Immunogenicity Of Factor VIII Research Articles

Factor VIII trafficking to CD4+ T cells shapes its immunogenicity and requires several types of antigen-presenting cells

AbstractDespite >80 years of clinical experience with coagulation factor VIII (FVIII) inhibitors, surprisingly little is known about the in vivo mechanism of this most serious complication of replacement therapy for hemophilia A. These neutralizing antidrug alloantibodies arise in ∼30% of patients. Inhibitor formation is T-cell dependent, but events leading up to helper T-cell activation have been elusive because of, in part, the complex anatomy and cellular makeup of the spleen. Here, we show that FVIII antigen presentation to CD4+ T cells critically depends on a select set of several anatomically distinct antigen-presenting cells, whereby marginal zone B cells and marginal zone and marginal metallophilic macrophages but not red pulp macrophages (RPMFs) participate in shuttling FVIII to the white pulp in which conventional dendritic cells (DCs) prime helper T cells, which then differentiate into follicular helper T (Tfh) cells. Toll-like receptor 9 stimulation accelerated Tfh cell responses and germinal center and inhibitor formation, whereas systemic administration of FVIII alone in hemophilia A mice increased frequencies of monocyte-derived and plasmacytoid DCs. Moreover, FVIII enhanced T-cell proliferation to another protein antigen (ovalbumin), and inflammatory signaling–deficient mice were less likely to develop inhibitors, indicating that FVIII may have intrinsic immunostimulatory properties. Ovalbumin, which, unlike FVIII, is absorbed into the RPMF compartment, fails to elicit T-cell proliferative and antibody responses when administered at the same dose as FVIII. Altogether, we propose that an antigen trafficking pattern that results in efficient in vivo delivery to DCs and inflammatory signaling, shape the immunogenicity of FVIII.

Coagulation Factor VIII Enhances the Cleavage of Von Willebrand Factor By ADAMTS13 In Vivo

Introduction: Coagulation factor VIII (FVIII) and von Willebrand factor (VWF) circulate as a noncovalent complex. This interaction helps increase the half-life of circulating FVIII and modulates FVIII immunogenicity. Our previous studies demonstrate that FVIII accelerates VWF proteolysis by ADAMTS13 in vitro under fluidic shear or under certain pathophysiological conditions. However, the physiological relevance of the FVIII-enhanced VWF proteolysis remains questionable. Here, we sought to determine if addition of recombinant FVIII at various concentrations to mice with or without circulating ADAMTS13 or to patients with hereditary thrombotic thrombocytopenic purpura (TTP) affects plasma VWF multimer distribution and its antigen levels. Methods: Recombinant human B-domain deleted FVIII (rFVIII) was intravenously administered into wild-type (WT),Adamts13+/-(A13+/-) and Adamts13-/- (A13-/-) mice. Blood samples were collected prior to and 24 hours following rFVIII infusion. In addition, FVIII was stably expressed in fviii-/-mice via administration of AAV8-BDDFVIII. Moreover, plasma was collected from patients with hereditary TTP who were treated with plasma derived FVIII concentrates. Plasma VWF multimers and antigen levels were determined by SDS-agarose gel electrophoresis followed by Western blotting and enzyme-linked immunoassay (ELISA), respectively. Paired T and Mann Whitney tests were performed to determine the statistical significance of the difference between two groups. Results: Our results demonstrated that an infusion of rFVIII at a low dose (40 ug/kg) and a high dose (160 ug/kg) into WT mice resulted in a dramatic reduction in plasma VWF multimer sizes (Fig. 1) and VWF antigen levels (not shown). FVIII at high dose but not at low dose could also significantly reduce the sizes of VWF multimers in Adamts13+/- and Adamts13-/- mice (Fig. 1). The enhanced degradation of VWF multimers in Adamts13-/- mice suggests that other VWF-cleaving enzymes may participate in the degradation of VWF-FVIII complex. In either Adamts13+/- or Adamts13-/- mice, administration of rFVIII at both low and high doses didn't result in significant changes in VWF antigen levels (not shown). Furthermore, a simultaneous infusion of both rFVIII and recombinant murine ADAMTS13 (rADAMTS13) into Adamts13-/- mice resulted in similar changes in plasma VWF multimer distribution (Fig. 1) and antigen levels (not shown). Importantly, all mice tolerated well to the high dose of rFVIII infusion without major thrombotic events during weeks of follow-up. The restoration of plasma FVIII to 100-200% of normal levels in the fviii-/- mice with AAV8-mediated gene therapy also led to a profound degradation of plasma VWF multimers and significantly reduced plasma levels of VWF antigen (not shown). Finally, a similar pattern of dramatically reduced plasma VWF multimers was observed in 3 out of 4 patients with hereditary TTP following treatment with plasma-derived FVIII concentrates (not shown). Conclusions: Our results demonstrate that supplementation of FVIII at physiological or super-physiological concentrations dramatically accelerates the degradation of VWF multimers in mice and human. These findings further support our hypothesis that FVIII is a physiological cofactor of ADAMTS13 in regulating VWF homeostasis. Our findings may also offer a scientific explanation how FVIII concentrates or perhaps rFVIII might work for the treatment of hereditary TTP and other arterial thrombosis. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Extra-Splenic Role of B Cell Activating Factor Blockade in Prevention of Factor VIII Inhibitors

The development of neutralizing antibodies (inhibitors) to factor VIII (FVIII) remains the most challenging complication of protein-based replacement therapy in hemophilia A (HA). Elevated levels of the B cell survival cytokine B cell activating factor (BAFF) are associated with inhibitors in HA patients and BAFF levels decrease with successful immune tolerance induction (Doshi BS et al, J Clin Invest 2021). In HA mice, localization of labeled FVIII to the spleen and subsequent depletion of splenic leukocytes has been shown to reduce and/or temporarily prevent FVIII inhibitors. In contrast, transient depletion of BAFF prevented inhibitors upfront and promoted long-term tolerance to FVIII in HA mice. Given BAFF's ability to modulate B cell pools in secondary lymphoid organs, here we compared the splenic versus extra-splenic role of BAFF in regulating the FVIII immune response in order to localize its mechanism of action.As BAFF could regulate survival of innate-like splenic-resident marginal zone B cells, initial experiments compared depletion of BAFF or marginal zone B cells in HA mice and showed more complete inhibitor prevention with BAFF depletion (median 0 vs 16.9 Bethesda Units [BU]/ml, respectively, p < 0.01). These data supported our hypothesis that BAFF's regulatory effect is not solely reliant on its modulation of the splenic-resident marginal zone B cell compartment in HA mice. To further understand the extra-splenic role of BAFF in FVIII immunogenicity, wild-type (WT) or severe HA mice (with F8 exon 16 knockout) on the C57Bl/6 background, were treated according to three groups (n=9-11/group): 1) sham controls, 2) splenectomy alone, and 3) anti-BAFF monoclonal antibody (2mg/kg IP once) 4 days prior to splenectomy (“combined therapy”). Following splenectomy, mice were injected with recombinant full length human FVIII at 80 IU/kg weekly for six weeks. Bethesda titers were measured one week following the final immunization. In order to provide hemostasis in HA mice without FVIII exposure prior to splenectomy, mice were given liver-directed gene therapy with AAV8-mFVIIa at 5e11 vg/mouse two weeks prior to splenectomy. Circulating mFVIIa levels of 5.8 ± 2.4 ug/ml at day 10 were observed. These levels normalized hemostasis allowing successful splenectomy in 85% of animals, which is similar to our experience in WT mice.In the combined therapy group, only a single HA mouse developed a high titer inhibitor (7.4 BU/ml) and no WT mice developed high titer inhibitors. As the presence of endogenous FVIII antigen in WT mice does not seem to significantly alter BAFF's role in preventing the alloantibody response to xenoantigen (human FVIII protein), data for WT and HA mice were combined for analysis. Although splenectomy decreased inhibitors titers compared to sham controls (23.1 vs 78.7 BU/ml), the addition of anti-BAFF antibody therapy to splenectomy significantly reduced the risk of high titer inhibitors (RR 0.15, 95% CI 0.02-0.66, p < 0.01). The median Bethesda titer in HA and WT splenectomized mice was 23.1 (0-139.4) versus 0 (0-7.4) with addition of BAFF antibody therapy. Of note, inhibitor prevention with combination splenectomy and anti-BAFF antibody therapy seen here is similar to our prior data using just anti-BAFF antibody monotherapy for FVIII inhibitor prevention in HA mice (RR 0.23, 95% CI 0.08-0.57) suggesting that splenectomy does not significantly improve FVIII inhibitor prevention in the setting of BAFF inhibition.Our data, using a challenging HA mouse model, suggest that anti-BAFF therapy may prevent FVIII inhibitors by inhibiting BAFF's function in non-splenic lymphoid compartments. As certain B cell subtypes, including marginal zone B cells, reside in non-splenic compartments in humans, our model mimics the anticipated human immune response to FVIII. Ongoing investigations into the B and T cell compartments in lymph nodes and bone marrow will provide further insights into the location of BAFF's regulatory role in the FVIII immune response and may provide insights into potential alternative pathways in the initial FVIII immune response. DisclosuresDoshi: Janssen: Consultancy; Spark Therapeutics: Speakers Bureau.

Ontogeny of the Alloimmune Anti-Canine Factor VIII Inhibitor Response in Severe Hemophilia Α Dogs

The development of inhibitors to Factor VIII (FVIII) is a major complication of hemophilia A (HA) treatment. Most preclinical studies in HA animals have been limited to a xenoprotein response, e.g., evaluating the murine immune response to human FVIII (hFVIII). Severe HA dogs are a naturally occurring outbred model that recapitulates the spontaneous bleeding phenotype of the disease; their severe HA is due to intron 22 inversion (INV22) of the canine F8 gene, analogous to the INV22 found in 45% of severe HA patients. Importantly, similar to HA INV22 patients, about 20% of these animals develop an anti-canine FVIII (cFVIII) inhibitor response. This ability of the HA dogs to develop inhibitors in a species-specific manner provides an opportunity to probe the molecular ontogeny of an alloimmune anti-FVIII response.Here, we use antibody phage display to capture the humoral anti-cFVIII IgG repertoire in a dog with severe HA. This dog developed and maintained high titers of cFVIII inhibitor (89 Bethesda Units, BU) after exposure to cFVIII protein. The dog then received AAV liver-directed gene therapy encoding a cFVIII transgene, and after an initial anamnestic response characterized by rapid increase of cFVIII inhibitor titer (peak titer of 182 BU), the dog exhibited successful inhibitor eradication and immune tolerance induction, similar to our previous report (Finn et al., Blood 2010).Canine peripheral blood mononuclear cells were used to construct a single-chain variable fragment (scFv) phage display library which was sequentially selected four times against immobilized recombinant B-domain deleted (BDD) cFVIII protein. The amount of enrichment per round of selection plateaued during the third round from which 55 scFv/phage clones were isolated for characterization. Individual phage clones were sequenced by Sanger sequencing and screened for binding to both canine and human BDD-FVIII by ELISA. ScFv/phage clones were tested for inhibitory activity in a modified Bethesda assay.We identified 26 distinct scFv clones binding cFVIII based on heavy chain/light chain composition comprising 16 distinct CDRH3 sequences and 23 distinct CDRL3 sequences. The heavy chains of the 26 clones were derived from six canine germline IGHV genes, namely IGHV3-2, IGHV3-5, IGHV3-9, IGHV3-19, IGHV3-38, and IGHV3-41. The 16 distinct CDRH3 sequences had a mean length of 10.1 ± 3.6 amino acids, shorter than the 13.5 ± 3.6 amino acids previously reported for total canine CDRH3 repertoires (Steiniger et al., Mol. Immunol. 2014). Only 10 of the 26 FVIII-specific clones contained a lambda light chain, despite canine IgG repertoires dominated by the use of lambda light chains (Steiniger et al.). Eight of the 26 clones exhibited inhibitory activity. Although this dog was never exposed to hFVIII, 12/26 (46%) clones bound both cFVIII and hFVIII, consistent with the high degree of homology between the two orthologues with 85% identity. For three clones, framework region mutations and different light chain pairings resulted in altered inhibitory activity and hFVIII binding. Interestingly, one clone in particular comprised the majority of randomly screened scFv from the third and fourth rounds of phage library selection.These results suggest that cFVIII-specific B cells arise from multiple germline VH genes and exhibit high CDRH3 diversity. While the IgG-FVIII interaction has previously been suggested to be mostly influenced by the IgG heavy chain, our data suggest that for some antibodies, the IgG light chain may influence both inhibitory activity and epitope reactivity. Future studies will assess B cell clonal evolution via deep sequencing and longitudinal epitope specificity during multi-year immune tolerance induction by continuous exposure to cBDD-FVIII gene therapy. This is the first in-depth assessment of FVIII immunogenicity in a large HA model that avoids the use of a non-species specific antigen. Direct comparison with ongoing studies in HA inhibitor patients will inform the immunodominant epitopes of FVIII across species and thus provide insights on FVIII immunogenicity. DisclosuresDoshi: Janssen: Consultancy; Spark Therapeutics: Speakers Bureau. Samelson-Jones: Spark: Research Funding; Pfizer: Consultancy, Research Funding. Siegel: Verismo Therapeutics, Inc: Other: Co-Founder and Equity Holder; Vetigenics, LLC: Other: Co-Founder and Equity Holder.

Characterization of the HLA-Class-II (HLAcII)/Therapeutic Factor VIII (FVIII)-Derived Peptidome By FVIII Domain and HLAcII Isomer

Abstract Background: Hemophilia A (HA) is caused by F8 mutations and resultant plasma deficiencies in FVIII activity. While bleeding can be arrested by infusing therapeutic FVIII proteins (tFVIIIs), ~30% of severe HA patients will develop neutralizing tFVIII antibodies (inhibitors) and become resistant to treatment. For inhibitors to develop, foreign peptides must be liberated from a tFVIII and displayed on dendritic cells (DCs) in self HLAcII molecules. Little is known about the inhibitor risk attributable to distinct isomers of individual HLAcII repertoires and different tFVIII domains. Aims: Determine the contributions to FVIII immunogenicity risk of the distinct 1) HLAcII isomers DP, DQ and DR, and 2) tFVIII domains A1, A2, A3, B, C1 and C2. Methods: We analyzed HLAcII/tFVIII peptidomic data generated in DC-protein processing assays (PPAs) that used monocyte-derived DCs from 12 healthy donors to compare the immunogenicity of a full length (FL) and B-domain truncated (BDT) recombinant FVIII (FL- and BDT-FVIII) ± von Willebrand Factor (VWF) and a plasma derived (pd)FVIII + VWF. All tFVIIIs contain 3 small acidic-residue-rich peptides (a1, a2 and a3) that connect A1 to A2, A2 to B, and B to A3. To simplify our analysis, any LC-MS/MS individually sequenced peptide (ISP) with an N-terminal residue in either the (i) C-terminal portion of A1, A2 or B that extends into the adjacent a1, a2 or a3 region, respectively, was placed into that domain, or (ii) a1, a2 or a3 region, whether or not it extends into A3, C1 or C2, was placed into that domain. We then calculated the expected frequency of peptides from each FVIII domain (including, when appropriate, the adjacent acidic regions) based on residue number. Finally, we compared the observed and expected peptide proportions and performed a Z-test on the difference of proportions per domain. We performed this analysis for all ISP data in which the observed frequencies were obtained by a weighting scheme jointly taking into account contributions by tFVIII & by domain (All-data). We also performed this analysis by tFVIII. Results: For the All-data comparisons, we found that A3 had a higher observed than expected proportion (dP = 22%; p = 0.006). For the FL-FVIII comparisons, the B domain had a lower observed than expected proportion (dP = -12.1%; p We also approached these data from the HLAcII perspective. We found that our 12 donors expressed 12 distinct DR molecules based on their DRB1 alleles. After tallying the per allele density of FVIII residues (for all 2332 residues) falling in the DR-bound fraction of peptides, we constructed FVIII residue-by-allele matrices of dimensions 2332 x 12 per tFVIII and performed singular value decompositions that yielded their respective matrices of right singular vectors also known as principal components (PCs). Such PCs are said to span or generate a corresponding vector subspace. Using matrix analytic methods, we can compare the therapeutic-specific subspaces to see if such contrasts reflect differences in their associated density profiles. To compare two subspaces, we compute their projection onto their intersection subspace, and use this projection to compute a subspace distance. We test the null hypothesis of a subspace distance equal to 0 (for statistically similar subspaces) by a permutation test. As an example, we compared the FL-FVIII + VWF and pdFVIII + VWF subspaces, and found that they are significantly different (subspace distance (SD) = 0.96; p Conclusions: Our findings demonstrate that the distinct domains of tFVIIIs and different alleles of individual HLAcII repertoires differentially contribute to inhibitor risk. Download : Download high-res image (175KB) Download : Download full-size image Disclosures Hofmann: CSL Behring: Employment. Escobar: Bayer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Shire: Honoraria, Membership on an entity's Board of Directors or advisory committees; CSL Behring: Honoraria, Membership on an entity's Board of Directors or advisory committees; NovoNordisk: Honoraria, Membership on an entity's Board of Directors or advisory committees; Genentech: Honoraria, Membership on an entity's Board of Directors or advisory committees. Dinh: Haplomics: Employment. Powell: CSL Behring: Employment. Howard: CSL Behring: Honoraria, Research Funding; Haplomics: Employment, Equity Ownership, Patents & Royalties, Research Funding.

Characterisation and application of recombinant FVIII-neutralising antibodies from haemophilia A inhibitor patients.

The development of anti-drug antibodies (ADAs) is a serious outcome of treatment strategies involving biological medicines. Coagulation factor VIII (FVIII) is used to treat haemophilia A patients, but its immunogenicity precludes a third of severe haemophiliac patients from receiving this treatment. The availability of patient-derived anti-drug antibodies can help us better understand drug immunogenicity and identify ways to overcome it. Thus, there were two aims to this work: (i) to develop and characterise a panel of recombinant, patient-derived, monoclonal antibodies covering a range of FVIII epitopes with varying potencies, kinetics and mechanism of action, and (ii) to demonstrate their applicability to assay development, evaluation of FVIII molecules and basic research. For the first objective we used recombinant antibodies to develop a rapid, sensitive, flexible and reproducible ex vivo assay that recapitulates inhibitor patient blood using blood from healthy volunteers. We also demonstrate how the panel can provide important information about the efficacy of FVIII products and reagents without the need for patient or animal material. These materials can be used as experimental exemplars or controls, as well as tools for rational, hypothesis-driven research and assay development in relation to FVIII immunogenicity and FVIII-related products.

Effects of Shear Stress on Production of FVIII and vWF in a Cell-Based Therapeutic for Hemophilia A.

Microfluidic technology enables recapitulation of organ-level physiology to answer pertinent questions regarding biological systems that otherwise would remain unanswered. We have previously reported on the development of a novel product consisting of human placental cells (PLC) engineered to overexpress a therapeutic factor VIII (FVIII) transgene, mcoET3 (PLC-mcoET3), to treat Hemophilia A (HA). Here, microfluidic devices were manufactured to model the physiological shear stress in liver sinusoids, where infused PLC-mcoET3 are thought to lodge after administration, to help us predict the therapeutic outcome of this novel biological strategy. In addition to the therapeutic transgene, PLC-mcoET3 also constitutively produce endogenous FVIII and von Willebrand factor (vWF), which plays a critical role in FVIII function, immunogenicity, stability, and clearance. While vWF is known to respond to flow by changing conformation, whether and how shear stress affects the production and secretion of vWF and FVIII has not been explored. We demonstrated that exposure of PLC-mcoET3 to physiological levels of shear stress present within the liver sinusoids significantly reduced mRNA levels and secreted FVIII and vWF when compared to static conditions. In contrast, mRNA for the vector-encoded mcoET3 was unaltered by flow. To determine the mechanism responsible for the observed decrease in FVIII and vWF mRNA, PCR arrays were performed to evaluate expression of genes involved in shear mechanosensing pathways. We found that flow conditions led to a significant increase in KLF2, which induces miRNAs that negatively regulate expression of FVIII and vWF, providing a mechanistic explanation for the reduced expression of these proteins in PLC under conditions of flow. In conclusion, microfluidic technology allowed us to unmask novel pathways by which endogenous FVIII and vWF are affected by shear stress, while demonstrating that expression of the therapeutic mcoET3 gene will be maintained in the gene-modified PLCs upon transplantation, irrespective of whether they engraft within sites that expose them to conditions of shear stress.

Characterization of Protein Unable to Bind Von Willebrand Factor in Recombinant Factor VIII Products: Can We Reduce Their Immunogenicity?

Introduction Therapeutic products with blood coagulation factor VIII (FVIII) have a wide range of protein contents per activity unit (or IU/mg, specific activity), available from previous studies (Lin et al, 2004; Butenas et al, 2009) and the prescribing information. The wide range of the specific activities seems to be more pronounced in recombinant FVIII products (rFVIII) compared to plasma-derived FVIII, implying presence of protein with altered structure and biochemical properties. In particular, rFVIII products were reported to contain a protein fraction (FVIII*), unable to bind von Willebrand factor (VWF) without functional activity (Lin et al, 2004;Ofosu et al 2012). Furthermore, FVIII* may represent a risk factor for development of FVIII inhibitors in Hemophilia A patients, as the binding to VWF was shown to reduce FVIII immunogenicity in a tissue culture and mice model systems (Gangadharan et al, 2017; Muczynski et al, 2018). Due to these reasons, FVIII* can be defined as an impurity, which needs to be understood and controlled in rFVIII products. Study objective To develop a methodology to isolate FVIII* from rFVIII samples and to characterize this fraction in various rFVIII products. Experimental design Using immobilized VWF affinity chromatography (IVAC) for analysis of FVIII samples, protein fractions collected from (i) the column flow-through (FVIIIFT, corresponding to FVIII*) and (ii) the column-bound and eluted fraction (FVIIIEL) were characterized using polyacrylamide gel electrophoresis (PAGE) gels followed by silver-staining and immunoblotting, FVIII activity test, surface plasmon resonance, mass spectrometry, and for plasma clearance in mice. Results A robust IVAC methodology was developed. Using this method, we isolated the FVIIIFT and FVIIIEL from all ten third-generation rFVIII products marketed in the USA by study time. These products represent all current pharmaceutical variants of rFVIII including full-size FVIII, B-domain deleted (truncated) FVIII, Fc-fused FVIII, single-chained FVIII, and PEGylated protein, including those with the extended plasma half-life. FVIIIFT was found in all rFVIII products at levels up to 22% of total protein. Compared to FVIIIEL, FVIIIFT had similar pattern of polypeptide bands by PAGE, but lower functional activity, significantly reduced sulfation at Tyr1680 important for VWF binding, moderately decreased interaction with recombinant cluster II of a low-density lipoprotein receptor related protein 1 (a major clearance receptor of FVIII), and approximately 3-times faster clearance in mice (Figure 1). Conclusions Our results show that the FVIII* structure is generally similar to that of the major fraction of rFVIII, while it differs by microheterogeneity in post-translational modifications and possibly local misfolding. The data suggest that upon administration of a rFVIII product in patients, its FVIII* fraction is rapidly removed from the circulation, resulting in a decrease of the effective dosage. Our findings demonstrate a potential of IVAC to control FVIII* fraction in rFVIII products, including its removal from the products during their manufacture. These applications may lead to achieving better quality and efficacy of rFVIII products including reduction of their immunogenicity for improving the care of Hemophilia A. Disclosures No relevant conflicts of interest to declare.

Tolerating Factor VIII: Recent Progress.

Development of neutralizing antibodies against biotherapeutic agents administered to prevent or treat various clinical conditions is a longstanding and growing problem faced by patients, medical providers and pharmaceutical companies. The hemophilia A community has deep experience with attempting to manage such deleterious immune responses, as the lifesaving protein drug factor VIII (FVIII) has been in use for decades. Hemophilia A is a bleeding disorder caused by genetic mutations that result in absent or dysfunctional FVIII. Prophylactic treatment consists of regular intravenous FVIII infusions. Unfortunately, 1/4 to 1/3 of patients develop neutralizing anti-FVIII antibodies, referred to clinically as “inhibitors,” which result in a serious bleeding diathesis. Until recently, the only therapeutic option for these patients was “Immune Tolerance Induction,” consisting of intensive FVIII administration, which is extraordinarily expensive and fails in ~30% of cases. There has been tremendous recent progress in developing novel potential clinical alternatives for the treatment of hemophilia A, ranging from encouraging results of gene therapy trials, to use of other hemostatic agents (either promoting coagulation or slowing down anti-coagulant or fibrinolytic pathways) to “bypass” the need for FVIII or supplement FVIII replacement therapy. Although these approaches are promising, there is widespread agreement that preventing or reversing inhibitors remains a high priority. Risk profiles of novel therapies are still unknown or incomplete, and FVIII will likely continue to be considered the optimal hemostatic agent to support surgery and manage trauma, or to combine with other therapies. We describe here recent exciting studies, most still pre-clinical, that address FVIII immunogenicity and suggest novel interventions to prevent or reverse inhibitor development. Studies of FVIII uptake, processing and presentation on antigen-presenting cells, epitope mapping, and the roles of complement, heme, von Willebrand factor, glycans, and the microbiome in FVIII immunogenicity are elucidating mechanisms of primary and secondary immune responses and suggesting additional novel targets. Promising tolerogenic therapies include development of FVIII-Fc fusion proteins, nanoparticle-based therapies, oral tolerance, and engineering of regulatory or cytotoxic T cells to render them FVIII-specific. Importantly, these studies are highly applicable to other scenarios where establishing immune tolerance to a defined antigen is a clinical priority.

Quantitative HLA‐class‐II/factor VIII (FVIII) peptidomic variation in dendritic cells correlates with the immunogenic potential of therapeutic FVIII proteins in hemophilia A

BackgroundPlasma‐derived (pd) or recombinant (r) therapeutic factor VIII proteins (FVIIIs) are infused to arrest/prevent bleeding in patients with hemophilia A (PWHA). However, FVIIIs are neutralized if anti‐FVIII‐antibodies (inhibitors) develop. Accumulating evidence suggests that pdFVIIIs with von Willebrand factor (VWF) are less immunogenic than rFVIIIs and that distinct rFVIIIs are differentially immunogenic. Since inhibitor development is T‐helper‐cell‐dependent, human leukocyte antigen (HLA)‐class‐II (HLAcII) molecules constitute an important early determinant. ObjectivesUse dendritic cell (DC)‐protein processing/presentation assays with mass‐spectrometric and peptide‐proteomic analyses to quantify the DP‐bound, DQ‐bound, and DR‐bound FVIII‐derived peptides in individual HLAcII repertoires and compare the immunogenic potential of six distinct FVIIIs based on their measured peptide counts. Patients/MethodsMonocyte‐derived DCs from normal donors and/or PWHA were cultured with either: Mix‐rFVIII, a VWF‐free equimolar mixture of a full‐length (FL)‐rFVIII [Advate® (Takeda)] and four distinct B‐domain‐deleted (BDD)‐rFVIIIs [Xyntha® (Pfizer), NovoEight® (Novo‐Nordisk), Nuwiq® (Octapharma), and Afstyla® (CSL Behring GmBH)]; a pdFVIII + pdVWF [Beriate® (CSL Behring GmBH)]; Advate ± pdVWF; Afstyla ± pdVWF; and Xyntha + pdVWF. ResultsWe showed that (i) Beriate had a significantly lower immunogenic potential than Advate ± pdVWF, Afstyla − pdVWF, and Mix‐rFVIII; (ii) distinct FVIIIs differed significantly in their immunogenic potential in that, in addition to (i), Afstyla + pdVWF had a significantly lower immunogenic potential than Beriate, while the immunogenic potential of Beriate was not significantly different from that of Xyntha + pdVWF; and (iii) rFVIIIs with pdVWF had significantly lower immunogenic potentials than the same rFVIIIs without pdVWF. ConclusionsOur results provide HLAcII peptidomic level explanations for several important clinical observations/issues including the differential immunogenicity of distinct FVIIIs and the role of HLAcII genetics in inhibitor development.

Elispots and Ex Vivo Expansion of FVIII-Specific T-Cell Clones Confirm Specificity of CD4+ T-Cell Responses to Self-FVIII in Healthy Non-Hemophilic Blood Donors

The existence of natural antibodies recognizing endogenous factor VIII (FVIII) and of FVIII-specific CD4+ T-cell responses in some healthy, non-hemophilic blood donors has been appreciated for &gt;20 years. The Conti-Fine group measured CD4+ T-cell proliferation following in vitro stimulation with FVIII protein or synthetic FVIII peptides. More recently, FVIII-specific CD4+ T-cell lines were expanded from PBMCs isolated from large blood volumes donated by healthy individuals, and estimates of specific precursor frequency (~2/million CD4+ T cells) were calculated on the basis of interferon (IFN)-gamma ELISPOT assays of FVIII-stimulated cells (Meuniere et al., Blood Advances 1(21): 1842-7). Escape of these self-reactive precursor cells from thymic editing via deletion or anergy and their subsequent persistence in the periphery may contribute to the rare but potentially severe autoimmune reactions to FVIII ("acquired hemophilia A") and to the unusual immunogenicity of therapeutic FVIII administered i.v. to hemophilia A patients. The present study sought to further characterize CD4+ T-cell responses to endogenous FVIII and to map epitopes recognized by these self-reactive cells. We were particularly interested to learn if these cells recognize multiple epitopes in FVIII or if they respond to only several immunodominant epitopes. Accordingly, IFN-gamma ELISPOT assays were carried out by stimulating CD4+ T cells with 15-mer FVIII peptides having 12-residue overlaps and spanning the FVIII A1, A2, A3, C1 and C2 domains. For efficient mapping, initial assays utilized large pools of peptides, and positive responses were then "decoded" by ELISPOTs using smaller peptide pools or individual peptides. Blood samples were obtained from healthy controls under approved IRB protocols. The ELISPOT assays utilized CD4+ T cells isolated by negative selection, with irradiated autologous PBMCs as antigen presenting cells. Anti-CD49d/CD28 monoclonal antibodies were added for co-stimulation to increase the sensitivity of the assay and cells were cultured with IL-7 to improve cell viability. As a result, this assay required smaller blood volumes, but it should be noted that lower-avidity T-cell responses were likely detected that might be missed in ELISPOT assays without these modifications. Relevance of such low-avidity self-reactive cells is provided by the clinical observation, consistent with basic immunological principles, that risk factors for autoimmune responses to FVIII include old age (pro-inflammatory), trauma, surgery and postpartum status, all of which up-regulate T-cell co-stimulatory factors. The first subject had HLA-DRB1*01:01 and HLA-DRB1*08:04 alleles. Stimulation with large peptide pools and rFVIII protein indicated recognition of epitopes in at least 3 FVIII domains. Additional ELISPOTs tested the immunogenicity of 15 peptides corresponding to FVIII peptides previously demonstrated to be presented on dendritic cells from 2 individuals with an HLA-DRB1*01:01 allele (van Haren et al., Mol Cell Proteomics. 2011;10(6)), ensuring that our assays included tests of naturally processed FVIII peptides. Two of these peptides, both from the FVIII A1 domain, produced ELISPOT readings above background levels. T cells were then stimulated with these peptides for 19 days, stained with peptide-loaded MHC Class II (HLA-DRB1*01:01) tetramers, sorted and expanded for another 14 days. Tetramer staining then confirmed isolation of CD4+ T-cell clones recognizing one of these peptides. T cells that recognize their cognate antigen with high avidity are significant drivers of allo- and autoimmune responses. Lower-avidity T cells, however, can play significant roles in pro-inflammatory settings. Tetramer staining validated our ELISPOT-based identification of specific epitopes in FVIII. We are now carrying out ELISPOT assays using pooled peptides followed by individual FVIII peptides as stimulants, to estimate the repertoire of FVIII-specific CD4+ T cells in healthy non-hemophilic individuals. Mapping of HLA-restricted T-cell epitopes will also enable future tetramer-based isolation and phenotypic characterization of these rare T cells without expanding them in culture. This will allow us to investigate the interesting question of what peripheral tolerance mechanisms prevent expansion of these self-reactive cells in vivo, except in rare cases of FVIII autoimmunity. . Disclosures Pratt: Bloodworks NW: Patents &amp; Royalties: inventor on patents related to FVIII immunogenicity; Grifols, Inc: Research Funding.

Neutralizing and Non-Neutralizing Anti-FVIII Antibodies in Black and White Hemophilia A Subjects: A Natural History Profile

The most common complication in hemophilia A (HA) treatment, affecting 25-30% of severe HA patients, is the development of alloimmune inhibitors that foreclose the ability of infused factor VIII (FVIII) to participate in coagulation. Inhibitors confer significant pathology on affected individuals and present major complexities in their management. Inhibitors are more common in African American patients, and it has been hypothesized that this is a consequence of haplotype (H)-treatment product mismatch. F8 gene haplotypes H1-H5 are defined by combinations of nonsynonymous SNPs encoding FVIII sequence variants D1241E, M2238V and R484H. F8 haplotypes H2-H5 are more prevalent in individuals with black African ancestry, while &gt;90% of the white population has the H1 haplotype. This study used a validated Luminex-based assay to determine total anti-FVIII antibody titers in plasma from 395 HA (189 black, 206 white) and 23 non-HA control subjects, measuring their binding to recombinant full-length H1 and H2 and B-domain-deleted (BDD) H1/H2, H3 and H4 FVIII proteins. Inhibitor titers were determined using a chromogenic Bethesda assay with the Nijmegen modification. Linear B-cell epitopes recognized by antibodies in human plasma samples were characterized using commercial peptide microarrays with imprinted 15-mer peptides spanning the FVIII A1, A2, C1 and C2 domains, with binding interactions detected using fluorescent-labeled anti-human IgG antibodies. Neither total nor inhibitory antibody titers correlated with F8 haplotype. FVIII peptides with the D1241E and M3348V polymorphisms showed low antibody reactivity, indicating they do not comprise linear B-cell epitopes. Similarly, antibodies from subjects with H3 and H5 haplotypes, who were necessarily infused with FVIII products having a different haplotype than that of their endogenous, (dysfunctional) F8 sequence, did not show haplotype-correlated differential binding to the three BDD-FVIII or full-length FVIII proteins, indicating the polymorphic M2238V or D1241E sites do not correspond to immunodominant, conformational B-cell epitopes. Interestingly, the BDD-FVIII proteins were significantly more reactive with antibodies in plasma than were two commercial full-length recombinant FVIII products. Overall, results of this study indicated that low-titer FVIII-reactive antibodies are readily detected in most HA subjects and in a majority of healthy non-HA controls. The observed stronger immunoreactivity of BDD-FVIII suggests that B-domain removal exposes novel B-cell epitopes, perhaps through conformational rearrangements of FVIII domains. Disclosures Pratt: Bloodworks NW: Patents &amp; Royalties: inventor on patents related to FVIII immunogenicity; Grifols, Inc: Research Funding. Peters:Sanofi: Employment. Mann:Haematologic Technologies: Other: Owner; Stago: Consultancy; Novo Nordisk: Consultancy; Takeda: Consultancy; Shire: Consultancy; Baxalta: Consultancy.

Comparative analysis of marketed factor VIII products: reply

Development of factor VIII (FVIII) inhibitors following replacement therapy with FVIII is one of the major challenges faced when treating patients with hemophilia A. Inhibitors develop in 20–32% of previously untreated patients with severe and in 3–13% with moderate or mild hemophilia A 1, 2. The cause of the immunogenicity is not well understood. There is evidence that both genetic and non-genetic factors influence patients’ susceptibility to develop these antibodies 3, 4. Novel hypothesis and research approaches are required to obtain more clarity on the molecular basis of FVIII immunogenicity and how FVIII triggers unwanted immune responses in some patients but not in others. The aim of our paper ‘Comparative analysis of marketed factor VIII products: recombinant products are not alike vis-a-vis soluble protein aggregates and subvisible particles’ 5 was to advance science and help to generate new hypotheses for future research on the molecular basis of FVIII immunogenicity. Our aim was to provide scientific transparency of our rationale and to stay on the level of a scientific debate rather than discussing brands or products. We believe that when it comes to treatment decisions clinical evidence on a robust basis is required. Disclosure of brand names as recently suggested by M Makris and A Farrugia in a letter to the editor of JTH 6 could influence treatment decisions by hypotheses rather than robust clinical evidence. Data from other protein products suggest that critical quality variables such as soluble protein aggregates (SPAs) and subvisible particles (SVPs) influence the immunogenicity of protein therapeutics 7. We analyzed SPAs and SVPs concentrations in commercially available recombinant FVIII (rFVIII) products to understand if there are differences between these products after reconstitution. Moreover, we wanted to know if and how levels of SPAs and SVPs change upon exposure of rFVIII products to relevant stress conditions such as agitation and sheer stress. Pre-existing SPAs and SVPs may act as seeds that nucleate further protein aggregation upon exposure to stress 8-10. In addition to the baseline levels of SPAs and SVPs in each rFVIII product, our data indicate that product mishandling after reconstitution can increase the concentration of SPAs and SVPs. Similar findings for FVIII products were recently published by Tsutomo et al. 15. Thus, it is important to educate end-users about proper product handling to avoid an amplification of potential adverse effects due to increases in SPAs and SVPs induced by mishandling. 15. In conclusion, we believe the research community needs to pay more attention to the presence of SPAs and SVPs in FVIII products and how these variables influence the immunogenicity of the products in patients. Our current understanding does not yet allow specific conclusions on how levels of SPAs and SVPs in FVIII products translate into product immunogenicity in patients but we believe that thorough assessment of these variables is important. B. M. Reipert wrote the manuscript. J. Anzengruber and F. Scheiflinger revised the manuscript and all authors approved the final version. We thank E. Langdon-Neuner for editing the English language of the manuscript. This work was funded by Shire. All authors are employees of Shire.

Cost analysis of plasma-derived factor VIII/von Willebrand factor versus recombinant factor VIII for treatment of previously untreated patients with severe hemophilia A in the United States

Background: Inhibitor development to factor VIII (FVIII) hemophilia therapy results in increased complications and substantial economic costs. The SIPPET study, the first randomized controlled trial to compare the immunogenicity of plasma-derived FVIII (pdFVIII)/von Willebrand factor (VWF) and recombinant-DNA-derived FVIII (rFVIII), demonstrated higher inhibitor rates in previously untreated patients (PUPs) treated with rFVIII than in PUPs treated with pdFVIII/VWF.Objective: To quantify the economic impact of treating PUPs with pdFVIII/VWF vs rFVIII.Methods: An Excel-based clinical and economic model was developed from a US healthcare payer perspective and run over a 5-year period. The analysis utilized a cohort approach to model patient treatment and outcomes over a monthly cycle to quantify differences in costs of FVIII, bypassing agents, and hospitalizations for serious bleeds. Rates of high-titer inhibitor development were obtained from the SIPPET study. Patients developing high-titer inhibitors were treated with immune tolerance induction (ITI). Patients who developed low-titer inhibitors and those who did not develop inhibitors continued their usual FVIII treatment. Patients who were successfully treated with ITI returned to FVIII treatment, while unsuccessfully treated patients received bypassing agents. Total costs per treated patient were estimated and a one-way sensitivity analysis was conducted to quantify the impact of parameter uncertainty on the model outcomes.Results: Total cumulative costs per patient over 5 years were $834,621 for pdFVIII/VWF patients and $1,237,163 for rFVIII patients, representing a total saving of $402,542 per patient over the 5-year period, for an average annual saving of $80,508 per patient.Conclusions: Based on data from the SIPPET study, this analysis found that initiating FVIII treatment in severe hemophilia A PUPs with pdFVIII/VWF has the potential to offer substantial cost savings to healthcare payers, amounting to a one-third reduction in costs.

Oxidation of factor VIII increases its immunogenicity in mice with severe hemophilia A

The development of antibodies against therapeutic factor VIII (FVIII) represents the major complication of replacement therapy in patients with severe hemophilia A. Amongst the environmental risk factors that influence the anti-FVIII immune response, the presence of active bleeding or hemarthrosis has been evoked. Endothelium damage is typically associated with the release of oxidative compounds. Here, we addressed whether oxidation contributes to FVIII immunogenicity. The control with N-acetyl cysteine of the oxidative status in FVIII-deficient mice, a model of severe hemophilia A, reduced the immune response to exogenous FVIII. Ex vivo exposure of therapeutic FVIII to HOCl induced a mild oxidation of the molecule as evidenced by the loss of free amines and resulted in increased FVIII immunogenicity in vivo when compared to native FVIII. The increased immunogenicity of oxidized FVIII was not reverted by treatment of mice with N-acetyl cysteine, and did not implicate an increased maturation of professional antigen-presenting cells. Our data document that oxidation influences the immunogenicity of therapeutic FVIII.

Differential Glycosylation Between Recombinant Factor VIII Produced in Baby Hamster Kidney and Chinese Hamster Ovary Cells Confers Differences in Immunogenicity in a Humanized Hemophilia Α Mouse Model

IntroductionThe formation of factor VIII (FVIII)- neutralizing antibodies is the most critical complication in the treatment of hemophilia A (HA). Recent clinical evidence suggests that recombinant FVIII (rFVIII) produced in baby hamster kidney (BHK-rFVIII) cells is more immunogenic than that produced in Chinese hamster ovary (CHO-rFVIII) cells. This difference in FVIII immunogenicity may be attributed to differences in protein glycosylation, which can impact the removal of FVIII from circulation through mechanisms leading to clearance and antigen presentation. Here, we document significant differences among the 25 potential N-linked glycans between these products, and we provide in vivo animal model-based evidence that supports these clinical observations.MethodsFactor VIII lectin binding was assessed by ELISA to detect exposed glycans. Commercially-available rFVIII products were adsorbed at 1 ug/mL and specific glycan moieties were detected using a panel of biotinylated lectins and HRP-conjugated streptavidin. Confirmation of differences and determination of N-linked glycan structures was conducted by LC-MS/MS.Eight to 12 week old transgenic C57BL/6 HA mice were used in these studies. This model contains a murine f8 exon 16 KO and additionally harbors a human F8 transgene with a R593C point mutation. While these mice have undetectable plasma levels of human FVIII antigen and activity, they are tolerant to intravenously infused human rFVIII. Clearance was assessed following a 6 IU (~240 IU/kg) infusion of each rFVIII product, and FVIII activity was measured by chromogenic assay and normalized to a 5-minute time point. The number of interferon (IFN)-γ secreting splenocytes from rFVIII-primed naïve mice was determined by ELISPOT.FVIII immune responses were elicited by subcutaneous infusion (6 IU twice-weekly for 2 weeks) and adjuvant-coupled intravenous infusion (1 ug lipopolysacharride with the first infusion as described above) with either rFVIII product. Week 5 plasma samples were assessed for FVIII-specific IgG by ELISA, and FVIII inhibitors by one-stage clotting assay.ResultsLectin binding showed that rFVIII produced in BHK cell lines exhibit lower proportions of high-mannose glycans (p<0.01), and greater levels of sialic acid capping (p<0.01) and fucosylated glycans (p<0.01). Mass spectra confirmed higher levels of sialic acid and identified two additional N-linked sites bearing high mannose glycans on CHO-rFVIII. In this mouse model we observed that BHK-rFVIII had a circulating half-life of 6.06 hours compared to the 10.01 hour half-life of CHO-rFVIII (p<0.0001).The immunogenicity of the BHK- and CHO-rFVIII products was next evaluated in vivo. In mice primed with a single 6 IU dose of BHK-rFVIII, we identified a higher proportion of FVIII-specific IFN-γ secreting splenocytes after seven days. Furthermore, long-term studies showed that 100% of mice subcutaneously exposed to BHK-rFVIII developed anti-FVIII IgG compared to the 47% that received CHO-rFVIII (p<0.01). Coincidently, when FVIII inhibitors were measured, we observed an incidence of 100% vs 37% (p<0.01), respectively. While the titres of FVIII-specific IgG were higher in mice exposed to BHK-rFVIII (p<0.01), there were no significant differences in the inhibitor concentrations. Similarly, we observed increased titres of FVIII-specific IgG in mice exposed intravenously (1 ug LPS with the first infusion) to BHK-rFVIII compared to CHO-rFVIII. However, there were no differences in the incidence of FVIII-specific IgG, nor in the incidence and concentration of inhibitors between the intravenously-infused mice.ConclusionsOur results demonstrate that BHK-rFVIII exhibits altered pharmacokinetic and immunogenic properties compared to CHO-rFVIII in humanized hemophilia A mice. The observed early increase in the proportion FVIII-specific IFN-γ producing cells in the spleen suggests an intrinsic immunogenic element of BHK-rFVIII. Similarly, the substantially increased immunogenicity of BHK-rFVIII in mice when treated subcutaneously complements the previously-reported clinical evidence. These differences may be attributed to the significant disparities in N-linked glycosylation, most notably high mannose and sialic acid containing glycans. Additional studies are underway to directly address the role of the these specific glycans and their potential impact on immunogenicity of rFVIII. DisclosuresLillicrap:Octapharma: Research Funding; Baxalta: Research Funding; Biogen-Idec: Research Funding; Bayer: Research Funding.

Blood Group O Protects against Inhibitor Development in Severe Hemophilia A Patients.

Increasing evidence supports the link between ABO(H) blood group determinants and hemostasis. In particular, the ABO-related different glycosylation patterns of von Willebrand factor strongly influence its clearance and functional levels, and this may contribute to the inter-individual variations in the half-life of infused Factor VIII (FVIII) in hemophilia A (HA) patients. We investigated the role of ABO blood groups in regulating FVIII immunogenicity by evaluating their distribution in patients with severe (FVIII < 1 IU/dL) HA according to inhibitor development and other known relevant factors. In a cohort of Italian severe HA patients (n = 209), the ABO blood group distribution was similar to that in the healthy general population. However, the distribution of inhibitors, developed in 56 patients overall (26.8%), was significantly different in the four ABO phenotypes (O, 18.2%; A, 31.9%; B, 39.1%, AB, 25%; p = 0.033); this difference seemed more pronounced when only high-titer inhibitors (overall, 21.1%) were considered (O, 11.4%; A, 27.7%; B, 34.8%; p = 0.011). Relative risks in O versus non-O blood group were 0.55 (95% CI: 0.33-0.92) and 0.40 (95% CI: 0.21-0.77) for any and high-titer inhibitors, respectively. In a multivariate logistic regression, O blood group was shown to lower (approximately twofold) inhibitor risk, similarly with plasma-derived FVIII, whereas high-risk F8 mutations were associated with increased risk. However, the estimated effect of O blood type on inhibitor development was free from any significant correlation to other covariates, including presence of high-risk F8 mutations and type of replacement FVIII used. In this retrospective cohort of severe hemophiliacs, blood group O appears to protect against inhibitor development, with independent effects from other covariates. Larger prospective studies are needed to confirm this finding and to delve deeper into its pathophysiologic mechanisms.

Concurrent influenza vaccination reduces anti-FVIII antibody responses in murine hemophilia A

AbstractInflammatory signals such as pathogen- and danger-associated molecular patterns have been hypothesized as risk factors for the initiation of the anti–factor VIII (FVIII) immune response seen in 25% to 30% of patients with severe hemophilia A (HA). In these young patients, vaccines may be coincidentally administered in close proximity with initial exposure to FVIII, thereby providing a source of such stimuli. Here, we investigated the effects of 3 vaccines commonly used in pediatric patients on FVIII immunogenicity in a humanized HA murine model with variable tolerance to recombinant human FVIII (rhFVIII). Mice vaccinated intramuscularly against the influenza vaccine prior to multiple infusions of rhFVIII exhibited a decreased incidence of rhFVIII-specific neutralizing and nonneutralizing antibodies. Similar findings were observed with the addition of an adjuvant. Upon exposure to media from influenza- or FVIII-stimulated lymph node or splenic lymphocytes, naïve CD4+ lymphocytes preferentially migrated toward media from influenza-stimulated cells, indicating that antigen competition, by means of lymphocyte recruitment to the immunization site, is a potential mechanism for the observed decrease in FVIII immunogenicity. We also observed no differences in incidence or titer of rhFVIII-specific antibodies and inhibitors in mice exposed to the live-attenuated measles-mumps-rubella vaccine regardless of route of administration. Together, our results suggest that concomitant FVIII exposure and vaccination against influenza does not increase the risk of inhibitor formation and may in fact decrease anti-FVIII immune responses.

The immunogenicity of platelet-derived FVIII in hemophilia A mice with or without preexisting anti-FVIII immunity

AbstractEvidence shows that factor VIII (FVIII) ectopically expressed in platelets (2bF8) is therapeutic in FVIIInull mice even with anti-FVIII inhibitory antibodies (inhibitors). If current efforts to generate platelets in vitro succeed, genetically manipulated platelets containing FVIII may be used therapeutically in hemophilia A patients with inhibitors. One important concern is the immunogenicity of platelet-derived FVIII. To address this concern, we infused 2bF8 transgenic (2bF8Tg) platelets into naïve FVIIInull mice weekly for 8 weeks. No anti-FVIII antibodies were detected in the infused animals during the study course. We then explored whether platelet-derived FVIII is immunogenic in FVIIInull mice with inhibitors. The 2bF8Tg platelets were transfused into rhF8-primed FVIIInull mice, resulting in no augmentation of anti-FVIII antibodies. To investigate whether preconditioning affects the immune response, animals were sublethally irradiated and subsequently transfused with 2bF8Tg platelets. No anti-FVIII antibodies were detected in the recipients after platelet infusions. Following further challenge with rhF8, the inhibitor titer in this group was significantly lower than in naïve FVIIInull mice utilizing the same immunization protocol. Thus, our data demonstrate that infusion of platelets containing FVIII triggers neither primary nor memory anti-FVIII immune response in FVIIInull mice and that sublethal irradiation plus 2bF8Tg platelet infusion suppresses anti-FVIII immune response in FVIIInull mice.

Potential for cellular stress response to hepatic factor VIII expression from AAV vector

Hemophilia A and B are coagulation disorders resulting from the loss of functional coagulation factor VIII (FVIII) or factor IX proteins, respectively. Gene therapy for hemophilia with adeno-associated virus vectors has shown efficacy in hemophilia B patients. Although hemophilia A patients are more prevalent, the development of therapeutic adeno-associated virus vectors has been impeded by the size of the F8 cDNA and impaired secretion of FVIII protein. Further, it has been reported that over-expression of the FVIII protein induces endoplasmic reticulum stress and activates the unfolded protein response pathway both in vitro and in hepatocytes in vivo, presumably due to retention of misfolded FVIII protein within the endoplasmic reticulum. Engineering of the F8 transgene, including removal of the B domain (BDD-FVIII) and codon optimization, now allows for the generation of adeno-associated virus vectors capable of expressing therapeutic levels of FVIII. Here we sought to determine if the risks of inducing the unfolded protein response in murine hepatocytes extend to adeno-associated virus gene transfer. Although our data show a mild activation of unfolded protein response markers following F8 gene delivery at a certain vector dose in C57BL/6 mice, it was not augmented upon further elevated dosing, did not induce liver pathology or apoptosis, and did not impact FVIII immunogenicity.