Improved kinetics of rIX‐FP,a recombinant fusion protein linking factor IX with albumin,in cynomolgus monkeys and hemophilia B dogs

Summary. Background: Prophylaxis of hemophilia B, at present, requires multiple infusions of human factor (F)IX concentrates per week. A FIX molecule with a prolonged half‐life has the potential to greatly improve the convenience of, and adherence to, prophylaxis. Objectives: The aim of our studies was to investigate the pharmacokinetic (PK) and pharmacodynamic (PD) profile of a recombinant fusion protein linking coagulation FIX with albumin (rIX‐FP). Methods: Cynomolgus monkeys and hemophilia B dogs received single intravenous doses of rIX‐FP (50–500 IU kg^?1). rIX‐FP plasma levels were determined by an activity‐based assay (dogs only) and anti‐FIX ELISA methods. Additionally, activated partial thromboplastin time (APTT) was determined in hemophilia B dogs. Data were compared with a direct study comparator (recombinant FIX [rFIX]) or previously published data. Results: The terminal half‐life of rIX‐FP was prolonged in both species compared with FIX reference data. In hemophilia B dogs, human FIX antigen levels remained above 0.05 IU mL^?1 more than three times longer after rIX‐FP (7.3 days) compared with rFIX (2.3 days), whereas respective calculations based on activity levels confirmed the observed superior profile. Prolonged PDs of rIX‐FP were demonstrated with APTT < 60 s sustained around four times longer with rIX‐FP (5.9 days) than rFIX (1.5 days). Conclusions: These studies indicate that the recombinant albumin fusion technology successfully improves the PK profile of FIX. Clinical studies will test whether the improved kinetics result in a significant half‐life extension in patients with hemophilia B.     

Efficient induction of immune tolerance to coagulation factor IX following direct intramuscular gene transfer

BACKGROUND: The formation of inhibitory anti-factor IX (anti-FIX) antibodies is a major complication of FIX protein replacement-based treatment for hemophilia B. It is difficult to treat patients with anti-FIX antibodies. Gene therapy is emerging as a potentially effective treatment for hemophilia. Direct i.m. injection of adeno-associated virus (AAV) is a safe and efficient procedure for hemophilia B gene therapy. However, the development of anti-FIX antibodies following i.m. of AAV may impede its application to patients. OBJECTIVE: We aimed to investigate induction of immune tolerance to human FIX (hFIX) by i.m. of AAV1, further validating i.m. of AAV1 for hemophilia B gene therapy. METHODS AND RESULTS: Cohorts of hemostatically normal and hemophilia B mice with diverse genetic and MHC backgrounds received i.m. of AAV-hFIX. Human FIX antigen and anti-hFIX antibodies were examined. I.m. of 1 x 10(11) vector genomes (VG) of AAV2 elicits formation of anti-hFIX antibodies comparable to those by hFIX protein replacement. I.m. of 1 x 10(11) VG of AAV1 results in expression of therapeutic levels of hFIX (up to 950 ng mL(-1), mean = 772 ng mL(-1), SEM +/- 35.7) and hFIX-specific immune tolerance in C57BL/6 mice. CONCLUSIONS: A single i.m. of AAV1 can result in efficient expression of therapeutic levels of hFIX and induction of hFIX tolerance in hemostatically normal and hemophilic B mice. Our results substantiate the prospect of i.m. of AAV1 for hemophilia B gene therapy and FIX tolerance induction.     

Laboratory testing in hemophilia: Impact of factor and non‐factor replacement therapy on coagulation assays

The advent of extended half‐life (EHL) recombinant clotting factors and innovative non‐factor replacement therapeutics, such as emicizumab, offers several advantages over existing products for the prophylactic treatment of people living with hemophilia (PwH). These include low annual bleeding rates with less frequent dosing, higher trough plasma concentrations, and a more convenient route of administration. However, increasing use of these therapies poses challenges to clinicians and coagulation laboratories due to the lack of standardized assays for monitoring of hemostatic parameters, and the potential for misinterpretation of test results, which may jeopardize patient safety. Definitive diagnosis of hemophilia and treatment monitoring is reliant on demonstrating factor VIII (FVIII; hemophilia A) or factor IX (FIX; hemophilia B) deficiency using a functional coagulation assay. The most frequently used assays are based on activated partial thromboplastin time, using a one‐stage or two‐stage process. While one‐stage and chromogenic assays have performed well with human‐derived FVIII and FIX and full‐length recombinant products, EHL recombinant factors are heterogeneous in structure and mode of action and therefore show wide variation in activity levels between different one‐stage assays, and between one‐stage and chromogenic assays. In the context of the recommended stepwise approach for laboratory diagnosis of hemophilia, we examine the diagnostic challenges associated with the use of EHL factors and novel non‐factor therapeutics and consider the optimal diagnostic approach in PwH who are receiving these treatments. Ultimately, accurate diagnostic solutions are a prerequisite for personalized therapy to minimize treatment burden and improve quality of life in PwH.     

Generation of a novel factor IX with augmented clotting activities in vitro and in vivo

Summary. Background: Hemophilia B is an X‐linked inherited disorder caused by the lack of functional factor IX (FIX). Currently, treatment of hemophilia B is performed by intravenous infusion of plasma‐derived or recombinant FIX. Objective: In an effort to reduce factor usage and cost, we investigated the potential use of FIX variants with enhanced specific clotting activity. Methods: Seven recombinant FIX variants using alanine replacement were generated and assayed for their activity in vitro and in vivo. Results: One variant containing three substitutions (V86A/E277A/R338A, FIX‐Triple) exhibited 13‐fold higher specific clotting activity and a 10‐fold increased affinity for human FVIIIa compared with FIX‐wild‐type (FIX‐WT) and was thus investigated systematically in vivo. Liver‐specific FIX‐Triple gene expression following hydrodynamic plasmid delivery revealed a 3.5‐fold higher specific clotting activity compared with FIX‐WT. Human FIX‐Triple and FIX‐WT knock‐in mice were generated and it was confirmed that FIX‐Triple has 7‐fold higher specific clotting activity than FIX‐WT under normal physiological conditions. Protein infusion of FIX‐Triple into hemophilia B mice resulted in greater improvement of hemostasis than that achieved with FIX‐WT. Moreover, tail‐vein administration of a serotype 8 recombinant Adeno‐associated vector (AAV8) expressing either FIX‐WT or FIX‐Triple in hemophilia B mice demonstrated a 7‐fold higher specific clotting activity of FIX‐Triple than FIX‐WT. Conclusions: Our results indicate that the FIX‐Triple variant exhibits significantly enhanced clotting activity relative to FIX‐WT due to tighter binding to FVIIIa, as demonstrated both in vitro and in vivo. Therefore, FIX‐Triple is a good candidate for further evaluation in protein replacement therapy as well as gene‐based therapeutic strategies.     

Two novel mutations in EGF-like domains of human factor IX dramatically impair intracellular processing and secretion.

We investigated the mechanisms responsible for severe factor IX (FIX) deficiency in two cross-reacting material (CRM)-negative hemophilia B patients with a mutation in the first and second epidermal growth factor (EGF) domains of FIX (C71Y and C109Y, respectively). We have determined the kinetics of mutant FIX biosynthesis and secretion in comparison with wild-type FIX (FIXwt). In transfected cells, FIXwt was retrieved as two intracellular molecular forms, rapidly secreted into the culture medium. One appeared to be correctly N-glycosylated, and corresponded to a form trafficking between the endoplasmic reticulum (ER) and Golgi apparatus. The other corresponded to the mature form, ready to be secreted, exhibiting correct N-glycosylation and sialylation. In contrast, the two mutants, FIXC71Y and FIXC109Y, were not secreted from the cells and did not accumulate intracellularly. Relative to FIXwt, they were retained longer in the ER and were only N-glycosylated. In addition, the intracellular concentration of the FIX mutants increased when ALLN, an inhibitor of cysteine proteases and of the proteasome degradation pathway, was added to the culture medium. Both the FIX mutants and FIXwt were associated in the ER with the 78-kDa glucose-regulated protein (GRP78/BiP) and calreticulin (CRT), though the amount of CRT associated with the two mutants was twice as strong as with FIXwt. These results strongly suggest that chaperone and lectin molecules act in concert to ensure both proper folding of FIXwt and the retention of mutant molecules.     

Enhancement of the enzymatic activity of activated coagulation factor IX by anti-factor IX antibodies

Summary.  Background:  Factor VIIIa (FVIIIa) binds to activated FIX and enhances the activation of FX by several orders of magnitude. Deficiency of FVIII causes the bleeding disorder hemophilia A and is treated by i.v. infusion of FVIII concentrates. Objectives:  To explore whether or not FVIII activity can be supplied by alternative molecules, e.g. molecules with FIXa-binding activity. Methods:  Conventional hybdridoma technology was used to discover antibodies exhibiting FVIII-like activity. Results:  We identified a series of antibodies specific for human FIX that mimicked the stimulatory effect of FVIIIa on FIXa. Upon binding to human FIXa, these antibodies enhanced the protease activity of FIXa towards its natural substrate FX about tenfold. A similar enhancement was also achieved with 5 p m FVIIIa (i.e . 16 mU mL⁻¹ or 1.6% activated FVIII). Procoagulant activity of these anti-FIXa antibodies was observed in model systems containing purified proteins as well as in plasma. Conclusion:  Our findings show that FVIII can, at least partially, be replaced by an unrelated molecule. Procoagulant antibodies might potentially aid the development of an FVIII substitute for hemophilia A treatment.     

The measurement of low levels of factor VIII or factor IX in hemophilia A and hemophilia B plasma by clot waveform analysis and thrombin generation assay

BACKGROUND: Precise assessment of clotting function is essential for monitoring of hemostatic treatment for hemophilias A and B. MATERIALS AND METHODS: Clot waveform analysis and thrombin generation assays were performed on factor (F) VIII- and FIX-deficient plasmas, which had been reconstituted with known amounts of recombinant FVIII (rFVIII) and affinity-purified FIX respectively. Clot waveforms were assessed qualitatively and quantitatively by measuring the parameters clotting time, maximum coagulation velocity (Min1), and maximum coagulation acceleration (Min2). The thrombin generation assay was also assessed qualitatively and measurements made of time to peak and peak height. RESULTS: Overall results obtained with both assays showed good correlation for both clotting factors confirming that the changes in clotting waveform reflected changes in thrombin generation. Both assays demonstrated a predictable dose response to the addition of FVIII or IX. However, clot waveform analysis was more sensitive than the thrombin generation assay, particularly in detecting very low levels (0-0.1 IU dL(-1)) of both factors. CONCLUSIONS: These data suggest that the application of clot waveform analysis to the routine management of the hemophiliacs could increase our understanding of the clinical significance of low levels of FVIII and FIX that cannot be measured by assays in current use. This may be particularly useful in the management of hemophiliacs with inhibitors or undergoing gene therapy.     

The chaperone‐like sodium phenylbutyrate improves factor IX intracellular trafficking and activity impaired by the frequent p.R294Q mutation

Essentials

• Missense mutations often impair protein folding, and thus intracellular trafficking and secretion.
• Cellular models of severe type I hemophilia B were challenged with chaperone‐like compounds.
• Sodium phenylbutyrate improved intracellular trafficking and secretion of the frequent p.R294Q.
• The increased coagulant activity levels (～3%) of p.R294Q would ameliorate the bleeding phenotype.

Summary

Background

Missense mutations often impair protein folding and intracellular processing, which can be improved by small compounds with chaperone‐like activity. However, little has been done in coagulopathies, where even modest increases of functional levels could have therapeutic implications.

Objectives

To rescue the expression of factor IX (FIX) variants affected by missense mutations associated with type I hemophilia B (HB) through chaperone‐like compounds.

Methods

Expression studies of recombinant (r)FIX variants and evaluation of secreted levels (ELISA), intracellular trafficking (immunofluorescence) and activity (coagulant assays) before and after treatment of cells with chaperone‐like compounds.

Results

As a model we chose the most frequent HB mutation (p.R294Q, ~100 patients), compared with other recurrent mutations associated with severe/moderate type I HB. Immunofluorescence studies revealed retention of rFIX variants in the endoplasmic reticulum and negligible localization in the Golgi, thus indicating impaired intracellular trafficking. Consistently, and in agreement with coagulation phenotypes in patients, all missense mutations resulted in impaired secretion (< 1% wild‐type rFIX). Sodium phenylbutyrate (NaPBA) quantitatively improved trafficking to the Golgi and dose dependently promoted secretion (from 0.3 ± 0.1% to 1.5 ± 0.3%) only of the rFIX‐294Q variant. Noticeably, this variant displayed a specific coagulant activity that was higher (~2.0 fold) than that of wild‐type rFIX in all treatment conditions. Importantly, coagulant activity was concurrently increased to levels (3.0 ± 0.9%) that, if achieved in patients, would ameliorate the bleeding phenotype.

Conclusions

Altogether, our data detail molecular mechanisms underlying type I HB and candidate NaPBA as affordable ‘personalized’ therapeutics for patients affected by the highly frequent p.R294Q mutation, and with reduced access to substitutive therapy.

     

Complete and sustained phenotypic correction of hemophilia B in mice following hepatic gene transfer of a high-expressing human factor IX plasmid

Summary.  Therapeutic correction of hemophilia B was achieved by rapid infusion of a large-volume solution containing a high-expressing human factor IX (hFIX) plasmid into the tail vein of hemophilia B mice. hFIX circulated at therapeutic levels (1–5 µg mL⁻¹) in all animals for more than 1 year as determined by both species-specific antigen assay and an activated partial thromboplastin time (APTT)-based clotting assay. There was acute, transient hepatic tissue damage by the infusion procedure and no significant inhibitory anti-hFIX antibodies developed. No bleeding episode was observed during or after treatment. Immunohistochemical studies indicated that the hFIX gene was exclusively expressed in hepatocytes, and that transduced cells had readily detectable hFIX protein at 4 h postinfusion, and stainable protein persisted for up to 1 year. Repeated infusions of hFIX plasmids boosted the hFIX expression to higher levels. These results demonstrate that hemophilia B can be treated by gene transfer of naked hFIX plasmids.     

Population pharmacokinetic modeling for dose setting of nonacog beta pegol (N9‐GP), a glycoPEGylated recombinant factor IX

Summary. Background: nonacog beta pegol (N9‐GP) is a glycoPEGylated recombinant factor IX (rFIX) molecule with a prolonged half‐life. Objectives: To provide information on potential dose regimens for N9‐GP for phase 3 pivotal and surgery trials. Methods: A population pharmacokinetic model was developed from single‐dose data derived from the first human‐dose trial with N9‐GP in hemophilia B patients, and was used to extrapolate to steady‐state conditions for different N9‐GP dose regimens for prophylaxis. The model was also used to compare prophylaxis using N9‐GP with standard prophylactic regimens using rFIX or plasma‐derived (pd) FIX (40 IU kg^?1 every third day). Plasma activity following dosing with N9‐GP, rFIX and pdFIX for surgery and on‐demand treatment of bleeds was also simulated. Results: A linear two‐compartmental model best described the pharmacokinetic profiles of N9‐GP, rFIX and pdFIX. A prophylactic regimen of 10 U kg^?1 N9‐GP once weekly predicted mean peak and trough levels of 18 and 4.2 U dL^?1, while 40 U kg^?1 once weekly predicted values of 72 and 17 U dL^?1, respectively. Standard prophylactic regimens with rFIX and pdFIX predicted mean peak and trough levels of 34 and 3.9 IU dL^?1 for rFIX, and mean values of 43 and 2.1 IU dL^?1 for pdFIX. Additional simulations predicted significantly reduced dosing frequency and factor concentrate consumption for N9‐GP vs. rFIX and pdFIX for surgery and the treatment of bleeds. Conclusions: N9‐GP may allow prophylaxis, surgical dosing regimens and on‐demand treatment of bleeding episodes with less frequent injections and lower factor concentrate consumption; this possibility is being investigated in prospective clinical trials.