WO1997003193A1 - Novel factor viii:c polypeptide analogs with altered metal-binding properties - Google Patents

Abstract

Factor VIII:C polypeptide analogs are provided that are native Factor VIII:C polypeptides that contain modifications wherein the modifications comprise the alteration of the metal-binding properties of the molecule. Nucleic acid molecules encoding Factor VIII:C polypeptide analogs, vectors and host cells containing such nucleic acid molecules are also provided. Further provided are analog complexes that contain at least two such analogs. Methods of producing the analog, the analog complex, the nucleic acids, 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 VIII:C POLYPEPTIDE ANALOGS WITH ALTERED METAL-BINDING PROPERTIES

Field of the Invention This invention relates generally to active

Factor VIII:C polypeptide analogs having improved properties. More particularly, the invention pertains to Factor VIII:C polypeptide analogs having altered metal- binding properties. This invention further relates to analog complexes comprising at least two such analogs, or one such analog and a native Factor VIII:C polypeptide, nucleic acid molecules encoding such analogs, vectors and host cells comprising the nucleic acid molecules, pharmaceutical compositions comprising the analogs or analog complexes, methods of making the analogs, nucleic acid molecules, vectors and host cells, and methods of prevention or treatment of active Factor VIII:C deficiency using the analogs, complexes, and/or nucleic acid molecules, vectors and host cells.

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 consists of the heavy and light chains associated by a metal ion bridge, and lacking amino acids 741-1648 (the B domain) .

The mature Factor VIII:C molecule consists of a triplicated A domain of 330 amino acids, a unique B domain of 980 amino acids, and a duplicated C domain of 150 amino acids with the structure 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 described 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 VIII:C. A difficulty in such treatment is the relatively short half-life of externally administered Factor VIII:C, lasting about 8 to 12 hours.

It would be advantageous, therefore, to produce Factor VIII:C polypeptides with improved properties.

Summarv 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 the presence of altered etal- binding properties in the analog molecule.

Such a modification can be achieved by one or more amino acid substitutions, additions or deletions.

In accordance with a further object of the present invention, there is provided the 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 VIIIiC polypeptide analog and a Factor VIII:C polypeptide, together with a metal ion. For example, the analog complex herein can comprise two Factor VIIIiC polypeptide analogs, and the two analogs can be selected from the group consisting of analogs of molecular weights of about (a) 80 kD and 90 kD; (b) 73 kD and 90 kD; (c) 80 kD and 50 kD; (d) 80 kD and 43 kD; (e) 73 kD and 50 kD; and (f) 73 kD and 43 kD. In accordance with yet another object of the present invention, there is provided a method of producing a Factor VIIIiC polypeptide analog as above by (a) providing a native Factor VIII:C polypeptide that contains an amino acid sequence, and (b) modifying at least one amino acid residue in the amino acid sequence to produce an analog as above.

In accordance with still another object of the present invention, there is provided a nucleic acid molecule that contains a nucleotide sequence that encodes the 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 the 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 VIIIiC 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 the nucleic acid molecule as above, comprisingi (a) providing a nucleic acid molecule that encodes a native Factor VIIIiC polypeptide as above and (b) modifying at least one codon to provide the analog.

There is also provided, in accordance with another object of the present invention, a method of producing the recombinant vector that contains the 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 the recombinant vector.

There is also provided, in accordance with an object of the present invention, a pharmaceutical composition that contains the active Factor VIIIiC 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

VIIIiC deficiency in a mammal comprising administering thereto a therapeutically effective amount of (a) an active Factor VIIIiC polypeptide analog as above, or (b) an active Factor VIIIiC polypeptide analog complex as above, or (c) the nucleic acid molecule as above, or (d) the recombinant vector as above, or (e) the nucleic acid molecule as above together with and an active Factor

VIIIiC polypeptide analog, or (f) the recombinant vector as above together with an active Factor VIIIiC 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.

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 inventor herein has discovered that Factor VIIIiC polypeptide analogs can be made that have improved properties. These analogs carry one or more amino acid residues that are modified from the native structure such that the molecule displays altered metal-binding properties. The modification can be at least one amino acid substitution, addition or deletion, at any portion of the native Factor VIIIiC molecule, so long as Factor VIII:C activity is not destroyed. Definitions

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 substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring. A Factor VIII:C polypeptide includes but is not limited to, for example, the following Factor VIII:C polypeptides: (a) a full-length Factor VIII:C molecule comprising a signal peptide and all A, B, and C domains; (b) a mature Factor VIIIiC molecule comprising all A, B, and C domains and lacking the signal peptide; (c) a truncated Factor VIIIiC molecule lacking the signal peptide and at least a portion of the B domain; (d) a cleaved Factor VIIIiC molecule comprising a light chain subunit of about 80 kD; (e) a cleaved Factor VIII:C molecule comprising a heavy chain fragment of about 90 kD; (f) a cleaved Factor

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 utein or derivative is made. Such muteins or derivatives may have, for example, amino acid insertions, deletions, or substitutions in the relevant molecule that do not substantially affect its properties. 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 procoagulation activity. Such activity is measured using standard assays for blood plasma samples, such as, for example, the Coatest assay or the activated partial, thromboplastin time test (APTT) . An "active" Factor VIII:C polypeptide analog will have at least about 50% of the coagulation or procoagluation activity displayed by the native molecule, preferably at leaεt 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.

By "alteration" of the metal-binding properties of native Factor VIII:C is meant, for example, that the metal-binding properties of native Factor VIIIiC are modified such that binding affinity for a particular metal is increased relative to the native molecule at a particular site or portion of the molecule, or that additional metal-binding sites are present in the analog, or that sites are present in the analog which confer the ability to bind metals not normally bound by the native molecule. Other modifications to the metal-binding properties of the Factor VIIIiC molecule will be readily apparent to one of skill in the art when read in light of the present disclosure and are also captured by the definition. 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 virus or viral vector or from a linear or circular plasmid. The term "regulatory control" refers to control of expression of a polynucleotide sequence by a regulatory element to which the polynucleotide sequence is operably linked. The nature of such regulatory control differs depending upon the host organism. In prokaryotes, such regulatory control is effected by regulatory sequences which generally include, for example, a promoter, and/or a transcription termination sequence. In eukaryotes, generally, such regulatory sequences include, for example, a promoter and/or a transcription termination sequence. Additionally, other components which control of expression, for example, a signal peptide sequence or a secretory leader sequence for secretion of the polypeptide, and a terminator for transcriptional termination, may be attached thereto to facilitate regulatory control of expression.

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 VIIIiC analogs of the invention may be determined by means known in the art, for example, by using the commercially available Coatest assay.

Preferably, the effective amount is sufficient to bring about prevention of further deterioration or treatment to improve coagulation, that is, to enhance coagulation properties such that hemostasis is achieved. The exact amount necessary will vary depending on the subject being treated; the age and general condition of the individual to be treated; the functionality of the endogenous Factor VIII:C gene present in the individual; and the mode of administration, among other factors. An appropriate effective amount can be readily determined by one of skill in the art. For example, depending on the severity of active Factor VIII:C polypeptide deficiency, up to about 1000 to about 3000 U of Factor VIIIiC polypeptide analog can be given to an average person such as a 70 kg male patient. Alternatively, sufficient Factor VIIIiC polypeptide analog or analog complex can be given to establish a plasma level of about 0.5 to about 2 U/ml of Factor VIIIiC analog or combination analog and native polypeptide. See U.S. Patent Nos. 3,631,018; 3,652,530, and 4,069,216 for methods of administration and amounts. 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 substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles. Suitable carriers may also be present and are generally large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles. Such carriers are well known to those of ordinary skill in the art. A thorough discussion of pharmaceutically acceptable excipients is 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 VIIIiC polypeptide analogs with improved properties. The analogs include modifications to the native Factor VIIIiC amino acid sequence which comprise amino acid substitutions, deletions or additions that confer altered metal-binding properties to the analog molecule. Such modifications can result in the addition of metal-binding sites or can include conformational changes which allow the molecule to bind metals more avidly. For example, the modifications can include the addition of metal binding sites to the heavy and light chains, particularly to the Al and A3 domains, to result in increased affinity between the heavy and light chains, and particularly between the Al and A3 domains thereof.

Similarly, additional metal-binding sites can be produced in the heavy chain domains, such as in the Al and A2 domains, to result in increased affinity between components of the heavy chain, such as between the Al and A2 domains. The augmented affinity will therefore result in decreased dissociation of the Factor VIIIiC molecule into inactive forms.

Furthermore, the modifications can be such that other divalent cation binding motifs result which allow for the association of a variety of other metals, not normally associated with the Factor VIIIiC molecule. For example, the primary and secondary structure of a protein together affect the propensity of a given portion of the protein to bind to a given metal. The conformational factors and amino acids associated with binding to particular metals are known. See, e.g., Tainer et al., Current Opin . Biotech . (1991) 2i582-591; Vallee and Auld, Biochem . (1990) 29i5647-5659; Toma et al., Biochem . (1991) 30197-106; Pantoliano et al., Biochem . (1988) 27ι8311-8317. Hence, amino acid residues can be added or substituted in appropriate portions of the native Factor VIIIiC molecule, based on, e.g., computer generated models of the native molecule, to produce new metal- binding sites. A particularly useful model of the Factor VIIiC molecule is described in Pan et al., Nature

Structural Biology (1995) 2i740-744. In this way, intramolecular bridging between domains of the Factor VIIIiC polypeptide analog can occur with a wide variety of divalent cations, including Mn²⁺, Fe²⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Ca²⁺ and Mg²⁺. Such a modification can be accomplished by the addition of EF hands, zinc fingers, His-X-X-X-His motifs, and the like, to the native Factor VIIIiC sequence. See, e.g., Tainer et al., Current Opin . Biotech . (1991) 2ι582-591; Vallee and Auld, Biochem . (1990) 29 5647-5659; Toma et al., Biochem . (1991) 30ι97- 106. For example, a particularly preferred modification involves creating a new type II copper binding site between the Al and A3 domains of the native Factor VIIIiC sequence, such as by substituting His or Met for any of Phe652, Tyrl786, Lysl818, Aspl840 and/or Asnl864, numbered relative to the native sequence as described in, e.g., U.S. Patent no. 5,045,455 and Truett et al., DNA (1985) 41333-349. Preferably, His will be substituted for at least two of these amino acids and more preferably His will be substituted for any two amino acids selected from the group of amino acids consisting of Phe652, Tyrl786 and Aspl840, also numbered relative to the native Factor VIIIiC sequence.

It is readily apparent that modifications can be made to the native Factor VIIIiC sequence by the addition, deletion or substitution of several amino acid residues, or by the addition, deletion or substitution of as little as one amino acid residue. The above-described modifications can be accomplished using, e.g., site- directed mutagenesis. See, e.g., Toma et al., Biochem . (1991) 30:97-106; Pantoliano et al., Biochem . (1988) 27:8311-8317. Such modifications result in an analog with improved qualities, such as enhanced stability, increased plasma half-lives, decreased antigenicity and the like. Nucleic acid molecules encoding the present

Factor VIIIiC polypeptide analogs can be made by modifying the native nucleic acid sequence that encodes the Factor VIIIiC polypeptide or cDNA sequences that encode the Factor VIIIiC polypeptides. In this regard, the DNA sequence and corresponding amino acid sequence of native Factor VIIIiC is known. See, e.g., U.S. Patent no. 5,045,455 and Truett et al., DNA (1985) 4ι333-349. Modifications can be made to the native Factor VIIIiC sequence by conventional techniques such as site-directed mutagenesis. For example, the M13 method for site directed mutagenesis is known, as described in Zoller and Smith, Nucleic Acids Res . (1982) lO 6487-6500, Methods Enzymol . (1983) lOOi 468-500, and DNA (1984) 3ι 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 mutagenesis, one or more of the codons in the selected portion of Factor VIIIiC, can be mutated by substitution, deletion or addition, to one or more codons encoding a metal-binding site or to codons which encode a molecule with greater affinity for a particular metal. A description of a protocol suitable for use herein for mutagenesis of specific sites of a Factor VIIIiC expression plasmid can be found in WO 87/07144.

The nucleic acid molecules of the present invention can also be made synthetically by piecing together nucleic acid molecules encoding heavy and light chain fragments derived from cDNA clones or genomic clones containing Factor VIIIiC coding sequences, preferably cDNA clones, using known linker sequences. Alternatively, the entire sequence or portions of nucleic acid sequences encoding analogs described above may be prepared by synthetic methods (e.g. using DNA synthesis machines) . Once made, the nucleic acid molecules can be inserted in vectors for production of recombinant vectors for transcription and translation of the nucleic acid molecules. One skilled in the art of DNA cloning and in possession of the DNA encoding native Factor VIIIiC polypeptide will be able to prepare suitable DNA molecules for production of the present analogs using known cloning procedures (e.g. restriction enzyme digestion, exonuclease digestion, ligation, and other appropriate procedures) outlined in any of the followingi Sambrook, et al, MOLECULAR CLONINGI A LABORATORY MANUAL 2nd ed. (Cold Spring Harbor Laboratory Press, 1989) ; DNA CLONING, Vol. I and II, D.N. Glover ed. (IRL Press, 1985); OLIGONUCLEOTIDE SYNTHESIS, M.J. Gait ed. (IRL Press, 1984) ; NUCLEIC ACID HYBRIDIZATION, B.D. Hames & S.J. Higgins eds. (IRL Press, 1984) ; TRANSCRIPTION AND TRANSLATION, B.D. Hames & S.J. Higgins eds., (IRL Press, 1984) ; ANIMAL CELL CULTURE, R.I. Freshney ed. (IRL Press, 1986); IMMOBILIZED CELLS AND ENZYMES, K. Mosbach (IRL Press, 1986); B. Perbal, A PRACTICAL GUIDE TO MOLECULAR CLONING, Wiley (1984); the series, METHODS IN ENZYMOLOGY, Academic Press, Inc.; GENE TRANSFER VECTORS FOR MAMMALIAN CELLS, J.H. Miller and M.P. Calos eds. (Cold Spring

Harbor Laboratory, 1987) ; METHODS IN ENZYMOLOGY, Vol. 154 and 155, Wu and Grossman, eds., and Wu, ed. , respectively (Academic Presε, 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) , 24:810-817; Munemitsu et al, Mol . Cell . Biol . , (1990) 20:5977-5982. 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 Presε, 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 sites into which the present nucleic acid molecule of the invention can be inserted. This vector also typically contains a selection marker for detection of the presence of the vector in the host cell. The vector can also provide, if desired, one or more regulatory elementε or control sequences for expresεion of the nucleic acid molecule. For example, the preεent vector can be derived from a plaε id, a virus, a cosmid, or a bacteriophage. This vector is typically capable of behaving as an autonomouε unit of replication when introduced into a hoεt cell. Moreover, the vector may be one that is capable of episomal existence or of integration into the host cell genome. A wide variety of replication systems are available, typically derived from viruseε that infect mammalian host cells. Illuεtrative replication systems include the replication syεtemε from Simian viruε 40, adenoviruε, bovine papilloma virus, polyoma virus, Epstein Barr virus, and the like. Thuε, the nucleic acid molecule of the preεent invention can be inεerted at an appropriate reεtriction 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 uεed for tranεfection or transformation of a host cell.

The host cellε of the invention can be, for example, prokaryotic or eukaryotic hoεt cellε, including bacterial, yeast, insect and mammalian expreεεion εyεtemε. Preferably the analogε of the preεent invention are expressed in mammalian host cell systemε.

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 hoεt cell can be used for expresεion of the Factor VIII:C polypeptide analogε.

The expreεεion caεsettes are introduced into the host cell by conventional methods, depending on the expresεion system used, as described further below.

Where viruseε are involved, transfection or transduction may be employed. The particular manner in which the host cell is transformed is not critical to thiε invention, depending substantially upon whether the expresεion cassetteε are joined to a replication εyεtem and the nature of the replication εyεtem and aεεociated geneε. Coexpreεsion of more than one Factor VIII:C polypeptide analog may be desired. For example, it may be desirable to express the light and heavy chains using separate constructs. In this regard, either or both of the light and heavy chainε may include modificationε as described above. "Coexpresεion" as used herein refers to the expreεsion of two or more Factor VIII:C polypeptides in a single host cell. Thus, for example, the expresεion of the 90 kD species and the 80 kD specieε in a εingle hoεt cell, would conεtitute "coexpression" as used herein. The polynucleotideε encoding for the polypeptideε can be harbored in a εingle vector, either under the control of the εame regulatory elementε or under the control of εeparate elements. Thus, the production of a fusion protein including active portionε of the two or more Factor VIII:C polypeptideε would be conεidered "coexpreεεed" for purpoεeε of the present definition as would the expreεεion of two geneε as a dicistronic conεtruct employing an internal riboεome entry site. Similarly, proteins expreεεed from the same vector but driven by separate regulatory elements, would alεo be conεidered "coexpreεεed." The term also refers to the expression of two or more proteins from separate conεtructε. Thuε, the expression of proteinε encoded from genes present on separate vectorε in a hoεt cell would also be considered "coexpresεion" for purposes of the present invention.

The tranεformed/tranεfected cellε 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, εo that the media or cell lyεate may be iεolated and the Factor VIII:C active complex extracted and purified. Similarly, the full-length molecule can be iεolated and treated under complex-forming conditionε, e.g., with the addition of calcium and the appropriate enzymeε, to form the active complex. Variouε meanε 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 iε 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.

Expresεion in Bacterial Cellε

Bacterial expreεεion εyεtemε can be uεed to produce the subject Factor VIII:C polypeptide analogs and nucleic acid molecules encoding the analogs. Control elements for use in bacterial syεtemε include promoters, optionally containing operator εequenceε, and ribosome binding siteε. Useful promoterε include εequenceε derived from εugar metabolizing enzymes, such aε galactoεe, lactose (lac) and maltose. Additional examples include promoter sequenceε derived from bioεynthetic enzymes such as tryptophan ( trp) , the β- lactamaεe (jbla) promoter εyεtem, bacteriophage λPL, and T7. In addition, εynthetic promoters can be used, εuch aε the tac promoter. The β-lactamaεe and lactoεe promoter systems are described in Chang et al., Nature (1978) 275 : 615, and Goeddel et al., Nature (1979) 281 : 544; the alkaline phoεphataεe, tryptophan (trp) promoter εystem are described in Goeddel et al., Nucleic Acids Res . (1980) 8 : 4057 and EP 36,776 and hybrid promoters such as the tac promoter is described in U.S. Patent No. 4,551,433 and deBoer et al., Proc. Natl. Acad. Sci. USA (1983) 80 : 21-25. However, other known bacterial promoterε useful for expresεion of eukaryotic proteinε are alεo suitable. A perεon skilled in the art would be able to operably ligate such promoters to the present Factor VIII:C polypeptide analog coding sequenceε, for example, aε deεcribed in Siebenliεt et al., Cell (1980) 20 : 269, uεing linkerε or adapterε to εupply any required reεtriction εiteε. Promoterε for uεe in bacterial εyεtemε alεo generally will contain a Shine-Dalgarno (SD) εequence operably linked to the DNA encoding the Factor VIII:C analog polypeptide. For prokaryotic hoεt cellε that do not recognize and proceεε the native polypeptide εignal sequence, the signal sequence can be εubεtituted by a prokaryotic εignal εequence εelected, 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 syεtemε are particularly compatible with Escherichia coli . However, numerous other εyεtemε for uεe in bacterial hoεts including Gram- negative or Gram-positive organiεmε εuch as Bacillus spp . , Streptococcus spp . , Streptomyces spp . , Pseudomonas specieε such as P . aeruginosa, Salmonella typhimurium, or

Serratia marcescans, 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 cells by electroporation, nuclear injection, or protoplast fusion as described generally in Sambrook et al. (1989), cited above. Theεe examples are illustrative rather than limiting. Preferably, the host cell should secrete minimal amounts of proteolytic enzymes. Alternatively, in vitro methodε of cloning, e.g., PCR or other nucleic acid polymeraεe reactionε, are εuitable.

Prokaryotic cellε uεed to produce the Factor VIII:C analog polypeptideε of thiε invention are cultured in suitable media, aε described generally in Sambrook et al., cited above.

Expreεεion in Yeaεt Cellε

Yeaεt expreεεion εyεtemε can alεo be uεed to produce the subject Factor VIII:C polypeptide analogs and nucleic acid moleculeε encoding the analogs. Expresεion and transformation vectorε, either extrachromoεomal repliconε or integrating vectorε, have been developed for tranεformation into many yeaεtε. For example, expression vectors have been developed for, among others, the following yeastε: Saccharomyces cerevisiae ,aε deεcribed in Hinnen et al., Proc . Natl . Acad . Sci . USA (1978) 75 : 1929; Ito et al., J . Bacteriol . (1983) 153 : 163; Candida albicans aε deεcribed in Kurtz et al., Mol . Cell . Biol . (1986) 6 : 142; Candida maltosa, aε deεcribed in Kunze et al., J. Basic Microbiol . (1985) 25 : 141; Hansenula polymorpha, aε deεcribed in Gleeεon 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, aε deεcribed in De Louvencourt et al., J . Bacteriol . (1983) 254: 737 and Van den Berg et al., Bio /Technology (1990) 8: 135; Pichia guillerimondii, as described in Kunze et al., J. Basic Microbiol . (1985) 25 : 141; Pichia pastoris, aε deεcribed in Cregg et al., Mol . Cell . Biol . (1985) 5 : 3376 and U.S. Patent Noε. 4,837,148 and 4,929,555; Schizosaccharomyces pombe, aε deεcribed in Beach and Nurεe, Nature (1981) 300 : 706; and Yarrowia lipolytica, aε deεcribed in Davidow et al., Curr. Genet . (1985) 10 : 380 and Gaillardin et al., Curr . Genet . (1985) 20: 49, Aspergillus hosts such aε A. nidulans, as described in Ballance et al., Biochem . Biophys . Res . Commun . (1983) 222: 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 reesia, aε deεcribed in EP 244,234, and filamentouε fungi εuch aε, e.g, Neurospora, Penicillium, Tolypocladium, aε described in WO 91/00357. Control εequenceε for yeaεt vectorε are known and include promoter regionε from genes such as alcohol dehydrogenase (ADH) , as described in EP 284,044, enolase, glucokinase, glucoεe-6-phoεphate isomerase, glyceraldehyde-3-phosphate-dehydrogenase (GAP or GAPDH) , hexokinaεe, phoεphofructokinaεe, 3-phoεphoglycerate mutase, and pyruvate kinase (PyK) , aε deεcribed in EP 329,203. The yeaεt PH05 gene, encoding acid phoεphataεe, also provides useful promoter sequences, as deεcribed in Myanohara et al., Proc. Natl . Acad . Sci . USA (1983) 80:1. Other εuitable promoter sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase, as described in Hitzeman et al., J . Biol . Chem . (1980) 255 : 2073, or other glycolytic enzymeε, such as pyruvate decarboxylase, trioεephosphate iεomerase, and phosphoglucose iεomeraεe, as described in Hess et al., J^". Adv . Enzyme Reg . (1968) 7: 149 and Holland et al., Biochemistry (1978J 17 : 4900. Inducible yeast promoters having the additional advantage of transcription controlled by growth conditionε, include from the liεt above and others the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphataεe, degradative enzymeε associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactoεe utilization. Suitable vectorε and promoterε for uεe in yeast expresεion are further described in Hitzeman, EP 073,657. Yeaεt enhancers also are advantageously uεed with yeast promoters. In addition, synthetic promoters which do not occur in nature also function as yeaεt promoterε. For example, upεtream activating sequences (UAS) of one yeast promoter may be joined with the tranεcription activation region of another yeaεt promoter, creating a εynthetic hybrid promoter. Exampleε of εuch hybrid promoters include the ADH regulatory sequence linked to the GAP transcription activation region, as described in U.S. Patent Noε. 4,876,197 and 4,880,734. Other exampleε of hybrid promoterε include promoters which consist of the regulatory sequenceε of either the ADH2, GAL4 , GAL10, or PH05 genes, combined with the transcriptional activation region of a glycolytic enzyme gene such as GAP or PyK, as described in EP 164,556. Furthermore, a yeast promoter can include naturally occurring promoterε of non-yeaεt origin that have the ability to bind yeaεt RNA polymeraεe and initiate tranεcription. Other control elementε which may be included in the yeaεt expreεεion 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 sequenceε which encode signal sequenceε for secretion. DNA encoding suitable signal εequenceε can be derived from genes for secreted yeast proteins, εuch aε the yeast invertase gene as described in EP 012,873 and JP 62,096,086 and the α-factor gene, as described in U.S. Patent Noε. 4,588,684, 4,546,083 and 4,870,008; EP 324,274; and WO 89/02463. Alternatively, leaderε of non-yeast origin, such as an interferon leader, alεo provide for εecretion in yeast, aε deεcribed in EP 060,057.

Methodε of introducing exogenous DNA into yeast hostε are well known in the art, and typically include either the tranεformation of εpheroplaεtε or of intact yeaεt cellε treated with alkali cationε. Tranεfor ationε into yeast can be carried out according to the method described in Van Solingen et al., J. Bact . (1977) 230: 946 and Hsiao et al., Proc . Natl . Acad . Sci . USA (1979) 76 : 3829. However, other methods for introducing DNA into cells εuch aε by nuclear injection, electroporation, or protoplaεt fusion may also be used as described generally in Sambrook et al., cited above.

For yeaεt secretion the native polypeptide signal sequence may be subεtituted by the yeast invertase, α-factor, or acid phosphatase leaders. The origin of replication from the 2μ plasmid origin iε suitable for yeaεt. A εuitable εelection gene for uεe in yeast is the trpl gene preεent in the yeaεt plasmid described in Kingsman et al., Gene (1979) 7: 141 or Tεchemper et al., Gene (1980) 20: 157. The trpl gene provideε a selection 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 εequence encoding a yeast protein can be linked to a coding sequence of a Factor VIIIiC polypeptide analog to produce a fusion protein that can be cleaved intracellularly by the yeast cellε upon expression. An example, of such a yeast leader sequence is the yeast ubiquitin gene.

Expresεion in Insect Cells

The Factor VIIIiC polypeptide analogs and nucleic acid molecules encoding the analogs can alεo be produced in inεect expreεεion εystems. For example, baculovirus expresεion vectorε (BEVε) are recombinant inεect viruεeε in which the coding sequence for a foreign gene to be expresεed is inserted behind a baculovirus promoter in place of a viral gene, e.g., polyhedrin, aε deεcribed in Smith and Summerε, U.S. Pat. No., 4,745,051. An expreεεion conεtruct herein includes a DNA vector useful as an intermediate for the infection or transformation of an inεect cell system, the vector generally containing DNA coding for a baculovirus transcriptional promoter, optionally but preferably, followed downstream by an insect signal DNA sequence capable of directing secretion of a deεired protein, and a site for insertion of the foreign gene encoding the foreign protein, the signal DNA sequence and the foreign gene being placed under the transcriptional control of a baculovirus promoter, the foreign gene herein being the coding sequence of a Factor VIIIiC polypeptide analog of this invention.

The promoter for use herein can be a baculovirus transcriptional promoter region derived from any of the over 500 baculoviruses generally infecting insects, such as, for example, the Orderε Lepidoptera, Diptera, Orthoptera, Coleoptera and Hymenoptera including, for example, but not limited to the viral DNAε of Autographo calif ornica MNPV, Bombyx mori NPV, rrichoplusia ni MNPV, Rachlplusia ou MNPV or Galleria mellonella MNPV, Aedes aegypti, Drosophila melanogaster, Spodoptera frugiperda , and Trichoplusia ni . Thuε, the baculoviruε transcriptional promoter can be, for example, a baculovirus immediate-early gene IEI or IEN promoter; an immediate-early gene in combination with a baculoviruε delayed-early gene promoter region εelected from the group conεisting 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 tranεcriptional enhancer elementε.

Particularly εuitable for uεe herein iε the εtrong polyhedrin promoter of the baculoviruε, which directε a high level of expreεεion of a DNA insert, as described in Frieεen 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 plaεmid for uεe herein usually alεo contains the polyhedrin polyadenylation signal, as described in Miller et al., Ann. Rev. Microbiol . (1988) 42 : 117 and a procaryotic ampicillin- reεiεtance (amp) gene and an origin of replication for selection and propagation in E. coli . DNA encoding suitable signal sequences can also be included and is generally derived from genes for secreted inεect or baculoviruε proteinε, εuch as the baculovirus polyhedrin gene, as described in Carbonell et al., Gene (1988) 73 : 409, aε well aε mammalian εignal εequenceε εuch as thoεe derived from geneε encoding human α-interferon aε deεcribed in Maeda et al., Nature (1985) 325: 592-594; human gastrin-releasing peptide, aε deεcribed in Lebacq- Verheyden et al., Mol . Cell . Biol . (1988) 8: 3129; human IL-2, aε deεcribed in Smith et al., Proc . Natl . Acad . Sci . USA (1985) 82 : 8404; mouεe IL-3 , as described in Miyajima et al., Gene (1987) 58 : 273; and human glucocerebroεidaεe, aε deεcribed in Martin et al., DNA (1988) 7:99.

Numerouε baculoviral εtrains and variantε and correεponding permiεεive insect host cells from hosts such as Spodoptera frugiperda (caterpillar) , Aedes aegypti (mosquito) , Aedes albopictus (mosquito) , Drosophila melanogaster (fruitfly) , and Bombyx mori host cells have been identified and can be uεed herein. See, for example, the description in Luckow et al.,

Bio/Technology (1988) 6: 47-55, Miller et al., in GENETIC ENGINEERING (Setlow, 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 geneε in addition to the polyhedrin promoter may be employed to advantage in a baculoviruε expreεsion syεtem. Theεe include immediate-early (alpha) , delayed-early (beta) , late (gamma) , or very late (delta) , according to the phaεe of the viral infection during which they are expreεεed. The expreεεion of theεe genes occurs εequentially, probably aε the result of a "cascade" mechanism of transcriptional regulation. Thus, the immediate-early genes are expresεed immediately after infection, in the abεence of other viral functionε, and one or more of the reεulting gene productε induces transcription of the delayed-early genes. Some delayed-early gene productε, in turn, induce tranεcription of late geneε, and finally, the very late geneε are expreεεed under the control of previously expresεed gene products from one or more of the earlier classes. One relatively well defined component of this regulatory cascade iε IEI, a preferred immediate-early gene of Autographo calif ornica nuclear polyhedrosis virus (AcMNPV) . IEI is presεed in the absence of other viral functions and encodes a product that εtimulates the transcription of several genes of the delayed-early clasε, including the preferred 39K gene, as described in Guarino and Summers, J . Virol . (1986) 57 : 563-571 and J^". Virol . (1987) 61 : 2091-2099 aε well aε late genes, as deεcribed in Guanno and Summerε, Virol. (1988) 262ι 444-451. Immediate-early geneε aε described above can be used in combination with a baculovirus gene promoter region of the delayed-early category. Unlike the immediate-early geneε, such delayed-early genes require the presence of other viral geneε or gene productε εuch aε thoεe of the immediate-early genes. The combination of immediate-early genes can be made with any of several delayed-early gene promoter regions such aε 39K or one of the delayed-early gene promoterε 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 such that expreεεion can be further controlled by the preεence of IEI, as described in L. A. Guarino and Summers (1986a) , cited above; Guarino & Summers (1986b) J . Virol . , (1986) 60ι 215-223, and Guarino et al. (1986c), J. Virol. (1986) 60 : 224-229. Additionally, when a combination of immediate-early geneε with a delayed-early gene promoter region iε uεed, enhancement of the expreεεion of heterologouε genes can be realized by the presence of an enhancer εequence in direct ciε linkage with the delayed-early gene promoter region. Such enhancer sequences are characterized by their enhancement of delayed-early gene expresεion in εituationε where the immediate-early gene or itε product iε limited. For example, the hr5 enhancer sequence can be linked directly, in ciε, to the delayed-early gene promoter region, 39K, thereby enhancing the expreεεion of the cloned heterologouε DNA aε deεcribed in Guarino and Summerε (1986a) , (1986b) , and Guarino et al. (1986) .

The polyhedrin gene iε claεεified aε a very late gene. Therefore, transcription from the polyhedrin promoter requireε the previouε expresεion of an unknown, but probably large number of other viral and cellular gene productε. Becauεe of thiε delayed expreεεion of the polyhedrin promoter, εtate-of-the-art BEVs, such as the exemplary BEV syεtem deεcribed by Smith and Summerε in, for example, U.S. Pat. No., 4,745,051 will expreεε foreign genes only as a result of gene expression from the rest of the viral genome, and only after the viral infection is well underway. Thiε repreεentε a limitation to the uεe of exiεting BEVε. The ability of the host cell to proceεε newly εyntheεized proteins decreaseε aε the baculovirus infection progreεεeε. Thuε, gene expreεεion from the polyhedrin promoter occurs at a time when the hoεt cell's ability to process newly synthesized proteins is potentially diminished for certain proteinε such as human tissue plasminogen activator. As a consequence, the expression of secretory glycoproteins in BEV syεtems iε complicated due to incomplete secretion of the cloned gene product, thereby trapping the cloned gene product within the cell in an incompletely proceεεed form.

While it haε been recognized that an inεect εignal sequence can be uεed to expreεs a foreign protein that can be cleaved to produce a mature protein, the present invention is preferably practiced with a mammalian signal sequence for example the Factor VIII signal sequence. An exemplary insect εignal εequence εuitable herein iε the εequence encoding for a Lepidopteran adipokinetic hormone (AKH) peptide. The AKH family conεiεts of short blocked neuropeptides that regulate energy subεtrate mobilization and metaboliεm in inεectε. In a preferred embodiment, a DNA εequence coding for a Lepidopteran Manduca εexta AKH εignal peptide can be uεed. Other insect AKH signal peptides, such as those from the Orthoptera Schistocerca gregaria locus can alεo be employed to advantage. Another exemplary inεect signal sequence is the sequence coding for Drosophila cuticle proteins such as CPl, CP2, CP3 or CP4.

Currently, the most commonly used transfer vector that can be uεed herein for introducing foreign genes into AcNPV is pAc373. Many other vectors, known to those of skill in the art, can alεo be uεed herein. Materials and methods for baculovirus/insect cell expresεion εyεtemε are commercially available in a kit form from companieε εuch aε Invitrogen (San Diego CA) ("MaxBac" kit) . The techniqueε utilized herein are generally known to those skilled in the art and are fully described in Summers and Smith, A MANUAL OF METHODS FOR BACULOVIRUS VECTORS AND INSECT CELL CULTURE PROCEDURES, Texas Agricultural Experiment Station Bulletin No. 1555, Texas A&M University (1987); Smith et al., Mol . Cell . Biol . (1983) 3ι 2156, and Luckow and Summers (1989) . These include, for example, the use of pVL985 which alterε the polyhedrin εtart codon from ATG to ATT, and which introduceε a BamHl cloning site 32 basepairε downεtream from the ATT, aε deεcribed in Luckow and Summerε, Virology (1989) 17 : 31.

Thuε, for example, for insect cell expresεion of the preεent polypeptideε, the desired DNA sequence can be inserted into the transfer vector, using known techniques. An inεect cell hoεt can be cotranεformed with the transfer vector containing the inserted desired DNA together with the genomic DNA of wild type baculoviruε, uεually by cotranεfection. The vector and viral genome are allowed to recombine reεulting in a recombinant virus that can be easily identified and purified. The packaged recombinant virus can be uεed to infect insect hoεt cellε to expreεε a Factor VIIIiC polypeptide analog.

Other methods that are applicable herein are the standard methods of inεect cell culture, cotranεfection and preparation of plaεmids are set forth in Summers and Smith (1987) , cited above. Thiε reference also pertains to the εtandard methodε of cloning genes into AcMNPV transfer vectorε, plaεmid DNA iεolation, tranεferring genes into the AcmMNPV genome, viral DNA purification, radiolabeling recombinant proteinε and preparation of inεect cell culture media. The procedure for the cultivation of viruεeε and cellε are deεcribed in

Volkman and Summers, J. Virol . (1975) 29:820-832 and Volkman, et al., J . Virol . ( 1976) 29:820-832.

Expresεion in Mammalian Cellε Mammalian expression syεtemε can alεo be uεed to produce the Factor VIIIiC polypeptide analogs and nucleic acid molecules encoding the analogs. Typical promoterε for mammalian cell expreεεion include the SV40 early promoter, the CMV promoter, the mouεe mammary tumor viruε LTR promoter, the adenovirus major late promoter (Ad MLP) , and the herpes εimplex viruε promoter, among others. Other non-viral promoterε, such as a promoter derived from the murine metallothionein gene, will also find use in mammalian constructε. Mammalian expreεεion may be either conεtitutive or regulated (inducible) , depending on the promoter. Typically, tranεcription termination and polyadenylation sequenceε 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 alεo present. Exampleε of tranεcription terminator/polyadenylation εignalε include thoεe derived from SV40, aε 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 sites, may also be designed into the conεtructε of the preεent invention.

Enhancer elementε can also be uεed herein to increaεe expression levels of the mammalian constructε. Exampleε include the SV40 early gene enhancer, aε 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 Viruε, aε described in Gorman et al., Proc . Natl . Acad . Sci . USA (1982b) 79 : 6777 and human cytomegaloviruε, aε deεcribed in Boεhart et al., Cell (1985) 42: 521. A leader εequence can alεo be preεent which includeε a εequence encoding a εignal peptide, to provide for the εecretion of the foreign protein in mammalian cellε. Alternatively, the Factor VIII εignal peptide can be used. Preferably, there are proceεεing εiteε encoded between the leader fragment and the gene of intereεt εuch 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 provideε for εecretion of a foreign protein in mammalian cells.

There exist expression vectors that provide for the transient expression in mammalian cells of DNA encoding the Factor VIII:C analog polypeptideε. In general, tranεient expreεεion involveε the uεe of an expression vector that is able to replicate efficiently in a host cell, such that the host cell accumulateε many copieε of the expreεεion vector and, in turn, εyntheεizes high levelε of a deεired polypeptide encoded by the expression vector. Transient expresεion systemε, comprising a εuitable expreεεion vector and a host cell, allow for the convenient positive identification of polypeptides encoded by cloned DNAs, as well aε for the rapid εcreening of εuch polypeptideε for deεired biological or phyεiological properties. Thus, tranεient expreεεion εyεtemε are particularly useful for purposes of identifying additional polypeptides that have Factor VIII:C-like activity.

Once complete, the mammalian expression vectors can be used to transform any of εeveral mammalian cells. Methodε for introduction of heterologouε polynucleotideε into mammalian cellε are known in the art and include dextran-mediated tranεfection, calcium phoεphate precipitation, polybrene mediated tranεfection, protoplaεt fuεion, electroporation, encapsulation of the polynucleotide(ε) in lipoεomeε, and direct microinjection of the DNA into nuclei. General aspects of mammalian cell host syεtem transformationε have been deεcribed by Axel in U.S. 4,399,216. A εynthetic lipid particularly uεeful for polynucleotide tranεfection is N-[l-(2,3- dioleyloxy)propyl]-N,N,N-trimethylammonium chloride, which is commercially available under the name Lipofectin® (available from BRL, Gaithersburg, MD) , and is described by Feigner et al., Proc . Natl . Acad . Sci . USA (1987) 84:7413. Mammalian cell lines available as hosts for expression are also known and include many immortalized cell lines available from the American Type Culture Collection (ATCC) , including but not limited to, Chineεe hamster ovary (CHO) cells, HeLa cellε, baby hamεter kidney (BHK) cellε, monkey kidney cells (COS) , human hepatocellular carcinoma cells (e.g., Hep G2) , human embryonic kidney cells, baby hamster kidney cells, mouεe sertoli cells, canine kidney cellε, buffalo rat liver cells, human lung cells, human liver cells, mouse mammary tumor cellε, aε well as others. The mammalian host cells uεed to produce the target polypeptide of thiε invention may be cultured in a variety of media. Commercially available media such aε Ham's F10 (Sigma), Minimal Essential Medium ([MEM], Sigma) , RPMI-1640

(Sigma), and Dulbecco's Modified Eagle's Medium ([DMEM1, Sigma) are εuitable for culturing the hoεt cellε. In addition, any of the media deεcribed in Ham and Wallace, Meth . Enz. (1979) 58 : 44, Barneε and Sato, Anal . Biochem . (1980) 102 : 255, U.S. Patent Noε. 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 aε culture media for the hoεt cells. Any of these media may be supplemented as necessary with hormones and/or other growth factors such aε inεulin, tranεferrin, or epidermal growth factor, εaltε (such aε εodium chloride, calcium, magneεium, and phoεphate) , buffers (εuch aε HEPES) , nucleosides (such as adenosine and thymidine) , antibiotics (εuch aε Gentamycin(tm) M drug), trace elementε (defined aε inorganic compoundε uεually preεent at final concentrations in the micromolar range) , and glucose or an equivalent energy source. Any other necessary supplementε may alεo be included at appropriate concentrations that would be known to thoεe skilled in the art. The culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artiεan.

The active Factor VIII:C analogε produced according to the invention have a variety of uses. For example, the analogs can be used aε immunogenε for the production of antibodieε. The analogε can also be used for the treatment of hemophiliacs and other hoεtε having blood clotting diεorderε. The Factor VIII:C analogε may diεplay increased plasma half-life or specific activity. Thus, the analogs may allow for lower dosageε or alternative modeε of adminiεtration and may improve hemoεtaεiε in hemophiliacs.

Alternatively, nucleic acid molecules or vectors comprising polynucleotide εequenceε encoding the Factor VIII:C analogε can be uεed directly for gene therapy and adminiεtered uεing εtandard gene delivery protocolε. In thiε regard, the nucleotide sequences encoding the Factor VIII:C analogs can be εtably integrated into the hoεt cell genome or maintained on a stable episomal element in the host cell. Methods for gene delivery are known in the art. See, e.g., U.S. Patent No. 5,399,346.

A number of viral based syεtemε have been developed for gene tranεfer into mammalian cellε. For example, retroviruεeε provide a convenient platform for gene delivery εyεtems. A selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo . A number of retroviral syεtemε have been deεcribed (U.S. Patent No. 5,219,740; Miller and Roεman, BioTechniques (1989) 7:980-990; Miller, A.D., Human Gene Therapy (1990) 2:5-14; Scarpa et al., Virology (1991) 180:849-852; Burnε et al., Proc . Natl . Acad . Sci . USA (1993) 90:8033-8037; and Boriε- 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 extrachromosomally thuε minimizing the riεkε aεεociated with insertional mutagenesis (Haj-Ahmad and Graham, J . Virol . (1986) 57:267-274; Bett et al., J . Virol . (1993) 67l5911-5921; Mittereder et al., Human Gene Therapy (1994) 5:717-729; Seth et al., J . Virol . (1994) 68:933-940; Barr et al., Gene Therapy (1994) 1:51-58; Berkner, K.L. BioTechniques (1988) 6:616-629; and Rich et al., Human Gene Therapy (1993) 41461-476) .

Additionally, various adeno-associated virus (AAV) vector εyεtemε have been developed for gene delivery. Such εyεtemε can include control εequences, such aε promoter and polyadenylation εiteε, aε well aε selectable markers or reporter genes, enhancer sequences, and other control elements which allow for the induction of tranεcription. AAV vectors can be readily constructed using techniques well known in the art. See, e.g., U.S. Patent Nos. 5,173,414 and 5,139,941; International Publication Noε. WO 92/01070 (publiεhed 23 January 1992) and WO 93/03769 (publiεhed 4 March 1993) ; Lebkowski et al., Molec . Cell . Biol . (1988) 8 3988-3996; Vincent et al., Vaccines 90 (1990) (Cold Spring Harbor Laboratory Presε) ; Carter, B.J. Current Opinion in Biotechnology (1992) 31533-539; Muzyczka, N. Current Topics in

Microbiol . and Immunol . (1992) 158ι97-129; Kotin, R.M. Human Gene Therapy (1994) 5i793-801; Shelling and Smith, Gene Therapy (1994) 1 165-169; and Zhou et al., J. Exp . Med. (1994) 179ι1867-1875. Additional viral vectorε which will find uεe for delivering the nucleic acid moleculeε encoding the Factor VIIIiC analog polypeptides for gene transfer include those derived from the pox family of viruses, including vaccinia virus and avian poxvirus. By way of example, vaccinia viruε recombinantε expreεεing the novel Factor VIIIiC analogs can be constructed as follows. The DNA encoding the particular analog is firεt inεerted 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 iε then used to transfect cellε which are εimultaneously infected with vaccinia. Homologouε recombination serveε to inεert 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 preεence of 5- bromodeoxyuridine and picking viral plaqueε reεistant thereto.

A vaccinia based infection/transfection εystem can be conveniently used to provide for inducible, transient expression of the Factor VIIIiC analogε in a host cell. In this εyεtem, cellε are firεt infected in vitro with a vaccinia virus recombinant that encodeε the bacteriophage T7 RNA polymeraεe. Thiε polymerase displays exquisite specificity in that it only transcribeε templateε bearing T7 promoterε. Following infection, cellε are transfected with the polynucleotide of interest, driven by a T7 promoter. The polymerase expressed in the cytoplasm from the vaccinia viruε recombinant transcribes the transfected DNA into RNA which iε then tranεlated into protein by the hoεt tranεlational machinery. The method provideε for high level, tranεient, cytoplaεmic production of large quantitieε of RNA and itε tranεlation productε. See, e.g., Elroy-Stein and Moεε, Proc . Natl . Acad . Sci . USA (1990) 8716743-6747; Fuerst et al., Proc . Natl . Acad . Sci . USA (1986) 8318122-8126.

Alternatively, avipoxviruseε, such as the fowlpox and canarypox viruses, can also be uεed to deliver the Factor VIIIiC analog geneε. Recombinant avipox viruses, expresεing immunogens from mammalian pathogens, are known to confer protective immunity when adminiεtered to non-avian species. The use of an avipox vector is particularly deεirable in human and other mammalian εpecieε εince members of the avipox genus can only productively replicate in susceptible avian species and therefore are not infective in mammalian cells. Methods for producing recombinant avipoxviruses are known in the art and employ genetic recombination, as described above with respect to the production of vaccinia viruεeε. 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 εyεtem can be uεed that will lead to high level expreεεion following introduction into hoεt cellε. Specifically, a T7 RNA polymeraεe 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 expresεion iε under the control of the T7 promoter. Thus, some of the T7 RNA polymerase generated from tranεlation of the amplification template RNA will lead to transcription of the desired gene. Because some T7 RNA polymerase iε 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 techniqueε. However the uεe of a plaε id iε preferred. Since high level expreεεion 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 repreεεor in the appropriate bacterial εtrain would interfere with the tranεcription 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 beginε 3' of the T7 gene and continueε through the 5' end of the T7 promoter. Such tranεcription will generate an antiεenεe tranεcript and reduce or eliminate tranεlation of T7 RNA polymerase RNAs. The second tranεcription unit conεisting of the T7 promoter preceding the gene of interest can be provided by a εeparate plaεmid or can be engineered onto the amplification plaεmid. Colocalization of the two transcription units is beneficial for ease of manufacturing and ensureε that both tranεcription unitε will alwayε be together in the cellε into which the plasmid is introduced. The T7 RNA polymerase plasmidε may include UTRε which comprise an Internal Ribosome Entry Site (IRES) present in the leader sequenceε of picornaviruεeε such aε the encephalomyocarditiε viruε

(EMCV) UTR (Jang et al. J . Virol . (1989) 63 : 1651-1660) . This εequence serves to enhance expresεion of εequences under the control of the T7 promoter. For a further discussion of T7 syεtemε and their uεe for tranεforming cellε, εee, e.g., International Publication No. WO 94/26911; Studier and Moffatt, J. Mol . Biol . (1986) 2891113-130; Deng and Wolff, Gene (1994) 243ι245-249; Gao et al., Biochem . Biophys . Res . Commun . (1994) 200:1201- 1206; Gao and Huang, Nuc . Acids Res . (1993) 21:2867-2872; Chen et al., Nuc . Acids Res . (1994) 22:2114-2120; and U.S. Patent No. 5,135,855.

Vectorε encoding the εubject Factor VIIIiC analogε can also be packaged in liposomeε prior to delivery to the εubject or to cellε 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:micromoleε lipid) , or more of lipid. For a review of the use of lipoεomeε aε carrierε 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 uεe in the inεtant invention include cationic (poεitively charged) , anionic (negatively charged) and neutral preparationε, with cationic liposomes particularly preferred. Cationic liposomes have been εhown to mediate intracellular delivery of plaεmid 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 tranεcription factorε (Debε et al., J . Biol . Chem . (1990) 265:10189-10192) , in functional form.

Cationic lipoεomeε are readily available. For example, N[1-2, 3-dioleyloxy)propyl]-N,N,N-triethy1- ammonium (DOTMA) lipoεomeε are available under the trademark Lipofectin, from GIBCO BRL, Grand Iεland, 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 lipoεomes can be prepared from readily available materialε 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 deεcription of the synthesis of DOTAP (1, 2-bis(oleoyloxy) -3- (trimethylammonio)propane) lipoεomeε.

Similarly, anionic and neutral lipoεo es are readily available, εuch as from Avanti Polar Lipids (Birmingham, AL) , or can be easily prepared uεing readily available materialε. Such materialε include phoεphatidyl choline, choleεterol, phoεphatidyl ethanolamine, dioleoylphoεphatidyl choline (DOPC) , dioleoylphoεphatidyl glycerol (DOPG) , dioleoylphoεhatidyl ethanolamine (DOPE) , among otherε. Theεe materialε can alεo be mixed with the DOTMA and DOTAP starting materialε in appropriate ratios. Methods for making liposomeε uεing theεe materialε are well known in the art. The lipoεomes can comprise multilammelar vesicleε (MLVε) , small unilamellar veεicleε (SUVε) , or large unilamellar veεicleε (LUVε) . The variouε lipoεome- nucleic acid complexes are prepared uεing 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; Papahadjopouloε et al., Biochim . Biophys . Acta (1975) 394:483; Wilεon et al., Cell (1979) 17 : 77 ) ; Deamer and Bangham, Biochim . Biophys . Acta (1976) 443:629; Oεtro 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 Papahadjopouloε, Proc . Natl . Acad . Sci . USA (1978) 75:145; and Schaefer-Ridder et al., Science (1982) 215:166.

The recombinant vectors (whether or not encapεulated in lipoεomeε) , may be administered in pharmaceutical compositionε aε described above. The pharmaceutical compositions will comprise sufficient genetic material to produce a therapeutically effective amount of the analog or analogs, as described above. For purpoεeε of the present invention, an effective dose will be from about 0.05 mg/kg to about 50 mg/kg of the DNA constructε in the individual to which it iε adminiεtered. Once formulated, the compoεitions of the invention can be adminiεtered directly to the εubject or, alternatively, in the caεe of the vectorε 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 tranεfection, calcium phoεphate precipitation, polybrene mediated tranεfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomeε, and direct microinjection of the DNA into nuclei, all well known in the art.

Direct delivery of the compoεitionε will generally be accompliεhed by injection, either εubcutaneouεly, intraperitoneally, intravenouεly or intramuεcularly. Other modes of administration include oral and pulmonary adminiεtration, εuppoεitorieε, and tranεdermal applicationε. Dosage treatment may be a single dose schedule or a multiple dose εchedule.

Although any εimilar or equivalent methodε and materialε may be employed in the practice or teεting of the present invention, the preferred methods and materials are now described.

The present invention will now be illuεtrated by reference to the following exampleε which εet forth particularly advantageouε embodimentε. However, it εhould be noted that these embodimentε are illuεtrative and are not to be conεtrued aε reεtricting the invention in any way.

Exampleε

The Factor VIII:C polypeptide analogs are made uεing conventional mutageneεiε techniqueε. In thiε regard, mutagenesis of the Factor VIII:C nucleotide sequences can be performed utilizing plaεmidε which include εequences encoding the full-length molecule, plaεmidε encoding the light and heavy chains and various modificationε of theεe molecules, depending on the Factor VIII:C analog desired. Such plasmidε are known and described in, e.g., U.S. Patent no. 5,045,455. The plasmids are first linearized e.g., by using restriction endonucleases which cleave at unique restriction sites. Once linearized, the plasmids are treated with calf intestine phoεphataεe and εeparated on low melting temperature triε-acetate agaroεe gelε. The linearized band iε extracted by adεorption to εilica dioxide and eluted in triε-EDTA. The plaεmid iε then denatured and the deεired phosphorylated mutagenic oligonucleotide is added. The mixture iε 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 triphoεphate, 3-4 unitε/μL of Klenow fragment of E . coli DNA polymeraεe I and 400 units/μL of T4 DNA ligase.

The reactions are terminated using phenolchloroform extraction and ethanol precipitation. DNA obtained iε used to transform bacterial host cells and positive cloneε εelected. DNA from the clones iε tranεferred to nitrocelluloεe, filters prepared, and hybridized to screening probes to ensure that the mutagenic oligonucleotide iε introduced into the correct fragment. Final mutationε are confirmed by DNA εequencing.

The DNA can be prepared by banding in CsCl and can be used to transfect COS-1 monkey cellε aε described in Kaufman, PNAS (1982) 82:689. After transfection, the polypeptide analog is isolated and Factor VIII:C activity is assayed by the Kabi Coatest chro agenic asεay method for the ability to clot Factor VIII deficient plaεma before and after thrombin activation.

The preεent invention haε been deεcribed with reference to εpecific embodiments. However, this application is intended to cover those changes and subεtitutions which may be made by those skilled in the art without departing from the spirit and the scope of the appended claimε.

Claims

WHAT IS CLAIMED IS:

1. An active Factor VIII:C polypeptide analog comprising a native Factor VIII:C polypeptide that is modified, wherein the modification comprises alteration of the metal-binding properties of native Factor VIII:C.

2. The analog of claim 1, wherein the modification is at least one amino acid addition, deletion or substitution, or a combination thereof.

3. The analog of claim 1, wherein the modification compriseε increaεing the metal ion-binding affinity of metal-binding sites of native Factor VIIIiC.

4. The analog of claim 1, wherein the modification comprises the addition of metal-binding siteε to the native Factor VIIIiC molecule.

5. The analog of claim 3, wherein the metal- binding εiteε are in the Al and/or A2 domains.

6. The analog of claim 4, wherein the metal- binding siteε are added to the Al and/or A2 domains.

7. The analog of claim 3, wherein the metal- binding sites are in the Al and/or A3 domains.

8. The analog of claim 4, wherein the metal- binding εiteε are added to the Al and/or A3 domains.

9. The analog of claim 8 wherein the modification comprises a substitution of His or Met for at least one amino acid selected from the group consiεting of Phe652, Tyrl786, Lyεl818, Aspl840 and Asnl864, numbered with reεpect to the native Factor VIIIiC polypeptide εequence.

10. The analog of claim 9 wherein the modification compriseε a εubεtitution of Hiε for at leaεt two amino acidε selected from the group consisting of Phe652, Tyrl786 and Aεpl840.

11. The analog of claim 1, wherein the native Factor VIIIiC polypeptide iε εelected from the group conεiεting ofi a) a full-length Factor VIIIiC molecule compriεing 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 εignal peptide and at leaεt a portion of the B domain; d) a cleaved Factor VIII:C molecule compriεing 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 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 compriεing a light chain fragment of molecular weight of about 73 kD.

12. An active Factor VIII:C polypeptide analog complex compriεing at least two Factor VIII:C polypeptide analogε aε claimed in claim 11, or at least one Factor VIII:C polypeptide analog and at least one Factor VIII:C polypeptide, wherein the complex compriseε a metal ion.

13. The analog complex of claim 11, wherein the complex compriεeε two Factor VIIIiC polypeptide analogε, and the Factor VIIIiC polypeptideε that are modified to form the two analogε are εelected from the group consisting 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.

14. The analog complex of claim 12, wherein the metal ion is a divalent cation.

15. The analog complex of claim 14, wherein the divalent cation iε a Ca⁺⁺ ion or a Cu⁺⁺ ion.

16. A method of producing a Factor VIIIiC polypeptide analog, compriεingi a) providing a native Factor VIIIiC polypeptide that compriεeε an amino acid sequence; and b) modifying at leaεt one amino acid reεidue in the amino acid εequence to produce the analog of claim 1.

17. A nucleic acid molecule compriεing a nucleotide εequence that encodeε a Factor VIIIiC polypeptide analog, wherein the analog compriεeε a native

Factor VIIIiC polypeptide that is modified, wherein the modification compriseε alteration of the metal-binding propertieε of native Factor VIIIiC.

18. A recombinant vector compriεing the nucleic acid molecule of claim 17 and a regulatory element, wherein the nucleic acid molecule is under regulatory control of the regulatory element.

19. A recombinant host cell comprising the nucleic acid molecule of claim 17.

20. A recombinant hoεt cell comprising the recombinant vector of claim 18.

21. A method of producing an active Factor

VIII:C polypeptide analog comprising: a) providing the recombinant host cell of claim 19; and b) allowing the recombinant host cell to expresε the analog.

22. A method of producing an active Factor VIII:C polypeptide analog compriεing: a) providing the recombinant hoεt cell of claim 20 and b) allowing the recombinant hoεt cell to expreεε the analog.

23. A method of producing a nucleic acid molecule that encodeε a Factor VIII:C polypeptide analog compriεing: a) providing a nucleic acid molecule that encodeε a native Factor VIII:C polypeptide, wherein the nucleic acid molecule compriεeε a codon for each amino acid reεidue in the native Factor VIII:C polypeptide; and b) modifying at leaεt one codon, to produce the nucleic acid molecule of claim 17.

24. The method of producing a nucleic acid molecule as claimed in claim 23, wherein the modification is performed by site directed mutagenesis or by use of polymerase chain reaction techniques.

25. 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 17.

26. A method of producing a recombinant host cell that comprises a nucleic acid molecule that compriεeε a nucleotide εequence that encodeε a Factor VIII:C polypeptide analog, comprising transforming a host cell with the nucleic acid molecule of claim 17.

27. A method of producing a recombinant host cell that comprises a recombinant vector that compriεeε a nucleotide εequence that encodeε a Factor VIII:C polypeptide analog, compriεing tranεforming a host cell with the recombinant vector of claim 18.

28. A pharmaceutical composition compriεing the active Factor VIII:C polypeptide analog of claim 1 and a pharmaceutically acceptable excipient.

29. A pharmaceutical compoεition comprising the active Factor VIII:C polypeptide analog complex of claim 12 and a pharmaceutically acceptable excipient.

30. A method for prevention or treatment of active Factor VIII:C deficiency in a mammal comprising administering thereto a therapeutically effective amount of an active Factor VIII:C polypeptide analog as claimed in claim 1.

31. A method for prevention or treatment of active Factor VIII:C deficiency in a mammal comprising administering thereto a therapeutically effective amount of an active Factor VIIIiC polypeptide analog complex aε claimed in claim 12.

32. A method for prevention or treatment of active Factor VIIIiC deficiency in a mammal comprising administering thereto a therapeutically effective amount of the nucleic acid molecule of claim 17.

33. 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 18.

34. 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 17 and an active Factor VIII:C polypeptide analog.

35. 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 18 and an active Factor VIIIiC polypeptide analog.

36. A method for prevention or treatment of active Factor VIIIiC deficiency in a mammal comprising administering thereto a therapeutically effective amount of the nucleic acid molecule of claim 17 and an active Factor VIIIiC analog complex.

37. A method for prevention or treatment of active Factor VIIIiC deficiency in a mammal compriεing adminiεtering thereto a therapeutically effective amount of the recombinant vector of claim 18 and an active Factor VIIIiC analog complex.