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• • C—CHEMISTRY; METALLURGY
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  • 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
  • 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)
  • C12Y304/21069—Protein C activated (3.4.21.69)

Definitions

• This invention relates to the use of recombinant DNA techniques to produce therapeutic proteins, in particular to the use of such techniques to produce novel, modified human uterine tissue plasminogen activator (mtPA) genes and plasmids containing such genes, host cells transformed or transfected thereby, and mtPA molecules produced therefrom.
• mtPA tissue plasminogen activator
• Tissue plasminogen activator is a multi-domain serine protease which catalyzes conversion of plasminogen to plasmin. As such, tPA is of therapeutic value. When administered exogenously, tPA can effect a lysis of blood clots (thrombolysis). tPA has been proven effective in clinical trials for treatment of myocardial infarction. Other indications being examined include pulmonary embolism, deep vein thrombosis and stroke.
• the tPA molecule contains five discrete structural domains. In the presence of plasmin, single-chain tPA or zymogen enzyme, can be cleaved into an activated two-chain form.
• the heavy chain contains four of these domains: a "finger” domain which is homologous to a portion of fibronectin; a "growth factor” domain which is homologous to epidermal growth factor; and two non-equivalent "kringle” domains. Plasmin cleavage to form two-chain tPA occurs C-terminal to Kringle 2 (at Arg 275 ).
• the light chain contains the serine protease domain, which is homologous to trypsin and chymotrypsin.
• tPA is a relatively, clot-specific plasminogen activator due to its affinity for fibrin, in turn responsible for forming the clot matrix. This fibrin affinity is believed to be due to interactions of the finger and Kringle 2 domains with fibrin. The participation of other domains in fibrin interactions is not well understood.
• tPA secreted by human melanoma cells was purified and characterized by Rijken et al. (J. Biol. Chem. 256, 7035 (1981). Therapeutic utility of exogenous tPA was demonstrated with the melanoma-derived material (Collen et al., J. Clin. Inv. 71, 368 (1983); Korninger et al., J . Clin Inv. 69, 573 (1982)). Differences between tPA derived from melanoma and normal uterine tissue have been reported (Pohl et al., FEBS Lett. 168, 29 (1984)).
• wild-type tPA as a human therapeutic is somewhat limited due to the large dose required (Verstraete et al. , Lancet 1, 842 (1985)). and may be accompanied by an unacceptably high incidence of bleeding complications due to non clot-specific activation of plasminogen (Verstraete et al. , J. Pharm. Exp. Ther. 235, 506 (1985)). Additional undesirable properties of wild-type tPA include its short in vivo half-life which can be lengthened by alteration of glycosylation of the protein by site directed mutagenesis (Lau et al., Bio/Technology 5, 953 (1987), fully incorporated herein by reference.
• mtPA' s generated by addi tions or del etions of ami no acids from the parent tPA mol ecul e at preexi sti ng retri ction sites .
• These modified cDNA's are then advantageously manipulated to generate DNA sequences with partial deletions of domains or deletions of entire domains.
• Vectors for expression of these sequences in mammalian cells are disclosed along with characterization of the resultant mtPA proteins.
• the most preferred embodiments of the present invention include cDNA's coding for utPA comprising one or more of the following modifications: Xho I restriction endonuclease site inserted at position 115, Xho I site inserted at position 277, Xho site inserted at position 515, Xho I site inserted at position 729, Xho I site inserted at position 879, Xho I site inserted at positio 1201, Xho I site inserted at position 1345, Xho I site inserted at position 1652, and Spe I site inserted at position 115 where the position numbers are from the nucleotide sequence.
• Additional preferred embodiments of the instant invention include host organisms for maintenance and replication of the sequences. Still other preferred embodiments include expression vectors for expression of said mtPA's in COS cells, C127 cells, CHO cells, and the mtPA proteins derived from these expression systems.
• Figure 1 shows utPA wild-type amino acid sequence in a two-dimensional representation showing location of domains and of restriction enzyme insertion sites
• Figure 2 depicts plasmid construction showing deletion of the
• Figure 3 shows the expression vector LK 444 BHS used for transient expression of modified tPA
• Figure 4 shows a vector for stable expression of mtPAs in CHO cells
• Figure 5 shows a vector for stable expression of mtPAs in C127 cells.
• cell culture refers to the containment of growing cells derived from either a multicellular plant or animal which allows for the cells to remain viable outside the original plant or animal .
• the term "host cell” refers to a microorgansim including yeast, bacteria and mammalian cells which can be grown in cell culture and transfected or transformed with a plasmid or vector containing a gene encoding a molecule having a tPA biological characteristic and expressing such molecule.
• domain refers to a discrete continuous part of an amino acid sequence that can be associated with a particular function. With respect to tPA, suitable references describing the domain regions include (Banyai, L. et al., Common evolutionary origin of the fibrin-binding structures of fibronectin and tissue-type plasminogen activator, FEBS Lett. 163(1), 37-41 (1983) and Ny, T. et al. The structure of the Human Tissue-type Plasminogen Activator Gene: Correlation of Intron and Exon Structures to Functional and Structural Domains, Proc. Natl. Acad.
• Table 2 illustrates the locations of the domain regions as used herein.
• downstream identifies sequences proceeding farther in the direction of expression; for example, the coding region is downstream from the initiation codon.
• interdomain refers to the regions of a protein's amino acid sequence that lie between the domains.
• the term "maintained” refers to the stable presence of a plasmid within a transformed host wherein the plasmid is present as an autonomously replicating body or as an integrated portion of the host's genome.
• microorganism includes both single cellular prokaryote and eukaryote organisms such as bacteria actinomycetes, yeast, and mammalian cells.
• non-native endonuclease restriction sites refers to endonuclease restriction sites that are not normally present in the native cDNA and are synthesized at pre-existing restriction sites of the native cDNA sequence.
• the term “operon” is a complete unit of gene expression and regulation, including structural genes, regulator genes, and control elements in DNA recognized by regulator gene product.
• plasmid refers to an autonomous self-replicating extrachromosomal circular DNA and includes both the expression and nonexpression types.
• expression plasmid includes both extrachromosomal circular DNA and DNA that has been incorporated into the host chromosome(s).
• promoter is a region of DNA involved in binding the RNA polymerase to initiate transcription.
• DNA sequence refers to a single- or double-stranded DNA molecule comprised of nucleotide bases, adenosine, thymidine, cytosine and guanosine and further includes genomic and copy DNA (cDNA).
• suitable host refers to a cell culture or microorganism that is compatible with a recombinant plasmid and will permit the plasmid to replicate, to be incorporated into its genome or to be expressed.
• upstream identifies sequences proceeding in the opposite direction from expression; for example, the bacterial promoter is upstream from the transcription unit, the initiation codon is upstream from the coding region.
• restriction endonuclease alternatively referred to herein as a restriction enzyme refers to one of a class of enzymes which cleave double-stranded DNA (dsDNA) at locations or sites characteristics to the particular enzyme.
• dsDNA double-stranded DNA
• EcoR1 cleaves dsDNA only at locations: 5'GAATTC3' to form 5' G and AATTC3' fragments 3'CTTAAG5' 3'CTTAA G5'
• Each linker contains the recognition sequence for the restriciton enzyme Xho I (CTCGAG), which is unique to the tPA cDNA and the SP65 vector.
• CTCGAG restriciton enzyme Xho I
• Sfanl site (nucleotide 16) at the 5' end of the clone near the ATG start codon for tPA and BglII site (2090) was cleaved, filled in with Klenow in the presence of dNTP's and Sal I linkers lysated to the blunt ends.
• the cDNA was recloned into pBR322 as a Sal I fragment and subsequently recloned into other vectors using the Sal I sites.
• 1ug of SP6-tPA was cleaved with Bgl II at the unique Bgl II recognition site (nucleotide 115) using the standard protocol.
• the linearized DNA was precipitated with ethanol and resuspended in nick-translation buffer (40mM KPO 4 (pH 7.5), 6.6mM MgCl 2 , 1.0mM mercaptoethanol, 250 ⁇ M dATP, dCTP, dTTP and dGTP together with 5 ⁇ of DNA polymerase I (Klenow fragment). This procedure fills in the 5' cohesive ends to generate "blunt ended" linearized DNA.
• E. coli bacterial strain DH5 using standard protocols and the transfected bacteria plated on LB agar amp plates.
• Bacterial colonies were picked, grown in LB media and DNA prepared on a small scale by standard procedures.
• the plasmid DNA was analyzed by restriction enzyme analysis and the loss of the unique Bgl II site and its replacement by a unique Xho I site was confirmed.
• the numbers in parenthesis reflect the nucleotide position.
• mutants encoding the heavy chain deletions were appropriately restriction enzyme digested and ligated as in the example given in Figure 2.
• plasmids were appropriately restriction enzyme digested and ligated as in the example given in Figure 2.
• a plasmid containing an 8 mer XhoI linker at the BglII (115) site was ligated with a plasmid containing an 8 mer XhoI linker at the Sty l (277) site via their now common XhoI cut cohesive ends.
• Bgl/Bstx del (5-139); 4 PLE 140 BglII (115) XhoI (12 mer) BstXI (515) XhoI (12 mer)
• Bgl/Eco del (5-207); 4 PRG 208 BglII (115) XhoI (10 mer) EcoRI (729) XhoI (10 mer) Bgl/Sea del (5-258); 4 LED 259 BglII (115) XhoI (8 mer) ScaI (879) XhoI (8 mer)
• Mutant-encoded protei ns wi th suffi ci ent fi bri nolyti c activi ty were analyzed by zymography rel ative to wi ld-type tPA.
• Table 3 shows the sequences of these and other mutants in detail.
• Table 4 provides Specific Activity data for the preferred embodiments of the present invention.
• Mutant-encoded proteins with sufficient fibrinolytic activity were analyzed by zymography relative to wild-type tPA.
• the SP65.tPA vector was also a convenient vector to use during the manipulation of the inserted cDNA e.g. deletion generation.
• Mutated cDNA molecules were recloned into the LK444BHS vector as shown in Figure 3.
• the BamHI, HindIII fragment of tPA cDNA or mutant derivative contained in the SP65 vector was obtained by restriction enzyme cleavage and gel filtration. This fragment was li gated to a BamHI, HindIII cleavage vector, LK444BHS. This mutation allowed for the transient expression of the tPA analogue in a COS 7 cell line driven by the human ß-actin promoter.
• the Sal I fragment was isolated from SP65.tPA, a mutated derivative by restriction enzyme cleavage and gel purification. This fragment was ligated to an Xho I cleaved vector CLH3AXBPV as shown in Figure 5. The orientation was determined and selected such that the inserted sequence was under the driving force of the metallothionine promoter in C127 cells.
• the Sal I fragment containing the tPA cDNA or mutated derivative was isolated from the SP65 vector by restriction enzyme cleavage and gel purification. This fragment was ligated into Xho I cleaved vector ELH3AXSV2DHFR as shown in Figure 4. The orientation was determined and selected such that the inserted sequence was under the driving force of the mettallothionein promoter in CHO cells. Transfection of COS cells
• a transient expression system was used wherein the expression vector (LK444BHS) was used to transfect COS-7 cells (ATCC # CRL1651). Two to three days after introduction of foreign DNA, conditioned medium was analyzed to characterize the activity of the secreted modified tPA protein.
• 3 ⁇ 10 cells were grown in 100 mm plates in DMEM + 10% glutamine for 1 day preceding transfection.
• Ten to 20 ⁇ g of DNA was added to 2.0 ml of tris-buffered saline (pH 7.5).
• 1 ml of 2 mg/ml DEAE-dextran (made just before transfection by adding 50 mgDEAE-dextran + 25 ml TBS) was added to this solution.
• Cells were washed 2 times with phosphate-buffered saline (PBS) and the transfection solution added. Cells were incubated at 37°C for 15-30 minutes. Dextran solution was then removed and cells washed again with PBS 2 times.
• PBS phosphate-buffered saline
• DUKX CHO cells were obtained from Lawrence Chasin of Columbia University. THese cells are deficient in dihydrofolate reductase.
• This gene is present in vector CLH3AXSV2DHFR.
• Cells were plated in alpha plus media 107. FBS, 1% glutamine medium at a density of 7 ⁇
• Modified tPA proteins were purified from conditioned medium by previously reported procedures (Lau et al., Bio/Technology 5, 953 (1987)).
• Quantitation of mtPA proteins in conditioned medium was performed with a commercially available ELISA Kit for determination of tPA from American Diagnostica (Greenwich, CT, USA).
• the coating and detection antibody is a goat anti-human tPA IgG.
• Activity was determined by a published spectrophotometric assay for the rate of activation of plasminogen (Verheijen et al., Thromb. Haemostas. 48, 266 (1982)).
• the absorbance change ;mearure in the assay is converted to Units by reference to a WHO melanoma tPA standard. Specific activity of the mtPA proteins is determined by dividing Units by protein, the latter as determined in the ELISA assay.
• the mtPAs of the invention may advantageously be admixed with a pharmaceutically acceptable carrier substance, e.g., saline, and administered orally, intravenously, or by injection into affected arteries of the heart. Administration will be generally as is carried out for two currently used blood clot lysing enzymes, streptokinase and urokinase.
• a pharmaceutically acceptable carrier substance e.g., saline
• the mtPA's of the invention may also be used therapeutically to lyse clots in human patients needing treatment of embolisms, e.g., post-operative patients, patients who have recently suffered myocardial infarction resulting in clots, and patients suffering from deep vein thrombi.
• embolisms e.g., post-operative patients, patients who have recently suffered myocardial infarction resulting in clots, and patients suffering from deep vein thrombi.
• embolisms e.g., post-operative patients, patients who have recently suffered myocardial infarction resulting in clots, and patients suffering from deep vein thrombi.
• embolisms e.g., post-operative patients, patients who have recently suffered myocardial infarction resulting in clots, and patients suffering from deep vein thrombi.
• the following examples are illustrative.
• mtPA lyophilized mtPA
• saline a syringe
• mtPA lyophilized mtPA
• intravenously over a period of about one hour
• intravenous infusion of about 50 mg/hr over a period of about three more hours.
• Example 2 For infusion treatment for the rapid lysis of coronary thrombi, the protocol of Example 2 is followed, except that infusion is preceded by the intravenous injection of a bolus of about 10 mg mtPA in saline.
• GAC GAC. TGG. ACG. GAG. TGT. GCC. CTC. GAG. GGC. TCC. GGC. TAC e 1 ) asp trp thr glu cys ala l eu glu gly ser gly tyr 408 409 410 411 412 415 416 417
• Bgl/Bstx del (5-139):4PLE140 106 120 539 543 i ) AGA.GGA.GCC.AGA.TCC.CCC.CTC.GAG.GGG.CTG.GGG.AAC.CAC.AAC i 1 ) arg gly ala arg ser pro leu gly gly leu gly asn his asn

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