WO1991017250A1

WO1991017250A1 - Method for the recombinant production of hiridius and hirudin-like polypeptides

Info

Publication number: WO1991017250A1
Application number: PCT/EP1991/000643
Authority: WO
Inventors: Luca Benatti; Paolo Carminati; Jacqueline Lansen; Guy Mazue; Romeo Roncucci; Paolo Sarmientos; Emanuela Scacheri; Philippe De Taxis Du Poet
Original assignee: Farmitalia Carlo Erba S.R.L.
Priority date: 1990-05-10
Filing date: 1991-04-05
Publication date: 1991-11-14
Also published as: HU9200088D0; EP0482144A1; PT97604A; NO920116D0; KR920703817A; IL98074A0; ZA913503B; GB9010552D0; NO920116L; CN1057294A; FI920058A0; CS133191A3; CA2063575A1; JPH05500615A; HUT59961A; IE911578A1; AU7652591A

Abstract

A hirudin or hirudin-like polypeptide having anti-thrombin activity is prepared by recombinant DNA methodologies. The hirudin or hirudin-like polypeptide is preferably expressed in E. coli or insect cells. The hirudin-like polypeptide is preferably a hybrid protein from HV1 and HV2 having the following amino acid sequence: HV12: Val-Val-Tyr-Thr-Asp-Cys-Thr-Glu-Ser-Gly-Gln-Asn-Leu-Cys-Leu-Cys-Glu-Gly-Ser-Asn-Val-Cys-Gly-Gln-Gly-Asn-Lys-Cys-Ile-Leu-Gly-Ser-Asp-Gly-Glu-Lys-Asn-Gln-Cys-Val-Thr-Gly-Glu-Gly-Thr-Pro-Asn-Pro-Glu-Ser-His-Asn-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-Gln where the underlined sequence is the HV2 portion.

Description

METHOD FOR THE RECOMBINANT PRODUCTION OF HIRIDIUS AND HIRUDIN-LIKE POLYPEPTID

The present invention relates to the preparation of hirudin, which was originally isolated from the leech Hirudo 5 medicinalis. as well as to the preparation of derivatives thereof.

The most popular anticoagulant peptides are probably those belonging to the family of hirudins. Hirudin, originally isolated from the medicinal leech,

¹⁰ Hirudo medicinalis_r is a well known and well characterize_d polypeptidic inhibitor of thrombin¹'². More particularly, it binds thrombin by ionic interactions thus preventing the cleavage of fibrinogen to fibrin and the subsequent fibrin- clot formation. In animal studies hirudin has demonstrated 5 efficacy in preventing venous thrombosis, vascular shunt occlusion and thrombin-induced disseminated intravascular coagulation. In addition, hirudin exhibits low toxicity, little or no antigenicity and a very short clearance time from circulation³. ⁰ Three natural variants of hirudin are known. The sequence of a first variant designated HV1 was determined by Dodt et al, FEBS 165 (1984) 180-184. The sequence of HV1 is, according to the three letter code (Eur. J. Biochem. 13£, 9-37, 1984):

5 Val-Val-Tyr-Thr-Asp-^Cys-Thr-Glu-Ser-Gly-Gln-Asn-Leu-Cys-Leu- Cys-Glu-Gly-Ser-Asn-Val-Cys-Gly-Gln-Gly-Asn-Lys-Cys-Ile-Leu- Gly-Ser-Asp-Gly-Glu-Lys-Asn-Gln-Cys-Val-Thr-Gly-Glu-Gly-Thr- Pro-Lys-Pro-Gln-Ser-His-Asn-Asp-Gly-Asp-Phe-Glu-Glu-Ile-Pro- Glu-Glu-Tyr-Leu-Gln. 0 so₃H

A second variant designated HV2 has been described by Dodt et al, Biol. Chem. Hoppe-Seyler 367 (1986) 803-811. HV2 differs from HV1 in the following respects: _lie at position 1 instead of Val, Thr at position 2 instead of Val, Lys at position 24 instead of Gin, Asn at position 33 instead of Asp, Lys at position 35 instead of Glu, Gly at position 36 instead of Lys, Asn at position 47 instead of Lys, Glu at position 49 instead of Gin and Asn at position 53 instead of Asp. A third variant designated HV3 has been described by Harvey et al, Proc. Natl. Acad. Sci. USA (1986) 1084- 1088. HV3 is identical to HV2 from positions 1 to 32 and then differs from HV1 in the following respects: Gin at position 33 instead of Asp, Lys at position 35 instead of Glu, Asp at position 36 instead of Lys, Gin at position 53 instead of Asp, Pro at position 58 instead of Glu, Asp at position 62 instead of Glu, Ala at position 63 instead of Tyr (SO₃H) , Asp at position 64 instead of Leu and Glu at position 65 instead of Gin. A new approach to the preparation of hirudins and hirudin-like polypeptide has now been devised. This approach is based on chemical synthesis of a nucleotide sequence encoding a hirudin or a hirudin-like polypeptide, and expression of the hirudin or hirudin-like polypeptide in recombinant organisms. The cultivation of the genetically modified organisms leads to the production of the desired product displaying full biological activity.

Accordingly, the present invention provides an expression vector comprising a DNA sequence encoding a hirudin or hirudin-like polypeptide. The invention further provides a host transformed with a compatible expression vector according to the invention, and also provides a synthetic DNA encoding a hirudin or hirudin-like polypeptide. A DNA fragment encoding a hirudin or hirudin- like polypeptide may be single or double stranded. A host in which a hirudin or hirudin-like polypeptide is able to be expressed is prepared by transforming a host with a compatible expression vector of the invention. The expression vector is generally prepared by:

(a) chemically synthesising DNA encoding a hirudin or hirudin-like polypeptide; and

(b) inserting the said DNA into an expression vector.

A hirudin or hirudin-like polypeptide is consequently prepared by providing a transformed host according to the invention under such conditions that a hirudin or hirudin-like polypeptide is expressed therein. The hirudin or hirudin-like polypeptide can then be isolated. In this way, a hirudin or hirudin-like polypeptide may be obtained in pure form.

The term "a hirudin or hirudin-like polypeptide" as used herein refers to hirudin in its natural HV1, HV2 and HV3 forms as well as derivatives thereof, e.g. by way of amino acid substitutions, deletions, insertions, extensions, functionalisations and chemical modifications. The invention can therefore also be applied to derivatives of HV1, HV2 or HV3 having anti-thrombin activity.

The amino acid sequence HVl, HV2 or HV3 may be modified by one or more amino acid substitutions, insertions and/or deletions and/or by an extension at either or each end. A derivative composed of such a modified sequence must of course still exhibit anti-thrombin activity. Typically there is a degree of homology of 75% or more between the amino acid sequence of HVl, HV2 or HV3 and the amino acid sequence of a derivative thereof. The degree of homology may be 85% or more or 95% or more.

For example, one or more amino acid residues of the sequence of HVl, HV2 or HV3 may be substituted or deleted or one or more additional amino acid residues may be inserted. The physicochemical character of the original sequence can be preserved, i.e. in terms of charge density, hydrophobicity/hydrophilicity, size and configuration. Candidate substitutions are, based on the one-letter code (Eur. J. Biochem. 138. 9-37, 1984): A for G and vice versa, V by A, L or G; K by R;

S by T and vice versa; E for D and vice versa; and Q by N and vice versa. As far as extensions are concerned, a short sequence of up to 50 amino acid residues may be provided at either or each terminal. The sequence may have up to 30, for example up to 20 or up to 10, amino acid residues. The hirudin or hirudin-like polypeptide may be subjected to one or more post-translational modification such as glycosylation, sulphation, COOH-amidation, acylation or chemical alterations of the polypeptide chain. The Tyr residue at position 63, for example, may be sulphated. A recombinant hirudin or hirudin-like polypeptide obtained according to the invention would not normally be sulphated at this position, unlike the natural HVl, HV2 and HV3. Further, the invention may be applied to the production of lower molecular weight derivatives which do not have the N- terminal or C-terminal portions of HVl, HV2 or HV3. The invention is particularly applicable to the production of HVl and of a HVl derivative composed of the first 46 residues of HVl followed by the amino acid sequence from residue 47 to 65 of the HV2 variant: HVl: Val-Val-Tyr-Thr-Asp-Cys-Thr-Glu-Ser-Gly-Gln-Asn-Leu-Cys-Leu- Cys-Glu-Gly-Ser-Asn-Val-Cys-Gly-Gln-Gly-Asn-Lys-Cys-Ile-Leu- Gly-Ser-Asp-Gly-Glu-Lys-Asn-Gln-Cys-Val-Thr-Gly-Glu-Gly-Thr- Pro-Lys-Pro-Gln-Ser-His-Asn-Asp-Gly-Asp-Phe-Glu-Glu-Ile-Pro- Glu-Glu-Tyr-Leu-Gln

Hybrid HV1/HV2 (designated HV12) :

Val-Val-Tyr-Thr-Asp-Cys-Thr-Glu-Ser-Gly-Gln-Asn-Leu-Cys-Leu- Cys-Glu-Gly-Ser-Asn-Val-Cys-Gly-Gln-Gly-Asn-Lys-Cys-Ile-Leu- Gly-Ser-Asp-Gly-Glu-Lys-Asn-Gln-Cys-Val-Thr-Gly-Glu-Gly-Thr- Pro-Asn-Pro-Glu-Ser-His-Asn-Asn-Glv-Asp-Phe-Glu-Glu-Ile-Pro- Glu-Glu-Tyr-Leu-Gln where the underlined sequence is the HV2 portion. The polypeptide HV12 as depicted above and derivatives thereof form another aspect of the invention.

The hirudins and hirudin-like polypeptides are prepared by recombinant DNA technology. A synthetic gene encoding a hirudin or hirudin-like polypeptide is prepared. The DNA coding sequence typically does not include introns. The synthetic gene is inserted in an expression vector able to drive production of the recombinant product. The synthetic gene is typically prepared by chemically synthesising oligonucleotides which, in total, correspond to the desired gene. The oligonucleotides are then assembled to obtain the gene.

A gene may therefore be constructed from four chemically synthesised oligonucleotides, each oligonucleotide representing about half of one strand of a double-stranded DNA gene. The oligonucleotides are ligated and annealed to obtain the desired gene. If desired, the gene sequence may be modified by site-directed mutagenesis to introduce one or more codon changes. Typically, a gene is constructed with restriction sites at each end to facilitate its subsequent manipulation.

A preferred DNA sequence encoding HVl is shown in Figure 1 of the accompanying drawings. A preferred DNA sequence encoding HV12 is shown in Figure 3. Either sequence may be modified to code for a derivative.

A DNA sequence may be provided which further encodes a leader peptide. The leader peptide is capable of directing secretion of the hirudin or hirudin-like polypeptide from cells in which the hirudin or hirudin-like polypeptide is to be expressed. The sequence encoding the leader peptide is typically fused to the 5'-end of the DNA sequence encoding the hirudin or hirudin-like polypeptide. The leader peptide is preferably the OmpA leader peptide when expression in a bacterial host, such as E. coli. is required. The leader peptide is preferably the leader peptide of vesicular stomatitis virus G protein (VSV G protein) where expression is to be in insect cells. Appropriate DNA sequences encoding the OmpA and VSV G protein leader sequences are shown in Figures 5 and 8 respectively.

A DNA sequence may be provided which encodes a fusion protein which is cleavable to release a hirudin or hirudin-like polypeptide. A DNA sequence may be used which encodes a carrier polypeptide sequence fused via a cleavable linkage to the N-terminus of a hirudin or hirudin-like polypeptide. The cleavable linkage may be one cleavable by cyanogen bromide.

For expression of a hirudin or hirudin-like polypeptide, an expression vector is constructed which comprises a DNA sequence encoding a hirudin or hirudin-like polypeptide and which is capable of expressing the hirudin or hirudin-like polypeptide when provided in a suitable host. Appropriate transcriptional and translational control elements are provided, including a promoter for the DNA sequence, a transcriptional termination site, and translational start and stop codons. The DNA sequence is provided in the correct frame such as to enable expression of the polypeptide to occur in a host compatible with the vector. The expression vector typically comprises an origin of replication and, if desired, a selectable marker gene such as an antibiotic resistance gene. A promoter is operably linked to the DNA sequence encoding a hirudin or hirudin-like polypeptide. The expression vector may be a plasmid. In that case, preferably a promoter selected from the P-trp ^{anc p}lcc/lac promoters is operably linked to the DNA sequence. Alternatively, the expression vector may be a virus. The virus may be a recombinant baculovirus in which the polyhedrin promoter is operably linked to the DNA sequence encoding a hirudin or hirudin-like polypeptide. An expression vector capable of expressing a hirudin or hirudin-like polypeptide may be prepared in any convenient fashion. A DNA fragment encoding hirudin or hirudin-like polypeptide may be inserted into an appropriate restriction site of an expression vector, for example a plasmid vector. A recombinant baculovirus may be prepared by:

(i) cloning a gene encoding a hirudin or hirudin- like polypeptide into a baculovirus transfer vector at a restriction site downstream of the polyhedrin promoter; and (ii) co-transfecting insect cells susceptible to baculovirus infection with the recombinant transfer vector from step (i) and intact wild-type baculovirus DNA.

Homologous recombination occurs, resulting in a recombinant baculovirus harbouring the hirudin gene or gene encoding a hirudin-like polypeptide downstream of the polyhedrin promoter. The baculovirus transfer vector may be one having a unique cloning site downstream of the polyhedrin ATG start codon. The product that is then expressed by the resulting recombinant baculovirus will be a fusion protein in which a N-terminal portion of the polyhedrin protein is fused to the N-terminus of a hirudin or hirudin-like polypeptide. As indicated above, a cleavable linkage may be provided at the fusion junction. The insect cells employed in step (ii) are typically Spodoptera fruqiperda cells. The wild-type baculovirus is typically Autoσrapha californica nuclear polyhedrosis virus (AcNPV) .

An expression vector encoding a hirudin or hirudin- like polypeptide is provided in an appropriate host. Cells are transformed with the gene for a hirudin or hirudin-like polypeptide. A transformed host is provided under such conditions that the hirudin or hirudin-like polypeptide is expressed therein. Transformed cells, for example, are cultivated so as to enable expression to occur. Any compatible host-vector system may be employed.

The transformed host may be a prokaryotic or eukaryotic host. A bacterial or yeast host may be employed, for example E. coli or S. cerevisiae. Gram positive bacteria may be employed. A preferred bacterial host is a strain of E. coli type B. Insect cells can alternatively be used, in which case a baculovirus expression system is appropriate. The insect cells are typically Soodoptera fruqiperda cells. As a further alternative, cells of a mammalian cell line may be transformed. A transgenic animal, for example a non-human mammal, may be provided in which a hirudin or hirudin-like polypeptide is produced. The hirudin or hirudin-like polypeptide that is expressed may be isolated and purified.

The hirudin or hirudin-like polypeptide prepared according to the invention may be used in a pharmaceutical formulation, together with a pharmaceutically acceptable carrier or excipient therefor. Such a formulation is typically for intravenous administration (in which case the carrier is generally sterile saline or water of acceptable purity) . The hirudin or hirudin-like polypeptide prepared according to the invention is an anti-thrombin and is suitable for treatment of thromboembolic events, such as the coagulation of blood, typically in a human patient. In one embodiment of the invention, the hirudin or hirudin-like polypeptide is coadministered with a plasminogen activator, such as tissue plasminogen activator. The hirudin or hirudin-like polypeptide prepared according to the invention has been found to be compatible with the latter.

The following Examples illustrate the invention.

In the accompanying drawings:

Figure 1 shows the nucleotide sequence of the four oligonucleotides coding for most of the hirudin HVl chain. The sequence underlined indicates the Ball site which has been used for further constructions. The lower part of the

Figure shows the mode of assembling of the four oligos. Hindlll and PstI sites were included to allow subsequent manipulations.

Figure 2 shows the scheme of the construction of the intermediate plasmid M13-HV1, which is the source of a Ball-BamHI DNA fragment for all further bufrudin constructions.

Figure 3 shows the nucleotide sequence of the four oligonucleotides coding for most of the hirudin HV12 chain.

The sequence underlined indicates the Ball site which has been used for further constructions. The lower part of the figure shows the mode of assembling of the four oligos.

Hindlll and PstI sites were included to allow subsequent manipulations.

Figure 4 shows the scheme of the construction of the intermeidate plasmid M13-HV12, which is the source of a Ball-BamHI DNA fragment for all further hirudin constructions.

Figure 5 shows schematically the construction of new recombinant M13s, named OMP-HV1 and OMP-HV12, which carry respectively the complete HVl gene and the complete HV12 gene linked to the OmpA leader peptide. The leader peptide sequence is underlined twice while the Ball blunt end and the Hindlll sticky end are underlined once.

Figure 6 shows schematically the construction of pFC-HVl and pFC-HV12 which are the plasmids used for the production of HVl and HV12 in E. coli.

Figure 7 shows the general structure of the plasmid pOMP-HVl used for the production of hirudin in E. coli. We employed traditional gene manipulation techniques to prepare this new plasmid where the hirudin gene is under transcriptional control of the hybrid promoter Pipp/iac* Even in this case, the OmpA leader peptide drives secretion of bufrudin to the periplasm of E. coli.

Figure 8 shows the nucleotide sequence and assembling of the synthetic oligos used for the secretion of hirudin from insect cells. The sequence underlined indicates the VSV G protein leader peptide.

Figure 9 is a schematic representation of the construction of a new recombinant M13, named VSV-HV12, where the complete bufrudin gene is linked to the VSV G protein leader peptide. Figure 10 shows schematically the construction of pAc-HV12 which has been used as a transfer vector to the baculovirus genome. pAcYMl is the starting plasmid widely used as an acceptor of heterologous sequences to be transferred to the virus. Figure 11 shows the nucleotide sequence and assembling of the synthetic oligos coding for the beginning of the hirudin chain. The ATG codon coding for the additional methionine residue is underlined.

Figure 12 shows schematically the construction of pAcFTl which has been used for intracellular hirudin expression.

Figure 13 is a schematic representation of the new transfer plasmids, named pAcFTl-HVl and pAcFTl-HV12, which carry respectively the complete HVl and HV12 sequences linked to the first 18 amino acids of polyhedrin. These plasmids have been used to transfer the heterologous sequence to the baculovirus genome.

Example 1: Chemical synthesis of the HVl and HV12 genes

The nucleotide coding sequences were designed on the basis of the Escherichia coli preferred codons⁴.

Moreover, a Ball restriction site was engineered very close to the 5' end of the synthetic genes to allow insertion of the coding sequences in different expression vectors. Indeed, the same synthetic genes were used for expression in bacterial and insect cells. In the case of insect cells methods were developed which yielded secreted or cytoplasmic products.

All plasmid DNA manipulations were carried out as described by Maniatis e£ al⁵. A HVl gene was synthesised and assembled as follows. Four synthetic complementary oligonucleotides were prepared using an automated DNA synthesiser (Applied Biosystems) and their sequence is shown in Figure 1. Following enzymatic phosphorylation the four oligos were assembled using DNA ligase and the resulting double-strand sequence was inserted in the M13 phage vector mpl8, obtaining the recombinant plasmid M13-HV1 which is shown in Figure 2. In order to enable insertion of the hirudin gene in the M13 vector, Hindlll and PstI sites were also added in the synthetic oligos. The correct nucleotide sequence has been verified by the Sanger method carried out on the single strand phage DNA⁶.

The recombinant plasmid M13-HV1 was used as the source of the HVl gene for all the expression vectors used in the Examples.

A HVl2 gene was synthesised and assembled in the same way. The oligos used to assemble the gene are shown in Figure 3. Oligos 3 and 4 code for different amino acids than oligos 3 and 4 of Figure 1. A recombinant plasmid M13- HV12, shown in Figure 3, was obtained.

Example 2: Expression and secretion of hirudin from E. coli cells

In order to obtain secretion to the periplasm of the recombinant product, it is necessary to synthesize the HVl and HV12 molecules each in the form of a pre-protein. More particularly, an amino acid sequence named "leader peptide", responsible for an efficient secretion must be present at the NH₂ end of HVl or HV12⁷'⁸. This extra sequence is then cleaved off. In vivo, during secretion, by a specific E. coli leader peptidase, yielding the correct mature sequence⁹.

Many examples of secretion systems have been described in the literature¹⁰'¹¹. Among them, we have selected the system based on the secretion signal of the Outer Membrane Protein of E. coli (Omp A) previously published¹². We therefore designed two additional complementary oligonucleotides coding for the OmpA leader peptide preceded by the OmpA Shine-Dalgarno sequence known to be responsible for an efficient translation of the messenger RNA¹³.

Their sequence, shown in Fig. 5, includes also the beginning of the HVl gene coding for the first 10 amino acids. The presence of the Ball site allowed the joining of this synthetic piece to the rest of the HVl coding sequence while the presence of the upstream Hindlll site allowed the joining to the M13 vector. Thus, the synthetic Hindlll-Ball fragment was ligated to a Ball-BamHI piece from M13-HV1 and inserted in M13mpl8, obtaining a new plasmid named OMP-HV1. The schematic representation of this new plasmid construction is also shown in Figure 5. Equivalent manipulations starting from M13-HV12 gave OMP-HV12 (Figure 5).

From 0MP-HV1 the hirudin gene can be excised as a Hindlll-BamHI fragment which codes for the OmpA Shine- Dalgarno and leader peptide followed by the hirudin coding sequence. This restriction fragment is now ready to be inserted in an appropriate expression vector. Several expression systems could, theoretically, be employed to obtain high level production of heterologous proteins in bacteria. The system based on the promoter Ptrp ^^as bee used with success in our laboratory in the past¹³. Again, even in the case of the selected promoter, the levels of expression of a given polypeptide cannot be predicted. The use of the promoter P rp °^{r t}*^ιe expression of hirudin has never been reported to date.

Plasmid pFC33, shown in Figure 6, has already been described in the literature¹³. It carries the resistance to the antibiotic ampicillin and the bacterial promoter Ptrp which drives expression of proapolipoprotein Al. Following digestion of pFC33 with Hindlll and BamHI, the large Hindlll-BamHI fragment, carrying the antibiotic resistance gene and the promoter, was isolated and joined to the Hindlll-BamHI fragment from OMP-HV1 or from OMP-HV12 coding for the hirudin HVl or the hirudin derivative HV12. The details of this construction are shown in Figure 6. We isolated new plasmids, named pFC-HVl and pFC-HV12, which are the final plasmids for the production of HVl and HV12 in E. coli.

A main object of the present invention is the use of E. coli strains of the type B for the expression and secretion to the periplasm of hirudin and its derivatives. Indeed, we have found that insertion of plasmid pFC-HVl in type B strains of the bacterium E. coli brings high level production of hirudin. Interestingly, different strain types of E. coli do not work as efficiently and it seems, therefore, that the host strain type is crucial for the successful production of hirudins.

Several type B strains of E. coli are available and can be used for the production of a hirudin or hirudin-like polypeptide. Preferred strains are ATCC 12407, ATCC 11303, NCTC 10537. Below is an example of transformation of strain NCTC 10537 with plasmid pFC-HVl and subsequent cultivation of the transformant.

Competent cells of strain NCTC 10537 were prepared using the calcium chloride procedure of Mandel and Higa¹⁴. Approximately 200μl of a preparation of these cells at 1 x 10⁹ cells per milliliter were transformed with 2μl of plasmid DNA (approximate concentration 5μg/ml) . Transformants were selected on plates of L-agar containing lOOμg/ml ampicillin. Two small colonies were streaked with wooden tooth picks (each as three streaks about 1 cm long) onto L-agar containing the same antibiotic. After 12 hours incubation at 37°C, portions of the streaks were tested for hirudin production by inoculation onto 10 ml of LB medium (containing ampicillin at a concentration of 150μg/ml) and incubated overnight at 37°C. The following day the cultures were diluted 1:100 in M9 medium, containing the same concentration of ampicillin, and incubated for 6 hours at 37°C.

20 ml of such culture were centrifuged at 12000xg, 4°C, for 10 minutes. The bacterial pellet was resuspended in 2 ml of 33 mM HCl Tris pH 8; an equal volume of a second solution 33 mM EDTA, 40% sucrose was then added and the total mixture was incubated under mild shaking conditions at 37°C for 10 minutes. Following centrifugation, the permeabilized cells were resuspended in 2 ml of cold water and left for 10 minutes in ice. The resulting supernatant was isolated by centrifugation and represents the periplasmic fraction of the bacterial cell.

Using a chromogenic assay that is based on the inhibition of the thrombin ability to cleave a synthetic substrate S-2238¹⁵, we have measured the presence of anti- thrombin activity in the periplasmic fraction of hirudin- produσing cells. In these samples, hirudin activity was present at the level of about 50μg/ml. This activity was absent in control periplasmic fractions. In the case of pFC-HVl^ the productivity of the hirudin variant HV12 was equivalent to 80 μg/ml.

With the similar approach we have also constructed a new expression/secretion plasmid for hirudin where the promoter Pipp/iac¹⁶ ^^s present instead of the promoter Ptrp* This different plasmid, named pOMP-HVl, is shown in Figure 7. Following insertion of this plasmid in E. coli strains of the type B, high levels of active hirudin were also obtained (40-80 μg/ml) . As starting plasmid for the construction of pOMP-HVl we used the plasmid pIN-III-ompA3 described by Ghrayb et al¹⁶. Conditions for cultivation and induction of expression with isopropyl-3-D- thiogalactopyranoside (IPTG) were as previously described¹⁶.

Example 3: Anticoagulant activity of recombinant HVl obtained from E. coli

The anticoagulant activity of the hirudin variant HVl was also tested in an activated partial thromboplastin time (aPTT) test and in a thrombin time (T.T.) test. Both tests were performed using an automatic coagulometer (ACL300 Research, Instrumentation Laboratory, Milan, Italy) . To normal citrated human plasma, were added increasing concentrations of the recombinantly produced hirudin HVl. The samples were then assayed with the automatic coagulometer which permits determination of aPTT and T.T. by adding automatically to the plasma the appropriate reagents and recording the rate of formation of the clot. aPTT was determined using cephaline and calcium chloride (automated APTT reagent, General Diagnostic, USA) and T.T. was determined using human thrombin (Fibrindex, Ortho Diagnostic, Milan, Italy) at a concentration of 5IU/ml. The reagents were prepared, stored and used according to the manufacturer's instructions.

The clotting times obtained in the aPTT and T.T. tests were plotted against the concentrations of the recombinant protein. In each test, the concentration which doubled clotting times relative to a normal plasma was calculated. The value obtained for the aPTT test was 210 ng/ml, whereas for the T.T. test the value was 90 ng/ml.

Example 4: Expression and secretion of HV12 from insect cells

To obtain secretion of HV12 from recombinant insect cells we had to join the HV12 coding sequence to a leader peptide that is efficiently recognized by these cells. We have used the leader peptide of the Vescicular Stomatitis Virus (VSV) G protein¹⁷. The use of such sequence for the production of hirudin or its derivatives in insect cells has never been reported to date. Similarly to what is described above, a synthetic DNA sequence coding for the VSV G protein leader peptide followed by the beginning of the HV12 gene has been prepared and the nucleotide sequence is given in Figure 8. Also in this case we provided convenient restriction sites (Hindlll, BamHI and Ball) to allow joining to the rest of the HV12 gene and to the expression vector.

The synthetic Hindlll-Ball fragment was joined to a purified Ball-BamHI fragment from M13-HV12 carrying the HV12 gene and inserted in M13mpl8 previously cut with Hindlll and BamHI. This construction which yielded a new plasmid named VSV-HV12 is schematically shown in Figure 9. From VSV-HV12 we have excised a BamHI-BamHI DNA fragment carrying the HV12 gene fused to the VSV leader peptide which was then inserted into the vector pAcYMl¹⁸, as shown in Figure 10. The resulting plasmid was named pAc-HV12.

To obtain expression in insect cells, the VSV-HV12 coding sequence must be transferred to the baculovirus genome under the transcriptional control of the polyhedrin promoter. For this purpose, we co-transfected insect cells with a wild-type baculovirus DNA and with the transfer vector pAc-HV12. As insect cells, Spodoptera frugiperda cells were chosen as host cells. Experimental details are as follows: S. frugiperda cells were transfected with a mixture of infectious AcNPV DNA and plasmid DNA representing the individual recombinant transfer vectors by a modification of the procedure described by Summers et al¹⁹. One microgram of viral DNA was mixed with 25-100 μg of plasmid DNA and precipitated with (final concentrations) 0.125 M calcium chloride in the presence of 20 mM HEPES buffer, pH 7.5, 1 mM disodium hydrogen orthophosphate, 5mM potassium chloride, 140 mM sodium chloride and 10 mM glucose (total volume 1ml) . The DNA suspension was inoculated onto a monolayer of 10⁶ S. fruqiperda cells in a 35-mm tissue culture dish, allowed to adsorb to the cells for 1 h at room temperature, then replaced with 1 ml of medium. After incubation at 28°c for 3 days the supernatant fluids were harvested and used to produce plaques in S. fruqiperda cell monolayers. Plaques containing recombinant virus were identified by their lack of polyhedra when examined by light microscopy. Virus from such plaques was recovered and after further plaque purification was used to produce polyhedrin-negative virus stocks.

The above procedure allowed us to isolate a recombinant baculovirus whose genome carried the HV12 gene under control of the polyhedrin promoter and of the VSV G protein leader peptide. We used this virus to infect S. frugiperda cells according to well-established procedures¹⁹, at a multiplicity of infection of 10. Infected cells were then cultivated in spinner culture or in monolayers in the presence of 10% foetal calf serum according to published methods¹⁹. At different times post-infection, anti-thrombin activity (ATU) was measured in the supernatant using the S- 2238 chromogenic assay. The following Table summarizes the results:

Table: HIRUDIN ACTIVITY ATU/10⁶ cells

time post-infection 0 hours 24 hrs 48 hrs 72 hrs Spinner culture 0.8 0.9 1.4 1.8 Monolayers 0.8 0.8 2.0 5.1

Example 5: Expression of HVl and HV12 in the cytoplasm of insect cells

Hirudin and its derivatives could also be produced and accumulated in the cytoplasm of S. fruqiperda cells.

This approach generally gives a better yield of heterologous proteins since it utilizes the expression signals of polyhedrin which is a non-secreted viral protein.

Our approach to obtain large quantities of recombinant HVl and HV12 is based on the expression of a fusion polypeptide where the first 18 amino acids of polyhedrin are joined in frame to the 65 amino acids of HVl or HV12. The presence of the NH₂ end sequence of polyhedrin allows high level expression²⁰. In addition, between the polyhedrin portion and the HVl or HV12 sequence we put a methionine residue which allows the release of the HVl or HV12 moiety by treatment of the hybrid protein with CNBr.

Similarly to the previous approaches, we prepared a synthetic DNA fragment which could allow the joining of the Ball-BamHI fragment from M13-HV1 or M13-HV12 to an appropriate transfer vector. The new synthetic piece, shown in Figure 11, includes also BamHI and Ball sites for subsequent manipulations. A different transfer vector, pAcFTl, carrying the nucleotide sequence coding for the first 18 amino acids of polyhedrin has been obtained (Figure 12) . Briefly, the EcoRV-BamHI fragment of pAcYMl¹⁸ has been replaced by a synthetic oligonucleotide containing the polyhedrin gene sequence from nucleotide -92 to nucleotide +55. A convenient BamHI site is present after this sequence and it has been used for insertion of the complete HVl or HV12 coding sequence according to a scheme illustrated in Figure 13. Through this construction, we obtained two new plasmids, named pAcFTl-HVl and pAcFTl-HV12, which have been used to transfer the hybrid genes to the baculovirus genome.

The recombinant baculoviruses were obtained as described in Example 4. Infection of S. fruqiperda cells was carried out according to standard procedures¹⁹. Cultivation of infected insect cells lead to the cytoplasmic accumulation of the fusion protein. This hybrid protein was the source of recombinant HVl or HV12. Several methods are available from the literature which can be used to cleave the hybrid with CNBr²¹'²². The application of the method of Olson et al²². has allowed us to obtain the HVl and HV12 of the correct polypeptide sequences. These two molecules displayed anti-thrombin activity.

References

1) Markwardt, F. 1970, Methods in Enzymology, .19, P« 92

2) Markwardt, F. 1985, Biomed. Biochim. Acta. 4j4_, p. 1007 3) Markwardt, F. Hauptmann, J. , Nowak, G. , Klessen, C, and Walsmann, P. 1982. Thromb. Haeroostasis 47, p. 226.

4) Grosjeans H. and Fiers W. 1982. Gene, ifJ, p. 199

5) Maniatis T. , Fritsch E.F. and Sambrook J. 1982. Cold Spring Harbor, NY 6) Sanger, F. , Nicklen, S., and Coulson, A.R. 1977, Proc. Natl. Acad. Sci. USA 74, p. 5463.

7) Blobel G. and Dobberstain B. 1975. J. Cell Biology, 67. p. 83

8) Pages J.M. 1983, Biochimie, 65., p. 531 9) Wolfe P.B. 1983. J. Biol. Chem. Z58., P« 12073

10) Talmadge K. , Stahl S. and Gilbert W. 1980. Proc. Natl. Acad. Sci. USA, 77, p. 3369

11) Oka T., Sakamoto S., Miyoshi K. , Fuwa T. , Yoda K. , Yamasaki M. , Ta ura G. and Miyake K. 1985. Proc. Natl. Acad. Sci. USA, 81, p. 7212

12) Henning V., Royer H.D., Teather R.M. , Hindennach I. and Hollenberg C.P. 1979. Proc. Natl. Acad. Sci. USA, 76. p. 4360

13) Isacchi A., Sarmientos P., Lorenzetti R. and Soria M. 1989, Gene £1, p. 129

14) Mandel M. and Higa A.J. 1970. J. Mol. Biology, 53, p. 154

15) Krstenansky, J.K. , and Mao, S.J.T. 1987. FEBS Lett. 211_f p. 10 16) Ghrayeb J., Kimura H. , Takahara M. , Hsiung H. , Masui Y. and Inouye M. 1984. EMBO Journal 3., p. 2437

17) Bailey, M.J. , McLeod, D.A. , Kang, C. , and Bishop, D.H.L. 1989. Virology 169, p. 323

18) Matsuura, Y. , Possee, R.D., Overton, H.A. and Bishop. D.H.L. 1987. J. Gen. Virol. 68_/ P- 1233 19) Summers, M.D., and Smith, G.E. 1987, Texas Agricultural Experiment Station Bulletin No. 1555

20) Luckow, V.A. and Summers, M.D. 1988, Virology, 167. p.56

21) Gross E. 1967. Methods in Enzymology, XX, p. 238 22) Olson H. , Lind P., Pohl G. , Henrichson C. , Mutt V., Jornvall H. , Josephson S., Uhlen M_% and Lake M. 1987, Peptides, , p. 301

Claims

1. An expression vector comprising a DNA sequence encoding a hirudin or a hirudin-like polypeptide.

2. A vector according to claim 1, which is a plasmid.

3. A vector according to claim 2, wherein a promoter selected from the Ptrp ^{and p}lpp/lac promoters is operably linked to the said DNA sequence.

4. A vector according to claim 1, which is a virus.

5. A vector according to claim 4, wherein the virus is a recombinant baculovirus in which the polyhedrin promoter is operably linked to the said DNA sequence.

6. A vector according to any one of the preceding claims, wherein the said DNA sequence further encodes a leader peptide capable of directing secretion of the said hirudin or hirudin-like polypeptide from cells in which the said hirudin or hirudin-like polypeptide is expressed.

7. A vector according to claim 6, wherein the leader peptide is the OmpA or VSV G protein leader peptide.

8. A vector according to any one of claims 1 to 5, wherein the said DNA sequence encodes a fusion protein which is cleavable to release the said hirudin or hirudin- like polypeptide.

9. A vector according to claim 8 when dependent upon claim 5, wherein the fusion protein comprises a N- terminal portion of the polyhedrin protein fused via a cleavable linkage to the N-terminus of a hirudin or hirudin- like polypeptide.

10. An expression vector according to any one of the preceding claims, wherein the said hirudin or hirudin- like polypeptide is: HVl: Val-Val-Tyr-Thr-Asp-Cys-Thr-Glu-Ser-Gly-Gln-Asn-Leu-Cys-Leu- Cys-Glu-Gly-Ser-Asn-Val-Cys-Gly-Gln-Gly-Asn-Lys-Cys-Ile-Leu- Gly-Ser-Asp-Gly-Glu-Lys-Asn-Gln-Cys-Val-Thr-Gly-Glu-Gly-Thr- Pro-Lys-Pro-Gln-Ser-His-Asn-Asp-Gly-Asp-Phe-Glu-Glu-Ile-Pro- Glu-Glu-Tyr-Leu-Gln; or

HV12 :

Val-Val-Tyr-Thr-Asp-Cys-Thr-Glu-Ser-Gly-Gln-Asn-Leu-Cys-Leu- Cys-Glu-Gly-Ser-Asn-Val-Cys-Gly-Gln-Gly-Asn-Lys-Cys-Ile-Leu-

Gly-Ser-Asp-Gly-Glu-Lys-Asn-Gln-Cys-Val-Thr-Gly-Glu-Gly-Thr-

Pro-Asn-Pro-Glu-Ser-His-Asn-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-

Glu-Glu-Tyr-Leu-Gln where the underlined sequence is the HV2 portion.

11. A host transformed with a compatible expression vector according to any one of the preceding claims.

12. A host according to claim 11, which is a bacterium.

13. A host according to claim 12, which is a strain of E. coli type B.

14. A host according to claim 11, which is a eucaryotic host selected from yeasts, mammalian cell lines, insect cell lines and animals.

15. A host according to claim 14, which is a

Spodoptera fruqiperda cell line.

16. A synthetic DNA encoding a hirudin or a hirudin-like polypeptide.

17. DNA according to claim 16, which further encodes a leader peptide capable of directing secretion of the said hirudin or hirudin-like polypeptide from cells in which the said hirudin or hirudin-like polypeptide is expressed.

18. DNA according to claim 17, wherein the leader peptide is the OmpA or VSV G protein leader peptide.

19. DNA according to claim 16, wherein the said DNA sequence encodes a fusion protein which is cleavable to release the said hirudin or hirudin-like polypeptide.

20. DNA according to any one of claims 16 to 19, wherein the said hirudin or hirudin-like polypeptide is HVl or HV12 as depicted in claim 10.

21. A process for the preparation of a hirudin or a hirudin-like polypeptide, which process comprises providing a host according to any one of claims 11 to 15 under such conditions that the said hirudin or hirudin-like polypeptide is expressed therein.

22. A process for the preparation of a host in which a hirudin or hirudin-like polypeptide is able to be expressed, which process comprises transforming a host with a compatible expression vector according to any one of claims 1 to 10.

23. A process according to claim 22, wherein the said expression vector has been prepared by:

(a) chemically synthesising DNA encoding the said hirudin or hirudin-like polypeptide; and

(b) inserting the said DNA into an expression vector.

24. The polypeptide HV12 as depicted in claim 10 or a derivative thereof.

25. A pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent and, as active ingredient, a hirudin or hirudin-like polypeptide which has been produced by the process of claim 21 or a polypeptide as claimed in claim 24.