WO1989007145A1

WO1989007145A1 - Modified gene sequences encoding modified tpa and products therefrom

Info

Publication number: WO1989007145A1
Application number: PCT/US1989/000464
Authority: WO
Inventors: David J. Livingston; William Markland
Original assignee: Integrated Genetics, Inc.
Priority date: 1988-02-05
Filing date: 1989-02-03
Publication date: 1989-08-10
Also published as: EP0400054A1; AU3064989A; JPH04500752A

Abstract

Novel modified DNA sequences are provided encoding for modified tissue plasminogen molecules. The sequences are modified by insertion of restriction site containing linkers into pre-existing restriction sites in the tPA DNA. The inserted restriction sites are unique to the tPA DNA thereby allowing for predictable deletions, modifications, and spacing changes in the tPA amino acid sequence.

Description

MODIFIED GENE SEQUENCES ENCODING MODIFIED TPA AND PRODUCTS THEREFROM

Background of the Invention

This invention relates to the use of recombinant DNA techniques to produce therapeutic proteins, in particular to the use of such techniques to produce novel, modified human uterine tissue plasminogen activator (mtPA) genes and plasmids containing such genes, host cells transformed or transfected thereby, and mtPA molecules produced therefrom.

Tissue plasminogen activator (tPA) is a multi-domain serine protease which catalyzes conversion of plasminogen to plasmin. As such, tPA is of therapeutic value. When administered exogenously, tPA can effect a lysis of blood clots (thrombolysis). tPA has been proven effective in clinical trials for treatment of myocardial infarction. Other indications being examined include pulmonary embolism, deep vein thrombosis and stroke.

The tPA molecule contains five discrete structural domains. In the presence of plasmin, single-chain tPA or zymogen enzyme, can be cleaved into an activated two-chain form. The heavy chain contains four of these domains: a "finger" domain which is homologous to a portion of fibronectin; a "growth factor" domain which is homologous to epidermal growth factor; and two non-equivalent "kringle" domains. Plasmin cleavage to form two-chain tPA occurs C-terminal to Kringle 2 (at Arg₂₇₅). The light chain contains the serine protease domain, which is homologous to trypsin and chymotrypsin. tPA is a relatively, clot-specific plasminogen activator due to its affinity for fibrin, in turn responsible for forming the clot matrix. This fibrin affinity is believed to be due to interactions of the finger and Kringle 2 domains with fibrin. The participation of other domains in fibrin interactions is not well understood.

Inter domain effects are also poorly understood, do partially to the lack of a three-dimensional structure for tPA.

It is one aspect of the present invention to provide methods for increasing the spacing between tPA domains for increasing the rate of fibrinolysis or the resistance to inhibition by endogenous tPA inhibitors present in human plasma.

tPA secreted by human melanoma cells was purified and characterized by Rijken et al. (J. Biol. Chem. 256, 7035 (1981). Therapeutic utility of exogenous tPA was demonstrated with the melanoma-derived material (Collen et al., J. Clin. Inv. 71, 368 (1983); Korninger et al., J . Clin Inv. 69, 573 (1982)). Differences between tPA derived from melanoma and normal uterine tissue have been reported (Pohl et al., FEBS Lett. 168, 29 (1984)).

Rijken et al., Biochem. Biophys. Acta 580, 140 (1979) describes the partial purification, from human uterine tissue, of human tissue plasminogen activator (utPA).

Recombinant DNA techniques have been used previously to obtain mRNA from a line of cancer cells (Bowes melanoma cells), this mRNA being used to produce cDNA encoding Bowes tPA, as described in Goeddel et al., European Pat. Appln. No. 0093619. Copending, commonly assigned U.S.S.N. 782,686 to Wei et al., fully incorporated herein by reference, describes DNA sequences encoding utPA and further describes site-directed mutagenesis of the DNA sequence at any one or more of the three positions which code for amino acids which in turn normally become glycosylated in post-translation processing steps by mammalian cells. The resultant modified tPA molecules having altered amino acid sequences, fail to exhibit glycosylation at the mutagenized site. The work has also been reported by Wei et al., DNA 4, 76 (1985), and in EPA 178,105.

Expression vectors for expression of secreted tPA in mouse cells were subsequently reported by Reddy et al., 3. Cell Biochem. 10D, 154 (1986).

The utility of recombinant wild-type tPA as a human therapeutic is somewhat limited due to the large dose required (Verstraete et al. , Lancet 1, 842 (1985)). and may be accompanied by an unacceptably high incidence of bleeding complications due to non clot-specific activation of plasminogen (Verstraete et al. , J. Pharm. Exp. Ther. 235, 506 (1985)). Additional undesirable properties of wild-type tPA include its short in vivo half-life which can be lengthened by alteration of glycosylation of the protein by site directed mutagenesis (Lau et al., Bio/Technology 5, 953 (1987), fully incorporated herein by reference.

It is an aspect of the present invention to engineer new mtPA possessing improvement of the fibrin affinity and catalytic ability, most preferably, in combination with other improved characteristics.

European Patent Application No. 0,234,051 to Pannekoek et al., discuss tPA molecules having rearranged domains but unaltered light chains. Bowes melanoma cells served as source of tPA for the work. It is noted, however, that while the application purports to provid the understanding and tools necessary for designing and actual production of tPA mutants, the description fails to provide a reproducible or predictable method for altering the melanoma cell derived cDNA for providing desired mtPAs. It is another aspect of the present invention to provide novel methods for predictably insuring the tPA cDNAs are altered in the desired manner to produce the desired mtPAs.

European Patent Application No. 0,231,624 by Marotti et al., describe other human tissue plasminogen activator analogs having rearranged or deleted native domain regions. The Marotti application describes complex and time consuming procedures for the generation of specified tPA cDNAs by complete chemical synthesis of oligonucleotides. Further, the synthesis was based on native tPA derived from human melanoma cells (Bowes cells).

It is yet another aspect of the present invention to provide simplified, more direct methods for the predictable rearrangement of domains with a tPA like molecule based on utPA.

It is a still further aspect of the present invention to provide unique cDNA sequences encoding tPA like molecules having unique restriction endonuclease sites located at predetermined positions and to provide noval molecules resulting therefrom.

It is a further aspect of the present invention to provide novel approaches for generating new molecules having a biological activity associated with tissue plasminogen activator.

Summary of the Invention

In accordance wi th the principl es and objects of the present i nvention there are provided improved mtPA' s generated by addi tions or del etions of ami no acids from the parent tPA mol ecul e at preexi sti ng retri ction sites . Surprisi ngly, some of these mtPA' s di splay substantial ly improved properti es over wi Id-type tPA with respect to fibrin affinity, fibri nolysis and resi stance to human pl asma tPA i nhi bi tors . Al so provided are DNA sequences modifi ed to contai n uni que restri ction si tes whi ch do not occur elsewhere in the cDNA and do not occur in the cDNA of mature or wild-type tPA. These modified cDNA's are then advantageously manipulated to generate DNA sequences with partial deletions of domains or deletions of entire domains. Vectors for expression of these sequences in mammalian cells are disclosed along with characterization of the resultant mtPA proteins.

The most preferred embodiments of the present invention include cDNA's coding for utPA comprising one or more of the following modifications: Xho I restriction endonuclease site inserted at position 115, Xho I site inserted at position 277, Xho site inserted at position 515, Xho I site inserted at position 729, Xho I site inserted at position 879, Xho I site inserted at positio 1201, Xho I site inserted at position 1345, Xho I site inserted at position 1652, and Spe I site inserted at position 115 where the position numbers are from the nucleotide sequence. It is important to recognize that thes manipulations may directly lead to either insertions or deletions of amino acids in the resultant sequences, and may have the effect of altering spacing between the domains of the tPA protein. The results of such alterations cannot be predicted. Also described herein are cDNA's derived from those listed above where sequence corresponding to nucleotide bases between Xho I sites in one mtPA and another mtPA have been deleted. In one preferred embodiment the mtPA with Xho I site at position 11 and the mtPA with Xho I site at position 277 has been used to construct a cDNA with nucleotides 121-276 deleted. Other deletion mutant embodiments have been similarly constructed and are detailed below.

Additional preferred embodiments of the instant invention include host organisms for maintenance and replication of the sequences. Still other preferred embodiments include expression vectors for expression of said mtPA's in COS cells, C127 cells, CHO cells, and the mtPA proteins derived from these expression systems. Brief Description of the Drawings

Further understanding of the invention may be had by reference to the figures wherein:

Figure 1 shows utPA wild-type amino acid sequence in a two-dimensional representation showing location of domains and of restriction enzyme insertion sites;

Figure 2 depicts plasmid construction showing deletion of the

DNA encoding the finger domain;

Figure 3 shows the expression vector LK 444 BHS used for transient expression of modified tPA;

Figure 4 shows a vector for stable expression of mtPAs in CHO cells; and

Figure 5 shows a vector for stable expression of mtPAs in C127 cells.

Detailed Description and Best Mode

Definitons

The term "cell culture" refers to the containment of growing cells derived from either a multicellular plant or animal which allows for the cells to remain viable outside the original plant or animal .

The term "host cell" refers to a microorgansim including yeast, bacteria and mammalian cells which can be grown in cell culture and transfected or transformed with a plasmid or vector containing a gene encoding a molecule having a tPA biological characteristic and expressing such molecule. The term "domain" refers to a discrete continuous part of an amino acid sequence that can be associated with a particular function. With respect to tPA, suitable references describing the domain regions include (Banyai, L. et al., Common evolutionary origin of the fibrin-binding structures of fibronectin and tissue-type plasminogen activator, FEBS Lett. 163(1), 37-41 (1983) and Ny, T. et al. The structure of the Human Tissue-type Plasminogen Activator Gene: Correlation of Intron and Exon Structures to Functional and Structural Domains, Proc. Natl. Acad.

Sci. USA 81, 5355-5359 (1984)). Table 2 illustrates the locations of the domain regions as used herein.

The term "downstream" identifies sequences proceeding farther in the direction of expression; for example, the coding region is downstream from the initiation codon.

The term "interdomain" refers to the regions of a protein's amino acid sequence that lie between the domains.

The term "maintained" refers to the stable presence of a plasmid within a transformed host wherein the plasmid is present as an autonomously replicating body or as an integrated portion of the host's genome.

The term "microorganism" includes both single cellular prokaryote and eukaryote organisms such as bacteria actinomycetes, yeast, and mammalian cells.

The phrase "non-native endonuclease restriction sites" refers to endonuclease restriction sites that are not normally present in the native cDNA and are synthesized at pre-existing restriction sites of the native cDNA sequence. The term "operon" is a complete unit of gene expression and regulation, including structural genes, regulator genes, and control elements in DNA recognized by regulator gene product.

The term "plasmid" refers to an autonomous self-replicating extrachromosomal circular DNA and includes both the expression and nonexpression types. When a recombinant microorganism or cell culture providing expression of a molecule is described as hosting an expression plasmid, the term "expression plasmid" includes both extrachromosomal circular DNA and DNA that has been incorporated into the host chromosome(s).

The term "promoter" is a region of DNA involved in binding the RNA polymerase to initiate transcription.

The term "DNA sequence" refers to a single- or double-stranded DNA molecule comprised of nucleotide bases, adenosine, thymidine, cytosine and guanosine and further includes genomic and copy DNA (cDNA).

The term "suitable host" refers to a cell culture or microorganism that is compatible with a recombinant plasmid and will permit the plasmid to replicate, to be incorporated into its genome or to be expressed.

The term "upstream" identifies sequences proceeding in the opposite direction from expression; for example, the bacterial promoter is upstream from the transcription unit, the initiation codon is upstream from the coding region.

The term "restriction endonuclease", alternatively referred to herein as a restriction enzyme refers to one of a class of enzymes which cleave double-stranded DNA (dsDNA) at locations or sites characteristics to the particular enzyme. For example, the restriction endonuclease EcoR1 cleaves dsDNA only at locations: 5'GAATTC3' to form 5' G and AATTC3' fragments 3'CTTAAG5' 3'CTTAA G5'

Although many such enzymes are known, the most preferred embodiments of the present inventions are primarily concerned with only selected restriction enzymes having specified characteristics.

All cited references are fully incorporated herein by reference.

Conventions used to represent plasmids and fragments are meant to be synonymous with conventional representations of plasmids and their fragments. Unlike the conventional circular figures, the single line figures on the charts represent both circular and linear double-stranded DNA with initiation or transcription occurring from left to right (5' to 3'). Numbering of nucleotides and amino acids correspond to the particular amino terminal form shown in Table 1 although it will be readily understood that obvious numbering modifications will apply to molecules with different NH₂ terminal forms. The table below provides the standard conventional abbreviations for amino acids as they are used herein.

Abbreviations for amino acids

Three-letter One-lette

Amino Acid abbreviation symbol

Alanine Ala A

Arginine Arg R

Asparagine Asn N

Aspartic acid Asp D

Asparagine or aspartic acid Asx B Cysteine Cys C

Glutamine Gin Q

Glutamic acid Glu E

Glutamine or glutamic acid Glx Z

Glycine Gly G

Histidine His H

Isoleucine He I

Leucine Leu L Lysine Lys K

Methionine Met M

Phenylalanine Phe F

Proline Pro P

Serine Ser S Threonine Thr T

Tryptophan Trp W

Tyrosine Tyr Y

Valine Val V

General Methods

Methods of DNA preparation, restriction enzyme cleavage, restriction enzyme analysis, gel electrophoresis, DNA fragment isolation, DNA precipitation, DNA fragment ligation, bacterial transformation, bacterial colony selection and growth are as detailed in Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York 1982 (hereafter referred to as Maniatis). Methods of in vitro RNA transcription in a buffered medium and in vitro protein translation in rabbit reticulocyte lysate are as detailed in the manufacturers instructions (Promega Biotech). DNA sequencing was performed using the Sanger dideoxy method using either single-stranded DNA or denatured double-stranded DNA. Synthetic Oligonucleotide Linkers

The following oligonucleotide linkers were obtained from Biolabs Inc.

1. d(CCTCGAGG) 8 mer

2. d(CCCTCGAGGG) 10 mer

3. d(CCCCTCGAGGGG) 12 mer

Each linker contains the recognition sequence for the restriciton enzyme Xho I (CTCGAG), which is unique to the tPA cDNA and the SP65 vector. The linkers were utilized in the generation of linker insertion mutants and subsequently for the generation of deletion mutants.

tPA cDNA Source

The cloning of the full-length cDNA of human uterine tPA is described by Reddy et al. (1987). Essentially mRNA was made from human uterine tissue by the guanidine thiocyanate procedure followed by CsCl gradient purification and oligo-dT affinity purification. Reverse transcriptase and Klenow were used to convert the message into double-stranded cDNA which was cloned into the Pst I site of pBR322. The tPA cDNA clone was screened for with oligonucleotides deduced from the sequence of Bowes melanoma tPA. A 2455 base pair cDNA was isolated, sequenced and found to be in good agreement with published sequences (Pennica et al., 1983). The uterine tPA cDNA differed from melanoma tPA at several sites (predominantly in the 3' untranslated region of the clone) (from Reddy et al., 1987).

An Sfanl site (nucleotide 16) at the 5' end of the clone near the ATG start codon for tPA and BglII site (2090) was cleaved, filled in with Klenow in the presence of dNTP's and Sal I linkers lysated to the blunt ends. The cDNA was recloned into pBR322 as a Sal I fragment and subsequently recloned into other vectors using the Sal I sites.

Generation of Mutants

Usefully positioned restriction enzyme recognition sites within the tPA cDNA were replaced with a synthetic oligonucleotide linker containing a different and unique restriction enzyme recognizition site (Xho I). A variety of linker lengths (8 mer, 10 mer and/or 12 mer) were introduced such that the reading frame of mutant protein product was maintained either as a simple linker-insert mutant or following deletion mutagenesis utilizing the original linker insertion mutants. As an example, a detailed method for the construction of the BglII (nucleotide 115) Xho (8 mer) mutant will be given with reference to Figure 2.

1ug of SP6-tPA was cleaved with Bgl II at the unique Bgl II recognition site (nucleotide 115) using the standard protocol. The linearized DNA was precipitated with ethanol and resuspended in nick-translation buffer (40mM KPO₄ (pH 7.5), 6.6mM MgCl₂, 1.0mM mercaptoethanol, 250 μM dATP, dCTP, dTTP and dGTP together with 5μ of DNA polymerase I (Klenow fragment). This procedure fills in the 5' cohesive ends to generate "blunt ended" linearized DNA.

For those restriction enzymes generating 3' cohesive ends the ends are made blunt by utilizing the 3' to 5' exonuclease activity of T4 DNA polymerase as per the manufacturers instructions.

After incubating at room temperature for one hour the Klenow was heat inactivated at 65ºC for five minutes. To this mixture was added 100 pmoles of phosphorylated 8 mer Xho I linker (commercially available), ligation buffer (final concentration of 50mM Tris (pH 7.8), 10mM MgCl₂, 20mM DTT, 1mM ATP and 50μg ml^-1 bovine serum albumin) and 200u of T₄ DNA ligase. The ligation was allowed to proceed overnight at 22°C. The ligated DNA was phenol:chloroform:1AA extracted and ethanol precipitated. This was resuspended in restriction enzyme buffer, overdigested with Xho I and run on a one percent agarose gel to remove multiple linkers and the excess linkers from the relinearized DNA. The relinearized DNA was extracted from the agarose and ethanol precipitated. The precipitated DNA was resuspended in ligation buffer and T. ligase and allowed to ligate overnight at 16°C.

A small aliquot of the religated DNA was transfected into the

E. coli bacterial strain DH5 using standard protocols and the transfected bacteria plated on LB agar amp plates.

Bacterial colonies were picked, grown in LB media and DNA prepared on a small scale by standard procedures. The plasmid DNA was analyzed by restriction enzyme analysis and the loss of the unique Bgl II site and its replacement by a unique Xho I site was confirmed.

Using this protocol Xho I linkers of various sizes (8 mer, 10 mer and/or 12 mer) were introduced into the following sites of the tPA cDNA.

Native Restriction Site Relative Location

Bgl II (115) - between the beginning of the mature processed tPA protein and the start of the finger domain

Sty I (527) - at the N-terminal beginning of the growth factor domain BstX I (515) at the approximate center of the Kringle 1 domain

EcoRI (720 internal to the Kringle 2 domain

Sea I (879) at the C-terminal of the Kringle 2 domain

EcoRI (1201 internal to the protease domain

Sac I (1345) internal to the protease domain

Sty l (1652) internal to the protease domain

The numbers in parenthesis reflect the nucleotide position.

In the construction of the mutants encoding the heavy chain deletions the following plasmids were appropriately restriction enzyme digested and ligated as in the example given in Figure 2. For example in the generation of deletion mutant Bgl/Sty a plasmid containing an 8 mer XhoI linker at the BglII (115) site was ligated with a plasmid containing an 8 mer XhoI linker at the Sty l (277) site via their now common XhoI cut cohesive ends.

Mutant Lesion Plasmids Utilized

Bgl/Sty del (5-57); 4 LEA 58 BglII (115) XhoI (8 mer) Styl (277) XhoI (8 mer)

Bgl/Bstx del (5-139); 4 PLE 140 BglII (115) XhoI (12 mer) BstXI (515) XhoI (12 mer)

Bgl/Eco del (5-207); 4 PRG 208 BglII (115) XhoI (10 mer) EcoRI (729) XhoI (10 mer) Bgl/Sea del (5-258); 4 LED 259 BglII (115) XhoI (8 mer) ScaI (879) XhoI (8 mer)

Sty/Bstx del (58-139); 58 SPR 140 StyI (277) XhoI (10 mer) BstXI (515) XhoI (10 mer

Sty/Eco del (58-207); 57 SPRG 208 StyI (277) XhoI (10 mer) EcoRI (729) XhoI (10 mer

Sty/Sea del (58-258); 57 SLED 259 StyI (277) XhoI (8 mer) ScaI (879) XhoI (8 mer)

Verification of mutants

All mutants were verified by restriction enzyme analysis, sequencing and/or in vitro transcription/translation analysis,

Mutant-encoded protei ns wi th suffi ci ent fi bri nolyti c activi ty were analyzed by zymography rel ative to wi ld-type tPA.

Del etion mutagenesi s of the heavy chai n of tPA

The utilization of appropriately positioned linker inserts (Xho I linkers) made it possible to generate a series of in-frame deletion mutants within the DNA encoding the heavy chain of tPA. An example of this deletion of the DNA encoding the "finger" domain of tPA is depicted in Figure 2 and shown on Table 3h). The Bgl II (115) and Sty I (277) sites are situated near to the boundaries of this domain. By the appropriate restriction enzyme digestion, gel electrophoresis and ligation, it was possible to generate a tPA cDNA which lacked the region of DNA encoding the finger domain (mutant Bgl/Sty on del (5-57); 4LEA58). Bgl II ( 115) XhoI (8) Sty I (277 Xho I (8)

i.e. a deletion of amino acids 5 to 57 and an insertion of leu; glu and ala. (N.B. the finger domain includes amino acids 9 to 46). The same procedure was used to generate the following heavy chain deletion mutants:

Mutant Lesion Domains Deleted

Bgl /Sty del (5-57); 4LEA58 F p G

Bgl/BstX del (5-139); 4PLE140 F G pKl

Bgl/Eco del (5-207); 4PRG208 F G Kl pK2

Bgl /Sea del (5-258); 4LED259 F G Kl K2

Sty/BstX del (58-139); 58SPR140 G pkl

Sty/Eco del (58-207); 57SPRG208 G Kl pK2

Sty/Sea del (58-258); 57SLED259 G Kl K2

Table 3 shows the sequences of these and other mutants in detail. Table 4 provides Specific Activity data for the preferred embodiments of the present invention.

Verification of mutations

All mutants were verified by restriction enzyme analysis, sequencing and/or in vitro transcription/translation analysis.

Mutant-encoded proteins with sufficient fibrinolytic activity were analyzed by zymography relative to wild-type tPA.

Vectors

Sp65-tPA

The BamHI-HindIII fragment containing the tPA cDNA sequence isolated from M13MP18.tPA by restriction enzyme analysis and gel electrophoresis and ligated into the Bam HI, HindIII cleaved SP65 vector (Promega Biotech). This orientation (with the 5' end of the cDNA adjacent to the SP6 promoter) enabled an analysis of the mutant protein product by in vitro RNA synthesis and in vitro protein synthesis. The SP65.tPA vector was also a convenient vector to use during the manipulation of the inserted cDNA e.g. deletion generation.

LK444BHS.tPA

Mutated cDNA molecules were recloned into the LK444BHS vector as shown in Figure 3. The BamHI, HindIII fragment of tPA cDNA or mutant derivative contained in the SP65 vector was obtained by restriction enzyme cleavage and gel filtration. This fragment was li gated to a BamHI, HindIII cleavage vector, LK444BHS. This mutation allowed for the transient expression of the tPA analogue in a COS 7 cell line driven by the human ß-actin promoter.

CLH3AXBPV.tPA

The Sal I fragment was isolated from SP65.tPA, a mutated derivative by restriction enzyme cleavage and gel purification. This fragment was ligated to an Xho I cleaved vector CLH3AXBPV as shown in Figure 5. The orientation was determined and selected such that the inserted sequence was under the driving force of the metallothionine promoter in C127 cells.

CLH3AXSV2DHFR-tPA

The Sal I fragment containing the tPA cDNA or mutated derivative was isolated from the SP65 vector by restriction enzyme cleavage and gel purification. This fragment was ligated into Xho I cleaved vector ELH3AXSV2DHFR as shown in Figure 4. The orientation was determined and selected such that the inserted sequence was under the driving force of the mettallothionein promoter in CHO cells. Transfection of COS cells

A transient expression system was used wherein the expression vector (LK444BHS) was used to transfect COS-7 cells (ATCC # CRL1651). Two to three days after introduction of foreign DNA, conditioned medium was analyzed to characterize the activity of the secreted modified tPA protein.

3 × 10 cells were grown in 100 mm plates in DMEM + 10% glutamine for 1 day preceding transfection. Ten to 20 μg of DNA was added to 2.0 ml of tris-buffered saline (pH 7.5). 1 ml of 2 mg/ml DEAE-dextran (made just before transfection by adding 50 mgDEAE-dextran + 25 ml TBS) was added to this solution. Cells were washed 2 times with phosphate-buffered saline (PBS) and the transfection solution added. Cells were incubated at 37°C for 15-30 minutes. Dextran solution was then removed and cells washed again with PBS 2 times. This solution was replaced with 10 ml DMEM medium (no serum) plus 100 μl chloroquine (10 mM). The cells were then incubated at 37°C for 4 hours. The cells were washed twice with PBS and fed with GIT serum free medium (10 ml).

Transfection of DHFR-CHO Cells

DUKX CHO cells were obtained from Lawrence Chasin of Columbia University. THese cells are deficient in dihydrofolate reductase.

This gene is present in vector CLH3AXSV2DHFR. Cells were plated in alpha plus media 107. FBS, 1% glutamine medium at a density of 7 ×

5 10 cells per 100 mm dish 24 hrs. before transfection. 100-50 μg of plasmid DNA in 0.5 ml transfection buffer (the composition of which is 4 g NaCl, 0.185 g KCl, 0.05 g Na₂HPO₄, 0.5 g dextrose, and 2.5 g HEPES, pH 7.5 per 500 ml total volume). 30 μl of 2M CaCl₂ is added to the above solution and the mixture allowed to equilibrate for 45 minutes at room temperature. The medium is removed from the dishes cells washed twice with PBS, and the DNA solution added to the cells. The cells are allowed to incubate at room temperature for 20 minutes. 5 ml of medium is then added and the cells incubated for four hours at 37ºC. The media was removed and the cells were then shocked with 15% glycerol in transfection buffer at 37ºC for 3.5 minutes. After 48 hours, the cells were split at a 1\3 ratio and fed with a selection medium containing 0.02 μM methotrexate. Cell colonies which survive the treatment appear 10 to 14 days after transfection.

Thereafter, selected colonies were amplified with increasing levels of methotrexate according to published procedures (e.g. Michel et al., Bio/Technology 3, 561 (1985)). Modified tPA proteins produced by these cells was purified by previously reported procedures (Lau et al., Bio/Technology 5, 953 (1987) and U.S. 4,656,134 to Ringold).

Transfection of C127 Cells

Mouse C127 cells were transfected with DNA prepartions according to methods previously published by researchers in

Assignees laboratories (Hsiung et al., J . Mol. Appl. Genetics 2, 497 (1984)). Genes encoding modified tPA's were cloned into BPV-based vector CLH3AVBPV and these plasmids used for tranfections.

Modified tPA proteins were purified from conditioned medium by previously reported procedures (Lau et al., Bio/Technology 5, 953 (1987)).

Assays of Modified tPA's

Quantitation of mtPA proteins in conditioned medium was performed with a commercially available ELISA Kit for determination of tPA from American Diagnostica (Greenwich, CT, USA). The coating and detection antibody is a goat anti-human tPA IgG. Activity was determined by a published spectrophotometric assay for the rate of activation of plasminogen (Verheijen et al., Thromb. Haemostas. 48, 266 (1982)). The absorbance change ;mearure in the assay is converted to Units by reference to a WHO melanoma tPA standard. Specific activity of the mtPA proteins is determined by dividing Units by protein, the latter as determined in the ELISA assay.

Pharmaceutical Applications

The mtPAs of the invention may advantageously be admixed with a pharmaceutically acceptable carrier substance, e.g., saline, and administered orally, intravenously, or by injection into affected arteries of the heart. Administration will be generally as is carried out for two currently used blood clot lysing enzymes, streptokinase and urokinase.

The mtPA's of the invention may also be used therapeutically to lyse clots in human patients needing treatment of embolisms, e.g., post-operative patients, patients who have recently suffered myocardial infarction resulting in clots, and patients suffering from deep vein thrombi. The following examples are illustrative.

Example 1

For emergency treatment of thrombi by bolus injection, 5-10mg of lyophilized mtPA are mixed together with saline and placed in the chamber of a syringe, which is used to inject the mtPA bolus into the patient intravenously.

Example 2

For infusion treatment for the rapid lysis of coronary thrombi, about 100mg/hr of lyophilized mtPA are infused intravenously over a period of about one hour, followed by intravenous infusion of about 50 mg/hr over a period of about three more hours.

Example 3

For infusion treatment for the rapid lysis of coronary thrombi, the protocol of Example 2 is followed, except that infusion is preceded by the intravenous injection of a bolus of about 10 mg mtPA in saline.

Example 4

For infusion treatment for the slow lysis of deep vein thrombi about 15 mg/hr of lyophilized mtPA dissolved in saline are infused intravenously over a period of about 12-24 hours.

It will now be readily recognized by those skilled in the art that the foregoing amounts are merely representative and are subject to variation depending on the individual characteristics of the particular mtPA selected. It will also be readily apparent that numerous modifications based on the teachings within may be made without departing from the spirit or scope of the present invention, and in particular but without limitation, the mtPAs of the present invention may be used for diagnostic purposes including in vitro assays and in vivo imaging applications.

TABLE 2

DESIGNATION OF TPA DOMAINS (after Degan et al. 1986)

Propeptide/signal 22 to 108 - 29 to - 1

Finger domain 133 to 246 9 to 46

Growth factor domain 268 to 367 54 to 87

Kringle 1 domain 391 to 634 95 to 176

Kringle 2 domain 655 to 900 183 to 264

Protease domain 943 to 1698 279 to 530

TABLE 3

(All non-primed letters (e.g., a)) refer to nucleotide sequences, all primed letters (e.g., a¹)) refer to corresponding amino acid sequences)

BglII (115) Xho I (8 mer) 4LEGS5

103 126 a ) AGA.AGA.GGA. GCC.AGA.TCC.CTC.GAG.GGA.TCT.TAC.CAA a ) arg arg gly ala arg ser leu glu gly ser tyr gln -2 -1 1 2 3 4 5 6

StvI(277)Xho I (8 mer) 57SLEA58

268 288 b ) TGC.AGC.GAG.CCA.AGC.CTC.GAG.GCA.AGG.TGT.TTC b¹) cys ser glu pro ser leu glu ala arg cys phe 54 55 56 57 58 59 60

BstXI(515)Xho I (10 mer) del(138-139) 137 PR140

505 540 c ) AGG.CCA.GAC.GCC.ATC.CCT.CGA.GGG.CTG.GGG.AAC.CAC c¹) arg pro asp ala ile pro arg gly leu gly asn his 133 134 135 136 137 140 141 142 143 144 EcoRI(1201)Xho I (8 mer) 365 FLEE 366

1192 1215 d ) GTC.CAT.AAG.GAA.TTC.CTC.GAG.GAA.TTC.GAT.GAT.GAC d¹) val his lys glu phe leu glu glu phe asp asp asp

362 363 364365 366 367 368 369

SacI(1345)Xho I (10 mer) del (413-414): 412 ALEG415

1330 1359 e ) GAC. TGG. ACG. GAG. TGT. GCC. CTC. GAG. GGC. TCC. GGC. TAC e¹ ) asp trp thr glu cys ala l eu glu gly ser gly tyr 408 409 410 411 412 415 416 417

StyI(1652) Xho I (10 mer) 516 PRGK 517

1639 1665 f ) CCG.GGT.GTG.TAC.ACC.AAG.CCT.CGA.GGC.AAG.GTT.ACC.AAC f¹) pro gly val tyr thr lys pro arg g ly Iys val thr asn

511 512 513 514 515 516 517 518 519

Sca I(879)Xho I (12 mer) del 258: 257 SPRGD 259

868 891 g ) CTG.ACG.TGG.GAG.TCC.CCT.CGA.GGG.GAC.TGT.GAT.GTG g¹) leu thr trp glu ser pro arg gly asp lys asp val

254 255 256 257 259 260 261 Bgly/Sty del (5-57):4LEA58

106 120 280 288 h ) AGA.GGA.GCC.AGA.TCC.CTC.GAG.GCA.AGG.TGT.TTC h¹) arg gly ala arg ser leu glu ala arg cys phe

-1 1 2 3 4 58 59 60

Bgl/Bstx del (5-139):4PLE140 106 120 539 543 i ) AGA.GGA.GCC.AGA.TCC.CCC.CTC.GAG.GGG.CTG.GGG.AAC.CAC.AAC i¹) arg gly ala arg ser pro leu gly gly leu gly asn his asn

-1 1 2 3 4 140 141 142 143 144 145

Bgl/Eco del (5-207) :4PRG208

106 120 730 741 j ) AGA.GGA.GCC.AGA.TCC.CCT.GCA.GGG.AAT.TCC.ATG.ATC j¹) arg gly ala arg ser pro arg gly asn ser met ile

-1 1 3 4 208 209 210 21 1

Bgl/Sca del (5-258):4LED259

106 120 883 894 k ) AGA.GGA.GCC.AGA.TCC.CTC.GAG.GAC.TGT.GAT.GTG. CCC k¹) arg gly ala arg ser leu glu asp cys asp val pro

-1 1 2 3 4 259 260 261 262

Sty/Bstx del (58-139):58 SPR140

268 281 539 553 l ) TGC.AGC.GAG.CCA.AGC.CCT.CGA.GGG.CTG.GGG.AAC.CAC.AAC l¹) cys ser glu pro ser pro arg gly leu gly asn his asn

54 55 56 57 140 141 142 143 144 145 Sty/Eco del (58-207);57SPRG208

268 279 730 741 m ) TGC.AGC.GAG.CCA.AGC.CCT.CGA.GGG.AAT.TCC.ATG.ATC m¹) cys ser glu pro ser pro arg gly asn ser met ile

54 55 56 57 208 209 210 211

Sty/Sca del(58-258);57SLED259 268 279 883 891 n ) TGC.AGC.GAG.CCA.AGC.CTC.GAG.GAC.TGT.GAT.GTG n¹) cys ser glu pro ser leu glu asp cys asp val

54 55 56 57 259 260 261

(mer specifications in paranthesis indicate length of necessary linker which, when inserted at the specified site, results in correct reading frame for translation.)

TABLE 4

Mutant Speci fi c Acti vi ty#

Wild type 693 BglII (115) 657 StyI (277) 247 BstXI (515) 217 EcoRI (729) 159 Scal (879) 237 EcoRI (1201) 76 SacI (1345) 8 StyI (1652) NPD Bgl/Sty 126 Bgl/BstX 141 Bgl/RI 195 Bgl/Sea 13 Sty/RI NPD* Sty/Sca NPD*

# - IU/μg in indirect amidolytic assay

NPD - Not Possible to Determine

*These molecules demonstrated low fibrinolysis activity by I¹²⁵ fibrin lysis assay

Claims

ClaimsWhat is claimed is:

1. DNA sequence comprising the sequence:

103 126

AGA.AGA.GGA.GCC.AGA.TCC.CTC.GAG.GGA.TCT.TAC.CAA

and all sequences which hybridize thereto under stringent conditions.

2. DNA sequence comprising the sequence:

268 288

TGC.AGC.GAG.CCA.AGC.CTC.GAG.GCA.AGG.TGT.TTC

and all sequences which hybridize thereto under stringent conditions.

3. DNA sequence comprising the sequence:

505 540

AGG.CCA.GAC.GCC.ATC.CCT.CGA.GGG.CTG.GGG.AAC.CAC

and all sequences which hybridize thereto under stringent conditions.

4. DNA sequence comprising the sequence:

1192 1215

GTC.CAT.AAG.GAA.TTC.CTC.GAG.GAA.TTC.GAT.GAT.GAC

and all sequences which hybridize thereto under stringent conditions.

5. DNA sequence comprising the sequence:

1330 1359 GAC.TGG.ACG.GAG.TGT.GCC.CTC.GAG.GGC.TCC.GGC.TAC

and all sequences which hybridize thereto under stringent conditions.

6. DNA sequence comprising the sequence:

1639 1665

CCG.GGT.GTG.TAC.ACC.AAG.CCT.CGA.GGC.AAG.GTT.ACC.AAC

and all sequences which hybridize thereto under stringent conditions.

7. DNA sequence comprising the sequence:

106 126

AGA.GGA.GCC.AGA.TCG.ACT.AGT.CGA.TCT.TAC.CAA

and all sequences which hybridize thereto under stringent conditions.

8. DNA sequence comprising the sequence:

106 120 280 288

AGA.GGA.GCC.AGA.TCC.CTC.GAG.GCA.AGG.TGT.TTC

and all sequences which hybridize thereto under stringent conditions.

9. DNA sequence comprising the sequence:

106 120 539 543

AGA.GGA.GCC.AGA.TCC.CCC.CTC.GAG.GGG.CTG.GGG.AAC.CAC.AAC

and all sequences which hybridize thereto under stringent conditions.

10. DNA sequence comprising the sequence:

106 120 730 741

AGA.GGA.GCC.AGA.TCC.CCT.GCA.GGG.AAT.TCC.ATG.ATC

and all sequences which hybridize thereto under stringent conditions.

11. DNA sequence comprising the sequence:

106 120 883 894 AGA.GGA.GCC.AGA.TCC.CTC.GAG.GAC.TGT.GAT.GTG.CCC

and all sequences which hybridize thereto under stringent conditions.

12. DNA sequence comprising the sequence:

268 281 539 553

TGC.AGC.GAG.CCA.AGC.CCT.CGA.GGG.CTG.GGG.AAC.CAC.AAC

and all sequences which hybridize thereto under stringent conditions.

13. DNA sequence comprising the sequence:

268 279 730 741

TGC.AGC.GAG.CCA.AGC.CCT.CGA.GGG.AAT.TCC.ATG.ATC

and all sequences which hybridize thereto under stringent conditions.

14. DNA sequence comprising the sequence:

268 279 883 891

TGC.AGC.GAG.CCA.AGC.CTC.GAG.GAC.TGT.GAT.GTG

and all sequences which hybridize thereto under stringent conditions.

15. The product produced by a host cell transformed or transfected with an expression vector comprising the sequence of Claim 1.

16. The product produced by a host cell transformed or transfected with an expression vector comprising the sequence of Claim 2.

17. The product produced by a host cell transformed or transfected with an expression vector comprising the sequence of Claim 3.

18. The product produced by a host cell transformed or transfected with an expression vector comprising the sequence of Claim 4.

19. The product produced by a host cell transformed or transfected with an expression vector comprising the sequence of Claim 5.

20. The product produced by a host cell transformed or transfected with an expression vector comprising the sequence of Claim 6.

21. The product produced by a host cell transformed or transfected with an expression vector comprising the sequence of Claim 7.

22. The product produced by a host cell transformed or transfected with an expression vector comprising the sequence of Claim 8.

23. The product produced by a host cell transformed or transfected with an expression vector comprising the sequence of Claim 9.

24. The product produced by a host cell transformed or transfected with an expression vector comprising the sequence of Claim 10.

25. The product produced by a host cell transformed or transfected with an expression vector comprising the sequence of Claim 11.

26. The product produced by a host cell transformed or transfected with an expression vector comprising the sequence of Claim 12.

27. The product produced by a host cell transformed or transfected with an expression vector comprising the sequence of Claim 13.

28. The product produced by a host cell transformed or transfected with an expression vector comprising the sequence of Claim 14.

29. A host cell transformed or transfected with a vector comprising the DNA sequence of Claim 1.

30. A host cell transformed or transfected with a vector comprising the DNA sequence of Claim 2.

31. A host cell transformed or transfected with a vector comprising the DNA sequence of Claim 3.

32. A host cell transformed or transfected with a vector comprising the DNA sequence of Claim 4.

33. A host cell transformed or transfected with a vector comprising the DNA sequence of Claim 5.

34. A host cell transformed or transfected with a vector comprising the DNA sequence of Claim 6.

35. A host cell transformed or transfected with a vector comprising the DNA sequence of Claim 7.

36. A host cell transformed or transfected with a vector comprising the DNA sequence of Claim 8.

37. A host cell transformed or transfected with a vector comprising the DNA sequence of Claim 9.

38. A host cell transformed or transfected with a vector comprising the DNA sequence of Claim 10.

39. A host cell transformed or transfected with a vector comprising the DNA sequence of Claim 11.

40. A host cell transformed or transfected with a vector comprising the DNA sequence of Claim 12.

41. A host cell transformed or transfected with a vector comprising the DNA sequence of Claim 13.

42. A host cell transformed or transfected with a vector comprising the DNA sequence of Claim 14.

43. A method for providing an altered DNA sequence encoding a molecule having a tPA biological property, said sequence capable of being rearranged in a predetermined manner comprising the steps of: a) providing a DNA sequence encoding a molecule having a tPA biological activity; and

b) inserting at at least two restriction sites normally present in said sequence a linker containing at least one unique restriction site to form a modified DNA sequence and wherein said linker has a mer length selected to ensure that correct translational reading frame is maintained.

44. The method as provided in claim 43 further comprising the steps of: a) cleaving said modified DNA sequence at at least two of said unique restriction sites to form fragments; and

b) ligating said fragments together.

45. The method as provided in claim 44 wherein said normal restriction sites are selected from the group of nucleotide positions consisting of 115, 277, 515, 1201, 1345 and 1652.

46. The method as provided in claim 45 wherein said unique restriction site is Xhol.