Background Fidanacogene elaparvovec, an adeno-associated virus-based gene ther-apy vector expressing the high-activity factor IX (FIX) variant FIX-R338L, is in develop-ment for hemophilia B. One-stage clotting (OS) assays and chromogenic substrate (CS)assays are commonly used to measure FIX-R338L variant activity. Data from ongoingtrials suggest FIX activity varies between different OS and CS assays. Material and Methods To better understand FIX-R338L activity in clinical samples, aninternational multisitefield study was conducted across a central laboratory and 18local laboratories, using standard protocols, reagents, and instrumentation, within dividual participant samples from a phase 1/2a study offidanacogene elaparvovec. Results Unlike the wild-type FIX control, FIX-R338L activity was higher with the OS silica-based assay versus OS ellagic acid-based and CS assays. Variation in FIX activity was greater at the lowest activity levels. Activated FIX (FIXa) in plasma could result in higher OS assay activity or increased thrombin generation, which could overestimateFIX activity. However, FIXa was not detected in the participant samples, indicating thatit was not contributing to the OS assay differences. Since individuals on gene therapy may receive exogenous replacement FIX products, replacement products were spiked into patient plasma samples to target a therapeutic concentration. Exogenous FIX was additive to endogenous FIX-R338L, with no interference from FIX-R338L. ConclusionThese results demonstrate FIX-R338L activity can be measured with OS andCS assays in clinical laboratories and provide insight into assay variability when measuringTHIEMECoagulation and FibrinolysisArticle published online: 2024-06-11

Background: Patients with hemophilia have deficiencies in intrinsic coagulation factors and can develop inhibitors that limit the effectiveness of replacement coagulation factors. Marstacimab, a human monoclonal antibody, binds and inhibits the human tissue factor pathway inhibitor. Marstacimab is currently under development as a potential prophylactic treatment to prevent bleeding episodes in patients with hemophilia A and B. Objective: To assess the effects of marstacimab alone or in combination with the bypassing agent recombinant factor FVlla (rFVlla) or activated prothrombin complex concentrate (aPCC) on thrombin generation and bleeding. Methods: Marstacimab and/or rFVlla or aPCC were added to hemophilic A or B plasma or nonhemophilic plasma in vitro. Hemostatic activity was measured using the thrombin generation assay. In vivo effects were assessed using a mouse acute bleeding model. Male hemophilia A mice were dosed with marstacimab plus aPCC before tail clip; blood loss was quantified by measuring hemoglobin. Results: Marstacimab plus rFVlla or aPCC slightly increased peak thrombin levels compared with either agent alone. This increase was within the reported range for nonhemophilic plasma and did not exceed levels observed in nonhemophilic plasma treated with marstacimab alone. Hemophilia A mice that received 200 U/kg aPCC had significantly reduced bleeding (62%) compared with vehicle-treated mice (p < 0.05), and marstacimab plus aPCC reduced bleeding by 83.3% compared with vehicle (p= 0.0009). Conclusions: Marstacimab alone or with bypassing agents increased hemostasis in hemophilia plasma without generating excessive thrombin. The hemostatic activity of marstacimab plus aPCC was confirmed in hemophilia A mice.

Classic galactosemia (CG) is a rare disorder of autosomal recessive inheritance. It is caused predominantly by point mutations as well as deletions in the gene encoding the enzyme galactose‐1‐phosphate uridyltransferase (GALT). The majority of the more than 350 mutations identified in the GALT gene cause a significant reduction in GALT enzyme activity resulting in the toxic buildup of galactose metabolites that in turn is associated with cellular stress and injury. Consequently, developing a therapeutic strategy that reverses both the oxidative and ER stress in CG cells may be helpful in combating this disease. Recombinant adeno‐associated virus (AAV)‐mediated gene therapy to restore GALT activity offers the potential to address the unmet medical needs of galactosemia patients. Here, utilizing fibroblasts derived from CG patients we demonstrated that AAV‐mediated augmentation of GALT protein and activity resulted in the prevention of ER and oxidative stress. We also demonstrate that these CG patient fibroblasts exhibit reduced CD109 and TGFβRII protein levels and that these effectors of cellular homeostasis could be restored following AAV‐mediated expression of GALT. Finally, we show initial in vivo proof‐of‐concept restoration of galactose metabolism in a GALT knockout mouse model following treatment with AAV‐GALT.

Limited information exists regarding the factor IX (FIX) coagulant activity (FIX:C) measured by different assays following FIX‐Padua gene therapy.

Galactosemias are a family of autosomal recessive genetic disorders resulting from impaired enzymes of the Leloir pathway of galactose metabolism including galactokinase, galactose uridyltransferase, and UDP-galactose 4-epimerase that are critical for conversion of galactose into glucose-6-phosphate. To better understand pathophysiological mechanisms involved in galactosemia and develop novel therapies to address the unmet need in patients, it is important to develop reliable assays to measure the activity of the Leloir pathway enzymes. Here we describe in-depth methods for indirectly measuring galacose-1-phosphate uridyltransferase activity in cell culture and animal tissues.

Sickle cell disease (SCD) is a severe genetic disorder that impacts approximately 20 million people worldwide.1 The causative β6 Glu-Val substitution is a gain of function mutation; in the deoxygenated state, the mutant protein, Hb S, can form polymers, leading to red blood cell sickling and precipitating downstream consequences, including vaso-occlusion (pain crisis), hemolytic anemia, stroke and related pathophysiology.2, 3 Polymerization is exponentially dependent on deoxy Hb S concentration.4 Thus, relatively small changes in deoxyHb S concentration will significantly impact polymerization, red blood cell sickling, and ultimately the clinical course of the disease. Pharmacologic evidence for the benefit of reducing the concentration of deoxyHb S arises from studies of covalent modification of Hb S, where stabilization of the oxygenated conformation increases Hb O2 affinity, reduces RBC sickling, extends RBC half-life, and ultimately reduces the frequency of vaso-occlusive crisis (VOC).5-8 In clinical trials, ex vivo carbamylation of patient blood led to improvements in hemolytic anemia; treated patients exhibited a 2.7 g/dl increase in hemoglobin, a 58% decrease in reticulocytes, a 65% decrease in irreversibly sickled cells, and an 80% decrease in frequency of painful crises.8 Subsequent oxyHb S stabilizing molecules were developed based on the observation that benzaldehyde derivatives formed stable, covalent Schiff bases with hemoglobin. The most advanced of this class of molecules is the covalent compound Voxelotor (GBT 440, Oxbryta), which was approved by the FDA in 2019 for the treatment of SCD. In the pivotal study, 59% of patients in the higher dose group (1500 mg/day) had increases of 1 g/dl or greater in hemoglobin, with a mean hemoglobin modification of 26.5%.9 While the impact of covalent Hb S modifiers on hemolytic anemia is well established, the rise in hemoglobin observed for voxelotor falls short of the effects observed by ex vivo carbamylation, and was not accompanied by a significant effect on the frequency of VOC. This suggests that the therapeutic potential of hemoglobin modification has not been fully realized. PF-7059013 is a non-covalent modifier of hemoglobin that stabilizes the oxygenated state (see Gopalsamy et al.). Here we present the impact this molecule has on a well-established mouse model of sickle cell disease. Treatment with PF-7059013 demonstrated robust changes in key markers of hemolytic anemia in the Townes mouse model,10 suggesting it has the potential to be a potent and efficacious therapy for SCD. PF-07059013 was orally administered to Townes SCD model mice twice daily at a dose of 200 mg/kg for 15 days. This dose was selected as it was expected to result in approximately 25% hemoglobin coverage.11 At 30 minutes post the initial dose, total blood concentrations for individual animals were 2–4 mM, consistent with the low total blood clearance observed in the single dose administration studies. These values translate to approximately 40%–60% hemoglobin coverage, based on measured hemoglobin concentrations. The high total blood concentrations observed following the initial dose were maintained for the duration the 15-day dose period (Supporting Information S1). Animals treated with PF-07059013 show a significant stabilization of the oxygenated state. The average p50 decreased by 53.7% (±21.2%) in the treated group, relative to vehicle, and the average p20, a more sensitive marker of compound occupancy, decreased by 84.4% (±2.6%) in the treated group relative to vehicle (Supporting Information S1). As expected from the large shifts in oxygen affinity, blood from animals in the treated group showed significant reductions in RBC sickling. Under stringent hypoxic conditions, treatment with PF-07059013 resulted in a 37.8% (±9%) decrease in RBC sickling (Figure 1(A)). Consistent with reduction of RBC sickling, following 15 days of dosing, mice treated with PF-07059013 showed significant improvement in markers of hemolytic anemia. PF-07059013 treated animals showed a 42.4% (±4.2%) increase in hemoglobin, with a mean increase in hemoglobin of 5 g/dl, as well as a 30.9% (±0.7%) increase in hematocrit, and a 39.2 (±9.3%) increase in red blood cells relative to vehicle. All of the changes were statistically significant, and the increases restored the hemoglobin, hematocrit, and RBC counts of the treated group to values similar to wild type (C7BL/6) mice (Figure 1(B)). In addition, treatment with PF-07059013 resulted in a 54.7% (±2.4%) decrease in reticulocytes (Figure 1(B)). Note, PF-07059013 achieves consistently high levels of hemoglobin occupancy in in vivo studies using the Townes SCD murine model, which were sustained for the duration of dosing. Taken together, these results indicate that a non-covalent molecule has the potential to be efficacious for the treatment of sickle cell disease, and that the presence of a reactive aldehyde is not a requirement for potency. Comparing the activity of early covalent modifiers in the Townes model with PF-07059013 is not possible, as the development of many of those molecules predated the development of the transgenic mouse models. However, it is possible to compare the performance of PF-07059013 in the Townes SCD mouse with previously published Voxelotor pre-clinical data in the Townes model. Oksenberg et al. report that twice-daily oral dosing of Townes SCD mice with Voxelotor/GBT 440 at 100 or 150 mg/kg for 9–12 days resulted in hemoglobin occupancies ranging from 11%–39.7%.12 The pharmacodynamic effects of Voxelotor were strongly correlated with the degree of hemoglobin occupancy, as only the animals attaining >30% occupancy (4/14) showed changes in reticulocyte count or red blood cell half-life relative to vehicle. Thus, PF-07059013 achieved high degrees of hemoglobin coverage upon twice-daily oral dosing at 200 mg/kg for 15 days. As PF-07059013 binds ditopically to Hb (two compound: one tetramer), the dose is comparable to the doses used in the Voxelotor animal studies,12 as PF-07059013 requires twice as much compound to achieve the same hemoglobin occupancy percentages. In contrast with Voxelotor preclinical studies12 all of the animals dosed with PF-07059013 (n = 7) achieved >40% hemoglobin occupancy. The consistently high occupancy level across all animals leads to a uniform improvement in markers of hemolytic anemia. Similarly, all PF-07059013 treated animals showed decreases in RBC sickling, ranging from 35.3%–45.5%. The observed decrease in RBC sickling with PF-07059013 treatment is consistent with the decrease observed for Voxelotor in Townes SCD mice that had high Hb occupancy.12 The role of increases in hemoglobin in the overall pathology of sickle cell disease, particularly as it relates to VOC, is not completely understood. In the Voxelotor pivotal trial, 59% of patients treated with 1500 mg/day experienced hemoglobin increases of 1 g/dl or greater (average = 1.1 g/dl), and showed reductions in reticulocytes and bilirubin, consistent with improvements in hemolytic anemia, following 24 weeks of dosing.9 Post-hoc analysis of the pivotal trial results indicated that patients who achieved a hemoglobin level of 10 g/dl or greater had reduced incidence of VOC (50/179), with the greatest benefit observed in the small group of patients that had hemoglobin levels of 12 g/dl or greater (10/179).13 These data are consistent with the observation that increased hemoglobin can lead to reductions in VOC, provided RBC sickling is sufficiently impeded; Diedrich et al. demonstrated a substantial reduction in VOC frequency following weekly extracorporeal carbamylation.8 After 3 months of treatment, hemoglobin had increased by an average of 2.7 g/dl to an average of 8.8 g/dl and occurrences of VOC decreased by 80%.8 An increase in hemoglobin alone is likely not sufficient, as SCD patients undergoing exchange transfusions still experience VOC.14 Ex vivo carbamylation was most efficacious when hemoglobin occupancy was above 35%, suggesting achieving and maintaining high levels of hemoglobin occupancy may be crucial for making the maximum reduction in RBC sickling, reducing hemolysis, and increasing hemoglobin. These data indicate that it is possible to correlate the hemoglobin increase mediated by stabilization of the oxygenated state to resolution of VOC, and further suggest that the size of the increase in hemoglobin may be an important influencer of other clinical outcomes. Based in part on the magnitude and consistency of the response in the Townes SCD mouse model presented here, clinical studies of PF-07059013 are currently underway. The authors wish to thank Dr. Carlo Brugnara (Boston Children's Hospital) for assistance in obtaining SCD patient blood, and for valuable project discussions. We thank Dr. John Kelly (Northeastern University Co-op program), Joseph Nneji (Northeastern University Co-op program), and Victoria Ball (Northeastern University Co-op Program) for assistance in hemoglobin purification. We thank Jazmyne Lopez (Pfizer Occupational Health and Wellness) for coordinating healthy human blood sample collection, and Dr. David Karanian and Dr. Jamie DaSilva (Pfizer Drug Safety Research and Development) for providing whole blood from relevant toxicology species. All authors listed were employees of Pfizer Inc and declare no conflict of interest. Kelly M. Knee: designed research, performed research, analyzed data, wrote the paper. Reema Jasuja: designed research, performed research, analyzed data. Amey Barakat: performed research, analyzed data. Dharani Rao: performed research, analyzed data. Zane Wenzel: performed research, analyzed data. Jayasankar Jasti: performed research, analyzed data. Jonathan Novak: performed research, analyzed data. Kevin Beaumont: designed research, analyzed data. David W. Piotrowski: designed research, analyzed data. Phil Jeffery: designed research, analyzed data. Christine Bulawa: designed research, analyzed data. John E. Murphy: analyzed data, designed research. Jay M. Janz: performed research, designed research, analyzed data, wrote the paper. The data that support the findings of this study are available from the corresponding author upon reasonable request. Appendix S1. Supporting information Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Erythroferrone (Erfe) is produced by erythroblasts in response to erythropoietin (EPO) and acts in the liver to prevent hepcidin stimulation by BMP6. Hepcidin suppression allows for the mobilization of iron to the bone marrow for the production of red blood cells. Aberrantly high circulating Erfe levels in conditions of stress erythropoiesis, such as in patients with β-thalassemia, promote the tissue iron accumulation that decisively contributes to morbidity in these patients. Here we developed neutralizing antibodies against Erfe to prevent hepcidin suppression and correct the iron loading phenotype in a mouse model of β-thalassemia (Hbb Th3/+ mice) and used these antibodies as tools to further characterize Erfe's mechanism of action. We demonstrate that Erfe binds to BMP6 with low nanomolar affinity, but also binds BMP2 and BMP4 with lower affinities. We further show that BMP6 binds the N-terminal domain of ERFE. This domain in itself was sufficient to cause hepcidin suppression in Huh7 hepatoma cells and in vivo in wildtype mice. Concurrently, anti-Erfe antibodies targeting the N-terminal domain prevented hepcidin suppression in Erfe-treated Huh7 cells and in EPO-treated mice. Crystal structure of the antibodies in contact with an N-terminal peptide of Erfe demonstrated critical contacts in the Erfe N-terminal domain imparting antibody selectivity to human and murine protein. Finally, we tested these antibodies in vivo in a mouse model of thalassemia. We observed a decrease in serum and liver iron in antibody-treated Hbb Th3/+ mice. In addition, treatment with anti-Erfe antibodies increased the number of red blood cells, hemoglobin concentration and hematocrit, while decreasing the number of reticulocytes and the red cell distribution width. These changes were more pronounced when mice are treated for eight weeks. Anti-Erfe treatment caused an increase in hepatic hepcidin mRNA expression, red blood cells, hemoglobin and hematocrit, while reticulocytes levels were lower and peripheral red cell lifespan was increased. In summary, we demonstrate that antibodies targeting the N-terminal domain of Erfe constitute a potential therapeutic tool for iron-loading anemias. Disclosures Arezes: UCB: Employment. Foy:Pfizer Inc.: Employment. Benard:pfizer: Employment. Sawant:Pfizer Inc.: Employment. Tam:Pfizer Inc.: Employment. Maben:Pfizer Inc.: Employment. LaVallie:Pfizer Inc.: Employment. Cunningham:Pfizer Inc.: Employment. Lambert:Pfizer Inc.: Employment. Pittman:Pfizer Inc.: Employment. Murphy:Pfizer Inc.: Employment. Draper:Pfizer: Research Funding. Jasuja:Pfizer Inc.: Employment. Drakesmith:Pfizer: Consultancy, Research Funding; Kymab: Other: Scientific Advistory; La Jolla Pharmaceutical: Research Funding.

Background: Adeno-Associated Virus (AAV) based liver transduction has emerged as a potentially viable gene therapy approach for the treatment of hemophilia patients. Fidanacogene elaparvovec (previously SPK-9001) is a hepatotropic bioengineered AAV based vector that delivers a high activity factor IX (FIX) transgene driven by a liver specific promoter. The Phase 1/2a development consists of a dosing study where patients are followed for 52 weeks post vector infusion followed by a long-term follow-up study for an additional 5 years. Data on the first 10 patients were previously published and demonstrated safe and sustained expression of a high activity FIX protein with an associated decreased requirement for exogenous factor administration and markedly reduced annualized bleeding rate. Here we present data on 15 patients infused with fidanacogene elaparvovec with ≥ 1 year of follow-up, which represents the largest cohort of Hemophilia B (HB) patients treated with the same vector at the same dose. Methods: Fifteen (15) adult HB patients were infused with 5 x 1011 vg/kg of fidanacogene elaparvovec and followed for at least 1 year as part of the Phase 1/2a dosing study. FIX activity (FIX:C) levels were measured using a one-stage assay. Endpoints include: Safety and tolerability, steady-state activity calculated as the geometric mean of all observed FIX:C activity levels from week 12 through week 52; annualized bleeding rate (ABR) prior to and 52 weeks after vector infusion; T cell response to fidanacogene elaparvovec capsid and transgene monitored post-infusion using an interferon-γ enzyme-linked immunospot (ELISpot) assay. Results: Three of fifteen patients were treated with corticosteroids for elevations in hepatic transaminases of which 2 were positive for capsid reactive T cells by interferon-γ ELISpot. There were otherwise no treatment related adverse events. The mean post-infusion steady-state FIX:C was 22.9%±9.9% at 1 year post vector infusion as measured in a central laboratory by one-stage assay utilizing Actin-FSL. Mean ABR during the first 52 weeks following fidanacogene elaparvovec infusion was 0.4±1.1 compared to 8.9±14.0 in the 52 weeks preceding infusion (p<0.001). Twelve (12) out of 15 patients reported zero bleeds in the 52 weeks post-vector infusion. Five of 15 subjects infused factor for a total of 20 infusions. Additional follow-up data will be presented for all patients enrolled in the long-term follow-up study. Conclusions: Fidanacogene elaparvovec was well tolerated in 15 patients with no serious adverse events. Data for all patients at 52 weeks post-infusion demonstrated a marked reduction in bleeding frequency and exogenous FIX use. All hepatic transaminase elevations responded to treatment with corticosteroids. Collectively, to date, this represents the largest cohort of HB patients treated with the same AAV based gene therapy and at the lowest dose. Treatment has been efficacious for all patients with manageable immune responses when present. These data support progression to a pivotal Phase 3 study at the dose evaluated. Disclosures George: University of Pennyslvania: Employment; Avrobio: Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy. Sullivan:Octapharma: Consultancy, Other: Advisory Board. Rasko:bluebird bio: Honoraria; Celgene: Honoraria; Novartis: Honoraria; FSHD Global Research Foundation: Membership on an entity's Board of Directors or advisory committees; Rarecyte: Consultancy, Equity Ownership; Gene Technology Technical Advisory, Australian Government: Other: Advisory committee; GSK: Honoraria; Takeda: Honoraria; Cynata: Honoraria; Genea: Equity Ownership; Cure The Future Foundation: Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria; Pfizer: Honoraria; Spark: Honoraria; Imago: Consultancy; Advisory Committee on Biologics, Australian Government: Other: Advisory Committee; NHMRC Mitochondrial Donation Expert Working Committee: Other: Advisory Committee; Australian Cancer Research Scientific Advisory Board: Membership on an entity's Board of Directors or advisory committees. Giermasz:Genentech/Roche: Consultancy, Other: Research, Speakers Bureau; uniQure: Consultancy, Other: Research; Bioverativ/Sanofi: Consultancy, Speakers Bureau; BioMarin: Consultancy, Other: Research; Sangamo: Other: Research. Samelson-Jones:The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania: Employment. Ducore:Bayer: Consultancy, Honoraria, Other: speaker (not bureau); Spark Therapeutics: Research Funding; Shire: Consultancy, Honoraria; Octapharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; HEMA Biologics: Consultancy, Honoraria; BioMarin: Research Funding; Bioverativ: Research Funding. Teitel:BioMarin: Consultancy; Bayer: Consultancy, Research Funding; Shire: Consultancy; Pfizer: Consultancy, Research Funding; Novo Nordisk: Consultancy; Octapharma: Consultancy; CSL Behring: Consultancy. McGuinn:Biogen: Research Funding; Roche/Genetech: Research Funding; Spark: Research Funding; Shire/Baxalta: Consultancy, Research Funding. Wright:Solid Biosciences: Consultancy; Yposkesi: Other: Senior Advisor, SAB; LogicBio Therapeutics: Other: Member, SAB; Memorial Sloan Kettering Cancer Center: Consultancy; Agilis Biotherapeutics: Consultancy; Axovant Sciences: Other: Chief Technology Officer, Gene Therapies; Akous Therapeutics: Consultancy; National Institutes of Health: Consultancy; Leland Stanford Junior University: Consultancy; Wright Biologics: Other; Sanofi Genzyme: Consultancy; Spark Therapeutics: Consultancy, Other: co-founder, Chief Technology Advisor/Officer, Member, SAB; Adrenas Therapeutics: Other: Member, SAB; Ambys Medicines: Consultancy; CEVEC Pharmaceuticl: Other: Member, SAB. Anguela:Spark Therapeutics: Employment, Equity Ownership, Patents & Royalties. High:Spark Therapeutics: Employment, Equity Ownership, Patents & Royalties. Rybin:Pfizer: Employment. Murphy:Pfizer Inc.: Employment. Rupon:Pfizer: Employment.

Hemophilia is a family of rare bleeding disorders characterized by inadequate levels of intrinsic coagulation factors, Factor VIII (FVIII) in hemophilia A and Factor IX (FIX) in hemophilia B. This leads to insufficient thrombin generation for the conversion of fibrinogen to fibrin for development of a stable clot. Replacement factor therapies are provided as a prophylactic treatment to prevent bleeds or for on-demand treatment of an active bleed. Some patients develop inhibitory antibodies making them refractory to replacement therapies. Although hemophilic patients have defects in the intrinsic pathway, the extrinsic pathway remains intact. Augmenting the extrinsic tissue factor pathway is an attractive alternate approach to maintain hemostasis in hemophilic patients. Tissue factor pathway inhibitor (TFPI) is a Kunitz domain type inhibitor that negatively regulates thrombin generation within the extrinsic pathway of coagulation by rapidly inactivating protease functions of Factor Xa and Factor VIIa/Tissue Factor complex and as such, TFPI inhibition has been explored as an innovative option to restore thrombin generation. Previously, we demonstrated that antibody mediated TFPI inhibition restores hemostasis in mouse models of hemophilia (Jasuja et al, 2016). Here we investigated the impact of an adeno-associated virus (AAV) vector expressed anti-TFPI antibody in correcting bleeding disorders in hemophilic mice as a novel approach to restore hemostasis. An AAV vector expressing heavy and light chains of a neutralizing anti-TFPI monoclonal antibody was designed and constructed. In vitro evaluations confirmed that vector expressed antibody was functional using a dilute Prothrombin Time (dPT) assay. Next, male hemophilic A mice were systemically administered (intravenous) 1x1011 AAV vector genomes (vg) encoding anti-TFPI or control and had hemostasis evaluated 16 days post-treatment. Following recalcification of whole blood, thromboelastography (TEG) was used to measure clot formation times (R value), speed of clot formation (K value), as well as maximum amplitude. In hemophilia A mice treated with AAV-anti-TFPI, clot formation time (R value) was normalized to wild type levels, with a 10-fold reduction compared to hemophilia A mice administered a control Green fluorescent protein (GFP) encoding AAV vector. Mice administered the control AAV-GFP vector failed to correct additional parameters, including speed of clot formation (K value), angle and maximum amplitude, all of which remained similar to untreated hemophilia A mice. In contrast, for AAV-anti-TFPI dosed Hem A mice, all of these parameters were in the normal range similar to wild type mice. Our data demonstrate AAV mediated delivery of anti-TFPI antibody corrects coagulation abnormalities observed in a mouse model of Hemophilia A. Disclosures Rakhe: Pfizer Inc.: Employment. Jasuja:Pfizer Inc.: Employment. Shelke:Pfizer Inc.: Employment. Sawant:Pfizer Inc.: Employment. Somanathan:Pfizer Inc.: Employment. Murphy:Pfizer Inc.: Employment. Pittman:Pfizer Inc.: Employment.

INTRODUCTION:Tissue factor pathway inhibitor (TFPI) is an endogenous inhibitor of the extrinsic pathway that negatively regulates thrombin production during coagulation. Under haemophilic conditions, where the intrinsic coagulation pathway is impaired, inhibition of TFPI may improve clotting.AIM:We investigated the ex vivo effects of a human TFPI neutralizing antibody, marstacimab (previously PF-06741086), in coagulation assays including rotational thromboelastometry (ROTEM), thrombin generation assay (TGA) and the dilute prothrombin time (dPT) assay, performed in haemophilic whole blood and plasmas. We compared the effects of marstacimab to the effects of recombinant coagulation factors and investigated the reproducibility of marstacimab in restoring haemostasis by comparing its effect in whole blood collected from the same study participants on differing days.METHODS:Citrated whole blood and plasmas obtained from haemophilia participants were supplemented ex vivo with vehicle, marstacimab, recombinant FVIII (rFVIII) or recombinant factor IX (rFIX) and analysed in ROTEM, TGA and the dPT assay using low tissue factor concentrations to trigger coagulation.RESULTS:Marstacimab induced pro-coagulant responses in ROTEM parameters including reduction in clotting times and increases in angle. Similarly, participant plasmas supplemented with marstacimab exhibited improvements in TGA parameters, including reduced lag times, increased peak thrombin concentrations and reductions in dPT clotting time. Concentrations of marstacimab tested showed activity comparable to addition of rFVIII or rFIX and were reproducible.CONCLUSIONS:These studies show the ex vivo potency of marstacimab in restoring haemostasis in whole blood and plasmas from haemophilia participants and comparability to ex vivo reconstitution with recombination coagulation factors.

Hemophilia A and B are hereditary bleeding disorders caused by intrinsic coagulation pathway deficiencies of Factor VIII or Factor IX, respectively. Tissue factor pathway inhibitor (TFPI) is a Kunitz-type serine protease inhibitor that negatively regulates thrombin generation within the extrinsic pathway of coagulation. PF-06741086 is a fully human monoclonal antibody which binds the Kunitz-2 domain and neutralizes the inhibitory activity of human tissue factor pathway inhibitor and is currently under development as a potential prophylactic treatment to prevent bleeding episodes in hemophilia A and hemophilia B patients with and without inhibitors. Activated prothrombin complex concentrate (aPCC) is used as bypass treatment for the resolution of bleeding in some hemophilia patients with inhibitors. Hemophilia inhibitor patients receiving PF-06741086 have a possibility to also receive treatment with aPCC. The aim of the current study was to assess the potential additive effect of PF-06741086 with aPCC added in vitro to Hemophilia A and B inhibitor plasmas using a thrombin generation assay (TGA). Thrombin generation in the presence of 1 pM tissue factor and 4 µM phospholipid, was measured using the calibrated automated thrombogram (CAT) system in citrated platelet poor hemophilia A inhibitor (88-160 Bethesda Units) donor plasma or hemophilia B inhibitor (FIX immune-depleted and spiked with FIX neutralizing antibody, 14 Bethesda Units) plasma following the addition of PF-06741086 or aPCC (FEIBA) either alone or in combination. All donors had less than 1% coagulation factor activity. Non-hemophilic plasma from healthy donors alone or spiked in vitro with 16 µg/mL of PF-06741086 was also included in the analysis. Non-hemophilic plasma would have the full complement of coagulation factors. Dose-dependent increases in peak thrombin were observed with the addition of aPCC alone or PF-06741086 alone to the hemophilia plasmas. For combination studies, the aPCC concentration of 1 Unit/mL was selected to correspond to plasma levels that could be achieved clinically post-dosing. The concentration of PF-06741086 at 16µg/mL in these studies was chosen to approximate the Cmax concentration following a single 300 mg subcutaneous dose. Both PF-06741086 (16 µg/mL) and aPCC (1 Unit/mL) decreased the lag time in hemophilia plasma, however, there was not an additive decrease in the lag time with the combination of PF-06741086 and aPCC. The addition of PF-06741086 in combination with aPCC to hemophilia plasma resulted in an increase in thrombin generation including a higher peak thrombin concentration compared to the addition of either alone, but was within the range reported in studies for non-hemophilic normal plasma. To summarize, the addition of aPCC (1 Unit/mL) in combination with PF-06741086 (16µg/mL) in vitro resulted in increased thrombin generation in hemophilia A and hemophilia B inhibitor plasmas without inducing excessive coagulation. Rakhe: Pfizer: Employment. Bowley:Pfizer: Employment. Murphy:Pfizer: Employment. Pittman:Pfizer: Employment.

BACKGROUND: Tissue Factor Pathway inhibitor (TFPI) is a plasma serine protease inhibitor that modulates the initiation of coagulation by directly binding and inhibiting the Tissue Factor (TF)/Factor VIIa/Factor Xa complex. TFPI is a multi-Kunitz domain protein that directly binds to and inhibits both activated Factor Xa (FXa) and FVIIa. Blocking TFPI can act as a bypass therapy by facilitating hemostasis initiated by tissue factor/FVIIa, thereby, compensating for loss of Factor VIII or Factor IX (in hemophilia A or B). PF-06741086, a fully human antibody engineered to inhibit TFPI, exhibits broad cross reactivity to TFPI from numerous species, including mouse. PF-06741086 is being developed as a potential treatment for bleeding disorders including hemophilia A and hemophilia B with and without inhibitors. aPCC (activated Prothrombin complex concentrates or FEIBA, Factor Eight Inhibitor Bypass Agent) is a bypass agent for to control bleed in Hemophilia patients with inhibitors. Since it is a plasma-derived concentrate containing various prothrombin complex coagulation factors in their enzymatic or zymogen form, it is possible that FEIBA could potentially impact the activity of PF-06741086.

Hemophilia A and B are hereditary bleeding disorders that result from deficiencies in the intrinsic coagulation pathway leading to insufficient generation of Factor Xa (FXa) and thrombin to promote stable hemostasis. Coagulation defects are also observed in other inherited rare factor bleeding disorders. The extrinsic pathway of coagulation in these disorders cannot generate sufficient levels of FXa due to the regulation by Tissue Factor Pathway Inhibitor (TFPI). TFPI is a Kunitz-type serine protease inhibitor that negatively regulates thrombin generation by inhibiting the FXa/tissue factor (TF)/Factor VIIa (FVIIa) complex. PF-06741086, a fully human inhibitory monoclonal antibody, binds the Kunitz-2 domain and is currently under development as a potential prophylactic treatment to prevent bleeding episodes in hemophilia A and hemophilia B patients with and without inhibitors. The addition of PF-06741086 in vitro to donor plasma from both healthy normal volunteers and hemophilia patients promoted thrombin generation and restored hemostasis in vivo in murine hemophilia bleeding models. Pharmacological effects of PF-06741086 on thrombin generation were also observed in a healthy volunteer Phase 1 study. Other rare disease coagulopathies also result in the insufficient generation of thrombin. In this study, the potential of PF-06741086 to restore thrombin generation in rare disease plasma was explored.