EP2714909A1 - Procédé de production de protéines du facteur viii par des procédés recombinants - Google Patents

Procédé de production de protéines du facteur viii par des procédés recombinants

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Publication number
EP2714909A1
EP2714909A1 EP12796718.0A EP12796718A EP2714909A1 EP 2714909 A1 EP2714909 A1 EP 2714909A1 EP 12796718 A EP12796718 A EP 12796718A EP 2714909 A1 EP2714909 A1 EP 2714909A1
Authority
EP
European Patent Office
Prior art keywords
factor viii
fviii
domain
protein
chain
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP12796718.0A
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German (de)
English (en)
Other versions
EP2714909A4 (fr
Inventor
Randal J. Kaufman
Steven W. Pipe
Michael Griffith
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Cangene Corp
University of Michigan System
Original Assignee
University of Michigan System
Inspiration Biopharmaceuticals Inc
University of Michigan Ann Arbor
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Priority claimed from US13/153,040 external-priority patent/US20120028900A1/en
Application filed by University of Michigan System, Inspiration Biopharmaceuticals Inc, University of Michigan Ann Arbor filed Critical University of Michigan System
Publication of EP2714909A1 publication Critical patent/EP2714909A1/fr
Publication of EP2714909A4 publication Critical patent/EP2714909A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • hemophilia treatment in the United States is estimated to cost about $50,000 per patient per year for the commercial product required for routine, on-demand, care.
  • this cost could be much higher insofar as the Medical and Scientific Advisory Committee for the National Hemophilia Foundation has
  • protein made in foreign recombinant cells must be successfully secreted out of the cell. Deficiencies in any one of a number of intracellular trafficking or enzymatic activities can result in the formation of a large percentage of non-functional protein and limit the usefulness of a genetically engineered cell system for the economical production of a biopharmaceutical product intended for commercial use.
  • This method may further comprise: f) recloning the at least one subclone identified in step e); and g) identifying at least one subclone expressing a higher level of FVIII relative to the at least one subclone selected in step e).
  • the isolated nucleic acid comprises a nucleotide sequence encoding a modified human FVIII polypeptide operably linked to a Chinese hamster elongation factor 1 -alpha promoter, wherein said modification comprises a truncated B domain comprising 226 amino acids from the amino-terminal end of the B domain containing 6 N-linked glycosylation site, a SQ linker, and a Pace/Furin cleavage site, characterized in that upon Pace/Furin cleavage and secretion the protein becomes a two-chain form comprising an A1-A2 -truncated B domain chain and an A3-C1-C2 chain.
  • FIG. 16 is a graph illustrating the results of an ELISA assay demonstrating antibody- inducible vWF binding of the inactivation resistant FVIII of the present invention following thrombin activation, and retained FVIII activity;
  • FIG. 34 depicts the presence of the FVIII B domain variants in cell extract and cell media.
  • FIG. 35 shows a schematic representation of the domain structure of FVIII BDD-SQ and FVIII B226/N6-SQ. Pace/Furin cleaves after the Arginine at position 1648.
  • biological activity or “biologically active” is determined with reference to a Factor VIII standard derived from human plasma.
  • Biological activity of a Factor VIII protein may be determined using the commercially available Factor VIII assay, COATEST (Kabi Pharmaceuticals) or other assay in the art.
  • COATEST measures the FVIII-dependent generation of Factor Xa from Factor X, with one unit defined as the amount of FVIII activity in one ml of pooled human plasma, 100 to 200 ng/ml (Vehar et al., Biotechnology of Plasma Proteins, Albertini et al, eds. pg. 2155, Basel, Karger, 1991).
  • the truncated B domain comprises 163 amino acids from the amino terminal end of the B domain containing at least four N-linked glycosylation sites (also referred to herein as the "FVIII B163/N4-SQ"). In yet another embodiment, the truncated B domain comprises 226 amino acids from the amino terminal end of the B domain containing at least six N-linked glycosylation sites (also referred to herein as the "FVIII B226/N6-SQ").
  • the Factor VIII SQ cDNAs may be assembled into transcription units and introduced into a suitable host system for expression.
  • the Factor VIII SQ expression vector includes a CHEF-1 promoter, a Factor VIII heavy chain coding sequence, a partial B domain sequence, a SQ linker, a Pace/furin cleavage site, and a Factor VIII light chain coding sequence.
  • the vector may be inserted or transfected (stably or transiently) into mammalian cell lines.
  • the cell lines can be grown on a large scale in suspension culture, such as serum-free medium supplemented with human albumin and recombinant insulin, or on solid support to produce commercial amounts of the recombinant FVIII.
  • Particular amino acid sequence variants may differ from the SQ linker peptide by insertion, addition, substitution or deletion of 1 amino acid, 2, 3, 4, 5-10, 10-20, 20-30, 30-40, 40-50, 50-100, 100-150, or more than 150 amino acids.
  • the present invention features methods which utilize CHEF- la promoters to producing high yields and commercially viable Factor VIII for use as human
  • FVIII nucleic acid sequence may vary from the wild-type FVIII by containing additional modifications such as those disclosed in U.S. Pat. No. 5,004,803, WO 86/06101, and WO 87/07144.
  • FVIII analogs have been developed to better understand the specific structural requirements for FVIII activatibility, inactivatibility, and in vivo efficacy and are also within the scope of the present invention. Included among the features to be optimized are simplified preparation, ease of administration, stability, improved clearance/distribution characteristics, reduced immunogenicity, and prolonged half-life.
  • variant FVIII nucleic acid sequences in accordance with the present invention also include allelic variations, i.e. variations in sequence due to natural variability from individual to individual, or with other codon substitutions or deletions which still retain FVIII-type procoagulant activity.
  • Prokaryotic and eucaryotic cell expression vectors containing and capable of expressing the nucleic acid sequences of the present invention may be synthesized by techniques well known to those skilled in this art.
  • the components of the vectors such as the bacterial replicons, selection genes, enhancers, promoters, and the like, may be obtained from natural sources or synthesized by known procedures (see, e.g. Kaufman et al, J. Mol. Biol. 159:601-621 (1982) and Kaufman, PNAS 82:689-693 (1995)).
  • Expression vectors useful in producing proteins of this invention may also contain inducible promoters or comprise inducible expression systems as are known in the art.
  • Established cell lines including transformed cell lines, are suitable as hosts.
  • Normal diploid cells cell strains derived from in vitro culture of primary tissue, as well as primary explants (including relatively undifferentiated cells such as hematopoietic stem cells) are also suitable.
  • Candidate cells need not be genotypically deficient in the selection gene so long as the selection gene is dominantly acting.
  • mammalian host cells provides for such post-translational modifications, e.g. proteolytic processing, glycosylation, tyrosine, serine, or threonine phosphorylation, as may be made to confer optimal biological activity on the expression products of the invention.
  • Established mammalian cell lines may be used, e.g. CHO (Chinese Hamster Ovary) cells.
  • the vector may include all or part of the bovine papilloma virus genome (Lusky et al, Cell 36:391-401 (1984)) and be carried in cell lines such as C127 mouse cells as a stable episomal element.
  • the nucleic acid further comprises a substitution of the Phenylalanine residue at 309 with a Serine residue.
  • the nucleic acid further comprises a substitution of the Phenylalanine residue at 309 with a Serine residue.
  • the nucleic acid comprises truncated B domain comprising 226 amino acids from the amino terminal end of the B domain containing 6 N-linked glycosylation sites.
  • a method for treating a patient for hemophilia comprises the step of administering to the patient a therapeutically effective amount of a protein comprising a modified human FVIII polypeptide operably linked to a Chinese hamster elongation factor 1 -alpha promoter, wherein said promoter is characterized by the ability to produce commercially viable Factor VIII protein and wherein said modification comprises a truncated B domain comprising at least 29 amino acids from the amino-terminal end of the B domain containing at least one N-linked glycosylation site, a SQ linker, and a Pace/Furin cleavage site, characterized in that upon Pace/Furin cleavage and secretion the protein becomes a two-chain form comprising an A 1-A2 -truncated B domain chain and an A3-C1- C2 chain.
  • the modified human FVIII polypeptide further comprises a substitution of the Phenylalanine residue at 309 with a Serine residue.
  • the modified human FVIII polypeptide comprises a truncated B domain comprising 226 amino acids from the amino terminal end of the B domain containing 6 N- linked glycosylation sites.
  • Methods for overrexpressing or producing Factor VIII proteins by co-expression with a processing factor can include the following techniques. First, a single vector containing coding sequences for more than one processing factor and a Factor VIII protein can be inserted into a selected host cell. Alternatively, two or more separate vectors encoding a Factor VIII protein plus one or more other processing factors, can be inserted into a host. Upon culturing under suitable conditions for the selected host cell, the two or more proteins are produced and interact to provide cleavage and modification of the proprotein into the mature protein.
  • Another alternative is the use of two transformed host cells wherein one host cell expresses the Factor VIII protein and the other host cell expresses one or more processing factor which will be secreted into the medium. These host cells can be co-cultured under conditions which allow expression and secretion or release of the recombinant Factor VIII protein and the co-expressed recombinant polypeptides.
  • a method for producing a recombinant Factor VIII protein comprising the steps of: (a) introducing into a cell a nucleic acid molecule encoding a modified human Factor VIII protein operably linked to a Chinese hamster elongation factor 1 -alpha promoter, wherein said promoter is characterized by the ability to produce commercially viable Factor VIII protein and wherein said modification comprises a deletion of the B domain, and an added SQ linker and a Pace/Furin cleavage site, characterized in that upon Pace/Furin cleavage and secretion the protein becomes a two-chain form comprising an Al- A2 chain and an A3-C1-C2 chain; and (b) incubating the cell under appropriate conditions for producing commercially viable Factor VIII protein.
  • a method for producing a recombinant Factor VIII protein comprising the steps of: (a) introducing into a cell a nucleic acid molecule encoding a modified human Factor VIII protein operably linked to a Chinese hamster elongation factor 1 -alpha promoter, wherein said promoter is characterized by the ability to produce commercially viable Factor VIII protein and wherein said modification comprises a truncated B domain comprising at least 29 amino acids from the amino-terminal end of the B domain containing at least one N-linked glycosylation site, a SQ linker, and a Pace/Furin cleavage site, characterized in that upon Pace/Furin cleavage and secretion the protein becomes a two- chain form comprising an A 1-A2 -truncated B domain chain and an A3-C1-C2 chain; and (b) incubating the cell under conditions for producing commercially viable Factor VIII protein.
  • Suitable host cells include prokaryote, yeast or higher eukaryotic cells such as mammalian cells and insect cells. Cells derived from multicellular organisms such as mammals are suitable as host cells for recombinant Factor VIII protein synthesis.
  • Expression vectors for such cells ordinarily include (if necessary) an origin of replication, a promoter located upstream from the DNA encoding Factor VIII protein(s) to be expressed and operatively associated therewith, along with a ribosome binding site, an RNA splice site (if intron-containing genomic DNA is used), a polyadenylation site, and a transcriptional termination sequence.
  • an origin of replication a promoter located upstream from the DNA encoding Factor VIII protein(s) to be expressed and operatively associated therewith, along with a ribosome binding site, an RNA splice site (if intron-containing genomic DNA is used), a polyadenylation site, and a transcriptional termination sequence.
  • a baculovirus expression vector comprises a baculovirus genome containing the gene to be expressed inserted into the polyhedrin gene at a position ranging from the polyhedrin transcriptional start signal to the ATG start site and under the transcriptional control of a baculovirus polyhedrin promoter.
  • Prokaryote host cells include gram negative or gram positive organisms, for example Escherichia coli (E. coli) or Bacilli. Higher eukaryotic cells include established cell lines of mammalian origin as described below. Exemplary host cells are E. coli W3110 (ATCC 27,325), E. coli B, E. coli XI 776 (ATCC 31,537), E. coli 294 (ATCC 31,446). A broad variety of suitable prokaryotic and microbial vectors are available. E. coli is typically transformed using ColEl replicons derived from pBR322.
  • Promoters most commonly used in recombinant microbial expression vectors include the betalactamase (penicillinase) and lactose promoter systems (Chang et al, Nature 275, 615 (1978); and Goeddel et al, Nature 281, 544 (1979)), a tryptophan (trp) promoter system (Goeddel et al, Nucleic Acids Res. 8, 4057 (1980) and EPO App. Publ. No. 36,776) and the tac promoter (H. De Boer et al, Proc. Natl. Acad. Sci. USA 80, 21 (1983)).
  • the promoter and Shine-Dalgarno sequence are operably linked to the DNA encoding the Factor VIII protein(s), i.e., they are positioned so as to promote transcription of Factor VIII Protein(s) messenger RNA from the DNA.
  • Eukaryotic microbes such as yeast cultures may also be transformed with Factor
  • Saccharomyces cerevisiae is the most commonly used among lower eukaryotic host microorganisms, although a number of other strains are commonly available.
  • Yeast vectors may contain an origin of replication from the 2 micron yeast plasmid or an autonomously replicating sequence (ARS), a promoter, DNA encoding one or more Factor VIII proteins, sequences for polyadenylation and transcription termination, and a selection gene.
  • ARS autonomously replicating sequence
  • Suitable promoting sequences in yeast vectors include the promoters for metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem. 255, 2073 (1980) or other glycolytic enzymes (Hess et al, J. Adv. Enzyme Reg. 7, 149 (1968); and Holland et al, Biochemistry 17, 4900 (1978)).
  • Suitable vectors and promoters for use in yeast expression are further described in R. Hitzeman et al., EPO Publn. No. 73,657.
  • the present invention provides a method of providing a functional Factor VIII.
  • the strategy may include co-expressing Factor VIII protein along with one or more processing factors in a single host cell.
  • the method comprises culturing a host cell which expresses a Factor VIII protein and/or processing factors; and then harvesting the proteins from the culture.
  • the culture can be carried out in any suitable fermentation vessel, with a growth media and under conditions appropriate for the expression of the Factor VIII protein(s) by the particular host cell chosen.
  • the Factor VIII protein can be collected directly from the culture media, or the host cells lysed and the Factor VIII protein collected therefrom. Factor VIII protein can then be further purified in accordance with known techniques.
  • Illustrative examples of such substances that may be removed by purification include thrombin and vonWillebrand factor; other protein contaminants, such as modification enzymes; proteins, such as hamster and mouse proteins, which are released into the tissue culture media from the production cells during recombinant protein production; non-protein contaminants, such as lipids; and mixtures of protein and non-protein contaminants, such as lipoproteins.
  • Also provided herein are methods for identifying a cell expressing commercially viable Factor VIII protein comprising: a) introducing into cells nucleic acid molecules encoding a Factor VIII protein operably linked to a promoter, wherein the promoter is characterized by the ability to overexpress or produce commercially viable Factor VIII protein; b) incubating the cells under conditions for overexpressing or producing Factor VIII protein; c) selecting clones expressing high levels of FVIII relative to the other clones; d) recloning the cells selected in step c); and e) identifying at least one subclone expressing a higher level of FVIII relative to those selected in step c).
  • a method for identifying a cell expressing commercially viable Factor VIII protein comprising: a) introducing into cells a nucleic acid molecule encoding a modified human Factor VIII protein operably linked to a Chinese hamster elongation factor 1 -alpha promoter, wherein said promoter is characterized by the ability to produce commercially viable Factor VIII protein and wherein said modification comprises a truncated B domain comprising at least 29 amino acids from the amino-terminal end of the B domain containing at least one N-linked glycosylation site, a SQ linker, and a Pace/Furin cleavage site, characterized in that upon Pace/Furin cleavage and secretion the protein becomes a two-chain form comprising an A 1-A2 -truncated B domain chain and an A3-C1- C2 chain; b) incubating the cells under conditions for producing commercially viable Factor VIII protein; c) selecting clones expressing high levels of FVIII
  • the cells are mammalian cells.
  • the mammalian cell is selected from the group consisting of a COS-1, CHO, and HEK 293 cell.
  • the truncated B domain comprises 226 amino acids from the amino terminal end of the B domain containing 6 N-linked glycosylation sites.
  • the modification further comprises a substitution of the Phenylalanine residue at 309 with a Serine residue
  • Stably transfected CHO cell lines were engineered that express the F309S mutant.
  • 35 original transfected CHO cell clones selected for dihydro folate reductase expression 5 clones were obtained that express significant levels of FVIII (approximately 1 U/ml/10 6 cells/day).
  • Two of these clones express the same level of FVIII as the original 10A1 cell line that was obtained by screening over 1000 original transfected cell clones.
  • methotrexate the mutation permits high level FVIII expression to be obtained more readily.
  • FVIII deficient plasma and normal pooled human plasma were obtained from George King Biomedical, Inc. (Overland Park, KS). Monoclonal antibody to the heavy chain of FVIII (F8) coupled to CL4B-sepharose was used and may be prepared by known methods.
  • Activated partial thromboplastin Automated APTT reagent was purchased from General Diagnostics Organon Teknika Corporation (Durham, NC). Soybean trypsin inhibitor, phenylmethylsulfonylfluoride (PMSF) and aprotinin were purchased from
  • DMEM Dulbecco's modified eagle medium
  • a-MEM Eagle's Medium
  • methionine-free DMEM obtained from Gibco BRL (Gaithersburg, MD).
  • Fetal bovine serum was purchased from PAA Laboratories Inc. (Newport Beach, CA).
  • Samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions and visualized by autoradiography after fluorography by treatment with En3hance (Dupont; Boston, MA).
  • SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis
  • FVIII APC inactivation of FVIII.
  • Purified FVIII samples diluted to 3 U/ml in buffer B were mixed with 100 ig/ml inosithin and human APC 100 ng/ml or buffer alone as a control. After increasing periods of time at 37°C, aliquots were diluted and the residual FVIII was determined.
  • R336I, R562K, and R336I/R562K mutant FVIII molecules are efficiently secreted with FVIII activity similar to wild-type FVIII.
  • the activity and secretion of FVIII mutants were measured by transient DNA transfection of COS-1 monkey cells.
  • the FVIII clotting activity in the conditioned medium demonstrated that all mutants had FVIII activity similar to wild- type FVIII, approximately 300 mU/ml (see Table 1).
  • Thrombin activation of the conditioned medium samples indicated that there was no difference in the rate of thrombin activation and decay of procoagulant activity. As shown in Figure 3, all samples were immediately activated (3-5 fold) at 10 seconds after thrombin addition and were immediately inactivated.
  • the symbols represent wild-type FVIII (X), R336I ( ⁇ ), R562K (0) and
  • R562K is completely resistant and R336I is mostly resistant to APC cleavage at the mutated site.
  • APC cleavage of FVIIIa was evaluated by treating [ 35 S]-methionine labeled immunoprecipitated FVIII with APC.
  • Analysis of APC cleavage products of wild-type FVIII analyzed by SDS-PAGE on a 5-15% gradient gel detected the heavy chain fragments of 50 kDa and 45 kDa representing the Al -domain, and of 43 kDa representing the A2-domain, that were not present in the conditioned medium of cells that did not receive DNA.
  • lane 2 a lower molecular weight product at 25 kDa was detectable, representing the carboxy-terminus of A2-domain.
  • lane 3 R336I FVIII was partially resistant to cleavage at residue 336, as indicated by an increase in the 50 kDa and a reduction of the 45 kDa cleavage products compared to wild-type.
  • the R336I displayed no change in the amount of the 25 kDa species indicating efficient cleavage at residue 562.
  • lane 4 R562K mutant FVIII was resistant to cleavage at residue 562 as indicated by the increase in the 43 kDa fragment and loss of the 25 kDa fragment.
  • the R562K mutant was efficiently cleaved at 336 as indicated by an intense 45 kDa fragment.
  • APC treatment of the R336I/R562K double mutant yielded an increase in the 50 kDa and 43 kDa species, and the reduction of 45 kDa and loss of 25 kDa species compared to wild-type FVIII (see Figure 5A, lane 5).
  • the migration of the 45 kDa fragment derived from APC cleavage of the R336I mutant was slightly reduced upon analysis by SDS-PAGE on an 8% polyacrylamide gel (see Figure 5B, compare lanes 7 and 8).
  • an R336I and K338I double mutant was made by site-directed mutagenesis.
  • R336I/K338I mutant did not generate the 45 kDa fragment upon APC digestion (see Figure 5B, lane 9).
  • molecular size markers are shown on the left and "Mock" represents cells that did not receive DNA.
  • vWF von Willebrand Factor
  • R336I/R562K (A) double mutant was resistant to inactivation and retained 76% activity after 60 min.
  • APC resistance assay kit to detect APC resistant FVIII.
  • a commercially available APC resistance assay kit (Coatest APC Resistance; Chromogenix, Molndal, Sweden) is used to screen the plasma of patients with thrombotic disease associated with the FV R506Q mutation.
  • the ability of this kit to detect APC resistant FVIII was tested by reconstitution of FVIII deficient plasma with either purified wild-type or purified mutant FVIII.
  • the APC resistance ratio was calculated by the measure of the clotting time in the presence of APC divided by the clotting time in the absence of APC (see Table 2). Only the R336I/R562K double mutant demonstrated a lower APC resistance ratio than 2, a value indicative of an APC resistance phenotype. Svensson, P.J. et ah, N. Engl. J. Med. 336:517 (1994).
  • R336I/R562K was partially resistant to cleavage at residue 336 and completely resistant at residue 562.
  • the cleavage of R336I likely occurred at an adjacent residue, Lys 338, since a double mutant R336I/K338I was completely resistant to cleavage at this site.
  • Anti-heavy chain factor VIII monoclonal antibody (F-8), F-8 conjugated to CL-4B Sepharose and purified recombinant factor VIII protein were obtained from
  • DMEM Dulbecco's modified Eagle's medium
  • methionine-free DMEM OptiMEM
  • Biotin N-hydroxy succinimide ester Biotin N-hydroxy succinimide ester
  • streptavidin-horseradish peroxidase conjugate were obtained from Gibco BRL (Gaithersburg, MD).
  • Oligonucleotide- directed mutagenesis was used to create a PCR fragment, KpnI/R740K/ApaI, and was ligated into Kpnl/Apal digested pMT 2 90/73. Construction 2 - 90/b/73 R740K. Vector pMT 2 VIII was used as the DNA template.
  • Oligonucleotide-directed mutagenesis was used to create a PCR fragment, KpnI/b/1689 Mlul (where b represents a DNA sequence encoding for amino acid residues 741 to 793 of the wild-type sequence followed by an Mlul site predicting amino acids threonine and arginine at residues 794 and 795/1689), which was ligated into Kpnl/Mlul digested vector pMT 2 VIII/1689/MluI.
  • the following amino acid sequence (SEQ ID NO.2) (and nucleotide sequence encoding same (SEQ ID NO: 1)) may be used as an amino acid sequence spacer, wherein residue 794 may be threonine or leucine:
  • Vector 90/b/73 was used as the DNA template (wherein b is described above and encodes threonine at residue 794). Oligonucleotide- directed mutagenesis was used to create a PCR fragment, KpnI/R740A/b/ApaI, which was ligated into Kpnl/Apal digested pMT 2 90/73.
  • KpnI/R740A/b/R1689A/ApaI which was ligated into Kpnl/Apal digested pMT 2 90/73.
  • the plasmid containing the wild-type FVIII cDNA sequence was designated FVIII WT. All plasmids were purified by centrifugation through cesium chloride and characterized by restriction endonuclease digestion and DNA sequence analysis.
  • Plasmid DNA was transfected into COS-1 cells by the DEAE-dextran method.
  • Conditioned medium was harvested at 64 hours post-transfection in the presence of 10% fetal bovine serum.
  • FVIII activity was measured by one-stage APTT clotting assay on a ML A Electra 750.
  • Protein synthesis and secretion were analyzed by metabolically labeling cells at 64 hours post-transfection for 30 minutes with [ 35 S]- methionine (300 mCi/ml in methionine-free medium), followed by a chase for 4 hours in medium containing 100-fold excess unlabeled methionine and 0.02% aprotinin.
  • Cell extracts and conditioned medium containing labeled protein were harvested.
  • WT and mutant FVIII proteins were immunoprecipitated from equal proportions of cell extract and conditioned medium with F-8 coupled to CL-4B Sepharose. Immunoprecipitates were washed and resuspended in Laemmli sample buffer. Samples were analyzed by electrophoresis on a reducing SDS-low bis-8% polyacrylamide gel. The gels were treated with En 3 Hance and the proteins visualized by autoradiography.
  • Partially purified IR8 protein was obtained from 200 mis of conditioned medium from transiently transfected COS-1 cells by immunoaffinity
  • FVIII WT protein was obtained from 200 mis of conditioned medium from stably transfected CHO cells and immunoaffinity purified in the same manner.
  • the proteins eluted into the ethylene glycol-containing buffer were dialyzed and concentrated against a polyethylene glycol (MW ⁇ 15-20,000)-containing buffer and stored at -70° C.
  • FVIII antigen determination FVIII antigen was quantified using a sandwich ELISA method utilizing anti-light chain antibodies ESH-4 and ESH-8. Purified recombinant FVIII protein was used as a standard.
  • amino acid sequence spacer represents the amino acid sequence spacer which is of a sufficient length to allow the protein to be activated by thrombin to achieve a heterodimer, wherein the A2-domain remains covalently associated with the light chain.
  • the amino acid sequence spacer is the amino portion of the wild-type B-domain, i.e. amino acid residues 741 to 793 followed by an Mlul site (for cloning purposes) predicting amino acids threonine or leucine, at residue 794 and arginine at 795/1689.
  • Figure 8 sets forth a model of activation of the constructs of the present invention. Wild-type FVIII and the mutant 90/73 both achieve a heterotrimer upon thrombin activation.
  • the mutant 90/80 is a BDD FVIII mutant (del741-1648) previously characterized, that migrates at -170 kDa and demonstrates an increased intensity from pulse-labeled cell extracts consistent with increased efficiency of synthesis ( Figure 10, lane 3). 90/73 migrates slightly faster due to the additional deletion of the residues of the acidic region ( Figure 10, lane 5). All the 90/b/73 based constructs including IR8 exhibited similar band intensity to the 90/80 and 90/73 constructs suggesting that the multiple missense mutations did not interfere with efficient protein synthesis. Additional bands within the cell extract are not observed in mock cell extract immunoprecipitated with an anti-FVIII specific antibody and represent both FVIII specific proteins and co-immunoprecipitating intracellular proteins.
  • IR8 did not reach peak activity until 30 seconds incubation with thrombin, suggesting a modestly reduced sensitivity to thrombin activation compared to FVIII WT.
  • IR8 still retained -38% of peak activity after 4 hours incubation with thrombin.
  • IR8 demonstrates increased FVIII specific activity in vitro. Immunoaffinity purified FVIII WT and IR8 were assayed for FVIII activity utilizing a standard one stage APTT clotting assay, wherein the first time point was 10 seconds. Antigen determinations were made utilizing a FVIII light chain based ELISA. Figure 13 shows the activation and reduced rate of inactivation expressed as specific activity. The specific activity values for IR8 were calculated based on a correction for its molecular weight. IR8 was observed to have a 5 -fold increased specific activity compared to FVIII WT (102 ⁇ 43 versus 18.6 ⁇ 7.4 U/mg of protein).
  • Wild-type FVIII continued to generate increasing amounts of FXa throughout 16 minutes of the first stage incubation.
  • the R531H, A284E and S289L could generate no more FXa after 8 and 16 minutes than that observed at 4 minutes, consistent with increased rate of inactivation of the mutant FVIIIa molecules early within the first stage of the assay.
  • Missense mutations N694I, R698L and R698W were expressed within a B-domainless FVIII vector by transient expression in COS-1 cells. Each of the mutations resulted in a secreted protein with l-st/2-st activity discrepancy similar to that reported from patient plasmas. Results
  • an A2-A3 disulfide bond may be obtained based on a molecular model of the A domains of FVa (Pellequer et al., Thrombosis Haemostatis, 84:849-57 (2000)), indicating that the molecular model could not predict which cysteine mutations would work, as only one successful disulfide bond resulted from several strategies attempted.
  • Immulon 2 microtiter wells (Dynatech Laboratories, Inc., Chantilly, VA) were coated with FVIII antibody at a concentration of 2 ig/ml overnight at 4°C in a buffer of 0.05 M sodium carbonate/bicarbonate pH 9.6.
  • FIG 14 shows the results of the FVIII-vWF binding ELISA. An anti-A2 domain trap was used. After a 4 hour incubation with FVIII-deficient plasma (1 : 100 dilution), binding was detected by perioxidase conjugated anti-vWFab. As shown in Figure 14, a 10-fold lower binding affinity of IR8 to vWF is observed in the absence of ESH8 compared to wild-type FVIII, and a 2-fold lower binding affinity is observed in the presence ofESH8.
  • Figure 15 shows the results of the FVIII-vWF binding ELISA with thrombin (Ila) and/or ESH8.
  • the same ELISA method was used however a 2-fold molar excess of ESH8 was employed as well as a 4 hour incubation with Ila (1 U/ml) in the presence of FVIII deficient plasma.
  • IR8 retains activity for vWF after thrombin activation suggesting that the heterodimer is intact after thrombin cleavage and ESH8 stabilizes the light chain confirmation such that it retains some affinity for vWF.
  • the binding assays described above utilize a "trap" antibody that only recognizes the A2-domain of FVIII, it will only detect FVIII-vWF complexes that recognize the A2-domain in association with the rest of the protein. Therefore, following the 4 hour incubation of the protein in the presence of excess thrombin, FVIII wild-type will not only have been fully activated but it will have also have been completely inactivated through A2 dissociation and/or further proteolytic cleavages, and will no longer associate with vWF in a complex that will be recognized by this assay.
  • the inactivation resistant FVIII of the present invention thus retains inducible binding even following complete activation by thrombin.
  • IR8 Affinity for von Willebrand factor and phospholipid.
  • ELISA and affinity biosensor analysis demonstrated IR8 had a 20-fold reduced affinity for von Willebrand factor (vWF), but a 34-fold increased affinity for phospholipid (PL) compared to rFVIII. These changes were attributed to deletion of the AR. In contrast to wild-type FVIII, these affinities were not changed upon thrombin activation of IR8.
  • the monoclonal antibody ESH8 increases the affinity of the thrombin-cleaved FVIII LC to vWF by preventing a LC conformational change that follows proteolytic removal of the AR in vitro ( Figure 22).
  • ESH8 inhibits FVIII activity in vitro by reducing the rate of vWF dissociation from FVIII upon thrombin activation.
  • Anti-F VIII antibodies specific for the PL binding site were still able to bind, suggesting that the PL binding site and the vWF binding site do not overlap within this LC conformation.
  • thrombin activation of IR8/ESH8 does not alter vWF dissociation (Figure 19).
  • FVIII/ESH8 is detected partially complexed with vWF in an inactive form, which may be due to A2 subunit dissociation or the PL binding site is blocked while the FVIII LC is bound to vWF.
  • ESH8 induces an IR8-vWF interaction similar to FVIII WT that does not change upon thrombin activation.
  • ESH8 induces a conformation of the LC that retains high affinity for vWF that is independent of the presence of the AR.
  • the AR may be responsible for regulating FVIII cofactor activity as the presence of the AR induces a high affinity vWF binding LC conformation and blocks that PL binding site and the absence of the AR results in a LC conformation that has low affinity for vWF thus the PL binding site is not blocked.
  • NMC-VIII/5 completely blocks the vWF binding site.
  • dissociation of vWF from IR8 was initiated ( Figure 23).
  • the rate of spontaneous dissociation of IR8 or FVIII WT from NMC-VIII/5 is negligible (Figure 23).
  • SPIII is a 340 kDa homodimeric disulfide-linked vWF fragment (residues 1-1365 of vWF) and has affinity for FVIII similar to intact vWF. Saenko, E.L. et al., J. Biol. Chem. 270: 13826-13833 (1995).
  • the effect of the increasing concentrations of SPIII (no SPIII in curve 1, 10 nM SPIII in curve 2, 25 nM SPIII in curve 3, 50 nM SPIII in curve 4) on binding of FVIII/ESH8 complex to PSPC monolayer is set forth in Figure 24. Results
  • IR8/ESH8/SPIII complex Preparation of the IR8/ESH8/SPIII complex.
  • the IR8/ESH8/SPIII complex was prepared by incubation (30 min, RT) of 200 nM SPIII, 200 nM ESH8 with varying concentrations of IR8 (0.1 nM-6.4 nM). Association of IR8/ESH8/SPIII with PSPC (25/75) was measured in HBS, 5 mM CaCl 2 until equilibrium was approached.
  • concentration of the IR8/ESH8/SPIII complex corresponding to curves 1-8 are 0, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2 and 6.4 nM, respectively (Figure 25, Panel A).
  • K d value K d value. Determination of the 3 ⁇ 4 value for IR8/ESH8/SPIII binding to PSPC monolayer is set forth in Panel B of Figure 25.
  • the open symbols are the values of equilibrium binding (B e ) determined from curves 1-7.
  • F is the concentration of unbound ligand, R max - maximal binding capacity of the PSPC monolayer.
  • the predominant Furin cleavage Heavy Chain product for Full Length ending at R1313 is about the same size as single chain B226/N6, while the Heavy Chain for B226/N6-SQ migrates more rapidly, and is subject to further processing to mature heavy chain and to domain A2. Similarly, while B226/N6 remains a single chain, mature Light Chain is evident in Full Length and B226/N6-SQ samples.
  • the FVIII proteins of the present invention can be formulated into pharmaceutically acceptable compositions with parenterally acceptable vehicles and excipients in accordance with procedures known in the art.
  • the pharmaceutical compositions of this invention suitable for parenteral administration, may conveniently comprise a sterile lyophilized preparation of the protein which may be reconstituted by addition of sterile solution to produce solutions such as isotonic with the blood of the recipient.
  • the preparation may be presented in unit or multi-dose containers, e.g. in sealed ampoules or vials.
  • nucleotide sequences encoding the FVIII proteins of the present invention may be associated with a gene therapy delivery system in accordance with procedures known in the art.
  • delivery systems include, without limitation, adenoviral, retroviral and adeno-associated viral vectors, as well as liposomes and DNA-protein complexes.
  • the sequences of the present invention are contained in or operatively-linked to such delivery systems in a manner which allows for transcription, e.g., through the use of sufficient regulatory elements. It will be appreciated that a variety of strategies and methodology for creating such gene therapy delivery systems are well known to those skilled in the art.
  • a pharmaceutical composition for intravenous injection may contain, in addition to the proteins, an isotonic vehicle such as sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, lactated Ringer's injection, or other vehicles as known in the art.
  • the pharmaceutical composition according to the present invention may also contain stabilizers, preservatives, buffers, anti-oxidants, or other additives known to those of skill in the art.
  • the proteins of the present invention will be in the form of pyrogen-free, parenterally acceptable aqueous solutions.
  • gene therapy delivery systems or vehicles containing nucleotide sequences of the present invention may also be used to treat patients suffering form hemophilia caused by deficiency of FVIII.
  • a therapeutically effective amount of such gene therapy delivery vehicles is administered to a mammal having a hemophiliac condition caused by FVIII deficiency.
  • administration of the vehicles of the present invention will be by procedures well established in the pharmaceutical arts, e.g. by direct delivery to the target tissue or site, intranasally, intravenously, intramuscularly, subcutaneously,

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Abstract

La présente invention concerne des procédés et des compositions pour la production de protéines du facteur VIII. De tels procédés comprennent l'introduction à l'intérieur d'une cellule d'une molécule d'acide nucléique codant pour une protéine du facteur VIII fonctionnellement liée à un promoteur, le promoteur étant caractérisé par la capacité à produire une protéine du facteur VIII commercialement viable ; et l'incubation de la cellule dans des conditions pour la production d'une protéine du facteur VIII commercialement viable. L'invention concerne également de molécules d'acide nucléique qui codent pour une protéine du facteur VIII fonctionnellement liée à un promoteur du facteur 1-a d'élongation de hamster de Chine (CHEF1) qui peut être utilisé dans les procédés de la présente invention.
EP12796718.0A 2011-06-03 2012-06-01 Procédé de production de protéines du facteur viii par des procédés recombinants Withdrawn EP2714909A4 (fr)

Applications Claiming Priority (2)

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US13/153,040 US20120028900A1 (en) 2006-06-30 2011-06-03 Method of producing factor viii proteins by recombinant methods
PCT/US2012/040376 WO2012170289A1 (fr) 2011-06-03 2012-06-01 Procédé de production de protéines du facteur viii par des procédés recombinants

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WO2014127215A1 (fr) 2013-02-15 2014-08-21 Biogen Idec Ma Inc. Gène du facteur viii optimisé
US11008561B2 (en) 2014-06-30 2021-05-18 Bioverativ Therapeutics Inc. Optimized factor IX gene
MX388739B (es) 2015-02-06 2025-03-20 Univ North Carolina Chapel Hill Casetes optimizados de expresión del gen humano del factor viii de coagulación y su uso.
JP2018524995A (ja) * 2015-08-06 2018-09-06 グリコトープ ゲーエムベーハー 抗体ドメイン融合タンパク質を含むヘテロマー
CA3012695A1 (fr) 2016-02-01 2017-08-10 Bioverativ Therapeutics Inc. Genes du facteur viii optimises
US11299533B2 (en) 2017-06-23 2022-04-12 Takeda Pharmaceutical Company Limited Purification of factor VIII subspecies
JP7672334B2 (ja) * 2018-10-23 2025-05-07 ザ・チルドレンズ・ホスピタル・オブ・フィラデルフィア 第ｖｉｉｉ因子機能を調節するための組成物および方法
EP3785726A1 (fr) * 2019-09-02 2021-03-03 Biotest AG Protéine de facteur viii à demi-vie prolongée
EP4017521A1 (fr) * 2019-09-02 2022-06-29 Biotest AG Protéine du facteur viii à demi-vie accrue
KR20220097891A (ko) 2019-09-30 2022-07-08 바이오버라티브 테라퓨틱스 인크. 렌티바이러스 벡터 제형
MX2023000156A (es) 2020-06-24 2023-02-16 Bioverativ Therapeutics Inc Metodos para la eliminacion de factor viii libre de preparaciones de vectores lentivirales modificados para expresar dicha proteina.
CN117192132B (zh) * 2023-11-03 2024-02-02 庄亚(北京)生物科技有限公司 一种vWF片段残留检测试剂盒及方法

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CA2656558A1 (fr) * 2006-06-30 2008-01-10 The Regents Of The University Of Michigan Procede de production de proteines de type facteur viii par des procedes recombinants

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