The IgG-degrading enzyme, Imlifidase, restores the therapeutic activity of FVIII in inhibitor-positive hemophilia A mice

Abstract

Neutralizing anti-factor VIII (FVIII) antibodies, known as FVIII inhibitors, represent a major drawback of replacement therapy in persons with congenital hemophilia A (PwHA), rendering further infusions of FVIII ineffective. FVIII inhibitors can also appear in non-hemophilic individuals causing acquired hemophilia A (AHA). The use of non-FVIII bypassing agents in cases of bleeds or surgery in inhibitor-positive patients is complicated by the lack of reliable biological monitoring and increased thrombotic risk. Imlifidase (IdeS) is an endopeptidase that degrades human immunoglobulin G (IgG); it was recently approved for hyperimmune patients undergoing renal transplants. Here we investigated the ability of IdeS to eliminate FVIII inhibitors in vitro and in a model of inhibitor-positive HA mice. IdeS cleaved anti-FVIII plasma IgG from PwHA and AHA patients, and hydrolyzed recombinant human anti-FVIII IgG independently from their subclass or specificity for the A2, A3, C1 or C2 domains of FVIII. In HA mice passively immunized with recombinant human anti-FVIII IgG, IdeS restored the hemostatic efficacy of FVIII, as evidenced by the correction of the bleeding tendency. Our results provide the proof of concept for the transient removal of FVIII inhibitors by IdeS, thereby opening a therapeutic window for efficient FVIII replacement therapy in inhibitor-positive patients.

Introduction

Up to 30% of the persons with hemophilia A (PwHA) may develop neutralizing anti-factor VIII (FVIII) allo-antibodies (FVIII inhibitors) after replacement therapy,¹ with approximately 60% exhibiting high inhibitory titers. The onset of FVIII inhibitors is favored by genetic (ethnicity, mutations in the F8 gene) and environmental (exposure) factors.² Neutralizing auto-antibodies against FVIII can also appear in individuals with no previous history of bleeding, typically in elderly individuals or in the postpartum period,³ causing acquired hemophilia A (AHA).

The management of clinically relevant acute bleeds and/or surgeries in patients with high FVIII inhibitor titers is particularly challenging. Bypassing agents (BPA), such as re-combinant activated FVII (rFVIIa) and activated prothrombin complex concentrates (aPCC), or recombinant porcine FVIII, are recommended as first-line treatments.

Apart from their proven efficacy, BPA have major drawbacks, including the need for frequent dosing, the lack of reliable biomarkers for hemostatic efficacy other than clinical improvement, and the increased thrombotic risk.^4–7 The development of emicizumab, a humanized bispecific antibody that mimics the co-factor function of FVIII, has revolutionized prophylaxis for PwHA and inhibitors.^8,9 Emicizumab dramatically reduces annualized bleeding rates with once-weekly or fewer subcutaneous injections.¹⁰ However, emicizumab does not completely restore hemo-stasis, and standard hemostatic treatments are still required for persons undergoing breakthrough bleeds or surgery.^11,12 Further, the concomitant use of emicizumab and BPA, particularly aPCC, carries an increased risk of thrombotic microangiopathies and thromboembolic events.¹³ Elderly hospitalized patients with acquired HA (PwAHA) with multiple comorbidities are also at increased risks of arterial and venous thrombotic events while receiving high BPA doses.^3,7 As a result, on-demand replacement therapy with exogenous FVIII remains the best option for managing acute bleeds or surgery in PwHA and PwAHA. Eliminating neutralizing anti-FVIII antibodies to temporarily restore the hemostatic efficacy of FVIII while avoiding the use of BPA is an appealing new therapeutic option in patients with FVIII inhibitors.

Streptococcus pyogenes, an important human pathogen, produces IdeS (immunoglobulin G [IgG]-degrading enzyme of Streptococcus pyogenes) as a defense mechanism against antibody attack and complement activation.¹⁴ IdeS is a cysteine proteinase that can cleave all four human IgG subclasses with a unique degree of specificity below the disulfide bridge in the hinge region.¹⁵ However, IdeS only partially hydrolyzes mouse IgG.¹⁶ IdeS sequentially cleaves the two heavy chains of IgG with different kinetics, thus releasing the F(ab')₂ fragment from the Fc fragment. A recombinant IdeS is commercially available (Imlifidase, Ideferix^®) and is the only desensitization treatment European Medicines Agency-approved for kidney transplant patients with donor-specific antibodies.¹⁷ IdeS is also being studied for its therapeutic potential in several autoimmune diseases^18,19,20 as well as in oncology and gene therapy.^21,22

Here, we hypothesized that the cleavage of circulating IgG by IdeS, leading to the fast, though temporary, clearance of IgG, may provide a new therapeutic opportunity for patients with FVIII inhibitors. We demonstrate that IdeS efficiently hydrolyzes polyclonal anti-FVIII IgG in patients’ plasma and monoclonal recombinant human anti-FVIII IgG (anti-FVIII rhIgG) in vitro. We developed a mouse model of inhibitor-positive severe HA by passively immunizing HA mice with anti-FVIII rhIgG. IdeS restored the hemostatic efficacy of FVIII infusions in inhibitor-positive HA mice. Our results provide the proof of concept for temporarily removing FVIII inhibitors by IdeS and opening a therapeutic window for efficient FVIII replacement therapy and better management of patients with FVIII inhibitors.

Methods

Plasma samples from patients with congenital or acquired hemophilia A

Plasma from 102 PwHA was obtained from the MIBS registry (Malmö International Brother Study) that includes siblings with and without a history of inhibitors.²³ Plasma from 43 PwAHA was obtained from the SACHA (Surveillance des Auto antiCorps au cours de l’Hémophilie Acquise) French registry at the time of inclusion with titers ≥1 Bethesda units (BU)/mL.⁷ Procedures were in accordance with the ethical standards of the responsible committees on human experimentation for both cohorts and with the Declaration of Helsinki. MIBS and SACHA are registered (clinicaltrails. gov. Identifier: NCT00231751 and NCT00213473, respectively).^7,23

Generation of recombinant human anti-FVIII immunoglobulin G

Four anti-FVIII rhIgG_k were produced: BOIIB2 (patent US20070065425A1), KM41,²⁴ LE2E9²⁵ and BO2C11²⁶ that are specific for the A2, A3, C1 and C2 domains of FVIII, respectively. The genes encoding the VH regions of IgG and the VL regions of Ig_k were cloned in eukaryotic expression vectors (kindly provided by Dr. Hugo Mouquet, INSERM, Paris). The corresponding IgG1_k and IgG4_k were produced in HEK293 cells using the Expi293 protocol (Thermo Scientific) and purified from the culture supernatant by affinity chromatography on protein G-agarose beads (GE Healthcare). Monoclonal IgG were validated by SDS-PAGE, enzyme-linked immunosorbant assay (ELISA) and modified Nijmegen-Bethesda assay.

Determination of anti-FVIII antibody inhibitory titers

The inhibitory activity of the anti-FVIII rhIgG was measured using the modified Nijmegen-Bethesda assay (MNBA).²⁷ Monoclonal IgG in phosphate-buffered saline (PBS, pH 7.4, Life Technologies) or in mouse plasma were serially diluted in veronal buffer and incubated vol/vol with a standard pool of human plasma (Siemens Healthcare), used as a source of FVIII, for 2 hours (h) at 37°C. The residual pro-coagulant FVIII activity (FVIII:C) was measured using a chromogenic assay following the manufacturer’s instructions (Siemens Healthcare). In the case of purified IgG, the inhibitory activity of the IgG was expressed in BU/mg IgG, defined as the inverse of the concentration of IgG needed to inhibit 50% of FVIII:C. In the case of IgG in mouse plasma, the inhibitory titers were expressed in BU/mL, defined as the plasma dilution that neutralizes 50% of normal plasma FVIII:C. Titers ≥0.6 BU/mL were considered as positive.

Generation of Imlifidase

The DNA sequence encoding IdeS from S. pyogenes was obtained from Geneart (Thermo Scientific). It was cloned into a pEX-N-His-tagged expression vector for expression in E. coli strain BL21. Protein expression was induced by 0.5 mM IPTG for 4 h at 37°C. Proteins were purified by immobilized metal affinity chromatography (HisTrap FF column, GE Healthcare). Buffer was exchanged with PBS using a PD-10 desalting column (GE Healthcare) and endotoxins were removed using the Pierce endotoxin removal kit (Thermo Scientific). Integrity of IdeS was confirmed by SDS-PAGE and concentration was determined using NanoDrop™ with a 50,880 M^-1cm^-1 extinction coefficient.

Hydrolysis of immunglobulin G by Imlifidase

For IgG in patients’ plasma, 10-fold diluted plasma was incubated in PBS alone or with 0.54 mM IdeS (yielding an approximate 12:1 molar ratio of IgG:IdeS) for 24 h at 37°C. For anti-FVIII rhIgG1_k and rhIgG4_k, IgG (1.66 mM) were incubated alone or with IdeS (0.14 mM) at a 12:1 IgG:IdeS molar ratio for 24 h at 37°C.

Mouse model of inhibitor-positive severe hemophilia A

Eight- to 12-week-old male and female exon 16 FVIII-deficient mice²⁸ on a C57BL/6 background (HA mice) were housed and handled in accordance with French regulations and the experimental guidelines of the European Community (Comité d’éthique en expérimentation animale no.005, protocol APAFIS#24748-2020032014465347). Naive HA mice were passively immunized by intravenous injection of the human recombinant BO2C11 IgG1_k (600 BU/kg). For determination of IgG half-life, blood was collected at 5 minutes (min), 4 h, 1, 2, 5, and 7 days post-injection. Inhibitory titers were measured in plasma using MNBA.

In vivo efficacy of Imlifidase in inhibitor-positive hemophilia A mice

HA mice were passively immunized by intravenous injection of BO2C11 IgG1_k alone at 1,200 BU/kg or 24,000 BU/kg, or of equimolar amounts of BOIIB2, KM41, LE2E9 and BO2C11 in IgG1_k format (2,800 BU/kg). Mice were treated 1 day later by intravenous injection of IdeS (0.6 mg/kg or 0.29 mM) or PBS as control. When indicated, mice received a second injection of IdeS 24 h later. Residual levels of intact anti-FVIII rhIgG, partially single-cleaved intermediate IgG, F(ab’)₂ fragments, and inhibitory activities were determined by ELISA and MNBA in plasma collected up to 6 days after IdeS or PBS injection.

Evaluation of bleeding tendency and hemostasis

Inhibitor-positive HA mice treated with PBS or IdeS were injected with therapeutic recombinant human FVIII (Helixate^®, 200 Ul/kg) via the retro-orbital route 3 days after IdeS or PBS injection. The bleeding tendency and hemostatic parameters were analyzed 2 h later. The bleeding tendency was evaluated using a standardized tail clipping assay in isoflurane-anesthetized mice (2% isoflurane in 30% O₂ and 70% N₂O; flow: 1 L/min) maintained at 37°C on a heating pad. Three mm of the distal tail was amputated and blood was collected over 10 min. Blood loss in each sample was calculated from a standard curve, as already described.²⁹ The FVIII:C was measured in plasma using a chromogenic test (Siemens Healthcare). Thrombin generation in platelet-poor plasma (PPP) was measured using the Calibrated Automated Thrombrogram and PPP Reagent Low (Stago) as already described,³⁰ except that PPP was diluted 1/6 in HEPES-buffered saline containing 0.5% bovine serum albumin (BSA).

SDS-PAGE and western blot

Purified IgG or IgG in human plasma (5 mg), incubated alone or with IdeS, were separated by SDS-PAGE in NuPAGE 4-12% gradient Bis-Tris protein gels (Thermo Scientific) under non-reducing conditions, and transferred to nitrocellulose membranes using a semi-dry iBlot system (Invitrogen). Membranes were blocked and incubated with a polyclonal goat anti-human F(ab’)₂ fragment-specific antibody (Invit-rogen) or a polyclonal rabbit anti-human Fc-specific antibody (Sigma-Aldrich). Bound antibodies were revealed using appropriate secondary antibodies: an horseradish peroxidase (HRP)-coupled rabbit anti-goat IgG (R&D System) or an HRP-coupled goat anti-rabbit IgG (Cell signaling), and the Pierce™ ECL Western Blotting Substrate and iBright™ FL1000 Imaging System (Thermo Scientific).

Human anti-FVIII immunoglobulin G enzyme-linked immunosorbant assay

ELISA plates (Maxisorp, Nunc) were coated with rhFVIII (Ad-vate^®, 2.5 mg/mL). Patients’ plasma or purified anti-FVIII rhIgG were added to the wells. Bound anti-FVIII IgG or F(ab’)₂ were revealed using an HRP-labeled mouse monoclonal antibody specific for human Fcγ (Southern Biotech) or an HRP-labeled goat anti-human IgG F(ab')₂ fragment secondary antibody (Thermo Scientific), respectively, and the o-phenylenediamine dihydrochloride (OPD, Sigma-Aldrich) substrate. Absorbances were read at 492 nm. The titers of anti-FVIII IgG in patients’ plasma were defined as the highest dilution of plasma yielding an optical density (OD) ≥cutoff. The cutoff was computed as the mean OD calculated for the plasma from 22 healthy individuals + 95% percentile^*standard deviation.³¹

Human immunoglobulin G and F(ab’)2 fragments enzyme-linked immunosorbant assay

ELISA plates were coated with a goat anti-human Ig k antibody (2.5 mg/mL; Southern Biotech). Purified anti-FVIII rhIgG or mouse plasma containing anti-FVIII rhIgG were added to the wells. Bound IgG were revealed using an HRP-labeled mouse monoclonal antibody specific for human Fcγ (Southern Biotech). Bound F(ab')₂ fragments were detected using an HRP-labeled goat anti-human IgG F(ab')₂ secondary antibody (Thermo Scientific). Absorbance was read at 492 nm after addition of the OPD substrate. Concentrations were calculated in mg/mL using BO2C11 as a standard.

Results

Imlifidase hydrolyzes anti-FVIII immunglobulin G in plasma from persons with hemophilia A and persons with acquired hemophilia A

We investigated whether IdeS hydrolyzes IgG in the plasma from 43 PwAHA and 102 PwHA. Twenty-two of the 102 PwHA plasma tested positive for FVIII inhibitors (mean ± standard deviation [SD]: 9.8±15.6 BU/mL, ranging from 0.6 to 63 BU/mL; Figure 1). Inhibitor-negative PwHA had titers below 0.6 BU/mL. Ten-fold diluted plasma was incubated alone or with IdeS (0.54 mM) for 24 h at 37°C. Samples from five randomly selected PwHA were analyzed by western blot to detect F(ab’)₂ and Fc fragments before and after IdeS treatment. As expected,³² incubation in the presence of IdeS led to a close to complete degradation of total IgG and the detection of traces of scIgG, together with the accumulation of F(ab’)₂ and Fc fragments at 100 and 25 kDa, respectively (Figure 1A).

We confirmed the cleavage of anti-FVIII IgG in plasma from PwHA and PwAHA using an anti-FVIII IgG ELISA. As re-ported,³³ some inhibitor-negative PwHA had detectable levels of FVIII-binding IgG, but at significantly lower levels than inhibitor-positive PwHA (Figure 1B; P<0.0001). Treatment with IdeS resulted in undetectable anti-FVIII IgG titers in the plasma from inhibitor-positive and inhibitornegative PwHA, and PwAHA (P<0.0001 in all cases). This is consistent with the release of the Fc fragments from the F(ab’)₂ fragments of the IgG upon IdeS-mediated cleavage and the associated loss of detection of the bound anti-FVIII F(ab’)₂ fragments by the anti-human Fc antibody in ELISA.

Imlifidase hydrolyzes anti-FVIII immunoglobulin G irrespective of their subclass and epitope specificity

Anti-FVIII IgG in PwHA and PwAHA belong in the large majority to the IgG1 and IgG4 subclasses.³³ In order to further decipher the action of IdeS on anti-FVIII IgG, we generated four monoclonal anti-FVIII rhIgG expressed in both the IgG1_k and IgG4_k formats, specific for the A2, A3, C1 or C2 domain of human FVIII.

The recombinant IgG1_k and IgG4_k versions of each monoclonal IgG exhibited identical dose-dependent binding to FVIII in ELISA (Figure 2A) and neutralized FVIII:C within identical orders of magnitude (Table 1). The four IgG were cleaved equally by IdeS, irrespective of their epitope specificity or IgG subclass. Indeed, incubation of each IgG with IdeS at a 12:1 molar excess for 24 h at 37°C resulted in the complete disappearance of the intact IgG and the generation of the F(ab’)₂ and Fc fragments (Figure 2B). Time-dependent analyses of IgG cleavage by IdeS, performed using BO2C11, demonstrated that IgG1_k and IgG4_k are cleaved with similar kinetics. More than 90% of the IgG were hydrolyzed as scIgG within the first 5 min of in vitro incubation and fully hydrolyzed F(ab’)₂ fragments were detected from 20 min onwards (Figure 2C). The physical dissociation between F(ab’)₂ and Fc fragments upon IdeS cleavage was confirmed by ELISA (Figures 3A; Online Supplementary Figure S1). Under static conditions (i.e., in a test tube), the F(ab’)₂ fragments of neutralizing anti-FVIII IgG, generated upon IdeS cleavage, are not eliminated and are presumably still able to neutralize the pro-coagulant activity of FVIII. Indeed, samples of native or IdeS-cleaved BO2C11 IgG neutralized FVIII:C to a similar extent in vitro in a MNBA, irrespective of the IgG subclass (Figure 3B). Accordingly, plasma from an inhibitor-positive PwHA neutralized FVIII:C to similar extent in vitro following incubation alone or with IdeS (Figure 3C).

Figure 1.Hydrolysis of immunglobulin G in the plasma from patients with congenital and acquired hemophilia A. (A) Plasma samples obtained from 5 congenital hemophilia A (HA) patients (P1-P5, MIBS cohort) were pre-incubated for 24 hours (h) at 37°C with Imlifidase (IdeS) (0.54 µM) or phosphate-buffered saline (PBS), and subjected to western blot. Immunoglobulin G (IgG) were recognized with a F(ab’)₂-specific antibody (top) or a Fc-specific antibody (bottom). Molecular weight markers are shown at the left of the blot. The predicted molecular weights of intact IgG, scIgG, F(ab’)₂ and Fc fragments are shown. (B) Ten-fold diluted plasma from 43 patients with acquired HA (PwAHA) and from 102 patients with mild, moderate or severe HA, with (Inh+ PwHA, n=22) or without (Inh- PwHA, n=80) FVIII inhibitors, were incubated for 24 h at 37°C with IdeS (0.54 µM) or PBS. Inhibitor-positive patients were defined by inhibitory titers ≥ 0.6 Bethesda units (BU)/mL. The graph depicts the titers of FVIII-specific IgG. Plasma samples were diluted at least 1:20. Samples that did not give a positive signal at this minimum dilution were considered as negative (ND: not detectable). Statistical differences were assessed using the two-sided Mann-Whitney test.

Figure 2.Cleavage of human monoclonal anti-FVIII immunglobulin G by Imlifidase. (A) Validation of human re-combinant monoclonal anti-FVIII im-munoglobulin G (IgG). Four human monoclonal neutralizing anti-FVIII IgG were cloned and expressed as IgG1_k (left panel) or IgG4_k (right panel). Their binding to FVIII was validated in a human anti-FVIII IgG enzyme-linked immunosorbant assay. Results are expressed in arbitrary units (AU, mean ± standard deviation from 3 independent experiments) using the optical densities measured at 492 nm. (B, C) IdeS-mediated hydrolysis of human monoclonal anti-FVIII IgG. The monoclonal anti-FVIII IgG1_k (left panel) or IgG4_k (right panel) were incubated with Imlifidase (IdeS) at a 12 IgG:1 IdeS molar ratio (1.66 µM IgG vs. 0.14 µM IdeS) for 24 hours (panels B) or for different periods of time (C) at 37°C. Samples were separated by SDS-PAGE under non-reducing conditions. Molecular weight markers and the predicted molecular weights of intact IgG, scIgG, F(ab’)₂ and Fc fragments are shown on the left and right of each gel, respectively.

Table 1.Inhibitory activity of monoclonal anti-FVIII IgG1_k and IgG4_k.

Validation of a mouse model of inhibitor-positive hemophilia A

In order to develop a mouse model of inhibitor-positive HA, we first determined the half-life of BO2C11 IgG1_k, used as model IgG, in FVIII-deficient mice. The intravenous injection of 600 BU/kg of BO2C11 IgG1_k was followed by a two-phase elimination pattern. Fitting the experimental data to a double exponential decay curve yielded fast and slow elimination half-lives of 0.2 and 9 days, respectively (Figure 4A). Inhibitory titers measured in mice plasma were 12.8±1.2 BU/mL at 5 min and 5.7±1.1 BU/mL at 24 h, representing a 45% reduction. The inhibitory titers remained relatively stable for the next 6 days (i.e., 3.3±1.9 BU/mL at day 7).

Figure 3.Inhibitory activity of F(ab’)₂ fragments generated upon Imlifidase cleavage. (A) Binding of BO2C11 immunoglobulin G (IgG) to FVIII following cleavage by Imlifidase (IdeS). BO2C11 IgG1_k (left panel) and IgG4_k (right panel) at 1.66 µM were incubated alone or with IdeS (0.14 µM) for 24 hours (h) at 37°C (12 IgG:1 IdeS molar ratio). The binding of the intact IgG and/or scIgG to FVIII and that of F(ab’)₂ fragments (showed as insets) was validated by enzyme-linked immunosorbant assay. Results are expressed in arbitrary units (AU, representative of 2 experiments) from optical density measured at 492 nm. (B) Inhibitory activity of IdeS-cleaved BO2C11 IgG. The inhibitory activity of BO2C11 IgG1_k and IgG4_k incubated in phosphate-buffered saline (PBS) alone (-) or with IdeS (+) was measured in a Bethesda assay. As a control, IdeS was introduced alone in the assay. Values depict the respective % of residual inhibitory activities as compared to the activity measured in the absence of IdeS for IgG1_k and for IgG4_k (means ± standard deviation of 3 independent experiments). (C) Inhibitory activity of IdeS-cleaved polyclonal anti-FVIII IgG. Plasma (1/10) from an inhibitor-positive patient with hemophilia A (PwHA) was incubated for 24 h at 37°C with IdeS (0.54 µM) or PBS. The binding to FVIII of intact IgG/scIgG and F(ab’)₂ fragments was measured by enzyme-linked immunsorbant assay. Results are expressed in AU using the optical densities measured at 492 nm. The inhibitory titer was measured in the plasma treated with PBS or IdeS using a modified Nijmegen-Bethesda assay (n=2, mean ± standard deviation).

In vivo Imlifidase efficacy and pharmacokinetics

In a first series of experiments, HA mice were passively immunized with 1,200 BU/kg of BO2C11 IgG1_k. This amount of IgG1_k achieved reproducible inhibitory titers of 9.4±2.3 BU/mL and 5.2±2.7 BU/mL 24 and 96 h later, respectively (Figure 4D), titers for which administration of therapeutic FVIII is inefficient in patients. Inhibitor-positive HA mice were treated with 0.6 mg/kg IdeS 24 h after the injection of BO2C11 IgG1_k (Figure 4B). As compared to PBS-treated control mice, IdeS-treated mice experienced a drastic 94% drop in IgG levels (either intact IgG or scIgG that are both detected in the human IgG ELISA) 6 h after IdeS injection (Figure 4C). The rapid loss of detection of IgG in mouse plasma was associated with a slower disappearance of the inhibitory activity towards FVIII that was still detectable at least 24 h following IdeS injection (Figure 4D). Interestingly, the progressive decrease in inhibitory activity in plasma demonstrated a statistically significant linear correlation with the disappearance of the F(ab’)₂ fragments of BO2C11 from the circulation (Figures 4E, F; r²=0.93; P<0.0001). The inhibitory activity was below the detection threshold of the assay 2-3 days after IdeS injection. Similar results were obtained when HA mice were passively immunized with a pool of BOIIB2, KM41, LE2E9 and BO2C11 IgG1_k (Online Supplementary Figure S2).

Fitting the experimental data of F(ab’)₂ catabolism (Figure 4E), from 6 h following IdeS injection onwards, to a one-phase decay curve yielded a 11.7 h half-life of human F(ab’)₂ fragments in mice (range, 10.1-13.1 h).

Figure 4.Imlifidase-mediated elimination of a FVIII inhibitor in inhibitor-positive hemophilia A mice. (A) Half-life of BO2C11 immunoglobulin G (IgG1_k) in hemophilia A (HA) mice. C57BL/6 HA mice (n=5) were passively immunized with BO2C11 IgG1_k (600 Bethesda units [BU]/kg). The graph depicts the inhibitory activity towards FVIII measured in plasma over time. (B) HA mice (n=6 per group) were passively immunized with 1,200 BU/kg of BO2C11 IgG1_k to reach 10 BU/mL after 24 hours (h), and injected with Imlifidase (IdeS) (0.6 mg/kg, 0.29 µM) or phosphate-buffered saline (PBS) 24 h later. (C-E). The levels of intact IgG and/or scIgG (C, IgG concentration at 24 h: 5.1±0.3 nM), the inhibitory titers (D) and the levels of F(ab’)₂ fragments (E) were determined over time by enzyme-linked immunosorbant assay and Bethesda assay (n=4, mean ± standard deviation). The dotted lines represent the respective detection thresholds: 0.03 µg/mL, 0.6 BU/mL and 0.08 µg/mL. (F) The graph shows, for the condition where mice were treated with IdeS, the plasma levels of IgG (C) and F(ab’)₂ fragments (E) as a function of the inhibitory activity in plasma (D) at 30, 48, 72 and 96 h following BO2C11 injection. The experimental data were interpolated using a linear curve (R²: goodness of fit). The grey zone in panel (D) depicts inhibitory titers below 5 BU/mL, a titer that is compatible with the hemostatic efficacy of exogenous FVIII.

Imlifidase corrects the bleeding tendency and restores FVIII hemostatic efficacy

In order to provide proof of concept towards the transient removal of FVIII inhibitors by IdeS, thereby opening a therapeutic window for efficient FVIII replacement therapy, inhibitor-positive HA mice were given 200 IU/kg of FVIII 96 h (3 days) after IdeS or PBS treatment (Figure 5A). Two hours later, the bleeding tendency and hemostatic efficacy of therapeutic FVIII were evaluated. The blood loss that followed tail tip amputation of IdeS-treated mice was significantly lower than that measured in PBS-treated mice (Figure 5B; 13±26 mL vs. 74±65 mL; P=0.0047), but was not different from that measured in naive inhibitor-negative HA mice that had received FVIII alone (21±16 mL). The reduction in blood loss was explained by a restoration of the hemostatic efficacy of therapeutic FVIII. FVIII:C recovery in IdeS-treated mice was significantly higher than that in PBS-treated mice (Figure 5C; 84.2±29.7% vs. 2.0±1.5%; P=0.0015) and did not differ from that in naive inhibitornegative mice injected with FVIII alone (112.4±58.7%). Accordingly, thrombin generation was significantly increased in IdeS-treated mice as compared to PBS-treated mice (Figures 5D, E; thrombin peak: 52±8 nM vs. 25±19 nM; P=0.0386).

Imlifidase efficacy in the context of very high inhibitory titers

In order to mimic the situation of patients with very high inhibitory titers, we passively immunized HA mice with 24,000 BU/kg of BO2C11 IgG1_k to reach inhibitory titers of 171±48 BU/mL and 97±7 BU/mL 24 and 168 h later, respectively. Mice then received either one or two injections of IdeS (0.6 mg/kg) with a 24-h interval. The circulating levels of IgG/scIgG and F(ab’)₂ fragments and the inhibitory titers were followed over time. The loss of detection of IgG/scIgG was faster than the decrease in detection of circulating F(ab’)₂ fragments and inhibitory activity (Figure 6). As compared to PBS-treated mice, the decrease in inhibitory activity was 27-fold and 68-fold 3 and 6 days after a single IdeS injection, respectively. Redosing of IdeS yielded a further reduction in inhibitory activity below 5 BU/mL (P<0.05 at 96 and 168 h).

Discussion

The promising therapeutic effect of IdeS has already been suggested in several preclinical models of human autoimmune diseases,^34–37 and in the context of gene therapy.²¹ In humans, IdeS potency has been explored in patients with anti-HLA allo-antibodies undergoing kidney transplant^38,39 and in patients with Goodpasture syndrome and auto-antibodies directed against the non-collagenous domain of the a3 chain of type IV collagen.¹⁹ Our work further substantiates the efficacy of IdeS treatment in both alloand auto-immune settings. There are alternatives to IdeS for removing pathogenic antibodies, such as plasmapheresis,⁴⁰ molecules that block the neonatal Fc receptor (FcRn),^41,42 immunosuppressive drugs,^43,44 or therapeutic antibodies that deplete B cells.⁴⁵ However, IdeS offers several benefits in terms of specificity and efficacy, fast elimination rate, and long-lasting effects. Importantly, the presence of pre-existing anti-IdeS IgG or the onset of an anti-IdeS immune response, which peaks around 2 weeks after IdeS administration, do not preclude repeated dosing of IdeS for several consecutive days, or at a 6-month distance from the first treatment.⁴⁶ Furthermore, IdeS can cleave anti-IdeS antibodies with an IgG isotype,²¹ and neutralization of IdeS by anti-IdeS antibodies has never been proven convincingly.

All pathogenic IgG hydrolyzed by IdeS in the disorders and disease models listed above are specific for antigens exposed at the surface of cells, platelets or viruses. In contrast, FVIII circulates in the blood. The soluble/membrane location of the antigen targeted by the pathogenic IgG determines the functional outcome of IdeS-mediated cleavage. Indeed, IdeS hydrolyzes IgG in two steps, starting with a rapid cleavage of one of the two heavy chains to generate a scIgG, followed by a slow cleavage of the second heavy chain that releases the F(ab’)₂ fragment from the Fc fragment.^14,15 While scIgG lose their capacity to bind and activate complement, as well as to mediate antibody-dependent cell cytotoxicity (ADCC), they retain their capacity to bind their target antigen and have a normal half-life owing to the preserved binding to the FcRn.¹⁴ In contrast, the F(ab’)₂ fragments of completely digested IgG lose all Fc fragment-mediated functions but maintain antigen-binding (and possibly neutralizaing capacity during their life span in the circulation. As a result, IdeS-mediated IgG cleavage has an immediate functional repercussion when the pathogenic IgG are directed against membrane antigens and exert their pathogenic effects by complement activation, phagocytosis or ADCC. In contrast, when the pathogenic IgG neutralize soluble antigens, as is the case of inhibitory anti-FVIII IgG, the functional consequence of IdeS-mediated cleavage is delayed until elimination of the F(ab’)₂ fragments from the circulation. Hence, in test tubes, the mere in vitro cleavage of monoclonal and polyclonal anti-FVIII IgG failed to abrogate the neutralizing activity of the residual F(ab’)₂ fragments towards FVIII:C. In vivo, the disappearance of the FVIII inhibitory titers from the plasma of passively immunized inhibitor-positive HA mice required 48 h after dosing with IdeS, which correlated with changes in plasma levels of F(ab’)₂ fragments and is consistent with the 12-h half-life of F(ab’)₂ fragments that we determined in HA mice.

Different preclinical models of HA have been developed, including dogs, rats and minipigs. FVIII-deficient mice, however represent the most widely used model owing to the convenience of breeding and availability of tools for studying the immune system and hemostasis. Most importantly, the immune response to human FVIII in mice resembles that seen in allo-immunized PwHA.⁴⁷ IdeS hydrolyzes IgG from a variety of species, including rabbits, pigs, humans and non-human primates, but not mouse IgG1 and IgG2b.¹⁶ As a result, the use of mouse models to study the effect of IdeS on induced endogenous IgG-mediated immune responses is not feasible. In order to tackle this limitation, we validated a model of passive transfer to FVIII-deficient HA mice of a neutralizing anti-FVIII rhIgG. In our study, we validated similar hydrolysis profiles in vitro for four different monoclonal anti-FVIII rhIgG, irrespective of their specificity for different FVIII domains and of their IgG1/4 subclass.

Figure 5.Efficacy of therapeutic FVIII in inhibitor-positive hemophilia A mice treated with Imlifidase. (A) Hemophilia A (HA) mice were passively immunized with BO2C11 immunoglobulin (Ig) G1_k (1,200 Bethesda units [BU]/kg) and treated with Imlifidase (IdeS) (0.6 mg/kg) or phosphate-buffered saline (PBS) 24 hours (h) later. The mice were then administered intravenously with therapeutic FVIII (Helixate^®, 200 IU/kg) at day 4. Control mice (wild-type [WT]) were injected with FVIII in the absence of passive immunization with BO2C11 IgG1_k and treatment with IdeS. (B-E) Two h after FVIII injection, the mice tails were amputated at the terminal 3 mm and the blood loss was evaluated over 10 minutes (min). (B) In parallel, plasma was collected to determine the restoration of hemostatic efficacy of therapeutic FVIII by measuring the FVIII:C in a chromogenic assay (C), and the levels of thrombin generation (nM) over time (min) and thrombin peak (nM) using a thrombin generation test (D, E). In the graphs, the horizontal bars represent means ± standard deviation (SD) and each symbol depicts an individual animal. Statistical differences were assessed using the non-parametric Kruskal-Wallis test corrected for multiple comparisons using the Dunn’s test (ns: non-significant). In panel (D), the means ± standard error of the means are depicted as plain line and dotted line curves, respectively (n=5-8 mice per group).

The administration of anti-FVIII rhIgG to HA mice has already been performed to confirm their inhibitory activity towards FVIII in vivo,⁴⁸ or to study the effect of antibodies on the pharmacokinetics⁴⁸ or immunogenicity of human therapeutic FVIII.⁴⁹ Here, we followed the kinetics of one of the anti-FVIII rhIgG (BO2C11 IgG1_k) in mice and determined that circulating IgG levels are rather stable 24 h following injection and for up to 5 to 6 days. We also showed that this model allows the precise adjustment and monitoring of the circulating FVIII inhibitory titers. The lack of endogenous production of human IgG is an obvious major limitation of the model, which renders it artificially favorable to IdeS treatment. However, in humans, IdeS administration results in the rapid elimination of IgG from the circulation within 2 to 6 h and de novo production of endogenous IgG is detected only after 1 to 2 weeks.^32,38 Taken together, the data suggest that IdeS is expected to achieve a FVIII inhibitor-free time window in PwHA and PwAHA that is wide enough to ensure hemostatic efficacy of FVIII replacement therapy in cases of breakthrough bleeds or major surgeries. In our experiments, mice with FVIII inhibitory titers of 8.3±2.0 BU/mL were successfully treated with IdeS, and therapeutic FVIII hemostatic efficacy was restored within 72 h. Interestingly, despite the persistence of the neutralizing F(ab’)₂ fragments during the first 48 h after IdeS dosing, inhibitory titers were reduced by 37±13% and 84±8%, respectively, 6 and 24 h after IdeS injection. The inhibitory titers measured at the latter time points, i.e., 5.4±1.1 BU/mL and 1.7±0.6 BU/mL, correspond to the situation of patients with low inhibitory titers who may benefit from high-dose FVIII replacement therapy. Similar observations were made when mice with very high inhibitory titers (i.e., 200 BU/mL) were treated with two doses of IdeS, albeit with a further delay to reach an inhibitory titer <5 BU/mL.

The anti-FVIII antibody responses in PwHA and PwAHA are dominated by IgG antibodies.^33,50 Indeed, anti-FVIII IgG titers of 1:20 or more were found in all plasma from the MIBS and SACHA cohorts. Although the presence of FVIII-binding IgM, IgA and IgE was not investigated in our study, the latter iso-types may be found in 3-10% of PwHA and 8-36% of PwAHA.^33,50 Although the importance of the latter isotypes in FVIII neutralization in vivo is uncertain, their presence may preclude a substantial percentage of patients from receiving IdeS therapy. These observations argue for pre-screening patients to determine their eligibility for IdeS treatment.

Figure 6.Imlifidase-mediated elimination of very high titer inhibitor. Hemophilia A (HA) mice (n=5 per group) were passively immunized with 24,000 Bethesda units (BU)/kg of BO2C11 immunoglobulin (Ig) G1_k to reach 200 BU/mL after 24 hours (h). Twenty-four h later, a group of mice was treated with a single dose of Imlifidase (IdeS) (0.6 mg/kg, 0.29 µM, full green circles) or phosphate-buffered saline (PBS), open circles. Another group of mice was treated twice with IdeS (full blue circles) 24 and 48 h after BO2C11 injection. The levels of intact IgG and/or scIgG ((A), IgG concentration at 24 h: 261±8 nM), the levels of F(ab’)₂ fragments (B) and the inhibitory titers (C) were determined over time by enzyme-linked immunosorbant assay and Bethesda assay (n=2, mean ± standard deviation). The dotted lines represent the respective detection thresholds: 0.03 µg/mL, 0.08 µg/mL and 0.6 BU/mL. The grey zone depicts inhibitory titers below 5 BU/ml. Statistical differences were assessed between mice treated with one or two injections of IdeS (at the 96- and 168-h time points) using the two-tailed non-parametric Mann-Whitney test (*P<0.05; otherwise non-significant).

The injection of IdeS to PwAHA requiring hemostatic treatment would minimize the need for BPA and the associated thrombotic risk while restoring the efficiency of FVIII treatment and monitoring. Based on our in vivo results, redosing IdeS 24 h after a first dose, as described in other pathologies,⁵¹ could be indicated for patients with the highest anti-FVIII levels. The use of IdeS as an immediate first-line therapy may be complementary to the use of immunosuppressive agents (i.e., corticosteroids, cyclophosphamide) to remove the inhibitors for a longer time period. On the other hand, the administration of IdeS to PwHA receiving emicizumab should presumably lead to the simultaneous elimination of both neutralizing anti-FVIII IgG and the drug. This would not only restore the clinical hemostatic efficacy of FVIII replacement but would also eliminate emicizumabrelated biological interference,⁵² ensuring accurate FVIII:C measurement in plasma. Notably, the majority of IdeS will be cleared from circulation within 24 to 48 h, allowing rapid re-administration of emicizumab for prophylaxis.⁵³ Furthermore, due to limited experience and a lack of guidelines, the management of surgeries in PwHA receiving emicizumab remains an open question. It is further complicated in patients who have inhibitors with variable clinical responses to rFVIIa.⁵⁴ In these patients, IdeS would provide a brief but beneficial inhibitor-free therapeutic window for high-risk major surgery or breakthrough bleeds. Finally, our in vitro and in vivo findings pave the way for a new therapeutic option to improve the management of FVIII inhibitor patients.

Footnotes

• Received August 5, 2022
• Accepted January 11, 2023

Correspondence

S. Lacroix-Desmazes sebastien.lacroix-desmazes@inserm.fr

V. Proulle Valerie.proulle@aphp.fr

Disclosures

SLD and JDD are inventors on patent EP18305971.6 related to the use of IdeS in the context of AAV-mediated gene therapy. All other authors have no conflicts of interest to disclose.

Contributions

MBJ, SD, CD, VP, and SLD designed the research. MBJ, VD, SD, and VP performed experiments. JA and HL contributed essential material. MBJ, SD, CD, VP, and SLD analyzed the results and made the figures. MBJ, VP, and SLD wrote the paper.

Data-sharing statements

Original data and protocols are available upon request to the first and corresponding authors.

Funding

This work was supported by the Institut National de la Santé et de la Recherche Médicale (INSERM), Centre National de la Recherche Scientifique (CNRS), Sorbonne Université, Université de Paris, Assistance Publique des Hôpitaux de Paris and by grants from the Bayer Hemophilia Award Program (BHAP 2019 and BHAP 2021), from Agence National de la Recherche (ANR-18-CE17-0010-02-Exfiltrins and ANR-21-CE17-0043-Persia), from Spark Therapeutics Inc. (Philadelphia, PA), from coordination médicale pour l'étude et le traitement des maladies hémorragiques constitutionnelles (CoMETH) and by the Innovative Medicines Initiative 2 Joint Undertaking ARDAT (Accelerating research & development for advanced therapies) project, under grant agreement No 945473. This joint undertaking received support from the European Union’s Horizon 2020 research and innovation program and EFPIA. MBJ was the recipient of a fellowship from Ministère de l'enseignement supérieur et de la recherche and of the 2022 Martin Villard Haemostasis Award (Grifols Scientific Awards) in basic research.

Acknowledgments

We wish to thank Dr. Carmen Coxon (National Institute for Biological Standards and Control, Hertfordshire, UK) for sharing the cDNA encoding KM41, as well as the staf from “Centre d'Expérimentation Fonctionnelle” at Centre de Recherche des Cordeliers (Paris) for assistance. Helixate® was a kind gift from CSL-Behring.