BAX 335 hemophilia B gene therapy clinical trial results: potential impact of CpG sequences on gene expression.

Gene therapy has the potential to maintain therapeutic blood clotting factor IX (FIX) levels in patients with hemophilia B by delivering a functional human F9 gene into liver cells. This phase 1/2, open-label dose-escalation study investigated BAX 335 (AskBio009, AAV8.sc-TTR-FIXR338Lopt), an adeno-associated virus serotype 8 (AAV8)-based FIX Padua gene therapy, in patients with hemophilia B. This report focuses on 12-month interim analyses of safety, pharmacokinetic variables, effects on FIX activity, and immune responses for dosed participants. Eight adult male participants (aged 20-69 years; range FIX activity, 0.5% to 2.0%) received 1 of 3 BAX 335 IV doses: 2.0 × 1011; 1.0 × 1012; or 3.0 × 1012 vector genomes/kg. Three (37.5%) participants had 4 serious adverse events, all considered unrelated to BAX 335. No serious adverse event led to death. No clinical thrombosis, inhibitors, or other FIX Padua-directed immunity was reported. FIX expression was measurable in 7 of 8 participants; peak FIX activity displayed dose dependence (32.0% to 58.5% in cohort 3). One participant achieved sustained therapeutic FIX activity of ∼20%, without bleeding or replacement therapy, for 4 years; in others, FIX activity was not sustained beyond 5 to 11 weeks. In contrast to some previous studies, corticosteroid treatment did not stabilize FIX activity loss. We hypothesize that the loss of transgene expression could have been caused by stimulation of innate immune responses, including CpG oligodeoxynucleotides introduced into the BAX 335 coding sequence by codon optimization. This trial was registered at www.clinicaltrials.gov as #NCT01687608.

The soluble cytoplasmic tail of CD45 regulates T-cell activation via TLR4 signaling.

The soluble cytoplasmic tail of CD45 (ct-CD45) is a cleavage fragment of CD45, that is generated during the activation of human phagocytes. Upon release to the extracellular space, ct-CD45 binds to human T cells and inhibits their activation in vitro. Here, we studied the potential role of TLR4 as a receptor for ct-CD45. Treatment of Jurkat TLR4/CD14 reporter cells with ct-CD45 induced the upregulation of the reporter gene NFκB-eGFP and could be blocked by inhibitors of TLR4 signaling. Conversely, ct-CD45 did not promote the NFκB-controlled eGFP induction in reporter cells expressing TLR1, TLR2, and TLR6 transgenes and did not lead to the activation of the transcription factors NFκB, AP-1, and NFAT in a Jurkat reporter cell line expressing endogenous TLR5. Moreover, ct-CD45 binds to recombinant TLR4 in an in vitro assay and this association was reduced in the presence of oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine. Blockade of TLR4 with mAb HTA125 partially reversed the ct-CD45-mediated inhibition of T-cell proliferation. Interestingly, targeting of TLR4 with mAb W7C11 also suppressed T-cell proliferation. In summary, the results of this study demonstrate that ct-CD45 acts via a noncanonical TLR4 activation pathway on T cells, which modulates TCR signaling.

IVIG induces apoptotic cell death in CD56dim NK cells resulting in inhibition of ADCC effector activity of human PBMC.

The mechanism of the efficacy of Intravenous immunoglobulins (IVIG) in autoimmune and inflammatory diseases is not well understood. This study aimed at understanding mechanisms of IVIG-mediated suppression of effector cell activities of peripheral blood mononuclear cells (PBMC) in antibody-dependent cellular cytotoxicity (ADCC). We were particularly interested in CD56dim NK cells, the main ADCC effector cells in PBMC. Exposure of PBMC to IVIG for at least 48 h induced a caspase-3-dependent apoptotic cell death of CD56dim NK cells without affecting CD56bright NK cells. Induction of apoptosis in CD56dim NK cells and concomitant suppression of ADCC effector activities of PBMC was associated with the monomer fraction of IVIG. Moreover, it was independent of IgG sialyation, did not depend on engagement of FcγRIII and could not be mimicked by IVIG (Fab')2 or IVIG Fc preparations. The described effect could contribute to the reduction of peripheral NK cells observed during IVIG therapy in patients.

Evaluation of Factor VIII Polysialylation: Identification of a Longer-Acting Experimental Therapy in Mice and Monkeys.

Extended half-life (EHL) factor therapies are needed to reduce the burden of prophylaxis and improve treatment adherence in patients with hemophilia. BAX 826 is a novel polysialylated full-length recombinant factor VIII [polysialyic acid (PSA) rFVIII] with improved pharmacokinetics (PK), prolonged pharmacology, and maintained safety attributes to enable longer-acting rFVIII therapy. In factor VIII (FVIII)-deficient hemophilic mice, PSArFVIII showed a substantially higher mean residence time (>2-fold) and exposure (>3-fold), and prolonged efficacy in tail-bleeding experiments (48 vs. 30 hours) compared with unmodified recombinant FVIII (rFVIII), as well as a potentially favorable immunogenicity profile. Reduced binding to a scavenger receptor (low-density lipoprotein receptor-related protein 1) and von Willebrand factor (VWF) as well as a largely VWF-independent circulation time in mice provide a rationale for prolonged BAX 826 activity. The significantly improved PK profile versus rFVIII was confirmed in cynomolgus monkeys [mean residence time: 23.4 vs. 10.1 hours; exposure (area under the curve from time 0 to infinity): 206 vs. 48.2 IU/ml⋅h] and is in line with results from rodent studies. Finally, safety and toxicity evaluations did not indicate increased thrombogenic potential, and repeated administration of BAX 826 to monkeys and rats was well tolerated. The favorable profile and mechanism of this novel experimental therapeutic demonstrated all of the requirements for an EHL-rFVIII candidate, and thus BAX 826 was entered into clinical assessment for the treatment of hemophilia A. SIGNIFICANCE STATEMENT: Prolongation of FVIII half-life aims to reduce the burden of prophylaxis and improve treatment outcomes in patients with hemophilia. This study shows that polysialylation of PSArFVIII resulted in prolongations of rFVIII circulation time and procoagulant activity, together with a favorable nonclinical safety profile of the experimental therapeutic.

How Full-Length FVIII Benefits from Its Heterogeneity - Insights into the Role of the B-Domain.

PURPOSE: To explore how the natural heterogeneity of human coagulation factor VIII (FVIII) and the processing of its B-domain specifically modulate protein aggregation. METHODS: Recombinant FVIII (rFVIII) molecular species containing 70% or 20% B-domain, and B-domain-deleted rFVIII (BDD-rFVIII), were separated from full-length recombinant FVIII (FL-rFVIII). Purified human plasma-derived FVIII (pdFVIII) was used as a comparator. Heterogeneity and aggregation of the various rFVIII molecular species, FL-rFVIII and pdFVIII were analysed by SDS-PAGE, dynamic light scattering, high-performance size-exclusion chromatography and flow cytometry-based particle analysis. RESULTS: FL-rFVIII and pdFVIII were heterogeneous in nature and demonstrated similar resistance to aggregation under physical stress. Differences were observed between these and among rFVIII molecular species. FVIII molecular species exhibited diverging aggregation pathways dependent on B-domain content. The propensity to form aggregates increased with decreasing proportions of B-domain, whereas the opposite was observed for oligomer formation. Development of cross-ß sheet-containing aggregates in BDD-rFVIII induced effective homologous seeding and faster aggregation. Naturally heterogeneous FL-rFVIII and pdFVIII displayed the lowest propensity to aggregate in all experiments. CONCLUSIONS: These results demonstrate that pdFVIII and FL-rFVIII have similar levels of molecular heterogeneity, and suggest that heterogeneity and the B-domain are involved in stabilising FVIII by modulating its aggregation pathway.

FVIII-binding IgG modulates FVIII half-life in patients with severe and moderate hemophilia A without inhibitors.

The substantial variability in pharmacokinetic parameters in hemophilia patients A poses a challenge for optimal treatment with factor VIII (FVIII) products. We investigated the effect of FVIII-specific immunoglobulin G (IgG) on FVIII half-life in a cohort of 42 adult patients with severe and moderate hemophilia A without inhibitors. Fifteen (35.7%) of 42 patients tested positive for FVIII-binding IgG with titers ≥1:20 in the initial antibody screen, 9 of these 15 patients had FVIII-specific antibodies with titers ≥1:40, mostly low-to-moderate-affinity IgG1 and IgG3, and 1 had high-affinity IgG4 and later developed low-titer FVIII inhibitors. His brother with low-to-moderate-affinity IgG1 and IgG3 also later developed low-titer FVIII inhibitors. The presence of FVIII-specific IgG subclass titer ≥1:40 antibodies was significantly associated with shorter FVIII half-life (median, 7.8 hours [interquartile range, 6.6-9.2 hours]) vs 10.4 hours [interquartile range, 8.9-13.8 hours]); the regression coefficient adjusted for log age and log von Willebrand factor (VWF) antigen was -0.32 (P = .004), accounting for 16.9% of the observed variability of FVIII half-life in our cohort. Our data indicate a significant contribution of non-neutralizing FVIII-specific IgG to FVIII half-life reduction in hemophilia A patients. Thus, screening for FVIII-specific IgG could be beneficial in tailoring FVIII prophylactic regimens.

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

Inflammatory 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.

Anti-factor VIII IgA as a potential marker of poor prognosis in acquired hemophilia A: results from the GTH-AH 01/2010 study.

Neutralizing autoantibodies against factor VIII (FVIII), also called FVIII inhibitors, are the cause of acquired hemophilia A (AHA). They are quantified in the Bethesda assay or Nijmegen-modified Bethesda assay by their ability to neutralize FVIII in normal human plasma. However, FVIII inhibitors do not represent the whole spectrum of anti-FVIII autoantibodies. Here, we studied isotypes, immunoglobulin G subclasses, and apparent affinities of anti-FVIII autoantibodies to assess their prognostic value for the outcome in AHA. We analyzed baseline samples from patients enrolled in the prospective GTH-AH 01/2010 study. Our data suggest that anti-FVIII immunoglobulin A (IgA) autoantibodies are predictors of poor outcome in AHA. Anti-FVIII IgA-positive patients achieved partial remission similar to anti-FVIII IgA-negative patients but had a higher risk of subsequent recurrence. Consequently, IgA-positive patients achieved complete remission less frequently (adjusted hazard ratio [aHR], 0.35; 95% confidence interval [CI], 0.18-0.68; P < .01) and had a higher risk of death (aHR, 2.62; 95% CI, 1.11-6.22; P < .05). Anti-FVIII IgA was the strongest negative predictor of recurrence-free survival after achieving partial remission and remained significant after adjustment for baseline demographic and clinical characteristics. In conclusion, anti-FVIII IgA represents a potential novel biomarker that could be useful to predict prognosis and tailor immunosuppressive treatment of AHA.

Dexamethasone promotes durable factor VIII-specific tolerance in hemophilia A mice via thymic mechanisms.

The development of inhibitory antibodies to factor VIII is the most serious complication of replacement therapy in hemophilia A. Activation of the innate immune system during exposure to this protein contributes to inhibitor development. However, avoidance of factor VIII exposure during innate immune system activation by external stimuli (e.g., vaccines) has not been consistently shown to prevent inhibitors. We hypothesized that dexamethasone, a drug with potent anti-inflammatory effects, could prevent inhibitors by promoting immunologic tolerance to factor VIII in hemophilia A mice. Transient dexamethasone treatment during ainitial factor VIII exposure reduced the incidence of anti-factor VIII immunoglobulin G in both a conventional hemophilia A mouse model (E16KO, 77% vs 100%, P=0.048) and a hemophilia A mouse model with a humanized major histocompatibility complex type II transgene (E17KO/hMHC, 6% vs 33%, P=0.0048). More importantly, among E17KO/hMHC mice that did not develop anti-factor VIII immunoglobulin G after initial exposure, dexamethasone-treated mice were less likely to develop a response after re-exposure six (7% vs 52%, P=0.005) and 16 weeks later (7% vs 50%, P=0.097). Similar results were obtained even when factor VIII re-exposure occurred in the context of lipopolysaccharide (30% vs 100%, P=0.069). The ability of these mice to develop immunoglobulin G to human von Willebrand factor, a structurally unrelated antigen, remained unaffected by treatment. Transient dexamethasone administration therefore promotes antigen-specific immunologic tolerance to factor VIII. This effect is associated with an increase in the percentage of thymic regulatory T cells (12.06% vs 4.73%, P<0.001) and changes in the thymic messenger ribonucleic acid transcription profile.

Affinity of FVIII-specific antibodies reveals major differences between neutralizing and nonneutralizing antibodies in humans.

Recently, we reported that distinct immunoglobulin (Ig) isotypes and IgG subclasses of factor VIII (FVIII)-specific antibodies are found in different cohorts of patients with hemophilia A and in healthy individuals. Prompted by these findings, we further investigated the distinguishing properties among the different populations of FVIII-specific antibodies. We hypothesized that the affinity of antibodies would discriminate between the neutralizing and nonneutralizing antibodies found in different study cohorts. To test this idea, we established a competition-based enzyme-linked immunosorbent assay technology to assess the apparent affinities for each isotype and IgG subclass of FVIII-specific antibodies without the need for antibody purification. We present a unique data set of apparent affinities of FVIII-specific antibodies found in healthy individuals, patients with congenital hemophilia A with and without FVIII inhibitors, and patients with acquired hemophilia A. Our data indicate that FVIII-specific antibodies found in patients with FVIII inhibitors have an up to 100-fold higher apparent affinity than that of antibodies found in patients without inhibitors and in healthy individuals. High-affinity FVIII-specific antibodies could be retrospectively detected in longitudinal samples of an individual patient with FVIII inhibitors 543 days before the first positive Bethesda assay. This finding suggests that these antibodies might serve as potential biomarkers for evolving FVIII inhibitor responses.

B-cell memory against factor VIII.

The development of persistent neutralizing antibodies against factor VIII (FVIII) is the most severe complication in the treatment of congenital hemophilia A patients with FVIII replacement therapies. Recently, we presented data which indicate that neutralizing antibodies are high-affinity antibodies which are mostly of the IgG1 and IgG4 subclasses. However, there are also FVIII-specific antibodies of low to moderate affinity which are found in some patients without neutralizing antibodies and in some healthy individuals. The underlying immune mechanisms which regulate the development of these different populations of FVIII-specific antibodies are poorly understood. In this review, we discuss potential mechanisms for the development of low-affinity and high-affinity antibodies. In particular, we discuss the role of memory B cells which are essential in maintaining long-lasting antibody responses and in driving the rapid anamnestic antibody response after re-exposure to the same antigen. We also present available data on the potential role of FVIII-specific memory B cells in the maintenance of neutralizing antibodies against FVIII.

Distinct characteristics of antibody responses against factor VIII in healthy individuals and in different cohorts of hemophilia A patients.

Neutralizing antibodies against factor VIII (FVIII) remain the major complication in the replacement therapy of hemophilia A patients. To better understand the evolution of these antibodies it is important to generate comprehensive datasets which include both neutralizing and nonneutralizing antibodies, their isotypes, and IgG subclasses. We developed sensitive ELISAs to analyze FVIII-binding antibodies in different cohorts of hemophilia A patients and in healthy individuals. Our data reveal the prevalence of FVIII-binding antibodies among healthy individuals (n = 600) to be as high as 19%, with a prevalence of antibody titers > or =1:80 of 2%. The prevalence of FVIII-binding antibodies was 34% (5% for titers > or =1:80) in patients without FVIII inhibitors (n = 77), 39% (4% for titers > 1:80) in patients after successful immune tolerance induction therapy (n = 23), and 100% (n = 20, all titers > or =1:80) in patients with FVIII inhibitors. We found significant differences for IgG subclasses of FVIII-binding antibodies between the different study cohorts. IgG4 and IgG1 were the most abundant IgG subclasses in patients with FVIII inhibitors. Strikingly, IgG4 was completely absent in patients without FVIII inhibitors and in healthy subjects. These findings point toward a distinct immune regulatory pathway responsible for the development of FVIII-specific IgG4 associated with FVIII inhibitors.

A Flow-Cytometry-Based Approach to Facilitate Quantification, Size Estimation and Characterization of Sub-visible Particles in Protein Solutions.

PURPOSE: Sub-visible particles were shown to facilitate unwanted immunogenicity of protein therapeutics. To understand the root cause of this phenomenon, a comprehensive analysis of these particles is required. We aimed at establishing a flow-cytometry-based technology to analyze the amount, size distribution and nature of sub-visible particles in protein solutions. METHODS: We adjusted the settings of a BD FACS Canto II by tuning the forward scatter and the side scatter detectors and by using size calibration beads to facilitate the analysis of particles with sizes below 1 µM. We applied a combination of Bis-ANS (4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid dipotassium salt) and DCVJ (9-(2,2-dicyanovinyl)julolidine) to identify specific characteristics of sub-visible particles. RESULTS: The FACS technology allows the analysis of particles between 0.75 and 10 µm in size, requiring relatively small sample volumes. Protein containing particles can be distinguished from non-protein particles and cross-ß-sheet structures contained in protein particles can be identified. CONCLUSIONS: The FACS technology provides robust and reproducible results with respect to number, size distribution and specific characteristics of sub-visible particles between 0.75 and 10 µm in size. Our data for number and size distribution of particles is in good agreement with results obtained with the state-of-the-art technology micro-flow imaging.

PEGylated Biopharmaceuticals: Current Experience and Considerations for Nonclinical Development.

PEGylation (the covalent binding of one or more polyethylene glycol molecules to another molecule) is a technology frequently used to improve the half-life and other pharmaceutical or pharmacological properties of proteins, peptides, and aptamers. To date, 11 PEGylated biopharmaceuticals have been approved and there is indication that many more are in nonclinical or clinical development. Adverse effects seen with those in toxicology studies are mostly related to the active part of the drug molecule and not to polyethylene glycol (PEG). In 5 of the 11 approved and 10 of the 17 PEGylated biopharmaceuticals in a 2013 industry survey presented here, cellular vacuolation is histologically observed in toxicology studies in certain organs and tissues. No other effects attributed to PEG alone have been reported. Importantly, vacuolation, which occurs mainly in phagocytes, has not been linked with changes in organ function in these toxicology studies. This article was authored through collaborative efforts of industry toxicologists/nonclinical scientists to address the nonclinical safety of large PEG molecules (>10 kilo Dalton) in PEGylated biopharmaceuticals. The impact of the PEG molecule on overall nonclinical safety assessments of PEGylated biopharmaceuticals is discussed, and toxicological information from a 2013 industry survey on PEGylated biopharmaceuticals under development is summarized. Results will contribute to the database of toxicological information publicly available for PEG and PEGylated biopharmaceuticals.

CD4+ T-cell epitopes associated with antibody responses after intravenously and subcutaneously applied human FVIII in humanized hemophilic E17 HLA-DRB1*1501 mice.

Today it is generally accepted that B cells require cognate interactions with CD4(+) T cells to develop high-affinity antibodies against proteins. CD4(+) T cells recognize peptides (epitopes) presented by MHC class II molecules that are expressed on antigen-presenting cells. Structural features of both the MHC class II molecule and the peptide determine the specificity of CD4(+) T cells that can bind to the MHC class II-peptide complex. We used a new humanized hemophilic mouse model to identify FVIII peptides presented by HLA-DRB1*1501. This model carries a knockout of all murine MHC class II molecules and expresses a chimeric murine-human MHC class II complex that contains the peptide-binding sites of the human HLA-DRB1*1501. When mice were treated with human FVIII, the proportion of mice that developed antibodies depended on the application route of FVIII and the activation state of the innate immune system. We identified 8 FVIII peptide regions that contained CD4(+) T-cell epitopes presented by HLA-DRB1*1501 to CD4(+) T cells during immune responses against FVIII. CD4(+) T-cell responses after intravenous and subcutaneous application of FVIII involved the same immunodominant FVIII epitopes. Interestingly, most of the 8 peptide regions contained promiscuous epitopes that bound to several different HLA-DR proteins in in vitro binding assays.

Immunogenicity profile of rurioctocog alfa pegol in previously treated patients with severe congenital hemophilia A.

ABSTRACT: Rurioctocog alfa pegol is an extended-half-life full-length recombinant factor VIII (FVIII) bound to 20-kDa polyethylene glycol (PEG) that has been shown to be well tolerated and efficacious in the treatment and prevention of bleeding events in previously treated patients with severe hemophilia A. Here, we present a comprehensive analysis of immunogenicity data collected during 6 clinical studies of rurioctocog alfa pegol, including a total of 360 unique previously treated patients with severe hemophilia A. The analysis included treatment-emerging FVIII-neutralizing antibodies (FVIII inhibitors); preexisting and treatment-emerging antibodies binding to FVIII, PEG-FVIII, or PEG; and treatment-emerging antibodies binding to Chinese hamster ovary host cell proteins. Moreover, the potential association between the presence of these binding antibodies and adverse events (AEs) observed in patients was investigated, and the potential impact of these antibodies on the incremental recovery of rurioctocog alfa pegol in patients was analyzed. Overall, the data indicate that rurioctocog alfa pegol is not associated with any unexpected immunogenicity characteristics. Of 360 patients, 1 patient developed a transient FVIII inhibitor with a titer of 0.6 Bethesda units per mL, which was not associated with any serious AEs. Antibodies binding to FVIII, PEG-FVIII, or PEG were not detected at the time when the inhibitor was present. Moreover, 54 of 360 patients either entered the clinical studies with preexisting binding antibodies or developed these antibodies after exposure to rurioctocog alfa pegol. These antibodies were transient in most patients and did not show any causal relationship to either AEs or spontaneous bleeding episodes.

Stimulation and inhibition of FVIII-specific memory B-cell responses by CpG-B (ODN 1826), a ligand for Toll-like receptor 9.

Factor VIII (FVIII)-specific memory B cells are essential components for regulating anamnestic antibody responses against FVIII in hemophilia A with FVIII inhibitors. We asked how stimulation and inhibition of FVIII-specific memory B cells by low and high concentrations of FVIII, respectively, are affected by concurrent activation of the innate immune system. Using CD138(-) spleen cells from hemophilic mice treated with FVIII to study restimulation and differentiation of memory B cells in vitro, we tested modulating activities of agonists for Toll-like receptors (TLRs) 2, 3, 4, 5, 7, and 9. Ligands for TLR7 and 9 were most effective. They not only amplified FVIII-specific memory responses in the presence of stimulating concentrations of FVIII, but also countered inhibition in the presence of inhibitory concentrations of FVIII. Notably, CpG oligodeoxynucleotide (CpG-ODN), a ligand for TLR9, expressed biphasic effects. It amplified memory responses at low concentrations and inhibited memory responses at high concentrations, both in vitro and in vivo. Both stimulatory and inhibitory activities of CpG-ODN resulted from specific interactions with TLR9. Despite their strong immunomodulatory effects in the presence of FVIII, ligands for TLR induced negligible restimulation in the absence of FVIII in vitro and no restimulation in the absence of FVIII in vivo.

T cell-independent restimulation of FVIII-specific murine memory B cells is facilitated by dendritic cells together with toll-like receptor 7 agonist.

Memory B cells are involved in long-term maintenance of antibody-dependent immunologic disorders. Therefore, it is essential to understand how the restimulation of FVIII-specific memory B cells in hemophilia A with FVIII inhibitors is regulated. We asked whether concurrent activation of the innate immune system by an agonist for toll-like receptor (TLR) 7 is able to facilitate the differentiation of FVIII-specific memory B cells in the absence of T-cell help. TLR7 recognizes single-stranded RNA as contained in RNA viruses such as influenza, Sendai, and Coxsackie B viruses. Our results indicate that highly purified murine memory B cells do not differentiate into FVIII-specific antibody-secreting cells in the presence of FVIII and the TLR7 agonist when cultured in the absence of CD4(+) T cells. However, CD11c(+) dendritic cells facilitate the T cell-independent differentiation of FVIII-specific memory B cells but only in the presence of FVIII and the TLR7 agonist. In contrast to T cell-dependent restimulation, the antibody response after T cell-independent restimulation of FVIII-specific memory B cells is skewed toward IgG2a, an antibody subclass that is efficient in activating the complement system and in inducing Fc-receptor-mediated effector functions, both are required for effective immune responses against pathogens.

Maintenance and break of immune tolerance against human factor VIII in a new transgenic hemophilic mouse model.

Replacement of the missing factor VIII (FVIII) is the current standard of care for patients with hemophilia A. However, the short half-life of FVIII makes frequent treatment necessary. Current efforts focus on the development of longer-acting FVIII concentrates by introducing chemical and genetic modifications to the protein. Any modification of the FVIII protein, however, risks increasing its immunogenic potential to induce neutralizing antibodies (FVIII inhibitors), and this is one of the major complications in current therapy. It would be highly desirable to identify candidates with a high risk for increased immunogenicity before entering clinical development to minimize the risk of exposing patients to such altered FVIII proteins. In the present study, we describe a transgenic mouse line that expresses a human F8 cDNA. This mouse is immunologically tolerant to therapeutic doses of native human FVIII but is able to mount an antibody response when challenged with a modified FVIII protein that possesses altered immunogenic properties. In this situation, immunologic tolerance breaks down and antibodies develop that recognize both the modified and the native human FVIII. The applicability of this new model for preclinical immunogenicity assessment of new FVIII molecules and its potential use for basic research are discussed.

Development of a transgenic mouse model with immune tolerance for human coagulation factor VIIa.

PURPOSE: Human factor VIIa (FVIIa) is commonly used as bypassing therapy to treat bleeding episodes in hemophilia patients with neutralizing antibodies to factors VIII (FVIII) or IX (FIX). There is a need for a suitable animal model to assess the immunogenicity of new FVIIa products during preclinical development. The aim of this study was the design of a novel transgenic mouse model with immune tolerance to human FVIIa. METHODS: The model was generated by transgenic expression of human F7 cDNA. FVIIa-specific immune responses after treatment with human FVIIa were assessed by analyzing circulating antibodies, antibody producing plasma cells and CD4(+) T cells. RESULTS: In contrast to wild-type mice, human FVII transgenic mice did not develop antibodies when treated with human FVIIa. The immune tolerance was specific and could be broken by application of human FVIIa together with a strong stimulus of the innate immune system. Break of tolerance was associated with increased numbers of pro-inflammatory FVIIa-specific CD4(+) T cells. CONCLUSIONS: The new mouse model is suitable to study the influence of the innate immune system on maintenance and break of immune tolerance against FVIIa and could be used to assess the immunogenicity of new FVIIa products during pre-clinical development.