Evaluation of Recombinant Fixfc-Xten Bleeding Efficacy in Hemophilia-B Mouse Models

Introduction: Prophylactic treatment for hemophilia has demonstrated clear benefit over on demand treatment in hemophilic patients. Alprolix (rFIXFc) is the first of a new generation of long acting clotting factors we made to reduce the frequency of intravenous infusions (Powell, NEJM, 2013). Nevertheless, intravenous injections can be particularly challenging for young patients and patients with limited venous access and are time consuming. We are developing a novel rFIXFc fusion protein, using XTEN recombinant technology, which is suitable for prophylactic subcutaneous dosing in hemophilia B. In the current studies we explore the in vivo bleeding efficacy of subcutaneously dosed rFIXFc-XTEN in hemophilia B mouse model. Methods: recombinant rFIXFc-XTEN was produced in HEK293 cells and affinity purified. The subcutaneous PK profile of rFIXFc-XTEN was compared to that of intravenously dosed rFIXFc in hemophilia B mice. The prolonged and acute bleeding efficacies were evaluated in HemB mouse tail-vein transection (TVT) and tail-clip bleeding models. Results and conclusions: Subcutaneous dosing of rFIXFc-XTEN shows a tmax around 20 hours post dosing in mice, and improved plasma activity levels compared to similar (IU/kg) intravenously dosed rFIX or rFIXFc. Using the TVT bleeding model in HemB mice we show that at 72 hours post dosing, subcutaneously dosed rFIXFc-XTEN has improved in vivo efficacy compared to intravenously dosed rFIXFc at all tested doses. Similarly, acute efficacy testing in the HemB mouse tail-clip bleeding model showed improved efficacy of intravenously dosed rFIXFc-XTEN compared to rFIXFc. These data support the potential of once weekly or less frequent subcutaneous prophylactic dosing of rFIXFc-XTEN in humans. Disclosures Liu: biogen: Employment, Equity Ownership. van der Flier:Biogen: Employment, Equity Ownership. Ismail:Biogen: Employment, Equity Ownership. Simpson:Biogen: Employment, Equity Ownership. Kurt:Biogen: Employment, Equity Ownership. Schellenberger:Amunix: Employment, Equity Ownership. Loh:Biogen: Employment, Equity Ownership, Other: share holder. Peters:Biogen: Employment, Equity Ownership, Other: share holder.

Prophylactic efficacy of BeneFIX vs Alprolix in hemophilia B mice

Key Points Because extravascular FIX is physiologically important, FIXs circulating levels do not independently predict hemostatic potential. A saphenous vein hemophilia B mouse model shows that 7 days postinfusion FIX-Fc and FIX provide equal hemostatic protection.

Hemophilia B Mouse Lines Doubly Humanized for Human F9 and MHC Class II Genes Advance Specificity of Inhibitor Study

Abstract 200 The development of neutralizing alloantibodies (inhibitors) against factor IX (FIX) in humans with hemophilia B is a potentially devastating complication; clinical investigation and evidence-based intervention for FIX inhibitors is complicated by their rarity. Current hemophilia B mouse models are not ideal for investigating the potential immunogenicity of hemophilia B therapies due to differences in mouse and human MHC class II structure and FIX epitope recognition. This recognition depends on CD4+ T cells and their direct interaction with surface-expressed MHC class II molecules complexed with antigen. We have developed a series of mouse lines that do not express mouse FIX or mouse MHC II, but instead express human FIX genes and humanized MHC II. The potential immunogenicity of human FIX and specific hFIX epitopes for CD4+ T cell recognition have been characterized in each of these “double humanized” (2H) mouse lines. Our derivation of the 2H mouse lines, all having first been bred through a complete knockout of mouse MHCII (mMHC II−/−), resulted in four lines of human HLA-DRB1*1501 mice. These four lines include 1) mouse FIX−/− (complete deletion; Cross-reacting material negative (CRM-)); 2) human R29X (expresses human FIX gene with early nonsense mutation; CRM-); 3) human R333Q (expresses human factor IX defective circulating protein with missense mutation; CRM+), and 4) human WT FIX (WT2 expresses hemostatic human FIX; CRM+). All lines were first shown to have similar numbers of MHCII and CD4+ expressing T cells in peripheral blood based on flow cytometry when compared with conventional (mouse MHC H2b) hemophilia B mice. To determine whether (hu-) mice have the same incidence and degree of immune response to hFIX compared to conventional hemophilia B mice, we immunized subcutaneously with hFIX emulsified in CFA. Importantly, control mMHC II−/− mice did not develop anti-FIX antibodies. Both strains of CRM- 2H hu-mice developed inhibitors and anti-hFIX IgG antibodies with similar intensity. CD4+-enriched splenocytes proliferated in vitro in response to hFIX challenge and secreted high amount of Th1-derived IFN-γ and moderate Th2 (IL-4 and IL-10) cytokines. However, when compared to the inhibitor-prone strains, conventional (mouse MHC H2b) and hu-DRB1*1501 CRM+ mice (both R333Q and WT2) tolerated hFIX, had larger total Treg populations, increased TGF-β1 cytokine, and demonstrated regulatory apoptosis of CD4+ Teffector cells. Additional lymphocyte response studies mapped four peptides derived from FIX catalytic domain as responsible for anti-FIX antibodies in the CRM- lines. (CD4+ T cells from CRM+ animals (hu-R333Q or hu-WT2 mice) did not proliferate to these four or any other hFIX peptides). The peptides identified in 2H hu- mice differed from immunostimulatory peptides identified in reported epitope mapping studies using FIX−/− mice having mouse MHC H2b, H2d, and H2kbackgrounds. These differences may reflect the fundamental structural and epitope recognition differences between murine and human MHC class II complexes. Finally, we have examined systemic and muscle-directed AAV-mediated gene therapy expressing either WT hFIX or a hFIX with a single amino acid gain of function variant (FIX Padua, R338LFIX). We have confirmed that expression of this single amino acid change does not increase inhibitor risk, as modeled in this double humanized hemophilia B model. This observation appears consistent with the extreme rarity of clinical inhibitor development in patients having underlying FIX missense mutations. In summary, the series of double humanized hemophilia B mouse models have hFIX recognition profiles that make them important tools for preclinical evaluation of novel hemophilia B therapeutics and to advance understanding of FIX inhibitors. Disclosures: No relevant conflicts of interest to declare.

Factor IX ectopically expressed in platelets can be stored in α-granules and corrects the phenotype of hemophilia B mice

We developed 2bF9 transgenic mice in a hemophilia B mouse model with the expression of human factor IX (FIX) under control of the platelet-specific integrin αIIb promoter, to determine whether ectopically expressing FIX in megakaryocytes can enable the storage of FIX in platelet α-granules and corrects the murine hemophilia B phenotype. FIX was detected in the platelets and plasma of 2bF9 transgenic mice by both antigen and activity assays. Approximately 90% of total FIX in blood was stored in platelets, most of which is releasable on activation of platelets. Immunostaining demonstrated that FIX was expressed in platelets and megakaryocytes and stored in α-granules. All 2bF9 transgenic mice survived tail clipping, suggesting that platelet-derived FIX normalizes hemostasis in the hemophilia B mouse model. This protection can be transferred by bone marrow transplantation or platelet transfusion. However, unlike our experience with platelet FVIII, the efficacy of platelet-derived FIX was limited in the presence of anti-FIX inhibitory antibodies. These results demonstrate that releasable FIX can be expressed and stored in platelet α-granules and that platelet-derived FIX can correct the bleeding phenotype in hemophilia B mice. Our studies suggest that targeting FIX expression to platelets could be a new gene therapy strategy for hemophilia B.

Restoring Initial Thrombin Generation Does Not Normalize Cutaneous Wound Healing in Hemophilia B.

We recently described abnormal wound healing in a mouse model of hemophilia B (Hoffman et al, Blood 2006; DOI 10.1182/blood-2006-05-020495). Specifically: epithelial closure was delayed in hemophilia B mice; hemophilic animals, unlike wild type, developed subcutaneous hematomas; macrophage influx was delayed compared to wild-type mice; and, surprisingly, angiogenesis was enhanced in the hemophilia B mice. We hypothesized that restoring the initial hemostatic burst of thrombin generation following wounding by administration of a single dose of factor IX (FIX) replacement or factor VIIa (FVIIa) bypassing therapy would not only prevent bleeding, but correct the subsequent wound healing process. One dose of therapy was given thirty minutes prior to placement of a single three mm punch biopsy wound on the dorsal skin of each wild type, untreated hemophilia B, and treated hemophilia B mouse. The size of the wounds was measured daily until full epithelial closure. The time course of epithelial closure in treated hemophilia B was intermediate between wild type and untreated hemophilia B. FVIIa-treated hemophilia B began to heal earlier than FIX-treated. Skin from the wound site was collected at different days and examined histologically. Macrophage influx was earlier in treated hemophilia B mice compared to untreated hemophilia B, likely a due to the increased thrombin and fibrin acting as chemotactic agents. The macrophage influx in FVIIa-treated HB was significantly greater at certain time points than in FIX-treated mice, possibly reflecting some signaling effect of TF/FVIIa in addition to its effects on thrombin generation. With the earlier influx in macrophages, hemoglobin was degraded to storage iron at earlier time points. However, tissue iron continued to persist in treated hemophilia B mice similar to untreated hemophilia B, suggesting continued rebleeding. FIX treatment led to significantly more angiogenesis than FVIIa. The reasons for this difference remain to be determined. Untreated and some treated hemophilia B mice developed subcutaneous hematomas both before and after wound closure. The early hematomas are likely caused by a combination of the initial wounding trauma and vulnerability to bleeding related to the high level of vascularity within the granulation tissue. We propose the late hematomas are due to a cycle of bleeding, leading to more inflammation with production of more pro-angiogenic cytokines, leading to greater angiogenesis with its attendant risk of bleeding. In conclusion, restoring initial hemostatic thrombin generation did not normalize cutaneous wound healing in a hemophilia B mouse model. While the time frame of healing may not be the same in human and murine hemophiliacs, our findings suggest that this model could be helpful in rational determination of treatment schedules for replacement or bypassing therapy following injury or surgery.