WO2018037217A1 - Facteur de coagulation - Google Patents

Facteur de coagulation Download PDF

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Publication number
WO2018037217A1
WO2018037217A1 PCT/GB2017/052468 GB2017052468W WO2018037217A1 WO 2018037217 A1 WO2018037217 A1 WO 2018037217A1 GB 2017052468 W GB2017052468 W GB 2017052468W WO 2018037217 A1 WO2018037217 A1 WO 2018037217A1
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seq
fusion polypeptide
polypeptide
amino acid
factor
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PCT/GB2017/052468
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English (en)
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Jude AKINWALE
Ian Wilkinson
Michael Makris
Richard Ross
Peter Artymiuk
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University Of Sheffield
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • C07K14/755—Factors VIII, e.g. factor VIII C (AHF), factor VIII Ag (VWF)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site

Definitions

  • the disclosure relates to long acting fusion polypeptides comprising Factor VIII [FVIII] and Factor IX [FIX] and their use in the treatment of blood clotting disorders.
  • Haemophilia A is a sex-linked clotting disorder characterised by excessive bleeding due to the impaired clotting ability of the blood and predominantly affects males caused by non-functional or insufficient expression of the blood-clotting-factor VIII.
  • Factor VIII is a large multi-domain glycoprotein, comprising six domains A1-A2-B-A3-C1-C2 with a molecular mass of 330 kDa.
  • FVIII loses its signal peptide and undergoes extensive post-translational modifications such as N- and O-linked glycosylation, and sulfation of essential tyrosine residues.
  • FVIII undergoes an initial intracellular proteolytic processing by thrombin at variable sites (mapped to Arg1313 and Arg1648 ) within the B domain, resulting in FVIII heterodimer formation that consists of an N-terminal heavy chain (A1-a1-A2-a2-B) and a C-terminal light chain (a3-A3-C1-C2), which are non-covalently linked by divalent metal ions.
  • Further activation at arginine 372 and 740 results in complete truncation of the B-domain and formation of a fully activated FVIII heterotrimer.
  • the B-domain region of FVIII has been shown to have no effect on its biological activity. Hence, the new generation rFVIII products often do not include the B-domain whilst retaining activity.
  • FVIII In circulation, FVIII interacts with von Willebrand Factor [vWF] through the C-terminal light chain and this association stabilizes FVIII by protecting the heavy-light chain interactions as well as protecting FVIII from proteolysis by limiting its interaction with phospholipid surfaces and inactivating proteases (e.g. activated protein C and FXa).
  • proteases e.g. activated protein C and FXa.
  • the plasma level of FVIII is regulated by macrophages that express certain cell surface clearance receptors such as Siglec-5 and macrophage-LRP1 which bind to the FVIII/vWF complex, facilitates its rapid endocytosis and the degradation of the internalized FVIII/vWF protein complex.
  • proteolytic activation of FVIII at R1689 causes the dissociation of FVIII from vWF and facilitates its interaction with both the phospholipid membrane and its serine-protease enzyme
  • Haemophilia A Treatment of Haemophilia A is by replacement therapy with recombinant factor VIII (rFVIII) on demand or as a prophylaxis (Berntorp et al., 2003, Gouider et al., 2015).
  • rFVIII recombinant factor VIII
  • the relatively short circulating half-life of current therapeutic products necessitates multiple weekly intravenous injections which are expensive and inconvenient (Schimpf et al., 1977).
  • the commercially available rFVIII products in clinical use are either full length (e.g. Advate, Kogenate) or B- domain deleted (e.g. Refacto), and there are a number of other rFVIII products with delayed clearance in development.
  • FVIII is unstable in vitro, particularly in aqueous solutions.
  • lyophilised formulations US2006/0205661 or WO2010/026186; the addition of stabilisers such as sucrose, trehalose, raffinose, arginine or complex formations of FVIII with other plasma proteins; see US6586573.
  • the administration of lyophilized compositions is a complex procedure as it requires reconstitution, a precise dosage regimen and often intravenous injection, steps restricting self- administration by patients.
  • WO2010/027152 discloses a FVIII stable at 25°C for a minimum of 18 weeks aqueous solution composition with the excess of Ca 2+ ions in the presence of EDTA.
  • the addition of Ca 2+ ions is commonly thought to contribute to stability and is found in most marketed compositions.
  • US6599724 describes improved stability of factor VIII in the presence of Cu 2+ or Zn 2+ or Mn 2+ .
  • FVIII is proposed the addition of histidine to increase stability; see US5605884 and EP-A-1016673.
  • others describe a destabilisation effect on FVIII by histidine; see US5874408.
  • addition of polyethylene glycol has also found to be beneficial for the stability of FVIII and is disclosed in US5565427.
  • WO2007090584 and US2008/0260755 each disclose therapeutic fusion proteins in which a coagulating factor such as VII, Vila is fused to a half-life enhancing polypeptide via a cleavable or non-cleavable linker.
  • This disclosure relates to a long acting fusion protein comprising FVIII fused as an in-frame fusion protein to an inactivated FIX.
  • recombinant FVIII is removed from the circulation relatively rapidly therefore requiring multiple administrations to those suffering from blood clotting disorders.
  • the disclosure illustrates enhanced in vivo PK by fusing FVIII to an inactivated FIX and that the fusion protein has in vivo activity using a bioactivity test.
  • the FVIII-FIX proteins are optionally linked via a flexible peptide linker molecule, allowing protection of Factor VIII against proteolytic cleavage whilst maintaining active FVIII in a temporarily inactive state.
  • This will provide an intravascular pool of inert FVIII that is readily available for activation by circulating thrombin upon the initiation of the coagulation process, and a method to prolong the half-life of the molecule leading to enhanced in vivo biological potency.
  • a fusion polypeptide comprising an amino acid sequence of a Factor VI 11 polypeptide, or a functional fragment or functional variant thereof, wherein said Factor VIII polypeptide is fused, either directly or indirectly, to a Factor IX polypeptide comprising an amino acid sequence wherein said amino acid sequence is modified by addition, deletion or substitution of at least one amino acid residue and wherein said modified Factor IX polypeptide has reduced or undetectable proteolytic activity.
  • Factor VIII is synthesized as a precursor polypeptide chain comprising of 2351 amino acids.
  • the first 19 amino acids code for the signal peptide while the mature single chain FVIII molecule consists of 2332 amino acids residues that are organised into six structural domains: A1-A2-B-A3-C1-C2.
  • the A2 and B domains are flanked by acidic residues thus segregating the A and B domains.
  • the B domain starts with the amino acid at position 763 and ends with the amino acid at position 1656 in the sequence of Factor VIII.
  • said amino acid sequence of Factor VIII polypeptide comprises or consist of a sequence set forth in SEQ ID NO: 1 or a sequence polymorphic sequence variant thereof.
  • polypeptide polymorphic sequence variant polypeptide has at least, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the full length amino acid sequence set forth in SED ID NO: 1 or SEQ ID 22.
  • amino acid sequence of Factor VIII polypeptide comprises or consists of a B-Domain deletion.
  • amino acid sequence of Factor VIII polypeptide comprises a sequence set forth in SEQ ID NO: 2 or SEQ ID 23.
  • said amino acid sequence of Factor IX polypeptide comprises a modified sequence of the sequence set forth in SEQ ID NO 3.
  • said modified polypeptide has at least, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the full length amino acid sequence set forth in SED ID NO: 3.
  • said amino acid sequence of Factor IX is modified by addition, deletion or substitution of at least one amino acid polypeptide in the protease domain comprising the sequence set forth in SEQ ID NO: 3 wherein said protease has reduced or undetectable proteolytic activity.
  • said Factor IX polypeptide proteolytic activity is reduced by 99% when compared to the proteolytic activity of an unmodified Factor IX polypeptide as set forth in SEQ ID NO: 3.
  • proteolytic activity is reduced by 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10% when compared to the proteolytic activity of an unmodified Factor IX polypeptide as set forth in SEQ ID NO: 3.
  • said modification comprises a deletion or substitution of amino acid residue serine 41 1 as set forth in SEQ ID NO: 3.
  • said amino acid sequence of Factor IX polypeptide comprises the sequence set forth in SEQ ID NO: 4 (S41 1 N)
  • said Factor VIII polypeptide is fused directly to said Factor IX polypeptide as an in frame translational fusion.
  • said Factor VIII polypeptide is fused indirectly via a peptide linker molecule to said Factor IX polypeptide as an in frame translational fusion.
  • said peptide linker is a flexible linker.
  • said peptide linker molecule comprises at least one copy of the peptide Gly Gly Gly Gly Ser [SEQ ID NO: 5].
  • said peptide linking molecule comprises 2, 3, 4, 5, 6, 7, 8, 9 or 10 copies of the peptide Gly Gly Gly Gly Ser.
  • said translational fusion polypeptide comprises a proteolytic cleavage site between said Factor VIII polypeptide and Factor IX polypeptide.
  • said proteolytic cleavage site comprises or consist of the amino acid sequence selected from the group consisting of DDDDK [SEQ ID NO 17], LVPRGS [SEQ ID NO 18], LVPRGS [SEQ ID NO 19], ENLYFQG [SEQ ID NO 20], or LEVLFQGP [SEQ ID NO 21].
  • the amino terminal amino acid of said Factor VIII polypeptide is linked to the carboxyl terminal amino acid of said Factor IX polypeptide.
  • the carboxyl terminal amino acid of said Factor VIII polypeptide is linked to the amino terminal amino acid of said Factor IX polypeptide.
  • said fusion polypeptide comprises or consist of the amino acid sequence set forth in SEQ ID NO: 6 or 24. In a preferred embodiment of the invention said fusion polypeptide comprises or consist of the amino acid sequence set forth in SEQ ID NO: 13 or 25. In a preferred embodiment of the invention said fusion polypeptide comprises or consist of the amino acid sequence set forth in SEQ ID NO: 14 or 26.
  • said fusion polypeptide comprises or consist of the amino acid sequence set forth in SEQ ID NO: 15 or 27.
  • said fusion polypeptide comprises or consist of the amino acid sequence set forth in SEQ ID NO: 16 or 28.
  • the in vivo pharmacokinetic stability of the fusion polypeptide according to the invention is improved by greater than 1.6 fold when compared to recombinant or native FVIII.
  • the in vivo pharmacokinetic stability of the fusion polypeptide according to the invention is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or at least 20 fold improved when compared to recombinant or native FVIII.
  • a nucleic acid molecule encoding the fusion polypeptide according to the invention is provided.
  • said nucleic acid molecule comprises the nucleotide sequence set forth in SEQ ID NO: 7, or a polymorphic nucleotide sequence variant thereof, wherein said variant comprises a nucleic acid molecule the complementary strand of which hybridizes under stringent hybridization conditions to the nucleotide sequence set forth in SEQ ID NO: 7 and wherein said nucleic acid molecule encodes Factor VIII polypeptide as herein disclosed.
  • said nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 8 or a polymorphic nucleotide sequence variant thereof, wherein said variant comprises a nucleic acid molecule the complementary strand of which hybridizes under stringent hybridization conditions to the nucleotide sequence set forth in SEQ ID NO: 8 and wherein said nucleic acid molecule encodes Factor IX polypeptide as herein disclosed.
  • said variant nucleic acid molecule comprises a nucleotide sequence set forth in SEQ ID NO: 9. In a preferred embodiment said variant nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 10.
  • said variant nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 11.
  • said nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 12.
  • Hybridization of a nucleic acid molecule occurs when two complementary nucleic acid molecules undergo an amount of hydrogen bonding to each other.
  • the stringency of hybridization can vary according to the environmental conditions surrounding the nucleic acids, the nature of the hybridization method, and the composition and length of the nucleic acid molecules used. Calculations regarding hybridization conditions required for attaining particular degrees of stringency are discussed in Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001); and Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology— Hybridization with Nucleic Acid Probes Part I, Chapter 2 (Elsevier, New York, 1993).
  • the T m is the temperature at which 50% of a given strand of a nucleic acid molecule is hybridized to its complementary strand.
  • Hybridization 5x-6x SSC at 65°C-70°C for 16-20 hours ii) Wash twice: 2x SSC at RT for 5-20 minutes each iii) Wash twice: 1x SSC at 55°C-70°C for 30 minutes each
  • an expression vector adapted to express the nucleic acid molecule according to the invention.
  • an isolated cell transformed or transfected with a vector according to the invention.
  • said microbial cell is a bacterial or yeast cell.
  • said cell is a mammalian cell
  • a method for the manufacture of the fusion polypeptide comprising: i) providing a cell according to the invention:
  • said cell is a microbial cell.
  • said cell is a mammalian cell.
  • a pharmaceutical composition comprising the polypeptide according to the invention including an excipient or carrier
  • said pharmaceutical composition is combined with a further therapeutic agent.
  • said further therapeutic agent is native Factor VIII.
  • compositions of the present invention are administered in pharmaceutically acceptable preparations.
  • Such preparations may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers and supplementary therapeutic agents'.
  • compositions of the invention can be administered by any conventional route, including injection.
  • the administration may be subcutaneous, intramuscular, intravenous, intracavity or intra-articular and may be prepared by any methods well known in the art of pharmacy.
  • the compositions of the invention are administered in effective amounts.
  • An "effective amount" is that amount of a composition that alone, or together with further doses, produces the desired response. This can be monitored by routine methods or can be monitored according to diagnostic methods of the invention discussed herein. Such amounts will depend, of course, on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner.
  • a maximum dose of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art, however, that a patient may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reasons.
  • a fusion polypeptide according to the invention for use as a medicament.
  • a fusion polypeptide according to the invention for use in the treatment of a blood coagulation disorder
  • said blood coagulation disorder is haemophilia A, or haemophilia B, a liver disorder or a surgery related haemorrhagic episode.
  • a fusion polypeptide according to the invention for use in a method of treating a blood coagulation disorderwherein said composition is administered i) prophylactic in a weekly or monthly dosage regimen and/or ii) in response to an acute haemorrhage.
  • said blood coagulation disorder is haemophilia A, or haemophilia B or a liver disorder or a surgery related haemorrhagic episode.
  • a process for the manufacture of Factor VIII comprising the steps i) providing a cell comprising a nucleotide sequence encoding a fusion polypeptide according to the invention wherein the nucleotide sequence comprises a proteolytic cleavage site between the amino acid sequence encoding the FVIII polypeptide and FIX polypeptide;
  • said protease is an endopeptidase.
  • Endopeptidases such as enteropeptidase, thrombin, Factor Xa, TEV protease or Rhinovirus 3C protease are routinely used in protein expression systems such as for the removal of affinity tags from recombinant proteins.
  • said proteolytic cleavage site comprises or consist of the amino acids selected from the group consisting of DDDDK [SEQ ID NO 17], LVPRGS [SEQ ID NO 18], LVPRGS [SEQ ID NO 19], ENLYFQG [SEQ ID NO 20], or LEVLFQGP [SEQ ID N0 21].
  • Figure 1 illustrates the unprocessed full length FVIII ;
  • FIG. 1 illustrates the fully processed mature full length FVIII
  • Figure 3 illustrates the B-Domain deleted FVIII (Refacto) structural arrangement
  • Figure 4 illustrates the B-Domain Deleted FVIII-FIXi (13A1) structural arrangement
  • Figure 5 illustrates the FVIII-FIXi hypothetical interaction with native FIX in a tenase complex.
  • Figure 6 illustrates a Western blot of FVIII-FIXi Protein
  • FIG. 7 Silver Stain Gel of FVIII-FIXi protein
  • FIG. 8 ELISA Analyses of Purified FVIII-FIXi Protein
  • FIG. 9 Coamatic Analyses of Purified FVIII-FIXi Protein
  • Figure 10 Thrombin Generation Curve of Purified FVIII-FIXi Protein
  • Figure 1 1 is a comparison of FVIII/FIX fusion polypeptide 13A1_FVIII and Advate FVIII Clearance in vWF/FVIII knockout (DKO) Mice.
  • This assay is routinely used to measure FVIII cofactor activity based on FX activation to FXa by FIXa, in the presence of factors reagents (Ca++ ions, phospholipids and thrombin).
  • FXa hydrolyses the chromogenic substrate S-2765, liberating the chromogenic group p-nitroaniline (pNA).
  • pNA produces a yellow colour that can be read photometrically at 405 nm wavelength. The colour produced is linearly related to the amount of FVIII in the test sample, with a detection limit of 0.005 lU/mL for FVIII using the Coamatic assay (Chromogenix).
  • ELISA technique is routinely used to determine the concentration of FVIII protein in a test sample against a standard (or reference sample) of known concentration.
  • Sandwiched-ELISA technique was employed to quantitate FVIII using a matched-Pair monoclonal antibody set for the ELISA of human Factor VIII antigen (Affinity Biologicals).
  • the plate is coated with a capture antibody, and after a period of incubation the test sample containing the protein of interest is added (along with the reference sample) and further incubated to facilitate binding to the coated antibody.
  • the excess/unbound protein is washed off and the detection antibody is added, and the captured is detected by visualization using a chromogenic substrate that could be measured at a specific wavelength.
  • the colour intensity when measured is directly proportional to the amount of sample captured, and this could be estimated by correlating with the reference sample of known concentration.
  • a paired antibody from Affinity Biologicals is used for the ELISA analyses of test proteins.
  • the aPTT test is used as a screening coagulation test to determine how long it takes for clotting formation to occur in vitro.
  • coagulation factors in the contact coagulation pathway are activated in vitro in the presence of a plasma, phospholipids and calcium ions.
  • the test could be used to determine any abnormality in the clotting pathway due to deficiency of any of the coagulation factors.
  • This test has been adapted to determine the FVIII deficiency or abnormality in vitro by using a FVIII-deficient plasma in the assay which contains all the clotting factors (constant and in excess) except FVIII.
  • the FVIII product to be tested is added to a FVIII-deficient plasma and incubated at 37°C. Thereafter, pre-warmed phospholipids (to 37°C), contact activator and calcium are added. Calcium initiates the clotting, and the time taken from the calcium addition to clotting formation is recorded.
  • the clotting time is inversely proportional to the activity of FVIII present in the assay. While the normal clotting time may vary depending on the instrument or reagents used, it is generally accepted that FVIII clotting time in a normal individual is approximately 30 seconds.
  • Affinity purification by FVIII antibody was used for the purification of 13A1_FVIII protein sample.
  • the VIII Select antibody bead is based on highly cross-linked agarose base matrix, which enables rapid processing of large sample volumes.
  • the ligand a 13 kD recombinant protein, is attached to the porous base matrix via a hydrophilic spacer arm making it easily available for binding to recombinant B-domain-depleted factor VIII.
  • 13A1_FVIII culture media are collected, concentrated using Vivaflow 200 concentrator, and centrifuged at 21 ,000g to pellet cells debris.
  • the clear 13A1_FVIII media is incubated with equilibrated VIII Select beads in a sterile column for >12 hours.
  • the unbound 13A1_FVIII are collected and the beads are washed with several column volume of equilibration buffer (HEPES supplemented with Tween 80, CaCI2 and NaCI).
  • HEPES equilibration buffer
  • the bound 13A1_FVIII is eluted in HEPES buffer containing NaCI, CaCI2, histidine and L-arginine.
  • TGA Thrombin Generation Assay
  • Thrombin Generation Assay is an in vitro laboratory test used to measure the ability of a plasma sample to form thrombin after the initiation of coagulation with tissue factor (TF) or another trigger. During haemostasis, thrombin is generated during the initiation, propagation and termination phases of the coagulation process culminating in the formation of a stable fibrin clot that halts the bleeding.
  • the TGA assay measures the overall thrombin generated during the coagulation process and thus reflects the total reactions that regulates thrombin formation and inhibition during the process.
  • the concentration of thrombin is determined by enzymatic reaction with a thrombin-specific chromogenic substrate, which produces a yellow coloured product proportional to the amount of thrombin. In this study a fluorogenic substrate was used to assess the amount of thrombin generated by 13A1_FVIII in the assay, in the presence/absence of all other clotting factors, using the calibrated automated thrombogram (CAT) method of
  • mice The efficacy and pharmacokinetics of the purified 13A1_FVIII coagulation factor was assessed in double knockout mice with deletion of FVIII and VWF genes (DKO). These mice have no detectable plasma FVIII procoagulant activity and do not bleed spontaneously, only with trauma.
  • the mice were infused with a single intravenous administration of the test and control Advate FVIII products at a dose of 200 lU/kg ( ⁇ 6 lU/mouse) independently and plasma samples were collected at intervals up to 48hours post-infusion.
  • Figure 1 is a diagrammatic representation of the full length factor VI II Structural Arrangement featuring the Signal Peptide (S), Heavy Chain (A1 , A2 and B Domains) and Light Chain (A3, C1 and C2 Domains).
  • Figure 2 is a diagrammatic representation of fully processed FVI II .
  • the full length FVI II is cleaved at arginine 372, 740 and 1689 and loses the B-domain to form a heterotrimer protein.
  • the resulting non-covalent heterotrimeric FVI II (A1/A2/A3-C1-C2) interacts with FIX as a cofactor for activation of FX to FXa.
  • FIG. 3 and Figure 4 respectively represent a B-Domain Deleted FVI II (Refacto) and a polypeptide according to the invention referred to as 13A1 or 13A1 FVII I .
  • FIG 6 is a western blot of a fusion polypeptide according to the invention.
  • the fusion polypeptide is 13A1_FVI I I.
  • Culture media containing secreted FVI I I-FIX protein from stable CHOFIpln cells were purified on a VII I select antibody column. Purified protein was resolved on 4% SDS-Page gel, transferred onto PVDF membrane and probed with FIX and FVI II A2 monoclonal antibodies to detect the full-length (FL) light (LC) and the heavy chains (HC) of FVII I-FIXi respectively.
  • Figure 7 is a silver stained gel. Culture media containing secreted FVII I-FIX protein from stable CHOFIpln cells were purified on VII I Select antibody column. Purified protein was resolved on 4% SDS-Page gel, and thereafter silver stained with Protogel silver stain kit according to manufacturer's specification.
  • Figure 8 is an ELISA.
  • Reconstituted B-domain deleted FVIII (Refacto) of known concentration was used to generate a standard curve in a plate-method sandwich ELISA with match-paired monoclonal antibodies (Affinity biologicals) (left image).
  • FVIII-FIXi samples were analysed simultaneously along with the standards, and the concentration of the FVIII antigens present in the FVIII-FIXi sample was interpolated from the standard curve
  • Figure 9 represents a coamatic analysis of the biological activity.
  • Reconstituted B-domain deleted FVIII (Refacto) of known concentration was used to generate a standard curve in a coamatic chromogenic assay using a plate-method (left image).
  • FVIII-FIXi samples were analysed simultaneously along with the standards, and the biological activities of the FVIII-FIXi proteins were interpolated from the standard curve.
  • Figure 10 represents a measure of the sensitivity of FVIII-FIXi fusion polypeptides to generate thrombin.
  • Congenital FVIII deficient plasma spiked with different concentrations (25, 50 and 75%) of approximately 3U/mL purified FVIII-FIXi were analysed in a thrombin generation test.
  • Two negative controls comprising of 100% of 13A1_FVIII (void of FVII l-deficient plasma) and 100% FVIII-deficient plasma were included in the assay, while normal plasma was used as the positive control (in orange).
  • the 100% control which was the 13A1_FVIII by itself generated no thrombin.
  • 100% FVIII-deficient plasma (0% product) showed a small thrombin peak.
  • Figure 1 1 shows PK of FVIII-FIXi fusion polypeptide in an animal model.
  • Peak (5 mins) plasma concentration of Advate FVIII was nearly twice that of 13A1_FVIII, then Advate fell to low levels very quickly whereas 13A1_FVIII continued to show mean activity of > 10% or 10 lU/dl at 48 hours.
  • the lower peak for the fusion molecule may represent a different volume of distribution as FIX binds to collagen IV (Wolber, AS et al., JBC 1997) which may provide an intravascular depot.
  • FIX binds to collagen IV (Wolber, AS et al., JBC 1997) which may provide an intravascular depot.
  • the fusion remains in the circulation at a much higher activity over the study whereas the Advate falls to low levels quickly.
  • the fusion has delayed clearance and prolonged in vivo biological activity over Advate.
  • Table 2 illustrates that 13A1_FVIII showed a reduced blood loss when bled at 24 hours post- injection when compared to Advate FVIII. In contrast, Advate showed a reduced blood loss when bled at 15 minutes post-injection when compared to 13A1_FVIII.
  • the total blood loss (mg Hg) for 13A1_FVIII was comparable at both bleeding time points of 15 minutes and 24 hrs, whereas for Advate there was a sharp increase in total blood loss at 24hrs (17.68 mg Hg) compared to 15 minutes (2.039 mg Hg).
  • BERNTORP E., ASTERMARK, J., BJORKMAN, S., BLANCHETTE, V. S., FISCHER, K., GIANGRANDE, P. L. F., GRINGERI, A., LJUNG, R. C, MANCO-JOHNSON, M. J., MORFINI, M., KILCOYNE, R. F., PETRINI, P., RODRIGUEZ-MERCHAN, E. C, SCHRAMM, W., SHAPIRO, A., VAN DEN BERG, H. M. & HART, C. 2003. Consensus perspectives on prophylactic therapy for haemophilia: summary statement. Haemophilia, 9, 1-4.
  • DHILLON S. 2012. Octocog Alfa, Antihaemophilic Factor (Recombinant), Plasma/Albumin Free Method (Advate (R)) A Review of its Use in the Management of Patients with Haemophilia A. Drugs, 72, 987-1007.
  • DUMONT J. A., LIU, T. Y., LOW, S. C, ZHANG, X., KAMPHAUS, G., SAKORAFAS, P., FRALEY, C, DRAGER, D., REIDY, T., MCCUE, J., FRANCK, H. W. G., MERRICKS, E. P., NICHOLS, T. C, BITONTI, A.
  • STENNICKE H. R., KJALKE, M., KARPF, D. M., BALLING, K. W., JOHANSEN, P. B., ELM, T., OVLISEN, K., MOLLER, F., HOLMBERG, H. L, GUDME, C. N., PERSSON, E., HILDEN, I., PELZER, H., RAHBEK-NIELSEN, H., JESPERSGAARD, C, BOGSNES, A., PEDERSEN, A. A., KRISTENSEN, A.
  • TURECEK P. L, BOSSARD, M. J., GRANINGER, M., GRITSCH, H., HOLLRIEGL, W., KALIWODA, M., MATTHIESSEN, P., MITTERER, A., MUCHITSCH, E. M., PURTSCHER, M., ROTTENSTEINER, H., SCHIVIZ, A., SCHRENK, G., SIEKMANN, J., VARADI, K., RILEY, T., EHRLICH, H. J., SCHWARZ, H. P. & SCHEIFLINGER, F. 2012. BAX 855, a PEGylated rFVIII product with prolonged half-life Development, functional and structural characterisation. Hamostaseologie, 32, S29-S38.

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Abstract

L'invention concerne des polypeptides de fusion à action prolongée comprenant le facteur VIII [FVIII] et le facteur IX [FIX] et leur utilisation dans le traitement de troubles de la coagulation sanguine.
PCT/GB2017/052468 2016-08-24 2017-08-21 Facteur de coagulation WO2018037217A1 (fr)

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GBGB1614462.8A GB201614462D0 (en) 2016-08-24 2016-08-24 Clotting factor
GB1614462.8 2016-08-24

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110950964A (zh) * 2018-09-26 2020-04-03 安源医药科技（上海）有限公司 突变型单链人凝血因子viii融合蛋白及其制备方法与用途

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1038959A1 (fr) * 1999-03-17 2000-09-27 Aventis Behring Gesellschaft mit beschränkter Haftung Facteur VIII sans domaine B, comprenant une ou plusieurs insertions d'intron I tronqué du facteur IX
WO2013016454A1 (fr) * 2011-07-25 2013-01-31 Biogen Idec Hemophilia Inc. Dosages pour la surveillance de troubles hémostatiques
EP2848253A1 (fr) * 2006-06-19 2015-03-18 Asklepios Biopharmaceutical, Inc. Facteur VIII modifié et gènes de facteur IX et vecteurs pour thérapie génique
WO2015085276A1 (fr) * 2013-12-06 2015-06-11 Biogen Idec Ma Inc. Outils pharmacocinétiques de population et leurs utilisations

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1038959A1 (fr) * 1999-03-17 2000-09-27 Aventis Behring Gesellschaft mit beschränkter Haftung Facteur VIII sans domaine B, comprenant une ou plusieurs insertions d'intron I tronqué du facteur IX
EP2848253A1 (fr) * 2006-06-19 2015-03-18 Asklepios Biopharmaceutical, Inc. Facteur VIII modifié et gènes de facteur IX et vecteurs pour thérapie génique
WO2013016454A1 (fr) * 2011-07-25 2013-01-31 Biogen Idec Hemophilia Inc. Dosages pour la surveillance de troubles hémostatiques
WO2015085276A1 (fr) * 2013-12-06 2015-06-11 Biogen Idec Ma Inc. Outils pharmacocinétiques de population et leurs utilisations

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
AMY SHAPIRO: "Development of long-acting recombinant FVIII and FIX Fc fusion proteins for the management of hemophilia", EXPERT OPINION ON BIOLOGICAL THERAPY, vol. 13, no. 9, 10 August 2013 (2013-08-10), ASHLEY, LONDON; GB, pages 1287 - 1297, XP055417162, ISSN: 1471-2598, DOI: 10.1517/14712598.2013.819339 *
LIND ET AL: "Novel forms of B-domain-deleted recombinant factor VIII molecules. Construction and biochemical characterization", EUROPEAN JOURNAL OF BIOCHEMISTRY, WILEY-BLACKWELL PUBLISHING LTD, GB, vol. 232, no. 1, 1 August 1995 (1995-08-01), pages 19 - 27, XP002111900, ISSN: 0014-2956, DOI: 10.1111/J.1432-1033.1995.TB20776.X *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110950964A (zh) * 2018-09-26 2020-04-03 安源医药科技（上海）有限公司 突变型单链人凝血因子viii融合蛋白及其制备方法与用途

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