EP0398962A1 - Rearranged tissue plasminogen activators and method for producing same - Google Patents

Abstract

The new methods described serve to create modified DNA coding for molecules having a biological property for tissue plasminogen activators. Modified DNAs allow for a wide variety of predictable modifications to the tissue plasminogen deactivator molecule including domain regions. New tissue plasminogen activator molecules obtained by these methods are also described.

Description

REARRANGED TISSUE PLASMINOGEN ACTIVATORS AND METHOD FOR PRODUCING SAME

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 Arg_27g). The light chain contains the serine protease domain, which is homologous to trypsin and chymotrypsin.

tPA i s a relati vely clot-speci fi c plasmi nogen acti vator due to its affinity for fibri n , whi ch forms the clot matrix . Thi s fi bri n affinity is believed to be due to interactions of the finger and Kringle 2 domains with fibrin. The lower affinity for fibrin by Kringle 1 is not well undestood.

It is one aspect of the present invention to produce an mtPA with higher fibrin specificity and one which can achieve higher rates of fibrinolysis than the wild-type tPA molecule.

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 fit al- (J- Biol. Chem. 256, 7035 (1981). Therapeutic utility of exogenous tPA was demonstrated with the melanoma-derived material (Collen it al., 3. Clin. Inv. 71, 368 (1983); Koringer ≤t al.. -3- Clin Inv. £2, 573 (1982)). Differences between tPA derived from melanoma and normal uterine tissue have been reported (Pohl £± 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-transl tion processing steps by mammalian cells. The resultant modified tPA molecules having altered amino acid sequences fail to exhibit glycosylation at the utagenized 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. , J. Cell Biochem. TOP. 154 (1986).

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 proports to provide the understanding and tools necessary for designing an 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 noval 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 novel molecules resulting therefrom. 5

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

0 Summary of the Invention

In accordance with the principles and objects of the present invention there are provided mtPA's which are generated from a parent tPA molecule by deletion, rearrangement or duplication of

15 domains. It was surprising to discover that such alterations still result in molecules having a biological activity associated with w ld- ype or unmodified tPA. It was totally unexpected that some of these novel mtPA's are superior to the parent molecule with respect to their rate of fibrinolysis.

Z0

A preferred method of the present invention comprises introducing at least two restriction enzyme sites into the tPA cDNA. These sites are ideally positioned at the positions in the tPA cDNA corresponding to boundaries of the tPA protein domains

25 although other locations may also serve.

The exact positioning of these sites is critical for preserving desirable activities of the parent tPA molecule. The most preferred embodiments of the present invention have introduced 30 into separate cDNAs an Avr II restriction site, an Nhe I restriction site , an Spe I restriction site , and an Xba I restriction site . The resultant preferred amino acid sequences are listed in Table 3 described below.

35 The altered cDNA's described above can then be advantageously manipulated to generate deletions or duplications of tPA domains as shown in Table 4. These manipulations are described in additional detail below and generally involve cutting the modified cDNA's with the appropriate restriction enzymes and thereafter ligating the resultant cDNA fragments to form new preferred cDNAs.

In the most preferred embodiments, the above modifications have been combined with other modifications for the purpose of obtaining molecules with extended in vivo half-life, for example the conversion of a asparagine to a glutamine at nucleotide 451.

Additional preferred embodiments of the mutant invention include host organisms for maintenance and replication of the sequences, 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 principles and objects of the present invention may be had by studying the accompanying Figures wherein:

Figure 1 shows the position of Avr II, Nhe land Spe I sites in the tPA cDNA;

Figure 2 is a schematic diagram of construction of vector expressing mtPA with K, deletion;

Figure 3 is a representation of the LK444BHS vector for transient expression of mtPA;

Figure 4 is a representation of CLH3AXS2DHFR vector for stable expression of mtPA in CHO cells; Figure 5 is a representation of CLH3AXBPV vector for stable expression of mtPA in C127 cells;

Figure 6 is a schematic diagram of the construction of vector containing mtPA havingK;]^ duplication;

Figures 7a-l and 7a-2 show the construction of the SP.SNA vector

10. containing the Ult rearrangment mtPA sequence; and

Figures 7b-l and 7b-2 show the construction of the SP.ULT containing the ULT rearrangement mtPA sequence.

15

Detailed Description and Best Mode

Definitions

The term "cell culture" refers to the containment of growing cells

Z0f^< 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

25 transfected or transformed with a plasmid or vector containing a gene encoding a molecule having a tPA biological characteristic and expression of such molecule.

The term "domain" refers to a discrete continuous part of an amino

3Q< acid sequence that can be equipped with a particular function. With respect to tPA, references (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

35 and Exon Structures to Functional and Structural Domains, Proc. Natl. Acad. Sci. USA 81, 5355-5359 (1984)) have been defined the domain regions and Figure 1 herein discloses the approximate locations of the domain regions.

SUBSTITUTESHEET 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 and yeast.

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. Where a recombinant microorganism of cell

^■ culture provides expression of such 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, thy idine, 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 w ll 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 as restriction enzymes refers to enzymes which cleave double- stranded DNA (dsDNA) at locations or sites characteristics to the particular enzyme. For example, the restriction endonuclease EcoRl cleaves dsDNA only at locations:

5'GAATTC3' to form 5' G and AATTC3' fragments.

3'CTTAAG5' 3'CTTAA G5'

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

Conventions used to represent plas ids 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 the obvious numbering modifications may apply with different NH₂ terminal forms. The table below provides the standard abbreviations for amino acids.

Abbreviations for amino acids

General Methods

Methods of DNA preparation, restriction enzyme cleavage, 5 restriction enzyme analysis, gel electrophoresis, DNA fragment isolation, DNA precipitation, DNA fragment ligation, bacterial transformation, bacterial colony selection and grov/th are as detailed in Maniatis et al- , Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York 1982 (hereafter referred to on 0 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 T5 DNA.

Synthetic Oliqonucleotide Linkers

The following oligonucleotide linkers were obtained from zσ^: Biolabs Inc.

1. d(CCTCGAGG) 8 raer

2. d(CCCTCGAGGG) 10 mer

3. d(CCCCTCGAGGGG) 12 mer 25

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 30 deletion mutants.

tPA cDNA Source

The cloning of the full-length cDNA of human uterine tPA is 35 described by Reddy et al. (1987). Essentially mRNA was made from human uterine tissue by the guanidine triocyanate 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 e± al., 1987).

An Sfanl site (nucleotide 16) at the 5' end of the clone near the ATG start codon for tPA and Bglll 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 a Linker Insertion Mutant

A Spe I linker was placed at the Bgl II (115) site of the tPA cDNA such that it could be used with the other introduced Avr II, Nle I and Spe I sites

A detailed method for the construction of the Bgl II (115) Spe

I (nucleotide 8) mutant is given below.

Iμg 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 KP0₄ (pH 7.5), 6.6mM M^'gCl₂, l.OmM 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. After incubating at room temperature for one hour the Klenow was heat inactivated at 65°C for five minutes. To this mixture was added 100 pmoles of phosphorylated 8 mer Xho I linker (commercially 5 available), ligation buffer (final concentration of 50mM Tris (pH 7.8), lOmM MgCl₂, 20mM DTT, I M ATP and 50μg ml^" bovine serum albumin) and 200u of T. DNA ligase. The ligation was allowed to proceed overnight at 22°C.

0 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

15 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 al quot of the religated DNA was transfected into the Zϋ. ___ 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 25 was analyzed by restriction enzyme analysis and the loss of the unique Bgl II site was confirmed:

Bgl II (115) Spe I (8 mer) 4TSRS5

30 106 126

AGA.GGA.GCC.AGA.TCG.ACT.AGT.CGA.TCT.TAC.CAA arg gly ala arg ser thr ser aro ser tyr gin -1 1 2 3 4 5 6

35 Synthesis of Primers for Site-Specific Mutagenesis

The human uterine tPA cDNA was modified by site-specific mutagenesis using synthetic oligonucleotides prepared by the solid 5 phase phosphotriester method .

The following primers were synthesized and used for such mutagenesis. They are in the anti-sense sequence.

10 1. Primer EGAV

5' GCA.ACT.TTT.CCT.AGG.CAC.TGA 3' for

i ntroduction of an Avr II si te (CCT-AGG) at nucl eoti des 256-261 15

2. Primer SWS

5' GCA.CGT.GGC.ACT.AGT.ATC.TAT.TTC 3' for

20. introduction of a Spe I site (ACT.AGT) at nucleotides 379-384

3. Primer KN1

5' CCCCTGTAACTAGTGCCCTG 3' for 25 introduction of a SPe I site (ACTAGT) at nucleotides 409-444

4. Primer KC1

30 5' GGGTGCTAGCGAACTCTGAG 3' for

introduction of an Nhel (GCTAGC) site at nucleotides 619-624

35 5. Primer KCA1

5 ' GTGCTFCAGCJAGCTGAGCTGTAC 3 ' for

i ntroduction of an Nhe I (GCTAGC) at nucl eoti des 613-618

6. Primer KNB2

5 ' GCCACGGTAGCJAGCCCCATTCCC 3 ' for

i ntroduction of an Nhe I (GCTAGC) si te at nucl eoti des 673-678

7. Primer KC2

5' GTACTCCCIAGGCAGCCTGC 3' for

introduction of an Avr II (CCTAGG) site at nucleotides 871-876

8. Primer KCA2

5' CGTCAGCCTAGGGTTCTTCAGC 3^r for

introduction of an Avr II (CCTAGG) site at nucleotides 862-867

9. Primer SWN

5' CAGGCCGCAGCTAGCGCAGGAGGG 3^l for

introduction of an Nhe 1 (GCTAGC) at nucleotides 901-906

10. Primer OMT

5' CCCTCCACTAGTGCGAAACTG 3' for

introduction of a SPe I site (ACTAGT) at nucleotides 943-948. 11. Primer Pen

5' CTGGTCACCTAGGCATGTTG 3' for 5 introduction of an Avr II (CCTAGG) at nucleotides 1693-1698

Site Specific Mutagenesis using above Synthesized Oligonucleotides

TO The M13 based oligonucleotide directed mutagenesis procedure is essentially that detailed by Kunkel et al (Proc. NaT'!. Acad. Sci. USA S2, 488 (1985)). Phage containing the M13tPA.MGA vector (the tPA cDNA which has the Asn 451 gin mutation described in the aforementioned copending application and publication of Wei fit al.)

Ϊϊ5 were grown in an E.coli strain CJ236 (which is dut^~ ung^~) in media in the presence of 0.5 ug ml uridine. The single stranded DNA produced contained uracil residues instead of thymine. The single stranded M13tPA.MGA was prepared by normal procedures.

ZQ 10 ng of the phosphorylated mutating oligonucleotide was annealed with 1 ug of single stranded DNA in a total volume of 20 ul containing 1 x SSC (0.15 M NaCl, 15 mM sodium citrate). The annealing mixture was heated to 70°C then allowed to cool slowly in a water bath for several hours. The annealed fragments were

25 converted in covalently cured circular DNA by the action of DNA polymerase and T4 ligase. The annealed mixture was made up to 100 ul containing 20 mM Hepes (pH7.8), 2 mM DTT, 10 mM MgCl₂, 500 uM each of dATP, dTTP, dCTP, and dGTP, ImM ATP, 2.5 units of Klenow and 2 units of T4 DNA ligase. Incubation started at 0°C for 5 minutes,

30 room temperature for 5 minutes, 37°C for 2 hours, and overnight at 16°C.

10 ul of the above reactive mixture was added to 300 ul of competent DH₅ bacterial cells and left in ice for 1 hour. The 35 bacteria were heat shocked at 37°C for 5 minutes then serially diluted into 3 ml of soft agar (0.6%) containing 200 ul of mid-way phase JM101 and poured onto LB agar plates and allowed to solidify. The plates were then incubated overnight at 37^βC.

5^"- Nitrocellulose lifts were taken from the plates, baked at

32

80°C, under vacuum, for 2 hours and hybridized with the 6 P ATP labeled mutant oligonucleotide. Mutant positive isolates were determined by thermal denaturation of the DNA:oligo duplexes and further grown in OM101 on a larger scale for maxi-prep Rf DNA 10 isolation. The Bam HI-Hind III fragment containing the tPA cDNA including the Asn 451 gin mutation and the new mutation was further recloned into the SP65 vector and/or mammalian cell expression vector using normal procedures of restriction enzyme cleavage and ligation.

I

An example of this method is the generation of mutant KCA2 (R252P;N451Q) in which an Avrll site is introduced into the 3' boundary of the cDNA encoding the Kringle 2 domain.

zα

559 570

AAC.CGC.AGG.CTG

(251) N R R L (254)

25 site-specifi c, ol igonecl eoti de directed mutagenesi s yi el ds

ACC.CCI.AGG.CTG

30 N P R L

Avrll site (CCTAGG).

35 Other mutants generated using this method are as follows: EGAV (V51R; N451Q)

SWS (R92S; N451Q)

KN1 (I101T; N451Q) KC1 (C171S; N451Q)

KCA1 (E169A; FT70S; N451Q)

KNB2 (S189A; A190S; N451Q)

KC2 (T255P; W256R; N451Q)

KCA2 (R252P; N451Q) SWN (S265A; T266S; N451Q)

OMT (I279T; K280S; N451Q)

Pen (R529P; P530R: N451Q)

(N.B. The unique restriction enzyme sites were introduced with a mutant (MGS (N451Q) hence each contains this mutant also)

The positions of the restriction enzyme sites relative to the domain structure of the tPA protein are as follows (see Figure 1):

Bgl II (115) Spe(8) - between the N-terminus of the mature processed tPA protein and the N-terminus of the finger domain

EGAV - between the C-terminus of the finger domain and the N-terminus of the growth factor domain

SWS - between the C-terminus of the growth factor domain and the

N-terminus of the Kringle 1 domain

KN1 - at the N-terminus of the Kringle 1 domain KC1 - at the C-terminus of the Kringle 1 domai n

KCA1 - - at the C-terminus of the Kringle 1 domai n

KNB2 - at the N-terminus of the Kringle 2 domai n

KC2 - at the C-terminus of the Kringle 2 domai n

KCA2 - at the C-terminus of the Kringle 2 domai n

SWN - between the C-terminus of the

Kringle 2 domain and the N-terminus of the protease domain

OMT - at the N-terminus of the protease domain

Pen - at the C-terminus of the protease domain

Manipulations of the tPA cDNA:

Avrll, Nhel, Spel and Xbal restriction enzymes recognize unique and separate hexanucleotide sequences ie.

Avrll CCTAGG

Nhel GCTAGC

Spel ACTAGT

Xbal TCTAGA When cleaved by the appropriate restriction enzyme the 5' overhang is common to all four sites ie.

Avrll

Nhel

Spel

Xbal

Hence fragments with the above ends can be easily ligated and as an extra benefit when the ends from the differently cut sites are ligated the resulting hybrid site is no longer recognized by either restriction enzyme making it diagnostic for the correct ligation and/or orientation of the fragment.

For example

Avrll 5' overhang Nhel 5' overhang

C CTAGC GGATC G i ligate

CCTAGC GGATCG Since it has lost its palindromic feature it is no longer cleaved by either Avrll or Nhel. The same finding applies to other possible combinations.

Manipulations of the tPA cDNa can be accomplished using the above introduced sites to generate deletions and duplications.

A) Deletions

The Avrll, Nhel and Spel sites located in the following mutants; EGAV, SWS, KNI, KCl, KCAl, KNB2, KC2, KCA2, SWN, OMT and Pen have been utilized to generate deletions in the tPA cDNa which cause a loss of domain(s) in the expressed protein product.

An example of such a manipulation is the deletion of the cDNA encoding the kringle 1 domain using mutants KNI and KCAl.

KNI KCAl

400 420 607 630 GAC-CAG-GGC-ACT-AGT-TAC-AGG AGC-TCA-GCT-AGC-TGC-AGC-ACC-CCT asp gin gly thr ser tyr arg ser ser ala ser cys ser thr pro

98 i 105 167 I 174

Spel cut Nhel cut

1 1 ^~ GAC.CAG.GCC.ACT.AG- C.TGC.AGC.ACC.CCT

1 ligate (digest with Spel and Nhel to remove any contaminating KNI or KCAl 400 630

GAC.CAG.GGC.ACT.AGC.TGC.AGC.ACC.CCT asp gin gly thr ser cys ser thr pro

98 99 100 171 172 173 174

ie. Kl del or del (101-170); 100TS171; N451Q

Details of the Kl deletion construction is as follows: 1 μg of plasmid SP6 KNI was digested with Hindlll and Spel and 1 μg of plasmid SP6 KCAl was digested with Hindlll and Nhel. The DNA fragments generated were separated and isolated by gel electrophoresis. The large fragment from SP6 KNI and the small fragment from SP6 KCAl were ligated using normal procedures; redigested with Spel and Nhel and transfected into the E. coli strain DH5 and plated onto LB media plates containing amplicillin. Resistant colonies were picked and grown. Plasmid DNA was prepared on a small scale and analyzed by restriction enzyme analysis for the deletions and loss of the Spel and Nhel sites. The procedure is shown schematically in Figure 2.

The same procedure was used to generate the following series of mutants.

Mutant Lesion Domains Deleted

del (171-190); 170AS191 N451Q Interkringle del (101-170); 100TS171; N451Q Kringle 1 del (189-256); 188AR257; N451Q Kringle 2 del (101-256); 100TR257; N451Q Kringles 1 and 2 del (5-51) 4TR52; N451Q Finger del (51-92) 50S93; N451Q Growth factor del (5-92) 4TS93; N451Q Finger & Growth factor del (2-266); 1TS267; N451Q The heavy chain. (Finger, growth factor, both kringles) ^• In the construction of the mutants encoding the deletions, the following plasmids were appropriately restriction enzyme digested and ligated as in Figures 2, 6 and 7.

For example, in the generation of the deletion mutant Kldel, a plasmid containing the unique Spe I site (KNI) was ligated with a plasmid containing the unique Nhe I site (KCAl) via their common cohesive ends.

Plasmids and Restriction Enzyme

Mutants Sites Utilized

N del KCAl (Nhel) and KNB2 (Nhel) Kl del KNI (Spel) and KCAl (Nhel) K2 del KNB2 (Nhel) and KCA2 (Avrll) K1K2 del KNI (Spel) and KCA2 (Avrll) F del Bgl/Spe (Spel) and EGAV (Avrll) G del EGAV (Avrll) and SWS (Spel) FG del Bgl/Spe (Spel) and SWS (Spel) Protease Bgl/Spe (Spel) and SWN (Nhel)

B. Duplications

Referring to Figure 1 utilizing the Avrll, Nhel and Spel sites listed and described in the previous sections it was possible to duplicate the DNA encoding domain(s) and hence duplicate the same domain(s) in the protein product. An example of such a duplication is the generation of a tPA analogue containing four kringle domains (a duplication of kringles 1 and 2) using mutants SWS and SWN (see Figure 6).

The following duplications have been constructed in a similar fashion. The amino acid sequences are shown in Tables 4 - 9 and 12 - 15 Mutant Duplicated^' Domains

2K1, 1K2 Kringle 1

1K1, 2K2 Kringle 2

2 1, 2 2 Kringles 1 and 2

S+N Kringles 1 and 2

2F Finger

2G Growth factor

2FG Finger and growth factor

2 Prot 1CV Protease domain

2 Prot 2CV Protease domain

In the construction of the mutants encoding the duplications the following plasmids were appropriately restriction enzyme digested and ligated as in Figures 2, 6 and 7a-l, 7a-2, 7b-l and 7b-2.

For example in the generation of the duplication mutant, 2F, a plasmid containing the introduced unique Avrll (EGAV) was ligated with a plasmid containing the introduced Spel site at the Bglll (115) site (Bgl/Spe) via their common adhesive ends.

Mutant Plasmids and restriction enzymes utilized

EGAV (Avrll) and Bgl/Spe (Spel) SWS (Spel) and EGAV (Avrll) SWS (Spel) and Bgl/Spe (Spel)

KCAl (Nhel) and KNI (Spel) KCA2 (Avrll) and KNB2 (Nhel) KCA2 (Avrll) and KNI (Spel)

SWN (Nhel) and SWS (Spel) OMT (Spel) and SWS (Spel)

SUBSTITUTESHEET Mutant Plasmids and restriction enzymes utilized

2 Prot 1CV Pen (Avrll) and OMT (Spel)

2 Prot 2CV Pen (Avrll) and SWN (Nhel)

10 C) Rearrangements

Utilizing the Avrll, Spel and Nhel sites either individually or in combination within a plasmid it was possible to rearrange domains or blocks of domains within the parent molecule such that the final product has a

15 shuffled domain structure.

As an example it was possible to rearrange the tPA cDNA such that the DNA encoding the heavy chain and light chain are switched ie. the the light chain at the amino terminal half of the protein and the heavy chain at the C-terminal half of the protein (see Figures 7a-l, 7a-2, 7b-l and 7b-2).

20

The final product was designated the ult rearrangement mutant.

In a similar manner other mutants were generated with rearranged

25 domains.

2K1 encodes for a tPA analogue in which the kringle 2 domain has been replaced by another kringle 1 domain generating a protein with two kringle 1 domains but no kringle 2. zσ

2K2 encodes for a tPA analogue which contains two kringle 2 domains and no kringle 1 domain.

The amino acid sequences of the rearranged mutants are shown in Tables 10, 11 and 16.

35

SUBSTITUTESHEET In the construction of the mutants encoding the rearrangements, the following plasmids were appropriately restriction enzyme digested and ligated as in Figures 2, 6 and 7a-l, 7a-2, 7b-l and 7b-2.

For an example, the ULT mutant was generated as in Figures 7a- 1, 7a-2, 7b-l and 7b-2.

10

Plasmids and restriction

Mutant enzymes utilized

15

2K1 KCAl (Nhel), KNI (Spel), KCA2 (Avrll) and KNB2 (Spel)

2K2 KCAl (Nhel), KNI (Spel),, KCA2 (Avrll) and KNB2 (Spel)

ZQ'

ULT Bgl/Spe (Spel), Swn (Nhel) and Pen (Avrll)

25 Verification of mutants

The mutations 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. 0

Vectors

Sp65-tPA 5

The BamHI-Hindlll fragment containing the tPA cDNA sequence isolated from M13MP18.tPA by restriction enzyme analysis and gel electrophoresis and ligated into the Bam HI, Hindlll cleaved SP65

SUBSTITUTE 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 i_n vitro RNA synthesis and i_n 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

Referring to Figure 3, mutated cDNA molecules were recloned into the LK444BHS vector. The BamHI, Hindlll fragment was isolated from SP65.tPA, a mutant derivative obtained by restriction enzyme cleavage and gel filtration. This fragment was ligated to a BamHI, Hindlll cleavage vector, LK444BHS. This mutation allowed for the transient expression of the tPA analogue in a COS 7 cell line driven by the human β-actin promoter.

CLH3AXBPV.tPA

Referring to Figure 5, 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

The tPA cDNA or mutants were restriction enzyme digested with Sail and recloned with the Xhol site cleaved in the vector. The orientation of the tPA cDNA or mutant is such that expression is driven by the metal!othionein promoter. The CLH3AXSV2DHFR vector is used for the stable expression of tPA or modified tPA in CHO cells with the ability for ethatrexate amplification. 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 x 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 mg DEAE-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^βC 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 10% FBS, 1% glutamine medium at a density of 7 x

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 Na₂HP0₄, 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 37^βC. The media was removed and the cells were then shocked with 15% glycerol in transfection 5 buffer at 37°C for 3.5 minutes. After 48 hours, the cells were split at a H3 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.

O 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.

T5 4,656,134 to Ringold).

Transfection of C127 Cells

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

Assignees laboratories (Hsiung et al . , 0. Mol. Appl. Genetics 2, 497 (1984)). Genes encoding modified tPA's were cloned into BPV-based vector CLH3AVBPV and these plasmids used for tranfections.

Z5 Modified tPA proteins were purified from conditioned medium by previously reported procedures (Lau et al . , Bio/Technology 5, 953 (1987)).

Assays of Modi fi ed tPA' s

30

Quanti tation of mtPA protei ns i n condi tioned medi um was performed wi th a commercial ly avai labl e ELISA Ki t for determi nation of tPA from Ameri can Diagnosti ca (Greenwi ch, CT, USA) . The coati ng and detection anti body i s a goat anti -human tPA IgG.

35 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 ;measured 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

10

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 1.5 carried out for two currently used blood clot lysine 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, 20; 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

25

For emergency treatment of thrombi by bolus injection, 5-1Omg 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.

30

Example 2

For infusion treatment for the rapid lysis of coronary thrombi, about lOOmg/hr of lyophilized mtPA are infused

35 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 i_n vitro assays and In vivo imaging application. T E

TGTGAAGCAATCATGGATGCAATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGTGGA 1 _{+ +} + + + + 60

ACACTTCGTTAGTACCTACGTTACTTCTCTCCCGAGACGACACACGACGACGACACACCT

START SIGNAL aa M K R G L C C V L L L C G -

GCAGTCTTCGTTTCGCCCAGCCAGGAAATCCATGCCCGATTCAGAAGAGGAGCCAGATCT

61 + + + + + + 120

CGTCAGAAGCAAAGCGGGTCGGTCCTTTAGGTACGGGCTAAGTCTTCTCCTCGGTCTAGA PROPEPTIDE «i aa A V F V S P S Q E I H A R F R R G A R S

TACCAAGTGATCTGCAGAGATGAAAAAACGCAGATGATATACCAGCAACATCAGTCATGG

121 + + + + + + 180

ATGGTTCACTAGACGTCTCTACTTTTTTGCGTCTACTATATGGTCGTTGTAGTCAGTACC aa Y Q V I C R D E K T Q M I Y Q Q H Q S W -

CTGCGCCCTGTGCTCAGAAGCAACCGGGTGGAATATTGCTGGTGCAACAGTGGCAGGGCA

181 + + + + + + 240

GACGCGGGACACGAGTCTTCGTTGGCCCACCTTATAACGACCACGTTGTCACCGTCCCGT

FINGER DOMAIN aa L R P V L R S N R V E Y C W C N S G R A -

CAGTGCCACTCAGTGCCTGTCAAAAGTTGCAGCGAGCCAAGGTGTTTCAACGGGGGCACC

241 + + + + + + 300

GTCACGGTGAGTCACGGACAGTTTTCAACGTCGCTCGGTTCCACAAAGTTGCCCCCGTGG aa Q C H S V P V K S C S E P .R C F N G G T

TGCCAGCAGGCCCTGTACTTCTCAGATTTCGTGTGCCAGTGCCCCGAAGGATTTGCTGGG

301 + + + + + + 360

ACGGTCGTCCGGGACATGAAGAGTCTAAAGCACACGGTCACGGGGCTTCCTAAACGACCC

GROWTH FACTOR DOMAIN aa C Q Q A L Y F S D F V C Q C P E G F A G

AAGTGCTGTGAAATAGATACCAGGGCCACGTGCTACGAGGACCAGGGCATCAGCTACAGG

TTCACGACACTTTATCTATGGTCCCGGTGCΔCGATGCTCCTGGTCCCGTAGTCGATGTCC aa K C C E I D T R A T C Y E D Q G I S Y R - GGCACGTGGAGCACAGCGGAGAGTGGCGCCGAGTGCACCAACTGGAACAGCAGCGCGTTG CCGTGCACCTCGTGTCGCCTCTCACCGCGGCTCACGTGGTTGACCTTGTCGTCGCGCAAC aa G T W S T A E S G A E C T N W N S S A L

GCCCAGAAGCCCTACAGCGGGCGGAGGCCAGACGCCATCAGGCTGGGCCTGGGGAACCAC

481 + + + + + + 540

CGGGTCTTCGGGATGTCGCCCGCCTCCGGTCTGCGGTAGTCCGACCCGGACCCCTTGGTG

KRINGLE 1 DOMAIN aa A Q K P Y S G R R P D A I R L G L G N H - AACTACTGCAGAAACCCAGATCGAGACTCAAAGCCCTGGTGCTACGTCTTTAAGGCGGGG 541 + + + + + +. 600

TTGATGACGTCTTTGGGTCTAGCTCTGAGTTTCGGGACCACGATGCAGAAATTCCGCCCC aa N Y C R N P D R D S K P W C Y V F K A G

AAGTACAGCTCAGAGTTCTGCAGCACCCCTGCCTGCTCTGAGGGAAACAGTGACTGCTAC

601 + + + + + + 660

TTCATGTCGAGTCTCAAGACGTCGTGGGGACGGACGAGACTCCCTTTGTCACTGACGATG aa K Y S S E F C S T P A C S E G N S D C Y

TTTGGGAATGGGTCAGCCTACCGTGGCACGCACAGCCTCACCGAGTCGGGTGCCTCCTGC

661 + + + +--- + + 720

AAACCCTTACCCAGTCGGATGGCACCGTGCGTGTCGGAGTGGCTCAGCCCACGGAGGACG aa F G N G S A Y R G T H S L T E S G A S C

CTCCCGTGGAATTCCATGATCCTGATAGGCAAGGTTTACACAGCACAGAACCCCAGTGCC

GAGGGCACCTTAAGGTACTAGGACTATCCGTTCCAAATGTGTCGTGTCTTGGGGTCACGG KRINGLE 2 DOMAIN aa L P W N S M I L I G K V Y T A Q N P S A -

CAGGCACTGGGCCTGGGCAAACATAATTACTGCCGGAATCCTGATGGGGATGCCAAGCCC

781 _{+ + +} + + + 840

GTCCGTGACCCGGACCCGTTTGTATTAATGACGGCCTTAGGACTACCCCTACGGTTCGGG aa Q A L G L G K H N Y C R N P D G D A K P

TGGTGCCACGTGCTGAAGAACCGCAGGCTGACGTGGGAGTACTGTGATGTGCCCTCCTGC

841 + + + + + + 900

ACCACGGTGCACGACTTCTTGGCGTCCGACTGCACCCTCATGACACTACACGGGAGGACG

«1 aa W C H V L K N R R L T W E Y C D V P S C

TCCACCTGCGGCCTGAGACAGTACAGCCAGCCTCAGTTTCGCATCAAAGGAGGGCTCTTC

901 _{+ + +} + + + 960

AGGTGGACGCCGGACTCTGTCATGTCGGTCGGAGTCAAAGCGTAGTTTCCTCCCGAGAAG aa S T C G L R Q Y S Q P Q F R I K G G L F

GCCGACATCGCCTCCCACCCCTGGCAGGCTGCCATCTTTGCCAAGCACAGGAGGTCGCCC

961 + + + + + + 1020

CGGCTGTAGCGGAGGGTGGGGACCGTCCGACGGTAGAAACGGTTCGTGTCCTCCAGCGGG

PROTEASE DOMAIN aa A D I A S H P W Q A A I F A K H R R S P- -

GGAGAGCGGTTCCTGTGCGGGGGCATACTCATCAGCTCCTGCTGGATTCTCTCTGCCGCC

1021 + + + + _{+ +} 1080

CCTCTCGCCAAGGACACGCCCCCGTATGAGTAGTCGAGGACGACCTAAGAGAGACGGCGG aa G E R F L C G G I L I S S C W I L S A A

CACTGCTTCCAGGAGAGGTTTCCGCCCCACCACCTGACGGTGATCTTGGGCAGAACATAC

1081 + + ₊ + _{+ +} 1140

GTGACGAAGGTCCTCTCCAAAGGCGGGGTGGTGGACTGCCACTAGAACCCGTCTTGTATG aa H C F Q E R F P P H H L T V I L G R T Y CGGGTGGTCCCTGGCGAGGAGGAGCAGAAATTTGAAGTCGAAAAATACATTGTCCATAAG 1141 + ₊ + + _{+ +} 1200

GCCCACCAGGGACCGCTCCTCCTCGTCTTTAAACTTCAGCTTTTTATGTAACAGGTATTC aa R V V P G E E E Q K F E V E K Y I V H K -

GAATTCGATGATGACACTTACGACAATGACATTGCGCTGCTGCAGCTGAAATCGGATTCG

1201 + + + + + + 1260

CTTAAGCTACTACTGTGAATGCTGTTACTGTAACGCGACGACGTCGACTTTAGCCTAAGC aa E F D D D T Y D N D I A L L Q L K S D S

TCCCGCTGTGCCCAGGAGAGCAGCGTGGTCCGCACTGTGTGCCTTCCCCCGGCGGACCTG

1261 + + + + + + 1320

AGGGCGACACGGGTCCTCTCGTCGCACCAGGCGTGACACACGGAAGGGGGCCGCCTGGAC aa S R C A Q E S S V V R T V C L P P A D L

CAGCTGCCGGACTGGACGGAGTGTGAGCTCTCCGGCTACGGCAAGCATGAGGCCTTGTCT

1321 + + + + + + 1380

GTCGACGGCCTGACCTGCCTCACACTCGAGAGGCCGATGCCGTTCGTACTCCGGAACAGA aa Q L P D W T E C E L S G Y G K H E A L S

CCTTTCTATTCGGAGCGGCTGAAGGAGGCTCATGTCAGACTGTACCCATCCAGCCGCTGC

1381 _{+ + +} + ₊ + 1440

GGAAAGATAAGCCTCGCCGACTTCCTCCGAGTACAGTCTGACATGGGTAGGTCGGCGACG aa P F Y S E R L K E A H V R L Y P S S R C

ACATCΔCAACATTTACTTAACAGAACAGTCACCGACAACATGCTGTGTGCTGGAGACACT

1441 _{+ + + + + +} 1500

TGTAGTGTTGTAAATGAATTGTCTTGTCAGTGGCTGTTGTACGACACACGACCTCTGTGA aa T S Q H L L N R T V T D N M L C A G D T -

CGGAGCGGCGGGCCCCAGGCAAACTTGCACGACGCCTGCCAGGGCGATTCGGGAGGCCCC

1501 + + + + + + 1560

GCCTCGCCGCCCGGGGTCCGTTTGAACGTGCTGCGGACGGTCCCGCTAAGCCCTCCGGGG aa R S G G P Q A - L H D A C Q G D S G G P

CTGGTGTGTCTGAACGATGGCCGCATGACTTTGGTGGGCATCATCAGCTGGGGCCTGGGC

1561 _{+ + + + + +} 1620

GACCACACAGACTTGCTACCGGCGTACTGAAACCACCCGTAGTAGTCGACCCCGGACCCG aa L V C L N D G R M T L V G I I S W G L G

TGTGGACAGAAGGATGTCCCGGGTGTGTACACCAAGGTTACCAACTACCTAGACTGGATT

1621 + + ^■-+ + + + 1680

ACACCTGTCTTCCTACAGGGCCCACACATGTGGTTCCAATGGTTGATGGATCTGACCTAA aa C G Q K D V P G V Y T K V T N Y L D W I

CGTGACAACATGCGACCGTGACCAGGAACACCCGACTCCTCAAAAGCAAATGAGATCCCG 1681 + + + + + +

GCACTGTTGTACGCTGGCACTGGTCCTTGTGGGCTGAGGAGTTTTCGTTTACTCTAGGGC aa R D N M R P END CCTCTTCTTCTTCAGAAGACACTGCAAAGGCGCAGTGCTTCTCTACAGACTTCTCCAGAC 1741 _{+ + + + + +} 1800

GGAGAAGAAGAAGTCTTCTGTGACGTTTCCGCGTCACGAAGAGATGTCTGAAGAGGTCTG

CCACCACACCGCAGAAGCGGGACGAGACCCTACAGGAGAGGGAAGAGTGGCATTTTCCCA

1801 + + + + + + 1860

GGTGGTGTGGCGTCTTCGCCCTGCTCTGGGATGTCCTCTCCCTTCTCACCGTAAAAGGGT

GATACTTCCCATTTTGGAAGATTTCAGGACTTGGTCTGATTTCAGGATACTCTGTCAGAT

1861 ^'■+ + + + + + 1920

CTATGAAGGGTAAAACCTTCTAAAGTCCTGAACCAGACTAAAGTCCTATGAGACAGTCTA

GGGAAGACATGAATGCACACTAGCCTCTCCAGGAATGCCTCCTCCCTGGGCAGAAATGGC

1921 + + + + + + 1980

CCCTTCTGTACTTACGTGTGATCGGAGAGGTCCTTACGGAGGAGGGACCCGTCTTTACCG

CATGCCACCCTGTTTTCAGCTAAAGCCCAACCTCCTGACCTGTCACCGTGAGCAGCTTTG

1981 _{+ + +} + ₊ -₊ 2040

GTACGGTGGGACAAAAGTCGATTTCGGGTTGGAGGACTGGACAGTGGCACTCGTCGAAAC

GAAACAGGACCACAAAAATGAAAGCATGTCTCAATAGTAAAAGATAACAAGATCTTTCAG

2041 + + + +- + + 2100

CTTTGTCCTGGTGTTTTTACTTTCGTACAGAGTTATCATTTTCTATTGTTCTAGAAAGTC

GAAAGACGGATTGCATTAGAAATAGACAGTATATTTΔTAGTCΔCAAGAGCCCAGCAGCGG

2101 + + + + + + 2160

CTTTCTGCCTAACGTAATCTTTATCTGTCATATAAATATCAGTGTTCTCGGGTCGTCGCC

CTCAAAGTTGGGGCAGGCTGGCTGGCCCGTCATGTTCCTCAAAAGAGCCCTTGACGTCAA

2161 + + + + + + 2220

GAGTTTCAACCCCGTCCGACCGACCGGGCAGTACAAGGAGTTTTCTCGGGAACTGCAGTT

GTCTCCTTCCCCTTTCCCCACTCCCTGGCTCTCAGAAGGTATTCCTTTTGTGTACAGTGT

2221 + + + + + + 2280

CAGAGGAAGGGGAAAGGGGTGAGGGACCGAGAGTCTTCCATAAGGAAAACACATGTCACA

GTAAAGTGTAAATCCTTTTTCTTTATAAACTTTAGAGTAGCATCGAGAGAATTGTATCAT 2281 + + + + + + 2340

CATTTCACATTTAGGAAAAAGAAATATTTGAAATCTCATCGTAGCTCTCTTAACATAGTA TTGAACAACTAGGCTTCAGCAATATTTATAGCAATCCATAGTTAGTTTTTΔCTTTTCGTT

2341 + + + -_{+ + +} 2400

AACTTGTTGATCCGAAGTCGTTATAAATATCGTTAGGTATCAATCAAAΔATGAAAAGCAA GCCACAACCCTGTTTTATACTGTACTTAATAAATTCAGATATATTTTTCACAGTTTTTCC

2401 + + _{+ + + +} 2460

CGGTGTTGGGACAAAATATGACΔTGAATTATTTAAGTCTATATAAAAAGTGTCAAAAAGG TABLE 2

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

10

Propeptide/signal 22 to 108 - 29 to - 1

Finger domain 133 to 246 9 to 46

15

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

Z0<

25

30

TABLE 3

(All non primed letters (eg a)). refer to nucleotide sequences, all pprriimmeedd lleettttiers (eg a )) refer to corresponding amino acid sequences.)

Ayrll. Nhel. Spel. Xbal Mutants

EGAV V51R: N451Q

241 273 a ) CAG.TGC.CAC.TCA.GTG.CCT.AGG.AAA.AGT.TGC.AGC a¹) gin cys his ser val pro arg. lys ser cys ser 45 46 47 48 49 50 51 52 53 54 55

SWS R92S: N4510

364 396 b ) TGC.TGT.GAA.ATA.GAT.ACI.AGI.GCC.ACG.TGC.TAC b ) cys cys glu ile asp thr er ala thr cys tyr 86 87 88 89 90 91 92 93 94 95 96

KNI I101T/ N4510

394 423 c ) TAC.GAG.GAC.CAG.GGC.ACT.AGT.TAC.AGG.GGC c ) tyr glu asp gin gly thr ser tyr arg gly

96 97 98 99 100 101 102 103 104 105 KCl C171 S : N4510

607 636 d ) AGC . TCA . GAG . TTC . GCT . AGC . ACC . CCT . GCC . TGC d¹ ) ser ser gl u phe al_a ser thr pro al a cys 107 168 169 170 171 172 173 174 175 176

KCAl E169A: F170S: N4510

598 633 e ) GGG.AAG.TAC.AGC.TCA.GCT.AGC.TGC.AGC.ACC.CCT.GCC e¹) gly lys tyr ser ser ala er cys ser thr pro ala 164 165 166 167 168 169 170 171 172 173 174 175

KNB2 S189A: A190S: N4510

655 687 f ) TGC.TAC.TTT.GGG.AAT.GGG.GCT.AGC.TAC CGT GGC f ) cys try phe gly asn gly ala ___r tyr arg gly 183 184 185 186 187 188 189 190 191 192 193

KC2 T255P: W256R: N4510

853 888 g ⁾ CTG.AAG.AAC.CGC.AGG.CTG.CCT.AGG.GAG.TAC.TGT.GAT g ⁾ leu lys asn arg arg leu βro arg glu tyr cys asp

249250251 252 253 254255256257 258 259260 KCA2 R252P: N4510

844 879 h ) TGC.CAC.GTG.CTG.AAG.AAC.CCT.AGG.CTG.ACG.TGG.GAG h¹) cys his val leu lys asn pro arg leu thr trp glu 246 247 248 249 250 251 252 253 254255 256 257

SWN S265A: T266S: N4510

883 921 i ) TGT.GAT.GTG.CCC.TCC.TGC.GCT.AGC.TGC.GGC.CTG.AGA.CAG i ) ays asp val pro ser cys ala ser cys gly leu arg gin

259 260 261 262 263 264 265 266 267 268 269 270 271

OMT I279T: K280S: N4510

928 963 j ) CAG.CCT.CAG.TTT.CGC.ACT.AGI.GGA.GGG.CTC.TTC.GCC j ) ^' gin pro gin phe arg thr fir gly gly leu phe ala 274 275 276 277 278 279 280281 282 283 284 285

PEN R529P: P530R: N4510

1678 1713 k ) ATT.CGT.GAC.AAC.ATG.CCT.AGG.TGA.CCA.GGA.ACA.CCC k ) ile arg asp asn met ___o arg end - 524525 526 527 528 529 530 HEAVY CHAIN DELETIONS

N Del de1(171-190):170AS191:N4510

601 618 679 . 690

1 ) AAG.TAC.AGC.TCA.GAG.TTC.GCLAGC.TAC.CGT.GGC.ACG 1 ) cys tyr ser ser glu phe ala sfir tyr arg gly thr 165 166 167 168 169 170 191 192 193 914

K₁ Del del(101-170):100TS171:N4510

394 408 619 633 m ) TAC.GAG.GAC.CAG.GGC.ACT.AGC.TGC.AGC.ACC.CCT.GCC ) tyr glu asp gin gly thr ifir cys ser thr pro ala 96 97 98 99 100 171 172 173 174 175

K_∑ Del del(189-256):188AR257:N45TO

658 672 877 891 n ⁾ TAC.TTT.GGG.AAT.GGG.GCLAGG.GAG.TAC.TGT.GAT.GTG n ) tyr phe gly asn gly al_a arg glu try cys asp val 184 185 186 187 188 257 258 259260261

K K₂ Del del(101-256):100TR257:N4510

394 408 877 891 o ) TAC.GAG.GAC.CAG.GGC.ACT AGG.GAG.TAC.TGT.GAT.GTG o ) tyr glu asp gin gly thr arg glu try cys asp val 96 97 98 99 100 257 258259260261 F Del del(5-51) ; 4TR52;N451Q

109 120 262 273 p ) GGA.GCC.AGA.TCG.ACT.AGG.AAA.AGT.TGC.AGC p ) gly ala arg ser thr arg lys ser cys ser 1 2 3 4 52 53 54 55

G Del del(51-92);50S93;N45lQ

244 258 285 396 q ) TGC.CAC.TCA.GTG.CCT.AG ♦GCC.ACG.TGC.TAC q ) cys his ser val pro ser ala thr cys tyr 46 47 48 49 50 93 94 95 96

FG Del del(5-92);4TS93;N451Q

109 120 285 396 r ) GGA.GCC.AGA.TCG.ACT.AGT.GCC.ACG.TGC♦TAC r ) gly ala arg ser thr ser ala thr cys tyr -3 -2 -1 1 93 94 95 96

Protease del(2-226);lTS267;N451Q

109 - 120 907 921 s ) GGA.GCC.AGA.TCG.ACT.AGC.TGC♦GGC.CTG.AGA.CAG s ) gly ala arg ser thr ser cys gly leu arg glu -3 -2 -1 1 267 268 269 270 271 2K1 DEL ( 1 90-252 INS ( 102-169)

661 675 412 616 t ) TTT . GGG . AAT . GGG . GCT AGC . TAC . AGG . GGC TAC.AGC. TCA. GCT

865 879

AGG.CTG.ACG.TGG.GAG

t¹) phe gly asn gly ala ser tyr arg gly tyr ser ser ala 185 186 187 188 189 102 103 104 105 166 167 168 169

arg leu thr trp gl-u 253 254255256257

2K2 DEL (102-169) INS (190-252)

400 411 676 864 u ) GAC.CAG.GGC.ACT AGC.TAC.CGT.GGC CTG.AAG.AAC.CCT

616 633

ACG.TGC.AGC.ACC.CCT.GCC

u ) asp gly gly thr ser tyr arg gly leu cys asn pro

98 99 100 101 190 191 192 193 249250251 252

ser cys ser thr pro ala 170 171 172 173 174 175 TABLE 4

MUTANT 2F

GARSYQVICRDEKTQMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVPRSRSYQVICRDEKTQMIYQQ HQSWLRPVLRSNRVEYCWCNSGRAQCHSVPVKSCSEPRCFNGGTCQQALYFSDFVCQCPEGFAGKCCEID TRATCYEDQGISYRGTWSTAESGAECTNWNSSALAQKPYSGRRPDAIRLGLGNHNYCRNPDRDSKPWCYV FKAGKYSSEFCSTPACSEGNSDCYFGNGSAYRGTHSLTESGASCLPWNSMILIGKVYTAQNPSAQALGLG KHNYCRNPDGDAKPWCHVLKNRRLTWEYCDVPSCSTCGLRQYSQPQFRIKGGLFADIASHPWQAAIFAKH RRSPGERFLCGGILISSCWILSAAHCFQERFPPHHLTVILGRTYRWPGEEEQKFEVEKYIVHKEFDDDT YDNDIALLQLKSDSSRCAQESSWRTVCLPPADLQLPDWTECELSGYGKHEALSPFYSERLKEAHBRLYP SSRCTSQHLLNRTVTDNMLCAGDTRSGGPQANLHDACQGDSGGPLVCLNDGRMTLVGIISWGLGCGQKDV PGVYTKVTNYLDWIRDNMRP

,

TABLE 5

MUTANT 2G

GARSYQVICRDEKTQMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVPVKSCSEPRCFNGGTCQQALY FSDFVCQCPEGFAGKCCEIDTRKSCSEPRCFNGGTCQQALYFSDFVCQCPEGFAGKCCEIDTRATCYEDQ GISYRGTWSTAESGAECTNWNSSALAQKPYSGRRPDAIRLGLGNHNYCRNPDRDSKPWCYVFKAGKYSSE FCSTPACSEGNSDCYFGNGSAYRGTHSLTESGASCLPWNSMILIGKVYTAQNPSAQALGLGKHNYCRNPD GDAKPWCHVLKNRRLTWEYCDVPSCSTCGLRQYSQPQFRIKGGLFADIASHPWQAAIFAKHRRSPGERFL CGGILISSCWILSAAHCFQERFPPHHLTVILGRTYRWPGEEEQKFEVEKYIVHKEFDDDTYDNDIALLQ LKSDSSRCAQESSVVRTVCLPPADLQLPDWTECELSGYGKHEALSPFYSERLKEAHBRLYPSSRCTSQHL LNRTVTDNMLCAGDTRSGGPQANLHDACQGDSGGPLVCLNDGRMTLVGIISWGLGCGQKDVPGVYTKVTN YLDWIRDNMRP

TABLE 6

Mutant 2FG

GARSYQVICRDEKTQMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVPVKSCSEPRCFNGGTCQQALY FSDFVCQCPEGFAGKCCEIDTSRSYQVICRDEKTQMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVP VKSCSEPRCFNGGTCQQALYFSDFVCQCPEGFAGKCCEIDTRATCYEDQGISYRGTWSTAESGAECTNWN SSALAQKPYSGRRPDAIRLGLGNHNYCRNPDRDSKPWCYVFKAGKYSSEFCSTPACSEGNSDCYFGNGSA YRGTHSLTESGASCLPWNSMILIGKVYTAQNPSAQALGLGKHNYCRNPDGDAKPWCHVLKNRRLTWEYCD VPSCSTCGLRQYSQPQFRIKGGLFADIASHPWQAAIFAKHRRSPGERFLCGGILISSCWILSAAHCFQER FPPHHLTVILGRTYRWPGEEEQKFEVEKYIVHKEFDDDTYDNDIALLQLKSDSSRCAQESSVVRTVCLP PADLQLPDWTECELSGYGKHEALSPFYSERLKEAHBRLYPSSRCTSQHLLNRTVTDNMLCAGDTRSGGPQ ANLHDACQGDSGGPLVCLNDGRMTLVGIISWGLGCGQKDVPGVYTKVTNYLDWIRDNMRP

TABLE 7

MUTANT 2K1 1K2

GARSYQVICRDEKTQMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVPVKSCSEPRCFNGGTCQQALY TO FSDFVCQCPEGFAGKCCEIDTRATCYEDQGISYRGTWSTAESGAECTNWNSSALAQKPYSGRRPDAIRLG LGNHNYCRNPDRDSKPWCYVFKAGKYSSASTPACSEGNSDCYFGNGSAYRGTHSLTESGASCLPWNSMIL IGKVYTAQNPSAQALGLGKHNYCRNPDGDAKPWCHVLKNRRLTWEYCDVPSCSTCGLRQYSQPQFRIKGG LFADIASHPWQAAIFAKHRRSPGERFLCGGILISSCWILSAAHCFQERFPPHHLTVILGRTYRVVPGEEE QKFEVEKYIVHKEFDDDTYDNDIALLQLKSDSSRCAQESSVVRTVCLPPADLQLPDWTECELSGYGKHEA T5 LSPFYSERLKEAHBRLYPSSRCTSQHLLNRTVTDNMLCAGDTRSGGPQANLHDACQGDSGGPLVCLNDGR MTLVGIISWGLGCGQKDVPGVYTKVTNYLDWIRDNMRP

∑σ

25

3:0

5 TABLE 8

MUTANT 1K1 2K2

GARSYQVICRDEKTQMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVPVKSCSEPRCFNGGTCQQALY FSDFVCQCPEGFAGKCCEIDTRATCYEDQGISYRGTWSTAESGAECTNWNSSALAQKPYSGRRPDAIRLG LGNHNYCRNPDRDSKPWCYVFKAGKYSSEFCSTPACSEGNSDCYFGNGSAYRGTHSLTESGASCLPWNSM ILIGKVYTAQNPSAQALGLGKHNYCRNPDGDAKPWCHVLKNPSYRGTHSLTESGASCLPWNSMILIGKVY TAQNPSAQALGLGKHNYCRNPDGDAKPWCHVLKNRRLTWEYCDVPSCSTCGLRQYSQPQFRIKGGLFADI ASHPWQAAIFAKHRRSPGERFLCGGILISSCWILSAAHCFQERFPPHHLTVILGRTYRWPGEEEQKFEV EKYIVHKEFDDDTYDNDIALLQLKSDSSRCAQESSVVRTVCLPPADLQLPDWTECELSGYGKHEALSPFY SERLKEAHBRLYPSSRCTSQHLLNRTVTDNMLCAGDTRSGGPQANLHDACQGDSGGPLVCLNDGRMTLVG IISWGLGCGQKDVPGVYTKVTNYLDWIRDNMRP

TABLE 9

MUTANT 2K1 2K2

TO

GARSYQVICRDEKTQMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVPVKSCSEPRCFNGGTCQQALY FSDFVCQCPEGFAGKCCEIDTRATCYEDQGISYRGTWSTAESGAECTNWNSSALAQKPYSGRRPDAIRLG LGNHNYCRNPDRDSKPWCYVFKAGKYSSEFCSTPACSEGNSDCYFGNGSAYRGTHSLTESGASCLPWNSM ILIGKVYTAQNPSAQALGLGKHNYCRNPDGDAKPWCHVLKNPSYRGTWSTAESGAECTNWNSSALAQKPY

T5 SGRRPDAIRLGLGNHNYCRNPDRDSKPWCYVFKAGKYSSEFCSTPACSEGNSDCYFGNGSAYRGTHSLTE SGASCLPWNSMILIGKVYTAQNPSAQALGLGKHNYCRNPDGDAKPWCHVLKNRRLTWEYCDVPSCSTCGL RQYSQPQFRIKGGLFADIASHPWQAAIFAKHRRSPGERFLCGGILISSCWILSAAHCFQERFPPHHLTVI LGRTYRWPGEEEQKFEVEKYIVHKEFDDDTYDNDIALLQLKSDSSRCAQESSVVRTVCLPPADLQLPDW TECELSGYGKHEALSPFYSERLKEAHBRLYPSSRCTSQHLLNRTVTDNMLCAGDTRSGGPQANLHDACQG

ZO DSGGPLVCLNDGRMTLVGIISWGLGCGQKDVPGVYTKVTNYLDWIRDNMRP

Z5

30

35 TABLE 10

MUTANT 2K1

O

GARSYQVICRDEKTQMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVPVKSCSEPRCFNGGTCQQALY FSDFVCQCPEGFAGKCCEIDTRATCYEDQGISYRGTWSTAESGAECTNWNSSALAQKPYSGRRPDAIRLG LGNHNYCRNPDRDSKPWCYVFKAGKYSSEFCSTPACSEGNSDCYFGNGASYRGTWSTAESGAECTNWNSS ALAQKPYSGRRPDAIRLGLGNHNYCRNPDRDSKPWCYVFKAGKYSSARLTWEYCDVPSCSTCGLRQYSQP

T QFRIKGGLFADIASHPWQAAIFAKHRRSPGERFLCGGILISSCWILSAAHCFQERFPPHHLTVILGRTYR WPGEEEQKFEVEKYIVHKEFDDDTYDNDIALLQLKSDSSRCAQESSVVRTVCLPPADLQLPDWTECELS GYGKHEALSPFYSERLKEAHBRLYPSSRCTSQHLLNRTVTDNMLCAGDTRSGGPQANLHDACQGDSGGPL VCLNDGRMTLVGIISWGLGCGQKDVPGVYTKVTNYLDWIRDNMRP

ZO

Z5

30

35 TABLE 11

MUTANT ZKZ

GARSYQVICRDEKTQMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVPVKSCSEPRCFNGGTCQQALY FSDFVCQCPEGFAGKCCEIDTRATCYEDQGTSYRGTHSLTESGASCLPWNSMILIGKVYTAQNPSAQALG LGKHNYCRNPDGDAKPWCHVLKNPSCSTPACSEGNSDCYFGNGSAYRGTHSLTESGASCLPWNSMILIGK VYTAQNPSAQALGLGKHNYCRNPDGDAKPWCHVLKNRRLTWEYCDVPSCSTCGLRQYSQPQFRIKGGLFA DIASHPWQAAIFAKHRRSPGERFLCGGILISSCWILSAAHCFQERFPPHHLTVILGRTYRVVPGEEEQKF EVEKYIVHKEFDDDTYDNDIALLQLKSDSSRCAQESSVVRTVCLPPADLQLPDWTECELSGYGKHEALSP FYSERLKEAHBRLYPSSRCTSQHLLNRTVTDNMLCAGDTRSGGPQANLHDACQGDSGGPLVCLNDGRMTL VGIISWGLGCGQKDVPGVYTKVTNYLDWIRDNMRP

TABLE 12

MUTANT S+N

GARSYQVICRDEKTQMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVPVKSCSEPRCFNGGTCQQALY FSDFVCQCPEGFAGKCCEIDTRATCYEDQGISYRGTWSTAESGAECTNWNSSALAQKPYSGRRPDAIRLG LGNHNYCRNPDRDSKPWCYVFKAGKYSSEFCSTPACSEGNSDCYFGNGSAYRGTHSLTESGASCLPWNSM ILIGKVYTAQNPSAQALGLGKHNYCRNPDGDAKPWCHVLKNRRLTWEYCDVPSCASATCYEDQGISYRGT WSTAESGAECTNWNSSALAQKPYSGRRPDAIRLGLGNHNYCRNPDRDSKPWCYVFKAGKYSSEFCSTPAC SEGNSDCYFGNGSAYRGTHSLTESGASCLPWNSMILIGKVYTAQNPSAQALGLGKHNYCRNPDGDAKPWC HVLKNRRLTWEYCDVPSCSTCGLRQYSQPQFRIKGGLFADIASHPWQAAIFAKHRRSPGERFLCGGILIS SCWILSAAHCFQERFPPHHLTVILGRTYRVVPGEEEQKFEVEKYIVHKEFDDDTYDNDIALLQLKSDSSR CAQESSVVRTVCLPPADLQLPDWTECELSGYGKHEALSPFYSERLKEAHBRLYPSSRCTSQHLLNRTVTD NMLCAGDTRSGGPQANLHDACQGDSGGPLVCLNDGRMTLVGIISWGLGCGQKDVPGVYTKVTNYLDWIRD NMRP

TABLE 13

MUTANT OMS

GARSYQVICRDEKTQMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVPVKSCSEPRCFNGGTCQQA FSDFVCQCPEGFAGKCCEIDTRATCYEDQGISYRGTWSTAESGAECTNWNSSALAQKPYSGRRPDAIR LGNHNYCRNPDRDSKPWCYVFKAGKYSSEFCSTPACSEGNSDCYFGNGSAYRGTHSLTESGASCLPWN ILIGKVYTAQNPSAQALGLGKHNYCRNPDGDAKPWCHVLKNRRLTWEYCDVPSCSTCGLRQYSQPQFR ATCYEDQGISYRGTWSTAESGAECTNWNSSALAQKPYSGRRPDAIRLGLGNHNYCRNPDRDSKPWCYV AGKYSSEFCSTPACSEGNSDCYFGNGSAYRGTHSLTESGASCLPWNSMILIGKVYTAQNPSAQALGLG NYCRNPDGDAKPWCHVLKNRRLTWEYCDVPSCSTCGLRQYSQPQFRIKGGLFADIASHPWQAAIFAKH SPGERFLCGGILISSCWILSAAHCFQERFPPHHLTVILGRTYRVVPGEEEQKFEVEKYIVHKEFDDDT NDIALLQLKSDSSRCAQESSWRTVCLPPADLQLPDWTECELSGYGKHEALSPFYSERLKEAHBRLYP RCTSQHLLNRTVTDNMLCAGDTRSGGPQANLHDACQGDSGGPLVCLNDGRMTLVGIISWGLGCGQKDV VYTKVTNYLDWIRDNMRP

TABLE 14

MUTANT 2 Prot 1 CV

GARSYQVICRDEKTQMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVPVKSCSEPRCFNGGTCQQALY FSDFVCQCPEGFAGKCCEIDTRATCYEDQGISYRGTWSTAESGAECTNWNSSALAQKPYSGRRPDAIRLG LGNHNYCRNPDRDSKPWCYVFKAGKYSSEFCSTPACSEGNSDCYFGNGSAYRGTHSLTESGASCLPWNSM ILIGKVYTAQNPSAQALGLGKHNYCRNPDGDAKPWCHVLKNRRLTWEYCDVPSCSTCGLRQYSQPQFRIK GGLFADIASHPWQAAIFAKHRRSPGERFLCGGILISSCWILSAAHCFQERFPPHHLTVILGRTYRVVPGE EEQKFEVEKYIVHKEFDDDTYDNDIALLQLKSDSSRCAQESSWRTVCLPPADLQLPDWTECELSGYGKH EALSPFYSERLKEAHBRLYPSSRCTSQHLLNRTVTDNMLCAGDTRSGGPQANLHDACQGDSGGPLVCLND GRMTLVGIISWGLGCGQKDVPGVYTKVTNYLDWIRDNMPSGGLFADIASHPWQAAIFAKHRRSPGERFLC GGILISSCWILSAAHCFQERFPPHHLTVILGRTYRWPGEEEQKFEVEKYIVHKEFDDDTYDNDIALLQL KSDSSRCAQESSWRTVCLPPADLQLPDWTECELSGYGKHEALSPFYSERLKEAHBRLYPSSRCTSQHLL NRTVTDNMLCAGDTRSGGPQANLHDACQGDSGGPLVCLNDGRMTLVGIISWGLGCGQKDVPGVYTKVTNY LDWIRDNMRP

TABLE 15

MUTANT 2 Prot 2 CV

GARSYQVICRDEKTQMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVPVKSCSEPRCFNGGTCQQAL FSDFVCQCPEGFAGKCCEIDTRATCYEDQGISYRGTWSTAESGAECTNWNSSALAQKPYSGRRPDAIRL LGNHNYCRNPDRDSKPWCYVFKAGKYSSEFCSTPACSEGNSDCYFGNGSAYRGTHSLTESGASCLPWNS ILIGKVYTAQNPSAQALGLGKHNYCRNPDGDAKPWCHVLKNRRLTWEYCDVPSCSTCGLRQYSQPQFRI GGLFADIASHPWQAAIFAKHRRSPGERFLCGGILISSCWILSAAHCFQERFPPHHLTVILGRTYRVVPG EEQKFEVEKYIVHKEFDDDTYDNDIALLQLKSDSSRCAQESSVVRTVCLPPADLQLPDWTECELSGYGK EALSPFYSERLKEAHBRLYPSSRCTSQHLLNRTVTDNMLCAGDTRSGGPQANLHDACQGDSGGPLVCLN GRMTLVGIISWGLGCGQKDVPGVYTKVTNYLDWIRDNMPSCGLRQYSQPQFRIKGGLFADIASHPWQAAI FAKHRRSPGERFLCGGILISSCWILSAAHCFQERFPPHHLTVILGRTYRWPGEEEQKFEVEKYIVHKE DDDTYDNDIALLQLKSDSSRCAQESSVVRTVCLPPADLQLPDWTECELSGYGKHEALSPFYSERLKEAH RLYPSSRCTSQHLLNRTVTDNMLCAGDTRSGGPQANLHDACQGDSGGPLVCLNDGRMTLVGIISWGLGC QKDVPGVYTKVTNYLDWIRDNMRP

TABLE 16

MUTANT Ult

GARSTSCGLRQYSQPQFRIKGGLFADIASHPWQAAIFAKHRRSPGERFLCGGILISSCWILSAAHCFQER FPPHHLTVILGRTYRVVPGEEEQKFEVEKYIVHKEFDDDTYDNDIALLQLKSDSSRCAQESSWRTVCLP PADLQLPDWTECELSGYGKHEALSPFYSERLKEAHBRLYPSSRCTSQHLLNRTVTDNMLCAGDTRSGGPQ ANLHDACQGDSGGPLVCLNDGRMTLVGIISWGLGCGQKDVPGVYTKVTNYLDWIRDNMPSRSYQVICRDE KTQMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVPVKSCSEPRCFNGGTCQQALYFSDFVCQCPEGF AGKCCEIDTRATCYEDQGISYRGTWSTAESGAECTNWNSSALAQKPYSGRRPDAIRLGLGNHNYCRNPDR DSKPWCYVFKAGKYSSEFCSTPACSEGNSDCYFGNGSAYRGTHSLTESGASCLPWNSMILIGKVYTAQNP SAQALGLGKHNYCRNPDGDAKPWCHVLKNRRLTWEYCDVPSCAR

TABLE 17

Mutant Specific Activity (mlU/ug)

MSI 350

KNI * 485

10 KCl * 584

KCAl 517

KNB2* 418

KC2 745

KCA2* 617

15

Ndel v.l. Kl del 120 K2del 400 K1 K2del v.l.

20^~

2K1, 1K2 320 1K1, 2k2 50 2K1, 2K2 v.l.

25 where v.l. = very low activity and * signifies the most preferred molecules

30

35

Claims

Cl ims

1. A method for producing an altered DNA sequence encoding a molecule having a biological activity associated with tissue plasminogen activator (tPA), which_.sequence is adapted for predictable rearrangement or deletion of deoxyribonucleic acids by the use of at least one endonuclease restriction enzyme comprising the steps of:

a) providing a sequence to be altered, said sequence encoding a molecule having a tPA biological activity; and

b) altering said sequence at at least two pre-existing restriction sites for creating restriction endonuclease sites susceptible of cleavage by at least one restriction endonuclease selected from the group consisting of Avrll, Nhel, Spel and Xbal.

2. A DNA sequence produced by the method of Claim 1

3. A host cell transformed or transfected by a sequence comprising the DNA sequence of Claim 2.

4. The method of Claim 1 wherein one of said sites is altered to create a cleavage site for an enzyme selected from the group consisting of Avrll, Nhel, Spel and Xbal and a second of said at least two sites is altered to create a cleavage site for an enzyme selected from the remaining members of said enzyme group.

5. A DNA sequence comprising:

241 273

CAG.TGC.CAC.TCA.GTG.CCT.AGG.AAA.AGT.TGC.AGC

and all sequences hybridizing thereto under stringent conditions.

A DNA sequence comprising:

364 396

TGC.TGT.GAA.ATA.GAT.ACI.AGI.GCC.ACG.TGC.TAC

and all sequences hybridizing thereto under stringent conditions.

7. A DNA sequence comprising:

394 423 TAC.GAG.GAC.CAG.GGC.ACT.AGC.TAC.AGG.GGC

and all sequences hybridizing thereto under stringent conditions.

8. A DNA sequence comprising:

607 636

AGC.TCA.GAG.TTC.GCl.AGC.ACC.CCT.GCC.TGC

and all sequences hybridizing thereto under stringent conditions.

9. A DNA sequence compri si ng:

598 633 GGG.AAG.TAC.AGC.TCA.GCT.AGC.TGC.AGC.ACC.CCT.GCC

and all sequences hybridizing thereto under stringent conditions.

10. A DNA sequence comprising:

655 687

TGC.TAC.TTT.GGG.AAT.GGG.GCI.AGC.TAC CGT GGC

and all sequences hybridizing thereto under stringent conditions.

11. A DNA sequence comprising:

853 888

CTG.AAG.AAC.CGC.AGG.CTG.CCI.AGG.GAG.TAC.TGT.GAT

and all sequences hybridizing thereto under stringent conditions.

12. A DNA sequence comprising:

844 879

TGC.CAC.GTG.CTG.AAG.AAC.CCT.AGG.CTG.ACG.TGG.GAG

and all sequences hybridizing thereto under stringent conditions.

13. A DNA sequence compri si ng:

883 921

TGT . GAT . GTG . CCC . TCC . TGC . GCT . AGC . TGC . GGC . CTG . AGA . CAG

and al l sequences hybri dizi ng thereto under stri ngent condi tions .

14. A DNA sequence comprising:

928 963

CAG.CCT.CAG.TTT.CGC.ACT.ACT.GGA.GGG.CTC.TTC.GCC

and all sequences hybridizing thereto under stringent conditions.

15. A DNA sequence comprising:

1678 1713 : ATT.CGT.GAC.AAC.ATG.CCT.AGG.TGA.CCA.GGA.ACA.CCC

and all sequences hybridizing thereto under stringent conditions.

16. A DNA sequence comprising:

601 618 679 690

AAG.TAC.AGC.TCA.GAG.TTC.GCT.AGC.TAC.CGT.GGC.ACG

and all sequences hybridizing thereto under stringent

. conditions.

17. A DNA sequence compri si ng :

394 408 619 633

TAC.GAG.GAC.CAG.GGC.AC AGC.TGC.AGC.ACC.CCT.GCC

and all sequence^'s hybridizing thereto under stringent conditions.

18. A DNA sequence comprising:

658 672 877 891

TAC.TTT.GGG.AAT.GGG.GCLAGG.GAG.TAC.TGT.GAT.GTG

and all sequences hybridizing thereto under stringent conditions.

19. A DNA sequence comprising:

394 408 877 891 Dt TAC.GAG.GAC.CAG.GGC.ACT AGG.GAG.TAC.TGT.GAT.GTG

and all sequences hybridizing thereto under stringent conditions.

5 20. A DNA sequence comprising:

109 120 262 273 GGA.GCC.AGA.TCG.ACTVAGG.AAA.AGT.TGC.AGC

0 and all sequences hybridizing thereto under stringent conditions.

5

21. A DNA sequence comprising:

244 258 285 396

TGC.CAC.TCA.GTG.CC .AGT.GCC.ACG.TGC.TAC

and all sequences hybridizing thereto under stringent conditions.

22. A DNA sequence comprising:

109 120 285 396

GGA.GCC.AGA.TCG.ACT.AGT.GCC.ACG.TGC.TAC

and all sequences hybridizing thereto under stringent conditions.

23. A DNA sequence comprising:

109 120 907 921

GGA.GCC.AGA.TCG.ACT.AGC♦TGC.GGC.CTG.AGA.CAG

and all sequences hybridizing thereto under stringent conditions.

24. A DNA sequence comprising:

661 675 412 616

TTT.GGG.AAT.GGG.GCT AGC.TAC.AGG.GGC TAC.AGC.TCA.GCT

865 879

AGG.CTG.ACG.TGG.GAG

and all sequences hybridizing thereto under stringent conditions.

25. A DNA sequence comprising:

400 411 676 864 GAC.CAG.GGC.ACT AGC.TAC.CGT.GGC CTG.AAG.AAC.CCT

and all sequences hybridizing thereto under stringent conditions.

26. A DNA sequence of Claim 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 further comprising the substitution of glutamine for asparagine at position 451.

27. A host cell transformed or transfected by a sequence comprising the sequence of Claim 5, 6, 7, 8, 9, 10, 11, 12,

13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25.

28. A host cell transformed or transfected by a sequence comprising the sequence of Claim 26.

29. The host cell of Claim 27 wherein said host cell is C1Z7.

30. The host cell of Claim Σ8 wherein said host cell is C1Z7.

31. The molecule produced by the host cell of Claim 27.

32. The molecule produced by the host cell of Claim 28.

33. The molecule produced by the host cell of Claim 29.

34. The molecule produced by the host cell of Claim 30.