WO1989010401A1 - Plasminogene activators with increased fibrin selectivity - Google Patents

Abstract

A modified plasminogen activator having greater fibrin selectivity than the unmodified plasminogen activator from which it is derived is provided. The modified plasminogen activator includes at least one of the following domains: a kringle that is more homologous with a kringle of plasminogen than is any kringle of the umodified plasminogen activator; a first hybrid t-PA kringle, the first hybrid kringle being a fusion of a portion of t-PA kringle 1 and a portion of t-PA kringle 2; or a second hybrid t-PA kringle, the second hybrid kringle being a fusion of a portion of t-PA kringle 2 and a portion of an scu-PA kringle.

Description

PLASMINOGEN ACTIVATORS WITH INCREASED FIBRIN SELECTIVITY

Fibrin Selectivity of Currently Available Plasminogen Activators

Control of the fibrinolytic system has been considered an important therapeutic goal because of the number of human disease states which could then be treated or prevented. For example, myocardial infarct, deep vein thrombosis, and pulmonary embolism all appear to involve undesirable fibrin clot formation. While many proteins interact in complex ways in fibrinolysis, some success in the treatment of myocardial infarct has been attained by use of various proteins termed plasminogen activators.

Recently, a second generation of plasminogen activators has replaced the original agents, urokinase and streptokinase, because these new agents offer relatively more fibrin selective plasminogen activation. In other words, these agents activate plasminogen in the vicinity of the fibrin clot but only minimally elsewhere in the general circulation. Thus, they spare other important circulating proteins such as alpha-2-antiplasmin and fibrinogen from degradation by the rather non-specific protease plasmin. Perhaps as a mechanism of control of thromolysis, both of these naturally-occurring plasminogen activators exhibit half-lives in the circulation of only two to five minutes.

These fibrin selective plasminogen activators have been extracted from normal and tumor tissues and are produced by certain cells in culture. There are two types of plasminogen activators with high fibrin selectivity - - a single-chain urinary plasminogen activator (scu-PA) also known as prourokinase, and tissue-type plasminogen activator (t-PA) - - which are easily distinguished immunologically. The single-chain urinary plasminogen activator is a trypsin-like serine protease 411 amino acid residues in length. In addition to the serine protease domain, the molecule contains two other domains: a cysteine-rich amino-terminal region of 45 amino acid residues which is homologous with epidermal growth factor (EGF) and thus is termed the EGF domain, and immediately adjacent to that domain is an 87 amino acid residue so-called "kringle" region which is highly homologous to the multiply disulfide bonded "kringle" domains of plasminogen and prothrombin. Hydrolysis of the lysine 158-isoleucine 159 bond by plasmin converts scu-PA into urokinase, a two-chain u-PA (tcu-PA) in which the chains are linked to one another by at least one disulfide bridge. Urokinase (plasmin-produced tcu-PA) is known to exist in either of two forms, a high molecular weight form (HMW urokinase) consisting of amino acid residues 1-411, and a low molecular weight form (LMW urokinase) consisting of amino acid residues 136-411 or 137-411. Cleavage by plasmin may play an important physiological role and has been demonstrated to produce a molecule with quite different properties as measured both in vitro and in vivo. For example, plasmin-produced tcu-PA is more active than scu-PA on small molecule substrates such as S2444 (N-pyro-Glu-Gly-p-nitroanilide; Kabi Vitrum, Sweden).

The properties of plasmin-produced tcu-PAs (both LMW and HMW urokinase), as measured in vivo, reveal them to be inferior to scu-PA with regard to efficiency of clot lysis (potency) and fibrin selectivity (Stump, D.C., Stassen, J.M., Demarsin, E., and Collen, D., 1987, Blood 69: 592-596; Collen et al., 1985, Circulation 72: 384-388). Current theory holds that fibrin-selectivity is a property of single-chain u-PA having an intact lysine 158-isoleucine 159 peptide bond. However, it is also clear that scu-PA is fibrin-selective whether it is full length, or truncated having an amino terminus at leucine 144 and lacking the EGF-like and kringle domains (Stump et al., 1987, supra; Stump, D.C., Lijnen, H.R., and Collen, D., 1986, J. Biol. Chem. 261: 17120-17126). Thus, the kringle domain of scu-PA appears to play no role in f ibrin selectivity; instead, the connecting region between the kringle and the serine protease domains seems important for this property. Consistent with this notion, plasmin-produced tcu-PA (ie., urokinase), which has suffered a proteolytic cleavage of the polypeptide chain in the connecting region, lacks fibrin-selectivity in its initiation of clot lysis in vitro and in vivo.

Tissue-type plasminogen activator (t-PA) is also a trypsin-like serine protease and is composed of a single polypeptide chain of 527 amino acids. The primary structure of t-PA shares a high degree of homology with that of scu-PA. For example, t-PA is converted by plasmin cleavage of the arginine 275-isoleucine 276 bond to a two-chain form also linked by at least one disulfide bond. However, current theory holds that there are no substantial differences between the activities of two-chain t-PA and single-chain t-PA. T-PA is also composed of several domains, including not only the EGF-like, the kringle, and serine protease domains analogous to those of u-PA, but also a second kringle and a so-called "finger" domain resembling the fibrin-binding region of fibronectin. The second kringle domain and the "finger" domain are both considered to contribute fibrin affinity to t-PA. However, recent evidence suggests that the finger domain is principally responsible for the fibrin affinity of t-PA, and the second kringle domain alone may be responsible for the fibrin-selectivity of t-PA action (Larsen et al., 1988., J. Biol. Chem. 263: 1023-1029).

Plasminogen, the substrate of both scu-PA and t-PA, is another multi-domain trypsin-like serine protease involved in thrombolysis. It is composed of two types of domains - - namely, kringle domains, of which there are five, and a serine protease domain. Recently, human plasminogen cDNA was cloned and sequenced (Forsgren, M., Raden, B., Israelsson, M., Larsson, K., and Lars-Olof, H., 1987, FEBS Lett. 213: 254-260). Previous studies have demonstrated that kringle 1 and kringle 4 of plasminogen bind lysine and the lysine analog epsilon-aminocaproic acid tightly (Vali, Z. and Patthy, L., 1982, J. Biol. Chem. 257: 2104-2110). According to current theory, the binding of these small molecules occurs in the same part of the protein which binds to fibrin, and lysine or epsilon-aminocaproic acid binding is an accurate predictor of fibrin affinity. For this reason, plasminogen kringles 1 and 4 are thought to contribute fibrin affinity to plasminogen.

While the amino acid sequences of all kringles are quite homologous, certain amino acid residues are thought to be important for high affinity binding to epsilon-aminocaproic acid and presumably to fibrin as well (Ramesh, V., Petros, A.M., Llinas, M., Tulinsky, A., and Park, C.H., 1987, J. Mol. Biol. 198: 481-498). Accordingly, the different kringles found on different proteins are known to differ in their affinity for fibrin. As mentioned previously the scu-PA kringle and the first kringle of t-PA exhibit no measurable affinity for fibrin, while the second kringle of t-PA and the first and fourth kringles of plasminogen exhibit high affinity for lysine-Sepharose and fibrin.

Clearly, both t-PA and scu-PA exhibit fibrin selective plasminogen activation in vivo, but they appear to do so by different mechanisms. For example, binding studies demonstrate an affinity of t-PA for fibrin, and kinetic studies indicate that interaction with soluble fibrin fragments dramatically decreases the K_m of t-PA for plasminogen, thus rendering it a more efficient plasminogen activator. In contrast, scu-PA exhibits little or no affinity for fibrin and soluble fibrin fragments have only modest affects on its kinetic parameters of plasminogen activation, and yet it activates plasminogen with a fibrin selectivity virtually indistinguishable from that of t-PA, sparing alpha-2-antiplasmin and fibrinogen in vivo. Finally, cleavage to the two chain form eliminates the fibrin selectivity of scu-PA action but has no significant effect on t-PA fibrin selectivity. Thus, the fibrin-selectivity of scu-PA activity appears to be due to a mechanism different from that of t-PA. This difference in mechanisms has been offerred as an explanation for the apparent synergism between t-PA and scu-PA during clot dissolution (Pannell, R., Black, J., and Gurewich, V., 1988, J. Clin. Invest. 81: 853-859; Collen, D., Stassen, J., Stump, D.C., and Vestraete, M., 1986, Circulation 14: 838-842).

Several models have been proposed to account for scu-PA fibrin selectivity. For example, one model suggests that scu-PA binds more efficiently to plasminogen which is bound to fibrin (Peltz, S.W., Hardt, T.A., and Mangel, W.F., 1982, Biochemistry 21: 2798-2804; Pannell R. and Gurewich, V., 1986, Blood 67: 1215-1223). By this model, plasminogen is the mediator for scu-PA fibrin selectivity and it is scu-PA's resistance to interaction with circulating plasminogen-activator inhibitors which permits it to reach the fibrin clot. According to another model, plasminogen or plasminogen bound to fibrin is both a substrate and an allosteric effector which stabilizes a more active form of scu-PA (Ellis, V., Scully, M.F., and Kakkar, V.V., 1987, J. Biol. Chem. 262: 14998-15003). In any case, it is clear that scu-PA is less active on small molecule substrates than tcu-PA, but it appears to be sufficiently active on the large substrate plasminogen to initiate plasminogen activation.

The Need for Increased Fibrin Selectivity While both t-PA and scu-PA exhibit fibrin selectivity in their activation of plasminogen, the discrimination is not complete and significant amounts of plasmin may be generated in the general circulation when the high doses necessary for rapid clearance of thrombi are administered (Verstraete et al., 1985, Lancet 2: 965-969; Acar et at., 1987, Seminars in Thromb. and Hemost. 13: 186-200). Even modest levels of plasmin are sufficient to diminish circulating alpha-2-antiplasmin and fibrinogen levels measureably, thereby compromising the patient's ability to form clots which might be needed in the period immediately following treatment with the plasminogen activator. What is needed is a plasminogen activator which exhibits better discrimination between plasminogen bound to, or in the vicinity of, the fibrin clot versus plasminogen in the general circulation. If such an agent were available, then larger doses could be administered without fear of breakdown of the patient ' s thrombogenic system, thereby permitting more rapid dissolution of the thrombus.

Fibrin-selectivity in plasminogen activation is understood to mean that plasminogen activation occurs primarily at the site of the fibrin clot so that little active plasmin is distributed in the general circulation where it can damage other thrombogenic proteins. Thus, fibrin-selectivity could arise by either or both of at least two mechanisms: (a) a plasminogen activator may activate plasminogen more efficiently at the surface of a fibrin clot, or (b) a plasminogen activator may activate plasminogen less efficiently in the general circulation. Clearly, the second mechanism would be expected to play a role in a plasminogen activator which bound to fibrin and thus spent less time in the general circulation. The t-PA molecule appears to have features required for both mechanisms to operate, while the scu-PA molecule appears to have only a feature for the first mechanism. Recently, several groups have attempted to provide u-PA with kringle domains from other proteins. For example, both Nakayama et al. (1986, Thrombosis & Haemostasis 56: 364-370) and Robbins and Tanaka (1986, Biochemistry 25: 3603-3611) have built hybrids of the heavy chain of human plasminogen (containing all five kringle domains) with the serine protease domain of LMW urokinase by fusing the two chains by means of disulfide bonds between existing cysteine residues. While these authors describe modest fibrin affinity and fibrin selectivity for their products, both incorporated the plasmin-cleaved version of u-PA (ie, LMW urokinase) as the source of the serine protease domain. This LMW form of u-PA exhibits severe depletion of circulating fibrinogen and antiplasmin in both in vitro and in vivo clot lysis experiments. Perhaps because of this fact, the protein fusions described by Nakayama et al. (1986, supra) and by Robbins and Tanaka (1986, supra) exhibited only modest fibrin selectivity. By contrast, the kringle-fusions to u-PA described in the present invention were made at the cDNA level and represent fusions to the truncated single-chain form of u-PA, the form which exhibits excellent fibrin selectivity in its plasminogen activation. As a result, the plasminogen kringle-scu-PA hybrids of this invention provide increased fibrin selectivity beyond that demonstrated for the molecules made by Nakayama et al. (1986, supra) and Robbins and Tanaka (1986, supra).

Finally, Nelles et al. (1987, J. Biol. Chem. 262: 10855-10862) have built a fusion of the finger, EGF, kringle 1, and kringle 2 domains of t-PA with the connecting region and serine protease domain of truncated scu-PA in an attempt to produce a scu-PA with increased fibrin selectivity. However, the result is a modified scu-PA with better fibrin selectivity than plasmin-produced tcu-PA but poorer fibrin selectivity than scu-PA itself or t-PA.

SUMMARY OF THE INVENTION A modified plasminogen activator having greater fibrin selectivity than the unmodified plasminogen activator from which it is derived is provided. The modified plasminogen activator includes at least one of the following domains: a kringle that is more homologous with a kringle of plasminogen than is any kringle of the unmodified plasminogen activator; a first hybrid t-PA kringle, the first hybrid kringle being a fusion of a portion of t-PA kringle 1 and a portion of t-PA kringle 2; or a second hybrid t-PA kringle, the second hybrid kringle being a fusion of a portion of t-PA kringle 2 and a portion of a scu-PA kringle. According to several embodiments of the invention, scu-PA is modified to provide increased fibrin selectivity in its activation of plasminogen as compared to unmodified scu-PA. We have discovered that by providing kringle domains from plasminogen or t-PA, or by modifying the scu-PA kringle, we have created a scu-PA molecule which exhibits more fibrin selectivity than previously known plasminogen activators. For example, we have found that fusion of plasminogen kringles 1 and/or 4 to a truncated scu-PA lacking its own EGF-like and kringle domains, or replacement of most of the scu-PA kringle with most of kringles 1 or 4 of plasminogen or with kringle 2 of t-PA results in a plasminogen activator with increased fibrin selectivity. In addition, replacement of the scu-PA EGF-like and kringle domains with the t-PA EGF-like and finger domains together with a t-PA kringle 1-kringle 2 fusion domain also provides a more fibrin-specific plasminogen activator. While the precise sites of fusion are not absolutely critical, preferred embodiments of this invention is shown in TABLE 1.

In another example, we have discovered that alteration of a few amino acid residues in the scu-PA kringle to make them more similar to the corresponding residues in the plasminogen kringles results in a scu-PA with increased fibrin selectivity in its activation of plasminogen. Comparison of the amino acid sequence of the scu-PA kringle (Holmes, W.E., Pennica, D., Blaber, M., Rey, M.W., Guenzler, W.A., Steffens, G.J., and Heyneker, H.L., 1985, Biotechnology 3: 923-929) with the sequences of the plasminogen kringles 1 and 4 (Forsgren et al., 1987, supra) reveals a number of differences despite the fact that all kringles are highly conserved. Specifically, we have changed leucine-80 to histidine, glutamine-81 to arginine, glutamine-82 to proline, threonine-83 to arginine, arginine-108 to aspartate, leucine-122 to arginine, and valine-123 to tryptophan (see TABLE 1). Other changes may be made but these suffice to demonstrate improved fibrin selectivity of the plasminogen activator and the feasibility of further improvements. The location of these amino acid residues in the folded protein has not been determined at this time, because the x-ray crystallographic structure is only known for the kringle of prothrombin. However, judging from the homology between prothrombin, plasminogen, and scu-PA kringles, it appears that many of the amino acid residues which we have altered in the scu-PA kringle may play a role in binding of the kringle to lysine or epsilon-aminocaproic acid. Naturally-occurring scu-PA and t-PA both exhibit very short half-lives in the circulatory system. This may serve as a mechanism for termination of plasminogen activation under normal conditions of maintaining the hemostatic condition; however, for purposes of thrombolytic therapy, such a short half-life requires administration of large doses just to keep circulating levels sufficiently high to accomplish the goal. Therefore, a fibrinspecific plasminogen activator with a longer circulating half-life would be useful for therapeutic purposes.

Recent published results demonstrate that modifications of the t-PA molecule affect its circulating half-life. Both Larsen et al. (1988, supra) and Lau, D., Kuzma, G., Wei, C., Livingston, D.J., and Hsiung, N. (1987, Bio/Technology 5: 953-958) have shown that removal of glycosylation sites prolong circulating half-life, and Larsen et al. (1988, supra) have shown that removal of the finger domain and the EGF-like domain extend the half-life. Surprisingly, we have found that the alterations to the scu-PA kringle described above result in scu-PA molecules with extended circulating half-lives. Thus, the same thrombolytic goals can be accomplished with significantly lower doses of plasminogen activator, while at the same time preserving circulating fibrinogen and alpha-2-antiplasmin levels.

The molecules of the present invention are produced by using recombinant DNA- methodology to provide new cDNA genes encoding new plasminogen activators. In one embodiment of the present invention, the first and/or fourth kringles from plasminogen or a hybrid of t-PA kringle l and kringle 2 together with the t-PA finger and EGF-like domains are added to a truncated scu-PA lacking its own kringle. Truncated scu-PA is a relatively fibrin selective plasminogen activator (Stump et al., 1986, supra), but the addition of a plasminogen kringle domain provides increased fibrin selectivity. In a second embodiment of the present invention, most of either plasminogen kringle 1 or 4 or t-PA kringle 2 replaces most of the scu-PA kringle to provide a scu-PA with increased fibrin selectivity in its activity. In a third embodiment of this invention, the scu-PA kringle itself is modified at between four and seven amino acid residues resulting in a scu-PA molecule with improved fibrin selectivity in its activation of plasminogen.

The production of both t-PA and scu-PA by recombinant DNA techniques has produced sufficient amounts of materials to permit tests of their effectiveness in the treatment of pulmonary embolism, deep vein thrombosis, myocardial infarct, and strokes. New fusions of plasminogen and scu-PA cDNAs and modifications of the scu-PA cDNA yield plasminogen activators with unexpected advantages in thrombolytic therapy, and these new activators are the subject of the present invention. The advantages include more efficient plasminogen activation with increased fibrin selectivity over that of the parent activator and longer circulating half-lives in vivo. Thus, new therapeutic agents useful for thrombolysis have been discovered.

It is an object of the present invention to provide more efficient plasminogen activators which are capable of acting with greater fibrin selectivity than previously available. Thus, higher doses may be used for more rapid dissolution of unwanted clots without fear of a general thrombogenic deterioration.

It is a further object of this invention to provide plasminogen activators with longer circulating half-lives than previously available.

Another object of the invention is to provide novel DNA sequences encoding the foregoing plasminogen activators.

Another object of the invention is to provide cloned vectors in transformed host cells capable of expressing the foregoing plasminogen activators.

Another object of this invention is to provide a modified urinary plasminogen activator in accordance with the preceding objects having altered amino acid residues in the kringle domain.

Still another object of this invention is to provide a modified plasminogen activator in accordance with the first and second objects with most of the first or fourth kringle from human plasminogen.

A further object of this invention is to provide a modified scu-PA in accordance with the first and second objects with a new kringle domain from human plasminogen or from human t-PA replacing the kringle and EGF domains of scu-PA.

A further object of the present invention is to provide means and methods of producing the new plasminogen activators of the preceding objects.

Yet another object of the invention is to provide improved methods and products for treating certain medical conditions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic drawing of plasmid pCGM16;

FIG. 2 is a schematic drawing of plasmid pCGM38; FIGS. 3A and 3B show the nucleotide sequences of synthetic oligodeoxy-nucleotides encoding plasminogen kringles 1 and 4;

FIG. 4 is a schematic drawing of plasmids pCGE242 and pCGE241 which contain the plasminogen kringle DNA of FIGS. 3A and 3B respectively cloned between the HindIII and Bglll sites of pSV2-gpt;

FIG. 5 is a schematic drawing of plasmids pCGM61 and pCGM74 which contain the plasminogen kringle fusions to truncated scu-PA;

FIG. 6 is a schematic drawing of plasmid pCGM34 which contains a portion of t-PA encoding DNA fused to DNA coding for scu-PA amino acid residues 132-411; and

FIG. 7 shows the nucleotide sequences of a synthetic oligodeoxy-nucleotide encoding a modified scu-PA kringle, with lines denoting the individual oligodeoxynucleotides synthesized, annealed, and ligated for the construction of plasmid pCGM99.

DESCRIPTION OF PREFERRED EMBODIMENTS In the specific examples of this invention, sc-uPA was obtained from human kidney cells (Kohno, T., Hopper, P., Lillquist, J.S., Suddith, R.L., Greenlee, R. and Moir, D.T., 1984, Biotechnology 2: 628-634). Sc-uPA is defined as that plasminogen activator having a single polypeptide chain and exhibiting plasminogen activator activity expressed in terms of CTA (Committee on Thrombolytic Agents) units determined by means of a fibrin plate assay (Brakman, P., 1967, "Fibrinolysis: a standardized fibrin plate method and a fibrinolytic assay of plasminogen", Scheltema and Holkema, Amsterdam, pp. 1-124) and having an amino acid and encoding nucleotide sequence substantially as shown in Fig. 1 of Holmes, W.E., et al. (1985; supra). The cells were grown to confluency in Dulbecco's Modified Eagles medium supplemented with 5% heat-inactivated NuSerum (Collaborative Research, Inc., Bedford, Massachusetts). Confluent cells were harvested by centrifugation after treatment with 0.25 percent trypsin for 15 minutes at 37°C and were frozen in liquid nitrogen.

The poly (A) RNA was isolated according to the method of Maniatis, T., Fritsch, E. F. and Sambrook, J. (1982, "Molecular Cloning. A Laboratory Manual", Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, pp. 191-198). Briefly, 5 grams of frozen cells were lysed with NP-40 and the nuclei were removed by ultra-centrifugation. The cytoplasmic fraction was treated with proteinase K, and the protein was removed by repeated phenol: chloroform:isoamyl alcohol extractions. The total cytoplasmic RNA was recovered by precipitation with ethanol with a yield of about 16 milligrams. The poly (A) RNA was isolated by oligo (dT) cellulose chromatography with a yield of about 200 micrograms. The intactness of the isolated poly (A) RNA was verified by in vitro translation in a rabbit reticulocyte lysate system. Conversion of poly (A) mRNA to double stranded cDNA was by standard procedures (see Wickens, M. P., Buell, G. N. and Schimke, R. T., 1978, J. Biol. Chem. 253: 2483-2495) using oligo (dT) as the primer of the first strand synthesis by reverse transcriptase, DNA polymerase I to synthesize the second strand, nuclease SI treatment to insure that the ends are flush and chromatography over Biogel A150M (Bio-Rad, Richmond, California) for size selection.

The size-selected double-stranded cDNA was ligated first to Xbal synthetic oligonucleotide linkers (Collaborative Research, Inc., Bedford, MA) and then into a single-stranded f1 phage vector (Zinder, N.D. and Boeke, J.D., 1982, Gene 19: 1-10; Bowden, D.W., Mao, J., Gill, T., Hsiao, K., Lillquist, J.S., Testa, D. and Vovis, G.F., 1984, Gene 27: 87-99). The cDNA library of recombinant fl phage was transfected into E. coli by standard methods to yield fl phage plaques suitable for preparing nitrocellulose filter lifts and probing (see below).

For screening of the phage cDNA library, fifteen oligodeoxynucleotide probes, each 15 or 20 bases in length and homologous to portions of the scu-PA coding region (Holmes, W.E., et al., 1985; supra), were synthesized by the automated phosphoramidite procedure (automated DNA synthesizer, Applied

Biosystems, Foster City, California). The probes were labeled with 32P by means of polynucleotide kinase and gamma- 32P-ATP and isolated from the residual label by means of a polyacrylamide gel. The resultant labeled probes were mixed and used to probe nitrocellulose plaque lifts from the library essentially according to the method of Benton, W. D. and Davis, R. W. (1977, Science 196: 180-182). The use of multiple probes insured that unexpected occasional polymorphisms within the gene or poor hybridization by certain probes due to unpredictable structure or sequence context problems would not prevent identification of the clones containing prourokinase. In this manner, approximately 50,000 clones were screened for prourokinase sequences. One of the colonies showed hybridization with eleven of the probes. Dideoxy-sequencing and restriction analysis of the latter clone showed that the clone contains a 2.2 kilobase insert and starts in the middle of the signal sequence. Further screening using a probe complementary to a region of the signal sequence revealed a second clone which harbors the missing part of the signal sequence.

These two overlapping inserts, each bounded by Xbal restriction endonuclease sites, were subcloned into another vector by fusing them at a unique Bglll restriction site which is common to both inserts. Restriction analysis was carried out on the resultant subclone pCGE194, and the latter cDNA was shown to encode full length scu-PA. FIG. 1 depicts the introduction of this full-length cDNA encoding scu-PA into the eukaryotic expression vector pSV2-gpt (Mulligen, R. and Berg, P., 1981, Proc. Nat'l. Acad. Sci. USA 78: 2072-2076). Briefly, the full length scu-PA cDNA was isolated from plasmid pCGE194 after restriction with Xbal followed by filling-out the ends with E. coli DNA polymerase I (Klenow fragment) and all four deoxynucleotide triphosphates. DNA of plasmid pSV2-gpt was cut with restriction endonucleases HindIII and Bglll, and the ends were filled out with E. coli DNA polymerase (Klenow fragment) and all four deoxynucleotide triphosphates. The approximately 2.3 kb scu-PA encoding fragment from pCGEl94 was ligated to the approximately 5.25 kb vector fragment from pSV2-gpt, and the ligation mixture was used to transform competent E. coli cells to ampicillin resistance. Cells carrying the desired plasmid pCGM16 were identified by restriction endonuclease digestion analysis of plasmid DNA isolated from them. Plasmid pCGM16 carrys the full length scu-PA encoding cDNA inserted in pSV2-gpt with the Xbal site but not the HindIII site regenerated at the SV40 promoter end of pSV2-gpt and the Xbal and Bglll sites lost at the other end of the insert. Restriction endonuclease sites also are shown throughout the figures. Relative positions of restriction endonuclease cleavage sites are depicted by straight lines and are labeled in italics. Sites which were present in the parental vectors but lost upon ligation are shown in parentheses.

After transformation of mammalian cells and integration into the chromosomes, this vector is capable of replication in mammalian cells and directing the expression of the added scu-PA cDNA from the SV40 early promoter. The secretion of sc-uPA from Chinese hamster ovary (CHO)cells which had been transformed with pCGM16 was verified by analysis of conditioned medium in the fibrin plate assay (Brakman, 1967, supra) and by assaying it for amidolytic activity (Kohno et al., 1984, supra).

In order to facilitate construction of expression/secretion vectors for genes encoding hybrid plasminogen activators, a plasmid was constructed having a unique Bglll restriction endonuclease access site at the end of the scu-PA secretion signal coding region. By using this vector, designated pCGM38 (FIG 2), new DNA fragments encoding useful portions of proteins with fibrin affinity may be inserted between DNA sequences coding for the scu-PA secretion signal and portions of the scu-PA structural gene. In order to construct pCGM38, plasmid pCGM16 was cleaved with BssHII, which cuts within the alanine codon at position -15 in the secretion signal region (see FIG. 1, and also Holmes, W.E. et al., 1985; supra), and then with Bglll, which cuts in the codon for arg88 (FIG. 1), to generate the desired 7.1 kilobase BssHII-Bglll fragment, and a 306 basepair BssHII-Bglll fragment which was discarded. A synthetic oligodeoxynucleotide pair described below was ligated between the BssHII and the Bglll sites.

The two synthetic deoxyoligonucleotides, each 44 nucleotides in length, were synthesized as described previously. They replace the portion of the signal sequence coding region which was deleted above and provide a new Bglll site at the end of the signal sequence codons. The sequences of the synthetic nucleotides are as follows:

5' CG CGC CTG CTT CTC TGC GTA CTA GTC GTG AGC GAC TCC AAG GGA 3' 3' G GAC GAA GAG ACG CAT GAT CAG CAC TCG CTG AGG TTC CCT CTA G 5'

These synthetic deoxyoligonucleotides were constructed so as to have a BssHII "sticky" end complementary to the BssHII cleaved end of the 7.1 kilobase fragment and a Bglll "sticky" end complementary to the Bglll cleaved end of the 7.1 kilobase fragment. In addition, an Spel (ACTAGT) restriction site is found within the complementary oligodeoxynucleotide sequences. The oligodeoxynucleotides were annealed, and then ligated to the 7.1 kilobase fragment, described above, for 5 hours at 15°C to create a new plasmid vector designated pCGM38 (FIG. 2) which was identified in an E. coli HB101 clone following introduction of the ligation mix into E. coli by standard methods of bacterial transformation. Digestion of the plasmid with various restriction endonucleases and determination of the DNA sequence of the scu-PA encoding region of the plasmid confirmed that the ligation of the fragments was correct since the complete signal sequence of scu-PA and the Bglll and BssHII restriction sites were recreated, and a new unique Spel restriction site is found.

Examples 1 and 2

As examples of this invention, the first or fourth kringle from human plasminogen was fused to human scu-PA lacking its own EGF and kringle domains. This was accomplished in these examples at the DNA level by building the fusion gene for expression in living cells.

Lines denote the individual oligodeoxynucleotides synthesized, annealed, and ligated for the construction of plasmids pCGE242 (FIG. 3A) and pCGE241 (FIG. 3B). Schematic diagrams of the entire plasmids pCGE242 and pCGE241 are shown in FIG. 4. DNA sequences encoding the amino acid sequences of the first (Plg-K1) and fourth (Plg-K4) kringles of human plasminogen were derived by synthesis of overlapping oligodeoxynucleotide fragments corresponding to the published amino acid sequence of the respective regions of plasminogen (Forsgren, M., Raden, B., Israelsson, M., Larsson, K, and Lars-Olof, H., 1987, FEBS Lett. 213: 254-260). Using standard methods known in the art, sixteen such synthetic oligodeoxynucleotides (FIG. 3A) were ligated together and cloned between the HindIII and Bglll sites of the mammalian expression vector pSV2-gpt (Mulligan and Berg, supra) to yield a DNA sequence initiating at a Bglll site and encoding the first kringle of plasminogen (amino acid residues 79-162; our numbering of sequence of Forsgren et al., supra, beginning with the glutamate encoded by nucleotides 133-135 as amino acid number 1). The resulting ligation mixture was used to transform competent E. coli cells, and a transformant carrying the desired plasmid, pCGE242 (FIG. 4), containing the Plg-K1 inserted between the HindiII and Bglll sites of pSV2-gpt, was isolated.

Similarly, sixteen synthetic oligodeoxynucleotides (FIG. 3B) were ligated together between the HindIII and Bglll sites of pSV2-gpt to yield a DNA sequence encoding the fourth plasminogen kringle (amino acid residues 354-435; numbering from sequence of Forsgren, et al., as described above) cloned in a suitable vector. An E. coli strain carrying the desired plasmid pCGE241 (FIG. 4) was isolated. Shown in Fig. 3 are the preferred sequences of the oligodeoxynucleotides used to make pCGE242 and pCGE241; however, many other sequences would suffice provided that they encode the same amino acid sequence, that they are cohesive in the proper order, and that they provide the necessary restriction endonuclease sites for the remainder of the constructions described below.

The Plg-K1-scu-PA and Plg-K4-scu-PA fusions were constructed by fusing the plasminogen kringle nucleotide sequences built as described above to the scu-PA nucleotide sequence in vector pCGM38 (FIG. 2). The fusions were performed at the common Fspl and Bglll sites. Plasmid DNA from pCGE242 (FIG. 4) and from pCGE241 (FIG. 4) was cut with restriction enzymes Fspl, which cuts in the synthetic kringle region, and Bglll which cuts at the junction of the kringle with pSV2-gpt, and the DNA fragments encoding Plg-K1 and Plg-K2 were isolated. Plasmid pCGM38 (FIG. 2) was cut with restriction endonucleases Bglll which cuts uniquely in the scu-PA cDNA, and with Kpnl which cuts in the vector sequences. The resulting approximately 5.17 kb fragment carrying most of the pSV2-gpt vector DNA, the SV40 early promoter and the scu-PA secretion signal coding DNA was isolated. A second aliquot of pCGM38 (FIG. 2) DNA was cut with restriction endonucleases Fspl, which cuts between codons 131 and 132 in the scu-PA cDNA, and with Kpnl, which cuts in the pSV2-gpt vector DNA. The resulting approximately 2 kb DNA fragment carrying scu-PA cDNA (codons 88-411) fused to a portion of pSV2-gpt vector DNA was isolated. In two separate reactions, the DNA encoding each of the plasminogen kringles (K1 and K4) were ligated to the two fragments derived from pCGM38. Both ligation mixtures were used to transform competent E. coli cells, and transformant strains carrying the desired plasmids were identified. These plasmids carry either Plg-K1 (pCGE242) or Plg-K4 (pCGE241) encoding DNA fused to truncated scu-PA(132-411) cDNA in the mammalian expression vector pSV2-gpt, but the kringles are out of reading frame with the scu-PA DNA.

The kringle-scu-PA junctions were placed in translational-reading-frame by restriction with BglII, blunting the ends by digestion with S1 nuclease, restriction with SnaBI, and religation. The resulting plasmids pCGM74 (FIG. 5) and pCGM61 (FIG. 5) carry functional transcriptional units encoding Plg-K1-scu-PA and Plg-K4-scu-PA fusions, respectively, both transcribed from the mammalian SV40 promoter and containing the scu-PA secretion signal for efficient secretion of the resulting protein (see Summary in Table 1).

Example 3 In addition, as another example of this invention, both Plg-K1 and Plg-K4 encoding sequences were fused to the scu-PA encoding cDNA at the codon for amino acid ala-132 to yield a scu-PA containing both plasminogen kringles known to bind to fibrin. DNA of plasmid pCGM74 (FIG. 5) was cut partially with restriction endonuclease Fspl (the desired site is at the Plg-K1 DNA scu-PA cDNA junction) and completely with Kpnl , which cuts in the pSV2-gpt vector sequences, and an approximately 5.43 kb fragment carrying most of the pSV2-gpt vector sequences together with the SV40 early promoter, the scu-PA secretion signal coding DNA, and the Plg-K1 encoding DNA was isolated. An approximately 2.14 kb fragment encoding most of Plg-K4 fused to scu-PA cDNA at codon 132 plus a portion of vector sequence was isolated following restriction endonuclease digestion of plasmid pCGM61 (FIG. 5) with SacI and Kpnl. These two isolated DNA fragments are ligated together with a third fragment carrying the remaining portion of Plg-K4 to yield plasmid pCGM75. The third fragment, an 89 bp piece of DNA, was derived from plasmid pCGE241 (FIG. 4) by digestion with Snal and SacI. Plasmid pCGM75 carrys the SV40 promoted gene for the scu-PA secretion signal fusion to Plg-K1(101-162)-Plg-K4(375-438)-scu-PA(132-411), all contained in vector pSV2-gpt (Mulligan and Berg, 1981, supra; see summary Table I below)

Example 4 In another example of this invention, the major part of plasminogen kringle 1 (Plg-K1; amino acid residues 102-162) replaces most of the kringle of scu-PA (amino acid residues 68-131) to yield a hybrid PA with improved fibrin selectivity. DNA of plasmid pCGM16 (FIHut to completion with restriction endonuclease Xbal at the SV40 promoter/scu-PA junction and partially with Ncol (desired site is at scu-PA amino acid codon 66), and an approximately 320 bp fragment containing scu-PA codons 1-66 was isolated. Plasmid pCGM74 (FIG. 5) was cut with restriction endonucleases Xbal at the SV40 promoter/scu-PA cDNA junction and SacI in Plg-K1 at codon 110 (see FIG. 3A), and the large fragment containing the vector sequences, the promoter and most of the DNA encoding Plg-K1 fused to scu-PA cDNA was isolated. Both fragments were ligated together in the presence of the following synthetic oligodeoxynucleotide pair which provides Ncol and SacI sites and a bridging sequence between scu-PA and Plg-K1 coding for amino acid residue 67 of scu-PA and residues 102-109 of Plg-K1.

67 102 103 104 105 106 107 108 109 met gly ile thr cys gln lys trp ser 5' C ATG GGT ATC ACT TGT CAA AAA TGG AGC T 3' 3' CCA TAG TGA ACA GTT TTT ACC 5'

The resulting ligation mixture was transformed into competent E. coli cells, and a transformant was identified carrying the desired plasmid pCGM107, containing the scu-PA(SS)-scu-PA(1-67)-Plg-K1 (102-162)-scu-PA(132-411) coding sequence.

Example 5 In a related additional example of this invention, the major part of plasminogen kringle 4 (Plg-K4; amino acid residues 376-435) replaces most of the kringle of scu-PA (amino acid residues 68-131) to yield another hybrid PA with improved fibrin selectivity. A strategy identical to that described above in Example 4 was used, except that the second DNA fragment encoded most of Plg-K4 fused to scu-PA(132-411) and derived from plasmid pCGM61 (FIG. 5) instead of pCGM74. Also, the synthetic oligodeoxy-nucleotides are of a different sequence, and that is shown below.

67 376 377 378 379 380 381 382 383 met gly lys lys cys gin ser typ ser 5' C ATG GGT AAG AAG TAC CAG TCG TGG AGC T 3' 3' CCA TTC TTC ACG GTC AGC ACC 5'

Both DNA fragments were ligated together in the presence of the synthetic oligodeoxynucleotides, and the resulting ligation mixture was used to transform competent E. coli cells. A transformant was identified which contained the desired plasmid pCGM109 encoding scu-PA(SS)-scu-PA(1-67)-Plg-K4(376-435)-SCU-PA(132-411).

Example 6

In another related example of this invention, most of the second kringle of t-PA (t-PA-K2; amino acid residues 198-261) replaces most of the scu-PA kringle (amino acid residues 68-131). As a first step in constructing this modified scu-PA cDNA, a fusion of t-PA cDNA (encoding amino acid residues 1-261) to scu-PA cDNA (encoding amino acid residues 132-411) was made as follows.

Plasmid pCGM16 (FIG. 1) was cut with restriction endonucleases Xbal at the promoter/scu-PA junction and Kpnl in the vector sequences, and a 5.1 kb fragment containing most of the vector sequences and the SV40 promoter region was isolated. Another aliquot of pCGM16 was cut completely with restriction endonuclease Kpnl and partially with Fspl (desired site is between codons 131 and 132), and a 1.99 kb fragment carrying cDNA encoding scu-PA from codons 132 to 411 and a portion of vector sequences was isolated. These two isolated fragments were ligated together in the presence of two synthetic oligodeoxynucleotides having a sequence encoding t-PA from codons 255 through 261 as shown below:

255 256 257 258 259 260 261 cys asp val pro ser cys 5' CTAG AC TGT GAC GTC CCC AGC TGC 3' 3" TG ACA CTG CAG GGG TCG ACG 5'

Competent E. coli cells were transformed with the ligated DNA, and a clone carrying plasmid pKHlll containing t-PA (codons 255-261) fused to scu-PA (codons 132-411) inserted adjacent to the SV40 promoter in pSV2-gpt was obtained.

Plasmid pCGM33 was used as the source for the remainder of the t-PA coding sequence. This plasmid carrys cDNA encoding full-length t-PA flanked by Xbal DNA linkers and located adjacent to the SV40 promoter in pSV2-gpt. The t-PA cDNA was obtained from RNA isolated from Bowes melanoma cells by standard methods of cDNA cloning. The t-PA sequence contained in pCGM33 is essentially the same as that described by others (Pennica, D., Holmes, W.E., Kohr, W. J., et al., 1983, Nature 301: 214-221). Plasmid pCGM33 was cut with restriction endonucleases Xbal at the promoter/t-PA junction and with Seal at codon 255 in the t-PA coding sequence, and an approximately 0.88 kb DNA fragment encoding amino acid residues 1-255 of t-PA was isolated. Plasmid pKHlll was cut with restriction endonuclease Xbal at the SV40-t-PA junction, blunted with S1 nuclease, and cut further with endonuclease Kpnl in the vector sequences. An approximately 2.01 kb fragment encoding t-PA (codons 255-261) fused, to scu-PA (codons 132-411) and pSV2-gpt vector sequences was isolated. These two isolated fragments were ligated to each other and to pSV2-gpt vector sequences (5.1 kb fragment) derived from Xbal and Kpnl restriction of pCGM16 as described above. The resulting plasmid pCGM34 carrying t-PA sequences (codons 1-261) fused to scu-PA sequences (codons 132-411) under control of the SV40 promoter in vector pSV2-gpt was isolated from an E. coli clone which had been transformed with that plasmid in the ligation mixture.

A gene encoding a hybrid scu-PA having most of its kringle replaced with kringle 2 of t-PA was constructed from plasmids pCGM34 and pCGM16 as follows. Plasmid pCGM16 was cut to completion with restriction endonuclease Kpnl and partially with Ncol (desired site is at codon 66), and an approximately 5.43 kb DNA fragment carrying most of the vector sequences, the SV40 promoter, and scu-PA cDNA (codons 1-66) was isolated. Plasmid pCGM34 was cut completely with Kpnl and partially with EcoRI (desired site is at t-PA codon 205), and a 2.16 kb DNA fragment carrying t-PA cDNA (codons 205-261) fused to scu-PA cDNA (codons 132-411) together with a small portion of vector sequence was isolated. These two isolated fragments were ligated to each other and to two synthetic oligodeoxy-nucleotides carrying codon 67 of scu-PA fused to codons 198-205 of t-PA as shown below.

67 198 199 200 201 202 203 204 205 met gly ala ser cys leu pro trp asn 5' C ATG GGT GCC AGC TGT CTA CCT TGG 3' 3' CCA CGG TCG ACA GAT GGA ACC TAA A 5'

The ligation mixture was used to transform competent E. coli cells, and a clone containing the desired plasmid pCGM105 carrying SV40-promoted scu-PA(codons 1-67)-t-PA(codons 198-261)-scu-PA(codons 132-411) was identified (see Summary in Table 1). Plasmid DNA was prepared and used to transform mammalian cells so that they produce the recombinant protein. Example 7

In another example of this invention, a hybrid kringle consisting of a portion of t-PA-K1 and a portion of t-PA-K2 was fused to truncated scu-PA(132-411), thereby replacing the scu-PA kringle. First, plasmid pCGM34 was cut with restriction endonuclease Narl at t-PA codon 110 and at Kpnl in the vector. An approximately 5.29 kb DNA fragment containing most of the pSV2-gpt vector, the promoter, and t-PA codons 1-110 was isolated. A second aliquot of pCGM34 was cut partially with EcoRI (desired site is at t-PA codon 205) and completely with Kpnl in the vector, and an approximately 2.2 kb DNA fragment containing t-PA codons 205-261 fused to scu-PA codons 132-411 was isolated.

These two DNA fragments were ligated together in the presence of a synthetic oligodeoxynucleotide pair having a Narl cohesive end and an EcoRI cohesive end and encoding amino acid residue 111 of t-PA followed by 200-205 of t-PA. The sequence of the oligodeoxynucleotides is shown below.

111 200 201 202 203 204 205 ala ser cys leu pro trp asn

5' C GCC AGC TGT CTA CCT TGG 3'

3' GG TCG ACA GAT GGA ACC TTA A 5' The resulting ligation mixture was used to transform competent E. coli cells, and a transformant containing the desired plasmid pCGM103 was identified. Plasmid pCGM103 encodes t-PA(SS)-t-PA-K1(1-111)-t-PA-K2-(200-261)-scu-PA(132-411) transcribed from the SV40 early promoter and carried on vector pSV2-gpt (see Summary in Table 1).

Examples 8 & 9 In two additional examples of this invention, specific amino acid residues of the scu-PA kringle were altered to yield a modified scu-PA with increased fibrin selectivity. First, threonine-83, arginine-108, leucine-122, and valine-123 were changed to arginine, aspartate, arginine, and tyrosine, respectively. This was accomplished by replacing the scu-PA codons for amino acid residues 67-131 in plasmid pCGM16 with synthetic oligodeoxy-nucleotides incorporating new codons for the altered residues described above. Briefly, plasmid pCGM16 (FIG. 1) was cut partially with restriction endonuclease Ncol (desired site at codon 66) and completely with Kpnl, and the resulting approximately 5.4 kb DNA fragment representing the most of the vector sequences together with the promoter and scu-PA codons 1-66 was isolated. Another aliquot of pCGM16 plasmid DNA was cut partially with Fspl (the site between codons 131 and 132) and completely with Kpnl to provide an approximately 2.0 kb fragment containing scu-PA codons 132-411 and the rest of the vector sequences. These two fragments were ligated together along with a set of synthetic oligodeoxynucleotides (see FIG. 7 below) which join the Ncol site with the Fspl site and provide the scu-PA coding region for codons 67-131 containing the altered amino acid codonsdescribed above (ie., T83R, R108D, L122R, and V123Y, where the first letter denotes the original amino acid at the numbered position and the last letter represents the new amino acid at that position). The resulting ligation mixture was used to transform competent E. coli cells, and transformants were found which contain the desired plasmid pCGM99 (see Summary in Table 1).

A second modified scu-PA cDNA containing three additional altered amino acid codons was produced by modification of plasmid pCGM99 as follows. DNA from plasmid pCGM99 was digested with restriction endonucleases AlwNI and Bglll (see FIG. 7), and the large fragment was isolated. It contains the entire plasmid except for the small sequence encoding the twelve amino acids between the AlwNI and Bglll sites. Two synthetic oligodeoxynucleotides of sequence shown below provide a modified set of codons for the twelve amino acids such that leucine-80 is changed to histidine, glutamine-81 is changed to arginine, and glutamine-82 is changed to proline .

77 78 79 80 81 82 83 84 85 86 87 88 89 ala thr val his arg pro arg tyr hit ala his trg ser 5' CT GTG CAC AGA CCA AGG TAC CAT GCA CAC A 3'

3' GA TGA CAC GTG TCT GGT TCC ATG GTA CGT GTG TCT AG

Alw NI Bgl II

These oligodeoxynucleotides were annealed and ligated together between the AlwNI and Bglll sites of the isolated large fragment from pCGM99. The resulting ligation mixture was used to transform competent E. coli HB101 cells to ampicillin resistance, and a clone carrying the desired plasmid pCGMlOl (see Summary in Table 1) was isolated.

Example 10 In another example of this invention, plasminogen kringles 1 and 4 are fused to t-PA residues 262 through 529 to create a new plasminogen activator molecule with greater fibrin specificity and longer in vivo half-life than naturallyoccurring t-PA or scu-PA. A gene for the new hybrid plasminogen activator is constructed as follows for expression in mammalian cells. First, plasmid . pCGM75 (see Example 3, supra) is cut completely with restriction endonuclease Kpnl in the pSV2-gpt vector sequence and partially with Fspl (desired site is at the Plg-K4-scu-PA junction), and an approximately 5.7 kb DNA fragment carrying most of the vector sequence together with all of Plg-Kl and Plg-K4 is isolated. Second, DNA encoding t-PA (codons 280-529) is isolated by cutting plasmid pCGM33 (see Example 6, supra) with restriction endonucleases Kpnl in the pSV2-gpt sequences and with Banll in the codon for amino acid residue 280. An approximately 1.69 kb DNA fragment is isolated. Finally, both of these isolated fragments are ligated together in the presence of two annealed synthetic oligodeoxy-nucleotides of the following sequence.

262 270 279 280 ser thr cys gly leu arg gin tyr ser gin pro gin phe arg lle lys gly gly leu5' TCC ACC TGC GGC CTG AGA CAG TAC AGC CAG CCT CAG TTT CGC ATC AAA GGA GGG CT 3'3' AGG TGG ACG CCG GAC TCT GTC ATG TCG GTC GGA GTC AAA GCT TAG TTT CCT C 5'

Banll end

These oligodeoxynucleotides span the gap between the Fspl and Kpnl sites of the isolated DNA fragments from plasmids pCGM75 and pCGM33, and they provide the coding sequence for amino acid residues 262 through 280 of t-PA. The ligation mixture is used to transform competent E. coli cells to ampicillin resistance, and a transformed clone is identified carrying the desired plasmid pCGMlll. Plasmid pCGM111 is pSV2-gpt carrying a fusion of scu-PA secretion signal-Plg-K1(70-162)-Plg-K4(354-438)-t-PA(262-529) to the SV40 early promoter (see summary in Table 1).

Example 11

Demonstration of fibrin selectivity during clot lysis in vitro initiated by mutant plasminogen activators. Plasmid pCGM74 is used in conjunction with a second plasmid supplying a selectable marker to co-transform Chinese hamster ovary cell line DG44 (Urlaub, G., Kas, E., Carothers, A.M., and Chasin, L.A., 1983, Cell 33: 405-412) which completely lacks the diploid DHFR locus. The co-transformed selectable plasmid is either pSV2-DHFR (Subramani, S., Mulligan, R., and Berg, P., 1981, Mol. Cell. Biol. 1: 854-864) or pdhfr2.9 (Crouse, G.F., MeEwan, R.N., and Pearson, M.L., 1983, Mol. Cell. Biol. 3: 257-266). Co-transformation is accomplished by means of the calcium precipitation procedure of Graham, F.L. and van der Eb (1973, Virology 52: 456-467). Transformants are selected in Ham's F12 medium supplemented with 10% fetal bovine serum, but lacking glycine, hypoxanthine and thymidine.

To increase the plasminogen activator expression level, resulting co-transformants are carried through a step-wise gene amplification procedure which involves challenging the cells with increasing concentrations of methotrexate (MTX) (Kaufman, R. and Sharp, P., 1982, J. Mol. Biol. 159: 601-621). Suitable high level plasminogen activator producing clones are identified by amidolytic assay (Kohno et al, 1984, supra) of cultures of cells which are resistant to high levels of MTX. Clones are grown in medium consisting of 1:1 DME:F12 which lacks thymidine and hypoxanthine (Gibco, Grand Island, NY) but is supplemented with 10% fetal bovine serum. Following growth in T-flasks and roller bottles, conditioned medium containing between 2 and 20 ug/ml of plasminogen activator is harvested.

The mutant plasminogen activators are purified by means of the following procedure. Conditioned medium is filtered through a 0.2 micron pleated capsule filter (Gelman, Ann Arbor, MI) to remove particulate material, titrated to pH7, if necessary, and applied to a column of anti-scu-PA-Sepharose equilibrated with a buffer consisting of 10 mM sodium phosphate (pH7.4), 0.14 M sodium chloride, 10 KlU/ml Aprotinin (Sigma, St. Louis, MO).

Anti-scu-PA-Sepharose was made by coupling scu-PA-specific monoclonal antibody to CNBr-activated Sepharose (purchased from Pharmacia, Piscataway, NJ). The monoclonal antibody was prepared by fusion of scu-PA immunized mouse spleen cells with antibody-secreting myeloma cells according to published procedures (Oi, V. & Herzenberg, L., 1980 in Selected Methods in Cellular Immunology, pp. 351-372. W.H. Freeman & Co., San Francisco). Analysis of the antibody showed that it is about 30 times more specific for scu-PA than tcu-PA. The coupling procedure was done essentially according to standard methods (see Pharmacia publication - Affinity Chromatography, Principles and Methods; also see Axen, R., Porath, J., & Ernback, S., 1967 Nature 214:1302-1304, and March, S.C., Parikh, I., & Cuatrecasas, P., 1974, Analytical Biochemistry 60:149-152) except that 20 mg of purified monoclonal antibody was coupled per ml of gel. Briefly, the procedure for coupling was as follows. A specified mass of dried cyanogen bromide-activated Sepharose was measured and swollen in 1 mM hydrochloric acid solution (200ml/g). The swollen gel was washed with coupling buffer (0.1 M sodium bicarbonate, pH 8.3, 0.5 M NaCl, 0.8 mM CaCl₂, 0.5 mM MgCl,) to equilibrate it before adding protein. The monoclonal antibody was dissolved in the same coupling buffer so that approximately 20 mg would couple per ml of gel. The protein solution was added to the cyanogen bromide-activated Sepharose suspension and then mixed end-over-end at room temperature for 3 hrs. The amount of monoclonal antibody added at the beginning of the reaction was estimated by absorbancy at 280 nm. After the reaction, the coupled Sepharose was filtered to remove the coupling buffer containing unreacted protein (estimated again by absorbancy at 280 nm). Remaining active groups on the cyanogen bromide-activated Sepharose were blocked next by adding a solution of 1M ethanolamine (in coupling buffer) and mixing the suspension at room temperature for 2 hours. The coupled Sepharose was filtered again and washed with 0.1 M acetate, pH 4, 1 M NaCl followed by 10 mM Na phosphate, pH 7.2, 1 M NaCl for a total of three times. The coupled resin was washed again with 3 M sodium thiocyanate dissolved in 10 mM Na phosphate, pH 7.2, 0.14 M NaCl, and then washed finally with 10 mM Na phosphate, pH 7.2, 0.14 M NaCl. The resin was then used as an affinity matrix for purification of scu-PA related proteins. After washing the column with the equilibration buffer, it is developed with 50 mM glycine (pH2) to elute the protein which had been bound to the antibody-Sepharose. The eluate is diluted with one-fourth volume of 100 mM sodium acetate (pH5.3), 1 M sodium chloride, adjusted to a pH of 5.3, and applied to a column of p-aminobenzamidine-Sepharose (Collaborative Research, Inc., Bedford, MA) equilibrated with a buffer consisting of 20 mM sodium acetate (pH5.3), 0.1 M sodium chloride. Protein flowing through the column is monitored by absorbancy at 280 nm, collected and concentrated to about 1 mg/ml for further analyses described below.

The purified mutant plasminogen activator is examined in the in vitro fibrin clot lysis model of Gurewich, V., Pannell, R., Louie, S., Kelley, P., Suddith, R.L., and Greenlee, R. (1983, J. Clin. Invest. 73: 1731-1739). Briefly, aliquots of citrated human plasma containing 1.5 uCi IBRIN

( 125I-labeled fibrm; Amersham Corp, Arlington

Heights, IL) are supplemented with 20 mM calcium chloride and 10 ul thromboplastin (T-0263; Sigma, St. Louis, MO) and incubated in 5 mm(i.d.) glass tubes for four hours at 37°C. The clots are removed from the glass tubes and bathed in 2.5 ml pooled human plasma containing the mutant plasminogen activators or purified wild-type scu-PA as a control at concentrations of about 10 to 50 nM. Potency is judged by the time and dose dependence of clot lysis; fibrin specificity is determined by measurement of residual plasma fibrinogen and alpha-2-antiplasmin levels by means of standard assays (Clauss, A., 1957, Acta. Hematol. 17: 237-246; Vermylen, C., deVreker, R.A., and Verstraete, M., 1963, Clinica Chemica Acta 8: 418-424; Edy, J., DeCook, F., and Collen, D., 1976, Thromb. Res. 8: 513-518). The mutant plasminogen activator exhibits potency equal to that of wild-type scu-PA, but shows increased fibrin specificity over that of wild-type scu-PA.

Example 12 Demonstration of fibrin selectivity during clot lysis in vitro initiated by a mutant plasminogen activator. Example 12 is identical to Example 11 except plasmid pCGM61 is used in place of plasmid pCGM74.

Example 13 Demonstration of fibrin selectivity during clot lysis in vitro initiated by a mutant plasminogen activator. Example 13 is identical to Example 11 except plasmid pCGM75 is used in place of plasmid pCGM74.

Example 14 Demonstration of fibrin selectivity during clot lysis in vitro initiated by a mutant plasminogen activator. Example 14 is identical to Example 11 except plasmid pCGM107 is used in place of plasmid pCGM74.

Example 15 Demonstration of fibrin selectivity during clot lysis in vitro initiated by a mutant plasminogen activator. Example 15 is identical to Example 11 except plasmid pCGM109 is used in place of plasmid pCGM74.

Example 16 Demonstration of fibrin selectivity during clot lysis in vitro initiated by a mutant plasminogen activator. Example 16 is identical to Example 11 except plasmid pCGM105 is used in place of plasmid pCGM74.

Example 17 Demonstration of fibrin selectivity during clot lysis in vitro initiated by a mutant plasminogen activator. Example 17 is identical to Example 11 except plasmid pCGM103 is used in place of plasmid pCGM74.

Example 18 Demonstration of fibrin selectivity during clot lysis in vitro initiated by a mutant plasminogen activator. Example 18 is identical to Example 11 except plasmid pCGM99 is used in place of plasmid pCGM74.

Example 19 Demonstration of fibrin selectivity during clot lysis in vitro initiated by a mutant plasminogen activator. Example 19 is identical to Example 11 except plasmid pCGM101 is used in place of plasmid pCGM74.

Example 20 Demonstration of fibrin selectivity during clot lysis in vitro initiated by a mutant plasminogen activator. Example 20 is identical to Example 11 except plasmid pCGM111 is used in place of plasmid pCGM74.

Example 21 Demonstration of fibrin specificity during clot lysis in vivo. The mutant plasminogen activator of Example 11 is also examined for potency and specificity during clot lysis in the rabbit jugular venous thrombosis model according to the protocol of Collen, D., Stassn, J.M., and Verstraete, M. (1983, J. Clin. Invest. 71: 368-376). Potency is judged by the time and dose dependence of clot lysis; fibrin specificity is determined by measurement of residual plasma fibrinogen and alpha-2-antiplasmin levels by means of standard assays (Clauss, A., 1957, Acta. Hematol. 17: 237-246; Vermylen, C, deVreker, R.A., and Verstraete, M., 1963, Clinica Chemica Acta 8: 418-424; Edy, J., DeCook, F., and Collen, D., 1976, Thromb. Res. 8: 513-518). The mutant plasminogen activators exhibit potency approximately equal to that of wild-type scu-PA, but show increased fibrin specificity over that of wild-type scu-PA.

Example 22 Demonstration of fibrin specif icity during clot lysis in vivo. Example 22 is identical to Example 21 except that the purified plasminogen activator is encoded by plasmid pCGM61 and is derived from Example 12.

Example 23 Demonstration of fibrin specificity during clot lysis in vivo. Example 23 is identical to Example 21 except that the purified plasminogen activator is encoded by plasmid pCGM75 and is derived from Example 13.

Example 24 Demonstration of fibrin specificity during clot lysis in vivo. Example 24 is identical to Example 21 except that the purified plasminogen activator is encoded by plasmid pCGM107 and is derived from Example 14.

Example 25 Demonstration of fibrin specificity during clot lysis in vivo. Example 25 is identical to Example 21 except that the purified plasminogen activator is encoded by plasmid pCGM109 and is derived from Example 15.

Example 26 Demonstration of fibrin specificity during clot lysis in vivo. Example 26 is identical to Example 21 except that the purified plasminogen activator is encoded by plasmid pCGM105 and is derived from Example 16.

Example 27 Demonstration of fibrin specificity during clot lysis in vivo. Example 27 is identical to Example 21 except that the purified plasminogen activator is encoded by plasmid pCGM103 and is derived from Example 17.

Example 28 Demonstration of fibrin specificity during clot lysis in vivo. Example 28 is identical to Example 21 except that the purified plasminogen activator is encoded by plasmid pCGM99 and is derived from Example 18.

Example 29 Demonstration of fibrin specificity during clot lysis in vivo. Example 29 is identical to Example 21 except that the purified plasminogen activator is encoded by plasmid pCGM101 and is derived from Example 19.

Example 30 Demonstration of fibrin specificity during clot lysis in vivo. Example 30 is identical to Example 21 except that the purified plasminogen activator is encoded by plasmid pCGM111 and is derived from Example 20.

Example 31 Demonstration of a longer half-life for a mutant plasminogen activator in the circulation of a rabbit. The purified mutant plasminogen activator of Example 11 is also analyzed for rate of disappearance from the circulation of a rabbit. During and post infusion of the plasminogen activator into the rabbit described in Example 21, aliquots of rabbit serum are withdrawn, processed to obtain platelet-poor plasma, and analyzed for the level of plasminogen activator according to the ELISA described by Stump, D.C., Kieckens, L. DeCook, F., and Collen, D.(1987, J.Pharmacol. Exp. Ther. 242: 245-250). Samples are analyzed immediately prior to infusion, at 1, 2, 3, and 4 hrs of infusion, and at 1, 3, 5, 7, 9, 15, 30 and 60 minutes after infusion.

In addition, the rate of disappearance of the mutant plasminogen activator following bolus injection into the circulation of a rabbit is also determined as follows. The purified mutant plasminogen activator of Example 11 is injected as a single bolus into the circulation of a rabbit to achieve an initial concentration of about 1 microgram per ml. Immediately prior to bolus injection, and at times of 1, 3, 5, 7, 9, 15, 30 and 60 minutes after bolus injection, samples are taken, platelet-poor plasma prepared, and the amount of plasminogen activator antigen present in the plasma quantitated by ELISA described by Stump et al. (1987, supra). Plasma plasminogen activator levels are plotted as a function of time and the harmacokinetic parameters are calculated by the method of Welling (1986, in "Pharmacokinetic Processes and Mathematics," ACS Monograph 185, Washington, D .C.).

The mutant plasminogen activator encoded by plasmid pCGM74 and purified as in Example 11 is found to exhibit a longer circulating half-life than natural scu-PA.

Example 32 Demonstration of a longer half-life for a. mutant plasminogen activator in the circulation of a rabbit. Example 32 is identical to Example 31 except that the purified plasminogen activator is encoded by plasmid pCGM61 and is derived from Example 12.

Example 33 Demonstration of a longer half-life for a mutant plasminogen activator in the circulation of a rabbit. Example 33 is identical to Example 31 except that the purified plasminogen activator is encoded by plasmid pCGM75 and is derived from Example 13. Example 34 Demonstration of a longer half-life for a mutant plasminogen activator in the circulation of a rabbit. Example 34 is identical to Example 31 except that the purified plasminogen activator is encoded by plasmid pCGM107 and is derived from Example 14.

Example 35 Demonstration of a longer half-life for a mutant plasminogen activator in the circulation of a rabbit. Example 35 is identical to Example 31 except that the purified plasminogen activator is encoded by plasmid pCGM109 and is derived from Example 15.

Example 36 Demonstration of a longer half-life for a mutant plasminogen activator in the circulation of a rabbit. Example 36 is identical to Example 31 except that the purified plasminogen activator is encoded by plasmid pCGM105 and is derived from Example 16.

Example 37 Demonstration of a longer half-life for a mutant plasminogen activator in the circulation of a rabbit. Example 37 is identical to Example 31 except that the purified plasminogen activator is encoded by plasmid pCGM103 and is derived from Example 17.

Example 38 Demonstration of a longer half-life for a mutant plasminogen activator in the circulation of a rabbit. Example 38 is identical to Example 31 except that the purified plasminogen activator is encoded by plasmid pCGM99 and is derived from Example 18.

Example 39 Demonstration of a_ longer half-life for a mutant plasminogen activator in the circulation of a rabbit. Example 39 is identical to Example 31 except that the purified plasminogen activator is encoded by plasmid pCGM101 and is derived from Example 19.

Example 40 Demonstration of a longer half-life for a_ mutant plasminogen activator in the circulation of a rabbit. Example 40 is identical to Example 31 except that the purified plasminogen activator is encoded by plasmid pCGMlll and is derived from Example 20.

TABLE 1 Summary of Examples

• Plasmid Plasminogen Activator

1 pCGM74 scu-PA(SS)-Plg-K1(79-162)-scu-PA(132-411)

2 pCGM61 scu-PA(SS)-PIg-K4(354-435)-scu-PA(132-411)

3 pCGM75 scu-PA(SS)-Plg-K1(79-162)-PlgK4(354-438)-scu-PA(132-411)

4 pCGM107 scu-PA(SS)-SCU-PA(1-67)-Plg-K1(102-162)-scu-PA(132-411)

5 pCGS109 scu-PA(SS)-SCU-PA(1-67)-Plg-K4(376-435)-scu-PA(132-411)

6 pCGM105 scu-PA(SS)-scu-PA(1-67)-t-PA(198-261)-seu-PA(132-411)

7 pCGM103 t-PA(SS)-t-PA(1-111)-t-PA(200-261)-scu-PA(132-411)

8 pCGM99 scu-PA(SS)-SCU-PA(T83R; R108D; L122R; V123Y)

9 pCGM101 scu-PA(SS)-SCU-PA(L80H; 081R; 082P; T83R; R108D; L122R; V123Y)

10 pCGM111 scu-PA(SS)-Plg-K1(79-162)-Plg-K4(354-438)-t-PA(262-529)

Notes: Numbering of amino acid codons and residues is according to the following published sequences. Scu-PA: Holmes, W.E., Pennica, D., Blaber, M., et al., 1985, Biotechnology 3: 923-929, t-PA: Pennica, D., Holmes, W.E., Kohr, W.J., et al., 1983, Nature 301: 214-221; Pig: Forsgren, M. , Raden, B., Israelsson, M., et al., 1987, FEBS Lett 213: 254-260 (with glutamate encoded by nucleotides 133-135 denoted as codon #1). The abbreviation "SS" refers to the DNA encoding the secretion signal sequences of either scu-PA (nucleotides -20 through -1 of sequence of Holmes et al. (supra) or t-PA (nucleotides -35 through -1 of sequence of Pennica et al. supra). Abbreviations for amino acid substitutions in Examples 8 and 9 are by the single letter code; the first letter designates the original amino acid at the numbered position, and the letter following the number designates the new amino acid at that position.

It is intended that all matter contained in the above description or shown in the accompanying drawings shall be interpreted in an illustrative and not limiting sense.

What is claimed is:

Claims

Claims

1. A modified plasminogen activator having greater fibrin selectivity than the unmodified plasminogen activator from which it is derived, said modified plasminogen activator including at least one of the following domains: kringle that is more homologous with a kringle of plasminogen than is any kringle of the unmodified plasminogen activator; a first hybrid t-PA kringle, said first hybrid kringle being a fusion of a portion of t-PA kringle 1 and a portion of t-PA kringle 2; or a second hybrid t-PA kringle, said second hybrid kringle being a fusion of a portion of t-PA kringle 2 and a portion of a scu-PA kringle.

2. A modified plasminogen activator as characterized in claim 1 and having most of plasminogen kringle 1 (amino acid residues 102-162).

3. A modified plasminogen activator as characterized in claim 1 and having most of plasminogen kringle 4 (amino acid residues 376-435).

4. A modified plasminogen activator as characterized in claim 1 and having most of t-PA kringle 2 ( amino acid residues 198-261.

5. A modified plasminogen activator as characterized in claim 1 wherein said first hybrid t-PA kringle has approximately amino acid residues 1-111 of t-PA kringle 1 and amino acid residues 200-261 of t-PA kringle 2.

6. A modified plasminogen activator as claimed in claim 1 further characterized by alterations to the kringle of the unmodified plasminogen activator which alterations provide increased fibrin selectivity.

7. A modified plasminogen activator as claimed in claim 1 further characterized by alterations to the kringle of the unmodified plasminogen activator which alterations provide a longer half-life to the modified plasminogen activator.

8. A modified form of single-chain urinary plasminogen activator (scu-PA) having alterations in a kringle domain which provide increased fibrin selectivity in its activation of plasminogen as compared to unmodified scu-PA.

9. A modified form of single-chain urinary plasminogen activator in accordance with claim 8 in which amino acid residues of the scu-PA kringle are altered to provide increased fibrin selectivity.

10. A modified form of single-chain urinary plasminogen activator in accordance with claim 9 in which amino acid residues thr-83, arg-108, leu-122, and val-123 are changed to arginine, aspartate, arginine, and tryptophan, respectively.

11. A modified form of single-chain urinary plasminogen activator in accordance with claim 9 in which amino acid residues leu-80, gln-81, gln-82, thr-83, arg-108, leu-122, and val-123, are changed to histidine, arginine, proline, arginine, aspartate, arginine, and tryptophan, respectively.

12. A modified form of single-chain urinary plasminogen activator in accordance with claim 8 in which a portion of kringle l or kringle 4 of plasminogen or a portion of kringle 2 of t-PA is fused to a portion of the kringle of scu-PA to provide a plasminogen activator with a hybrid kringle and increased fibrin selectivity.

13. A modified form of single-chain urinary plasminogen activator in accordance with claim 12 in which most of plasminogen kringle 1 (amino acid residues 102 to 162) is fused to scu-PA in place of most of the scu-PA kringle (amino acid residues 68 to 131).

14. A modified form of single-chain urinary plasminogen activator in accordance with claim 12 in which most of plasminogen kringle 4 (amino acid residues 376 to 435) is fused to scu-PA in place of most of the scu-PA kringle (amino acid residues 68 to 131).

15. A modified form of single-chain urinary plasminogen activator in accordance with claim 12 in which most of plasminogen kringle 2 (amino acid residues 198 through 261) is fused to scu-PA in place of most of the scu-PA kringle (amino acid residues 68 to 131).

16. A modified form of single-chain urinary plasminogen activator in accordance with claim 8 in which kringle 1 or kringle 4 of plasminogen or both kringle 1 and kringle 4 of plasminogen substitute for the EGF-like domain and kringle of scu-PA.

17. A modified form of single-chain urinary plasminogen activator in accordance with claim 16 in which plasminogen kringle 1 (amino acid residues 79 through 162) is fused to a portion of scu-PA (amino acid residues 132 through 411).

18 A modified form of single-chain urinary plasminogen activator in accordance with claim 16 in which plasminogen kringle 4 (amino acid residues 354 through 435) is fused to a portion of scu-PA (amino acid residues 132 through 411).

19 A modified form of single-chain urinary plasminogen activator in accordance with claim 16 in which both plasminogen kringle 1 (amino acid residues 79 through 162) and plasminogen kringle 4 (amino acid residues 354 through 438) are fused to a portion of scu-PA (amino acid residues 132 through 411).

20 A modified form of single-chain urinary plasminogen activator in accordance with claim 8 in which the EGF-like and kringle domains of scu-PA ( amino acid residues 1 through 132) are replaced by the finger, EGF-like and hybrid kringle 1/kringle 2 domains of t-PA (amino acid residues 1 through 111 followed by amino acid residues 200 through 261).

21. A modified form of single-chain urinary plasminogen activator having alterations in the kringle domain which provide longer circulating half-life in man.

22. A DNA sequence encoding for a modified plasminogen activator having greater fibrin selectivity than the unmodified plasminogen activator from which it is derived, said modified plasminogen activator including at least one of the following domains: kringle that is more homologous with a kringle of plasminogen than is any kringle of the unmodified plasminogen activator; a first hybrid t-PA kringle, said first hybrid kringle being a fusion of a portion of t-PA kringle 1 and a portion of t-PA kringle 2; or a second hybrid t-PA kringle, said second hybrid kringle being a fusion of a portion of t-PA kringle 2 and a portion of a scu-PA kringle.

23. A DNA sequence encoding for a modified scu-PA having alterations in the kringle domain which provide increased fibrin selectivity in its activation of plasminogen as compared to unmodified scu-PA.

24. A DNA sequence encoding for a modified scu-PA as claimed in claim 23, said DNA sequence further characterized by encoding for amino acid alterations of a kringle of the unmodified scu-PA which alterations provide increased fibrin selectivity.

25. A DNA sequence encoding for a modified scu-PA as claimed in claim 23 further characterized in that said DNA sequence encodes for a plasminogen activator in which a portion of kringle 1 or kringle 4 of plasminogen or a portion of kringle 2 of t-PA is fused to a portion of the kringle of scu-PA to provide a plasminogen activator with a hybrid kringle and increased fibrin selectivity.

26. A DNA sequence encoding for a modified scu-PA as claimed in claim 23 further characterized in that said DNA sequence encodes for a plasminogen activator in which kringle 1 or kringle 4 of plasminogen or both kringle 1 and kringle 4 of plasminogen substitute for the EGF-like domain and kringle of scu-PA.

27. A DNA sequence encoding for a modified scu-PA as claimed in claim 23 further characterized in that said DNA sequence encodes for a plasminogen activator in which the EGF-like and kringle domains of scu-PA (amino acid residues 1-132) are replaced by the finger, EGF-like and hybrid kringle 1/kringle 2 domains of t-PA (amino acid residues l-111 followed by amino acid residues 200-261).

28. A cloned vector capable of expressing a modified plasminogen activator having greater fibrin selectivity than the unmodified plasminogen activator from which it is derived, said modified plasminogen activator including at least one of the following domains: kringle that is more homologous with a kringle of plasminogen than is any kringle of the unmodified plasminogen activator; a first hybrid t-PA kringle, said first hybrid kringle being a fusion of a portion of t-PA kringle 1 and a portion of t-PA kringle 2; or a second hybrid t-PA kringle, said second hybrid kringle being a fusion of a portion of t-PA kringle 2 and a portion of a scu-PA kringle.

29. A cloned vector capable of expressing a modified form of single-chain urinary plasminogen activator (scu-PA) having alterations in the kringle domain which provide increased fibrin selectivity in its activation of plasminogen as compared to unmodified scu-PA.

30. The vector of claim 28 cloned in a mammalian host cell.

31. The vector of claim 29 cloned in a yeast or fungal host cell.

32. A method for making an improved plasminogen activator for use in treating various disease states comprising, selecting a plasminogen activator and modifying the plasminogen activator to provide regions of increased homology between the modified plasminogen activator and a plasminogen kringle.

33. The modified urinary plasminogen activator of claim 8 which when administered to catalyze dissolution of an unwanted clot in the body causes loss of circulating fibrinogen that is less than of the loss caused by administration of an equivalent mass of native single-chain urinary plasminogen activator.

34. The modified urinary plasminogen activator of claim 8 which when administered to catalyze dissolution of an unwanted clot in the body causes less degradation of circulating fibrinogen than that caused by administration of an equivalent mass of native urinary plasminogen activator.

35. The composition comprising a therapeutically effective amount of the modified plasminogen activator of claim 1 in admixture with a pharmacologically acceptable excipient.

36. The composition of claim 35 in which the amount is from 0.1 mg/ml to 10.0 mg/ml.