Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/745—Blood coagulation or fibrinolysis factors
  • C07K14/755—Factors VIII, e.g. factor VIII C (AHF), factor VIII Ag (VWF)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• This invention relates to a method for effecting an increased expression of recombinant proteins, especially proteins having Factor VIII:c activity.
• Hemophilia A is an X-chromosome-1inked inherited disease which afflicts 1-2 males per 10,000. The desease is caused by an absence of deficiency of Factor VIII:C.
• Factor VIII:C is a very large glycoprotein (native M r 330 K - 360 K) , which is present in plasma at extremely low concentrations. It is a necessary element in the proteolytic cascade which converts soluble fibrinogen to insoluble fibrin, forming a clot to prevent blood loss from traumatized tissue. In the bloodstream, it is found in noncovalent association with von Willebrand factor (vWF) , which acts as a stabilizing carrier protein.
• vWF von Willebrand factor
• Factor VIII:C is very susceptible to cleavage by thrombin, plasmin, activated protein C, and other serine proteases. It is generally isolated from plasma or plasma products as a series of related polypep- tides ranging from M r 160 K-40 K with predominant species of M r 92 K and M r 80 K-77 K. This complex pattern has made the analysis of the structure of active Factor VIII:C very diffi ⁇ cult.
• the full-length protein contains three repeats of one sequence (I) , and two repeats of a second sequence (III) .
• a third, heavily glycosylated sequence (II) is present between the second and third I repeats, and is ap- parently cleaved proteolytically to form the M r 92 K and M r 80 K polypeptides.
• the first two I repeats form the A domain, while the third I repeat and the two III repeats form the C domain.
• the II sequence forms the B domain.
• the full- length protein has the structure I 1 -I 2 -II-l 3 -III 1 -IIl2 (A-B-C)
• the M r 92 K and M r 80 K polypeptides have the structures I ⁇ - ⁇ and 1 3 -1112-1112, respectively.
• R.L. Burke et al, J Biol Chem (1986) have shown by expression of the 92 K and 80 K polypeptides that both peptides are necessary for Factor VIII:C activity.
• Factor VIII:C has historically been isolated from blood in a concentrated form for therapeutic treatment of hemophilia. However, concerns regarding transmission of HIV and other blood-borne diseases have stimulated activity to provide alternative supplies of Factor VIII:C. It is of substantial interest to be able to supply compositions having Factor VIII:C activity without concerns as to the transmission viral diseases associated with the native Factor VIII:C.
• the recombinant proteins having Factor VIII:C activity which are prepared according to the present invention may be full length Factor VIII:C corresponding to the protein isolated from plasma, or a derivative thereof having the capability of normalizing the insufficient blood clotting caused by deficien ⁇ cy of Factor VIII:C.
• the derivatives of Factor VIII:C may be shortened single chain forms or derivatives comprising two chains. Even fragments of Factor VIII:C which may not per se show coagulant activity, but which may be used in the treatment of haemophiliacs e.g. for saturation of antibodies against Fac ⁇ tor VIII:C present in inhibitor patients.
• the proteins produced in accordance with the present invention show homology with all or a part of the natural Factor VIII:C molecule.
• EP 294 910 disclose recombi ⁇ nant expression of shortened single chain forms or subunits of Factor VIII:C or co-expression of subunits for the production of complexes showing coagulant activity or binding affinity to antibodies inhibiting Factor VIII:C.
• heparin only "showed a very limited to no effect" on the expression of Factor VIII delta 2 having a deletion of amino acids 771 to 1666, as disclosed in EP 303 540 as compared to serum free medium.
• the expression level is slightly increased with increased concentration of heparin.
• the expression level is far below the expression level in the presence of serum or vWF.
• the present invention relates to a method for effecting an increased expression of recombinant proteins having Factor VIII:C activity in a host cell being able to express said protein comprising culturing said host i a cell growth medium comprising heparin in a very low concentration below lOIU/ml so as to express said protein.
• heparin in small amounts below 10IU heparin/ml increases the expression of recombinant proteins showing Factor VIII:C activity and stabilizes the product to an extent leading to an increased yield of more than 50%.
• the stabilizing effect of heparin is indicated through the observed reduction of the activation of the 92 kD subunit by proteases.
• Such addition may, according to the invention, be made to cell growth medium comprising serum, preferably in the form of fetal or new born calf serum, and to serum free medium comprising lipoprotein, vWF, or phospholipid or other additional constituents used for increasing the expression in serum free media in order to obtain the improved expression.
• the lipoprotein used in the method of the invention may e.g. be lipoproteins as described in EP 254076. Such lipoproteins are commercially available under the Trade Mark EX-CYTE.
• the lipoprotein may also be isolated from egg yolk, for example by the method described in Immunological Communications (5) , 475-493 (1980).
• Phospholipids used in the method of the invention may e.g. be such phospholipids as described in WO87/04187.
• additional constituents used for increasing the expression in serumfree media may e.g. be an egg yolk fraction being free of lipoprotein and lipids.
• heparin is added to a concentration of from 0.5 to 8 IU/ml, a concentra ⁇ tion of from 1 to 2 IU/ml being most preferred.
• heparin to a cell growth medium comprising serum is a preferred aspect of the invention giving rise to a considerable increase of the Factor VIII:C level.
• heparin to a cell growth medium comprising serum and further added lipoprotein giving rise to an extremely high level of Factor VIII:C on expression in suspension.
• serum free cell culture medium is used in order to avoid the addition of constituents which might give rise to transference of blood borne diseases, such medium being supplemented with lipopro ⁇ tein, von Willebrand Factor, phospholipid, or a combination of two or more of these.
• lipoprotein and heparin are added to a serum free medium.
• about 5% of lipoprotein fraction and about 2IU of heparin are added giving rise to a very high expression of Fator VIII:C in the term of expressed Factor VIII:C ac ⁇ tivity.
• heparin is added to a growth medium for culturing a host cell for expressing a complex of the 92kD and 80/77kD subunits of Factor VIII:C.
• heparin does not only increase the level of expression of the individual subunits of Factor VIII:C, but also increases the degree of complex formation and stabilizes the produced complex and thus, the yield of product showing coagulant activity.
• a domain refers to that portion of human Factor VIII:C which constitutes the M r 92 K protein subunit or the Factor VIII:C heavy chain (FVIII-HC) .
• the A domain contains from about 740 to about 760 amino acids, and is found at the N- terminus of the native human Factor VIII:C.
• Of particular interest is an N-terminal chain having the entire sequence to the thrombin cleavage site at Arg 74Q -Ser 741 «
• B domain refers to that portion of native human Factor VIII:C which is generally removed by intracellular cleavage, and which is heavily glycosylated in human plasma and when expressed in mammalian cells such as C0S7, CHO and BHK cells.
• the B domain contains an N-terminal sequence, which allows cleavage of the A domain from the B domain by thrombin.
• the B domain also has a C-terminal processing site which allows cleavage of the C domain from the A-B precursor by an enzyme located in the Golgi apparatus of the mammalian cell.
• C domain refers to that portion of native human Factor VIII:C which constitutes the C-terminus of the full length protein, and is cleaved intracellularly to form the Factor VIII:C light chain (FVIII-LC) .
• the light chain will have an amino acid sequence substantially the same as the amino acid sequence of the C-terminus of a Factor VIII:C polypeptide, usually at least about 80%, more usually at least about 90% of the Factor VIII:C M r 80 K chain, particularly beginning with amino acid 1640, preferably at about amino acid 1649, ⁇ 10 amino acids, more particularly ⁇ 1 amino acid, and continuing to at least about amino acid 2300, usually 2310, ⁇ 10 amino acids, preferably 2325, ⁇ 5 amino acids, more preferably to the terminal amino acid (2332) .
• the light chain will have at least about 85%, more usually at least 95%, of the 111,-111 2 domains, desirably the I3-III1-III2 domains.
• co-expressing refers to simultaneous expression of an A domain polypeptide (92 K) and a C domain polypeptide (80 K) within the same host cell.
• the poly- nucleotide sequences encoding the A and C domains may be on the same or on different expression cassettes or plasmids. Co-ex ⁇ pression of the A and C domains permits proper folding to oc ⁇ cur, which in turn provides an A-C complex having activity and efficiency of secretion.
• production medium refers to any medium suitable for culturing host cells, and includes media suitable for obtaining expression of recombinant products whether or not actual cell "growth" occurs.
• Production media generally include nutrients and a metabolizable energy source in an aqueous solution.
• production media may also include a compound which induces expression of the recombinant polypeptides of the invention. Selection of such an inducing compound depends upon the promoter selected to control expres- sion.
• Other typical additives include selection compounds (i.e., drugs or other chemicals added to the media to insure that only transformed host cells survive in the medium) and serum, such as fetal bovine serum (FBS) .
• FBS fetal bovine serum
• serum-free medium is a solution which has been sup- plemented to such an extent that the necessary trace factors present in serum need not be added in the form of serum.
• the serum free medium may be a synthetical medium not comprising components isolated from animal tissues or body fluids. There are many suitable cell growth media available from commercial sources.
• IU as used herein in connection with heparin is defined by standardization against the 4th International
• homology means identity or sub ⁇ stantial similarity between two polynucleotides or two poly ⁇ peptides. Homology is determined on the basis of the nucleotide or amino acid sequence of the polynucleotide or polypeptide. In general terms, usually not more than 10, more usually not more than 5 numberl, preferably not more than about 1 number% of the amino acids in the chains will differ from the amino acids naturally present in the Factor VIII:C A and C domains. Par- ticularly, not more than about 5%, more usually not more than about 1% will be nonconservative substitutions. Conservative substitutions include:
• Nonconservative changes are generally substitutions of one of the above amino acids with an amino acid from a different group (e.g., substituting Asn for Glu), or substituting Cys, Met, His, or Pro for any of the above amino acids.
• the specific activity of a protein complex prepared according to the invention may be determined by means known in the art, as described below (e.g., by using the commercially available Coatest assay) .
• the structural genes typically include a leader sequence coding for the signal peptide which directs the polypeptide into the lumen of the endoplasmic reticulum for processing and matura ⁇ tion.
• additional sequences encoding propeptides which are processed post-translationally by endopeptidases, where the endopeptidases cleave a peptide bond, removing the propeptide to generate the mature polypeptide.
• the signal peptide may be the naturally occurring one, particularly for the N-terminal peptide, or may be any signal peptide which provides for the processing and maturation of the polypeptides.
• Various mammalian host cells may be employed in which the regulatory sequences and replication system are functional.
• Such cells include C0S7 cells, Chinese hamster ovary (CHO) cells, mouse kidney cells, hamster kidney cells, HeLa cells, HepG2 cells, or the like, e.g VERO cells, W-138 or MDCK cell lines.
• the expressed product can be purified by affinity chromato ⁇ graphy using antibodies, particularly monoclonal antibodies directed against the FVIII-LC or FVIII-HC, chromatography, e.g. HPLC, electrophoresis, or extraction.
• affinity chromato ⁇ graphy using antibodies, particularly monoclonal antibodies directed against the FVIII-LC or FVIII-HC, chromatography, e.g. HPLC, electrophoresis, or extraction.
• the subject method provides for production of a complex of the active chains (92 K and 80 K) which has Factor VIII:C activity. Production is evidenced by conditioned media as described in the experimental section, which will have at least about 1, usually at least about 5 U/mL, more usually at least about 10 U/mL of Factor VIII:C activity in the Coatest assay.
• the proteins having Factor VIII:C activity produced according to the invention are primarily intended for treatment of hemophiliacs and patients suffering from other conditions involving blood clotting disorders.
• the subject proteins may be administered in physiologically acceptable carrier, such as water, saline, phosphate buffered saline, and citrate buffered saline, at concentrations in the range of about 10-200 U/mL. See U.S. Patent Nos. 3,631,018; 3,652,530, and 4,069,216 for methods of administration and amounts.
• Other conventional addi ⁇ tives may also be included. They also have a variety of uses as immunogens for the production of antibodies, for isolation of von Willebrand factor by affinity chromatography and in diagnostic assays for Factor VIII:C.
• Fig. 1 shows a titration of the heparin effect on level of Factor VIII:C units in suspension culture
• Fig. 2 shows a gel illustrating the stabilizing effect of heparin on Factor VIII-HC
• Lipoprotein was isolated from egg yolk in the form of a fraction being rich in lipoprotein. The fractionation was carried out as disclosed in detail in Example 6.
• PEG 6000 from Merck, Catalogue No. 807491 was used for the fractionation of egg yolk.
• PBS used for the fractionation of egg yolk was made by dis ⁇ solving 8.0 g of NaCl, 0.2 g of KCl, 1.15 g of Na 2 HP0 4 and 0.2 g of KH 2 PO. in deionized water, adjusting the pH to 7.3 by adding 1M HCl/NaOH and adding deionized water ad 1 liter. All chemicals were from Merck.
• the DHFR " CHO cell line DG44 (G. Urlaub et al., So Cell Mol Genet (1986) 12:555-566) was first transfected with the plasmid pCMF8-80AT: In this plasmid the CMV promoter (described in example 7 of W091/07490) transcribes the FVIII-LC cDNA derived from pSVF8-80AT (described in example 6 of W091/07490) and downstream is placed the Ad-MLP/dhfr casette derived from pAd- DHFR (described in example 4 of WO91/07490) . The transfection method used was the polybrene method of W. CHaney et al. (Som Cell Mol Genet (1986) 12:237-244). By selection of DHFR + cells (DMEM + 10% DFCS) several FVIII-LC producers were isolated; one of these was designated 11W.
• the cells selected in this way on the basis of the expression level were seeded into T-flasks or spinners for cultivation in the absence of heparin or in the presence of heparin in various concentrations.
• transfection referred to in WO91/07490 is hereby incorporated by reference, including the reference to the plasmide pSVF8-92, pSVF8-80 and pSVF8-200 deposited under the accession number ATCC 40222, ATCC 40223 and ATCC 40190, respectively.
• the cell line designated "45" co-expressing Factor VIII:C Heavy Chain and Light Chain was cultivated in suspension at 37°C in cell factories in DMEM + 10% dialysed fetal calf serum in the conventional manner, and cells were harvested by trypsinization and resuspended in 100 ml TECHNE spinners at a density of 2 million cells per ml in DMEM (Gibco 074-90024T) supplemented with 110 mg/1 Na-pyruvate, 150 mg/1 1-proline, 3.7 g/1 NaCH0 3 , 1.4 g/1 tryptose phosphate, 5 mg/1 insulin, 0.5 g/1 6-amino- hexanoic acid and 2% NBS heat inactivated at 56°C for 30 minutes. The cultures were incubated at 27°C. Samples were taken over a period of 9 days. Various amounts of heparin were added.
• the isolated cell line designated "57" co-expressing Factor VIII:C Heavy Chain and Light Chain was cultivated in t-flasks in DMEM + 10% dialysed fetal calf serum in the conventional manner to confluence. Confluent t-flasks were shifted to the same medium as in Example 2, and incubated at the same tempera ⁇ ture in the presence and absence of heparin. The cultures were fed fresh medium with 3 days intervals. Samples were taken for FVIII assays prior to media change.
• Table 1 shows the increased FVIII:C levels obtained from t- flask cultures when heparin is added in low concentration (2 IU/ml) .
• the increase is to a level of 113 to 138% of the FVIII level of the control without heparin addition as a function of the cultivation time in the presence of heparin.
• the isolated cell lines designated "45” and "57” co-expressing Factor VIII:C Heavy Chain and Light Chain were cultivated in suspension as explained above in the presence and absence of heparin.
• the increase in FVIII level is even more pronounced when the cells are cultured in suspension.
• the increase in FVIII level caused by addition of heparin is 120 to 193% of the control, where no heparin is added.
• Tables 2 and 3 also indicate that the increase of the Factor VIII:C level is in excess of the increased expression of the Factor VIII:LC and Factor VIII:HC when co-expression in the presence of heparin.
• the media were collected for immuno- 5 precipitation with a dog polyclonal antiserum to human FVIII; this antiserum binds both the complex of the subunits and free heavy and light chains of FVIII.
• the precipitated samples were loaded on a 10% SDS gel. The resulting exposure is shown in
• Fig. 2 The 92 kD HC and the 80 kD LC doublet are seen in all 0 lanes. Comparing lanes 1 and 2 it is seen that the amounts of the 50/43 kD bands originating from the HC are much more pronounced in lane 1 than in lane 2, indicating that heparin has suppressed the activating cleavage of the heavy chain and hence, a greater fraction of the subunit-complex is found as the more stable complex of the 92 kD and 80 kD subunits.
• the sediment was dissolved in 200 ml PBS, stirred for 30 min. and spun down at 10,000 RPM for 20 min.
• the sediment was redissolved in 200 ml 1 M NaCl and stirred overnight at 40° C. The mixture was then centrifuged at 10,000 RPM for 20 min. and the supernatant was sterilised by filtering through 0.2 ⁇ pore size filter.
• This lipoprotein fraction was used in the below culturing.
• the cell line designated "57" co-expressing Factor VIII:C Heavy Chain and Factor VIII:C Light Chain was cultured in T-80 flasks in serum containing medium. After reaching confluency the cells were adapted to production conditions for three days. Produc ⁇ tion medium and conditions were described in Example 3, except that there was no addition of serum and the basal medium was DMEM/F-12. 5% addition of the egg yolk fraction containing lipoprotein was tested alone and together with 1, 2 and 5 IU heparin, and for comparison the same medium without lipoprotein was tested using 1, 2 and 5 IU heparin. Serum free medium without lipoprotein and heparin was used as control. The medium was changed and samples were taken on day 2 and day 4 and assayed for FVIII:C (coa) activity.
• the supernatant is the lipoproteinfree egg yolk protein fraction (SUP 0) .
• This proteinfraction was used in the below culturing.
• the cell line designated "57" co-expressing Factor VIII:C Heavy Chain and Factor VIII:C Light Chain was cultured in T-80 flasks in serum containing medium. After reaching confluency the cells were adapted to production conditions for three days. Produc ⁇ tion medium and conditions were described in Example 3, except that there was no addition of serum and the basal medium was DMEM/F-12. A 5% addition of the egg yolkprotein fraction was tested alone and together with 1, 5 and 10 IU heparin. The medium was changed and samples were taken on day 2 and day 4 and assayed for FVIII:C (coa) activity.

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• 1993
  • 1993-11-17 EP EP94900069A patent/EP0679192A1/de not_active Withdrawn
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