CA1341444C - Modified tissue plasminogen activator - Google Patents

Abstract

A DNA construct containing a nucleotide sequence consisting essentially of a first region encoding a fibrin-binding domain and a second region positioned downstream of the first region, with the second region encoding a catalytic domain for the serine protease activity of tissue-type plasminogen activator is disclosed. The nucleotide sequence codes for a protein which has substantially the same biological activity as t-PA. The regions may be derived from genomic clones or cDNA clones of t-PA, or may be constructed by conventional DNA synthesis techniques. Expression vectors capable of directing the expression of the t-PA-like protein, and transfected or transformed cells producing such a protein, are also disclosed.

Description

MODIFIED TISSUE PLASMINOGEN ACTIVATOR
The present invention relates to fibrinolytic factors in general, and more specifically, to modified tissue-type plasminogen activators.
Blood coagulation is a process consisting of a complex interaction of various blood components which even-tually gives rise to a fibrin network or clot. Degradation of the fibrin network can be accomplished by activation of the zymogen plasminogen into plasmin, a serine protease which acts directly to degrade the fibrin network. Conver-sion of plasminogen into plasmin can be catalyzed by tissue-type plasminogen activator (t-PA), a fibrin-specific serine protease.
T-PA is believed to be the physiological vascular activator of plasminogen and normally circulates as a single polypeptide chain (Mr, 72,000). Urokinase-type plasminogen activator (u-PA) is another member of the class of plasminogen activators characterized as serine proteases.
U-PA is functionally and immunologically distinguishable from t-PA.
In the presence of fibrin, t-PA is activated by cleavage at a single site in the central region of the molecule. The heavy chain of t-PA (two variants of Mr 40,000 and 37,000) is derived from the amino terminus, while the light chain (Mr, 33,000) is derived from the carboxy-terminal end of the t-PA molecule.
A two-dimensional model of the potential precursor t-PA protein has been established (Ny et al., PNAS 81: 5355-5359, 1984). From this model, it was deter mined that the heavy chain contains two triple disulfide structures known as "kringles." These kringle structures also occur in prothrombin, plasminogen and urokinase and are believed to be important for binding to fibrin (Ny et al., ibid). The second kringle (K2) of t-PA is believed to have a higher affinity for fibrin than the first kringle (K1) (Ichinose, Takio, and Fujikawa, J. Clin. Invest. 78:
163-169 (19i~5)).
The heavy chain of t-PA also contains a "finger"
domain that is homologous to the finger domains of fibro nectin. Fibronectin has been implicated in a variety of biological activities, including fibrin binding, and such biological activity has been correlated to four or five of the nine finger domains possessed by fibronectin. The heavy chain of t-PA also contains a growth factor-like domain.
T-PA's light chain contains the active site for serine protease activity, which is highly homologous to the active sites of other serine proteases.
Native t-PA additionally comprises a pre-region followed downstream by a pro-region, which are collectively referred to as the "pre-pro" region. The pre-region con-tains a signal peptide which is important for secretion of t-PA by vascular endothelial cells (Ny et al., ibid). The pre sequence is believed responsible for secretion of t-PA
into the lumen of the endoplasmic reticulum, a necessary step in extracellular secretion. The pro sequence is believed to be cleaved from the t-PA molecule following transport from the endoplasmic reticulum to the Golgi apparatus.
The biological activity of t-PA is substantially enhanced in the presence of fibrin (Sherry, New Eng. J.

Med. 313: 1014-1017, 1985). Unlike the less specific plasminogen activators streptokinase and urokinase, t-PA
has relatively little serine protease activity except at the site of the clot. It is theorized that plasminogen and t-PA are initially bound to the fibrin clot, with the result that enzymatic degradation of the plasminogen into plasmin is sterically facilitated.
The use of t-PA for fibrinolysis in animal and human subjects has highlighted several shortcomings of the native molecule. The half-life of t-PA in vivo has been shown to be as short as three minutes in humans (Nilsson et al., Scand. J. Haematol. 33: 49-53, 1984). Injected t-PA
was rapidly cleared by the liver, and most of the injected material was metabolized to low molecular weight forms within 30 minutes. This short half-life may limit the effectiveness of t-PA as a thrombolytic agent by necessitating high dosages and prolonged infusion. Fuchs et al. (Blood 65: 539-544, 1985) concluded that infused t-PA is cleared by the liver in a process independent of the proteolytic site, and that infused t-PA will not accumulate in the body. Furthermore, doses of t-PA
sufficient to lyse coronary thrombi are far larger than normal physiological levels, and lead to systemic degradation of fibrinogen (Sherry, ibid).
Consequently, for clinical applications it would be advantageous to employ fibrinolytic agents possessing enhanced fibrin-binding capability, increased biological half-life or increased solubility as compared to native t-PA. Such agents would preferably lack structural and functional features which may not play an active role in fibrinolysis. For example, it would be desirable to produce a fibrinolytic molecule which did not contain an epidermal growth factor (EGF) domain since it has been discovered that the EGF domain is not necessary for fibrinolytic activity. Deletion of all or part of the growth factor domain may also increase the solubility of the molecule, due to the hydrophobic nature of that domain.

.. . 1 3 41 44 4 Increased solubility would permit use of smaller (injectable) amounts of t-PA and permit faster administration to patients. Because the active site is not involved in the physiological clearing of t-PA, removal of extraneous domains may also increase the half-life of the resultant modified t-PA molecule in vivo without inactivating it. Specific activity may also be increased, thereby allowing the use of smaller doses. Furthermore, enhancement of t-PA fibrin binding could be achieved by adding additional kringle structures and/or finger domains.
Additionally, a smaller molecule may be more easily secreted by recombinant cells.
In light of the facts that native t-PA is a composite mosaic polypeptide and the t-PA gene comprises multiple exons encoding the aforementioned structural and functional domains (Ny et al., ibid), it would be desirable to construct a DNA sequence which optimally expresses the specific fibrinolytic activity of t-PA. Optimization of the protein product could be accomplished by cloning only the nucleotide sequences which encode the desired structur al and functional properties of t-PA, while eliminating those sequences which do not contribute to the desired biological activity. Multiple copies of the desired structures may also be incorporated into the optimized proteins.
Clearly, there is a need in the art for a fibrinolytic agent which combines the clinical efficacy of t-PA with ease of administration and minimal undesirable side effects. The present invention fulfills this need by providing modified forms of t-PA which may be produced in relatively large quantities. Through the use of recombinant DNA technology, a consistent and homogeneous source of modified t-PAs is provided. The modified t-PAs can be utilized to lyse existing clots in heart attack and stroke victims and in others where the need to lyse or suppress the formation of fibrin matrices is therapeutically desirable.

Briefly stated, the present invention discloses a DNA construct containing a nucleotide sequence consisting essentially of a first region encoding a fibrin-binding domain, and a second region positioned downstream of the first region, with the second region encoding a catalytic domain for the serine protease activity of tissue-type plasminogen activator. The sequence codes for a protein which has substantially the same biological activity as t-PA. The first and second regions may be derived either from genomic clones or cDNA clones of t-PA or may be constructed by conventional DNA synthesis techniques.
Preferably, the first region, which encodes the fibrin-binding domain, encodes one or more kringle struc tures. In particular, the first region encodes the K1 and K2 kringle structures of t-PA, a duplicated K2 structure or a single. K2 structure; or kringle structures from other proteins may be substituted. The first region encoding the fibrin-binding domain may additionally encode one or more finger domains.
The second region encodes a catalytic domain which is essentially the serine protease domain of t-PA. A
particularly preferred second region encodes the light chain of t-PA extending from amino acid number 276 and continuing through amino acid number 527.
In addition, the present invention discloses expression vectors capable of directing the expression of a protein having substantially the same biological activity as t-PA. The vectors include a promoter which is operably linked to a nucleotide sequence, the nucleotide sequence consisting essentially of a first region encoding a fibrin-binding domain, and a second region positioned downstream from the first region, the second region encoding a catalytic domain for the serine protease 1 ~ 41 44 4 activity of t-PA. The sequence codes for a protein which has substantially the same biological activity as t-PA.
A third aspect of the invention discloses cells transfected or transformed to produce a protein having substantially the same biological activity as t-PA.
The cells contain a DNA construct comprising a promoter operably linked to a nucleotide sequence consisting essentially of a first region encoding a fibrin-binding domain and a second region positioned downstream of the first region, the second region encoding a catalytic domain for the serine protease activity of t-PA. The sequence codes for a protein which has substantially the same biological activity as t-PA.
The cells may be mammalian cells or microorgan isms. Preferred microorganisms include bacteria, particu larly E. coli, and eukaryotic microorganisms, particularly the yeast Saccharomyces cerevisiae and filamentous fungi such as As ergillus.
The present invention further provides a method of producing a protein which has substantially the same biological activity as t-PA. The method comprises the steps of inserting into host cells a DNA construct which contains a promoter operably linked to a nucleotide sequence consisting essentially of a first region encoding a fibrin-binding domain and a second region positioned downstream of said first region, the second region encoding a catalytic domain for the serine protease activity of t-PA.
The sequence codes for a protein which has substantially the same biological activity as t-PA. The second step involves growing the host cells in an appropriate medium, followed by the step of isolating the protein product encoded by the DNA construct that is produced by the host cells. The host cells may be mammalian cells or microorganisms. Preferred microorganisms include bacteria, particularly E. coli, and eukaryotic microorganisms, parti-cularly the yeast Saccharomyces cerevisiae and filamentous fungi such as Aspergillus.

Still a further aspect of the present invention discloses proteins having substantially the same biological activity as t-PA which consist essentially of an amino terminal fibrin-binding domain and a carboxyl terminal serine protease domain.
Other aspects of the invention will become evident upon reference to the following detailed description and attached drawingsi in which Figure 1 illustrates the pre-pro t-PA coding sequence constructed from cDNA and synthesized oligo-nucleotides, together with the amino acid sequence of the encoded protein. Numbers above the lines refer to nucleotide position and numbers below the lines refer to amino acid position.
Figure 2 illustrates the construction of the vector Zem99.
Figure 3 illustrates the construction of the t-PA
expression vector pDR817.
Figure 4 shows a comparison of the amino termini of several modified t-PA molecules described herein.
Figure 5 illustrates the construction of plasmid pDR3002 and t-PA vectors derived from pDR3002.
Figure 6 illustrates the amino acid sequence of a mutant t-PA protein lacking the finger and growth factor domains, and the nucleotide sequence encoding the protein.
Figure 7 illustrates the amino acid sequence of a mutant t-PA protein lacking the finger, growth factor and Kringle 1 domains, together with the nucleotide sequence encoding the protein.
Prior to setting forth the invention, it may be helpful to an understanding thereof to set forth defini tions of certain terms used herein.

a Complementary DNA or cDNA: A DNA molecule or sequence which has been enzymatically synthesized from the sequences present in an mRNA template.
DNA Construct: A DNA molecule, or a clone of such a molecule, either single- or double-stranded, which may be isolated in partial form from a naturally occurring gene or which has been modified to contain segments of DNA
which are combined and juxtaposed in a manner which would not otherwise exist in nature.
Plasmid or Vector: A DNA construct containing genetic information which provides for its replication when inserted into a host cell. Replication may be autonomous or by integration into the host genome. A plasmid general-ly contains at least one gene sequence to be expressed in the host cell, as well as sequences encoding functions which facilitate such gene expression, including promoters and transcription initiation sites and terminators. It may be a linear or a closed, circular molecule.
Pre-pro Region: An amino acid sequence which generally occurs at the amino termini of the precursors of certain proteins, and which is generally cleaved from the protein, at least in part, during secretion. The pre-pro region comprises, in part, sequences directing the protein into the secretory pathway of the cell.
Domain: A three-dimensional, self-assembling array of specific amino acids of a protein molecule, which contains structural elements necessary for a specific biological activity of that protein.
Fibrin-binding Domain: That portion of a protein necessary for the binding of that protein to fibrin. In native t-PA, the kringle structures and finger domain indi vidually and collectively contribute to the fibrin binding.
According to the present invention, it has been found that the EGF domain does not significantly contribute to fibrin binding of t-PA or modified t-PAs.
Biological Activity: The function or set of functions performed by a molecule in a biological context ( i . e. , in an organism, a cell, or an in vitro facsimile) .
Biological activities of proteins may be divided into catalytic and effector activities. Catalytic activities of fibrinolytic factors often involve the activation of other proteins through specific cleavage of precursors. In contrast, effector activities include specific binding of the biologically active molecule to other molecules, such as fibrin, or to cells. Effector activity frequently augments, or is essential to, catalytic activity under physiological conditions. Catalytic and effector activities may, in some cases, reside in the same domain of the protein. For native t-PA, biological activity is characterized by the conversion, in the presence of fibrin, of the pro-enzyme or zymogen plasminogen into plasmin, which in turn degrades fibrin matrices. Because fibrin acts as a cofactor in the activation of plasminogen, native t-PA has little activity in the absence of fibrin. As used herein the phrase "substantially the same biological activity as t-PA" includes conversion of, in the presence of fibrin, the pro-enzyme or zymogen plasminogen into plasmin.
As noted above, t-PA is known to play a major role in the degradation of fibrin clots. Because native t-PA is a mosaic protein possessing regions within the mole-rule which have been found to be unnecessary for the fibrinolytic activity of t-PA, it is desirable to provide modified t-PA molecules wherein the fibrin-binding and serine protease activities are retained while excluding all or part of the EGF domain and other non-fibrinolytic functional domains from such modified t-PA. Additionally, it would be desirable to produce modified t-PA having enhanced fibrin-binding activity or increased in vivo half-life.
According to the present invention, it is preferred to produce these novel proteins through the use of recombinant DNA technology, using cDNA clones or genomic clones as starting materials. Suitable DNA sequences can to also be synthesized according to standard procedures. It is preferred to use cDNA clones because, by employing the full-length cDNA encoding native t-PA as starting material for producing modified t-PA, introns are removed so that all exons of the native t-PA are present and correctly oriented with respect to one another. Full-length cDNA
also provides the advantage of easily generating modified molecules by "chewing back" the t-PA cDNA from the 5' end, thus providing a multiplicity of cDNA fragments which can be ultimately inserted into host cells. Alternatively, the cDNA can be used as a template for deletion or insertion of sequences via oligonucleotide-directed mutagenesis.
Utilization of t-PA cDNA allows the convenient enhancement of the fibrin-binding domain of native t-PA by the insertion of additional kringle structures and finger domains. This methodology provides a means for selecting the optimum combination of functional domains found in native t-PA or other proteins to provide fibrinolytic agents with enhanced biological activity with respect to fibrin-binding and serine protease activity.
Accordingly, the present invention provides a method of producing novel proteins having biological activity that is substantially the same as t-PA. Novel proteins described herein have been shown to have a five- to ten-fold greater in vivo half-life than native t-PA. These novel proteins are expressed in transfected mammalian cells, and in transformed fungi and bacteria.
A cDNA clone containing sequences encoding mature human t-PA has been previously identified. The t-PA cDNA
sequence has been inserted into plasmid pDR1296, and this plasmid has been introduced into E. coli JM83. This transformant has been deposited with the American Type Culture Collection, Bethesda, MD, and assigned Accession No. 53347.
Strain pDR1296/JM83 was used as the source of t-PA cDNA. Plasmid pDR1296 was isolated, and the t-PA cDNA
was excised by restriction endonuclease digestion. The t-PA restriction fragment was then incubated with Bal 31, and the enzymatic reaction time was controlled in order to produce a continuum of t-PA nucleotide sequences exhibiting size heterogeneity. The size heterogeneity results from the progressive shortening of the 5' terminus of the t-PA
cDNA.
The Bal 31 digested fragments were separated by gel electrophoresis and the desired fragment size range was selected. The fragments were eluted from the gel according to conventional techniques.
In a second approach, part of the cDNA sequence from pDR1296 was used as a template for oligonucleotide directed deletion mutagenesis. By this method, sequences encoding specific domains of native t-PA are precisely deleted or altered.
In a third approach, the 5' coding region of a modified t-PA sequence was constructed from synthesized oligonucleotides and joined to the 3' region of the cDNA.
The modified t-PA fragments were then joined to an appropriate pre-pro sequence. The pre-pro sequence of t-PA may be isolated from cDNA or genomic libraries, or may be constructed from synthesized oligonucleotides. Oligo nucleotides are preferably machine-synthesized, purified, and annealed to construct double-stranded fragments. The resultant double-stranded fragments are ligated as necessary to produce the pre-pro sequence. This pre-pro sequence comprises a 105 by sequence, as depicted in Figure 1. The synthesized pre-pro sequence was then joined to the 5' termini of the modified t-PA fragments. Other pre-pro sequences may be used, depending on the host cell type selected. In some instances it may be desirable to use a pre-pro sequence which is endogenous to the host species.
The pre-pro-t-PA fragments are inserted into a suitable expression vector, which in turn is inserted into appropriate host cells. The method of insertion will depend upon the particular host cell chosen. Methods for transfecting mammalian cells and transforming bacteria and fungi with foreign DNA are well known in the art. Suitable expression vectors will comprise a promoter capable of directing the transcription of a foreign gene in a host cell.
In some instances it is preferred that expression vectors further comprise an origin of replication, as well as sequences which regulate and/or enhance expression levels, depending on the host cell selected. Suitable expression vectors may be derived from plasmids, RNA and DNA viruses or cellular DNA sequences, or may contain elements of each.
Preferred prokaryotic hosts for use in carrying out the present invention are strains of the bacteria Escherichia coli, although Bacillus and other genera are also useful. Techniques for transforming these hosts and expressing foreign DNA sequences cloned in them are well known in the art (see, for example, Maniatis et al., Molecular. Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 1982). Vectors used for expressing foreign DNA
in bacterial hosts will generally contain a selectable marker, such as a gene for antibiotic resistance, and a promoter which functions in the host cell. Appropriate promoters include the trp (Nichols and Yanofsky, Meth. in Enzymology 101: 155, 1983), lac (Casadaban et al., J. Bact.
143: 971-980, 1980), TAC (Russell et al., Gene 20: 231-243, 1982), and phage promoter systems. Plasmids useful for transforming bacteria include pBR322 (Bolivar et al., Gene 2: 95-113, 1977), the pUC plasmids (Messing, Meth. in Enzymology 101: 20-77, 1983: and Vieira and Messing, Gene 19: 259-268, 1982), pCQV2 (Queen, J. Mol. Appl. Genet. 2:
1-10, 1983), and derivatives thereof.
Eukaryotic microorganisms, such as the yeast Saccharomyces cerevisiae, or filamentous fungi including Asperqillus, may also be used as host cells. Particularly preferred species of Aspergillus include A. nidulans, A. niger, A. o~~zae, and A. terreus. Techniques for transforming yeast are described by Beggs (Nature 275:

104-108, 1978). Expression vectors for use in yeast include YRp7 (Struhl et al., Proc. Natl. Acad. Sci. USA 76:
1035-1039, 1979), YEpl3 (Broach et al., Gene 8: 121-133, 1979), pJDB248 and pJDB219 (Beggs, ibid), and derivatives thereof. Such vectors will generally comprise a selectable marker, such as the nutritional marker TRP, which allows selection in a host strain carrying a trill mutation.
Preferred promoters for use in yeast expression vectors include promoters from yeast glycolytic genes (Hitzeman et al., J. Biol. Chem. 255: 12073-12080, 1980; Alber and Kawasaki, J. Mol. Appl. Genet. 1: 419-434, 1982) or alcohol dehydrogenase genes (Young et al., in Genetic Engineering of Microorganisms for Chemicals, Hollaender et al., eds., p. 335, Plenum, New York, 1982: and Ammerer, Meth. in Enzymology 101: 192-201, 1983). To facilitate purification of a modified t-PA protein produced in a yeast transformant and to obtain proper disulphide bond formation, a signal sequence from a yeast gene encoding a secreted protein may be substituted for the t-PA pre-pro sequence. A particu-larly preferred signal sequence is the pre-pro region of the MF 1 gene (Kurjan and Herskowitz, Cell 30: 933-943, 1982). Aspergillus species may be transformed according to known procedures, for example, that of Yelton et al. (Pros.
Natl. Acad. Sci. USA 81: 1740-1747, 1984).
Higher eukaryotic cells may also serve as host cells in carrying out the present invention. Cultured mammalian cells, such as the BHK, CHO, and J558L cell lines, are preferred. Tk- BHK cells are particularly preferred. Expression vectors for use in mammalian cells will comprise a promoter capable of directing the transcription of a foreign gene introduced into a mammalian cell. Particularly preferred promoters include the SV40 (Subramani et al., Mol. Cell Biol. 1:854-64, 1981) and MT-1 promoters (Palmiter et al., Science 222: 809-814, 1983).
Also contained in the expression vectors is a transcription terminator, located downstream of the insertion site for the DNA sequence to be expressed. A preferred terminator is .a. 1341444 the human growth hormone (hGH) gene terminator (DeNoto et al., Nuc. Acids Res. 9: 3719-3730, 1981).
For expression of mutant t-PAs in cultured mammalian cells, expression vectors containing cloned t-PA
sequences are introduced into the cells by appropriate transfection techniques, such as calcium phosphate-mediated transfection (Graham and Van der Eb, Virology 52: 456-467, 1973: as modified by Wigler et al., Proc. Natl. Acad. Sci.
USA 77: 3567-3570, 1980). A DNA-calcium phosphate precipi-tate is formed, and this precipitate is applied to the cells in the presence of medium containing chloroquine (100um). The cells are incubated for four hours with the precipitate, followed by a two-minute, 15~ glycerol shock.
A portion of the cells take up the DNA and maintain it inside the cell for several days. A small fraction of the cells integrate the DNA into the genome of the host cell or maintain the DNA in non-chromosomal nuclear structures.
These transfectants are identified by cotransfection with a gene that confers a selectable phenotype (a selectable marker). A preferred selectable marker is the DHFR gene, which imparts cellular resistance to methotrexate (MTX), an inhibitor of nucleotide synthesis. After the host cells have taken up the DNA, drug selection is applied to select for a population of cells that are expressing the selectable marker in a stable fashion.
Coamplification as a means to increase expression levels can be accomplished by the addition of MTX to the culture medium, preferably by sequentially increasing the concentration of MTX in the medium, followed by repeated cloning by dilution of the drug-resistant cell lines.
Variations in the ability to amplify relate both to the initial genomic configuration (i.e. extra-chromosomal vs.
chromosomal) of the cotransfected DNA sequences as well as to the mechanism of amplification itself, in which variable amounts of DNA rearrangements can occur. This is noticed upon further amplification of clones previously shown to be capable of frequent and stable coamplification. For this reason, it is necessary to clone by dilution after every amplification step. Cells which express the DHFR marker are then selected and screened for production of t-PA.
Screening may be done by enzyme linked immunosorbent assay (ELISA) or by biological activity assays.
Additionally, it has been observed that under certain conditions production of t-PA has a deleterious effect on mammalian cells in culture. This is believed to be due in part to the plasmin (a nonspecific protease) generated when cells which have been transfected to produce t-PA are cultured in media containing plasminogen, a component of serum. The t-PA produced by the transfected cells activates the plasminogen to plasmin, which attacks cell membranes and contributes to cell detachment. Other proteolytic activities are also believed to be involved.
It has been found that including protease inhibitors in the culture media blocks the activation of plasminogen and facilitates t-PA production. A particularly preferred protease inhibitor is aprotinin, which is included in the culture media at a concentration of from approximately 100 units/ml to 50,000 units/ml, preferably 100 units/ml, in medium containing plasminogen-free serum, or from approxi-mately 1000 units/ml to 50,000 units/ml, preferably 1000 units/ml, if using normal fetal calf serum. Additional useful protease inhibitors include tranexamic acid and epsilon amino caproic acid, which are included in the media at mM concentrations.
The t-PA so produced is recovered from the cultured cells by removing the culture medium and fraction sting it. A preferred method of fractionation is affinity chromatography using an anti-t-PA antibody, a fibrin-celite column or a lysine-sepharose column. Other conventional purification methods, such as ion-exchange chromatography, high-performance liquid chromatography or gel filtration, may also be used.
In summary, the present invention provides modified t-PA proteins and a method for production of modified t-PA proteins having substantially the same biological activity as native t-PA through use of stably transfected or transformed host cells. The protein products thus expressed are then purified from the cells or cell growth media and assayed for biological activity. The assay may monitor conversion of plasminogen to plasmin, fibrin-binding affinity or plasma half-life characteristics.
Immunological assays may also be used.
To summarize the examples which follow, Example 1 discloses a t-PA cDNA clone made from mRNA from the Bowes melanoma cell line. The cDNA was used to construct the plasmid pDR1296 which was utilized to transform E. coli strain JM83. The t-PA sequence of pDR1296 was then joined to a synthesized pre-pro sequence which was constructed from oligonucleotides. An MT-1 promoter and human growth hormone terminator were added and the vector Zem99, as depicted in Figure 2, was constructed.
Example 2 discloses the cloning and sequencing of human genomic t-PA obtained from a DNA library derived from normal liver tissue.
Example 3 discloses the preparation of t-PA
sequences having random 5' end deletions utilizing the full-length pDR1296 t-PA clone. A TAC promoter and t-PA
pre-pro sequence were ligated to the randomly deleted t-PA
cDNA sequences. A pDR$17 construct directed the production of t-PA fibrinolytic activity ten- to thirty-fold higher than that obtained using a full-length t-PA clone.
Expression of the truncated sequences in cultured mammalian cells is also disclosed.
Examples 4 and 5 describe methods of looping out the coding sequences for the finger domain and growth factor domain of t-PA.
Examples 6 and 7 disclose methods of looping out the coding sequences for the finger domain, growth factor domain and Kringle 1 structure to produce modified t-PA.
Example 8 discloses the construction of a mutant t-PA sequence utilizing synthesized oligonucleotides.

Example 9 discloses a mutant sequence lacking the growth factor domain coding sequence which is constructed by deletion mutagenesis.
Example 10 describes the method of expressing mutant t-PAs in transformed E. coli JM105 utilizing bacterial expression vector pDR816 (described in Example 3).
Example 11 teaches a method of expressing mutant t-PAs in mammalian cells by transfecting baby hamster kidney (BHK) cells with expression vectors comprising mutant t-PA sequences.
Example 12 describes the expression of mutant t-PAs in S. cerevisiae cells which are transformed with a vector comprising a mutant t-PA nucleotide sequence.
The following examples are offered by way of illustration and not by way of limitation.
~vrnnnr ~e Example 1 - Construction of a Full-Length t-PA Clone The sequence of a human t-PA cDNA clone has been reported (Pennica et al., Nature 301: 214-221, 1983). The sequence encodes a pre-pro peptide of 32-35 amino acids followed by a 527-530 amino acid mature protein.
A cDNA clone comprising the coding sequence for mature t-PA was constructed using as starting material mRNA
from the Bowes melanoma cell line (Rijken and Collen, J.
Biol. Chem. 256: 7035-7041, 1981). This cDNA was then used to construct the plasmid pDR1296. Escherichia coli strain JM83 transformed with pDR1296 has been deposited with the American Type Culture Collection under Accession No. 53347.
Because the pre-pro sequence was not present in the cDNA clone pDR1296, it was constructed from synthesized oligonucleotides and subsequently joined to the cDNA. In the synthesized t-PA pre-pro sequence, cleavage sites for Bam HI and Nco I were introduced immediately 5' to the first codon (ATG) of the pre-pro sequence, and a Bgl II
(Sau 3A, Xho II) site was maintained at the 3' end of the pre-pro sequence. The naturally occurring pre-pro sequence lacks a convenient restriction site near the middle;
however, the sequence GGAGCA (coding for amino acids -20 and -19, Gly-Ala) can be altered to GGCGCC to provide a Nar I site without changing the amino acid sequence.
To construct the pre-pro sequence, the following oligonucleotides were synthesized using an Applied Biosystems Model 380-A DNA synthesizer:
ZC131: 5~GGA TCC ATG GAT GCA ATG AAG AGA GGG CTC TGC

TGT GTG3~

ZC132: S~TGG CGC CAC ACA GCA GCA GCA CAC AGC AGAG3~

ZC133: S~GGC GCC GTC TTC GTT TCG CCC AGC CAG GAA ATC

CATG3~

ZC134: S AGA TCT GGC TCC TCT TCT GAA TCG GGC ATG GAT

Following purification, oligomers ZC131 and ZC132 were annealed to produce an overlap of 12 base pairs (Section 1). Oligomers ZC133 and ZC134 were similarly annealed (Section 2).
The oligomers were mixed in Pol I buffer (Bethesda Research Labs), heated to 65°C for five minutes, and slowly cooled to room temperature for four hours to anneal. Ten units of DNA polymerase I were added and the reaction proceeded for two hours at room temperature. The mixtures were electrophoresed on an 8% polyacrylamide-urea sequencing gel at 1,000 volts for 2~- hours in order to size fractionate the reaction products. The correct size fragments (those in which the polymerase reaction went to completion) were cut from the gel and extracted.
After annealing, Section 1 was cut with Bam HI
and Nar I and cloned into Bam HI + Nar I - cut pUC8 (Vieira and Messing, Gene 19: 259-268, 1982; and Messing, Meth. in Enzymology 101: 20-77, 1983). Section 2 was reannealed and cut with Nar I and Bgl II and cloned into Bam HI + Nar (Sau 3A, Xho I - cut pUC$. Colonies were screened with the appropriate labelled oligonucleotides. Plasmids identified as positive by colony hybridization were sequenced to verify that the correct sequence had been cloned.
Section 1 was then purified from a Bam HI + Nar I
double digest of the appropriate pUC clone. Section 2 was purified from a Nar I + Xho II digest. The two fragments were joined at the Nar I site and cloned into Bam HI - cut pUC8.
The t-PA sequence of pDR1296 was then joined to the synthesized pre-pro sequence in the following manner (Figure 2). Plasmid pICl9R (Marsh et al., Gene 32:
481-486, 1984) was digested with Sma I and Hind III. The on region of SV40 from map position 270 (Pvu II) to position 5171 (Hind III) was then ligated to the linearized pICl9R to produce plasmid Zem67. This plasmid was then cleaved with Bgl II and the terminator region from the human growth hormone gene (De Noto et al., Nuc. Acids Res.
9: 3719-3730, 1981) was inserted as a Bgl II - Bam HI
fragment to produce plasmid Zem86. The synthesized t-PA
pre-pro sequence was removed from the pUC8 vector by digestion with Sau 3A. This fragment was inserted into Bgl II-digested Zem86 to produce plasmid Zem88. Plasmid pDR1296 was digested with Bgl II and Bam HI and the t-PA
cDNA fragment was isolated and inserted into Bgl II - cut Zem88. The resultant plasmid was designated Zem94.
The vector, Zem99, comprising the MT-1 promoter, complete t-PA coding sequence, and the hGH terminator was then assembled in the following manner (Figure 2). A Kpn I-Bam HI fragment comprising the MT-1 promoter was isolated from MThGHlll (Palmiter et al., Science 222: 809-814, 1983) and inserted into pUCl8 to construct Zem93. Plasmid MThGH112 (Palmiter et al., ibid) was digested with Bgl II
and religated to eliminate the hGH coding sequence. The MT-1 promoter and hGH terminator were then isolated as an Eco RI fragment and inserted into pUCl3 to construct Zem4.
Zem93 was then linearized by digestion with Bam HI and Sal I. Zem4 was digested with Bgl II and Sal I and the hGH

terminator was purified. The t-PA pre-pro sequence was removed from the pUCB vector as a Bam HI-Xho II fragment.
The three DNA fragments were then joined and a plasmid having the structure of Zem97 (Figure 2) was selected.
5 Zem97 was cut with Bgl II and the Xho II t-PA fragment from Zem94 was inserted. The resultant vector is Zem99.
Example 2 - Cloning and Sequencing of Human Genomic t-PA
('l r,no 10 A genomic t-PA clone was obtained from a DNA
library derived from normal liver tissue. The library was constructed by insertion of fetal human liver DNA fragments into bacteriophage lambda (Lawn et al., Cell 15: 1157-1174, 1978).
15 The library was used to infect E. coli strain LE392 (ATCC 33572) (Maniatis et al., Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory, 1982, p.
504). An overnight culture of cells in L-broth containing 0.2~ maltose, 10 mM MgS04, and 50 ug/ml thymidine was con-20 centrated two-fold in 10 mM MgS04. 750 u1 of the concen-trate was plated, together with 1 u1 of the phage library (200,000 phage/ul) on L-broth agar in a NZY amine soft agar overlay using 22 cm x 22 cm plates. Approximately 105 colonies were obtained per plate following an overnight incubation at 37°C. Colonies were transferred to nitro-cellulose and the lifts were treated with 0.1 M NaOH, 1.5 M
NaCl, air-dried, and baked two hours at 80°C. Pre-hybridi-zation and hybridization (to a full-length, nick-translated t-PA cDNA probe) were carried out in SET buffer (SET con-tains, per liter, 175.2 g NaCl, 72.7 g Tris, 14.8 g EDTA, pH 8.0 with HC1) at 65°C. Filters were washed in 2 x SSC, O.lo SDS, dried, and autoradiographed. Thirteen prelimi nary positives were identified. Two rounds of plaque purification (screened as above) identified nine positives from the group of thirteen.
The nine positive genomic clones were plated on E. coli LE392, grown overnight, and lysates were prepared.

The phage were purified on CsCl gradients and mapped by hybridization to oligonucleotide probes shown in Table 1.
mnur~ i ZC46 (5' TCG TTT ACT CTA GG3') ZC88 (5' TGC AGC GAG CCA AGG3') ZC89 (5' ACG TGG AGC ACA GCG3') ZC91 (5' CCC TCC TGC TCC ACC3') ZC94 (5' TAG GAT CCA TGG ATG CAA TGA AGA GAG GGC3') ZC96 (5' CTG CTG TGT GGA GCA GTC TTC GTT TCG CCC3') ZC98 (5' TGC CCG ATT CAG AAG AGG AGC CAG ATC TTC3') Probes ZC94, ZC96 and ZC98 will hybridize to the signal peptide coding region; the remaining probes hybridize to the mature peptide coding region. The nine positive isolates were found to fall into three distinct classes which together span the entire t-PA coding region.
Insert size was determined by digestion with Eco RI.
For each class, the clone having the largest insert was digested with Eco RI, Bgl II, and Eco RI + Bgl II and the restriction fragments were probed on Southern blots (Southern, J. Mol. Biol. 98: 503-517, 1975) using representative oligonucleotide probes. Clone number 9 was found to contain the entire coding sequence for mature t-PA, but not the pre-pro region. Eco RI and Bgl II-Eco RI
fragments of the insert from clone number 9 were inserted into M13 and pUCl3 vectors for sequencing and further analysis. The fragments were sequenced by the dideoxy method (Sanger et al., J. Mol. Biol. 143: 161, 1983, and Sanger et al., Proc. Natl. Acad. Sci. USA 74: 5463, 1977).
Example 3 - Preparation of t-PA Sequences Having Random 5' End Deletions A. Deletion of 5' Coding Sequences Ten ug of pDR1296 were digested with either five units of Bgl II or five units of Nar I to completion. The resultant linearized DNAs were electrophoresed on a 0.7%
agarose gel. The DNA fragments were eluted from the gel using TE buffer (10 mM Tris, pH 7.4, 5 mM EDTA) . The DNA
fragments were resuspended in 46 u1 of H20 and 12 u1 of 5x Ba131 buffer (3M NaCl, 62.5 mM CaCl2, 62.5 mM MgCl2, 100 mM
Tris-HCl, pH 8.0, 50 mM EDTA, pH 8.0) plus 2 u1 of Ba131 (0.5 units/ l, obtained from Bethesda Research Labora-tories). The reaction mixtures were incubated at 30°C and u1 aliquots were removed at one minute intervals for six 10 minutes. The digested samples were combined, extracted with phenol-CHC13, and precipitated with ethanol. The fragment ends were filled in with DNA polymerise I (Klenow fragment) and dNTPs. Following the fill-in reaction, samples were digested with Xba I and separated on a 0.7%
agarose gel. Fragments smaller than 1700 by were cut out and extracted with TE buffer. Subsequent mapping identified 55 truncated sequences.
B. Preparation of pDR817 Expression Vector Referring to Figure 3, the 28 by trpA terminator (obtained from P-L Biochemicals) was ligated into pUCl8 which had been cut with Sst I and filled in using T4 DNA
polymerise to form plasmid pDR813. The 28 by terminator fragment was similarly inserted into the filled-in Cla I
site of pICl9R to construct plasmid pDR812. Plasmid pDR540 (Russell et al., Gene 20: 231-243, 1982), which contains the TAC promoter and lac operator (lac0), was cut with Hind III, filled in with Klenow polymerise, and digested with Bam HI. The resulting ~92 by fragment, comprising the TAC
promoter and lac0, was ligated into Bam HI + Sma I cut pDR813 to form pDR814. Plasmid pDR814 was then digested with Eco RI and Hind III and the fragment comprising the trpA terminator, TAC promoter, and lac0 was isolated and ligated into pDR812 which had been digested to completion with Hind III and partially digested with Eco RI. The resultant plasmid was designated pDR815.

Plasmid Zem99 (see Example 1) was digested with Bam HI and Xba I and the fragment comprising the t-PA cDNA
(including the pre-pro sequence) was isolated. Plasmid pDR815 was cut with Bam HI and Xba I and ligated to the t-PA fragment to construct plasmid pDR816.
Ten ug of pDR816 were digested to completion with five units of Bgl II and the ends filled in using DNA
polymerase I (Klenow fragment). The DNA was then digested to completion with five units of Xba I, extracted with phenol-CHC13, ethanol precipitated, and electrophoresed on a 0.7% agarose gel. A 3050 by fragment (comprising pICl9R, TAC promoter, and t-PA pre-pro sequences) was eluted from the gel.
The 3050 by fragment and the randomly deleted t-PA cDNA sequences were ligated overnight at 16°C.
Ligation mixtures were used to transform competent E. coli JM83 cells. Transformed cells were plated on Ampicillin and resistant colonies were selected and transferred to fresh Ampicillin-containing plates in triplicate. The plates were incubated overnight at 37°C, and colonies were transferred to nitrocellulose filters. The filters were placed on Ampicillin plates and incubated four hours at 37°C, then treated with CHC13 vapor for 10 minutes. The filters were air-dried for 10 minutes and placed directly onto a fibrin plate and incubated at 37°C overnight. The filters were lifted off the plates and colonies capable of causing fibrin lysis were picked for further analysis. A
duplicate set of filters was similarly processed for colony immuno blot assay. Colonies capable of producing t-PA-like polypeptide were identified.
One plasmid, designated pDR817, was characterized in detail. The plasmid was digested with Bam HI and Xba I
and a 1270 by t-PA fragment was gel purified and subcloned into Ml3mpl8 (replicative form). The DNA sequence of the insert was determined by the dideoxy method. Results indicated that pDR817 lacked nucleotides 192-524 (numbering based on that of Pennica et al., ibid). It thus encodes a ~ 341 44 4 mature protein consisting of 416 amino acids with an amino terminus as shown in Figure 4A. Amino acids 2-112 of naturally occurring t-PA were deleted.
E. coli strain JM105 was transformed with pDR816 and pDR817. Untransformed cells and the transformants were grown overnight in M9 medium supplemented with 0.4~ glucose and 0.2~ casamino acids. Ten ml aliquots were removed from each culture and the cells harvested. The inducer IPTG
(isopropylthiogalactoside) was added to the remainder of the cultures to a final concentration of 1 mM. After induction for 120 minutes and 240 minutes, 10 ml aliquots were removed and the cells harvested. The cell pellets were washed once with water and resuspended in 450 u1 of 20°s sucrose in 100 mM Tris, pH 8Ø The cells were lysed by the addition of 50 u1 of 5 mg/ml lysozyme in 50 mM EDTA.
The mixtures were incubated 10 minutes at room temperature, 500 u1 of lysis buffer (0.3% Triton X-100, 150 mM Tris, pH
8.0, 0.2 M EDTA) were added, and the mixtures placed on ice for 30 minutes. The lysates were centrifuged one hour at 35,000 rpm in a Beckman SW50 rotor. The supernatants were removed and assayed by the fibrin lysis method. Results (see Table 2) indicated that the pDR817 construct directed the production of t-PA activity 10- to 30-fold higher than that obtained using a full-length t-PA clone.

* tra3e-mark Fibrin Lysis Strain Time* Activity {ug/1) JM105 120 min. 0 JM105 240 min. 0 pDR816:JM105 - 7.4 10 pDR816:JM105 120 min. 5.0 pDR816:JM105 240 min. 7.4 pDR817:JM105 - 160.0 pDR817:JM105 120 min. 150.0 15 pDR817:JM105 240 min. 120.0 * Time after addition of IPTG.
(-) indicates sample taken prior to addition of IPTG.
20 Aliquots of the transformed cells and the untransformed control were analyzed for protein production by Western blot assay. DNA sequence analysis indicated that the pDR817 t-PA polypeptide is approximately 100 amino acids shorter than the polypeptide produced by pDR816 25 (Figure 4A). E. coli JM105 transformed with pDR817 has been deposited with American Type Culture Collection under Accession No. 53446.
C. Expression of t-PA Deletion Mutants in Mammalian Cells Expression vectors were constructed in the following manner. Plasmid Zem86 {described in Example 1) was digested with Hind III and the ends filled in using DNA
polymerase I (Klenow fragment). The linearized DNA was then digested with EcoRI, and a 450 by fragment, comprising the SV40 on sequence, was gel purified and ligated to Sma I + Eco RI digested pUCl3. The resultant vector was designated pDR3001. Plasmid pDR3001 was digested with Sal I and Eco RI and the '450 by fragment, comprising SV40 on and polylinker sequences, was gel purified. Zem 86 was partially digested with EcoRI and completely digested with Xho I to remove the SV40 on sequence. The SV40 fragment from pDR 3001 was then joined to the linearized Zem 86. The resultant plasmid was designated pDR3002 (Figure 5).
Mammalian cell expression vectors were then constructed. Plasmid pDR3002 was digested with Bam HI and Xba I. The bacterial vectors described in Example 3B were digested with Bam HI and Xba I, and the t-PA sequences are gel purified and ligated to the linearized pDR3002. The resultant vectors (Tab.le 3) contain expression units of SV40 promoter - mutant t-PA sequence - hGH terminator. The vector pDR3004 (Figure 5) comprises the mutant t-PA
sequence from pDR817. The primary translation product from pDR3004 i.s predicted to have the sequence shown in Figure 4A. E. coli LM1035 transformed with pDR3004 has been deposited with American Type Culture Collection under Accession No. 53445.

Summary of Sequential Deletion Mutants Plasmid Sequence at Pre-pro Junction Domains*
pDR3004 AGA TCG TGCACC AAC K1 (partial), K2, SP

Arg Ser CysThr Asn pDR3006 AGA TCC CTG GGG AAC K1 (partial), K2, SP

Arg Ser Leu Gly Asn ._ 1 341 44 4 pDR3007 AGA TCC ACCAAC AGT K1 (partial), K2, SP

Arg Ser ThrAsn Trp pDR3008 AGA TCG GGAAAC AGT K2, SP

Arg Ser GlyAsn Ser pDR3010 AGA TCG GGTGCC TCC K2 (partial), SP

Arg Ser GlyAla Ser pDR3011 AGA TCT GAGGGA ACC K2, SP

Arg Ser GluGly Asn pDR3012 AGA TCG AAGTAC AGC K1 (partial), K2, SP

Arg Ser LysTyr Ser pDR3013 AGA TCC TGCCAG CAG GF (partial), K1, 1 62 63 64 K2, SP

Arg Ser CysGln Gln pDR3014 AGA TCG AATGGG TCA K2, SP

1 1$4185 186 Arg Ser AsnGly Ser pDR3016 AGA TCG GGCACC TGC GF (partial), Kl, 1 60 61 62 K2, SP

Arg Ser GlyThr Cys * GF - growth 1 Kringle K2 Kringle factor, - l, - 2, K

SP - serine protease The described Table vectors in 3 are used to transfect cultu red mammalian cells according to standard 1 ~414~4 procedures. Logarithmically growing Tk- baby hamster kid-ney (BHK) cells were used for cotransfection of a mixture (1:1 ratio) of expression vector DNA coding for the mouse wild-type DHFR gene (pSV2-DHFR, disclosed by Subramani et al., Mol. Cell. Biol. l: 854-864, 1981) and an expression vector encoding a mutant t-PA protein. Twenty-four hours after transfection, the cells were supplemented with Dulbecco's modified Eagle's medium (DME) containing 10%
fetal bovine serum (depleted of plasminogen by passage over a lysine-sepharose column), aprotinin (100 units/ml), penicillin, and 250 mM MTX. Cells were fed with this selective medium several times over the next 10 to 14 days.
Drug-resistant colonies were screened for t-PA activity by the fibrin plate method. Cell lines producing active protein at levels greater than 1 pg/cell/day were further amplified, cloned by dilution, and scaled up for protein isolation and characterization.
Three of the above-described deletion mutants were chosen for further characterization. These correspond to mutants 3016, 3004 and 3008, mutants representing deletions into three different domains (see Table 3). A
monoclonal antibody which was capable of detecting all three of the truncated molecules examined was used as primary antibody for detection of truncated tPAs. The standard ELISA protocol for detection of truncated tPAs is outlined below.

ELISA Procedure for Truncated tPA
Plate out 100 u1 of 1 ug/ml of monoclonal antibody in Buffer A
Buffer A = 100 mM Na2C03 pH 9.6 Incubate overnight at 4°C
wash 3x with Buffer B
Buffer B = 10 mM Na Phosphate pH 7.2; 150 mM NaCl:
0.5% Tween 20*
Block for two hours at 37°C with Buffer C
Buffer C = Buffer B + 1% BSA
Load samples in Buffer C
Incubate at 37°C for two hours Wash 3x with Buffer B
Load 100 u1 of 4.5 ug/ml of polyclonal rabbit anti-tPA
Incubate at 37°C for one hour Wash 3x with Buffer B
Load HRP conjugated goat anti-rabbit Incubate 1 hour at room temperature Wash 4x with Buffer B
Color development Mutant proteins 3004 and 3008 were purified on a lysine-sepharose column. Cell culture media were dialyzed overnight against loading buffer (50 mM sodium phosphate pH
7.3, 0.1 M NaCl, 0.005% Tween 80, 0.003 M NaN3).
The dialyzed solution was loaded onto a lysine-sepharose*
column at 0.5 ml per minute. The column was washed with loading buffer, then bound material was eluted with loading buffer containing 0.4 M arginine. Eluate fractions were assayed by ELISA and fibrin lysis assay.
The fibrin lysis assay is based on the method of Binder et al. (J. Biol. Chem. 254: 1998, 1979). Ten ml of a bovine fibrinogen solution (3.0 mg/ml in 0.036 M sodium acetate pH 8.4, 0.036 M sodium barbital, 0.145 M NaCl, 10'4 M CaCl2, 0.02% NaN3) were added to 10 ml of a 1.5% solution of low melting temperature agarose in the same buffer at 40°C. To this solution was added 10 u1 of bovine thrombin (500 U/ml). The mixture was poured onto a Gelbond*agarose * ~ra~~P-mark support sheet (Marine Colloids) and allowed to cool. Wells were cut in the agarose and to the wells was added 10 u1 of the sample to be tested plus 10 u1 of phosphate-buffered saline containing 0.1~ bovine serum albumin. Results were 5 compared to a standard curve prepared using purified tPA.
The development of a clear halo around the well indicates the presence of biologically active plasminogen activator.
Example 4 - Loop-out of Finger and Growth Factor Domain 10 Coding Sequences The sequences encoding the finger and growth factor domains of t-PA were deleted from the cDNA by site-specific mutagenesis, essentially as described by Zoller et al., Manual for Advanced Techniques in Molecular Cloning 15 Course, Cold Spring Harbor Laboratory, 1983. Oligonucle-otides were synthesized on an Applied Biosystems 380-A DNA
synthesizer and purified by electrophoresis on denaturing gels.
Precise deletions were designed based on a 20 comparison of the genomic and cDNA t-PA sequences. cDNA
sequences corresponding to the exons encoding the finger and growth factor domains were deleted.
To prepare a template for mutagenesis of the t-PA
sequence, approximately 1 ug of Zem 99 was digested with 25 one unit each of BamHI and EcoRI. The DNA fragments were separated on a 1°s agarose gel and the 730 by BamHI-EcoRI
fragment was electro-eluted onto NA-45 DEAE membrane (Schleicher & Schuell) as directed by the supplier. The DNA was extracted with phenol-CHC13 and EtOH precipitated.
30 The purified fragment was then ligated to Bam HI + Eco RI
digested M13mp8 (replicative form) by incubating the two fragments in the presence of T4 DNA ligase for twelve hours at 12°C. The recombinant phage were transfected into competent E. coli JM101. Phage DNA was purified from plaques and sequenced by the dideoxy method to confirm the presence of the correct cDNA sequence.

Single-stranded M13 template DNA was prepared and site-specific mutagenesis was carried out using the oligonucleotide ZC490 (S~TAC CAA GTG ACC AGG GCC3~) as mutagenic primer. The universal M13 primer was used as second primer. Twenty pmoles phosphorylated mutagenic primer and 20 pmoles second primer were combined with one pmole single-stranded template in 10 u1 of 20 mM Tris, pH
7.5, 10 mM MgCl2, 50 mM NaCl, 1 mM DTT and incubated at 65°C for ten minutes, then five minutes at room temperature and placed on ice. Ten u1 of 20 mM Tris, pH 7.5, 10 mM
MgCl2, 2 mM ATP, 10 mM DTT containing 1 mM dNTPs, 2.5 units Klenow polymerase, and 3.5 units DNA ligase were added to the annealed DNA and the mixture incubated three hours at 15°C. The DNA was then transfected into competent E. coli JM101, and the cells plated on YT agar and incubated at 37°C. Plaques were transferred to nitrocellulose and pre-hybridized at the Tm-4°C of the mutagenic primer for one hour in 6x SSC, lOx Denhardt's and hybridized to 32p-labeled mutagenic primer at Tm-4°C in the same solution.
After three washes at Tm-4°C, filters were exposed to X-ray film overnight. Additional wash steps were performed at 5°C higher increments as necessary to identify mutant plaques. The mutated inserts were sequenced by the dideoxy method and a clone having the desired loop-out was selected.
When joined to the remainder of the t-PA coding sequence, this sequence encodes a protein with the amino terminus shown in Figure 4B.
Example 5 - Deletion of Finger and Growth Factor Sequences A second strategy for deleting the finger and growth factor sequences used as starting material the plasmid pDR1496, which comprises the t-PA coding sequence.
S. cerevisine strain E8-11C transformed with pDR1496 has been deposited with American Type Culture Collection under Accession No. 20728. To prepare a template for mutagenesis of the t-PA sequence, 1 g of pDR1496 was digested with 5 units each of Sph I and Xba I for two hours at 37°C. The .. 1341444 DNA was electrophoresed on a 0.7o agarose gel and a frag-ment of 2100 by was purified. This fragment was ligated to Sph I + Xba I digested M13tg130 (replicative form;
obtained from Amersham; Kieny et al., Gene 26:91, 1983) to construct M13tg130W. The recombinant phage were transfected into E. coli JM103 and single-stranded template DNA was prepared. Oligonucleotide-directed deletion mutagenesis was carried out using 20 pmoles phosphorylated mutagenic primer (sequence: 5'CGT GGC CCT GGT ATC TTG GTA
AG3') and 1 pmole template DNA in 20 mM Tris pH 7.5, 10 mM
MgCl2, 50 mM NaCl, 1 mM DTT at 65°C for 10 minutes. The mixture was then incubated for 5 minutes at room tempera-ture and placed on ice. Ten 1 of 20 mM Tris pH 7.5, 10 mM MgCl2, 2 mM ATP, 10 mM DTT containing 1 mM dNTPs, 2.5 units Klenow fragment and 3.5 units T4 DNA ligase were added and the annealed DNA mixture was incubated for 3 hours at 15°C. The DNA was transfected into E. coli JM103 and the cells were plated on YT agar and incubated at 37°C.
Plaques were screened as described in Example 4. A mutant sequence having the desired deletion of finger and growth factor sequences was designated clone #2600. The sequence of the encoded protein is shown in Figure 6.
Example 6 - Loop-out of Finger, Growth.Factor and Kringle 1 Coding Sequences Sequences encoding the finger, growth factor, and Kringle 1 sequences were deleted from the cloned cDNA in a manner analogous to the deletion described in Example 4.
The loop-out precisely joined codons for amino acids 4 and 176 of mature t-PA, resulting in a deletion of DNA
sequences corresponding to the exons encoding the finger, growth factor and Kringle 1 regions (Figure 4C).
The "'730 by BamHI-EcoRI t-PA fragment comprising the pre-pro and 5' end of the mature sequence from zem 99 was prepared and cloned in M13mp19 (replicative form).
Single-stranded template DNA was prepared and mutagenesis carried out using oligonucleotide ZC722 (5'ACT GTT TCC CAC

.. 1 341 44 4 TTG GTA3') and the M13 universal primer. Mutant plaques were screened and sequenced to identify clones having the desired mutation.
Example 7 - Deletion of Finer, Growth Factor and Kringle 1 Coding Sequences The finger, growth factor and Kringle 1 sequences were deleted in a second mutagenesis procedure using the single-stranded M13tg130W template described in Example 5.
Mutagenesis was carried out as described in Example 5 using an oligonucleotide primer having the sequence 5'GCA GTC ACT
GTT TCC TTG GTA AG3'. Mutant plaques were screened as previously described. A clone having the correct deletion was designated #2700. The sequence of the encoded protein is shown in Figure 7.
Example 8 - Construction of a Mutant t-PA Sequence Comprising Synthesized Oliqonucleotides The 5' portion of a DNA sequence encoding a mature t-PA with a deletion for the finger, growth factor, and Kringle 1 regions is constructed from synthesized oligonucleotides. The resultant fragment is then joined to the 3' cDNA and pre-pro sequences. The following oligonucleotides are used:
ZC636: 5'GAT CTT ACC AAG TGG GAA ACA GTG ACT GCT ACT TTG
GGA ATG GGT CAG3' ZC637: 5'TAG GCT GAC CCA TTC CCA AAG TAG CAG TCA CTG TTT
CCC ACT TGG TAA3' ZC638: 5'CCT ACC GTG GCA CGC ACA GCC TCA CCG AGT CGG GTG
CCT CCT GCC TCC CGT GG3' ZC639: 5'AAT TCC ACG GGA GGC AGG AGG CAC CCG ACT CGG TGA
GGC TGT GCG TGC CAC GG3' The four oligonucleotides were separately phosphorylated using T4 kinase. Oligonucleotides ZC636 and ZC637 are annealed under conditions described in Example 1 with an overlap of 44 bp. Oligonucleotides ZC638 and ZC639 are similarly annealed with an overlap of 49 bp.
To construct a mammalian cell expression vector containing the mutant t-PA sequence, Zem99 is digested to completion with Bgl II and partially digested with Eco RI
to remove the 600 by 5' portion of the t-PA gene. The fragment comprising the vector, t-PA pre-pro and 3' t-PA
sequences is gel purified and joined, in a three-part ligation, to the paired oligonucleotides. The amino-terminal sequence of the encoded protein is shown in Figure 4C.
Example 9 - Deletion of Growth Factor Coding Sequence A mutant sequence lacking the growth factor domain coding sequence was constructed by deletion mutagenesis using oligonucleotide ZC820 (S~GTA GCA CGT GGC CCT GGT TTT
GAC AGG CAC TGA GTG3~) and the M13 template described in Example 6. This deletion joined the codons for amino acids 49 and 88 of mature t-PA (Figure 4D). A correct clone was identified by sequencing and designated M13mp19-820.
Replicative form DNA was prepared and digested with PvuII
and HindIII. A 713 by fragment comprising the mutated t-PA
sequence was isolated and ligated to SmaI and HindIII cut pUCl8. The ligation mixture was transformed into E. coli HB101. A correct clone was identified and designated pUCl8-820.
Exam 1e 10 - Expression of Mutant t-PAs in Bacteria For expression in transformed bacterial cells, mutant t-PA sequences described above are removed from their respective M13 vectors by digesting replicative form DNA to completion with Bgl II and partially with Eco RI.
The mutant t-PA sequences are purified by standard procedures.
The bacterial expression vector pDR816 (described in Example 3) is digested with Bgl II and Xba I, and the fragment comprising the pICl9R, TAC promoter, t-PA pre-pro, and trpA terminator sequences is purified. The 3' t-PA
coding sequence is purified from an Eco RI + Xba I digest of pDR816.
The three fragments are joined in a triple 5 ligation to construct bacterial expression vectors containing the entire mutant t-PA sequences.
Similarly, the phosphorylated, annealed oligonu-cleotide pairs described in Example 8 are inserted in pDR816 which has been digested to completion with Bgl II
10 and partially digested with Eco RI to remove the 600 by 5' portion of the mature t-PA sequence.
The resultant vectors are used to transform E. coli JM105. Transformed cells are grown in M9 medium supplemented with 0.4o glucose and 0.2~ casamino acids.
15 Cells are harvested and lysed, and the cell debris was removed by centrifugation. The supernatants are assayed for t-PA activity by fibrin lysis assay and for protein production by Western blot assay.
20 Example 11 - Expression of Mutant t-PAs in Mammalian Cells The mutant sequences descr ibed above in Examples 4, 5, 6, 7 and 9 were inserted into mammalian cell expression vectors comprising the SV40 or MT-1 promoter and the DHFR selectable marker. The resultant expression 25 vectors were cotransfected into Tk- BHK cells, drug selection was applied, and transfected cell lines were selected, expanded for scale-up and protein production and characterization.
The mutant sequences were removed from clones 30 #2600 and #2700 as Bgl II-Apa I fragments and were inserted into Zem99 (Example 1). The resultant vectors were cotransfected with pSV2-DHFR DNA into Tk- BHK cells, as described in Example 3C.
Plasmid pUCl8-820 was digested with EcoRI and 35 BamHI and the mutant t-PA sequence (620 bp) was inserted into pDR3002. The resultant vector, designated p820, was transfected into Tk- BHK cells, as previously described.

.. ' 34~ 444 The mutant protein #2600 was purified on a 2.6 x 20 cm column of monoclonal antibodies immobilized on Sepharose~(Pharmacia). Media from transfected BHK cells were applied to the column at a rate of 200 ml/hr at 4°C.
The column was equilibrated with 0.1 M Tris-HC1 buffer pH7.5, containing 0.5 M NaCl and 20 KIU/ml of Aprotinin.
The column was washed with 1000 ml of the above buffer and the t-PA was eluted with the same buffer containing 5 M
KSCN. The t-PA fractions were concentrated by ultra-filtration to a volume of 5m1 and loaded on a column (2.6 x 90 cm) of Sephacryl S-200~T(Pharmacia) equilibrated with 50 mM Tris-HCl buffer pH7.5, containing 1.5 M KSCN and 0.5 M NaCl. The column was developed with the same buffer at a rate of 25 ml/hr. The t-PA fractions were concentrated by ultrafiltration and subjected to gel filtration on a column of Sephadex G-25~(PD10, Pharmacia) equilibrated with 1 M ammonium bicarbonate. The resultant purified protein was lyophilized in the presence of mannitol.
Media containing mutant t-PA 2700 were applied to a column (5 x 20 cm) of zinc-chelating Sepharose (Pharmacia) equilibrated with 50 mM Tris-HC1 buffer pH 7.5, containing 1 M NaCl and 20 KIU/ml of Aprotinin at a rate of 400 ml/hr. at 4°C. The column was washed with 2000 ml of the same buffer. The t-PA was eluted by gradually increasing the concentration of imidazole from 0 to 50 mM
in the same buffer. The t-PA fractions were directly applied to a column (2.6 x 20 cm) of concanavalin A coupled Sepharose 4B (Pharmacia) equilibrated with 10 mM sodium phosphate buffer pH 7.5, containing 1M NaCl at a rate of 30 ml/hr. The column was washed with the same buffer. The t-PA was eluted by gradually increasing the concentration of ~-methylamannoside from 0 to 0.4 M on 10 mM sodium phosphate buffer pH 7.5, containing 1M NaCl, 20 KIU/ml of Aprotinin and 2 M KSCN. The t-PA fractions were concentrated by ultra-filtration to about 5 ml and loaded on a column (2.6 x 90 cm) of Sephacryl S-200 equilibrated ~- !~Y'C7C.~~ ~f~~~c with 50 mM Tris-HC1 pH 7.5, containing 1.5 M KSCN and 1M
NaCI. The column was developed with the same buffer at a rate of 25 ml/hr and the t-PA fractions were pooled, concentrated, desalted and lyophilized as described above.
Plasma clearance of the 2600 and 2700 mutant t-PA
molecules was tested in rats. Male Sprague Dawley rats (230 g - 270 g body weight) were injected with 0.4 mg/kg body weight of 125z_labelled mutant t-PA or authentic recombinant t-PA purified from transfected BHK cells.
Injection was via the femoral vein. Blood samples (0.5 ml) were withdrawn from the jugular vein and assayed for t-PA
protein by a sandwich ELISA using affinity purified polyclonal. rabbit antibody. Results, shown in Table 4, indicate that the mutant proteins have a plasma half-life up to five times that of authentic t-PA.

Plasma Half-Life of Mutant t-PAs Protein ~ Phase (minutes) ~ Phase (minutes) Authentic t-PA 1.7 40 2700 9.6 ~ 84 Mutant proteins 3008 and 2600 were purified and assayed for in vivo half-life. Subconfluent 75 cm2 flasks containing cells producing mutant protein 3008, 2600 or authentic t-PA (control) were washed in 10 ml methionine-free Dulbecco's MEM containing 100 U/ml penicillin, 100~4g/ml streptomycin and 10 ~ 9/m1 aprotinin.
The cells were incubated in 10 ml of the same medium containing 1 mci 35S-methionine (New England Nuclear) for 20 hours at 37°C. The supernatant was centrifuged at 2000 rpm for 10 minutes and stored at -20°C. Non-radioactive t-PA's were produced from cell layers in 1200 cm2 trays (NUNC) maintained in Dulbecco's containing 100 U/ml penicillin, 100~1.(g/ml streptomycin and 100~Gtg/ml aprotinin.
Approximately 400 ml medium was harvested. NaCl and Tween 80* were added to the combined cell culture media to concentrations of 1M and 0.2~, respectively. The pH was adjusted to 7.5. After filtration on a 0.45~t m filter the mutants were purified using immunosorbent chromatography on monoclonal antibodies.
The purified t-PA mutants were found to be predominantly in the single-chain form. The specific activities were determined by the fibrin plate assay and are shown in Table 5.

Protein Specific Activity (IU/mg protein) authentic.t-PA 400,000 3008 590,000 2600 380,000 In vivo half-life of the mutant proteins 3008 and 2600 was determined. Female Wistar rats were anaesthetized and catheters were placed in the jugular vein for intravenous administration and in the carotid artery for sampling. Heparin (1 mg/kg body weight) was given i.v. ten minutes before administration of the test solution. Blood samples of approximately 2501 were taken before and 2, 3, 4, 6 and 8 minutes after the administration of 0.25-0.5 ml test solutions. Radioactivity in plasma was determined by liquid scint illation and elimination half-lives were calculated by linear regression using a one compartment model. Half-lives (mean or 3-5 experiments) were: native t-PA, 2.3 min.: 3008, 12 min. and 2600, 17 min.
* trade-mark Example 12 - Expression of Mutant t-PAs in Yeast For expression in yeast, the mutant t-PA
sequences are excised from the bacterial vectors described in Example 10 as Bgl II-Xba I fragments.
Plasmid pDR1496 is a yeast expression vector comprising the S. cerevisiae TPI promoter (Alber and Kawasaki, J. Mol. Appl. Genet. 1:419-434, 19$2), the S.
cerevisiae MF 1 signal sequence (Kurjan and Herskowitz, Cell 30: 933-943, 1982: and U.S. Patent 4,546,082), t-PA
coding sequence, and the S. cerevisiae TPI terminator (Alber and Kawasaki, ibid). S, cerevisiae strain E8-llc transformed with pDR1496 has been deposited with American Type Culture Collection under Accession No. 20728. The plasmid is isolated from the transformant by standard methods. The t-PA sequence is removed from pDR1496 by partial digestion with Bgl II and Xba I. The 12.2 kb vector fragment is purified and ligated to the Bgl II-Xba I
mutant t-PA sequence.
Expression vectors are then used to transform S.
cerevisiae strain E8-llc. Cells are grown at 30° in -leu medium supplemented with 2~ glucose and 0.1 M potassium hydrogen phthalate, pH 5.5. Cells are removed during log phase growth, harvested by centrifugation, disrupted, and the lysates assayed for t-PA activity.
From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

Claims (45)

1. A DNA construct containing a nucleotide sequence consisting essentially of a first region encoding fibrin-binding domain having at least one finger domain and/or at least one kringle domain, said first region being substantially free of an EGF domain, and a second region positioned downstream of said first region, said second region encoding a catalytic domain for the serine protease activity of tissue-type plasminogen activator (t-PA), said first region and said second region being derived from a nucleotide sequence encoding t-PA, the sequence (i) coding for a protein which has substantially the same biological activity as t-PA
and (ii) comprising less than the complete sequence for naturally occurring t-PA.

2. The DNA construct of claim 1 wherein the first region encoding the fibrin-binding domain encodes at least one kringle structure.

3. The DNA construct of claim 1 wherein the first region encoding the fibrin-binding domain encodes two kringle structures.

4. The DNA construct of claim 3 wherein the two kringle structures each have essentially the amino acid sequence of the K2 kringle structure of t-PA.

5. The DNA construct of claim 1 wherein the first region encoding the fibrin-binding domain encodes the K1 and K2 kringle structures of t-PA.

6. The DNA construct of claim 1 wherein the first region encoding the fibrin-binding domain encodes at least one finger domain.

7. The DNA construct of claim 1 wherein the catalytic domain encoded by the second region is essentially the serine protease domain of t-PA.

8. The DNA construct of claim 1 wherein the second region encoding the catalytic domain encodes essentially the amino acid sequence of t-PA extending from amino acid number 276 and continuing through amino acid number 527, according to Figure 1.

9. An expression vector capable of directing the expression of a protein having substantially the same biological activity as t-PA, said vector including a promoter, said promoter being operably linked to a nucleotide sequence comprising less than the complete sequence for naturally occurring t-PA and consisting essentially of a first region encoding a fibrin-binding domain having at least one finger domain and/or at least one kringle domain, said first region being substantially free of an EGF domain, and a second region positioned downstream of said first region, said second region encoding a catalytic domain for the serine protease activity of tissue-type plasminogen activator (t-PA), said first region and said second region being derived from a nucleotide sequence encoding t-PA, the sequence (i) coding for a protein which has substantially the same biological activity as t-PA
and (ii) comprising less than the complete sequence for naturally occurring t-PA.

10. The vector of claim 9 wherein the first region encoding the fibrin-binding domain encodes at least one kringle structure.

11. The vector of claim 9 wherein the first region encoding the fibrin-binding domain encodes two kringle structures.

12. The vector of claim 11 wherein the two kringle structures each have essentially the amino acid sequence of the K2 kringle structure of t-PA.

13. The vector of claim 11 wherein the first region encoding the fibrin-binding domain encodes the K1 and kringle structures of t-PA.

14. The vector of claim 9 wherein the first region encoding the fibrin-binding domain encodes at least one finger domain.

15. The vector of claim 9 wherein the catalytic domain encoded by the second region is essentially the serine protease domain of t-PA.

16. The vector of claim 9 wherein the second region encoding the catalytic domain encodes essentially the amino acid sequence of t-PA extending from amino acid number 276 and continuing through amino acid number 527, according to Figure 1.

17. Cells containing a DNA construct containing a promoter operably linked to a nucleotide sequence comprising less than the complete sequence for naturally occurring t-PA
and consisting essentially of a first region encoding a fibrin-binding domain having at least one finger domain and/or at least one kringle domain, said first region being substantially free of an EGF domain, and a second region positioned downstream of said first region, said second region encoding a catalytic domain for the serine protease activity of tissue-type plasminogen activator (t-PA), said first region and said second region being derived from a nucleotide sequence encoding t-PA, the sequence (i) coding for a protein which has substantially the same biological activity as t-PA
and (ii) comprising less than the complete sequence for naturally occurring t-PA.

18. The cells of claim 17 wherein the first region encoding a fibrin-binding domain encodes at least one kringle structure.

19. The cells of claim 17 wherein the first region encoding the fibrin-binding domain encodes two kringle structures.

20. The cells of claim 19 wherein the two kringle structures each have essentially the amino acid sequence of the K2 kringle structure of t-PA.

21. The cells of claim 17 wherein the first region encoding the fibrin-binding domain encodes the K1 and K2 kringle structures of t-PA.

22. The cells of claim 17 wherein the first region encoding the fibrin-binding domain encodes at least one finger domain.

23. The cells of claim 17 wherein the catalytic domain encoded by the second region is essentially the serine protease domain of t-PA.

24. The cells of claim 17 wherein the second region encoding the catalytic domain encodes essentially the amino acid sequence of t-PA extending from amino acid number 276 and continuing through amino acid number 527, according to Figure 1.

25. The cells of claim 17 wherein said cells are yeast cells.

26. The cells of claim 17 wherein said cells are bacterial cells.

27. The cells of claim 17 wherein said cells are mammalian cells.

28. A method of producing a protein which has substantially the same biological activity as t-PA, comprising:

inserting into cells a DNA construct containing a promoter operably linked to a nucleotide sequence comprising less than the complete sequence for naturally occurring t-PA
and consisting essentially of a first region encoding a fibrin-binding domain having at least one finger domain and/or at least one kringle domain, said first region being substantially free of an EGF domain, and a second region position downstream of said first region, said second region encoding a catalytic domain for the serine protease activity of tissue-type plasminogen activator (t-PA), said first region and said second region being derived from a nucleotide sequence encoding t-PA, the sequence (i) coding for a protein which has substantially the same biological activity as t-PA
and (ii) comprising less than the complete sequence for naturally occurring t-PA.

29. The method of claim 28 wherein the first region encoding the fibrin-binding domain encodes at least one kringle structure.

30. The method of claim 28 wherein the first region encoding the fibrin-binding domain encodes two kringle structures.

31. The method of claim 30 wherein the two kringle structures each have essentially the amino acid sequence of the K2 kringle structure of t-PA.

32. The method of claim 30 wherein the first region encoding the fibrin-binding. domain encodes the K1 and K2 kringle structures of t-PA.

33. The method of claim 28 wherein the first region encoding the fibrin-binding domain encodes at least one finger domain.

34. The method of claim 28 wherein the catalytic domain encoded by the second region is essentially the serine protease domain of t-PA.

35. The method of claim 28 wherein the second region encoding the catalytic domain encodes essentially the amino acid sequence of t-PA extending from amino acid number 276 and continuing through amino acid number 527, according to Figure 1.

36. The method of claim 28 wherein said cells are yeast cells.

37. The method of claim 28 wherein said cells are bacterial cells.

38. The method of claim 28 wherein said cells are mammalian cells.

39. A protein having substantially the same biological activity as t-PA, consisting essentially of (i) an amino terminal fibrin-binding domain comprising at least one finger domain and/or at least one kringle domain, said amino terminal fibrin-binding domain being substantially free of an EGF domain and (ii) a carboxyl terminal serine protease domain; said amino terminal fibrin-binding domain and said carboxyl terminal serine protease domain being derived from t-PA.

40. The protein of claim 39 wherein the fibrin-binding domain comprises at least one kringle structure.

41. The protein of claim 39 wherein the fibrin-binding domain comprises two kringle structures.

42. The protein of claim 41 wherein the two kringle structures each have essentially the amino acid sequence of the K2 kringle structure of t-PA.

43. The protein of claim 41 wherein the fibrin-binding domain comprises K1 and K2 kringle structures of t-PA.

44. The protein of claim 39 wherein the fibrin-binding domain comprises at least one finger domain.

45. The protein of claim 39 wherein the serine protease domain has essentially the amino acid sequence of the serine protease domain of t-PA.