The animals received 10−5 to 1 IU FIX (Benefix, Pfizer) or 0.01 to 1 IU FIX-Fc (Alprolix, Sanofi) intradermally (ID) in the groin area twice per week for 4 weeks and continued throughout at the same frequency after initiation of intraperitoneal (IP) and i.v. administration of 1 IU FIX (Benefix, Pfizer) ±50 μg triprolidine (antihistamine; Sigma) and 10 μg ABT-491 (platelet-activating factor receptor antagonist; Sigma) in the tail vein once per week for 5 to 6 weeks (1 IP followed by 4 or 5 i.v. injections). IP administration before continuing with i.v. injections ensured more consistent inhibitor formation in the control groups with lower nonresponse rates. triprolidine and ABT-491 were coinjected to prevent anaphylaxis-related mortality.
Benefix
Manufactured by Pfizer
Sourced in France, United States
Benefix is a blood clotting factor IX concentrate used to treat hemophilia B, a genetic disorder that prevents proper blood clotting. It is a laboratory-produced version of the human factor IX protein, which is essential for normal blood clotting.
Automatically generated - may contain errors
Lab products found in correlation
Complete freund s adjuvant, by Merck Group (1 mentions)
Fetal calf serum (fcs), by Atlanta Biologicals (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Elispot aec substrate set, by BD (1 mentions)
Rmpi 1640, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase conjugated goat anti monkey igg, by Rockland Immunochemicals (1 mentions)
Immunospot analyzer, by Cellular Technology (1 mentions)
Freund s adjuvant, by Merck Group (1 mentions)
Refacto af, by Pfizer (1 mentions)
Start hemostasis analyzer, by Diagnostica Stago (1 mentions)
Gapdh antibody, by Cell Signaling Technology (1 mentions)
Actin fs aptt reagent, by Siemens (1 mentions)
Imidazole, by Merck Group (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
Dylight 633, by Thermo Fisher Scientific (1 mentions)
Saponin, by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Facsdiva software, by BD (1 mentions)
Triprolidine, by Merck Group (1 mentions)
18 protocols using benefix
1
Hemophilia FIX Inhibitor Prevention
2
Induction of Immune Tolerance to hF.IX
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Male C57BL/6 mice were treated i.v. with an AAV8-luc vector (4 × 1012 vg kg−1) together with SVP[Rapa] or with SVP[empty], followed three weeks later by the i.v. injection of 4 × 1012 vg kg−1 of an AAV5-hF.IX vector or an AAV8-hF.IX vector. Additionally, mice were treated with same initial injection of AAV8-luc vector as described previously and challenged three weeks later with the subcutaneous injection of 100 μg of recombinant hF.IX protein (BeneFix®, Pfizer, Paris, France) formulated in complete Freund’s adjuvant (Sigma-Aldrich, St. Louis, MO).
3
Quantifying AAV-Encoded hFIX Antibody-Secreting B Cells
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To determine the frequencies of B cells that secreted antibodies against AAV-encoded hFIX, we performed a B cell ELISpot assay.45 (link),46 (link) Briefly, 0.1 U of BeneFIX (Pfizer) in PBS was overnight coated onto surfactant-free multiscreen filter plates (Millipore). As a positive control, monkey IgG (whole molecule) (Rockland, Limerick, PA, USA) was coated at 500 ng/mL in PBS. Only medium was used as a negative control. Cryopreserved splenocytes from each non-human primate were revived in RMPI 1640 (Life Technologies) containing 10% fetal calf serum (Atlanta Biologicals, Norcross, GA, USA), 10,000 U/mL penicillin, and 100 mg/mL streptomycin (Life Technologies). One million splenocytes were added to each coated well and incubated for 16 h. IgG bound to hFIX was probed with horseradish peroxidase-conjugated goat anti-monkey-IgG (Rockland, Limerick, PA, USA). Spots were developed using BD ELISpot AEC substrate set (BD Biosciences, San Diego, CA, USA) and were counted using the ImmunoSpot analyzer (Cellular Technology, Shaker Heights, OH, USA).
4
Generation of 2bFXa-Hemophilia B Mouse Model
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We used a F9 knockout mouse purchased from Jackson Lab (Bar Harbor, ME, USA) as the HB mouse model. 2bFXa transgenic mice with a C57BL/6 background were constructed by Cyagen (Guangzhou, China). In the mice, the FXa cassette was constructed by replacing the activation peptide of the human FX with an Arg–Lys–Arg sequence as previously reported10 (link). and the platelet-specific promoter αIIb was applied to control the expression of FXa. The αIIb promoter was from Dr. David A. Wilcox(BloodCenter of Wisconsin, WI, USA). We crossed 2bFXa mice with HB mice and screened for 2bFXa-HB transgenic mice. All mice were maintained in special pathogen-free rooms at the Animal Center of Hangzhou Normal University. All methods were approved by the Experimental Animal Ethics Committee of Hangzhou Normal University, and they were performed in accordance with the relevant guidelines and regulations. We followed the guidelines of the Animal Research: Reporting of In Vivo Experiments (ARRIVE). FIX inhibitors in 2bFXa-HB mice were developed through intraperitoneal injection of recombinant human FIX (BeneFIX, Pfizer, China) as previously described16 (link). Briefly, 200 U/kg recombinant hFIX mixed with Freund’s adjuvant (SigmaAldrich, China) was injected into the mice twice with an interval of 3 weeks. The mouse plasma was collected 10 days after the last injection for inhibitor determination.
5
Hemophilia B Mouse Model Generation
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All animal studies were carried out in accordance with French guidelines regarding the care and use of experimental animals and approved by the French Ethics Committee C2EA‐26 for animal experiments under number 509 1 2015062215414016. HB mouse farming (Jackson Laboratory, strain name B6; 129P2‐F9tm1Dws/J[20 ]) was established under pathogen‐free conditions, and newborn mice from the mating of F9KO heterozygous female and F9KO male mice were used for this study. Forty‐eight hours after birth, each newborn mouse from the same litter received a transcutaneous injection into the liver using a 29‐gauge needle of both 105‐corrected HB‐iPSCs at day 11 of hepatocyte differentiation and 1U of recombinant FIX (BeneFIX; Pfizer), to ensure the survival of hemophilic mice. After 2 weeks, the mice were sacrificed and genotyped by PCR using the primers listed in Table S2 . The livers and plasma were collected for further study.
See Supporting Information for additional materials and methods.
See Supporting Information for additional materials and methods.
6
Bleeding Rate Comparison for Hemophilia
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Efficacy was assessed by comparing the treated annualised bleeding rate (ABR) during the study treatment period with the treated ABR from an external control group of 65 individuals who received on‐demand moroctocog alfa (AF‐CC) (ReFacto AF; Pfizer Inc.) or nonacog alfa (BeneFIX; Pfizer Inc.) in previous studies3 , 19 , 20 and matched key inclusion/exclusion criteria of the current study (aged 18 to <65 years; factor activity ≤1%). Pfizer internal studies were selected for this comparison group to provide individual participant‐level data that were prospectively collected.
The ABR during the three‐month study treatment period was also compared with the pretreatment ABR calculated retrospectively from the medical record. On‐study ABR was defined as the number of treated bleeding episodes within nine days after last dose/[(last dose date + 9 − first dose date + 1)/365.25] and pretreatment ABR was the number of treated bleeding episodes within six months pre‐enrolment × 2.
The ABR during the three‐month study treatment period was also compared with the pretreatment ABR calculated retrospectively from the medical record. On‐study ABR was defined as the number of treated bleeding episodes within nine days after last dose/[(last dose date + 9 − first dose date + 1)/365.25] and pretreatment ABR was the number of treated bleeding episodes within six months pre‐enrolment × 2.
7
Oral Tolerance Induction for Hemophilia B
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All procedures in dogs were approved by the University of North Carolina at Chapel Hill’s Institutional Animal Care and Use Committee. Outbred hemophilia B dogs (20–24 kg) of the UNC-Chapel Hill strain were used. These dogs have a F9 missense mutation, resulting in a lack of circulating antigen and severe hemophilia B.26 (link), 27 (link) To induce oral tolerance, lyophilized lettuce cells were mixed into dog chow and fed twice per week for 13 weeks. Each dose was adjusted to deliver CTB-FIX antigen at 0.3 mg/kg. Starting at 4 weeks into the experiment, recombinant human FIX (Benefix; Pfizer) was given i.v. at 10 IU/kg (once per week for 8 weeks). The four dogs undergoing oral tolerance studies were S12 (male; 2 years old), S14 and S15 (males; 15 months old), and P44 (male; 4 years old). Control dogs received FIX i.v. but no oral antigen: P08 and P10 (females; 3 years old) were injected weekly at 10 IU/kg for 4 weeks and S13 (male; 15 months old) and P05 (male; 5 years old) were injected weekly at 10 IU/kg for 8 weeks. Finally, two additional dogs (O07 and O67; female; 3 years old) received oral CTB-FIX antigen at 0.03–0.12 mg/kg twice per week for ∼6 months.
8
Adoptive Transfer of hCD20-tg B Cells for hFIX Immunogenicity
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BALB/c-HB mice were adoptively transferred with 5–10 × 106 primary murine hCD20-tg B cells transduced with CD20-LVhFIX-IgG. This was followed by either SC immunization with 1 IU hFIX (Benefix, Pfizer) in adjuvant (Sigma Adjuvant System) or 3 IU hFIX replacement therapy by the IV route, once a week, for 8 weeks. Plasma samples were collected by tail bleed into 0.38% sodium citrate, and inhibitory antibodies to FIX were measured by Bethesda assay, as described.43 (link) Measurements were carried out in a Diagnostica Stago STart Hemostasis Analyzer. ELISA-based measurements of IgG1 antibodies to hFIX were carried out as described.43 (link)
9
Benefix® Addition to Plasma with Concizumab
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rFIX (Benefix®, Pfizer) was added to a HB plasma pool (George King Bio‐Medical Inc.) in the absence or presence of concizumab.
10
Quantitative FIX Protein Analysis
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For detection of FIX protein, the F9 monoclonal antibody (M01), clone 2C9 (Abnova) was employed in a dilution of 1:500. GAPDH protein expression was detected using GAPDH antibody (1:2000; Cell Signaling Technology). Detection was performed via licor Odyssey infrared imaging system. Recombinant FIX (BeneFix, Pfizer) was employed as positive control. Supernatant obtained from CHO cells was employed for ELISA-based quantification of FIX antigen (FIX:Ag) using ZYMUTEST Factor IX from HYPHEN BioMed according to the manufacturer’s protocol including a standard FIX calibrator concentration set to 100% which equals 100 U/dl. Thus, FIX:Ag levels are expressed as % FIX protein. FIX activity was determined using a one-stage coagulation assay. Samples were diluted 1:5 in imidazol buffer and mixed with an equal amount of FIX deficient plasma and Actin FS APTT reagent (all from Siemens Healthcare). After incubation at 37°C for 2 min, 0.025 M calcium-chloride was added, and the coagulation time recorded in an Amelung KC10 coagulometer. Calibration curves were generated with human standard plasma diluted in FIX deficient plasma.
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