WO1997003194A1

WO1997003194A1 - Lysine 1689 factor viii:c polypeptide analogs

Info

Publication number: WO1997003194A1
Application number: PCT/US1996/011441
Authority: WO
Inventors: Rae Lyn Burke; Steven Rosenberg
Original assignee: Chiron Corporation
Priority date: 1995-07-11
Filing date: 1996-07-09
Publication date: 1997-01-30
Also published as: AU6456096A

Abstract

Factor VIII:C polypeptide analogs are provided that are native Factor VIII:C polypeptides that include an Arg to Lys substitution at position 1689. Nucleic acid molecules encoding the Factor VIII:C polypeptide analogs, vectors and host cells containing such nucleic acid molecules are also provided. Further provided are analog complexes that contain the analog. 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

LYSINE 1689 FACTOR VIII . C POLYPEPTIDE ANALOGS

Field of the Invention This invention relates generally to active Factor VHI.C polypeptide analogs having improved properties. More particularly, the invention pertains to a Factor VIII:C polypeptide analog wherein Argl689 is substituted with Lys. This invention further relates to analog complexes comprising 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 analog 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 NIII.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 cDΝA 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 Ν-terminal residues comprise the signal sequence. Therefore, the mature Factor NIII: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.

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 ΝH2-A 1-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.

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. Summary of the Invention It is, therefore, an object of the present invention to provide a Factor VIII.C polypeptide analog or analog complex that has improved properties. In accordance, therewith, there is provided an active Factor VII C polypeptide analog that is substantially the same as a native Factor VIILC polypeptide, except for the presence of Lys at position 1689, numbered relative to the native molecule as reported in U.S. Patent no. 5,045,455 and Truett et al , DNA (1985) 4:333-349.

In accordance with a further object of the present invention, there is provided the analog as above where the native Factor VIILC polypeptide that is modified is selected from the group consisting of (a) a full-length Factor VIILC molecule comprising a signal peptide and all A, B, and C domains; (b) a native Factor VIILC molecule comprising all A, B, and C domains and lacking a signal peptide; (c) a truncated Factor VIILC molecule lacking a signal peptide and at least a portion of the B domain; and (d) a cleaved Factor VIILC molecule containing a light chain subunit of molecular weight of about 80 kD.

In accordance with a further object of the present invention, there is provided an active Factor VIILC analog complex that contains a light chain Factor VIILC polypeptide analog having a Lys at position 1689, numbered relative to the native molecule, and a heavy chain Factor VIILC polypeptide, together with a metal ion.

In accordance with yet another object of the present invention, there is provided a method of producing a Factor VIILC polypeptide analog as above by (a) providing a native Factor VIILC polypeptide that contains Arg 1689, numbered relative to the native molecule, and (b) substituting Arg 1689 with a Lys residue, 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 VIILC 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, comprising: (a) providing a nucleic acid molecule that encodes a native Factor VIILC polypeptide having an Arg at position 1689, numbered relative to the native molecule; and (b) modifying the codon encoding the Arg at position 1689 to encode a Lys, 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 VIILC 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 VIILC deficiency in a mammal comprising administering thereto a therapeutically effective amount of (a) an active Factor VIILC polypeptide analog as above, or (b) an active Factor VIILC 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 VIILC polypeptide analog, or (f) the recombinant vector as above together with an active Factor VIILC 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 incoφorated by reference.

The inventor herein has discovered that a Factor VIILC polypeptide analog comprising the substitution of Arg 1689 with a Lys, can be made that has improved properties.

Definitions

The term "Factor VIILC 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, glycosylation s, 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 VIILC polypeptide includes but is not limited to, for example, the following Factor VIILC polypeptides: (a) a full-length Factor VIILC molecule comprising a signal peptide and all A, B, and C domains; (b) a mature Factor VIILC molecule comprising all A, B, and C domains and lacking the signal peptide; (c) a truncated Factor VIILC molecule lacking the signal peptide and at least a portion of the B domain; and (d) a cleaved Factor VIILC molecule comprising a light chain subunit of about 80 kD.

Factor VIILC 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 analog 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 VIILC polypeptide analog will have at least about 50% of the coagulation or pro-coagluation 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 VIILC 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 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 VIILC 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 VIILC 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 VIILC polypeptide deficiency, up to about 1000 to about 3000 U of Factor VIILC polypeptide analog can be given to an average person such as a 70 kg male patient. Alternatively, sufficient Factor VIILC polypeptide analog or analog complex can be given to establish a plasma level of about 0.5 to about 2 U/ml of Factor VIILC 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. , NJ. 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 for a Factor VIILC polypeptide analog with improved properties. The analog includes an amino acid substitution at position 1689 of the native Factor VIILC amino acid sequence, such that Argl689 is substituted with Lys. Other modifications to the native Factor VIILC sequence can also be present so long as Factor VIILC activity is not destroyed. The above-described modifications can be readily accomplished using, e.g. , site- directed mutagenesis to result in an analog with improved qualities, such as enhanced activity, increased stability, increased plasma half-lives, decreased antigenicity and the like.

Nucleic acid molecules encoding the present Factor VIILC polypeptide analogs can be made by modifying the native nucleic acid sequence that encodes the Factor VIILC polypeptide or cDNA sequences that encode the Factor VIILC polypeptides. In this regard, the DNA sequence and corresponding amino acid sequence of native Factor VIILC is known. See, e.g. , U.S. Patent no. 5,045,455 and Truett et al , DNA (1985) 4:333-349. Modifications can be made to the native Factor VIILC 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) 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 mutagenesis, the codon for Arg 1689 can be mutated by substitution, to a codon encoding Lys, such as AAA or AAG. A description of a protocol suitable for use herein for mutagenesis of specific sites of a Factor VIILC 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 VIILC 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 VIILC 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 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 Press, 1985); OLIGONUCLEOTIDE SYNTHESIS, MJ. Gait ed. (IRL Press, 1984); NUCLEIC ACID HYBRIDIZATION, B.D. Hames & SJ. Higgins eds. (IRL Press, 1984); TRANSCRIPTION AND TRANSLATION, B.D. Hames & SJ. Higgins eds. , (IRL Press, 1984); ANIMAL CELL CULTURE, R.L 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 Press, 1987), IMMUNOCHEMICAL METHODS IN CELL AND MOLECULAR BIOLOGY, RJ. 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), 74:810-817; Munemitsu et al, Mol. Cell. BioL , (1990) 70:5977-5982. 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 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 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 episomal 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 systems include the replication systems from Simian virus 40, adenovirus, bovine papilloma virus, polyoma virus, Epstein Barr virus, 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 cells, including bacterial, yeast, insect and mammalian expression systems. 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 VIILC polypeptide analogs.

The expression cassettes are introduced into the host cell by conventional methods, depending on the expression system used, as described 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.

Coexpression of more than one Factor VIILC polypeptide may be desired. For example, it may be desirable to express the light and heavy chains using separate constructs. "Coexpression" as used herein refers to the expression of two or more Factor VIILC polypeptides in a single host cell. Thus, for example, the expression of the 90 kD species 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 VIILC polypeptides would be considered "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 expressed from the same vector but driven by separate regulatory elements, would also be considered "coexpressed." The term also refers to the expression 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 chains have been used for coexpression, the product can be obtained as a complex of the two Factor VIILC chains, so that the media or cell lysate may be isolated and the Factor VIILC 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-solvent 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 VIILC polypeptide analogs and nucleic acid molecules encoding the analogs. Control elements for use in bacterial systems include promoters, optionally containing operator sequences, 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 such as tryptophan (trp), the β- lactamase (bla) promoter system, bacteriophage λPL, and T7. In addition, synthetic promoters can be used, such as the tac promoter. The 3-lactamase and lactose promoter systems are described in Chang et al , Nature (1978) 275: 615, and Goeddel et al. , Nature. (1979) 281: 544; the alkaline phosphatase, tryptophan (tφ) promoter system 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 promoters useful 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 VIILC polypeptide analog coding sequences, for example, as described in Siebenlist et al, Cell (1980) 20: 269, using linkers or adapters to supply any required restriction sites. Promoters for use in bacterial systems also generally will contain a Shine-Dalgarno (SD) sequence operably linked to the DNA encoding the Factor VIILC analog polypeptide. For prokaryotic host cells that do not recognize and process the native polypeptide signal 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 Escherichia coli. However, numerous other systems for use in bacterial hosts including Gram- negative or Gram-positive organisms such as Bacillus spp. , Streptococcus spp. , Streptomyces spp. , Pseudomonas species such as P. aeruginosa, Salmonella typhimurium, or Serratia marcescans, among others. Methods for introducing exogenous DNA into these hosts typically include the use of CaC^ 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. These examples are illustrative rather than limiting. Preferably, the host cell should 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 VIILC analog polypeptides of this invention are cultured in suitable media, as described generally in Sambrook et al. , cited above.

Expression in Yeast Cells

Yeast expression systems can also be used to produce the subject Factor VIILC polypeptide analogs and nucleic acid molecules encoding the analogs. Expression and transformation vectors, either extrachromosomal replicons or integrating vectors, have been developed for transformation into many yeasts. For example, expression vectors have been developed for, among others, the following yeasts: Saccharomyces cerevisiae ,as described in Hinnen et al. , Proc. Natl. Acad. Sci. USA (1978) 75: 1929; Ito et al. , J. Bacteriol. (1983) 753: 163; Candida albicans 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: 731 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, as described in Cregg et aL , Mol. Cell. BioL (1985) 5: 3376 and U.S. Patent Nos. 4,837, 148 and 4,929,555; Schizosaccharomyces 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, Aspergillus hosts such as A. nidulans, as described in Ballance et aL , Biochem. Biophys. Res. Commun. (1983) 772: 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, as described in EP 244,234, and filamentous fungi such as, e.g, Neurospora, 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 isomerase, 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 suitable 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 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 , Biochemistry (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 as 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 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 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 sequences 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 described 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 Hsiao et aL , Proc. Natl. Acad. Sci. USA (1979) 76: 3829. However, other methods for introducing DNA into cells such as by nuclear injection, electroporation, 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 substituted by the yeast invertase, α-factor, or acid phosphatase leaders. The origin of replication from the 2μ plasmid origin is suitable for yeast. A suitable 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 Tschemper et al. , Gene (1980) 10: 157. The trp] gene provides a selection marker for a mutant strain of yeast 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 VIILC 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 is the yeast ubiquitin gene.

Expression in Insect Cells

The Factor VIILC polypeptide analogs and nucleic acid molecules encoding teh analogs can also be produced in insect expression systems. 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 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 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 VIILC 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, rrichoplusia ni MNPV, Rachlplusia ou MNPV or Galleria mellonella MNPV, Aedes 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 HmdIII 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 suitable for use 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, as described in Miller et al. , Ann. Rev. Microbiol. (1988) 42: 11 and a procaryotic ampicillin-resistance (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 insect or baculovirus 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 glucocerebrosidase, 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 (cateφillar), Aedes aegypti (mosquito), Aedes albopictus (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 (Setlow, J.K. et al. eds.), Vol. 8 (Plenum Publishing, 1986), pp. 277-279, and Maeda et aL , Nature, (1985) 575: 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 products, in turn, induce transcription of late genes, 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 pressed 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 described in Guarino and Summers, J. Virol. (1986) 57: 563-571 and J. Virol. (1987) 61: 2091-2099 as well as late genes, 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 expressed such that expression can be further controlled by the presence of IEI, as described in L. A. Guarino and Summers (1986a), cited above; Guarino & Summers (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 is used, 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 sequences 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 described in Guarino and Summers (1986a), (1986b), and Guarino et al. (1986).

The polyhedrin gene is classified 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 system described by Smith and Summers in, for example, U.S. Pat. No. , 4,745,051 will express 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. This represents a limitation to the use of existing BEVs. The ability of the host cell to process newly synthesized proteins decreases as the baculovirus 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 processed form.

While it has been recognized that an insect signal sequence 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 signal 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 insects. 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 used transfer vector that can be used herein for introducing foreign genes into AcNPV is pAc373. Many other vectors, known to those of skill in the art, can also be used herein. Materials and methods for baculovirus/insect cell expression systems 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 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) 3: 2156, and Luckow and Summers (1989). These include, for example, the use of pVL985 which alters the polyhedrin start codon from ATG to ATT, and which introduces a BamΑl cloning site 32 basepairs downstream from the ATT, as described in Luckow and Summers, Virology (1989) 77:31. Thus, for example, for insect cell expression of the present polypeptides, the desired DNA sequence can be inserted into the transfer vector, using known techniques. An insect cell host can be cotransformed with the transfer vector containing the inserted desired DNA together with the genomic DNA of wild type baculovirus, usually by cotransfection. 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 VIILC polypeptide analog.

Other methods that are applicable herein are the standard methods of insect cell culture, cotransfection and preparation of plasmids 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, transferring 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) 79:820-832. Expression in Mammalian Cells

Mammalian expression systems can also be used to produce the Factor VIILC polypeptide analogs and nucleic acid molecules encoding the analogs. Typical promoters for mammalian cell expression include the SV40 early promoter, the CMV promoter, the mouse mammary tumor virus LTR promoter, the adenovirus major late promoter (Ad MLP), and the heφes 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 constructs. 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 VIILC polypeptide analog coding sequence, is also present. Examples of transcription 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 sites, may also be designed into the constructs of the present invention.

Enhancer elements can also be used herein to increase expression levels of the mammalian constructs. Examples 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 described in Boshart et al. , Cell (1985) 41: 521. A leader sequence 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 sites 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 expression vectors that provide for the transient expression in mammalian cells of DNA encoding the Factor VIILC analog polypeptides. In general, transient expression 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 expression 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 polypeptides for desired biological or physiological properties. Thus, transient expression systems are particularly useful for puφoses of identifying additional polypeptides that have Factor VIILC-like activity.

Once complete, the mammalian expression vectors 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 fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei. General aspects of mammalian cell host system transformations have been described by Axel in U.S. 4,399,216. A synthetic lipid particularly useful for polynucleotide transfection is N-[l-(2,3-dioleyloxy)propyl]-N,N,N-tri- methylammonium 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, Chinese hamster 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 host cells used to produce the target polypeptide of this invention may be cultured in a variety of media. Commercially available media such as Ham's F10 (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) 702: 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 necessary with hormones and/or other growth factors such as insulin, transferrin, or epidermal growth factor, salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucieosides (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 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 artisan.

The active Factor VIILC 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. The Factor VIILC 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 VIILC analogs can be used directly for gene therapy and administered using standard gene delivery protocols. In this regard, the nucleotide sequences encoding the Factor VIILC analogs can be stably integrated into the host 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 systems 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 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 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) 7:5-14; Scaφa et aL , Virology (1991) 780:849-852; 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 extrachromosomally thus minimizing the risks associated with insertional mutagenesis (Haj-Ahmad and Graham, J. Virol. (1986) 57:267-274; Bett et aL , J. Virol. (1993) 67:591 1-5921 ; 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) 7:51-58; 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 selectable markers or reporter genes, enhancer sequences, and other control elements which allow for the induction of transcription. 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 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 Press); Carter, BJ. Current Opinion in Biotechnology (1992) 3:533-539; Muzyczka, N. Current Topics in Microbiol. and Immunol. (1992) 758:97-129; Kotin, R.M. Human Gene Therapy (1994) 5:793-801 ; Shelling and Smith, Gene Therapy (1994) 7: 165-169; and Zhou et al. , J. Exp. Med. (1994) 779: 1867- 1875.

Additional viral vectors which will find use for delivering the nucleic acid molecules encoding the Factor VIILC 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 virus recombinants expressing the novel Factor VIILC 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 TKJecombinant 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 VIILC 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 specificity in that it only transcribes templates bearing T7 promoters. Following infection, cells are transfected with the polynucleotide of interest, driven by a T7 promoter. The polymerase expressed in the cytoplasm from the vaccinia virus recombinant transcribes the transfected 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, avipox viruses, such as the fowlpox and canarypox viruses, can also be used to deliver the Factor VIILC 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 species since 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 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 expression 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, some of the T7 RNA polymerase generated from translation of the amplification template RNA will lead to transcription 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 repressor 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 transcription unit consisting of the T7 promoter preceding the gene of interest can be provided by a separate plasmid or can be engineered onto the amplification plasmid. Colocalization of the two transcription units is beneficial for ease of manufacturing and ensures that both transcription units will always be together in the cells into which the plasmid is introduced. The T7 RNA polymerase plasmids may include UTRs which comprise an Internal Ribosome Entry Site (IRES) present in the leader sequences of picomaviruses such as the encephalomyocarditis virus (EMCV) UTR (Jang et al. J. Virol. (1989) 63: 1651-1660). This sequence serves to enhance expression of sequences under the control of the T7 promoter. For a further discussion of T7 systems and their use for transforming cells, see, e.g., Intemational Publication No. WO 94/2691 1 ; Studier and Moffatt, J. Mol. Biol. (1986) 789: 1 13-130; Deng and Wolff, Gene (1994) 743:245-249; Gao et al. , Biochem. Biophys. Res. Commun. (1994) 200: 1201-1206; Gao and Huang, Nuc. Acids Res. (1993) 27:2867-2872; Chen et al. , Nuc. Acids Res. (1994) 22:2114- 2120; and U.S. Patent No. 5, 135,855.

Vectors encoding the subject Factor VIILC analogs can also be packaged in liposomes prior to delivery to the subject or to cells derived therefrom. Lipid encapsulation 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) 7097: 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 liposomes have been shown 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-triethylammonium (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 liposomes include transfectace (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 synthesis 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 using readily available materials. Such materials include phosphatidyl choline, cholesterol, phosphatidyl 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 liposomes can comprise multilammelar vesicles (MLVs), small 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) 77:77); 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) 275: 166.

The recombinant vectors (whether or not encapsulated in liposomes), may be administered in pharmaceutical compositions as 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 puφoses 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, altematively, 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. , Intemational 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, electroporation, 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 subcutaneously, intraperitoneally, intravenously or intramuscularly. Other modes of administration include oral and pulmonary administration, suppositories, and transdermal applications. Dosage treatment may be a single dose schedule or a multiple dose schedule.

Although any similar 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. Examples The present invention will now be illustrated by reference to the following examples which set forth particularly advantageous embodiments. However, it should be noted that these embodiments are illustrative and are not to be construed as restricting the invention in any way.

Example 1 Construction of Plasmids An expression cassette for the production of the Factor VIILC polypeptide analog was prepared using plasmid pSVF8-80 which is an expression plasmid for the 80 kD Factor VIILC light chain. Plasmid pSVF8-80 was derived from plasmid pSV7d (Truett et al , DNA (1985) 4:333-349) and was constructed as follows. Starting from the Sail site in the polylinker pSV7d, pSVF8-80 consists of a 201 bp fragment of a tissue plasminogen activator cDNA from nucleotides -98 to + 103 (relative to the start codon) terminating at a Bglll site (tPA sequences given in Degan et al, J. Biol. Chem. (1986) 267:6972-6985), a 29 bp synthetic Bglll to Bell linker-adaptor encoding nucleotides +5002 to +5031 of Factor VIILC ligated to a 2464 bp Bell fragment of factor VIILC spanning from a Bell site created at nucleotide 5028 of the factor VIILC cDNA through in vitro mutagenesis (Zoller and Smith, Meth. Enzymol (1983) 700:468) (pF8GM7), to a Bell site in the 3' untranslated region, at nucleotide 7492, and a 400 bp fragment of tPA 3' untrans¬ lated sequence spanning from a Bglll site to a synthetic PstI site generated from the cDNA cloning, followed by the polylinker from the vector M13mp9 (Vieira and Messing, Gene (1982) 79:259) and then pSV7d. Plasmid pSVF8-80R→K is a derivative of plasmid pSVF8-80, where the codon encoding Arg 1689 was changed by site-directed mutagenesis, to a codon encoding Lys (indicated by *). - pSVF8-80ΔR is also a derivative of plasmid pSVF8-80 and encodes for a Factor VIILC polypeptide analog where Argl689 is deleted. Example 2 Transfection and Expression in COS7 Cells Plasmid pSVF8-80R→K, encoding the Arg to Lys Factor VIILC polypeptide analog, and plasmid pSVF8-80ΔR, which encodes for a Factor VIILC polypeptide analog where Arg 1689 is deleted, were co-transfected along with a wild type heavy chain plasmid, pSVF8-92E, into COS7 cells (Guzman, Cell (1981) 23: 175) to test for activity as follows. COS7 cells were transfected using the calcium phosphate coprecipitation method (van der Eb and Graham, Meth. Enzymol. (1980) 65:826-39) coupled with treatment with chloroquine diphosphate (Luthman and Magnusson, Nuc. Acids Res. (1983) 77: 1295-1308) using 50 μg of plasmid DNA per 5x10 cells for 14 hr. Cells may also be transfected by the DEAE-dextran method of Sompayrac and Danna, Proc. Nat. Acad. Sci. USA (1981) 78:7575-78.

The COS7 cells were cultured in Dulbecco's modified Eagle medium supplemented with 10% fetal calf serum, 100 U/mL penicillin, 100 μg/mL streptomycin, 292 μg/mL giutamine, and 110 μg/mL sodium pyruvate. Samples were obtained from a 48-hour collection of serum-containing medium at 88 hours post transfection and tested for activity using the Coatest assay (Rosen et al , Thromb. and Haemostasis (1985) 54:818-823), which measures the generation of activated Factor X (Xa) as a linear function of the concentration of exogenously supplied Factor VIILC. As shown in Table 1 , both light chain mutants are active. The Arg to Lys substitution derivative shows a two-fold higher specific activity than the wild type chain.

To determine the fate of these chains, both in the medium and after exposure to thrombin, radioimmunoprecipitations of the conditioned medium before and after treatment with thrombin were performed as follows. Samples from a 24 hr pulse labeling of the COS7 cells above, transfected with the various factor VIILC plasmids at approximately 86 hr post-transfection, were immunoprecipitated with antisera according to standard protocols. The samples tested included (1) cell lysate and medium, respectively, from cells transfected with pSVF8-92ΔR plus pSVF8-80; (2) cell lysate and medium from expression of pSVF8-92R→K plus pSVF8-80; (3) cell lysate and medium from expression of pSVF8-92 plus pSVF8-80; (4) cell lysate and medium from expression of pSVF8- 92 plus pSVF8-80ΔR; and (5) cell lysate and medium from expression of pSVF8- 92 plus pSVF8-80R→K. These samples were electrophoresed on a 10% acrylamide gel which was then fluorographed and exposed for 1 week. The wild type molecule showed substantial processing to a 73 kd doublet and to a smaller 68 kd species in the medium. The 80-R→K molecule showed little cleavage and the 80ΔR mutant showed no chain cleavage.

Essentially the same results were obtained when the medium was pretreated with thrombin as follows. The samples were as described above. Samples containing approximately 150 munit of light chain by ELISA assay were incubated with 15 munits of thrombin for 3 min. at 20°C, then thrombin inhibitor was added to a final concentration of I mM to stop the reaction. Samples were also treated with thrombin inhibitor alone. Finally, an attempt was made to thrombin-activate each of these molecules as follows. For the thrombin activation, a medium sample containing wild type heavy and light chains with a total of 37 mU of coagulation activity was incubated in a volume of 200 μl in 20 M imidazole, 150 mM NaCl, 2.5 mM CaCl₂, 100 mM lysine HCl, pH 6.8 at 4°C. The reaction was started at time 0 by the addition of either 25 mU of thrombin or buffer alone and transferred to room temperature. Samples were removed at selected times and immediately assayed for coagulation activity. The same experiment was also performed on medium containing the wild type 92 chain and the mutant 80R→K chain. The 80ΔR, 92ΔR and 92R→K species were essentially not thrombin activatable under the assay conditions. However the 80R→K peptide was thrombin activatable to the same extent as the wild type chain although at a slower rate.

In summary, these results show that the 80R→K Factor VIILC polypeptide analog has increased stability in the medium, higher specific activity, and slower activation kinetics. Table 1

Analysis of Mutant Light Chain Activities by Transient Expression³

DNAs CAg Coagulation Coatest Coatest/CAg mu/ml mu/ml mu/ml

80 + 92 950 240 160 0.168

80Δ + 92 1300 17 33 0.025

80R→K + 92 550 171 205 0.373

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 VIILC polypeptide analog comprising a native Factor VIILC polypeptide that is modified, wherein the modification comprises substitution of the Arg residue at position 1689, numbered with respect to the native Factor VIILC polypeptide sequence, with Lys.

2. The analog of claim 1 , wherein the native Factor VIILC polypeptide is selected from the group consisting of: a) a full-length Factor VIILC molecule comprising a signal peptide and all A, B, and C domains; b) a mature Factor VIILC molecule comprising all A, B, and C domains and lacking a signal peptide; c) a truncated Factor VIILC molecule lacking a signal peptide and at least a portion of the B domain; and d) a cleaved Factor VIILC molecule comprising a light chain subunit of molecular weight of about 80 kD.

3. An active Factor VIILC polypeptide analog complex comprising a Factor VIILC polypeptide analog as claimed in claim 2, and a Factor VIILC polypeptide, wherein the complex comprises a metal ion.

4. The analog complex of claim 3, wherein the metal ion is a divalent cation.

5. The analog complex of claim 4, wherein the divalent cation is a Ca^{+ +} ion or a Cu ^{+ +} ion.

6. A method of producing a Factor VIILC polypeptide analog, comprising: a) providing a native Factor VIILC polypeptide that comprises an Arg at position 1689, numbered with respect to the native Factor VIILC polypeptide sequence; and b) substituting the Arg at position 1689 with a Lys residue, to produce the analog of claim 1.

7. A nucleic acid molecule comprising a nucleotide sequence that encodes the Factor VIILC polypeptide analog, wherein the analog comprises a native Factor VIILC polypeptide that is modified, wherein the modification comprises substitution of the Arg residue at position 1689, numbered with respect to the native Factor VIILC polypeptide sequence, with Lys.

8. A recombinant vector comprising the nucleic acid molecule of claim 7 and a regulatory element, wherein the nucleic acid molecule is under regulatory control of the regulatory element.

9. A recombinant host cell comprising the nucleic acid molecule of claim 7.

10. A recombinant host cell comprising the recombinant vector of claim 8.

11. A method of producing an active Factor VIILC polypeptide analog comprising: a) providing the recombinant host cell of claim 9; and b) allowing the recombinant host cell to express the analog.

12. A method of producing an active Factor VIILC polypeptide analog comprising: a) providing the recombinant host cell of claim 10 and b) allowing the recombinant host cell to express the analog.

13. A method of producing a nucleic acid molecule that encodes a Factor VIILC polypeptide analog comprising: a) providing a nucleic acid molecule that encodes a native Factor VIILC polypeptide comprising an Arg residue at position 1689, numbered with respect to the native Factor VIILC polypeptide sequence; and b) modifying a codon encoding the Arg residue at position 1689 to encode a Lys, to produce the nucleic acid molecule of claim 7.

14. The method of producing a nucleic acid molecule as claimed in claim 13, wherein the modification is performed by site directed mutagenesis or by use of polymerase chain reaction techniques.

15. A method of producing a recombinant vector that comprises a nucleic acid molecule that comprises a nucleotide sequence that encodes a Factor VIILC polypeptide analog, comprising linking a regulatory element to the nucleic acid molecule of claim 7.

16. A method of producing a recombinant host cell that comprises a nucleic acid molecule that comprises a nucleotide sequence that encodes a Factor VIILC polypeptide analog, comprising transforming a host cell with the nucleic acid molecule of claim 7.

17. A method of producing a recombinant host cell that comprises a recombinant vector that comprises a nucleotide sequence that encodes a Factor VIILC polypeptide analog, comprising transforming a host cell with the recombinant vector of claim 8.

18. A pharmaceutical composition comprising the active Factor VIILC polypeptide analog of claim 1 and a pharmaceutically acceptable excipient.

19. A pharmaceutical composition comprising the active Factor VIILC polypeptide analog complex of claim 3 and a pharmaceutically acceptable excipient.

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

21. A method for prevention or treatment of active Factor VIILC deficiency in a mammal comprising administering thereto a therapeutically effective amount of an active Factor VIILC polypeptide analog complex as claimed in claim 3.

22. A method for prevention or treatment of active Factor VIILC deficiency in a mammal comprising administering thereto a therapeutically effective amount of the nucleic acid molecule of claim 7.

23. A method for prevention or treatment of active Factor VIILC deficiency in a mammal comprising administering thereto a therapeutically effective amount of the recombinant vector of claim 8.

24. A method for prevention or treatment of active Factor VIILC deficiency in a mammal comprising administering thereto a therapeutically effective amount of the nucleic acid molecule of claim 7 and an active Factor VIILC polypeptide analog.

25. A method for prevention or treatment of active Factor VIILC deficiency in a mammal comprising administering thereto a therapeutically effective amount of the recombinant vector of claim 8 and an active Factor VIILC polypeptide analog.

26. A method for prevention or treatment of active Factor VIILC deficiency in a mammal comprising administering thereto a therapeutically effective amount of the nucleic acid molecule of claim 7 and an active Factor VIILC analog complex.

27. A method for prevention or treatment of active Factor VIILC deficiency in a mammal comprising administering thereto a therapeutically effective amount of the recombinant vector of claim 8 and an active Factor VIILC analog complex.