AU619803B2 - Rearranged tissue plasminogen activators and method for producing same - Google Patents

Description

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OPI DATE 25/08/89 w( AOJP DATE 28/09/89 APPLN. I D 30534 89

PCT

PCT NUMBER PCT/US89/00465 (51) International Patent Classification 4 International Publication Number: WO 89/ 07146 C12N 15/00, C07H 21/04 Al(43) International Publication Date: 10 Augs 99(00 9 (22) International Filing Date: 3 February 1989 (03.02,89) pean patent), FR (European patent), GB (European patent), IT (European patent), JIP, LU (European pa- (31)Pririt AplictionNumer:152692 tent), NL (European patent), SR, (European patent).

(32) Priority Date: 5 February 1988 (05.02,88) Published With international search report.

(33) Priority Country: us Before the expiration of the time limit for amending the ~in the event of the receipt (71) SECTION 34 DIREC I0N SEE ErUO1 RM DIECTE 0( N?'iI (72) K,4 15 It' d >e~I (74) Agent: HOFER, Mark, Integrated Genetics, Inc., :9' One Mountain Road, Framingham, MA 01701 (US).

(54) Title: REARRANGED TISSUE PLASMINOGEN ACTIVATORS AND METHOD FOR PRODUCING SAIVIE ZZ <Z 0 i:2 S/PP F G K1 K2

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S=Spel site A=Avrll site N=NheI site Plasminogen Cleavage Site (57) Abstract Novel Methods aire provided for creating altered DNAs encoding molecules having a tPA biological propert.- The altered DNAs allow for a wide variety of predictable altl.ations or' the tPA molecule including the domain regkcns. Addi' tionally, novel tPA molecules resulting from the methods ate dksclosed..

S- 1 c" 'rll-1- WO 89/07146 PCT/US89/00465 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 j 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 'ringle 2 (at Arg 275 The light chain contains the serine protease domain, which is homologous to trypsin and chymotrypsin.

V tPA is a relatively clot-specific plasminogen activator due to its affinity for fibrin, which forms the clot matrix. This fibrin x

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WO89/071 2 PCT/US89/00465 WO 89/07145 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 Srates 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 et al. Biol. Chem. 256, 7035 (1981).

Therapeutic utility of exogenous tPA was demonstrated with the melanoma-derived material (Collen ei al., J. Clin. Inv. 71, 368 (1983); Koringer 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 sequnces 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 W O 89/07146 3 PCT/US89/00465 cells. The resultant modified tPA molecules having altered ami no 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 mosue cells were subsequently reported by Reddy et al., 3. Cell Biochem.

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 jt a1., 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.

r -I I WO 89/07146 4 PCT/US89/00465 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.

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.

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 domains. It was surprising to discover that such alterations still result in molecules having a biological activity associated with wild-type 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.

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 positi.rns in the tPA cDNA corresponding to boundaries of the tPA protein domains 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 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.

1 I WO'89/07146 -5 PCT/US89/00465 The altered cDNA's described above can then be advantageously ji manipulated to generate deletions or duplications of tPA domains as Sshown in Table 4. These manipulations are described in additional detail below and generally involve cutting the modil'ied cDNA's with 5 the appropriate restriction enzymes and thereafter l1gating the S' 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 j 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.

Ii Brief Description of the Drawings Further understanding of the principles and objects of the i present invention may be had by studying the accompanying Figures 1 wherein: Figure 1 shows the position of Avr II, Nhe land Spe I sites in the tPA cDNA; I Figure 2 is a schematic diagram of construction of vector expressing mtPA with K 1 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; i r WO 89/07146 PCT/US89/00465 -6- Figure 5 is a representation of CLH3AXBPV vector for stable of mtPA in C127 cells; Figure 6 is a schematic diagram of the construction of vector containing mtPA havingK 1

K

2 duplication; Figures 7a-1 and 7a-2 show the construction of the SP.SNA vector 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.

Detailed Description and Best Mode Definitions 20 The term "cell culture" refers to the containmenr 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, Sbacteria and mammalian cells which can be grown in cell culture and 2 transfected or transformed with a plasmid or vector containing a gene encoding a molecule having a tPA biological characteristic and expression of such molecule.

(1 The term "domain" refers to a discrete continuous part of an amino acid sequence that can be equipped with a particular function. With respect to tPA, references (Banyal, 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)) have been defined the domain regions and Figure 1 herein discloses the approximate locations of the domain regions.

SUBSTITUTE SHEET WO 89/07146 7 PCT/US89/00465 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).

WO 89/07146 8 PCT/US89/00465 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 S 15 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 S 20 as restriction enzymes refers to enzymes which cleave doublestranded 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'CTTAA Althouqh 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 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 g~r~ i WO 89/07146 9 PCT/US89/00465 double-stranded DNA with initiation or transcription occurring from left to right to Numbering of nucleotides and amino acids correspond I:o the particular amino terminal form shown in Table 1, although it will be readily understood the obvious numbering modifications may apply with different NH 2 terminal forms. The table below provides the standard abbreviations for amino acids.

Abbreviations for amino acids Three-letter One-letter 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 G1n Q Glutamic .c id Glu E Glutamine or glutamic acid Glx Z Glyine Gly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe

F

Proline Pro

P

Serine Ser S Threonine Thr

T

Tryptophan Trp H Tyrosine Tyr Y Valine Val

V

*L--_IL

I I WO 89/07146 0 PCT/US89/00465 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 on Maniatis). Methods of in itro 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 rostriciton 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 t al. (1987). Essentially mRNA was made from VVO 89/07146 1 PCT/US89/00465 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 convett 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 goe; zreement 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 1987).

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

i Generation of a Linker Insertion Mutant A Spe I linker was placed at the Bgl II (115) site of the tPA cDNA sucti 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, 1pg 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 KPO 4 (pH 6.6mM MgC12, lOmM mercaptoethanol, 250 pM dATP, dCTP, dTTP and dGTP together with 8p of ONA polymerase I (Klenow fragment). This procedure fills in the 5' cohesive ends to generate "blunt ended" linearized DNA.

WO 89/07146 12 PCT/US89/00465 After incubating at room temperature for one hour the Klenow was heat inactivated at 65 0 C 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 10mM MgC1 2 20mM DTT, ImM ATP and 50pg m 1 bovine serum albumin) and 200u of T 4 DNA ligase. The ligation was allowed to proceed overnight at 22°C.

The ligated DNA was phenol:chloroform:IAA 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 4 ligase and allowed to ligate overnight at 16*C, A small aliquot of the religated DNA was transfected into the E. cli 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 was confirmed: Bl ,T (115) Spe I (8 mer) 106 126 AGA.GGA.GCCAGA.TCG.ACT.AGT.CGATCT TAC.CAA arg gly ala arg ser thr ser arq ser tyr gln 2 3 4 5 6 1 13, WO 89/07 146 3-PCT/US89/00465 Synthesis of Primers for Site-Specific Mutagenesis The human uterine tPA cONA was modified by site-specific mutagenesis using synthetic oligonucleotides prepared by the solid phase phosphotriester method The following primzrs were synthesized and used for such mutagenesis. They are in the anti-sense sequence.

1. Primer EGAV GCA.ACTTT.CCTAGG.CAC.TGA 3' for introduction of an Avr II site (CCT-AGG) at nucleotides 256-261 2. Primer SkIS GCA.CGT.GGC.ACT.AGT.ATC.TAT.TTC 3' for introduction of a Spe I site (ACT.AGT) at nucleotides 379-384 3. Primer KNI CCCCTGTAACTAGTGCCCTG 31 for introduction of a SPe X site (AGTAGT) at nuc~eotjdes 409-444 4. Primer KC1 5' GGGTGCTA..G-CGAACTCTGAG 3' for introduction of an NheI (GCTAGC) site at nucleotides 619-6324 14, WO089/07146 4-PCT/US89/00465 Primer KCAI GTGCTFCAGCTAGCTGAGCTGTAC 3' for introduction of an Nhe I (GCTAGC) at nucleotides 613-518 6. Primer KNB2 GCCACGGTAGCTAGCCCCATTCCC 3' for introduction of an Nhe I (GCTAGC) site at nucleo-'Ides 673-678 7. Primer KC2 5' GTACTCCCIAGLCAGCCTGC 3' for introduction of an Avr II (CCTAGG) site at nucleotides 871-876 8. Primer KCA2 CGTCAGCCT8AGGGTTCTTCAGC 3' for U introduction of an Avr 11 (CCTAGG) site at nucleotides 862-867 259. Primer SWN CAGGCCGCAG-CTAGCGCAGGAGGG 3' for intro3duction of an Nhe 1 (GCTAGC) at nucleotides 901-906 Primer OT 5'1 CCCTCCACTA.GTGCGAAACTG 3' for Introduction of a SPe I site (ACTAGT) at nucleotides 943-948.

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drTIU i >;/n046 WO 89/07146 15 11. Primer Pen CTGGTCACCTAGGCATGTTG 3' for introduction of an Avr II (CCTAGG) at nucleotides 1693-1698 Site Specific Mutagenesis using above Synthesized Oligonucleotides The M13 based oligonucleotide directed mutagenesis procedure is essentially that detailed by Kunkel et al (Proc. NaT'1. Acad.

Sci. USA 82, 488 (1985)). Phage containing the M13tPA.MGA vector (the tPA cDNA which has the Asn 451 gln mutation described in the aforementioned copending application and publication of Wei et al.) were grown in an E.coli strain C3236 (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.

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 sldwly in a water oath for several hours. The annealed fragments were 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 MgC1 2 500 uM each of dATP, dTTP, dCTP, and dGTP, 1mM ATP, 2.5 units of Klenow and 2 units of T4 DNA ligase. Incubation started at 0OC for 5 minutes, room temperature for 5 minutes, 37°C for 2 hours, and overnight at 16°C.

ul of the above reactive mixture was added to 300 ul of competent DH 5 bacterial cells and left in ice for 1 hour. The bacteria were heat shocked at 37*C for 5 minutes then serially

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41 WO 89/07146 PCT/US89/00465 16 diluted into 3 ml of soft agar 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°C.

Nitrocellulose lifts were taken from the plates, baked at 32 0 C, under vacuum, for 2 hours and hybridized with the 6 P32 ATP labeled mutant oligonucleotide. Mutant positive isolates were determined by thermal denaturation of the DNA:oligo duplexes and further grown in JM101 on a larger scale for maxi-prep Rf DNA isolation. The Bam HI-Hind III fragment containing the tPA cDNA including the Asn 451 gln mutation and the new mutation was further recloned into the SP65 veLtor and/or mammalian cell expression vector using normal procedures of restriction enzyme cleavage and ligation.

T

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

559 570

AAC.CGC.AGG.CTG

(251) N R R L (254) site-specific, oligonecleotide directed mutagenesis yields

ACC.CCI.AGG.CTG

N E R L AvrII site (CCTAGG).

Other mutants generated using this method are as follows: WO089/07146 PCT/US89/00465 17

EGAV

sWs KN 1 KC1 KCA 1 KN B2 KC2 KCA2

SN

OMT

Pen (V51R; N451Q) (R92S; N451Q) (MOlT N451Q) (Cl71S; N451Q) (E169A; F17OS; (S189A; A19OS; (T255P; W256R; (R252P; N451Q) (S265A; T266S; (1279T; K280S; (R529P; P530R: N451Q) N451Q) N451Q) N451Q) N451 Q) N451 Q) The unique restriction enzyme sites were introduced with a mutant (MGS (N4Sl.Q) 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 0); Bgl 11 (115) Spe(8)

EGAV

between the N-terminus of the mature processed tPA protein and the N-terminus of the finger domain between the C-terminus of the finger domain and the N-terminus of the growth factor domain between the C-terminus of the growth factor domain and the N-terminus of the Kringle I domain at the N-terminus of the Kringle I domain sWs ~j WO 89/07146 PCT/US89/00465 18 KCA 1 KN 82 at the domai n at the domai n at the domai n at the domai n at the domai n C-~terminus of the Kringle 1 C~terminus of the Kringle 1 N-terminus of the Kringle 2 C-terminus of the Kringle 2 C-terminus of the Kringle 2 KCA2 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 doma' n Pen at the C-terminus of the protease domain Manipulations of the tPA cDNA; AvrII, NheI, SpeI and XbaI restriction enzymes recognize unique and separate hexanucleotide sequences le.

AvrII CCTAGG NheI GCTAGC SPOI ACTAGT XbaI TCTAGA

V

U

WO 89/07146 PCT/US89/00465 19 When cleaved by overhang is common to AvrII CCTAGG

GGATCC

the appropriate restriction all four sites ie.

enzyme the

TO

Nhel Spel Xbal

GCTAGC

CGATCG

ACTAGT

TGATCA

TCTAGA

AGATCT

C

GGATC

G

CGATC

A

TGATC

T

AGATC

CTAGG

C

CTAGC

G

CTAGT

A

CTAGA

T

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 AvrII 5' overhang

C

GGATC

Nhel 5' overhang

CTAGC

G

ligate

CCTAGC

GGATCG

PCT/US89/00465 WO 89/07146 20 Since it has lost its palindromic feature it is no longer cInaved by either AvrII or NheI. The same finding applies to other possible ccmbi nations.

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

A) Deletions The AvrII, NheI and SpeI sites located in the following mutants; EGAV, S1WS, KNI, KG1, KCAl, KNB2, 'KcQ', 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 proteW~ product.

An example of such a manipulation is the deletion of the cONA encoding the kringle 1 domain using mutants KNl and KCA1..

400 420

GAC-.CAG-GGC-ACT-AGT-TAC-AGG

asp gln gly thr ser tyr arg 98 1 105 SpeI cut

I

GAG. GAG. GCC-ACT .AG KCA 1 607 630

AGC-TCA-.GCT-AGC-TGC-AGC-ACC-CCT

ser ser ala ser cys ser thr pro 167 1 174 NheI cut

I

C.TGC.AGC.ACC.CCT

Si gate (digest with SpeI and NheI to remove any contaminating KNl or KCAl WO 89/07146 PC!/US89/00465

A

U

t

I

I

I

p

I

21- 400 630 GAG. GAG GGC .ACT .AC TGC -AGG. ACC. CCr asp gin gly thr ser cys ser thr pro 98 99 100 171 172 173 174 ie, Ki del or del (101-170); iOOTSi7i; N451Q Details of the KI deletion construction is as follows: 1 jig of plasmid SP6 KNI was digested with HIindIII adSe n go plasmid SP6 KCAI was digested with HindIII and NheI. The DNA fragments generated were separated and isolated by gel electrophoresis. The large fragment from SP6 KNI and the small fragment from SP6 KCA1 were ligated using normal procedures; redigested With SpeI and NheI and transfected into the E. coi strain DH5 and plated onto LB media plates containing amplicillin.

Resistant, colonies were picked and grown. Plasmid DNA was prepared on a sm~all scale ,~nd analyzed by restriction enzyme analysis for the deletions and loss of the SpeI and NheI sites. The procedure is shown schematically in Figure 2.

The same procedure was used to generate of mutants.

Mutant Lesion N del Kl del K2 del KlK2 del F del G del EG del Protease (171-190); 170AS191 N451Q (101-170); 1OOTSl7l;, N451 Q (189-256); 188AR257; N451 Q (101-256); 100TR257; N451 Q (5-51) 4TR52; N451Q (51-92) 50593; N451Q (5-92) 4TS93; N451Q 1TS267: N451Q the following series Domains Deleted Interkringie Kringle 1 Kringle 2 Kringles I and 2 Finger Growth factor Finger Growth factor The heavy chain.

(Finger, growth factor, both kringles) WO 89/07146 PCT/US89/00465 22 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 (KN1) was ligated with a plasmid containing the unique Nhe I site (KCA1) via their common cohesive ends.

Plasmids and Restriction Enzyme Mutants Sites Utilized N del KCA1 (Nhel) and KNB2 (NheI) K1 del KN1 (Spel) and KCA1 (Nhel) K2 del KNB2 (Nhel) and KCA2 (AvrII) K1K2 del KNI (Spel) and KCA2 (AvrII) F del Bgl/Spe (Spel) and EGAV (AVrII) G del EGAV (AvrII) and SNS (Spel) FG del Bgl/Spe'(SpeI) and SWS (Spel) Protease Bgl/Spe (Spel) and SWN (NheI) B, Duplications Referring to Figure 1 utilizing the AvrII, 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 doiain(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 SNS and SWN (see Figure 6).

The following duplications h',ve been constructed in a similar fashion. The amino acid sequences are shown in Tables 4 9 and 12 WO 89/07146 PCT/US89/00465 -23- Mutant Duplicated Domains 2Kl1 11(2 W~i 2K(2 2K1? 2K(2 S-sN 2F 2G 2FG 2 Prot 1CV 2 Prot 2CV Kringle 1 Kringle 2 Kringles 1 and, 2 Kringles 1 and, 2 Finger Growth factor Finger and growth factor Protease domain Protease domain In the construction of the mutants encoding the duplications the following plasmids were appropriately restriction enzyme digested and ligated as in Figures 2t 6 and 7a-1, 7a-2, 7b-1 and 7b-2.

For example in the generation of the duplication mutant, 2F, at plasmid containing the introduced unique Avr1tI (EGAV) was ligated with a plasrnid containing the introduced Spel site at, the BglII (115) site (Bgl/Spe) via their common adhesive ends.

Mvjtant Plasrnids and restriction enzymes utilized EGAV (AvrII) aj~ Bgl/Spe (SpeI) SWS (SpeI) and, E-CAV (AvrII) SWS (SpeI) and Bgl,/Spe (Spel) KCAl KCA2 KCA2 (Nhel) and KNI (Spel) (AvrII) and O(B2 (NheI) (AvriI) and KNI (Spel) 35 S+14I

OMS

SUN (NheI) and SWS (Spel) GOlT (Spel) and SWS (Spel) SUBSTITUTE SHEET I- I PCT/US89/00465 WO 89/07146 -24- Mutant 2 Prot 1CV 2 Prot 2CV Plasmids and restriction enzymes utilized Pen (AvrII) and OMT (Spel) Pen (AvrII) and SVN (Nhel) C) Rearrangements Utilizing the AvrII, 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 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-1, 7a-2, 7b-1 and 7b-2).

The final product was designated the ult rearrangement mutant, In gq similar manner other mutants were generated with rearranged 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.

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.

SUBSTITUThr SHEET Vrt WO 89/07146 PCT/US89/00465 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-1, 7a-2, 7b-l and 7b-2.

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

Plasmids and restriction enzymes utilized Mutant KCA1 (NheI)! KNi (SpeI), KCA2 and KNB2 (SpeI) (AvrII KCAl (rqheI)t KNI (SpeI)tp KCA2 (AvrII) and KNB2 (SpeI) Bgl/Spe (Spel), Swn (NheI) and Pen (AvrII) liLT Ve rif icat ionof 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.

Vectors The BamlIX-Hindlll fragment containing the tPA cDNA sequence isolated from M13MP18.t'A by restriction enzyme analysis and gel electrophoresis and ligated into the tam III, HindZIll cleaved SUEaSTIIUTIE SH4ET WC) 89/07 146 PCT/US89/0.046S -26v'ector I Promega Biotech). This orientation (with the 5' end of the LL)NA adjacent to the SP6 promoter) enabled an analysis of the mutant protein product by i-n 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 cONA deletion generation, LK4448ffS, tPN Referring to Figure 3, mutated cDNA miolecuies were recloned Into the LK444IBHS vector. The BanHI, HindIII fragmcnt was isolated from SKI-, tPA, a mvtant derivative obtained by restriction enzyme Lcleavage and gel filtration. This fragment was ligated to a BamHI, Hin(IIII cleavage vector, LK444BHS, ThIs mutaton allowed for the transient expression of the tPA analogue in a COS 7 cell line driven by the human 13-actin, promoter.

CLH3AXBPV.tPA Referring to Figure 5, the Sal I fragment was isolated from a Mutated derivative by striction enzyme cleavage and gel purification. This fragment was ligated to an Xho I cleavedI [Ivector CLfI3AXBPV as shown in Figure 5, The orientation was determined and selected such that the inserted sequence was under 11 25 the drivir'g force of the metal lothioriine promoter in C127 cells.

CAS~2iE The tPA cDNA or mutants were restriction enzyme digested with Salt and reeloaied with the XhQI si~te cleaved in the vector. The orientation of the tPA cOMA or mutant i such that expritssion is dr.ven, by the metallothionein promoter, The CLH3AX.V20MF vector is used for the stable expression of tPA or tModirled tPA in CHO cells With the ability for methatreXate amplification.

u-rr. I 'I WO 89/07146 PCT/US89/00465 27 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.

1' 3 x 106 cells were grown in 100 mm plates in DMEM glutamine for 1 day preceding transfection. Ten to 20 pg of DNA was added to 2.0 ml of tris-buffered saline (pH 1 ml of 2 mgiml 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 -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 pl 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 fre'e 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 101 FBS, 1% glutamine medium at a'density of 7 x 105 cells per 100 mm dish 24 hrs. before transfection. 100-50 pg of plasmid DNA in 0.5 ml transfection buffer (the composition of which is 4 g NaC1, 0.185 g KC1, 0.05 g Na 2

HPO

4 0.5 g dextrose, and 2.5 g HEPES, pH 7.5 per 500 ml total volume). 30 p1 of 2M CaC1 2 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 WO 89/07146 PCT/US89/00465 28 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°C. The media was removed and the cells were then shocked with 15% glycsrol in transfection buffer at 37°C 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 pM methotrexate. Cell colonies which survive the treatment appear 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 a, 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.

WO 89/07146 PCT/US89/00465 29 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 The mtPAs of the invention may advantageously be admixed with a pharmaceutically acceptable carrier substance, saline, and administered orally, intravenously, or by injection into affected arteries of the heart. Administration will be generally as is TS 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, 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 chaaiber 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 WO 89/07146 PCT/US89/00465 30 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 application.

r i WO 89/07146 PCT/US89/00465 TABLE 1

TGTGAAGCAATCATGGATGCAATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGTGGA

1

ACACTTCGTTAGTACCTACGTTACTTCTCTCCCGAGACGACACACGACGACGACACACCT

START SIGNAL aa MMKRGLCCVLLLCG

GCAGTCTTCGTTTCGCCCAGCCAGGAAATCCATGCCCGATTCAGAAGAGGAGCCAGATCT

61

CGTCAGAAGCAAAGCGGGTCGGTCCTTTAGGTACGGGCTAAGTCTTCTCCTCGGTCTAGA

PROPEPTIDE -11 a 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 0 V I C R D E K T Q M I Y Q If Q S W

CTGCGCCCTGTGCTCAGAAGCAACCGGGTGGAATATTGCTGGTGCAACAGTGGCAGGGCA

181 +240

GACGCGGGACACGAGTCTTCGTTGGCCCACCTTATAACGACCACGTTGTCACCGTCCCGT

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

CAGTGCCACTCAGTGCTGTCAAAAGTTGCAGCGAGCCAAGGTGTTTCAACGGGGGCACC

241 300

GTCACGGTGAGTCACGGACAGTTTTCAACGTCGCTCGGTTCCACAAAGTTGCCCCCGTGG

+1 aa 0 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 A L Y F SD F V CO C PEG FAG

AAGTGCTGTGAAATAGATACCAGGGCCACGTGCTACGAGGACCAGGGCATCAGCTACAGG

420

TTCACGACACTTTATCTATGGTCCCGGTGCACGATGCTCCTGGTCCCGTAGTCGATGTCC

aa K C C E I D T R A T C Y E DO0 G I S Y R

GGCACGTGGAGCACAGCGGAGAGTGGCGCCGAGTGCACCAACTGGAACAGOAGCGCGTTG

+480

CCGTGCACCTCGTGTCGCCTCTCACCGCGGCTCACGTGGTTGACCTTGTCGTCGCGCAAC

a G T W S T A E S G A E C T N W N S S A L

GCCCAGAAGCCCTACAGCGGGCGGAGGCCAGACGCCATCAGGCTGGGCOTGGGGAACCAC

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 WO 89/07146 PCT/US89/00465

AACTACTGCAGAAACCCAGATCGAGACTCAAAGCCCTGGTGCTACGTCTTTAAGGCGGG

541 600

TTGATGACGTCTTTGGGTCTAGCTCTGAGTTCGGGACCACGATGCAGAAATTCCGCCCC

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

4,, aa KY S SE F Cs T P A CS E G N S DC Y

TTTGGGAATGGGTCAGCCTACCGTGGCACGCACAGCCTCACCGAGTCGGGTGCCTCCTGC

661 720

AAACCCTTACCCAGTCGGATGGCACCGTGCGTGTCGGAGTGGCTCAGCCCACGGAGGACG

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

CTCCCGTGGAATTCCATGATCCTGATAGGCAAGGTTTACACAGCACAGAACCCCAGTGCC

721 780

GAGGGCACCTTAAGGTACTAGGACTATCCGTTCCAAATGTGTCGTGTCTTGGGGTCACG

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

CAGGCACTGGGCCTGGGCAAACATAATTACTGCCCGAATCCTGATGGGGATGCCAACCC

840

GTCCGTGACCCGGACCCGTTTGTATTAATGACGGCCTTAGGACTACCCCTACGGTTCGGG

aa Q A L G L G K H N Y CR N P D G D A K P

TGGTGCCACGTGCTGAAGAACCGCAGGCTGACGTGGGAGTACTGTGATGTGCCCTCCTGC

841 900

ACCACGGTGCACGACTTCTTGGCGTCCGACTGCACCCTCATGACACTACACGGGAGGACG

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

TCCACCTGCGGCCTGAACAGTACAGCCAGCCTCAGTTTCGCATCAAAGGAGGGCTCTTC

960

AGGTGGACGCCGGACTCTGTCATGTCGGTCGGAGTCAAAGCGTAGTTTCCTCCCGAGAAG

41t aa S T C G L R 0 Y S P P Q F R I K G G L F

GCCGACATCGCCTCCCACCCCTGGCAGCTGCCATCTTTGCCAAGCACAGGAGGTCGCCC

1020

CGGCTGTAGCGGAGGGTGGGGACCGTCCGACGGTAGAAACGGTTCGTGTCCTCCAGCGGG

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

GGAGAGCGGTTCCTGTGCGGGGGCATACTCATCAGCTCCTGCTGGATTCTCTCTGCCGCC

1021 1080

CCTCTCGCCAAGGACACGCCCCCGTATGAGTAGTCGAGGACGACCTMCAGAGACGGCGG

aa G E R F' L C G G I L I S SC W I L S A A

CACTGCTTCCAGGAGAGGTTTCCGCCCCACCACCTGACGGTGATCTTGGGCAGMCATAC

1140

GTACGAAGGTCCTCTCCAAAGGCGGGGTGGTGGACTGCCACTAGMCCCGTCTTGTATG

aa, H C F Q E R F P P H H L T V I L G R T Y il Iil WO 89/07 146 PCT/US89/00465

CGGGTGGTCCCTGGCGAGGAGGAGCAGAAATTTGAAGTCGAAAAATACATTGTCCATAAG

1141 1200

GCCCACCAGGGACCGCTCCTCCTCGTCTTTAAACTTCAGCTTTTATATGTAACAGGTATTC

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

GAATTCGATGATGACACTTACGACAATGACATTGCGCTGCTGCAGCTGAAATCGGATTCG

1201 1260

TCAGCTGCCGGAGACTGGACGGAGCTGTGAGCTCTCCGGCTCGGCGGGCCTG

16

AGCGACGGCCTCTGTCGAG(GTACACTCGAGAGGCCGAGCCGTCCGCGAC

aa SQR PDAWQ E CS ELSGYRGKVHEL PA DL

CACTTCTCGGAGGCGGATGTGAGCTCATCGCTACCCATAGCCTGCT

1381 1440

GTGAGTACCTGCCACTCTCCGAGTAGCGATGGGTAGTCGGACGA

aa P F Y S E R L K E A H V R L Y P S S R C ACATCACAACATTTACTTAACAGAACAGTCACCGACAACATGCTGTGT GCTGGAGACACT 1441

TGTAGTGTTGTAAATGAATTGTCTTGTCAGTGGCTGTTGTACGACACACGACCTCTGTGA

aa T S Q H L L. N R T V T D N M L C A G D T '4 CGGAGCGGCGGGCCCCAGGCAAACTTGCACGACGCCTGCCAGGGCGATTCGGGAGGCCCC 1501 1560 K GCCTCGCCGCCCGGGGTCCGTTTGAACGTGCTGCGGACGGTCCCGCTAAGCCCTCCGGGG aa R 9 G G P Q A N' C D S

CTGGTGTGTCTGAACGATGGCCGCAT(ACTTTGGTGGGCATCATCAGCTGGGOCCTIGGGC

14 1561 1620

~GACCACACAGACTTGCTACCGGCGTACTGAAACCACCCGTAGTAGTCGACCCCGGACCCG

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

TGTGGACAGAAGGATGTCCCGGGTGTGTACACCA.AGCTTACCAACTACCTAGACTGGATT

1621

ACACCTGTCTTCCTACAGGGCCCACACATGTGGTTCCAATGGTTLGATGGATCTGACCTMA

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

CGTGACAACATGCGACCGTGACCAGGAACACCCGACTCCTCAAGCAAATGAGATCCCG

181 GCACTGTTGTACGCTGGCACTGGTCCTTGTGGGCTGAGGAGTTTTCGTTTAkCTCTAGGGC +1 aa R D N M R P END WOQ89/07146 PCT/US89/00465

CCTCTTCTTCTTCAGAAGACACTGCAAAGGCGCAGTGCTTCTCTACAOACTTCTCCAGAC

1741 1800

GGAGAAGAAGAAGTCTTCTGTGACGTTTCCGCGTCACGAAGAGATGCTCTGAAGAGGTCTG

CCACCACACCGCAGAAGCGGGACGAGACCCTACAGGAGAGGGAAGAGTGGCATTTTCCCA

1801 1860

GGTGGTGTGGCGTCTTCGCCCTGCTCTGGGATGTCCTCTCCCTTCTCACCGTAAAAGGGT

GATACTTCCCATTTTGGAAGATTTCAGGACTTGGTCTGATTTCAGGATACTCTGTCAGAT

1861 1920

CTATGAAGGGTAAAACCTTCTAAAGTCCTGAACCAGACTAAAGTCCTATGAGACAGTCTA

GGGAAGACATGAATGCACACTAG(CTCTCCAGGAATGCCTCCTCCCTGGGCAGAAATGGC

1921 1980 CCCT'rCTGTACTTACGTGTGATCGGAGA(7GTCCTTAcCGGAGGAGCGACCCGTCTTTACCG

CATGCCACCCTGTTTTCAGCTAAAGCCCAACCTCCTGACCTGTCACCGTGAGCAGCTTTG

1981 2040

GTACGGTGGGACAAAAGTCGATTTCGGGTTGGAGGACTGGACAGTGGCACTCGTCGAAAC

GAAACAGGACCACAAAAATGAAAGCATGTCTCAATAGTAAAAGATAACMAGATCTTTCAG

CTTTGTCCTGGTGTTTTTACTTTCGTACAGAGTTATCATTTTCTATTGTTCTAGAAAGTC

GAAAGACGGATTGCATTAGAAATAGACAGTATATTTATAGTCACMAGAGCCCAGCAGCGG

CTTTCTGCCTAACGTAATCTTTATCTGTCATATAAATATCAGTGTTCTCGGGTCGTCGCC

CTCAAAGI'TGGGGCAGLGCTGGCTGGCCCGTCATGTTCCTCAAAAGAGCCCTTGACGTCAA

2220

GAGTTTCAACCCCGTCCGACCGACCGGGCAGTACMAGGAGTTTTCTCGGGAACTGCAGTT

GTC TCCTTCCCCTTTCCCCACTCCCTGGCTCTCAGAAGGTATTCCTTTTGTGTACAGTGT 2221 2280

CAGAGGAAGGGGAAAGGGGTGAGGGACCAGAGTCTTCCATAAGGAAAACACATGTCACA

GTAAAGTGTAAATCCTTTTTCTTTATAAACTTTAGAGTAGCATCGAGAGAATrGTATCAT 2340

CATTTCACATTTAGGAAAAAGAAATATTTGAAATCTCATCGTAGCTCTCTTAACATAGTA

TTGAACAACTAGGCTTCAGCAATTTAAACTCCATAGTTAGTTTTTACTTTTCGTT

2400

AACTTGTTGATCCGAAGTCGTTATAATATCGTTAGGTATCTCAAATGAAAAGCAA

GCCACMACCCTGTTTTATACTGTACTTAATAAATTCAGATATATTTTTCACAGTTTTTCC

2460

CGGTGTTGGGACAAAATATGACATGATTATTTGTCTATATAAGTGTCAAGG

WOx;c 89/07146 35 PCT/US89/00465 TABLE 2 DESIGNATION OF TPA DOMAINS (after Degan et al. 1986) Propeptide/signal Finger domain Growth factor domain [(tingle 1 domain [(ringle 2 domain Pro tease domain 108 246 367 634 900 1698 29 9 54 183 279

I

.36 WO 89/07146 PCT/USS9100465 TABLE 3 (All non primed letters (eg refer to nucleotide sequences, all primed letters (eg refer to corresponding amino acid sequences.) AvrII. NheI, Spel. XbaI Mutants EGAV V51R, N4510Q 241 273 a CAG.TGC.CAC.TCA.GTG.CCT.AGGAAA.AGT,TGC.AGC al) gln cys his sei val pro ara lys ser cys ser 46 47 48 49 50 51 52 53 54 swS R92S: N4510 364 396 b TGC, TGT AA 4AA. GAT. ACIAG. GCC. AG TGC TAG b) cys cys glu ile asp thr se~r ala thr cys tyr 86 87 88 89 90 91 92 93 94 95 96 -KN 1 1-101T/ 394 423 c TAC. GAG. GAC. CAG. GGC. AQ, AGT, TAC. AGG. GX c tyr glu asp gin gly liar ser tyr arg gly 96 97 98 99 100 101 102 103 104 105 I WO 89/07146 37 PCT/US89/00465 C171S: -N4510 607 636 d AGC.TCA.GAG.TTC.GCT,AGC.ACC.CCT.GCC.TGC di) ser ser glu phe j ser thr pro ala cys 107 168 169 170 171 172 173 174 175 176 KCA 1 E169A: F170S: N451Q 598 633 e) GGGAAG.TAC,AGC.TCA.GCT.AGC.TGC.AGC.ACC.CCT.GCC e 1 gly lys tyr ser ser ala ser cys ser thr pro ala 164 165 166 167 168 169 170 171 172 173 174 175 KNB2Z S189A: A190S: N4510 655 687 f TGC.TAC.TTT,GGG.AAT.GGG.$CI.AGCTAC CGT GGC f) cys try phe gly asn gly ala ieir tyr arg gly 183 184 185 186 187 188 189 190 191 192, M9 T255Pt W256Rt N4510 853 888 g CTG.AAGAAC.CGC.AGG.CTG.gCI.AGG.GAG.TAC.TGT.GAT g1) leu lys asn arg arg 1eu M r rgu tyr cys asp 249 250 251 252 253 254 255 256 257 258 259 260 WO 89/07146 38 PCT/US89/00465 KCA2 R252P: N4510 844 879 h 'fCC.CAC.GTG.CTG.AAG.AAC.CCT.AGG.CTG.ACG.TGG.GAG h 1 cys his val leu lys asn pro arg leu thr trp glu 246 247 248 249 250 251 252 253 254 255 256 257 SN S265A: T266S: N4510 883 921 i TGT.GAT.GTG,CCC.TCC.TGC.GCT.AGC.TGC.GGC.CTG.AGACAG i 1 ays asp val pro ser cys alk cys gly leu arg gin 259 260 261 262 263 264 265 266 267 268 269 270 271 QMT 1279T: K280S N4510 928 963 J CAG,CCTqCAG.TTT,C-GCqACT.AGT.GGA,GGG*CTC.TTC.GCC gin pro gin phe arg ±is .ig gly gly ieu phe ala 274 275 276 277 278 279 280 281 282 283 284 285

MN~

R529P: P530R,: 1678 1713 k ATThCGT.GAC.AACATG.C -jAGG,TGACCA.GGA.ACA.CCC k 1 lie arg asp asn" met Q end- 524 525 526 527 528 529 530 WO 89/07146 101 39 HEAVY CHAIN. DELETIONS PCT/US89/004,65 N Del dPI(171-'IQn),17nAqI91 -N4510 601 618 679 690 AAG. TAC.AGC. TCA GAG. TTC.CT2--'I" ACCII4 GGC.ACG )cys tyr ser sev glu phe A-1 e tyr- arg gly thr 165 166 167 168 169 170 191 192 193 914 K, Del f]PI(101-170)!10OTqI7I !N4510 394 408 619 633 mn TAC,GAG.GACCAGGGC.ACT.AG~QTGC.AGC.ACCqCCT.GCC mn tyr glu asp gin gly thr ur_ cys sev' thr pro ala 96 97 98 99 100 171 172 173 174 175 9 2 -Del del (189-256) :188AR257:,N4510 658 672 877 891 n TAC.TTT.CiGG,AAT.GGG.GCT.AGG.GAG.TAC.TGT.GAT.GTG n tyr phe gly asn gly al arg glu try cys asp val 184 185 186 187 188 257 258 259 260 261 K D1K l del(101-256)!100TR257,:N4510 394 408 87''7 891 o TAC.GAG.GAGCAG.GGCAL Agg.GAG.TAC.TGT.GAT.GTG o) tyr glu asp gin gly tk gu try cys asp Val 96 97 98 99 100 257 258 259 260 261 r ii lj 4 it WO 89/071A6 F Del del 4TR5Z; N451JQ 262 273 G. AAA .AGT.TGC. AGC glys sercys ser 52 53 54 del(51-92) ;50S93;N451Q PCT/US89/00465 109 120 p GGA. GCC.-AGA .TCS. ACT. AG p) gly ala arg sez thr ar, 1 2 3 4 G3 Del 1~44 258 285 3 q OC. CAC. TCA. GTG iCCT. AGT. GCC -ACG, TGC. TAC qC z'ys his ser va, pro ser ala thr cys tyr 46 47 48 49 50 9i 94 95 96 FG Del del 4TS93; ;N451Q 109 120 2853 r) GGAGCC.AGA.TCG ACT.AGT.GCCXACG.TOC.TAC r) gly ala arg .9er tht' ser ala thr cys tyr -3 -2 -1 1 93 94 95 96 6 del(2-226) TS267 ;N451Q Pro tease 109 120 907 921 s)GGA -GCC..'CA TCG. ACT. AGC. TGC.GCGC. CTG, AGA -cAG s) gly ala arg ser thr seir cys gly leu arg glti -3 -2 -1 1 267 268 269 270 271

L

WO 89/07146 41 VO 8/0716 1 -PCT/US89/00465 2 K]nEL (190-252 INS (102-169) 661 675 t TTT.GGG.AAT.GGG.GCT 412 61

AGC.TAC.AGG.GGC-------TAC.AGC.TCA.GCT

865 8

AGG.CTG.ACG.TGG.GAG

phe gly asn gly ala 185 186 187 188 1189 ser tyr arg ser ser ala 102 103 104 105 166 167 168 169 arg leu Ohr trp glu 253 254 255 256 257 2 K2 DEL (102-169) INS (190-252) 400 4111 676 864 U GAC.CAG.GGC.ACT AGC.TAC.CGT.GGC-------CTG.AAGAACCCT 616 633

ACG.TGC.AGC.ACC.CCT.GCC

asp gly ly thr 98 99 100 101 ser tyr arg gly 190 191 192 193 leu cys asn pro 249 250 251 252 ser cys ser thr pro ala 170 171 172 173 174 175 r3iA4;~~.

I I i WO 89/07 146 PCT/US89/00465 42 TABLE 4 MUTANT 2F GARSYQVI CRDEKTQMIYQQHQSWLRPVLRSNRVEYC WCNSGRAQCHS VPRS RSYQVI CR0 EKTQMI YQQ HQSNLRPVLRSN RVEYCMCNSGRAQCHSVPVKSCS EPRCFNGGTCQQALYFSDFVCQCP EGFAGKCCEI 0 TRATCY EDQGI SYRGTWSTAESGA ECTNWN SSA LAQKPYSGR RPDA IRLGLGNHN vCRNPDfWSKPNCYV FKAGKYSSEFCSTPACSEGNSDCYFGNGSAYRGTHSLTESGASCLPMNSMI LIGKVYTAQNPSAQALGLG KHNYCRN PDGDAKPWCHVLKNRRLTWEYCDVPSCSTCGLRQYSQPQFRI KGGLvADI ASH PWQAA T FAKH RRSPGERFLCGGI LISSCWI LSAAHCFQERFPPHHLTVI LGRTYRVVPGEEEQKFEVEKYIVHKEFD00T YDNDIALLQLKSDSSRCAQESSVVRTVCLPPADLQLPDWTECELSGYGKH EALSPFYS ELKAH RPLYP SSRCTSQHLLNRTVTDNMLCAGDTRSGGPQANLHDACQGDSGGPLVCLNDGRMTLVGI ISWGLGCGQKDV

PGVYTKVTNYLDWIRDNMRP

U

WO 89/07146 PCT/US89/00465 43

TABLE

MUTANT 2G GARSYQVICRDEKTQMIYQQHQSHLRPVLRSNRVEYCNCNSGRAQCHSVPVKSCS EPRCFNGGTCQQALY

FSDFVCQCPEGFAGKCCEIDTRKSCSEPRCFNGGTCQQALYFSDFVCQCPEGFAGKCCEIDTRATCYEDQ

GISYRGTWSTAESGAECTNWJNSSALAQKPYSGRRPDAI RLGLGNHNYCRNPDRIJSKPNCYVFKAGKYSSE FCSTPALS EGNSDCYFGNGSAYRGTIS LTESGASCLPHNSMI LIGKVYTAQN PSAQALGLGKHNYCR~iPD GDAKPNCHVLKN RRLTWEYCDVPSCSTCGLRQYSQPQFR IKGGL FAD IASH PWQAA I FAKHRRSPGERFL CGGILISSCWI LSAAHCFQERFPPHHLTVI LGRTYRVVPGEEEQKFEVEKYIVHKEP'DDDTYDNDIALLQ LKSDSSRCAQESSVVRTVCLPPADLQLPDHTEGELSGYGKHEALSPFYS ERLKEAHBRLYPSSRCTSQH L LNRTVTDNMLCAGDTRSGGPQANLHDACQGDSGGPLVCLNDGRMTLVGI ISWGLGCGQKDVPGVYTKVTN' YLDNI RDNMRP r ii -41 PCT/US89/ 00465 WO 89/07146 44 TABLE 6 Mutant 2FG

GARSYQVICRDEKTQMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVPVKSCSEPRCFNGGTCQQALY

FSD FVCQCPEGFAGr,-NCCE IDTS RSYQVI CRDEKTQM IYQQHQSW L RPVLRS NRVEYCWCN SG RAQC{SVP VKSCSEPR CFNIGGTt.QQALYFSDFVCQCPEGFAGKCCEI DTRATCY EDQGI SYRGTWSTA ESGAECTNN SSALAQKPYSGRRPDAI RLGLGNHNYCRNPDRDSKPHCYVFKAGKYSS EFCSTPACSEGNSDCYFGNGSA YRGTHSLTESGASCLPHNSMI LIGKVYTAQNPSAQALGLGKHNYCRN PDGDAKPWCHVLKN RR LTHEYCD VPSCSTCGLRQYSQPQFRIKGGLFAOIASHPWQAAI FAKHRRSPGERFLCGGI LISSCNI LSAAHCFQER FPPHHLTVI LGRTYRWPGEEEQKFEVEKYIVHKEFDDDTYDNDIALLQLKSDSSRCAQESSVVRTVCLP PADLQLPDWTECELSGYGKH EA LS PFYS ERLKEAH BR LYPSS RClTSQH LLN RTVTDNMLCAGDTRSGGPQ AN LHDACQ'-PJSGGPLVCLNDGRMTLVGI ISWGLGCGQKDVPGVYTKVTNYLDWI RDNMRP WO 89/07146 PCT/US89/00465 45 TABLE 7 MUTANT 2Kl lK2 GARSYQVI CRDEKTQMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHS VPVKSCS EPRGFNGGTCQQA LY

FSDFVCQCPEGFAGKCCEIDTRATCYEDQGISYRGTWSTAESGAECTNHNSSALAQKPYSGRRPDAIRLG

LGNHNYCRNPDRDSKPWCYVFKAGKYSSASTPACSEGNSDCYFGNGSAYRGTHSLTESGASCLPHNSMI

L

I GKVYTAQNPSAQALGLGKHNYCRNPDGDAKPWCH VLKNRR LTWEYCO VPSCSTCGLRQYSQPQFRI KGG LFADIASHPHQAAI FAKHRRSPGERFLCGGI LISSCNI LSAAHCFQERFPPHHLTVI LGRTYRVVPGEEE QKFEVEKYIVHKEFDDDTYDNDIALLQLKSDSSRCAQESSVVRTVCLPPADLQLPDWTECELSGYGKH

EA

LSPFYSERLKEAHBRLYPSSRCTSQHLILNRTVTDNMLCAGDTRSGGPQANLHOACQGDSGGPLVCLNJGR

MTLVGI I SkGLGCGQKD VPG VYTKVTNYLDWTA RDNMRP 1\ 1'

V

I WO 89/07146 PCT/US89/00465 46 TABLE 8 MUTANT iKI 2K2 GARSYQVI CRDEKTQMIYQQHQSWLRP VLRSNRVEYCWCNSGRAQCHSVPVKSCS EPRCFNGGTCQQA LY FSD FVCQCP EGFAGKCCE IDTRATCY EDQGI SYRGTHS TA ESGA ECTNHNS SA LAQKPYSGR RPDA I R LG LGNHNYCRNPDRDSKPWCYVFKAGKYSSEFCSTPACS~cGNSDCYFGNGSAYRGTHSLi ESGASCLPNNSM I LIGKWYTAQNPSAQALGLGKHNYCRNPDGDAKPWCH VLKiHPSYRGTHSLTESGASCLPWNSMI LIGKVY TAQNPSAQALGLGKHNYCRNPflGQDAKPWCHVLKN RRLTWEYCDVPSCSTCGLRQYSQPQFR IKGGL FADI ASHPWQAAI FAKHRRSPGERFLCGGI LISSCWI LSAAHCFQERFPPHHLTVI LGRTYRVVPGEEEQKFEV EKYIVHKEFDDDTYDNDIALLQLKSQSSRCAQESSVVRTVCLPPADLQLPDHTECELSGYGKH EALSPFY S RLKEAHBRLYPSSRCTSQH LLNRT VTDNMLCAGDTRSGGPQAN LHDACQGOSGGPLVCLN DGRMTLVG I ISWGLGCGQKDVPGVYTKVTNYLDWIRDNMRP POO-

K

I~i

I

I

I

t WO 89/07146 PCT/US89/00465 47 TABLE 9 MUTANT 2Kl WK GARSYQVICRDEKTQMIYQQHQSNLRPVLRSNRVEYCHCNSGRAQCHSVPVKSCS EPCFNGGTCQQALY

FSDFVCQCPEGFAGKCCEIDTIRATCYEDQGISYRGTWSTAESGAECTNWNSSALAQKPYSGRRPDAIRLG

LGNHNYCRNPDRDSKPWCYVFKAGKYSSEFC STPACSEGNSDCYFGNGSAYRGTHS LTESGASCLPNNSM I LIGKVYTAQNPSAQALGL GKHNYCRNPOGDAKP WCH VLKNPSYRGTIISTAESGAECTNNNSSA LAQKPY SGRRPDAI RLGLGNHNYCRNPDRDSKPHCYVFKAGKYSSEFCSTPACSEGNSDCYFGNGSAYRGTHS LTE SGASCLPWNSIII LIGKVYTAQNPSAQALGLGKHNYCRNPDGDAKPWHIVLKNRRLTHEYCDVPSCSTCGL RQYSQPQFRIKGGLFADIASHPWQAAI FAKHRRSPGERFLCGGI L7SSCHI LSAAHCFQERFPPHH LTVI

LGRTYRVVPGEEEQKFEVEKYIVHKEFDDDTYDNDIALLQLKSDSSRCAQESSVVRTVCLPPADLQLPDH

TECELSGYGKHEALSPFYSERLKEAHBRLYPSSRCTSQHLLNRTVTDNMLCAGDTRSGGPQANLHDACQG

DSGGPLVCLNiOGRMTLVGI I SWGLGCGQKO VPGVYTKVTNYLDJI RDNMRP WO 89/07146 WO 89/7 146PCT/US89/00465 48 TABLE MUTANT 201 GARSYQVICRDEKTQMIYQQHQSHLRPVLRSNRVEYCWCNSGRAQCHSVPVKSCS EPRCFNGGTCQQALY FSDFVCQCPEGFAGKCCEIDTRATCYEDQGISYRG'rHSTAESGAECTNN~SSALAQKPYSGRRPDAiRLG LGNHNYCRNPDRDSKPWCYVFKAGKYSSEFCSTPACS EGNSDCYFGNGASYRGTHSTAESGAECTNNNS S ALAQKPYSGRRPDAI RLGLGNHNYCRNPDRDFI-PWCYVFKAGKYSSARLTHEYCDVPSCSTCGLRQYSQP QFRIKGGLFADIASIPMQAAI FAKHRRSPGERFLCGGI LI SSCWI LSAAHCFQERFPPHHLTVI LGRTYR

VVPGEEEQKFEVEKYIVHKEFDDDTYDNDIALLQLKSDSSRCAQESSVVRTVCLPPALQLPDNJTECELS

GYGKHEALSPFYSERLKEAHBiRLYPSSRCTSQHLLNRTVTDNMLCAGDTRSGGPQANLHDACQGDSGGPL VCLNDGRMTLVGI I SWGLGCGQKDVPGVYTKVTNYLDWI RDNMRP r7 WO 89/07 146 PCT/US89/00465 49 TABLE I11 MUTANT 2K2 GARSYQViCRDEKTQMIYQQHQSkHLRPVLRSN RVEYCkJCNSGRAQCHSVPVKSCS EPRCFNGGTCQQA LY

FSDFVCQCPEGFAGKCCEIDTRATCYEDQGTSYRGTHSLTESGASCLPWNSMILIGKVYTAQNPSAQALG

LGKHNYCRNPDGDAKPHCHVLKNPSCSTPACS EGNSDCYFGNGSAYRGTHSLTESGASCL.PMNSMI LI GK VYTAQNPSAQALGLGKHNYCRNPDGDAKPWCHVLKNRRLTNEYGDVPSGSTGGLRQYSQPQFRI KGGLFA IIASHPWQAAI FAKHRRSPGERFLCGGI LISSCHI LSAAHCFQERFPPHHLTVI LGRTYRVVPGEEEQKF

EVEKYIVHKEFDDDTYDNDIALLQLKSDSSRCAQESSVVRTVCLPPADLQLPDHTEGELSGYGKHEALSP

FYSERLKEAHBRLYPSSRCTSQHLLNRTVTDNMLCAGDTRSGGPQANLHDACQGDSGGPLVCLNDGRMTL

VGI ISWGLGCGQKDVPGVYTKVTNYLDWI RDNMRP WO 89/07146 WO 8907146PCT/US89/00465 TABLE 12 MUTANT Si-N GARSY~QVI CRDEKTQMIYQQHQSWLRPVLRSNRVEYCW.CN SGRAQCHS VPVKSCS EPRCFNGGTCQQA LY FSDFVCQCPEGFAGKCCEIDTRATCYEDQGI SYRGTNSTAESGAECTNWNSSALAQKPYSGRRPDAI RLG LGN HNYCRN PD RDSKPWJCYVFKAGKYSS EFCSTPACS EGN SDCY FGN GSAYRGTH SLT ESGASC LPN SM I LIGKVYTAQNPSAQALGLGKHNYCRNPDGDAKPNCHVLKNRRLThEYCDVPSCASATCYEDQGISYRGT NSTAESGAECTNMNSSALAQKPYSGRRPDAI RLGLGNHNYCRNPDRDSKPNCYVFKAGKYS S E FGSTPAC SEGNSDCYFGNGSAYRGTHSLTESGASCLPWJNSMI LIGKVYTAQNPSAQALGLGKHNYCRNPOGDAKPNC HVLKNRRLTWEYCDVPSCSTCGLRQYSQPQFRIKGGLFADIASHPNQAAI FAKHRRSPGER FLCGGI LIS SCWAI LSAAHCFQERFPPHHLTVI LGRTYRVVPGEEEQKFEVEKYIVHKEFDDDTYDNDIALLQLKSDSSR CAQESSVVRTVCLPPADLQLPONTECELSGYGKHEALSPFYS ERLKEAHiBRLYPSSRCTSQN LLNRTVTO NMLCAGDTRSGGPQANLHDACQGDSGGPLVCLNDGRMTLVGI I SkGLGCGQKDVPGVYTKVTNYLDWI RD

NMRP

VwQ89/07146 -5 51 PCT/US89/00465 TABLE 13 MUTANT OMS GARSYQVI CR0 EKTQMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVP VKSCS EPRCFNGGTCQQA LY FSDFVCQCPEGFAGKCCEIDTRATCYEDQGI SYRGTWSTAESGAECTNWNSSALAQKPYSGRRPDAI RLG LGN HNYCRN PDRSKPHCYVF KAGKYSS EFCSTPACS EGNSDCY FGNGSAYRGTHS LTE SGASCLPHN SM I LI GKVYTAQN PSAQALGLGKH NYCRN PDGDAKPHCH VLKNRRLTWEYCDVPSCSTCGLRQYSQPQF RTS ATCYEDQGI SYRGTWSTAESGAECTNHNSSALAQKPYSGRRPDAI RLGLGNHNYCRN PDRDSKPfICYVFK AGKYSSEFCSTPACSEGNSDCYrGNGSAYRGTHSLTESGASCLPMNSMI

LIGKVYTAQNPSAQALGLGKK

NYCRNPDGDAKPMCHVLKNRRLTWEYCDVPSCSTCGLRQYSQPQFRIKGGLFADIASHPWQAAI

FAKHRR

SPGERFLCGGILISSCHILSAAHCFQERFPPHHLTVI

LGRTYRVVPGEEEQKFEVEKYIVHKEFDDDTYD

NDIALLQLKSDSSRCAQESSVVRTVCLPPADLQLPOWTECELSGYGKH

EALSPFYSERLKEAHBRLYPSS

RCTSQHLLNRTVThNMLCAGDTRSGGPQANLHDACQGDSGGPLVCLNDiRMTLVGI

ISMGLGCGQKDVPG

VYTKVTNYLDHI RDNMRP WO 89/07146 -52 -PCT/US89/00465 TABLE 14 MUTANT 2 Prot 1 CV 110 GARSYQVICRDEKTQMIYQQHQSMLRPVLRSNRVEYCHCNSGRAQCHSVPVKSCSEPRCFNGGTCQQALY FSDFVCQCPEGFAGKCCEI DTRATCYEDQGISYRGTHSTAESGAECTNHNSS&LAQKPYSGRRP0 I RLG LGMHNYCRNPDRDSKPkJCYVFKAGKYSSEFCSTPACSEGNSDCYFGNGSAYRTH4SLTESGASCLPHNSM

ILIGKVYTAQNPSAQALGLGKHNYCRNPDGDAKPHCHVLKNRRLTHEYCDVPSCSTCGLRQYSQPQFRIK

1 GGLFADIASHPHQAAI FAKHRRSPGERFLCGGILISSCWILSAAHCFQERFPPHHLTVI LGRTYRVVPGE E QKFEVEKYIVHKEFDDDTYDNDIALLQLKSDS$RCAQESSVVRTVCLPPADLQLPDHTECELSQYGKH

EALSPFYSERLKEAHBRLYPSSRCTSQHLLNRTVTDNMLCAGDTRSGGPQANLHDACQGDSGGPLVCLND

GRMTLVGIISNGLGCGQKDVPGVYTKVTNYLDMIRDNMPSGGLFADIASHPHQAAIFAKHRRSPGE.RFLC

GGLSCISACQRPHLV RYVVGEQFVKIHEDDYNILQ V 20 KSDSSRCAQESSVVRTVCLPPADLQLPDW1TECELSGYGKHEALSPFYS ERLKEAHBRLYPSSRCTSQHLL NRTVTDNMLCA GDTRSGGPQAN LH VACQGDSGGPLVCLNQCRMTLVGI tSNGLiCGQKDVPGVYTKVTNY i LDWIRDNMVRP ii WO 89/07146 53 WO 8/07 46 -53 -PCT/US89/00465 .1 TABLE~ MUTANT 2 Prot 2 CV

GARSYQVICRDEKTQMIYQQHQSMLRPVLRSNRVEYCNCNSGRAQCHSVPVKSCSEPRCFNGGTCQQALY

FSDFVCQCPEGFAGKCCEIODTRATCYEDQGI SYRGTNSTA ESGAECTNMNSSALAQKPYSGRRPDAI RLG LGNHNYCRN PDRDSKPWCYVFKAGKYSS EFCSTPACS EGNSDCYFGNGSAYRGTHS LTESGASCLPWNSM I LIGKVYTAQNPSAQALGLGKHNYCRNPDGDAKPCHVLKNRRLTWAEYCDVPSCSTCGLRQYSQPQFRIK GGLFADIASHPWQAAI FAKHRRSPGERFLICGGI LISSCWI LSAAHCFQERFPPKjLT IILGRTYRVVPGE EEQKFEVFKYIVHKEFDDDTYDNOlALLQLKSDSSRCAQESSVVRTVCLPPADLQLPDNTECELSGYGKH EALSPFYSERL.KEAHBRLYPSSRCTSQHLLNRTVTDNMLCAGDTRSGGPQANLIIDACQGoSGGPLVCLND GRMTLVGIISHGLGCGQKDVPGVYTrKVTNYLDHI RDNMPSCGLRQYSQPQFRIKGGLFAOIASHPMQAAI FAKHRRSOGERFLCGGI LISSCHI LSAAHCFQERFPPHH LlVI I GR'rYRVVPGUEEQKFEVEKYIVHKu, DDDTYDNDIALLQ%4 DSSRCAQESSVVRTVCLPPAOLQLPDWCELSGYGKIWL-ALSPFYSERLK AHB RLYPSSRCTSQHLLNRTVTDNMCAGJTRSGGPQANLHDACQGDSGGPLVCLNJGRMTLVGI I SNGLGCG QKDVPGVYTKVTNY LDWI RDNMF,,P *1 54 WO 89/07146 PCT/US89/00465 TABLE 16 MUTANT Ult GA RSTSCGLRQYSQPQFR IKGGL FADI ASH PHQAAI FAKH RRS PGER FLCGGI LISSCNILSAAHCFQER FPPHH-LTVI LGRTYRVVPGEEEQKFEVEKYIVH-KEFDDDTYDNDIALLQLKSDSSRCAQESSVVRTVCLP PAIM'.QLPDWTECEL YGKHEALSPFYSERLKEAHBRLYPSSRCTSQHLLNRTVTDNMLCAGDTRSGGPQ AN U]DACQGDSGGPLVCLNOGRMTLiGI ISNGLGCGQKDVPGVYTKVTNYLDNIRDNMPSRSYQVICRDE KTQMIYQQHQSHLRPVLRSN RVEYCWCNSGRAQCHS VPVKSCS EPRGFNGGTCQQA LYFSOFVCQCPEGF AGKCCEIDTRATCYEDQGI SYRGTWSTAESG AECTNWJNSSALAQKPYSGRRPDAI RLGLGNHNYCRNPDR QSKPWCYVFKAGKYSSEFCSTPACSEGNSDCYFGNGSAYRGTHSLTESGASCLPHNSMI LIGKVYTAQNP SAQALGLGKHNYCRNPOAKP' 1'CHVLKNRRLTWEYCDVPSCAR W- i WO 89/07146 55 PCT/L S89/00465 TABLE 17 Mutant MS 1 KN 1I* KC1 KCA 1 KNB2 KC2 KCA2* Ndel Kidel K2 del Kl K2del 2K1, IK2* IKI, Wk 2K1, WK Specific Activity (MIU/ug) 350 485 584 517 418 745 617 v.1.I 120 400 v.1 320 s0 v.1 where v.1I, =very low acti vi ty and pr Lf'rred molecules signifies the most

Claims (6)

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 AvrII, 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 AvrII, NheI, 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. WO089/07146 -57 -PCT/US89/00465 A DNA sequence comprising: 241 273 CAG. TGC. CAC. TCA. GTG. CCT. AGG. AAA. AGT. TGC. AGC and all tsequences hybridizing thereto under stringent conditions.

106. A DNA sequence comprising: 364 396 TGC.TGT.GAA.ATA.GAT.ACT.AGT.GCC.ACG.TGC.TAC and all sequences hybridizing thereto under stringent conditions. 7. A DNA sequernc,;- comprising: V394 423 TAC. GAG. GAC. CAG. GGC. ACT. AGC. TAC. AGG. GGC and all sequences hybridizing thereto under str-ingent u conditions. 8. A DNA sequence comprising: 607 636 I AGC.TCA.GAG.TTC.GCTAGC.ACC.CCT.GCC.TGC and all sequences hybridizing thereto under stringent conditions. j 'I WO 89/071' 6 Z)B PCT/US89/00465 9. A DNA sequence comprising: 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.gCT.AGC.TAC CGT GGC and all sequences hybridizing thereto under stringent conditions. 11. A DNA sequenc. comprising: 853 888 CTG.-AAG. AAC. CGC. AGG. CTG. CT -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 -Ci AGG. CTG -ACG, TGG -GAG and all sequences hybridizing thereto under stringent conditions. WO 89/07146 59 PCT/US89/00465 13. A DNA sequence comprising: 883 921 TGT.GAT.GTG.CCC.TCC.TGC.GCT.AGC.TGC.GGC.CTG.AGA.CAG and all sequences hybridizing thereto under stringent conditions. 14. A DNA sequence comprising: 928 963 CAG.CCT.CAG.TTT.CGC.AC-T.AGT.GGA.GGG.GTC.TTC.GCC and all sequences hybridizing thereto under stringent conditions. 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.TAG.AGC.TCA.GAG.,rTC.GCT.AGC.TAC.CGT.GGC.ACG and all sequences hybridizing thereto under stringent conditions. WO 89/07146 60 PCT/US89/00465 17. A DNA sequence comprising: 394 408 619 633 TAC.GAG.GAC.CAG.GGC.ACT.AGC.TGC.AGC.ACC.CCT.GCC and all sequences hybridizing thereto under stringent conditions. 18. A DNA sequence comprising: 658 672 877 891 TAC.TTT.GGG.AAT.GGG.GCT.AGG.GAG.TAC.TGT.GAT.GTG and all sequences hybridizing thereto under stringent conditions. 19. A DNA sequence comprising: 394 408 877 891 TAC.GAG.GACCAGGGC.ACT AGG.GAG.TAC.TGT.GAT.GTG and all sequences hybridizing thereto under stringent conditions. 20. A DNA sequence comprising: 109 120 262 273 GGA.GCC.AGA.TCG.ACTAGa.AAA.AGT.TGC.AGC and all sequences hybridizing thereto under stringent conditions. 'Wo'89/07146 61 -PCT/US89/00465 21. A DNA sequence comprising: 244 258 285 396 TGC.CAC.TCA.GTG.CCT.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.

2023. 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 cotidi tions.- r r m f p. 0 C C 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 any of claims 5 to 25 which is part of a larger DNA sequence having a nucleotide at position 451 which has been substituted so that in the protein encoded by the DNA sequence a glutamine residue is encoded by the DNA sequence instead of an asparagine residue. 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 28, A host cell transformed or transfected by a sequence comprising the sequence of claim 26, 20 29. The host cell of Claim 27 wherein said host cell is C127. 30, The host cell of Claim 28 wherein said 1i:st cell is C127, 31. An expressed protein produced by the host cell of Claim 27. 32, An expressed protein produced by the host cell of Claim 28. *i S C S C. C 5 r (I 63 33. An expressed protein produced by the host cell of 'Claim 29. 34. An expressed protein produced by the host cell of Claim Dated this 11th day of November, 1991. GENZYMB CORPORATION By Its Patent Attorneys DAVIES COLLISON CAVE, 6*S* *6U *6 @6 6 6 6 6* 6 6 *6S 6 *6*6 6 6~@6 6% 66 66 6 6 6 6 *666 6* 66 6 606666 6 66 6 6* 6* 6 666666 6 9111? 1,Jratic.14Je430$534Jdt,63 L