New IGG-degrading enzyme-FC fusion protein and use thereof

Abstract

The present invention relates to a new IgG-degrading enzyme. Here, the inventors investigated whether the Fc fusion technology could be applied to a therapeutic enzyme of bacterial origin. Particularly, they developed IdeS-Fc fusion proteins as a divalent homodimer (IdeS-Fcdiv) or a monovalent heterodimer (IdeS-Fcmonov), in order to extend the IgG-depleting action of IdeS over time. Both IdeS-Fc efficiently separated monoclonal and polyclonal human IgG into F(ab')2 and Fc fragments, although with slower kinetics than their native counterpart. IdeS-Fcmonov exhibited a seven-fold half-life extension in vivo as compared to IdeS, and a significantly better residual cleavage of human IgG at later time points after injection. Their results provide the proof of concept for the use of an IdeS with extended IgG-hydrolyzing functions in vivo that could rapidly translate to the clinic. Thus, the present invention relates to an IgG-degrading enzyme-Fc fusion protein comprising at least one IgG-degrading enzyme fused to an Fc part of an antibody wherein the Fc part comprises at least one mutation.

Description

[0001] NEW IGG-DEGRADING ENZYME-FC FUSION PROTEIN AND USE THEREOF

[0002] FIELD OF THE INVENTION:

[0003] The present invention relates to an IgG-degrading enzyme-Fc fusion protein comprising at least one IgG-degrading enzyme fused to an Fc part of an antibody wherein the Fc part comprises at least one mutation.

[0004] BACKGROUND OF THE INVENTION:

[0005] IdeS (Immunoglobulin G-degrading enzyme of Streptococcus pyogenes, or imlifidase) is a cysteine proteinase produced by Streptococcus pyogenes, a human pathogenic bacterium, as a defense mechanism against host antibodies and complement (1). IdeS cleaves all four human immunoglobulin G (IgG) subclasses with a unique degree of specificity below the disulfide bridge in the hinge region (2). IdeS sequentially cleaves the two heavy chains of IgG with different kinetics, thus releasing the F(ab')2 fragment from the Fc fragment. Decoupling of the F(ab')2 and Fc fragments dramatically reduces their circulating half-lives.

[0006] A recombinant IdeS is commercially available (Idefirix®) as a desensitization treatment for kidney transplant patients with donor-specific antibodies (3). The therapeutic potential of IdeS was demonstrated in several preclinical models of IgG-mediated autoimmune diseases, including rheumatoid arthritis, immune thrombocytopenic purpura, neuromyelitis optica, Goodpasture disease, Guillain-Barre syndrome or heparin-induced thrombocytopenia (1,4-9). In a model of congenital hemophilia B, IdeS also allowed efficient gene therapy by removing neutralizing IgG directed against the viral vector containing the F9 transgene (10). Recently, we demonstrated in a preclinical model of hemophilia A that IdeS efficiently restores the procoagulant activity of therapeutic factor VIII (FVIII) by removing neutralizing anti-FVIII IgG allo-antibodies (4,11). IdeS, however, has a very short half-life, dramatically limiting its action since novel antibody production can be seen as soon as 7 days following its administration in humans (12). This suggests that weekly administrations are necessary for a continuous IgG depletion, which may be required for patients with IgG-mediated autoimmune diseases during the acute phase or when they are refractory to other treatments. The addition of the Fc domain of an IgG to therapeutic proteins has been successfully used to improve their short half-lives by recycling upon binding to the FcRn (neonatal Fc receptor), giving rise to over 13 market- approved drugs (13), among which several enzyme-Fc proteins. SUMMARY OF THE INVENTION:

[0007] Here, the inventors investigated whether the Fc fusion technology could be applied to a therapeutic enzyme of bacterial origin. Particularly, they developed IdeS-Fc fusion proteins as a divalent homodimer (IdeS-Fcdlv) or a monovalent heterodimer (IdeS-Fcmonov), in order to extend the IgG-depleting action of IdeS over time. Both IdeS-Fc efficiently separated monoclonal and polyclonal human IgG into F(ab')2 and Fc fragments, although with slower kinetics than their native counterpart. IdeS-Fcmonovexhibited a seven-fold half-life extension in vivo as compared to IdeS, and a significantly better residual cleavage of human IgG at later time points after injection. Their results provide the proof of concept for the use of an IdeS with extended IgG-hydrolyzing functions in vivo that could rapidly translate to the clinic.

[0008] Thus, the present invention relates to an IgG-degrading enzyme-Fc fusion protein comprising at least one IgG-degrading enzyme fused to an Fc part of an antibody wherein the Fc part comprises at least one mutation.

[0009] Particularly, the invention is defined by its claims.

[0010] DETAILED DESCRIPTION OF THE INVENTION:

[0011] IgG-degrading enzyme-Fc fusion protein of the invention

[0012] A first aspect of the invention relates an IgG-degrading enzyme-Fc fusion protein comprising at least one IgG-degrading enzyme fused to an Fc part of an antibody wherein the Fc part comprises at least one mutation.

[0013] In a particular embodiment, the IgG-degrading enzyme-Fc fusion protein according to the invention comprises one IgG-degrading enzyme.

[0014] In a particular embodiment, the IgG-degrading enzyme-Fc fusion protein according to the invention comprises two IgG-degrading enzymes.

[0015] In a particular embodiment, the IgG-degrading enzyme-Fc fusion protein according to the invention is fused to the Fc part with a linker and a hinge region.

[0016] 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.

[0017] In one embodiment, the hinge region of the IgG-degrading enzyme-Fc fusion protein of the invention is a hinge which is not cleaved by an IgG-degrading enzyme like the endopeptidase IdeS. Particularly, the term IgG-degrading enzyme denotes an IgG protease which is a cysteine protease, a thiol protease or a serin 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.

[0018] More particularly, the IgG protease is the IdeS from Streptococcus pyogenes.

[0019] In a particular embodiment, the IdeS from Streptococcus pyogenes comprises at least one mutation in the site of N-glycosylation. Particularly the mutation is N297A or Q.

[0020] In a particular embodiment, the amino acid at the position 297 is not mutated (still N).

[0021] In one embodiment, the hinge region of the IgG-degrading enzyme-Fc fusion protein of the invention is a hinge that is not hydrolyzed by an IgG-degrading enzyme and particularly IdeS.

[0022] In one embodiment, the hinge region of the IgG-degrading enzyme-Fc fusion protein of the invention is a hinge of an antibody from mouse or rat.

[0023] In a particular embodiment, the hinge region is the hinge region of mouse IgG.

[0024] In a particular embodiment, the hinge region is not the hinge region of human IgG.

[0025] In another particular embodiment, the hinge region comprises the peptidic sequence as set for SEQ ID NO: 1 : KPCICTVPEVS.

[0026] In one embodiment, the linker of IgG-degrading enzyme-Fc fusion protein has the following sequence ((G)nS)x wherein n is between 1 and 10 and x between 1 and 5.

[0027] In a particular embodiment, the linker has the sequence as set for in SEQ ID NO: 2 :GGGS, or SEQ ID NO: 3: GGGGS or SEQ ID NO: 4: GGGGGS.

[0028] In one embodiment, the linker of the IgG-degrading enzyme-Fc fusion protein of the invention has the following sequence ((G)nR)x wherein n is between 1 and 10 and x between 1 and 5.

[0029] In a particular embodiment, the linker has the sequence as set for in SEQ ID NO: 5: GGGGR. A combination of peptidic sequences SEQ ID NO: 2 and 4 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: 6: GGGSGGGSGGGSGGGR.

[0030] According to the invention, the Fc part comprises the two constant CH2 domains and the two constant CH3 domains of an antibody.

[0031] As used herein, the term “CH2 domain” includes the portion of a heavy chain molecule that extends, e.g., from about EU positions 239-340. The CH2 domain is unique in that it is not closely paired with another domain. Rather, two N-linked branched carbohydrate chains are interposed between the two CH2 domains of an intact native IgG molecule. As used herein, the term “CH3 domain” includes the portion of a heavy chain molecule that extends approximately 110 residues from N-terminus of the CH2 domain, e.g., from about residue 341-447, EU numbering system). The CH3 domain typically forms the C-terminal portion of the antibody.

[0032] In a particular embodiment, the Fc region consists or comprises CH2 and CH3 constant domains of the antibody’s two heavy chain, that extends, e.g., from residue 239 EU numbering system and ending at C-terminus of the antibody.

[0033] In some embodiments, the amino acid residues of the Fc part of the IgG-degrading enzyme-Fc fusion protein of the invention are numbered according to the EU numbering system.

[0034] In a particular embodiment, the Fc part derives from the human IgGl (Uniprot reference P01857).

[0035] In one embodiment, the mutations present in the Fc part of the IgG-degrading enzyme- Fc fusion protein are described for example in the article Gunasekaran K. et al. the journal of biological chemistry, 2010.

[0036] 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, Y407V M252Y, S254T, T256E, G237D, P238D, H268D, P271G or A330R or a combination thereof.

[0037] 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. 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

[0038] Y349C:T366S:L368A:Y407V and T366W:S354C.

[0039] In a particular embodiment, the IgG-degrading enzyme has a 6-His Tag (HHHHHH: SEQ ID NO: 7) at its N-terminal end or C-Terminal end.

[0040] More particularly, the IgG-degrading enzyme is the Ides and the 6-His Tag is as its N- Terminal end.

[0041] In a particular embodiment, the IgG-degrading enzyme-Fc fusion protein is the IdeS- Fcdlv, a divalent homodimer with the following structure: HHHHHH (SEQ ID NO: 7)-IdeS- GGGGS-KPCICTVPEVS (SEQ ID 8)-FcN297A / HHHHHH (SEQ ID NO: 5)-IdeS-GGGGS- KPCICTVPEVS (SEQ ID NO:8)-FCN297Aor is the IdeS-Fcmonov, a monovalent heterodimer with the following structure: HHHHHH (SEQ ID NO: 7)-IdeS-GGGGS-KPCICTVPEVS (SEQ ID NO: 8)- FCN297A / K392D / K409D / KPCICTVPEVS (SEQ ID NO: l)-FcN297A / E356K / D399K.

[0042] Uses and methods related to the fusion protein of the invention

[0043] A second aspect of the invention relates to an IgG-degrading enzyme-Fc fusion protein for use as a medicament.

[0044] In a particular embodiment, the IgG-degrading enzyme-Fc fusion protein is used in the treatment of a disease caused by a loss of function or activity of a protein.

[0045] Particularly, the disease caused by a loss of function or activity of a protein is induced by pathogenic antibodies, more specifically by IgG.

[0046] In another particular embodiment, the IgG-degrading enzyme-Fc fusion protein is used in situations where the treatment of a disease is prevented by pathogenic anti-drug antibodies.

[0047] For example, the disease is caused by IgG leading to a loss of function or activity of a protein, or leading to the elimination of a protein. The diseases can be due to neutralizing antibody and can be autoimmune diseases or IgG4-mediated diseases for example.

[0048] The IgG-degrading enzyme-Fc fusion protein can also be used in gene therapy to remove anti-AAV antibodies for example.

[0049] The IgG-degrading enzyme-Fc fusion protein can also be used to remove antibodies specific for molecules expressed at the cell surface in order to prevent antibody-dependent cell cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) for example. In a particular embodiment, The IgG-degrading enzyme-Fc fusion protein can also be used in the cases of kidney transplant, post-operative treatment, acquired haemophilia or autoimmune diseases.

[0050] 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-Barre syndrome, 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

[0051] In a third aspect, the invention relates to a method to prepare F(ab’)2 from Immunoglobulin G (IgG) comprises the steps of: a. incubating the IgG of interest with the IgG-degrading enzyme-Fc fusion protein of the claim 1 for at least 1 hour; b. incubating the sample resulting from the step 1 on a mini-column of protein G or protein A beads during 10 to 60 min; c. centrifugating and recovering of the purified F(ab’)2. Particularly, said method of the invention can comprise a first step of purification of a sample containing IgG (like serum or plasma) by known method (like Melon Thermo Fisher method).

[0052] 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.

[0053] FIGURES:

[0054] Figure 1. Production of IdeS-Fc variants. Panel A. Schematic representation of the structures of IdeS-Fcdlv(left) and Ides-Fcmonov(right). — / ++ represent the K392D / K409D and E356K / D399K mutations respectively. Panel B. The different purified IdeS variants (5 pg / lane) were separated on a 4-12% SDS-PAGE under non-reducing conditions and proteins were revealed using Coomassie blue staining. The molecular weights are shown on the left of the gel. Panel C. Binding of IdeS-Fcdlv(100 nM, left) and Ides-Fcmonov(100 nM, right) to human (dotted line curves) and mouse (plain line curves) FcRn, as assessed by surface plasmon resonance. Panel D. The human monoclonal anti-factor VIII IgG, BO2C11, or therapeutic human polyclonal IgG (IVIG) were incubated in PBS alone or with the different IdeS variants at E:S 1 :12 molar ratios for 24 hours at 37°C, prior to separation on a 4-12% gradient SDS- PAGE (5 pg / lane) under non-reducing conditions. Proteins were revealed using Coomassie blue staining. Molecular weight standards are shown on the left of each gel. Data are representative of at least two independent experiments.

[0055] Figure 2. Kinetics of IgG hydrolysis mediated by the different IdeS variants. The human monoclonal anti -factor VIII IgG (BO2C11, panel A) and therapeutic human polyclonal IgG (IVIG, panel B) were incubated with IdeSWT(left panels), IdeS-Fcdlv(middle panels) or IdeS-Fcmonov(right panels) at a E: S 1 : 12 ratio for various durations at 37°C. Samples (5 pg / lane) were then separated by SDS-PAGE in 4-12% gradient gel under non-reducing conditions. Proteins were revealed using Coomassie blue staining. Molecular weight markers are indicated on the left of the gels, and the bands corresponding to intact IgG; scIgG, F(ab’)2 fragments and Fc fragments are identified at the right of the gels. Data are representative of at least two independent experiments.

[0056] Figure 3. In vivo half-life and efficacy of IdeS variants. Panel A. Mice were injected intravenously with IdeSWT(0.6 mg / kg, 17.1 nmol / kg, full circles), IdeS-Fcdlv(1.75 mg / kg, 14.6 nmol / kg, empty circles) or IdeS-Fcmonov(1.75 mg / kg, 20,6 nmol / kg, empty squares). Blood was recovered 5 minutes after injection and at the indicated time points. IdeS and Fc-fused IdeS were detected in serum by ELISA. The graph depicts the variation over time in the amounts of the different IdeS variants expressed as a percentage of the serum concentration measured at 5 minutes. Data represent means±SD of 3-11 mice per group depending on the time point. The experimental data were fitted to a non-linear one-phase decay equation with the plateau fixed at 0, and the half-lives were calculated. The inset depicts the half-lives with 95% confidence intervals (CI) and robustness of fits (R2). Panels B and C. Mice were passively immunized intravenously with 10 mg of IVIG. Twenty -four hours later, mice were treated with IdeS (17.1 nmol / kg, full squares), IdeS-Fcmonov(20.6 nmol / kg, empty squares) or PBS (empty circles). Panel B. Blood was recovered at the indicated time points and the levels of intact or scIgG measured in serum by ELISA. Statistical differences in IgG levels between mice treated with IdeS or IdeS-Fcmonovwere compared using a two-way ANOVA using the 24-hour to 288- hour time points (**: PO.01). Panel C. Mice were passively immunized a second time with 10 mg IVIG. Levels of intact or scIgG were measured 48 hours later by ELISA. Data represent means±SD of 5-10 mice per group depending on the time point. Statistical differences in IgG levels were compared between mice treated with IdeS or IdeS-Fcmonovusing a two-sided nonparametric Mann-Whitney at the 48-hour and at the 96-hour time-points (***: P<0.001). All data are representative of two independent experiments.

[0057] Figure 4. Preparation of F(ab’)i fragments using Fc-fused IdeS. A. Protocol for removal of Fc-fragment from human IgG or Fc-fused proteins. Fc-fused proteins or IgG are incubated with IdeS-Fc for 45 min to 6 hours at a 1 : 12-1 : 15 enzyme: substrate molar ratio (Step 1). The mixture is then incubated with protein G-coupled agarose beds for 15 min (Step 2). The pure Fc-free protein or F(ab’)2 fragments are recovered following a quick centrifugation (Step 3), undiluted in the starting buffer and without contamination with IdeS. B. Efficacy of IdeS-Fc on human polyclonal IgG (IVIg). IVIg (XX nM) was incubated alone (lanes 4, 7, 10), with IdeS (XX nM, lanes 5, 8, 11) or with IdeS-Fc (XX nM, lanes 6, 9, 12) for 6 hours at 37°C with an enzyme-to-substrate ratio of 1 : 12. Samples were then run through sepharose- coated beads. Pre-purification samples are shown on lanes 4-6. Flow through fractions are shown in lanes 7-9. Eluates are shown in lanes 10-12. The migration profiles of IdeS and IdeS- Fc alone are shown on lanes 2 and 3, respectively. Proteins (3 pg per lane) were separated by 4-12% SDS-PAGE under non-reducing conditions and stained with Coomassie blue. Molecular weight markers are shown on lane 1 and indicated on the left of the gel. Bands corresponding to intact IVIg, F(ab’)2 fragments, IdeS-Fc, IdeS, and Fc fragments are shown on the right. Data a representative of >3 independent experiments.

[0058] Figure 5. Preparation of F(ab’)2 fragments from a therapeutic monoclonal IgG (i.e., Rituximab) using IdeS-Fc. Panels A and B. Hydrolysis of Rituximab (Rtxmb) by IdeS- Fc. Rtxmb (XX nM) was incubated alone (lanes 4, 7, 10), with IdeS (XX nM, lanes 5, 8, 11) or with IdeS-Fc (XX nM, lanes 6, 9, 12) for 6 hours at 37°C with an enzyme-to-substrate ratio of 1 : 12. Samples were then run through sepharose-coated beads. Pre-purifi cation samples are shown on lanes 4-6. Flow through fractions are shown in lanes 7-9. Eluates are shown in lanes 10-12. The migration profiles of IdeS and IdeS-Fc alone are shown on lanes 2 and 3, respectively. Proteins (3 pg per lane) were separated by 4-12% SDS-PAGE under non-reducing conditions: proteins were stained using Coomassie blue (panel A), or IdeS and IdeS-Fc were recognized by Western blot using a biotinylated anti-His tag antibody followed by streptavidin- HRP (panel B). Molecular weight markers are shown on lane 1. Bands corresponding to intact Rtxmb, Rtxmb F(ab’)2 fragments, IdeS-Fc, IdeS, and Fc fragments are shown on the right. Data a representative of >3 independent experiments. Panels C and D. Binding of Rituximab- derived F(ab’)2 fragments to CD20 on Raji cells. Native Rtxmb, or purified IdeS-Fc generated Rtxmb F(ab’)2 fragments were incubated with CD20-positive Raji cells at concentrations of 7, 35, and 70 nM for 30 min at 37°C. The binding was detected using an Alexa Fluor 647-coupled goat anti-human IgG (H+L) antibody, or a PE-coupled goat anti-human IgGFc antibody. Panels C and D depict the mean fluorescence intensity (MFI) measured for the F(ab’)2 fragments or for intact IgG, respectively, the case of detection using the anti-human IgG (H+L) and antihuman IgG Fc antibodies. Each condition was tested in triplicates and results are means±SD. Data are representative of 2 independent experiments.

[0059] Figure 6. Isolation of functional factor IX from therapeutic Fc-fused factor IX using IdeS-Fc. Panel A. Hydrolysis of factor IX by IdeS-Fc. Factor IX (FIX, XX nM) was incubated alone (lanes 4, 7, 10), with IdeS (XX nM, lanes 5, 8, 11) or with IdeS-Fc (XX nM, lanes 6, 9, 12) for 6 hours, at 37°C with an enzyme-to-substrate ratio of 1 : 12. Samples were then run through sepharose-coated beads. Pre-purifi cation samples are shown on lanes 4-6. Flow through fractions are shown in lanes 7-9. Eluates are shown in lanes 10-12. Panel B. Time-dependent hydrolysis of factor IX by IdeS-Fc. FIX (XX nM) was incubated with IdeS- Fc for 10 to 180 min. Migration profiles of the incubated samples are shown in lanes 4-8. On both gels, the migration profiles of IdeS and IdeS-Fc alone are shown on lanes 2 and 3, respectively. Proteins (3 pg per lane) were separated by 4-12% SDS-PAGE under non-reducing conditions and proteins were stained using Coomassie blue. Molecular weight markers are shown on lane 1. Bands corresponding to FIX-Fc, single-chain FIX-Fc (FIX-scFc), FIX, IdeS- Fc, IdeS, and Fc fragments are shown on the right. Data a representative of >3 independent experiments.

[0060] EXAMPLE 1:

[0061] Material & Methods

[0062] Production of IdeS variants.

[0063] Wild-type IdeS (IdeSWT) and inactive IdeSC94S(25) were produced and purified as described. (4) Two versions of human Fc-fused IdeS were produced. In both constructs, the cDNA of IdeS was fused with that of the human Fcyl portion starting at residue S239. IdeS had a 6-His Tag at its N-terminal end. IdeS and the Fcyl were separated by a GGGGS (SEQ ID NO:3) linker. The human hinge region was replaced by the KPCICTVPEVS (SEQ ID NO: 1) sequence that corresponds to hinge region of the mouse Fcyl, to avoid autodigestion of the fusion proteins. The N297A mutation was introduced in the Fc sequence to avoid engagement of the FcyR. IdeS-Fcdlvis a divalent homodimer with the following structure: HHHHHH (SEQ ID NO: 7)-IdeS-GGGGS-KPCICTVPEVS (SEQ ID NO: 8)-FcN297A / HHHHHH (SEQ ID NO: 7)-IdeS-GGGGS-KPCICTVPEVS (SEQ ID NO: 8)-FcN297A. IdeS-Fcmonovis a monovalent heterodimer with the following structure: HHHHHH (SEQ ID NO: 7)-IdeS-GGGGS- KPCICTVPEVS (SEQ ID NO: 8)- FCN297A / K392D / K409D / KPCICTVPEVS (SEQ ID NO: 1)- pcN297A / E356K / D399K jn the case of ideS-Fcmonov, the KK and DD mutations in the CH3 domains where introduced to favor electrostatic steering and production of the heterodimeric IdeS-Fc Fc. The Fc-fused proteins were expressed using the Expi293™ Expression System (Thermofisher) and purified from supernatant on a nickel-based Histrap IMAC resin (TALON® Superflow™, Cytiva). Buffer exchange was performed using PD-10 desalting columns against phosphate buffer saline (PBS).

[0064] Surface plasmon resonance.

[0065] The binding of Fc-fused IdeS to the human (h) and mouse (m) FcRn was studied by surface plasmon resonance using Biacore 2000 (Cytiva). Biotinylated human FcRn or murine FcRn (Immuni track, Denmark) were diluted with tris-citrate buffer (100 mM Tris, 100 mM NaCl, 5% glycerol and 0.1% tween 20 at pH 6) and immobilized on a streptavidin sensor chip at a protein surface density not exceeding 0.5 ng / mm2(450-550 RU). Fc-fused IdeS were diluted to 100 nM in running buffer (100 mM Tris, 100 mM NaCl, 5% glycerol and 0.1% tween 20, pH 6). Association and dissociation were monitored for 4 and 5 minutes, respectively, with flow rates of 30 pl / minute at 25°C. Chips were regenerated upon injection of 15 pl of 100 mM Tris, 100 mM NaCl pH 7.8. In vitro hydrolysis of IgG by IdeS.

[0066] In an end-point assay, IdeSWT, IdeS-Fcdlv, IdeS-Fcmonovor IdeSC94S(0.16 pM) were added to pooled human therapeutic polyvalent IgG (IVIG, Gammagard, Baxter, 2 pM) or to a human monoclonal anti-FVIII IgG, BO2C11 (2 pM)(4) at a 1: 12 molar ratio in 100 pL of PBS- 0.025% tween 20, for 24 hr at 37°C. In time course experiments, IVIG or BO2C11 (2.4 pM) were incubated with 4.8 nM of IdeSWT, IdeS-Fcdlv, IdeS-Fcmonovor IdeSC94Sat 37°C. At various time points, the reactions were stopped by addition of 25 mM iodoacetamide.

[0067] In vivo efficacy of IdeS variants.

[0068] All animal experimentations were conducted in accordance with French regulations and the experimental guidelines of the European Community (APAFIS#36357- 2022040513338571). To determine the half-life of IdeS and IdeS-Fc fusion variants, isoflurane- anesthetized male or female C57BL / 6 mice (8-14 weeks-old) received either IdeSWT(0.6 mg / kg, 17.1 nmol / kg), IdeS-Fcdlv(1.75 mg / kg, 14.6 nmol / kg) or IdeS -Fcmonov(1.75 mg / kg, 20,6 nmol / kg) through the retro-orbital venous plexus. Blood was collected at regular time points up to 144 hours and serum was isolated and stored at -20°C until use. Following quantification of intact IgG / scIgG by ELISA, the half-lives were calculated by fitting the experimental data to a non-linear one-phase decay equation with the plateau fixed at 0 (Prism Version 9.4.1, GraphPad).

[0069] To validate the enzymatic efficacy of the IdeS-fusion variants, mice were passively immunized with 10 mg human polyvalent IgG for therapeutic use (IVIG, Gammagard, Baxter). Twenty -four hours later, the mice were injected intravenously with PBS (100 pl), IdeS (17.1 nmol / kg, 100 pl) or IdeS-Fcmonov(20.6 nmol / kg, 100 pl). When indicated, mice were readministered with 10 mg IVIG 48 hours after the injection of IdeS / IdeS-Fcmonov. Blood was collected at regular time points up to 288 hours and serum was isolated and stored at -20°C until use. Intact IgG / scIgG were quantified by ELISA.

[0070] Intact / scIgG and IdeS-Fc ELISA.

[0071] Serum was prepared and analyzed for the presence of intact / scIgG, IdeS or IdeS-Fc variants using immunosorbent assays. Briefly, diluted serum samples were incubated in wells coated with a mouse anti-human Ig kappa (2.5 pg / ml; Southern Biotech #2060-01) or with mouse anti-human IgG Fc (5 pg / ml; Clinisciences #LS-C69574) for the detection of intact / scIgG or IdeS-Fc variants, respectively. Bound proteins were probed using a peroxidase- labeled mouse anti-human IgG Fc-HRP (1 / 4000; SB Cat. No. 9040-05) and detected via peroxi dase-mediated hydrolysis of OPD (o-phenylenediamine dihydrochloride). In the case of IdeS, the molecule was detected by anti-HisTag (2pg / mL; R&D Systems, #BAM050), and bound proteins were revealed using a goat anti-IdeS (1 / 500; Genovis Fabricator), detected with an anti-goat IgG-HRP (1 / 1000; Sigma-Aldrich, #HAF017). Optical densities were measured at 492 nm. Purified IVIG or purified IdeS-Fc variants were used for the standard curve, respectively.

[0072] Results

[0073] Generation and characterization of IdeS-Fc fusion protein.

[0074] Two Fc-fused IdeS constructs were generated. The first one, referred to as IdeS-Fcdlv, is a divalent homodimer with the IdeS-hinge-CH2-CH3 / IdeS-hinge-CH2-CH3 structure (Fig 1A). The second construct, referred to as IdeS-Fcmonov, is a monovalent heterodimer IdeS-hinge- CH2-CH3 / hinge-CH2-CH3, wherein the association of the two polypeptide chains is strengthened by the introduction of complementary charged residues in the CH3 domains (Fig 1A), a strategy referred to as electrostatic steering. In both constructs, i) to avoid self-digestion, the human hinge region was replaced by the hinge region of mouse IgGl, which is not cleaved by IdeS (4), ii) to hamper binding to the FcyR, asparagine 297 was mutated to an alanine to prevent N-glycosylation, and iii) to provide spatial flexibility to IdeS, the latter was separated from the hinge region using a GGGGS (SEQ ID NO: 3) linker. IdeS-Fcdlvand IdeS-Fcmonovpurity and migration at the expected molecular weights were validated by SDS-PAGE as compared to the 37 kDa molecular weights of wild-type (WT) IdeS and of an enzymatically inactive IdeSC94S(Fig IB) The binding of IgG and Fc-fused proteins to the FcRn is critical to ensure the recycling of the molecules at the cell surface following endocytosis and to route the molecules away from the lysosomal degradation pathway. This property accounts for the long half-lives of human IgGl and albumin in the circulation (i.e., 3 weeks). Both IdeS-Fcdlvand IdeS-Fcmonovbound to human and mouse FcRn, although with a higher affinity to mouse FcRn, in line with the literature (14), as assessed by surface plasmon resonance at pH 6 (Fig 1C).

[0075] IdeS-Fc and IdeS hydrolysis activities on monoclonal and polyclonal human IgG.

[0076] In order to assess the catalytic activity of IdeS-Fc constructs, we incubated either IdeS- Fc, IdeSWT, or IdeSC94Swith a monoclonal human anti-FVIII IgG (clone BO2C11) (4) or with polyclonal intravenous immunoglobulins (IVIG) (Fig ID). IgG incubated alone or with IdeSC94Sretained their native structure with a major band at 150 kDa in a non-reducing SDS- PAGE. In contrast, both IdeS-Fc variants cleaved human IgG, generating identical migration profiles with major bands at 25 kDa (Fc fragments) and 100 kDa ((Fab’)2 fragments), similarly to IdeSWT. F(ab’)2 fragments alone lack Fc fragment-mediated functions, which induces a dramatically reduced half-life, but maintains antigen-binding (and possibly neutralizing) capacity during their life span in the circulation (4). As a result, IdeS-mediated IgG cleavage has an immediate functional repercussion when the pathogenic function of IgG is mediated by Fey receptors or complement, as is the case in immune thrombocytopenia and autoimmune hemolytic anemia (15). The functional repercussion is however delayed when the pathogenic functions are directly mediated by the antigen-binding Fab. To further study the catalytic activity of IdeS-Fc constructs, IdeSWT, IdeS-Fcdlvand IdeS-Fcmonovwere incubated for various durations with either IVIG or BO2C11.

[0077] All three constructs performed the cleavage of the first heavy chain within 5 minutes of incubation, as shown by the disappearance of the intact IgG band and the shift to the partially cleaved single chain (sc) IgG (Fig 2A and 2B). The three constructs also completely hydrolyzed both IVIG and BO2C11 after 24 hours of incubation as shown by the disappearance of the scIgG band. However, in the case of the hydrolysis of IVIG, IdeS-Fcdlvand IdeS-Fcmonovexhibited lower hydrolysis activity, with a longer persistence of the scIgG protein band and a longer time to generate the fully hydrolyzed F(ab’)2 fragments (most obvious at the 40- and 60- minute time points). Whether the apparent decrease in catalytic efficacy is a consequence of the fusion of the Fc fragment to IdeS remains to be confirmed. Such an impact has been reported previously in the case of fusion of the Fc fragment to enzymes (16). Alternatively, anti-IdeS IgG that are present in IVIG could reduce the proteolytic efficacy of IdeS (10).

[0078] Extension of IgG hydrolysis in vivo using IdeS-Fc.

[0079] In order to decipher whether the fusion of the Fc portion to IdeS has an impact on the half-life of the enzyme, we injected naive C57B1 / 6 mice either with IdeS-Fc variants or with IdeSWTat doses similar to those given to patients (0.6 mg / kg or 17 nmol / kg for IdeSWT, Fig 3A) Mice were bled at different time points over 144 hours and the levels of IdeS were measured in serum using dedicated ELISAs. IdeS exhibited a half-life of 1.1 hours with a 95% confidence interval (CI) of 0.8-1.4 hours. This is substantially lower than the 5 hours-long halflife reported for IdeS in humans (17), but parallels the reduced half-life of human IgG found in mice as compared with humans (i.e., 7-9 days versus 21 days). Interestingly, IdeS-Fcdlvexhibited a half-life that did not differ from that of IdeSWT. One may speculate that the divalency of the protein perturbs its intracellular routing or hastens its catabolism for reasons yet to be deciphered. In contrast, the IdeS-Fcmonovpresented a half-life of 7.9 [CI: 5.7-11.2] hours, which corresponds to a 7.2-fold increase compared to native IdeS. The fusion technology has been applied with success to several proteins, including enzymes (factor IX) or cofactors (procoagulant factor VIII), resulting in increases in circulating half-lives of 2.4-3.7 and 1.4-1.8 folds in the patients, respectively (18,19). These results are within the range of what we observed with Fc-fused IdeS in mice. Future experiments will decipher whether the half-life of IdeS may be further extended by the introduction of the M252Y / S254T / T256E mutations in the fused Fc fragment, as previously shown for monoclonal antibodies (20).

[0080] Next, we compared the efficacy of IdeSWTand IdeS-Fcmonovin eliminating human IgG in vivo. IdeSWTand IdeS-Fcmonovwere administered to mice passively immunized with 10 mg of IVIG (Fig 3B). The injection of both molecules was followed by a 10-fold decrease of intact or scIgG within the first 6 hours, followed by a slower decrease in the next days, which paralleled the spontaneous elimination of IVIG detected in mice that had not received IdeS (PBS group). From the 24-hour time point onwards, IdeS-Fcmonovdemonstrated a 1.7-2.3-fold enhanced degradation of human IgG compared to native IdeS (P<0.01), which may be related to its extended residence time in the blood.

[0081] Treatment with IdeS in humans leads to the almost immediate elimination of IgG. However, a rebound of the antibodies may occur within 6 to 13 days, as shown in kidney transplant patients (12) or patients affected by the anti-GBM disease (21), thus jeopardizing the long-term beneficial effect of IdeS. Presumably, an Fc-fused IdeS with an extended half-life could curb the rebound of pathological antibodies. To test this hypothesis, passively immunized mice treated with IdeSWTand IdeS-Fcmonovwere dosed with IVIG a second time 24 hours later. Following redosing with IVIG, mice initially treated with IdeSWTexhibited similar IgG kinetics during the next 50 hours as control mice treated with PBS (Fig 3C). Conversely, mice administered with IdeS-Fcmonovdemonstrated a 5.4-fold decrease in IgG levels at 96 hours compared to IdeSWT-treated mice (Fig 3C, <0.01). The data confirm that enzymatically proficient IdeS was still present in the circulation at the time of IVIG redosing. Such an extent of functional half-life represents a potential benefit in the context of autoimmune diseases, where it should allow a reduction in the frequency of IdeS administrations, thus improving patient care and reducing the associated costs while optimally delaying the rebound of pathogenic autoantibodies.

[0082] Several strategies exist to foster the elimination of pathogenic IgG. In addition to the use of depleting anti-CD20 antibodies or corticosteroids, which impact the whole B cell compartment or induce wide immune suppression, respectively, pathogenic antibodies may be eliminated by plasmapheresis. More recently, monoclonal antibodies (rozanolixizumab, nipocalimab and batoclimab) have been developed to block the FcRn and prevent the recycling of IgG, thus accelerating IgG clearance. They are currently undergoing clinical trials in various IgG-mediated pathologies. In addition, efgartigimod, a genetically engineered human Fc fragment with augmented affinity towards FcRn, recently obtained FDA approval for the treatment of myasthenia gravis (22). The use of FcRn antagonists is associated with an important decrease in the levels of circulating IgG, without impacting levels of IgA, IgE and IgM, and globally improves the course of the disease. In humans, a single dose of efgartigimod or rozanolixizumab leads to a 50% and 43% reduction in circulating IgG, respectively (23,24), and the time required to reach maximum IgG elimination spans from 5 to 7 days. Conversely, depletion of IgG by FcRn antagonists lasts up to 8 weeks, probably owing to the long half-life of the antagonists. This contrasts with IdeS, which achieves nearly 100% reduction after a single administration within a few hours of injection (3), a critical advantage for autoimmune patients during the acute phase of their disease when treatment timing is crucial. A long-acting Fc-fused IdeS would advantageously complete the panoply of IgG-depl eting drugs.

[0083] Conclusion.

[0084] Here, we developed monovalent or divalent IdeS-Fc fusion proteins. Both constructs bound mouse and human FcRn at acidic pH, a key mechanism for the longer half-life of circulating IgG, and hydrolysed monoclonal and polyclonal human IgG with efficacy comparable to that of IdeSWT. IdeS-Fcmonovexhibited a seven -fold increase in its half-life, which translated into a 1.7-2.3-fold enhanced degradation of human IgG in passively immunized mice 48 hours following injection of the enzyme. In view of the number of pre-clinical studies that recently validated IdeS for the treatment of IgG-mediated auto-immune pathologies, our proof- of-concept is of importance and could lead to a reduction in the frequency of IdeS dosing.

[0085] EXAMPLE 2:

[0086] Generation of F(ab’)i fragments from polyclonal human IgG by IdeS-Fc

[0087] We first validated that IdeS-Fc is as efficient as IdeS at proteolyzing human IgG. We incubated a therapeutic preparation of pooled human IgG (IVIg) with IdeS or with IdeS-Fc for 6 hours at 37°C at an 1 : 12 enzyme: substrate molar ratio. Both avatars of the enzyme cleaved IVIg yielding separated F(ab’)2 and Fc fragments (Fig. 4B, lanes 4-6). Following incubation of the mixture on agarose-coupled protein G, the F(ab’)2 fragments were recovered in the flowthrough (Fig. 4B, lanes 8-9) while the Fc fragments were retained on the affinity matrix (Fig. 4B, lanes 11-12) IdeS and IdeS-Fc incubated alone migrated at 37 kDa and 90 kDa, respectively. IdeS-Fc showed additional contaminating bands at 65 kDa and above 160 kDa. Of note, a faint band of IdeS was detectable at 37 kDa in the F(ab’)2 fragment-containing flow- through fraction (lane 8) while the IdeS-Fc flow-through contained only F(ab’)2 fragments, with IdeS-Fc and Fc fragments being retained on the column (lane 9). The eluate from the IdeS- Fc mixture contained IdeS-Fc (not seen on the gel), some contaminating F(ab’)2 fragments that were not fully recovered in the flow-through, and Fc fragments (lane 12). Comparison of the starting and end concentrations of total IgG and F(ab’)2 fragments indicated a recovery yield of the F(ab’)2 fragments of 91.4% (Table 1).

[0088] Table 1. Yields of recovery of Fc-free proteins

[0089] TgG and F(ab’)2 fragments were quantified by ELISA; FIX-Fc and FIX was quantifier by nanodrop.

[0090] ■^Calculated as: (CFT X VFT) / (Ci x Vi) x 100, where C, FT, V and i stand for concentration, flow-through, volume, and initial, respectively. Vi was 500 or 250 pl, and VFT was 550 or 275 pl, for IgG or FIX-Fc, respectively.

[0091] IdeS and IdeS-Fc were separated alone as controls, and had molecular weights of 37 kDa and 90 kDa, respectively. IdeS-Fc showed additional contaminating bands at 65 kDa and above 160 kDa. Of note, the hinge region of IdeS-Fc originates from the mouse IgGl and is hence not a substrate for IdeS. This explains why IdeS-Fc is not hydrolyzed in the process and remains as a major protein bands of 90 kDa in all conditions.

[0092] Generation of pure F(ab’)i fragments from a therapeutic monoclonal IgG by IdeS- Fc

[0093] We then compared the capacity of IdeS-Fc and IdeS to proteolyze the therapeutic human monoclonal IgG Rituximab that is specific for CD20. As in the case of IVIg, the F(ab’)2 and Fc fragments of Rituximab were readily separated after a 6-hour long incubation at 37°C (Figure 5A, lanes 5-6). Similarly to IVIg, IdeS was recovered with the F(ab’)2 fragments of Rituximab (Figure 5A, lane 8). The presence of IdeS-Fc with the Fc fragments in the eluted fraction was however hardly distinguishable owing to the close migration of the monoclonal IgG and of IdeS-Fc in the gel (Figure 5B, lane 12). To more clearly investigate the putative contamination of the purified F(ab’)2 fragments by IdeS, the samples were revealed by Western blot using an anti-His tag antibody that detects both IdeS and IdeS-Fc. Indeed, both enzymes were identified in the pre-purifi cation samples (Figure 5B, lanes 5-6). While IdeS was found in the flow through fraction containing the F(ab’)2 fragments of Rituximab (Figure 5B, lane 8), IdeS-Fc was not (Figure 5B, lane 9). Conversely, IdeS-Fc, but not IdeS, co-eluted with the fraction containing the Fc fragments of Rituximab (Figure 5B, lanes 12 and 11, respectively). Taken together, the results confirm that the use of Fc-fused IdeS provides appropriate proteolysis of Rituximab and yields a preparation of F(ab’)2 that is free of adverse contaminating proteins. In the case of Rituximab digested by IdeS-Fc, the recovery yield of the F(ab’)2 fragments was 87.4% (Table 1).

[0094] We then investigated whether the F(ab’)2 fragments generated upon hydrolysis by IdeS- Fc conserve their antigen binding specificity. To this end, CD20+ Raji cells were incubated with increasing concentrations of the native Rituximab IgGl or of the Rituximab F(ab’)2 fragments recovered in the protein G flow-through following IdeS-Fc cleavage. Raji cells are a human Burkitt’s lymphoma line that express high levels of surface CD20. When detected with an anti-Fc secondary antibody, only the native IgGi but not the F(ab’)2 fragments were evidenced at the surface of Raji cells (Figures 5C-5D), which confirms the removal of the Fc fragments from Rituximab following incubation in the presence of IdeS-Fc. In contrast, when detected by an anti-(H+L) secondary antibody, both the F(ab’)2 fragments and the native IgGl were found at the surface of Raji cells with identical saturation profiles (Figure 2C-2E). A549 cells, a human lung epithelial cell line lacking CD20 expression, was used as a negative control. No binding signal was seen either for Rituximab and the purified F(ab’)2 fragments (data not shown). Together, these data confirm the hydrolysis of Rituximab by IdeS-Fc and the following purification process neither altered the binding specificity of the purified F(ab’)2 fragments, nor conferred non-specific binding properties. In the experiments, trastuzumab, an unrelated human therapeutic monoclonal IgGi that targets the HER2 receptor, was used as an isotype control.

[0095] Generation of pure human FIX from therapeutic Fc-fused FIX

[0096] Next, we explored whether IdeS-Fc may be used to isolate the pure enzymatic moiety from an Fc-fused therapeutic enzyme. Therapeutic FIX-Fc (Alprolix®) is an extended half-life therapeutic FIX used for the treatment or prophylactic prevention of bleeds in patients with hemophilia B . FIX-Fc (98 kDa) was incubated with IdeS-Fc or IdeS at a 1 : 15 enzyme: substrate molar ratio for 6 hours at 37°C. Both enzymes efficiently cleaved FIX-Fc and generated protein bands at 25 and ~52 kDa corresponding to the molecular weights of Fc fragments and Fc-free FIX, respectively (Figure 6A). While IdeS-Fc was absent from the flow-through and recovered in the eluate together with the Fc fragments, IdeS was still present in the flow-through together with FIX, and was absent from the Fc fragment eluate (Figure 6A, lanes 8, 9, 11 and 12). We also analyzed the cleavage of FIX-Fc by IdeS-Fc at multiple time points (i.e., 10, 20, 45, 90 min, and 3 hrs). Short incubation times of 10 and 20 minutes yielded an intermediate protein band at ~78 kDa (Figure 6B), that, in all likelihood, corresponds to the partially digested IdeS- Fc devoid of one of the Fc monomers, and reminds of the situation seen with IgG and generation of a single chain (sc) IgG. Interestingly, the incubation time required to generate the Fc-free FIX could be reduced to 45 min (Figure 6B), which brings the total process time required to obtain the pure FIX moiety (Figure 4A) to less than 90 minutes. The recovery yield of the Fc- free FIX was estimated to 97.8% (Table 1) and no FIX was retained on the protein G-agarose beads.

[0097] Three different batches of Factor IX (FIX) cleaved by IdeS-Fc were tested and compared to the native FIX-Fc before digestion. The enzymatic activity of the samples was measured using the BIOPHEN™ Factor IXa chromogenic assay kit, which quantifies activated FIX (FIXa) activity by measuring the release of p-nitroaniline (pNA) from a chromogenic substrate at 405 nm. Assays were performed at 37°C according to the manufacturer’s instructions, with calibration curves established using the FIXa calibrator. Activity values of cleaved FIX-Fc batches were consistently xi± yi mIU / mL, X2± yz mIU / mL, and X3± yz mIU / mL (mean ± SD, n=3), comparable to the native FIX-Fc activity of xo± yO mIU / mL. These results demonstrate that IdeS-Fc cleavage does not impair the functional activity of FIX. Specifically, the enzymatic removal of the Fc portion preserves the integrity of the Factor IX moiety, ensuring it retains its ability to interact with essential cofactors such as activated Factor VIII and phospholipid surfaces, confirming that the cleavage process does not disrupt the coagulation cascade functions. This preservation of activity supports the utility of IdeS-Fc cleavage in preparing functional fragments of Fc-fused proteins.

[0098] These results confirm the robustness and reproducibility of the IdeS-Fc cleavage and purification process for both antibodies and Fc-fused proteins.

[0099] Taken together, the use of Fc-fused IdeS allows the generation of pure uncontaminated and undiluted Fc-free proteins in a short amount of time. The Fc-free proteins may be F(ab’)2 fragments prepared from polyclonal or monoclonal human and non-human primate IgG, or any other protein such as an enzyme (FIX for Alprolix), a receptor (VEGF receptor or TNF receptor for Afhbercept and Etanercept, respectively), a check point inhibitor (CTLA-4 or LFA-3 for Belatacept and Alefacept, respectively), or a cofactor (factor VIII for Eloctate).

[0100] REFERENCES:

[0101] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

[0102] 1. Johansson BP, Shannon O, Bjbrck L. IdeS: a bacterial proteolytic enzyme with therapeutic potential. PloS One. 27 fevr 2008;3(2):el692.

[0103] 2. Vincents B, von Pawel -Rammingen U, Bjbrck L, Abrahamson M. Enzymatic characterization of the streptococcal endopeptidase, IdeS, reveals that it is a cysteine protease with strict specificity for IgG cleavage due to exosite binding. Biochemistry. 14 dec 2004;43(49): 15540-9.

[0104] 3. Jordan SC, Lorant T, Choi J, Kjellman C, Winstedt L, Bengtsson M, et al. IgG Endopeptidase in Highly Sensitized Patients Undergoing Transplantation. N Engl J Med. 3 aout 2017;377(5):442-53.

[0105] 4. Bou-Jaoudeh M, Delignat S, Daventure V, Astermark J, Levesque H, Dimitrov JD, et al. The IgG-degrading enzyme, Imlifidase, restores the therapeutic activity of FVIII in inhibitor-positive hemophilia A mice. Haematol ogica. 1 mai 2023; 108(5): 1322-34.

[0106] 5. Nandakumar KS, Johansson BP, Bjbrck L, Holmdahl R. Blocking of experimental arthritis by cleavage of IgG antibodies in vivo. Arthritis Rheum, oct 2007;56(10):3253-60.

[0107] 6. Tradtrantip L, Asavapanumas N, Verkman AS. Therapeutic cleavage of anti- aquaporin-4 autoantibody in neuromyelitis optica by an IgG-selective proteinase. Mol Pharmacol, juin 2013;83(6): 1268-75.

[0108] 7. Yang R, Otten MA, Hellmark T, Collin M, BjbrckL, Zhao MH, et al. Successful treatment of experimental glomerulonephritis with IdeS and EndoS, IgG-degrading streptococcal enzymes. Nephrol Dial Transplant Off Publ Eur Dial Transpl Assoc - Eur Ren Assoc, aout 2010;25(8):2479-86.

[0109] 8. Takahashi R, Yuki N. Streptococcal IdeS: therapeutic potential for Guillain- Barre syndrome. Sci Rep. 21 juill 2015;5:10809. 9. Kizlik-Masson C, Deveuve Q, Zhou Y, Vayne C, Thibault G, McKenzie SE, et al. Cleavage of anti-PF4 / heparin IgG by a bacterial protease and potential benefit in heparin- induced thrombocytopenia. Blood. 30 mai 2019;133(22):2427-35.

[0110] 10. Leborgne C, Barbon E, Alexander JM, Hanby H, Delignat S, Cohen DM, et al. IgG-cleaving endopeptidase enables in vivo gene therapy in the presence of anti-AAV neutralizing antibodies. Nat Med. juill 2020;26(7): 1096-101.

[0111] 11. Bou-Jaoudeh M, Mimoun A, Delignat S, Peyron I, Capdevila L, Daventure V, et al. Imlifidase, a new option to optimize the management of patients with hemophilia A on emicizumab. J Thromb Haemost JTH. oct 2023;21(10):2776-83.

[0112] 12. Winstedt L, Jarnum S, Nordahl EA, Olsson A, Runstrbm A, Bockermann R, et al. Complete Removal of Extracellular IgG Antibodies in a Randomized Dose-Escalation Phase I Study with the Bacterial Enzyme IdeS - A Novel Therapeutic Opportunity. PLoS ONE. 15 juill 2015;10(7):e0132011.

[0113] 13. Duivelshof BL, Murisier A, Camperi J, Fekete S, Beck A, Guillarme D, et al. Therapeutic Fc-fusion proteins: Current analytical strategies. J Sep Sci. 2021;44(l):35-62.

[0114] 14. Ober RJ, Radu CG, Ghetie V, Ward ES. Differences in promiscuity for antibody-FcRn interactions across species: implications for therapeutic antibodies. Int Immunol. 1 dec 2001 ; 13(12): 1551-9.

[0115] 15. Chalayer E, Gramont B, Zekre F, Goguyer-Deschaumes R, Waeckel L, Grange L, et al. Fc receptors gone wrong: A comprehensive review of their roles in autoimmune and inflammatory diseases. Autoimmun Rev. mars 2022;21(3): 103016.

[0116] 16. Grubb JH, Vogler C, Tan Y, Shah GN, MacRae AF, Sly WS. Infused Fc-tagged beta-glucuronidase crosses the placenta and produces clearance of storage in utero in mucopolysaccharidosis VII mice. Proc Natl Acad Sci U S A. 17 juin 2008;105(24):8375-80.

[0117] 17. Lorant T, Bengtsson M, Eich T, Eriksson BM, Winstedt L, Jamum S, et al. Safety, immunogenicity, pharmacokinetics, and efficacy of degradation of anti-HLA antibodies by IdeS (imlifidase) in chronic kidney disease patients. Am J Transplant Off J Am Soc Transplant Am Soc Transpl Surg. nov 2018; 18(11):2752-62.

[0118] 18. Frampton JE. Efmoroctocog Alfa: A Review in Haemophilia A. Drugs. 2021;81(17):2035-46.

[0119] 19. Lamb YN, Hoy SM. Eftrenonacog Alfa: A Review in Haemophilia B. Drugs. 2023;83(9):807-18. 20. Zalevsky J, Chamberlain AK, Horton HM, Karki S, Leung IWL, Sproule TJ, et al. Enhanced antibody half-life improves in vivo activity. Nat Biotechnol. fevr 2010;28(2): 157-9.

[0120] 21. Soveri I, Mblne J, Uhlin F, Nilsson T, Kjellman C, Sonesson E, et al. The IgG- degrading enzyme of Streptococcus pyogenes causes rapid clearance of anti-glomerular basement membrane antibodies in patients with refractory anti-glomerular basement membrane disease. Kidney Int. nov 2019;96(5): 1234-8.

[0121] 22. Heo YA. Efgartigimod: First Approval. Drugs, fevr 2022;82(3):341-8.

[0122] 23. Ulrichts P, Guglietta A, Dreier T, van Bragt T, Hanssens V, Hofman E, et al. Neonatal Fc receptor antagonist efgartigimod safely and sustainably reduces IgGs in humans. J Clin Invest. 128(10):4372-86.

[0123] 24. Kiessling P, Lledo-Garcia R, Watanabe S, Langdon G, Tran D, Bari M, et al. The FcRn inhibitor rozanolixizumab reduces human serum IgG concentration: A randomized phase 1 study. Sci Transl Med. nov 2017;9(414):eaanl208. 25. Wenig K, Chatwell L, von Pawel -Rammingen U, Bjbrck L, Huber R,

[0124] Sondermann P. Structure of the streptococcal endopeptidase IdeS, a cysteine proteinase with strict specificity for IgG. Proc Natl Acad Sci U S A. 14 dec 2004; 101(50): 17371-6.

Claims

- 23 -CLAIMS:

1. An IgG-degrading enzyme-Fc fusion protein comprising at least one IgG-degrading enzyme fused to an Fc part of an antibody wherein the Fc part comprises at least one mutation.

2. The IgG-degrading enzyme-Fc fusion protein according to the claim 1 wherein the IgG- degrading enzyme-Fc fusion protein comprises two IgG-degrading enzymes.

3. The IgG-degrading enzyme-Fc fusion protein according to the claim 1 wherein the IgG- degrading enzyme-Fc fusion protein is fused to the Fc part with a linker and a hinge region.

4. The IgG-degrading enzyme-Fc fusion protein according to the claims 1 to 3 wherein the IgG enzyme is the IdeS from Streptococcus pyogenes.

5. he IgG-degrading enzyme-Fc fusion protein according to the claims 1 to 4 wherein the hinge region comprises the peptidic sequence as set for SEQ ID NO: 1 : KPCICTVPEVS.

6. The IgG-degrading enzyme-Fc fusion protein according to the claims 1 to 4 wherein the linker of IgG-degrading enzyme-Fc fusion protein has the following sequence ((G)nS)x wherein n is between 1 and 10 and x between 1 and 5.

7. The IgG-degrading enzyme-Fc fusion protein according to the claims 1 to 4 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, Y407V M252Y, S254T, T256E, G237D, P238D, H268D, P271G or A330R or a combination thereof.

8. The IgG-degrading enzyme-Fc fusion protein according to the claim 1 for use as a medicament.

9. The IgG-degrading enzyme-Fc fusion protein for use according to the claim 7 wherein the IgG-degrading enzyme-Fc fusion protein is used for the treatment of a disease caused by a loss of function or activity of a protein.

10. The IgG-degrading enzyme-Fc fusion protein for use according to the claim 7 wherein the IgG-degrading enzyme-Fc fusion protein is used in gene therapy to remove anti- AAV antibodies for example.

11. A method to prepare F(ab’)2 from Immunoglobulin G (IgG) comprises the steps of: a. incubating the IgG of interest with the IgG-degrading enzyme-Fc fusion protein of the claim 1 for at least 1 hour; b. incubating the sample resulting from the step 1 on a mini-column of protein G or protein A beads during 10 to 60 min; c. centrifugating and recovering of the purified F(ab’)2.

Images