AU609337B2 - Tissue plasminogen activator (t-pa) muteins and processes for the preparation thereof - Google Patents

Description

310 /00 COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952 DECLARATION IN SUPPORT OF CONVENTION OR NON-CONVENTION APPLICATION FOR A PATENT

In support of the Application made for a patent for an invention Insert title of invention. entitled: "TISSUE PLASMINOGEN ACTIVATOR (t-PA) MUTEINS AND PROCESSES FOR THE PREPARATION THEREOF" Insert full name(s) and address(es) of declarant(s) being the appli- We Martin Daum and Bernd Kolb cant(s) or person(s) authorized to sign on behalf of an applicant BOTH of Boehringer Mannheim GmbH company. of Sandhofer Strage 112-132 D-6800 Mannheim-Waldhof Federal Republic of Germany Cross out whichever of paragraphs do solemnly and sincerely declare as follows:- 1(a) or 1(b) does not apply 1(a) relates to application made I. W~ Xhtla tiaolxxx)ornaitcx by individual(s) 1(b) relates to application made or(b) XXK authorized by by company; insert name of applicant company. Boehringer Mannheim GmbH

the for the patent to make this declaration on its ehalf. Crpss out whichever of paragraphs *2(a)'or 2(b) does not apply S2J,) relates to application made 2. tXbhe>extatSexXXeolaxxxxx ftbrimitox by lnventor(s) relates to application made or(b) 1. Anne STERN b' company(s) or person(s) who areonot inventor(s); insert full of KarwendelstraBe 10, D-8122 Penzberg name(s) and address(es) of inven- tc:s. 2. Ulrich WEIDLE of Landwehrstra3e 56, D-8000 Munchen 2 3. Ralf MATTES of Friedrich-Zundel-Straae 14, D-7000 Stuttgart All of Federal Republic of Germany

aree the actual inventor.. of the invention and the facts upon which the aientitled to make the application are as follows

SSlat Inanner in which applicant(s) The applicant would, if a patent were granted on ,JZ:r\'e title from Inventor(s) and application made by the said actual inventors, be entitled to have the patent assigned to it.

Cross out paragraphs 3 and 4 3. The basic as defined by Sectvl:n 141 of the Act was made for ncn-convention applications. i'or convention applications, FEderal Republic of Cermanf 2 4 October 1988 n Mannh im G mb insert basic country(s) followed by date(s) and basic applicant(s). by...Bo.ehring.er...Mannheim GmbH on the in on the by in on the

4. The basic application.......... referred to in paragraph 3 of this Declaration was the first application......... made in a Convention country in respect of the invention the subject of the application. Insert place and date of signature. Declared at Mannheim this 21 st day of Tuly, 1989 BOEHR GER MANNH IM GMBt Signature of declarant(s) (no ppa i attestation required)

Note: Initial all alterations. Dr. Martin Daum DAVIES COLLISON, MELBOURNE and CANBERRA.

_e I

(12) PATENT ABRIDGMENT (11) Document No. AU-B-43657/89 (19) AUSTRALIAN PATENT OFFICE (10) Acceptance No. 609337

(54) Title TISSUE PLASMINOGEN ACTIVATOR (T-PA) MUTEINS AND PROCESSES FOR THE PREPARATION THEREOF International Patent Classification(s) C12N 009/64 C12N 016/00 C12N 015/58 C12P 021/02

(21) Application No. 43657/89 (22) Application Date 23.10.89 Priority Data (31) Number (32) Date (33) Country 3836200 24.10.88 DE FEDERAL REPUBLIC OF GERMANY

(43) Publication Date 26.04.90 (44) Publication Date of Accepted Application 26.04.91 (71) Applicant(s) BOEHRINGER MANNHEIM GMBH

(72) Inventor(s) ANNE STERN; ULRICH WEIDLE; RALF MATTES (74) Attorney or Agent DAVIES COLLISON, 1 Little Collins Street, MELBOURNE VIC 3000

(56) Prior Art Documents AU 55514/86 AU 67701/87 AU 17410/88

(57) Therefore, it is an object of the present

invention to provide t-PA muteins which, in comparison

with natuTal t-PA, display an increased fibrin binding

and stimulatability and, as before, display all the

characteristic properties of biological t-PA.

CLAIM

1. Tissue plasminogen activator mutein, wherein it has the natural amino acid sequence of at least one of the domains fibrin finger and loop II in multiple form.

2. Tissue plasminogen activator (t-PA) mutein according to claim 1, further comprising a deletion of the amino acid sequence of the domain EGF and/or loop I.

3. Tissue plasminogen activator (t-PA) mutein according to claim 1 or 2, wherein the fibrin finger domain, amino acids 4 to 49, is present twice. /2 pq o t u %AXAfl Awuu 9 ole Z SII i2 5 i 1.4 1. Z XM/\nistr dONWINFIHlEL333Q:8t/d 01 II11 1.216

(11) AU-B-43657/89 -2- (10)609337

9. Process for the preparation of a t-PA mutein which has the natural amino acid sequence of at least one of the domains fibrin finger and loop II in multiple form, wherein a) several complete t-PA genes are inserted directly behind one another in an appropriate vector, b) those sequences which code for amino acids which are not to be present 3everal times in the product are removed by oligonucleotide-directed mutagenesis, c) the so obtained mutated vector is introduced into an appropriate cell and amplified, d) those vectors in which the deletion according to step b) has taken place are recovered and e) the vector is expressed in an appropriate host cell.

ALLOWED A

602 COMMON WEALTI OF AUSTRALIA PATENTS ACT 1952 COMPLETE SPECIFICATION

NAME ADDRESS OF APPLICANT:

Boeliringer Mannheimi GinbH Sancihofer Strisse 112-132 D-6800 Mairnheirn-.Walclhof Federal Republic of Germany

NAME(S) OF INVENTOR(S):

Anne STERN Ulrich WEIDLE Ralf MATTES

ADDRESS FOR SERVICE: I his do,'2ulanet contain s the DAVIES COLLISON amnendmients made Under Patent Attorneys ion 49 and is correcct for 1 Little Collins Street, Melbourne, 3000. printin tY

COMPLETE SPECIFICATION FOR THE INYENTION ENTITLED:

Tissue plasminogen activator (t-PA) muteins and processes for the preparation thereof

The following statemnent is a full description of this invention, including the best miethod of performing it known to m-e/us:..

Insert place and date of signature. Declared at Mannheim this 21 St. day of July, 1 989 BOEHR GER MANNH M GMBH Signature of declarant(s) (no p pa HIY i M attestation required) pp a

Note: Initial all alterations. Dr. Martin Daum g

DAVIES COLLISON, MELBOURNE and CANBERRA.

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The present invention is concerned with new t-PA

muteins and processes for the preparation thereof, as

well as with agents for dissolving coagula containing

these t-PA muteins.

Coagulated blood contains, as main component, the

protein matrix polymeric fibrin. This fibrin is broken

down proteolytically by plasmin. Plasmin is formed

from plasminogen via activation by the so-called

plasminogen activators, for example by the tissue

plasminogen activator t-PA (tissue-type plasminogen

activatory). The enzymatic activity of natural t-PA or

t-PA obtained gene-technologically from mammalian cells

(catalytic activation of plasminogen to plasmin) is very

low in the absence of fibrin or fibrin cleavage products

(FCP) but can be substantially increased in the presence

of these stimulators (by more than the factor of

i The working mechanism of t-PA in vivo is described,

for example, in Korniger and Collen, Thromb.Hmostasis

46 (1981), 561-565. The stimulatability of the activity

of t-PA by fibrinogen is a decisive advantage in

comparison with other known plasminogen activators, for

example urokinase or streptokinase (cf. for example

B.M. Hoylaerts et al., J. Biol. Chem., 257, 2912-2919/

1982; W. Nieuwenhuizen et al., Biochem. Biophys. Acta,

755, 531-533/1983).

In the clinic, t-PA has already proved to be an

effective agent for the treatment of thrombi, for which

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reason attempts have been made to prepare it in the

largest amounts possible.

However, clinical investigations have shown that,

because of the rapid breakdown rates in the liver,

large amounts of t-PA are necessary in order to bring

about an effective thrombolysis in vivo. However, high

treatment doses result in side effects, for example

haemorrhages. Therefore, it is desirable to improve

this thrombolytic agent and to reduce the side effects,

such as systemic fibrinolysis. A reduction of the

treatment dose is also to be achieved.

Detailed investigations have shown that t-PA

consists of the domains which are shown in Fig. 1 of

the accompanying drawings.

European Patent Application No. 0,241,210

describes t-PA muteins which contain several fibrin

finger domains. The DNA sequence for the fibrin finger

domain was hereby isolated as a restriction fragment

and placed, via a synthetic linker, before the first

amino acid of the protein. However, as a result of

the use of the synthetic linker, an amino acid sequence

is obtained which differs from that of the correspond-

ing natural fibrin finger domain.

European Patent Application No. 0,231,624

describes a synthetically prepared t-PA gene and the

t-PA muteins prepared therefrom which contain each of

the domains at most twice. This DNA was synthesised

3U3ntPPnMCC C~ -n~ilrXL~3~C~FX

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in such a manner that new restriction cleavage points

were introduced into the sequence in order to simplify

the cloning of the individual domains. The amino acid

positions, especially at tL domain transitions, were

thereby greatly modified. Thus, for these t-PA

muteins, too, the amino acid sequence differs from the

sequence of the natural protein.

Therefore, it is an object of the present

invention to provide t-PA muteins which, in comparison

with natural t-PA, dizplay an increased fibrin binding

and stimulatability and, as before, display all the

characteristic properties of biological t-PA.

Thus, according to the present invention, there

is provided a tissue plasminogen activator (t-PA)

mutein which has the natural amino acid sequence of at

least one of the domains fibrin finger and loop II in

multiple form.

By natural amino acid sequence is understood, in

the scope of the present invention, the amino acid

sequence for the individual domains known from Pennica

et al., Nature, 301, 214/1983. The multiplication takes

place according to the exon-intron limits of the

natural t-PA DNA sequence. Consequently, there is

obtained a protein which admittedly contains at least

one of the two domains in multiple form but contains no

additional foreign amino acids in its sequence and also

no deletion of an amino acid. Preferably, the multiple sequences are present doubled but they can be present three or four fold or more often.

In a preferred embodiment of the present

invention, in the case of the t-PA mutein, the amino

acid sequence of the domain EGF and/or loop I is

additionally deleted. Due to the deletion of the EGF

domain, there is obtained a substantially prolonged

half life time in comparison with natural t-PA since

this domain is needed for the rapid breakdown of the

protein in the liver. The loop I domain can be deleted

without loss of activity and there is thereby obtained

the advantage that the amino acid sequence is not

excessively elongated by the doubling.

In a preferred embodiment of the present invention,

in the t-PA mutein, the fibrin finger domain, namely

amino acids 4 to 49, is present doubled. In an espec-

ially preferred embodiment of the present invention,

the amino acids of the EGF domain, namely amino acids

to 87, and the amino acids of the loop I domain,

amino acids 88 to 175, are additionally deleted.

In another preferred embodiment of the present

9 invention, the amino acid sequence of the

amino acids 176 to 261, is doubled. loop II domain,

In a further espec-

ially preferred embodiment, the amino acids of the EGF

domain and of the loop I domain are additionally deleted.

In a third preferred embodiment, the EGF and the

loop I domains are deleted and the fibrin finger and c~-

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the loop II domains are doubled. Depending upon whether the t-PA mutein according to the present invention has been expressed in prokaryotes or eukaryotes, it is present glycosylated or unglycosylated. In a preferred embodiment of the present invention, the t-PA mutein is glycosylated and, therefore, resembles natural t-PA especially strongly. The present invention also provides a process for the preparation of a t-PA mutein which has the natural 00 o 000 amino acid sequence of at least one of the domains fibrin 0 o o. 0 finger and loop II in multiple form, wherein 0o 0 a) several complete t-PA genes are inserted directly 00 0 00 0 0 behind one another in an appropriate vector, 0 00 b) those sequences which code for amino acids which are o0 0 not to be present several times in the product are o 0 a removed by oligonucleotide-directed mutagenesis, c) the so obtained mutated vector is introduced into an appropriate cell and amplified, d) those vectors in which the deletion according to step b) has taken place are recovered and e) the vector is expressed in an appropriate host cell. o o

The oligonucleotide-directed mutagenesis is thereby S0 0 0 carried out according to per se known methods, for example Morinaga et al., Biotechnology, 2, 634/1984. S 0 00 In a preferred embodiment of the present invention, S~ 0@00 tt before the insertion of the two t-PA gene into a vector, the EGF domain and/or loop I domain is removed therefrom, also by oligonucleotide-directed mutagenesis. For the oligonucleotide-directed mutagenesis, oligonucleotides are used which are complementary to

910108,ejhspe.014,43657.spe,6

I-

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nucleotides of the 3' or 5' sequence of the DNA

sequences to be bound, an oligonucleotide with a length

of 18 to 26 nucleotides preferably being used.

In a further preferred embodiment of the present

invention, the EGF and loop I domains are first removed

and then the DNA sequence for the domain fibrin finger

loop II (K2) and the light chain (protease) (L)

introduced into the vector twice behind one another,

the sequence FK2LFK2L thereby being obtained and by

oligonucleotide-directed mutagenesis, the sequences

between the two F domains, i.e. K2 and L, or the

sequences between the two K2 domains, i.e. L and F, are

removed therefrom. Vectors are obtained therefrom which

contain the DNA sequence for a mutein with the domains

FFK2L or FK2K2L.

In a preferred embodiment of the present invention,

t-PA cDNA can be used as t-PA DNA. The vectors obtained

are preferably amplified in prokaryote cells, an

Escherichia coli strain preferably being used which

contains a lac Iq plasmid. After selection of the

vectors in which the deletions have taken place, in a

preferred embodiment of the present invention,-the

vector is expressed in eukaryotes. The expression is

admittedly also possible in prokaryotes but a non-

glycosylated product is obtained therefrom. However,

since a glycosylated product corresponds to the natural.

t-PA, the expression in eukaryotes is prefer:red. In an

L especially preferred embodiment, the expression is carried out in CHO cells. For the expression in eukaryotes, as vector there is preferably used a vector amplifiable on dihydrofolate reductase or based on bovine papilloma virus.

In especially preferred embodiments, the plasmids

pAl8/l, pAl9/l are expressed in Escherichia coli. The

present invention also provides the plasmils pA18/l and

pAl9/l, which contain the mutein DNA for the domains

FFK2L and FK2K2L, respectively, the plasmids pA24 and

which contain the DNA sequence for the same

domains but additionally contain the eukaryotic signal

sequence, and the plasmids pSV-FFK2L-dhfr and pSV-

FK2K2L-dhfr, which represent amplifiable eukaryotic

expression vectors for FFK2L and FK2K2L.

The present invention also provides an agent for

dissolving coaguli containing at least one tissue

plasminogen activator (t-PA) mutein according to the

present invention which contains at least the domains

fibrin finger and/or loop II multiplied. Especially

preferably, the agent contains a t-PA derivative in

which the EGF and the loop I domains are additionally

deleted. In an especially preferred embodiment, the

agent contains the mutein with the domains FFK2L or

FK2K2L.

The present invention provides new pharmacologic-

ally active t-PA muteins which display increased fibrin binding and fibrin stimulatability and, in preferred embodiments, display a distinctly lowered breakdown rate in the liver. In addition, in the case of the t-PA muteins, there is obtained an increased rate of production in eukaryotes.

Therefore, it is possible to provide medicaments

for dissolving coaguli in the case of which a substant-

ially lower content of t-PA shows a clearly increased

effectiveness.

The following Examples are given for the purpose

of illustrating the present invention, with reference

to the accompanying drawings, in which

Fig. 1 shows the arrangement of the domains of t-PA;

Fig. 2 shows schematically the preparation of the

plasmids pA18/l and pAl9/l; and

Fig. 3 shows schematically the preparation of the

plasmids pSV-FFK2L-dhfr and pSV-FK2K2L-dhfr.

Example 1.

Preparation of FFK2L.

a) Preconstruction of FK2L/FK2L.

The starting plasmid pREM 7685, described in

European Patent Application No. 0,242,836, contains

the following components: tac promotor, lac operator

region with an ATG start codon, the coding region for

the mutein FK2L, the transcription terminator from

pKK223-3, a 3-lactamase gene, a kanamycin-resistant

gene and the replication origin of the plasmid pACYC177.

~aE arlSna~rim~sssry~

The sequence of the mutein FK2L is composed of the

nucleotides 190-336 domain), 715-1809 (K2 domain,

protease, small part of 3'UT (nori-translated 3' region))

and an ATG start codon. The nucleotide positions are

given according to the sequence described by Pennica

et al., (Nature, 301, 214/1983).

For the duplication of the t-PA mutein expression

cassette, consisting of promotor/operator region and

the region coding for the mutein, including the trans-

S 10 cription signals, the plasmid pREM7685 was cleaved

with the restriction enzyme Xho II. The restriction

batch was separated electrophoretically in agarose gel

and an about 1480 bp sized fragment isolated.

In a second batch, pREM 7685 was linearised with

the enzyme Bam HI and subsequently treated with alkaline

phosphatase fron calf intestine.

After ligation of the two DNA fragments obtained,

the ligation batch was transformed in lac 1q plasmid-

containing Escherichia coli cells, DSM 3689. Plasmid-

carrying transformants are selectioned by the addition

of 25 mg./ml. kanamycin to the nutrient medium.

By restriction enzyme analysis, those clones

could be selected from the transformants which contained

the plasmid pA15. This plasmid differs from the start-

ing plasmid pREM 7685 inter alia by the appearance of an

about 1480 bp sized additional Sma I fragment which

confirms not only the incorporation but also the desired rririscaa~irrar~~

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oritentation of the Xho II fragment. pA15 thus contains

the above-mentioned expression cassette twice, namely

in tandem arrangement.

b) Mutagenesij..

pAl5 -rved as starting plasmid for the construct-

ion of the mutein gene FFK2L. For the deletion of the

desired regions from pA15, there was used the method of

Morinaga et al., Biotechnology, 2, 634/1984. For the

heteroduplex formation two fragments were isolated from

Fragment A: pA15 was cleaved with the two restriction

enzymes Bam HI and Sal I (polylinker region). The

cleavage products were separated gel electrophoretically

and the large Bam HI/Sal I fragment eluted from the gel.

Fragment B: pAl5 was linearised with the restriction

enzyme Xho I. The linearised plasmid was also obtained

preparatively by gel electrophoresis. For the

mutagenesis, the following oligonucleotide was prepared

synthetically:

5' GCCTGTCAAAGTGATCTGCA 3'

For the heteroduplex formation, fragment A,

fragment B (in each case 450 fmol) and the oligonucleo-

tide (75 pmol) were mixed and first incubated for 3

minutes at 100 0 C. in the presence of 50 mM sodium

chloride, 10 mM Tris HC1 (pH 7.5) and 10 mM magnesium

sulphate and immediately transferred to ice. The

renaturing of the DNA took place for 30 minutes at 60 0 C.

For the repair synthesis, the reaction batch was

adjusted to: desoxynucleotide triphosphate (0.25 mM),

ATP (1 mM), sodium chloride (100 mM), Tris HC1 pH

mM), magnesium chloride (8 mM), -mercaptoethanol

(1 mM), Klenow fragment of the DNA polymerase from

Escherichia coli (0.125 u/pl. batch) and T4 ligase

(0.1 U/pl. batch). The repair synthesis took place for 0 4 hours at 16 C. Subsequently, this batch was trans-

formed in Escherichia coli cells with a lac Iq plasmid,

DSM 3689, and the transformants selectioned by the

addition of 25 pg./ml. kanamycin to the nutrient medium.

With the help of the colony hybridisation

technique with the use of the above-described

mutagenesis oligonucleotide as probe, those clones could

be selected which carry the plasmid pAl8/l. This plasmid

differs from the starting plasmid pA15 inter alia by the

absence of an Sma I cleavage position which had first

been introduced by the duplication of the mutein

expression cassette. Figure 1 of the accompanying

drawings shows schematically the preparation of pAl8/l.

c) Expression of FFK2L in Escherichia coli.

The plasmid pAl8/l was transformed in Escherichia

3 coli cells, DSM 3689, which contain a lac Iq plasmid.

The plasmid-carrying cells were cultured in the nutrient

medium to OD5 0.4 with aeracinn 0 50 at 37 C. and induced

for three hours by the addition of 0.2% lactose or 5 mM

IPTG (isopropyl-l-thio-P-D-galactopyranoside). According to the protocoll described in Federal Republic of

Germany Patent Application No. 35 37 708, the so-

called inclusion bodies were separated off from the

cell protein and the mutated t-PA protein renatured.

The so-obtained protein has a molecular weight of about

49 kD and binds to polyclonal antibodies against human

t-PA (produced in the goat). The renatured mutein

(renaturing as described in European Patent Application

No. 0,219,874) showed not only amidolytic activity but

also fibrin stimulatabilitv in the plasminogenolytic

test.

d) Construction of FFK2L with eukarvotic signal

sequence.

In order to provide the mutein gene with the leader

sequence and the 3'UT untranslated region), corres-

ponding to the original cDNA sequence, the following

fragments were isolated: from plasmid p7.1, DSM 4719,

which in the polylinker pUC12 contains the 5'UT

untranslated region), the leader sequence and the N-

terminal sequence of t-PA up to and including nucleotide

position 208, ~as obtained en about 2.7 kb sized

Pst I/Hind III fragment. From the plasmid pePA9S.1

(see European Patent Application No. 0 242 835) was

isolated a Hind III/Scal fragment which coirprised about

120 bp and contained the coding region for the protease

and the 3'UT. From pAl8/l was first prepared a Bam HI/

Sca I fragment from which, by partial restriction with

I 1 i 1 ~"II~L~YI

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Pst I, was obtained an about 470 bp-containing Pst I/

Sca I fragment. This fragment contained the coding

region for the duplicated domain F and the K2 domain.

The fragments were ligated and transformed in

Escherichia coli with lac Iq plasmid, DSM 3689.

Kanamycin-resistant transformants were screened with

the above-mentioned mutagenesis primer. The correct

plasmid, pA 25, gives, in the case of restriction with

Pst I, fragments of the lengths 2700 bp (pUC 12 vector);

790 bp, 730 bp, 140 bp and 70 bp (t-PA fragments).

Example 2.

Preparation of FK2K2L.

a) Mutagenesis and expression in Escherichia coli.

From the FK2L double gene plasmid pA15 were

isolated, according to Example 1, the two fragments A

and B. However, in this case, for the heteroduplex

formation there was used the following oligonucleotide:

GCCCTCCTGCGGAAACAGTG 3'

All experimental steps .ere carried out as already

described in Example 1. By colony hybridisation with

the above-mentioned mutagenesis oligonucleotide were

determined those clones which contain the desired

plasmid pA19/l (5.2 kb). This plasmid was characterised

by restriction enzyme analysis and tested for the absence

of an Sma I cleavage position. Figure 2 of the

accompanying drawings shows schematically, inter alia,

the preparation of pAl9/1. pAl9/l was transfec-ed in

Escherichia coli, DSM 3689, and the mutein FK2K2L

expressed. The protein has a size of about 52000

Dalton. The mutein binds to polyclonal human t-PA

antibodies (produced in the goat) and shows amidolytic

activity and fibrin stimultability in the plasminogen-

olytic test.

b) Recunititution of FK2K2L with eukarvotic signal

sequence.

For the attachment of the original leader sequence

to the mutein gene FK272L, the following fragments were

Sisolated and ligated: a 2.7 kp-sized BamHI/Hind III

fragment from p 7.1, DSM 4719, an about 0.2 kb-sized

Bam HI/Ssp I from pEZO (in the Pst I cleavage position

of p 7.1 was introduced a 450 bp Pst I fragment from

the t-PA coding sequence; and an about 1.3 kb-sized

Ssp I/Hind III fragment irom pAl9. The ligation batch

was transformed in Escherichia coli cells, DSM 3689,

and the plasmid-containing transformants selectioned by

o the addition of 50 pg./ml. ampicillin to the nutrient

medium. The desired plasmid pA24 was characterised by

detailed restriction enzyme analysis.

Example 3.

Construction of amplifiable eukarvotic expression

vectors for FFK2L and FK2K2L.

FFK2L and FK2K2L cDNA were isolated as Xba-Hind III

fragments from pA25 and pA24 and ligated into the Bam HI

position of the eukaryotic expression vector pKCR

(O'Hare et al., Proc. Natl. Acad. Sci., USA, 78,

1527-1531/1981) after the ends had been made blunt

not only of FFK2L and FK2K2L c-DNA but also

of the vector with nuclease Sl. The ligation batch

was transfected in Escherichia coli, HB101 (DSM 1607),

p-lasmid-carrying colonies isolated on nutrient medium

with 50 pg./ml. ampicillin and plasmids with the correct

orientation (syn) of FFK2L and FK2K2L with regard to

the early promotor of SV40 determined by restriction

mapping. The expression vectors are designated as

pKCR-FFK2L and pKCR-FK2K2L. From these plasmids was

isolated the particular expression cassette as Aat II-

Sal I fragment. For this purpose, pKCR-FFK2L and

pKCR-FK2K2L were partially cleaved with Sal I and

subsequently with Aat II, the ends made blunt with

nuclease Sl and the corresponding fragments (shown

in the drawing with a broken line) isolated after

separation on a low melting agarose gel. These frag-

ments were ligated into the Eco RI position of the

amplifiable vector pAdD26SV(A) filled with Klenow

(Kaufman and Sharp, J. Mol. Biol., 159, 601-621/1982).

The ligated reaction mixtures were transfected-in

Escherichia coli HB 101 and colonies isolated on

tetracyclin-containing nutrient medium (10 pg./ml.).

The corresponding recombinants were determined by

restriction enzyme analysis which also ascertained the

orientation of the expression cassettes for the t-PA

IrYI-L

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derivatives relative to the expression vector. The

isolated expression vectors were designated as

pSV-FFK2L-dhfr and pSV-FK2K2L-dhfr (Fig. 3).

pSV-FFK2L-dhfr is characterised in that, in the

case of restriction with Sal I, two fragments are

obtained with the lengths of 2470 bp and 7100 bp.

pSV-FK2K2L-dhfr is characterised in that, in the

case of restriction with Sal I, two fragments are

obtained with the lengths of 2470 bp and 6860 bp.

Example 4.

Establishment of CHO cells which constitutively

secrete FFK2L and FK2K2L.

CHO dhfr- cells (DUKX, DHFR-), ECACC 88072103,

were propagat.d as described by Urlaub and Chasin (Proc.

Natl. Acad. Sci. USA, 77, 4216-4220/1980). The cells

were propagated in alpha medium which was supplemented

with adenosine, deoxyadenosine and thymidine and

foetal calf serum with penicillin and streptomycin.

For the permanent expression, calcium phosphate

precipitates were prepared with 20 pg. of the appropriate

expression constructions, as described (Graham and

Van der Eb, Virology, 52, 456-467/1973), in an.end

volume of 4 ml. 1 ml. of the precipitate was added to

3 x 105 to 1 x 106 cells in 10 ml. of medium in 9 cm.

culture dishes. The cells were incubated with the

precipitate for 6 to 18 hours, the medium was removed

and washed with 10 ml. TBS (25 mmol/l. Tris-HCl (pH 7.4),

1 r-l~ i' 'IC I 1- i D

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137 mmol/l. sodium chloride, 5 mmol/l. potassium

chloride, 0.6 mmol/l. disodium hydrogen phosphate

(pH 7.4)),incubated with 2 ml. 20% glycerol in tris-

buffered saline (TBS) for 3 to 3.5 minutes, washed

with TBS and then incubated in an appropriate medium.

The CHO cells were transformed 1:10 48 hours after

transfection and multiplied in selection medium

(Kaufman and Sharp, J. Mol. Biol., 159, 601-621/1982).

The selection medium was alpha medium with dialysed

o oQ 10 foetal calf serum, i.e. it contained no addition of

adenosine, deoxyadenosine and thymidine. Clones were

isolated 2 to 3 weeks after transfection after trypsin-

ation, cultured for mass culturing and the supernatants

investigated by ELISA for t-PA immunoreactivity.

Positive clones were then pooled and 4 plates with, in

each case, 1 x 106 cells were selectioned with 20 nmol/l.

C methotrexate.

After 2 weeks, methotrexate-resistant clones

appeared. They were allowed to grow to confluence and

exposed to a medium with 100 nmol/l. methotrexate.

Resistant cells were exposed to a stepwise increase of

the methotrexate concentration (300 nmol/l.,

500 nmol/l., 1 pmol/l. and 5 pmol/l.). The clones were

finally isolated by limited dilution and the best

producers of the t-PA mutein determined by investigation

of the supernatants for t-PA immunoreactivity by ELISA.

J_ I I I .L

Y~ Li--i

-19-

Example

Determination of t-PA, FFK2L and FK2K2L by ELISA

(enzyme linked immunosorbent assay).

Microtitre plates (Nunc) with 96 cups were coated

with anti-t-PA-IgG (8 pg./ml.) in 50 mmol/l. potassium

phosphate buffer (pH 7.5) and washed three times with

PBS (0.15 mol/l. sodium chloride, 10 mmol/l. sodium

phosphate; pH 0.5% BSA (bovine serum albumin). 0 The cups were incubated for 12 hours at 4 C. with

200 pl. tissue culture supernatant. The supernatants

were carefully removed and the cups washed three times

with PBS (phosphate buffered saline). The cups were

then incubated with peroxidase-conjugated goat anti-

t-PA-IgG, hydrogen peroxide and ABTS® [2,2'-azino-di-

(3-ethylbenzthiazoline-sulphonate The absorption

was measured at 405 nm and the evaluation carried out

by comparison of the absorption with a calibration

curve.

Example 6.

Determination of the fibrinogen-stimulated

plasminogenolytic activity of t-PA, FFK2L and FK2K2L.

Plasminogen activator activity was determined by

an indirect spectrophotometric test (Verheijen et al.,

Thromb. Haemostasis, 48, 266-269/1I82). t-PA converts

plasminogen into the active serine protease plasmin

which hydrolyses a peptidic bond in a chromogenic

substrate, the absorption of which was monitored at

CF I _II r ii I

I_1IY~ i

405 nm over a period of time of up to 3 hours. As

chromogenic substrate, there was used tosyl-glycyl-

prolyl-lysine-4-nitroanilide acetate (Chromozym PL). 0 The tests were carried out at 25 C. in 0.1 mol/l.

Tris-HCl (pH 0.15 mol/l. Tween 80 containing

0.13 pmol/l. plasminogen, CNBr-digested fibrinogen R (120 pg./ml.) and 0.30 mmol/1. Chromozym PL.

Example 7.

Culture substrates of CHO cells which constitut-

ively secrete t-PA or FK2K2L were investigated with

regard to the fibrinogen-stimulating proteolytic activity

as described in Example 6 and simultaneously the amount

of t-PA or FK2K2L was determined in the supernatants by

ELISA. It was thereby shown thzt the proteolytic

activity of FK2K2L was stimulated by CNBr fragments of

fibrinogen about just as strongly (about 20 to 30 fold)

as it was by t-PA. Surprisingly, it was shown that

FK2K2L, in comparison with t-PA, shows a specific

activity increased by a factor of 3. CHO cells which

cor ,titutively secrete FK2K2L dissolve off from the

C Petri dishes even after the first amplification step.

This is to be attributed to the activation of

plasminogen present in the serum to the active serine

protease plasmin by FK2K2L. In the presence of the

protease inhibitor aprotonin in t:he culture supernatants

was preponderantly detected single-chained FK2K2L and

in the case of its aLsence exclusivly two-chained t-PA, the expression in eukaryotes is preferred. In an

-21-

FK2K2L after gel electrophoretic separation of immuno-

adsorbed culture supernatants and Lbe visualisation

thereof with peroxidase-labelled antibodies against

human t-PA.

Supernatants of CHO cells which have been trans-

fected with the amplifiable expression vector pSV-

FFK2L-dhfr significantly increase the proteolytic

activity of t-PA in the presence of CNBr fragments of

fibrinogen. This derivative displays about the same

specific activity as t-PA.

gene and the replication origin of the plasmid pACYUI//.

Ii-- e

-22-

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. Tissue plasminogen activator (t-PA) mutein, wherein it has the natural amino acid sequence of at least one of the domains fibrin finger and loop II in multiple form.

2. Tissue plasminogen activator (t-PA) mutein according to claim 1, further comprising a deletion of the amino acid sequence of the domain EGF and/or loop I.

o. 3. Tissue plasminogen activator (t-PA) mutein according 0 to claim 1 or 2, wherein the fibrin finger domain, amino 0 o °o acids 4 to 49, is present twice.

4. Tissue plasminogen activator (t-PA) mutein according o o to claim 1 or 2, wherein the loop II domain, amino acids "176 to 261, is present twice.

Tissue plasminogen activator (t-PA) mutein according

000 0o o to claim 1 or 2, wherein the fibrin finger domain and the loop II domain are present twice.

o 6. Tissue plasminogen activator (t-PA) mutein according to claim 1, further comprising a deletion of the EGF domain, amino acids 50 to 87, and the loop I domain, o oo amino acids 88 to 175.

7. Tissue plasminogen activator (t-PA) mutein according to any of the preceding claims, wherein it has been expressed in eukaryotes and is glycosylated.

8. Tissue plasminogen activator (t-PA) muteins according to claim 1, which are hereinbefore specifically exemplified.

910108,ejhspc.014,43657.spe,22

TS

Claims (1)

1. ~rll~ll

   -23

   9. Process for the preparation of a t-PA mutein which has the natural amino acid sequence of at least one of the domains fibrin finger and loop II in multiple form, wherein a) several complete t-PA genes are inserted directly behind one another in an appropriate vector, b) those sequences which code for amino acids which are not to be present several times in the product are removed by oligonucleotide-directed mutagenesis, c) the so obtained mutated vector is introduced into an 0 appropriate cell and amplified, d) those vectors in which the deletion according to 09 fo step b) has taken place are recovered and e) the vector is expressed in an appropriate host cell. o 0

   10. Process according to claim 9 for the preparation of a t-PA mutein according to any of claims 2 'o 8, wherein, before the insertion of the t-PA genes into a vector, the sequences coding for the EGF and/or loop I domain are ,o also removed by oligonucleotide-directed mutagenesis. 0

   on11. Process according to claim 9 or 10, wherein an oligonucleotide is used having 18 to 26 nucleotides.

   o 0°0 12. Process according to any of claims 9 to 11, wherein, 0o 0 by oligonucleotide-directed mutagenesis, there are first Sremoved the DNA sequences for the amino acids of the EGF and loop I domain and then, after insertion of the t-PA genes, by oligonucleotide-directed mutagenesis, the sequences between the two sequences between the two sequence coding for the fibrin finger domain or the sequences coding for the loop II domain are removed.

   13. Process according to any of claims 9 to 12, wherein the t-PA cDNA is used as t-PA DNA.

   S 910108,ejhspe.014,43657.spe,23

   SLSTC/ ~VrQ~

   -z ,r 1 renaturing of the DNA took place for 30 minutes at 60 C.

   -11-3

   i 7 i~ Ipgfn~xrr~---

   -24-

   14. Process according to any of claims 9 to 13, wherein the expression is carried out in eukaryotes.

   Process according to claim 14, wherein the expression is carried out in CHO cell!;.

   16. Process according to any of claims 10 to 15, wherein the amplification and possibly the expression is carried out in an Escherichia coli strain which contains a lac Iq plasmid.

   17. Process according to any of claims 14 to 16, wherein, as vectors for the expression in eukaryotes, there are used vectors amplifiable on dihydrofolate reductase or based on bovine papilloma virus.

   18. Process according to claim 16, wherein the lac Iq plasmid expressed in Escherichia coli is plasmid pAl8/1 as hereinbefore defined or the plasmid pAl9/1 as hereinbefore defined. fl I

   19. Process for the preparation of a t-PA mutein according to cl-im 1, substantially as hereinbefore described and exemplified.

   t-PA mutein, whenever produced by the process according to any of claims 9 to 19.

   21. Plasmid pA18/1 as hereinbefore defined.

   22. Plasmid pAl9/l as hereinbefore defined.

   23. Plasmid pA24 as hereinbefore defined.

   24. Plasmid pA25 as hereinbefore defined.

   910108,ejhspc.014,43657.spe,24 lkvfu (1sopropyi-1-ct-tio-IP-L-gaiactopyranoside)- According mw~ .me

   25

   Plasmid pSV-FFK2L-dhfr as hereinbef ore defined.

   26. Plasmid pSV-FK2K2L-dhfr as hereinbefore defined.

   27. Agent for dissolving coagula, wherein it contains at least one tissue plasminogen activator (t-PA) mutein according to any of claims 1 to 8 and

   DATED this 16th day of January, 1991

   A BOEHRINGER MANNHEIM GmbH C~l By Its Patent Attorneys A DAVIES COLLISON

   0

   0 0

   910116,ejhspe.014,43657.spe,25 k3k~1J~

   Fig.-1 PLASMINOGEN-ACTIVATOR OF THE TISSUE TYPE

   C 0 0 ot~y 0

   0

   CDOH C C

   0 0' COOH

   NH 2

   HEAVY CHAIN LIGHT CHAIN sF--4- FIBRIN- EGF- LOOP I LOOP II PROT EASE FINGER DOMAIN* DOMAIN DOMAIN

   o o 0 0 DOMAIN WITH HOMOLOGY TO EPIDERMAL GROWTH FACTOR IEGFI CQ 0 C ~0

   A-9 1 C> 6,51

   Fig Ap R

   )FK2L TM TERMINATOR 1)tac Apoz AMPICILLIN-RESISTANCE ,BcmHI (XhoI[) KmR =KANAMYCIN-RESISTANCE 11M~ t-PA SEQUENCE XBamHI. Ptoc =TAC-PROMOTOR FK2 L Ptac

   FINGER DOMAIN K2= LOOP Il DOMAIN iTh LIGASEL xiThUR XhoU1 L PROTEASE PART OF o 00 t-PA (LIGHT CHAIN) *0 0

   FK21L

   I P'tac 00033

   fFK2 L 0

   C, 00 0 U 03)

   (30'.3,3 o .30 0 MUTAGENESE PRIMER Ptac MUTAGENESE PRIMER 0 5' GCCTGTCAAAGTGATCTGCA 5' 13CCCTCCTGGAAACAG 3' 3. 0

   SITE DIRECTED

   o C, 03) 33 MUTAGENESIS 0

   00 33 00

   FK2K2L

   4~1t~

   Fig .3

   Sal'32

   x Sal PARTIEL x EcoRI o x AatI[ xKLENOW 0, XNUCLEASE Si o 0 ISOLATION OF 0000 THE MARKED FRAGMENT +Tke LIGASE p8R 322 S 4VS KK2 pBR 322 pSV FK2 K2L- dhfr

   0 0

   IpBR 322Ry FFK2 L IVS pBR 322' pSV -FFK2 L Of r oo 0

   0

   ADENO- MAJOR-LATE PROMOT ER dhtr: OIHYDROFOLATE REDUCTASE

   RABBI] p OBIN t- PA IVS INTERV~ENING SEQUENCE OF RAE ~BIT 0- GLOBIN