Immune Tolerance Induction Protocols Research Articles

VCA supercooling in a swine partial hindlimb model

Vascularized composite allotransplantations are complex procedures with substantial functional impact on patients. Extended preservation of VCAs is of major importance in advancing this field. It would result in improved donor-recipient matching as well as the potential for ex vivo manipulation with gene and cell therapies. Moreover, it would make logistically feasible immune tolerance induction protocols through mixed chimerism. Supercooling techniques have shown promising results in multi-day liver preservation. It consists of reaching sub-zero temperatures while preventing ice formation within the graft by using various cryoprotective agents. By drastically decreasing the cell metabolism and need for oxygen and nutrients, supercooling allows extended preservation and recovery with lower ischemia–reperfusion injuries. This study is the first to demonstrate the supercooling of a large animal model of VCA. Porcine hindlimbs underwent 48 h of preservation at − 5 °C followed by recovery and normothermic machine perfusion assessment, with no issues in ice formation and favorable levels of injury markers. Our findings provide valuable preliminary results, suggesting a promising future for extended VCA preservation.

CPA toxicity screening of cryoprotective solutions in rat hearts

In clinical practice, donor hearts are transported on ice prior to transplant and discarded if cold ischemia time exceeds ∼5 h. Methods to extend these preservation times are critically needed, and ideally, this storage time would extend indefinitely, enabling improved donor-to-patient matching, organ utilization, and immune tolerance induction protocols. Previously, we demonstrated successful vitrification and rewarming of whole rat hearts without ice formation by perfusion-loading a cryoprotective agent (CPA) solution prior to vitrification. However, these hearts did not recover any beating even in controls with CPA loading/unloading alone, which points to the chemical toxicity of the cryoprotective solution (VS55 in Euro-Collins carrier solution) as the likely culprit. To address this, we compared the toxicity of another established CPA cocktail (VEG) to VS55 using ex situ rat heart perfusion. The CPA exposure time was 150 min, and the normothermic assessment time was 60 min. Using Celsior as the carrier, we observed partial recovery of function (atria-only beating) for both VS55 and VEG. Upon further analysis, we found that the VEG CPA cocktail resulted in 50 % lower LDH release than VS55 (N = 4, p = 0.017), suggesting VEG has lower toxicity than VS55. Celsior was a better carrier solution than alternatives such as UW, as CPA + Celsior-treated hearts spent less time in cardiac arrest (N = 4, p = 0.029). While we showed substantial improvement in cardiac function after exposure to vitrifiable concentrations of CPA by improving both the CPA and carrier solution formulation, further improvements will be required before we achieve healthy cryopreserved organs for transplant.

Levels of Anti-FVIII IgG4 and Total IgG Are Biomarkers of Immune Tolerance Induction Failure in Patients with Inherited Hemophilia a and High-Responding Inhibitors

Background: The development of inhibitors in patients with inherited hemophilia A (PwHA) is an important complication of the treatment with exogenous factor VIII (FVIII), leading to inefficient replacement therapy. Immune tolerance induction (ITI) is the standard treatment to eradicate inhibitors in HA. The immune biology and biomarkers related with ITI outcome are still unclear. Few studies have investigated this, of which most included few PwHA. Furthermore, such studies have not assessed samples in specific time-points, but have rather grouped patients into categories of inhibitors and non-inhibitors. Aims: This study aimed to investigate specific anti-FVIII immunoglobulins IgG1, IgG4 and total IgG as biomarkers of ITI outcome at the completion of ITI. Methods: We included PwHA from 15 hemophilia treatment centers, participants of the Brazilian Immune Tolerance (BrazIT) Cohort Study. Inclusion criteria were severe and moderately-severe (<2% FVIII) PwHA, with high-responding (>5.0BU/mL) inhibitors, who completed a first course of ITI. All patients were treated with the same ITI protocol, i.e., 50 international units (IU)/kilogram (kg) of FVIII. In case ITI titers did not decrease by at least 20% first 3-6 months after inhibitor peak, FVIII was increased to 100 IU/kg daily. We collected socio-demographic, clinical and laboratory data using standardized forms. Outcome of ITI was defined according to published classification as failure, partial and total successes. For this analysis, plasma samples were collected in citrate at completion of ITI and frozen at -80°C until assessment. We measured plasma levels of anti-FVIII IgG1, IgG4 and total IgG in ELISA using immobilized recombinant (r) FVIII (alfaoctocogue, Takeda, USA). Local ethics committees approved this study and all patients/guardians signed informed consent. Results: We included 98 PwHA, median age of 11.0 years (interquartile range [IQR] 7.0-23.0), 94 (95.9%) severe HA (<1%). According to ITI outcome, 40 (40.8%) had ITI failure, 37 (37.8%) had partial success and 21 (21.4%) had total success (Table 1). Levels of total IgG in patients who failed ITI (median optical density [OD], 0.37; IQR 0.15-0.73) were significantly higher when compared to the levels of patients with partial (median OD, 0.03; IQR 0.00-0.14; p<0.0001) and complete (median OD, 0.04; IQR 0.00-0.07; p<0.0001) success. Levels of total IgG anti-FVIII were not different between partial and complete success groups. Levels of anti-FVIII IgG4 were significantly higher in PwHA who failed ITI (median OD, 2.19; IQR 0.74-2.52) compared with those who had partial (median OD, 0.39; IQR 0.09-1.11; p<0.0001) and complete success (median OD, 0.07; IQR 0.06-0.40; p<0.0001). Anti-FVIII IgG4 levels were higher in PwHA presenting partial success compared with those with complete success (p=0.02). Levels of anti-FVIII IgG1 were not different between PwHA who failed ITI (median OD, 0.03; IQR 0.01-0.21), in comparison with the ones who achieved partial success (median OD, 0.03; IQR 0.01-0.08; p=0.35) and achieved complete success (median OD, 0.03; IQR 0.02-0.05; p=0.72) (Table 2). Conclusion: We conclude that anti-FVIII IgG4 and IgG total are good biomarkers of ITI outcome. Levels of IgG4 are associated with ITI outcome in a dose-dependent manner, with failure>partial>complete success. Anti-FVIII IgG1 is not a useful biomarker of ITI outcome. This study was fully supported by Brazilian Governmental grants from Fapemig, CNPq and Ministry of Health of Brazil (Fundo Nacional de Saúde).

Low-Dose Immune Tolerance Induction Therapy for Severe Hemophilia a Inhibitor Patients: Immunosuppressants Improve Outcomes but Are Generally Not Necessary for Inhibitor-Titer below 200 BU/Ml

Background We previously showed that low-dose immune tolerance induction (ITI) incorporating immunosuppressants (IS) for severe hemophilia A patients with inhibitor titer ≥40 Bethesda Units (BU)/mL (LD-ITI-IS 40 regimen) had relatively high success rate with lower cost compared to those on high-dose ITI. Recent evidence suggested that patients with inhibitor titer <200 BU/mL do not need high-dose ITI raising the question that IS may also not be necessary. Objectives To compare the efficacy of LD-ITI incorporating IS only for inhibitor titer ≥200 BU/mL (LD-ITI-IS 200 regimen) with that of LD-ITI-IS 40 regimen. Methods A prospective, single-arm, single-center cohort study on patients receiving LD-ITI-IS 200 beginning 2021, compared to those receiving LD-ITI-IS 40 before 2021. The inclusion criteria were: (i) patients with an established diagnosis of SHA [FVIII clotting activity (FVIII: C) <1% before inhibitor development]; (ii) patients ≤14 years age at the ITI-start; (iii) patients with high-titer inhibitors (≥5BU/mL). All study patients received a LD-ITI with pd-FVIII/VWF (at 50 FVIII IU/kg) every-other-day. IS (rituximab + prednisone) were added for peak inhibitor-tier ≥40 BU/mL in LD-ITI-IS 40 regimen and ≥200 BU/mL in LD-ITI-IS 200 regimen. IS used for each regimen included rituximab 375 mg/m 2/week (maximum 600 mg) for 4 weeks, together with prednisone 2 mg/kg/day (maximum 60 mg) for one month, then tapered over 3 months. For patients receiving rituximab, intravenous immunoglobulin (200 mg/kg/month for 6 months) was administered for infection prophylaxis. Success is defined as negative inhibitor plus FVIII recovery ≥66% of the expected. Partial success is defined as achieving inhibitor elimination but with persistently abnormal FVIII recovery. Results We enrolled 30 patients on LD-ITI-IS 200 and 64 patients on LD-ITI-IS 40, with similar baseline clinical characteristics (Table 1). Given that IS was initiated at a higher peak inhibitor-titer in the LD-ITI-IS 200 regimen, IS in the LD-ITI-IS 200 group was introduced at a significantly lower rate than that in the LD-ITI-IS 40 regimen group (30% vs. 62.5%, p = 0.003). The 2 regimens (LD-ITI-IS 200 vs.LD-ITI-IS 40) had similar success rate (66.7% vs. 79.7%), median time to success (7.4 vs. 10.6 months) (Figure 1), and ABR during ITI (3.7 vs. 2.8). The per kg treatment cost from ITI-start to ITI success in the LD-ITI-IS 200 (US$2167) was significantly lower than that in the LD-ITI-IS 40 (US$3349). Among patients taking IS in the two regimens rituximab infusion-related side effects were similar, being 22.2% (2/9) for the LD-ITI-IS 200 patients and 22.5% (9/40) for the LD-ITI-IS 40 patients. All these side effects could be resolved and subsequently prevented by antihistamine drugs. No severe adverse events occurred. Among patients with peak inhibitor-titer 40-199 BU/mL, 10 non-IS-using (on LD-ITI-IS 200 regimen) and 28 IS-using (on LD-ITI-IS 40 regimen) had similar success rate (60% vs. 78.6%) and time to success (9.0 vs. 8.8 months). The per kg treatment cost from ITI-start to ITI partial success in the LD-ITI-IS 200 (US$891) was significantly lower than that in the LD-ITI-IS 40 (US$2819). Between the two protocols, there were 12 patients with peak inhibitor-titer ≥200BU/mL (all using IS, 7 in LD-ITI-IS 200, 5 in LD-ITI-IS 40). Among them, 8 patients (66.7%, 4 on each regimen) achieved success over a median 10.1 months, and none relapsed at median 13.5 months follow-up since achieving success. Between the 2 protocols, there were 44 patients (13 in LD-ITI-IS 200, 31 in LD-ITI-IS 40) with peak inhibitor-titer 5-39 BU/mL. Their overall success rate was 79.5%. Nine of the 44 patients (2 in LD-ITI-IS 200, 7 in the LD-ITI-IS 40) received IS because they failed to have adequate inhibitor titer decline in the first 3 months of ITI. All 9 achieved success at median 11.3 months from ITI-start with no relapsed after a median 22.6 months follow-up since success. Conclusions We have optimized the use of IS in our low-dose ITI protocol for SHA patients with high-titer inhibitors. We showed that more restrictive use of IS only for patients with peak inhibitor-titer ≥200 BU (instead of at a lower titer of ≥40 BU) maintained the same efficacy while decreasing the cost. Decreasing use of IS would decrease its potential side effects including infection risks.

Eradication of FIX inhibitor in haemophilia B children using low‐dose immune tolerance induction with rituximab‐based immunosuppressive agent(s) in China

Development of haemophilia B inhibitors (HBI) results in the ineffectiveness of FIX replacement therapy. Inhibitor eradication by immune tolerance induction (ITI) is therefore necessary. In HBI, ITI even at high FIX dose is less effective and has a higher risk of severe complications. To characterize clinical features and outcome of ITI on HBI. This retrospective study was conducted in Haemophilia Paediatric Comprehensive Care Centre of China. We used low-dose ITI (25-50 FIX IU/kg/three-times-weekly to every-other-day) with domestic prothrombin complex concentrate (PCC), combined with two successive immunosuppressive (IS) regimens. Sixteen HBI children, representing 5.7% of all and 14.4% of our severe registered HB patients, were enroled. Seven cases reported allergic reactions (ARs) proximal to inhibitor development. The historic peak inhibitor titre was median 54.2 (range 4.7-512) BU, and 15 (93.8%) had high-titre inhibitors. Twelve patients adherent to ITI were analysable. Of the nine ITI patients who received rituximab/prednisone (IS Regimen-1), four achieved tolerization in 1.4-43.3 months. Two subsequently relapsed but re-tolerized after a second course of IS Regimen-1. During ITI, the median treated bleed was .39/month (82.7% reduction from before ITI), and the incidence of AR and nephrotic syndrome (NS) complications was each at 22% (2/9). Three ITI patients received modified 'Beutel' protocol (IS Regimen-2) using multiple-IS-drugs, and two had rapid tolerization (.8 and 1.8 months). Inhibitor eradication could be achieved by low-dose ITI protocol using PCC combined with IS. Larger studies are needed to confirm if ITI with IS Regimen-2 is more effective with less complications.

Antigen-specific immunotherapy with apitopes suppresses generation of FVIII inhibitor antibodies in HLA-transgenic mice

AbstractHemophilia A (HA) is a blood clotting disorder that is caused by various genetic deficiencies in the factor VIII (FVIII)-encoding F8 gene. Patients receiving FVIII-replacement therapy are at risk for developing neutralizing antibodies (FVIII inhibitors), rendering the FVIII-replacement therapy ineffective. Immunological tolerance toward FVIII can be achieved through immune tolerance induction protocols in some patients, but this is a lengthy and costly desensitization program. Long-term eradication of inhibitors in patients with HA could be achieved by antigen-specific immunotherapy targeting CD4+ T-cells, because formation of FVIII inhibitors is T-cell dependent. Here, we report a peptide-based antigen-specific immunotherapy that is designed to specifically reestablish immune tolerance to FVIII through the development of antigen-processing-independent epitopes (apitopes). We identified 2 FVIII immunodominant peptides in immunized HLA-DRA*0101/DRB1*1501 transgenic (HLA-DR2tg) mice that were optimized for tolerogenicity. These modified peptide analogs were initially screened for recognition using FVIII-specific T-cell hybridoma clones from FVIII-immunized HLA-DR2tg mice. The FVIII apitopes were promiscuous and bound common human HLA-DRB1* allelic variants. The combination of these 2 FVIII apitopes (ATX-F8-117), administered according to a dose-escalation protocol, promoted T-cell tolerance toward FVIII in HLA-DR2tg mice. Furthermore, treatment with ATX-F8-117 significantly reduced FVIII inhibitor formation. ATX-F8-117 regulates anti-FVIII T-cell and B-cell responses, specifically the generation of FVIII inhibitors, revealing peptide-based antigen-specific immunotherapy as a promising approach to suppress and treat inhibitor formation in susceptible patients with HA.

Costs and management of patients with hemophilia A in France: the Hemraude study

The Hemraude study was conducted to describe the profile of patients with HA, disease management, and economic burden in a collective perspective. This retrospective study was conducted using the French administrative healthcare claims database SNIIRAM/SNDS. Male patients treated for hemophilia A with a long-term illness (ALD) status or invalidity were included in the study between January 1, 2016 and December 31, 2017. Patients were classified in six treatment groups: no treatment, on-demand FVIII, prophylactic FVIII, FVIII in immune tolerance induction (ITI) protocol, on-demand bypassing agents, and prophylactic bypassing agents. Patients treated with FVIII in ITI protocol and those treated with bypassing agents are deemed to have developed inhibitors. HA patients were compared to a control population without coagulation disorder and matched (ratio 1:3) on age and sex. A total of 4172 patients were included in the analysis, aged on average 35.2years, 5.3% had HIV infection, and 8.8% had hepatitis B or C. In 2017, half of the patients received no treatment for HA, 46.7% were treated with FVIII (25% on demand, 20.6% with prophylaxis, and 1.1% ITI), 1.5% with bypassing agents. Patients treated with prophylactic treatments, either inhibitor or non-inhibitor, were less likely to be hospitalized for severe bleeding compared to patients receiving on-demand treatments. The average annual costs for HA management per patient were 72,209.60 €. The highest costs were observed in patients treated with FVIII in ITI protocol and those receiving prophylactic bypassing agents. Direct costs of HA treatments for HA may be very high especially in the small percentage of patients developing inhibitors or treated with ITI protocol.

Evaluation of Predictors of Outcome in Immune Tolerance Induction Treatment of Hemophilia Α Patients with High-Responding Inhibitors

Introduction: Inhibitor development remains a major complication for patients with hemophilia A. Inhibitors are alloantibodies that neutralize factor (F) VIII activity, which impairs the hemostatic effect of infused FVIII concentrate. Immune tolerance induction (ITI) consists of regular FVIII administration in an attempt to eliminate the presence of inhibitors. There are several ITI protocols with similar success rates around 70%. Still, the ideal ITI regimen and the factors that can influence its success are not well defined.Objective: This study aimed to evaluate the predictors factors for ITI response among Brazilian hemophilia A patients with high-responding inhibitors who have undergone ITI protocol for the first time.Patients and Methods: We retrospectively analyzed clinical and laboratory data of hemophilia A patients with high-responding inhibitors (peak titer >5 Bethesda Unit (BU) and anamnestic response to FVIII) from eight Brazilian Hemophilia Treatment Centers, who underwent ITI treatment for the first time. ITI outcome was defined as: (i) complete success, negative inhibitor (<0.6 BU) on at least two consecutive measurements, FVIII recovery >66%, and half-life >7 h, with absence of anamnestic response to FVIII; (ii) partial success, negative inhibitor titer and absence of one or two of the PK parameters of ITI success (FVIII recovery > 66% or half-life ≥7 h); (iii) clinical response, inhibitor titer <5 BU and/or FVIII recovery <66% and/or FVIII half-life <7 h, with clinical response to FVIII infusions and no increase of inhibitor titer >5 BU; and (iv) failure, lack of criteria for complete or partial success or clinical response within 33 months of treatment. Statistical analysis were carried out using the Kruskal-Wallis H test.Results: In this study, we accessed data from 88 patients, from which 61 were analyzed for ITI outcome, having achieved ITI complete success or reached 33 months under ITI protocol. Low-dose ITI protocol was initially used for all patients and started with FVIII concentrate 25-50 IU/kg 3x/week, with dose increment to up to 100 IU/kg daily when a minimum of 20% decrease in the inhibitor titer was not achieved in 6 months. 41/61 (67.2%) achieved complete ITI success criteria, in a median period of 17 months (interquartile range (IQR) 10.3-22.6) after starting ITI. 4 (6.5%) remained as partial ITI success over 33 months of treatment. Among the remaining 16 patients, 3 (5%) had clinical response to FVIII concentrate, without requirement of bypassing agents to achieve hemostatic response, and 13 (21.3%) failed ITI treatment. When comparing groups with different outcomes, statistical analysis revealed that the historical peak titer (p= 0.002), inhibitor titer at the moment of ITI protocol started (p= 0.013) and the peak titer during ITI protocol (p< 0.0001) were important predictors factors of ITI success (table 1). Other clinical factors that possibly interfered with ITI outcome were bleeding rates during ITI treatment, both annualized bleeding rate (ABR) (p=0.047) and annualized joint bleeding rate (AJBR) (p=0.014). Although there was no difference in age at ITI onset, a statistically significance was also observed in the difference of central venous catheter use between complete success and failure groups (5% vs 38.5%; p= 0.023). A possible explanation could be the need for a more intense treatment in the failure group, with a higher demand for venous access. However, the higher risk of infection and thrombosis in this group can also result in lower ITI success. In the group with no ITI outcome analyzed (27/88), 10 (11.4%) discontinued ITI treatment for several reasons and at least 4 of them are no longer using bypassing agents. 16 are still under ITI treatment, from which 7 (43.8%) already achieved clinical response to FVIII concentrate.Conclusion: In this study, we observed a complete ITI success rate of 67.2% among hemophilia A patients with inhibitor submitted to ITI treatment for the first time. In this cohort, inhibitor titer influenced ITI success throughout different treatment phases, such as historical peak, titer before ITI onset and peak during ITI. The frequency of bleeding episodes during ITI and the use of central venous access were also important predictors of ITI success. This knowledge should contribute to the stratification of prognosis and contribute to decision making in clinical care of patients with high-responding inhibitors under ITI treatment. [Display omitted] DisclosuresOzelo:Pfizer: Consultancy, Research Funding, Speakers Bureau; Roche: Consultancy, Speakers Bureau; Biogen: Consultancy, Research Funding, Speakers Bureau; CSL Behing: Consultancy; Shire: Consultancy, Research Funding, Speakers Bureau; Grifols: Speakers Bureau; Novo Nordisk: Consultancy, Research Funding, Speakers Bureau.

Tolerance induction with donor hematopoietic stem cell infusion in kidney transplantation: a single-center experience in China with a 10-year follow-up.

BackgroundImmunosuppressive therapy after life-saving kidney transplantation increases the risk of infection, cardiovascular diseases, metabolic diseases, and cancer. To date, four centers (three in the USA and one in South Korea) have reported clinical tolerance trials in kidney transplantation. We performed the first Chinese clinical trial in which kidney transplantation was combined with donor hematopoietic stem cell (DHSC) infusion to induce tolerance. This study summarizes the 10-year follow-up results.MethodsFrom 2009 to 2017, 11 donor/recipient pairs underwent living-related kidney transplantation combined with DHSC infusion. Two of the pairs were human leukocyte antigen (HLA)-matched, and nine were HLA-mismatched. DHSCs were mobilized using granulocyte colony-stimulating factor (G-CSF) and harvested 1 day before transplantation. The recipients received consecutive total lymphoid irradiation (TLI) for 3 days before kidney transplantation. The induction drug was anti-thymocyte globulin (ATG). DHSCs were infused on days 2, 4, and 6 post surgery. All patients were followed-up until Dec 2019. Routine laboratory examinations, chimerism, biopsies, and mixed lymphocyte reactions were performed.ResultsOne HLA-matched recipient had 30–50% chimerism, while the other patients had less than 1% chimerism. Recipients had donor-specific hyporesponsiveness (DSH) while sustaining normal reactivity to non-donors in mixed lymphocyte reactions. All recipients were followed up for 717–3,918 days. One recipient lost allograft function, and 10 recipients had stable renal function. None of the 11 recipients developed myelosuppression or graft-versus-host disease (GVHD) post transplantation. Our protocol did not increase the risk of infection. Allograft biopsy confirmed that one patient had mild rejection with Banff grade IA, while the other ten recipients did not develop rejection. Five patients were able to reduce the dose of their immunosuppressive therapy.ConclusionsOur immune tolerance induction protocol, which used DHSC infusion and TLI, achieved low dose immunosuppression with long-term stable kidney allograft survival in Chinese patients.

Practical Guidance of the GTH Haemophilia Board on the Use of Emicizumab in Patients with Haemophilia A.

Emicizumab has been approved for bleeding prophylaxis in patients with haemophilia A (PWHAs) with or without inhibitors. Because of substantial differences between factor VIII (FVIII) and Emicizumab, the 'Ständige Kommission Hämophilie' of the German, Austrian, Swiss Society for Thrombosis and Haemostasis Research (GTH) established a practical guidance for the use of Emicizumab in PWHAs. A systematic literature research was conducted in PubMed. Based on this and on personal experience, this practical guidance has been developed. Each single statement has been discussed among members of the 'Ständige Kommission Hämophilie' and revised accordingly. The final set of recommendations has been approved by all authors analogous to the Delphi method. This practical guidance is provided for physicians treating PWHAs with regard to general aspects, patient education, bleeding treatment, surgery, use of Emicizumab in previously untreated patients (PUPs), patients with newly diagnosed inhibitors and elderly patients. Patients should be treated in expert centres and adequate laboratory tests to monitor Emicizumab levels, FVIII replacement and inhibitors should be available. Early experience of immune tolerance induction protocols integrating Emicizumab is reviewed, and the limited experience in PUPs and very young children is described. So far, no thromboembolic complications have been reported with the concomitant use of FVIII or recombinant activated FVII for bleeding treatment or surgery. Activated prothrombin complex concentrate doses of >100 U/kg for >24 hours should be avoided whenever possible because of the high risk of thrombosis and/or thrombotic microangiopathy. In conclusion, this study is designed to support haemophilia physicians using Emicizumab in physicians treating hemophilia and using (PWHAs). With further post-marketing experience and trials, regular updates are necessary.

Escape or Fight: Inhibitors in Hemophilia A.

Replacement therapy with coagulation factor VIII (FVIII) represents the current clinical treatment for patients affected by hemophilia A (HA). This treatment while effective is, however, hampered by the formation of antibodies which inhibit the activity of infused FVIII in up to 30% of treated patients. Immune tolerance induction (ITI) protocols, which envisage frequent infusions of high doses of FVIII to confront this side effect, dramatically increase the already high costs associated to a patient's therapy and are not always effective in all treated patients. Therefore, there are clear unmet needs that must be addressed in order to improve the outcome of these treatments for HA patients. Taking advantage of preclinical mouse models of hemophilia, several strategies have been proposed in recent years to prevent inhibitor formation and eradicate the pre-existing immunity to FVIII inhibitor positive patients. Herein, we will review some of the most promising strategies developed to avoid and eradicate inhibitors, including the use of immunomodulatory drugs or molecules, oral or transplacental delivery as well as cell and gene therapy approaches. The goal is to improve and potentiate the current ITI protocols and eventually make them obsolete.

Clinical Care of Bone Health in Patients on the Immune Tolerance Induction's Protocols With an Immunosuppressive Agent for Inhibitor Eradication in Hemophilia.

Nowadays, the development of factor VIII and IX inhibitors in patients with hemophilia is considered as the most challenging in the treatment of hemophilia. Immune tolerance induction (ITI) therapy is an approach for eradication of inhibitors. Some ITI protocols are routinely in use for the eradication of inhibitors in patients with hemophilia. Moreover, such a therapeutic regimen may facilitate the tendency to reduced bone density in patients with inhibitor. This study scheduled to investigate whether that predisposing role of ITI protocols with an immunosuppressive agent has considered or not. By a literature review, published ITI protocols in hemophilia with inhibitors were evaluated. Among them, 51 papers found and studied thoroughly. None of them had performed the bone mineral examination in patients with hemophilia and inhibitor under treatment. Since there are 2 coexisting facilitating factors in these protocols, considering the bone mineral density study for patients with inhibitor who are undergoing ITI protocols with an immunosuppressive agent is recommended.

Factor VIII: Perspectives on Immunogenicity and Tolerogenic Strategies for Hemophilia A Patients.

A major complication in treating hemophilia A is the development of neutralizing antibodies (inhibitors) against therapeutic administered factor VIII (FVIII), which occurs in approximately 20-30% of patients with severe disease. These inhibitors render FVIII replacement therapy ineffective and increase the morbidity and mortality risk. The currently accepted method to eradicate inhibitors is immune tolerance induction (ITI), and frequent intensive administration of FVIII until inhibitor titers drop. Current ITI protocols are extremely costly and not effective in all patients. During the last decade, many types of research have been accomplished to clarify the mechanisms that mediate immune tolerance induction. Novel experimental therapies including monoclonal antibodies, viral vector-mediated gene therapy, regulatory T cell induction using immunosuppressive drugs, and nanoparticle-based immune modulation show promising results in hemophilia A clinical trials. This review focuses on treatment options towards the anti-FVIII immune responses and current novel therapies in clinical trials.

Origins and organization of the NHLBI State of the Science Workshop: Generating a national blueprint for future research on factor VIII inhibitors.

The major complication of protein replacement therapy for haemophilia A is the development of anti-FVIII antibodies or inhibitors that occur in 25%-30% of persons with severe haemophilia A. Alternative therapeutics such as bypassing agents or immune tolerance induction protocols have additional challenges and are not always effective. Assemble a National Heart, Lung and Blood Institute (NHLBI) State of the Science (SOS) Workshop to generate a national blueprint for research on inhibitors to solve the problem of FVIII immunogenicity. An Executive Steering Committee was formed in October 2017 to establish the scientific focus and Scientific Working Groups for the SOS Workshop in May 2018. Four working groups were assembled to address scientific priorities in basic, translational and clinical research on inhibitors. Working Group 1 was charged with determining the scientific priorities for clinical trials to include the integration of non-intravenous, non-factor therapeutics including gene therapy into the standard of care for people with haemophilia A with inhibitors. Working Group 2 established the scientific priorities for 21st-century data science and biospecimen collection for observational inhibitor cohort studies. The scientific priorities for acquiring an actionable understanding of FVIII immunogenicity and the immunology of the host response and FVIII tolerance were developed by Working Group 3. Working Group 4 designed prospective pregnancy/birth cohorts to study FVIII immunogenicity, inhibitor development and eradication. The NHLBI SOS Workshop generated a focused summary of scientific priorities and implementation strategies to overcome the challenges of eradicating and preventing inhibitors in haemophilia A.

Innovative Approaches for Immune Tolerance to Factor VIII in the Treatment of Hemophilia A.

Hemophilia A (coagulation factor VIII deficiency) is a debilitating genetic disorder that is primarily treated with intravenous replacement therapy. Despite a variety of factor VIII protein formulations available, the risk of developing anti-dug antibodies (“inhibitors”) remains. Overall, 20–30% of patients with severe disease develop inhibitors. Current clinical immune tolerance induction protocols to eliminate inhibitors are not effective in all patients, and there are no prophylactic protocols to prevent the immune response. New experimental therapies, such as gene and cell therapies, show promising results in pre-clinical studies in animal models of hemophilia. Examples include hepatic gene transfer with viral vectors, genetically engineered regulatory T cells (Treg), in vivo Treg induction using immune modulatory drugs, and maternal antigen transfer. Furthermore, an oral tolerance protocol is being developed based on transgenic lettuce plants, which suppressed inhibitor formation in hemophilic mice and dogs. Hopefully, some of these innovative approaches will reduce the risk of and/or more effectively eliminate inhibitor formation in future treatment of hemophilia A.

Oral Tolerance Induction in Hemophilia B Dogs Fed with Transplastomic Lettuce

Anti-drug antibodies in hemophilia patients substantially complicate treatment. Their elimination through immune tolerance induction (ITI) protocols poses enormous costs, and ITI is often ineffective for factor IX (FIX) inhibitors. Moreover, there is no prophylactic ITI protocol to prevent anti-drug antibody (ADA) formation. Using general immune suppression is problematic. To address this urgent unmet medical need, we delivered antigen bioencapsulated in plant cells to hemophilia B dogs. Commercial-scale production of CTB-FIX fusion expressed in lettuce chloroplasts was done in a hydroponic facility. CTB-FIX (∼1 mg/g) in lyophilized cells was stable with proper folding, disulfide bonds, and pentamer assembly after 30-month storage at ambient temperature. Robust suppression of immunoglobulin G (IgG)/inhibitor and IgE formation against intravenous FIX was observed in three of four hemophilia B dogs fed with lyophilized lettuce cells expressing CTB-FIX. No side effects were detected after feeding CTB-FIX-lyophilized plant cells for >300 days. Coagulation times were markedly shortened by intravenous FIX in orally tolerized treated dogs, in contrast to control dogs that formed high-titer antibodies to FIX. Commercial-scale production, stability, prolonged storage of lyophilized cells, and efficacy in tolerance induction in a large, non-rodent model of human disease offer a novel concept for oral tolerance and low-cost production and delivery of biopharmaceuticals.

Durable and sustained immune tolerance to ERT in Pompe disease with entrenched immune responses.

Enzyme replacement therapy (ERT) has prolonged survival and improved clinical outcomes in patients with infantile Pompe disease (IPD), a rapidly progressive neuromuscular disorder. Yet marked interindividual variability in response to ERT, primarily attributable to the development of antibodies to ERT, remains an ongoing challenge. Immune tolerance to ongoing ERT has yet to be described in the setting of an entrenched immune response. Three infantile Pompe patients who developed high and sustained rhGAA IgG antibody titers (HSAT) and received a bortezomib-based immune tolerance induction (ITI) regimen were included in the study and were followed longitudinally to monitor the long-term safety and efficacy. A trial to taper the ITI protocol was attempted to monitor if true immune tolerance was achieved. Bortezomib-based ITI protocol was safely tolerated and led to a significant decline in rhGAA antibody titers with concomitant sustained clinical improvement. Two of the 3 IPD patients were successfully weaned off all ITI protocol medications and continue to maintain low/no antibody titers. ITI protocol was significantly tapered in the third IPD patient. B cell recovery was observed in all 3 IPD patients. This is the first report to our knowledge on successful induction of long-term immune tolerance in patients with IPD and HSAT refractory to agents such as cyclophosphamide, rituximab, and methotrexate, based on an approach using the proteasome inhibitor bortezomib. As immune responses limit the efficacy and cost-effectiveness of therapy for many conditions, proteasome inhibitors may have new therapeutic applications. This research was supported by a grant from the Genzyme Corporation, a Sanofi Company (Cambridge, Massachusetts, USA), and in part by the Lysosomal Disease Network, a part of NIH Rare Diseases Clinical Research Network (RDCRN).

Innovating immune tolerance induction for haemophilia.

Haemophilia A is an X-linked bleeding disorder characterized by a deficiency of coagulation protein factor VIII (FVIII). A challenging complication of therapeutic FVIII infusions is the formation of neutralizing alloantibodies against the FVIII protein defined as inhibitors. The development of FVIII inhibitors drastically alters the quality of life of the patients and is associated with tremendous increases in morbidity as well as treatment costs. Current clinical immune tolerance induction protocols to reverse inhibitors are lengthy, costly and not effective in all patients. Prophylactic protocols to prevent inhibitor formation have not yet been developed in the clinical setting. However, there has been ample progress towards this goal in recent years in preclinical studies using animal models of haemophilia. Here, we review the mechanisms that lead to inhibitor formation against FVIII and two promising new strategies for antigen-specific tolerance induction. CD4+ T cells play an important role in the FVIII-specific B cell response. Immune tolerance can be induced based on transplacental delivery of FVIII domains fused to Fc or on oral delivery of leaf cells from chloroplast transgenic crop plants. Recent literature suggests that prophylactic tolerance induction protocols for FVIII may be feasible in haemophilia A patients.

688. Supplemental Immune Suppression Is Required for AAV-F8 ITI in Hemophilia A Mice with Pre-Existing Inhibitors

Hemophilia is an inherited coagulation disorder resulting in the loss of functional clotting factors VIII (hemophilia A) or IX (hemophilia B). Patients with the severe hemophilia (less than 1% residual clotting factor activity) develop spontaneous bleeds into the joints and closed spaces and have a high incidence of morbidity if not treated. Current treatment involves frequent infusion of short-lived exogenous plasma derived or recombinant clotting factors. A major complication with protein therapy is the development of inhibitory antibodies in 25-30% hemophilia A and 3-5% hemophilia B patients that prevent normalization of hemostasis with continued protein therapy. Inhibitor patients are treated with either bypassing agents or immune tolerance induction (ITI) therapy requiring daily infusions (months to years) of supraphysiological levels of clotting factor protein. ITI therapy is effective in up to 70% of hemophilia A patients, while it often fails in hemophilia B patients due to the development of pathogenic antibodies that induce anaphylaxis and nephrotic syndrome. Both treatment options for inhibitor patients have high associated financial and health related costs for the patient. We have previously shown that hemophilia B mice with pathogenic inhibitory antibodies are effectively treated with a novel immune tolerance induction protocol using a liver directed adeno-associated virus expressing the gene encoding for human factor IX protein (AAV8-F9). AAV8-F9 ITI rapidly eliminates pre-existing inhibitors and provided sustained therapeutic levels of FIX protein even following repeated exposure to recombinant FIX protein. Based on our success with hemophilia B, we sought to develop a complementary protocol for hemophilia A mice with pre-existing inhibitors. Our initial studies were conducted using a codon-optimized F8 (coF8) gene, which has been shown to enhance the levels of transcribed FVIII protein. In the first set of experiments we tested two different vector doses (1×1011 and 1×1012 vg) of an AAV8-cohF8 vector for factor VIII expression levels and tolerance induction in hemophilia A mice on two different genetic backgrounds (BALB/c-F8−/Y and 129/BL6-F8−/Y). Since BALB/c-F8−/Y mice develop lower titer FVIII inhibitors following intravenous hFVIII protein challenge compared to 129/BL6-F8−/Y mice we expected better outcomes with AAV8-cohF8 vector treated BALB/c-F8−/Y mice. Surprisingly BALB/c-F8−/Y mice spontaneously developed FVIII inhibitors approximately 4 weeks following vector delivery. In contrast,129/BL6-F8−/Y mice expressed vector dose dependent therapeutic levels FVIII protein without inhibitors and 75% of the mice remained inhibitor free following four weekly IV FVIII protein challenges. Subsequent studies for AAV8-cohF8 ITI were conducted in 129/BL6-F8−/Y mice. We generated a group of inhibitor mice and treated a subset of these mice with 1×1012 vg AAV8-coF8. AAV8-cohF8 ITI alone was not sufficient to eliminate inhibitors. Therefore we designed two additional studies to determine if 1×1012 vg AAV8-coF8 ITI would be more effective if combined with transient immune suppression using oral rapamycin (4mg/kg) or anti-CD20 (10mg/kg) mediated B cell depletion. While rapamycin adjunct therapy failed to lower inhibitors, preliminary data show that anti-CD20 combined with AAV8-coF8 ITI leads to a sharp reduction in high titer inhibitors. Given that FVIII protein invokes a stronger immune response in mice and patients, our data suggests that successful gene therapy for hemophilia A may require supraphysiological levels of FVIII protein to prevent inhibitor formation and transient immune suppression to be effective as an ITI therapy.

Immune Tolerance Induction Treatment Is Cost-Effective in Adult Patients with Long-Standing High-Responding Inhibitors

Introduction: Despite treatment of hemophilia having achieved considerable improvement over the last decades, inhibitor development remains a major complication for these patients. Inhibitors are alloantibodies that neutralize factor (F) VIII coagulation activity, and occur in approximately 25% of hemophilia A patients. Immune tolerance induction (ITI) is the choice therapy to eradicate these antibodies. There are several ITI protocols with similar success rates, approximately 70%. The presence of long-standing inhibitors in the adult population has usually been associated with poor ITI outcome. In Brazil, as in other countries with constrained resources, limited access to factor concentrates prevented the use of ITI. Thus, almost all patients with inhibitors remained a long time with this complication. Only after 2013 with increased availability of factor concentrates in the country, has assured long-term prophylaxis and ITI treatment been possible.Objective: This study aimed to describe the Brazilian experience in managing ITI in adult patients from seven Hemophilia Treatment Centers with long-standing inhibitor using bypassing agents to control bleeding episodes, considering bleeding frequency, treatment barriers and economic impact of therapy.Patients and Methods: We retrospectively analyzed clinical and laboratory data of hemophilia A patients with high-responding inhibitors (peak titer > 5 Bethesda Unit (BU), anamnestic response to FVIII) over 18 yo who underwent ITI treatment and were using bypass agents before ITI. The initial ITI protocol used was low-dose FVIII concentrate (25-50 IU/kg 3x/week)Results: In Brazil, since the availability of ITI, 39 adult hemophilia A patients with inhibitors, have been submitted to ITI for the first time. In this study, we accessed data from 26 patients, of these 13 (50%) already achieved complete ITI success criteria (inhibitor titer < 0.6 BU; FVIII recovery ≥ 66%, and half-live ≥ 6 h). Another 4 patients with ongoing ITI achieved inhibitor titer < 5 BU and are no longer using bypassing agents. Thus, 17/26 (65%) patients discontinued the bypassing agents in a median period of 2 mo (range 0 to 18 mo) after starting ITI. After 24 months of irregular treatment 2 patients abandoned ITI protocol. Analyses of the 13 patients with complete success revealed mean age at first reported inhibitor of 16.21y (SD ± 11.6; median 21y, range 0.7 to 37y), and mean age at ITI onset of 31.2y (SD ± 10.6; median 27y, range 19 to 47y). The mean period from the first inhibitor detected until starting ITI was 15y (SD ± 5.1), with a 16y median (range 7 to 22y). The historical peak inhibitor mean was 53.1 BU (SD ± 49.5; median 39.4 BU, range 5.5 to 163 BU). The mean pre ITI inhibitor titer was 6.6 BU (SD ± 5.5; median 5.5 BU, range 0.6 to 20.8 BU). Low-dose ITI protocol was initially used for all patients, however 3 patients had the dose increased during treatment (100 IU/kg from 3 to 7 x/week). During ITI, 10/13 patients received prophylaxis with bypassing agents, and 9 began prophylaxis before ITI started. The mean time to achieve inhibitor titer < 0.6 BU was 11.6 mo (SD ± 12.5; median 7 mo, range 0.5 to 44 mo). All 13 patients achieved other ITI success criteria. However, a delay was observed in the majority of patients in FVIII recovery and half-life. Regarding clinical outcome, we observed a significant reduction in both, annualized bleeding rate (ABR), and annualized joint bleeding rate (AJBR), comparing 12 months before ITI, during ITI, and after achieving complete success (figure 1). Regarding economic evaluation, we observed a significant reduction in the median cost of clotting factor consumption, comparing 12 months before starting ITI, and the following years after achieving ITI success. No significant difference was observed between the median cost of 12 months before, and ITI period (figure 2).Conclusion: This study indicated that this treatment is worthwhile even for inhibitor patients with poor prognostic factors for ITI considering both effectiveness to control and prevent bleeding episodes, and economic impact. In this group, 65% of adult hemophilia A high-responding inhibitors patients stopped using bypassing agents after a median period of 2 months after starting ITI. Despite being a high cost treatment, ITI can be cost-effective, especially for patients using bypassing agents. These results reinforce the positive impact of the ITI even in long time inhibitor adult patients. [Display omitted] [Display omitted] DisclosuresOzelo:Baxter: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novo Nordisk: Research Funding, Speakers Bureau; Biogen: Research Funding.