WO1997003195A1 - Novel factor viii:c polypeptide analogs with altered protease sites - Google Patents

Novel factor viii:c polypeptide analogs with altered protease sites Download PDF

Info

Publication number
WO1997003195A1
WO1997003195A1 PCT/US1996/011444 US9611444W WO9703195A1 WO 1997003195 A1 WO1997003195 A1 WO 1997003195A1 US 9611444 W US9611444 W US 9611444W WO 9703195 A1 WO9703195 A1 WO 9703195A1
Authority
WO
WIPO (PCT)
Prior art keywords
residue
factor viii
deletion
pro
analog
Prior art date
Application number
PCT/US1996/011444
Other languages
French (fr)
Inventor
David T. Hung
Fred E. Cohen
Michael Innis
Original Assignee
Chiron Corporation
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Chiron Corporation filed Critical Chiron Corporation
Priority to AU64861/96A priority Critical patent/AU6486196A/en
Publication of WO1997003195A1 publication Critical patent/WO1997003195A1/en

Links

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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to the discovery that an active Factor VIII.C polypeptide analog can be made that is modified at a site adjacent to an arginine ("Arg") residue, where the Arg is at a site other than at an activating site, creating an arginine-proline (“Arg-Pro”) linkage or a proline-arginine (“Pro-Arg”) linkage.
  • Arg arginine residue
  • Pro-Arg proline-arginine
  • This invention also relates to a Factor V II.C polypeptide analog with a modification that comprises creation of a tripeptide having the formula P 3 -P 2 ⁇ p i wherein P 3 is a residue selected from the group consisting of Phe, Glu, and Pro; P 2 is any amino acid residue except Ser and P2 is not Leu335 and is not Asnl720; and P is Arg. Additionally, the invention pertains to substitutions at the non-activating Arg residues occurring at positions Arg336, Argl719 and/or Argl721.
  • This invention further relates to an analog complex 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 10,000 males.
  • 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 consi ⁇ t ⁇ of the heavy and light chains as ⁇ ociated by a metal ion bridge, and lacking amino acid ⁇ 741-1648 (the B domain) .
  • the mature Factor VIII.C molecule con ⁇ i ⁇ ts 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.
  • 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”)
  • Factor VlllrC Patients suffering from hemophilia A are conventionally treated with purified or substantially purified Factor VlllrC.
  • a difficulty in such treatment is the relatively short half-life of externally administered Factor VIII.C, lasting about 8 to 12 hours.
  • This instability of Factor VIII:C derives in part from its susceptibility to proteolytic cleavage by plasma protease.
  • plasma proteases for example, thrombin, Factor Xa, and activated protein C (“APC”) , inactivate Factor VIII.C by cleaving the molecule at multiple sites. It would be advantageous, therefore, to produce Factor VIII:C polypeptides with improved properties.
  • an active Factor VIII.C polypeptide analog that is substantially the same as a native Factor VIII.C polypeptide, except for modification at a site that is adjacent to a non-activating Arg residue, that is, adjacent to an Arg residue that is not at an activation site, such as Argl689 or Arg372.
  • the modification of the present invention creates an Arg-Pro linkage or a Pro-Arg linkage. Such a modification can be achieved by one or more amino acid substitutions, additions or deletions.
  • non-activating Arg residue is at least one selected from the group consisting of amino acid residues 220, 226, 250, 279, 282, 336, 359, 562, 747, 776, 1310, 1313, 1645, 1648, 1719, and 1721, numbered with respect to the native Factor VIII:C polypeptide sequence, as depicted in Figures 1A-1F.
  • an analog with a modification that comprises creation of a tripeptide having the formula P 3 ⁇ P 2 -p ⁇ # wherein P 3 is a residue selected from the group consisting of Phe, Glu, and Pro; P 2 is any amino acid residue except Ser and P2 is not Leu335 and is not Asnl720; and P ⁇ is Arg.
  • an active Factor VIII:C polypeptide analog comprising a native Factor VIII.C polypeptide that is modified at at least one non- activating Arg residue selected from the group consisting of Arg336, Argl719 and Argl721, wherein the modification comprises a substitution of any of amino acids Pro, Glu, Asp, Asn, Gin, Ser and Tyr for Arg336, a substitution of any of amino acids Pro, Glu, Asp, Asn, Gin, Ser and Tyr for Argl719 and/or a substitution of any of amino acids Glu, Asp, Asn, Gin, Ser and Tyr for Argl721, numbered with respect to the native Factor VIII:C polypeptide sequence.
  • 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
  • Factor VIII.C molecule containing a heavy chain fragment of molecular weight of about 50 kD (h) a cleaved Factor VIII.C molecule containing a heavy chain fragment of molecular weight of about 43 kD; and (i) a cleaved Factor VIII.C molecule containing a light chain fragment of molecular weight of about 73 kD.
  • an active Factor VIII.C analog complex that contains either at least two Factor VIII.C polypeptide analogs as above, or a Factor VIII.C polypeptide analog and a Factor VIII.C polypeptide, together with a metal ion.
  • the analog complex herein can comprise two Factor VIII.C 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 VIII.C 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 re ⁇ idue in the amino acid ⁇ equence to produce an analog as above.
  • nucleic acid molecule that contains a nucleotide sequence that encodes an 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 a nucleic acid molecule or recombinant vector as above.
  • a method of producing an active Factor VIII.C polypeptide analog as above comprising: (a) providing the recombinant host cell as above, and (b) allowing the recombinant host cell to express the analog.
  • a method of producing a nucleic acid molecule as above comprising: (a) providing a nucleic acid molecule that encodes a native Factor VIII:C polypeptide as above and (b) modifying at least one codon to provide the analog.
  • a method of producing a recombinant vector that contains a nucleic acid molecule as above comprising linking a regulatory element to the nucleic acid molecule.
  • 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 a recombinant vector.
  • composition that contains an active Factor VIII:C polypeptide analog or analog complex as above, and a pharmaceutically acceptable excipient.
  • methods for prevention or treatment of active Factor VIII:C deficiency in a mammal comprising administering thereto a therapeutically effective amount of (a) an active Factor VIII:C polypeptide analog as above, or (b) an active Factor VIII:C polypeptide analog complex as above, or (c) a nucleic acid molecule as above, or (d) a recombinant vector as above, or (e) a nucleic acid molecule as above together with and an active Factor VIII:C polypeptide analog, or (f) a recombinant vector as above together with an active Factor VIII:C polypeptide analog.
  • Factor VIII:C polypeptide analogs can be made that have improved properties. These analogs include one or more amino acid residues that are modified from the native structure. The modification can be at least one amino acid substitution, addition or deletion, at an amino acid residue adjacent to an Arg residue so as to generate, for example, an Arg-Pro linkage or a Pro-Arg linkage.
  • the Factor VIII:C polypeptide analog can also include a modification that comprises creation of a tripeptide having the formula P 3 _ P 2 ⁇ p ⁇ , wherein P 3 is a residue selected from the group consisting of Phe, Glu, and Pro; P 2 is any amino acid residue except Ser and P2 is not Leu335 and is not Asnl720; and P 2 is Arg.
  • P 3 is a residue selected from the group consisting of Phe, Glu, and Pro
  • P 2 is any amino acid residue except Ser and P2 is not Leu335 and is not Asnl720
  • P 2 is Arg.
  • the above modifications do not occur at a site of Factor VIII:C activation.
  • non-activating Arg residues, Arg336 and Argl719 can be substituted with amino acids Pro, Glu, Asp, Asn, Gin, Ser and Tyr and/or the non-activating Arg residue Argl721 can be substituted with Glu, Asp, Asn, Gin, Ser and Tyr, to impart a Factor VIII:C analog with improved properties.
  • 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 sub ⁇ tituted 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 VIII:C molecule comprising all A, B, and C domains and lacking the signal peptide; (c) a truncated Factor VIII:C molecule lacking the signal peptide and at least a portion of the B domain; (d) a cleaved Factor VIII:C 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 VIII:C molecule comprising a heavy chain fragment of about 50 kD; (g) a cleaved Factor VIII:C molecule comprising a heavy chain fragment of about 43 k
  • 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 mutein 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 pro-coagulation activity. Such activity is measured by 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 pro-coagulation activity displayed by the native molecule, preferably at least about 60% to 80%, and more preferably at least about 90% or more of the coagulation or procoagulation activity displayed by the native Factor VIII:C molecule.
  • a "nucleic acid molecule” as used herein, 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 viru ⁇ or viral vector or from a linear or circular plasmid.
  • regulatory control refers to control of expres ⁇ ion of a polynucleotide ⁇ equence by a regulatory element to which the polynucleotide sequence is operably linked.
  • the nature of such regulatory control differs depending upon the host organism.
  • prokaryotes such regulatory control is effected by regulatory sequences which generally include, for example, a promoter, and/or a transcription termination sequence.
  • eukaryotes generally, such regulatory sequences include, for example, a promoter and/or a transcription termination sequence.
  • 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 VIII:C analogs of the invention may be determined by means known in the art, for example, by using the commercially available Coate ⁇ t assay.
  • the effective amount is ⁇ ufficient to bring about prevention of further deterioration or treatment to improve coagulation, that is, to enhance coagulation properties such that hemostasis is achieved.
  • 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.
  • Pharmaceutically acceptable 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.
  • auxiliary ⁇ ubstances 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, polysaccharide ⁇ , 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. A thorough discussion of pharmaceutically acceptable excipients i ⁇ available in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J. 1991) .
  • the therapeutic 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 VIII:C polypeptide analogs with improved properties.
  • the analogs include Pro-Arg bonds or Arg-Pro bonds at non- activating Arg residues.
  • the modification will not introduce an Arg-Ala, Arg-Met, Arg-Gly, Arg-Ser or Arg-Thr bond at the modified site.
  • the modification can comprise creation of a tripeptide having the formula 3 _ p 2 ⁇ p i' wherein P 3 is a residue selected from the group consisting of Phe, Glu, and Pro; P 2 is any amino acid residue except Ser and P2 is not Leu335 and is not Asnl720; and P-L is Arg.
  • Arg residue when the non-activating Arg residue is Arg220, representative modification ⁇ can compri ⁇ e an insertion of at least one Pro residue between Asp219 and Arg 220; an insertion of at least one Pro residue between Arg220 and Asp221; a deletion comprising residues Asp221, Ala222, Ala223, Ser224, Ala225, Arg226, Ala227, and Trp228; an insertion of at least one Phe residue between Gln218 and Asp219; an insertion of at least one Glu residue between Gln218 and Asp219; an insertion of at least one Pro residue between Gln218 and Asp219; a deletion comprising residues Thr212, Lys213, Asn214, Ser215, Leu216, Met217, and Gln218; a ⁇ ub ⁇ titution of at least one Phe residue at Gln218; a substitution of at least one Glu residue at Gln218; and/
  • representative modifications can comprise an insertion of at least one Pro residue between Ala225 and Arg226; an insertion of at least one Pro residue between Arg226 and Ala227; a deletion comprising residues Ala227 and Trp228; a deletion comprising residues Ala227 and Trp228 and insertion of at least one Pro residue to replace the deleted residues; a deletion comprising residues Ala227, Trp228, Pro229, and Lys230, and an insertion of at least one Pro residue to replace the deleted residues; an insertion of at least one Phe residue between Ser224 and Ala225; an insertion of at least one Glu residue between Ser224 and Ala225; an insertion of at least one Pro residue between Ser224 and Ala225; (d) a substitution of at least one Phe residue at Ser224; a substitution of at least one Glu residue at Ser224; and/or a substitution of at least one Pro residue at Ser224.
  • Arg250 When the non-activating Arg residue is Arg250, representative modifications can comprise an insertion of at least one Pro residue between His249 and Arg250; an insertion of at least one Pro residue between Arg250 and Lys251; a deletion comprising residues Lys251 and Ser252 and insertion of at least one Pro residue to replace the deleted residues; a substitution of Lys251 with at lea ⁇ t one Pro residue; a deletion comprising residues Gly244, Leu245, Ile246, Gly247, Cys248, and His249; a deletion comprising residues Arg240, Ser241, Leu242, Pro243, Gly244, Leu245, Ile246, Gly247, Cys248, and Hi ⁇ 249 and an in ⁇ ertion of at least one Pro residue to replace the deleted residue ⁇ ; an in ⁇ ertion of at lea ⁇ t one Phe re ⁇ idue between Cy ⁇ 248 and His249; an insertion of at least one Glu residue between Cys248 and Hi ⁇
  • Arg279 When the non-activating Arg residue is Arg279, representative modifications can comprise an insertion of at least one Pro residue between Val278 and Arg279; an insertion of at least one Pro residue between Arg279 and Asn280; a deletion comprising residues Asn280, His281, and Arg282 and an insertion of at least one Pro residue to replace the deleted residues; a deletion comprising Asn280, His281, Arg282, Gln283, Ala284, and Ser285, and an insertion of at least one Pro residue to replace the deleted residues; an insertion of at lea ⁇ t one Phe re ⁇ idue between Leu277 and Val278; an in ⁇ ertion of at least one Glu residue between Leu277 and Val278; an insertion of at least one Pro residue between Leu277 and Val278; a deletion comprising residue Leu277; a deletion comprising residue Val278; a deletion comprising residues Gly273, His274, Thr275, Phe27
  • Arg282 When the non-activating Arg residue is Arg282, representative modifications can comprise an insertion of at least one Pro residue between His281 and Arg282; an insertion of at least one Pro residue between Arg282 and Gln283; a deletion comprising residues Arg279, Asn280, and His281 and an insertion of at least one Pro residue to replace the deleted residues; a deletion comprising residues Gln283, Ala284, and Ser285, and an insertion of at least one Pro residue to replace the deleted residue ⁇ ; a deletion comprising residues Gln283, Ala284, Ser285, Leu286, Glu287, Ile288, and Ser289; an insertion of at least one Phe residue between Asn280 and His281; an insertion of at least one Glu residue between Asn280 and His281; an in ⁇ ertion of at least one Pro residue between Asn280 and His281; a deletion comprising residues Leu277, Val278, Arg279, and Asn
  • Arg336 When the non-activating Arg re ⁇ idue is Arg336, representative modifications can compri ⁇ e an insertion of at least one Pro residue between Leu335 and Arg336; a deletion comprising residues Gln334, and Leu335; an insertion of at least one Pro residue between Arg336 and Met337; a deletion comprising residues Met337, and Lys338 and insertion of at least one Pro residue in place of the deleted residues; a deletion comprising residue Leu335 and an in ⁇ ertion of at least one Phe residue between Pro333 and Gln334; a deletion comprising re ⁇ idue Leu335 and an in ⁇ ertion of at least one Glu residue between Pro333 and Gln334; a deletion comprising residue Leu335 and an insertion of at least one Pro residue between
  • Pro333 and Gln334 a deletion comprising residue Leu335; a deletion comprising residue ⁇ Gln334, and Leu335; a deletion comprising residues Pro333, Gln334, and Leu335; a deletion comprising residues Glu332, Pro333, Gln334, and Leu335; a deletion comprising residue Leu335 and a sub ⁇ titution of at least one Phe residue at Pro333; and/or a deletion comprising residue Leu335 and a substitution of at least one Glu residue at Pro333.
  • Arg359 When the non-activating Arg residue is Arg359, representative modifications can comprise an insertion of at least one Pro residue between Val358 and Arg359; an insertion of at least one Pro residue between Arg359 and Phe360; a deletion comprising residues Phe360, Asp361, A ⁇ p362, A ⁇ p363, A ⁇ n364, and Ser365; a deletion compri ⁇ ing residues Phe360, Asp361, A ⁇ p362, A ⁇ p363, Asn364, Ser365, Pro366, and Ser367, and an insertion of at least one Pro residue to replace the deleted residues; an insertion of at least one Phe residue between Val357 and Val358; an insertion of at least one Glu residue between Val357 and Val358; an insertion of at least one Pro residue between Val357 and Val358; a deletion comprising residues Met355, Asp356, and Val357; a substitution of at least one Phe residue at Val357; a substitution of at least one Glu
  • Arg562 When the non-activating Arg residue is Arg562, representative modifications can comprise an insertion of at least one Pro residue between Gln561 and Arg562; an insertion of at least one Pro residue between Arg562 and Gly563; a deletion comprising residues Ser558, Val559, Asp560, Gln561, and an insertion of at least one Pro residue to replace the deleted residues; a deletion comprising residues Lys556, Glu557, Ser558, Val559, Asp560, Gln561, and an insertion of at least one Pro residue to replace the deleted residues; a deletion comprising residues Gly563, Asn564, Gln565, Ile566, Met567, and Ser568, and an insertion of at least one Pro residue to replace the deleted residues; a deletion comprising residues Gly563, Asn564, Gln565, Ile566, Met567, Ser568, Asp569, Lys570, and Arg571, and an insertion of at
  • representative modifications can comprise an insertion of at least one Pro residue between Ser746 and Arg747; a deletion comprising residue Ser746 and an insertion of at least one Pro residue to replace the deleted residue; a deletion comprising residue His748; a deletion comprising residue His748 and an insertion of at least one Pro residue to replace the deleted residue; an insertion of at least one Pro residue between Arg747 and His748; a deletion comprising residues His748, Pro749, and Ser750 and an insertion of at least one Pro residue to replace the deleted residues; a deletion comprising Ser743, Gln744, Asn745, Ser746; a deletion comprising His748, Pro749, Ser750, Thr751, Arg752, Gln753, and Lys754, and an insertion of at least one Pro residue to replace the deleted residues; a deletion comprising residue Ser746 and an insertion of at least one Phe residue between Gln744 and Asn745;
  • Arg776 When the non-activating Arg residue is Arg776, representative modifications can comprise an insertion of at least one Pro residue between His775 and Arg776; an insertion of at least one Pro residue between Arg776 and Thr777; a deletion compri ⁇ ing re ⁇ idue Thr777; a deletion compri ⁇ ing re ⁇ idue Thr777 and an insertion of at least one Pro residue to replace the deleted residue; a deletion comprising residues Trp772, Phe773, Ala774, and His775; a deletion comprising residues Trp772, Phe773, Ala774, and His775, and an insertion of at least one Pro residue to replace the deleted residues; a deletion comprising residues Lys768, Thr769, Asp770, Pro771,
  • Argl310 When the non-activating Arg residue is Argl310, representative modifications can comprise an insertion of at least one Pro residue between Glnl309 and Argl310; an insertion of at least one Pro residue between Argl310 and Serl311; a deletion comprising residue Serl311 and an insertion of at least one Pro residue to replace the residue; a deletion comprising residues Ser1311, Lysl312, and Argl313 and an insertion of at least one Pro residue to replace the deleted residues; a deletion comprising residues Serl311, Lysl312, Argl313, Alal314, Leul315, and Lysl316, and an insertion of at least one Pro residue to replace the deleted residues; a deletion comprising residues Serl311, Lysl312, Argl313, Alal314, Leul315, Lysl316, Glnl317, Phel318, Argl319, and Leul320; a deletion comprising residues Serl311, Lysl312,
  • Argl313 When the non-activating Arg residue is Argl313, representative modifications can comprise an insertion of at least one Pro residue between Lysl312 and Argl313; a deletion comprising residue Lysl312 and an insertion of at least one Pro residue to replace the deleted residue; a deletion compri ⁇ ing residues Argl310, Serl311, and Lysl312, and an insertion of at least one Pro residue to replace the deleted residues; an insertion of at lea ⁇ t one Pro re ⁇ idue between Argl313 and Alal314; a deletion comprising residues Alal314, Leul315, and Lysl316, and an in ⁇ ertion of at least one Pro residue to replace the deleted residues; a deletion comprising residues Alal314, Leul315, Lysl316, Glnl317, Phel318, and Argl319 and an insertion of at least one Pro residue to replace the deleted residues; a deletion comprising residues Alal314, Leul315,
  • representative modifications can comprise a deletion comprising residues Vall642, Leul643, and Lysl644; a deleteion compri ⁇ ing residue Lysl644 and an in ⁇ ertion of at lea ⁇ t one Pro residue to replace the deleted residue; an insertion of at least one Pro residue between Argl645 and Lysl644; an insertion of at least one Pro re ⁇ idue between Argl645 and Hisl646; a deletion comprising residues Hisl646, Glnl647, and Argl648 and an in ⁇ ertion of at least one Pro residue to replace the deleted residues; a deletion comprising residues Hisl646, Glnl647, Argl648, Glul649, Ilel650, Thrl651, and Argl652, and an insertion of at least one Pro residue to replace the deleted residues; an insertion of at least one Phe residue between Leul643 and Ly ⁇
  • Argl648 When the non-activating Arg residue is Argl648, representative modifications include a deletion comprising residues Vall642, Leul643, Lysl644, Argl645, Hisl646, and Glnl647; an insertion of at least one Pro residue between Glnl647 and Argl648; a deletion comprising residues Lysl644, Argl645, Hisl646, and Glnl647 and an insertion of at least one Pro residue to replace the deleted residues; a deletion comprising residues Glul649, Ilel650, Thrl651, and Argl652 and an insertion of at least one Pro residue to replace the deleted residues; an insertion of at least one Pro residue between Argl648 and Glul649; an insertion of at least one Phe residue between Hisl646 and Glnl647; an insertion of at least one Glu residue between Hisl646 and Glnl647; an in ⁇ ertion of at least one Pro residue between His
  • Argl719 When the non-activating Arg residue is Argl719, representative modifications a deletion comprising residues Hisl716, Vall717, and Leul718; an in ⁇ ertion of at least one Pro residue between Leul718 and Argl719; a deletion comprising residues Asnl720, and Argl721 and an in ⁇ ertion of at least one Pro residue to replace the deleted residues; an insertion of at least one Pro residue between Argl719 and Asnl720; an insertion of at least one Phe residue between Vall717 and Leul718; an insertion of at least one Glu residue between Vall717 and Leul718; an insertion of at least one Pro residue between Vall717 and Leul718; a deletion comprising residue ⁇
  • Argl721 representative modifications include a deletion comprising residues Hisl716, Vall717, Leul718, Argl719, and Asnl720; an insertion of at least one Pro residue between A ⁇ nl720 and Argl721; an insertion of at least one Pro residue between Argl721 and Alal722; a deletion comprising residues Alal722, Glnl723, and Serl724 and an insertion of at least one Pro residue to replace the deleted residues; a deletion comprising residues Alal722, Glnl723, Serl724, Glyl725, Serl726, and Vall727; a deletion comprising residues Alal722, Glnl723, Serl724, Glyl725, and Serl726, and an
  • polypeptide analogs contemplated by the present invention are tho ⁇ e analogs including substitutions of the non-activating Arg residues found at positions Arg336, Argl719 and Argl721, numbered with respect to the native Factor VIII:C polypeptide ⁇ equence.
  • tho ⁇ e analogs including substitutions of the non-activating Arg residues found at positions Arg336, Argl719 and Argl721, numbered with respect to the native Factor VIII:C polypeptide ⁇ equence.
  • any of amino acids Pro, Glu, Asp, Asn, Gin, Ser and Tyr can be substituted at position Arg336 and/or position Argl719, to generate a Factor VIII:C polypeptide analog with improved propertie ⁇ .
  • any of amino acids Glu, Asp, Asn, Gin, Ser and Tyr can be substituted at position Argl721.
  • the Factor VIII:C polypeptide analog of this embodiment will include a substitution of Arg336 with Pro, a substitution of Arg 1719 with Pro and/or a substitution of Argl721 with Glu. Nucleic acid molecules encoding the present
  • Factor VIII:C polypeptide analogs can be made by modifying the native nucleic acid sequences that encode the Factor VIII:C polypeptide or cDNA sequences that encode the Factor VIII:C polypeptides. Such modification can be done by conventional techniques such as site- directed mutagenesis. For example, the M13 method for site directed mutagene ⁇ is is known, as described in Zoller and Smith, Nucleic Acids Re ⁇ . (1982) 10: 6487- 6500, Methods Enzymol . (1983) 100: 468-500, and DNA (1984) 3: 479-488, using single stranded DNA, and the method of Morinaga et al . Bio/technol .
  • one or more of the codons encoding a residue adjacent to an arginine, preferably, at the carboxy side can be mutated by substitution, deletion or addition, to a codon encoding proline, thereby creating polypeptides with either Pro-Arg bonds or Arg-Pro bonds where none existed before, provided that the modification does not affect an activation cleavage site, such as Arg372 and Arg740.
  • the codons encoding proline include CCU, CCT, CCG, CCA, and CCC.
  • the nucleic acid molecule ⁇ of the pre ⁇ ent invention can al ⁇ o be made ⁇ ynthetically by piecing together nucleic acid molecules encoding heavy and light chain fragments derived from cDNA clones or genomic clones containing Factor VIII:C coding sequences, preferably cDNA clones, using known linker sequences. Alternatively, the entire sequence or portions of nucleic acid sequence ⁇ encoding analogs described above may be prepared by synthetic methods (e.g. using DNA synthe ⁇ i ⁇ 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.
  • DNA cloning and in posses ⁇ ion of the DNA encoding native Factor VIII:C polypeptide will be able to prepare suitable DNA molecules for production of the present analogs using known cloning procedure ⁇ (e.g. restriction enzyme digestion, exonuclease digestion, ligation, and other appropriate procedures) outlined in any of the following: Sambrook, et al , MOLECULAR CLONING: A LABORATORY MANUAL 2nd ed. (Cold Spring Harbor Laboratory Press, 1989) ; DNA CLONING, Vol. I and II, D.N. Glover ed. (IRL Pres ⁇ , 1985); OLIGONUCLEOTIDE SYNTHESIS, M.J. Gait ed.
  • cloning procedure e.g. restriction enzyme digestion, exonuclease digestion, ligation, and other appropriate procedures
  • 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 site ⁇ 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 elements or control sequences for expression of the nucleic acid molecule.
  • the present vector can be derived from a plasmid, a virus, a cosmid, or a bacteriophage. This vector is typically capable of behaving as an autonomous unit of replication when introduced into a host cell.
  • the vector may be one that is capable of epi ⁇ omal existence or of integration into the host cell genome.
  • replication systems typically derived from viruses that infect mammalian host cells.
  • Illustrative replication sy ⁇ tems include the replication systems from Simian virus 40, adenoviru ⁇ , bovine papilloma viru ⁇ , polyoma viru ⁇ , Ep ⁇ tein Barr viru ⁇ , and the like.
  • the nucleic acid molecule of the present invention can be inserted at an appropriate restriction 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 used for transfection or transformation of a host cell.
  • the host cells of the invention can be, for example, prokaryotic or eukaryotic host cell ⁇ , including bacterial, yeast, insect and mammalian expression sy ⁇ tems.
  • the analogs of the present invention are expressed in mammalian host cell systems.
  • 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
  • an eukaryotic host cell can be used for expression of the Factor VIII:C polypeptide analogs.
  • the expression cassettes are introduced into the host cell by conventional methods, depending on the expression system used, as de ⁇ cribed further below. Where viruses are involved, transfection or transduction may be employed.
  • the particular manner in which the host cell is transformed is not critical to this invention, depending substantially upon whether the expression cassettes are joined to a replication system and the nature of the replication system and associated genes.
  • Coexpres ⁇ ion of more than one Factor VIII:C polypeptide analog may be de ⁇ ired. For example, it may be de ⁇ irable to expre ⁇ the light and heavy chain ⁇ u ⁇ ing ⁇ eparate constructs. In this regard, either or both of the light and heavy chains may include modifications as described above.
  • Coexpression refers to the expression of two or more Factor VIII:C polypeptides in a ⁇ ingle ho ⁇ t cell. Thus, for example, the expression of the 90 kD specie ⁇ and the 80 kD species in a single host cell, would constitute “coexpression” as used herein.
  • the polynucleotides encoding for the polypeptides can be harbored in a single vector, either under the control of the same regulatory elements or under the control of separate elements.
  • a fusion protein including active portions of the two or more Factor VIII:C polypeptides would be con ⁇ idered "coexpressed" for purposes of the present definition as would the expression of two genes as a dicistronic construct employing an internal ribosome entry site.
  • proteins expres ⁇ ed from the same vector but driven by separate regulatory elements would also be considered “coexpressed.”
  • the term also refers to the expres ⁇ ion of two or more proteins from separate constructs.
  • the expression of proteins encoded from genes present on separate vectors in a host cell would also be considered “coexpression" for purposes of the present invention.
  • the transformed/transfected cells 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, so that the media or cell lysate may be isolated and the Factor
  • VIII:C active complex extracted and purified.
  • the full-length molecule can be isolated and treated under complex-forming conditions, e.g., with the addition of calcium and the appropriate enzymes, to form the active complex.
  • Various means are available for extraction and purification, such as affinity chromatography, ion exchange chromatography, hydrophobic chromatography, electrophoresis, solvent- ⁇ olvent extraction, selective precipitation, and the like.
  • affinity chromatography ion exchange chromatography
  • hydrophobic chromatography hydrophobic chromatography
  • electrophoresis solvent- ⁇ olvent extraction
  • selective precipitation and the like.
  • the particular manner in which the product is 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 expression systems can be used to produce the subject Factor VIII:C polypeptide analog ⁇ and nucleic acid sequences encoding the analogs.
  • Control elements for use in bacterial systems include promoters, optionally containing operator ⁇ equences, and ribosome binding sites.
  • Useful promoters include sequences derived from sugar metabolizing enzymes, such as galactose, lactose (lac) and maltose. Additional examples include promoter sequences derived from biosynthetic enzymes ⁇ uch as tryptophan (trp) , the ⁇ - lactamase (Jla) promoter system, bacteriophage ⁇ PL, and T7.
  • synthetic promoters can be used, such as the tac promoter.
  • the ⁇ -lactamase and lactose promoter sy ⁇ tems are described in Chang et al . , Nature (1978) 275 : 615, and Goeddel et al . , Nature (1979) 281 : 544; the alkaline phosphatase, tryptophan (trp) promoter system are described in Goeddel et al . , Nucleic Acid ⁇ Re ⁇ . (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 .
  • the signal sequence can be substituted by a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase, Ipp, or heat stable enterotoxin II leaders.
  • a prokaryotic signal sequence selected, 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.
  • the foregoing systems are particularly compatible with E ⁇ cherichia coli .
  • numerous other systems for use in bacterial hosts including Gram- negative or Gram-positive organisms such as Bacillu ⁇ ⁇ pp. , Streptococcus ⁇ pp. , Streptomyce ⁇ ⁇ pp . , Pseudomonas species such as P . aerugino ⁇ a, Salmonella typhimurium, or Serratia marce ⁇ cans, 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 cell ⁇ by eiectroporation, nuclear injection, or protoplast fusion as described generally in Sambrook et al . (1989), cited above. These examples are illustrative rather than limiting.
  • the host cell ⁇ hould secrete minimal amounts of proteolytic enzymes.
  • in vitro methods of cloning e.g., PCR or other nucleic acid polymerase reactions, are suitable.
  • Prokaryotic cells used to produce the Factor VIII:C analog polypeptides of this invention are cultured in suitable media, as described generally in Sambrook et al . , cited above.
  • Yeast expression systems can also be used to produce the subject Factor VIII:C polypeptide analogs and nucleic acid sequences encoding the analogs.
  • Expression and transformation vectors either extrachromosomal replicons or integrating vectors, have been developed for transformation into many yeasts.
  • expres ⁇ ion vectors have been developed for, among others, the following yeast ⁇ : Saccharomyces cerevisiae ,as described in Hinnen et al . , Proc . Natl . Acad . Sci . USA (1978) 75 : 1929; Ito et al . , J . Bacteriol . (1983) 153 : 163; Candida albican ⁇ as described in Kurtz et al . , Mol .
  • Control sequences for yeast vectors are known and include promoter regions from genes such as alcohol dehydrogenase (ADH), as described in EP 284,044, enolase, glucokinase, glucose-6-phosphate isomera ⁇ e, glyceraldehyde-3-phosphate-dehydrogenase (GAP or GAPDH) , hexokinase, phosphofructokinase, 3-phosphoglycerate mutase, and pyruvate kinase (PyK) , as described in EP 329,203.
  • the yeast PH05 gene, encoding acid phosphatase also provides useful promoter sequences, as described in Myanohara et al., Proc.
  • ⁇ uitable promoter sequences for use with yeast host ⁇ include the promoters for 3-phosphoglycerate kina ⁇ e, a ⁇ described in Hitzeman et al . , J . Biol . Chem . (1980) 255 : 2073, or other glycolytic enzymes, such as pyruvate decarboxylase, triosephosphate isomerase, and phosphoglucose isomerase, as described in Hess et al . , J. Adv . Enzyme Reg . (1968) 7: 149 and Holland et al . ,
  • Inducible yeast promoters having the additional advantage of transcription controlled by growth conditions, include from the list above and others the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in Hitzeman, EP 073,657. Yeast enhancers also are advantageously used with yeast promoters. In addition, synthetic promoters which do not occur in nature also function a ⁇ yeast promoters.
  • upstream activating sequences (UAS) of one yeast promoter may be joined with the transcription activation region of another yeast promoter, creating a synthetic hybrid promoter.
  • hybrid promoters include the ADH regulatory sequence linked to the GAP transcription activation region, as described in U.S. Patent Nos. 4,876,197 and 4,880,734.
  • Other examples of hybrid promoters include promoters which consist of the regulatory sequences of either the ADH2 , GAL4 , GAL10 , or PH05 genes, combined with the tran ⁇ criptional 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 promoters of non-yeast origin that have the ability to bind yeast RNA polymerase and initiate transcription.
  • Other control elements which may be included in the yeast expression vectors are terminators, for example, from GAPDH and from the enolase gene, as described in Holland et al . , J . Biol . Chem . (1981) 256 : 1385, and leader sequences which encode signal sequences for secretion.
  • DNA encoding suitable signal sequence ⁇ can be derived from genes for secreted yeast proteins, such as the yeast invertase gene as described in EP 012,873 and JP 62,096,086 and the ⁇ -factor gene, as de ⁇ cribed in U.S. Patent Nos.
  • leaders of non-yeast origin such as an interferon leader, also provide for secretion in yeast, as described in EP 060,057.
  • Methods of introducing exogenous DNA into yeast hosts are well known in the art, and typically include either the transformation of spheroplasts or of intact yeast cells treated with alkali cations. Transformations into yeast can be carried out according to the method described in Van Solingen et al . , J . Bact . (1977) 130 : 946 and H ⁇ iao et al . , Proc . Natl . Acad . Sci . USA (1979) 76 : 3829. However, other methods for introducing DNA into cells such as by nuclear injection, eiectroporation, or protoplast fusion may also be used as described generally in Sambrook et al . , cited above.
  • the native polypeptide signal sequence may be ⁇ ub ⁇ tituted by the yeast invertase, ⁇ -factor, or acid phosphatase leaders.
  • the origin of replication from the 2 ⁇ plasmid origin is ⁇ uitable for yea ⁇ t.
  • a ⁇ uitable selection gene for use in yeast is the trpl gene present in the yeast plasmid described in Kingsman et al . , Gene (1979) 7: 141 or T ⁇ che per et al . , Gene (1980) 10 : 157.
  • the trpl gene provide ⁇ a ⁇ election 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 sequence encoding a yeast protein can be linked to a coding sequence of a Factor VIII:C polypeptide analog to produce a fusion protein that can be cleaved intracellularly by the yeast cells upon expression.
  • An example, of such a yeast leader sequence i ⁇ the yeast ubiquitin gene.
  • Insect expression sy ⁇ tems can be used to produce the Factor VIII:C polypeptide analogs and nucleic acid sequences encoding the analogs.
  • baculovirus expression vectors are recombinant insect viruses in which the coding sequence for a foreign gene to be expressed is inserted behind a baculovirus promoter in place of a viral gene, e.g., polyhedrin, as described in Smith and Summers, U.S. Pat. No., 4,745,051.
  • An expression construct herein includes a DNA vector useful as an intermediate for the infection or transformation of an insect cell sy ⁇ tem, the vector generally containing DNA coding for a baculoviru ⁇ transcriptional promoter, optionally but preferably, followed downstream by an insect signal DNA sequence capable of directing secretion of a desired 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 VIII:C 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 Orders Lepidoptera, Diptera, Orthoptera, Coleoptera and Hymenoptera including, for example, but not limited to the viral DNAs of Autographo californica MNPV, Bombyx mori NPV, rrichoplu ⁇ ia ni MNPV, Rachlplu ⁇ ia ou MNPV or Galleria mellonella MNPV, Aede ⁇ aegypti, Drosophila melanogaster, Spodoptera frugiperda , and Trichoplusia ni .
  • the baculovirus transcriptional promoter can be, for example, a baculovirus immediate-early gene IEI or IEN promoter; an immediate-early gene in combination with a baculovirus delayed-early gene promoter region selected from the group consisting 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 transcriptional enhancer elements. Particularly ⁇ uitable for u ⁇ e herein is the strong polyhedrin promoter of the baculovirus, which directs a high level of expression of a DNA insert, as described in Friesen et al . (1986) "The Regulation of Baculovirus Gene Expression" in: THE MOLECULAR BIOLOGY OF BACULOVIRUSES (W.Doerfler, ed.); EP 127,839 and EP
  • the plasmid for use herein usually also contains the polyhedrin polyadenylation signal, a ⁇ described in Miller et al . , Ann . Rev .
  • Microbiol . (1988) 42 111 and a procaryotic ampicillin- resistance (amp) gene and an origin of replication for selection and propagation in E. coli .
  • DNA encoding suitable signal sequence ⁇ can also be included and is generally derived from gene ⁇ for ⁇ ecreted in ⁇ ect or baculoviru ⁇ proteins, such as the baculovirus polyhedrin gene, as described in Carbonell et al . , Gene (1988) 73 : 409, as well as mammalian signal sequences such as those derived from genes encoding human ⁇ -interferon as described in Maeda et al .
  • viruses may be used as the virus for transfection of host cells such as Spodoptera frugiperda cells.
  • baculovirus genes in addition to the polyhedrin promoter may be employed to advantage in a baculovirus expression system. These include immediate-early (alpha) , delayed-early (beta) , late (gamma) , or very late (delta) , according to the phase of the viral infection during which they are expressed. The expression of these genes occurs sequentially, probably as the result of a "cascade" mechanism of transcriptional regulation. Thus, the immediate-early genes are expressed immediately after infection, in the absence of other viral functions, and one or more of the resulting gene products induces transcription of the delayed-early genes.
  • immediate-early genes are expressed immediately after infection, in the absence of other viral functions, and one or more of the resulting gene products induces transcription of the delayed-early genes.
  • IEI a preferred immediate-early gene of Autographo californica nuclear polyhedrosis virus
  • AcMNPV Autographo californica nuclear polyhedrosis virus
  • IEI is pre ⁇ ed in the absence of other viral functions and encodes a product that stimulates the transcription of several genes of the delayed-early class, including the preferred 39K gene, as de ⁇ cribed in Guarino and Summers, J . Virol . (1986) 57 : 563-571 and J. Virol . (1987) 61 : 2091-2099 as well a ⁇ late gene ⁇ , as described in Guanno and Summers, Virol . (1988) 162 : 444-451.
  • Immediate-early genes as described above can be used in combination with a baculovirus gene promoter region of the delayed-early category. Unlike the immediate-early genes, such delayed-early genes require the presence of other viral genes or gene products such as those of the immediate-early genes.
  • the combination of immediate-early genes can be made with any of several delayed-early gene promoter regions such as 39K or one of the delayed-early gene promoters found on the Hindlll fragment of the baculovirus genome. In the present instance, the 39 K promoter region can be linked to the foreign gene to be expres ⁇ ed ⁇ uch that expre ⁇ ion can be further controlled by the pre ⁇ ence of IEI, a ⁇ described in L. A.
  • enhancement of the expression of heterologous genes can be realized by the presence of an enhancer sequence in direct cis linkage with the delayed-early gene promoter region.
  • enhancer ⁇ equences are characterized by their enhancement of delayed-early gene expression in situations where the immediate-early gene or its product is limited.
  • the hr5 enhancer sequence can be linked directly, in cis, to the delayed-early gene promoter region, 39K, thereby enhancing the expression of the cloned heterologous DNA as de ⁇ cribed in Guarino and Summer ⁇ (1986a), (1986b), and Guarino et al .
  • an insect ⁇ ignal ⁇ equence can be used to express 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 ⁇ ignal sequence.
  • An exemplary insect signal sequence suitable herein is the sequence encoding for a Lepidopteran adipokinetic hormone (AKH) peptide.
  • the AKH family consists of short blocked neuropeptides that regulate energy substrate mobilization and metabolism in insect ⁇ .
  • a DNA sequence coding for a Lepidopteran Manduca sexta AKH signal peptide can be used.
  • Other insect AKH signal peptides, such as those from the Orthoptera Schistocerca gregaria locus can also be employed to advantage.
  • Another exemplary insect signal sequence is the sequence coding for Drosophila cuticle proteins such as CPI, CP2, CP3 or CP4.
  • the desired DNA sequence can be in ⁇ erted into the tran ⁇ fer vector, u ⁇ ing known technique ⁇ .
  • An insect cell host can be cotran ⁇ formed with the transfer vector containing the inserted de ⁇ ired DNA together with the genomic DNA of wild type baculoviru ⁇ , u ⁇ ually by cotran ⁇ fection.
  • the vector and viral genome are allowed to recombine resulting in a recombinant virus that can be easily identified and purified.
  • the packaged recombinant virus can be used to infect insect host cells to express a Factor VIII:C polypeptide analog.
  • Mammalian expression systems can also be used to produce the Factor VIII:C polypeptide analogs and nucleic acid sequences encoding the analogs.
  • Typical promoters for mammalian cell expres ⁇ ion include the SV40 early promoter, the CMV promoter, the mou ⁇ e mammary tumor virus LTR promoter, the adenovirus major late promoter (Ad MLP) , and the herpes simplex virus promoter, among others.
  • Other non-viral promoters such as a promoter derived from the murine metallothionein gene, will also find use in mammalian con ⁇ tructs.
  • Mammalian expression may be either constitutive or regulated (inducible) , depending on the promoter.
  • transcription termination and polyadenylation sequences 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 also present.
  • tran ⁇ cription terminator/polyadenylation signals include those derived from SV40, as described in Sambrook et al. (1989) MOLECULAR CLONING: A LABORATORY MANUAL, 2d edition, (Cold Spring Harbor Press, Cold Spring Harbor, N.Y.).
  • Introns, containing splice donor and acceptor ⁇ ite ⁇ may al ⁇ o be designed into the constructs of the present invention.
  • Enhancer elements can also be used herein to increase expres ⁇ ion levels of the mammalian construct ⁇ .
  • Example ⁇ include the SV40 early gene enhancer, as 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 Virus, as described in Gorman et al . , Proc . Natl . Acad . Sci . USA (1982b) 79 : 6777 and human cytomegalovirus, as de ⁇ cribed in Bo ⁇ hart et al . , Cell (1985) 41 : 521.
  • LTR long terminal repeat
  • a leader ⁇ equence can also be present which includes a sequence encoding a signal peptide, to provide for the secretion of the foreign protein in mammalian cells.
  • the Factor VIII signal peptide can be used.
  • the adenovirus tripartite leader is an example of a leader sequence that provides for secretion of a foreign protein in mammalian cells.
  • transient expres ⁇ ion involves the use of an expression vector that is able to replicate efficiently in a host cell, such that the host cell accumulates many copies of the expression vector and, in turn, synthesizes high levels of a desired polypeptide encoded by the expression vector.
  • Transient expre ⁇ sion systems comprising a suitable expression vector and a host cell, allow for the convenient positive identification of polypeptides encoded by cloned DNAs, as well as for the rapid screening of such polypeptide ⁇ for desired biological or physiological properties.
  • transient expression systems are particularly useful for purposes of identifying additional polypeptides that have Factor VIII:C-like activity.
  • the mammalian expres ⁇ ion vector ⁇ can be used to transform any of several mammalian cells.
  • Methods for introduction of heterologous polynucleotides into mammalian cells include dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fu ⁇ ion, eiectroporation, encapsulation of the polynucleotide(s) in lipo ⁇ omes, and direct microinjection of the DNA into nuclei.
  • General a ⁇ pect ⁇ of mammalian cell ho ⁇ t system transformations have been described by Axel in U.S. 4,399,216.
  • a ⁇ ynthetic lipid particularly u ⁇ eful for polynucleotide transfection 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 de ⁇ cribed 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, Chinese ham ⁇ ter ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS) , human hepatocellular carcinoma cells (e.g., Hep G2) , human embryonic kidney cells, baby hamster kidney cells, mouse sertoli cells, canine kidney cells, buffalo rat liver cells, human lung cells, human liver cells, mouse mammary tumor cells, as well as others.
  • the mammalian ho ⁇ t cell ⁇ used to produce the target polypeptide of this invention may be cultured in a variety of media.
  • any of the media described in Ham and Wallace, Meth . Enz . (1979) 58 : 44, Barnes and Sato, Anal. Biochem . (1980) 102 : 255, U.S. Patent Nos. 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 as culture media for the host cells.
  • any of these media may be supplemented as neces ⁇ ary with hormones and/or other growth factors such as insulin, transferrin, or epidermal growth factor, salt ⁇ ( ⁇ uch a ⁇ sodium chloride, calcium, magnesium, and phosphate) , buffers (such as HEPES) , nucleosides (such as adenosine and thymidine) , antibiotics (such as Gentamycin(tm) M drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range) , and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those ⁇ killed in the art.
  • hormones and/or other growth factors such as insulin, transferrin, or epidermal growth factor, salt ⁇ ( ⁇ uch a ⁇ sodium chloride, calcium, magnesium, and phosphate) , buffers (such as HEPES) , nucleosides (such as adenosine and thymidine) , antibiotics
  • the culture condition ⁇ such as temperature, pH, and the like, are those previously used with the ho ⁇ t cell selected for expres ⁇ ion, and will be apparent to the ordinarily ⁇ killed arti ⁇ an.
  • the active Factor VIII:C analogs produced according to the invention have a variety of uses.
  • the analogs can be used as immunogens for the production of antibodies.
  • the analogs can also be used for the treatment of hemophiliacs and other hosts having blood clotting disorders.
  • the Factor VIII:C analogs may display increased plasma half-life or specific activity.
  • the analogs may allow for lower dosages or alternative modes of administration and may improve hemostasis in hemophiliacs.
  • nucleic acid molecules or vectors comprising polynucleotide sequences encoding the Factor VIII:C analogs can be used directly for gene therapy and administered using standard gene delivery protocols.
  • the nucleotide sequences encoding the Factor VIII:C analog ⁇ can be stably integrated into the host cell genome or maintained on a stable episomal element in the ho ⁇ t cell.
  • Methods for gene delivery are known in the art. See, e.g., U.S. Patent No. 5,399,346.
  • retroviral based ⁇ ystems have been developed for gene transfer into mammalian cells.
  • retroviruses provide a convenient platform for gene delivery systems.
  • a selected gene can be inserted into a vector and packaged in retroviral particles u ⁇ ing 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 systems have been described (U.S. Patent No. 5,219,740; Miller and Rosman, BioTechniques (1989) 7:980-990; Miller, A.D. , Human Gene Therapy (1990) 1:5-14; Scarpa et al .
  • adenovirus vectors have also been described. Unlike retroviruses which integrate into the host genome, adenoviruses persist extrachromo ⁇ omally thu ⁇ minimizing the risks associated with insertional mutagenesis (Haj-Ahmad and Graham, J . Virol . (1986) 57:267-274; Bett et al . , J. Virol .
  • AAV vector systems have been developed for gene delivery. Such systems can include control sequences, such as promoter and polyadenylation sites, as well as ⁇ electable markers or reporter genes, enhancer sequence ⁇ , and other control elements which allow for the induction of transcription.
  • AAV vectors can be readily constructed u ⁇ ing technique ⁇ well known in the art. See, e.g., U.S. Patent No ⁇ . 5,173,414 and 5,139,941; International Publication Nos. WO 92/01070 (published 23 January 1992) and WO 93/03769 (published 4 March 1993) ; Lebkowski et al . , Molec. Cell . Biol .
  • Additional viral vectors which will find use for delivering the nucleic acid molecules encoding the Factor VIII:C analog polypeptides for gene transfer include those derived from the pox family of viruse ⁇ , including vaccinia virus and avian poxvirus.
  • vaccinia virus recombinants expressing the novel Factor VIII:C analogs can be constructed as follows. The DNA encoding the particular analog is first inserted 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 is then used to transfect cells which are simultaneously infected with vaccinia.
  • TK thymidine kinase
  • Homologous recombination serves to insert 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 presence of 5- bromodeoxyuridine and picking viral plaques resistant thereto.
  • a vaccinia based infection/transfection system can be conveniently used to provide for inducible, transient expression of the Factor VIII:C analogs in a host cell. In this system, cells are first infected in vitro with a vaccinia virus recombinant that encodes the bacteriophage T7 RNA polymerase. This polymerase displays vibrant ⁇ pecificity in that it only tran ⁇ cribe ⁇ templates bearing T7 promoters.
  • RNA RNA
  • the method provides for high level, transient, cytoplasmic production of large quantities of RNA and its translation products. See, e.g., Elroy-Stein and Moss, Proc . Natl . Acad . Sci . USA (1990) 87:6743-6747; Fuerst et al . , Proc . Natl . Acad . Sci . USA (1986) 83:8122-8126.
  • avipoxviruses such as the fowlpox and canarypox viruses, can also be used to deliver the Factor VIII:C analog genes.
  • Recombinant avipox viruses expressing immunogens from mammalian pathogens, are known to confer protective immunity when administered to non-avian species.
  • the use of an avipox vector is particularly desirable in human and other mammalian specie ⁇ since members of the avipox genus can only productively replicate in susceptible avian specie ⁇ and therefore are not infective in mammalian cell ⁇ .
  • Methods for producing recombinant avipoxviruses are known in the art and employ genetic recombination, as described above with respect to the production of vaccinia viruses. 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 system can be used that will lead to high level expres ⁇ ion following introduction into host cells.
  • T7 RNA polymerase 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 expression is under the control of the T7 promoter. Thus, ⁇ ome of the T7 RNA polymerase generated from translation of the amplification template RNA will lead to tran ⁇ cription of the desired gene. Because some T7 RNA polymerase is 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 PCR techniques. However the use of a plasmid is preferred. Since high level expression of T7 RNA polymerase appears to be lethal to host cells, the plasmid should be one where expression of T7 RNA polymerase can be controlled. For example, a lac operator can be engineered distal or proximal (or both) to the T7 promoter. The binding of the preexisting lac repres ⁇ or in the appropriate bacterial strain would interfere with the transcription of the template by blocking access to the promoter by T7 RNA polymerase. Alternatively, or in combination with the above, a plasmid can be constructed where transcription from a bacterial promoter begins 3' of the T7 gene and continues through the 5' end of the T7 promoter.
  • Such transcription will generate an antisense transcript and reduce or eliminate translation of T7 RNA polymerase RNAs.
  • the second tran ⁇ cription unit con ⁇ i ⁇ ting of the T7 promoter preceding the gene of interest can be provided by a separate pla ⁇ mid or can be engineered onto the amplification plasmid. Colocalization of the two transcription units i ⁇ beneficial for ea ⁇ e of manufacturing and en ⁇ ures that both transcription units will always be together in the cells into which the plasmid is introduced.
  • the T7 RNA polymera ⁇ e pla ⁇ mid ⁇ may include UTRs which comprise an Internal Ribosome Entry Site (IRES) present in the leader sequence ⁇ of picornaviru ⁇ e ⁇ ⁇ uch a ⁇ the encephalomyocarditi ⁇ virus (EMCV) UTR (Jang et al. J. Virol . (1989) 63:1651-1660).
  • IRS Internal Ribosome Entry Site
  • EMCV encephalomyocarditi ⁇ virus
  • Vectors encoding the subject Factor VIII:C analogs can also be packaged in liposomes prior to delivery to the subject or to cells 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:micromoles lipid) , or more of lipid.
  • Liposomal preparations for use in the instant invention include cationic (positively charged) , anionic (negatively charged) and neutral preparations, with cationic liposomes particularly preferred.
  • Cationic liposome ⁇ have been ⁇ hown to mediate intracellular delivery of plasmid DNA (Feigner et al . , Proc . Natl .
  • Cationic liposomes are readily available.
  • N[1-2, 3-dioleyloxy)propyl]-N,N,N-triethyl- ammonium (DOTMA) liposomes are available under the trademark Lipofectin, from GIBCO BRL, Grand Island, NY.
  • liposome ⁇ examples include tran ⁇ fectace (DDAB/DOPE) and DOTAP/DOPE (Boerhinger) .
  • DOTAP/DOPE tran ⁇ fectace
  • Other cationic liposomes can be prepared from readily available materials 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 description of the synthe ⁇ i ⁇ of DOTAP (1,2-bis(oleoyloxy) -3-(trimethylammonio)propane) liposomes.
  • anionic and neutral liposomes are readily available, such as from Avanti Polar Lipids (Birmingham, AL) , or can be easily prepared u ⁇ ing readily available material ⁇ .
  • material ⁇ include phosphatidyl choline, cholesterol, pho ⁇ phatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC) , dioleoylphosphatidyl glycerol (DOPG) , dioleoylphoshatidyl ethanolamine (DOPE) , among others.
  • DOPC dioleoylphosphatidyl choline
  • DOPG dioleoylphosphatidyl glycerol
  • DOPE dioleoylphoshatidyl ethanolamine
  • These materials can also be mixed with the DOTMA and DOTAP starting materials in appropriate ratios. Methods for making liposomes using these materials are well known in the art.
  • the liposome ⁇ can compri ⁇ e multilammelar vesicles (MLVs) , ⁇ mall unilamellar vesicles (SUVs) , or large unilamellar vesicles (LUVs) .
  • MLVs multilammelar vesicles
  • SUVs ⁇ mall unilamellar vesicles
  • LUVs large unilamellar vesicles
  • the various liposome- nucleic acid complexes are prepared using methods known in the art. See, e.g., Straubinger et al . , in METHODS OF IMMUNOLOGY (1983), Vol. 101, pp. 512-527; Szoka et al . , Proc . Natl . Acad . Sci . USA (1978) 75:4194-4198; Papahadjopoulos et al .
  • the recombinant vectors may be administered in pharmaceutical compositions as de ⁇ cribed 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 constructs in the individual to which it is administered.
  • the compositions of the invention can be administered directly to the subject or, alternatively, in the case of the vectors described above, delivered ex vivo , to cells derived from the subject. Methods for the ex vivo delivery and reimplantation of transformed cells into a subject are known in the art and described in e.g., International Publication No. WO 93/14778 (published 5 August 1993) .
  • Such methods will include dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, eiectroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei, all well known in the art.
  • Direct delivery of the compositions will generally be accomplished by injection, either subcutaneou ⁇ ly, intraperitoneally, intravenously or intramuscularly.
  • Other modes of administration include oral and pulmonary administration, suppositories, and transdermal application ⁇ .
  • Do ⁇ age treatment may be a ⁇ ingle do ⁇ e schedule or a multiple dose ⁇ chedule.
  • the present invention will now be illu ⁇ trated by reference to the following examples which set forth particularly advantageou ⁇ embodiment ⁇ .
  • these embodiments are illustrative and are not to be construed as restricting the invention in any way.
  • the Factor VIII:C polypeptide analogs are made using conventional mutagenesi ⁇ techniques.
  • mutagenesis of the Factor VIII:C nucleotide sequence ⁇ can be performed utilizing plasmids which include sequence ⁇ encoding the full-length molecule, plasmids encoding the light and heavy chains and various modifications of these molecules, depending on the Factor VIII:C analog desired.
  • Such plasmid ⁇ are known and de ⁇ cribed in, e.g., U.S. Patent no.
  • the plasmids are first linearized e.g., by using restriction endonucleases which cleave at unique restriction site ⁇ . Once linearized, the plasmids are treated with calf intestine phosphatase and separated on low melting temperature tris-acetate agarose gels. The linearized band is extracted by adsorption to silica dioxide and eluted in tris-EDTA. The pla ⁇ mid is then denatured and the desired phosphorylated mutagenic oligonucleotide i ⁇ added. The mixture is 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 triphosphate, 3-4 units/ ⁇ L of Klenow fragment of E. coli DNA polymera ⁇ e I and 400 unit ⁇ / ⁇ L of T4 DNA ligase.
  • the reactions are terminated using phenolchloroform extraction and ethanol precipitation.
  • DNA obtained is used to transform bacterial host cells and positive clones selected.
  • DNA from the clones is transferred to nitrocellulo ⁇ e, filters prepared, and hybridized to screening probes to ensure that the mutagenic oligonucleotide is introduced into the correct fragment.
  • Final mutations are confirmed by DNA sequencing.
  • the DNA can be prepared by banding in C ⁇ Cl and can be u ⁇ ed to tran ⁇ fect COS-l monkey cell ⁇ a ⁇ de ⁇ cribed in Kaufman, PNAS (1982) 82:689. After transfection, the polypeptide analog is isolated and Factor VIII:C activity is assayed by the Kabi Coatest chromagenic assay method for the ability to clot Factor VIII deficient plasma before and after thrombin activation.
  • Residue 221 of Factor VIII:C is mutated using the oligonucleotide TTC ATG CAG GAT AGG CCX GCT GCA TCT GCT CGG.
  • the GAT encoding for Asp which normally occurs at the underlined position is mutated to form the codon for Pro which can be CCX where X is A, T, G or C.
  • the mutagenesis i ⁇ carried out, the correct sequence is confirmed, and the analog produced and tested for activity, as described above.
  • Factor VIII:C polypeptide analogs can be constructed and assayed as described above.

Abstract

Factor VIII:C polypeptide analogs are provided that are native Factor VIII:C polypeptides that contain amino acid modifications at one or more amino acid residues adjacent an Arg residue, provided that the Arg residue is not at a Factor VIII:C activation site. Such modifications create one or more Arg-Pro or Pro-Arg linkages. Nucleic acid molecules encoding Factor VIII:C polypeptide analogs, vectors and host cells containing such nucleic acid molecules are also provided. Additional modifications include the creation of a tripeptide having the formula P3-P2-P1, wherein P3 is a residue selected from the group consisting of Phe, Glu, and Pro; P2 is any amino acid residue except Ser and P2 is not Leu335 and is not Asn1720; and P1 is Arg. Other modifications include substitutions at the non-activating Arg residues occurring at positions Arg336, Arg1719 and/or Arg1721. Further provided are analog complexes that contain at least two such analogs. Methods of producing the analogs, the analog complexes, nucleic acids encoding the same, vectors, and host cells are also provided as well as methods of using such compositions for prevention or treatment of active Factor VIII:C polypeptide deficiencies.

Description

NOVEL FACTOR VHIrC POLYPEPTIDE ANALOGS WITH ALTERED PROTEASE SITES
Field of the Invention This invention relates to the discovery that an active Factor VIII.C polypeptide analog can be made that is modified at a site adjacent to an arginine ("Arg") residue, where the Arg is at a site other than at an activating site, creating an arginine-proline ("Arg-Pro") linkage or a proline-arginine ("Pro-Arg") linkage. This invention also relates to a Factor V II.C polypeptide analog with a modification that comprises creation of a tripeptide having the formula P3-P2~pi wherein P3 is a residue selected from the group consisting of Phe, Glu, and Pro; P2 is any amino acid residue except Ser and P2 is not Leu335 and is not Asnl720; and P is Arg. Additionally, the invention pertains to substitutions at the non-activating Arg residues occurring at positions Arg336, Argl719 and/or Argl721. This invention further relates to an analog complex 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.
Background of the Invention 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 10,000 males. 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 consiεtε of the heavy and light chains asεociated by a metal ion bridge, and lacking amino acidε 741-1648 (the B domain) .
The mature Factor VIII.C molecule conεiεts 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 NH2-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. During activation, the mature Factor VIII.C polypeptide is cleaved to generate heavy and light chain fragments that are further cleaved. For example, cleavage of the light chain after arginine residue 1689 ("Argl689") yields a light chain fragment of about 73 kD ("the 73 kD fragment") , and cleavage of the heavy chain after arginine residue 372
("Arg372") yields smaller heavy chain fragments of about 50 kD and 43 kD ("the 50 kD and 43 kD fragments," respectively), as deεcribed in Eaton et al . (1986), Biochem . 25 : 505-512. At a minimum, the complex formed by the 50 kD and 73 kD polypeptides appears to be required for Factor VIII.C coagulant activity. Following activation, the heavy and light chain fragments of Factor VIII:C are inactivated by plasma proteases.
Patients suffering from hemophilia A are conventionally treated with purified or substantially purified Factor VlllrC. A difficulty in such treatment is the relatively short half-life of externally administered Factor VIII.C, lasting about 8 to 12 hours. This instability of Factor VIII:C derives in part from its susceptibility to proteolytic cleavage by plasma protease. Such plasma proteases, for example, thrombin, Factor Xa, and activated protein C ("APC") , inactivate Factor VIII.C by cleaving the molecule at multiple sites. It would be advantageous, therefore, to produce Factor VIII:C polypeptides with improved properties.
Summary of the Invention It is, therefore, an object of the present invention to provide a Factor VIII:C polypeptide analog that has improved properties.
In accordance, therewith, there is provided an active Factor VIII.C polypeptide analog that is substantially the same as a native Factor VIII.C polypeptide, except for modification at a site that is adjacent to a non-activating Arg residue, that is, adjacent to an Arg residue that is not at an activation site, such as Argl689 or Arg372. The modification of the present invention creates an Arg-Pro linkage or a Pro-Arg linkage. Such a modification can be achieved by one or more amino acid substitutions, additions or deletions. In accordance with another object of the present invention, there is provided an analog as above where the non-activating Arg residue is at least one selected from the group consisting of amino acid residues 220, 226, 250, 279, 282, 336, 359, 562, 747, 776, 1310, 1313, 1645, 1648, 1719, and 1721, numbered with respect to the native Factor VIII:C polypeptide sequence, as depicted in Figures 1A-1F.
In accordance with another object of the present invention, there is provided an analog with a modification that comprises creation of a tripeptide having the formula P3~P2 -pι# wherein P3 is a residue selected from the group consisting of Phe, Glu, and Pro; P2 is any amino acid residue except Ser and P2 is not Leu335 and is not Asnl720; and Pλ is Arg.
In accordance with another object of the invention, there is provided an an active Factor VIII:C polypeptide analog comprising a native Factor VIII.C polypeptide that is modified at at least one non- activating Arg residue selected from the group consisting of Arg336, Argl719 and Argl721, wherein the modification comprises a substitution of any of amino acids Pro, Glu, Asp, Asn, Gin, Ser and Tyr for Arg336, a substitution of any of amino acids Pro, Glu, Asp, Asn, Gin, Ser and Tyr for Argl719 and/or a substitution of any of amino acids Glu, Asp, Asn, Gin, Ser and Tyr for Argl721, numbered with respect to the native Factor VIII:C polypeptide sequence.
In accordance with a further object of the present invention, there is provided an analog as above where 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 molecular weight of about 90 kD; (g) a cleaved
Factor VIII.C molecule containing a heavy chain fragment of molecular weight of about 50 kD, (h) a cleaved Factor VIII.C molecule containing a heavy chain fragment of molecular weight of about 43 kD; and (i) a cleaved Factor VIII.C molecule containing a light chain fragment of molecular weight of about 73 kD.
In accordance with a further object of the present invention, there is provided an active Factor VIII.C analog complex that contains either at least two Factor VIII.C polypeptide analogs as above, or a Factor VIII.C polypeptide analog and a Factor VIII.C polypeptide, together with a metal ion. For example, the analog complex herein can comprise two Factor VIII.C 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.
In accordance with yet another object of the present invention, there is provided a method of producing a Factor VIII.C 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 reεidue in the amino acid εequence to produce an analog as above.
In accordance with still another object of the present invention, there is provided a nucleic acid molecule that contains a nucleotide sequence that encodes an analog as above.
In accordance with another object of the present invention, there is provided 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.
In accordance with yet another object of the present invention, there is provided a recombinant host cell that contains a nucleic acid molecule or recombinant vector as above.
There is also provided, in accordance with another object of the present invention, a method of producing an active Factor VIII.C polypeptide analog as above, the method comprising: (a) providing the recombinant host cell as above, and (b) allowing the recombinant host cell to express the analog.
There is further provided, in accordance with another object of the present invention, a method of producing a nucleic acid molecule as above, comprising: (a) providing a nucleic acid molecule that encodes a native Factor VIII:C polypeptide as above and (b) modifying at least one codon to provide the analog. There is also provided, in accordance with another object of the present invention, a method of producing a recombinant vector that contains a nucleic acid molecule as above, comprising linking a regulatory element to the nucleic acid molecule. In accordance with a further object of the present invention, there is provided 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 a recombinant vector.
There is also provided, in accordance with an object of the present invention, a pharmaceutical composition that contains an active Factor VIII:C polypeptide analog or analog complex as above, and a pharmaceutically acceptable excipient.
Furthermore, there is also provided, in accordance with another object of the present invention, methods for prevention or treatment of active Factor VIII:C deficiency in a mammal comprising administering thereto a therapeutically effective amount of (a) an active Factor VIII:C polypeptide analog as above, or (b) an active Factor VIII:C polypeptide analog complex as above, or (c) a nucleic acid molecule as above, or (d) a recombinant vector as above, or (e) a nucleic acid molecule as above together with and an active Factor VIII:C polypeptide analog, or (f) a recombinant vector as above together with an active Factor VIII:C polypeptide analog.
Further objects, features, and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description, while indicating the preferred embodiments of the invention, is given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
Brief Description of the Figure Figures 1A-1F depict the amino acid sequence of native Factor VIII:C.
Detailed Description of the Invention The invention described herein draws on previously published work and pending patent applications. By way of example, such work consists of scientific papers, patents or pending patent applications. All of these publications and applications, cited previously or below are hereby incorporated by reference.
The inventors herein have discovered that Factor VIII:C polypeptide analogs can be made that have improved properties. These analogs include one or more amino acid residues that are modified from the native structure. The modification can be at least one amino acid substitution, addition or deletion, at an amino acid residue adjacent to an Arg residue so as to generate, for example, an Arg-Pro linkage or a Pro-Arg linkage.
Furthermore, the Factor VIII:C polypeptide analog can also include a modification that comprises creation of a tripeptide having the formula P3 _P2~pι, wherein P3 is a residue selected from the group consisting of Phe, Glu, and Pro; P2 is any amino acid residue except Ser and P2 is not Leu335 and is not Asnl720; and P2 is Arg. The above modifications do not occur at a site of Factor VIII:C activation. Additionally, the non-activating Arg residues, Arg336 and Argl719 can be substituted with amino acids Pro, Glu, Asp, Asn, Gin, Ser and Tyr and/or the non-activating Arg residue Argl721 can be substituted with Glu, Asp, Asn, Gin, Ser and Tyr, to impart a Factor VIII:C analog with improved properties.
Definitions
The term "Factor VIII:C polypeptide" refers to a polymer of amino acids and does not refer to a specific length of the product. Thus, 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 subεtituted 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 VIII:C molecule comprising all A, B, and C domains and lacking the signal peptide; (c) a truncated Factor VIII:C molecule lacking the signal peptide and at least a portion of the B domain; (d) a cleaved Factor VIII:C 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 VIII:C molecule comprising a heavy chain fragment of about 50 kD; (g) a cleaved Factor VIII:C molecule comprising a heavy chain fragment of about 43 kD; and (h) a cleaved Factor VIII:C molecule comprising a light chain fragment of about 73 kD.
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 mutein 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. For example, 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. The term "active" in reference to the polypeptide analogs herein refers to biological activity, such as coagulation or pro-coagulation activity. Such activity is measured by 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 pro-coagulation activity displayed by the native molecule, preferably at least about 60% to 80%, and more preferably at least about 90% or more of the coagulation or procoagulation activity displayed by the native Factor VIII:C molecule. A "nucleic acid molecule" as used herein, refers to either RNA or DNA or its complementary strands thereof, that contains a nucleotide sequence.
The term "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. For example, in prokaryotes, such a sequence can include one or more of a promoter sequence, a ribosomal binding site, and a transcription termination sequence. In eukaryotes, for example, such a sequence can include one or more of a promoter sequence, and a transcription termination sequence. If any 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 viruε or viral vector or from a linear or circular plasmid. The term "regulatory control" refers to control of expresεion of a polynucleotide εequence 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.
By "therapeutically 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 VIII:C analogs of the invention may be determined by means known in the art, for example, by using the commercially available Coateεt assay. Preferably, the effective amount is εufficient 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 VIII:C polypeptide analog can be given to an average perεon εuch aε a 70 kg male patient. Alternatively, sufficient Factor VIII:C polypeptide analog or analog complex can be given to establiεh a plasma level of about 0.5 to about 2 U/ml of Factor VIII:C 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.
The term "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. Pharmaceutically acceptable 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 εubstances, 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, polysaccharideε, 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. A thorough discussion of pharmaceutically acceptable excipients iε available in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J. 1991) . Typically, the therapeutic 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 VIII:C polypeptide analogs with improved properties. The analogs include Pro-Arg bonds or Arg-Pro bonds at non- activating Arg residues. Preferably, the modification will not introduce an Arg-Ala, Arg-Met, Arg-Gly, Arg-Ser or Arg-Thr bond at the modified site.
Additionally, the modification can comprise creation of a tripeptide having the formula 3_p2~pi' wherein P3 is a residue selected from the group consisting of Phe, Glu, and Pro; P2 is any amino acid residue except Ser and P2 is not Leu335 and is not Asnl720; and P-L is Arg.
Thus, when the non-activating Arg residue is Arg220, representative modificationε can compriεe an insertion of at least one Pro residue between Asp219 and Arg 220; an insertion of at least one Pro residue between Arg220 and Asp221; a deletion comprising residues Asp221, Ala222, Ala223, Ser224, Ala225, Arg226, Ala227, and Trp228; an insertion of at least one Phe residue between Gln218 and Asp219; an insertion of at least one Glu residue between Gln218 and Asp219; an insertion of at least one Pro residue between Gln218 and Asp219; a deletion comprising residues Thr212, Lys213, Asn214, Ser215, Leu216, Met217, and Gln218; a εubεtitution of at least one Phe residue at Gln218; a substitution of at least one Glu residue at Gln218; and/or a substitution of at least one Pro residue at Gln218.
When the non-activating Arg residue is Arg226, representative modifications can comprise an insertion of at least one Pro residue between Ala225 and Arg226; an insertion of at least one Pro residue between Arg226 and Ala227; a deletion comprising residues Ala227 and Trp228; a deletion comprising residues Ala227 and Trp228 and insertion of at least one Pro residue to replace the deleted residues; a deletion comprising residues Ala227, Trp228, Pro229, and Lys230, and an insertion of at least one Pro residue to replace the deleted residues; an insertion of at least one Phe residue between Ser224 and Ala225; an insertion of at least one Glu residue between Ser224 and Ala225; an insertion of at least one Pro residue between Ser224 and Ala225; (d) a substitution of at least one Phe residue at Ser224; a substitution of at least one Glu residue at Ser224; and/or a substitution of at least one Pro residue at Ser224.
When the non-activating Arg residue is Arg250, representative modifications can comprise an insertion of at least one Pro residue between His249 and Arg250; an insertion of at least one Pro residue between Arg250 and Lys251; a deletion comprising residues Lys251 and Ser252 and insertion of at least one Pro residue to replace the deleted residues; a substitution of Lys251 with at leaεt one Pro residue; a deletion comprising residues Gly244, Leu245, Ile246, Gly247, Cys248, and His249; a deletion comprising residues Arg240, Ser241, Leu242, Pro243, Gly244, Leu245, Ile246, Gly247, Cys248, and Hiε249 and an inεertion of at least one Pro residue to replace the deleted residueε; an inεertion of at leaεt one Phe reεidue between Cyε248 and His249; an insertion of at least one Glu residue between Cys248 and Hiε249; an insertion of at least one Pro residue between Cys248 and Hiε249; a deletion comprising residues Gly244, Leu245, Ile246, Gly247, and Cys248; a εubstitution of at least one Phe residue at Cys248; a subεtitution of at leaεt one Glu residue at Cyε248; and/or a εubstitution of at least one Pro residue at Cyε248.
When the non-activating Arg residue is Arg279, representative modifications can comprise an insertion of at least one Pro residue between Val278 and Arg279; an insertion of at least one Pro residue between Arg279 and Asn280; a deletion comprising residues Asn280, His281, and Arg282 and an insertion of at least one Pro residue to replace the deleted residues; a deletion comprising Asn280, His281, Arg282, Gln283, Ala284, and Ser285, and an insertion of at least one Pro residue to replace the deleted residues; an insertion of at leaεt one Phe reεidue between Leu277 and Val278; an inεertion of at least one Glu residue between Leu277 and Val278; an insertion of at least one Pro residue between Leu277 and Val278; a deletion comprising residue Leu277; a deletion comprising residue Val278; a deletion comprising residues Gly273, His274, Thr275, Phe276, and Leu277; a deletion compriεing residues Leu271, Glu272, Gly273, His274, Thr275, Phe276, and Leu277; a substitution of at least one Phe residue at Leu277; a substitution of at least one Glu residue at Leu277; and/or a substitution of at least one Pro reεidue at Leu277.
When the non-activating Arg residue is Arg282, representative modifications can comprise an insertion of at least one Pro residue between His281 and Arg282; an insertion of at least one Pro residue between Arg282 and Gln283; a deletion comprising residues Arg279, Asn280, and His281 and an insertion of at least one Pro residue to replace the deleted residues; a deletion comprising residues Gln283, Ala284, and Ser285, and an insertion of at least one Pro residue to replace the deleted residueε; a deletion comprising residues Gln283, Ala284, Ser285, Leu286, Glu287, Ile288, and Ser289; an insertion of at least one Phe residue between Asn280 and His281; an insertion of at least one Glu residue between Asn280 and His281; an inεertion of at least one Pro residue between Asn280 and His281; a deletion comprising residues Leu277, Val278, Arg279, and Asn280; a deletion comprising residueε Gly273, His274, Thr275, Phe276, Leu277, Val278, Arg279, and Aεn280; a substitution of at least one Phe residue at Asn280; a substitution of at least one Glu residue at Asn280; and/or a substitution of at least one Pro residue at Asn280.
When the non-activating Arg reεidue is Arg336, representative modifications can compriεe an insertion of at least one Pro residue between Leu335 and Arg336; a deletion comprising residues Gln334, and Leu335; an insertion of at least one Pro residue between Arg336 and Met337; a deletion comprising residues Met337, and Lys338 and insertion of at least one Pro residue in place of the deleted residues; a deletion comprising residue Leu335 and an inεertion of at least one Phe residue between Pro333 and Gln334; a deletion comprising reεidue Leu335 and an inεertion of at least one Glu residue between Pro333 and Gln334; a deletion comprising residue Leu335 and an insertion of at least one Pro residue between
Pro333 and Gln334; a deletion comprising residue Leu335; a deletion comprising residueε Gln334, and Leu335; a deletion comprising residues Pro333, Gln334, and Leu335; a deletion comprising residues Glu332, Pro333, Gln334, and Leu335; a deletion comprising residue Leu335 and a subεtitution of at least one Phe residue at Pro333; and/or a deletion comprising residue Leu335 and a substitution of at least one Glu residue at Pro333.
When the non-activating Arg residue is Arg359, representative modifications can comprise an insertion of at least one Pro residue between Val358 and Arg359; an insertion of at least one Pro residue between Arg359 and Phe360; a deletion comprising residues Phe360, Asp361, Aεp362, Aεp363, Aεn364, and Ser365; a deletion compriεing residues Phe360, Asp361, Aεp362, Aεp363, Asn364, Ser365, Pro366, and Ser367, and an insertion of at least one Pro residue to replace the deleted residues; an insertion of at least one Phe residue between Val357 and Val358; an insertion of at least one Glu residue between Val357 and Val358; an insertion of at least one Pro residue between Val357 and Val358; a deletion comprising residues Met355, Asp356, and Val357; a substitution of at least one Phe residue at Val357; a substitution of at least one Glu residue at Val357; and/or a substitution of at least one Pro residue at Val357.
When the non-activating Arg residue is Arg562, representative modifications can comprise an insertion of at least one Pro residue between Gln561 and Arg562; an insertion of at least one Pro residue between Arg562 and Gly563; a deletion comprising residues Ser558, Val559, Asp560, Gln561, and an insertion of at least one Pro residue to replace the deleted residues; a deletion comprising residues Lys556, Glu557, Ser558, Val559, Asp560, Gln561, and an insertion of at least one Pro residue to replace the deleted residues; a deletion comprising residues Gly563, Asn564, Gln565, Ile566, Met567, and Ser568, and an insertion of at least one Pro residue to replace the deleted residues; a deletion comprising residues Gly563, Asn564, Gln565, Ile566, Met567, Ser568, Asp569, Lys570, and Arg571, and an insertion of at least one Pro residue to replace the deleted residueε; an insertion of at least one Phe residue between Asp560 and Gln561; an inεertion of at leaεt one Glu residue between Asp560 and Gln561; an insertion of at least one Pro residue between Asp560 and Gln56l; a deletion comprising residues Ser558, Val559, and Asp560; a substitution of at least one Phe residue at Asp560; a substitution of at least one Glu residue at Asp560; and/or a subεtitution of at least one Pro residue at Asp560.
When the non-activating Arg residue is Arg747, representative modifications can comprise an insertion of at least one Pro residue between Ser746 and Arg747; a deletion comprising residue Ser746 and an insertion of at least one Pro residue to replace the deleted residue; a deletion comprising residue His748; a deletion comprising residue His748 and an insertion of at least one Pro residue to replace the deleted residue; an insertion of at least one Pro residue between Arg747 and His748; a deletion comprising residues His748, Pro749, and Ser750 and an insertion of at least one Pro residue to replace the deleted residues; a deletion comprising Ser743, Gln744, Asn745, Ser746; a deletion comprising His748, Pro749, Ser750, Thr751, Arg752, Gln753, and Lys754, and an insertion of at least one Pro residue to replace the deleted residues; a deletion comprising residue Ser746 and an insertion of at least one Phe residue between Gln744 and Asn745; a deletion comprising residue Ser746 and an insertion of at least one Glu residue between Gln744 and Asn745; a deletion comprising residue Ser746 and an insertion of at least one Pro residue between Gln744 and Asn745; a deletion compriεing reεidue Ser746 and a substitution of at least one Phe residue at Gln744; a deletion comprising residue Ser746 and a substitution of at least one Glu residue at Gln744; and/or a deletion comprising residue Ser746 and a substitution of at least one Pro residue at Gln744.
When the non-activating Arg residue is Arg776, representative modifications can comprise an insertion of at least one Pro residue between His775 and Arg776; an insertion of at least one Pro residue between Arg776 and Thr777; a deletion compriεing reεidue Thr777; a deletion compriεing reεidue Thr777 and an insertion of at least one Pro residue to replace the deleted residue; a deletion comprising residues Trp772, Phe773, Ala774, and His775; a deletion comprising residues Trp772, Phe773, Ala774, and His775, and an insertion of at least one Pro residue to replace the deleted residues; a deletion comprising residues Lys768, Thr769, Asp770, Pro771,
Trp772, Phe773, Ala774, and His775, and an inεertion of at least one Pro residue to replace the deleted residues; a deletion comprising residues Thr777, Pro778, met779, Pro780, and Lys781, and an insertion of at least one Pro residue to replace the deleted residues; an insertion of at least one Phe residue between Ala774 and His775; an insertion of at least one Glu residue between Ala774 and His775; an inεertion of at leaεt one Pro residue between Ala774 and His775; a deletion comprising residue Ala774; a deletion comprising residues Trp772, Phe773 and Ala774; a deletion comprising residues Lys768, Thr769, Asp770, Pro771, Trp772, and Phe773; a substitution of at least one Phereεidue at Ala774; a εubstitution of at least one Glu residue at Ala774; and/or a substitution of at least one Pro residue at Ala774.
When the non-activating Arg residue is Argl310, representative modifications can comprise an insertion of at least one Pro residue between Glnl309 and Argl310; an insertion of at least one Pro residue between Argl310 and Serl311; a deletion comprising residue Serl311 and an insertion of at least one Pro residue to replace the residue; a deletion comprising residues Ser1311, Lysl312, and Argl313 and an insertion of at least one Pro residue to replace the deleted residues; a deletion comprising residues Serl311, Lysl312, Argl313, Alal314, Leul315, and Lysl316, and an insertion of at least one Pro residue to replace the deleted residues; a deletion comprising residues Serl311, Lysl312, Argl313, Alal314, Leul315, Lysl316, Glnl317, Phel318, Argl319, and Leul320; a deletion comprising residues Serl311, Lysl312, Argl313, Alal314, Leul315, Lysl316, Glnl317, Phel318, Argl319, and Leul320, and an insertion of at least one Pro residue to replace the deleted residues; an insertion of at least one Phe reεidue between Thr1308 and Glnl309; an inεertion of at least one Glu residue between Thr1308 and Glnl309; an insertion of at least one Pro residue between Thrl308 and Glnl309; a deletion comprising residues Vall307, and Thrl308; a substitution of at least one Phe residue at Thrl308; a substitution of at least one Glu residue at Thr1308; and/or a substitution of at least one Pro residue at Thr1308. When the non-activating Arg residue is Argl313, representative modifications can comprise an insertion of at least one Pro residue between Lysl312 and Argl313; a deletion comprising residue Lysl312 and an insertion of at least one Pro residue to replace the deleted residue; a deletion compriεing residues Argl310, Serl311, and Lysl312, and an insertion of at least one Pro residue to replace the deleted residues; an insertion of at leaεt one Pro reεidue between Argl313 and Alal314; a deletion comprising residues Alal314, Leul315, and Lysl316, and an inεertion of at least one Pro residue to replace the deleted residues; a deletion comprising residues Alal314, Leul315, Lysl316, Glnl317, Phel318, and Argl319 and an insertion of at least one Pro residue to replace the deleted residues; a deletion comprising residues Alal314, Leul315, Lysl316, Glnl317, Phel318, Argl319, and Leul320; an insertion of at least one Phe residue between Ser1311 and Lysl312; an insertion of at least one Glu residue between Serl311 and Lysl312; an insertion of at least one Pro reεidue between Serl311 and Lysl312; a deletion comprising residues Vall307, Thrl308, Glnl309, Argl310, and Serl311; a εubεtitution of at least one Phe residue at Ser1311; a substitution of at least one Glu residue at Ser 1311; and/or a substitution of at least one Pro residue at Ser 1311.
When the non-activating Arg reεidue iε Argl645, representative modifications can comprise a deletion comprising residues Vall642, Leul643, and Lysl644; a deleteion compriεing residue Lysl644 and an inεertion of at leaεt one Pro residue to replace the deleted residue; an insertion of at least one Pro residue between Argl645 and Lysl644; an insertion of at least one Pro reεidue between Argl645 and Hisl646; a deletion comprising residues Hisl646, Glnl647, and Argl648 and an inεertion of at least one Pro residue to replace the deleted residues; a deletion comprising residues Hisl646, Glnl647, Argl648, Glul649, Ilel650, Thrl651, and Argl652, and an insertion of at least one Pro residue to replace the deleted residues; an insertion of at least one Phe residue between Leul643 and Lyεl644; an insertion of at least one Glu residue between Leul643 and Lysl644; an insertion of at least one Pro residue between Leul643 and Lysl644; a deletion comprising residues Vall642, and Leul643; a deletion comprising residues Prol641, Vall642, and Leul643; a substitution of at least one Phe residue at Leul643; a substitution of at least one Glu residue at Leul643; and/or a substitution of at least one Pro residue at Leul643.
When the non-activating Arg residue is Argl648, representative modifications include a deletion comprising residues Vall642, Leul643, Lysl644, Argl645, Hisl646, and Glnl647; an insertion of at least one Pro residue between Glnl647 and Argl648; a deletion comprising residues Lysl644, Argl645, Hisl646, and Glnl647 and an insertion of at least one Pro residue to replace the deleted residues; a deletion comprising residues Glul649, Ilel650, Thrl651, and Argl652 and an insertion of at least one Pro residue to replace the deleted residues; an insertion of at least one Pro residue between Argl648 and Glul649; an insertion of at least one Phe residue between Hisl646 and Glnl647; an insertion of at least one Glu residue between Hisl646 and Glnl647; an inεertion of at least one Pro residue between Hisl646 and Glnl647; a deletion comprising residues Vall642, Leul643, Lysl644, Argl645, and Hisl646; a deletion comprising residues Prol641, Vall642, Leul643, Lysl644, Argl645, and Hisl646; a substitution of at least one Phe residue at Hisl646; (g) a substitution of at least one Glu reεidue at Hisl646; and/or a subεtitution of at leaεt one Pro residue at Hisl646.
When the non-activating Arg residue is Argl719, representative modifications a deletion comprising residues Hisl716, Vall717, and Leul718; an inεertion of at least one Pro residue between Leul718 and Argl719; a deletion comprising residues Asnl720, and Argl721 and an inεertion of at least one Pro residue to replace the deleted residues; an insertion of at least one Pro residue between Argl719 and Asnl720; an insertion of at least one Phe residue between Vall717 and Leul718; an insertion of at least one Glu residue between Vall717 and Leul718; an insertion of at least one Pro residue between Vall717 and Leul718; a deletion comprising residueε
Hisl716, and Vall717; a substitution of at least one Phe residue at Vall717; a substitution of at least one Glu residue at Vall717; and/or a substitution of at least one Pro residue at Vall717. When the non-activating Arg residue is Argl721, representative modifications include a deletion comprising residues Hisl716, Vall717, Leul718, Argl719, and Asnl720; an insertion of at least one Pro residue between Aεnl720 and Argl721; an insertion of at least one Pro residue between Argl721 and Alal722; a deletion comprising residues Alal722, Glnl723, and Serl724 and an insertion of at least one Pro residue to replace the deleted residues; a deletion comprising residues Alal722, Glnl723, Serl724, Glyl725, Serl726, and Vall727; a deletion comprising residues Alal722, Glnl723, Serl724, Glyl725, and Serl726, and an insertion of at least one Pro residue to replace the deleted residues; a deletion comprising residue Asnl720 and an insertion of at least one Phe residue between Leul718 and Argl719; a deletion compriεing residue Asnl720 and an insertion of at least one Glu reεidue between Leul718 and Argl719; a deletion comprising residue Asnl720 and an insertion of at least one Pro residue between Leul718 and Argl719; a deletion comprising residues Vall717, Leul718, Argl719, and Asnl720; a deletion compriεing residue Asnl720 and a substitution of at least one Phe residue at Leul718; a deletion comprising residue Asnl720 and a substitution of at least one Glu residue at Leul718; and/or a deletion comprising residue Asnl720 and a εubεtitution of at least one Pro residue at Leul718.
Other polypeptide analogs contemplated by the present invention are thoεe analogs including substitutions of the non-activating Arg residues found at positions Arg336, Argl719 and Argl721, numbered with respect to the native Factor VIII:C polypeptide εequence. For example, based on the model of Factor VIII:C described in Pan et al., Nature Structural Biology (1995) 2:740-744, any of amino acids Pro, Glu, Asp, Asn, Gin, Ser and Tyr can be substituted at position Arg336 and/or position Argl719, to generate a Factor VIII:C polypeptide analog with improved propertieε. Similarly, any of amino acids Glu, Asp, Asn, Gin, Ser and Tyr can be substituted at position Argl721. Preferably, the Factor VIII:C polypeptide analog of this embodiment will include a substitution of Arg336 with Pro, a substitution of Arg 1719 with Pro and/or a substitution of Argl721 with Glu. Nucleic acid molecules encoding the present
Factor VIII:C polypeptide analogs can be made by modifying the native nucleic acid sequences that encode the Factor VIII:C polypeptide or cDNA sequences that encode the Factor VIII:C polypeptides. Such modification can be done by conventional techniques such as site- directed mutagenesis. For example, the M13 method for site directed mutageneεis is known, as described in Zoller and Smith, Nucleic Acids Reε . (1982) 10: 6487- 6500, Methods Enzymol . (1983) 100: 468-500, and DNA (1984) 3: 479-488, using single stranded DNA, and the method of Morinaga et al . Bio/technol . 636-639 (July 1984) , using heteroduplexed DNA. According to the method of the invention, by site-directed mutagenesiε, one or more of the codons encoding a residue adjacent to an arginine, preferably, at the carboxy side, can be mutated by substitution, deletion or addition, to a codon encoding proline, thereby creating polypeptides with either Pro-Arg bonds or Arg-Pro bonds where none existed before, provided that the modification does not affect an activation cleavage site, such as Arg372 and Arg740. The codons encoding proline include CCU, CCT, CCG, CCA, and CCC. A description of a protocol suitable for use herein for mutagenesis of specific sites of a Factor VIII:C expression plasmid can be found in WO 87/07144.
The nucleic acid moleculeε of the preεent invention can alεo be made εynthetically by piecing together nucleic acid molecules encoding heavy and light chain fragments derived from cDNA clones or genomic clones containing Factor VIII:C coding sequences, preferably cDNA clones, using known linker sequences. Alternatively, the entire sequence or portions of nucleic acid sequenceε encoding analogs described above may be prepared by synthetic methods (e.g. using DNA syntheεiε 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.
One skilled in the art of DNA cloning and in possesεion of the DNA encoding native Factor VIII:C polypeptide will be able to prepare suitable DNA molecules for production of the present analogs using known cloning procedureε (e.g. restriction enzyme digestion, exonuclease digestion, ligation, and other appropriate procedures) outlined in any of the following: Sambrook, et al , MOLECULAR CLONING: A LABORATORY MANUAL 2nd ed. (Cold Spring Harbor Laboratory Press, 1989) ; DNA CLONING, Vol. I and II, D.N. Glover ed. (IRL Presε, 1985); OLIGONUCLEOTIDE SYNTHESIS, M.J. Gait ed. (IRL Preεε, 1984); NUCLEIC ACID HYBRIDIZATION, B.D. Hames & S.J. Higgins edε. (IRL Preεε, 1984); TRANSCRIPTION AND TRANSLATION, B.D. Hames & S.J. Higgins eds., (IRL Press, 1984); ANIMAL CELL CULTURE, R.I. Freshney ed. (IRL Preεε, 1986); IMMOBILIZED CELLS AND ENZYMES, K. Mosbach (IRL Presε, 1986); B. Perbal, A PRACTICAL GUIDE TO MOLECULAR CLONING, Wiley (1984); the εeries, METHODS IN ENZYMOLOGY, Academic Presε, Inc. ; GENE TRANSFER VECTORS FOR MAMMALIAN CELLS, J.H. Miller and M.P. Caloε edε. (Cold Spring
Harbor Laboratory, 1987); METHODS IN ENZYMOLOGY, Vol. 154 and 155, Wu and Grossman, eds., and Wu, ed. , respectively (Academic Press, 1987) , IMMUNOCHEMICAL METHODS IN CELL AND MOLECULAR BIOLOGY, R.J. Mayer and J.H. Walker, eds. (Academic Press London, Harcourt Brace U.S., 1987), PROTEIN PURIFICATION: PRINCIPLES AND PRACTICE, 2nd ed. (Springer-Verlag, N.Y. (1987), and HANDBOOK OF
EXPERIMENTAL IMMUNOLOGY, Vol. I-IV, D.M. Weir et al , (Blackwell Scientific Publications, 1986); Kitts et al , Biotechniques, (1993) , 14:810-817; Munemitsu et al, Mol . Cell . Biol . , (1990) 10:5977-5982. Finally, a preferred method of preparing nucleic acid molecules encoding the described analogs is by use of PCR techniques, especially overlapping PCR, as described in PCR PROTOCOLS: A GUIDE TO METHODS AND APPLICATIONS, Innis, Gelfand, Sninsky, and White (eds.) (Academic Press, 1990) . 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 siteε 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 elements or control sequences for expression of the nucleic acid molecule. For example, the present vector can be derived from a plasmid, a virus, a cosmid, or a bacteriophage. This vector is typically capable of behaving as an autonomous unit of replication when introduced into a host cell. Moreover, the vector may be one that is capable of epiεomal existence or of integration into the host cell genome. A wide variety of replication systems are available, typically derived from viruses that infect mammalian host cells. Illustrative replication syεtems include the replication systems from Simian virus 40, adenoviruε, bovine papilloma viruε, polyoma viruε, Epεtein Barr viruε, and the like. Thus, the nucleic acid molecule of the present invention can be inserted at an appropriate restriction 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 used for transfection or transformation of a host cell. The host cells of the invention can be, for example, prokaryotic or eukaryotic host cellε, including bacterial, yeast, insect and mammalian expression syεtems. Preferably the analogs of the present invention are expressed in mammalian host cell systems. The regulatory elements to be used in the vector depend on the host system that is to be utilized. For example, a prokaryotic host cell can be used for amplification of the nucleic acid molecule of the present invention, while an eukaryotic host cell can be used for expression of the Factor VIII:C polypeptide analogs.
The expression cassettes are introduced into the host cell by conventional methods, depending on the expression system used, as deεcribed further below. Where viruses are involved, transfection or transduction may be employed. The particular manner in which the host cell is transformed is not critical to this invention, depending substantially upon whether the expression cassettes are joined to a replication system and the nature of the replication system and associated genes. Coexpresεion of more than one Factor VIII:C polypeptide analog may be deεired. For example, it may be deεirable to expreεε the light and heavy chainε uεing εeparate constructs. In this regard, either or both of the light and heavy chains may include modifications as described above. "Coexpression" as used herein refers to the expression of two or more Factor VIII:C polypeptides in a εingle hoεt cell. Thus, for example, the expression of the 90 kD specieε and the 80 kD species in a single host cell, would constitute "coexpression" as used herein. The polynucleotides encoding for the polypeptides can be harbored in a single vector, either under the control of the same regulatory elements or under the control of separate elements. Thus, the production of a fusion protein including active portions of the two or more Factor VIII:C polypeptides would be conεidered "coexpressed" for purposes of the present definition as would the expression of two genes as a dicistronic construct employing an internal ribosome entry site. Similarly, proteins expresεed from the same vector but driven by separate regulatory elements, would also be considered "coexpressed." The term also refers to the expresεion of two or more proteins from separate constructs. Thus, the expression of proteins encoded from genes present on separate vectors in a host cell would also be considered "coexpression" for purposes of the present invention. The transformed/transfected cells 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, so that the media or cell lysate may be isolated and the Factor
VIII:C active complex extracted and purified. Similarly, the full-length molecule can be isolated and treated under complex-forming conditions, e.g., with the addition of calcium and the appropriate enzymes, to form the active complex. Various means are available for extraction and purification, such as affinity chromatography, ion exchange chromatography, hydrophobic chromatography, electrophoresis, solvent-εolvent extraction, selective precipitation, and the like. The particular manner in which the product is 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. Expression in Bacterial Cells
Bacterial expression systems can be used to produce the subject Factor VIII:C polypeptide analogε and nucleic acid sequences encoding the analogs. Control elements for use in bacterial systems include promoters, optionally containing operator εequences, and ribosome binding sites. Useful promoters include sequences derived from sugar metabolizing enzymes, such as galactose, lactose (lac) and maltose. Additional examples include promoter sequences derived from biosynthetic enzymes εuch as tryptophan (trp) , the β- lactamase (Jla) promoter system, bacteriophage λPL, and T7. In addition, synthetic promoters can be used, such as the tac promoter. The β-lactamase and lactose promoter syεtems are described in Chang et al . , Nature (1978) 275 : 615, and Goeddel et al . , Nature (1979) 281 : 544; the alkaline phosphatase, tryptophan (trp) promoter system are described in Goeddel et al . , Nucleic Acidε Reε . (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. However, other known bacterial promoterε uεeful for expression of eukaryotic proteins are also suitable. A person skilled in the art would be able to operably ligate such promoters to the present Factor VIII:C polypeptide analog coding sequences, for example, as described in Siebenlist et al . , Cell (1980) 20 : 269, uεing linkers or adapters to εupply any required reεtriction εites. Promoters for use in bacterial systemε alεo generally will contain a Shine-Dalgarno (SD) sequence operably linked to the DNA encoding the Factor VIII:C analog polypeptide. For prokaryotic host cells that do not recognize and procesε the native polypeptide εignal sequence, the signal sequence can be substituted by a prokaryotic signal sequence selected, 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.
The foregoing systems are particularly compatible with Eεcherichia coli . However, numerous other systems for use in bacterial hosts including Gram- negative or Gram-positive organisms such as Bacilluε εpp. , Streptococcus εpp. , Streptomyceε εpp . , Pseudomonas species such as P . aeruginoεa, Salmonella typhimurium, or Serratia marceεcans, among others. Methods for introducing exogenous DNA into these hosts typically include the use of CaCl2 or other agents, such as divalent cations and DMSO. DNA can also be introduced into bacterial cellε by eiectroporation, nuclear injection, or protoplast fusion as described generally in Sambrook et al . (1989), cited above. These examples are illustrative rather than limiting. Preferably, the host cell εhould secrete minimal amounts of proteolytic enzymes. Alternatively, in vitro methods of cloning, e.g., PCR or other nucleic acid polymerase reactions, are suitable.
Prokaryotic cells used to produce the Factor VIII:C analog polypeptides of this invention are cultured in suitable media, as described generally in Sambrook et al . , cited above.
Expreεsion in Yeast Cells
Yeast expression systems can also be used to produce the subject Factor VIII:C polypeptide analogs and nucleic acid sequences encoding the analogs. Expression and transformation vectors, either extrachromosomal replicons or integrating vectors, have been developed for transformation into many yeasts. For example, expresεion vectors have been developed for, among others, the following yeastε: Saccharomyces cerevisiae ,as described in Hinnen et al . , Proc . Natl . Acad . Sci . USA (1978) 75 : 1929; Ito et al . , J . Bacteriol . (1983) 153 : 163; Candida albicanε as described in Kurtz et al . , Mol . Cell . Biol . (1986) 6: 142; Candida maltosa, as described in Kunze et al., J. Basic Microbiol. (1985) 25: 141; Hansenula polymorpha, as described in Gleeson et al., J. Gen. Microbiol. (1986) 132: 3459 and Roggenkamp et al., Mol. Gen. Genet. (1986) 202 :302); Kluyveromyces fragilis, as described in Das et al., J. Bacteriol. (1984) 158: 1165; Kluyveromyces lactis, as described in De Louvencourt et al., J. Bacteriol. (1983) 154: 737 and Van den Berg et al., Bio /Technology (1990) 8: 135; Pichia guillerimondii, as described in Kunze et al . , J. Baεic Microbiol. (1985) 25: 141; Pichia paεtoriε, as described in Cregg et al . , Mol. Cell. Biol. (1985) 5: 3376 and U.S. Patent Nos. 4,837,148 and 4,929,555; Schizoεaccharomyceε pombe, as described in Beach and Nurse, Nature (1981) 300: 706; and Yarrowia lipolytica, as described in Davidow et al . ,
Curr. Genet. (1985) 10: 380 and Gaillardin et al . , Curr. Genet. (1985) 10: 49, Aεpergilluε hosts such as A. nidulans, as described in Ballance et al., Biochem. Biophys. Reε. Commun. (1983) 112: 284-289; Tilburn et al., Gene (1983) 26: 205-221 and Yelton et al . , Proc.
Natl. Acad. Sci. USA (1984) 81: 1470-1474, and A. niger, as described in Kelly and Hynes, EMBO J. (1985) 4: 475479; Trichoderma reeεia, as described in EP 244,234, and filamentous fungi such as, e.g, Neuroεpora, Penicillium, Tolypocladium, as described in WO 91/00357. Control sequences for yeast vectors are known and include promoter regions from genes such as alcohol dehydrogenase (ADH), as described in EP 284,044, enolase, glucokinase, glucose-6-phosphate isomeraεe, glyceraldehyde-3-phosphate-dehydrogenase (GAP or GAPDH) , hexokinase, phosphofructokinase, 3-phosphoglycerate mutase, and pyruvate kinase (PyK) , as described in EP 329,203. The yeast PH05 gene, encoding acid phosphatase, also provides useful promoter sequences, as described in Myanohara et al., Proc. Natl. Acad. Sci. USA (1983) 80:1. Other εuitable promoter sequences for use with yeast hostε include the promoters for 3-phosphoglycerate kinaεe, aε described in Hitzeman et al . , J . Biol . Chem . (1980) 255 : 2073, or other glycolytic enzymes, such as pyruvate decarboxylase, triosephosphate isomerase, and phosphoglucose isomerase, as described in Hess et al . , J. Adv . Enzyme Reg . (1968) 7: 149 and Holland et al . ,
Biochemiεtry (1978; 17 : 4900. Inducible yeast promoters having the additional advantage of transcription controlled by growth conditions, include from the list above and others the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in Hitzeman, EP 073,657. Yeast enhancers also are advantageously used with yeast promoters. In addition, synthetic promoters which do not occur in nature also function aε yeast promoters. For example, upstream activating sequences (UAS) of one yeast promoter may be joined with the transcription activation region of another yeast promoter, creating a synthetic hybrid promoter. Examples of such hybrid promoters include the ADH regulatory sequence linked to the GAP transcription activation region, as described in U.S. Patent Nos. 4,876,197 and 4,880,734. Other examples of hybrid promoters include promoters which consist of the regulatory sequences of either the ADH2 , GAL4 , GAL10 , or PH05 genes, combined with the tranεcriptional activation region of a glycolytic enzyme gene such as GAP or PyK, as described in EP 164,556. Furthermore, a yeast promoter can include naturally occurring promoters of non-yeast origin that have the ability to bind yeast RNA polymerase and initiate transcription. Other control elements which may be included in the yeast expression vectors are terminators, for example, from GAPDH and from the enolase gene, as described in Holland et al . , J . Biol . Chem . (1981) 256 : 1385, and leader sequences which encode signal sequences for secretion. DNA encoding suitable signal sequenceε can be derived from genes for secreted yeast proteins, such as the yeast invertase gene as described in EP 012,873 and JP 62,096,086 and the α-factor gene, as deεcribed in U.S. Patent Nos. 4,588,684, 4,546,083 and 4,870,008; EP 324,274; and WO 89/02463. Alternatively, leaders of non-yeast origin, such as an interferon leader, also provide for secretion in yeast, as described in EP 060,057.
Methods of introducing exogenous DNA into yeast hosts are well known in the art, and typically include either the transformation of spheroplasts or of intact yeast cells treated with alkali cations. Transformations into yeast can be carried out according to the method described in Van Solingen et al . , J . Bact . (1977) 130 : 946 and Hεiao et al . , Proc . Natl . Acad . Sci . USA (1979) 76 : 3829. However, other methods for introducing DNA into cells such as by nuclear injection, eiectroporation, or protoplast fusion may also be used as described generally in Sambrook et al . , cited above.
For yeast secretion the native polypeptide signal sequence may be εubεtituted by the yeast invertase, α-factor, or acid phosphatase leaders. The origin of replication from the 2μ plasmid origin is εuitable for yeaεt. A εuitable selection gene for use in yeast is the trpl gene present in the yeast plasmid described in Kingsman et al . , Gene (1979) 7: 141 or Tεche per et al . , Gene (1980) 10 : 157. The trpl gene provideε a εelection marker for a mutant εtrain of yeaεt lacking the ability to grow in tryptophan. Similarly, Leu2-deficient yeast strains (ATCC 20,622 or 38,626) are complemented by known plasmids bearing the Leu2 Gene. For intracellular production of the present polypeptides in yeast, a sequence encoding a yeast protein can be linked to a coding sequence of a Factor VIII:C polypeptide analog to produce a fusion protein that can be cleaved intracellularly by the yeast cells upon expression. An example, of such a yeast leader sequence iε the yeast ubiquitin gene.
Expression in Insect Cells
Insect expression syεtems can be used to produce the Factor VIII:C polypeptide analogs and nucleic acid sequences encoding the analogs. For example, baculovirus expression vectors (BEVs) are recombinant insect viruses in which the coding sequence for a foreign gene to be expressed is inserted behind a baculovirus promoter in place of a viral gene, e.g., polyhedrin, as described in Smith and Summers, U.S. Pat. No., 4,745,051. An expression construct herein includes a DNA vector useful as an intermediate for the infection or transformation of an insect cell syεtem, the vector generally containing DNA coding for a baculoviruε transcriptional promoter, optionally but preferably, followed downstream by an insect signal DNA sequence capable of directing secretion of a desired 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 VIII:C 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 Orders Lepidoptera, Diptera, Orthoptera, Coleoptera and Hymenoptera including, for example, but not limited to the viral DNAs of Autographo californica MNPV, Bombyx mori NPV, rrichopluεia ni MNPV, Rachlpluεia ou MNPV or Galleria mellonella MNPV, Aedeε aegypti, Drosophila melanogaster, Spodoptera frugiperda , and Trichoplusia ni . Thus, the baculovirus transcriptional promoter can be, for example, a baculovirus immediate-early gene IEI or IEN promoter; an immediate-early gene in combination with a baculovirus delayed-early gene promoter region selected from the group consisting 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 transcriptional enhancer elements. Particularly εuitable for uεe herein is the strong polyhedrin promoter of the baculovirus, which directs a high level of expression of a DNA insert, as described in Friesen et al . (1986) "The Regulation of Baculovirus Gene Expression" in: THE MOLECULAR BIOLOGY OF BACULOVIRUSES (W.Doerfler, ed.); EP 127,839 and EP
155,476; and the promoter from the gene encoding the plO protein, as described in Vlak et al . , J. Gen . Virol . (1988) 69 : 765-776. The plasmid for use herein usually also contains the polyhedrin polyadenylation signal, aε described in Miller et al . , Ann . Rev .
Microbiol . (1988) 42 : 111 and a procaryotic ampicillin- resistance (amp) gene and an origin of replication for selection and propagation in E. coli . DNA encoding suitable signal sequenceε can also be included and is generally derived from geneε for εecreted inεect or baculoviruε proteins, such as the baculovirus polyhedrin gene, as described in Carbonell et al . , Gene (1988) 73 : 409, as well as mammalian signal sequences such as those derived from genes encoding human α-interferon as described in Maeda et al . , Nature (1985) 315 : 592-594; human gastrin-releasing peptide, as described in Lebacq- Verheyden et al . , Mol . Cell . Biol . (1988) 8 : 3129; human IL-2, as described in Smith et al . , Proc . Natl . Acad . Sci . USA (1985) 82 : 8404; mouse IL-3, as described in Miyajima et al . , Gene (1987) 58 : 273; and human glucocerebrosidaεe, as described in Martin et al . , DNA (1988) 7:99. Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar) , Aedes aegypti (mosquito) , Aedes albopictuε (mosquito) , Drosophila melanogaster (fruitfly) , and Bombyx mori host cells have been identified and can be used herein. See, for example, the description in Luckow et al., Bio/Technology(1988) 6 : 47-55, Miller et al . , in GENETIC ENGINEERING (Setiow, J.K. et al . eds.), Vol. 8 (Plenum Publishing, 1986), pp. 277-279, and Maeda et al . , Nature, (1985) 315 : 592-594. A variety of such viral strains are publicly available, e.g., the L-l variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV. Such viruses may be used as the virus for transfection of host cells such as Spodoptera frugiperda cells.
Other baculovirus genes in addition to the polyhedrin promoter may be employed to advantage in a baculovirus expression system. These include immediate-early (alpha) , delayed-early (beta) , late (gamma) , or very late (delta) , according to the phase of the viral infection during which they are expressed. The expression of these genes occurs sequentially, probably as the result of a "cascade" mechanism of transcriptional regulation. Thus, the immediate-early genes are expressed immediately after infection, in the absence of other viral functions, and one or more of the resulting gene products induces transcription of the delayed-early genes. Some delayed-early gene productε, in turn, induce tranεcription of late geneε, and finally, the very late genes are expressed under the control of previously expressed gene products from one or more of the earlier classes. One relatively well defined component of this regulatory cascade is IEI, a preferred immediate-early gene of Autographo californica nuclear polyhedrosis virus (AcMNPV) . IEI is preεεed in the absence of other viral functions and encodes a product that stimulates the transcription of several genes of the delayed-early class, including the preferred 39K gene, as deεcribed in Guarino and Summers, J . Virol . (1986) 57 : 563-571 and J. Virol . (1987) 61 : 2091-2099 as well aε late geneε, as described in Guanno and Summers, Virol . (1988) 162 : 444-451.
Immediate-early genes as described above can be used in combination with a baculovirus gene promoter region of the delayed-early category. Unlike the immediate-early genes, such delayed-early genes require the presence of other viral genes or gene products such as those of the immediate-early genes. The combination of immediate-early genes can be made with any of several delayed-early gene promoter regions such as 39K or one of the delayed-early gene promoters found on the Hindlll fragment of the baculovirus genome. In the present instance, the 39 K promoter region can be linked to the foreign gene to be expresεed εuch that expreεεion can be further controlled by the preεence of IEI, aε described in L. A. Guarino and Summers (1986a) , cited above; Guarino & Summerε (1986b) J. Virol . , (1986) 60 : 215-223, and Guarino et al . (1986c), J . Virol . (1986) 60 : 224-229.
Additionally, when a combination of immediate-early genes with a delayed-early gene promoter region iε uεed, enhancement of the expression of heterologous genes can be realized by the presence of an enhancer sequence in direct cis linkage with the delayed-early gene promoter region. Such enhancer εequences are characterized by their enhancement of delayed-early gene expression in situations where the immediate-early gene or its product is limited. For example, the hr5 enhancer sequence can be linked directly, in cis, to the delayed-early gene promoter region, 39K, thereby enhancing the expression of the cloned heterologous DNA as deεcribed in Guarino and Summerε (1986a), (1986b), and Guarino et al . (1986). The polyhedrin gene iε claεεified as a very late gene. Therefore, transcription from the polyhedrin promoter requires the previous expression of an unknown, but probably large number of other viral and cellular gene products. Because of this delayed expression of the polyhedrin promoter, state-of-the-art BEVs, such as the exemplary BEV syεtem deεcribed by Smith and Summers in, for example, U.S. Pat. No., 4,745,051 will express foreign genes only as a result of gene expreεεion from the reεt of the viral genome, and only after the viral infection is well underway. This represents a limitation to the use of existing BEVs. The ability of the host cell to process newly synthesized proteins decreases as the baculoviruε infection progresses. Thus, gene expression from the polyhedrin promoter occurs at a time when the host cell's ability to process newly synthesized proteins is potentially diminished for certain proteins such as human tissue plasminogen activator. As a consequence, the expression of secretory glycoproteins in BEV systems is complicated due to incomplete secretion of the cloned gene product, thereby trapping the cloned gene product within the cell in an incompletely procesεed form.
While it has been recognized that an insect εignal εequence can be used to express 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 εignal sequence.
An exemplary insect signal sequence suitable herein is the sequence encoding for a Lepidopteran adipokinetic hormone (AKH) peptide. The AKH family consists of short blocked neuropeptides that regulate energy substrate mobilization and metabolism in insectε. In a preferred embodiment, a DNA sequence coding for a Lepidopteran Manduca sexta AKH signal peptide can be used. Other insect AKH signal peptides, such as those from the Orthoptera Schistocerca gregaria locus can also be employed to advantage. Another exemplary insect signal sequence is the sequence coding for Drosophila cuticle proteins such as CPI, CP2, CP3 or CP4.
Currently, the most commonly uεed tranεfer vector that can be used herein for introducing foreign genes into AcNPV is pAc373. Many other vectorε, known to thoεe of εkill in the art, can also be used herein. Materials and methods for baculovirus/insect cell expression syεtems are commercially available in a kit form from companies such as Invitrogen (San Diego CA) ("MaxBac" kit) . The techniques utilized herein are generally known to those skilled in the art and are fully described in Summerε and Smith, A MANUAL OF METHODS FOR BACULOVIRUS VECTORS AND INSECT CELL CULTURE PROCEDURES, Texaε Agricultural Experiment Station Bulletin No. 1555, Texas A&M University (1987); Smith et al . , Mol . Cell . Biol . (1983) 3: 2156, and Luckow and Summers (1989) . These include, for example, the uεe of pVL985 which alterε the polyhedrin εtart codon from ATG to ATT, and which introduces a BamHl cloning site 32 basepairs downstream from the ATT, as described in Luckow and Summers, Virology (1989) 17:31.
Thus, for example, for insect cell expression of the present polypeptides, the desired DNA sequence can be inεerted into the tranεfer vector, uεing known techniqueε. An insect cell host can be cotranεformed with the transfer vector containing the inserted deεired DNA together with the genomic DNA of wild type baculoviruε, uεually by cotranεfection. The vector and viral genome are allowed to recombine resulting in a recombinant virus that can be easily identified and purified. The packaged recombinant virus can be used to infect insect host cells to express a Factor VIII:C polypeptide analog.
Other methods that are applicable herein are the standard methods of insect cell culture, cotranεfection and preparation of plaεmids are set forth in Summers and Smith (1987) , cited above. This reference also pertains to the standard methods of cloning genes into AcMNPV transfer vectors, plasmid DNA isolation, tranεferring genes into the AcmMNPV genome, viral DNA purification, radiolabeling recombinant proteins and preparation of insect cell culture media. The procedure for the cultivation of viruses and cells are described in Volkman and Summers, J . Virol . (1975) 19:820-832 and Volkman, et al . , J . Virol . ( 1976) 19:820-832.
Expression in Mammalian Cells
Mammalian expression systems can also be used to produce the Factor VIII:C polypeptide analogs and nucleic acid sequences encoding the analogs. Typical promoters for mammalian cell expresεion include the SV40 early promoter, the CMV promoter, the mouεe mammary tumor virus LTR promoter, the adenovirus major late promoter (Ad MLP) , and the herpes simplex virus promoter, among others. Other non-viral promoters, such as a promoter derived from the murine metallothionein gene, will also find use in mammalian conεtructs. Mammalian expression may be either constitutive or regulated (inducible) , depending on the promoter. Typically, transcription termination and polyadenylation sequences will also be present, located 3' to the translation stop codon. Preferably, a sequence for optimization of initiation of translation, located 5' to the Factor VIII:C polypeptide analog coding sequence, is also present. Examples of tranεcription terminator/polyadenylation signals include those derived from SV40, as described in Sambrook et al. (1989) MOLECULAR CLONING: A LABORATORY MANUAL, 2d edition, (Cold Spring Harbor Press, Cold Spring Harbor, N.Y.). Introns, containing splice donor and acceptor εiteε, may alεo be designed into the constructs of the present invention. Enhancer elements can also be used herein to increase expresεion levels of the mammalian constructε. Exampleε include the SV40 early gene enhancer, as 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 Virus, as described in Gorman et al . , Proc . Natl . Acad . Sci . USA (1982b) 79 : 6777 and human cytomegalovirus, as deεcribed in Boεhart et al . , Cell (1985) 41 : 521. A leader εequence can also be present which includes a sequence encoding a signal peptide, to provide for the secretion of the foreign protein in mammalian cells. Alternatively, the Factor VIII signal peptide can be used. Preferably, there are processing siteε encoded between the leader fragment and the gene of interest such that the leader sequence can be cleaved either in vivo or in vitro . The adenovirus tripartite leader is an example of a leader sequence that provides for secretion of a foreign protein in mammalian cells.
There exist expreεsion vectors that provide for the transient expresεion in mammalian cellε of DNA encoding the Factor VIII:C analog polypeptideε. In general, transient expresεion involves the use of an expression vector that is able to replicate efficiently in a host cell, such that the host cell accumulates many copies of the expression vector and, in turn, synthesizes high levels of a desired polypeptide encoded by the expression vector. Transient expreεsion systems, comprising a suitable expression vector and a host cell, allow for the convenient positive identification of polypeptides encoded by cloned DNAs, as well as for the rapid screening of such polypeptideε for desired biological or physiological properties. Thus, transient expression systems are particularly useful for purposes of identifying additional polypeptides that have Factor VIII:C-like activity.
Once complete, the mammalian expresεion vectorε can be used to transform any of several mammalian cells. Methods for introduction of heterologous polynucleotides into mammalian cells are known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fuεion, eiectroporation, encapsulation of the polynucleotide(s) in lipoεomes, and direct microinjection of the DNA into nuclei. General aεpectε of mammalian cell hoεt system transformations have been described by Axel in U.S. 4,399,216. A εynthetic lipid particularly uεeful for polynucleotide transfection 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 deεcribed 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, Chinese hamεter ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS) , human hepatocellular carcinoma cells (e.g., Hep G2) , human embryonic kidney cells, baby hamster kidney cells, mouse sertoli cells, canine kidney cells, buffalo rat liver cells, human lung cells, human liver cells, mouse mammary tumor cells, as well as others. The mammalian hoεt cellε used to produce the target polypeptide of this invention may be cultured in a variety of media. Commercially available media such as Ham's FIO (Sigma), Minimal Essential Medium ([MEM], Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ([DMEM1, Sigma) are suitable for culturing the host cells. In addition, any of the media described in Ham and Wallace, Meth . Enz . (1979) 58 : 44, Barnes and Sato, Anal. Biochem . (1980) 102 : 255, U.S. Patent Nos. 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 as culture media for the host cells. Any of these media may be supplemented as necesεary with hormones and/or other growth factors such as insulin, transferrin, or epidermal growth factor, saltε (εuch aε sodium chloride, calcium, magnesium, and phosphate) , buffers (such as HEPES) , nucleosides (such as adenosine and thymidine) , antibiotics (such as Gentamycin(tm) M drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range) , and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those εkilled in the art. The culture conditionε, such as temperature, pH, and the like, are those previously used with the hoεt cell selected for expresεion, and will be apparent to the ordinarily εkilled artiεan. The active Factor VIII:C analogs produced according to the invention have a variety of uses. For example, the analogs can be used as immunogens for the production of antibodies. The analogs can also be used for the treatment of hemophiliacs and other hosts having blood clotting disorders. In this regard, due to their resistance to proteolytic cleavage, the Factor VIII:C analogs may display increased plasma half-life or specific activity. Thus, the analogs may allow for lower dosages or alternative modes of administration and may improve hemostasis in hemophiliacs.
Alternatively, nucleic acid molecules or vectors comprising polynucleotide sequences encoding the Factor VIII:C analogs can be used directly for gene therapy and administered using standard gene delivery protocols. In this regard, the nucleotide sequences encoding the Factor VIII:C analogε can be stably integrated into the host cell genome or maintained on a stable episomal element in the hoεt cell. Methods for gene delivery are known in the art. See, e.g., U.S. Patent No. 5,399,346.
A number of viral based εystems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. A selected gene can be inserted into a vector and packaged in retroviral particles uεing 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 . A number of retroviral systems have been described (U.S. Patent No. 5,219,740; Miller and Rosman, BioTechniques (1989) 7:980-990; Miller, A.D. , Human Gene Therapy (1990) 1:5-14; Scarpa et al . , Virology (1991) 180:849-852; Burns et al . , Proc . Natl . Acad . Sci . USA (1993) 90:8033-8037; and Boris- Lawrie and Temin, Cur . Opin . Genet . Develop . (1993) 3:102-109. A number of adenovirus vectors have also been described. Unlike retroviruses which integrate into the host genome, adenoviruses persist extrachromoεomally thuε minimizing the risks associated with insertional mutagenesis (Haj-Ahmad and Graham, J . Virol . (1986) 57:267-274; Bett et al . , J. Virol . (1993) 67:5911-5921; 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) 4:461-476) .
Additionally, various adeno-associated virus (AAV) vector systems have been developed for gene delivery. Such systems can include control sequences, such as promoter and polyadenylation sites, as well as εelectable markers or reporter genes, enhancer sequenceε, and other control elements which allow for the induction of transcription. AAV vectors can be readily constructed uεing techniqueε well known in the art. See, e.g., U.S. Patent Noε. 5,173,414 and 5,139,941; International Publication Nos. WO 92/01070 (published 23 January 1992) and WO 93/03769 (published 4 March 1993) ; Lebkowski et al . , Molec. Cell . Biol . (1988) 8:3988-3996; Vincent et al . , Vaccines 90 (1990) (Cold Spring Harbor Laboratory Preεε) ; Carter, B.J. Current Opinion in Biotechnology (1992) 3:533-539; Muzyczka, N. Current Topics in Microbiol . and Immunol . (1992) 158:97-129; Kotin, R.M. Human Gene Therapy (1994) 5:793-801; Shelling and Smith, Gene Therapy (1994) 1:165-169; and Zhou et al . , J. Exp. Med . (1994) 179:1867-1875.
Additional viral vectors which will find use for delivering the nucleic acid molecules encoding the Factor VIII:C analog polypeptides for gene transfer include those derived from the pox family of viruseε, including vaccinia virus and avian poxvirus. By way of example, vaccinia virus recombinants expressing the novel Factor VIII:C analogs can be constructed as follows. The DNA encoding the particular analog is first inserted 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 is then used to transfect cells which are simultaneously infected with vaccinia. Homologous recombination serves to insert 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 presence of 5- bromodeoxyuridine and picking viral plaques resistant thereto. A vaccinia based infection/transfection system can be conveniently used to provide for inducible, transient expression of the Factor VIII:C analogs in a host cell. In this system, cells are first infected in vitro with a vaccinia virus recombinant that encodes the bacteriophage T7 RNA polymerase. This polymerase displays exquisite εpecificity in that it only tranεcribeε templates bearing T7 promoters. Following infection, cells are transfected with the polynucleotide of interest, driven by a T7 promoter. The polymerase expresεed in the cytoplaεm from the vaccinia virus recombinant transcribeε the tranεfected DNA into RNA which is then translated into protein by the host translational machinery. The method provides for high level, transient, cytoplasmic production of large quantities of RNA and its translation products. See, e.g., Elroy-Stein and Moss, Proc . Natl . Acad . Sci . USA (1990) 87:6743-6747; Fuerst et al . , Proc . Natl . Acad . Sci . USA (1986) 83:8122-8126.
Alternatively, avipoxviruses, such as the fowlpox and canarypox viruses, can also be used to deliver the Factor VIII:C analog genes. Recombinant avipox viruses, expressing immunogens from mammalian pathogens, are known to confer protective immunity when administered to non-avian species. The use of an avipox vector is particularly desirable in human and other mammalian specieε since members of the avipox genus can only productively replicate in susceptible avian specieε and therefore are not infective in mammalian cellε. Methods for producing recombinant avipoxviruses are known in the art and employ genetic recombination, as described above with respect to the production of vaccinia viruses. 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.
As an alternative approach to infection with vaccinia or avipox virus recombinants, or to the delivery of genes using other viral vectors, an amplification system can be used that will lead to high level expresεion following introduction into host cells.
Specifically, a T7 RNA polymerase 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 expression is under the control of the T7 promoter. Thus, εome of the T7 RNA polymerase generated from translation of the amplification template RNA will lead to tranεcription of the desired gene. Because some T7 RNA polymerase is 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 PCR techniques. However the use of a plasmid is preferred. Since high level expression of T7 RNA polymerase appears to be lethal to host cells, the plasmid should be one where expression of T7 RNA polymerase can be controlled. For example, a lac operator can be engineered distal or proximal (or both) to the T7 promoter. The binding of the preexisting lac represεor in the appropriate bacterial strain would interfere with the transcription of the template by blocking access to the promoter by T7 RNA polymerase. Alternatively, or in combination with the above, a plasmid can be constructed where transcription from a bacterial promoter begins 3' of the T7 gene and continues through the 5' end of the T7 promoter. Such transcription will generate an antisense transcript and reduce or eliminate translation of T7 RNA polymerase RNAs. The second tranεcription unit conεiεting of the T7 promoter preceding the gene of interest can be provided by a separate plaεmid or can be engineered onto the amplification plasmid. Colocalization of the two transcription units iε beneficial for eaεe of manufacturing and enεures that both transcription units will always be together in the cells into which the plasmid is introduced. The T7 RNA polymeraεe plaεmidε may include UTRs which comprise an Internal Ribosome Entry Site (IRES) present in the leader sequenceε of picornaviruεeε εuch aε the encephalomyocarditiε virus (EMCV) UTR (Jang et al. J. Virol . (1989) 63:1651-1660). This εequence serves to enhance expression of sequenceε under the control of the T7 promoter. For a further diεcuεεion of T7 systems and their use for transforming cells, see, e.g., International Publication No. WO 94/26911; Studier and Moffatt, J. Mol . Biol . (1986) 189:113-130; Deng and Wolff, Gene (1994) 143:245-249; Gao et al . , Biochem . Biophys . Reε . Commun . (1994) 200:1201- 1206; Gao and Huang, iVuc. Acids Reε . (1993) 21:2867-2872; Chen et al . , Nuc . Acids Reε . (1994) 22:2114-2120; and U.S. Patent No. 5,135,855.
Vectors encoding the subject Factor VIII:C analogs can also be packaged in liposomes prior to delivery to the subject or to cells derived therefrom. Lipid encapεulation 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:micromoles lipid) , or more of lipid. For a review of the use of liposomes as carriers for delivery of nucleic acids, see, Hug and Sleight, Biochim . Biophys . Acta . (1991) 1097:1-17; Straubinger et al . , in METHODS OF ENZYMOLOGY (1983), Vol. 101, pp. 512-527. Liposomal preparations for use in the instant invention include cationic (positively charged) , anionic (negatively charged) and neutral preparations, with cationic liposomes particularly preferred. Cationic liposomeε have been εhown to mediate intracellular delivery of plasmid 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 transcription factors (Debs et al . , J . Biol . Chem . (1990) 265:10189-10192), in functional form. Cationic liposomes are readily available. For example, N[1-2, 3-dioleyloxy)propyl]-N,N,N-triethyl- ammonium (DOTMA) liposomes are available under the trademark Lipofectin, from GIBCO BRL, Grand Island, NY. (See, also, Feigner et al . , Proc . Natl . Acad . Sci . USA (1987) 84:7413-7416). Other commercially available liposomeε include tranεfectace (DDAB/DOPE) and DOTAP/DOPE (Boerhinger) . Other cationic liposomes can be prepared from readily available materials 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 description of the syntheεiε of DOTAP (1,2-bis(oleoyloxy) -3-(trimethylammonio)propane) liposomes.
Similarly, anionic and neutral liposomes are readily available, such as from Avanti Polar Lipids (Birmingham, AL) , or can be easily prepared uεing readily available materialε. Such materialε include phosphatidyl choline, cholesterol, phoεphatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC) , dioleoylphosphatidyl glycerol (DOPG) , dioleoylphoshatidyl ethanolamine (DOPE) , among others. These materials can also be mixed with the DOTMA and DOTAP starting materials in appropriate ratios. Methods for making liposomes using these materials are well known in the art.
The liposomeε can compriεe multilammelar vesicles (MLVs) , εmall unilamellar vesicles (SUVs) , or large unilamellar vesicles (LUVs) . The various liposome- nucleic acid complexes are prepared using methods known in the art. See, e.g., Straubinger et al . , in METHODS OF IMMUNOLOGY (1983), Vol. 101, pp. 512-527; Szoka et al . , Proc . Natl . Acad . Sci . USA (1978) 75:4194-4198; Papahadjopoulos et al . , Biochim . Biophys . Acta (1975) 394:483; Wilson et al . , Cell (1979) 17 : 11 ) ; Deamer and Bangham, Biochim . Biophys . Acta (1976) 443:629; Ostro et al . , Biochem . Biophys . Res . Commun . (1977) 76:836; Fraley et al . , Proc . Natl . Acad . Sci . USA (1979) 76:3348); Enoch and Strittmatter, Proc . Natl . Acad . Sci . USA (1979)
76:145); Fraley et al . , J. Biol . Chem . (1980) 255:10431; Szoka and Papahadjopoulos, Proc . Natl . Acad . Sci . USA (1978) 75:145; and Schaefer-Ridder et al . , Science (1982) 215:166. The recombinant vectors (whether or not encapsulated in liposomes) , may be administered in pharmaceutical compositions as deεcribed above. The pharmaceutical compositions will comprise sufficient genetic material to produce a therapeutically effective amount of the analog or analogs, as described above. For purposes of the present invention, an effective dose will be from about 0.05 mg/kg to about 50 mg/kg of the DNA constructs in the individual to which it is administered. Once formulated, the compositions of the invention can be administered directly to the subject or, alternatively, in the case of the vectors described above, delivered ex vivo , to cells derived from the subject. Methods for the ex vivo delivery and reimplantation of transformed cells into a subject are known in the art and described in e.g., International Publication No. WO 93/14778 (published 5 August 1993) . Generally, such methods will include dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, eiectroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei, all well known in the art. Direct delivery of the compositions will generally be accomplished by injection, either subcutaneouεly, intraperitoneally, intravenously or intramuscularly. Other modes of administration include oral and pulmonary administration, suppositories, and transdermal applicationε. Doεage treatment may be a εingle doεe schedule or a multiple dose εchedule.
Although any εimilar or equivalent methods and materials may be employed in the practice or testing of the present invention, the preferred methods and materials are now described.
The present invention will now be illuεtrated by reference to the following examples which set forth particularly advantageouε embodimentε. However, it should be noted that these embodiments are illustrative and are not to be construed as restricting the invention in any way. Examples The Factor VIII:C polypeptide analogs are made using conventional mutagenesiε techniques. In this regard, mutagenesis of the Factor VIII:C nucleotide sequenceε can be performed utilizing plasmids which include sequenceε encoding the full-length molecule, plasmids encoding the light and heavy chains and various modifications of these molecules, depending on the Factor VIII:C analog desired. Such plasmidε are known and deεcribed 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 siteε. Once linearized, the plasmids are treated with calf intestine phosphatase and separated on low melting temperature tris-acetate agarose gels. The linearized band is extracted by adsorption to silica dioxide and eluted in tris-EDTA. The plaεmid is then denatured and the desired phosphorylated mutagenic oligonucleotide iε added. The mixture is 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 MgCl2, ImM beta-mercaptoethanol, 400 μM ATP, 100 μM deoxynucleotide triphosphate, 3-4 units/μL of Klenow fragment of E. coli DNA polymeraεe I and 400 unitε/μL of T4 DNA ligase.
The reactions are terminated using phenolchloroform extraction and ethanol precipitation. DNA obtained is used to transform bacterial host cells and positive clones selected. DNA from the clones is transferred to nitrocelluloεe, filters prepared, and hybridized to screening probes to ensure that the mutagenic oligonucleotide is introduced into the correct fragment. Final mutations are confirmed by DNA sequencing. The DNA can be prepared by banding in CεCl and can be uεed to tranεfect COS-l monkey cellε aε deεcribed in Kaufman, PNAS (1982) 82:689. After transfection, the polypeptide analog is isolated and Factor VIII:C activity is assayed by the Kabi Coatest chromagenic assay method for the ability to clot Factor VIII deficient plasma before and after thrombin activation.
Example 1 Conεtruction of a Factor VIII:C Polvpeptide Analog Pro221
Residue 221 of Factor VIII:C is mutated using the oligonucleotide TTC ATG CAG GAT AGG CCX GCT GCA TCT GCT CGG. The GAT encoding for Asp which normally occurs at the underlined position is mutated to form the codon for Pro which can be CCX where X is A, T, G or C. The mutagenesis iε carried out, the correct sequence is confirmed, and the analog produced and tested for activity, as described above.
Other Factor VIII:C polypeptide analogs can be constructed and assayed as described above.
The present invention has been described with reference to specific embodiments. However, this application is intended to cover those changes and substitutions which may be made by those skilled in the art without departing from the spirit and the scope of the appended claims.

Claims

WHAT IS CLAIMED IS:
1. An active Factor VIII:C polypeptide analog comprising a native Factor VIII:C polypeptide that is modified at a site adjacent to a non-activating Arg reεidue, wherein the modification compriεes creation of an Arg-Pro or a Pro-Arg dipeptide.
2. The analog of claim 1, wherein the non- activating Arg residue is an Arg reεidue εelected from the group conεiεting of amino acid reεidueε 220, 226, 250, 279, 282, 336, 359, 562, 747, 776, 1310, 1313, 1645, 1648, 1719, and 1721, numbered with reεpect to the native Factor VIII:C polypeptide εequence.
3. The analog of claim 1, wherein the modification iε an amino acid addition, substitution, or deletion, or a combination thereof.
4. The analog of claim 1, wherein the modification is amino acid substitution and the substitution is of about one to about 7 amino acid residues.
5. The analog of claim 1, wherein the modification is amino acid deletion, and the deletion is of about one to about 7 amino acid residueε.
6. An active Factor VIII:C polypeptide analog comprising a native Factor VIII:C polypeptide that is modified at a site adjacent to a non-activating Arg residue, wherein the modification comprises creation of a tripeptide having the formula P3~P2~Pi wherein P3 is a residue εelected from the group conεiεting of Phe, Glu, and Pro; P2 is any amino acid residue except Ser and P2 is not Leu335 and is not Asnl720; and Px is Arg.
7. The analog of claim 2, wherein the non- activating Arg residue is Arg220, and the modification comprises at least one selected from the group consisting of: (a) an insertion of at least one Pro residue between Asp219 and Arg 220; (b) an insertion of at least one Pro residue between Arg220 and Aεp221; and (c) a deletion comprising residues Asp221, Ala222, Ala223, Ser224, Ala225, Arg226, Ala227, and Trp228.
8. The analog of claim 6, wherein the non- activating Arg residue is Arg220, and the modification comprises at least one selected from the group consisting of: (a) an insertion of at leaεt one Phe residue between Gln218 and Aεp219; (b) an insertion of at least one Glu residue between Gln218 and Asp219; (c) an inεertion of at least one Pro residue between Gln218 and Asp219; (d) a deletion comprising residues Thr212, Lys213, Asn214, Ser215, Leu216, Met217, and Gln218; (e) a substitution of at least one Phe residue at Gln218; (f) a subεtitution of at leaεt one Glu residue at Gln218; and (g) a substitution of at least one Pro residue at Gln218.
9. The analog of claim 2, wherein the non- activating Arg residue is Arg226, and the modification compriseε at leaεt one selected from the group consiεting of: (a) an insertion of at least one Pro residue between Ala225 and Arg226; (b) an insertion of at least one Pro residue between Arg226 and Ala227; (c) a deletion comprising reεidues Ala227 and Trp228; (d) a deletion comprising residues Ala227 and Trp228 and insertion of at least one Pro residue to replace the deleted residues; and (e) a deletion comprising residues Ala227, Trp228, Pro229, and Lys230, and an insertion of at least one Pro residue to replace the deleted residues.
10. The analog of claim 6, wherein the non- activating Arg residue is Arg226, and the modification comprises at least one selected from the group consisting of: (a) an insertion of at least one Phe residue between Ser224 and Ala225; (b) an insertion of at least one Glu residue between Ser224 and Ala225; (c) an insertion of at least one Pro residue between Ser224 and Ala225; (d) a substitution of at least one Phe residue at Ser224; (e) a substitution of at least one Glu residue at Ser224; and (f) a substitution of at least one Pro residue at Ser224.
11. The analog of claim 2, wherein the non- activating Arg residue is Arg250, and the modification comprises at least one selected from the group consisting of: (a) an insertion of at least one Pro residue between Hiε249 and Arg250; (b) an insertion of at least one Pro residue between Arg250 and Lys251; (c) a deletion comprising residues Lys251 and Ser252 and insertion of at least one Pro residue to replace the deleted residues; (d) a substitution of Lys251 with at least one Pro residue; (e) a deletion comprising residues Gly244, Leu245, Ile246, Gly247, Cys248, and His249; and (f) a deletion comprising residues Arg240, Ser241, Leu242, Pro243, Gly244, Leu245, Ile246, Gly247, Cys248, and His249 and an insertion of at least one Pro residue to replace the deleted residues.
12. The analog of claim 6, wherein the non- activating Arg residue is Arg250, and the modification comprises at least one selected from the group consisting of: (a) an insertion of at least one Phe residue between Cys248 and Hiε249; (b) an inεertion of at least one Glu residue between Cyε248 and His249; (c) an insertion of at least one Pro residue between Cys248 and His249; (d) a deletion compriεing residues Gly244, Leu245, Ile246, Gly247, and Cys248; (e) a substitution of at least one Phe reεidue at Cys248; (f) a substitution of at least one Glu residue at Cys248; and (g) a εubεtitution of at leaεt one Pro reεidue at Cys248.
13. The analog of claim 2, wherein the non- activating Arg reεidue iε Arg279, and the modification compriεeε at leaεt one selected from the group consisting of: (a) an insertion of at least one Pro residue between Val278 and Arg279; (b) an insertion of at least one Pro residue between Arg279 and Asn280; (c) a deletion comprising residues Asn280, His281, and Arg282 and an insertion of at least one Pro residue to replace the deleted residues; and (d) a deletion comprising Asn280, His281, Arg282, Gln283, Ala284, and Ser285, and an insertion of at least one Pro residue to replace the deleted residues.
14. The analog of claim 6, wherein the non- activating Arg residue is Arg279, and the modification comprises at least one εelected from the group consisting of: (a) an insertion of at least one Phe residue between Leu277 and Val278; (b) an insertion of at least one Glu residue between Leu277 and Val278; (c) an insertion of at least one Pro residue between Leu277 and Val278; (d) a deletion compriεing residue Leu277; (e) a deletion comprising residue Val278; (f) a deletion comprising residues Gly273, His274, Thr275, Phe276, and Leu277; (g) a deletion comprising residues Leu271, Glu272, Gly273, His274, Thr275, Phe276, and Leu277; (h) a substitution of at least one Phe residue at Leu277; (i) a substitution of at least one Glu residue at Leu277; and (j) a subεtitution of at leaεt one Pro residue at Leu277.
15. The analog of claim 2, wherein the non- activating Arg residue iε Arg282, and the modification comprises at least one selected from the group consiεting of: (a) an insertion of at least one Pro residue between His281 and Arg282; (b) an insertion of at least one Pro residue between Arg282 and Gln283; (c) a deletion comprising residueε Arg279, Asn280, and His281 and an insertion of at least one Pro residue to replace the deleted residues; (d) a deletion comprising residues Gln283, Ala284, and Ser285, and an insertion of at least one Pro residue to replace the deleted residues; and (e) a deletion comprising residues Gln283, Ala284, Ser285, Leu286, Glu287, Ile288, and Ser289.
16. The analog of claim 6, wherein the non- activating Arg residue is Arg282, and the modification compriεeε at leaεt one selected from the group consiεting of: (a) an insertion of at least one Phe residue between Asn280 and His281; (b) an insertion of at least one Glu residue between Asn280 and His281; (c) an insertion of at least one Pro residue between Asn280 and His281; (d) a deletion comprising residueε Leu277, Val278, Arg279, and Aεn280; (e) a deletion comprising residueε Gly273, His274, Thr275, Phe276, Leu277, Val278, Arg279, and Asn280; (f) a substitution of at least one Phe reεidue at Aεn280; (g) a substitution of at least one Glu residue at Asn280; and (h) a substitution of at least one Pro residue at Asn280.
17. The analog of claim 2, wherein the non- activating Arg residue iε Arg336, and the modification comprises at least one selected from the group consisting of: (a) an insertion of at least one Pro residue between Leu335 and Arg336; (b) a deletion comprising residues Gln334, and Leu335; (c) an insertion of at least one Pro residue between Arg336 and Met337; and (d) a deletion comprising residues Met337, and Lys338 and insertion of at least one Pro reεidue in place of the deleted reεidueε.
18. The analog of claim 6, wherein the non- activating Arg reεidue is Arg336, and the modification compriseε at least one selected from the group consiεting of: (a) a deletion compriεing residue Leu335 and an insertion of at leaεt one Phe residue between Pro333 and Gln334; (b) a deletion comprising residue Leu335 and an insertion of at least one Glu residue between Pro333 and Gln334; (c) a deletion comprising residue Leu335 and an insertion of at least one Pro residue between Pro333 and Gln334; (d) a deletion comprising residue Leu335; (e) a deletion comprising residues Gln334, and Leu335; (f) a deletion comprising residues Pro333, Gln334, and Leu335; (g) a deletion comprising residues Glu332, Pro333, Gln334, and Leu335; (h) a deletion comprising residue Leu335 and a substitution of at least one Phe residue at Pro333; and (i) a deletion comprising residue Leu335 and a substitution of at least one Glu residue at Pro333.
19. The analog of claim 2, wherein the non- activating Arg residue iε Arg359, and the modification comprises at least one selected from the group consiεting of: (a) an insertion of at least one Pro residue between Val358 and Arg359; (b) an insertion of at least one Pro residue between Arg359 and Phe360; (c) a deletion comprising residues Phe360, Asp361, Asp362, Asp363, Asn364, and Ser365; and (d) a deletion compriεing residues Phe360, Asp361, Aεp362, Aεp363, Asn364, Ser365, Pro366, and Ser367, and an insertion of at leaεt one Pro reεidue to replace the deleted residues.
20. The analog of claim 6, wherein the non- activating Arg residue is Arg359, and the modification comprises at least one selected from the group consisting of: (a) an insertion of at least one Phe residue between Val357 and Val358; (b) an insertion of at least one Glu residue between Val357 and Val358; (c) an insertion of at least one Pro reεidue between Val357 and Val358; (d) a deletion comprising residues Met355, Asp356, and Val357; (e) a substitution of at least one Phe residue at Val357; (f) a substitution of at least one Glu residue at Val357; and (g) a substitution of at least one Pro residue at Val357.
21. The analog of claim 2, wherein the non- activating Arg residue is Arg562, and the modification comprises at least one selected from the group consisting of: (a) an insertion of at leaεt one Pro residue between Gln561 and Arg562; (b) an insertion of at least one Pro residue between Arg562 and Gly563; (c) a deletion comprising residues Ser558, Val559, Asp560, Gln561, and an insertion of at least one Pro residue to replace the deleted residueε; (d) a deletion compriεing reεidues Lys556, Glu557, Ser558, Val559, Asp560, Gln561, and an inεertion of at least one Pro residue to replace the deleted residues; (e) a deletion comprising residues Gly563, Asn564, Gln565, Ile566, Met567, and Ser568, and an insertion of at least one Pro residue to replace the deleted residueε; and (f) a deletion comprising residues Gly563, Asn564, Gln565, Ile566, Met567, Ser568, Aεp569, Lys570, and Arg571, and an insertion of at least one Pro residue to replace the deleted residues.
22. The analog of claim 6, wherein the non- activating Arg residue is Arg562, and the modification comprises at least one selected from the group consisting of: (a) an insertion of at least one Phe residue between Asp560 and Gln561; (b) an insertion of at least one Glu residue between Asp560 and Gln561; (c) an insertion of at least one Pro residue between Asp560 and Gln561; (d) a deletion compriεing reεidueε Ser558, Val559, and Aεp560;
(e) a εubεtitution of at least one Phe residue at Asp560;
(f) a εubεtitution of at leaεt one Glu reεidue at Aεp560; and (g) a εubεtitution of at least one Pro residue at
Asp560.
23. The analog of claim 2, wherein the non- activating Arg reεidue iε Arg747, and the modification compriεeε at leaεt one selected from the group consiεting of: (a) an inεertion of at leaεt one Pro reεidue between Ser746 and Arg747; (b) a deletion comprising residue Ser746 and an insertion of at least one Pro residue to replace the deleted residue; (c) a deletion comprising residue His748; (d) a deletion compriεing reεidue Hiε748 and an insertion of at least one Pro residue to replace the deleted reεidue; (e) an insertion of at least one Pro residue between Arg747 and His748; (f) a deletion comprising residues His748, Pro749, and Ser750 and an insertion of at least one Pro residue to replace the deleted residues; (g) a deletion comprising Ser743, Gln744, Asn745, Ser746; and (h) a deletion comprising His748, Pro749, Ser750, Thr751, Arg752, Gln753, and Lys754, and an inεertion of at least one Pro residue to replace the deleted residues.
24. The analog of claim 6, wherein the non- activating Arg residue is Arg747, and the modification comprises at least one selected from the group consiεting of: (a) a deletion comprising residue Ser746 and an insertion of at least one Phe residue between Gln744 and Asn745; (b) a deletion comprising reεidue Ser746 and an insertion of at least one Glu residue between Gln744 and Asn745; (c) a deletion comprising residue Ser746 and an insertion of at least one Pro residue between Gln744 and Asn745; (d) a deletion comprising residue Ser746 and a substitution of at least one Phe residue at Gln744; (e) a deletion comprising residue Ser746 and a substitution of at least one Glu residue at Gln744; and (f) a deletion comprising residue Ser746 and a substitution of at least one Pro residue at Gln744.
25. The analog of claim 2, wherein the non- activating Arg residue iε Arg776, and the modification comprises at least one selected from the group conεiεting of: (a) an inεertion of at least one Pro residue between His775 and Arg776; (b) an insertion of at least one Pro residue between Arg776 and Thrill (c) a deletion comprising residue Thrill ; (d) a deletion comprising residue Thrill and an insertion of at least one Pro residue to replace the deleted residue; (e) a deletion comprising reεidues Trp772, Phe773, Ala774, and His775; (f) a deletion compriεing residues Trp772, Phe773, Ala774, and His775, and an insertion of at least one Pro residue to replace the deleted residues; (g) a deletion comprising residues Lys768, Thr769, Asp770, Pro771, Trp772, Phe773, Ala774, and His775, and an insertion of at leaεt one Pro reεidue to replace the deleted reεidues; and (h) a deletion comprising residues Thrill , Pro778, met779, Pro780, and Lys781, and an inεertion of at least one Pro residue to replace the deleted residues.
26. The analog of claim 6, wherein the non- activating Arg residue is Argil 6 , and the modification comprises at least one selected from the group consiεting of: (a) an insertion of at least one Phe residue between Ala774 and His775; (b) an insertion of at least one Glu residue between Ala774 and His775; (c) an insertion of at least one Pro residue between Ala774 and His775; (d) a deletion comprising residue Ala774; (e) a deletion comprising residueε Trp772, Phe773 and Ala774; (f) a deletion compriεing residues Lys768, Thr769, Aεp770, Pro771, Trp772, and Phe773; (g) a substitution of at least one Phe reεidue at Ala774; (h) a εubεtitution of at leaεt one Glu residue at Ala774; and (i) a subεtitution of at leaεt one Pro reεidue at Ala774.
27. The analog of claim 2, wherein the non- activating Arg residue is Argl310, and the modification comprises at least one εelected from the group conεisting of: (a) an insertion of at least one Pro residue between Glnl309 and Argl310; (b) an insertion of at least one Pro residue between Argl310 and Serl311; (c) a deletion comprising residue Serl311 and an insertion of at least one Pro reεidue to replace the reεidue; (d) a deletion compriεing reεidueε Ser1311, Lyεl312, and Argl313 and an insertion of at leaεt one Pro reεidue to replace the deleted reεidueε; (e) a deletion compriεing residues Serl311, Lysl312, Argl313, Alal314, Leul315, and Lyεl316, and an insertion of at least one Pro residue to replace the deleted reεidues; (f) a deletion comprising residueε Serl311, Lyεl312, Argl313, Alal314, Leul315, Lyεl316, Glnl317, Phel318, Argl319, and Leul320; and (g) a deletion compriεing residues Serl311, Lysl312, Argl313, Alal314, Leul315, Lysl316, Glnl317, Phel318, Argl319, and Leul320, and an insertion of at least one Pro residue to replace the deleted residues.
28. The analog of claim 6, wherein the non- activating Arg residue is Argl310, and the modification compriseε at leaεt one selected from the group consisting of: (a) an insertion of at least one Phe residue between Thrl308 and Glnl309; (b) an insertion of at least one Glu residue between Thr1308 and Glnl309; (c) an insertion of at least one Pro residue between Thr1308 and Glnl309; (d) a deletion comprising reεidues Vall307, and Thrl308; (e) a substitution of at least one Phe reεidue at Thrl308; (f) a εubεtitution of at leaεt one Glu reεidue at Thrl308; and (g) a εubεtitution of at leaεt one Pro reεidue at Thrl308.
29. The analog of claim 2, wherein the non- activating Arg reεidue iε Argl313, and the modification compriεeε at least one selected from the group consisting of: (a) an insertion of at leaεt one Pro residue between Lysl312 and Argl313; (b) a deletion compriεing reεidue Lyεl312 and an inεertion of at leaεt one Pro reεidue to replace the deleted reεidue; (c) a deletion comprising residueε Argl310, Ser1311, and Lyεl312, and an inεertion of at least one Pro residue to replace the deleted residues; (d) an insertion of at least one Pro reεidue between Argl313 and Alal314; (e) a deletion comprising residues Alal314, Leul315, and Lysl316, and an insertion of at least one Pro residue to replace the deleted residues; (f) a deletion comprising residues Alal314, Leul315, Lysl316, Glnl317, Phel318, and Argl319 and an inεertion of at least one Pro residue to replace the deleted residueε; and (g) a deletion compriεing reεidues Alal314, Leul315, Lysl316, Glnl317, Phel318, Argl319, and Leul320.
30. The analog of claim 6, wherein the non- activating Arg residue is Argl313, and the modification compriseε at leaεt one εelected from the group consisting of: (a) an insertion of at least one Phe residue between Serl311 and Lysl312; (b) an insertion of at least one Glu residue between Serl311 and Lyεl312; (c) an insertion of at least one Pro residue between Serl311 and Lysl312; (d) a deletion comprising residues Vall307, Thr1308, Glnl309, Argl310, and Serl311; (e) a substitution of at least one Phe reεidue at Serl311; (f) a εubεtitution of at leaεt one Glu reεidue at Ser 1311; and (g) a substitution of at least one Pro reεidue at Ser 1311.
31. The analog of claim 2 , wherein the non- activating Arg residue is Argl645, and the modification comprises at least one selected from the group consisting of: (a) a deletion comprising residues Vall642, Leul643, and Lysl644; (b) a deleteion compriεing reεidue Lysl644 and an insertion of at least one Pro reεidue to replace the deleted reεidue; (c) an insertion of at least one Pro residue between Argl645 and Lysl644; (d) an inεertion of at least one Pro residue between Argl645 and Hisl646; (e) a deletion comprising residues Hisl646, Glnl647, and Argl648 and an inεertion of at leaεt one Pro residue to replace the deleted residues; and (f) a deletion comprising residueε Hiεl646, Glnl647, Argl648, Glul649, Ilel650, Thrl651, and Argl652, and an inεertion of at least one Pro residue to replace the deleted residues.
32. The analog of claim 6, wherein the non- activating Arg residue is Argl645, and the modification comprises at least one selected from the group consiεting of: (a) an inεertion of at least one Phe residue between Leul643 and Lysl644; (b) an inεertion of at least one Glu residue between Leul643 and Lysl644; (c) an insertion of at least one Pro residue between Leul643 and Lysl644; (d) a deletion comprising residues Vall642, and Leul643; (e) a deletion comprising residues Prol641, Vall642, and Leul643; (f) a subεtitution of at least one Phe residue at Leul643; (g) a subεtitution of at least one Glu residue at Leul643; and (h) a substitution of at least one Pro residue at Leul643.
33. The analog of claim 2, wherein the non- activating Arg residue is Argl648, and the modification comprises at least one selected from the group consiεting of: (a) a deletion compriεing reεidues Vall642, Leul643, Lysl644, Argl645, Hiεl646, and Glnl647; (b) an inεertion of at least one Pro residue between Glnl647 and Argl648; (c) a deletion comprising residues Lysl644, Argl645, Hisl646, and Glnl647 and an insertion of at least one Pro residue to replace the deleted residues; (d) a deletion comprising residueε Glul649, Ilel650, Thrl651, and Argl652 and an inεertion of at least one Pro residue to replace the deleted residues; and (e) an insertion of at leaεt one Pro reεidue between Argl648 and Glul649.
34. The analog of claim 6, wherein the non- activating Arg residue is Argl648, and the modification compriseε at leaεt one selected from the group consisting of: (a) an insertion of at least one Phe residue between Hisl646 and Glnl647; (b) an insertion of at least one Glu residue between Hisl646 and Glnl647; (c) an inεertion of at least one Pro residue between Hisl646 and Glnl647; (d) a deletion comprising residues Vall642, Leul643, Lysl644, Argl645, and Hisl646; (e) a deletion compriεing residues Prol641, Vall642, Leul643, Lysl644, Argl645, and Hisl646; (f) a substitution of at least one Phe residue at Hisl646; (g) a substitution of at least one Glu residue at Hisl646; and (h) a substitution of at least one Pro reεidue at Hisl646.
35. The analog of claim 2, wherein the non- activating Arg residue is Argl719, and the modification compriseε at leaεt one εelected from the group conεiεting of: (a) a deletion compriεing reεidueε Hisl716, Vall717, and Leul718; (b) an insertion of at least one Pro residue between Leul718 and Argl719; (c) a deletion compriεing reεidueε Aεnl720, and Argl721 and an inεertion of at leaεt one Pro reεidue to replace the deleted reεidues; and (d) an insertion of at least one Pro residue between Argl719 and Asnl720.
36. The analog of claim 6, wherein the non- activating Arg residue is Argl719, and the modification compriεes at least one selected from the group consisting of: (a) an insertion of at least one Phe residue between Vall717 and Leul718; (b) an insertion of at least one Glu residue between Vall717 and Leul718; (c) an insertion of at least one Pro residue between Vall717 and Leul718; (d) a deletion comprising residues Hisl716, and Vall717; (e) a subεtitution of at leaεt one Phe reεidue at Vall717; and (f) a εubεtitution of at leaεt one Glu residue at Vall717; (g) a subεtitution of at leaεt one Pro residue at Vall717.
37. The analog of claim 2, wherein the non- activating Arg residue is Argl721, and the modification comprises at least one selected from the group consisting of: (a) a deletion comprising residues Hisl716, Vall717, Leul718, Argl719, and Aεnl720; (b) an insertion of at least one Pro residue between Asnl720 and Argl721; (c) an insertion of at least one Pro residue between Argl721 and Alal722; (d) a deletion comprising residues Alal722, Glnl723, and Serl724 and an insertion of at leaεt one Pro residue to replace the deleted residues; (e) a deletion comprising residueε Alal722, Glnl723, Serl724, Glyl725, Serl726, and Vall727; and (f) a deletion comprising residues Alal722, Glnl723, Serl724, Glyl725, and Serl726, and an insertion of at least one Pro residue to replace the deleted residues.
38. The analog of claim 6, wherein the non- activating Arg residue is Argl721, and the modification compriseε at leaεt one selected from the group consisting of: (a) a deletion comprising residue Asnl720 and an insertion of at least one Phe residue between Leul718 and Argl719; (b) a deletion comprising residue Asnl720 and an inεertion of at least one Glu residue between Leul718 and Argl719; (c) a deletion comprising residue Aεnl720 and an insertion of at least one Pro residue between Leul718 and Argl719; (d) a deletion comprising residueε Vall717, Leul718, Argl719, and Asnl720; (e) a deletion comprising reεidue Aεnl720 and a εubεtitution of at leaεt one Phe residue at Leul718; (f) a deletion comprising residue Asnl720 and a εubεtitution of at least one Glu residue at Leul718; and (g) a deletion comprising residue Asnl720 and a substitution of at least one Pro residue at Leul718;
39. An active Factor VIII:C polypeptide analog comprising a native Factor VIII:C polypeptide that is modified at at least one non-activating Arg reεidue εelected from the group conεiεting of Arg336, Argl719 and Argl721, wherein the modification comprises a subεtitution of any of amino acids Pro, Glu, Asp, Asn, Gin, Ser and Tyr for Arg336, a substitution of any of amino acids Pro, Glu, Aεp, Aεn, Gin, Ser and Tyr for
Argl719 and/or a substitution of any of amino acids Glu, Asp, Asn, Gin, Ser and Tyr for Argl721, numbered with respect to the native Factor VIII:C polypeptide sequence.
40. The analog of claim 39, wherein the modification comprises at leaεt one amino acid substitution selected from the group conεiεting of a εubεtitution of Pro for Arg336, a εubstitution of Pro for Argl719 and a substitution of Glu for Argl721, numbered with reεpect to the native Factor VIII:C polypeptide εequence.
41. The analog of claim 1, wherein the native Factor VIII:C polypeptide iε selected from the group consisting of: a) a full-length Factor VIII:C molecule comprising a signal peptide and all A, B, and C domainε; b) a mature Factor VIII:C molecule compriεing 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 comprising a light chain εubunit of molecular weight of about 80 kD; e) a cleaved Factor VIII:C molecule comprising a heavy chain fragment of molecular weight in a range of about 90 kD to 200 kD; f) a cleaved Factor VIII:C molecule comprising a heavy chain fragment of molecular weight of about 90 kD; g) a cleaved Factor VIII:C molecule comprising a heavy chain fragment of molecular weight of about 50 kD; h) a cleaved Factor VIII:C molecule comprising a heavy chain fragment of molecular weight of about 43 kD; and i) a cleaved Factor VIII:C molecule comprising a light chain fragment of molecular weight of about 73 kD.
42. An active Factor VIII:C polypeptide analog complex compriεing at leaεt two Factor VIII:C polypeptide analogs as claimed in claim 41, or at least one Factor VIII:C polypeptide analog and at least one Factor VIII:C polypeptide, wherein the complex comprises a metal ion.
43. The analog complex of claim 41, wherein the complex compriseε two Factor VIII:C polypeptide analogε, and the Factor VIII:C polypeptideε that are modified to form the two analogs are selected from the group consiεting 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.
44. The analog complex of claim 42, wherein the metal ion is a divalent cation.
45. The analog complex of claim 44, wherein the divalent cation is a Ca++ ion or a Cu++ ion.
46. The analog of claim 6, wherein the native Factor VIII:C polypeptide is εelected from the group consisting of: a) a full-length Factor VIII:C molecule comprising a εignal peptide and all A, B, and C domainε; b) a mature 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 leaεt a portion of the B domain; d) a cleaved Factor VIII:C molecule comprising a light chain εubunit of molecular weight of about 80 kD; e) a cleaved Factor VIII:C molecule compriεing a heavy chain fragment of molecular weight in a range of about 90 kD to 200 kD; f) a cleaved Factor VIII:C molecule compriεing a heavy chain fragment of molecular weight of about 90 kD; g) a cleaved Factor VIII:C molecule comprising a heavy chain fragment of molecular weight of about 50 kD; h) a cleaved Factor VIII:C molecule comprising a heavy chain fragment of molecular weight of about 43 kD; and i) a cleaved Factor VIII:C molecule comprising a light chain fragment of molecular weight of about 73 kD.
47. An active Factor VIII:C polypeptide analog complex comprising at least two Factor VIII:C polypeptide analogs aε claimed in claim 46, or at leaεt one Factor VIII:C polypeptide analog and at least one Factor VIII:C polypeptide, wherein the complex compriseε a metal ion.
48. The analog complex of claim 46, wherein the complex compriεes two Factor VIII:C polypeptide analogs, and the Factor VIII:C polypeptides that are modified to form the two analogε are selected from the group consiεting of molecular weightε 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.
49. The analog complex of claim 47, wherein the metal ion iε a divalent cation.
50. The analog complex of claim 49, wherein the divalent cation is a Ca++ ion or a Cu++ ion.
51. The analog of claim 40, wherein the native Factor VIII:C polypeptide 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 mature 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 comprising a light chain subunit of molecular weight of about 80 kD; e) a cleaved Factor VIII:C molecule comprising a heavy chain fragment of molecular weight in a range of about 90 kD to 200 kD; f) a cleaved Factor VIII:C molecule comprising a heavy chain fragment of molecular weight of about 90 kD; g) a cleaved Factor VIII:C molecule comprising a heavy chain fragment of molecular weight of about 50 kD; h) a cleaved Factor VIII:C molecule comprising a heavy chain fragment of molecular weight of about 43 kD; and i) a cleaved Factor VIII:C molecule comprising a light chain fragment of molecular weight of about 73 kD.
52. An active Factor VIII:C polypeptide analog complex compriεing at least two Factor VIII:C polypeptide analogs as claimed in claim 51, or at least one Factor VIII:C polypeptide analog and at least one Factor VIII:C polypeptide, wherein the complex comprises a metal ion.
53. The analog complex of claim 51, wherein the complex compriseε two Factor VIII:C polypeptide analogε, and the Factor VIII:C polypeptides that are modified to form the two analogs are selected from the group conεiεting of molecular weightε 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.
54. The analog complex of claim 52, wherein the metal ion iε a divalent cation.
55. The analog complex of claim 54, wherein the divalent cation iε a Ca++ ion or a Cu++ ion.
56. A method of producing a Factor VIII:C polypeptide analog, comprising: a) providing a native Factor VIII:C polypeptide that comprises an amino acid sequence; and b) modifying an amino acid residue in the amino acid sequence to produce the analog of claim 1.
57. A method of producing a Factor VIII:C polypeptide analog, comprising: a) providing a native Factor VIII:C polypeptide that compriseε an amino acid εequence; and b) modifying an amino acid reεidue in the amino acid εequence to produce the analog of claim 6.
58. A method of producing a Factor VIII:C polypeptide analog, compriεing: a) providing a native Factor VIII:C polypeptide that comprises an amino acid sequence; and b) modifying an amino acid residue in the amino acid sequence to produce the analog of claim 40.
59. A nucleic acid molecule comprising a nucleotide sequence that encodes the Factor VIII:C polypeptide analog of claim 1.
60. A nucleic acid molecule comprising a nucleotide sequence that encodes the Factor VIII:C polypeptide analog of claim 6.
61. A nucleic acid molecule comprising a nucleotide sequence that encodes the Factor VIII:C polypeptide analog of claim 40.
62. A recombinant vector comprising the nucleic acid molecule of claim 59 and a regulatory element, wherein the nucleic acid molecule is under regulatory control of the regulatory element.
63. A recombinant vector comprising the nucleic acid molecule of claim 60 and a regulatory element, wherein the nucleic acid molecule is under regulatory control of the regulatory element.
64. A recombinant vector comprising the nucleic acid molecule of claim 61 and a regulatory element, wherein the nucleic acid molecule is under regulatory control of the regulatory element.
65. A recombinant host cell comprising the nucleic acid molecule of claim 59.
66. A recombinant hoεt cell comprising the nucleic acid molecule of claim 60.
67. A recombinant host cell comprising the nucleic acid molecule of claim 61.
68. A recombinant host cell comprising the recombinant vector of claim 62.
69. A recombinant hoεt cell compriεing the recombinant vector of claim 63.
70. A recombinant host cell comprising the recombinant vector of claim 64.
71. A method of producing an active Factor VIII:C polypeptide analog comprising: a) providing the recombinant host cell of claim 65; and b) allowing the recombinant host cell to express the analog.
72. A method of producing an active Factor
VIII:C polypeptide analog comprising: a) providing the recombinant host cell of claim 66; and b) allowing the recombinant host cell to expresε the analog.
73. A method of producing an active Factor VIII:C polypeptide analog compriεing: a) providing the recombinant hoεt cell of claim 67; and b) allowing the recombinant hoεt cell to expreεε the analog.
74. A method of producing an active Factor VIII:C polypeptide analog comprising: a) providing the recombinant host cell of claim 68; and b) allowing the recombinant host cell to express the analog.
75. A method of producing an active Factor VIII:C polypeptide analog comprising: a) providing the recombinant host cell of claim 69; and b) allowing the recombinant host cell to expreεs the analog.
76. A method of producing an active Factor VIII:C polypeptide analog comprising: a) providing the recombinant hoεt cell of claim 70; and b) allowing the recombinant host cell to expresε the analog.
77. A method of producing a nucleic acid molecule that encodeε a Factor VIII:C polypeptide analog comprising: a) providing a nucleic acid molecule that encodes a native Factor VIII:C polypeptide, wherein the nucleic acid molecule comprises a codon for each amino acid residue in the native Factor VIII:C polypeptide; and b) modifying at leaεt one codon that iε adjacent to a codon encoding a non-activating Arg residue, to produce the nucleic acid molecule of claim 59.
78. The method of producing a nucleic acid molecule as claimed in claim 77, wherein the modifying is performed by εite directed mutagenesis or by use of polymerase chain reaction techniqueε.
79. A method of producing a nucleic acid molecule that encodes a Factor VIII:C polypeptide analog comprising: a) providing a nucleic acid molecule that encodes a native Factor VIII:C polypeptide, wherein the nucleic acid molecule comprises a codon for each amino acid residue in the native Factor VIII:C polypeptide; and b) modifying at leaεt one codon that iε adjacent to a codon encoding a non-activating Arg residue, to produce the nucleic acid molecule of claim 60.
80. The method of producing a nucleic acid molecule as claimed in claim 79, wherein the modifying is performed by site directed mutagenesiε or by uεe of polymeraεe chain reaction techniqueε.
81. A method of producing a nucleic acid molecule that encodes a Factor VIII:C polypeptide analog comprising: a) providing a nucleic acid molecule that encodes a native Factor VIII:C polypeptide, wherein the nucleic acid molecule comprises a codon for each amino acid residue in the native Factor VIII:C polypeptide; and b) modifying at least one codon that is adjacent to a codon encoding a non-activating Arg residue, to produce the nucleic acid molecule of claim 61.
82. The method of producing a nucleic acid molecule as claimed in claim 81, wherein the modifying is performed by site directed mutagenesiε or by use of polymerase chain reaction techniqueε.
83. A method of producing a recombinant vector that comprises a nucleic acid molecule that comprises a nucleotide sequence that encodes a Factor VIII:C polypeptide analog, comprising linking a regulatory element to the nucleic acid molecule of claim 59.
84. A method of producing a recombinant vector that comprises a nucleic acid molecule that comprises a nucleotide sequence that encodes a Factor VIII:C polypeptide analog, comprising linking a regulatory element to the nucleic acid molecule of claim 60.
85. A method of producing a recombinant vector that comprises a nucleic acid molecule that comprises a nucleotide sequence that encodes a Factor VIII:C polypeptide analog, comprising linking a regulatory element to the nucleic acid molecule of claim 61.
86. A method of producing a recombinant host cell that compriεes a nucleic acid molecule that comprises a nucleotide sequence that encodeε a Factor VIII:C polypeptide analog, compriεing transforming a host cell with the nucleic acid molecule of claim 59.
87. A method of producing a recombinant host cell that comprises a nucleic acid molecule that comprises a nucleotide sequence that encodeε a Factor VIII:C polypeptide analog, comprising transforming a hoεt cell with the nucleic acid molecule of claim 60.
88. A method of producing a recombinant host cell that comprises a nucleic acid molecule that comprises a nucleotide sequence that encodes a Factor VIII:C polypeptide analog, compriεing tranεforming a hoεt cell with the nucleic acid molecule of claim 61.
89. A method of producing a recombinant host cell that compriseε a recombinant vector that comprises a nucleotide sequence that encodes a Factor VIII:C polypeptide analog, comprising transforming a host cell with the recombinant vector of claim 62.
90. A method of producing a recombinant host cell that comprises a recombinant vector that comprises a nucleotide sequence that encodes a Factor VIII:C polypeptide analog, comprising transforming a hoεt cell with the recombinant vector of claim 63.
91. A method of producing a recombinant hoεt cell that comprises a recombinant vector that comprises a nucleotide sequence that encodes a Factor VIII:C polypeptide analog, comprising transforming a host cell with the recombinant vector of claim 64.
92. A pharmaceutical composition comprising the active Factor VIII:C polypeptide analog complex of claim 42 and a pharmaceutically acceptable excipient.
93. A pharmaceutical composition comprising the active Factor VIII:C polypeptide analog complex of claim 47 and a pharmaceutically acceptable excipient.
94. A pharmaceutical composition comprising the active Factor VIII:C polypeptide analog complex of claim 52 and a pharmaceutically acceptable excipient.
95. A method for prevention or treatment of active Factor VIII:C deficiency in a mammal comprising administering thereto a therapeutically effective amount of the pharmaceutical compoεition of claim 92.
96. A method for prevention or treatment of active Factor VIII:C deficiency in a mammal compriεing administering thereto a therapeutically effective amount of the pharmaceutical composition of claim 93.
97. A method for prevention or treatment of active Factor VIII:C deficiency in a mammal compriεing adminiεtering thereto a therapeutically effective amount of the pharmaceutical composition of claim 94.
98. A method for prevention or treatment of active Factor VIII:C deficiency in a mammal comprising adminiεtering thereto a therapeutically effective amount of the nucleic acid molecule of claim 59.
99. A method for prevention or treatment of active Factor VIII:C deficiency in a mammal comprising administering thereto a therapeutically effective amount of the nucleic acid molecule of claim 60.
100. A method for prevention or treatment of active Factor VIII:C deficiency in a mammal compriεing administering thereto a therapeutically effective amount of the nucleic acid molecule of claim 61.
101. A method for prevention or treatment of active Factor VIII:C deficiency in a mammal comprising administering thereto a therapeutically effective amount of the recombinant vector of claim 62.
102. A method for prevention or treatment of active Factor VIII:C deficiency in a mammal comprising administering thereto a therapeutically effective amount of the recombinant vector of claim 63.
103. A method for prevention or treatment of active Factor VIII:C deficiency in a mammal compriεing adminiεtering thereto a therapeutically effective amount of the recombinant vector of claim 64.
104. A method for prevention or treatment of active Factor VIII:C deficiency in a mammal comprising administering thereto a therapeutically effective amount of the nucleic acid molecule of claim 59 and an active Factor VIII:C polypeptide analog.
105. A method for prevention or treatment of active Factor VIII:C deficiency in a mammal comprising administering thereto a therapeutically effective amount of the nucleic acid molecule of claim 60 and an active Factor VIII:C polypeptide analog.
106. A method for prevention or treatment of active Factor VIII:C deficiency in a mammal comprising adminiεtering thereto a therapeutically effective amount of the nucleic acid molecule of claim 61 and an active Factor VIII:C polypeptide analog.
107. A method for prevention or treatment of active Factor VIII:C deficiency in a mammal compriεing adminiεtering thereto a therapeutically effective amount of the recombinant vector of claim 62 and an active Factor VIII:C polypeptide analog.
108. A method for prevention or treatment of active Factor VIII:C deficiency in a mammal compriεing adminiεtering thereto a therapeutically effective amount of the recombinant vector of claim 63 and an active Factor VIII:C polypeptide analog.
109. A method for prevention or treatment of active Factor VIII:C deficiency in a mammal comprising administering thereto a therapeutically effective amount of the recombinant vector of claim 64 and an active Factor VIII:C polypeptide analog.
110. A method for prevention or treatment of active Factor VIII:C deficiency in a mammal comprising administering thereto a therapeutically effective amount of the nucleic acid molecule of claim 59 and an active Factor VIII:C analog complex.
111. A method for prevention or treatment of active Factor VIII:C deficiency in a mammal compriεing adminiεtering thereto a therapeutically effective amount of the nucleic acid molecule of claim 60 and an active Factor VIII:C analog complex.
112. A method for prevention or treatment of active Factor VIII:C deficiency in a mammal compriεing administering thereto a therapeutically effective amount of the nucleic acid molecule of claim 61 and an active Factor VIII:C analog complex.
113. A method for prevention or treatment of active Factor VIII:C deficiency in a mammal comprising administering thereto a therapeutically effective amount of the recombinant vector of claim 62 and an active Factor VIII:C analog complex.
114. A method for prevention or treatment of active Factor VIII:C deficiency in a mammal comprising administering thereto a therapeutically effective amount of the recombinant vector of claim 63 and an active Factor VIII:C analog complex.
115. A method for prevention or treatment of active Factor VIII:C deficiency in a mammal comprising administering thereto a therapeutically effective amount of the recombinant vector of claim 64 and an active Factor VIII:C analog complex.
PCT/US1996/011444 1995-07-11 1996-07-09 Novel factor viii:c polypeptide analogs with altered protease sites WO1997003195A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU64861/96A AU6486196A (en) 1995-07-11 1996-07-09 Novel factor viii:c polypeptide analogs with altered protease sites

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US102595P 1995-07-11 1995-07-11
US60/001,025 1995-07-11

Publications (1)

Publication Number Publication Date
WO1997003195A1 true WO1997003195A1 (en) 1997-01-30

Family

ID=21694023

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1996/011444 WO1997003195A1 (en) 1995-07-11 1996-07-09 Novel factor viii:c polypeptide analogs with altered protease sites

Country Status (2)

Country Link
AU (1) AU6486196A (en)
WO (1) WO1997003195A1 (en)

Cited By (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000028021A1 (en) * 1998-11-10 2000-05-18 Baxter Aktiengesellschaft A factor viii-polypeptide with factor viii:c-activity
WO2000071714A2 (en) 1999-05-24 2000-11-30 The American National Red Cross Methods of reducing factor viii clearance and compositions therefor
US6221349B1 (en) 1998-10-20 2001-04-24 Avigen, Inc. Adeno-associated vectors for expression of factor VIII by target cells
WO2003087161A1 (en) * 2002-04-18 2003-10-23 Merck Patent Gmbh Modified factor viii
US6838437B2 (en) 1996-04-24 2005-01-04 University Of Michigan Inactivation resistant factor VIII
WO2006027111A1 (en) * 2004-09-06 2006-03-16 Zlb Behring Gmbh Modified coagulation factor viii with enhanced stability
US7211559B2 (en) 2003-10-31 2007-05-01 University Of Maryland, Baltimore Factor VIII compositions and methods
US7351577B2 (en) 1998-10-20 2008-04-01 Genzyme Corporation Adeno-associated vector compositions for expression of Factor VIII
US7615622B2 (en) 2001-01-12 2009-11-10 University Of Maryland, Baltimore Methods and compositions for reducing heparan sulfate proteoglycan-mediated clearance of factor VIII
US7632921B2 (en) 2004-11-12 2009-12-15 Bayer Healthcare Llc Site-directed modification of FVIII
EP2206785A1 (en) 1998-12-31 2010-07-14 Novartis Vaccines and Diagnostics, Inc. Improved expression of HIV polypeptides and production of virus-like particles
EP2292780A2 (en) 2003-09-30 2011-03-09 The Trustees of the University of Pennsylvania Adeno-associated virus (AAV) clades, sequences, vectors containing same, and uses thereof
EP2357010A1 (en) 2005-04-07 2011-08-17 The Trustees of The University of Pennsylvania Method of increasing the function of an AAV vector
WO2011126808A2 (en) 2010-03-29 2011-10-13 The Trustees Of The University Of Pennsylvania Pharmacologically induced transgene ablation system
US8183344B2 (en) 1996-04-24 2012-05-22 University Of Michigan Inactivation resistant factor VIII
WO2012112832A1 (en) 2011-02-17 2012-08-23 The Trustees Of The University Of Pennsylvania Compositions and methods for altering tissue specificity and improving aav9-mediated gene transfer
US20120297494A1 (en) * 2009-10-16 2012-11-22 Tommy Eugene Howard Compositions and methods of treatment of black hemophiliac patients
WO2013049493A1 (en) 2011-09-28 2013-04-04 The Trustees Of The University Of Pennsylvania Inducible adeno -associated virus vector mediated transgene ablation system
WO2016168728A2 (en) 2015-04-16 2016-10-20 Emory University Recombinant promoters and vectors for protein expression in liver and use thereof
US9719106B2 (en) 2013-04-29 2017-08-01 The Trustees Of The University Of Pennsylvania Tissue preferential codon modified expression cassettes, vectors containing same, and uses thereof
US10272163B2 (en) 2012-12-07 2019-04-30 The Regents Of The University Of California Factor VIII mutation repair and tolerance induction
US11185573B2 (en) 2004-12-06 2021-11-30 Haplomics, Inc. Allelic variants of human factor VIII
US20220033475A1 (en) * 2018-10-23 2022-02-03 The Children's Hospital Of Philadelphia Compositions and methods for modulating factor viii function
WO2022032140A2 (en) 2020-08-07 2022-02-10 Amicus Therapeutics, Inc. Vesicle targeting proteins and uses of same
CN114106146A (en) * 2015-02-06 2022-03-01 北卡罗来纳大学查珀尔希尔分校 Optimized human coagulation factor VIII gene expression cassette and application thereof

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1987007144A1 (en) * 1986-05-29 1987-12-03 Genetics Institute, Inc. Novel procoagulant proteins
WO1988008035A1 (en) * 1987-04-06 1988-10-20 Genetics Institute, Inc. IMPROVED METHOD FOR PRODUCING FACTOR VIII:c-TYPE PROTEINS
EP0309237A1 (en) * 1987-09-25 1989-03-29 Genentech, Inc. A transient expression system for producing recombinant protein

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1987007144A1 (en) * 1986-05-29 1987-12-03 Genetics Institute, Inc. Novel procoagulant proteins
WO1988008035A1 (en) * 1987-04-06 1988-10-20 Genetics Institute, Inc. IMPROVED METHOD FOR PRODUCING FACTOR VIII:c-TYPE PROTEINS
EP0309237A1 (en) * 1987-09-25 1989-03-29 Genentech, Inc. A transient expression system for producing recombinant protein

Cited By (47)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6838437B2 (en) 1996-04-24 2005-01-04 University Of Michigan Inactivation resistant factor VIII
US8183344B2 (en) 1996-04-24 2012-05-22 University Of Michigan Inactivation resistant factor VIII
US7459534B2 (en) 1996-04-24 2008-12-02 The Regents Of The University Of Michigan Inactivation resistant factor VIII
US6221349B1 (en) 1998-10-20 2001-04-24 Avigen, Inc. Adeno-associated vectors for expression of factor VIII by target cells
US7351577B2 (en) 1998-10-20 2008-04-01 Genzyme Corporation Adeno-associated vector compositions for expression of Factor VIII
WO2000028021A1 (en) * 1998-11-10 2000-05-18 Baxter Aktiengesellschaft A factor viii-polypeptide with factor viii:c-activity
US7544660B2 (en) 1998-11-10 2009-06-09 Stichting Sanquin Bloedvoorziening Factor VIII polypeptide having factor VIII:C activity
EP2206785A1 (en) 1998-12-31 2010-07-14 Novartis Vaccines and Diagnostics, Inc. Improved expression of HIV polypeptides and production of virus-like particles
WO2000071714A2 (en) 1999-05-24 2000-11-30 The American National Red Cross Methods of reducing factor viii clearance and compositions therefor
US7615622B2 (en) 2001-01-12 2009-11-10 University Of Maryland, Baltimore Methods and compositions for reducing heparan sulfate proteoglycan-mediated clearance of factor VIII
WO2003087161A1 (en) * 2002-04-18 2003-10-23 Merck Patent Gmbh Modified factor viii
EP2298926A1 (en) 2003-09-30 2011-03-23 The Trustees of The University of Pennsylvania Adeno-associated virus (AAV) clades, sequences, vectors containing same, and uses thereof
EP2292780A2 (en) 2003-09-30 2011-03-09 The Trustees of the University of Pennsylvania Adeno-associated virus (AAV) clades, sequences, vectors containing same, and uses thereof
EP2292779A2 (en) 2003-09-30 2011-03-09 The Trustees of the University of Pennsylvania Adeno-associated virus (AAV) clades, sequences, vectors containing same, and uses thereof
EP2345731A1 (en) 2003-09-30 2011-07-20 The Trustees of the University of Pennsylvania Adeno-associated virus (AAV) clades, sequences, vectors containing same, and uses thereof
US7211559B2 (en) 2003-10-31 2007-05-01 University Of Maryland, Baltimore Factor VIII compositions and methods
WO2006027111A1 (en) * 2004-09-06 2006-03-16 Zlb Behring Gmbh Modified coagulation factor viii with enhanced stability
US7632921B2 (en) 2004-11-12 2009-12-15 Bayer Healthcare Llc Site-directed modification of FVIII
US9364520B2 (en) 2004-11-12 2016-06-14 Bayer Healthcare Llc Factor VIII conjugates
US9096656B2 (en) 2004-11-12 2015-08-04 Bayer Healthcare Llc Factor VIII conjugates
US11185573B2 (en) 2004-12-06 2021-11-30 Haplomics, Inc. Allelic variants of human factor VIII
EP2383346A1 (en) 2005-04-07 2011-11-02 The Trustees of the University of Pennsylvania Method of increasing the function of an AAV vector
EP2357010A1 (en) 2005-04-07 2011-08-17 The Trustees of The University of Pennsylvania Method of increasing the function of an AAV vector
EP2359866A1 (en) 2005-04-07 2011-08-24 The Trustees of The University of Pennsylvania Method of increasing the function of an AAV vector
EP4282956A2 (en) 2005-04-07 2023-11-29 The Trustees of the University of Pennsylvania Method of increasing the function of an aav vector
EP4282957A2 (en) 2005-04-07 2023-11-29 The Trustees of the University of Pennsylvania Method of increasing the function of an aav vector
EP3603676A1 (en) 2005-04-07 2020-02-05 The Trustees of the University of Pennsylvania Method of increasing the function of an aav vector
EP2359865A1 (en) 2005-04-07 2011-08-24 The Trustees of The University of Pennsylvania Method of increasing the function of an AAV vector
EP2359867A1 (en) 2005-04-07 2011-08-24 The Trustees of The University of Pennsylvania Method of increasing the function of an AAV vector
EP3085389A1 (en) 2005-04-07 2016-10-26 The Trustees Of The University Of Pennsylvania Method of increasing the function of an aav vector
US20120297494A1 (en) * 2009-10-16 2012-11-22 Tommy Eugene Howard Compositions and methods of treatment of black hemophiliac patients
WO2011126808A2 (en) 2010-03-29 2011-10-13 The Trustees Of The University Of Pennsylvania Pharmacologically induced transgene ablation system
US10918658B2 (en) 2011-02-17 2021-02-16 The Trustees Of The University Of Pennsylvania Compositions and methods for altering tissue specificity and improving AAV9-mediated gene transfer
US10406173B2 (en) 2011-02-17 2019-09-10 Trustees Of The University Of Pennsylvania Compositions and methods for altering tissue specificity and improving AAV9-mediated gene transfer
WO2012112832A1 (en) 2011-02-17 2012-08-23 The Trustees Of The University Of Pennsylvania Compositions and methods for altering tissue specificity and improving aav9-mediated gene transfer
US9884071B2 (en) 2011-02-17 2018-02-06 The Trustees Of The University Of Pennsylvania Compositions and methods for altering tissue specificity and improving AAV9-mediated gene transfer
US11766448B2 (en) 2011-02-17 2023-09-26 The Trustees Of The University Of Pennsylvania Compositions and methods for altering tissue specificity and improving AAV9-mediated gene transfer
WO2013049493A1 (en) 2011-09-28 2013-04-04 The Trustees Of The University Of Pennsylvania Inducible adeno -associated virus vector mediated transgene ablation system
US10272163B2 (en) 2012-12-07 2019-04-30 The Regents Of The University Of California Factor VIII mutation repair and tolerance induction
US11083801B2 (en) 2012-12-07 2021-08-10 Haplomics, Inc. Factor VIII mutation repair and tolerance induction
US10647998B2 (en) 2013-04-29 2020-05-12 The Trustees Of The University Of Pennsylvania Tissue preferential codon modified expression cassettes, vectors containing same, and uses thereof
US9719106B2 (en) 2013-04-29 2017-08-01 The Trustees Of The University Of Pennsylvania Tissue preferential codon modified expression cassettes, vectors containing same, and uses thereof
CN114106146A (en) * 2015-02-06 2022-03-01 北卡罗来纳大学查珀尔希尔分校 Optimized human coagulation factor VIII gene expression cassette and application thereof
EP3626274A2 (en) 2015-04-16 2020-03-25 Emory University Recombinant promoters and vectors for protein expression in liver and use thereof
WO2016168728A2 (en) 2015-04-16 2016-10-20 Emory University Recombinant promoters and vectors for protein expression in liver and use thereof
US20220033475A1 (en) * 2018-10-23 2022-02-03 The Children's Hospital Of Philadelphia Compositions and methods for modulating factor viii function
WO2022032140A2 (en) 2020-08-07 2022-02-10 Amicus Therapeutics, Inc. Vesicle targeting proteins and uses of same

Also Published As

Publication number Publication date
AU6486196A (en) 1997-02-10

Similar Documents

Publication Publication Date Title
WO1997003195A1 (en) Novel factor viii:c polypeptide analogs with altered protease sites
WO1997003193A1 (en) Novel factor viii:c polypeptide analogs with altered metal-binding properties
CA1341229C (en) Procoagulant proteins derived from factor viii: c
JP3048387B2 (en) Methods for increasing viral protein expression
US7855269B2 (en) Method for treating inflammation
US7361738B2 (en) Megakaryocyte stimulating factors
US5726147A (en) Human mutant tissue factor compositions useful as tissue factor antagonists
AU646014B2 (en) Novel platelet-derived growth factor B chain analogs and method for homogeneous production thereof
CA2084514A1 (en) O-glycosylated ifn-alpha
US7556941B2 (en) Materials and methods for preparing dimeric growth factors
US5359033A (en) Cleaved dimers of mullerian inhibiting substance-like polypeptides
AU2002337901A1 (en) Nucleic acid and amino acid sequences encoding high-level expressor factor VIII polypeptides and methods of use
EP1417231B1 (en) Modified human thymic stromal lymphopoietin
JP2554418B2 (en) Human interleukin-5 receptor α-chain or a part thereof, particularly DNA encoding shIL5Rα and a vector containing the same, host cell transformed with this vector, and method for producing and using the same
WO1997003191A1 (en) Novel factor viii:c polypeptide analogs comprising factor v domains or subdomains
AU651083B2 (en) Production of biologically active platelet-derived growth factor from high expression host cell systems
WO1997003194A1 (en) Lysine 1689 factor viii:c polypeptide analogs
US20020165358A1 (en) TCF mutant
EP0905234A2 (en) Allelic variant of human STAT3
EP0648268A1 (en) Therapeutic fragments of von willebrand factor
JPH0144317B2 (en)
GB2258234A (en) Soluble kit ligands
WO2001004307A1 (en) Alpha protein - 27
JPH10271998A (en) Production of polypeptide
JPH08173172A (en) Method of synthesizing inhibin alpha chain and/or beta chain

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA CN IL JP MX US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA