WO1992004450A1 - Hybrid plasminogen activators - Google Patents

Hybrid plasminogen activators Download PDF

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Publication number
WO1992004450A1
WO1992004450A1 PCT/GB1991/001455 GB9101455W WO9204450A1 WO 1992004450 A1 WO1992004450 A1 WO 1992004450A1 GB 9101455 W GB9101455 W GB 9101455W WO 9204450 A1 WO9204450 A1 WO 9204450A1
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plasminogen
hybrid
chain
plasminogen activator
sequence
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PCT/GB1991/001455
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English (en)
French (fr)
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Ian Dodd
Michael Joseph Brown
Jeffery Hugh Robinson
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Beecham Group Plc
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Publication of WO1992004450A1 publication Critical patent/WO1992004450A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6435—Plasmin (3.4.21.7), i.e. fibrinolysin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6456—Plasminogen activators
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21007—Plasmin (3.4.21.7), i.e. fibrinolysin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to a nycrid fic ⁇ nolyti enzyme and derivatives thereof, its preparation, pnarmaceuticai compositions containing it and its use in the treatment of thrombotic disease, in particular acute myocardial infarction.
  • tissue-type plasminogen activator t-PA
  • nucleotide sequence for the cDNA which codes for t-PA are known (see Pennica et. al.. 1983; Nature, 301, 214) .
  • t-PA is known to have fibrinolytic activity.
  • tissue-type plasminogen activator denotes a plasminogen activator of the group having the immunological properties defined for t-PA at the XXVIII Meeting of the International Committee on Thrombosis and Haemostasis, Bergamo, Italy, 27 July 1982.
  • the numbering system for the amino acid sequence of t-PA used herein is that described in the Nature 1983 reference for mature (S-chain) t-PA in which the N-terminal serine is numbered 1.
  • L-chain t-PA has an N-terminal glycine residue at position -3 and U-chain t-PA has an N-terminal valine at position 4.
  • References to t-PA herein are understood to include all such variant forms.
  • Native t-PA is composed of a B or light and an A or heavy chain.
  • the B-chain contains the active site of the enzyme.
  • the cleavage site for the conversion of t-PA from the single to the two-chain form is located between residues arg-275 5 and ile-276. In the two-chain form the chains are held together by a disulphide bridge formed between residues cys-264 in the A-chain and cys-395 in the B-chai,n.
  • u-PA urokinase-type plasminogen activator
  • Urokinase-type plasminogen activator is known to have fibrinolytic activity.
  • the two chains of u-PA are termed the A- and
  • the B-chain contains the active site of the enzyme.
  • the cleavage site for the conversion of u-PA from the single to the two chain form is located between residues lys-158 and ile-159. In the two chain form the chains are held together by a disulphide bridge formed between residues
  • urokinase-type plasminogen activator denotes a plasminogen activator of the group having the immunological properties defined for u-PA 35 at the XXVIII Meeting of the International Committee on Thrombosis and Haemostasis, Bergamo, Italy, 27 July 1982.
  • the numbering system for the amino acid and nucleotide sequence of u-PA used herein is that described in Holmes, W. E. et al, 1985 (op_. cit.) in which the N- terminal serine residue is numbered 1.
  • references herein to t-PA and u-PA species include both native forms and muteins.
  • Plasmin is a two-chain serine protease which may be obtained by the cleavage of the single chain precursor, plasminogen, at a specific internal peptide bond.
  • the amino acid sequence of human plasminogen is known (Wiman and Walters (1975) Eur.J. Biochem. 50., 489-494 and 58., 539-547; Wiman
  • the cleavage site of human plasminogen is located between residues arg-560 and val-561 (according to the sequence numbering of Sottrup-Jensen e a_l. (1978) Atlas of Protein Sequence (op.cit.)).
  • Two species of plasminogen have been identified ( F.J. Castellino, Chemical Reviews Vol. 81 p431 (1981)): glu 1 which has an N-terminal glutamic acid residue at position 1 and lys 77 which has an N-terminal lysine residue at position 77.
  • Glu-plasminogen is also easily converted by limited plasmic digestion to other modified forms with N-terminal valine (val-yg) or methionine (metgg) (C. Miyashita, E. Wenzel and M. Heiden, Haemostasis J 3., supp.l pp 7-13 (1988)) . References to plasminogen herein are understood to include all these species.
  • Plasminogen has five kringle structures.
  • the region from the first to the last cysteine residue of each kringle structure, residues 84 to 162, 166 to 243, 256 to 333, 358 to 435 and 462 to 541 inclusive will be referred to herein as the domains respectively.
  • a hybrid plasminogen activator which comprises kringle 5 or kringles 4 and 5 of plasminogen linked to the B-chain of t-PA or u-PA via an amino acid sequence comprising, respectively, the t-PA cleavage site between residues 275 and 276 and the cysteine residue 264 of t-PA or the u-PA cleavage site between residues 158 and 159 and the cysteine residue 148 of u-PA.
  • 'B-chain' is meant at least that portion of the B-chain containing the functional active centre of t-PA or u-PA, and preferably comprises residues 276-527 or 159-411 respectively.
  • the linking sequence of amino acids may be introduced synthetically during the preparation-of the hybrid plasminogen activator (PA) and/or derived from native sequences.
  • PA hybrid plasminogen activator
  • Native plasminogen includes cysteine residues at positions 548 and 558, C-terminal to plasminogen kringle 5, which participate in the interchain disulphide bonds of the two-chain plasmin form. In the preferred embodiment these residues are not present in the linking sequence.
  • the linking sequence should be chosen so as to avoid the presence of a site susceptible to trypsin-like proteolytic cleavage N-terminal to residue cys-264 of t-PA or cys-148 of u-PA, as appropriate.
  • the linking sequence of amino acids preferably comprises t-PA residues 264 to 275 inclusive, more preferably residues 262 to 275 inclusive.
  • the linking sequence of amino acids preferably comprises u-PA residues 148 to 158 inclusive, more preferably residues 137 to 158 inclusive.
  • the hybrid PA may be represented symbolically as: (Z 3 K 4 P) m ⁇ 4 K 5 Pz 5 Bt
  • B*- comprises residues 276-527 of t-PA
  • m is 0 or 1
  • K ⁇ P and Kt j P represent kringle domains 4 and 5 derived from plasminogen and each of Z3, Z 4 and Z5 represents, as appropriate, an optional N-terminal amino acid sequence or a bond or a linking sequence of amino acids which may be introduced synthetically during the preparation of the hybrid PA and/or derived from native plasminogen and/or t-PA sequences, the sequence Z5 comprising at least residues cys-264 and arg-275 of t-PA.
  • the hybrid PA may be represented symbolically as:
  • B u comprises residues 159-411 of u-PA and each of Z3, Z 4 and Z5 represents, as appropriate, an optional N-terminal amino acid sequence or a bond or a linking sequence of amino acids which may be introduced synthetically during the preparation of the hybrid PA and/or derived from native plasminogen and/or u-PA sequences
  • the sequence Z5 comprising at least residues cys-148 and lys-158 of u-PA and m, K ⁇ P and K5 are as previously defined.
  • the sequence Z3 preferably has at its N-terminus the sequence [GARSYQ] or [SYQ] corresponding to the L- and S-chain forms of t-PA, and comprises some or all of the native plasminogen inter-domain sequence between plasminogen kringle domains 3 and 4, preferably plasminogen residues 347-357.
  • the sequence Z 4 preferably has at its N-terminus the sequence [GARSYQ] or [SYQ] corresponding to the L- and S-chain forms of t-PA, and comprises some or all of the native plasminogen inter-domain sequence between plasminogen kringle domains 4 and 5, preferably plasminogen residues 443-461.
  • Z ⁇ preferably represents the native plasminogen inter-domain sequence between plasminogen kringle domains 4 and 5.
  • Suitable sequences (Z ⁇ ) linking the plasminogen kringle 5 domain to the t-PA B-chain include:
  • sequences 1 and 2 consist of residues 542-544 of plasminogen and residues 263 to 275 of t-PA linked by a serine residue.
  • the interposed serine residue can be identified with ser-545 of plasminogen or ser-262 of t-PA.
  • residue 275 of t-PA has been replaced by glutamine in accordance with EP-A-0233013.
  • the preferred sequence 3 consists of residues 262 to 275 of t-PA.
  • the preferred sequence (Z ) linking the plasminogen kringle 5 domain to the u-PA B-chain is:
  • the preferred hybrid PA' s of the invention have the following structures:
  • Pig x-y represents residues x-y of plasminogen
  • B t is as previously defined and the symbols in brackets represent amino acid residues according to the single letter amino acid notation, including one and two chain variants, L- and S-chain variants, and mixtures thereof.
  • the hybrid PA of the invention may be derivatised to provide pharmaceutically useful conjugates analogous to known PA-containing conjugates, for example:
  • an enzyme-protein conjugate as disclosed in EP-A-0152 736 comprising at least one optionally blocked fibrinolytic enzyme linked by way of a site other than the catalytic site responsible for fibrinolytic activity to at least one human protein;
  • the hybrid PA of the invention may take the place of a PA as the enzyme or (human) protein component, as appropriate, of any of the conjugates described above.
  • the invention provides a process for preparing hybrid plasminogen activator according to the invention which process comprises expressing DNA encoding said hybrid plasminogen activator in a recombinant host cell and recovering the hybrid plasminogen activator product.
  • the DNA polymer comprising a nucleotide sequence that encodes the hybrid PA also forms part of the invention.
  • the process of the invention may be performed by conventional recombinant techniques such as described in Maniatis t_. aJL., Molecular Cloning - A Laboratory Manual; Cold Spring Harbor, 1982 and DNA Cloning vols I, II and III (D.M. Glover ed., IRL Press Ltd).
  • the process may comprise the steps of:
  • the invention also provides a process for preparing the DNA polymer by the condensation of appropriate mono-, di- or oligomeric nucleotide units.
  • the preparation may be carried out chemically, enzymatically, or by a combination of the two methods,in vitro or in. vivo as appropriate.
  • the DNA polymer may be prepared by the enzymatic ligation of appropriate DNA fragments, by conventional methods such as those described by D. M. Roberts et al in Biochemistry 1985, 2_4, 5090-5098.
  • the DNA fragments may be obtained by digestion of DNA containing the required sequences of nucleotides with appropriate restriction enzymes, by chemical synthesis, by enzymatic polymerisation, or by a combination of these methods.
  • Digestion with restriction enzymes may be performed in an appropriate buffer at a temperature of 20°-70°C, generally in a volume of 50 ⁇ l or less with 0.1-10 ⁇ g DNA.
  • Enzymatic polymerisation of DNA may be carried out jLn. vitro using a DNA polymerase such as DNA polymerase I (Klenow fragment) in an appropriate buffer containing the nucleoside triphosphates dATP, dCTP, dGTP and dTTP as required at a temperature of 10°-37°C, generally in a volume of 50 ⁇ l or less.
  • Enzymatic ligation of DNA fragments may be carried out using a DNA ligase such as T4 DNA ligase in an appropriate buffer at a temperature of 4°C to ambient, generally in a volume of 50 ⁇ l or less.
  • the chemical synthesis of the DNA polymer or fragments may be carried out by conventional phosphotriester, phosphite or phosphoramidite chemistry, using solid phase techniques such as those described in 'Chemical and Enzymatic Synthesis of
  • the DNA polymer is preferably prepared by ligating two or more DNA molecules which together comprise a DNA sequence encoding the hybrid PA.
  • the DNA molecules may be obtained by the digestion with 30 suitable restriction enzymes of vectors carrying the required coding sequences.
  • the precise structure of the DNA molecules and the way in which they are obtained depends upon the structure of the 35 desired hybrid PA product.
  • the design of a suitable strategy for the construction of the DNA molecule coding for the hybrid PA is a routine matter for the skilled worker in the art.
  • the expression of the DNA polymer encoding the hybrid PA in a recombinant host cell may be carried out by means of a replicable expression vector capable, in the host cell, of expressing the DNA polymer.
  • the expression vector is novel and also forms part of the invention.
  • the replicable expression vector may be prepared in accordance with the invention, by cleaving a vector compatible with the host cell to provide a linear DNA segment having an intact replicon, and combining said linear segment with one or more DNA molecules which, together with said linear segment, encode the hybrid PA, under ligating conditions.
  • the ligation of the linear segment and more than one DNA molecule may be carried out simultaneously or sequentially as desired.
  • the DNA polymer may be preformed or formed during the construction of the vector, as desired.
  • the choice of vector will be determined in part by the host, which may be a prokaryotic cell, such as E_;_ coli or Streptomyces sp., or a eukaryotic cell, such as a mouse C127, mouse myeloma, human HeLa, Chinese hamster ovary, filamentous or unicellular fungi or insect cell.
  • the host may also be a transgenic animal.
  • Suitable vectors include plasmids, bacteriophages, cosmids and recombinant viruses, derived from, for example, baculoviruses and vaccinia.
  • the preparation of the replicable expression vector may be carried out conventionally with appropriate enzymes for restriction, polymerisation and ligation of the DNA, by procedures described in, for example, Maniatis et al., cited above. Polymerisation and ligation may be performed as described above for the preparation of the DNA polymer. Digestion with restriction enzymes may be performed in an appropriate buffer at a temperature of 20°-70°C, generally in a volume of 50 ⁇ l or less with 0.1-10 ⁇ g DNA.
  • the recombinant host cell is prepared, in accordance with the invention, by transforming a host cell with a replicable expression vector of the invention under transforming conditions.
  • Suitable transforming conditions are conventional and are described in, for example, Maniatis et al. , cited above, or ''DNA Cloning'' Vol. II, D.M. Glover ed., IRL Press Ltd, 1985.
  • a bacterial host such as E. coli may be treated with a solution of CaCl2 (Cohen et al, Proc. Nat. Acad. Sci., 1973, jS9_, 2110) or with a solution comprising a mixture of RbCl, MnCl 2 , potassium acetate and glycerol, and then with 3-[N-morpholino]-propane-sulphonic acid, RbCl and glycerol.
  • Mammalian cells in culture may be transformed by calcium co-precipitation of the vector DNA onto the cells.
  • the invention also extends to a host cell transformed with a replicable expression vector of the invention.
  • Culturing the transformed host cell under conditions permitting expression of the DNA polymer is carried out conventionally, as described in, for example, Maniatis et al and ''DNA Cloning'' cited above.
  • the cell is supplied with nutrient and cultured at a temperature below 45°C.
  • the hybrid PA expression product is recovered by conventional methods according to the host cell.
  • the host cell is bacterial, such as E. coli it may be lysed physically, chemically or enzymatically and the protein product isolated from the resulting lysate.
  • the product may generally be isolated from the nutrient medium.
  • the DNA polymer may be assembled into vectors designed for isolation of stable transformed mammalian cell lines expressing the hybrid PA; e.g. bovine papillomavirus vectors or amplified vectors in Chinese hamster ovary cells (DNA cloning Vol.II D.M. Glover ed. IRL Press 1985; Kaufman, R.J. et al., Molecular and Cellular Biology 5, 1750-1759, 1985; Pavlakis G.N. and Hamer, D.H., Proceedings of the National
  • the hybrid PA prepared in accordance with the invention may be glycosylated to varying degrees. Furthermore, as observed by Pohl et.al.. Biochemistry, 1984, 23, 3701-3707, varying degress of glycosylation may also be found in unmodified, naturally occurring t-PA. Plasminogen also exhibits varying degrees of glycosylation (Hayes M.L, J.
  • hybrid PA is understood to include such glycosylated variations.
  • the hybrid PA prepared in accordance with the invention may exist in the single or two chain forms or mixtures thereof.
  • the invention extends to all such forms .
  • the hybrid PA of the invention comprises the B-chain of native t-PA or u-PA linked to an A-chain comprising kringle 5 or kringles 4 and 5 derived from plasminogen via a linking sequence of amino acids comprising residues 264 and 275 of t-PA or residues 158 and 148 of u-PA.
  • This hybrid PA A-chain may be employed as one chain of a fibrinolytically active hybrid protein such as disclosed in EP-0 155 387.
  • the hybrid A-chain may be prepared by separation from the B-chain thereof by mild reduction. Alternatively the hybrid A-chain may be prepared by expressing DNA coding therefor in a recombinant host cell and recovering the hybrid A-chain product.
  • the hybrid protein comprising the hybrid A-chain linked to the B-chain of a fibrinolytically active protease may be prepared by (a) mixing said A- and B-chains under oxidative conditions; or (b) ligating DNA encoding said A-chain to DNA encoding said B-chain and expressing the ligated DNA in a prokaryote or eukaryote host; and thereafter optionally blocking the catalytic site of the hybrid protein with a removable blocking group.
  • the oxidation and reduction conditions are as generally described in EP-A-0 155 387.
  • the resulting hybrid protein may be used in any of the methods and compositions described hereinafter for the hybrid PA itself.
  • the hybrid PA of the invention or conjugate thereof can be further derivatised such that any catalytic site essential for fibrinolytic activity is optionally blocked by a removable blocking group.
  • the expression 'removable blocking group' includes groups which are removable by hydrolysis at a rate such that the pseudo-first order rate constant for hydrolysis is in the range of 10 — (, sec—i to 10—_? sec—1 , more preferably 10 " ⁇ sec -1 to 10 ⁇ 3 sec “ , in isotonic aqueous media at pH 7.4 at 37°C.
  • blocking groups and blocking reactions are described in European Patent No.0009879 and EP 0297882 and include acyl groups such as optionally substituted benzoyl or optionally substituted acryloyl.
  • Suitable optional substituents for benzoyl blocking groups include halogen, C ⁇ _ alkyl, C ⁇ _g alkoxy, C- ⁇ . alkanoyloxy, C- ⁇ _ alkanoylamino, amino or p-guanidino.
  • Suitable optional substituents for acryloyl blocking groups include C ⁇ _ alkyl, furyl, phenyl or C- j __g alkylphenyl.
  • the removable blocking group is a
  • the pseudo first order rate constant for hydrolysis of the derivative is in the range 6.0 x 10 -5 to 4.0 x 10 ⁇ 4 sec -1 when measured in a buffer system consisting of 0.05M sodium phosphate, 0.1M sodium chloride, 0.01% v/v detergent comprising polyoxyethylenesorbitan monoleate having a molecular weight of approximately 1300, at pH 7.4 at 37°C.
  • the pseudo first order rate constant for hydrolysis of the derivative is in the range 6.0 x 10 to 2.75 x 10 "4 s “1 , preferably 6.0 x 10 "5 to 2.5 x 10 "4 s “1 , more preferably 6.0 x 10 — ⁇ to 2.0 x 10—4 s—1, still more preferably 6.0 x 10 ⁇ 5 to 1.5 x 10 s -1 and most preferably 7.0 x 10 ⁇ 5 to 1.5 x 10 "4 s "1 .
  • the 2-aminobenzoyl group is substituted with a halogen atom in the 4-position.
  • the halogen atom is fluorine, chlorine or bromine.
  • preferred groups include C ] __ alkyl, C ⁇ _ alkoxy and C- j __ alkenyl substituents in the 3- or 5-positions of the ring.
  • blocking group examples include 4-fluoro-2- aminobenzoyl, 4-chloro-2-aminobenzoyl, 4-bromo-2-aminobenzoyl and p-methoxybenzoyl.
  • hybrid PA and derivatives of the invention are suitably administered in the form of a pharmaceutical composition.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a hybrid PA or derivative of the invention in combination with a pharmaceutically acceptable carrier.
  • compositions according to the invention may be formulated in accordance with routine procedures as pharmaceutical compositions adapted for intravenous administration to human beings.
  • compositions for intravenous administration are solutions of the sterile enzyme in sterile isotonic aqueous buffer.
  • the composition may also include a solubilising agent to keep the hybrid PA or derivative in solution and a local anaesthetic such as lignocaine to ease pain at the site of injection.
  • the hybrid PA or derivative will be supplied in unit dosage form for example as a dry powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of protein in activity units.
  • composition comprises a derivative of the invention or where the hybrid PA includes a removable blocking group, an indication of the time within which the free protein will be liberated may be given.
  • the protein is to be administered by infusion, it will be dispensed with an infusion bottle containing sterile pharmaceutical grade 'Water for Injection' or saline. Where the protein is to be administered by injection, it is dispensed with an ampoule of sterile water for injection or saline.
  • the injectable or infusable composition will be made up by mixing the ingredients prior to administration.
  • the quantity of material administered will depend upon the amount of fibrinolysis required and the speed with which it is required, the seriousness of the thromboembolic condition and position and size of the clot.
  • the precise dose to be employed and mode of administration must per force in view of the nature of the complaint be decided according to the circumstances by the physician supervising treatment.
  • a patient being treated for a thrombus will generally receive a daily dose of from 0.01 to 10 mg/kg of body weight, such as 0.10 to 2.0mg/kg, either by injection in for example up to five doses or by infusion.
  • a method of treating thrombotic diseases which comprises administering to the sufferer an effective non-toxic amount of hybrid PA or derivative of the invention.
  • the invention provides the use of a hybrid PA or derivative of the invention for the manufacture of a medicament for the treatment of thrombotic diseases.
  • the invention also provides a hybrid PA or derivative of the invention for use as an active therapeutic substance and in particular for use in the treatment of thrombotic diseases.
  • cleavage of about l ⁇ g of plasmid DNA or DNA fragments was effected using about 5 units of a restriction enzyme (or enzymes) in about 20 ⁇ l of an appropriate buffer solution.
  • DNA fragments were isolated from LMP agarose gels as described by Maniatis et al, (Molecular Cloning-A Laboratory Manual, Cold Spring Harbor Laboratory, 1982) .
  • the excised gel band was purified using GENECLEAN tm , (Stratech Scientific, London) used according to the manufacturers instructions.
  • Oligonucleotides were made on Applied Biosystems 381A DNA Synthesizer according to the manufacturers instructions. When used in plasmid construction the oligonucleotides were annealed by heating together at 95°C for 5 minutes and cooling slowly to room temperature. The annealed oligonucleotides were then ready for ligation.
  • Cell preparation cells were trypsinised and plated out at approx. 2.4 x 10 5 cells per 35mm dish and incubated in 1.5ml growth medium (this is Hepes buffered RPM1 1640 medium (041-02400) containing 10% Serum (021-06010), 2% sodium bicarbonate solution (043-05080),; Gibco, Paisley, Scotland) at 37°C in a humidified incubator in an atmosphere of 5% C ⁇ 2/95% air. After 72h the cells were refed, and used for transfection 24h later.
  • 1.5ml growth medium this is Hepes buffered RPM1 1640 medium (041-02400) containing 10% Serum (021-06010), 2% sodium bicarbonate solution (043-05080),; Gibco, Paisley, Scotland
  • Transfection procedure Cultures were changed to Eagles MEM (041-01095), 10% serum (021-06010), and 1% non-essential amino acids (043-01140) 3h before transfection. The transfections used calcium coprecipitation as described in 'DNA Cloning' Ed. D.M. Glover (Chap. 15, C. Gorman). Glycerol shock and 5mM butyrate treatments were used. Plasminogen activator(s) secreted by transfected cells was harvested in 1.0ml RPMI 1640 medium (as above, but lacking serum) + 4% Soybean Peptone.
  • Cell preparation cells were trypsinised and plated out at a density of approx. 2.5 x 10° cells per 175cm flask in 30ml growth medium (above) . After 72h an extra 25ml of growth medium was added and the cells were used for DNA transfection 24h later (as above) . 25ml of harvest medium were used per flask.
  • the cells were plated at a density of approximately 2.0 x 10 6 cells per flask and 25ml of growth medium was added after 96h incubation and the cells used as above.
  • Hybrid was assayed against the chromogenic substrate S-2288 (KabiVitrum, Sweden) at a substrate concentration of ImM in 0.1 M triethanolamine.HCl pH 8.0 at 25°C.
  • An SU is defined as the amount of activity that gives an O.D. increase at 405nm of 0.001/min in 0.5 ml substrate in a 1 cm pathlength cell.
  • SDS PAGE was carried out to determine the apparent molecular weight (s) of the hybrid plasminogen activators using essentially the method of Laemmli (Nature 1970 227 680-685) .
  • the activators were identified either by staining for protein or by a fibrin zymography technique (Dodd, I. et al Thromb. Haemostasis 1986, 55. 94-97) . Using these methods it was generally possible to determine chain nature (sc v tc) .
  • a Q is the initial activity of the acyl-enzyme and A ma ⁇ is the maximum activity possible after deacylation and was determined by deacylation of an aliquot of acyl-enzyme in 0.1M Tris. HCl, 20% w/v glycerol, 0.14M NaCl, 0.01% w/v Tween 80 pH 7.4 at 37°C for lh followed by amidolytic assay with S-2288 under the above conditions (i.e. phosphate buffer pH 7.4, 37°C) .
  • a and K the first order deacylation rate constant, were treated as unknowns in the fitting process and were derived by non-linear regression analysis on a VAX 11/750 computer.
  • B t t-PA amino acid residues 276 to 527 inclusive.
  • K ⁇ P plasminogen amino acid residues 358 to 435 inclusive.
  • K5P plasminogen amino acid residues 462 to 541 inclusive.
  • sc indicates that the protein is in single chain form.
  • tc indicates that the protein is in two chain form.
  • This plasmid comprises a cDNA encoding the t-PA signal sequence (-35 to -1) linked to the above hybrid plasminogen activator.
  • the restriction sites used below were located as follows:-
  • Fragment 1 was the large fragment from an Sstl/Bglll digest of a ⁇ UC8 derivative containing a modified multiple cloning site.
  • Fragment 2 was an approximately lkb Styl/Sstl fragment from pTRH37 (as described in EP-A 0297 882) encoding most of K 4 ?,
  • Linker 3 oligonucleotide linker
  • Linker 3 (designed to encode the tripeptide SYQ and amino-acid residues 347-359 of plasminogen) was formed by annealing two oligonucleotides (A) and (B) of sequence:-
  • the DNA was transformed into E.coli HB101 cells.
  • a plasmid (pDH55i) was isolated which has the structure shown in Fig.l.
  • Fragment 4 was an approximately lkb Bglll/Sstl fragment from pDH55i encoding the first three amino acids (SYQ) of t-PA, residues 347-357 of plasminogen, K 4 P K 5 P and part of Fragment 5: was an approximately 1.6kb Sstl/BamHl fragment from pTRH37 encoding the C-terminal part of B- and vector sequences.
  • Fragment 6 was a BamHl/Bglll fragment derived from pTRE15 encoding vector sequences and the t-PA signal sequence. These three fragments were ligated together and transformed into E.coli HB101 cells.
  • a plasmid was isolated which has the structure shown in Fig.2.
  • the plasmid when introduced into HeLa cells, directed the expression of a novel plasminogen activator.
  • Conditioned medium from twenty 175c ⁇ r- HeLa cultures transfected with the plasmid pDH55 was centrifuged at approximately 9000g for 30 min.
  • the supernatant (480ml) was buffer-exchanged into PBS'A' (Dulbecco) /0.01% Tween 80 pH 7.4 using a column (i.d., 90mm;h,226 mm) of Sephadex G25 and a 710ml fraction eluting immediately after the void volume of the column was obtained.
  • the 710 ml fraction was then purified in a similar way to that described for t-PA (Dodd, I. et al FEBS Lett., (1986) 2JJ9.13-17).
  • the zinc chelate and lysine Sepharose Fast Flow columns had volumes of 90ml and 10ml respectively.
  • Protein H55 was dissociated from the lysine Sepharose column using a 0.5M arginine-containing buffer; peak H55 - containing fractions were identified by a microtitre plate S2288 assay and were pooled and were ultrafiltered using a membrane with a nominal molecular weight cut off of 10,000 (YM10, Amicon) to a final volume of 2.2ml. This retentate was regarded as the H55 product.
  • Fibrin plate assay showed that the original, conditioned harvest medium contained approximately 17000 IU and that the 5 product contained approximately 22000 IU. This difference is believed to be within the natural error in the assay.
  • This plasmid comprises a cDNA encoding the t-PA signal sequence (-35 to -1) linked to the above hybrid plasminogen activator.
  • the restriction sites used during construction were as follows:-
  • HinFl plasminogen nucleotide 1498 AlwNl: t-PA nucleotide 1130 Sstl : t-PA nucleotide 1417 BamHl: located in SV40 polyA/t intron fragment of pTRE15 Bglll: t-PA nucleotide 187
  • Fragment 1 was from an Sstl/Bglll digest of a pUC8 derivative containing a modified linker region, (as described in Example 1) .
  • Fragment 7 was an approximately 411bp HinFl/AlwNl fragment from pTRH37 (as described in EP-A-0297 882) encoding part of the K 4 P-K 5 P bridge, the whole of K 5 P and part of B t .
  • Fragment 8 was an approximately 292bp AlwNl/Sstl fragment from pTRH37 (as described in EP-A-0297 882) encoding part of B fc .
  • Linker 9 (designed to encode the tripeptide SYQ and amino-acid residues 443-455 of plasminogen) was formed by annealing two oligonucleotides C and D of sequence:-
  • the DNA was transformed into E.coli HB101 cells.
  • a plasmid (pDH56i) was isolated which has the structure shown in Fig.3.
  • Fragment 10 was an approximately 750bp Bglll/Sstl fragment from pDH56i encoding the first three amino acids of t-PA (SYQ), residues 443 to 461 of plasminogen, K ⁇ P and part of
  • Fragment 5 was an approximately 1.6kb Sstl/BamHl fragment from pTRH37 encoding the C-terminal part of B 1 - and vector sequences. (As described in Example 1)
  • Fragment 6 was a BamHl/Bglll fragment derived from pTRE15 encoding vector sequences and the t-PA signal sequence. (As described in Example 1)
  • a plasmid was isolated (pDH56) which has the structure shown in Fig.4.
  • the plasmid when introduced into HeLa cells, directed the expression of a novel plasminogen activator.
  • the 780ml sample was chromatographed on zinc chelate Sepharose (see Example 2) and aminohexyl Sepharose 4B (AH Sepharose; Sigma chem.Co.). The latter chromatography was carried out as follows.
  • the column (i.d., 15 mm; h, 45 mm; Vt, 8.0 ml) was equilibrated with PBS/TW.
  • the imidazole-eluted fraction from the zinc chelate column was applied and was washed through with PBS/TW.
  • H56 protein was desorbed using 0.02M Tris/0.5M NaCl/0.5M L-arginine/0.01% Tween 80 pH 7.0. All parts of the chromatography were at 4°C at approximately 100 cm h ⁇ . Active fractions (containing H56) were identified using S2288 and then concentrated by stirred-cell ultrafiltration (YM10, Amicon Ltd) . The ultrafiltered retentate was regarded as the product.
  • the product showed a dose-response relationship on human fibrin plates slightly different to that of t-PA and exhibited a single major band of fibrinolytic activity at apparent M r 40,000 on SDS PAGE followed by fibrin zymography.
  • This band had the same M r as a doublet, possibly triplet, of polypeptides detected after probing Western blots of H56 with a monoclonal directed at the B-chain of t-PA (ESP2, BioScot, U.K.) or an anti-t-PA B chain Ig G preparation (Dodd, I. et. al, Thrombos. Haemostos., 1986 55. 94) .
  • the supernatant was buffer-exchanged into PBS/TW using Sephadex G25 (PD10) and then purified on freshly autoclaved pABA Sepharose (Vt 11ml) using the same protocol as for AH Sepharose described in (a) .
  • Material that was eluted from the pABA Sepharose column by the 0.5M arginine buffer was concentrated (stirred-cell ultrafiltration; YM10) and buffer-exchanged into 0.05M sodium phosphate/0.IM sodium chloride/lOmg ml mannitol/50 ⁇ M E-amino caproic acid/0.01% Tween 80 ⁇ H7.4 (Sephadex G25, PD10) .
  • the buffer-exchanged material was regarded as the product.
  • the dose-response of the product on fibrin plates was approximately parallel to that of t-PA; the product contained approximately 3000 IU/ml.
  • SDS PAGE/fibrin zymography and Western blotting studies revealed similar pictures to those obtained for product (a) .
  • SDS PAGE (non-reduced) followed by silver staining also showed a major band in the approximate M r 40,000 region.
  • the cDNA encoding H55 was recovered from pDH55 as a 3.2kb BamHI/MluI fragment. This fragment was subcloned into pTRHll (EPA 0297 882) replacing the original 4.lkb MluI/BamHI fragment (which encoded protein H204) .
  • the new plasmid was called pDH17. In pDH17, the hybrid and dhfr transcription cassettes are opposed i.e. converge at their 3' ends.
  • a second plasmid, pDH16 was also prepared.
  • the whole Xhol fragment carrying the H55 transcription cassette (including RSVLTR and SV40 elements : depicted in EPA 0297 882, pTRH71) is reversed with respect to that in pDH17.
  • the transcription cassettes for dhfr and H55 are therefore transcribed in tandem.
  • CHO cells were trypsinised and plated out at 6 x 10 5 per 90 mm dish and left in growth medium [Hams F12 nutrient media (041-1765) with 1% stock penicillin/streptomycin (043-5070) and 10% foetal calf serum (013-6290); Gibco, Paisley, Scotland] at 37°C in a humidified incubator, in an atmosphere of 5% C0 2 /95% air. After 18 hrs the growth medium was replaced with transfection medium [Eagles MEM (041-1095) with 1% non-essential amino acids (043-1140), 1% stock penicillin/streptomycin (043-5070) , and 10% newborn calf serum (021-6010) ; Gibco, Paisley, Scotland] . After a further 2 hrs the cells were used for DNA transfection.
  • Hams F12 nutrient media (041-1765) with 1% stock penicillin/streptomycin (043-5070) and 10% foetal calf serum (013-6290); Gibco
  • transfection procedure carried out in transfection medium, used calcium coprecipitation and glycerol shock as described in DNA Cloning Volume II (Ed. D.M. Glover; chapter 6, C. Gorman) . Following transfection the cells were maintained in growth medium for 48 hrs under growth conditions (as above), prior to the selection procedure.
  • the cells were medium changed into selective medium [ ⁇ MEM (041-2561) with 2% stock glutamine (043-5030) , 1% stock penicillin/streptomycin (043-5070) and 10% dialysed foetal calf serum (063-6300) ; Gibco, Paisley, Scotland] .
  • the cells were maintained in selective medium for 8-10 days until colonies of dhfr+ cells appeared.
  • Isolated colonies were grown to confluency in 25 cm flasks and harvested in serum-free medium for 24 hours. Fibrinolytically active protein was detected by fibrin plate assay.
  • the material was buffer-exchanged using Sephadex G25 (PD10) into 0.02M Tris/0.2M NaCl/0.2M L-Arginine/0.01% Tween 80 pH 7.0 and stored at -40°C.
  • the material was buffer-exchanged into 0.02M Tris/0.2M NaCl/0.2M L-Arginine/0.01% Tween 80 pH 7.0 using Sephadex G25 (PD10) and stored at -40°C.
  • pAcCL29 Vector this is based on pAcYMl (an expression vector in which a unique Bam HI cloning site has been positioned so as to maximise expression using the polyhedrin promoter: Matsura, Y, Possee R.D. Overton, H.A. and Bishop D.H.L [1987] J.Gen.Virol 68. 1233-1250) .
  • pAcCL29 (Livingstone, C. and Jones I (1989) NAR 17. 2366) was derived from pAcYMl as follows; an approximately 5Kb EcoRI-XhoI fragment coding for all the signals necessary for efficient expression and recombination were removed from pAcYMl, blunt ended and ligated into in-filled EcoRI Hind III sites in pUCll ⁇ (Vieira. J. and Messing J. [1987] Meth. Enzy. I ⁇ 3_3-ll) .
  • Wild type virus is: ⁇ nt.n ⁇ rapha californica nuclear polyhedrosis virus (AcNPV) .
  • IPLB Sf21 derived from Spodoptera frn ⁇ iperda . (Vaughan, J.L., Goodwin, R.H., Thompkins, G.J. and McCawley, P. 1977: In Vitro 13, 213-217)
  • the tac expression vector pDB525 was derived from pKK223-3
  • PTR550 The 3.28 kb Sphl-Sca I fragment of pKK223-3 was replaced with the equivalent fragment from pAT153 (Twigg, A.J. and Sherratt, D.J. (1980) Nature, 283. 216-218) to render the plasmid non-mobilisable; this new vector was called PTR550.
  • PTR550 was restricted with Eagl and a 1.7kb blunt-ended EcoRI fragment from ptac-1-Iq, encoding the laclq gene, was ligated in to give pDB525 (figure 5A) .
  • laclq gene (Calos, M.P. (1978) Nature, 274 762-765) ensures tight repression of the tac promoter under non-induced conditions.
  • H55 expression plasmid pDB549 was transformed into E.coli HB101.
  • the transformed host was grown in -Broth at 37°C to an OD550 of 0.8-1.0 and expression was induced with ImM IPTG (isopropyl- ⁇ -D-thiogalactopyranoside) .
  • Construction of the plasmid pDH55U was accomplished by substituting a 620b ⁇ MluI-BstXI fragment from pDH55
  • Example 1 Mlul site located in RSVLTR promoter, BstXI site at nucleotide 1209 in plasminogen cDNA sequence
  • Example 2 Mlul site located in RSVLTR promoter, BstXI site at nucleotide 1209 in plasminogen cDNA sequence
  • the novel hybrid was expressed using HeLa cell system (Methods) .
  • Fibrinolytically active protein as determined by fibrin plate assay, was recovered from the HeLa cell harvest medium. £;-;ample 1
  • kb Mlul - BspMII (Mlul in RSVLTR, BspMII at nucleotide 1060 in u-PA cDNA) fragment and a 593 bp Avail - BspMII (Avail at 1693 in plasminogen, BspMII at 1060 in u-PA) fragment were isolated from pDH55U (Example 12) . These were ligated with a 816 bp Mlul - Avail (Mlul in RSVLTR, Avail at 1693 in plasminogen) fragment from ⁇ DH56 (Example 4) to give pDH56U. Fibrinolytically active protein was expressed as for H55U.
  • Example 7 The transfections described in Example 7 were carried out with either 10 or 20 ⁇ g of pDH16 or ⁇ DH17. All were then selected as described in Example 7 and were amplified as follows.
  • methotrexate concentration was initially 0.05 ⁇ M and was increased stepwise to 5 or 10 ⁇ M.
  • methotrexate the best cell line, as judged by activity on a fibrin plate, was the pDH17-transfected mass culture (17MC) .
  • methotrexate the 3 best cell lines (the pDH16-and pDH17-transfected mass cultures [16MC and 17MC] and the pDH17-transfected clone [17.1]) were sub-cloned, giving 12 sub-clones per cell line.
  • the best cell line from these 36 sub-clones plus the 3 parental lines was sub-clone #1 isolated from 16MC i.e., 16MC.1.
  • a column (i.d., 16 mm; h, 12mm) of Benzamidine Sepharose was equilibrated with PBS 'A 1 (Dulbecco) /0.01% Tween 80.
  • the conditioned media was applied to the column and was washed through with equilibration buffer followed by 0.02M Tris/0.5M NaCl/0.01% Tween 80 pH 7.4.
  • H55U was then dissociated from the matrix by washing with 0.02M Tris/0.5M NaCl/0.5M L-arginine/0.01% Tween 80 pH7.4.
  • the chromatography was at 4°C at a flow rate of 100 cmh -1 .
  • the eluant from the column was collected as discrete fractions.
  • Fractions containing the protein H55U were identified using the microtitre-plate based chromogenic substrate assay (General example (ix) ) except that S2444 was used instead of S2288.
  • the most active fractions were pooled and were ultrafiltered (YM10, Amicon Ltd) to 2.0 ml (the 'product' ) .
  • Assay of the product by fibrin plate assay with reference to a u-PA standard showed it contained 100 IU/ml.
  • a column (i.d., 16mm; h, 15mm) of Benzamidine Sepharose was equialibrated with PBS "A' (Dulbecco) /0.01% Tween 80.
  • the conditional media was applied to the column and was washed through with equilibration buffer followed by 0.02M Tris/0.5M NaCl/0.01% Tween 80 pH 7.4.
  • H56U was then dissociated from the matrix by washing with 0.02M Tris/0.5M NaCl/0.5M L-arginine/0.01% Tween 80 pH 7.4.
  • the chromatography was at 4°C at a flow rate of 100 cm h -1 .
  • the eluant from the column was collected as discrete fractions.
  • Fractions containing the protein H56U were identified using th microtitre-plate based chromogenic substrate assay (General example (ix) ) except that S2444 was used instead of S2288.
  • the most active fractions were pooled and were ultrafiltered (YM10, Amicon Ltd) to 2.9 ml (the * product' ) .
  • H56 was purifed from the media using two chromatography columns in series.
  • the media was passed down a column (i.d., 41mm; h, 38mm) of S-Sepharose Fast Flow that had been equilibrated in 20mM succinate, lOmM EACA pH 5.0.
  • the media was washed through with equilibration buffer followed by a gradient (in equilibration buffer) of 0 to IM NaCl.
  • a final rinse with the IM NaCl-containing buffer was then carried out.
  • the eluant was fractionated and assayed using the S2288 chromogenic substrate assay.
  • the peak H56-containing fractions were those collected during the development of the NaCl gradient and were pooled. The pH of this pool was adjusted to 7.0 using NaOH.
  • the pH 7.0 - adjusted pool was then chromatographed on a column (i.d., 16mm; h, 55mm) of zinc chelate Sepharose that had been equilibrated in PBS * A' (Dulbecco) /0.01% Tween 80. After application of the pool from the S- Sepharose the zinc chelate was washed with equilibration buffer followed by 0.02M sodium phosphate/0.3M NaCl/0.01% Tween 80 pH 7.4 and then 0.02M sodium phosphate/0.3M NaCl/0.05M imidazole/0.01% Tween 80 pH 7.4.
  • H56-containing fractions were identified by S2288 substrate assay and were pooled.
  • the pool had a volume of 44 mi and contained 100,000 IU by fibrin plate assay with reference to a u-PA standard curve. This particular pool was not analysed by SDS PAGE.
  • other purified H56 batches, prepared under almost identical conditions, were analysed under non-reducing conditions. On silver- staining, a single major band at apparent M r approximately
  • the second purification example relates to 12 litres conditioned media from the CHO cell line 16MC.1
  • the column was then washed with PBS containing IM NaCl at a linear velocity of lOOcmh -1 for 5 bed volumes.
  • the column was then eluted using a linear gradient of 0-lOmM EACA in PBS/1M NaCl (over 6 bed volumes) . Elution was monitored by following 280nm adsorption of the protein eluted by the EACA. 73mg, as determined by fibrin plate analysis, of H55 was eluted in IL of solution.
  • Plasmid pDH55 signal t-PA signal sequence
  • Plasmid pDH56 signal t-PA signal sequence.

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US5801146A (en) * 1996-05-03 1998-09-01 Abbott Laboratories Compound and method for inhibiting angiogenesis
US5972896A (en) * 1996-05-03 1999-10-26 Abbott Laboratories Antiangiogenic peptides and methods for inhibiting angiogenesis
WO2000032759A1 (en) * 1998-12-02 2000-06-08 Oklahoma Medical Research Foundation Human plasminogen activator
US6699838B1 (en) 1996-05-03 2004-03-02 Abbott Laboratories Antiangiogenic peptides
US7442763B2 (en) * 1999-12-06 2008-10-28 Hopital Sainte-Justine Compositions for treating abnormalities in glomerular filtration, patent ductus arteriosus and osteoporosis

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GB9013345D0 (en) * 1990-06-14 1990-08-08 Beecham Group Plc Novel compounds

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US5801146A (en) * 1996-05-03 1998-09-01 Abbott Laboratories Compound and method for inhibiting angiogenesis
US5972896A (en) * 1996-05-03 1999-10-26 Abbott Laboratories Antiangiogenic peptides and methods for inhibiting angiogenesis
US5981484A (en) * 1996-05-03 1999-11-09 Abbott Laboratories Antiangiogenic peptides and methods for inhibiting angiogenesis
US6057122A (en) * 1996-05-03 2000-05-02 Abbott Laboratories Antiangiogenic peptides polynucleotides encoding same and methods for inhibiting angiogenesis
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US6699838B1 (en) 1996-05-03 2004-03-02 Abbott Laboratories Antiangiogenic peptides
US7495068B2 (en) 1996-05-03 2009-02-24 Abbott Laboratories Antiangiogenic peptides, polypeptides encoding same and methods for inhibiting angiogenesis
WO2000032759A1 (en) * 1998-12-02 2000-06-08 Oklahoma Medical Research Foundation Human plasminogen activator
US7442763B2 (en) * 1999-12-06 2008-10-28 Hopital Sainte-Justine Compositions for treating abnormalities in glomerular filtration, patent ductus arteriosus and osteoporosis

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