence emission upon cleavage, modification, or conformational change induced by the activity of a target molecule or enzyme, thereby enabling detection and / or quantification of said activity.In one embodiment, the detecting agent of the IgG-degrading enzyme substrate is a fluorochrome or any means using a fluorochrome. In a particular embodiment, the technology ADAP STAR (using complementary oligomers or fluorescent molecules) can be used (see for example https: / / www.hamiltoncompany.com / automated-liquid-handling / assay-ready-workstations / adap-star).

[0038] In one embodiment, the mutations present on each monomer of the IgG-degrading enzyme substrate of the invention are described for example in the article Gunasekaran K. et al. the journal of biological chemistry, 2010.

[0039] For example, the mutations of the inventions can be selected in the group consisting in but not limited to E356K, D399K, K409D, K392D, K409E, K370D, D357K, T366W, T366S, L368A or Y407V.

[0040] In another embodiment, the mutations present in the Fc part of the IgG-degrading enzyme-Fc fusion protein are described for example in the articles Ridgway JB et al, ProtEngin 1996, Macor P et al, Leukemia 2015, Moore G et al, Mabs 2011 and Merchant AM et al, PNAS 1998.

[0041] For examples the mutations of the inventions can be selected in the group consisting in but not limited to T366Y, Y349T, T366S, T366W, S354C, Y349C, L368A, T394F, S364H, T394W, F405A, Y407T, Y407V, T366Y:F405A, T394W:Y407T Y349C:T366S:L368A:Y407V and T366W:S354C.

[0042] According to the invention, each monomer will comprise at least one mutation different from the other monomer.

[0043] In a particular embodiment, the mutations will be present on the CH3 domain of the monomer.

[0044] In a particular embodiment, one CH3 domain comprises the E356K and D399K mutations and the other CH3 domain comprises the K392D and K409D mutations.

[0045] In a particular embodiment, at least one CH3 domain comprises a Tag. In a particular embodiment the Tag is an HQ tag, HN tag, HAT tag or and his Tag.

[0046] According to the invention, the tag is useful to purify the substrate of the invention.

[0047] Uses and methods related to the substrate of the invention

[0048] A second aspect of the invention relates to a method for detecting the activity of an IgG-degrading enzyme comprising the following steps:a. Incubating the IgG-degrading enzyme substrate of the invention with a sample likely to contain an IgG protease for at least 10 seconds at 37°C in the dark; and b. Detecting the emission fluorescence emitted by the two fluorochromes wherein the ratio of two fluorescence is reflecting the IgG-degrading enzyme activity. In a particular embodiment the invention relates to a method for detecting the activity of an IgG-degrading enzyme comprising the following steps:

[0049] a. Incubating the IgG-degrading enzyme substrate of the invention with an IgG protease for at least 10 seconds at 37°C in the dark; and

[0050] b. Detecting the emission fluorescence emitted by the two fluorochromes wherein the ratio of two fluorescence is reflecting the IgG protease activity.

[0051] Particularly, the concentration of the substrate is between 0.05 and 10 pM. Particularly, the concentration of the substrate is 1, 2, 3, 4, 5, 6, 7, 8 or 9 pM. Particularly, the concentration of the substrate is 0.0625 pM.

[0052] Particularly, the IgG protease substrate will be incubated during 10 seconds to 48 hours. Particularly, the IgG protease substrate will be incubated between 10 minutes, 15 minutes, 20 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 18 hours, 24 hours, 36 hours or 48 hours.

[0053] According to the invention, the method for detecting the activity of an IgG-degrading enzyme can be used for:

[0054] - screening IgG-degrading enzyme mutants with preserved catalytic activity;

[0055] - selecting IgG-degrading enzyme-fused molecules with preserved specific / catalytic activity;

[0056] - screening for IdeS orthologs from other bacterial strains or IdeS mutants generated by combinatorial libraries.

[0057] A third aspect of the invention relates to a method for detecting the presence or not of neutralising anti-IgG-degrading enzyme antibody in a sample, comprising the following steps in the dark:

[0058] a. Incubating a sample in the presence of an IgG-degrading enzyme for at least 30 minutes;

[0059] b. adding of the IgG-degrading enzyme substrate of the invention for at least one hour;c. blocking the IgG-degrading enzyme catalytic activity by addition of iodoacetamide for at least 30 min;

[0060] d. detecting the emission fluorescence emitted by the two fluorochromes wherein the ratio of two peaks of fluorescence is indicative of the residual IgG- degrading enzyme activity present in the samples, and thus of the presence of neutralizing anti-IgG protease antibodies in the sample.

[0061] In particular embodiment, the sample can be incubate with the IgG-degrading enzyme fore at least 1, 2, 3, 4, 5, 6 , 7, 8 , 9 or 10 hours.

[0062] In a particular embodiment, the step a. can be preceded by the purification of the IgG from the sample, using the Melon technology.

[0063] In a particular embodiment, the sample according to the invention is blood, serum, plasma, supernatant or other biological source.

[0064] Particularly the concentration of the IgG-degrading enzyme is between 0 and 20 nM. Particularly, the concentration of the IgG-degrading enzyme substrate is 1 pM.

[0065] Particularly, the concentration of the iodoacetamide is between 100 nM and 1 pM. Particularly, the IgG-degrading enzyme or IgG-degrading enzyme substrate or the iodoacetamide will be incubated during 1 to 4 hours.

[0066] According to the invention, the method for detecting the presence or not of anti-IgG-degrading enzyme antibody can be used for:

[0067] - prognostic value: this method can be used to determine levels of anti-IgG-degrading enzyme neutralizing antibodies prior to IgG-degrading enzyme treatment like kidney transplant, gene therapy, post-operative treatment, acquired haemophilia, alloimmune diseases or autoimmune diseases.

[0068] According to the invention, autoimmune diseases are selected from Addison’s disease, alopecia areata, ankylosing spondilitis, antiphospholipid syndrome, aplastic anaemia, autoimmune gastritis, autoimmune hearing loss, autoimmune haemolytic anaemias, autoimmune hepatitis, autoimmune hypoparathyroidism, autoimmune hypophysitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome, autoimmune myocarditis, autoimmune oophoritis, autoimmune orchitis, autoimmune polyendocrinopathy, Beghet’s disease, bullous pemphigoid, cardiomyopathy, chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, coeliac disease, Crohn’s disease, CREST syndrome, Degos disease, epidermolysis bullosa acquisita, essential mixed cryoglobulinaemia, giant cells arteritis, glomerulonephritis, Goodpasture’s syndrome, Graves’ disease, Guillan-Barresyndrome, Hashimoto’s thyroiditis, idiopathic thrombocytopenic purpura, inflammatory bowel disease, Kawasaki’s disease, Meniere’s syndrome, mixed connective tissue disease, Mooren’s ulcer, multiple sclerosis, myasthenia gravis, pemphigus foliaceous, pemphigus vulgaris, pernicious anaemia, polyarteritis nodosa, polyglandular autoimmune syndrome type 1 (PAS-1), polyglandular autoimmune syndrome type 2 (PAS-2), polyglandular autoimmune syndrome type 3 (PAS-3), polymyositis / dermatomyositis, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud’s syndrome, Reiter’s syndrome, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren’s syndrome, subacute thyroiditis, sympathetic opthalmia, systemic lupus erythematosus, Takayasu’s arteritis, type 1 diabetes mellitus, vitiligo, Vogt-Koyanagi -Harada disease or Wegener’s granulomatosis.

[0069] More particularly, the IgG protease of the methods of the invention is the IdeS from Streptococcus pyogenes.

[0070] 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.

[0071] FIGURES:

[0072] Figure 1: A. Schematic representation of the structure of the VIC2R FRET substrate. The VIC2R FRET substrate is a heterodimer. One of the monomers includes the basic cyan fluorescent protein ECFP, derived from Aequorea victoria, fused to a single GGGS (SEQ ID NO: 7) linker, to the hinge region, APELLGGPSVF (SEQ ID NO: 3), CH2 and CH3 of the human IgGl and to a 6-His Tag. The second monomer includes the basic (yellow fluorescent protein Venus, derived from Aequorea victoria, fused to a single GGGS (SEQ ID NO: 7) linker, and to the hinge region, APELLGGPSVF (SEQ ID NO: 3), CH2 and CH3 of the human IgGl. The E356K / D399K and K392D / K409D mutations were introduced in the CH3 domains of the eCFP-Fc and Venus-Fc monomers to foster electrostatic steering and assembly of the heterodimer over that of the individual homodimers upon expression in eukaryotic cells.

[0073] B. Size-exclusion chromatography of purified the VIC2R FRET substrate. The VIC2R FRET substrate was produced using the Expi293 technology (ThermoFisher) and purified from supernatant by cobalt affinity chromatography and size-exclusion chromatography. The graph depicts the elution profile of the substrate size-exclusion chromatography (1 ml / min) with a single peak at 170 ml, detected by absorbance at 280 nm (plain line curve), 434 nm (dotted linecurve) and 515 nm (broken line curve). C. Purity of the VIC2R FRET substrate and individual monomers. The VIC2RFRET substrate and the individual eCFP-Fc and Venus-Fc monomers were produced using the Expi293 technology. After purification by affinity and sizeexclusion chromatography, the proteins (5 pg) were separated by SDS-PAGE in NuPAGE 4-12% gradient Bis-Tris protein gels (Thermo Scientific) under non-reducing conditions, and revealed using Coomassie blue staining. The molecular weight standard is shown on the lane of the left of each gel. D. Fluorescence profiles of the VIC2R FRET substrate and individual monomers. The fluorescence emission profiles of the purified VIC2R FRET substrate and individual monomers were recorded after excitation at 434 nm (eCFP-Fc monomer and VIC2R substrate) or at 480 nm (Venus-Fc).

[0074] Figure 2: A. End-point cleavage of the VIC2R FRET substrate by IdeS. The VIC2R FRET substrate (2 pM) was incubated with IdeS (0.16 pM) in PBS at a E:S 1:12 molar ratio for 24 hours at 37°C. Proteins (5 pg) were separated by SDS-PAGE in NuPAGE 4-12% gradient Bis-Tris protein gels under non-reducing conditions. Proteins were revealed using Coomassie blue staining. B. Dose-dependent cleavage of the VIC2R FRET substrate by IdeS. The VIC2R FRET substrate (2 pM) was incubated alone or with increasing concentrations of IdeS (0-2 pM) in PBS For 45 minutes at 37°C. Proteins were then separated by SDS-PAGE. C. Time-dependent cleavage of the VIC2R FRET substrate by IdeS. The VIC2R FRET substrate (2 pM) was incubated alone or with IdeS (0.16 pM) in PBS for 5, 10, 20, 40, 60, 80 minutes, or 6 or 24 hours at 37°C. Proteins were separated by SDS-PAGE. D. End-point cleavage of the VIC2R FRET substrate by IdeS. The VIC2R FRET substrate (2 pM) was incubated alone (empty circles) or with 0.16 pM IdeS (full circles) in PBS for 24 hours at 37°C. The fluorescence emission profiles were measured following excitation at 434 nm.

[0075] Figure 3: A. Determination of the kinetic parameters of IdeS-mediated VIC2R cleavage. The VIC2R substrate (5 to 0.0625 pM) was incubated with 1 nM IdeS for up to 400 sec with a reading of the fluorescence at 530 and 484 nm and excitation at 434 nm every 20 seconds. The concentration of hydrolyzed substrate was calculated for each time point and each substrate concentration, and was plotted as a function of the total substrate concentration. Representative of two independent experiments. B. Michaelis-Menten curve of IdeS-mediated VIC2R cleavage. The initial hydrolysis rates V0 (pM / s) were calculated for each substrate concentration (0.0625-15 pM), using the linear phase of the curves depicting the concentration of hydrolyzed substrate as a function of time. The graph depicts the V0 (pM / s) plotted as a function of the total substrate concentration. Experimental data were fitted to theMichaelis-Menten equation to derive the kinetic parameters. Data are pooled from two independent experiments.

[0076] Figure 4: Neutralization of IdeS activity by IVIg. IVIg (0, 2.5, 5, 10 mg / ml) was incubated alone or with IdeS (8, 16, 20 and 24 nM) for 1 hour at 37°C prior to addition of a synthetic FRET substrate for an additional hour (see Fig 2). Following excitation at 434 nm, the ratios of fluorescence emission measured at 530 and 484 nm were used to determine the % of IdeS neutralization as compared to the conditions where IdeS and the substrate were incubated alone (0%) or where the substrate was incubated without IdeS (100%).

[0077] Figure 5: A. Anti-IdeS IgG in the plasma from 7 healthy individuals. Plasma from 7 healthy individuals was incubated in serial dilutions on plates coated with an inactive IdeS mutant wherein Cysteine 94 is mutated into a Serine. Bound anti-IdeS IgG were detected using a polyclonal anti -human Fey IgG coupled to peroxydase. An engineered humanized monoclonal anti-IdeS IgG was used as a standard. B. Anti-IdeS IgG in the plasma from 6 patients with acquired hemophilia. Retrospective plasma samples from 6 patients with acquired hemophilia were collected and incubated in serial dilutions on plates coated with IdeSC94S. Bound anti-IdeS IgG were detected using a polyclonal anti -human Fey IgG coupled to peroxydase. An engineered humanized monoclonal anti-IdeS IgG was used as a standard.

[0078] Figure 6: A. Schema of IdeS neutralization assay. IdeS is incubated in PBS-0.025% tween 20 alone, with a control human monoclonal or with diluted plasma or serum in a final volume of 50 pl for 1 hr at 37°C. The samples are then mixed with 200 pl of VIC2R FRET substrate for an additional hour at 37°C. lodoacetamide is added to block the enzyme. The fluorescence is measured at 530 and 484 nm after excitation at 434 nm. B. Neutralizing IdeS activity in plasma from healthy individuals. Plasma from 7 healthy individuals was diluted 1 / 2, 1 / 3 and 1 / 9 in PBS-0.025% tween 20 and incubated with IdeS (4 nM). The samples were then mixed with the substrate. The ratios of fluorescence measured at 530 and 484 nm were used to determine the residual percentage of IdeS activity as compared to the conditions where IdeS was incubated alone (100%) or when the substrate was incubated without IdeS (0%). As a control, IdeS was incubated with the irrelevant human monoclonal IgG Trastuzumab prior to the addition of the substrate. C. Dose-dependency of IdeS neutralizing activity in plasma from 3 individual healthy individuals. Plasma from 3 healthy individuals was diluted 1 / 2, 1 / 3 and 1 / 9 in PBS-0.025% tween 20 and incubated with different concentration of IdeS (4, 16, 64 nM). The ratios of fluorescence measured at 530 and 484 nm were used to determine the residual percentage of IdeS activity as compared to the conditions where IdeS was incubated alone (100%) or when the substrate was incubated without IdeS (0%). D. Neutralizing IdeSactivity in plasma from patients with acquired hemophilia. Plasma from 6 patients with acquired hemophilia was then diluted 1 / 10 to 1 / 90 folds in PBS-0.025% tween 20 and incubated with IdeS (4 nM). The ratios of fluorescence measured at 530 and 484 nm were used to determine the residual percentage of IdeS activity as compared to the conditions where IdeS was incubated alone (100%) or when the substrate was incubated without IdeS (0%).

[0079] Figure 7. Blood from healthy donors contains IdeS-neutralizing IgG. A-B. Gender distribution of IdeS neutralizing activity. A. Correlation between the levels of anti-IdeS IgG and IdeS neutralizing activity. The correlation between the two sets of data was evaluated by two-tailed non-parametric Spearman correlation. The dotted line depicts the LOQ of anti-IdeS IgG (2.34 pg / ml). The dashed line represents the lowest anti-IdeS IgG concentration (9.95 pg / ml) yielding >70% IdeS neutralizing activity. B. Correlation between the levels of anti-IdeS IgA and IdeS neutralizing activity. The dotted line depicts the LOQ of anti-IdeS IgA (0.023 In review 23 pg / ml). The dashed line represents the lowest anti-IdeS IgA concentration (0.05 pg / ml) yielding >70% IdeS neutralizing activity. C. IdeS neutralization depends on IgG. IgG was purified from the plasma and serum from 4 donors with detectable IdeS neutralizing activity. Plasma / serum (diluted 1 / 10), purified IgG (1 mg / ml), and the IgG-depleted fractions (flow through, diluted 1 / 10) were analyzed for their ability to neutralize IdeS. Samples were pre-incubated for 1 hour with 8 nM IdeS, prior to addition of the FRET substrate for an additional hour. The ratios of fluorescence measured at 530 and 484 nm after excitation at 434 nm, were used to determine the percentage of IdeS neutralization, as described previously. The inset depicts IdeS (8 nM) neutralization as a function of the concentration of IgG (0.4-5 mg / ml)) purified from the plasma of donor 7.

[0080] Figure 8: Anti-IdeS IgG in IdeS-treated Cynomolgus macaques. Two to 3-year-old Cynomolgus macaques were injected intravenously with 500 pg / kg IdeS. Blood samples were collected 85 or 92 days later (full circles), or 112 or 126 days later (empty square and empty circle). Anti-IdeS IgG (left panel) and IdeS neutralizing antibodies (right panel) were detected by ELISA and using the FRET substrate, respectively.

[0081] Figure 9: Induction of anti-IdeS IgG in naive animals. (A) IgG anti-IdeS induced in naive mice by intravenous injection of IdeS at a therapeutic dose (0.25 mg / kg) (B) Detection of IdeS neutralizing antibodies. Serum from 17 IdeS-treated mice and from 5 PBS-treated mice was diluted 1 :2.5 in PBS-3% BSA and incubated with 4 nM IdeS for 1 hour at 37 °C. Samples were subsequently mixed with a synthetic FRET substrate for an additional hour. The ratio of the fluorescence emitted at 530 nm versus that emitted at 484 nm was used to determine the residual IdeS activity. The values were then compared to controls where IdeS and the substratewere incubated alone (100% activity) or where the substrate was incubated without IdeS (0% activity).

[0082] EXAMPLE:

[0083] Material & Methods

[0084] Cloning, expression and purification of the VIC2R FRET substrate.

[0085] The coding sequences for eCFP and Venus were obtained from Addgene (#105293 and #39813, respectively). The coding sequence for the hinge-CH2-CH3 of human IgGl was obtained from (20). The gene encoding eCFP was fused with that encoding the Fc portion of human IgGl from residue A231, containing two mutations at E356K and D399K. The gene encoding Venus was fused with that encoding the Fc portion of human IgGl from residue A231, containing two mutations at K392D and K409D. The introduced mutations favor the heterodimerization of the eCFP-Fc / Venus-Fc construct by virtue of electrostatic steering. A GGGS (SEQ ID NO: 7) linker was added between each fluorescent protein and the Fc fragment. A 6-his tag was added at the C-terminus of the eCFP-Fc monomer. The two cDNA were cloned independently in the pALL eukaryote expression vector under the control of the CMV promoter and using the murine IgGl signal peptide (murine IgGl signal peptide, GenBank Accession Number DQ407610). The VIC2R FRET substrate was expressed following co-transfection of HEK293 cells using the Expi293™ Expression System (Thermofisher). Occasionally, cells were transfected with each independent vector alone. After 6 days of production, the supernatant was harvested. The Venus expressed alone were purified on protein A-sepharose. To this end, the supernatant was mixed with binding buffer (20 mM Na2HPO4, 150 mM NaCl pH 7). The recombinant protein was eluted with 0.1 M citric acid pH 3, neutralized with 1 M Tris pH 9. The eCFP-Fc / Venus-Fc VIC2R substrate and eCFP-Fc expressed alone were purified by immobilized metal ion chromatography. The supernatants were mixed with binding buffer (20 mM Na2HPO4, 150 mM NaCl, 20 mM imidazole pH 7) before purification and the recombinant proteins were eluted with elution buffer (20 mM Na2HPO4, 150 mM NaCl, 500 mM imidazole pH 7). All proteins were finally purified by size exclusion chromatography using Superdex 200 column against PBS to obtain only the heterodimer forms of the substrate or the monomeric form of the controls. The absorbance of the purified proteins was measured using a UV-Vis spectrophotometer (Agilent Technologies).

[0086] Digestion of the VIC2R FRET substrate by IdeS.

[0087] For end-point digestion experiments, IdeS (0.16 pM) was added to 2 pM of the VIC2R substrate in PBS. The reaction was incubated for 24 hours at 37°C in 100 pL: 20 pL were keptfor separation by SDS-PAGE 4-12% Bis-tris; 50 pL were diluted in 200 pL PBS to reach a final substrate concentration of 500 nM and were transferred to 96-well black polystyrene plates. The cleavage was monitored with a TEC AN Infinite M200 Pro. Emission spectra were recorded after excitation at 434 nm. For dose-dependent digestion experiments, IdeS (0-2 pM) was incubated with 2 pM of the VIC2R substrate in PBS at 37°C for 45 min. For timedependent digestion assays, IdeS (0.16 pM) and the VIC2R substrate (2 pM) were incubated in PBS at 37°C. Aliquots were recovered after 0, 5, 10, 20, 40 and 80 minutes and for 6 and 24 hours. Samples were then analyzed by SDS-PAGE 4-12% Bis-tris.

[0088] Determination of the kinetic parameters governing the digestion of VIC2R FRET substrate by IdeS.

[0089] The fluorescence ratio F530 nm / F484 nm was calculated for the undigested VIC2R substrate (R0) and for the VIC2R substrate incubated with 1 nM IdeS 24 hours at 37°C (i.e., fully digested substrate, R24) following excitation at 434 nm.

[0090] The VIC2R substrate (15 to 0.0625 pM) was then incubated with 1 nM IdeS for up to 580 sec with a reading of the fluorescence at 530 and 484 nm every 20 seconds. At each time point and for each substrate concentration, the fluorescence ratios F530 nm / F484 nm (R) were calculated. The concentrations of digested substrate were finally calculated using the ratiometric FRET analysis method as described in Liu et al (21): C=(R-R0) / (R24-R0)xCi, where Ci is the initial VIC2R substrate concentration. The initial velocity V0 (pM / sec) was computed during the linear phase by using the fluorescence ratio R measured at 380 seconds. The Km and Vmax were calculated following plotting of the V0 as a function of the substrate concentration, and fitting the experimental data to the Michaelis-Menten equation (Prism, version 9.4.1).

[0091] Plasma from patients with acquired hemophilia and healthy individuals.

[0092] Plasma from 6 patients with acquired hemophilia and 7 healthy donors was obtained from the SACHA (Surveillance des Auto antiCorps au cours de 1’Hemophilie Acquise) French registry. Plasma from patients had been collected at the time of inclusion with titers >1 Bethesda units (BU) / mL.(22) Procedures were in accordance with the ethical standards of the responsible committees on human experimentation and with the Declaration of Helsinki. SACHA is registered (NCT00213473) at www.clinicaltrials.gov. (22, 23).

[0093] Treatment of mice with IdeS.

[0094] Sixteen to 31 week-old male and female C57BL / 6 mice were injected intravenously once a week for 5 weeks. with lOOpl endotoxin-free (<0.3 lU / ml) IdeS (0.6 mg / kg). Blood was obtained before each IdeS injection and 4 days after the last IdeS dosing. Serum was preparedand stored at -20°C until use. Mice were housed and handled in agreement with French and European law (APAFIS protocol n°2022040513338571).

[0095] Anti-IdeS IgG ELISA.

[0096] ELISA plates (Maxisorp, Nunc) were coated with an inactive IdeS variant wherein the Cys94 is mutated to a Ser (IdeSC94S) (2 pg / ml in PBS) overnight at 4°C. After blocking using PBS-3% bovine serum albumin (BSA) for 1 hour at 37°C, plasma / serum was incubated in dilutions for an additional hour at 37°C. Bound IgG were detected using a polyclonal anti human Fey antibody (Southern Biotech, USA) conjugated to peroxydase in the case of human samples. The bound detection antibodies were revealed using the peroxydase OPD substrate (Sigma-Aldrich). Optical density was read at 492 nm.

[0097] IdeS neutralization assay.

[0098] IdeS (4 nM or as indicated) was incubated in 50 pl PBS-0.025% tween 20 alone, or with the irrelevant human monoclonal Trastuzumab IgGl (1 mg / ml) used as a negative control, or with dilutions of human plasma, for 1 hr at 37°C. The mixture was then added to 200 pl of 500 nM in PBS and incubated 1 hr at 37°C in the dark. The reaction was stopped by addition of 10.4 pl 500 nM iodoacetamide and an additional hour of incubation at room temperature in the dark. The emission of the substrate was read using a TEC AN Infinite M200 Pro at 484 nm and 530 nm after excitation at 434 nm. The “residual IdeS activity” was calculated as the ratio of measured fluorescence ratio F530 nm / F484 nm for each condition over the fluorescence ratio measured in the absence of IdeS multiplied by 100. The percentage of IdeS neutralization was calculated as 100-“ residual IdeS activity”.

[0099] Digestion B02C11 and IVIg by IdeS

[0100] For in vitro cleavage, IVIg and BO2C11 (2000 nM each) were incubated in PBS alone or with IdeS (1-8 nM) for 6 hours at 37°C, at pH 7.4. For competitive digestion experiment, IVIg and BO2C11 (2000 nM) were incubated with 1 nM IdeSC94S for 1 hour at 37°C in PBS pH 7.4, prior to the addition of 0.5 nM IdeS for an additional 6 hours. Samples were analyzed by SDS-PAGE In review 6 using 12% gradient Bis-Tris protein Gel (NuPAGE, Thermosfisher) under non-reducing conditions followed by a coloration with Coomassie Blue.

[0101] Purification of IgG from plasma / serum

[0102] For IgG purification, plasma / serum (25 pl) was diluted 1:10 in purification buffer (Pierce™ Melon™ Gel IgG Spin Purification Kit) and incubated with 100 pl of Melon Gel for 10 min at room temperature. Purified IgG was collected. For IgG depletion, plasma / serum samples (25 pl) were diluted 1:10 in PBS prior to incubation with 100 pl Protein A-coupled agarose beads (Pierce Protein A / G Agarose) for 20 min at room temperature. The IgG-depletedflowthrough was collected by centrifugation. Samples were stored at -20°C until use. IgG was In review 8 quantified in plasma / serum, in the purified IgG fraction and in the IgG-depleted flow through by ELISA using an unlabeled goat anti -human Ig kappa antibody (2.5 pg / mL; 2060-01 Southern Biotech) for capture, and an HRP-conjugated mouse anti-human IgG Fc (1 / 3000, 9040-05 Southern Biotech) for detection. A standard curve was established using serial dilutions of IVIg.

[0103] Source of antibodies and enzyme

[0104] BO2C11, KM41, BOIIB2 and LE2E9 are recombinant human IgGl specific for factor VIII originating from patients with hemophilia A (15). Trastuzumab is a humanized monoclonal IgG specific for the human epidermal growth factor receptor 2 (Herceptin®, Roche). D02M03F05, D03M01D07 and D03M02C09 are recombinant human IgGl originating from healthy individuals generated in our lab. Wild-type IdeS and the inactive IdeSC94S variant were produced and purified as described (15). Intravenous immunoglobulins (IVIg) are pooled polyclonal IgG from healthy donors and were from Takeda Pharmaceuticals U.S.A., Inc. (Cambridge, MA).

[0105] Results

[0106] IdeS-neutralizing IgG are present in IVIg

[0107] We first confirmed the presence of anti-IdeS IgG in IVIg, a pool of normal human IgG. IVIg bound in a dose-dependent manner to the immobilized inactive IdeSC94S variant by ELISA (data not shown). The binding of IVIg to immobilized IdeSC94S was inhibited in a dose-dependent manner by soluble IdeSC94S with an IC50 of 108.6±39.8 nM (data not shown). As controls, 7 human monoclonal IgGl that are not specific for IdeS exhibited no binding to IdeS (data not shown), indicating that the binding of IVIg to IdeSC94S occurs through the Fab and not through the hinge-CH2CH3 domains in a substrate-like manner. We then examined the hydrolysis of IVIg by IdeS. We first used BO2C11, one of the human monoclonal IgG, as a control. BO2C11 IgG was entirely hydrolyzed into scIgG and F(ab’)2 fragments after 6 hours at 37°C in the presence of 1 nM IdeS, the lowest concentration tested (data not shown). The scIgG was undetectable in the presence of 6 nM IdeS, indicating complete hydrolysis. In contrast, the hydrolysis of IVIg was incomplete in the presence of 1 nM Ides, with a residual band of intact IgG and no generation of F(ab’)2 (data not shown). Residual scIgG was still detectable after incubation in the presence of 8 nM IdeS. The complete hydrolysis of IVIg by 1 nM IdeS was however restored when IVIg was preincubated with IdeSC94S prior to addition of IdeS (Fig IE), suggesting saturation of the neutralizing anti-IdeS IgG fraction within IVIg. Conversely, addition of IdeSC94S had no effect on IdeS-mediated BO2C11 hydrolysis. Takentogether, the data indicate that IVIg contains anti-IdeS IgG and that the latter are able to neutralize the proteolytic activity of IdeS.

[0108] Engineering of a synthetic FRET substrate for IdeS.

[0109] IdeS specifically hydrolyses the human IgG in the hinge region between G / A236 and G237 (24). IgG cleavage occurs in two steps with the first rapid hydrolysis of one of the two heavy chains followed by the slower hydrolysis of the second heavy chain. We sought to engineer a FRET substrate that mimics the first cleavage step of human IgG by IdeS. To this end, we generated a heterodimer the sequence of which is inspired by the structure of the human IgGl (Figure 1A). The heterodimer includes:

[0110] a monomer, wherein eCFP is fused through a GGGS (SEQ ID NO: 7)linker to the hinge-CH2-CH3 fragment of the human IgGl followed by a 6-His tag. While the substrate could theoretically be purified on a protein G-sepharose column, the 6-His tag was introduced to allow purification of the protein without the need for an acid-elution step,

[0111] a monomer wherein the Venus fluorochrome is fused through a GGGS (SEQ ID NO: 7) linker to the hinge-CH2-CH3 fragment of the human IgGl.

[0112] To favor the production of the heterodimer following transfection of eukaryotic cells, we introduced the E356K / D399K and K392D / K409D mutations in the CH3 domains of the eCFP-Fc and Venus-Fc monomers, respectively, to foster electrostatic steering (25). Each monomer was cloned in a different expression vector. HEK 293 cells were transiently transfected and the produced protein was purified from culture supernatant using a cobalt matrix followed by size-exclusion chromatography. Size exclusion chromatography (Figure IB) yielded a single protein band that was detected upon excitation at 280 nm (detection of proteins), at 434 nm (detection of eCFP) and at 515 nm (detection of Venus). The two individual monomers were also produced and purified. The purity of the heterodimer and of the single monomers (theoretical molecular weights for the eCFP-Fc and Venus-Fc monomers: 51.15 and 50.49 kDa) was further confirmed by SDS-PAGE (Figure 1C). The fluorescence emission profiles of the heterodimer and of the monomers were recorded after excitation at 434 nm for the heterodimer and eCFP-Fc monomer, and at 480 nm for the Venus-Fc monomer. The results demonstrate the anticipated emission spectra, with peaks at 484 nm for eCFP-Fc, at 530 nm for Venus-Fc and for the synthetic substrate (Figure ID). The results demonstrate efficacy of the energy transfer in the heterodimer from the eCFP fluorochrome to the Venus fluorochrome, yielding to a low emission at 484 nm and higher emission peak at 530 nm. The potential IdeS FRET substrate is referred to “VIC2R substrate” in the rest of the text.Validation of the hydrolysis of the synthetic VIC2R FRET substrate by IdeS.

[0113] The VIC2R substrate and IdeS were first incubated at a 12: 1 molar ratio for 24 hours at 37°C. Separation of the end-point digestion product by SDS-PAGE yielded 2 protein bands with molecular weights between 23 and 24 kDa, that correspond to the digested Fc fragments of eCFP-Fc and Venus-Fc, and at about 27 kDa, that corresponds to the eCFP-Hinge and Venus-Hinge fragments. A trace of IdeS was detected at 37 kDa (Figure 2A). We then performed a dose-dependent analysis of VIC2R hydrolysis in the presence of increasing concentrations of IdeS. The molecules were incubated at molar VIC2R:IdeS ratios between 1 :0.00002 and 1 :0.25, for 45 min at 37°C and digestion was followed by SDS-PAGE. There was a clear dosedependent hydrolysis of the VIC2R substrate with increasing amounts of enzyme (Figure 2B). Likewise, incubation of fixed concentrations of VIC2R and IdeS for different amounts of time at 37°C, lead to a time-dependent hydrolysis of the substrate with about half of the substrate being cleaved within 5-10 minutes at the VCR2:IdeS ratio used here, and close to complete hydrolysis being reached after 6 hours, as assessed by SDS-PAGE (Figure 2C). Last, changes in fluorescence profiles were measured in end-point digestion experiments following incubation of VIC2R (2 pM) and IdeS (0.16 pM) at a 12:1 molar ratio, for 24 hours at 37°C. The cleavage observed by western blot (Figure 3A) and the physical separation of the two fluorochrome was confirmed by loss of the energy transfer from the eCFP fluorochrome to the Venus fluorochrome as measure at 530 nm and gain in emission of the eCFP fluorochrome at 484 nm (Figure 2D). The ratios of fluorescence intensities F530 nm / F484 nm changed from 1.63±0.26 before hydrolysis to 0.68±0.03 (mean±SD, n=3-4) after complete cleavage.

[0114] Kinetic parameters governing IdeS-mediated VIC2R hydrolysis.

[0115] To determine the initial velocity of the reaction, IdeS (1 nM) was incubated with different concentrations of VICR2 substrate and the fluorescence was measured at 484 nm (F484) and 530 nm (F530) after excitation at 434 nm every 20 seconds. The ratio of fluorescence F530 / F484 was calculated at each time point and for each substrate concentration and was translated into pM of hydrolyzed substrate using the ratiometric method as described by Liu et al (21) (Figure 3A). The initial velocity V0 was calculated over the linear phase (i.e., 400 seconds) and plotted as a function of the initial substrate concentration (Figure 3B). Fitting the experimental data to the Michaelis-Menten equation allowed calculation of the kinetic parameters. The Vmax was 0.012 pM / s and Km was 6.074 pM. The calculated Kcat was 12.25 s-1. Interestingly, these values were in the same order of magnitude as the one previously published for the cleavage of human IgG by IdeS, as studied by densitometric analysis of SDS-PAGE migration profiles (5) or surface plasmon resonance (4) (Table 1). The FRET substrate was used to develop a functional neutralization assay: IVIg (0.5-10 mg / ml) was incubated with IdeS (8-24 nM) for 1 hour prior to addition of the substrate. IVIg neutralized IdeS in a dosedependent manner with >85% neutralization at the highest IgG concentration (Fig 4). Conversely, IdeS overcame the neutralizing activity in a dose-dependent manner. Calculation of the IgG concentrationsleading to 50% neutralization yielded 1.4, 5.7, 7.1 and 9.1 pg / ml for 8, 16, 20 and 24 nM IdeS, respectively. Taken together, these data confirm that IdeS-neutralizing IgG are present in pools of normal IgG.

[0116] IdeS-specific ADA and NAbs in the plasma from healthy individuals and patients with acquired hemophilia.

[0117] Using the VIC2R substrate, we then developed an assay to detect and quantify IdeS neutralizing antibodies. The presence of anti-IdeS IgG was first investigated in a home-made anti-IdeS ELISA wherein an engineered humanized anti-IdeS IgG is used as a standard and ELISA plates are coated with the inactive IdeSC94S variant. Anti-IdeS IgG were detected at high titers in the plasma from all tested healthy donors - the 50% binding was reached at dilutions 1 / 640 and 1 / 2500, Figure 5A) - and patients with acquired hemophilia - 50% binding reached at dilutions 1 / 100 and 1 / 2000, Figure 5B).

[0118] Quantification of neutralizing anti-IdeS antibodies.

[0119] In order to measure IdeS-neutralizing antibodies, we developed an assay wherein the plasma is incubated with IdeS for 1 hours at 37°C, following which the VIC2R substrate is added for an additional hour at 37°C (Figure 6A). The reaction is then stopped by addition of iodoacetamide and fluorescence is read at 484 nm and 530 nm after excitation at 434 nm.

[0120] The first incubation step of individuals’ plasma with IdeS for 1 hours at 37°C allows digestion of the endogenous plasma IgG, thus avoiding competition for cleavage between IgG and the VIC2R substrate. Importantly, the F(ab’)2 fragment that result from the digestion of plasma IgG by IdeS conserve their neutralizing capacity towards their cognate antigen (26).

[0121] The F530 / F484 ratio measured when IdeS incubated in PBS-0.025% tween 20 alone prior to addition of the substrate was defined as 100% IdeS activity. Conversely, the F530 / F484 ratio measured when the VIC2R substrate was incubated alone without IdeS, was defined as 0% of IdeS activity. An additional control was used occasionally wherein IdeS was incubated with an irrelevant monoclonal human IgG (i.e., trastuzumab) prior to the addition of the substrate.Plasma from healthy donors and patients was incubated with IdeS at different dilutions, namely 1 / 4, 1 / 12 and / or 1 / 18. When tested at 1 / 4 dilution, plasma from 3 donors showed complete IdeS (4 nM) neutralization, plasma from 3 donors showed between 10 and 70% residual IdeS activity, and plasma from 1 donor showed no neutralization towards IdeS at all (Figure 6B). The neutralizing activity decreased for all donors with further dilution of the plasma, with only one donor showing 50% residual IdeS activity at 1 / 18 dilution. Incidentally, the donor with the highest titer of anti-IdeS IgG (Figure 5A) was showing the highest neutralizing activity towards IdeS at a 1 / 18 dilution. Levels of anti -IdeS IgG and IgA were higher when measured in serum than when measured in plasma, which is consistent with the highest IgG concentration in serum (data not shown). In plasma, healthy donors presented with heterogenous levels of anti-IdeS IgG: 23.89±22.03 pg / ml [min: 0.80 pg / ml-max: 131.30 pg / ml]; and anti-IdeS IgA: 0.993±3.007 pg / ml [0.010-23.160 pg / ml]. In serum, levels of anti-IdeS IgG were 36.91±26.00 pg / ml [3.47-115.30 pg / ml] and levels of anti-IdeS IgA were 2.201±4.595 pg / ml [0.0-25.740 pg / ml]. There was no difference in anti-IdeS IgG or IgA titers between males and females, or according to the age of the donors (data not shown). Five healthy donors (3 females and 2 males) had anti-IdeS IgG levels below the LOQIgG, and 20 donors (13 females and 7 males) had anti-IdeS IgA levels below the LOQIgA (data not shown). Hence, the prevalence of anti -IdeS IgG and IgA were 96.3% and 85.3% in our cohort, respectively. Combining data from plasma and serum samples revealed a weak but statistically significant positive correlation between the levels of anti-IdeS IgG and IgA.

[0122] We then investigated the effect of IdeS concentration on its neutralization by the donors’ plasma diluted 1 / 2, 1 / 3 or 1 / 9 in PBS (Figure 6C). For Donor 1, IdeS at 4 nM was systematically blocked for all tested plasma dilutions. For Donor 6, in agreement with Figure 5B, no neutralizing activity towards IdeS was detected in plasma. The plasma of Donor 4 neutralized IdeS for plasma dilutions of 1 / 2 and 1 / 3 and only for the lowest IdeS concentration. Taken together, the data indicate that the neutralizing activity meaured in plasma is dependent both on the plasma dilution and enzyme concentration, and different from donor to donor.

[0123] Plasma from patients were tested at the dilution closest to their respective plateau (Figure 5B). Under such conditions, one patient with high anti-IdeS IgG titer showed 50% neutralization of IdeS and one patient with intermediate anti -IdeS IgG titer showed 75% neutralization of IdeS (Figure 6D). IdeS neutralization above 70% were detected for samples that had anti-IdeS IgG>9.95 pg / ml or IgA>0.05 pg / ml (Figure 7A, 7B), although most samples with IgG / IgA concentrations greater than the latter threshold did not show IdeS neutralization, at least in the present experimental conditions. Of note, with a serum / plasma dilution of 1 / 10,only one sample showed IdeS neutralizing activities >70%. There was a positive correlation, albeit with poor goodness-of-fit, between the IdeS neutralizing activity and the levels of anti-IdeS IgG (R2 =0.042, P=0.0164), or of anti-IdeS IgA (R2 =0.076, P=0.0012). To investigate whether the IdeS neutralizing activity is carried by the IgG or IgA fraction of plasma / serum, we purified IgG from 2 serum and from 2 plasma samples on protein G-agarose beads. The purification of the IgG and depletion from plasma / serum was confirmed by SDS- PAGE and by ELISA (data not shown). The starting material (plasma / serum), purified IgG and IgG-depleted fractions (flow-through) were then assessed for IdeS neutralizing activity using 8 nM IdeS. While IdeS neutralization was readily detected in plasma / serum and in the purified IgG fractions of the four donors, it was completely absent from the flow-through (Figure 7C).

[0124] Taken together, the results indicate that anti-IdeS IgG and anti-IdeS IgA are present in a large majority of healthy individuals. The presence of circulating anti -IdeS antibodies however translates into in vitro detectable IdeS neutralizing activity in about 1% of the normal population. Whether the prevalence of IdeS neutralizing antibodies is the same among individual from different geographical areas or increases in some pathologies, such as chronic kidney disease, remains to be determined. Taken together, these observations underscore the need for a prospective clinical trial to assess IdeS-binding and IdeS-neutralizing antibody levels in highly sensitized kidney transplant recipients, both prior to and following IdeS administration. In this respect, the assays validated in the present work represent indispensable tools.

[0125] EXAMPLE 2:

[0126] We confirm that our assay is capable of detecting and quantifying the neutralizing activity of anti-IdeS IgG in non-human primates (Figure 8), and mice (Figure 9). Anti-IdeS IgG was detected in in IdeS-treated Cynomolgus macaques, and the IdeS neutralization was measured with our assay (Figure 8). We also study the induction of anti-IdeS IgG in naive mice by intravenous injection of IdeS at therapeutic doses (Figure 9A). The serum from IdeS-treated mice demonstrated heterogenous levels of neutralizing activity towards IdeS, with a mean residual IdeS proteolytic activity of 42±10% (range: 2 to 100%) as compared to PBS-treated mice (89±12%, range: 43 to 100%, Figure 9B, P=0.013). Nine of 17 IdeS-treated mice had residual IdeS proteolytic activity below 30%.

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Claims

-25-CLAIMS:

1. An IgG-degrading enzyme substrate comprising or consisting of two different monomers fused to a detecting agent, whereina) each monomer consists of the hinge-CH2-CH3 proteins of an immunoglobulin;b) each monomer is fused to one detecting agent at its N-terminal or C-terminal part;c) the CH3 domain of the monomer comprises at least one mutation; andd) each monomer is link to one detecting agent thanks to a linker and the hinge region or fragment thereof wherein the hinge region comprises the peptidic sequence GPSVF (SEQ IDNO: 1).

2. An IgG-degrading enzyme substrate according to the claim 1 wherein the hinge region of the IgG-degrading enzyme substrate is a hinge cleaved by an IgG-degrading enzyme like the endopeptidase IdeS.

3. An IgG-degrading enzyme substrate according to the claim 1 wherein the hinge region has the sequence as set for SEQ ID NO: 3 : APELLGGPSVF.

4. An IgG-degrading enzyme substrate according to the claims 1 to 3, wherein the linker of the IgG-degrading enzyme substrate has the following sequence ((G)nS)x wherein n is between 1 and 10 and x between 1 and 5.

5. An IgG-degrading enzyme substrate according to the claim 4 wherein the linker has the sequence as set for in SEQ ID NO: 7: GGGS.

6. An IgG-degrading enzyme substrate according to the claims 1 to 5 wherein the mutations can be selected in the group consisting in but not limited to E356K, D399K, K409D, K392D, K409E, K370D, D357K, T366W, T366S, L368A or Y407V.

7. A method for detecting the activity of an IgG-degrading enzyme comprising the following steps:a) incubating the IgG-degrading enzyme substrate according to the claims 1 to 6 with a sample likely to contain an IgG protease for at least 10 seconds at 37°C in the dark; andb) detecting the emission fluorescence emitted by the two fluorochromes wherein the ratio of two fluorescences is reflecting the IgG-degrading enzyme activity.

8. A method for detecting the presence or not of neutralising anti-IgG-degrading enzyme antibody in a sample comprising the following steps in the dark:a) incubating a sample in the presence of an IgG-degrading enzyme for at least 30 minutes;b) adding of the IgG-degrading enzyme substrate according to the claims 1 to 6 for at least one hour;c) blocking the IgG-degrading enzyme catalytic activity by addition of iodoacetamide for at least 30 min;d) detecting the emission fluorescence emitted by the two fluorochromes wherein the ratio of two peaks of fluorescence is indicative of the residual IgG-degrading enzyme activity present in the samples, and thus of the presence of neutralizing anti- IgG protease antibodies in the sample.

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