WO1997003193A1 - Nouveaux analogues du polypeptide du facteur viii:c presentant des proprietes modifiees de liaison avec des metaux - Google Patents

Nouveaux analogues du polypeptide du facteur viii:c presentant des proprietes modifiees de liaison avec des metaux Download PDF

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Publication number
WO1997003193A1
WO1997003193A1 PCT/US1996/011438 US9611438W WO9703193A1 WO 1997003193 A1 WO1997003193 A1 WO 1997003193A1 US 9611438 W US9611438 W US 9611438W WO 9703193 A1 WO9703193 A1 WO 9703193A1
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factor viii
analog
polypeptide
viiiic
factor
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PCT/US1996/011438
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English (en)
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David T. Hung
Fred E. Cohen
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Chiron Corporation
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Priority to AU64558/96A priority Critical patent/AU6455896A/en
Publication of WO1997003193A1 publication Critical patent/WO1997003193A1/fr

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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)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • Factor VIII:C polypeptide analogs having improved properties. More particularly, the invention pertains to Factor VIII:C polypeptide analogs having altered metal- binding properties. This invention further relates to analog complexes comprising at least two such analogs, or one such analog and a native Factor VIII:C polypeptide, nucleic acid molecules encoding such analogs, vectors and host cells comprising the nucleic acid molecules, pharmaceutical compositions comprising the analogs or analog complexes, methods of making the analogs, nucleic acid molecules, vectors and host cells, and methods of prevention or treatment of active Factor VIII:C deficiency using the analogs, complexes, and/or nucleic acid molecules, vectors and host cells.
  • Hemophilia A is an X-chromosome-linked inherited bleeding diathesis that results from the deficiency of an active blood clotting factor termed Factor VIII:C.
  • the disease afflicts approximately 1 in
  • Factor VIII:C is a large glycoprotein that participates in the blood coagulation cascade that ultimately converts soluble fibrinogen to insoluble fibrin clot, effecting hemostasis.
  • the deduced primary amino acid sequence of human Factor VIII:C determined from the cloned cDNA indicates that Factor VIII:C is a heterodimer processed from a larger precursor polypeptide consisting of 2351 amino acids, referred to herein as the precursor or full- length Factor VIII:C molecule, of which the first 19 N- terminal residues comprise the signal sequence. Therefore, the mature Factor VIII:C molecule, starting with Alal, which does not contain the signal peptide sequence, includes a sequence of 2332 amino acids.
  • Amino acids from about 1 to about 1648 of the mature Factor VIII:C molecule give rise to "heavy chain” fragments with molecular weights ranging from approximately 90 kD to 200 kD.
  • Amino acids from about 1649 to about 2331 of the mature Factor VIII:C molecule comprise a "light chain” with a molecular weight of approximately 80 kD ("the 80 kD subunit").
  • the heterodimeric mature Factor VIII:C molecule consists of the heavy and light chains associated by a metal ion bridge, and lacking amino acids 741-1648 (the B domain) .
  • the mature Factor VIII:C molecule consists of a triplicated A domain of 330 amino acids, a unique B domain of 980 amino acids, and a duplicated C domain of 150 amino acids with the structure NH 2 -A1-A2-B-A3-C1-C2- COOH. See, e.g., Kaufman, R. J., Structure and Biology of Factor VIII, in Part VI, Hemostasis and Thrombosis, pp. 1276-1284; and Pan et al., Nature Structural Biology (1995) 2:740-744.
  • Factor VIII:C is known to be activated by plasma proteases such as thrombin.
  • the mature Factor VIII:C polypeptide is cleaved to generate heavy and light chain fragments that are further cleaved.
  • cleavage of the light chain after arginine residue 1689 yields a light chain fragment of about 73 kD ("the 73 kD fragment")
  • cleavage of the heavy chain after arginine residue 372 yields smaller heavy chain fragments of about 50 kD and 43 kD (“the 50 kD and 43 kD fragments," respectively) , as described in Eaton et al. (1986) ,
  • an active Factor VIII:C polypeptide analog that is substantially the same as a native Factor VIII:C polypeptide, except for the presence of altered etal- binding properties in the analog molecule.
  • Such a modification can be achieved by one or more amino acid substitutions, additions or deletions.
  • the native Factor VIII:C polypeptide that is modified is selected from the group consisting of (a) a full-length Factor VIII:C molecule comprising a signal peptide and all A, B, and C domains; (b) a native Factor VIII:C molecule comprising all A, B, and C domains and lacking a signal peptide; (c) a truncated Factor VIII:C molecule lacking a signal peptide and at least a portion of the B domain; (d) a cleaved Factor VIII:C molecule containing a light chain subunit of molecular weight of about 80 kD; (e) a cleaved Factor VIII:C molecule containing a heavy chain fragment of molecular weight in a range of about 90 kD to about 200 kD; (f) a cleaved Factor VIII:C molecule comprising a heavy chain fragment of a mo
  • an active Factor VIII:C analog complex that contains either at least two Factor VIII:C polypeptide analogs as above, or a Factor VIIIiC polypeptide analog and a Factor VIII:C polypeptide, together with a metal ion.
  • the analog complex herein can comprise two Factor VIIIiC polypeptide analogs, and the two analogs can be selected from the group consisting of analogs of molecular weights of about (a) 80 kD and 90 kD; (b) 73 kD and 90 kD; (c) 80 kD and 50 kD; (d) 80 kD and 43 kD; (e) 73 kD and 50 kD; and (f) 73 kD and 43 kD.
  • a method of producing a Factor VIIIiC polypeptide analog as above by (a) providing a native Factor VIII:C polypeptide that contains an amino acid sequence, and (b) modifying at least one amino acid residue in the amino acid sequence to produce an analog as above.
  • nucleic acid molecule that contains a nucleotide sequence that encodes the analog as above.
  • a recombinant vector that contains the nucleic acid molecule as above and a regulatory element, where the nucleic acid molecule is placed under regulatory control of the regulatory element.
  • a recombinant host cell that contains the nucleic acid molecule or recombinant vector as above.
  • a method of producing an active Factor VIIIiC polypeptide analog as above comprising: (a) providing the recombinant host cell as above, and (b) allowing the recombinant host cell to express the analog.
  • nucleic acid molecule as above, comprisingi (a) providing a nucleic acid molecule that encodes a native Factor VIIIiC polypeptide as above and (b) modifying at least one codon to provide the analog.
  • a method of producing a recombinant host cell that comprises a nucleic acid molecule as above, comprising transforming a host cell with the nucleic acid molecule, or transforming a host cell with the recombinant vector.
  • composition that contains the active Factor VIIIiC polypeptide analog or analog complex as above, and a pharmaceutically acceptable excipient.
  • Factor VIIIiC polypeptide analogs can be made that have improved properties. These analogs carry one or more amino acid residues that are modified from the native structure such that the molecule displays altered metal-binding properties. The modification can be at least one amino acid substitution, addition or deletion, at any portion of the native Factor VIIIiC molecule, so long as Factor VIII:C activity is not destroyed. Definitions
  • Fractor VIII:C polypeptide refers to a polymer of amino acids and does not refer to a specific length of the product.
  • peptides, oligopeptides, and proteins are included within the definition of polypeptide. This term also does not exclude post- expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like. Included within the definition are, for example, polypeptides containing one or more analogs of an amino acid, including, for example, unnatural amino acids, polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring.
  • a Factor VIII:C polypeptide includes but is not limited to, for example, the following Factor VIII:C polypeptides: (a) a full-length Factor VIII:C molecule comprising a signal peptide and all A, B, and C domains; (b) a mature Factor VIIIiC molecule comprising all A, B, and C domains and lacking the signal peptide; (c) a truncated Factor VIIIiC molecule lacking the signal peptide and at least a portion of the B domain; (d) a cleaved Factor VIIIiC molecule comprising a light chain subunit of about 80 kD; (e) a cleaved Factor VIII:C molecule comprising a heavy chain fragment of about 90 kD; (f) a cleaved Factor
  • Factor VIII:C polypeptides also include muteins or derivatives of the polypeptides with conservative amino acid changes that do not alter the biological activity of the polypeptide from which the utein or derivative is made.
  • Such muteins or derivatives may have, for example, amino acid insertions, deletions, or substitutions in the relevant molecule that do not substantially affect its properties.
  • the mutein or derivative can include conservative amino acid substitutions, such as substitutions which preserve the general charge, hydrophobicity/hydrophilicity, and/or stearic bulk of the amino acid substituted, for example Gly/Ala; Val/Ile/Leu; Asp/Glu; Lys/Arg; Asn/Gln; and Phe/Trp/Tyr.
  • the mutein or derivative should exhibit the same general structure as the native polypeptide, and may also include polypeptides having one or more peptide mimics or peptoids.
  • active in reference to the polypeptide analogs herein refers to biological activity, such as coagulation or procoagulation activity. Such activity is measured using standard assays for blood plasma samples, such as, for example, the Coatest assay or the activated partial, thromboplastin time test (APTT) .
  • An "active" Factor VIII:C polypeptide analog will have at least about 50% of the coagulation or procoagluation activity displayed by the native molecule, preferably at lea ⁇ t about 60% to 80% and more preferably at least about
  • nucleic acid molecule refers to either RNA or DNA or its complementary strands thereof, that contains a nucleotide sequence.
  • regulatory element refers to an expression control sequence that is conventionally used to effect expression of a gene.
  • a regulatory element includes one or more components that affect transcription or translation, including transcription and translation signals.
  • Such a sequence can be derived from a natural source or synthetically made, as in hybrid promoters and includes, for example, one or more of a promoter sequence, an enhancer sequence, a combination promoter/enhancer sequence, an upstream activation sequence, a downstream termination sequence, a polyadenylation sequence, an optimal 5' leader sequence to optimize initiation of translation, and a Shine- Dalgarno sequence.
  • the expression control sequence that is appropriate for expression of the present polypeptide differs depending upon the host system in which the polypeptide is to be expressed.
  • such a sequence in prokaryotes, can include one or more of a promoter sequence, a ribosomal binding site, and a transcription termination sequence.
  • such a sequence in eukaryotes, for example, can include one or more of a promoter sequence, and a transcription termination sequence.
  • a component that is necessary for transcription or translation is lacking in the nucleic acid molecule of the present invention, such a component can be supplied by a vector.
  • Regulatory elements suitable for use herein may be derived from a prokaryotic source, an eukaryotic source, a virus or viral vector or from a linear or circular plasmid.
  • regulatory control refers to control of expression of a polynucleotide sequence by a regulatory element to which the polynucleotide sequence is operably linked. The nature of such regulatory control differs depending upon the host organism. In prokaryotes, such regulatory control is effected by regulatory sequences which generally include, for example, a promoter, and/or a transcription termination sequence. In eukaryotes, generally, such regulatory sequences include, for example, a promoter and/or a transcription termination sequence. Additionally, other components which control of expression, for example, a signal peptide sequence or a secretory leader sequence for secretion of the polypeptide, and a terminator for transcriptional termination, may be attached thereto to facilitate regulatory control of expression.
  • terapéuticaally effective amount is meant an amount of analog that will improve the blood coagulation properties as compared to coagulation in the absence of the analog.
  • a “therapeutically effective amount” will fall within a relatively broad range that can be determined through routine trials.
  • the activity of the Factor VIIIiC analogs of the invention may be determined by means known in the art, for example, by using the commercially available Coatest assay.
  • the effective amount is sufficient to bring about prevention of further deterioration or treatment to improve coagulation, that is, to enhance coagulation properties such that hemostasis is achieved.
  • the exact amount necessary will vary depending on the subject being treated; the age and general condition of the individual to be treated; the functionality of the endogenous Factor VIII:C gene present in the individual; and the mode of administration, among other factors.
  • An appropriate effective amount can be readily determined by one of skill in the art. For example, depending on the severity of active Factor VIII:C polypeptide deficiency, up to about 1000 to about 3000 U of Factor VIIIiC polypeptide analog can be given to an average person such as a 70 kg male patient.
  • sufficient Factor VIIIiC polypeptide analog or analog complex can be given to establish a plasma level of about 0.5 to about 2 U/ml of Factor VIIIiC analog or combination analog and native polypeptide. See U.S. Patent Nos. 3,631,018; 3,652,530, and 4,069,216 for methods of administration and amounts.
  • pharmaceutically acceptable excipient refers to an excipient for administration of a therapeutic agent, in vivo , and refers to any pharmaceutical agent that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity.
  • Pharmaceutically acceptable carriers in therapeutic compositions may contain liquids such as water, saline, glycerol and ethanol.
  • salts can be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles. Suitable carriers may also be present and are generally large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles. Such carriers are well known to those of ordinary skill in the art.
  • compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared.
  • the present invention provides Factor VIIIiC polypeptide analogs with improved properties.
  • the analogs include modifications to the native Factor VIIIiC amino acid sequence which comprise amino acid substitutions, deletions or additions that confer altered metal-binding properties to the analog molecule. Such modifications can result in the addition of metal-binding sites or can include conformational changes which allow the molecule to bind metals more avidly.
  • the modifications can include the addition of metal binding sites to the heavy and light chains, particularly to the Al and A3 domains, to result in increased affinity between the heavy and light chains, and particularly between the Al and A3 domains thereof.
  • additional metal-binding sites can be produced in the heavy chain domains, such as in the Al and A2 domains, to result in increased affinity between components of the heavy chain, such as between the Al and A2 domains.
  • the augmented affinity will therefore result in decreased dissociation of the Factor VIIIiC molecule into inactive forms.
  • the modifications can be such that other divalent cation binding motifs result which allow for the association of a variety of other metals, not normally associated with the Factor VIIIiC molecule.
  • the primary and secondary structure of a protein together affect the propensity of a given portion of the protein to bind to a given metal.
  • the conformational factors and amino acids associated with binding to particular metals are known. See, e.g., Tainer et al., Current Opin . Biotech . (1991) 2i582-591; Vallee and Auld, Biochem . (1990) 29i5647-5659; Toma et al., Biochem . (1991) 30197-106; Pantoliano et al., Biochem .
  • amino acid residues can be added or substituted in appropriate portions of the native Factor VIIIiC molecule, based on, e.g., computer generated models of the native molecule, to produce new metal- binding sites.
  • a particularly useful model of the Factor VIIiC molecule is described in Pan et al., Nature
  • a particularly preferred modification involves creating a new type II copper binding site between the Al and A3 domains of the native Factor VIIIiC sequence, such as by substituting His or Met for any of Phe652, Tyrl786, Lysl818, Aspl840 and/or Asnl864, numbered relative to the native sequence as described in, e.g., U.S. Patent no. 5,045,455 and Truett et al., DNA (1985) 41333-349.
  • His will be substituted for at least two of these amino acids and more preferably His will be substituted for any two amino acids selected from the group of amino acids consisting of Phe652, Tyrl786 and Aspl840, also numbered relative to the native Factor VIIIiC sequence.
  • Factor VIIIiC polypeptide analogs can be made by modifying the native nucleic acid sequence that encodes the Factor VIIIiC polypeptide or cDNA sequences that encode the Factor VIIIiC polypeptides.
  • the DNA sequence and corresponding amino acid sequence of native Factor VIIIiC is known. See, e.g., U.S. Patent no. 5,045,455 and Truett et al., DNA (1985) 4 ⁇ 333-349.
  • Modifications can be made to the native Factor VIIIiC sequence by conventional techniques such as site-directed mutagenesis.
  • site-directed mutagenesis For example, the M13 method for site directed mutagenesis is known, as described in Zoller and Smith, Nucleic Acids Res .
  • one or more of the codons in the selected portion of Factor VIIIiC can be mutated by substitution, deletion or addition, to one or more codons encoding a metal-binding site or to codons which encode a molecule with greater affinity for a particular metal.
  • a description of a protocol suitable for use herein for mutagenesis of specific sites of a Factor VIIIiC expression plasmid can be found in WO 87/07144.
  • the nucleic acid molecules of the present invention can also be made synthetically by piecing together nucleic acid molecules encoding heavy and light chain fragments derived from cDNA clones or genomic clones containing Factor VIIIiC coding sequences, preferably cDNA clones, using known linker sequences. Alternatively, the entire sequence or portions of nucleic acid sequences encoding analogs described above may be prepared by synthetic methods (e.g. using DNA synthesis machines) . Once made, the nucleic acid molecules can be inserted in vectors for production of recombinant vectors for transcription and translation of the nucleic acid molecules.
  • a vector suitable for use herein for the production of a recombinant vector comprises a nucleic acid sequence with one or more restriction enzyme recognition sites into which the present nucleic acid molecule of the invention can be inserted. This vector also typically contains a selection marker for detection of the presence of the vector in the host cell.
  • the vector can also provide, if desired, one or more regulatory element ⁇ or control sequences for expres ⁇ ion of the nucleic acid molecule.
  • the pre ⁇ ent vector can be derived from a pla ⁇ id, a virus, a cosmid, or a bacteriophage. This vector is typically capable of behaving as an autonomou ⁇ unit of replication when introduced into a ho ⁇ t cell.
  • the vector may be one that is capable of episomal existence or of integration into the host cell genome.
  • a wide variety of replication systems are available, typically derived from viruse ⁇ that infect mammalian host cells.
  • Illu ⁇ trative replication systems include the replication sy ⁇ tem ⁇ from Simian viru ⁇ 40, adenoviru ⁇ , bovine papilloma virus, polyoma virus, Epstein Barr virus, and the like. Thu ⁇ , the nucleic acid molecule of the pre ⁇ ent invention can be in ⁇ erted at an appropriate re ⁇ triction site in the vector so as to be placed under the control of one or more regulatory elements in the vector to form a recombinant vector that can be u ⁇ ed for tran ⁇ fection or transformation of a host cell.
  • the host cell ⁇ of the invention can be, for example, prokaryotic or eukaryotic ho ⁇ t cell ⁇ , including bacterial, yeast, insect and mammalian expre ⁇ ion ⁇ y ⁇ tem ⁇ .
  • the analog ⁇ of the pre ⁇ ent invention are expressed in mammalian host cell system ⁇ .
  • the regulatory elements to be used in the vector depend on the host system that is to be utilized.
  • a prokaryotic host cell can be used for amplification of the nucleic acid molecule of the present invention, while an eukaryotic ho ⁇ t cell can be used for expres ⁇ ion of the Factor VIII:C polypeptide analog ⁇ .
  • the expre ⁇ ion ca ⁇ settes are introduced into the host cell by conventional methods, depending on the expres ⁇ ion system used, as described further below.
  • transfection or transduction may be employed.
  • the particular manner in which the host cell is transformed is not critical to thi ⁇ invention, depending substantially upon whether the expres ⁇ ion cassette ⁇ are joined to a replication ⁇ y ⁇ tem and the nature of the replication ⁇ y ⁇ tem and a ⁇ ociated gene ⁇ .
  • Coexpre ⁇ sion of more than one Factor VIII:C polypeptide analog may be desired.
  • either or both of the light and heavy chain ⁇ may include modification ⁇ as described above.
  • "Coexpres ⁇ ion" as used herein refers to the expre ⁇ sion of two or more Factor VIII:C polypeptides in a single host cell.
  • the expres ⁇ ion of the 90 kD species and the 80 kD specie ⁇ in a ⁇ ingle ho ⁇ t cell would con ⁇ titute "coexpression" as used herein.
  • the polynucleotide ⁇ encoding for the polypeptide ⁇ can be harbored in a ⁇ ingle vector, either under the control of the ⁇ ame regulatory element ⁇ or under the control of ⁇ eparate elements.
  • a fusion protein including active portion ⁇ of the two or more Factor VIII:C polypeptide ⁇ would be con ⁇ idered "coexpre ⁇ ed" for purpo ⁇ e ⁇ of the present definition as would the expre ⁇ ion of two gene ⁇ as a dicistronic con ⁇ truct employing an internal ribo ⁇ ome entry site.
  • proteins expre ⁇ ed from the same vector but driven by separate regulatory elements would al ⁇ o be con ⁇ idered "coexpre ⁇ ed.”
  • the term also refers to the expression of two or more proteins from separate con ⁇ truct ⁇ . Thu ⁇ , the expression of protein ⁇ encoded from genes present on separate vector ⁇ in a ho ⁇ t cell would also be considered “coexpres ⁇ ion" for purposes of the present invention.
  • the tran ⁇ formed/tran ⁇ fected cell ⁇ are then grown in an appropriate nutrient medium. If separate constructs encoding heavy and light chain analogs have been used for coexpression, the product can be obtained as a complex of the two Factor VIII:C chains, ⁇ o that the media or cell ly ⁇ ate may be i ⁇ olated and the Factor VIII:C active complex extracted and purified. Similarly, the full-length molecule can be i ⁇ olated and treated under complex-forming condition ⁇ , e.g., with the addition of calcium and the appropriate enzyme ⁇ , to form the active complex.
  • complex-forming condition ⁇ e.g., with the addition of calcium and the appropriate enzyme ⁇
  • Variou ⁇ mean ⁇ are available for extraction and purification, such as affinity chromatography, ion exchange chromatography, hydrophobic chromatography, electrophoresis, solvent- ⁇ olvent extraction, selective precipitation, and the like.
  • the particular manner in which the product i ⁇ isolated is not critical to this invention, and is selected to minimize denaturation or inactivation and maximize the isolation of a high-purity active product.
  • Bacterial expre ⁇ ion ⁇ y ⁇ tem ⁇ can be u ⁇ ed to produce the subject Factor VIII:C polypeptide analogs and nucleic acid molecules encoding the analogs.
  • Control elements for use in bacterial sy ⁇ tem ⁇ include promoters, optionally containing operator ⁇ equence ⁇ , and ribosome binding site ⁇ .
  • Useful promoter ⁇ include ⁇ equence ⁇ derived from ⁇ ugar metabolizing enzymes, such a ⁇ galacto ⁇ e, lactose (lac) and maltose.
  • promoter sequence ⁇ derived from bio ⁇ ynthetic enzymes such as tryptophan ( trp) , the ⁇ - lactama ⁇ e (jbla) promoter ⁇ y ⁇ tem, bacteriophage ⁇ PL, and T7.
  • trp tryptophan
  • jbla ⁇ - lactama ⁇ e
  • ⁇ ynthetic promoters can be used, ⁇ uch a ⁇ the tac promoter.
  • the ⁇ -lactama ⁇ e and lacto ⁇ e promoter systems are described in Chang et al., Nature (1978) 275 : 615, and Goeddel et al., Nature (1979) 281 : 544; the alkaline pho ⁇ phata ⁇ e, tryptophan (trp) promoter ⁇ ystem are described in Goeddel et al., Nucleic Acids Res . (1980) 8 : 4057 and EP 36,776 and hybrid promoters such as the tac promoter is described in U.S. Patent No. 4,551,433 and deBoer et al., Proc. Natl. Acad. Sci. USA (1983) 80 : 21-25.
  • Promoter ⁇ for u ⁇ e in bacterial ⁇ y ⁇ tem ⁇ al ⁇ o generally will contain a Shine-Dalgarno (SD) ⁇ equence operably linked to the DNA encoding the Factor VIII:C analog polypeptide.
  • SD Shine-Dalgarno
  • the signal sequence can be ⁇ ub ⁇ tituted by a prokaryotic ⁇ ignal ⁇ equence ⁇ elected, for example, from the group of the alkaline phosphatase, penicillinase, Ipp, or heat stable enterotoxin II leaders.
  • the origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria.
  • sy ⁇ tem ⁇ are particularly compatible with Escherichia coli .
  • numerous other ⁇ y ⁇ tem ⁇ for u ⁇ e in bacterial ho ⁇ ts including Gram- negative or Gram-positive organi ⁇ m ⁇ ⁇ uch as Bacillus spp . , Streptococcus spp . , Streptomyces spp . , Pseudomonas specie ⁇ such as P . aeruginosa, Salmonella typhimurium, or
  • Serratia marcescans among others. Methods for introducing exogenous DNA into these hosts typically include the use of CaCl 2 or other agents, such as divalent cations and DMSO. DNA can also be introduced into bacterial cells by electroporation, nuclear injection, or protoplast fusion as described generally in Sambrook et al. (1989), cited above. The ⁇ e examples are illustrative rather than limiting. Preferably, the host cell should secrete minimal amounts of proteolytic enzymes. Alternatively, in vitro method ⁇ of cloning, e.g., PCR or other nucleic acid polymera ⁇ e reaction ⁇ , are ⁇ uitable.
  • Prokaryotic cell ⁇ u ⁇ ed to produce the Factor VIII:C analog polypeptide ⁇ of thi ⁇ invention are cultured in suitable media, a ⁇ described generally in Sambrook et al., cited above.
  • Yea ⁇ t expre ⁇ ion ⁇ y ⁇ tem ⁇ can al ⁇ o be u ⁇ ed to produce the subject Factor VIII:C polypeptide analogs and nucleic acid molecule ⁇ encoding the analogs.
  • Expres ⁇ ion and transformation vector ⁇ either extrachromo ⁇ omal replicon ⁇ or integrating vector ⁇ , have been developed for tran ⁇ formation into many yea ⁇ t ⁇ .
  • expression vectors have been developed for, among others, the following yeast ⁇ : Saccharomyces cerevisiae ,a ⁇ de ⁇ cribed in Hinnen et al., Proc . Natl . Acad . Sci . USA (1978) 75 : 1929; Ito et al., J . Bacteriol .
  • Control ⁇ equence ⁇ for yea ⁇ t vector ⁇ are known and include promoter region ⁇ from genes such as alcohol dehydrogenase (ADH) , as described in EP 284,044, enolase, glucokinase, gluco ⁇ e-6-pho ⁇ phate isomerase, glyceraldehyde-3-phosphate-dehydrogenase (GAP or GAPDH) , hexokina ⁇ e, pho ⁇ phofructokina ⁇ e, 3-pho ⁇ phoglycerate mutase, and pyruvate kinase (PyK) , a ⁇ de ⁇ cribed in EP 329,203.
  • ADH alcohol dehydrogenase
  • GAP or GAPDH glyceraldehyde-3-phosphate-dehydrogenase
  • hexokina ⁇ e pho ⁇ phofructokina ⁇ e
  • 3-pho ⁇ phoglycerate mutase 3-pho ⁇ pho
  • the yea ⁇ t PH05 gene encoding acid pho ⁇ phata ⁇ e, also provides useful promoter sequences, as de ⁇ cribed in Myanohara et al., Proc. Natl . Acad . Sci . USA (1983) 80:1.
  • Other ⁇ uitable promoter sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase, as described in Hitzeman et al., J . Biol . Chem .
  • glycolytic enzyme ⁇ such as pyruvate decarboxylase, trio ⁇ ephosphate i ⁇ omerase, and phosphoglucose i ⁇ omera ⁇ e, as described in Hess et al., J " . Adv . Enzyme Reg . (1968) 7: 149 and Holland et al., Biochemistry (1978J 17 : 4900.
  • Inducible yeast promoters having the additional advantage of transcription controlled by growth condition ⁇ , include from the li ⁇ t above and others the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphata ⁇ e, degradative enzyme ⁇ associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galacto ⁇ e utilization.
  • Suitable vector ⁇ and promoter ⁇ for u ⁇ e in yeast expres ⁇ ion are further described in Hitzeman, EP 073,657.
  • Yea ⁇ t enhancers also are advantageously u ⁇ ed with yeast promoters.
  • synthetic promoters which do not occur in nature also function as yea ⁇ t promoter ⁇ .
  • up ⁇ tream activating sequences (UAS) of one yeast promoter may be joined with the tran ⁇ cription activation region of another yea ⁇ t promoter, creating a ⁇ ynthetic hybrid promoter.
  • UAS up ⁇ tream activating sequences
  • Example ⁇ of ⁇ uch hybrid promoters include the ADH regulatory sequence linked to the GAP transcription activation region, as described in U.S. Patent No ⁇ . 4,876,197 and 4,880,734.
  • hybrid promoter ⁇ examples include promoters which consist of the regulatory sequence ⁇ of either the ADH2, GAL4 , GAL10, or PH05 genes, combined with the transcriptional activation region of a glycolytic enzyme gene such as GAP or PyK, as described in EP 164,556.
  • a yeast promoter can include naturally occurring promoter ⁇ of non-yea ⁇ t origin that have the ability to bind yea ⁇ t RNA polymera ⁇ e and initiate tran ⁇ cription.
  • Other control element ⁇ which may be included in the yea ⁇ t expre ⁇ ion vectors are terminators, for example, from GAPDH and from the enolase gene, as described in Holland et al., J . Biol . Chem .
  • leader sequence ⁇ which encode signal sequence ⁇ for secretion.
  • DNA encoding suitable signal ⁇ equence ⁇ can be derived from genes for secreted yeast proteins, ⁇ uch a ⁇ the yeast invertase gene as described in EP 012,873 and JP 62,096,086 and the ⁇ -factor gene, as described in U.S. Patent No ⁇ . 4,588,684, 4,546,083 and 4,870,008; EP 324,274; and WO 89/02463.
  • leader ⁇ of non-yeast origin such as an interferon leader, al ⁇ o provide for ⁇ ecretion in yeast, a ⁇ de ⁇ cribed in EP 060,057.
  • Method ⁇ of introducing exogenous DNA into yeast host ⁇ are well known in the art, and typically include either the tran ⁇ formation of ⁇ pheropla ⁇ t ⁇ or of intact yea ⁇ t cell ⁇ treated with alkali cation ⁇ . Tran ⁇ for ation ⁇ into yeast can be carried out according to the method described in Van Solingen et al., J. Bact . (1977) 230: 946 and Hsiao et al., Proc . Natl . Acad . Sci . USA (1979) 76 : 3829. However, other methods for introducing DNA into cells ⁇ uch a ⁇ by nuclear injection, electroporation, or protopla ⁇ t fusion may also be used as described generally in Sambrook et al., cited above.
  • the native polypeptide signal sequence may be sub ⁇ tituted by the yeast invertase, ⁇ -factor, or acid phosphatase leaders.
  • the origin of replication from the 2 ⁇ plasmid origin i ⁇ suitable for yea ⁇ t.
  • a ⁇ uitable ⁇ election gene for u ⁇ e in yeast is the trpl gene pre ⁇ ent in the yea ⁇ t plasmid described in Kingsman et al., Gene (1979) 7: 141 or T ⁇ chemper et al., Gene (1980) 20: 157.
  • the trpl gene provide ⁇ a selection marker for a mutant ⁇ train of yea ⁇ t lacking the ability to grow in tryptophan.
  • Leu2-deficient yeast strains are complemented by known plasmids bearing the Leu2 Gene.
  • a ⁇ equence encoding a yeast protein can be linked to a coding sequence of a Factor VIIIiC polypeptide analog to produce a fusion protein that can be cleaved intracellularly by the yeast cell ⁇ upon expression.
  • An example, of such a yeast leader sequence is the yeast ubiquitin gene.
  • the Factor VIIIiC polypeptide analogs and nucleic acid molecules encoding the analogs can al ⁇ o be produced in in ⁇ ect expre ⁇ ion ⁇ ystems.
  • baculovirus expres ⁇ ion vector ⁇ BEV ⁇
  • BEV ⁇ baculovirus expres ⁇ ion vector ⁇
  • a baculovirus promoter in place of a viral gene, e.g., polyhedrin, a ⁇ de ⁇ cribed in Smith and Summer ⁇ , U.S. Pat. No., 4,745,051.
  • An expre ⁇ ion con ⁇ truct herein includes a DNA vector useful as an intermediate for the infection or transformation of an in ⁇ ect cell system, the vector generally containing DNA coding for a baculovirus transcriptional promoter, optionally but preferably, followed downstream by an insect signal DNA sequence capable of directing secretion of a de ⁇ ired protein, and a site for insertion of the foreign gene encoding the foreign protein, the signal DNA sequence and the foreign gene being placed under the transcriptional control of a baculovirus promoter, the foreign gene herein being the coding sequence of a Factor VIIIiC polypeptide analog of this invention.
  • the promoter for use herein can be a baculovirus transcriptional promoter region derived from any of the over 500 baculoviruses generally infecting insects, such as, for example, the Order ⁇ Lepidoptera, Diptera, Orthoptera, Coleoptera and Hymenoptera including, for example, but not limited to the viral DNA ⁇ of Autographo calif ornica MNPV, Bombyx mori NPV, rrichoplusia ni MNPV, Rachlplusia ou MNPV or Galleria mellonella MNPV, Aedes aegypti, Drosophila melanogaster, Spodoptera frugiperda , and Trichoplusia ni .
  • the baculoviru ⁇ transcriptional promoter can be, for example, a baculovirus immediate-early gene IEI or IEN promoter; an immediate-early gene in combination with a baculoviru ⁇ delayed-early gene promoter region ⁇ elected from the group con ⁇ isting of a 39K and a Hindlll fragment containing a delayed-early gene; or a baculovirus late gene promoter.
  • the immediate-early or delayed-early promoters can be enhanced with tran ⁇ criptional enhancer element ⁇ .
  • the pla ⁇ mid for u ⁇ e herein usually al ⁇ o contains the polyhedrin polyadenylation signal, as described in Miller et al., Ann. Rev. Microbiol . (1988) 42 : 117 and a procaryotic ampicillin- re ⁇ i ⁇ tance (amp) gene and an origin of replication for selection and propagation in E. coli .
  • DNA encoding suitable signal sequences can also be included and is generally derived from genes for secreted in ⁇ ect or baculoviru ⁇ protein ⁇ , ⁇ uch as the baculovirus polyhedrin gene, as described in Carbonell et al., Gene (1988) 73 : 409, a ⁇ well a ⁇ mammalian ⁇ ignal ⁇ equence ⁇ ⁇ uch as tho ⁇ e derived from gene ⁇ encoding human ⁇ -interferon a ⁇ de ⁇ cribed in Maeda et al., Nature (1985) 325: 592-594; human gastrin-releasing peptide, a ⁇ de ⁇ cribed in Lebacq- Verheyden et al., Mol . Cell . Biol .
  • baculovirus gene ⁇ in addition to the polyhedrin promoter may be employed to advantage in a baculoviru ⁇ expre ⁇ sion sy ⁇ tem.
  • the ⁇ e include immediate-early (alpha) , delayed-early (beta) , late (gamma) , or very late (delta) , according to the pha ⁇ e of the viral infection during which they are expre ⁇ ed.
  • the expre ⁇ ion of the ⁇ e genes occurs ⁇ equentially, probably a ⁇ the result of a "cascade" mechanism of transcriptional regulation.
  • the immediate-early genes are expres ⁇ ed immediately after infection, in the ab ⁇ ence of other viral function ⁇ , and one or more of the re ⁇ ulting gene product ⁇ induces transcription of the delayed-early genes.
  • Some delayed-early gene product ⁇ induce tran ⁇ cription of late gene ⁇ , and finally, the very late gene ⁇ are expre ⁇ ed under the control of previously expres ⁇ ed gene products from one or more of the earlier classes.
  • One relatively well defined component of this regulatory cascade i ⁇ IEI, a preferred immediate-early gene of Autographo calif ornica nuclear polyhedrosis virus (AcMNPV) .
  • IEI is pres ⁇ ed in the absence of other viral functions and encodes a product that ⁇ timulates the transcription of several genes of the delayed-early clas ⁇ , including the preferred 39K gene, as described in Guarino and Summers, J . Virol . (1986) 57 : 563-571 and J " . Virol . (1987) 61 : 2091-2099 a ⁇ well a ⁇ late genes, as de ⁇ cribed in Guanno and Summer ⁇ , Virol. (1988) 262 ⁇ 444-451.
  • Immediate-early gene ⁇ a ⁇ described above can be used in combination with a baculovirus gene promoter region of the delayed-early category.
  • delayed-early genes require the presence of other viral gene ⁇ or gene product ⁇ ⁇ uch a ⁇ tho ⁇ e of the immediate-early genes.
  • the combination of immediate-early genes can be made with any of several delayed-early gene promoter regions such a ⁇ 39K or one of the delayed-early gene promoter ⁇ found on the Hindlll fragment of the baculovirus genome.
  • the 39 K promoter region can be linked to the foreign gene to be expres ⁇ ed such that expre ⁇ ion can be further controlled by the pre ⁇ ence of IEI, as described in L. A. Guarino and Summers (1986a) , cited above; Guarino & Summers (1986b) J .
  • the hr5 enhancer sequence can be linked directly, in ci ⁇ , to the delayed-early gene promoter region, 39K, thereby enhancing the expre ⁇ ion of the cloned heterologou ⁇ DNA a ⁇ de ⁇ cribed in Guarino and Summer ⁇ (1986a) , (1986b) , and Guarino et al. (1986) .
  • Thi ⁇ repre ⁇ ent ⁇ a limitation to the u ⁇ e of exi ⁇ ting BEV ⁇ .
  • the expression of secretory glycoproteins in BEV sy ⁇ tems i ⁇ complicated due to incomplete secretion of the cloned gene product thereby trapping the cloned gene product within the cell in an incompletely proce ⁇ ed form.
  • an in ⁇ ect ⁇ ignal sequence can be u ⁇ ed to expre ⁇ s a foreign protein that can be cleaved to produce a mature protein
  • the present invention is preferably practiced with a mammalian signal sequence for example the Factor VIII signal sequence.
  • An exemplary insect ⁇ ignal ⁇ equence ⁇ uitable herein i ⁇ the ⁇ equence encoding for a Lepidopteran adipokinetic hormone (AKH) peptide.
  • the AKH family con ⁇ i ⁇ ts of short blocked neuropeptides that regulate energy sub ⁇ trate mobilization and metaboli ⁇ m in in ⁇ ect ⁇ .
  • a DNA ⁇ equence coding for a Lepidopteran Manduca ⁇ exta AKH ⁇ ignal peptide can be u ⁇ ed.
  • Other insect AKH signal peptides, such as those from the Orthoptera Schistocerca gregaria locus can al ⁇ o be employed to advantage.
  • Another exemplary in ⁇ ect signal sequence is the sequence coding for Drosophila cuticle proteins such as CPl, CP2, CP3 or CP4.
  • the desired DNA sequence can be inserted into the transfer vector, using known techniques.
  • An in ⁇ ect cell ho ⁇ t can be cotran ⁇ formed with the transfer vector containing the inserted desired DNA together with the genomic DNA of wild type baculoviru ⁇ , u ⁇ ually by cotran ⁇ fection.
  • the vector and viral genome are allowed to recombine re ⁇ ulting in a recombinant virus that can be easily identified and purified.
  • the packaged recombinant virus can be u ⁇ ed to infect insect ho ⁇ t cell ⁇ to expre ⁇ a Factor VIIIiC polypeptide analog.
  • Expres ⁇ ion in Mammalian Cell ⁇ Mammalian expression sy ⁇ tem ⁇ can al ⁇ o be u ⁇ ed to produce the Factor VIIIiC polypeptide analogs and nucleic acid molecules encoding the analogs.
  • Typical promoter ⁇ for mammalian cell expre ⁇ ion include the SV40 early promoter, the CMV promoter, the mou ⁇ e mammary tumor viru ⁇ LTR promoter, the adenovirus major late promoter (Ad MLP) , and the herpes ⁇ implex viru ⁇ promoter, among others.
  • Other non-viral promoter ⁇ such as a promoter derived from the murine metallothionein gene, will also find use in mammalian construct ⁇ .
  • Mammalian expre ⁇ ion may be either con ⁇ titutive or regulated (inducible) , depending on the promoter.
  • tran ⁇ cription termination and polyadenylation sequence ⁇ will also be present, located 3' to the translation stop codon.
  • a sequence for optimization of initiation of translation located 5' to the Factor VIII:C polypeptide analog coding sequence, is al ⁇ o present.
  • Example ⁇ of tran ⁇ cription terminator/polyadenylation ⁇ ignal ⁇ include tho ⁇ e derived from SV40, a ⁇ described in Sambrook et al.
  • Enhancer element ⁇ can also be u ⁇ ed herein to increa ⁇ e expression levels of the mammalian construct ⁇ .
  • Example ⁇ include the SV40 early gene enhancer, a ⁇ described in Dijkema et al., EMBO J . (1985) 4: 761 and the enhancer/promoter derived from the long terminal repeat (LTR) of the Rous Sarcoma Viru ⁇ , a ⁇ described in Gorman et al., Proc . Natl . Acad . Sci . USA (1982b) 79 : 6777 and human cytomegaloviru ⁇ , a ⁇ de ⁇ cribed in Bo ⁇ hart et al., Cell (1985) 42: 521.
  • LTR long terminal repeat
  • a leader ⁇ equence can al ⁇ o be pre ⁇ ent which include ⁇ a ⁇ equence encoding a ⁇ ignal peptide, to provide for the ⁇ ecretion of the foreign protein in mammalian cell ⁇ .
  • the Factor VIII ⁇ ignal peptide can be used.
  • the adenovirus tripartite leader is an example of a leader sequence that provide ⁇ for ⁇ ecretion of a foreign protein in mammalian cells.
  • tran ⁇ ient expre ⁇ ion involve ⁇ the u ⁇ e of an expression vector that is able to replicate efficiently in a host cell, such that the host cell accumulate ⁇ many copie ⁇ of the expre ⁇ ion vector and, in turn, ⁇ ynthe ⁇ izes high level ⁇ of a de ⁇ ired polypeptide encoded by the expression vector.
  • Transient expres ⁇ ion system ⁇ comprising a ⁇ uitable expre ⁇ ion vector and a host cell, allow for the convenient positive identification of polypeptides encoded by cloned DNAs, as well a ⁇ for the rapid ⁇ creening of ⁇ uch polypeptide ⁇ for de ⁇ ired biological or phy ⁇ iological properties.
  • tran ⁇ ient expre ⁇ ion ⁇ y ⁇ tem ⁇ are particularly useful for purposes of identifying additional polypeptides that have Factor VIII:C-like activity.
  • the mammalian expression vectors can be used to transform any of ⁇ everal mammalian cells.
  • Method ⁇ for introduction of heterologou ⁇ polynucleotide ⁇ into mammalian cell ⁇ are known in the art and include dextran-mediated tran ⁇ fection, calcium pho ⁇ phate precipitation, polybrene mediated tran ⁇ fection, protopla ⁇ t fu ⁇ ion, electroporation, encapsulation of the polynucleotide( ⁇ ) in lipo ⁇ ome ⁇ , and direct microinjection of the DNA into nuclei.
  • General aspects of mammalian cell host sy ⁇ tem transformation ⁇ have been de ⁇ cribed by Axel in U.S. 4,399,216.
  • a ⁇ ynthetic lipid particularly u ⁇ eful for polynucleotide tran ⁇ fection is N-[l-(2,3- dioleyloxy)propyl]-N,N,N-trimethylammonium chloride, which is commercially available under the name Lipofectin® (available from BRL, Gaithersburg, MD) , and is described by Feigner et al., Proc . Natl . Acad . Sci . USA (1987) 84:7413.
  • Mammalian cell lines available as hosts for expression are also known and include many immortalized cell lines available from the American Type Culture Collection (ATCC) , including but not limited to, Chine ⁇ e hamster ovary (CHO) cells, HeLa cell ⁇ , baby ham ⁇ ter kidney (BHK) cell ⁇ , monkey kidney cells (COS) , human hepatocellular carcinoma cells (e.g., Hep G2) , human embryonic kidney cells, baby hamster kidney cells, mou ⁇ e sertoli cells, canine kidney cell ⁇ , buffalo rat liver cells, human lung cells, human liver cells, mouse mammary tumor cell ⁇ , a ⁇ well as others.
  • the mammalian host cells u ⁇ ed to produce the target polypeptide of thi ⁇ invention may be cultured in a variety of media. Commercially available media such a ⁇ Ham's F10 (Sigma), Minimal Essential Medium ([MEM], Sigma) , RPMI-1640
  • any of the media de ⁇ cribed in Ham and Wallace, Meth . Enz. (1979) 58 : 44, Barne ⁇ and Sato, Anal . Biochem . (1980) 102 : 255, U.S. Patent No ⁇ . 4,767,704, 4,657,866, 4,927,762, or 4,560,655, WO 90/103430, WO 87/00195, and U.S. RE 30,985, may be used a ⁇ culture media for the ho ⁇ t cells.
  • any of these media may be supplemented as necessary with hormones and/or other growth factors such a ⁇ in ⁇ ulin, tran ⁇ ferrin, or epidermal growth factor, ⁇ alt ⁇ (such a ⁇ ⁇ odium chloride, calcium, magne ⁇ ium, and pho ⁇ phate) , buffers ( ⁇ uch a ⁇ HEPES) , nucleosides (such as adenosine and thymidine) , antibiotics ( ⁇ uch a ⁇ Gentamycin(tm) M drug), trace element ⁇ (defined a ⁇ inorganic compound ⁇ u ⁇ ually pre ⁇ ent at final concentrations in the micromolar range) , and glucose or an equivalent energy source.
  • hormones and/or other growth factors such as a ⁇ in ⁇ ulin, tran ⁇ ferrin, or epidermal growth factor, ⁇ alt ⁇ (such a ⁇ ⁇ odium chloride, calcium, magne ⁇ ium, and pho ⁇ phate) , buffers ( ⁇ uch a ⁇ HEPES)
  • any other necessary supplement ⁇ may al ⁇ o be included at appropriate concentrations that would be known to tho ⁇ e skilled in the art.
  • the culture conditions such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled arti ⁇ an.
  • the active Factor VIII:C analog ⁇ produced according to the invention have a variety of uses.
  • the analogs can be used a ⁇ immunogen ⁇ for the production of antibodie ⁇ .
  • the analog ⁇ can also be used for the treatment of hemophiliacs and other ho ⁇ t ⁇ having blood clotting di ⁇ order ⁇ .
  • the Factor VIII:C analog ⁇ may di ⁇ play increased plasma half-life or specific activity.
  • the analogs may allow for lower dosage ⁇ or alternative mode ⁇ of admini ⁇ tration and may improve hemo ⁇ ta ⁇ i ⁇ in hemophiliacs.
  • nucleic acid molecules or vectors comprising polynucleotide ⁇ equence ⁇ encoding the Factor VIII:C analog ⁇ can be u ⁇ ed directly for gene therapy and admini ⁇ tered u ⁇ ing ⁇ tandard gene delivery protocol ⁇ .
  • nucleotide sequences encoding the Factor VIII:C analogs can be ⁇ tably integrated into the ho ⁇ t cell genome or maintained on a stable episomal element in the host cell. Methods for gene delivery are known in the art. See, e.g., U.S. Patent No. 5,399,346.
  • retroviru ⁇ e ⁇ provide a convenient platform for gene delivery ⁇ y ⁇ tems.
  • a selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art.
  • the recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo .
  • retroviral sy ⁇ tem ⁇ have been de ⁇ cribed (U.S. Patent No.
  • adenoviruses persist extrachromosomally thu ⁇ minimizing the ri ⁇ k ⁇ a ⁇ ociated with insertional mutagenesis (Haj-Ahmad and Graham, J . Virol . (1986) 57:267-274; Bett et al., J . Virol . (1993) 67l5911-5921; Mittereder et al., Human Gene Therapy (1994) 5:717-729; Seth et al., J . Virol . (1994) 68:933-940; Barr et al., Gene Therapy (1994) 1:51-58; Berkner, K.L. BioTechniques (1988) 6:616-629; and Rich et al., Human Gene Therapy (1993) 41461-476) .
  • AAV vector ⁇ y ⁇ tem ⁇ can include control ⁇ equences, such a ⁇ promoter and polyadenylation ⁇ ite ⁇ , a ⁇ well a ⁇ selectable markers or reporter genes, enhancer sequences, and other control elements which allow for the induction of tran ⁇ cription.
  • AAV vectors can be readily constructed using techniques well known in the art. See, e.g., U.S. Patent Nos. 5,173,414 and 5,139,941; International Publication No ⁇ .
  • Additional viral vector ⁇ which will find u ⁇ e for delivering the nucleic acid molecule ⁇ encoding the Factor VIIIiC analog polypeptides for gene transfer include those derived from the pox family of viruses, including vaccinia virus and avian poxvirus.
  • vaccinia viru ⁇ recombinant ⁇ expre ⁇ ing the novel Factor VIIIiC analogs can be constructed as follows.
  • the DNA encoding the particular analog is fir ⁇ t in ⁇ erted into an appropriate vector so that it is adjacent to a vaccinia promoter and flanking vaccinia DNA sequences, such as the sequence encoding thymidine kinase (TK) .
  • This vector i ⁇ then used to transfect cell ⁇ which are ⁇ imultaneously infected with vaccinia. Homologou ⁇ recombination serve ⁇ to in ⁇ ert the vaccinia promoter plus the gene encoding the instant protein into the viral genome.
  • the resulting TK ⁇ recombinant can be selected by culturing the cells in the pre ⁇ ence of 5- bromodeoxyuridine and picking viral plaque ⁇ re ⁇ istant thereto.
  • a vaccinia based infection/transfection ⁇ ystem can be conveniently used to provide for inducible, transient expression of the Factor VIIIiC analog ⁇ in a host cell.
  • cell ⁇ are fir ⁇ t infected in vitro with a vaccinia virus recombinant that encode ⁇ the bacteriophage T7 RNA polymera ⁇ e.
  • Thi ⁇ polymerase displays extraordinar specificity in that it only transcribe ⁇ template ⁇ bearing T7 promoter ⁇ .
  • cell ⁇ are transfected with the polynucleotide of interest, driven by a T7 promoter.
  • the polymerase expressed in the cytoplasm from the vaccinia viru ⁇ recombinant transcribes the transfected DNA into RNA which i ⁇ then tran ⁇ lated into protein by the ho ⁇ t tran ⁇ lational machinery.
  • the method provide ⁇ for high level, tran ⁇ ient, cytopla ⁇ mic production of large quantitie ⁇ of RNA and it ⁇ tran ⁇ lation product ⁇ . See, e.g., Elroy-Stein and Mo ⁇ , Proc . Natl . Acad . Sci . USA (1990) 8716743-6747; Fuerst et al., Proc . Natl . Acad . Sci . USA (1986) 8318122-8126.
  • avipoxviruse ⁇ such as the fowlpox and canarypox viruses
  • Recombinant avipox viruses, expres ⁇ ing immunogens from mammalian pathogens, are known to confer protective immunity when admini ⁇ tered to non-avian species.
  • the use of an avipox vector is particularly de ⁇ irable in human and other mammalian ⁇ pecie ⁇ ⁇ ince members of the avipox genus can only productively replicate in susceptible avian species and therefore are not infective in mammalian cells.
  • Methods for producing recombinant avipoxviruses are known in the art and employ genetic recombination, as described above with respect to the production of vaccinia viru ⁇ e ⁇ . See, e.g., WO 91/12882; WO 89/03429; and WO 92/03545.
  • Molecular conjugate vectors such as the adenovirus chimeric vectors described in Michael et al., J . Biol . Chem . (1993) 268 ⁇ 6866-6869 and Wagner et al., Proc. Natl. Acad. Sci. USA (1992) 89 : 6099-6103 , can also be used for gene delivery.
  • an amplification ⁇ y ⁇ tem can be u ⁇ ed that will lead to high level expre ⁇ ion following introduction into ho ⁇ t cell ⁇ .
  • a T7 RNA polymera ⁇ e promoter preceding the coding region for T7 RNA polymerase can be engineered. Translation of RNA derived from this template will generate T7 RNA polymerase which in turn will transcribe more template. Concomitantly, there will be a cDNA whose expres ⁇ ion i ⁇ under the control of the T7 promoter.
  • T7 RNA polymerase generated from tran ⁇ lation of the amplification template RNA will lead to transcription of the desired gene. Because some T7 RNA polymerase i ⁇ required to initiate the amplification, T7 RNA polymerase can be introduced into cells along with the template(s) to prime the transcription reaction.
  • the amplification template can be generated by
  • the plasmid should be one where expression of T7 RNA polymerase can be controlled.
  • a lac operator can be engineered distal or proximal (or both) to the T7 promoter. The binding of the preexisting lac repre ⁇ or in the appropriate bacterial ⁇ train would interfere with the tran ⁇ cription of the template by blocking access to the promoter by T7 RNA polymerase.
  • a plasmid can be constructed where transcription from a bacterial promoter begin ⁇ 3' of the T7 gene and continue ⁇ through the 5' end of the T7 promoter. Such tran ⁇ cription will generate an anti ⁇ en ⁇ e tran ⁇ cript and reduce or eliminate tran ⁇ lation of T7 RNA polymerase RNAs.
  • the second tran ⁇ cription unit con ⁇ isting of the T7 promoter preceding the gene of interest can be provided by a ⁇ eparate pla ⁇ mid or can be engineered onto the amplification pla ⁇ mid. Colocalization of the two transcription units is beneficial for ease of manufacturing and ensure ⁇ that both tran ⁇ cription unit ⁇ will alway ⁇ be together in the cell ⁇ into which the plasmid is introduced.
  • the T7 RNA polymerase plasmid ⁇ may include UTR ⁇ which comprise an Internal Ribosome Entry Site (IRES) present in the leader sequence ⁇ of picornaviru ⁇ e ⁇ such a ⁇ the encephalomyocarditi ⁇ viru ⁇
  • IRS Internal Ribosome Entry Site
  • Vector ⁇ encoding the ⁇ ubject Factor VIIIiC analog ⁇ can also be packaged in liposome ⁇ prior to delivery to the ⁇ ubject or to cell ⁇ derived therefrom.
  • Lipid encap ⁇ ulation is generally accomplished using liposomes which are able to stably bind or entrap and retain nucleic acid.
  • the ratio of condensed DNA to lipid preparation can vary but will generally be around 1:1 (mg DNA:micromole ⁇ lipid) , or more of lipid.
  • Liposomal preparations for u ⁇ e in the in ⁇ tant invention include cationic (po ⁇ itively charged) , anionic (negatively charged) and neutral preparation ⁇ , with cationic liposomes particularly preferred.
  • Cationic liposomes have been ⁇ hown to mediate intracellular delivery of pla ⁇ mid DNA (Feigner et al., Proc . Natl . Acad . Sci . USA (1987) 84:7413-7416) ; mRNA (Malone et al., Proc . Natl . Acad . Sci . USA (1989) 86:6077-6081); and purified tran ⁇ cription factor ⁇ (Deb ⁇ et al., J . Biol . Chem . (1990) 265:10189-10192) , in functional form.
  • Cationic lipo ⁇ ome ⁇ are readily available.
  • N[1-2, 3-dioleyloxy)propyl]-N,N,N-triethy1- ammonium (DOTMA) lipo ⁇ ome ⁇ are available under the trademark Lipofectin, from GIBCO BRL, Grand I ⁇ land, NY.
  • liposome ⁇ examples include tran ⁇ fectace (DDAB/DOPE) and DOTAP/DOPE (Boerhinger) .
  • DOTAP/DOPE tran ⁇ fectace
  • DOTAP/DOPE DOTAP/DOPE
  • Other cationic lipo ⁇ omes can be prepared from readily available material ⁇ using techniques well known in the art. See, e.g., Szoka et al., Proc . Natl . Acad . Sci . USA (1978) 75:4194-4198; PCT Publication No. WO 90/11092 for a de ⁇ cription of the synthesis of DOTAP (1, 2-bis(oleoyloxy) -3- (trimethylammonio)propane) lipo ⁇ ome ⁇ .
  • anionic and neutral lipo ⁇ o es are readily available, ⁇ uch as from Avanti Polar Lipids (Birmingham, AL) , or can be easily prepared u ⁇ ing readily available material ⁇ .
  • material ⁇ include pho ⁇ phatidyl choline, chole ⁇ terol, pho ⁇ phatidyl ethanolamine, dioleoylpho ⁇ phatidyl choline (DOPC) , dioleoylpho ⁇ phatidyl glycerol (DOPG) , dioleoylpho ⁇ hatidyl ethanolamine (DOPE) , among other ⁇ .
  • the ⁇ e material ⁇ can al ⁇ o be mixed with the DOTMA and DOTAP starting material ⁇ in appropriate ratios.
  • Methods for making liposome ⁇ u ⁇ ing the ⁇ e material ⁇ are well known in the art.
  • the lipo ⁇ omes can comprise multilammelar vesicle ⁇ (MLV ⁇ ) , small unilamellar ve ⁇ icle ⁇ (SUV ⁇ ) , or large unilamellar ve ⁇ icle ⁇ (LUV ⁇ ) .
  • the variou ⁇ lipo ⁇ ome- nucleic acid complexes are prepared u ⁇ ing methods known in the art. See, e.g., Straubinger et al., in METHODS OF IMMUNOLOGY (1983), Vol. 101, pp.
  • the recombinant vectors may be administered in pharmaceutical composition ⁇ a ⁇ described above.
  • the pharmaceutical compositions will comprise sufficient genetic material to produce a therapeutically effective amount of the analog or analogs, as described above.
  • an effective dose will be from about 0.05 mg/kg to about 50 mg/kg of the DNA construct ⁇ in the individual to which it i ⁇ admini ⁇ tered.
  • the compo ⁇ itions of the invention can be admini ⁇ tered directly to the ⁇ ubject or, alternatively, in the ca ⁇ e of the vector ⁇ described above, delivered ex vivo , to cells derived from the subject.
  • Direct delivery of the compo ⁇ ition ⁇ will generally be accompli ⁇ hed by injection, either ⁇ ubcutaneou ⁇ ly, intraperitoneally, intravenou ⁇ ly or intramu ⁇ cularly.
  • Other modes of administration include oral and pulmonary admini ⁇ tration, ⁇ uppo ⁇ itorie ⁇ , and tran ⁇ dermal application ⁇ .
  • Dosage treatment may be a single dose schedule or a multiple dose ⁇ chedule.
  • the Factor VIII:C polypeptide analogs are made u ⁇ ing conventional mutagene ⁇ i ⁇ technique ⁇ .
  • mutagenesis of the Factor VIII:C nucleotide sequences can be performed utilizing pla ⁇ mid ⁇ which include ⁇ equences encoding the full-length molecule, pla ⁇ mid ⁇ encoding the light and heavy chains and various modification ⁇ of the ⁇ e molecules, depending on the Factor VIII:C analog desired.
  • pla ⁇ mid ⁇ which include ⁇ equences encoding the full-length molecule, pla ⁇ mid ⁇ encoding the light and heavy chains and various modification ⁇ of the ⁇ e molecules, depending on the Factor VIII:C analog desired.
  • Such plasmid ⁇ are known and described in, e.g., U.S. Patent no. 5,045,455.
  • the plasmids are first linearized e.g., by using restriction endonucleases which cleave at unique restriction sites.
  • the plasmids are treated with calf intestine pho ⁇ phata ⁇ e and ⁇ eparated on low melting temperature tri ⁇ -acetate agaro ⁇ e gel ⁇ .
  • the linearized band i ⁇ extracted by ad ⁇ orption to ⁇ ilica dioxide and eluted in tri ⁇ -EDTA.
  • the pla ⁇ mid i ⁇ then denatured and the de ⁇ ired phosphorylated mutagenic oligonucleotide is added.
  • the mixture i ⁇ heated and allowed to slowly cool at room temperature.
  • a heteroduplex oligonucleotide mixture can be used and the reactions made with, e.g., 2 mM MgCl 2 , ImM beta-mercaptoethanol, 400 ⁇ M ATP, 100 ⁇ M deoxynucleotide tripho ⁇ phate, 3-4 unit ⁇ / ⁇ L of Klenow fragment of E . coli DNA polymera ⁇ e I and 400 units/ ⁇ L of T4 DNA ligase.
  • the reactions are terminated using phenolchloroform extraction and ethanol precipitation.
  • DNA obtained i ⁇ used to transform bacterial host cells and positive clone ⁇ ⁇ elected.
  • Final mutation ⁇ are confirmed by DNA ⁇ equencing.
  • the DNA can be prepared by banding in CsCl and can be used to transfect COS-1 monkey cell ⁇ a ⁇ described in Kaufman, PNAS (1982) 82:689. After transfection, the polypeptide analog is isolated and Factor VIII:C activity is assayed by the Kabi Coatest chro agenic as ⁇ ay method for the ability to clot Factor VIII deficient pla ⁇ ma before and after thrombin activation.

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Abstract

On décrit des analogues du polypeptide du facteur VIII:C, lesquels sont des polypeptides du facteur VIII:C natif qui contiennent des modifications consistant en l'altération des propriétés de fixation aux métaux de la molécule. On décrit également des molécules d'acide nucléique codant des analogues du polypeptide du facteur VIII:C, ainsi que des vecteurs et cellules hôtes contenant de telles molécules. En outre, on décrit encore des complexes d'analogues qui contiennent au moins deux de ces analogues. On décrit enfin des procédés de production de cet analogue, du complexe d'analogues, d'acides nucléiques, de vecteurs et de cellules hôtes, ainsi que des procédés d'utilisation de telles compositions dans la prévention ou le traitement de déficiences en polypeptide du facteur VIII:C.
PCT/US1996/011438 1995-07-11 1996-07-09 Nouveaux analogues du polypeptide du facteur viii:c presentant des proprietes modifiees de liaison avec des metaux WO1997003193A1 (fr)

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AU64558/96A AU6455896A (en) 1995-07-11 1996-07-09 Novel factor viii:c polypeptide analogs with altered metal-binding properties

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US60/000,871 1995-07-11

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Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5859204A (en) * 1992-04-07 1999-01-12 Emory University Modified factor VIII
US6180371B1 (en) 1996-06-26 2001-01-30 Emory University Modified factor VIII
US6458563B1 (en) 1996-06-26 2002-10-01 Emory University Modified factor VIII
EP1598367A1 (fr) * 2004-05-18 2005-11-23 ZLB Behring GmbH Facteurs de coagulation modifiés de stabilité ameliorée et leur dérivés
WO2008077616A1 (fr) 2006-12-22 2008-07-03 Csl Behring Gmbh Facteurs de coagulation modifiés présentant une demi-vie in vivo prolongée
US7560107B2 (en) 1996-06-26 2009-07-14 Emory University Modified factor VIII
US7576181B2 (en) 2004-05-03 2009-08-18 Ipsen Biopharm Limited Method of administering porcine B-domainless fVIII
EP2206785A1 (fr) 1998-12-31 2010-07-14 Novartis Vaccines and Diagnostics, Inc. Expression améliorée de polypeptides HIV et production de particules de type virus
WO2013120939A1 (fr) 2012-02-15 2013-08-22 Csl Behring Gmbh Variants du facteur de von willebrand ayant une affinité de liaison au facteur viii améliorée
US8575104B2 (en) 2008-06-24 2013-11-05 Csl Behring Gmbh Factor VIII, von willebrand factor or complexes thereof with prolonged in vivo half-life
US8765915B2 (en) 2006-02-06 2014-07-01 Csl Behring Gmbh Modified coagulation factor VIIa with extended half-life
EP2796145A1 (fr) 2013-04-22 2014-10-29 CSL Behring GmbH Complexe
US9150637B2 (en) 2010-11-05 2015-10-06 Baxalta Inc. Variant of antihemophilic factor VIII having increased specific activity
WO2016000039A1 (fr) 2014-07-02 2016-01-07 Csl Limited Facteur de von willebrand modifié
WO2016142288A1 (fr) 2015-03-06 2016-09-15 Csl Behring Recombinant Facility Ag Facteur von willebrand modifié présentant une demi-vie améliorée
WO2016188907A1 (fr) 2015-05-22 2016-12-01 Csl Behring Recombinant Facility Ag Polypeptides du facteur von willebrand tronqué pour le traitement de l'hémophilie
WO2016188905A1 (fr) 2015-05-22 2016-12-01 Csl Behring Recombinant Facility Ag Procédés de préparation du facteur willebrand modifié
WO2017117631A1 (fr) 2016-01-07 2017-07-13 Csl Limited Facteur de von willebrand tronqué muté
WO2018087271A1 (fr) 2016-11-11 2018-05-17 Csl Behring Recombinant Facility Ag Polypeptides du facteur de von willebrand tronqué pour une administration extravasculaire dans le traitement ou la prophylaxie d'un trouble de la coagulation du sang
WO2018087267A1 (fr) 2016-11-11 2018-05-17 Csl Behring Recombinant Facility Ag Polypeptides du facteur de von willebrand tronqué pour le traitement de l'hémophilie
US10808023B2 (en) 2016-01-07 2020-10-20 CSL Behring Lengnau AG Mutated von Willebrand factor

Citations (1)

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Non-Patent Citations (1)

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Title
PAN ET AL: "PROPOSED STRUCTURE OF THE A DOMAINS OF FACTOR VIII BY HOMOLOGY MODELLING", NATURE STRUCTURAL BIOLOGY, vol. 2, no. 8, September 1995 (1995-09-01), pages 740 - 744, XP000611617 *

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US5859204A (en) * 1992-04-07 1999-01-12 Emory University Modified factor VIII
US7560107B2 (en) 1996-06-26 2009-07-14 Emory University Modified factor VIII
US6180371B1 (en) 1996-06-26 2001-01-30 Emory University Modified factor VIII
US6458563B1 (en) 1996-06-26 2002-10-01 Emory University Modified factor VIII
US8951515B2 (en) 1996-06-26 2015-02-10 Emory University Modified factor VIII
US7012132B2 (en) 1996-06-26 2006-03-14 Emory University Modified factor VIII
US7122634B2 (en) 1996-06-26 2006-10-17 Emory University Modified factor VIII
EP2206785A1 (fr) 1998-12-31 2010-07-14 Novartis Vaccines and Diagnostics, Inc. Expression améliorée de polypeptides HIV et production de particules de type virus
US7576181B2 (en) 2004-05-03 2009-08-18 Ipsen Biopharm Limited Method of administering porcine B-domainless fVIII
US8101718B2 (en) 2004-05-03 2012-01-24 Emory University Methods of administering porcine B-domainless fVIII
US8501694B2 (en) 2004-05-03 2013-08-06 Emory University Method of administering porcine B-domainless fVIII
WO2005111074A1 (fr) * 2004-05-18 2005-11-24 Zlb Behring Gmbh Facteurs de coagulation modifies dotes d'une stabilite amelioree, et leurs derives
EP1598367A1 (fr) * 2004-05-18 2005-11-23 ZLB Behring GmbH Facteurs de coagulation modifiés de stabilité ameliorée et leur dérivés
US8765915B2 (en) 2006-02-06 2014-07-01 Csl Behring Gmbh Modified coagulation factor VIIa with extended half-life
WO2008077616A1 (fr) 2006-12-22 2008-07-03 Csl Behring Gmbh Facteurs de coagulation modifiés présentant une demi-vie in vivo prolongée
US8754194B2 (en) 2006-12-22 2014-06-17 Csl Behring Gmbh Modified coagulation factors with prolonged in vivo half-life
EP3231440A1 (fr) 2006-12-22 2017-10-18 CSL Behring GmbH Facteurs de coagulation modifiés avec une demi-vie in vivo prolongée
EP2865760A1 (fr) 2008-06-24 2015-04-29 CSL Behring GmbH Facteur Vlll, facteur von Willebrand ou complexes associés avec demivie in vivo prolongée
US8575104B2 (en) 2008-06-24 2013-11-05 Csl Behring Gmbh Factor VIII, von willebrand factor or complexes thereof with prolonged in vivo half-life
US9290561B2 (en) 2008-06-24 2016-03-22 Csl Behring Gmbh Factor VIII, von Willebrand factor or complexes thereof with prolonged in vivo half-life
US10053500B2 (en) 2010-11-05 2018-08-21 Baxalta Incorporated Variant of antihemophilic factor VIII having increased specific activity
US9150637B2 (en) 2010-11-05 2015-10-06 Baxalta Inc. Variant of antihemophilic factor VIII having increased specific activity
WO2013120939A1 (fr) 2012-02-15 2013-08-22 Csl Behring Gmbh Variants du facteur de von willebrand ayant une affinité de liaison au facteur viii améliorée
US9458223B2 (en) 2012-02-15 2016-10-04 Csl Behring Gmbh Von willebrand factor variants having improved factor VIII binding affinity
WO2014173873A1 (fr) 2013-04-22 2014-10-30 Csl Behring Gmbh Complexe
EP2796145A1 (fr) 2013-04-22 2014-10-29 CSL Behring GmbH Complexe
US9878017B2 (en) 2013-04-22 2018-01-30 Csl Ltd. Covalent complex of von Willebrand Factor and factor VIII, compositions, and uses relating thereto
WO2016000039A1 (fr) 2014-07-02 2016-01-07 Csl Limited Facteur de von willebrand modifié
US10253088B2 (en) 2014-07-02 2019-04-09 CSL Behring Lengnau AG Modified von Willebrand Factor
WO2016142288A1 (fr) 2015-03-06 2016-09-15 Csl Behring Recombinant Facility Ag Facteur von willebrand modifié présentant une demi-vie améliorée
US11155601B2 (en) 2015-03-06 2021-10-26 CSL Behring Lengnau AG Modified von Willebrand factor having improved half-life
WO2016188905A1 (fr) 2015-05-22 2016-12-01 Csl Behring Recombinant Facility Ag Procédés de préparation du facteur willebrand modifié
EP4089109A2 (fr) 2015-05-22 2022-11-16 CSL Behring Lengnau AG Procédés de préparation du facteur willebrand modifié
US11564976B2 (en) 2015-05-22 2023-01-31 CSL Behring Lengnau AG Methods for preparing modified von Willebrand Factor
WO2016188907A1 (fr) 2015-05-22 2016-12-01 Csl Behring Recombinant Facility Ag Polypeptides du facteur von willebrand tronqué pour le traitement de l'hémophilie
US10688157B2 (en) 2015-05-22 2020-06-23 CSL Behring Lengnau AG Truncated von Willebrand factor polypeptides for treating hemophilia
US10772936B2 (en) 2015-05-22 2020-09-15 CSL Behring Lengnau AG Methods for preparing modified von Willebrand factor
US10905747B2 (en) 2015-05-22 2021-02-02 CSL Behring Lengnau AG Methods for preparing modified von Willebrand factor
US10806774B2 (en) 2016-01-07 2020-10-20 CSL Behring Lengnau AG Mutated truncated von Willebrand Factor
US10808023B2 (en) 2016-01-07 2020-10-20 CSL Behring Lengnau AG Mutated von Willebrand factor
WO2017117631A1 (fr) 2016-01-07 2017-07-13 Csl Limited Facteur de von willebrand tronqué muté
WO2018087267A1 (fr) 2016-11-11 2018-05-17 Csl Behring Recombinant Facility Ag Polypeptides du facteur de von willebrand tronqué pour le traitement de l'hémophilie
WO2018087271A1 (fr) 2016-11-11 2018-05-17 Csl Behring Recombinant Facility Ag Polypeptides du facteur de von willebrand tronqué pour une administration extravasculaire dans le traitement ou la prophylaxie d'un trouble de la coagulation du sang
US11814421B2 (en) 2016-11-11 2023-11-14 CSL Behring Lengnau AG Truncated von Willebrand Factor polypeptides for treating hemophilia
US11890327B2 (en) 2016-11-11 2024-02-06 CSL Behring Lengnau AG Truncated von Willebrand factor polypeptides for extravascular administration in the treatment or prophylaxis of a blood coagulation disorder

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