WO1990013640A1 - Procedes et substances d'expression du plasminogene humain dans un systeme de cellules eukaryotiques - Google Patents

Procedes et substances d'expression du plasminogene humain dans un systeme de cellules eukaryotiques Download PDF

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WO1990013640A1
WO1990013640A1 PCT/US1990/002296 US9002296W WO9013640A1 WO 1990013640 A1 WO1990013640 A1 WO 1990013640A1 US 9002296 W US9002296 W US 9002296W WO 9013640 A1 WO9013640 A1 WO 9013640A1
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plasminogen
recited
cell
hpg
cells
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PCT/US1990/002296
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Francis J. Castellino
Joann L. Whitefleet-Smith
Elliot D. Rosen
James H. Mclinden
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The University Of Notre Dame Du Lac
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Priority to AU56596/90A priority Critical patent/AU647391B2/en
Publication of WO1990013640A1 publication Critical patent/WO1990013640A1/fr
Priority to FI915149A priority patent/FI915149A0/fi
Priority to NO91914269A priority patent/NO914269L/no

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    • C—CHEMISTRY; METALLURGY
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
    • C07K14/3153—Streptokinase
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
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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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • 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
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/99—Enzyme inactivation by chemical treatment
    • 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
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/14011—Baculoviridae
    • C12N2710/14111—Nucleopolyhedrovirus, e.g. autographa californica nucleopolyhedrovirus
    • C12N2710/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2710/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • Metho d an d Materials for Expression of Human Plasminogen in a Eukaryotic Cell System
  • the present application relates in general to methods and materials for expression of plasminogen and in particular to methods and materials for expression of human plasminogen in a baculovirus vector-infected insect cell system and products thereof which are substantially free of tissue plasminogen activator, urokinase and streptokinase.
  • Deleterious accumulations in blood vessels of the clot protein fibrin are prevented by proteolytic degradation (fibrinolysis) of fibrin or of its precursor fibrinogen by the enzyme plasmin (Pm).
  • proteolytic degradation fibrinolysis
  • Pm plasmin
  • pathological fibrin deposits are not degraded spontaneously, resulting in thrombosis, the presence of a blood clot (thrombus) in a blood vessel.
  • thrombolytic therapy i.e., dissolution of the blood clot by Pm, is the only feasible treatment.
  • Pm is produced in the circulation by activation of a precursor, the "proenzyme” or “zy ogen” called plasminogen (Pg).
  • plasminogen Pg
  • Thrombolytic therapy is conducted by the administration of a plasminogen activator.
  • plasminogen activators are streptokinase (SK), urokinase (UK) and tissue plasminogen activator (TPA).
  • SK streptokinase
  • UK urokinase
  • TPA tissue plasminogen activator
  • HPg Human Pg exists in the circulation as a single-chain glycoprotein containing 791 amino acids having an amino-terminal amino acid of Glu (circulating HPg may thus be referred to as [Glu 1 ] plasminogen).
  • HPg is activated by cleavage of a Arg 5 " 1 - Valgg2 peptide bond to produce the two-chain, disulfide- linked serine protease human Pm (HPm) .
  • This cleavage may be catalyzed by a variety of activators, among which are SK, UK and TPA. [See, Castellino et al., Chem. Rev., 81, 431-446 (1981)].
  • thrombolytic therapy Although thrombolytic therapy is useful, its therapeutic potential is constrained by the availability of plasminogen at the site of the thrombus.
  • concentration of plasminogen may be limited due to consumption of plasminogen as a result of thrombolytic therapy, to an inadequate amount of plasminogen being present in thrombi, or to a local plasminogen depletion related to the age of the thrombus and ischemia (a localized anemia due to a reduction of blood flow) .
  • ischemia a localized anemia due to a reduction of blood flow
  • supplementation of the locally available amount of plasminogen is desirable.
  • expression of large amounts of plasminogen in a recombinant expression system is a convenient way to obtain plasminogen for use in thrombolytic therapy, there have been great difficulties in expression of intact HPg in mammalian expression
  • the present invention provides eukaryotic cells lacking a site-specific plasminogen activator, preferably invertebrate cells, provides an expression
  • the insect cell expression vector may be a baculovirus vector, and is preferably an
  • the gene encoding human plasminogen encode [Glu 1 ]plasminogen and that the invertebrate cell is a Spodoptera frugiperda cell.
  • the present invention also provides
  • plasminogen which differs from circulating plasminogen in- a post-translational modification and which may be expressed by a cell according to the present invention, preferably a Spodoptera frugiperda cell.
  • Plasminogen according to the present invention has an active conformation (i.e., the molecule is properly folded into a tertiary structure permitting activation to plasmin), is substantially free of plasminogen activators and is entirely free of UK, SK and TPA.
  • the term "substantially free” is employed herein to refer to plasminogen which does not exhibit degradation (i.e., is at least 99% intact protein) on a Western blot performed as in Example 6 (below) after incubation for 48 hours at 27°C.
  • the present invention further provides a pharmaceutical composition including a plasminogen, preferably a human plasminogen. It is preferred that a pharmaceutical composition be isotonic and sterile-filtered.
  • the pharmaceutical composition may also include a fibrinolytic enzyme such as TPA or UK, or such as SK complexed with the plasminogen.
  • the active site of the fibrinolytic enzyme may be acylated.
  • a method for expression of human plasminogen according to the present invention includes the step of culturing eukaryotic cells lacking a site-specific plasminogen activator, preferably invertebrate cells, containing a gene encoding a plasminogen under conditions which permit expression of the gene, and preferably includes the step of infecting a Spodoptera frugiperda cell with an Autographa californica virus including a gene encoding a human plasminogen.
  • the method may also involve cotransfecting an insect cell with a transfer plasmid including a gene encoding HPg and with a wild type Autographa californica viral DNA. Plasminogen according to the present invention may be produced according to these methods.
  • the present invention also includes isolated DNA which encodes plasminogen and which is operably linked to a promoter, preferably wherein the DNA has all or a functional part of the coding sequence set forth in Figure 2.
  • the DNA may be geno ic DNA.
  • the DNA may include a promoter operably linked to the DNA and/or a signal sequence for secretion of the plasminogen from the cell. It is presently preferred that the DNA include a signal sequence recognized by invertebrate host cells.
  • a method according to the present invention includes administering to a patient in need of thrombolytic therapy an effective amount of a pharmaceutical composition according to the present invention, and preferably includes administering to a patient in need of thromobolytic therapy an effective amount of a pharmaceutical composition including a complex of a plasminogen and streptokinase.
  • a further method according to the present invention involves preparing a binary complex between a fibrinolytic enzyme, preferably streptokinase, and plasminogen, preferably human plasminogen, the complex having the catalytic site essential for fibrinolytic activity blocked by a group that is removable by hydrolysis.
  • a fibrinolytic enzyme preferably streptokinase
  • plasminogen preferably human plasminogen
  • This method includes the steps of: culturing invertebrate cells which do not produce an endogenous plasminogen activator and are transformed with an expression vector including a nucleic acid encoding plasminogen under conditions that permit expression of the nucleic acid; recovering the plasminogen from the host cell culture, preferably from the cell culture medium; and mixing the fibrinolytic enzyme with the plasminogen in the presence of an excess of a blocking agent of the formula A-B or E-F, wherein A is a group that is selective for the catalytic site essential for fibrinolytic activity and is capable of transferring from the group B to the catalytic site, and B is a group that facilitates the attachment of A to the enzyme, E is a locating group that locates the agent in the catalytic site and F is a group that is capable of transferring from the locating group to the catalytic site.
  • agent AB be p-nitrophenyl-p'-guanidinobenzoate, that the method further include the step of isolating the binary complex so formed, that group E is p-amidinophenyl or p- acetamidophenyl group, and that group F be a b nzoyl or acryloyl group.
  • the group removable by hydrolysis be an acyl group, more preferably a benzoyl, substituted benzoyl, acryloyl, or substituted acryloyl group.
  • "Operably linked” as used herein refers to juxtaposition such that the normal function of the components can be performed.
  • a coding sequence "operably linked" to control sequences refers to a configuration wherein the coding sequence may be expressed under the control of these sequences, and wherein the DNA sequences being linked are contiguous and, in the case of a secretory leader, contiguous and in reading phase.
  • DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned to facilitate translation of the coding sequence.
  • Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, then synthetic oligonucleotide adaptors or linkers may be used in accordance with conventional practice.
  • cell As used herein, "cell,” “cell line,” and “cell culture” are used interchangeably and all such designations include progeny. Thus, “transformants” or “transformed cells” includes the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that all - 1 -
  • progeny may not be precisely identical in DNA content due to deliberate or inadvertent mutations. Also included in these terms are mutant progeny which have the same function for which a primary subject cell is f 5 screened. Where distinct designations are intended, it will be clear from the context.
  • viral DNA and a transfer vector are taken up by a host cell. DNA from the plasmid may be transferred to. he viral DNA by
  • Imaging refers to the taking up of a viral expression vector by a host cell, and leads to expression of any coding sequences and to the production
  • Transformation means introducing DNA into an organism so that the DNA is replicable, either as an extrachromosomal element or by chromosomal integration. Depending on the host cell used,
  • a calcium treatment employing calcium chloride as described by Cohen, Proc. Natl. Acad. Sci. (USA), 6£, 2110 (1972), is generally used for prokaryotes or other cells that contain
  • Site-directed mutagenesis is a technique standard in the art, and is conducted using a synthetic oligonucleotide primer complementary to a single- stranded phage DNA to be mutagenized except for limited
  • the synthetic oligonucleotide is used as a primer to direct synthesis of a strand complementary to the phage, and the resulting double-stranded DNA is transformed into a phage-supporting host bacterium. Cultures of transformed bacteria are plated in top agar, permitting plaque formation from single cells which harbor the phage. Theoretically, 50% of new plaques contain the phage having, as a single strand, the mutated form; 50% will have the original sequence. The plaques are hybridized with kinased synthetic primer at a temperature which permits hybridization of an exact match, but at which temperature the mismatches with the original strand are sufficient to prevent hybridization. Plaques which hybridize with the probe are then selected and cultured, and the DNA is recovered.
  • control sequences refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism.
  • expression system refers to DNA containing a desired coding sequence and control sequence in operable linkage, so that hosts transformed with these sequences are capable of producing the encoded proteins. To effect transformation, the expression system may be included on a vector called herein an "expression vector.” However, the relevant DNA may also be integrated into the host chromosome.
  • Post-translational modification refers primarily to glycosylation but may also involve prote ⁇ lysis, phosphorylation, acylation, sulfation, ⁇ -carboxylation or ⁇ -hydroxylation.
  • Figure 1 is restriction map of the vector pll9PN127.6 according to the present invention
  • Figures 2 is a nucleotide sequence for vector pll9PN127.6, including a deduced amino acid sequence for human plasminogen;
  • Figure 3 is a flow chart which schematically depicts the construction of a transfer vector according to the present invention.
  • Figure 4 is a restriction map which schematically depicts a baculovirus transfer vector according to the present invention.
  • Figure 5 depicts Western blots of materials from the host cells according to the present invention
  • Figure 6 is a graphic depiction of the results of an activation assay of HPg produced according to the present invention
  • Figure 7 is a depiction of an immunodot assay for HPg in fractions recovered from a purification column according to the present invention.
  • Figure 8 illustrates a Western blot of samples from various stages of purification according to the present invention
  • Figure 9 is an illustration of an electrophoretic gel run on aliquots of HPg or recombinant HPg according to the present invention after activation by urokinase.
  • a functional form of [Glu ⁇ jPg is obtained by expression of [Glu ⁇ ]Pg DNA in invertebrate cells or in eukaryotic cells lacking plasminogen activators.
  • expression is obtained in insect cells infected with a recombinant baculovirus containing a Pg gene. The expression process in these cells is linked to production of viral occlusions including a major viral structural protein, polyhedrin.
  • a cDNA which encodes the human plasminogen has been inserted adjacent the polyhedrin promoter in the genome of the baculovirus Autographa californica nuclear polyhedrosis virus. The virus was then used to infect cultured cells of the farm armyworm, Spodoptera frugiperda. Recombinant HPg (rec-HPg) was secreted into the medium by 24 hours post-infection (p.i.), at which point virtually no rec-HPg antigen remained inside the cells. At 48 hours p.i., a maximal level of intact rec- HPg was present in the medium. The rec-HPg found in the medium is at least 99% intact (full length) protein.
  • Proteolytic digestion observed after 48 hours p.i. appears to occur when the cells are crowded (i.e. at 3.5 X 10 5 cells/cm 2 ), and it is presently preferred that the cells be cultured at a lower density (1.33 X 10 ⁇ cells/cm 2 ). As may be seen in Figure 5, there is no apparent degradation even at 93 hours p.i. when cells are plated at a lower density.
  • the protein portion of the rec-HPg produced by this expression system possessed a molecular weight substantially equivalent to that of plasma [Glu 1 ]plasminogen.
  • the rec-HPg adsorbed to Sepharose- lysine, and was eluted with ⁇ -aminocaproic acid.
  • the recombinant protein interacted with polyclonal antibodies generated to plasma HPg, as well as with a monoclonal antibody directed against a distinct region (kringles 1-3) of the plasma HPg molecule, in a manner equivalent to that of the human plasma protein.
  • insect cell-expressed rec-HPg was activatable to plasmin by urokinase.
  • RecHPg recombinant plasminogen
  • RecHPg may be converted to Pm by an activator, and that interact with anti-plasma Pg polyclonal and monoclonal antibodies, as well as with the ligand, ⁇ -aminocaproic acid.
  • a Pg gene incorporated into a baculovirus genome, may be expressed in a fully functional form in insect cells infected with a recombinant virus. It is believed that such expression in fully functional form has not been achieved to date in mammalian expression systems.
  • plasminogen refers to a plasminogen, such as the human plasminogen having the a ino acid sequence shown in Figure 2, together with analogues and variants thereof having the biological activity of native human Pg.
  • the biological activity of native Pg is shared by any analog or variant thereof that is capable of being cleaved by a plasminogen activator (e.g., streptokinase, urokinase, or tissue plasminogen activator) to produce plasmin or that possesses an. immune epitope which is immunologically cross-reactive with an antibody raised against at least one epitope of native Pg.
  • a plasminogen activator e.g., streptokinase, urokinase, or tissue plasminogen activator
  • an immune epitope which is immunologically cross-reactive with an antibody raised against at least one epitope of native Pg.
  • Analogs or variants are defined as molecules in which .
  • amino acid sequence variants include not only alleles of the Figure 2 sequence, but also predetermined mutations thereof. Generally, amino acid sequence variants have an amino acid sequence with at least about 80% homology, and more typically at least about 90% homology, to that of the native Pg of Figure 2. Henceforth, the term Pg shall mean either the native sequence or variant form unless otherwise appropriate.
  • a latent plasmin heavy chain which includes residues 1-561, contains five highly homologous regions called “kringles” [Sottrup-Jensen et al.. Prog. Chem. Fibrinolysis Thrombolysis, 3_, 191-209 (1977)], each containing approximately 80 amino acids. These kringles most likely exist as independent domains [Castellino et al., J. Biol. Chem., 256 4778-4782 (1981)] and are of importance to the functional properties of HPg and HPm.
  • the kringle 1 domain (amino acid residues 84-162) may be important in the interaction of plasmin or plasminogen with fibrin and fibrinogen [Lucas et al., J. Biol. Chem., 258 4249- 4256 (1983)], and to contain the strong [Glu 1 ]Pg binding site for the positive effector ⁇ -aminocaproic acid (EACA) [Markus et al., J.Biol. Chem., 253, 727-732 (1978)]. Additionally, this same segment is responsible for the initial rapid binding of HPm to its major plasma inhibitor, ⁇ 2 -antiplasmin [Morol et al., J. Biol.
  • the kringle 4 region appears to contain weak EACA binding site(s) present on [Glu ⁇ ]Pg, which may be involved in the very large ligand-induced conformational alteration of [Glu ⁇ Pg [Violand et al., J. Biol. Chem., 251, 3906-3912 (1976)] and in a concomitant increase in the activation rate of the zymogen in the presence of the positive effector EACA, [Claeys et al., Biochim. Biophys. Acta, 342, 351-359 (1974)].
  • Pg expressed in a eukaryotic cell including Pg having native glycosylation and the amino acid sequence as set forth in Figure 2, analogous Pg proteins from other animal species such as bovine, equine, porcine, ovine, canine, murine, feline, and the like, deglycosylated or nonglycosylated derivatives of such Pg proteins, and biologically active amino acid sequence variants of Pg, including alleles and in vitro-generated covalent derivatives of Pg proteins that demonstrate plasminogen activity.
  • Amino acid sequence variants of Pg include, for example, deletions from, or insertions or substitutions of, residues within the amino acid Pg sequence shown in Figure 2.
  • deletion, insertion and substitution may also be made to arrive at the final construct, provided that the final construct possesses the desired activity.
  • the mutations made in the DNA encoding the variant Pg do not place the sequence out of reading frame and it is further preferred that they do not create complementary regions that could produce secondary mRNA structure (see, e.g., European Patent Publication No. 075,444).
  • Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions of from one residue to polypeptides of essentially unrestricted length, as well as intrasequence insertions of single or multiple amino acid residues.
  • Intrasequence insertions i.e., insertions within the mature HPg sequence
  • An example of a single terminal insertion is mature HPg having an N-terminal methionyl residue. This variant may result as an artifact of the direct expression of rec-Pg in recombinant cell culture, i.e., expression without a signal sequence to direct the secretion or cell membrane association of mature rec-Pg.
  • terminal insertions include: (1) fusions of signal sequences, whether heterologous or homologous, to the N-terminus of mature rec-Pg to facilitate the secretion of mature rec-Pg from recombinant hosts, (2) fusions of immunogenic polypeptides (i.e., polypeptides sufficiently large to confer immunogenicity to the target sequence), e.g., bacterial polypeptides such as 8-lactamase, 8- galactosidase, or an enzyme encoded by the E. coli trp r locus, and (3) fusions with cell surface binding 5 substances, such as membrane anchors. Fusions with cell surface binding substances need not be produced by recombinant methods, but may be the product of covalent or non-covalent association with rec-Pg.
  • immunogenic polypeptides i.e., polypeptides sufficiently large to confer immunogenicity to the target sequence
  • bacterial polypeptides such as 8-lactamase, 8- galactosidase, or
  • the third group of variants are those in which
  • Substantial changes in function or immunological " identity may be made by selecting substitutions which are less conservative than those in Table 1, i.e., selecting residues that differ more significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.
  • substitutions that in general are expected to produce the greatest changes in rec-Pg properties will be those in which (a) glycine and/or proline is substituted by another amino acid or is deleted or inserted; (b) a hydrophilic residue, e.g.
  • seryl or threonyl is substituted for (or by) a hydrophobic residue, e.g. leucyl, isoleucyl, phenylalanyl, valyl, or alanyl; (c) a cysteine is substituted for (or by) any other residue; (d) a residue having an electropositive side chain, e.g. lysyl, arginyl, or histidyl, is substituted for (or by) an electronegative residue, e.g. glutamyl or aspartyl; or (e) a residue having a bulky side chain, e.g. phenylalanine, is substituted for (or by) one not having such a side chain, e.g. glycine.
  • a hydrophobic residue e.g. leucyl, isoleucyl, phenylalanyl, valyl, or alanyl
  • a cysteine is substituted for (
  • the variants may be prepared by site-directed mutagenesis of nucleotides in the DNA encoding the Pg, thereby producing DNA encoding the variant, and by thereafter expressing the DNA in invertebrate cell culture.
  • DNA encoding Pg or variants thereof may also be chemically synthesized and assembled by any of a number of techniques, prior to expression in a host cell. [See, e.g., Caruthers, U.S. Patent No. 4,500,707; Balland et al., Biochimie, 67 725-736 (1985); Edge et al.. Nature, 292, 756-762 (1982)].
  • Variant Pg fragments having up to about 100 residues may be conveniently prepared by in vitro synthesis. The variants typically exhibit the same qualitative biological activity as the naturally occurring form.
  • the site for introducing an amino acid sequence variation is predetermined, the variation itself need not be predetermined.
  • random mutagenesis may be conducted at the target codon or region and the expressed Pg variants may be screened for the optimal combination of desired activity.
  • Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, such as M13 primer mutagenesis.
  • Amino acid sequence deletions generally range from about 1 to 30 residues, more preferably 1 to 10 residues, and typically are contiguous.
  • deletions and insertions, and substitutions in particular are not expected to produce radical changes in the characteristics of the rec-Pg molecule.
  • substitution, deletion, or insertion in advance of doing so, for example, when modifying the active site of Pg or an immune epitope, one skilled in the art appreciates that the effect may be evaluated by routine screening assays.
  • a variant typically may be made by site-specific mutagenesis of the native Pg-encoding nucleic acid, expression of the variant nucleic acid in recombinant cell culture, and, optionally, purification from the cell culture, for example by immunoaffinity adsorption on a rabbit polyclonal anti-Pg column (to absorb the variant by at least one remaining epitope).
  • the activity of the cell lysate or purified Pg variant may then be screened in a suitable screening assay for the desired characteristic.
  • a change in the immunological character of the Pg such as affinity for a given antibody, may be measured by a competitive immunoassay. Changes in activation levels are measured by the appropriate assay. Modifications of such protein properties as redox or thermal stability, hydrophobicity, susceptibility to proteolytic degradation, or the tendency to aggregate with carriers or into multimers are assayed by methods well known to the ordinarily skilled artisan.
  • any cell culture is workable, whether from vertebrate or invertebrate culture, if the cells do not contain endogenous plasminogen activators.
  • plasminogen activators are enzymes that activate the zy ogen plasminogen to generate the serine proteinase plasmin (by cleavage at Arg 560 -Val 561 ) that degrades various proteins, including fibrin.
  • the plasminogen activators are streptokinase, a bacterial protein, urokinase, an enzyme synthesized in the kidney and elsewhere and originally extracted from urine, and human tissue plasminogen activator, an enzyme produced by the cells lining blood vessel walls.
  • Eukaryotic microbes such as yeast cultures
  • Saccharomyces cerevisiae or common baker's yeast
  • yeast is the most commonly used among eukaryotic microorganisms, although a number of other strains are commonly available.
  • the plasmid YRp7 for example [Stinchcomb et al.. Nature, 282, 39 (1979); Kingsman et al., Gene, 7, 141 (1979); Tschemper et al.. Gene, 10, 157 (1980)] is commonly used.
  • This plasmid contains the trpl gene that provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No. 44,076 or PEP4-1 [Jones, Genetics, 5, 12 (1977)].
  • the presence of the trpl lesion as a characteristic of the yeast host cell genome provides an effective environment for detecting transformation by growth in the absence of tryptophan.
  • Suitable promoting sequences in yeast vectors include the promoters for 3-phosphoglycerate kinase [Hitzeman et al., J. Biol. Chem., 255, 12073-12080 (1980)] or other glycolytic enzymes [Hess et al., J. Adv. Enzyme Re . , ⁇ , 149 (1968); Holland et al..
  • Biochemistry, 17, 4900 (1978)] such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6- phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.
  • the termination sequences associated with these genes are also ligated into the expression vector 3' of the sequence desired to be expressed to provide polyadenylation of the mRNA and termination.
  • promoters which have the additional advantage of transcription controlled by growth conditions, are the promoter region for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, and the aforementioned glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization.
  • Any plasmid vector containing yeast-compatible promoter, origin of replication and termination sequences is suitable.
  • cultures of cells derived from multicellular organisms may also be used as hosts.
  • any such cell culture is workable, whether from vertebrate or invertebrate culture.
  • Cells which naturally do not contain endogenous plasminogen activators, such as invertebrate cells are preferred.
  • plasminogen activators are enzymes which activate the zymogen plasminogen to generate the serine proteinase plasmin (by cleavage of plasminogen at Arg 560 -Val 5 ⁇ 1 ) which in turn degrades various proteins, including fibrin.
  • Site-specific plasminogen activators as referred to herein are streptokinase (a bacterial protein), urokinase (an enzyme synthesized in the kidney and elsewhere and originally extracted from urine) , and human tissue plasminogen activator (an enzyme produced by the cells lining blood vessel walls).
  • Vertebrate cells may also be employed as host cells, even if they have endogenous plasminogen activators in nature, provided that the genes encoding such activators are deleted prior to use according to the present invention. Propagation of vertebrate cells in culture (tissue culture) has becomes a routine procedure in recent years. Examples of such useful host cell lines which may be mutated include VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, and W138, BHK, COS-7, 293, and MDCK cell lines.
  • Expression vectors for such cells ordinarily include (as necessary) an origin of replication, a promoter located in front of the gene to be expressed, along with any necessary ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences.
  • the control functions on the expression vectors are often provided by viral material.
  • commonly used promoters are derived from polyomavirus, adenovirus 2, and most frequently simian virus 40 (SV40).
  • SV40 simian virus 40
  • the early and late promoters of SV40 virus are particularly useful because both are easily obtained from the virus as a fragment which also contains the SV40 viral origin of replication [Fiers et al.. Nature, 273, 113 (1978)].
  • Smaller or larger SV40 fragments may also be used, provided there is included the approximately 250-bp sequence extending from the Hind III site toward the Bgl I site located in the viral origin of replication. Further, it is also possible, and often desirable, to utilize promoter or control sequences normally associated with the desired gene sequence, provided such control sequences are compatible with the host cell systems.
  • An origin of replication may be provided either by construction of the vector to include an exogenous origin, such as may be derived from SV40 or other viral (e.g., polyomavirus, adenovirus, VSV, BPV) source, or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter is often sufficient.
  • an exogenous origin such as may be derived from SV40 or other viral (e.g., polyomavirus, adenovirus, VSV, BPV) source, or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter is often sufficient.
  • a preferred host cell for transfection by the vectors of the invention which comprise DNA sequences encoding both Pg and DHFR protein it is appropriate to select the host according to the type of DHFR protein employed. If wild-type DHFR protein is employed, it is preferable to select a host cell which is deficient in DHFR, thus permitting the use of the DHFR coding sequence as a marker for successful transfection in selective medium which lacks hypoxanthine, glycine, and thymidine.
  • An appropriate host cell in this case is the Chinese hamster ovary (CHO) cell line deficient in DHFR activity [prepared and propagated as described by Urlaub et al., Proc. Nat'l. Acad. Sci.
  • plasminogen activator genes may be performed generally according to the procedures of: Thomas et al.. Nature, 324, 34-38 (1988); Sedivy, Bio/Technology, ⁇ _, 1192-1196 (1988); Rauth et al., Proc. Nat'l. Acad. Sci. (USA), 83, 5587- 5591 (1986); Ayares et al., Proc. Nat'l. Acad. Sci. (USA), 83, 5199-5203 (1986); Roizman et al., U.S. Patent No., 4,769,331; and Maniatis, Nature, 317, 205-206 (1985).
  • DHFR protein with low binding affinity for MTX is used as the controlling sequence, it is not necessary to use DHFR-deficient cells. Because the 5 mutant DHFR is resistant to methotrexate, MTX-containing media may be used as a means of selection provided that the host cells are themselves methotrexate sensitive. Most eukaryotic cells which are capable of absorbing MTX appear to be methotrexate sensitive. One such useful
  • 10 cell line is a CHO line, CH0-K1, CHO-K1 (ATCC No. CCL 61).
  • viruses may be used as the virus herein according to the present invention, particularly for transfection of Spodoptera frugiperda cells.
  • an enzyme derivative may be prepared which comprises a binary complex between streptokinase and plasminogen, which complex has a catalytic site essential for fibrinolytic activity blocked by a group that is removable by hydrolysis such that the pseudo-
  • 35 first order rate constant for hydrolysis of the derivative is in the range of 10 ⁇ ° sec “1 to 10 ⁇ 3 sec “"1 in isotonic aqueous media at pH 7.4 at 37°C, provided that the group that blocks the catalytic site is not a p-guanidino-benzoyl group.
  • suitable such groups include acyl groups such as benzoyl, substituted benzoyl, acryloyl, or substituted acryloyl groups.
  • a method for preparing the complexes includes mixing streptokinase with plasminogen in the presence of an excess of a blocking agent of the formula A-B or E-F, wherein A is a group that is selective for the catalytic site essential for fibrinolytic activity and that is capable of transferring from the group B to the catalytic site, and B is a group that facilitates the atachment of A to the enzyme; E is a locating group that locates the agent in the catalytic site and F is a group capable of transferring from the locating group to the catalytic site, and thereafter optionally isolating the derivatives so formed.
  • A is a group that is selective for the catalytic site essential for fibrinolytic activity and that is capable of transferring from the group B to the catalytic site
  • B is a group that facilitates the atachment of A to the enzyme
  • E is a locating group that locates the agent in the catalytic site
  • F is a group capable of transferring from the locating group
  • the group removable by hydrolysis is an acyl group, most preferably a benzoyl, substituted benzoyl, acryloyl, or substituted acryloyl group, e.g., benzoyl substituted with halogen,
  • AB is p-nitrophenyl-p'-guanidinobenzoate
  • group E is p- a idinophenyl or p-acetamidophenyl
  • group F is a benzoyl or acryloyl group.
  • compositions which includes human plasminogen.
  • a pharmaceutically acceptable carrier such as isotonic aqueous buffer or pharmaceutical grade "Water for Injection.”
  • the invention encompasses a pharmaceutical formulation comprising a pharmaceutically acceptable carrier together with a fibrinolytic enzyme, preferably a complex of the enzyme with the plasminogen, more preferably a binary complex of streptokinase and plasminogen, and most preferably a p-anisoyl streptokinase/plasminogen complex without internal peptide bond cleavage, as in Smith et al., U.S. Patent No. 4,808,405.
  • the active site of the complex responsible for fibrinolytic activity is blocked by a group that is removable by hydrolysis such that the pseudo-first order rate constant for hydrolysis of the complex is in the range of 10 ""6 sec *"1 to 10 "3 sec " in isotonic aqueous media at pH 7.4 at 37°C.
  • the compositions according to this invention are formulated in accordance with standard procedures to be adapted for parenteral administration to humans.
  • the compositions for intravenous administration are solutions of the sterile derivative in sterile isotonic aqueous buffer.
  • the compostion also includes a solubilizing agent for the complex.
  • the complex is supplied in unit dosage form, for example, as a dry powder or water- free concentrate in a sealed container such as an ampoule.
  • the complex is dispensed from an infusion bottle containing sterile pharmaceutical grade Water for Injection.
  • the complex is dispensed from a vial of sterile Water for Injection.
  • the injectable or infusible composition will be made up by mixing the ingredients prior to administration.
  • the effective amount of complex administered will depend on many factors, including the amount of fibrinolysis required and the speed with which it is required, the extent of thromboembolism, and the position and size of the clot, but the amount is generally dictated by the result to be obtained, i.e., lysis of the clot.
  • lysis i.e., lysis of the clot.
  • a patient with a pulmonary embolism or a life-threatening thrombus will require admnistration of a bolus of rapidly acting material.
  • a small quantity of slow-acting material is particularly useful.
  • the precise dose to be employed and the mode of administration may be decided according to the* circumstances as seen by the physician.
  • a patient being treated for a medium-size thrombus receives a dose of from 0.10 to 1.0 mg/kg of body weight daily either by injection (in up to eight doses) or by infusion. More specific descriptions of methods, materials, and products according to the present invention appear in the following Examples.
  • Example 1 the construction of a transfer vector containing HPg is described.
  • Example 2 an experiment is described in which host cells are cotransfected with the transfer vector of Example 1 and a wild type baculovirus.
  • Example 3 the DNA of a viral vector is examined for the presence of HPg DNA by Southern blotting.
  • Example 4 is a description of the culture of cells infected with the viral vector containing HPg and of the purification of rec-HPg expressed by those cells.
  • Example 5 is a description of an experiment examining the time-course of expression of rec-HPg in the host cells.
  • Example 6 a Western blot analysis of the cells and media of Example 5 is described.
  • Example 7 is a description of a quantitavive immunoassay of the cells and media of Example 5.
  • Example 8 includes a description of an activation assay performed on HPg from materials from the cultures of Example 5.
  • Example 9 preparation of rec-HPg on a larger scale than that of Example 4 is described.
  • Example 10 is a description of activation of Pg according to the present invention by urokinase.
  • Example 11 is a description of the construction of a streptokinase-plasminogen complex using the HPg prepared according to the present invention.
  • Example 12 methods for constructing cells lacking a site-specific plasminogen activator and cells which may be produced by such methods are disclosed.
  • baculovirus vector including DNA encoding HPg, was constructed.
  • a plasmid designated pUC119PN127.6 was prepared as follows. A first cDNA was isolated from an n-oligo(dT)-primed cDNA library constructed from liver mRNA isolated from five individuals. [Okyama et al., Mol. Cell. Biol., 2 , 161-170 (1982) and Gubler et al..
  • a ⁇ gtlO cDNA clone was recovered with a 75- base oligonucleotide probe (PL.l), corresponding to nucleotides 1,306-1,380 of human plasminogen cDNA [Forsgren et al., FEBS Lett., 213, 254-260 (1987)].
  • Filters were hybridized in 5 X SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH6.7), 5 X Denhardt's solution, 20% formamide, 10% dextran sulphate and 20 ⁇ g/ml of boiled, sonicated salmon sperm DNA at 42°C.
  • a ⁇ gtlO clone (designated ⁇ gtl0:pmgn#127) was cut with SstI and ligated to Sstl-cut p ⁇ C119 to give pUC119PN127.6, a restriction map for which is illustrated in Figure 1 and a nucleotide and a deduced amino acid sequence for which are illustrated in Figure 2.
  • the clone was sequenced to completion. DNA sequence analysis was performed by the dideoxy chain termination method on both strands of the subcloned double-stranded cDNA [Sanger et al., Proc. Nat'l. Acad.
  • a transfer vector pAV6 was constructed to include DNA sequences surrounding the AcMNPV polyhedrin (PH) gene.
  • the plasmid pAV6 contains the polyhedrin promoter in 1.8 kb from an Xho I site located 5' of the PH gene to nucleotide -8 of the polyhedrin initiation codon (ATG) .
  • the plasmid pAV6 also includes a 1.5 kb fragment extending from a Kpn I site within the polyhedrin gene to a Bam HI site 3' of this same gene. The Xho I and Bam HI sites were lost during cloning of these fragments.
  • Transfer vector pAV6 was constructed as follows and as illustrated in Figure 3.
  • the resulting Xho I/Bam HI fragment was ligated into a Sal I- and Bam Hi-cut mpl9 vector (Bethesda Research Laboratories, Gaitherburg, Maryland) to form a construction designated mpl9Xho-Bam.
  • the plasmid mpl9Xho-Bam was cut with Eco RV and Kpn I, and a first synthetic oligonucleotide (constructed, as were all oligonucleotides referred to below, on a 380 A model automated DNA synthesisizer available from Applied Biosystems, Foster City, California)- was ligated into the cut mpl9Xho-Bam to produce a vector designated mpl9AlD.
  • the first synthetic oligonucleotide had the following sequence, including the indicated restriction and transcription initiation sites.
  • the first synthetic oligonucleotide replaced a sequence at .the 5' end of the Xhol/Bam HI fragment in mp 19Xho- Bam from an Eco RV site to a putative CAP site and includes a multiple cloning site having Bam HI, Eco RI, Sal I and Kpn I sites.
  • a pUC12 vector (Bethesda Research Laboratories) was cut with Hind III and SstI, mp 19AID was cut with Hind III and Kpn I, and a 1.8 kb fragment containing sequences flanking the 5' end of the polyhedrin gene was isolated.
  • the plasmid pEcoI-I was cut with Bam HI and Kpnl and, from among the digestion products, a 1.5 kb fragment, extending from the Kpnl site within the polyhedrin gene to a Bam HI site in the 3' flanking region thereof, was isolated.
  • a plasmid, pDS was constructed by cutting pBR322 (the 4.4 kb plasmid available from Bethesda Research Laboratories) with Hind III and Sal I, filling in with Klenow fragment, and ligating.
  • the plasmid pDS lacks the sequences between Hind III and Sal I and loses the Sal I site, but maintains the Hind III site.
  • the plasmid pAV2 was cut with Hind III and Bam HI, and a 1.5 kb Hind III/Bam HI fragment (containing the 5' flanking sequence) was isolated.
  • pAV2 was cut with Bam HI and Eco RI, and a 1.5 kb Eco RI/Bam HI fragment extending from the Bam HI site in the multiple cloning site to the Eco RI site adjacent to the 3' flanking sequence was isolated.
  • the plasmid pDS was cut with Hind III and Eco RI and ligated to the two fragments made from pAV2.
  • the resulting plasmid was designated pAV3.
  • the plasmid pAV3 was cut with Bam HI and Sal I.
  • a vector pAC373 [Smith et al., Mol. Cell., Biol., 3_, 2156-2165 (1983)] (containing NPV viral DNA from a Sal I site about 1 kb 5' of the polyhedrin gene ⁇ 5 to a Bam HI site inserted at nucleotide -8 (the "A" of i the "ATG" initiation site being +1) was cut with Bam HI and Sal I and ligated to the Bam Hi/Sal I-cut pAV3 to produce a vector designated pAV4.
  • the vector pAV4 was cut with EcoRI, the ends 10 were filled with Klenow fragment and ligated to produce the vector designated pAV6 (lacking the Eco RI site of pAV4) .
  • a Bam HI/Nae I fragment containing a cDNA encoding the entire HPg amino acid sequence in the form 15 of [Glu- ⁇ HPg was excised from the plasmid pll9PN127.6 and inserted into the Bam HI and Sma I sites of plasmid pAV6.
  • a restriction map of the resulting recombinant transfer plasmid, designated pAV6HPg, directing the expression of HPg is illustrated in Figure 4.
  • restriction sites have been abbreviated as follows: R, Eco RI; X, Xho I; V, Eco RV; H, Hind III; S, Sma I; K, Kpn I; and N, Nae I.
  • the plasmid pAV6HPg contains the AcMNPV polyhedrin (PH) promoter linked to the HPg signal and 25 mature [Glu ⁇ -jPg coding sequence.
  • the HPg insert contained (from 5' to 3') 20 bases from pUC119, 38 bases of linker plus 5' untranslated sequences, 57 bases from the HPg signal sequence, 2373 bases of the final translated and processed HPg sequence, 322 bases of 3' 30 untranslated sequences plus the poly-A sequence and a linker, and 367 bases from pUC119.
  • the construct pAV6HPg and wild type viral DNA were used to cotransfect cultured Spodoptera frugiperda cells.
  • crossover between homologous polyhedrin flanking regions of the transfer vector and the virus provides a full length recombinant virus carrying the HPg gene in place of the PH gene.
  • the AcTR temperature inactivation resistant strain of Autographa californica nuclear polyhedrosis virus was used as the host virus for recombinant constructions.
  • a cloned e.g. Sf9 cells, as available from the American Type Culture Collection, Rockville, Maryland under accession number A.T.C.C. CRL 1711
  • uncloned Spodoptera frugiperda cell line may be employed to provide host cells for all virus growth and manipulations [Vaughn et al.. In Vitro, 13, 213-217
  • the medium was removed and the cells were incubated in a 0.5 ml suspension of wild type DNA from AcMNPV (0.1 ⁇ g) plus pAV6HPg transfer plasmid DNA (1 ⁇ g) in NaCl (0.8 g/1), KC1 (0.37 g/l)/Na 2 H2P ⁇ 4 + 2H 2 0(0.125 g/1), dextrose (1 g/1) and Hepes-NaOH (5 g/1) at pH 7.2. After incubation overnight at about 27°C, the DNA suspension was removed and the cells were incubated for 5 days in Hinks medium, plus 10% FBS.
  • Spodoptera frugiperda cells may also be cultured in suspension by employing Corning (New York, New York) slow-speed stirring vessels on a Cellgro slow- speed magnetic stirrer (Thermolyne Corp., Dubuque, Iowa). To each 1000 ml stirring vessel, 300 ml of incomplete Hink's medium (supplemented with 8.3% FBS and the penicillin/streptonycin/amphotericin B mixture as above) are added. The vessel is then inoculated with 5 X 10 6 cells and stirred at 80 rpm. Cells are subcultured when they attain a density of 2 -3 X 10° cells/ml by removing 150-250 ml of cell suspension and replacing it with fresh medium. Suspension-grown cells attach to flasks and may thus be used in procedures requiring monolayers.
  • Cells may also be grown in a defined, low- protein medium EX-CELL 400 produced by JR. Scientific, Woodland, California. This medium may be used in place of complete Hink's medium for culturing cells in monolayers, in spinner flask suspension cultures or in air-lift bioreactors [q.v. Maiorella et al.. Biotechnology, 6 , 1406-1410 (1988)]
  • EX-CELL 400 produced by JR. Scientific, Woodland, California. This medium may be used in place of complete Hink's medium for culturing cells in monolayers, in spinner flask suspension cultures or in air-lift bioreactors [q.v. Maiorella et al.. Biotechnology, 6 , 1406-1410 (1988)]
  • OB ⁇ occlusion body-negative plaques
  • Southern blots were prepared from Eco RI digests of DNA preparations from three plaque-purified OB- viruses.
  • Cells (3 X 10 ⁇ ) were allowed to attach to 60 mm petri dishes and were then infected with 3 different plaque-purified OB " viruses at 10X multiplicity. At 72 hours p.i., cells were resuspended by gentle pipetting and pelleted at 3500 g, for 20 minutes at 16°C. The cell pellet was suspended and lysed in 4 M guanidinium isothiocyanate/0.5% sarcosyl/5 mM sodium citrate/0.1 mM 8-mercaptoethanol. The resulting solution was extracted four times with phenol/chloroform, and the DNA was precipitated twice with ethanol.
  • the three virus DNA samples, wild type virus DNA and the original pAV6HPg transfer vector were digested with Eco RI and subjected to Southern blot analysis.
  • the digested samples were separated on an 0.85% agarose gel, and electroblotted onto a nylon membrane using a Bio-Rad transfer unit (Bio-Rad, Richmond, California) and Bio-Rad protocol for non- denatured gels.
  • Copies of HPg-containing plasmid P119PN127.6 and pUC13 were labeled with P 32 by nick translation [generally as described in Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 109-112, 387-389 (1982); and Southern, J. Mol.
  • Hybridized bands were visualized by 5 autoradiography. fa One plaque displayed the presence of a new band at a position corresponding to the 2.2 kb Eco RI fragment from the HPg gene in the transfer vector (data not shown). This virus clone, designated Pg3A, was used 10 in the following Examples. Southern blots of DNA from Pg3A indicated that the [Glu- ⁇ Pg gene was inserted in the viral DNA.
  • lanes 1 and 2 represent cells and media, respectively, from a wild-type viral infection
  • lanes 3 and 4 are cells and media, respectively, 24 hours, p.i., with the recombinant virus, pAV6HPg
  • lanes 5 and 6 represent cells and media, respectively, 48 hours, p.i., with pAV6HPg
  • lanes 7-11 are media samples from cells infected with pAV ⁇ HPg at times of 66 hours, 70 hours, 74 hours, 80 hours and 93 hours, p.i.. respectively.
  • the HPg signal peptide is recognized by the ⁇ . insect cells. This is readily seen at the higher cell
  • HPg recognizable by its reactivity toward a polyclonal antibody pool generated against plasma HPg, is secreted into the medium.
  • the band for rec-HPg consists of a doublet.
  • human plasma plasminogen such as
  • ELISA enzyme-linked im unoadsorbant assay
  • a rabbit anti-HPg pool was obtained from Boehringer Mannheim (Indianapolis, Indiana).
  • the second antibody was detected with an anti-rabbit IgG-alkaline phosphatase conjugate, after hydrolysis of the indicator, p- nitrophenyl phosphate, by the adsorbed alkaline phosphatase.
  • the lower limit of detection was approximately 30 ng/ l relative to a [Glu 1 ]Pg standard obtained by purifying fresh human plasma on sepharose- lysine generally according to the procedures of Brockway et al.. Arch. Biochem. Biophys., 151, 194-199 (1972) .
  • Example 5 For determination of rec-HPg function for materials derived from the cultures of Example 5, two activation assays were developed in which the zymogen was removed from the media prior to activation by reagents that relied upon specific folding of the protein molecule.
  • a mouse-anti-HPg kringle 1-3 monoclonal antibody designated 35-J-4 was adsorbed to the wells of a polyvinyl plate, followed by the test samples.
  • the wells of an immunoblot apparatus were blocked with 3 MM paper and 50 ⁇ l of Sepharose-lysine was added, again followed by the test samples.
  • Hybridoma cell lines producing monoclonal antibodies to HPg were generated, screened and stabilized as described in Ploplis et al.. Biochemistry, 21.' 5891-5897 (1982).
  • Monoclonal antibody 35-J-4 was raised against the kringle 1-3 region of HPg, and was purified by affinity chromatography on HPg-Sepharose.
  • the kringle 1-3 region of HPg (residues 79-355) was isolated by limited elastase digestion, followed by affinity chromatography on Sepharose-lysine.
  • plasminogen activity has been determined between 66 and 72 hours. It is presently preferred that media be harvested between 48 and 54 hours.
  • Rec-HPg is activatable to plasmin, after its removal from the medium by two specific methods, i.e., reactivity against a monoclonal antibody that is directed conformationally to the kringle 1-3 domain regions of the molecule ( Figure 6), and by the affinity
  • a larger scale rec-HPg preparation was accomplished in which a total of 1.4 X 10 9 cells were infected with a 3X multiplicity of the virus-encoded HPg gene. At 48 hours, p.i., a total of 1300 ml of medium was decanted from the cells and tested for the presence of rec-HPg. After extensive dialysis against 5 mM sodium phosphate (pH 8.0) and concentration of the medium by lyophilization, the sample was redissolved in H 0 and percolated over a column of Sepharose-lysine, as above and rec-HPg was eluted with a gradient of 0-20 mM EACA.
  • Rec-HPg was identified by an immunodot assay with polyclonal rabbit-anti-human HPg.
  • Figure 7 illustrates the results of an immunodot assay for the presence of HPg in the fractions recovered from a Sepharose-lysine affinity chromatography separation (after elution with a gradient of EACA of the culture media harvested from pAV6HPg- infected insect cells. Aliquots from fractions obtained from the column were adsorbed onto nitrocellulose paper, through the wells of a dot-blot apparatus. The presence of HPg on the paper was detected by immunoblot analysis, after addition of rabbit-anti-HPg followed by goat-anti- rabbit-IgG conjugated to alkaline phosphatase. A goat- anti-human polyclonal antibody pool to plasma HPg was purchased from Sigma Chemical Company, and a similar rabbit-anti-human antibody pool was obtained from Boehringer-Mannheim. In Figure 7, the start of the EACA gradient
  • Figure 8 illustrates a Western blot of samples obtained from the various stages of the large scale procedure purified by the affinity chromatography column described in Figure 7. The samples, electrophoretically transferred from the NaDodS04/PAGE gel to the nitrocellulose paper, were either stained for total protein (Janssen Auro Dye forte stain) or detected by immunoassay for HPg as described with reference to Figures 5 and 7 above. Lane 1, a standard mixture of affinity variants I and II of HPg.
  • Lane 2 the culture media from pAV6HPg-infected insect cells, stained for total protein.
  • Lane 3 HPg purified from the culture media represented by lane 2, stained for total protein.
  • total protein staining shows that the conditioned insect cell culture fluid contains three major protein bands (lane 2), one of which is identified as HPg, by immunostaining (lane 5).
  • the material eluted from the Sepharose-lysine column was identified as HPg, by both total protein stain (lane 3), and by immunostaining (lane 6).
  • the gels of Figure 8 demonstrate that the isolated HPg possesses approximately the same molecular weight as affinity chromatography variant 1 of plasma HPg which is its most fully glycosylated form.
  • the data of Figure 8 (lanes 2 and 5) show that HPg is one of three major products secreted by the pAV6HPg-infected cells, under the given conditions.
  • Two chain human kidney urokinase was a gift from Abbott Laboratories and existed mainly in the low molecular weight form. Aliquots were removed at 10, 20, 30, 60, 90 and 120 min., and the activation was terminated by immediate addition to 15 ⁇ l of gel loading buffer, as above. Samples were separated on a 9% (w/v) NaDodS0 4 /PAGE gel under reducing conditions, and visualized by silver staining. A continuous coupled assay for the determination of the activation rate of rec-HPg was performed generally as in [Urano et al., J. Biol. Chem.
  • Figure 9 shows a reduced NaDodS0 4 -PAGE analysis of the activation of plasma HPg and rec-Pg by low molecular weight urokinase. Both forms of Pg show a time dependent conversion from a one-chain form (Pg) to a two-chain form (Pm). Western analysis of this same
  • HPm chains of the two preparations are the same. Some lower molecular weight HPm heavy chains are observed that are similar in the two plasminogens and result from activation of the low molecular weight forms of HPg. Urokinase activation of rec-Pg was also
  • lanes 1-7 are plasma HPg samples, and lanes 8-14 are rec-HPg samples.
  • the following time points are shown in Figure 9: 0 min., lanes 1 and 8; 10 min., lanes 2 and 9; 20 min., lanes 3 and 10; 30 min., lanes 4 and 11; 60 min., lanes 5 and- 12; 90 min., lanes 6 and 13; 120 min., lanes 7 and 14.
  • the heavy and light chains of the two-chain form are indicated by H and L, respectively.
  • fibrinolytic enzymes may be used as thrombolytic agents.
  • the catalytic site of fibrinolytic enzymes may be blocked by a group which is removable by hydrolysis under certain conditions. Smith et al., U.S. Patent No. 4,808,405; and Smith et al.. Nature, 290, 505-508 (1981).
  • HPg according to the present invention may be employed as a thrombolytic agent alone, as a complex with a fibrinolytic enzyme, as a complex with an acylated fibrinolytic enzyme, as an acylated proenzyme or as an acylated proenzyme in a complex with a fibrinolytic enzyme or an acylated fibrinolytic enzyme.
  • An acylated streptokinase/acylated plasminogen complex according to the present invention may be prepared as follows. Streptokinase (about 451 mg; as available from
  • a pharmaceutical composition according to the present invention may be prepared from the above as follows.
  • Human serum albumin (clinical grade) (18.9 ml 20% w/v) may be then added to the mixture with stirring for 2 minutes at 4 ⁇ C.
  • Lysine/mannitol buffer may be added to bring the volume to about 400 ml.
  • the fluid may then be diafiltered for about 2 _ hours at 18°C until about 2400 ml of diafiltrate is collected.
  • the fluid may then be filtered through a 0.22 ⁇ sterile filter and transferred to a sterile reservoir from which aliquots may be dispensed into sterile freeze-drying vials followed by freeze drying.
  • Cells lacking plasminogen activators may be constructed by applying UV irradiation to cells expressing such activators and selecting progeny of the cells which do not produce products which exhibit immunological or biological (e.g. fibrinolytic) properties of a plasminogen activator in screening tests of the sort well known to those skilled in the art.
  • Cells lacking site-specific plasminogen activators may be constructed by any of a number of techniques based upon homologous recombination. For such techniques see, e.g., Roizman et al., U.S. Patent No.
  • site-specific plasminogen activators such portions of the genes encoding these activators between the homologous regions may be deleted or rendered inactive.
  • a cell in which such a site-specific plasminogen activator gene has been deleted or inactivated may be employed as a host cell according to the present invention and a mammalian cell in which UK and TPA genes have been partially or totally deleted or rendered inactive is preferred.

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Abstract

Du plasminogène humain recombinant peut être produit dans des cellules d'invertébrés ou dans d'autres cellules eukaryotiques manquant d'un activateur de plasminogène avec spécificité du site, par expression d'un vecteur contenant un gène codant pour le plasminogène humain. Le plasminogène humain peut par conséquent être exprimé sans dégradation par plasmine produite par des activateurs de plasminogène intracellulaire se trouvant dans des cellules de mammifères. Le plasminogène produit par une telle expression peut être purifié, acylé, mélangé pour former un complexe avec des enzymes fibrinolytiques acylées ou non acylées et formé en compositions pharmaceutiques destinées à être utilisées dans la thrombolyse thérapeutique.
PCT/US1990/002296 1989-05-01 1990-04-26 Procedes et substances d'expression du plasminogene humain dans un systeme de cellules eukaryotiques WO1990013640A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
AU56596/90A AU647391B2 (en) 1989-05-01 1990-04-26 Methods and materials for expression of human plasminogen in a eukaryotic cell system
FI915149A FI915149A0 (fi) 1989-05-01 1991-10-31 Foerfarande och material foer expression av humant plasminogen i ett eukaryotiskt cellsystem som saknar en laegesspecifik plasminogenaktivator.
NO91914269A NO914269L (no) 1989-05-01 1991-10-31 Fremgangsmaate og materialer for ekspresjon av humanplasminogen i et eukaryot cellesystem

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US34580189A 1989-05-01 1989-05-01
US345,801 1989-05-01

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JP (1) JPH05500748A (fr)
AU (1) AU647391B2 (fr)
FI (1) FI915149A0 (fr)
PT (1) PT93933A (fr)
WO (1) WO1990013640A1 (fr)

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991009118A2 (fr) * 1989-12-07 1991-06-27 British Bio-Technology Limited Proteines fibrinolytiques et antithrombotiques activables
WO1993006223A1 (fr) * 1991-09-27 1993-04-01 Centre National De La Recherche Scientifique (C.N.R.S.) Vecteurs recombinants viraux pour l'expression dans des cellules musculaires
US6099831A (en) * 1992-09-25 2000-08-08 Centre National De La Recherche Scientifique Viral recombinant vectors for expression in muscle cells
US6743623B2 (en) 1991-09-27 2004-06-01 Centre National De La Recherche Scientifique Viral recombinant vectors for expression in muscle cells
EP1343903B1 (fr) * 2000-12-21 2007-11-14 Thromb-X N.V. Vecteur d'expression de levure et production d'une proteine recombinee d'une cellule de levure
US7365159B2 (en) 1994-04-26 2008-04-29 The Children's Medical Center Corporation Angiostatin protein
US7420036B2 (en) 2000-11-28 2008-09-02 Waisman David M Anti-angiogenic polypeptides
WO2010138631A1 (fr) * 2009-05-26 2010-12-02 Biolex Therapeutics, Inc. Compositions et procédés permettant la production de plasminogène aglycosylé
WO2011004011A1 (fr) 2009-07-10 2011-01-13 Thrombogenics Nv Variantes du plasminogène et de la plasmine
WO2012093132A1 (fr) 2011-01-05 2012-07-12 Thrombogenics Nv Variantes de plasminogène et de plasmine
US8304400B2 (en) 2001-11-28 2012-11-06 Waisman David M Compositions and methods for inhibiting tumor growth and metastasis
WO2013024074A1 (fr) 2011-08-12 2013-02-21 Thrombogenics N.V. Variants du plasminogène et de la plasmine
US8637010B2 (en) * 2001-09-06 2014-01-28 Omnio Healer Ab Method of accelerating wound healing by administration of plasminogen

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US3943245A (en) * 1974-02-14 1976-03-09 Armour Pharmaceutical Company Purification of plasminogen
US4745051A (en) * 1983-05-27 1988-05-17 The Texas A&M University System Method for producing a recombinant baculovirus expression vector
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US3943245A (en) * 1974-02-14 1976-03-09 Armour Pharmaceutical Company Purification of plasminogen
US4808405A (en) * 1979-11-05 1989-02-28 Beecham Group P.L.C. P-anisoyl streptokinase/plasminogen complex
US4769331A (en) * 1981-09-16 1988-09-06 University Patents, Inc. Recombinant methods and materials
US4745051A (en) * 1983-05-27 1988-05-17 The Texas A&M University System Method for producing a recombinant baculovirus expression vector

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Advances in Protein Chemistry (New York, USA) Volume 38, Issued 1986, BRIGGS et al., "Molecular Mechanisims of Protein Secretion: The Role of the Signal Sequence", pages 109-180, (See especially pages 113-115 and 117-118). *
Biochemistry, (Washington, D.C., USA) vol. 23, Issued 1984, MALINOWSKI et al., "Characterization of a Complementary deoxyribonucleic acid coding for Human and Bovine plasminogen", pages 4243-4250. *
CASTELLINO et al., "Methods in Enzymology", Vol.80, 1981, Academic Press (New York, USA), see pages 365-378. *
FEBS LETTERS Volume 213, March 1987. FORSGREN et al., "Molecular cloning and characterization of a full-length cDNA clone for human plasminogen", pages 254-260,(See the entire document). *
gene Volume 73 (Amsterdam, The Netherlands), STEINER et al., "Human a tissue-type plasminogen activator synthesized by using a baculovirus vector in insect cells compared with human plasminogen activator produced in mouse cells", pages 449-457 (See entire document). *
GOODBOURN et al., Current Communications in Molecular Biology, 1988, Cold Spring Harbor Laboratory, (Cold Spring Harbor, USA), see pages 17-22. *
Proceedings of the National Academy of Sciences (Washington, USA) Volume 78, Issued April 1981, MULLIGAN et al., "Selection for animal cells that express the E. coli gene coding for xanthineguanine phosphoribosyltransferase", pages 2072-2076. (See entire document). *
Proceedings of The National Academy of Sciences, (Washington, USA) Volume 81, Issued March 1984, DEANS et al., "Expression of immunoglobulin heavy chain gene transfected into lymphocytes", pages 1292-1296, (See entire document). *
Science, (Washington, D.C. USA) Vol. 209, Issued 19 September 1980, MULLIGAN et al., "Expression of a bacterial gene in mammalian cells", pages 1422-1427, (See entire document). *
See also references of EP0467987A4 *

Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991009118A3 (fr) * 1989-12-07 1991-10-31 British Bio Technology Proteines fibrinolytiques et antithrombotiques activables
WO1991009118A2 (fr) * 1989-12-07 1991-06-27 British Bio-Technology Limited Proteines fibrinolytiques et antithrombotiques activables
WO1993006223A1 (fr) * 1991-09-27 1993-04-01 Centre National De La Recherche Scientifique (C.N.R.S.) Vecteurs recombinants viraux pour l'expression dans des cellules musculaires
US6743623B2 (en) 1991-09-27 2004-06-01 Centre National De La Recherche Scientifique Viral recombinant vectors for expression in muscle cells
US6099831A (en) * 1992-09-25 2000-08-08 Centre National De La Recherche Scientifique Viral recombinant vectors for expression in muscle cells
US7365159B2 (en) 1994-04-26 2008-04-29 The Children's Medical Center Corporation Angiostatin protein
US7420036B2 (en) 2000-11-28 2008-09-02 Waisman David M Anti-angiogenic polypeptides
US7445775B2 (en) 2000-12-21 2008-11-04 Thromb-X Nv Yeast expression vector and a method of making a recombinant protein by expression in a yeast cell
EP1343903B1 (fr) * 2000-12-21 2007-11-14 Thromb-X N.V. Vecteur d'expression de levure et production d'une proteine recombinee d'une cellule de levure
US8637010B2 (en) * 2001-09-06 2014-01-28 Omnio Healer Ab Method of accelerating wound healing by administration of plasminogen
US8304400B2 (en) 2001-11-28 2012-11-06 Waisman David M Compositions and methods for inhibiting tumor growth and metastasis
WO2010138631A1 (fr) * 2009-05-26 2010-12-02 Biolex Therapeutics, Inc. Compositions et procédés permettant la production de plasminogène aglycosylé
WO2011004011A1 (fr) 2009-07-10 2011-01-13 Thrombogenics Nv Variantes du plasminogène et de la plasmine
US9226953B2 (en) 2009-07-10 2016-01-05 Thrombogenics Nv Variants of plasminogen and plasmin
WO2012093132A1 (fr) 2011-01-05 2012-07-12 Thrombogenics Nv Variantes de plasminogène et de plasmine
US9121014B2 (en) 2011-01-05 2015-09-01 ThromboGenies NV Plasminogen and plasmin variants
WO2013024074A1 (fr) 2011-08-12 2013-02-21 Thrombogenics N.V. Variants du plasminogène et de la plasmine
US9644196B2 (en) 2011-08-12 2017-05-09 Thrombogenics Nv Plasminogen and plasmin variants

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EP0467987A4 (en) 1992-07-08
PT93933A (pt) 1991-01-08
AU647391B2 (en) 1994-03-24
AU5659690A (en) 1990-11-29
EP0467987A1 (fr) 1992-01-29
JPH05500748A (ja) 1993-02-18

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