Human Factor VII Research Articles

Comparing the Yield of Recombinant Human Factor VII Protein Expressed by the rDNA-Promoter with the CMV-Promoter in Iranian Lizard Leishmania.

Iranian Lizard Leishmania (I.L.L) is a nonpathogenic Leishmania strain. Due to its advantages, several recombinant proteins have been produced in this host. However, I.L.L shows a lower yield of recombinant protein expression compared to other commercial hosts. Considering the role of protease enzymes in protein digestion, we selected cysteine protease B (CPB) to investigate its impact on recombinant protein yield in I.L.L. we generated gene knockouts by utilizing homologous recombination (HR) and CRISPR methods. To assess the efficacy of the designed construct, we compared the yield of recombinant human factor VII (rhFVII) production between cells transfected with the pLEXSY-hyg2-FVII vector and the CMV-promoter-based construct (pF7cmvneo). The knockout of a single CPB gene allele through the HR method or the complete knockout of all alleles through the CRISPR method led to cell death. This outcome suggests that even the deletion of a single CPB gene allele diminishes the protein to a level insufficient for the survival of I.L.L, indicating a critical dependency on the presence of this protein for the organism's viability. rhFVII exhibited a greater expression yield with the pLEXSY construct compared to the pF7cmvneo construct in I.L.L. The lower expression rate of pF7cmvneo may be influenced by epigenetic factors related to the CPC gene or the RNA polymerase used for the expression of that promoter. Therefore, considering alternative integration targets for CMV-promoter-based constructs and incorporating UTR sequences of I.L.L high-expression proteins in the vector may enhance recombinant protein expression rates.

Elevated plasma levels of factor VIII enhance arterial thrombus formation on erosive smooth muscle cell-rich neointima but not normal intima in rabbits

BackgroundPlaque erosion, a type of coronary atherothrombosis, involves superficial injury to smooth muscle cell (SMC)-rich plaques. Elevated levels of coagulation factor VIII (FVIII) correlate with an increased ischemic heart disease risk. FVIII may contribute to thrombus formation on eroded plaques. AimsWe aimed to elucidate the role of elevated FVIII in arterial thrombus formation within SMC-rich neointima in rabbits. Methods and resultsWe assessed the effect of recombinant human FVIII (rFVIII) on blood coagulation in vitro and platelet aggregation ex vivo. An SMC-rich neointima was induced through balloon injury to the unilateral femoral artery. Three weeks after the first balloon injury, superficial erosive injury and thrombus formation were initiated with a second balloon injury of the bilateral femoral arteries 45 min after the administration of rFVIII (100 IU/kg) or saline. The thrombus area and contents were histologically measured 15 min after the second balloon injury. rFVIII administration reduced the activated partial thromboplastin time and augmented botrocetin-induced, but not collagen- or adenosine 5′-diphosphate-induced, platelet aggregation. While rFVIII did not influence platelet-thrombus formation in normal intima, it increased thrombus formation on SMC-rich neointima post-superficial erosive injury. Enhanced immunopositivity for glycoprotein IIb/IIIa and fibrin was observed in rFVIII-administered SMC-rich neointima. Neutrophil count in the arterial thrombus on the SMC-rich neointima correlated positively with thrombus size in the control group, unlike the rFVIII group. ConclusionsIncreased FVIII contributes to thrombus propagation within erosive SMC-rich neointima, highlighting FVIII's potential role in plaque erosion-related atherothrombosis.

The identification of a novel compound heterozygous mutation in hereditary human coagulation factor VII deficiency following a bamboo leaf green snake bite.

Hereditary factor VII (FVII) deficiency is an uncommon autosomal recessive disorder associated with mutations in the F7 gene, and laboratory investigations usually reveal isolated prolongation in prothrombin time (PT)/international normalized ratio (INR). Venom-induced consumptive coagulopathy (VICC) is distinguished by the activation of the coagulation pathway, which is triggered by procoagulant toxins in snake venom. Diagnosing snakebites in patients with hereditary FVII deficiency presents a challenge because prolonged time PT/INR is considered the most valuable diagnostic method for VICC. Therefore, it is possible that certain patients may not promptly receive an accurate diagnosis of hereditary FVII deficiency. We present a pedigree featuring hereditary FVII deficiency, which was diagnosed through Sanger sequencing, following a bamboo leaf green snake bite.

Eptacog beta for the management of patients with haemophilia A and B with inhibitors: A European perspective.

Eptacog beta (activated), a recombinant human factor VIIa (rFVIIa), was approved by the US Food and Drug Administration (FDA) in 2020 (SEVENFACT®, LFB & HEMA Biologics) and the European Medicines Agency (EMA) in 2022 (CEVENFACTA®, LFB). In Europe, eptacog beta is indicated for the treatment of bleeds and the prevention of bleeds during surgery or invasive procedures in adults and adolescents (≥12 years old) with congenital haemophilia A or B with high-titre inhibitors (≥5 BU) or with low-titre inhibitors who are expected to have a high anamnestic response to factor VIII or factor IX, or to be refractory to increased dosing of these factors. The efficacy and safety of eptacog beta were evaluated in three Phase III clinical studies, PERSEPT 1, 2 and 3. For the EMA filing dossier, the analysis of data from PERSEPT 1 and 2 differed from the analysis used to support the filing in the US. In this review, we summarise current data regarding the mode of action, clinical efficacy and safety of eptacog beta for the management of haemophilia A and B in patients with inhibitors from a European perspective. In addition to providing a valuable summary of the analyses of the clinical data for eptacog beta conducted for the EMA, our review summarises the potential differentiators for eptacog beta compared with other current bypassing agents.

Simoctocog alfa (Nuwiq®) in children: early steps in life's journey for people with severe hemophilia A.

People with severe hemophilia A usually experience their first bleed early in life. In children with severe hemophilia A, primary prophylaxis is recommended to prevent recurrent and potentially life-threatening bleeds that significantly impact day-to-day life. Factor VIII (FVIII) prophylaxis is well-established in children and has been shown to reduce the development of hemophilic arthropathy. However, a major challenge of FVIII therapy is the development of neutralizing anti-FVIII antibodies (FVIII inhibitors). Simoctocog alfa (Nuwiq®) is a human cell line-derived recombinant FVIII (rFVIII) whose immunogenicity, efficacy, and safety have been studied in 167 children with severe hemophilia A across two prospective clinical trials and their long-term extensions. In 105 previously untreated children, the inhibitor rate of 16.2% for high-titer inhibitors (26.7% for all inhibitors) was lower than published rates for hamster cell line-derived rFVIII products. There was no inhibitor development in previously untreated children with non-null F8 mutations and in previously treated children. In a case series of 10 inhibitor patients, 8 (80%) underwent successful immune tolerance induction with simoctocog alfa with a median time to undetectable inhibitor of 3.5 months. In an analysis of 96 children who enrolled in the extension studies and received long-term simoctocog alfa prophylaxis for up to 5 years, median spontaneous, joint, and total annualized bleeding rates were 0.3, 0.4, and 1.8, respectively. No thromboembolisms were reported in any of the 167 children, and there were no treatment-related deaths. Optimal care of children should consider several factors, including minimization of inhibitor development risk, maintaining tolerance to FVIII, highly effective bleed prevention and treatment, safety, and impact on long-term outcomes such as bone and joint health. In this context we review the pediatric clinical data and ongoing studies with simoctocog alfa.

GO-8: Stable Expression of Factor VIII over 5 Years Following Adeno-Associated Gene Transfer in Subjects with Hemophilia a Using a Novel Human Factor VIII Variant

Background: GO-8 (ClinicalTrials.gov: NCT03001830) is a study of liver-directed adeno-associated virus (AAV) gene therapy for haemophilia A (HA) that uses a factor VIII (FVIII) variant containing a 17 amino-acid peptide comprising six N-linked glycosylation motifs from the human FVIII B-domain (AAV-HLP-hFVIII-V3). In preclinical studies, AAV-HLP-hFVIII-V3 mediated a 3-fold higher FVIII expression when compared to an identical AAV construct encoding the hFVIII-SQ variant used in most HA gene therapy trials. Methods: In a multi-centre, open-label, non-randomised, phase I/II clinical trial, we assessed the safety and efficacy of escalating doses of AAV-HLP-hFVIII-V3 pseudotyped with an AAV8 capsid in adults with severe haemophilia A (FVIII activity ≤1%). All participants received prophylactic glucocorticoids, with or without tacrolimus, with the aim of reducing the risk of vector-related transaminase elevation. The primary endpoints were safety and efficacy. Efficacy was assessed by measuring FVIII activity (FVIII: C) using both chromogenic and one-stage clotting assays and factor consumption pre and post-gene therapy. Results: As of May 31 2023, 12 participants were enrolled sequentially into one of four vector doses: 6×1011 vector genomes (vg)/kg body weight (n=1), 2×1012 vg/kg (n=3), 4×1012 vg/kg (n=3), or 6×1012 vg/kg (n=5). All participants were on FVIII prophylaxis prior to gene therapy. The most common vector-related adverse event was an elevation in liver aminotransferase levels, which occurred in 10 of 12 participants. In 7 of the 8 participants treated at doses ≥4×1012 vg/kg, recurrent elevation in aminotransferase levels was observed during the first 12 months, often associated with tapering of immunosuppression. This resulted in a reduction in transgene expression from peak levels in all participants, with a complete loss of transgenic protein in one participant. Vector-related elevation in aminotransferase was not observed after the 12-month time point in long-term follow-up. Mean chromogenic FVIII: C levels at 12 months after gene therapy were 3 IU/dL in the 6×1011 vg/kg cohort, 13±9IU/dL (range: 2-19 IU/dl) in the 2×1012 vg/kg cohort, 8±1IU/dl in the 4×1012 vg/kg cohort (range: 7-9 IU/dl) and 22±34 IU/dl in the 6×1012 vg/kg cohort (range 1-82 IU/dl). Transgene expression was then stably maintained over a median follow-up of 3 years (range: 0.2-5 years) from the level achieved 1-year post-infusion, best illustrated by the data from the 2×1012 and 4×1012 vg/kg cohorts shown in Figure 1. FVIII: C was, on average 2-fold higher when measured using a one-stage clotting assay compared to the chromogenic method. Nine of the 12 participants remained off prophylaxis after gene therapy for the duration of the follow-up period. Baseline mean and median annualised factor VIII use was 4097 and 4657 IU/kg per year before gene therapy. Following gene therapy, the mean and median annualised factor VIII concentrate use reduced across all participants to 1186 and 61 IU/kg (One sample t-test p=0.0009), respectively. No FVIII inhibitors or thrombotic events were reported for the duration of the study. Conclusion: A single infusion of AAV-HLP-hFVIII-V3 resulted in stable FVIII expression over a follow-up period of up to 5 years in participants with severe haemophilia A. A high rate of liver aminotransferase elevation following gene transfer impacted transgene expression. However, 9 of the 12 participants were able to discontinue FVIII prophylaxis over the duration of the study, resulting in a significant reduction in FVIII concentrate usage.

Low Rebleed Incidence with Eptacog Beta Treatment through 24 and 48 Hours in Children (

Introduction Persons with hemophilia A or B and inhibitors (PwHABI) who are not on prophylaxis may have high rates of bleeding, and, if not treated effectively, may have significant morbidity related to rebleeding. Dosing strategies of bypassing agents (BPAs) are variable in clinical practice, and following a period of initial bleed control, rebleeding may occur. The development of additional bypassing agents that can minimize the incidence of rebleeding in this patient population is an important medical need. Eptacog beta is a recombinant activated human factor VII approved in the US and Mexico (Sevenfact®, 2020), as well as the EU and UK (Cevenfacta®, 2022) as a BPA for the treatment of bleeding episodes (BEs) in PwHABI aged 12 years and older using two initial dose regimens (IDRs) of 75 or 225 µg/kg. In the EU and UK, eptacog beta is also indicated for preventing bleeding in the setting of surgery and other invasive procedures. Approval in the US and Mexico was based on demonstrated efficacy in the pivotal phase 3 PERSEPT 1 trial (NCT02020369), which included 27 adolescent and adult PwHABI who treated 465 mild or moderate BEs. Bleed resolution at 24 hours post-initial infusion was 96.7% and 99.5% for 75 and 225 µg/kg IDRs, respectively, with a single bleed recurrence observed before 24 hours. The subsequent phase 3 PERSEPT 2 trial (NCT02448680) enrolled 25 PwHABI <12 years of age who treated 546 mild or moderate BEs. Bleed resolution at 24 hours post-initial infusion in these pediatric patients was 97.4% and 98.0% for 75 and 225 µg/kg IDRs, respectively. The objective of this analysis is to evaluate the incidence of rebleeding when treating mild or moderate BEs in PwHABI <12 years of age with eptacog beta during PERSEPT 2 at 24 and 48 hours post-initial dose. Methods PwHABI in PERSEPT 2 were randomized to either the 75 or the 225 µg/kg IDR to treat BEs and were crossed over to the alternate IDR every 3 months. No subjects in PERSEPT 2 were using prophylaxis. Subjects assigned to the 75 µg/kg IDR treated BEs with initial doses of 75 µg/kg eptacog beta followed by 75 µg/kg q3h as needed. Subjects assigned to the 225 µg/kg IDR treated BEs with an initial 225 µg/kg eptacog beta dose, followed after 9 hours by 75 µg/kg q3h as needed. Mild or moderate BE treatment success at a given time point was assessed by the patient or caregiver and was defined as obtaining a hemostasis evaluation of “excellent” or “good” on a 4-point scale with no additional eptacog beta being infused, no alternative hemostatic agents or blood products being used, and no increase in pain following the first “excellent” or “good” assessment. For this analysis, a BE was considered a rebleed if occurring at the same anatomical location as the original bleed, and within 24 or 48 hours of the initial infusion for the original bleed. Results During the study, subjects treated 546 BEs. Nine rebleeds occurred among 4 of the 25 children within 24 hours, and 13 rebleeds occurred among 6 children within 48 hours. The rebleed incidence and 95% confidence intervals (CIs) through 24 hours was 1.3% [95% CI: 0.0%, 3.6%] for the 75 µg/kg IDR and 2.0% [95% CI: 0.0%, 3.9%] for the 225 µg/kg IDR. The corresponding rebleed incidence and 95% CIs through 48 hours for the 75 and 225 µg/kg IDRs were 2.1% [95% CI: 0.0%, 4.5%] and 2.6% [95% CI: 0.4%, 4.9%], respectively. The differences in rebleed incidence between the two IDRs were not statistically significant at either 24 or 48 hours (Figure 1). The overall rebleed incidence in PERSEPT 2 was 1.6% [95% CI: 0.0%, 3.7%] through 24 hours and 2.4% [95% CI: 0.2%, 4.5%] through 48 hours. No treatment-related adverse events or thrombotic complications were observed. Conclusions Eptacog beta demonstrated a low overall incidence of rebleeding through 24 hours (1.6%) and 48 hours (2.4%) in PwHABI <12 years of age during PERSEPT 2. Rebleeding was similarly low for either IDR at both time points. These findings align with those previously reported for PERSEPT 1 in adolescent and adult patients. Combined with the high efficacy at 24 hours (>96%) observed in both trials and both IDRs, these results suggest that eptacog beta can provide clinicians and caregivers with a treatment option that can establish and maintain hemostatic control of mild or moderate BEs in PwHABI of all ages.

In Utero Cell Treatment of Hemophilia A Mice via Human Amniotic Fluid Mesenchymal Stromal Cell Engraftment.

Hemophilia is a genetic disorder linked to the sex chromosomes, resulting in impaired blood clotting due to insufficient intrinsic coagulation factors. There are approximately one million individuals worldwide with hemophilia, with hemophilia A being the most prevalent form. The current treatment for hemophilia A involves the administration of clotting factor VIII (FVIII) through regular and costly injections, which only provide temporary relief and pose inconveniences to patients. In utero transplantation (IUT) is an innovative method for addressing genetic disorders, taking advantage of the underdeveloped immune system of the fetus. This allows mesenchymal stromal cells to play a role in fetal development and potentially correct genetic abnormalities. The objective of this study was to assess the potential recovery of coagulation disorders in FVIII knockout hemophilia A mice through the administration of human amniotic fluid mesenchymal stromal cells (hAFMSCs) via IUT at the D14.5 fetal stage. The findings revealed that the transplanted human cells exhibited fusion with the recipient liver, with a ratio of approximately one human cell per 10,000 mouse cells and produced human FVIII protein in the livers of IUT-treated mice. Hemophilia A pups born to IUT recipients demonstrated substantial improvement in their coagulation issues from birth throughout the growth period of up to 12 weeks of age. Moreover, FVIII activity reached its peak at 6 weeks of age, while the levels of FVIII inhibitors remained relatively low during the 12-week testing period in mice with hemophilia. In conclusion, the results indicated that prenatal intrahepatic therapy using hAFMSCs has the potential to improve clotting issues in FVIII knockout mice, suggesting it as a potential clinical treatment for individuals with hemophilia A.

Transplacental delivery of therapeutic proteins by engineered immunoglobulin G: a step toward perinatal replacement therapy

BackgroundTransplacental delivery of maternal immunoglobulin G (IgG) provides humoral protection during the first months of life until the newborn’s immune system reaches maturity. The maternofetal interface has been exploited therapeutically to replace missing enzymes in the fetus, as shown in experimental mucopolysaccharidoses, or to shape adaptive immune repertoires during fetal development and induce tolerance to self-antigens or immunogenic therapeutic molecules. ObjectivesTo investigate whether proteins that are administered to pregnant mice or endogenously present in their circulation may be delivered through the placenta. MethodsWe engineered monovalent immunoglobulin G (FabFc) specific for different domains of human factor VIII (FVIII), a therapeutically relevant model antigen. FabFc was injected with exogenous FVIII into pregnant severe hemophilia A mice or pregnant mice expressing human FVIII following AAV8-mediated gene therapy. FabFc and FVIII were detected in the pregnant mice and/or fetuses by enzyme-linked immunosorbent assay and immunohistochemistry. ResultsAdministration of FabFc to pregnant mice allowed the maternofetal delivery of FVIII in a FcRn-dependent manner. FVIII antigen levels achieved in the fetuses represented 10% of normal plasma levels in the human. We identified antigen/FabFc complex stability, antigen size, and shielding of promiscuous protein patches as key parameters to foster optimal antigen delivery. ConclusionOur results pave the way toward the development of novel strategies for the in utero delivery of endogenous maternal proteins to replace genetically deficient fetal proteins or to educate the immune system and favor active immune tolerance upon protein encounter later in life.

Maladaptive lymphangiogenesis is associated with synovial iron accumulation and delayed clearance in factor VIII–deficient mice after induced hemarthrosis

BackgroundMechanisms of iron clearance from hemophilic joints are unknown. ObjectivesTo better understand mechanisms of iron clearance following joint bleeding in a mouse model of hemophilia. MethodsHemarthrosis was induced by subpatellar puncture in factor VIII (FVIII)–deficient (FVII−/−) mice, +/− periprocedural recombinant human FVIII, and hypocoagulable (HypoBALB/c) mice. HypoBALB/c mice experienced transient FVIII deficiency (anti-FVIII antibody) at the time of injury combined with warfarin-induced hypocoagulability. Synovial tissue was harvested weekly up to 6 weeks after injury for histological analysis, ferric iron and macrophage accumulation (CD68), blood and lymphatic vessel remodeling (αSMA; LYVE1). Synovial RNA sequencing was performed for FVIII−/− mice at days 0, 3, and 14 after injury to quantify expression changes of iron regulators and lymphatic markers. ResultsBleed volumes were similar in FVIII−/− and HypoBALB/c mice. However, pronounced and prolonged synovial iron accumulation colocalizing with macrophages and impaired lymphangiogenesis were detected only in FVIII−/− mice and were prevented by periprocedural FVIII. Gene expression changes involved in iron handling (some genes with dual roles in inflammation) and lymphatic markers supported proinflammatory milieu with iron retention and disturbed lymphangiogenesis. ConclusionAccumulation and delayed clearance of iron-laden macrophages were associated with defective lymphangiogenesis after hemarthrosis in FVIII−/− mice. The absence of such findings in HypoBALB/c mice suggests that intact lymphatics are required for removal of iron-laden macrophages and that these processes depend on FVIII availability. Studies to elucidate the biological mechanisms of disturbed lymphangiogenesis in hemophilia appear critical to develop new therapeutic targets.

Design of a Real-World Observational Study in Previously Untreated and Minimally Treated Hemophilia A Patients: Protect-NOW.

Background The efficacy, safety, and immunogenicity of each of Octapharma's factor VIII (FVIII) products, Nuwiq, octanate, and wilate, have been investigated in previously untreated patients (PUPs) with severe hemophilia A in prospective clinical trials. The aim of the Protect-NOW study is to evaluate the effectiveness, safety, and utilization patterns of Nuwiq, octanate, and wilate in PUPs and minimally treated patients (MTPs; <5 exposure days [EDs] to FVIII concentrates or other blood products containing FVIII) with severe hemophilia A in a real-world setting. Real-world data provide valuable information that complement data obtained from interventional clinical trials. Methods Protect-NOW (ClinicalTrials.gov identifier: NCT03695978; ISRCTN identifier: 11492145) is a real-world study in PUPs and MTPs treated with either the human cell line-derived recombinant FVIII Nuwiq (simoctocog alfa) or a plasma-derived FVIII concentrate containing von Willebrand factor (octanate or wilate). It is a prospective and (partly) retrospective, observational, international, noncontrolled, noninterventional study. A total of 140 PUPs and MTPs with severe hemophilia A will be enrolled across around 50 specialized centers worldwide and followed for either 100 EDs or a maximum period of 3 years from ED1. The primary objectives are to assess effectiveness in the prevention and treatment of bleeding episodes and overall safety, including inhibitor development. The secondary objectives are to assess utilization patterns (including dosage and frequency of administration) and the effectiveness in surgical prophylaxis. Conclusions The Protect-NOW study will provide information on the treatment of PUPs and MTPs in routine clinical practice, which will help guide clinical decision making for treating these patients in the future.

A comprehensive evaluation of stable expression “hot spot” in the ScltI gene of Chinese hamster ovary cells

The Chinese hamster ovary (CHO) cell is the most widely used biopharmaceutical expression system, but its long-term expression is unstable. This issue can be effectively addressed by site-specific integration of exogenous genes into the genome. Therefore, exogenous protein sites with stable expression in the CHO cell genome must be identified. CRISPR/Cas9 technology was used in this study to integrate various exogenous genes into the ScltI site as a "hot spot" at the CHO-K1 cell genome NW_003614095.1, and the stability and adaptability of exogenous genes expressed at the site were investigated. Flow cytometry sorting technology was used to obtain positive monoclonal cell lines that expressed either intracellular protein green fluorescent protein (EGFP) or secretory protein human serum albumin (HSA). For 60 passages, the positive monoclonal cell lines' cell growth cycles and exogenous protein expression were both observed. The results demonstrated that integrating the gene encoding exogenous proteins into the ScltI site had no effect on cell growth. The fluorescence intensity of EGFP was similar after 60 passages, and the expression of HSA increased slightly. Additionally, the super-monomeric protein VWF hydrolase (ADAMTS13) (190kDa), human coagulation factor VII (FVII) (55kDa), and interferon α2b (12kDa) were integrated into the ScltI site for expression. In conclusion, the site located in the first exon of the ScltI gene within the CHO-K1 cell genome NW_003614095.1 is an ideal "hot spot" for the stable expression of various exogenous proteins. KEY POINTS: • The site-specific integration strategy of an exogenous gene in CHO cells was established for the ScltI site. • The genes for EGFP and HSA were site-directed integrated and stably expressed at the ScltI site. • The ScltI site fulfills the expression of exogenous proteins of different molecular weight sizes (15-190kDa).

COMPARING THE EFFECTIVENESS OF CONCENTRATE OF ALL PROTHROMBIN COMPLEX FACTORS WITH RECOMBINANT HUMAN COAGULATION FACTOR VIIa IN THE TREATMENT OF BLEEDING AFTER CARDIAC SURGERY

Aim: The authors evaluated the effectiveness of treatment with recombinant human coagulation factor VIIa and concentrate of all prothrombin complex factors in patients with massive postoperative bleeding that could not be controlled with traditional therapy. Materials and Methods: In the period from 2020 to 2021, recombinant human coagulation factor VIIa was administered to 18 patients after cardiac surgery (group I), while the concentrate of all prothrombin complex factors was administered to 16 patients postoperatively (group II). During this period, 647 patients were operated on. The patients had normal coagulation screening tests (APTT, INR, TT, fibrinogen level, and PLT level) before surgery. Mean blood loss before and after administration of eptacog alfa and the total prothrombin complex concentrate was assessed. The mean dose of eptacog alfa was 30.95 mcg/kg b.w., and the total prothrombin complex factor concentrate dose was 14.17 mcg/kg b.w. After transfusion with red blood cell concentrate, fresh frozen plasma, and platelet concentrate, in the absence of improvement in the dynamics of postoperative drainage, it was decided to include recombinant human coagulation factor VIIa or a concentrate of all prothrombin complex factors in the treatment. Results: After administration of recombinant human coagulation factor VIIa at a dose of 30.95 mcg/kg b.w., bleeding stopped in 12 patients, but the remaining 6 patients required reoperation due to persistently high drainage. The decision to perform a rethoracotomy was made by a team of cardiothoracic surgeons and anesthesiologists, taking into account the dynamics of drainage (bleeding) and the hemodynamic stability of the patient. After the administration of concentrate of all prothrombin complex factors at a dose of 14.17 U/kg b.w., bleeding stopped in 12 patients. Four patients required reoperation due to persistent bleeding. Conclusions: Treatment with recombinant human coagulation factor VIIa and concentrate of all prothrombin complex factors is effective and safe for cardiac surgery patients.

The Pharmacokinetics and Pharmacodynamics of A Novel Recombinant Activated Human Factor VII, GEN-0828, in Hemophilia B Mice

GEN-0828, a proposed clinical candidate for hemophilia and trauma hemorrhage treatment, is a novel recombinant activated human factor VII (rFVIIa). The purpose of this paper is to compare the pharmacokinetics and pharmacodynamics of GEN-0828 in hemophilia B mice with those of NovoSeven®, the only marketed rFVIIa product worldwide., GEN-0828 and NovoSeven® showed similar affinity bioactivity to recombinant tissue factor (rTF) in vitro. Pharmacodynamics data indicated a generally similar hemostatic efficacy (ED50) of GEN-0828 (10.91 KIU·kg−1) and NovoSeven® (18.91 KIU·kg−1) at the doses studied in hemophilia B mice, while GEN-0828 represented a lower initial effective dosage compared with that of NovoSeven® in terms of both blood loss and APTT. GEN-0828 exhibited linear pharmacokinetic profiles in hemophilia B mice at the 30–338 KIU·kg−1 dose range, the comparative pharmacokinetic study with NovoSeven® indicated better characteristics than NovoSeven® in terms of the appropriate higher maximal concentration (Cmax) and area under the plasma concentration–time curve (AUClast) and longer mean residence time (MRT). In conclusion, GEN-0828 was a promising new type of rFVIIa compound with favourable pharmacokinetic and pharmacodynamic profiles.

Biological activity of a new recombinant human coagulation factor VIII and its efficacy in a small animal model

Deficiency in human coagulation factor VIII (FVIII) causes hemophilia A (HA). Patients with HA may suffer from spontaneous bleeding, which can be life-threatening. Recombinant FVIII (rFVIII) is an established treatment and prevention agent for bleeding in patients with HA. Human plasma-derived FVIII (pdFVIII), commonly used in clinical practice, is relatively difficult to prepare. In this study, we developed a novel B-domain-deleted rFVIII, produced and formulated without the use of animal or human serum-derived components. rFVIII promoted the generation of activated factor X and downstream thrombin, and, similar to that of other available FVIII preparations, its activity was inhibited by FVIII inhibitors. In addition, rFVIII has ideal binding affinity to human von Willebrand factor. Activated FVIII (FVIIIa) could be degraded by activated protein C and lose its procoagulant activity. In vitro, commercially available recombinant FVIII (Xyntha) and pdFVIII were used as controls, and there were no statistical differences between rFVIII and commercial FVIII preparations, which demonstrates the satisfactory efficacy and potency of rFVIII. In vivo, HA mice showed that infusion of rFVIII rapidly corrected activated partial thromboplastin time, similar to Xyntha. Moreover, different batches of rFVIII were comparable. Overall, our results demonstrate the potential of rFVIII as an effective strategy for the treatment of FVIII deficiency.

Determination of rFVII Concentration in Cell Culture Supernatant Using VIISelect Resin and RP-HPLC-UV

Background: Recombinant activated human coagulation factor VIIa (rFVIIa), a vitamin K-dependent serine protease, was originally developed by Novo Nordisk® for the treatment of patients with FVII deficiency as well as patients with hemophilia A and B with inhibitors against FVIII or FIX. The gene for human FVII is cloned and expressed in baby hamster kidney (BHK) cells, and the protein is secreted into the culture media, which is then converted to the active form during chromatographic purification steps. When secreted into the culture media, measuring the concentration levels of FVII is needed in order to monitor, control and optimize the production processes. However, because of the high complexity of such media, reliable analytical techniques are required for this purpose. Objective: This work focuses on the analytical application of VIISelect resin (GE Healthcare) as a highly selective adsorbent for cleanup and preconcentration of rFVII in culture supernatant before analysis by reversed-phase high-performance liquid chromatography (RP-HPLC). Method: Empty 1 ml SPE cartridges were packed with VIISelect resin. Four types of solutions were used for the sample preparation. The RP-HPLC separation was conducted on a C4 column with UV detection. Results: The method shows recoveries greater than 97% in culture medium with relative standard deviations (RSDs) of less than 2%. The limits of detection and quantification were 0.039 mg/L and 0.12 mg/L, respectively. Conclusion: The method showed better performance in terms of precision and accuracy for rFVIIa determination in cell culture supernatant compared to other techniques, like ELISA and SDSPAGE.

Guiding Pups with Severe Hemophilia a and Their Parents from Diagnosis to a Full Prophylactic Schedule Mitigating the Risk of Bleed and the Risk for Inhibitor Formation - a Single Centre Approach

Introduction Pediatricians treating newly diagnosed severe hemophilia A patients are confronted with the challenge to protect the children against bleeds that are potentially life-threatening or have long-term damaging effects on joints. A well-established protective effect has early prophylaxis with coagulation factor VIII (FVIII). However, higher doses and "peak treatment moments” have been associated with a rate of inhibitor formation against factor VIII in up to 40%1,2. Another still controversial topic is the impact of the product choice on inhibitor risk. There is some evidence that plasma-derived factor VIII, or certain concentrates, are less immunogenic3,4. We report our experience with an individualized approach to managing PUPs at a single hemophilia treatment center (HTC) in Germany. Methods PUPs with severe hemophilia A (FVIII:C <1%) treated at the Gerinnungszentrum Rhein-Ruhr (GZRR), Germany, between January 2013 and July 2022 were followed prospectively. Our treatment approach includes a thorough training of parents to early detect signs of bleeds and avoid injuries with special focus on the first 50 exposure days (EDs). Patients are treated with a human plasma-derived FVIII (pdFVIII/VWF) concentrate for at least the first 50 EDs with an individually tailored treatment schedule that leads to a full prophylactic schedule. Initial doses are chosen low, given weekly or every 10 days and escalated according to bleeding tendency. In case of more severe bleeds, high doses of 60-80 IU/kg are chosen to avoid treatment for more than 2 days. Minor bleeds or hematomas are not always treated. The parents stay in close contact with the center so that the decision to treat with pdFVIII/VWF can be done on a case-by-case basis. We monitor joint health by prophylactic physiotherapy and regular ultrasound every 3-6 months. Non-urgent surgical procedures are postponed, and venous access system implants are avoided within the first 100 EDs. Inhibitor levels are measured using the modified Bethesda assay every 3-4 EDs until ED 100, and every 3 months thereafter for 2 years. Results Data from 38 consecutive PUPs with severe hemophilia A were collected, of which 35 (92.1%) have been treated with FVIII. Of the 38 patients, 34 started regular treatments within the first 10 EDs. The schedule was tailored individually ranging from 21 IU/kg every 10 days up to 40 IU/kg twice per week. One patient was treated on-demand for an intracranial bleed from ED 1-100. Only 3 spontaneous bleeds in 3 patients were treated; those bleeds had led to diagnosis. All other patients remained free of spontaneous bleeds requiring treatment. One patient developed a high titer inhibitor that was diagnosed at ED 4. He had received 35 IU/kg for treatment of a hematoma and 2 further equal doses in 2 separate weeks. The mother had reported unusual hematoma formation with a dark and more extended middle part following a slow resolution already before the Bethesda assay was positive. Discussion Our approach to managing PUPs includes treatment with human pdFVIII/VWF for at least the first 50 EDs. During this time, we treat bleeds only when considered necessary but then with high doses to minimize further infusions. We report 1 inhibitor development in a prospective cohort of 38 patients. In contrast, between 2003 and 2012, four of nine (44%) PUPs treated at our center developed inhibitors during the first 20 EDs (2 low-, 2 high-titer inhibitors). At that time, we used recombinant FVIII (rFVIII) for prophylaxis in most patients and were less selective about which and how to treat bleeds with FVIII. After at least 50 EDs we usually discuss the option to switch patients from pdFVIII/VWF to rFVIII as the injection volume is generally lower. Conclusion With a rate of 1/30 (3.3%) patients with more than 20 EDs developing an inhibitor, these data support the use of our individualized management approach allowing for a tolerization phase with FVIII on the way to a full prophylactic schedule. We consider the close contact and thorough training of the parents/guardians crucial in order to closely monitor the patients and assure adequate protection from bleeds. 1. Gouw SC et al. J Thromb Haemost 2007; 5(7): 1383-90. 2. Gouw SC et al. Blood 2013; 121(20): 4046-55. 3. Klukowska A, et al. Haemophilia 2018; 24(2): 221-8. 4. Peyvandi F et al. A. New Engl J Med 2016; 374(21): 2054-64. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Augmenting the Persistence and Efficacy of Engineered Regulatory T Cells to Suppress Anti-Drug Antibody Formation

Background Adoptive cell therapy (ACT) using engineered antigen specific regulatory T cells (Tregs) is a powerful tool for delivering immune tolerance in autoimmune disease, transplantation and in suppressing undesirable anti-drug antibody (ADA) responses against therapeutic proteins. An important example of the latter is hemophilia A (HA), a genetic disorder characterized by deficiency of the blood clotting protein, factor VIII (FVIII). Replacement therapy with recombinant FVIII can be neutralized by ADA development in up to 30% of severe HA patients. We previously showed that ACT with Tregs expressing a chimeric CD3ε based T cell receptor fusion construct (TRuC), encoding extracellular antibody fragments with specificity to human FVIII, suppressed ADA formation to FVIII in a preclinical HA model [Mol Ther. 2021 29(9):2660-2676]. Ensuring adequate in vivo persistence and a suppressive phenotype without risk of plasticity are major concerns that impede translation of ACT with engineered Tregs. Here we evaluate a synthetic single chain antibody fusion IL2 cytokine product (IL2-IC) to selectively expand adoptively transferred FVIII-TRuC Tregs in vivo. We then combine IL2-IC delivery with FVIII-TRuC Tregs expressing the co-inhibitory receptor PD-L1 to test improved suppression to ADA formation. Methods IL2-IC was generated by fusing IL-2 cytokine to the anti-IL-2 monoclonal antibody F5111 via a flexible linker. This complex specifically delivers IL2 to cells that express high levels of IL2 receptor α (IL2Rα) (i.e., Tregs) while avoiding interaction with cells that express low levels (i.e., Tconv, NK and CD8+ T cells). To assess whether IL2-IC could expand TRuC Tregs in vivo, 2x106 FVIII TRuC Tregs were injected intravenously (IV) into HAmice followed 5 hrs later with low dose IL2-IC or IL2 fused to an irrelevant Ab (FITC-IC) as control. 24 hrs later, mice received 2 international units (IU) of FVIII, and spleens were assayed on days 3 and 6. Murine PD-L1 was co-expressed in FVIII TRuC Tregs and in vitro assessed by stimulating TRuC PDL1 Tregs with 2IU FVIII alone or in combination with 2 ug/ml of PD1-Fc to understand the impact of PDL1 signaling on the Treg phenotype. In vivo, 0.5x106 FVIII TRuC PD-L1 Tregs were IV injected in HAmice followed 5 hrs later by mock, IL2-IC or FITC-IC IV injections. 24 hrs later, mice received weekly IV injections of 1IU FVIII. Results IL2-IC treated FVIII TRuC Tregs were preferentially expanded over endogenous Tregs (fold expansion 7.47 vs 4.56 respectively) in spleens on day 3 post ACT. Whereas both IL2-IC and FITC-IC treated FVIII TRuC Tregs had higher division indices over mock treatment (4.047 ± 0.03 and 5.070 ± 0.07 over 2.00 ± 0.00), only IL2-IC treated FVIII TRuC Tregs showed increased frequencies (1.72% ± 0.376% of CD4+ T cells) as compared to FITC-IC (0.28% ± 0.005% of CD4+ T cells) or mock treatment (0.23% ± 0.023% of CD4+ T cells). FITC-IC treatment nonspecifically expanded CD8+ Tconv cells as well as NK cells, whereas IL2-IC selectively expanded Tregs. PD1-Fc triggered PD-L1 signals in FVIII stimulated TRuC Tregs resulted in significant upregulation of Treg associated activation markers FoxP3, CD69, LAP, CTLA4, PD1, ICOS and BCL2 in vitro. In vivo, IL2-IC administration combined with FVIII TRuC PD-L1 ACT led to complete suppression of ADA formation in HA mice. At 4 weeks, mice that only received FVIII infusions developed high functional ADA titers (29.53 ± 19.58 BU/mL), while IL2-IC, TRuC PDL1 Treg and TRuC PDL1 Treg + IL2-IC groups had significantly lower titers of 1.117 ± 0.952 BU/mL, 1.805 ± 0.702 BU/mL and 0.423 ± 0.243 BU/mL, respectively. FITC-IC combined with PDL1 TRuC Tregs resulted in high ADAs of average 109 ± 21.31 BU/mL, confirming the requirement for the IL2Rα targeting antibody to selectively expand Tregs. Conclusion We show that the PD-L1/PD1 axis is an important pathway that can be exploited to improve the suppressive capacity of engineered Tregs. We also demonstrate that exogenous administration of the novel IL2-IC can robustly expand FVIII TRuC Tregs. These 2 strategies can be readily combined to selectively expand and enhance the suppressive capacity of engineered Tregs. The concepts defined here can be extended for improving the persistence and suppressive potential of engineered Tregs for various autoimmune disorders and to prevent transplant rejection. We are currently evaluating long term suppression of ADA responses using this approach.

Eptacog Beta Efficacy in Treating Mild or Moderate Bleeds in Target Joints of Individuals with Hemophilia A or B and Inhibitors in PERSEPT 1

Introduction Recurrent bleeding in the same joint of a patient with hemophilia can lead to painful and chronic inflammation of the synovium, joint swelling, reduced range of motion, and in the long term joint arthropathy and irreversible joint damage. Early bleed resolution in the susceptible joint (known as a target joint) is thought to be crucial for preventing joint arthropathy and other incapacitating sequelae, and thus ultimately preserving joint function and health in persons with hemophilia. Eptacog beta was FDA-approved in 2020 as a recombinant activated human factor VII for the treatment and control of bleeding episodes (BEs) in persons with hemophilia A or B and inhibitors (PwHABI) ages 12 years and older using two initial dose regimens (IDRs) of 75 or 225 µg/kg eptacog beta that were shown to be safe and efficacious. The pivotal phase 3 trial (PERSEPT 1; NCT02020369) included 27 PwHABI ages 12 to 54 years who treated 465 mild or moderate BEs, with treatment success proportions at 12 hours post-initial infusion of 82% and 91% for 75 and 225 µg/kg IDRs, respectively. Objective To evaluate eptacog beta efficacy when treating target and non-target joint BEs in PwHABI from PERSEPT 1, at 12 and 24 hours post-initial dose of eptacog beta. Methods PERSEPT 1 was a global, multicenter, open-label, prospective phase 3 trial using a crossover design (Wang et al., Haemophilia 23, pp. 832-43 [2017]). PwHABI who were not using prophylaxis were randomized to either the 75 or the 225 µg/kg IDR to treat BEs, and were crossed over to the alternate IDR every 3 months. Subjects in the 75 µg/kg IDR treated BEs with initial doses of 75 µg/kg eptacog beta followed by 75 µg/kg q3h as needed. Subjects in the 225 µg/kg IDR treated BEs with an initial 225 µg/kg eptacog beta dose, followed after 9 hours by 75 µg/kg q3h as needed. Mild or moderate BE treatment success at a given timepoint was defined as obtaining a hemostasis evaluation of "excellent" or "good" on a 4-point scale ("excellent" or "good" indicating hemostatic efficacy; "moderate" or "none" indicating lack of efficacy) with no additional eptacog beta being infused, no alternative hemostatic agents or blood products being used, and no increase in pain following the first "excellent" or "good" assessment. A target joint was defined in this trial as a joint in which recurrent bleeding had occurred on 4 or more occasions during the previous 6 months, or a joint in which 20 lifetime BEs had occurred. Results Twenty-seven PwHABI treated 396 mild or moderate joint BEs in PERSEPT 1; 17 of these subjects had been previously diagnosed with target joints. Of the 396 mild or moderate joint BEs treated, 135 were target joint bleeds (in 15 subjects) and 261 were non-target joint bleeds (in 25 subjects). At 12 hours post-initial infusion, target joint treatment success proportions and 95% confidence intervals (CIs) were 82% (95% CI: [63%, 100%]) and 93% (95% CI: 82%, 100%]) in the 75 and 225 µg/kg IDRs, respectively (Figure 1). Corresponding success proportions and CIs for non-target joint bleeds were 86% (95% CI: [75%, 97%]) and 95% (95% CI: [90%, 100%]), respectively. The increase in treatment success proportion observed at 12 hours with the 225 µg/kg IDR over the 75 µg/kg IDR was statistically significant for both target joint and non-target joint groups (p < 0.05). For both IDRs, the difference in treatment success proportions between target and non-target joint BEs was not statistically significant. Nearly all target (97-100%) and non-target joint bleeds (96-100%) were successfully treated at 24 hours post-initial eptacog beta infusion in each IDR (Figure 1). Conclusions The increase in both target and non-target joint bleed treatment success seen at 12 hours for the 225 µg/kg IDR over the 75 µg/kg IDR was statistically significant, and is consistent with a dose-dependent thrombin burst driving effective clot formation at the site of injury. While the 75 µg/kg IDR may be appropriate for certain patients, the 225 µg/kg IDR shows improved treatment success at 12 hours and may be the preferred choice in most circumstances. With the high efficacy seen at 24 hours, eptacog beta offers an important treatment option for treating both target and non-target joint BEs in PwHABI. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Modulating the microenvironment during FVIII uptake influences the nature of FVIII-peptides presented by antigen-presenting cells.

Hemophilia A is a severe bleeding disorder caused by the deficiency of functionally active coagulation factor VIII (FVIII). The induction of neutralizing anti-drug antibodies is a major complication in the treatment of hemophilia A patients with FVIII replacement therapies. Why some patients develop neutralizing antibodies (FVIII inhibitors) while others do not is not well understood. Previous studies indicated that the induction of FVIII inhibitors requires cognate interactions between FVIII-specific B cells and FVIII-specific CD4+ T cells in germinal center reactions. In this study, we investigated the FVIII peptide repertoire presented by antigen-presenting cells (APCs) under different microenvironment conditions that are expected to alter the uptake of FVIII by APCs. The aim of this study was to better understand the association between different microenvironment conditions during FVIII uptake and the FVIII peptide patterns presented by APCs. We used a FVIII-specific CD4+ T cell hybridoma library derived from humanized HLA-DRB1*1501 (human MHC class II) hemophilic mice that were treated with human FVIII. APCs obtained from the same mouse strain were preincubated with FVIII under different conditions which are expected to alter the uptake of FVIII by APCs. Subsequently, these preincubated APCs were used to stimulate the FVIII-specific CD4+ T cell hybridoma library. Stimulation of peptide-specific CD4+ T-cell hybridoma clones was assessed by analyzing the IL-2 release into cell culture supernatants. The results of this study indicate that the specific microenvironment conditions during FVIII uptake by APCs determine the peptide specificities of subsequently activated FVIII-specific CD4+ T cell hybridoma clones. Incubation of APCs with FVIII complexed with von Willebrand Factor, FVIII activated by thrombin or FVIII combined with a blockade of receptors on APCs previously associated with FVIII uptake and clearance, resulted in distinct peptide repertoires of subsequently activated hybridoma clones. Based on our data we conclude that the specific microenvironment during FVIII uptake by APCs determines the FVIII peptide repertoire presented on MHC class II expressed by APCs and the peptide specificity of subsequently activated FVIII-specific CD4+ T cell hybridoma clones.