Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P21/00—Preparation of peptides or proteins
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/745—Blood coagulation or fibrinolysis factors
  • C07K14/755—Factors VIII, e.g. factor VIII C (AHF), factor VIII Ag (VWF)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• This invention relates to the production of factor VIII in high yields. More particularly, this invention relates to a method for increasing yield of factor VIII by growing factor VIII-producing cells in the presence of the von Willebrand factor.
• Factor VIII the procoagulant component of factor VIII/vWf, is initially synthesized as a single chain macromolecular precursor, which is later cleaved to yield the fragments which constitute "mature" factor VIII [see generally, W. J. Williams et al., Hematology, pp. 1085-90, McGraw-Hill, New York (1972)].
• Mature factor VIII is composed of two chains bridged by a calcium ion; an amino-terminal heavy chain of 740 amino acids, and a carboxy-terminal light chain of 684 amino acids.
• the primary translation product of factor VIII is a single chain in which the heavy chain of mature factor VIII is separated from the light chain by a "maturation polypeptide" of 908 amino acids.
• vWf Von Willebrand factor
• vWf is a large multimeric plasma protein, comprised of at least two subunits linked together by disulfide bonds. It is a protein important in the hemostatic process, and is associated with normal platelet aggregation and adhesive properties. It mediates the attachment of platelets to the basement membrane after vascular injury.
• the larger polymers of vWf function in platelet aggregation at the site of vascular injury [T. S.
• the mature vWf protein is synthesized in several steps.
• a 240,000-260,000 molecular weight prepro-vWf subunit of 2813 amino acids is synthesized in vascular endothelial cells and megakaryocytes [E. A. Jaffe et al., "Synthesis of Antihemophilic Factor Antigen By Cultured Human Endothelial Cells," J. Clin. Invest., 52, pp. 2757-64 (1973); R. Nachman et. al., "Synthesis of Factor VIII Antigen By Cultured Guinea Pig Megakaryocytes," J. Clin. Invest. , 60, pp. 914-21 (1977); C. L.
• factor VIII is synthesized and secreted by the hepatocyte [M. G. Zelechowska et al., "Ultra structural Localization of Factor VIII Procoagulant Antigen In Human Liver Hepatocytes, " Nature, 317, pp. 729-30 (1985); K. L. Wion et al., "Distribution of Factor VIII mRNA and Antigen in Human Liver and Other Tissues," Nature, 3T7, pp. 726-28 (1985)].
• the secreted factor VIII is endocytosed by the neighboring sinusoidal endothelial cell [H. V.
• Haemophilia A is a sex-linked hemorrhagic disease which is caused by a deficiency, either in amount or in biological activity, of factor VIII.
• the symptoms of acutely bleeding haemophilia patients are treated with factor VIII traditionally purified from normal sera.
• Various methods of purification have been described in the literature [see, Zimmerman et al., United States patent 4,361,509; Saundrey et al. United States patent 4,578,218; E. G. D. Tuddenham et al., "The Properties of Factor VIII Coagulant Activity Prepared By Immunoadsorbent Chromatography, Journal of Laboratory Clinical Medicine, 93, pp. 40-53 (1979); D. E. G. Austen, "The Chromatographic Separation of Factor VIII on
• the present invention solves the problems referred to above by providing a means of producing factor VIII in high yields. More specifically, it provides a method of increasing the yield of factor VIII by growing factor VIII-producing cells in the presence of human vWf. According to this method, 5 to 30 ⁇ g of vWf are present in the culture medium ' in which the factor VIII-producing cells are grown, either by supplementing the medium with vWf or by cotransforming recombinant factor VIII-producing cells with the DNA sequence encoding vWf. According to this invention, this method can be used to increase the yield of both native and recombinant factor VIII.
• Figure 1 °compares the results of several induction experiments with clone K using a culture fluid containing 10 ⁇ g/ml vWf to those obtained using a culture fluid containing no vWf.
• Figure 2 depicts the levels of factor VIII accumulated in the culture fluid when clone K is induced to secrete factor VIII into culture fluid containing 0, 5, 10, 20, or 30 ⁇ g/ml human vWf.
• Figure 3 depicts the factor VIII yield in two other clones tested, Aat2.10 and Aat2.27.
• Figure 4 is a schematic representation of plasmid RE.neo, which contains a (modified) factor VIII gene, under the transcriptional control * of the adenovirus-2 major late promoter.
• Factor VIII A polypeptide having a mole ⁇ cular weight of 265,000, and upon maturation and activation, being capable of functioning as cofactor for the factor IXa-dependent maturation of factor X in the blood coagulation cascade.
• factor VIII includes the glycoproteins also known as factor VIII:C, factor VIII procoagulant activity protein, or factor VIII-clotting activity [see W. J. Williams et al., Hematology, pp. 1056, 1074 and 1081] .
• factor VIII also refers to polypeptides characterized by a dele ⁇ tion of a major portion of the maturation polypeptide of factor VIII.
• factor VIII includes proteins that comprise the N-terminal mature heavy chain and the C-terminal mature light chain of factor VIII either linked together as a single chain or bridged by a calcium or other metal ion bridge. It also includes proteins having an amino terminal methionine, e.g., f-Met-factor VIII, and proteins that are characterized by other amino acid deletions, additions or substitutions so long as those proteins substantially retain the biological activity of factor VIII. It also includes polypep ⁇ tides having natural allelic variations that may exist and occur from individual to individual. Fur ⁇ thermore, it includes factor VIII polypeptides whose degree .and location of glycosylation, or other post- translation modifications, may vary depending on the cellular environment of the producing host or tissue.
• Von Willebrand Factor a polypeptide which is responsible for platelet adhesion to blood vessel walls and acts as a carrier for factor VIII in plasma.
• the present invention relates to a method for increasing the yield of factor VIII. More par ⁇ ticularly, it provides a method for the production of recombinant and native factor VIII produced in culture in high yields through the addition of vWf to the culture medium of the growing factor VIII- producing host or cell-line.
• vWf may be added to fresh culture fluid for host cells which constituitively produce factor VIII or, in the case of host cells which inducibly produce factor VIII, vWf may be added to culture fluid immediately prior to the induction of those cells.
• vWf may be co-synthesized with factor VIII in hosts which are transformed with DNA sequences encoding both vWf and factor VIII.
• vWf affinity-purified human vWF to demon ⁇ strate the increase in factor VIII yield.
• recombinant vWf may also be used.
• vWf present in the culture fluid may bind to factor VIII as it is released from the exocytosed Golgi vesicles of a mammalian host cell, thereby protecting the factor VIII molecule from extracellular proteolytic degradation.
• vWf may be endocytosed into either the endoplasmic reticulum or some compartment of the Golgi apparatus of the mammalian host, where it binds to newly synthesized factor VIII and facilitates its export, perhaps by protecting it from lysosomal proteolysis.
• the factor VIII produced according to the methods of this invention may be purified by a variety of conventional steps and strategies.
• Useful puri ⁇ fication steps include those used to purify natural and recombinant factor VIII [see, for example, L.-O. Andersson et al., PNAS (USA), 83, pp. 2979-83 (1986)].
• the intermediate factor Vlll-vWf complex formed according to the methods of this invention may be dissociated to form factor VIII according to Anderson's method.
• the factor VIII produced by the methods of this invention are useful in compo- sition and methods for treatment of haemophilia A and in a variety of agents useful in treating uncon ⁇ trolled bleeding.
• the factor VIII produced according to the methods of this invention may be formulated using known methods to prepare pharmaceutically useful compositions.
• Such compositions also will preferably include conventional pharmaceutically acceptable carriers and may include other medicinal agents, carriers, adjuvants, excipients, etc., e.g., human serum albumin or plasma preparations. See, e.g.,
• the resulting formulations will contain an amount of modified factor VIII effective in the recipient to treat uncontrolled bleeding.
• Administration of these polypeptides, or pharmaceutically acceptable deriva ⁇ tives thereof, may be via any of the conventional accepted modes of administration of factor VIII. These include parenteral, subcutaneous, or intra ⁇ venous administration.
• compositions of this invention used in the therapy of haemophilia A may also be in a variety of forms.
• the preferred form depends on the intended mode of administration and therapeutic application.
• the dosage and dose rate will depend on a variety of factors, for example, whether the treatment is given to an acutely bleeding patient or as a prophylactic treatment.
• the factor VIII level should be high enough to prevent hemorrhage and promote epithelialization [see discussion in Williams, Hematology, pp. 1335-43].
• EXAMPLE We describe in this example the increased production of a modified factor VIII using the method of this invention. First, we constructed cDNA sequences which encode a modified factor VIII molecule having a deletion of a major part or all of the maturation polypeptide of native factor VIII.
• RE.neo was constructed by insert ⁇ ing into RE a transcription unit directing the expres ⁇ sion of agpt in animal cells.
• Plasmid pSVneo2911 was con ⁇ structed as follows: first, the SV40 Hpall-BamHI restriction fragment containing the entire SV40 early region was inserted between Clal and BamHI into pBRd [F. A. M. Asselbergs et al. "A Recombinant Chinese Hamster Ovary Cell Line Containing A 300-fold Ampli ⁇ fied Tetramer of the Hepatitis B Genome Together With A Double Selection Marker Expresses High Levels of Viral Protein," J. Mol. Biol., 189, pp. 401-11 (1986)].
• the resultant plasmid, RE.neo contained two SV40 replication origins, one 5' to the adeno- virus-2 major late promoter and the other overlapping the SV40 early promoter which controls transcription of the aminoglycoside 3 '-phosphotransferase (G418/neo) gene.
• G418/neo aminoglycoside 3 '-phosphotransferase
• BSC40 cells which are BSC1 African green monkey kidney cells which have been adapted to grow at 40°C [W. W. Brockman and D. Nathans, PNAS (USA) , 71, pp. 942-46 (1974)].
• BSC40 cells which contain a SV40 origin of replication, a transcription unit for factor VIII, and a transcription unit for amino- glycoside phosphotransferase and (2) a 10-fold molar excess of plasmid LTRtsA58, containing a transcrip ⁇ tion unit for a temperature sensitive SV40 T antigen allele.
• the mutant tsA58 virus is a temperature- sensitive mutant of SV40 which does not produce progeny at 39°C.
• the large T antigen protein specified by the tsA58 mutant is much more labile at the non-per ⁇ missive temperature than wild type large T antigen protein [P. Tegtmeyer et al., Journal of Virology, 16, pp. 168-78 (1975)].
• Our preferred cell lines, clones K, Aat2.10, and Aat2.27 are BSC40 transfectants which inducibly synthesize and secrete recombinant factor VIII.
• Clone K [ATCC No. CRL 9206] was obtained by co-trans- fecting BSC40 with supercoiled RE.neo and supercoiled LTRtsA58 and selecting at 39.5°C for G418 resistance.
• Clones Aat2.10 and Aat2.27 were obtained by co-trans- fecting BSC40 with RE.neo linearized at the unique Aat2 site and supercoiled LTRtsA58 and selecting at 39.5°C for G418 resistance.
• We made our preferred cell lines by co- transfecting confluent cultures of BSC40 cells in 100 mm Petri dishes with 10 micrograms pLTRtsA58 and 1.5 micrograms RE.neo using the standard technique of calcium phosphate precipitation.
• Factor VIII assay using conditioned medium for each line at both temperatures. We selected clones exhibiting the highest levels of activity at 33°C for further study. No clone expressed factor VIII at 39.5°C at non-negligible levels.
• Figure 3 demonstrates that the increased yield of factor VIII activity in the presence of human vWf is not specific only to clone K. We also observed increases in factor VIII yield in clones Aat2.10 and Aat2.27.

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• 1988
  • 1988-01-29 AU AU13409/88A patent/AU606925B2/en not_active Expired
  • 1988-01-29 JP JP63501908A patent/JP2872255B2/ja not_active Expired - Lifetime
  • 1988-01-29 WO PCT/US1988/000292 patent/WO1988005825A1/en not_active Application Discontinuation
  • 1988-01-29 EP EP19880902010 patent/EP0302925A4/en not_active Withdrawn
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• 1989
  • 1989-01-29 KR KR1019880701229A patent/KR890700671A/ko not_active Application Discontinuation

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