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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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  • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
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  • 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
  • C12N9/6459—Plasminogen activators t-plasminogen activator (3.4.21.68), i.e. tPA
• • 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
• • 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)
  • C12Y304/21069—Protein C activated (3.4.21.69)
• • 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 hybrid fibrinolytic enzyme- its preparation, pharmaceutical compositions containing it and its use in the treatment of thromboembolic diseases, in particular acute myocardial infarction.
• European Patent No 0009879 discloses derivatives of in vivo fibrinolytic enzymes which are useful therapeutic agents for treating venous thrombosis.
• the derivatives are characterised by the active catalytic site on the enzymes being blocked by a group which is removable by hydrolysis such that the pseudo first order rate constant for hydrolysis is in the range 10 — Pi to 10 — J - ⁇ sec—1.
• EP-0155387 discloses a fibrinolytically active hybrid protein which comprises one chain of a 2-chain protease linked to a chain of a different 2-chain protease, or to the same chain of the same protease*, at least one of the chains in the hybrid protein being derived from a fibrinolytically active protease, such that the hybrid protein has a catalytic site essential for fibrinolytic activity which is optionally blocked by a removable blocking group.
• EP-A-0297882 discloses a hybrid plasminogen activator which comprises the five kringle domains 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, the catalytic site of the t-PA or u-PA B-chain being optionally blocked by a removable blocking group.
• EP-A-0297882 A large number of possible blocking groups were mentioned in EP-A-0297882 as suitable, particular emphasis being placed on haloanthraniloyl groups, i.e. the 2-aminobenzoyl group substituted in the 3- or 4- position with a halogen atom and optionally further substituted with one or more weakly electron-withdrawing or electron-donating groups.
• Particular blocking groups taught in EP-A-0297882 included 2-aminobenzoyl substituted in the 4-position with fluorine, chlorine or bromine.
• 4-methoxybenzoyl plasminogen 1-5 1/t-PA 262-527 including one and two chain variants, lys ⁇ g and glu- ⁇ variants, and mixtures thereof.
• t-PA and plasminogen have the meanings and a ino acid numbering system given in EP-A-0297882.
• acyl hybrid PA of the invention is the glu- variant, especially the single chain form thereof.
• the acyl hybrid PA preferably comprises at least 60%, more preferably at least 80% of the preferred glu lf single chain variant relative to the other chain variants.
• the invention provides glu- ⁇ , single chain plasminogen 1-541/t-PA 262-527.
• This variant is preferably present at a level of at least 60%, more preferably 80% relative to the other chain variants.
• the acyl hybrid PA of the invention may be prepared as generally described in EP-A-0297882.
• the unacylated hybrid PA is first prepared by expressing DNA encoding the hybrid PA in host cell and recovering the hybrid PA product.
• the DNA is prepared conventionally by the condensation of appropriate mono-, di- or oligomeric nucleotide units as described in EP-A-0297882.
• the host cell is prepared conventionally by transformation with a replicable expression vector capable, in the host cell, of expressing the coding DNA.
• the choice of vector will be determined in part by the host cell, which may be prokaryotic, • such as E_;_ coli, or eukaryotic, such as .mouse C127, mouse myeloma, Chinese hamster ovary, fungi e.g. filamentous fungi or unicellular 'yeast' or an insect cell such as Drosophila.
• the host cell may also be in a transgenic animal.
• Suitable vectors include plasmids, bacteriophages, cosmids and recombinant viruses, derived from, for example, baculoviruses or vaccinia.
• the preferred gl lf single chain form of the hybrid PA may be obtained by appropriate choice of vector, host cell, expression conditions and purification conditions.
• Chinese hamster ovary (DXB11) cells may be harvested in serum-free medium to prevent proteolysis.
• DXB11 Chinese hamster ovary
• protease inhibitor(s) e.g. aprotinin
• protease inhibitors may be required in all or some of the purification buffers. 5
• the hybrid PA is reacted with a suitable acylating agent in order to prepare the 4-methoxybenzoyl derivative.
• the invention therefore also provides a process for 0 preparing the acyl hybrid PA of the invention, which process comprises reacting the hybrid PA with a blocking agent JK in which J is a locating group which mediates binding of the agent to the catalytic site of the enzyme and K is a p-methoxybenzoyl group. 5
• group J examples include 4-amidinophenoxy and
• a preferred acylating agent is 4-amidinophenyl 04'-methoxybenzoate, normally used in the form of the HC1 salt. This compound is disclosed in EP-0009879.
• the blocking reactions are preferrably carried out in aqueous media, at a pH in the range 6.75 to 8.5, more 5 preferably 6.75 to 7.25 and at temperatures in the range 0°C to 30°C more preferably 15°C to 30°C.
• the preferred concentration range for the blocking agent is 0.01 to 2.0 millimolar, and the preferred enzyme 0 concentration range is 1 to 500 micromolar.
• the acyl hybrid PA or the glu ⁇ , single chain plasminogen 1- 541/t-PA 262-527 of this invention is preferably administered as a pharmaceutical composition for the treatment of thrombotic diseases.
• the present invention also provides a pharmaceutical composition
• a pharmaceutical composition comprising the acyl hybrid PA or glu-i, single chain plasminogen 1-541/t-PA 262-527 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 hybrid PA in sterile isotonic aqueous buffer. Where necessary the composition may also include a solubilising agent to keep the hybrid PA in solution and a local anaesthetic such as lignocaine to ease pain at the site of injection.
• the hybrid PA 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, as well as, for the acyl hybrid PA, an indication of the time within which the free protein will be liberated.
• the hybrid PA is to be administered by infusion, it will be dispensed with an infusion bottle containing sterile pharmaceutical grade 'Water for Injection' .
• the hybrid PA or is to be administered by injection it is dispensed with an ampoule of sterile water for injection.
• 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 mature, fresh or nascent thrombus will generally receive a daily dose of from 0.01 to 10 mg/kg of body weight either by injection in 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 an acyl hybrid PA or glu- ⁇ , single chain plasminogen 1-541/t-PA 262-527 of the invention.
• an acyl hybrid PA or glu- ⁇ , single chain plasminogen 1-541/t-PA 262- 527 of the invention for the manufacture of a medicament for the treatment of thrombotic diseases.
• the invention also provides an acyl hybrid PA or glu ⁇ , single chain plasminogen 1-541/t-PA 262-527 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.
• blunt ends were required they were produced by treating the DNA preparation with DNA Polymerase 1, Klenow fragment as described by Maniatis et al, (Molecular Cloning-A Laboratory Manual, Cold Spring Harbor Laboratory, 1982) .
• sticky ends were removed by digestion using Mung Bean nuclease (Maniatis et al) .
• Short kinased oligonucleotide linkers encoding single or multiple restriction sites were ligated onto blunt ended fragments, the linker(s) was digested with the appropriate restriction endonuclease producing the required 'sticky end' necessary for further manipulation (Maniatis et al) .
• Transformation of plasmid DNA into E. coli HBlOl used competent HBlOl supplied by Gibco BRL (Paisley, Scotland) , according to the manufacturers instructions.
• DNA fragments were isolated from LMP agarose gels as described by Maniatis et al. Alternatively the excised gel band was purified using GENECLEAN (Stratech Scientific, 5 London) used according to the manufacturer's instructions.
• Oligonucleotides were either purchased or were made on 0 Applied Biosystems 381A DNA Synthesizer according to the manufacturer's instructions and were kinased as described in Maniatis et al.
• Hybrid was assayed against the chromogenic substrate S2288 (KabiVitrum, Sweden) at a substrate concentration of lmM 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
• hybrid plasminogen activator solutions were measured on human plasminogen-containing fibrin plates as described (Dodd, I., and Carr, K., Thrombosis Res. 1989 55.79 - 85) .
• Dose-responses of hybrid plasminogen activators had slightly different slopes to those of the tissue-type plasminogen activator standard so all activities are approximate. Activities are expressed in IU with reference to the 2nd International standard for t-PA, Lot 86/670.
• Based on the activity on fibrin plates the hybrid protein is estimated* to have a specific activity of approximately 150,000 - 250,000 IU/mg protein, equivalent to a molar specific activity, of approximately 1.5 - 2.5 x 10 13 IU/mole.
• the pseudo first order rate constant was determined by hydrolysing the acyl-enzyme under physiological conditions, i.e. in isotonic aqueous media at pH 7.4 and at 37°C. At regular intervals aliquots were withdrawn and incubated with a chromogenic substrate and the rate of conversion of the substrate measured as indicated above.
• the hydrolysis was followed until such time as the rate of conversion of substrate reached a maximum.
• the rate constant k may then be calculated by plotting:
• a ma ⁇ is the maximum rate at which an aliquot converts substrate and t is the rate at which an aliquot converts substrate at time t.
• rate.constants are calculated by computerised non-linear regression analysis, fitting the A t and time data to the equation:-
• a Q is the activity of the acyl-enzyme preparation before deacylation.
• 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. HC1, 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.
• the deacylation half-life for some compounds was determined in human plasma rather than in phosphate buffered saline. Measurement of plasma deacylation rate of acyl enzymes relies on the property of rapid, irreversible inhibition of hybrid plasminogen activators in plasma following their deacylation. Acyl enzyme (5nM) was incubated in plasma at pH7.4 and 37°C and lOO ⁇ l aliquots removed at various time points.
• the deacylation half-life is defined as the time at which 50 per cent of the initial theoretical concentration of acyl enzyme had deacylated.
• 35 GIU-L indicates the protein is believed to comprise the native (nascent) plasminogen N-terminus i.e. amino acid residues 1 onwards.
• Lys-rg indicates the protein is believed to comprise the processed lys 7 g N-terminus.
• Alternative processed N-termini e.g. metgg and val 7 c- are known in nature (Miyashita et al 1988, Haemostasis 1J3 supp. 1, pp 7-13) .
• sc. indicates that the protein is in single chain form
• tc. indicates that the protein is in two chain form
• the plasmid pSV 2 dhfr was obtained from the American Type Culture Collection (ATCC 37146: Molec. Cell. Biol. .1: 854-864 1981).
• the plasmid BPV-MT-Xho was obtained from D. Hamer (National Institutes of Health, Bethesda, Maryland) and contains a version of the mouse metallothionein-1 gene (Hamer and Walling (1982) J. Mol. Appl. Genet. 1 273-288) in which the Bglll site just 5' to the translation start point has been converted to an Xhol site by linkering.
• the dhfr- Chinese hamster ovary cell line (CHO-DXB11) has been described previously (Urlaub, G. and L.A. Chasin, Proc. Natl. Acad. Sci. 77.4216-4220).
• the plasmid pTRH69 was constructed from two fragments A and B isolated from the plasmid PSV 2 dhfr and the plasmid BPV-MT-Xhol respectively. Fragment A - The plasmid PSV 2 dhfr (Fig. 1) was linearised * with EcoRI, blunt ended by infilling and linkered with Xhol linkers. The linearised plasmid was then digested with Xhol and Bglll releasing fragment A (a 3.4kb fragment encoding the dihydrofolate reductase cDNA and SV 4Q early promoter sequences) .
• Fragment B The plasmid BPV MT-Xhol was linearised with Sstl, blunt-ended by nuclease digestion and linkered using Bglll linkers. The linear plasmid was then digested with Hindlll, blunt ended by infilling and linkered with Xhol linkers. The plasmid was then digested with Bglll and Xhol, releasing fragment B (0.3kb encoding the 3 metallothionein polyadenylation sequences) (Fig. 2) . Fragments A and B were isolated by LMP agarose electrophoresis and ligated together. The ligated DNA was transformed into E coli HBlOl and the plasmid PTRH69 was obtained (Fig. 3) .
• the plasmid ⁇ TRH69 was linearised with Xhol and phosphatased.
• the LMP agarose-purified fragment was ligated with a 3.3Kb Xhol fragment derived from the plasmid pTRE24, which encodes a modified t-PA protein.
• E. coli HBlOl was then transformed with the ligated DNA to obtain pTRH70 (Fig. 4) .
• the plasmid pTRH70 was digested with Mlul and BamHl; a 4.5Kb fragment (comprising the vector functions) was isolated by LMP agar se gel purification and ligated with a 4.1Kb Mlul/BamHl fragment isolated from pTRH37 (encoding hybrid protein H37) . E. coli HBlOl was transformed with the ligated DNA and the plasmid pTRH13 was obtained (Fig. 5) .
• CHO cells were trypsinised and plated out at 5 x 10 * ⁇ per
• the transfection procedure carried out in growth medium used calcium coprecipitation and glycerol shock as described in: DNA Cloning, Ed D.M. Glover (Chap. 15. C. Gorman) . Following transfection with pTRH13 the cells were maintained in growth medium for 46 hrs under growth conditions (as above) prior to the selection procedure.
• the selection and co-amplification procedure was carried out essentially as described by R. J. Kaufman (1985) Molecular and Cellular Biology 5, 1750-1759. 46hrs post transfection the cells were medium changed into selective medium MEM ALPHA (041-02571) , 1% stock glutamine (043-05030) , 1% stock penicillin/streptomycin (043-05070) and 10% dialysed bovine foetal calf serum (220-6300AJ) -(Gibco, Paisley, Scotland) . The cells were maintained in selective medium for 8-10 days until colonies of dhfr + cells appeared. When colonies were established the cells were medium changed into selective medium containing methotrexate, (A6770; Sigma, England) .
• the methotrexate concentration was initially 0.02 ⁇ M and was increased stepwise to 5 ⁇ M.
• Early batches of the hybrid protein H37 were produced from a mass culture (CHOZ3) amplified to 3 ⁇ M methotrexate. Later batches were produced either from two cell lines (CH0Z5-C4 and CHOZ5-C13) which were isolated by ring cloning from a mass culture amplified to 5 ⁇ M methotrexate (CHOZ5) , or CHOZ5-C4E12, which is a subclone (dilution plated) of CHOZ5-C4.
• Example 10 selected for survival in 3 ⁇ M methotrexate (Example 1.2c) were harvested (lOOmls per 175c ⁇ - flask) for 3 or 4 days in Hams F12 nutrient mixture: Dulbecco's modified Eagles medium (50:50)and 1% pencillin/streptomycin (GIBCO, Paisley, Scotland) (note, serum free) .
• the ultrafiltered retentate (10.7ml) contained approximately 220,000 IU/ml and 23,000 SU/ml. These figures generate an IU:SU ratio of 9.6 (but see comments in Methods:assay) ; other purifications typically yielded good quality sc H37 with lower ratios.
• Single chain H37 (lOmg ml -1 in 0.02M Tris/0.5M NaCl/0.5M L-arginine/0.01% Tween 80 pH 7.4; 0.5ml) isolated from CHOZ5-C4 and -C13 cell lines was mixed with human lys plasminogen (0.5mg ml -1 barbitone/saline buffer; 3 ⁇ l) and incubated for 2h at 37°C and then stored at -40°C. Analysis by SDS PAGE, followed by protein staining and fibrin zymography showed that the majority of the incubate was in the glu- j ⁇ two-chain form.
• the 2.0ml was buffer-exchanged into 0.02M Tris/0.2M NaCl/0.2M L-arginine/0.02% Tween 80 pH 7.4 using Sephadex G25 (PDIO) and the product stored at -40°C.
• PDIO Sephadex G25
• the mixture was buffer-exchanged into 0.02M Tris/0.2M NaCl/0.2M L-arginine/0.01% Tween 80 pH 7.4 using a Sephadex G25 PD10 column.
• the buffer-exchanged product was stored at -40°C.
• the deacylation half-life of the product in human, plasma at 37°C was 34 min.
• BPV Bovine papillomavirus sequences
• MT-1 Mouse metallothionein gene sequences - including
• Sstl Restriction enzyme site: Sstl was linkered with Bglll linkers after Sstl digestion

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