Fusion of Factor IX to Factor XIII-B Sub-Unit Improves the Pharmacokinetic Profile of Factor IX

AbstractProphylaxis is currently considered the optimal care for severe haemophilia. For patients and their families one of the major difficulties with prophylaxis is the need for frequent venipunctures. The half-life of standard factor IX (FIX) concentrates is approximately 18 hours, which requires 2 or 3 intravenous infusions per week to achieve bleeding prevention in patients with severe haemophilia B. Prolonging the half-life of FIX can therefore reduce the frequency of infusions. Recently, extended half-life recombinant FIX (rFIX) concentrates have been developed. We designed a new rFIX molecule fused to coagulation FXIII-B sub-unit. This sub-unit is responsible for the long half-life of the FXIII molecule (10–12 days). The rFIX-LXa-FXIIIB fusion protein contains a short linker sequence cleavable by activated FX (FXa), to separate rFIX from the carrier protein as soon as traces of FXa are generated, leaving rFIX free to perform its enzymatic role in the tenase complex. The rFIX-LXa-FXIIIB fusion protein was expressed in human hepatic Huh-7 cells and Chinese hamster ovary cells, and both wild-type rFIX (rFIX-WT) and rFIX-LXa-FXIIIB showed similar clotting activity and thrombin generation capacity in vivo after injection in haemophilia B mice compared with rFIX-WT. The half-life of the rFIX-LXa-FXIIIB molecule in WT mice and rats was 3.9- and 2.2-fold longer, respectively, compared with rFIX-WT. A potential advantage of this new molecule is its capacity to bind to fibrinogen via FXIII-B, which might accelerate fibrin clot formation and thus improve haemostatic capacity of the molecule.

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A Pharmacokinetic Study of the Plasma-Derived Factor IX AlphaNine® and Its Comparison with the Recombinant Factor IX BeneFIX®, in Previously Treated Patients with Severe Hereditary Hemophilia B.

Blood ◽

10.1182/blood.v110.11.3955.3955 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3955-3955

Author(s):

Vicente R. Cortina ◽

T. Lissichkov ◽

K. Zavilska ◽

M. Matysiak ◽

L. Gercheva ◽

...

Keyword(s):

Half Life ◽

Factor Ix ◽

Hemophilia B ◽

Severe Haemophilia ◽

Open Label ◽

Recombinant Factor Ix ◽

Previously Treated Patients ◽

Previously Treated ◽

In Vivo Recovery

Abstract Objectives The objective of the present study was two fold: first, to determine the pharmacokinetic (PK) profile of the plasma-derived FIX concentrate AlphaNine® in patients with congenital severe haemophilia B (FIX:C 2%). To do this, two PK studies were carried out one six months after the first. The second objective was a comparison of the Alphanine® PK profile with the recombinant Factor IX, BeneFIX®. Patients and methods The first study was a prospective, five-center, open-label, comparative, PK study carried out in 25 severe hemophilia B patients who received 2 single doses of 65–75 IU/kg of AlphaNine® within 6 months (t=0 and t=6). The following parameters were assessed: in vivo recovery, half-life, AUC, mean residence time and clearance. As an extension of the study, a single dose of 65–75 IU/kg of BeneFIX® was administered in 9 out of 25 patients, after a wash-out period of 7–15 days. Results Table 1 summarizes the results obtained when comparing AlphaNine® within a period of time of 6 months (PK1 vs PK2) in 25 patients. Table 2 shows the results obtained when comparing the in vivo recovery of AlphaNine ® vs BeneFIX ® in the 9 patients studied. Conclusions These results confirm that AlphaNine® PK has similar profile as other plasma derived FIX products presently available to treat Hemophilia B patients. In addition, our results show that the recombinant FIX studied, BeneFIX® has a reduced in vivo recovery when is compared to AlphaNine®. Table 1 Parameter AlphaNine® (PK1) t=0 m AlphaNine® (PK2) t=6 m Results are expressed as Mean (SD) In vivo recovery (IU/dl:IU/kg) 1.0 (0.2) 1.2 (0.4) Half-life (h) 34.5 (6.2) 33.7 (5.4) Clearance (ml/min) 0.07 (0.01) 0.07 (0.01) AUC0-inf (IUxh/dl) 1602 (312) 1644 (360) MRT0-inf (h) 35.8 (5.4) 34.6 (5.2) Table 2 Parameter AlphaNine® (PK2) BeneFIX® Results are expressed as Mean (SD); * p<0.05 for the comparison of the in vivo recovery for the BeneFIX® group with the AlphaNine® PK2 In vivo recovery (IU/dl:IU/kg) 1.3 (0.5) 0.8 (0.2)*

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Immune tolerance induction using Fc‐fusion‐protein recombinant factor IX in severe haemophilia B

Haemophilia ◽

10.1111/hae.14424 ◽

2021 ◽

Author(s):

Ali Amid ◽

Heather Perkins ◽

Julie Gauthier ◽

Arnaud Bonnefoy ◽

Manuel Carcao ◽

...

Keyword(s):

Fusion Protein ◽

Immune Tolerance ◽

Tolerance Induction ◽

Factor Ix ◽

Severe Haemophilia ◽

Immune Tolerance Induction ◽

Haemophilia B ◽

Fc Fusion Protein ◽

Recombinant Factor Ix

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Characterisation of factor IX with a glycine-to-valine missense mutation at residue 190 in a patient with severe haemophilia B

Thrombosis and Haemostasis ◽

10.1160/th10-11-0762 ◽

2011 ◽

Vol 105 (04) ◽

pp. 616-625 ◽

Cited By ~ 1

Author(s):

Chung-Yang Kao ◽

Chia-Ni Lin ◽

Yung-Li Yang ◽

Nobuko Hamaguchi ◽

Shu-Jhu Yang ◽

...

Keyword(s):

Missense Mutation ◽

Conformational Changes ◽

Protein Function ◽

Mammalian Species ◽

Active Form ◽

Factor Ix ◽

Severe Haemophilia ◽

Haemophilia B

SummaryA patient with severe haemophilia B with a glycine-to-valine missense mutation at residue 190 (c25, chymotrypsin numbering) in factor IX (FIX; FIX-G190V or FIX-FuChou) had <1% of normal FIX clotting activity and 36% of normal FIX antigen levels (cross-reacting material-reduced, CRMr). Residue 190 in the C-terminal protease domain of human FIX is highly conserved in mammalian species and the serine protease family, suggesting that it has an indispensable role in protein function. To explore the pathological mechanism by which this mutation contributes to dysfunction of the FIX molecule, we functionally characterised FIX-G190V in vitro and in vivo. Liver-specific FIX-G190V gene expression following hydrodynamic plasmid delivery into haemophilia B mice revealed a 5.7-fold reduction in specific clotting activity compared with FIX-WT (wild type) and a two-fold decrease in plasma FIX-G190V concentration. Pulse-chase analysis demonstrated that FIX-G190V was secreted at a significantly slower rate than was FIX-WT. Purified FIX-G190V and FIX-WT displayed normal calcium-dependent conformational changes as shown by intrinsic fluorescence quenching. The in vivo half-lives of FIX-G190V and FIX-WT were indistinguishable. FIX-G190V was, however, more readily degraded than FIX-WT, especially after being activated by the active form of FXI. The vulnerable sites were mapped to the peptide bonds at Arg116-Leu117, Lys265-Tyr266, Arg327-Val328, and Arg338-Ser339, which are in the exposed loops of the FIX molecule. Also, failure of FXIa-activated FIX-G190V to bind p-aminobenzamidine indicated an abnormal conformation of the active-site pocket. Thus, the mutation at residue 190 of FIX may result in protein misfolding that affects secretion, clotting function, and hydrolysis.

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Order of intron removal during splicing of endogenous adenine phosphoribosyltransferase and dihydrofolate reductase pre-mRNA.

Molecular and Cellular Biology ◽

10.1128/mcb.13.10.6211 ◽

1993 ◽

Vol 13 (10) ◽

pp. 6211-6222 ◽

Cited By ~ 55

Author(s):

O Kessler ◽

Y Jiang ◽

L A Chasin

Keyword(s):

Dihydrofolate Reductase ◽

Chinese Hamster Ovary ◽

Half Life ◽

Chinese Hamster Ovary Cells ◽

Chinese Hamster ◽

Pairwise Comparison ◽

Adenine Phosphoribosyltransferase ◽

Ovary Cells ◽

Intron 1

Using a strategy based on reverse transcription and the polymerase chain reaction, we have determined the order of splicing of the four introns of the endogenous adenine phosphoribosyltransferase (aprt) gene in Chinese hamster ovary cells. The method involves a pairwise comparison of molecules that retain one intron and have either retained or spliced another intron(s). A highly preferred order of removal was found: intron 3 > 2 > 4 = 1. This order did not represent a linear progression from one end of the transcript to the other, nor did it correlate with the conformity of the splice site sequences to the consensus sequences or to the calculated energy of duplex formation with U1 small nuclear RNA. By using actinomycin D to inhibit RNA synthesis, the in vivo rate of the first step in splicing was estimated for all four introns; a half-life of 6 min was found for introns 2, 3, and 4. Intron 1 was spliced more slowly, with a 12-min half-life. A substantial amount of RNA that retained intron 1 as the sole intron was exported to the cytoplasm. In the course of these experiments, we also determined that intron 3, but not intron 4, is spliced before 3'-end formation is complete, probably on nascent transcripts. This result is consistent with the idea that polyadenylation is required for splicing of the 3'-most intron. We applied a similar strategy to determine the last intron to be spliced in a very large transcript, that of the endogenous dihydrofolate reductase (dhfr) gene in Chinese hamster ovary cells (25 kb). Here again, intron 1 was the last intron to be spliced.

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Clinical, humanistic, and economic burden of severe haemophilia B in adults receiving factor IX prophylaxis: findings from the CHESS II real-world burden of illness study in Europe

Orphanet Journal of Rare Diseases ◽

10.1186/s13023-021-02152-1 ◽

2021 ◽

Vol 16 (1) ◽

Author(s):

Tom Burke ◽

Sohaib Asghar ◽

Jamie O’Hara ◽

Margaret Chuang ◽

Eileen K. Sawyer ◽

...

Keyword(s):

Quality Of Life ◽

Real World ◽

Economic Burden ◽

Half Life ◽

Burden Of Illness ◽

Indirect Costs ◽

Factor Ix ◽

Severe Haemophilia ◽

Haemophilia B

Abstract Background Real-world studies of the burden of severe haemophilia B in the context of recent therapeutic advances such as extended half-life (EHL) factor IX (FIX) products are limited. We analysed data from the recent CHESS II study to better understand the clinical, humanistic, and economic burden of severe haemophilia B in Europe. Data from male adults with severe haemophilia B receiving prophylaxis were analysed from the retrospective cross-sectional CHESS II study conducted in Germany, France, Italy, Spain and the United Kingdom. Inhibitors were exclusionary. Patients and physicians completed questionnaires on bleeding, joint status, quality of life, and haemophilia-related direct and indirect costs (2019–2020). All outcomes were summarised using descriptive statistics. Results A total of 75 CHESS II patients were eligible and included; 40 patients (53%) provided self-reported outcomes. Mean age was 36.2 years. Approximately half the patients were receiving EHL versus standard half-life (SHL) prophylaxis (44% vs 56%). Most patients reported mild or moderate chronic pain (76%) and had ≥ 2 bleeding events per year (70%), with a mean annualised bleed rate of 2.4. Mean annual total haemophilia-related direct medical cost per patient was €235,723, driven by FIX costs (€232,328 overall, n = 40; €186,528 for SHL, €290,620 for EHL). Mean annual indirect costs (€8,973) were driven by early retirement or work stoppage due to haemophilia. Mean quality of life (EQ-5D) score was 0.67. Conclusions These data document a substantial, persistent real-world burden of severe haemophilia B in Europe. Unmet needs persist for these patients, their caregivers, and society.

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Order of intron removal during splicing of endogenous adenine phosphoribosyltransferase and dihydrofolate reductase pre-mRNA

Molecular and Cellular Biology ◽

10.1128/mcb.13.10.6211-6222.1993 ◽

1993 ◽

Vol 13 (10) ◽

pp. 6211-6222

Author(s):

O Kessler ◽

Y Jiang ◽

L A Chasin

Keyword(s):

Dihydrofolate Reductase ◽

Chinese Hamster Ovary ◽

Half Life ◽

Chinese Hamster Ovary Cells ◽

Chinese Hamster ◽

Pairwise Comparison ◽

Adenine Phosphoribosyltransferase ◽

Ovary Cells ◽

Intron 1

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Genetic fusion to albumin improves the pharmacokinetic properties of factor IX

Thrombosis and Haemostasis ◽

10.1160/th09-04-0255 ◽

2009 ◽

Vol 102 (10) ◽

pp. 634-644 ◽

Cited By ~ 77

Author(s):

Thomas Weimer ◽

Ulrich Kronthaler ◽

Wiegand Lang ◽

Stefan Schulte ◽

Hubert J. Metzner

Keyword(s):

Half Life ◽

Fusion Proteins ◽

Mammalian Cells ◽

Factor Ix ◽

Recurrent Bleeding ◽

Haemophilia B ◽

Pharmacokinetic Properties ◽

Genetic Fusion ◽

In Vivo Recovery

SummaryHaemophilia B is a X-chromosome linked disease characterised by a deficiency of functionally active coagulation Factor IX (FIX). Patients with severe haemophilia B at risk of recurrent bleeding are treated approximately twice a week in a prophylactic setting by application of FIX concentrates.To increase convenience and compliance of the therapy it is desirable to reduce the dosing frequency by improving the pharmacokinetic properties of FIX. Here a concept of rFIX (recombinant factor IX) albumin fusion proteins (rIX-FPs) with cleavable linker peptides derived from the FIX activation sequence is presented. Constructs of the genetic fusion of FIX to albumin via cleavable linkers were expressed in mammalian cells and characterised after purification. In vitro activation studies with FXIa demonstrated that cleavage of the linker and the activation peptide proceeded comparably well. In a clotting assay the rIX-FPs with cleavable linker showed a 10- to 30-fold increase in the molar specific clotting activity compared to fusion proteins with non-cleavable linkers. Furthermore, in-vivo recovery, terminal half-life and the AUC of rIX-FPs in rats and rabbits as determined by FIX antigen measurements were significantly increased compared to rFIX (BeneFIX®). In FIX deficient (FIX−/−) mice the in-vivo recovery and the AUC were also significantly increased.The efficacy in reducing bleeding time was shown in FIX−/−mice by a tail tip bleeding model. The results suggest that rIX-FPs with a cleavable linker between FIX and albumin are a promising concept that may support the use of the albumin fusion technology to extend the half-life of FIX.

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IN VIVO RECOVERY AND HALF-LIFE OF A STEAM-TREATED FACTOR IX (FIX) CONCENTRATE

10.1055/s-0038-1644067 ◽

1987 ◽

Author(s):

S Mörsdorf ◽

E Seified ◽

M Köhler ◽

F Fasco ◽

P Hellstern ◽

...

Keyword(s):

Heat Treatment ◽

Body Weight ◽

Half Life ◽

Factor Ix ◽

Risk Of Infection ◽

Haemophilia B ◽

Before And After ◽

The Mean ◽

In Vivo Recovery

The introduction of heat treatment of FVIII or FIX concentrates has reduced the risk of infection, however, has raised the question of a reduced haemo-statical effect. Therefore, the in vivo recovery and half-life of a steam-treated FIX concentrate (S-TIM4, Immuno) were investigated in 10 haemophilia B patients from two haemophilia centers. Patients mean age was 33 y (range 17-51 y) and the mean body weight (BW> was 67 kg (range 44-81 kg). Basal FIX levels ranged from 0.007 to 0.03 (median 0,007) U/ml. The patients had not received FIX concentrate at least 7 d prior to the study. Patients 1-4 received 4 different lots, patients 5-10 received one single lot. Blood was drawn before and after 15, 30 min, 1h, 4 h, 8 h, 10 h, 12 h, 24 h and additionally 48 h in patients 1-4. FIX levels were measured using FIX deficient plasma from Immuno (patients 1-10) in center 1, additionally in patients 5-10 using FIX deficient plasma from MerzDade. In vivo recovery and half-life were calculated according to Allain (1980, 1984) and given in % and h, respectively. Results: The table shows the dose and the calculated in vivo recovery and half-life, according to the FIX measurements in center 1 (C1) or center 2 (C2).Although the apparently longer half-life of patients 1-4 may in part be explained by the longer period of FIX measurements in center 1, the exclusive use of one single lot of FIX concentrate suggests an influence of the lot transfused in these patients. However, laboratory signs of DIC were not present.

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Long-acting recombinant fusion protein linking coagulation factor IX with albumin (rIX-FP) in children

Thrombosis and Haemostasis ◽

10.1160/th16-03-0179 ◽

2016 ◽

Vol 116 (10) ◽

pp. 659-668 ◽

Cited By ~ 19

Author(s):

Hervé Chambost ◽

Christoph Male ◽

Thierry Lambert ◽

Susan Halimeh ◽

Tatiana Chernova ◽

...

Keyword(s):

Fusion Protein ◽

Coagulation Factor ◽

Factor Ix ◽

Recombinant Fusion Protein ◽

Severe Haemophilia ◽

Spontaneous Bleeding ◽

Haemophilia B ◽

Coagulation Factor Ix ◽

Routine Prophylaxis ◽

Bleeding Episodes

SummaryA global phase 3 study evaluated the pharmacokinetics, efficacy and safety of a recombinant fusion protein linking coagulation factor IX with albumin (rIX-FP) in 27 previously treated male children (1–11 years) with severe and moderately severe haemophilia B (factor IX [FIX] activity ≤2 IU/dl). All patients received routine prophylaxis once every seven days for up to 77 weeks, and treated any bleeding episodes on-demand. The mean terminal half-life of rIX-FP was 91.4 hours (h), 4.3-fold longer than previous FIX treatment and clearance was 1.11 ml/h/kg, 6.4-fold slower than previous FIX treatment. The median (Q1, Q3) annualised spontaneous bleeding rate was 0.00 (0.00, 0.91) and was similar between the <6 years and ≥6 years age groups, with a weekly median prophylactic dose of 46 IU/kg. In addition, patients maintained a median trough level of 13.4 IU/dl FIX activity on weekly prophylaxis. Overall, 97.2 % of bleeding episodes were successfully treated with one or two injections of rIX-FP (95 % CI: 92 % to 99 %), 88.7 % with one injection, and 96 % of the treatments were rated effective (excellent or good) by the Investigator. No patient developed FIX inhibitors and no safety concerns were identified. These results indicate that rIX-FP is safe and effective for preventing and treating bleeding episodes in children with haemophilia B with weekly prophylaxis. Routine prophylaxis with rIX-FP at treatment intervals of up to 14 days are currently being investigated in children with severe and moderately severe haemophilia B. Clinicaltrials.gov (NCT01662531)

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