Recombinant Human FVIII Research Articles

The first recombinant FVIII produced in human cells – an update on its clinical development programme

The development of inhibitors and the need for frequent venous access for FVIII injection are major challenges in current haemophilia treatment. Presently available recombinant FVIII (rFVIII) products produced in hamster cell lines are associated with inhibitor formation in up to 32% of previously untreated patients. The new human cell line-derived recombinant human FVIII (Human-cl rhFVIII) protein is the first native, unmodified truly human rFVIII product produced in a human cell line without additive animal proteins. The aim of using a human cell line for the production of rFVIII is the avoidance of non-human epitopes on rFVIII, thereby potentially reducing the rate of inhibitor development, avoiding allergic reactions and allowing personalized prophylaxis with the chance of fewer infusions. Studies to date show that prophylaxis with Human-cl rhFVIII prevents 96% of bleeding events in adults with severe haemophilia A when compared to on-demand treatment. Available pharmacokinetic data with a mean half-life of 17.1 h allow personalized prophylaxis with the chance of fewer infusions. Studies in previously treated children and adults indicate that Human-cl rhFVIII is efficacious and safe in the prevention and treatment of bleeding episodes and that none of the treated patients developed inhibitors or allergic reactions thus far.

Development of lentiviral vectors for transient and stable protein overexpression in mammalian cells. A new strategy for recombinant human FVIII (rhFVIII) production

BackgroundRecombinant protein overexpression in mammalian cells constitutes a real challenge in therapeutic protein production. Following the discovery of intron functionality in gene expression, various expression vectors that include them in their sequences have been developed. In this study, the main goal was to develop new lentiviral vectors (LVs) carrying different promoter and intron-containing 5′UTR (5′ untranslated region) combinations and the design of LVs for rhFVIII production in Chinese hamster ovary (CHO) cells. ResultsBy combining the human cytomegalovirus (CMV) or the elongation factor 1α (EF-1α) promoters along with different 5′UTRs that included leader introns, between 2 and 12-fold increases were reached, when transient and stable expression of the enhanced green fluorescent protein (EGFP) and rhFVIII were analyzed. Also, new LVs provided with promoters and 5′UTRs from high expression genes, according to a gene database, were designed. Three of them were shown to be superior to the EF-1α promoter in three widely used cell lines. ConclusionIn the present work, LVs containing different promoters and 5′UTRs were designed. In transient and stable assays some of these constructs have shown higher activity compared with commercial promoters and, therefore, constitute promising candidates for therapeutic protein production.

Induction Of Tolerance To FVIII Using Nanoparticles In a Murine Model Of Hemophilia A

Anti-FVIII inhibitor development is currently considered the most serious side effect in the treatment of hemophilia A patients. In this study, we sought to address this problem by determining the efficacy of a novel tolerogenic protocol utilizing nanoparticle formulations provided by Selecta Biosciences. Thus, hemophilia A mice (FVIII KO, n = 6-8) were treated weekly for five weeks through intravenous route with 1 µg recombinant human FVIII (rFVIII), plus immune modulating agent coupled nanoparticles or control nanoparticles (coded F1 or F2 or F3). An additional control group was included, in which the mice were treated on the same schedule with high dose IVIG + rFVIII. As expected, all the mice in the control group (later revealed to be F3) developed high levels anti-FVIII IgG after the five treatments, with the average levels at 21.2 ± 4.4 µg/ml. Both the F1 and F2 nanoparticle treatments effectively prevented the anti-FVIII inhibitory antibody development throughout the course of the 5-week treatment. One week after the final treatment, the majority (5/7) of both F1 and F2 groups remained undetectable for anti-FVIII IgG antibodies. Consistent with this pattern, the anti-FVIII inhibitor titers for the F1, F2, F3 and IVIG groups were 42.3 ± 9.9, 14.3 ± 7.2, 92.9 ± 2.5, and 30.3 ± 5.6 Bethesda Units (BU)/ml, respectively after another 7 days (p < 0.01 for F1 and F2 vs. F3). To determine the duration of the FVIII-tolerance, the mice were challenged twice with rFVIII (1µg) 29 and 53 days after the final nanoparticle treatment. After the first FVIII challenge, the inhibitor titers for the F1, F2, F3 and IVIG groups were 81.3 ± 10.8, 33.1 ± 13.2, 84.1 ± 2.2 (p = 0.41 for F1 vs. F3; and p < 0.01 for F2 vs. F3), and 50.6 ± 9.7 BU/ml, respectively. Remarkably, the FVIII-tolerance in F2 group was sustained even after additional FVIII challenges ∼ 2 months later, when the effect in IVIG group was completely lost. A therapeutic protocol using mice pre-immunized with FVIII also led to substantial reductions in anti-FVIII titers. In conclusion, the concomitant Selecta F2 nanoparticle treatments not only significantly prevented the anti-FVIII inhibitory antibody development during the treatments, but also resulted in long-term FVIII-tolerance, suggesting a potential clinical application. (Supported in part by a grant-in-aid from Selecta Bioscience and by HL061883) Disclosures:Griset:Selecta Biosciences: Employment, Equity Ownership. Maldonado:Selecta Biosciences: Employment, Equity Ownership. Kishimoto:Selecta Biosciences: Employment, Equity Ownership. Scott:Selecta Biosciences: Research Funding.

Repression of Factor VIII Inhibitor Development with Apoptotic Factor VIII-Expressing Embryonic Stem Cells

Development of factor VIII (fVIII)-neutralizing antibodies, called inhibitors, is a challenging problem in the management of hemophilia A patients. We explored the possibility of pretreatment with apoptotic fVIII-expressing embryonic stem (ES) cells to prevent the development of fVIII inhibitors. Murine ES cells integrated with the human F8 gene were differentiated into embryoid bodies, dissociated to a single cell suspension, subjected to hypo-osmotic shock to induce apoptosis, and intraperitoneally injected into hemophilia A mice. Inhibitors were induced by periodic intraperitoneal injections of recombinant human fVIII (rhfVIII). In the groups in which intraperitoneal injections of rhfVIII began at 1-3 weeks after pretreatment, the titers of inhibitors were significantly lower after the third administration of rhfVIII compared with that in the control group in which apoptotic Ainv18 ES cells (without the human F8 gene) were used for pretreatment, and continued to show lower levels until the sixth administration of rhfVIII. These results suggest that pretreatment with apoptotic hfVIII-expressing ES cells might be promising for the prevention of fVIII inhibitor development in hemophilia A patients.

Reduced Antigenicity of Recombinant Ovine Factor VIII in Hemophilia A Inhibitor Patient Plasmas

AbstractAbstract 1124Hemophilia A is an X-linked recessive disorder caused by deficiencies or functional defects in coagulation factor VIII (fVIII). Approximately 20–30% of patients with severe hemophilia A develop antibodies against fVIII (inhibitors) following fVIII replacement therapy, which presents significant complication to the control of subsequent bleeding episodes. State of the art treatment options for patients with inhibitors include fVIII-bypassing agents such as recombinant factor VIIa or activated prothrombin-complex concentrate. Previously, plasma-derived porcine fVIII was a treatment option for inhibitor patients and was effective due to the reduced antigenicity of porcine fVIII toward anti-human fVIII inhibitors. However due to concerns regarding viral contamination, the plasma-derived porcine fVIII products were discontinued and no alternative fVIII products have been made available to patients with inhibitors. Presently, a recombinant porcine fVIII product (OBI-1, Inspiration Biopharmaceuticals) is being investigated in two phase 3 clinical trials for congenital and acquired hemophilia A. Rationale for the development of such a product consists of the prior success of plasma-derived porcine fVIII and the concept that the most effective and lowest risk treatment for fVIII deficiency, even in the presence of inhibitors, remains a fVIII product. Recently, a line of hemophilia A sheep was reestablished from banked frozen sperm and the ovine fVIII (ofVIII) gene, causal mutation, and protein were genetically and biochemically characterized. B-domain deleted (BDD) ovine fVIII shares 86% identity to human fVIII at the amino acid level and confers phenotypic correction, in vivo, to hemophilia A mice using a tail transaction bleeding model. Recombinant ofVIII was expressed in baby-hamster kidney cells and purified to > 95% homogeneity using a two-step ion exchange chromatography procedure. Highly purified ofVIII displays a specific activity of 18,300 units/mg, which is approximately twice that of recombinant BDD human fVIII. Furthermore, the decay of ofVIII activity following thrombin activation is slower than BDD human fVIII suggesting prolonged activity in vivo. Lastly, ofVIII demonstrates equivalent binding to human von Willebrand factor at physiological concentrations in vitro. A translational aim of the present study was to test the hypothesis that unique sequences within ofVIII confer differential antigenicity compared to human and/or porcine fVIII in congenital and acquired inhibitor patient plasmas. To address this hypothesis, the reactivity of 28 samples (22 congenital patient samples designated 1–22, and 6 acquired hemophilia A patient samples designated A1-A6) from the Emory IRB approved inhibitor bank towards recombinant BDD human, porcine, and ovine fVIII were assessed by enzyme-linked immunosorbant assay (Figure 1). When normalized to the reactivity towards human fVIII, the data revealed reduced reactivity towards ofVIII in 27 of 28 total samples. In only one patient was the reactivity towards ofVIII greater than that towards human fVIII and, in this sample, the reactivity towards porcine fVIII also was greater than 100%. Furthermore, plasma reactivity to ovine fVIII was significantly reduced compared to porcine fVIII (P = 0.025; Mann-Whitney U Test). Median values of the relative cross reactivity towards porcine and ovine fVIII were 54 and 38%, respectively. Preliminary inhibitor analysis (Bethesda assay) of three samples shown to contain titers against human fVIII of 25, 19, and 68 BU/ml, revealed undetectable inhibitor titers towards ofVIII in 2 samples, and a titer of 5 BU/ml in the third, respectively. These results suggest that additional orthologous recombinant fVIII molecules may be enabling to the treatment of patients harboring pathogenic inhibitors to human fVIII. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Development and Characterization of Recombinant Ovine Coagulation Factor VIII

Animal models of the bleeding disorder, hemophilia A, have been an integral component of the biopharmaceutical development process and have facilitated the development of recombinant coagulation factor VIII (fVIII) products capable of restoring median survival of persons with hemophilia A to that of the general population. However, there remain several limitations to recombinant fVIII as a biotherapeutic, including invasiveness of intravenous infusion, short half-life, immunogenicity, and lack of availability to the majority of the world's population. The recently described ovine model of hemophilia A is the largest and most accurate phenocopy. Affected sheep die prematurely due to bleeding-related pathogenesis and display robust adaptive humoral immunity to non-ovine fVIII. Herein, we describe the development and characterization of recombinant ovine fVIII (ofVIII) to support further the utility of the ovine hemophilia A model. Full-length and B-domain deleted (BDD) ofVIII cDNAs were generated and demonstrated to facilitate greater biosynthetic rates than their human fVIII counterparts while both BDD constructs showed greater expression rates than the same-species full-length versions. A top recombinant BDD ofVIII producing baby hamster kidney clone was identified and used to biosynthesize raw material for purification and biochemical characterization. Highly purified recombinant BDD ofVIII preparations possess a specific activity nearly 2-fold higher than recombinant BDD human fVIII and display a differential glycosylation pattern. However, binding to the carrier protein, von Willebrand factor, which is critical for stability of fVIII in circulation, is indistinguishable. Decay of thrombin-activated ofVIIIa is 2-fold slower than human fVIII indicating greater intrinsic stability. Furthermore, intravenous administration of ofVIII effectively reverses the bleeding phenotype in the murine model of hemophilia A. Recombinant ofVIII should facilitate the maintenance of the ovine hemophilia A herd and their utilization as a relevant large animal model for the research and development of novel nucleic acid and protein-based therapies for hemophilia A.

Characterisation of the post-translational modifications of a novel, human cell line-derived recombinant human factor VIII

IntroductionHost cell lines used for recombinant protein expression differ in their ability to perform post-translational modifications (PTMs). The currently available recombinant human FVIII (rhFVIII) products are produced in mammalian, non-human cell lines. For rhFVIII, glycosylation and sulfation are vital for functionality and von Willebrand factor (VWF)-binding affinity. Here we present the characterisation of the PTMs of a novel, human cell line-derived recombinant human FVIII (human-cl rhFVIII). rhFVIII expression in a human cell line avoids expression of undesirable mammalian glycoforms like Galα1-3Galβ1-GlcNAc-R (α-Gal) and N-glycolylneuraminic acid (Neu5Gc), which constitute epitopes antigenic to humans. Materials and methodsWe describe sulfation analysis, glycan profiling and characterisation using liquid chromatography-mass spectrometry and high performance anion exchange chromatography with pulsed amperometric detection. Results and conclusionsHuman-cl rhFVIII is confirmed to be sulfated and glycosylated comparable to human plasma-derived FVIII. Most importantly, human-cl rhFVIII is devoid of the antigenic Neu5Gc or α-Gal epitopes observed in Chinese Hamster Ovary- and Baby Hamster Kidney-derived rFVIII products. Both the avoidance of non-human glycan structures and the achievement of complete sulfation are proposed to lower the intrinsic immunogenicity of human-cl rhFVIII compared with current rFVIII products.

Antidote strategies to reverse anticoagulation with TB‐402, a long‐acting partial inhibitor of factor VIII

TB-402 is a partially inhibiting antibody of factor VIII that is under development as a long-acting anticoagulant. The reversibility of FVIII inhibition by TB-402 was evaluated in vitro after spiking with recombinant human FVIII (rhFVIII), human plasma-derived FVIII (hpdFVIII), recombinant activated human FVII (rhFVIIa), FVIII inhibitor bypassing activity (FEIBA), and prothrombin complex concentrate (PCC). Twelve subjects were randomized to placebo or 35 or 70 IU kg(-1) rhFVIII 48 h after a single dose of 620 μg kg(-1) TB-402. TB-402 concentrations, FVIII activity (FVIII:C), activated partial thromboplastin time (APTT) and thrombin generation were measured over a period of 8 weeks. In spiked samples, TB-402 inhibited FVIII:C by 30%, prolonged APTT by 4.5 s, and reduced the peak height in the thrombin generation assay to 56% ± 13% of the control value. In the presence of 10 μg mL(-1) TB-402, rhFVIII restored FVIII:C and APTT to the values obtained in the absence of TB-402. The inhibitory effect of TB-402 on thrombin generation was entirely reversed by rhFVIII, hpdFVIII, rhFVIIa, FEIBA, and PCC. In men, the mean half-life (t(1/2) ) of TB-402 was 14.2 days. TB-402 lowered the endogenous thrombin potential by 23% for ~ 35 days. Infusion of 35 IU kg(-1) rhFVIII had a marginal effect, whereas 70 IU kg(-1) rhFVIII restored FVIII:C, reduced APTT back to baseline for 9 h, and restored thrombin generation for ~ 3 h. TB-402 resulted in a stable long-term anticoagulant effect. rhFVIII and other procoagulants counteracted the effect of TB-402 temporarily, and may be effective antidotes for future clinical practice.

Targeting FVIII Expression to Platelets Induces Immune Tolerance in Hemophilia A Mice with or without Pre-Existing Anti-FVIII Immunity,

Abstract 4170Our previous studies have shown that targeting FVIII expression to platelets (2bF8) can correct murine hemophilia A phenotype even in the presence of inhibitory antibodies. In the present study, we wanted to explore 1) whether platelets containing FVIII can act as an immunogen; and 2) whether platelet-derived FVIII can induce immune tolerance in a hemophilia A mouse model. To investigate whether platelets containing FVIII can act as an immunogen in hemophilia A mice, we infused transgenic mouse platelets with a level of platelet-FVIII of 6 mU/108 platelets to naïve FVIIInull mice weekly for 8 weeks. These platelets were between 30 to 50% of total platelets upon infusion and the levels of platelet-FVIII in the infused animals were 0.11 ± 0.01 mU/108 platelets (n = 6) one week after infusion. No anti-FVIII inhibitory antibodies were detected in the infused mice during the study course. All animals developed inhibitors following further challenged with recombinant human FVIII (rhFVIII) at a dose of 50 U/kg by intravenous injection weekly for 4 weeks, indicating that infusion of platelets containing FVIII does not trigger immune response in hemophilia A mice.To explore whether platelet-derived FVIII will act as an immunogen in the presence of primed spleen cells (from mice already producing inhibitory antibody), we co-transplanted splenocytes from highly immunized FVIIInull mice and bone marrow (BM) cells from 2bF8 transgenic mice into 400 cGy sub-lethal irradiated FVIIInull recipients. We monitored the levels of inhibitory antibodies in recipients for up to 8 weeks and found that inhibitor titers declined with time after transplantation. We then challenged co-transplantation recipients with rhFVIII and found that inhibitor titers in the control group co-transplantat of FVIIInull BM cells increased 103.55 ± 64.83 fold (n = 4), which was significantly more than the group receiving 2bF8 transgenic BM cells (14.34 ± 18.48, n = 5) (P <.05). The inhibitor titers decreased to undetectable in 40% of 2bF8 transgenic BM cells co-transplantation recipients even after rhFVIII challenge, indicating immune tolerance was induced in these recipients.To further explore the immune response in the lentivirus-mediated platelet-derived FVIII gene therapy of hemophilia A mice, we transduced hematopoietic stem cells from pre-immunized FVIIInull mice with 2bF8 lentivirus (LV) followed by syngeneic transplantation into pre-immunized lethally irradiated FVIIInull recipients and monitored the levels of inhibitor titers in recipients. After full BM reconstitution, platelet-FVIII expression was sustained (1.56 ± 0.56 mU/108platelets, n = 10), but inhibitor titers declined with time, indicating that platelet-derived FVIII does not provoke a memory response in FVIIInullmice that had previously mounted an immune response to rhFVIII. The t1/2 of inhibitor disappearance in 2bF8 LV-transduced recipients (33.65 ± 11.12 days, n = 10) was significantly shorter than in untransduced controls (66.43 ± 22.24 days, n = 4) (P <.01). We also transplanted 2bF8 LV-transduced pre-immunized HSCs into 660 cGy sub-lethal irradiated naïve FVIIInull mice. After BM reconstituted, recipients were assessed by platelet lysate FVIII:C assay and tail clip survival test to confirm the success of genetic therapy. Animals were then challenged with rhFVIII. Only 2 of 7 2bF8 LV-transduced recipients developed inhibitory antibodies (55 and 87 BU/ml), while all untransduced control developed high titer of inhibitors (735.50 ± 94.65 BU/ml, n = 4).In conclusion, our results demonstrate that 1) platelets containing FVIII are not immunogenic in hemophilia A mice; and 2) platelet-derived FVIII may induce immune tolerance in hemophilia A mice with or without pre-existing inhibitory antibodies. This tolerance induction would add an additional significant benefit to patients with platelet-derived FVIII gene therapy strategy because protein infusion could be administered in some special situations (e.g. surgery in which a greater levels of FVIII may be required) with minimized risk of inhibitor development. Disclosures:No relevant conflicts of interest to declare.

Co-Administration Therapy Modulates Immunity to FVIII In Hemophilia A Mice

Abstract 1219Our long-term objective is the development of novel therapies for the safe and effective treatment of hemophilia. In particular a safe and economic prophylactic modality to prevent inhibitors development is therefore highly desirable. To this end we have explored strategies to modulate inhibitor development based on the co-administration of FVIII with other proteins, specifically albumin and IVIG. We hypothesise that such co-administration may modulate immunity to FVIII and result in a safe and cost-effective therapy. Based on some published evidence that FIX and vWF may have a modulatory effect on FVIII, we reasoned that perhaps albumin might also play an immunomodulatory effect on FVIII. We have developed a protocol for infusing recombinant FVIII in hemophilia A mice modelled on the current therapy of hemophiliacs, and in response to this protocol hemophilia mice develop high titre of inhibitors. Thus, this model can be used to assess potential immununomodulatory strategies.Groups of hemophilia A mice (n=5) were infused weekly IV with 2IU rFVIII for a total of 4 weeks. Three of these groups of mice were also administered human albumin (either 25mg IV, 25mg IP, or 500mg IV) at the same time as FVIII. Mice were bled before the treatment and weekly for the 4 weeks, and total antibodies to FVIII were detected by ELISA. Importantly, while mice receiving FVIII had high antibody titer and were in good health, there was significant mortality in the mice treated with FVIII and albumin. Interestingly, the surviving mice had a very low antibody titre, statistically significant from mice receiving only rFVIII.Given the mortality, another experiment was designed to test the effect of reduced doses of albumin. Groups of hemophilia A mice (n=5) were infused IV with a weekly injection of 2IU rFVIII for a total of 4 weeks supplemented with either 25μg, 50μg or 100μg of human albumin. Mice were bled at weekly intervals until the end of the experiment (day 28), and the titre of anti-FVIIII antibodies was determined. Most All mice survived the entire treatment and were found in good health. Further, the FVIII antibody titre in the group that received albumin was detectable, but significantly reduced with respect to the mice that received only FVIII. Marked variability among individual mice was observed.A group of hemophilia A mice (n=5) received weekly IP injections of 2IU rFVIII. Another group of mice received the same regimen of FVIII in addition to human IVIG (1g/kg). A third group of hemophilia mice received 2IU rFVIII and a higher dose of human IVIG (2g/kg). These doses mimic the established human IVIG regimen. Mice were bled before treatment and at weekly intervals thereafter. The titre of anti-FVIII antibodies in mice was determined by ELISA. By day 21, the antibody titre in mice injected with FVIII was high, as expected. In contrast, the titre in mice receiving FVIII and IVIG was low, and comparable to that of all the mice prior to the treatment (na•ve mice), strongly suggesting an immune modulatory effect by IVIG (Figure 1). Our data supports the limited use of IVIG to treat inhibitors. [Display omitted] Our findings suggest that co-administration of FVIII with albumin and IVIG can modulate the humoral immune response to recombinant human FVIII in hemophilia mice. Further, the observed effect appears to be dependent on the dose of albumin co-administered. It is hypothesized that the observed modulation is due to antigen competition, as previously speculated. Importantly, while IVIG was well tolerated by mice, very serious adverse effects were observed in mice treated with high doses of albumin. Interestingly, even the high dose used represents a small fraction of the physiological levels of albumin in human plasma, which is 3–5 g/dL. IVIG, in contrast, is a known modulator of the immune response in humans, and the study of this modulatory effect in a hemophilia mouse may lead to novel therapeutic strategies, relevant for the management of hemophilia. Disclosures:No relevant conflicts of interest to declare.

Induction of tolerance to factor VIII by transient co‐administration with rapamycin

Formation of inhibitory antibodies is a frequent and serious complication of factor (F) VIII replacement therapy for the X-linked bleeding disorder hemophilia A. Similarly, hemophilia A mice develop high-titer inhibitors to recombinant human FVIII after a few intravenous injections. Using the murine model, the study sought to develop a short regimen capable of inducing tolerance to FVIII. A 1-month immunomodulatory protocol, consisting of FVIII administration combined with oral delivery of rapamycin, was developed. The protocol effectively prevented formation of inhibitors to FVIII upon subsequent intravenous treatment (weekly for 3.5 months). Control mice formed high-titer inhibitors and had CD4(+) T effector cell responses characterized by expression of IL-2, IL-4 and IL-6. Tolerized mice instead had a CD4(+)CD25(+)FoxP3(+) T cell response to FVIII that suppressed antibody formation upon adoptive transfer, indicating a shift from Th2 to Treg if FVIII antigen was introduced to T cells during inhibition with rapamycin. CD4(+) T cells from tolerized mice also expressed TGF-β1 and CTLA4, but not IL-10. The presence of FVIII antigen during the time of rapamycin administration was required for specific tolerance induction. The study shows that a prophylactic immune tolerance protocol for FVIII can be developed using rapamycin, a drug that is already widely in clinical application. Immune suppression with rapamycin was mild and highly transient, as the mice regained immune competence within a few weeks.

Enhanced Biosynthesis of Coagulation Factor VIII through Diminished Engagement of the Unfolded Protein Response

Human and porcine coagulation factor VIII (fVIII) display a biosynthetic efficiency differential that is being exploited for the development of new protein and gene transfer-based therapies for hemophilia A. The cellular and/or molecular mechanism(s) responsible for this phenomenon have yet to be uncovered, although it has been temporally localized to post-translational biosynthetic steps. The unfolded protein response (UPR) is a cellular adaptation to structurally distinct (e.g. misfolded) or excess protein in the endoplasmic reticulum and is known to be induced by heterologous expression of recombinant human fVIII. Therefore, it is plausible that the biosynthetic differential between human and porcine fVIII results from differential UPR activation. In the current study, UPR induction was examined in the context of ongoing fVIII expression. UPR activation was greater during human fVIII expression when compared with porcine fVIII expression as determined by ER response element (ERSE)-luciferase reporter activity, X-box-binding protein 1 (XBP1) splicing, and immunoglobulin-binding protein (BiP) up-regulation. Immunofluorescence microscopy of fVIII expressing cells revealed that human fVIII was notably absent in the Golgi apparatus, confirming that endoplasmic reticulum to Golgi transport is rate-limiting. In contrast, a significant proportion of porcine fVIII was localized to the Golgi indicating efficient transit through the secretory pathway. Overexpression of BiP, an integral UPR protein, reduced the secretion of human fVIII by 50%, but had no effect on porcine fVIII biosynthesis. In contrast, expression of BiP shRNA increased human fVIII expression levels. The current data support the model of differential engagement of UPR by human and porcine fVIII as a non-traditional mechanism for regulation of gene product biosynthesis.

Differential Engagement of the Unfolded Protein Response by Human and Porcine Factor VIII

AbstractAbstract 2224Despite 83% amino acid sequence identity, human factor VIII (fVIII) and porcine fVIII display a striking species-specific expression differential (10 – 100- fold) that may be relevant to each species' maintenance of hemostasis. This expression differential has been exploited for the development of improved recombinant fVIII therapeutics and gene-transfer-based therapies (Doeringet al., JBC 2002, JBC 2004; Gangadharanet al., Blood 2006; Doeringet al., Mol. Ther. 2007; Doeringet al., Mol. Ther. 2009; Doorisset al., Hum. Gene Ther. 2009). The exact biochemical or cellular mechanisms responsible for this phenomenon has yet to be uncovered. However, the expression differential has been temporally and spatially localized to post-translational steps that occur in the endoplasmic reticulum (ER)/golgi apparatus (Doeringet al., JBC 2004). The unfolded protein response (UPR) is a coordinated cellular mechanism designed to regulate the build-up and flow of proteins in the ER and is sometimes referred to as the “quality control pathway”. Early studies of recombinant fVIII production in heterologous cell culture systems uncovered that human fVIII biosynthesis induces up-regulation of the UPR through interaction with immunoglobulin binding protein (BiP)/glucose regulated protein 78 (GRP78). These studies prompted the speculation that human fVIII may not undergo correct secondary and tertiary folding efficiently or, alternatively, may display thermodynamic instability subsequent to initial folding. Either event could serve to induce UPR activity and affect overall fVIII biosynthesis. In the current study, we hypothesized that the differential in biosynthesis between recombinant human and porcine fVIII results from differential engagement of the UPR pathway. To test this hypothesis, we studied the UPR response during expression of recombinant human and porcine fVIII in heterologous baby hamster kidney cells. In experiments utilizing a ER response element (ERSE)-luciferase reporter construct, human fVIII expression was shown to increase luciferase activity 2-fold over that observed with porcine fVIII despite the secretion of 10-fold more porcine fVIII based on activity measurement of the conditioned medium. Human fVIII expression also induced X-box protein 1 (XBP-1) splicing, another marker of UPR activation, to a greater extent than did porcine fVIII expression (2-fold versus 1.1-fold over naïve BHK cells, respectively). Immunofluorescence microscopy of fVIII expressing cells revealed that human fVIII almost exclusively was localized to the ER and displayed significant co-localization with BiP, a resident ER luminal protein. In contrast, a significant proportion of porcine fVIII antigen was localized to the Golgi apparatus and co-localized with the lectin, wheat germ agglutinin (r2 = 0.8 versus 0.6 for human fVIII), suggesting more efficient transit through the secretory pathway. Perturbation of the intracellular BiP levels by transduction of fVIII expressing BHK cells with recombinant lentivector encoding BiP revealed that, under normal conditions, BiP levels may actually be limiting in terms of the interaction with human fVIII. Over-expression of BiP (6.5-fold increased BiP mRNA levels) reduced the secretion of human fVIII by 50%, whereas the same BiP over-expression had no affect on porcine fVIII biosynthesis. In neither case were fVIII mRNA levels negatively affected by BiP over-expression. In contrast, transduction of fVIII expressing BHK cells with lentivector encoding shRNA targeting BiP led to 2-fold improvement in human fVIII expression levels. Again, these data support a role of BiP in the impediment of human fVIII biosynthesis that is not equivalent for porcine fVIII. Therefore, differential engagement of the UPR by human and porcine fVIII was observed in the current study, thus supporting a dominant role of protein folding/stability characteristics in the expression differential present among porcine and human fVIII. This study also demonstrates that the investigation of orthologous protein biosynthesis can yield important insights into the regulation of gene product levels through non-traditional mechanisms. Disclosures:No relevant conflicts of interest to declare.

BAY 94–9027, a PEGylated Recombinant Human FVIII, Shows Less Immunogenicity Compared to Un-PEGylated Recombinant FVIII

AbstractAbstract 2214 Background:BAY 94–9027, a recombinant FVIII candidate containing a single, large, branched polyethylene glycol (PEG) molecule conjugated to a specific amino acid, is intended for replacement therapy in hemophilia A. It has been demonstrated to have extended efficacy due to prolonged half life compared to un-PEGylated FVIII in preclinical models(Mei et al. Blood, 2010 118(2) 270–279). This may allow less frequent treatment than with current FVIII products. Methods:Binding (bAB) and activity neutralizing antibodies (nAB) were measured in 3 animal models: hemophilia A mice (FVIII deficient), normal rats and normal rabbits, both with a normal coagulation system. Male hemophilia A mice received once weekly intravenous (IV) injections of BAY 94–9027 for 5 weeks. An un-PEGylated rFVIII molecule (rFVIII) was injected as comparator at the same frequency to give comparable exposure by AUC or dose. Normal rats and rabbits were dosed IV every other day for 2 weeks and blood samples were analyzed for antibodies during treatment and after the end of treatment. Analysis of bABs was based on an ELISA assay. The analysis of nABs was based on a modified FVIII Chromogenic assay (Coatest SP FVIII, Dia Pharma) assay. nAB titers were defined as 50% inhibition of 1 IU/mL rFVIII (in accordance to the definition of FVIII Bethesda units). Results:Previous nonclinical experiments show that animals are likely to have a much higher frequency of anti-FVIII antibody formation than is seen in humans due to the foreignness of this human protein in animals. As expected, antibodies to BAY 94–9027 or rFVIII developed in hemophilia A mice, rats and rabbits since the human protein acts as antigen. In the day 21 and day 36 mouse samples, bABs and nABs against BAY 94–9027 and rFVIII were detected in a time- and dose-dependent manner. By day 21 (after 3 administrations), mice treated with rFVIII showed statistically higher mean titers and more mice had measurable antibodies compared to animals treated with BAY 94–9027 at the same dose. By day 36 (after 5 administrations), animals treated with rFVIII showed statistically higher mean titers than those treated with BAY 94–9027 when comparing both, doses or overall exposure (AUC). At the end of the study, 17/36 mice (47%) treated with BAY 94–9027 had bAB titers, of which 8 (47% of animals with binding antibodies or 22% of all mice treated with BAY 94–9027) showed neutralizing (inhibitory) potential. Whereas, 20/24 (83%) mice treated with rFVIII (un-PEGylated comparator) had detectable bAB titer, of which 18 (90% of animals with binding antibodies or 75% of all animals treated with rFVIII) showed neutralizing potential. In normal rats and rabbits anti-drug bAB and nABs were assessed on days 7, 9 and 15 during treatment and twice after the end of the 2 week treatment. Results confirmed the findings in Hemophilia A mice that, generally more animals responded with bAB and nABs to rFVIII than with BAY 94–9027, the PEGylated protein. Conclusions:The results indicate that BAY 94–9027, which has the same acute efficacy and prolonged duration of protection from bleeding, as seen in hemophilia A mouse efficacy studies (Mei et al. Blood, 2010 118(2) 270–279), was significantly less immunogenic in hemophilia A mice, normal rats and normal rabbits when compared to un-PEGylated rFVIII. This confirms findings with other PEGylated proteins, which indicate that specially branched PEGs may shield antigenic epitopes on the protein surface and can make it less immunogenic (BN Novicov et al. J Control Release, 2010. doi:10.1016/j.jconrel.2010. 06.003). Clinical studies need to assess if these findings can be confirmed in humans. Disclosures:Ivens: Bayer HealthCare Pharmaceuticals: Employment, Equity Ownership. Zierz: Bayer Schering Pharma: Employment, Equity Ownership. Haaning: Bayer HealthCare Pharmaceuticals: Employment, Equity Ownership. McDonald: Bayer HealthCare Pharmaceuticals: Employment, Equity Ownership.

Functional aspects of factor VIII expression after transplantation of genetically‐modified hematopoietic stem cells for hemophilia A

Major complications with respect to the development of gene therapy treatments for hemophilia A include low factor VIII (fVIII) expression and humoral immune responses resulting in inhibitory anti-fVIII antibodies. We previously achieved sustained curative fVIII activity levels in hemophilia A mice after nonmyeloablative transplantation of genetically-modified hematopoietic stem cells (HSCs) encoding a B-domain deleted porcine fVIII (BDDpfVIII) transgene with no evidence of an immune response. Mouse HSCs were transduced using MSCV-based recombinant virus encoding BDDpfVIII and transplanted into hemophilia A mice. Transplanted mice were followed for donor cell engraftment, fVIII expression and activity, and generation of anti-fVIII immune response. We now show that: (i) the protein expressed by hematopoietic cells has a specific activity similar to that of purified protein; (ii) BDDpfVIII expressed from hematopoietic cells effectively induces thrombus formation, which is shown using a new method of in vivo analysis of fVIII function; (iii) naïve and pre-immunized mice receiving HSC gene therapy are nonresponsive to challenges with recombinant human fVIII; (iv) nonresponsiveness is not broken after stringent challenges with BDDpfVIII; and (v) T cells from these mice are unresponsive to BDDpfVIII presentation. Furthermore, stem cells isolated from donors with high titer anti-human fVIII antibodies show no defects in donor cell engraftment or the ability to express BDDpfVIII. These results demonstrate that HSC gene therapy can be an effective alternative treatment for individuals with hemophilia A and may benefit patients by inducing immunological nonresponsiveness to fVIII replacement products.

Cooperative Interaction Between Classical and Non-Classical Factor VIII C2 Domain Antibody Epitopes.

Abstract 219The immune response to factor VIII (fVIII) currently is the most significant complication in the management of patients with hemophilia A. In addition, antibodies to fVIII can develop in non-hemophiliacs, producing acquired hemophilia A, which frequently produces life- or limb-threatening bleeding. In either congenital or acquired hemophilia A, the majority of inhibitory antibodies are directed at either the 40-kDa A2 or the 15-kDa C2 domains of fVIII. Classical anti-C2 antibodies inhibit the binding of fVIII and activated fVIII to phospholipid membranes. These antibodies bind to hydrophobic feet in the C2 domain that include loops containing M2199-F2000 and L2251-L2252. Non-classical anti-C2 antibodies inhibit the activation of fVIII by thrombin and factor Xa. They bind to the beta sheet that comprises the front face of the C2 domain that is remote from the phospholipid binding region. In the Bethesda assay, non-classical anti-C2 antibodies inhibit fVIII incompletely at saturating concentrations and thus are type II inhibitors. However, they have inhibitory titers on an equimolar basis that are usually at least 10-fold higher than classical anti-C2 antibodies. von Willebrand factor (VWF) competes with classical but not non-classical anti-C2 bodies for binding to fVIII. We recently found that the binding of classical anti-C2 antibodies to fVIII in a direct ELISA was increased in the presence non-classical anti-C2 antibodies, suggesting the presence of a cooperative interaction between fVIII antibody epitopes. In the present study, we characterized the interaction of highly-purified recombinant full-length human fVIII with a classical anti-C2 antibody, ESH4, in the presence and absence of a non-classical anti-C2 antibody, G99, by surface plasmon resonance spectroscopy. The ligand, ESH4, was covalently attached to a Biacore CM5 biosensor chip and the kinetics of binding of the analyte, fVIII, at concentrations of 10, 20 and 30 nM were measured at 25 °C. When present, G99 (60 nM) was pre-mixed with fVIII for 30 min before the start of the kinetic experiment. In both the presence and absence of G99, global nonlinear least-squares analysis indicated that the binding of fVIII to ESH4 was first-order in both the adsorption and desorption phases, consistent with a 1:1 reversible bimolecular interaction (Fig. 1). Both the association and dissociation rates were slower in the presence of G99. Estimated phenomenological association and dissociation rate constants were 0.5 × 105 M−1 sec−1 and 1.5 × 10−4 sec−1 and 1.7 × 105 M−1 sec−1 and 4.4 × 10−4 sec−1 in the presence and absence of G99, respectively. The clinical implication of these results is that infusion of recombinant fVIII (which does not contain VWF) or plasma-derived fVIII (which is bound to VWF) may result in complex kinetic interactions that are significantly influenced by cooperative interactions between anti-C2 antibodies and competitive interactions involving VWF. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Directed Engineering of a High-expression Chimeric Transgene as a Strategy for Gene Therapy of Hemophilia A

Human coagulation factor VIII (fVIII) is inefficiently biosynthesized in vitro and has proven difficult to express at therapeutic levels using available clinical gene-transfer technologies. Recently, we showed that a porcine and certain hybrid human/porcine fVIII transgenes demonstrate up to 100-fold greater expression than human fVIII. In this study, we extend these results to describe the use of a humanized, high-expression, hybrid human/porcine fVIII transgene that is 89% identical to human fVIII and was delivered by lentiviral vectors (LVs) to hematopoietic stem cells for gene therapy of hemophilia A. Recombinant human immunodeficiency virus-based vectors encoding the fVIII chimera efficiently transduced human embryonic kidney (HEK)-293T cells. Cells transduced with hybrid human/porcine fVIII encoding vectors expressed fVIII at levels 6- to 100-fold greater than cells transduced with vectors encoding human fVIII. Transplantation of transduced hematopoietic stem and progenitor cells into hemophilia A mice resulted in long-term fVIII expression at therapeutic levels despite <5% genetically modified blood mononuclear cells. Furthermore, the simian immunodeficiency virus (SIV) -derived vector effectively transduced the human hematopoietic cell lines K562, EU1, U.937, and Jurkat as well as the nonhematopoietic cell lines, HEK-293T and HeLa. All cell lines expressed hybrid human/porcine fVIII, albeit at varying levels with the K562 cells expressing the highest level of the hematopoietic cell lines. From these studies, we conclude that humanized high-expression hybrid fVIII transgenes can be utilized in gene therapy applications for hemophilia A to significantly increase fVIII expression levels compared to what has been previously achieved.

A review of current literature on second-generation, sucrose-formulated, full-length recombinant factor VIII

Recombinant factor VIII formulated with sucrose (rFVIII-FS) is a full-length recombinant human coagulation FVIII product which is formulated without the addition of human serum albumin. It is approved for the treatment and prophylaxis of bleeding events in patients with hemophilia A in various countries including the U.S. and the E.U. In substitution therapy, rFVIII-FS is well tolerated by patients who lack FVIII, including patients undergoing major surgery. To date there are no head-to-head studies evaluating the therapeutic profile of rFVIII-FS in comparison to other octocog or morocog alfa products. However, available data of noncomparative trials, with a favorable safety profile, indicate that rFVIII-FS is a viable alternative to other FVIII products for the treatment and prophylaxis of bleeding episodes in patients with hemophilia A.

Unraveling the Danger Signal Necessary for the Immune Response to Therapeutic Factor VIII.

It is well established that mice which do not produce endogenous factor VIII (fVIII−/−) can manifest a robust immune response to exogenous fVIII treatments. They form B-cell and T-cell responses even when they encounter fVIII through traditionally tolerogenic routes (e.g., intravenous or intraperitoneal). In the fVIII−/− mouse, repeated administration of recombinant human fVIII has emerged as a useful model for studying the physiologic response in hemophilic patients iatrogenically immunized to therapeutic factor VIII treatments. While environmental factors likely offer some co-stimulatory signals, nonetheless, the ability to respond effectively in the absence of extrinsic adjuvant begs the questions of what is the “danger signal” required for immune responsiveness to fVIII? We have previously shown that when factor VIII is heat inactivated (56°, 30′), it completely losses function and much of its immunogenicity (Skupsky and Scott, Blood 110: 2685Abstract, 2007). Heated fVIII lacks several of its B-cell epitopes (we did not find a subsequent response to neo-epitopes), but retains its T-cell epitopes. We concluded that fVIII's immunogenicity is inherently tied to its function. To explore this topic further, we have immunized hemophilic mice with rfVIII and compared the response to mice treated with both rfVIII and Hirudin. Hirudin is the reactive agent found in medicinal leech saliva and its anti-coagulant activity is based on its ability to inhibit thrombin. We found that T cell responses to rfVIII in mice protected with Hirudin are significantly reduced (p<0.05) and the anti-fVIII antibody concentration has decreased by 25%. As a control, we injected a third group of mice i.v. with an equivalent amount of another foreign protein, ovalbumin (OVA) in PBS. As expected, the mice did not respond to this historically tolerogenic treatment. Interestingly, when mice were injected simultaneously with rfVIII and OVA, they did form a humoral response to both the fVIII (200 μg/ml) and the OVA (30μg/ml). This suggests that fVIII may have adjuvant properties remaining to be discovered. Overall, these data suggest that the activation of thrombin provides co-stimulatory signals necessary for the immune response. Activated thrombin does this directly or indirectly through the activation of other blood components, including platelets.

Passive Transfer of Polyethylene Glycol to Liposomal-Recombinant Human FVIII Enhances its Efficacy in a Murine Model for Hemophilia A

The replacement therapy using recombinant human FVIII (rFVIII) is the first line of therapy for hemophilia A. Approximately 15-30% of the patients develop inhibitory antibodies. Recently, we reported that liposomes composed of phosphatidylserine (PS) could reduce the immunogenicity of rFVIII. However, PS containing liposomal-rFVIII is likely to reduce the systemic exposure and efficacy of FVIII due to rapid uptake of the PS containing liposomes by the reticuloendothelial system (RES). Here, we investigated whether phosphatidylserine (PS) liposomes containing Polyethylene glycol (PEG) (PEGylated), could reduce the immunogenicity of rFVIII and reverse the reduction in systemic exposure of rFVIII. Animals given PEGylated liposomal-rFVIII had lower total and inhibitory anti-rFVIII antibody titers, compared to animals treated with rFVIII alone. The mean stimulation index of CD4+ T-cells from animals given PEGylated liposomal-rFVIII also was lower than for animals that were given rFVIII alone. Pharmacokinetic studies following intravenous dosing indicated that the systemic exposure (area under the activity curve, AUAC(0-24h)) of PEGylated liposomal-rFVIII was approximately 59 IU/mL x h and significantly higher than that of non-PEGylated liposomal-rFVIII (AUAC(0-24h) approximately 36 IU/mL x h). Based on these studies, we speculate that PEGylated PS-containing liposomal rFVIII may improve efficacy of rFVIII.