AU647391B2 - Methods and materials for expression of human plasminogen in a eukaryotic cell system - Google Patents

Description

OPI DATE 29/11/90 APPLN. ID 56596 PC AOJP DATE 10/01/91 PCT NUMBER PCT/US90/02296 INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (51) International Patent Classification 5 (11) International Publication Number: WO 90/13640 C12N 15/00, 15/12, 9/68 C12N 9/70, 9/96, 15/52 A1 (43) International Publication Date: 15 November 1990 (15.11.90) C12N 7/01 (21) International Application Number: (22) International Filing Date: Priority data: 345,801 1 May 19 PCT/US90/02296 26 April 1990 (26.04.90) 89 (01.05.89) (74) Agent: GRUBER, Lewis, Marshall, O'Toole, Gerstein, Murray Bicknell, Two First National Plaza, Suite 2100, Chicago, IL 60603 (US).

(81) Designated States: AT (European patent), AU, BE (European patent), CH (European patent), DE (European patent), DK '.European patent), ES (European patent), FI, FR (European patent), GB (European patent), IT (European patent), JP, LU (European patent), NL (European patent), NO, SE (European patent).

Published With international search report.

Before the expiration of the time limit for amending the claims and to be republished in the event of the receipt of amendments.

(7 Applicant: THE UNIVERSITY OF NOTRE DAME DU LAC [US/US]; An Indiana Corporation, Not'e Dame, IN 46556 (US).

(72) Inventors: CASTELLINO, Francis, J. 16056 Baywood Lane, Granger, IN 46530 WHITEFLEET-SMITH, Joann, L. 36 Dorrance Street, Danielson, CT 06239 ROSEN, Ellibt, D. 1219 Leeper Avenue, South Bend, IN 46617 McLINDEN, James, H. 4232 Hickory Road, Apt.3A, Mishawaka, IN 46545 (US).

I /1I 4 Ig (54)Title: METHODS AND MATERIALS FOR EXPRESSION OF HUMAN PLASMINOGEN IN A EUKARYOTIC CELL SYSTEM (57) Abstract Recombinant human plasminogen may be produced in invertebrate cells or in other eukaryotic cells lacking a site-specific plasminogen activator by expression of a vector including a gene encoding human plasminogen. Human plasminogen may thus be expressed without degradation by plasmin produced by intracellular plasminogen activators found in mammalian cells. The plasminogen product of such expression may be purified, acylated, complexed with acylated or non-acylated fibrinolytic enzymes and formulated into pharmaceutical compositions for use in thrombolytic therapy.

See back of page VO 90/13640 PC/US90/02296 1 Methods and Materials for Expression of Human Plasminogen in a Eukaryotic Cell System Background 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 In a large variety of disorders, pathological fibrin deposits are not degraded spontaneously, resulting in thrombosis, the presence of a blood clot (thrombus) in a blood vessel.

In many cases, thrombolytic therapy, 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 "zymogen" called plasminogen Thrombolytic therapy is conducted by the administration of a plasminogen activator. Among such plasminogen activators are streptokinase urokinasa (UK) and tissue plasminogen activator (TPA).

Human Pg (HPg) exists in the circulation as a single-chain glycoprotein containing 791 amino acids WO 90/13640.

PCT/US90/02296 2 having an amino-terminal amino acid of Glu (circulating HPg may thus be referred to as [Glul] plasminogen).

[Forsgren et al., FEBS Lett., 213, 254-260 (1987); Malinowski et al., Biochem., 23 4243-4250 (1984); McLean et al., Nature, 330, 132-137 (1987); Sottrup-Jensen et al., Prog. Chem. Fibrinolysis Thrombolysis, 3, 191-209 (1977); Wiman, Eur. J. Biochem., 39, 1-9 (1973); and Wiman, Eur. J. Biochem., 76, 129-137 (1977)]. Analysis of the carbohydrate sequence of HPg reveals that there are two glycosylation variants, a first having two glycosylation sites (Asn 289 and Thr 346 and a second having one glycosylation site (Thr 346 with subforms exhibiting incomplete silation [Castellino, Chem. Rev., 81, 431-446 (1981)]. These forms and subforms are examples of post-translational modifications exhibited by circulating plasminogen.

HPg is activated by cleavage of a Arg 561 Va1 562 peptide bond to produce the two-chain, disulfidelinked 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)].

Although thrombolytic therapy is useful, its therapeutic potential is constrained by the availability of plasminogen at the site of the thrombus. The 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).

[Anderle et al., Haemostasis, 18, (Suppl. 165-175 (1988)]. Thus, supplementation of the locally available amount of plasminogen is desirable.

Although expression of large amounts of plasminogen in a recombinant expression system is a WO 90/13640 PCr/US90/02296 3 convenient way to obtain plasminogen for use in thrombolytic therapy, there have been great difficulties in expression of intact HPg in mammalian expression systems due to the nearly ubiquitous presence of intracellular plasminogen activators among mammalian cell types. The presence of these activators results in the appearance of a degraded form of HPg in conditioned cell media of such expression systems, possibly from autodigestion of plasminogen by the HPm produced [Busby et al., Fibrinolysis, 2, 64 (1988)].

Therefore, it is desirable to have an expression system for plasminogen which avoids the degradation present in reported systems.

Summary of the Invention The present invention provides eukaryotic cells lacking a site-specific plasminogen activator, preferably invertebrate cells, provides an expression vector comprising a gene encoding plasminogen, preferably human plasminogen, and provides, in particular, an insect cell expression vector including a gene encoding HPg. The insect cell expression vector may be a baculovirus vector, and is preferably an Autographa californica nuclear polyhedrosis virus. It is presently preferred that the gene encoding human plasminogen encode (Glul]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. Human plasminogen, specifically (Glul]plasminogen, is presently preferred.

Plasminogen according to the present invention WO 90/13640 PMT/US9VO2/0296 4 has an active conformation 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 is at least 99% intact protein) on F Western blot performed as in Example 6 (below) after incubation for 48 hours at 27 0 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 genomic DNA. The DNA may include a promoter operably linked to the DNA and/or a WO 90/13640 PCT/US90/02296 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. 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 fibrinolytie 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. It is presently preferred that agent AB be WO 9C/13640 PC/US90/02296 6 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 pacetamidophenyl group, and that group F be a benzoyl or acryloyl group.

It is presently preferred that 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. Thus, 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 conti-' ous and, in the case of a secretory leader, contiguous and in reading phase. For example: 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.

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 WVO 9/13640 PCT/US90/02296 7 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 screened. Where distinct designations are intended, it will be clear from the context.

In "cotransfection," viral DNA and a transfer vector (plasmid) are taken up by a host cell. DNA from the plasmid may be transferred to the viral DNA by homologous recombination leading to the production of recombinant viruses. Numerous methods of transfection are known to the ordinarily skilled artisan, for example, methods employing CaPO 4 and electroporation.

Successful transfection is generally recognized when the operation of the vector within the host cell is detected.

"Infection" 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 of the virus.

"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, transformation is done using standard techniques appropriate to such cells. A calcium treatment employing calcium chloride, as described by Cohen, Proc.

Natl. Acad. Sci. (USA), 69, 2110 (1972), is generally used for prokaryotes or other cells that contain substantial cell-wall barriers.

"Site-directed mutagenesis" is a technique standard in the art, and is conducted using a synthetic oligonucleotide primer complementary to a singlestranded phage DNA to be mutagenized except for limited mismatching which represents the desired mutation.

Briefly, the synthetic oligonucleotide is used as a WO 90/13640 PCT/US90/02296 8 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. Plaque which hybridize with the probe are then selected and cultured, and the DNA is recovered.

The term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism.

The term "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" as used herein refers primarily to glycosylation but may also involve proteolysis, phosphorylation, acylation, sulfation, y-carboxylation or a-hydroxylation.

WO 90/13640 PCT/US9002296 9 Brief Description of the Drawings Figure 1 is restriction map of the vector pll9PN127.6 according to the present invention; Figures 2 is a nucleotide sequence for vector pl19PN127.6, 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; and 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.

WO 90/13640 PCT/US90/02296 10 Detailed Description In order to effectively apply recombinant DNA methodology to production of Pg, it is important to have an expression system for this large and complex molecule which does not degrade the expression product. An invertebrate expression system is useful in this regard because plasminogen activators do not appear to be present in such cells.

According to the present invention, a functional form of [GluL)Pg is obtained by expression of '[Glul]Pg DNA in invertebrate cells or in eukaryotic cells lacking plasminogen activators. Preferably 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 at which point virtually no rec-HPg antigen remained inside the cells. At 48 hours 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.

By contrast, in a baby hamster kidney (BHK) cell system [Busby et al., Fibrinolysis, 2, 64 (1988)1 less than 10% of the synthesized plasminogen is reported to have appeared in the medium. Although this was originally full-length plasminogen it was quickly degraded to smaller forms. Only degraded forms of WO 90/13640 PC/US90/02296 11 plasminogen were found inside the BHK cells.

In crowded cultures according to the present invention after 48 hours p.i. the rec-HPg underwent substantial proteolytic digestion. This digestion was detected by observation of immuno-reactive bands in a Western blot analysis of samples taken from cultures that had been seeded with 3.5 X 10 .cells/cm 2 These bands occurred at lower molecular weights than the native rec-HPg. With time, the intensity of the native band decreased and the intensity of the lower molecular weight bands increased. In addition, the observed molecular weight of the lower bands also decreased.

Proteolytic digestion observed after 48 hours p.i. appears to occur when the cells are crowded (i.e.

at 3.5 X 105 cells/cm 2 and it is presently preferred that the cells be cultured at a lower density (1.33 X 105 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 [Glul]plasminogen. The rec-HPg adsorbed to Sepharoselysine, and was eluted with e-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. Finally, insect cell-expressed rec-HPg was activatable to plastin by urokinase.

These results demonstrate that the expression system according to the present invention produces a full-length functional single-chain recombinant plasminogen (rec-Pg), which may be isolated intact from the culture medium, with some consideration for the WO 90/13640 PCT/US90/02296 12 temporal events that occur in secretion and longer-term degradation of the protein. RecHPg according to the present invention 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, e-aminocaproic acid. These results indicate that the molecule retains the recited important functional properties that it is functional Pg), and is correctly folded.

Thus, 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.

As used herein, "plasminogen," or refers to a plasminogen, such as the human plasminogen having the amino 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 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. Analogs or variants are defined as molecules in which.the amino acid sequence, glycosylation, or other feature of native Pg has been modified covalently or noncovalently. 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 WO 90/13640 PCT/US90/02296 13 mean either the native sequence or variant form unless otherwise appropriate.

In [Glul]Pg, 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. Biol0 Chem., 256 4778-4782 (1981)] and are of importance to the functional properties of HPg and HPm. As examples, 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 (Glul]Pg binding site for the positive effector e-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, a2-antiplasmin [Morol et al., J. Biol. Chem., 251, 5956-5965 (1976)]. The kringle 4 region (residues 358-435) appears to contain weak EACA binding site(s) present on [Glul]Pg, which may be involved in the very large ligand-induced conformational alteration of [Glul]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)].

Included within the scope of the present invention is 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 WO 90/13640 PC'r/US0/02296C 14 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. Any combination of deletion, insertion and substitution may also be made to arrive at the final construct, provided that the final construct possesses the desired activity. Obviously, it is preferred that 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, European Patent Publication No. 075,444).

Amino acid sequence insertions include aminoand/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 insertions within the mature HPg sequence) may range gen2rally from about 1 to 10 residues, more preferably 1 to 5. 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 rer-Pg in recombinant cell culture, i.e., expression without a signal s quence to direct the secretion or cell membrane association of mature rec-Pg. Other examples of terminal insertions include: 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, fusions of immunogenic pbiypeptides polypeptides sufficiently large to WO 90/13640 PCr/US90/02296 15 confer immunogenicity to the target sequence), e.g., bacterial polypeptides such as B-lactamase, 8galactosidase, or an enzyme encoded by the E. coli trp locus, and fusions with cell surface binding 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.

The third group of variants are those in which at least one amino acid residue in the rec-Pg molecule, and preferably only one, has been removed and a different residue has been inserted in its place. Such substitutions generally are made in accordance with the following Table 1 when it is desired to modulate finely the characteristics of rec-Pg.

WO 90/ 13640 PTU9/29 PCY/US90/02296 16 TABLE 1 Original Residue Exemplary Substitutions Ala Arg As n Asp Cys Gin Glu Gly His Ile Leu Lys Met Phe Ser Thr Trp Tyr Val Gly; Ser Lys Gin; His Giu Se r Asn Asp Ala; Pro Asn; Gin Leu; Val Ile; Val Arg; Gin; Glu Leu; Tyr; Ile Met; Leu; Tyr Thr Ser Tyr Trp; Phe Ile; Leu WO 90/13640 PCT//US90/02296 17 Substantial changes in function or immunological 'identity may be made by selecting substitutions which are less conservative than those in Table 1, selecting residues that differ more significantly in their effect on maintaining the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, the charge or hydrophobicity of the molecule at the target site, or the bulk of the side chain. -The substitutions that in general are expected to produce the greatest changes in rec-Pg properties will be those in which glycine and/or proline is substituted by another amino acid or is deleted or inserted; a hydrophilic residue, e.g. seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g. leucyl, isoleucyl, phenylalanyl, valyl, or alanyl; a cysteine is substituted for (or by) any other residue; 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 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.

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, 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 WO 90/13640 PCT/US90/02296 18 synthesis. The variants typically exhibit the same qualitative biological activity as the naturally occurring form.

While the site for introducing an amino acid sequence variation is predetermined, the variation itself need not be predetermined. For example, to optimize the performance of a mutation at a given site, 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 residues, and typically are contiguous.

Most deletions and insertions, and substitutions in particular, are not expected to produce radical changes in the characteristics of the rec-Pg molecule. However, when it is difficult to predict the exact effect of the 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. For example, 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. For example, a change in the immunological character of the Pg, such as affinity for WO 90/13640 PCT/US90/02296 19 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.

In principle, according to the present invention any cell culture is workable, whether from vertebrate or invertebrate culture, if the cells do not contain endogenous plasminogen activators. For purposes herein, "plasminogen activators" are enzymes that activate the zymogen plasminogen to generate the serine proteinase plasmin (by cleavage at Arg 560 -Val 561 that degrades various proteins, including fibrin. Among 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, may be employed according to the present invention.

Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among eukaryotic microorganisms, although a number of other strains are commonly available. For expression in Saccharomyces, the plasmid YRp7, for example [Stinchcomb et al., Nature, 282, 39 (1979); Kingsman et al., Gene, 1, 141 (1979); Tschemper et al., Gene, 10, 157 (1980)] is commonly used. This plasmid contains the trpl gene that provides 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, 85, 12 (1977)]. The presence of the trpl lesion as a characteristic of the yeast host cell genome provides an effective environment WO 90/13640 PCr/US90/02296 20 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 Reg., 7, 149 (1968); Holland et al., Biochemistry, 17, 4900 (1978)], such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinaser glucose-6phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. In constructing suitable expression plasmids, 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. Other 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.

In addition to microorganisms, cultures of cells derived from multicellular organisms may also be used as hosts. In principle, 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. Herein, "plasminogen activators" are enzymes which activate the zymogen plasminogen to generate the serine proteinase plasmin (by cleavage of WO 90/13640 PC/US90/02296 21 plasminogen at Arg 560 -Val 561 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.

For use in mammalian cells, the control functions on the expression vectors are often provided by viral material. For example, commonly used promoters are derived from polyomavirus, adenovirus 2, and most frequently simian virus 40 (SV40). 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 '-ite located in WO 90/13640 PCT/US90/02296 22 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 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.

In selecting 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. (USA), 77(7), 4216-4220 (1980)) and as made deficient in plasminogen activator activity according to the present invention. Deletion of plasminogen activator genes may be performed generally according to the procedures of: Thomas et al., Nature, 324, 34-38 (1988); Sedivy, Bio/Technology, 6, 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 WO 90/13640 PC/US90/02296 23 (1985).

If 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 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 cell line is a CHO line, CHO-K1, CHO-K1 (ATCC No. CCL 61).

Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosphila melanogaster (fruitfly), and Bombyx mori host cells, have been identified. [See, Luckow et al., Bio/Technology, 6, 47-55 (1988); and Maeda et al., Nature, 315, 592-594 (1985)]. A variety of such viral strains are publicly available, the L-l variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used as the virus herein according to the present invention, particularly for transfection of Spodoptera frugiperda cells.

Complexes formed between fibrinolytic enzymes and plasminogen may be used as thrombolytic agents, as described further in Smith et al., U.S. Patent No.

4,808,405 the disclosure of which is incorporated herein by reference and which is illustrated in Example 11 below. Briefly, 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 pseudofirst order rate constant for hydrolysis of the derivative is in the range of 10 6 sec-1 to 10 3 sec-1 WO 90/13640 PC/US90/02296 24 in isotonic aqueous media at pH 7.4 at 370C., provided that the group that blocks the catalytic site is not a p-guanidino-benzoyl group. Examples of suitable such groups include acyl groups such as benzoyl, substituted benzoyl, acryloyl, or substituted acryloyl groups.

A method for preparing the complexes includes mixing streptokinast 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. Preferably the group removable by hydrolysis is an acyl group, most preferably a benzoyl, substituted benzoyl, acryloyl, or substituted acryloyl group, benzoyl substituted with halogen,

C

1 -6 alkyl, C 1 -6 alkoxy, C 1 -6 alkanoyloxy, or C_-6 alkanoylamino or acryloyl substituted with C 1 -6 alkyl, furyl, phenyl, or C 1 -6 alkyl phenyl. Also, preferably AB is p-nitrophenyl-p'-guanidinobenzoate, group E is pamidinophenyl or p-acetamidophenyl, and group F is a benzoyl or acryloyl group.

Also contemplated as part of this invention is a pharmaceutical composition which includes human plasminogen. Preferably such composition comprises a pharmaceutically acceptable carrier such as isotonic aqueous buffer or pharmaceutical grade "Water for Injection." In addition, the invention encompasses a pharmaceutical formulation comprising a pharmaceutically acceptable carrier together with a fibrinolytic enzyme, preferably a complex of the eazyme with the plasminogen, more preferably a binary complex of streptokinase and WO 90/13640 PCr/US90/02296 25 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. In a further embodiment 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 1 in isotonic aqueous media at pH 7.4 at 37 0

C.

The compositions according to this invention are formulated in accordance with standard procedures to be adapted for parenteral administration to humans.

Typically, the compositions for intravenous administration are solutions of the sterile derivative in sterile isotonic aqueous buffer. Where necessary, the compostion also includes a solubilizing agent for the complex. In general, the complex is supplied in unit dosage form, for example, as a dry powder or waterfree concentrate in a sealed container such as an ampoule. For administration by infusion, the complex is dispensed from an infusion bottle containing sterile pharmaceutical grade Water for Injection. For administration by 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. For example, a patient with a pulmonary embolism or a life-threatening thrombus will require admnistration of a bolus of rapidly acting material. On the other hand, where it is desired to WO 90/13640 PC/US90/02296 26 prevent the formation of thrombi after an operation, 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. However, in general, a patient being treated for a tedium-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.

In Example 1, the construction of a transfer vector containing HPg is described.

In Example 2, an experiment is described in which host cells are cotransfected with the transfer vector of Example 1 and a wild type baculovirus.

In 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.

In 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 Example 8 includes a description of an activation assay performed on HPg from materials from the cultures of Example In 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 WO 90/13640 PC/US90/02296 27 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.

In Example 12, methods for constructing cells lacking a site-specific plasminogen activator and cells which may be produced by such methods are disclosed.

Example 1 Using restriction endonucleases purchased from Promega (Madison, Wisconsin) under the conditions specified by the supplier, a 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., Gene, 25, 263-269 (1983)]. Size-selected cDNAs (greater than 600 base pairs) were ligated into XgtlO bacteriophage vectors (Stratagene, San Diego, California), and screened without amplification [Drayna et al., Nature, 327, 632-634 (1987)]. The cDNA was ligated to the xgtl0 vector using a linker as follows.

Eco RI Sac I/SstI Sal I XgtlO: GAATTCT CGAGCTC GTCGACC: cDNA A XgtlO cDNA clone was recovered with a base oligonucleotide probe 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 NaCI, 15 mM trisodium citrate), 50 mM sodium phosphate (pH6.7), 5 X WO 90/13640 PCr/ US90/02296 28 Denhardt's solution, 20% formamide, 10% dextran sulphate and 20 pg/ml of boiled, sonicated salmon sperm DNA at 42 0 C. overnight and washed for 1 hour in 2 X SSC, 0.1% SDS at 550 and exposed to X-ray film. A xgtl0 clone (designated gtlO:pmgn#127) was cut with SstI and ligated to Sstl-cut pUC119 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.

Sci. (USA), 74, 5463-5467 (1977)) either directly on double-stranded DNA or single-stranded templates [Chen et al., DNA, 4, 165-170 (1985)] after subcloning into a pUC119 vector.

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. A 7.2 kb Eco RI fragment of Autographa californica DNA designated pEcoRI-I [Smith et al., J. Virol., 45, 215-225 (1983); Smith et al., J. Virol., 46, 584-593 (1983)], containing the polyhedrin gene and flanking sequences, was cut with Xho I and Bam HI (Sal I and Xho I digestions produce compatible ends). The resulting Xho I/Bam HI fragment was ligated into a Sal I- and Bam HI-cut mpl9 vector WO 90/13640 PCT/US90/02296 29 (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.

transcription initiation 3'-CTATAGTACCTCTATTAATTTTACTATTGGTAGAGCGTTTCCTAGGCTTA Eco RV Bam HI Eco RI

TCGTCGACGGTACC

AGCAGCTGCCTAGG

Sal I Kpn I The first synthetic oligonucleotide replaced a sequence at the 5' end of the XhoI/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.

Next, a pUC12 vector (Bethesda Research Laboratories) was cut with Hind III and SstI, mp 19A1D 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 KpnI 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 31 flanking region thereof, was isolated. These three WO 90/13640 PCT/US90/02296 30 fragments (pUC12, 1.8 kb and 1.5 kb) were ligated along with a second synthetic oligonucleotide having the sequence 5'-GATCAGCT, to produce a construction designated pAVl. After cutting pAV1 with Bam HI and Kpn I, the resulting fragment was ligated to a third synthetic oligonucleotide to produce a construction designated pAV2. The thiird synthetic oligonucleotide had a nucleotide sequence as follows.

Nru I Bgl II Sst I Bam HI Bst EII Xba I Sma I CTA GAT CTG AGC TCG CGA TGG ATC CCG GGT AAC 3'-AT CTA GAC TCG AGC GCT ACC TAG GGC CCA TTG Kpn I CGG TAC

GC

A plasmid, pDS, was constructed by cutting pBR322 (the 4.4 kb plasmid available from Bethesda Research Labortories) with Hind III and Sal I, filling in with Klenow fragment, and ligating. Thus, 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. Next, 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.

WO 90/13640 PC/US90/02296 31 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 to a Bam HI site inserted at nucleotide -8 (the of the "ATG" initiation site being 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 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 of [Glul]HPg was excised from the plasmid pll9PN127.6 and ir.,erted 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. 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 6t the HPg signal and mature [Glul)Pg coding sequence. The HPg insert contained (from 5' to 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' untranslated sequences plus the poly-A sequence and a linker, and 367 bases from pUC119. The Bam HI site in pAV6 was located at a position corresponding to nucleotide-8 of the PH gene (8 nucleotides located 5' of the PH-initiating methionine residue).

Transfer vector pAV6HPg was deposited in the host E. coli DH5a on April 18, 1989 with the American WO 90/13640 P~r/US90/02296 32 Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, under Accession No. 67929.

Example 2 The construct pAV6HPg and wild type viral DNA were used to cotransfect cultured Spodoptera frugiperda cells. In a cell, 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 (AcMNPV) was used as the host virus for recombinant constructions. A cloned Sf9 cells, as available from the American Type Culture Collection, Rockville, Maryland under accession number A.T.C.C. CRL 1711) or 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 (1977)]. The procedures employed for culturing insect cells are described in Summers et al., Texas Agricultural Experiment Station Bulletin No. 1555, (1987), at pages 10-31 and 38, except that Gibco powdered Grace's Anteraea medium, Hink's medium (instead of TNM-KH medium) and a penicillin/streptomycian/amphotericin B antibiotic mixture were employed in the procedure at page 38 thereof.

Specifically, Spodoptera frugiperda cells (3 X 106), in Hink's medium [Hink, Nature, 226, 466-467 (1970)] plus 10% fetal bovine serum (FBS) (Smith et al., Mol. Cell. Biol., 3, 2156-2165 (1983)], were allowed to adhere to a 60 mm culture dish. After two hours, the medium was removed and the cells were incubated in a ml suspension of wild type DNA from AcMNPV (0.1 pg) plus WO 90/13640 PCT/US90/02296 33 pAV6HPg transfer plasmid DNA (1 Ug) in NaC1 (0.8 g/l), KC1 (0.37 g/l)/Na 2

H

2

PO

4 +2H 2 0(0.125 dextrose (1 g/l) 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 slowmpeed magnetic stirrer (Thermolyne Corp., Dubuque, Zowa). To each 1000 ml stirring vessel, 300 ml of incomplete Hink's medium (supplemented with 8.3% FBS and the penicillin/streptonycin/amphctericin B mixture as above) are added. The vessel is then inoculated with 5 X 106 cells and stirred at 80 rpm. Cells are subcultured when they attain a density of 2 -3 X 106 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, lowprotein 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 Maiorella et al., Biotechnology, 6, 1406-1410 (1988)) Progeny viruses from the cotransfection were examined in plaque assays. Viruses from occlusion body-negative plaques observed by light microscopy, were chosen and purified by two additional rounds of plaque assays. The presence of such plaques indicated that the infecting virus is no longer producing PH protein and that the [Glul)Pg gene may have replaced the viral PH sequences.

WO 90/13640 PCr/US9/02296 34 Example 3 Southern blots were prepared from Eco RI digests of DNA preparations from three plaque-purified OB- viruses.

Cells (3 X 106) were allowed to attach to mm petri dishes and were then infected with 3 different plaque-purified OB- viruses at 10X multiplicity. At 72 hours cells were resuspended by gentle pipetting and pelleted at 3500 g, for 20 minutes at 16 0 C. The cell pellet was suspended and lysed in 4 M guanidinium sarcosyl/5 mM sodium citrate/0.1 mM a-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 nondenatured gels. Copies of HPg-containing plasmid pl19PN127.6 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. Biol., 98, 503-517 (1975)1 and were used as hybridization probes.

Filters were incubated at 37 0 C. for 3 hours in prehybridization fluid (generally described in Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 109-112, 387-389 (1982)], washed with H20, then incubated for 18 hours at 37°C., each probe being dissolved in prehybridization fluid. Labeled probe was removed and non-specific PCT/US90/02296 WO 90/13640 35 radioactivity was eliminated by gently rocking the filter for 45 minutes at 37 0 C. in 10 mM EDTA/0.2% (w/v) NaDodSO 4 pH 8.0. The wash solution was changed four times. Hybridized bands were visualized by autoradiography.

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 in the following Examples. Southern blots of DNA from Pg3A indicated that the [Glul]Pg gene was inserted in the viral DNA.

'Example 4 A total of 45 tissue culture flasks (150 cm 2 containing 20 ml Hinks Medium plus 10% fetal bovine serum were inoculated with 2 X 105 cells/cm 2 and the cells were infected with a 3X multiplicity of Pg3A virus. After 48 hours, the growth medium was decanted, units/ml aprotinin (Sigma Chemical Co., St. Louis, Missouri) was added and residual cells and debris were removed by centrifuging at 13,000 g for 10 minutes at 4 0 C. Unless otherwise noted, all purification steps were performed at 4 0 C. The supernate was retained and dialyzed against four changes of 5 mM sodium phosphate (pH then lyophilized.

After dissolving in a minimal amount of the supernatant was passed over a 5 ml column of a Sepharose-lysine affinity support [Brockway et al., Arch. Biochem. Biophys., 151, 194-199 (1972)] at a flow rate of 0.5 ml/min. The column was then washed with 0.1 M sodium phosphate (pH and eluted with 100 mM sodium phosphate/25 mM EACA (pH 8.0) or with a linear (30 ml X 30 ml) gradient of EACA (0 EACA, start concentration; 20 mM EACA, limit concentration) in 0.1 1 PCT/US90/02296 WO 90/13640 36 sodium phosphate (pH The fractions shown to contain Pg (by dotblot analysis) were pooled, dialyzed against three changes of H 2 0, lyophilized and stored at In immunedot analysis, nitrocellulose membranes were used with a BioRad microfiltration apparatus. Samples (50 pl) were added to wells and the analysis was continued as described in the Bio-Rad instruction manual. Rabbit anti-HPg was used to detect the presence of Pg, followed by a goat anti-rabbit IgGalkaline phosphatase conjugate. After the membrane was removed from the apparatus, positive wells were visualized as described for Western analysis below.

Small scale cultures (in 60 mm petridishes) of the Pg3A-infected insect cells were analyzed for rec-HPg expression by ELISA, Western blotting and activation assays. Activatable rec-HPg was observed on at least three occasions as shown by positive results in an ELISA antigen assay and by positive results in an activation assay, but degraded forms of the protein were also seen. Therefore, a time course study for expression was performed.

Example In a time course experiment, samples of cells and cell supernate were removed at 24, 48, 66, 70, 74e and 93 hours, p.i. Eight 60 mm plates were provided for each time point: wild-type (AcMNPV) and mockinfected plates (at 1.8 X 105 cells/cm 2 and two plates for each of three cell densities 7 X 104 cells/cm 2 1.8 X 105 cells/cm 2 and 3.5 X 105 cells/cm 2 each infected, as described above, with either a 3X or 10X multiplicity of Pg3A virus.

When the virus suspension was removed the cells were washed with Grace's Insect Medium (Gibco, WO 90/13640 PCr/US90/02296 37 Grand Island, New York) and the incubation was continued in Grace's Medium. At the times indicated, working solutions for each sample were obtained by resuspending the relevant cells in the resident growth medium with gentle pipetting, followed by centrifugation to separate the medium from the cell pellet. The washed pellet was resuspended in PBS, and lysed by vortexing in t' e presence of glass beads. The resulting debris was pelleted by centrifugation, and the supernate retained. At 24, 48, 66, 70, 74, 80 and 93 hours, p.i.

both the medium and the cell pellet supernate for each sample were tested for the presence of HPg antigen by ELISA and Western blotting and for the level of activatable protein by an activation assay.

Example 6 For Western analysis of cells and media of Example 5, protein samples were separated by NaDodSO 4 /PAGE [Laemmli, Nature, 227, 680-685 (1970)] on 9% polyacrylamide gels under non-reducing conditions. Separated protein bands were transferred to nitrocellulose according to the general principles of Burnette, Anal. Biochem., 112, 195-203 (1981). The conditions Sor transfer were 4 0 C. in 20 mM Tris-HC1/150 mM glycine/20% methanol, pH 8.3, at 19 volts for 18 hours. The filters were washed with 20 mM Tris- HC1/500 mM NaC1 at pH 7.5 (TBS), and then incubated at 37 0 C for one hour in 1% gelatin (Bio-Rad EIA grade) in TBS (blocking buffer). This solution was replaced with another containing 2 pg/ml rabbit anti-HPg (Boehringer Mannheim, Indianapolis, Indiana) in blocking buffer, and incubated at room temperature for minutes, with mixing. The filter was washed with changes of 0.05% Tween-20 in TBS (wash buffer) at room temperature over 30 minutes. The filter was next WO 90/13640 PCr/IJ~S90/02296 38 incubated with rabbit anti-goat IgG-alkaline phosphatase conjugate in blocking buffer for 90 minutes at room temperature, with mixing, and then washed as above.

Positive bands were visualized after incubations, at room temperature, with the substrate solution [16.5 mg nitro blue tetrazolium/0.5 ml of 70% aqueous mg bromochloroindolyl phosphate in 1 ml H 2 0, which was added to 50 ml of 0.1 M Tris-HCl/0.1 M NaCl/0.005 M MgC12 at pH The reaction was terminated by washing with several changes of H 2 0.

The results obtained from the Western analysis at a 10X viral multiplicity are illustrated in Figure 2 in which Western blots show the time course of sacretion of HPg into the medium from three respective cell cultured Spodoptera frugiperda cells infected with the recombinant virus, pAV6HPg, which contains the signal peptide and coding sequence for HPg. Three cell densities [0.7 X 105, 1.8 X 105 and 3.5 x 105 cells/cm 2 were infected with a 10 X multiplicity of pAV6HPg.

In Figure 5, Western blots of host cells at a density of 0.7 X 105 cells/cm 2 and secreting HPg according to the present invention, of materials from the host cells according to the present invention at a density of 1.8 X 105 cells/cm 2 and secreting HPg according to the present invention, and of materials from the host cells according to the present invention at a density of 3.5 X 105 cells/cm 2 and secreting HPg according to the present invention are depicted. In all three cases 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; and lanes 7-11 are media samples from cells infected with pAV6HPg at times of 66 hours, hours, 74 hours, 80 hours and 93 hours, p.i., WO 90/13640 PCT/ US9)0/02296 39 respectively.

As indicated by the results illustrated i Figure 5, the HPg signal peptide is recognized by cn.

insect cells. This is readily seen at the higher cell density where at 24 hours, rec-HPg antigen is observed in the cell sample. By 48 hours, p.i., approximately 90% of the rec-HPg antigen is observed to be in the medium. At no time was a positive result obtained for either the AcMNPV or mock-infected cells.

At the two lower cell densities there is no visible degradation of rec-HPg, even at 93 hours, p.i. However, at the highest cell density, where cells are severely crowded, degradation is clearly visible, beginning at 48 hours, p.i. Since conditions had been found under which the rec-HPg remained intact and was secreted into the medium, the basis for this degradation was not pursued further.

From the time-course studies of expression illustrated in Figure 5, it is clear that conditions can be readily found such that HPg, recognizable by its reactivity toward a polyclonal antibody pool generated against plasma HPg, is secreted into the medium.

As also seen in Figure 5, the band for rec-HPg consists of a doublet. In human plasma plasminogen such a doublet is observed due to the presence of two differentially-glycosylated forms of the protein [Koltringer et al., Atherosclerosis, 58, 187-198 (1985); Rhoads et al., JAMA, 256, 2540-2544 (1986); Brown et al., Science, 232, 34-37 (1986)].

SWO 90/13640 PCT/US90/02296 40 Example 7 For determination of the levels of rec-HPg present in cells and supernates from the cultures of Example 5, an enzyme-linked immunoadsorbant assay (ELISA) was employed in a variation of a published procedure [Ploplis et al., Biochemistry, 21, 5891-5897 (1982)]. A goat anti-HPg polyclonal antibody pool (Sigma Chemical Company, St. Louis, Missouri) was coated on a polyvinyl plate, the test sample added, and incubation allowed to proceed at room temperature for 1 hour. After washing the plates with 10 mM sodium phosphate/150 mM NaCI (PBS), pH 8.0, a rabbit anti-HPg antibody (2 ug/ml in 1% BSA-PBS) was added. 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, pnitrophenyl phosphate, by the adsorbed alkaline phosphatase. The lower limit of detection was approximately 30 ng/ml relative to a [Glul)Pg standard obtained by purifying fresh human plasma on sepharoselysine generally according to the procedures of Brockway et al., Arch. Biochem. Biophys., 151, 194-199 (1972).

In a quantitative analysis of the level of antigen present in the cells and supernates from these cultures as illustrated in Figure 5, little measurable rec-HPg antigen was found in the cells at times as early as 24 hours p.i. While there is some scatter in the results obtained upon analysis of the media, antigen is secreted, to levels of 0.7-1.0 ug/10 6 cells, by approximately 66 hours, p.i. Small differences of significance in the rec-HPg antigen levels were observed upon comparison of the cell infections with 3X and multiplicities of virus.

WO 90/13640 PCT/US90/02296 41 From the time-course studies of expression, it is clear that conditions can be readily found such that HPg, recognizable by its reactivity toward a polyclonal antibody pool generated against plasma HPg, is secreted into the medium, at levels of approximately 1 ug/ml.

Example 8 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. In the first assay, 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. In the second assay, the wells of an immunoblot apparatus were blocked with 3 MM paper and ul 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. Using ELISA assays [Ploplis et al., Biochemistry, 21, 5891-5897 (1982)], the following dissociation (avidity) constants have been determined for this antibody: [GlullPg, 2 X 10 8 M; the plasmin heavy chain (residues 1-561), 3 X 10 8

M;

[Lys 7 8 ]Pg, 2.7 X 10 8 M; the kringle 1-4 region (residues 80-440), 4.1 X 10- 8 M; and the kringle 1-3 region (residues 80-338/354), 3.9 X 10 8 M. No binding WO 90/13640 PCT/US90/02296 42 was observed to reduced and carboxymethylated HPg, to intact kringle 4, to prothrombin, or to tissue plasminogen activator, all kringle-containing proteins.

In each assay, after incubation with the sample for 1 hour at room temperature, each well was washed with 0.1 M sodium phosphate, at pH 8.0, followed by 10 mM Hepes-NaOH/100 mM NaOAc at pH 7.4 (reaction buffer), and activated with urokinase (about 100 nM in reaction buffer). Human kidney two-chain urokinase was a gift of Abbott Laboratories and existed mainly in the low molecular weight form. A solution of the plasmin chromogenic substrate, D-val-leu-lys-p-nitroanilide (S2251) (0.5 mM, final concentration), in reaction buffer, was added and allowed to incubate for at least 6 hours at 37 0 C. The absorbance at 405 nm, resulting from release of p-nitroanilide from S2251, was measured and compared to the absorbance produced by a set of 2-fold serial dilutions with standard [Glul)Pg in place of the test samples.

Figure 6 illustrates the results of an activation assay performed on the same samples which were used to generate Figure 5. In Figure 6, the activatability of the samples of Figure 5 by urokinase, after interaction of the test samples with a monoclonal antibody to the kringle 1-3 region of HPg, adsorbed to the wells of a polyvinylchloride microtiter plate, is assayed. The ordinate represents the net hydrolysis of the plasmin substrate, S2251, measured for a period of 6 hours after activation as absorbance at a wave length of 405 nm. In Figure 6, solid symbols denote results for media samples, open symbols indicate results for cells, circles indicate results for cells infected with a 3-fold multiplicity of virus and squares indicate results for cells infected with a 10-fold multiplicity of virus.

WO 90/13640 PCT/US90/02296 43 Some activity is present in the cell samples at 24 hours, p.i. that decreases to zero by 66 hours, p.i. At the same time the majority of activatable rec- HPg is secreted into the media, which increases, slightly from 70 hours to 90 hours. Qualitatively similar results have been obtained without regard to whether mouse anti-human-HPg (kringle 1-3) monoclonal antibody 35-J-4 or a Sepharose-lysine affinity support was employed to selectively remove rec-HPg from the test sample prior to activation.

In activation assays, a maximum level of 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 and by the affinity of HPg for the ligand EACA (data not shown), which is also dependent on the integrity of the conformations of the kringle regions of HPg. Thus, not only is the rec-HPg secreted by the insect cells in a conformation suitable for its activation, but it is also properly folded for interaction with a specific monoclonal antibody, as well as a small ligand, which exerts an important regulatory role in the activation of HPg.

WO 90/13640 PCT/US90/02296 44 Example 9 A larger scale rec-HPg preparation was accomplished in which a total of 1.4 X 109 cells were infected with a 3X multiplicity of the virus-encoded HPg gene. At 48 hours, 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

2 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 pAV6HPginfected 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-antirabbit-IgG conjugated to alkaline phosphatase. A goatanti-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 (0-20 mM) is indicated by a symbol. The EACA concentration at the start and end of consecutive fractions containing the rec-HPg is shown to the left of 2F and 4E, respectively. Dots 5A-E are from a 100 mM EACA column wash and 5F and G are blanks. The last dot shown is a plasma HPg standard at a concentration of WO 90/13640 PCriUS9/02296 45 approximately 200 ng/ml. The progress of elution from the column can be followed by reading the plate from top-to-bottom and left to right The results, shown in Figure 7, demonstrate that rec-HPg antigen was eluted at EACA concentrations ranging from 8 mM 12 mM, which is approximately the same position as affinity chromatography variant 1 [Brockway et al., Arch. Biochem. Biophys., 151, 194-199 (1972)] of human plasma HPg.

The strength of the interaction of the insectcell secreted rec-HPg with EACA is comparably similar to that of native plasma HPg, since rec-HPg is eluted at a similar concentration range of EACA that is required to specifically elute plasma HPg from this same affinity chromatography column (Figure Thus, in combination with the results of the previous Examples, these data strongly suggest that the detectable rec-HPg secreted from invertebrate cells exists in a conformation very similar to that of native HPg.

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 NaDodSO 4 /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. Lanes 4-6, as in lanes 1-3, respectively immunostained, as described above in connection with Figures 5 and 8.

In Figure 8, total protein staining shows that WO 90/13640 PCT/US90/02296 46 the conditioned insect cell culture fluid contains three major protein bands (lane one of which is identified as HPg, by immunostaining (lane The material eluted from the Sepharose-lysine column was identified as HPg, by both total protein stain (lane and by immunostaining (lane 6).

The gels of Figure 8 (lanes 3 and 6) 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. In fact, 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.

Example The activation of recHPg by a plasminogen activator was examined. Two reaction mixtures were prepared, containing either 100pg/ml rec-HPg or 0l0ug/ml plasma HPg in 10 mM Hepes-NaOH, pH 7.4. At time 0, a pl aliquot was removed from each reaction and added to 15 4l of NaDodSO 4 /PAGE loading buffer [Laemmli, Nature, 227, 680-685 (1970)] containing B-mercaptoethanol. Urokinase (UK) was then added to the reaction mixtures, at a final concentration of 2 nM.

Two chain human kidney urokinase was a gift from Abbott Laboratories and existed imainly in the low molecular weight form. Aliquots were removed at 10, 20, 30, 90 and 120 min., and the activation was terminated by immediate addition to 15 ul of gel loading buffer, as above. Samples were separated on a 9% (w/v) NaDodSO 4 /PAGE gel under reducing conditions, and visualized by silver staining.

WO 90/13640 PCT/US90/02296 47 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., 262, 15959-15964 (1987)] in 10 mM Hepes-NaOH, pH 7.4/100 mM NaOAc at 37°C. Assay components weze rec-HPg (0.22- 0.66 mM), 0.5 mM of the HPm substrate, D-Val-Leu-Lys-pnitroanilide (S2251), and 2 mM EACA in a final volume of 0.8 ml. The reaction was initiated by the addition of UK to a final concentration 2 nM.

Figure 9 shows a reduced NaDodSO 4

-PAGE

analysis of the activation of plasma HPg and rec-Pg by Slow molecular weight urokinase. Both forms of Pg show a time dependent conversion from a one-chain form (Pg) to a two-chain form Western analysis of this same experiment demonstrates that all of the same protein bands are present (data not shown), indicating that all originate from HPg. A greater number of low-molecular weight HPg (and their corresponding heavy chains) are seen with the recombinant material than with the plasma HPg. These bands have also been seen in numerous laboratories with human plasma HPg, their relative amounts depend upon conditions of activation, and result from feedback limited and specific proteolysis of HPg by the HPm generated in the activation. However, the final 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 analyzed using a continuous coupled assay in which turnover of the plasmin substrate S2251 is followed.

With 2.0 nM UK and 0.33 pM plasma HPg, the activation rate was found to be 7 X 10 12 M/sec. NaOAc and EACA were employed as buffer components so that the rates of activation of [Glul]Pg functionally equaled those of the more rapid activating [Lys 78 )Pg. This assured that PCT/US90/02296 WO 90/13640 48 activation rate differences between plasma [Glul]Pg and rec-HPg would not be due to possible influence of the actual or functional presence of [Lys 78 ]Pg. The rate data obtained indicate that UK activates rec-HPg at a rate similar to, and apparently slightly greater than, that seen for plasma HPg [Burnette, Anal. Biochem., 112, 195-203 (1981)).

The results of the activation analyses are shown in Figure 9. In Figure 9, 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; 2U 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.

The results presented in Figure 9 indicate that rec-HPg can be converted to a two-chain form by a catalytic amount of UK at a rate similar to, or even greater than, that of plasma HPg. There are likely some differences in the recombinant protein and its human plasma counterpart, indicated in Figure 9 by the presence of lower molecular weight PHgs at early activation times, that are fully activatable to HPm.

Similar low molecular weight forms of plasma HPg have been reported, isolated and characterized [Sottrup- Jensen et al., Prog. Chem. Fibrinolysis Thromobolysis, 3, 191-209 (1977); Paoni et al., J. Biol. Chem., 252, 7725-7732 (1977) and Powell et al., Biochem. Biophys.

Res. Comm., 102, 46-52 (1981)], indicating that only the rates of intermediate processes leading to their formation may be different in the two HPg preparations. This suggests that small conformational differences exist in the HPgs, perhaps due to differences in glycosylation, the result of which is to allow more facile HPm feedback limited proteolysis to WO~ 90/13640 PCT/US90/02296 49 occur.

Example 11 Complexes formed between fibrinolytic enzymes and plasminogen 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).

Therefore, 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 AB Kabi, Stockholm, Sweden) may be mixed with a lysine/mannitol buffer (about 110 ml) at pH 7.0 and sterile glycerol (about 60 ml) and stirred for 5 minutes at 4 0 C. A sterile filtered solution of p-amidino-phenyl p'-anisate in DMSO (about 15 ml, about 20 mM) may be added over 2 minutes and the mixture stirred for minutes at 4°C. HPg according to the present invention (about 809 mg) may be added over 2 minutes and the mixture stirred for 60 minutes at 4 0

C.

A pharmaceutical composition according to the present invention may be prepared from the above as follows. Human serum albumin (clinical grade) (18.9 ml w/v) may be then added to the mixture with stirring for 2 minutes at 4 0 C. Lysine/mannitol buffer may be added to bring the volume to about 400 ml. The fluid may then be diafiltered for about 21 hours at 18 0 C until WO 90/13640 PCT/US90/02296 50 about 2400 ml of diafiltrate is collected. The fluid may then be filtered through a 0.22 p sterile filter and transferred to a sterile reservoir from which aliquots may be dispensed into sterile freeze-drying vials followed by freeze drying.

Example 12 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 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 hotologous recombination. For such techniques see, Roizman et al., U.S. Patent No. 4,769,331; Smithies et al., Nature, 317, 23v (1985); Maniatis, Nature, 317, 205-206 (1985); Ayares et al., Proc. Natl. Acad. Sci. (USA), 83, 5199-5203 (1986); Rath et al., Proc. Natl. Acad. Sci. (USA), 83, 5587-5591 (1986); and Thomas, Nature, 324, 34-38 (1986).

By transfection of a cell with oligonucleotides or recombinant constructions synthesized to include termini homologous to the known portions or nucleotide sequences (for UK, Heynecker et al., EPO Publication No. 92,182; and for TPA, Goeddel et al., EPO Publication No. 93,619) of part or all of sitespecific 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 invntion and a WO 90/13640 PCr/US90/02296 51 mammalian cell in which UK and TPA genes have been partially or totally deleted or rendered inactive is preferred.

Although the present invention has been described in terms of a preferred embodiment, it is expected that modifications and variations will occur to those skilled in the art upon consideration of the present invention. Therefore, it is intended that all such modifications and variations be encompassed within the scope of the invention as claimed.

Claims (17)

1. An invertebrate cell expression vector comprising a gene encoding a plasminogen.

2. The invertebrate cell expression vector as recited in claim 1 wherein said invertebrate cell expression vector is an insect cell expression vector.

3. The invertebrate cell expression vector as recited in claim 2 wherein said insect cell expression vector is a baculovirus vector.

4. The invertebrate cell expression vector as recited in claim 3 wherein the baculovirus vector is selected from the group consisting of Autographa californica and Bombyx mori nuclear polyhedrosis virus vectors.

5. The invertebrate cell expression vector as recited in claim I wherein said gene encodes human plasminogen.

6. The invertebrate cell expression vector as recited in claim 5 wherein the invertebrate cell expression vector is an Autographa californica nuclear polyhedrosis virus vector.

7. The invertebrate cell expression vector as recited in claim 6 wherein said vector comprises a nucleotide sequence coding for human plasminogen as shown in Figure 2,

8, The invertebrate cell expression vector as recited in claim 7 and as deposited under accession No.

67929. r 53

9. An invertebrate cell expression vector comprising a gene encoding plasminogen wherein said cell expression vector is selected from the group consisting of virus, polyomavirus, adenovirus, VSV and BPV.

10. An eukaryotic cell lacking a site-specific plasminogen activator, said cell comprising an expression vector comprising a gene encoding a plasminogen.

11. An eukaryotic cell as recited in claim 10 wherein said cell is an invertebrate cell.

12. The invertebrate cell as recited in claim 11 wherein said cell is a Spodoptera frugiperda cell.

13. The invertebrate cell as recited in claim 11 wherein the vector is an Autographa californica nuclear polyhedrosis virus.

14. Plasminogen expressed by a cell as recited in claim 10 wherein said plasminogen differs from circulating plasminogen in a post-translational modification.

15. The plasminogen as recited in claim 15 wherein said cell is a Spodoptera frugiperda cell.

16. The plasminogen as recited in claim 14 wherein said plasminogen is a human plasminogen,

17. A pharmaceutical composition comprising a plasminogen as recited in claim 14. t8. The pharmaceutical composition as recited in claim 17 wherein said composition further comprises a fibrinolytic enzyme. 54 19. The pharmaceutical composition as recited in claim 18 wherein the active site of said fibrinolytic enzyme is acylated. The pharmaceutical composition as recited in claim 18 wherein said fibrinolytic enzyme is complexed with said human plasminogen. 21. The pharmaceutical composition as recited in claim 20 wherein the fibrinolytic enzyme is selected from the group consisting of streptokinase, urokinase, tissue plasminogen activator or a combination thereof. 22. The pharmaceutical composition as recited in claim 21 wherein said fibrinolytic enzyme is a panisoyl streptokinase/plasminogen complex without internal peptide bond cleavage. 23. The pharmaceutical composition as recited in claim 17 wherein said pharmaceutical composition is isotonic. 24. The pharmaceutical composition as recited in claim 17 wherein said pharmaceutical composition is sterile-filtered. A method for producing plasminogen comprising the step of culturing an eukaryotic cell as recited in claim 26. The method for producing plasminogen as recited in claim 25 wherein said culturing step comprises the step of maintaining invertebrate cells containing a gene encoding a human plasminogen under conditions which permit expression of the gene and further comprising the 55 step of recovering plasminogen from the cell culture. 27. The method for producing a plasminogen as recited in claim 26 wherein the invertebrate cells are Spodoptera frugiperda cells, said method further comprising the step of infecting the Spodoptera frugiperda cell with an Autographa californica virus comprising a gene encoding a human plasminogen. 28. The method for producing plasminogen as recited in claim 27 further comprising the step of cotransfecting a Spodoptera frugiperda cell with a transfer vector comprising a gene encoding a human plasminogen and with a wild type Autographa californica viral DNA. 29. A product of the method as recited in claim wherein said plasminogen differs from circulating plasminogen in a post-translational modification. A product of the method as recited in claim 27. 31. Isolated DNA encoding plasminogen and comprising an eukaryotic promoter operably linked to said DNA. 32. The isolated DNA of claim 31 wherein said DNA further comprises a signal sequence operably linked to said DNA for secretion of the plasminogen outside the cell. 33. The isolated DNA as recited in claim 32 wherein the signal sequence is recognized by invertebrate host cells. 34. The isolated DNA as recited in claim 31 wherein said DNA is ge.;:mic DNA. 56 The isolated DNA as recited in claim 33 wherein said DNA has a nucleotide sequence as shown in Figure 2. 36. A method for thrombolytic therapy comprising the step of administering to a patient in need of thrombolytic therapy an effective amount of the composition as recited in claim 17. 37. A method for thrombolytic therapy comprising the step of administering to a patient in need of thrombolytic therapy an effective amount of composition as recited in claim 21. 38. The method for thrombolytic therapy as recited in claim 37 wherein the composition comprises a p-anisoyl streptokinase/plasminogen complex without internal peptide bond cleavage. 39. A method for preparing a binary complex between a fibrinolytic enzyme and plasminogen, the complex having a catalytic site essential for fribrinolytic activity blocked by a group removable by hydrolysis, comprising the steps of: culturing invertebrate cells lacking site-specific plasminogen activators, said invertebrate cells being transformed with an expression vector comprising a nucleic acid encoding plasminogen under conditions which permit expression of the nucleic acid; recovering the plasminogen from the cell culture; and mixing a fibrinolytic enzyme with the plasminogen to form a binary complex in the presence of an excess of -57 a blocking agent of the formula selected from the group consisting of A-B and E-F, wherein A is a hydrolytically labile blocking group which is selective for the catalytic site essential for fibrinolytic activity and S 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 hydrolytically labile blocking group which is capable of transferring from the locating group to the catalytic site. The method as recited in claim 39 further comprising the step of isolating the binary complex. 41. The method as recited in claim 39 wherein the plasminogen is human plasminogen and the fibrinolytic enzyme is streptokinase. 42. The method as recited in claim 39 wherein the hydrolytically labile blocking group is an acyl group. 43. The method as recited in claim 42 wherein the acyl group is a benzoyl, substituted benzoyl, acryloyl, or substituted acryloyl group. 44. The method as recited in claim 41 wherein AB is p-nitrophenyl-p'-guanidinobenzoate. The method as recited in claim 41 wherein E is a p-amidinophenyl or a p-acetamidophenyl group. 46. The method as recited in claim 41 wherein F is a benzoyl or acryloyl group. 47. The plasminogen of claim 14 wherein the 0 58 plasminogen is a mammalian plasminogen having an active conformation and being substantially free of plasminogen activators. 48. The mammalian plasminogen as recited in claim 47 wherein said plasminogen is entirely free of urokinase, streptokinase and tissue plasminogen activator. 49. The mammalian plasminogen as recited in claim 47 wherein said mammalian plasminogen is free of other mammalian proteins. 50. The mammalian plasminogen as recited in claim 49 wherein said mammalian plasminogen is a human plasminogen. 51. The plasminogen substantially as hereinbefore described with reference to the Examples. 52. A method of producing a plasminogen substantially as hereinbefore described with reference to the Examples. 53. A pharmaceutical composition comprising of plasminogen substantially as hereinbefore described with reference to the Examples. DATED this 14th day of JANUARY 1994 UNIVERSITY OF NOTRE DAME DU LAC Attorney: IAN T. ERNST Fellow Institute of Patent Attorneys of Australia of SHELSTON WATERS t r k P*" V