Prof Marc Jacquemin

Gilles Thibault

Dr Roseline D'

Srini K Jag

S Lacroix-Desmazes
email: sebastien.lacroix-desmazes@inserm.frv.proulle

My most profound appreciation goes to Dr. Sébastien Lacroix-Desmazes, my Ph.D. supervisor. Thank you for believing in me, letting me work on so many different projects, supporting me whenever I needed your help, carefully monitoring my progress, and giving me invaluable advice throughout my Ph.D. I learned a

Table of contents

List of figures

Introduction

My research focused on the pathogenesis of FVIII inhibitors in hemophilia A patients. I pursued two complementary goals: 1) gain a better understanding of the pathogenic process at play and look for the prognostic markers for the successful eradication of FVIII inhibitors, and 2) develop novel strategies to eradicate FVIII inhibitors. My research project is thus at the cross-road between different research fields: coagulation and hemophilia, immunology and immunoglobulins, and immune intervention.

The introduction will be subdivided into three chapters to provide the background required for a better understanding of my Ph.D. work. Chapter 1 introduces hemostasis and factor VIII, a key factor in the coagulation process. Chapter 2 introduces hemophilia A, a coagulation disorder resulting from defects in factor VIII, its different treatment options and the associated complications, notably the onset of an anti-drug neutralizing humoral response. Chapter 3 covers the IgG-degrading enzyme of S. Pyogenes (IdeS), its potential as an emerging drug, and its relevance in the setting of hemophilia A.

Chapter I: Factor VIIIa key factor in hemostasis 1. Hemostasis

Hemostasis is the physiological process that stops bleeding at the site of an injury while maintaining normal blood flow throughout the body. Blood loss is controlled by the formation of a stable platelet and fibrin plug that closes the damaged site after a series of reactions involving plasma proteins, platelets, extracellular membrane components, and vascular smooth muscle. At the same time, hemostasis inhibitors and the fibrinolytic system are activated to limit the size of the clot and clear it once the blood vessel structure has been repaired. From zebrafish to humans, hemostasis is a highly conserved machinery that involves three concurrent processes: primary, secondary, and tertiary hemostasis.

Primary hemostasis

The initial response to vascular injury is vasoconstriction which limits blood flow and leads to endothelial cells' activation. When activated, endothelial cells secrete von Willebrand factor (VWF), a large multimeric protein, with the primary function of recruiting platelets to the injured endothelium and subendothelial matrix (Appendix 1). Under high shear stress conditions, VWF binds to exposed subendothelial collagen first, then to the glycoprotein (GP) Ib-IX-V complex through the GPIbα receptor abundantly expressed at the platelet surface [START_REF] Sakariassen | Role of Platelet Membrane Glycoproteins and Von Willebrand Factor in Adhesion of Platelets to Subendothelium and Collagen[END_REF]. The binding of VWF to platelets is the first step in the platelet adhesion process, acting as a bridge between the platelets and the subendothelial matrix. Adhesion of platelets to the subendothelial matrix at the site of injury is reinforced by the binding of the platelet receptor GPVI to exposed collagen. Adhesion and signaling induced by these different interactions initiate the activation of platelets. Activated platelets first undergo an irreversible change in shape from smooth discs to multi-pseudopodal plugs, significantly increasing their surface area. Then, platelets release their cytoplasmic granules, such as thromboxane A2 and adenosine diphosphate (ADP), which mediate a feedback activation of nearby platelets, recruiting them to the site of injury. The binding of adenosine diphosphate to its platelet purinergic receptors induces the activation and conformational of the platelet receptor complex GPIIb/IIIa, also known as integrin αIIbβ3 [START_REF] Weiss | Platelet adhesion and thrombus formation on subendothelium in platelets deficient in glycoproteins IIb-IIIa, Ib, and storage granules[END_REF][START_REF] Jackson | Signaling events underlying thrombus formation[END_REF]. The activated integrin αIIbβ3 binds to its ligands (i.e., fibrinogen, VWF) and promotes platelet-platelet aggregation and the formation of an unstable platelet plug. Another critical step in platelet activation is the redistribution of anionic phospholipids, such as phosphatidylserine (PS), from the inner to the outer leaflet of the platelet plasma membrane. This process is essential for recruiting and activating coagulation factors on the surface of activated platelets, which is required for secondary hemostasis to begin.

Secondary hemostasis (Coagulation)

The classic coagulation "cascade" or "waterfall model" described by Macfarlane and Davie in the early 1960s [START_REF] Davie | Waterfall Sequence for Intrinsic Blood Clotting[END_REF] resulted in the development of in vitro models of coagulation known today as the routine coagulation tests, prothrombin time (PT), and activated partial thromboplastin clotting time (aPTT). It described a sequential interaction of serine proteases and other clotting factors known as the extrinsic and intrinsic pathways, which converge on a final common pathway (Figure 1). However, there are several discrepancies and limitations of these routine tests in describing in vivo hemostasis, which led to the development of a cell-based model of coagulation in the early 1990s [START_REF] Broze | Why do hemophiliacs bleed?[END_REF]. In this model, in vivo coagulation is characterized by replacing "independent pathways" with "co-dependent stages" of plasma protein interaction with vascular cells, which are the initiators and regulators of the cascade (Figure 2). According to this new model, coagulation can be divided into three overlapping stages: 1) an initiation phase, 2) an amplification phase, and 3) a propagation phase.

Initiation phase

The initiation phase, classically referred to as the extrinsic coagulation pathway, starts when the vasculature is disrupted, and subendothelial cells like smooth muscle cells and fibroblasts become exposed to the bloodstream. These cells expose a key initiator of the coagulation cascade, the tissue factor (TF), which binds to both coagulation factor VII (FVII) and a small circulating amount of its activated form FVIIa (1-2% of total FVII). By acting as a cofactor for FVII, TF promotes proteolysis and activation of FVIIa [START_REF] Kirchhofer | Initiation of blood coagulation: the tissue factor/factor VIIa complex[END_REF]. The TF/FVIIa complex proteolytically cleaves traces of FIX and FX into FIXa and FXa, respectively. This allows FXa to associate with cofactor FVa to form a prothrombinase complex on TF-expressing cells, which converts small amounts of prothrombin (FII) into thrombin (FIIa). Thrombin plays a crucial role in the amplification and propagation phases of coagulation, the so-called intrinsic pathway [START_REF] Furie | Mechanisms of thrombus formation[END_REF].

Figure 1: Classic coagulation cascade. Schematic representation of the intrinsic (blue), extrinsic (red), and common (purple) pathways of the coagulation cascade as well as the regulatory (black) and fibrinolytic (orange) system components. PK, prekallikrein; HMWK, highmolecular-weight kininogen; TF, tissue factor; PL, phospholipid; TFPI, tissue factor pathway inhibitor; PS, protein S; APC, activated protein C; PC, protein C; TM, thrombomodulin; FDP, fibrin degradation product; TAFI, thrombin activatable fibrinolysis inhibitor; α2AP, alpha 2-antiplasmin; tPA, tissue plasminogen activator; PAI-1, plasminogen activator inhibitor-1. From [START_REF] Godby | Congenital and acquired disorders of secondary hemostasis[END_REF].

Amplification phase

The slowly accumulating amounts of thrombin further activate platelets that have adhered to the site of injury. In parallel, thrombin converts FV into FVa, thus amplifying prothrombinase activity, and converts FVIII into its activated form (FVIIIa), dissociating it from its chaperone VWF. FVIIIa acts as a cofactor to FIXa on the surface of activated platelets to support FXa generation. In addition, thrombin converts FXI into FXIa.

Propagation phase

The propagation phase occurs on surfaces enriched with procoagulant anionic phospholipids, such as activated platelets, away from TF-expressing surfaces, where the initiation phase takes place in current models. Activated FXI converts FIX into FIXa, which then associates with thrombin-cleaved FVIII (Figure 2). Absence or near-absence of FVIII or FIX leads to severe bleeding complications (hemophilia A and B, respectively), thus underlining the importance of these coagulation factors for normal hemostasis [START_REF] Godby | Congenital and acquired disorders of secondary hemostasis[END_REF]. On PS-exposing cell membranes, the tenase complex of FIXa/FVIIIa catalyzes the conversion of FX to FXa, after which the FXa/FVa complex produces sufficient amounts of thrombin, the thrombin burst, to form fibrin fibers massively. As a final step, FXIII is activated by thrombin, and the FXIIIa catalyzes the formation of covalent crosslinks between adjacent fibrin chains to yield a stable fibrin clot.

Hemostasis inhibitors

To avoid overactivation, coagulation is regulated by several physiological anticoagulant mechanisms (Figure 1). The most important ones are 1) antithrombin (AT), which inactivates thrombin as well as FVIIa, FIXa, FXa, FXIa, and FXIIa [START_REF] Quinsey | Antithrombin: in control of coagulation[END_REF], 2) tissue factor pathway inhibitor (TFPI), which constrains the activity of the TF/FVIIa and prothrombinase complexes, limiting the initiation of coagulation [START_REF] Jr | Tissue factor pathway inhibitor: structure-function[END_REF] and3) protein C and S systems, which inactivate FVa and FVIIIa, effectively dampening thrombin generation amplification [START_REF] Esmon | Anticoagulation proteins C and S[END_REF].

Tertiary hemostasis (Fibrinolysis)

The fibrinolytic system dissolves blood clots after the vasculature has been repaired and prevents blood clots from forming in healthy blood vessels. Plasmin is the main player in this process because it cleaves the fibrin polymers, as well as fibrinogen, and produces fibrin degradation products that interfere with platelet aggregation and fibrin polymerization, destabilizing the platelet clot, which dissolves under high shear stress.

Plasmin is generated from plasminogen by the proteases tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA), both secreted by endothelial cells (Figure 1). The fibrinolytic system can be regulated by inhibitors targeting plasminogen activators (e.g., or plasmin (e.g., ⍺2-antiplasmin) [START_REF] Gale | Current Understanding of Hemostasis[END_REF][START_REF] Hoffman | Hematology: Basic Principles and Practice[END_REF][START_REF] Rau | Serpins in thrombosis, hemostasis and fibrinolysis[END_REF] or removing carboxy-terminal lysine residues of partially degraded fibrin (e.g., TAFIa). Activated thrombin-activatable fibrinolysis inhibitor (TAFIa) leads to the elimination of plasminogen binding sites, inhibition of plasminogen activation, and impaired fibrinolysis [START_REF] Mosnier | Regulation of Fibrinolysis by Thrombin Activatable Fibrinolysis Inhibitor, an Unstable Carboxypeptidase B That Unites the Pathways of Coagulation and Fibrinolysis[END_REF].

Under normal physiological conditions, the body maintains a balance of clotting and bleeding. Any pathological scenario, however, will tilt the balance in favor of hemorrhagic or thrombotic complications. FVIII is a critical component of secondary hemostasis; insufficient levels result in inefficient thrombin production and impaired hemostasis.

Factor VIII biology

The human FVIII gene (F8) was cloned in 1984 [START_REF] Gitschier | Characterization of the human factor VIII gene[END_REF]. It is one of the largest genes known, spanning about 186 kb, and is located at Xq28, near the terminus of the long arm of the X chromosome. One of the peculiarities of F8 is the presence of two additional genes, F8A and F8B, located within intron 22 and expressed ubiquitously (Figure 3). Moreover, F8A has two additional copies that are not part of the F8 gene. The functions of the F8A and F8B genes are unknown. F8 comprises 26 exons that encode a precursor polypeptide chain of 2351 amino acids, including a signal peptide of 19 amino acids. The mature full-length FVIII glycoprotein is synthesized as a large monomer (~300 kDa) of 2332 amino acid residues with the domain structure: A1-a1-A2-a2-B-a3-A3-C1-C2 [START_REF] Lenting | The Life Cycle of Coagulation Factor VIII in View of Its Structure and Function 15[END_REF][START_REF] Vehar | Structure of human factor VIII[END_REF]. [START_REF] Graw | Haemophilia A: from mutation analysis to new therapies[END_REF].

The three A domains of FVIII share approximately 35-40% sequence homology with each other and with the A domains of FV and copper-binding protein ceruloplasmin [START_REF] Church | Coagulation factors V and VIII and ceruloplasmin constitute a family of structurally related proteins[END_REF]. The A domains are surrounded by short spacers (a1, a2, and a3) that contain aspartic acid (Asp) and glutamic acid (Glu) residue clusters, known as acidic regions, which function as binding sites for thrombin and facilitate thrombin-catalyzed activation.

In addition, the a3 region and regions within both C1 and C2 domains have been suggested to mediate the tight interaction of the FVIII heterodimer with its carrier protein VWF [START_REF] Bos | Blood coagulation factors V and VIII: Molecular Mechanisms of Procofactor Activation[END_REF]. Similarly, the C domains are structurally related to the C domains of FV and phospholipid-binding protein. It has been well-documented that both the A and C domains are required for FVIII activity. On the contrary, the large B domain (~40% of the mass of FVIII) does not exhibit any significant homology with other known proteins, has a variable sequence, even among FVIII from different species, and is highly glycosylated. The B domain is dispensable for FVIII procoagulant activity; however, there are several controversies concerning its function [START_REF] Pahl | Effect of F8 B domain gene variants on synthesis, secretion, activity and stability of factor VIII protein[END_REF][START_REF] Pipe | Functional roles of the factor VIII B domain[END_REF]. According to a recent study by Jun et al., different B domain regions may have varying degrees of influence on the expression, intracellular transport, secretion, and stability of FVIII [START_REF] Jun | Functional identification of factor VIII B domain regions in hepatocyte cells[END_REF].

The primary source of circulating FVIII is liver sinusoidal endothelial cells, not hepatocytes, as previously thought, though extrahepatic sources also contribute to FVIII plasma levels. Recent studies found that FVIII was expressed and secreted by both mouse and human hematopoietic cells (e.g., monocytes, macrophages, and megakaryocytes) and extrahepatic endothelial cells (e.g., in the spleen, lungs, and kidneys) [START_REF] Zanolini | Extrahepatic sources of factor VIII potentially contribute to the coagulation cascade correcting the bleeding phenotype of mice with hemophilia A[END_REF]. Moreover, FVIII was found to be synthesized by lymphatic and fenestrated, but not conventional blood capillary, endothelial cells [START_REF] Pan | Patterns of expression of factor VIII and von Willebrand factor by endothelial cell subsets in vivo[END_REF].

The FVIII protein is secreted as a heterogeneous mixture of heterodimers following variable post-translational intracellular proteolysis at the B-A3 junction plus additional cleavage at sites within the B domain. Thus, FVIII contains a constant-sized light chain (A3-C1-C2; 80 kDa) and a heavy chain (A1-A2-B; 90-210 kDa) variable in size due to the presence of some or all of the contiguous B domain. The binding of the two chains is noncovalent and requires a metal ion-dependent linkage with the residues contained within the A1 and A3 domains (Figure 3). FVIII is one of the largest and least stable coagulation factors, circulating in plasma in a non-covalent complex with VWF (Appendix 2). The latter chaperone protects FVIII from premature clearance and/or proteolytic degradation, yielding a half-life of 12 hours in adults (shorter in children, around 9 hours) [START_REF] Versloot | Terminal half-life of FVIII and FIX according to age, blood group and concentrate type: Data from the WAPPS database[END_REF]. It also prevents its binding to phospholipids and FIX and concentrates it at sites of vascular injury (Lacroix-Desmazes et al., 2008). After specific thrombin or FXa cleavages that mainly remove the remainder of the B domain and one of the high-affinity VWF binding sites, FVIIIa dissociates from VWF as a heterotrimeric assembly (A1/A2/A3-C1-C2). FVIIIa binds to activated platelet surfaces, likely through embedding several solvent-exposed hydrophobic loops on the C1 and C2 domains [START_REF] Hsu | The factor VIII C1 domain contributes to platelet binding[END_REF]. FVIIIa also binds to FIXa, forming the intrinsic tenase complex, which amplifies the generation of FXa and, subsequently, thrombin. Inactivation of FVIIIa occurs by spontaneous A2 domain dissociation or by specific cleavages within A1 and A2 domains by activated protein C. It is finally cleared from the circulation by pinocytosis, phagocytosis, or receptor interactions with scavenger cells such as macrophages.

The occurrence of thrombotic and bleeding disorders brought on by altered cofactor, inhibitor or proteolytic activities highlights the hemostatic importance of the correct regulation of the activation and inactivation of these components. Hemophilia A, a bleeding disorder caused by a FVIII shortage or abnormality, is one such disorder.

Chapter II: Hemophilia A

History of hemophilia

The first written reference and earliest description of hemophilia are attributed to Egyptian papyri and Hebrew texts from the 2nd century A.D. [START_REF] Aledort | History of haemophilia[END_REF][START_REF] Ingram | The history of haemophilia[END_REF]. The latter referred to the exemption from circumcision for children with a family history of at least two deceased brothers due to hemorrhage after this procedure [START_REF] Rosner | Hemophilia in the Talmud and rabbinic writings[END_REF].

However, the first clinical report describing this disease as a hereditary disorder dates from 1803, when Dr. JC Otto (Philadelphia) described an inheritable bleeding disorder that affects only males and is transmitted through unaffected women in several families [START_REF] Otto | An account of an hemorrhagic disposition existing in certain families[END_REF]. In 1828, Hopff and his professor Dr. Schonlein (Zurich), coined the term "hemophilia", which refers to an affinity to bleed [START_REF] Hopff | Ueber die Haemophilie, oder die erbliche Anlage zu tödtlichen Blutungen: Inaugural-Abhandlung[END_REF]. According to data published in The Lancet in 1840, an 11-year-old boy who had been bleeding for six days following surgery healed after receiving a whole blood transfusion [START_REF] Lane | HAEMORRHAGIC DIATHESIS.: SUCCESSFUL TRANSFUSION OF BLOOD[END_REF]. This was the first successful medical therapy for hemophilia. Then, hemophilia became popular after Queen Victoria of England, believed to have been the carrier of hemophilia B (FIX deficiency), "transmitted" the disease to her son Leopold, who died from bleeding after a fall at the age of 30 [START_REF] Rogaev | Genotype analysis identifies the cause of the "royal disease[END_REF]. By that time, hemophilia had become known as the "royal disease" and spread in the 19th and 20th centuries to other royal families (Germany, Spain, and Russia) through the Queen's daughters, Alice and Beatrice [START_REF] Ingram | The history of haemophilia[END_REF].

The discovery of an "anti-hemophilic globulin", nowadays known as FVIII, that could speed up the clotting of hemophilic blood in the middle of the 20th century marked a turning point in the history of hemophilia [START_REF] Patek | HEMOPHILIA. II. SOME PROPERTIES OF A SUBSTANCE OBTAINED FROM NORMAL HUMAN PLASMA EFFECTIVE IN ACCELERATING THE COAGULATION OF HEMOPHILIC BLOOD[END_REF]. It led to the development of cryoprecipitate, and the first clotting factor concentrates for the treatment of bleeding disorders. Classical hemophilia, known as hemophilia A, is a rare bleeding disorder caused by a FVIII deficiency or abnormality. Fifteen years later, in 1952, Rosemary Biggs (Oxford) distinguished a different disease entity called "Christmas disease" after the name of the first child described with this FIX deficiency condition [START_REF] Biggs | Christmas Disease[END_REF][START_REF] Hynes | Development of the present concept of hemophilia[END_REF]. It is now called hemophilia B (HB). This was followed one year later by the discovery of the FXI deficiency, also called hemophilia C [START_REF] Rosenthal | New Hemophilia-like Disease Caused by Deficiency of a Third Plasma Thromboplastin Factor[END_REF].

This dissertation focuses on hemophilia A, the most common type, accounting for around 80% of all hemophilia cases reported in the World Federation of Hemophilia (WFH) 2021

Annual Global Survey (https://www1.wfh.org/). The following paragraphs will describe two different HA pathologies, congenital and acquired, in detail.

Congenital hemophilia A

Presentation and diagnosis

Etiology and epidemiology

Hemophilia A (HA), also known as classic hemophilia as described in the previous paragraph, is a congenital X-linked recessive bleeding disorder caused by missing or defective FVIII. Lacking sufficient procoagulant activity, HA patients bleed longer than other people and are consequently exposed to increased morbidity and mortality. Normal plasma FVIII (FVIII:C) levels range from 50% to 150%, with levels below 40% determining a person's symptoms. The severity of HA is determined by these levels and can be divided into three categories: mild, moderate, and severe, which correspond to factor levels of 6-40%, 1-5%, and <1%, respectively [START_REF] Srivastava | the WFH Guidelines for the Management of Hemophilia panelists and co-authors[END_REF]. Inherited mutations account for 2/3 of HA cases; however, the remaining 1/3 of cases are sporadic and stem from spontaneous mutations in people with no family history of the disease. Mothers of sporadic patients may be non-carriers, carriers with de novo mutations, or carriers who have acquired the mutation from their parents [START_REF] Lu | Spectrum and origin of mutations in sporadic cases of haemophilia A in China[END_REF]. HA occurs in approximately 1 in 5000 male births; females are usually hemophilia carriers presenting with one affected X chromosome (Iorio et al., 2019) (Figure 4). Nevertheless, females can also have HA, though much rarer than males, and may bleed as significantly as affected males. When a woman or girl has hemophilia, both X chromosomes are affected, or one is affected and the other is absent or non-functional [START_REF] Van Galen | A new hemophilia carrier nomenclature to define hemophilia in women and girls: Communication from the SSC of the ISTH[END_REF][START_REF] Miller | Genetic causes of haemophilia in women and girls[END_REF]. In an effort to improve the diagnosis and treatment of HA in girls and women, experts now classify hemophilia carriers into five clinically relevant categories. This classification was based on FVIII levels and the presence or absence of a bleeding phenotype, according to a recent report from the International Society on Thrombosis and Haemostasis (ISTH) Scientific and Standardization Committee (SSC) [START_REF] Van Galen | A new hemophilia carrier nomenclature to define hemophilia in women and girls: Communication from the SSC of the ISTH[END_REF]. HA affects individuals from different origins and ethnic groups.

From the 118 countries that submitted their data to the WFH Annual Global Survey in 2021, the number of people with HA around the world is approximately 185,318, with India reporting the highest number (21,350), followed by China (17,779) and the United States (14,098) (https://www1.wfh.org/).

Bleeding patterns

The typical serious bleeding sites include joints (hemarthrosis), muscles, especially deep compartments, and mucous membranes in the mouth, gums, nose, and genitourinary tract. Life-threatening bleeding sites include intracranial, neck/throat, or gastrointestinal regions [START_REF] Srivastava | Guidelines for the management of hemophilia[END_REF]. The frequency of bleeding varies depending on the site: joints (70-80%), muscle (10-20%), other sites (major bleeds; 5-10%), and central nervous system (< 5%). Patients with mild HA may not excessively bleed until they experience serious injury, trauma, or surgery; spontaneous bleeding is rare. In these patients, the first bleeding episode might not occur until adulthood. Women with mild HA often experience heavy menstrual bleeding and can experience hemorrhages after childbirth [START_REF] Kadir | The obstetric experience of carriers of haemophilia[END_REF]. Patients with moderate HA experience occasional spontaneous bleeding and tend to have prolonged bleeding in case of minor trauma or surgery. On the other hand, patients with severe HA experience frequent spontaneous bleeding into joints or muscles, often in the absence of identifiable hemostatic challenges. Many males with severe hemophilia are diagnosed due to bleeding after circumcision. Though most children with severe HA experience the first bleeding in their first year of life, it is important to note that in some cases, bleeding symptoms may not appear until later, when they start walking or running [START_REF] Srivastava | Guidelines for the management of hemophilia[END_REF].

Clinical and biological diagnosis

An accurate diagnosis of HA is essential to inform appropriate management. HA should be suspected in patients presenting with a history of 1) easy bruising in early childhood, 2) spontaneous bleeding, particularly into the joints, muscles, and soft tissues, or 3) excessive bleeding following trauma or surgery. If hemophilia is suspected, the clinician should obtain the patient's bleeding history and family history of abnormal or unexplained bleeding experienced by siblings or maternal male relatives. A family history of bleeding is obtained in about 2/3 of all HA patients. The specialist performs blood coagulation screening tests such as PT and aPTT (and fibrinogen dosage) to identify the potential cause of bleeding. HA patients will present with a normal PT and fibrinogen tests but an isolated prolonged aPTT, with no acquired cause of prolonged aPTT, such as the presence of a circulating lupus anticoagulant antibody [START_REF] Srivastava | the WFH Guidelines for the Management of Hemophilia panelists and co-authors[END_REF]. Factor assays (i.e., FVIII, FIX, FXI, and FXII) are performed to determine the underlying factor deficiency. In case of an isolated factor deficiency (except for FXII), clinicians must assess the presence of specific inhibitors (the modified Nijmegen-Bethesda assay is used to detect FVIII inhibitors, cf. paragraph 2.2.1.3) [START_REF] Verbruggen | Improvements in factor VIII inhibitor detection: From Bethesda to Nijmegen[END_REF]. Due to high variability in reagents' sensitivity, aPTT may be normal or minimally prolonged in mild HA, therefore highlighting the need for a careful clinical examination to further investigate factor assays in case of suspicion of a bleeding tendency. Importantly, for girls with low FVIII:C levels, there is frequent confusion between genetic and bleeding disorder diagnosis, which delays age of diagnosis and limits access to appropriate care compared to boys [START_REF] Van Galen | A new hemophilia carrier nomenclature to define hemophilia in women and girls: Communication from the SSC of the ISTH[END_REF][START_REF] Di Michele | Severe and moderate haemophilia A and B in US females[END_REF].

Genetic diagnosis

Currently, two approaches are being used to determine the genetic cause of HA. The first involves tracking the defective X-chromosome in the family by analyzing single nucleotide polymorphisms or microsatellite variable number tandem repeat markers in F8. The second is direct mutation analysis in F8. Every year, new mutations in the F8 gene are discovered, and their relationship with disease severity is reevaluated to gain a better understanding of HA and the reasons for varying degrees of severity. As of March 2022, 3052 unique variants in the F8 gene (excluding inversions) were compiled within the EAHAD database (https://f8-db.eahad.org/), corresponding to 10144 individual cases.

Genetic defects causing impaired FVIII function are deletions, inversions, insertions, and point mutations leading to premature peptide chain termination (nonsense), amino acid replacement (missense), or mRNA splicing defects. All these mutations can be found in patients with severe HA, though inversion of intron 22 accounts for 45% of cases [START_REF] Lakich | Inversions disrupting the factor VIII gene are a common cause of severe haemophilia A[END_REF]. In mild-moderate patients with HA, missense mutations are the most prevalent, accounting for 79.5% of cases (Figure 5). Adapted from [START_REF] Johnsen | Novel approach to genetic analysis and results in 3000 hemophilia patients enrolled in the My Life, Our Future initiative[END_REF].

Not all patients show a clear correlation between F8 mutations and FVIII:C. About 2% of patients who develop HA do not have any F8 mutations, despite the absence of the FVIII protein in their blood and autoantibodies [START_REF] El-Maarri | Analysis of mRNA in hemophilia A patients with undetectable mutations reveals normal splicing in the factor VIII gene[END_REF][START_REF] Johnsen | Novel approach to genetic analysis and results in 3000 hemophilia patients enrolled in the My Life, Our Future initiative[END_REF].

Therefore, post-translational inhibition of the F8 mRNA, or direct repression of the FVIII protein (e.g., non-coding RNA), as well as epigenetic repression of the F8 gene, were proposed as a non-genetic influence mechanisms of HA onset and severity [START_REF] Jankowska | Further Evidence That MicroRNAs Can Play a Role in Hemophilia A Disease Manifestation: F8 Gene Downregulation by miR-19b-3p and miR-186-5p[END_REF][START_REF] Zimta | The Possible Non-Mutational Causes of FVIII Deficiency: Non-Coding RNAs and Acquired Hemophilia A[END_REF]. However, more detailed research is required.

Treatments

Replacement therapies 2.2.1.1. Standard factor replacement therapies

Treatment focuses on restoring FVIII activity, and the globally accepted standard of care for HA is FVIII replacement. Initially, in the 1960s-1970s, the use of cryoprecipitates and intermediate-purity factor concentrates revolutionized hemophilia care [START_REF] Pool | Production of high-potency concentrates of antihemophilic globulin in a closed-bag system[END_REF][START_REF] Webster | Clinical use of a new glycine-precipitated antihemophilic fraction[END_REF] but this resulted in the transmission of bloodborne pathogens, including hepatitis C and human immunodeficiency virus (HIV) [START_REF] Mannucci | AIDS, hepatitis and hemophilia in the 1980s: memoirs from an insider[END_REF]. In the 1990s, treatment for hemophilia advanced, and factor products became safer as tighter screening methods and advanced viral inactivation methods were implemented for purified plasma-derived FVIII (pd-FVIII). Furthermore, recombinant technologies were used to produce synthetic factor products (Figure 6). Schematic structure of native coagulation factor VIII (FVIII, top panel), which undergoes intracellular processing before secretion to generate a heterogeneous mixture of proteins with truncated B domains. Summary of conventional FVIII molecules (which have a half-life similar to that of native FVIII, as demonstrated in clinical trials), as well as extended half-life (EHL) FVIII molecules that are approved or in clinical trials. Schematics of structures and the differences are highlighted. The cell line used to produce each product is in parentheses. BDD, B domain deleted; BHK, baby hamster kidney; CHO, Chinese hamster ovary; Fc, crystallizable fragment; HEK293, human embryonic kidney 293; VWF, von Willebrand factor. From [START_REF] Peters | Advances and innovations in haemophilia treatment[END_REF].

In 1992, the first recombinant FVIII (rFVIII) product was approved by the Food and Drug Administration (FDA) [START_REF] Schwartz | Human Recombinant DNA-Derived Antihemophilic Factor (Factor VIII) in the Treatment of Hemophilia A[END_REF][START_REF] Boedeker | The manufacturing of the recombinant factor VIII, Kogenate[END_REF]. In addition to addressing the issue of relying on the availability of human plasma sources, recombinant technologies facilitated the development of novel products.

Prophylaxis, a preventive treatment regimen performed 2-3 times per week in children with HA, became more common by 1995, reducing the risk of life-threatening bleeding, hemarthroses, and joint damage, compared to on-demand therapy [START_REF] Aledort | A longitudinal study of orthopaedic outcomes for severe factor-VIII-deficient haemophiliacs. The Orthopaedic Outcome Study Group[END_REF][START_REF] Nilsson | Twenty-five years' experience of prophylactic treatment in severe haemophilia A and B[END_REF]. However, replacement therapy faces various challenges, including the requirement of frequent intravenous injections due to the short half-life of standard FVIII products (i.e., 8-12 hours), associated with poor venous access, the immunogenicity of infused FVIII products, and high cost (Peyvandi et al., 2016). Several generations of standard half-life products have evolved to address these challenges in the last two decades (Figure 6). Six commercially available third-generation rFVIII products are free of human plasma or albumin [START_REF] Shapiro | Anti-hemophilic factor (recombinant), plasma/albumin-free method (octocog-alpha; ADVATE) in the management of hemophilia A[END_REF][START_REF] Mahlangu | BAY 81-8973, a full-length recombinant factor VIII for the treatment of hemophilia A: product review[END_REF][START_REF] Lissitchkov | Novel, human cell line-derived recombinant factor VIII (human-cl rhFVIII; Nuwiq® ) in adults with severe haemophilia A: efficacy and safety. Haemoph. Off[END_REF][START_REF] Kelley | An improved manufacturing process for Xyntha/ReFacto AF[END_REF][START_REF] Mahlangu | Efficacy and safety of rVIII-SingleChain: results of a phase 1/3 multicenter clinical trial in severe hemophilia A[END_REF]. Two of them are full-length rFVIII products (Advate®, Takeda; BAY 81-8973, Kovaltry®, Bayer), and the four others have bioengineered B-domain deleted (refactor AF, Xyntha®, Pfizer; Human-cl rhFVIII, Nuwiq®, Octapharma; and Turoctocog alfa, Novoeight®, Novo Nordisk) or B-domain truncated rFVIII (rFVIII SingleChain, Afstyla®, CSL Behring). Overall, the efficacy of these products in treating bleeding events is greater than 80%, with comparable efficacy in bleed prevention when used prophylactically. These products have 90-100% efficacy in perioperative hemostasis in patients undergoing major and minor surgeries.

Extended half-life factor replacement therapies

Treatment advances continued with modifications to extend the factor half-life, improving its pharmacokinetic profile, thus maximizing the time between infusions while minimizing infectious risks (Figure 6). Four commercially available extended half-life rFVIII products are registered with the FDA and the European Medicines Agency (EMA) [START_REF] Mahlangu | Phase 3 study of recombinant factor VIII Fc fusion protein in severe hemophilia A[END_REF][START_REF] Konkle | Pegylated, full-length, recombinant factor VIII for prophylactic and on-demand treatment of severe hemophilia A[END_REF][START_REF] Lentz | Results from a large multinational clinical trial (guardian TM 1) using prophylactic treatment with turoctocog alfa in adolescent and adult patients with severe haemophilia A: safety and efficacy[END_REF][START_REF] Reding | Safety and efficacy of BAY 94-9027, a prolonged-half-life factor VIII[END_REF]. The technologies used are fragment crystallizable (Fc) fusion (rFVIIIFc, Eloctate®, Sobi) or attachment of a polyethylene glycol (PEG) molecule to the FVIII protein (BAX 855,Adynovate®,Takeda;Esperoct®,Novo Nordisk;Jivi®,Bayer). The four products had comparable 90-100% hemostatic efficacy, and no neutralizing antibodies were detected in any of the clinical trials. In addition, while preexisting and transient nonneutralizing antibodies were detected before and during exposure to the extended halflife rFVIII products, they had no effect on FVIII activity, and many disappeared during continued exposure to the drugs. Unfortunately, these products could not extend the halflife of FVIII more than 1.5 folds due to their interaction with endogenous plasma VWF, which have a similar affinity as native FVIII and are therefore subjected to the VWFimposed half-life ceiling. To overcome this imposed ceiling, the group of Seth Chhabra et al. engineered a stabilized FVIII that does not bind to circulating VWF, the rFVIIIFc-VWF-XTEN (Efanesoctocog Alfa, BIVV001) (Seth [START_REF] Chhabra | BIVV001, a new class of factor VIII replacement for hemophilia A that is independent of von Willebrand factor in primates and mice[END_REF]. This drug increases up to 4 folds (38 hours) the hemostatic control compared to standard products. Currently, its long-term safety and efficacy in previously treated patients are being assessed in a phase 3 open-label, multicenter study expected to be completed by February 2027 (NCT04644575).

Major complication of factor replacement therapies

Unfortunately, replacement therapies can be complicated by the development of neutralizing antibodies directed against FVIII (FVIII inhibitors). Patients with the severe form of the disease are at the highest risk of developing FVIII inhibitors during their first 20 exposures to FVIII concentrates, with one exposure defined as all FVIII concentrates administered within 24 hours. FVIII inhibitors develop at a cumulative incidence of around 30% of previously untreated patients (PUPs) with severe HA, and 5-10% of those with mild-to-moderate HA (Gouw et al., 2013b;[START_REF] Van Den Berg | Timing of inhibitor development in more than 1000 previously untreated patients with severe hemophilia A[END_REF]. When present, FVIII inhibitors neutralize the biological activity of infused FVIII, making the patient refractory to treatment. Therefore, morbidity and treatments costs are considerably increased for patients with inhibitors. FVIII inhibitors can be quantified by their ability to inhibit FVIII:C using the functional Bethesda assay or the Nijmegen modification of the Bethesda assay.

An inhibitor titer is represented as Bethesda unit per milliliter (BU/mL). It is defined as the dilution of patient plasma sample required to achieve a 50% neutralization of FVIII:C in an equivalent volume of normal plasma (source of FVIII). Among these inhibitors, almost onethird are transient and spontaneously disappear without sequelae and need for specific treatment regimens. In cases of persistent low titer inhibitors (i.e., <5 BU/ml), treatment with standard FVIII products is still feasible by adjusting the doses to saturate the inhibitors. However, for patients with high titer inhibitors (i.e., ≥5 BU/ml), standard FVIII replacement therapy is no longer effective, and recurrent joint bleeds are commonly managed by on-demand treatment with bypassing agents. These agents eliminate the need for FVIII while supplementing other coagulation factors.

In the past few decades, the treatment of HA patients has come a long way. New approaches have been developed to address the challenges of replacement FVIII products (i.e., intravenous injections, short-half life, and inhibitor development) and to improve the hemostatic potential independently of their administration. In addition to bypassing agents that are blood products, new products include non-factor agents promoting the coagulation pathway (e.g., emicizumab, mim8) and others blocking the anticoagulant pathway (e.g., concizumab, fitusiran, seprinPC).

Non-replacement therapies

Bypassing agents

Bypassing agents (BPAs) include recombinant activated FVII (rFVIIa, NovoSeven®, Novo Nordisk) and plasma-derived activated prothrombin complex concentrate (aPCC, FEIBA®, Shire). rFVIIa promotes coagulation through TF-dependent and independent pathways, binds to TF to activate FX and FIX, and allows the coagulation cascade to resume. aPCC is plasma-derived and contains mainly prothrombin, FIX, FX, and FVIIa.

While aPCC has a longer half-life than rFVIIa (8-12 hours vs. 2-3 hours), some patients treated with aPCC may experience allergic reactions and anamnestic responses because aPCC contains FIX and traces of FVIII [START_REF] Dimichele | Inhibitors: resolving diagnostic and therapeutic dilemmas[END_REF][START_REF] Kempton | Toward optimal therapy for inhibitors in hemophilia[END_REF].

Despite their suboptimal hemostatic efficacy compared to FVIII, the development of BPAs, since 1997, offered HA patients with inhibitors alternative products to treat and reduce bleeding events and prevent or delay the development of joint damage [START_REF] López-Fernández | Spanish Consensus Guidelines on prophylaxis with bypassing agents in patients with haemophilia and inhibitors[END_REF]. In the FEIBA NovoSeven Comparative (FENOC) Study, similar hemostatic efficacies were found for both agents in treating joint bleeds in HA patients with inhibitors (Astermark et al., 2006a). However, neither rFVIIa nor aPCC could predictably achieve hemostasis in all patients or all bleeds. A better efficacy with one product over the other was reported in a range of 9.8-43.8% at 2 to 36-hour time points. In addition, in around 10% of the cases, neither treatment effectively stopped bleeds. Additionally, it should be highlighted that BPAs do not currently have reliable biomarkers for hemostatic efficacy besides clinical improvement to help with HA patient management.

Desmopressin

Synthetic drugs, such as desmopressin (DDAVP), were introduced to treat mild HA.

DDAVP can be administered intravenously, subcutaneously, and intranasally. It temporarily stimulates the endogenous release of FVIII and VWF by endothelial cells; however, not all patients respond to this treatment [START_REF] Mannucci | 1-DEAMINO-8-D-ARGININE VASOPRESSIN: A NEW PHARMACOLOGICAL APPROACH TO THE MANAGEMENT OF HAEMOPHILIA AND VON WILLEBRAND'S DISEASE[END_REF].

Emicizumab

Emicizumab (Hemlibra®, Roche) is a recombinant bispecific antibody with humanized binding sites to both human FIX/FIXa and FX/FXa (Figure 7) designed to mimic the cofactor activity of FVIII (Shima et al., 2016). Since 2018, it has been the first and only FDA-and EMA-licensed non-factor replacement therapy for routine prophylaxis in HA patients with and without inhibitors (J. Mahlangu et al., 2018;Oldenburg et al., 2019).

Emicizumab offers the advantage of a subcutaneous route of administration, which makes it easier to start prophylaxis in pediatric patients at a young age and does not require central venous access devices, significantly improving patients' quality of life. It also has the advantage of a long half-life of 4-5 weeks; therefore, less frequent administrations are required, a high efficacy in bleed prevention, and a reduction in the frequency of bleeding episodes (an annualized bleeding rate (ABR) reduced by 96-97%). Prophylaxis dosing with emicizumab consists of an induction period of 3 mg/kg/week for 4 weeks followed thereafter by 1.5 mg/kg/week or alternative dosing schedules including 3 mg/kg every 2 weeks or 6 mg/kg every 4 weeks. Importantly, emicizumab interferes with hemostasis screening tests, particularly those based on an aPTT, rendering them inadequate to reflect hemostatic efficacy. Therefore, appropriate tests must be used to monitor hemostasis when exogenous FVIII is used for surgery or bleeding events in patients receiving emicizumab [START_REF] Nougier | Emicizumab treatment: Impact on coagulation tests and biological monitoring of haemostasis according to clinical situations (BIMHO group proposals)[END_REF]. To ensure reliable results in such situations and to measure FVIII inhibitors, chromogenic assays containing non-human (i.e., bovine) coagulation factors IX and X, which are insensitive to emicizumab, must be used. The one-size-fits-all approach suggested for the use of this drug generates a number of concerns about how much this approach will stand up in real-life experience after treating patients, with different lifestyles and risk factors, for several years. From (Lenting et al., 2017).

The HAVEN study program results and several additional real-world evidence data sets showed that emicizumab had a favorable pharmacokinetic and pharmacodynamic profile.

For instance, phase 3 studies HAVEN 1,HAVEN 3,HAVEN 4,and HAVEN 5 assessed safety and efficacy profiles of different doses regimens in HA adults and adolescents with and/or without inhibitors [START_REF] Mancuso | Emicizumab Prophylaxis in Adolescent/Adult Patients with Hemophilia A Previously Receiving Episodic or Prophylactic Bypassing Agent Treatment: Updated Analyses from the HAVEN 1 Study[END_REF]J. Mahlangu et al., 2018;Pipe et al., 2019b;[START_REF] Yang | Prophylactic emicizumab for hemophilia A in the Asia-Pacific region: A randomized study (HAVEN 5)[END_REF]. The latter profiles were comparable to those obtained in the pediatric HAVEN 2 study (2 to 12-year-old) [START_REF] Young | Emicizumab Prophylaxis Provides Flexible and Effective Bleed Control in Children with Hemophilia Α with Inhibitors: Results from the HAVEN 2 Study[END_REF]. Furthermore, according to a recently published summary of long-term follow-up data from the HAVEN 1-4 clinical trials, emicizumab has an acceptable long-term safety profile and is associated with significant improvements in health-related outcomes (Callaghan et al., 2021). However, experience of emicizumab in infants (from birth to ≤12 months of age) is limited. Currently, a phase 3 HAVEN 7 study (NCT04431726) and several real-world studies are ongoing to reinforce previous promising results. In addition, another clinical trial HAVEN 6 is ongoing in HA patients with mild or moderate levels of FVIII (NCT04158648).

Though emicizumab mimics FVIII activity, these molecules are biochemically different and are not regulated by the same mechanisms. Therefore, emicizumab is used to prevent, but not to treat, acute bleeds in HA patients with inhibitors. Breakthrough bleeds require treatment with FVIII concentrates for patients without inhibitors (J. Mahlangu et al., 2018) or hemostatic BPAs for patients with inhibitors. Importantly, rFVIIa should be the first-line treatment in the latter patients. The use of aPCC in doses >100 U/kg for more than 24 hours was associated with a risk of thrombotic events and thrombotic microangiopathy (TMA). In HAVEN 1, two thrombotic and three TMA occurred (Oldenburg et al., 2017) and an additional TMA was reported post-marketing [START_REF] Howard | Evaluation of the Safety of Emicizumab Prophylaxis in Persons with Hemophilia A: An Updated Summary of Thrombotic Events and Thrombotic Microangiopathies[END_REF]. Thus, aPCC should only be used if no other treatment options are available or effective. As per the 2020 practical guidance of the GTH hemophilia board, an initial dose of maximum 50 U/kg should not be exceeded (Holstein et al., 2020a). Lower doses have been described as effective and safe in single cases [START_REF] Young | Emicizumab Prophylaxis Provides Flexible and Effective Bleed Control in Children with Hemophilia Α with Inhibitors: Results from the HAVEN 2 Study[END_REF]. If aPCC use is planned to cover major surgery, Emicizumab should be stopped approximately 6 months upfront because of the long half-life of the drug and possible interactions during that period (Holstein et al., 2020a). Interactions of aPCC with emicizumab lead to a synergistic effect and excessive thrombin generation since aPCC contains FIXa and FX, the targets of emicizumab.

Emicizumab, like FVIII replacement therapy or any other therapeutic molecule, has the potential for immunogenicity. According to the emicizumab prescribing information, 3.5% of patients in the HAVEN clinical trials developed anti-emicizumab antibodies, though less than 1% developed neutralizing antibodies [START_REF] Paz-Priel | Immunogenicity of Emicizumab in People with Hemophilia A (PwHA): Results from the HAVEN 1-4 Studies[END_REF].

Despite the great therapeutic efficacy of emicizumab, its favorable safety profile, outstanding pharmacokinetics, and adaptable dose options, there are a few unmet needs related to its administration. These include 1) its indication for preventing bleeding rather than treating it, 2) the interference of emicizumab with clotting-based laboratory assays, 3) hemostatic monitoring in the perioperative context and 4) the lack of drugs to reverse its impact. A number of studies are currently being conducted to address some of these issues. These include the MOTIVATE study, which examines the use of emicizumab prophylaxis combined with low-dose FVIII to induce immune tolerance. This protocol intends to reduce the burden of standard immune tolerance induction protocols (ITI), described in paragraph 2.4.3, and achieve inhibitor eradication (NCT04023019). The PRIORITY study also examines the potential role of emicizumab in preventing inhibitor recurrence (NCT04030052).

Mim8

Mim8 is a next-generation FVIII-mimetic bispecific antibody for subcutaneous prophylactic treatment of HA patients with and without inhibitors. It outperforms emicizumab in mouse models and in vitro assays. Preclinical data showed a 15-fold increase in potency as compared to emicizumab [START_REF] Kjellev | Mim8a Next-Generation FVIII Mimetic Bi-Specific Antibody -Potently Restores the Hemostatic Capacity in Hemophilia a Settings in Vitro and In Vivo[END_REF]. It was generated using Duobody®, an in vitro technology using the controlled antigen-binding fragment (Fab)-arm exchange. After screening more than 30 000 pairs of anti-FIXa and anti-FX antibodies, Mim8 was created by optimization of functional and biophysical properties. The anti-FIXa arm of Mim8 recognizes epitopes different from those recognized by the emicizumab arm. Mim8 assembles efficiently with FIXa and FX on membranes supporting FX activation. In addition, it has minimal target binding in the blood to avoid systemic coagulation, low immunogenicity, and low viscosity (Østergaard et al., 2021). Currently, phase 1 and 2 clinical trials are ongoing to evaluate the efficacy and safety of Mim8 (NCT05127473; NCT04204408). In addition, two phase 3 trials are recruiting HA patients (NCT05053139) and children with HA (NCT05306418), with or without inhibitors. Importantly, preliminary results from in vitro studies show that chromogenic bovine FIXa/FX assays are insensitive to Mim8 and can be used to precisely measure additional rFVIII therapy or FVIII inhibitors in Mim8-treated patients (Bowyer A. et al., ISTH 2021 Abstract: PB0679). Moreover, a modified chromogenic test can be used to precisely measure Mim8 levels.

Inhibitors of natural anticoagulants

The traditional method of treating HA has been to replace the missing procoagulant protein or, in cases where inhibitors are present, to use BPAs. However, hemostasis can be recovered by blocking the natural anticoagulants. This has first been demonstrated by the influence of natural anticoagulant and fibrinolytic factors' levels in modulating HA clinical severity in a co-inheritance setting [START_REF] Shetty | Contribution of natural anticoagulant and fibrinolytic factors in modulating the clinical severity of haemophilia patients[END_REF]. In the latter setting, thrombin generation was found to be increased (e.g., protein C or TFPI deficiency).

Therefore, several novel non-replacement therapies have been developed and have shown promise in early-phase clinical trials. Like emicizumab, a major potential advantage of these agents is their indifference to the presence of inhibitors. [START_REF] Gualtierotti | Updates on Novel Non-Replacement Drugs for Hemophilia[END_REF].

• Fitusiran

The primary natural anticoagulant AT functions by inactivating thrombin and FXa.

Therefore, inhibiting AT is an interesting strategy. Fitusiran is an investigational small interfering RNA (siRNA) agent that specifically targets AT messenger RNA to suppress AT production in the liver [START_REF] Pasi | Targeting of Antithrombin in Hemophilia A or B with RNAi Therapy[END_REF] (Figure 8). Due to its mechanism of action that enhances thrombin generation potential, this therapy could restore sufficient hemostasis in both HA and HB patients with or without inhibitors [START_REF] Pipe | Long-Term Durability, Safety and Efficacy of Fitusiran Prophylaxis in People with Hemophilia a or B, with or without Inhibitors -Results 202 from the Phase II Study[END_REF]. It also has the advantage of subcutaneous administration and a prolonged duration of action.

It was found that the most effective way to prevent bleeding is to reduce AT by 75%.

Breakthrough bleeds are not expected to be treated by fitusiran; these can be treated with BPAs or FVIII replacement, but lower dosages must be used to reduce the risk of excessive procoagulant activity (Pipe et al., 2019a).

As of today, five thromboembolism events have been reported during clinical trials. The safety and efficacy of fitusiran were assessed in HA and HB patients with [START_REF] Young | Efficacy and Safety of Fitusiran Prophylaxis, an siRNA Therapeutic, in a Multicenter Phase 3 Study (ATLAS-INH) in People with Hemophilia A or B, with Inhibitors (PwHI)[END_REF]) ( ATLAS-INH; NCT03417102) or without inhibitors [START_REF] Srivastava | Fitusiran, an Investigational siRNA Therapeutic Targeting Antithrombin for the Treatment of Hemophilia: First Results from a Phase 3 Study to Evaluate Efficacy and Safety in People with Hemophilia a or B without Inhibitors (ATLAS-A/B)[END_REF] (ATLAS-A/B; NCT03417245). In both patients, prophylactic fitusiran administration (80 mg, once per month) resulted in an ABR reduction of around 90%. However, there are still concerns about the thromboembolic risk and appropriate dosing regimen for factor replacement in the setting of bleeding or surgery. The long-term safety and efficacy of fitusiran are currently being investigated by the ATLAS-OLE study (NCT03754790) as well as phase 3 studies in adolescents and a dose-confirmation study in children (ATLAS-PEDS, NCT03974113).

• SerpinPC

Another approach is to target activated protein C (aPC). aPC, in collaboration with its cofactor protein S, catalyzes the rapid degradation of the cofactors FVa and FVIIIa, effectively inhibiting prothrombinase and tenase complex activity (Figure 8). Thus, inhibiting aPC may prolong thrombin propagation and restore hemostatic balance in HA patients. Inhibitors of aPC can be found endogenously and are members of the serpin family, but these might cross-react with other procoagulant proteases such as FXa, FVIIa, and thrombin. Therefore, Polderdijk et al. designed a modified serpin that potently inhibits aPC but cross-reacts minimally with procoagulant enzymes [START_REF] Polderdijk | Design and characterization of an APC-specific serpin for the treatment of hemophilia[END_REF]. Its efficacy is currently being investigated in a phase 1/2 clinical trial in patients with severe HA and HB (AP-0101; NCT0473498). For now, there were no SerpinPC-related adverse events and no elevation in D-dimer levels, an early sign of excessive thrombin generation associated with other investigational hemophilia treatments.

• Anti-TFPI

The first anti-TFPI tested was BAX499 (ARC19499), a polyethylene glycol-conjugated aptamer that binds to TFPI and blocks TFPI inhibition of FXa and FVIIa [START_REF] Waters | Aptamer ARC19499 mediates a procoagulant hemostatic effect by inhibiting tissue factor pathway inhibitor[END_REF]. Aptamers are single-stranded oligonucleotides that bind a specific target protein with high affinity. Unfortunately, this drug did not reach phase 2 and 3 studies and was stopped at phase 1 (NCT01191372) due to increased bleeding events.

After the aptamer's failure, attention turned to the development of monoclonal anti-TFPI antibodies: concizumab [START_REF] Chowdary | Safety and pharmacokinetics of anti-TFPI antibody (concizumab) in healthy volunteers and patients with hemophilia: a randomized first human dose trial[END_REF], marstacimab [START_REF] Cardinal | A first-in-human study of the safety, tolerability, pharmacokinetics and pharmacodynamics of PF-06741086, an anti-tissue factor pathway inhibitor mAb, in healthy volunteers[END_REF], befovacimab [START_REF] Chowdary | Pharmacodynamics, Pharmacokinetics and Safety of Bay 1093884, an Antibody Directed Against Human TFPI, in Patients with Factor VIII or IX Deficiency (With and Without Inhibitors): A Phase 1 Study[END_REF], and MG1113 [START_REF] Kwak | MG1113, a specific anti-tissue factor pathway inhibitor antibody, rebalances the coagulation system and promotes hemostasis in hemophilia[END_REF] (Figure 8).

A rate-limiting step in the hemostasis inhibition process is the binding of the TFPI Kunitz-2 (K2) domain to FXa, which makes the latter domain an attractive target for anti-TFPI drug development. TFPI has three tandem Kunitz-type inhibitory domains (K1, K2, and K3). Therefore, the four antibodies were developed to target the K2 domain of the TFPI, with the befovacimab also targeting the K1 TFPI domain. Under physiological conditions, the TFPI molecule binds FXa via its K2 domain to form the TFPI-FXa complex which inhibits FXa. The TF-FVIIa complex then binds the TFPI-FXa through the TFPI K1 domain to form the TFPI-FXa-TF-FVIIa quaternary complex with resultant inhibition of FVIIa.

Concizumab and Marstacimab are currently under phase 3 clinical development for the treatment of patients with HA and HB with and without inhibitors (NCT05135559 and NCT05611801, respectively). However, non-fatal thrombotic events in 3 participants under concizumab treatment with distinct thrombotic risk factors were observed [START_REF] Seremetis | Risk Mitigation Strategy for Concizumab Clinical Trials after Pause Due to Non-Fatal Thrombotic Events[END_REF]. Additionally, the Befovacimab phase 2 trial (NCT03597022) was terminated due to thrombotic events, while MG1113 is currently undergoing a phase 1 investigation (NCT05493631).

Thrombotic events have been described with several non-replacement therapies. These events underscore the subtle line separating hemostasis and thrombosis. The potential thrombotic risk in patients with HA receiving non-replacement therapy is particularly tricky when administered in combination with replacement factors or BPAs. Long-term monitoring and patient education to recognize and report signs and symptoms of thrombosis should be undertaken to limit these risks.

Gene therapy

Hemophilia is an ideal candidate for gene therapy as a disease attributed to the deficiency or dysfunction of a single protein. Lentiviral vectors are very challenging to manufacture; therefore, adeno-associated viral (AAV) vectors are more commonly used in gene therapy. However, one limitation of AAV vectors is their limited packaging capacity (~4.7 kb). Thus, initial gene therapy studies initiated in 2013 were conducted in patients with HB to deliver the FIX gene (~2.8 kb) to hepatocytes. Similar studies were initiated at a later stage for HA patients since the F8 gene is larger (~7 kb) than the vector's capacity and has a poor expression profile at therapeutically effective levels. Multiple strategies are being evaluated to address these drawbacks: 1) increasing transcription through promoter engineering, 2) improving translation through codon optimization of FVIII mRNA, and 3) promoting FVIII secretion by amino-acid substitution [START_REF] Brown | Target-Cell-Directed Bioengineering Approaches for Gene Therapy of Hemophilia A[END_REF]Pipe et al., 2022).

Currently, 16 clinical studies of gene therapy for HA developed by 8 companies are registered at ClinicalTrials.gov. BioMarin has reported the largest phase 3 gene therapy study (NCT03370913) in adults with severe HA with Roctavian™ (Valoctocogene Roxaparvovec) [START_REF] Ozelo | Valoctocogene Roxaparvovec Gene Therapy for Hemophilia A[END_REF]. To date, a single 6x10 13 vector genomes (vg)/kg dose of Roctavian has been well tolerated, with a mean ABR reduction of 80% from baseline and a 94% reduction in FVIII usage in year 3 compared to baseline. Furthermore, at the end of year 3, 92% of patients were no longer receiving prophylaxis (biomarinannouncement-2023).

An important limitation of AAV treatment is preexisting neutralizing anti-AAV antibodies specific to the various AAV serotypes resulting in reduced treatment efficacy. These antibodies are prevalent in the population due to natural infection with wild-type AAV during childhood ranging from 20% to 70%, depending on the specific AAV serotype and subject population [START_REF] Klamroth | Global Seroprevalence of Pre-existing Immunity Against AAV5 and Other AAV Serotypes in People with Hemophilia A[END_REF]. Therefore, BioMarin is conducting a phase 1/2 clinical trial to evaluate the effect of administering high doses (6x10 13 vg/kg) of the construct to patients with HA with preexisting anti-AAV capsid antibodies (NCT02576795).

Such results are crucial to determine whether readministration of the same vector is possible and whether the expression of FVIII will be maintained and protected from silencing, degradation, or loss due to cellular turnover. Moreover, whether an episome-derived liver expression will be sustained in a setting of substantial liver proliferation, as in pediatric patients (the liver quadruples in size during the first 4-5 years of development) or those with liver disease (e.g., hepatitis and/or cirrhosis) is still not known. For these patients, site-specific integration of the transgene following a single vector dose to avoid AAV dilution and loss of expression could be especially beneficial. Recently, the therapeutic potential of site-specific integration of therapeutic transgenes within the albumin locus has restored hemostasis and long-term expression of human FVIII and human FIX in mouse models of both HA and HB [START_REF] Sharma | In vivo genome editing of the albumin locus as a platform for protein replacement therapy[END_REF].

FVIII inhibitors -Pathophysiology

Immune response to FVIII and development of inhibitors

The immune response to the exogenously administered therapeutic FVIII follows a classical pattern wherein some of the intravenously administered FVIII accumulates in secondary lymphoid organs, as observed in spleens of FVIII-deficient mice [START_REF] Navarrete | Splenic marginal zone antigen-presenting cells are critical for the primary allo-immune response to therapeutic factor VIII in hemophilia A[END_REF], and is endocytosed by antigen-presenting cells (APCs) (i.e., dendritic cells (DCs), macrophages, B cells). FVIII is then processed and peptides are presented on the major histocompatibility complex (MHC) class II molecules to naive FVIII-specific CD4+ T helper (Th) cells (Figure 9). Upon activation, Th cells provide help to naive FVIII-specific B cells that proliferate and differentiate into memory B cells and plasma cells. Interestingly, T follicular helper cells (Tfh) have recently been identified as a subset of CD4+ T cells that help activate FVIII-specific B cells and are responsible for a T cell-dependent humoral response [START_REF] Jing | Induction of activated T follicular helper cells is critical for anti-FVIII inhibitor development in hemophilia A mice[END_REF]. VWF was shown to play an immune-protective role towards therapeutic FVIII by reducing the amount of FVIII internalized by APCs and, consequently, presented to CD4+ T cells (Dasgupta et al., 2007). Conversely, activation of complement C3 was shown to enhance FVIII uptake and boost the anti-FVIII immune response, at least in a mouse model of severe HA [START_REF] Rayes | Complement C3 is a novel modulator of the anti-factor VIII immune response[END_REF]. From [START_REF] Prezotti | Immunogenicity of Current and New Therapies for Hemophilia A[END_REF].

Detailed description of FVIII inhibitors

FVIII inhibitors are high-affinity polyclonal antibodies mainly belonging to the IgG isotype.

The anti-FVIII IgG are predominantly IgG4 in patients with high levels of inhibitors and IgG1 in patients with low inhibitory titers (Whelan et al., 2013;Hofbauer et al., 2015).

Inhibitors can affect FVIII procoagulant function and catabolism in several ways [START_REF] Ananyeva | Inhibitors in hemophilia A[END_REF]Batsuli et al., 2018;[START_REF] Lacroix-Desmazes | Catalytic activity of antibodies against factor VIII in patients with hemophilia A[END_REF], preventing FVIII from 1) interacting with FIXa and FX; 2) adhering to VWF and/or activated platelet surfaces; 3) being activated by thrombin (or FXa) and/or from being released from VWF; or 4) endocytosis by antigen-presenting cells.

Inhibitory antibodies are thus directed to the FVIII light chain, its heavy chain, or both chains. They are primarily directed against the A2, A3, C1, and C2 domains of FVIII [START_REF] Scandella | Localization of Epitopes for Human Factor VIII Inhibitor Antibodies by Immunoblotting and Antibody Neutralization[END_REF][START_REF] Prescott | The Inhibitor Antibody Response Is More Complex in Hemophilia A Patients Than in Most Nonhemophiliacs With Factor VIII Autoantibodies[END_REF]. FVIII inhibitors may also accelerate the clearance of therapeutic FVIII (Kazatchkine et al., 1980;Batsuli et al., 2018) or hydrolyze FVIII [START_REF] Lacroix-Desmazes | Catalytic activity of antibodies against factor VIII in patients with hemophilia A[END_REF]. Non-neutralizing anti-FVIII IgG are also detected in treated patients without inhibitors (Whelan et al., 2013) as well as in healthy donors with normal hemostasis (Algiman et al., 1992). Albeit to a lesser extent than neutralizing antibodies, the non-neutralizing anti-FVIII IgG can affect the half-life of FVIII in circulation and the effectiveness of replacement therapy [START_REF] Hofbauer | FVIII-binding IgG modulates FVIII half-life in patients with severe and moderate hemophilia A without inhibitors[END_REF]. FVIII inhibitors are classified based on the kinetics and extent of inhibition of FVIII. Type I inhibitors follow second-order kinetics (dose-dependent linear inhibition), completely inactivate FVIII, and are more common in severe HA. In contrast, type II inhibitors have complex kinetics, incompletely inactivate FVIII, and are more common in patients with mild HA or in patients without hemophilia who develop an acquired FVIII inhibitor (cf. paragraph 3).

Risk factors in the development of FVIII inhibitors

The etiology of FVIII inhibitor development is multifactorial and still unclear. Although the risks have been divided into those that are inherited (genetic risk factors) and those that are connected to therapy (non-genetic risk factors), there is still, to date, no reliable or consistent indicator of whether or not a PUP would develop FVIII inhibitors.

Genetic risk factors

Established genetic risk factors (unmodifiable risk factors) include the F8 gene mutation and ensuing severity of HA, the family history of inhibitor development, ethnicity, and some immune response genes. Null variants or variants that result in total absence of the FVIII protein (large deletions, duplications, insertions, inversions, nonsense mutations, and splice-site variants), have shown the strongest association with inhibitor development as compared to other variants (small in-frame deletions, duplications, insertions, missense mutations). According to empirical research, the inhibitor risk ranges from >75% in multidomain deletions to 20-30% in the intron 22 inversion mutation and <10% in missense mutations and small FVIII deletions and insertions [START_REF] Goodeve | The Molecular Basis of Hemophilia A: Genotype-Phenotype Relationships and Inhibitor Development[END_REF][START_REF] Gouw | F8 gene mutation type and inhibitor development in patients with severe hemophilia A: systematic review and metaanalysis[END_REF]. These results highlight the importance of the education of HA patients' immune system against endogenous FVIII, whether centrally in the thymus or at the periphery. In principle, the greater the similarity between endogenously produced FVIII and exogenously administered therapeutic FVIII, the lower the risk of developing a neutralizing immune response. However, the situation in HA patients is more complex. Some missense mutations causing mild-to-moderate HA are associated with inhibitor development rates comparable to those in patients with severe HA [START_REF] Eckhardt | Factor VIII gene (F8) mutation and risk of inhibitor development in nonsevere hemophilia A[END_REF].

The poor antigen presentation and absence of tolerance induction in such cases can be explained, for instance, by impaired secretion following retention of the nascent FVIII protein in the endoplasmic reticulum for some mutated forms of FVIII [START_REF] Malhotra | Antioxidants reduce endoplasmic reticulum stress and improve protein secretion[END_REF]. On the other hand, missense mutations may result in T-cell epitopes in the mutated endogenous FVIII that differ from those in the therapeutic FVIII. In the latter case, the immune system education does not fit, and regulatory mechanisms cannot be adequately engaged [START_REF] Jacquemin | CD4+ T-cell clones specific for wild-type factor VIII: a molecular mechanism responsible for a higher incidence of inhibitor formation in mild/moderate hemophilia A[END_REF][START_REF] Pashov | In silico calculated affinity of FVIIIderived peptides for HLA class II alleles predicts inhibitor development in haemophilia A patients with missense mutations in the F8 gene. Haemoph. Off[END_REF].

According to several studies, members of families with a history of inhibitor development are more likely to develop FVIII inhibitors than members of families without such a history. Data from the Malmö International Brother Study (MIBS) clearly supports this idea. The risk for inhibitor development in families with a previous inhibitor history was found to be 48%, compared to only 15% in families with no history of known inhibitors (Astermark et al., 2001).

Numerous studies have consistently found variations in inhibitor risk among various ethnic groups; however, the causes of these variations are still unknown [START_REF] Aledort | Inhibitors occur more frequently in African-American and Latino Haemophiliacs[END_REF]Astermark et al., 2001;[START_REF] Miller | F8 and F9 mutations in US haemophilia patients: correlation with history of inhibitor and race/ethnicity[END_REF]. For example, patients of non-Caucasian ethnicity, particularly those of African descent, had a 1.9-4.7-fold higher risk of developing inhibitors (Black African descent > Latin American > Caucasian).

Moreover, several polymorphisms in immune response genes such as interleukin 10 (IL-10), tumor necrosis factor (TNF), and cytotoxic T-lymphocyte associated antigen 4 (CTLA-4) have been proposed as genetic risk factors (Astermark et al., 2006c(Astermark et al., , 2006b;;[START_REF] Pavlova | Impact of polymorphisms of the major histocompatibility complex class II, interleukin-10, tumor necrosis factor-α and cytotoxic T-lymphocyte antigen-4 genes on inhibitor development in severe hemophilia A[END_REF]. Human leukocyte antigen (HLA) type, a factor determining which peptides are presented to naive T helper cells, could theoreticallythough consistent reports are lackingbe a risk factor in inhibitor development. Notably, conflicting reports associate HLA class II alleles, such as HLA-DRB1*1501, DQB1*0602, and DQA1*0102, with a higher risk of inhibitor development [START_REF] Hay | HLA class II profile: a weak determinant of factor VIII inhibitor development in severe haemophilia A. UKHCDO Inhibitor Working Party[END_REF][START_REF] Oldenburg | HLA genotype of patients with severe haemophilia A due to intron 22 inversion with and without inhibitors of factor VIII[END_REF][START_REF] Pavlova | Impact of polymorphisms of the major histocompatibility complex class II, interleukin-10, tumor necrosis factor-α and cytotoxic T-lymphocyte antigen-4 genes on inhibitor development in severe hemophilia A[END_REF].

A few years ago, our team described a correlation between a GT repeat polymorphism in the promoter of the gene encoding the anti-inflammatory enzyme heme-oxygenase-1 (hmox1 gene) and inhibitor development in patients with severe HA (Repesse et al., 2013). More recently, Matino et al. demonstrated that following in vitro stimulation by CpG, dendritic cells from HA patients with inhibitors are less prone to express indoleamine 2,3dioxygenase 1 (IDO1), a key regulatory enzyme that supports regulatory T-cell functions and peripheral tolerance in adult life (Matino et al., 2015). A recent study described an association of the cytokine B cell activating factor (BAFF) gene polymorphism with the development of FVIII inhibitors [START_REF] Hodeib | BAFF rs9514828 gene polymorphism and the risk of the development of inhibitors in children with severe haemophilia A[END_REF]. BAFF is implicated in B cell survival, maturation, and differentiation. Therefore, elevated BAFF levels may serve as a helpful prognostic marker for the development of FVIII inhibitors in pediatric HA patients [START_REF] Doshi | B cell-activating factor modulates the factor VIII immune response in hemophilia A[END_REF].

Non-genetic risk factors

The non-genetic or potentially modifiable risk factors consist of the treatment regimen employed (including prophylactic versus on-demand), treatment intensity at first exposure, the type of FVIII concentrate used, and danger signals at the moment of administration (i.e., inflammation caused by major bleeds or surgery).

The importance of the type of FVIII product in inhibitor development remains highly debated. Studies report that rFVIII products have a higher risk of inhibitor development than pd-FVIII products containing VWF. This increased immunogenicity is thought to be due to alterations in FVIII post-translational modifications and in VWF binding, resulting in a loss of protection against inhibitor formation. Importantly, VWF has been demonstrated to reduce FVIII endocytosis by DCs (Dasgupta et al., 2007). In 2016, the Survey of Inhibitors in Plasma Product Exposed Toddlers (SIPPET) study, an international randomized prospective clinical trial, compared the incidence of inhibitor development in PUPs with severe HA treated with different FVIII products (Peyvandi et al., 2016). Patients treated with pd-FVIII had a significantly lower inhibitor incidence than those treated with rFVIII (26.8% vs. 44.5%). Despite concerns about the study's design and generalizability, it remains the only randomized controlled comparative trial conducted to date.

Most inhibitors develop at a very young age, with the highest incidence occurring between the ages of 0 and 4 years, according to the United Kingdom Hemophilia Center Doctors' Organization (UKHCDO) database (Hay et al., 2011). Two small cohort studies found an inverse correlation between the early age at first exposure and inhibitor formation, but they were not controlled for risk factors for inhibitor formation [START_REF] Bom | Age at first treatment and immune tolerance to factor VIII in severe hemophilia[END_REF][START_REF] Lorenzo | Incidence of factor VIII inhibitors in severe haemophilia: the importance of patient age[END_REF]. On the contrary, other studies could not demonstrate any association with age (Gouw et al., 2007;[START_REF] Santagostino | Environmental risk factors for inhibitor development in children with haemophilia A: a case-control study[END_REF]. However, the intensity of the first FVIII exposure is postulated as a risk factor for inhibitor formation later in life. It might explain the association between age at first exposure and inhibitor development seen in the CANAL study (Gouw et al., 2007b). This was further suggested in the prospective Research Of Determinants of INhibitor Development among PUPs with haemophilia (RODIN) study (Gouw et al., 2013b).

Another conclusion from the RODIN study is that except during the initial period of treatment (first 20 exposure days), prophylaxis was found to have a protective effect against inhibitor development (Gouw et al., 2013b).

Eradication of FVIII inhibitors

Immune tolerance induction

While it is now possible to restore hemostasis in HA patients with inhibitors using various drugs described in the previous paragraphs (see paragraph 2.2.2), the eradication of inhibitors is currently best achieved through immune tolerance induction (ITI) therapy. It entails the regular and prolonged administration of FVIII concentrates, usually at high doses, to suppress the anti-FVIII antibody response, leading to immunological tolerance and desensitization [START_REF] Brackmann | Massive factor-VIII infusion in haemophiliac with factor-VIII inhibitor, high responder[END_REF]. ITI is prohibitively expensive, requires extreme compliance from the patients, and is successful only in 60-80% of the patients with severe HA (DiMichele et al., 2007;[START_REF] Coppola | Optimizing management of immune tolerance induction in patients with severe haemophilia A and inhibitors: towards evidence-based approaches[END_REF]. It generally requires several months before reaching success (9 to 33 months). Response to ITI is even less favorable in patients with mild-to-moderate HA [START_REF] Castaman | Molecular and clinical predictors of inhibitor risk and its prevention and treatment in mild hemophilia A[END_REF].

Children with recently diagnosed inhibitors are the best candidates for ITI therapy.

Depending on their clinical profile and bleeding pattern, adults with persistent inhibitors may also be good candidates. Patients presenting with transient inhibitors or low-titer antibodies can typically continue prevention and treatment of bleeds using replacement therapy instead of ITI. However, those with high-titers (≥ 5 BU) are advised to start ITI as soon as possible. It was previously recommended that ITI therapy in patients with titers exceeding 10 BU should be delayed until the titers drop to less than 10 BU; nevertheless, some evidence suggests that starting ITI immediately improves the success rate in these patients [START_REF] Collins | First-line immune tolerance induction for children with severe haemophilia A: A protocol from the UK Haemophilia Centre Doctors' Organisation Inhibitor and Paediatric Working Parties[END_REF]. Successful ITI is defined as a persistently negative Bethesda titer, accompanied by normal pharmacokinetics, including factor recovery >66% and half-life >6 hours for standard FVIII concentrates. In the event of ITI success, it is recommended that patients initiate or resume long-term prophylaxis to help maintain tolerance and avoid rapid changes in dose. Interestingly, while ITI refers to the induction of immune tolerance, the only criterion in favor of tolerance induction was the disappearance of the inhibitory titer. Yet, evidence in favor of an actual induction of FVIIIspecific active immune tolerance that relies on the induction of regulatory T and B cells just started to emerge (Schep et al., 2021a).

Proposed mechanisms of action of ITI

The production of anti-idiotypic antibodies (directed to anti-FVIII antibodies), suppression of FVIII-specific memory B cell differentiation into antibody-secreting plasma cells, T cell exhaustion through overstimulation leading to T cell anergy, and induction of regulatory T cells are some of the proposed mechanisms for tolerance induction (Figure 10). However, FVIII-specific T cells, as well as anti-FVIII IgG with neutralizing potential, may still be detected in patient's blood after ITI success (Pautard et al., 2011;Gilles et al., 1996). Hence, the immunological mechanisms underlying the establishment of a tolerogenic immune response in ITI are unclear.

Hausl et al. reported that high doses of FVIII inhibit the re-stimulation of FVIII-specific memory B cells and prevent their differentiation into antibody-secreting plasma cells in vitro and in vivo in a murine model of HA (FVIII-deficient mice). The inhibition of memory B cell responses was irreversible, involved the activation of caspases, and was not mediated by FVIII-specific T cells [START_REF] Hausl | High-dose factor VIII inhibits factor VIII-specific memory B cells in hemophilia A with factor VIII inhibitors[END_REF]. Reipert et al. further confirmed these findings by reporting that co-stimulatory interactions with activated T cells are essential for re-stimulating FVIII-specific memory B cells in in vivo studies using HA mice.

However, the concentration of FVIII required for inhibition of memory B cell re-stimulation (100-300 ng/ml; with an almost complete inhibition at 1 µg/ml) and the concentration required for inhibition of T cell re-stimulation (100 µg/ml) are very different. This makes it unlikely that the inhibition of memory B cell re-stimulation is caused by an inhibition of T cell stimulation [START_REF] Reipert | Modulation of factor VIII-specific memory B cells[END_REF]. The net result of these events is that (long-living) plasma cells are not replenished, which eventually leads to the eradication of inhibitors. Finally, also the development of anti-idiotypic anti-FVIII antibodies might be involved in this process by neutralizing the effect of anti-FVIII inhibitors. CTLA-4: cytotoxic T-lymphocyte associated antigen 4; PD1: Programmed cell death 1. From (Schep et al., 2018).

However, whether this is what actually happens in patients undergoing ITI is still unknown.

Of note, the concentration needed to induce a complete inhibition of FVIII-specific memory B cells in mouse models is 10 times higher than the physiological levels of FVIII and those that can be achieved even with high-dose ITI protocols. Nevertheless, 0.05-0.24% out of all IgG-producing memory B cells were FVIII-specific in inhibitor patients, as showed by Van Helden et al., whereas very few or none were found in patients successfully treated with ITI ( [START_REF] Van Helden | Factor VIII-specific memory B cells in patients with hemophilia A[END_REF].

Most of the knowledge about the role of T cells in inducing peripheral tolerance is based on fundamental research; clinical data are scarce. It has been established that the chronic exposure of a naive immune system to an antigen in the absence of danger signals leads to the induction of regulatory T cells (Tregs). Thus, the chronic exposure of HA patients to FVIII during ITI could also induce FVIII-specific Tregs, which in turn would be able to suppress FVIII-specific effector T cells essential for FVIII-specific B cells stimulation.

However, in patients with FVIII inhibitors, the immune system is already primed and questions remain on whether modulation of pre-existing memory B and T cells is achievable. The contribution of naturally occurring Tregs to the suppression of FVIIIspecific T cell responses in humans was first assessed by Kamate et al., who found that the FVIII-dependent response was markedly increased in Treg-depleted peripheral blood mononuclear cells (PBMCs) samples from healthy individuals (Kamaté et al., 2007). To determine whether ITI leads to the depletion of FVIII-specific T cells, Pautard et al. expanded FVIII-specific T cells obtained from successfully desensitized patients by coculture with autologous DCs loaded with FVIII and then detected the cells releasing interleukins following stimulation with FVIII (Pautard et al., 2011). A significant number of FVIII-specific T cells were detected in one out of five patients, indicating that the elimination of FVIII-specific T cells is not required to restore tolerance towards FVIII.

Additional information was provided by a study from Hu et al., wherein CD4+ T cell blasts from healthy individuals and HA patients with and without inhibitors were analyzed for cytokine production responding to rFVIII [START_REF] Hu | Cytokine production by CD4+ T cells specific for coagulation factor VIII in healthy subjects and haemophilia A patients[END_REF]. Twenty-three of the 44 healthy subjects significantly responded to rFVIII, and their CD4+ T cell blasts produced interferon-gamma (IFN-γ) and transforming growth factor-β (TGF-β), whereas the CD4+ T cell blasts of the other subjects produced only TGF-β. Similarly, to healthy responders, HA patients without inhibitors also produced IFN-γ and TGF-β. In contrast, rFVIII exposure of cells from HA patients with inhibitors induced the production of IFN-γ and IL-4, but not TGF-β. These findings suggest a role of Th1 cells (illustrated by the IFN-γ production) in initiating the immune response to rFVIII and of Th2 cells (illustrated by the IL-4 production) in the development of strong inhibitor production. Whereas a regulatory Th3 response (illustrated by TGF-β) may prevent inhibitors synthesis and maintain tolerance to FVIII in healthy individuals and HA patients without inhibitors remains to be confirmed.

Recent studies by Schep et al. have associated inhibitor eradication in patients monitored

during ITI with an increase in regulatory B cells (Bregs), Tregs, and CD4+ T cell expression of CTLA-4 and programmed cell death protein 1 (PD1) (Schep et al., 2021a).

These results suggest that deficits in peripheral tolerance mechanisms are associated with the existing anti-FVIII immune response and that Bregs and changes in immunomodulatory properties of CD4+ T cells probably contribute to the induction of tolerance to FVIII in HA patients with inhibitors.

Finally, basic research studies demonstrated the implication of anti-idiotypic antibodies in ITI-mediated reestablishment of tolerance to FVIII in HA patients; clinical data is lacking. This implication dates back to 1984 when anti-idiotypic antibodies present in intravenous immunoglobulins (IVIg) preparations were found to inhibit anti-FVIII autoantibodies in IVIg-treated patients with high-titer anti-FVIII autoantibodies [START_REF] Sultan | ANTI-IDIOTYPIC SUPPRESSION OF AUTOANTIBODIES TO FACTOR VIII (ANTIHAEMOPHILIC FACTOR) BY HIGH-DOSE INTRAVENOUS GAMMAGLOBULIN[END_REF]. Later, both anti-FVIII antibodies and their counteracting anti-idiotypic antibodies inhibiting binding to FVIII were found in healthy individuals (Algiman et al., 1992;Gilles and Saint-Remy, 1994). Gilles et al. also demonstrated that successful ITI in two HA patients was associated with the formation of anti-idiotypic antibodies, which neutralized the inhibitory activity of anti-FVIII antibodies (Gilles et al., 1996). Indeed, plasma from patients treated with ITI, both successful and unsuccessful, contained an anti-FVIII antibody-neutralization factor (i.e., anti-idiotypic antibodies) in the IgG fraction, which increased during successful ITI (Sakurai et al., 2004). A mouse anti-idiotypic monoclonal antibody (mAb), 14C12, specifically prevented a high-affinity human mAb, BO2C11, directed toward the C2 domain of FVIII from binding to FVIII. This anti-idiotypic mAb restored normal FVIII:C in a murine HA model reconstituted with rFVIII by neutralizing the functional inhibitory activity of BO2C11 [START_REF] Gilles | In vivo neutralization of a C2 domain-specific human anti-Factor VIII inhibitor by an anti-idiotypic antibody[END_REF]. These data were further supported by in vitro and in vivo results demonstrating the neutralization capacity of anti-idiotypic antibodies towards human monoclonal and polyclonal anti-FVIII antibodies [START_REF] Gilles | Role of anti-idiotypic antibodies in immune tolerance induction[END_REF]. Whether antiidiotypic antibodies could work as alternative ligands to FVIII in binding to B cell receptors (BCR) and induce inhibition of FVIII-specific B cells, as seen in the work of Hausl et al. presented in the previous paragraph, needs to be investigated [START_REF] Hausl | High-dose factor VIII inhibits factor VIII-specific memory B cells in hemophilia A with factor VIII inhibitors[END_REF].

ITI protocols

Different dosing regimens and combined therapy (additional immunosuppressive agents) have been described, yet, the choice of an ITI regimen is often determined on a case-bycase basis. The three most well-known protocols are the Bonn protocol, the Van Creveld protocol, and in difficult-to-treat patients, the Malmö protocol, but multiple adaptations of these regimens are used (Table 1).

In the International ITI study, there was no statistically significant difference in efficacy between patients randomly assigned to low-dose (50IU/kg FVIII, 3 times a week) or highdose (200 IU/kg FVIII, daily) regimens. However, the low-dose regimen required a longer time to achieve tolerance, and more bleeds occurred during that period [START_REF] Hay | The principal results of the International Immune Tolerance Study: a randomized dose comparison[END_REF]. Patients on ITI might experience frequent bleeding; thus, BPA or emicizumab prophylaxis can be initiated, though emicizumab prophylaxis showed a significantly greater reduction in bleeding rates than BPA (Oldenburg et al., 2017). Clinical trials are needed to evaluate whether emicizumab should be initiated before, during, after, Table 1: Immune tolerance induction protocols in patients with hemophilia A. Adapted from (Schep et al., 2018).

or instead of ITI (Le [START_REF] Quellec | Emicizumab should be prescribed independent of immune tolerance induction[END_REF]. As well, trials are needed to define the role of immunosuppressive and immunomodulatory drugs in HA patients with inhibitors.

Potential predictors of ITI outcome

Epidemiological studies have identified several clinical parameters associated with ITI outcomes. Patient-related factors include inhibitor titer at the start of ITI, the time elapsed between inhibitor diagnosis and ignition of ITI, historical peak inhibitory titer, and inhibitor peak titer during ITI (Ljung et al., 2019). Other patient-related factors include age at the start of ITI, patient ethnicity, and FVIII genotype, but data are limited and impact unclear.

The disease-causing non-null variants (i.e., small insertions/deletions and missense mutations) have been associated with a favorable outcome [START_REF] Coppola | Factor VIII gene (F8) mutations as predictors of outcome in immune tolerance induction of hemophilia A patients with high-responding inhibitors[END_REF].

Interestingly, some properties of the anti-FVIII IgG were suggested as predictive markers of ITI outcome. Thus, patients who fail ITI have high anti-FVIII IgG4/IgG1 ratios [START_REF] Helden | IgG subclasses of anti-FVIII antibodies during immune tolerance induction in patients with hemophilia A[END_REF]Minno et al., 2014). Furthermore, antibodies specific for the heavy chain or A2 domain are found in most patients who fail ITI, while most patients successfully treated with ITI have anti-C2 or anti-light chain antibodies (Helden et al., 2010;Minno et al., 2014).

Nevertheless, there is no molecular explanation today on how these properties may dictate ITI outcomes. My Ph.D. research included investigating how the subclass and/or epitope specificity of the predominant circulating anti-FVIII IgG may dictate the course of the immune response and favor the induction of tolerance.

In addition, the impact of treatment-related factors, such as replacement factor dose and product, on ITI outcome is currently unclear. However, there have been promising reports about the potentially beneficial role of VWF and the recently introduced rFVIII-Fc in the induction of tolerance (Di [START_REF] Di Minno | A role for von Willebrand factor in immune tolerance induction in patients with haemophilia A and inhibitors?[END_REF][START_REF] Malec | Extended half-life factor VIII for immune tolerance induction in haemophilia[END_REF]. According to newly published results from the global prospective VerITI-8 study, rFVIII-Fc was well tolerated and showed a quick time to tolerance (median of 2.7 months) with persistent responses in nearly two-thirds of participants [START_REF] Malec | Efficacy of rFVIIIFc for First-Time Immune Tolerance Induction (ITI) Therapy: Final Results from the Global, Prospective VerITI-8 Study[END_REF].

Novel strategies for immune tolerance induction in hemophilia A

New strategies to eradicate FVIII inhibitors are being developed, given the invasive, lengthy, and expensive nature of ITI protocols. In patients who fail ITI, for instance, the use of rituximab and other immunomodulatory (e.g., IVIg) or immunosuppressive drugs (e.g., cyclophosphamide) are already being used individually or concomitantly in clinical practice [START_REF] Collins | Novel therapies for immune tolerance in haemophilia A[END_REF]. However, rather than inducing FVIII-specific tolerance, these agents have a general immunosuppressive effect. Therefore, several novel strategies focus on targeted therapies that are mainly directed to key players of the FVIII immune response (i.e., FVIII-specific B-and T-cell, Treg and effector T cells) [START_REF] Sherman | Innovative Approaches for Immune Tolerance to Factor VIII in the Treatment of Hemophilia A[END_REF].

Immune complexes in patients undergoing ITI

The injection of large amounts of FVIII in patients with circulating anti-FVIII IgG leads to the formation of FVIII-containing immune complexes (FVIII-IC). Indeed, the presence of circulating FVIII-IC was documented in multi-transfused HA patients (Kazatchkine et al., 1980). Likewise, FVIII-IC were detected in patients with acquired HA, an autoimmune disease characterized by the development of anti-FVIII autoantibodies, and were hypothesized to accelerate FVIII clearance from the circulation by the reticuloendothelial system (Nogami et al., 2001).

Immune complexes (ICs) are three-dimensional structures formed by the interaction of an antibody (Ab) with its cognate antigen (Ag). The Ag:Ab ratio varies according to i) the size of the antigen, the density of antigenic epitopes, ii) the isotype, subclass, and affinity of the antibody, and iii) the oligoclonality of the Ab response. ICs are present either in systemic circulation or formed locally in tissue. The accumulation of abnormal IC levels triggers activation of the complement system and Fcγ receptors (FcγRs) signaling, leading to mononuclear cell degranulation, antibody-dependent cell-mediated cytotoxicity (ADCC), and/or release of pro-inflammatory cytokines [START_REF] Wen | Immunoregulatory functions of immune complexes in vaccine and therapy[END_REF]. Uncontrolled IC deposition was implicated in pathologies such as nephropathies, autoimmunity, and lung injury. The biological effects of IgG-containing ICs are, however, modulated by several parameters 1) the IgG subclass, 2) the capacity to bind complement, 3) their respective affinities for different activating FcγRs (i.e., CD64/FcγRI, CD32a/FcγRIIa, CD16/FcγRIII) or inhibitory FcγRs (i.e., CD32b/FcγRIIb), 4) the engagement of lectin-based type II FcRs (DC-SIGN and CD23) sensitive to the glycosylation state of IgG constant region (Fc), and 5) the relative expression of the FcγRs on different cell types at different locations in the organism [START_REF] Nimmerjahn | Fcγ receptors as regulators of immune responses[END_REF]) (Figure 11). In HA patients with inhibitors, the formation of FVIII-containing ICs (FVIII-IC) may have different repercussions on the innate and adaptive cells implicated in the anti-FVIII immune response. In striking contrast to results obtained by Hausl et al., wherein the injection of high doses of FVIII to inhibitor-positive mice (theoretically leading to FVIII-IC formation) led to the elimination of FVIII-specific memory B cells [START_REF] Hausl | High-dose factor VIII inhibits factor VIII-specific memory B cells in hemophilia A with factor VIII inhibitors[END_REF], FVIII-IC were recently found to promote the differentiation of murine FVIII-specific memory B cells into antibody-secreting cells through a mechanism implicating FcγRIIB [START_REF] Vollack | Anti-FcγRIIB (CD32) Antibodies Differentially Modulate Murine FVIII-Specific Recall Response in vitro[END_REF][START_REF] Werwitzke | Deletion or inhibition of Fc gamma receptor 2B (CD32) prevents FVIII-specific activation of memory B cells in vitro[END_REF]. The latter data are, however, not in agreement with seminal works on IC-mediated feedback inhibition of Ab production [START_REF] Takai | Augmented humoral and anaphylactic responses in FcγRII-deficient mice[END_REF].

Similarly, studies on the effect of FVIII-IC at the level of innate APCs have brought conflicting results. The formation of FVIII-IC was shown to increase the endocytosis of FVIII by mouse immature bone marrow-derived DCs in an FcγR-dependent manner (Hartholt et al., 2017). The effect of the binding of IgG to FVIII on its endocytosis may, however, depend on the very properties of the anti-FVIII IgG. Indeed, co-incubation of FVIII with the human monoclonal anti-C1 IgG1, KM33, or anti-C2 IgG4, BO2C11, independently blocks FVIII uptake by human monocyte-derived DCs (MoDCs) (Wroblewska et al., 2012;Gangadharan et al., 2017). Conversely, co-incubation of FVIII with the mouse monoclonal anti-C1 IgG, F156, or anti-C2 IgG2a, ESH8, increases FVIII uptake (Gangadharan et al., 2017;Batsuli et al., 2018).

Acquired hemophilia A

Presentation and Diagnosis

Etiology and epidemiology

In previous paragraphs, we discussed congenital HA, which affects mainly males, though females might as well be affected, with a long history or family history of bleeding. Up to 30% of patients with congenital HA develop anti-FVIII alloantibodies after repeated exposures to plasma-derived or recombinant FVIII products administered as replacement therapy. In contrast to congenital HA, acquired hemophilia A (AHA) is a rare bleeding disorder that affects both males and females without previous family or personal history of bleeding. It is caused by autoantibodies against coagulation FVIII:C that develop after an immune tolerance breakdown as a result of a combination of genetic and environmental factors. Compared to autoantibodies against other coagulation factors, those directed against FVIII are the most prevalent. According to the literature, the overall incidence is 1-4 cases per million/year [START_REF] Franchini | Acquired factor VIII inhibitors[END_REF] or 1-1.5 cases per million/year [START_REF] Charlebois | Management of acquired hemophilia A: Review of current evidence[END_REF]. AHA is likely to be underdiagnosed in the elderly, but the incidence increases as awareness grows. It is important to note that the incidence of AHA increases with age: while pediatric cases have been reported with an estimated incidence of 0.045 per million/year [START_REF] Franchini | Acquired factor VIII inhibitors[END_REF][START_REF] Charlebois | Management of acquired hemophilia A: Review of current evidence[END_REF], the vast majority of patients are adults with a median age of 64-78 years. AHA has also been related to pregnancy occurring in young women aged 20-40 years (Delgado et al., 2003;[START_REF] Collins | Acquired hemophilia A in the United Kingdom: a 2-year national surveillance study by the United Kingdom Haemophilia Centre Doctors[END_REF]. [START_REF] Leebeek | New Developments in Diagnosis and Management of Acquired Hemophilia and Acquired von Willebrand Syndrome[END_REF].

Although most cases are idiopathic (44-67% of AHA cases), AHA has been associated with several underlying conditions (Table 2). These typically consist of hematological or solid malignancies [START_REF] Napolitano | Acquired haemophilia in cancer: A systematic and critical literature review[END_REF] as well as autoimmune diseases such as systemic lupus erythematosus or rheumatoid arthritis [START_REF] Knoebl | Demographic and clinical data in acquired hemophilia A: results from the European Acquired Haemophilia Registry (EACH2)[END_REF][START_REF] Tiede | Prognostic factors for remission of and survival in acquired hemophilia A (AHA): results from the GTH-AH 01/2010 study[END_REF]. Less common underlying conditions include pregnancy [START_REF] Tengborn | Pregnancy-associated acquired haemophilia A: results from the European Acquired Haemophilia (EACH2) registry[END_REF], drug exposure (e.g., antibiotics, alemtuzumab) [START_REF] Sborov | Acquired hemophilia a: a current review of autoantibody disease[END_REF][START_REF] Van Der Zwan | Acquired haemophilia A after alemtuzumab therapy[END_REF][START_REF] Kramer | Hematological immune related adverse events after treatment with immune checkpoint inhibitors[END_REF], and viral infections (e.g., HIV, influenza, and SARS-Cov-2) [START_REF] Rattanathammethee | Acquired hemophilia A in the HIVinfected patient: a case report and literature review[END_REF][START_REF] Franchini | The first case of acquired hemophilia A associated with SARS-CoV-2 infection[END_REF].

Bleeding patterns

The bleeding phenotype in AHA is variable, ranging from mild to life-threatening bleeding, and poorly correlates with FVIII:C or inhibitor titer at diagnosis [START_REF] Delgado | Acquired Haemophilia: Review and Meta-Analysis Focused on Therapy and Prognostic Factors[END_REF][START_REF] Knoebl | Demographic and clinical data in acquired hemophilia A: results from the European Acquired Haemophilia Registry (EACH2)[END_REF], unlike the congenital HA bleeding phenotype. Patients with AHA typically present with subcutaneous hematomas (80%) followed by bleeding in muscular (45%), gastrointestinal (21%), genitourinary (9%), and retroperitoneal (9%) spaces. The bleeding pattern in AHA patients is strikingly different from that in patients with congenital HA, who mainly suffer from joint bleeds and only present with mild cutaneous hematomas after trauma. In post-partum AHA, soft-tissue, muscular, and vaginal bleeding are the common presentations. Furthermore, in a case report transplacental transfer of inhibitory anti-FVIII IgG from mothers diagnosed with AHA postpartum may result in bleeding in the neonate [START_REF] Lulla | Transplacental transfer of postpartum inhibitors to factor VIII[END_REF]. Several registry studies reported up to 10% of AHA patients without bleeding symptoms, in whom the diagnosis was made because of an isolated prolonged aPTT. In the past, bleeding-related mortality was reported to be 22-31%.

However, an incidence of 3% to 9% has recently been found, with 3.2% in the European EACH2 cohort and respectively 3, 7.7, and 9% in large German (Holstein et al., 2020b), Dutch (Schep et al., 2021b), and British [START_REF] Collins | Acquired hemophilia A in the United Kingdom: a 2-year national surveillance study by the United Kingdom Haemophilia Centre Doctors[END_REF] studies. This decrease in fatal bleeds could be attributed to improved therapeutic approaches. However overall mortality rates are still high (i.e., 38.2% in the Dutch cohort), and primarily due to treatment-related infections (19.2%) (Schep et al., 2021b).

Laboratory diagnosis

The current recommendations for diagnosis and treatment of AHA are only based on registry data and clinical experience rather than controlled clinical trials because of the rarity of the condition and its unanticipated onset (Tiede et al., 2020). Therefore, in the setting of spontaneous bleeding without previous personal or family history of bleeding, initial screening should include an aPTT and PT screening tests. AHA diagnosis should be considered in the case of a prolonged isolated aPTT, not corrected by mixing study consisting of the incubation of equal volumes of patient and normal plasma, and normal PT. For the diagnosis to be confirmed, levels of intrinsic coagulation factors and inhibitors should be measured. Autoantibodies often displaying type II kinetics are typically found in AHA patients using the Nijmegen modification of the Bethesda assay and/or an anti-FVIII ELISA, along with reduced FVIII activity (FVIII:C): <1% in 50% of cases, <5% in 75% of the cases, and <40% in 100% of the cases [START_REF] Tiede | Prognostic factors for remission of and survival in acquired hemophilia A (AHA): results from the GTH-AH 01/2010 study[END_REF]. Of note, testing of antiporcine inhibitors using a modified Bethesda assay should also be considered if treatment with recombinant porcine FVIII (rpFVIII) is an option. It is crucial to consider the presence of lupus anticoagulants, which sometimes coexist with FVIII inhibitors [START_REF] Taher | Coexistence of lupus anticoagulant and acquired haemophilia in a patient with monoclonal gammopathy of unknown significance[END_REF].

Heparins, heparinoids, and direct oral anticoagulants may interfere with inhibitor test results and mimic circulating inhibitors. In summary, AHA is defined by an isolated prolonged aPTT along with the presence of a neutralizing FVIII inhibitor >0.6 BU/mL and a FVIII:C <40% (Tiede et al., 2020).

Pathophysiology

There is currently no recognized cause for the spontaneous development of neutralizing anti-FVIII IgG in AHA patients. Previous studies (Algiman et al., 1992;[START_REF] Moreau | Antibodies to the FVIII light chain that neutralize FVIII procoagulant activity are present in plasma of nonresponder patients with severe hemophilia A and in normal polyclonal human IgG[END_REF]Whelan et al., 2013;Hofbauer et al., 2015) demonstrated that around 20% of nonhemophilic individuals may generate non-neutralizing anti-FVIII IgG which are often of low affinity and IgG1 subclass. According to the first prospective cohort study (HIPS), the alloantibody response in congenital HA develops from low-affinity, mostly FVIII-specific IgG1 antibodies, to high-affinity FVIII-specific IgG4 antibodies [START_REF] Reipert | The prospective Hemophilia Inhibitor PUP Study reveals distinct antibody signatures prior to FVIII inhibitor development[END_REF].

Following the latter result, one may wonder whether non-neutralizing IgG antibodies in non-hemophilic patients are the predecessors of the neutralizing autoantibodies that develop in AHA. It is important to note that autoantibodies of AHA are identical to alloantibodies of HA with IgG4 being the major component of the anti-FVIII antibody population, though this subclass accounts only for 5% of total IgG in normal plasma. FVIIIspecific IgG1, IgG2, and less frequently IgG3 subclasses are also present. Results from the GTH-AH 01/2010 study [START_REF] Tiede | Anti-factor VIII IgA as a potential marker of poor prognosis in acquired hemophilia A: results from the GTH-AH 01/2010 study[END_REF], evaluated the most common subtypes with the highest titers and affinities to be IgG1 (88%, KA 5.8 × 10 10 M -1 ) and IgG4 (98%, KA 1.3 × 10 10 M -1 ), though IgG2 (77%), IgG3 (41%), IgA (46%), and IgM (9%) antibodies were also detected. IgG1 and IgG4 antibodies correlated with the highest inhibitor titers whereas IgA antibodies did not correlate with FVIII neutralizing activity. Interestingly, IgA antibodies correlated with the outcome of AHA as opposed to IgG antibodies. Limited research on AHA has shown that IgG antibodies are typically polyclonal and primarily bind to the A2 and C2 domains of FVIII, as in congenital HA [START_REF] Scandella | In Hemophilia A and Autoantibody Inhibitor Patients: The Factor VIII A2 Domain and Light Chain Are Most Immunogenic[END_REF]. However, autoantibodies in AHA are either directed to the A2 or C2 domain, not both [START_REF] Prescott | The Inhibitor Antibody Response Is More Complex in Hemophilia A Patients Than in Most Nonhemophiliacs With Factor VIII Autoantibodies[END_REF], with the C2 domain being more frequently targeted. Recent research identified anti-C1 domain antibodies in 78% of patients with AHA and 57% of patients with HA, respectively [START_REF] Kahle | Frequency and epitope specificity of anti-factor VIII C1 domain antibodies in acquired and congenital hemophilia A[END_REF]. However, the clinical significance of these findings remains unknown. In a study comparing the epitope specificity of anti-FVIII autoantibodies in plasma samples from postpartum AHA and other AHA patients, it has been shown that the A1a1 domain of FVIII is the primary target of IgG autoimmune response in postpartum AHA [START_REF] Lapalud | The IgG autoimmune response in postpartum acquired hemophilia A targets mainly the A1a1 domain of FVIII[END_REF]. Albeit the fact that IgG4 was predominant in all groups, IgG1 and IgG3 subclasses were more frequently detected in postpartum AHA than in the other groups. This might be due to the postpartum greater involvement of Th1-driven response, which may contribute to the successful outcome of these patients, unlike the other AHA groups, where Th2-driven IgG4 seem to predominate [START_REF] Reding | Distribution of Th1-and Th2-induced anti-factor VIII IgG subclasses in congenital and acquired hemophilia patients[END_REF].

Data from studies reviewing the role of the immune system in AHA showed an association of polymorphisms in CTLA-4, non-hemophilic F8 gene variants, and HLA-DRB1*16 and HLA-DQB1*0502 with the development of autoantibodies [START_REF] Oldenburg | Genetic markers in acquired haemophilia[END_REF][START_REF] Tiede | Acquired haemophilia caused by non-haemophilic factor VIII gene variants[END_REF]. Both HLA and CTLA-4 play an important role in the maintenance of peripheral T cell tolerance [START_REF] Mackay | Tolerance and autoimmunity[END_REF]. Interestingly, these variations in immune response genes, specifically the latter HLA types, have been demonstrated to protect against inhibitors in congenital HA [START_REF] Hay | HLA class II profile: a weak determinant of factor VIII inhibitor development in severe haemophilia A. UKHCDO Inhibitor Working Party[END_REF][START_REF] Oldenburg | HLA genotype of patients with severe haemophilia A due to intron 22 inversion with and without inhibitors of factor VIII[END_REF].

Furthermore, some studies have identified FVIII-reactive CD4+ T cells in both patients with congenital or acquired HA and healthy individuals; though cell responses in the latter group were less intense and less stable over time (Reding et al., 2000). Therefore, there is a chance that a trigger will cause a breach in tolerance, allowing these CD4+ T cells to proliferate and, in the right context, cause AHA.

Management of acquired hemophilia A

Given the rarity of the disease, research examining the best course of treatment for AHA is limited. Nevertheless, the international consensus AHA guidelines, which were initially published in 2009 and updated in 2020, provide extensive recommendations based on professional experience and registry data. Clinicians are required to refer their patients to a specialized care center for management and if an immediate referral is not possible, professional guidance should be sought.

Hemostatic therapies

Hemostatic treatment of clinically relevant bleeds in patients with AHA should be initiated irrespective of inhibitor titer and residual FVIII:C since these parameters do not correlate with the bleeding phenotype [START_REF] Collins | Acquired hemophilia A in the United Kingdom: a 2-year national surveillance study by the United Kingdom Haemophilia Centre Doctors[END_REF][START_REF] Knoebl | Demographic and clinical data in acquired hemophilia A: results from the European Acquired Haemophilia Registry (EACH2)[END_REF]. According to data from the UK and EACH2 registries [START_REF] Collins | Acquired hemophilia A in the United Kingdom: a 2-year national surveillance study by the United Kingdom Haemophilia Centre Doctors[END_REF]Baudo et al., 2012), around 70% of patients with AHA require hemostatic treatment, while the rest were untreated because they have no bleeds or only minor subcutaneous bleeds. However, close monitoring is required since fatal bleeding can occur up to 5 months after the initial bleed.

The use of BPAs or rpFVIII is the appropriate first-line treatment for AHA patients who present with clinically relevant bleeding (Figure 12). There was no clear efficacy or safety advantage of one drug over the others. However, in the EACH2 registry, bleeding control was significantly higher in patients treated with BPAs (93.3%) than those treated with FVIII/DDAVP (68.3%). Thus, pd-FVIII or human rFVIII should be considered only if BPAs or rpFVIII are unavailable or ineffective and the inhibiter titer is low (<5BU). DDAVP should not be used in elderly patients who are at risk of fluid overload, heart failure, or hyponatremia.

Both BPAs, rFVIIa and aPCC, have been successfully used for decades, and treatment efficacy was >90% at both patients and bleed levels (Baudo et al., 2012). However, these products require frequent dosing. rFVIIa is administered intravenously in a dose of 90 µg/kg every 2-3 hours until hemostasis is achieved. Whereas, aPCC is given at 50-100 U/kg every 8-12 hours, with a maximum of 200 U/kg/day. The frequency and intensity of dosing are solely based on clinical improvement in bleeding symptoms since neither rFVIIa nor aPCC can be monitored using standard laboratory assays (i.e., aPTT).

Moreover, according to the EACH2 study, these drugs may present a thrombotic risk, with 2.9% and 4.8% thrombotic events reported in patients treated with rFVIIa and aPCC, respectively (Baudo et al., 2012). This risk can be increased if both BPAs are used in combination or sequential manner and thus must be avoided [START_REF] Ingerslev | Parallel use of by-passing agents in haemophilia with inhibitors: a critical review[END_REF].

If the anti-porcine titer is low (<20 BU) or undetectable, rpFVIII is appealing as its activity can be monitored with a one-stage FVIII assay. A B-domain-deleted rpFVIII was approved by the FDA for the treatment of AHA in 2014 (Obizur®, Baxter). For initial treatment, the recommended and approved dose is 200 U/kg to achieve 100-200% FVIII activity followed by additional doses every 4-12 hours to maintain FVIII through 50% after acute bleeding is controlled [START_REF] Kruse-Jarres | Efficacy and safety of OBI-1, an antihaemophilic factor VIII (recombinant), porcine sequence, in subjects with acquired haemophilia A[END_REF]. Retrospective studies reported the use of a lowerthan-approved dose, 100 U/kg instead of 200 U/kg, with FVIII monitoring every 2-3 hours and re-dosing based on FVIII level [START_REF] Martin | Lower doses of recombinant porcine factor VIII maintain excellent haemostatic efficacy[END_REF]. It is important to note that rpFVIII inhibitors can develop de novo and decrease the product's efficiency. Therefore, titration of anti-rpFVIII inhibitors should be carried out throughout the treatment. Kruse-Jarres et al. found that 5 out of 28 patients treated with rpFVIII developed de novo anti-rpFVIII inhibitors within 8-85 days after the first infusion [START_REF] Kruse-Jarres | Efficacy and safety of OBI-1, an antihaemophilic factor VIII (recombinant), porcine sequence, in subjects with acquired haemophilia A[END_REF]. Another appealing drug for use in patients with AHA is emicizumab (described in the previous section 2.2.2.3), which is FDA-approved for prophylaxis in patients with moderate to severe congenital HA. However, patients with AHA are generally older and have more comorbidities than those with congenital HA, raising concerns about the safety and efficacy of emicizumab in these patients. Early reports of thrombotic complications such as pulmonary emboli and thrombotic stroke, as well as mortality, were associated with the off-label and compassionate use of emicizumab in AHA patients (Makris et al., 2019). More recently, Knoebl et al. published a case series of 12 AHA patients in whom emicizumab proved to have hemostatic efficacy and was well tolerated [START_REF] Knoebl | Emicizumab for the treatment of acquired hemophilia A[END_REF]. Patients received a 3 mg/kg subcutaneous dose once a week for 2-3 weeks, followed by a 1.5 mg/kg dose every 3 weeks. Emicizumab treatment resulted in immediate aPTT normalization and rapid cessation of bleeding in a median of 3 days. It is important to note that aPCC treatment was held or switched to rFVIIa for 2 days before initiating emicizumab treatment due to the association of thrombotic microangiopathy in patients with congenital HA treated concurrently with emicizumab and aPCC (Oldenburg et al., 2017). Even though emicizumab has been shown to reduce the duration of initial and recurrent bleeding, a prospective trial in AHA patients treated with emicizumab is lacking.

Two ongoing clinical trials (NCT05345197 and NCT04188639) are assessing the safety and efficacy of emicizumab use in AHA.

Inhibitor eradication

In AHA patients, it is critical not only to achieve and maintain hemostasis but also to restore FVIII immune tolerance by removing the inhibitor. Immunosuppressive therapy (IST) is recommended in the majority of AHA patients, except frail patients, according to the 2020 update of the AHA guidelines (Tiede et al., 2020). Indeed, since most patients with AHA are elderly, the potential side effects of the chosen therapy should be carefully considered. The recommended first-line therapy is corticosteroids alone, such as prednisone, for 3-4 weeks for patients with FVIII levels ≥1% and inhibitor titer ≤ 20 BU at baseline; if the patient does not respond a second agent might be added. Whereas for patients with FVIII levels <1% or inhibitor titer >20 BU the combination of corticosteroids with a second agent such as rituximab or cyclophosphamide is recommended. Indeed, recent studies (Schep et al., 2021b) found that combination therapy had a higher complete remission rate than corticosteroids alone (67.7-83.3% vs. 35.2%, respectively). However, when combination therapy is used, infection rates increase and are responsible for a significant proportion of mortality (around 20%). The choice of the second agent depends mainly on the patient's comorbidities. It was demonstrated that after a median of 5-6 weeks, 60-80% of patients treated with IST experience remission [START_REF] Kruse-Jarres | Acquired hemophilia A: Updated review of evidence and treatment guidance[END_REF]. However, relapses may happen during corticosteroid withdrawal as well as after total cessation of IST in 15% and 25% of cases, respectively (Schep et al., 2021b). In a recent analysis of pregnancy-associated AHA, the incidence of relapse in subsequent pregnancies was found to be 22% [START_REF] Dewarrat | Acquired haemophilia A in the postpartum and risk of relapse in subsequent pregnancies: A systematic literature review[END_REF]. The benefits and risks of treatment must be evaluated for each patient, particularly given the fact that a percentage of patients will have spontaneous remission without IST.

Patients who do not respond to first-line therapy after 3-5 weeks should receive secondline therapy with either cyclophosphamide or rituximab, depending on which drug was not used in first-line therapy. For patients refractory to all these treatments, other drugs can be used but there is currently limited data on them [START_REF] Mcfadyen | Factor VIII inhibitor eradication with bortezomib in acquired haemophilia A[END_REF][START_REF] Obaji | Mycophenolate mofetil as adjunctive therapy in acquired haemophilia A[END_REF]. High-dose IVIg is not recommended for inhibitor eradication in patients with AHA; though efficient in other autoimmune diseases [START_REF] Crenier | Low response to high-dose intravenous immunoglobulin in the treatment of acquired factor VIII inhibitor[END_REF]. High-dose FVIII has been combined with IST for immune tolerance induction in AHA in several trials, but efficacy in addition to that of standard IST is unclear [START_REF] Zeitler | Treatment of acquired hemophilia by the Bonn-Malmo Protocol: documentation of an in vivo immunomodulating concept[END_REF]. ITI is therefore not used unless first-line hemostatic therapies have failed in patients with life-threatening bleeding. Finally, immunoadsorption protocols can be used in patients who fail IST to successfully remove the inhibitor (Esteves [START_REF] Pereira | Immunoadsorption for the Treatment of Acquired Hemophilia: New Observational Data, Systematic Review, and Meta-Analysis[END_REF].

Due to the stated risks of recurrence and adverse events, AHA patients should be closely monitored until they achieve complete remission. Afterward, a follow-up for several months is recommended by monitoring FVIII:C, inhibitor titers, and aPTT. More prospective research is needed to determine optimal management and identify biomarkers of response in AHA.

Gram-positive bacteria Streptococcus pyogenes

The Gram-positive bacteria Streptococcus pyogenes, also known as group A Streptococcus (GAS), is a preeminent human pathogen responsible for a broad spectrum of superficial and invasive disease conditions [START_REF] Cunningham | Pathogenesis of Group A Streptococcal Infections[END_REF]. Although asymptomatic carriage of S. pyogenes is common, it causes over 700 million cases of pharyngitis or skin infection each year worldwide, as well as over 650,000 invasive infections [START_REF] Avire | A Review of Streptococcus pyogenes: Public Health Risk Factors, Prevention and Control[END_REF]. S. pyogenes can cause infection in nearly every human organ or tissue, and severe life-threatening conditions such as necrotizing fasciitis, endocarditis, sepsis, and toxic shock syndrome. S. pyogenes infections are commonly associated with an intense inflammatory state reflecting an increased vascular permeability and recruitment of neutrophils to the site of infection. The propensity of GAS to cause systemic infections in otherwise healthy children and adults defines the pathogen's ability to resist host innate immune clearance mechanisms. Typically, upon GAS detection, neutrophils, macrophages, and dendritic cells are recruited and activated by pattern recognition receptors (PRRs) and GAS-derived pathogen-associated molecular patterns (PAMPs), which usually prevent microbial dissemination beyond epithelial surfaces [START_REF] Fieber | Responses of innate immune cells to group A Streptococcus[END_REF].

However, to divert these immune responses, S. pyogenes expresses a whole array of immunomodulating proteins that contribute to its pathogenesis, both surface-associated and secreted. Briefly, two distinct stages of S. pyogenes pathogenesis can be distinguished: (1) the initial attachment to host tissues, which facilitates colonization and requires numerous surface proteins, such as the M protein family and pili, which interact with a wide variety of host extracellular proteins and cell receptors; and ( 2 Resistance to cationic antimicrobial peptides is afforded by D-alanyl modification of cell wall teichoic acids, cysteine protease SpeB-mediated degradation, and binding/inactivation by protein streptococcal inhibitor of complement (SIC). M protein and protein H bind Fc domains of Igs in a non-opsonic manner, and proteolytic inactivation of Ig is a property of SpeB, Mac-1/2, and endopeptidase S. DNase production facilitates escape from NETs, and the pore-forming cytolysins streptolysin S (SLS) and streptolysin O (SLO) exhibit lytic activity against host neutrophils and macrophages. From [START_REF] Nizet | Understanding how leading bacterial pathogens subvert innate immunity to reveal novel therapeutic targets[END_REF].

The cell wall of GAS hosts a wide range of molecules and serves various functions, the majority of which are critical to the cell's viability (Figure 13). Although the cell wall's primary function is to provide a rigid exoskeleton for protection against mechanical and osmotic lysis, it can serve as well in GAS as an attachment site for proteins that interact with the bacterial environment. Over the last decade, it has become clear that GAS has evolved a variety of unique mechanisms for immobilizing abundant extracellular proteins on their surface, such as albumin, immunoglobulins (IgG, IgA), fibrinogen, complement inhibitors and α2-macroglobulin [START_REF] Navarre | Surface Proteins of Gram-Positive Bacteria and Mechanisms of Their Targeting to the Cell Wall Envelope[END_REF]. Many of these proteinprotein interactions are mediated by members of the M protein family, which are the major surface proteins and virulence factors of GAS, with over 200 distinct serotypes identified [START_REF] Ellen | M protein-associated adherence of Streptococcus pyogenes to epithelial surfaces: prerequisite for virulence[END_REF][START_REF] Fischetti | Streptococcal M protein: molecular design and biological behavior[END_REF][START_REF] Cunningham | Pathogenesis of Group A Streptococcal Infections[END_REF][START_REF] Sanderson-Smith | A systematic and functional classification of Streptococcus pyogenes that serves as a new tool for molecular typing and vaccine development[END_REF].

Antibodies directed toward microorganisms are key players in the adaptive immune response. They recruit complement factors and direct leukocytes to the site of infection, ultimately leading to phagocytosis and killing of the microorganism. In addition to the cellwall anchored Ig-binding proteins described above, S. pyogenes produces enzymes that cleave or inactivate Igs. These enzymes have been suggested to contribute to GAS pathogenesis and help evade recognition and killing mediated by antibodies.

Although S. pyogenes frequently colonizes mucosal surfaces, the bacteria lacks IgA proteases that cleave the hinge region of IgA antibodies and are typically secreted by several mucosal colonizing/infecting bacteria [START_REF] Plaut | The IgA1 proteases of pathogenic bacteria[END_REF]. Instead, it employs an IgAbinding M protein to interfere with the binding of IgA to its receptors and a streptococcal cysteine proteinase, the Streptococcal pyrogenic exotoxin B (SpeB), capable of degrading the COOH-terminal part of IgA [START_REF] Collin | Effect of SpeB and EndoS from Streptococcus pyogenes on human immunoglobulins[END_REF]. SpeB, the classic cysteine protease, was shown to cleave the heavy chains of all isotypes of human Igs: IgG, IgM, IgA, IgD and IgE. However, SpeB is not expressed until S. pyogenes reaches the stationary growth phase, at least under laboratory conditions [START_REF] Chaussee | Temporal production of streptococcal erythrogenic toxin B (streptococcal cysteine proteinase) in response to nutrient depletion[END_REF], raising doubts about its potential role as an enzyme that cleaves opsonizing IgG antibodies, for instance. Additionally, proteolytic cleavage of IgG by SpeB is extremely slow. Since SpeB has other redundant substrates, it does not seem appropriate as firstline defense against specific IgG under certain environmental conditions.

As mentioned previously, when infection and inflammation occur, the pathogen must cope with specific IgG and neutrophils recruited to the infection site. Thus, S. pyogenes secretes several IgG-degrading/targeting enzymes, two of which -EndoS and IdeSshow remarkable specificity for IgG [START_REF] Collin | Extracellular Enzymes with Immunomodulating Activities: Variations on a Theme in Streptococcus pyogenes[END_REF]. EndoS (Endoglycosidase in S. pyogenes) efficiently and specifically hydrolyzes the functionally important N-linked glycan of IgG, thus hampering the effector functions of IgG, including altered binding to Fc-receptors on leukocytes and activation of the complement system. IdeS (Immunoglobulin G-degrading enzyme of S. pyogenes) will be extensively described in the following paragraphs. Of the two immune evasion factors, IdeS is the most advanced in clinical development. S. pyogenes expresses two variants of this enzyme often distinguished by naming the first variant, IdeS/Mac-1, and the second variant, Mac-2.

These variants display less than 50% sequence identity within the middle third of the protein [START_REF] Lei | Opsonophagocytosis-Inhibiting Mac Protein of Group A Streptococcus: Identification and Characteristics of Two Genetic Complexes[END_REF]Agniswamy et al., 2004). Except for Mac-2 proteins of streptococcal M28 serotype, both IdeS and Mac-2 protein variants are bifunctional proteins that combine both Fc receptor-binding activity and IgG endopeptidase activity to achieve efficient protection against IgG-mediated phagocytosis (von Pawel-Rammingen, 2012).

Mechanism of action of IdeS

IdeS, discovered in 2002, is unique among prokaryotic cysteine proteinases, as the 35 kDa protein is secreted in its mature form and does not require processing or activation to exert its enzymatic activity, in contrast to SpeB (von Pawel-Rammingen et al., 2002).

IdeS hydrolyzes all four human IgG subclasses with a unique degree of specificity below the disulfide bridge between the two glycine residues in positions 236 and 237 in the lower hinge region. It, however, does not cleave other Ig isotypes (i.e., IgM, IgE, IgD, IgA), and no other substrate has yet been identified. Its catalytic efficiency follows the order of IgG1=IgG4>IgG3>IgG2. IgG2 lacks glycine at position 236 (Figure 14). In addition to human IgG, IdeS cleaves monkey and rabbit but not mouse, sheep, or pig IgG (Agniswamy et al., 2004). Only poor cleavage was observed for mouse IgG2a and IgG3.

Sequence comparison revealed that the human, monkey, and rabbit IgG had a conserved lower hinge sequence of PELLGG at positions 232-237 near the cleavage site. This suggests that the latter sequence serves as a binding site for IdeS. Interestingly, residues in the lower hinge region of IgG have been implicated as being important for Fc receptor recognition and complement binding [START_REF] Duncan | The binding site for C1q on IgG[END_REF][START_REF] Radaev | Recognition of immunoglobulins by Fcγ receptors[END_REF]. Step two generates a fully cleaved product that cannot mediate complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC) or antibody-dependent cell-mediated phagocytosis (ADCP) by means of Fcγ receptor. Adapted from (Jordan et al., 2017). B. Representation of the amino acid sequences of the hinge region of the four IgG subclasses with the cleavage sites for papain and IdeS. Adapted from [START_REF] Falkenburg | Anti-Hinge Antibodies Recognize IgG Subclass-and Protease-Restricted Neoepitopes[END_REF].

However, proteins containing putative IdeS cleavage sites in their primary sequences and natural and synthetic peptides containing amino acid segments similar to that of the IdeS cleavage site in IgG could not be hydrolyzed by IdeS (Vincents et al., 2004). These results stress the extremely high degree of substrate specificity of IdeS and raise the question about the presence of a second substrate binding site or an exosite on IdeS for enzymatic activity. To answer the latter question, IdeS was incubated with only the Fc part of IgG generated by papain cleavage (Fcpapain). Papain, a cysteine protease, cleaves antibodies above the hinge region containing the disulfide bonds (Figure 14) resulting in the generation of two separate monovalent Fab fragments and one intact Fc fragment [START_REF] Van Eyk | Fragmentation of human IgG globulin with papain, trypsin and pepsin[END_REF]. IdeS was capable of hydrolyzing Fcpapain under non-reducing as well as reducing conditions. However, IdeS could not cleave the hinge region peptide on its own without the presence of the remaining Fc molecule (Vincents et al., 2004). Thus, Fcpapain is considered the smallest known substrate for IdeS, and this strongly indicates that IdeS binds to the heavy chain domain CH2 or CH3 or their interface.

The crystal structure of IdeS was first solved at a 1.9 Å resolution and revealed that IdeS adopts a canonical papain-like cysteine protease fold [START_REF] Wenig | Structure of the streptococcal endopeptidase IdeS, a cysteine proteinase with strict specificity for IgG[END_REF]. However, IdeS displays different features, such as significant insertions in its structure and its distinct substrate-binding site. The most notable site is the unusual RGD motif (Arg-Gly-Asp), which is involved in the interaction of IdeS with vitronectin (αvβ3) and platelet receptors (αIIbβ3), suggesting additional, yet unknown properties of the enzyme [START_REF] Lei | Opsonophagocytosis-Inhibiting Mac Protein of Group A Streptococcus: Identification and Characteristics of Two Genetic Complexes[END_REF].

The active site encompasses a classic catalytic triad in which the catalytic Cys94 is hydrogen-bonded to His262 and completed by a hydrogen bond between Asp284 and His262. This conformation, together with Lys84 and Asp286, forms a negatively charged region that has been shown to be important for the proper positioning of IgG in the active site [START_REF] Wenig | Structure of the streptococcal endopeptidase IdeS, a cysteine proteinase with strict specificity for IgG[END_REF]. A catalytically inactive IdeS is obtained by mutating the cysteine with a serine (IdeS-C94S) or an alanine (IdeS-C94A).

Recently, a new crystal structure of IdeS in complex with its IgG1 Fc substrate was presented with a resolution of 2.34 Å (Sudol et al., 2022). The latter shows asymmetric binding of IdeS across the CH2 domains of both Fc chains and their lower hinge regions.

IdeS appears to clamp down over the lower hinge region of the Fc chain being cleaved, creating a cavity in which the catalytic residues are brought into close proximity to the cleavage site (Figure 15). However, the upper hinge region of IgG and its Fab regions did not significantly contribute to complex formation. Nevertheless, the possible interaction of IdeS with the IgG Fab regions cannot be excluded. The 1:1 stoichiometry observed in the latter crystal structure is consistent with previous kinetic analyses showing that IdeS functions predominantly in a monomeric form and that its enzyme activity follows classical Michaelis-Menten kinetics (Vindebro et al., 2013). Furthermore, very few interactions between IdeS and the Fc N-linked glycans could be predicted, suggesting that IdeS can accommodate IgG with heterogeneous glycosylation.

A study characterizing IdeS enzymatic activity revealed activity in the pH range of 5. 1-7.6 with an optimum pH of 6.6. IdeS was stable at high basic pH (i.e., pH 10) but not at low acidic pH. Maximum activity was achieved around 37°C, and decreased thermal stability was observed at 42°C (Vincents et al., 2004). Iodoacetate and iodoacetamide inhibit enzymatic activity, while typical cysteine protease inhibitors (i.e., E-64 and cystatins) fail to do so. IdeS must bind to the Fc part of IgG to initiate cleavage, and the remarkable specificity lies in this initial protein-protein interaction. The cleavage is a two-step process involving both antigen-bound and freely circulating human IgG. In the first step, one of the two heavy chains of the intact IgG molecule is rapidly cleaved [START_REF] Ryan | Proteolysis of purified IgGs by human and bacterial enzymes in vitro and the detection of specific proteolytic fragments of endogenous IgG in rheumatoid synovial fluid[END_REF]. This results in the intermediate product, single-cleaved IgG (scIgG) that has a significantly reduced capacity to mediate Fc-dependent effector functions. In a physiological environment, scIgG is indistinguishable from intact IgG and has the same half-life in vivo (i.e., ~3 weeks in humans and 6-8 days in mice) (Brezski et al., 2009). Due to a conformational change in scIgG, the second step of the reaction, in which the second heavy chain is cleaved and a complete separation into an F(ab')2 fragment and a noncovalently linked homodimeric Fc fragment is achieved, requires a longer time to complete compared to the first step (catalytic constant (kcat) of 0.1 s -1 vs. 10 s -1 , respectively) (Vindebro et al., 2013). The F(ab')2 fragments retain the full binding capacity to epitopes but are unable to participate in Fc-mediated activities (i.e., complement fixation, immune cells' recruitment, recycling). Thus, Fc-dependent effector functions, such as antibodydependent cell-mediated phagocytosis (ADCP), ADCC, or complement-dependent cytotoxicity (CDC), are efficiently abrogated (Brezski et al., 2009;von Pawel-Rammingen et al., 2002). Interestingly, IdeS hydrolyzes not only soluble IgG but also IgG-type BCR, thereby preventing antigen binding to the BCR. This may further enhance the immunosuppressive effect of IdeS by reducing the activation of memory B cells [START_REF] Järnum | The Bacterial Enzyme IdeS Cleaves the IgG-Type of B Cell Receptor (BCR), Abolishes BCR-Mediated Cell Signaling, and Inhibits Memory B Cell Activation[END_REF]. IdeS C94A is colored blue; IgG1 Fc E382A is colored in grey. N-linked glycans and the IgG1 Fc E382A hinge peptide in the focused panel are shown as sticks and colored, with oxygen, nitrogen and sulfur atoms in red, blue, and yellow, respectively. b. Superposition of complexed IdeS C94A with three published apo structures of IdeS (PDB IDs 1Y08, 2AU1 and 2AVW, colored in red, orange, and yellow, respectively) and focused view of the prosegment binding loop. c. Binding of IgG1 Fc E382A hinge peptide within the IdeS C94A active site. Fc E382A peptide and IdeS C94A active site residues are depicted as sticks and colored by heteroatom; catalytic triad residues are colored purple. The scissile peptide bond is colored in cyan; hydrogen bonds are depicted as green dashes. From (Sudol et al., 2022).

Preclinical settings

Pathogenic IgG can be involved in various alloimmune and autoimmune diseases. It is estimated that autoimmune diseases (rheumatoid arthritis, systemic lupus, myasthenia gravis, immune thrombocytopenic purpura, Goodpasture's syndrome, etc.) affect about 5% of the population. Furthermore, although performing according to its intended function, the action of IgG is not desirable in different conditions such as acute transplant rejection following organ transplantation, inactivation of life-saving drugs (anti-drug antibodies), limiting successful viral vector-based gene therapies, as well as engineered cell therapies.

Thus, the efficient removal of pathogenic IgG is an important clinical matter. Because of its unique substrate specificity and fast and efficient enzymatic reaction, IdeS has the potential to function as a therapeutic drug for clinical disorders involving pathogenic IgG.

To provide proof of concept for the use of IdeS as a therapeutic agent for the treatment of IgG-mediated disorders, animal models have been used (i.e., mouse, rabbit, and monkey). IdeS showed promise in animal models of autoimmune arthritis, immune thrombocytopenic purpura (ITP), antiglomerular basement membrane (anti-GBM; Goodpasture's disease), and neuromyelitis optica (NMO). More recently, it also showed promise in animal models of heparin-induced thrombocytopenia (HIT), an immune mediated adverse drug reaction, as well as in gene therapy and engineered-cell therapies.

• Collagen-induced and collagen antibody-induced arthritis IdeS hydrolyzed IgG in vivo and protected mice against collagen antibody-induced arthritis (CAIA). It also delayed the onset and reduced the severity of collagen-induced arthritis (CIA) and significantly inhibited antibody-induced relapses in a model of chronic arthritis (Nandakumar et al., 2007).

• Immune thrombocytopenic purpura A single injection of IdeS was able to rescue mice from a lethal dose of polyclonal rabbit anti-mouse platelet surface antigens in a model of immune thrombocytopenic purpura (ITP) (Johansson et al., 2008). F(ab')2 fragments were cleared from the circulation within 2 days and IgG started to reappear after 4-5 days and were back to normal levels on day 10. IdeS was not detectable after day 3. When IdeS injection was repeated on day 40, a similar pattern was observed.

• Goodpasture's disease

In a mouse model of Goodpasture's disease, known as anti-glomerular basement membrane (GBM) disease, Yang et al. found that IdeS hydrolyzed both circulating and kidney-bound anti-GBM IgG, prevented the development of proteinuria and inhibited complement deposition in the kidney (Yang et al., 2010).

• Neuromyelitis optica

IdeS efficiently cleaved autoantibodies directed towards astrocyte water channel aquaporin-4 (AQP4) in vivo in a mouse model of neuromyelitis optica, a severe demyelinating disease, and greatly reduced the lesions [START_REF] Tradtrantip | Therapeutic Cleavage of Anti-Aquaporin-4 Autoantibody in Neuromyelitis Optica by an IgG-Selective Proteinase[END_REF].

• Gene therapy

In mice passively immunized with neutralizing amounts of IVIg, IdeS administration decreased anti-AAV antibodies contained in the IVIg pool and enabled efficient liver gene transfer to correct HA and HB (Leborgne et al., 2020). The approach was scaled up to non-human primates, a natural host for wild-type AAV. IdeS treatment prior to AAV vector infusion was safe and resulted in enhanced liver transduction, even in the setting of vector re-administration.

• Engineered cell therapy Chimeric antigen receptor (CAR) T cells were programmed to either express transmembrane IdeS or secrete IdeS. These cells retained their original immune functions, cleaved IgG in vitro and in vivo, covered themselves in a protective coating of non-functional F(ab')2, thereby preventing new attacks by other IgG, and had improved persistence and efficacy over traditional CAR T cell therapies in the presence of antibodies directed to their cell surfaces [START_REF] Peraro | Incorporation of bacterial immunoevasins to protect cell therapies from host antibody-mediated immune rejection[END_REF].

Clinical settings

IdeS has been used in a series of studies that showed rapid and efficient cleavage of the entire IgG pool in human blood in vitro, complete but temporary IgG removal in rabbits, and recovery from various IgG-driven autoimmune diseases in mice. All these results favor using IdeS as a new therapeutic drug to eliminate pathogenic IgG in patients and led to the initiation of several clinical trials. Imlifidase (Idefirix™), a recombinant cysteine protease produced in Escherichia coli and derived from the streptococcal enzyme IdeS, is being developed by Hansa Biopharma AB for the treatment of transplant rejection and rare IgG-mediated autoimmune conditions. The EMA approved intravenous imlifidase in 2020 for desensitization treatment of highly sensitized adult kidney transplant patients with positive crossmatch against an available deceased donor (Al-Salama, 2020). In the USA, Australia, France, and Austria, imlifidase is now undergoing clinical evaluation to prevent kidney transplant rejection. In addition, clinical development for anti-GBM disease and Guillain-Barré syndrome is also ongoing in France, the UK, and the Netherlands. Some insights into these clinical trials are discussed below.

• Healthy volunteers

In 2015, Winstedt et al. published the results of a first-in-man, double-blinded and randomized phase 1 study wherein 20 healthy male volunteers received single ascending intravenous doses of imlifidase (0.01, 0.04, 0.12, or 0.24 mg/kg body weight [BW]) (Winstedt et al., 2015). Because S. pyogenes is a ubiquitous infection, nearly all adults have antibodies to IdeS. As such, participants with anti-IdeS IgG titers in the top 20% of a reference population (i.e., >15 mg/L) were excluded from this study to minimize the risk of infusion reactions or reduced efficacy from neutralizing anti-IdeS antibodies. This limitation might have impacted the generalizability of the study results, but it is noteworthy that subsequent studies have not applied this exclusion criterion with similar overall results. Results demonstrated that the plasma IgG pool was completely cleaved within minutes of dosing for the highest dose groups (0.12 and 0.24 mg/kg), reaching a nadir at 2-6 hours before slowly recovering afterward by 1-2 weeks. However, a large interindividual variation in the rate of IgG recovery was observed. The elimination of F(ab')2 fragments was somewhat faster than that of Fc fragments.

Moreover, imlifidase treatment rapidly and effectively cleaved the pre-existing antigenspecific IgG (i.e., against diphtheria, tetanus, pertussis, polio, and Haemophilus influenzae type b) in all participants, and the reappearance of antigen-specific IgG followed the reappearance of total IgG. In individuals that received a 0.24 mg/kg dose, the half-life of imlifidase was 4.9 (±2.8) hours, with the main fraction eliminated within 24 hours.

However, the present study did not quantitate the distribution and elimination phase halflives. The description sheet included in the product package states an average distribution-phase half-life of 1.8 hours and an elimination-phase half-life of 89 hours.

Importantly, 2 hours post-dosing, the phagocytic capacity of IgG fragments was significantly reduced, reaching background levels for at least 7 days before returning to original levels. Imlifidase was generally well-tolerated, and no serious adverse events were reported. All individuals produced antibodies to IdeS within 1-2 weeks of administration, with anti-IdeS titers increasing approximately 20-fold and peaking at 2 weeks before normalizing by 6 months. The significance of these anti-IdeS antibodies on repeat dosing is yet unknown.

The integrity of membrane-bound IgG-type BCR on circulating CD19+ B cells, pre-dose and at different time points after imlifidase administration, has also been studied as an exploratory part of the trial (NCT01802697). The IgG-BCR was as sensitive and efficiently cleaved by imlifidase as circulating IgG. The data show a significant loss of the F(ab')2 staining on the surface of CD19+ cells 2 hours post-dosing and gradual recovery over days following treatment (time to recovery was patient-dependent). Experiments on B cell lines and PBMCs treated with IdeS showed that cleavage by IdeS completely prevents IgG-BCR signaling that usually follows receptor ligation, likely impacting antigen presentation to CD4+ T cells [START_REF] Järnum | The Bacterial Enzyme IdeS Cleaves the IgG-Type of B Cell Receptor (BCR), Abolishes BCR-Mediated Cell Signaling, and Inhibits Memory B Cell Activation[END_REF]. Moreover, memory B cells were temporarily silenced in vitro after IdeS treatment, preventing them from responding to antigenic stimulation and their transition into IgG-secreting plasma cells. The latter result suggests a possible long-term implication of IdeS. However, IdeS had no blocking effect on cells' differentiation if it was used after the initiation of activation. Importantly, in the latter study, IdeS had no impact on cell viability.

• HLA-sensitized kidney transplant patients

The preferred course of treatment for patients with end-stage renal disease is kidney transplantation. However, approximately 30% of the patients on transplantation waiting lists show evidence of sensitization in the form of anti-HLA alloantibodies [START_REF] Jordan | Evaluation of intravenous immunoglobulin as an agent to lower allosensitization and improve transplantation in highly sensitized adult patients with end-stage renal disease: report of the NIH IG02 trial[END_REF]. These alloantibodies were brought on by exposure to blood transfusions, previous transplants, pregnancy, or other events. Thus, in the latter patients, transplantation is challenged by the risk of antibody-mediated rejection (AMR) and poor graft survival.

Desensitization protocols exist which combine plasmapheresis with IVIg and B lymphocyte-depleting agents (e.g., rituximab and anti-CD20) [START_REF] Vo | Rituximab and intravenous immune globulin for desensitization during renal transplantation[END_REF][START_REF] Ishida | Desensitization Regimen Consisting of High-Dose Intravenous Immunoglobulin, Plasmapheresis, and Rituximab (an Anti-CD20 Antibody), Without Eculizumab and/or Bortezomib, in 41 Highly Sensitized Kidney Transplant Recipients[END_REF]. However, these typically require several weeks of preparation, limiting their use to living donor transplants. They also frequently lead to incomplete removal of donor-specific antibodies, rebound antibody production, and an elevated risk of acute and chronic AMR.

These are the leading causes of early graft loss and return to dialysis.

In 2017, Jordan et al. reported using imlifidase therapy to desensitize 25 highly HLA-sensitized kidney transplant candidates (Jordan et al., 2017). The study combined data from two separate phase 1/2 studies. Imlifidase was given intravenously 4-6 hours before transplantation at a dose of 0.24 mg/kg BW (in the U.S. study; n=14) or at a dose of 0.25 mg/kg or 0.50 mg/kg (in the Swedish study; n=11). All patients received standard immunosuppression and prophylactic antibiotic agents to prevent bacterial infection in the absence of IgG antibodies. The data show a significant reduction of total IgG and anti-HLA IgG levels after treatment with IdeS, which allowed for successful transplantation in 24 out of 25 patients. The one allograft loss was due to hyperacute rejection in a patient with non-HLA IgM and IgA antibodies that cannot be cleaved by IdeS. Ten patients experienced AMR between 2 weeks and 5 months following transplantation. In total, 38 serious adverse events occurred in 15 patients, with 5 possibly related to IdeS.

More recently, Jordan et al. reported on a small series of 18 highly sensitized kidney transplant candidates in an open-label, single-arm, phase 2 trial conducted at five transplant centers (Jordan et al., 2021). Donor-specific antibodies (DSAs) were present in all patients at baseline, and the ability of imlifidase to create a negative crossmatch test within 24 h was assessed. All patients received one dose of imlifidase (0.25 mg/kg BW) on day 0. Conversion of baseline positive crossmatch to negative within 24 h after imlifidase treatment was achieved in 89.5% of the transplanted patients. DSAs most often rebounded 3-14 days post-treatment, with substantial interpatient variability. Patient survival was 100%, with a graft survival of 88.9% at 6 months. With this, 38.9% had early biopsy-proven AMR with an onset at 2-19 days post-transplantation that was treated using standard therapies. Seven adverse events in 6 patients were classified as possibly or probably related to treatment and were mild-moderate in severity. At 3 years, death-censored allograft survival was 93% for patients who developed AMR compared to 77% for those who did not develop AMR. Patient survival was 94% vs. 85% respectively. The onset of AMR remains a concern throughout the post-transplant course in highly sensitized recipients. Nonetheless, the pre-transplant DSA diminished after imlifidase administration and reoccurred as IgG titers repopulated. DSA did not increase in strength during rebound relative to pre-transplant levels nor increase over time. Though further studies may define the optimal use of imlifidase, it is a potent option to enable transplantation among highly sensitized patients for whom lifetime dialysis and its accompanying morbidity may be the only alternative (Kjellman et al., 2021).

Finally, a new trial started recently (NCT05049850) to investigate DSA rebound in highly HLA-sensitized kidney transplant patients treated with imlifidase prior to transplantation. This study will assess whether imlifidase, in combination with B-cell and/or plasma-cellinterfering drugs (i.e., bortezomib, belatacept, rituximab, and IVIg), can suppress DSA rebound within 3 months post-transplantation and the occurrence of AMR in highly HLA-sensitized kidney transplant candidates with a pre-transplant positive crossmatch against their living donor.

• Kidney transplant recipients presenting with AMR A randomized controlled trial (NCT03897205) enrolled 30 patients to compare the reduction in DSA levels between imlifidase (0.25 mg/kg) versus 5-10 sessions of plasmapheresis given to kidney transplant recipients presenting with active or chronic active AMR. The recruitment has just been completed as of March 2022.

Another trial is currently enrolling (NCT04711850) and aims to provide a better understanding of the long-term outcome of imlifidase treatment on active or chronic active AMR in kidney transplant recipients. Data are gathered during 3 years of an extended follow-up in a clinical trial where patients were treated for AMR with either imlifidase or plasmapheresis.

• Goodpasture's disease Goodpasture's disease is related to the presence of anti-glomerular basement membrane (GBM) autoantibodies directed against the non-collagenous domain of type IV collagen that bind to the basement membrane in the kidney glomeruli and lungs [START_REF] Hellmark | Diagnosis and classification of Goodpasture's disease (anti-GBM)[END_REF]. This causes renal and, in some cases, pulmonary failure. Despite plasma exchange, most patients, particularly those with poor prognostic factors (i.e., acute kidney injury) at diagnosis, become dependent on dialysis. According to current guidelines, the primary treatment is the rapid removal of pathogenic autoantibodies (https://kdigo.org/guidelines/gd/).

The FDA and the European Commission granted imlifidase orphan drug designation for anti-GBM disease in 2018.

Recently, Uhlin et al. reported on a phase 2a study wherein 15 patients with circulating anti-GBM autoantibodies and acute kidney failure, of which 10 were dialysis-dependent, received a single dose of 0.25 mg/kg of imlifidase [START_REF] Uhlin | Endopeptidase Cleavage of Anti-Glomerular Basement Membrane Antibodies in vivo in Severe Kidney Disease: An Open-Label Phase 2a Study[END_REF]. In addition, all patients received standard treatment with cyclophosphamide and corticosteroids, but therapeutic plasma exchanges (TPE) were only performed if autoantibodies rebounded.

Six hours after imlifidase infusion, anti-GBM antibodies disappeared in all patients, but a return after a median of 7 days prompted investigators to re-initiate extracorporeal measures in 10 patients. Results showed that 67% of patients (10 out of 15) achieved dialysis independence 6 months after imlifidase treatment. This is significantly higher than 18% of patients (9 out of 50) in a historical control cohort study. Also, 5 out of 10 patients recovered renal function by 6 months compared to 2 out of 83 patients in a previous cohort-based study [START_REF] Huart | Outcomes of patients with Goodpasture syndrome: A nationwide cohort-based study from the French Society of Hemapheresis[END_REF], and no patients who were dialysis-independent at the beginning progressed to dialysis. There were 8 serious adverse events, including 1 death; however, none were related to Imlifidase. Imlifidase seems to outperform TPE/immunoadsorption in terms of rapid anti-GBM antibody reduction, but over 50% will need extracorporeal measures after the rebound of antibodies. A phase 3 clinical study is ongoing to compare standard-of-care alone or combined with imlifidase (NCT05679401).

• Guillain-Barré Syndrome Guillain-Barré Syndrome (GBS) is the most common disease leading to acute paralysis. This rare and inflammatory disease of the peripheral nervous system is usually preceded by an infection or other immune stimulation. It induces an aberrant autoimmune response targeting peripheral nerves and their spinal roots. Despite treatment with the current standard of care, including IVIg or TPE, around 70% of patients have severe symptoms resulting in the inability to walk unaided, and 20-30% require mechanical ventilation for a period ranging from weeks to months [START_REF] Yuki | Guillain-Barré Syndrome[END_REF].

Animal models showed promising results wherein IdeS prevented disease progression and facilitated improvement in a rabbit model of GBS (Wang et al., 2017).

In 2018, imlifidase was granted orphan drug designation by the FDA for treating GBS. The safety, tolerability, and efficacy of imlifidase in combination with standard-of-care IVIg in GBS patients are being assessed in an open-label, single-arm, multi-center phase 2 study that has been initiated and is currently recruiting participants (NCT03943589).

• Immune-mediated thrombotic thrombocytopenic purpura Immune thrombotic thrombocytopenic purpura (iTTP) is a rare life-threatening thrombotic microangiopathy. It is caused by a severe deficiency of ADAMTS13 (A Disintegrin And Metalloproteinase with Thrombospondin type 1 motif, 13), the VWF-cleaving protease, primarily caused by IgG autoantibodies directly inhibiting the activity of ADAMTS13 and/or accelerating its clearance from circulation. This medical emergency requires TPE as firstline therapy, combined with corticosteroids, and a conditional recommendation to add the anti-VWF nanobody caplacizumab [START_REF] Zheng | ISTH guidelines for treatment of thrombotic thrombocytopenic purpura[END_REF]. Therefore, in 2019, Stubbs et al. published a study wherein imlifidase was tested as a potential therapy for iTTP [START_REF] Stubbs | Administration of immunoglobulin Gdegrading enzyme of Streptococcus pyogenes (IdeS) for persistent anti-ADAMTS13 antibodies in patients with thrombotic thrombocytopenic purpura in clinical remission[END_REF]. In this open-label phase 2 trial, imlifidase was first given to 2 individuals with iTTP who were in clinical remission but had persistently decreased ADAMTS13 activity (≤10%) and elevated anti-ADAMTS13 IgG despite numerous lines of immunosuppressive therapy, including rituximab.

Both patients experienced a rapid decrease in total IgG and specific anti-ADAMTS13 IgG levels. However, there was no recovery in ADAMTS13 activity. This was probably due to residual F(ab')2 fragments that circulate for several days and likely continue to bind ADAMTS13 and inhibit its activity. Intriguingly, this study was the first to describe acute serum sickness-like reactions in both patients on days 6 and 7 and several other complications that led to early study termination.

Nevertheless, it is important to note that this study has several limitations. The most significant is the choice of study population. The 2 individuals had persistently low ADAMTS13 activity despite several lines of therapy, which is a rare circumstance, as most individuals in clinical remission with low ADAMTS13 activity respond well to rituximab [START_REF] Westwood | Rituximab prophylaxis to prevent thrombotic thrombocytopenic purpura relapse: outcome and evaluation of dosing regimens[END_REF]. Therefore, the pathophysiology of these 2 individuals is poorly understood, and their clinical remission before the study implies that their bodies were in a delicate homeostasis that was disrupted by the sudden loss of IgG after treatment with imlifidase. Furthermore, serum sickness is an immune-complex-mediated hypersensitivity reaction. According to the latter study results, it is unexpected that an immune-complexmediated phenomenon would result on days 6 and 7 when IgG levels are decreased, and IdeS is undetectable. However, other immunoglobulin subclasses were not investigated, e.g., anti-IdeS IgM.

For now, and with the available information, imlifidase is unlikely to replace the combination of TPE/corticoids/caplacizumab for the management of acute TTP, particularly as a monotherapy. TPE is valuable in such pathology because, in addition to removing autoantibodies, the administered plasma contains ADAMTS13, which replenishes endogenous levels. Moreover, the use of caplacizumab, a nanobody targeting the A1 domain of VWF, prevents VWF interaction with the platelet glycoprotein Ib-IX-V receptor and the ensuing microvascular thrombosis [START_REF] Scully | Caplacizumab Treatment for Acquired Thrombotic Thrombocytopenic Purpura[END_REF].

As described in the previous paragraphs, the complete, rapid, though temporary removal of IgG by IdeS provides a new potential therapeutic opportunity in IgG-mediated pathogenic conditions. Thus, the potential efficacy of IdeS treatment in both allo-and autoimmune settings of hemophilia A is of clinical interest.

Summary

Persons with congenital hemophilia A (PwHA) lack or have an altered procoagulant factor VIII (FVIII) protein. To replace the missing or defective factor, PwHA are exposed to factor concentrates. However, in 30% of the cases neutralizing alloantibodies called 'FVIII inhibitors' develop and render factor replacement therapy ineffective. FVIII inhibitors may also arise as autoantibodies in persons with no previous history of bleeding; this is commonly referred to as acquired hemophilia A (AHA). It typically concerns elderly individuals or women in the postpartum period. Managing clinically relevant bleeds and surgeries in patients with high titer inhibitors is challenging. Bypassing agents and nonfactor replacement therapies (i.e., emicizumab) are generally used. However, the former agents have several drawbacks, such as frequent dosing, the lack of reliable biomarkers for hemostatic efficacy other than clinical improvement, and the potential thrombotic risk, especially in elderly people with comorbidities. The latter agents, particularly emicizumab, do not completely restore hemostasis, and standard hemostatic treatments are still required for patients undergoing breakthrough bleeds or surgery. Further, the concomitant use of emicizumab and BPAs, particularly aPCC, carries an increased risk of thrombotic microangiopathies and thromboembolic events. Therefore, for PwHA and PwAHA, the eradication of FVIII inhibitors is a significant unmet need.

In the present study, we took advantage of the newly commercialized Immunoglobulin G (IgG)-degrading enzyme of Streptococcus pyogenes (IdeS; Imlifidase) that was shown to efficiently and quickly cleave all four human IgG subclasses. IdeS is approved for desensitization of kidney transplant patients with donor-specific antibodies and is currently being studied for its potential use in several autoimmune diseases, oncology, and gene therapy. We hypothesized that the fast, though temporary, cleavage of circulating IgG by IdeS may provide a new therapeutic option for patients with FVIII inhibitors. We demonstrate that IdeS efficiently hydrolyzes polyclonal anti-FVIII IgG in plasma obtained from PwHA and PwAHA and monoclonal recombinant human anti-FVIII with different subclasses and epitope specificities in vitro. We then developed a mouse model of inhibitor-positive severe HA by passively immunizing HA mice with recombinant human anti-FVIII IgG at different inhibitory titers . This model was developed because IdeS poorly cleaves mouse IgG. As a result, IdeS cleaved the human anti-FVIII IgG in vivo, reaching low to nadir IgG levels and restoring the hemostatic efficacy of FVIII infusions, as evidenced by the correction of the bleeding tendency in inhibitor-positive HA mice. Re-dosing of IdeS within 24-hours of the first injection was possible and essential for very high titers.

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. For instance, in PwAHA requiring hemostatic treatment, the injection of IdeS would minimize the need for BPAs and the associated thrombotic risk while restoring the efficiency of FVIII treatment and monitoring. Moreover, 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 in PwAHA. 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 emicizumab-related biological interference, ensuring accurate FVIII:C measurement in plasma. Importantly, the majority of IdeS will be cleared from circulation within 24-48 hr, allowing rapid re-administration of emicizumab for prophylaxis.

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

Melissa Bou-Jaoudeh, 1 Sandrine Delignat, 1 Victoria Daventure, 1 Jan Astermark, 2 Hervé Lévesque, 3 Jordan D Dimitrov, 1 Claire Deligne, 1 Valérie Proulle 1,4 and Sébastien Lacroix-Desmazes 1

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 recombinant 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 cofactor 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 hemostasis, and standard hemostatic treatments are still required for patients 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 LE2E9 25 and BO2C11 26 that are specific for the A2, A3, C1 and C2 domains of FVIII, respectively. The genes encoding the VH regions of the IgG and the VL regions of the 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/μg 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 50880 M -1 cm -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.

Evaluation of bleeding tendency and hemostasis

Inhibitor-positive HA mice treated with PBS or IdeS were injected with therapeutic recombinant human FVIII (Helixate ® , 200 U/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% O 2 and 70% N 2 O; 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 plateletpoor 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 μg), 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 (Invitrogen) or a polyclonal rabbit anti-human Fc © -specific antibody (Sigma-Aldrich). Bound antibodies were revealed using appropriate secondary antibodies: an 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 (Advate ® , 2.5 μg/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 enzymelinked immunosorbant assay ELISA plates were coated with a goat anti-human Ig κ antibody (2.5 μg/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 horseradish peroxidase (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 μg/mL using BO2C11 as a standard.

Results

Imlifidase hydrolyzes anti-FVIII immunglobulin G in plasma from persons with hemophilia A and patients 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 μM) 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 reported, 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 ma-jority 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 procoagulant activity of FVIII. Indeed, The inhibitory activity of the 4 monoclonal IgG1 and IgG4 antibodies was measured in a modified Nijmegen Bethesda assay. The table depicts, for each immunglobulin G (IgG), the domain specificity, the inhibitory activity in the IgG1 and IgG4 formats and the original reference. mAB: monoclonal antibody.

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).

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).

A B C

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; r 2 =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 onephase decay curve yielded a 11.7 h half-life of human F(ab') 2 fragments in mice (range, 10.4 to 13.1 h). 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 μL vs. 74±65 μL; P=0.0047), but was not different from that measured in naive inhibitor-negative HA mice that had received FVIII alone (21±16 μL). The reduction in blood loss was explained by a restoration of the hemostatic efficacy of therapeutic FVIII. FVIII:C recovery in IdeStreated 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 α3 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 neutralization) 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 neutralizing anti-FVIII IgG, the functional consequence of IdeS-mediated cleavage is delayed until elimination of the F(ab') ever 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 nonhuman 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.

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 pharmacokinetics 48 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 prescreening patients to determine their eligibility for IdeS treatment.

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.

Disclosures

Data-sharing statements

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

Summary

In the past few decades, the treatment of hemophilia A (HA) patients has come a long way. New approaches have been developed to address the challenges of replacement factor VIII (FVIII) products (i.e., intravenous injections, short-half life, and inhibitor development) and to improve the hemostatic potential independently of their administration. Among these, emicizumab is a bispecific humanized IgG4 that mimics FVIII pro-coagulant activity by bridging activated factor IX (FIXa) with its substrate, factor X (FX). Emicizumab efficiently reduces the bleeding tendency in HA patients with and without FVIII inhibitors. However, emicizumab only partially mimics FVIII activity, and its association with bypassing agents (BPA) in the context of surgery or major bleeds was associated with thrombotic events and thrombotic microangiopathy (TMA). Importantly, the 4-5 week-long half-life of emicizumab and the absence of an antidote to the drug may compromise emergency bleeding management, particularly in patients with FVIII inhibitors.

The aim of this study was to investigate whether Imlifidase, the IgG-degrading enzyme produced by Streptococcus pyogenes (IdeS), might transiently eliminate emicizumab.

This would set the stage for the use of procoagulant molecules for surgery and major bleeds while preventing the risk of thrombotic events.

Our results show an efficient time-and dose-dependent hydrolysis of emicizumab in vitro and in vivo. Cleavage, however, occurred with a slower kinetic than that of a recombinant human monoclonal anti-FVIII IgG4 used as control. In severe HA mice, F(ab')2 fragments resulting from cleaved emicizumab were systematically undetectable within 18 hours of IdeS treatment. Background levels of scIgG were still detected. However, a double injection of a 10-fold excess of IdeS reduced the procoagulant activity of emicizumab by 88.1%.

In conclusion, IdeS may be administered to HA patients with and without inhibitors to rapidly eliminate emicizumab. This would allow better management of programmed surgery with high hemorrhagic risk and breakthrough bleeds using high-dose BPA without exposing the patients to significant thrombotic risk. In addition, the concomitant elimination by IdeS of anti-FVIII IgG could restore the therapeutic efficacy of replacement FVIII. Alternatives to therapeutic FVIII have been developed in the recent years to enhance thrombin generation or mimic the procoagulant activity of FVIII. Among these, emicizumab is an engineered humanized monoclonal IgG4 that bridges activated factor IX (FIXa) with its substrate [1], factor X (FX), and reduces the bleeding tendency in HA patients with and without anti-FVIII inhibitory antibodies [2,3]. However, emicizumab only partially mimics FVIII activity, and its association with bypassing agents to correct breakthrough bleeds in patients has been associated with thrombotic events and thrombotic microangiopathy (TMA) [4].

Importantly, the 4-5 week-long half-life of emicizumab and the absence of an antidote to the drug may compromise the treatment of acute bleeds or major surgeries, particularly in patients with FVIII inhibitors. Here, we investigated whether Imlifidase, the IgG-degrading enzyme produced by Streptococcus pyogenes (IdeS) [5,6], may be used to quickly remove emicizumab and set the stage for the use of procoagulant molecules without triggering deleterious prothrombotic events.

Results and Discussion

We first validated that IdeS, which hydrolyzes all human IgG subclasses, cleaves emicizumab in vitro. As shown by SDS-PAGE (Figure 1A), IdeS hydrolyzed emicizumab in a time and dose-dependent manner. At a 10-fold molar excess of IdeS, emicizumab was completely hydrolyzed as early as after 30 min of incubation, with disappearance of the band corresponding to intact IgG and appearance of major bands migrating at 100 kDa and 25 kDa, which correspond to the digested F(ab') 2 and Fc fragments, respectively. IdeS cleaves IgG in two steps, a first fast cleavage (k cat 10 s -1 ) of one of the heavy chains followed by slower hydrolysis (k cat 0.1 s -1 ) of the second heavy chain [7]. At a 1:0.1 molar ratio of IgG versus IdeS, singlecleaved intermediate IgG (scIgG, ~140 kDa, wherein one heavy chain is proteolyzed) was detected even after incubation for 24 hours, indicating incomplete hydrolysis of emicizumab.

Accordingly, detection of the intact IgG4 or scIgG4 in a sandwich ELISA showed complete hydrolysis of emicizumab at 6 hours only for IgG:IdeS molar ratio above or equal to 1 (Figure 1B).

We have recently shown that IdeS cleaves the human monoclonal anti-FVIII IgG, BO2C11, both in its IgG1 and IgG4 subclasses [8]. Comparison of the proteolytic kinetics of emicizumab and BO2C11 IgG4 by IdeS (incubated at an IgG:IdeS molar ratio of 12:1) revealed a slower digestion of emicizumab evidenced on both the heavy chains. Indeed, at 5 min, only about half of emicizumab was digested into scIgG, while most of BO2C11 was hydrolyzed (Figure 1C).

Similarly, traces of emicizumab scIgG were still detected after 24 hours, while BO2C11 scIgG became undetectable after 1 hour of incubation. IgG with a single-cleaved heavy chain exhibits reduced binding to complement and Fc receptors and reduced capacity to mediate complement and antibody-dependent cellular cytotoxicity or to facilitate phagocytosis [9,10]. scIgG, however, retain essential properties of intact IgG, including the ability to bind the neonatal Fc receptor (FcRn) and, hence, be endowed with a half-life of 3 weeks in humans and 9 days in mice and to bind to their cognate antigen(s).

Several point mutations have been introduced in the CH1, hinge, CH2 and CH3 domains of both the heavy chains of emicizumab. The introduction of the mutations aimed at improving its stabilization and downstream processing, reducing C-terminal heterogeneity and fostering heterodimerization of the heavy chains (Figure 1D) [11,12]. The introduced amino-acid changes may account for the reduced cleavage efficacy of emicizumab by IdeS. IdeS was recently shown to cover the hinge regions and several loops of the CH2 domains of both heavy chains of a human monoclonal IgG1 [13]. Interestingly, the two mutations located in the hinge that of an IgG1, suggesting that they are not responsible for the lower cleavage efficacy of emicizumab by IdeS. Alternatively, the mutations introduced in the CH3 domain may affect the Fc regions of emicizumab involved in the interaction with IdeS. Indeed, IgG molecules exhibit long-distance structural coupling between their domains, and amino-acid replacements may induce structural/electrostatic modifications at distance from the mutated residue [14][15][16][17][18]. For instance, the introduction of mutations in the Fc portion at a distance from the FcRn contact surface was shown to improve the binding affinity between the two molecules [19,20].

Likewise, a single mutation in the variable region of the broadly neutralizing antibody NIH45-46 increased by three folds the affinity of the IgG for the FcRn receptor [21]. We cannot rule out however the possibility that the nature of the Fab fragments plays a role in determining whether an IgG is a suitable substrate for IdeS. Indeed, therapeutic monoclonal antibodies that differ only in the sequence of their Fab domains and, hence, recognize different cognate antigens, but share identical Fc fragments, demonstrated different sensitivities to IdeS proteolysis in vitro [22].

We then investigated whether the use of IdeS could rapidly remove emicizumab from the circulation in a preclinical model of severe hemophilia A. FVIII-deficient mice were first injected intravenously with different doses of emicizumab, and the plasma concentration of emicizumab was followed for up to 24 hours (Figure 2A). Irrespective of the dose administered, the circulating concentration of emicizumab stabilized 6 hours following injection (Figure 2B).

The concentration of emicizumab was almost linearly correlated with the injected dose, both 5 min and 24 hours after injection (Figure 2C), as shown previously [23]. Thus, a dose of 3 mg/kg yielded a plasma concentration of 32.4±10.2 µg/ml 6 hours later, which is in the same order of magnitude as trough levels measured in patients with hemophilia A dosed once-weekly with 1.5 mg/kg (i.e., 45 µg/ml) [4]. In a second series of experiments, mice were treated with PBS or with one or two injections of equimolar or 10-fold molar excess IdeS within the first 9 hours that followed the injection of 3 mg/kg emicizumab (Figure 3A). Emicizumab was detected in mouse plasma by Western blot 6, 9, 24, 48 and 72 hours later. In all cases, the treatment with IdeS of mice passively immunized with emicizumab 6 hours or 6 and 9 hours earlier resulted in the hydrolysis of emicizumab (Figure 3B). Cleaved Fc fragments were readily detectable as early as 3 hours following the first IdeS treatment. F(ab') 2 fragments were systematically undetectable within 18 hours of IdeS treatment, irrespective of the dose of IdeS administered and the number of IdeS administrations.

This suggests a faster clearance of the F(ab') 2 fragments of emicizumab compared to the typical ~12-hour long half-life of human F(ab') 2 fragments in mice [8,24]. In stark contrast, scIgG, that retain binding capacity to the FcRn and, hence, an unmodified in vivo half-life [9], were still detected at 72 hours (Figure 3B). Whether the rat-mouse hybrid nature of the VH and VL domains of emicizumab [25], that are chimerized with a human IgG4 heavy chain or IgG kappa light chain, respectively, explains such a rapid clearance of the F(ab') 2 fragments of emicizumab is not clear. Alternatively, we cannot completely exclude the presence of lowaffinity cross-reactivity of the anti-FIX and/or anti-FX variable regions of emicizumab with endogenous murine FIX or FX, which can potentially accelerate the catabolism of the F(ab') 2 fragments.

The plasma levels of intact IgG or scIgG were further quantified by sandwich ELISA at different time points. The levels of emicizumab were reduced by 70.5% 66 hours after 2 injections of equimolar amounts of IdeS (Figure 3C). The decrease was enhanced when a 10fold excess of IdeS was injected once or twice (by 86.9% and 90.3%, respectively), indicating that a 10-fold molar excess of the enzyme compensates for the lesser susceptibility of emicizumab to IdeS-mediated hydrolysis. Interestingly, 3 hours after the first injection of IdeS, the circulating levels of total IgG or scIgG were reduced by 27.2, 69.7 and 71.0% (Figure 3C) Circulating levels of emicizumab F(ab') 2 fragments showed a rapid decrease within 18 hours after the injection of IdeS (Figure 3D). The decrease culminated when IdeS was injected a second time at a 10-fold molar excess over the concentration of emicizumab. Our ELISA setup does not discriminate between cleaved F(ab') 2 fragments and scIgG. However, the absence of F(ab') 2 fragments shown in Western blot from the 24-hour time point onwards (Figure 3B), together with the steady signal detected by ELISA over this time period (Figure 3D), indicate that the emicizumab isoform that remains is the scIgG. Measure of the procoagulant activity of emicizumab in a chromogenic assay confirmed elimination of the functionally active molecule (Figure 3E). Hence, the reduction in procoagulant activity of emicizumab ranged between 23.4% and 44.2% after a first injection of IdeS, depending on the amounts of IdeS injected, and peaked at 88.1% when IdeS was injected twice in 10-fold molar excess.

The management of acute bleeds and major/emergency surgeries is complicated in patients under emicizumab prophylaxis. This is particularly true in patients with FVIII inhibitors that cannot be treated with exogenous FVIII. The hemostatic management in these patients relies on the use of bypassing agents, activated prothrombin complex concentrates (aPCC) or recombinant activated factor VII (rFVIIa). rFVIIa has a very short half-life, thus requiring frequent dosing, and is inefficient in some patients [26]. Conversely, the association of emicizumab with aPCC, and to a lesser extent with rFVIIa, exposes the patients to a risk for TMA/thrombotic events. High doses rFVIIa or aPCC should thus be avoided as first-line therapy in these patients [27]. Our results provide the proof-of-concept for the rapid elimination of emicizumab using IdeS. IdeS-mediated removal of emicizumab in inhibitor-negative patients with uncontrollable bleeding would open the possibility of using bypassing agents without exposing the patients to thrombotic risk. In line with this, our data show that a double injection 4±3.9 µg/ml to 10.1±1.3 µg/ml in 3 hours and to 3.6±0.7 µg/ml over 21 hours (Figure 3C).

At these concentrations, emicizumab has no substantial effect on the activated partial thromboplastin clotting time, at least in vitro [28].

We have recently reported that IdeS efficiently eliminates inhibitory anti-FVIII IgG in a mouse model of inhibitor-positive severe hemophilia A [8]. IdeS thus opens a time window for the transient use of therapeutic FVIII to control excessive bleeding. In contrast to emicizumab, however, the elimination of the F(ab') 2 fragments of the hydrolyzed anti-FVIII IgG required 12 to 72 hours to reach a BU titer below 5 BU/ml, depending on the starting inhibitory titer [8].

Our present data highlight the potential of IdeS in inhibitor-positive patients under emicizumab therapy. In the latter patients, IdeS would remove both emicizumab and FVIII inhibitors, albeit with different kinetics. The rapid removal of emicizumab by IdeS would set the stage for the use of high-dose bypassing agents to control acute bleeds for a period of 2-3 days without exposing the patients to major thrombotic risk. Over the period, the progressive elimination of inhibitory F(ab') 2 fragments following the rapid cleavage of FVIII inhibitors by IdeS would restore the therapeutic efficacy of FVIII. Importantly, the rapid clearance of IdeS (half-life of 4.9±2.8 hours in the human) [29] should allow to quickly resume emicizumab prophylaxis as soon as the severe acute bleeds have been resolved.

Authors

Summary

FVIII inhibitors are a major concern for effectively treating persons with hemophilia A (PwHA). The primary strategy to eradicate these inhibitors is immune tolerance induction (ITI) therapy, which relies on the daily injection of high-dose factor VIII (FVIII) to desensitize the immune system. Tolerance is achieved after chronic and prolonged administration of factor VIII (FVIII) concentrates (9-33 months), usually at high doses.

However, ITI is successful in only 60-80% of severe PwHA and is expensive and cumbersome.

Several clinical parameters have been associated with ITI outcome both treatment-related and patient-related. Interestingly, anti-FVIII IgG subclasses and epitope specificities may be predictive of clinical outcome. Indeed, PwHA who fail ITI have high anti-FVIII IgG4/IgG1 ratios and these antibodies are mainly specific for the heavy chain or A2 domain of FVIII.

On the contrary, successfully treated PwHA have antibodies specific for the light chain or C2 domain of FVIII. However, it remains unclear how properties of anti-FVIII IgG may be predictive of ITI outcome.

In the context of ITI, the injection of large amounts of FVIII to PwHA with inhibitors leads to the formation of FVIII-containing immune complexes (FVIII-IC). The latter were hypothesized to accelerate FVIII clearance from the circulation by the reticuloendothelial system. However, these FVIII-IC may have an effect on innate and adaptive immune cells involved in tolerance induction. To our knowledge, this has not been thoroughly studied in a complete human system where the impact of human FVIII-IC on human cells is examined.

In this study, we generated synthetic FVIII-IC using Our findings contribute to a better understanding of the mechanisms underlying ITI outcome in PwHA, particularly those related to different properties of anti-FVIII IgG.

Introduction

The eradication of factor VIII (FVIII) inhibitors is currently best achieved through immune tolerance induction (ITI) therapy in hemophilia A (HA) patients. It entails the regular and prolonged administration of FVIII concentrates, usually at high doses, to suppress the anti-FVIII antibody response, leading to immunological tolerance and desensitization.

Unfortunately, ITI is prohibitively expensive, requires extreme patient compliance, and is successful only in 60-80% of the patients with severe HA (DiMichele et al., 2007) and generally requires several months before reaching success (9 to 33 months).

Epidemiological studies have identified several clinical parameters associated with ITI outcome. Patient-related factors include inhibitory titer at the start of ITI, the time elapsed between inhibitor detection and initiation of ITI, historical peak inhibitory titer and peak inhibitory titer during ITI (Ljung et al., 2019). Other patient-related factors include age at the start of ITI, patient ethnicity and FVIII genotype, but their impact on ITI outcome remains unclear. Interestingly, properties of the anti-FVIII IgG were associated with ITI outcome. Thus, patients who fail ITI have high anti-FVIII IgG4/IgG1 ratios [START_REF] Helden | IgG subclasses of anti-FVIII antibodies during immune tolerance induction in patients with hemophilia A[END_REF]Minno et al., 2014). Furthermore, antibodies specific for the heavy chain or A2 domain are found in most patients who fail ITI, while most successfully treated patients have anti-C2 or anti-light chain antibodies (Helden et al., 2010;Minno et al., 2014).

Nevertheless, there is no molecular explanation today on how the properties of anti-FVIII IgG dictate ITI outcome.

The injection of large amounts of FVIII in patients with circulating anti-FVIII IgG leads to the formation of FVIII-containing immune complexes (FVIII-IC). Indeed, the presence of circulating FVIII-IC was documented in multi-transfused PwHA (Kazatchkine et al., 1980).

Likewise, FVIII-IC were detected in patients with acquired HA, an autoimmune disease characterized by the development of anti-FVIII autoantibodies (Nogami et al., 2001). FVIII-IC were hypothesized to accelerate FVIII clearance from the circulation by the reticuloendothelial system (Nogami et al., 2001;Batsuli et al., 2016Batsuli et al., , 2018)). In the context of HA patients with inhibitors under ITI therapy, the formation of FVIII-IC may have different repercussions on the innate and adaptive immune cells implicated in the anti-FVIII immune response. To our knowledge, this has not been extensively investigated in a complete human system wherein the effect of human FVIII-IC is analyzed on human cells.

Indeed, the use of mouse models or in vitro experiments have yielded contradictory results. The formation of FVIII-IC using murine monoclonal antibodies was shown to increase FVIII endocytosis by mouse bone marrow-derived dendritic cells (DCs) (Hartholt et al., 2017). Conversely, the co-incubation of FVIII with the human monoclonal anti-C1

IgG, KM33, or anti-C2 IgG, BO2C11, independently blocked FVIII uptake by human monocyte-derived DCs (MoDCs) (Wroblewska et al., 2012;Gangadharan et al., 2017). In contrast, the co-incubation of FVIII with the mouse monoclonal anti-C2 IgG, ESH8, drastically increased FVIII uptake by MoDCs. Thus, properties of FVIII-IC might impact FVIII endocytosis by DCs in a species-dependent manner.

In this study, we hypothesized that different properties of FVIII-IC that form in HA patients

Methods

Generation of recombinant human anti-FVIII IgG

Four recombinant human monoclonal anti-FVIII IgGk (rhIgG) were produced: BOIIB2, KM41, LE2E9, and BO2C11, which are specific for the A2, A3, C1, and C2 domains of FVIII, respectively. The specificity and inhibitory activity of the 8 rhIgG has been described

previously (Bou-Jaoudeh et al., 2023).

Human anti-FVIII IgG ELISA

ELISA plates (Maxisorp, Nunc) were coated with rhFVIII (Advate®, 2 μg/mL) diluted in phosphate buffer saline (PBS, pH 7.4, Life Technologies). Plates were then blocked with 3% bovine serum albumin (BSA) in PBS. Serial dilutions (0-1 µg/ml) of anti-FVIII rhIgG were added to the wells. Bound anti-FVIII rhIgG were revealed using an HRP-labeled mouse monoclonal antibody specific for human Fcγ (Southern Biotech) and the ophenylenediamine dihydrochloride (OPD, Sigma-Aldrich) substrate. Absorbance was read at 492 nm.

Measure of the neutralizing activity of the anti-FVIII rhIgG

The inhibitory activity of the anti-FVIII rhIgG was measured using the modified Nijmegen-Bethesda assay (MNBA). Monoclonal IgG in PBS 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 hr at 37°C. The residual pro-coagulant FVIII activity (FVIII:C) was measured using a chromogenic assay following the manufacturer's instructions (Siemens Healthcare). The inhibitory activity of the IgG was expressed in BU/µg, defined as the inverse of the concentration of IgG needed to inhibit 50% of FVIII:C.

Binding of anti-FVIII rhIgG to FcRn by surface plasmon resonance

The kinetic constants of the interactions between BO2C11, BOIIB2, LE2E9 and KM41 in

Human FcγR reporter cell assays

Mouse BW5147 are stably transduced to express human FcγR on the cell surface with the cytosolic signaling domain replaced with the murine CD3ζ chain. Thus, engagement of the FcγR at the cell surface triggers signaling leading to the secretion of murine interleukin (IL)-2 (Kolb et al., 2019). FcγR reporter cell lines were maintained in RPMI-1640, 10% fetal calf serum (FCS), 250 µM 2-mercaptoethanol, 100 U/mL penicillin, 100 µg/mL streptomycin and 1 µg/ml of puromycin (Invivogen). The expression of CD16A (FcγRIIIA), CD32A (FcγrIIA) and CD32B (FcγRIIB) by the transduced cells was validated by surface staining and flow cytometry with PE or FITC conjugated anti-human CD16 (clone 3G8) or CD32 (clone 3D3) and isotype controls (BD Biosciences) (supplementary

Figure S2).

For binding assays, IgG preincubated alone or with recombinant full-length FVIII (Helixate, 10 nM final concentration) for 30 min at 37°C were incubated with the FcγR transduced cell lines in RPMI-1640, 0.5% FCS, 100 U/mL penicillin, and 100 µg/mL streptomycin for 90 min at 4°C. After washing in ice-cold PBS, bound rhIgG were detected with a PEconjugated goat anti-human Fc antibody (eBiosciences). FACS analysis was performed on live cells using the fixable viability dye eFluor780 (eBiosciences) staining and BD LSRII and FACS Diva software (BD Biosciences).

For FcγR signaling assays, 96 well-culture plates were blocked with RPMI-1640, 10%

FCS for 1 hr at 37°C to avoid the immobilization of soluble rhIgG and/or soluble FVIII-IC on the plate surface. FcγR reporter cells (2x10 5 cells/well) were then incubated with rhIgG alone or pre-incubated with FVIII in RPMI-1640, 10% FCS, 100 U/mL penicillin, and 100 µg/mL streptomycin for 20 hr at 37°C. Secreted mouse IL-2 was measured in supernatants by ELISA (OptEIA kit, BD Biosciences).

Generation of immature human monocyte-derived dendritic cells

Monocytes were isolated from the blood of healthy donors using anti-CD14 magnetic microbeads (Miltenyi Biotec). Agreement was obtained for the use of buffy bags from healthy donors (Etablissement Français du sang, N°18/EFS/033). Monocytes

Maturation of monocyte-derived dendritic cells

Full-length FVIII (Kogenate®, 10 nM) was pre-incubated alone or with a 5-molar excess of the pool of the four IgG1k or IgG4k for 30 min at 37°C in X-VIVO 15 serum free medium (Lonza). Pre-formed FVIII-IC were then incubated for 48 hr at 37°C with 5-days-old immature MoDCs (0.25x10 6 cells/well) in 48-well culture plates in X-VIVO 15 medium supplemented with GM-CSF (1000 IU/10 6 cells) and IL-4 (500 IU/10 6 cells) (Miltenyi Biotec). Lipopolysaccharide (LPS, 100 ng/ml) was used as a positive control. LPS (0.2 ng/ml or 100 ng/ml) was added to FVIII-IC conditions in some experiments as additional stimuli for MoDCs. Cells were collected after 48 hr and stained with the fixable viable dye eFluor 780 (Thermo Fisher Scientific) and for different maturation surface markers (CD40, CD80, CD86, and CD83). Cells were then analyzed by flow cytometry.

NK stimulation assay

NK-92 and PTA-6967 cells (ATCC #CRL-2407 and #PTA-6967, respectively) were maintained in complete medium: alpha-MEM medium, supplemented with 0.2 mM inositol, 0.02 mM folic acid, 0.1 mM 2-mercaptoethanol, 12.5% horse serum (Thermo Fisher Scientific) and 12.5% FCS (Biowest). To measure the secretion of IFNγ, NK-92 or PTA-6967 cells were incubated in 96-well round-bottom plates (1x10 5 cells/well) with pools of the four IgG1k or the four IgG4k (2.5 nM of each IgG) pre-incubated or not with FVIII (Helixate, 10 nM) in complete medium. Secreted IFNγ was measured in the supernatant 24 hr later by ELISA (Thermo Fisher Scientific). For degranulation assay, PTA-6967 cells were incubated in 24-well plates (5x10 5 cells/well) with the pools of IgG1k

or IgG4k as described above, in the presence of a BV421-conjugated anti-LAMP1 antibody (Biolegend) for 18 hr at 37°C. Brefeldin A and Monensin were added after 1 hr of incubation to inhibit endocytic trafficking. Cells were harvested and stained with the fixable viable dye eFluor 780 (Thermo Fisher Scientific), a FITC-labeled anti-CD16

antibody and an Alexa 700 anti-CD56 antibody (BD Biosciences). The surface expression of LAMP1 was measured with BD LSRII cytometer and analyzed with the FACSDiva™ software.

(CD32A) lead to a dose-dependent binding of the rhIgG to the cell surface, as compared to incubation of the rhIgG with the parental non-transduced cells. The IgG1k bound to CD32A with a higher intensity than the IgG4k (Figure 1C). Interestingly, the different IgG1k and IgG4k demonstrated some degree of intra-subclass binding heterogeneity, which may relate to the differences in their variable domains (Grevys et al., 2022;Kosuge et al., 2023). As expected (Bruhns et al., 2009), incubation of the rhIgG with the CD16Aexpressing cell line yielded binding only in the case of IgG1k and only a residual signal in the case of IgG4k.

In vitro generation of FVIII-containing immune complexes

The formation of FVIII-IC upon incubation of FVIII with a 5-molar excess of anti-FVIII rhIgG was validated by the co-immunoprecipitation using protein G-coated agarose beads, shown in the case of BO2C11 IgG1k and IgG4k (Figure 2A). FVIII was detected when it was pre-incubated with BO2C11 IgG1k and IgG4k prior to immunoprecipitation. The migration profile matched that of untouched FVIII (Figure 2A, 'FVIII alone', lane 3). No FVIII was detected in the eluate when beads were incubated alone or with FVIII alone, showing that FVIII does not stick non-specifically to the beads. Likewise, no FVIII was detected when it was incubated in the presence of a non-specific IgG1, the anti-human epidermal growth factor receptor 2 protein antibody (trastuzumab). The Western blot confirmed the presence of IgG in the eluates, only in samples containing IgG. A band was visible at 50 kDa, which corresponds to the reduced heavy chain of IgG (Figure 2A, lower panel).

To further validate the in vitro formation of FVIII-IC, we first evaluated the binding of the pooled IgG1k or the pooled IgG4k, pre-incubated alone or with FVIII at three different IgG:FVIII ratio (0.25:1, 1:1, and 5:1) to the different reporter cell lines.

In the absence of FVIII, pool of IgG1k bound to CD16A and CD32A-expressing cells, though at different levels. The binding to CD16A, CD32A and CD32B was markedly enhanced when IgG1k were pre-incubated with FVIII (Figure 2B). Conversely, IgG4k exhibited binding to the cells expressing CD32A and CD32B only in the presence of FVIII, and failed to bind to CD16A both in the presence and absence of FVIII.

induced the overexpression of CD40, CD80 and CD86 (supplementary Figure S4). The addition of FVIII-IC had no effect on the maturation profile of MoDCs incubated with low or high concentrations of LPS.

Activation of natural killer cells by FVIII-IC

As shown in Figure 2, IgG4-containing FVIII-IC do not engage the CD16A at the surface of transduced cells. Most human NK cells only express the CD16A. Interestingly, the commercially available therapeutic Fc-fused FVIII, Eloctate®, that contains a Fcγ1, was found to trigger the activation of NK cells (Lagassé et al., 2021). This was not the case for the therapeutic Fcγ1-fused FIX, suggesting that FVIII may influence the activation of NK cells independently from or in synergy with CD16 engagement. We therefore investigated In contrast, the secretion of IFNγ and expression of LAMP1 were unchanged in the absence of FVIII, when CD16 was lacking at the cell surface or when IgG4k-containing FVIII-IC were used to stimulate the cells.

Tables Table 1. Inhibitory activity of human recombinant anti-FVIII IgG

The inhibitory activity of the 8 monoclonal IgG1k and IgG4k was measured in a modified Nijmegen Bethesda assay. The table depicts, for each IgG, the domain specificity and the inhibitory activity in the IgG1 and IgG4 formats. Values in the table depict mean±SD for 3-4 independent measures. As mentioned earlier, this thesis report is based on three articles, two of which relate to the potential use of the bacterial enzyme IdeS for the treatment of HA patients, and the third one, which is in preparation, assesses the impact of FVIII-IC on the immune response to FVIII in the context of ITI. Therefore, I will start by discussing the results obtained in this last article in the first part and discuss the former articles in the second part.

Legends to Figures

FVIII-containing immune complexes: impact on the immune response to FVIII during ITI therapy

The major complication of congenital HA is the development of FVIII inhibitors in up to 30% of severe HA cases (Gouw et al., 2013a), rendering further FVIII replacement therapy inefficient. Until today, the only proven efficient strategy to eradicate inhibitors is ITI which only succeeds in 60-80% of severe HA cases (DiMichele et al., 2007;[START_REF] Valentino | US Guidelines for immune tolerance induction in patients with haemophilia a and inhibitors[END_REF]. ITI consists of reeducating the immune system to tolerate FVIII. Yet, the only criterion in favor of tolerance induction is the disappearance of the inhibitory titer. The immunological mechanisms underlying the establishment of a tolerogenic immune response are still unknown. However, evidence in favor of an actual induction of FVIIIspecific active immune tolerance relying on the induction of regulatory T and B cells is just starting to emerge (Schep et al., 2021a).

Moreover, 20-40% of patients unresponsive to ITI remain without efficient therapy for the eradication of FVIII inhibitors. Interestingly, some immunologic properties of the anti-FVIII IgG (i.e., epitope specificity and subclass) were suggested as predictive markers of ITI outcome. Thus, patients who fail ITI have high anti-FVIII IgG4/IgG1 ratios [START_REF] Helden | IgG subclasses of anti-FVIII antibodies during immune tolerance induction in patients with hemophilia A[END_REF]. Furthermore, antibodies specific for the heavy chain or A2 domain are found in most patients who fail ITI, while most patients successfully treated with ITI have anti-C2

or anti-light chain antibodies (Helden et al., 2010;[START_REF] Lapalud | Anti-A2 and anti-A1 domain antibodies are potential predictors of immune tolerance induction outcome in children with hemophilia A[END_REF]. Notably, the injection of large amounts of FVIII in patients with circulating anti-FVIII IgG leads to the formation of FVIII-IgG immune complexes (FVIII-IC). Nevertheless, there is no molecular explanation today on whether and how properties of FVIII-IC may dictate ITI outcome.

The aim of this study was to understand the biological relevance of different properties of FVIII-IC (i.e., IgG subclass and epitope specificity), that form in inhibitor-positive patients undergoing ITI and receiving high doses of FVIII.

This was investigated using synthetic FVIII-IC formed with recombinant FVIII (rFVIII) and a panel of recombinant monoclonal human anti-FVIII IgG1 and IgG4 directed to different domains of the FVIII (i.e., A2, A3, C1 and C2). Using synthetic FVIII-IC allowed me to control the composition of preformed immune complexes, such as IgG subclass and epitope specificity, and size of the immune complexes (i.e., FVIII:IgG ratio). Moreover, it gave me the tools to compare my work with previous work with a slight twist: using a complete human system (i.e., human recombinant IgG and FVIII and human cells) instead of a mouse system or heterologous mouse/human systems.

Validation of FVIII-IC formation

I first validated the formation of the synthetic FVIII-IC by i) Western blot analysis following co-immunoprecipitation on protein G beads and ii) by binding and signaling assays using the BW5147:FcγR-ζ reporter cell lines. My results correlate with previously reported data on Fc-FcγR interactions (Bruhns et al., 2009). FVIII-IgG1 immune complexes signaled through all FcγR. FVIII-IgG4 immune complexes signaled through all FcγR, except CD16, with the most significant signal observed with CD32B receptor for which IgG4 are known to retain a relatively high affinity compared to activating FcγR (Bruhns et al., 2009).

However, the high-affinity CD64-expressing cell line showed unexpected low signaling results and the expression of CD64 at the cell surface was not stable even after treatment with the selection antibiotic puromycin. Ideally, this cell line should be subcloned but, due to time constraints, it was excluded from the experiments. Importantly, CD32A has two allelic variants: CD32A-H131 has an overall higher affinity for the Fc fragment than CD32A-R131 [START_REF] Shashidharamurthy | Dynamics of the interaction of human IgG subtype immune-complexes with cells expressing R and H allelic forms of a low affinity Fc gamma receptor CD32A[END_REF]. Results shown in my manuscript in preparation were obtained with the CD32A-R131 variant. I was nevertheless interested in this polymorphism because Eckhardt et al. showed several years ago that HA patients with the HH genotype of the CD32A polymorphism 131R > H are at a 3-fold higher risk of developing inhibitors than patients with the RR genotype (Eckhardt et al., 2014). I thus assessed binding and signaling through the CD32AH variant. I found a higher interaction of both IgG1 and IgG4-containing IC with the CD32AH variant compared to the CD32AR variant (data not shown). However, I observed better signaling through the CD32AR variant compared to the CD32AH variant. This experiment was not done for the FVIII:IgG ratio of 1:5 used in other assays and the signaling assay was only done once and needs to be repeated.

Impact of FVIII-IC on the effector functions of immune cells

In physiology, inappropriate generation and clearance of ICs are commonly associated with chronic viral or bacterial infections [START_REF] Wang | Immune Complexes: Not Just an Innocent Bystander in Chronic Viral Infection[END_REF][START_REF] Yamada | Suppression of Fcγ-receptor-mediated antibody effector function during persistent viral infection[END_REF] and autoimmune diseases, such as systemic lupus erythematosus or rheumatoid arthritis [START_REF] Antes | C1q-bearing immune complexes detected by a monoclonal antibody to human C1q in rheumatoid arthritis sera and synovial fluids[END_REF][START_REF] Koffler | SYSTEMIC LUPUS ERYTHEMATOSUS: PROTOTYPE OF IMMUNE COMPLEX NEPHRITIS IN MAN[END_REF]). In the latter pathologies, uncontrolled IC deposition in tissues results in inflammatory responses, classified as type III hypersensitivity, causing local damage [START_REF] Rajan | The Gell-Coombs classification of hypersensitivity reactions: a reinterpretation[END_REF]. However, the biological effects of IgG-containing ICs can be modulated by several parameters such as the valency and IgG subclass composition, the capacity to bind complement and engage different activating (CD64, CD32A, CD32C, CD16) or inhibitory (CD32B) FcγR, etc. Moreover, depending on the cell type and the expression level of different FcγR, the interaction of FVIII-IC with FcγR at the cell surface can have various immunological consequences [START_REF] Nimmerjahn | Fcγ receptors as regulators of immune responses[END_REF]. All human FcγR are activating-type receptors except CD32B, which is an inhibitory receptor.

For this study I initially planned to use different types of immune cell such as MoDCs, bDCs, NK and B cells, each expressing a different panel of FcγR. Immature MoDCs, for example, express the activating CD32A and inhibitory CD32B FcγRs. Immature MoDCs can thus provide an excellent model for studying the modulation and function of CD32A and CD32B on the same cells. CD64 expression can be increased on immature MoDCs by in vitro stimulation with IFN-γ (Boruchov et al., 2005). Conventional blood DCs express mostly CD32A and CD32B, but also CD64 [START_REF] Fanger | Type I (CD64) and type II (CD32) Fc gamma receptor-mediated phagocytosis by human blood dendritic cells[END_REF], whereas B cells mainly express CD32B and NK cells mainly express CD16A [START_REF] Bruhns | Mouse and human FcR effector functions[END_REF], though a fraction of NK and B cells can express CD32C (7-15% of individuals) [START_REF] Metes | Ligand binding specificities and signal transduction pathways of Fc gamma receptor IIc isoforms: the CD32 isoforms expressed by human NK cells[END_REF][START_REF] Li | Allelic Dependent Expression of an Activating Fc receptor on B cells Enhances Humoral Immune Responses[END_REF].

The presence of circulating FVIII-IC in HA patients was documented a long time ago, first in multi-transfused HA patients following blood precipitation with low concentrations of polyethylene glycol (Kazatchkine et al., 1980), and later on in patients with AHA after purification on protein G-sepharose beads (Nogami et al., 2001). However, except for their potential role in accelerating FVIII clearance from the circulation by the reticuloendothelial system as documented in the work of Nogami et al., there is not much consistent data on how FVIII-IC might modulate FVIII immune responses. Seminal work by Eibl et al.

documented many years ago that the in vitro co-incubation of human monocytes with FVIII-IC is followed by a general decrease of monocyte functions, particularly surface expression of FcγR, release of O2 radicals and bactericidal activity [START_REF] Eibl | A component of factor VIII preparations which can be separated from factor VIII activity down modulates human monocyte functions[END_REF].

Nevertheless, the formation of FVIII-IC might have repercussions on different cell types.

They might for instance: 1) facilitate internalization by professional antigen presenting cells (APCs) such as DCs, macrophages, or B cells, as described in general for antigen:

antibody immune complexes [START_REF] Amigorena | Fc receptors for IgG and antigen presentation on MHC class I and class II molecules[END_REF][START_REF] Schuurhuis | Antigen-Antibody Immune Complexes Empower Dendritic Cells to Efficiently Prime Specific CD8+ CTL Responses In Vivo1[END_REF] ;

2) influence the maturation state of APCs and their capacity to polarize T-cell responses in vitro [START_REF] Regnault | Fcγ Receptor-mediated Induction of Dendritic Cell Maturation and Major Histocompatibility Complex Class I-restricted Antigen Presentation after Immune Complex Internalization[END_REF]; or 3) cross-link FcRγIIB and B-cell receptors (BCR) at the surface of FVIII-specific memory B cells mediating inhibitory signals, thus inhibiting downstream biological responses of the B cell including activation, antigen presentation, proliferation and antibody production [START_REF] Kurosaki | Regulation of B-cell signal transduction by adaptor proteins[END_REF].

• Endocytosis of FVIII-IC by dendritic cells

Conflicting results were obtained regarding FVIII uptake by DCs. Recent work by Hartholt et al. showed that the formation of FVIII-IC increases the endocytosis of FVIII by mouse immature bone marrow-derived DCs and that this process involves Fc-FcγR interactions.

The effect of the binding of IgG to FVIII on its endocytosis may, however, depend on the very properties of the anti-FVIII IgG. Indeed, co-incubation of FVIII with the human monoclonal anti-C1 IgG1, KM33, or anti-C2 IgG4, BO2C11, independently blocked FVIII uptake by human MoDCs (Wroblewska et al., 2012;Gangadharan et al., 2017).

Conversely, co-incubation of FVIII with the mouse monoclonal anti-C1 IgG, F156, or anti-C2 IgG2a, ESH8, increased FVIII uptake (Batsuli et al., 2016;Gangadharan et al., 2017).

To my knowledge, a systematic analysis of different properties of FVIII-IC in terms of epitope specificity and IgG subclass on FVIII uptake in a complete human in vitro system (i.e., human IgG and human DCs) is lacking.

To answer this question, I started endocytosis experiments using bDCs. However due to difficulties in purifying sufficient amounts of bDCs and time constraints, I switched to MoDCs, which are easier to purify and culture and with which our lab has years of experience. Experiments were pursued based on the molar ratio used by Hartholt et al., which is a 5-molar excess of antibodies over FVIII. My results show that irrespective of the epitope specificity or IgG subclass, FVIII-IC reduce the uptake of FVIII by immature MoDCs compared to FVIII alone. The highest reduction was observed after incubation with the pool of IgG1 or IgG4-containing FVIII-IC (63.6 ± 14.4 % and 60.9 ± 13.2 %, respectively). These results are in accordance with previously published results with anti-C2 and anti-C1 antibodies since residues in the C1 and/or C2 domains of FVIII were shown to be implicated, at least in vitro, in FVIII uptake (Gangadharan et al., 2017).

However, our results are not in accordance with those obtained by Hartholt et al., and this can have several explanations: the different panels of FcγR expressed on mouse bonemarrow-derived DCs (i.e., FCγRI, FcγRIIB and FCγRIII), species variability, etc. I was, however, surprised to see a low albeit statistically significant reduction of FVIII endocytosis in the presence of the anti-A2 or anti-A3 recombinant human IgG1 or IgG4.

This observation may point towards the identification of unknown receptors on MoDCs that mediate FVIII capture and internalization, at least in vitro.

• Complement as a modulator of FVIII-IC uptake FVIII endocytosis experiments has been performed in vitro using serum-free medium thus ignoring a potential role for the complement system. Complement activation occurs through three different pathways [START_REF] Nesargikar | The complement system: history, pathways, cascade and inhibitors[END_REF]: 1) the classical pathway triggered by C1q binding to immune complexes, 2) the lectin pathway triggered by the binding of mannose-binding lectin to carbohydrates structures on pathogens, and 3) the alternative pathway spontaneously and continuously activated at a low rate (i.e., C3 tickover). Inappropriate complement activation has been linked to autoimmune reactions.

Recent work by Rayes et al. documented the importance of complement activation on

FVIII immunogenicity [START_REF] Rayes | Complement C3 is a novel modulator of the anti-factor VIII immune response[END_REF]. Indeed, activation of complement C3 allowed a 2-fold increase in the uptake of FVIII in vitro and the exhaustion of C3 drastically reduced the immune response to therapeutic FVIII in FVIII-deficient mice. Furthermore, while the mutation of 3 charged amino acids in the C1 domain of FVIII was shown to abolish FVIII uptake by MoDCs (Gangadharan et al., 2017), complement activation rescued the internalization of the non-immunogenic FVIII C1 mutant [START_REF] Rayes | Complement C3 is a novel modulator of the anti-factor VIII immune response[END_REF]. The effect of complement activation on IC-mediated FVIII uptake has never been addressed. This is relevant since IgG4, in contrast to IgG1, does not bind or activate complement. I wanted to address this potential effect; however, in vitro experiments showed counterintuitive results owing to the binding of FVIII per se to complement molecules such as C1q (Figure 16). Binding of FVIII to C1q introduced a bias in the assessment of FVIII-IC binding to C1q via the Fc portion of IgG. As a result, I decided to stop the experiments.

• Maturation of dendritic cells

Furthermore, I explored the impact of synthetic FVIII-IC on the maturation state of MoDCs in the presence or absence of additional stimuli targeting pattern recognition receptors on DCs (i.e., LPS at 0.2 ng/ml and 100 ng/ml). However, none of the tested conditions yielded changes in the expression of surface markers associated with the maturation of MoDCs.

The supernatant was collected to explore cytokine production as a readout, but these measurements have not been done yet. These results are, however, in agreement with those published by Hartholt et al., where no major changes in cytokine production profile were observed (Hartholt et al., 2017). In our case, since MoDCs express both CD32A and Figure 16: FVIII binding to C1q. ELISA plates were coated with human C1q at 10 µg/ml in PBS. FVIII (Advate) was then incubated in serial dilutions (0-100 nM) in borate buffer. Bound FVIII was detected using a biotinylated mouse monoclonal anti-human A2 antibody (GMA-8015), an HRPlabeled streptavidin and TMB substrate. Absorbance was read at 450 nm.

CD32B, the response might be balanced between activating and inhibitory signals.

Measurement of cytokine production levels and blockage of one of the receptors might give us answers and will be performed in the coming weeks.

Interestingly, unpublished results from the lab showed changes in the maturation state of bDCs after co-incubation with inhibitor-positive HA patients' plasma spiked with rFVIII. For these experiments retrospective plasma samples from inhibitor-positive (IP) and inhibitornegative (IN) patients with severe HA obtained from the ABIRISK cohort, as well as plasma from healthy donors (HD) were used. Significant increase in the expression of surface markers (i.e., CD80, CD86, CD40) and secretion of pro-inflammatory cytokines (i.e., IL1-β and TNF-α) reflected the induced maturation of bDCs (Figure 17). Importantly, the plasma contains not only immunoglobulins but also molecules of the complement systems. Whether the results observed are solely associated with the formation of FVIII-IC following the spiking of the plasma with FVIII or are also linked to complement activation or other mechanisms needs further investigation.

• Activation of natural killer cells

Natural killer (NK) cells are a type of innate cytotoxic lymphocyte that mediate anti-tumor and anti-viral responses and represent 5-20% of all circulating lymphocytes in humans [START_REF] Langers | Natural killer cells: role in local tumor growth and metastasis[END_REF]. They do not require prior sensitization or affinity maturation to eliminate their targets. NK cells express inhibitory (e.g., KIR) and activating (e.g., CD16) receptors at their surface, and the balance between the corresponding signals determines the activation status of NK cells. In the context of HA, available data on NK cells date back to the 20 th century when the use of contaminated cryoprecipitates resulted in the transmission of bloodborne pathogens [START_REF] Mannucci | AIDS, hepatitis and hemophilia in the 1980s: memoirs from an insider[END_REF]. In HA patients with the acquired immune deficiency syndrome, circulating levels of T cells, B cells and NK cells were very low and NK cells were nonfunctional [START_REF] Landay | Immunologic studies in asymptomatic hemophilia patients. Relationship to acquired immune deficiency syndrome (AIDS)[END_REF].

NK cells are potent effector cells, interesting from the point of view that they mainly bear the activating CD16 receptor at their surface, a receptor with low to medium affinity for IgG1 Fc and no affinity for IgG4 Fc. Depending on the predominant IgG subclass present in the blood at the time of FVIII administration in the course of ITI, FVIII-IC might have different repercussions on innate and adaptive immune cells. Interestingly, HA patients successfully treated by ITI have very low or undetectable levels of FVIII-specific memory B cells ( [START_REF] Van Helden | Factor VIII-specific memory B cells in patients with hemophilia A[END_REF]. These frequencies are identical to those observed in patients without inhibitors, but they are lower than those seen in HA patients with inhibitors, which range from <0.01 to 0.40% of total IgG+ B cells. We can thus speculate that FVIII-IgG1 immune complexes bind to the BCR on FVIII-specific memory B-cells while the Fc moieties engage with the CD16 receptor on NK cells, leading to NK cells' activation and destruction of target B cells. This might explain the greater probability of ITI success in patients with anti-FVIII IgG1 compared to those with anti-FVIII IgG4. However, one of the proposed mechanisms that prevent NK cells from attacking self-cells is the expression of inhibitory receptors (KIR) specific for self-MHC class I molecules, expressed on almost all nucleated cells. Whether FVIII-IC engagement with CD16 receptors may overcome inhibitory stimuli mediated by KIR receptors, for instance, remains to be determined.

With the help of Sandrine Delignat, we investigated such a possibility based on the approach recently used by Lagassé et al. wherein recombinant FVIIIFc protein was able to activate NK cells and induce the lysis of a FVIII-specific B cell line (BO2C11) (Lagassé et al., 2021). BO2C11 is an Epstein-Barr virus (EBV)-transformed memory B cell line specific for the C2 domain of FVIII generated by Prof. Jacquemin and colleagues from an inhibitor-positive HA patient (Jacquemin et al., 1998). We thus compared the effects of IgG1-and IgG4-containing immune complexes on the activation of CD16-negative (NK92) and CD16-positive (PTA-6967) NK cell lines. These cell lines, however, do not express KIR at their surface. Our preliminary results (shown in Article 3), demonstrate the activation of CD16-expressing NK cells by IgG1-containing FVIII-IC but not by IgG4-containing FVIII-IC, as expected. Activation was validated by increased IFN-γ secretion and increased expression of LAMP1, a marker of NK cell activation and function.

In the next month, I will test in an antibody-dependent cellular cytotoxicity (ADCC) assay the potential of NK cells activated by IgG1-containing FVIII-IC to lyse the FVIII-specific memory B-cell hybridoma, BO2C11. At a late stage, the experiment will be reproduced using patients' plasma in which high anti-FVIII IgG1 or IgG4 levels are detected. These in vitro findings might provide an underlying molecular mechanism explaining at least in part the link between IgG subclass and ITI outcomes.

• FVIII-IC repercussions on FVIII-specific memory B cells FVIII-specific memory B cells represent an additional type of professional APCs and stimulators of CD4+ T cells, the activation and differentiation of which may be altered by the nature of circulating FVIII-IC. In a primed immune system, upon re-exposure to FVIII, these cells will be rapidly re-stimulated to proliferate and differentiate into antibodysecreting plasma cells (ASCs) that secrete high-affinity antibodies. In the context of ITI, ideally, FVIII-specific memory B cells must be eradicated or inactivated for successful therapy in HA patients with FVIII inhibitors. Recent work from Georgescu et al.

demonstrated that the cross-linking by a therapeutic Fc-fused FVIII of the FVIII-specific Bcell receptors (BCR) and of the inhibitor FcγRIIB (CD32B) at the surface of FVIII-specific memory B cells triggered inhibitory signaling [START_REF] Georgescu | Recombinant Factor VIII Fc Inhibits B Cell Activation via Engagement of the FcγRIIB Receptor[END_REF]. The latter are splenic B-cell hybridomas obtained from FVIII-exposed HA mice [START_REF] Healey | The humoral response to human factor VIII in hemophilia A mice[END_REF].

This is in agreement with the seminal finding that administration of high dose FVIII (supraphysiological) to inhibitor-positive FVIII-KO mice, which presumably leads to the formation of FVIII-IC, irreversibly prevented the differentiation of memory B cells into ASCs by inducing B-cell apoptosis [START_REF] Hausl | High-dose factor VIII inhibits factor VIII-specific memory B cells in hemophilia A with factor VIII inhibitors[END_REF]. However, the opposite effect was observed with physiological (0.1 µg/ml) or lower doses of FVIII. More recently, FVIII-IC were found to be unable to promote the differentiation of mouse FVIII-specific memory B cells into ASCs upon FVIII restimulation, irrespective of FVIII dose, in CD32B knock-out mice [START_REF] Werwitzke | Deletion or inhibition of Fc gamma receptor 2B (CD32) prevents FVIII-specific activation of memory B cells in vitro[END_REF]. This was explained by the fact that when CD32B is depleted or blocked with an anti-CD32B antagonist, the inhibitory signal transmitted by FVIII-IC is lacking thus FVIII-specific T cell stimulation and ASC formation are suppressed [START_REF] Vollack | Anti-FcγRIIB (CD32) Antibodies Differentially Modulate Murine FVIII-Specific Recall Response in vitro[END_REF]. FVIII-IC formation was validated in the latter worker but variable levels were observed between mice. Although caution is needed to extrapolate data from mice to humans, we can speculate the same scenario in HA patients, wherein some will develop FVIII-IC enhancing ASCs while others will not, thus suppressing ASC formation.

In an attempt to reconcile these findings, one of my initial objectives was to study the effect of the synthetic FVIII-IC with different epitope specificities and IgG subclasses on the activation and differentiation of human FVIII-specific memory B cells. My preliminary results suggested that FVIII-IC containing IgG1 or IgG4 antibodies triggered equivalent signaling levels through human FcγRIIB-expressing cell lines (see Article 3).

Studying the effect of FVIII-IC on human FVIII-specific memory B cells from HA patients is particularly challenging owing to the scarcity of these cells in blood (from <0.01 to 0.40% of total IgG+ B cells) (van Helden et al., 2010) and difficulty to collect large volumes of blood from the patients. The only FVIII-specific human memory B cell line available in our lab is the previously described EBV-immortalized BO2C11. These cells are, therefore, not appropriate to study the activation of the cells and their differentiation into ASCs. For my Ph.D. work, I had decided to exploit a recently described technology to generate FVIIIspecific human memory B cells. In collaboration with Prof. François-Loïc Cosset (INSERM U1111, ENS Lyon, France), I have cloned in the FAM0 lentiviral vector the sequences of the light and heavy variable chains (VL and VH) from two human anti-FVIII IgG, the anti-C2 domain IgG BO2C11 and the anti-A2-domain IgG BOIIB2, upstream of the human κ chain constant and secretory Fcγ1 constant regions (CH1-CH2-CH3), respectively. In the FAM0 vector, the variable and constant domains are linked via the F2A/furin peptide and subcloned downstream of the FEEK promoter [START_REF] Fusil | A Lentiviral Vector Allowing Physiologically Regulated Membrane-anchored and Secreted Antibody Expression Depending on B-cell Maturation Status[END_REF]. The CH3 domain is linked to the transmembrane and cytoplasmic domains M1 and M2 of the human IgG1 BCR (Figure 18). The vector thus encodes the membrane-anchored form of the BO2C11 or BOIIB2 IgG1. Lentiviral vectors were generated by the team in Lyon by transient transfection of 293T cells using an envelope plasmid, a Gag-Pol packaging plasmid and FAM0 vector encoding BO2C11 or BOIIB2. My plan was to join the lab in Lyon to perform the transduction of naïve B cells and validate the expression and functionality of the transgenic BCR. However, the covid pandemic resulted in a halt in this aspect of my research project. In the future, the aim would be to investigate the effect of FVIII-IC loaded DCs and FVIII-specific memory B cells on the polarization of human FVIII-specific naïve CD4+ T cells. [START_REF] Fusil | A Lentiviral Vector Allowing Physiologically Regulated Membrane-anchored and Secreted Antibody Expression Depending on B-cell Maturation Status[END_REF].

IdeS: a new potent tool to eradicate FVIII inhibitors

Interest in using IdeS in HA setting

IdeS has been used in a series of studies that showed rapid and efficient, though temporary, cleavage of IgG in different preclinical and clinical settings of IgG-mediated pathogenic conditions (Jordan et al., 2017;Kizlik-Masson et al., 2019;Leborgne et al., 2020). A recombinant IdeS is commercially available (Imlifidase, Ideferix®) and is the only desensitization treatment approved by the EMA for kidney transplant patients with donorspecific antibodies (Al-Salama, 2020). IdeS is currently being studied for its therapeutic potential in several autoimmune diseases, as well as in oncology and gene therapy. Thus, I thought of exploring the potential use of IdeS to eradicate FVIII inhibitors that develop in congenital (HA) and acquired HA (AHA).

FVIII inhibitors are polyclonal IgG that mainly belong to the IgG1 and IgG4 subclasses and are directed to different domains of the FVIII (Whelan et al., 2013;Hofbauer et al., 2015). They expose patients to high risk morbidity and mortality. The only efficient strategies to eliminate these inhibitors are ITI for HA patients and immunosuppressive therapy for AHA patients. Unfortunately, ITI is not efficient in 20-40% of the cases (DiMichele et al., 2007) in addition to being a very expensive and demanding therapy.

Furthermore, immunosuppression in AHA patients is accompanied by a high risk of serious and fatal infections (Borg et al., 2013;Tiede et al., 2020). Thus, a better option for inhibitor eradication is of clinical interest. To prevent and treat bleeds in patients with hightiter inhibitors, BPA (i.e., rFVIIa and aPCC) or recombinant porcine FVIII (for AHA patients), are recommended. However, 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.

The development of emicizumab has revolutionized prophylaxis for HA patients with inhibitors (Shima et al., 2016). However, emicizumab cannot prevent all bleeds, thus standard hemostatic treatments are still required for patients undergoing breakthrough bleeds or surgery: FVIII (for patients with low-titer inhibitors) or BPA (for patients with hightiter inhibitors). The latter combination leads to potential thrombotic events (Oldenburg et al., 2017). Moreover, no antidote has yet been proposed for emicizumab. This thesis work brought the proof of concept for the efficient transient removal of FVIII inhibitors by IdeS, thereby opening a therapeutic window for efficient FVIII replacement therapy in inhibitorpositive patients.

Mouse model used

To

Importance of antigen location in determining functional consequences of IdeS

A critical point in HA or AHA disorders is that pathogenic IgG are directed toward a soluble antigen. This is different from all the other disease settings in which IdeS treatment was proven efficient, except for ADAMTS13, wherein target antigens are exposed at the surface of cells, platelets or viruses. Indeed, the functional consequence of IdeS-mediated cleavage depends on whether the pathogenic IgG is targeting a soluble or membranebound antigen. This is explained by the fact that IdeS cleaves soluble or antigen-bound IgG and transmembrane IgG-type BCR in a two-step process. A rapid first cleavage in the lower hinge of one heavy chain results in an intermediate scIgG. The latter shows impaired Fcγ-mediated effector functions (i.e., ADCP, ADCC and CDC) but no impact on antigen binding and has a similar half-life as intact IgG due to the maintained binding to the FcRn (Brezski et al., 2009). The following cleavage on the second heavy chain is slower and results in the complete dissociation of F(ab)'2 and Fc fragments. F(ab′)2 fragments have no Fcγ-mediated effector functions and a decreased half-life relative to intact IgG since they can no longer interact with FcRn. However, they retain the ability to bind to antigens and, potentially, neutralize them throughout their lifespan in circulation. Therefore, when the pathogenic IgG are directed against membrane antigens and exercise their pathogenic effects by complement activation, phagocytosis or ADCC, IdeS-mediated IgG cleavage has an immediate functional consequence (Brezski et al., 2009;Kizlik-Masson et al., 2019). In contrast, the functional consequence of IdeS-mediated cleavage is postponed until the removal of the F(ab')2 fragments from the circulation when pathogenic IgG neutralize soluble antigens, as in the case of neutralizing anti-FVIII IgG. This explains why I had to wait at least 2-3 days before treating with FVIII the passively immunized inhibitor-positive HA mice, which received an IdeS dose. In the experiments I performed, I found a 12-hour half-life of F(ab')2 fragments, consistent with previous findings (Sehlin et al., 2017). The complete disappearance of the FVIII inhibitory titers, related to remaining F(ab')2 fragments, from the plasma of mice with very high titer inhibitors (i.e., 200 BU/ml) required a longer time after dosing with IdeS (~6 days). However, inhibitory titers <5 BU/ml were reached 3-4 days after dosing IdeS twice within 24 hours, which is compatible with FVIII treatment as can be performed in patients with lower-titer inhibitors.

Future directions (scaling-up to patients)

• Other anti-FVIII Ig subclasses

Although the IgG isotype predominates the anti-FVIII immune response in HA and AHA patients, other Ig isotypes (i.e., IgA, IgM and IgE) can be found in 3-10% and 8-36%, respectively (Bonnefoy et al., 2021;Whelan et al., 2013). The importance of the latter isotypes in FVIII neutralization in vivo is uncertain. Nevertheless, their presence may render a number of inhibitor-positive patients ineligible for IdeS therapy. Therefore, neutralizing Ig isotypes may need to be pre-screened in advance to determine IdeS treatment eligibility.

• Interest of IdeS for AHA patients

The only efficient strategy to eradicate FVIII inhibitors in most AHA patients is immunosuppressive therapy. ITI is not used in these individuals, unless first-line hemostatic therapies have failed in patients with life-threatening bleeding, because its efficacy in combination with immunosuppressive drugs is unclear (Tiede et al., 2020).

Since patients with AHA are typically older and have more concomitant conditions than those with congenital HA, there are also questions concerning the safety and effectiveness of emicizumab in these patients (Makris et al., 2019). Therefore, by administering IdeS to AHA patients who need hemostatic treatment, the use of BPA and the thrombotic and infectious risk it carries could be reduced, and the effectiveness of FVIII treatment and monitoring restored. Based on my in vivo results, redosing IdeS 24 hours after a first dose, as described in other pathologies (Jordan et al., 2021), could be recommended for patients with very high titer inhibitors. The use of IdeS as an immediate first-line therapy may be complementary to the use of immunosuppressive drugs (i.e., corticosteroids, cyclophosphamide) to remove the inhibitors for a longer time period.

• Cleavage of emicizumab: a potential risk or benefit for HA patients?

In inhibitor-positive HA patients under emicizumab prophylaxis, IdeS administration would presumably cleave both the drug and the neutralizing anti-FVIII IgG, though with different kinetics (see Article 2). Though emicizumab cleavage is slower than that of a recombinant anti-FVIII IgG, its corresponding F(ab)'2 fragments are eliminated faster from mice circulation. As mentioned previously, emicizumab cannot prevent all bleeds and therefore BPA must be used for the management of emergency situations. One might think that eliminating emicizumab is more risky than beneficial. However, in inhibitor-positive patients, the combined elimination of emicizumab and anti-FVIII IgG is beneficial in terms of: i) using high-dose BPA to control acute bleeds in the first 2-3 days without thrombotic risk ii) efficiently using FVIII replacement therapy afterwards once anti-FVIII F(ab')2

fragments have been eliminated and iii) ensuring correct FVIII:C measurement by removing the biological interference brought in by emicizumab. Furthermore, due to limited experience and a lack of guidelines, the management of surgeries in HA receiving emicizumab prophylaxis is still up for debate [START_REF] Lewandowska | Management of people with haemophilia A undergoing surgery while receiving emicizumab prophylaxis: Real-world experience from a large comprehensive treatment centre in the US[END_REF][START_REF] Kruse-Jarres | Surgical outcomes in people with hemophilia A taking emicizumab prophylaxis: experience from the HAVEN 1-4 studies[END_REF]. It is further complicated in inhibitor-positive patients with inconsistent clinical responses to rFVIIa. For these patients, IdeS would offer a brief but valuable therapeutic window free of inhibitors for a better management of emergency situations. Importantly, once the risk is over, emicizumab prophylaxis could be quickly resumed since IdeS is rapidly cleared from human circulation (half-life of 5 hours).

• Interest of IdeS for ITI therapy

According to the recently published experience of Mahlangu et al., treating bleeds with FVIII in inhibitor-negative patients on emicizumab did not induce any thrombotic complication [START_REF] Mahlangu | Emicizumab state-of-the-art update[END_REF]. This suggests that treating bleeds with FVIII in patients on emicizumab, in which inhibitors are absent or have been efficiently eradicated, is likely to be both safer and more convenient than treating bleeds with current BPA in patients on emicizumab whose inhibitors persist. Therefore, we might think that injecting IdeS to quickly eradicate inhibitors before starting ITI with a combination of emicizumab and FVIII might be beneficial. Moreover, though the consequences of IdeS-cleaved BCR on the functions of B cells in vivo are still unclear, the temporary blockade of memory Bcell signaling seen in vitro may lead to a damped memory response (Järnum et al., 2017).

This should be beneficial for tolerance induction. One may wonder whether administering IdeS over time could keep IgG degraded while also dampening the IgG memory B cell response. However, we need to keep in mind that IgM, IgA, and IgE B cells would be unaffected.

• Potential toxicity following IdeS treatment

One concern in IdeS treatment of HA patients is the potential infections that might arise when the whole IgG pool, and not only FVIII-specific IgG, are depleted. However, in previous clinical studies, IdeS was generally well-tolerated, and no serious adverse events were observed (most common side effects were mild and self-limiting). The only study reporting major adverse events is the one in the setting of immune thrombotic thrombocytopenic purpura (iTTP) [START_REF] Stubbs | Administration of immunoglobulin Gdegrading enzyme of Streptococcus pyogenes (IdeS) for persistent anti-ADAMTS13 antibodies in patients with thrombotic thrombocytopenic purpura in clinical remission[END_REF], however, this study has several limitations that were discussed in the introduction part of the present thesis report (see chapter II paragraph 4).

Antihistamines and corticosteroids have been used prophylactically in some studies to prevent infusion reactions, though uncommon. Particularly in patients undergoing renal transplantation and receiving immunosuppressive drugs, antibiotics were administered as prevention to the anticipated risk of bacterial infections; however, no serious adverse events were reported. This might be explained by the persistence of IgM and IgA isotypes that are not affected by IdeS. Therefore, the degree of immunosuppression could be less drastic than that of therapies that globally suppress immunoglobulins (e.g., rituximab).

Moreover, IVIg has been used to replenish sufficient amounts of IgG and restore humoral immunity, with a recommended 12-hour delay between the administration of IdeS and the subsequent IVIg administration (Huang et al., 2022). Furthermore, the depletion of endogenous IgG induced by IdeS is only temporary since the half-life of IdeS in humans was estimated to be around 5 hours and the endogenous IgG pool reappear within 1-2 weeks (Winstedt et al., 2015;[START_REF] Lorant | Safety, immunogenicity, pharmacokinetics, and efficacy of degradation of anti-HLA antibodies by IdeS (imlifidase) in chronic kidney disease patients[END_REF].

• Immunogenicity of IdeS (Repetitive dosing / chronic use)

Since S. pyogenes is a pathogen that only infects humans, it is not surprising that preexisting anti-IdeS IgG may be found in individuals prior to administration. These individuals might have been pre-exposed to IdeS during prior streptococcal infections, therefore mounting a memory response against IdeS would be expected. This is consistent with a typical recall immune response wherein substantial levels of anti-IdeS IgG develop in individuals after IdeS dosing within a couple of days. These antibodies might increase the risk of infusion reactions or reduce the efficacy of IdeS if subsequent doses are required. However, to my knowledge, the potentially neutralizing capacity of anti-IdeS IgG has never been assessed. Moreover, the development or presence of other Ig isotypes (i.e., IgM, IgA, and IgE) has not been considered, except in the study by Lorant et al., where no anti-IdeS IgE were detected [START_REF] Lorant | Safety, immunogenicity, pharmacokinetics, and efficacy of degradation of anti-HLA antibodies by IdeS (imlifidase) in chronic kidney disease patients[END_REF]. Part of this concern may stem from experience with other bacterial enzymes, like streptokinase, for which antistreptokinase antibodies can drastically reduce the activity of the enzyme following repeated dosing [START_REF] Buchalter | Are streptokinase antibodies clinically important?[END_REF]. However, infusion side effects are typically minor and do not prevent repeated dosing.

Furthermore, redosing IdeS after 40 days in rabbits has been accomplished successfully without detectable infusion responses or activity loss (Johansson et al., 2008). Also, despite a wide possible range of anti-IdeS antibodies at baseline, IdeS was always effective in depleting IgG in patients and healthy individuals. Interestingly, the anti-IdeS IgG levels decreased below the detection limit immediately after dosing due to the cleavage of anti-IdeS IgG by IdeS itself. The anti-IgG levels start to rise on day 7 and peak on day 14, after which they return to their baseline levels 6 to 12 months later (Winstedt et al., 2015). The anti-IdeS IgG levels were not significantly different between patients that received one or two doses of IdeS within 48 hours [START_REF] Lorant | Safety, immunogenicity, pharmacokinetics, and efficacy of degradation of anti-HLA antibodies by IdeS (imlifidase) in chronic kidney disease patients[END_REF]. Therefore, with our current knowledge, redosing after the 6-12 months' time frame should be possible but needs more investigation.

Another very interesting point is that IdeS cleaves, with the same efficacy as circulating IgG, IgG-type BCR at the surface of CD19+ B cells. In patients with chronic kidney disease, BCR expression by circulating B lymphocytes was decreased for at least 3 days following IdeS injection [START_REF] Lorant | Safety, immunogenicity, pharmacokinetics, and efficacy of degradation of anti-HLA antibodies by IdeS (imlifidase) in chronic kidney disease patients[END_REF]. IdeS has also been shown to temporarily silence memory B cells signaling, at least in vitro (Järnum et al., 2017). Whether IdeS can as well silence IdeS-specific memory B cells is unknown.

• Next generation enzyme

Imlifidase may not be appropriate for chronic conditions like HA that require repeated injections due to its intrinsic immunogenicity. Therefore, developing an enzyme with a longer half-life and/or lower immunogenicity is needed. These variations may widen the therapeutic window and, more importantly, allow the use of a modified IdeS in a variety of conditions where repeated dosing is advantageous, including hemophilia A, autoimmune conditions where patients may experience flares or post-transplant, as well as a number of gene therapy and oncology indications. Hansa Biopharma is currently developing IgGcleaving enzymes under the NiceR initiative that are less likely to trigger immunity, but no results have been made public yet (hansabiopharma.com/nicer). In our lab, I have helped Victoria Daventure to check whether we could extend the half-life of IdeS by coupling it to the Fc portion of an IgG1. This was done using the electrostatic steering approach by introducing mutations that install opposite charges at the CH3/CH3 interface (DD/KK) favoring heterodimerization. With this approach, we could increase IdeS half-life in mice by ~6 fold (Figure 19). Further experiments are ongoing to see whether we can further extend the half-life and whether with we can prolong IgG depletion.

Reconnaissance du FVIII endogène par les lymphocytes T en conditions physiologiques

La présence de LT CD4 + anti-FVIII dans le sang d'individus sains a été suggérée par la capacité de ces cellules à proliférer in vitro en présence de FVIII ou de peptides couvrant les différents domaines de la molécule [15,16]. Les LT CD4 + spécifiques du FVIII ont été récemment quantifiés avec précision chez des individus sains par le groupe de Bernard Maillère (CEA, Saclay) : la fréquence de cellules spécifiques du FVIII chez un individu sain est de 2 cellules par million de LT CD4 + [17] ; cette proportion est proche de celle des cellules spécifiques de protéines exogènes, telles que l'ovalbumine ou l'hémocyanine (respectivement 1,3 l'un des TLR, le TLR-2 [3], et n'induit pas la maturation de cellules dendritiques humaines immatures in vitro [4]. Des travaux ont, par ailleurs, suggéré que le FVIII, dont le rôle dans la cascade de la coagulation est d'induire une explosion de génération de thrombine, permettrait l'activation indirecte des récepteurs activés par les protéases (proteinase-activated receptors, PAR), via la thrombine alors produite en excès, récepteurs qui, à leur tour, induiraient la libération de cytokines et de chimiokines pro-inflammatoires [5]. Ces données ont cependant été contredites par des études plus récentes utilisant un mutant de FVIII, portant la mutation V634M qui génère une protéine inactive. Le mutant V634M, non-actif, présente une activité immunogène similaire à celle du FVIII sauvage chez les souris FVIII-KO : la génération de thrombine induite par le FVIII comme signal de danger, n'apparaît donc pas suffisante pour justifier de l'immunogénicité du FVIII [6]. Des résultats similaires ont également été obtenus dans des expériences où l'activation de la voie extrinsèque de la coagulation avait été altérée [7]. L'incapacité du FVIII à activer directement ou indirectement le système immunitaire suggère donc qu'il n'est pas impliqué dans la génération des signaux de danger à l'origine de la réponse immunitaire primaire nécessaire à la formation des anticorps.

Statut inflammatoire des patients

Les infections, les actes chirurgicaux ou les vaccinations qui peuvent être concomitants à l'injection du FVIII thérapeutique chez les patients ont souvent été évoqués comme des évènements susceptibles de générer des signaux de danger capables d'activer la réponse immunitaire contre le FVIII. Cependant, des expériences récentes démontrent que l'exposition de souris FVIII-KO au vaccin vivant atténué contre la rougeole, les oreillons et la rubéole, au moment de l'administration du FVIII, n'a aucune influence sur l'incidence ou l'intensité de la réponse immunitaire anti-FVIII [8]. Ces données expérimentales ont été confirmées par des observations cliniques rétrospectives (registre PEDNET) montrant que la vaccination pédiatrique réalisée de façon concomitante à l'injection du FVIII thérapeutique n'est pas associée à une incidence accrue d'allo-immunisation contre le FVIII [9]. L'influence de l'inflammation sur la réponse immunitaire anti-FVIII a également été examinée. Chez la souris FVIII-KO, une inflammation chronique, provoquée par une hémorragie récurrente [10] ou par une hémarthrose aiguë induite par une blessure [11], n'augmente pas la réponse immunitaire contre du FVIII exogène administré. Ces observations obtenues dans un modèle pré-clinique trouvent, encore une fois, un écho chez les patients atteints d'HA, mais de manière indirecte. En effet, l'administration de FVIII thérapeutique à distance d'évènements à l'origine d'une réponse inflammatoire (vaccinations, infections virales ou bactériennes, chirurgie) à des patients HA n'ayant jamais été exposés auparavant au FVIII (previously untreated patients, ou PUPs) est associée à une incidence d'inhibiteurs du FVIII de l'ordre de 40 %3 [12,13].

n'exprimant aucun FVIII:Ag circulant, présentent la plus forte incidence de développement d'inhibiteurs [28] [24]. Bien que le risque de développement d'inhibiteurs chez les patients atteints d'HA sévère soit plus élevé au cours des 20 premiers jours cumulés d'exposition (2-3 premières années de vie) [30], l'incidence des inhibiteurs du FVIII présente un pic à un âge avancé [31]. Incidemment, le grand âge est caractérisé par un affaiblissement des mécanismes de tolérance immunitaire et le risque de développer des manifestations autoimmunes s'accroît [32]. La survenue de ce pic et 20 cellules/million de cellules T CD4 + ) [18]. Elle est en revanche 10 fois plus élevée que celle des LT spécifiques de protéines thérapeutiques immunogènes comme l'adalimumab, le rituximab, l'infliximab ou l'érythropoïétine (0,1 à 1 cellule/million de cellules T CD4 + ) [18,19]. Ces données suggèrent l'existence d'une sélection négative inefficace des LT qui reconnaissent le FVIII endogène, et cela malgré l'expression d'ARN messagers codant le FVIII par les cellules épithéliales thymiques médullaires qui expriment cet autoantigène [20]. La moitié des LT CD4 + anti-FVIII détectés dans le sang des individus sains ont un phénotype de cellules mémoires [17]. Ceci suggère l'existence d'une réponse immunitaire contre le FVIII endogène chez les individus en conditions physiologiques, mais aussi une régulation concomitante destinée à éviter une auto-réactivité qui pourrait être délétère. La présence simultanée de lymphocytes Treg et de LT réactifs au FVIII avait été suggérée dans deux études indépendantes, même si la spécificité des Treg pour le FVIII n'avait pas été démontrée [21,22].

Reconnaissance du FVIII endogène par les immunoglobulines en conditions physiologiques

La présence d'IgG naturelles (ne provenant pas d'une immunisation délibérée) anti-FVIII dans le sang de donneurs sains a été décrite pour la première fois par le groupe de Michel Kazatchkine [23]. Le groupe de Birgit Reipert a récemment confirmé la présence de ces anticorps chez environ 19 % des donneurs sains, des anticorps majoritairement d'isotypes IgG1, IgG3 et IgA, et présentant de faibles affinités de liaison au FVIII, avec des constantes d'affinité à l'équilibre de l'ordre du nanomolaire [24,25]. La reconnaissance physiologique du FVIII endogène par les immunoglobulines est associée à une régulation qui implique des anticorps anti-idiotypiques [26,27]

SUMMARY

Origin and nature of the neutralizing immune response against therapeutic factor VIII The use of therapeutic proteins induces in some patients the appearance of neutralizing antibodies. This is the case of pro-coagulant factor VIII (FVIII) used in patients with hemophilia A. Several parameters related to the protein itself, to the type of pathology or to the patients, condition the immunogenicity of a therapeutic protein. Understanding these parameters would help to anticipate or prevent the development of neutralizing antibodies. In the case of FVIII, we propose that the development of neutralizing antibodies does not result from an unpredicted immune response but rather from the inability of the patient's organism to develop an anti-inflammatory or regulatory response. ‡ I ntravenous and subcutaneous immunoglobulin (IVIG/SCIG) are the therapeutic normal IgG preparations obtained from the pooled plasma of many thousands of healthy donors. In addition to its application in the replacement therapy of primary and secondary immune deficiencies, high-dose (1-2 g/kg) IVIG is used for the immunotherapy of large number of systemic autoimmune and inflammatory diseases like immune thrombocytopenic purpura, Kawasaki disease, Guillain-Barré syndrome, transplantation, inflammatory myopathies, and many others. In addition, IVIG therapy has been explored over 100 different pathologies as an off-label manner 1,2 .

Based on the studies performed both in human and experimental models, several mechanisms have been proposed for IVIG. The current evidence suggests that IVIG suppresses the activation and function of various innate immune cells (like dendritic cells (DCs), neutrophils, monocytes, macrophages) and effector T and B cells. On the other hand, IVIG promotes the anti-inflammatory processes such as enhancing the tolerogenic properties of antigen presenting cells, increasing the secretion of anti-inflammatory molecules like IL-1RA and IL-10, and expansion of regulatory T cells (Tregs) 3-6 . The signaling pathways implicated in anti-inflammatory actions of IVIG are however not completely known and are the subjects of intense research.

β-catenin, the central component of the canonical Wnt signaling pathway plays a major role in balancing the immune aggression verses tolerance 7-13 . In addition to nonimmune cells, β-catenin is expressed in all immune cells including DCs, macrophages and T cells, and regulate their functions 10,14-16 . In fact, conditioning of DCs with Wnt ligands promotes the expansion of Tregs and limit the expansion of Th1/Th17 cells 12,17 . In experimental autoimmune encephalomyelitis (EAE) model, lack of βcatenin in DCs leads to increased severity of the disease 12,17,18 . Conversely, treatment with β-catenin agonists protects the mice from EAE with reduced frequency of IFN-γ and IL-17-expressing cells 12 .

It is interesting to note that IVIG has been reported to suppress the inflammatory cytokines in DCs and reciprocally regulate Tregs and effector Th1/Th17 responses in vitro, in vivo in EAE model, and in treated autoimmune patients 19-33 . In view of reports on the central role of β-catenin/Wnt pathway in mediating these anti-inflammatory effects, we hypothesized that IVIG induces the activation of β-catenin/Wnt pathway to exert antiinflammatory effects.

In the present report, we show that IVIG indeed activates the β-catenin pathway along with Wnt5a secretion in human DCs in a sialylation independent manner. Mechanistically, β-catenin activation by IVIG requires low-density lipoprotein receptor related proteins (LRP) 5 and 6 co-receptors but FcγRII and Syk pathway are not implicated. However, despite induction of βcatenin/Wnt signaling, we found that this pathway is dispensable for the anti-inflammatory action of IVIG both in vitro, and in vivo in mediating the protection against EAE in mice and reciprocal regulation of effector Th17/Th1 and Tregs.

Results

IVIG stimulates β-catenin signaling in DCs. Glycogen synthase kinase-3 beta (GSK-3β) acts as a negative regulator of β-catenin pathway by phosphorylating β-catenin at Ser33, Ser37 and Thr41, and directing it to proteasomal degradation 34 . Nonphosphorylated forms of β-catenin are functionally active and translocate to nucleus and regulate the expression of target genes. In view of critical role of DCs in mediating both immune response and tolerance, we first investigated whether IVIG activates β-catenin signaling in these innate immune cells. Monocytederived DCs were treated with 25 mg/ml of IVIG (equivalent of concentrations of infused IgG reaches in the blood of autoimmune patients immediately following IVIG immunotherapy) for 24 h and activation of β-catenin signaling was analyzed by immunoblotting for active form of β-catenin (non-phospho βcatenin).

We found that IVIG significantly increased the levels of active β-catenin, which is not phosphorylated at Ser33, Ser37, and Thr41 residues (Fig. 1a). On the other hand, equimolar concentrations of human serum albumin (HSA), an irrelevant protein control for IVIG, did not alter the basal levels of active βcatenin and were similar to untreated cells.

We have also assessed the levels of triple phosphorylated βcatenin (Ser33, Ser37, and Thr41). Consistent with the data on active β-catenin (Fig. 1a), IVIG significantly decreased the phospho-β-catenin (Fig. 1b). The effect of IVIG was dosedependent. Furthermore, IVIG promoted the translocation of βcatenin into the nucleus, thus validating the activation of βcatenin pathway in IVIG-treated DCs (Fig. 1c).

We explored if increase in the levels of active β-catenin by IVIG was due to enhancement in the β-catenin levels. For this purpose, we first performed CTNNB1 gene (that encodes βcatenin) expression analyses by quantitative reverse transcriptionpolymerase chain reaction (RT-PCR) and found that IVIG significantly enhanced the CTNNB1 mRNA in DCs (Fig. 1d). However, analyses of β-catenin protein by western blot did not reveal significant differences between various experimental conditions (Fig. 1e). These data thus imply that IVIG-mediated increase in the levels of active β-catenin is not because of enhancement in the β-catenin protein levels.

As GSK-3β negatively regulates active β-catenin, we wondered if the positive effect of IVIG on β-catenin is associated with concomitant inhibition of GSK-3β. Of note, GSK-3β protein was significantly downregulated by IVIG (Fig. 2), thus signifying that IVIG-induced activation of β-catenin is coupled with reduced GSK-3β levels. Together, these results show that IVIG activates βcatenin signaling in human DCs.

IVIG activates β-catenin in DCs in an IgG-sialylation independent manner. We then explored the mechanisms of activation of β-catenin in DCs by IVIG. Several reports have shown the importance of terminal α- (2,6) sialic acid linkages of Fc region in mediating the anti-inflammatory actions of IVIG 31,35-37 . Therefore, we examined if IVIG-induced activation of β-catenin is dependent on the sialylation content of IgG. IVIG was desialylated by treatment with recombinant neuraminidase. The sialylation content in neuraminidase-treated IVIG was <2.5 μg/25 mg of IgG as assessed by reverse phase high performance liquid chromatography 38,39 . Lectin-blot analyses also confirmed the desialylation of IVIG 38 . By using desialylated IVIG, we found that IVIG-induced activation of β-catenin in DCs is independent of sialylation status of IVIG (Fig. 3). In fact, desialylated IVIGinduced activation of β-catenin similar to that of native IVIG.

β-catenin activation by IVIG does not imply C-type lectin and FcγRIIA receptors. C-type lectin receptors like Dectin-1 and FcγR-mediated signaling have been reported to induce β-catenin activation and its stabilization 40,41 . To investigate if IVIG induces β-catenin activation via C-type lectin receptors, we treated the DCs with Ca 2+ chelating agent ethylenediaminetetraacetic acid (EDTA) followed by IVIG treatment. However, IVIG-induced βcatenin activation remained intact upon treatment of DCs with EDTA (Fig. 4a), implying that IVIG stimulates β-catenin activation in a Ca 2+ -independent manner.

Monocyte-derived DCs mainly express FcγRII, while FcγRIII and FcγRI are either absent or marginal 42,43 . However, FcγRIIA-blockade with monoclonal antibodies did not affect IVIG-induced β-catenin activation in DCs (Fig. 4b). As C-type lectin receptors and activating FcγR mediate signaling via Syk pathway 44,45 , to rule out the implication of both the receptors, we employed Syk inhibitor in the experiments. DCs were pre-treated with Syk inhibitor followed by culture with IVIG. Consistent with the previous results, Syk inhibition had no effect on the ability of IVIG to induce β-catenin activation (Fig. 4c).

Intact IgG is mandatory for the β-catenin activation by IVIG.

We next aimed at dissecting whether induction of β-catenin activation by IVIG implicates F(ab′) 2 or Fc fragments. DCs were treated with IVIG (25 mg/ml) and equimolar concentrations of F (ab′) 2 or Fc fragments. In line with the data on the lack of implications of sialylation of IgG, Syk and FcγRIIA in mediating β-catenin activation by IVIG, Fc fragments of IVIG failed to induce β-catenin activation (Fig. 5a). These results raised the prospect that IVIG might induce βcatenin activation via F(ab′) 2 fragments. However, we did not observe β-catenin activation even with F(ab′) 2 fragments of IVIG (Fig. 5b). Together our data indicate that intact IgG is mandatory for the β-catenin activation by IVIG.

Wnt5a and LRP5/6 interaction is critical for β-catenin activation by IVIG. We then explored if Wnt-receptor interaction is required for the activation of β-catenin by IVIG. Signaling by Wnt ligands upon binding to transmembrane Frizzled (Fz) receptor and its co-receptors, LRP5/6 leads to the transduction of signals and accumulation of β-catenin in the cytoplasm by preventing GSK-3β-mediated phosphorylation and proteosomal degradation of β-catenin. Eventually, β-catenin translocate into the nucleus to activate Wnt target genes (canonical Wnt/β-catenin pathway) 8,9,14,46,47 .

We first screened for the expression of canonical ligands by quantitative RT-PCR. Levels of mRNA for canonical ligands like WNT3A, WNT1, and WNT10A were significantly upregulated upon 12 h of IVIG treatment of DCs (Fig. 6a).

Wnt ligands are also known to mediate noncanonical signaling pathway independent of β-catenin to regulate the cytoskeleton and intracellular calcium levels 8,48 . We hence investigated the expression of several non-canonical ligands-induced in IVIGtreated DCs. Interestingly, IVIG also enhanced the expression of several Wnt ligands implicated in non-canonical pathway like WNT5A, WNT7A, and WNT7B (Fig. 6b) though significant expression was observed only with WNT5A.

These data encouraged us to assess the secretion of prototype canonical ligand protein Wnt3a and noncanonical ligand protein Wnt5a in the culture supernatants. In contrast to transcript analyses, however, Wnt3a was not secreted in IVIGtreated DCs. Only Wnt5a was significantly secreted by these DCs (over 10 ng/ml) (Fig. 6c). These results thus highlight the possible involvement of noncanonical Wnt5a in the IVIGinduced activation of β-catenin signaling in human DCs.

Though Wnt5a is typically associated with noncanonical Wnt signaling, it could activate Wnt/β-catenin pathway in the presence of Fz4 and LRP5 49,50 . Therefore, to unequivocally prove that IVIG triggered β-catenin activation in DCs through LRP5mediated signaling of Wnt5a, we resorted to silence LRP5 gene by siRNA method. Although LRP6 might not be binding to Wnt5a, to prevent the binding of other canonical Wnt ligands, we decided to knock-down both LRP5 and 6 co-receptor genes to ensure complete inhibition of β-catenin activation signals. In line with our hypothesis, the ability of IVIG to activate β-catenin was compromised when DCs are treated with siRNA against LRP5/6 (Fig. 7).

Together, these data imply that IVIG-induced activation of βcatenin pathway in DCs is dependent on the Wnt5a-LRP5/6 coreceptor interaction.

β-catenin is dispensable for the anti inflammatory effects of IVIG on DCs. The critical question was whether β-catenin signaling is important for the anti-inflammatory actions of IVIG. We addressed this by using FH535, a small molecule inhibitor of Wnt/β-catenin signaling 51 . We first examined the inhibitory effect of FH535 on the levels of IVIG-induced active β-catenin (non-phospho β-catenin) in DCs. As shown in Fig. 8a, FH535 significantly reduced the levels of active β-catenin thus confirming that FH535 is functional 52 . Previous reports have shown that β-catenin pathway regulates the inflammatory cytokine response in toll-like receptor 4 (TLR4)-activated DCs 53,54 . Therefore, by using TLR4-activated DCs (lipopolysaccharide, LPS stimulation), we investigated if βcatenin signaling is implicated in the anti-inflammatory actions of IVIG. As depicted in Fig. 8b, IVIG reduced the production of IL-6 and IL-8 in activated DCs, thus validating the suppressive effects of IVIG on DCs 19 . However, inhibition of Wnt/β-catenin signaling by FH535 had no repercussion on the inhibitory actions of IVIG on the production of these inflammatory cytokines. These results suggest that β-catenin though induced by IVIG, this pathway is dispensable for the anti-inflammatory action of IVIG.

β-catenin is dispensable for the anti inflammatory effects of IVIG in vivo. To validate the role of β-catenin signaling in the anti-inflammatory actions of IVIG in vivo, we resorted to EAE model. Previous reports have shown that IVIG protects the mice from the disease and is associated with the peripheral expansion of Tregs and suppression of pathogenic Th1 and Th17 cells 24,26 .

We first wanted to confirm that IVIG induces activation of βcatenin in mouse immune cells. Culturing of splenocytes from C57/BL6 mice with IVIG for 24 h induced active-β-catenin while HSA had no such effect (Fig. 9a). Thus, irrespective of species (human or mouse), IVIG stimulates β-catenin signaling.

Furthermore, inhibition of β-catenin signaling in vivo in EAE model by daily injection of β-catenin antagonist (FH535) along with IVIG did not compromise the protective effects of IVIG. The clinical scores were similar to IVIG injected in combination with dimethyl sulfoxide (DMSO, solvent control) (Fig. 9b). The control mice developed the signs of EAE by day 12, whereas the onset of the disease was delayed in both IVIG + solvent control and IVIG + β-catenin antagonist FH535 groups. Further, the severity of disease remained mild irrespective of mice received inhibitor or not (Fig. 9b).

We analyzed the various subsets of CD4 + T cells in the spleen of mice on the day of onset of disease. Confirming the previous reports, IVIG suppressed both Th1 and Th17 cells and reciprocally enhanced Tregs (Fig. 9c,d). Importantly, β-catenin antagonist had no effect on the IVIG-mediated reciprocal regulation of Tregs and effector Th1 and Th17 cells (Fig. 9c,d). Together, these data demonstrate that β-catenin signaling is dispensable for IVIG-mediated anti-inflammatory effects in vivo.

Discussion

Despite its widespread use over the last 35 years as an immunotherapeutic agent in autoimmune and inflammatory diseases, our knowledge on the mechanisms by which IVIG benefits such a vast number of pathologies is still incomplete. IVIG interacts with various arms of the immune system and exerts antiinflammatory effects by diverse mechanisms that appear to function in coordination. While mechanisms like saturation of neonatal Fc receptor (FcRn), neutralization of pathogenic autoantibodies and inflammatory cytokines, and complement scavenging are functioning during early phase of IVIG therapy; regulation of immune cells like innate cells (like DCs, neutrophils, monocytes, macrophages, basophils) and adaptive immune cells (T and B cells) occur during subsequent phase to ensure immune homeostasis 3,4,19,26,55-60 . DCs play a crucial role as professional antigen presenting cells in balancing the immune response and tolerance. Investigations on the mode of action of IVIG have revealed that it inhibits the activation of both human and murine DCs and the production of inflammatory cytokines 19,23,27 The ability of IVIG to suppress DC activation also had impact on the DC-mediated T cell responses and the pathogenesis of autoimmune diseases 19,20,55,61,62 . The signaling pathway(s) that are implicated in the regulation of DC functions by IVIG is not well understood. Previous reports have shown that preconditioning of human monocyte-derived DCs with IVIG impairs TLR4-mediated activation of extracellular signal-regulated kinases 1/2 (ERK1/2) 63 . Although ERK1/2 was not inhibited by IVIG in murine bone marrow-derived DCs, when these cells were stimulated with LPS along with IVIG, p38 mitogen activated protein kinase (p38MAPK) activation was suppressed 23 . Possibly, differences in the concentrations of IVIG and/or experimental conditions might have contributed to the differences observed between human and mouse DCs. Type II Ctype lectin receptors appear to play an important role in transducing the signaling events by IVIG in DCs. However, the nature of the lectin receptor varies depending on the subset of DCs 64,65 . By signaling via DC-SIGN (Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin), IVIG and its F (ab') 2 fragments induced prostaglandin E2 in human DCs by activating cyclooxygenase-2 pathway and mediated Treg expansion 26 . The IL-3-produced from these activated Tregs might license basophils to undergo activation by IVIG leading to the secretion of IL-4 and further suppression of effector Th17 and Th1 cells 56,66 . On the other hand, in the humanized DC-SIGN mice model, Fc fragments containing terminal α-(2,6) sialic acids interacted with DCs and induced IL-33 35 though in human, DC-SIGN-positive DCs failed to produce IL-33 upon exposure to IVIG 67 . Ligation of DCIR (Dendritic cell immunoreceptor) by I-VIG induced phosphorylation of immunoreceptor tyrosine-based inhibition motif (ITIM)-linked phosphatases Src homology 2 (SH2) domain-containing inositol polyphosphate 5-phosphatase-1 (SHIP-1) and SH2-containing tyrosine phosphatase-2 (SHP-2) in pulmonary CD11c + DCs of mice 68 . Thus, IVIG activates various signaling pathways in DCs to mediate anti-inflammatory actions.

Though Wnt/β-catenin pathway is generally studied for their role in embryonic development and tumorigenesis 8 , several recent studies have emphasized the role of Wnt/β-catenin pathway in the regulation of systemic and mucosal inflammatory immune responses by mediating tolerogeneic properties in DCs and ensuing T cell responses 13,18,41,69-71 . β-catenin signaling in DCs suppresses inflammatory cytokines like IL-6, induces antiinflammatory cytokines, and reciprocally regulates Treg and Th1/ Th17 responses in vivo in experimental models of autoimmune and inflammatory diseases like EAE and inflammatory bowel disease 18,69 . In line with these data on the role of β-catenin in promoting tolerance in DCs, treatment of these cells with Wnt3a proteins that trigger β-catenin activation also suppress TLRinduced pro-inflammatory cytokines in DCs 17 . As both IVIG and Wnt/β-catenin pathway mediate similar anti-inflammatory actions on DCs and T cell responses, inspired us to explore if normal IgG (IVIG) targets Wnt/β-catenin pathway to exert therapeutic benefits. In line with our proposition, IVIG induced activation of βcatenin in DCs. However, inhibition of β-catenin pathway did not compromise the ability of IVIG to suppress IL-6 and IL-8 production in DCs, suggesting that β-catenin pathway is redundant for IVIG. Our data thus affirm that IVIG is not bound by a single mechanism to exert anti-inflammatory effects. The mutually nonexclusive pathways induced by IVIG ensure that a deficiency in a particular pathway could be compensated by others. βcatenin pathway however is not only the pathway dispensable for anti-inflammatory actions of IVIG. In fact, heme oxygenase-1 (HO-1) pathway that regulates inflammatory responses by catabolizing the free heme and releasing carbon monoxide and biliverdin, is also found be not essential for the anti-inflammatory effects of IVIG 72 .

How does IVIG induce β-catenin activation? Mechanistically, our data imply that IVIG induces Wnt5a in DCs that signals via frizzled and LRP5 receptors to activate β-catenin pathway. Though Wnt5a is a noncanonical Wnt ligand, various reports have indicated its involvement to induce β-catenin pathway 49,50 . In the presence of Fzd4 and LRP coreceptors, Wnt5a could indeed activate β-catenin-mediated canonical pathway 49,50 . Secretion of Wnt5a in IVIG-treated DCs and abrogation of IVIGinduced β-catenin activation upon silencing of LRP 5/6 indicate Wnt5a-induced β-catenin activation by IVIG.

Several reports have demonstrated that β-catenin could be activated by Dectin-1, TLR2, or FcγR-mediated signaling 18,40,41,73 . In our report, pre-treatment of DCs with EDTA that chelates Ca 2+ and inhibits C-type lectin receptor-mediated binding of ligands Fig. 8 β-catenin signaling is dispensable for the anti-inflammatory actions of intravenous immunoglobulin on dendritic cells. DCs were stimulated with LPS for 24 h. The cells were then treated with β-catenin antagonist FH535 for 2 h followed by culture with IVIG (25 mg/ml) for 24 h. a Levels of active βcatenin in the total cell lysate as analyzed by immunoblot. Representative immunoblot and % difference in the active β-catenin are presented (mean ± SEM, n = 3 donors). Images are cropped for the presentation. b The amounts of IL-6 and IL-8 in the cell-free culture supernatants as assessed by ELISA (mean ± SEM, n = 7 donors). *P < 0.05; **P < 0.01; ns, not significant; as determined by one-way ANOVA followed by Tukey's multiple comparisons test. CA cells alone treated with LPS, HSA human serum albumin.

including Dectin-1, DC-SIGN and DCIR, did not prevent the ability of IVIG to induce β-catenin activation. The retention of ability to induce β-catenin activation by desialylated IVIG suggest that type II Fc receptors are also not implicated in the process 31,35,74 . The involvement of TLR2 was also precluded in view of the fact that IVIG-could induce β-catenin activation in DCs independent of TLR signaling.

We found that induction of β-catenin activation by IVIG requires intact IgG while neither Fc nor F(ab′) 2 fragments could recapitulate these functions. As human monocyte-derived DCs mainly express low affinity FcγRII, explains the inability of Fc fragments of IVIG to induce β-catenin activation. However, despite having the ability to bind DCs 19 , F(ab′) 2 fragments did not promote β-catenin activation. Therefore, it is likely that cooperation between Fab and Fc regions lead to β-catenin activation by IVIG. As reported earlier, IVIG contains antibodies to various self-motifs including those antigens expressed on DCs 4-6 . Thus, binding of Fab region of IVIG to DCs might license Fc region to bind FcγRII of adjacent DCs and signal β-catenin activation in those cells. Human DCs display a balanced expression of activating FcγRIIA and inhibitory FcγRIIB 43 . Data from the FcγRIIA blocking experiments and Syk inhibition assays provide a pointer towards lack of participation of activating FcγRs including FcγRIIA in IVIG-induced β-catenin activation. These data thus suggest a possible role for FcγRIIB in inducing βcatenin activation although, we do not exclude the implication of yet another non-identified receptor. In fact, reports have demonstrated that interaction of IgG with certain receptors like FCRL5 (Fc receptor-like protein 5) on B cells requires intact IgG 3,4,75 .

To explore the importance of β-catenin signaling in IVIGmediated anti-inflammatory effects in vivo, we used EAE model. Our previous data show that IVIG has the ability to delay onset of the disease and to reduce the severity of EAE. The protection was associated with the inhibition of Th1/Th17 responses and peripheral expansion of Tregs 24,26 . Activation of the β-catenin pathway is also reported to suppress the severity of the EAE and to mediate reciprocal regulation of effector CD4 + T cells and Tregs 12 . EAE has been used by several groups to explore the antiinflammatory mechanisms of IVIG. Although a recent report suggested possible neutralization of Mycobacterial antigens by F (ab′) 2 fragments as a mechanism of IVIG-mediated protection in EAE 76 , other lines of evidences suggest that IVIG mechanisms in EAE go beyond mere neutralization of Mycobacterial antigens. For inducing EAE, Mycobacterial antigens are emulsified in Freund's adjuvant and then injected to the mice. Mycobacterial antigens in emulsion are not freely accessible to antibodies to get neutralized. It is contrary to the report of Quast et al. 76 who used free Mycobacterial antigens in ELISA to test binding of IVIG. Furthermore, other reports have shown the importance of Tregs, IL-11 receptor, IL-33 receptor in IVIG-mediated protection against EAE. Depletion of Tregs or deficiency of IL-11R, IL-33R, SIGN-R1 (Specific ICAM-3 grabbing nonintegrin-related 1), all led to abrogation of protection by IVIG 26,31,77,78 .

Our data also advocate IgG structure-dependent diversity in the mechanisms of IVIG. While some mechanisms are dependent on F(ab′) 2 or Fc fragments 3-6,79 , others like β-catenin activation as shown here requires intact IgG that were either not damaged or subjected to structural modifications. Moreover, the structural integrity of IgG is critical for the half-life of IVIG in the treated patients. Though both IVIG and β-catenin mediate similar actions, from the current perspective it is evident that IVIG does not link β-catenin to exert therapeutic benefits, as IVIG is able to reduce the severity of EAE and reciprocal regulation of Th1/Th17 and Treg responses independent of β-catenin. These data thus highlight that a single mechanism might not be entirely responsible for the sustained beneficial effects of IVIG in autoimmune and inflammatory diseases.

Methods

Reagents. CD14 MicroBeads, basophil isolation kit II, rhGM-CSF, rhIL-4 were from Miltenyi Biotec (Paris, France). Rabbit MAbs to non-phospho (active) β-catenin (Ser33/37/Thr41) (clone D13A1, 1:500 dilution), GSK-3β (clone 27C10, 1:1000 dilution), GAPDH (HRP-conjugated) (clone 14C10, 1:1000 dilution), β-Actin (HRP-conjugated) (clone 13E5, 1:5000 dilution); polyclonal protein A and peptide affinity chromatography purified rabbit antibodies to phospho-β-catenin (Ser33/37/Thr41) (#9561, 1:500 dilution), β-catenin (#9562, 1:1000 dilution), mouse PCNA MAb (clone PC10, 1:1000 dilution) and HRP-conjugated affinity purified horse anti-mouse IgG secondary antibody (#7076, 1:2000 dilution) were from Cell Signaling Technology (Ozyme, Saint Quentin Yvelines, France). HRPconjugated human adsorbed goat anti-rabbit IgG secondary antibody (#4010-05; 1:5000 dilution) was purchased from Southern Biotech (Birmingham, LA).

FITC-conjugated anti-mouse MAbs to IFN-γ (clone XMG1.2, 1:75 dilution for 0.1 million cells) and CD25 (clone 7D4, 1:30 dilution), anti-human CD32 (clone FLI8.26, 1:50 dilution), anti-human CD86 (clone FUN-1, 1:50 dilution); PEconjugated anti-mouse MAb to IL-17A (clone TC11-18H10, 1:50 dilution); Per-CP/Cy5.5-conjugated anti-mouse MAb to CD4 (clone RM4-5, 1:50 dilution); APC/ Cy7-conjugated anti-human MAb to CD69 (clone FN50, 1:100 dilution) were from BD Biosciences (Le Pont de Claix, France). APC-conjugated MAb to FOXP3 (clone FKJ-16s, 1:30 dilution), APC-conjugated anti-human HLA-DR (clone G46-6, 1:50 dilution) and Fixable Viability Dye eFlour506 (1:500 dilution) were from eBioscience (Paris, France). Alexa Flour 488-conjugated goat anti-rabbit IgG (H + L) was from Invitrogen (ThermoFisher Scientific, Illkirch, France #A11034, 1:5000 dilution). Blocking MAb to FcγRIIA (clone IV.3) was purchased from Stem Cell Technologies (Grenoble, France).

Syk inhibitor R406 and β-Catenin/TCF Inhibitor, FH535 were obtained from InvivoGen (San Diego, CA) and Merck Chimie (Fontenay-sous-Bois, France) respectively. LPS (E. coli 055:B5) was purchased from Sigma-Aldrich (St. Quentin Fallavier, France).

Plasma-derived HSA and chitin were kind gifts from LFB Biomedicaments (Les Ulis, France) and Dr. V Aimanianda (Institut Pasteur, Paris, France). IL-3 was purchased from ImmunoTools (Friesoythe, Germany).

Therapeutic normal IgG or IVIG. As a source of IVIG, Privigen® and Hizentra® (CSL Behring) were used. IVIG was dialyzed against large volumes of RPMI 1640 at 4 °C for 4 h. For desialylation of IgG, IVIG was treated with recombinant neuraminidase (New England BioLabs, USA) as previously described 38,39 . Lectinblot analyses was performed to validate the desialylation of IVIG (Supplementary Fig. 1). The sialylation content was measured by reverse phase high performance liquid chromatography 38,39 . F(ab′) 2 and Fc fragments of IVIG were prepared by pepsin and papain digestion respectively 56 . The F(ab′) 2 and Fc fragments were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses for the verification of purity.

Generation of human DCs. Peripheral blood mononuclear cells (PBMCs) were obtained by subjecting the buffy coats of healthy donors to Ficoll density gradient centrifugation (Centre Necker-Cabanel, L'Établissement Français du Sang, Paris; EFS-INSERM ethical committee permission 15/EFS/012; 18/EFS/033). CD14 + cells were positively selected using CD14 MicroBeads (Miltenyi Biotec). Monocytes were cultured for 5 days in the presence of GM-CSF (1000 IU/10 6 cells) and IL-4 (500 IU/10 6 cells) to obtain DCs 80 . The gating strategy for DCs is provided in the Supplementary Fig. 2a. Induction of EAE in mice. C57BL/6J female mice were obtained from Envigo laboratories, France. All the experiments on mice were done after approval from the ethical committee for animal experimentation and French Ministry of Higher Education and Research (APAFIS#10539-2017070715163055 V4). EAE was Fig. 9 β-catenin signaling is dispensable for the IVIG-mediated protection from EAE and reciprocal regulation of Tregs and effector CD4 + T cells. a Splenocytes from C57/BL6 mice were treated with IVIG or HSA for 24 h followed by immunoblot analysis for the active β-catenin. Images are cropped for the presentation. b EAE was induced in three groups of C57/BL6 mice by immunizing with MOG 35-55 in complete Freund's adjuvant. First group of mice received no injections. Second group mice were injected with IVIG and DMSO and the third group with IVIG along with β-catenin antagonist FH535 daily until peak of the disease. Development of disease was monitored every day. Mean clinical scores from one of the two independent experiments (n = 9 mice) are presented. Error bars represent SEM. ***p < 0.001; ****p < 0.0001; ns, not significant; as determined by two-way ANOVA with Bonferroni post-ttest. c On the day of onset of clinical signs, mice were sacrificed and splenocytes were collected for the analysis of CD4 + T cell subsets. Th1, Th17, and Treg population was analyzed by flow cytometry. Representative dot plots showing the frequency (% positive cells) of CD4 + IFN-γ + Th1, CD4 + IL-17A + Th17, and CD4 + CD25 + FOXP3 + Tregs in various groups are presented. d Frequency of CD4 + T cell subsets (Th1, Th17, and Treg) in the spleen from two independent experiments (mean ± SEM, n = 8 mice). *p < 0.05; **p < 0.01; ns, not significant; as determined by one-way followed by Tukey's multiple comparisons test. induced in 8-week-old mice as described in our earlier studies 24,26 . Briefly, mice were immunized on day 0 by subcutaneously injecting 200 µg of MOG 35-55 peptide (MEVGWYRSPFSRVVHLYRNGK; synthesized at Polypeptide group, Strasbourg, France; 95% purity) emulsified in complete Freund's adjuvant, containing 880 µg of killed Mycobacterium tuberculosis strain H37RA. Further, all mice received 300 ng pertussis toxin on day 0 and day 2. Mice were observed every day for the development of clinical signs of EAE. Clinical signs in control mice typically appeared between 10-12 days post immunization. Mice were scored based on the following scoring system where, 0, no signs; 1, tail paresis; 2, hind limb paresis; 3, hind limb paralysis; 4, tetraplegia; and 5, moribund. Any animal reached the score 4 was euthanized for ethical reasons.

Mice were divided into three groups. Control group, IVIG + DMSO group and IVIG + inhibitor group. Mice received by intraperitoneal route, 0.8 g/kg of IVIG in combination with either 50 μl of DMSO (Sigma-Aldrich) or 15 mg/kg of β-catenin inhibitor (FH535) every day until peak of the disease.

Isolation of mouse splenocytes and CD4 + T cell analysis. On the day of onset of the disease, mice were sacrificed to collect spleen. Splenocytes were isolated and red blood cells were lysed using ACK (Ammonium-Chloride-Potassium) lysis buffer. Cells (0.5 million/ml) were stimulated with phorbol myristate acetate (PMA) (50 ng/ml/0.5 million cells) and ionomycin (500 ng/ml/0.5 million cells), along with GolgiStop for 4 h. Th1, Th17, and Treg populations were analyzed by combination of surface and intracellular staining for various markers. Surface staining was performed with fluorescence-conjugated MAbs to CD4 and CD25. After fixation and permeabilization by intracellular staining kit (eBioscience), intracellular staining with fluorescence-conjugated MAbs to IFN-γ, IL-17A, and FOXP3 were carried out. Samples were acquired using LSR-II flow cytometer (BD Biosciences) and data were analyzed by FACS-DIVA (BD Biosciences). The gating strategy for CD4 + T cells is provided in the Supplementary Fig. 2b.

Treatment of DCs and mice splenocytes. DCs (0.5 million cells/ml) were cultured in RPMI-1640 containing 10% fetal calf serum (FCS) alone or with 25 mg/ml or 15 mg/ml of IVIG or desialylated IVIG (25 mg/ml) or equimolar concentration of F(ab′) 2 fragments, Fc fragments or HSA (irrelevant protein control) for 24 h. Activation of the β-catenin signaling was analyzed by immunoblotting.

For the experiments involving treatment of pharmacological inhibitors, concentrations were chosen after titration. β-Catenin/TCF inhibitor, FH535 was used to inhibit β-Catenin at the concentration of 15 μM, 2 h prior to the treatment of IVIG.

In other experiments, DCs were pretreated with pre-determined concentrations of EDTA (0.5 mM) or with Syk inhibitor R406 (10 μM) or with blocking MAb to FcγRIIA (10 μg/0.5 million cells) for one hour before culturing with IVIG. We confirmed that aforementioned concentrations of EDTA, Syk inhibitor and FcγRIIA MAb were functional (Supplementary Fig. 3).

RNA isolation and quantitative RT-PCR. For the analyses of gene expression, cells were treated with IVIG (25 mg/ml) or HSA for 12 h. Untreated cells were used as control. Total RNA from the different conditions was isolated using the RNeasy Mini Kit (Qiagen, Hilden, Germany). cDNA was synthesized using High-Capacity cDNA Reverse Transcription kit (ThermoFisher Scientific, Courtaboeuf, France). Quantitative RT-PCR for CTNNB1 was done by TaqMan Universal Master Mix II with UNG (Applied Biosystems, Foster City, CA), and expression was measured with TaqMan Gene Expression Assays (Applied Biosystems, Hs00355045_m1 (CTNNB1) and Hs02786624_g1 (glyceraldehyde 3-phosphate dehydrogenase, GAPDH).

For the analyses of expression of Wnt genes, SYBR TM Green PCR Master Mix (ThermoFisher Scientific) was used. Gene expression was calculated relative to the housekeeping gene GAPDH. The primers used in the study are as follows:

WNT1-F: CAGCGACAACATTGACTTCG, R: GCCTCGTTGTTGTGAAGGTT;

WNT3A-F: CCTGCACTCCATCCAGCTACA, R: GACCTCTCTTCCTACCTTTCCCTTA;

WNT5A-F: CTCACTGAAATGCGTGTTGG, R: AATGCCCTCTCCACAAAGTG;

WNT7A-F: CCCACCTTCCTGAAGATCAA, R: GTCCTCCTCGCAGTAGTTGG; WNT7B-F: GGCACAAGGACCTACCAGAG, R: CCTGATGTGTTCTCCCAGGT;

WNT10A-F: CCACTCCGACCTGGTCTACTTTG, R: TGCTGCTCTTATTGCACAGGC;

GAPDH-F: CGACCACTTTGTCAAGCTCA, R: GGTGGTCCAGGGGTCTTACT.

Transfection with siRNA. LRP5 and LRP6 predesigned Accell SMART Pool siRNA was purchased from Dharmacon (Thermo Fisher Scientific) and introduced into cells according to manufacturer's protocol. Briefly, DCs were cultured in 12 well plate at 0.5 × 10 6 cells/0.5 ml of Accell Delivery media. One micrometer of nontargeting control siRNA or LRP5 and LRP6 siRNA were introduced into cells for 72 h. After 72 h, cells were cultured in RPMI with 10% FCS and IVIG for 24 h followed by FACS analysis. Surface staining of DC was performed with fluorescence-conjugated MAb to HLA-DR. For active-β-catenin detection, cells were stained with rabbit MAb to non-phospho (active) β-catenin (Ser33/37/Thr41) and followed by Alexa Flour 488-conjugated goat anti-rabbit IgG (H + L) by using Cell Signaling Buffer Set A (Miltenyi Biotec).

Immunoblotting. Total cell lysates were obtained by lysing cells in RIPA buffer (radioimmunoprecipitation assay buffer, Sigma-Aldrich) containing protease and phosphatase inhibitors (Sigma-Aldrich). Proteins were quantified using Bio-Rad protein assay reagent (Bio-Rad, Marnes-la-Coquette, France) and an equal amount of proteins were resolved on SDS-PAGE and transferred on to nitrocellulose, iBlot™ Transfer Stack (ThermoFisher Scientific). The membrane was blocked using 5% bovine serum albumin (BSA) in TBST (Tris-buffered saline (TBS) and Polysorbate 20 or Tween 20) for 60 min. The blots were incubated overnight at 4 °C with various primary antibodies diluted in TBST-5% BSA followed by incubation with secondary antibody for 2 h. After washing blots in TBST thrice with 10 min interval, blots were developed with SuperSignal TM WestDura Extended Duration substrate (Thermo Fisher Scientific). β-actin or GAPDH were used as a loading control. The western blot images were analyzed by myImageAnalysis software v 2.0 (Thermo Fisher Scientific). The full western blot images are provided in the Supplementary Fig. 4.

Analyses of nuclear translocation of β-catenin. DCs were cultured alone or in presence of IVIG or equimolar concentration of HSA for 24 h. Cells were harvested and pellets were washed twice with ice cold PBS and resuspended in hypotonic lysis buffer (20 mM Tris pH 7.5, 5 mM MgCl 2 , 1 mM EDTA, and 240 mM Sucrose). After incubating for 10 min on ice, cells were centrifuged at 13,000 rpm for 10 min at 4 °C to pellet down the nuclei. Supernatant was collected and used as a cytosolic extract. Pellets were resuspended in ice cold Buffer C (20 % Glycerol, 20 mM HEPES pH7.9, 420 mM NaCl, 1.5 mM MgCl 2 and 0.2 mM EDTA). Pellets were incubated on ice for 30 min followed by centrifugation at 13,000 rpm for 20 min at 4 °C to collect the nuclear extract. Immunoblotting of cytosolic and nuclear extracts was performed as detailed above.

ELISA for measuring the cytokines and Wnt ligands. DCs were stimulated with LPS (100 ng/0.5 million cells /ml) for 24 h. The cells were hen treated with βcatenin antagonist FH535 for 2 h followed by culture with IVIG (25 mg/ml) for additional 24 h. Cell-free supernatants were analyzed for the secretion of IL-8 and IL-6 by ELISA (ELISA Ready-SET-Go, eBioscience).

Secretion of Wnt3a and Wnt5a was analyzed in cell-free supernatants from IVIG-treated DCs with the help of Human Protein Wnt-3a and Wnt-5a ELISA Kit (MyBioSource, San Diego, CA).

Statistical analyses.

As highlighted in the figure legends, the experiments were repeated several times by using independent donors or mice to ensure reproducibility. Human data were from three to seven donors except for nuclear translocation of β-catenin that was representative of two donors. Mice data were from two independent experiments. Statistical analyses were performed by using one-way ANOVA followed by Tukey's multiple comparisons test or by two-way ANOVA with Bonferroni post-t-test for in vivo experiments. P < 0.05 was considered significant.

Reporting summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article. complications hémorragiques, de complications infectieuses qui grèvent la morbimortalité (8-20%) de la maladie.

Figure 1 :

 1 Figure 1: Classic coagulation cascade. .........................................................................

Figure 2 :

 2 Figure 2: Cell-based model of coagulation. ...................................................................

Figure 3 :

 3 Figure 3: The coagulation factor VIII gene and protein. .................................................

Figure 4 :

 4 Figure 4: Identified patients with bleeding disorders over time (1999-2021). .................

Figure 5 :

 5 Figure 5: Frequencies of different types of F8 DNA variants. ........................................

Figure 6 :

 6 Figure 6: Structural differences in selected rFVIII products and clinical candidates. .....

Figure 7 :

 7 Figure 7: Comparison of FVIIIa and emicizumab. ..........................................................

Figure 8 :

 8 Figure 8: Mechanism of action of natural anticoagulants inhibitors. ..............................

Figure 9 :

 9 Figure 9: Mechanisms of anti-FVIII alloantibody development. .....................................

Figure 10 :

 10 Figure 10: Proposed working mechanisms of ITI. ..........................................................

Figure 11 :

 11 Figure 11: The human Fcγ receptor family. ...................................................................

Figure 12 :

 12 Figure 12: Choice and monitoring of hemostatic therapy in acquired hemophilia A. .....

Figure 13 :

 13 Figure 13: Mechanisms by which the bacterial pathogen GAS subverts host innate immune defense. ...........................................................................................................

Figure 14 :

 14 Figure 14: Cleavage of intact IgG by IdeS. ....................................................................

Figure 15 :

 15 Figure 15: Crystal structure of IgG1 Fc E382A -IdeS C94A complex. .....................................

Figure 16 :

 16 Figure 16: FVIII binding to C1q. ...................................................................................

Figure 17 :

 17 Figure 17: In vitro activation of blood DCs in the presence of plasma and therapeutic FVIII. ....................................................................................................................................

Figure 18 :

 18 Figure 18: Schematic representation of antibody-encoding lentiviral vector design. ...

Figure 19 :

 19 Figure 19: Half-life of IdeS and IdeSKK-DD variant in mice. ............................................

Figure 2 :

 2 Figure 2: Cell-based model of coagulation. Representation of the initiation, amplification, and propagation phases generating a local thrombin burst. TF, tissue factor; PAR, protease-activated receptor; vWF, von Willebrand factor. From (Godby et al., 2022).

Figure 3 :

 3 Figure 3: The coagulation factor VIII gene and protein. The diagram shows the principal features of the coagulation factor VIII (F8) gene, as well as the production and processing of the encoded protein. Intron 22 of F8 gene contains two further genes (F8A and F8B); colored arrows (red and blue, respectively) indicate the orientation of their transcription. Black arrows pointing upwards indicate thrombin cleavage sites. Adapted from[START_REF] Graw | Haemophilia A: from mutation analysis to new therapies[END_REF].

Figure 4 :

 4 Figure 4: Identified patients with bleeding disorders over time (1999-2021). From the WFH Annual Global Survey of 2021 (https://www1.wfh.org/)

Figure 5 :

 5 Figure 5: Frequencies of different types of F8 DNA variants. A. Variants detected in males with severe hemophilia A. B. Variants detected in males with mild-moderate hemophilia A.Adapted from[START_REF] Johnsen | Novel approach to genetic analysis and results in 3000 hemophilia patients enrolled in the My Life, Our Future initiative[END_REF].

Figure 6 :

 6 Figure 6: Structural differences in selected rFVIII products and clinical candidates.Schematic structure of native coagulation factor VIII (FVIII, top panel), which undergoes intracellular processing before secretion to generate a heterogeneous mixture of proteins with truncated B domains. Summary of conventional FVIII molecules (which have a half-life similar to that of native FVIII, as demonstrated in clinical trials), as well as extended half-life (EHL) FVIII molecules that are approved or in clinical trials. Schematics of structures and the differences are highlighted. The cell line used to produce each product is in parentheses. BDD, B domain deleted; BHK, baby hamster kidney; CHO, Chinese hamster ovary; Fc, crystallizable fragment; HEK293, human embryonic kidney 293; VWF, von Willebrand factor. From[START_REF] Peters | Advances and innovations in haemophilia treatment[END_REF].

Figure 7 :

 7 Figure 7: Comparison of FVIIIa and emicizumab. The differences in the interactions with enzyme and substrate between FVIIIa (left panel) and emicizumab (right panel) are presented.From(Lenting et al., 2017).

Figure 8 :

 8 Figure 8: Mechanism of action of natural anticoagulants inhibitors. PC, protein C; TF, tissue factor, TFPI, TF pathway inhibitor. From[START_REF] Gualtierotti | Updates on Novel Non-Replacement Drugs for Hemophilia[END_REF].

Figure 9 :

 9 Figure 9: Mechanisms of anti-FVIII alloantibody development. APC, Antigen presenting cells; Th, T helper cells; Tfh, T follicular helper cells; TCR, T-cell receptor; CD40L, CD40 ligand; IL, interleukin; TNF-α, tumor necrosis factor-alpha; IFNγ, interferon-γ; TGF-β, transforming growth factor beta; IgG, immunoglobulin G; MHC-II, major histocompatibility complex class II molecule.From[START_REF] Prezotti | Immunogenicity of Current and New Therapies for Hemophilia A[END_REF].

Figure 10 :

 10 Figure 10: Proposed working mechanisms of ITI. Repeated administration of factor VIII (FVIII) in a non-inflammatory state leads to presentation of FVIII without costimulatory signals (A) and/or upregulation of inhibitory T-cell molecules (B), such as CTLA-4 or PD1. This in turn causes anergy of FVIII-specific effector T-cells (Teffs) and induction of regulatory T-cells (Tregs), indicated by the balance shifting towards the Treg site. These Tregs exhibit inhibitory effect on both T-and B-cells. At the same moment high doses of FVIII induce apoptosis of FVIII-specific B-memory cells (FVIII B-mem). The elimination of these FVIII B-mems reduces antigen presentation and subsequent activation of FVIII T-mem, which further shifts the balance from Teff towards Treg.The net result of these events is that (long-living) plasma cells are not replenished, which eventually leads to the eradication of inhibitors. Finally, also the development of anti-idiotypic anti-FVIII antibodies might be involved in this process by neutralizing the effect of anti-FVIII inhibitors. CTLA-4: cytotoxic T-lymphocyte associated antigen 4; PD1: Programmed cell death 1. From(Schep et al., 2018).

Figure 11 :

 11 Figure 11: The human Fcγ receptor family. β2m, Beta-2 microglobulin; GPI, glycosyl phosphatidyl inositol; ITAM, immunoreceptor tyrosine-activating motif; ITIM, immunoreceptor tyrosine-inhibitory motif; NK, natural killer. Adapted from (Shock et al., 2020).

  Nevertheless, a systematic evaluation of the repercussion of the different properties of FVIII-IC on the evolution of an ongoing anti-FVIII immune response is lacking. Such knowledge should, at term, provide a better understanding of the mechanisms underlying ITI in HA patients and potentially lead to the identification of predictive markers of ITI outcome and the development of novel tools to foster ITI success. Part of my Ph.D. project has been to understand how the nature of FVIII-IC that form in HA patients at the start of ITI influences the course of the anti-FVIII immune response.

Figure 12 :

 12 Figure 12: Choice and monitoring of hemostatic therapy in acquired hemophilia A. rFVIIa, recombinant activated factor VII (eptacog alfa); APCC, activated prothrombin complex concentrate; rpFVIII: recombinant porcine factor VIII (susoctocog alfa), hFVIII, human (plasmaderived or recombinant) factor VIII; h: hour; d: day. From(Tiede et al., 2020)

  ) resistance to host immunity, in which S. pyogenes uses mechanisms to evade or inhibit complement and/or antibody-mediated opsonization, phagocytosis, neutrophil killing, and destruction by antimicrobial peptides.The following paragraphs provide insight into the different mechanisms employed.

Figure 13 :

 13 Figure 13: Mechanisms by which the bacterial pathogen GAS subverts host innate immune defense. Phagocyte recruitment is reduced by peptidases ScpA and SpyCEP/ScpC that degrade the chemokines C5a and IL-8, respectively. Complement deposition is limited by M protein binding of host counter-regulatory factors C4BP and FH. Phagocytic uptake is reduced by Mac-1/2 binding of phagocyte Fc receptors.Resistance to cationic antimicrobial peptides is afforded by D-alanyl modification of cell wall teichoic acids, cysteine protease SpeB-mediated degradation, and binding/inactivation by protein streptococcal inhibitor of complement (SIC). M protein and protein H bind Fc domains of Igs in a non-opsonic manner, and proteolytic inactivation of Ig is a property of SpeB, Mac-1/2, and endopeptidase S. DNase production facilitates escape from NETs, and the pore-forming cytolysins streptolysin S (SLS) and streptolysin O (SLO) exhibit lytic activity against host neutrophils and macrophages. From[START_REF] Nizet | Understanding how leading bacterial pathogens subvert innate immunity to reveal novel therapeutic targets[END_REF].

Figure 14 :

 14 Figure 14: Cleavage of intact IgG by IdeS.Intact human IgG, regardless of isotype, is cleaved by IdeS in two steps. Step one results in a single cleavage of the IgG molecule (scIgG) in which one heavy chain remains intact.Step two generates a fully cleaved product that cannot mediate complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC) or antibody-dependent cell-mediated phagocytosis (ADCP) by means of Fcγ receptor. Adapted from(Jordan et al., 2017). B. Representation of the amino acid sequences of the hinge region of the four IgG subclasses with the cleavage sites for papain and IdeS. Adapted from[START_REF] Falkenburg | Anti-Hinge Antibodies Recognize IgG Subclass-and Protease-Restricted Neoepitopes[END_REF].

Figure 15 :

 15 Figure15: Crystal structure of IgG1 Fc E382A -IdeS C94A complex.Overall structure of the complex. IdeS C94A is colored blue; IgG1 Fc E382A is colored in grey. N-linked glycans and the IgG1 Fc E382A hinge peptide in the focused panel are shown as sticks and colored, with oxygen, nitrogen and sulfur atoms in red, blue, and yellow, respectively. b. Superposition of complexed IdeS C94A with three published apo structures of IdeS (PDB IDs 1Y08, 2AU1 and 2AVW, colored in red, orange, and yellow, respectively) and focused view of the prosegment binding loop. c. Binding of IgG1 Fc E382A hinge peptide within the IdeS C94A active site. Fc E382A peptide and IdeS C94A active site residues are depicted as sticks and colored by heteroatom; catalytic triad residues are colored purple. The scissile peptide bond is colored in cyan; hydrogen bonds are depicted as green dashes. From(Sudol et al., 2022).

•

  Heparin-induced thrombocytopenia IdeS prevented heparin-induced thrombocytopenia (HIT) engendered by an injection of a monoclonal chimeric IgG1, directed against platelet factor 4 (PF4), with heparin (H) in transgenic mice expressing human PF4 and the FcγRIIA receptor. The cleavage of anti-PF4/H IgG by IdeS abolished FcγRIIA-dependent cellular activation and prevented thrombus formation and thrombocytopenia induced by anti-PF4/H antibodies (Kizlik-Masson et al., 2019).

  Kjellman et al. have recently reported on the outcomes at 3 years post-transplant in imlifidase-desensitized kidney transplant recipients. In 4 single-arm, open-label, phase 2 studies, 39 crossmatch-positive patients received imlifidase before kidney transplantation.

  My Ph.D. work has addressed two complementary goals: 1) to gain a better understanding and decipher the mechanisms underlying the outcome of ITI in persons with congenital HA (PwHA) and look for prognostic markers for the successful eradication of FVIII inhibitors, and 2) to develop novel strategies to eradicate FVIII inhibitors in PwHA and persons with acquired HA (PwAHA), using the IgG-degrading enzyme of S.pyogenes (IdeS) to transiently remove FVIII inhibitors from inhibitor-positive patients, thus opening a time window for treatment with therapeutic FVIII in situations of major bleeds or surgery.To address the first goal, I formed synthetic FVIII-IC using recombinant human monoclonal anti-FVIII IgG (anti-FVIII rhIgG) with different epitope specificities and subclasses. Because anti-FVIII IgG mainly belong to the IgG1 and/or IgG4 subclasses, I cloned, purified, and validated four anti-FVIII rhIgG in both IgG1 and IgG4 formats: the anti-A2 domain BOIIB2, the anti-A3 domain KM41, the anti-C1 domain LE2E9, and the anti-C2 BO2C11. In addition, FVIII-IC binding and signaling through FcγR were evaluated using BW5147:FcγR-ζ reporter cell lines. My initial plan was also to test the effect of these synthetic FVIII-IC on the activation or differentiation of different populations of immune cells (MoDCs, bDCs, NK cells, and transduced FVIII-specific B cells). To address the second goal, I first validated the ability of IdeS to cleave anti-FVIII IgG present in patient's plasma and the recombinant neutralizing IgG produced in the lab. I also assessed the efficacy of IdeS to restore the hemostatic efficacy of therapeutic FVIII in vivo in tail clipping assays in FVIII-deficient mice reconstituted with human anti-FVIII IgG and treated with IdeS. By injecting different doses of inhibitors, I determined the adequate frequency of dosing needed to transiently reduce neutralizing antibody titers to levels wherein FVIII replacement therapy can be efficiently administered. Since a large majority of PwHA are currently under emicizumab prophylaxis, assessing the cleavage efficacy of IdeS over this humanized IgG4 drug is a must. The main objective of clearing emicizumab from the circulation is to avoid interference with standard hemostatic agents needed for breakthrough bleeding and major surgeries, sometimes leading to lethal thrombosis. Thus, I validated the ability of IdeS to cleave emicizumab in vitro and in vivo using FVIII-deficient mice reconstituted with a therapeutic dose of emicizumab. In addition, cleavage kinetics were compared to those obtained with the in-lab-produced BO2C11 IgG4. This thesis is based on the following publications: Article 1: Bou-Jaoudeh M, Delignat S, Daventure V, Astermark J, Lévesque H, Dimitrov JD, Deligne C, Proulle V, Lacroix-Desmazes S. The IgG-degrading enzyme, Imlifidase, restores the therapeutic activity of FVIII in inhibitor-positive hemophilia A mice. Haematologica. 2023 Jan 19. doi: 10.3324/haematol.2022.281895. Epub ahead of print. PMID: 36655430. Article 2: Bou-Jaoudeh M, Mimoun A, Delignat S, Peyron I, Daventure V, Deligne C, Dimitrov JD, Christophe OD, Denis CV, Lenting PJ, Proulle V, Lacroix-Desmazes S.et al. The IgG-degrading enzyme IdeS as an antidote to emicizumab in experimental hemophilia A. (Under revision at to the Journal of Thrombosis and Haemostasis) Article 3: Bou-Jaoudeh M, Delignat S, Daventure V, Dimitrov JD, Lagassé D, Kolb P, Sauna ZE, Hengel H, Lacroix-Desmazes S. FVIII-containing immune complexes reduce FVIII uptake by antigen-presenting cells and activate NK cells. (In progress)

Figure 1 .

 1 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 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 3 .

 3 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 Imlifidaes (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 immunsorbant assay. Results are expressed in arbitrary units (AU, representative of 2 experiments) from optical density measured at 492 nm. (B) Inhibitory activity of IdeScleaved 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).

Figure 4 .

 4 Figure 4. Imlifidase-mediated elimination of a FVIII inhibitor in inhibitor-positive hemophila A mice. (A) Half-life of BO2C11 immunoglobulin G (IgG1 k ) in hemophila 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 (panel 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 immunsorbant 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 the plasma levels of IgG (C) and F(ab') 2 fragments (E) as a function of the inhibitory activity in plasma (D) for the condition where mice were treated with IdeS at 30, 48, 72 and 96 h following BO2C11 injection. The experimental data were interpolated using a linear curve (R 2 : 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.

Figure 5 .

 5 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).

Figure 6 .

 6 Figure6. 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). Twentyfour hours (h) later, a group of mice was then treated with a single dose of Imlifidase (IdeS) (0.6 mg/kg, 0.29 µM, full green circles) or phosphate-buffered saline (PBS). 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 immunsorbant 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 2-tailed non-parametric Mann-Whitney test (*P<0.05; otherwise non-significant).

10 ARTICLE

 10 -Removal of FVIII inhibitors by IdeS in hemophilia A M. Bou-Jaoudeh et al. ARTICLE -Removal of FVIII inhibitors by IdeS in hemophilia A M. Bou-Jaoudeh et al.

1 Title.

 1 The IgG-degrading enzyme IdeS as an antidote to emicizumab in experimental hemophilia A Authors. Melissa Bou-Jaoudeh,* Angelina Mimoun,* Sandrine Delignat,* Ivan Peyron, † Victoria Daventure,* Claire Deligne,* Jordan D Dimitrov,* Olivier D. Christophe, † Cécile V. Denis, † Peter J. Lenting, † Valérie Proulle,* , ‡ Sébastien Lacroix-Desmazes* Affiliations. *Institut National de la Santé et de la Recherche Médicale, Centre de Recherche des Cordeliers, CNRS, Sorbonne Université, Université Paris Cité, Paris, France; † Laboratory for Hemostasis, Inflammation & Thrombosis, Unité Mixte de Recherche 1176, Institut National de la Santé et de la Recherche Médicale, Université Paris-Saclay, 94276 Le Kremlin-Bicêtre, France; ‡ Service d'Hématologie Biologique et Unité Fonctionnelle d'Hémostase, Hôpital Cochin, AP-HP.Centre, Université Paris Cité, Paris, France.Running head. Removal of emicizumab by IdeS in hemophilia ACorresponding author. Sébastien Lacroix-Desmazes (sebastien.lacroix-desmazes@inserm.fr) bispecific chimeric humanized IgG4, mimics the procoagulant activity of factor VIII (FVIII). Its long half-life and subcutaneous injection route have been life-changing in treating hemophilia A patients with and without FVIII inhibitors. However, emicizumab only partially mimics FVIII activity and is inefficient in treating acute bleeds. Emergency management is particularly complicated in patients with FVIII inhibitors under emicizumab prophylaxis, wherein therapeutic FVIII is inefficient and high-dose bypassing agents are associated with thrombotic risk. Thus, temporarily eliminating emicizumab for better emergency management is of clinical interest. Imlifidase, an IgG-degrading enzyme (IdeS) of bacterial origin, was used to eliminate pathogenic IgG in patients undergoing organ transplant, in preclinical models of alloimmune responses to drugs, autoimmune diseases and gene therapy hampered by the presence of preexisting immunity to adeno-associated viruses. Here, we investigated the potential of IdeS to quickly remove emicizumab from the circulation of FVIIIdeficient mice. IdeS hydrolyzed emicizumab in vitro and in vivo, albeit at slower rates than a recombinant human monoclonal IgG4. The persistence for several days in mice blood of low amounts of the single-cleaved intermediate IgG was compensated by the rapid clearance of the F(ab') 2 fragments and, hence, rapid loss of emicizumab procoagulant activity. We propose that IdeS could be administered to hemophilia A patients to rapidly eliminate emicizumab. This would allow for controlling acute bleeds using high-dose bypassing agents without exposing the patients to major thrombotic risk. In inhibitor-positive patients, the concomitant elimination by IdeS of anti-FVIII IgG would restore the therapeutic efficacy of FVIII. hemophilia A (HA) by administration of exogenous factor VIII (FVIII) is complicated by the elevated cost, immunogenicity and short half-life of the molecule.

  and CH2 domains (F296Y) of emicizumab revert the corresponding IgG4 sequence to

7 when

 7 IdeS was injected twice at an equimolar IdeS:IgG ratio, once at a 10-fold molar IdeS:IgG ratio or twice at a 10-fold molar ratio, respectively.

  excess of IdeS reduced the circulating concentration of emicizumab from 35.

Figure 1 .

 1 Figure 1. In vitro cleavage of emicizumab by IdeS. Panel A. Emicizumab was incubated with

14 Figure 2 .

 142 Figure 2. Half-life of emicizumab in FVIII-deficient mice. Panel A. C57BL/6 exon 16 FVIII-

Figure 3 .

 3 Figure 3. In vivo elimination of emicizumab upon hydrolysis by IdeS in FVIII-deficient

  Figure 1 310x230mm (300 x 300 DPI)

  8 recombinant human IgG (rhIgG) and recombinant human FVIII (rhFVIII). These rhIgG are IgG1 and IgG4 antibodies are directed against different domains of FVIII (A2, A3, C1 and C2). FVIII-IC containing IgG1 and IgG4 signaled differently through FcγR-expressing reporter cells. FVIII-IC inhibited FVIII endocytosis by immature monocyte-derived dendritic cells (MoDCs) regardless of epitope specificity or IgG subclass. Moreover, FVIII-IC did not impact the maturation state of MoDCs. In contrast to IgG4-containing FVIII-IC, IgG1-containing FVIII-IC induced the activation of a CD16+ NK cell line in an FcγR-dependent manner.

  at the start of ITI influence the course of the anti-FVIII immune response. We generated four recombinant human IgG specific for different domains of FVIII in either an IgG1k or an IgG4k format. We then formed synthetic FVIII-IC. IgG1 and IgG4-containing FVIII-IC signalled differently through FcγR-expressing reporter cells. The synthetic human FVIII-IC reduced FVIII endocytosis by immature MoDCs independently of epitope specificity or subclass of the IgG. Moreover, FVIII-IC did not impact the maturation state of MoDCs. In contrast to IgG4-containing FVIII-IC, IgG1-containing FVIII-IC induced the activation of a CD16+ NK cell line in an FcγR-dependent manner. At term, our findings should provide a better understanding of the mechanisms underlying ITI in HA patients and potentially lead to the identification of predictive markers of ITI outcome and the development of novel tools to foster ITI success.

  IgG1k and IgG4k formats, and the biotinylated human FcRn (ITF01, Immunitrack) were determined by surface plasmon resonance (SPR)-based technology (Biacore 2000 system; Biacore Cytiva, Uppsala, Sweden). Biotinylated FcRn was immobilized on a streptavidin sensor chip (Biacore Cytiva) activated using 1 M NaCl, 0.05 M NaOH solution at a flow rate of 30 μl/min. In brief, FcRn was diluted in running buffer (100 mM Tris, 100 mM NaCl, 5% glycerol, 0.1% tween 20, pH 6) to a final concentration of 5 μg/ml and injected at a flow rate of 10 μl/min. IgG(25-0.19 nM) were injected at 30 μl/min. The association and dissociation phases were monitored for 4 and 5 min, respectively.Regeneration of the chip surface was achieved using a solution of 100 mM Tris, 100 mM NaCl, pH 7.7. Binding to the surface of an uncoupled control flow cell was subtracted from the binding to the FcRn-immobilized flow cells. Calculation of the kinetic rate constants was performed by global analysis of the experimental data using the kinetic models included in the BIAevaluation version 4.1 software (Biacore Life Sciences), fitting the data with the lowest value of χ 2 .Immunoprecipitation of FVIII-ICFVIII (200 nM) was incubated with or without single IgG (1000 nM) for 30 min at 37°C in 250 μL reaction buffer (Hepes 20 mM, NaCl 150 mM, CaCl2 5 mM, tween 20 0.05%). After incubation, 50 μL of protein G-coupled agarose beads (Roche) were added and incubated for 1 hr at 4°C on a rotating wheel. The non-retained fraction was removed and eliminated through consecutive washing and centrifugation steps. FVIII-IC were eluted by the addition of Laemmli's sample buffer and heating for 10 min at 80°C. After centrifugation, eluates were collected, loaded on NuPAGE 4-12% bis-Tris gels (Thermo Scientific) and transferred to nitrocellulose membranes. For IgG, samples were run under reducing conditions (0.1M Dithiothreitol). IgG on nitrocellulose membranes were detected using an HRP-labeled mouse anti-human IgG (Fc) antibody. For FVIII, samples were run under non-reducing conditions. FVIII was detected using a biotinylated sheep anti-human FVIII:C polyclonal antibody (SAF8C-APBIO, Affinity Biologicals) and HRP-coupled streptavidin. Proteins were revealed with the Pierce™ ECL Western Blotting Substrate (Thermo Scientific) and iBright™ FL1000 Imaging System (Thermo Scientific).

( 0 .

 0 5x10 6 cells/ml) were cultured in RPMI-1640 (Thermo Scientific) with 10% FCS, supplemented with GM-CSF (1000 IU/10 6 cells) and IL-4 (500 IU/10 6 cells) (Miltenyi Biotec) for 5 days to generate immature MoDCs. After 5 days, the differentiation of MoDCs (> 90% purity) was confirmed by flow cytometry upon loss of CD14 staining (M5E2 clone, BD Pharmingen), expression of major histocompatibility complex (MHC) class II and CD1a (HI149 and G46.6 clones, respectively, BD Pharmingen) (supplementary FigureS3). The expression of CD32A (FcγRIIA/B, clone 3D3, BD Pharmingen), CD64 (FcγRI, clone 10.1, BD Pharmingen) and CD16 (FcγRIIIA/B clone 3G8, BD Pharmingen) were assessed. The acquisition was performed on an LSR II cytometer with FACSDiva™ software (BD Biosciences). The results are expressed as fold change of mean fluorescent intensity (MFI) of the tested conditions versus MFI of cells incubated with isotypic controls.FVIII-IC uptake by immature monocyte-derived dendritic cells FITC-coupled full-length FVIII (Kogenate®, 20 nM) was pre-incubated alone or with a 5molar excess of single IgG1k or IgG4k (BO2C11, BOIIB2, LE2E9, and KM41) or 5-molar excess of the pool of the four IgG1k or IgG4k for 30 min at 37°C. Samples were then incubated with 5-day-old immature MoDCs (0.2x10 6 cells/100 μl) in Iscove's Modified Dulbecco's Medium for 30 min at 37°C or 4°C. Cells were washed with ice-cold PBS and fixed with 3.7% formaldehyde (Sigma) for 20 min at 4°C. Cells were then analyzed by flow cytometry. The uptake was quantified as the difference in MFI between 37°C and 4°C to remove the signal generated by the binding of FVIII to the cell surface.

  whether FVIII-IC with different IgG subclasses would differently trigger NK cells. The CD16A-expressing PTA-6967 cell line and the parental CD16-negative NK-92 cell line (Figure 4A) were incubated with FVIII alone or pre-treated with pooled anti-FVIII IgG1k or IgG4k. The secretion of IFNγ was measured in the supernatant and the expression of LAMP1 at the surface of NK cells was assessed as an indirect marker of NK cell degranulation. Incubation of PTA-6967 cells led to the secretion of IFNγ and increased expression of LAMP1 upon incubation with IgG1k-containing FVIII-IC (Figure 4B and 4C).

Figure 1 :

 1 Figure 1: Validation and characterization of human recombinant monoclonal anti-FVIII IgG. A. Purity and expected molecular weight. IgG1k and IgG4k (5 μg) were separated by SDS-PAGE under non-reducing conditions. Molecular weight markers are shown on the left lane of the gel. B. Interaction of IgG1κ and IgG4κ with FcRn by SPR. Real-time profiles of the interaction of BOIIB2 IgG1k (left panel) and IgG4k (right panel) (25-0.19 nM) with FcRn. The binding response is depicted as resonance units (RU). C. Binding of anti-FVIII IgG1k and IgG4k to BW5147:FcγRs reporter cells. BOIIB2 (red), BO2C11 (black), KM41 (green) or LE2E9 (purple) in the IgG1 (full circles) or IgG4 (empty circles) formats were incubated at different concentration (0.67, 6.7 and 67nM) with CD32A or CD16A reporter cells or wild-type (WT) parental BW5147 cells at 4°C. The binding of IgG to FcγRs expressed at the cell surface was evaluated by flow cytometry after staining of the cells with an anti-human Fc antibody conjugated to PE. The results express the fold change in the mean fluorescence intensity (MFI) of the anti-human Fc-PE compared to the MFI measured in the case of WT cells. Graphs depict mean±SD of 3 independent experiments.

Figure 2 :

 2 Figure 2: Generation and detection of FVIII-containing immune complexes. A. Eluates obtained by immunoprecipitation were collected and analyzed by Western blot. FVIII-IC were identified by revelation with a biotinylated sheep anti-human FVIII antibody (SAF8C) under non-reducing conditions (top panel) and an HRP-labeled mouse antihuman IgG (Fc) antibody under reducing conditions with DTT (bottom panel). Non-eluted FVIII (FVIII alone) was used as a positive control. Trastuzumab, a monoclonal Ab that does not recognize FVIII, was used as a negative control as well as eluate of protein G alone or incubated with FVIII. Molecular weight markers is shown on the left lane of the blots. B. The pool of the four IgG1k or IgG4k (BOIIB2, KM41, LE2E9, BO2C11) were preincubated alone (white and black bars, respectively) or with 10nM of FVIII (dashed white and black bars, respectively) at different IgG:FVIII molar ratio (0.25:1, 1:1, 5:1) prior to incubation with FcγR reporter cells at 4°C for 90min. The binding of IgG1k or IgG4k to FcγR reporter cells was measured by flow cytometry after staining of the cells with an antihuman Fc conjugated to PE. The graphs depict the MFI of the anti-human Fc (mean±SD

Figure 3 :

 3 Figure 3: FVIII-IC impact on MoDCs. A. FVIII-IC uptake by immature MoDCs. FVIII-FITC (20 nM) was pre-incubated alone or with a 5-molar excess of single IgG1k or IgG4k (BOIIB2, KM41, LE2E9, BO2C11) or with a pool of the four IgG1k/IgG4k. Uptake by MODCs was analyzed by flow cytometry. Results are expressed as percentages of the difference of mean fluorescence intensity measured at 37°C and 4°C (ΔMFI (37°C -4°C)), whereby 100% corresponds to ΔMFI obtained with FVIII-FITC alone. Graphs are representative of the mean±SD obtained from 4-9 different donors. Statistical significance was assessed using the non-parametric Kruskal-Wallis test corrected for multiple comparisons using the Dunn's test (**: p<0.01). B. FVIII-IC effect on maturation phenotype of MoDCs. Cells were harvested at 48 hr and DC phenotype was assessed by flow cytometry. The MFI of surface expression of CD40, CD80, CD83, and CD86 are reported.Results are representative of 4 independent experiments (mean ± SD). Statistical significance was assessed using the non-parametric Kruskal-Wallis test corrected for multiple comparisons using the Dunn's test (*: p<0.05).

Figure 4 :

 4 Figure 4: FVIII-immune complexes formed with IgG1 activate NK cells through CD16A. A. Phenotypic analysis of CD16A expression by NK cell lines. Filled grey histogram: unstained PTA-6967 cells, open black histogram: Stained NK92 cells, open red histogram: stained PTA-6967 cells. B. Activation of CD16A negative (NK92, white bars) and CD16A positive NK cells (PTA-6967, black bars) was evaluated by quantification of secreted IFNγ after 24 hr of incubation with the FVIII-IC formed with IgG1 or IgG4 or the pool of IgG1 or IgG4 alone. The graph depicts the mean±SD of the fold

FVIII

Figure 17 :

 17 Figure 17: In vitro activation of blood DCs in the presence of plasma and therapeutic FVIII. Immature blood DCs (bDCs) purified from the blood of healthy individuals were incubated alone or with FVIII in the presence of plasma from healthy donors (HD), inhibitor-negative patients (IN) or inhibitor-positive-patients (IP) for 40 hours. A. Change in expression of surface markers. The results are expressed as the fold change of mean fluorescence intensity (MFI) of CD86 expression on the surface of bDCs in the tested conditions versus incubation of the bDCs with serum AB. B. Change in cytokine secretion profile. TNF-α levels (pg/ml) were measured in the supernatant collected at 40 hours. Statistical differences were assessed using the non-parametric Kruskal-Wallis test corrected for multiple comparisons using the Dunn's test (*:p<0.05; **: p<0.01; ***:p<0.0001).

Figure 18 :

 18 Figure 18: Schematic representation of antibody-encoding lentiviral vector design. The expression of membrane anchored IgG is driven by Ig light chain promoter (FEEK). The constant IgG1 heavy chain (CH) and kappa light chain (CL) genes are fused with the IgG variable regions (VL and VH). Co-expression of CH and VH is obtained by the introduction of the F2A peptide sequences. F: HIV-1 flap element; WPRE: woodchuck hepatitis virus posttranscriptional regulatory element; pAM: polyAdenylation sequence. Adapted from[START_REF] Fusil | A Lentiviral Vector Allowing Physiologically Regulated Membrane-anchored and Secreted Antibody Expression Depending on B-cell Maturation Status[END_REF].

  investigate the potential use of IdeS in HA patients, I used a mouse model of severe HA. However, IdeS does not efficiently cleave mouse IgG. Therefore, I passively injected mice with recombinant human IgG1 at different inhibitory titers to mimic different situations observed in patients (i.e. 10, 30, and 200 BU/ml). IgG1 subclass was randomly chosen, since the same cleavage was observed in vitro for both subclasses. The lack of endogenous production of human IgG is a limitation of this model rendering it artificially favorable for IdeS treatment. However, this model allows precise adjustment and monitoring of the circulating FVIII inhibitory titers. Moreover, in humans, IdeS was shown to cleave the whole IgG pool within 2 to 6 hours (Winstedt et al., 2015) and de novo production of endogenous IgG was detected after 1-2 weeks (though kinetics were different between individuals). In the HA animal model, intact IgG were depleted as soon as 6 hours post-dosing.

Figure 19 :

 19 Figure 19: Half-life of IdeS and IdeSKK-DD variant in mice. The levels of IdeS variants were measured in mice plasma over time. Results depict the % of the levels measured for each variant at different time points compared to the level measured at 5 minutes after injection (t0).

Fig. 1

 1 Fig. 1 Intravenous immunoglobulin (IVIG) stimulates β-catenin signaling in dendritic cells. Monocyte-derived DCs (0.5 million cells/ml) were cultured either alone (cells alone, CA), with IVIG (15 or 25 mg/ml) or with an equimolar concentration of human serum albumin (HSA) for 24 h. a Immunoblot analysis of active-β-catenin (n = 7 donors) and b Immunoblot analysis of p-β-catenin (n = 5 donors). Representative immunoblots and densitometric analyses of the blots (mean ± SEM) are presented. β-Actin was used as a loading control. Images are cropped for the presentation. c Investigation of nuclear translocation of β-catenin protein by immunoblot. Representative immunoblot from DCs of two donors is presented. d DCs were treated with IVIG or HSA for 12 h and the mRNA levels of CTNNB1 was analyzed by quantitative real-time RT-PCR (mean ± SEM, n = 7 donors). e Changes in the levels βcatenin protein in IVIG-treated DCs (n = 3) as analyzed by immunoblot. *P < 0.05; **P < 0.01; ns, not significant; as determined by one-way ANOVA followed by Tukey's multiple comparisons test.

Fig. 3 Fig. 2

 32 Fig.3Intravenous immunoglobulin (IVIG) activates β-catenin in human dendritic cells in an IgG-sialylation independent manner. DCs (0.5 million cells/ml) were cultured either alone (cells alone, CA), treated with IVIG (25 mg/ml) or with desialylated IVIG (25 mg/ml) for 24 h. Activation of βcatenin was analyzed by immunoblotting. β-Actin was used as a loading control. Representative immunoblot and densitometric analyses of the blots (mean ± SEM, n = 3 donors) are presented. *P < 0.05; ns, not significant; as determined by one-way ANOVA followed by Tukey's multiple comparisons test.

Fig. 4

 4 Fig. 4 Lack of implications of C-type lectin receptors and activating FcγRIIA in mediating β-catenin activation by intravenous immunoglobulin (IVIG) in dendritic cells. DCs were pre-treated with (a) EDTA or (b) anti-CD32A MAb or (c) Syk inhibitor for one hour followed by treatment with IVIG for 24 h. Representative immunoblots and densitometric analyses of the blots (mean ± SEM, n = 4-5 donors) are presented. GAPDH (a) or β-Actin (b and c) were used as loading controls. *P < 0.05; **P < 0.01; ns, not significant; as determined by one-way ANOVA followed by Tukey's multiple comparisons test. CA cells alone.

Fig. 5

 5 Fig.5Intact IgG is mandatory for the β-catenin activation by intravenous immunoglobulin (IVIG). DCs (0.5 million cells/ml) were cultured either alone (cells alone, CA), treated with IVIG (25 mg/ml) or equimolar concentrations of (a) Fc or (b) F(ab′) 2 fragments of IVIG for 24 h. Activation of β-catenin was analyzed by immunoblotting. β-Actin was used as a loading control. Representative immunoblots and densitometric analyses of the blots (mean ± SEM, n = 3-4 donors) are presented. *P < 0.05; **P < 0.01; ns, not significant; as determined by one-way ANOVA followed by Tukey's multiple comparisons test.

Fig. 6

 6 Fig. 6 Effect of intravenous immunoglobulin (IVIG) on the induction of Wnt ligands in dendritic cells. Monocyte-derived DCs were treated with IVIG at 25 mg/ml concentration or with equimolar concentration of HSA for 12 h. a The expression of various canonical Wnt ligands (mean±SEM, n = 6 donors) and b noncanonical Wnt ligands (mean±SEM, n = 5 donors) was analyzed by quantitative real-time RT-PCR. c The amount of secreted Wnt 3a and Wnt 5a in DCs treated with IVIG for 24 h (mean ± SEM, n = 4 donors). *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant; as determined by one-way ANOVA followed by Tukey's multiple comparisons test. CA cells alone, HSA human serum albumin.

  ARTICLE COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-020-0825-4 10 COMMUNICATIONS BIOLOGY | (2020) 3:96 | https://doi.org/10.1038/s42003-020-0825-4 | www.nature.com/commsbio

  En présence d'anticorps anti-FVIII, les traitements hémostatiques de première intention sont les agents court-circuitant (« agents by-passant » ou ABP) comme le FVII activé recombinant ou les concentrés de facteurs activés du complexe prothrombinique d'origine plasmatique (rFVIIa et CCPa). A l'opposé des traitements par FVIII, les ABP n'ont aucun marqueur biologique prédictif d'efficacité hémostatique et peuvent être responsables d'accidents thrombotiques, notamment chez les patients avec comorbidités. L'apparition de l'emicizumab, un anticorps monoclonal bispécifique humanisé qui reconnait les FIX et FX et « mime » l'action pro-coagulante du FVIII, a révolutionné la prévention des hémorragies des patients HA avec inhibiteurs. Néanmoins, les épisodes hémorragiques aigus spontanés ou post-traumatiques, ou les chirurgies/gestes invasifs programmés nécessitent toujours le recours aux traitements hémostatiques habituels. Mon travail de thèse repose sur deux objectifs principaux. Le premier a été de comprendre les mécanismes associés aux résultats de l'ITI chez les PwHA et de rechercher des marqueurs pronostiques pour une éradication efficace des inhibiteurs du FVIII. Le second a été de développer une nouvelle stratégie pour éradiquer efficacement les inhibiteurs du FVIII chez les PwHA et les PwAHA. Plusieurs paramètres cliniques ont été associés aux résultats de l'ITI, qu'ils soient liés au traitement ou au patient. Il est intéressant de noter que les sous-classes d'IgG anti-FVIII et leurs spécificités épitopiques peuvent potentiellement prédire le résultat clinique. En effet, les PwHA qui échouent à l'ITI ont des ratios IgG4/IgG1 anti-FVIII élevés et ces anticorps sont principalement dirigés contre la chaîne lourde ou le domaine A2 du FVIII. Au contraire, les PwHA traités avec succès ont des anticorps dirigés contre la chaîne légère ou le domaine C2 du FVIII. Cependant, on ne sait toujours pas comment les propriétés des IgG anti-FVIII peuvent être prédictives du résultat de l'ITI. Dans le contexte de l'ITI, l'injection de grandes quantités de FVIII à des PwHA avec des inhibiteurs conduit à la formation de complexes immuns contenant du FVIII (FVIII-IC). Ces derniers ont été supposés accélérer l'élimination du FVIII de la circulation par le système la corrélation clinico-biologique qui permet l'adaptation des doses et de la fréquence des injections. Créer une fenêtre thérapeutique en éliminant rapidement les anticorps anti-FVIII pour restaurer temporairement l'efficacité hémostatique du FVIII est une alternative innovante pour optimiser la prise en charge de patients avec anticorps anti-FVIII. Dans la présente étude, j'ai exploité l'enzyme dégradant l'immunoglobuline G (IgG) de Streptococcus pyogenes (IdeS ; Imlifidase), récemment commercialisée, qui s'est révélée capable de cliver efficacement et rapidement les quatre sous-classes d'IgG humaines. Le clivage par IdeS est un processus en deux étapes. Un premier clivage rapide au niveau de la région charnière basse de l'une des deux chaines lourdes de l'IgG entraine la formation d'un produit intermédiaire, une IgG simple chaine (scIgG). Ce dernier est suivi d'un clivage plus lent au niveau de la seconde chaine lourde résultant par la dissociation complète des fragments F(ab')2 et fragments Fc. L'IdeS est approuvé pour la désensibilisation des patients ayant subi une transplantation rénale et présentant des anticorps spécifiques au donneur. Cette enzyme fait actuellement l'objet d'études pour son utilisation potentielle dans plusieurs maladies auto-immunes, en oncologie et en thérapie génique. J'ai émis l'hypothèse que le clivage rapide, bien que temporaire, des IgG circulantes par l'IdeS pourrait constituer une nouvelle option thérapeutique pour les patients souffrant d'inhibiteurs du FVIII. J'ai d'abord démontré que l'IdeS hydrolyse efficacement les IgG anti-FVIII polyclonales dans le plasma obtenu à partir de PwHA et PwAHA et des anticorps anti-FVIII monoclonaux recombinants humains de différentes sous-classes et spécificités épitopiques in vitro. J'ai ensuite développé un modèle murin d'HA sévère avec inhibiteurs en immunisant passivement des souris HA avec des IgG anti-FVIII humaines recombinantes à différents titres inhibiteurs (10-200 UB/ml). Ce modèle a été mis au point parce que l'IdeS ne clive pas toutes les sous-classes d'IgG de souris et le clivage n'est pas aussi efficace que pour les IgG humaines. Mes résultats montrent que l'IdeS est capable de cliver de façon efficace les IgG anti-FVIII humaines in vivo, atteignant des niveaux d'IgG faibles ou nuls et rétablissant l'efficacité hémostatique des perfusions de FVIII, comme le montre la correction de la tendance hémorragique chez les souris HA avec inhibiteurs. Une re-administration d'IdeS dans les 24 heures suivant la première injection était possible et nécessaire pour les titres inhibiteurs très élevés. Cependant, dans les pays développés, la plupart des personnes atteintes d'HA sont sous prophylaxie avec emicizumab. En ce qui concerne le clivage d'emicizumab, une IgG4 recombinante, mes résultats montrent une hydrolyse efficace in vitro et in vivo, dépendante du temps et de la dose d'IdeS. Cependant, le clivage se produit avec une cinétique plus lente que celle d'une autre IgG4 monoclonale recombinante humaine anti-FVIII utilisée comme contrôle. Chez les souris atteintes d'HA sévère, les fragments F(ab')2 résultant du clivage de l'emicizumab étaient systématiquement indétectables dans les 18 heures suivant le traitement par IdeS. Des niveaux faibles de scIgG étaient encore détectés. Cependant, une double injection d'un excès de 10 fois d'IdeS a réduit l'activité procoagulante de l'emicizumab de 88,1%, testées ex vivo par ajout de réactifs humains. Mes résultats apportent la preuve de concept de l'élimination temporaire des inhibiteurs du FVIII par l'IdeS et l'ouverture d'une fenêtre thérapeutique pour un traitement substitutif efficace avec du FVIII exogène et une meilleure gestion des patients avec des inhibiteurs du FVIII. Par exemple, chez les PwAHA nécessitant un traitement hémostatique, l'injection d'IdeS minimiserait le besoin d'APB et le risque thrombotique associé tout en rétablissant l'efficacité du traitement et de la surveillance du FVIII. De plus, l'utilisation de l'IdeS comme traitement immédiat de première ligne peut être complémentaire à l'utilisation d'agents immunosuppresseurs (c.-à-d. corticostéroïdes, cyclophosphamide) pour éliminer les inhibiteurs pendant une période plus longue chez les PwAHA. D'autre part, l'administration d'IdeS aux PwHA avec inhibiteurs recevant de l'emicizumab devrait vraisemblablement conduire à l'élimination simultanée des IgG anti-FVIII neutralisantes et du médicament. Cela permettrait non seulement de restaurer l'efficacité hémostatique clinique du remplacement du FVIII, mais aussi d'éliminer les interférences biologiques liées à l'emicizumab, garantissant ainsi une mesure précise de l'activité pro-coagulante du FVIII dans le plasma. Cela permettrait une meilleure prise en charge des interventions chirurgicales et des hémorragies majeures. Il est important de noter que la majorité de l'IdeS administré sera éliminé de la circulation dans les 24-48 heures, ce qui permettra une re-administration rapide de l'emicizumab en prophylaxie.

Table 2 : Etiology and underlying disorders of AHA. Adapted from

 2

  FVIII rhIgG1 k and rhIgG4 k , IgG (1.66 μM) were incubated alone or with IdeS (0.14 μM) 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-defi-
	cient mice 28 on a C57BL/6 background (HA mice) were
	housed and handled in accordance with French regulations
	and the experimental guidelines of the European Commu-
	nity (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 deter-
	mination 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

54

μM IdeS (yielding an approximate 12:1 molar ratio of IgG:IdeS) for 24 h at 37°C. For Haematologica | 108 XXXXXXX 2023 antik format (2,800 BU/kg). Mice were treated 1 day later by intravenous injection of IdeS (0.6 mg/kg or 0.29 μM) or PBS as control. When indicated, mice received a second injection of IdeS 24 h later. Residual levels of intact anti-FVIII rhIgG, partially hydrolyzed single-chain (sc) IgG, F(ab') 2 fragments, and inhibitory activities levels were determined by ELISA and MNBA in plasma collected up to 6 days after IdeS or PBS injection.

Table 1 .

 1 Inhibitory activity of monoclonal anti-FVIII IgG1k and IgG4k.

	A

A B C

  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, how-Haematologica | 108 XXXXXXX 2023

Table 2 . Binding kinetics to FcRn measured by surface plasmon resonance

 2

	IgG1 k	IgG4 k
	Association (ka) and dissociation (kd) kinetic rate constants, characterizing the interaction
	of FcRn with IgG1k and IgG4k are depicted in the table. The data were obtained by
	applying global kinetic analysis on the interactions measured by SPR at 25°C.

mAb k a (10 5 M -1 s -1 ) k d (10 -3 s -1 ) K D (nM) χ 2 k a (10 5 M -1 s -1 ) k d (10 -3 s -1 ) K D (nM) χ

									2
	BO2C11	7.7	6.4	8.2	1.5	4.6	10.7	23.4	0.1
	BOIIB2	7.2	7.8	10.8	0.3	8.8	17.1	19.4	0.1
	LE2E9	9.4	4.4	4.7	3.2	8.6	11.9	13.8	0.2
	KM41	4.1	1.1	2.6	1.9	11.2	2	1.78	2.1

Facteurs de risques potentiellement impliqués dans l'initiation de la réponse immunitaire contre le FVIII thérapeutique La dose administrée

  Le facteur VIII, de masse moléculaire 330 kDa, est composé de 2 332 acides aminés. Principalement synthétisé par le foie mais également par le système réticuloendothélial, il est ensuite clivé en hétérodimère où la chaîne lourde (210 kDa) et la chaîne légère (80 kDa) sont reliées par un ion calcium.obtenues soit par extraction à partir de pools de plasma de donneurs sains (dérivés plasmatiques), soit par bio-ingénierie génétique (produits recombinants). Les concentrés de FVIII plasmatiques et recombinants présentent des différences qualitatives selon leur structure, leur profil de glycosylation, leur capacité de liaison au facteur de Willebrand (VWF), la protéine chaperonne du FVIII. Néanmoins, quelle que soit leur origine, ils partagent tous la capacité d'induire une réponse « anticorps » avec la production d'immunoglobulines G (IgG) neutralisantes, que l'on appelle « inhibiteurs du FVIII », chez 5 à 30 % des patients. La survenue de ces inhibiteurs du FVIII rend les patients réfractaires au traitement, ce qui entraîne une dégradation de leur qualité de vie ainsi qu'une complication de leur prise en charge clinique. Plusieurs facteurs de risque ont été proposés pour expliquer l'apparition des inhibiteurs du FVIII, en particulier des facteurs de risques génétiques, tels que des antécédents familiaux, le type d'anomalie génétique causant l'HA et la sévérité de la maladie qui en découle, les haplotypes HLA-DR (human leukocyte antigen-DR) et les polymorphismes de certains gènes liés à l'immunité et à l'inflammation qu'ils présentent. Il est à ce jour toutefois impossible de prédire avec certitude si un patient donné développera ou non des inhibiteurs du FVIII.

		Ainsi, la grande majorité des études effectuées au
		cours des dix dernières années n'a pas permis d'iden-
		tifier d'évènements particuliers, source de l'induction
		de signaux de danger, dont le contrôle préviendrait ou
		réduirait l'intensité de la réponse immunitaire contre
		La dose de FVIII administrée est l'un des rares para-mètres qui a été associé de façon consistante au déve-loppement d'une réponse immunitaire anti-FVIII. Des travaux réalisés sur des souris FVIII-KO ont clairement démontré que l'intensité de la réponse immunitaire le FVIII thérapeutique. Il est donc possible que ces signaux de danger ne soient pas si importants pour le déclenchement de la réponse anti-FVIII, et que le niveau d'activation basal du système immunitaire soit, lui, suffisant pour permettre la stimulation de LT naïfs	REVUES
		était directement corrélée à la dose de facteur injecté spécifiques du FVIII, à condition toutefois qu'une quan-
		par voie intraveineuse. Ces données expérimentales tité suffisante de FVIII soit prise en charge par les CPA,
		ont été confirmées par l'étude RODIN qui montre que internalisée et présentée aux LT.
		l'intensité du traitement FVIII et la dose moyenne de
		FVIII administrée sont corrélées à l'incidence du déve-loppement d'inhibiteurs chez les patients atteints d'HA sévère (activité du FVIII < 0,01 UI/mL ou < 1 % de l'acti-vité dans le plasma normal). L'administration quoti-La reconnaissance du soi : le péché originel du système immunitaire Les lymphocytes B et T subissent, respectivement	SYNTHÈSE
		dienne de fortes doses de FVIII aux patients ayant déve-dans la moelle osseuse et le thymus, des processus de
		loppé des inhibiteurs (un protocole appelé « induction sélection positive et négative. Cette sélection garantit
		de tolérance immunitaire » ou ITI) permet néanmoins la fonctionnalité des lymphocytes émergents et de
		l'éradication des inhibiteurs du FVIII chez deux tiers des leurs récepteurs de l'antigène respectifs, BCR (B cell
	Réponse immunitaire contre le FVIII	patients, ce qui suggère que la relation entre la dose receptor) et TCR (T cell receptor). Lors de cette sélec-
		de FVIII administrée et son immunogénicité n'est pas tion, les lymphocytes présentant une auto-réactivité
	La réponse immunitaire contre le FVIII thérapeutique est considérée	linéaire et que ce seul paramètre de dose n'est pas suf-importante sont éliminés et les lymphocytes peu auto-
	comme une réponse immunitaire classique semblable à celle que l'on	fisant pour expliquer l'immunogénicité du FVIII. réactifs sont retenus. Dans le cas des LT, les cellules
	observe pour un antigène exogène [1]. Administré par voie intravei-	ayant une affinité intermédiaire pour des antigènes
	neuse, une partie du FVIII s'accumule dans les organes lymphoïdes	Immunogénicité intrinsèque du FVIII du soi (auto-antigènes) peuvent se différencier en LT
	secondaires, notamment la rate. Ceci a été mis en évidence dans un	Comme proposé dans la « théorie du danger », une régulateurs (Treg) dits naturels. Ces cellules inhibe-
	modèle d'HA sévère : les souris déficientes en FVIII (FVIII-KO) traitées	réponse immunitaire ne survient que si les CPA sont ront les LT auto-réactifs ayant échappé à la sélection
	avec du FVIII exogène. Le FVIII est ensuite internalisé par des cellules	activées par des signaux de danger via des récepteurs négative [14]. La reconnaissance du soi est donc le
	présentatrices d'antigènes (CPA) et est présenté aux lymphocytes T	qui appartiennent à la famille des PRR (pattern reco-fondement même de la sélection des lymphocytes qui
	(LT) CD4 + naïfs mais spécifiques du FVIII. Après avoir reçu un signal	gnition receptors), dont les Toll-like receptors (TLR), peuplent l'organisme. Elle fait partie de l'homéosta-
	d'activation adéquat, les CPA activent alors ces LT qui, à leur tour, sti-	des récepteurs de l'immunité innée spécifiques d'anti-sie du système immunitaire et peut être considérée
	mulent des lymphocytes B (LB) spécifiques du FVIII qui se différencient	gènes microbiens (ou PAMP, pour pathogen-associated comme le péché originel du système immunitaire. Ceci
	en LB mémoires et en plasmocytes sécréteurs d'anticorps anti-FVIII.	molecular pattern) ou de molécules produites à la suite est notamment vrai pour la reconnaissance du FVIII
	Bien que les étapes effectrices de la réponse immunitaire dirigée	de stress cellulaires (ou DAMP, pour damage associated par les cellules de l'immunité adaptative.
	contre le FVIII soient désormais connues, les causes de l'initiation	molecular pattern). Il a été proposé que la molécule
	de cette réponse restent encore à élucider. Ceci est important afin	FVIII pouvait être elle-même à l'origine de ces signaux
	de comprendre pourquoi 5 à 30 % des patients développent une telle	de danger. L'immunogénicité intrinsèque du FVIII a ainsi
	réponse anti-FVIII neutralisante à la suite d'une administration de	été suggérée par sa capacité à conférer à d'autres
	FVIII thérapeutique, alors que 70 à 95 % des patients restants ne déve-	molécules, immunologiquement inertes, une immuno-
	lopperont pas de réponse.	génicité. Par exemple, l'injection à des souris FVIII-KO
	Au cours des 20 dernières années, une grande partie de la recherche	du premier domaine de l'hémagglutinine (HA1) fusionné
	consacrée à comprendre les mécanismes associés à l'immunogé-	à la chaîne légère du FVIII 2 augmente considérablement
	nicité du FVIII s'est fondée sur la « théorie du danger » proposée	la réponse immunitaire anti-hémagglutinine [2]. Tou-
	par Polly Matzinger. Selon cette théorie, les CPA ne s'activent et ne	tefois, le FVIII ne peut être considéré lui-même comme
	deviennent matures que si elles reçoivent des « signaux de danger ».	un signal de danger puisqu'il est incapable d'activer
	Examinons donc les données cliniques et expérimentales disponibles
	concernant les potentielles sources de signaux de danger adjuvants
	de la réponse immunitaire anti-FVIII chez ces patients : saignements
	aigus/chroniques, traumatismes, chirurgie, infections ou vaccinations.

2

Survenue d'inhibiteurs anti-FVIII : réponse immunitaire exacerbée ou tolérance immunitaire avortée

  exogène entraîne une réponse immunitaire dirigée contre le FVIII thérapeutique qui se traduit par la production d'IgG anti-FVIII neutralisantes. Plusieurs facteurs de risque permettent d'expliquer le développement des inhibiteurs du FVIII. Le type d'anomalie génétique responsable de l'HA, et plus précisément la présence ou non de la protéine endogène FVIII:Ag dans la circulation, est le facteur de risque le mieux démontré. Les patients HA sévères, et particulièrement ceux SYNTHÈSE REVUES impliquée dans le renforcement des fonctions régulatrices des LT[36]. Ces résultats suggèrent que la capacité de l'organisme des patients à activer une machinerie anti-inflammatoire et/ou des voies régulatrices joue un rôle clef dans leur capacité à contrôler la réponse immunitaire dirigée contre le FVIII thérapeutique. FVIII après administration de FVIII thérapeutique. Cette réponse potentiellement neutralisante est contrôlée par une tolérance immunitaire active. C

	Figure 1. Réponses immuni-
	taires dirigées contre le FVIII
	endogène ou exogène. A.
	Reconnaissance du FVIII en
	physiologie. L'homéostasie de
	la réponse anti-FVIII en condi-
	tions physiologiques repose sur
	un équilibre entre une recon-
	naissance immunitaire du FVIII
	endogène par les lymphocytes
	T (LT) spécifiques et des immu-
	noglobulines (Ig) G anti-FVIII
	naturelles autoréactives, et
	une régulation impliquant des
	LT régulateurs et des anticorps
	anti-idiotypiques B. Réponse
	anti-FVIII tolérisée chez les
	patients hémophiles A sans
	inhibiteur. Nous proposons que
	tous les patients développent
	une réponse immunitaire anti-
	, rappelant ainsi la situation
	des lymphocytes T anti-FVIII : les anticorps anti-idiotypiques pro-
	tecteurs « naturels » sont produits spontanément chez les individus
	sains et contrôlent les IgG « naturelles » anti-FVIII potentiellement
	neutralisantes.
	Le FVIII endogène n'est donc pas ignoré par le système immunitaire en
	conditions physiologiques. Au contraire, l'homéostasie de la recon-
	naissance du FVIII par un système immunitaire sain repose sur un
	équilibre entre une réaction immunitaire contre le FVIII impliquant
	les lymphocytes T et B spécifiques du FVIII, ainsi que les anticorps anti-FVIII, et une réponse tolérogène qui s'y oppose, qui repose sur les lymphocytes Treg et les anticorps anti-idiotypiques. Reconnaissance du FVIII exogène thérapeutique chez les patients HA Chez 5 à 30 % des patients atteints d'HA, l'administration intravei-Importance de la réponse anti-inflammatoire La comparaison de polymorphismes génétiques portés par des patients ayant ou n'ayant pas développé d'inhibiteurs du FVIII suite à son administration a révélé l'existence d'associations entre la survenue d'inhibiteurs et certains polymorphismes de promoteurs de gènes codant des cytokines, le TNF-α (tumor necrosis factor alpha), l'IL(interleukine)-10, et un point de contrôle immunitaire, CTLA-4 (cytotoxic T-lymphocyte-associated protein 4). Nous avons égale-neuse de FVIII Les travaux réalisés ces 15 dernières années ont cher-ché à expliquer les raisons pour lesquelles 5 à 30 % des patients atteints d'HA développent une réponse immu-nitaire neutralisant le FVIII thérapeutique, alors que 70 à 95 % de ces patients ne répondent pas au FVIII. La nature des signaux de danger, qui jouent le rôle d'adju-vant de la réponse naïve anti-FVIII, a également été examinée. Chez l'individu sain, le FVIII endogène n'est pas « ignoré » par le système immunitaire en conditions ment décrit une corrélation entre un polymorphisme (répétitions GT)

physiologiques, mais il est bel et bien reconnu. Le maintien de l'homéostasie de la réponse anti-FVIII repose sur un équilibre entre une reconnaissance potentiellement pathogène (inhibitrice) et un contrôle actif de cette reconnaissance (Figure

1A

). Les IgG anti-FVIII sont détectables chez un bon nombre de patients atteints l' induction concomitante d'anticorps anti-idiotypiques bloquants

[34]

. La disparition d'inhibiteurs détectables dans le plasma lors de l'ITI ne repose donc pas simplement sur l'élimination des cellules et des anticorps spécifiques du FVIII. Collectivement, ces observations suggèrent l'existence d'un équilibre entre les cellules ou les molécules spécifiques du FVIII exogène et les éléments régulateurs de la réponse immunitaire ; cela rappelle les mécanismes de maintien de l'homéostasie immunitaire vis-àvis du FVIII endogène qui sont mis en oeuvre dans des conditions physiologiques. du promoteur du gène hmox1, codant l'enzyme anti-inflammatoire heme-oxygenase 1 (HO-1), et le développement d'inhibiteurs chez les patients HA sévères

[35]

. Plus récemment, Matino et al. ont démontré que, après stimulation in vitro par du CpG, les cellules dendritiques de patients ayant développé des inhibiteurs du FVIII sont moins susceptibles d'exprimer l'indoléamine 2,3 dioxygénase 1 (IDO1), une enzyme

. Réponse neutralisante anti-FVIII chez les patients hémophiles A avec inhibiteurs

  . L'organisme de 5 à 30 % des patients ne parvient pas à développer une réponse anti-inflammatoire et/ou immunitaire tolérogène capable de contrebalancer la réponse neutralisante initiée par l'administration de FVIII thérapeutique.

A Reconnaissance

du FVIII en physiologie

	Anticorps	IgG anti-FVIII
	anti-idiotypiques	naturelles
	LT régulateurs	LT anti-FVIII
		naturels
	B

Réponse anti-FVIII tolérisée chez les patients sans inhibiteur C Réponse neutralisante anti-FVIII chez les patients avec inhibiteurs d

  'HA. Des anticorps inhibiteurs sont détectés de manière transitoire chez un grand nombre de patients après leur première exposition au FVIII thérapeutique. Ces inhibiteurs du FVIII peuvent apparaître à un âge où les mécanismes de régulation immunitaire sont compromis. L'apparition d'inhibiteurs du FVIII est, de plus, associée à une capacité réduite de l'organisme à activer les mécanismes anti-inflammatoires/ tolérogènes qui reposent sur HO-1 ou IDO1. Sur la base de ces observations, nous proposons l'hypothèse selon laquelle tous les patients hémophiles A traités avec du FVIII thérapeutique développent une réponse immunitaire anti-FVIII. Dans le cas des patients qui ne développent pas de réponse neutralisante, le FVIII thérapeutique n'est pas « ignoré » par le système immunitaire ; il est reconnu et évalué « immunologiquement » comme tout antigène exogène (Figure1B). Un corollaire de cette hypothèse est qu'il n'existe pas de signaux de dangers spécifiques de la réponse anti-FVIII neutralisante : la maturation des CPA conduisant à l'activation de LT naïfs spécifiques du FVIII thérapeutique est permise par un système immunitaire dynamique confronté à un environnement en constante évolution. Dans ce contexte, nous ne pensons pas que l'apparition d'anticorps neutralisant le FVIII résulte de l'activation exacerbée du système immunitaire au moment de l'administration du FVIII. Au contraire, nous proposons que l'apparition d'anticorps neutralisant le FVIII résulte de l'incapacité de l'organisme des patients à développer une réponse anti-inflammatoire et/ou immunitaire tolérogène spécifique du FVIII qui contrebalancerait la réponse neutralisante déclenchée (Figure1C).

	Un tel changement de perspective devrait avoir des répercussions sur
	la prise en charge clinique des patients. Par exemple, chez les patients
	développant une réponse avec inhibiteurs, l'utilisation d'immunosup-
	presseurs [37,38] favorisant la mise en place de réponses tolérogènes
	devrait être privilégiée à celle de médicaments qui suppriment simple-
	ment la réponse adaptative. Il devrait également encourager à éla-
	borer des stratégies d'immuno-interventions destinées à établir une
	tolérance immunitaire spécifique du FVIII. Reste à déterminer si un tel
	changement de paradigme est aussi valable pour les réponses immu-
	nitaires neutralisantes qui se développent contre d'autres protéines
	thérapeutiques, telles que les anticorps monoclonaux ou les protéines
	thérapeutiques n'ayant pas d'équivalent endogène. ‡

ARTICLE -Removal of FVIII inhibitors by IdeS in hemophilia AM. Bou-Jaoudeh et al.

Journal of Thrombosis and Haemostasis

Le facteur VIII est synthé tisé sous forme d'une pré proté ine de 330 kDa. Dans le plasma, il circule sous forme d'un dimè re stabilisé par le facteur de Willebrand. Activé par la thrombine, le facteur VIII catalyse la ré action d'activation du facteur X par le facteur IX activé , en pré sence d'ions calcium et de phospholipides.

m/s n°4, vol. 36, avril 2020

Étude EPIC, ClinicalTrials.gov Identifier: NCT01376700.

COMMUNICATIONS BIOLOGY | (2020) 3:96 | https://doi.org/10.1038/s42003-020-0825-4 | www.nature.com/commsbio

Acknowledgments

I would like to express my gratitude and appreciation to my thesis committee members for their guidance through this process; your suggestions and feedback have been invaluable. Prof. Bertrand Belier and Dr. Sophie Sibéril, my master's instructors at the Sorbonne University, laid the basis for my scientific eagerness. Dr. Sophie Sibéril was Acknowledgments ........................................................................................................... iii Abstract ............................................................................................................................ v 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 staff from "Centre d'Expérimentation Fonctionnelle" at Centre de Recherche des Cordeliers (Paris) for assistance. Helixate® was a kind gift from CSL-Behring. Acknowledgments. We wish to thank the staff from "Centre d'Expérimentation Fonctionnelle" at Centre de Recherche des Cordeliers (Paris) for assistance. Funding. This work was supported by 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 Nationale 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'Etude 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 receives 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. PJL receives research support from Pfizer, Roche, Sanofi and Sobi. Acknowledgements We wish to thank Dr. Carmen Coxon (National Institute for Biological Standards and Control, Hertfordshire, UK) for sharing the cDNA encoding KM41. Helixate® and Kogenate® were kind gifts from Bayer Healthcare (Dr Pedro Paz, Berkeley, USA) and CSL-Behring (Dr Hasan Katovic, Paris, France). Biotinylated FcRn was a kind gift from Immunitrack (Dr Sune Justesen, Copenhagen, Denmark).

Acknowledgements

Supported by Institut National de la Santé et de la Recherche Médicale, Sorbonne Université and Université Paris Descartes, France; A.K. was a recipient of fellowship from Indo-French Center for Promotion of Advanced Research (CEFIPRA) and La Fondation pour la Recherche Médicale (FDT201805005552). M.B.J. was a recipient of a fellowship from Ministère de l'enseignement supérieur et de la recherche (Ecole doctorale 394, Sorbonne Université, France). We thank Dr. C Galeotti and Dr. E Stephen-Victor for the support. We also thank the staff of core facilities at Centre de Recherche des Cordeliers for the help: Centre d'Histologie, d'Imagerie et de Cytométrie (CHIC), Centre d'Explorations Fonctionnelles (CEF), and Le Centre de Génotypage et de Biochimie (CGB).

L'hémophilie A (HA) est un trouble hémorragique congénital lié à un déficit en facteur VIII (FVIII) pro-coagulant fonctionnel. La complication majeure du traitement substitutif par FVIII exogène est le développement d'anticorps anti-FVIII neutralisants, appelés inhibiteurs du FVIII, chez 30% des personnes atteintes d'HA sévère (PwHA). Ces inhibiteurs rendent inefficace la thérapie substitutive et exposent les patients à un risque élevé de morbidité et de mortalité. Une forme acquise d'HA, due à l'apparition d'anticorps inhibant le FVIII endogène, existe également. Les personnes atteintes d'HA acquise (PwAHA) n'ont aucun antécédent de trouble hémorragique et présentent soudainement des épisodes hémorragiques graves mettant leur vie en danger. Les seules stratégies qui éliminent efficacement les inhibiteurs du FVIII sont l'induction de la tolérance immune (ITI) pour les PwHA et le traitement immunosuppresseur (IST) pour les PwAHA. Malheureusement, l'ITI est inefficace dans 20 à 40% des cas et est extrêmement coûteuse et exigeante, tandis que l'IST s'accompagne d'un risque élevé d'infections graves et mortelles. Les mécanismes sous-jacents à l'induction de la tolérance immunitaire par ITI chez les PwHA sont encore mal connus.

Le premier objectif de mon travail de thèse a été de comprendre les mécanismes associés aux résultats de l'ITI en évaluant l'impact biologique de complexes immuns synthétiques contenant du FVIII (FVIII-IC), modélisant ceux qui peuvent se former au cours de l'ITI chez les patients, sur différentes cellules immunitaires. Le second objectif était d'estimer l'intérêt de l'enzyme bactérienne de Streptococcus pyogenes (IdeS) dégradant les immunoglobulines G comme nouvel outil efficace pour l'éradication des inhibiteurs du FVIII.

J'ai tout d'abord utilisé des cellules transduites équipées d'un système rapporteur de signalisation via les FcγRs par sécrétion d'interleukine 2. Nos résultats montrent que les FVIII-IC humains synthétiques signalisent efficacement et à différents niveaux par le biais des FcγRs humains. Les FVIII-IC réduisent l'endocytose du FVIII par les cellules dendritiques dérivées de monocytes humains (MoDCs), indépendamment de la sousclasse des IgG et de leur spécificité épitopique. Ces FVIII IC n'ont aucun impact sur la maturation des MoDCs, même en présence de signaux pro-inflammatoires. Cependant, les FVIII-IC contenant des IgG1 induisent l'activation des cellules NK via le CD16 (FcγRIII). Les résultats sur IdeS montrent que l'administration de cette enzyme à des souris HA sévères avec inhibiteurs du FVIII, permet d'ouvrir une fenêtre thérapeutique pour l'administration du FVIII et le rétablissement de l'hémostase. De plus, IdeS élimine efficacement in vitro et in vivo l'emicizumab, un anticorps mimétique du FVIII qui présente un risque prothrombotique quand il est combiné à des agents procoagulants pour traiter les saignements d'urgence.

Mes résultats montrent que les FVIII-IC pourraient avoir un impact biologique sur les fonctions des cellules immunitaires. D'autre part, IdeS apparaît comme un nouvel outil thérapeutique pour éradiquer les inhibiteurs du FVIII chez les PwHA et les PwAHA, ouvrant ainsi une fenêtre thérapeutique pour l'utilisation efficace du FVIII. En outre, dans le cas des PwHA sous prophylaxie avec emicizumab, IdeS élimine le médicament, ce qui permet d'utiliser en toute sécurité les agents procoagulants à haute dose ou le FVIII sans exposer les patients à des événements thrombotiques potentiellement mortels.

Hémophilie A -FVIIIinhibiteurs du FVIII -Enzyme dégradant les IgG

Ph.D. and the members of the Bayer BHAP Awards, CoMETH Awards, and Martin Villar Haemostasis Awards committees who believed in my research projects and awarded them financial support. It has been an honor to be named the winner of the 15 th Martin Villar Haemostasis Award in Basic Research. I would also like to thank all our collaborators.

Funding

Funding

This work was supported by 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). 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.

Title. FVIII-containing immune complexes reduce FVIII uptake by antigen-presenting cells and activate NK cells.

Discussion

Please refer to the discussion of the Ph.D. report.

Data availability

The authors declare that all data supporting the findings of this study are available within the paper and its supplementary information. Source data underlying the graphs presented in the main figures is available in Supplementary Data 1

List of tables

List of appendices

Article 3

Authors. Melissa Bou-Jaoudeh, 1 Sandrine Delignat, 1 Victoria Daventure, 1 Jordan D.

Dimitrov, 1 H.A. Daniel Lagassé, 2 Philipp Kolb, 3,4 Zuben E. Sauna, 2 Hartmut Hengel, 3,4 Sébastien Lacroix-Desmazes 1

Affiliations. 1 Institut National de la Santé et de la Recherche Médicale, Centre de Recherche des Cordeliers, CNRS, Sorbonne Université, Université Paris Cité, Paris, France; 2 Hemostasis Branch, Division of Plasma Protein Therapeutics, Office of Tissues and Advanced Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, MD, United States; 3 Institute of Virology, University Medical Center, Albert-Ludwigs-University Freiburg, Freiburg, Germany; 4 Faculty of Medicine, Albert-Ludwigs-University Freiburg, Freiburg, Germany.

Corresponding author.

Sébastien Lacroix-Desmazes (sebastien.lacroix-desmazes@inserm.fr) Content. 4 Figures,2 Tables;4 Supplementary Figures

Results

Validation and characterization of the recombinant human monoclonal IgG

The anti-FVIII humoral response in PwHA is represented in its large majority by polyclonal anti-FVIII antibodies of the IgG isotype. Among these, IgG1 represent the primary subclass in inhibitor-negative patients and in patients with low-titer inhibitors; conversely, IgG4 are more prominent in patients with high-titer inhibitors (Whelan et al., 2013). Here, we cloned and produced eight recombinant human monoclonal anti-FVIII IgG (rhIgG), using the VH and VL genes obtained from anti-FVIII IgG isolated from inhibitor-positive patients. BOIIB2, KM41, LE2E9 and BO2C11 are rhIgG specific for the A2, A3, C1 and C2 domains of FVIII, respectively. They were expressed both in the IgG1k and IgG4k formats. The purity and expected molecular weight of the rhIgG were validated by SDS-PAGE under non-reducing (Figure 1A) and reducing conditions (supplementary Figure S1A). For all rhIgG, one band around 150 kDa corresponding to non-reduced IgG was visible. In the case of the IgG4k, an expected additional band of low intensity was observed at 75 kDa (Figure 1A). Under reducing conditions, two bands were visible at 25 and 50 kDa corresponding to the light and heavy chains of the reduced IgG, respectively (supplementary Figure S1A).

The dose-dependent binding of the 8 rhIgG to immobilized FVIII confirmed the integrity of the Fab regions of the antibodies (supplementary Figure S1B). In agreement, the 8 rhIgG demonstrated high inhibitory activities towards FVIII in Bethesda assays, with no influence of the IgG subclass (Table 1). We then validated the integrity of the Fc fragment of the rhIgG. All IgG1k and IgG4k bound to the neonatal Fc receptor (FcRn) with affinities in the nanomolar range as measured by SPR (Table 2). Calculation of the association, dissociation and equilibrium affinity constants indicated that IgG1k bound FcRn with about 2 to 3-fold higher affinities than IgG4k, except for KM41. The difference was due to the increased dissociation rates associated with the IgG4 subclass. This is depicted in the case of BOIIB2 in Figure 1B.

To further confirm the structural integrity of the Fc fragments, we took advantage of transduced reporter cell lines (BW5147:FcγR-ζ) expressing different human FcγRs.

Incubation of the 8 rhIgG with the BW5147:FcγR-ζ cell line expressing the human FcγRIIIA FcγR-signalling was then evaluated upon measuring the secretion of IL-2 in the culture supernatant. IgG1k triggered IL-2 secretion only in the presence of FVIII for the three tested FcγR. IgG4k triggered IL-2 secretion only in the case of CD32A/B and in the presence of FVIII (Figure 2C). IgG4k failed to trigger IL-2 secretion in the case of CD16A.

FVIII-IC uptake by immature monocyte-derived dendritic cells

We then studied the effect of the engagement of FVIII in IC on its endocytosis by antigenpresenting cells. DCs were differentiated from monocytes of healthy individuals. Immature MoDCs expressed CD32 (supplementary Figure S3) to a greater extent than CD64 and failed to express CD16, as previously reported (Boruchov et al., 2005). FITC-labelled FVIII was pre-incubated alone or with a 5-molar excess of each single IgG1k or IgG4k, as well as with pools of the four IgG1k or the four IgG4k antibodies (BOIIB2, KM41, LE2E9, or BO2C11).

The endocytosis of FVIII-FITC by MoDCs was dose-dependent, as assessed by flow cytometry (data not shown) (Dasgupta et al., 2007).

The incubation of FVIII with individual anti-FVIII rhIgG resulted in a systematic and statistically significant decrease in FVIII uptake, irrespective of the epitope specificity or IgG1/IgG4 subclass of rhIgG (Figure 3A).

Thus, the levels of intracellular FVIII were reduced by 31±14, 23±9, 37±15, 50±12% in the case of BOIIB2, KM41, LE2E9 and BO2C11 IgG1k, and by 38±11, 27±16, 37±13, 53±10% in the case of the recombinant IgG4k, respectively. Interestingly, both the pools of rhIgG1k and IgG4k demonstrated increased reduction in FVIII uptake, yielding 64±14 % and 61±13 % inhibition for the IgG1k and IgG4k pools, respectively (Figure 3A).

Effect of FVIII-IC on the maturation of monocyte-derived dendritic cells

We then assessed whether FVIII-IC had an impact on the maturation phenotype of MoDCs following a 48-hr incubation (Figure 3B). There was no change in the surface expression of CD40, CD80, CD83 and CD86 when FVIII-IC were added to MoDCs. We also investigated whether the presence of FVIII-IC may alter the maturation of MoDCs in the context of stimulation of the cells with low (0.2 ng/ml) or high (100 ng/ml) levels of LPS.

Low levels of LPS did not induce the maturation of MoDCs. In contrast, 100 ng/ml LPS Impact of anti-FVIII IgG on the interaction between VWF and FVIII. A) FVIII consists of domains A1, A2, B, A3, C1 and C2. The A domains are bordered by acidic regions a1, a2 and a3. On the scheme are indicated the binding sites of the anti-FVIII antibodies BOIIB2, KM41, LE2E9 and BO2C11 directed against residues in the A2, A3, C1 and C2 domains of FVIII, respectively (Jacquemin et al., 1998(Jacquemin et al., , 2000;;van den Brink et al., 2001;[START_REF] Spiegel | Structure of a factor VIII C2 domain-immunoglobulin G4κ Fab complex: identification of an inhibitory antibody epitope on the surface of factor VIII[END_REF]. B) In the standard view and in accordance with crystal structures, FVIII domains are illustrated with the polypeptide chain running clockwise from A1 to C2. FVIII circulates in complex with VWF. Binding to VWF is approximated by its dimeric D′D3 fragment as illustrated, with major contacts between D′ and the C1 domain (three regions: W2062-S2069, T2086-S2095, and S2157-L2166). Additional interactions occur with the A3 domain (V1857-N1861 and E1875) and the a3 acidic peptide (V1670-D1678) as well as the C2 domain (Q2235-T2244) [START_REF] Chiu | Mapping the interaction between factor VIII and von Willebrand factor by electron microscopy and mass spectrometry[END_REF]. Sulfation of FVIII-Tyr1680 is required for efficient VWF binding, increasing the FVIII-VWF affinity fivefold. Values in the table depict mean ± SD.

REMERCIEMENTS

Ce travail a bénéficié du soutien de l'Inserm et du CNRS, de financements ANR-18-CE17-0010-02 et H2020-MSCA-ITN-2019 (number 859974). MBJ bénéficie d'une allocation de recherche de Sorbonne Université (École doctorale ED394).

LIENS D'INTÉRÊT

Les auteurs déclarent n'avoir aucun lien d'intérêt concernant les données publiées dans cet article.

BON DE COMMANDE

Competing interests

F.K. is an employee of CSL Behring AG, Switzerland. Other authors have no relevant competing interests.

Summary in French

Thesis Results show that the synthetic human FVIII-IC signaled efficiently and at different levels through individual human FcγRs using a cell-based reporter system. Moreover, FVIII-IC reduced FVIII uptake by human monocyte-derived dendritic cells (MoDCs) irrespective of IgG subclass and epitope specificity. These FVIII-IC had no impact on the maturation of MoDCs, even with additional stimuli. However, IgG1-containing FVIII-IC could activate NK cells via the CD16 (FcγRIII). Results on IdeS show that giving IdeS to high-titer inhibitorpositive HA mice opened a time window for FVIII to be administered efficiently and restore hemostasis. Moreover, IdeS was efficient in vitro and in vivo in eliminating emicizumab, a FVIII mimetic agent with a potential risk of thrombotic events when combined with pro-thrombotic bypassing agents to manage emergency bleeds.

Together these results demonstrate that FVIII-IC could have a biological impact on the functions of immune cells. Interestingly, IdeS appears as a potent new tool for the eradication of FVIII inhibitors in PwHA and PwAHA, thus opening a therapeutic window for the efficient use of FVIII. In PwAHA, which are typically elderly individuals with multiple comorbidities, IdeS may allow the use of FVIII instead of bypassing agents involving high thrombotic risk. In PwHA with inhibitors on emicizumab prophylaxis, IdeS can efficiently remove the drug and anti-FVIII IgG allowing FVIII replacement therapy for adequate follow-up of hemostatic efficacy. This approach is useful for programmed surgeries with a high hemorrhagic risk where no guidelines are available and patients are treated with rFVIIa without reliable biomarkers for hemostatic efficacy. It can also be useful in cases of breakthrough bleeds where rFVIIa is inefficient and aPCC needs to be used for more than 24 hours without exposing the patients to thrombotic events.

Hemophilia A -FVIII -FVIII inhibitors -IgG-degrading enzyme