Uptake Of Factor VIII Research Articles

Modulating the microenvironment during FVIII uptake influences the nature of FVIII-peptides presented by antigen-presenting cells.

Hemophilia A is a severe bleeding disorder caused by the deficiency of functionally active coagulation factor VIII (FVIII). The induction of neutralizing anti-drug antibodies is a major complication in the treatment of hemophilia A patients with FVIII replacement therapies. Why some patients develop neutralizing antibodies (FVIII inhibitors) while others do not is not well understood. Previous studies indicated that the induction of FVIII inhibitors requires cognate interactions between FVIII-specific B cells and FVIII-specific CD4+ T cells in germinal center reactions. In this study, we investigated the FVIII peptide repertoire presented by antigen-presenting cells (APCs) under different microenvironment conditions that are expected to alter the uptake of FVIII by APCs. The aim of this study was to better understand the association between different microenvironment conditions during FVIII uptake and the FVIII peptide patterns presented by APCs. We used a FVIII-specific CD4+ T cell hybridoma library derived from humanized HLA-DRB1*1501 (human MHC class II) hemophilic mice that were treated with human FVIII. APCs obtained from the same mouse strain were preincubated with FVIII under different conditions which are expected to alter the uptake of FVIII by APCs. Subsequently, these preincubated APCs were used to stimulate the FVIII-specific CD4+ T cell hybridoma library. Stimulation of peptide-specific CD4+ T-cell hybridoma clones was assessed by analyzing the IL-2 release into cell culture supernatants. The results of this study indicate that the specific microenvironment conditions during FVIII uptake by APCs determine the peptide specificities of subsequently activated FVIII-specific CD4+ T cell hybridoma clones. Incubation of APCs with FVIII complexed with von Willebrand Factor, FVIII activated by thrombin or FVIII combined with a blockade of receptors on APCs previously associated with FVIII uptake and clearance, resulted in distinct peptide repertoires of subsequently activated hybridoma clones. Based on our data we conclude that the specific microenvironment during FVIII uptake by APCs determines the FVIII peptide repertoire presented on MHC class II expressed by APCs and the peptide specificity of subsequently activated FVIII-specific CD4+ T cell hybridoma clones.

Practical Application of the Athndataset: Evaluating Uptake of Extended Half-Life Factor Concentrates in Patients with Moderate and Severe Hemophilia

IntroductionHemophilia patients and providers have more treatment options than ever before. Integration of extended half-life (EHL) factor products into routine use for hemophilia A or B prophylaxis varies across hemophilia treatment centers (HTCs) and has not been formally described. The ATHNdataset is a national database of bleeding disorder and thrombosis patients who opt-in from 138 ATHN-affiliated HTCs and includes required core data elements. The primary aim of this longitudinal, observational study is to characterize the evolving real-world use of factor VIII (FVIII) and factor IX (FIX) concentrates, including rate of switching from one factor replacement product to another (switch rate), comparative annual factor consumption, and infusion frequency using serial queries of the ATHNdataset. A sub-study of detailed site-level questionnaires, above and beyond the ATHNdataset, including 8 HTCs evaluated the ability of the ATHNdataset to capture changes in EHL product use in real-time and demonstrate the fidelity of the ATHNdataset.MethodsAll patient level data was collected and analyzed under the patient authorization for the ATHNdataset. The selected population for analysis included all patients with moderate and severe hemophilia A or B without active inhibitors. EHL products were defined as those with moieties added with intention of half-life extension. The ATHNdataset was queried at baseline (June 2016) and follow-up 9 months later (March 2017). Sub-study site-level switching data was captured via quarterly RedCap™ questionnaires.ResultsATHNdataset analysisAt baseline, 7,552 patients were analyzed (5,504 FVIII, 1978 FIX). Of 3,163 FVIII patients on prophylaxis 87% were prescribed conventional FVIII concentrates and 13% EHL concentrates. Of the 699 FIX patients on prophylaxis 70% were prescribed conventional FIX concentrates, 30% EHL concentrates. Median dose and infusion frequency for FVIII products were 40 IU/kg/dose and 156 annual infusions and for FIX were 65 IU/kg/dose and 104 annual infusions.The 9-month follow-up analysis yielded 7,864 patients eligible (5,764 FVIII, 2,100 FIX). Frequency of use for both EHL FVIII and FIX is increasing. Baseline use of EHL FIX was substantially higher than that of EHL FVIII and continues to rise. Growth in use of EHL FVIII for prophylaxis was 3 times that of conventional FVIII. In aggregate 17% of patients changed from an on-demand to prophylaxis regimen with 40% of these patients initiating prophylaxis with an EHL product.Table 1: Comparative use of conventional and EHL factor concentrate captured in ATHNdataset.Table 2: Median dose and infusion frequency of conventional and EHL factor concentrates prescribed for prophylaxis.Sub-study analysisEight sites representing 1009 patients with moderate and severe hemophilia A or B from across the U.S. participated in the sub-study analysis. At baseline 8% of these patients were prescribed EHL concentrates. Across sites, the median was 10 patients (range: 0-18). Of the patients on an EHL product participating in the ATHNdataset, 63% were on EHL FVIII. At the 9-month follow-up an additional 33 patients had switched to an EHL product (17 FVIII, 16 FIX) equating to an overall EHL use of only 11.6%. Two patients switched among EHL products and 2 returned to a conventional product. Half of sub-study sites reported routinely obtained blood samples for PK analysis; 6 of 8 sites reported obtaining blood samples for PK analysis for patient switching to EHL products during follow-up period.ConclusionThe ATHNdataset presents a unique opportunity to investigate practice changes among U.S. HTCs. Uptake of EHL FVIII has been modest despite the first product being licensed in 2014. In contrast, the use of EHL FIX has now risen to greater than 40% of patients receiving prophylaxis. The median usage of EHL FVIII results in 27% reduction of annual units of factor prescribed for prophylaxis, and 46% reduction for EHL FIX compared to conventional products. The incremental increase in per unit price of EHL products, 30-72% greater than conventional products, exceeds the annual reduction of product usage. Consistent reporting of bleed events and adherence are not currently available for comparison. The contrast between sub-study uptake results and the ATHNdataset results highlight the practice variability among HTCs with respect to integration of EHL products into clinical care. [Display omitted] DisclosuresCroteau:ATHN/Hemophilia of Georgia: Research Funding; Octapharma: Honoraria; Baxalta, Dimension Therapeutics, Genentech, Pfizer: Research Funding; Aptevo, Baxalta/Shire, Bayer, CSL Behring, Genentech, Novo Nordisk, Octapharma, Pfizer: Consultancy. Raffini:Green Cross Inc: Consultancy; Genetech: Consultancy; CSL Behring: Consultancy; Bayer: Consultancy. Silvey:Pfizer, CSL Behring, Octapharma: Consultancy. Wheeler:NovoNordisk: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees; Octapharma: Membership on an entity's Board of Directors or advisory committees; Shire: Membership on an entity's Board of Directors or advisory committees. Neufeld:Novartis: Membership on an entity's Board of Directors or advisory committees; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees; Octapharma: Consultancy, Honoraria.

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.

Complement C3 is a novel modulator of the anti-factor VIII immune response.

Development of neutralizing antibodies against therapeutic Factor VIII (FVIII) is the most serious complication of the treatment of hemophilia A. There is growing evidence to show the multifactorial origin of the anti-FVIII immune response, combining both genetic and environmental factors. While a role for the complement system on innate as well as adaptive immunity has been documented, the implication of complement activation on the onset of the anti-FVIII immune response is unknown. Here, using in vitro assays for FVIII endocytosis by human monocyte-derived dendritic cells and presentation to T cells, as well as in vivo complement depletion in FVIII-deficient mice, we show a novel role for complement C3 in enhancing the immune response against therapeutic FVIII. In vitro, complement C3 and its cleavage product C3b enhanced FVIII endocytosis by dendritic cells and presentation to a FVIII-specific CD4+ T-cell hybridoma. The C1 domain of FVIII had previously been shown to play an important role in FVIII endocytosis, and alanine substitutions of the K2092, F2093 and R2090 C1 residues drastically reduce FVIII uptake in vitro. Interestingly, complement activation rescued the endocytosis of the FVIII C1 domain triple mutant. In a mouse model of severe hemophilia A, transient complement C3 depletion by humanized cobra venom factor, which does not generate anaphylatoxin C5a, significantly reduced the primary anti-FVIII immune response, but did not affect anti-FVIII recall immune responses. Taken together, our results suggest an important adjuvant role for the complement cascade in the initiation of the immune response to therapeutic FVIII.

Enhanced uptake of blood coagulation factor VIII containing immune complexes by antigen presenting cells

Background A major complication in the treatment of hemophilia A is the development of inhibitory antibodies targeting coagulation factor VIII (FVIII). Eradication of these inhibitors can be established by immune tolerance induction (ITI), which consists of daily administration of high dosages of FVIII. FVIII immune complexes (FVIII-IC) could be formed following FVIII infusion in patients with pre-existing anti-FVIII antibodies. Objectives Here we studied endocytosis of FVIII-IC by bone marrow-derived dendritic cells (BMDCs). Methods BMDCs were pulsed with FVIII/FVIII-IC and uptake was assessed by flow cytometry and confocal imaging. Results BMDCs were able to efficiently internalize FVIII-IC in a dose-dependent manner, 3-4-fold more efficiently when compared with equimolar concentrations of non-complexed FVIII. Uptake of FVIII-IC, but not FVIII alone, could be inhibited with anti-Fcγ receptor (FcγR) antibody 2.4G2, indicating functional involvement of FcγR. No internalization of FVIII-IC was observed in BMDCs lacking FcγRI, FcγRIIb, FcγRIII and FcγRIV. Genetic ablation of FcγRIIb, FcγRIII or FcγRIV individually did not affect the ability of anti-FVIII IgG to promote the uptake of FVIII. BMDCs lacking FcγRI showed lower FVIII-IC uptake levels when compared with other single FcγR null BMDCs. Expression of the inhibitory FcγRIIb alone was sufficient to internalize FVIII-IC more efficiently than FVIII. Conclusions FcγR are critical in the internalization of FVIII-IC by BMDCs and multiple FcγR can contribute independently to this process. Our findings provide a basis for future studies to address whether the outcome of ITI is dependent on the interplay between FVIII-IC and inhibitory and activating FcγR.

Factor VIII C2 Domain Epitopes Recognized By Classical and Non-Classical Anti-C2 Domain Monoclonal Antibodies Help Mediate FVIII Uptake By Dendritic Cells

Introduction: Dendritic cells are the predominant antigen presenting cells (APCs) responsible for recognition and processing of pathogens and other proteins to induce a primary immune response. Factor VIII (FVIII) is a highly immunogenic protein and the formation of anti-FVIII antibodies remains the most common complication of hemophilia treatment. The A2 and C2 domains of FVIII account for a significant portion of antibodies formed in patients with severe hemophilia A. Prior studies have shown that C1 domain epitopes mediate FVIII uptake by human and murine monocyte-derived dendritic cells (MDDCs). In contrast, in-vitro uptake of FVIII bound by a human-derived anti-A2 monoclonal antibody (MAb) directed against epitopes Arg484-Ile508 did not alter FVIII intake, suggesting that interactive residues within the A2 domain do not participate in FVIII presentation to APCs. However, in light of the functional and structural similarities between the C1 and C2 domains and current knowledge of the immunogenic B cell epitopes within the C2 domain, further investigation into the C2 domain's role in FVIII uptake by APCs is essential.Methods:Human peripheral blood mononuclear cells were harvested from healthy donors after written consent. Monocytes were isolated by positive selection using CD14 microbeads. CD14+ monocytes were stimulated and expanded into immature MDDCs in medium containing 1000 U/ml GM-CSF and 800 U/ml IL-4 for 4-6 days. To evaluate FVIII uptake by dendritic cells, MDDCs were incubated with 10 nM DyLight650-conjugated recombinant full length FVIII (DyL650-FVIII) in the absence or presence of a saturating concentration of 80 nM of murine-derived anti-human FVIII MAbs following a 30 minute pre-incubation period of FVIII/MAb for optimal binding at 37°C. MAbs tested were classical anti-C2 MAbs 3D12 and I89, non-classical anti-C2 MAb B9, and anti-A2 MAb 4A4. Cells were subsequently washed, fixed, and analyzed by flow cytometry. Internalization of FVIII and median fluorescence intensity of FVIII uptake by MDDCs in the presence of MAbs were compared to MDDCs exposed to FVIII or serum free medium alone.Results: Both classical anti-C2 MAbs 3D12 and I89 reduced the percentage of FVIII internalized by MDDCs by 68% and 70% respectively compared to MDDCs treated with FVIII alone similar to the decrease seen with anti-C1 MAbs. Classical anti-C2 MAbs potently inhibit the interaction of FVIII with key binding ligands of Von Willebrand factor (VWF) and phospholipids. The proposed binding epitopes of 3D12, a group B MAb, includes Leu2251, Leu2252, and Phe2196. MAb I89 is a group AB MAb and the proposed binding epitope encompasses residues Met2199 and Phe2200. Non-classical anti-C2 MAb B9 reduced FVIII uptake by 31%, which is comparably less than the classical anti-C2 MAbs. B9 is a group BC antibody that inhibits FVIII binding to phospholipids but does not inhibit binding to VWF. Prior studies have shown that Lys2227 is a critical structural epitope for group BC anti-C2 MAbs. Anti-A2 MAb 4A4, a MAb that binds the Asp403-His444 epitope, did not significantly alter FVIII uptake demonstrating similar internalization of FVIII to the FVIII control. Representative histograms of the percentage of internalized FVIII by untreated MDDCs (shaded gray), MDDCs treated with FVIII alone (black solid line), and MDDCs treated with FVIII in the presence of the anti-C2 or A2 MAbs tested (black dotted line) are shown (Figure 1). Observed reductions in FVIII uptake in the presence of the anti-C2 MAbs suggests that the binding sites recognized by these MAbs play a role in FVIII uptake by dendritic cells.Conclusion: Binding epitopes recognized by classical and non-classical anti-C2 MAbs mediate FVIII uptake by dendritic cells similar to anti-C1 MAbs. Better understanding of the specific epitopes that promote FVIII uptake and inhibitor formation could ultimately lead to development of mechanisms for inhibitor prediction and prevention. [Display omitted] DisclosuresMeeks:Genentech: Membership on an entity's Board of Directors or advisory committees; Bayer Healthcare: Membership on an entity's Board of Directors or advisory committees; Biogen: Membership on an entity's Board of Directors or advisory committees; Grifols: Membership on an entity's Board of Directors or advisory committees; CSL Behring: Membership on an entity's Board of Directors or advisory committees; Shire: Membership on an entity's Board of Directors or advisory committees.

Hunting down factor VIII in the immunopeptidome

Major histocompatibility complex class II (MHCII)-restricted peptide presentation is crucial for the selection and subsequent proliferation of antigen specific CD4+ T cells. While selection of antigen-specific CD4+ T cells is beneficial in the context of vaccination, emergence of antigen CD4+ T cells following administration of therapeutic proteins like factor VIII (FVIII) is not desirable. The mechanism of uptake, processing and presentation of FVIII by antigen-presenting cells (APCs) has been the subject of intense study over the past 10years. Multiple receptors have been implicated in the uptake of FVIII by APCs. A crucial determinant directing its entry in APCs resides in the C1 domain of FVIII. Until recently, our knowledge on the repertoire of FVIII derived presented on MHCII was limited. Peptide sequences on FVIII recognized by CD4+ T cells have been identified using MHCII tetramers as well as by directly monitoring peptide-induced proliferation of CD4+ T cells. More recently, the repertoire of naturally presented peptides derived from FVIII has been identified by pulsing of immature dendritic cells with FVIII. In a complementary approach HLA-DRB1∗15 transgenic mice were used to identify HLA-DRB1∗15 restricted CD4+ T cells reactive towards human FVIII. In this review we summarize our current knowledge on FVIII derived peptides that are presented on MHCII and discuss the relevance of these findings for the etiology of inhibitor development in patients with hemophilia A.

Modification of an exposed loop in the C1 domain reduces immune responses to factor VIII in hemophilia A mice

AbstractDevelopment of neutralizing Abs to blood coagulation factor VIII (FVIII) provides a major complication in hemophilia care. In this study we explored whether modulation of the uptake of FVIII by APCs can reduce its intrinsic immunogenicity. Endocytosis of FVIII by professional APCs is significantly blocked by mAb KM33, directed toward the C1 domain of FVIII. We created a C1 domain variant (FVIII-R2090A/K2092A/F2093A), which showed only minimal binding to KM33 and retained its activity as measured by chromogenic assay. FVIII-R2090A/K2092A/F2093A displayed a strongly reduced internalization by human monocyte-derived dendritic cells and macrophages, as well as murine BM-derived dendritic cells. We subsequently investigated the ability of this variant to induce an immune response in FVIII-deficient mice. We show that mice treated with FVIII-R2090A/K2092A/F2093A have significantly lower anti-FVIII Ab titers and FVIII-specific CD4+ T-cell responses compared with mice treated with wild-type FVIII. These data show that alanine substitutions at positions 2090, 2092, and 2093 reduce the immunogenicity of FVIII. According to our findings we hypothesize that FVIII variants displaying a reduced uptake by APCs provide a novel therapeutic approach to reduce inhibitor development in hemophilia A.

Elucidation of the Roles of Individual Asparagine-Linked Glycans Outside of the B Domain on Factor VIII Secretion

Abstract 2238Post-translational modifications play vital roles in the secretion, function, intermolecular interactions and degradation of most secreted and transmembrane proteins. Factor VIII (FVIII) is a heavily glycosylated protein with up to 25 asparagine (Asn)-linked glycans, the bulk of which are present within its B domain. However, deletion of the B domain is not deleterious to FVIII expression and function. In addition, FVIII has several potential Asn-linked glycosylation sequons in its other domains, of which four have been experimentally deduced to be glycosylated: Asn41 and Asn239 in the A1 domain, Asn1810 in the A3 domain and Asn2118 in the C1 domain. Of these, Asn239 and Asn2118 have been determined to comprise complex oligomannose structures. Such complex oligomannose structures have been proposed to play a role in mediating interaction with immunomodulatory cells (i.e. dendritic cells). The present study was aimed at delineating the role(s) of these four Asn-linked glycans in the expression of FVIII in vitro and in vivo and to identify possible bioengineering targets to influence FVIII expression, clearance and processing by immunomodulatory cells. Individual Asn residues were mutated to glutamine (Gln) to create single and multiple glycosylation mutants in both full length (FVIII-WT) and B domain-deleted (BDD)–FVIII, by site-directed mutagenesis. A variant of BDD-FVIII completely devoid of Asn-linked glycans, designated as Degly-BDD-FVIII, was also generated. Transient transfections of the mutants were carried out in COS-1 and CHO cells and their secretion and function were analyzed and compared to that of the respective native FVIII proteins. Antigen and activity assays revealed that the secretion and function of Asn41Gln and Asn1810Gln mutants were only modestly affected (85–90% of WT) but a more significant reduction was observed in the case of Asn239Gln mutant (35–50% of WT). Interestingly, there was no significant difference in secretion or function for Asn2118Gln in either FVIII-WT or BDD-FVIII protein backbones. The double mutants, Asn41/239Gln and Asn239/2118Gln behaved similarly to that of Asn239Gln mutant (30–45% of WT). The triple mutants, Asn41/239/2118Gln and Asn239/1810/2118Gln showed a further decline in secretion (∼30-40% of WT) while Degly-BDD-FVIII demonstrated secretion of only about 15–20% of BDD-FVIII. The FVIII specific activity of each of these glycosylation mutants was similar to the native FVIII proteins. An ELISA-based Von Willebrand Factor (VWF) binding assay revealed no significant differences between immunoaffinity-purified FVIII-WT and Asn2118Gln mutant in their ability to bind VWF. Findings from in vivo expression (via hydrodynamic tail vein injection of plasmid DNA) of these glycosylation mutants in a F8−/− (exon 16 knock-out) hemophilia A mouse model were similar to the in vitro results in the cell lines. Plasma FVIII activity levels were measured 24 hrs post-injection via orbital bleed. While Asn2118Gln (5.2 – 6 U/mL) did not exhibit any difference from BDD-FVIII (4.8 – 5.9 U/ml), Asn239Gln (1.9 – 2.4 U/ml) was expressed at less than 50% of BDD-FVIII levels. The expression of Degly-BDD-FVIII (0.4 – 0.7 U/ml) was further reduced to ∼10% of BDD-FVIII levels. Taken together, these results indicate that of the four Asn-linked glycans, Asn239 was the most crucial for proper secretion of FVIII whereas, Asn2118 did not contribute to the efficiency of FVIII expression. The oligosaccharide structure on Asn239 is positioned at the A1-A2 interface and likely contributes to proper protein folding. However, the sugar moieties on Asn2118 have been shown to be positioned at the A3-C1 domain interface and postulated to participate in packing and stabilization (Shen et al, 2008). This would have suggested that disruption of this residue within the C1 domain might have a deleterious effect on protein secretion or function. Our results with Asn2118Gln in both FVIII-WT and BDD-FVIII protein backbones suggest that this Asn-linked glycosylation can be eliminated without any impact on FVIII expression or function including no impact on FVIII-VWF interaction. This Asn-linked glycan, therefore, could be targeted in bioengineering strategies to determine if eliminating this particular oligomannose structure might impact mannose-receptor mediated uptake of FVIII by dendritic cells. Disclosures:No relevant conflicts of interest to declare.

Uptake of blood coagulation factor VIII by dendritic cells is mediated via its C1 domain

The uptake and processing of blood coagulation factor VIII (FVIII) by antigen-presenting cells and the subsequent presentation of FVIII-derived peptides to CD4(+) Tcells direct the immune response to FVIII in patients with hemophilia A. Multiple receptors including mannose receptor and low-density lipoprotein receptor-related protein-1 (LRP1) have been implicated in FVIII uptake. This work studies the involvement of receptor candidates in FVIII uptake by dendritic cells (DCs). Furthermore, we explore FVIII residues that mediate endocytosis. FVIII uptake was performed with human monocyte-derived and murine bone marrow-derived DCs. To investigate FVIII endocytosis, competition assays with soluble receptor ligands, binding studies with recombinant receptor fragments, and small-interfering RNA-induced gene silencing were performed. In addition, FVIII-targeting monoclonal antibodies KM33 and VK34 were used. To confirm invitro results, hemophilic E17 knockout mice were pretreated with antibodies prior to FVIII injections and anti-FVIII titers were determined. Upon treatment of DCs with mannan or LRP ligand α2-macroglobulin, we observed only a minor decrease in FVIII internalization. In addition, small interfering RNA-mediated knockdown of LRP, mannose receptor, or DC-SIGN expression in monocyte-derived dendritic cells did not prevent FVIII uptake. Binding studies using Fc chimeras revealed that LRP, DC-SIGN, and mannose receptor can bind to FVIII; however, we did not observe a critical role for these receptors in FVIII uptake. Previous studies have shown that human antibodies targeting the C1 (KM33) and A2 (VK34) domains of FVIII interfere with binding to endocytic receptors. Preincubation of FVIII with VK34 did not influence FVIII uptake; however, KM33 completely inhibited FVIII endocytosis by both monocyte-derived dendritic cells and bone marrow-derived dendritic cells. Accordingly, anti-FVIII antibody titers were greatly reduced following the preadministration of KM33 invivo. Together, our observations emphasize the physiological significance of KM33-targeted residues within theC1 domain in the uptake of FVIII by DCs invitro and invivo.

C1 domain residues Lys 2092 and Phe 2093 are of major importance for the endocytic uptake of coagulation factor VIII

Factor VIII (FVIII) catabolism has been demonstrated to involve LDL receptor-related protein (LRP). We have established that antibody fragment KM33 inhibits cofactor function of FVIII by interacting with the membrane binding region 2092–2093 of the C1 domain. As KM33 also inhibits LRP-dependent uptake of FVIII, we now assessed the role of region 2092–2093 for LRP-dependent endocytosis. For this purpose, we employed functional fluorescent FVIII-YFP or -GFP derivatives and U87MG cells which express high levels of LRP. Confocal microscopy studies and flow cytometry analysis combined with siRNA technology showed that the fluorescent FVIII derivatives are indeed effectively internalized by U87MG cells in a LRP-dependent manner. Competition experiments employing an antagonist of the LDL receptor family members revealed that there is a cell surface binding event for FVIII, which is independent of LRP. Cell surface binding proved to be less effective for the FVIII-YFP variants K2092A, F2093A and K2092A/F2093A. Surface plasmon resonance analysis showed that these substitutions affect LRP binding as well. Finally, flow cytometry analysis revealed a major reduction of endocytic uptake of these FVIII-YFP variants. Our results demonstrate that C1 domain residues 2092–2093 are of major importance for FVIII endocytosis by contributing to cell surface binding and receptor binding.

Investigating the Mechanisms Underlying FVIII Antibody Production in Hemophilic Mice Following Recombinant and Plasma-Derived FVIII Infusion

The formation of Factor VIII (FVIII) antibodies after FVIII treatment in hemophilic patients is currently the major treatment-related complication faced by hemophilic patients. It has been reported that the use of plasma-derived FVIII (pdFVIII) in patients and animal models of hemophilia A will result in reduced anti-FVIII antibody formation compared to recombinant FVIII (rFVIII). In an attempt to explain this phenomenon, an initial hypothesis relating to the uptake of FVIII by antigen presenting cells has been formulated. In contrast, here we examine the hypothesis that the cytokine microenvironment induced by pdFVIII has a critical influence on the reduction of anti-FVIII antibody titers in hemophilia A mice. Micro-array and confirmatory quantitative RT-PCR experiments performed on CD11c+ dendritic cells, isolated 24 hours after rFVIII or pdFVIII infusion, show that pdFVIII causes a different immune gene expression profile than rFVIII administration. Both treatments caused the up regulation of pro-inflammatory gene expression. However, the rFVIII and pdFVIII treatments caused the up regulation of genes that induce Th1 and Th2 responses, respectively. Moreover, after administering rFVIII or pdFVIII concentrates to mice, we observed that each treatment induced a distinct T cell splenic cytokine micro-environment. Recombinant FVIII induced the release of Th1 cytokines and IL10, while pdFVIII induced the release of Th2 cytokines and TGF-α. In this mouse model, we observed the formation of high titers of anti-human VWF antibodies in the pdFVIII-treated mice and we propose that in these animals, VWF competes with FVIII for antigenic presentation. We further investigated the potential of antigenic competition by treating mice with FVIII and increasing concentrations of another unrelated protein, recombinant human factor IX (FIX). Our studies have shown an inverse relationship between increasing concentrations of FIX and the production of anti-FVIII antibodies. In summary, these studies allude to new and additional mechanisms that may contribute to the reduction of anti-FVIII antibody development in hemophilia A mice treated with pdFVIII.

Factor VIII bypasses CD91/LRP for endocytosis by dendritic cells leading to T-cell activation

The development of factor VIII (FVIII) inhibitors remains the major hurdle in the clinical management of patients with hemophilia A. FVIII uptake by professional antigen-presenting cells (APC) is the first step involved in initiation of immune responses to FVIII. Studies on FVIII catabolism have highlighted the role played by CD91/LRP as a potential target for increasing FVIII half-life in patients and prolonging treatment efficiency. We investigated the involvement of CD91 in FVIII endocytosis by human dendritic cells (DC), a model of professional APC. Immature DC were generated from circulating monocytes from healthy donors. Surface expression of CD91 was assessed by flow cytometry. Uptake of fluorescein isothiocyanate-conjugated ligands by immature DC was studied in the presence of various blocking agents. CD91 was expressed on approximately 20% of DC and mediated the internalization of its model ligand, alpha2-macroglobulin. DC internalized FVIII and activated a human FVIII-specific T-cell clone in a dose-dependent manner. FVIII uptake by DC and subsequent T-cell activation were not inhibited by receptor-associated protein. Our results indicate that CD91 and other members of the LDL receptor family are not strongly implicated in FVIII internalization by monocyte-derived DC, and suggest the involvement of alternative divalent ion-dependent endocytic receptors.

Effective Factor VIII Endocytosis Is under Conformational Control of von Willebrand Factor.

Von Willebrand Factor (VWF) protects Factor VIII (FVIII) from rapid clearance of the cofactor from the circulations. This notion may agree with the observation that VWF blocks FVIII binding to LDL receptor (LDLR) and LDL receptor-related protein (LRP) in vitro. In spite of the presence of VWF, however, we have previously demonstrated that LRP and LDLR are involved in the catabolism of FVIII in vivo. This suggests that dissociation of the FVIII-VWF complex in plasma or at the cell surface drives the LRP/LDLR dependent clearance of FVIII. We therefore first assessed the effect of VWF on the binding of FVIII to the cell surface employing confocal microscopy. To this end, a functional FVIII derivative containing the yellow fluorescent protein (FVIIIYFP) was incubated with LRP-expressing or LDLR-expressing CHO cells at 4 °C in the presence and absence of VWF. The result showed that there was a distinct yellow fluorescent staining of the cell surface irrespective of the presence of VWF. When the same experiment was performed at 37 °C, however, yellow fluorescent focal spots rapidly appeared inside the cells but only in the absence of VWF. Co-localization studies demonstrated that these spots originated from FVIIIYFP present in early endosomes. These findings suggest that VWF blocks the transfer of FVIII to its endocytic receptors but not the binding of FVIII to the cell surface. Intriguingly, in the presence of ristocetin, FVIIIYFP was localized inside the cells not only in the absence of VWF but also in its presence. In agreement with this finding, flow cytometric analysis confirmed that VWF was no longer able to prevent endocytosis of FVIIIYFP in the presence of ristocetin. These results suggest that switching VWF in the VWF-FVIIIYFP complex into its active conformation triggers the endocytic uptake of FVIII. The restored endocytosis of FVIII was not the consequence of a reduced affinity of “active-VWF” for FVIII. The latter was concluded from solid phase binding studies that showed that FVIII-VWF complex formation is indistinguishable in the presence and absence of ristocetin. We propose that switching VWF in the VWF-FVIII complex into its active conformation initiates at the cell surface a sequence of molecular events, which ultimately lead to the endocytic uptake of FVIII by cells expressing LRP or LDLR.

Von Willebrand Factor Protects Factor VIII from Endocytosis by Human Monocytes−Derived Dendritic Cells and Subsequent Presentation to Immune Effectors.

The role of Von Willebrand Factor (VWF) as a chaperone molecule for procoagulant factor VIII (FVIII) has been extensively documented. Under physiological conditions, VWF binds to FVIII after its release in the circulation. VWF protects FVIII from proteolysis by lipid−bound proteases, stabilizes FVIII and further regulates its elimination by lipoprotein receptors. We investigated whether VWF might modulate FVIII endocytosis by professionnal antigen presenting cells (APCs). Immature dendritic cells (DCs), utilised as model of APCs, were generated from circulating monocytes of healthy blood donors and incubated with FITC−labeled FVIII (FVIII−FITC). T cell assays were performed by co−culturing the human FVIII−specific CD4+ T cell clone, D9E9, with immature DCs from MHC−matched donors in presence of unconjugated FVIII and increasing concentrations of VWF (1:1 to 1:130). Preincubation of FVIII−FITC with VWF at 1 to 130−fold molar excess resulted in a dose−dependent inhibition of FVIII endocytosis (12 ± 2% to 94 ± 18%, respectively). Human serum albumin used in equivalent molar ratios did not prevent FVIII internalization. In contrast, the protective effect of VWF on FVIII uptake was significantly restored by co−incubation of FVIII and VWF in the presence of F(ab')2 fragments of BO2C11, an anti−FVIII IgG, and Ac418, an anti−VWF IgG, that both disrupt the interaction between FVIII and VWF. In addition, blocking DC entry of FVIII by VWF resulted in a dose−dependent reduction of the activation of D9E9 (up to 75%). Interestingly, D9E9 activation by DCs loaded with a synthetic FVIII−derived peptide, I2144–T2161, was not altered in the presence of equivalent concentrations of VWF, indicating that VWF does not have a direct inhibitory effect on T cell activation. The administration of exogenous FVIII to patients with hemophilia A, results in up to 30% of the cases, in the developement of FVIII−specific antibodies that inhibit FVIII procoagulant activity. In vivo experimental evidences and clinical observations have documented that the presence of VWF in FVIII therapeutic concentrates may be associated with a lower incidence of FVIII inhibitors. Here, we demonstrate that VWF may reduce the immunogenicity of FVIII by preventing its internalization by professionnal APCs, and reducing the subsequent presentation of FVIII−derived peptides to T lymphocytes.