Kathleen ; Pratt

Valder R Arruda
email: arruda@email.chop.edu

Sébastien Lacroix-Desmazes

Kathleen P Pratt

Inhibitors -Recent Insights

Keywords: Factor VIII, haemophilia A, protein immunogenicity, FVIII inhibitors, immune tolerance, gene therapy Haemophilia Factor VIII, haemophilia A, protein immunogenicity, FVIII inhibitors, immune tolerance, gene therapy

teaching and research institutions in France or abroad, or from public or private research centers.

We have modified the sentence, in roder to avoid the used of "waned" and mellow down the statement We hope that the MS is now ready for acceptance at Haemophilia. And I want to apologize once again for all the back and forth and missing deadlines.

Introduction

The problem of inhibitory antibody development against factor VIII (FVIII) has been central to the treatment of hemorrhages in haemophilia A (HA) patients using exogenous therapeutic FVIII. The field has recently evolved with the advent of FVIIIfree hemostatic agents, notably the bispecific antibody emicizumab (Genentech, Inc.), which does not induce neutralizing anti-FVIII antibodies (inhibitors) and is efficacious for prophylaxis even in the presence of inhibitory anti-FVIII IgG. However, therapeutics that can partially "bypass" the need for FVIII, such as emicizumab, activated Prothrombin Coagulant Complex (aPCC), or recombinant FVIIa, are all less effective than FVIII itself in treating major traumatic bleeds or as a therapeutic to support patients during surgery. In addition, administration of aPCC at higher doses (1), or aPCC plus emicizumab, carries a risk of thrombotic events (2,3). It is the shared feeling of the authors of this review that, unless proven wrong by future data, no non-FVIII molecule is or will be as fully efficacious as FVIII in performing all of the functions of FVIII. Hence, therapeutic FVIII will often remain the optimal agent to arrest serious bleeding. Therefore, as most patients will experience significant bleeding events over the course of their lifetime, preventing and reversing inhibitor development should remain a clinical priority.

Immune responses to FVIII and FIX

Therapies for HA and haemophilia (HB) share a common goal of replacing the patient's missing or defective clotting factor at levels sufficient to maintain haemostasis.

Worldwide, most patients with access to haemophilia care are treated by intravenous infusions of human plasma-derived FVIII or factor IX (FIX), respectively. Prophylactic administration to maintain trough (minimum) levels of the missing factor requires frequent infusions, placing a burden on patients and their families, although factors with increased half-lives have recently become available, with further improvements expected in the future (Chhabra et al Blood 2020, in press PMID:32078672). For patients with severe HA or HB, whose genetic mutation precludes expression of any FVIII or FIX protein, respectively, the infused clotting factor may be perceived by their immune system as "foreign". Patients who circulate a dysfunctional FVIII or FIX protein, e.g. due to missense mutations, may also develop an immune response, although inhibitor incidences in these patients are lower (4). Approximately half of severe HA (SHA) patients who develop an inhibitor do so within the first 20 FVIII infusions (5), reminiscent of the sequential "prime + boost" injections required for effective vaccination responses, and their inhibitor risk declines to insignificant after 50 exposure days (6,7). It should be noted, however, that lower inhibitor incidences in mild or moderately severe HA are due in part to their lower exposure to therapeutic FVIII. Inhibitors have been reported in these patients after 20-50 exposure days, and those with >50 exposure days have a similar risk to that of multiply infused patients with SHA (8), while the risk of developing an inhibitor after 50 exposure days is quite low. Risks increase again in the aging HA population, especially in those undergoing surgery requiring more intensive FVIII therapy (9). This review focuses primarily on HA and FVIII, but keep in mind that many of the principles of FVIII immunogenicity apply to FIX immunogenicity as well. One major difference is that most mutations resulting in haemophilia B cause only minor structural changes to the FIX protein, although these defects are sufficient to reduce or completely abrogate its clotting activity through disabling its serine protease function.

In contrast, almost half of SHA cases are caused by inversion mutations that seriously significant inhibitors afflict a larger fraction of severe SHA patients (~1 in 4) than severe haemophilia B patients. It is not known if this is due to the larger number of potential epitopes in FVIII administered to SHA patients, compared to epitopes in FIX administered to HB patients who circulate a defective FIX protein, or if the higher inhibitor is incidence is due to a greater inherent immunogenicity of FVIII compared to FIX. Finally, some HB patients develop anaphylactic responses to therapeutic FIX, whereas such responses to FVIII are extremely rate in HA patients.

It remains undetermined whether the immune systems of inhibitor-negative patients ignore the therapeutic molecule or actively tolerate it. FVIII is administered intravenously, which is considered a relatively tolerogenic delivery route due to its rapid clearance as blood is filtered and cleared by the spleen and liver, with (usually) a lack of significant inflammation at the injection site. In the absence of danger/adjuvant, foreign substances encountered in the gut, venous or lymphatic system are typically ignored or tolerated by the immune system. In fact, most HA patients can successfully achieve haemostasis through intravenous FVIII administration. Why, then, do inhibitors develop in some but not all patients? The type of genetic mutation is somewhat predictive of risk (10). However, discordant immune responses have been noted between patients with the same haemophilia-causing mutation, and even in identical twins with SHA (11), strongly indicating that environmental factors are also important.

Inflammation surely plays a role, and indeed, trauma, and surgery are acknowledged risk factors. Higher doses of FVIII are often required in these settings, further increasing inhibitor risk, although this intensive FVIII therapy is unavoidable because of the need to prevent excessive blood loss. Interestingly, one study using blood samples from a SHA inhibitor research subject (volunteer blood donor) indicated that high-affinity CD4 + T cells recognized only a very limited number of FVIII epitopes (13). This in turn suggested that many potentially pathogenic FVIII-specific T-effector cells may actually be deleted following initial FVIII exposures, and that inhibitors may develop when this tolerance-promoting mechanism breaks down, as the remaining FVIII-responsive T-effector cells continue to circulate, contributing to ongoing B-cell stimulation (14). This hypothesis requires further testing. FVIII epitopes recognized by B cells and by inhibitory antibodies have also been mapped using various methods (15), and in addition to defining specific immunogenic sites, these studies have identified functionally important regions of the FVIII protein that are critical for its binding to platelets and other components of the blood clotting cascade reaction. An exciting, recent clinical breakthrough has been the introduction of a bispecific therapeutic antibody, emicizumab, which mimics the pro-coagulant function of activated FVIII. This therapeutic agent has transformed HA care by providing effective hemostasis to patients even in the presence of a high-responding inhibitor (2,3).

FVIII-free hemostatic "bypass" agents

Initially approved only for inhibitor patients, emicizumab is increasingly being considered or adopted by non-inhibitor patients as well, offering dual advantages of subcutaneous administration and less frequent infusions compared to standard prophylactic FVIII therapy. We are still learning about relative benefits and limitations as "real-world" experience with this therapeutic approach accumulates (16), and pharmaceutical companies are currently developing potential next-generation bispecific antibodies. emicizumab is less effective than FVIII in supporting major surgery or controlling serious traumatic bleeds, and careful administration of appropriate alternative bypass agents is still required in cases where an inhibitor precludes effective use of FVIII. Activated prothrombin complex concentrates (aPCC) are not recommended for patients on emicizumab because of potentially increased risk of thrombosis when these therapeutics are combined (16,17), while recombinant FVIIa has been shown to have a better safety profile, indicating it may be administered together with emicizumab to control serious bleeds in inhibitor patients (17). Additional non-FVIII hemostatic agents, most still preclinical and beyond the scope of this review, are also unlikely to fully compensate for FVIII in all situations. For these reasons, maintaining or restoring tolerance to FVIII in non-inhibitor and inhibitor patients, respectively, including those successfully utilizing a non-FVIII therapeutic alone for prophylactic therapy, remains a high priority. 8 Some compelling research questions, which will require future well-designed clinical and basic science studies, are: how much FVIII will be required to maintain tolerance in patients receiving prophylactic non-FVIII therapies, how often must it be administered and at what dose, and what is the best route and formulation? One current approach to address these questions in the case of patients receiving emicizumab prophylaxis is the "Atlanta protocol", in which ITI is carried out by infusing FVIII at 50-100 IU/kg, 3X/week (18). This protocol built on the International ITI study (19) in which there were no significant differences in rates of overall successful tolerance between groups of patients that received a low dose (50 IU/kg 3X/week) versus high dose (100 IU/kg daily) FVIII regimen; the low-dose arm experienced more bleeds, but administering the lowest-dose ITI regimen to patients on emicizumab prophylaxis would not be expected to incur an increased bleeding risk. A prospective clinical trial is now being conducted, but at present there are no conclusive data indicating the relative effectiveness of this or similar protocols in producing either shortterm or durable peripheral tolerance to FVIII. Yet, if FVIII desensitization is achieved in inhibitor-positive patients receiving hemostasis through bispecific antibody therapy, should the clinicians, and the patients, continue FVIII infusions to maintain active tolerance in case therapeutic FVIII is needed in the future? Because of less bleeding, inhibitor patients on bispecific antibody prophylaxis are expected to show less inflammation, and this may actually improve their ITI success rates; this possibility will be answered by longer-term studies of patient outcomes. Oral tolerance methods, which are in the preclinical pipeline, may eventually prove a more acceptable alternative compared to ongoing FVIII infusions, as they do not require frequent needle pokes (20). (21). This selection process also generates natural CD4 + CD25 + Foxp3 + regulatory T cells (nTregs), which down-regulate T-effector and B-cell responses (22), thereby preventing immune responses and associated inflammatory processes from becoming pathogenic. HA patients who circulate a dysfunctional FVIII protein (generally resulting in mild or moderate severity HA) are expected to have acquired central tolerance to this selfprotein, which decreases but does not eliminate their risk of developing an immune response to regions of therapeutic FVIII that differ from their endogenous protein.

Patients with SHA are heterogenous, with ~45% having a F8 intron-22 inversion mutation, and the rest having nonsense, frameshift, missense, etc. mutations that preclude production of a functional FVIII protein. Depending on the presence or absence of a non-functional or dysfunctional endogenous FVIII molecule, HA patients are categorised as cross-reactive material-positive (CRM pos ) or CRM neg . The type of HA-causing gene abnormality, and the associated presence/absence of the FVIII antigen, are thought to be predominant risk factors for inhibitor development (10).

Peripheral tolerance develops primarily in organized secondary lymphoid organs, e.g., lymph nodes, spleen, and lamina propria. The primary clearance site for FVIII is the liver, which also provides a mostly tolerogenic environment. Additional types of Tregs may also be induced at the periphery upon encounter with a self-or non-self antigen, F o r P e e r R e v i e w 10 including CD4 + CD25 + Foxp3 + induced Tregs (iTregs), and CD4 + cells that secrete the tolerogenic cytokines IL-10, TGFß and/or IL-35 and that express inhibitory receptors such as CTLA-4 and PD-1 (23). These iTregs can promote peripheral tolerance to foreign proteins in the periphery, especially if the initial encounter is in a noninflammatory context (absence of immunologic "danger" signals (24)). For example, we are all exposed to numerous foreign substances in our diet, but most of them do not provoke a pathogenic immune response. There is tremendous interest in potentially harnessing the power of iTregs to promote tolerance of HA patients to FVIII, as in principle such strategies would avoid undesirable side effects of more general immunosuppression therapies (25). For example, "oral tolerance induction" has been achieved in animal models by feeding HA mice plants that were engineered to produce FVIII (20).

Is FVIII inherently immunogenic, even in non-HA individuals?

One would expect the healthy, non-HA population to develop central tolerance to FVIII, which is of course a self-protein. However, recent work demonstrated the presence of FVIII-specific CD4 + T cells in non-HA individuals (26). Importantly, half of the circulating FVIII-specific CD4 + T cells had a memory phenotype, which may explain why anti-FVIII IgG can be found in many healthy donors (27)(28)(29). Together, these observations suggest that central tolerance towards FVIII is not complete. It further suggests that an active, albeit peripherally-regulated, anti-FVIII immune response is at play in non-HA individuals. In agreement with this, an increased prevalence of FVIII-reactive TGFß + Th3 cells was shown in healthy donors (30) as well as the presence, among peripheral blood cells of healthy donors, of CD4 + CD25 + Foxp3 + Tregs able to control the in vitro expansion of effector CD4 + T cells in the presence of FVIII (31).

T-cell tolerance to FVIII in HA

As explained above, central tolerance to FVIII is impaired in healthy individuals. While not at play in CRM neg patients (because they do not produce FVIII), we hypothesize that central tolerance to FVIII is also impaired in CRM pos patients. In this context, inhibitor risk should reflect the ability to develop (or not) peripheral tolerance, similar to that of the non-HA population, with this tolerization directly influenced by the quantity and/or nature of the endogenous dysfunctional FVIII protein (if any). This hypothesis was tested and somewhat validated in the case of patients with missense mutations (32)(33)(34), but with the caveat as mentioned above that increased exposure to therapeutic FVIII increases their risk substantially. Yet, about 70% of SHA patients, most of whom are CRM neg , never develop clinically significant FVIII inhibitors. This indicates that the presence of the endogenous molecule is not an absolute necessity to develop tolerance and, conversely, that absence of FVIII does not warrant the inevitable triggering of a high-titer antibody response.

Danger theory

The danger theory posits that the immune system does not discriminate per se between self-and non-self-antigens, as the decision to develop immune responses also requires a pro-inflammatory context that prevails in the microenvironment where the antigen is encountered by immune cells. In the context of HA, several triggers able to evoke danger signals have been investigated. The possibility that vaccination at the time or close to administration of therapeutic FVIII signals danger has been invalidated both in preclinical and epidemiological studies (35,36). Likewise, experiments in FVIIIdeficient mice suggest that acute bleeds or surgery do not increase inhibitor risk or the intensity of the immune response (37). Conversely, the dose or intensity of FVIII was confirmed as a risk factor both in mice (38) and in patients (39). Yet, the fact that highdose FVIII used in ITI is able to eliminate FVIII inhibitors in up to 80% of patients indicates that the relationship between FVIII dose and inhibitor risk is far from linear.

In agreement with this, the administration of therapeutic FVIII to SHA patients at distance from presumed danger triggers (major bleeds, surgery, infection, vaccination) did not show consistency in preventing inhibitor development (40,41). Taken together, the available evidence suggests that the decision for the immune system to develop a neutralizing FVIII-specific immune response is not merely under the control of exogenous or endogenous inflammatory insults/danger signals. Instead, for reasons which remain unclear (tropism of FVIII to the spleen (42), or presence on the FVIII molecule of molecular patterns mediating endocytosis by antigen-presenting cells (43)(44)(45)), FVIII can trigger activation of the immune system even without adjuvant/"danger" signals.

Anti-danger theory

In an attempt to decipher whether hemolysis and heme release foster the development of FVIII inhibitors by providing danger signals, FVIII-deficient mice were injected with heme prior to administration of FVIII (46). Unexpectedly, heme injection almost completely abrogated the onset of the anti-FVIII immune response, due to antiinflammatory enzyme heme-oxygenase-1 (HO-1) induction and the ensuing catabolism of heme into the anti-inflammatory molecules carbon monoxide and biliverdin (46). The distribution of long guanine-thymine (GT) repeats in the hmox1 gene promoter region, which encodes HO-1, in a retrospective cohort of SHA patients was associated with a reduced capacity of the cells to express HO-1 in vitro (47) and (IDO-1) following in vitro TLR9 stimulation by CpG-ODN (49). In contrast, dendritic cells from inhibitor-negative patients could differentiate CD4 + T cells into regulatory T cells in vitro when stimulated by CpG-ODN (49). Thus, the risk for a patient to develop clinically relevant inhibitory anti-FVIII IgG may not merely rely on the presence of particular danger signals during FVIII administration, but rather on the intrinsic (genetic) capacity of the patient to mount protective anti-inflammatory and/or immunoregulatory responses.

Novel tolerogenic cellular therapies

Multiple preclinical models aimed at the use of immune therapy to either prevent or eradicate inhibitors in hemophilia are currently under development (25,(50)(51)(52). Some of these approaches are similar to recent novel cancer therapies, in which cells from a patient's blood are engineered to recognize specific targets on tumors, multiplied in cell culture, and then infused back into the patient (53). For example, re-targeting of CD8 + T cells to recognize CD19 + B cells from patients with acute or chronic leukemia/lymphomas culminated in recent approvals by US and EU regulatory agencies (54). In addition to cancer immunotherapies, there has been great interest in adapting cellular engineering strategies based on similar principles to develop novel therapies for benign auto/allo-immune diseases. For example, proof-of-principle studies have characterized chimeric antigen receptors on CD8 + T cells targeting desmoglin 3-expressing B cells, which are the target of the underlying pathology of (55). CD8 + T cells are not a feature of immune responses to FVIII, however, the properties of engineered CD8 + T cells may be exploited in attempts to eliminate inhibitors, e.g. by targeting FVIII-specific B cells (56).

In applications to the FVIII inhibitor problem, the goals are to either promote tolerance to FVIII via engineered Tregs, or to eliminate FVIII-specific B cells. Because the engineered cells are highly specific for FVIII, such approaches should in principle avoid causing general immunosuppression, which can increase risks of infection and even some cancers. Promising results have been obtained using appropriate animal models, and there is hope that one or more of these approaches may eventually be tested in clinical trials. The more that scientists learn about these immune mechanisms, the likelier we are to see advances in tolerizing HA patients to therapeutic FVIII, delivered either intravenously or via gene therapy.

Gene therapy as a tolerogenic strategy

An early report by Ashanti et al. showed the efficacy of orthotropic liver transplant (OLT) in an adult SHA patient complicated by a rapid, progressive hepatocellular carcinoma and a high-titer inhibitor (57). Progressively increasing FVIII activity levels were detected post-transplant, with normalization of infused FVIII recovery (Fig 1 ) and inhibitor eradication during follow up (~4 months post-transplant). Notably, the patient's inhibitor titer increased immediately after the transplant, a finding that resembled the anamnestic response to FVIII noted in many inhibitor patients upon initiation of ITI.

However, induction of tolerance to FVIII was evidenced by disappearance of the inhibitor and normalization of FVIII recovery. However, subsequent OLT for inhibitor patients were associated with lethal systemic microangiopathy or lack of efficacy (58, ). Moreover, a non-HA OLT patient who received a liver from a HA donor with no evidence of a recent inhibitor unexpectedly developed a high-titer inhibitor posttransplant. Re-examination of the donor's medical history showed an inhibitor had been detected earlier (but not close to the OLT), indicating the primary resident FVIIIspecific lymphocytes were still viable, and expansion after FVIII exposure resulted in inhibitor formation. This patient required a second liver transplant (60). Despite these disparate clinical outcomes, these early results encouraged animal model studies to evaluate the potential tolerogenic delivery of FVIII to the liver through gene therapy, rather than OLT.

Collective data from mouse models for genetic or acquired diseases indicate that liver gene therapy using AAV vectors for hepatocyte-restricted transgene expression could prevent the formation of pathological antibodies to the transgene and eradicate preexisting antibodies. This apparent immune tolerance induction was transgene-specific, without causing systemic down-regulation of host immune competence. The underlying mechanisms of immune tolerance in this model involve complex and diverse pathways, and some of these strategies could be "mimicked" by pharmacologic or molecular modifications (61)(62)(63). The question of whether the scientific, regulatory and patient communities would jump from results obtained in rodent models, which have shown efficacy and reproducibility, directly to human gene therapy trials for inhibitor eradication has focused attention on the need for careful assessment of available information from large-animal disease models as well.

Large animal models for the assessment of immune tolerance following AAV liver gene therapy for hemophilia

Inhibitor eradication in severe HA and HB dogs by AAV liver gene therapy

In 2010, Finn et al reported the proof-of-principal use of AAV serotype 8 for liver gene therapy in HA dogs with inhibitors to cFVIII (65), as summarized in Table 1. cFVIII transgene expression was achieved using a hepatocyte-specific promoter delivered by IV injection. The primary endpoint was inhibitor disappearance in all 4 dogs in ~5 weeks for the UNC-CH colony, and in ~18 months for the Queen's University colony.

Circulating cFVIII activity and antigen levels increased steadily following inhibitor eradication. Subsequent challenges with cFVIII protein showed no evidence of an immune response and demonstrated normalization of FVIII recovery. The latter is a clinical surrogate test for the presence of both neutralizing and non-neutralizing antibodies (which may accelerate clearance of the protein). A substantial reduction of spontaneous bleeds further confirmed the success of this strategy in improving the disease phenotype. Together, these findings demonstrate the potential of AAV liver gene therapy for inhibitor eradication in large animal HA models. Moreover, successful and safe eradication of FIX inhibitors was demonstrated in severe HB dogs using an AAV vector encoding a hyper-functional cFIX (64). Future studies in canine models with preexisting high titer inhibitors will be required to test the feasibility of this strategy. This is a critical requirement for proposed clinical studies in human subjects with high titer and/or refractory inhibitors.

Is an intensive immune suppression regimen a safe strategy coupled with liver gene therapy to eradicate inhibitors?

There is evidence that T-regulatory cells (Tregs) were generated at early time points following AAV liver gene therapy (62). It is conceivable that combining intensive immune suppression with AAV liver gene therapy could interfere with the generation and/or function of these cells. For example, Mingozzi et al. showed that administering a specific antibody to CD25, which was meant to target T-effector cells at early time points following AAV liver gene therapy in a NHP model, actually decreased the numbers and function of the Tregs, thus facilitating inhibitor formation (67). Recent AAV liver gene therapy efforts have coupled vector administration with transient immunosuppression methods to prevent or reverse AAV capsid-mediated liver toxicity and transgene expression loss. More recently, it was observed that the use of rabbit antithymocyte globulin (ATG) administration at the time of AAV delivery results in a strong anti-transgene-product antibody response, while delaying ATG by only ~5 weeks does not. The underlying mechanism of this anti-transgene-product antibody response is, at least in part, associated with a skewing of the ratio of Th17/Treg cells (Finn et al, Mol Ther 2020 in press). Combined, these results suggest that there is a critical time window around vector administration for transgene-product tolerance that is Treg-dependent, which can be disrupted by intense immunosuppression. Thus, extensive further testing prior to contemplating clinical gene therapy studies employing

Questions and future directions for gene therapy to promote tolerance

In deciding how to select the best population to test a given gene therapy for inhibitor eradication, the following aspects should be taken into consideration: a. Children and subjects with recently developed lower-titer inhibitors have higher ITI success rates than adults with longer-duration inhibitors. Should only patients with more encouraging prognoses be enrolled? What about pediatric subjects who failed ITI? By definition, adults have a poorer-risk prognosis (duration of inhibitor, high titers, etc.).

b. What is the optimal vector design and transgene levels strategy for a successful ITI?

Although AAV vectors using a hepatocyte-specific promoter is a common requirement for ITI, the relative importance of vector capsid, vector dose and transgene (FVIII wild type or variants) are debatable. Overall, high transgene levels are desirable for eradication of inhibitors, but the use of strategies associated with low levels of expression may be also beneficial. In both systems, could the continuous noninterrupted endogenous expression ("maximum compliance") be the main determinant of the ITI success? As noted in the International-ITI study, the high-dose cohort was (29,69). The majority of multiply-infused HA patients develop peripheral tolerance to FVIII, while the immune system of the remaining patients is incapable of maintaining active immune tolerance, and therefore clinically-relevant neutralizing antibodies arise. Protective immune tolerance may, however, be triggered in inhibitor patients upon appropriate stimulation of their immune system, for instance by ITI. In the near future, additional options will become available to foster FVIII-specific peripheral immune tolerance in inhibitor-positive patients, possibly including administration of FVIII orally or during fetal life (70) or induction of FVIII production at immuno-tolerogenic sites (i.e., liver targeted gene therapy). Whether naturally-induced or enforced upon the organism, it is important to keep in mind that the established peripheral immune tolerance to FVIII is a homeostatic equilibrium. It is prone to perturbations; risks of developing inhibitors may re-appear, for instance at an older age when the immune system declines and the potency of Tregs weakens (71). Therefore, establishing and maintaining durable tolerance to FVIII remains a strong priority.

F

Introduction

The problem of inhibitory antibody development against factor VIII (FVIII) has been central to the treatment of hemorrhages in haemophilia A (HA) patients using exogenous therapeutic FVIII. The field has recently evolved with the advent of FVIIIfree hemostatic agents, notably the bispecific antibody emicizumab (Genentech, Inc.), which does not induce neutralizing anti-FVIII antibodies (inhibitors) and is efficacious for prophylaxis even in the presence of inhibitory anti-FVIII IgG. However, therapeutics that can partially "bypass" the need for FVIII, such as emicizumab, activated

Prothrombin Coagulant Complex (aPCC), or recombinant FVIIa, are all less effective than FVIII itself in treating major traumatic bleeds or as a therapeutic to support patients during surgery. In addition, administration of aPCC at higher doses (1), or aPCC plus emicizumab, carries a risk of thrombotic events (2, 3). It is the shared feeling of the authors of this review that, unless proven wrong by future data, no non-FVIII molecule is or will be as fully efficacious as FVIII in performing all of the functions of FVIII. Hence, therapeutic FVIII will often remain the optimal agent to arrest serious bleeding. Therefore, as most patients will experience significant bleeding events over the course of their lifetime, preventing and reversing inhibitor development should remain a clinical priority.

Immune responses to FVIII and FIX

Therapies for HA and haemophilia (HB) share a common goal of replacing the patient's missing or defective clotting factor at levels sufficient to maintain haemostasis.

Worldwide, most patients with access to haemophilia care are treated by intravenous infusions of human plasma-derived FVIII or factor IX (FIX), respectively. Prophylactic administration to maintain trough (minimum) levels of the missing factor requires This review focuses primarily on HA and FVIII, but keep in mind that many of the principles of FVIII immunogenicity apply to FIX immunogenicity as well. One major difference is that most mutations resulting in haemophilia B cause only minor structural changes to the FIX protein, although these defects are sufficient to reduce or completely abrogate its clotting activity through disabling its serine protease function.

In It remains undetermined whether the immune systems of inhibitor-negative patients ignore the therapeutic molecule or actively tolerate it. FVIII is administered intravenously, which is considered a relatively tolerogenic delivery route due to its rapid clearance as blood is filtered and cleared by the spleen and liver, with (usually) a lack of significant inflammation at the injection site. In the absence of danger/adjuvant, foreign substances encountered in the gut, venous or lymphatic system are typically ignored or tolerated by the immune system. In fact, most HA patients can successfully achieve haemostasis through intravenous FVIII administration. Why, then, do inhibitors develop in some but not all patients? The type of genetic mutation is somewhat predictive of risk (10). However, discordant immune responses have been noted

between patients with the same haemophilia-causing mutation, and even in identical twins with SHA (11), strongly indicating that environmental factors are also important.

Inflammation surely plays a role, and indeed, trauma, and surgery are acknowledged risk factors. Higher doses of FVIII are often required in these settings, further increasing inhibitor risk, although this intensive FVIII therapy is unavoidable because of the need to prevent excessive blood loss. Interestingly, one study using blood samples from a SHA inhibitor research subject (volunteer blood donor) indicated that high-affinity CD4 + T cells recognized only a very limited number of FVIII epitopes (13). This in turn suggested that many potentially pathogenic FVIII-specific T-effector cells may actually be deleted following initial FVIII exposures, and that inhibitors may develop when this tolerance-promoting mechanism breaks down, as the remaining FVIII-responsive T-effector cells continue to circulate, contributing to ongoing B-cell stimulation (14). This hypothesis requires further testing.

FVIII epitopes recognized by B cells and by inhibitory antibodies have also been mapped using various methods (15), and in addition to defining specific immunogenic sites, these studies have identified functionally important regions of the FVIII protein that are critical for its binding to platelets and other components of the blood clotting cascade reaction. Initially approved only for inhibitor patients, emicizumab is increasingly being considered or adopted by non-inhibitor patients as well, offering dual advantages of subcutaneous administration and less frequent infusions compared to standard prophylactic FVIII therapy. We are still learning about relative benefits and limitations as "real-world" experience with this therapeutic approach accumulates ( 16), and pharmaceutical companies are currently developing potential next-generation bispecific antibodies. emicizumab is less effective than FVIII in supporting major surgery or controlling serious traumatic bleeds, and careful administration of appropriate alternative bypass agents is still required in cases where an inhibitor precludes effective use of FVIII. Activated prothrombin complex concentrates (aPCC)

FVIII-free hemostatic "bypass" agents

are not recommended for patients on emicizumab because of potentially increased risk of thrombosis when these therapeutics are combined (16,17), while recombinant FVIIa has been shown to have a better safety profile, indicating it may be administered together with emicizumab to control serious bleeds in inhibitor patients (17). Additional non-FVIII hemostatic agents, most still preclinical and beyond the scope of this review, are also unlikely to fully compensate for FVIII in all situations. For these reasons, maintaining or restoring tolerance to FVIII in non-inhibitor and inhibitor patients, respectively, including those successfully utilizing a non-FVIII therapeutic alone for prophylactic therapy, remains a high priority. current approach to address these questions in the case of patients receiving emicizumab prophylaxis is the "Atlanta protocol", in which ITI is carried out by infusing FVIII at 50-100 IU/kg, 3X/week (18). This protocol built on the International ITI study (19) in which there were no significant differences in rates of overall successful tolerance between groups of patients that received a low dose (50 IU/kg 3X/week) versus high dose (100 IU/kg daily) FVIII regimen; the low-dose arm experienced more bleeds, but administering the lowest-dose ITI regimen to patients on emicizumab prophylaxis would not be expected to incur an increased bleeding risk. A prospective clinical trial is now being conducted, but at present there are no conclusive data indicating the relative effectiveness of this or similar protocols in producing either shortterm or durable peripheral tolerance to FVIII. Yet, if FVIII desensitization is achieved in inhibitor-positive patients receiving hemostasis through bispecific antibody therapy, should the clinicians, and the patients, continue FVIII infusions to maintain active tolerance in case therapeutic FVIII is needed in the future? Because of less bleeding, inhibitor patients on bispecific antibody prophylaxis are expected to show less inflammation, and this may actually improve their ITI success rates; this possibility will be answered by longer-term studies of patient outcomes. Oral tolerance methods, which are in the preclinical pipeline, may eventually prove a more acceptable alternative compared to ongoing FVIII infusions, as they do not require frequent needle pokes (20). including CD4 + CD25 + Foxp3 + induced Tregs (iTregs), and CD4 + cells that secrete the tolerogenic cytokines IL-10, TGFß and/or IL-35 and that express inhibitory receptors such as CTLA-4 and PD-1 (23). These iTregs can promote peripheral tolerance to foreign proteins in the periphery, especially if the initial encounter is in a noninflammatory context (absence of immunologic "danger" signals (24)). For example, we are all exposed to numerous foreign substances in our diet, but most of them do not provoke a pathogenic immune response. There is tremendous interest in potentially harnessing the power of iTregs to promote tolerance of HA patients to FVIII, as in principle such strategies would avoid undesirable side effects of more general immunosuppression therapies (25). For example, "oral tolerance induction" has been achieved in animal models by feeding HA mice plants that were engineered to produce FVIII (20).

Is FVIII inherently immunogenic, even in non-HA individuals?

One would expect the healthy, non-HA population to develop central tolerance to FVIII, which is of course a self-protein. However, recent work demonstrated the presence of FVIII-specific CD4 + T cells in non-HA individuals (26). Importantly, half of the circulating FVIII-specific CD4 + T cells had a memory phenotype, which may explain why anti-FVIII IgG can be found in many healthy donors (27)(28)(29). Together, these observations suggest that central tolerance towards FVIII is not complete. It further suggests that an active, albeit peripherally-regulated, anti-FVIII immune response is at play in non-HA individuals. In agreement with this, an increased prevalence of FVIII-reactive TGFß + Th3 cells was shown in healthy donors (30) as well as the presence, among peripheral blood cells of healthy donors, of CD4 + CD25 + Foxp3 + Tregs able to control the in vitro expansion of effector CD4 + T cells in the presence of FVIII (31).

T-cell tolerance to FVIII in HA

As explained above, central tolerance to FVIII is impaired in healthy individuals. While not at play in CRM neg patients (because they do not produce FVIII), we hypothesize that central tolerance to FVIII is also impaired in CRM pos patients. In this context, inhibitor risk should reflect the ability to develop (or not) peripheral tolerance, similar to that of the non-HA population, with this tolerization directly influenced by the quantity and/or nature of the endogenous dysfunctional FVIII protein (if any). This hypothesis was tested and somewhat validated in the case of patients with missense mutations (32)(33)(34), but with the caveat as mentioned above that increased exposure to therapeutic FVIII increases their risk substantially. Yet, about 70% of SHA patients, most of whom are CRM neg , never develop clinically significant FVIII inhibitors. This indicates that the presence of the endogenous molecule is not an absolute necessity to develop tolerance and, conversely, that absence of FVIII does not warrant the inevitable triggering of a high-titer antibody response.

Danger theory

The danger theory posits that the immune system does not discriminate per se between self-and non-self-antigens, as the decision to develop immune responses also requires a pro-inflammatory context that prevails in the microenvironment where the antigen is encountered by immune cells. In the context of HA, several triggers able to evoke danger signals have been investigated. The possibility that vaccination at the time or close to administration of therapeutic FVIII signals danger has been invalidated both in preclinical and epidemiological studies (35,36). Likewise, experiments in FVIIIdeficient mice suggest that acute bleeds or surgery do not increase inhibitor risk or the intensity of the immune response (37). Conversely, the dose or intensity of FVIII was confirmed as a risk factor both in mice (38) and in patients (39). Yet, the fact that highdose FVIII used in ITI is able to eliminate FVIII inhibitors in up to 80% of patients indicates that the relationship between FVIII dose and inhibitor risk is far from linear.

In agreement with this, the administration of therapeutic FVIII to SHA patients at distance from presumed danger triggers (major bleeds, surgery, infection, vaccination)

did not show consistency in preventing inhibitor development (40,41). Taken together, the available evidence suggests that the decision for the immune system to develop a neutralizing FVIII-specific immune response is not merely under the control of exogenous or endogenous inflammatory insults/danger signals. Instead, for reasons which remain unclear (tropism of FVIII to the spleen (42), or presence on the FVIII molecule of molecular patterns mediating endocytosis by antigen-presenting cells (43)(44)(45)), FVIII can trigger activation of the immune system even without adjuvant/"danger" signals.

Anti-danger theory

In an attempt to decipher whether hemolysis and heme release foster the development of FVIII inhibitors by providing danger signals, FVIII-deficient mice were injected with heme prior to administration of FVIII (46). Unexpectedly, heme injection almost completely abrogated the onset of the anti-FVIII immune response, due to antiinflammatory enzyme heme-oxygenase-1 (HO-1) induction and the ensuing catabolism of heme into the anti-inflammatory molecules carbon monoxide and biliverdin (46). The distribution of long guanine-thymine (GT) repeats in the hmox1 gene promoter region, which encodes HO-1, in a retrospective cohort of SHA patients was associated with a reduced capacity of the cells to express HO-1 in vitro (47) and

Novel tolerogenic cellular therapies

Multiple preclinical models aimed at the use of immune therapy to either prevent or eradicate inhibitors in hemophilia are currently under development (25,(50)(51)(52). Some of these approaches are similar to recent novel cancer therapies, in which cells from a patient's blood are engineered to recognize specific targets on tumors, multiplied in cell culture, and then infused back into the patient (53). For example, re-targeting of CD8 + T cells to recognize CD19 + B cells from patients with acute or chronic leukemia/lymphomas culminated in recent approvals by US and EU regulatory agencies (54). In addition to cancer immunotherapies, there has been great interest in adapting cellular engineering strategies based on similar principles to develop novel therapies for benign auto/allo-immune diseases. For example, proof-of-principle studies have characterized chimeric antigen receptors on CD8 AAV vector encoding a hyper-functional cFIX (64). Future studies in canine models with preexisting high titer inhibitors will be required to test the feasibility of this strategy. This is a critical requirement for proposed clinical studies in human subjects with high titer and/or refractory inhibitors.

Is an intensive immune suppression regimen a safe strategy coupled with liver gene therapy to eradicate inhibitors?

There is evidence that T-regulatory cells (Tregs) were generated at early time points following AAV liver gene therapy (62

Questions and future directions for gene therapy to promote tolerance

In deciding how to select the best population to test a given gene therapy for inhibitor eradication, the following aspects should be taken into consideration:

a. Children and subjects with recently developed lower-titer inhibitors have higher ITI success rates than adults with longer-duration inhibitors. Should only patients with more encouraging prognoses be enrolled? What about pediatric subjects who failed ITI? By definition, adults have a poorer-risk prognosis (duration of inhibitor, high titers, etc.).

b. What is the optimal vector design and transgene levels strategy for a successful ITI?

Although AAV vectors using a hepatocyte-specific promoter is a common requirement for ITI, the relative importance of vector capsid, vector dose and transgene (FVIII wild type or variants) are debatable. Overall, high transgene levels are desirable for eradication of inhibitors, but the use of strategies associated with low levels of expression may be also beneficial. In both systems, could the continuous noninterrupted endogenous expression ("maximum compliance") be the main determinant of the ITI success? As noted in the International-ITI study, the high-dose cohort was (29,69). The majority of multiply-infused HA patients develop peripheral tolerance to FVIII, while the immune system of the remaining patients is incapable of maintaining active immune tolerance, and therefore clinically-relevant neutralizing antibodies arise. Protective immune tolerance may, however, be triggered in inhibitor patients upon appropriate stimulation of their immune system, for instance by ITI. In the near future, additional options will become available to foster FVIII-specific peripheral immune tolerance in inhibitor-positive patients, possibly including administration of FVIII orally or during fetal life (70) or induction of FVIII production at immuno-tolerogenic sites (i.e., liver targeted gene therapy). Whether naturally-induced or enforced upon the organism, it is important to keep in mind that the established peripheral immune tolerance to FVIII is a homeostatic equilibrium. It is prone to perturbations; risks of developing inhibitors may re-appear, for instance at an older age when the immune system declines and the potency of Tregs weakens (71). Therefore, establishing and maintaining durable tolerance to FVIII remains a strong priority. Please find herewith our review article submitted in frame with the WFH 2020 as a state-ofthe-art review, and entitled "Inhibitors -think differently".

F

In this review, we expose the ideas and discussions that will be presented during the WFH meeting in Kuala Lumpur. The review briefly summarizes our understanding of anti-FVIII immune responses. We propose that the prevailing observed active tolerance to FVIII in both HA and non-HA individuals rests to greater or lesser extents on peripherally-induced immune tolerance. We also propose that the immune systems of inhibitor-negative HA patients do not merely ignore therapeutic FVIII, but rather have immunologically assessed and actively tolerized the patients to exogenous FVIII. We also provide evidence in favour of the fact that Induction of such peripheral immune tolerance may be triggered in HA patients who failed to do so upon initial FVIII exposure by 'appropriate' stimulation of the immune system, including immune tolerance induction therapy via intensive FVIII therapy, oral administration of FVIII, cellular therapies, or gene therapy directed to immuno-tolerogenic sites such as the liver.

We hope that our review will be of interest to the readership of Haemophilia.

With my kindest regards

Sebastien Lacroix-Desmazes

Done 2 .

 2 the second revised manuscript I have modified the text according to Reviewer 2 comments, as indicated here:1. need to use generic emicizumab rather than Hemlibra. Emicizumab is not indicated for treating bleeds while rfVIIa and aPCC are so would work on the wording of those sentences in the introduction Emicizumab is mentioned along with FVIIa and aPCC in the introduction (page 3)3. on page 36 emicizumab mimics the pro-coagulant function of FVIIIaCorrected4. I wouldn't say that the anti-CD 20 enthusiasm waned. The initial study used Ritux alone without ITI. Many patients had significant responses (even if not fully tolerized) and were treated with Ritux if ITI alone failed prior to advent of emicizumab.

  Kind regards and thank you for your support and patienceSebastien

  F8 gene and thereby prevent circulation of any FVIII protein. Clinically

6

 6 Development of HA inhibitor responses follows recognition of linear epitopes in FVIII by CD4 + T-effector cells, which proliferate and secrete cytokines, and T-follicular cells migrate to germinal centers to provide help for FVIII-specific B-cells. These B cells then develop into memory B cells or antibody-secreting plasma cells, which may be very long-lived. Further exposures to FVIII (e.g. later in life) can re-activate these FVIIIspecific B cells, restoring or prolonging the inhibitor response. Multiple FVIII epitopes recognized by T cells, B cells and antibodies have been characterized, and although not yet comprehensively catalogued, this information has proven quite useful in mechanistic studies to better understand FVIII immunogenicity and tolerance(12).

  Do most HA patients become tolerant to exogenous FVIII?Central immune tolerance to self-proteins is established in the primary lymphoid organs in which T-and B-lymphocyte precursors arise, i.e., the thymus and the bone marrow, respectively. Central T-cell tolerance relies on positive selection by cortical thymic epithelial cells (TECs) of thymocytes having functional T-cell receptors and negative selection by medullary TECs of autoreactive thymocytes

  reduced anti-inflammatory potential. The presence of a long GT repeat in at least one allele was associated with a 2-fold increased prevalence of FVIII inhibitors in SHA patients(48). More recently, Matino et al. demonstrated the impaired ability of dendritic cells from inhibitor-positive patients to express indoleamine-dioxygenase-1

  ) models of severe hemophilia have utilized distinct breeds that mount immune responses to canine transgenes. The availability of recombinant or plasmaderived canine proteins (FVIII and FIX), and specific antibodies against these proteins including subclasses of IgG(64)(65)(66) are major assets for the use of the canine model for inhibitor prevention and/or eradication. The use of a human (h) transgene in nonhuman primates (NHP) in other studies showed limited the duration of transgene expression due to the formation of cross-species antibodies to hFVIII or hFIX and prevented eradication of these inhibitors by AAV gene therapy. Thus, the canine model may be an ideal model for ITI by gene therapy.

  intensive immunosuppressive therapies is needed. The use of AAV liver gene therapy for inhibitor eradication could eventually emerge as a novel and efficacious therapy that is feasible for more patients, since clinical experience has clearly demonstrated it to be relatively efficacious and (so far) safe, either alone or coupled with cellular-based therapies.

  eradication and better bleeding control at early time points, but after 3 years there was no difference in ITI success rates. c. What is the risk of anaphylactic reaction due to FVIII or FIX expression following gene therapy, since the transgene expression, to date, cannot be efficiently ceased? This is likely to be a more significant safety concern regarding FIX inhibitors, based on the rare clinical evidence of anaphylaxis in these patients. d. T-cell therapy prior to gene therapy could lower inhibitor titers in subjects with high and refractory inhibitor titers, thus favoring ITI. The use of an anti-CD20 antibody may not be as efficient in view of its failure to provide full tolerance to FVIII when used as rescue following ITI failure.e. What about assessing the cost effectiveness of liver gene therapy for ITI? ITI is less expensive than bypass products (used prophylactically and/or on demand). The costs associated with preclinical, novel approaches are not yet sufficiently defined to allow firm conclusions to be drawn.ConclusionsRecent studies of anti-FVIII immune responses, and of immunologic aspects of cellular and gene therapy approaches to promote immune tolerance, have provided new insights that lead us to re-think several longstanding assumptions about FVIII immunogenicity and tolerance. Therapeutically administered FVIII is immunologically assessed by the immune systems of all FVIII-infused patients. This is illustrated by the development of transient inhibitors in up to 26% of the patients(68) and by the non-neutralizing anti-FVIII IgG in inhibitor-negative patients

Fig. 1 .

 1 Fig. 1. Comparison of gene therapy in an orthotopic liver transplantation (OLT)

4 1

 4 Figure 1

  placing a burden on patients and their families, although factors with increased half-lives have recently become available, with further improvements expected in the future (Chhabra et al Blood 2020, in press PMID:32078672). For patients with severe HA or HB, whose genetic mutation precludes expression of any FVIII or FIX protein, respectively, the infused clotting factor may be perceived by their immune system as "foreign". Patients who circulate a dysfunctional FVIII or FIX protein, e.g. due to missense mutations, may also develop an immune response, although inhibitor incidences in these patients are lower(4). Approximately half of severe HA (SHA) patients who develop an inhibitor do so within the first 20 FVIII infusions (5), reminiscent of the sequential "prime + boost" injections required for effective vaccination responses, and their inhibitor risk declines to insignificant after 50 exposure days(6, 7). It should be noted, however, that lower inhibitor incidences in mild or moderately severe HA are due in part to their lower exposure to therapeutic FVIII. Inhibitors have been reported in these patients after 20-50 exposure days, and those with >50 exposure days have a similar risk to that of multiply infused patients with SHA (8), while the risk of developing an inhibitor after 50 exposure days is quite low. Risks increase again in the aging HA population, especially in those undergoing surgery requiring more intensive FVIII therapy(9).

6

 6 Development of HA inhibitor responses follows recognition of linear epitopes in FVIII by CD4 + T-effector cells, which proliferate and secrete cytokines, and T-follicular cells migrate to germinal centers to provide help for FVIII-specific B-cells. These B cells then develop into memory B cells or antibody-secreting plasma cells, which may be very long-lived. Further exposures to FVIII (e.g. later in life) can re-activate these FVIIIspecific B cells, restoring or prolonging the inhibitor response. Multiple FVIII epitopes recognized by T cells, B cells and antibodies have been characterized, and although not yet comprehensively catalogued, this information has proven quite useful in mechanistic studies to better understand FVIII immunogenicity and tolerance(12).

7

 7 An exciting, recent clinical breakthrough has been the introduction of a bispecific therapeutic antibody, emicizumab, which mimics the pro-coagulant function of activated FVIII. This therapeutic agent has transformed HA care by providing effective hemostasis to patients even in the presence of a high-responding inhibitor(2, 3).

8

 8 Some compelling research questions, which will require future well-designed clinical and basic science studies, are: how much FVIII will be required to maintain tolerance in patients receiving prophylactic non-FVIII therapies, how often must it be administered and at what dose, and what is the best route and formulation? One

1 (

 1 reduced anti-inflammatory potential. The presence of a long GT repeat in at least one allele was associated with a 2-fold increased prevalence of FVIII inhibitors in SHA patients(48). More recently, Matino et al. demonstrated the impaired ability of dendritic cells from inhibitor-positive patients to express indoleamine-dioxygenase-IDO-1) following in vitro TLR9 stimulation by CpG-ODN(49). In contrast, dendritic cells from inhibitor-negative patients could differentiate CD4 + T cells into regulatory T cells in vitro when stimulated by CpG-ODN(49). Thus, the risk for a patient to develop clinically relevant inhibitory anti-FVIII IgG may not merely rely on the presence of particular danger signals during FVIII administration, but rather on the intrinsic (genetic) capacity of the patient to mount protective anti-inflammatory and/or immunoregulatory responses.

20 detection

 20 eradication and better bleeding control at early time points, but after 3 years there was no difference in ITI success rates. c. What is the risk of anaphylactic reaction due to FVIII or FIX expression following gene therapy, since the transgene expression, to date, cannot be efficiently ceased? This is likely to be a more significant safety concern regarding FIX inhibitors, based on the rare clinical evidence of anaphylaxis in these patients. d. T-cell therapy prior to gene therapy could lower inhibitor titers in subjects with high and refractory inhibitor titers, thus favoring ITI. The use of an anti-CD20 antibody may not be as efficient in view of its failure to provide full tolerance to FVIII when used as rescue following ITI failure.e. What about assessing the cost effectiveness of liver gene therapy for ITI? ITI is less expensive than bypass products (used prophylactically and/or on demand). The costs associated with preclinical, novel approaches are not yet sufficiently defined to allow firm conclusions to be drawn.ConclusionsRecent studies of anti-FVIII immune responses, and of immunologic aspects of cellular and gene therapy approaches to promote immune tolerance, have provided new insights that lead us to re-think several longstanding assumptions about FVIII immunogenicity and tolerance. Therapeutically administered FVIII is immunologically assessed by the immune systems of all FVIII-infused patients. This is illustrated by the development of transient inhibitors in up to 26% of the patients(68) and by the of non-neutralizing anti-FVIII IgG in inhibitor-negative patients

5 ModifiedFig. 1 .

 51 Figure Legend

TableTable 1 : Eradication of inhibitors to canine FVIII in adult male dogs with severe HA following AAV liver gene therapy.

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**Collective bleed episodes/months prior to and post gene therapy for UNC-CH dogs were 10 /40 and 2/68, respectively (more detailed in (65))

Table count

 count

	Abstract
	The development of inhibitory antibodies to therapeutic factor VIII (FVIII) in
	haemophilia A (HA) patients is the major complication in treatment/prevention of
	hemorrhages. The reasons some HA patients develop inhibitors while others do not
	remain unclear. This review briefly summarizes our understanding of anti-FVIII immune
	responses, the roles of T cells, both effector and regulatory, and generally discusses
	the interplay between FVIII and the immune system, both in factor replacement therapy
	and gene therapy, with some comparisons to factor IX and haemophilia B therapies.
	F o r F o r
	P e e P e e
	r r
	R R
	: 1 Reference count: 69	e v i e w e v i e w
	Keywords: Factor VIII, haemophilia A, protein immunogenicity, FVIII inhibitors,
	immune tolerance, gene therapy
		2

Notably, we propose that the prevailing observed active tolerance to FVIII in both HA and non-HA individuals rests to greater or lesser extents on peripherally-induced immune tolerance. We also propose that the immune systems of inhibitor-negative HA patients do not merely ignore therapeutic FVIII, but rather have immunologically assessed and actively tolerized the patients to exogenous FVIII. Induction of such peripheral immune tolerance may further be triggered in HA patients who failed to tolerize upon initial FVIII exposure by 'appropriate' stimulation of their immune system, e.g., by immune tolerance induction therapy via intensive FVIII therapy, by oral administration of FVIII, by cellular therapies, or by gene therapy directed to immunotolerogenic sites such as the liver.

  F8 gene and thereby prevent circulation of any FVIII protein. Clinically significant inhibitors afflict a larger fraction of severe SHA patients (~1 in 4) than severe haemophilia B patients. It is not known if this is due to the larger number of potential epitopes in FVIII administered to SHA patients, compared to epitopes in FIX administered to HB patients who circulate a defective FIX protein, or if the higher inhibitor is incidence is due to a greater inherent immunogenicity of FVIII compared to FIX. Finally, some HB patients develop anaphylactic responses to therapeutic FIX, whereas such responses to FVIII are extremely rate in HA patients.

	disrupt the
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	5

contrast, almost half of SHA cases are caused by inversion mutations that seriously

Large animal models for the assessment of immune tolerance following AAV liver gene therapy for hemophilia

  CD8 + T cells are not a feature of immune responses to FVIII, however, the properties of engineered CD8 + T cells may be exploited in attempts to eliminate inhibitors, e.g. by targeting FVIII-specific B cells(56).In applications to the FVIII inhibitor problem, the goals are to either promote tolerance to FVIII via engineered Tregs, or to eliminate FVIII-specific B cells. Because the engineered cells are highly specific for FVIII, such approaches should in principle avoid causing general immunosuppression, which can increase risks of infection and even some cancers. Promising results have been obtained using appropriate animal models, and there is hope that one or more of these approaches may eventually be tested in clinical trials. The more that scientists learn about these immune mechanisms, the likelier we are to see advances in tolerizing HA patients to therapeutic FVIII, delivered either intravenously or via gene therapy. Canine (c) models of severe hemophilia have utilized distinct breeds that mount immune responses to canine transgenes. The availability of recombinant or plasmaderived canine proteins (FVIII and FIX), and specific antibodies against these proteins including subclasses of IgG (64-66) are major assets for the use of the canine model for inhibitor prevention and/or eradication. The use of a human (h) transgene in nonhuman primates (NHP) in other studies showed limited the duration of transgene expression due to the formation of cross-species antibodies to hFVIII or hFIX and prevented eradication of these inhibitors by AAV gene therapy. Thus, the canine model may be an ideal model for ITI by gene therapy.

	F o r P e e r R e v i e w An early report by Ashanti et al. showed the efficacy of orthotropic liver transplant (OLT) in an adult SHA patient complicated by a rapid, progressive hepatocellular carcinoma and a high-titer inhibitor (57). Progressively increasing FVIII activity levels F o r R e v i e w 59). Moreover, a non-HA OLT patient who received a liver from a HA donor with no evidence of a recent inhibitor unexpectedly developed a high-titer inhibitor post-transplant. Re-examination of the donor's medical history showed an inhibitor had been detected earlier (but not close to the OLT), indicating the primary resident FVIII-specific lymphocytes were still viable, and expansion after FVIII exposure resulted in inhibitor formation. This patient required a second liver transplant (60). Despite these disparate clinical outcomes, these early results encouraged animal model studies to evaluate the potential tolerogenic delivery of FVIII to the liver through gene therapy, rather than OLT. existing antibodies. This apparent immune tolerance induction was transgene-specific, without causing systemic down-regulation of host immune competence. The underlying mechanisms of immune tolerance in this model involve complex and diverse pathways, and some of these strategies could be "mimicked" by pharmacologic or molecular modifications (61-63). The question of whether the scientific, regulatory F o r R w e i v e pemphigous vulgaris (55). Gene therapy as a tolerogenic strategy P e e r Collective data from mouse models for genetic or acquired diseases indicate that liver gene therapy using AAV vectors for hepatocyte-restricted transgene expression could r prevent the formation of pathological antibodies to the transgene and eradicate pre-e e P Inhibitor
	were detected post-transplant, with normalization of infused FVIII recovery (Fig 1) and and patient communities would jump from results obtained in rodent models, which
	inhibitor eradication during follow up (~4 months post-transplant). Notably, the patient's have shown efficacy and reproducibility, directly to human gene therapy trials for
	inhibitor titer increased immediately after the transplant, a finding that resembled the inhibitor eradication has focused attention on the need for careful assessment of
	anamnestic response to FVIII noted in many inhibitor patients upon initiation of ITI. available information from large-animal disease models as well.
	However, induction of tolerance to FVIII was evidenced by disappearance of the
	+ T cells targeting inhibitor and normalization of FVIII recovery. However, subsequent OLT for inhibitor
	desmoglin 3-expressing B cells, which are the target of the underlying pathology of patients were associated with lethal systemic microangiopathy or lack of efficacy (58,
	14 15 16

eradication in severe HA and HB dogs by AAV liver gene therapy In

	F o r
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	R
	Circulating cFVIII activity and antigen levels increased steadily following inhibitor eradication. Subsequent challenges with cFVIII protein showed no evidence of an e v i e w
	immune response and demonstrated normalization of FVIII recovery. The latter is a
	clinical surrogate test for the presence of both neutralizing and non-neutralizing
	antibodies (which may accelerate clearance of the protein). A substantial reduction of
	spontaneous bleeds further confirmed the success of this strategy in improving the
	disease phenotype. Together, these findings demonstrate the potential of AAV liver
	gene therapy for inhibitor eradication in large animal HA models. Moreover, successful
	and safe eradication of FIX inhibitors was demonstrated in severe HB dogs using an
	17

2010, Finn et al reported the proof-of-principal use of AAV serotype 8 for liver gene therapy in HA dogs with inhibitors to cFVIII

(65)

, as summarized in Table

1

. cFVIII transgene expression was achieved using a hepatocyte-specific promoter delivered by IV injection. The primary endpoint was inhibitor disappearance in all 4 dogs in ~5 weeks for the UNC-CH colony, and in ~18 months for the Queen's University colony.

  ). It is conceivable that combining intensive immune suppression with AAV liver gene therapy could interfere with the generation and/or function of these cells. For example, Mingozzi et al. showed that administering a specific antibody to CD25, which was meant to target T-effector cells at early time points following AAV liver gene therapy in a NHP model, actually decreased the

	more intensive immunosuppressive therapies is needed. The use of AAV liver gene
	therapy for inhibitor eradication could eventually emerge as a novel and efficacious
	therapy that is feasible for more patients, since clinical experience has clearly
	demonstrated it to be relatively efficacious and (so far) safe, either alone or coupled
	with cellular-based therapies.
	F o r
	P e e
	r
	numbers and function of the Tregs, thus facilitating inhibitor formation (67). Recent R
	AAV liver gene therapy efforts have coupled vector administration with transient immunosuppression methods to prevent or reverse AAV capsid-mediated liver toxicity and transgene expression loss. More recently, it was observed that the use of rabbit antithymocyte globulin (ATG) administration at the time of AAV delivery results in a e v i e w
	strong anti-transgene-product antibody response, while delaying ATG by only ~5
	weeks does not. The underlying mechanism of this anti-transgene-product antibody
	response is, at least in part, associated with a skewing of the ratio of Th17/Treg cells
	(Finn et al, Mol Ther 2020 in press). Combined, these results suggest that there is a
	critical time window around vector administration for transgene-product tolerance that
	is Treg-dependent, which can be disrupted by intense immunosuppression. Thus,
	extensive further testing prior to contemplating clinical gene therapy studies employing

TableTable 1 : Eradication of inhibitors to canine FVIII in adult male dogs with severe HA following AAV liver gene therapy.

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Haemophilia

Acknowledgements

We would like to thanks Dr. Ben Samelson-Jones for help in revising the manuscript and insightful commnents. Supported by NIH/NHLBI U54 HL142012-01 (Arruda), R01 HL137335-01 (Arruda), Institut National de la Santé et de la Recherche Médicale, Centre National de la Recherche Scientifique, and Sorbonne Université (Lacroix-Desmazes), and R01 HL130448-05 (Pratt).

Acknowledgements

We would like to thanks Dr. Ben Samelson-Jones for help in revising the manuscript and insightful commnents. Supported by NIH/NHLBI U54 HL142012-01 (Arruda), R01 HL137335-01 (Arruda), Institut National de la Santé et de la Recherche Médicale, Centre National de la Recherche Scientifique, and Sorbonne Université (Lacroix-Desmazes), and R01 HL130448-05 (Pratt).

Authors contributions

KP, VA and SLD B wrote the paper

Disclaimer

The opinions or assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the Department of Defense or the Uniformed Services University of the Health Sciences (USUHS).

Do most HA patients become tolerant to exogenous FVIII?

Central immune tolerance to self-proteins is established in the primary lymphoid organs in which T-and B-lymphocyte precursors arise, i.e., the thymus and the bone marrow, respectively. Central T-cell tolerance relies on positive selection by cortical thymic epithelial cells (TECs) of thymocytes having functional T-cell receptors and negative selection by medullary TECs of autoreactive thymocytes (21). This selection process also generates natural CD4 + CD25 + Foxp3 + regulatory T cells (nTregs), which down-regulate T-effector and B-cell responses (22), thereby preventing immune responses and associated inflammatory processes from becoming pathogenic. HA patients who circulate a dysfunctional FVIII protein (generally resulting in mild or moderate severity HA) are expected to have acquired central tolerance to this selfprotein, which decreases but does not eliminate their risk of developing an immune response to regions of therapeutic FVIII that differ from their endogenous protein.

Patients with SHA are heterogenous, with ~45% having a F8 intron-22 inversion mutation, and the rest having nonsense, frameshift, missense, etc. mutations that preclude production of a functional FVIII protein. Depending on the presence or absence of a non-functional or dysfunctional endogenous FVIII molecule, HA patients are categorised as cross-reactive material-positive (CRM pos ) or CRM neg . The type of HA-causing gene abnormality, and the associated presence/absence of the FVIII antigen, are thought to be predominant risk factors for inhibitor development (10).

Peripheral tolerance develops primarily in organized secondary lymphoid organs, e.g., lymph nodes, spleen, and lamina propria. The primary clearance site for FVIII is the liver, which also provides a mostly tolerogenic environment. Additional types of Tregs may also be induced at the periphery upon encounter with a self-or non-self antigen,

Authors contributions

KP, VA and SLD B wrote the paper

Disclaimer

The opinions or assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the Department of Defense or the Uniformed Services University of the Health Sciences (USUHS).