FVIII Protein Is Not Detectable in Human PBMCs or Livers from Dogs with an Intron-22 Inversion Mutation: Implications for FVIII Immunogenicity and Tolerance

A clinically relevant question is whether partial FVIII proteins are expressed in tissues from patients with severe hemophilia A (HA) due to an intron-22 inversion mutation. If so, this could in principle confer central immune tolerance to the expressed FVIII regions, thereby lowering the risk of anti-FVIII immune responses. In 2013, Pandey et al. reported detection of FVIII proteins in peripheral blood mononuclear cells (PBMCs), B cells and liver tissues from an intron-22 inversion subject and non-HA controls. They concluded that partial FVIII proteins were translated from inverted mRNA encoding F8 exons 1-22, and also from the F8B transcript, which contains F8 exons 23-26 (Nature Medicine 19(10), 1318-24). In 2014, the Montgomery and Ginsburg labs reported that FVIII protein is expressed principally, and possibly exclusively, in endothelial cells (ECs). These studies utilized mouse models with EC-directed deletion of F8 exons 17-18, or of the FVIII transporter protein LMAN1, respectively. The F8-EC-knockout mice had a severe hemophilia A (HA) phenotype. Earlier studies from several labs, including Pandey et al., relying primarily on antibody staining, had reported FVIII expression in additional cell types, including PBMCs and hepatocytes. The present study tests the hypothesis that partial FVIII proteins are expressed from inverted mRNA encoding F8 exons 1-22 and/or from F8B mRNA. A panel of FVIII-specific polyclonal and monoclonal antibodies (mAbs) was tested for FVIII specificity by staining permeabilized PBMCs, monocytes, T cells, blood outgrowth ECs, monocyte-derived macrophages and dendritic cells, HUVECs and B-cell lines. Positive and negative controls included FVIII-expressing BHK cells and non-engineered BHK cells. Specificity was further tested by immunoprecipitation (IP) of cell lysates followed by SDS-PAGE, Western blots and mass spectrometry to define proteins pulled down by the anti-FVIII antibodies. In addition, immunohistochemistry (IHC) staining was carried out for liver sections from HA dogs with an intron-22 inversion mutation and from otherwise normal (non-HA) control dogs. IP followed by SDS-PAGE, Westerns and/or mass spec revealed that a significant fraction of the anti-FVIII antibodies bound to other proteins besides FVIII in the tissues and cells examined. A cocktail of 4 anti-FVIII mAbs that were validated using (+) and (-) control samples was used to stain isolated and cultured cells and tissues. Cross-recognition of canine FVIII was confirmed by IP + Western blots. IHC using the mAb cocktail followed by HRP-conjugated secondary antibodies produced variable staining of liver tissues from HA and non-HA dogs, regardless of tissue preparation methods. However, use of fluorescent-labeled secondary mAbs produced signals well above background in ECs of control dog livers but not in the HA livers. Multiple confocal images were selected randomly, and average MFI values of 10 non-HA liver sections were well above those of 10 HA sections (p = 0.006). A second staining method, the Duolink Proximity Ligation Assay, was employed as an independent test to detect interactions between FVIII and VWF. Again, only normal control liver sections showed fluorescence above background (p =0.0004). F8B mRNA was not detected in canine liver and was not expected based on the canine intron-22 DNA sequence. We conclude that if protein is expressed from inverted mRNA encoding F8 exons 1-22, it is below the detection limit of these assays. Permeabilized human cells were tested for FVIII expression by staining using the mAb cocktail, or validated anti-FVIII-C2 mAbs to detect the putative protein encoded by F8B, followed by fluorescent detection. Cells were also stimulated with histamine followed by a chromogenic FVIII activity assay of supernatants. Results indicated that if FVIII or F8B protein are expressed in non-ECs, they are below the detection limit of these assays. We also note that if the putative F8B-encoded protein confers tolerance, immune responses of HA patients to the FVIII C2 domain would be quite rare, which is not the case. Although neonatal thymic expression of partial FVIII proteins may occur, immune tolerance to self-antigens requires repeated exposures of the immune system to the antigens. We conclude it is unlikely that HA patients with an intron-22 inversion mutation have universally acquired central tolerance to partial proteins encoded by inverted F8 mRNA or F8B. Disclosures Ragni: Shire/Takeda: Consultancy, Other: Study drug; Sangamo: Research Funding; OPKO: Research Funding; Bioverativ/Sanofi: Consultancy, Research Funding; Bayer: Consultancy; ICER: Consultancy; Biomarin: Consultancy, Research Funding; Alnylam/Sanofi: Consultancy, Research Funding; Spark Therapeutics: Consultancy, Research Funding. Pratt:Bloodworks NW: Patents & Royalties: inventor on patents related to FVIII immunogenicity; Grifols, Inc: Research Funding.

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French Previously Untreated Patients with Severe Hemophilia A after Exposure to Recombinant Factor VIII : Incidence of Inhibitor and Evaluation of Immune Tolerance

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615358 ◽

1998 ◽

Vol 80 (11) ◽

pp. 779-783 ◽

Cited By ~ 60

Author(s):

Y. Laurian ◽

E. P. Satre ◽

A. Borel Derlon ◽

H. Chambost ◽

P. Moreau ◽

...

Keyword(s):

Factor Viii ◽

Immune Tolerance ◽

Hemophilia A ◽

High Titer ◽

Recombinant Factor Viii ◽

High Dose ◽

Severe Hemophilia ◽

Inhibitor Development ◽

Intron 22 Inversion ◽

Median Period

SummaryFifty French previously untreated patients with severe hemophilia A (factor VIII <1%), treated with only one brand of recombinant factor VIII (rFVIII), were evaluated for inhibitor development, assessment of risk factors and outcome of immune tolerance regimen. The median period on study was 32 months (range 9-74) since the first injection of rFVIII. Fourteen patients (28%) developed an inhibitor, four of whom (8%) with a high titer (≥10 BU). All inhibitor patients but one continued to receive rFVIII either for on-demand treatment or for immune tolerance regimen (ITR). Among these patients, inhibitor was transient in 2 (4%), became undetectable in 6 and was still present in 6. The prevalence of inhibitor was 12%. Presence of intron 22 inversion was found to be a risk factor for inhibitor development. Immune tolerance was difficult to achieve in our series despite a follow-up period of 16 to 30 months: immune tolerance was complete in only one out of the 3 patients undergoing low dose ITR and in one out of the 5 patients with high dose ITR.

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Neither Factor VIII Nor the Putative F8B Protein Is Detectable in Human Peripheral Blood Mononuclear Cells

Blood ◽

10.1182/blood.v126.23.3507.3507 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3507-3507

Author(s):

Devi Gunasekera ◽

Kenneth B. Lewis ◽

Alexis A. Thompson ◽

Ronald R, Louie ◽

David W. Scott ◽

...

Keyword(s):

Mononuclear Cells ◽

Hemophilia A ◽

Human Peripheral Blood ◽

Positive Control ◽

Human Pbmcs ◽

Peripheral Blood Mononuclear ◽

Western Blots ◽

Bhk Cells ◽

Sds Page ◽

Secondary Antibodies

Abstract Factor VIII (FVIII) is encoded by 26 exons comprising the F8 gene. The F8 mRNA transcript encodes the full-length FVIII protein, which consists of 2332 amino acids. A CpG island within intron 22 of F8 includes an initiation site for a second, shorter transcript termed F8B. F8B contains a short exon spliced in-frame to F8 exons 23-26, and this mRNA transcript is expressed in multiple human tissues. The putative protein encoded by this gene corresponds to 8 amino acids encoded by the exon within F8 intron 22, followed by the FVIII C2 domain sequence. Earlier studies have unambiguously demonstrated FVIII expression in human endothelial cells, including liver and lung. Animal model studies have indicated FVIII is expressed, possibly exclusively, in endothelial cells. Other recent reports have presented evidence for FVIII and F8B protein expression in human peripheral blood mononuclear cells (PBMCs). In this study, we utilized several methods to test the hypothesis that FVIII and/or the putative F8B protein is expressed in human PBMCs. Frozen PBMCs from healthy human subjects were thawed and then washed 5-10X to ensure removal of plasma-derived FVIII. Samples containing 50 million PBMCs were lysed in buffer containing protease inhibitors, centrifuged, and the supernatants analyzed by immunoprecipitation (IP) followed by SDS-PAGE and Western blots. The IP experiments utilized both polyclonal anti-FVIII antibodies and cocktails of monoclonal antibodies (mAbs) specific for different FVIII domains, and Western blots used several combinations of primary and secondary antibodies. Negative controls consisted of BHK cell lysates, and positive controls were purified FVIII and lysates of BHK cells transduced with a plasmid encoding FVIII. Western blots carried out with sensitive chemiluminescence detection identified the expected bands in the positive control lanes. Bands at expected positions for FVIII or F8B proteins (~240kDa, 75 kDa, 25-30 kDa) visualized by Coomassie-blue staining of lysates analyzed by SDS-PAGE were cut from the gels, subjected to trypsin digestion and then analyzed by mass spectrometry (LC-MS). FVIII-derived peptides were detected only in the positive control samples. Intracellular staining (ICS) of permeabilized PBMCs was carried out using mAbs specific for the FVIII A1, A2, C1, and C2 domains and a mAb specific for the FVIII light chain. The mAbs were covalently labeled with Alexa-Fluor 488 to avoid possible nonspecific binding of labeled secondary antibodies. PBMCs from (1) normal control subjects; (2) a severe hemophilia A subject with an intron-22 inversion mutation and (3) a severe hemophilia A subject with a deletion of F8 exons 3-6 were analyzed. All 5 anti-FVIII mAbs detected FVIII protein in FVIII-expressing BHK cells, while none detected FVIII in BHK cells or in any of the human PBMC samples. Our results are consistent with several recent studies reporting proteomics analysis of human PBMCs, which have identified no FVIII peptides or proteins. We conclude that if FVIII or F8B proteins are expressed in human PBMCs, their concentration is below the detection limits of these assays. Disclosures No relevant conflicts of interest to declare.

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Administration of Dexamethasone during Initial Exposure to Factor VIII Induces Durable and Antigen-Specific Tolerance to Factor VIII in Hemophilia Α Mice with a Humanized Major Histocompatibility Complex II Transgene

Blood ◽

10.1182/blood.v124.21.1489.1489 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1489-1489 ◽

Cited By ~ 2

Author(s):

Paul C. Moorehead ◽

Maria T. Georgescu ◽

Alice S. van Velzen ◽

Kate Sponagle ◽

Barbara Vidal ◽

...

Keyword(s):

Immune Responses ◽

Factor Viii ◽

Research Funding ◽

Hemophilia A ◽

Severe Hemophilia ◽

Humanized Mouse ◽

Humanized Mouse Model ◽

Advisory Committees ◽

Initial Exposure ◽

Histocompatibility Complex

Abstract Background For persons with severe hemophilia A receiving exogenous factor VIII (FVIII) replacement therapy, antibodies that interfere with the coagulant function of FVIII (inhibitors) are the most severe complication of treatment. Although avoidance of immunologic “danger signals” during initial FVIII exposure has been proposed as a strategy to reduce the risk of inhibitors, its effectiveness is not proven or reproducible. We have previously reported that the active elimination of danger signals, using the potent anti-inflammatory and immunosuppressive corticosteroid dexamethasone (Dex), during initial exposure to FVIII decreased the anti-FVIII immune response in an immunologically humanized mouse model of severe hemophilia A. We sought to demonstrate the effectiveness of Dex in preventing anti-FVIII immune responses in a larger cohort of mice, and to determine the durability and antigen-specificity of the immunologic tolerance conferred by Dex. Materials and Methods Mice with a hemophilia A phenotype due to knockout of exon 17 of the F8gene and with a chimeric human/murine transgene for Major Histocompatibility Complex II allele DRB1*1501 on a C57Bl/S129 background were used for all experiments. (These mice have previously been demonstrated to have an approximately 30% incidence of anti-FVIII IgG antibodies after treatment with recombinant human FVIII). Recombinant human FVIII (Advate, approximately 0.1 mcg/unit) was given by tail vein injection (IV) at 6 units/dose. Dex was given intraperitoneally at 75 mcg/dose. Lipopolysaccharide (LPS) was given IV with FVIII at 2 mcg/dose. Plasma-derived human von Willebrand Factor (VWF, approximately 10 mcg/unit) was given IV at 2 units/dose. Blood samples were collected retro-orbitally or via cardiac puncture, and plasma was obtained by centrifugation with samples frozen at -80C until analysis. Anti-FVIII immunoglobulin G (IgG) and anti-VWF IgG were detected by ELISA, and FVIII inhibitory activity was measured using the Bethesda assay. Statistical comparisons were made using the chi-square or Fischer’s exact test, as appropriate. Results In the first experiment, mice were treated with either FVIII or FVIII and Dex (FVIII+Dex) daily x 5, and then sampled 5 weeks later. The incidence of anti-FVIII IgG was higher (21% vs. 6%, p = 0.084) in FVIII mice (n=34) than in FVIII+Dex mice (n=35). IgG negative FVIII mice were then given FVIII daily x 3 (FVIII/FVIII), while IgG negative FVIII+Dex mice were given either FVIII (FVIII+Dex/FVIII) or FVIII with LPS (FVIII+Dex/FVIII+LPS) daily x 3. Mice were sampled 3 weeks later. The incidence of anti-FVIII IgG was higher in FVIII/FVIII mice (n=23) than in FVIII+Dex/FVIII mice (n=15) (52% vs. 7%, p=0.005), but not higher than in FVIII+Dex/FVIII+LPS mice (n=10) (52% vs. 30%, p=0.28). While 8 of 23 FVIII/FVIII mice had a positive Bethesda assay, 0 of 15 FVIII+Dex/FVIII mice had inhibitors (35% vs 0%, p=0.01). In the second experiment, mice (n=30) had FVIII+Dex daily x 5, and were all anti-FVIII IgG negative when sampled 4 and 14 weeks later. Mice then had either a re-exposure with FVIII daily x 3 (FVIII+Dex/FVIII) 16 weeks after first FVIII exposure, or an intermittent exposure to FVIII one day every 4 weeks followed by FVIII daily x 3 (FVIII+Dex/intFVIII) at 16 weeks. The incidence of anti-FVIII IgG was low (17% overall) and not statistically different between FVIII+Dex/FVIII (n=15) and FVIII+Dex/intFVIII (n=15) mice (7% vs. 27%, p=0.33). Bethesda assays showed a similar pattern. Mice in both groups were then given VWF weekly x 4, and sampled 1 week later: all mice developed anti-VWF IgG, except 1 mouse that had had FVIII+Dex/intFVIII. Conclusions When given during initial exposure to FVIII, Dex decreases the incidence of anti-FVIII IgG in an immunologically humanized mouse model of severe hemophilia A. The incidence of new anti-FVIII IgG is low after re-exposure to FVIII, either 6 weeks or 16 weeks after initial exposure. Importantly, Dex-treated mice were able to form antibodies to an unrelated antigen. These results suggest that Dex induces durable immunologic tolerance to FVIII specifically, and that anti-FVIII immune responses are not reduced simply by generalized immunosuppression. Further studies are warranted, both to determine the specific mechanisms by which Dex induces tolerance to FVIII and to investigate the feasibility and effectiveness of using Dex for this purpose in the clinical setting. Disclosures Moorehead: Baxter: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria. Reipert:Baxter Innovation GmbH: Employment. Steinitz:Baxter: Employment. Hough:Bayer: Research Funding. Lillicrap:Baxter: Research Funding; Bayer: Research Funding; CSL Behring: Research Funding; Biogen Idec: Research Funding.

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Immune Tolerance Induction Using Rfviiifc (Eloctate)

Blood ◽

10.1182/blood.v126.23.3531.3531 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3531-3531 ◽

Cited By ~ 2

Author(s):

Lynn M. Malec ◽

Margaret V Ragni ◽

Janna M. Journeycake ◽

Michelle Alabek

Keyword(s):

Board Of Directors ◽

Immune Tolerance ◽

Half Life ◽

Research Funding ◽

Hemophilia A ◽

Central Venous Access ◽

Tolerance Induction ◽

Severe Hemophilia ◽

Advisory Committees ◽

Immune Tolerance Induction

Abstract Introduction: Inhibitor formation affects approximately 30% of individuals with severe hemophilia A. The eradication of inhibitors using immune tolerance induction (ITI) remains the mainstay of therapy, although typically requires daily high-dose factor VIII via a port for up to a year. Extended half-life recombinant factor VIII Fc fusion protein (rFVIIIFc, Eloctate¨) has a half-life extension 1.5-fold longer than standard recombinant FVIII (rFVIII), reducing treatment frequency, and also induces regulatory T cell response to FVIII in animal models. We hypothesized that rFVIIIFc would provide more effective ITI, specifically shortening ITI, than rFVIII. We describe ITI with rFVIIIFc in three patients with severe hemophilia A. Methods: Immune tolerance induction was initiated with rFVIIIFc (Eloctate) in three children with severe hemophilia A and an anti-FVIII inhibitor. Dosing was per MD discretion with family agreement, and performed by central venous access device or intravenous infusion via heplock. Follow-up was scheduled every 6-8 weeks, with planned determination of FVIII half-life once the anti-FVIII fell to <0.6 B.U. Tolerance was a priori defined as achieving anti-FVIII <0.6 B.U. and half-life, t½ >6 hours. FVIII half-life was determined by one-stage FVIII:C assay on citrate samples drawn pre- and 10 minutes, 1, 2, 4, and 6 hours post-infusion of a single dose of rFVIIIFc. Once a t½ >6 hours was documented, incremental reduction to 50 IU/kg every other day or three times weekly, once there was evidence of maintenance of inhibitor neutralization and a >6 hour FVIII:C half-life. Results: Immune tolerance induction was initiated with rFVIIIFc at a dose of 100-200 IU/kg rFVIIIFc via central venous access device every other day or three times weekly per MD discretion in three children with severe hemophilia A and in anti-FVIII inhibitor > 5 B.U. (Table 1). Two patients had F8 genetic testing. In two patients, Pt 1 and Pt 3, this was the initial ITI course, and in the third child (Pt 2) this was salvage ITI after failing to achieve tolerance due to noncompliance with daily rFVIII ITI taper regimen. In two rFVIIIFc ITI was begun when anti-FVIII was < 10 B.U. Historic peak titers were 16-422 B.U. The time to anti-FVIII tolerance was 4-12 weeks Discussion: Immune tolerance induction was successful in three children with inhibitors using rFVIIIFc, including a child previously failing rFVIII ITI. The time to anti-FVIII=0 was 4-12 weeks, significantly shorter than with current rFVIII ITI. There were no adverse effects. These data indicate that rFVIIIFc safely and effectively induced immune tolerance to FVIII in children with inhibitors. Whether ITI may be accomplished more rapidly with rFVIIIFc, and the optimal dose for ITI will require prospective studies. A prospective observational study of rFVIIIFc ITI pre- and post-ITI T cell responses in children with hemophilia and inhibitors, the H emophilia I nhibitor R esponse to E loctate (HIRE) Study, is underway. Table 1. Immune Tolerance Induction with rFVIIIFc in Hemophilia A Inhibitor Patients Patient (Pt) Hemophilia Severity F8 Gene Mutation Age at Anti-FVIII Detection Peak Anti-FVIII Titer Initial ITI Dose Time toAnti-FVIII = 0 Current Anti-FVIII 1 <0.01 IU/ml Intron 22 inversion 13 months 32 B.U. 200 IU/kg QOD 12 weeks 0 B.U. 2 < 0.01 IU/ml Exon 18 nonsense variant 9 months 422 B.U. 200 IU/kg 3x/week 4 weeks 0 B.U. 3 <0.01 IU/ml Not available 10 years 16 B.U. 100 IU/kg QOD 11 weeks 0 B.U. Disclosures Malec: Baxter: Research Funding; Biogen: Research Funding. Ragni:Pfizer: Research Funding; Tacere Benitec: Membership on an entity's Board of Directors or advisory committees; Baxalta: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Myers Squibb: Research Funding; Dimension: Research Funding; Vascular Medicine Institute: Research Funding; Shire: Membership on an entity's Board of Directors or advisory committees, Research Funding; CSL Behring: Research Funding; SPARK: Research Funding; Biomarin: Research Funding; Genentech Roche: Research Funding; Bayer: Research Funding; Biogen: Research Funding; Alnylam: Research Funding. Journeycake:CSL, Baxalta, NovoNordisk: Consultancy; ATHN: Research Funding; Biogen: Speakers Bureau; ATHN: Membership on an entity's Board of Directors or advisory committees.

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Durability of ITI Utilizing Rfviiifc

Blood ◽

10.1182/blood.v128.22.3793.3793 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3793-3793

Author(s):

Margaret V. Ragni ◽

Lynn M. Malec ◽

Janna M. Journeycake

Keyword(s):

Immune Tolerance ◽

Half Life ◽

Research Funding ◽

Hemophilia A ◽

Tolerance Induction ◽

Severe Hemophilia ◽

Immune Tolerance Induction ◽

Viii Inhibitor ◽

Inhibitor Titer

Introduction: The eradication of inhibitors using immune tolerance induction (ITI) remains the mainstay of therapy in patients with severe hemophilia A who develop inhibitors. The long-acting recombinant factor VIII Fc fusion protein, rFVIIIFc (Eloctate™), which is safe and effective in the prevention and treatment of bleeding events, may promote tolerance to FVIII as shown in preclinical animal models and an inhibitor prone child, as Fc suppresses immunoregulatory Tcells to proteins to which Fc is attached. We therefore previously hypothesized rFVIIIFc would provide effective ITI, specifically shortening and simplifying ITI, and have previously described successful inhibitor eradication in three patients. Long-term follow-up data after successful ITI in patients with severe hemophilia remains limited. In the International Immune Tolerance Induction study, at 1-year follow-up, 6 of 66 subjects who had achieved tolerance demonstrated evidence of relapse at a median of 9.5 months. Of these 6 subjects, 1 had a measurable inhibitor titer and 5 had reduced FVIII recovery. We aim to provide follow-up data on our cohort of patients who had successful inhibitor eradication utilizing rFVIIIFc for ITI. Methods: Immune tolerance induction was initiated in three patients with severe hemophilia A and anti-VIII >5 B.U., in two as initial ITI (Pt. 1, 3), and one as salvage (Pt. 2) after failing to achieve ITI with standard rFVIII due to poor compliance. Follow-up was scheduled every 6-8 weeks, with planned determination of FVIII half-life once the anti-FVIII fell to <0.6 B.U. Tolerance was a priori defined as achieving anti-FVIII <0.6 B.U., FVIII recovery of at least 60%, and half-life (t½) >6 hours. Once a t½ >6 hours was documented, incremental reduction to rFVIIIFc occurred. Patients continued to be followed by their local HTC as per standard of care. Results: ITI was initiated with rFVIIIFc at a dose of 100-200 IU/kg rFVIIIFc every other day or three times weekly per MD discretion. The time to initial anti-FVIII <0.6 B.U. was 4-12 weeks. Patient 1 and 2 were able to achieve tolerance, with a FVIII recovery of at least 60%, and half-life (t½) >6 hours, at weeks 18 and 17, respectively, after initiation of ITI. Patient 3 has improved but is not yet fully tolerized, as evidenced by 57% recovery and a t½ of approximately 7 hours. Anti-VIII inhibitor titers remain negative at 15, 16 and 15 months, from the initiation of ITI in patients 1, 2, and 3 respectively. Patients 1 and 2 have been able to decrease their post ITI prophylaxis dosing regimen to 80 IU/kg and 65 IU/kg three times a week while maintaining a FVIII trough of >1%. No patients were maintained on bypassing prophylaxis during ITI and no patients have experienced hemarthroses or other major bleeding event since the initiation of ITI. Discussion: Immune tolerance induction was successful in three children with inhibitors using rFVIIIFc, including a child previously failing rFVIII ITI. The time to anti-FVIII=0 was 4-12 weeks, significantly shorter than with current rFVIII ITI. At a mean duration of follow up of 15.3 months, all patients achieved an anti-VIII inhibitor titer of 0 B.U. Repeat pharmacokinetics studies will be available at planned subsequent follow-up visit. To date, these data indicate that rFVIIIFc safely and effectively induced immune tolerance to FVIII in three children with inhibitors, and has provided durable and continuing immune tolerance to FVIII. Whether rFVIIIFc ITI will be successful and durable in a larger cohort of children with severe hemophilia A will require prospective studies. A prospective observational study of rFVIIIFc ITI pre- and post-ITI T cell responses in children with hemophilia and inhibitors, the Hemophilia Inhibitor Response to Eloctate (HIRE) Study, has begun recruitment. Disclosures Ragni: SPARK: Research Funding; Shire: Consultancy; Novo Nordisk: Research Funding; Genentech: Research Funding; CSL Behring: Research Funding; Biomarin: Consultancy; Biogen: Consultancy, Research Funding; Baxalta: Research Funding; Alnylam Pharmaceuticals: Consultancy, Research Funding; Tacere Benitec: Consultancy; Vascular Medicine Institute: Research Funding; OPKO: Research Funding. Malec:Vascular Medicine Institute: Research Funding; Biogen: Research Funding; Baxalta: Research Funding; Biogen: Consultancy. Journeycake:CSL: Consultancy; Biogen: Consultancy; Baxalta/Shire: Consultancy.

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Novel Severe Hemophilia a Mouse Model with Factor VIII Intron 22 Inversion

Biology ◽

10.3390/biology10080704 ◽

2021 ◽

Vol 10 (8) ◽

pp. 704

Author(s):

Jeong Pil Han ◽

Dong Woo Song ◽

Jeong Hyeon Lee ◽

Geon Seong Lee ◽

Su Cheong Yeom

Keyword(s):

Mouse Model ◽

Hemophilia A ◽

Clinical Symptoms ◽

Structural Similarity ◽

Coagulation Disorder ◽

Severe Hemophilia ◽

Spontaneous Bleeding ◽

Intron 22 Inversion ◽

Fviii Activity

Hemophilia A (HA) is an X-linked recessive blood coagulation disorder, and approximately 50% of severe HA patients are caused by F8 intron 22 inversion (F8I22I). However, the F8I22I mouse model has not been developed despite being a necessary model to challenge pre-clinical study. A mouse model similar to human F8I22I was developed through consequent inversion by CRISPR/Cas9-based dual double-stranded breakage (DSB) formation, and clinical symptoms of severe hemophilia were confirmed. The F8I22I mouse showed inversion of a 391 kb segment and truncation of mRNA transcription at the F8 gene. Furthermore, the F8I22I mouse showed a deficiency of FVIII activity (10.9 vs. 0 ng/mL in WT and F8I22I, p < 0.0001) and severe coagulation disorder phenotype in the activated partial thromboplastin time (38 vs. 480 s, p < 0.0001), in vivo bleeding test (blood loss/body weight; 0.4 vs. 2.1%, p < 0.0001), and calibrated automated thrombogram assays (Thrombin generation peak, 183 vs. 21.5 nM, p = 0.0012). Moreover, histological changes related to spontaneous bleeding were observed in the liver, spleen, and lungs. We present a novel HA mouse model mimicking human F8I22I. With a structural similarity with human F8I22I, the F8I22I mouse model will be applicable to the evaluation of general hemophilia drugs and the development of gene-editing-based therapy research.

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Efficacy of rFVIIIFc for First-Time Immune Tolerance Induction (ITI) Therapy: Final Results from the Global, Prospective VerITI-8 Study

Blood ◽

10.1182/blood-2021-155156 ◽

2021 ◽

Vol 138 (Supplement 2) ◽

pp. LBA-5-LBA-5

Author(s):

Lynn Malec ◽

An Van Damme ◽

Anthony Chan ◽

Mariya Spasova ◽

Nisha Jain ◽

...

Keyword(s):

Half Life ◽

Stock Options ◽

Research Funding ◽

Hemophilia A ◽

High Titer ◽

High Dose ◽

Severe Hemophilia ◽

First Time ◽

Inhibitor Titer ◽

Privately Held

Abstract Introduction: Inhibitor development is a major complication of factor VIII (FVIII) replacement therapy, affecting approximately 30% of people with severe hemophilia A (Peyvandi et al Lancet 2016). Inhibitor eradication is the standard of care to restore responsiveness to FVIII; however, ITI regimens often require frequent high-dose factor injections over a long period (DiMichele et al Haemophilia 2007; Carcao et al Haemophilia 2021). Median (interquartile range [IQR]) time (months) to negative titer in the International ITI Study with high-dose FVIII was 4.6 (2.8-13.8) (n=31); negative titer to normal recovery was 6.9 (3.5-12.0) (n=23); and normal recovery to tolerance was 10.6 (6.3-20.5) (n=22) (Hay and DiMichele Blood 2012). Recombinant factor VIII Fc fusion protein (rFVIIIFc) is an extended half-life (EHL) FVIII that showed potential benefits for ITI in retrospective clinical data and case reports (Malec et al Haemophilia 2016; Groomes et al Pediatr Blood Cancer 2016; Carcao et al Haemophilia 2021). VerITI-8 (NCT03093480) is the first prospective study of rFVIIIFc in first-time ITI and follows on from the reITIrate (NCT03103542) study of rFVIIIFc for rescue ITI (Königs et al Res Pract Thromb Haemost, ISTH 2021). Aim: Describe outcomes in the verITI-8 study of first-time ITI with rFVIIIFc over 48 weeks in subjects with severe hemophilia A and high-titer inhibitors. Methods: VerITI-8 is a prospective, single-arm, open-label, multicenter study exploring efficacy of rFVIIIFc for first-time ITI in people with severe hemophilia A with high-titer inhibitors. Initial screening was followed by an ITI period in which all subjects received rFVIIIFc 200 IU/kg/day until tolerization or 48 weeks had elapsed (Figure). This was followed by tapered dose reduction to standard prophylaxis and follow-up. Key inclusion criteria included males with severe hemophilia A, high-titer inhibitors (historical peak ≥5 Bethesda units [BU]/mL), and prior treatment with any plasma-derived or recombinant standard half-life or EHL FVIII. Key exclusion criteria included coagulation disorder(s) other than hemophilia A and previous ITI. The primary endpoint was time to tolerization (successful ITI) with rFVIIIFc defined by inhibitor titer <0.6 BU/mL, incremental recovery (IR) ≥66% of expected IR (IR ≥1.32 IU/dL per IU/kg) (both at 2 consecutive visits), and t ½ ≥7 hours (h) within 48 weeks. Secondary endpoints included number of subjects achieving ITI success, annualized bleed rates (ABR), and adverse events (AEs). Results: Sixteen subjects were enrolled and received ≥1 rFVIIIFc dose. Median (range) age at baseline was 2.1 (0.8-16.0) years, and historical peak inhibitor titer was 22.4 (6.2-256.0) BU/mL (Table). Twelve (75%), 11 (69%), and 10 (63%) subjects, respectively, achieved a negative inhibitor titer, an IR >66%, and a t½ ≥7 h (ie, tolerance) within 48 weeks. Median (IQR) times in weeks to achieve these markers of success were 7.4 (2.2-17.8), 6.8 (5.4-22.4), and 11.7 (9.8-26.2) (ie, 2.7 [2.3-6.0] months to tolerance), respectively. One subject achieved partial success (negative inhibitor titer and IR ≥66%), and 5 subjects failed ITI, of which 2 had high inhibitors throughout, 2 experienced an increase in inhibitor levels, and 1 recorded a negative inhibitor titer at 282 days. Most bleeds occurred in the ITI period when median (IQR) ABRs (n=13) were 3.8 (0-10.1) overall, 0 (0-2.6) for spontaneous, 1 (0-4) for traumatic, and 0 (0-3.1) for joint. During tapering, median (IQR) ABRs (n=10) were overall, 0 (0-2.4); spontaneous, 0 (0-0); traumatic, 0 (0-1.3); and joint, 0 (0-0). All 16 subjects experienced ≥1 treatment-emergent AE (TEAE), the most frequent of which was pyrexia in 7 subjects (44%). One subject reported ≥1 related TEAE (injection site pain). Nine subjects (56%) experienced ≥1 treatment-emergent serious AE (TESAE). TESAEs occurring in ≥2 subjects included vascular device infection, contusion, and hemarthrosis. No treatment-related TESAEs, discontinuations due to AEs, or deaths were reported. Conclusions: rFVIIIFc is the first EHL FVIII with prospective data for first-time ITI in patients with severe hemophilia A with historical high-titer inhibitors. Evaluated within a 48-week timeframe, rFVIIIFc offered rapid time to tolerization (median 11.7 weeks; 2.7 months) with durable responses in almost two-thirds of subjects and was well tolerated. Optimizing ITI to eradicate inhibitors remains a priority. Figure 1 Figure 1. Disclosures Malec: CSL Behring: Consultancy; Genentech: Consultancy; HEMA Biologics: Consultancy; Pfizer: Consultancy; Sanofi: Consultancy, Research Funding; Takeda: Consultancy; Bioverativ: Consultancy, Research Funding, Speakers Bureau; Shire: Consultancy; Bayer: Consultancy. Van Damme: Pfizer: Consultancy; Shire: Consultancy; Bayer: Consultancy. Chan: Bioverativ: Consultancy. Jain: Sanofi: Ended employment in the past 24 months; Takeda: Current Employment, Current holder of stock options in a privately-held company. Sensinger: Sanofi: Current Employment, Current holder of stock options in a privately-held company. Dumont: Sanofi: Current Employment, Current holder of stock options in a privately-held company. Lethagen: Sobi: Current Employment, Current holder of stock options in a privately-held company. Carcao: Bayer, Bioverativ/Sanofi, CSL Behring, Novo Nordisk, Octapharma, Pfizer, Roche, and Shire/Takeda: Research Funding; Bayer, Bioverativ/Sanofi, CSL Behring, Grifols, LFB, Novo Nordisk, Pfizer, Roche, and Shire/Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees. Peyvandi: Roche: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Sobi: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Ablynx, Grifols, Kedrion, Novo Nordisk, Roche, Shire, and Sobi: Other: Personal Fees. OffLabel Disclosure: adheres to routine clinical practice

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Immune Tolerance in Two High Responder, Severe Hemophilia A Patients with Inhibitors: The Importance of High Doses and the Duration of the Treatment

Vox Sanguinis ◽

10.1159/000462148 ◽

1996 ◽

Vol 70 (1) ◽

pp. 72-73 ◽

Cited By ~ 2

Author(s):

N. Ciavarella ◽

M. Schiavoni ◽

A. Fasano ◽

M.G. Giliberti ◽

C. Ettorre ◽

...

Keyword(s):

Immune Tolerance ◽

Hemophilia A ◽

High Responder ◽

Severe Hemophilia ◽

High Doses

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Analysis of intron 22 inversions of the factor VIII gene in severe hemophilia A: implications for genetic counseling

Blood ◽

10.1182/blood.v84.7.2197.2197 ◽

1994 ◽

Vol 84 (7) ◽

pp. 2197-2201 ◽

Cited By ~ 33

Author(s):

PV Jenkins ◽

PW Collins ◽

E Goldman ◽

A McCraw ◽

A Riddell ◽

...

Keyword(s):

Factor Viii ◽

Genetic Counselling ◽

Hemophilia A ◽

Family Members ◽

Rflp Analysis ◽

Homologous Region ◽

Severe Hemophilia ◽

Factor Viii Gene ◽

Intron 22 Inversion ◽

Blotting Technique

Abstract Intrachromosomal recombinations involving F8A, in intron 22 of the factor VIII gene, and one of two homologous regions 500 kb 5′ of the factor VIII gene result in large inversions of DNA at the tip of the X chromosome. The gene is disrupted, causing severe hemophilia A. Two inversions are possible, distal and proximal, depending on which homologous region is involved in the recombination event. A simple Southern blotting technique was used to identify patients and carriers of these inversions. In a group of 85 severe hemophilia A patients, 47% had an inversion, of which 80% were of the distal type. There was no association with restriction fragment length polymorphism (RFLP) haplotypes. The technique has identified a definitive genetic marker in families previously uninformative on RFLP analysis and provided valuable information for genetic counselling information may now be provided for carriers without the need to study intervening family members and the diagnosis of severe hemophilia A made in families with only a nonspecific history of bleeding. Analysis of intron 22 inversion should now be the first-line test for carrier diagnosis and genetic counselling for severe hemophilia A and may be particularly useful when there is no affected male family member or when intervening family members are unavailable for testing.

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